Stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane by alkene-utilizing bacteria

Resting cells of ethene grown Mycobacterium 2W produced 1,2-epoxypropane stereospecifically from propene as revealed by optical rotation, ¹H n.m.r. using a chiral shift reagent, and also by complexation gas chromatography involving a glass capillary column coated with an optically active metal chelate. The gas-liquid chromatography method allowed the rapid screening of 11 strains with regard to stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane. Bacteria grown on either ethene, propene or butadiene all predominantly produced the R form of 1,2-epoxypropane from propene and 1,2-epoxybutane from 1-butene while the strains tested for 1-chloro-2,3-epoxypropane production from 3-chloro-1-propene predominantly accumulated the S enantiomer.     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Stereospecificγ-lactamase activity in aPseudomonas fluorescens species

Twenty five environmental isolates enriched for their ability to grow onN-acetylphenylalanine as sole carbon source were investigated for their hydrolytic action on (+)-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one). Strain CMC 3060, a mucoidal Gram-negative rod identified as a strain ofPseudomonas fluorescens, produced high levels of (+)lactamase, and was subsequently found to produce two distinct intracellular enantiomer-selective, -lactamases, one for each isomer. The (+)lactamase was produced constitutively whereas the (-lactamase was produced only in the presence of the substrate. The (+)lactamase was stable when stored as a frozen cell paste but unstable as a protein solution, losing activity during purification and storage. This enzyme was highly selective for the (+)lactam and showed no activity against a wide range of similar compounds. By use of rapid purification techniques and the inclusion of protease inhibitors and protein stabilisers, the (+)lactamase was purified to homogeneity by FPLC and found to be a monomer of molecular weight 61000 Da.     

NAD+-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

An NAD⁺-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K _m and V _max for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K _m and V _max for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH₂-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Synthesis and enantioselectivity of the enantiomers of PG9 and SM21, new potent analgesic and cognition-enhancing drugs

The enantiomers of two α-tropanyl esters, SM₂₁ (1) and PG₉ (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(−)-S-1, (−)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM₂₁, (+)-S-PG₉]. The ACh-releasing effect of the enantiomers of SM₂₁ in rats is in agreement with the results in mice, while PG₉ enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT₄ antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the α-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds. © 1996 Wiley-Liss, Inc.     

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin

Microbial reduction of α-chloroketone to α-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide. About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the α-chloroketone stereospecifically. Many strains provided the R-α-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835. Few microorganisms showed preferential formation of S-α-chlorohydrin after reduction. Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-α-chlorohydrin with ee of 88%, 79% and 78%, respectively. The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-α-chlorohydrin with ≥95% ee was obtained by appropriate modification of reaction conditions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 259–262. Received 23 June 2000/ Accepted in revised form 06 November 2000     

Effect of molecular structure on thermodynamic properties of carbohydrates. A calorimetric study of aqueous di- and oligosaccharides at subzero temperatures

For aqueous solutions of di- and oligosaccharides thermodynamic properties have been investigated at subzero temperatures using differential scanning calorimetry. The amount of unfrozen water observed is found to increase linearly with the glass transition temperatures of anhydrous carbohydrates. Furthermore, the amount of unfrozen water shows a linear relationship with known solution properties of aqueous carbohydrates, such as partial molar compressibility and heat of solution. The different effectiveness among various di- and oligosaccharides to avoid ice formation is associated with the combination of constitutive monosaccharides and attendant molecular structure features including the position and type of the glycosidic linkage between the constituent units. More unfrozen water is induced in the presence of a carbohydrate having a poorer compatibility with the three-dimensional hydrogen-bond network of water. A series of these results obtained imply that there is a common key of carbohydrate stereochemistry governing several different thermodynamic amounts of a given system involving carbohydrates. In this context, a modified stereospecific-hydration model can be used to interpret the present results in terms of stereochemical effects of carbohydrates.     

Stereoselective metabolism of pentoxifylline in vitro and in vivo in humans

Pentoxifylline increases erythrocyte flexibility, reduces blood viscosity, and inhibits platelet aggregation and is thus used in the treatment of peripheral vascular disease. It is transformed into at least seven phase I metabolites, of which two, M1 and M5, are active. The reduction of the keto group of pentoxifylline to a secondary alcohol in M1 takes place chiefly in erythrocytes, is rapidly reversible, and creates a chiral center. The aims of this study were: to develop HPLC methods to separate the enantiomers of M1, to investigate the kinetics of the reversible biotransformation of pentoxifylline to (R)- and (S)-M1 in hemolysed erythrocyte suspension, and to quantify the formation of the enantiomers of M1 (as well as M4 and M5) after intravenous and oral administration of pentoxifylline to human volunteers. (R)- and (S)-M1 could be separated preparatively on a cellobiohydrolase column, while determination in blood or plasma was by HPLC after chiral derivatization with diacetyl-L-tartaric acid anhydride. The metabolism of pentoxifylline to (R)-M1 in suspensions of hemolysed erythrocytes followed simple Michaelis-Menten kinetics (K(m) = 11 mM), while that to (S)-M1 was best described by a two-enzyme model (K(m) = 1.1 and 132 mM). Studies with inhibitors indicated that the enzymes were of the carbonyl reductase type. At a therapeutic blood concentration of pentoxifylline, the calculated rate of formation of (S)-M1 is 15 times higher than that of the (R)-enantiomer. Back-conversion of M1 to pentoxifylline was 3-4 times faster for the (S)- than for the (R)-enantiomer. In vivo, the R:S plasma concentration ratio of M1 ranged from 0.010-0.025 after intravenous infusion of 300 or 600 mg of pentoxifylline, and from 0.019-0.037 after oral administration of 600 mg. The biotransformation of pentoxifylline to M1 was thus highly stereoselective in favor of the (S)-enantiomer both in vitro and in vivo.     

Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND

The new computer algorithm FOUND, which is implemented as an integrated module of the DYANA structure calculation program, is capable of performing systematic local conformation analyses by exhaustive grid searches for arbitrary contiguous fragments of proteins and nucleic acids. It uses torsion angles as the only degrees of freedom to identify all conformations that fulfill the steric and NMR-derived conformational restraints within a contiguous molecular fragment, as defined either by limits on the maximal restraint violations or by the fragment-based DYANA target function value. Sets of mutually dependent torsion angles, for example in ribose rings, are treated as a single degree of freedom. The results of the local conformation analysis include allowed torsion angle ranges and stereospecific assignments for diastereotopic substituents, which are then included in the input of a subsequent structure calculation. FOUND can be used for grid searches comprising up to 13 torsion angles, such as the backbone of a complete -helical turn or dinucleotide fragments in nucleic acids, and yields a significantly higher number of stereospecific assignments than the precursor grid search algorithm HABAS.