Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.     

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine

Stereoselective synthesis of 3′-C-methylene- and 2′-methyl-3′-C-methylene-3′-deoxythymidine is described, the key reaction being the formation of 3-C-methylene function by catalytic isomerization of a chiral epoxyalcohol, prepared from commercially available 3-methyl-2-butenal and 3-methyl-2-pentenal.     

3′-Fluoro-3′-deoxyribonucleoside 5′-triphosphates: Synthesis and use as terminators of RNA biosynthesis

3′-Fluoro-3′-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5′- triphosphates, NTP(3′-F). It was shown that NTP(3′-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.     

Synthesis and serotonin receptor binding properties of 5-substituted 3-(1′,2′,5′,6′-tetrahydropyridin-3′-yl) indoles

A semi-rigid 5-hydroxytryptamine (5-HT) analogue, RU28253 [5-methoxy-3-(1′,2′,5′,6′-tetrahydropyridin-3′-yl) indole], is a potent 5-HT₁ and 5-HT₂ agonist. It is isomeric to RU24969 [5-methoxy-3-(1′,2′,5′,6′-tetrahydropyridin-4′-yl) indole], a conformationally restricted 5-HT homologue, which has been extensively used in the study and classification of 5-HT receptors. A series of RU28253 derivatives with diverse substituents on indole 5-position were synthesized and their dissociation constants determined at the 5-HT₁ and 5-HT₂ receptors.     

Twitches in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Single muscle fibers continue to twitch for up to 20 min when immersed in ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) solutions containing less than 10⁻⁸ M free calcium. Failure of the twitch results from reversible depolarization, which occurs after 15–20 min in EGTA. The results make it clear that external calcium or calcium in the transverse tubules play no essential part in action potential propagation or excitation-contraction coupling.     

Synthesis and evaluation of novel daunomycin-phosphate-sulfate -β-glucuronide and -β-glucoside prodrugs for application in adept

The synthesis, antiproliferative effect and enzymatic hydrolysis of daunomycin-3′-N- and -4′-O-phosphate and -sulfate derivatives and of daunomycin-3′-N-CO-β-glucuronide and -β-glucoside, designed to be prodrugs in ADEPT are described. The phosphate derivatives were almost as toxic as the parent drug whereas the sulfates were not hydrolyzed by aryl sulfatases. Glucuronyl and glucosyl prodrugs were found to be useful for application in ADEPT.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

Synthesis of 3-methylpseudouridine and 2′-deoxy-3-methyl-pseudouridine

The first chemical synthesis of 3-methyl-ψ-uridine (5) and its 2′-deoxy analogue (9) has been achieved. ψ-Uridine was trimethylsilylated and the crude product was treated with acetyl chloride, to give the 1-acetyl derivative (3). Crude 3 was methylated with dimethoxymethyldimethylamine and then saponified, to give crystalline 5 in 82% overall yield. Treatment of 5 with 1,3-dichloro-1,1,3,3-tetraiso-propyldisiloxane afforded the 3′,5′-protected product, which was converted into the 2′-O-[(imidazol-1-yl)thiocarbonyl] derivative 7. Reduction of 7 with tributyltin hydride followed by deblocking of the product gave crystalline 2′-deoxy-3-methyl-ψ-uridine (9) in 35% yield from 5.     

Carbocyclic phosphonate analogs of 2′,3′-dideoxyadenosine-5′-monophosphate as substrates of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetate

Several carbocyclic phosphonate analogs of 2′,3′-dideoxyadenosine-5′-monophosphate (ddAMP) were pyrophosphorylated by E. coli 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase in the presence of PRPP. Structure-activity relationships are discussed.     

Synthesis of 4′-methoxyadenosine and related compounds

Addition of iodine and methanol to N⁶,N⁶-dibenzoyl-9(2,3-O-carbonyl-5-deoxy-β-d-erythro-pent-4-enofuranosyl)adenine (4) selectively gives N⁶,N⁶-dibenzoyl-2′,3′-O-carbonyl-5′-deoxy-5′-iodo-4′-methoxyadenosine (5). Compound 5 can be converted into 4′-methoxyadenosine via hydrolysis of the carbonate followed by benzoylation, displacement of the 5′-iodo function by benzoate ion, and hydrolysis with ammonia. Configurational assignments are based upon comparisons of ¹H- and ¹³C-n.m.r. spectra with those of previously characterised analogues in the uracil series and by borate electrophoresis. Intermediates in the above scheme have also been converted into 5′-amino-5′-deoxy-4′-methoxyadenosine, 4′-methoxy-5′-O-sulfamoyladenosine, and ethyl 4′-methoxyadenosine-5′-carboxylate, each of which is a 4′-methoxy analogue of biologically active derivatives of adenosine.     

Syntheses and molecular structures of ruthenium(II) complexes of the atropisomeric ligands 1,1′-biphenyl-2,2′-diamine and 3,3′-diamino-2,2′-bipyridine

The unique ligands of [Ru(bipy)₂(bpda)](PF₆)₂ (1, BPDA=1,1′-biphenyl-2,2′-diamine) and [Ru(bipy)₂(dabipy)](PF₆)₂ (2, DABIPY=3,3′-diamino-2,2′-bipyridine) are atropisomeric (exhibit hindered rotation about the sigma bonds that connect the two aromatic groups), so the complexes are diasteromeric with conformation isomers possible for the atropisomeric ligands and configurational isomers possible at the metal centers. Only one diastereomer is observed in the solid-state in both cases. The seven- (1) and five-membered (2) chelate ring of dabipy and bpda (the ligand is bound through its pyridyl groups) ligands are δ when the configuration at the metal is Δ. No evidence for atropisomerization is found in solution. For 1, we conclude bpda binds stereospecifically; however, the atropisomerization barrier of dabipy may be sufficiently low for 2 to preclude the observation of diastereomers by low-temperature NMR spectroscopy.     

Imidazo[2′-3′-:6,5]dipyrido[3,2-b:2′,3′-e]-1,4-diazepines: non-nucleoside HIV-1 reverse transcriptase inhibitors with greater enzyme affinity than nevirapine

The chemistry and SAR of a new series of imidazo[2′,3′:6,5]dipyrido[3,2-b:2′,3′-e]-1,4-diazepines is described. These compounds show improved affinity for HIV-1 RTase and antiviral activity over nevirapine, which has undergone clinical trials.     

Design, synthesis and preliminary evaluation of novel 3′-Substituted carboxycyclopropylglycines as antagonists at group 2 metabotropic glutamate receptors

Two novel 3′-substituted carboxycylopropylglycines, (2S,1′S,2′S,3′R)-2-(3′-xanthenylmethyl-2′-carboxycyclopropyl)glycine (8a) and (2S,1′S,2′S,3′R)-2-(3′-xanthenylethyl-2′-carboxycyclopropyl)glycine (8b), were synthesized and evaluated as mGluR ligands. Compound 8b showed to be a potent group II antagonist with submicromolar activity.     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

A new method for the 3′-deoxygenation of butirosins A and B

An aziridine ring-formation involving the reaction of adjacent amino and alcohol groups with triphenylphosphine, carbon tetrachloride, and triethylamine was applied at the 2′ and 3′ positions of butirosin A (1a) and B (1b). The amino groups at the 2′ position of 1a and 1b were p-methoxybenzylated to increase the nucleophilicity of the nitrogen atom and to avoid the formation of a P-N linkage, and the N-p-methoxybenzyl derivatives were converted into the aziridine derivatives, which were then subjected to hydrogenolysis and removal of the protecting groups to give 3′-deoxybutirosin A (7a) and B (7b), respectively. This new method is compared with the conventional N, O-protecting method that involves several complex steps.     

Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT

Phospholipid conjugates of 3′-azido-3′-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112–6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with ³¹P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC₅₀ values of AZTTP-DSG were 0.33 and 0.79 μM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 μM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Identification of a highly conserved 3′ UTR in the translationally regulated mRNA for prostaglandin synthase

Prostaglandin H synthase (E.C. 1.14.99.1) is induced by growth factors and lymphokines such as EGF and IL-1, and is suppressed by anti-inflammatory glucocorticoids. Inhibition of enzyme synthesis by glucocorticoids is mediated by a novel translational control that appears to involve conversion of the PG synthase mRNA into a cryptic non-hybridizable form.

In order to understand expression of the enzyme in more detail, a full length 2.8 Kb cDNA was cloned from a human embryonic lung cell cDNA library and the complete mRNA including the 3′ untranslated region (3′ UTR), was sequenced. The coding sequence for the human PG synthase shows greater than 90% homology with the sheep and mouse enzymes. A high degree of conservation (70%), however, was also observed in the approximately 750 nucleotide sequence that comprises the 3′ non-coding domain of both sheep and human PG synthase mRNA's and with the approximately 900 nucleotide 3′ UTR of the mouse RNA (68% sheep vs mouse; 47% human vs mouse). Extensive microregions of 10–30 nucleotides are distributed throughout the 3′ UTR where homology between species is 95–100%.

This high degree of conservation in a non-coding region and recent evidence from other genes suggests that these 3′ UTR sequences have important regulatory functions possibly related to translational control of this mRNA by growth factors and glucocorticoids.