Production of (R)-3-amino-3-phenylpropionic acid and (S)-3-amino-3-phenylpropionic acid from (R,S)-N-acetyl-3-amino-3-phenylpropionic acid using microorganisms having enantiomer-specific amidohydrolyzing activity

(R)-3-Amino-3-phenylpropionic acid ((R)-beta-Phe) and (S)-3-amino-3-phenylpropionic acid ((S)-beta-Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure beta-Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac-beta-Phe) in an enantiomer-specific manner was performed. A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)-beta-Phe (>99.5% ee) and (S)-beta-Phe (>99.5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.     

Bile acid amides derived from chiral amino alcohols: novel antimicrobials and antifungals

Cholic and deoxycholic acid amides 10-17 have been synthesised from (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, (1S,2S)-1-phenyl-2-amino-1,3-propanediol 4, (1R,2R)-1-para-nitrophenyl-2-amino-1,3-propanediol 3, (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5. Amide 12 derived from N-succinimidyl ester 9 of deoxycholic acid and (1R,2R)-1-phenyl-2-amino-1,3-propanediol 2, found to be active against Cryptococcus neoformans and the amide 17 obtained from N-succinimidyl ester 9 of deoxycholic acid and (1S,2S)-1-para-nitrophenyl-2-amino-1,3-propanediol 5, is found to be potent against various gram-positive bacteria.     

X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+)-1-amino-2-phenylethyl phosphonic acid.

The structure of the carboxypeptidase A complex with the inhibitor (S)-(+)-1-amino-2-phenylethylphosphonic acid has been determined at 0.23 nm resolution. The delta F map shows electron-density peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc ion in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations). A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)-(+)-1-amino-2-phenylethylphosphonic acid is essentially in agreement with our results.     

Comparative stability of 1-alkoxyalkyl alpha-D-glucosides in the presence of acid or alpha-D-glucosidase from yeast

The aminated 1-alkoxyalkyl glycosides [(S)-2-amino-1-methoxyethyl] 6-amino-6-deoxy-alpha-D-glucopyranoside (3) and [(R,S)-1-ethoxyethyl] 6-amino-6-deoxy-alpha-D-glucopyranoside (4) have been synthesised and characterised. These compounds as well as [(R)-2-amino-1-methoxyethyl] alpha-D-glucopyranoside (1) prepared earlier are resistant against alpha-D-glucosidase (maltase, alpha-D-glucoside glucohydrolase, E.C. 3.2.1.20) from yeast, yet undergo hydrolysis under relatively mild acidic conditions. The kinetic parameters of the interaction with alpha-D-glucosidase and with acid were determined. The relative rates of acid hydrolysis of aminated 1-alkoxyalkyl glycosides compared with aminated ordinary glycosides suggest essential differences in the mechanism of acid-catalysed hydrolysis.     

Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid

Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.     

Biochemical and spectroscopic studies on (S)-2-hydroxypropylphosphonic acid epoxidase: a novel mononuclear non-heme iron enzyme

The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase. Results reported herein provided initial biochemical evidence revealing that HPP epoxidase is an iron-dependent enzyme and that both NAD(P)H and a flavin or flavoprotein reductase are required for its activity. The 2 K EPR spectrum of oxidized iron-reconstituted fosfomycin epoxidase reveals resonances typical of S = (5)/(2) Fe(III) centers in at least two environments. Addition of HPP causes a redistribution with the appearance of at least two additional species, showing that the iron environment is perturbed. Exposure of this sample to NO elicits no changes, showing that the iron is nearly all in the Fe(III) state. However, addition of NO to the Fe(II) reconstituted enzyme that has not been exposed to O(2) yields an intense EPR spectrum typical of an S = (3)/(2) Fe(II)-NO complex. This complex is also heterogeneous, but addition of substrate converts it to a single, homogeneous S = (3)/(2) species with a new EPR spectrum, suggesting that substrate binds to or near the iron, thereby organizing the center. The fact that NO binds to the ferrous center suggests O(2) can also bind at this site as part of the catalytic cycle. Using purified epoxidase and (18)O isotopic labeled HPP, the retention of the hydroxyl oxygen of HPP in fosfomycin was demonstrated. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require alpha-ketoglutarate for activity. In contrast, HPP epoxidase is alpha-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and C-O bond formation.     

Site-directed mutagenesis and spectroscopic studies of the iron-binding site of (S)-2-hydroxypropylphosphonic acid epoxidase

(S)-2-Hydroxylpropanylphosphonic acid epoxidase (HppE) is a novel type of mononuclear non-heme iron-dependent enzyme that catalyzes the O2 coupled, oxidative epoxide ring closure of HPP to form fosfomycin, which is a clinically useful antibiotic. Sequence alignment of the only two known HppE sequences led to the speculation that the conserved residues His138, Glu142, and His180 are the metal binding ligands of the Streptomyces wedmorensis enzyme. Substitution of these residues with alanine resulted in significant reduction of metal binding affinity, as indicated by EPR analysis of the enzyme-Fe(II)-substrate-nitrosyl complex and the spectral properties of the Cu(II)-reconstituted mutant proteins. The catalytic activities for both epoxidation and self-hydroxylation were also either eliminated or diminished in proportion to the iron content in these mutants. The complete loss of enzymatic activity for the E142A and H180A mutants in vivo and in vitro is consistent with the postulated roles of the altered residues in metal binding. The H138A mutant is also inactive in vivo, but in vitro it retains 27% of the active site iron and nearly 20% of the wild-type activity. Thus, it cannot be unequivocally stated whether H138 is an iron ligand or simply facilitates iron binding due to proximity. The results reported herein provide initial evidence implicating an unusual histidine/carboxylate iron ligation in HppE. By analogy with other well-characterized enzymes from the 2-His-1-carboxylate family, this type of iron core is consistent with a mechanism in which both oxygen and HPP bind to the iron as a first step in the in the conversion of HPP to fosfomycin.     

青霉菌立体选择性环氧化顺丙烯磷酸产生磷霉素

由土壤中分离出一株青霉 (Penicilliumsp .) ,编号F5,能选择性的将顺丙烯磷酸环氧化为磷霉素 ,在pH7 5、2 8℃、2 80r min条件下培养 6d ,底物浓度 0 3%时 ,产物浓度达 2 2mg mL ,产率 41 % ;底物浓度 0 6%时产率 8%。转化产物经磷霉素敏感菌生物检测 ,TLC检测 ,并与标准品比较 ,确证为磷霉素。     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Substrate stereospecificity and active site topography of gamma-aminobutyric acid aminotransferase for beta-aryl-gamma-aminobutyric acid analogues

The substrate and inhibitory properties of (R)- and (S)-4-amino-3-phenylbutanoic acid, (R)- and (S)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofens), (E)-4-amino-3-phenylbut-2-enoic acid, and (E)-4-amino-3-(4-chlorophenyl)but-2-enoic acid are determined and compared with those of 4-aminobutanoic acid, 4-aminobut-2-enoic acid (4-aminocrotonic acid), and the racemic mixtures of 4-amino-3-arylbutanoic acids. All compounds in both series were found to be substrates, except for the R-isomers, which were identified as competitive inhibitors. These results are compared with known pharmacological data regarding the appropriate isomers.     

Efficient chemoenzymatic synthesis of (S)-α-amino-4-fluorobenzeneacetic acid using immobilized penicillin amidase

An efficient chemoenzymatic route was developed for synthesis of (S)-α-amino-4-fluorobenzeneacetic acid, a valuable chiral intermediate of Aprepitant, using immobilized penicillin amidase catalyzed kinetic resolution of racemic N-phenylacetyl-4-fluorophenylglycine. The optimum temperature, pH and agitation rate of the reaction were determined to be 40 °C, 9.5 and 300 rpm, respectively. Kinetic resolution of 80 g L⁻¹ N-phenylacetyl-4-fluorophenylglycine by immobilized amidase 20 g L⁻¹ resulted in 49.9% conversion and >99.9% e.e. within 3 h. The unreacted N-phenylacetyl-4-fluorophenylglycine can be easily racemized and then recycled as substrate. The production of (S)-α-amino-4-fluorobenzeneacetic acid was further amplified in 1 L reaction system, affording excellent conversion (49.9%) and enantioselectivity (99.9%). This chemoenzymatic approach was demonstrated to be promising for industrial production of (S)-α-amino-4-fluorobenzeneacetic acid.     

Isoform-selective thiazolo[5,4-b]pyridine S1P1 agonists possessing acyclic amino carboxylate head-groups

Replacement of the azetidine carboxylate of an S1P(1) agonist development candidate, AMG 369, with a range of acyclic head-groups led to the identification of a novel, S1P(3)-sparing S1P(1) agonist, (-)-2-amino-4-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo[5,4-b]pyridin-2-yl)phenyl)-2-methylbutanoic acid (8c), which possessed good in vivo efficacy and pharmacokinetic properties. A 0.3mg/kg oral dose of 8c produced a statistically significant reduction in blood lymphocyte counts 24h post-dosing in female Lewis rats.     

2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Identification of the reaction products

In a previous report we have described some properties of a novel flavoenzyme from a denitrifying Pseudomonas species which catalyzes the oxygen- and NAD(P)H-dependent conversion of 2-aminobenzoyl-CoA [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur. J. Biochem. 185, 637-634]. In this paper, we report on the identification of the three products formed from 2-aminobenzoyl-CoA in this reaction. The spectroscopic data and the chemical properties of these compounds and those of their degradation products are compatible with the structures of 2-amino-5-hydroxybenzoyl-CoA, 2-amino-5-hydroxycyclohex-1-enecarboxyl-CoA and of 2-amino-5-oxocyclohex-1-enecarboxyl-CoA. The latter is the main product and was found to be rather unstable since it hydrolyzes and decarboxylates readily at pH less than 5. Ammonia is released from the decarboxylation product in the neutral pH range to yield 1,4-cyclohexanedione. Conditions were optimized such that the CoA thioester of 2-amino-5-hydroxybenzoate is the product obtained at greater than 98% yield. 2-amino-5-hydroxycyclohex-1-enecarboxyl-CoA is the product which is formed when the mixture of the reaction products is treated with sodium borohydride before separation.     

Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-like binding mode and the ligand adopts two different conformations. The K(i) of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 +/- 29 nM and at full-length GluR2(R)o it was 175 +/- 8 nM. (S)-4-AHCP appears to elicit partial agonism at GluR2 by inducing an intermediate degree of domain closure (17 degrees). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)i, relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain D2 prevents full D1-D2 domain closure, which is limited by steric repulsion, especially between Leu704 and the ligand.     

Enantioselective synthesis of (S)-2-amino-4-phenylbutanoic acid by the hydantoinase method

Biosynthesis of (S)-(+)-2-amino-4-phenylbutanoic acid (1) was performed by nonenantioselective hydantoinase and L-N-carbamoylase using racemic 5-[2-phenylethyl]-imidazolidine-2,4-dione (rac-2) as a substrate. The compounds involved in this biocatalysis process could be simultaneously resolved by high-performance liquid chromatography using Chirobiotic T column with a mobile phase of EtOH/H(2)O = 10/90 at pH 4.2-4.5. To our knowledge, this is the first report of the successful production of 1 by the combination of recombinant hydantoinase and L-N-carbamoylase.     

Chiral discrimination promoted by (1S,2S)-1-phenyl-2-amino-1,3-propanediol derivatives in the asymmetric Reformatsky reaction

Some 3-t-butyldimethylsilyloxy derivatives, synthesized from the cheap commercially available (1S,2S)-2-amino-1-phenyl-1,3-propanediol [(1S,2S)- 1 ], have been successfully employed as new chiral ligands in the asymmetric Reformatsky reaction on aldehydic substrates. The influence both of the substrate and of the ligand on the stereochemical pathway has been investigated by varying the structure of the carbonyl substrate and of the optically active aminodiols. © 1995 Wiley-Liss, Inc.     

Design and optimization of novel (2S,4S,5S)-5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxy-2-isopropylhexanamides as renin inhibitors

Introduction of the 2,2-dimethyl-4-phenylpiperazin-5-one scaffold into the P(3)-P(1) portion of the (2S,4S,5S)-5-amino-6-dialkylamino-4-hydroxy-2-isopropylhexanamide backbone dramatically increased the renin inhibitory activity without using the interaction to the S(3)(sp) pocket. Compound 31 exhibited >10,000-fold selectivity over other human proteases, and 18.5% oral bioavailability in monkey.     

Click synthesis and biologic evaluation of (R)- and (S)-2-amino-3-[1-(2-[18F]fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid for brain tumor imaging with positron emission tomography

The (R)- and (S)-enantiomers of 2-amino-3-[1-(2-[18F]fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid (4) were synthesized and evaluated in the rat 9L gliosarcoma brain tumor model using cell uptake assays, biodistribution studies, and micro-positron emission tomography (microPET). The (R)- and (S)-enantiomers of [18F]4 were radiolabeled separately using the click reaction in 57% and 51% decay-corrected yields, respectively. (S)-[18F]4 was a substrate for cationic amino acid transport and, to a lesser extent, system L transport in vitro. In vivo biodistribution studies demonstrated that (S)-[18F]4 provided higher tumor uptake and higher tumor to brain ratios (15:1 at the 30- and 60-minute time points) compared to the (R)-enantiomer (7:1 at the 30- and 60-minute time points). MicroPET studies with (S)-[18F]4 confirmed that this tracer provides good target to background ratios for both subcutaneous and intracranial 9L gliosarcoma tumors. Based on these results, the 1H-[1,2,3]triazole-substituted amino acid (S)-[18F]4 has promising PET properties for brain tumors and represents a novel class of radiolabeled amino acids for tumor imaging.     

Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.