PL170146B1 - Method of defecting presence of a given oligonucleotide sequence - Google Patents

Abstract

This invention relates to a method for detecting the presence of an oligonucleotide sequence of interest and to new reagents useful in a variety of biochemical and chemical contexts, including nucleic acid hybridization assays and chemical phosphorylation of compounds containing hydroxyl. The reagents are particularly useful for introducing cleavage sites and/or abasic sites into oligonucleotide and polynucleotide chains.

Description

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

Detection of the presence of a given oligonucleotide sequence is possible thanks to the introduction of cleavable sites selectively into the oligonucleotide and polynucleotide chains. These techniques are described in U.S. Patent Application Serial Number 251,152 and in U.S. Patent No. 4,775,619, which is evidently cited in this specification. Selectively cleavable sites are useful in many different types of hybridization techniques. For example, in one type where hybridization results in a solid support duplex of the tagged probe and the DNA sample, a cleavage site selectively contained in the hybrid structure allows the tagged probe to be directly separated from the solid support. US Patent No. 4,775,619 is primarily concerned with the use of restriction endonuclease cleavage sites in this type of assay. Chemically cleavable sites can also be used, tadiejud disarray bonds,

1,2-diols and the like. Such sites are introduced during the synthesis of olonudleotide. 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 cleavable site by other methods, including the use of chemical or enzymatic reagents, e.g., reducing agents. Cleavage sites are created by introducing chemical moieties, preferably photosensitive moieties, into the chains of oligonudleotidones or pnlinudleotyes. Tadie new photosensitive moieties are useful in many different types of hybridization assays, including those described in the above-cited patent applications as well as in the nucleic acid amplification and hybridization assay disclosed in European Patent Application No. 88.309697.6 by the applicants of the present invention.

Another use of these above-mentioned and other reagents in general is the formation of non-basic sites within the structure of oligonucleotides. The term non-basic site herein denotes an -OR ether residue at a position in which the hydroxyl group -OH or a nucleobase would normally occur.

Yet another use for such reagents is in chemical phosphorylation. Chemical phosphorylation of hydroxyl groups is essential in many different aspects of oligonucleotide chemistry. An example is the synthesis of oligonucleotides where the steps of synthesis and deprotection must be phosphorylated on the free 5'-hydroxyl group of the oligonudleotad in order to make it suitable for use in most biological processes. It also requires phosphorylation of the 3'-functional group for hadrodsalowej / 1 / prevent extension of the 3 'polymerase and 2 in the chemical ligation of DNA, which generally wymaganajest presence of residues 3 ^/-phosphate during the coupling of oligonucleotides using chemical methods.

The 5'-phosphatation reaction is generally performed using a T4 polynucleotide kinase and ATP. This reaction is neither very precise nor very 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 in Tetrahedron Lett. 23, 4793-4796 (1982), Marugg 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 either unstable reagents or complicated modification of standard deprotection and purification procedures. Similar difficulties exist with monofunctional and bifferential reagents for 3'-phosphoralα (see Sonveaux, previous entry, p. 297).

Thus, in addition to being useful in the process of introducing cleavable and / or non-basic sites into olieonudleotide or polynudleotide chains, many compounds may be used as phosphorylation reagents which overcome the limitations of the prior phosphorylation procedures / they may also be useful in phosphorylation reactions which are used in commonly accepted purification schemes by dimethodsytratylone derivatives (DMT). References generally relating to methods for synthesizing oligonudleotads include those in which 5 'to 3' syntheses based on the use of β-giveanoethalphosphate protecting groups are described. These include, for example, 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). 5 'to 3' syntheses in solution 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 v

J. Am. Chem. Soc. 103, 3185-3191 / 1981 / the use of phosphite chlorides in the production of oligonudleotides. The use of phosphoramidate in 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 automated solid phase synthesis of oligodeoxyribonucleotide. It is also the subject of the above-cited publications, and of Warner et al. In DNA, 3, 401-411 (1984), the disclosures of which are cited herein.

Horn and Urdea describe in DNA, 5, 5: 421-425 (1986) the phosphorylation of solid support DNA fragments using bis (cyanoethoxy) -N, N-diisopropylaminophosphine. This method is also reported 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) provide 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 for the detection of specific oligonucleotide sequences. Ranki et al. In Gene 21, 77-85, describe a technique they refer to as sandwich-type hybridization for the detection of oligonucleotide sequences. Pfeuffer and Helmrich in J. Biol. Chem. 250, 867-876! X) -15I, report coupling of guanosine-5'-O- (3-thiotriphosphate) with 4B sepharose. In J.Histochem.and Cytochem. 29,227-237, Bauman et al. Describe the labeling of 3'-RNA with fluorophores.

PCT patent application WO / 8302277 discloses the addition of modified ribonucleotides to DNA fragments for labeling purposes and methods for analyzing such DNA fragments. Renz i Kurz in Nucl.Acids Res. 12, 3435-3444 describe the covalent linkage of enzymes to oligonucleotides. Wallace, in DNA Recombinant Technology / Ed. Woo S. CRC Press, Boca Raton, Florida, reviews the use of probes in diagnostics in general. Chou and Merigan in N.Eng.J.of Med. 308, 921-925, report the use of a radiolabeled probe for the detection of cytomegalovirus. Inman in Methods in Enzymol., 34B, 24, 77-102 / 1974 / describes binding procedures to polyakylamides and Parikh et al. In Methods in Enzymol, 34B, 24.77-102 I)) 4 describe agarose coupling reactions, 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 support. Chu et al. In Proc. Natl. Acad. Sci / USA 11, 6513-6529, describe a technique for derivatizing 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 for 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 photosensitive protecting groups.

The publication of K. Groebke et al. In Helvetica Chim.Acta 73, 608-617 / I)) 0 / is related in that 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 test nucleic acid, which consists in combining said nucleic acid sample with a polynucleotide reagent of the formula under hybridization conditions.

OH

OH where DNA1 represents the first DNA segment, DNA2 represents the second DNA segment and one of the symbols x and y represents zero, while the other one represents an integer from 1-12 inclusive, with either the sample or the reagent being previously deposited on the support and as a result hybridization of the nucleic acid to be tested with the polynucleotide reagent creates a tag bound to the carrier via a cleavage site

-O- / CH ₂ / _a -CH- / CH ₂ / _b -O-,

I- ^NO 2 and then substantially liberating this carrier from the carrier bound label other than through this selectively cleavable site, whereafter this cleavage site is cleaved by photolysis using light of at least 350 nm wavelength and the label released from the carrier is detected . Preferably, this method uses a polynucleotide reagent of the formula I, wherein the symbol x is zero, the symbol y is an integer from 1-4 inclusive, or the symbol y is 1 or zero. Preferably, a reagent of formula I is used in which the symbol x represents an integer of 1-4 inclusive or in which the symbol x is 1.

In another embodiment of the invention, a method for detecting a given oligonucleotide sequence in a test nucleic acid present in a nucleic acid sample is by combining the nucleic acid sample with a polynucleotide reagent of formula in an aqueous medium under hybridization conditions.

0.

S 'V II He' 3

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

OH

J.

OH tor

HO,

AND

OH where DNA1 represents the first DNA segment, DNA2 represents the second DNA segment and one of the symbols x and y represents zero, while the second one represents an integer from 1-12 inclusive, wherein either the sample or the reagent component is previously deposited on the support and by hybridization the nucleic acid under study creates a tag attached to the carrier through the cleavage site with the polynucleotide reagent,

-0- / 0H ₂ / _a -CH- / CH ₂ / _b -0-

ho _2, and then the carrier with the bound polynucleotide reagent and the nucleic acid to be tested is separated from the aqueous environment, then this carrier is washed with a medium of a different hybridization strength than this aqueous medium in order to remove the label bound to the carrier other than through the cleavage site, then cleaved this fissile site is photolyzed using light having a wavelength of at least 350 nm and the label released from the support is detected. In this embodiment, the polynucleotide reagent of formula 2 is preferably used, in which the symbol x is zero and the symbol y is an integer from 1-4, inclusive, or the symbol y is 1 or zero.

In another preferred embodiment, a reagent is used in which, in the above formula, x can represent an integer of 1-4 inclusive. Preferably, in the above formula, y is an integer from 1-4 inclusive, especially 1. Preferably, y is the number 1.

Another embodiment of the method of detecting the oligonucleotide sequence in the test nucleic acid present in the sample is a nucleic acid that are combined under hybridization conditions said nucleic acid sample with the polynucleotide reagent of the formula 3 '3 ¹¹

-HO ⁹ / DNA - / ⁹ -0-P-0

-1 l

OH

IS '3' _H0 -p—— O- ⁹ / DNA _O / ⁹ -OH

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

170 146 where R 'is hydrogen, an aryl or an oryolkyl group, R _t may be the same or different and represent an amino, nitro, halogen, hydroxyl, lower alkyl, or lower alkoxy group, Rj may be the same or different i denote amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy group, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R and _n is containing 1-16 carbon atoms alkylene or oxyethylene oligomer - / CH2CH2OA - where z is an integer from 1-16 inclusive and Rn is a group

or ch ₃ o-ch ₂ ch ₂ -o-ch ₂ -.

wherein either the sample or the reagent is previously deposited on the support and hybridization of the nucleic acid to be tested with the polynucleotide reagent creates a label attached to the support via a cleavage site of formula

where R is 2-nitrobenzyl, 4-penten-1-yl,

-ch ₂ ch ₂ s- / Q

CH ₂ CH ₂ Si / CH _3/3,

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

0

II II

CH ₂ -C-CH ₂ CH ₂ -C-,

170 146

Ο

0Η ₃ 0-0Η2-σΗ ₂ -0-σΗ ₂ and thereafter essentially frees this carrier from the carrier bound tag other than through this selectively cleavable site and then cleaves this cleavage site by photolysis using wavelength light at least 350 nm and the label released from the support is detected. Preferably, in the process according to the invention, a reagent according to the given formula is used, in which R is a 2-nitrobenzyl group or a group

- ^{0H 0H} 2 2 ^S - <(O / 'or wherein R is

or wherein R is 2-mgtdlgur-9,10anthraquinruracgtal, or R is 4-οthen-1-yl, or R is 4-pgntgn-1-yl, or wherein R is

-ch ₂ ch ₂ -ZQ \ - no ₂ or also R is

AND

After

And oh **

In a further variant, the method of detecting a given sequence of oliornuę | gotydrwgj in the tested acid nuę | grurvic present in the sample of uuklginrwegr acid consists in combining under hybridization conditions, in an aqueous medium, this sample of nuuguic acid with the reagent prliuuęlgotdowdm of formula

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

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

0

II II

GH ₂ -C-GH ₂ OH ₂ -C-, o

0 ₂ N—,

V /

S-CHoCHo-O-Cc c.

Q \ -s-ch ₂ ch ₂ -oc-

or

CH ₃ O-CH2CH ₂ -O-CH ₂ wherein either the sample or the reagent component is previously deposited on the support and hybridization of the nucleic acid to be tested with the polynucleic acid reagent creates a label bound to the support via a cleavage site of formula

where R is a 2-niirobenzyl group, 4-ppnten-1-yl group, groups of the formulas:

-P-0

L o

or

CH ₂ CH ₂ Si / CH _3/3

-R

0-R,

CH ₂ CH ₂

no ₂

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

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

and then isolating the carrier with the bound polynucleotide reagent and the test nucleic acid from the aqueous medium, then washing this carrier with a medium with a different hybridization strength than the aqueous medium in order to remove the label bound to the carrier other than through the cleavage site, then cleaving this a cleavage site by photolysis using light with a wavelength of at least 350 nm and the label released from the support is detected. In preferred embodiments, a polynucleotide reagent and the formula given are used, wherein R is a 2-nitrobenzyl group, or

CH ₂ CH ₂ S

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

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

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

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

Additional advantages of the invention are set forth in part hereinafter, and in part will become apparent to those skilled in the art upon examination of the invention, or may be learned by practicing the invention.

The new reagents used in the process of the invention are light-sensitive chemicals of the general formula

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

Other new reagents used are compounds of the general formula:

wherein R 1 R ² and R are as defined below. Such compounds are useful for creating non-basic sites in oligonucleotide chains. These sites are cleavable or non-cleavable.

Still other new reagents used are compounds of the general formula

CH _O - 0 - R ¹

I ² '

CH-, - C - CH _O - 0 - R ' ³ I ²

Cl ₂ - 0 - R ^

2 wherein R, R and Rn are as defined below. Such compounds are used to create branch points during the synthesis of nucleic acid multimers.

The method of carrying out the invention.

A. Definitions.

The term selectively cleavable site denotes a functional or multifunctional site that is selectively cleavable. As mentioned above, the sites which are specifically cleavable by photolysis are of primary interest in the invention.

The terms oligonucleotide and polynucleotide as used herein are general terms, including polydesoxyribonucleotides / containing 2'-deoxy-D-ribose or its modified forms /, polyribonucleotides / containing D-ribose or its modified forms / and any other type of polynucleotide that is an N-glucoside base a purine or pyrimidine base, or a modified purine or pyrimidine base. Similarly, the term nucleoside is generic to ribonucleosides, deoxyribonucleosides, and any other nucleoside that is an N-glucoside of a purine or pyrimidine base or a modified purine or pyrimidine base. There is no predetermined delimitation in length between the terms oligonucleotide and polynucleotide, and throughout this specification the terms are interchangeable. These oligonucleotides and polynucleotides can be single-stranded and double-stranded, typically single-stranded. The oligonucleotides used in the present invention generally consist of from about 2 to about 2000 monomeric units, and typically, for most probe applications, from about 2 to about 100 monomeric units.

The term nucleic acid sample denotes a sample in which the presence of a nucleic acid sequence to be tested is suspected. The analyzed nucleic acid means that the DNA or RNA in the nucleic acid sample contains the sequence being tested.

The term phosphorylation reagents denote compounds which, upon reaction or a series of reactions with a hydroxyl-containing compound, yield the phosphoric acid monoester.

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

By aromatic substituents is meant any aromatic ring, optionally substituted on one or more carbon atoms with moieties which do not substantially alter its function or reactivity.

B. Chemical structure of the new light-sensitive reagents:

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

R ¹ - 0 - / 0Η ₂ / _χ - CH - / CH ₂ / _y - 0 - R ² where R ¹ is an alkaline and acid labile protecting group, R ² is a phosphorus derivative allowing the addition of the reagent to position 5 'of the chain uuęlgrzydowgor or oligonulgotddrwgoo, 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 where x is a number other than zero and y is zero 1, never called NP-type reagents in this description1) and (2) structures where x is zero andy is another number than zero (called NP2-type reagents). The two types of structures, as can be easily deduced from the general formula above, are quite similar. Both types are suitable for introducing specific sites in the rligonui chains. Due to the presence of a nitrifinous residue, these sites are readily soluble by photolysis. However, as described in greater detail hereinafter, 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 novel light-sensitive magentacia group is as follows: R 1 as stated above is a protective group which is alkaline stable and acid sensitive. Such protecting groups are known in the synthesis of gotdd. These include uigprdutavir or substituted aryl or arylalkyl groups where aryl is e.g. phenyl, naphthyl, furanyl, phenyl or a similar group in which the substituents of 0 to 3, typically 0 to 2, are any, not interacting with each other stable uigpolarue groups or polar, electron donating or donating. Examples of such groups are dimethocytrdtyl (DMT), monomgtocytrityl (MMT), trityl, and 9-phenyl-9-xatenyl-1puyl).

A moiety which is particularly preferred for use herein is dimgtokuytrdtyl (1DMT). ₂

Rp is a phosphorus derivative selected to facilitate the condensation of the reagent with the 5'-hydroxyl group of the nucleoside chain or oligonelotide chain. These types of groups include fouforduoamides, phosphotrigstrid, foufodigstrid, phosphites, hydrogenphosphonia, thiofouforauy and the like (see, inter alia, European Patent Application Publication No. 0225807, inventor: Urdea et al., Title: Solution Sandwich Test of Nucleic Acid and Used in the probe polynucleus. The disclosure contained in this publication is cited in this description). Particularly preferred groups as substituents for R 2 are the phosphite amides of the formula:

N (lPr) ₂ where Y is a methyl or β-cyjauoglutyl group and iPr is an isopropyl group. NajęoredstuigJ, Y is a β-cyanrgtyl group.

From the definitions given above, it can readily be concluded that the substituents R 1 and R 2 are chosen so as to allow the introduction of a light-sensitive reagent into the DNA fragment using a standard phosphinamide technique. That is, for the synthesis of the ollgouucleotide, the substituent r2 is chosen to react with the 5'-hddroxyl group of the uctoside chain or the oligonucleotide chain, and the residue R1 is selected to allow reaction with the 3'-hydroxyl group of the nucleoside chain or the ollgouucleotide chain.

With respect to the symbols x and y, one 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 falling under the above general category are the following compounds:

170 146

"ΝΡ1"

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

C. Synthesis of the above reagents:

NP1-type reactants, that is, where x is a number other than zero and y is zero, are synthesized by following the reactions outlined in Scheme 1. NP2-type reactants are synthesized by following the reactions outlined in Scheme 2.

Scheme 1

HO - CH_ ²

CH - OH

DMT - 0 -

DMT-Cl pyridine? ^H 2

^ N (iPr) ₂

Cl - P ₂ CH ₂ CH ₂ CN

DiPEA ch ₂ oi _ł

WITH

CH.CH.CN

170 146

Scheme 2

HO - OH,

TBDMS - O - CH ₂

CH - OH

0Γ '

TBDMS-Cl _ dmap / tea ch ₂ c and ₂

DMT-C1 ~ dmap / tea oh ₂ ci ₂

TBDMS

TBAF

THF ~

- DMT

WELL,

Cl - P yi (iPr).

OCH ₂ CH ₂ CN

DiPEA ch ₂ ci ₂ (iPr) ₉ N _x ncch ₂ ch ₂ o

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

DMT "= dimethodsaeratyl; dMT-C1 = eimethodsatal chloride; iPr = isopropyl; DiPEA = diisopropyl chloride; αmna; TBDMS-Cl = t-yutalndimethalsillyl chloride; dMAp = 4-dimethyl-monopyridine; TEA = triethylamine; TBAF = tetrαyytaloαmonion fluoride.

The synthesis of NP1-type reagents consists in blocking the hydroxyl terminal group of 2- (O-nitrophen) -1,2-ethanndiol with compounds with R 1 group, e.g. DMT or the like, and then reacting the remaining hydroxyl group with the appropriate phosphorus derivative, leaving the R "residue. In Scheme 1, an exemplary reagent for the latter purpose is chloro-N, N-diisopropalαmino-2-dylαnoetndsphosphine. One can easily deduce narratives of this basic scheme. For example, monomethodsatalium chloride, tratalium chloride, 9-dichloro-9-phenalxanthene, or the like can be used as an alternative to dimethroditatal chloride to introduce other R1 substituents.

170 146

Similarly, in order to introduce other substituents R ^2, in a second reaction step, alternative substituted phosphines. For the change of x, the starting material should contain additional methylene groups.

In order to synthesize the NP2-type reagent, m is those in which x is zero and y is a number other than zero, a similar sequence of reactions, with the proviso that the order of introduction of the substituents R ¹ and R 2 is reversed. Thus, initially, a 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 where the remainder is the free hydroxyl group is reacted with a base-stable and acid labile blocking group, e.g. dimethoxytrityl chloride (DMT-Cl) to introduce the R1 substituent. The terminal hydroxyl group is then deprotected, e.g. with tetrabutylammonium fluoride and, as in scheme 1, reacted with an appropriately substituted phosphine derivative introducing the residue R2.

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

The novel, light-sensitive reagents used in the method of the invention are readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite technique, well known and described in many of the references cited above.

Most generally, the introduction of this new reagent into a DNA fragment requires the formation of a 5'-hydroxyl bond at R2 and a 3'-hydroxyl bond at R1.

Thus, after introducing the light-sensitive reagent, the hybrid oligonucleotide chain will have the following structure:

"" HO ⁵ /? ³ DNA / DNA ^_2/3 - OH

p - o I

OH where DNA1 is the first DNA segment, DNA2 is the second DNA segment, and x and y are as defined above. DNA1 and DNA2 may be linear or branched. Such a polynucleotide reagent may be used in hybridization assays, such as those described in European Patent Application Publication No. 88,309,203.3 by the present applicants and in U.S. Patent No. 4,775,619. These assays use linear polynucleotide reagents containing selectively cleavable sites, that is, those in which DNA1 and DNA2 are linear. A polynucleotide reagent containing a photosensitive moiety can also be used in the amplification assays disclosed in U.S. Patent Application Serial Nos. 07 / 252,638 and 07 / 340,031, incorporated herein (see also PCT Publication No. WO89 / 03891). As reported in these applications, cleavable linker molecules are introduced into amplification multimers at specific sites either to analyze the structure of the multimer or as a means of releasing specific segments (such as a portion of the multimer that binds to the tagged oligonucleotide). In such applications, DNA1 and / or DNA2 are branched polynucleotide segments. After synthesis and purification of the multimer, the branched polynucleotide structure of the multimer can be specifically cleaved without further degrading the nucleotide structure. It is preferred that the cleavage sites be introduced at or near the linkage sites of the multimer as this allows quantification of the individual multimer branches.

Depending on whether the light-sensitive reagent incorporated into the oligonucleotide or polynucleotide belongs to the NP1 type / i.e. where x is mu than 0 and y is 0 / or to NP2 type / i.e. where x is zero and y represents a number other than zero /, two different types of fragments are produced after cleavage. As shown in Scheme 3, cleavage of an oligonucleotide containing an NP1 residue results in a first fragment having a 5'-phosphate group and a second fragment having a 3'-end 2-nitrosophyll residue.

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

photolysis (UV light> 350 nm; mercury lamp)

5'-HO ⁵ (DNA.) ⁵ -OP- (CH) ¹ IX

OH 0 = 0 in

c ^z x ^z

5'-H0-P-O- (DNA-) ⁹ -OH

OH

WELL

17 (0 146

Scheme 4

5'-HO- ⁵ \ BNA1) ^3, -0-i> -0-CH- (CH2) _y -0-P-0- ⁵ - (DNAg) -OH

OH

OH .NO, photolysis (UV light> 350 nm mercury lamp)

Since the cleavage 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 free from contamination by externally introduced cleavage agents is obtained. Moreover, the polynucleotide reagent itself is more stable as such, cleavable only by exposure to UV light of the appropriate wavelength.

E. Phosphorylation using the reagents described above.

The above-described reagents, in addition to being useful in the procedure for incorporating photosensitive cleavage sites, are also suitable as reagents for chemical phosphorylation. Phosphorylation with the use of these reagents consists in their condensation with a compound containing a hydroxyl group and subsequent photochemical cleavage to release the nitrophenyl group. In this respect, these new reagents are quite versatile as they can be used for both the 5 'and 3'-phosphorylation of a nuclpodide or ligonuclpotide chain.

For 5'-phosphorylation, an NP1 type reagent is used, that is, a reagent in which x is other than zero and y is zero. As shown in Scheme 3, cleavage of the polynucleide reagent containing the NP1 type molecule produces a nucleoside or DNA fragment containing a 5'-phosphate group.

An NP2-type reagent is used for the 3'-phosphorylation as illustrated in Scheme 4. Cleavage of the NP2-type polynuclearide reagent results in cleavage fragments, one of which contains a 3'-phosphate group and the other one contains a niirose phenyl moiety.

170 146

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

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

wherein R 1 and R ² are as defined above in part A of this section, and R is 2-nitrobenzyl, 4-penten-1-yl and groups of the formulas:

-ch ₂ ch ₂ sZ Q

-CH ₂ CH ₂ Si / CH _3/3,

in which R 'represents hydrogen, an aryl or arylalkyl residue, where the aryl or arylalkyl residue preferably contains 1-8 carbon atoms, the R substituents may be the same or different and represent the following groups: amino, nitro, halogen, hydroxyl, lower group alkyl and lower alkoxy, Rj may be the same or different and represent: 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 replaced with hydrogen without disturbing the R1 substituent, such as those represented by the formulas:

170 146

Ο

CM = ν »ΑCH3-O-CH ₂ CH ₂ -O-CH ₂ and Rm is either an alkylene oligomer containing 1 to 16 carbon atoms, more preferably 2 to 12 carbon atoms or an oxyethylene - (CH2CH2O) oligomer _with -, where z is an integer of 1 to 16, more usually 2 to 12 inclusive. In an optimal embodiment, if R is a 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 an oligonucleotide or polynucleotide chain, but also, like the reagents described above, are useful for introducing cleavage sites.

If R represents a remainder represented by the formula:

then it is preferred that R 'is hydrogen or phenyl. As noted above, Ri and Rj may be any of a wide variety of substituents. In a particularly preferred embodiment, the above structure is 2-methylene-9,10-anthraquinone carbonate ester, that is, a group in which Ri and Rj as well as R 'are hydrogen.

Reagents of formula

170 146

is readily synthesized from deoxyribose and an alcohol derivative containing the R residue, i.e., 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 readily converted to a 5'- and 3'-protected analog by standard methods, e.g. by introducing a dimethoxytrityl (DMT) group or an anologic group at the -5'fR ¹ position) and a phosphorus derivative such as a phosphoramidite, phosphotriester or the like in 3 'position (R ² ).

As noted in Part D of this section, these reagents are readily incorporated into an oligonucleotide or polynucleotide chain using the standard phosphoramidite method. Upon introduction of 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

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

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

170 146 for example 2-methylene-9,10-anthraquinone-acetal, cleavage is carried out by oxidation with NA2S2O41 followed by treatment with a base. Where R is

-ch ₂ ch ₂ -

the cleavage 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 by N-bromosuccinimide followed by treatment with a base.

As mentioned above, the reagents which allow the cleavage of an oligonucleotide or polynucleotide chain are suitable for use in the amplification assays disclosed in European Patent Application No. 88,309,697.6 by the present Applicants and cited above. With the deoxyribose-based reagents described in this section, nucleic acid multimer branch points can be formed using multifunctional nucleic acid monomers of the structure

wherein: R1 is an alkaline and acid labile blocking group;

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

R ³ is hydrogen, methyl, iodine, bromine or fluoro;

R is hydrogen or methyl;

R ⁵ is a levulinyl group,

170 146 or

where:

R ', Ri and Rj are as defined above, k is zero, 1, 2, 3 or 4 and Ri may be the same or different and represent an amino, nitro, halogen, hydroxy, lower alkyl and lower alkoxy group ;

Z is a group selected from the following formulas:

(2) (2) (2) (2) (2) (0Η ₂ ) _χ - NH - i - O (1) (CH ₂ ) _x - NH - C - (CH ₂ ) _y - 0 (D (CH ₂ ) _x - NH - C - (CH _? ) _V -SS - (CH ₂ ) _v - O (1) e

2'y ^A 2'y (CHJ '- NH - (Π) - Wy ~ ⁰ (1) (1) (CH ₂ - CH ₂ - ⁰ ) _x - and (2) (1), - (ch ₂ ) _x - 0 where x and y can be the same or different and represent integers in the range from 1 to 8.

Subsequently, such nucleic acid monomers are introduced into the oligouucleotide or polynucleotide chain as described above, with a cleavable or easily removable R ⁵ residue defining the site at which secondary oligonucleotide chains are synthesized.

The branching sites of nucleic acid multimers are also formed using polyfunctional non-nucleotide compounds of the general formula:

CH _O - 0 - R ¹ l ² 9

CH, - C - CH? - O - R ⁵ I ²

CH ₂ - 0 - R _{n where} ^:

R, R and Rn are as defined above. In a particularly preferred wykonmiu, R is DMT, R ² represents β-cyanoethyl fodfęrynoamid and R is the residue of lewulinylową. 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) reacting the compound so protected with a R salt, e.g. with dimoxyirinyl chloride in such a manner to convert one of the two free hydroxyl groups into -OR2 (3) by reacting the resulting compound with Rn-OH or with a salt of Rn, e.g. with levulinic acid or its salt, with simultaneous displacement of the blocking group from steps (1) and (4) reaction of an intermediate compound of formula

CH _O - 0 - HI ² ,

CH ₃ - C - CH ₂ -o -r '

CH ₂ - 0 - R _n with a reagent by which the remaining free hydroxyl group is converted to -OR1, e.g. with β-cyanoeiocdy-N, N-d-disopropylamenochlorophosine.

Such non-standard sites are particularly useful both for the possibility of cleaving the oligonucleotide chain at a specific site and for other purposes, for example for the synthesis of branched nucleic acid multimers.

G. Additional selectively cleavable linker residues.

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

iPr

Wherein DMT is dimethoxytrityl, Bz is benzyl, iPr is isopropyl and R ⁶ is either methyl or β-cyanoethyl. As with the reagents described above, this residue is also easily introduced into the oligonucleotide chain by known methods. The cleavage at the site containing this residue is carried out chemically in two steps: (1) by oxidation with an aqueous solution of sodium periodate for one hour and then (2) by treatment with an aqueous solution of n-propylamine.

It is understood that the invention has been described with reference to its preferred specific embodiments and that the foregoing description as well as the examples provided below are intended to illustrate and not limit the scope of the invention.

Example 1 Synthesis of [2- (2-nitrophenyl) -2- (O-dimethoxytrytyloxy) ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine (BP1):

2- (O-nitrophenyl) -1,2-ethanediol (2.5 g, 13.6 mmol) was dried by evaporation once 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 of 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 (2x250 mL each) and 80% saturated aqueous NaCl 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 of toluene and once with 200 ml of CH3CN. The product was purified on a silica gel column (eluting with methylene chloride CH2Cl2 with 0.5% triethylamine) to give 6.5 g (13.6 mmol) of pure product (100% yield).

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

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

Example IL Synthesis of [2- (2-nitrophenyl) -1- (O-dimethoxytrytyloxy) ethoxy] -N, N-diisopropylamino-2-cyanoethoxyphosphine (NP2):

2- (O-nitrophenyl) -1,2-ethanediol (2.5 g, 13.6 mmol) was 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 mmol). Stirring was continued for 18 hours at 20 ° C. Further TBDMS-Cl (200 mg) was then 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 in vacuo, the residue was evaporated with toluene (200 ml) and CH 3 CN (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 the solvent was removed 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 (1 x 250 ml) and dried over solid Na2SO4. After the solvent was removed 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. The removal of the 1-O-TBDMS group was complete after 30 minutes. The product was purified on a silica gel column to give pure 2-O-DMT-2- (O-nitrophenyl) -1,2-ethane 170 146 diol (2.4 g, 4.5 mmol). This material was converted into 2-caianoethalphosphite amine as described above in quantitative yield.

Example ΙΠ. A test fragment was prepared using a standard phosphite-alpha synthesis procedure.

5 '- T15 - 3' - p - NP1 - p - 5 '- T20 - 3' - OH / where p is phosphate /. After complete deblocking, the purified DNA oligomer was dissolved in water and photolyzed for 15 minutes (mercury lamp, λ> 350 nm). Analysis by polyadrylamide gel electrophoresis (PAGE) of the sample after fused photolysis showed that the test fragment was completely cleaved into new fragments that migrated at the rate expected for the T20 and Ti 5 segments.

Example IV. Synthesis of 5'-DMT-1 A) - / 2- ^ tnrobenzyjo / 2-deoxyrib0 / .o-3 '“C> -! Tlet.alofosίnrannαrsidy:

Desndsyrinase (10 millimeter), 2-nitrobenzyl alcohol (30 millimeter) and dldhlorohydric acid (DCA, 100 ml) in 100 ml of dry acetonitrile were gently refluxed for 2 hours. After cooling to 20 ° C, pyridine was added to neutralize the 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 chromatography on silidonam, eluting with a gradient of methanol from 0 to 6%. The fractions containing the product (a 1: 1 mixture of α - and β isomers) were collected and the solvent was removed under reduced pressure to give 2.5 g of a light yellow solid. (5.2 mmol, 52% yield).

The residue of the O-nitrobenzyl deoxyribose derivative was dissolved in 25 ml of CH2Cl2 containing 200 mg of dimethylnopyridine (DMAP) and 1.4 ml of triethylamine. To this solution was added dropwise DMT-Cl (1.7 g, 5 mmol), dissolved in 25 mL of CH 2 Cl 2. After all starting materials had consumed, 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 silicon dioxide gel, eluting with 5'-DMT-Γ-0-2-nitrobenzyl-2'-deodsibose isomers with a gradient of 0-3% methanol. 2.3 g of the product are obtained in the form of a yellow foam (2.65 mmol).

3-Methylphosphoramidite was prepared in a known manner.

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

This blocked non-basic phosphoramidite nucleotide was incorporated under standard conditions into the 3'-T20- [1'-C- (2-nitroyenzyl / -2'-deodsyrayose] -Tlo oligomer on a solid support. This fragment was deprotected with DCA (to remove the 5'-DMT residue), 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, in order to cleave the 3'-succinate bond). The supernatant was heated at 60 ° C for 18 hours. In a stability test of the 5'-DMT-r-O- (2-nitrobenzyl / -2'-deoxyribose residue), virtually no degradation was observed. A sample of this material in water was photolyzed for 2C minutes with high intensity mercury lamp light to remove the O-nitroyenzyl group from the 5'-DMT-Γ-O- / 2-nitroyenzyl / -2'-deodsrabose residue. No cleavage of this oligomer was observed during this photolysis step. A previously photolyzed sample of this oligomer was incubated in NH4OH at 60 ° C for 2 hours. The treatment with the base resulted in complete cleavage of the oligomer into the two component oligomers, Thio-3'-p and 5'-p-T20.

These reactions are shown in Schemes 5 and 6.

Scheme 5

Scheme 6 τ ~

Η + 1 m

about

AND

Cleavage of DNA fragments bound by 2-nltrobenzyl-2-deoxyriboside.

Example 5 Preparation of N-4- (O-N, N-diisopropylaminomethoxyphosphinyl-6-oxyhexyl) -5'-DMT-2 ', 3'-dibenzoyl cytidine.

Uridine (24.5 g, 100 mmol) was dried by evaporation with pyridine (2 x 150 ml). The residue was dissolved in 150 ml of pyridine and dimethoxytritrhium chloride (34 g, 100 mmol) was added dropwise with stirring. Stirring was 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% NalCO., 3 times with 800 ml of 80% saturated aqueous NaCl solution, dried over solid N2SO4, filtered and evaporated to dryness, the 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-ribouridine, which was dried by evaporation with pyridine, and the residue was dissolved in 250 ml of pyridine . Benzoyl chloride (20 ml, 170 mmol /) in 100 ml methylene chloride was added dropwise to the pyridine solution at 0 ° C. 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 work-up 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-Triazole (88.19 g) was suspended in 400 ml of acetonitrile at 0 ° C and POCb (27.53 ml) was added with 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 stirred suspension at 0 ° C. The ice-water bath was removed and stirring was 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 under reduced pressure, evaporated with toluene then with acetonitrile to give 4- (triazo! O (-1b-D-5'-O-DMT-2 ', 3-dibenzoylorybofuranosyl) -2 / 1H) -pyrimidinone in white foam with quantitative yield.

To a stirred solution of this compound in 350 ml of acetonitrile was added directly 11.89 g (101.5 mmol) of 6-aminohexanol. Stirring was continued for 18 hours. The reaction mixture was diluted with 700 ml of ethyl acetate and extracted as above. After the organic layer was dried 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, yielding 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 appropriate methylphosphoramidite was prepared using standard procedures. The modified nucleotide, N-4- (6-hydroxyhexyl-5'-DMT-2 ', 3-dibenzoyl cytidine (8.7 g, 10.2 mmol) was dissolved in 50 ml of methylene chloride containing 8.8 ml (50 mmol) of diizziprooylethyleneamine and then at 0 ° C 1.94 ml / 10 mmol / N, N-diisopropylaminomethoxychlorophosphine are slowly added. 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% NaHCO3 solution, twice with 80% saturated aqueous NaCl solution, dried over Na2SO4 and filtered. The solvent was removed under reduced pressure and the residue was evaporated with toluene and acetonitrile. The crude phosphite material was purified on a silica gel column eluting with a gradient of 50-70% ethyl acetate in methylene chloride containing 2% triethylamine to yield 7.25 g of N-4- / 0, N, N-duzopropylaminomethoxyphosphyl-6-oxyhexyl / -5 ^/ -DMT-2 ', 3 - dibenzoyl cytidines.

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

Modified ribonucleoside R containing an exocyclic alkylhydroxy group was synthesized from uridine. The protected ribonucleoside phosphite R was incorporated under standard conditions into a 5'-Thio-R-Ti5-3 'oligomer on a solid support. The samples of the purified product were subjected to 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 for 30 minutes. Subsequent treatment of the periodate-treated oligomer with n-propylamine in triethylammonium acetate at 60 ° C for 90 minutes resulted in complete cleavage of the oligomer into T w-3'-p and T15 fragments, modified at the 5 'end.

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

Scheme 7 of bottom ₂ -o

B n + 1

LiTA ₂ —O

N

B.

o / dna ₂ -o

N - ^ 0

CHO CHO n-PrNH ₂

TEAA 1 hour 60 ° C n-1 p

n + 1

ΗΟζ ^ Χν ^^ ΟΗ '

Propyl

170 146

The above cleavage scheme was applied to a series of branched dNa oligomers where the blocked ribonucleoside phosphite R-phosphite was introduced during the first cycle of secondary synthesis of solid support linear oligomers containing 10.201 branch points. In each case, the secondary synthesis was carried out using the T10 oligomer, producing branched oligomers of the following structure: 3'-T2o-En-5 '/ branch point -3'-R-Rio-5' / _n n = 10, 20, 30. These molecules were subjected to cleavage conditions. Analysis by polyacrylamide gel electrophoresis showed that all of the branching oligomers (side arms) were cleaved and in all cases the major product was Tn) -3'-p. This analysis also showed that the distribution of the cleavage products depends on the number of branches in the branched DNA molecule, with the number of shorter oligomers increasing as the number of branches per molecule increases. The reduction in homogeneity appears to be mainly due to the ionic spatial forces within the solid support during chemical synthesis.

Example VII. In a minor example, the preparation of the DMT-E '/ Lev / BCE-phosphoramidite multifunctional linker is described by the reactions outlined in Scheme 8.

Scheme 8

Cff ₂ 0H CH ^ - C - ch ₂ oh CHgOH E ' chloride toluenesulfonyl (TsCl). CH _o 0H 1 <- CH, - C 1 You -CHgOH S ₂ - 0 - Ts E 7Ta / <HU ₂ 0H levulic acid > DKTtCI t h ₂ oh CH, - C - CH "-O-DHT ³ 1 ² CH ₂ -0- Lev." ^ t, cesium salt ob ₃ -0 - CHg - 0-DMT CH ₂ - 0 - Ts DOT-E7Lev / DHT-E7TS / CNCHoCHoO-P ^^ ^{1 2 2} \ n / 1Pt / ₂

<N / l PRE I - 0CH ₂ CH ₂ CN

CH, - C - CH _o 0DMT 3 | 2

CH ₂ -0-Lev

DMT-E '/ Lev / BCE phosphoramidite

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, a solution of toluenesulfonyl chloride (TsCl 50 mmol) in 125 ml of CH2Cl2 was added dropwise.

The reaction mixture was allowed to warm to room temperature and stirring was continued for a total of 5 hours. The solvents were then evaporated under reduced pressure. The residue was dissolved in 500 ml of ethyl acetate and the organic layer was washed twice with 500 ml of 5% NaHCO3 solution, once with 500 ml of 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 entire amount of E '(Ts) was dissolved in 250 ml of CH2G2 and triethylamine / 14 ml was added; 100 millimoles (and N, N-mgtyl amylpyridine (100 ml). To this solution was added DMT-Cl / 13.6 g; 40 mmol / 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 workup as described above.

The crude reaction product was purified on a standard silica gel column (800 ml silica), eluting with a gradient of methanol in a mixture of CH2G2 and 0.5% methylamine. 14.8 g (25 mmol) of pure DMT-E '(Ts) are 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 of DMF. The solution was heated on a hot plate in a sealed vial (heat setting to position 3, temperature approximately 100 ° C) for 18 hours. Analysis showed that the reaction was complete after this time. 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) of pure DMT-E '(Lev) product, which was converted into 2-cyuoethylfouphorinamide as follows:

DMT-E '(Lev) was dissolved in 20 ml of CH 2 Cl 2 containing 2.6 ml (15 mmol) of N, N-lisopropdlethylamine and cooled to 0 ° C; to this solution was added by syringe 1.1 ml (5 mmoles) of 2-cyjauoethoxy-N, N-diisopropylamluochlorophosphide. After about 30 minutes, the reaction was complete; the reaction mixture was then 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 dryness to give 3.6 g of product as a white foam. The crude product, DMT-E '/ Lev / BCE phosphoramidite, was purified on a silica gel column and eluted with a mixture of methylene chloride, ethyl acetate and triethylamluyl (45:45:10 v / v) to give 3.24 g (4.5 mmol) of pure DMT-E '' / Lev / BCE fouforyuoamldu in the form of a white foam. NMR? P / 6 = 148.5 ppm; Coupling efficiency: 98%.

Example VIII. In this example, an alternative synthesis of the multi-functional linker DMT-E '/ Lev / BCE fouforduamide is described and depicted in Scheme 9.

170 146

Scheme 9

CH, CH, OH I ²

C - CH? OH I.

CH ₂ OH become _{6 3} ci

CH.

C - CH, - O ² CH ₂ - O - Lev

DMT

4r

CLOTH

CH, - C ch ₂ oh ch ₂ - O - Si {b ₅ E * (TPS)

DMT-C1

DMT-E '(lev)

CH _O - OH I ²

CH, - C - CH _O - O - DMT ⁵ I ²

CH ₂ -0-Lev levulinic acid

EDIC *

CH,

CH _o OH AND ²

-C - CH ₂ - O - DMT ch ₂ - O - si <£> ₅ dmt-e '(tps)

X cnch_ch _o cp ^ ^{2 2 x} N (iPr), ch ₂ - O - P

N (iPr) ₂

CH, - C - CH _o 0-DMT ⁵ and _T

CH ₂ - o - Lev och ₂ ch ₂ cn

DMT-E '/ Lev / BCE phosphoramidite

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

170 146

Tris- (hydroxymethyl / ethane (200 mmoles) was evaporated from 250 ml of pyridine and the residue was dissolved in 125 ml of pyridine. To this solution, cooled to 0 ° C, a solution of 50 mmoles of triphenylchlorosilane (TPS) in 125 ml of CH2Cl2 was added dropwise. to warm to room temperature with continued stirring for a total of 18 hours.

The solvents were then evaporated under reduced pressure, the residue was dissolved in 500 ml of ethyl acetate and the solution was washed twice with 500 ml of 5% NaHCO. 3, once with 500 ml of 80% saturated aqueous NaCl solution and dried over solid Na2SO4. After filtration, the solvent was evaporated in vacuo to give 18 g of crude E '(TPS) which was used without further purification. The entire amount (46 mmol of E') TPS was dissolved in 250 ml of CH2G2 and 14 ml of 100 mmol of triethylamine and 100 mg N, N-dimethylaminopyridine. To this solution was added 50 mMol DMT-Cl dissolved in 125 ml CH2G2. 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.

The crude product was purified on a standard silica gel column (800 mL silica), eluting with a gradient of methanol in a mixture of CH2Cl2 and 0.5% triethylamine. 14.8 g (25 mmol) of pure DMT-E '(TPS) are obtained. Either 10 mMol DMT-E7TPS was 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) of levulinic acid were added.

Then, to this solution was added dropwise 3.83 g (20 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in 50 ml of CH 2 Cl 2. 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 workup as described above. The residue from this treatment was dissolved in 50 ml of THF and 40 ml of pyridine were added first, then 10 ml of concentrated acetic acid, and finally 20 ml of 1M tetrabutylammonium fluoride in THF (Aldrich).

Thirty-layer chromatography performed after 30 minutes showed that all starting materials were consumed. Then most of the solvent was evaporated under reduced pressure and the residue was subjected to the following aqueous treatment: 250 mg of ethyl acetate was added to dissolve most of the organic material, and then 250 ml of 5% aqueous sodium bicarbonate solution (CO2 was evolved) was slowly added.

Solid NaHCOb was then added while stirring and dissolving it until a precipitate of salt remained and no CO2 was evolved. The combined aqueous / organic solution was transferred to a separating funnel and the organic layer was washed as above. After removal of the solvent, 5.96 g of crude DMT-E '(Lev) were obtained as a clear oil. This product was isolated by chromatography on silica gel using about 500 g of silica and eluting with a mixture of CH2Cl2 and 0.25% triethylamine with the addition of C% and 1% methanol. 2.7 g (5.2 mmol of (DMT-E '(Lev)) are obtained as a clear, colorless oil.

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

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

2.4 g (3.3 mmoles) of pure DMT-E '/ Lev / BCE phosphoramidite in the form of a white foam.

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

170 146

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Claims (29)

Patent claims A method of extracting an olinonocleotide obtn carrier in a test nucleic acid present in a nucleic acid sample, characterized by combining said nucleic acid sample with the nucleic acid sample of the formula under hybridization conditions. ABOUT II 5'-HO ⁵ (DNA1) ³ -OPO- (CH2) _x -CH- (CH ₂ ) _y -OPO- ⁵ (DNA ₂ ) ³ -OH OH OH where DNAi represents the first DNA segment, DNA2 represents the second DNA segment, and one of the symbols x and y represents zero, while the other one represents an integer from 1-12 inclusive, wherein either the sample or the reagent is previously deposited on the support, and as a result of hybridization of the nucleic acid under study with the polynucleotide reagent, a tag is created which is bound to the carrier through a cleavage site -O- (CH ₂ ) _a -CH- (CH ₂ ) _b -0-, ror ^N ° ² then substantially liberating this carrier from the carrier bound tag other than through this selectively cleavable site, then cleaving this site cleavable by photolysis using light of at least 350 nm wavelength and detection of the label released from the support. 2. The method according to p. The method of claim 1, wherein the polynudleotide reagent is of the formula according to claim 1 1 where x is zero. 3. The method according to p. A process according to claim 2, characterized in that the pnhnudlentium reagent of formula according to claim 2 is used. 4. The compound of claim 1, wherein y represents an integer from 1-4 inclusive. 4. The method according to p. 3. The process of claim 3, wherein the polynudkeota reagent of the formula according to claim 3 is used. 1 where y is 1. 5. The method according to p. A method according to claim 1, characterized in that the peptide longotide reagent of the formula according to claim 1 is used. Wherein y is zero. 170 146 6. The method according to p. The method of claim 4, wherein the polynucleotide reagent of the formula according to claim 4 is 4. The compound of claim 1, wherein x represents an integer from 1-4 inclusive. 7. The method according to p. The method of claim 6, wherein the polynucleotide reagent of the formula according to claim 6 is used. 1 where x is 1. A method for detecting the presence of a given oligonucleotide sequence in a test nucleic acid present in a nucleic acid sample, characterized by combining said nucleic acid sample with a polynucleotide reagent of the formula in an aqueous medium under hybridization conditions. 5 '3' ° «5 '3' 5'-HO ⁵ (DNA © -CPO- (CH ₂ ) _x -CH - (CH ₂ ) -0-P-O- (DNA © -OH OH WELL, OH where DNA1 represents the first DNA segment, DNA2 represents the second DNA segment and one of the symbols x and y represents zero, while the other one represents an integer from 1-12 inclusive, with either the sample or the reagent component being previously deposited on the support and as a result hybridization of the test nucleic acid with the polynucleotide reagent creates a tag bound to the carrier via a cleavage site, -O- (CH ₂ ) _a -CH- (CH,) _b -O- no ₂ and then the carrier with the bound polynucleotide reagent and the tested nucleic acid is separated from the aqueous medium, and this carrier is washed with a medium of a different hybridization strength than this aqueous medium to remove the label bound to the support other than through the cleavage site, in turn this cleavable site is cleaved by photolysis using light with a wavelength of at least 350 nm and the label released from the support is detected. 9. The method according to zestrz. z, characterized in that the polynucleotide reagent y of formula according to claim 1 is used. 8, where x is zero. 10. The method according to the above. The method of claim 9, wherein the polynucleotide reagent of the formula according to claim 9 is used. Is an integer ranging from 1-4 inclusive. 11. The method according to p. 10. The process according to claim 10, wherein the polynuclide reagent is of the formula according to claim 10. 8 where y represents the number one. 12. The method according to p. The method of claim 8, wherein the polynucleotide reagent of the formula according to claim 8 is used. 8. Where y is 0. 13. The method according to p. The method of claim 11, wherein the polynucleotide reagent of the formula according to claim 11 is used. Wherein x represents an integer in the range 1-4 inclusive. 14. A method of detecting the presence of a given oligonucleotide sequence in a test nucleic acid present in a nucleic acid sample, characterized by combining said nucleic acid sample with a polynucleotide reagent of the formula under hybridization conditions. 5'-H0 ⁵ (DNAj) ³ -0-P-0 OH HO-p — O- ⁵ (DNA,) ³ -OH II where DNAi is the first DNA segment, DNA2 is the second DNA segment and R is 2-nitrobenzyl, 4-penten-1-yl, -ch ₂ ch ₂ p -CH ₂ CH ₂ Si (CH ₃ ) ₃ , where R 'is hydrogen, aryl or arylalkyl, R may be the same or different, 1 may be amino, nitro, halogen, hydroxyl, lower alkyl, or lower alkoxy, Rj may be the same or different and represent amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, and is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4 , R _m is an alkylene or oxyethylene oligomer containing 1-16 carbon atoms - (CH2CH2O) _z - where z is an integer of 1-16 inclusive and R _n is the group Θ O CH ^ CH ^ -C-CH ₂ -C-, 0 ₂ N o II CH ₂ CH ₂ -0-C- S-CH ₂ CH ₂ oc · S-CH? CH II or CH ₃ O-CH ₂ CH ₂ -0-CH ₂ -. wherein either the sample or the reagent is previously deposited on the support and hybridization of the nucleic acid to be tested with the polynucleotide reagent creates a label attached to the support via a cleavage site of formula R -0-i 0 0. wherein R is 2-nitrobenzyl, 4-penten-1-yl. -CH ₂ CH ₂ S '~ CH ₂ CH ₂ Si (CH ₅ ) ₃ , -R -O-R m n O where R 'is hydrogen, an aryl or arylalkyl group, R1 may be the same or different and represent an amino, nitro, halogen, hydroxyl, lower alkyl or lower alkoxy group, Rj may be the same or different and represent a group amino, nitro, halogen, hydroxyl, lower alkyl or lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R _m is an alkylene oligomer containing 1-16 carbon atoms or oxyethylene - (CH2CH2O) _z - where z is an integer from 1-16 inclusive and R _n is a group of formula ABOUT II CH ₂ -C-CH ₂ CH ₂ -CO 170 146 CH ₃ O-CH ₂ -CH ₂ -O-CH ₂ and thereafter substantially liberating this carrier from the carrier bound label other than through this selectively cleavage site, whereafter this cleavage site is cleaved by photolysis using light of a wavelength? at least 350 nm and the label released from the support is detected. 15. The method according to p. 14. The method according to claim 14, wherein the polynucleotide reagent of the formula according to claim 14 is used. 14. The compound of claim 14, wherein R is 2-nitrobenzyl. 16. The method according to p. 14. The method according to claim 14, wherein the polynucleotide reagent of the formula according to claim 14 is used. 14, wherein R is -CH ₂ CH ₂ S ' 17. The method according to p. 14. The method according to claim 14, wherein the polynucleotide reagent of the formula according to claim 14 is used. 14, wherein R is ABOUT 18. The method according to p. 14. The method of claim 14, wherein the polynucleotide reagent according to claim 14 is used. 14. wherein R is 2-methylene-9,10-anthraquinone acetal. 19. The method according to p. 14, characterized in that the reagent according to claim 14 is used 14. The compound of claim 14, wherein R is 4-penten-1-yl. 20. The method according to p. As claimed in claim 14, characterized in that the reagent according to the present invention is used. 14, wherein R is -ch ₂ ch ₂ _ / Q • no ₂ 21. The way to go to essrrz.l4, characterizing that there is an issue of II - P 0 ' AND 22. A method for selecting one nucleic acid for a given olieonucloyride present in a nucleic acid sample, characterized by combining under hybridization conditions, in an aqueous medium, said nucleic acid sample with a polynucleotide reagent of formula -HO (DNA.,) -O-P-0-r AND OH 0 ' -CH ₂ CH ₂ S-e Q -CH ₂ CH ₂ Si (CH ₃ ) ₃ I et 'z' HO —P - O- ⁹ (DNA ^ ⁹ -OH II • (Fr. where DNA1 is the first DNA segment, DNA2 is the second DNA segment with R is 2-niirobenzyl, 4-penten-1-yla, 170 146 Where R 'is hydrogen, an aryl or arylalkyl group, R1 may be the same or different and represent an amino, nitro, halogen, hydroxy, lower alkyl, or lower alkoxy group, Rj may be the same or different and represent amino, nitro, halogen, hydroxy, lower alkyl or lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R _m is an alkylene ligomer containing 1-16 carbon atoms or oxyethylene - (CH2CH2O) _Z - where z is an integer from 1-16 inclusive and R _n is a group O II II ch ₂ -c-ch ₂ ch ₂ -c-, ° 2 ^N_ aO o Π CH ₂ CH ₂ -O-Co Loam S-CH2CH2 -O-C 170 146 CH ₃ O-CH ₂ CH ₂ -O-CH ₂ Ο ί »or wherein either the sample or the reagent component is previously deposited on the support and hybridization of the nucleic acid to be tested with the polynucleotide reagent creates a tag bound to the support via a cleavage site of formula w wherein R is a 2-nitrobenzyl, 4-penten-1-yl group, groups of the formulas: where R 'is hydrogen, an aryl or arylalkyl group, Rj may be the same or different and represent an amino, nitro, halogen, hydroxyl, lower alkyl or lower alkoxy group, Rj may be the same or different and represent an amino group , nitro, halogen, hydroxyl, lower alkyl or lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R _m is 1-16 carbon atoms, alkylene or oxyethylene oligomer - (CH2CH2O) _z - where z is an integer from 1-16 inclusive and R _n is a group of formula 0 O 1 'JJ ch ₃ -c-ch ₂ ch ₂ -c-, O tl s-ch ₂ ch ₂ -oc- oo «11 S-CH ₉ CH ₉ -OC- or o CH ₃ -O-CH ₂ -CK ₂ "0-CH _2", and then the carrier with the bound polynucleotide reagent and the nucleic acid to be tested is separated from the aqueous medium, and then the carrier is washed with a medium of different hybridization strength than the aqueous medium in order to remove label attached to the carrier other than through the cleavage site, in turn, cleavage of the cleavage site by photolysis using light having a wavelength of at least 350 nm and detecting the label released from the carrier. 23. The method according to p. 22, characterized in that the polynucleotide reagent of the formula according to claim 22 is used. 22, wherein R is 2-nitrobenzyl. 24. The method according to p. 22, characterized in that the polynucleotide reagent of the formula according to claim 22 is used. 22, wherein R is -ch ₂ ck ₂ p 170 146 25. The method according to p. 22, wherein the polynuclearide reagent of the formula according to claim 22 is 22, wherein R is 26. The method of claim 22, wherein the polynucleide reagent of claim 1 is 22, wherein R is 2-mptylene-9,10-onironhinone-anpyial. 27. The method according to p. 22, characterized in that the reagent of formula according to claim 22 is used. 22, wherein R is 4-penien-1-yl. 28. The method according to p. A process as claimed in claim 22, characterized in that the reagent of formula according to formula 22 is used 22, wherein R is -ch ₂ ch ₂ -no ₂ 29. The method downstream of Law 22, with the fact that Oożpj is eeagnłapvzoękz according to claim 22, wherein R is II _ -P —0 L o