Introduction of 5′-Terminal Amino and Thiol Groups into Synthetic Oligonucleotides

Abstract

Oligonucleotides terminating in a 5′-primary amine group are synthesized using solid phase phosphoramidite chemistry. The 5′-terminal amine group in the deprotected oligonucleotide is further derivatized with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) followed by treatment with dithiothreitol (DTT) to produce 5′-thiol terminated oligonucleotides. Introduction of 5′-thiol group is further confirmed by reading the absorbance of the released chromophore, pyridine-2-thione at 343 nm; ?₃₄₃=8080/M.     

Synthesis and Anti-HIV Activity of Novel 4′-Trifluoromethylated 5′-Deoxycarbocyclic Nucleoside Phosphonic Acids

Efficient synthetic route to novel 4′-trifluoromethylated 5′-deoxycarbocyclic nucleoside phosphonic acids was described from α-trifluoromethyl-α,β-unsaturated ester. Coupling of purine nucleosidic bases with cyclopentanol using a Mitsunobu reaction gave the nucleoside intermediates which were further phosphonated and hydrolyzed to reach desired nucleoside analogs. Synthesized nucleoside analogs were tested for anti-HIV activity as well as cytotoxicity. Adenine analog 22 shows significant anti-HIV activity (EC₅₀ = 8.3 μM) up to 100 μM.     

Synthesis and Antiviral Evaluation of Novel 4′-Trifluoromethylated 5′-Deoxyapiosyl Nucleoside Phosphonic Acids

On the basis of the discovery that the threosyl nucleoside phosphonate PMDTA is a potent anti-HIV compound, we synthesized several 4′-trifluoromethyl-5′-deoxyapiosyl nucleoside phosphonic acids and evaluated their anti-HIV activity. An efficient synthetic route was optimized, starting from an α-trifluoromethyl-α,β-unsaturated ester. Glycosylation of the purine nucleosidic bases with a glycosyl donor yielded modified nucleoside intermediates, which were then phosphonated and hydrolyzed to provide the targeted nucleoside analogs. Once synthesized, the anti-HIV and cytotoxic activities of each analog were evaluated. None of the analogs showed significant anti-HIV activity at concentrations up to 100 μM.     

Synthesis of purine and pyrimidine 2′-deoxynucleosides from a 1,2-dithio sugar precursor

9-(2-S-Ethyl-2-thio- and α-D-mannofuranosyl)adenine (8α and 8β) were synthesized from ethyl 3,5,6-tri-O-acetyl-2-S-ethyl-1,2-dithio-α-D-mannofuranoside (1) by bromination followed by coupling of the resultant bromide (2) with 6-benzamido-(chloromercuri)purine. The 2-chloro analogues (10α and 10β) of 8α and 8β were obtained by way of a fusion reaction between 1,3,5,6-tetra-O-acetyl-2-S- ethyl-2-thio-α-D-mannofuranose (5) and 2,6-dichloropurine. Fusion of the bromide 2 with 2,4-bis(trimethylsilyloxy)pyrimidine and its 5-methyl derivative led to 1-(2-S- ethyl-2-thio-β-D-mannofuranosyl)uracil (16) and its thymine analogue (15). The action of Raney nickel led to rapid dechlorination of 10α and 10β, and all of the 2′-thio-nucleosides underwent desulfurization to give the corresponding 2′-deoxynucleosides. Sequential periodate oxidation-borohydride reduction converted the hexofuranosyl nucleosides into their pentofuranosyl analogues. Thus prepared were 9-(2-deoxy-α-and β-D-arabino-hexofuranosyl)adenine (11α and 11β) and their 2-deoxy-D-threo-pentofuranosyl counterparts (6α and 2′-deoxy-3′-epiadenosine, 6β), and 1-(2-deoxy- β-D-arabino-hexofuranosyl)-thymine (17) and -uracil (18) and their 2-deoxy-D-threo-pentofuranosyl counterparts (3′-epithymidine, 21, and 2′-deoxy-3′-epiuridine, 20). Detailed n.m.r.-spectral correlations are described for the series, and various derivatives of the nucleosides are reported.     

Novel Synthesis and Anti-HIV Activity of 4′-Branched Exomethylene Carbocyclic Nucleosides Using a Ring-Closing Metathesis of Triene

The exomethylene of 6 was successfully constructed from the aldehyde 5 using Eschenmoser's reagents. A triene compound 7 was cyclized successfully using Grubbs’ II catalyst to give an exomethylene carbocycle nucleus for the target compound. A Mitsunobu reaction was successfully used to condense the natural bases (adenine, thymine, uracil, and cytosine). The synthesized cytosine analogue 20 showed moderate anti-HIV activity (EC₅₀ = 10.67 μM).     

One-pot Microbial Synthesis of 2′-deoxyribonucleoside from Glucose, Acetaldehyde, and a Nucleobase

A one-pot enzymatic synthesis of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker’s yeast (Saccharomyces cerevisiae) generated ATP which was used to produce d-glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The d-glyceraldehyde 3-phosphate produced was transformed to 2′-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2′-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2′-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.     

Synthesis of 6,3′-Methanothymidine from a Ribofuranos-3-Ulose and 2,4-Dimethoxy-5,6-Dimethylpyrimidine

Abstract

6,3′-Methanothymidine, a thymidine analogue conformationally fixed by the 6,3′-methano-bridge, was synthesized by the sequence of condensation of a ribbofuranos-3-ulose and a pyrimidinylmethyllithium, intramolecular glycosylation, 2′-deoxygenation, and final deprotection.     

Antimutagenic Effect of Amino Acids on the Mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

The antimutagenic activity of protein-constituting amino acids except histidine on the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was investigated in vitro using Salmonella typhinurium TA-100 as an indicator bacterium (Ames test), and concentrations (IC₅₀) of amino acids that inhibit 50% of the mutagenecity were measured. Cysteine was found to be most active and glycine, tryptophan, lysine, and arginine were strong antimutagenic amino acids. Other amino acids showed moderate or weak antimutagenic activities, depending on the amino acids. The results indicate that amino acids play a substantial role in chemoprevention of N-nitroso amine-induced mutagenicity.     

2′- and 3′- Cholesterol-Conjugated Adenosine and Cytosine Nucleoside Building Blocks: Synthesis of Lipidic Nucleic Acids

Abstract

Recently our laboratory introduced¹ chemistries to synthesize 2′- and 3′- cholesteroluridine conjugates which were incorporated into several antisense oligonucleotides. We have now extended this chemistry to other nucleosides (adenosine and cytosine) and synthesized antisense oligonucleotide conjugates for several disease targets. Synthesis of these cholesterol nucleosides was carried out hy condensing choleskrol chloroformate with 2′-O-alkylamine or 3′-O-alkylamine of the appropriate nucleoside. The 2′-O-alkylamines were deiived from direct alkylation procedure.     

Synthesis of 3- and 2′-fucosyl-lactose and 3,2′-difucosyl-lactose from partially benzylated lactose derivatives

O-β-d-Galactopyranosyl-(1→4)-O-[α-l-fucopyranosyl-(1→3)]-d-glucose has been synthesised by reaction of benzyl 2,6-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-β-d-galactopyranosyl)-β-d-glucopyranoside with 2,3,4-tri-O-benzyl-α-l-fucopyranosyl bromide in the presence of mercuric bromide, followed by hydrogenolysis. Benzylation of benzyl 3′,4′-O-isopropylidene-β-lactoside, via tributylstannylation, in the presence of tetrabutylammonium bromide or N-methylimidazole, gave benzyl 2,6-di-O-benzyl-4-O-(6-O-benzyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-β-d-glucopyranoside (6). α-Fucosylation of 6 in the presence of tetraethylammonium bromide provided either benzyl 2,6-di-O-benzyl-4-O-[6-O-benzyl-3,4-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-α-l-fucopyransoyl)-β-d- galactopyranosyl]-β-d-glucopyranoside (13, 73%) or a mixture of 13 (42%) and benzyl 2,6-di-O-benzyl-4-O-[6-O-benzyl-3,4,-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d- galactopyranosyl-3-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d-glucopyranoside (16, 34%). α-Fucosylation of 13 in the presence of mercuric bromide and 2,6-di-tert-butyl-4-methylpyridine gave 16 (73%). Hydrogenolysis and acid hydrolysis of 13 and 16 afforded O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→4)-d-glucose and O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→4)-O-[α-l-fucopyranosyl-(1→3)]-d-glucose, respectively.     

Polymerization of β-amino Acids in Aqueous Solution

We have compared carbonyl diimidazole (CDI) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as activating agents for the oligomerization of negatively-charged - and -amino acids in homogeneous aqueous solution. -Amino acids can be oligomerized efficiently using CDI, but not by EDAC. -Amino acids can be oligomerized efficiently using EDAC, but not by CDI. Aspartic acid, an - and -dicarboxylic acid is oligomerized efficiently by both reagents. These results are explained in terms of the mechanisms of the reactions, and their relevance to prebiotic chemistry is discussed.     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Synthesis of 4′-C-Ethynyl and 4′-C-Cyano Purine Nucleosides from Natural Nucleosides and Their Anti-HIV Activity

Abstract

Purine 2′-deoxynucleosides bearing an ethynyl or a cyano group at C-4′ of the sugar moiety were synthesized from the corresponding 2′-deoxynucleosides. These compounds exhibited very potent anti-HIV activity, and remained active against drug resistant HIV strains.     

Synthesis and Antiviral Evaluation of 3′-Substituted Thymidine Analogues Derived from 3′-Amino-3′-deoxythymidine

Abstract

To assess the structure-activity relationship for antiviral activity, a series of 3′-deoxy-3′-N-functionalized thymidine analogues were synthesized. Several of these thymidine analogues show moderate in vitro activity against HIV-1 and HIV-2.     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

Synthesis of RNA Fragments Using the H-Phosphonate Method and 2′-(2′-Chlorobenzoyl) Protection

Abstract

The performance of 2′-(2-chlorobenzoyl) protected ribonucleoside H-phosphonates in the synthesis of oligoribonucleotides has been studied.     

Synthesis and Evaluation of Biological Activity of Antimicrobial – Pro-Proliferative Peptide Conjugates

Skin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.     

Synthesis and antibacterial activity of a series of novel 9-O-acetyl- 4′-substituted 16-membered macrolides derived from josamycin

A series of novel 9-O-acetyl-4′-substituted 16-membered macrolides derived from josamycin has been designed and synthesized by cleavage of the mycarose of josamycin and subsequent modification of the 4′-hydroxyl group. These derivatives were evaluated for their in vitro antibacterial activities against a panel of Staphylococcus aureus and Staphylococcus epidermidis. 15 (4′-O-(3-Phenylpropanoyl)-9-O-acetyl-desmycarosyl josamycin) and 16 (4′-O-butanoyl-9-O-acetyl-desmycarosyl josamycin) exhibited comparable activities to josamycin against S. aureus (MSSA) and S. epidermidis (MSSE).