Synthesis of Novel 2′,3′-Difluorinated 5′-Deoxythreosyl Phosphonic Acid Nucleosides as Antiviral Agents

A novel route for the synthesis of 2′,3′-difluorinated 5′-deoxythreosyl phosphonic acid nucleosides from glyceraldehyde using the Horner-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate is described. The second fluorination at the 2′-position was an electrophilic reaction performed using N-fluorodibenzenesulfonimide. Glycosylation reactions between the nucleosidic bases and glycosyl donor 9 generated nucleosides that were further phosphonated and hydrolyzed to produce the desired nucleoside analogues. The synthesized nucleoside analogues 13, 16, 20, and 23 were tested for anti- human immunodeficiency virus (HIV) activity as well as cytotoxicity. Adenine derivative 16 showed significant anti-HIV activity up to 100 μM.     

Synthesis and Comparative Study of Anti-Adenoviral Activity of 6-Azacytidine and Its Analogues

This paper presents the results of synthesis and study of cytotoxicity and the anti-adenoviral activity of new N4-derivatives of 6-azacytidine and its α-L-glycopyranosyl analogues obtained by the simplified one-pot version of the silyl condensation method. The resulting acylated 4-methylmercapto-1,2,4-triazin-3(2Н)-one glycosides then underwent the amination and/or ammonolysis to provide 6-azacytidine glycoside analogues (2–6, 12, 15, 17) and compounds with modifications at both base and sugar fragments (11, 15). The evaluation of cytotoxicity and antiviral activity of new compounds against AdV5 showed high selectivity indexes for N4-methyl-6-azacytidine (2) and N,O-tetraacetyl-6-azacytidine (8). High anti-adenoviral activity of N4-methyl-6-azacytidine as well as very low cytotoxicity may suggest its further investigation as potential compound for the therapy of AdV infection.     

Synthesis of Novel 3′-Hydroxymethyl 5′-Deoxythreosyl Phosphonic Acid Nucleoside Analogues as Potent Antiviral Agents

A very efficient synthetic route to novel 3′-hydroxymethyl 5′-deoxythreosyl phosphonic acid nucleosides was described. The discovery of threosyl phosphonate nucleoside (PMDTA, EC₅₀ = 2.53 μM) as a potent antihuman immunodeficiency virus (anti-HIV) agent has led to the synthesis and biological evaluation of 3′-modified 5′-deoxy versions of the threosyl phosphonate nucleosides. 3′-Hydroxymethyl 5 ′-deoxythreosyl phosphonic acid nucleoside analogues 15, 19, 24, and 28 were synthesized from 1,3-dihydroxyacetone and tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 19 exhibits moderate in vitro anti-HIV-1 activity (EC₅₀ = 10.2 μM).     

Human polymerase α inhibitors for skin tumors. Part 2. Modeling,synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives

Recently, the three-dimensional structure of the active site of human DNA polymerase α (pol α) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol α in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.     

Synthesis of Six-Membered Nucleoside Analogs. Part 1: Pyrimidine Nucleosides Based on the 1,3-Dioxane Ring System

Abstract

The synthesis of pyrimidine nucleosides, cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]uracil (4) cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]thymine (5) and cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]cytosine (6) and their corresponding trans isomers is described. Compound 4 showed modest, selective activity against human immunodeficiency virus in acutely infected primary human lymphocytes.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Synthesis and chemiluminescence of copolymers of 5-amino-8-vinyl-phthalazine-1, 4(2H,3H)-dione with methyl methacrylate or styrene,and of α, ω-bis[5-amino-phthalazine-1,4 (2H,3H)-dion-]8-yl alkanes [=α, ω-bis(6-luminyl) alkanes]: Investigations on an intramolecular ‘distance effect’

Oligomers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione exhibit about 0.05% of the chemiluminescence quantum yield of the corresponding ‘monomer unit’, i.e. 5-amino-8-ethyl-phthalazine-1,4(2H,3H)-dione which has a similar quantum yield to luminol. The quantum yields of copolymers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione (1a) with methyl methacrylate or with styrene increase up to 1000-fold, relative to the quantum yield of oligomers of (1a). Thus the monomer units of methyl methacrylate or styrene appear to act as ‘spacers’ between the lumigenic groups. α,ω-Bis[(5-amino-phthalazine-1,4(2H,3H)-dion-)8-yl] alkanes show an analogue ‘distance’ effect: the chemiluminescence quantum yield increases with increasing alkane chain length. As the fluorescence of the corresponding amino phthalates (which are intermediates in the synthesis of the phthalazine diones) is only slightly influenced by the distance between the lumigenic groups it is suggested that a mainly chemical ‘distance effect’ is working here: the smaller the intramolecular distance between the hydrazide groups the more inhibition exists in respect of the oxidative reaction producing the luminol-type chemiluminescence.     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Synthesis of an Epoxide Derivative of Thymidine as a Potential Enzyme Inhibitor

Abstract

Attempts to prepare I -[7,8-anhydro-2,5,6-trideoxy-α-L-lyxo-(and β-n-ribo)-octofuranosyl]thymine (10) by treatment of halohydrins 6–9 with sodium hydride in DMF or sodium methoxide in methanol gave mixtures of the epoxides 10 or 11 and the 3′,8′-anhydronucleoside 12. The structure of 12 was confirmed by oxidation to the cyclic ketone 14. The successful synthesis of 10 involved a Wittig reaction on the thymidine-5′-aldehyde 16 to give the unsaturated ketoacetate 18 which was reduced in two steps to the diacetate 20. The 7′-O-tosyl derivative 21 upon treatment with sodium methoxide in chloroform gave the pure epoxide 10 which was marginally toxic to L1210 cells in culture (I₅₀=25 μM) and demonstrated borderline in vivo activity (24% ILS) against P388 murine leukemia.     

Design and Synthesis of Novel Carbocyclic Versions of 2′-spirocyclopropyl Ribonucleosides as Potent Anti-HCV Agents

The discovery of 2′-spirocyclopropyl-ribocytidine as a potent inhibitor of RNA synthesis by NS5B (IC₅₀ = 7.3 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of carbocyclic versions of 2′-spiropropyl-nucleosides from cyclopentenol 6. Spirocyclopropylation of enone 7 was completed by using (2-chloroethyl)-dimethylsulfonium iodide and potassium t-butoxide to form the desired intermediate 9a. The synthesized nucleoside analogues, 18, 19, 26, and 27, were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. The synthesized cytosine nucleoside 19 showed moderate anti-HCV activity (IC₅₀ = 14.4 μM).     

Utilization of 1,3-Dioxolanes in the Synthesis of α-branched Alkyl and Aryl 9-[2-(Phosphonomethoxy)Ethyl]Purines and Study of the Influence of α-branched Substitution for Potential Biological Activity

Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.     

5′-Nor Carbocyclic Ribavirin

Abstract

An efficient synthesis of 5′-nor carbocyclic ribavirin (4) is described in 13 steps from conveniently available (+)-(1R,4S)-4-hydroxy-2-cyclopenten-1-yl acetate (6). Compound 4 was evaluated against the following viruses: herpes simplex type 1 and 2, vaccinia, cowpox, smallpox, Ebola, hepatitis B, hepatitis C, adenovirus type 1, influenza A (H1N1 and H3N2), influenza B, parainfluenza type 3, Pichinde, Punta Toro A, respiratory syncytial, rhinovirus type 2, Venezuelan equine encephalitis, yellow fever, and West Nile. No activity was found nor was there any cytotoxicity to the viral host cells.     

Stereoselective Synthesis of 2-Deoxy-2-Fluoroarabinofuranosyl-α-1-Phosphate and Its Application to the Synthesis of 2′-Deoxy-2′-Fluoroarabinofuranosyl Purine Nucleosides by a Chemo-Enzymatic Method

Stereoselective introduction of a phosphate moiety into 2-deoxy-2-fluoroarabinofuranose derivatives at the anomeric position was investigated by two methods. One involved a stereoselective hydrolysis of 1-bromo-derivative, and the consecutive phosphorylation of 2-deoxy-2-fluoro-α-D-arabinofuranose via a phosphoramidite derivative. The other method involved stereoselective α-phosphorylation of the 1-bromo-derivative at the 1-position. The resulting α-1-phosphate was utilized to prepare 2′-deoxy-2′-fluoroarabinofuranosyl purine nucleosides by an enzymatic glycosylation reaction. This chemo-enzymatic method will be applicable to the synthesis of some 2′F-araNs, and three important 2′F-araNs were actually obtained in 30–40% yields from 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinose with high purity.     

Production of α-glucosidase by Bacillus sp. strains

α-Glucosidase was detected in four wild-type amylolytic strains belonging to the Bacillus genus. The strains showed α-glucosidase activity in extracellular and membrane-bound fractions. Kinitic studies of the α-glucosidase synthesis in the batch cultures of four strains of the Bacillus genus showed two profiles: partially and totally growth-linked synthesis. The presence of different activities and production profiles of α-glucosidase in the strains at high or low glucose concentrations in the medium would indicate that α-glucosidase may have a role in the regulation of the metabolism of α-polysaccharides.     

Lifespan regulation in α/β posterior neurons of the fly mushroom bodies by Rab27

Brain function has been implicated to control the aging process and modulate lifespan. However, continuous efforts remain for the identification of the minimal sufficient brain region and the underlying mechanism for neuronal regulation of longevity. Here, we show that the Drosophila lifespan is modulated by rab27 functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure that shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/βp neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade‐offs in major life functions. Within the α/βp neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant‐negative S6K in the α/βp neurons increases lifespan. Furthermore, the expression of phospho‐mimetic S6 in α/βp neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR‐mediated protein synthesis in α/βp neurons is sufficient to promote longevity.     

Semisynthesis of 6-Chloropurine-2′-deoxyriboside 5′-Dimethoxytrityl 3′-(2-Cyanoethyl-N,N-diisopropylamino)Phosphoramidite and its Use in the Synthesis of Fluorescently Labeled Oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2′-deoxyriboside from the reaction of 6-chloropurine with 2′-deoxycytidine catalyzed by nucleoside-2′-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2′-deoxyriboside 5′-dimethoxytrityl 3′-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

Phosphopentomutase of Bacillus stearothermophilus TH6-2: The Enzyme and Its Gene ppm

Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis. We identified a sequence 5′-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene. The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735. The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide. The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.     

The Biosynthesis and Metabolism of Acarbose in Actinoplanes sp. SE 50/110: A Progress Report

Abstract

The α-glucosidase inhibitor acarbose produced by Actinoplanes sp. SE50/110 is a pseudotetrasaccharide, which consists of an unsaturated cyclitol (carba-sugar), 4-amino-4,6-dideoxyglucose and maltose. The cyclitol (valienol) and the 4-amino-4,6-dideoxyglucose are linked via an N-glycosidic (imino) bond, forming the so-called acarviosyl moiety, which is primarily responsible for the inhibitory effect on α-glucosidases. The gene cluster encoding the biosynthetic genes for the synthesis of acarbose (acb-genes) was sequenced and 25 open reading frames belonging to the acb-gene cluster were identified. Based on the analysis of the enzymes encoded by the acb-cluster, the biosynthesis and ecological role of acarbose is described. The gene cluster includes genes which encode: proteins for the synthesis of the cyclitol; the enzymes for the synthesis of dTDP-4-amino-4,6-dideoxyglucose; glycosyltransferases for the condensation reactions; ATP-dependent exporters and importers; extracellular starch degrading enzymes; and intracellular acarbose modifying enzymes. Acarbose has a dual role for the producer: it inhibits α-glucosidic enzymes of competitors and functions as a carbophor for the uptake of glucose or starch molecules.