CA2088257A1 - Incorporation of selectably clevable and/or abasic sites into oligonucleotide chains and reagents therefor - Google Patents

Abstract

Novel reagents useful in a variety of biochemical and chemical contexts, including nucleic hybridization assays and chemical phosphorylation of hydroxyl-containing compounds. The reagents are particularly useful for introducing cleavable sites and/or abasic sites into oligonucleotide or polynucleotide chains.

Description

W092/0252X PCT/~IS91/052X-~8~7 .

INCORPORATION OF SELECTPBLY CLEAVABLE AND/OR
- ABASIC SITES INTO OLIGONUCLEOTIDE
CHAINS AND REA&ENTS THEREFOR

Description Technical Field The invention relates generally to the incorporation of selectably cleavable and/or abasic sites into oligonucleotide chains, and more particularly relates to novel reagents useful for those purposes. The invention also relates to methods of using the novel reagents in biochemical assays and in phosphorylation reactions.

~ 20 Bac~qround ;~ Incorporation of selectably cleavable sites into oligonucleotide and polynucleotide c~ains has been ~' described in related U.S. Patent Application Serial No.
251,152 and in great-grandparent U.S. Patent No.
4,775,619, the disclosures of which are incorporated by reference herein. Selectably cleavable sites are useful in a number of different types of hybridization assay formats. For example, in one type of assay in which hybridization gives rise to a solid-supported duplex of a labeled probe and sample DNA, a selectably cleavable site contained within the hybrid structure will enable ready `- separation of the label from the solid support. U.S.
Patent No. 4,775,619 is primarily directed to the use of restriction endonuclease-cleavable sites in such assays.
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W092/0252~ PC~/~'S91/0;2g-~ 2-Chemically cleavable sites, e.g., disulfide linkages, 1,2-diols, and the like, may also be used, and can be introduced durina oligonucleotide syn~hesis, and are cleavable with partlcular chemical reagents, e.g., with thiols, periodate, or the li~e.
The present invention is also directed to selectably cleavable sites. However, the present method involves introduction o sites which are cleavable by photolysis as well as sites which are cleavable by other means, e.g., using chemical or enzymatic reagents, e.g., reducing agents. The cleavable sites of the invention are created by incorporation of chemical moieties, preferably photolabile moieties, ir.to oligonucleotide or polynucleotide chains. The novel photolabil~ moieties are useful in a number of different types of hybridization assay formats, including those described in the above-cited applications, as well as in the amplification nucleic acid hybridization assay described 20 in applicants' EPo Application No. 88.309697.6.
Another use of the reagents of the invention is, in general terms, the creation of abasic sites within oligonucleotides. By "abasic site" is meant an ether moiety -OR at a position which normally contains a hydroxyl group -OH or a nucleobase. The utility of such derivatization is extensive as will be disclosed in detail hereinbelow.
' Still another use of the reagents of the invention is in chemical phosphorylation. In many different aspects of oligonucleotide chemistry, che~ical phosphorylation of hydroxyl groups is necessary. For example, in oligonucleotide synthesis, after synthesis and deprotection, the free 5'-hydroxyl group of the ~ oligonucleotide must be phosphorylated for use in most '~

, WO92/02528 PCT/~'S91/052~--3- `2~8~2~7 biological processes. Also, phosphorylatlon of the 3'-hydroxyl functionality is necessary: (1) to prevent - extension of the 3' terminus by a polymerase; and (2) in the chemical ligation of DNA, i.e., a 3' phosphate moiety is typically required in the coupling of oligonucleotides using chemical means.
5'-phosphorylation has conventionally been carried out with T4 polynucleotide kinase and ATP, a reaction that is not particularly reliable or ef f icient.
Several methods for chemical 5'-phosphorylation are also known, including those described by Nadeaux et al., BiochemistrY 23:6153-6159 (1984), van der Marel et al., Tetrahedror. Lett. 22:1463-1466 (1981), Himmelsbach and Pfleiderer, Tetrahedron Letters 23:4793-4796 (1982), Marugg et al., Nucleic Acids Research 12:8639-8651 ( 198~ ), and Kondo et al., Nucleic ~cids Research ; Svmposium Series 16:161_164 tl985). However, most of these methods involve the use of unstable reagents or require extensive modification of standard deprotection ~ and purification procedures. Similar problems have been ;' found with monofunctional and bifunctional 3'-; phosphorylating reagents (see Sonveaux, supra, at 297).
; Thus, in addition to utility in providing cleavable and/or abasic sites within oligonucleotide or polynucleotide chains, many of the compounds of the present invention are additionally use~ul as phosphorylating reagents which ove.rcome the limitations of current phosphorylation procedures (and may also be useful in phosphorylation reactions that are used in conventional dimethoxytrityl ~DMT] purification schemes).
Background references which rel~te generally to methods for synthesizing oligonucleotides include those related to 5'-to-3' syntheses based on the use of ~-cyanoethyl ' ' .

~:
~, ` .

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W092/0252~ PCr/~S91/052X-2088~7 --phosphate protecting groups, e.g., de Napoli et al., Gazz Chim Ital 1l4:6~ (1984), Rosenthal et al., TetrahedrOn Letters 24:1691 (1983), Belaga je and Brush, Nucleic Acids Research 10:6295 (1977), in references which descrlb~
solution-phase 5'-to-3' syntheses include Hayatsu and Xhorana, J American Chemical SociPt~ 89:3880 (1957), Gait and Sheppard, Nucleic Acids_Research 4:1135 (1977), Cramer and Xoster, Anqew. Chem. Int. Ed. Enql. 7:473 (1968), and Blackburn et al., Journal of the Chemical Society, Part C, 2438 (~967).
In addition to the above-cited art, Matteucci and Caruthers, J. American Chemical society 103:3185-3191 tl981), describe the use of phosphochloridites in the preparation of oligonucleotides. Beaucage and Caruthers, Tetrahedron Letters 22:18S9-1862 (1981), and U.S. Patent No. 4,415,732 describe the use of phosphoramidites in the preparation of oligonucleotides.
Smith, ABL 15 24 (December 1983), describes automated solid-phase oligodeoxyribonucleotide synthesis. See also the references cited therein, and Warner et al., DNA
3:401-411 (1984), whose disclosure is incorporated herein by reference.
Horn and Urdea, DNA 5.5:421-425 (1986), describe phosphorylation or solid-supported DNA fragments using bis(cyanoethoxy)-N,N-diisopropyl-aminophosphine.
See also, Horn and Urdea, Tetrahedron Letters 27:4705-4708 (1986).
References which relate to hybridization techniques in general ir,clude the following: Meinkoth and Wahl, Anal. BiochemistrY 138:267-284 (1984), provide an excellent review of hybridization techniques. Leary et al., Proc. Natl. Acad. Sci. (USA) 80:4045-4049 (1983), describe the use of biotinylated DNA in conjunction with .

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W092/0252X PC~ S91/Oi2X-,: , ~5~ æu8~7 an avidin enzyme conjugate for detection of specific oligonucleotide sequences. Ranki et al., Gene 21:77-85, describe what they refer to as a "sandwich~ hybridization for detection of oligonucleotide sPquences . Pf euffer and Helmrich, J. Biol. Chem. 250:867-876 (1975), describe the coupling of guanosine-5'-0-(3-thiotriphosphate) to Sepharose 4B. Bauman et al., J. Histochem. and Cvtochem.
29:227-237, describe the 3' -labeling of RNA with fluorescers. PCT Application W0/8302277 describes the addition to DNA fraqments of modified ribonucleotides for labeling and meth~ds for analyzing such DNA frasments.
Renz and Kurz, Nucl. Acids. Res. i2:3435-3444, describe the covalent linking of Pnzymes to oligonu~leotides.
Wallace, DNA Recombinant Technoloay (Woo, S., ed.) CRC
Press, Boca Raton, Florida, provides a general background of the use of probes in diagnosis. Chou and Merigan, N.
Ena._J. of Med. 308:921-92S, describe the use of a radioisotope-labeled probe for the detection of CMV.
Inman, Methods in Enzvmol. 34B, 24:77-102 (1974), describes procedures for linking to polyacrylamides, while Parikh et al., Methods in Enz~mol. 34B, 24:77-102 (1974), describe coupling reactions with agarose. Alwine et al., Proc. Natl. Acad. Sci. (USA) 74:5350-5354 (1977), describe a method of transferring oligonucleotides from gels to a solid support for hybridization. Chu et al., Proc. Natl. Acad. Sci. (USA) 11:6513-6529, describe a technique for derivatizing terminal nucleotides. ~o et ~ al., Biochemistrv 20:64-67 (1981), describe derivatizing ;- 30 terminal nucleotides through phosphate to form esters.
Ashley and MacDonald, Anal. Biochem. 140:95-103 (1984), report a method for preparing probes 4rom a surface-bound template.

. .
. . .

- . -W092/0252X PCT/~S91/052~-2~X57 -6-Hebert and Gravel, Can. J. Chem. ~2:187-189 (1974), and Rubinstein et al., Tetrahedron Lett., No. 17, pp. 1445-1448 (1975), describe the use of 2-nitrophenyl-containing compounds as light-sensitive protecting groups.
K. Groebke et al. Helvetica Chemica Acta.
73:608-617 (1990) is relevant insofar as the reference describes the use of the t-butyldimethylsilyl moiety to pro~ect a hydroxyl functionality.
summarv of t~e 3isclosure Accordingly, it is a primary object of the invention to address the above-mentioned needs in the art, and to provide methods and reagents for introducing selectably cleavable and/or abasic sites into oligonucleotide chains.
It is another object of the invention to provide such methods and reagents for introducing selectably cleavable sites into oli~onucleotide chains, wherein the selectably cleavable sites are chemically cleavable.
It is still another object of the invention to provide such methods and reagents for introducing selectably cleavable sites into oligonucleotide chains, wherein the sel ctably cleavable sites are cleavable by liyht.
It is a further object of the invention to provide methods and reagents for introducing abaslc sites ~` 30 into oligonucleotide chains.
It is still a further object of the invention to provide methods and reagents for chemically phosphorylating hydroxyl groups.
!~

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W~92/02~2x PCT/~'S91/05~-2~882a7 It is yet another object of the inventiOn to provide reagents for incorporating abasic sites into oligonucleotide chains which may then be used to create a branched nucleic acid ~ultimer.
It is another object of the invention to provide such reagents wherein the abasic sites are non-nucleotidic.
Additional o~j ects, ~dvantages and novel ~eatures of the invention will be set forth in part in the description which follows, and in part will beecome apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
- l5 In one aspect, then, novel reagents are provided which are photolabile chemical compounds having the general structure :
Rl- O-(CH,~- CH-(CH~)y- O-R' ~ NO, ' wnerein Rl, R2, x and y are as deined below. These ~ c~mpo~nds may be incorporated into oligonucleotide chains ; sa ~5 to enable cleavage ~y light.
In another aspect, novel reagents are provided having ~2 general structure ,: ~
, ~ `' , .,.~, . . .

,.. -.~. :
`

:

W092/0252~ PCT/~S91/OS2~-2 0 ~ 7 l~o~f OR~

wherein R1, R2 and R are as defined below~ Such compounds are useful in creating abasic sites ~ithin oligonucleotide chains, which may or may not be cleavable.
In still another aspect, novel reagents are provided having the general structure .' CEI -O-Rl ~ 20 1 2 2 3-C-CH;~--O-R
,. CH2-0-Rn :~ .
.wherein Rl, R2 and Rn are as defined below. Such ~ 25 compounds are useful to create branch points in the :~ synthesis of nucleic acid multimers.
In other aspects, methods of using these reagents in a variety of contexts are provided as well.

~` 30 Modes for carryinq out the Invention A. Definitions:
~:~ By "selectably cleavable site" .'i meant a functionality or plurality of functional'.~ies which can ~: `
: ~

WO 92/0252X PCr/- S91 /052X-_g_ ~82~7 be 5electively cleaved . The f ocus of the present inv~ntion, as noted hereinabo~e, is primarily on sites which are specifically clea~able using photolysiS.
As used herein the terms "oligonucleotide" and "polynucleotide" shall be generic to polydeoxy-ribonucleotides (containing 2 ~-deoxy-D-ribose or modified forms thereof), to polyribonucleotides ~containing D-ribose or modified forms thereof), and to any other type ` 10 of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine base. The term "nucleoside" will similarly be generic to ribonucleosides, d~oxyribonu cleosides, or to any other nucleoside which is an N-glycoside of a purine or-pyrimidine base, or of a modified purine or pyrimidinebase. There is no intended distinction in length between the term ~'oligo nucleotide" and "polynucleotide" and these terms will be used inter changeably. These oligonucleotides and polynucleotides may be single-stranded or double-stranded, typically single-stranded.
Also, the oligonucleotides of the present invention are normally of from about 2 to about 2000 monomer units, and more typically, for most probe-based applications, from about 2 to about 100 monomer units.
By "nucleic acid sample" is intended a sample suspected of cont~inin~ a nucleic acid sequence of ; interest.
- E3y "nucleic acid analyte" is intended DNA or RNA in said nucleic acid sample containing the sequence o~ interest.
By "pho~phorylating reagents" as used herein are intended compou~ds which, upon a reaction or series of reactions with a ..~droxyl-containing compound, will yield a phosphate mon -ster.

.~ :

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.

W092/0~52X PCT/~S91/0528-2~8~25~ lo-~ . ;. .
By "lower alkyl" and "lower alkoxy~ are meant alkyl and alkoxy substituents, respectivelyr having from about 1 to 8, more typically from about 1 to 6, carbon atoms.
Where aro~atic substituents are indicated, it is to be understood that each individual aromatic ring may be substituted at one or more carbon ato~s with moieties which do not substantially affect function or 10 reactivity.
B. Structure of the Novel Photolabile Reaq~nts:
In one embodiment of the invention, novel - reagents are provided which are photola~ile chemical compounds having the structure:

R~--O~ CH--~ CH~)y O-R' ~ NO, : . 25 wherein R1 is a base-stable, acid-sensitive blockin~
group, R2 is a phosphorus derivative selected to enable addition of the reagent to the 5' position of a nucleoside or an oligonucleotide chain, and one of x and y is zero while the other is an integer in the range of 1 to 12 inclusive. Two ~asic types of structures fall within the above generic formula: (1) those wherein x is nonzero and y is zero (som~times referred tc herein as "NP1-type" reagents)i and (2) those wherPin x is zero and y is nonzero (sometimes -:eferred to herein as "NP2-type"

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WO 92/0252g PC~/~iS91/05~
8 2 ~ 7 reagents). These two types of structures are, as may be - readily inferred from the above generic formula, quite similar. They are each ~seful for introducing specific sites into oligonucleotide chains, which, because of the nitrophenyl moiety, are readily cleavable via photolysis.
; However, as will be discussed in more de~ail below, ~he two families of chemical reagents are distinguishable insofar as they are useful in slightly dif~erent contextS-Turning now in more detail to the various substituents of the novel photolabile reagents:
R1 is, as noted above, a base-stable, acid-sensitive blocki~g group. such blocking groups are well known in the ar~ of oligonucleoti~e synthesis and include unsubstituted or substituted aryl or aralkyl groups, where the aryl is, e.g., phenyl, naphthyl, furanyl, biphenyl, or the like, and where the substituents are from 0 to 3, usually 0 to 2, and include any non-interfering stable groups, neutral or polar, electron-donating or withdrawing. Examples of such groups are dimethoxytrityl (DMT), monomethoxytrityl (MMT), trityl and pixyl. A particularly preferred moiety for use herein is DMT.
R2 is a phosphorus derivative which is selected so as to facilitate condensation of the reagent with the . ,.
5'-hydroxyl group of a nucleoside or an oligonucleotide chain. Such groups include phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, phosphorothioates, and the like (sae, e.g., EP Publication No. 0225807 by Urdea et al., "Solution Phase Nucleic Acid Sandwich ~ssay anl Polynucleotide Probes Useful Therein", the disclosur. of which is incorporated by reference herein). Par.icularly preferred :- :
, .~. ; ~

W092/0252~ PcT/~:s91/Oj2g-~8~2~ -12-groups useful as R2 are phosphoramidites having the structure:

N(iPr 1 --?~
' ' O--Y

wherein Y is selected from the group consisting of methyl - and ~-cyanoethyl, and "iPr" represents isopropyl. Most preferably, Y is ~-cyanoethyl.
As may be readily deduced from the above : 15 definitions, the Rl and R2 substi~uents are generally selected so as to allow lncorporation of the pho~olabile reagent into a DNA fraqment using standard phosphoramidite chemistry protocols. That is, during oligonucleotide synthesis, the R2 substituent is selected ` 20 so as to react with the 5'-hydroxyl group of a nucleoside ~ or an oligonucleotide chain, while the R1 moiety is :~ selected so as to enable reaction with the 3'-hydroxyl of a nucleoside or an oligonucleotide chain.
With respect to the subscripts x and y, one of 25 x and y is zero while the other is an integer in the range of l to 12 inclusive, more preferably in the range o~ 1 to 4 inclusive, and most preferably 1.
: Exemplary reagents falling within the a~orementioned general category are the following.
3b : 35 .
. ~;, . . .
. ;~
~'~
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wos2/02s2~ PcTl~!s91/o528--13- 2~2~7 ~lT~ CH. ~ "NP~"
CE~ -O--?~
~hO, \p~
~CCH~G~20 CH~ D~lT
O ~

~ "NP2"
i ' .
.~....

As indicated, these specific structures, [2-(2-nitrophenyl)-2-(0-dimethoxytrltyloxy)ethoxy]-N,N-diisopropylamino-2-cyanoethoxyphosphine and [2-(2-nitrophenyl)-1-(0-dimethoxytrityloxy)ethoxy]-N,N-diisopropylamino-2-cyanoethoxyphosphine, are designated herein as compounds "NP1" and "NP2", respectively, and are the specific reagents syn~hesized in Examples 1 and 2 below.
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W092/02528 PCT/~1591/052~/
2088'~7 ~14-C. Svnthesis of the Above Reaaents:
Reagents of NPl-type, that is, wherein x is nonzero and y is zero, are synthesized according to the reaction sequence outlined in Scheme 1. Reagents of the - NP2-type are synthesized according to the set of reactions outlined in Scheme 2.

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~ 3S

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PCl lI,'S'~l /0528-wo 92/0252~ -- -15- ` 2a~2~7 ~hç~n~ !

DMT--O--CH, H9--fH2 CH--OH

~re ' D~IT--O~CX~ N(iPr), ~'(iPr), CH--O ?\
Cl--P\ I OCE~CHtC~J

O _ ~ t~ NO2 ,"

~':

:
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`,~

wo g~/0252~ PCr/-S91/0;2~-20882~7 -16-Sch~me ' HO~ IS--O--~
CH--OH CH--OH
~NOl TBDMS--Cl ~~

~ CH2 ., .

TBD~IS--O--fH, CH--O--DMT
DMT--Cl ¦ TBAF
,~ ~0~
DMAP~EA ~` ~ - THF
; CH2CI~ 1~, :` 20 ( Pr),)~
HO--CH. P--O
CH--O--DMT Cl--?~ ~CCH.CH.O CH--O--I:)MT
,~ NO~ OCH2CHtC~I ~ NO~
.;~; 30 l~ DiPEA

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W092/0252X PCT/~91/052~--17- ~8~2~7 Abbreviations in Schemes 1 and 2: "DMT" =
dimethoxytrityl; "DMT-Cl" = dimethoxytrityl chloride;
"iPr" = isopropyl; "DiPEA" = diisopropylethylamine;
"TBDMS-Cl" = t-butyldimethylsllyl chloride; ~DMAP" = 4-dimethylaminopyridine; "TEA" - triethylamine; "TBAF" =
tetrabutylammonium fluoride.
Synthesis of NP1-type reagents involves capping the terminal hydroxyl group of 2-~O-nitrophenyl)-1,2-ethanediol with the R1 species, e.g., with DMT or the like, followed by reaction of the remaining hydroxyl group with a selected phosphorus derivative to give rise to the R2 moiety. As shown in Scheme l, an exemplary reagent for this latter purpose is chloro-N,N-diisopropylamino-2-cyanoethoxyphospnine. Variations on this basic scheme may be readily deduced. For example, to provide different Rl substituents, one would use monomethoxytrityl chloride, trityl chloride, pixyl chloride, or the like, as an alternative to dimethoxytrityl chloride. Similarly, to give rise to different R2 substituents, alternative substituted phosphines would be employed in the second step of the reaction. To vary x, additional methylene groups are required in the initial starting material.
To synthesi~e reagents of the NP2-type, i.e., wherein x is zero and y is nonzero, a similar synthetic sequence is carried out, except that the order in which the Rl and R2 substituents are introduced is reversed.
Thus, initially, the terminal hydroxyl group of the 2-(O-nitrophenyl)-1,2-ethanediol starting material is reacted with t-butyldimethylsilyl chloride ("TBDMS-Cl") to block that hydroxyl group during the n~xt reaction step, in which the remaining free hydroxyl group, is reacted with a base-stable, acid-sensitive blocking , :~`
'`' . .
';

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WO 92/0252X PC~/~'S91/05~-~Q~57 -18-group, e.g., dimethoxytrityl chloride ( "DMT-Cl~ ), to provide the R1 substituent. The termin~l hydroxyl group is then deprotected, e.g., with tetrabutylammonium fluoride, and, as in Scheme l, reacted with a suitable substituted phosphine derivative to give rise to the R2 moiety.

D. Use of the Above Reaqents to Create Selectably Cleavable Sites:
The novel photolabile reagents of the invention are readily incorporated into an oligonucleotide or poly~ucleotide chai~ using standard phosphoramidite chemistry, well known in the ar~, and as described, for example, in a nu~Der of the references cited hereinabove.
In general terms, incorporation of the novel reagent into a DNA fragment involves linkage to a 5'-hydroxyl group at R2, and linkage to a 3'-hydroxyl group at R1.
Thus, after incorporation of the pho~Qlabile reagent, the hybrid oligonucleotide chain will have the following structure:

5'- Hcy~DNA!l3-o-p-o-(cH~ CH-(CH,jV-O-P-O-'~A~ OH
OH ~ YO2 ' in which DNAl represents a first seqment of DNA, DNA2 represents a second segment of DNA, and x and y are as defined earlier. DNAl and DNA2 may be either linear or branched. This polynucleotide reagent may be used in hybridization assays such as those described in applicants' EPO Application No. 88.309203.3 and U.S.
Patent No. 4,775,619. These assays involve the use of .` .
'~

' '~
:,, W0 92tO252X PCI/l_IS91/0528-~)8~32~7 linear polynucleotide reagents haYing selectable cleavage sites, i.e., wherein DNAl and DNA2 are linear. The polynucleotide reagent containing the photolabile moiety of the invention may also be used in the amplification - assays of U.S. Patent Applications Serial Nos. 07/252,638 and 07/340,031, both incorporated by reference herein (see also PCT ~ublication No. WO89/03891). As described in those applications, cleavable "linker" molecules may be incorporated into amplification multimers at predetermined sites for the purpose of analyzing the structure of the multimer or as a means for releasing predetermined sesments (such as the portion of the multimer that binds to the labeled oligonucleotide). In such an application DNA1 andtor DNA2 are branched polynucleotide segments. Subsequent to multimer synthesis and purification, the branched polynucleotide structure of the multimer can be cleaved specifically without additional degradation of the nucleotide structure. It is preferred, clearly, that the cleavable sites be introduced at or near the junctions of the multimer to enable quantitation of the individual multimer "branches".
Depending on whether the photolabile reagent incorporated into the oligonucleotide or polynucleotide ~9 an NPl-type (i.e., wherein x is nonzero and y is zero) o~ an NP2-type (i.e., wherein x is zero and y is ~ nonzero), two dlfferent types of fragments will result ; up~n cleavage. That is, as illustrated in Scheme 3, cleavage of an oligonucleotide containing an NP1-type moiety will result in a first fragment having a terminal 5'-phosphate and a second fragment which at its 3'-terminus contains the residue 2-nitrosophenyl species.
By contrast, as illustrated in Scheme 4, cleavage of a ~; 35 ' :

., ~
, ~ .

.
. -: - . ~ .

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W092/0252X PCT/~S91/0;2~-% o 88~7 -20-polynucleotide containing the NP2~type moiety will give rise to a first fraqment containing the residual 2-nitrosophenyl group at its 5' terminus and a second fragment having a terminal 3'-phosphate.

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PCI t~S91/052~-WO 92/0~52X
-21- ~82~7 O O
--HO5~ A,]3--O--?--O--(CH,~--CH O--3--O--5~DNA2~3--OH
0 OH ~ ~

pnotoivsis ~uv light ~ i50 nm: H~ np : 20 ., O O
--~ICY~ A~ O-P--tCH~ , c--HO--~--O--5 ~ j3--OH
OH C--O OH

~NO

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:, :: , :,, :::

~ :: - : '- :

WO 92/0252X PCr/~Ssl/0;2g~

2 ~ ~ 8 2 i 7 ~h~

o o ~--HC) ' IDNA, j3--O--P--O~ (CH~)y--O--P--O--' [DNA,31 OH

OH ~ O7 OH

phololYsis (uY li~hs ~ ~co nm: H l~sno . ' . .
;~
~ 2 0 : O
~--~10~ D~lA, j3--O--P--OH
.:; . I
OH
`` 25 ,.:

~C--(CH,)yO--l o ~ 'A~ OH

; 3 0 ~O
~' " ' .

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~: ` 35 .

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~'092/0252~ PcT/~Isgl/os28--23- 2~88~7 As cleavage is effec~ed via photolysis, using uv lisht having a wavelength of at least about 350 n~, no enzymatic or chemical reagents are re~uired. Thus, a cleaner procedure is provided, resulting in a product that is necessarily free of contamination with external cleavage reagents. In addition, the polynucleotide reagent itself is inherently more stable, cleavable as it is only by treatment with ultraviolet light of a suitable wavelength.
E. PhosPhorvlation Usina the Above Reaaents:
The reagents described above, in addition to their utility in providing photolabi~e cleavage sites, are also useful as chemical phosphorylation reagents.
Phosphorylation using these reagents involves condensation with a hydroxyl-containing compound, followed by photochemical cleavage and release Gf the nitrophenyl group. The novel reagents are quite versatile in this regard, as they may be used for either 5'- or 3'ophosphorylation of a nucleoside or an oligonucleotide chain.
For 5'-phosphorylation, an NP1-type reagent is required, i.e., a reagent wherein x is nonzero and y is zero. As illustrated in Scheme 3 above, cleavage of a polynucleotide reagent containing the NP1-type molecule results in a nucleoside or DNA fragment containing a 5'-phosphate group.
For 3'-phosphorylation, an NP2-type reagent is necessary, as illustrated in Scheme 4. Cleavage of a polynucleotide reagent containing the NP2-type molecule gives rise to cleavage frag~en~:. in which one of the fragments contains a 3'-phospha~ : group and the remaining fragment contains the nitrosophe.~l res.idue.

wo s2/02s2~ PC~ S~l/052~-2~882~7 -24-F. IncorPoration of Abasic Sites and Sites for Svnthesizinq Secondarv Oliqonucleotide Chains:
In another embodiment of the invent1on, reagents are provided which are useful for incorporating abasic sites into oligonucleotide chains, which sites may or may not be "cleavable." These reagents have the structure R
~0 ,~

OR' , , wherein R1 and R2 are as described in part A of this section, above, and wherein R is selected from the group ~ consisting of 2-nitroben~yl, 4-penten-1-yl, : 25 --CH,CH,S~ --CH.CH~Si(CH3)3 R' I ll R,. -R~-O-RD, R; O

", . ` ~ ~ ~, " ` .

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WO92/02;2~ pCT/~S91/052~-2 ~ ~

nd ~ ~
O-'10 in Which R' is hydrogen, aryl or aral~yl, if aryl oraralkyl, preferably C1-C8 aryl or aralkyl, the Ri may ~e the same or different and are selected from the grou~
consisting of amino, nitro, haloge~o, hydroxyl, lower . alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting Of amino, nitro, halogeno, hydroxyl, lower alXyl and lower ; alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, ~ or 4.
Rn represents the levulinyl group -(CO)CH2CH2(C0) CH3 or .: any other blocking or protective group that can be .~ removed and replaced with hydrogen without affecting Rl, such as :~ O

: ~ . O

:~ 30 2~ C~2cH2--c-' O

~S-CH2cH2-O-c- t : 35 W092/0252X PCT/ ~! S91/0528 2~8~ ~
~3S-CHzC~2~0~C- ~ and CH30-CH2 -C~2-0-C~2- ~

and ~ is either alkylene of 1 to 16, more preferably 2 to 12, carbon atoms, or an oxyethyl~ne oligomer -(CH2C~2O)z-, where z is an integer in the range of 1 to 16, more typically 2 to 12, inclusive. Optimally, when R
is - ~ -O- ~ , Rn is levulinyl and Rm is -~CH2CH20)4-.
- These deoxyribose-based reagents not only introduce abasic sites into an oligonucleotide or polynucleotide chain, but are also, like the reagents described above, useful for providing sites which are cleavable.
- ~ Where R is , 20 `~ R' O

R; O
:~
:
:~ 30 it is preferred that R' be hydrogen or phenyl. The Ri and Rj, as indicated, may represent any one of a number ~ .~ . of different substituents. In a particularly preferred .':' .

wo 92/02~ pCl / ~lS~) 1 /05~, 2~882~ ~

em~odiment, the aforementioned structure is the 2-methylene-9,10-anthraquinone carbonate ester, i.e., Ri and Rj are hydrogen, as is ~1.
Reagents of the formula ~ .
R

OR' ~' may be readily synthesized from deoxyribose and the alcohol derivative of the R moiety, i.e., R-OH. In the case of 2-nitrobenzyl, for example, deoxyribose would be reacted with 2-nitrobenzyl alcohol to give the 1'-0-(2-nitrobenzyl) derivative. This intermediate may be readily converted into the 5'- and 3'-protected analog using standard methods, e.g., for the incorporation of the dimethoxytrityl (DMT) group or an analogous group at the 5'-position (Rl) and a phosphorus derivative such as a phosphoramidite, phosphotriester or the like at the 3'-position (R2).
These reagents may be readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite chemistry a~ nGt~d in part D Gf 'his section. After incorporatisn of these deoxyri~ose-based cleavable moieties into the oligonucleotide or :

, W0~2/0252~ PcT/~91/0;2X
2~88%~ ~ -28-polynucleotide chain, the cleavable chaln, containinq the abasic sites -OR, will have the struc~ure o/ R
~ ~ HCY~D~A1~3 0-~ O - ~ O ~
\~/

HO -~- O-' p~A ~`- OH

in which DNA1 and DNA2 are first and second segments of DNA as described earlier. Such a polynucleotide reagent may be used in a variety oî hybridization assays.
Cleavage of the oligonucleotide or polynucleo-20 tide chains containing these reagents may be carried out as follows. Where R is 2-nitrobenzyl, cleavage may be effected via photolysis using uv light haviny a wave length of at least about 350 nm, followed by basic hydrolysis with, e.g., ammonium hydroxide or the like.
Where R is -CH2CH2S-~ (wherein ~ represents phenyl), cleavage is effected by oxidation of the sulfur atom to ; -SO- or SO2- with, e.g., sodium periodate, followed by treatment with base. Where R is -CH2CH2Si(CH3)3, the oligonucleotide may be cleaved by treatment with, for example, fluoride ion, again followed by base. Where R
is ::~

-. ~

WO 9~/02~2~ PC T/ - S'~ 1 /052~--25- ~!8g2~7 ~' O

R;

: .

for example, the 2-methylene-9-10-anthraquinone acetal, cleavage may be carried out by oxidation with Na2S204, followed by treatment with base. ~here R is , - CH~CH, ~ NO, cleavage may be ef~ected using DBU (1,8-diazabicyclo ~5 4 0 undec-7-ene~. Where R is phosphate, removal may be effected with alkaline phosphatase followed by treatment with base, while where R is 4-penten-1-yl, cleavage will be carried out typically using N-bromosuccinimide, followed by treatment with base.
As noted above, the reagents of the present invention which ena~le cleavage of an oligonucleotide or polynucleotide cnain may b~ used in the amplirication assay described in applicants' EPO Application No.
88.309697.6, referenced earlier herein. With the ' .

, WO 92/0252X Pcr/~is91/0s2~-2~882~ ~
deoxyribose based reagents described in this section, the branch points of the nucleic acid ~multimer~ may be created using multifunctional nucleic acid monomers having the structure 1 o R5 Z

~:: R4--O~ N

-1~~
y 0~
. .

, .
~ wherein , ~ .

:`;~;
. .

" :

, -PCT/~'S91/052 W092/02~2X

2 ~ 7 Rl is a base-stable, acid-sensitive blocking group, ~2 is a phosphorus derivative that enables _ addition of the nucleic acid to the 5~-position of an oligonucleotide chain during chemical synthesis;
R3 is selacted from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl;
R5 is selected from the group consisting of levulinyl, .

R~:

~ ~

R O

in Which R', Ri and Rj are as defined earlier and in 3 0 which k is O, 1, 2, 3 or 4, and the R~ may be the same or different and are selected from the group consisting of ~:: , amino, nitro, halogeno, hyd,ox~l, lower alkyl ar,d lower ` alkoxy; and ~:; Z is selected from the group consisting of ~5 .

' I'Cr/~ S91 /0;2g-wo 92/0252~

2088%~7 o (2~ ,1 (.) --(CH2) ~

.
O

{CH2) 2~ C ~CH.j y--O-- ;

" ' ' . (1) 1 i 1) --(CH~) % ~H--C CH2) y ~ CH~ y tCH~j %~ ~ (CH.) V--O--(2~ (1) ~CH~--CH2--O) ~ ; ~nd :
(-)(1) (CH,~ ,~.--O

`~ , ~ 35 ``~'``
. .

:
~'`
:
. -~

: ~: - . ' . , -wo9~/02s28 PCT/~S91/0528-.
_33_ ~g2~7 wherein x and y may be the same or different and are integers in the range of l to 8.
These nucleic acid monomers may thPn be incorporated into an oligonucleotide or polynucleotide chain as described above, With the cleavable, or ; removable, moiety R5 defining the site at which secondary o oligonucleotide chains are synthesized.
Branch points of nucleic acid "multimers~ may also be created using multifunctional, non-nucleotidic compounds having the general structure CH2 -O-Rl CH2--0 Rn where Rl, R2 and ~ are as defined earlier here n. In a particularly preferred embodiment, R is DMT, R is ~
cyanoethyl phosphoramidite, and ~n is leSrulinyl. Such compounds may be synthesized from tris-hydroxymethyl ethane by: (l) protecting one of the hydroxyl groups by reaction with, e.g., triphenylchlorosilane or tosyl 2S ch~o~ide; ~2) reacting the protected compound with a salt of R2 ~ g., dimethoxytrityl chloride, so that one of the two free hydroxyl groups is converted to -oR2; (3) reacting the compound so provided with Rn~OH or a salt of ItRn", e.g., ~evulinic acid or a salt thereof, thereby displacing the protecting group of step (l); and (4) reacti~g the intermediate compound ~ 35 : .
, .

'`:~ ' :~' W092/0252X PCT/~s91/os2~-2~ 88 2 5~ ~34~

cH3-c-c~2-O-R
CH2 Rr, with a reagent effective to convert the remaining free hydroxyl group to -OR1, e.g., ~-cyanoethoxy-N,N-diisopropylaminochlorophosphine.
These abasic sites are extremely useful both in enabling cleavage of an oligonucleotide chain at a particular point as wall as for other purposes, e.g., synthesis of a branched nucleic acid multimer.
G. Additional Selectable Cleava~e LinXer Moieties:
Still a further reagent useful ~or providing a selectably cleavable site within an oligonucleotide chain is represented by the structure .~ .

H~ ~ ~ 0~
. , . I I
0-~ ~ J o ~ R6 DMT- 0 ~ i 'r( ~ 30 O~z GBz : .
, `3 . .
~ .

~ :

W092/0252N PCT/-~S91/052~, . ~35~ ~08~2~7 whereln DM~ representS dimethoxytrityl, Bz represents benzyl, iPr represents isopropyl, and R6 is either methyl or ~-cyanoethyl. As with the reagen~s described earlier, this moiety may be readily incorporated into an oligonucl20tide chain using conventional methods.
Cleavage at ~he site containing this moiety is achieved with a two-step chemical procedure: (l) oxidation wlth aqueous sodium periodate for 1 hour followed by ~2) treatment with aqueous n-propylamine.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing descr1ption, as well as the examples which follo~, are intended to illustrate and not limit the scope of the invention.

ExamPle 1 Synthesis of [2-(2-nitrophenyl)-2-(o-dimethoxytrityloxy)ethoxy]-NlN-diisopropylamino-2-cyanoethoxyphosphine ("NP1"): 2-(O-Nitrophenyl)-1,2-ethanediol (2.S g, 13.6 mmole) was dried by coevaporation once with pyridine. The residue was dissolved in pyridine (50 ~l) and 4,4'-dimethoxytrityl chloride (DMT-Cl) 13.6 mmole was added. The reaction mixture wasstirred for 18 hours ~t 20c. Most of the pyridine was then distilled off and ~e oily residue dissolved in 250 ml ethyl acetate. The organic phase was washed with 5%
Na~CO3 (2x 250 ml), 80% saturated aqueous NaCl (lx 250 ml), and dried over solid Na25~4. After filtration, the solvent was removed in vacuo and the residue coevaporated , with toluene (lx 200 ml) and CX3CN (lX 200 ml). The product was purified on a olumn of silica gel (eluted - ~ ! , :

-WO 92/02~28 PCT/I 591/0528-~2088'~st~ -36-with CH2Cl2-o.s~ trie~hylamlne) to give 6.5 g ( 13 . 6 mmole) pure produc~ (100% yield).
The purified produc~ of 1-0-3MT-2-(0-nitrophenyl)-l, 2-ethane diol was converted to ~-cyanoethyl phosphoramidite by reaction in c~2cl2 (50 ml) with chloro-N,N-diisopropylamino-2-cyanoet~Oxy phosphine ~1~ mmole) in the presence of diisopropylethylamine (30 mmole) at 10C for 30 min. Ethyl acetate (200 ml) was then added and the combined organic phase washed with 80%
saturated ~queous NaCl (2x 250 ml) and dried over solid Na2504. After removal of the solvent in vacuo, the residue was coevaporated with toluene (100 ml) and CH3CN
(lOo ml) to give 9.5 g of the 2-0-phosphoramidite of 1-0-dimethoxytrityl-2-(0-nitrophenyl)-1,2-ethanediol (100 yield).

ExamPle-2 Synthesis of ~2-(2-nitrophenyl)-1-(o~dimethoxy-trityloxy)ethoxy]-N,N-diisopropylamino-2 cyano eth~y-phosphine ("NP2"): 2-(0-Nitrophenyl) 1,2-ethane diol (2.5 g, 13.6 mmole) was dried by coevaporation with CH3CN. The dried compound was then dissolved in C~2C12 (100 ml)-CH3Cl (10 ml). N,N-Dimethylaminopyridine (100 mg) and triethylamine (3.6 ml, 26 mmole) were added, and, with stirring, solid t-~utyldimethylsilyl chloride (TBDMS-Cl) (2.6 g, 15 mmole) was added. The stirring was ~; continued for 18 hours at 20C. Then more ~BDMS-Cl (200 mg) was added. After one hour the reaction mixturP was diluted with 400 ml ethyl acetate. The organic phase was washed with 5% NaHC03 (2X 250 ml) 2nd 80% saturated aqUeous NaCl (lx 250 rl), and dried over solid Na2S04.
After remaval of the solvents in vacuo, the residue was coevaporated With t~lUene (200 ml) and C~3CN (200 ml) to .
.

w092/0252~ PCT/-S9110528--37- 2~8~2~7 give 2.5 g of crude 1-O-TBDMS-2-(O-nitrophenyl)-1,2-ethanediol. The crude material was c~evaporated with pyridine, and the residue was dissolved in pyridine (50 ml). DMT-Cl (30 mmole) ws added and the reaction mixture stirred at 20C for 48 hours. After removal of the solvent in vacuo, the residue was dissolved in ethyl ac~tate (250 ml). The organic phase was washed With 5%
NaHCO3 (2x 250 ml) and 80% saturated aqueous NaCl (lx 250 ml), and dried over solid Na2S04. After removal of t~e solvent in vacuo, the residue was coevaporated with toluene and CH3CN. The residue was dissolved in THF (100 ml) and 10 ml of a lM solution of tetrabutylammonium fluoride in ~HF was added. The removal of the 1-O-TBDMS
group was complete in 30 min. The product was purified on a silica gel column to give pure 2-O-DMT-2-(O-nitrophenyl)-1,2-ethanediol (2.4 g, .5 mmole). This material was converted to the 2-cyanoethylphosphor-amidite, as described above, in quantitative yield.

Example 3 A test fragment 5'-T15-3'-p-NPl-p-5'-T2o-3'-OH
( I~pll a phosphate) was assem~led using standard phosphoramidite synthetic procedures. After complete deprotection, the purified DNA oligomer dissolved in H2O
was subjected to photolysis for 15 minutes (Hg lamp, > 350 nm). PAGE analysis of the photolyzed sample showed that the treatment had-resulted in complete cleavage of the test fragment into new fragments that migrated as would be expected for segments T20 and T15.
Exam~le 4 Synthesis of 5'-DMT-l'-O-t2-nit~ ~enzyl)-2-deoxyribose 3'-O-methylphosphoramidite:

. :: - - .: -- , ; ~

W092/02528 Pc~/~s9l/Oj28-~88~57 -38-Deoxyribose (10 mmole), 2-nitrobenzyl alcohol (30 mmole) and dichloroacetic acid (~CA~; 100 ~1) in 100 ml of dry acetonitrile were heated to gentle reflux for 2 hours. After cooling to 20OC pyridine was added to - neutralize the DCA and the solvent removed in VacUo. The residue was dissolved in 500 ml ethyl acetate and the organic phase washed with 400 ml 5% NaHC03, 400 ml 80%
saturated aqueous NaCl and dried over solid Na2S04.
After filtration the solvent was removed in vacuo and the residue coevaporated with toluene and acetonitrile. The crude reaction mixture was dissolved in CH2C12 and the product was isolated by silica gel chrom~tography using a 0-6% me~hanol gradient. The frac~.ons containin~ the product (mixture of ~- and ~-isomers; ratio l:1) were pooled and the solvent removed in vacuo to give 2.5 g of a slightly yellow solid (S.2 mmole; 52~ yield).
The residue of deoxyribose-O-nitrobenzyl was dissolved in 25 ml CH2Cl2 containing 200 mg dimethylamino pyridine ("DMAP") and 1.4 ml triethylamine. To this solution was added dropwise DMT-Cl (1.7 g; 5 mmole) dissolved in 25 ml CH2Cl2. When all starting material had been consumed, the reaction mixture was diluted with 250 ml ethyl acetate and extracted, dried and coevaporated as described above. The crude reaction mixture was subjected to silica gel chromatography and the 5~-DMT-1~-0-2-nitrobenzyl-2'-deoxyribose isomers were eluted with a 0-3% methanol gradient to give 2.3 g yellow foam t2.65 mmole).
The 3-methylphosphoramidite was prepared using standard procedures. 5'-DMT-1'-0-(2 nitrobenzyl)-2'-deox~ribose was dissolved in 40 ml CH2C12 containing 2.8 ml i,iPEA and N,N-diisopropylaminomethylchlorophosphine (2 ~ mmole) was added at 0C. After 30 minutes the . .

:, .
. .

WO 92~0~52~ pC~t~l~i91/05~, -39- 20g~2a7 reaction mixture was diluted with 200 ml ethyl acetate which was washed with 3x 200 ml 80% saturated aqueous NaC1, dried over solid Na25O4, and filt~red. ~he solvent was removed in vacuo and the residue coevaporated with toluene and acetonitrile. This material was used without ~urther purification.
This protected abasic nucleoside phosphor-amidite was incorporated under standard conditions into an oligomer 3~-T2o-[ll-o-(2-nitrobenzyl)-2~-deoxyribose~-T10 on a solid support. The fragment was deprotected with DCA (to remove 5'-DMT), thiophenol (thiophenol/triethylamino/dioxane, 1:1;2 v/v for 1 hour at 23C, to remove methyl) and NH40H (aqueous ammonium hydroxide for 1 hour at 20C, to cleave the 3~-succinate linkage). The supernatant was heated at 600c for 18 hours. No cleavage was observed demonstrating the base stability of the 5'-DMT-1'-0-(2 nitrobenzyl)-2'-deoxyribose moiety. A sample of this material in water was subjected to photolysis for 20 minutes using a high intensity Hg lamp to remove the o-nitrobenzyl group from the 5'-DM1'-1'-0-(2 nitrobenzyl)-2'-deoxyri~ose moiety.
No cleavage of the oligomer was observed during the photolysis step. A sample of the oligomer Which had been subjected to photolysis was incubated in NH40H at 60C
for 2 hours. The basic treatment resulted in complete cleavage of the oligomer into the two component oligomers Tlo-3~-P and 5 P T20~
These reactions are outlined in Schemes 5 and 6.
.

, .
: 35 :, .

.` . . ;
. .

!

WO 92/02528 PCI / I S9 1/0;28-~ o 8~ 7 )--I /~
o ) o C ,.

_ j C~ I

: ' O _ I
tu _ ;~0 .'', ' ' ~<
,:'`~ _ O~

~. 25 ~
,, -- ~ Z
~ ~ ~-0 ;
~ t ::
~
~ 35 .' .
.:

: ~' ~` : ~ : .

' ' 'S9 1 /052X--41- 2~257 Scheme 6 o~ ~c~
C;7~ \ ~ Z
~, o o _ I ~D Z

15 F j O
'o7 1 / Z
~ J
o~ ~b o ~o ~ L~

_ ~
I tq Z
~o~ , O
~~~ e ~
: 30 ~ ~/ Z
.. ~ o ~ ~ ~ O
~ C
. ~o~
:

wos2to~528 PCr/~lS91/05~8-2~8~c~5~
Ex2m~1e_s Preparation of N-4-(O-~,N-diisopropylamlno methoxyphosphinyl-6-oxyhexyl)-s~-DMT-2~,3~-dibenzoyl cytidine:
Uridine (24.5 g, 100 mmole) was dried by coevaporation with pyridine (2 x 150 ml). The residue was dissolved in 150 ml of pyridine and dimethoxytrityl chloride-Cl (34 g, 100 mmole) added dropwise with s~irring. The reaction mixture was left stirring for 48 hours. Methanol (100 ml) was addPd and after 30 minutes the solvents were removed in vacuo. The residue was dissolved in 800 ml ethyl acetate and the organic phase was washed with 3 x 800 ml 5% NaHCO3, 3 x 800 ~1 80%
saturated aqueous NaCl, dried over solid Na2SO4, filtered and evaporated to dryness, followed by coevaporation with toluene and acetonitrile. Silica gel chromatography of the crude product using a 0-7% methanol/1% triethylamine - gradient afforded 46.36 g, 84.9 mmole of 5'-DMT-ribouridine) was dried by coevaporation with pyridine and the residue was dissolved in 250 ml pyridine. Benzoyl - chloride (20 ml, 170 mmole) in 100 ml methylene chloride was added dropwise to the pyridine solution at 0C.
After stirring at 20C for 2 hours, the solvent was . 25 removed in vacuo and the residue coevaporated wlth toluene. The residue was dissolved in ethyl acetate and subjected to the same aqueous workup as described above for 5'-DMT-uridine.
The crude 5'-DMT-2',3'-dibenzoyl-uridine~ which was used without further purification, was dissolved in 150 ml acetonitrile. 1,2,4-~riazole (88.19 g) was suspended in 400 ml of acetonitrile at 0c and POC13 (27.56 ml) was added with rapid stirring. Then triethylamine (106.7 ml) was added dropwise over 15 `.

.

: , . .

WO 92/02~;2X PCr/l_'S91/0~2f~-2~882~7 minutes to the stirred slurry at 0C. After 30 minutes, 5'-DMT-2',3'-dibenzoyl-uridine dissolved in 1~0 ml acetonitrile was added dropwise to the above stirred slurry at 0C. The ice-wa~er bath was removed and stirring continued for 1 hour at room tempe~ature. The reaction mixture was diluted with 1400 ml ethyl acetate, ~xtracted and dried as above. The solvents were removed in vacuo, coevaporated with toluene then acetonitrile to give 4-(triazolo)-1-b-D-5'-O- DMT- 2 ',3'-di~enzoyl-ribofuranosyl)-2(lH)-pyrimidinone as a white foam in quantitative yield. To a stirred solution of this latter compound in 350 ml CH3CN was directly added solid 6-aminohexanol (11.89 g, 101.5 mmole). Stirring was continued for 18 hours. The reaction mixture was then diluted with 700 ml of ethyl acetate and extracted as above. After drying of the organic phase over Na2S04, the solvent was removed in vacuo. The product was purified on a silica 60H column eluted with 0-50% of ethyl acetate in CH2Cl2 to give 35.4 g (41.5 mmole) yellow foam of N-4-(6-hydroxyhexyl)-sl-o-DMT-2l~3 dibenzoylcytidine.
The corresponding methylphosphoramidite was prepared using standard procedures. The modi~ied nucleoside N-4-(6-hydroxyhexy-5'-DMT-2',3'-di~enzoyl cytidine (8.7 g, 10.2 mmole) was dissolved in 50 ml methylene chloride containing 8.8 ml (50 mmole) disopropylethylamine and N,N-diisopropylaminomethoXy chlorophosphine (1.94 ml, 10 mmole) was added slowly at 0C. After 30 minutes the reaction mixture was diluted with 2S0 ml ethyl acetate and the organic phase was washed with 2 x 250 ml 5% NaHC03; 2 x 80% saturat2d aq.
NaCl, dried over solid Na2S04 and filtered. The solvent was removed in vacuo and the residue coevaporated with ., ,.. , ~ -, , , , -W092/0252~ P~T/~'S91/0528-2~82~7 ~44~
toluene and acetonitrile. The crude phosphitylated material was purified on a column of silica gel using a gradient of S0-70~ ethyl acetate in methylene ch~oride containing 2~ triethylamine to give 7.25 g of N-4-(O-N,N-diisopropylaminomethoxy phosphinyl-6-oxyhexyl)-5'-DMT-2',3'-dibenzoyl cytidine.

Exam~_e 6 ~0 Oxidative cleavage of the cis-diol system with sodium periodate readily occurs in the terminal ribonucleoside of RNA molecules. In the presence of amines the resulting dialdehyde readily eliminates both the base moiety and the phosphate at the 5'-carbon. This example describes the use of this concept in the design of a cleavable site molecule where two DNA oligomers are linked via the 5'- and the side-chain hydroxyl groups of an N-4-(6-hydroxyhexyl)-cytidine molecule.
The modified ribonucleoside R containing an exo-cyclic alkyl hydroxyl group was synthesized from uridine. The protected R ribonucleoside ph~sphoramidite was incorporated under standard conditions into an oligomer 5'-Tlo-R-T15-3' on a solid support. Purified samples of the product were subjected to a series of chemical treatments and the samples analyzed by PAGE. No cleavage of the oligomer was o~served after treatment with ammonium hydroxide at 60C for 18 hours. Treatment with sodium periodate in water at 4C for 30 minutes resulted in partial cleavage. Further exposure of periodate-treated oligomer to n-propylamine in triethylammonium acetate at 60C for 90 minutes resulted in complete cleavage of the oligomer into Tlo 3'-p and a T15 species modified at the 5' end. Scheme 7 outlines the cleavage of R ribonucleoside linked DNA fragments.

... .

., .

:

wos2/02~2~ PCTt~S91/0;2~--45- 208~2~

The cleavage scheme has been applied to several branched DNA oligomers, where the protected R
ribonucleoside phosphoramidite was incorporated during the first cycle of the secondary synthesis of solid-supported linear oligomers containing 1o, 20, and 30 comb branch points, respectively. In each case the secondary synthesis was a T1o oligomer resultin~ in branched oligomers of the following structure:

3~-T2o-En-5~branch-point-3l-R-Tlo-5l] n, n=10, 20, 30.

-These molecules were subjected to the cleavage conditions. PAGE analysis indicated that all the side arm oli~omers were cleaved off, and Tlo 3'-p was the main product in all cases. The analysis further showed that the product distribution depends on the number of branches in the branched DNA molecule, where the quantity ; 20 of shorter oligomers increases as more branches are present in the molecule. The decrease in product homogeneity appears to be mainly the result of steric constraints within the solid support during chemical ~, . synthesis.

: 35 ,. . . . . . . . . . .

.~. .

W0 92/02~3 Pcr/~;ssl/os~s-Scheme 7 rJ N R 2 ~ ~ ''~ 3 3 ~0 1~ONPi1 ~0 ^H

H~ ?/~3 ONR~ 9~-L I ~3' 3 ~a O N R
~'`1~3~j CllO CH0 r N H ~ ~ r E a ~ o ur ~ 6 0 C

H~
o N RZ -o~3~

3 0 ~ O N R .
~: ' ~o~ . , ' .
H~N~ ~ ~
3 5 l~oP~ l .l ' :

WO9~/0252~ PCT/~IS91/052~-~47~ ~08~257 Exam~le 7 This example describes preparation of the multifunctional linker "DMT-E'(Lev)BCE amidite" as shown in scheme 8-Scheme 8 C~2OH CH2~
CH3_c_cH2oH TsCl CH -C-CH20H
CH20H C~2-O-Ts - E' E'(Ts) . 1 DMT-Cl 2H levulinic acid CH2oH
CH3-C-CH2-o-D~T < cesium salt CH3-C-CH2-O-DMT
lH2 - o - Lev cH2_O-Ts DMT-E' (Lev) . DMT-E'(Ts) Cl ! cNcH2cH2o p ~ N(iPr)2 ¦ N(iPr)2 CH O-P
: 30 1 2 OcH2cH2cN

;, ~ CH2-0-Lev ~ ~ .
; ~ 35 DMT-E~ (Lev)BCE ami.~ te , . . -W092/02~28 PcT/ll.S91Jo52 8~ 2cj ~ -48-Tris-hydroxymethyl ethane (E'; 200 mmole) was co-evaporated with 250 ml pyridine and the residue dissolved in 125 ml pyridine. To this solution, cooled to 0C, was added dropwise a solution of tosyl chloride ("TsCl"; 50 mmole) in 125 ml CH2Cl2. The reaction - mixture was allowed to warm to room temperature and stirring was continued for a total of 5 hours. Then the solvents were removed in vacuo. The residue was lo dissolved in 500 ml ethyl acetate which was washed with : 2x500 ml 5% NaHCO3 solution, lx500 ml 80% sat. aq. NaCl solution and ~inally dried over solid Na25O4. After filtration the solvent was removed in vacuo to give 13.
g crude E'(Ts). This material was used without further 15 purification. All El(Ts) was dissolved in 250 ml CH2Cl2 and triethylamine (14 ml; loo mmole) and N,N-dimethylaminopyridine (loo mg) were added. To this solution was added DMT-Cl tl3.6 g; 40 mmole) dissolved in 125 ml CH2Cl2. After 18 hours at room temperature, the reaction mixture was diluted with 500 ml ethyl acetate and subjected to the same aqueous wor~up as described above.
The crude reaction product was purified on a -; standard silica column ("800 ml" silica) eluted with a gradient of methanol in CH2Cl2/0.5% triethylamine to give 14.8 g ~25 mmole) of pure DMT-E'(Ts). All of this material was treated with 50 mmole of freshly prepared levulinic acid cesium salt (prepared according to M.
Bodanszky and A. Bodanszky, in The Practice of Pe~tide 30 SYnthesis, p. 37, Springer Verlag (1984)), in 50 ml DMF.
The solution was heated on a hot plate in a sealed vial (setting 3; temperature ca 100C) for 18 hours, at which time the analy~is showed t le reaction to be complete.
The DMF was removed in v.cuo and the residue dissolved in W092/0252x PCT/~S91/0528--49~ '~o g~ 2~7 ethyl acetate. The organic phase was washed as described a~ove. The crude product was subjected to silica gel chromatography and the pure product was eluted with s CH2C12/0.5~ triethylamine to give 2.5 g (4.8 mmole) pure product of DMT-E'(Lev~. The pure DMT-E'(Lev) was converted to the 2-cyanoethyl phosphoramidite as follows.
DMT-E'(~ev) was dissolved in 20 ml CH2ClZ containing N~N-diisopropylethylamine (2.6 ml; 15 mmole) and cooled to 0C; to this solution was added under argon with a syringe 2-cyanoethoxy-N,N-diisopropylaminochlOrOphOSphine (1.1 ml; 5 mmole). After ca. 30 minutes the reaction was complete and the reaction mixture was diluted with 150 ml ethyl acetate. The organic phase was washed with 2X150 ml 5% NaHC03 and 2X150 ml 80% saturated NaCl solution.
After drying over solid Na2SO4 the solution was filtered and evaporated to dryness to give 3.6 g white foam of DMT-E'(Lev) BCE amidite. The crude amidite was purified on a column of silica gel eluted with CH2Cl2/ethyl 20 acetate/triethylamine (45:45:10 v/v) to give a white foam of pure DMT-E'(Lev) BCE amidite (3.24 g; 4.5 mmole). NMR
(31p~ ~ 148.5 ppm and coupling efficiency 98%.

Exam~le 8 Thls example describes an alternative synthesis of the multifunctional linker "DMT-E'(Lev) BCE amidite"
as shown in Scheme 9.

;
:"

,: - ~ : .

: ~ . : . , ~ . .

wo 92/0'~2X PCr/l.~S9~/0528-2a8~2~rl Sche~ne g C~ OH
Cl ~2 OHS i~p 3 C 1 1 2 CH -C--CH20H ~ 3 1 CH2-0-S1~3 E' (TPS) - . E' I
I

¦ DMT-Cl H2 S ~3levul nic a CH3-C-CH2-O DMT

CH 2 -o-Lev 1~
D~T-E' (TPS) D~IT-E ' ( Lev ) F
CH 2-OH CNc~2cH2o ~N(iPr)2 : 3 1 ~ CH2 O Lev "~` ~
2 5 y N ( iPr ) 2 ~: C~I2O-P
¦ oc~l2cH2cN

CH3 _C-CH20DMT
I

C}I2-0-Lev : 30 DMT-E ' ( Lev ) 8CE amidite *EDIC = ethyl-3-(3 dimethylaminopropyl) carbodii~ide hydrochl:,ride.
: 35 ~, :' :
;::

. .; .
, . . .

W092/0252~ PCT/IS91/0;28, -51- 208~2~7 Tris-hydroxymethyl ethane (200 mmole) was co-evaporated with 250 ml pyridine and the residue diss~lved in 125 ml pyridine. To this solution, cooled to 0C, was added dropwise a solution of triphenylchlorosilane ("TPS"; 50 mmole) in 125 ml CH2C12. The reaction mixture was allowed to warm to room temperature and stirring was continued for a total of 18 hours. Then the solvents - were removed in ~acuo. The residue was dissolved in 500 10 ml ethyl acetate which was washed with 2x500 ml 5% NaHC~3 solution, lx500 ml 80% sat. aq. NaCl solution and finally dried over solid Na2S04. After filtration the solvent was removed in vacuo to give 18 g crude E'(TPS). This material was used without further purification. All 15 E'(TPS) ~46 mmole) was dissolved in 250 ml CH2Cl2 and triethylamine (14 ml; 100 mmole) and N,N-dimethylamino-pyridine (100 mg) were added. To this solution was added DMT-C1 (50 mmole) dissolved in 125 ~l cH2Cl2. After 18 hours at room temperature, the reaction mixture was 20 diluted with 500 ml ethyl acetate and subjected to the same aqueous wor~up as described above to give 35.8 g yellow foam.
The crude reaction product was purified on a standard silica column ("800 ml" silica) eluted with a 25 gradient of methanol in CH2C12/0.5% triethyla~ine to give 14.8 g (25 mmole) of pure D~T-E'(TPS). Purified DMT-E'-(TPS) (10 mmole) was dissolved in 50 ml containing N,N-dimethylaminopyridine (lO0 mg) and 2,6-lutindine (~.3 ml, 20 mmole), and levulinic acid (2.3 g, 20 mmole) was added. To this solution was added dropwise 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ~3.83 g, 20 mmole~ dissolved in 50 ml CY.2Cl2. Af_~r 18 ho:~s the reaction was complete (tlc analysis), and the, reaction mixture was diluted with 500 ml ethyl acetate ".~
: `
.

. . ' '` .
, , ~ . ~,., . . : : . :

W0~2/0252X PCT/~'.S91/0;2~-~ ~ 8 8 ~ 52-and sub~ec~ed to ~he same aqueous worXup as descrlbed above. The residue fro~ this workup was dissolved in ~HF
(50 ml) and first 40 ml pyri~ine and t~en lo ml concentrated acetic acid were added, followed by ~O ml lM
tetrabutylammonium fluoride in THF (Aldrich~. Tlc analysis after 30 minutes showed that all starting material had been consumed. Most of the solvent was thPn removed in vacuo and the remaining residue subjected to the following aqueous workup: ethyl acatate (250 ml) was added to dissolve most organic material and 250 ml 5%
sodium bicarbonate solution was added slowly (C02 evolution). Then solid NaHC03 was added with stirri~g - and dissolved until solid salt remained and C02 evolution lS ceased. The combined aqueous/organic solution was transferred to a separatory funnel and the organic phase washed as described above. Removal of the solvent yielded 5.96 g crude DMT-E'(Lev), as a clear oil. ~he product was isolated by silica gel chromatography using ca. 500 g silica and CH2Cl2/0.25% triethylamine containing 0% and 1% methanol as the eluent to give 2.7 g (5.2 mmole) DMT-E'(Lev) as a clear, colorless oil.
The pure DMT-E'(Lev) was converted to the 2-cyanoethyl phosphoramidite as follows: DMT-EI(Lev) was dissolved in 20 ml CH2Cl2 containing N,N-diisopropyl-ethylamine (2.6 ml; 15 mmole) and cooled to 0~; to this solution was added under argon with a syringe 2-cyanoethoxy-N,N-diisopropylamino-chlorophosphine (1.1 ml;
5 mmole). After ca. 30 minutes the reaction was complete and the reaction mixture was diluted with 150 ml ethyl acetate. The organic phase was washed with 2xlS0 ml 5%
Na~C03 and 2x150 ml 80% saturated NaCl solution. After drying over solid Na2S04 the solution was filtered and evaporated to dryness to give 3.4 g white foam of DMT-' ~ . . .
. . .
:

I'CT/~S91/052~-wo92/~252X
~8~2~
E'tLev) scE amidite. The crude amidite was purified on a column of silica gel eluted with CH2C12/ethyl acetate/triethylamine (45:45:10 v/v) to give a white foam _ of pure DMT-E'(Lev) BCE amidite (2.4 g; 3.3 mmole). NMR
t31~) ~ 148 . 5 ppm and coupling eff iciency 9896.

~ ' . ~ .
~ : .
''.'.' `~ ';' ':

Claims (65)

Claims

1. A reagent having the structure wherein R1 is a base-stable, acid-sensitive blocking group;
R2 is a phosphorus derivative selected to enable addition of the reagent to the 5' position of an oligonucleotide chain; and one of x and y is zero while the other is an integer in the range of 1 to 12 inclusive.

2. The reagent of claim 1, wherein x is zero.

3. The reagent of claim 2, wherein y is an integer in the range of 1 and 4 inclusive.

4. The reagent of claim 3, wherein R1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl.

5. The reagent of claim 3, wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

6. The reagent of claim 4, wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

7. The reagent of claim 5, wherein R2 is a phosphoramidite having the structure wherein Y is selected from the group consisting of methyl and .beta.-cyanoethyl, and iPr represents isopropyl.

8. The reagent of claim 6, wherein R2 is a phosphoramidite having the structure wherein Y is selected from the group consisting of methyl and .beta.-cyanoethyl, and iPr represents isopropyl.

9. The reagent of claim 1, wherein y is zero.

10. The reagent of claim 9, wherein x is an integer in the range of 1 and 4 inclusive.

11. The reagent of claim 10, wherein R1 is selected from the group consisting of dimethoxytrltyl, monomethoxytrityl, trityl and pixyl.

12. The reagent of claim 10, wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

13. The reagent of claim 11, wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites,. H-phosphonates, and phosphorothioates.

14. The reagent of claim 12, wherein R2 is a phosphoramidite having the structure wherein Y is selected from the group consisting of methyl and .beta.-cyanoethyl, and iPr represents isopropyl.

15. The reagent of claim 13, wherein R2 is a phosphoramidite having the structure wherein Y is selected from the group consisting of methyl and .beta.-cyanoethyl, and iPr represents isopropyl.

16. A reagent having the structure wherein R1 is a base-stable, acid-sensitive blocking group;
R2 is a phosphorus derivative selected to enable addition of the reagent to the 5' position of an oligonucleotide chain; and R is selected from the group consisting of 2-nitrobenzyl, 4-penten-1-yl, -CH2CH3Si(CH3)3 , and in which R' is hydrogen, aryl or aralkyl, the R1 may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, is C1-C16 alkylene or an oxyethylene oligomer -(CH2CH20)z- where z is an integer in the range of 1 to 16 inclusive, and Rn is selected from the group consisting of , , , and

17. The reagent of claim 16 wherein R1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl.

18. The reagent of claim 16 wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

19. The reagent of claim 16 wherein R is 2-nitrobenzyl.

20. The reagent of claim 16 wherein R is

21. The reagent of claim 16 wherein R is -CH2CH2Si(CH3)3

22. The reagent of claim 16 wherein R is

23. The reagent of claim 22 wherein R is the 2-methylene-9,10-anthraquinone acetal.

24. The reagent of claim 22 wherein R is 4-penten-1-yl.

25. The reagent of claim 22 wherein R is

26. The reagent of claim 22 wherein R is

27. A polynucleotide reagent having the structure wherein DNA1 is a first segment of DNA;
DNA2 is a second segment of DNA; and one of x and y is zero while the other is an integer in the range of l to 12 inclusive.

28. The polynucleotide reagent of claim 27, wherein x is zero.

29. The polynucleotide reagent of claim 28, wherein y is an integer in the range of l to 4 inclusive.

30. The polynucleotide reagent of claim 29, wherein y is one.

31. The polynucleotide reagent of claim 27, wherein y is zero.

32. The polynucleotide reagent of claim 30, wherein x is an integer in the range of 1 to 4 inclusive.

33. The polynucleotide reagent of claim 32, wherein x is one.

34. A polynucleotide reagent having the structure wherein DNA1 is a first segment of DNA;
DNA2 is a second segment of DNA; and R is selected from the group consisting of 2-nitrobenzyl, 4-penten-1-yl, , -CH2CH2Si(CH3)3 , and in which R' is hydrogen, aryl or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, haloqeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl an lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, Rm is C1-C16 alkylene or an oxyethylene oligomer -(CH2CH2O)z- where Z is an integer in the range o-f 1 to 16 inclusive, and Rn is selected from the group consisting of , , , and

35. The polynucleotide reagent of claim 34 wherein R is 2-nitrobenzyl.

36. The polynucleotide reagent of claim 34 wherein R is

37. The polynucleotide reagent of claim 34 wherein R is

38. The polynucleotide reagent of claim 34 wherein R is the 2-methylene-9,10-anthraquinone acetal.

39. The reagent of claim 34 wherein R is 4-penten-1-yl.

40. The reagent of claim 34 wherein R is

41. The reagent of claim 34 wherein R is

42. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:
combining under hybridizing conditions said nucleic acid sample with the polynucleotide reagent of claim 27, wherein one of said sample or said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site substantially freeing said support of label bound to said support other than through said selectable cleavage site;
cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm;
and detecting label free of said support.

43. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:
combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with the polynucleotide reagent of claim 27, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;
washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said cleavage site;
cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm;
and detecting label free of said support.

44. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:
combining under hybridizing conditions said nucleic acid sample with the polynucleotide reagent of claim 34, wherein one of said sample or said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site R is selected from the group consisting of 2-nitrobenzyl, 4-penten-1-yl, -CH2CH2Si(CH3)3 , and in which R' is hydrogen, aryl or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or -different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, Rm is C1-C16 alkylene or an oxyethylene oligomer -(CH2CH2O)z- where z is an integer in the range of 1 to 16 inclusive, and Rn is selected from the group consisting of , , and CH3O-CH2-CH2-O-CH2-, substantially freeing said support of label bound to said support other than through said selectable cleavage site;
cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm;
and detecting label free of said support.

45. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, said method comprising:

combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with the polynucleotide reagent of claim 34, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through the cleavage site R is selected from the group consisting of 2-nitrobenzyl, 4-penten-1-yl, -CH2CH2Si(CH3)3 , , and in which R' is hydrogen, aryl or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, Rm is C1-C16 alkylene or an oxyethylene oligomer -(CH2CH2O)z- where z is an integer in the range of 1 to 16 inclusive, and Rn is selected from the group consisting of , , , and separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;

washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said cleavage site;
cleaving said cleavage site via photolysis using light having a wavelength of at least about 350 nm;
and detecting label free of said support.

46. A multifunctional nucleic acid monomer having the structure wherein R1 is a base-stable, acid-sensitive blocking group;

R2 is a phosphorus derivative that enables addition of the nucleic acid to the 5'-position of an oligonucleotide chain during chemical synthesis;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl;
R5 is selected from the group consisting of levulinyl, and in which R' is hydrogen, aryl or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, nalogeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, and k is 0, 1, 2, 3 or 4, and z is selected from the group consisting of ;

;

;

; and .

wherein x and y are integers which may be the same or different and are integers in the range of 1 to 8.

47. A polynucleotide reagent having the structure wherein DNA1 is a first segment of DNA;
DNA2 is a second segment of DNA;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl; and R5 is selected from the group consisting of levulinyl, and in which R' is hydrogen, aryl or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, and k is 0, 1, 2, 3 or 4, and Z is selected from the group consisting of ;

;

;

, ; and .

wherein x and y are integers which may be the same or different and are integers in the range of 1 to 8.

48. A method of phosphorylating a compound containing a hydroxyl moiety, comprising reacting the hydroxyl moiety of the compound with the reagent of claim 9, followed by photolysis using light of a wavelength of at least about 350 nm.

49. The method of claim 48, wherein the hydroxyl moiety is the 5'-hydroxyl of a nucleoside.

50. The method of claim 49, wherein the hydroxyl moiety is the terminal 5'-hydroxyl of an oligonucleotide chain.

51. The method of claim 49, wherein the nucleoside is bound to a solid support.

52. The method of claim 50, wherein the oligonucleotide chain is bound to a solid support.

53. A method of phosphorylating the 3'-hydroxyl of a nucleoside, comprising reacting the nucleoside with the reagent of claim 2, followed by photolysis using light of a wavelength of at least about 350 nm.

54. The method of claim 53, wherein the nucleoside is present at the 3'-terminus of an oligonucleotide chain.

55. The method of claim 53, wherein the nucleoside is bound to a solid support.

56. The method of claim 54, wherein the oligonucleotide chain is bound to a solid support.

57. A cleavable linker molecule having the structural formula wherein DMT represents dimethoxytrityl, Bz represents benzyl, iPr represents isopropyl, and R6 is methyl or .beta.-cyanoethyl.

58. The cleavable linker molecule of claim 57 wherein R6 is methyl.

59. The cleavable linker molecule of claim 57 wherein R6 is .beta.-cyanoethyl.

60. A compound having the structure wherein R1 is a base-stable, acid-sensitive blocking group;
R2 is a phosphorus derivative selected to enable addition of the reagent to the 5' position of an oligonucleotide chain; and Rn is a blocking or protective group that can be removed and replaced with hydrogen without affecting R1.

61. The compound of claim 60 wherein R1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl.

62. The compound of claim 60 wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

63. The compound of claim 61 wherein R2 is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates, and phosphorothioates.

64. The compound of claim 60 wherein Rn is selected from the group consisting of , , and

65. The compound of claim 63 wherein Rn is selected from the group consisting of , , , and CH3O-CH2-CH2-O-CH2- .