8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTIES OF NUCLEOSIDES AND OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT POSITION-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N⁸-(2′-deoxy-ribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C⁸-(2′-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2′-deoxyisoguanosine.     

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     

A One Step Synthesis of O6-Methyl-2′-deoxyguanosine

Abstract

A single step chemical synthesis of N⁷-methyl-2′-deoxyguanosine (m⁷dG), N¹-methyl-2′-deoxyguanosine (m¹dG) and O⁶-methyl-2′-deoxyguanosine (m⁶dG) is described. The products were separated on the silical gel plates and characterized by nuclear magnetic resonance and mass spectrometry.     

Oligonucleotides Incorporating N7-(2′-Deoxy-β-d-erythro-pentofuranosyl)isoguanine

Abstract

The H-phosphonate and the phosphoramidite of N⁷-2′-deoxyisoguanosine (2) were prepared and incorporated into oligonucleotide duplexes. Their base pairing properties were investigated and compared with those of the parent purine nucleosides.     

Design,Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A₃ adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N ⁶-(3-iodobenzyl)- and 2-chloro-N ⁶-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K _i = 0.38 nM and 0.28 nM, respectively) at the human A₃AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.     

N2-Substituted-2′-deoxyguanosine 5′-Triphosphates as Substrates for E. coli DNA Polymerase I

Abstract

Several N²-alkyl and N²-phenyl 2′-deoxyguanosine 5′-triphosphates and 2-bromo-2′-deoxyinosine 5′-triphosphate were synthesized and tested as substrates for E. coli DNA polymerase I with a template: primer system requiring incorporation of 85 nucleotides. N²-Methyl-dGTP and N²-ethyl-dGTP were found to be efficiently incorporated in place of dGTP to give full length product. N²-n-Hexyl-dGTP supported limited full length synthesis at high concentration, but N²-phenyl- and N²-(p-n-butylphenyl)-dGTP were poor substrates. 2-Bromo-2′-deoxyinosine 5′-triphosphate was a good substrate for pol I, and it was a replacement only for dGTP. Melting temperatures of oligodeoxyribonucleotides containing N²-alkyl-dG residues, annealed to complementary single stranded DNA, were lower than that of the normal oligomer.     

Synthesis of Some Nucleoside Cyclic Ethylene Phosphothionates

Abstract

This communication describes the synthesis of 5′-deoxy-5′-chloro-3′-(2-thio-1,3,2-dioxaphosphorinanyl)thymidine, N⁴,2′,3′-triacetyl-5′-(2-thio-1,3,2-dioxaphosphorinanyl)-1-β-D-arabinosyl-cytosine and N⁴-acetyl-5′-(2-thio-1,3,2-dioxaphosphorinanyl)-1-β-D-arabinosylcytosine.     

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract

A procedure was developed for the chemical synthesis of P¹,P²-dinucleoside-5′-diphosphates (N₁(5′)pp(5′)N₂) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm⁷G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.     

8-Aza-1-deazapurine Nucleosides as Antiviral Agents

Abstract

2′,3′-Dideoxy-8-aza-1-deazaadenosine (21) and its α-anomer (20) were synthesized via glycosylation of 7-chloro-3H-1,2,3-triazolo[4,5-b]pyridi-ne with 2,3-dideoxy-5-O-[(1, 1)-dimethylethyl)diphenylsilyl]-D-glycero-o-pen-tofuranosyl chloride. The reaction gave a mixture of α- and β-anomers of N³-, N⁴- and N¹-glycosylated regioisorners (12–15). The α- and β-anomers of the N⁴-glycosylated isomer 26 and 27 were also synthesized through the glycosylation of 8-aza-1-deazaadenine with 1-acetoxy-2,3-dideoxy-5-O-f(1,1-di-methylethyl)dimethylsilyl]-D-glycero-pentouranose. These dideoxynucleo-sides and a series of previously synthesized 8-aza-1-deazapurine nucleosidcs were tested for activity against several DNA and RNA viruses, HIV-1 included. The α- and β-anomers of 7-chloro-3-(2-deoxy-D-erythro-pentofuranosyl)-3H-1,2,3-triazolo[4,5-b]pyridine (3a and 4) showed activities against Sb-1 and Coxs viruses. The α- and β-anomers of 2′,3′-dideoxy-8-aza-1-deazaadenosine (20 and 21) were found active as inhibitors of adenosine deaminase.     

Synthesis of 4′-(Hydroxymethyl)Guanosine and a Phosphonate Analogue of Guanylic Acid

Abstract

The synthesis of 4′-(hydroxymethyl)guanosine (7) and the phosphonate analogue 8 of guanylic acid proceed from a common intermediate, 2′, 3′-O-isopropylidene-N ²-(monomethoxytrityl)-guanosine-5′-aldehyde (13).     

Syntheses of 2′-O-Methylisocytidine Phosphoramidite and Methylphosphonamidite Synthons

Abstract

The 2′-O-methylisocytidine phosphoramidite synthon 7 and methylphosphonamidite synthon 8 are synthesized from 2′-O-methyluridine. The N² -(N′, N′-dimethylformamidine) protected 2′-O-methylisocytidine is stable to basic deamination and acidic depyrimidination. Synthon 7 and synthon 8 have been incorporated into oligomers via the automated solid state procedure.

     

Synthesis of Base-Modified Oligonucleotides Containing 6- and 7-Aryl Lumazines

Abstract

6-Phenyl, 7-phenyl, 6-(4-biphenyl)-, 7-(4-biphenyl)lumazine N¹-(2′-deoxy-D-ribofuranosides) were synthesized and incorporated in the different positions of self-complementary oligodeoxyribonucleotides, and the influence of modifications on the melting points of duplexes was studied.     

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.     

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).     

Process Development for the Synthesis of 5′-O-(4,4′-Dimethoxytrityl)-N 2-isobutyryl-2′-O-(2-methoxyethyl)-guanosine—A Potential Precursor for the Second Generation Antisense Oligonucleotides: An Improved Process for the Preparation of 2′-O-Alkyl-2,6-diaminopurine Riboside

Abstract

An efficient four step process for the preparation of 5′-O-(4,4′-dimethoxytrityl)-N ²-isobutyryl-2′-O-(2-methoxyethyl)-guanosine 1 was developed. Direct 2′-O-alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive and commercially available reagents such as KOH, DMSO and alkyl halides at room temperature in 4–6 hrs. Pure 2′-O-(2-methoxyethyl)-DAPR 3 was isolated by crystallization from methanol. Enzymatic deamination of 3 followed by selective N ²-isobutyrylation and 5′-O-dimethoxytritylation furnished desired 1 in high yield and purity. Fully optimized four step synthetic process has been scaled up to the pilot plant level.     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

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.     

Asymmetric Synthesis of α-Methylglutamic Acid and α-Methylornithine by a Chiral Isocyano Amide Reagent

An efficient approach to the asymmetric syntheses of α-methylglutamic acid and α-methylornithine is described. Two chiral reagents, (2′S)-N-(2′-methoxymethylpyrrolidine)-2-isocyanopropionamide 4 and (2′S)-N-(2′-hydroxymethylpyrrolidine)-2-isocyanopropionamide 5, were employed for the asymmetric induction. α-Methylglutamic acid 7 was synthesized by the asymmetric Michael-addition of methyl acrylate to 4 and 5 as the key step. The optical yield of 7 was 10~45% (R-form). α-Methylornithine 12 was also synthesized by the reaction of 4 with acrylonitrile as the key step. The optical yield of 12 was 31.7% (R-form).     

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.