5-methoxy. 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing.

(54) Title: NUCLEOTIDES FOR FAST TERMINATION OF PHOTODES COMPOSITION 5-ΜΕΤΟΧΙ, 3 '-OH UNLOCKED AND METHODS FOR SEQUENCING NUCLEIC ACID.

(54) Title: 5-ΜΕΤΗΟΧΥ. 3'-OH UNBLOCKED, FAST PHOTOCLEAVABLE TERMINATING NUCLEOTIDES AND METHODS FOR NUCLEIC ACID SEQUENCING.

(57) Summary

The present invention generally relates to 5-methoxy-substituted nitrobenzyl-substituted nitrobenzyl-labeled and unlabelled 3'-OH nucleotides and nucleotides that can be photodecomposed for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds can be used as reversible terminators as they exhibit rapid nucleotide uptake kinetics, single base termination, high nucleotide selectivity, and rapid termination group decomposition resulting in naturally occurring nucleotide.

(57) Abstract

The present invention relates generally to 3'-OH unblocked nucleotides and nucleosides labeled and unlabeled with 5methoxy-substituted nitrobenzyl-based photocleavable terminating groups for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.

_SE_ «CRÍTAiifA Ot ¡ta® *

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Mexican Institute of Industrial Property

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PATENT TITLE NO. 342195

Headlines):

Home:

Denomination:

Classification:

Inventor (s):

LASERGEN, INC.

8052 El Rio Street, Houston, Texas, 77054, USA

QUICK PHOTODES COMPOSITION NUCLEOTIDES 5METOXI COMPOSITION, 3'-OH UNLOCKED AND METHODS FOR SEQUENCING NUCLEIC ACID.

lnt.CI.8: C07H19 / 073; C07H19 / 10; C07H19 / 173; C07H19 / 20

BRIAN PHILIP STUPI: HONG Ll; WEIDONG WU; MEGAN N. HERSH; DAVID

HERTZOG; SIDNEY E. MORRIS; MiCHAEL L. METZKER

REQUEST

International filing date!

September 2012

PRIORITY

Number:

MX / a / 2014/003075

Country:

US

US

Validity: Twenty years

Date:

September 2011 October 7, 2011

Number:

61/534,347

61/627,211

Expiration Date: September 13, 2032 'The reference patent is granted based on articles 1, 2nd section V, 6th section III, and 59 of the Industrial Property Law.

In accordance with article 23 of the Industrial Property Law, this patent has a validity of twenty non-expendable years, counted from the date of filing of the international application and will be subject to the payment of the fee to maintain the rights in force. .

Who subscribes to the present title Jo does based on the provisions of toa sMfoutoá 8 * íáeciones III and 7 ° bis 2 of the Industrial Property Law (Official Mario de la Federación (DO F.) 27/0671881, rafbRMHKtte 08080994, 25 / 10/1996, 12/26/1987, 05/17/1999, 01/26/2004, 06/16/2005, 01/25/2006, 06/05/2009, 06/01/2010, 06/18/2010 / 2010, 06/28/2010, 01/27/2012 and 04/09/2012); Articles 1, 3, fraction V, subsection a), 4, and 12, sections I and III of the Regulations of the Mexican Institute of Industrial Property (DOF 12/14/1939, amended on 07/01/2002, 07/15/19) 2004, 28 / C7 / 2004 and 7/09/2007), articles 1, 3, 4, 5, section V, subsection a), 16 sections »I and III and 30 of the Organic Statute of the Mexican Property Institute Industrial (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1, 3 and 5 Clause a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Holders of the Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

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Sand; No 550. Step 1,

Cok Puebic Santa Maris Pepepán, Xoehimilco CP 16020.

Mexico City

Tel. (55) 53 34 G7 00 ww-v.impi goPrn *

Issue Date: September 20, 2016

THE DIVISIONAL DIRECTOR OF PATENTS

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NAHANNY CANAL REYES

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MX / 2016/75385

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NUCLEOTIDES OF TERMINATION OF

MEXICAN INSTITUTE

PHOTODESCOMPOSITION 5-METOXI, 3'-QH NO BWffifflaiÁI

AND SEQUENCING METHODS OF ÁCinn miici Firn

Related Requests

This application receives the benefit of the priority of the North American Provisional Application 61 / 627,21 1, filed on October 7, 2011, and the North American Provisional Application 61 / 534,347, filed on September 13, 2011, where the contents Both applications are incorporated into the present invention by reference.

Field of the Invention

The present invention relates generally to compositions and methods for DNA sequencing and other types of DNA analysis. More particularly, the present invention relates in part to rapid unblocked 3'-OH nucleotides and nucleosides with photochemical decomposition groups and to methods for use in a number of DNA sequencing methods, including applications in biomedical research.

Background of the Invention

Methods for rapid DNA sequencing are necessary to analyze diseases and mutations in the population and developing therapies (Metzker Publication, 2010, which is incorporated in the present invention as

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reference). Commonly observed forms of human sequence variation are single nucleotide polymorphisms (SNPs), occurring in approximately 1 in 300 to 1 in 1000 genomic sequence base pairs and structural variants (SVs) including block substitutions, insertions / deletions, inversions , segmental duplications, and copy number variants. Structural variants may encompass 22% of all variable events and more variant bases than those with SNP contribution (Levy et al., 2001, which is incorporated by reference in the present invention). This discovery is consistent with that of Scherer, Hurles, and colleagues, who analyzed 270 individuals using microformation-based methods (Publication of Redon et al., 2006, which is incorporated by reference in the present invention). In constructing the entire sequence of the human genome, efforts have been made to identify the underlying genetic link to common diseases and cancer through SNP and SV mapping or direct association. Technology developments focused on fast, high-performance, and low-cost DNA sequencing can facilitate the understanding and use of genetic information, such as SNP and SV in applied medicine.

In general, 10% to 15% of SNPs will affect protein function by altering amino acid residues

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IMPI

INsTIT '. SPOUT

Of. The specific i -ivMfOAO, will affect the adequate processing dB'Wí ge by changing the mechanisms of divio'iéw or affect the level of normal expression of the gene or protein, through various regulatory mechanisms. SVs also play an important role in human biology and diseases (Publications of lafrate et al., 2004; Sebat et al., 2004; Tuzun et al., 2005; Stranger et al., 2001, which are incorporated herein invention for reference). The identification of the informative SNPs and SVs is considered to lead to a more accurate diagnosis of inherited disease, a better prognosis of risk susceptibilities or identity of sporadic tissue mutations. An application of an individual's SNP and SV profile can significantly delay disease generation or progression with prophylactic drug therapies. Additionally, a SNP and SV profile of the genes that metabolize the drug can be used to prescribe a specific drug regimen to provide safer and more effective results. To achieve these ambitious goals, genome sequencing will move into the resequencing phase with the potential for partial sequencing of a large majority of the population, which may involve sequencing specific regions in parallel, which are distributed throughout the genome. human to obtain the SNP and SV profile for a given complex disease.

The sequence variations underlying the

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More common diseases, probably involving SNPs, SVs, and a number of combinations of them are scattered throughout the associated genes and exist at low frequencies. Therefore, DNA sequencing technologies that employ de novo sequencing strategies are more likely to detect and / or discover these rare, widely dispersed variants than technologies that target only known SNPs.

An example of how NGS technologies can be applied in the detection of SNP variants, SVs, single nucleotide variants (SNVs) and a number of combinations thereof, is cancer diagnostics. These assays have traditionally been a single marker method, a single trial that has recently progressed to testing multiple markers with a single experimental method. However, each cancer is often genetically complex with many mutations occurring simultaneously in numerous genes. Accordingly, traditional methods lead to expensive and time-consuming tests, while providing information only with respect to a few select select sequence variants. Recent advances in NGS technologies have enabled specific methods that target many medically actionable gene targets associated with various cancers (See Publications in Su et al., 2011; Beadling et al.

IMPI

INSTITUTE «ÍXICANO Di LA?« CWÍiMO

2012). Due to recent successes in eo sequencing, such as the The Canüéf béliume Atlas (TCGA) project, the International Cancer Genome Consortium project (Consortium

Cancer Genome International) (ICGC), and the Catalog of Somatic Mutations in Cancer (COSMIC) database, there is a vast compendium of knowledge regarding these gene targets in many types of cancer, and the result of therapeutics in cancers containing such mutations (See Publication of Futreal et al., 2004). Further work, in part as a result of the Pediatric Cancer Genome Project, has shown that pediatric cancers have different genetic profiles marked by fewer mutations and a prevalence of mutations in alternative molecular pathways (See Wu et al. Publications, 2012; Meldrum et al. 2011). The greatest unmet need today in cancer diagnostics is a fast, high-performance technology with the precision and sensitivity necessary for early-stage detection, to identify rare sequence variants belonging to a limited subpopulation of cells passing through a carcinogenic transformation.

Traditionally, DNA sequencing has been achieved through the “Sanger” or “dideoxi” method, which involves the, O pf IVA £ 5 ι «ϊτ; τυτ &lt;, macano

Of the t'k'OPJtOAp chain termination of DNA synthesis HféWanfí incorporation of 2 ', 3'-dideoxynucleotides (ddÑ ik¿T DNA utilization (Metzker et al., 2005, which is incorporated herein invention for reference.) Since 2005, there has been a fundamental change outside of the application of automatic Sanger sequencing for genome analysis. The benefits of Next Generation Sequencing Technologies (NGS) include the ability to produce a huge volume of data at a low price, in some cases in excess of hundreds of millions of short sequence reads per instrument run.

Many of these methods are commonly referred to as synthesis sequencing (SBS), which does not clearly define the different DNA sequencing mechanics (see Metzker Publications, 2010; Metzker 2005, which are incorporated into the present invention by reference). DNA polymerase dependent strategies have been classified as cyclic reversible termination (CRT), single nucleotide addition (SNA, eg pyrosequencing), and real-time sequencing. A method by which DNA polymerase is replaced by DNA ligase, is referred to as ligation sequencing (SBL). These methods have been described in the Metzker Publication (2010), which is incorporated into the present invention by reference.

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MEXICAN INSTITUTE,

Sequencing technologies Include widely grouped methods γ * ιτι? - (ό) template preparation, (or) sequencing and imaging, and (c) data analysis. The only combination of specific protocols distinguishes one technology from the other, and determines the type of data produced from each platform. These differences in data production present challenges when comparing platforms based on data quality and cost. Although quality ratings and precision estimates are provided by each manufacturer, there is no consensus on a "quality basis" from one platform that is equivalent to that of another platform.

Two methods used to prepare templates for NGS reactions include: clonally amplified templates that originate from simple DNA molecules and templates from simple DNA molecules. Sequencing methods using DNA polymerases are classified as cyclic reversible termination (CRT), single nucleotide addition (SNA), and real-time sequencing (See Metzker Publication 2010). Ligation sequencing (SBL), a method in which DNA polymerase is replaced by DNA polymerase, has also been used in NGS technologies, (see for example the Publications of Shendure et al., 2005; Valouev et al ., 2008). Imaging methods coupled with these sequencing strategies

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ΙΜΓίΤ ·,; τθ ,,.: Ν, „.. 7;

I heard Λ rí ,.^;&gt;: v ^ 3 fluctuate from measuring bioluminescérite signals'S '¹four-color images of mo'TécuIaI events The voluminous data produced by these NGS platforms imposes substantial demands on information technology, in terms of data storage, monitoring and quality control (see Pop &amp; Salzberg Publication, 2008).

The need for robust methods that produce a non-biased nucleic acid material source, representative of the genome under investigation, remains an important goal. Current methods generally involve random break genomic DNA in smaller sizes of which are created in either fragment templates or pairing templates. A common theme among NGS technologies is that the template adheres to or immobilizes a solid surface or support. Immobilization of spatially separated template sites allows thousands to billions of sequencing reactions to be carried out simultaneously.

Although clonally amplified methods offer certain advantages over bacterial cloning, some of the protocols are usually complicated to implement, and require a large amount of genomic DNA material (3 to 20 pg). Preparation of the single molecule templates is simpler and requires less material than

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INSTITUTO MEXICANO de ia iWf '/ ír) * /?

heading (&lt;1 pg). Furthermore, these methods did not require 'Ff'Pi which creates mutations in the clonal amjjTílióüUdb templates, which are disguised as sequence variants. Target sequences with high AT content and high GC content also show amplification trends in product performance, resulting in their underrepresentation in genome assemblies and assemblies. Quantitative applications such as RNA (See Wang et al., 2009 Publication), are most effectively carried out with unamplified template sources, which do not alter the abundance of representation of mRNA molecules.

An important aspect of the CRT method is the reversible terminator, of which there are two main types: 3'-Blocked and 3'-OH unlocked (Metzker, 2010). The use of a ddNTP, which acts as a chain terminator in Sanger sequencing, provided the basis for the initial development of reversible blocking groups attached to the 3 'end of nucleotides (Metzker et al. 1994; Canard &amp; Sarfati, 1994). Blocking groups such as 3'-O-alildNTPs (Metzker et al., 1994; US Patent 6,664,079; Ju et al., 2006; US Patent 7,057,026;

US Patent 7,345, 159; US Patent 7,635,578; North American Patent 7,713,698) and 3'-Oazidomethyl-dNTPs (North American Patent 7,057,026; Guo et al., 2008; Bentley et al., 2008; North American Patent

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MEXICAN INSTITUTE Ot LA TROBÉCAT ·

7,414,116; North American Patent 7,541.4 ^^¹North American 7,592,435; North American Patent /, ΰ'β' Norte North American Patent 7,771,973) have been used in CRT. 3'-0-Blocked terminators require the decomposition of two chemical bonds to remove fluorophore from the nucleobase and restore the 3'-OH group. A drawback in the use of these reversible terminators is that the blocking group attached to the 3 'end usually causes a trend against DNA polymerase incorporation. DNA polymerase mutagenesis is often required to facilitate the incorporation of 3'-Blocked terminators. A large number of DNA polymerases designed «

They have been genetically engineered either by site-directed or random mutagenesis containing one or more amino acid substitutions, insertions and / or deletions and subsequently identified by high-throughput classification with the goal of more efficiently incorporating 3'-blocked nucleotides .

The difficulty in identifying a modified enzyme that efficiently incorporates 3'-0-blocked terminators by classification of large mutant DNA polymerase libraries has led to the development of reversible 3'-unlocked terminators. It was shown that a small group of photodecomposition attached to the base of a 3'-OH-unlocked nucleotide can act as a

ΙΜΡϊ

INSTITUTO MEXICANA DE LA t-ROHECAU INUUSTKIAL effective reversible terminator and is incorporated efficiently through wild type DNA polymerases (Wu et al., 2007; Metzker, 2010; Litosh et al., 2011, Gardner et al., 2012; Patents North American 7,897,737, 7,964,352; and 8, 148,503,

Publication of North American Patent Application

201 1/0287427). For example, 5-hydroxymethyl-2'deoxyuridine (HOMedU) was found naturally in the genomes of numerous lower bacteriophages and eukaryotes (Gommers-Ampt Publication, 1995, which is incorporated by reference in the present invention). Its hydroxymethyl group can serve as molecular management to adhere a small group of photodecomposition termination. Other naturally-derived hypermodified bases that can be further modified to function as reversible terminators include 5-hydroxymethyl-2'-deoxycytidine (HOMedC), which are found naturally in the genomes of bacteriophages T2, T4, and T6 (Wyatt &amp; Cohen, 1953; Gommers-Ampt, 1995) and of mammals (Kriaucionis &amp; Heintz, 2009; Tahiliani et al., 2009, Ito et al., 2010). The pyrrolopyrimidine (7deazapurine) ring structure is also naturally found in nucleoside antibiotics (Carrasco &amp; Vázquez Publication, 1984, which is incorporated by reference in the present invention) and tRNA bases (Limbach, et al., 1994, which is incorporated into the present invention by reference), and the 7deaza-7-hydroxymethyl-2'-deoxyadenosine compounds (C⁷-HOMedA) (Rockhill

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institute ^ íxicamo and^?DE LA ΓΚΟΗΕΟΑΟ V et al., 1997) and 7-deaza-7-hydroxymethyl-2'-d oxigoanosin HOMedG) (McDougall et al., 2001) have been reported.

One aspect of the present invention is the use of a modified 2-nitrobenzyl group attached to the nucleoside nucleobase and hydroxymethyl nucleotides. Described half a century ago, 2-nitrotoluene solutions (Wettermark, 1962) and its derivatives (Wettermark, 1962; Hardwick et al., 1960; Mosher et al., 1960; Sousa &amp; Weinstein, 1962; Weinstein et al.,

1966) were reported as exhibiting the property of photochromism, a phenomenon considered to be the result of the temporary formation of an ac / '- nitro anion intermediate (Weinstein et al., 1966; Morrison, 1969). Without intending to be bound by theory, it is generally accepted that the absorption of a photon by the nitro group results in the hydrogen abstraction of the α-carbon (Mosher et al., 1960; Berson &amp; Brown, 1955; De Mayo, 1960) , the formation of the ac / '- nitron anion intermediate, and subsequently the release of the "caged" effector molecule and the creation of a nitrosocarbonyl by-product (Corrie, 2005). These early studies suggest that an α-substitution of benzyl carbon (Wettermark, 1962) or the substitution of the 4-position of the benzene ring with an electron donation group (Sousa &amp; Weinstein, 1962; Weinstein et al., 1966) , increased the range of photochromic effect. These discoveries lead to the development of 2-nitrobenzyl protecting groups

<img file="MX342195B_D0017.tif" />

Γ. Λ. ~

INSTITUTE «ΕΛΙΟ.Ν '»

FROM THE PP.OHF.OAlS \ photosensitive (Barltrop et al., 1966; Patchorru ^? '*¹· 1

Patchornik et al., 1970). The degree to which the lunyu is altered¹ - Photochemical decomposition normally depends, however, on numerous factors that are reported to include benzyl carbon substitution (Walker et al., 1986; Hasan et al., 1997; Giegrich et al., 1998), the group ( s) functional adhered to the benzyl ring (Wootton &amp; Trentham, 1989; Hasan et al.,

1997; Giegrich et al., 1998), and the starting group (Walker et al., 1986) as well as the pH (McCray et al., 1980; Walker et al.,

1986; Wootton &amp; Trentham, 1989), solvent (Sousa &amp; Weinstein,

1962; McGall et al., 1997; Giegrich et al., 1998), and light intensity (McCray et al., 1980; McGall et al., 1997). A property, however, that has not been studied is stereochemistry, whereby the substitution of the 2-nitrobenzyl α-carbon or benzyl carbon results in a surgical center. For nucleotide synthesis, coupling of racemic α-substituted 2-nitrobenzyl alcohol can result in two diastereomers, differing only by the absolute (R or S) configuration on the benzyl carbon.

Another class of unblocked 3'-OH nucleotides has been described in the Publications of Mitra et al. (2003) and Turcatti et al. (2008), who rely on the spherical obstacle of the bulky ink group to stop incorporation after addition of the first nucleotide. It should be noted that

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<img file="MX342195B_D0019.tif" />

MEXICAN INSTITUTE

DE H3 ·: p | Er&gt; AD substituted 2-nitrobenzyl nucleotide analogs' ^^JSTd^lis by Wu et al. (2007), Lltosh et al. (2011), and Caniliui U al, 7Q1.2. cause termination of DNA synthesis without the requirement for bulky substituents such as fluorescent inks. A further class of 3'-unlocked nucleotides has been described by Helicos Biosciences. These nucleotides use a second nucleoside or nucleotide analog that acts as an inhibitor of DNA synthesis (Bowers et al., 2009; US Patent 7,476,734). A significant difference in termination properties is observed when comparing the compounds of the present invention with those described by Bowers. For example, Bowers et al. described constant prestate kinetics employing two two-base homopolymer templates, for which ranges k were measured_p0|₍₊₂₎ for the three unlocked 3'-OH “virtual” terminators. Bowers et al. he conducted his termination experiments in termination assays of submicromolar nucleotide concentrations (ie, 100 to 250 nM). In contrast, various compounds of the present invention performed at 10 µΜ over a time course of 0.5 to 20 minutes. Both compounds dU.V and dU.VI, were quickly incorporated in the first base position (100% for 2 minutes) and subsequently the finished DNA synthesis in that position. No appreciable signal can be detected at the expected second base position in the times of

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MEXICAN INSTITUTE OF PROPERTY 20 minute incubation. For more details, c'SfflWKar

Publication of Gardner et al., 2012. '...... - -

Unlocked reversible 3'-OH terminators normally have several advantages over 3'-O-blocked nucleotides.

reversible terminators

For example, for many unlocked 3'-OHs the decomposition of only one single bond removes both termination and fluorophore groups from the nucleobase. This in turn results in a more efficient strategy to restore the nucleotide for the next CRT cycle. A second advantage of unlocked 3'-OH reversible terminators is that many of these compounds show more favorable enzyme incorporation, and in some cases, can be incorporated just like a natural nucleotide with wild type DNA polymerases (Wu et al., 2007; Litosh et al., 2011; Gardner et al., 2012; North American Patent 7,897,737;

US Patent 7,964,352; US Patent 8,148,503; North American Patent Application Publication 201 1/0287427), although in other cases, this efficiency has not been observed (Bowers et al., 2009; North American Patent 7,476,734). A challenge for unlocked 3'-OH terminators is to create the appropriate modifications to the base, leading to termination of DNA synthesis after the addition of a single base. This is important because an unlocked 3'-OH group is the substrate

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Eí LA ΓΒΟΠεϋΑΟ natural to incorporate the following nucí ótldo entra'r? F &lt;í?^IAL

Next-generation sequencing technologies (NGS) have facilitated important biomedical discoveries, although chemical improvements are still needed for a number of reasons, including reduced error ranges, reduced slow cycle times. To be effective in NGS assays, it is typically desirable that reversible terminators exhibit a number of ideal properties including, for example, rapid nucleotide uptake kinetics, single base termination, high nucleotide selectivity, and / or rapid decomposition of the pool of termination. For example, there is a need to develop new nucleosides and nucleotides that meet these challenges.

Brief Description of the Invention

In some respects, the present disclosure provides novel compounds and compositions that are useful in efficient sequencing of genomic information in high throughput sequencing reactions. In another aspect, reagents and reagent combinations are provided that can efficiently and producibly provide genomic information. In further aspects, the present invention provides libraries and reagent formations for diagnostic methods and for developing targeted therapeutics for individuals.

In some respects, this description provides ______ _

INSTITUTO MEXtCAN.&gt; DE La _WW. »Ap 'industrial new compounds that can be used in DNA sequencing. For example, the present disclosure provides compounds of the formula:

<img file="MX342195B_D0021.tif" />

<img file="MX342195B_D0022.tif" />

(V),

OH R₂ (VI), or (VII),

<img file="MX342195B_D0023.tif" />

thiothryphosphate or polyphosphate;

R₂ it is hydrogen or hydroxy;

R₃ it's alkyl_(cs8) or alkyl (_cs8) replaced;

R₄ it is hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

I rent₍cs6), acyl_(cs6), alkoxy_(cs6), acyloxy_(Cs6), alkylamino_(CS6), dialkylaminO (_Cs₆). amido_(Cs6), or a substituted version of any of these groups;

R5 and Re each are independently:

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

I rent_(Cs6), alkenylO (_Cs6), alkynyl_(Cs6), aryl_(cs6), aralkyl (_cs8), heteroaryl_(cs6), acyl_(Cs6), alkoxy_(cs6), acyloxy_(Cs6), alkylamino₍cs6), dialkylaminO (_CS6), amido_(cs6), or a substituted version of any of these groups;

a group of the formula:

n

<img file="MX342195B_D0024.tif" />

mn

where

<img file="MX342195B_D0025.tif" />

X is ......... ¹ -------- O-, -S-, or -NH-; or alkanediyl₍csi2), alkenodiyl_(Csi2), alkynediyl₍csi2) · or a substituted version of any of these groups;

Y is -O-, -NH-, alkanediyl_(Csi2) or alkanedi yl_(Cif 2) substituted;

n is an integer from 0 to 6; and m is an integer from 0 to 6; ou ne n lazad o r-reporter;

or a salt, tautomer, or optical isomer thereof.

In some embodiments, the compounds are further defined as a compound of formulas I, II, III, IV, V, VI, or Vil. In some embodiments, Ri is hydroxy, monophosphate, diphosphate, triphosphate, α-thiothriphosphate, or polyphosphate.

In some modalities, R₂ it is hydrogen, hydroxy. In some modalities, R₃ is alkylole), for example alkyl_(C34), including isopropyl or tert-butyl. In some modalities, R₄ it is hydrogen, nitro. In some modalities, R₅ it is hydrogen, iodine or alkoxy_(cs6) i including, for example, methoxy. In some modalities R₅ is a group of the formula:

n where

X is

<img file="MX342195B_D0026.tif" />

alkanediyl_(cs12)alkenodiyl_(cs12), a í &lt;^ ü7TrCTCI 111 u 2'yr arenod¡ilO (_CS; i₂), heteroarenodülO (_CSi2), or a substituted version of any of these groups;

n is is an integer from 0 to 6.

In some modalities, X is a I quinodii I or _(c2.₈₎For example, -C = C- In some modes, n is zero. In some modalities, R₅ is a group of the formula:

<img file="MX342195B_D0027.tif" />

where

X is

-O-, -S-, or -NH-; or alkanediyl_(cs12)alkenodiyl_(Csi2), arenodülo_(cs12), heteroarenodi¡lo₍csi2), or one of any of these groups;

Y is -O-, -NH-, alkanediyl₍c £ i2) substituted;

n is an integer from 0 to 6; and m is an integer from 0 to 6.

alkynediyl_(cSi2), substituted version or alkanediyl₍csi2)

In some embodiments, X is alkynediyl_(c2-8), for example, -C = C- In some embodiments, Y is -CH₂- In some modalities, ns zero. In some embodiments, m is zero. In

<img file="MX342195B_D0028.tif" />

some modalities, R₅ is a-reporter-linker '?^DAND^Tfi^IA&amp; l modalities, the linker is: .....

where

X is

-O-, -S-, or -NH-; or alkanediyl_(cs12), alkenodiyl_(cS12)alkynediyl_{Cyes₂₎, a re n od ii I o ₍1₂), heteroarenodiil_(csi₂₎, or a substituted version of any of these groups;

and n is an integer from 0 to 6.

In some embodiments, X is alkynediyl_(c2_₈), for example, -C = C- In some embodiments, n is zero. In some modalities, the linker is:

where

X is

-O-, -S-, or -NH-; or alkanediyl_(0S12), alkenodiyl_(cs12)alkynediyl_(Cái2). arenediyl_(csi₂), heteroarenodiil_(CSi₂₎, or a substituted version of any of these groups;

Y is -O-, -NH-, alkanediylO (_CS12) or an iilO alkan (_{CSl 2)}

<img file="MX342195B_D0029.tif" />

INSTHVT J MEXICANO Dfc LA PROPIEDAD INDUSTRIAL replaced;

n is an integer from 0 to 6; and m is an integer from 0 to 6.

In some modalities, X is a I quinodii I or _(c2.8), for example, -C ^ C- In some embodiments, Y is -CH₂-, In some modes, n is zero. In some embodiments, m is zero. In some embodiments, the reporter is ink-based, where the ink is zantene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or squuaine ink. In some modalities, the reporter is:

<img file="MX342195B_D0030.tif" />

<img file="MX342195B_D0031.tif" />

In some modalities, R₆ it is hydrogen. In some embodiments, the featured carbon atom is in the S configuration. In some embodiments, the featured carbon atom is in the R configuration. In some embodiments, the compound is further defined as:

<img file="MX342195B_D0032.tif" />

Oh

<img file="MX342195B_D0033.tif" />

<img file="MX342195B_D0034.tif" />

<img file="MX342195B_D0035.tif" />

<img file="MX342195B_D0036.tif" />

OH OH

<img file="MX342195B_D0037.tif" />

Oh

<img file="MX342195B_D0038.tif" />

or a salt and / or protonated form of any of these formulas. &lt;

&gt; In some embodiments, the compound is further defined as:

<img file="MX342195B_D0039.tif" />

OH, or

OH where R is = 0 or = S, or any of these formulas.

In some additional modalities such as:

<img file="MX342195B_D0040.tif" />

the compound is in the form

<img file="MX342195B_D0041.tif" />

Oh

<img file="MX342195B_D0042.tif" />

Oh

<img file="MX342195B_D0043.tif" />

where R is = 0 or = S, or a salt and / or protonated form of any of these formulas.

In some embodiments, the compound is further defined as:

<img file="MX342195B_D0044.tif" />

<img file="MX342195B_D0045.tif" />

Oh

<img file="MX342195B_D0046.tif" />

<img file="MX342195B_D0047.tif" />

IMSTilUT-l MEXICAN PROPERTY

INDUSTRIAL

<img file="MX342195B_D0048.tif" />

<img file="MX342195B_D0049.tif" />

<img file="MX342195B_D0050.tif" />

-4 Λ

OT .Xtí-4, vr. 'í⁵'. '

<img file="MX342195B_D0051.tif" />

ÍA • 'OAJ F.CA/}

IN JUSTfGAL

<img file="MX342195B_D0052.tif" />

<img file="MX342195B_D0053.tif" />

OMe f-Bu '

HO.

-TO

-CK

Ό O '

O ^^ O.

Λ- &amp;

i NNH N ^ NH₂

<img file="MX342195B_D0054.tif" />

&gt;

<img file="MX342195B_D0055.tif" />

or a salt and / or protonated form of any of these formulas.

In another aspect of the present invention, methods are provided for sequencing a target nucleic acid comprising the following steps:

(i) adhering the 5 'end of a primer to a solid surface;

<img file="MX342195B_D0056.tif" />

(ii) hybridize a target nucleic acid to the solid adhered to the solid surface, to form a hybridized primer / target nucleic acid complex;

(iii) obtain a polymerase and one or more compounds described here, provided that the compound of the different formulas l-VII has different fluorophores;

(iv) reacting the hybridized primer / target nucleic acid complex with a polymerase and one or more compounds from step (iii), to form a growth primer strand through a polymerase reaction;

(v) generating images of the growth primer strand to identify the incorporated compound of step (iv) through its fluorophore;

(vi) exposing the solid surface with the growth primer strand to a light source to remove a photodecomposition termination portion of the formula:

<img file="MX342195B_D0057.tif" />

O me

OR₂N the variables as defined in the present invention referenced in step (iii), resulting in an extended primer with components of natural origin; and

1NSTITUΤΟ ΜEx | CA Νο &lt;

FROM PROPERTY (vii) repeat steps (iv) to (vi) one or more SP've-oessCSS: identify a plurality of bases in IT acid ρ^{, ιηΙοί}Target ςη, where the extended primer from step (vi) of the previous cycle reacts in place of the hybridized primer / target nucleic acid complex in step (iv) of the subsequent cycle.

In some embodiments, step (vi) is carried out in the presence of a sodium azide. In some embodiments, the sodium azide concentration is 0.1mM to 10mM, for example, about 1mM. In some embodiments, step (vi) is carried out in the presence of sodium acetate. In some embodiments, the sodium acetate concentration is 0.1mM to 10mM, for example, about 1mM.

In some embodiments, steps (v) or (vi) are carried out in the presence of thiourea. In some embodiments, the concentration of thiourea is from 10mM to 500mM, for example, approximately 100mM.

In some embodiments, step (vi) is carried out in the presence of dithiothreitol (DTT). In another aspect, sequencing methods of a target nucleic acid are provided comprising the following steps:

(i) adhering the 5 'end of a nucleic acid to a solid surface;

(ii) hybridize a primer for nucleic acid attached to

<img file="MX342195B_D0058.tif" />

the solid surface to form a com, hybridized / target nucleic acid; - (iii) obtain a polymerase and one or more compounds described here, provided that the compound of the different formulas I to Vil have different fluorophores (iv) react the hybridized primer / target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growth primer strand by a polymerase reaction;

(v) generating images of the growth primer strand to identify the incorporated compound of step (iv) through its fluorophore;

(vi) exposing the solid surface with the growth primer strand to a light source to remove a photodecomposition termination portion of the formula:

O Me with the variables as defined here, resulting in an extended primer with naturally occurring components; and (vii) repeating steps (iv) to (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer from step ^^^ of'1 above, reacts in place of oo myrh · jndn — cahad or hybridized / target nucleic acid in step (iv) of the subsequent cycle.

In some embodiments, step (vi) is carried out in the presence of a sodium azide. In some embodiments, step (vi) is carried out in the presence of dithiothreitol (DTT).

In some embodiments, the incorporation of at least one compound according to step (iv) occurs at from about 70% to about 100% of the incorporation efficiency of its natural nucleotide counterpart. In some modalities, incorporation efficiency occurs at from about 85% to about

100%.

In some embodiments, the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and DNA polymerase. In some embodiments, from about 85% to about 100% of the photodecomposition termination portions are removed by exposure to a light source in step (vi). In some embodiments, the incorporation of at least one compound according to step (iv) is followed by termination of strand growth with an efficiency of from about 90% to about 100%.

In some modes, a _______ ΡI detector is used

INSTITUTO MEXICANO pulsed multiple line excitation for gene ^ aOferiác ^

<img file="MX342195B_D0059.tif" />

step (v). - ...........

In some embodiments, the method further comprises washing the growth primer strand after step (iv) or (vi).

In some embodiments, the method further comprises, prior to step (iv), covering any growth primer and primer strands that do not react in step (iv).

In some embodiments, the method further comprises sequencing multiple-target nucleic acids synchronously.

In another aspect of the present invention, methods are provided for converting a naturally occurring component into a nucleic acid molecule, into a naturally occurring component, wherein the methods comprise:

(i) incorporate a compound described herein;

<td colspan="2">(I) exposing the resulting nucleic acid to a</td><td>source</td><td>of</td>

<td>light to remove a portion</td><td colspan="2">termination</td><td>of</td>

<td>photo decomposition of the formula:</td><td></td><td></td><td></td>

<td>r₅I</td><td></td><td></td><td></td>

<td></td><td>OMe</td><td></td><td></td>

<td>ο₂ν ^ Λ</td><td>X</td><td></td><td></td>

<td>^H^ A r₃</td><td></td><td></td><td></td>

<td>with variables just like here</td><td>are defined</td><td>depart</td><td>of the</td>

nucleic acid.

- -a- 'Λ jar

INSTITUTO MU.ICan&gt; jgl

D £ LA í'P.OFfEPAD

In some embodiments, the compWfíHie &amp;% JSíwjs method synchronously convert natural component gUu uiigeii-wo found in multiple nucleic acid molecules into naturally occurring components. In some embodiments, the method further comprises synchronously terminating multiple nucleic acid syntheses.

In another aspect, the present invention provides methods for terminating a nucleic acid synthesis comprising the step of placing an unlocked 3'-OH nucleotide or nucleoside described above in the environment of a polymerase, and allowing for the incorporation of 3 'nucleotide or nucleoside -OH unlocked in a nucleic acid molecule. In some embodiments, the completion efficiency of DNA synthesis at unlocked nucleotide or nucleoside 3'-OH incorporation ranges from about 90% to about 100%. In some embodiments, the incorporation efficiency of the unlocked 3'-OH nucleotide or nucleoside ranges from about 70% to about 100% compared to the incorporation efficiency of a naturally occurring nucleotide or nucleoside with the same base as the nucleotide or nucleoside 3'-OH unlocked.

In another aspect, the present invention provides methods for carrying out Sanger sequencing or Sanger-type sequencing which comprises using a compound described herein as a nucleotide analog of FéVwiwiafc? 2K§ ^

In another aspect, mWodus pwrn rlntarminax are provided with the sequence of a target nucleic acid, wherein the methods comprise:

(i) adding a target nucleic acid to a Sanger or Sanger type sequencing apparatus, (ii) adding one or more compounds described herein to the sequencing apparatus, provided that when more than one type of base is present, each base adheres to a different fluorophore;

(iii) adding a complementary primer and a polymerase enzyme;

(iv) carry out a polymerase reaction to incorporate at least one of the compounds from step (ii) into a growth nucleic acid strand, and (v) analyze the result of the Sanger sequencing reaction with sequencing instrumentation fluorescence or by pulsed multiple line excitation fluorescence, where steps (i) to (iii) can be carried out in any order.

In some embodiments, the incorporation of at least one compound according to step (iv), is followed by termination of the strand growth at an efficiency of from about 90% to about 100%. In some modalities, the incorporation of at least one

<img file="MX342195B_D0060.tif" />

compound according to step,, approximately 70% up to approximately iTrerritei 100 ·%¹ of te · efficiency of incorporation of a native substrate with the same base in the polymerase reaction. In some modalities, the incorporation of efficiency occurs in approximately 85% to approximately 100%. In some embodiments, the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and

DNA.

In another aspect, the present invention provides methods for incorporating a non-naturally occurring component into a nucleic acid, wherein the methods comprise:

(i) adding a target nucleic acid to a sequencing apparatus;

(ii) adding one or more compounds described herein to the sequencing apparatus, provided that when more than one type of base is present, each base adheres to a different fluorophore;

(Ii) add a polymerase enzyme; and (iv) carrying out a polymerase reaction to incorporate at least one of the compounds of step (ii) in a growth nucleic acid strand where steps (i) to (iii) can be carried out in any order.

In some modalities, the method further comprises:

<img file="MX342195B_D0061.tif" />

(v) analyze the result of the polymerase chain reaction for incorporation of at least one compound from step (i).

In some embodiments, the incorporation of at least one compound according to step (iv) is followed by termination of the strand growth at an efficiency of from about 90% to about 100%. In some embodiments, the incorporation of at least one compound according to step (iv) occurs at from about 70% to about 100% of the efficiency of incorporation of the native substrate with the same base in the polymerase reaction, with a substrate native with the same base in the polymerase reaction.

In another aspect, the present invention provides methods for carrying out minisequencing or minisequencing-type sequencing comprising adding a compound described herein to a minisequencing or minisequencing-type sequencing method.

In some embodiments of any of the methods described above, the compound is further defined as a compound of formula I, II, III, IV, V, VI or Vil.

In another aspect, the present invention provides a system comprising:

a flow cell comprising a plurality of granules, wherein:

<img file="MX342195B_D0062.tif" />

INSTITUTO MEXICANO DE Ι.Λ l'XOTIFCA'.l Each granule is adhered to a molecule where a compound described here has been used for a polymerase; and the flow cell is at least partially transparent for visible and UV light;

an image generating device configured to capture images of the flow cell;

a filter wheel comprising at least four spectrum filters, wherein the filter wheel is configured to cycle between each filter;

a lamp configured to create a light path from the flow cell through a filter on the filter wheel for the image generating device; and an ultraviolet light source configured to provide ultraviolet light to DNA molecules in the flow cell.

In some embodiments, the flow cell is a microfluidic cell. In some embodiments, the system further comprises an objective lens between the filter wheel and the flow cell. In some embodiments, the system further comprises a mirror configured to direct the light path to the image generating device.

In some respects, the present disclosure provides cancer diagnoses that are rapid, high-throughput, accurate, and sensitive for early stage detection,

<img file="MX342195B_D0063.tif" />

INSTITUTO MEXICANO OSLA EROME:) AI&gt; ra a iheusTBiei

<img file="MX342195B_D0064.tif" />

to . -_α - · ». | · W · Ι · Λ Γ Μ¹ r 11&gt; 'A 1 »j to identify rare sequence variants that involve a limited population of cells. that., pass —- pxu. carcinogenic transformation.

Other objects, features and advantages of the present invention will be appreciated from the following detailed description. However it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present invention, are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention may be appreciated by those skilled in the art from this detailed description. It should be noted that simply because a particular compound is attributed to a particular generic formula, it does not mean that it cannot also belong to another generic formula.

Brief Description of the Figures

The drawings that follow are part of this specification and are included to further demonstrate certain aspects of it. The present invention may be better understood through reference to one of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1 - HOMedNTP Analog Structures

Rented by 2-Nitrob ncilo. "R" is H, / 'so-propyl or ter42

ΪΜΡΙίξΒ »

Π-iS * ί^J: '7'λ' .PZíCaíIo butyl. R^{, B} is H, 4-OMe, 5-OMe, 4,5-di-OMe keys for the specific examples. indicates two di stereochemical configurations on this carbon atom. The part of the formulas inside the dotted ellipsoids 5 indicates the termination of functional groups that decompose upon exposure to UV light.

Figure 2 - Elimination of Temporary Product (TP) with DTT. Fluorescent gel image of a dU.VI UV photochemical decomposition time series incorporated by Therminator ™ 10 polymerase in the presence of (A) 1 mM NaN₃ and (B) 1 mM NaN₃, 50 mM DTT. Columns: "P" (primer) contains Therminator ™ bound to one or I igo PI anti 11 a-4 hybridized to primer-1 labeled with BODIPY-FL in 1x ThermoPol buffer (Wu et al., 2007; Litosh et al., 2011), “I” 15 (incorporation) contains what was found in column “P” plus 100 nM dU.VI, and the time point columns contain what was found in column “I” plus samples of described time that they were exposed to light 0.70 W / cm² 365 nm. "IP" indicates the incorporated product and "CP" indicates the decomposed product.

Figure 3 - X-Ray Crystal Structure of (1S) canphanate of (S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1propyl. Crystallographic measurements were made on a (1 S)-(S) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl 25 crystal with dimensions of 0.50 mm x 0.05 mm x 0.05 mm such

<img file="MX342195B_D0065.tif" />

DJ: ^_{¡: I}

DE r. &lt;&gt; £.; ·:&gt;, And as described in the Publication of Litosh et '^ Sf ^^ Q which is incorporated into the present invention ÓÓHTO' ltíftíieiieia. · 'Data collection', CuKa radiation, λ = 1.54178 Á, T = 110 ± 2 ° K, 20_max = 120.0 °, 32,513 reflections collected, 2,913 unique (/? ¡Nt = 0.0517). Final GooF = 1.091, R1 = 0.0681, wR2 = 0.1695, R indices based on 2,913 reflections with l&gt; 2sigma (l) (refinement in F²), 290 parameters, 43 restrictions. Lp and applied absorption corrections, μ = 0.819 mm '¹. Absolute structure parameter: 0.05 ± 0.09. X-ray crystallography data: C22H2gNO7, M = 419.46. Orthorhombic, a = 6.29, b = 15.00, c = 22.27 A (α, β, γ = 90 °), V = 2,099.29 A³, space group P212Í2Í, Z = 4, Dc = 1,327 g / cm '³, F (000) = 896.

Figure 4 - DTT Eliminates Nitrous Intermediary (TP). Fluorescent gene image from dU.VI UV photochemical decomposition experiment incorporated by Therminator ™ Polymerase Lens: “P” (primer) contains Therminator ™ linked to oIigopIanti11a-4 hybridized to primer-1 labeled with BODIPY-FL in 1x ThermoPol buffer , “I” (incorporation) contains what was found in the column “P plus 100 nM dU.VI. AG reagents described as final concentrations in the key were added, and the samples were exposed to light 0.70 W / cm² 365 nm for 10 seconds. "IP" indicates incorporated product, "CP" indicates decomposed product and "TP" indicates temporary product.

Figures 5A and B - Optical Preparation for D d Measurements Photochemical composition. Figure 5A shows a

<img file="MX342195B_D0066.tif" />

<img file="MX342195B_D0067.tif" />

iíísti τ υτο μ sx icah or eppendorf tube cut schma of 0. {&gt; 'cut in half, power meter ΡΜ100 ·, - ^ · η · 1,000 pm perforation cartridge using a manual translation stage of 3 axes to align the arc beam. Figure 5B shows a sample holder and the modified 0.5 mL Eppendorf tube with an internal alignment card to align the arc beam at the center of a 10 pL or 20 pL reaction sample.

Figure 6 - Example of photochemical dissociation reaction. Upon UV-induced photochemical dissociation, the terminating 2-nitrobenzyl derivative is released to produce a natural hydroxymethyl nucleotide. The combination of a stereospecific (S) -ter-butyl group attached on the benzyl carbon coupled with a 2-nitrobenzyl ring modified 5-OMe group substantially increased the range of photochemical decomposition reaction. In the case of C⁷-HOMedG, the range increased by more than an order of magnitude with respect to its corresponding analog of origin.

Figure 7 - tert-Butyl Substitution in α-Carbon and Methoxy Substitution in Position 5 Correlates with what

Enhanced Photochemical Decomposition Ranges. This figure compares the photochemical decomposition ranges of the origin, (S) -a-tert-butyl and (S) -a-tert-butyl-5-0 Me 2nitrobyl-alkyls groups on nucleosides C⁷-HOMedA, HOMedC, _ ......

MÍXJCaw INSTITUTE, Ti

DE LA TROIJEDaí,

C⁷-HOMedG and HOMedU. DT values₅₀ inferno§^{TO THE} inMe? mr faster photochemical decomposition ranges. ——

Figure 8. A schematic representation of a system for generating images of fluorescent granules in a flow cell.

Figure 9. Example of Granule Preparation and Immobilization Method. Schematic illustration of the steps of an example preparation of a granule sample in a flow cell.

Figure 10. Schematic illustration of the steps of incorporation, fluorescence images and photochemical decomposition in a CRT cycle.

Figure 11. Illustration of multi-resolution images of three CRT cycles and subsequent base designation of individual granules.

Figure 12 - Terminator Chemical Formulas

Reversible 3'-OH Adhered to Generic Inks (“Fluor”). The part of the formulas comprised by the dotted ellipsoid indicates the functional groups of termination marked with ink and which are decomposed upon exposure to UV light.

Figure 13 - Diagram of the Random Nick Sequencing (RNS) method. The lighting Lightning Terminators ™ indicated in the figure are comprised of the reversible terminators of the present invention.

<img file="MX342195B_D0068.tif" />

Description D carved from the Inv ntion

I. Reversible Terminators and MeYó'dóé Cié SIIILUSls of * the Same

In one aspect, the present disclosure provides novel compounds that can be used to function as reversible terminators in a variety of different DNA sequencing applications. The compounds provided in the present disclosure are also referred to as reversible terminators, 3'-OH unlocked reversible terminators and as Lightning Terminators ™. In some embodiments, the compounds of the following formulas are provided:

<img file="MX342195B_D0069.tif" />

(IV), (V),

<img file="MX342195B_D0070.tif" />

<img file="MX342195B_D0071.tif" />

where:

it is hydroxy, monophosphate, diphosphate, triphosphate, atiotriphosphate or polyphosphate;

R₂ it is hydrogen or hydroxy;

R₃ is rent_(Cs8) or alkyl_(C&lt;s) substituted;

R₄ it is hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; rented), acyl_(Cs6), alkoxy_(Cs6), acyloxy_(Cs6), alkylamino₍cs6), dialkylamino₍cs6) amido₍cs6}, or a substituted version of any of these groups;

R₅ and R₆ each are independently:

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; alkynyl)_(Cs6)&gt; a I quini I _(Cse), aril_(Cs6), aralquil₍cs8), heteroaryl_(Cs6), acil_(Cs6). alkoxy₍cs6)&gt; acyloxy (cse), alkylamino (_Cs6), dialkylamino₍cs6), amido_(CS6), or a substituted version of any of these groups;

a group of the formula:

<img file="MX342195B_D0072.tif" />

where

X is

-O-, -S-, or -NH-; or alkan!_(Csi2), alkenodiil₍csi2), alkynediyl₍csi2), or a substituted version of any of these groups;

Y is -O-, -NH-, alkanediil₍csi2) or an alkane-diyl₍csi2) substituted;

n in an integer from 0-6; and m in an integer from 0-6; or a -linker-reporter;

or a salt, tautomer, or optical isomer thereof.

The ink-labeled a-fBu-5OMe-2-nitrobenzyl alkylated hydroxymethyl nucleotides can be synthesized according to the following schemes and procedures.

A. Synthesis of 7 - [(S) -1 - (5-methoxy-2-n-trophenyl) -2,2-dim tilpropyloxy] methyl-7-deaza-2'-deoxyadenosine na-5'-triphosphates labeled with ink

ΟΜβ

IMPI

MEXICAN INSTITUTE OF PROPERTY I INDUSTRIAL

X ^ OMe

<img file="MX342195B_D0073.tif" />

ο, ν

<img file="MX342195B_D0074.tif" />

<img file="MX342195B_D0075.tif" />

<img file="MX342195B_D0076.tif" />

Oh

<img file="MX342195B_D0077.tif" />

Oh

Scheme 2a. Synthesis of 7 - [(S) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethyl-propylloxy] methyl-7-deaza-2'-deoxyadenosine-5 '-triphosphate marked with ink. Reagents and conditions: (i) fS) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1 propanol, 110 ° C; (ii) n-Bu₄NF, THF, room temperature; (iii) NH₃, 1,4-dioxane / MeOH, 100 ° C; (iv) Npropargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF; (v) POCI₃, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1M

HNEt₃HCO₃.

<img file="MX342195B_D0078.tif" />

Scheme 2b. (vi) Alexa Fluor 488 NHS, Na damper₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

<img file="MX342195B_D0079.tif" />

Scheme 2c. (vii) 6-FAM NHS, Na damper₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

<img file="MX342195B_D0080.tif" />

Scheme 2d. (v / '/ Z) CF488A NHS, damper

Na₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

B. Synthesis of ink-labeled 5 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxyur * idina-5'-triphosphate.

<img file="MX342195B_D0081.tif" />

OTBS OH OH

<img file="MX342195B_D0082.tif" />

Oh

<img file="MX342195B_D0083.tif" />

E qu ma 3a. Synthesis d ni trophene il) -2,2-d imeti l-propyloxy] meti I-2 'triphosphate ink marked. Reagents and conditions: (i) (S) 1 - (4-iodo-5-methoxy-2-n ytrophen yl) -2,2-di methyl-1-pro panol, 11 0 ° C;

(//) NH₄F, MeOH, 50 ° C; (/ 77) / V-propargyltrifluoroacetamide,

Pd (PPh₃) 4 (0), Cul, Et₃N, DMF; (iv) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1M

HNEt₃HCO₃;

<img file="MX342195B_D0084.tif" />

Scheme 3b. (v) Alexa Fluor 532 NHS, Na damper₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

<img file="MX342195B_D0085.tif" />

Na₂CO₃/ NaHCO₃

<img file="MX342195B_D0086.tif" />

Scheme 25 0.1 M (pH 9.2).

3d. O) Cy3 NHS, damper Na₂CO₃/ NaHCO₃

<img file="MX342195B_D0087.tif" />

'Jx. ... Λ f «NS-nruTCfmexicano V ', D £ uA ί / ΟΡ.ΈΡΛ'Ί C

C. Sínt id 7 - [(S) -1- (5-m toxi-2-níWWéni dimethyl-propi lox¡] methyl-7-deaza-2'-deoxygimTCrSTrrér^r3^to^ ink-labeled triphosphate

<img file="MX342195B_D0088.tif" />

Scheme 4a. Synthesis of ink-labeled 7 - [(S) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7-deaza-2'-deoxyguanosine-5'-triphosphate. Reagents and conditions: (i) MsCI, DMAP, CH₂CI₂, 0 ° C; (//) (S) -1- (4-iodo-5methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol, 115 ° C; (/ 77) n-Bu₄NF,

<img file="MX342195B_D0089.tif" />

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL _

THF, room temperature; (iv) sin-pyridine-2-aldoxime, 1,1,3,3-tetramethyl guanidine, 1,4-dioxane / DMP,? 0⁶or; (v) Npropargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF; (vi) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P20₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

<img file="MX342195B_D0090.tif" />

Scheme 4b. (v // J Alexa Fluor 594 NHS, damper Na₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

<img file="MX342195B_D0091.tif" />

Scheme 4c. (vi i) 6-ROX NHS, damper

Na₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

D. Synthesis of ink-labeled 5 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxycytidine-5'-triphosphate

<img file="MX342195B_D0092.tif" />

(i¡¡)

<img file="MX342195B_D0093.tif" />

<img file="MX342195B_D0094.tif" />

<img file="MX342195B_D0095.tif" />

Scheme 5a. Synthesis of ink-labeled 5 - [(S) -1 - (5-methoxy-2-nitrophenyl) -2,2-d imethyl-propyloxy] methyl-2'-deoxycytidi na-5 'triphosphate. Reagents and conditions: (i) TBSCI, imidazole, DMF, room temperature; (// ') 2,4,6-triisopropylbenzenesulfonyl hydrochloride, DMAP, Et₃N, CH₂CI₂, room temperature; (///) NH₃, 1,4-dioxane, 90 ° C; (iv) nBu₄NF, THF, room temperature, 82%; (v) Npropargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF; (vi) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (z? -Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

<img file="MX342195B_D0096.tif" />

Scheme 5b. (vi) Cy5 NHS, damper Na₂CO₃/ NaHCO₃

0.1 M (pH 9.2)

<img file="MX342195B_D0097.tif" />

Scheme 5c. (vi) Alexa Fluor 647 NHS, Na damper₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

In some embodiments, it was observed that the stereochemistry of the α-carbon-substituted alkyl can increase the photochemical decomposition properties of

INSTITUTO MÍ .'- JCTNO K ^ ÉiajCeí DE i. A, ΒΙΟίΊΖΟΛΟ

INDUSTRIAL a 2-nitrobenyl group. In some modalities, ^ qTTe photochemical decomposition ranges or A Γ3 S3 ^ 0 S were observed to result from combining the stereospecific group in the carbonate with another chemical group attached to the 2-nitrobenzyl ring. See for example, figure 7.

The compounds of the present disclosure can be made using the methods described above and in the Examples section below. For example, a summary of a synthesis for making α-tBu-5-OMe-2-nitrobenzyl alcohol, including an enantiopurate thereof, is provided in Example 8. These methods can be further modified and optimized using the principles and techniques of organic chemistry, as applied by those skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated in the present invention. as reference.

The compounds used in the methods of the present invention can contain one or more symmetrically substituted carbon or nitrogen atoms, and can be isolated in optically active or racemic form. Therefore, all chiral, diastereomeric, racemic, epimeric forms, and all geometric isomeric forms of a structure.

<img file="MX342195B_D0098.tif" />

<img file="MX342195B_D0099.tif" />

IH-5 T t T VT Ο Μ Ϊ X 'C Λ * Ο

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INDUSTRIAL are projected unless specific stereochemistry or specific isomeric form is indicated. The compounds can arise as racemates or racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a simple diastereomer is obtained. The surgical centers of the compounds of the present invention may have the S or R configuration. For example, in some aspects of the present invention, the substitution and its stereochemistry of the benzyl ether carbone pyrimidine bases of modified 5-hydroxymethyl or 7-hydroxymethyl-7-deazapurine affect the biological function and decomposition rates of the reaction of Base modified dNTPs, 3'-OH unlocked.

The compounds of the present invention may also have the advantage that they may be more effective, less toxic, have a longer action, be more potent, produce fewer side effects, be more easily absorbed and / or have a better pharmacokinetic profile (for example , greater oral bioavailability and / or less clearance), and / or have other pharmacological, physical or chemical properties with respect to the compounds known in the art, either to be used in the indications stated here or otherwise.

The chemical formulas used to present compounds of the present invention will normally show

IMPIOS li-ÍST! ΟΠΟ MEXICAN only one of the various tautomers possible merit tiVIer ^ rrres ^ For example, many types of smi • wniuultfoy group of ketones because they exist in equilibrium with corresponding enol groups. Similarly, there are many imine groups in equilibrium with enamine groups. Regardless of which tautomer is illustrated for a given compound, and regardless of which is the most prevalent, all tautomers of a given chemical formula are screened.

Furthermore, the atoms for making the compounds of the present invention are designed to include all the isotopic forms of said atoms. Isotopes, as used herein, include atoms that have the same atomic number, but different mass numbers. By way of general example and without limitation, hydrogen isotopes include tritium and deuterium, and carbon isotopes include¹³C and ¹⁴C. Similarly, it is contemplated that one or more of the carbon atoms of a compound of the present invention can be replaced by a silicone atom. Furthermore, it is contemplated that one or more of the oxygen atoms of a compound of the present invention may be replaced by a sulfur or selenium atom (s).

In some embodiments, the unlocked 3'OH reversible terminators provided herein have an alpha-thiophosphate group, preferably an alpha-thiothriphosphate group. See for example, compounds 54b, 55b, 59b, 60b, 63b, 64b, 68b, and

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69b, in example 8 found at continuSRyfÜW. known in the art that DNA polymerases' exhibit · »¹ 3'-5 'activity. The function of 3'-5 'activity is to remove only the incorporated nucleotide from the primer strand. Many commercially available DNA polymerases are deleted or mutated from the 3'-5 'exonuclease domain to reduce this activity to below detectable levels. However, even the low level activity of some DNA polymerases can result in poor sequence data quality due to the mismatch of the primary signal. In some embodiments, unlocked 3'-OH reversible terminators having alpha-thiothriphosphate groups can be used to reduce, minimize, and / or eliminate residual 3'-5 'exonuclease activity. Without intending to be bound by theory, it is well known in the art that alpha-thiothriphosphates are resistant to exonuclease activity. See for example, European Patent EP 0 640 146 to Rosental and Brenner, which is incorporated into the present invention by reference.

The compounds of the present invention may also exist as a prodrug. Since prodrugs are known to increase the numerous desirable qualities of pharmacists (eg, solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the present invention can, if desired, be supplied as a prodrug. . Therefore, the

MEXICAN INSTITUTE ΕΪ The Ι-? Ω?; ΕΕΆΓ, present invention contemplates prodrugs of IÓ ^^ VnpifesTos of the present invention, as well as methods for δüΜIήI¿ΓΓ3Τ prodrugs. Prodrugs of the compounds used in the present invention can be prepared by modifying functional groups present in the compound such that the modifications are decomposed, either by routine manipulation or in vivo, for the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which the hydroxy, amino, or carboxy group is linked to any group, which when the prodrug is administered to a subject, breaks down into the form of a hydroxy, amino, or carboxylic acid, respectively.

The present disclosure further provides nucleotide and nucleoside compounds, as well as salts, esters, and phosphates thereof that can be used in rapid DNA sequencing technology. However, it should be recognized that the particular anion or cation that is part of any salt of the present invention is not important, as long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in

Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated by reference in the present invention. The compounds are optionally in the form of

<img file="MX342195B_D0100.tif" />

ribonucleoside triphosphates (NTPs) and deoxyribonucleoside triphosphates (dNTP). Nucleotide and nucleoside compounds in some cases include a group that can be chemically or enzymatically decomposed labeled with a reporter group, such as a fluorescent ink. Nucleotide and nucleoside compounds include chemically or enzymatically removable protection groups that are designed to terminate DNA synthesis as well as to decompose rapidly, so that these monomers can be used for rapid sequencing in a parallel format. The presence of such rapidly decomposable groups labeled with fluorescent dyes in the nucleotide and nucleoside compounds can increase the speed and precision of sequencing of large DNA oligomers in parallel, to allow, for example, rapid sequencing of the entire genome , and the identification of polymorphisms and other valuable genetic information.

These unlocked 3'-OH terminators as well as well tolerated through a number of commercially available DNA polymerases represent a key advantage over blocked 3'-0 terminators.

The benzyl group of the compounds described herein can also be derived to include a selected fluorescent ink or other reporter group.

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<img file="MX342195B_D0101.tif" />

II. Property d T rminador s R vr ibl

As described above, in one aspect, novel alkylated 2-nitrobenzyl nucleotides with rapid photochemical decomposition properties are provided that can be used as improved reversible terminators for cyclic reversible terminator (CRT) sequencing applications. Such applications are described in the Metzker Publication (2005, 2010), which is incorporated by reference in the present invention. In some embodiments, 7-deaza-7-hydroxymethyl-2'-deoxyadenosine (C⁷HOMedA) (Rockhill et al, 1997) and 7-deaza-7-hydroxymethyl-2'-deoxyguanosine (C⁷-HOMedG) (McDougall et al, 2001) modified together with HOMedC and HOMedU with a variety of substituted 2-nitrobenzyl groups. See Figure 1. In some embodiments, the reversible terminators described herein exhibit a number of suitable properties, including rapid nucleotide incorporation kinetics, single base termination, high nucleotide selectivity, and / or rapid termination group decomposition.

Chromatographic conditions were identified to separate C analogs⁷-HOMedA in simple diastereomeric nucleotides, with the first elution isomer indicated as ds1, and the second as ds2. To assess the photochemical dissociation effect of stereochemistry of an isoopropyl group substitution with the 2-nitrobenzyl ring modifications of 466

ΙΜΡΓ ^

INSTITUTE Μ = Χ! ΟΑΚΟ 'DE LA FÍ'.G^:; tD? -.^:'í methoxy (4-OMe) and 6-nitro (6-NO₂), be syntet¡zaroH^NltW'á^L analogues C⁷-HOMedA dA.lll.a - dA.lll.c, as well as eI -dA.I Utí U11¿JSTi ”(veTTT Examples section below). Incorporation tests were carried out with these analogues C⁷-HOMedATP alkylated with 2-nitrobenzyl and subsequently subjected to UV photochemical decomposition experiments or in sodium azide solutions (Table 1).

Table 1. Ranges of Photochemical Decomposition of Analogs C⁷-HOMEDA

<td rowspan="2">Analog C⁷-HOMEDA</td><td colspan="2">DT₅₀ in 1mm NaN₃</td>

<td>Without DTT</td><td>50m DDT</td>

<td>dA.I</td><td> 3.6 ±0.1</td><td> 3. 5 ±0.1</td>

<td>dA.lll.a dsl</td><td> 4.5 ±0.2</td><td> 4.4 ±0.2</td>

<td>dA.lll.a ds2</td><td> 2.2 ±0.1</td><td> 2.1 ±0.1</td>

<td>dA.lll.bds1</td><td> 7.0 ±0.3</td><td> 6.1 ±0.4</td>

<td>dA.lll.b ds2</td><td> 1.1 ±0.1</td><td> 1.0 ±0.1</td>

<td>dA.lll.c dsl</td><td> 3.4 ±0.2</td><td> 3.0 ±0.2</td>

<td>dA.lll.c ds2</td><td> 2.8 ±0.2</td><td> 2.5 ±0.1</td>

In all cases, the ds2 isomers of dA.lll.a - dA.lll.c showed faster photochemical decomposition rates (i.e., lower DT values).₅₀) by factors of 2.0x, 6 Ax, and 1.2x, respectively, compared to their counterparts. Interestingly, the ds1 isomers exhibited DT values₅₀ similar (dA.lll.c) or higher (dA.lll.ao

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MEXICAN INSTITUTE OF PROPERTY

<img file="MX342195B_D0102.tif" />

dA.lll.b) compared to the original dAJ analog. Ézsfo ^ da provide evidence that the stereochemistry of the substituted isoopropyl group is an important determinant and coupled with a 4-OMe substitution, the analog dA.lll.b ds2 produced the value DT₅₀ lowest for the α-isopropyl C group⁷-HOMedA.

Previous work has shown that the α-terbutyl analogue dU.V exhibited excellent CRT properties such as single base termination and high nucleotide selectivity (Litosh et al, 2011). This allowed for further review of the stereospecific effect using a different a-substitution group coupled with various OMe ring substitutions by synthesizing four α-tert-butyl analogues C⁷-HOMedG, dG.Va - dG.Vd, along with the source dG.I (figure 1). Consistently with the a-isopropyl-C analogs⁷-HOMedATP, UV photochemical decomposition experiments revealed that the ds2 isomers of dG.Va - dG.Vd showed faster ranges by factors of 3.1 x, 4.5 x, 4.4x, and 3.0x, respectively, compared to their counterparts (Table 2).

<img file="MX342195B_D0103.tif" />

Table 2. Ranges of Photochemical Decomposition of Analogs C

- Η OM ed G.

<td rowspan="2">Analog C⁷-HOMedG</td><td colspan="2">DT_SW in 1mm NaN₃</td>

<td>Without DTT</td><td>50m DDT</td>

<td>dG.I</td><td> 9.2 ± 0.3</td><td> 8.1 ±0.2</td>

<td>dG.Va ds1</td><td> 11.0 ±0.4</td><td> 10.7 ±0.2</td>

<td>dG.Va ds2</td><td> 3.6 ±0.3</td><td> 3.5 ±0.3</td>

<td>dG.Vb ds1</td><td> 4.9 ± 0.3</td><td> 4.6 ±0.3</td>

<td>dG.Vb ds2</td><td> 1.1 ±0.1</td><td> 1.3 ±0.2</td>

<td>dG.Vc ds1</td><td> 3.5 ±0.3</td><td> 3.0 ±0.1</td>

<td>dG.Vc ds2</td><td> 0.8 ±0.1</td><td> 0.8 ±0.1</td>

<td>dG.Vd ds1</td><td> 2.4 ±0.1</td><td> 2.3 ± 0.2</td>

<td>dG.Vd ds2</td><td> 0.8 ±0.1</td><td> 0.8 ±0.1</td>

Both the ds1 and ds2 5-OMe isomers exhibited faster photochemical decomposition rates of 1.4x times each compared to the corresponding 4-OMe isomers. The bis-substituted 4,5-di-OMe isomer ds1 showed faster decomposition rates compared to the mono-substituted 4-OMe (2.0x) or 5-OMe (1.5x) isomers. Conversely, the 5-OMe ds2 and 4,5-di-OMe ds2 isomers exhibited DT values.₅₀ Identical in just 0.8 seconds. In the absence of an α-substitution group, Hasan et al (1997) reported an increase in range of only 1.2x for a 5OMe-2-nitrobyl analogue with respect to its corresponding origin.

<img file="MX342195B_D0104.tif" />

Comparison of the ds1 and ds2 isomers of dG.Vc with dG.Va revealed greater range increases of 3.6x and 4.4x, respectively, suggesting that the stereospecific tert-butyl group increases the effect of the 5-OMe group. With four-color CRT applications, this combination provides good flexibility in ring system utility, as a linker structure that can also adhere to the 4-position to create the ink-labeled analogues (US Patent Nos. 7,897,737 7,964,352, and 8,

148,503; North American Patent Application Publication No. 201 1/0287427; Metzker, 2010).

To determine the stereochemistry of these afer-butyl C analogs⁷-HOMedG, the (1S) -camphanate of (R / S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol was resolved into its enantipouro (S) alcohol by fractional crystallization Corrie et al, 1992) (Figure 3). This (S) and (S) -a- / er-butyl-2-nitrobenzyl alcohol (US Patent No. 8,148,503; Litosh et al, 2011), each were coupled to C⁷-HOMedG (figure 1). RP-HPLC analysis of their corresponding triphosphates revealed that both ds2 isomers of dG.Va and dG.Vc had identical peak retention times as that of dG.V and dG.V1, respectively, thus allowing us to determine that both ds2 isomers they may have the same (S) configuration on the α-carbon. By inference, the corresponding ds1 isomers of dG.Va and dG.Vc have been assigned the

<img file="MX342195B_D0105.tif" />

configuration (R).

These alcohols (S) were subsequently coupled to the remaining nucleosides to review the effect of the starting group on the photochemical decomposition range. For example, UV photochemical decomposition experiments revealed that DT values₅₀ for the 2-nitrobenzyl analogues of origin they ranged from 2.0 sec for dC.I to 9.2 sec for dG.I (Figure 7 and Table 3).

Table 3. Ranges of Photochemical Decomposition of

Reversible Terminators

<td rowspan="2">Nucleotide analog</td><td colspan="2">DT_S0 in 1mm NaN₃</td>

<td>Without DTT</td><td>50m DDT</td>

<td>dA.I</td><td> 3.6 ±0.1</td><td> 3.5±0.1</td>

<td>dA.V</td><td> 2.1 ±0.1</td><td> 2.0 ±0.2</td>

<td>dA.VI</td><td> 0.8±0.1^{[to the}</td><td> 0.8 ±0.1</td>

<td>dC.I</td><td> 2.0 ±0.3</td><td> 1.6 ±0.2</td>

<td>AD</td><td> 1.2±0.1</td><td> 1.0 ±0.2</td>

<td>AD C.VI</td><td> 0.6±0.1^{[to the}</td><td> 0.6 ±0.1</td>

<td>dG.I</td><td> 9.2 ±0.3</td><td> 8.1 ±0.2</td>

<td>dG.V</td><td> 3.0 ±0.1</td><td> 2.9 ± 0.2</td>

<td>dG.VI</td><td> 0.8 ±0.1</td><td> 0.8 ±0.1</td>

<td>dU.I</td><td> 2.1 ±0.1</td><td> 1.7 ±0.1</td>

<td>dU.V</td><td> 1.4 ±0.1</td><td> 1.3+0.1</td>

<td>dU.VI</td><td> 0.7±0.1^[θ1</td><td> 0.7 ±0.1</td>

^{[to the}Temporal product (TP) observed by gel electrophoresis; considered as the decomposed product in the DT value₅₀

Substitution of benzyl carbon with (S) -ter-butyl resulted in decomposition ranges increased by

<img file="MX342195B_D0106.tif" />

onal 'OTTan factors of 1.5x - 3.1 x and 5-OMe substitution further increased the ranges by iaÓTdTSS from 3.0x - 11.5x compared to the source analogs. The largest range increase was observed when C analogs were compared⁷-HOMedG, reducing DT values₅₀ 9.2 to 0.8 sec (figure 7, black bars). The complete set of reversible (S) -5-OMe-a-ter-butyl terminators showed a narrower range of DT values₅₀ 0.6 to 0.8 sec. These data suggest that the combined effects of the (S) -ater-butyl and 5-OMe groups play an important role in decreasing the dissociation range variation observed with particular nucleotide starting groups, having the practical application of providing conditions and faster dissociation rates for the CRT cycle. Unexpectedly, temporary products of the incorporation assays were observed for (S) -5-OMe-a-tert-butyl-C⁷HOMedA, -HOMedC and -HOMedU, but not -C⁷-HOMedG, after a brief exposure to UV light (HOMedU was shown only in figure 2, left side). Since the only difference was the incorporated nucleotide, we hypothesized that the faster breakdown of the (S) -5-OMe-a-tert-butyl-2-nitrobenzyl group produces a more reactive 2-nitrosoketone by-product that attacks the 3'-terminal nucleotide of the growth primer strand.

To investigate the conditions that exterminate the

<img file="MX342195B_D0107.tif" />

<img file="MX342195B_D0108.tif" />

INSTITUTO MEXICANO intermediate of nitrous, a number of amino acids and thiol were tested during the 4-doo-epoxy experiments. UV photochemistry (figure 5). Of these, only dithiothreitol (DTT) (Cleland, 1964) removed the temporary product (Figure 2, right side). In some embodiments, the effective DTT concentration is 1mM to 1M. In some embodiments, the effective DTT concentration is 5mM to 100mM. In some embodiments, the effective DTT concentration is 10mM to 50mM. In some embodiments, the effective DTT concentration is approximately 50mM. In some embodiments, the photochemical decomposition step takes place in the presence of sodium azide. In some embodiments, the effective sodium azide concentration is 0.1 mM to 1M. In some embodiments, the effective sodium azide concentration is from mM to 100mM. In some embodiments, the effective sodium azide concentration is 1mM to 50mM. In some embodiments, the effective sodium azide concentration is approximately 1 mM.

To test the effects of the range, UV photochemical decomposition experiments were repeated for all compounds in the presence of DTT, from which DT values ​​were reduced₅₀ of various isomers of origin and ds1 (Tables 1, 2, and 3). Bart et al. (2005) proposed that DTT attacks the nitrous group by nucleophilic addition, although a latest review by Corrie (2005) describes protective thiols as

INSTITUTO MEXICO, \ H'J X¿í £ Í «íSÍi Di LA FROflEOAi?

unnecessary, as evidence of the + a ^ biaro ^ iSs interference of the nitrosoketone by-product remains-minimal. In other examples described herein, DTT plays an important protective role against such unwanted reactions.

The stereo-specific (S) configuration of the substituted group combined with the 5-methoxy group was found to be a determining factor in the creation of fast composition reversible terminators. The reactive nitrosoketone by-product can be effectively removed during photochemical decomposition in the presence of DTT, providing suitable conditions to maintain the biological integrity of the CRT reaction.

III. Nucleotide and Nucleoside Compounds and Their Use n

DNA sequencing

The reversible terminators of the present invention can be used in DNA sequencing methods based on a variety of methods, including:

• "Sanger" or "dideoxy" methods, which involve DNA synthesis strand termination by incorporation of 2 ', 3'-dideoxynucleotides (ddNTPs) using DNA polymerase. See Metzker et af, 2005 Publication, which is incorporated by reference in the present invention.

Sequencing by synthesis (SBS), which usually does not clearly indicate the different mechanics

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<img file="MX342195B_D0109.tif" />

DNA sequencing. Consult the Publications ae

Metzker, 2010; Metzker 2005, which the present invention is incorporated by reference.

• DNA polymerase dependent strategies, which are also classified as reversible cyclic termination (CRT), single nucleotide addition (SNA, eg pyrosequencing) and real time sequencing. See Metzker Publication, 2010, which is incorporated by reference in the present invention.

• Sequencing of simple molecules using the Random Nick Sequencing (RNS) method.

In some embodiments, the present invention provides methods for sequencing a target nucleic acid where the methods comprise the following steps:

(i) adhere the 5 'end of the primer to a solid surface;

(ii) hybridize a target nucleic acid to the primer attached to the solid surface;

(iii) add a compound according to any of the structures described here, as long as more than one type of base is present, where each base is adhered to a different reporter group.

(iv) adding a nucleic acid replication enzyme to the hybridized primer / target nucleic acid complex to incorporate the composition of step (iii) into the primer strand

<img file="MX342195B_D0110.tif" />

<img file="MX342195B_D0111.tif" />

<img file="MX342195B_D0112.tif" />

INSTITUTO νΖΧϊΟΛΝΟ De Lá F / .Or: tDAl&gt;

growth, where the composition incorporates ^ a 'of (iii) terminates the polymerase reaction with a track 3 of between about 70% to about 100%;

(v) washing the solid surface to remove unincorporated components;

(vi) detect the incorporated reporter group to identify the incorporated composition of step (iii);

(vii) optionally adding one or more chemical compounds to permanently cap the unexpanded primers;

(viii) removing the termination portion comprising the photochemical decomposition of the termination portion, resulting in an extended primer, with pyrimidine bases of 5-hydroxymethyl or 7-hydroxymethyl-7-deazapurine;

(ix) washing the solid surface to remove the decomposed termination group; and (x) repeating steps (iii) to (viii) one or more times to identify the plurality of bases in the target nucleic acid.

In some variations, the order of steps (iii) and (iv) is reversed. In further variations, the polymerase and the compound are added at the same time. In modalities, they are in the same solution.

In another aspect, the present invention provides a method for sequencing a target nucleic acid comprising the following steps:

IM F*

<img file="MX342195B_D0113.tif" />

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INDUSTRIAL _ __ (i) adhere the 5 'end of the target nucleic acid to a solid surface;

(ii) hybridize a primer to the target nucleic acid attached to the solid surface;

(iii) adding a compound according to any of the structures described here, as long as more than one type of base is present, each base adheres to a different reporter group;

(iv) adding a nucleic acid replication enzyme to the hybridized primer / target nucleic acid complex to incorporate the composition from step (iii) into the growth primer strand, wherein the incorporated composition from step (iii) terminates the polymerase reaction with an efficiency of between about 70% to about 100%;

(v) washing the solid surface to remove the incorporated components;

(vi) detect the incorporated reporter group to identify the incorporated composition of step (iii);

(vii) optionally adding one or more chemical compounds to permanently cap non-extended primers;

(viii) removing the termination portion comprising the photochemical decomposition of the termination portion, resulting in an extended primer with 5-hydroxymethyl or 7-hydroxymethyl-7-deazapurine bases of pyrimidine;

(ix) washing the solid surface to remove the decomposed termination group; and

<img file="MX342195B_D0114.tif" />

INSTITUTE * · O ¹¿&gt; E LA zkOHí'Úx.O INDUSTRIAL (x) repeat steps (iii) to (ix) one or more times to identify the plurality of bases in the target nucleic acid.

In some variations, the order of steps (iü) and (¡v) is reversed.

In some embodiments the compound is incorporated into an enzyme that replicates nucleic acid which is a polymerase of

DNA. In some embodiments, DNA polymerase is selected from the group consisting of Taq DNA polymerase, Klenow DNA polymerase (exo-), Bst DNA polymerase, VENT® DNA polymerase (exo-) (Thermococcus cloned DNA polymerase A littoralis containing the D141A and E143A mutations), Pfu DNA polymerase (exo-) and DEEPVENT ™ DNA polymerase (exo-) (DNA polymerase A, cloned from the Pyrococcus GB-D species, and containing the D141A and E143A). In some embodiments, DNA polymerase is selected from the group consisting of AMPLITAQ® DNA polymerase, FS (Taq DNA polymerase containing the G46D and F667Y mutations), THERMOSEQUENASE ™ DNA polymerase (Taq DNA polymerase containing the F667Y mutation). ), THERMOSEQUENASE ™ II DNA polymerase (combination of THERMOSEQUENASE ™ DNA polymerase and T acidophilum pyrophosphatase), THERMINATOR ™ DNA polymerase (DNA polymerase A, cloned from the species Thermococcus 9 ° N-7 and containing mutations D141A, E143A, · / -ί&gt; r- η i J. Λ jX ΒβΤΐη · ΤΟ ¡'' «•• '• C -'. HO

Dt La; &lt;; r «iCAO £ Ít

i.NL'k and A485L), THERMINATOR ™ DNA polymerase, THERMINATOR ™ DNA polymerase containing additional lYIUTUUIÜIl Y409V- », and VENT® DNA polymerase (exo-) A488L (VENT® DNA polymerase (exo -) containing the mutation

A488L).

The compounds of the present description can be made using the methods indicated in the section on

Examples. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by one skilled in the art. Such principles and techniques are taught for example in the Publications of Wu et al. (2007; Litosh et al. (2011); Stupi et al. (2012); Gardner et al., 2012; North American Patents 7,897,737, 7,964,352 and 8,148,503; Application Publication for

North American Patent 2011/0287427, which are incorporated into the present invention by reference.

In some modalities, the sample components allow the determination of SNPs. The method may be for high-throughput identification of informational SNPs.

SNPs can be obtained directly from genomic DNA material, amplified PCR material, or cloned DNA material and can be assayed using a single nucleotide primer extension method. The single nucleotide primer extension method may comprise the use of single unlabeled dNTPs, single labeled dNTPs, 3.79

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O-modified with simple dNTPs, 2'-dNTPs single base, alpha-thio-simple dNTPs or 7 'ai niidgnwin.ir.ioA + irinc single marked. The minisequencing method may comprise the use of simple unlabeled dNTPs, simple unlabeled dNTPs, 3'-O-modified with simple dNTPs, 2'dNTPs modified with simple bases, alpha-thio-dNTPs simple or 2 ', 3'- simple labeled dideoxynucleotides. SNPs can be obtained directly from genomic DNA material, PCR amplified material, or cloned DNA materials.

A. Nucleotide and Nucleoside Compounds and Their Use in

CRT

In some aspects of the present invention, the nucleotide and nucleoside compounds provided herein (reversible terminators) can be used in cyclic reversible termination (CRT) based DNA sequencing technology. CRT is a cyclical method of detecting synchronous, single-base additions of multiple templates. The method differs in itself from the Sanger method (Metzker, 2005, which is incorporated into the present invention by reference) in that it can be carried out without the need for gel electrophoresis, a major bottleneck in the advancement of this field. However, like Sanger sequencing, longer reading lengths result in fewer sequencing runs required to cover the entire genome. The ν Λ^{Μ ϊ?}

MEXICAN INSTITUTE 'ί' DI LA HkCPlEÍMD C

<img file="MX342195B_D0115.tif" />

CRT cycle normally comprises three steps, iMwó®irp&gt; imaging and deprotection —EJ — Uáuxüjxo, “deprotection” can be used synonymously with “dissociation”, so that the three steps can be described as incorporation, generation of images and decomposition. For this procedure, cycle efficiency, cycle time, and sensitivity are important factors. Cycle efficiency is the product of deprotection and incorporation efficiencies, and determines the CRT reading length. The CRT cycle time is the sum of the incorporation, imaging, and deprotection time. For rapid CRT for genome-wide sequencing, nucleotide and nucleoside compounds such as those described herein can be used, which exhibit fast and efficient deprotection properties. These compounds can be labeled with reporter groups such as fluorescent inks, directly attached to the benzyl group having an azido substitution on alpha carbon, provided, for example, reversible, fluorescent terminators with similar deprotection properties. It has remained difficult to achieve the goal of long CRT readings, because reversible terminators normally act as deficient substrates with commercially available DNA polymerases. The modified nucleotide analogs of the present invention can be used to enhance this technology by providing substrates í &lt;⁴

STftlAL. Nuce

<img file="MX342195B_D0116.tif" />

1N5TITLE. ·;

Which incorporate as well or better than a natural with commercially available DNA polymerases?

Photodecomposable groups adhere to the base of an unlocked 3'-OH nucleotide, such as the groups described herein, can act as an effective reversible terminator, and can be efficiently incorporated by wild-type DNA polymerases. Consult the Publications of Wu et al., 2007; Metzker, 2010; Litosh et al.,

2011; Gardner et al., 2012; North American Patents 7,897,737, 7,964,352 and 8,148,503; North American Patent Application Publication 201 1/0287427, which are incorporated into the present invention by reference. For example, 5-hydroxymethyl-2'-deoxidine (HOMedU) is found naturally in the genomes of numerous bacteriophages and lower eukaryotes (Gommers-Ampt, 1995, which is incorporated herein invention for reference). Its hydroxymethyl group can serve as a molecular handle to adhere a small termination group that can be photodecomposed. Other naturally modified hypermodified bases that can be further modified in the manner described herein, to function as reversible terminators, Include 5-hydroxymethyl-2'-deoxycytidine (HOMedC), which is naturally found in the genomes of T2 bacteriophages, T4 and T6 (Wyatt &amp; Cohén, 1953; Gommers-Ampt, 1995) and of mammals (Kriaucionls &amp; Heintz, 2009; Tahiliani et al., 2009;

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Ito et al., 2010). The ring structure of pyrroIó ^ hTWid deazapurina) is also of natural origin 'éll arnibiülibus-'ée nucleósido (Carrasco &amp; Vázquez, 1984, which is incorporated into the present invention by reference) and tRNA bases (Limbach, et al. , 1994, which is incorporated into the present invention by reference), and the compounds 7-deaza-7hydroxymethyl-2'-deoxyadenosine (C⁷-HOMedA) (Rockhill et al., 1997) and 7-deaza-7-hydroxymethyl-2'-deoxyguanosine (C⁷-HOMedG) (McDougall et al., 2001) can also be further modified in the manner described herein to function as reversible terminators.

In some embodiments described herein, the group that can be photodecomposed is a substituted 2-nitrobenzyl nucleotide, which can be efficiently photochemically decomposed, for example, with 365 nm of UV light.

Consult the Patent Application Publication

North American 2010/0041041, which is incorporated into the present invention by reference. It is generally understood that wavelengths&gt; 300nm are used to minimize DNA and protein damage (Corrie, 2005) with various specific wavelengths in addition to 365nm, with 340nm being (Kaplan et al., 1978) and 355 nm (Seo, 2005).

In some embodiments, the unlocked 3'OH reversible terminators described herein typically have a number of advantages, including, for example, photodecomposition of

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Say LA PaCrtEGAD C unicam nt a single bond, removes both termination and fluorophore groups from the nucleobase. This in turn can be used to more efficiently restore the nucleotide for a subsequent CRT cycle. A second advantage of the unlocked 3'-OH reversible terminators provided herein is that many of these compounds show more favorable enzymatic incorporation, and in some embodiments, can be incorporated as easily as a natural nucleotide with wild-type DNA polymerases.

A challenge for unlocked 3'-OH terminators is to create suitable base modifications that lead to termination of DNA synthesis after simple base addition. This is normally important because an unlocked 3'-OH group is the natural substrate for incorporating the next input nucleotide. The compounds described here address this challenge. For example, in some embodiments, there are the reversible terminators provided herein that lead to DNA synthesis termination after simple base addition.

In some embodiments, the compounds described herein can be used in CRT to read directly from genomic DNA. Fragmented genomic DNA can be hybridized to a high-density oligonucleotide chip that contains primer sites that span selected chromosomes. Each primer sequence is separated by the reading length

<img file="MX342195B_D0117.tif" />

INSTITUTO MEXICANO DELANiOHÍDAD estimated by the CRT method. Among the additions^WAbase; A fluorescent imager can simultaneously image the entire high-density chip, marking significant improvements in speed and sensitivity. In specific embodiments, a fluorophore, which adheres to the benzyl group having an azido substitution on the alpha carbon or its derivatives described herein, is removed through a specific enzymatic or chemical reaction releasing the benzyl group for the next round of base addition. In other specific embodiments, a fluorophore, which adheres to the benzyl group having an amide substitution on the alpha carbon or its derivatives described herein, is removed through a specific enzymatic or chemical reaction that liberates the benzyl group for the following turn of base addition. After approximately 500 CRT cycles, the complete and contiguous genome sequence information can be compared to the reference human genome to determine the degree and type of sequence variation in an individual sample. Reversible terminators that feature higher incorporation and deprotection efficiency will typically achieve greater cycle efficiencies, and therefore greater read lengths.

CRT efficiency is defined through the formula: (RL)^EC// = 0.5, where RL is the base reading length and Ceff is the overall cycle efficiency. In other words, the

INSTITUTE OF THE I · Λ. Λ το μεχχλνο tí? '®'®? -? *' ··; inüuti'kíal »c.uj ^ _5 reading length of 7 bases can be achieved with an efficiencies of preferably 90% general cycle efficiency, 70 bases can be achieved with a cycle efficiency of 99% and 700 bases with a efficiency of 99.9% cycle. The incorporation efficiency of the compounds according to the present invention can range from about 70% to about 100% of the incorporation of the analog native nucleoside. Preferably, the incorporation efficiency will range from about 85% to about 100%. Photochemical dissociations will fluctuate from about 85% to about 100%. Furthermore, the termination of nucleic acid extension will range from about 90% to about 100% at the time of incorporation of the compounds according to the present invention. The nucleotide and nucleoside compounds in one embodiment have a cycle efficiency of at least 80%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

Another aspect of the present invention is directed towards the use of pyrosequencing, which is a non-electrophoretic, bioluminescence method that measures the release of inorganic pyrophosphate (PPi), proportionally converting it into visible light through a series of enzymatic reactions (Ronaghi et al., 1998, which is incorporated herein

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invention for reference). Not like others^uusrn £ todcrs — sequencing using modified nucleotides ¹ To complete DNA synthesis, the pyrosequencing assay manipulates DNA polymerase through a single addition of a dNTP in limiting amounts. Subsequently, the DNA polymerase is extended to the primer at the time of incorporation of the dNTP and complementary pauses. DNA synthesis is restarted after addition of the next complementary dNTP in the delivery cycle. The order and intensity of the light peaks are recorded on flow charts, which reveal the underlying DNA sequence. For homopolymer repeats of up to six nucleotides, the number of dNTPs added is directly proportional to the light signal. Homopolymer repeats greater than six nucleotides can result in insertion errors, which are the most common type of error in pyrosequencing. The modified nucleotide analogs of the present invention can enhance this technology by precise sequencing through homopolymer repeats. Particularly those that are greater than six nucleotides in length.

Another aspect of the present invention is directed to the use of Sanger sequencing, for example, as applied to heterozygote detection. Despite the breakthrough, improvements in dideoxy-BigDye terminator sequencing chemistry are needed for accurate detection of

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SAY LA ffiOW¹. AD heterozygous. It is generally considered that a uniform peak height in the pΓItriarl'ffS ñSCé 'IYi᧠data is reliable and accurate detection of heterozygote and base calling. The Sanger sequencing termination pattern is primarily due to the incorporation of sequence dependent trend by DNA polymerase, which can selectively incorporate natural nucleotides into modified nucleotides (Metzker et al., 1998, which is incorporated into the present invention as reference). These examples of trend incorporation are more pronounced with ink-terminator chemistry than primer-ink chemistry. This can be attributed to the effects of large fluorescent ink structures attached to the termination nucleotide, which decrease enzyme activity by at least 10-fold compared to that of the natural substrate. Therefore, reduction of the trend incorporation effects by DNA polymerase towards the ink-labeled terminators can lead to improved detection of heterozygote. The modified nucleotide analogs of the present invention can enhance this technology by incorporation as well as or better than a natural nucleotide, thereby eliminating incorporation trends in Sanger sequencing.

Another aspect of the present invention is directed towards the use of clonally amplified templates and template templates.

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Simple DNA molecule. The front end of NGS technologies can be divided into two fields: clonally amplified templates of simple DNA molecules and simple DNA molecule templates. It is well recognized in the art that DNA can be immobilized to a solid surface by either attaching a primer to the surface and hybridizing a target nucleic acid to that primer (US Patent 5,770,367; Harris et al., 2008, which are incorporated in the present invention by reference) or by attaching a target nucleic acid to the surface by clonal amplification and hybridizing a primer for the target nucleic acid (Dressman et al., 2003; Margulies et al., 2005, which are incorporated into the present Invention for reference). Any immobilization configuration can be used in the present invention to subsequently bind a DNA polymerase to initiate either the CRT method or the pyrosequencing method.

In one aspect, the present invention is directed to the use of single template molecules, consisting of large DNA fragments (eg, 0.1 to 0.5 megabases). In some embodiments, an adapter-free strategy, called Random Nick Sequencing (RNS), can be used. It has several advantages including (a) no requirement for PCR or adapter ligation, (or) redundant sequencing of the same template to improve precision, and (c) sites of

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U-JiTITVTOMEXICANO * --- DE LA PROI IEDAÜ sequencing reaction visible through Ia^INDa simple molecule that provides localized ñ'ó Vfl 'assemblies. For example, the PCR process creates mutations in the clonally amplified templates that are masked as sequence variants. Target sequences with high AT content and high GC content also show amplification trends in product performance, resulting in their underrepresentation in genome assemblies and assemblies. Knowing the position of the sequencing reactions of a given single molecule template will simplify the positioning assignment and organization of the genomic complex and framework regions in the genome assembly.

In the RNS method, sugar non-specific nucleases, such as those isolated from Vibrio vulnificus (vvn), create random single-stranded nicks in dsDNA and digest both ssDNA and RNA (See Hsia et al., 2005). Vvn binding occurs in the minor groove of DNA, preventing sequence-dependent nucleobase recognition. This represents an advantage in generating indentations along the DNA molecule in a randomized manner with no sequence dependent trend. The nick updraft strand becomes a primer site for the polymerase to start the RNS reaction. For this method to work in some modalities, the polymerase must have the ability to

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move the downdraft strand while extending the downdraft strand. Various well-known DNA polymerases that possess this property include polymerases &lt;p29 (See Publication of Soengas et al., 1995, which is incorporated into the present invention for reference) and Bst (See Publication of Aliotta et al., 1996 , which is incorporated into the present invention by reference). Vent (exo-) polymerases (see Gardner et al., 1999, which is incorporated by reference in the present invention) and Therminator ™ t also exhibit strand displacement properties, although they may be limited to 50 bases before stopping. After UV decomposition, hydroxymethyl nucleotides are created that serve as excellent template bases for subsequent RNS turns (Figure 13). Strand displacement during RNS creates a bifurcated dsDNA (flap) structure similar to that created with the Invader assay (See Publication Lyamichev et al., 1999). Fin endonucleases (FEN1) are known to decompose these bifurcated structures as shown in Figure 13, generating a 5'-PO end strand₄ downstream without a nucleotide gap (Kaiser et al., 1999). This creates a substrate that can be ligated to repair the dsDNA molecule for subsequent RNS turns.

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B. Tests of Polymer to .noustrui

Another aspect of the present invention is di-diige di ub'OTfee polymerase assays. Natural and modified nucleotides were tested for efficiency incorporation using the "polymerase endpoint assay" (Wu et al., 2001, which is incorporated into the present invention by reference). This trial reviews the incorporation efficiency in coupled and uncoupled template bases. Incorporation efficiency is measured by determining the concentration at which the compound incorporates half of the template primer complexes (IC₅₀). Titrations of the increasing compound concentration were carried out to generate curves, from which the IC can be determined₅oThe DNA template sequence is selected depending on which compound will be tested. For example, the first interrogation base after the primer in the template sequence is the complement base of the compound when the incorporation efficiency is measured, and one of three decoupled bases when the decoupling discrimination properties are measured.

For the hardened reaction, a DNA polymerase (eg THERMINATOR ™ DNA polymerase, 0.25 units per reaction, New England Biolabs), Thermopol 1x Buffer and a known concentration of either natural or modified nucleotide are added to every 10 pL of r action and s incubate to

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INSTITUTO MEXICANO ¡&gt; E LA I ROPIED at a temperature of 75 ° C for 10 minutes, SW '^ ewfn ice, and are extinguished with 10 pL of the eleventh-century treatment. (98% formamide: 10 mM Na₂EDTA, pH = 8.0, 25 mg / ml Blue Dextran). The stopped reactions are heated at a temperature of 75 ° C for 30 seconds to denature the DNA, and are then placed on ice. Extension products are analyzed on a 10% Long Ranger polyacrylamide gel (Lonza), using a Model 377 ABI DNA sequencer. Additional details are provided in Example 1 below.

C. Discrimination and Termination of Decoupling

Another aspect of the present invention is directed at increased discrimination against decoupling incorporation, for example, through the use of the reversible terminators described herein. Substitution on the α-carbon of the 2-nitrobenzyl group has been reported to increase the decomposition reaction range (Reichmanis et al., 1985; Cameron and Frechet, 1991; Hasan et al., 1997, which are incorporated into the present invention for reference). Without intending to be bound by any theory, the results presented here suggest that the substitution of 2-nitrobenzyl group in carbonate can also affect the completion of DNA synthesis of unlocked 3'-OH nucleotide triphosphates and improve discrimination against incorporation of decoupling. Ad plus, and based

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In the results described in greater detail, I-MPI Mexican Institute D £ LA FXONECAD found that the stereochemistry of the 2-nitrobenzyl group of ercarbon substitutes can have a significant impact on the degree of decoupling discrimination and the range of the reaction of decomposition. Without intending to be bound by theory, at least two factors were found that normally influence the termination of UV synthesis after a single incorporation: a) substitution on the α-carbon group

2-nitrobenzyl, and b) substitution at position 2 of the benzyl ring.

D. UV Decomposition Ranges

Another aspect of the present invention is directed toward providing reversible terminators with improved UV decay rates. Decomposition of the terminally substituted 2-nitrobenzyl group when the analogs were incorporated into the primer strand with 365 nm UV light allows the next cycle of incorporation to resume. Without intending to be bound by theory, at least two factors were found that normally influence the UV decomposition ranges of the incorporated nucleotide analogs: a) stereochemistry of the substitution of the α-carbon of the 2-nitrobenzyl group, and b) substitution in the benzyl ring .

E. Next Generation Sequencing Technologies (NGS)

Another aspect of the present invention is directed to ν * ** 'Α Λ.

FROM THE INDUSTRIAL PROMÉOÜ application of reversible terminators and methods provided here to the next generation sequencing methods. Sequencing technologies include a number of methods that are grouped as broadly as (a) template preparation, (or) sequencing and imaging, and (c) data analysis. The unique combination of specific protocols distinguishes one technology from another and determines the type of data produced from each platform. These differences in data production present challenges when comparing platforms based on data quality and cost. Although quality ratings and precision estimates are provided by each manufacturer, there is no consensus that a "quality basis" for one platform is equivalent to that for another platform. The compounds and methods described herein can be used in combination with and / or applied to one or more template formats described below.

Methods used to prepare templates for NGS reactions include: clonally amplified templates originating from single DNA molecules, and single DNA molecule templates. Sequencing methods using DNA polymerases are classified as cyclic reversible termination (CRT), single nucleotide addition (SNA), and real-time sequencing. Ligation sequencing (SBL), a method in which

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been used in NGS technologies. See for example Isrs—- "Publications of Shendure et al., 2005 and Valouev et al., 2008, which are incorporated by reference into the present invention. Imaging methods coupled with these sequencing strategies range from measuring bioluminescent signals to four-color images of simple molecular events. In some embodiments, these combined methods are additionally combined with appropriate information technology systems with the ability to handle the voluminous data produced by NGS platforms, including aspects related to data storage, tracking, and quality control. See the Pop &amp; Salzberg Publication, 2008, which is incorporated by reference in the present invention.

a) Template Preparation

In some embodiments, the present invention is directed to applying and / or combining reversible terminators and sequencing methods with one or more templates or template preparation methods. For example, in some modalities, robust template preparation methods are used. These produce trendless, representative sources of nucleic acid material from the genome under investigation. In some embodiments, the method involves randomly breaking genomic DNA into smaller sizes than is already created

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MEXICAN INSTITUTE OF PROPERTY, either fragment templates or templates^{INl&gt; lJ &lt;}dⁱandⁱ corresponding. In some modalities; For example, associated with NGS technologies, the template adheres or immobilizes to a solid surface or support. Immobilization 5 of the spatially separated template sites can be used to allow thousands to billions of sequencing reactions to be carried out simultaneously.

Clonally amplified templates. In some embodiments, the present invention comprises the use of clonally amplified templates or methods of preparing clonally amplified templates. For example, such templates can be used with imaging systems that are not designed to detect simple fluorescent events. For example, two common amplification methods are emulsion PCR (also called emPCR) and solid phase amplification. Consult the Publications of Dressman et al., 2003 and Fedurco et al., 2006, both of which are incorporated into the present invention by reference. In some embodiments, emPCR can be used to prepare sequencing templates in a cell-free system, which has the advantage of preventing arbitrary loss of genomic sequences - a problem that is normally inherent in bacterial cloning methods. In some modes, a library of fragment targets or corresponding pairs is created, r and

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FROM PtL «'ISDA'I C' and bind adapters containing siteS^{, I}'"of'^lc (universal to target ends, allowing complex genomes to be amplified with common PCR primers. For example, after ligation, DNA is separated into single strands and captured into granules under conditions that favor one DNA molecule per granule. Consult Metzker Publication 2010, Figure 1a, which is incorporated by reference in the present invention. For example, after successful amplification and enrichment of the emPCR granules, millions of polyacrylamide gel can be immobilized on a standard microscope slide (used with a Polonator instrument, see Publication of Shendure et al., 2005, which is incorporated into the present invention by reference), chemically crosslinked to an amino coated glass surface (used with the Life / APG SOLiD and Polonator instruments; for example see Kim et al., 2007, which is incorporated by reference into the present invention) or deposited either into individual PicoTiterPlate (PTP) tanks (used with the Roche / 454 instrument; Margulies et al., 2005 , which is incorporated into the present invention by reference) or lonChip deposit (used with the Ion Torrent instrument; Romberg et al., 2011, which is incorporated into the present invention by reference) where NGS chemistry can be carried out. In some

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INDUSTRIAL _ modalities, solid phase amplification can be used to produce clonally amplified groups, randomly distributed from fragment templates or corresponding pairs in a glass slide. Consult the

Metzker Publication 2010, Figure 1b, which is incorporated into the present invention by reference. In some embodiments, the high-density forward and reverse primers covalently adhere to the slide, and the ratio of the primers to the template on the support defines the surface density of the amplified groups. In some embodiments, solid phase amplification can be used to produce 100 to 200 million spatially separated template groups (Illumin / Solexa), which provide free ends at which a universal sequencing primer can be hybridized to start the NGS reaction. See Bentley et al., 2008, which is incorporated by reference in the present invention.

Simple molecule templates. In some embodiments, the present invention comprises the use of single molecule templates or single molecule template preparation methods. Although clonally amplified methods offer certain advantages over bacterial cloning, some of the protocols are complicated to implement and require a large amount of genomic DNA material (3 to

twenty pg). Preparation of simple molecule templates

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INSTITUTO Mf.XICAUO. ,, ...^DS '-Λ ΡΚΤίΊΕΟΛΟ, is usually simpler and requires less^,WILDEBEESTPirate game (&lt;1 pg). In some embodiments, these methods do not require PCR, which typically creates clonally amplified template mutations that are masked as sequence variants. Target sequences with high AT content and high GC content may also show amplification trends in product performance, resulting in their underrepresentation in genome alignments and assemblies. In some embodiments, quantitative applications can be used, such as RNA sequence. See Wang et al., 2009, which is incorporated into the present invention by reference. Such applications typically perform most effectively with unamplified template sources, which do not alter the abundance of representation of mRNA molecules. In some embodiments, and before the NGS reaction is carried out, single molecule templates are normally immobilized on solid supports using one of at least three different methods. In the first method, which can be used in some embodiments, the spatially distributed single primer molecules covalently adhere to the solid support (See Harris et al., 2008 Publication). The template, which can be prepared, for example, by randomly fragmenting the starting material into small sizes (eg, ~ 200-250 bp) and

100

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By adding common adapters to the fragment ends, it subsequently hybridizes to the immobilized primer. Refer to 'Metzker Publication 2010, Figure 1c, which is incorporated by reference in the present invention. In a second method, which can be used in some embodiments, the spatially distributed single molecule templates are covalently adhered to the solid support (See Harris et al., 2008 Publication) by priming and spreading the single-molecule, single-strand templates from immobilized primers. Consult Metzker's Publication

2010, Figure 1c, which is incorporated into the present invention by reference. In some embodiments, a common template primer is subsequently hybridized. See Metzker Publication 2010, Figure 1d, which is incorporated into the present invention by reference. In either method, DNA polymerase can be used, for example, to bind to the immobilized primed template configuration to initiate the NGS reaction. In a third method, which can be used in some embodiments, spatially distributed simple polymerase molecules are adhered to the solid support (see Publication Eid et al., 2009, which is incorporated into the present invention for reference), to the which bound a printed template molecule (see Metzker Publication 2010, Figure 1e, which is incorporated into the present invention by reference). In general, consult

101

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US Patents 7,329,492 and 6,255.08 ^^ are incorporated into the present invention as a reference ™ ™ "Larger DNA molecules (up to ten thousand base pairs) can be used with this technique, for example, and unlike the first two methods, the third method can be used with real-time methods, resulting in potentially longer read lengths.

b) Sequencing and image generation

There are fundamental differences in the sequencing of clonally amplified and single molecule templates. Clonal amplification can be used in some modalities to produce identical template populations, each of which has undergone the sequencing reaction. At the time of imaging, the observed signal is a consensus of the nucleotides or probes added to the identical templates for a given cycle. This typically places a greater demand on the efficiency of the addition process, as incomplete extension of the template assembly results in a lag strand mismatch (also called type 2 mismatch). The addition of multiple nucleotides or probes can also occur in a given cycle, resulting in a major strand mismatch (also called a type 1 mismatch). The concept of lag was described by Cheeseman (Consult US Patent 5,302,509, which is incorporated into the present invention as reference).

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giving rise to base naming errors and shorter lÉCLUiys (See Publication Erlich et al., 2008). Because lag is not an aspect with single molecule templates, the requirement for cycle efficiency is relaxed. However, single molecules are susceptible to multiple nucleotide and probe additions in any given cycle. Here, it can be seen that deletion errors arise, in some embodiments, due to the quenching effects between adjacent ink molecules and the signal is not detected due to the incorporation of the dark nucleotides or probes. The following sections describe sequencing and imaging strategies that use both single molecule and clonally amplified templates. In some aspects of the present invention, reversible terminators of the method of use provided herein, can be applied and / or used in combination with any one or more DNA polymerase dependent strategies, including, for example, CRT, SNA, and DNA sequencing. real time. In some embodiments, the compounds of the present invention and their method of use can be applied and / or used in combination with the CRT method.

There are a number of commercially available NGS systems that generate fluorescent signal images for simple DNA molecules (See Publications of

103

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Harris et al., 2008; Eid et al., 2009, both of which β§Τ3η incorporated into the present invention by reference). ET solving single molecules in formation can be carried out at 100x magnification with a highly sensitive CCD camera, provided that the individual DNA molecules are separated by a distance approaching the light diffraction limit (i.e. 250 nm). Variations may occur that may depend on the magnification or horizontality of the surface, which should be obvious to one skilled in the art. A technique that is widely used for detection of single molecule fluorescent signals is a total internal reflection fluorescence microscope (TIRF). (See Axelrod Publication 1989, which is incorporated into the present invention by reference). Other techniques that can be used in the present invention are known in the art include, but are not limited to, near-field scanning light microscope (SNOM; Consult Moyer et al., 1993, which is incorporated by reference in the present invention. ).

F. Imaging System

FIG. 8 illustrates an embodiment of an imaging system 100 for generating fluorescent signal images derived from a clonally amplified template. System 100 is configured to generate images of a microfluidic flow cell 50 that has been

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A range of imaging technologies, such as standard four-color imaging or blind color pulsing multiple line excitation, can be used in various modalities in combination with the unlocked 3'-OH reversible terminators. The illustrated mode is configured to generate standard four-color images.

System 100 comprises an imaging device 10 (eg, digital camera, photocell, etc.) configured to capture fluorescent signals derived from emPCR-amplified template granules immobilized in microfluidic flow cell 50.

Lamp 14 (eg, a xenon lamp) creates a light path 30 between microfluidic flow cell 50 and imaging device 10. Light from lamp 14 travels to filter wheel 16. In the embodiment illustrated, filter wheel 16 is motorized and comprises four spectrum filters so that four-color images can be captured. Filter wheel 16 is configured to switch between each of the four spectrum filters at each tile position so that the imaging device 10 can capture four-color images of the unlocked 3'-OH reversible terminators

105 incorporated.

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A part of the light from the lamp 14 travels from the wheel of the filter 16 through the objective lens 18 to the microfluidic flow cell 50. Another part of the light from the lamp 14 travels from the filter wheel 16 to the device. Imaging 10. In the illustrated embodiment, the light path 30 is directed to the imaging 10 using the mirror 12. In other embodiments, such mirrors may not be required, while in other embodiments, two or more mirrors may be required.

System 100 also comprises an ultraviolet (UV) light source 20 configured to provide UV light to flow cell 50. A UV light source 20 may be a light emitting diode (LED) in some embodiments, or it may be any other UV light source.

G. Minimizing Ozone Pollution

In another aspect, the present invention provides sequencing methods that minimize the effects of ozone pollution. Ozone (O₃) is an allotrope of oxygen that has attributes both beneficial and detrimental to life on earth. For example, ozone is created in the stratosphere using high-energy radiation from the sun that divides molecular oxygen (OR₂) into two atoms, which subsequently combine with different O molecules₂ to form O₃. This stratospheric layer protects the

106 living organisms in the

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dangerous produced by the sun. However, ozone is considered as an environmental pollutant in the land in the troposphere. Ozone is also created under smoggy conditions where sunlight acts on the combination of nitrogen oxides and volatile organic compounds that are produced by industrial facilities, electrical utilities, motor vehicle exhausts, gasoline vapors and chemical solvents (Consult Publication EPA, 2011). Ozone levels increase during the hot summer months, and the effects of ozone increase damage with increasing relative humidity levels. Tropospheric ozone causes various damages to crops and forests (See Publication of Hewitt et al., 1990), numerous respiratory problems in animals and humans (See Publication Fairchild et al., 1959; Bhalla, 1994), as well as adverse effects with many consumer products including automobile tires (See Crabtree and Kemp Publication, 1946), inks found in textile materials (See Salvin and Walker Publication, 1955), as well as fluorescent inks used in molecular biology.

From a chemical perspective, ozone is an electrophilic agent that is mostly reactive with electron pairs commonly found in olefinic compounds.

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FROM PROPERTY 'O ^ ewoSSLfí (for example, chemicals containing lacé ^'^ll'^rtfóbi? ñs ^ - € re carbon-carbon). There has been extensive dedicated research on ozone chemistry, which is called ozonation. An extensive two-volume book series provides a comprehensive review describing the properties of ozone and the many reactions it can go through with organic substrates. Volume 1 of this series is devoted mostly exclusively to the reaction of ozone with olefinic compounds (See Bailey Publication, 1978; Bailey 1982), for which Volumes 1 and 2 are incorporated by reference.

Inks and ink intermediates are well known in the literature, and most types of inks used today have been discovered more than a century ago (Gordon and Gregory, 1983). Inks can be grouped into classes based on how they are used, or based on their chemical structures (Gordon, 2009). For the latter, the inks have been classified into the general classes of (/) azo, (//) anthraquinone, (iii) benzodifuranone, (/ V) polycyclic aromatic carbonyl, (v) indigoid, (vi) polymetin and related inks (for example, cyanine), (vii) styryl, (viii) di and triaryl carbonium and related inks (for example, fluorescein, rhodamine and its sulphonated derivatives), (zx) phthalocyanine, (x) quinophthalone, (x /) sulfur, (xii) nitro and nitroso, and (xiii) miscellaneous (for example, Coumarin and BODIPY) (Gregory,

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2009). Most, if not all^wSo^Tto^1ALti7f ink intermediates have a multiplicity of carbon-carbon doubles that makes them sensitive to ozone. In the mid-1970s, Lofqulst and colleagues taught the general conclusion that most inks can be susceptible to ozonolysis (See US Patent 3,822,996; US Patent 3,859,045; US Patent 3,917,499. The effect of ozone exposure to fabrics with inks is a fading of the ink (for example, loss of ink fastness), which was first reported in 1955. Salvln and Walker coined the term "O-fading", which in their upholstery service test revealed that ozone caused significant ink fading when exposed to various anthraquinone blue inks (eg, Eastman Blue GLF, Amacel Blue and Interchemical Blue B) as well as yellow and red anthraquinone inks (See Salvin Publication, 1955). Other examples of anthraquinone inks, such as Cl Basic Blue 47 (See North American Patent 3,822,996), and Disperse Blue (Disperse Blue) 3 (See North American Patent 3,859,045) have been reported as susceptible to O-fading. High humidity increases fading-0 (Consult North American Patent 3,917,449; North American Patent 4,737,155; North American Patent 3,822,996; North American Patent 4,304,568),

109

INSTITUTO MEXICANO Ui LA í'XOF7 £ ;; aD industrial »-. and for fabrics, it has been suggested that moisture provide sufficient mobility of the ink to diffuse into the surface of the material where the ozone reaction occurs (See US Patent 4,737,155).

Fluorescent inks, such as those belonging to the polymethine class, have also been reported as susceptible to ozone reactions, which reduces their fluorescent signal intensities. For example, Cy3 and Cy5 inks have been widely used in micro-formation technologies for gene expression, genotyping, and resequencing applications (See Gershon, 2004). Fare and colleagues showed the results that Cy5 and its sulfonated derivative Alexa Fluor 647 were susceptible to ozone damage at exposure levels of 5 to 10 ppb at 10 to 30 sec (Consult Fare et al., 2003). Fluorescent intensity levels were also reduced for Cy3 and its sulfonated derivative Alexa Fluor 555 at higher ozone levels (&gt; 100 ppb). Kadushin and Getts observed that the start signal can degrade up to ~ 10% in 1 to 5 minutes (See

Publication of North American Patent Application

2004/01 10196).

There are a large number of chemical reagents, which act as an antiozonant. Examples include paraphenyldiamine, dihydroquinoline, thiourea (See US Patent 4,737,155; US Patent 4,631,066),

110

ΙΜΡϊ

MEXICAN INSTITUTE

ÜE THE PROPERTY

<img file="MX342195B_D0139.tif" />

thiorea substituted with saturated alkyl, alkyl '^ Tfors f alkyl (Consult North American Patent, 0.022,006), ethoxylated aliphatic tertiary aminm (Consult North American Patent 3,859,045), substituted piperidine thiourea (Consult North American Patent 4,304,568), oxadiazide thiones substituted thiazine thiones (See US Patent 4,304,568), acrylic polymerase, methacrylate or ethylacrylate copolymers (See North American Patent Application Publication 2004/01 10196), and polythiourea (See North American Patent 3,917,449). For the present invention, thiourea can be used in some embodiments, in one or more solutions to prevent ozonolysis of fluorescent inks in the sequencing reaction. Patent Publication WO 2012/037394, which is incorporated into the present invention by reference, provides methods for using thiourea in combination with methods involving NGS technologies. In some embodiments, the imaging and / or photochemical decomposition steps can be carried out in the presence of thiourea, for example, at the concentrations described in the brief description of the foregoing invention.

H. Sample Preparation

Methods for preparing DNA of interest for NGS analysis include, for example, clonally amplified and unamplified templates (eg, single molecules). In some

111

<img file="MX342195B_D0140.tif" />

Modalities, PCR emulsion can be used as illustrated in Figure 9, samples TJ'Uecferr-pfopara.

as follows. First, the genomic DNA is isolated. The DNA is subsequently broken down into smaller pieces. Later, common adapters are attached to the ends of said fragments. The adapter-ligated DNA molecules are subsequently separated into single strands and captured into 1 pm size granules under conditions that favor one DNA molecule per granule. An aqueous oil emulsion creates individual aqueous drops that encapsulate these DNA-granule complexes. PCR amplification is carried out within these drops to create granules containing 10⁴-10⁶ copies of the same template sequence. After successful amplification and enrichment, millions to hundreds of millions of emPCR granules are chemically immobilized for a microfluidic flow cell 50. In some embodiments, the flow cell 50 can comprise eight channels and can be made of glass.

I. Operation System

Once the flow cell 50 is prepared with the granules, the flow cell 50 can be placed in the sequencing system 100. Figure 10 illustrates the typical steps in a CRT cycle. A single DNA molecule is illustrated for illustrative purposes, although those skilled in the art will understand that this process is carried out in many

112

IMPI

INSTITUTO MEXICANO et LA FXOFISLjao industrial

<img file="MX342195B_D0141.tif" />

DNA molecules. industrial

First, in the incorporagTPTl step, "Be" 0'rporfrF "reversible 3'-OH terminators using DNA polymerase (illustrated as a closure) as described above.

Subsequently, images of the fluorescently labeled DNA molecules are generated. Lamp 14 is activated such that light path 30 is created from flow cell 50 to image generating device 10. Image generating device 10 captures images through each of the four filters spectrum of filter wheel 16. Using filter wheel 16, the image of each spectrum channel is generated in mosaic form to capture the fluorescent signals within the microfluidic flow cell 50. The base designation is then carried out from the processed fluorescent intensities of the individual granules (eg, a purified blue signal may be designated as an "A" base since the reversible 3'-OH terminator was marked with a blue ink). The reading length of the CRT method is a direct function of the number of cycles that are executed (see Metzker 2010; Metzker, 2005, which are incorporated into the present invention for reference).

Subsequently, photochemical dissociation can be carried out. Using a UV light source 20, the UV light shines in the flow cell 50. The UV light decomposes

113

MÍXiCANO INSTITUTE

<img file="MX342195B_D0142.tif" />

in photochemical form outside the group of termincílS'rf ^ the fluorescent. In this way, the acid nucTeTcó ll'lUll111uaele&gt; ~ -s-erestaura to its native state.

A wash is then supplied, which washes the termination group and the fluorescent groups. Incorporation, imaging, decomposition steps and washing can be carried out for many CRT cycles as desired. Figure 11 illustrates four-color mosaic images from three cycles, and the subsequent base designation of the individual granules.

IV. Definitions

When used within the context of a chemical group, the term "hydrogen" means -H; "Hydroxy" means -OH; "Oxo" means = O; "Halo" independently means -F, -Cl, -Br or I; "Amino" means -NH₂; "Hydroxyamino" means -NHOH; "Nitro" means -NO₂; imino means = NH; "Cyano means -CN; "Isocyanate" means -N = C = O; "Azido" means -N₃; in a monovalent context "phosphate" means -OP (O) (OH)₂ or an unprotonated form thereof; in a divalent context "phosphate means -OP (O) (OH) O- or a deprotonated form thereof; "Mercapto" means -SH; and "uncle" means = S; "Sulfonyl" means -S (O)₂-; and "sulfinyl" means -S (O) -.

Within the context of chemical formulas, the symbol means a single bond, "=" means a double bond, and "=" means a triple bond. The symbol represents a link

114

IMPI

<img file="MX342195B_D0143.tif" />

Mexican INSTITUTE OF THE rXOWEIJAD optional, which if present is either sim fW^STor^A1The symbol represents a single link 0 Ull uiilace eloL4e ·.

So, for example, the structure

<img file="MX342195B_D0144.tif" />

includes the structures' »» and. As will be appreciated by those skilled in the art, none of said ring atoms forms part of more than one double bond. The symbol "7vw" when drawn perpendicularly through a link indicates a point of group attachment. It should be noted that the point of adherence is normally identified only in this way for larger groups, in order to assist the reader in rapid and unambiguous identification of a point of adherence. The symbol means a simple link where the group attached to the thick end of the slice is "off the page." The symbol "'&lt;111" means a simple link where the group attached to the thick end of the slice is "inside the" page ". The symbol means a single bond where the conformation (eg either R or S) or geometry is undefined (eg either E or Z).

Any undefined valence in an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to the atom. When a group "R is illustrated as a" floating group "in a ring system, for example, in the formula:

115

<img file="MX342195B_D0145.tif" />

<img file="MX342195B_D0146.tif" />

then R can replace any hydrogen atom attached to any of the ring atoms, including an illustrated, implied or explicitly defined hydrogen, provided that a stable structure is formed. When an "R" group is illustrated as a "floating group" in a fused ring system, such as in the formula:

H then R can replace any hydrogen attached to any of the ring atoms of any of the fused rings unless otherwise specified. Replaceable hydrogens include illustrated hydrogens (eg, hydrogen attached to nitrogen in the formula above), implicated hydrogens (eg, a hydrogen in the formula above that is not shown but understood to be present), expressly defined hydrogens and optional hydrogens whose presence depends on the identity of a ring atom (eg, a hydrogen attached to group X, when X equals -CH-), as long as a stable structure is formed. In the illustrated example, R can reside on either the 5-membered or 6-membered ring of the

116

MEXICAN INSTITUTE

FROM OWNERSHIP Λ »_2ΐϊίϊ ^ $ 3 fused ring system. In the previous formula ^ S ^ letí subscript “y” followed immediately by the group 'N élllltí lus · parenthesis, represents a numeric variable. Unless otherwise specified, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

For the groups and classes below, the following subscripts in parentheses further define the group / class as indicated: "(Cn)" defines the exact number (n) of carbon atoms in the group / class . "(C ^ n) defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number being as small as possible for the group in question, for example, it is understood that the minimum number of carbon atoms in the group "alkenyl &lt;_ca8)”Or the“ alkene ”class_(cs6) ”. it's two. For example, "alkoxy_(Csio) ”designates alkoxy groups having 1 to 10 carbon atoms (eg 1, 2, 3, 4, 5,

6, 7, 8, 9, or 10, or any range derived from it (eg, 3 to 10 carbon atoms). (Cn-n ') defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Similarly, it designates the groups "alkyl (_C2-io) having 2 to 10 carbon atoms (eg 2, 3, 4, 5, 6,

7, 8, 9, or 10, or any range derived from it (for example, 3 to 10 carbon atoms).

117

MJPί

MRXICAH INSTITUTE ·&gt;

OSLA FROP.icD.Aa f **

The term "saturated" as used in ^ 'lá ^' f ^ re

<img file="MX342195B_D0147.tif" />

Invention means the compound or mocTrfiesrdu group having a carbon-carbon double bond or carbon-carbon triple bonds, except as indicated below. The term does not exclude heteroatom carbon multiple bonds, for example, an oxygen carbon double bond or a nitrogen carbon double bond. Furthermore, it does not exclude a carbon-carbon double bond that may arise as part of keto-enol tautomerism or imine / enamine tautomerism.

The term "aliphatic" when used without the "substituted" modifier means that the modified compound / group is an acyclic or cyclic hydrocarbon group or compound, but not aromatic. In aliphatic compounds / groups, carbon atoms can be attached in straight chains, branched chains, or non-aromatic (alicyclic) rings. The aliphatic compounds / groups can be saturated, that is, linked by single bonds (alkanes / alkyl), or unsaturated, with one or more double bonds (alkenes / alkenyl) or with one or more triple bonds (alkynes / alkynyl). When the term "aliphatic" is used without the "substituted" modifier, then only carbon and hydrogen atoms are present. When the term is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, F, -Cl, -Br, -I, -NH₂, -NOT_2i "CO₂H, -CO₂CH_3j -CN, -SH, -OCH_3i OCH₂CH₃, -C (O) CH₃, -NHCH₃, -NHCH₂CH_3i -N (CH₃)₂, -C (O) NH_2i

118

<img file="MX342195B_D0148.tif" />

Τ5! Τ X 1

ÍN5TITUTC ME.'ICAÍJÍ)

OF INDUSTRIAL PROPERTY

-OC (O) CH_3i bear)₂NH₂.

The term "alkyl" when used without the "substituted" modiTic ^ crcJT refers to a monovalent saturated aliphatic group with one carbon atom as the point of adhesion, a linear or branched cyclic, cyclic or acyclic structure, and no atoms in addition of carbon and hydrogen. Therefore, as used in the present invention, cycloalkyl is a subset of alkyl. -CH groups₃ (Me), -CH₂CH₃ (Et), CH₂CH₂CH₃ (z? -Pr or propyl), -CH (CH₃)₂ (/ -Pr, 'Pr or isopropyl), CH (CH₂)₂ (cyclopropi lo), -CH₂CH₂CH₂CH₃ (n-Bu), CH (CH₃) CH₂CH₃ (sec-butyl), -CH₂CH (CH₃)₂ (isobutyl), C (CH₃)₃ (fer-butyl, f-butyl, f-Bu or 'Bu), -CH₂C (CH₃)₃ (neopentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic group, with one or two carbon atoms as the point of adhesion (s), a linear or branched cyclic, cyclic or acyclic structure, no carbon or carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH₂(methylene), -CH₂CH₂-, -CH₂C (CH₃)₂CH₂-, -CH₂CH₂CH₂- and * ^ * ,, are non-limiting examples of alkanediyl groups. The term "alkylidene" when used without the "substituted" modifier refers to the divalent group = CRR 'where R and R' are independently hydrogen, alkyl or R and R 'are taken together

<img file="MX342195B_D0149.tif" />

119

IMPI ^ S&gt;

'Τ5τιτ&gt; σαmexican to represent an alkanediyl having meritOO' of carbon. Non-limiting examples of alauylidene groups include: = CH₂, = CH (CH₂CH₃) y = C (CH₃)₂. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NOT₂, -CO₂H, CO₂CH₃, -CN, -SH, -OCH_3i -OCH₂CH_3i -C (O) CH_3i -NHCH_3i NHCH₂CH₃, -N (CH₃)_2i -C (O) NH_2i -OC (O) CH₃ bear)₂NH₂. The following groups are non-limiting examples of substituted alkyl groups: -CH₂OH, -CH₂CI, -CF_3i -CH₂CN, -CH₂C (O) OH, CH₂C (O) OCH₃, -CH₂C (O) NH_2i -CH₂C (O) CH₃, -CH₂OCH_3i CH₂OC (O) CH₃, -CH₂NH_2i -CH₂N (CH₃)₂ and -CH₂CH₂CI. The term "haloalkyl" is a subset of substituted alkyl, where one or more hydrogen atoms have been substituted with or without a halo group other than carbon, hydrogen and halogen present. The group, -CH₂CI is a non-limiting example of a haloalkyl. An "alkane" refers to the compu HR, where R is alkyl. The term "fluoroalkyl" is a subset of substituted alkyl wherein one or more hydrogens have been substituted with a fluoro group and without other atoms besides carbon, hydrogen and fluoro being present. -CH groups₂F, -CF₃ and -CH₂CF₃, are non-limiting examples of fluoroalkyl groups. An "alkane" refers to the compound HR, where R is alkyl.

The term "alkenyl" when used without the modifier

120 substituted "refers to a group

<img file="MX342195B_D0150.tif" />

monovalent with a carbon atom as the point cte · - · adhesion, a linear or branched cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, without carbon-carbon triple bonds and without atoms other than carbon and hydrogen. Non-limiting examples of carbon-carbon groups include: -CH = CH₂ (vinyl),

CH = CHCH₃, -CH = CHCH₂CH₃, -CH₂CH = CH₂ (allyl),

CH₂CH = CHCH₃ and -CH = CH-C₆H₅. The term "alkenodiyl" when used without the "substituted" modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as adhesion points, a linear or branched cyclic, cyclic or acyclic structure, at least one carbon-carbon double bond non-aromatic, without carbon-carbon triple bonds and without atoms other than carbon and hydrogen. The groups, -CH = CH-,

-CH = C (CH₃) CH₂-, -CH = CHCH₂- and are non-limiting examples of alkenodiyl groups. When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH,

<img file="MX342195B_D0151.tif" />

OCH₂CH₃, -C (O) CH_3i -NHCH₃, -NHCH₂CH_3i -N (CH₃)_2i -C (O) NH_2i

-OC (O) CH₃ bear)₂NH₂. The groups, -CH = CHF, -CH = CHCI and CH = CHBr, are non-limiting examples of substituted alkenyl groups. An "alkene" refers to the compound HR, where

R is alkenyl.

121

IΜ ΡI

MEXICAN INSTITUTE Λ

I heard the property

The term "alkylin" when used without '$ T^STiW6diHc ^ rof "substituted" refers to an aliphatic groupff lliualui atho — monovalent with a carbon atom as the point of adhesion, a linear or branched cyclic, cyclic or acyclic structure, at least one carbon-carbon triple bond and no atoms in addition carbon and hydrogen. As used in the present invention, the term "alkynyl" does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups, -C = CH, -C = CCH₃ and -CH₂ChCCH₃, are non-limiting examples of alkynyl groups. When alkynyl is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, F, -Cl, -Br, -I, -NH₂, -NOT_2i -CO₂H, -CO₂CH₃, -CN, -SH, -OCH₃, OCH₂CH₃, -C (O) CH₃, -NHCH₃, -NHCH₂CH_3i -N (CH₃)₂, -C (O) NH_2i-OC (O) CH₃ bear)₂NH₂. An "alkyne" refers to the compound HR, where R is alkynyl.

The term "aryl" when used without the "substituted" modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of adhesion, forming part of the carbon atom of one or more aromatic ring structures of six members, where the ring atoms are all carbon, and where the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings can be fused or unfused. As used herein

122

<img file="MX342195B_D0152.tif" />

Invention, the term does not exclude the presence of che ^ Tepn- or alkyl groups (allowing a limitation of ...... carbon number) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl) phenyl, C₆H₄CH₂CH3 (ethylphenyl), naphthyl and the monovalent group derived from biphenyl. The term "arenediyl" when used without the "substituted" modifier refers to a divalent aromatic group with two aromatic carbon atoms as adhesion points, the carbon atoms being part of one or more six-membered aromatic ring structures, where the ring atoms are all carbon, and where the monovalent group consists of having no atoms other than carbon and hydrogen. As used in the present invention, the term does not exclude the presence of one or more alkyl groups (allowing carbon number limitation) adhered to the first aromatic ring or any additional aromatic ring present. IF more than one ring is present, the rings can be fused or unfused. Non-limiting examples of arenediyl groups include:

When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂,

123

<img file="MX342195B_D0153.tif" />

-ΝΟ_2ι "CO₂H, -CO₂CH_3i -CN, -SH, -OCH3, -ocff₂W ^ £ Íci -NHCH₃, -NHCH₂CH₃, -N (CH₃)₂, -C (O) NHt, OC (Q) CH_toSW)₂NH₂. An "arene" refers to compound HR, where R is aryl.

The term "aralkyl" when used without the "substituted" modifier refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Examples of non-limiting aralkyls are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the modifier "substituted", one or more hydrogen atoms of the alkanediyl and / or the aryl has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NOT₂, CO₂H, -CO₂CH_3i -CN, -SH, -OCH₃, -OCH₂CH_3i -C (O) CH_3i NHCH₃, -NHCH₂CH_3i -N (CH₃)_2i -C (O) NH_2i -OC (O) CH₃ bear)₂NH₂. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl) -methyl and 2-chloro-2-phenyl-et-1-yl.

The term "heteroaryl" when used without the "substituted" modifier refers to a monovalent aromatic group with one carbon atom or aromatic nitrogen atom as the point of adhesion, the carbon atom or nitrogen atom being part of a or more aromatic ring structures, where at least one of the atoms is nitrogen, oxygen or sulfur, and where the heteroaryl group consists of no atom other than carbon, hydrogen,

124

ΙΡι

MEXICAN INSTITUTE &amp;

FROM THE PROPERTY aromatic nitrogen, aromatic oxygen or sulfur á'FüfTfí'áti as used in the present invention, eTtéfifllPlü nu excludes the presence of one or more alkyl, aryl and / or aralkyl groups (allowing limitation of carbon numbers) attached to the aromatic ring or aromatic ring system. If more than one ring is present, the rings can be fused or unfused. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, ndolilo, indazolyl (lm), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl and triazolyl.

The term "N-heteroaryl" refers to a heteroaryl group with a nitrogen atom as the point of adhesion. The term "heteroarenodiyl" when used without the "substituted" modifier refers to a divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom such as the two points of adhesion, the atoms forming part of one or more aromatic ring structures, where at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atom other than carbon, hydrogen, non-aromatic nitro, aromatic oxygen, or aromatic sulfur. As used in the present invention, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups.

125

ΙΑ

<img file="MX342195B_D0154.tif" />

MEXICAN INSTITUTE

PROPERTY &lt;\ (allowing limitation of carbon numbers)^&gt;'^I'efip'l^sl^Iin aromatic ring or aromatic ring system "&lt;5; ' IF more than one ring is present, the rings can be merged or unmerged. Non-limiting examples of heteroarenodiyl groups include:

When these terms are used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, NH₂, -NOT₂, -CO₂H, -CO₂CH_3i -CN, -SH, -OCH₃, -OCH₂CH_3j C (O) CH₃, -NHCH_3i -NHCH₂CH₃, -N (CH₃)_2i -C (O) NH_2i -OC (O) CH₃bear)₂NH₂.

The term "heterocycloalkyl" when used without the "substituted" modifier refers to a monovalent non-aromatic group with one carbon atom or nitrogen atom as the point of adhesion, the carbon atom or nitrogen atom being part of one or plus non-aromatic ring structures where at least one of the ring atoms is nitrogen, oxygen or sulfur, and where the heterocycloalkyl group consists of no atom other than carbon, hydrogen, nitrogen, oxygen and sulfur. As used in the present invention, the term does not exclude the presence of one or more alkyl groups (allowing limitation of carbon numbers) attached to the ring or ring system.

126

<img file="MX342195B_D0155.tif" />

MEXICAN INSTITUTE OE THE PROPERTY)

As used in the present invention, é'P'Wrmi excludes the presence of one or more ring system bonds, provided that the resulting group remains non-aromatic. If more than one ring is present, the rings can be fused or unfused. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahldrotiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "Nheterocycloalkyl" refers to a heterocycloalkyl group with a ring atom as the point of adhesion. When the term "heterocycloalkyl" is used with the "substituted" modifier, one or more hydrogen atoms has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NOT₂, -CO₂H, -CO₂CH₃, -CN, -SH, -OCH_3i -OCH₂CH_3i -C (O) CH_3i-NHCH_3i -NHCH₂CH_3i -N (CH₃)_2i -C (O) NH_2i -OC (O) CH_3i SW)₂NH₂ or -C (O) OC (CH₃)₃ (fer-butyloxycarbonyl, BOC).

The term "acyl" when used without the "substituted" modifier refers to the group -C (O) R, where R is a hydrogen, alkyl, aryl, aralkyl, or heteroaryl, as defined above. The groups, -CHO, C (O) CH₃ (acetyl, Ac), -C (O) CH₂CH₃, -C (O) CH₂CH₂CH₃, C (O) CH (CH₃)₂, -C (O) CH (CH₂)₂, -C (O) C₆H₅, -C (O) C₆H₄CH_3i C (O) CH₂C₆H₅, -C (0) (imidazolyl) are non-limiting examples of acyl groups. A "thioacyl" is defined in an analogous way,

127

MEXICAN PROPERTY INSTITUI!) Except that the oxygen atom of the group -CfüSyH '*¹It has been replaced with a sulfur atom, -¿ (¿) R. When 5ΤΓ uses any of these terms with the "substituted" modifier, one or more hydrogen atoms (including a hydrogen atom attached directly to the carbonyl or thiocarbonyl group, if it exists) has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NOT₂, CO₂H, -CO₂CH_3i -CN, -SH, -OCH_3i -OCH₂CH₃, -C (O) CH₃, NHCH₃, -NHCH₂CH_3i -N (CH₃)_2i -C (O) NH₂, -OC (O) CH₃ bear)₂NH₂. Groups, -C (O) CH₂CF₃, -CO₂H (carboxyl), CO₂CH₃ (methylcarboxyl), -CO₂CH₂CH₃, -C (O) NH₂ (carbamoilo) and -CON (CH₃)₂, are non-limiting examples of acyl groups.

The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, where R is an alkyl, as the term is defined above. Non-limiting examples of alkoxy groups include: -OCH₃ (methoxy), -OCH₂CH₃(ethoxy), -OCH₂CH₂CH₃, -OCH (CH₃)₂ (isopropoxy), -O (CH₃)₃ (terbutoxy), -OCH (CH₂)₂, -O-cyclopentyl and -O-cyclohexyl. The terms "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy," heteroaryloxy "," heterocycloalkoxy "and" acyloxy "when used without the" substituted "modifier refer to groups defined as -OR, where R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloa Iq uyl and acyl, respectively. The term "alkoxidiyl" refers to the divalent group -O-alkanediyl, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The term

128

<img file="MX342195B_D0156.tif" />

INSTITUTO MEXICANO OE LA PROPIEDAD industrial _ without modifying "alkylthio" and "acylthio" when using "substituted", they refer to the group -SR, where R is alkyl and acyl, respectively. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH₂, -NOT_2i -CO₂H, -CO₂CH₃, -CN, -SH, -OCH₃, OCH₂CH₃, -C (O) CH₃, -NHCH_3i -NHCH₂CH₃, -N (CH₃)₂, -C (O) H_2i OC (O) CH₃ bear)₂NH₂. The term "alcohol" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms has been replaced with a hydroxy group.

The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, where R is an alkyl, as defined above. Non-limiting examples of alkylamino groups include: -NHCH₃ and NHCH₂CH₃. The term "dialkylamino" when used without I "substituted" modifier refers to the group -NRR ', where R and R' can be the same or different alkyl groups, or R and R 'can be taken together to represent an alkanediyl. Non-limiting examples of dialkylamino groups include: N (CH₃)₂, -N (CH₃) (CH₂CH₃) and Λ / -pir ro I idin ¡I o. The terms "alkoxyamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino," heteroarylamino "," heterocycloalkylamino "and" alkylsulfonylamino ", when used without the modifier" their substituted "refers to groups defined as - NHR, where

129

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• .VriTuroMsxiCANO Say THE PROPERTY

R is alkoxy, alkenyl, alkynyl, aryl, aralkylO ',^DUSyouⁱtea ^_heterocycloalkyl alkylsulfonyl, respectively. An example. non-limiting of an arylamino group is -NHC₆H₅. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl, as defined above. A non-limiting example of an amido group is -NHC (O) CH₃. The term "alkylimino" when used without the "substituted" modifier refers to a divalent group = NR, where R is an alkyl, as that term was defined above. The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -N H-alkanediyl-NH- or - to I ca n od i ¡IN Ha I ca n od ¡¡o-. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms has been independently replaced by -OH, -F, -Cl, -Br, -I, - H₂, -NOT₂, -CO₂H, -CO₂CH₃, -CN, -SH, -OCH₃, -OCH₂CH_3i -C (O) CH_3i -NHCH₃, -NHCH₂CH₃, -N (CH₃)_2i C (O) NH₂, -OC (O) CH₃ bear)₂NH₂. -NHC (O) OCH groups₃ and NHC (O) NHCH₃ they are non-limiting examples of substituted amido groups.

The term "alkylphosphate" when used without the "substituted" modifier refers to the group -OP (O) (OH) (OR), where R is an alkyl, as that term was defined above. Non-limiting examples of alkyl phosphate groups include: -OP (O) (OH) (OMe) and -OP (O) (OH) (OEt). The term "dialkylphosphate" when used without the modifier

130

<img file="MX342195B_D0158.tif" />

"Substituted" refers to the group -OP (O) (OR) (OR '), in aonae ~ TCy R' can be the same alkyl groups or dITé'F'é'rtfes, or kyR · "* can be taken together to represent an alkanediyl. Non-limiting examples of dialkyl phosphate groups include: OP (O) (OMe)₂, -OP (O) (OEt) (OMe) and -OP (O) (OEt)₂. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms has been replaced by -OH, -F, -Cl, -Br, -I, - H₂, -NOT₂, -CO₂H, CO₂CH₃, -CN, -SH, -OCH₃, -OCH₂CH₃, -C (O) CH_3i -nhch₃, NHCH₂CH₃, -N (CH₃)_2i -C (O) NH_2i -OC (O) CH₃ bear)₂NH₂.

The terms "alkylsulfonyl" and "alkylsulfinyl" when used without the "substituted" modifier refers to S (O) groups₂R and -S (O) R, respectively, where R is an alkyl, as said term was defined above. The terms "alkenylsulfonyl", "alkynylsulfonyl," arylsulfonyl "," aralqullsulfonyl "," heteroarylsulfonyl "and" heterocycloalkylsulfonyl "are defined in an analogous manner. When any of these terms is used with the "substituted" modifier, one or more hydrogen atoms has been independently replaced by -OH, -F, -Cl, -Br, -I, NH₂, -NOT₂, -CO₂H, -CO₂CH₃, -CN, -SH, -OCH₃, -OCH₂CH₃, C (O) CH₃, -NHCH_3i -NHCH₂CH_3i -N (CH₃)_2i -C (O) NH_2i -OC (O) CH₃bear)₂NH₂.

As used in the present invention, a "surgical assistant" refers to a removable surgical group that has the

131

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Ability to Influence Compounds Experts in the art are familiar with CTÓT, ITTC1TÜS ~~~ compounds, and many of them are commercially available.

The use of the word "a" or "one, one" when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one", although it is also consistent with the meaning of "one or more ”,“ at least one ”and“ one or more than one ”.

Throughout this application, the term "approximately" is used to indicate that a value includes the inherent error variation of the device, the method being used to determine the value or the variation that exists among the study subjects.

The terms "comprise", "has" and "includes" are open-ended linking verbs. Any of the ways in which one or more of these verbs are used, such as comprise ”,“ comprising ”,“ has ”,“ which has ”,“ includes ”and“ which includes ”, are also open-ended. For example, any method that "understands", "has" or "includes" one or more steps, is not limited to having only one or more steps, and also covers other steps not described.

The term "effective", as said term is used in the specification and / or claims, means suitable to achieve a desired, expected or projected result. The

132

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Mexicano ΐϊΝϊτηυτο Mexican ot LA PXOPIKL'AD “effective amount”, “therapeutically amount i saw“ pharmaceutically effective amount ”when using · dentre · from the context of treating a patient or subject with a compound, means the amount of the compound which , when administered to a subject or patient to treat a disease, it is sufficient to effect treatment for the disease.

The term "hydrate" when used as a modifier for a compound means that the compound has less than one (eg, hemihydrate), one (eg, monohydrate), or more than one (eg, dihydrate) associated water molecule with each molecule of compound, such as in solid forms of the compound.

As used in the present invention, the term "IC₅₀"Refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates that both a particular drug and another substance (inhibitor) are needed to inhibit a given biological, biochemical, or chemical process (or component of a process, for example, an enzyme, cell, cell receptor, or microorganism) to the half.

An "isomer in a first compound is a separate compound where each molecule contains the same constituent atoms as the first compound, but where the configuration of these atoms differs in three dimensions.

As used in the present invention, the term

133

1Y1 Jr

MEXICAN INSTITUTE Df. THE PROPERTY industrial mammalian organism

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"Patient" or "subject" refers to such as human, monkey, cow, sheep, goat, dog, cat, ίδϊΟΠ'Γ rat, Guinea pig, or transgenic species thereof. In certain modalities, the patient or subject is a primate. Non-limiting examples of human subjects are adults, youth, infants, or fetuses.

As used generally in the present invention the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms which, within the scope of medical judgment, are suitable for use in contact with tissues, organs and / or body fluids of humans and animals without toxicity, irritation, allergic response, or other excessive problems or complications with a reasonable benefit / risk ratio.

"Pharmaceutically acceptable salts" means salts of the compounds of the present invention that are pharmaceutically acceptable, as defined above, and that possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'134 acid

IMPI

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methylenebis (3-hydroxy-2-ene-1-carboxylic), methyl ib [[2.2.2] oct-2-ene-1-carboxylic acid, acetic acid, mono- and dicarboxylic aliphatic acids, aliphatic sulfuric acids, sulfuric acids aromatics, benzenesulfonic acid, benzoic acid, camphor sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopenta nopropionic acid, ethanpsulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o- (4-hydroxybenzoyl) benzoic acid, oxalic acid, pclorobenzenesulfonic acid, substituted phenyl-alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present have the ability to react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. You must recognize that the particular anion or cation that forms

135

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INSTiruTAMiXICANO starts from any salt of the present and important, provided that the salt, in whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Sa / ts: Properties, and Use (PH Stahl &amp; CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

The term "pharmaceutically acceptable carrier" as used in the present invention means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting an agent. chemical.

The term "prevention" or "prevent" includes: (1) inhibit the generation of a disease in a subject or patient who is at risk and / or predisposed to the disease but who does not yet experience or show any or all of the pathology or symptomatology of the disease, and / or (2) stops generating the pathology or symptomatology of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who still experiences or displays any or all of the pathology or symptomatology of the disease.

A "stereoisomer" or "optical isomer" is an isomer of a given compound where they are linked

136

X JLVJL JL JL tNSTITUT, MEXICAN PROPERTY atoms to the same other atoms, but in doiTO'é^You!'tfifieT ^ 3í9 configuration of said atoms in t γΤΤΠ III tí i rs iu 11 e s.— “enantiomers” are stereoisomers of a certain compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chirical center, also referred to as a stereocenter or stereogenic center, which is any point, although not necessarily an atom, in a group-containing molecule such that an exchange of either group leads to a stereoisomer . In organic compounds, the center of surgery is usually a carbon, phosphorus, or sulfur atom, although other atoms may also be stereocenters in organic or inorganic compounds. A molecule can have multiple stereocenters, providing many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (eg, tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocentres.

Molecules with symmetry often have less than a maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture.

Alternatively, a mixture of enantiomers can sr

137

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Nantiomerically enriched so that the enantiomer is greater than 50%: 't + o-rmalme ^ to. The _enantiomers and / or diasteromers can be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or axis of chirality for which stereochemistry has not been defined, said stereocenter or axis of chirality may be present in its R-form, S-form, or as a mixture of R and S forms, including racemic mixtures and not racemic. As used in the present invention, the phrase "substantially free of other stereoisomers" means that the composition contains £ 15%, more preferably £ 10%, even more preferably £ 5%, or more preferably £ 1%, of another stereoisomer ( s).

The term "treatment" or "treat includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (eg, stopping further development of the pathology and / or symptomatology), (2 ) alleviating a disease in a subject or patient who is experiencing or showing the pathology or symptomatology of the disease (for example, reversing the pathology and / or symptomatology) and / or (3) effecting any measurable decrease in a disease in a subject or patient who is experiencing or displaying the pathology or symptomatology of the disease.

The terms "nucleotide base", "nucí obase" or

138

IMPIAS *

INSTITUTO MEXICANO Zteicíí DE LA TKf.lPIEDAD simply “base”, as used in 'Tcf ^ rerew? Invention refers to a heteroaromatic CIO uiiyeii qiH * ring containing substituted or unsubstituted nitrogen of a type that is commonly found in nucleic acids, as well as natural, substituted, modified, or designed or analogue variants thereof. In a typical embodiment, the nucleobase has the ability to form Watson-Crick and / or Hoogsteen hydrogen bonds with a suitably complementary nucleobase. Example nucleobases include, but are not limited to, purines such as 2aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N⁶TO²-isopentenyladenine (6 i A), / V⁶-TO²-isopentenyl-2-methylthioadenine (2ms6iA), / V⁶-met¡laden¡na, guanine (G), isoguanine, N²dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6thioguanine (6sG), hypoxanthine, and O⁶-methylguanine;

7-deaza-purines such as 7-deazaadenine (7-deaza-A), 7-deazaguanine (7-deaza-G), 7-deaza-7-hydroxymethyladenine, 7deaza-7-aminomethyladenine and 7-deaza-7-hydroxymethylguanine ;

pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, 5-hydroxymethylcytosine (HOMeC), 5-aminomethylcytosine, thymine (T), 4-thothymine (4sT), 5,6-dihydrothimine, OR⁴methylthymine, uracil (U), 4-thiouracil (4sU), 5-hydroxymethyluracil (HOMeU), 5-aminomethyl-uracil and 5,6-dihydrouracil (dihydrouracil; D);

indoles such as nitroindole and 4-methylindole; such pyrroles

139 ΐΜΡΙ • 'STITUrO MEXICANO DC THE INDUSTRIAL PROPERTY

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like nitropyrrole; nebularin; base (Y); tea. industrial

Additional sample nucleobases * ss can be found in the Lehninger Publication, 2005, which is incorporated by reference in the present invention, and the references mentioned therein.

The term "nucleoside" as used in the present invention refers to a glycosylamine consisting of a nucleobase linked to a five carbon sugar, usually a ribose or deoxyribose. Examples of these include, but are not limited to, cytidine, 2'-deoxycytidine, 5h id roxy I meti I cytidine, 2^,-deoxy-5-hidroxilmetilcitidina, 5aminometilcitidina, 2'-deoxy-5aminometilcitidina, uridine, 2'deoxyuridine, 5hidroxilmetiluridina, 2'-deoxy-5hidroxilmetiluridina, 5aminometiluridina, 2'-deoxy-5aminometiluridina, adenosine, 2 ' -deoxydenosine, 7-deaza-7hydroxymethyladenosine, 2'-deoxy-7-deaza-7hydroxymethyladenosine, 7-deaza-7-aminomethyladenosine, 2'-deoxy-7-deaza-7-amino-methyladenosine, guanos i na, 2'-deoxyguanosine, 7-deaza-7-hydroxymethyl guanosine, 2'-deoxy-7-deaza-7-hydroxymethyl, 7-deaza-7aminomethyl guanosine, 2'-deoxy-7-deaza-7-aminomethyl guanosine, thymidine and 2'-deoxythymidine.

A "nucleotide" is composed of a nucleoside with one, two, three, or more phosphate groups chain-linked to the 5-carbon sugar of the nucleoside.

140

The term lag is a phenomenon,

<img file="MX342195B_D0166.tif" />

staged addition methods, including but without methods, CRT, SNA and SBL, when growing primers move out of synchronicity of any given cycle. The lag or type 2 strand offset (for example, n + 1 of the expected cycle) results from incomplete extension, and the main or type 1 strand offset (for example, n + 1) results from the addition of multiple nucleotides or probes in a population of identical templates.

The term "dark nucleotide" or "dark probe" refers to a nucleotide or probe that does not contain a fluorescent tag. It can be generated from its decomposition or carried from the previous cycle or it can be hydrolyzed in situ from its ink-labeled counterpart in the current cycle.

Unless otherwise specified, a "linker" refers to one or more divalent groups (link members) that function as a covalently linked molecular bridge between two other groups. A linker can contain one or more link members and one or more types of link members. Example link members include: C (O) NH- -C (O) O-, -NH-, -S-, -S (O)_n~ where n is 0, 1 or 2, -O, -OP (O) (OH) O-, -OP (O) (O ') O-, alkanediyl, alkenodiyl, alkynediyl, arenediyl, heteroanediyl or a combination of the same. Some linkers have pending side chains or pending functional groups (or both). The

141

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INSTITUTE Μ £ λ) CANO «Λ

Say LA PROWSJAIt examples of such pending portions are motíTWWdoree ^ -tfé · ^ hydrophilicity, for example, groups of soiu bi H i ó nti μ u, μ υ · ι ·. example -SO₃H or -SO₃'. In some embodiments, a linker may connect a reporter to another portion such as a chemically or enzymatically reactive group (eg, a dissociable or non-dissociable termination portion). In other embodiments, a linker connects a reporter to a biological or non-biological component, for example, a nucleobase, a nucleoside, or a nucleotide. In additional embodiments, a linker connects chemically reactive groups to a nucleobase, nucleoside, or nucleotide. The portion made up of a linker linked to a reporter can be designated as -L-Reporter. Depending on factors such as the molecules to be linked and the conditions under which the strand synthesis method is carried out, the linker can vary in length and composition to optimize properties such as stability, length, FRET efficiency, resistance to certain chemical and / or temperature parameters, and being of sufficient stereoselectivity or size to operably bind a tag to a nucleotide such that the resulting conjugate is useful in optimizing a polymerization reaction. Linkers can be employed using standard chemical techniques including, but not limited to, amine linkers for attaching tags to nucleotides (see, eg, Publication

142

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MEXICAN INSTITUTE Pt LA RRÜPIFDAD

INDUSTRIAL

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Hobbs and Trainor, US Patent 5,151,507, which is incorporated by reference into the present invention); a linker typically contains a primary or secondary amine to operably link a tag to a nucleotide; and a rigid hydrocarbon arm added to a nucleotide base (see, eg, Service Publication, 1998, which is incorporated by reference in the present invention). Some exemplary binding methodologies for reporter adhesion to base molecules are provided in US Patents 4,439,356 and 5,188,934; European Patent Application 87310256.0; International Application PCT / US90 / 05565 and in the Publication of Barone et al., 2001, each of which is incorporated in its entirety by reference to the present invention.

A "decomposable linker" is a linker that has one or more groups that can decompose or break as a result of a reaction or condition. The term "group that can be decomposed" refers to a portion that allows the release of a portion, for example, a fluorogenic or fluorescent portion. Said decomposition is normally transmitted in chemical, photochemical or enzymatic form. Exemplary enzymatically decomposable groups include phosphates, or groups attached via a peptide bond.

As used in the present invention, the term

143

MEXICAN INSTITUTE ifCj · -M

PROPERTY gg / “IC50” refers to but is not limited to the ntYclW &amp; n che ^ -tFr nucleotide analog where its ¡n¿D'l μυι dbiúii tu un&gt;. primer-template complex produces equal numbers of moles of substrate and product and / or can be defined, but not limited to the incorporation efficiency measured by determining the concentration at which the compound is incorporated on behalf of the primer-template complexes.

As used in the present invention, the term "oligonucleotide" refers to DNA fragments of 2 to 200 nucleotides covalently linked.

As used in the present invention, the term "reporter" refers to a chemical portion that has the ability to produce a detectable signal directly or indirectly. Examples of reporters include fluorescent ink clusters, radioactive labels, or clusters that produce a signal by chemiluminescent or bioluminescent means.

Examples of fluorescent ink groups include zantene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthaloclanin, phycobiliprotein, ALEXA FLUOR® 350, ALEXA FLUOR® 430, ALEXA FLUOR® 488,

ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR®

546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA

FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610,

ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR®

660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA

144

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FLUOR® 750, and a squarin ink.

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Additional fluorescent ink groups that · 88 can be used in some embodiments of the present invention are described throughout this specification and in Haugland Publication 2005 and in US Patents 4,439,356 and 5,188,934, which are incorporated in the present invention for reference. Examples of radioactive labels that can be used as reporters in some embodiments of the present invention are well known in the art, such as ³⁵Yes, ³H ³²P o ³³P. The examples of reporters who operate by chemiluminescent or bioluminescent means and who may be used as reporters in some embodiments of the present invention are described in the Nieman Publications, 1989; Given &amp; Schowen, 1989; Orosz et al., 1996; and Hastings, 1983, which are incorporated by reference in the present invention.

As used in the present invention, the term "template" may refer to an oligonucleotide that serves as the complementary strand for DNA synthesis (incorporation) or a recombinant DNA molecule that is made from a known region, usually a sequence of vector or adapter to which a universal primer can be linked, and the target sequence, which is normally an unknown portion to be sequenced.

145

IM F!

ΓΟ MEXICAN

The term "fragment templates" is a library of fragments that have been prepared by mprlia.nle. Small-section genomic DNA randomized into small sizes &lt;1 kb and ligating adapters to each end of the fragment. These templates generally require less DNA than might be required for a corresponding pair library.

The term "matching pair template" refers to a genomic library that has been prepared by circulating the cut DNA fragment that has been selected for a given size (examples include 2kb or 5kb or 10kb or 20kb or any other size desired), thus bringing the ends that were previously distant from each other in close proximity. Cutting these circles into linear DNA fragments creates corresponding pair templates.

As used in the present invention, the term "primer" refers to an oiigonucleotide that hybridizes to a complement sequence in the template strand (usually a known sequence) used to initiate DNA synthesis (incorporation).

When used in the scientific or technical sense in the present invention, the term "incorporation" refers to a nucleotide or nucleotide analog that forms a complement base pair with the template strand and a covalent bond to a primer strand through of a polymerase. The complex

INSTO UTO MEXICANO Λ&gt;

OF THE PROEJEHAn

146

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MEXICAN ~ '^£ Primer-template primer PRüPIEL'AD extends one or more ba^sá鹧^1AL Initial primer strand.

As used in the present invention, the term "decomposition" refers to the removal of the termination group by chemical cleavage, enzymatic cleavage, or the like.

As used in the present invention, the term "incorporation cycle" refers to the incorporation of a nucleotide or nucleotide analog through a polymerase, the detection and identification of the nucleotide or nucleotide analog, and whether it is an analog nucleotide, the breakdown of the termination group, and if originally present in the nucleotide analog, the fluorescent ink group of said analog.

As used in the present invention, the term "lack of incorporation" refers to a nucleotide or nucleotide analog that forms an unsupplemented base pair with the template strand, and a covalent bond to a primer through a polymerase. . The primer-template complex extends one or more bases from the initial primer strand.

As used in the present invention, the term "discrimination" refers to differences in IC concentration₅₀ for lack of incorporation versus incorporation of the nucleotide or nucleotide analogs through a polymerase.

As used in this invention, the term

147

<img file="MX342195B_D0170.tif" />

Termination refers to the incorporation of a nucleotide or nucleotide that forms a complement or non-complement base pair with the template strand and a covalent bond to a primer through a polymerase. The primer-template complex extends only one base from the starting primer strand or growing primer strand for any given cycle of incorporation.

The terms "termination portion" and "termination group" as used in the present invention are synonyms, they refer to a small chemical group (eg, &lt;500 daltons, excluding any modifications, such as a linker or linker / ink) than when adhering to at least part of a nucleoside (for example, sugar or nucleobase) or nucleotide (for example, sugar, nucleobase or phosphate group) confers substantial termination properties on the nucleoside or nucleotide. In some embodiments, the termination portion is further modified with a linker and / or an ink-bonded linker. In preferred embodiments, the termination properties of said modified termination group are not substantially altered.

As used in the present invention, the term "DT₅₀"Refers to the amount of time required to dissociate 50% of the base analog incorporated in the primer-template complex.

148

<img file="MX342195B_D0171.tif" />

The term "analogous" as invention is understood to be a^to -nuptinria φιρ _nnit comprises the same basic carbon skeleton and carbon functionality in its structure as a "given compound", but may mimic the given compound by incorporating one or more suitable substitutions such as, for example, substitution of carbon for heteroatoms.

The above definitions exceed any conflicting definition in any of the references that are incorporated into the present invention. The fact that certain terms are defined, however, should not be construed as indicating that any term is undefined or undefined. Rather, all terms used are considered to describe the invention in terms so that one skilled in the art can appreciate the scope and practice of the present invention.

V. Examples

The following examples are included to demonstrate the preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques described in the examples below represent the techniques discovered by the inventor to function well in the practice of the present invention, and therefore may be considered as constituting preferred modes for your practice. However, those skilled in the art, in light of the present

149

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IMPI

MEXICAN INSTITUTE

...... . FROM the PROPERTY description, it should be appreciated that changes in the specific modalities that they describe can be reatazavAL and still obtain a similar or similar result without departing from the spirit and scope of the present invention.

Example 1 - Methods and Materials

Reagents and materials. All reagents were purchased from commercial sources and used as received, unless otherwise indicated.

Spectroscopic and analytical instrumentation. Spectra were recorded¹H NMR, ¹³C NMR and ³¹P NMR on a Bruker DPX 400 spectrometer as previously described in the Publication of Wu et al. (2007), which is incorporated into the present invention by reference. Mass spectrum analysis was provided through the Mass Spectrometry Laboratory at the MD Anderson Cancer Center (Houston, TX) and the Core Mass Spectrometry Facility at Rice University ( Houston, TX). X-ray crystallography was performed through the X-ray Diffraction Laboratory at Texas A&amp;M University (College Station, TX). UV / Vis measurements were taken using a Beckman DU-800 spectrophotometer. Anion exchange chromatography was carried out using a Q Sepharose FF column (2.5 x 20 cm) with a linear gradient of 75% triethylammonium bicarbonate (TEAB, 0.1 M) in 25% acetonitrile for TEAB 75% (1.5

150

<img file="MX342195B_D0173.tif" />

MEXICAN INSTITUTE Df LA ΚΚΟΠεΡΛΟ

M) in 25% acetonitrile for 240 min err^Dim &lt;* rafrg45 = -cr ^ flow of 4.5 mL per min. Of om ο · ίο§4 &lt;ι4 »» ··· Ι4 · ^ Μ.κΙο ·· High Performance Reverse Phase (RP-HPLC) was performed using a Beckman System Gold equipped with a 128 5 solvent module and a UV 166 detector or 168 photodiode formation UV / Vis detector. RP-HPLC for nucleoside and nucleotide analogs was performed using a 4.6 mm x 250 mm Aquapore OD-300 Ci column.₈, with buffer A containing 100 mM triethylammonium acetate (TEAA), pH 7.0, and buffer 10 B containing 100 mM TEAA, pH 7.0, 70% (v / v) acetonitrile. Example 2 - Synthesis of substituted 2-nitrobenzyl alcohols (R / S) -1 - (2-Nitrophenyl) -2-methyl-1 -propanol ·. The synthesis of (/? / S) -1- (2-nitrophenyl) -2-methyl-115 propanol was previously reported. See the Publication of Litosh et al. (2011), which is incorporated into the present invention by reference.

(R / S) -1 - (2-Nitrophenyl) -2,2-dimethyl-1 -propanol ·. Synthesis of (RZS) - 'i - (2-nitropheniI) -2,2-dimethiI-1 propanol was previously reported. See the Publication of Litosh et al. (2011), which is incorporated into the present invention by reference.

(R / S) -1 - (2,6-Dinitrophenyl) -2-methyl-1-propanol:

NO (i)

HO '

OR₂N

1-iodine-2,6dinitr b ncen (R / S) -1- (2,6-dinitrophenyl) 2-m til-1-propanol

151

<img file="MX342195B_D0174.tif" />

Esqu ma S1. Synthesis of (R / S) -1- (2,6-WTf1tfonfrSÍlprmetil-1 -propanol. Reagents and conditions? * '(Yf PhMgDr, TWff._rminus 50 ° C; / '- PrCHO, minus 50 ° C at room temperature,

30%.

To a solution of 1-iodine-2,6-dinitrobenzene (Smith and Ho

1990), which is incorporated into the present invention by reference) (1.55 g, 5.27 mmol) in anhydrous THF (18 mL) at minus 50 ° C under a nitrogen atmosphere, phenylmagnesium bromide was added dropwise ( 2 M in THF, 3.2 mL,

6.4 mmol) in a range so that the temperature did not exceed minus 45 ° C. At the end of the addition, the mixture was stirred at minus 50 ° C for five minutes, followed by the addition of isobutyraldehyde (0.96 mL, 1 1 mmol). The mixture was gradually warmed to room temperature, quenched with NH solution₄CI saturated (10 mL), and subsequently diluted with water (50 mL). The mixture was extracted with CH₂CI₂ (100 mL) three times. The combined organic phase was washed with brine (50 mL), dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield (F? / S) -1- (2,6-dinitrophenyl) -2-methyl-1-propanol (0.375 g, 30 %) in the form of a yellow oil. ¹H NMR (400 MHz, CDCI₃): δ 7.82 (d, 2 H, J = 8.0 Hz, Ph-H), 7.59 (t, 1 H, J = 8.0 Hz, Ph-H), 4.83 (dd, 1 H, J = 9.2 and 7.6 Hz , PhCH), 2.87 (d, 1 H, J = 7.6 Hz, OH), 2.19 (m, 1 H, CH), 1.12 (d, 3

H, J = 6.4 Hz, CH₃), 0.76 (d, 3 H, J = 6.8 Hz, CH₃).

152

IMPÍ (R / S) -1 - (4-Meto-2-nitrophenyl) -2-methyl-1 -propan

INSTITUTO MÍXICANÍ ot la pm '. rostiRD iDan ovmstriaí

<img file="MX342195B_D0175.tif" />

not₂

<img file="MX342195B_D0176.tif" />

4-iodo3-nitroanisole

OMe (i)

<img file="MX342195B_D0177.tif" />

OMe (R / S) - 1- (4-methoxy2-n-trophenyl) -2-methyl 1-propanol

Scheme S2. Synthesis of (R / S) -1- (4-methoxy-2-nitrophenyl) 2-methyl-1-propanol. Reagents and conditions ·, (i) PhMgCI, THF, minus 40 ° C; / -PrCHO, minus 40 ° C at room temperature,

67%.

To a solution of 4-iodo-3-nitroanisole (2.79 g, 10.0 mmol) in anhydrous THF (20 mL) at minus 40 ° C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF, 6.0 mL, 12 mmol) in a range so that the temperature did not exceed minus 35 ° C. At the end of the addition, the mixture was stirred at minus 40 ° C for five minutes, followed by the addition of isobutyraldehyde (1.8 mL, 20 mmol). The mixture was gradually warmed to room temperature, quenched with NH solution₄CI saturated (5.0 mL), diluted with CH₂CI₂ (100 mL) and washed with water (100 mL). The organic phase was separated, and the aqueous phase was extracted with CH₂CI₂ (50 mL) three times. The combined organic phase was washed with brine (40 mL), dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield (R / SJ-1 - (4-methoxy-2-nitrophenyl) -2-methyl-1-propanol (1.5 g, 67% ) in the form of a

153 light yellow oil.

<img file="MX342195B_D0178.tif" />

J = 8.8 Hz, Ph-H), 7.34 (d, 1 H, J = 2.8 Hz, Ph.H ·), ....... 7.15, (ddy-4

H, J = 8.8 and 2.8 Hz, Ph-H), 4.92 (dd, 1 H, J = 5.6 and 3.2 Hz, PhCH), 2.46 (br s, 1 H, OH), 2.00 (m, 1 H, CH ), 0.97 (d, 3H, J =

6.4 Hz, CH₃), 0.86 (d, 3 H, J = 6.8 Hz, CH₃).

(R / S) -1 - (4-Methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol

<img file="MX342195B_D0179.tif" />

4-iodo- (R / S) - 1- (4-metOX¡3-nitroanisol 2-nitrophenyl) 2,2-dimethyl-1 -propanol

Scheme S3. Synthesis of (/? / S) -1 - (4-methoxy-2-nitrophenyl) 2,2-d imeti I-1-propanol. Reagents and conditions', (i) PhMgCI, THF, minus 40 ° C; (CH₃)₃CCHO, minus 40 ° C at room temperature, 74%.

To a solution of 4-iodo-3-nitroanisole (2.38 g, 8.50 mmol) in anhydrous THF (10 mL) at minus 40 ° C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF) was added dropwise , 4.7 mL, 9.4 mmol) in a range so that the temperature did not exceed minus 35 ° C. At the end of the addition, the mixture was stirred at minus 40 ° C for one hour, followed by the addition of trimethylacetaldehyde (1.13 mL, 10.2 mmol). The mixture was stirred at minus 40 ° C for two hours and then at room temperature for another hour. The reaction was quenched with brine (100 mL), and the

154

IMPI

MEXICAN INSTITUTE. ._Λ1,,. . . PROPERTY mix was extracted with CH2CI2 (40 mL) three vwjsek combined organic MSBPre dried over Na₂SO4 and &amp; oo-oncentrated ¿/ 3 · vacuo, and the residue was purified by silica gel column chromatography to produce (R / S) -] - (4-methoxy-2n it ro feni I) -2, 2-di me ti I -1-racemic propanol (1.52 g, 74%). ¹H NMR (400 MHz, CDCI₃): δ 7.67 (d, 1 H, J = 9.2 Hz, Ph-H), 7.22 (d, 1 H, J = 2.4 Hz, Ph-H), 7.12 (dd, 1 H, J = 8.8 and 2.8 Hz , PhH), 5.27 (d, 1H, J = 4.0Hz, Ph-CH), 3.86 (s, 3H, OCH₃), 2.01 (d, 1 H, J = 4.0 Hz, OH), 0.86 (s, 9 H, C (CH₃)₃).

(R / S) -1 - (5-Methoxy-2-nitrophenyl) -2, 2-dimethyl-1 -propanol and (S) -1 - (5-methoxy-2-nitro-phenyl) -2,2- dimetU-1 -propanol no₂ (i)

OMe

<img file="MX342195B_D0180.tif" />

(¡¡)

<img file="MX342195B_D0181.tif" />

OMe (/ ySl-HS-methoxy2-n-trophenyl) -2,2-d-methyl1-propyl (1 S) -camfanat

3- Iodine4- Nitroanisole (R / S) -1 - (5-methoxy2-nitrophenyl) -2,2-dimethyl1-propanol

<img file="MX342195B_D0182.tif" />

OMe (S) -1- (5-methoxy, 2-nitrophenyl) -2,2-dimethyl1-propyl (IS) -camfanate (S) -1- (5-methoxy2-nitrophenyl) 2,2 -di m eti I -1 -p rop an ol

155

<img file="MX342195B_D0183.tif" />

dimethyl-1-propanol. Reagents and conditions: (i) PhMgCI, THF anhydrous, minus 40 ° C; (CH₃)₃CCHO, minus 40 ° C at room temperature, 88%; (I) (1 S) -camphanic acid chloride, DMAP, CH2CI2, room temperature; (/ 77) fractional crystallization from ethyl acetate / hexane, 43%; (iv) K₂CO₃, MeOH, reflux, 99%.

To a solution of 3-iodo-4-nitroanisole (2.79 g, 10.0 mmol) in anhydrous THF (10 mL) at minus 40 ° C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF, 4.2 mL, 8.3 mmol) in a range so that the temperature did not exceed minus 35 ° C. At the end of the addition, the mixture was stirred at minus 40 ° C for two hours, followed by the addition of trimethylacetaldehyde (1.1 mL, 10 mmol). The mixture was stirred at minus 40 ° C for two hours and then at room temperature for another hour. The reaction was subsequently quenched with brine (100 mL), and the mixture was extracted with CH₂CI₂ (40 mL) three times. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by chromatography on a silica gel column to yield (R / S) -1 - (5-methoxy-2n it ro feni I) - 2,2 - di met i I Racemic -1-propanol (1.76 g, 88%). ¹H NMR (400 MHz, CDCI₃): δ 7.89 (d, 1 H, J = 9.2 Hz, Ph-H), 7.27 (d, 1 H, J = 2.8 Hz, Ph-H), 6.84 (dd, 1 H, J = 8.8 and 2.8 Hz , Ph156

<img file="MX342195B_D0184.tif" />

Η), 5.62 (d, 1 Η, J = 4.0 Hz, PhCH), 3.89 (s, (d, 1 H, J = 4.0 Hz, OH), 0.89 (s, 9 H, C (CH₃)^-—

To a solution of racemic (R / S) -1- (5-methoxy-2-nitrophenyl) -2,2dimethyl-1-propanol (1.75 g, 7.3 mmol) and DMAP (2.92 g, 23.9 mmol) in CH₂CI₂ anhydrous (10 mL), (fS) -camphanic chloride (Corrie et al, 1992), which is incorporated into the present invention by reference) (2.6 g, 12 mmol) was added, and the mixture was stirred during the overnight at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (50 mL) and washed with a NaHCO solution₃saturated (50 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by a silica gel column chromatography to produce (1S) (R / S) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethll- 1 propyl (2.5 g, 85%, 1: 1 mixture of diastereomers). The camphanate was dissolved in ethyl acetate (30 mL) followed by the slow addition of hexane (120 mL) with stirring. The needle crystals gradually formed from the solution over a period of two hours. The crystals were collected by filtration to produce the pure simple (1S) diastereomer of (S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl. The filtrate was concentrated In vacuo, and the crystallization process was repeated twice to provide additional (S) -1 (5) methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl (total 1.08 g, 43%).¹Ή NMR (400 MHz, CDCI₃): δ 8.04 (d, 1 H, J = 9.2 Hz,

157

<img file="MX342195B_D0185.tif" />

<img file="MX342195B_D0186.tif" />

<img file="MX342195B_D0187.tif" />

MEXICAN INSTITUTE

Ph-H), 7.27 (d, 1 H, J = 2.8 Hz, Ph-H), 6.88 (dff / e ^

8.8 Hz, Ph-H), 6.81 (3, 1 H, Ph-CH), 3.87 ($ ,, ... 3 H, DGH_to), 2 (m, 1H, CH), 1.92 (m, 2H, CH₂), 1.66 (m, 1H, CH), 1.12 (s, 3

H, CH₃), 1.06 (s, 3H, CH₃), 1.02 (s, 3H, CH₃), 0.95 (s, 9H, C (CH₃)₃).

Method for obtaining X-ray crystallography data: Crystallographic measurements were taken on a crystal of (1S) (S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propylophane with dimensions of 0.50 mm x 0.05 mm x 0.05 mm as described in the Litosh et al (2011) Publication, which is incorporated by reference in the present invention. See figure 3.

Data collection ', CuKa radiation, λ = 1.54178 A, T = 110 ± 2 ° K, 26_max = 120.0 °, 32,513 reflections collected, 2,913 unique (R_int = 0.0517). Final GooF = 1.091, R1 = 0.0681, \ nR2 = 0.1695, R indices based on 2,913 reflections with l&gt; 2sigma (l) (refinement in F²), 290 parameters, 43 restrictions. Lp and absorption corrections applied, μ = 0.819 mm '¹. Absolute structure parameter: 0.05 ± 0.09.

X-ray crystallography data: C₂₂H₂9NO₇, M = 419.46. Orthorombic, a = 6.29, b = 15.00, c = 22.27 A (α, β, y = 90 °), V = 2,099.29 A³, space group Ρ2ι2ι2₁₍ Z = 4, D_c =

I. 327 g / cm '³, F (000) = 896.

A mixture of (1 S) -camphanate of (S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl (590 mg, 1.4 mmol) and K2CO₃

158

Jtvmvro MtxtCAwo (389 mg, 2.8 mmol) in methanol (MeOH, 25 mPI refluxed for one hour, then ** cooled, m rnnr. »Ntrft in vacuo, and diluted with CH₂CI₂ (50 mL). The organic phase was washed with brine (50 mL), dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce (S) -1- (5-methoxy-2-nitropheniI) -2,2-dimethi-1 -propanol enantiopuro (333 mg, 99 %). ¹H NMR was identical to that of racemic alcohol.

(R / S) -1 - (4,5-Dimethoxy-2-nitrophenyl) -2,2-dimethyl-1 -propanol

<img file="MX342195B_D0188.tif" />

1,2-dimethoxybenzene

<td></td><td>not₂</td><td>i-Bu NO₂</td><td></td>

<td></td><td></td><td>(V hoAA</td><td></td>

<td>l '^^ OMe</td><td>η ^ οΜθ</td><td>AND</td><td>OMe</td>

<td></td><td>OMe</td><td>OMe</td><td></td>

<td>4,5-diiodO-1,2- dimethoxy- benzene</td><td>4-iodine-5-nitro- 1,2-dimethoxy- benzene</td><td colspan="2">(R / S) · 1 - (4,5-dim toxi- 2-nitrophenyl) -2,2- dimethyl-1-propanol</td>

Scheme S5. Synthesis of (R / S) -1 - (4,5-dimethoxy-2-nitrophenyl) 2,2-dimethyl-1-propanol. Reagents and conditions: (i) ICI, CH3COOH, 100 ° C, 62%; (ii) HNO₃, CH₃COOH, room temperature, 75%; (iii) PhMgCI, THF, minus 40 ° C; (CH₃)₃CCHO, minus 40 ° C at room temperature, 20%.

1,2-Dimethoxybenzene (5.0 g, 36 mmol) was dissolved in acetic acid (10 mL), and the solution was cooled in an ice-water bath followed by dropwise addition of iodine chloride (8.7 g, 54 mmol ). After 10 minutes, the reaction mixture was heated to a temperature of 100 ° C for two hours and subsequently cooled to room temperature. The

159

IMPI needle crystals that rushed out of the solution¹ Fil and washed with acetic acid (5.0 mL) three VTíUtíü. Use busi dried overnight under high vacuum to produce 4,5-diiodo-1,2-dimethoxybenzene (8.8 g, 62%).¹H NMR (400 MHz, CDCI₃): δ 7.28 (s, 2H, Ph-H), 3.85 (s, 6H, OCH₃).

4,5-Diiodo-1,2-dimethoxybenzene (8.8 g, 23 mmol) in acetic acid (300 mL) was added, and the mixture was heated to a temperature of 100 ° C to dissolve the solid. The clear mixture was subsequently cooled to room temperature followed by portionwise addition of nitric acid (68-70%, 120 mL). The reaction mixture was stirred at room temperature overnight, and then poured into ice-water (200 mL). The mixture was extracted by CH₂CI₂ (100 mL) three times. The combined organic phase was washed with a NaHCO solution₃ saturated (200 mL), brine (100 mL), and dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 4-iodo5-nitro-1,2-dimethoxybenzene (5.25 g, 75%). ¹H NMR (400 MHz, CDCI₃): δ 7.60 (s, 1H, Ph-H), 7.38 (s, 1H, Ph-H), 3.98 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃).

To a solution of 4-iodo-5-nitro-1,2-dimethoxybenzene (4.6 g, 15 mmol) in anhydrous THF (10 mL) at minus 40 ° C under a nitrogen atmosphere, chloride was added dropwise of phenylmagnesium (2 M in THF, 7.5 mL, 15 mmol) in a range so that the temperature did not exceed minus 35 ° C. At the end

160

<img file="MX342195B_D0189.tif" />

INSTITUTO MEXICAN of the addition, the mixture was stirred in less hours, followed by the addition of acetaldehyde-do-tr-methyl ... (. 2..Q mL, 18 mmol). The mixture was stirred at minus 40 ° C for two hours and then at room temperature for another hour. The reaction was quenched with brine (100 mL), and the mixture was extracted with CH₂CI₂ (40 mL) three times. The combined organic phase was dried over Na₂SW₄ and concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield racemic (R / S) - ^ - (4,5-dimethoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol (0.8 g, 20%). ¹H NMR (400 MHZ, CDCI₃): δ 7.41 (1,1 H, Ph-H), 7.21 (s, 1 H, Ph-H), 5.60 (s, 1 H, PhCH), 3.95 (s, 3 H, OCH3), 3.92 (s , 3 H, OCH₃), 0.90 (s, 9H, (CH₃)₃).

Example 3 - Synthesis of 7-HOM -7Deaza-2'-Deoxyadenosine Triphosphate Analogs

7- (2-nitrobenzyloxy) methyl-7-deaza-2'-deoxyadenosine-5'tri phosphate

161

Η O.

OH (i¡¡)

<img file="MX342195B_D0190.tif" />

TBSO.

Cl ^H&gt; or w ^N

TBSO.

<img file="MX342195B_D0191.tif" />

= * · «'Mu * ¡rs“ sa »»

OTBS

OTBS (¡v)

O, N

TBSO.

<img file="MX342195B_D0192.tif" />

OTBS

O, N

<img file="MX342195B_D0193.tif" />

OTBS

<img file="MX342195B_D0194.tif" />

V-V ió

HO ^ zO \_D&gt;'^v'' Ϊ _ Λ _ Λ _ Λ \ UoJ OOO or O or γ_7

OH dA.I

Scheme S6. Synthesis of 7- (2-nitrobenzyloxy) methyl-7-deaza2'-deoxyadenosine-5'-triphosphate. Reagents and conditions', (/) TBSCI, imidazole, DMF, room temperature, 87%; (ii) CO, PdCI₂[PhCN]₂, MeOH / 1,4-dioxane, 50 ° C, 98%; (iii) LiBH₄, MeOH, THF, reflux, 45%; (iv) 2-nitrobenzyl bromide, z? -Bu + NBr, CH₂CI₂/ aq. NaOH, room temperature, 50%; (v) n-Bu₄NF,

THF, 0 ° C at room temperature; (vi) NH₃, 1,4-dioxane / MeOH, 100 ° C, 91% in two steps; (vii) POCI₃, (MeO)₃PO, minus 40 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

Compound 1 (Seela et ai. (2005), which is

162

<img file="MX342195B_D0195.tif" />

MEXICAN INSTITUTE

DE LA MOHEDAL '_ incorporated into the present invention as refer nrft ^^ O?

2.0 mmol) was evaporated from anhydrous pyridine dissolved in anhydrous DMF (4.0 mL). Te rb u ti chloride Id imeti I si I i lo (0.90 g, 6.0 mmol) and imidazole (0.82 g, 12 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The reaction was concentrated in vacuo and purified by silica gel chromatography to yield 9- [pD-3 ', 5'-O-bis (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] -6-chloro-7-iodo- 7deazapurine 2 (1.08 g, 87%) in the form of a white foam.¹H NMR (400 MHz, CDCI₃): δ 8.61 (s, 1 Η, H- 2), 7.81 (s, 1 H, H-8), 6.74 (t, 1 H, J = 6.4 Hz, H-1 '), 4.56 (m, 1 Η, H-4 '), 4.01 (m,

Η, H-3 '), 3.87 (dd, 1 H, H-5'a), 3.79 (dd, 1 H, H-5'b), 2.39 (m,

H, Η-2'a and H-2'b), 0.96 (s, 9 H, (CH₃)₃CSi), 0.91 (s, 9H, (CH₃)₃CS¡), 0.18 (2s, 6 H, (CH₃)₂Yes), 0.15 (s, 6H, (CH₃)₂And it is).

To a solution of compound 2 (1.55 g, 2.48 mmol) in

Anhydrous 1,4-dioxane (42 mL) and anhydrous MeOH (42 mL), triethylamine (0.87 mL) was added. After stirring for 10 minutes under a CO atmosphere, bis (benzonitrile) dichloropalladium (l I) (0.05 g, 0.13 mmol) was added, and the reaction was stirred at a temperature of 50 ° C for 48 hours under a CO atmosphere . The mixture was subsequently concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 9- [PD-3 ', 5'-O-bis (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] -6-chloro -7methoxycarbonyl-7-deazapurine 3 (1.36 g, 98%) in the form of a

163

IMPI

<img file="MX342195B_D0196.tif" />

ΙΓ &lt;2 1 IIUI \ J ΜΕΛΗ „Api .7 viscous oil. ¹H NMR (400 MHz, CDCI₃): δ 8.6 ^^LW ^ alH

8.31 (s, 1 Η, H-8), 6.77 (t, 1 H, J = 6.8 Hz,. ^ .. 1 /. ^ ... 53., (M, 1 JdL ·., ^

H-4 '), 4.06 (m, 1 Η, H-3'), 3.90 (s, 3 H, CH₃0), 3.87 (dd, 1 Η, H5'a), 3.81 (dd, 1 H, H-5'b), 2.42 (m, 2 H, H-2'a, and H-2'b), 0.93 ( yes

H, (CH₃)₃CS¡), 0.13 (s, 6 H, (CH₃)₂Yes), 0.12 (s, 6H, (CH₃)₂Yes!)

To a solution of compound 3 (0.28 g, 0.50 mmol) in anhydrous THF (4.0 mL), lithium borohydride (44 mg, 2.0 mmol) was added, followed by MeOH (0.1 mL). The reaction mixture was stirred at room temperature for 10 minutes and then heated under reflux for 45 minutes. On cooling to room temperature, the mixture was diluted with CH₂CI₂ (20 mL) and water (2.0 mL). The organic layer was separated, washed with brine (5.0 mL) twice, dried over Na₂SW₄, and concentrated in vacuo. The residue was purified by silica gel chromatography to produce 9- [β-ϋ-3 ', 5'-O-bis (fer-butyldimethylsilyl) -2'-deoxyribofuranosyl] -6-chloro-7hydroxymethyl-7-deazapurine 4 (0.12 g, 45%) as a white foam.¹H NMR (400 MHz, CDCI₃): δ 8.62 (s, 1 Η, H-8), 7.61 (s, 1 Η, H-2), 6.75 (dd, 1 H, J = 6.0 and 7.2 Hz, Hl '), 4.96 (AB d, 1 H, J = 1 1.6 Hz, 7-CH₂a), 4.91 (AB d, 1 H, J = 11.6 Hz, 7-CH₂b), 4.57 (m, 1 Η, H-4 '), 4.00 (m, 1 H, H-3'), 3.80 (m, 2 H, H-5'a, and H-5'b), 2.44 ( m, 1H, H-2'a), 2.04 (m, 1H, H-2'b), 0.91 (2s, 18H, (CH₃)₃CS¡), 0.11 (2 s, 12 H, (CH₃)₂Yes!)

To a solution of compound 4 (30 mg, 0.057 mmol) in

164

IMPI

INSTITUTO MEXICANO OS LA PROPIEDAD

CH₂CI₂ (2.0 mL), n-Bu was added₄NBr (9 mg, (T.OTS^lME 2-Nitrobenzyl Bromide (37mg, 0.17nmul) and ana'TülCTClúrr NaOH (1M, 2.0 mL). The reaction mixture was vigorously stirred at room temperature for 48 hours in the dark. The organic layer was separated, dried over Na2SO4, concentrated in vacuo, and the residue was purified by chromatography on silica gel to yield 9- [pD-3 ', 5'-O-bis (tert-butyldimethylsilyl) -2 '-deoxyribofuranosyl] -6-chloro-7- (2-nitrobenzyloxy) methyl-7-deazapurine 5 (19 mg, 50%) in the form of a viscous oil. ¹H NMR (400 MHz, CDCI3): δ 8.63 (s, 1 H, H-2), 8.06 (dd, 1 H, J = 8.4 and 1.2 Hz, Ph-H), 7.84 (d, 1 H, J = 7.6 Hz, Ph-H), 7.64 (s, 1 Η, H-8), 7.62 (m, 1 H, Ph-H), 7.43 (t, 1 H, Ph-H), 6.75 (dd, 1 H , J = 7.2 and 6.0 Hz, Hl '), 5.03 (s, 2 H, PhCH₂), 4.95 (AB d, 1 H, J = 12.0 Hz, 7-CH₂a), 4.88 (AB d, 1 H, J = 12.0 Hz, 7-CH₂b), 4.59 (m, 1 Η, H-4 '), 4.00 (m, 1 Η, H-3'), 3.80 (m, 2 H, H-5'a, and H-5'b), 2.48 ( m, 1H, H-2'a), 2.37 (m, 1H, H-2'b), 0.92 (2s, 18H, (CH₃)₃CSi), 0.1 1 (s, 6 H, (CH₃)₂Yes),

<img file="MX342195B_D0197.tif" />

0.10 (s, 6H, (CH₃)₂Yes!)

An n-Bu solution₄NF (17 mg, 0.054 mmol) in THF (1.0 mL) was added to a solution of compound 5 (18 mg, 0.028 mmol) in THF (1.0 mL) at a temperature of 0 ° C. The reaction mixture was gradually warmed to room temperature and stirred for two hours. The mixture was concentrated in vacuo, dissolved in 1,4-dioxane (2.0 mL), followed by the addition of NH₃in a MeOH solution (7M, 4.0 mL). The mixture was transferred into

165

IMPI ιηττΓτυτ!&gt; Mexican I heard THE INDUSTRIAL PROPERTY

100 ° C last

<img file="MX342195B_D0198.tif" />

a sealed tube and stirred at a temperature 16 hours, then cooled to room temperature, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 7- (2-nitrobenzyloxy) methyl-7-deaza-2 ' -deoxydenosine 6 (10 mg, 91%) in the form of a white foam. ¹H NMR (400 MHz, DMSOd₆): δ 8.08 (s, 1 Η, H-2), 8.06 (m, 1 H, Ph-H), 7.75 (m, 2 H, PhH), 7.58 (m, 1 H, Ph-H), 7.42 (s, 1 Η, H-8), 6.64 (bs, 2 H, D₂Or interchangeable, 6-NH₂), 6.48 (dd, 1 H, J = 2.0 and 6.0 Hz, H-1 '), 5.25 (d, 1 H, J = 4.0 Hz, D₂Interchangeable, 3'-OH), 5.08 (t, 1 H, J = 5.6 Hz, D₂Interchangeable O, 5'-OH), 4.90 (s, 2H, PhCH₂), 4.75 (AB dd, 2 H, 7-CH₂), 4.33 (m, 1 Η, H-3 '), 3.81 (m, 1 Η, H4'), 3.54 (m, 2 H, H-5'a, and H-5'b), 2.47 (m, 1 H, H-2'a), 2.15 (m, 1H, H-2'b).

Compound 6 (6 mg, 0.014 mmol) was phosphorylated with POCI₃(2.6 pl, 0.028 mmol) in proton sponge (6 mg, 0.028 mmol) in trimethylphosphate (0.25 mL) at minus 40 ° C for four hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (66 mg, 0.14 mmol) and tri-nbutylamine (28 μΙ) in anhydrous DMF (0.28 mL) was added. After 30 minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 1.0 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in water (2.0 mL), filtered, and purified using RP-HPLC (see above) to

166

IMPI hWWUTO «UtCxwj» 1L * FROTO f A »« OWSfTHIAl __ produce 7- (2-nitro-benzyloxy) methyl-7-deaza-2'-deoxyadenosine-

<img file="MX342195B_D0199.tif" />

5'-triphosphate dA.I. HRMS (ESI): For molecular ion C₁₉H₂₃N₅OR₁₅P₃ [MH] -, the calculated mass was 654.0403, and the observed mass was 654.0397.

7- [1 - (2-nitro phenyl) -2-methyl-propyloxy] deoxyadenosine-5'-tr i phosphate methyl-7-deaza-2'-

<img file="MX342195B_D0200.tif" />

S7 scheme. Synthesis of 7- [1 - (2-n¡trofeníl) -2-m tilpropi I ox¡] metíl-7-deaza-2'-desoxiadenosi na-5'-triphosphate.

Reagents and conditions: (i) TsCI, DMAP, CH₂CI₂, room temperature, 39%; (//) (ft / S) - (2-nitrophenyl) -2-methyl-propanol racemic, pure, 105 ° C, 54%; (/// ') n-Bu₄NF, THF, 0 ° C at room temperature; (vi) NH₃, 1,4-doxane / MeOH, 100 ° C, 76% in two steps; (v) POCI₃, (MeO)₃PO, minus 40 ° C to 0 ° C; (n167

<img file="MX342195B_D0201.tif" />

WITUTO MEXICANO DE IA INDUSTRIAL PROPERTY

Bu₃NH)₂H₂P2O7, / 7-Bu₃N, DMF; 1 Μ H Et₃HCO₃.

To a solution of compound 4 (0.26 0.49 mmoi) eh

CH₂CI₂ anhydrous (12 mL), 4-dimethylaminopyridine (DMAP; 0.15 g, 1.2 mmol) and tosyl chloride (0.11 g, 0.58 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and then concentrated in vacuo. The residue was purified by silica gel chromatography to produce 9- [8-D-3 ', 5'-O-bis- (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] -6-chloro-7-chloromethyl-7- deazapurine 7 (0.103 g, 39%) as a viscous oil.¹H NMR (400 MHz, CDCI₃): δ 8.64 (s, 1 Η, H-2), 7.72 (s, 1 Η, H-8), 6.73 (t, 1 H, J = 6.8 Hz, H-1 '), 4.95 (AB d, J = 12.4 Hz, 7-CH_2nd), 4.91 (AB d, J = 12.0 Hz, 7-CH_2b), 4.58 (m, 1 Η, H-3 '), 4.00 (m, 1 Η, H-4'), 3.82 (m, 2 H, H-5'a, and H-5'b), 2.41 (m , 2 H, H-2'a and H-2'b), 0.95 (s, 9 H, (CH₃)₃CS¡), 0.93 (s, 9 H, (CH₃)₃CS¡), 0.12 (s, 6 H, (CH₃)₂S¡), 0.1 1 (s, 6 H, (CH₃)₂Yes!)

Compound 7 (54 mg, 0.10 mmol) and racemic (R / SJ-1- (2-nitrophenyl) -2-methyl-propanol (191 mg, 0.98 mmol) were dissolved in CH₂CI₂ anhydrous (10 mL). The solvent was removed in vacuo, and the residue was heated for one hour under a nitrogen atmosphere, then dissolved in a minimal amount of ethyl acetate and purified by silica gel chromatography to produce 9- [6-D- 3 ', 5'-O-bis (fer-butyldimethylsilyl) -2'-deoxyribofu ranosyl] -6-chloro-7- [1 - (2-nitrophenyl) -2-methyl-propyloxy] methyl-7-deazapurine 8 ( 38 mg, 54%)

168

IMPI ^ ΐΜΤίτυτσ mexicana D £ LAPROmiMÜ as a 1: 1 mixture of the two diastereomers. 'TFWIUNTÍW ^ MHz, CDCI₃) for diastereomers: δ 8.60 and Qr5'9 {'£' S, ····! Ή ·, - lt * r 7.83 (m, 1 H, Ph-H), 7.79 (m, 1 H, Ph-H ), 7.56 (m, 1 H, Ph-H), 7.48 and 7.47 (2 s, 1 Η, H-8), 7.38 (m, 1 H, Ph-H), 6.70 (m, 1 H,

H-1 '), 4.81 (m, 1H, Ph-CH), 4.70 (m, 1H, 7-CH₂a), 4.58 (m, 2H,

7-CH₂b and H-3 '), 3.99 (m, 1 Η, H-4'), 3.78 (m, 2 H, H-5'a and H5'b), 2.48 (m, 1 H, H-2'a) , 2.35 (m, 1H, H-2'b), 1.96 (m, 1H,

CH), 0.98 and 0.96 (2 d, 3 H, CH₃), 0.93 (2 s, 9 H, (CH₃)₃CSi), 0.89 (2 s, 9 H, (CH₃)₃CS¡), 0.82 and 0.78 (2 d, 3 H, CH₃), 0.12 (2 s, 6 H, (CH₃)₂YES), 0.08 and 0.07 (2 s, 3 H, (CH₃)₂YES), 0.06 and 0.05 (2 s, 3 H, (CH₃)₂Yes!)

An n-BU solution₄NF (44 mg, 0.14 mmol) in THF (2.0 mL) was added to a solution of compound 8 (38 mg, 0.05 mmol) in THF (2.0 mL) at a temperature of 0 ° C. The reaction was gradually warmed to room temperature and stirred for two hours. The mixture was concentrated in vacuo, dissolved in 1,4-dioxane (4.0 mL), followed by the addition of NH₃ in a MeOH solution (7M, 8.0 mL). The mixture was transferred to a sealed tube, stirred at a temperature of 100 ° C for 24 hours, cooled to room temperature, and then concentrated in vacuo. The residue was purified by silica gel chromatography to produce 7- [1- (2-nitrophenyl) 2-methyl-propyloxy] methyl-7-deaza-2'-deoxyadenosine 9 (19 mg,

76%) in the form of a 1: 1 mixture of two diastereomers.¹ H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.06 and 8.04

169

IMPI

MfclICAN INSTITUTE)

DE IA mOriEOAD (2 s, 1 Η, H-2), 7.90 (m, 1 H, Ph-H), 7.67 (m, 2 H, fTTIT 7 ^ (m, 2 H, Ph-H), 7.19 and 7.16 (2 s, 1 Η, H-8), 6? 63 (bs, 2 H, U₂OR

<img file="MX342195B_D0202.tif" />

interchangeable, 6-NH₂), 6.39 (m, 1 Η, H-1 '), 5.23 (m, 1 H, D₂Interchangeable O, 3'-OH), 5.00 (m, 1 H, D₂Interchangeable O, 5'OH), 4.72 (2 d, 1 H, Ph-CH), 4.45 (s, 2 H, 7-CH₂), 4.30 (m, 1 H, H-3 '), 3.77 (m, 1 Η, H-4'), 3.49 (m, 2 H, H-5'a, and H-5'b), 2.40 (m , 1 H, H-2'a), 2.12 (m, 1 H, H-2'b), 1.94 (m, 1 H, CH), 0.87 (m, 3 H, CH₃), 0.74 (m, 3H, CH₃).

Compound 9 (19 mg, 0.041 mmol) was phosphorylated with POCI₃ (16 μί, 0.16 mmol) and a proton sponge (18 mg, 0.082 mmol) in trimethylphosphate (0.4 mL) at minus 40 ° C for five hours under a nitrogen atmosphere. A solution of bis-tri- / 7-butylammonium pyrophosphate (97 mg, 0.20 mmol) and tri-n-butylamine (40 μΙ_) in anhydrous DMF (0.40 mL) was added. After 30 minutes of stirring, a triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The R residue was dissolved in water (5.0 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to produce 7- [1- (2-nitrophenyl) -2methyl-propyloxy] methyl-7-deaza-2'-deoxyadenosine-5'-triphosphate dA.lll.a in the in the form of a 1: 1 mixture of two diastereomers, which were separated using RP-HPLC to produce the simple diastereomers dA.lll.a ds1 and dA.lll.a ds2. In all the

170

<img file="MX342195B_D0203.tif" />

INSTITUTO MEXICANO cases, the diast reomer (ds1) was eluted more'íiSEíSfflftS diastereomer 2 (ds2) using RP-HPLC. frv mpmr For molecular ion C₂₂H₂₉N₅Oi₅P₃ [MH] ', the calculated mass was 696.0873, and the observed mass was 696.0864.

7- [1 - (4-Methox i-2-nitrof in yl) -2-methyl-p ropiloxy] methyl-7-deaza2'-deox i adenosine-5'-tri phosphate

<img file="MX342195B_D0204.tif" />

R = H or TBS

10 11 (iv)

<img file="MX342195B_D0205.tif" />

Scheme S8. Synthesis of 7- [1- (4-methoxy-2-nitrophenyl) -2-methyp ropiloxy] methi-7-deaza-2'-deoxy to denosi na-5'-triphosphate.

Reagents and conditions', (i) (R / S) -1- (4-methoxy-2-nitrophenyl) -2metll-1-propanol racemic, 108 ° C; (ii) n-Bu₄NF, THF, 0 ° C at room temperature; (iii) NH₃, 1,4-dloxane / MeOH, 100 ° C, 32%

171

<img file="MX342195B_D0206.tif" />

INSTITUTO MEXICANO PE Lfi fEOPJEPAU in three steps; (iv) POCI_3l (MeO)₃PO, 0 ° C; (n-Bu₃

Bu₃N, DMF; 1 M HNEt₃HCO₃. --Compound 7 (103 mg, 0.19 mmol) and (R / S) -1 - (4-methoxy2-n-trophenyl) -2-methyl-1-propanol racemic (428 mg, 1.9 mmol) were dissolved in CH₂CI₂ anhydrous (3.0 mL). The solvent was removed in vacuo, and the residue was heated at a temperature of 108 ° C for 30 minutes under a nitrogen atmosphere, cooled to room temperature, dissolved in a minimal amount of ethyl acetate, and purified by flash chromatography. Silica gel to produce 6-chloro-7- [1- (4methoxy-2-nitrophenyl) -2-methyl-propyloxy] methyl-7-deazapurine 2'-deoxyribonucleosides 10. The sample was dissolved in THF (8.0 mL), cooled to a temperature of 0 ° C, and subsequently added to a solution of n-Bu₄NF (68 mg, 0.22 mmol) in THF (2.0 mL). The reaction was gradually warmed to room temperature and stirred for minutes. The mixture was concentrated in vacuo, dissolved in

1,4-dioxane (8.0 mL), followed by the addition of NH₃ in MeOH (7N, 24 mL). The mixture was transferred to a sealed tube and stirred at a temperature of 100 ° C for 16 hours, then cooled to room temperature, and concentrated in vacuo. The residue was purified by silica gel chromatography to produce 7- [1 - (4-methoxy-2-nitrophenyl) -2-methyl-propyloxy] methyl-7deaza-2'-deoxyadenosine 11 (30 mg, 32% in three steps) in the form of a 1: 1 mixture of two diastereomers.¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.06 and 8.05 (2 s, 1 Η, H172

IMPI

MEXICAN INSTITUTE '¿«frasesESÍ' DE LA FXOHEDAI&gt; O» »¡3sLJ

2), 7.57 and 7.54 (2 d, 1 H, J = 8.8 Hz, Ph-H), 7.47 and^NW4^l (2&gt; ^ P

<td>H, J = 2.6 Hz,</td><td>Ph-H), 7.33 and</td><td> 7.</td><td> 27</td><td>(2 dd, J = -er6-y g'.O</td><td>++ er</td><td>-4-4 + r</td>

<td>Ph-H), 7.18 and</td><td>7.15 (2 s,</td><td> 1</td><td>H</td><td>H-8), 6.63 (bs, 2</td><td>H</td><td>D₂OR</td>

<td>interchangeable,</td><td>6-NH₂), 6.43</td><td>(m</td><td> , 1</td><td>Η, H-1 '), 5.24 (m,</td><td>1 HOUR,</td><td>d₂or</td>

interchangeable, 3'-OH), 5.03 (m, 1 H, D₂Interchangeable O, 5'OH), 4.55 (m, 2H, Ph-CH, 7-CH₂a), 4.30 (m, 2H, 7-CH₂b and H3 '), 3.86 and 3.84 (2 s, 3 H, MeO), 3.78 (m, 1 Η, H-4'), 3.48 (m, 2 Η, H-5 '), 2.45 (m, 1 H , H-2'a), 2.12 (m, 1H, H-2'b), 1.93 (m, 1H, CH (CH₃)₂), 0.88 (m, 3H, CH₃), 0.74 and 0.71 (2 d, J = 6.8 Hz, 3 H, CH₃).

Compound 11 (28 mg, 0.06 mmol) was phosphorylated with POCI₃ (11 pl, 0.12 mmol) and proton sponge (25 mg, 0.12 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for two hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 μ in anhydrous DMF (1.0 mL).

After 10 minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to produce 7- (1- (4-me toxi-2-nitro fe nil) -2-methyl-propyloxy] methyl-7deaza-2'-deoxyadenosine-5'-triphosphate dA .lll.b in the form of

173

IMPl $

MEXICAN INSTITUTE

D £ PROPERTY Λ a 1: 1 mixture of two diastereomers, which using RP-HPLC to produce the diasfeTe ^ ñTSTBT 55'i 111 p I is

<img file="MX342195B_D0207.tif" />

dA.lll.b ds1 and dA.lll.b ds2. HRMS (ESI): For the molecular ion C₂₃H₃₁N₅O1₁₆P₃ [MH] ', the calculated mass was 726.0979, and the observed mass was 726.0984.

7- [1- (2,6-Dinitrophenyl) -2-methyl-propyloxy] metH-7-deaza-2'-deoxyadenosine-5'-tri phosphate

<img file="MX342195B_D0208.tif" />

OH dAJII.c

Scheme S9. Synthesis of 7- [1- (2,6-dinitrophenyl) -2-m tilpropiloxy] m eti l-7-deaza-2'-deoxy-adenosi na-5'-tri phosphate.

Reagents and conditions: (/ ') (R / S) -1 - (2,6-dinitrophenyl) -2-methyl-1 racemic propanol, 108 ° C; (// ') n-Bu₄NF, THF, 0 ° C at temperature

174

IMPI mexican twsTmrro of the ΡΐυηκοΑ ·

<img file="MX342195B_D0209.tif" />

environment; (iii) NH₃, 1,4-dioxane / MeOH, 100 ° C, 38% tr steps; (vi) POCI₃, (MeO)₃PO, 0 ° C; (n-Bu₃NΗ)₂ΤΤ7Ρ7θ77ΤΓβτιτΝ

DMF; 1 Μ H Et₃HCO₃.

Compound 7 (109 mg, 0.20 mmol) and racemic (R / S) -1 - (2,6-dinitrophenyl) -2-methyl-1-propanol (448 mg, 1.9 mmol) were dissolved in CH₂CI₂ anhydrous (10 mL). The solvent was removed in vacuo, and the residue was heated at a temperature of 108 ° C for 30 minutes under a nitrogen atmosphere, subsequently dissolved in a minimal amount of ethyl acetate and purified by silica gel chromatography to produce 6-chloro-7- [1 - (2,6-dinitrophenyl) -2-methylpropyloxy] methyl-7-deazapurine-2'-deoxyribo-nucleosides 12. The sample was dissolved in THF (5.0 mL), cooled to a temperature of 0 ° C, and subsequently added to a solution of / 7-BU4NF (31 mg, 0.10 mmol) in THF (2.0 mL). The reaction was gradually warmed to room temperature and stirred for two hours. The mixture was concentrated in vacuo, dissolved in 1,4-dioxane (4.0 mL), followed by the addition of NH₃ in MeOH (7N, 18 mL). The mixture was transferred to a sealed tube, stirred at a temperature of 100 ° C for 36 hours, cooled to room temperature, and then concentrated in vacuo. The residue was purified by silica gel chromatography to produce 7- [1- (2,6-dinitrophenyl) -2-methyl-propyloxy] methyl-7deaza-2'-deoxyadenosine 13 (38 mg, 38% in three steps) in the form of a 1: 1 mixture of two diastereomers.¹H NMR (400

175

IMPI

MEXICAN INSTITUTE ot industrial property

<img file="MX342195B_D0210.tif" />

MHz, DMSO-d₆) for diastereomers: δ 8.17 (m, 1 H, Ph-H), 8.07 and 8.06 (2 s, 1 Η, H-2), 7.85 (m, 1 H, Ph-H), 7.69 (m, 1 H , Ph-H), 7.20 and 7.18 (2 s, 1 Η, H-8), 6.57 (bs, 2 H, D₂Or interchangeable, 6-NH₂), 6.46 (m, 1 Η, H-1 '), 5.26 (d, J = 3.6 Hz, 1 H, D₂Interchangeable O, 3'-OH), 5.01 (m, 1 H, D₂Interchangeable O, 5'-OH), 4.60 (m, 2H, Ph-CH and 7-CH₂a), 4.29 (m,

H, 7-CH₂b), 4.13 (m, 1 Η, H-3 '), 3.80 (m, 1 Η, H-4'), 3.51 (m,

H, H-5'a and H-5'b), 2.49 (m, 1H, CH (CH₃)₃), 2.16 (m, 2 Η, H2'a and H-2'b), 0.91 (m, 3 H, CH₃), 0.65 (m, 3H, CH₃). ToF-MS (ESI): For the molecular ion C₂₂H₂₇N₆OR₈[M + H] ⁺ , the calculated mass was 503.1890, and the observed mass was

503.2029.

Compound 13 (30 mg, 0.06 mmol) was phosphorylated with POCI₃ (17 pl_, 0.18 mmol) and a proton sponge (26 mg, 0.12 mmol) in trimethylphosphate (0.4 mL) at a temperature of 0 ° C for four hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) and tri-n-butylamine (120 pL) in anhydrous DMF (1.2 mL) was added. After 30 minutes of stirring, a triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred for one hour at room temperature and was then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified by anion exchange chromatography. The fraction s containing the triphosphate were combined and

176

IMPIzg&gt;

THE MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY were lyophilized to produce 7- [1 - (2,6-dinitrophenyl) -2-meTnT propyloxy] methyl-7-deaza-2'-deoxyadenosine-5'-triphosphate dA.lllc in the form of a 1: 1 mixture of two diastereomers which were separated using RP-HPLC to produce the simple diastereomers dA.III.c ds1 and dA.lll.c ds2. HRMS (ESI): For the C22H molecular ion₂8NeO₁₇P3 [MH] ', the calculated mass was 741.0724, and the observed mass was 741.0731.

7 - [(S) -1 - (2-Nitropheni!) - 2,2-dimethyl-propyloxy] methyl-7-deaza2'-deoxyaden osi na-5'-triphosphate

<img file="MX342195B_D0211.tif" />

OH dA.V

Scheme S10. Synthesis of 7 - [(S) -1 - (2-nitrophen i I) -2,2-dim tilpropi loxi] metí l-7-deaza-2'-des oxi a de nos i na-5'-triphosphate.

Reagents and conditions: (i) (S) -1- (2-nitrophenyl) -2,2-dimethyl-1propanol, 110 ° C; (//) n-Bu₄NF, THF, t ambient temperature; 75%

177

<img file="MX342195B_D0212.tif" />

IMPI

INSTITUTO MEXICANO D £ LA PHOPIÉPAP INDUSTRIAL in two steps; (///) NH₃, 1,4-dioxane / MeOH, 100 ° C, 93%

POCI₃, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 'T “M HNEt₃HCO₃.

Compound 7 (130 mg, 0.24 mmol) and (S) 1- (2-n-trophenyl) -2,2-d-methyl-1-propanol (290 mg, 1.4 mmol) were heated to a temperature of 110 ° C for 45 minutes under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dissolved in THF (10 mL) followed by the addition of n-BU₄NF (189 mg, 0.60 mmol). The mixture was stirred at room temperature for two hours and was then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (20 mL) and washed with brine (30 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 6-chloro-9 [pD-2'-deoxyribofuranosyl] -7 - [(S) -1- (2-nitrophenyl) -2, 2-dimethylpropyloxy] methyl-7-deazapurine 14 (90 mg, 75%). ¹H NMR (400 MHz, CDCI₃): δ 8.51 (s, 1 Η, H-2), 7.68 (m, 2 H, Ph-H), 7.45 (t, 1 H, J = 7.2 Hz, Ph-H), 7.38 (s, 1 Η , H-8), 7.29 (t, 1 H, J = 7.2 Hz, Ph-H), 6.39 (dd, 1 H, J = 6.0 and 8.0 Hz, H-1 '), 4.97 (s, 1 H, Ph-CH), 4.70 (m, 3H, 7-CH₂ and H-3 '), 4.16 (m, 1 Η, H-4'), 3.83 (m, 2 Η, H-5 '), 2.80 (m, 1 H, Η-2'a), 2.35 (m , 1 H, H-2'b), 0.82 (s, 9 H, C (CH₃)₃).

Compound 14 (90 mg, 0.18 mmol) was dissolved in 1,4-dioxane (8.0 mL) followed by the addition of NH₃ in MeOH (7 N,

178

IMPI

INSTITUI '&gt; MEXICAN OF INDUSTRIAL PROPERTY

<img file="MX342195B_D0213.tif" />

mL). The mixture was transferred to a sealed tube and stirred at a temperature of 100 ° C for 24 hours, "cooled" to room temperature, and then concentrated in vacuo. The residue was purified by silica gel chromatography to yield 7 - [(S) -1- (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7deaza-2'-deoxyadenosine 15 (80 mg, 93%).¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (s, 1 Η, H-2), 7.91 (dd, 1 H, J = 1.2 and 8.0 Hz, Ph-H), 7.71 (m, 2 H, Ph-H), 7.58 (m, 1H, Ph-H), 7.24 (s, 1H, H-8), 6.68 (bs, 2H, D₂Or interchangeable, 6-NH₂), 6.46 (dd, 1 H, J = 6.0 and 8.0 Hz, H-1 '), 5.27 (d, 1 H, D₂Interchangeable O, 3'OH), 5.06 (t, 1 H, D₂Interchangeable O, 5'-OH), 4.87 (s, 1H, PhCH), 4.65 (d, 1H, J = 12.8 Hz, 7-CH₂a), 4.49 (m, 1 Η, H-3 '), 4.36 (d, 1 H, 7-CH₂b), 3.80 (m, 1 Η, H-4 '), 3.49 (m, 2 Η, H-5'), 2.45 (m, 1 H, H-2'a), 2.17 (m, 1 H, H-2'b), 0.75 (s, 9 H, C (CH₃)₃).

Compound 15 (25 mg, 0.053 mmol) was phosphorylated with POCI₃ (22 pL, 0.24 mmol) and proton sponge (23 mg, 0.11 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 4.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After stirring for 10 minutes, triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The siduo r dissolved in

179

<img file="MX342195B_D0214.tif" />

20% d ac aqueous tonitrile (20 mL), were chromatographed in an area of ​​aninnoc ι.α ^ triphosphate-containing fractions were combined and lyophilized to produce 7 - [(S) -1 - (2- nitrophenyl) -2,2-dimethyl-propyloxy!] methI-7deaza-2'-deoxyadenosine-5'-triphosphate dA.V, which was further purified using RP-HPLC. HRMS (ESI): For the molecular ion C₂₃H₃₁N₅OR₁₃P₃ [MH] ', the calculated mass was 710.1029, and the observed mass was 710.1032.

7 - [(S) -1 - (5-Methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxyJmethyl7-deaza-2'-deoxy adenosine- 5'-triphosphate

<img file="MX342195B_D0215.tif" />

OH dA.VI

Scheme S11. Synthesis of 7 - [(S) -1 - (5-methoxy-2-nitrophenyl) -2,2dimethyl-propyloxy] methyl-7-deaza-2'-deoxyadenosine-5^!triphosphate. Reagents and conditions: (/) (S) -1 - (5-methox¡-2180

ΙΜΡΪ @ ^

MEXICAN INSTITUTE OF PROPiF.DAl?

nitrophenyl) -2,2-dimethyl-1-propanol, 110 ° C; (ii) n - Tfflrr room temperature; 78% in two steps ·, (iii) Mita, dioxane / MeOH, 100 ° C, 74%; (iv) POCI₃, (MeO)₃PO, 0 ° C; (nBu₃NH)₂H₂P2O7, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

Compound 7 (165 mg, 0.30 mmol) and (S) 1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol (330 mg, 1.4 mmol) were heated to a temperature 110 ° C for 45 minutes under a nitrogen atmosphere. The reaction mixture was cooled to room temperature and dissolved in THF (10 mL), followed by the addition of n-Bu₄NF (236 mg, 0.75 mmol). The mixture was stirred at room temperature for two hours and was then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (40 mL) and washed with brine (50 mL), and the aqueous phase was extracted with CH₂CI₂ (40 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 6-chloro-9 [3-D-2'-deoxyribofuranosyl] -7 - [(S) -1- (5-methoxy-2 -nitrophenyl) -2,2dimethyl-propyloxy] methyl-7-deazapurine 16 (122 mg, 78%). ¹H NMR (400 MHz, CDCI₃): δ 8.55 (s, 1 H, H-2), 7.79 (d, 1 H, J = 9.2 Hz, Ph-H), 7.35 (s, 1 Η, H-8), 7.15 (d, 1 H , J = 3.2 Hz, Ph-H), 6.68 (dd, 1 H, J = 3.2 and 9.2 Hz, Ph-H), 6.33 (dd, 1 H, J = 5.6 and 8.8

Hz, H-1 '), 5.26 (s, 1H, Ph-CH), 4.85 (d, 1H, J = 8.8Hz, 7CH₂a), 4.75 (m, 1 Η, H-3 '), 4.70 (d, 1 H, J = 8.8 Hz, 7-CH₂b), 4.13 (m, 1 Η, H-4 '), 3.95 (m, 1 H, H-5'a), 3.83 (s, 3 H, OCH₃),

3.78 (m, 1H, Η-5'b), 2.86 (m, 1H, H-2'a), 2.30 (m, 1H, H-2'b),

181

<img file="MX342195B_D0216.tif" />

IMPÍ

MEXICAN INSTITUTE OF LA TROPIEPAI

0.83 (s, 9H, C (CH₃)₃). industrial

Compound 16 (120 mg, 9'.g9 iiiiiiüI) -in 1,4 dioxane (10 mL) was dissolved followed by the addition of NH₃ in MeOH (7 N, 10 mL). The mixture was transferred to a sealed tube and stirred at a temperature of 100 ° C for 24 hours, then cooled to room temperature, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 7- [ (s) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-7-deaza-2'-deoxyadenosine 17 (87 mg, 74%).¹H NMR (400 MHz, DMSO-d_and): δ 8.06 (s, 1 Η, H-2), 7.97 (d, 1 H, J = 9.2 Hz, Ph-H), 7.22 (s, 1 Η, H-8), 7.08 (d, 1 H , J = 2.8 Hz, PhH), 7.05 (dd, 1 H, J = 2.8 and 9.2 Hz, Ph-H), 6.66 (bs, 2 H, D₂Or interchangeable, 6-NH₂), 6.42 (dd, 1 H, J = 6.0 and 8.0 Hz, H-1 '), 5.25 (d, 1 H, D₂Interchangeable O, 3'-OH), 5.15 (s, 1H, Ph-CH), 5.03 (t, 1H, D₂Interchangeable O, 5'-OH), 4.64 (d, 1 H, J = 12.8 Hz, 7-CH₂a), 4.43 (d, 1H, J = 12.8 Hz, 7-CH₂b), 4.30 (m, 1H, H-3 '), 3.84 (s, 3H, OCH₃), 3.77 (m, 1 Η, H-4 '), 3.45 (m, 2 Η, H5'), 2.43 (m, 1 H, H-2'a), 2.14 (m, 1 H, H-2 'b), 0.75 (s, 9H, C (CH₃)₃).

Compound 17 (21 mg, 0.042 mmol) was phosphorylated with POCI₃ (40 pL, 0.43 mmol) and a proton sponge (18 mg, 0.084 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 7.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL in anhydrous DMF (1.0) was added.

182

<img file="MX342195B_D0217.tif" />

mL). After 10 minutes of triethylammonic bicarbonate buffer (0. IM, pH 7.5; 'l (J mL). The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in aqueous acetonitrile at 20% (20 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to produce 7 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2d¡methyl-propylloxy] methyl-7- deaza-2'-deoxyadenosine-5'-triphosphate dA.VI, which was further purified using RPHPLC. HRMS (ESI): For the molecular ion Cz ^ asNsChePs [MH] ', the calculated mass was 740.1 135, and the observed mass was

740.1156.

183

Example 4 - Synthesis of the Deaza-2'-deoxyguanosine analog of

<img file="MX342195B_D0218.tif" />

MEXICAN INSTITUTE OF PROPERTY

Trifo tato ¿rrr-Ήό

7- (2-nitrobenzyloxy) methyl · 7-deaza-2'-deoxyguanosine-5 triphosphate

<img file="MX342195B_D0219.tif" />

or₂not₂n

<img file="MX342195B_D0220.tif" />

Oh

<img file="MX342195B_D0221.tif" />

Oh

<img file="MX342195B_D0222.tif" />

Scheme S12. Synthesis d 7- (2-nitrob nzyloxy) methyl-7-deaza

184

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INSTITUTO MU n. &lt;λ., íxr- ~ .t'iJl ·. .. FROM PROPERTY V »» i&gt; JÍLá¿i

2'-d soxi g uanosi n a-5'-tri phosphate. Reagents and coft &amp; easy &amp; ne ^^ jr TBSCI, imidazole, DMF, room temperature;¹ 60%; ΟΘ ·, · PdCI₂[PhCN]₂, MeOH / 1,4-dioxane, 50 ° C, 91%; (///) LiBH₄, MeOH, THF, reflux, 54%; (iv) 2-nitrobenzyl bromide, n-Bu₄NBr, CH₂CI₂/ aq. NaOH, room temperature, 48%; (v) n-Bu₄NF, THF, 0 ° C at room temperature, 95%; (vi) DABCO, H₂Or, reflux, 30%; (_{i saw}) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

Compound 18 (Seela and Peng, 2005, which is incorporated into the present invention by reference) (1.35 g, 3.29 mmol) was evaporated from anhydrous pyridine (3.0 mL) three times and subsequently dissolved in anhydrous DMF (6.0 mL).

Te rb uti Id im et i Is i I i o o (5.95 g, 39.5 mmol) a nd imidazole (5.37 g, 78.9 mmol) were added and the mixture was stirred at a temperature of 50 ° C for 48 hours with chloride of terb utild i met Is il i lo additional (2.97 g, 19.7 mmol) and Imidazol (2.69 g , 39.4 mmol) being added every six hours. The reaction mixture was concentrated in vacuo and purified by silica gel chromatography to yield 9- [6-D-3 ', 5'-O-bis (te rb utild i metí Is i I i I) -2' -deoxyribof uranes yl ] -2- (ferbutyldimethylsilyl) amino-6-chloro-7-iodo-7-deazapurine 19 (1.48 g, 60% yield) in the form of a white foam. ¹H NMR (400 MHz, CDCI₃): δ 7.35 (s, 1 H, H-8), 6.53 (t, 1 H, J = 6.0 Hz, H-1 '), 4.70 (s, 1 Η, 2-NH), 4.47 (m, 1 H, H-3 '), 3.97 (m, 1 Η, H4'), 3.78 (m, 2 H, H-5'a and H-5'b), 2.23 (m, 2 H, H-2 ' a and H-2'b),

185 r I

MEXICAN INSTITUTE í ^ 'íxegKí-.ÍSS * Vil DELA «ΟΡΙίΟΑίΤ

0.98 (s, 9H, (CH₃)₃CS¡), 0.95 (s, 9 H, (CH₃)₃CSi), TW ^ S, (CH₃)₃CS¡), 0.29 (2 s, 6 H, (CH₃)₂Si), 0.13 (2 ~ s ', 8 H,' “CH3) 2 ^ 1), 0.09 (s, 6 H, CH₃)₂Yes!)

A solution of compound 19 (720 mg, 0.96 mmol) was dissolved in anhydrous 1,4-dioxane (30 mL). Anhydrous MeOH (30 mL) and triethylamine (0.58 mL) were added, and the mixture was stirred for 10 minutes under a CO atmosphere, followed by the addition of bis (benzonitrile) dichloropalladium (l I) (20 mg, 0.05 mmol) . The reaction was stirred at a temperature of 58 ° C for 24 hours under a CO atmosphere, and then concentrated in vacuo. The residue was purified by silica gel chromatography to produce 9- [3-D-3 ', 5'-O-bis (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] -2- (terbutyldimethylsilyl) amino-6-chloro -7-methoxycarbonyl-7-deazapurine 20 (600 mg, 91%) in the form of a viscous oil. ¹H NMR (400 MHz, CDCI₃): δ 7.92 (s, 1 Η, H-8), 6.57 (dd, 1 H, J = 8.0 and 6.0 Hz, H-1 '), 4.78 (s, 1 Η, 2-NH), 4.49 (m , 1 Η, H-3 '), 4.02 (m, 1 Η, H-4'), 3.85 (s, 3 H, CH₃), 3.81 (m, 2H, H-5'a and H-5'b), 2.25 (m, 2H, H-2'a and Η-2'b), 0.98 (s, 9H, (CH₃)₃CS¡), 0.93 (s, 9 H, (CH₃)₃CS¡), 0.92 (s, 9 H, (CH₃)₃CS¡), 0.31 (s, 6 H, (CH₃)₂S¡), 0.13 (2 s, 6 H, (CH₃)₂S¡), 0.11 (s, 6 H, (CH₃)₂Yes!)

To a solution of compound 20 (1.11 g, 1.63 mmol) in anhydrous THF (56 mL), lithium borohydride (143 mg,

6.5 mmol), followed by MeOH (0.94 mL). The reaction mixture was heated under reflux for one hour. By cooling off

186

T ¡P ^ T

MEXICAN INSTITUTE

Say THE PROflIUA:; - 'í¿--._; J »Λ room temperature, the reaction mixture slS ^ dlfíiyo¹* CH2CI2 rate (700 mL) and was extinguished with acuity '(70 irity.¹¹Organic was separated, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel chromatography to produce 9- [PD-3 ', 5'-O-bis- (tert-butyldimethylsilyl) -2'-deoxyribofurans!] -2- (tert-butyldimethylsilyl) ami n0-6-chloro-7hydroxymethyl-7-deazapurine 21 (0.58 g, 54%) in the form of a viscous oil.¹H NMR (400 MHz, CDCI3): δ 7.16 (s, 1 Η, H-8), 6.56 (t, 1 H, J = 6.4 Hz, H-1 '), 4.79 (AB d, J = 13.6 Hz, 7-CH₂a), 4.75 (AB d, J = 13.6 Hz, 7-CH₂b), 4.70 (s, 1 Η, 2-NH), 4.50 (m, 1 Η, H-3 '), 3.96 (m, 1 Η, H-4'), 3.76 (m, 2 H, H- 5'a and H-5'b), 2.23 (m, 2H, H-2'a and H-2'b), 0.98 (s, 9H, (CH₃)₃CS¡), 0.94 (s, 9 H, (CH₃)₃CS¡), 0.92 (s, 9 H, (CH₃)₃CS¡), 0.30 (s, 3 H, (CH₃)₂S¡), 0.29 (S, 3 H, (CH₃)₂S¡), 0.11 (s, 6 H, (CH₃)₂S¡), 0.10 (s, 6 H, (CH₃)₂Yes!)

To a solution of compound 21 (150 mg, 0.23 mmol) in CH₂CI₂ (3.0 mL), n-Bu was added₄NBr (37 mg, 0.12 mmol), 2-nitrobenzyl bromide (148 mg, 0.68 mmol) and a NaOH solution (1M, 3.0 mL). The reaction mixture was vigorously stirred at room temperature for two days in the dark. The organic phase was separated, dried over Na₂SW₄, concentrated in vacuo, and purified by silica I chromatography to produce 9- [β-D-3 ', 5' -Ob is - (te r- bu ti I dimet il si I i I) 2'- deoxyribofuranosyl] -2- (fer-butyldimethylsilyl) amino-6-chloro-7- (2-nitrobenzyloxy) methyl-7-deazapurine 22 (87 mg, 48%) in the form

187

<img file="MX342195B_D0223.tif" />

MEXICAN INSTITUTE faith &amp; viscous ac it. H NMR (400 MHz, CDCI₃):^{| f}®^or6^R.'66 J = 8.0 and 1.2 Hz, Ph-H), 7.87 (d, 1 H, J = 7.2 Ha, Ph II), 7.61-ftltr1 H, J = 7.6 and 1.2 Hz, Ph-H), 7.43 (m, 1H, Ph-H), 7.20 (s, 1H, H-8), 6.56 (dd, 1H, J = 7.6 and 6.0Hz, H-1 '), 4.99 (s, 2H , PhCH₂), 4.83 (AB d, 1 H, J = 1 1.4 Hz, 7-CH₂a), 4.75 (AB d, 1 H, J = 11.4 Hz, 7-CH₂b), 4.67 (s, 1 Η, 2-NH), 4.50 (m, 1 H, H-3 '), 3.96 (m, 1 Η, H-4'), 3.77 (m, 2 H, H- 5'a and H-5'b), 2.25 (m, 2H, H-2'a and H2'b), 0.98 (s, 9H, (CH₃)₃CSi), 0.92 (s, 18H, (CH₃)₃CSi), 0.30 (s, 3H, (CH₃)₂S¡), 0.29 (s, 3 H, (CH₃)₂Yes), 0.09 (m, 12H, (CH₃)₂Yes!)

N-Bu solution was added dropwise₄NF (123 mg, 0.39 mmol) in THF (2.0 mL), to a solution of compound 22 (105 mg, 0.13 mmol) in THF (3.0 mL) at a temperature of 0 ° C. The reaction mixture was stirred at a temperature of 0 ° C for one hour and then at room temperature for two hours. The mixture was concentrated in vacuo and purified by silica gel chromatography to produce 2-amino-6-chloro-9- [3-D-2'-deoxyribofuranosyl] -7- (2-nitrobenzyloxy) methyl-7-deazapurine 23 (57 mg, 95%) in the form of a yellow foam. ¹H NMR (400 MHz, DMSO-de): δ, 8.02 (m, 1 H, Ph-H), 7.74 (m, 2 H, Ph-H), 7.55 (m, 1 H, Ph-H), 7.41 (s, 1H, H-8), 6.73 (s, 2H, D₂Or swap, NH₂), 6.41 (dd, 1 H, J = 8.4 and 6.0 Hz, H-1 '), 5.26 (d, 1 H, D₂Interchangeable O, 3'-OH), 4.91 (t, 1 H, D₂Interchangeable O, 5'OH), 4.88 (s, 2H, Ph-CH₂), 4.66 (dd, 2H, J = 11.6 Hz, 7-CH₂),

188

INSTITUI · Μί-ΧίνΛΝΟ Give LA PROHEL'AÜ

<img file="MX342195B_D0224.tif" />

4.31 (m, 1 Η, H-3 '), 3.78 (m, 1 Η, H-4'), 3.50 (σι, ”Τ^τ^ H2.38 (m, 1H, Η-2'a), 2.15 (m, 1H, H-2'b). -

A mixture of 23 (38 mg, 0.085 mmol) and 1.4 dayzab [2.2.2] octane (11 mg, 0.1 mmol) in water (4.0 mL) was heated under reflux for four hours under an atmosphere nitrogen. The water was removed in vacuo, and the residue was evaporated from MeOH (3.0 mL) three times, and purified by silica gel chromatography to produce 7- (2-nitrobenzyloxy) methyl-7-deaza-2'-deoxyguanosine 24 ( 1 1 mg,

30%).¹Ή NMR (400 MHz, DMSO-d₆): δ 10.4 (s, 1 H, D₂Interchangeable, NH), 8.03 (dd, 1 H, J = 8.4 and 0.8 Hz, Ph-H), 7.83 (d, 1 H, J = 7.6 Hz, Ph-H), 7.73 (m, 1 H, Ph -H), 7.55 (m, 1 H, Ph-H), 6.92 (s, 1 Η, H-8), 6.28 (m, 1 Η, H-1 '), 6.26 (bs, 2 H, D₂Or interchangeable, NH₂), 5.21 (d, 1 H, D₂Interchangeable O, 3'OH), 4.89 (t, 1 H, D₂Interchangeable O, 5'-OH), 4.88 (s, 2H, PhCH₂), 4.60 (dd, 2H, 7-CH₂), 4.28 (m, 1 Η, H-3 '), 3.74 (m, 1 H, H-4'), 3.48 (m, 2 Η, H-5 '), 2.32 (m, 1 H, H- 2'a), 2.08 (m, 1 Η, H2'b).

Compound 24 (11 mg, 0.025 mmol) was phosphorylated with POCI₃ (15 pL, 0.05 mmol) and a proton sponge (11 mg, 0.05 mmol) in trimethylphosphate (0.3 mL) at a temperature of 0 ° C for two hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (118 mg, 0.25 mmol) and tri-n-butylamine (50 pL) in anhydrous DMF (0.5 mL) was added. After 30 minutes of stirring, a buffer was added.

189

<img file="MX342195B_D0225.tif" />

IMPI

MEXICAN INSTITUTE OF PROPERTY tri-tilamonium bicarbonate (1 M, pH 7.5; 5.0 mL and^DLTa^{TO THE}r was stirred at room temperature for one year and then concentrated in vacuo. The residue was dissolved in water (10 mL), filtered, and purified by anion exchange chromatography. The fractions containing the triphosphate were combined and lyophilized to produce 7- (2n io benzyloxy) meth l-7-deaza-2'-deoxyguine nosin-5'-triphosphate dG.I, which was further purified using RP-HPLC. HRMS (ESI): for the molecular ion &lt;3₁₉Η₂₃Ν5θι₆Ρ3 [MH] ', the calculated mass was 670.0353, and the observed mass was

670.0344.

7- [1 - (2-Nitrophenyl) -2,2-dimetU-propyloxy] methyl-7-deaza-2'-deoxy-nosine-5'-tri-phosphate

<img file="MX342195B_D0226.tif" />

190

<img file="MX342195B_D0227.tif" />

<img file="MX342195B_D0228.tif" />

Scheme S13. Synthesis of 7- [1 - (2-nitrophenyl) -2,2-dim tilpropyloxy] methyl-7-deaza-2'-deoxy-guanosine-5'-tr-phosphate.

Reagents and conditions: (/ ') MsCI, DMAP, CH₂CI₂, 0 ° C; (/ '/') (R / S) - * \ - (2-nitrophen yl) -2,2-d imetil-1 -propanol racemic, 115 ° C;

(iii) / 7-BU4NF, THF, room temperature, 26% in three steps;

(iv) sin-pyridine-2-aldoxime, 1,1,3,3-tetramethyl guanidine,

1,4-doxane / DMF, 70 ° C, 70%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1M

HNEt₃HCO₃.

DMAP (148 mg, 1.2 mmol) and MsCI (71 pL, 0.9 mmol) were added to a solution of compound 21 (200 mg, 0.30 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and diluted with CH₂CI₂(15 mL). The solution was applied to a short (2x3 cm) silica gel stopper and eluted quickly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio; 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with (ft / S) -1- (2-n-trophenyl) -2,2-dimyl-1-propanol

191

IMPI

MEXICAN INSTITUTE y'ZSxr'rtZS'J, \ “« Λ '!? ™ *?

racemic (500 mg, 2.4 mmol). The mixture is c ^^ra'^ALjAt a temperature of 115 ° C for 45 minutes, the temperature was lowered with nitrogen, cooled to room temperature and subsequently dissolved in THF (10 mL) followed by the addition of n-Bu₄NF (283 mg, 0.90 mmol). The mixture was stirred at room temperature for four hours and then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (25 mL) and washed with brine (25 mL), and the aqueous phase was extracted with CH₂CI₂ (25 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino6-chloro-9- [PD-2'-deoxyribofuranosyl] -7- [1 - (2-nitrofe ni 1) -2 , 2-dimethyl-propyloxy] methyl-7-deazapurine 25 (40 mg, 26% for three steps) in the form of a 1: 1 mixture of two diastereomers.

To a solution of compound 25 (40 mg, 0.08 mmol) n 1,4-dioxane (1.0 mL) and DMF (2.0 mL), sinpyrimidine-2-aldoxime (180 mg, 1.5 mmol) and guanidine of 1, were added. 1,3,3-tetramethyl (211 pL, 1.68 mmol). The mixture was heated overnight to a temperature of 70 ° C under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (20 mL) and washed sequentially with an acetic acid solution (0.1 M, 30 mL), a NaHCO solution₃ saturated (30 mL), and brine (30 mL). The organic phase was dried over Na₂SW₄, s concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 7- [1- (2-nitrof nil) 192

IMPI

MEXICAN INSTITUTE ¿A

FROM PXOP11: DA n

2,2-dimethyl-propyloxy] methyl-7-deaza-2'-d soxyguanosine ^ ™ '¹mg, 70%) as a 1: 1 mixture of two diastereomers. '* Η NMR * (400 MHz, MeOH-d₄) for diastereomers: δ 7.79 (m, 1 H, PhH), 7.73 (m, 1 H, Ph-H), 7.56 (m, 1 H, Ph-H), 7.39 (m, 1 H, PhH), 6.87 and 6.86 (2 s, 1 Η, H-8), 6.30 (m, 1 Η, H-1 '), 4.99 and 4.97 (2 s, 1 H, Ph-CH), 4.63-4.36 (m, 3 H , 7-CH₂ and H-3 '), 3.91 (m, 1 Η, H-4'), 3.69 (m, 2 Η, H-5 '), 2.48 (m, 1 H, H-2'a), 2.20 (m , 1H, H-2'b), 0.79 and 0.77 (2 s, 9 H, (CH₃)₃).

Compound 26 (25mg, 0.05mmol) was phosphorylated with POCI₃ (20 μΐ_, 0.21 mmol) and proton sponge (21 mg, 0.1 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 3.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to produce 7- [1 - (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7-deaza2'-deoxy guanosine-5'-triphosphate dG.Va in the form of a 1: 1 mixture of two diastereomers, which were separated using RP-HPLC to produce the simple diastereomers

193

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IN5T1TU

OF THE PRO l 1

F.XICANO · νδά ^ *. ·?): Οί · ΙίΙ) Α!) DG.Va ds1 and dG.Va ds2. HRMS (ESI): For the '1 W * 1h or letf &amp; rér C₂₃H3iN₅OR₁₆P₃ [M-Hf, the calculated mass was 776: 0979,¹ and the observed mass was 726.0992.

7 - [(S) -1- (2-Nitrophenyl) -2, 2-dimethyl-propyloxy] methyl-7-deaza2'-deoxyguanosine-5'-tri phosphate

<img file="MX342195B_D0229.tif" />

Scheme S14. Synthesis of 7 - [(S) -1- (2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-7-deaza-2'-deoxy-guanosi na-5'-triphosphate.

Reagents and conditions-. (/ ') MsCI, DMAP, CH₂CI₂, 0 ° C; (/ '/') (S) 1- (2-nitrophenyl) -2,2-dyl til-f-propanol, 115 ° C; (iii) n-Bu₄NF,

194

MEXICAN INSTITUTE AT-J

DE LA? RO »'I t uAiJ

THF, room temperature, 35% in three steps; to the

MeOH, reflux, 74%; (v) 1,4-dioxane, 2M NaO'M, rerfluju, 93%, '(vi) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (nBu₃NH)₂H₂P2O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

DMAP (224 mg, 1.8 mmol) and MsCI (107 pL,

1.4 mmol) to a solution of compound 21 (300 mg, 0.46 mmol) in CH₂CI₂ anhydrous (10 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and diluted with CH₂CI₂(20 mL). The solution was applied to a short (2x3 cm) silica gel plug and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with (S) -1 - (2-nitrophenyl) -2,2-dimethyl-1 -propanol (520 mg, 2.5 mmol). The mixture was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature and dissolved in THF (20 mL) followed by the addition of n-Bu₄NF (491 mg, 1.6 mmol). The mixture was stirred at room temperature for four hours and then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (20 mL) and washed with brine (30 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino-6-chloro-9- [PD-2'195

<img file="MX342195B_D0230.tif" />

IM F!

INSTITUTO MÍX.IC'.N.?

Dfc LA ►'KOPiíCAC INDUSTRIAL d soxir¡bofuranosil] -7 - [(S) -1- (2-nitrophenyl) -2,2-d¡metilpropiloxi] methyl-7-deazapurina 27 (81 mg, 35% para three steps). ¹H NMR (400 MHz, MeOH-d₄): δ 7.79 (m, 2 H, Ph-H), 7.60 (dt, 1 H, J = 1.2 and 8.0 Hz, Ph-H), 7.46 (dt, 1 H, J = 1.2 and 8.0 Hz, PhH) , 7.27 (s, 1 Η, H-8), 6.47 (dd, 1 H, J = 6.4 and 8.0 Hz, H-1 '), 4.98 (s, 1 H, Ph-CH), 4.71 (d, 1 H, J = 12.4 Hz, 7-CH2 a), 4.50 (m, 1 Η, H-3 '), 4.47 (d, 1 H, J = 12.4 Hz, 7-CH₂ b), 3.96 (m, 1 H, H-4 '), 3.73 (m, 2 Η, H-5'), 2.59 (m, 1 H, H-2'a), 2.30 (m, 1 Η, H2'b), 0.80 (s, 9H, (CH₃)₃).

Compound 27 (104 mg, 0.21 mmol) was dissolved in a solution of sodium methoxide in MeOH (0.5 M, 10 mL), and the mixture was refluxed for one hour under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, neutralized with acetic acid, and then concentrated in vacuo. The residue was purified by silica gel chromatography to produce 2-amino-6-methoxy9- [PD-2'-deoxyribofuranosyl] -7 - [(S) -1 - (2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-7-deazapurine 28 (75 mg, 74%). 'H NMR (400 MHz, CDCI₃): δ 7.74 (m, 2H, Ph-H), 7.52 (t, 1H, J = 8.0Hz, Ph-H), 7.36 (t, 1H, J = 8.0Hz, Ph-H), 6.71 (s, 1 Η, H-8), 6.47 (dd, 1 H, J = 5.6 and 9.6 Hz, H-1 '), 5.04 (s, 1 H, Ph-CH), 4.71 (m, 1 Η, H-3 '), 4.47 (dd, 2 H, J = 12 Hz, 7-CH₂ ), 4.15 (m, 1 Η, H4 '), 3.94 (s, 3 H, OCH₃), 3.76 (m, 2 Η, H-5 '), 3.01 (m, 1 Η, H2'a), 2.19 (m, 1 H, H-2'b), 0.82 (s, 9 H, (CH₃)₃).

S dissolved compound 28 (70 mg, 0.14 mmol) in 1.4196

<img file="MX342195B_D0231.tif" />

<img file="MX342195B_D0232.tif" />

watery

j. Ji

INSTITUTE, V. ¿X'CA ^ O DE LA PROPÍe.DAi)

INDUSTRIAL dioxane (6.0 mL) followed by the addition of a sodium hydroxide solution (2M, 12 mL). The mixture was heated under reflux for four days under a nitrogen atmosphere, cooled to room temperature, neutralized with dilute hydrochloric acid (1M), and concentrated in vacuo. The residue was evaporated from MeOH (5.0 mL) three times and subsequently purified by silica gel chromatography to produce 7 - [(S) -1- (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl -7deaza-2'-deoxyguanosine 29 (22 mg, 33%). Starting material 28 (42 mg, 60%) was also recovered from the reaction.¹H NMR (400 MHz, CDCI₃): δ 11.02 (br si, 1 Η, NH), 7.69 (m, 2 H, Ph-H), 7.52 (t, 1 H, J = 7.2 Hz, Ph-H), 7.33 (t, 1 H, J = 7.2 Hz, Ph-H), 6.66 (s, 1 Η, H-8), 6.13 (t, 1 H, J = 6.8 Hz, H- 1 '), 6.03 (br s, 2 H, 6- NH₂), 4.92 (s, 1 H, Ph-CH), 4.77 (m, 1 Η, H-3 '), 4.57 (d, 1 H, J = 12.8 Hz, 7-CH₂a), 4.12 (m, 1 Η, H-4 '), 3.05 (d, 1 H, J = 12.8 Hz, 7-CH₂ b), 3.75 (m, 2 Η, H-5 '), 2.87 (m, 1 Η, H2'a), 2.29 (m, 1 H, H-2'b), 0.76 (s, 9 H, ( CH₃)₃).

Compound 29 (16 mg, 0.033 mmol) was phosphorylated with POCI₃ (17 pL, 0.18 mmol) and a proton sponge (14 mg, 0.066 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for four hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a triethylammonium bicarbonate buffer (0.1 M, pH 7.5;

197

<img file="MX342195B_D0233.tif" />

mL). The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to produce 7 - [(S) -1- (2-nitrophenyl) -2,2-dimethylpropyloxyJmethyl-7-deaza-2'-deoxyguanosine-5'-triphosphate dG.V, which was further purified using RPHPLC conditions. The retention time of dG.V was identical to that of dG.Va ds2 by RP-HPLC analysis using the same condition (data not shown). HRMS (ESI): For the molecular ion C23H31N₅01₆P3 [MH] ', the calculated mass was 726.0979, and the observed mass was 726.0986.

7- [1- (4-Methoxy-2-nitrophenyl) -2, 2-dimethyl-propyloxy] methyl-7-deaza2'-deoxyguanosine-5'-triphosphate

TBSO

NHTBS

OTBS

QMe (¡)

<img file="MX342195B_D0234.tif" />

NHTBS

OTBS (¡i). (&lt;¡I)

<img file="MX342195B_D0235.tif" />

Oh

Oh

198

<img file="MX342195B_D0236.tif" />

<img file="MX342195B_D0237.tif" />

Scheme S15. Synthesis of 7- [1 - (4-methoxy-2-nitrophenyl 1) -2,2dimethyl-propyloxy] methyl-7-deaza-2'-deoxyguanosine-5'-triphosphate Reagents and conditions: (/) MsCI, DMAP, CH2CI2, 0 ° C; (ii) racemic (F? / S) -1- (4-methoxy-2-n-trophenyl) -2,2-dimethyl-1-propanol, 115 ° C; (7/7) n-Bu₄NF, THF, room temperature, 21% in three steps; (iv) sin-pyridine-2-aldoxime, guanidine d

1,1,3,3-tetramethyl, 1,4-dioxane / DMF, 70 ° C, 59%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (r? -Bu₃NH)₂H2P₂O7, nBu₃N, DMF; 1 M HNEt₃HCO₃.

DMAP (346 mg, 2.8 mmol) and MsCI (165 pL, 2.1 mmol) were added to a solution of compound 21 (470 mg, 0.72 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and diluted with CH₂CI₂(20 mL). The solution was applied to a short (2x3 cm) silica gel plug and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio n 80/20 / 0.5). The elu nt was concentrated in vacuo, and the

199

IMPÍ¿g *

MEXICAN INSTITUTE OF PROPERTY r siduo was mixed with (R / S) - ^ - (4-methoxy-2-niVPW ^ Vl^lil) -z72 ^ racemic dimethyl-1-propanol (1.6 g, 6.69 mrmj-l). Nitrite was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature, and dissolved in THF (20 mL) followed by the addition of n-Bu₄NF (788 mg, 2.5 mmol). The mixture was stirred at room temperature for four hours and then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (20 mL) and washed with brine (30 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino-6-chloro-9 [3-D-2'-deoxyribofuranosyl] -7 - [(S) -1 - (4 -methoxy-2-n itrofe ni 1) -2,2 dimethyl-propyloxy-methyl-7-deazapurine 30 (80 mg, 21% for three steps) in the form of a 1: 1 mixture of two diastereomers.

To a solution of compound 30 (80 mg, 0.15 mmol) in 1,4-dioxane (1.0 mL) and DMF (2.0 mL), sinpyrimidine-2-aldoxime (366 mg, 3.0 mmol) and guanidine of

1,1,3,3-tetramethyl (414 pL, 3.3 mmol), and the mixture was heated overnight to a temperature of 70 ° C under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (20 mL) and washed sequentially with acetic acid (0.1 M, 30 mL), a NaHCO solution₃ saturated (30 mL), and brine (30 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and I r siduo was purified by g I silica chromatography

200

IMPI

MEXICAN INSTITUTE OF FXOFltDAD to produce 7 - [(S) -1 - (4-methoxy-2-nitrof niI) cfimeVr propyloxy] methyl-7-deaza-2'-deoxy-guanosine 31 (45 mg, b and% j ΗΠ

<img file="MX342195B_D0238.tif" />

the form of a 1: 1 mixture of two diastereomers. ¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.31 (br s, 1 H, D₂Interchangeable, NH), 7.63 and 7.62 (2 d, 1 H, J = 2.8 Hz, PhH), 7.41 and 7.40 (2 d, 1 H, J = 2.8 Hz, Ph-H), 7.27 ( m, 1 H, PhH), 6.98 and 6.96 (2 s, 1 H, H-8), 6.28 (m, 1 H, H-1 '), 6.22 (br s, 2 H, D₂Or interchangeable, NH₂), 5.22 (d, 1 H, D₂Interchangeable O, 3'-OH), 4.88 (t, 1 H, D₂Interchangeable O, 5'-OH), 4.73 and 4.71 (2 s, 1 H, Ph-CH), 4.47-4.24 (m, 3 H, 7-CH₂ and H-3 '), 3.85 and 3.83 (2 s, 3 H, OCH₃), 3.74 (m, 1 H, H-4 '), 3.48 (m, 2 Η, H-5'), 2.28 (m, 1 H, H-2'a), 2.06 (m, 1 H, H -2'b), 0.80 and 0.78 (2 s, 9 H, (CH₃)₃).

Compound 31 (25 mg, 0.048 mmol) was phosphorylated with POCI₃ (15 pL, 0.18 mmol) and a proton sponge (21 mg, 0.10 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 3.5 hours under a nitrogen atmosphere. S added a solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL). After 10 minutes of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The fractions containing the triphosphate were combined and

201 n ¡trophe Wsií'fS - clYh ^

<img file="MX342195B_D0239.tif" />

INSTITUTO MEXICANO DE LA FROHCPAO lyophilized to produce 7- [1- (4-methoxy-2-propylloxy) methyl-7-deaza-2'-deoxyguanosine-fc'-triphosphate dO-V ·· fr en the form of a 1: 1 mixture of two diastereomers, which were separated using RP-HPLC to produce the simple diastereomers dG.Vb ds1 and dG.Vb ds2 HRMS (ESI): For the molecular ion C24H33N5O17P3 [MH] ' , the calculated mass was 756.1084, and the observed mass was 756.1101.

7- [1 - (5-Methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7deaza-2'-deoxy-nosine-5'-tri phosphate

<img file="MX342195B_D0240.tif" />

OH dG.Vc

202

<img file="MX342195B_D0241.tif" />

MEXICAN INSTITUTE OF THE 1'ROPItOAD

What about S16. Sínt si d 7- [1- (5-m Ιοχΐ-Σ-ηΠΤ ^ ΒΤόηΠ dimethyl-propyloxy] methyl-7-deaza-2'-deoxyyuaiiusinⁱ9'- * triphosphate. Reagents and conditions: (/ ') MsCI, DMAP, CH2CI2, 0 ° C; (/ '/) (R / S) -1 - (5-methoxy-2-ntrophe ni l) -2,2-d methane 1-1-racemic propanol, 115 ° C; (iii) n-Bu₄NF, THF, room temperature, 24% in three steps; (iv) sin-p¡r¡d¡na-2-aldox¡ma, guanidine of

1,1,3,3-tetramethyl, 1,4-dioxane / DMF, 70 ° C, 57%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂O7, nBu₃N, DMF; 1 M HNEt₃HCO₃.

DMAP (302 mg, 2.5 mmol) and MsCI (145 pL, 1.9 mmol) were added to a solution of compound 21 (410 mg, 0.62 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and diluted with CH₂CI₂(20 mL). The solution was applied to a short (2x3 cm) silica gel stopper and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio: 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with racemic (R / S) -1 - (5-methoxl-2-n-trophenyl) -2,2-d-methyl-1-propanol (800 mg, 2.2 mmol). The mixture was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature, and dissolved in THF (10 mL) followed by the addition of n-Bu₄NF (683 mg, 3.3 mmol). The mixture was stirred at room temperature for four hours and then concentrated in vacuo. The

203 ri

INSTITUTO .MEXICANO T ^ &lt;'- 77a? Xz DE LA PACTÍzPAD' L

INI 'U51 RiAL, 2 residue was dissolved in CH2CI2 (20 mL) and washed with brine (30 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino-6-chloro-9 [pD-2'-deoxyribofuranosyl] -7- [1- (5-methoxy-2-nitrophenyl ) -2,2dimethyl-propyloxyjmethyl-7-deazapurine 32 (80 mg, 24% for three steps) as a 1: 1 mixture of two diastereomers. ¹H NMR (400 MHz, CDCI₃) for diastereomers: δ 7.86 and 7.83 (2 d, 1 H, J = 8.8 Hz, Ph-H), 7.19 and 7.17 (2 d, 1 H, J = 2.8 Hz, Ph-H), 6.91 and 6.90 (2 s, 1 Η, H-8), 6.80 and 6.75 (2 dd, 1 H, J = .8 and 8.8 Hz, Ph-H), 6.17 (m, 1 Η, H-1 '), 5.23 and 5.21 ( 2 s, 1 H, Ph-CH), 5.01 and 5.00 (2 br s, 2 H, NH₂), 4.73 (m, 1 Η, H-3 '), 4.65-4.49 (m, 2 H, 7-CH2), 4.14 (m, 1 Η, H-4'), 3.84 (m, 5 Η, H -5 'and OCH₃), 2.78 (m, 1 H, H-2'a), 2.33 (m, 1 H, H-2'b), 0.82 and 0.81 (2 s, 9 H, (CH₃)₃).

To a solution of compound 32 (80 mg, 0.15 mmol) in 1,4-dioxane (1.0 mL) and DMF (2.0 mL), sinpyrimidine-2-aldoxlma (360 mg, 3.0 mmol) and guanidine of

1,1,3,3-tetramethyl (414 pL, 3.3 mmol), and the mixture was heated to a temperature of 70 ° C overnight under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (20 mL) and washed sequentially with acetic acid (0.1 M, 30 mL), a NaHCO solution₃ saturated (30 mL), and brine (30 mL). The organic phase was collected on Na₂SW₄, concentrated in vacuo, and

204

IMPI

MEXICAN INSTITUTE

OI LA FRCHf.OAD C * «¿Mi (£ áX ^{! ND}ra the residue was purified by dg chromatography to produce 7- [1 - (5-methoxy-2-ni troté nil) -2,2-dimétTT propyloxy] methyl-7-deaza-2'-deoxyguanosine 33 ( 43 mg, 57%) in the form of a 1: 1 mixture of two diastereomers. ¹H NMR (400 MHz, DMSO-de) for diastereomers: δ 10.34 (br s, 1 H, D₂Interchangeable, NH), 7.92 and 7.89 (2 d, 1 H, J = 8.8 Hz, PhH), 7.15 (m, 1 H, Ph-H), 6.95 (m, 1 H, Ph-H), 6.82 and 6.81 (2 s, 1 H, H-8), 6.22 (m, 3 H, 2 HD₂Or interchangeable, H-1 'and NH₂), 5.19 (d, 1 H, D₂Interchangeable O, 3'-OH), 5.12 and 5.10 (2 s, 1 H, PhCH), 4.84 (t, 1 H, D₂Interchangeable O, 5'-OH), 4.47-4.31 (m, 2H, 7-CH₂), 4.24 (m, 1H, H-3 '), 3.85 and 3.83 (2s, 3H, OCH₃),

3.71 (m, 1 Η, H-4 '), 3.44 (m, 2 Η, H-5'), 2.24 (m, 1 H, H-2'a),

2.01 (m, 1H, H-2'b), 0.76 and 0.74 (2s, 9H, (CH₃)₃).

Compound 33 (20 mg, 0.04 mmol) was phosphorylated with POCI₃ (25 pL, 0.27 mmol) and a proton sponge (16 mg, 0.08 mmol) in trimethylphosphate (0.30 mL) at a temperature of 0 ° C for six hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The

205

<img file="MX342195B_D0242.tif" />

fractions containing I triphosphate were lyophilized to produce 7- [1 - (5-methoxy-2-nilΙΌ ΓήπιΙ¹) ^! ·, 2- dfrrretr + · propyloxy] methi-7-deaza-2'-deoxyguan osi na-5'-triphosphate dG.Vc in the form of a 1: 1 mixture of two diastereomers, which were separated using RP-HPLC to produce the simple diastereomers dG.Vc ds1 and dG.Vc ds2. HRMS (ESI): For the molecular ion C24H₃₃N₅OR₁₇P₃ [MH] ', the calculated mass was 756.1084, and the observed mass was 756.1088.

7- [1- (4, 5-Dimethoxy-2-nitrophenyl) -2, 2-dimethyl-propyloxy] methyl7-deaza-2'-deoxyguan $ ida-5'-tri phosphate

TBSO

NHTBS

OTBS (i)

TBSO

NHTBS

OTBS (¡i). (H)

<img file="MX342195B_D0243.tif" />

Oh

Oh

<img file="MX342195B_D0244.tif" />

OH dG.Vd

206

<img file="MX342195B_D0245.tif" />

Esqu ma S17. Sínt is d 7- [1 - (4,5-dim toxi-2-nitrof nil) 2,2-di metí l-pro piloxi] metí I-7-deaza-2'-deoxig uanos i na-5'trifosfato . Reagents and conditions', (/) MsCI, DMAP, CH₂CI₂, 0 ° C; (//) (R / S) -l- (4,5-dimethoxy-2-nitrophenyl) -2,2-dimethyl-1 propanol racemic, 115 ° C; (iii) n-Bu₄NF, THF, room temperature, 23% in three steps; (iv) sin-pyridine-2-aldoxime, 1,1,3,3-tetramethyl guanidine, dioxane / DMF, 70 ° C, 68%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

DMAP (273 mg, 2.2 mmol) and MsCI (130 pL, 1.7 mmol) were added to a solution of compound 21 (370 mg, 0.56 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 30 minutes and diluted with CH₂CI₂(25 mL). The solution was applied to a short (2 x 3 cm) silica gel plug and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio: 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with racemic (ft / SJ-1- (4,5-dimethoxy-2-n-trophenyl) -2,2-dimethyl-1-propanol (800 mg, 3.0 mmol ). The mixture was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature and dissolved in THF (10 mL) followed by the addition of n-Bu₄NF (530 mg, 1.7 mmol). The mixture was stirred at room temperature for two hours and was then concentrated in vacuo. The

207

<img file="MX342195B_D0246.tif" />

Residue was dissolved in CH2CI2 (40 mL) and washed with brine (50 mL), and the aqueous phase was extracted with CH₂CI₂ (40 mL) twice. The combined organic phase was dried over Na₂SW₄ and concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino-6-chloro-9 [PD-2'-deoxyribofuranosyl] -7- [1- (4,5-dimethoxy-2 -nitrophenyl) -2,2dimethyl-propyloxymethyl-7-deazapurine 34 (70 mg, 23% for three steps) as a 1: 1 mixture of two diastereomers. ¹H NMR (400 MHz, CDCI₃) for diastereomers: δ 7.42 and 7.39 (2 s, 1 H, Ph-H), 7.15 and 7.13 (2 s, 1 H, Ph-H), 6.89 and 6.84 (2 s, 1 H, H-8), 6.12 (m, 1 H, H-1 '), 5.22 and 5.16 (2 s, 1 H, Ph-CH), 5.10 and 5.08 (2 bs, 2 H, NH₂), 4.71-4.41 (m, 3H, H-3 'and 7-CH2), 4.13 (m, 1

H, H-4 '), 3.94 (4 s, 7 H, OCH₃ x 2 y H-5'a), 3. 78 (m, 1 Η, H5'b), 2.90 (m, 1 H, H-2'a), 2.25 (m, 1 H, H-2'b ), 0.82 and 0.80 (2 s, 9 H, (CH₃)₃).

To a solution of compound 34 (65 mg, 0.1 1 mmol) in

I, 4-dioxane (1.0 mL) and DMF (2.0 mL), sinpirimidine-2-aldoxime (292 mg, 2.4 mmol) and guanidine were added to

1,1,3,3-tetramethyl (330 pL, 2.6 mmol), and the mixture was heated to a temperature of 70 ° C overnight under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (40 mL) and washed sequentially with acetic acid (0.1 M, 50 mL), a NaHCO solution₃ saturated (50 mL), and brine (50 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography

208

ΪΜΡΙ ^

MEXICAN INSTITUTE ·

OF THE PRORITY to produce 7- [1 - (4,5-dimethoxy-2-nitrophenyl) -27 ^^OR^^lrm tTi ^ propyloxy] methyl-7-deaza-2'-deoxyguanosine 35 (42 mg, 68%) in the form of a 1: 1 mixture of two diastereomers. ¹H NMR (400 MHz, DMSO-de) for diastereomers: δ 10.33 (br s, 1 H, D₂Interchangeable, NH), 7.47 and 7.44 (2 s, 1 H, Ph-H), 7.16 and 7.15 (2 s, 1 H, Ph-H), 6.83 and 6.82 (2 s, 1 H, H-8) , 6.22 (m, 3H, 2 HD₂Or interchangeable, NH₂ y H-1 '), 5.18 (br s, 1 H, D₂Interchangeable O, 3'-OH), 5.06 and 5.04 (2 s, 1 H, Ph-CH), 4.83 (t, 1 H, D₂Interchangeable O, 5'-OH), 4.44-4.23 (m, 3H, 7-CH₂ and H3 '), 3.82 (4 s, 6 H, OCH₃ x 2), 3.70 (m, 1H, H-4 '), 3.42 (m, 2H, H-5'), 2.22 (m, 1H, H-2'a), 2.01 (m, 1H , H-2'b), 0.77 and 0.75 (2 s, 9 H, (CH₃)₃).

Compound 35 (40 mg, 0.073 mmol) was phosphorylated with POCI₃ (14 pL, 0.15 mmol) and a proton sponge (31 mg, 0.15 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for two hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The fractions containing the triphosphate were combined and

209

<img file="MX342195B_D0247.tif" />

They were lyophilized to provide 7- [1- (4,5-dimethoxy-2-nitrophenyl) 2,2-dimethyl-propyloxy] methyl-7-deaza-2'-deoxyguanosine-5'-triphosphate dG.Vd in the form of a 1: 1 mixture of two diastereomers, which were separated using RP-HPLC to produce the simple diastereomers dG.Vd ds1 and dG.Vd ds2. HRMS (ESI): For the molecular ion C25H₃N₅OR₁₈P3 [MH] ', the calculated mass was 786.1190, and the observed mass was

786.1206.

7 - [(S) -1 - (5-Methoxy-2-nitrophenyl) -2, 2-dimethyl-propyloxy] methyl7-deaza-2'-deoxyguanosine-5'-triphosphate

TBSO ci

NHTBS (i¡), (iü)

Cl (i)

<img file="MX342195B_D0248.tif" />

NHTBS

OTBS

OTBS

<img file="MX342195B_D0249.tif" />

Oh

Oh

<img file="MX342195B_D0250.tif" />

OH dG.VI

210 • MEXICAN INSTITUTE OF LA mPHEDAD

INDUSTRIAL

E qu maS18. Yes if de7 - [(S) -1- (5-methoxy-2-nitrophenyl) -27 ± = dimethyl-propyloxy] methyl-7-deaza-2'-deoxyguanosine-5'-triphosphate. Reagents and conditions', (i) MsCI, DMAP, CH₂CI₂, 0 ° C; (//) (S) -1 - (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1 -propanol,

115 ° C; (/ 77) /? - Bu₄NF, THF, room temperature, 27% for three steps; (iv) sin-pyridine-2-aldoxime, 1,1,3,3-tetramethyl guanidine, 1,4-dioxane / DMF, 70 ° G, 76%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1M

HNEt₃HCO₃.

DMAP (224 mg, 1.8 mmol) and MsCI (106 pl_, 1.4 mmol) were added to a solution of compound 21 (300 mg, 0.46 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and diluted with CH₂CI₂(20 mL). The solution was applied to a short (2x3 cm) silica gel plug and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (volume ratio: 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with (S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol (500 mg, 2.1 mmol). The mixture was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature and dissolved in THF (10 mL) followed by the addition of n-Bu₄NF (507 mg, 1.6 mmol). The mixture was stirred at room temperature for four hours and then concentrated in vacuo. The residue

211

<img file="MX342195B_D0251.tif" />

dissolved n CH₂CI₂ (20 mL) and washed with brine? Ci^Tlt30 the aqueous phase was extracted with CH₂CI₂ (20 mL) ÜUy vulus. the combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce

2-amino-6-chloro-9- [PD-2'-deoxyribofuranosyl] -7 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethylpropyloxy] -methyl-7-deazapurine 36 (67 mg, 27% for three steps). ¹H NMR (400 MHz, CDCI₃): δ 7.82 (d, 1 H, J = 8.8 Hz, Ph-H), 7.16 (d, 1 H, J = 2.8 Hz, Ph-H), 6.90 (s, 1 Η, H-8), 6.72 (dd, 1

H, J = 8.8 and 2.8 Hz, Ph-H), 6.12 (dd, 1 H, J = 9.2 and 6.0 Hz, H1 '), 5.22 (s, 1 H, Ph-CH), 5.15 (br s, 2 H, NH₂), 4.69-4.55 (m, 3

H, H-3 'and 7-CH₂), 4.11 (m, 1 Η, H-4 '), 3.92 (m, 1 H, H-5'a), 3.82 (s, 3 H, OCH₃), 3. 73 (m, 1 H, H-5'b), 2.81 (m, 1 H, Η-2'a), 2.21 (m, 1 H, H-2'b), 0.82 (s, 9 H, (CH₃)₃).

To a solution of compound 36 (65 mg, 0.12 mmol) in

I, 4-dioxane (1.0 mL) and DMF (2.0 mL), sinpirlmidlin-2-aldoxime (292 mg, 2.4 mmol) and guanidine were added.

1,1,3,3-tetramethyl (331 pL, 2.6 mmol), and the mixture was heated overnight to a temperature of 70 ° C under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (20 mL) and sequentially washed with acetic acid (0.1 M, 30 mL), a NaHCO solution₃ saturated (30 mL), and brine (30 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 7 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl212

IMPI

MEXICAN INSTITUTE • and industrial property

<img file="MX342195B_D0252.tif" />

propyloxy] methyl-7-deaza-2'-deoxyguanosine 37 (48 mg, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.37 (br s, 1 H, D₂Interchangeable, NH), 7.95 (d, 1 H, J = 9.2 Hz, Ph-H), 7.18 (d, 1 H, J = 2.8 Hz, Ph-H), 7.03 (dd, 1 H, J = 9.2 and 2.8 Hz, Ph-H), 6.84 (s, 1 Η, H-8), 6.23 (m, 3 H, 2 HD₂Or interchangeable, NH₂ and H1 '), 5.20 (d, 1 H, D₂Interchangeable O, 3'-OH), 5.13 (s, 1H, PhCH), 4.84 (t, 1H, D₂Interchangeable O, 5'-OH), 4.48 (d, 1 H, J = 12.0 Hz, 7-CH₂a), 4.32 (d, 1H, J = 12.0 Hz, 7-CH₂b), 4.27 (m, 1 Η, H-3 '), 3.88 (s, 3 H, OCH₃), 3.73 (m, 1 Η, H-4 '), 3.46 (m, 2 H, H-5'), 2.30 (m, 1 H, H-2'a), 2.05 (m, 1 H, H -2'b), 0.77 (s, 9H, (CH₃)₃).

Compound 37 (10 mg, 0.02 mmol) was phosphorylated with POCI₃ (26 pL, 0.26 mmol) and a proton sponge (8 mg, 0.04 mmol) in trimethylphosphate (0.3 mL) at a temperature of 0 ° C for 6.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified by anion exchange chromatography. The triphosphate- containing fractions were combined and lyophilized to provide 7 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethyl213

<img file="MX342195B_D0253.tif" />

propyloxymyl til-7-deaza-2'-deoxyguanosine-5'-triphosphate dU.VT, which was further purified using RP-HPLc. tr retention time of dG.VI was identical to that of dG.Vc ds2 by RP-HPLC analysis under the same condition. HRMS (ESI): For the molecular ion C₂₄H₃₃N₅OR₁₇P₃ [MH] ', the calculated mass was 756.1084, and the observed mass was 756.1101. Example 5 - Synthesis of 5-HOMe-2 'Triphosphate Analog ·

Deoxyuridine

5 - [(S) -1 - (5-Methoxy-2-nitrophenyl) -2, 2-dimetH-propyloxy] methyl2'-deoxyuridine -'- triphosphate

TBSO ·

OTBS

<img file="MX342195B_D0254.tif" />

OH dU.VI

Oh

Scheme S19. Synthesis of 5 - [(S) -1- (5-methoxy-2-nitrophenii) -2,2dimethyl-propyloxy] methyl-2'-deoxyuridine-5'-triphosphate.

Reagents and conditions', (i) (S) -1 - (5-methoxy-2-nitrophenyl) -2,2dimethyl-1 -propanol, 110 ° C; (/ '/) NH₄F, MeOH, 50 ° C, 56% in two steps; (/ '//') POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (nBu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

Compound 38 (Litosh et al, 2011, which is incorporated into the present invention by reference) (315 mg, 0.49 mmol) and (S) -1- (5-m toxy-2-nitrophenyl) -2,2- dimethyl-1-propanol

214

MEXICAN INSTITUTE

Dt PROPERTY Ή industrial&gt; ϊΓ ·· £ 29 »(490 mg, 2.1 mmol) were heated at a temperature of 1TQ * C for 45 minutes under a nitrogen atmosphere. The mixture was cooled to room temperature, dissolved in MeOH (10 mL), and followed by the addition of NH₄F (400mg, 11mmol). The mixture was stirred at a temperature of 50 ° C for 12 hours, concentrated in vacuo, dissolved in CH₂CI₂ (50 mL), and washed with brine (50 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 5 - [(S) -1- (5-methoxy, 2-n-trophenyl, -2,2-dimethyl-propyloxy] methy-2'-deoxyuridine 39 (130 mg, 56%). ¹H NMR (400 MHz, CDCh): δ 9.14 (br s, 1 Η, NH), 7.90 (d, 1 H, J = 9.2 Hz, Ph-H), 7.67 (s, 1 Η, H-6), 7.17 (d, 1H,

J = 2.8 Hz, Ph-H), 6.84 (dd, 1 H, J = 9.2 and 2.8 Hz, Ph-H), 6.18 (t, 1 H, J = 6.4 Hz, H-1 '), 5.22 (s , 1 H, Ph-CH), 4.56 (m, 1 Η, H3 '), 4.24 (d, 1 H, J = 12.4 Hz, 5-CH₂a), 4.15 (d, 1H, J = 12.4 Hz, 5-CH₂b), 4.00 (m, 1H, H-4 '), 3.90 (m, 1H, H-5'a), 3.88 (s, 3H, OCH₃), 3.81 (m, 1 H, H-5'b), 2.35 (m, 2 Η, H-2), 0.83 (s, 9 H, C (CH₃)₃).

Compound 39 (30 mg, 0.065 mmol) was phosphorylated with POCI₃ (9 pL, 0.097 mmol) and proton sponge (28 mg, 0.13 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for one hour under an atmosphere of nitrogen. A solution of tri-nb ^ tilamonium pyrophosphate (147 mg, 0.32 mmol) and tri-n-butylamine (64 pL) in anhydrous DMF (0.64 mL) was added. After stirring for 10 minutes, a buffer was added.

215

<img file="MX342195B_D0255.tif" />

INDUSTRIAL triethylammonium bicarbonate (0.1 M, pH 7.5; 10 mL). The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to provide 5 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-2'-deoxyuridine-5'-triphosphate dU. VI, which was further purified using RP-HPLC. HRMS (ESI): For the molecular ion C22H31N₃OR₁₈P3 [MH] ', the calculated mass was 718.0815, and the observed mass was 718.0824.

Example 6 - Synthesis of 5-HOM 2'-Desoxicltid Triphosphate Analogues i na

5- (2-nitrobencHoxy) methyl-2'-deoxycytidine-5'-tri phosphate

<img file="MX342195B_D0256.tif" />

216

<img file="MX342195B_D0257.tif" />

OH dC.I

OTBS OH

43

Scheme S20. Synthesis of 5- (2-nitrobenzyloxy) methyl-2'-deoxiciti di na-5'-triphosphate. Reagents and conditions: (i) 2-nitrobenzyl alcohol, 110 ° C; (/ '/') n-BU₄NF, THF, room temperature, 53% in two steps; (// '/') TBSCI, imidazole, DMF, room temperature, 80%; (iv) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et₃N, CH₂CI₂, room temperature; (v) NH₃, 1,4-dioxane, 90 ° C, 69% in two steps; (vi) nBu₄NF, THF, room temperature, 96%; (vii) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃.

Compound 38 (300 mg, 0.46 mmol) and 2-nitrobenzyl alcohol (500 mg, 3.3 mmol) were heated at a temperature of 110 ° C for 45 minutes under a nitrogen atmosphere. The mixture was cooled to room temperature, dissolved in THF (20 mL) followed by the addition of n-Bu₄NF (362 mg, 1.2 mmol). The mixture was stirred at room temperature for four hours, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 5- (2-nitrobenzyloxy) methyl-2'-deoxyuridine 40 (Litosh et al., 2011, which is incorporated herein

217

IMPI

MEXICAN INSTITUTE 'fé ** ás3 ¿ZSéíJ? Í DE LA PROPERTY CL- ^ Nia f.4 inv nción as reference (95 mg, 53%). ¹H RMW'Y4Ü0 CDCI₃): δ 8.45 (br s, 1 Η, NH), 8.05 (s, 1 KH-bJ, 8.Ό2 (U, J 8.0 Hz, 1 H, Ph-H), 7.80 (d, 1 H, J = 8.0 Hz, Ph-H), 7.69 (t, 1 H,

J = 8.0 Hz, Ph-H), 7.43 (t, 1 H, J = 8.0 Hz, Ph-H), 6.21 (t, 1 H, J = 6.0 Hz, H-1 '), 4.94 (dd, J = 14.4 Hz, 2H, Ph-CH₂), 4.66 (m, 1H, H-3 '), 4.35 (s, 2H, 5-CH₂), 3.95 (m, 3H, H-4 'and H-5'), 2.42 (m, 1H, Η-2'a), 2.30 (m, 1H, Η-2'b).

To a solution of compound 40 (Litosh et al., 2011, which is incorporated by reference in the present invention) (70 mg, 0.18 mmol) in anhydrous DMF (2.0 mL), TBSCI (60 mg, 0.40 mmol) was added. and imidazole (54 mg, 0.80 mmol). The mixture was stirred at room temperature under a nitrogen atmosphere, concentrated in vacuo, dissolved in CH₂CI₂ (20 mL), and washed with a NaHC0 solution₃ saturated (30 mL). The organic and aqueous phases were separated, and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried with Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 3 ', 5'-O-bis- (tert-butyldimethylsilyl) -5- (2nitrobenzyloxy) -methyl-2'-deoxyuridine 41 (90 mg, 80%). ¹H NMR (400 MHz, CDCI₃): δ 8.04 (d, J = 8.0 Hz, 1 H, Ph-H), 7.98 (br s,

H, NH), 7.80 (d, 1 H, J = 8.0 Hz, Ph-H), 7.74 (s, 1 Η, H-6),

7.64 (q, 1H, J = 8.0Hz, Ph-H), 7.44 (t, 1H, J = 8.0Hz, Ph-H),

6.21 (q, 1H, J = 6.0Hz, H-1 '), 4.95 (s, 2H, Ph-CH₂), 4.41 (m, 1

H, H-3 '), 4.34 (dd, 2 H, J = 11.6 Hz, 5-CH₂), 3.96 (m, 1H, H-4 '),

218

IMPI

1NSTITVT.;. MS / JCANO ty &amp; áa'tTGZ &amp; Jñ

3.79 (m, 2H, H-5¹), 2.29 (m, 1 H, Η-2'a), 2.05 (Π ¥? Υφι&gt; Η,

0.89 (2 s, 18 H, C (CH₃)₃), 0.08 (4 s, 12 H, (34¼¼ --_

2,4,6-Triisopropyl benzenesulfonyl chloride (176 mg, 0.59 mmol) was added to a solution of compound 41 (85 mg, 0.14 mmol), DMAP (19 mg, 0.16 mmol), and triethylamine (0.18 mL, 1.3 mmol) in CH₂CI₂ anhydrous (5.0 mL). The mixture was stirred at room temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the residue was dissolved in a NH solution₃ in 1,4-dioxane (0.5 M, 15 mL). The mixture was transferred into a sealed tube and heated overnight to a temperature of 90 ° C. The mixture was cooled to room temperature, concentrated in vacuo, dissolved in CH₂CI₂ (30 mL), and washed with brine (30 mL). The organic and aqueous phases were separated, and the aqueous phase was extracted with CH₂CI₂ (30 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield 3 ', 5'-O-bis- (tert-butyldimethylsilyl) -5- (2nitrobenzyloxy) methyl-2'-deoxycytidine 42 ( 60 mg, 69% for two steps). ¹H NMR (400 MHz, CDCI₃): δ 8.08 (d, J = 8.0 Hz, 1 H, Ph-H), 7.81 (s, 1 Η, H-6), 7.65 (m, 2 H, Ph-H), 7.64 (q, 1 H , J = 8.0 Hz, Ph-H), 7.49 (m, 1 H, Ph-H), 6.29 (t, 1 H, J = 6.4 Hz, H1 '), 5.75 (br s, 1 H, NH₂), 4.85 (dd, 2H, J = 13.6 Hz, Ph-CH₂), 4.41 (s, 2H, 5-CH₂), 4.34 (m, 1 H, H-3 '), 3.95 (m, 1 H, H-4'), 3.89 (dd, 1 H, J = 2.8 Hz, Η-5'a), 3.76 (dd , 1 H, J = 2.8 Hz, H219

IMPI

MEXICAN INSTITUTE OF THE FtOPIEPAD

5'b), 2.46 (m, 1 H, Η-2'a), 1.98 (m, 1 H, Η-2'b), 0.92 'JW &amp; fc

H, C (CH₃)₃), 0.11-0.08 (4 s, 12 H, CH₃). To a solution of compound 42 was added (55 mg, 0.09 mmol) in THF (10 mL), n-Bu₄NF (63 mg, 0.20 mmol). The mixture was stirred at room temperature for four hours and concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 5- (2-nitrobenzyloxy) methyl-2'-deoxycytidine 43 (34 mg, 96%) .¹H NMR (400 MHz, DMSO-d₆): δ 8.05 (d, J = 8.0 Hz, 1 H, Ph-H), 7.89 (s, 1 Η, H-6), 7.74 (m, 2 H, Ph-H), 7.55 (m, 1 H , Ph-H), 7.39 (br s, 1 H, D₂Or interchangeable, NH₂), 6.74 (br s, 1 H, D₂Or interchangeable, NH₂), 6.12 (t, 1 H, J = 6.4 Hz, H-1 '), 5.21 (br s, 1 H, D₂Interchangeable O, 3'-OH), 4.99 (br s, 1 H, D₂Interchangeable O, 5'-OH), 4.81 (s, 2H, Ph-CH₂), 4.30 (dd, 2H, J = 11.6 Hz, 5-CH₂), 4.20 (m, 1 H, H-3 '), 3.76 (m, 1 Η, H-4'), 3.55 (m, 2 Η, H-5 '), 2.11 (m, 1 H, Η- 2'a), 1.95 (m, 1H, Η-2'b).

Compound 43 (32 mg, 0.081 mmol) was phosphorylated with POCI₃ (30 pL ,, 0.32 mmol) and a proton sponge (35 mg, 0.16 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for three hours under a nitrogen atmosphere. A solution of tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and

<img file="MX342195B_D0258.tif" />

220 ^ΤΜΡΙ

TTIJTO MEXICANO 'Έ La? KOPtEOAD

<img file="MX342195B_D0259.tif" />

it was subsequently concentrated in vacuo. The residue '§^orand^STπΓΑΡ-diso in 20% aqueous acetonitrile (20 mL), ΎΠΠΌy SH 'puirfre ^ · by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to provide 5- (2-nitrobenzyloxy) methyl-2'-deoxycytidine-5'-triphosphate dC.I, which was further purified using RP-HPLC. HRMS (ESI): For the molecular ion C₁₇H22N₄OR₁₆P₃ [MH] ', the calculated mass was

631.0244, and the observed mass was 631.0258.

5 - [(S) -1 - (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxycytidine-5'-tri phosphate

TBSO

Br

<img file="MX342195B_D0260.tif" />

ÓTBS

OTBS OTBS

Four. Five (i¡¡)

<img file="MX342195B_D0261.tif" />

OTBS OH

47

<img file="MX342195B_D0262.tif" />

OH dC.V

221

MEXICAN INSTITUTE

Phew. THE PROPERTY (V-ZSU

What about S21. Synt id 5 - [(S) -1 - (2-niWfén d¡met¡l-prop¡lox¡] met¡l-2^,-deoxycytidine-5^,-T'riTesfaTTJ ·. ··

Reagents and conditions: (i) (S) -1- (2-nitrophenyl) -2,2-dimethyl-1 propanol, 110 ° C, 21%; (I) 2,4,6-Triisopropylbenzenesulfonyl chloride, DMAP, Et₃N, CH₂CI₂, room temperature, 31%; (iii) NH₃, 1,4-dioxane, 90 ° C, 91%; (iv) n-Bu₄NF, THF, room temperature, 82%; (v) POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1M

HNEt₃HC0₃.

Compound 38 (Litosh et al., 2011, which is incorporated into the present invention by reference) (520 mg, 0.80 mmol) and (S) 1- (2-nitrophenyl) -2 were heated to a temperature of 110 ° C , 2-dimethyl-1-propanol (580 mg, 2.8 mmol) for one hour under a nitrogen atmosphere. The mixture was cooled to room temperature, dissolved in a minimal amount of ethyl acetate, and purified by silica gel chromatography to produce 3 ', 5'-O-bis- (tert-butylsimethylsilyl) -5 [(S ) -1- (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxyuridine 44 (115 mg, 21%). Also obtained from the reaction were (3 'or 5') - O- (tert-butylsimethylsilyl) -5 - [(S) -1 - (2-n itrofe ni 1) -2,2-di meth Ipropyloxy] methyl- 2'-deoxyuridine (78 mg, 17%) and 5 - [(S) -1- (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxyuridine (16 mg, 4%). ¹H NMR (400 MHz, CDCI₃): δ 8.97 (s, 1 Η, NH), 7.76 (d, 2 H, J = 8.0 Hz, Ph-H), 7.60 (m, 2 H, Ph-H and H-6), 7.41 (s, 1 H, Ph-H), 6.29 (dd, 1 H, J = 6.0 and 7.6 Hz, H-1 '), 4.97 (s 1 H, Ph222

BWJTOTO MSXJCANt fll

PE LA rRfWDAp .V—? * '&lt;·.' TO

CH), 4.42 (m, 1 Η, H-3 '), 4.28 (AB d, 1 H, J = 12.0

4.06 (AB d, 1 H, J = 12.0 Hz, 5-CH₂b), 3.92 (irr, ·· 1 H r44-4¿ ^ ~ a ~ £ &amp;. (m, 2 Η, H-5 '), 2.30 (m, 1 H, Η-2'a), 2.05 ( m, 1H, Η-2'b), 0.95 (s, 9H, (CH₃)₃CS¡), 0.90 (s, 9 H, (CH₃)₃CSi), 0.83 (s, 9H, (CH₃)₃C), 0.12 (s, 3H, CH₃YES), 0.09 (s, 3H, CH₃YES), 0.07 (s, 3H, CH₃YES), 0.06 (s, 3H, CH₃Yes!)

2,4,6-Triisopropyl benzenesulfonyl chloride (61 mg, 0.20 mmol) was added to a solution of compound 44 (110 mg, 0.16 mmol), DMAP (20 mg, 0.17 mmol), and triethylamine (63 pL, 0.45 mmol) in CH₂CI₂ anhydrous (3.0 mL). The mixture was stirred at room temperature for 36 hours under a nitrogen atmosphere, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 3 ', 5'-O-bis- (ter- butiIsimetiIsiIil) -5 - [(S) -1 - (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-0⁴- (2,4,6-trisisopropylbenzenesulfonyl) -2'-deoxyuridine 45 (47 mg, 31%).

¹H NMR (500 MHz, CDCI₃): δ 8.08 (s, 1 Η, H-6), 7.80 (dd, 1 H, J = 1.2 and 8.0 Hz, Ph-H), 7.78 (dd, 1 H, J = 1.6 and 8.0 Hz, Ph- H), 7.67 (m, 1 H, Ph-H), 7.46 (m, 1 H, Ph-H), 7.20 (s, 2 H, Ph-H), 6.09 (t, 1 H, J = 6.4 Hz , H-1 '), 4.98 (s, 1H, Ph-CH), 4.35 (m, 1 Η, H-3'), 4.25 (AB d, 1H, J = 11.6 Hz, 5-CH₂a), 4.11 (AB d, 1 H,

J = 11.6 Hz, 5-CH₂b), 3.97 (m, 1 Η, H-4 '), 3.79 (dd, 1 H, J = 3.6 and

11.6 Hz, Η-5'a), 3.74 (dd, 1 H, J = 11.6 and 3.6 Hz, Η-5'b), 2.90 (m,

H, CH), 2.50 (m, 2 Η, H-2 '), 1.98 (m, 2 H, CH), 1.31 - 1.22 (m,

H, (CH₃)₂CH x 3), 0.88 (2 s, 18 H, (CH₃)₃CSi x 2), 0.87 (s, 9

223

IMPI

MEXICAN INSTITUTE CB LA PWCRBDA ·

H, (CH₃)₃C), 0.07 (s, 6H, (CH₃)₂Yes), 0.06 (s, 6 Η, (Clffly

NH solution added₃ in 1,4-dioxane (Ó.S h / l, 2.0 mL) to a solution of compound 45 (47 mg, 0.05 mmol) in

I, anhydrous 4-dioxane (2.0 mL). The mixture was transferred to a sealed tube and heated at a temperature of 90 ° C for ten hours. The mixture was cooled to room temperature, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 3 ', 5'-Obis- (tert-butyldimethylsilyl) -5 - [(S) -1- (2-nitrophenyl) -2,2-dimethylpropyloxy] -methyl-2'-deoxycytidine 46 (31 mg, 91%).¹H NMR (400 MHz, CDCI₃): δ 7.67 (m, 3 H, Ph-H), 7.53 (s, 1 Η, H-6), 7.45 (m, 1 H, Ph-H), 6.30 (t, 1 H, J = 6.6 Hz , H-1 '), 5.72 (br s, 2 H, NH₂), 4.88 (s, 1H, Ph-CH), 4.32 (m, 1H, H-3 '), 4.28 (AB d, 1H, J = 12.8Hz, 5-CH₂a), 4.08 (AB d, 1 H, J = 12.8 Hz, 5-CH₂b), 3.87 (m, 1 H, H-4 '), 3.74 (dd, 1 H, J = 3.6 and 14.8 Hz, Η-5'a), 3.66 (dd, 1 H, J = 3.6 and 11.3 Hz , Η-5'b), 2.41 (m, 1 H, Η-2'a), 2.03 (m, 1 H, Η-2'b), 0.90 (s, 9 H, (CH₃)₃CSi), 0.87 (s, 9H, (CH₃)₃CS¡), 0.83 (s, 9 H, C (CH₃)₃), 0.09 (2 s, 6 H, (CH₃)₂Yes),

<img file="MX342195B_D0263.tif" />

0.06 (2 s, 6 H, (CH₃)₂Yes!)

N-Bu solution added₄NF (28 mg, 0.09 mmol) in THF (1.0 niL) to a solution of compound 46 (20 mg, 0.03 mmol) in THF (2.0 mL). The mixture was stirred at room temperature for 30 min and concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield 5 - [(S) -1 - (2-nitrophenyl) -2,2- dimethyl-propyloxy] methyl224

MEXICAN INSTITUTE. DE LA PRONE'JAD Q— &lt;3 j

2'-deoxycytidine 47 (11 mg, 82%). ¹H NMR (400 M

7.87 (s, 1 Η, H-6), 7.82 (dd, 1 H, J = 1.2 and · »: · 4 lie, Ph · H), 7.-7-6 (dd, 1 H, J = 1.6 y 8.0 Hz, Ph-H), 7.68 (m, 1 H, Ph-H), 7.51 (m, 1 H, Ph-H), 6.23 (t, 1 H, J = 6.6 Hz, H-1 ') , 4.94 (s, 1H, Ph-CH), 4.44 (AB d, 1H, J = 13.2Hz, 5-CH₂a), 4.34 (m, 1 H, H-3 '), 4.11 (AB d, 1 H, J = 13.2 Hz, 5-CH₂b), 3.88 (m, 1 H, H-4 '), 3.71 (dd, 1 H, J = 3.2 and 12.0 Hz, H-5'a), 3.63 (dd, 1 H, J = 4.0 and 12.0 Hz , H5'b), 2.35 (m, 1 H, Η-2'a), 2.14 (m, 1 H, Η-2'b), 0.80 (s, 9 H, C (CH₃)₃).

Compound 47 (11 mg, 0.025 mmol) was phosphorylated with POCI₃ (7 pL ,, 0.075 mmol) and proton sponge (11 mg, 0.05 mmol) in trimethylphosphate (0.3 mL) at a temperature of 0 ° C for three hours under a nitrogen atmosphere. A solution of tri-n-butylammonium pyrophosphate (59 mg, 0.125 mmol) and tri-n-butylamine (30 pL) in anhydrous DMF (0.25 mL) was added. After 5 minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 5.0 mL) was added. The reaction was stirred at room temperature for one hour and then lyophilized to dryness. The residue was dissolved in water (5.0 mL), filtered and purified by anion exchange chromatography. The triphosphate-containing fractions were combined and lyophilized to provide 5 - [(S) -1- (2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-2'-deoxycytidine-5'-triphosphate dC.V, which was purified further using RP-HPLC. HRMS (ESI): For the molecular ion C₂iH₃₀N4O₁₆P₃ [MH] ', the

225 calculated mass was 687.0870, and

687.0873.

5 - [(S) -1 - (5-Methoxy-2-nitrophenyl) -2,

2'-deoxycytidine-5'-tri phosphate

I neither pi

MEXICAN INSTITUTE

OF PROPERTY &lt;· * · _, INDUSTRIAL the observed mass ro

2-dimetH-propyloxy] meta-

<img file="MX342195B_D0264.tif" />

go

<img file="MX342195B_D0265.tif" />

OMe

O, N

<img file="MX342195B_D0266.tif" />

NH, f-Bu

<img file="MX342195B_D0267.tif" />

oo OO ~ OOX s

OH AD VI

Scheme S22. Synthesis of 5 - [(S) -1- (5-methoxy-2nit rofe nil) -2,2-dimeth-l-pro ploxi] m-ethyl-2'-de oxycytidine-5'-triphosphate. Reagents and conditions ·, (i) TBSCI, imidazole, DMF, room temperature, 70%; (ii) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et₃N, CH₂CI₂temperature

226 for

<img file="MX342195B_D0268.tif" />

MRXICAN INSTITUTE&gt;

FROM PRO / IEOaD C d eneras ol ambi nte; (ii¡) NH₃, 1,4-dioxane, 90 ° C, 65% n-Bu₄NF, THF, room temperature, 82%; proton, (MeO)₃PO, at a temperature of 0 ° C; (nBU3NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HC0₃.

Added to a solution of compound 39 (235 mg, 0.49 mmol) in anhydrous DMF (3.0 mL), TBSCI (320 mg, 0.8 mmol), and imidazole (109 mg, 1.6 mmol). The mixture was stirred at room temperature for six hours, concentrated in vacuo, dissolved in CH₂CI₂ (20 mL), and washed with a NaHCO solution₃saturated (50 mL). The organic and aqueous phases were separated, and the aqueous phase was extracted with CH₂CI₂ (30 mL) three times. The combined organic phase was dried with Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 3 ', 5' - Ob i s- (te rb u ti Id i meti I si I il) -5 [(S) - 1 - (5-methoxy-2-nitrophenyl) -2,2-di-meth-l-propyloxy] meth-I-2'-deoxyuridine 48 (245 mg, 70%). ¹H NMR (400 MHz, CDCI₃): δ 8.00 (br s, 1 Η, NH), 7.88 (d, J = 9.2 Hz, 1 H, Ph-H), 7.60 (s, 1 Η, H-6), 7.22 (d, 1 H, J = 2.8 Hz, Ph-H), 6.84 (dd, 1 H, J = 2.8 and 8.0 Hz, Ph-H), 6.25 (dd, 1 H, J = 5.6 and 8.0 Hz, H-1 '), 5.23 (s, 1H, Ph-CH), 4.40 (m, 1H, H-3 '), 4.26 (d, 1H, J = 12Hz, 5-CH₂a), 4.11 (d, 1 H, J = 12 Hz, 5-CH₂b), 3.89 (m, 4H, OCH₃ and H-4 '), 3.78 (m, 2H, H-5'), 2.27 (m, 1H, Η-2'a), 2.04 (m, 1H, Η-2'b), 0.90 and 88 (2 s, 18 H, S¡C (CH₃)₃), 0.84 (s, 9H, C (CH₃)₃), 0.08 (3 s, 12 H, CH₃).

2,4,6227 benzenesulfonyl chloride added

<img file="MX342195B_D0269.tif" />

MIXJONI INSTITUTE, I heard. the μ .-&gt;? ;; π_λγ&gt; l'V,

Triisopropyl (363 mg, 1.2 mmol) to a solution d'eT'Oompt&gt;

(170 mg, 0.24 mmol), DMAP (32 rrrgn — 8? G6 mm triethylamine (0.34 mL, 2.4 mmol) in CH₂CI₂ anhydrous (8.0 mL). The mixture was stirred at room temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the residue was dissolved in a NH solution₃ in 1,4-dioxane (0.5 M, 20 mL). The mixture was transferred to a sealed tube and heated overnight to a temperature of 90 ° C. The mixture was cooled to room temperature, concentrated in vacuo, dissolved in CH₂CI₂ (20 mL), and washed with brine (50 mL). The organic and aqueous phases were separated, and the aqueous phase was extracted with CH₂CI₂ (30 mL) three times. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 3 ', 5'-O-bis- (te rb ut i Id im et i Is i I il) -5 - [( S) -1 (5-methoxy-2-n ythro fe nil) -2,2-d meth-l-pro piloxy] meth-2'-deoxycytidine 49 (110 mg, 65% for two steps). ¹H NMR (400 MHz, DMSO-de): δ 7.96 (d, J = 8.8 Hz, 1 H, Ph-H), 7.50 (br s, 1 H, NH₂), 7.38 (s, 1 Η, H-6), 7.08 (dd, 1 H, J = 2.8 and 8.8 Hz, PhH), 7.04 (d, 1 H, J = 2.8 Hz, Ph-H), 6.80 ( br s, 1H, NH₂), 6.13 (t, 1 H, J = 6.4 Hz, H-1 '), 5.09 (s, 1 H, Ph-CH), 4.31 (m, 1 Η, H3'), 4.25 (d, 1 H, J = 12.8 Hz, 5-CH₂a), 4.08 (d, 1H, J = 12.8 Hz, 5-CH₂b), 3.87 (s, 3H, OCH₃), 3.76 (m, 1 H, H-4 '), 3.64 (m, 2 H, H-5'), 3.76 (dd, 1 H, J = 2.8 Hz, Η-5'b), 2.10 (m , 1 H, Η-2'a), 2.00 (m, 1 H, Η-2'b), 0.87 (s, 9 H, C (CH₃)₃), 0.78 and 0.76 (2 s, 18

228

<img file="MX342195B_D0270.tif" />

IMPI

MEXICAN INSTITUTE OF PROPERTY

H, S¡C (CH₃)₃), 0.07, 0.06, -0.01, and -0.04 (4 s, 12 H '^ SICH

Added to a solution of compound 49 (130 my, Of · ® · mmol) in THF (10 mL), n-Bu₄NF (141 mg, 0.44 mmol). The mixture was stirred at room temperature for four hours, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield 5 - [(S) 1 - (5-methoxy-2-nitrophenyl) -2 , 2-dimethyl-propyloxy] methyl-2'-deoxycytidine 50 (72 mg, 82%).¹H NMR (400 MHZ, DMSO-d₆): δ 7.99 (d, J = 8.0 Hz, 1 H, Ph-H), 7.65 (s, 1 Η, H-6), 7.42 (br s, 1 H, D₂Or interchangeable, NH₂a), 7.06 (m, 2 H, Ph-H), 6.72 (br s, 1 H, D₂Or interchangeable, NH₂b), 6.11 (t, 1 H, J = 6.4 Hz, H-1 '), 5.17 (d, 1 H, D₂Interchangeable, 3'-OH), 5.12 (s, 1H, Ph-CH), 4.78 (t, 1H, D₂Interchangeable O, 5'-OH), 4.25 (d, 1 H, J = 12.4 Hz, 5-CH₂a), 4.15 (m, 1H, H-3 '), 4.05 (d, 1H, J = 12.4Hz, 5CH₂b), 3.87 (s, 3H, OCH₃), 3.72 (m, 1 H, H-4 '), 3.44 (m, 2 Η, H5'), 3.76 (dd, 1 H, J = 2.8 Hz, Η-5'b), 2.08 (m, 1 H, Η-2'a), 1.95 (m, 1 H, Η-2'b), 0.77 (s, 9 H, C (CH₃)₃).

Compound 50 (20 mg, 0.043 mmol) was phosphorylated with POCI₃ (24 pL ,, 0.26 mmol) and a proton sponge (19 mg, 0.086 mmol) in trimethylphosphate (0.3 mL) at a temperature d 0 ° C for six hours under a nitrogen atmosphere. S was added to a solution of tri-n-butylammonium pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 \ L) in anhydrous DMF (1.0 mL). After 10 minutes of stirring, triethylammonium bicarbonate buffer (0.1M, pH 7.5; 10) was added.

229

IMPI

M2XJCAN INSTITUTE&gt;

BE INDUSTRIAL PROPERTY mL). The reaction was stirred at room temperature for one hour and was then concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified by anion exchange chromatography. The fractions having triphosphate were combined and lyophilized to provide 5 - [(S) -1- (5-methoxy-2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-2'-deoxycytidine-5'-triphosphate dC. VI, which was further purified using RP-HPLC. HRMS (ESI): For the molecular ion C₂2H32N₄Oi7P₃ [MH] ', the calculated mass was 719.0975, the observed mass was 719.0983.

Example 7-Synthesis of (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) 2,2-dimethyl-1-propanol and (S) -1- (4-iodo-5-methoxy -2-nitrophenyl) 2,2-dimethyl-1-propanol

<img file="MX342195B_D0271.tif" />

<img file="MX342195B_D0272.tif" />

3-iodoanisole

3,6-diiodo-4nitroanlsol (IV S) · 1- (4-iodo-5-methoxl2-nitrophenyl, -2,2-d imetll ·

1-Propanol (IS)-(R / S) -144-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl

<img file="MX342195B_D0273.tif" />

(IS)-(S) -1 - (7-iodo-5-methoxy-2-nitrophenyl) -2,2-d immethyl-propyl-campanate

<img file="MX342195B_D0274.tif" />

(S) -1 - (4-iodo-5-methoxy, 2-nitrophenyl) -2,2-dimethyl · 1-pr pan I

230 ? Ϊ

MEXICAN INSTITUTE Jrt delaí'Rt?: E05O

E qu ma S23. Synthesis of (R / S) ~ 1- (4-y οάθ⁶6^;^ ™ «ίό&gt; ί222 - ^&gt; ^ nitrophenyl) -2,2-dimethii-1 -propanol and (S) -1- (4 yedo 5 mot'Oxi-2-— ~ nitrophenyl) -2,2-dimethyl- 1-propanol. (i) NaNO₂, CH₃COOH, HNO₃, room temperature; l₂, 60 ° C, 25% for two steps (80% pure); (ii) PhMgCI, (CH₃)₃CCHO, THF, minus 40 ° C at room temperature, 72%; (¡Ii) (1S) caffeic acid chloride, DMAP, CH₂CI₂ , room temperature, 80%; (iii) fractional crystallization from ethanol, 63%; (iv) K₂CO₃,

MeOH, reflux.

Nitric acid (68-70%, 125 mL) was slowly mixed with glacial acetic acid (125 ml) at room temperature, followed by the addition of NaNO₂ (400mg, 5.8mmol) and 3-iodoanisole (10g, 42.7mmol). After the reaction was stirred at room temperature for 24 hours, (10.8 g, 42.7 mmol) was added and the mixture was stirred overnight at a temperature of 60 ° C. The reaction mixture was poured into ice-water (500 ml) and extracted by CH₂CI₂ (100 mi) three times. The combined organic phase was neutralized with a NaHCO solution₃saturated (500 ml), washed with an aqueous solution of Na₂S₂OR₃(20%, 100 ml), dried on Na₂SW₄, and concentrated in vacuo.

The residue was purified by silica gel column chromatography to produce crude 3,6-diiodo-4-nitroanisole (5.4 g), which was mixed with an unknown by-product (20%) and used in the following passage without further purification. ¹H NMR (400 MHz, CDCI₃): δ 8.42 (s, 1H, Ph-H),

231

MEXICAN INSTITUTE OF THE PRO? '£ OÁI&gt; INDUSTRIAL

7.33 (s, 1H, Ph-H), 3.97 (s, 3H, OCH₃).

To a solution of 3,6-diiodo-4-nitroan¡snl r.txutft,. (J70 mg ,. 80% purity, 1.52 mmol) in anhydrous THF (10 mL) at a temperature of minus 40 ° C under an atmosphere of nitrogen, phenylmagnesium chloride (2 M in THF, 0.46 mL, 0.92 mmol) was added dropwise in a range such that the temperature could not exceed less than 35 ° C. At the end of the addition, the mixture was stirred at a temperature below 40 ° C for two hours, followed by the addition of trimethylacetaldehyde (0.22 mL, 1.97 mmol). The mixture was stirred at a temperature of less than 30 ° C for two hours and then at room temperature for another hour. Subsequently the reaction was quenched with brine (1.0 mL), diluted with CH₂CI₂ (100 mL), and the solution was washed with CH₃COOH (0.1 N, 50 ml) and brine (50 ml) in sequences. The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield (R / S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1propanol racemic (399 mg, 72%). ¹H NMR (400 MHz, CDCI₃): δ 8.32 (s, 1 H, Ph-H), 7.17 (s, 1 H, Ph-H), 5.60 (d, 1 H, J = 4.0 Hz, PhCH), 3.98 (s, 3 H, OCH₃), 2.12 (d, 1 H, J = 4.0 Hz, OH), 0.89 (s, 9H, C (CH₃)₃).

To a solution of racemic (R / S) -1- (4-iodo-5-methoxy-2-nitrophenyl) 2,2-dimethyl-1-propanol (395 mg, 1.1 mmol) and DMAP (263 mg, 2.16 mmol ) in CH₂CI₂ anhydrous (5.0 mL), added

232

INI Ρ ϊ ^ 38 ^ 5

MEXICANc INSTITUTE

PROPERTY QoJSL ^ (1 S) -camphanyl chloride (Corrie et al., | ^ -5u-aT is incorporated into the present invention cuiiwi efeTcncJd) · (300 mg, 1.62 mmol), and the mixture was stirred for overnight at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (50 mL) and washed with a NaHCO solution₃ saturated (50 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce (1 S)-(R / S) -1- (4-iodo-5-methoxy-2n it ro feni) I) -2,2-dimethyl-1-propyl (490 mg, 80%, mixture of diastereomers 1: 1).

(R / S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl (3.4 g) was dissolved in boiling ethanol (150 ml), (1 S) -camfanate was dissolved. the solution was kept in a hot oil bath and cooled slowly to room temperature and kept overnight. Needle crystals gradually formed and were collected by filtration to produce the pure simple diastereomer (1 S) -camphanate of (R) -1 - (4-iodo-5-methoxy-2n it rofe ni I) -2,2 - tell me you I -1 -propyl (870 mg, 51%). The remaining mother liquor was concentrated in vacuo, and the residue was redissolved in boiling ethanol (150 ml), and the solution was rapidly cooled to room temperature and the needle crystals formed within two hours. The crystals were collected by filtration to produce the pure simple diastereomer (1 S) -camfanate of (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2233

MEXICAN INSTITUTE X

FROM ERGPÍEDA3 dimethyl-1-propyl. The crystallization process sd ^ 'fé'P'ttii ~ times to produce (S) -artictional pure diastereomer (HHal l-yQ-Tg, 63%).¹H NMR (400 MHz, CDCI₃) for (1 S) -camphanate of (R) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl: δ 8.48 (s, 1H, Ph-H ), 6.94 (s, 1H, Ph-H), 6.84 (s, 1H, Ph-CH), 3.93 (s, 3H, OCH₃), 2.42 (m, 1 H, CH), 2.11 (m, 1 H, CH), 1.92 (m, 1 H, CH₂), 1.75 (m, 1H, CH₂), 1.11 (s, 3H, CH₃), 1.05 (s, 3H, CH₃), 0.97 (s, 9H, C (CH₃)₃), 0-86 (s, 3H, CH₃). ¹H NMR (400

MHz, CDCI₃) for (1 S) -camphanate of (S) -1- (4-iodo-5-methoxy-2-nitropheniI) -2,2-dimethI-1-propyl: δ 8.48 (s, 1H, Ph-H), 6.95 (s, 1H, Ph-H), 6.80 (s, 1H, Ph-CH), 3.96 (s, 3H, OCH₃), 2.37 (m, 1H, CH), 1.92 (m, 2H, CH₂), 1.66 (m, 1H, CH), 1.14 (s, 3H, CH₃), 1.07 (s, 3H, CH₃), 1.06 (s, 3H, CH₃), 0.98 (s, 9H, C (CH₃)₃).

A mixture of (1 S) -camphanate of (S) -1 - (4-iodo-5-methoxy2-nitrophenyl) -2,2-dimethyl-1-propyl (1.1 g, 2.0 mmol) and K₂CO₃(552 mg, 4.0 mmol) in methanol (50 mL) was heated under reflux for one hour, and then cooled, concentrated in vacuo, and diluted with CH₂CI₂ (50 mL). The organic phase was washed with brine (50 mL), dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce (S) -1- (4-iodo-5-methoxy2-nitrophenyl) -2,2-dimethyl-1-propanol enantiopure ( 720 mg, 98%).

¹H NMR (400 MHz, CDCI₃): δ 8.32 (s, 1H, Ph-H), 7.17 (s, 1H,

Ph-H), 5.60 (d, 1H, J = 4.0Hz, PhCH), 3.98 (s, 3H, OCH₃), 2.12

234

INSTITUT

ί. PI $ 3 ^^.

· '? MEXICAN .W .A PAOPIÉDAi? O.teJeáLJS / (d, 1 H, J = 4.0 Hz, OH), 0.89 (S, 9H, C (CH₃)₃). ^{Df LA}| NDUS1 WAL ^{V, |}* «C

Example 8 - Synthesis of hydroxymethyl Nucleotides Alkylated with tBu-5-OMe-2-nitrobenzyl Tagged With

ink

Synthesis of (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2,2dimethyl-1-propanol and (S) -1- (4-iodo-5-methoxy-2-nitrophenyl ) -2,2dimethyl-1 -propanol

<img file="MX342195B_D0275.tif" />

Óme OMe

<img file="MX342195B_D0276.tif" />

(R / S) - 1- (4-iodo-5-methoxy 2-nitrophenyl) -2,2-dimethyl1-propane ol

<img file="MX342195B_D0277.tif" />

(IS) -camphanate of (R / S) -1 - (4-iodo-5-methoxy2-nltrofen¡l) -2,2-dimetll1 -propllo

3-iodoanisole 3,6-diyodo-4-nitroanisole

<img file="MX342195B_D0278.tif" />

(IS)-(S) -1 - (- 4-iodo-5-methoxy2-n ltrofenll) -2,2-d imetil * 1 -propyl-campanate

<img file="MX342195B_D0279.tif" />

(S) -1 - (4-iodo-5-methoxy2-nitrophenyl) -2,2-dimethyl1-propanol

Scheme S24. Synthesis of (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol and (S) -1 - (4-iodo-5-methoxy-2-nitrophenyl ) -2,2-dimethyl-1-propanol. (i) NaNO₂, CH₃COOH, HNO₃, room temperature; l₂, 60 ° C, 25% during two steps (80% pure); (¡I) PhMgCI, (CH₃)₃CCHO, THF, minus 40 ° C at

235

INSTITUTO MEX.CAN?

PROPERTY * room temperature, 72%; (iii) chloride of locus camfánica, DMAP, CH₂CI₂ , room temperature, 8 Ό%; (111? Fractional crystallization from ethanol, 63%; (iv) K₂CO₃, MeOH, reflux, 98%.

Nitric acid (68-70%, 125 mL) was slowly mixed with glacial acetic acid (125 ml) at room temperature, followed by the addition of NaNO₂ (400mg, 5.8mmol) and 3-iodoanisole (10g, 42.7mmol). After the reaction was stirred at room temperature for 24 hours, l was added₂ (10.8 g, 42.7 mmol) and the mixture was stirred overnight at a temperature of 60 ° C. The reaction mixture was poured into ice water (500 ml) and extracted by means of ΟΗ₂ΟΙ₂ (100 mi) three times. The combined organic phase was neutralized with a NaHCO solution₃ saturated (500 ml), washed with an aqueous solution of Na₂S₂OR₃ (20%, 100 ml), dried on Na₂SW₄, and concentrated in vacuo. The residue was purified by silica gel column chromatography to produce crude 3,6-diiodo-4-nitroanisole (5.4 g), which was mixed with an unknown by-product (20%) and used in the next step without further purification.¹H NMR (400 MHz, CDCI₃): δ 8.42 (s, 1H, Ph-H), 7.33 (s, 1H, Ph-H), 3.97 (s, 3H, OCH₃).

To a solution of crude 3,6-diiodo-4-nitroanisole (770 mg, 80% purity, 1.52 mmol) in anhydrous THF (10 mL) at a temperature of less than 40 ° C under a nitrogen atmosphere, was added in the form of chloride drops

<img file="MX342195B_D0280.tif" />

236

MEXICAN INSTITUTE * ¿

FROM THE ROW VW »phenylmagnesium (2 M in THF, 0.46 mL, 0.92 mmol) errtiwrant that the temperature cannot exceed m € w&gt; eo - 4e — 86 ° -6 ·. ·· - · Αί term of addition , the mixture was stirred at a temperature of less than 40 ° C for two hours, followed by the addition of trimethylacetaldehyde (0.22 mL, 1.97 mmol). The mixture was stirred at a temperature of less than 30 ° C for two hours and then overnight for one hour. . The reaction was subsequently quenched with brine (1.0 mL), diluted with CH₂CI₂ (100 mL), and the solution was washed with CH₃COOH (0.1 N, 50 ml) and brine (50 ml) in sequences. The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1 racemic propanol (399 mg, 72%). ¹H NMR (400 MHz, CDCI₃): δ 8.32 (s, 1 H, Ph-H), 7.17 (s, 1 H, Ph-H), 5.60 (d, 1 H, J = 4.0 Hz, PhCH), 3.98 (s, 3 H, OCH₃), 2.12 (d, 1 H, J = 4.0 Hz, OH), 0.89 (s, 9H, C (CH₃)₃).

To a solution of racemic (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) 2,2-dimethyl-1-propanol (395 mg, 1.1 mmol) and DMAP (263 mg, 2.16 mmol ) in CH₂CI₂ anhydrous (5.0 mL), (1 S) -camphanic chloride (Corrie et al., 1992, which is incorporated by reference in the present invention) (350 mg, 1.62 mmol) was added, and the mixture was stirred during the overnight at room temperature under a nitrogen atmosphere. The reaction mixture was diluted with CH₂CI₂ (50 mL) and washed with

237

<img file="MX342195B_D0281.tif" />

IMPI • MTITUTO MEXICANO Give LA PROPUPAU a NaHCo solution₃ saturated (50 mL). The fas ^ ofga dried on Na₂SW₄, concentrated in uactr », ¹ and the residue was purified by silica gel column chromatography to produce (1 S) -camphanate of (R / S) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2, 2-dimethyl-1-propyl (490 mg, 80%, mixture of diastereomers).

(R / S) -1- (4-Iodine-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl (3.4 g) was dissolved in (15) -caf. In boiling ethanol (150 ml), The solution was kept in a hot oil bath and slowly cooled to room temperature and kept overnight. Needle crystals were gradually formed and collected by filtration to produce the pure simple (1 S) -camfanate (R) -1 (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1 diastereomer. -propyl (870 mg, 51%). The remaining mother liquor was concentrated in vacuo, and the residue was redissolved in boiling ethanol (150 ml), and the solution was rapidly cooled to room temperature and needle crystals formed within two hours. The crystals were collected by filtration to produce a pure simple (1 S) -campanate of (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl. The crystallization process was repeated twice to produce additional pure (S) -diastereomer (total 1.07 g, 63%). ¹H NMR (400 MHz, CDCI₃) for (1 S) -camphanate of (R) -1 - (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl: δ 8.48 (s, 1H, Ph-H), 6.94 (s, 1H, Ph-H), 6.84

238 i JSi

MEXICAN INSTITUTE ϊ ^ 4 «· - &lt;ί? .'TO^{D £ LA}, S? ¿?

(s, 1H, Ph-CH), 3.93 (s, 3H, OCH₃), 2.42 (m, 1 W * ÓH) ^Hee2 ^ rr (m, 1 H, CH), 1.92 (m, 1 H, CH₂), 1.75 (m, 1 'Η7ΤΓΗ₂) ·, I.'l 1 ”TS, · 3

H, CH₃), 1.05 (s, 3H, CH₃), 0.97 (s, 9H, C (CH₃)₃), 0.86 (s, 3H, CH3). ¹H NMR (400 MHz, CDCI₃) for (1 S) -camphanate of (S) -1 (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propyl: δ 8.48 (s, 1 H , Ph-H), 6.95 (s, 1H, Ph-H), 6.80 (s, 1H, Ph-CH), 3.96 (s, 3H, OCH₃), 2.37 (m, 1H, CH), 1.92 (m, 2H, CH₂), 1.66 (m, 1H, CH), 1.14 (s, 3H, CH₃), 1.07 (s, 3H, CH₃), 1.06 (s, 3H, CH₃), 0.98 (s, 9H, C (CH₃)₃).

A mixture of (1 S) -camphanate of (S) -1 - (4-iodo-5-methoxy2-nitrophenyl) -2,2-dimethyl-1-propyl (1.1 g, 2.0 mmol) and K₂CO₃(552 mg, 4.0 mmol) in methanol (50 mL) was heated under reflux for one hour, then cooled, concentrated in vacuo, and diluted with CH₂CI₂ (50 mL). The organic phase was washed with brine (50 mL), dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce an (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1-propanol enantiopure (720 mg, 98%). ¹H NMR (400 MHz, CDCI₃): δ 8.32 (s, 1H, Ph-H), 7.17 (s,

H, Ph-H), 5.60 (d, 1H, J = 4.0Hz, PhCH), 3.98 (s, 3H, OCH₃), 2.12 (d, 1 H, J = 4.0 Hz, OH), 0.89 (s, 9H, C (CH₃)₃).

Synthesis of 7 - {(S) -1 - [4- (3-amino-1-propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl-propyloxy} methyl-7-deaza-2'-deoxyadenosine-5 '-triphosphate and 5'-a-thiothriphosphate labeled with ink

239

<img file="MX342195B_D0282.tif" />

54a R = O

54b R = S

Scheme S25a. Synthesis of 7 - {(S) -1 - [4- (3-amino-1 propinyl) -5-methoxy-2-n-trophenyl] -2,2-dimethyl-propylloxy} met L-7deaza-2 * -deoxydenosine-5'-triphosphate and ink-labeled 5'-a-thiothriphosphate. Reagents and conditions: (i) MsCI, DMAP, CH₂CI₂, (ii) (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1propanol, 110 ° C; (iii) n-Bu₄NF, THF, room temperature; 29% for three steps; (iv) NH₃, 1,4-dioxane / MeOH, 100 ° C, 80%; (v) N-propargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF, 98%; (vi) To 54a: POCI₃, proton sponge, (MeO)₃PO, 0 ° C;

240

Ο. Jj f '· «

MEXICAN INSTITUTE

FROM PROPERTY V&gt; e, ¿í7s7j ^ (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; iWV ^ H.

54b: PSCI₃, 2,4,6-collidine, (EtO)₃PO, 0 ° C at ΓδΙϊψΗΐ alurambiente; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; NH4OH.

(v¡)

<img file="MX342195B_D0283.tif" />

Oh

55a R = O 55b R = S

Scheme S25b. (vi) Alexa Fluor 488 NHS, damper 0.1 M Na₂CO₃/ NaHCO₃ (pH 9.2).

DMAP (463 mg, 3.80 mmol) and MsCI (177 pL,

2.28 mmol) to a solution of compound 4 (400 mg, 0.76 mmol) in CH₂CI₂ anhydrous (5.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere The reaction was stirred at a temperature of 0 ° C for 10 minutes and overnight at room temperature for 3 hours. Later the

241

<img file="MX342195B_D0284.tif" />

MEXICAN INSTITUTE ')

GIVE PROPERTY reaction was diluted with CH₂CI₂ (20 mL). The solution ict a short silica gel stopper (2 x 3 '”δΠΊ) ..... and sh eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (80 mL, volume ratio: 80 / 20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with (S) 1- (4-iodo-5-methoxy-2-n-trophenyl) -2,2-dimethi1-1-propanol (500 mg, 1.37 mmol). The mixture was heated at a temperature of 115 ° C for 45 minutes under a nitrogen atmosphere, cooled to room temperature and dissolved in THF (10 mL). N-Bu added₄NF (526 mg, 1.67 mmol) and the mixture was stirred at room temperature for 12 hours and then concentrated in vacuo. The residue was dissolved in CH₂CI₂ (50 mL) and washed with brine (50 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 6-chloro-9 (PD-2'-deoxyribofuranosyl) -7 - [(5) -1 - (4-iodo-5-methoxy -2-nitrophenyl) -2,2-dimethyl-propyloxy] -methyl-7-deazapurine 51 (135 mg, 29% for three steps). ¹H NMR (400 MHz, CDCI₃): δ 8.56 (s, 1 Η, H-2), 8.21 (s, 1 H, Ph-H), 7.34 (s, 1 H, H-8), 7.02 (s, 1 H, Ph-H) , 6.35 (dd, 1 H, J = 6.0 and 8.8 Hz, H-1 '), 5.20 (s, 1 H, PhCH), 4.76 (dd, 2 H, J = 12.4 and 36.4 Hz, 7-CH₂), 4.74 (m, 1 Η, H3 '), 4.13 (m, 1 H, H-4'), 3.96 (m, 1 H, Η-5'a), 3.92 (s, 3 H, OCH₃), 3.80 (m, 1 H, Η-5'b), 2.85 (m, 1 H, Η-2'a), 2.30 (m, 1 H, Η-2'b), 0.83 (s, 9 H , C (CH₃)₃).

242

IMPIOS

MEXICAN INSTITUTE OF PROPERTY

S dissolved Computo 51 (135 mg, 0.22'WrhÓl) dioxane (10 mL) followed by the addition of NFT₃ tíli MeOH (7 MrM »· mL). The mixture was transferred to a sealed tube and stirred at a temperature of 100 ° C for 24 hours, then cooled to room temperature, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 7- [ (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethylpropyloxy] methyl-7-deaza-2'-deoxyadenosine at 52 (110 mg, 80%).

¹H NMR (400 MHz, CDCI₃): δ 8.24 (s, 1 Η, H-2), 8.22 (s, 1H, Ph-

<td>H), 6.96</td><td>(s,</td><td>1 H, Ph-H)</td><td> , 6.79</td><td>(s,</td><td>1 H, H-8), 6.1</td><td>4 (d</td><td>d,</td><td> 1</td><td>H, J =</td>

<td>6.0 and 7.6</td><td>Hz</td><td>, H-1 '), 5.2</td><td>11 (s, 1</td><td>H</td><td>Ph-CH), 4.74</td><td>(m,</td><td> 1</td><td>H</td><td>H-3 '),</td>

<td>4.56 (dd,</td><td>2H</td><td>, J = 13.2</td><td>and 24.8</td><td>Hz,</td><td>7-CH₂), 4.17</td><td>(m,</td><td> 1</td><td>H</td><td>H-4 '),</td>

<td>3.93 (m,</td><td>1 HOUR,</td><td>Η-5'a), 3.</td><td>83 (s,</td><td>3H,</td><td>OCH₃), 3.79</td><td>(m,</td><td colspan="2">1 HOUR,</td><td>Η-5'b),</td>

<td>2.96 (m,</td><td> 1</td><td>H, Η-2'a),</td><td> 2.21</td><td>(m,</td><td>1 H, Η-2'b),</td><td> 0.8</td><td> 3</td><td>(s,</td><td>, 9 H,</td>

C (CH₃)₃).

A solution of compound 52 (183 mg, 0.29 mmol), Npropargyltrifluoroacetylamide (435 mg, 2.9 mmol), tetrakis (triphenylphosphine) -palladium (0) (66 mg, 0.057 mmol), Cul (21 mg, 0.11 mmol), and Et₃N (170 pL, 1.22 mmol) in anhydrous DMF (4.0 mL) was stirred at a temperature of 50 ° C for 24 hours. The mixture was concentrated in vacuo and purified by silica gel column chromatography to yield 7 - {(S) 1 - [5-methoxy-4- (3-trifluoroacetamido-1-propinyl) -2-nitrophenyl] -2 , 2dimethyl-propyloxy} methyl-7-deaza-2'-deoxyadenosine 53 (185 mg, 98%).¹H NMR (400 MHz, MeOH-d₄): δ 8.06 (s, 1 H, H-2), 7.89

243

<img file="MX342195B_D0285.tif" />

(s, 1 H, Ph-H), 7.17 (s, 1 Η, H-8), 7.15 (s, 1 1 H, J = 6.0 and 7.6 Hz, H-1 '), 5.23 (s, 1 H , Ph - ett ·), 4.00 (d, 1 = 12.8 Hz, 7-CH₂a), 4.49 (m, 1 Η, H-3 '), 4.34 (s, 2H, CH₂), 3.97 (m, 1H, H-4 '), 3.86 (s, 3H, OCH₃), 3.70 (m, 2 H, H-5 '), 2.59 (m, 1 H, Η-2'a), 2.2.8 (m, 1 H, Η-2'b), 0.85 (s, 9 H, C (CH₃)₃).

Compound 53 (52 mg, 0.08 mmol) was phosphorylated with POCI₃ (14 μΙ_, 0.15 mmol) and a proton sponge (34 mg, 0.16 mmol) in trimethylphosphate (0.5 mL) at a temperature of 0 ° C for 3 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a solution of triethylammonium bicarbonate (TEAB, 0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB / 25% acetonitrile (20 mL), filtered and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The triphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 7 {(S) -1- [4- (3-amino-1 -propinyl) -5-methoxy-2-nitrophenyl] -2,2-d-methylpropyloxy} methyl-7-deaza-2'-deoxyadenosine-5'-triphosphate 54a, the

244

INSTITUTO MEXICANO DE LA PROPERTY which was further purified by inverse straining on a PerkinElmer Aquapore UU-3ÜU column (T ”pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C₂7H3₆N₆OR₁₆P3 [MH] ', the calculated mass was 793.1401, and the observed mass was

793.1426.

Compound 53 (91 mg, 0.14 mmol) was thiophosphorylated with PSCI₃ (14 pL, 0.14 mmol) and 2,4,6-collidine (34 mg, 0.28 mmol) in triethylphosphate (1.0 mL) at a temperature of 0 ° C for 1 hour under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (332 mg, 0.7 mmol) and trin-butylamine (140 pL) in anhydrous DMF (1.4 mL) was added. After 2 minutes of stirring, a triethylammonium bicarbonate solution (TEAB, 1M, pH 7.5; 20 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue dissolved in 75% 0.1 M

TEAB / 25% acetonitrile (20 mL), was filtered, and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The thiothriphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 7 {(S) -1 - [4- (3-amino-1 - propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl245

IMPI wsrrnnx, Mexican ^Of tA MOPIEOa · propyloxy} methyl-7-d aza-2'-deoxyadenosine-5'-atryotriphos5? ato 54b, which was further purified by reverse phase HPLT loading on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C27H₃₆N₆OR₁₅P₃S [MH], the calculated mass was 809.1172, and the observed mass was 809.1 155.

A solution of Alexa Fluor 488 NHS (5 mg, 7.8 pmol) in anhydrous DMSO (200 pL) was added to a solution of triphosphate 54a (1.6 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, 30 pH 9.2, 0.4 mL). The mixture was left at room temperature in the dark for one hour. The mixture was first purified by anion exchange HPLC on a column

Dlonex ADNpac PA200 (250 x 4 mm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled triphosphate 55a were combined and concentrated to a small volume, and the product was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm) . Mobile phase; A, 0.1 M TEAB; B, acetonitrile.

A solution of Alexa Fluor 488 NHS (5 mg, 7.8 pmol} in anhydrous DMSO (200 pL) was added to a solution of thiothophosphate 54b (4.1 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 1.0 mL). The mixture was left at room temperature in the

<img file="MX342195B_D0286.tif" />

246 darkness for one hour using chromatography. The exchange mix

IMPI ¡TlTUTOMíiXiCíNf) φ of anions using se

<img file="MX342195B_D0287.tif" />

a Q Sepharose FF column (2.5 x 10 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled thiothophosphate 55b were combined and lyophilized to dryness, and the product was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pam, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile.

Synthesis of 7 - {(S) -1- [4- (3-amino-1-propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl-pro piloxy} metil -7-deaza-2 'deoxyguanosine -5'-triphosphate and 5'-a-thiothriphosphate

<img file="MX342195B_D0288.tif" />

Oh

247

IMPI

INSTITUTO MEJUCAN DI LA MONEDAD INDUSTRIAL

<img file="MX342195B_D0289.tif" />

<img file="MX342195B_D0290.tif" />

<img file="MX342195B_D0291.tif" />

59a R = O

59b R = S

Scheme S26a. Synthesis of 7 - {(S) -1 - [4- (3-amino-1-propynyl) -5methoxy-2-n-trophenyl] -2,2-d-methyl-propyloxy} methyl-7-deaza -2'-deoxyguanos ina-5'-triphosphate and ink-labeled 5'-α-thiothriphosphate. Reagents and conditions: (/) MsCI, DMAP, CH₂CI₂, 0 ° C; (ii) (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl-1 propanol, 115 ° C; (iii) n-Bu₄NF, THF, room temperature; 18% for three steps; (iv) sin-pyridine-2-aldoxime, 1,1,3,3-tetramethyl guanidine, 1,4-dioxane / DMF, 70 ° C, 72%; (v) Npropargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF, 96%; (vi) To 59th: POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (nBu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; NH₄Oh For 59b: PSCI₃, 2,4,6-collidine, (EtO)₃PO, 0 ° C at room temperature; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, 1 M HNEt₃HCO₃; NH₄Oh

248

IMPI

Mexican Institute of Industrial Property

<img file="MX342195B_D0292.tif" />

<img file="MX342195B_D0293.tif" />

60a R = O 60b R = S

Scheme S26b. (viii) Alexa Fluor 594 NHS, Na damper₂CO₃/ NaHCO₃ 0.1 M (pH 9.2).

DMAP (502 mg, 4.1 mmol) and MsCI (238 μΙ_, 3.1 mmol) were added to a solution of compound 18 (680 mg, 1.0 mmol) in CH₂CI₂ anhydrous (6.0 mL) at a temperature of 0 ° C under a nitrogen atmosphere. The reaction was stirred at a temperature of 0 ° C for 10 minutes and was subsequently diluted with CH₂CI₂ (20 mL). The solution was applied to a short (2x3 cm) silica gel stopper and eluted rapidly with a hexane / ethyl acetate / triethylamine solvent system (80 mL, volume ratio: 80/20 / 0.5). The eluent was concentrated in vacuo, and the residue was mixed with (S) -1 - (4-iodo-5-methoxy-2249

<img file="MX342195B_D0294.tif" />

nitropheniI) -2,2-dim til-1-propanol (500 mg, 2.1) was heated to a temperature of 115 ° C during 45 mlritrtvs under a nitrogen atmosphere, cooled to room temperature and dissolved in THF (10 mL N-BU added₄NF (1.07 g, 3.40 mmol) and the mixture was stirred at room temperature for 12 hours and then concentrated in vacuo. The residue was dissolved in CH2CI2 (50 mL) and washed with brine (50 mL), and the aqueous phase was extracted with CH₂CI₂ (20 mL) twice. The combined organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 2-amino-6-chloro-9- (3-D-2'-deoxyribofi iran osi l) -7 - [(S) -1 - (4-y odo-5-methoxy-2-nitrofe ni 1) -2,2dimethyl-propyloxy-methyl-7-deazapurine 56 (125 mg, 18% for three steps). ¹H NMR (400 MHz, CDCI₃): δ 8.24 (s, 1 H, Ph-H), 7.04 (s, 1 H, Ph-H), 6.91 (s, 1 H, H-8), 6.17 (dd, 1 H, J = 6.0 and 8.4 Hz, H-1 '), 5.18 (s, 1 H, Ph-CH), 5.11 (br s, 2 H, NH₂), 5 4.71 (m, 1H, H-3 '), 4.59 (dd, 2H, J = 12.4 and 24.4 Hz, 7-CH2), 4.13 (m, 1H, H-4'), 3.96 ( s, 3 H, OCH₃), 3.88 (m, 1 H, H-5'a), 3. 79 (m, 1 H, H-5'b), 2.76 (m, 1 H, H-2'a), 2.32 (m, 1H, H2'b), 0.81 (s, 9H) (CH₃)₃).

To a solution of compound 56 (100 mg, 0.16 mmol) in 1,4-dioxane (1.5 mL) and DMF (3.0 mL), sinpyrimidine-2-aldoxime (389 mg, 3.2 mmol) and guanidine of 1, were added. 1,3,3-tetramethyl (439 pL, 3.5 mmol), and the mixture was heated overnight at a temperature of 70 ° C, under a

250

ΤΓ&gt; ·

MEXICAN INSTITUTE »&lt;ZJ

OF THE PROPERTY -^F· ^ Nitrogen atmosphere. The reaction mixture is^{, N0}¿)^sÍTÍf * yó * ^rCH2CI2 (20 mL) and washed sequentially with an acetic acid solution (0.1 M, 50 mL), a saturated NaHCO3 solution (50 mL), and brine (50 mL). The organic phase was dried over Na2SO4, concentrated in vacuo, and the residue was purified by chromatography on silica gel to yield 7 - [(S) -1 (4^:Iodine-5-methoxy-2-nitrophenyl) -2,2-dimethyl-propyloxy] methyl-7deaza-2'-deoxyguanosine 57 (70 mg, 72%). ¹H NMR (400 MHz, MeOH-d4): δ 8.20 (s, 1 H, Ph-H), 7.17 (s, 1 H, Ph-H), 6.82 (s, 1 H, H-8), 6.18 ( m, 1H, H-1 '), 5.23 (s, 1H, Ph-CH), 4.71 (d, 1H,

J = 12.0 Hz, 7-CH₂a), 4.52 (d, 1H, J = 12.0 Hz, 7-CH₂b), 4.43 (m, 1H, H-3 '), 3.97 (s, 3H, OCH₃), 3.91 (m, 1H, H-4 '), 3.71 (m,

H, H-5 '), 2.49 (m, 1 H, H-2'a), 2.19 (m, 1 H, H-2'b), 0.85 (s, 9

H, (CH₃)₃).

A solution of compound 57 (50mg, 0.08mmol), Npropargyltrifluoroacetylamide (117mg, 0.8mmol), tetrakis (triphenylphosphine) -palladium (0) (18mg, 0.02mmol), Cul (5.9mg, 0.03mmol), and Et₃N (48 pL, 0.34 mmol) in anhydrous DMF (3.0 mL) was stirred at a temperature of 50 ° C for 12 hours. The mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography to yield 7 - {(S) 1- [5-methoxy-4- (3-trifluoroacetamido-1-propinyl) -2-nitrophenyl] -2,2dimethyl-propyloxy} methyl-7-deaza-2'-deoxyguanosine 58 (50 mg,

96%). ¹H NMR (400 MHz, MeOH-d₄): δ 7.87 (s, 1 H, Ph-H), 7.26 (s, 1 H, Ph-H), 6.84 (s, 1 H, H-8), 6.20 (m, 1 H, H-1 ' ), 5.25 (s, 1

251

IMPI Mexican institute OF THE PRCW.DA'J

H, Ph-CH), 4.67 (d, 1H, / = 12.0 Hz, 7-CH₂a), 4.S «P^LW 1Τθ ^ 12.0 Hz, 7-CH₂b), 4.43 (m, 1H, H-3 '), 4.33 (a,' W + rettzTrSrSfr-— (s, 3H, OCH₃), 3.89 (m, 1H, H-4 '), 3.70 (m, 2H, H-5'), 2.46 (m,

H, H-2'a), 2.18 (m, 1H, H-2'b), 30 0.86 (s, 9H, (CH₃)₃).

Compound 58 (52 mg, 0.08 mmol) was phosphorylated with POCI₃ (27 pL, 0.3 mmol) and a proton sponge (33 mg,

0.16 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 4 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a solution of triethylammonium bicarbonate (TEAB, 0.1 M, pH 7.5;

mL). The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB / 25% acetonitrile (20 5 mL), filtered and purified anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75%

0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The triphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 7 - {(S} -1 - [4- (3-min. -1-propin yl) -5-methoxy-2-n trophy yl] 2,2-dimethyl-propyloxy} methyl-7-deaza-2'-deoxyguanosine-5'-triphosphate 59a, which was further purified by

252

IMPI £ 3

MEXICAN INSTITUTE OE THE PROPERTY

Reverse phase HPLC on a PerkinElmeP'WifÍhapoY ^ OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, ΎΙ. IM TEAD, Dacetonitrile. HRMS (ESI): For the molecular ion C₂7H₃6N₆OR₁₇P3 [Μ-H] ', the calculated mass was 809.1350, and the observed mass was 15 809.1360.

Compound 59 (50 mg, 0.075 mmol) was thiophosphlated with PSCI₃ (9 pL, 0.09 mmol) and 2,4,6-collidine (18 mg, 0.15 mmol) in triethylphosphate (0.5 mL) at room temperature for 2.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) and tri-nbutylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 2 minutes of stirring, a solution of triethylammonium bicarbonate (TEAB, 1M, pH 7.5; 20 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% TEAB / 0.1

25% acetonitrile (20 mL) was filtered, and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75% 0.1 25 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The thiothriphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 7 {(S) -1- [4- (3-amino-1 -propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethylpropyloxy} methyl-7-deaza-2'-deoxyguanosine-5'-a-30 thiothriphosphate

253

<img file="MX342195B_D0295.tif" />

Mexican institute Ο € LA FMONEEJAD

59b, which was further purified by reverse phase media Lj on a PerkinElmer AdUy'pUTe OÜ-SGÚ column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile. HRMS (ESI): For molecular ion C₂7H₃6N₆OR₁₆P3S [MH], the calculated mass was 825.1121, and the observed mass was

825.1103.

A solution of Alexa Fluor 594 NHS (4.2 mg, 5.2 pmol) in anhydrous DMSO (170 pL) was added to a solution of 59a triphosphate (2.2 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 0.5 mL). The mixture was left at room temperature in the dark for one hour. The mixture was first purified by anion exchange HPLC on a column

Dionex ADNpac PA200 (250 x 4 mm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled triphosphate 60a were combined and concentrated to a small volume, and the product was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm) . Mobile phase: A, 0.1 M TEAB; B, acetonitrile.

A solution of Alexa Fluor 594 NHS (5 mg, 6.2 pmol) in anhydrous DMSO (200 pL) was added to a solution of thiothophosphate 59b (4.45 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 0.78 mL). The mixture was left at room temperature in the dark for one hour. The mixture was purified first

254

<img file="MX342195B_D0296.tif" />

by anion exchange chromatography using a Q Sepharose FF column (2.5 x 10 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled thiothriphosphate 60b were combined and concentrated to a small volume, and the product was further purified by reverse phase HPLC on a column.

PerkinElme r Aquapore OD-300 (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile.

Synthesis of 5 - {(S) -1 - [4- (3-amino-1-propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl-propUoxy} methyl-2'-deoxyuridine-5'-triphosphate and Ink-labeled 5'-a-thiothriphosphate

<img file="MX342195B_D0297.tif" />

II

TBSO.

OTBS

<img file="MX342195B_D0298.tif" />

OH OH

62

II (¡V)

<img file="MX342195B_D0299.tif" />

Oh

63a R = O 63b R = S

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MEXICAN INSTITUTE, 'T ^ Kl

OF PROPERTY 1 _ „ _r, z «- .INDUSTRIAL. W -i- '

Esqu ma S27a. Ink-marked 5- {S) -1 - [4- (3-amino-1 -propiniT methoxy-2-nitrophenyl] -2,2-dimethyl-propyloxy} methyl-¿'deoxyuridine-5'-triphosphate Reagents and conditions: (i) (S) -1- (4-iodo-5-methoxy-2-nitrophenyl) -2,2-dimethyl1-propanol, 110 ° C; (/ '/') NH₄F MeOH, 50 ° C, 28% for two steps; (///) A / -propargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF, 90%; (iv) For 63a: POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; NH₄Oh For 63b: PSCI₃, 2,6-lutidine, (EtO)₃PO, 0 ° C at room temperature; (n-Bu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; one

<img file="MX342195B_D0300.tif" />

64a R = O 64b R = S

What about S27b. (v) Alexa Fluor 532 NHS, damper

256

NaHCO₃/ Na₂CO₃ 0.1 Μ (pH 9.2).

INSTITUTO MtXICANO Klafroí'iídap k

INDUSTRIAL

<img file="MX342195B_D0301.tif" />

Compound 38 (350 mg-jQ-rE) was heated⁷! mm ^ i) y (£) 1 - (4-iodo-5-methoxy-2-n-trophenyl) -2,2-dimethyl-1-propanol (720 mg,

1.97 mmol) at a temperature of 110 ° C for 45 minutes under a nitrogen atmosphere. The mixture was cooled to room temperature, dissolved in MeOH (10 mL), and followed by the addition of NH₄F (400mg, 11.1mmol). The mixture was stirred at a temperature of 50 ° C for 12 hours, concentrated in vacuo, dissolved in CH₂CI₂ (50 mL), and washed with brine (50 mL). The organic phase was dried over Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 5 - [(S) -1- (4-iodo-5-methoxy-2-nitrophenyl) 2,2-dimethyl-propyloxy] methyl -2'-deoxyuridine 61 (90 mg, 28%). ¹H NMR (400 MHz, CDCI₃): δ 8.34 (s, 1 H, Ph-H), 7.65 (s, 1 Η, H6), 7.12 (s, 1 H, Ph-H), 6.17 (t, 1 H, / = 6.8 Hz, HF ), 5.18 (s, 1H, Ph-CH), 4.59 (m, 1H, H-3 '), 4.27 (d, 1H, J = 12.0Hz, 5CH₂a), 4.15 (d, 1H, J = 12.0 Hz, 5-CH₂b), 4.00 (m, 1H, H-4 '),

3.97 (s, 3H, OCH₃), 3.95 (m, 1 H, H-5'a), 3.82 (m, 1 H, H-5'b), 2.34 (m, 2 Η, H-2), 0.84 (s, 9 H, C (CH₃)₃).

A solution of compound 61 (80 mg, 0.13 mmol), Nproparglltrlfluoroacetllamide (196 mg, 1.30 mmol), tetrakis (triphenylphosphine) -palladium (0) (30 mg, 0.026 mmol), Cul (9.9 mg, 0.052 mmol ), and Et₃N (80 pL) in anhydrous DMF (3.0 mL) was stirred at a temperature of 50 ° C for 12 hours. The mixture was concentrated in vacuo and the residue was purified by

257

<img file="MX342195B_D0302.tif" />

MEXICAN INSTITUTE I heard THE PROPERTY silica g I column chromatography for prtRTOWr 1 - [5-methoxy-4- (3-trifluoroacetamido-1 - propiniI) -2^Jniliufen 11) -2,2- »dimethyl-propyloxy} methyl-2'-deoxyuridine 62 (75 mg, 90%). ¹H

NMR (400 MHz, MeOD-d₄): δ 8.11 (s, 1 Η, H-6), 8.08 (s, 1 H, Ph-H), 7.36 (s, 1 H, Ph-H), 6.27 (t, 1 H, y = 6.4 Hz , H-1 '), 5.33 (s, 1H, Ph-CH), 4.47 (m, 1 Η, H-3'), 4.44 (s, 2H, 5-CH₂), 4.32 (d, 2H, J = 2.0Hz, CH2), 4.08 (s, 3H, OCH₃), 3.99 (m, 1 Η, H4 '), 3.87 (m, 1 H, H-5'a), 5 3.79 (m, 1 H, H-5'b), 2.30 (m, 2 H, H -2), 0.93 (s, 9H, C (CH₃)₃).

Compound 62 (40 mg, 0.064 mmol) was phosphorylated with POCI₃ (21 pl_, 0.22 mmol) and proton sponge (27 mg, 0.13 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 4 hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) - and tri-n-butylamine (100 pL) in anhydrous DMF (1.0 mL) was added. After 10 minutes of stirring, a triethylammonium bicarbonate solution (TEAB, 0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB / 25% acetonitrile (20 mL), filtered, and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The fractions containing the triphosphate were combined and lyophilized to dryness. The residue s dissolved

258

MEXICAN INSTITUTE

FROM TXOPJÍOAJ vV-- SmTJÍ in water (10 mL) and treated with ammonium hydroxide '^ S'h'certt? 5cro (10 mL, 27%) at room temperature duiSTriTU a liwa pete · produce 5- { (SJ -1 - [4- (3-amino-1-propinyl) -5-methoxy-2-nitrophenyl] 2,2-dimethyl-propyloxy} methyl-2'-deoxyuridine-5'-triphosphate 63a, which is further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm) Mobile phase: A, 0.1 0.1 TEAB; B, acetonitrile.

Compound 62 (130 mg, 0.21 mmol) was thiophosphorylated with PSCI₃ (26 pL, 0.25 mmol) and 2,6-lutidine (89 mg, 0.84 mmol) in triethylphosphate (0.6 mL) at room temperature for 1 hour under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (474 ​​mg, 1.0 mmol) and tri-nbutylamine (200 pL) in anhydrous DMF (2.0 mL) was added. After 2 minutes of stirring, a solution of triethylammonium bicarbonate (TEAB, 1M, pH 7.5; 20 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB / 25% acetonitrile (20 mL), filtered, and purified by anion exchange chromatography using a column

Q Sepharose FF (2.5 x 20 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile.

The thiothriphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL,

27%) at room temperature for one hour to produce 5259

IΜ P ί

MEXICAN INSTITUTE OF PROPERTY {(S) -1- [4- (3-amino-1-propinyl) -5-m toxi-2-nitrophenyl^lF-^P^ T-dimeTfT^rj^ propyloxy} methyl-2'-deoxyuridin-5'-a-thiothryphosphate 63b, which was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C25H34O₁₇P3S [MH] ', the calculated mass was 787.0853, and the observed mass was 787.0884.

A solution of Alexa Fluor 532 NHS (2 mg, 2.76 pmol) in anhydrous DMSO (80 pL) was added to a solution of triphosphate 62a (1.07 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 0.3 mL). The mixture was left at room temperature in the dark for one hour. The mixture was first purified by anion exchange HPLC on a column

Dionex ADNpac PA200 (250 x 4 mm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile: B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled 63a triphosphate were combined and concentrated to a small volume, and the product was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm) . Mobile phase: A, 0.1 M TEAB; B, acetonitrile.

A solution of Alexa Fluor 532 NHS (2.5 mg, 3.45 pmol) in anhydrous DMSO (100 pL) was added to a solution of thiothophosphate 62b (1.03 pmol) in NaHCO buffer.₃/ Na₂CO₃(0.1 M, pH 9.2, 0.15 mL). The mixture was left at mperature

260

IMPI

INSTITUTO MEXICANO DELA PROPIEDAD room in the dark for one hour. The mixKa was first purified by G6 exchange chromatography

<img file="MX342195B_D0303.tif" />

anions using a Q Sepharose FF column (2.5 x 10 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled thiothriphosphate 63b were combined and lyophilized to dryness, and the product was further purified by reverse phase HPLC on a PerkinElmer column.

Aquapore OD-300 (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1

MTEAB; B, acetonitrile.

Synthesis of 5 - {(S) -1- [4- (3-amino-1-propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl-propyloxy} methyl-2'-deoxycytidine-5'-triphosphate and Ink-labeled 5'-a-thiothriphosphate

<img file="MX342195B_D0304.tif" />

i-Pr.

₀/^s-OR-'-^pr i-Pr

<img file="MX342195B_D0305.tif" />

<img file="MX342195B_D0306.tif" />

261

<img file="MX342195B_D0307.tif" />

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MEXICAN INSTITUTE

Οΐ THE PROPERTY ·

INDUSTRIAL

<img file="MX342195B_D0308.tif" />

68a R = O 68b R = S

Scheme S28a. Synthesis of 5 - {(SJ-1 - [4- (3-amino-1-propynyl) -5methoxy-2-nitrophenyl] -2,2-dimethyl-propyloxy} methyl-2'-deoxycytidine-5'-tr Ink marked phosphate.

Reagents and conditions: (i) TBSCI, imidazole, DMF, room temperature, 96%; (//) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et₃N, CH₂CI₂, room temperature; (iii) NH₃₎ 1,4-dioxane, 90 ° C; (iv) n-Bu₄NF, THF, room temperature, 83% for three steps; (v) Npropargyltrifluoroacetamide, Pd (PPh₃)₄(0), Cul, Et₃N, DMF, 91%; (vi) For 68a: POCI₃, proton sponge, (MeO)₃PO, 0 ° C; (nBu₃NH)₂H₂P₂OR₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; NH₄Oh For 68b: PSCI₃, 2,6-lutidine, (EtO)₃PO, 0 ° C; (n-Bu₃NH)₂H₂P2O₇, nBu₃N, DMF; 1 M HNEt₃HCO₃; NH₄Oh

262

<img file="MX342195B_D0309.tif" />

69b R = S

Scheme S28b. (vii) Cy5 NHS, damper Na₂CO₃/ NaHCO₃0.1 M (pH 9.2).

To a solution of compound 61 (295 mg, 0.49 mmol) in anhydrous DMF (5.0 mL), TBSCI (185 mg, 1.23 mmol) and imidazole (160 mg, 2.35 mmol) were added. The mixture was stirred at room temperature for 12 hours, concentrated in vacuo, and dissolved in CH₂CI₂ (50 mL). The solution was washed with a NaHCO solution₃ (50 mL) and the aqueous phase was extracted with CH₂CI₂ (30 mL) three times. The combined organic phase was dried with Na₂SW₄, concentrated in vacuo, and the residue was purified by silica gel chromatography to produce 3 ', 5'-Obis- (tert-butyldimethylsilyl) -5 - [(S) -1 - (4-iodo-5-methoxy -2-nitrophenyl) 263

<img file="MX342195B_D0310.tif" />

<img file="MX342195B_D0311.tif" />

MEXICAN INSTITUTE OF PROPERTY

2,2-dimethyl-propyloxy] methyl-2'-deoxyuridine 65 (400 NMR (400 MHz, CD0₃): δ 8.32 (s, 1 H, Ph-H - ('S', 1 II, H 6 · ^ -—

7.12 (s, 1 H, Ph-H), 6.12 (t, 1 H, y = 6.8 Hz, H-1 '), 5.20 (s, 1 H, Ph-CH), 4.60 (m, 1 H, H -3 '), 4.25 (d, 1H, J = 12.0 Hz, 5-CH₂to),

4.14 (d, 1H, J = 12.0 Hz, 5-CH₂b), 4.02 (m, 1H, H-4 '), 3.97 (s,

H, OCH₃), 3.94 (m, 1H, H-5'a), 3.83 (m, 1H, H-5'b), 2.34 (m,

H, H-2), 0.90 and 88 (2 s, 18 H, S¡C (CH₃)₃), 0.84 (s, 9H,

C (CH₃)₃), 0.08 (3 s, 12 H, CH₃).

2,4,6-Triisopropyl benzenesulfonyl chloride (581 mg, 1.92 mmol) was added to a solution of the compound (400 mg, 0.48 mmol), DMAP (64 mg, 0.53 mmol), and triethylamine (0.60 mL, 4.32 mmol) in CH₂CI₂ anhydrous (15 mL). The mixture was stirred at room temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the residue was dissolved in NH solution₃ in 1,4-dioxane (0.5 M, 20 mL). The mixture was transferred into a sealed tube and heated overnight to a temperature of 90 ° C. The reaction was subsequently cooled to room temperature, concentrated in vacuo, and the residue was dissolved in THF (8.0 mL) followed by the addition of n-Bu₄NF (333 mg, 1.06 mmol). The mixture was stirred at room temperature for four hours, concentrated in vacuo, and the residue was purified by silica gel column chromatography to produce 5 - [(S) -1- (4-iodo-5methoxy-2-nitrophenyl ) -2,2-dimethyl-propyloxy] methyl-2'-deoxycytidine (240 mg, 83% for three steps in total).¹H NMR (400 MHz,

264

Μ Ρ I, Μ Τ ·_γυτο MEXICAN

MeOD-d₄): δ 8.33 (s, 1 Η, Ph-H), 7.91 (s, 1 Η,

H, Ph-H), 6.16 (t, 1 H, J = 6.4 Hz, H-1 '), s' μ, Ph.rH)

4.41 (d, 1H, J = 12.4Hz, 5-CH₂a), 4.33 (m, 1 H, H-3 '), 4.26 (d, 1 H, J = 12.4 Hz, 5-CH₂b), 3.97 (s, 3H, OCH₃), 3.88 (m, 1 Η, H4 '), 3.70 (m, 2 Η, H-5'), 2.34 (m, 1 H, H-2'a), 2.17 (m, 1 Η, H2'b ), 0.84 (s, 9H, C (CH₃)₃).

A solution of compound 66 (245 mg, 0.4 mmol), npropargyltrifluoroacetylamide (603 mg, 4.0 mmol), tetrakis (triphenylphosphine) -palladium (0) (92 mg, 0.8 mmol), Cul (30 mg, 0.16 mmol), and Et₃N (240 µΐ_, 1.7 mmol) in anhydrous DMF (5.0 mL) was stirred at a temperature of 50 ° C for 12 hours. The mixture was concentrated in vacuo and purified by chromatography on a silica gel column to yield 5 - {(S} -1 - [5-methoxy-4 (3-trifluoroacetamido-1-propinyl) -2-nitrophenyl] -2 , 2-dimethylpropyloxy} methyl-2'-deoxycytidine 67 (230 mg, 91%).¹H NMR (400 MHz, MeOD-d₄): δ 7.98 (s, 1 H, Ph-H), 7.92 (s, 1 Η, H-6), 7.19

<td>(s, 1 H, Ph</td><td>iH),</td><td>6.15 (t, 1H, J =</td><td>6.4 Hz, H</td><td> -1’),</td><td> 5.21</td><td>(s</td><td> , 1</td><td>H, Ph-</td>

<td>CH), 4.41</td><td>(d, 1</td><td>H, J = 13.2 Hz,</td><td>5-CH₂to),</td><td> 4.33</td><td>(m,</td><td> 3</td><td>H</td><td>H-3 'and</td>

<td>CH₂), 4.27</td><td>(d,</td><td>1 H, J = 13.2 Hz,</td><td>5-CH₂b),</td><td> 3.96</td><td>(s,</td><td> 3</td><td>H</td><td>OCH₃),</td>

<td>3.88 (m, 1</td><td colspan="2">Η, H-4 '), 3.72 (m, 2</td><td>Η, H-5 '),</td><td> 2.30</td><td>(m,</td><td> 1</td><td>H</td><td>Η-2'a),</td>

<td>2.12 (m, 1</td><td>Η, H</td><td>-2'b), 0.84 (s, 9 h</td><td>I, C (CH₃)3</td><td> )</td><td></td><td></td><td></td><td></td>

Compound 67 (45 mg, 0.072 mmol) was phosphorylated with POCI₃ (15 pL, 0.16 mmol) and a proton sponge (31 mg, 0.14 mmol) in trimethylphosphate (0.35 mL) at a temperature of 0 ° C for 4 hours under a nitrogen atmosphere. I know

265

IMPI

MEXICAN INSTITUTE PE LA rKORIFJJAD

<img file="MX342195B_D0312.tif" />

added a solution of bis-tri-n-buflTáWttn mg pyrophosphate, 0.5 mmol) and tri-n-butylamine (100 pL) in anhydrous DMF (1.-Q · mL). After 10 minutes of stirring, a triethylammonium bicarbonate solution (TEAB, 0.1 M, pH 7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB / 25% acetonitrile (20 mL), filtered, and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The fractions containing the triphosphate were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 5 - {(S) -1- [4- (3-amino- 1-propynyl) -5-methoxy-2-nitrophenyl] -2,2-dimethyl-propyloxy} methyl-2'-deoxycytidine-5'-triphosphate 68a, which was further purified by reverse phase HPLC on a column PerkinElmer Aquapore OD-300 (7pm, 250 x 4.6mm). Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C₂5H35N₅OR₁₇P3 [MH] ', the calculated mass was 770.1241, and the observed mass was 770.1234.

Compound 67 (118 mg, 0.19 mmol) was thiophosphorylated with PSCI₃ (24 pL, 0.23 mmol) and 2,6-lutidine (80 mg, 0.75 mmol) n triethylphosphate (0.5 mL) at a temperature of 0 ° C for 1 hour

266

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MEXICAN INSTITUTE

PROPERTY &lt;= »under a nitrogen atmosphere. A '' S'ÓTWi i or bis-tri-n-butylammonium pyrophosphate (474 ​​10 mgTT.O rnmul) and lii'n-butylamine (200 pL) were added in anhydrous DMF (2.0 mL). After 2 minutes of stirring, a solution of triethylammonium bicarbonate (TEAB, 1M, pH 7.5; 20 mL) was added. The reaction was stirred at room temperature for one hour and then concentrated in vacuo. The residue dissolved in 75% 0.1 M

TEAB / 25% acetonitrile (20 mL), was filtered, and purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 20 15 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. The thiothriphosphate-containing fractions were combined and lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to produce 5 {(S) -1- [4- (3-amino-1 -propyl) -5-methoxy-2-nitrophenyl] -2,2-dimethylpropyloxy} methyl-2'-deoxycytidine-5'-a-thiothriphosphate 68b, which was further purified by ΗΡίφ of reverse phase on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C₂5H₃₅N5O_ieP3S [MH] ', the calculated mass was 786.1012, and the observed mass was 786.0983.

A solution of Cy5 NHS (5 mg, 6.3 pmol) in anhydrous DMSO (200 pL) was added to a solution of triphosphate 68a (1.59 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 0.35

<img file="MX342195B_D0313.tif" />

267

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MEXICAN INSTITUTE OF PROPERTY mL). The mixture was left at room temperature at rt^or:WburWW for an hour. The triphosphatermafocKlo was first purified with ink by anion exchange HPLC on a column

Dionex DNApac PA200 (250 x 4 mm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled triphosphate 69a were combined and concentrated to a small volume, and the product was purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 Μ TEAB; B, acetonitrile.

A solution of Cy5 NHS (5 mg, 6.3 pmol) in anhydrous DMSO (200 pL) was added to a solution of thiothophosphate 68b (2.96 pmol) in NaHCO buffer.₃/ Na₂CO₃ (0.1 M, pH 9.2, 0.53 mL). The mixture was left at room temperature in the dark for one hour. The ink-labeled thiothriphosphate was first purified by anion exchange chromatography using a Q Sepharose FF column (2.5 x 10 cm). Mobile phase: A, 75% 0.1 M TEAB / 25% acetonitrile; B, 75% 1.5 M TEAB / 25% acetonitrile. Fractions containing the ink-labeled thiothriphosphate 69b were combined and lyophilized to dryness, and the product was further purified by reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 MTEAB; B, acetonitrile.

All the methods described and claimed in the

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Undue experimentation and improper experimentation may be developed in light of the present invention. - Although the compositions and methods of the present invention have been described in terms of certain embodiments, those skilled in the art will appreciate that variations can be applied to the methods and steps or to the step sequences of the method described herein without departing from the concept. , essence and scope of the invention. More specifically, it will be appreciated that certain agents that are both chemically and physiologically related can be substituted by the agents described herein, while being able to achieve the same or similar results. All such substituents and similar modifications that may be appreciated by those skilled in the art, will be considered to be within the spirit, essence and concept of the present invention as defined in the appended claims.

Example 9 - UV Photo-decomposition Studies It was discovered that the range of UV photo-decomposition depends on a number of experimental factors including light intensity. See the Publications of McCray et al (1980) and McGall et al. (1997), which are incorporated into the present invention by reference. To compare the photochemical decomposition ranges between the nucleotide analogues described here, a protocol is developed to provide a daily light intensity output of 0.70 ± 0.01 W / cm² to

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INSTITUTO MEXICANO samples, see below. A checker^{LA |}éíX? T ^ s (as used in these studies - I have heard, previously in the Publication of Wu et al. (2007), which is incorporated into the present invention by reference, and the protocol implemented is described below .

UV Deprotector Configuration: The power supply was turned on for approximately 30 minutes before the lamp and recirculating bath as described by the manufacturer. The IR liquid filter was cooled to a temperature of 9 ° C. Light intensity was determined using a model PM100 energy meter (Thorlabs), a 1000 pm perforation (Edmund Optics), a modified 0.5 mL Eppendorf tube cut in half, and a 3-axis manual translation cap (Newport). , see figure 5. The half-cut Eppendorf tube was placed at the front of the bore and the energy meter detector above to encompass the distortion of the geometric shape of the light as it passes through the reaction solution. Subsequently, the translation stage was used to align the tube / perforation / detector device with the highest intensity from the arc ray.

Intensity adjustment at 0.7W / cm²: To stabilize its output, the lamp was left on for one hour before the intensity measurements. Subsequently, the measured energy was adjusted by increasing the current of the energy supply.

270

In order to achieve the adjusted mean energy (P) equation:

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ZK LA MOFfíUAE »indujtríal ₉__ intensity (/) of 0.70 W / cm, se at ~ 5.5 mW, according to TT / rxr² where r is the radius of the perforation. Energy readings were recorded over a five minute period (at one second intervals) and converted into intensity readings, which fluctuated over a six week period. Between 0.68 ± 0.01 and 0.72 ± 0.02 W / cm².

Beam Alignment with the 0.5 mL Tube Clamp: The modified Eppendorf tube, perforation, and energy meter were subsequently removed from the UV deprotector, and the rotating specimen clamp with a height of 67.18 ± 0.25 mm was installed. The beam was then focused by placing a 0.5 mL Eppendorf tube in a specimen holder, the tube of which was modified with an internal alignment card to provide reference lines for volume heights of 10 pL and 20 pL, see Figure 5. Reference lines allowed the beam to focus on a given reaction volume. The beam alignment was further verified by looking at the image of the lamp's mercury arc produced by the rear reflector. A second alignment card was placed in the rotating specimen holder to view the image, which when properly aligned using the reflector can produce an arc image

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inverted in the opening itself. This step ensured that the arc points were not superimposed, which could cause overheating, while maintaining a power output of ~ 5.5mW. The rotation specimen clamp speed was adjusted in a range of 1,200 to 1,350 rpm using a Nova-Strobe DA Plus (Monarch Instrument) stroboscope by adjusting the motor torque with an adjusting screw.

Photochemical decomposition assays: Nucleotide analogs were incorporated using 10 pL reactions, as described for PEP assays, at a final concentration of 100 nM. See the Publication of Litosh et al. (2011), which is incorporated into the present invention by reference. The o I ig op I anti 11 a-2, oligo-template-5, and I I igop I anti 11 a-4, each hybridized with primer-1 labeled with BODIPY-FL, were used for the C analogues.⁷-HOMedA, C⁷-HOMedG, and HOMedU, respectively. BODIPY-FL-labeled primer-3 hybridized oligoplant template-8 was used to assay the HOMedC analogs. The incorporated reactions were quenched with either a 1 mM sodium azide solution; Sodium azide; 50mM DTT solution; or CG reagents (see key in Figure 4), exposed to 365 nm ultraviolet (UV) light for various time points using a UV deprotector, and then placed on ice. Ten pL of the stop solution were added (98% deionized formamide; 10mM

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2,000,000), and samples were analyzed using ΟΊΤ ”” Model 377 AB DNA sequencer. Triplicate decomposition assays were performed to calculate the DT value.₅₀ ± average ISD, as described in the Publication of Litosh et al. (2011), which is incorporated into the present invention by reference.

REFERENCES

The following references, which are described in the Appendix, to the extent that they provide exemplary procedures or other supplemental details for those set forth herein, are specifically incorporated by reference in the present invention.

North American Patent No. 3,822,996

US Patent No. 3,859,045

US Patent No. 3,917,499

US Patent No. 4,737,155

US Patent No. 4,304,568

US Patent No. 4,631,066

US Patent No. 4,737,155

US Patent No. 4,439,356

US Patent No. 5,151,507

US Patent No. 5,188,934

US Patent No. 5,302,509

US Patent No. 5,770,367

273

Patent

North American No

6,664,079

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INDUSTRIAL

<img file="MX342195B_D0315.tif" />

North American Patent No. 7,057,026 .. ...

US Patent No. 7,345,159

US Patent No. 7,414,116

US Patent No. 7,476,734

US Patent No. 7,541,444

US Patent No. 7,556,537

US Patent No. 7,592,435

US Patent No. 7,635,578

US Patent No. 7,713,698

North American Patent No. 7,771,973

US Patent No. 7,897,737

US Patent No. 7,964,352

US Patent No. 8,148,503

North American Patent Application Publication No.

2004/0110196

North American Patent Application Publication No.

2006/0160081

North American Patent Application Publication No.

2010/0041041

WO 2012/037394

Aliotta eí a /., Genet Anal. Biomol Eng., 1 2: 185-95, 1996. Axelrod, Methods Cell Biol, 30: 245-70, 1989.

Bailey, Ozonation in organic chemistry Vol. 1, Academic

Press, Inc., New York, 1978.

274

IMPI

INSTITUTE Mf. X ¡CANO Dt LA PROH3DAO

Bailey, Ozonation in organic chemistry Vol.

Press, Inc., New York, 1982. ''

Barltrop et al, Chem. Commun., 822-23, 1966.

Barone et al, Nucleosides, Nucleotides, and Nucleic Acids,

20:1141-45, 2001.

Barth et al, J. Amer. Chem. Soc., 119: 4149-59, 1997.

Beadling et al, J. Mol. Diagn., 13: 504-13, 2012.

Bentley et al, Nature, 456: 53-59, 2008.

Berson &amp; Brown, J. Amer. Chem. Soc., 77: 447-50, 1955.

Bhalla, Toxicology Letters, 73: 91-101, 1994

Bowers et al, Nature Methods, 6: 593-95, 2009.

Cameron and Frechet, J. Am. Chem. Soc., 1 13: 4303-13,

1991.

Canard &amp; Sarfati, Gene, 148: 1-6, 1994.

Carrasco &amp; Vázquez, Med. Res. Rev., 4: 471-512, 1984.

Cleland, Biochemistry, 3: 480-82, 1964.

Corrie et al, J. Chem. Soc., Perkin Trans. 1, 1015-19,

1992.

Corrie, in Dynamics studies in biology, Goeldner and Givens (Eds.), Wiley-VCH Verlag GmbH &amp; Co. KGaA, Weinheim, pp. 128, 2005.

Crabtree and Kemp, Indus. Eng. Chem., 38: 278-296, 1946

De Mayo, in Advances in Organic Chemistry, Raphael et al. (Eds.), Interscience, NY, 2: 367-425,1960.

Dressman et al, Proc. Nati Acad. Sel USA, 100: 8817-

<img file="MX342195B_D0316.tif" />

275

8822, 2003.

IMPI

MEXICAN INSTITUTE THE PROPERTY

INDUSTRIAL

<img file="MX342195B_D0317.tif" />

Eid et al, Science, 323: 133-138, 2009.

EPA (2011) http://www.epa.gov/oar/oaqps/gooduph¡fil¡/bad.html#6. Last update on Tuesday, July 21, 21,2011.

European Patent Application. 87310256.0

European Patent EP 0 640 146

Fairchild et al., Science, 130: 861-862, 1959

Fare et al., Anal. Chem., 75: 4672-4675, 2003.

Futreal et al, Nature Rev. Cancer, 4: 177-83, 2004.

Gardner et al., Nucleic Acids Res., 27: 2545-53, 1999. Gardner et al, Nudeic Acids Res., 40: 7404-15, 2012. Gershon, Nature Methods, 1: 263-270, 2004.

Giegrich et al., Nucleosides Nucleotides, 17: 1987-1996,

1998.

Givens &amp; Schowen, In Chemiluminescence and

Photochemical Reaction Detection in Chromatography, JW Birks, Ed .; Photochemical Reaction Detection in Chromatography. VCH: New York, pp 125-47,

1989.

Gommers-Ampt et a !, FASEB J., 9: 1034-42, 1995.

Gordon and Gregory, Organic Chemistry of Color, SpringerVerlag, New York, 1983.

Gregory, Encyclopedia of Chemical Technology (Kirk, R.

E., and Othm r, DF, eds) pp. 1-66, 2009

276

IMPIM?

MEXICAN INSTITUTE OF PROPERTY

Guo et al., Proc. Nati. Acad. Sel EUA, 1 ^^ 45-5 ^ 2008. Handbook of Pharmaceutical Salts: PTó / üéftlé'é, P 'Use¹(Manual of Pharmaceutical Salts: Properties and Uses), Stahl &amp; Wermuth (Eds.), Verlag Helvética Chimica Acta, 2002.

Hardwick et al, Trans. Farad. Soc., 56: 44-50, 1960.

Harris et al, Science, 320: 106-109, 2008.

Hasan et al, Tetrahedron, 53: 4247-64, 1997.

Hastings, J. Mol Evo !. 19: 309-21, 1983.

Haugland, In The handbook. A guide of fluorescent probes and labeling technologies, Invitrogen, 2005.

Hewitt et al., Nature, 344: 56-58, 1990

Hsia et al, Current Opinion in StructuralBiology, 15: 126134, 2005.

lafrate et al, Nature Genet, 36: 949-51, 2004.

Ito et al, Nature, 466: 1 129-33, 2010.

Ju etal, Proc. Nati Acad. Sci. USA, 103: 19635-40, 2006. Kaiser et al, J. Biol Chem., 274: 21387-394, 1999. Kriaucionis &amp; Heintz, Science, 324: 929-30, 2009.

Lehninger, In: Principles of Biochemistry

Biochemistry), 273-305, WH Freeman and Co., NY, 2005.

Levy et al, PLoS Biol, 5: e254, 2007.

Limbach, et al., Nucleic Acids Res., 22: 2183-2196, 1994.

Litosh et al, Nucleic Acid Res., 39: e39.

Lyamichev et al, Nature Biotechnol, 17: 292-296, 1999.

277

IMPI

KOTTUTO MEXICANO OI LA WOFJfDAD

C hemist _rand'^wus™ ^ e aSrotTs?

March's Advanced Organic Mechanisms, and Structure (March Chemistry O rg áirl da A'V a I l'ZaOT '' *: Reactions, Mechanisms and Structure), 2007.

Margulies et al, Nature, 437: 376-80, 2005.

McCray et al, Proc. Nati Acad. Sel. USA, 77: 7237-41,

1980.

McDougall et al, Nucleosides, Nucleotides &amp; Nucleic Acids, 20: 501-6, 2001.

McGall et al, J. Amer. Chem. Soc., 7/9: 5081-90, 1997.

Meldrum et al. Clin. Biochem. Rev., 32: 177-95, 2011. Metzkereta et al, Biotechniques, 25: 446-462, 1998.

Metzker et al., Nucleic Acids Res., 22: 4259-67, 1994. Metzker, Cenóme Res., 15: 1767-76, 2005.

Metzker, Nature Rev. Genet., 11: 31-46, 2010.

Mitra et al, Anal Biochem., 320: 55-65, 2003.

Morrison, In: The chemistry of nitro and nitroso groups, Feuer (Ed.), Interscience publishers, NY, 165-213, 1969.

Mosher et al, J. Chem. Phys. 32: 1888-89, 1960.

Moyeret al., Laser Focus World, 29 (10), 1993.

Nieman, In Chemiluminescence and Photochemical

Reaction Detection in Chromatography, JW Bírks, Ed .; VCH: New York, pp 99-123, 1989.

Orosz et al, Crit. Rev. Anal Chem., 26: 1-27. nineteen ninety six.

278

IMPI

MEXICAN INSTITUTE OF PROPERTY

INDUSTRIAL

<img file="MX342195B_D0318.tif" />

Patchornik et al., J. Am. Chem. Soc., 92: 6333-35, 1970. Patchornik, En: Pharmacology of hormonal polypeptides and proteins (Back et al. (Eds.),

Plenum Press, NY, 11-16, 1968. Application

PCT / US90 / 05565.

Ramanathan et al, Anal Biochem., 330: 227-241, 2004.

Redon et al, Nature, 444: 444-454, 2006.

Reichmanis et al., J. Polymer Sel, 23: 1-8, 1985.

Rockhill et al, J. Am. Chem. Soc., 1 18: 10065-68, 1997. Ronaghietal., Science, 281: 363-365, 1998.

Salvin and Walker, Textile Research Journal, 25: 571 -581,

955

Sebat et al., Science, 305: 525-528, 2004.

Seela and Peng, In: Current Protocols in Nucleic Acid

Chemistry (Current Protocols in Nucleic Acid Chemistry), Beaucage et al. (Eds.), John Wiley &amp; Sons, Inc., 1.10.1, 2005.

Seo et al, Proc. Nati Acad. Sci USA, 102: 5926-5931,

2005.

Service, Science, 282: 1020-1021, 1998.

Smith and Ho, J. Org. Chem., 55: 2543, 1990.

Soengas et al, J. Mol Biol, 253: 517-29, 1995 Sousa &amp; Weinstein, J. Org. Chem., 27: 3155-59, 1962. Staudiner &amp; Meyer, Helv. Chim. Acta, 2: 635, 1919.

Stranger et al, Science, 315: 848-853, 2007.

279

IMPI

MEXICAN INSTITUTE

Stupi et al., Angew. Chem. Int. Ed., 51: 1724-1 2 ^^ = ^

Su et al., J. Mol Diagn., 13: 74-84, 201 1. ------------------------------------- -,,

Tahiliani et al, Science, 324: 930-35, 2009.

Turcatti et al, Nucleic Acids Res., 36: e25, 2008.

Tuzun et al, Nature Genet., 37: 727-32, 2005.

Walker et al., Biochemistry, 25: 1799-1805, 1986.

Weinstein et al, J. Org. Chem., 31: 1983-1985, 1966.

Wettermark, Nature, 194: 677, 1962.

Wootton &amp; Trentham, In: Photochemical probes in biochemistry (NATO Science Series C), Nielsen (Ed)., Kluwer Academic Publishers,

Ann Arbor, 272: 277-296, 1989.

Wu et al, Nature Genet., 44: 251-253, 2012.

Wu et al, Nucleic Acids Res., 35: 6339-6349, 2007. Wyatt &amp; Cohén, Biochem. J., 55: 774-782, 1953.

280

IMPI

MEXICAN INSTITUTE OF THE INDUSTRIAL PKONEDAP

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