Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Nucleosides and Nucleotides. 139. Stereoselective Synthesis of (2′S)-2′-C-Alkyl-2′-deoxyuridines

Abstract

A synthetic method for (2′S)-2′-C-alkyl-2′-deoxyuridines (9) has been described. Catalytic hydrogenation of 1-[2-C-alkynyl-2-O-methoxalyl-3,5-O-TIPDS-β-D-arabino-pentofuranosyl]uracils (5) gave 1-[2-C-(2-alkyl)-2-O-methoxalyl-3,5-O-TIPDS-β-D-arabino-pentofuranosyl]uracils (4) as a major product, which were then subjected to the radical deoxygenation, affording (2′S)-2′-alkyl-2′-deoxy-3′,5′-O-TIPDS-uridines (7) along with a small amount of their 2′R epimers.

     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

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     

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 2′-DEOXY-2′-FLUOROGUANYL-(3′,5′)-GUANOSINE

ABSTRACT

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac₂O/AcOH to give N ²,O ^3′,O ^5′-triacetyl-2′-deoxy-2′-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5′-OH by a 4,4′-dimethoxytrityl group, this nucleoside component was converted to 2′-deoxy-2′-fluoroguanyl-(3′,5′)-guanosine (6c, GfpG).     

Analysis of Carotenoid Composition in Petals of Calendula (Calendula officinalis L.)

Nineteen carotenoids were identified in extracts of petals of orange- and yellow-flowered cultivars of calendula (Calendula officinalis L.). Ten carotenoids were unique to orange-flowered cultivars. The UV–vis absorption maxima of these ten carotenoids were at longer wavelengths than that of flavoxanthin, the main carotenoid of calendula petals, and it is clear that these carotenoids are responsible for the orange color of the petals. Six carotenoids had a cis structure at C-5 (C-5′), and it is conceivable that these (5Z)-carotenoids are enzymatically isomerized at C-5 in a pathway that diverges from the main carotenoid biosynthesis pathway. Among them, (5Z,9Z)-lycopene (1), (5Z,9Z,5′Z,9′Z)-lycopene (3), (5′Z)-γ-carotene (4), and (5′Z,9′Z)-rubixanthin (5) has never before been identified. Additionally, (5Z,9Z,5′Z)-lycopene (2) has been reported only as a synthesized compound.     

Synthesis of L-3′-Hydroxymethylribonucleosides

Abstract

The target compounds were synthesized via the key intermediate carbohydrate 8, which was synthesized by first selectively protecting the 1′ - and 2′- hydroxyl groups followed by selective tosylation of the 5′ -hydroxyl group to obtain compound 3. The tosyl moiety was then replaced by a benzyl ether to obtain 4. Compound 4 underwent Dess-Martin oxidation to afford the ketone 5. Compound 5 was subjected to Wittig olefination to afford the alkene 6 followed by regioselective hydroboration to obtain 7. Compound 7 was fully acetylated using acetic acid, acetic anhydride and sulfuric acid to obtain the key intermediate 8.     

Synthesis of (E)-5-(2-cIOdovinyl)-3′-0-(1-Methyl-1,4-Dihydropyridyl-3 -Carbonyl)-2′-Fluoro-2′-Deoxyuridine (Ivfru-Cds) for Brain Targetted Delivery of Ivfru,an Antiviral Nucleoside

Abstract

(E)-5-(2-lodovinyl)-2′-fluoro-3′-0-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11) was synthesized for future evaluation as a lipophilic, brain-selective, pyrimidine phosphorylase-resistant, antiviral agent for the treatment of Herpes simplex encephalitis (HSE). Treatment of (E)-5-(2-iodovinyl)-2′-fluoro-2′-deoxyuridine (6) with TBDMSCI in the presence of imidazole in DMF yielded the protected 5′-O-t-butyldimethylsilyl derivative (7). Subsequent reaction with nicotinoyl chloride hydrochloride in pyridine afforded (E)-5-(-2-iodovinyl)-2′-fluoro-3′-O-(3-pyridylcarbonyl)-5′-O-t-butyldimethylsily-2′-deoxyuridine (8). Deprotection of the silyl ether moiety of 8 with n-Bu₄N⁺F^? and quaternization of the resulting 3′-O-(3-pyridylcarbonyl) derivative 9 using iodomethane afforded the corresponding 1-methylpyridinium salt 10. The latter was reduced with sodium dithionite to yield (E)-5-(2-iodovinyl)-2′-fluoro-3′-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11).     

Synthesis,In Vitro Anti-HIV Activity,and Biological Stability of 5′-O-Myristoyl Analogue Derivatives of 3′-Fluoro-2′,3′-Dideoxythymidine (FLT) as Potential Bifunctional Prodrugs of FLT

Abstract

A group of 5′-O-myristoyl analogue derivatives of FLT (2) were evaluated as potential anti-HIV agents that were designed to serve as prodrugs to FLT. 3′-Fluoro-2′,3′-dideoxy-5′-O-(12-methoxydodecanoyl)thymidine (4) (EC₅₀ = 3.8 nM) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-azidododecanoyl)thymidine (8) (EC₅₀ = 2.8 nM) were the most effective anti-HIV-1 agents. There was a linear correlation between Log P and HPLC Log retention time for the 5 ′-O-FLT esters. The in vitro enzymatic hydrolysis half-life (t½), among the group of esters (3–8) in porcine liver esterase, rat plasma and rat brain homogenate was longer for 3′-fluoro-2′,3′-dideoxy-5 ′-O-(myristoyl)thymidine (7), with t½ values of 20.3, 4.6 and 17.5 min, respectively.     

Conformationally Locked Nucleosides. Synthesis of Oligodeoxynucleotides Containing 3′-Amino-3′-deoxy-3′-N,5′(R)-C-ethylenethymidine

Abstract

3′-Amino-3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-N,5′(R)-C-ethylenethymidine (6) was synthesized starting from 3′-azido-3′-deoxythymidine. Condensation of 6 with 5′-O-(H-phosphonyl)thymidine and 5′-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

Synthesis and Absolute Configuration of Pyriculol

A total synthesis of optically active pyriculol is described. The Wittig reaction between an aldehyde 19 and a triphenylphosphonium ylide 12 gave an intermediate 20. Successive treatment of 20 with p-toluenesulfonic acid, active manganese dioxide, and potassium carbonate gave (3′R,4′S)-pyriculol (23), which was identical with natural pyriculol (1) in all respects. From this synthesis, the absolute stereochemistry of pyriculol (1) was determined to be 2-[(3′R,4′S)-3′,4′-dihydroxy- (1′E,5′E)-1′,5′-heptadienyl]-6-hydroxybenzaldehyde     

MECHANISM OF INACTIVATION OF S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE BY 5′-DEOXY-5′-S-ALLENYLTHIOADENOSINE AND 5′-DEOXY-5′-S-PROPYNYLTHIOADENOSINE

5′-Deoxy-5′-S-allenylthioadenosine 1 and 5′-deoxy-5′-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II “mechanism-based” inhibitors.     

SYNTHESIS OF 3′-THIOAMIDO-MODIFIED 3′-DEOXYTHYMIDINE-5′-TRIPHOSPHATES AND THEIR USE AS CHAIN TERMINATORS IN SANGER-DNA SEQUENCING

The thioamide derivatives 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-[(2-methyl-1-thioxo-propyl)amino]thymidine 1 and 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-{{6-{[(9H-(fluo-ren-9-ylmethoxy)carbonyl]-amino}-1-thioxohexyl}amino} thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphet-ane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5′-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.     

α-Hydroxyphosphonate Oligonucleotides: A Promising DNA Type?

Abstract

The synthesis of monomers ( S )-1, ( R )-1 and 2 derived from (5′ S )-, (5′ R )-2′-deoxythymidine-5′-C-phosphonic acids and 2′,5′-dideoxythymidine-5′-C-phosphonic acids was elaborated. The protection of the 5′-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers ( S )-3, ( S )-4, ( S )-5, ( R )-4 and ( R )-5 containing the chiral 3′-O-P-CH(OH)-5″ internucleotide linkage. Their hybridization properties with dA₁₅ and rA₁₅ were studied as well as their resistance against nuclease cleavage.     

Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Nucleosides and Nucleotides. 104. Radical and Palladium-Catalyzed Deoxygenation of the Allylic Alcohol Systems in the Sugar Moiety of Pyrimidine Nucleosides§,1

Abstract

New methods for the synthesis of 2′,3′-didehydro-2′,3′-dideoxy-2′ (and 3′)-methyl-5-methyluridines and 2′,3′-dideoxy-2′ (and 3′)-methylidene pyrimidine nucleosides have been developed from the corresponding 2′ (and 3′)-deoxy-2′ (and 3′)-methylidene pyrimidine nucleosides. Treatment of a 3′-deoxy-3′-methylidene-5-methyluridine derivative 8 with 1,1′-thiocarbonyldiimidazole gave the allylic rearranged 2′,3′-didehydro-2′,3′-dideoxy-3′-[(imidazol-1-yl)carbonylthiomethyl] derivative 24. On the other hand, reaction of 8 with methyloxalyl chloride afforded 2′-O-methyloxalyl ester 25. Radical deoxygenation of both 24 and 25 gave 26 exclusively. Palladium-catalyzed reduction of 2′,5′-di-O-acetyl-3′-deoxy-3′-methylidene-5-methyluridine (32) with triethylammonium formate as a hydride donor regioselectively afforded the 2′,3′-dideoxy-3′-methylidene derivative 35 and 2′,3′-didehydro-2′,3′-dideoxy-3′-methyl derivative 34 in a ratio of 95:5 in 78% yield. These reactions were used on the corresponding 2′-deoxy-2′-methylidene derivatives. An alternative synthesis of 2′,3′-dideoxy-2′-methylidene pyrimidine nucleosides (43, 52, and 54) was achieved from the corresponding 1-(3-deoxy-β-D-thero-pentofuranosyl)pyrimidines (44 and 45). The cytotoxicity against L1210 and KB cells and inhibitory activity of the pathogenicity of HIV-1 are also described     

Synthesis of Methylene-Bridged Analogues of Nicotinamide Riboside,Nicotinamide Mononucleotide and Nicotinamide Adenine Dinucleotide

Abstract

5-O-tert-Butyldimethylsilyl-1,2-O-isopropylidene-3(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose (11a) and ?3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (11b) were prepared by condensation of 5-O-tert-butyldimethylsilyl-1,2-O-isopropylidene-α-D-erythro-3-pentulofuranose (10) with lithiated (LDA) 2-methylnicotinamide and 6-methylnicotinamide, respectively, and then deprotected to give 1,2-O-isopropylidene-3-(R)-(nicotinamid-2-ylmethyl)-α-D-ribofuranose(12a) and 1,2-O-isopropylidene-3(R)-(nicotinamid-6-ylmethyl)-α-D-ribofuranose (12b). Benzoylation as well as phosphorylation of compounds 12 afforded the corresponding 5-O-benzoate (13b) and 5-O-monophosphates (14a and 14b). Treatment of 13b with CF₃COOH/H₂O caused 1,2-de-O-isopropylidenation with simultaneous cyclization to the corresponding methylene-bridged cyclic nucleoside - 3′,6-methylene-1-(5-O-benzoyl-β-D-ribofuranose)-3-carboxamidopyridinium trifluoro-acetate (8b) - restricted to the “anti” conformation. In a similar manner compounds 14a and 14b were converted into conformationally restricted 2,3′-methylene-1-(β-D-ribofuranose)-3-carboxamidopyridinium-5′-monophosphate (9a - “syn”) and 3′,6-methylene-1-(β-D-ribofuranose)-3-carboxamido -pyridinium-5′monophosphate (9b - “anti”) respectively. Coupling of derivatives 12a and 12b with the adenosine 5′-methylenediphosphonate (16) afforded the corresponding dinucleotides 17. Upon acidic 1,2-de-O-isopropylidenation of 17b, the conformationally restricted P¹-[6,3′-methylene-1-(β-D-ribofuranos-5-yl)-3-carboxamidopyridinium]-P²-(adenosin-5′-yl)methylenediphosphonate 18b -“anti” was formed. Compound 18b was found to be unstable. Upon addition of water 18b was converted into the anomeric mixture of acyclic dinucleotides, i. e. P¹-[3(R)-nicotinamid-6-ylmethyl-D-ribofuranos-5-yl]-P²-(adenosin-5′-yl)-methylenediphosphonate (19b). In a similar manner, treatment of 17a with CF₃COOH/H₂O and HPLC purification afforded the corresponding dinucleotide 19a.

     

A Short High Yielding Synthesis of the Potent Anti-VZV Carbocyclic Nucleoside Analogue Carba-BVDU

Abstract

A short high yielding synthesis of the potent anti-varicella-zoster virus (VZV) carbocyclic nucleoside analogue carba-BVDU 1 starting from aminodiol 2 is described. Reaction of 2 with acyl carbamate 3 and subsequent ring closure under acidic conditions afforded 5-ethyl-2′-deoxy-4′a-carbauridine 5. In situ acetylation of 5 afforded 3′,5′-di-O-acetyl-5-ethyl-2′-deoxy-4′a-carbauridine 6 in 78% overall yield from 2. Radical bromination of 6 with either bromine or NBS and subsequent treatment with triethylamine gave an efficient conversion to 3′,5′-di-O-acetyl-5-(E)-(2-bromovinyl)-2′-deoxy-4′a-carbauridine 7. Deacetylation of 7 afforded 1 in an overall 45–53% yield from 2.     

Synthesis of Homologated Halovinyl Derivatives from Aristeromycin and Their Inhibition of Human Placental S-Adenosyl-L-homocysteine Hydrolase¶

Abstract

Moffatt oxidation of 2′,3′-O-isopropylidenearisteromycin (1a) and treatment of the 5′-carboxaldehyde with [(p-tolylsulfonyl)methylene]triphenylphosphorane gave the homologated vinylsulfone 2. Treatment of 2 with tributylstannane/AIBN gave the (E/Z)-vinylstannanes which were converted into the E and Z fluoro- and iodovinyl analogs. Chain extension via the 5′-cyano-5′-deoxy derivative 10a gave the 6′-carboxaldehyde of homoaristeromycin. S-Adenosyl-L-homocysteine hydrolase was strongly inhibited by the fluorovinyl, 5b, and iodovinyl, 4b and 7b, compounds, and time-dependent kinetics were observed [1–2 μM (K_i) and 0.1–0.2 min^?1 (k _inact)]. The mechanism of inactivation was shown to involve addition of water at the vinyl 5′ or 6′ carbons with elimination of halide.