8-Aza-7-deazaadenine N8- and N8-(β-D-2′-Deoxyribofuranosides): Building blocks for automated DNA synthesis and properties of oligodeoxyribonucleotides

Oligonucleotides with alternating 8-aza-7-deaza-2′-deoxyadenosine (= c⁷z⁸A_d2) and dT residues (see 11, 14 and 16 ) or 4-aminopyrazolo [3,4-d] pyrimidine N²-(β-D -2′-deoxyribofuranoside) (= c⁷z⁸A′_d¹); ( 3 ) and dT residues (see 12 ) have been prepared by solid-phase synthesis using P(III) chemistry, Additionally, palindromic oligomers derived from d(C-T-G-G-A-T-C-C-A-G) but containing 2 or 3 instead of dA (see 18 – 22 ) have been synthesized. Benzoylation of 2 or 3 , followed by 4,4′-dimethoxytritylation and subsequent phosphitylation yielded the methyl or the cyanoethyl phosphoramidites 8a,b and 9 . They were employed in automated. DNA synthesis. Alternating oligomers containing 2 or 3 showed increase dT_m values compared to those with dA, in particular 12 with an unusual N²-glycosylic bond. The palindromic oligomers 18 - 22 containing 2 or 3 instead of dA outside of the enzymic recognition side reduced the hydrolysis rate, replacement within d(G-A-T-C) abolished phosphodiester hydrolysis.     

3-Deaza-2′-deoxyadenosine: Synthesis via 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxyribonucleosides and properties of oligonucleotides

The synthesis of 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxy-β-D -ribonucleosides 2 and 9 and the conversion of the N¹-isomer 2 into the 2′,3′-didehydro-2′,3′-dideoxyribonucleoside 3a or (via 7 ) 3-deaza-2′-deoxyadenosine ( 1 ) is described. Phosphonate building blocks of 1 were employed in solid-phase synthesis of self-complementary base-modified oligonucleotides. Their properties were studied with regard to duplex stability and hydrolysis by the restriction enzyme Eco RI.     

1,7-Dideaza-2′-deoxyadenosine: Building blocks for solid-phase synthesis and secondary structure of base-modified oligodeoxyribonucleotides

The 1,7-dideaza-2′-deoxyadenosine (c¹c⁷A_d; 1 ) was converted into building blocks 3a , b for solid-phase oligodeoxyribonucleotide synthesis. Testing various N-protecting groups – benzoyl, phenoxyacetyl, [(fluoren-9-yl)methoxy]carbonyl, and (dimethylamino)methylidene – only the latter two were found to be suitable ( 1 → 4b, d ). Ensuing 4,4′-dimethoxytritylation of 4d and phosphitylation afforded the 3′-phosphonate 3a or the 3′-[(2-cyanoethyl)diisopropylphosphoramidite] 3b . Self-complementary oligonucleotides with alternating dA or c¹c⁷A_d and dT residues ( 7 and 8 ) as well as palindromic oligomers such as d(C-G-C-G-c¹c⁷ A-c¹c⁷ A-T-T-C-G-C-G) ( 10 ) and d(G-T-A-G-c¹c⁷ A-c¹c⁷ A-T-T-C-T-A-C) ( 12 ) were synthesized. Duplex stability was decreased because 1 cannot form Watson-Crick or Hoogsteen base pairs if incorporated into oligonucleotides. On the other hand, the structural modifications in 10 and 12 forced these palindromic oligomers to form hairpin structures.     

2′-Deoxy-β-D-ribofuranosides of N6-Methylated 7-Deazaadenine and 8-Aza-7-deazaadenine: Solid-phase synthesis of oligodeoxyribonucleotides and properties of self-complementary duplexes

The syntheses of 7-deaza-N⁶-methyladenine N⁹-(2′-deoxy-β-D -ribofuranoside) ( 2 ) as well as of 8-aza-7-deaza-N⁶-methyladenine N⁸? and N⁹?(2′-deoxyribofuranosides) ( 3 and 4 , resp.) are described. A 4,4′-dimeth-oxylritylation followed by phosphitylation yielded the methyl phosphoramidites 12–14 . They were employed together with the phosphoramidite of 2′-deoxy-N⁶v-methyladenosine ( 15 ) in automated solid-phase oligonucleotide synthesis. Alternating or palindromic oligonucleotides derived from d(A-T)₆ or d(A-T-G-C-A-G-A*-T-C-T-G-C-A) but containing one methylated pyrrolo[2,3-d]pyrimidine or pyrazolo[3,4-d]pyrimidine moiety in place of a N⁶-methylaminopurine (A*) were synthesized. Melting experiments showed that duplex destabilization induced by a N⁶-Me group of 2′-deoxy-N⁶-methyladenosine is reversed by incorporation of 8-aza-7-deaza-2′-deoxy-N⁶-meihyladenosine, whereas 7-deaza-2′-deoxy-N⁶-methyladenostne decreased the T_m value further. Regiospecific phosphodiester hydrolysis of d(A-T-G-C-A-G-m⁶A-T-C-T-G1-C-A) by the endodeoxyribonuclease Dpn I, yielding d(A-T-G-C-A-G-m⁶A) and d(pT-C-T-G-C-A), was prevented when the residue c⁷m⁶A_d ( 2 ), c⁷m⁶z⁸A_d ( 3 ), or c⁷m⁶z⁸A_d′ ( 4 ) replaced m⁶A_d ( 1 ) indicating that N(7) of N⁶-methyladenine is a proton-acceptor site for the endodeoxyribonuclease.     

N7-DNA: Synthesis and Base Pairing of Oligonucleotides Containing N7-(2-Deoxy-β-D-erythro-pentofuranosyl)guanine (N7Gd)

The synthesis of oligonucleotides containing N⁷-(2-deoxy-β-D -erythro-pentofuranosyl)guanine (N⁷G_d; 1 ) is described. Compound 1 was prepared by nucleobase-anion glycosylation of 2-amino-6-methoxypurine ( 5 ) with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 6 ) followed by detoluoylation and displacement of the MeO group ( 8→10→1 ). Upon base protection with the (dimethylamino)methylidene residue (→ 11 ) the 4,4-dimethoxytrityl group was introduced at OH? C(5′) (→ 12 ). The phosphonate 3 and the phosphoramidite 4 were prepared and used in solid-phase oligonucleotide synthesis. The self-complementary dodecamer d(N⁷G? C)₆ shows sigmoidal melting. The T_m of the duplex is 40°. This demonstrates that guanine residues linked via N(7) of purine to the phosphodiester backbone are able to undergo base pairing with cytosine.     

N6-(Carbamoylmethyl)-2′-deoxyadenosine,a rare DNA Constituent: Phosphoramidite Synthesis and Properties of Palindromic Dodecanucleotides

N⁶-(Carbamoylmethyl)-2′-deoxyadenosine ( 1 ), a modified nucleoside occurring in bacteriophage Mu, was synthesized by two different routes. Glycinamide was introdued by nucleophilic displacement of(2,4,6,-triisopro-pylphenyl)sulfonyloxy or ethylsulfinyl groups at C(6) of the purine moiety. Compound 1 was converted into the protected phosphoramidite 6b and employed in solid-phase synthesis of the self-complementary oligonucleotides 7–14 . Replacement of 2′-deoxyadenosine by 1 led to a strong decrease of the T_m values of the oligomers d(A-T)₆ ( 7 ) and d(A-T-G-A-A-G-C-T-T-C-A-T)( 10 ), respectively. As the oligemer 10 contains the recognition site d(A-A-G-C-T-T) of the endodeoxyribonuclease Hind III, it was subjected to sequence-specific hydrolysis experiments. Replacement of the first or second A_d by 1 prevented enzymatic phosphodiester hydrolysis (results with 11 and 12 ). In contrast, slow hydrolysis was observed if the less bulky N⁶-methyl-2′-deoxyadenosine replaced the second A _d residue (results with 14 ).     

The Thermal N9/N7 Isomerization of N2‐Acylated 2′‐Deoxyguanosine Derivatives in the Melt and in Solution

The protected 2′‐deoxyguanosine derivatives 5a – c undergo N⁹→N⁷ isomerization in the melt and in solution. The rate of isomerization is much faster than in the case of the corresponding ribonucleosides and occurs even in the absence of a catalyst. In the melt (195°, 2 min), the N²,3′‐O,5′‐O‐tris(4‐toluoyl) derivative 5b and the N²‐acetyl‐3′,5′‐bis‐O‐[(tert‐butyl)dimethylsilyl] derivative 5c gave anomeric mixtures of the N⁷‐isomers 9b / 10b (43%) and 9c / 10c (55%), respectively. In addition, the N⁹‐α‐D ‐anomers 8b and 8c were obtained. Different from 5b , the isomerization of peracetylated 5a resulted in low yields. Compound 5b was also prone to isomerization performed in solution (toluene, 100°, 5 min; chlorobenzene, 120°, 5 min), furnishing the N⁷‐regioisomers in 24–53% yield. The highest yield of the N⁹→N⁷ isomerization occurred in the presence of 2‐deoxy‐3,5‐di‐O‐(4‐toluoyl)‐α‐D ‐erythro‐pentofuranosyl chloride.     

Studies on the Base-Pairing Properties of N7-(2-Deoxy-β-D-erythro-pentofuranosyl)guanine (N7Gd)

The base-pairing properties of N⁷-(2-deoxy-β-D -erythro-pentofuranosyl)guanine (N⁷G_d; 1 ) are investigated. The nucleoside 1 was obtained by nucleobase-anion glycosylation. The glycosylation reaction of various 6-alkoxy-purin-2-amines 3a - i with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 8 ) was studied. The N⁹/N⁷-glycosylation ratio was found to be 1:1 when 6-isopropoxypurin-2-amine ( 3d ) was used, whereas 6-(2-methoxyethoxy)purin-2-arnine ( 3i ) gave mainly the N⁹-nucleoside (2:1). Oligonucleotides containing compound 1 were prepared by solid-phase synthesis and hybridized with complementary strands having the four conventional nucleosides located opposite to N⁷G_d. According to T_m values and enthalpy data of duplex formation, a base pair between N⁷G_d and dG is suggested. From the possible N⁷G_d dG base pair motives, Hoogsteen pairing can be excluded as 7-deaza-2′-deoxyguanosine forms the same stable base pair with N⁷G_d as dG.     

Synthesis of Phosphonates and Oligodeoxyribonucleotides Derived from 2′-Deoxyisoguanosine and 2′-Deoxy-2-haloadenosines

Oligonucleotides containing 2′-deoxyisoguanosine ( 1 ) or 2-chloro-2′-deoxyadenosine ( 2a ) have been prepared by solid-phase synthesis. Suitably protected phosphonates 3a, 4a , and 4b as well as the phosphoramidite of 1 have been obtained from the nucleosides 1, 2a , or 2b via the (dimethylamino)methylidene derivatives 5–7 . 4,4′-Dimethoxytrityl groups were introduced to yield the base-protected derivatives 8–10 . Alternatively to the direct incorporation of 1 into oligonucleotides, they were also obtained by the photochemical conversion of a 2a residue within the oligonucleotide chain.     

7-Deazaguanosine: Synthesis of an oligorbonucleotide building block and disaggregation of the U-G-G-G-G-U G4 structure by the modified base

Building blocks derived from 7-deazaguanosine (c⁷G, 1 ) were prepared for solid-phase oligoribonucleotide synthesis. Compound 1 was converted into the isobutyurl derivative 2b and the (dimethylamino)methylidene compound 3 (Scheme 1). After tritylation (→ 4a , b ), silylation was studied with regard to regioselectivity. It was found that the triisopropylsilyl group in combination with the (dimethylamino)methylidene residue gave the highest 2′ -selectivity (→ 5e ). The 2′ -O -silyl derivative 5e was reacted with PCl₃ affording the 3′ -phosphonate 7 which was used in solid-phase oligoribonucleotide synthesis. Oligonucleotides derived from U-G-G-G-G-U with an increasing number of c⁷G residues instead of G were synthesized. Aggregation was studied by polyacrylamidegel electrophoresis and CD Spectroscopy. Disaggregation of the G₄-structure of U-G-G-G-G-U was observed when c⁷G replaced G, demonstrating that guanine N(7) participates in the aggregation process.     

Synthesis of 2′-Deoxyribofuranosides of 8-Aza-7-deazaguanine and Related Pyrazolo[3,4-d]pyrimidines

The N(1)- and N(2)-(2′-deoxyribofuranosides) 1 and 2 , respectively, of 8-aza-7-deazaguanine were prepared via phase-transfer glycosylation in the presence or absence of Bu₄NHSO₄ as catalyst of 6-amino-4-methoxy-lH-pyrazolo[3,4-d]pyrimidine ( 7c ) with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 10 ). On a similar route, but without catalyst and employing THF as organic phase, the 6-amino-4-chloronucleosides 11b and 12b were synthesized from 7a and converted into the N(1)-and N(2)-substituted 4-thioxo analogues 17a and 18a , respectively. The ratio of N(1)- to N(2)-glycosylation was 2:1 for 7c and 1:1 for 7a , viz. depending on the nucleobase structure. The rate of the H⁺-catalyzed N-glycosyl hydrolysis was strongly decreased for the N(2)-(β-D -2′-deoxyribofuranosides) as compared to the N(1)-compounds. However, the N(1)-nucleoside 1 , which is an isostere of 2′-deoxyguanosine, is sufficiently stable to be employed later in solid-phase oligonucleotide synthesis.     

Synthesis of 3-Deaza-2′-deoxyadenosine and 3-Deaza-2′,3′-dideoxyadenosine: Glycosylation of the 4-Chloroimidazo[4,5-c]pyridinyl Anion

The convergent syntheses of 3-deazapurine 2′-deoxy-β-D -ribonucleosides and 2′,3′-dideoxy-D -ribonucleosides, including 3-deaza-2′-deoxyadenosine ( 1a ) and 3-deaza-2′,3′-dideoxyadenosine ( 1b ) is described. The 4-chloro-lH-imidazo[4,5-c]pyridinyl anion derived from 5 was reacted with either 2′-deoxyhalogenose 6 or 2′,3′-dideoxyhalogenose 10 yielding two regioisomeric (N¹ and N³) glycosylation products. They were deprotected and converted into 4-substituted imidazo[4,5-c]pyridine 2′-deoxy-β-D -ribonucleosides and 2′,3′-dideoxy-D -ribonucleosides. Compounds 1a and 1b proved to be more stable against proton-catalyzed N-glycosylic bond hydrolysis than the parent purine nucleosides and were not deaminated by adenosine deaminase.     

Synthesis of 3-(2-chloro-5-nitroanilino and 4-nitroanilino)-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-ones

A new synthesis of 3-anilino-1-aryl-2-pyrazolin-5-ones in which the pyrazolinone ring is built via N? N bond formation is described. 2-Cyano-2′,4′,6′-trichloroacetanilide 1 was converted to imino ether hydrochloride 2 which was reacted with anilines in methanol to produce N-arylimino ether 3a,b. Reaction of these N-arylimino ethers with hydroxylamine gave N-arylamidoximes 4a,b . An 1,2,4-oxadiazol-5-one 6a was prepared from the N-arylamidoxime 4a and subjected to base-induced rearrangement. The desired 3-anilino-pyrazolinone 7a was obtained only in a very low yield. However, O-acetylation of the N-arylamidoximes 4a,b followed by acid-catalyzed ring closure and rearrangement in the presence of excess acetic anhydride gave a mixture of N-acetylanilinopyrazolinones (e.g. 10 ) and 4-acetyloxy-3-N-acetylanilinopyrazoles (e.g. 12 ) which upon acid hydrolysis afforded the 3-anilinopyrazolinones 7a,b in better yield.     

Reaction of N1,N2-diarylacetamidines with (2,4,7-trinitro-9H-fluoren-9-ylidene)propanedinitriles

Novel spiro[fluorene-9,4′-(1′,2′,3′,4′-tetrahydropyridine)]-5′-carbonitriles 6a-c have been obtained from the reaction of N¹,N²-diarylacetamidines 1a-c with (2,4,7-trinitro-9H-fluoren-9-ylidene)propanedinitrile ( 2 ) in ethyl acetate solutions at ambient temperature for 6a,b or under reflux for 6c , respectively.     

Azimine. V.. Untersuchungen zur stereoisomerie an der N(2), N(3)-bindung von 2, 3-dialkyl-1-phthalimido-aziminen

Azimines. V. Investigation on the Stereoisomerism Around the N (2), N (3) Bond in 2, 3-Dialkyl-1-phthalimido-azimines 2, 3-(cis-1, 3-Cyclopentylene)-1-phthalimido-azimine ( 7 ) and isomerically pure (2 Z)- and (2 E)-2, 3-diisopropyl-1-phthalimido-azimine ( 9a and 9b ) were prepared by the addition of phthalimido-nitrene ( 1 ) to 2, 3-diazabicyclo [2.2.1]hept-2-ene ( 6 ) and to (E)- and (Z)-1, 1′-dimethylazoethane ( 8a and 8b ), respectively. Comparison of their UV. spectra with those of two stereoisomeric azimines of known configuration, namely (1 E, 2 Z)- and (1 Z, 2 E)-2, 3-dimethyl-1-phthalimido-azimine ( 5a and 5b ), reveals that 2, 3-dialkyl-1-phthalimido-azimines with (2 Z)-configuration are characterized by a shoulder at about 258 nm (? ≈? 14,000) and those with (2 E)-configuration by a maximum at 270–278 nm (? ≈? 10,000). The (2 E)-azimine 9b isomerizes under acid catalysis as well as thermally and photochemically into the more stable (2 Z)-isomer 9a . Under the last two conditions the isomerization is accompanied by a slower fragmentation with loss of nitrogen into N, N′-diisopropyl-N, N′-phthaloylhydrazine ( 4 , R = iso-C₃H₇). The same fragmentation was also observed on thermolysis and photolysis of the (2 Z)-isomer 9a . The kinetic parameters for the thermal isomerization of 9b (they fit first-order plots) and for the fragmentation of 9a and 9b were determined by ¹H-NMR. spectroscopy in benzene, trichloromethane and acetonitrile. In the photolysis of 9a or 9b the fragmentation is accompanied by dissociation into the azo compounds 8a or 8b and the nitrene 1 , the latter being subject to trapping by cyclohexene. With the azimine 7 , an analogous thermal fragmentation was observed to give N, N′-(cis-1, 3-cyclo-pentylene)-N, N′-phthaloylhydrazine ( 15 ), but more energetic conditions were required than with 9 . Photolysis of 7 led exclusively to dissociation into the azo compound 6 and the nitrene 1 , perhaps because the fragmentation of 7 is prevented by ring strain.     

Synthese und chemische Eigenschaften heterocyclischer Tetracyanodimethane eines heterocyclischen N,N′-Dicyanodiimins sowie von voll substituierten monocyclischen Tetracyanodimethanen

Syntheses and Chemical Properties of Heterocyclic Tetracyanodimethanes of a Heterocyclic N,N′-Dicyanodiimine and of Fully Substituted Monocyclic Tetracyanodimethanes In the presence of different bases, 4,8-diethoxy-3H,7H-benzo[1,2-c:4,5-c′]diisoxazole-3,7-dione ( 3 ) reacts with malonodinitrile or β-oxobenzenepropanenitrile to the coloured salts 4a , b and 5 , respectively, which are alkylated to the tetracyanoquinodimethane-like heterocycles 2a , b and to the bis [benzoyl(cyano)methylidene]-substituted heterocycle 6 (Scheme 1). Hydrogenation of 2a , b affords the fully substituted 7a , b , and with 2-(1,3-dithiol-2-ylidene)-1,3-dithiole, 2a gives the 1:1 charge-transfer complex 8 . Heterocyclic quinone 9 is transformed to the monocyarioimino derivative 10 , and 3 reacts with cyanamide and NaH to the N,N′-dicyanodiimine salt 12.     

N7-DNA: Synthesis and Base Pairing of Oligonucleotides containing 7-(2-Deoxy-β -D-erythro-pentofuranosyl)adenine (N7Ad)

The synthesis of oligonucleotides containing 7-(2-deoxy-β -D -erythro-pentofuranosyl)adenine (N⁷A_d; 1 ) is described. Compound 1 was obtained from the precursor 4-amino-1H -imidazole-5-carbonitrile 2-deoxyribonucleoside 6 and was found to be much more labile than A_d. The N⁶-benzoyl protecting group (see 8 ) destabilized the N-glycosylic bond further and was difficult to remove by NH₃-catalyzed hydrolysis. Therefore, a (dimethyl-amino)methylidene residue was introduced (→ 9 ). Amidine 9 was blocked at OH? C(5′) with the dimethoxytrityl residue ((MeO)₂Tr), and phosphonate 4 as well as phosphoramidite 5 were prepared under standard conditions. Phosphonate 4 was employed in solid-phase oligonucleotide synthesis. Homooligonucleotides as well as self-complementary oligonucleotides were prepared. The oligomer d[(N⁷A)₁₁-A] ( 11 ) formed a duplex with d(T₁₂) ( 13 ). Antiparallel chain polarity and reverse Watson-Crick base pairing was deduced from duplex formation of the self-complementary d[(N⁷A)₈-T₈] ( 14 ).     

[2+3] Cycloadditions of ‘Thiocarbonyl Ylides’ (= (Alkylidenesulfonio)methanides) and diazoalkanes with N,N′-(thiocarbonyl)diimidazole (= 1,1′-(carbonothioyl)bis[1H-imidazole])

Heating of a mixture of N,N′-(thiocarbonyl)diimidazole (= 1,1′-(carbonothioyl)bis[1H-imidazole]; 1 ) and 2,5-dihydro-1,3,4-thiadiazole 2a or 2b gave the 1,3-dithiolanes 4a and 4b , respectively, via a regiospecific 1,3-dipolar cycloaddition of the corresponding ‘thiocarbonyl methanides’ 3a , b onto the C?S group of 1 (Schemes 1 and 2). The adamantane derivative 4b was not stable in the presence of 1H-imidazole and during chromatographic workup. The isolated 1,3-dithiole 5 is the product of a base-catalyzed elimination of 1H-imidazole from the initial cycloadduct 4b . The formation of the S,N-acetal 6 can be rationalized by a protonation of the ‘thiocarbonyl ylide’ 3b followed by a nucleophilic addition of 1H-imidazole. With the diazo compounds 8a–e (Scheme 3) 1 underwent a regiospecific 1,3-dipolar cycloaddition to give the corresponding 2,5-dihydro-1,3,4-thiadiazole derivatives 9 , which spontaneously eliminated 1H-imidazole to yield (1H-imidazol-1-yl)-1,3,4-thiadiazoles 10 . The structures of 10a and 10d were established by X-ray crystallography. In the case of diazodiphenylmethane ( 8f ), the initial cycloadduct 9f decomposed via a ‘twofold extrusion’ of N₂ and S to give 1,1′-(2,2-diphenylethenylidene)bis[1H-imidazole] ( 11 ; Scheme 3).     

Nucleosides XII.1 Synthesis of 5‐Modified Isoguanosines and Reinvestigation of 5′‐Deoxy‐N3,5′‐cycloisoguanosine

Isoguanosine ( 3 ) underwent a coupling reaction with diaryl disulfides in the presence of tri‐n‐butylphosphine when its 6‐amino group was protected by N,N‐dimethylaminomethylidene. The synthesis of 5′‐deoxy‐N³,5′‐cycloisoguanosine ( 6 ) and its 2′,3′‐O‐isopropylidene derivative ( 11 ) were accomplished in excellent yields from isoguanosines ( 3 & 10 ) in the presence of triphenylphospine and carbon tetrachloride in pyridine. Chlorination at the 5′‐position of isoguanosine ( 3 ) with thionyl chloride followed by the aqueous base‐promoted cyclization afforded the same product 6 . The structures were elucidated by spectroscopic analysis including IR, UV, 1‐D and 2‐D NMR.     

Synthesis and conformational properties of 2′-deoxy-2′-methylthio-pyrimidine and -purine nucleosides: Potential antisense applications

A convenient and shorter synthesis of 2′-deoxy-2′-methylthiouridine analogs 5 , ?5-methyluridine 6 , -cyti-dine 15 , ?5-methylcytidine 16 , -adenosine 27 and -guanosine 34 was accomplished. Successful conversion of ribonucleosides (5-methyl U, U, A, G) into the corresponding 2′-substituted nucleosides involves nucleophilic displacement (S_N2) of an appropriate leaving group at the 2′-position by methanethiol, a soft nucleophile. Reaction between 2,2′-anhydrouridine and methanethiol in the presence of N¹,N¹,N³,N³-tetramethylguani-dine in N,N-dimethylformamide gave 5 , in 75% yield. Preparation of 6 by a similar route was described. Acylated 5 and 6 were transformed into their triazole derivatives, which on ammonolysis furnished 15 and 16 , respectively in good yield. Similarly, tetraisopropyldisiloxanyl (TIPS) protected 2′-O-aratriflates- of-adenosine and -guanosine reacted with methanethiol in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene at - 25°, followed by deblocking of the TIPS protecting group furnished 27 and 34 , respectively. The confor-mational flexibility (N/S equilibrium) of the sugar moiety in nucleosides 5 , 15 , 27 and 34 was studied utilizing nmr spectroscopy, suggesting that the 2′-methylthio group influenced the sugar conformation to adopt a rigid S-pucker in all cases. The extra stiffness of the sugar moiety in these analogs is believed to be due to the electronegativity of the substituent and the steric bulk. The usefulness of these nucleosides to prepare uniformly modified 2′-deoxy-2′-methylthio oligonucleotides for antisense therapeutics is proposed.