WO1994001445A1

WO1994001445A1 - Base-protected nucleotide analogs with protected thiol groups

Info

Publication number: WO1994001445A1
Application number: PCT/US1993/006514
Authority: WO
Inventors: Michelle M. Hanna
Original assignee: Research Corporation Technologies, Inc.
Priority date: 1992-07-14
Filing date: 1993-07-12
Publication date: 1994-01-20

Abstract

The present invention is directed to protected thio analogs of the pyrimidine and purine bases for syntheses of DNA and RNA by chemical or enzymatic methods. The subject analogs include reagents suitable for DNA or RNA synthesis via phosphoramidite, H-phosphonate or phosphotriester chemistry as well as reagents suitable for use by RNA and DNA polymerase, including thermostable polymerases employed by PCR or other nucleic acid amplification techniques. Methods of synthesizing the nucleotide analogs are also provided by the present invention. The nucleotide analogs of this invention can thus be incorporated into oligonucleotides or polynucleotides, deprotected and derivatized with a functional group. A method of synthesizing oligonucleotides with a functional group using the subject nucleotide analogs is also provided.

Description

! BASE-PROTECTED NUCLEOTIDE ANALOGS

WITH PROTECTED THIOL GROUPS

The present invention is directed to protected thio analogs of the pyrimidine and purine bases for c syntheses of DNA and RNA by chemical or enzymatic methods. The subject analogs include reagents suitable for DNA or RNA synthesis via phosphoramidite, H- phosphonate or phosphotriester chemistry as well as reagents suitable for use by RNA and DNA polymerase, including thermostable polymerases employed by PCR or other nucleic acid amplification techniques. Methods of synthesizing the nucleotide analogs are also provided by the present invention. The nucleotide analogs of this invention can thus be incorporated into oligonucleotides

- c or polynucleotides, deprotected and derivatized with a functional group.

Oligonucleotides with a variety of modifications have found widespread utility for many purposes such as stabilizing oligonucleotides to

20 degradation, introducing reporter groups, allowing site- specific delivery of therapeutics, and introducing crosslinkers. Such modifications principally occur as modified internucleotide phosphate linkages or analogs of such linkages, modified sugars or modified bases.

25 Additionally, 5'- or 3'-end conjugates of the oligonucleotides represent another class of modified oligonucleotides. The present invention relates to base-modified nucleotide analogs with protected thiol groups; these analogs are intermediates for chemical or

30 enzymatic synthesis of oligonucleotides and polynucleotides.

35 The syntheses of oligoribonucleotides and oligodeoxyribonucleotides (i.e. oligonucleotides) containing base-modified nucleotides at specific positions provides apowerful tool in the analysis of protein-nucleic acid or nucleic acid-nucleic acid interactions. These oligonucleotides have many other uses such as the site-directed delivery of therapeutics, utility as anti-sense therapeutics, and utility as diagnostic probes. Nucleotide analogs can be introduced into nucleic acids either enzymatically, utilizing DNA and RNA polymerases, or chemically, utilizing manual or automated synthesis. Preparation of such oligonucleotides by automated synthesis, utilizing, e.g., phosphora idite nucleotides, allows for incorporation of a broad range of nucleotide analogs, without the restraints for specific substrate conformation of the nucleotides that is imposed by most polymerases. Often, nucleotide analogs containing photoreactive crosslinking groups are introduced into oligonucleotides to probe protein-nucleic acid interactions via photocrosslinking (for partial review, see Hanna, 1989, Methods Enzymol. 180:383-409) . Deoxyoligonucleotides containing 4-thiothymidine or 6- thiodeoxyguanosine have been prepared and used for photochemical crosslinking of proteins directly to the nucleotide bases (Nikiforov et al. , 1992, Nucleic Acids Res. j ):1209-14) . Similarly, oligonucleotides containing 5-(aminopropyl)-2'-deoxyuridine have been prepared and the amino group subsequently modified with fluorescent or photoactive groups. (Gibson et al. , 1987, Nucleic Acids Res. 1^:6455-66.) However, the former thiodeoxynucleotides suffer the disadvantage that they cannot be modified with thiol modifying reagents which require a sulfhydryl (SH) group. Further, at least the 4-thiothymidine and the 5-aminoprσpyl- deoxyuridine base analogs can interfere with normal Watson-Crick base pairing, making such analogs unsuitable for use in enzymatic nucleic acid synthetic methods.

A cleavable photocrosslinking nucleotide analog, 5-[ (4-azidophenacyl)thio]uridine-5'-triphoεphate (5-APAS-UTP) has been reported (Hanna et al. , 1989, Biochemistry 28:5814-5820). However, the synthetic route to this compound is difficult, has many disadvantages and does not involve an intermediate which has a protected thiol group. In this regard, the intermediates involved in the preparation of 5-APAS-UTP are unsuitable for chemical synthesis of DNA or RNA by either manual or automated methods.

Accordingly, the present invention provides base-protected nucleotide analogs, both ribonucleotides and deoxynucleotides, that contain masked thiol groups on base positions not involved in Watson-Crick base pairing (or at the 6 position of guanine which still appears to be capable of Watson-Crick base pairing) . These analogs can be incorporated into oligonucleotides via automated synthesis and isolated with the thiol- protecting group intact. After removal of the thiol protecting group many types of functional groups, such as photocrosslinking agents, fluorescent tags, radioisotopes, biotin, reporter molecules or other functional groups, can be site-specifically attached by utilizing thiol- odifying reagents. This feature adds a level of specificity to the oligonucleotide modifications not present with the amino-tagged analogs previously described (Gibson et al, 1987), and enables examination of molecular interactions that are not directly at the nucleotide base by allowing functional groups to be placed at varying distances from the base or helix strand. Since these analogs have the functional group attached via the sulfur atom, they have the further advantage of being cleavable.

This invention relates to nucleotide analogs which are modified bases with protected thiol groups attached at a position on the base which is not involved in Watson-Crick base pairing or which does not disrupt normal Watson-Crick base pairing. These nucleotide analogs are intermediates in chemical or enzymatic synthesis of DNA or RNA and are therefore stable under such conditions. However, after synthesis, the protecting group is removable to generate a reactive thiol group. Once generated, the thiol group can be treated with thiol modifying reagents to attach functional groups such as crosslinking agents or reporter molecules.

In particular, the nucleotide analogs of the present invention are intermediates for chemical synthesis of oligoribonucleotides or oligodeoxyribonucleotides of the formula:

wherein R_a. is -H or a protecting group; R₂ is -H, a protecting group, a solid support, or taken together with the attached oxygen forms a phosphoramidite group, a phosphorothioamidite group, a phosphonate group, or an O-substituted monophosphate group;

R₃ is -H, -OH, or -0R₄;

R₄ is lower alkyl or a protecting group; and B is a modified purine or pyrimidine base comprising a protected thiol group attached at a position on said base that is not involved in Watson- Crick base pairing or does not disrupt normal Watson- Crick base pairing, said protected thiol group being stable under conditions of chemical nucleic acid synthesis or conditions of enzymatic nucleic synthesis and being convertible to a reactive thiol (SH) after said synthesis. Preferred nucleotide analogs of the present invention are the protected phosphoramidites with modified adenine, cytosine, guanine or uridine bases for use in the chemical synthesis of DNA and RNA by the phosphoramidite method.

In addition, the nucleotide analogs of the present invention include nucleotide phosphates, that is those intermediates for enzymatic synthesis of oligodeoxyribonucleotides, oligoribonucleotides, or longer polynucleotides, of the formula:

wherein R together with the attached oxygen forms a phosphate group;

R₅ is -H, -OH, -OR₄; R₃ is -H, -OH, or -OR₄; R₄ is lower alkyl, a phosphate group, a protecting group or a solid support; and

B is a modified purine or pyrimidine base comprising a protected thiol group attached at a position on said base that is not involved in Watson- Crick base pairing, said protected thiol group being stable under conditions of chemical nucleic acid synthesis or conditions of enzymatic nucleic synthesis and being convertible to a reactive thiol after said synthesis. Preferred nucleotide analogs of the present invention are the nucleotide triphosphates with modified adenine, cytosine, guanine or uridine bases.

Another aspect of this invention provides nucleic acids and oligonucleotides containing the subject nucleotide analogs having a protected thiol group on a base moiety of that nucleic acid or oligonucleotide. A method is also provided to synthesize these nucleic acids or oligonucleotides, deprotect the thiol group and attach a functional group to the reactive thiol moiety. Yet another aspect of this invention is directed to a method of synthesizing the subject base- protected nucleotide analogs.

Fig. 1 depicts the reaction scheme for synthesis of the 2-cyanoethyldiisopropyl (CED) phosphoramidites of 5-SCN-dU and 5-SCN-U.

Abbreviations: DMTr, 4,4'-di ethoxytrityl; DMAP, 4- (N,N-dimethyl)-amino pyridine; tBu, tert-butyl; Me, methyl.

Fig. 2 outlines the steps for producing an oligonucleotide trimer with a photoactivatible crosslinker via the analog-substituted oligonucleotide trimer A-U(SCN)-C. Incorporation of 5-SCN-dU into the trimer is accomplished by automated DNA synthesis using phosphoramidite chemistry to produce the trimer A-

U(SCN)-C. This trimer is deprotected with DTT to produce A-U(SH)-C, followed by reaction with p- azidophenacyl bromide to yield the trimer with the covalently-linked photo crosslinker,

A-U(APAS)-C. Abbreviations: A, 2'-dA^toz; C, 2'-dC^bz;

T, 2'-dT; U(SCN), 5-SCN-2'-dU; U(SH), 5-SH-2'-dU; U(APAS), 5-thio-(S-4-azidophenacyl)-2'-dU; DTT, dithiothreitol; APB, p-azidophenacylbromide; Et₃N, triethylammonium bromide buffer.

Fig. 3 depicts a reaction scheme for the synthesis of 5-thiocyanotocytosine (5-SCN-C). Fig. 4 depicts a reaction scheme for the synthesis of (A) 8-SCN-dA or 8-SCN-A, (B) 8-SCN-dG or 8-

SCN-G and (C) 6-SCN-dG or 6-SCN-G.

The present invention relates to a series of nucleotide analogs which are masked synthons for use as intermediates in chemical or enzymatic synthesis of nucleic acids, including synthesis of both oligonucleotides and polynucleotides. The nucleotide analogs of this invention which contain a protected thiol group can thus be incorporated into DNA or RNA under standard synthetic conditions without loss of the thiol protecting group. This stability of the thiol protecting group permits site-selective introduction into a nucleic acid of the nucleotide analog in a manner which facilitates later addition of a functional group at that site. Thus, the subject oligonucleotides (or polynucleotides) can contain one (or more) of the subject nucleotide analogs.

Thus, functional groups which can not withstand the conditions for chemical nucleic acid synthesis, especially during automated synthesis, or which may be too bulky for enzymatic nucleic acid synthesis can be readily incorporated into the final oligonucleotide (or polynucleotide) product. Such derivatized nucleic acids have great utility in studying protein-nucleic acid or nucleic acid-nucleic acid interactions, as well as the potential for site-specific delivery of therapeutics. Similarly, these compounds can also serve as therapeutics. Hence, the present invention permits the skilled artisan to place chemical tags such as crosslinking groups, fluorescent molecules, radioisotopes or other reporter molecules at specific positions on nucleic acid molecules for analysis of molecular mechanisms, for creation of diagnostic probes, for therapeutics, for antisense therapeutics and for many other purposes.

In particular, the nucleotide analogs of the present invention are intermediates for the chemical synthesis of DNA and RNA by manual or automated techniques and are represented by the formula:

wherein R_x is -H or a protecting group;

R₂ is -H, a protecting group, a solid support, or taken together with the attached oxygen forms a phosphora idite group, a phosphorothioamidite group, a phosphonate group, or an O-substituted monophosphate group;

R₃ is -H, -OH, or -OR₄;

R₄ is lower alkyl or a protecting group; and B is a modified purine or pyrimidine base comprising a protected thiol group attached at a position on said base that is not involved in Watson- Crick base pairing or does not disrupt normal Watson- Crick base pairing, said protected thiol group being stable under conditions of chemical nucleic acid synthesis or conditions of enzymatic nucleic synthesis and being convertible to a reactive thiol after said synthesis.

As used herein, protecting groups defined by Ri, R₂ and R₄ include any known protecting group suitable for protection of the sugar hydroxyls (2' , 3' or 5' hydroxyls) of both ribose and deoxyribose sugars. In this regard, Greene et al. (1990) Protecting Groups in Organic Synthesis, 2nd ed. , John Wiley & Sons, Inc., New York, NY, provides a comprehensive review of protecting groups, and methods of preparing the corresponding protected compounds, which can be used for different reactive groups, including reactive hydroxyl groups.

Accordingly, protecting groups defined by R_{l r} R₂ and R₄ are lower alkyl, lower cyanoalkyl, lower alkanoyl, aroyl, aryloxy, aryloxy-lower alkanoyl, haloaryl, fluorenylmethyloxy-carbonyl (FMOC), levuloyl, 9-phenylxanthene-9-yl, trityl, £-monomethoxytrityl (MMTr), p-dimethoxytrityl (DMTr), isopropyl, isobutyl, 2-cyanoethyl, acetyl, benzoyl, phenoxyacetyl, halophenyl, l-(2-chloro-4-methylphenyl)-4-methoxy-4- piperidinyl, 2'-acetal, O-nitrobenzyl, tert- butyldi ethylsilyl (TBDMS), tetrahydrofuranyl, 4- methoxytetrahydropyranyl and related groups.

Preferred Rj_. protecting groups include 5'OH protecting groups especially DMTr, MMTr, FMOC, levuloyl and 9-phenylxanthene-9-yl groups, and most preferably DMTr. Preferred R₂ protecting groups include 3'OH protecting groups, especially the acetyl group. Preferred R₄ protecting groups include 2'OH protecting groups especially TBDMS, 2'-acetal, tetrahydrofuranyl, 4-methoxytetrahydropyranyl and l-(2-chloro-4- methylphenyl)-4-methoxy-4-piperidinyl groups, and most preferably TBDMS.

As defined herein, solid supports include controlled pore glass (CPG), polystyrene silica, cellulose, nylon, and the like. Preferred solid supports are CPG and polystyrene. An especially preferred solid support is CPG.

R₂ can be taken with the oxygen atom to which it is attached, to form a phosphoramidite, phosphorothioamidite, a phosphonate, an O-substituted onophosphate, or any other group compatible with chemical nucleic acid synthesis, especially automated DNA or RNA synthesis. As used herein, the phosphoramidite and phosphorothioamidite groups have the general formulas I and II, respectively

II

wherein R_e is lower alkyl, n-cyano alkyl, substituted lower alkyl, aryl, aralkyl, substituted aralkyl and the like, and R₇ and R_s are independently lower alkyl or when taken together with the nitrogen to which they are attached form

Preferably the R₆ group of the phosphoramidite group (Formula I) is 2-cyanoethyl (CED) or methyl. Accordingly, the preferred phosphoramidites of this invention are also referred to as CED phosphoramidites or methyl phosphoramidites.

As used herein, the term lower alkyl, when used singly or in combination, refers to alkyl groups containing one to six carbon atoms. Lower alkyl groups can be straight chain or branched and include such groups as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl and the like. The preferred alkyl groups contain one to four carbon atoms. The term aryl, when used alone or in combination, refers to an aromatic ring containing six to ten ring carbon atoms. The aryl group includes phenyl, and 1- or 2-naphthyl. The preferred aryl group is phenyl.

The term aralkyl refers to aryl groups as described above to which substituents are attached to the aryl by an alkylene bridge. The most preferred aralkyl group is benzyl.

When R-₇ and R_β are lower alkyl, the preferred groups are each isopropyl groups.

As used herein a phosphonate group (or H- phosphonate) is represented by the formula:

and are conveniently provided as salts, and preferably as triethylammonium salts.

As used herein, O-substituted onophosphates have the formula:

wherein R₉ is lower alkyl, haloalkyl, aryl, haloaryl, or heteroaromatic. By haloalkyl or haloaryl is meant alkyl or aryl groups, respectively, which have been substituted with one or more halogen atoms including F, Cl, Br or I. Preferred halo substituents are Cl and Br. Preferred R₉ groups include 2-chlorophenyl, 2,5- dichlorophenyl, 2,2,2-trichloroethyl and the N-oxide of 4-methoxypyridine-2-methylene groups.

Modified purine and pyrimidine bases have a protected thiol group attached at a position on the base which is not involved with Watson-Crick base pairing or which does not disrupt normal Watson-Crick base pairing. The protected thiol groups of this invention are stable, i.e. not removable, under the conditions used for chemical synthesis of DNA or RNA, and particularly, under the conditions employed in automated DNA or RNA synthesis. Furthermore, the protecting group of the thiol is removable under conditions which do not disrupt the integrity of the oligonucleotide or polynucleotide. In other words, after a nucleotide analog of the present invention has been incorporated into an oligonucleotide, for example, the protected thiol group can be converted to a reactive thiol (SH) to which functional groups can subsequently be added using thiol-modifying reagents. In accordance with this invention the protected thiol groups include thiocyanato (-SCN) and alkylthiocyanato (-R₁₀SCN) groups. The R₁₀ group of this invention is lower alkyl as herein defined. As used herein, Watson-Crick base pairing refers to the hydrogen bonding pattern of adenine-thymine (AT) base pairs or of guanine-cytosine (GC) base pairs. Accordingly, the preferred protected thiol position on purines (A,G) bases is the 8 position on the purine ring, whereas it is the 5 position on the pyrimidine (C,T,U) ring. Moreover, the 6 position of guanine substituted with sulfur appears to allow normal Watson- Crick base pairing (Taktakishvili et aJL. , 1990, Bioorg. Khim. lJ:59-68). Consequently, the protected thiol group can also be attached to the 6 position of the guanine ring; base pairing can occur after deprotection. The bases of the present invention are thus purines such as guanine (G) and adenine (A), pyrimidines such as cytosine (C), and uracil (U) . Purine and pyrimidine bases contain additional reactive groups, particularly exocyclic amines, which must also be protected during assembly of the nucleotide chain via chemical synthesis. Accordingly, bases of this invention can have additional protecting groups attached as needed to any of the ring positions. Such protected bases are well known in the art and include but are not limited to, N²-isobutyryl guanosine, N²-dialkyl-formadine guanosine, N⁴-anisoyl cytosine, N⁴-benzoyl cytosine, N⁴-isobutyryl cytosine, N^β benzoyl adenine and N⁶-dialkylformadine adenine. The dialkylformadine protected bases are preferably provided as dimethylformadine. The dimethylformadine protected bases together with N⁴-isobutyryl cytosine are known by the trade name FOD™ base protection (Applied Biosystems, Inc.). Use of these further protected bases is compatible with incorporation of the protected thiol groups as well as subsequent deprotection reactions.

The bases of this invention also include any related base analog that is capable of base pairing with a guanine, adenine, thy ine, cytosine, uracil or the corresponding protected analogs as set forth above for use in chemical synthetic methods to produce DNA and RNA. For example, such base analogs include pseudocytosine, isopseudocytosine, 3-aminophenyl- imidazole, 2'-O-methy1-adenine, 7-deazadenine, 7- deazaguanine, 4-acetylcytosine, 2'-O-methylσytosine, dihydrouracil, 2'-O-methyluracil, 2'-O-methyl- pseudouracil, β-D-galactosylqueonine, 2'-0- methylguanine, xanthine, hypoxanthine, N6-isopentenyl- adenine, 1-methyladenine, 1-methyl-pseudouracil, 1- methyl-guanine, 1-methylxanthine, 2,2-dimethylguanine, 2-methyl-adenine, 2-methylguanine, 3-methylcytosine, N6- methyladenine, 7-methylguanine, β-D-mannosylqueonine. Bases attached to the ribose or deoxyribose sugar in an α-anomeric configuration can also be present. In a preferred embodiment the bases of the invention are A, C, G, U, N^G protected A, N⁴ protected C, or N² protected G. Since U does not contain a reactive amine group, additional protection on this bases is not typically used in chemical DNA or RNA synthesis. The compounds of the present invention also include the phosphates of the formula:

wherein R_x together with the attached oxygen forms a phosphate group; R_s is -H, -OH, -0R₄;

R₃ is -H, -OH, or -0R₄; R₄ is lower alkyl, a phosphate group, a protecting group or a solid support; and

B is a modified purine or pyrimidine base comprising a protected thiol group attached at a position on said base that is not involved in Watson- Crick base pairing, said protected thiol group being stable under conditions of chemical nucleic acid synthesis or conditions of enzymatic nucleic synthesis and being convertible to a reactive thiol after said synthesis. The groups defined by R₃, R₄, R₅ and B are as set forth above unless otherwise noted. In the case of the modified bases the protected thiol group is also stable, i.e. not removable, under the conditions used for enzymatic synthesis of DNA or RNA. The preferred positions for attachment of the protected thiol group are the 5 position of the pyrimidine and the 8 position of the purines. Additionally, the purine and pyrimidine bases generally do not require protection of the additional reactive groups such as the amino.

Accordingly, the preferred bases for this class of compounds includes A, C, G and U.

In accordance with this invention the phosphate groups of R₅ and R₄ of this invention embody all the phosphorylated forms at the C₃ and C₅ positions of the sugar moiety and include onophosphates, diphosphates, triphosphates and tetraphosphates. Preferably the phosphate group is a triphosphate for R₅. The preferred compounds of this invention include the phosphoramidites and 5'triphosphates of 5- SCN-dU, 5-SCN-U, 5-SCN-dC, 5-SCN-C, 8-SCN-dA, 8-SCN-A, 8-SCN-dG and 8-SCN-G. In the case of the phosphoramidites, the base moieties can contain additional protecting groups on the exocyclic amines. The nucleotide analogs of the present invention can be prepared by adding a protected thiol group to the base moiety of the desired nucleoside. The so-modified nucleoside can then be phosphorylated to produce a nucleotide analog phosphate compound of this invention using conventional phosphorylation techniques. To produce a phosphoramidite, phosphonate, phosphothioamidite or O-substituted monophosphate of this invention, the 5'-OH or 2'-OH groups of above- modified nucleoside are protected as necessary by addition of the desired protecting group by standard methodology. This protection step(s) is (are) followed by conversion to the nucleotide-analog phosphoramidite, phosphorothioamidite, phosphonate or O-substituted monophosphate by reaction of the 3'OH of the nucleoside with the appropriate modifying group. Similarly, the 3'OH can be attached to a solid support, such as CPG, or another protecting group using conventional methodology available to the ordinarily skilled artisan in this field.

For example, to prepare protected thiol-groups at the 5 position of uridine or 2'-deoxyuridine nucleosides (5-SCN-U or 5-SCN-dU) the method of

Nagamachi et al. (1972, J.C.S. Chem. Commun. 18:1025-6) can be employed. Hence, lead thiocyanate or lead alkylthiocyanate is dissolved in glacial acetic acid which has been saturated with chlorine. Chlorine gas is bubbled through the acid mixture up to several hours

(about 1 to 3 h) to produce the thiocyanate chloride or alkylthiocyanate chloride in solution. If necessary, or desired, the solution can be used directly or filtered prior to the next step. In either case, the solution is degassed and an 0.5 equivalent (eq) of U or dU is added all at once with stirring. This reaction is maintained with stirring at room temperature for about 4 hours. If necessary, the reaction can be conducted at 10-50°C for varying amounts of time (from 1 h to 24 h) until the reaction is completed. An excess of cyclohexene or other quenching agent is then added to quench any remaining thiocyanate chloride or alkylthiocyanate chloride. Quenching is complete in 15 to 60 min, and usually in about 30 min. After removing solvents and organic residue in vacuo, the remaining residue can be dissolved in a minimum amount of acetonitrile or other compatible solvent, frozen and lyophilized overnight before purification by HPLC. Alternatively, the purification can be done directly from the acetonitrile solution using HPLC or other chromatographic methods. These thio-protected dU and U nucleosides can then be further derivatized as described herein to produce the nucleotide analogs of the present invention.

The 5-SCN-dUTP or 5-SCN-UTP compounds can be prepared by reacting 5-Br-dUTP or 5-Br-UTP with AgSCN or silver alkylthiocyanate in tetrahydrofuran (THF) or acetonitrile for about 1 to about 20 hours at room temperature or until the reaction is complete. The desired product is then isolated by HPLC. The mono-, di- or tetra phosphate compounds can be prepared by a similar reaction by beginning with the appropriate starting compound. If necessary, the 2'-OH of the ribose sugar can be protected prior to the reaction using any of the known 2'-OH protecting groups by conventional techniques.

One method to produce the nucleotide analog phosphoramidites of this invention is shown in Fig. 1. As depicted, 5-SCN-dU or 5-SCN-U is reacted with the DMTrCl, or another 5'OH protecting group, in an anhydrous organic solvent in the presence of an organic base for 2-24 hours until the reaction is complete. The resulting product can be isolated by HPLC and then converted to a CED phosphoramidite by reaction with 2- cyanoethyl-N,N-diisopropylchlorophosphor-amidite and ethyl-diisopropylamine under anhydrous conditions and in an inert atmosphere. This reaction is preferably stirred for 5 h, although this time can be varied, the solvents removed in vacuo and the residue lyophilized. Exposure to atmosphere is acceptable but should be minimized. This phosphoramidite can be stored dry under positive pressure of inert atmosphere (argon or nitrogen) in a tightly sealed container at low temperature. Other phosphoramidites of this invention can be prepared by reaction with the appropriate chlorophosphoro-amidite. For example, the O- methylphosphoramidites can be prepared by reacting the 5-SCN-5'-DMTr-dU with N,N-diisopropylmethyl- phosphonamidic chloride by conventional techniques. Similarly, the phosphorothioamidite, phosphonates, O- substituted monophosphate can be prepared using convention techniques with commercially available reagents. As mentioned above to avoid unwanted side reactions, protection of 5'OH and 2'OH on the sugar moiety and the exocyclic amine groups on the base moiety may be necessary before the final reaction step which produces the nucleotide analogs of this invention.

In general the chemical synthetic routes to nucleotide phosphoroamidites, phosphorothioamidites, phosphonates and O-substituted monophosphates as well as nucleotide phosphates are well known. In addition, nucleotide phosphates can be enzymatically synthesized (See, for example, Sambrook et al. , 1989, Molecular Cloning, 2nd ed. , Cold Spring Harbor Press, Cold Spring Harbor, NY or other manuals for recombinant DNA techniques) . Chemical synthesic techniques for these compounds as well as the common synthetic routes to prepare RNA and DNA have been described in many sources. Particularly, useful references include Gait (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford; Blackburn et al. (1990) Nucleic Acids in Chemistry and Biology, IRL Press, Oxford, especially Chap. 3; Chaps 13-16 of Methods in Enzymology, Vol. 154 (Wu et al. , eds.) Academic Press, San Diego, CA, 1987; Chaps. 13-14 in Methods in Molecular Biology, Vol. 4 (Walker, ed. ) , Humana Press, Clifton, NJ, 1988; and Uhlmann et al. (1990) Chem. Rev. 90:544-584.

In addition to providing methods for chemical synthesis of DNA and RNA, some of these references (particularly Gait and Uhlmann et aL. ) describe the reactions and methodology for adding protecting groups to 5'OH, 3'OH and 2'OH groups, and exocyclic amine groups (N^e of adenine, N⁴ of cytosine, N² of guanine). These references also provide information and protocols to attach nucleotides to solid supports which protocols are useful for attaching the base-protected nucleotide analogs of this invention.

The synthesis of 5-SCN-dC and 5-SCN-C can be accomplished in a manner analogous to the method of Nagamachi for synthesizing 5-SCN-dU as described above. Similarly, 5-Br-dC or 5-Br-C can be converted to 5-SCN- dC or 5-SCN-C using the silver thiocyanate reaction described above. In these cases, it may be necessary to first protect the 4 amine group of cytosine (e.g., as the i-butoxy amide) and complete the remaining protective stages (5'-0-DMTr and 2'-0-TBDMS, if needed) before preparation of a CED phosphoramidite or conversion to the triphosphate (Reese et al. , 1980, Tetrahedron Lett. Zl:2265; Sung, 1982, J. Org. Chem. 47:3623). Alternatively, 5-SCN-dU or 5-SCN-U can be converted to the corresponding cytosine compounds by acetylating the OH groups of these uridine analogs, followed by conversion to the amine at the 4 position. Reaction of triacetoxy uridine with pyridine under the right conditions (4-ClPhOP(0)Cl₂ in pyridine or 1- (2,4,6-triisopropylbenzene sulfonyl)3-nitro-l,2,4- triazole) gives 4-(3-nitro-l,2,4-triazolo)-2' ,3' ,5'- (triacetoxy)-uridine. The acetoxylation of deoxyuridine or uridine is done using acetic anhydride. After conversion to the cytosine analog, the acetyl groups are removed by treatment with base. This reaction scheme for synthesis of 5-SCN-C is shown in Fig. 3. This same reaction can be used to convert guanosine to the 2-amino adenosine analog.

Preparation of 8-SCN-dA and 8-SCN-A can be accomplished as depicted in Fig. 4A. Preparation of 8- SCN-dG and 8-SCN-G can be accomplished as depicted in Fig. 4B. In these reactions the 8-chloro (or other 8- halo) form of guanine or adenine are treated with silver or lead thiocyanate under conditions analogous to those for synthesis of 5-SCN-dU. Preparation of 6-SCN-dG and 6-SCN-G can be accomplished as depicted in Fig. 4C. Any other of these thiocyanate compounds can also be synthesized as alkylthiocyanate compounds by substituting the alkylthiocyanate silver or lead salt for the corresponding thiocyanate salt.

The conversion of 5-SCN-dU and 5-SCN-U to 5' phosphates, phosphoramidites, phosphorothioamidites, phosphonates, and O-substituted monophosphates has been described hereinabove. All of these reaction schemes can be used to produce the corresponding thiol protected adenine, guanine and cytosine analogs. If necessary, various protecting groups for the 5'OH, 3'OH or 2'OH groups as well as the exocylic amines can be added in accordance with the methodology described herein.

Another aspect of this invention relates to the oligonucleotides or polynucleotides containing the nucleotide analogs of this invention and a method of preparing such nucleic acids using the subject nucleotide analogs. Oligonucleotides and polynucleotides of this invention are made by standard methods of chemical (automated or manual) synthesis or enzymatic synthesis of DNA and RNA. Such methods are well known in the art. In chemical synthesis, the nucleotide analog of this invention is substituted for a particular nucleotide at the desired point in the synthesis. To achieve effective and high yield coupling of the subject analog can require extended coupling times from about 1 hour to about 24 hours. This time period can readily be determined by one of ordinary skill in the art.

After .incorporation of the nucleotide analog and complete synthesis of the oligonucleotide or polynucleotide, the thiol can be deprotected and reacted with any number of thiol-modifying reagents to attach a functional group at that point on the oligonucleotide or polynucleotide. Deprotection of thiocyanate or alkylthiocyanate can be accomplished by treatment with dithiothreitol (DTT) or by other means of reducing sulfides. In a preferred method, deprotection is accomplished by treating the oligonucleotide with 3 equivalents of DTT at 55°C for about 15 min (see Example 5).

Any variety of functional groups can then be added to the reactive thiols group generated by the deprotection step. Such functional groups include cross-linking groups, photoactivated cross-linking groups (e.g. arylazides), and reporter molecules such as radioisotopes, biotin, enzymes and fluorescent markers. The methods for adding functional groups are well known in the art.

Yet another aspect of this invention provides a method of preparing the nucleotide analogs of the invention. In particular, this method involves preparing a thiol protected nucleoside or nucleotide base wherein said thiol is attached to a position on said base that is not inovlved in Watson-Crick base pairing or does not disrupt normal Watson-Crick base pairing; reacting said nucleoside or nucleotide base under conditions to effect conversion of said base to a phosphoramidite, phosphorothioamidite, phosphonate, O- substituted monophosphate or phosphate nucleotide analog and under conditions which do not destroy the protected thiol; and recovering said analog. In accordance with this invention, this method is accomplished as described above for synthesis of the subject nucleotide analogs. Recovery of the analogs can be accomplished by HPLC, FPLC or other chromatographic separation technique.

A further aspect of this invention provides a method of synthesizing a nucleic acid with a functional group by incorporating a thiol protected nucleotide analog in accordance with this invention into a nucleic acid by a chemical or enzymatic method for nucleic acid synthesis; recovering the nucleic acid containing the analog; deprotecting the analog of that nucleic acid to produce a nucleic acid containing a reactive thiol group; reacting the reactive thiol group with a thiol- modifying reagent to thereby attach a functional group and produce the nucleic acid with the functional group; and recovering the nucleic acid with the functional group. In accordance with this invention, this method is accomplished as described hereinabove for synthesis of oligonucleotides and polynucleotides (See for example Gait or Sambrook et al) . As used herein, nucleic acids include oligonucleotides and polynucleotides. Preferably, the oligonucleotides range in size from about 5 to about 200 nucleotides. Polynucleotides range in size from 200 nucleotides to 10 kb or more. Recovery of the analogs can be accomplished by HPLC, FPLC, other chromatographic techniques, extraction, phase separation or precipitation.

The Examples further illustrate the invention.

Example 1

Synthesis of 5-thiocyanato-2' -deoxyuridine phosphoramidite

Synthesis of 5-SCN-dU { 1 of Fig. 1) was achieved using a modification of the method of Nagamachi et al. (1972). Lead thiocyanate (1.42 g, 4.4 mmol) was added to a chlorine-saturated glacial acetic acid solution (10 mL) and the reaction was stirred with a slow, active addition of chlorine gas for 1.5 hours, at which time the mixture can be filtered, or used without further purification. After repeatedly de-gassing the resulting yellow solution of SCNCl, 2'-deoxyuridine (1.0 g, 4.4 mmol) was added and the reaction stirred at room temperature for 4 hours. An excess of cyclohexane (5 ml, to quench any remaining SCNCl) was added to the reaction and allowed to stir for thirty minutes. After removing the solvents and organic residues in vacuo, the residue was taken up in a minimum amount of acetonitrile, frozen and lyophilized overnight.

Separation and purification by HPLC was conducted in a 21.2 x 150 mm ODS column with 6 /E 15% acetonitrile in 50 mM triethylammonium bicarbonate (TEAB), pH8 buffer over 30 min. The resultant yield of 5-SCN-dU was 58%. The corresponding synthesis using 2'-deoxyuridine-5'- monophosphate proved to be unworkable, giving an intractable mixture of products.

The 5-SCN-dU was further protected for automated DNA synthesis. DMTrCl 0.6 g, 1.75 mmol, in 15 mL pyridine was added dropwise to a suspension of 5-SCN- dU (0.5 g, 1.75 mmol) in anhydrous pyridine, and the reaction was stirred for 18 hours. HPLC purification gave 84% product 5-SCN-5'-DMTr-dU (3 of Fig. 1) after repeated lyophilization from acetonitrile. Use of acetonitrile as solvent and N,N-dimethylaminopyridine (DMAP) as base (3-5 mol eq. ) resulted in slight improvement of the yield (88%), and much shorter reaction time (2.5 hrs). The reaction product was purified by HPLC using 9 R 33%, 12 min, and 33 R 80%, 30 min, acetonitrile in 50 mM TEAB, pH 8 buffer and lyophilized overnight. The final step to produce the desired phosphoramidite was addition of 2-cyanoethyl- N,N-diisopropyl (CED) chlorophosphoramidite. Materials and glassware were dry, and all steps were accomplished under inert atmosphere. Oven dried 5-SCN-5'-DMTr-2'-dU (0.05 g, 0.085 mmol) and anhydrous ethyldiisopropyl amine (75 ml, 0.425 mmol) were dissolved in anhydrous dichloromethane (5 mL) . CED chloro-phosphoramidite (0.178 g, 0.085 mmol) was added via syringe, and the reaction was stirred under a nitrogen atmosphere for 5 hours. After removal of solvents in vacuo, the residue was lyophilized overnight. Exposure to atmosphere for short times is acceptable, but must be minimized for efficacy of the automated synthesis. The 5-SCN-5'-DMTr- dU CED phosphoramidite (5_ of Fig. 1) was taken up in a small quantity ( ^"1 mL) of anhydrous acetonitrile, and appropriate dilutions made for determination of concentration ( λ _max 273 ran, ε = 10400 M^_1cm^_:L). The phosphoramidite may be stored for some time as a dry solid under a positive pressure of inert atmosphere (nitrogen or argon) in a tightly sealed container at a low temperature. Example 2

Synthesis of 5-thiocyanato-2' -uridine phosphoramidite

The synthesis of 5-SCN-U (2_^ of Fig. 1) and the protection of the 5'OH group with DMTr was accomplished as described in Example 1. However, a step for protection of the 2'-hydroxyl position of the ribose sugar was necessary and accomplished by the methods of Hakimelahi et al. (1982, Can. J. Chem. 60:1106-13) , Ogilvie et al. (1978, Can. J. Chem. 5_6:2768-80) and Ogilvie et al. (1979, Can. J. Chem. 57:2230-38) .

After protection of the 5'OH group with DMTr, the 5-SCN-5'-DMTr-U (5.5 mol eq. ) and silver nitrate (1.5 eq. ) was dissolved in pyridine (5.5 mol equivalents) and stirred for 5-10 minutes (until the AgN0₃ is dissolved) . tert-Butyldimethylsilyl chloride (TBDMSCl) was added all at once and the mixture stirred at room temperature for 1.5 hours. The reaction mixture was filtered into 5% sodium bicarbonate solution (to prevent de-tritylation during workup) , extracted with methylene chloride, and evaporated to dryness. The residue was taken up in acetonitrile and purified by HPLC. The CED phosphoramidite (€> of Fig. 1) was then prepared as described in Example 1.

Exa ple 3

Deprotection of 5-SCN-dU with dithiothreitol (DTT)

Deprotection of 5-SCN-dU with 1 mol equivalent DTT at pH 8.9 (50 mM TrisHCl) over the prescribed 2 minutes (Nagamachi et al. , 1992) gave little deprotection, as shown by UV absorption at 328 nm (ε = 8.82 x 10^~3 M-^-cm^-1; Ho et al. , 1976 in Nucleic Acid Chemistry Vol.2 (Townsend et al. , eds) John Wiley and Sons, Inc., New York, pp. 813-816). Further, use of 2 mol equivalents DTT and warming at 37° C over 30 minutes gave 53% deprotection. However, nearly quantitative deprotection of 5-SCN-dU was achieved with 3 mol equivalents DTT and warming to 55° C for 15 minutes.

Example 4

Automated Synthesis of an Oligonucleotide Trimer Containing 5-SCN-dU

Automated incorporation of the nucleotide analog 5-SCN-dU phosphoramidite into an oligonucleotide trimer was accomplished using an Applied Biosystems, Inc. (ABI) Model 392 DNA Synthesizer using standard phosphoramidite chemistry as provided by ABI except the coupling time for the analog phosphoramidite was extended to 1 h. (Extension of the coupling time up to about 12-16 h gave similar results.) The oligonucleotide trimer A-U(SCN)-C was assembled using the nucleotides A-CED, U(SCN)-CED and C-CED. A control trimer A-T-C was assembled using the same nucleotides except that T-CED replaced U(SCN)-CED. Each of these trimers was purified by HPLC and lyophilized for 72 h prior to further analysis.

A larger oligonucleotide (31-mer) was also synthesized with 5-SCN-dU phosphoramidite by the same methods. The 31-mer was from the promoter of E. coli RNA polymerase and had the sequence 5'-AAAGC AAAGA AATGC T-U(SCN)-GAC TCTGT AGCGG G.

Abbreviations: A-CED, N⁶-benzoyl-5'-0-(4,4'- dimethoxytrityl)-2'-deoxyadenosine-3'-(2-cyanoethyl-N,N- diisopropyl)phosphoramidite; C-CED, N⁴-benzoyl-5'-0-(4- 4'-dimethoxytrityl)-2'-deoxycytidine-3'-(2-cyanoethyl- N,N-diisopropyl)phosphoramidite; T-CED, 5'-0-(4,4'- dimethoxytrityl-2-deoxythymidine-3'-(2-cyanoethyl-N,N- diisopropyl)phosphoramidite; U(SCN)-CED, 5-thiocyanato- 5'-O-(4-4'-dimethoxytrityl)-2'-deoxyuridine-3'-(2- cyanoethyl-N,N-diisopropyl)phosphoramide. Example 5

Deprotection of A-U(SCN)-C with Dithiothreitol (DTT)

Deprotection conditions for the analog- substituted oligonucleotide trimer were examined. The results of treating control and analog-substituted trimer with various amounts of DTT at various temperatures for various times are shown in Table 1. Nearly quantitative deprotection occurred when the analog-substituted trimer was treated with 3 mol equivalents of DTT at 55° C for 15 min. DTT had no effect on the integrity of the control trimer within the detection limits of diode array UV spectral analysis.

TABLE 1

Effects of DTT on Control and Thiocyanato Protected Trimers

Reactions which contained the analog-substituted oligonucleotide are indicated by letters with primes (Tubes A'-D'). Reactions without primes contained the control trimer.

Example 6

Deprotection and Functionalization of A-U(SCN)-C with a Photocrosslinker

The A-U(SCN)-C trimer was deprotected and reacted with a photocrosslinker as schematically outlined in Fig. 2. Quantitative deprotection of A- U(SCN)-C was achieved with 3 mol equivalents of DTT at 55°C for 15 minutes. The trimer dA(5-SH-dU)dC was isolated by HPLC and lyophilized for 72 hours prior to the analysis. Addition of a photocrosslinker was accomplished by the addition of 1.5 equivalents of p_- azidophenacyl bromide to a solution of the analog trimer in 50 mM TEAB pH 8 buffer in dim or red light. Concentration was determined by UV analysis at 300 nm ( ε = 20000 M-^-cπr³-).

Example 7

Production of a Fluorescein-labelled Oligonucleotide

The 31-mer of Example 4 was deprotected with 3 mol equivalents of DTT at 55°C for 15 min. The 31-mer was isolated by HPLC and reacted with 5- iodoacetimidofluorescein to produce the 31-mer with fluorescein attached on the dU at nucleotide 17. Similarly, the p-azidophenacyl photocrosslinker has been added to the dU by the method described in Example 6.

Claims

I Claim :

A nucleotide analog of the formula:

wherein R_x is -H or a protecting group; R₂ is -H, a protecting group, a solid support, or taken together with the attached oxygen forms a phosphoramidite group, a phosphorothioamidite group, a phosphonate group, an O-substituted monophosphate group or an O-substituted monothiophosphate group; R₃ is -H, -OH, or -OR₄;

2. The nucleotide analog of Claim 1 wherein said base is adenine, cytosine, guanine or uracil.

3. The nucleotide analog of Claim 2 wherein said base comprises an additional protecting group on a reactive moiety of said base.

4. The nucleotide analog of Claim 3 wherein said base is N²-isobutyryl guanosine, N⁴-anisoyl cytosine, N⁴-benzoyl cytosine, N⁶-benzoyl adenine, N⁴- isobutyryl cytosine, N⁶-dialkylformadine adenine or N²- dialkylformadine guanosine.

5. The nucleotide analog of Claim 1 wherein said protected thiol group is thiocyanate (-SCN) or alkylthiocyanate (-R₁₀SCN), wherein R₁₀ is lower alkyl.

6. The nucleotide analog of Claim 1, wherein said position is 8 when said base is a purine, wherein said position is 5 when said base is a pyrimidine, or wherein said position is 6 when said base is guanine.

7. The nucleotide analog of Claim 1, wherein said R protecting group is p-(dimethoxytrityl) , p- (monomethoxy-trityl) , fluorenylmethyloxycarbonyl, levuloyl or 9-phenylxanthene-9-yl.

8. The nucleotide analog of Claim 1, wherein said R₄ protecting group is l-(2-chloro-4-methylphenyl)- 4-methoxy-4-piperidinium, 2'-acetal, o-nitrobenzyl, tert-butyldimethyl silyl, tetrahydrofuranyl or 4- methoxytetrahydropyranyl.

9. The nucleotide analog of Claim 1 wherein

R_x is a protecting group and R₂ when taken together with the attached oxygen is a phosphoramidite group.

10. The nucleotide analog of Claim 9 wherein said base is adenine, cytosine, guanine or uracil.

11. The nucleotide analog of Claim 10 wherein said base comprises an additional protecting group on a reactive moiety of said base.

12. The nucleotide analog of Claim 11 wherein said base is N²-isobutyryl guanosine, N⁴-anisoyl cytosine, N⁴-benzoyl cytosine, N^β-benzoyl adenine, N⁴- isobutyryl cytosine, N^e-dialkylformadine adenine or N²- dialkylformadine guanosine.

13. The nucleotide analog of Claim 9 wherein said protected thiol group is thiocyanate (-SCN) or alkylthiocyanate (-R₁₀SCN), wherein R₁₀ is lower alkyl.

14. The nucleotide analog of Claim 9, wherein said position is 8 when said base is a purine, wherein said position is 5 when said base is a pyrimidine, or wherein said position is 6 when said base is guanine.

15. The nucleotide analog of Claim 9, wherein said R_x protecting group is p-(dimethoxytrityl) , p- (monomethoxy-trityl) , fluorenylmethyloxycarbonyl, levuloyl or 9-phenylxanthene-9-yl.

16. The nucleotide analog of Claim 9, wherein said R₄ protecting group is l-(2-chloro-4-methylphenyl)- 4-methoxy-4-piperidinyl, 2'-acetal, o-nitrobenzyl, tert- butyldimethyl silyl, tetrahydrofuranyl or 4- methoxytetrahydropyranyl.

17. The nucleotide analog of Claim 1 or 9, wherein said phosphoramidite is represented by the formula

R_s is lower alkyl, cyanoethyl or substituted lower alkyl; and

R-7 and R_B are independently lower alkyl, or when taken together with the nitrogen to which they are attached form the groups

18. A nucleotide analog of the formula:

wherein R_x is a protecting group;

R₂ taken together with the attached oxygen forms a phosphoramidite group;

R₃ is -H, -OH, or -OR₄? R₄ is lower alkyl or a protecting group; and

B is a modified purine or pyrimidine base comprising a thiocyanate or an alkylthiocyanate group attached at the 8 position on said purine base or the 5 position on said pyrimidine base.

19- The nucleotide analog of Claim 18 wherein said base is N²-isobutyryl guanosine, N⁴-anisoyl cytosine, N⁴-benzoyl cytosine, N⁶-benzoyl adenine, N⁴- isobutyryl cytosine, N⁶-dialkylformadine adenine or N²- dialkylformadine guanosine.

20. The nucleotide analog of Claim 18, wherein said R protecting group is p-(dimethoxytrityl) , p-(monomethoxytrityl) , fluorenylmethyloxycarbonyl, levuloyl or 9-phenylxanthene-9-yl.

21. The nucleotide analog of Claim 18, wherein said phosphoramidite is represented by the formula i I

R_e is lower alkyl, cyanoethyl or substituted lower alkyl; and 0 R-₇ and R_B are independently lower alkyl, or when taken together with the nitrogen to which they are attached form the groups

22. The nucleotide analog of Claim 21 wherein R₆ is 2-cyanoethyl, R_v is isopropyl and R_β is isopropyl. 0

23. The nucleotide analog of Claim 22 wherein said base is uridine or deoxyuridine.

24. The nucleotide analog of Claim 23 wherein R is p-(dimethoxytrityl) .

25. The nucleotide analog of Claim 1 wherein 5 said O-substituted monophosphate group is 0-2- chlorophenyl monophosphate, 0-2,5-dichlorophenyl monophosphate, 0-2,2,2-trichloroethyl monophosphate or the N oxide of 4-methoxy-pyridine-2-methylene monophosphate. 0

26. A nucleotide analog of the formula:

35

wherein

R together with the attached oxygen forms a phosphate group;

R₅ is -H, -OH, -OR₄; R₃ is -H, -OH, or -OR₄;

R₄ is lower alkyl, a phosphate group, a protecting group or a solid support; and

B is a modified purine or pyrimidine base comprising a protected thiol group attached at a position on said base that is not involved in Watson- Crick base pairing, said protected thiol group being stable under conditions of chemical nucleic acid synthesis or conditions of enzymatic nucleic synthesis and being convertible to a reactive thiol after said synthesis.

27. The nucleotide analog of Claim 26 wherein said base is adenine, cytosine, guanine or uracil.

28. The nucleotide analog of Claim 26 wherein said protected thiol group is thiocyanato (-SCN) or alkylthiocyanato (-R₁₀SCN), wherein R _o is lower alkyl.

29. The nucleotide analog of Claim 26, wherein said position is 8 when said base is a purine, or wherein said position is 5 when said base is a pyrimidine.

30. The nucleotide analog of Claim 26, wherein said phosphate group is a monophosphate, a diphosphate, a triphosphate or a tetraphosphate.

31. The nucleotide analog of Claim 26 wherein said analog is 5-thiocyanato-2'-uridine-5'-triphosphate or 5-thiocyanato-2'-deoxyuridine-5'-triphosphate.

32. An oligonucleotide containing the nucleotide analog of any one of Claims 1, 9, 18 or 26.

33. A method of producing the nucleotide analog of anyone of Claims 1, 9, 18 or 26 which comprises preparing a thiol-protected nucleoside or nucleotide base wherein said thiol is attached to a position on said base that is not involved in Watson- Crick base pairing or does not disrupt normal Watson- Crick base pairing; reacting said nucleoside or nucleotide base under conditions to effect conversion of said base to a phosphoramidite, phosphorothioamidite, phosphonate, O-substituted monophosphate or phosphate nucleotide analog and under conditions which do not destroy the protected thiol; and recovering said analog.

34. A method of synthesizing a nucleic acid having an attached functional group which comprises incorporating a thiol-protected nucleotide analog of any one of Claims 1, 9, 18 or 26 into a nucleic acid by a chemical or enzymatic method of nucleic acid synthesis; recovering said nucleic acid containing said analog; deprotecting the analog of said nucleic acid to produce a nucleic acid containing a reactive thiol group; treating the reactive thiol group with a thiol modifying reagent to thereby attach a functional group and produce said nucleic acid with an attached functional group; and recovering said nucleic acid with said attached functional group.

35. The method of Claim 34 wherein said functional group is a photocrosslinker, a crosslinker, a reporter molecule, a radioisotope or a fluorescent group.

36. The method of Claim 35 wherein said photocrosslinker is an aryl azide.

37. The method of Claim 35 wherein said reporter group is biotin, an enzyme, or a fluorescent molecule.

38. The method of Claim 35 wherein said fluorescent group is fluorescein.