IE912661A1 - Incorporation of selectably cleavable and/or abasic sites¹into oligonulceotide chains and reagents therefor - 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

INCORPORATION OF SELECTABLY CLEAVABLE AND/OR ABASIC SITES INTO OLIGONUCLEOTIDE CHAINS AND REAGENTS 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.

Background Incorporation of selectably cleavable sites into oligonucleotide and polynucleotide chains 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. -2Chemically cleavable sites, e.g., disulfide linkages, 1,2-diols, and the like, may also be used, and can be introduced during oligonucleotide synthesis, and are cleavable with particular chemical reagents, e.g., with thiols, periodate, or the like.

The present invention is also directed to selectably cleavable sites. However, the present method involves introduction of 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, into oligonucleotide or polynucleotide chains. The novel photolabile 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 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, chemical 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 -3biological processes. Also, phosphorylation 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. '-phosphorylation has conventionally been carried out with T4 polynucleotide kinase and ATP, a reaction that is not particularly reliable or efficient. 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., Tetrahedron Lett. 22:1463-1466 (1981), Himmelsbach and Pfleiderer, Tetrahedron Letters 23.:4793-4796 (1982), Marugg et al., Nucleic Acids Research 12:8639-8651 (1984), and Kondo et al., Nucleic Acids Research Symposium Series 16:161-164 (1985). 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 useful as phosphorylating reagents which overcome 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 relate generally to methods for synthesizing oligonucleotides include those related to 5'-to-3' syntheses based on the use of 0-cyanoethyl -4phosphate protecting groups, e.g., de Napoli et al., Gazz Chim Ital 114:65 (1984), Rosenthal et al., Tetrahedron Letters 2.4:1691 (1983), Belagaje and Brush, Nucleic Acids Research .10:6295 (1977), in references which describe solution-phase 5'-to-3’ syntheses include Hayatsu and Khorana, J American Chemical Society 89:3880 (1957), Gait and Sheppard, Nucleic Acids Research 4:1135 (1977), Cramer and Koster, Anqew. Chem. Int. Ed. Engl. 7:473 (1968), and Blackburn et al., Journal of the Chemical Society. Part C, 2438 (1967).

In addition to the above-cited art, Matteucci and Caruthers, J. American Chemical Society 103:31853191 (1981), describe the use of phosphochloridites in the preparation of oligonucleotides. Beaucage and Caruthers, Tetrahedron Letters 22.:18 59-1862 (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 of solid-supported DNA fragments using bis(cyanoethoxy)-N,N-diisopropyl-aminophosphine.

See also, Horn and Urdea, Tetrahedron Letters 27:47054708 (1986).

References which relate to hybridization techniques in general include the following: Meinkoth and Wahl, Anal. Biochemistry 138:267-284 (1984), provide an excellent review of hybridization techniques. Leary et al., Proc. Natl. Acad. Sci. (USA) 80:4045-4049 (1983), describe the use of biotinylated DNA in conjunction with -5an 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 sequences. Pfeuffer and Helmrich, J. Biol. Chem. 250:867-876 (1975), describe the coupling of guanosine-5'-O-(3-thiotriphosphate) to Sepharose 4B. Bauman et al., J. Histochem. and Cytochem. 29:227-237. describe the 3'-labeling of RNA with fluorescers. PCT Application WO/83O2277 describes the addition to DNA fragments of modified ribonucleotides for labeling and methods for analyzing such DNA fragments. Renz and Kurz, Nucl. Acids. Res. 12.:3435-3444, describe the covalent linking of enzymes to oligonucleotides. Wallace, DNA Recombinant Technology (Woo, S., ed.) CRC Press, Boca Raton, Florida, provides a general background of the use of probes in diagnosis. Chou and Merigan, N. Eng. J, of Med, 308:921-925. describe the use of a radioisotope-labeled probe for the detection of CMV. Inman, Methods in Enzymol. 34B, 24.:77-102 (1974), describes procedures for linking to polyacrylamides, while Parikh et al., Methods in Enzymol. 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. Ho et al., Biochemistry 20:64-67 (1981), describe derivatizing terminal nucleotides through phosphate to form esters. Ashley and MacDonald, Anal. Biochem. 140:95-103 (1984), report a method for preparing probes from a surfacebound template. -δ10 Hebert and Gravel, Can. J. Chem. 52:187-189 (1974), and Rubinstein et al., Tetrahedron Lett.. No. 17, pp. 1445-1448 (1975), describe the use of 2-nitrophenylcontaining 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 protect a hydroxyl functionality.

Summary of the Disclosure 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 oligonucleotide 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 selectably cleavable sites are cleavable by light.

It is a further object of the invention to provide methods and reagents for introducing abasic sites into oligonucleotide chains.

It is still a further object of the invention to provide methods and reagents for chemically phosphorylating hydroxyl groups. -7It 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 multimer.

It is another object of the invention to provide such reagents wherein the abasic sites are nonnucleotidic.

Additional objects, advantages and novel features 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.

In one aspect, then, novel reagents are provided which are photolabile chemical compounds having the general structure Rl — O-(CH2)x— CH-(CH2)y-O-R2 • 1 2 wherein R , R , x and y are as defined below. These compounds may be incorporated into oligonucleotide chains so as to enable cleavage by light.

In another aspect, novel reagents are provided having the general structure . 1 2 wherein R , R and R are as defined below. Such compounds are useful in creating abasic sites within oligonucleotide chains, which may or may not be cleavable.

In still another aspect, novel reagents are provided having the general structure CHq-C-CH9-O-R2 I CH2-0-Rn • 1 2 wherein R , R and Rn are as defined below. Such 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.

Modes for Carrying Out the Invention A. Definitions: By selectably cleavable site is meant a functionality or plurality of functionalities which can -9be selectively cleaved. The focus of the present invention, as noted hereinabove, is primarily on sites which are specifically cleavable using photolysis.

As used herein the terms oligonucleotide and polynucleotide shall be generic to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), to polyribonucleotides (containing Dribose or modified forms thereof), and to any other type 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, deoxyribonu cleosides, or to any other nucleoside which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine base. 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 singlestranded 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 containing a nucleic acid sequence of interest.

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

By phosphorylating reagents as used herein are intended compounds which, upon a reaction or series of reactions with a hydroxyl-containing compound, will yield a phosphate monoester. -10By lower alkyl and lower alkoxy are meant alkyl and alkoxy substituents, respectively, having from about 1 to 8, more typically from about 1 to 6, carbon atoms.

Where aromatic substituents are indicated, it is to be understood that each individual aromatic ring may be substituted at one or more carbon atoms with moieties which do not substantially affect function or reactivity.

B. Structure of the Novel Photolabile Reagents: In one embodiment of the invention, novel reagents are provided which are photolabile chemical compounds having the structure: R1 — O-iCH2)x—CH—CH2)—O-R2 no2 wherein R1 is a base-stable, acid-sensitive blocking 2 . . group, R 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 basic types of structures fall within the above generic formula: (1) those wherein x is nonzero and y is zero (sometimes referred to herein as NPl-type reagents); and (2) those wherein x is zero and y is nonzero (sometimes referred to herein as NP2-type -11reagents). These two types of structures are, as may be readily inferred from the above generic formula, quite similar. They are each useful 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 detail below, the two families of chemical reagents are distinguishable insofar as they are useful in slightly different contexts.

Turning now in more detail to the various substituents of the novel photolabile reagents: R1 is, as noted above, a base-stable, acidsensitive blocking group. Such blocking groups are well known in the art of oligonucleotide 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 noninterfering stable groups, neutral or polar, electrondonating or withdrawing. Examples of such groups are dimethoxytrityl (DMT), monomethoxytrityl (MMT), trityl and pixyl. A particularly preferred moiety for use herein is DMT.

R 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, Hphosphonates, phosphorothioates, and the like (see, e.g., EP Publication No. 0225807 by Urdea et al., Solution Phase Nucleic Acid Sandwich Assay and Polynucleotide Probes Useful Therein, the disclosure of which is incorporated by reference herein). Particularly preferred -122 ... groups useful as R are phosphoramidites having the structure: NiiPrb / □ \ 0—Y wherein Y is selected from the group consisting of methyl and β-cyanoethyl, and iPr represents isopropyl. Most preferably, Y is β-cyanoethyl.

As may be readily deduced from the above definitions, the R and R substituents are generally selected so as to allow incorporation of the photolabile reagent into a DNA fragment using standard phosphoramidite chemistry protocols. That is, during oligonucleotide synthesis, the R substituent is selected 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 x and y is zero while the other is an integer in the range of 1 to 12 inclusive, more preferably in the range of 1 to 4 inclusive, and most preferably 1.

Exemplary reagents falling within the aforementioned general category are the following: -1310 DMT—O—CHi CH—O— / —NO> NiPr-> A ' XOCH2CH2CN NPl tPr.N ' \ NCCH2CH2O' CH-O-DMT p— O— CH2 ON NP2 As indicated, these specific structures, [2-(2nitrophenyl)-2-(O-dimethoxytrityloxy)ethoxy]-N,Ndiisopropylamino-2-cyanoethoxyphosphine and [2-(2nitrophenyl)-1-(O-dimethoxytrityloxy)ethoxy]-N,Ndiisopropylamino-2-cyanoethoxyphosphine, are designated herein as compounds NPl and '’NP2, respectively, and are the specific reagents synthesized in Examples 1 and 2 below. -14C. Synthesis of the Above Reagents: 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. -15Schemc 1 DMT— Cl pyridine DMT—O—CH2 N(iPr)-> / \>ch2ch2cn DiPEA Ch2Cl2 -16HO—CHo io DMT— Cl DMAP/TEA CH2C12 HO—CH2 CH-O—DMT NO-, Sshsmc 2 TBDMS- Cl DMAP/TEA CH2C12 TBDMS—O—CH2 TBDMS—O—CH? I CH-O—DMT NO-, N(iPr)2 r xoch2ch2cn DiPEA CH2C12 (iPrbN NCCHnCH.O7 TBAF THF CH-O—DMT NO2 P—o -17Abbreviations in Schemes 1 and 2: DMT = dimethoxytrityl; DMT-Cl = dimethoxytrityl chloride; iPr = isopropyl; DiPEA = diisopropylethylamine; TBDMS-C1 = t-butyldimethylsilyl chloride; DMAP = 4dimethylaminopyridine; TEA = triethylamine; TBAF = tetrabutylammonium fluoride.

Synthesis of NPl-type reagents involves capping the terminal hydroxyl group of 2-(O-nitrophenyl)-1,2ethanediol 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 R moiety. As shown in Scheme 1, an exemplary reagent for this latter purpose is chloro-N,Ndiisopropylamino-2-cyanoethoxyphosphine. Variations on this basic scheme may be readily deduced. For example, to provide different R1 substituents, one would use monomethoxytrityl chloride, trityl chloride, pixyl chloride, or the like, as an alternative to dimethoxytrityl chloride. Similarly, to give rise to . 2 . different R 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 synthesize 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 1 2 . the R and R 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-C1) to block that hydroxyl group during the next reaction step, in which the remaining free hydroxyl group, is reacted with a base-stable, acid-sensitive blocking -18group, e.g., dimethoxytrityl chloride (DMT-C1), to provide the R1 substituent. The terminal hydroxyl group is then deprotected, e.g., with tetrabutylammonium fluoride, and, as in Scheme 1, reacted with a suitable substituted phosphine derivative to give rise to the R moiety.

D. Use of the Above Reagents to Create Selectably Cleavable Sites: The novel photolabile reagents of the invention are readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite chemistry, well known in the art, and as described, for example, in a number 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 · 1 R , and linkage to a 3'-hydroxyl group at R .

Thus, after incorporation of the photolabile reagent, the hybrid oligonucleotide chain will have the following structure: HO^fDNAjp-O-P-O-CCHa),—CH-(CH2)y-O-P-O--rDNAnJ3-OH OH OH in which DNA^ represents a first segment of DNA, DNA2 represents a second segment of DNA, and x and y are as defined earlier. DNA^ 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 -19linear polynucleotide reagents having selectable cleavage sites, i.e., wherein DNA^ 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 Publication No. W089/03891). 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 segments (such as the portion of the multimer that binds to the labeled oligonucleotide). In such an application DNA^ and/or 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 is an NPl-type (i.e., wherein x is nonzero and y is zero) or an NP2-type (i.e., wherein x is zero and y is nonzero), two different types of fragments will result upon cleavage. That is, as illustrated in Scheme 3, cleavage of an oligonucleotide containing an NPl-type moiety will result in a first fragment having a terminal 5'-phosphate and a second fragment which at its 31 terminus contains the residue 2-nitrosophenyl species.

By contrast, as illustrated in Scheme 4, cleavage of a -20polynucleotide containing the NP2-type moiety will give rise to a first fragment containing the residual 2nitrosophenyl group at its 5' terminus and a second fragment having a terminal 3'-phosphate. -21Schemc 3 O O ll II ' - HO5 [DN Aj )3-O-P—O- (CHt)x—CH-O—P-O-5 [DNA2]3-OH OH NOh OH photolysis (uv light > 350 nm; Hg lamp) Ο ’ —HO^TDNAJ3 —O-P—tCH?)* OH \=0 o II <· a 5’- HO—P-O-5 [DNA?j3 I OH —OH NO -22Scheme 4 O O II II — HO—[DNA,]3 -O-P-O— CH-(CH,)y-O-P-O-- [DNA,J3-OH OH NO, OH photolysis (uv light > 350 nm; Hg lamp) -HO—- [DNA, ]3 -O-P-OH I OH NO 0 C—(CH,)yO—Ρ—O—- [DNAJ3—OH OH -23As cleavage is effected via photolysis, using uv light having a wavelength of at least about 350 nm, no enzymatic or chemical reagents are required. 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. Phosphorylation Using the Above Reagents: The reagents described above, in addition to their utility in providing photolabile 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 of the nitrophenyl group. The novel reagents are quite versatile in this regard, as they may be used for either 5'- or 3’-phosphorylation of a nucleoside or an oligonucleotide chain.

For 5’-phosphorylation, an NPl-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 NPl-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 fragments in which one of the fragments contains a 3'-phosphate group and the remaining fragment contains the nitrosophenyl residue. -24F. Incorporation of Abasic Sites and Sites for Synthesizing Secondary Oligonucleotide Chains: In another embodiment of the invention, 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 • 12 wherein R and R are as described in part A of this section, above, and wherein R is selected from the group consisting of 2-nitrobenzyl, 4-penten-l-yl, O in which R' is hydrogen, aryl or aralkyl, if aryl or aralkyl, preferably C]_~C8 aryl or aralkyl, the R| 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.

Rn represents the levulinyl group -(CO)CH2CH2(CO)CH3 or any other blocking or protective group that can be removed and replaced with hydrogen without affecting R1, such as o2n,O, li -CH2CH2-O-C-, -0-C-, -26O0 Ο S-CH2CH2-O-C-, and ch3o-ch2-ch2-o-ch2-, and is either alkylene of 1 to 16, more preferably 2 to 12, carbon atoms, or an oxyethylene oligomer -(CH2CH2O)z~, where z is an integer in the range of 1 to 16, more typically 2 to 12, inclusive. Optimally, when R is “RmO“Rni Rn is levulinyl and Rm is -(CH2CH2O)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 R; The R^ it is preferred that R1 be hydrogen or phenyl, and Rj, as indicated, may represent any one oJ of different substituents. In a particularly preferred -27embodiment, the aforementioned structure is the 2methylene-9,10-anthraquinone carbonate ester, i.e., and Rj are hydrogen, as is R'.

Reagents of the formula 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 l’-0-(2nitrobenzyl) 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 (R1) and a phosphorus derivative such as a phosphoramidite, phosphotriester or the like at the 3'. . 2 position (R ).

These reagents may be readily incorporated into an oligonucleotide or polynucleotide chain using standard phosphoramidite chemistry as noted in part D of this section. After incorporation of these deoxyribose-based cleavable moieties into the oligonucleotide or -28polynucleotide chain, the cleavable chain, containing the abasic sites -OR, will have the structure HO—P-O-3 [DNA,]3:—oh II O 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 of hybridization assays.

Cleavage of the oligonucleotide or polynucleotide chains containing these reagents may be carried out as follows. Where R is 2-nitrobenzyl, cleavage may be effected via photolysis using uv light having 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-0 (wherein φ 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 for example, the 2-methylene-9-10-anthraquinone acetal, cleavage may be carried out by oxidation with Na2S2O4, followed by treatment with base. Where R is cleavage may be effected 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-l-yl, cleavage will be carried out typically using Nbromosuccinimide, followed by treatment with base.

As noted above, the reagents of the present invention which enable cleavage of an oligonucleotide or polynucleotide chain may be used in the amplification assay described in applicants' EPO Application No. 88.309697.6, referenced earlier herein. With the -30deoxyribose 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 wherein -31R1 is a base-stable, acid-sensitive blocking group; . . .

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

R is selected from the group consisting of hydrogen, methyl, I, Br and F; R4 is hydrogen or methyl; R is selected from the group consisting of levulinyl, in which R', R^ and Rj are as defined earlier and in which k is 0, 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, hydroxyl, lower alkyl and lower alkoxy; and Z is selected from the group consisting of -320 (2) II (I) —(CH2) x—NH —C —00 (2) || Ο —(CHi) x—NH —C-(CHn) y-010 (2) (1) -(CH2) — NH —C-(CH2) y —S —S -(CHj) y -O (2) (1) -(CH2) x — O30 -33wherein x and y may be the same or different and are 5 integers in the range of l to 8.

These nucleic acid monomers may then be incorporated into an oligonucleotide or polynucleotide chain as described above, with the cleavable, or . 5 removable, moiety R defining the site at which secondary oligonucleotide chains are synthesized.

Branch points of nucleic acid multimers may also be created using multifunctional, non-nucleotidic compounds having the general structure CHq-O-R115 I 2 CH3-C-CH2-O-Rz CH~-O-R_ n . · where R , R and Rp are as defined earlier herein. In a particularly preferred embodiment, R is DMT, R is βcyanoethyl phosphoramidite, and Rn is levulinyl. Such compounds may be synthesized from tris-hydroxymethyl ethane by: (1) protecting one of the hydroxyl groups by reaction with, e.g., triphenylchlorosilane or tosyl chloride; (2) reacting the protected compound with a salt of R , e.g., dimethoxytrityl chloride, so that one of the 2 two free hydroxyl groups is converted to -OR ; (3) reacting the compound so provided with Rn~OH or a salt of Rn, e.g., levulinic acid or a salt thereof, thereby displacing the protecting group of step (1); and (4) reacting the intermediate compound -34CH--O-H I 2 CH7-C-CHD-0-R I CH2-O-Rn with a reagent effective to convert the remaining free hydroxyl group to -OR1, e.g., /3-cyanoethoxy-N,Ndiisopropylaminochlorophosphine.

These abasic sites are extremely useful both in enabling cleavage of an oligonucleotide chain at a particular point as well as for other purposes, e.g., synthesis of a branched nucleic acid multimer.

G. Additional Selectable Cleavage Linker Moieties: Still a further reagent useful for providing a selectably cleavable site within an oligonucleotide chain is represented by the structure -35wherein DMT represents dimethoxytrityl, Bz represents benzyl, iPr represents isopropyl, and R6 is either methyl or 0-cyanoethyl. As with the reagents described earlier, this moiety may be readily incorporated into an oligonucleotide chain using conventional methods.

Cleavage at the site containing this moiety is achieved with a two-step chemical procedure: (1) oxidation with aqueous sodium periodate for 1 hour followed by (2) treatment with aqueous n-propylamine.

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

Example 1 Synthesis of [2-(2-nitrophenyl)-2-(0dimethoxytrityloxy)ethoxy]-N,N-diisopropylamino-2cyanoethoxyphosphine (NP1): 2-(O-Nitrophenyl)-1,2ethanediol (2.5 g, 13.6 mmole) was dried by coevaporation once with pyridine. The residue was dissolved in pyridine (50 ml) and 4,41-dimethoxytrityl chloride (DMTCl) 13.6 mmole was added. The reaction mixture was stirred for 18 hours at 20°C. Most of the pyridine was then distilled off and the oily residue dissolved in 250 ml ethyl acetate. The organic phase was washed with 5% NaHC03 (2x 250 ml), 80% saturated aqueous NaCl (lx 250 ml), and dried over solid Na2SO4· After filtration, the solvent was removed in vacuo and the residue coevaporated with toluene (lx 200 ml) and CH3CN (lx 200 ml). The product was purified on a column of silica gel (eluted -36with CH2C12-O.5% triethylamine) to give 6.5 g (13.6 mmole) pure product (100% yield).

The purified product of l-O-DMT-2-(Onitrophenyl)-1,2-ethane diol was converted to βcyanoethyl phosphoramidite by reaction in CH2Cl2 (50 ml) with chloro-N,N-diisopropylamino-2-cyanoethoxy phosphine (15 mmole) in the presence of diisopropylethylamine (30 mmole) at 10°C for 30 min. Ethyl acetate (200 ml) was then added and the combined organic phase washed with 80% saturated aqueous NaCl (2x 250 ml) and dried over solid Na2SO4. After removal of the solvent in vacuo, the residue was coevaporated with toluene (100 ml) and CH-jCN (100 ml) to give 9.5 g of the 2-O-phosphoramidite of 10-dimethoxytrityl-2-(O-nitrophenyl)-1,2-ethanediol (100% yield).

Example 2 Synthesis of [2-(2-nitrophenyl)-1-(O-dimethoxytrityloxy)ethoxy]-N,N-diisopropylamino-2 cyano ethoxyphosphine (”ΝΡ2): 2—(O-Nitrophenyl) l,2-ethane diol (2.5 g, 13.6 mmole) was dried by coevaporation with CH^CN. The dried compound was then dissolved in CH2C12 (100 ml)-CH3Cl (10 ml). N,N-Dimethylaminopyridine (100 mg) and triethylamine (3.6 ml, 26 mmole) were added, and, with stirring, solid t-butyldimethylsilyl chloride (TBDMS-Cl) (2.6 g, 15 mmole) was added. The stirring was continued for 18 hours at 20°C. Then more TBDMS-Cl (200 mg) was added. After one hour the reaction mixture was diluted with 400 ml ethyl acetate. The organic phase was washed with 5% NaHC03 (2x 250 ml) and 80% saturated aqueous NaCl (lx 250 ml), and dried over solid Na2SO4· After removal of the solvents in vacuo, the residue was coevaporated with toluene (200 ml) and CH^CN (200 ml) to -37give 2.5 g of crude 1-0-TBDMS-2-(O-nitrophenyl)-1,2ethanediol. The crude material was coevaporated with pyridine, and the residue was dissolved in pyridine (50 ml). DMT-Cl (30 mmole) ws added and the reaction mixture stirred at 20°C for 48 hours. After removal of the solvent in vacuo, the residue was dissolved in ethyl acetate (250 ml). The organic phase was washed with 5% NaHCO3 (2x 250 ml) and 80% saturated aqueous NaCl (lx 250 ml), and dried over solid Na2SO4· After removal of the solvent in vacuo, the residue was coevaporated with toluene and CH^CN. The residue was dissolved in THF (100 ml) and 10 ml of a IM solution of tetrabutylammonium fluoride in THF 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-(Onitrophenyl)-1,2-ethanediol (2.4 g, 4.5 mmole). This material was converted to the 2-cyanoethylphosphoramidite, as described above, in quantitative yield.

Example 3 A test fragment 5'-T15~3'-p-NPl-p-51-T2θ-3’-OH (p = phosphate) was assembled using standard phosphoramidite synthetic procedures. After complete deprotection, the purified DNA oligomer dissolved in H20 was subjected to photolysis for 15 minutes (Hg lamp, λ > 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 T2Q and T15· Example 4 Synthesis of 5'-DMT-1'-O-(2-nitrobenzyl)-2deoxyribose 3’-O-methylphosphoramidite: -38Deoxyribose (10 mmole), 2-nitrobenzyl alcohol (30 mmole) and dichloroacetic acid (DCA; 100 ml) in 100 ml of dry acetonitrile were heated to gentle reflux for 2 hours. After cooling to 20°C 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% NaHCO3, 400 ml 80% saturated aqueous NaCl and dried over solid Na2SO4· 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 chromatography using a 0-6% methanol gradient. The fractions containing the product (mixture of a- and /3-isomers; ratio 1:1) were pooled and the solvent removed in vacuo to give 2.5 g of a slightly yellow solid (5.2 mmole; 52% yield).

The residue of deoxyribose-O-nitrobenzyl was dissolved in 25 ml CH2C12 containing 200 mg dimethylamino pyridine (DMAP) and 1.4 ml triethylamine. To this solution was added dropwise DMT-C1 (1.7 g; 5 mmole) dissolved in 25 ml CH2C12· 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-21-deoxyribose isomers were eluted with a 0-3% methanol gradient to give 2.3 g yellow foam (2.65 mmole).

The 3-methylphosphoramidite was prepared using standard procedures. 5'-DMT-1'-O-(2 nitrobenzyl)-2'deoxyribose was dissolved in 40 ml CH2C12 containing 2.8 ml DiPEA and N,N-diisopropylaminomethylchlorophosphine (2.0 mmole) was added at 0°C. After 30 minutes the -39reaction mixture was diluted with 200 ml ethyl acetate which was washed with 3x 200 ml 80% saturated aqueous NaCl, dried over solid Na2SO4, and filtered. The solvent was removed in vacuo and the residue coevaporated with toluene and acetonitrile. This material was used without further purification.

This protected abasic nucleoside phosphoramidite was incorporated under standard conditions into an oligomer 3'-Τ2θ-[1'-O-(2-nitrobenzyl)-21deoxyribose] 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 2 0°C, to remove methyl) and NH^OH (aqueous ammonium hydroxide for 1 hour at 20°C, to cleave the 3'-succinate linkage). The supernatant was heated at 60°C for 18 hours. No cleavage was observed demonstrating the base stability of the 5'-DMT-1'-0-(2 nitrobenzyl)-21deoxyribose 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'-DMT-1'-O-(2 nitrobenzyl)-2'-deoxyribose 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 NH^OH at 60°C for 2 hours. The basic treatment resulted in complete cleavage of the oligomer into the two component oligomers T10-3’-p and 5*PT20· These reactions are outlined in Schemes 5 and 6. a -41Scheme 6 CLEAVAGE OE 2-N1TROBENZYL 2 ’-DEOXYR I BOS IDE LINKED DNA FRAGMENTS -42Example 5 Preparation of N-4-(0-N,N-diisopropylamino methoxyphosphinyl-6-oxyhexyl)-5'-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 stirring. The reaction mixture was left stirring for 48 hours. Methanol (100 ml) was added and after 30 minutes the solvents were removed in vacuo. The residue was dissolved in 800 ml ethyl acetate and the organic phase was washed with 3 x 800 ml 5% NaHCO3, 3 x 800 ml 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'-DMTribouridine) 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 O’C.

After stirring at 20°C for 2 hours, the solvent was removed in vacuo and the residue coevaporated with 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-Triazole (88.19 g) was suspended in 400 ml of acetonitrile at 0°C and POC13 (27.56 ml) was added with rapid stirring. Then triethylamine (106.7 ml) was added dropwise over 15 -43minutes to the stirred slurry at 0°C. After 30 minutes, '-DMT-2',3’-dibenzoyl-uridine dissolved in 150 ml acetonitrile was added dropwise to the above stirred slurry at 0°C. The ice-water bath was removed and stirring continued for 1° hour at room temperature. The reaction mixture was diluted with 1400 ml ethyl acetate, extracted and dried as above. The solvents were removed in vacuo, coevaporated with toluene then acetonitrile to give 4-(triazolo)-l-b-D-5’-O-DMT- 2·,3’-dibenzoylribofuranosyl)-2(1H)-pyrimidinone as a white foam in quantitative yield. To a stirred solution of this latter compound in 350 ml CH^CN was directly added solid 6aminohexanol (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 Na2SO4, the solvent was removed in vacuo. The product was purified on a silica 60H column eluted with 0-50% of ethyl acetate in CH2C12 to give 35.4 g (41.5 mmole) yellow foam of N-4-(6-hydroxyhexyl)-51-O-DMT-2',3'dibenzoylcytidine.

The corresponding methylphosphoramidite was prepared using standard procedures. The modified nucleoside N-4-(6-hydroxyhexy-5'-DMT-2',3'-dibenzoyl 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 0°C. After 30 minutes the reaction mixture was diluted with 250 ml ethyl acetate and the organic phase was washed with 2 x 250 ml 5% NaHCO3, 2 x 80% saturated aq. NaCl, dried over solid Na2SO4 and filtered. The solvent was removed in vacuo and the residue coevaporated with -44toluene and acetonitrile. The crude phosphitylated material was purified on a column of silica gel using a gradient of 50-70% ethyl acetate in methylene chloride containing 2% triethylamine to give 7.25 g of N-4-(ΟΝ, N-diisopropylaminomethoxy phosphinyl-6-oxyhexyl)-5'DMT-2’,3’-dibenzoyl cytidine.

Example 6 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 phosphoramidite was incorporated under standard conditions into an oligomer 5’-T10-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 observed after treatment with ammonium hydroxide at 60°C for 18 hours. Treatment with sodium periodate in water at 4°C for 30 minutes resulted in partial cleavage. Further exposure of periodate-treated oligomer to n-propylamine in triethylammonium acetate at 60°C for 90 minutes resulted in complete cleavage of the oligomer into T1Q-3’-p and a T15 species modified at the 5’ end. Scheme 7 outlines the cleavage of R ribonucleoside linked DNA fragments.

,E 912661 -45The 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 solidsupported linear oligomers containing 10, 20, and 30 comb branch points, respectively. In each case the secondary synthesis was a T10 oligomer resulting in branched oligomers of the following structure: 3’-Τ2θ-Εη-5’[branch-point-3'-R-T1Q-5’]n, n=10, 20, .

These molecules were subjected to the cleavage conditions. PAGE analysis indicated that all the side arm oligomers were cleaved off, and T1Q-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 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.

N*iO, Propyl -47Example 7 This example describes preparation of the multifunctional linker DMT-E'(Lev)BCE amidite as shown in Scheme 8.

Scheme 8 CHoOH I CH^-C-CH^OH I CH2OH TsCl CH-OH I CIK-C-CH-OH I CH2-O-TS E1(TS) DMT-Cl ch2oh CHt-C-CHo-O-DMT z j - z £ CH2-O-Lev DMT-E*(Lev) levulinic acid cesium salt Cl cnch2ch2o-p „ N(iPr)2 ch2o-p N(iPr)2 och2ch2cn ch3-c-ch2odmt DMT-E'(Lev)BCE amidite CH70H I ch,,-c-ch9-o-dmt ! CH2-O-Ts DMT-E'(Ts) CH2-O-Lev -48Tris-hydroxymethyl ethane (E1; 200 mmole) was co-evaporated with 250 ml pyridine and the residue dissolved in 125 ml pyridine. To this solution, cooled to 0°C, was added dropwise a solution of tosyl chloride (TsCl”; 50 mmole) in 125 ml CH2C12. 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 dissolved in 500 ml ethyl acetate which was washed with 2x500 ml 5% NaHCO3 solution, 1x500 ml 80% sat. aq. NaCl solution and finally dried over solid Na2SO4. After filtration the solvent was removed in vacuo to give 13.7 g crude E'(Ts). This material was used without further purification. All E'(Ts) was dissolved in 250 ml CH2C12 and triethylamine (14 ml; 100 mmole) and N,Ndimethylaminopyridine (100 mg) were added. To this solution was added DMT-C1 (13.6 g; 40 mmole) dissolved in 125 ml CH2C12. After 18 hours at room temperature, the reaction mixture was diluted with 500 ml ethyl acetate and subjected to the same aqueous workup as described above.

The crude reaction product was purified on a standard silica column (800 ml silica) eluted with a gradient of methanol in CH2C12/O.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 Peptide 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. 100°C) for 18 hours, at which time the analysis showed the reaction to be complete.

The DMF was removed in vacuo and the residue dissolved in ΙΕ 912661 -49ethyl acetate. The organic phase was washed as described above. The crude product was subjected to silica gel chromatography and the pure product was eluted with CH2C12/O·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'(Lev) was dissolved in 20 ml CH2C12 containing Ν,Ν-diisopropylethylamine (2.6 ml; 15 mmole) and cooled to 0°C; 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% NaHCO3 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 CH2C12/ethyl 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) δ 148.5 ppm and coupling efficiency 98%.

Example 8 This example describes an alternative synthesis of the multifunctional linker DMT-E'(Lev) BCE amidite as shown in Scheme 9. -50Scheme 9 ch2oh | Si03Cl CH3 -c-ch2 OH _CH2OH E' CHoOH I CH9-C-CH,OH I CH2-O-Si03 E'(TPS) CH2-O-Si03 | levulinic acid CH-.-C-CH--O-DMT <--3 | 2 EDIC* CH2-O-Lev DMT-E'(Lev) f“ DMT-Cl ch2oh ch3-c-ch2-o-dmt CH2-O-Si03 DMT-E’(TPS) CH--OH I CH-,-C-CH--O-DMT I CH2-O-Lev /Cl CNCH9CH70-P xN(iPr)2 CH2-O-Lev DMT-E'(Lev)BCE amidite *EDIC = l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. -51Tris-hydroxymethyl ethane (200 mmole) was coevaporated with 250 ml pyridine and the residue dissolved in 125 ml pyridine. To this solution, cooled to O’C, 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 vacuo. The residue was dissolved in 500 ml ethyl acetate which was washed with 2x500 ml 5% NaHCO3 solution, 1x500 ml 80% sat. aq. NaCl solution and finally dried over solid Na2SO4. After filtration the solvent was removed in vacuo to give 18 g crude E'(TPS). This material was used without further purification. All E’(TPS) (46 mmole) was dissolved in 250 ml CH2C12 and triethylamine (14 ml; 100 mmole) and N,N-dimethylaminopyridine (100 mg) were added. To this solution was added DMT-C1 (50 mmole) dissolved in 125 ml CH2C12. After 18 hours at room temperature, the reaction mixture was diluted with 500 ml ethyl acetate and subjected to the same aqueous workup 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 gradient of methanol in CH2C12/O.5% triethylamine to give 14.8 g (25 mmole) of pure DMT-E’(TPS). Purified DMT-E'(TPS) (10 mmole) was dissolved in 50 ml containing N,Ndimethylaminopyridine (100 mg) and 2,6-lutindine (2.3 ml, 20 mmole), and levulinic acid (2.3 g, 20 mmole) was added. To this solution was added dropwise l-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (3.83 g, 20 mmole) dissolved in 50 ml CH2C12. After 18 hours the reaction was complete (tic analysis), and the reaction mixture was diluted with 500 ml ethyl acetate -52and subjected to the same aqueous workup as described above. The residue from this workup was dissolved in THF (50 ml) and first 40 ml pyridine and then 10 ml concentrated acetic acid were added, followed by 20 ml IM tetrabutylammonium fluoride in THF (Aldrich). Tic analysis after 30 minutes showed that all starting material had been consumed. Most of the solvent was then removed in vacuo and the remaining residue subjected to the following aqueous workup: ethyl acetate (250 ml) was added to dissolve most organic material and 250 ml 5% sodium bicarbonate solution was added slowly (CO2 evolution). Then solid NaHCO3 was added with stirring and dissolved until solid salt remained and CO2 evolution 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. The 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 2cyanoethyl phosphoramidite as follows: DMT-E'(Lev) was dissolved in 20 ml CH2C12 containing N,N-diisopropylethylamine (2.6 ml; 15 mmole) and cooled to 0°C; to this solution was added under argon with a syringe 2cyanoethoxy-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 2x150 ml 5% NaHCO3 and 2x150 ml 80% saturated NaCl solution. After drying over solid Na2SO4 the solution was filtered and evaporated to dryness to give 3.4 g white foam of DMT35 -53E’(Lev) BCE amidite. The crude amidite was purified column of silica gel eluted with CH2Cl2/ethyl acetate/triethylamine (45:45:10 v/v) to give a white of pure DMT-E'(Lev) BCE amidite (2.4 g; 3.3 mmole).

O 1 ( P) on a foam NMR wherein

Claims (67)

Claims

1. A reagent having the structure R l —O-iCHA— CH—(CH,) V —O-R 2 o NO, R x is a base-stable, acid-sensitive blocking group; R z 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 R 1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl. . . 2 ·

5. The reagent of claim 3, wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates. -55. . 2

6. The reagent of claim 4, wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates. . . 2

7. The reagent of claim 5, wherein R is a phosphoramidite having the structure N(iPr), / \ 0—Y wherein Y is selected from the group consisting of methyl and β-cyanoethyl, and iPr represents isopropyl. . . 2

8. The reagent of claim 6, wherein R is a phosphoramidite having the structure N(iPr) 2 O—Y wherein Y is selected from the group consisting of methyl and β-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 R 1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl. -5610

12. The reagent of claim 10, wherein R z is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates. . . 2

13. The reagent of claim 11, wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates.

14. The reagent of claim 12, wherein R z is a phosphoramidite having the structure N(iPr) 2 0—Y wherein Y is selected from the group consisting of methyl and β-cyanoethyl, and iPr represents isopropyl. . . 2 .

15. The reagent of claim 13, wherein R is a phosphoramidite having the structure N(iPr) 2 -< 0—Y -57wherein Y is selected from the group consisting of methyl and β-cyanoethyl, and iPr represents isopropyl.

16. A reagent having the structure wherein R 1 is a base-stable, acid-sensitive blocking group; ο R 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 2nitrobenzyl, 4-penten-l-yl, in which R' is hydrogen, aryl or aralkyl, the R^ 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, R^ is alkylene or an oxyethylene oligomer -(CH 2 CH 2 O) z - where z is an integer in the range of 1 to 16 inclusive, and R n is selected from the group consisting of CH 3 - 2 CH 2 -59v-O-w-- CH 3 O-CH 2 -ch 2 -O-CH 2 15

17. The reagent of claim 16 wherein R 1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl. . . 2 ·

18. The reagent of claim 16 wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates.

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

20. The reagent of claim 16 wherein R is -60-ch 2 ch 2 s

21. The reagent of claim 16 wherein R is 10 -CH 2 CH 2 Si(CH 3 ) 3 .

22. The reagent of claim 16 wherein R is R j

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 4penten-l-yl.

25. The reagent of claim 22 wherein R is —CH 2 CH 2

26. The reagent of claim 22 wherein R is -61Ο II —Ρ —οI ο·

27. A polynucleotide reagent having the structure 5'— hoAna,? '-O-P-O-iCH2) x -CH-(CH,)v-O-P-O- 5 OH OH NO OH wherein DNA^ is a first segment of DNA; DNA 2 is a second segment of DNA; and one of x and y is zero while the other is an integer in the range of 1 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 1 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. Ε912661 -6232. The polynucleotide reagent of claim 30, wherein x is an integer in the range of 1 to 4 inclusive

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

33. 34. A polynucleotide reagent having the _ structure OH HO—P—O- s '[DNA 2 1 j —OH II 0 wherein DNA^ is a first segment of DNA; DNA 2 is a second segment of DNA; and R is selected from the group consisting of 2nitrobenzyl, 4-penten-l-yl, and —CH 2 CH 2 no 2 in which R’ is hydrogen, aryl or aralkyl, the R| 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 -64alkoxy, i is zero, l, 2 or 3, j is zero, 1, 2, 3 or 4, is c 2 -c 16 alkylene or an oxyethylene oligomer -(CH 2 CH 2 O) z - where Z is an integer in the range of 1 to 16 inclusive, and R n is selected from the group consisting of ch 3 o-ch 2 -ch 2 -o-ch 2 30

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

35. 36. The polynucleotide reagent of claim 34 wherein R is -65- CHjCHjS io

36. 37. The polynucleotide reagent of claim 34 wherein R is R ;

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

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

39. 40. The reagent of claim 34 wherein R is —CH 2 CH 2 NO-»

40. 41. The reagent of claim 34 wherein R is II —p —oo•Ε 912661 -6642. 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 —0—(CH-j) a —CH—(CH 2 ) b —0— 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.

41. 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 Ιε 912661 -67polynucleotide reagent results in a label being bound to said support through the cleavage site —0—(CH,) a — CH- 2 ) b -O— 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.

42. 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 -685 R is selected from the group consisting of 2 nitrobenzyl, 4-penten-l-yl, CH 2 CH 2 Si(CH 3 ) 3 ) in which R’ is hydrogen, aryl or aralkyl, the R| 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, R m is c i -c i6 alkylene or an oxyethylene oligomer -(CH 2 CH 2 O) z - where z is an integer in the range of 1 to 16 inclusive, and R n is selected from the group consisting of CH 0 o LI 11 -c-ch 2 ch 2 -c3 ch 3 o-ch 2 -ch 2 -o-ch 2 -x 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. 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: -71combining 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 2nitrobenzyl, 4-penten-l-yl, in which R' is hydrogen, aryl or aralkyl, the R^ 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 -73alkoxy, i is zero, 1, 2 or 3, j is zero, 1, 2, 3 or 4, R m is c i -c i6 alkylene or an oxyethylene oligomer -(CH 2 CH 2 O) z - where z is an integer in the range of 1 to 16 inclusive, and R n is selected from the group consisting of ch 3 o-ch 2 -ch 2 -o-ch 2 separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium; -74washing 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. 46. A multifunctional nucleic acid monomer having the structure wherein R 1 is a base-stable, acid-sensitive blocking group; -752 R is a phosphorus derivative that enables addition of the nucleic acid to the 5'-position of an oligonucleotide chain during chemical synthesis; 3 · . R is selected from the group consisting of hydrogen, methyl, I, Br and F; R is hydrogen or methyl; R is selected from the group consisting of levulinyl, in which R' is hydrogen, aryl or aralkyl, the R^ 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 -76amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy, the 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 (2) II (1) -(CH,) x —NH —C —O15 ,2) II (1) —(CH,) x —NH —C-(CH,) y-0O (2) || (1) 20 -(CH^j—NH—C—(CH 2 ) y —S —S -(CH 2 ) y -O- ; —(CH,-CH, —O) x - ; and W (1) —(CH,) x —035 -77wherein x and y are integers which may be the same or different and are integers in the range of 1 to 8.

45. 47. A polynucleotide reagent having the structure R 5 I z 5’ — ΗΦ’ΕΌΝΑ!] 3 ' R 3 HO—P-O- 5 [DNA,] 3 —OH II 0 wherein DNA 3 is a first segment of DNA; DNA 2 is a second segment of DNA; R 3 is selected from the group consisting of hydrogen, methyl, I, Br and F; R 4 is hydrogen or methyl; and R 5 is selected from the group consisting of levulinyl, in which R 1 is hydrogen, aryl or aralkyl, the R^ 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 R^ 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 -790 (2) II (1) —iCHo) x —NH —C —O10 (2) II ( 1 ) - 2 ) x —NH —C-(CHi) y-02) (1) -(CH 2 )— NH — C—(CH 2 )y —S —S -(CH 2 )y -0 (1) 25 -(CHt) x -O wherein x and y are integers which may be the same or different and are integers in the range of 1 to 8. •Ε 912661 -8048. 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.

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

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

48. 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.

49. 54. The method of claim 53, wherein the nucleoside is present at the 3 1 -terminus of an oligonucleotide chain.

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

51. 56. The method of claim 54, wherein the oligonucleotide chain is bound to a solid support. -8157. A cleavable linker molecule having the structural formula wherein DMT represents dimethoxytrityl, Bz represents benzyl, iPr represents isopropyl, and R 6 is methyl or /3cyanoethyl.

52. 58. The cleavable linker molecule of claim 57 wherein R 6 is methyl.

53. 59. The cleavable linker molecule of claim 57 25 wherein R 6 is β-cyanoethyl.

54. 60. A compound having the structure CH^-O-R 1 I 30 CH 3 -C-CH 2 -O-R 2 ch 9 -o-r„ 2 n wherein -82R 1 is a base-stable, acid-sensitive blocking group; R is a phosphorus derivative selected to enable addition of the reagent to the 5' position of an oligonucleotide chain; and R n is a blocking or protective group that can be removed and replaced with hydrogen without affecting R 1 .

55. 61. The compound of claim 60 wherein R 1 is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl. . . 2 ·

56. 62. The compound of claim 60 wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates. . . 2

57. 63. The compound of claim 61 wherein R is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, Hphosphonates, and phosphorothioates.

58. 64. The compound of claim 60 wherein R n is selected from the group consisting of ,E 912661 -83CH. 0 9 ii j· -C-CH 7 CH 2 -C- CH 3 O-CH 2 -CH 2 -O-CH 2 25

59. 65. The compound of claim 63 wherein R n is selected from the group consisting of ,E 912661 -8410 CH 3 O-CH 2 -CH 2 -O-CH 2 25 - 85

60. 66. A reagent having the structure given and defined in claim 1, substantially as hereinbefore described and exemplified.

61. 67. A reagent having the structure given and defined in claim 16, substantially as hereinbefore described and exemplified.

62. 68. A method according to any one of claims 42-45 detecting the presence of an oligonucleotide sequence of a nucleic acid analyte present in a nucleic acid sample, ly as hereinbefore described and exemplified. for interest in substantial

63. 69. A multifunctional nucleic acid monomer having the structure given and defined in claim 46, substantially as hereinbefore described and exemplified.

64. 70. A method according to claim 48 of phosphorylating a compound containing a hydroxyl moiety, substantially as hereinbefore described and exemplified.

65. 71. A phosphorylated compound containing a hydroxyl moiety, whenever obtained by a method claimed in any one of claims 48-56 or 70.

66. 72. A cleavable linker molecule having the structural formula given and defined in claim 57, substantially as hereinbefore described and exemplified.

67. 73. A compound having the structure given and defined in claim 60, substantially as hereinbefore described and exemplified.