Enzymatic synthesis of β-D-2', 3'-unsaturated-5-fluorocytidine from β-D-2',3'-unsaturated thymidine

We have used crude preparations of N-deoxyribosyl transferases (NdRT-II) from Lactobacillus helveticus to catalyze the transfer of a glycosyl moiety from a donor nucleoside to an acceptor base. Optimal conditions for the transglycosylation reaction to make D-D4FC starting from D-D4T and 5-FC were determined after the analysis of several experimental parameters including reaction time, concentration of substrate, pH and the type of buffer. For the first time, a practical procedure for enzymatic synthesis of β-D-2',3'-unsaturated-5-fluorocytidine (β-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, D-D4FC) from β-D-2',3'-unsaturated thymidine (D-D4T) has been established. This method will be useful in the manufacture of important nucleoside analogues for anti-viral therapy.     

Rapid synthesis of 2′,3′-dideoxy-3′β-fluoro-pyrimidine nucleosides from 2′-deoxypyrimidine nucleosides

A rapid synthesis of 2′,3′-dideoxy-3′-fluoro-β-d-threo-nucleosides bearing the pyrimidine canonical bases of nucleic acids has been developed in order to discover new nucleoside derivatives as potential antiviral drugs. However, when evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Conformational features of 2′-O-methyl-adenosylyl-adenosine

In order to obtain information about the conformational features of a 2′-O-methylated polyribonucleotide at the nearest neighbor level, a detailed nuclear magnetic resonance study of AmpA was undertaken. AmpA was isolated from alkali hydrolysates of yeast RNA, and proton spectra were recorded at 100 MHz in the Fourier transform mode in D₂O solutions, 0.01 M, pH 5.4 and 1.5 at 25°C. ³¹P spectra were recorded at 40.48 MHz. Complete, accurate sets of nmr parameters derived for each nucleotidyl unit by simulation iteration methods. The nmr data were translated into conformational parameters for all the bonds using procedures developed in earlier studies from these laboratories. It is shown that AmpA exists in aqueous solution with a flexible molecular framework, which shows preferences for certain orientations. The ribose rings exist as a ²E ? ³E equilibrium with the —pA ribose showing a bias for the ³E pucker. The C(4′)—C(5′) bonds of both nucleotidyl units show significant preference (75–80%) to exist in gg conformation. The dominant conformer (80%) about C(5′)—O(5′) of the 5′-nucleotidyl unit is g′g′. Even though an unambiguous determination of the orientation of the 3′-phosphate group cannot be made, tentative evidence shows that it preferentially occupies g⁺ domains [O(3′)—P trans to C(3′)—C(2′)] in which the H(3′) —C(3′)—O(3′)—P(3′) dihedral angle is about 31°. There is reasonable evidence that the 2′-O-methyl preferentially occupies the domain in which the O(2′)—CH₃ bond is trans to C(2′)—C(1′). Lowering of pH to 1.5, which results in protonation of both the adenine moieties, causes destacking of AmpA. Such destacking is accompanied by small, but real, perturbations in the conformations about most of the bonds in the backbone. A detailed comparison of the solution conformations of ApA and AmpA clearly shows that 2′-O-methylation strongly influences the conformational preference about the C(3′)—O(3′) bond of the 3′-nucleotidyl unit, in addition to inducing small changes in the overall ribophosphate backbone conformational equilibria. The effect of 2′-O-methylation is such that the C(3′)—O(3′) is forced to occupy preferentially the g⁺ domain rather than the normally preferred g^? domain [O(3′)—P trans to C(3′)—C(4′)] in ApA. The data on ApA and AmpA further reveal that the extent of stacking interaction is less in AmpA compared to ApA. It is suggested that stacked species of AmpA exist as right-handed stacks where the magnitude of ω and ω′ about O(5′)—P and P—O(3′) is about 290°. The reason for the lesser degree of stacking in AmpA compared to ApA is intramolecular interaction between 2′-O-methyl and the flexible O(3′)—P—O(5′) bridge, the interaction causing some perturbation in the magnitudes of ω/ω′, causing destacking. The destacking will lead to an increase in _χCN by a few degrees, causing an increase in ²E populations; the latter in turn will shift the 3′ phosphate group from g^? to g⁺ domains. In short, a coupled series of conformational events is envisioned at the onset of destacking, made feasible by the interaction between the 2′-O-methyl group and the swivel O(3′)—P—O(5′) bridge.     

Synthesis and biological activity of 1β-methyl-2-[5′-isoxazoloethenylpyrrolidin-3′-ylthio]carbapenems

A new series of 1β-methylcarbapenems 1a–i bearing isoxazoloethenyl groups on the pyrrolidine ring has been prepared and evaluated for in vitro antibacterial activity and stability to DHP-I. Most compounds showed excellent antibacterial activity and high stability to DHP-I superior to that of meropenem. Of these new carbapenems, 1a,b,h exhibited the best combination of antibacterial activity and DHP-I stability.     

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.     

Conformational study of poly(α,β-L-aspartic acid)

The conformation of several samples of poly(α,β-L -Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L -Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm^?1 was shifted to 620 cm^?1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.     

Theoretical studies on β-lactam antibiotics. IV. Conformational analysis of novel β-lactam antibiotics and the binding specificities of crosslinking enzyme(s) and β-lactamases

Conformational-energy calculations were carried out on the new family of β-lactam antibiotics (viz., thienamycin, PS-5, 1-oxa- and 1-thiapenems, and their close analogs); these exhibit broad-spectrum antibacterial activity and stability towards β-lactamase-producing strains. The bicyclic ring system in all the compounds studied was found to be highly rigid and to favor only one conformation. This is in contrast to findings in penicillins, where the five-membered ring assumes two puckered conformations. The relative orientations of the bicyclic ring system and the nature and configuration of the substituent at C-5 position, besides nonplanarity of the lactam peptide bond, are shown to be important for biological activity. The present study, in agreement with x-ray studies, predicts that the lactam peptide bond in 1-carbapenem is more nonplanar than in 1-thiapenem. These studies also suggest that the conformational requirement of bicyclic ring system to bind to crosslinking enzyme(s) and β-lactamases is very similar.     

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

Conformational studies of adenosine 5′-phosphorodiamidates in aqueous solution

CD and nmr studies show that the conformational equilibrium of N(P)-alkylated adenosine 5′-phosphorodiamidates 2 – 4 and, to a much lesser extent, adenosine 5′-phosphorodiamidate 1 differ at two parts from that of adenosine 5′-phosphate in water. The glycosidic bond conformation is slightly shifted into the direction of the syn range, and the ribose ring N-conformer population increases. The conformational changes increase with increasing degree of N(P)-alkylation and cause a change of the sign of the long-wavelength CD band for 2 – 4 . The new conformation may be characterized by a stacked arrangement for the adenine ring and the 5′-substituted phosphate group of 2 – 4 . Hydrophobic attraction between the head and the tail of the molecules, as well as the entropy effect (which promotes the clustering of apolar faces in water), may be jointly responsible for this stacked arrangement. The intramolecular attractive forces stabilizing the stacked conformation may act at the expense of the solubility decreasing intermolecular attractive forces, and may thus be responsible for the increased water solubility of 2 – 4 , which is higher than that of 1 by more than two orders of magnitude.     

Conformational behavior of α,α-dialkylated peptides

The preferred conformations of N-acetyl-N′-methyl amides of some dialkylglycines have been determined by empirical conformational-energy calculations; minimum-energy conformations were located by minimizing the energy with respect to all the dihedral angles of the molecules. The conformational space of these compounds is sterically restricted, and low-energy conformations are found only in the regions of fully extended and helical structures. Increasing the bulkiness of the substituents on the C^α, the fully extended conformation becomes gradually more stable than the helical structure preferred in the cases of dimethylglycine. This trend is, however, strongly dependent on the bond angles between the substituents on the C^α atom: In particular, helical structures are favored by standard values (111°) of the N-C^α-C′ angle, while fully extended conformations are favored by smaller values of the same angle, as experimentally observed, for instance, in the case of α,α-di-n-propylglycine.     

Synthesis and anti-HIV activity of novel 2′,3′-dideoxy-3′-thiacytidine prodrugs

We report here the synthesis of a novel series of 5′-O-carbonates of 3TC, using different aliphatic alcohols and N,N-carbonyldiimidazol. Its antiviral activity was determined in peripheral blood mononuclear cells (PBMCs) showing some carbonate derivatives with an activity similar to or better than 3TC, except 3TC-Metha and 3TC-2Pro with less activity. In vitro assays in PBMCs have demonstrated that cytotoxicity increases as the carbon chain length of the alcohol moiety increases, showing compounds with a normal chain length of n = 2–5 good selective index, compared to the parent drug. Thus, this work is an important contribution leading to the suppression of HIV replication.     

Genetic variation in the β, β‐carotene‐9′, 10′‐dioxygenase gene and association with fat colour in bovine adipose tissue and milk

β, β‐carotene‐9′, 10′‐dioxygenase (BCO2) plays a role in cleaving β‐carotene eccentrically, and may be involved in the control of adipose and milk colour in cattle. The bovine BCO2 gene was sequenced as a potential candidate gene for a beef fat colour QTL on chromosome (BTA) 15. A single nucleotide base change located in exon 3 causes the substitution of a stop codon (encoded by the A allele) for tryptophan⁸⁰ (encoded by the G allele) (c. 240G>A, p.Trp80stop, referred to herein as SNP W80X). Association analysis showed significant differences in subcutaneous fat colour and beta‐carotene concentration amongst cattle with different BCO2 genotypes. Animals with the BCO2 AA genotype had more yellow beef fat and a higher beta‐carotene concentration in adipose tissues than those with the GA or GG genotype. QTL mapping analysis with the BCO2 SNP W80X fitted as a fixed effect confirmed that this SNP is likely to represent the quantitative trait nucleotide (QTN) for the fat colour‐related traits on BTA 15. Moreover, animals with the AA genotype had yellower milk colour and a higher concentration of beta‐carotene in the milk.     

Localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes

The location of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2′,3′-cyclic nucleotide 3′-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2′,3′-cyclic nucleotide 3′-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.     

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.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.