Method of defecting presence of a given oligonucleotide sequence

The present invention relates to a method for detecting the presence of a given oligonucleotide sequence.

Detection of the presence of a given oligonucleotide sequence is possible by introducing selectively cleavable sites into oligonucleotide and polynucleotide chains. These techniques are described in U.S. Patent Application Serial Number 251.152 and U.S. Patent No. 4,775,619, which are cited herein. Selectively cleavable sites are useful in many different types of hybridization techniques. For example, in one type in which hybridization leads to a duplex of a labeled probe and a DNA sample deposited on a solid support, the cleavage site selectively contained in the hybrid structure allows direct separation of the labeled probe from the solid support. U.S. Patent No. 4,775,619 is primarily devoted to the use of restriction endonuclease cleavage sites in this type of assay. Chemically cleavable sites can also be used, such as biside bonds,

And, 2-diols and the like. Such sites are introduced during the synthesis of olieonudleotide. They are cleaved with suitable chemical reagents, e.g. thiols, periodates or the like.

It is also possible to introduce a site that can be cleaved by photolysis as well as a site cleavable by other means, among others using chemical or enzymatic reagents, e.g. reducing agents. Cleavage sites are created by introducing chemical residues, preferably light sensitive residues into oligonudleotidone or pnlinudleotye chains. Tadie new light sensitive residues are useful in many different types of hybridization assays, including those described in the patent applications cited above. as well as in an attempt to amplify and hybridize the nucleic acids disclosed in European Patent Application No. 88.309697.6 made by applicants of the present invention.

Another use of these above and other reagents is in general terms to create mismatched sites within the oligonucleotide structure. The term non-basic site here means the -OR ether residue at the position at which the -OH hydroxyl group or nucleic base is normally present.

Still another use of such reagents is chemical phosphorylation. In many different aspects of oligonucleotide chemistry, chemical phosphorylation of hydroxyl groups is necessary. An example is the synthesis of oligonucleotides, where after the steps of synthesis and removal of protective groups, the free 5'-hydroxyl group of the oligonudleotad should be phosphorylated so that it is suitable for use in most biological processes. It is also necessary to phosphorylate the 3'-hadrodsal functional group to / 1 / prevent polymerase 3 & apos; extension by chemicalase DNA ligation, where the presence of residue 3 is generally required^/phosphate during chemical coupling of oligonucleotides.

The 5'-phosphoration reaction is generally carried out using the T4 polynucleotide kinase and ATP. This reaction is neither very accurate nor efficient. A number of chemical 5'-phosphorylation methods are also known. They are described, among others by Nadeaux et al. in Biochemistry 23, 6153-6159 / 1984 /, van der Marel et al. in Tetrahedron Lett. 22 1463-1466 / 1981 /, Himmelsbach and Pfleiderer at Tetrahedron Lett. 23, 4793-4796 / 1982 /, Marugg'a et al. in Nucleic Acids Research 12,8639-8651 / 1984 / and by Kondo et al. in Nucleic Acids Research Symposium Series 16,161-164 / 1985 /. Most of these methods are due to the use of unstable reagents or the complex modification of standard deprotection and purification procedures. Similar difficulties occur with monofunctional and bifunctional reagents up to 3'-phosphoralase / see Sonveaux, previous entry, p. 297 /.

Thus, in addition to being useful in the processes of introducing fissile and / or non-basic sites into olieonudleotide or polynudkeotad chains, a number of compounds can be used as phosphorylation reagents that overcome the limitations of existing phosphorylation procedures / they may also be useful in phosphorylation reactions that are used in commonly accepted purification schemes through dimethylditratylone / DMT / derivatives. Literature references generally related to methods for synthesizing oligonudleads include those in which 5 'to 3' syntheses based on the use of β-daanethalophosphate protecting groups are described. These include, e.g., de Napoli et al., Gazz.Chim.Ital. 114.65 / 1984 /, Rosenthal et al., Tetrahedron Letters 24.1691 / 1983 /, Belagaje and Brush, Nukleic Acids Research 10.6295 / 1977 /. Solution 5 'to 3' syntheses are described by Hayatsu and Khorana in J.Am.Chem.Soc. 89, 3880/1957 /, Gait and Sheppard in Nucleic Acids Research 4, 1135/1977 /, Cramer and Koster in Angew.Chem.Int.Ed.Engl., 7, 473/1968 / and Blackbum et al. in J.Chem.Soc.Part C, 2438 1X9611.

In addition to the prior art cited above, Matteucci and Caruthers describe in

J. Am.Chem.Soc. 103, 3185-3191 / 1981 / use of phosphite chlorides for the production of oligonudleotides. The use of phosphinamidone for the production of oligonucleotides is described by Beaucage and Caruthers in Tetrahedron Letters 22, 1859-1862 / 1981 and in US Patent No. 4,415,732. Smith in ABL 15-24 / December 1983 / described the automatic synthesis of solid phase oligodeoxyribonucleotide. It is also the subject of the above-cited publications and publications of Warner et al. In DNA, 3, 401-411 / 1984 /, the disclosures of which are cited in the present description.

Horn and Urdea described in DNA, 5, 5: 421-425 / 1986 / phosphorylation of DNA-supported solid fragments using bis / cyanoethoxy / -N, N-diisopropylaminophosphine. This method is also given by Horn and Urdea in Tetrahedron Letters 27, 4705-4708 / 1986 /.

The following publications are devoted to hybridization techniques: Meinkoth and Wahl / Anal. Biochemistry 138, 267-284 / 1984 / give an excellent overview of hybridization techniques. Leary et al. In Proc.Natl.Acad.Sci. / USA / 80, 4045-4049 / 1983 / describe the use of biotinylated DNA in combination with an avidin-enzyme conjugate to detect specific oligonucleotide sequences. Ranki et al. in Gene 21, 77-85, describe a technique they call sandwich-type hybridization for the detection of oligonucleotide sequences. Pfeuffer and Helmrich in J. Biol. Chem. 250, 867-876! X) -I5I, have reported the coupling of guanosine-5'-O- (3-tri-triphosphate) with Sepharose 4B. In J.Histochem.and Cytochem. 29.227-237, Bauman et al. Describe 3'-RNA labeling with fluorophores.

In patent description of PCT, WO / 8302277 is disclosed the addition of modified ribonucleotides to DNA fragments for labeling and methods of analyzing such DNA fragments. Renz and Kurz in Nucl. Acids Res. 12, 3435-3444 describe the covalent binding of enzymes to oligonucleotides. Wallace at DNA Recombinant Technology / ed. Woo S. CRC Press, Boca Raton, Florida, makes a general review of the use of probes in diagnostics. Chou and Merigan in N.Eng.J.of Med. 308, 921-925, report the use of a radio-labeled probe for the detection of cytomegalovirus. Inman in Methods in Enzymol., 34B, 24, 77-102 / 1974 / describes binding procedures for polyakylamides and Parikh et al. In Methods in Enzymol, 34B, 24.77-102 I)) 4 describe coupling reactions with agarose, Alwine et al. in Proc.Natl.Acad.Sci. / USA /, 74, 5350-5354 / 1977 / report a method of transferring oligonucleotides from gels to a solid hybridization carrier. Chu et al. in Proc.Natl.Acad.Sci / USA 11.6513-6529, describe a technique for derivatization of terminal nucleotides. Ho et al. In Biochemistry 20, 64-67 / 1981 / report the derivatization of terminal nucleotides via phosphates to form esters. Ashley and MacDonald in Anal.Biochem. 140, 95-103 / 1984 / report a method of preparing probes on a surface-bound matrix.

Hebert and Gravel in Can J. Chem. 52, 187-189 / 1974 / and Rubinstein et al. In Tetrahedron Latters 17, pp. 1445-1448 / 1975 / describe the use of compounds containing a 2-nitrophenyl group as light sensitive protective groups.

The publication of K. Groebke et al. In Helvetica Chim.Acta 73.608-617 / I)) 0 / is related in so far as it concerns the use of a tert-butyldimethylsilyl residue to block a functional group.

The subject of the invention is a method for detecting a given oligonucleotide sequence in a nucleic acid being tested, consisting in combining this sample of nucleic acid with a polynucleotide reagent of formula under hybridization conditions

<img file="PL170146B1_D0001.tif" />

OH

OH in which DNA1 is the first DNA segment, DNA2 is the second DNA segment, and one of the symbols x and y is zero, while the second one is an integer 1-12 inclusive, wherein either the sample or reagent is previously deposited on the carrier, and as a result hybridization of the test nucleic acid with the polynucleotide reagent a label is formed bound to the carrier via a cleavage site

-O- / CH₂/_and-CH / CH₂/_b-ABOUT-,

<img file="PL170146B1_D0002.tif" />

AND-^N02 and then essentially releasing this carrier from the carrier-bound label other than via this selectively cleavable site, followed by this cleavage by photolysis using light with a wavelength of at least 350 nm, and the label released from the carrier. Preferably, the method uses a polynucleotide reagent of formula 1 in which the symbol x is zero, the symbol y is an integer 1-4 inclusive, or the symbol y is 1 or zero. Preferably a reagent of formula 1 is used in which the symbol x is an integer 1-4 inclusive or wherein the symbol x is 1.

In another embodiment of the invention, the method of detecting a given oligonucleotide sequence in a test nucleic acid present in a nucleic acid sample is that it combines under hybridization conditions in an aqueous environment this sample of nucleic acid with a polynucleotide reagent of formula

0.

S 'V II He' 3

-HO⁵ / DNA-j /⁵ -0-P-0- / CH2 / x-CH - / CH2 / y-0-P-0-⁵ / DHA2 /⁵

OH

J

OH track

HO

AND

OH in which DNA1 is the first DNA segment, DNA2 is the second DNA segment and one of the symbols x and y is zero, while the second one is an integer 1-12 inclusive, wherein either the sample or reagent component is previously deposited on the support and as a result of hybridization a test nucleic acid with a polynucleotide reagent, a label is formed bound to the carrier via a cleavage site,

-0- / 0H₂/_and-CH / CH₂/_b-0-

<img file="PL170146B1_D0003.tif" />

ho₂ and then the carrier with the bound polynucleotide reagent and the test nucleic acid is separated from the aqueous medium, after which the carrier is washed with a medium of a different hybridization force than this aqueous medium in order to remove the carrier-associated label other than via the fissile site, in turn it is split a fissile site by photolysis using light with a wavelength of at least 350 nm and a tracer released from the carrier is detected. This solution preferably uses a polynucleotide reagent of formula 2 in which the symbol x is zero and the symbol y is an integer 1-4 inclusive or the symbol y is 1 or zero.

In another preferred embodiment, a reagent is used wherein the x can be an integer 1-4 in the above formula. Preferably, in the above formula, y is an integer 1-4 inclusive, especially 1. Preferably, y is 1.

Another variant of the method for detecting a given oligonucleotide sequence in a test nucleic acid present in a nucleic acid sample is that under hybridization conditions this sample of the nucleic acid is combined with a 3 '3 formula polynucleotide reagent ¹¹

-HO⁹ /GOUT-,/⁹ -0-P-0

-1 years

<img file="PL170146B1_D0004.tif" />

OH

IS '3' _H0-after-⁹ /GOUT_ABOUT/⁹ OH

II ²in which DNA1 is the first DNA segment, DNA2 is the second DNA segment and R is the 2-nitrobenzyl, 4-penten-1-yl group,

<img file="PL170146B1_D0005.tif" />

170 Wherein R 'is hydrogen, an aryl or oryl yolkyl group, R substituents_t may be the same or different and represent an amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy group, the substituents Rj may be the same or different and represent an amino, nitro, halogen, hydroxy, lower or lower alkyl group alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R_in means an alkylene or oxyethylene oligomer containing 1-16 carbon atoms - / CH2CH2OĄ - where z is an integer 1-16 inclusive and Rn is a group

<img file="PL170146B1_D0006.tif" />

or ch₃OCH₂ch₂-O-CH₂- .

wherein either the sample or reagent is previously deposited on the support and as a result of hybridization of the nucleic acid tested with the polynucleotide reagent, a label bound to the support is formed via a cleavage site of formula

<img file="PL170146B1_D0007.tif" />

wherein R is a 2-nitrobenzyl, 4-penten-1-yl group,

analysis₂ch₂s- / Q

CH₂CH₂Si / CH₃/₃ ,

<img file="PL170146B1_D0008.tif" />

where R 'is hydrogen, aryl or arylalkyl, the substituents R1 may be the same or different and be amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, Rj may be the same or different and be amino , nitro, halogen, hydroxy, lower alkyl or lower alkoxy, and means zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, Rm is the alkylene or oxyethylene oligomer containing 1-16 carbon atoms - / CH2CH2O / Z - where z is an integer 1-16 inclusive and R_n means a group of formula

0

II II

CH₂-C-CH₂CH₂-C-,

170 146

Ο

<img file="PL170146B1_D0009.tif" />

<img file="PL170146B1_D0010.tif" />

0Η₃0-0Η2-σΗ₂-0-σΗ₂ and then substantially releases this carrier from the carrier-bound label other than via this selectively cleavable site, followed by the cleavage of the site cleavable by photolysis using a light of at least 350 nm wavelength and the tag released from the carrier is detected. Preferably, the process of the invention uses a reagent according to the formula given wherein R is a 2-nitrobenzyl group or a group

-^0H2^0H2^S- &lt;(O / 'or wherein R is

<img file="PL170146B1_D0011.tif" />

or in which R is 2-mgtdlgur-9,10anthraquinruracgtal, or in which R is a 4-yl group, or R is a 4-pgntgn-1-group, or in which R is

analysis₂ch₂ —ZQ \ - no₂ or R is also

AND

After

And about **

In a further variant, the method of detecting a given sequence of oliornine | gotydrwgj in the studied nuva | pearly acid present in the uuklginrwegr acid sample is based on the fact that it combines under hybridization conditions in an aqueous environment this sample of the low acid with a reagent with the formula

<img file="PL170146B1_D0012.tif" />

170 146 where DNA1 is the first DNA segment, DNA2 is the second DNA segment and R is the 2-nitrobenzyl, 4-penten-1-yl group,

<img file="PL170146B1_D0013.tif" />

where R 'is hydrogen, aryl or arylalkyl, R1 may be the same or different and be amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, Rj may be the same or different and be a group amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, and means zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R_m means an alkylene or oxyethylene ligomer containing 1-16 carbon atoms - / CH2CH2O / Z - where z is an integer 1-16 inclusive and Rn is a group

0

II II

GH₂-C GH₂OH₂-What

<img file="PL170146B1_D0014.tif" />

0₂N-

V /

S-CHoCHo-O-Cc c.

Q \ -s-ch₂ch₂OC-

<img file="PL170146B1_D0015.tif" />

or

CH₃0-CH 2 CH₂-0-CH₂wherein either the sample or reagent component is previously deposited on the support and as a result of hybridization of the test nucleic acid with the polynucleotide reagent a label bound to the support is formed via a cleavage site of formula

<img file="PL170146B1_D0016.tif" />

wherein R is a 2-niirobenzyl group, 4-ppnten-1-yl group with the formulas:

<img file="PL170146B1_D0017.tif" />

-P-0

L o

or

CH₂CH₂Si / CH₃/₃

-R

0-R

CH₂CH₂

<img file="PL170146B1_D0018.tif" />

Well₂

170 146 where R 'is hydrogen, aryl or aralkyl, R may be the same or different and amino, nitro, hydroxy, lower alkyl, or lower alkoxy, Rj may be the same or different and be amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, and means zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R_m means an alkylene or oxyethylene oligomer containing 1-16 carbon atoms - / CH2CH2O4 - where z is an integer 1-16 inclusive and Rn is a group of formula

<img file="PL170146B1_D0019.tif" />

<img file="PL170146B1_D0020.tif" />

λγ \ ^{11 11} / f) \ -S-CH_aboutCK, -0-C^{2 2} or

and then the carrier with the bound polynucleotide reagent and the test nucleic acid is separated from the aqueous medium, after which the carrier is washed with a medium of a different hybridization force than this aqueous medium in order to remove the carrier-associated label other than via the fissile site, in turn it is split a fissile site by photolysis using light with a wavelength of at least 350 nm and a tracer released from the carrier is detected. In preferred embodiments, a polynucleotide reagent and the formula is used wherein R is a 2-nitrobenzyl group, or

CH₂CH₂S

<img file="PL170146B1_D0021.tif" />

or R is 2-methylene-9,10-anthraquinone-acetal or R is 4-penten-1-yl or R is

<img file="PL170146B1_D0022.tif" />

There are various methods and reagents for incorporating selectively and / or non-basic cleavage sites into oligonucleotide chains, with selectively cleavable sites that are chemically or light cleavable often being used. It can also be the introduction of non-basic sites into oligonucleotide chains by various methods and reagents.

The reagents referred to above are also suitable as chemical phosphorylation reagents. In addition, reagents for incorporating non-basic sites into oligonucleotide chains can be used to form branched nucleic acid multimenes.

Reagents in which non-basic sites are not nucleotides are also used.

Additional advantages of the invention are set forth in the remainder of the description and partly will become apparent to those skilled in the art after examining the invention, or may be learned by practicing the invention.

The new reagents used in the process of the invention are light sensitive chemical compounds of the general formula

<img file="PL170146B1_D0023.tif" />

2 where R, R, x and y are defined below. These compounds are introduced into oligonucleotide chains to allow light cleavage.

Other new reagents used are compounds of the general formula:

<img file="PL170146B1_D0024.tif" />

where r1 R²and R are defined below. Such compounds are useful for creating non-basic sites in oligonucleotide chains. These sites are fissile or non-fissile.

Still other new reagents used are compounds of the general formula

CH_ABOUT - 0 - R¹

AND ² '

CH-, - C - CH_ABOUT - 0 - R ' ³ AND ²

ch₂ - 0 - R ^

2 where R, R and Rn have the meanings given below. Such compounds are used to form branch points during the synthesis of nucleic acid multimers.

Mode of carrying out the invention.

A. Definitions.

The term selectively cleavable site means a functional or multifunctional site selectively cleavable. As mentioned above, sites that are specifically cleavable by photolysis are of particular importance in the invention.

The terms oligonucleotide and polynucleotide as used herein are general terms, including polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), polyribonucleotides / containing D-ribose or modified forms thereof, and any other type of N-glucoside polynucleotide purine or pyrimidine or modified purine or pyrimidine base. Similarly, the term nucleoside is a generic term for ribonucleosides, deoxyribonucleosides, and any other N-glucoside purine or pyrimidine base nucleoside or modified purine or pyrimidine base. There is no predetermined demarcation as to the length between terms, oligonucleotide and polynucleotide, and these terms are interchangeable with each other. These oligonucleotides and polynucleotides may be single-stranded and double-stranded, usually single-stranded. The oligonucleotides used in the present invention generally consist of from about 2 to about 2000 monomeric units, and usually, for most applications as probes, from about 2 to about 100 monomeric units.

The term nucleic acid sample means a sample in which the nucleic acid sequence of interest is suspected. The analyzed nucleic acid means that the DNA or RNA in the nucleic acid sample contains the test sequence.

The term phosphorylation reagents means compounds which, by reaction or from a series of reactions with a hydroxyl group containing compound, give a phosphoric acid monoester.

The lower alkyl and lower alkoxy groups are respectively alkyl and alkoxy substituents containing 1-8 carbon atoms, generally 1-6 carbon atoms.

Aromatic substituents are understood to mean any aromatic ring optionally substituted on one or more carbon atoms by residues that do not significantly change its function or reactivity.

B. Chemical structure of new light-sensitive reagents:

The new reagents used are light-sensitive compounds with the formula:

R¹ - 0 - / 0Η₂/_χ - CH - / CH₂/_s - 0 - R² where R¹ means a protective group stable in alkaline environment and unstable in acidic environment, R² is a phosphorus derivative that allows the reagent to be added to the 5 'position of the acid chain or oligonucleotide, one of the symbols x and y is zero while the other is an integer in ranges 1 to 12 inclusive. The above general formula covers two basic types of structures: (1) structures in which x is a number other than zero and y is zero 1 never called NP1 reagents in this specification) and (2) structures in which x is zero and y is a number than zero (called NP2 type reagents). These two types of structures, as can easily be derived from the above general formula, are quite similar. Both types are suitable for entering specific places in roundabout chains. Due to the presence of the nitrous residue, these sites are easily soluble by photolysis. However, as described in more detail later in the description, these two families of chemical reagents differ from uigblg in that they are used in slightly different contexts.

The more precise meaning of the individual substituents in the new light-sensitive magentacids is as follows: R1 as above, is a protective group that is stable in an alkaline environment and sensitive in an acidic environment. Such protective groups are known in the synthesis of rligrnuę gotddów. These include uproputavirug or substituted aryl or arylalkyl groups wherein aryl is e.g. phenyl, naphthyl, furanyl, phenyl or similar group, in which groups from 0 to 3, usually from 0 to 2, are any non-interacting stable uigpolarue groups or polar, withdrawing or electron donating groups. Examples of such groups are dimethocytrdtyl (DMT), monomgtocythyl (MMT), trityl and 9-feuyl-9-xatenyl-piyl) groups.

The rest, the use of which is particularly preferred here, is the dimgtokuytrdtyl 1DMT group).₂

Rp is a phosphorus derivative selected so as to facilitate condensation of the reagent with the 5'-hydroxyl group of the nucleotide or oligonucleotide. These types of groups include fouforduoamides, phosphototrigstrd, phosphodigstrd, phosphites, hydrogen phosphonium, thiophouforau and the like (see, among others, European Patent Application Publication No. 0225807, inventor: Urdea et al., Title: Solution-tested nucleic acid sandwich assay and used in the probe polynesia eotydowg. The disclosures in this publication are cited in this description.) Particularly preferred groups as substituents r2 are phosphinamides of formula:

N (LPR)₂ where Y is a methyl or β-cyjaogtdl group and iPr is an isopropyl group. NajejoredstuigJ, Y is a β-cyanergyl group.

From the above definitions, it can easily be concluded that the substituents R1 and r2 are selected so as to allow the introduction of a light-sensitive reagent into the DNA fragment using the standard phosphinamidamide technique. That is, for the synthesis of ollgouucleotide, the r2 substituent is selected so that it reacts with the 5'-hdroxy group of the uuklgosidowggr or oligonucleotide chain and the residue R1 is selected so as to allow reaction with the 3'-hydroxyl group of the uucleoside or ollgouuklgotddowggo chain.

With respect to the symbols x and y, one of them is zero while the other is an integer from 1 to 12 inclusive, more preferably from 1 to 4 inclusive and most preferably -1.

Examples of reagents included in the above general category are the following compounds:

170 146

<img file="PL170146B1_D0025.tif" />

"ΝΡ1"

<img file="PL170146B1_D0026.tif" />

As you can see, these specific structures, [2- (2-nitrophenyl) -2- (O-dimethoxytrityloxy) ethoxy] N, N-diisopropylamino-2-cyanoethoxyphosphine and [2- (2-nitrophenyl) -1- (O-dimethoxytrityloxy) ) ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine, designated as NP1 and NP2, respectively. These are specific reagents whose synthesis is described in Examples 1 and 2.

C. Synthesis of the above reagents:

NP1 type reagents, i.e. those in which x is a number other than zero and y is zero, are synthesized in the reactions shown in Scheme 1. NP2 reagents are synthesized in the reactions shown in Scheme 2.

Diagram 1

HO - CH_ ²

CH - OH

DMT - 0 -

<img file="PL170146B1_D0027.tif" />

DMT-Cl pyridine?^H2

<img file="PL170146B1_D0028.tif" />

^ N (iPr)₂

Cl - P ^ 0CH₂CH₂CN

DiPEA ch₂oi_L

<img file="PL170146B1_D0029.tif" />

FROM

OCH.CH.CN

170 146

Diagram 2

<img file="PL170146B1_D0030.tif" />

HO - OH,

TBDMS - O - CH₂

CH - OH

0Γ '

TBDMS-Cl _ dmap / tea ch₂these₂

DMT-C1 ~ dmap / tea oh₂these₂

TBDMS

<img file="PL170146B1_D0031.tif" />

TBAF

THF ~

<img file="PL170146B1_D0032.tif" />

- DMT

WELL,

Cl - P yi (iPr).

OCH₂CH₂CN

DiPEA ch₂these₂ (IPr)₉N_x ncch₂ch₂about

<img file="PL170146B1_D0033.tif" />

In diagrams 1 and 2, the abbreviations have the following meanings:

DMT "= dimethodsaeratyl; dMT-C1 = eimethodsatratalene chloride; iPr = isopropyl; DiPEA = diisopropyl diamino; TBDMS-Cl = t-yutalndimethosyl chloride; dMAp = 4-dimethylmiminopyidine; TEA = triethylamine; TBAF = fluoride tetrαyytaloαmonions.

The synthesis of NP1 reagents consists in blocking the terminal hydroxyl group of 2- (O-nitrophenal) -1,2-ethanndiol with compounds with an R 'group, e.g. DMT or the like, and then reacting the remaining hydroxyl group with the appropriate phosphorus derivative, exposing the R "residue. In Scheme 1, an example of a reagent for the latter purpose is chloro-N, N-diisopropaloαmino-2-dylαnoethylphosphine. You can easily derive the narrative of this basic scheme. For example, monomethodatatratal chloride, tratal chloride, 9-chloro-9-phenoxyloxanthene or the like can be used as an alternative to dimethyserital chloride to introduce other R1 substituents.

170 146

Similarly, to introduce other R substituents², alternative substituted phosphines are used in the second reaction step. For change x, the starting substance should contain additional methylene groups.

In order to synthesize NP2-type reagents, rn is one in which x is zero and y is a number other than zero, a similar reaction sequence is carried out, except that the order of introduction of R substituents¹ and R2 is inverted. Thus, initially, the terminal hydroxyl group in the starting 2- (o-nitrophenyl) -1,2-ethanediol is reacted with tert-butyldimethylsilyl chloride (TBDMS-C1) to block this hydroxyl group during the next reaction step in which the remaining the free hydroxyl group is reacted with a basic stable and acid labile blocking group, e.g. dimethoxytrityl chloride (DMT-Cl), introducing the R1 substituent. Then the terminal hydroxyl group is unblocked, e.g. with tetrabutylammonium fluoride, and as in Scheme 1, the reaction is carried out with a suitably substituted phosphine derivative by introducing the residue R2.

D. Use of the reagents described above to create selectively fissile sites.

The new light-sensitive reagents used in the method of the invention are easily introduced into the oligonucleotide or polynucleotide chain using a standard phosphamidamide technique well known and described in many references cited above.

Generally, the introduction of this new reagent into a DNA fragment requires the formation of a 5'-hydroxyl group bond at r2 and a 3'-hydroxyl group bond at R1.

So, after the introduction of the light-sensitive reagent, the hybrid oligonucleotide chain will have the following structure:

'' HO⁵ / ^ DNA³ /GOUT₂/³ - OH

<img file="PL170146B1_D0034.tif" />

p - about I

OH where DNA1 is the first DNA segment, DNA2 is the second DNA segment, and y and y have the meanings given above. DNA1 and DNA2 can be linear or branched. Such a polynucleotide reagent can be used in hybridization assays, such as those described in European Patent Application Publication No. 88.309203.3 made by the present applicants and in US Patent No. 4775619. Linear polynucleotide reagents containing selectively cleavable sites, i.e., where DNA1 and DNA2 are linear, are used in these assays. A polynucleotide reagent containing a light sensitive residue may also be used in the amplification assays disclosed in U.S. Patent Application Serial Numbers 07 / 252.638 and 07 / 340.031 incorporated herein (see also PCT Publication No. W089 / 03891). As reported in these applications, cleavable linker molecules are introduced into amplification multimers at specific locations to analyze multimer structure or as a means of releasing specific segments (such as the portion of the multimer that binds to the tagged oligonucleotide). In this type of application, DNA1 and / or DNA2 are branched polynucleotide segments. After multimer synthesis and purification, the branched polynucleotide structure of this multimer can be specifically cleaved without further degradation of the nucleotide structure. Preferably, fissile sites are introduced at or near the sites of the multimer linker, as this allows quantification of individual multimer branches.

Depending on whether the light-sensitive reagent introduced into the oligonucleotide or polynucleotide is of the type NP1 / that is, where x is a number me than 0 ay means 0 / or to the type NP2 / that is, one where x is zero ay means a number other than zero /, after splitting two different types of fragments are formed. As shown in Scheme 3, cleavage of an oligonucleotide containing an NP1-type residue results in a first fragment having a 5'-phosphate terminal group and a second fragment having a 2'-nitrosophenyl residue at the 3 'end.

Conversely, as shown in Scheme 4, after cleavage of a polynucleotide containing an NP2 residue, the first fragment containing the 2-nitrosophenyl residue at the 5 'end and the second fragment with the 3'-phosphate end group is formed.

<img file="PL170146B1_D0035.tif" />

photolysis (UV light&gt; 350 nm; mercury lamp)

5'-HO⁵ (GOUT.)⁵ -OP- (CH) ¹ IX

OH 0 = 0 w

c^from x^from

5'-H0-P-0- (DNA-)⁹ OH

OH

<img file="PL170146B1_D0036.tif" />

WELL

17(0 146

Diagram 4

5'-HO⁵\ BNA1)^3,-0-i&gt; -0-CH- (CH2)_s-0-P-0-⁵ - (DNAg) -OH

OH

OH .NO, photolysis (UV light&gt; 350 nm mercury lamp)

<img file="PL170146B1_D0037.tif" />

Since the fission occurs by photolysis under the influence of ultraviolet with a wavelength of at least about 350 nm, no enzymatic or chemical reagents are needed. The procedure is therefore cleaner and a product is obtained free of contaminants introduced from the outside. In addition, the polynucleotide reagent itself is as such more stable, and only cleavable by exposure to UV light at the appropriate wavelength.

E. Phosphorylation using the reagents described above.

In addition to being useful in the procedure for incorporating light-sensitive fissile sites, the reagents described above are also suitable as chemical phosphorylation reagents. Phosphorylation using these reagents involves their condensation with a hydroxyl-containing compound and subsequent photochemical cleavage to release the nitrophenyl group. In this respect, these new reagents are quite versatile because they can be used for both the 5 '- and 3'-phosphorylation of a nuclodide or ligonuclide chain.

A reagent of NP1 type is used for 5'-phosphorylation, i.e. a reagent in which x is a number other than zero and y is zero. As shown in Scheme 3, the cleavage of a polynucleoide reagent containing an NP1 type molecule results in a nucleoside or DNA fragment containing a 5'-phosphate group.

For 3'-phosphorylation, an NP2 type reagent is used, which is illustrated in Scheme 4. As a result of cleavage of a polynucleotide reagent containing an NP2 type molecule, cleavage fragments are formed, one of which contains a 3'-phosphate group and the other fragment contains a niirosephenyl residue.

170 146

F. Introduction of non-base sites and sites for synthesizing secondary oligonucleotide chains.

The reagents below are useful for introducing non-basic sites into oligonucleotide chains, which sites may be cleavable or non-cleavable. These reagents have the structure represented by the formula

<img file="PL170146B1_D0038.tif" />

where R1 and R² are defined above, in Part A of this Section, and R is a 2-nitrobenzyl, 4-penten-1-yl group and groups having the formulas:

analysis₂ch₂sZ Q

CH₂CH₂Si / CH₃/₃ ,

<img file="PL170146B1_D0039.tif" />

in which formulas R 'is hydrogen, an aryl or arylalkyl residue, where the aryl or arylalkyl residue preferably has 1-8 carbon atoms, the R substituents may be the same or different and denote amino, nitro, halogen, hydroxy, lower group alkyl and lower alkoxy, Rj may be the same or different and represent the following groups: amino, nitro, halogen, hydroxy, lower alkyl and lower alkoxy, and is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4.

Rn is a levulinyl group - (CO) CH2CH2 (CO) CH3 or any other blocking or protecting group that can be removed or exchanged for hydrogen without violating the R1 substituent such as the groups represented by the formulas:

170 146

Ο

<img file="PL170146B1_D0040.tif" />

CM = ν »ΑCH3-O-CH₂CH₂-O-CH₂and Rm is either an alkylene oligomer containing 1 to 16 carbon atoms, more preferably 2 to 12 carbon atoms or an oxyethylene oligomer - (CH2CH2O)_from- where z is an integer 1 to 16, more often 2 to 12 inclusive. In an apthal embodiment, if R is the residue -Rm-O-Rn, then Rn is a levulinyl group and Rm is - (CH2CH2O) 4-.

These deoxyribose-based reagents not only introduce non-basic sites into the oligonucleotide or polynucleotide chain but also, like the reagents described above, are useful for introducing fissile sites.

If R is the remainder represented by the formula:

<img file="PL170146B1_D0041.tif" />

then it is preferred that R 'is hydrogen or phenyl. As stated above, Ri and Rj can be any of many different substituents. In a particularly preferred embodiment, the above structure is a 2-methylene-9,10-anthraquinone carbonate ester, i.e. a group in which R1 and Rj as well as R 'are hydrogen.

Reagents of the formula

170 146

<img file="PL170146B1_D0042.tif" />

easily synthesized from deoxyribose and an alcohol derivative containing an R residue, i.e. from R-OH. For example, in the case of 2-nitrobenzyl, deoxyribose is reacted with 2-nitrobenzyl alcohol to give the! -O- (2-nitrobenzyl) derivative. This intermediate is easily converted to the 5'- and 3'-protected analog by standard methods, e.g., introduction of dimethoxytrityl (DMT) or anologic group at the -5'fR position¹) and a phosphorus derivative such as phosphinamide, phosphotroxy or similar in the 3 'position (R²).

As reported in Part D of this section, these reagents are easily introduced into the oligonucleotide or polynucleotide chain using a standard phosphamidamide method. After introducing these deoxyribose-based cleavable residues into an oligonucleotide or polynucleotide chain, such a cleavable chain containing non-basic sites -OR will have the structure represented by the formula

<img file="PL170146B1_D0043.tif" />

wherein DNA1 and DNA2 are the first and second DNA segments described above. Such a polynucleotide reagent is suitable for use in many types of hybridization assays.

Cleavage of oligonucleotide or polynucleotide chains containing these reagents is carried out as follows. If R is a 2-nitrobenzyl group, the cleavage is carried out by photolysis with ultraviolet light at a wavelength of at least about 350 nm, followed by basic hydrolysis, e.g. with ammonium hydroxide or a similar reagent.

If R is -CH2CH2-S- (where φ is phenyl), the cleavage is carried out by oxidizing the sulfur atom to -SO- or -SO2- with e.g. sodium periodate and subsequent base treatment. If R is -CH2CH2-Si / CH3 / 3, then the oligonucleotide is cleaved by treatment, e.g. with fluoride ion and then with a base. In compounds in which R is:

<img file="PL170146B1_D0044.tif" />

170 For example, 2-methylene-9,10-anthraquinone-acetal, cleavage is carried out by oxidation with NA2S2O41 followed by base treatment. Where R is

analysis₂ch₂—

<img file="PL170146B1_D0045.tif" />

fission occurs under the action of DBU /1,8-diazabicyklo[5.4.0]undecenu-7/. If R is phosphate, this group is removed by treatment with alkaline phosphatase and then with a base, and if R is 4-penten-1-yl, then cleavage is generally carried out with N-bromosuccinimide followed by base treatment.

As mentioned above, reagents that allow cleavage of the oligonucleotide or polynucleotide chain are suitable for use in the amplification assays disclosed in European Patent Application No. 88.309697.6 made by the present Applicants and cited above. With the deoxyribose-based reagents described in this section, branching points of the nucleic acid multimer can be created using multifunctional nucleic acid monomers

<img file="PL170146B1_D0046.tif" />

where: R1 is an alkaline stable and acid labile blocking group;

r2 is a phosphorus derivative enabling addition of the nucleic acid to the -5 'position of the oligonucleotide chain during chemical synthesis;

R³ is hydrogen, methyl, iodine, bromine or fluorine;

R is hydrogen or methyl;

R⁵ is a levulinyl group

<img file="PL170146B1_D0047.tif" />

170 146 or

<img file="PL170146B1_D0048.tif" />

where:

R ', Ri and Rj have the meanings given above, k is zero, 1, 2, 3 or 4 and the substituents Ri can be the same or different and represent an amino, nitro, halogen, hydroxy group, lower alkyl group and lower alkoxy group ;

Z represents a group selected from the following formulas:

(2) (2) (2) (2) (2) (0Η₂)_χ - NH - and - O (1) (CH₂)_x - NH - C - (CH₂)_s - 0 (D (CH₂)_x - NH - C - (CH_?)_v -SS - (CH₂)_v - O (1) e

2'y ^AND2'y (CHJ „- NH - (Π) - Out ~ ⁰ (1) (1) (CH₂ - CH₂ - ⁰ )_x - and (2) (1), - (ch₂)_x - 0 in which x and y can be the same or different and represent whole numbers between 1 and 8.

Then, as described above, such nucleic acid monomers are introduced into the oligouucleotide or polynucleotide chain with a cleavable or easily removable residue R⁵ determining the site at which secondary oligonucleotide chains are synthesized.

Branches of nucleic acid multimers are also created using multifunctional non-nucleotide compounds of the general formula:

CH_ABOUT - 0 - R¹l ² 9

CH, - C - CH? - O - R⁵ AND ²

CH₂ - 0 - R_n gd^zie:

R, R and Rn have the meanings given above. In a particularly preferred embodiment, R is DMT, R² is β-cyanoethylfodfefrinamide and Rn is a levulinyl residue. Such compounds are synthesized from tris-hydroxymptylethane by: (1) blocking one of the hydroxyl groups by reaction with e.g. triphenylchlorosilane or tosyl chloride, (2) reaction of such a protected compound with an R salt, e.g. with dimeioxyir'yyl chloride, in such a way to convert one of the two free hydroxyl groups into -OR2 (3) by reacting the resulting compound with Rn-OH or with an Rn salt, e.g. with levulinic acid or its salt, with simultaneous displacement of the blocking group from step (1) and (4) reaction of the intermediate compound of formula

CH_ABOUT - 0 - HI ² ,

CH₃ - C - CH₂ -o -r '

CH₂- 0 - R_n with a reagent by which the remaining free hydroxyl group is converted to -OR1, e.g. with β-cyanoeiokdy-N, N-disopropylamenochlorophosine.

Such nonadditional sites are particularly useful both for the possibility of cleaving the oligonucleotide chain at a specific site as well as for other purposes, for example for the synthesis of branched nucleic multimers.

G. Additional selectively cleavable linker residues.

Still another reagent useful for incorporating a selectively cleavable site in an oligonucleotide chain is a compound represented by the formula

<img file="PL170146B1_D0049.tif" />

Pr

170 146 in which DMT is dimethoxytrityl, Bz is benzyl, iPr is isopropyl and R⁶is either methyl or β-cyanoethyl. As with the reagents described above, this residue is also easily introduced into the oligonucleotide chain by known methods. Cleavage at the site containing this residue is carried out chemically, in two stages: (1) by oxidation with an aqueous solution of sodium periodate for an hour and then (2) treatment with an aqueous solution of n-propylamine.

It is understood that the invention has been described with reference to its preferred, specific solutions and the above description and the examples given below are intended to illustrate and not limit the scope of the invention.

Example I. Synthesis of [2- (2-nitrophenyl) -2- / O-dimethoxytrityloxy / ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine / BP1 /:

2- / 0-nitrophenyl / -1.2-ethanediol / 2.5 g, 13.6 mmol / dried by one time evaporation with pyridine. The residue was dissolved in 50 ml of pyridine and 13.6 mmol of 4,4'-dimethoxytrityl chloride (DMT-Cl) was added. The reaction mixture was stirred for 18 hours at 20 ° C. Then almost all the pyridine content was distilled off and the oily residue was dissolved in 250 ml of ethyl acetate. The organic layer was washed with 5% NaHCCO solution (2 times 250 ml) and 80% saturated aqueous NaCl solution 9 (once, 250 ml) and dried over solid Na2SO4. After filtration, the solvent was removed under reduced pressure; the residue was evaporated once with 200 ml toluene and once with 200 ml CH3CN. The product was purified on a silica gel column (eluting with methylene chloride CH2Cl2 with 0.5% triethylamine), yielding 6.5 g (13.6 mmol) (pure product / 100 percent yield).

The purified product, 1-O-DMT-2- (O-nitrophenyl / -1,2-ethanediol, was converted into β-cyanoethylphosphinamide by reaction with 15 millimoles of chloro-N, N-diisopropylamino-2-cyanoethoxyphosphine in 50 ml CH2Cl2 in the presence of diisopropylethylamine / 30 millimoles / which was carried out at a temperature of 10 ° C for 30 minutes. Then 200 ml ethyl acetate was added and the combined organic layer was washed with 80% saturated aqueous NaCl solution (2 x 250 ml) and then dried over solid Na2SO4. After removal of the solvent under reduced pressure, the residue was co-evaporated with toluene (100 mL) and CH3CN (100 mL) to obtain

9.5 g of 2, O-phosphinamide 1-O-dimethoxytrityl-2- (O-nitrophenyl) -1,2-ethanediol (100 percent yield).

Example IL Synthesis of [2- / 2-nitrophenyl / -1- / O-dimethoxytrityloxy / ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine / NP2 /:

2- (O-nitrophenyl) -1,2-ethanediol / 2.5 g, 13.6 mmol / dried by evaporation from CH3CN. The dried compound was dissolved in a mixture of CH2Cl2 (100 ml) and CH3G (10 ml), then N, N-dimethylaminopyridine (100 mg) and triethylamine (3.6 ml, 26 mmol) were added and solid tert-butyldimethylsilyl chloride / TBDMS Cl was added with stirring. / / 2.6 g, 15 millimoles /. Stirring was continued for 18 hours at 20 ° C. Then 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% NaHCO 3 (2 x 250 ml) and 80% saturated aqueous NaCl (1 x 250 ml) and dried over solid Na 2 SO 4. After removal of the solvents under reduced pressure, the residue was evaporated with toluene (200 mL) and CH3CN (200 mL), to give 2.5 g of crude 1-O-TBDMS-2 / O-nitrophenyl / -1,2-ethanediol. The crude product was evaporated with pyridine and the residue was dissolved in pyridine (50 ml). DMT-Cl (30 mmol) was added and the reaction mixture was stirred at 20 ° C for 48 hours. After removal of the solvent under reduced pressure, the residue was dissolved in ethyl acetate (250 ml). The organic phase was washed with 5% NaHCO3 (2 x 250 ml) and 80% saturated aqueous NaCl solution (1 x 250 ml) and dried over solid Na2SO4. After removal of the solvent under reduced pressure, the residue was evaporated with toluene and CH3CN. The residue was dissolved in THF (100 ml) and 10 ml of a 1M solution of tetrabutylammonium fluoride in THF was added. Removal of the 1-O-TBDMS group was completed after 30 minutes. The product was purified on a silica gel column to give pure 2-O-DMT-2- (O-nitrophenyl) -1,2-ethane 170146 diol (2.4 g, 4.5 mmol). This material was carried out in 2-caanoethalphosphiteam as described above in quantitative yield.

Example ΙΠ. A test fragment was prepared using the standard phosphiteαmidone synthesis procedure.

5 '- T15 - 3' - p - NP1 - p - 5 '- T20 - 3' - OH / where p is phosphate /. After complete unblocking, the purified DNA oligomer was dissolved in water and photolyzed for 15 minutes (mercury lamp, λ&gt; 350 nm). Analysis by polyadrylamine gel electrophoresis / PAGE / samples after added photolysis showed that the test fragment was completely split into new fragments that migrated at the expected speed for the T20 and Ti 5 segments.

Example IV Synthesis of 5'-DMT-1 A) - / 2- ^ tnrobenzyjo / 2-deoxyryb0 / .o-3 '"C&gt; -! Tlet.alofosίnrannαrsidy:

Dezndsirinase (10 millimolar), 2-nitrobenzyl alcohol (30 millimolar) and dldhlornodtic acid / DCA, 100 ml / in 100 ml dry acetonitrile were kept under gentle boiling for 2 hours. After cooling to 20 ° C, pyridine was added to neutralize DCA and the solvent was removed under reduced pressure. The residue was dissolved in 500 mL of ethyl acetate, and the organic phase was washed with 400 mL of 5% NaHCO3, 400 mL of 80% saturated aqueous NaCl solution and dried over solid Na2SO4. After filtration, the solvent was removed under reduced pressure and the residue was evaporated with toluene and acetonitrile. The crude reaction mixture was dissolved in CH2CL2 and the product was isolated by silica gel chromatography, eluting with a methanol gradient of 0 to 6%. The fractions containing the product / mixture of α - and β- isomers in a 1: 1 ratio were collected and the solvent removed under reduced pressure to give 2.5 g of a light yellow solid. / 5.2 millimoles, 52% yield.

The residue of O-nitrobenzyl deoxyribose derivative was dissolved in 25 ml CH2Cl2 containing 200 mg dimethylnnopyridine (DMAP) and 1.4 ml triethylamine. To this solution, DMT-Cl (1.7 g, 5 mmol) dissolved in 25 mL CH2Cl2 was added dropwise. After reacting all the starting ingredients, the reaction mixture was diluted with ethyl acetate / 250 ml / extracted, then dried and evaporated as described above. The crude reaction mixture was chromatographed on silica gel, eluting 5'-DMT--2-0-2-nitrobenzyl-2'-desodisrabose isomers in a gradient of 0-3% methanol. 2.3 g of product were obtained in the form of a yellow foam (2.65 mmol).

3-Methylphosphinamide was prepared in a known manner.

5'-DMT-1'-0- (2-nitroyenzyl) -2'-desodsiryose was dissolved in 40 ml of CH2Cl2 containing 2.8 ml of DiPEA and N, N-diisopropylaminomethyl phosphosphine (2.0 mmol) was added at 0 ° C. After 30 minutes, the reaction mixture was diluted with 200 mL ethyl acetate and washed 3 times, 200 mL 80% saturated aqueous NaCl solution, dried over solid N2SO4 and filtered. The solvent was evaporated under reduced pressure, and the residue was co-evaporated with toluene and acetonitrile. This product was used without further purification.

This blocked, non-basic phosphamidamide nucleotide was introduced under standard conditions into the 3'-T20- [1'-C- / 2-nitroyenzyl / -2'-desodsyrayozo] ​​-T oligomer on a solid support. This fragment was unblocked with DCA (to remove residue-5'-DMT), then treated with thiophenol / a mixture of thiophenol, triethylamine and dioxane, 1: 1: 2 by volume, for one hour at 20 ° C to remove the methyl group / and NH4OH / aqueous ammonium hydroxide solution for one hour at 20 ° C to cleave the 3'-yurstate bond /. The supernatant was heated at 60 ° C for 18 hours. In the stability test of the 5'-DMT-r-0- / 2-nitrobenzyl / -2'-deoxyribose residue, virtually no decay was observed. A sample of this material in water was photolyzed for 2C minutes, exposure to high intensity mercury lamp light to remove the 0-nitroyenzyl group from the 5'-DMT-Γ-0- / 2-nitroyenzyl / -2'-desodisrabose residue. No cleavage of this oligomer was observed during this photolysis step. A sample of this oligomer was previously photolyzed incubated in NH4OH at 60 ° C for 2 hours. The base treatment resulted in complete cleavage of the oligomer into two component oligomers, Tio-3'-p and 5 '-p-T20.

These reactions are shown in schemes 5 and 6.

Diagram 5

<img file="PL170146B1_D0050.tif" />

Diagram 6 τ ~

Η + 1 m

<img file="PL170146B1_D0051.tif" />

about

AND

Cleavage of DNA fragments bound through 2-n-trobenzyl-2-deoxyriboside.

Example V. Preparation of N-4- / 0-N, N-diisopropylaminomethoxyphosphinyl-6-oxyhexyl / -5'-DMT-2 ', 3'-dibenzoyl cytidine.

Uridine (24.5 g, 100 millimoles) / dried by evaporation with pyridine (2x150ml). The residue was dissolved in 150 ml of pyridine and dimethoxythritol chloride (34 g, 100 mmol) was added dropwise with stirring. Stirring continued for 48 hours. Methanol (100 ml) was added and after 30 minutes the solvents were evaporated under reduced pressure. The residue was dissolved in 800 ml of ethyl acetate and the organic layer was washed three times with 800 ml of 5% NalcCO3, 3 times with 800 ml of saturated aqueous NaCl solution, dried over solid N2SO4, filtered and evaporated to a dry residue, which residue was then evaporated with toluene and acetonitrile. Chromatography of the crude product on silica gel, eluting with a gradient of 0-7% methanol and 1% triethylamine, gave 46.36 g, 84.9 mmol, 5'-DMT-riburidine, which was dried by evaporation with pyridine, and the residue dissolved in 250 ml of pyridine . To the pyridine solution, at 0 ° C, benzoyl chloride / 20 mL, 170 mmol / 100 mL methylene chloride was added dropwise. After stirring for two hours at 20 ° C, the solvent was removed under reduced pressure, and the residue was evaporated with toluene. This residue was dissolved in ethyl acetate and subjected to the same aqueous treatment as described above for 5'-DMT-uridine.

Crude 5'-DMT-2-, 3'-dibenzoyl-uridine, which was used without further purification, was dissolved in 150 ml of acetonitrile. 1,2,4-Triazol (88.19 g) was suspended in 400 ml acetonitrile at 0 ° C and POCb (27.53 ml) was added under rapid stirring. Then, triethylamine (106.7 ml) was added dropwise to the stirred suspension at 0 ° C over 15 minutes. After 30 minutes, 5'-DMT-2 ', 3'-dibenzoyluridine dissolved in 150 ml of acetonitrile was added to the above mixed suspension at 0 ° C. The ice-water bath was set aside and stirring continued for an hour at room temperature. The reaction mixture was diluted with 1400 mL of ethyl acetate, extracted and dried as above. The solvents were removed under reduced pressure, evaporated with toluene and then with acetonitrile to give 4- / triazo! -1b-D-5'-0-DMT-2 ', 3-dibenzoylorybofuranosyl / -2 / 1H / -pyrimidinone in white foam with quantitative efficiency.

11.89 g / 101.5 mmol / 6-aminohexanol were added directly to a stirred solution of this compound in 350 ml acetonitrile. Stirring continued for 18 hours. The reaction mixture was diluted with 700 mL of ethyl acetate and extracted as above. After drying the organic layer over NaiSO4, the solvent was removed under reduced pressure. The product was purified on a 60H silica gel column, eluting with a concentration gradient of 0 to 50% ethyl acetate in CH2Cl2, to give 35.4 g /41.5 mmol / N-4- / 6-hydroxyhexyl / -5'-0-DMT-2-, 3'-dibenzoyl cytidine in the form of a yellow foam.

The corresponding methylphosphinamide was prepared using standard procedures. Modified nucleotide, N-4- / 6-hydroxyhexyl-5'-DMT-2 ', 3 - dibenzoyl cytidine / 8.7 g, 10.2 mmol / dissolved in 50 ml of methylene chloride containing 8.8 ml / 50 mmol / diiszioyl ethyleneamine, followed by at 1.94 ml / 10 millimoles / N, N-diisopropylaminomethoxychlorophosphine was slowly added at 0 ° C. After 30 minutes, the reaction mixture was diluted with 250 ml of ethyl acetate, then the organic layer was washed twice with 250 ml of 5% NaHCOb solution, twice with 80% saturated aqueous NaCl solution, dried over Na2SO4 and filtered. The solvent was removed under reduced pressure pressure and the residue was evaporated with toluene and acetonitrile. The crude phosphinated material was purified on a silica gel column, eluting with a gradient of 50-70% ethyl acetate in methylene chloride containing 2% triethylamine, to obtain 7.25 g of N-4- / 0, N, N-disopropylaminomethoxyphosphoryl-6-oxyhexyl / -5^/-DMT-2 ', 3 - dibenzoyl cytidine.

Example VI. In the final ribonucleoside of RNA molecules, oxidative cleavage of the cis-diol system easily occurs under the action of sodium periodate. In the presence of amines, the resulting dialdehyde easily eliminates both the basic and phosphate residues at the 5 'carbon. This example describes the use of this phenomenon to build a fissile site molecule in which two DNA oligomers are linked through the 5 'end and side chain hydroxyl groups of the N-4- / 6-hydroxyhexyl /-cytidine molecule.

Modified ribonucleoside R containing an exocyclic alkylhydroxy group was synthesized from uridine. Protected ribonucleoside R phosphinamide was introduced under standard conditions into the 5 '-Tio-R-Ti5-3' oligomer on a solid support. Samples of the purified product were treated with a series of chemical reagents and then analyzed by polyacrylamide gel electrophoresis (PAGE). No oligomer cleavage was observed after treatment with ammonium hydroxide at 60 ° C for 18 hours. Treatment with sodium periodate in water at 4 ° C resulted in partial cleavage over 30 minutes. Further action on the periodate treated with periodate n-propylamine oligomer in triethylammonium acetate at 60 ° C for 90 minutes resulted in complete cleavage of the oligomer into T w-3'-pi fragments at T15, modified at the 5 'end.

Scheme 7 shows the cleavage of ribonucleoside R-linked DNA fragments.

7 bottom scheme₂-about

<img file="PL170146B1_D0052.tif" />

B n + 1

Lita₂ -ABOUT

<img file="PL170146B1_D0053.tif" />

N

B.

o / dna₂-about

<img file="PL170146B1_D0054.tif" />

N - ^ 0

<img file="PL170146B1_D0055.tif" />

CHO CHO n-PrNH₂

TEAA 1 hour 60 ° C n-1 p

n + 1

<img file="PL170146B1_D0056.tif" />

ΗΟζ Χν ^ ^^ ΟΗ '

propyl

170 146

The above cleavage scheme was applied to a number of branched dNa oligomers, where blocked ribonucleoside R phosphinamide was introduced during the first cycle of secondary synthesis of solid-supported linear oligomers containing 10, 20 branch points. In each case, secondary synthesis was carried out using the T10 oligomer, producing branched oligomers with the following structure: 3'-T20-En-5 '/ branch point -3'-R-Rio-5' /_n n = 10, 20, 30. These molecules were subjected to conditions under which fission took place. Analysis by polyacrylamide gel electrophoresis showed that all branch oligomers (side arms) were cleaved and in all cases the main product was Tn) -3'-p. This analysis also showed that the spread of fission products depends on the number of branches in the branched DNA molecule, with the number of shorter oligomers increasing as the number of branches in the molecule increases. The decrease in homogeneity appears to be mainly due to ionic spatial forces within the solid support during chemical synthesis.

Example VII. The smaller example describes the production of the multifunctional linker DMT-E '/ Lev / BCE-phosphinamidamide in the reactions shown in Scheme 8.

Diagram 8

<td rowspan="2">cff₂0H CH ^ - C - ch₂oh CHgOH E '</td><td rowspan="2">chloride toluenesulfonyl / TsCl /.</td><td colspan="2">CH_about0H 1 &lt;-</td>

<td>CH, - C 1 these</td><td>-CHgOH S₂ - 0 - Ts E 7Ta /</td>

<td>&lt;HU₂0H</td><td>levulic acid</td><td> &gt;</td><td>DKTtCI t h₂oh</td>

<td rowspan="2">CH, - C - CH "-0-DHT ³ 1 ²CH₂-0- Lev</td><td>cesium salt</td><td>ob₃ -0</td><td>- CHg - 0-DMT</td>

<td></td><td colspan="2">CH₂ - 0 - Ts</td>

<td>DOT-E7Lev /</td><td></td><td colspan="2">DHT-E7TS /</td>

<td colspan="2">CNCHoCHoO-P ^^¹^{2 2} \ N / 1Pt /₂</td><td></td><td></td>

&lt;N / l PRE I - 0CH₂CH₂CN

CH, - C - CH_about0DMT 3 2

CH₂-0-Lev

DMT-E '/ Lev / BCE phosphinamidamide

Tris-hydroxymethylethane (E ', 200 mmol) was evaporated from 250 ml of pyridine, and the residue was dissolved in 125 ml of pyridine. To this solution, cooled to 0 ° C, was added dropwise a solution of toluenesulfonyl chloride / TsCl 50 millimoles / in 125 mL CH2Cl2.

The reaction mixture was allowed to warm to room temperature and continued stirring for a total of 5 hours. The solvents were then evaporated under reduced pressure. The residue was dissolved in 500 ml ethyl acetate and the organic layer was washed twice with 500 ml 5% NaHCO3 solution, once with 500 ml 80% saturated aqueous NaCl solution and finally dried over solid Na2SO4. After filtration, the solvent was evaporated under reduced pressure. 13.7 g of crude E '/ Ts / were obtained. This product was used without further purification. The total amount of E '/ Ts / was dissolved in 250 mL CH2G2 and triethylamine / 14 mL added; 100 millimoles / and N, N-dlmgtylamine pyridine / 100 ml /. DMT-Cl / 13.6 g was added to this solution; 40 millimoles / dissolved in 125 ml CH2O2. 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.

The crude reaction product was purified on a standard column packed with silica gel (800 mL silica), eluting with a methanol gradient in a mixture of CH2G2 and 0.5% methylamine. 14.8 g / 25 millimoles / pure DMT-E '/ Ts / were obtained. The entire amount of this product was treated with 50 millimoles of freshly prepared cesium salt of levulmic acid / according to the recipe given by M. Bodanszky and A. Bodauszky in The Practice of Peptide Sduthesiu, p. 37, ed. Springer Verlag / 1984 / in 50 ml DMF. The solution was heated on a hot plate in a sealed vial / heating set to position 3, temperature about 100 ° C / for 18 hours. The analysis showed that after this time the reaction was complete. DMF was evaporated under reduced pressure and the residue was dissolved in ethyl acetate. The organic phase was washed as described above. The crude product was chromatographed on silica gel. The pure product was eluted with methylene chloride with the addition of 0.5% triethylamine to give 2.5 g / 4.8 mmol (pure DMT-E '/ Lev), which was converted into 2-cyanoylethyl-phosphinamide as follows:

DMT-E '/ Lev / was dissolved in 20 mL CH2Cl2 containing 2.6 mL / 15 millimol / N, N-dichloropropylethylamine and cooled to 0 ° C; to this solution was added, under an argon atmosphere, by syringe, 1.1 ml / 5 millimoles / 2-cyanoethoxy-N, N-diisopropylamluochlorophosphide. After about 30 minutes, the reaction was complete; then the reaction mixture was diluted with 150 ml of ethyl acetate. The organic phase was washed twice with 150 ml of 5% NaHCO3 and twice with 150 ml of 80% saturated NaCl solution. After drying over solid Na2SO4, the solution was filtered and evaporated to a dry residue, obtaining 3.6 g of product in the form of a white foam. The crude product, DMT-E '/ Lev / BCE phosphinamidamide, was purified on a silica gel column and eluted with a mixture of methylene chloride, ethyl acetate and triethylamluy / 45: 45: 10, by volume /, to give 3.24 g / 4.5 millimole / pure DMT-E '' / Lev / BCE fouforyuoamldu in the form of white foam. NMR ^ P / O = 148.5 ppm; Coupling efficiency: 98%.

Example VIII. This example describes and illustrates in Scheme 9 an alternative synthesis for the multifunctional linker DMT-E '/ Lev / BCE of flavofuoamide.

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Diagram 9

CH, CH, OH AND ²

C - CH? OH I

CH₂OH up 6₃these

CH.

C - CH, - O ²CH₂ - O - Lev

DMT

4r

CLOTH

CH, - C ch₂oh ch₂ - O - Si {b₅E * (TPS)

DMT-C1

DMT-E '(lev)

CH_ABOUT - OH AND ²

CH, - C - CH_ABOUT - O - DMT ⁵ AND ²

CH₂-0-Lev levulinic acid

EDIC *

CH,

CH_about0H AND ²

-C - CH₂ - O - DMT ch₂ - O - si &lt;£&gt;₅DMT-e '(TPS)

X cnch_ch_aboutcp ^ ^{2 2 x}N (iPr), ch₂- O - P

N (iPr)₂

CH, - C - CH_about0-DMT ⁵ and _T

CH₂- o - Lev oh₂ch₂cn

DMT-E '/ Lev / BCE phosphinamidamide

EDIC = 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. 0 is phenyl

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Tris- / hydroxymethyl / ethane / 200 millimoles / evaporated from 250 ml pyridine and the residue dissolved in 125 ml pyridine. To this solution cooled to 0 ° C was added dropwise a solution of 50 millimoles triphenylchlorosilane / TPS / in 125 ml CH2Cl2. The reaction mixture was left to warm to room temperature while continuing to stir for a total of 18 hours.

The solvents were then evaporated under reduced pressure, the residue dissolved in 500 ml ethyl acetate and the solution washed 2 times with 500 ml 5% NaHCO3, once with 500 ml 80% saturated aqueous NaCl solution and dried over solid Na2SO4. After filtration, the solvent was evaporated under reduced pressure to give 18 g of crude E '/ TPS / which was used without further purification. The whole amount (46 millimoles / E' / TPS) was dissolved in 250 ml CH2G2 and 14 ml / 100 millimoles / triethylamine and 100 were added mg N, N-dimethylaminopyridine. To this solution, 50 millimoles of DMT-Cl dissolved in 125 mL of CH2G2 were added. 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 a yellow foam product.

This crude product was purified on a standard silica gel column / 800 mL silica / eluting with a methanol gradient in a mixture of CH 2 Cl 2 and 0.5% triethylamine. 14.8 g / 25 millimoles (pure DMT-E '/ TPS) were obtained. Pretty much 10 mmol DMT-E7TPS / dissolved in 50 ml containing 10 mg N, N-dimethylaminopyridine and 2.3 ml / 20 mmol / 2,6-lutidine and 2.3 g / 20 mmol / levulinic acid were added.

Then, 3.83 g / 20 mmol / 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride dissolved in 50 ml CH 2 Cl 2 was added dropwise to this solution. After 18 hours, the reaction was complete / analysis by thin-plate chromatography (tlc). The reaction mixture was diluted with 500 ml of ethyl acetate and subjected to the same aqueous treatment as described above. The residue from this treatment was dissolved in 50 ml of THF and 40 ml of pyridine were added first, followed by 10 ml of concentrated acetic acid and finally 20 ml of 1 m tetrabutylammonium fluoride in THF (Aldrich).

Thin layer chromatography after 30 minutes showed that all starting materials had been consumed. Most of the solvent was then evaporated under reduced pressure and the residue was subjected to the following aqueous treatment: 250 mg of ethyl acetate were added to dissolve most of the organic material, followed by the slow addition of 250 ml of 5% aqueous sodium bicarbonate solution (CO2 evolved).

Solid NaHCO 3 was then added, stirring and dissolving it until salt precipitate began to form and CO2 evolution ceased. The combined aqueous-organic solution was transferred to a separatory funnel and the organic layer was washed as above. After removal of the solvent, 5.96 g of crude DMT-E '/ Lev / was obtained as a clear oil. This product was isolated by silica gel chromatography using approximately 500 g of silica, and a mixture of CH2Cl2 and 0.25% triethylamine with the addition of C% and 1% methanol for elution. 2.7 g /5.2 millimoles / DMT-E '/ Lev / were obtained in the form of a transparent, colorless oil.

Pure DMT-E '/ Lev / was converted to 2-cyanoethyl phosphite-amide as follows: DMT-E' / Lev / dissolved in 20 ml CH2G2 containing 2.6 ml / 15 mmol / N, N-diisopropylethylamine and cooled to 0 ° C. To this solution was added, under an argon atmosphere, by syringe, 1.1 ml / 5 mmol / 2-cyanoethoxy-N, N-diisopropylaminochlorophosphine. After about 30 minutes, the reaction was complete. The reaction mixture was then diluted with 150 mL ethyl acetate and the aqueous layer was washed 2 times with 150 mL 5% NaHCO2 and twice with 150 mL 80% saturated NaCl.

After drying over solid Na2SO4, the solution was filtered and evaporated to dryness. 3.4 g of DMT-E '/ Lev / BCE phosphinamidamide were obtained in the form of a white foam. This crude phosphinamide was purified on a silica gel column, eluting with a solvent mixture of CH 2 Cl 2 / ethyl acetate / triethylamine (45: 45: 10, by volume). received

2.4 g / 3.3 millimoles / pure DMT-E '/ Lev / BCE phosphinamidamide in the form of white foam.

NMR / 3 * P / δ: 148.5 ppm. Coupling efficiency: 98%.

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