CHEMICAL AND ENZYMATIC SYNTHESIS OF 4′-THIO-β-D-ARABINOFURANOSYLCYTOSINE MONOPHOSPHATE AND TRIPHOSPHATE

N⁴-Acetyl-1-(2, 3-di-O-acetyl-4-thio-β-D-arabinofuranosyl)cytosine (2) was synthesized in three steps from 1-(4-thio-β-D-arabinofuranosyl)cytosine (1). The reaction of this partially blocked 4′-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5′-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5′-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4′-thio-ara-C-5′-triphosphate 5. This triphosphate 5 was converted to 4′-thio-ara-C -5′-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.     

9-(2-Deoxy-ß-D-xylofuranosyl)adenine and 1-(2-Deoxy-ß-D-xylofuranosyl)thymine: Phosphorylation and Stability

Abstract

Phosphorylation of 1-(2-deoxy-β-D-xylofuranosyl)thymine (1) or 9-(2-deoxy-β-D-xylofuranosyl)adenine (3) with phosphoryl chloride gives the cyclic 3′,5′-phosphates (2 and 4a) but not the 5′-monophosphates 8a or 8b. The latter are obtained by phosphorylation of the 3′-0-benzoylated 2′-deoxy-β-D-xylonucleosides (7a, b) and subsequent base-catalyzed removal of the benzoyl groups. Compound 3, as the parent dA, depurinates in acidic medium, a reaction which is facilitated in the case of the N⁶-benzoyl derivative 9b and reduced after the introduction of an amidine protecting group. N-Glycosylic bond hydrolysis of 2′-deoxy-β-D-xylofuranosyl nucleosides is enhanced by a factor of two compared to 2′-deoxy-β-D-ribofuranosyl nucleosides.     

6-Azathymidine-4′-thionucleosides: synthesis and antiviral evaluation

The synthesis of dideoxy-6-azathymidine 4′-thionucleoside 1-(2,3-dideoxy-4-thio-β-D-erythro-pentofuranosyl)-(6-azathymidine) (2), and the L-nucleoside, 1-(4-thio-β-L-erythro-pentofuranosyl)-(6-azathymidine) (3) and their evaluation against a wide panel of antiviral assays are described. The L-thionucleoside (3) was devoid of antiviral activity. The dideoxy-thionucleoside (2) was moderately active against vaccinia virus (VV) and the herpes simplex virus strains HSV-1 (strain KOS) and HSV-2 (strain G) (MIC 12 μM) and retained inhibitory activity vs a thymidine kinase-deficient strain HSV-1/TK^–, suggesting that (2) is not dependent on viral TK-catalysed phosphorylation for antiviral activity and/or may use an alternative metabolic activation pathway.     

Design and Synthesis of Triazole-Linked xylo-Nucleoside Dimers

Three triazole-linked nonionic xylo-nucleoside dimers T^L-t-T^xL, T^L-t-A^BzxL and T^L-t-C^BzxL have been synthesized for the first time by Cu(I) catalyzed azide-alkyne [3 + 2] cycloaddition reaction (CuAAC) of 1-(3′-azido-3′-deoxy-2′-O,4′-C-methylene-β-D-ribo-furanosyl)thymine with different alkynes, i.e., 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)thymine, 9-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylo-furanosyl)-N6-benzoyladenine and 1-(5′-deoxy-5′-C-ethynyl-2′-O,4′-C-methylene-β-D-xylofuranosyl)-N4-benzoylcytosine in 90%–92% yields. Among the two Cu(I) reagents, CuSO₄.5H₂O-sodium ascorbate in THF:^tBuOH:H₂O (1:1:1) and CuBr.SMe₂ in THF used for cycloaddition (click) reaction, the former one was found to be better yielding than the latter one.     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

Modified Nucleosides. II.1 Economical Synthesis of 2′,3′-Dideoxycytidine

Abstract

An economical two pot synthesis of 2′,3′-dideoxycytidine (2) from N⁴-acetyl-cytidine (4) has been developed. The key feature of this sequence is the in situ reductive elimination of a mixture of 1-(3-bromo-3-deoxy-2,5-di-O-acetyl-β-D-xylofuranosyl)-N⁴-acetylcytosine (5) and 1-(2-bromo-3-deoxy-3,5-di-O-acetyl-β-D-arabinofuranosyl)-N⁴-acetylcytosine (6) and subsequent hydrogenation of the resultant olefin over palladised charcoal.     

Ribosylation of the Base Residue of Inosine Derivatives by Phase-Transfer Catalysis

Abstract

Reaction of 2′,3′,5′-O-silylated inosine derivative 1 with 2, 3-O-isopropylidene-5-O-tritylribosyl chloride (3) in a two-phase (CH₂Cl₂-aq. NaOH) system in the presence of Bu₄NBr gave three products, i. e., 6-O-α-, 6-O-β-, and N ¹-β-isomers of glycosides 4, 5a, and 5b. A similar PTC reaction of 1 with 2, 3, 5-tri-O-benzylribosyl bromide (9) gave four regio- and stereo-isomers involving the N¹-β-glycoside 10. Reaction of 1 with 2, 3, 5-tri-O-benzoylribosyl bromide (11) afforded three products involving the desired N¹-β-glycoside 12b, which could be deprotected to give N ¹-ribosylinosine (15b) as a useful intermediate for the synthesis of cIDPR.

     

Structure-Activity Relationships of Tiazofurin Analogs: Synthesis and Computational Studies of 4′-Thio Derivatives of Thiophenfurin and Furanfurin

Abstract

The synthesis and computational studies of 5-(4-thio-β-D-ribofuranosyl)-furan-3-carboxamide (furanthiofurin) and 5-(4-thio-β-D-ribofuranosyl)thiophene-3-carboxamide (thiophenthiofurin) are reported.     

Synthesis of 9-(2-Deoxy-2-Fukuoro-β-D- Abinoruranosyl)Hypoxhine. The First Direct Introduction of A 2′-β-Fldoro Substituent in Ppepormed Purine Nucleosides. Studies Directed Toward the Synthesis of 2′-DEOXY-2′-Substituted Arabppmocebosides. 8.1

Abstract

The 3′, 5′-di-O-acetyl-, 3′-, 5′-di-O-balzyl-, 3′-O-acety -5-O-trityl- and 3′-, 5′ -di-O-trityl-2′-O-triflyl-1-benzylhnosine (8c, 15, 20C, and 27, respectively) were prepared and subjected to nucleophilic reaction with TASF. Thus, 3′, 5′-O-(1, 1, 3, 3-tetraisopropyldisiloxanyl)-1-benzylinosine (5c) was triflylated, desilylated, and then acetylated to give 8c. Also, 5c was converted into the 2′-O-tetrahydropyrnyl (W) derivative 11 which was desilylated and then benzylated to give 2′-O-tetrahydropyranyl-O^3′, O^5′, N¹-tribenzylinosine (13). Removal of the THP group from 13 followed by triflylation afforded 2′-O-triflyld-O^3′,O^5′ N¹-tribenzylinosine (15). 3′-O-Acetyl-2′ -O-triflyl-,O^5′,N¹-inosine (20) was prepared frmn 5′ -O-trityl-1-benzylhh (18c) by conversion into the 2′-, 3′-O-(di-n-butylstannylene) derivative which was treated with triflyl chloride and then acetylated. Treatment of 1-benzyl-inosine (4c) with trityl chloride in pyridine containing p-dimethylamino-pyridine afforded a mixture of 2′-, 5′- and 3′-, 5′-di-O-trityl-l-benzylinosine (25 and 26, respectively). These regioiscums were chrcanato-graphically separated. Triflylation of 26 gave 2′-o-triflyl-3′-, 5′-di-O-trityl-1-benzylhoshe (27).

The triflates 8c and 15 only afforded elhination products upon treatment with TASF. However, the trif late group in 20c and 27 was displaced by fluoride with fornation of the 2′-fluoro-arabino nucleosides, 21c and 28, in 10 and 30% yield, respectively. After deprotection of 28, 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)hypowntkine (1, F-ara-H) was obtained in good yield. The conformational influence of the sugar protecting groups on the rate of nucleophilic substitution against elimination is discussed.     

An Improved Post-Synthetic Substitution Approach for Synthesis of Oligodeoxynucleotides Containing Labile 4-Substituted Thymines

Abstract

A simple procedure is described for the preparation of a versatile oligodeoxynucleotide which contains 4-phenylthiothymidine. This versatile oligomer has been successfully used for synthesis of oligonucleotides containing labile 5-methyl-N⁴, N⁴-ethanocytosine (7) or 4-azido-5-methyl-2-pyrimidinone-1-β-(2′-deoxyriboside) (8).     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.

     

Synthesis of Nucleoside 3′-Thiophosphates in One Step Procedure

Abstract

A mild and efficient one-step method of thiophosphorylation was devised for acid-sensitive nucleosides. The procedure is based on thiophosphorylation of nucleoside magnesium alkoxide by 2-chloro-2-thio-1,3,2-dioxaphospholane. The utility and efficiency of this method combined with deprotection of the resulting cyclic triester were demonstrated by its application to the synthesis of both adenosine 3′- and 5′-thiophosphates. The procedure does not require protection of the exocyclic amino group and can be successfully used for the thiophosphorylation of nucleosides that are unusually sensitive to depurination.     

Synthesis and Antiviral Activities of 5-Substituted 1-(2-Deoxy-2-C-methylene-4-thio-β-D-erythro-pentofuranosyl)uracilst†

Abstract

Various 5-substituted 1-(2-deoxy-2-C-methylene-4-thio-β-D-erythropentofuranosyl)uracils (4′-thioDMDUs) were synthesized from D-glucose via sila-Pummerer-type glycosylation. All of the β-anomers of 5-substituted 4′-thioDMDU, except the 5-hydroxyethyl derivative, showed potent anti-HSV-1 activity (ED₅₀ = 0.016–0.096 μg/mL). 5-Ethyl- and 5-iodo-4′-thioDMDUs were also active against HSV-2 (ED₅₀ = 0.17 and 0.86 μg/mL, respectively). 5-Bromovinyl-4′-thioDMDU was particularly active against VZV (ED₅₀ = 0.013 μg/mL).

     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

Formation of an Unexpected 2-Deoxy-α-D-Threo-Pentofuranosyl Azide by Reaction of O2,3′-Anhydro-5′-O-trityl-2′-deoxycytidine with Lithium Azide

Abstract

Reaction of 0²,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN₃s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4     

Interaction of (2′ -5′) and (3′ -5′) Linked 2-Aminoadenylyl-3-aminoadenosines with Polyuridylic Acid

Abstract

2′-5′ and 3′-5′ linked 2-aminoadenylyl-2-aminoadenosines [(2′-5′)n²Apn²A (1) and (3′-5′)n²Apn²A (2)] were synthesized by condensation of 5′-O-monomethoxytrityl-N ² N ⁶-dibenzoyl-2-aminoadenosine and N ²,N ⁶,2′,3′-O-tetrabenzoyl-2-aminoadenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). The conformational properties of these dimers 1 and 2 were examined by UV, NMR and CD spectroscopy. The results reveal that the 2′-5′-isomer 1 takes a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′-isomer 2. Interactions of 1 and 2 with polyuridylic acid (Poly (U)) were also examined by Tm, mixing curves, UV and CD spectra. Both the dinucleoside isomers 1 and 2 formed a complex of 1 : 2 stoichiometry with poly(U), which was much more stable than that of the corresponding ApA isomer     

Synthesis of New 5″-Sulfonylamido Derivatives of 3″-Azido-3″-Deoxythymidine (AZT)

Abstract

A series of 5′-N-methanesulfonyl derivatives of 3′-azido-5′-(alkylamino)-3′,5′-dideoxythymidine was synthesised. The first step of the synthesis involved the reaction of 1-(2,5-dideoxy-5-O-tosyl-β-D-threo-pentofuranosyl)thymine 1 with an appropriate amine to give 1-[5-(alkylamino)-2,5-dideoxy-β-D-threo-pentofuranosyl]thymines 2a-e and 1-(2,5-dideoxy-β-threo-pent-4-enofuranosyl)thymine 3 as a by-product. Compounds 2a-e were treated with an excess of methanesulfonyl chloride to yield intermediates 1-[5-(dimethylamino)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-pentofuranosyl]-thymine 4a and 1-[5-(N-alkyl-N-methanesulfonyl)-3-O-methanesulfonyl-2,3,5-trideoxy-β-D-threo-penfuranosyl]thymines 4b-e. The reaction of 4a-e with lithium azide in dimethyl-formamide afforded the final compounds 1-[3-azido-5-(N-methyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymine 5a and 1-[3-azido-5-(N-alkyl-N-methanesulfonyl)-2,3,5-trideoxy-β-D-erythro-penofuranosyl]thymines 5b-e. The independent synthesis of 4′,5′-unsaturated product 3 was also described.     

Synthesis of Some 2′,3′-Dideoxy-2′-C-Methyl-Substituted Nucleosides

Abstract

Nucleoside analogues analogues1-(2′,3′-dideoxy-2′-C-hydroxymethyl-β-D-erythro-pentofuranos-yl)thymine (1), 2′,3′-dideoxy-2′-C-hydroxymethylcytidine (2), 2′,3′-dideoxy-2′-C-hydroxymethyladenosine (3), 1-(2′-C-azidomethyl-2′,3′-dideoxy-β-D-erythro-pento-furanosyl)thymine (4), 2′-C-azidomethyl-2′,3′-dideoxycytidine (5), and 2′3′-dideoxy-2′-C-methylcytidine (6) have been synthesized from (S)-4-hydroxymethyl-y-butyro-lactone (7)     

Unexpected Dehalogenation of 3-Bromopyrazolo[3,4-d]pyrimidine Nucleosides During Nucleobase-Anion Glycosylation

Abstract

The anion-glycosylation (KOH, MeCN, TDA-1) of 3-bromopyrazolo[3,4-d]-pyrimidines 4a and 4b with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride (5) furnishes the regioisomeric N′-β-D-2′-deoxyribonucleosides 6a and 6b together with the dehalogenated N²-regioisomers 8a and 8b, stereoselectively. The dehalogenation takes place after the glycosylation and results from the sensitivity of the N-2 nucleosides toward aqueous base. An addition/elimination mechanism is suggested for the dehalogenation reaction.     

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d-glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate)-β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.