Method for detecting the presence of an oligonucleotide sequence of interest

DESCRIPTIVE MEMORY

Description ►

Technical field

The invention relates generally to the incorporation of non-basic and / or selectively cleavable sites into oligonucleotide chains and more particularly to novel reagents useful for these purposes. The invention also relates to methods of using the new reagents in biochemical assays and phosphorylation reactions.

I | Background of the Invention

Incorporation of selectively cleavable sites into oligonucleotide and polynucleotide chains has been described in U.S. Patent Application Serial No. 251,152 and elsewhere, U.S. Patent 4,775,619, the disclosures of which are incorporated herein by reference, Selective Cleavage Sites. They are useful in a number of types of hybridization assay formats. For example, in a type of

<img file="PT98488B_D0001.tif" />

Assay in which hybridization gives rise to a solid support duplex of a labeled probe and a DNA sample, a selective cleavage site contained in the hybridized structure, will allow ready separation of the marker from the solid support. U.S. Patent No. 4,775,619 is primarily directed to the use of cleavage sites for a restriction endonuclease in such assays. Chemically cleavable sites, e.g. disulfide bonds, 1,2-diols and the like may still be used and may be introduced during oligonucleotide synthesis, the cleavage being performed by particular chimeric reagents, eg with thiols, periodates or the like,

The present invention is also directed to selectively cleavable sites. However, the present method involves the introduction of sites that are cleavable by photolysis, as well as sites cleavable by other processes, eg using chemical or enzymatic reagents, eg reducing agents. The cleavable sites of the invention are created by incorporating chimeric radicals, preferably photolabile radicals, into oligonucleotide or polynucleotide chains. The novel photolabile radicals are useful in a number of different types of hybridization assay formats, including those described in the above Pedidods, as well as in nucleic acid amplification of the hybridization assay described by the applicants of EPO Publication No. 88,30967.6.

Another use for the reagents of the invention is generally to create non-basic sites within the oligonucleotides. By non-basic site is meant an ether radical -OR, at a position which normally contains a hydroxyl group, -OH or a nucleobase. The usefulness of this derivation is extensive as will be revealed in more detail below.

Still another use of the reagents of this invention is in chemical phosphorylation. In many different aspects of oligonucleotide chemistry, chemical phosphorylation of hydroxyl groups is required. For example, in oligonucleotide synthesis, after synthesis and deprotection, the free 5'-hydroxyl group of the oligonucleotide must be phosphorylated for use in more biological processes. Phosphorylation of the 3'-hydroxyl function is also required: (1) to prevent extension of the 3 'terminal by a polymerase? and (2) in chemical DNA binding, ie, a 3 'phosphate radical is typically required in the coupling of oligonucleotides using chemical means.

5 'end phosphorylation has conventionally been performed with T4 polynucleotide kinase and ATP, a reaction that is not particularly reliable or efficient. Various methods for 5 'chemical phosphorylation are also known, including those described by Nadeaux et al., Biochemistry 23: 6153-6159 (1984), Van der Marel et al., Tetrahedron Lett.22; 1463-1466 (1981), Himmelsbach and Pfleiderer, Tetrahedron Letters

23; 4793-4796 (1982), Marugg et al., Nucleic Acids Research

12: 8639-8651 (1984), and Kondo et al., Nucleic Acids Research Symposium Series 16: 161-164 (1985). 16: 161-164 (1985). However, most of these methods involve the use of unstable reagents or require extensive standard deprotection modifications and purification procedures. Similar problems have been encountered with monofunctional and bifunctional 3 'phosphorylation reagents (see Sonveaux, supra, 297).

Thus, in addition to the utility in providing cleavable and / or non-basic sites on oligonucleotide or polynucleotide chains, many of the compounds of the present invention are additionally useful as phosphorylation reagents that overcome the limitations of standard phosphorylation processes (and may also be useful in phosphorylation reactions which are used in conventional dimethoxytrityl purification schemes (DMT)). Source references generally relating to methods for the synthesis of oligonucleotides include those related to 5 'to 3' synthesis, based on the use of beta-cyanoethylphosphate protecting groups, eg, from Napoli et al., Gazz Chim Ital 114: 65 (1984), Rosenthal et al., Tetrahedron Letters 24: 1691 (1983), Belagaje and Brush, Nucleic Acids Research 10: 6295 (1977), in references describing the 5 'to 3' synthesis solution phase include Hayatsu and Khorana, J American Chemical Society 89: 3880 (1957), Gait and Sheppard, Nucleic Acids Research 4: 1135 (1977), Cramer and Koster, Angew. Chem. Int. Ed. Engl. 7: 473 (1968), and Blackburn et al., Journal of the Chemical Society, Part C, 2438 (1967).

In addition to the aforementioned technique, Matteucci and Caruthers, J. American Chemical Society 103: 3185-3191 (1981), describe the use of phosphochlorochlorites in the preparation of oligonucleotides. Beaucage and Caruthers, Tetrahedron Letters 22: 1859-1862 (1981), and U.S. Patent No. 4,415,732 describes the use of phosphoramidites in the preparation of oligonucleotides. Smith, ABL 15-24 (December 1983), describes the solid.

automatic synthesis of phase oligodeoxyribonucleotides See also references cited therein and in Warner et al., DNA 3; 401-411 (1984), the disclosure of which is incorporated herein by reference.

Horn and Urdea, DNA 5.5: 421-425 (1986), describe the phosphorylation of solid support DNA fragments using bis (cyanoethoxy) -N, N-diisopropyl aminophosphine, See also Horn and Urdea, Tetrahedron Letters 27: 4705-4708 (1986).

<img file="PT98488B_D0002.tif" />

References relating to hybridization techniques generally include the following: Meinkoth and Wahl, Anal. Biochemistry 138: 267-284 (1984), provide an excellent review of hybridization techniques. Leary et al., Proc. Natl. Acad. Know. (USA) 80: 4045-4049 (1983), describes the use of bionylated DNA in conjunction with an avidin enzyme conjugate for the detection of specific oligonucleotide sequences. Ranki et al., Gene 21: 77-85, describe what they refer to as sandwich hybridization for the detection of oligonucleotide sequences. Pfeuffer and Helmrich, J. Biol. Chem. 250: 867-876 (1975), describes the coupling of guanosine-5'-O- (3-thiotriphosphate) to sepharose 4B. Bauman et al., J. Histochem. and Cytochem. 29: 227-237, describes the labeling of the 3 'end of RNA with fluorescent substances. PCT Application WO / 8302277 describes the addition to tagged modified ribonucleotide DNA fragments and methods for analyzing such DNA fragments. Renz and Kurt, Nucl. Acids Res. 12: 34353444, describes covalent binding of enzymes to oligonucleotides. Wallace, DNA Recombinant Technology (Woo, S., ed.) CRC Press, Boca Raton, Florida, provides general information.

<img file="PT98488B_D0003.tif" />

<img file="PT98488B_D0004.tif" />

<img file="PT98488B_D0005.tif" />

about the use of probes in the diagnosis. Chou and Merigan, N. Eng. J. of Med. 308; 921-925, describes the use of a radioisotope-labeled probe for CMV detection. Inman, Methods in Enzymol. 34B, 24: 77-102 (1974), describes procedures for binding to polyacrylamides, while Parikh et al., Methods in Enzymol. 34B, 24: 77-102 (1974), describes coupling reactions with agarose. Alwine et al., Proc. Natl, Acad. Know. (USA) 74; 5350-5354 (1977), describes a method of transferring gel oligonucleotides to a solid support for hybridization. Chu et al., Proc, Natl. Acad. Know. (USA) 11; 6513-6529 describes a technique for terminal nucleotide derivatization. Ho et al., Biochemistry 20; 64-67 (1981), describes the derivatization of terminal nucleotides through phosphates for ester formation. Ashley and McDonald, Anal. Biochem. 140: 95-103 (1984), reports a method for preparing probes from a mold attached to a surface.

Hebert and Gravei, Can. J. Chem. 52: 187-189 (1974), and Rubinstein et al., Tetrahedron Lett. 1445-1448 (1975), discloses the use of 2-nitrophenyl containing compounds as light sensitive protecting groups.

K. Groebke et al., Helvetica Chemica Acta. 73: 608-617 (1990) is relevant to date as the reference describes the use of the t-butyldimethylsilyl radical to protect a hydroxyl function.

<img file="PT98488B_D0006.tif" />

Summary of Revelations

Accordingly, a first object of the invention is to meet the above-mentioned needs of the art and to provide methods and reagents for introducing selective and / or non-basic cleavage sites into oligonucleotide chains.

Another object of the invention is to provide such methods and reagents for introducing selectively cleavable sites into oligonucleotide chains, wherein said sites are chemically cleavable,

Still another object of the invention is to provide such methods and reagents for introducing selective cleavage sites into oligonucleotide chains, wherein said sites are cleavable by light.

Another object of the invention is to provide methods and reagents for introducing non-basic sites into oligonucleotide chains.

Still another object of the invention is to provide methods and reagents for the chemical phosphorylation of hydroxyl groups.

It is yet another object of the invention to provide reagents for incorporating non-basic sites into oligonucleotide chains that can then be used to create a branched nucleic acid multimer.

Another object of the invention is to provide certain reagents wherein non-basic sites are non-nucleotide.

Additional objects, advantages and novel features of the invention will be in part set forth in the following description and, on the other hand, will become apparent to those skilled in the art upon examination of the foregoing, or may be learned by practice of the invention.

Thus, in one aspect, novel reagents are provided, which are photolabile chemical compounds having the general structure:

<img file="PT98488B_D0007.tif" />

2 wherein R, R, x and y are defined below. These compounds may be incorporated into oligonucleotide chains to allow light cleavage.

On the other hand, new reagents are provided having the general structure:

R

<img file="PT98488B_D0008.tif" />

2 wherein where R, R, and R are defined below. These compounds are useful in creating non-basic sites on oligonucleotide chains, which may or may not be cleavable,

In another aspect, new reagents are provided having the general structure:

CHL-OR¹! ^THE

CH₃“C ~ CH₂-OR '' í

CH₂-O ~ R_no

<img file="PT98488B_D0009.tif" />

2 where R, R and R_no are defined below. These compounds are useful for creating branching points in synthesis.

I of nucleic acid multimers.

In other aspects, methods of using these reagents in a wide variety of contexts are further disclosed.

Modes of Carrying Out the Invention

A. Definitions:

By selectively cleavable site is meant a feature or plurality of features that can be selectively cleaved. The focus of the present invention, as noted above in the present text, is primarily sites that are specifically cleavable using photolysis.

The terms oligonucleotide and polynucleotide, as used herein, will be generic to polydeoxyrbonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), polyribonucleotides (containing D-ribose or modified forms), and to any other type. polynucleotide which is an N-glucoside of a purine or pyrimidine base or a modified purine or pyrimidine base. 0 The term nucleoside will similarly be generic to ribonucleosides, deoxyribonucleosides, or any other nucleoside that is an N-glucoside of a purine or pyrimidine base, or modifications of these bases. There is no intention in distinguishing length with respect to the use of the oligo nucleotide and polynucleotide terms and these terms will be used interchangeably. These oligonucleotides and polynucleotides may be single or double stranded, typically single stranded. Also, the oligonucleotides of the present invention are usually from about 2 to about 2000 monomer units and more typically for most probe-based applications, from about 2 to 100 monomer units.

By nucleic acid assay is meant the DNA or RNA in said nucleic acid sample containing the sequence of interest.

By phosphorylation reagents as used herein are meant compounds which, in a reaction or series of reactions with a compound containing a hydroxyl group, will produce monoester phosphates.

By lower alkyl and lower alkoxy are meant alkyl and alkoxy substituents, respectively, having from about 1 to 8, more typically from 1 to 6, carbon atoms.

Where aromatic substituents are indicated, it should be understood that each individual aromatic ring may be substituted on one or more carbon atoms with radicals that do not substantially affect function or reactivity.

B, Structure of New Photolabile Reagents:

In one embodiment of the invention, novel reagents which are photolabile chemical compounds having the following structure are provided:

OR '

<img file="PT98488B_D0010.tif" />

O

<img file="PT98488B_D0011.tif" />

wherein R is a base-stable, acid-sensitive blocking group, R is a phosphorus derivative selected to allow the reagent to be added to the 5 'position of a nucleoside or oligonucleotide chain, and one of two, x or y, is zero, while the other is an integer between 1 and 12 inclusive. Two basic types of structures are within the above generic formula (1) those where x is non-zero and yo zero (sometimes referred to herein as NP1-type reagents); and (2) those where x is zero and y is nonzero (sometimes referred to herein as NP2 type reagents. These two types of structures are, as can be readily deduced from the generic formula given above, quite similar. Both are useful for introducing specific sites into oligonucleotide chains, which, due to the nitrophenyl radical, are readily cleavable by photolysis. However, as will be discussed below in more detail, the two families of chemical reagents are distinguishable to date as they prove useful in slightly different contexts.

Now in greater detail for the various substituents of the new photolabile reagents:

R is, as noted above, an acid-sensitive base-stable blocking group. Such blocking groups are well known to those skilled in the art of ligonucleotide synthesis and include substituted or unsubstituted aryl or alkoxy groups, where the aryl group is, eg, phenyl, naphthyl, furanyl, biphenyl or the like and where the substituents are of 0 to 3, usually from 0 to 2, and include any stable non-interfering neutral or polar electron donor or acceptor group. Examples of such groups are dimethoxytrityl (DMT), monomethoxytrityl (MMT), trityl and pixil. A radical preferably used in these techniques is DMT.

R is a phosphorus derivative which is selected to facilitate condensation of the reagent with the 5'-hydroxyl group of a nucleoside or oligonucleotide chain. These groups include phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, phosphorothioates and the like (see

eg ,, Publication EP No. 0225807 by Urdea et al., Solution Phase Nucleic Acid Sandwich Assay and polynucleotide Probes Useful | Therein, the disclosures of which are incorporated herein by reference).

Particularly preferred groups useful as R are phosphoramidites having the following structure:

N (iPr)₂ /

Wherein Y is selected from the group consisting of methyl and beta-cyanoethyl, and iPr is isopropyl. Preferably,

I y is beta-cyanoethyl.

I

As can readily be deduced from the above definitions, the R and R substituents are generally selected to allow incorporation of the photolabile reagent into a fragment of

DNA using Standard Chemical Protocols for Phosphoramidite.

That is, during oligonucleotide synthesis, the R substituent is selected to react with the 5'-hydroxyl group of a nucleoside or an oligonucleotide chain, while the radical of R 2 is selected to allow reaction with the

<img file="PT98488B_D0012.tif" />

3'-hydroxyl group of a nucleoside or oligonucleotide chain.

As for the indexes x and y, while one is zero, the other is an integer between 1 and 12 inclusive, more preferably between 1 and 4 inclusive, preferably 1,

Examples of reagents belonging to the general category mentioned above are as follows:

<img file="PT98488B_D0013.tif" />

\

NiPri

OCH₂CH₂CN

NPl ”

InPriN

NCCH₂CH₂The p— o—— CHj

<img file="PT98488B_D0014.tif" />

CH-O-DMT

NP2

As indicated, these specific structures are [2- (2-nitrophenyl) -2- (O-dimethoxytrityloxy) ethoxy] -N, N-diisopropylamino-2cyanoethoxyphosphine and [2- (2-nitrophenyl) -1- (Q-dimethoxytrityloxy) ) ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine are designated herein as NP1 and NP2 compounds, respectively, and are the specific reagents synthesized in Examples 1 and 2 below,

C. Synthesis of Reagents Referred Above:

NP1-type reagents, that is, where x is nonzero and y is zero, are synthesized according to the reaction sequence given in scheme 1. NP2-type reagents are synthesized according to the reaction set shown in scheme 2.

SCHEME 1 ho-ch₂

<img file="PT98488B_D0015.tif" />

DMT- Clyridine

<img file="PT98488B_D0016.tif" />

N (iPrb

Cl — for

OCH₂CH₂CN

D1PEA

Ch, Cl₂

<img file="PT98488B_D0017.tif" />

SCHEME 2

HO-CH₂

TBDMS — O — CH₂

<img file="PT98488B_D0018.tif" />

TBDMS-- Cl ~

DMAP / TEA

CH₂C1₂

<img file="PT98488B_D0019.tif" />

DMT— Cl

DMAP / TEA ch₂ci₃

<img file="PT98488B_D0020.tif" />

tbdms it ~ ch₂

CH-O — DMT

<img file="PT98488B_D0021.tif" />

(iPr)₂K

ClN (iPr)₂OCH₂CH₂CN

NCCH1CH1O

DiPEA

CHiCU * * ♦

TBAF

THF

P-0 / I

CH-O-DMT NOj

<img file="PT98488B_D0022.tif" />

<img file="PT98488B_D0023.tif" />

Abbreviations in Schemes 1 and 2: DMT = dimethoxytrityl; DMTCl = dimethoxytrityl chloride; iPr = isopropyl; DiPEA = diisopropylethylamine; TBDMS-C1 = tbutyldimethylsilyl chloride; DMAP = 4-dimethylaminopyridine; TEA = triethylamine; TBAF = tetrabutylammonium fluoride.

Synthesis of NP1-type reagents involves protecting the 2- (O-nitrophenyl) -1,2-ethanediol terminal hydroxyl group by 1 cap with R species, eg, with DMT or the like, followed by reaction of the hydroxyl group Remaining with a phosphorus derivative selected to give the radical R, As shown in Scheme 1, an exemplary reagent for this latter purpose is Chloro-N, N-diisopropylamino-2-cyanoethoxyphosphine. Variations in this basic scheme can be easily deduced. For example, to provide different R substituents, monomethoxytrityl chloride, trityl chloride, pixyl chloride or the like would be used as an alternative to trimethoxytrityl chloride. Similarly, to yield different 2 R substituents, alternative substituted phosphines would be used in the second reaction step. To vary from x additional methylene groups are required in the starting material.

To synthesize NP2 type reagents, ie where x is zero and y is nonzero, a similar synthetic sequence is performed except that the order of

2 introduction of the substituents R and R be reverse. Thus, initially, the terminal hydroxyl group of 2- (O-nitrophenyl) -1,2-ethanedi.ol starting material reacts with t-butyldimethylsilyl chloride (TBDMS-Cl) to block that hydroxyl group during the next reaction step, where the rest

<img file="PT98488B_D0024.tif" />

free hydroxyl group reacts with a base-stable acid-sensitive blocking group, eg, dimethoxytrityl chloride (DMTCl) to provide the substituent R3. The terminal hydroxyl group is then deprotected, eg, with tetrabutylammonium fluoride and, as shown in scheme 1, reacts with a substituted phosphine 2 derivative suitable to give the radical R.

D. Use of the Above Reagents for

Selectively Cleavable Sites:

The novel photolabile reagents of the invention are readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite chemistry, well known to those skilled in the art, and as described, for example, in a number of references cited above herein. In general terms, incorporation of the new reagent 2 into a DNA fragment involves binding to the 5'-hydroxyl group at R and binding to the 3'-hydroxyl group at R '.

Thus, upon incorporation of the photolabile reagent, the hybrid oligonucleotide chain will have the following structures:

5 '- HO^5,CDNA0³~ OP ~ O - (CH2)_x-CH-iCH2) yO - PO-³'[DNAjl^3:-OH

OH

NO OH

<img file="PT98488B_D0025.tif" />

where jDNA represents a first segment of DNA, DNA2 represents a second segment of DNA, exey obey the previous definitions. DNAs, and DNA2 can be linear or branched. This polynucleotide reagent may be used in hybridization assays as described in EPO Applicant Applications No.88.309203.3 and US Patent No. 4,775,619. These assays involve the use of linear polynucleotide reagents having selective cleavage sites, ie, wherein DNA1 and DNA2 are linear. The polynucleotide reagent containing the photolabile radical of the invention may also be used in the amplification assays of US Patent Nos. 07 / 252,638 and 07 / 340,031, both incorporated by reference herein (see also PCT Publications No. WO89 / 03891). As described in these Applications, cleavable binding molecules may be incorporated into amplification multimers at predetermined sites for the purpose of analyzing the multimer structure or as a means for releasing predetermined segments (such as the multimer moieties that bind to the labeled oligonucleotide). In these Applications, DNA and / or DNA2 are branched polynucleotide segments. Subsequent to the synthesis and purification of the multimers, the branched polynucleotide structure of the multimer can be specifically cleaved without further degradation of the nucleotide structure. It is clearly preferable that cleavable sites be introduced at or near the multimer junctions to allow quantification of the individual multimer branches.

Depending on whether the photolabile reagent incorporated into the oligonucleotide or polynucleotide is of type NP1 (ie where y is zero and x is nonzero) or of type NP2 (ie where x is zero and y is nonzero), two results will result. types of different fragments upon cleavage. That is, as if

<img file="PT98488B_D0026.tif" />

As shown in Scheme 3, cleavage of an oligonucleotide containing an NP1-type radical will result in a first fragment having a 5 'terminal phosphate and a second fragment which at its 3' end contains a residue of the 2 nitrosophenyl species. In contrast, as shown in Scheme 4, cleavage of a polynucleotide containing an NP2-type radical will yield a first fragment containing the 2 'nitrosophenyl residual group at its 5' end and a second fragment having a 3'-terminal phosphate.

SCHEME 3

<img file="PT98488B_D0027.tif" />

Photolysis (UV light&gt; 350nm; Hg lamp)

<img file="PT98488B_D0028.tif" />

AT THE

<img file="PT98488B_D0029.tif" />

SCHEME 4 θ Η

5 '—- HO - ^' [DNAJ4 -OP-O-CH— (CHijy-OP-O- ^ (DNAJ³—OH

OH

<img file="PT98488B_D0030.tif" />

Photolysis (UV light&gt; 350nm; Hg lamp)

O

II

5'— HO —- [DNA,]³-OP — OH +

OH

<img file="PT98488B_D0031.tif" />

O _P „O_5 '[DNAJ³—OH

I

OH

Since cleavage is performed via photolysis using uv light having a wavelength of at least about 350 nm, no chemical or enzymatic reagents are required. This provides a cleaner procedure resulting in a product that is necessarily free of contamination with external cleavage reagents. In addition, the polynucleotide reagent itself is inherently more stable, self-cleaving by UV light treatment of a suitable wavelength.

i

IE · Phosphorylation Using Above Reagents;

The reagents described above, in addition to their utility in providing photolabile cleavable sites, are also useful as phosphorylation chemical reagents. Phosphorylation using these reagents involves Condensation with a compound containing a hydroxyl group, followed by photochemical cleavage and release of the nitrophenyl group. The novel reagents are quite versatile in this respect as they can be used for either 3 'or 5' phosphorylation of a nucleoside or an oligonucleotide chain.

For 5 'phosphorylation, a reagent of type NP1 is required, ie a reagent where x is nonzero and y is zero. As illustrated in Scheme 3 above, cleavage of a polynucleotide reagent containing NP1-like molecules results in a nucleoside or DNA fragment containing a 5 'phosphate group.

For 3 'phosphorylation, an NP2-type reagent as shown in Scheme 4 is required. Cleavage of a polynucleotide reagent containing NP2-type molecules gives rise to cleavage fragments wherein one of the fragments contains a 3'-phosphate group. and the remaining fragment contains the nitrosophenyl residue,

F. Incorporation of Non-Basic Sites and Sites for Secondary Oligonucleotide Chain Synthesis:

In another embodiment of the invention, reagents are provided which are useful for introducing non-basic sites into oligonucleotide chains whose sites may or may not be cleavable. These reagents have the structure:

<img file="PT98488B_D0032.tif" />

2 R and R are described in Part A of this section, above, and where R is selected from the group consisting of 2-nitrobenzyl, 4-penten-1-yl,

<img file="PT98488B_D0033.tif" />

—P — 0 '

I

0 '—ch₂ch₂ wherein R 'is hydrogen, aryl or aralkyl, and if aryl or aralkyl, preferably C 1 -C 6 aryl or alkyl, R R may be the same or different and are selected from the group consisting of amino, nitro, halogens, hydroxyl, low alkyl and low alkoxy, R1 may be the same or different and are selected from the group consisting of amino, nitro, halogens, hydroxyl, low alkyl and low alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4. R_norepresents the levulinyl - (CO) CH2CH2 (CO) CH3 group or any other protecting or blocking group which may be removed and substituted

<img file="PT98488B_D0034.tif" />

<img file="PT98488B_D0035.tif" />

S-CH₉CH ~ -0-C-, Xyl ^{2 2} s-ch₂ch₂-o-cCH₃Q-CH₂-gh₂-o-ch₂-, and Rm is either alkylene of 1 to 16 carbon atoms, preferably 2 to 12 carbon atoms, or an oxyethylene oligomer - (CH2CH2O)_z- where Z is an integer between 1 and 16, typically between 2 and 12, inclusive. Under optimal conditions, when R is -Rjfl-O-Rn, R_no is levulinyl and R_m ê - (CH 2 CH 2 Q 4 -) These deoxyribose-based reagents not only introduce non-basic sites into oligonucleotides or polynucleotide strands, but are also, like the reagents described below, useful for providing cleavable sites.

where R is:

<img file="PT98488B_D0036.tif" />

It is preferable that R 'is hydrogen or phenyl. R1 and R1, as indicated, may represent any of a number of different substituents. In a particularly preferred embodiment, the above-mentioned structure is 2-methylene-9,10-anthraquinone carbonate ester, ie, R 1 and R 3 are hydrogen as well as R '.

The reagents of the formula:

<img file="PT98488B_D0037.tif" />

may be readily synthesized from deoxyribose and the radical alcohol derivative R, ie, R-OH. In the case of 2-nitrobenzyl, for example, deoxyribose would react with 2-nitrobenzyl alcohol to give the derivative 1'-0- (2-nitrobenzyl),

This intermediate can be rapidly converted to analogs.

5'- and 3'- protected using Standard methods, eg, for incorporation of the dimethoxytrityl group (DMT) or an analogous group at the 5'-position (R) and a phosphorus derivative such as phosphoramidite, p-phosphotriester or the like. 3'- (R) position,

These reagents may be readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite chemistry as noted in part D of this section. Upon incorporation of these deoxyribose-based cleavable radicals into the oligonucleotide or polynucleotide chain, the cleavable chain containing the non-basic sites -OR will have the structure:

II

5'— Η ^ ΌΝΑ, Ρ-Ο-ΡOH

<img file="PT98488B_D0038.tif" />

pO -⁵'CDN Ao]³ OH wherein the DNA and pDNA are the first and second DNA segments as described above. A polynucleotide reagent such as this may be used in a variety of hybridization assays.

Cleavage of oligonucleotides or polynucleotide chains containing these reagents may be conducted as follows. When R is a 2-nitrobenzyl, cleavage may be performed by photolysis using UV light having a wavelength of at least 350 nm, followed by basic hydrolysis with eg ammonium hydroxide or the like. When R is CH 2 CH 2 S-0 (where 0 represents a phenyl group), the cleavage is effected by oxidizing the sulfur atom to -SO or -SQ 2 with,

eg, sodium periodate, followed by base treatment. When R is -CH 2 CH 2 Si (CH 3) 3, the oligonucleotide may be cleaved by treatment with, for example, fluoride ion, again followed by treatment with a base. When R is:

<img file="PT98488B_D0039.tif" />

for example, acetal cleavage of 2-methylene-9-10 anthraquinone may be performed by oxidation with Na 2 S 2 O 4, followed by treatment with a base. Where R is:

—-CH1CH1 —NOt cleavage can be performed using DBU (1,8diazabicyclo [5 · 4 * 0 'undec-7-eno]. When R is a phosphate, removal can be done with alkaline phosphatase followed by treatment with when R is a 4-penten-1-yl, cleavage will typically be performed using Nbromosuccinimide, followed by treatment with a base.

As noted above, reagents of the present invention that allow cleavage of an oligonucleotide or polynucleotide chain can be used in amplification assays described in Applicant EPO Application No. 88.309697.6,

<img file="PT98488B_D0040.tif" />

referenced earlier in this text. With the deoxyribose-based reagents described in this section, branching points of the nucleic acid multimer can be created using multifunctional nucleic acid monomers having the structure:

R⁵

Z

<img file="PT98488B_D0041.tif" />

on what:

R is a base stable acid sensitive blocking group;

R is a phosphorus derivative that allows the addition of nucleic acid to the 5 'position of an oligonucleotide chain during chemical synthesis;

R is selected from the group consisting of hydrogen, methyl, I, Br, and F;

R is hydrogen or methyl;

R is selected from the group consisting of levulinyl,

<img file="PT98488B_D0042.tif" />

7

<img file="PT98488B_D0043.tif" />

<img file="PT98488B_D0044.tif" />

R1 wherein R1, R1 and R1 are as defined above, and wherein k is O, 1, 2, 3, or 4, and R1 may be the same or different and are selected from the group consisting of amino, nitro, halogen, hydroxyl low alkyl and low alkoxy; ez is selected from the group consisting of;

(2) II (l) —CH₂) _x—- NH — C —0-;

p.) II 0) - (CH3) _x- ~ -NH —C - ÍCH.J _y-0 (2) - NH - C - (CH₂)_y - S —3-tCH ^ y 0 (D (2) (2) • (CH.J _x—

<img file="PT98488B_D0045.tif" />

&lt;,(% _y -O (D

and.

(2) (1) —— (CHj) x —Ό κ

are ch₂-or^j where x and y can be the same or different and integers between 1 and 8.

These nucleic acid monomers can then be incorporated into an oligonucleotide or polynucleotide chain as described above, with the cleavable or removable radical R, defining the site where secondary oligonucleotide chains will be synthesized.

Branching points of nucleic acid multimers can also be created using multifunctional, non-nucleotide compounds having the general structure:

1, 2 ch₃-c-ch₂-or '

CH₂-OR_no

2 where R, R and R_no are as defined above in this text, 1 2

In a particularly preferred embodiment, R is DMT, R is beta-cyanoethyl phosphoramidite, and R is_no is levulinyl. These compounds may be synthesized from trishydroxymethyl ethane by: (1) protecting one of the hydroxyl groups by reaction with, eg, triphenylchlorosilane or tosyl chloride; (2) reacting the

compound protected with a salt of R^z', eg, dimethoxytrityl chloride, such that one of the two free hydroxyl groups 2 is converted to -OR; (3) reacting the compound provided with R_no-OH or a salt of R_noeg, levulinic acid or a salt of this acid, thereby removing the protecting group from step (1); and (4) reacting the intermediate compound:

CH - OH

I ch₃-c-ch₂-or²

CH₂~ O ~ R_no

X with a reagent effective in converting the remaining free hydroxyl group to -OR, eg, beta-cyanoethoxy-N, Ndiisopropylaminochlorophosphine.

These non-basic sites are extremely useful for either cleavage of an oligonucleotide chain at a particular point or for other purposes, eg, synthesis of a branched nucleic acid multimer,

G. Selective Cleavage Additional Binding Radicals:

Yet another reagent useful for providing a selectively cleavable site within an oligonucleotide chain is represented by the structure;

<img file="PT98488B_D0046.tif" />

N-iPri

0,

R⁶ wherein DMT represents dimethoxytrityl, Bz represents a benzyl group, iPR represents an isopropyl and R0 is a methyl or beta-cyanoethyl group. Like the reagents described above, this radical can be readily incorporated into an oligonucleotide chain using conventional methods. Cleavage at the site containing this radical is accomplished with a two step chemical procedure; (1) oxidation with aqueous sodium periodate for 1 hour followed by (2) treatment with aqueous n-propylamine.

It is to be understood that while the invention has been described in conjunction with preferred specific embodiments thereof, the foregoing description as well as the following examples are intended to illustrate and not to limit the scope of the invention.

Synthesis of [2- (2-nitrophenyl) -2- (0-dimethoxytrityloxy) ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine (NP1); 2- (Nitro-phenyl) -1,2-ethanediol (2.5 g, 13.6 mmol) was dried once by coevaporation with pyridine. The residue was dissolved in pyridine (50 mL) and 13.6 mmol of 4,4'-dimethoxytrityl chloride (DMT-C1) was added. The reaction mixture was stirred for 18 h at 20 ° C. Most of the pyridine was then distilled off and the residual oil dissolved in 250 ml of ethyl acetate. The organic phase was washed with 5% NaHCO 3 (2 x 250 mL), 80% saturated aqueous NaCl solution (1 x 250 mL), and dried over solid Na 2 SO 4. After filtration, the solvent was removed in vacuo and the residue coevaporated with toluene (1 x 200 mL) and CH 3 CN (1 x 200 mL).

I ml). The product was purified on a silica gel column (eluted) with CH 2 Cl 2 - 0.5% triethylamine) to give 6.5 g (13.6 mmol) of pure product (100% yield).

10-DMT-2- (O-nitrophenyl) -1,2-ethanediol purified product was converted to beta-cyanoethyl phosphoramidite by reaction in CH 2 Cl 2 (50 ml) with chloro-N, N-diispropylamino-2cyanoethoxyphosphine (15 mmol) in the presence of of diisopropylethylamine (30 mmol) at 10 ° C for 30 min. Ethyl acetate (200 ml) was then added and the combined organic phase washed with solution.

<img file="PT98488B_D0047.tif" />

of 80% saturated aqueous NaCl (2 x 250 mL) and dried over solid Na 2 SO 4. After removal of the solvent in vacuo, the residue was coevaporated with toluene (100 mL) and CH 3 CN (100 mL) to give 9.5 g of Q-dimethoxytrityl-2- (O-nitrophenyl) -1-2-O-phosphoramidite, 2ethanediol (100% production)

Example 2

The synthesis of [2- (2-nitrophenyl) -1- (O-dimethoxytrityloxy) ethoxy] -N, N-diisopropylamino-2 cyano ethoxy phosphine (NP2); 2- (Nitrophenyl) 1,2-ethanediol (2.5 g, 13.6 mmol) was dried by coevaporation with CH 3 CN. The dried compound was then dissolved in | CH 2 Cl 2 (100 mL) -CH 3 Cl (10 mL). N, N-dimethylaminopyridine (100 mg) and triethylamine (3.6 ml, 26 mmol) were added, and with stirring, t-butyldimethylsilyl chloride (TBDMS-Cl) (2.6 g, 15 mmol) was added. Stirring was continued for 18 hours at 20 ° C. Then more TBDMS-Cl (200 mg) was added. After one hour the reaction mixture was diluted with 400 ml of ethyl acetate. The organic phase was washed with 5% NaHCOg (2 x 250 mL) and 80% saturated aqueous NaCl solution (1 x 250 mL) and dried over

Solid Na 2 SO 4. After removal of solvents in vacuo, the residue was coevaporated with toluene (200 mL) and CH 3 CN (200 mL) to give 2.5 g of 10-TBDMS-2- (O-nitrophenyl) -1,2-ethanediol. gross. The crude material was coevaporated with pyridine, and the residue was dissolved in pyridine (50 mL). DMT-C1 (30 mmol) was added and the reaction mixture stirred at 20 ° C for 48 hours. After removal of the solvent in vacuo, the residue was dissolved in ethyl acetate (250 mL). The organic phase was washed with 5% NaHCO 3 (2 x 250 mL) and 80% saturated aqueous NaCl solution (1 x 250 mL) and dried over solid Na 2 SO 4. After removal of the solvent in vacuo, the residue was

<img file="PT98488B_D0048.tif" />

coevaporated with toluene and CH 3 CN. The residue was dissolved in. THF (100 ml) and 10 ml of 1M solution of tetrabutylammonium fluoride in THF were also added. Removal of 1-0TBDMS group was complete within 30 minutes. The product was purified on a silica gel column to give pure 2-Q-DMT-2- (Q-nitrophenyl) -1,2-ethanediol (2.4 g, 4.5 mmol). This material was converted to 2-cyanoethylphosphoramidite as described above for quantitative production.

I Example 3 | A 5'-T15-3 test fragment⁷-p-NP1-p-5'-T20? 3'-OH (p = phosphate) was achieved using standard synthetic procedures for phosphoramidite. After complete deprotection, the purified DNA oligomer dissolved in water was photolysed for 15 minutes (Hg lamp,&gt; 350 nm). PAGE analyzes of the photolysis sample revealed that the treatment had resulted in complete cleavage of the test fragment into new fragments that migrated, as expected, to the segments.^T20 ^{and T}15·

Example 4

Synthesis of 5'-DTM-1'-O- (2-nitrobenzyl) -2-deoxyribose 3'-Q-methylphosphoramidite:

Deoxyribose (10 mmol), 2-nitrobenzyl alcohol (30 mmol) and dichloroacetic acid (DCA; 100 mL) in 100 mL of dry acetonitrile were heated at slow reflux for two hours. After cooling to 20 ° C pyridine was added to neutralize DCA and the solvent removed in vacuo. The residue was dissolved in 500 mL of ethyl acetate and the organic phase washed with 400 mL of 5% NaHCO 3, 400 mL of 80% saturated aqueous NaCl solution and dried over solid Na 2 SO 4. After filtration, the solvent was removed in vacuo and the residue coevaporated with toluene and acetonitrile. The crude reaction mixture was dissolved in CH 2 Cl 2 and the product was isolated by silica gel chromatography using a 0-6% methanol gradient. Fractions containing the product (alpha- and beta-isomers mixture, 1: 1 ratio) were mixed and the solvent removed in vacuo to give 2.5 g of a slightly yellow solid (5.2 mmol; 52% yield) .

The deoxyribose-O-nitrobenzyl residue was dissolved in 25 mL of CH 2 Cl 2 containing 200 mg of dimethylamino pyridine (DMAP) and 1.4 mL of triethylamine. To this solution was added dropwise DMT-C1 (1.7 g; 5 mmol) dissolved in 25 mL of CH 2 Cl 2. When all the starting material was consumed, the reaction mixture was diluted with 250 mL of ethyl acetate and extracted, dried and coevaporated as described above. The crude reaction mixture was subjected to silica gel chromatography and the 5'-DMT-1'-0-2-nitrobenzyl-2'-deoxyribose isomers were eluted with a 0-3% methanol gradient to give 2.3 g. of yellow foam (2.65 mmol).

3-methylphosphoramidite was prepared using standard procedures. 5'-DMT-1'-O- (2-nitrobenzyl) -2'deoxyribose was dissolved in 40 ml CH 2 Cl 2 containing 2.8 ml DiPEA and N, N-diisopropylaminomethylchlorophosphine (2.0 mmol) was added at 0 ° C. ° C. After 30 minutes the reaction mixture was diluted with 200 ml of ethyl acetate which¹ It was washed with 3 x 200 ml of 80% saturated aqueous NaCl solution, dried over Na 2 SO 4.

Λ ..... h is solid and filtered. The solvent was removed in vacuo and the residue evaporated with toluene and acetonitrile. This material was then used without further purification.

This non-basic protected phosphoramidite nucleoside was incorporated under Standard conditions into a 3'-T20- [1'-0- (2-nitrobenzyl) -2'-deoxyribose] -Τχθ oligomer on a solid support. The fragment was deprotected with DCA (to remove 5'-DMT), thiophenol (thiophenol / triethylamino / dioxane, 1: 1: 2 v / v for one hour at 20 ° C to remove methyl), and NH4OH ( aqueous ammonium for one hour at 20 ° C to cleave the 3 'succinate bond). The supernatant was heated at 60 ° C for 18 hours. No cleavage was observed demonstrating the base stability of the 5'-DMT-1'-0- (2-nitrobenzyl) -2'-deoxyribose radical. A sample of this material in water was photolysed for 20 minutes using a high density Hg lamp to remove the 0-nitrobenzyl group from the 5'-DMT-1'-O- (2nitrobenzyl) -2'-deoxyribose radical. No cleavage of the oligomer was observed during the photolysis step. A sample of the oligomer that had been photolysed was incubated in NH 4 OH at 60 ° C for 2 hours. Basic treatment resulted in complete cleavage of the oligomer into the two component oligomers.

5'-p-T20 ·

These reactions are presented in the diagrams and 6.

SCHEME 5

<img file="PT98488B_D0049.tif" />

<img file="PT98488B_D0050.tif" />

<img file="PT98488B_D0051.tif" />

SCHEME 6

<img file="PT98488B_D0052.tif" />

Cleavage of

2-NITROBENZIL 2 ALLOCATED DNA FRAGMENTS ¹-DESXYSIDE

Example 5

Preparation of N-4- (O-N, N-Diisopropylaminomethoxy fosfinyl-6-oxyhexyl) -5'-DMT-2 ', 3'-dibenzoyl cytidine:

Uridine (24.5 g, 100 mmol) was dried by coevaporation with pyridine (2 x 150 mL). The residue was dissolved in 150 mL of pyridine and dimethoxytrityl-Cl chloride (34g, 100 mmol) added dropwise with agitation. The reaction mixture was allowed to stir for 48 hours. Methanol (100 ml) was added and after 30 minutes the solvents were removed in vacuo. The residue was dissolved in 800 mL of ethyl acetate and the organic phase was washed with 3 x 800 mL of 5% NaHCQ3, 3 x 800 mL of 80% saturated aqueous NaCl solution, dried over Na 2 SO 4, filtered and evaporated. to dryness, followed by coevapopulation with toluene and acetonitrile. Silica gel chromatography of the crude product using a gradient of 0-7% methanol / 1% triethylamine yielded 46.36 g, 84.9 mmol of (5'-DMT-ribouridine) was dried by pyridine coevaporation and the residue It was dissolved in 250 ml of pyridine. Benzoyl chloride (20 mL, 170 mmol) in 100 mL of methylene chloride was added dropwise to the 0 ° C pyridine solution. After stirring at 20 ° C for 2 hours, the solvent was removed in vacuo and the residue coevaporated with toluene. 0 The residue was dissolved in ethyl acetate and subjected to the same aqueous treatment as described above for 5'-DMT-uridine.

Crude 5'-DMT-23'-dibenzoyl uridine, which was used without further purification, was dissolved in 150 ml of acetonitrile. 1,2,4-Triazole (88.19 g) was suspended in 400 'ml acetonitrile at 0 ° C, and POCl 3 (27.56 ml) was added with rapid stirring. Then triethylamine (106.7 ml) was added

<img file="PT98488B_D0053.tif" />

dropwise over 15 minutes to the semi-liquid mixture stirred at 0 ° C. After 30 minutes, 5'-DMT-23'-dibenzoyl uridine diluted in 150 ml acetonitrile was added dropwise to the above-mentioned semi-liquid stirred mixture at 0 ° C. The ice-water bath was removed and stirring continued for one hour at room temperature. The reaction mixture was diluted with 1400 ml of ethyl acetate, extracted and dried as above. The solvents were removed in vacuo, coevaporated with toluene and then acetonitrile to give 4- (triazolo) -1D-5'-0-DMT-2 ', 3'dibenzoyl (ribofuranosyl) -2 (1H) -pyrimidinone, as a white foam in quantitative production. To a stirred solution of the latter compound in 350 ml of CH 3 CN was added directly solid 6-aminohexanol (11.89 g, 101.5 mmol). Stirring was continued for 18 hours. The reaction mixture was then diluted with 700 mL of ethyl acetate and extracted as above. After drying the organic phase over Na 2 SO 4, the solvent was removed in vacuo. The product was purified on a 6OH silica column, eluted with a 0-5% gradient of ethyl acetate in CH 2 Cl 2 to give 35.4 g (41.5 mmol) of N-4- (6-hydroxyhexyl) yellow foam -5'-O-DMT-2 ', 3'-dibenzoylcytidine,

The corresponding methylphosphoramidite was prepared using standard procedures. N-4- (6-hydroxyhexy-5'-DMT-23'-dibenzoyl cytidine modified nucleoside (8.7 g, 10.2 mmol) was dissolved in 50 mL of methylene chloride containing 8.8 mL (50 mmol) diisopropylethylamine and N, Ndiisopropylaminomethoxy chlorophosphine (1/94 mL, 10 mmol) was slowly added at 0 ° C. After 30 minutes, the reaction mixture was diluted with 250 mL of ethyl acetate and the phase added.

<img file="PT98488B_D0054.tif" />

The organic layer was washed with 2 x 250 ml 5% NaHCO3, 2 x 80% saturated aqueous NaCl-solution, dried over solid Na2 SO4 and filtered. The solvent was removed in vacuo and the residue coevaporated with toluene and acetonitrile. The crude phosphorylated material was purified on a silica gel column using a 50-70% ethyl acetate gradient in methylene chloride containing 2% triethylamine to give 7.25 g of N-4- (ON, Ndiisopropylaminimethoxy phosphinyl). 6-oxyhexyl) -5'-DMT-2 ', 3'dibenzoyl cytidine.

i

I Example 6

Oxidative cleavage of the cis-diol system with sodium periodate readily occurs at the terminal ribonucleoside of RNA molecules. In the presence of amines, the resulting dialdehyde readily eliminates both the base radical and the 5 'carbon phosphate. This example describes the use of this concept in designing a cleavable site of a molecule where two DNA oligomers are linked via 5 'and the side chain of hydroxyl groups of an n-4- (6-hydroxyhexyl) cytidine molecule.

modified R ribonucleoside containing an exo-cyclic hydroxyl group I was synthesized from uridine. The protected R ribonucleoside phosphoramidite R was incorporated under Standard conditions into a 5'-TiO ”R-T1” 3 'oligomer on a solid support. Purified product samples were subjected to a series of chemical treatments, and the samples analyzed by PAGE. No cleavage of the oligomer was observed after treatment with ammonium hydroxide at 60 ° C for 18 hours. 0 Treatment with sodium periodate in water at 4 ° C for 30 minutes resulted in partial cleavage. Another exposure of the sodium periodate-treated oligomer, n-propylamine in triethylammonium acetate, at 60 ° C for 90 minutes resulted in complete cleavage of the oligomer in the 5 ', 10' end-modified Ti0 3 '-p and T15 species. Scheme 7 depicts the cleavage of ribonucleoside R bound to DNA fragments.

The cleavage scheme has been applied to several branched DNA oligomers, in which the protected phosphoramidite R ribonucleoside was incorporated during the first round of secondary synthesis of linear oligomers, attached to a solid support containing 10, 20 and 30 branch points in comb respectively. In each case the secondary synthesis was a Τχο oligomer resulting in branched oligomers with the following structure:

3'-T20 "En-5 '[branching point-3'-R-Tiq-5'] nn = 10, 20, 30

These molecules were subjected to cleavage conditions. PAGE analysis indicated that all oligomeric side arms were cleaved, and Tig-3'-p was the major product in all cases. Analyzes further revealed that the distribution of the product depends on the number of branches in the branched DNA molecules, in which the amount of smaller oligomers increases with increasing number of branches of the molecule. The decrease in product homogeneity appears to be mainly the result of steric pressures within the solid support during chemical synthesis.

SCHEME 7

<img file="PT98488B_D0055.tif" />

N * IO,

<img file="PT98488B_D0056.tif" />

π-PrNH, z rCflfl z χ ₃

60 ° C

<img file="PT98488B_D0057.tif" />

Example 7

This multifunctional scheme 8.

This example describes the preparation of the DMT-E '(Lev) BCE amidite ligand as shown in

SCHEME 8 ch₂oh

CH₃-C-CH_OOH

I ²

CH₂QH

TsCl ——

AND'

CH_?0H levulinic acid I

CH_what-C-CH - O-DMT Cesium Salt ³ I ² (: - CH₂-O-Lev

DMT-E '(Lev)

Cl cnch₂ch₂op

N (iPr)₂ | N (Pr)₂/ ^ύ

CH, OP \

och₉ch_Ocn ch₃-c-ch, odmt ¹I ²CH₂~ o-Lev

CH-OH

I ² ch₃-c-ch₉oh

I ²CH₂-O-Ts E '(Ts)

DMT-C1 v

ch₉oh

I ch₃-c ~ ch₂-o-dmt

CH₂-O-Ts

DMT-E '(Ts)

DMT-E '(Lev) BCE trishydroxymethyl ethane amidite (Ε'; 200 mmol) was coevaporated with 250 mL pyridine and the residue dissolved in 125 mL pyridine. To this solution, cooled to 0 ° C, was added dropwise a solution of tosyl chloride (TsCl; 50 mmol) in 125 mL of CH 2 Cl 2. The reaction mixture was allowed to reach room temperature with continued stirring for a total of 5 hours. Then, the solvents were removed in vacuo. The residue was dissolved in 500 ml of ethyl acetate which was washed with 2 x 500 ml of NaHCQ3 solution. ^The 5%, 1 x 500 ml 80% saturated aqueous NaCl solution, and finally dried over solid Na2 SO4. After filtration the solvent was removed in vacuo to give 13.7 g of crude E '(Ts). This material was used without further purification. All E '(Ts) was dissolved in 250 ml CH 2 Cl 2 and triethylamine (14 ml; 100 mmol) and N, N-dimethylaminopyridine (100 mg) was added. To this solution was added DMTCl (13.6 g, 40 mmol) dissolved in 125 mL of CH 2 Cl 2. After 18 hours at room temperature, the reaction mixture was diluted with 500 mL of ethyl acetate and subjected to the same aqueous treatment described above.

The reaction crude products were purified on a standard silica column (800 ml silica) eluted with a methanol gradient in CH 2 Cl 2 / 0.5% triethylamine to give 14.8 g (25 mmol) DMT-E '(Ts ) pure. All of this material was treated with 50 mmol of freshly prepared levulinic acid cesium salt (prepared according to M. Bodanszky and A. Bodanszky, in The Practice of Peptide Synthesis, p.37, Springer Verlag (1984)), 50 ml of DMF. The solution was heated on a hot plate in a sealed container (conditions 3; temperature ca, 100 ° C) for 18 hours.

<img file="PT98488B_D0058.tif" />

after this time the analyzes revealed that the reaction was complete. The DMF was removed in vacuo and the residue dissolved in ethyl acetate. The organic phase was washed as described above. The crude product was chromatographed on silica gel and the pure product eluted with CH 2 Cl 2 / triethylamine.

0.5% to give 2.5 g (4.8 mmol) of pure DMT-E '(Lev) product. O

Pure DMT-E '(Lev) was converted to 2-cyanoethyl phosphoramidite as follows. DMT-E '(Lev) was dissolved in 20 mL of CH 2 Cl 2 containing N, N-diisopropylethylamine (2.6 mL; 15 mmol) and cooled to 0 ° C; To this solution was added 2-cyanoethoxy-N, Ndiisopropylaminochlorophosphine (1.1 ml; 5 mmol) with a syringe and under argon. After about 30 minutes the reaction was complete and the reaction mixture was diluted with 150 mL of ethyl acetate. The organic phase was washed with 2 x 150 ml 5% NaHCO 3 and 2 x 150 ml 80% saturated NaCl solution. After drying over solid Na 2 SO 4 the solution was filtered and evaporated to dryness to give

3.6 g DMT-E '(Lev) BCE amidite white foam. The crude amidite was purified on a silica gel column eluted with

CH 2 Cl 2 / ethyl acetate / triethylamine (45:45:10 v / v) to give a pure white foam of DMT-E '(Lev) BCE amidite (3.24 g, 4.5 31 mmol). NMR (δ) δ 148.5 ppm, and coupling efficiency 98%.

Example o__8

This example describes an alternative synthesis of the multifunctional ligand DMT-E '(Lev) BCE amidite as shown in Scheme 9.

SCHEME 9 ch₂what

I sió₃cl ch₃-c-ch₂oh -&gt;

ch₂what

AND'

I ch₂-yes₃ ch₃-c-ch₂-o-dmt

CH₂-O-Lev

Levulinic Acid EDIC *

CH OH

I ch₃-c-ch₂oh CH₂^wQ * SY0₃E '(TPS)

DMT-C1 ch₂oh ch₃-c-ch₂-o-dmt

CH₂-Q-Si0₃

DMT-E '(Lev)

F ”

DMT-E '(TPS) v

CH «-OH

I

CH₃-C-CH₂-O-DMT

CH₂-O-Lev _XC1

CNCH₉CH ~ 0-P ^sN (iPr)₂

<img file="PT98488B_D0059.tif" />

CH ^ -O-Lev

DMT-E '(Lev) BCE amidite * EDIC = carbodiimide hydrochloride

1-ethyl-3 “(3-dimethylairiinopropyl)

<img file="PT98488B_D0060.tif" />

Trishydroxymethyl ethane (200 mmol) was coevaporated with 250 mL of pyridine and the residue dissolved in 125 mL of pyridine. To this solution, cooled to 0 ° C, was added dropwise a solution of triphenylchlorosilane (TPS; 50 mmol) in 125 mL of CH 2 Cl 2. The reaction mixture was allowed to warm to room temperature and stirring was continued for a total of 18 hours. Then the solvents were removed in vacuo. The residue was dissolved in 500 mL of ethyl acetate which was washed with 2 x 500 mL of 5% NaHCO 3 solution, 1 x 500 mL of 80% saturated aqueous NaCl solution, and finally dried over Na 2 SO 4. 4 solid. After filtration, the solvent was removed in vacuo to give 18 g of crude E '(TPS). This material was used without further purification. All E '(TPS) (46 mmol) was dissolved in 250 mL of CH 2 Cl 2 θ triethanolamine (14 mL; 100 mmol) and N was added._fN-dimethylaminopyridine (100 mg). To this solution was added DMT-C1 (50 mmol) dissolved in 125 mL of CH 2 Cl 2. After 18 hours at room temperature, the reaction mixture was diluted with 500 mL of ethyl acetate and subjected to the same aqueous treatment as described above to give 35.8 g of yellow foam.

The crude reaction product was purified on a column.

I

Standard silica gel (800 mL silica) eluted with a methanol gradient in 0.5% CH 2 Cl 2 / triethanolamine to give 14.8 g (25 mmol) of pure DMT-E '(TPS). Purified DMT-E '(TPS) (10 mmol) was dissolved in 50 mL containing N, N-dimethylaminopyridine (100 mg) and 2,6-lutindine (2.3 mL, 20 mmol) and levulinic acid (2 mL) was added. 3 g, 20 mmol). To this solution was added dropwise 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (3.83 g, 20 mmol) dissolved in 50 mL of CH2 Cl2. After 18

<img file="PT98488B_D0061.tif" />

Within hours the reaction was complete (TLC analysis), and the reaction mixture was dissolved with 500 mL of ethyl acetate and subjected to the same aqueous treatment described above. The residue obtained from this treatment was dissolved in THF (50 ml) and 40 ml of pyridine was first added and then 10 ml of concentrated acetic acid followed by 20 ml of 1M tetrabutylammonium fluoride in THF (Aldrich). TLC analysis after 30 minutes revealed that all starting material had been consumed. Most of the solvent was then removed in vacuo and the remaining residue subjected to the following aqueous treatment: Ethyl acetate (250 mL) to dissolve most of the organic material and 250 mL of 5% sodium bicarbonate solution was added. slowly (CO2 evolution). The solid NaHCO 3 was then added with stirring and dissolved until solid salt remained and until all evolution of CO 2 had ceased. The combined aqueous / organic solution was transferred to a separatory funnel and the organic phase washed as described above. Removal of solvent yielded 5.6 g of DMT-E '(^Lev) crude, as a clear oil. The product was isolated by silica gel chromatography using ca. 500 g of silica and 0.25% CH 2 Cl 2 / triethylamine containing 0% and 1% methanol as eluant to give 2.7 g (5.2 mmol) DMT-E '(Lev) as a clear, colorless oil .

DMT-E^fPure (Lev) was converted to 2-cyanoethyl phosphoramidite as follows: DMT-E '(Lev) was dissolved in 20 mL of CH 2 Cl 2 containing N, N-diisopropylethylamine (2.6 mL, 15 mmol) and cooled to 0 °. Ç; To this solution was added with a syringe and under argon 2-cyanoethoxy-N, N-diisopropylaminochlorophosphine (1.1 ml; 5 mmol). After ca. 30 minutes reaction

7th •Ç-

<img file="PT98488B_D0062.tif" />

<img file="PT98488B_D0063.tif" />

The reaction mixture was complete and the reaction mixture was diluted with 150 ml of ethyl acetate. The organic phase was washed with 2 x 150 ml of

5% NaHCO3 and 2 x 150 ml saturated 80% NaCl solution. After drying over solid Na 2 SO 4 the solution was filtered and evaporated to dryness to give 3.4 g of white DMT-E '(Lev) BCE amidite foam. The crude amidite was purified on a silica gel column, eluted with CH 2 Cl 2 / ethyl acetate / triethylamine (45:45:10 v / v), to give a white foam of pure DMT-E '(Lev) BCE ( 2.4 g (3.3 mmol). NMR (P) δ 148.5 ppm and coupling efficiency₍ 98%.