Dynamic resolution and stereoinversion of secondary alcohols by chemo-enzymatic processes.

To overcome the maximum 50% yield limitation of classical resolution methods, deracemization processes involving a racemization step (dynamic resolution) or a prochiral intermediate (stereoinversion) have been developed. The use of transition metal complexes as racemizing agents, in combination with an enzymatic reaction, has been successfully extended to the deracemization of a number of simple or functionalized sec-alcohols. A two-enzyme process has been also investigated for their sequential or simultaneous deracemization. Other prominent results arise from an (apparently general) oxidoreduction process catalyzed by a single whole-cell microorganism.

Expression and characterization of the naturally occurring mutation L394R in human gamma-glutamyl carboxylase.

Patients with mutation L394R in gamma-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IX's propeptide and vitamin KH(2). Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K(i) for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V(max)/K(m) toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K(m) for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K(m) for vitamin KH(2). The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH(2) binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.

Microbial hydroxylation/functionalization of terpenoid synthons derived from communic acids.

Incubation of a communic acid-derived synthon with Cunninghamella elegans quantitatively affords 1 beta, 3 beta- and 7 beta- monohydroxylated derivatives.

Microbial hydroxylation of natural drimenic lactones.

Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields the corresponding 3 beta-hydroxy derivatives. From isodrimenin, the known natural 7 alpha-hydroxy derivative (futronolide) was also obtained and its structure was definitely established by X-ray crystallographic study of its acetate derivative.

Microbial models for drug metabolism.

This review describes microbial transformation studies of drugs, comparing them with the corresponding metabolism in animal systems, and providing technical methods for developing microbial models. Emphasis is laid on the potential for selected microorganisms to mimic all patterns of mammalian biotransformations and to provide preparative methods for structural identification and toxicological and pharmacological studies of drug metabolites.

Microbial models of drug metabolism: microbial transformations of Trimegestone (RU27987), a 3-keto-delta(4,9(10))-19-norsteroid drug.

Screening microorganisms for the biotransformation of the 3-keto-delta(4,9(10))-19-norsteroid RU27987 (Trimegestone) resulted in the isolation of nine identified metabolites, some of them being selectively produced by different strains. Eight metabolites were found to be hydroxylated on various positions of the rings, and one was additionally epoxidized. These microbial metabolites could be used as chromatographic standards and two of them were found identical to the unknown major human metabolites. Moreover, most microbial metabolites were produced in sufficient amounts to be tested for their biological activities. All these features demonstrate the usefulness and versatility of microbial biotransformation systems as a tool for early identification and convenient production of potentially active mammalian and non-mammalian metabolites.

Aminobicyclo[2.2.1.]heptane dicarboxylic acids (ABHD), rigid analogs of ACPD and glutamic acid: synthesis and pharmacological activity on metabotropic receptors mGluR1 and mGluR2.

Isomeric norbornane-derived rigid analogs mimicking different potential conformations of ACPD (1-aminocyclopentane-1,3-dicarboxylic acid) and glutamic acid have been synthesized, via the hydantoin route, to be used as conformational probes for bioactive conformations at the glutamatergic receptors of the central nervous system. Activities on metabotropic receptors mGluR1 and mGluR2 are reported and discussed.

Synthesis and pharmacological characterization of aminocyclopentanetricarboxylic acids: new tools to discriminate between metabotropic glutamate receptor subtypes.

The four stereoisomers of 1-aminocyclopentane-1,3,4-tricarboxylic acid {ACPT-I (18) and -II (19), (3R, 4R)-III [(-)-20], and (3S,4S)-III [(+)-20]} have been synthesized and evaluated for their effects at glutamate receptors subtypes. ACPTs are ACPD analogues in which a third carboxylic group has been added at position 4 in the cyclopentane ring. None of the ACPT isomers showed a significant effect on ionotropic NMDA, KA, and AMPA receptors. On the other hand, ACPT-II (19) was found to be a general competitive antagonist for metabotropic receptors (mGluRs) and exhibited a similar affinity for mGluR1a (KB = 115 +/- 2 microM), mGluR2 (KB = 88 +/- 21 microM), and mGluR4a (KB = 77 +/- 9 microM), the representative members of group I, II and III mGluRs, respectively. Two other isomers, ACPT-I (18) and (+)-(3S,4S)-ACPT-III [(+)-20], were potent agonists at the group III receptor mGluR4a (EC50 = 7.2 +/- 2.3 and 8.8 +/- 3.2 microM) and competitive antagonists with low affinity for mGluR1a and mGluR2 (KB > 300 microM). Finally, (-)-(3R,4R)-ACPT-III [(-)-20] was a competitive antagonist with poor but significant affinity for mGluR4a (KB = 220 microM). These results demonstrate that the addition of a third carboxylic group to ACPD can change its activity (from agonist to antagonist) and either increase or decrease its selectivity and/or affinity for the various mGluR subtypes.

Microbial biotransformations of a synthetic immunomodulating agent, HR325.

The microbial biotransformation of HR325 [2-cyano-3-cyclopropyl-3-hydroxy-N-(4'-trifluoromethyl-3'-methylphenyl)- propenamide], a synthetic immunomodulating agent, has been investigated in order to be compared with animal metabolism and to prepare some metabolites which are difficult to obtain by chemical methods. Several fungal strains are able to completely metabolize this drug. Mortierella isabellina NRRL 1757 only achieves a benzylic hydroxylation on the aromatic methyl group, affording in high yield the corresponding hydroxymethyl derivative. In addition, other strains, such as Cunninghamella elegans ATCC 26269 or Beauveria bassiana ATCC 7159 can cleave both cyclopropyl and cyano groups in a new unknown oxidative biochemical reaction, which can be mimicked by m-chloroperbenzoate oxidation. The resulting cyanohydrin is hydrolyzed and reduced to a primary alcohol. In B. bassiana, the final incubation product is a beta-4-O-methylglucoside derivative of this alcohol, and has been fully characterized by independent synthesis. The different metabolic patterns of HR325 in the three fungal strains are discussed, and a mechanistic hypothesis about the oxidative cleavage of the right part of the molecule is proposed. The production of microbial metabolites is compared to animal metabolism in terms of structure and efficiency.

Solution conformation of alpha, beta or gamma-methylglutamyl-containing derivatives as probes of vitamin K-dependent carboxylase using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methyl substituted N-BOC glutamic acid methyl esters (2M, 3T, 3E, 4T, 4E) has been completely characterized through combined NMR and molecular modeling studies. Hetero- and homonuclear coupling constants were measured in order to assign the remaining diastereotopic methylene protons at C(3) and/or C(4), and used for comparison with theoretical data. In parallel, the complete conformational analysis of these analogues has been achieved using molecular mechanics and molecular dynamics (MD) methods. The conformation of the glutamyl residue is established by the excellent agreement between the experimental and calculated side chain scalar coupling constants. The theoretical NMR data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for internal motions. There is a significant influence of the methyl group on the conformational behaviour and on the biological relevance of these structures. Steric effect or electrostatic interaction may also have a considerable influence in stabilizing a conformational population in D2O solution. The conformational preferences of those different analogues in aqueous and methanol solution are discussed in the light of biological results obtained on the vitamin K-dependent carboxylase system.

Microbial models of mammalian metabolism. Fungal metabolism of phenolic and nonphenolic p-cymene-related drugs and prodrugs. I. Metabolites of thymoxamine.

This study was undertaken to validate the use of microbial biotransformation systems for drug metabolism studies. Thymoxamine 1 was rapidly hydrolyzed to desacetylthymoxamine (DAT) 2 by numerous fungi. Other known animal metabolites, such as N-desmethyl-desacetylthymoxamine 3 and desacetylthymoxamine-O-sulfate 6, were produced from DAT by Mucor rouxii and Mortierella isabellina. DAT-N-oxide 5, a putative animal microsomal metabolite, was also produced by M. isabellina, in addition, a few strains (such as Actinomucor elegans, Mucor hiemalis, and Mucor janssenii) produced a glycosylated metabolite that was identified by high-resolution 1H- and 13C-NMR, MS, and enzymatic hydrolysis as the corresponding [4-(2-dimethylaminoethoxy)-5-isopropyl-2-methyl-phenyl]-1-beta-D- glucopyranoside 7. A similar glucosylation reaction was observed when thymohydroquinone 10 was incubated with A. elegans. Several strains were able to produce transiently thymohydroquinone from DAT-N-oxide 5, possibly through a beta-elimination mechanism.

Microbial models of mammalian metabolism. Fungal metabolism of phenolic and nonphenolic p-cymene-related drugs and prodrugs. II. Metabolites of nonphenolic derivatives.

A cymene-derived drug, 3-[4'-(o-ethoxyphenyl)piperazin-1'-yl]-ethyloxy-p-cymene+ ++ (B1178), is not significantly metabolized by fungal microorganisms. On the contrary, one of its metabolites in rat, 3-(piperazin-1'-yl)ethoxy-p-cymene (B1071), is quantitatively converted by Cunninghamella echinulata NRRL 3655 into two hydroxylated products: the corresponding phenol derivative and a benzylic alcohol derivative. Other strains, such as Beauveria bassiana ATCC 7159 and Mortierella isabellina MMP 108, produce exclusively an N-acetyl derivative in high yield. Results obtained are discussed on the grounds of relative hydrophobicity of substrates vs. fungi metabolism and detoxification capabilities.

Conformational analysis of glutamic acid analogues as probes of glutamate receptors using molecular modelling and NMR methods. Comparison with specific agonists.

The activity of five glutamic acid analogues substituted in position 3 or 4 by a methyl (3T, 3E, 4T, and 4E) or a methylene group (4M) has been examined at one cloned Glu receptor subtype, mGluR1. These analogues interact with glutamate receptors of the central nervous system, especially the ligand 4T [(2S,4S)-4-methylglutamic acid] at the metabotropic glutamate receptor mGluR1. It was observed that only the 4T isomer is as potent an agonist as glutamic acid, whereas other isomers are less active. Furthermore, 4E [(2S,4R)-4-methylglutamic acid] exhibited an exceptional selectivity for the KA ionotropic receptor subtype while 4M [(2S)-4-methyleneglutamic acid] was active at the NMDA receptors. These molecules represent suitable tools among a population of similar glutamate analogues for a classical structure-function relationship study. We have undertaken a conformational analysis by 1H and 13C NMR spectroscopy and molecular modelling of these molecules. Hetero- and homonuclear coupling constants were measured in order to assign the diastereotopic methylene protons at C(3) or C(4), and used for comparison in molecular dynamics (MD) simulations. The hydrogen-bonding possibility, steric effects or electrostatic interactions may be a considerable influence in stabilizing a conformational population in D2O solution. The conformations may be grouped by the two backbone torsion angles, chi 1 [alpha-CO2(-)-C(2)-C(3)-C(4)] and chi 2 [+NC(2)-C(3)-C(4)-gamma CO2-] and by the two characteristic distances between the potentially active functional groups, alpha N(+)-gamma CO2- (d1) and alpha CO2(-)-gamma CO2- (d2). The conformational preferences in solution of 4T, 4E and (3T, 3E, 4M) are discussed in the light of the physical features known for a specific metabotropic agonist (ACPD) and specific ionotropic agonists (KA) and (NMDA), respectively.

Vitamin K-dependent carboxylase. In vitro inhibitory activity of cyclopentane and cyclohexane-derived analogues of glutamic acid and their conformational study by NMR and molecular dynamics in aqueous solution.

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a Boc-protected amino group and a methyl ester group and in position 3 by a free carboxylate group (6-9), has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). These compounds have been shown to be weak competitive inhibitors (Ki approximately 20-65 mM) of the vitamin K-dependent carboxylation of Boc-Glu-OMe in rat liver microsomes independently of their ring size and stereochemical features. However, the cyclic trans isomers have been found more active than the cis ones, and Boc-trans-C5-OMe (9) is the most potent inhibitor in the series (cis and trans isomers are defined by the relative arrangement of the carboxyl functions). Such cyclic glutamyl derivatives may provide valuable informations on the preferred bioactive conformations of synthetic glutamyl substrates at the active site of the carboxylase. In aqueous solution, the Boc-cis- and trans-C6 esters exhibit chair conformations with exclusively equatorial and axial substituent positions, while the Boc-cis- and -trans-C5 compounds may display envelope E or 'twist' T conformations with the substituents in the following positions, equatorial; axial and isoclinal. For each compound, the conformations resulting from NMR and MD data were analyzed and classified according to the dihedral angles chi 1 and chi 2, the distances of functional groups, and the spatial charge distribution involving the free carboxyl group. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. These results are discussed in relation with the carboxylase inhibitory activity of the analogues, and a spatial disposition of the glutamyl side chain that could be recognized by the carboxylase is deduced.

Conformational study in water by NMR and molecular modeling of cyclic glutamic acid analogues as probes of vitamin K dependent carboxylase.

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a BOC protected amino group and a methyl ester group and in position 3 by a free carboxylate group, has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). Their structural properties were under investigation for a structure-activity relationship analysis to determine the preferred conformation in the carboxylase active site. For each compounds, resulting conformations from NMR and MD data were analyzed and classified according to the dihedral angles chi 1 and chi 2, the distances and the spatial distribution involving charged or substituted C- and N-terminal groups. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. A comparison between these different classes of the active and nonactive derivatives was achieved.

Microbial transformation of steroids: contribution to 14 alpha-hydroxylations.

The regioselective and stereoselective hydroxylation of steroids by fungal strains previously known for their hydroxylation capabilities, such as Thamnostylum (= Helicostylum) piriforme ATCC 8992, Mucor griseocyanus ATCC 1207a, Actinomucor elegans (= Mucor parasiticus) MMP 3122 (Mucorales), and Zygodesmus sp. ATCC 14716, was investigated with special interest for the 14 alpha-hydroxylation reaction. A preliminary screening had shown that some of these microorganisms were adequate for the production of 14 alpha-hydroxylated derivatives of the following steroids: progesterone, 5 beta-pregnane-3,20-dione, 3 beta-hydroxy-5 beta-pregnane-20-one, 3 beta-hydroxy-5 beta-17 (alpha H)-etianic acid methyl ester, androst-4-ene-3,17-dione, and testosterone. About 20 metabolites have been isolated and purified by silicagel chromatography and semi-preparative reverse-phase HPLC. These metabolites have been fully characterized by 1H, 13C NMR and mass spectrometry. All the identified metabolites were hydroxylated at some distinct positions, such as 6 beta-, 7 alpha-, 9 alpha-, 14 alpha-, 15 beta-, or dihydroxylated at 6 beta,14 alpha-,7 alpha,14 alpha-, 9 alpha,14 alpha-, 14 alpha,15 alpha-, 14 alpha,15 beta-positions; nine of these metabolites have not been reported previously. The relationship between the structural features of the investigated steroids and the site-specific hydroxylation has been delineated, and progesterone was found to be the best substrate for the production of 14 alpha-hydroxylated derivative, using T. piriforme.

Pulse radiolysis study of vitamin K1 reduction in ethanolic solution.

A pulse radiolysis study of vitamin K1 reduction was carried out in argon-saturated ethanolic solutions. The alpha-hydroxyethyl radicals CH3C.HOH (R.) and the solvated electrons (e-solv) reduce vitamin K1, leading to the semiquinone transient K1H.. This species, characterized by its absorption spectrum, decays by disproportionation and leads to the formation of the hydroquinone K1H2. The rate constants of the monoelectronic exchanges involved in this reduction have been determined.

Chemoenzymatic access to enantiomeric bicyclo[2.2.1]heptan-2,5-diones.

A practical integrated process, combining an enzymatic resolution step with a few chemical transformations, is described for the synthesis of (1R, 4R)- and (2S, 4S)-bicyclo[2.2.1]heptan-2,5-diones 1 of high enantiomeric purity, starting from a standard mixture of (+/-)-endo- and exo-2-acetoxy-5-norbornene.

Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichia coli.

Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli. The best substrates were, in decreasing order of specific activity, D-erythro-4-methylglutamic acid, D-erythro-3-methylglutamic acid, DL-homocysteic acid, (+/-)-trans-1-amino-3-carboxy-cyclopentanecarboxylic acid and (+/-)-trans-1-amino-3-carboxy-cyclohexanecarboxylic acid. Among the different stereoisomers, only the D-erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates. Apart from the D-erythro-3- and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine.

Synthesis of diastereoisomeric peptides incorporating cycloglutamic acids. Substrate specificity of vitamin K-dependent carboxylation.

Tripeptides Boc-X-Glu-Val where X is alpha-methyl glutamic acid or various cyclic analogues of glutamic acid, such as 1-amino-1,3-dicarboxycyclohexane (cis or trans-CHGA) or -cyclopentane (cis or trans-CPGA) have been synthesized. Methods for the selective protection, activation, and coupling of such unnatural amino acids are described. The peptides, which are potential competitive inhibitors of the vitamin K-dependent carboxylation, have been preliminarily tested with the rat liver microsomal carboxylase and found to be effective substrates of the carboxylation reaction.