Microbial Oxidation of Amines: A Preliminary Investigation on the Resolution of Racemic 1-Phenylethyl Amine

The microorganisms Pichia pastoris, Pichia membranaefaciens, Candida boidinii and Aureobasidium sp. were screened for their ability to use amine as a nitrogen source. The range of amines accepted varied from organism to organism. P. pastoris used &#102 -unsubstituted amines, while the other three strains used transformed &#102 -substituted amines like cyclohexyl, 2-butyl and 1-phenylethyl amines. Biotransformation occurred via an oxidative pathway leading mainly to the production of the corresponding ketones. Investigation of the kinetic resolution of racemic 1-phenylethyl amine showed that the optically pure ( S ) enantiomer was obtained with Aureobasidium sp. experiments, and that with P. membranaefaciens or C. boidinii, the ( R ) enantiomer could also be produced, but with lower stereoselectivity (52%<ee<70%). This resolution process constitutes a new method for the production of optically active amines.     

(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.     

Aluminum chloride-mediated kinetic resolution of racemic gamma-substituted-gamma-lactones

Preparation of chiral gamma-substituted-gamma-lactones (1) through kinetic resolution is described. (S)-(-)-1-Phenylethylamine (2) in the presence of anhydrous AlCl(3) shows satisfactory levels of enantioselection in reaction with racemic gamma-substituted-gamma-lactones 1, where (R)-1 remains unreacted, while (S)-1 is enantioselectively converted to the ring-opened amide (S,S)-4. The enantiopurity of (R)-(+)- gamma-substituted gamma-lactones recovered ranges from 62-98% ee.     

Enhanced efficiency due to the use of achiral additives in the optical resolution of 1-phenylethylamine by its glutaric acid derivative

Racemic 1-phenylethylamine was optically resolved by its own derivative formed with glutaric acid namely (+)-(R)-N-(1-phenylethyl)glutaramic acid. The amide acid resolving agent was synthesized from (+)-(R)-1-phenylethylamine by N-derivatization. The glutaric acid derivative was the next in a homologous series of dicarboxilic acid derivatized resolving agents of racemic 1-phenylethylamine. Resolution results obtained with the oxalic, malonic, and succinic acid derivatives were previously discussed(1). Each of the above derivative resolving agents could be successfully applied as resolving agents of 1-phenylethylamine. The efficiency of the present optical resolution using (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent was remarkably inferior to the results obtained by its shorter chained homologues(1). Use of achiral additives, like urea, thiourea, N-methylurea, and N,N'-dimethylurea caused large increase in the efficiency of the resolution by (+)-(R)-N-(1-phenylethyl)glutaramic acid resolving agent. Precipitated salts obtained in the resolutions performed in the presence of the additives were investigated by thermoanalysis, X-ray powder diffraction, and optical microscopy. Based on the analytical data, the improvement of the resolution results was attributed to the influence of the additives on the crystal nucleation processes of the diasteromeric salts.     

Synthesis,further biological evaluation and pharmacodynamics of newly discovered inhibitors of TNF-alpha production

(1S,2R)-2-Acylamino-1-methyl-2-phenylethyl phosphate derivatives 2a, 2b, 3a, and 5a, which are conformationally restricted and metabolically stable analogues of (2R)-2-acylamino-2-phenylethyl phosphate derivatives 1a and 1b, are a new class of inhibitors of TNF-alpha production. More efficient alternative synthesis of a key intermediate, (1R,2S)-1-amino-1-(3-methoxyphenyl)propan-2-ol hydrochloride (9), was achieved using one-step, three-component coupling of 3-methoxyphenyl boronic acid (13), (5S)-2,2,5-trimethyl-1,3-dioxolan-4-ol (14), and amino diphenyl methane (15), [as reported in J. Am. Chem. Soc. 1998, 120, 11798]. Evaluation of the hypotensive activity of these compounds was done to assess one of their side effects. Among the compounds tested, the above-mentioned four compounds (2a, 2b, 3a, and 5a) were identified as inhibitors with both sufficient potency and an acceptable safety margin regarding their hypotensive activity. The pharmacodynamics of these compounds were also investigated. Single-dose pharmacokinetic data for compounds 2a, 2b, 3a, and 5a are displayed. These compounds were estimated to be mainly metabolized by the liver in the species tested based on their in vitro stability in tissue homogenates and plasma. A representative compound, 2a, showed good linearity of its plasma concentration after intravenous injection.     

Optical resolution of β-lactams on 1-phenylethylcarbamates of cellulose and amylose

Optical resolution of six β-lactams was examined by HPLC using chiral stationary phases consisting of tris((R)-, (RS)-, and (S)-1-phenylethylcarbamate)s of cellulose and amylose. All β-lactams were optically resolved at least by one of the carbamates. Amylose tris((S)-1-phenylethylcarbamate) showed high optical resolving abilities for some β-lactams.     

Thiorphan and retro-thiorphan display equivalent interactions when bound to crystalline thermolysin

The three-dimensional structures of (S)-thiorphan and (R)-retro-thiorphan bound to thermolysin have been determined crystallographically and refined to residuals of 0.183 and 0.187 at 1.7-A resolution. Thiorphan [N-[(S)-2-(mercaptomethyl)-1-oxo-3-phenylpropyl]glycine] [HSCH2CH(CH2C6H5)CONHC-H2COOH] and retro-thiorphan [[[(R)-1-(mercaptomethyl)-2-phenylethyl] amino]-3-oxopropanoic acid] [HSCH2CH(CH2C6H5)NHCOCH2COOH] are isomeric thiol-containing inhibitors of endopeptidase EC 24-11 (also called "enkephalinase"). The mode of binding of thiorphan to thermolysin is similar to that of (2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide [Monzingo, A.F., & Matthews, B.W. (1982) Biochemistry 21, 3390-3394] with the inhibitor sulfur atom coordinated to the active site zinc and the peptide portion forming substrate-like interactions with the enzyme. The isomeric inhibitor retro-thiorphan, which differs from thiorphan by the inversion of an amide bond, utilizes very similar interactions with enzyme. Despite the inversion of the -CO-NH- linkage the carbonyl oxygen and amide nitrogen display very similar hydrogen bonding, as anticipated by B.P. Roques et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3178-3182]. These results explain why thermolysin and possibly other zinc endopeptidases such as endopeptidase EC 24-11 fail to discriminate between these retro-inverso inhibitors.     

Combinations of chiral and prochiral singlet radical-pairs in reaction cavities of polyethylene films. Control and analysis of radical tumbling and translation.

The regio- and stereo-chemistries of combination products from chiral 1-naphthoxy/(R)-2-phenylpropanoyl and prochiral 1-naphthoxy/1-phenylethyl singlet radical-pairs (radical-pairs A and B, respectively) have been studied at different temperatures in polyethylene (PE) films with different crystallinities. The radical-pairs have been generated as intermediates along the photo-Fries reaction course of 1-naphthyl (R)-2-phenylpropanoate ((R)-1) and the photo-Claisen reaction course of 1-naphthyl (R)-1-phenylethyl ether ((R)-2). Radical-pair was produced directly upon lysis of the first excited singlet state of (R)-2 and indirectly after irradiation of (R)-1 and subsequent decarbonylation of the 2-phenylpropanoyl radical of radical-pair A. Comparison of the fates of the directly and indirectly formed radical-pairs provides detailed information about the nature of the reaction cavities within the polyethylene hosts and how the combinations of the radical-pairs are influenced by their initial locations within a cavity. The results, especially when taken with those from irradiations in n-alkanes, indicate that the cavities are "templated" by the (R)-1 and (R)-2 guest molecules and that the templated shapes are retained in some form for periods that are at least as long as the time required for decarbonylation of a 2-phenylpropanoyl radical. In addition, the enantiomeric excesses of the decarbonylated photoproducts from (R)-1(2, 2-(1-phenylethyl)-1-naphthol (2BN), and 4-(1-phenylethyl)-1-naphthol (4BN)) or 2BN and 4 BN from (R)-2 indicate different influences of temperature on translational and tumbling motions of the radicals of radical-pairs B within their polyethylene cages.     

Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase

Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS)     

Bicyclic glutamic acid derivatives

For the second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids via Strecker reaction of chiral ketimines, the cyanide addition as the key stereodifferentiating step produces mixtures of diastereomeric alpha-amino nitrile esters the composition of which is independent of the reaction temperature and the type of the solvent, respectively. The subsequent hydrolysis is exclusively achieved with concentrated H(2)SO(4) yielding diastereomeric mixtures of three secondary alpha-amino alpha-carbamoyl-gamma-esters and two diastereomeric cis-fused angular alpha-carbamoyl gamma-lactams as bicyclic glutamic acid derivatives, gained from in situ stereomer differentiating cyclisation of the secondary cis-alpha-amino alpha-carbamoyl-gamma-esters. Separation was achieved by CC. The pure secondary trans-alpha-amino alpha-carbamoyl-gamma-esters cyclise on heating and treatment with concentrated H(2)SO(4), respectively, to diastereomeric cis-fused angular secondary alpha-amino imides. Their hydrogenolysis led to the enantiomeric cis-fused angular primary alpha-amino imides. The configuration of all compounds was completely established by NMR methods, CD-spectra, and by X-ray analyses of the (alphaR,1R,5R)-1-carbamoyl-2-(1-phenylethyl)-2-azabicyclo[3.3.0]octan-3-one and of the trans-alphaS,1S,2R-2-ethoxycarbonylmethyl-1-(1-phenylethylamino)cyclopentanecarboxamide.     

Novel non-peptide inhibitors targeting death receptor-mediated apoptosis

We have previously reported that ECH, (2R, 3R, 4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one inhibits Fas-mediated apoptosis by blocking self-activation of pro-caspase-8 in the death-inducing signaling complex (DISC). A series of ECH derivatives were asymmetrically synthesized via key synthetic intermediates obtained from lipase-catalyzed kinetic resolution. Inhibitory activities of the derivatives towards death receptor-mediated apoptosis both in type I and type II cells were investigated, revealing that novel non-peptide inhibitors, RKTS-33 and RKTS-34, are effective as ECH.     

The first preparation of enantiopure 1-methyl-7-oxabicyclo[2.2.1]heptan-2-one, a versatile chiral building block for terpenoids

Methods for the resolution of (+/-)-1-methyl-7-oxabicyclo[2.2.1]heptan-2-one 1, a versatile chiral building block for terpenoids, have been investigated. While no efficient result was obtained with kinetic resolution methods, both enantiomers of 1 were prepared optically pure for the first time via esterification of the reductive products of 1 with (+)-mandelic acid and oxidation of the saponified products of diastereomer esters, in an overall yield of 70%. The absolute configurations of (-)-1 and (+)-1 were determined as (1S,4R)-(-)-1 and (1R,4S)-(+)-1 by the CD exciton chirality method and confirmed by Moshers (1)H-NMR method.     

The stereochemistry of NADH utilization by the flavoenzyme monooxygenase orcinol hydroxylase.

Ribbons et al. (Ribbons, D.W., Ohta, Y., and Higgins, I.J. (1972) in Molecular Basis of Electron Transport, Miami Winter Symposic Series (Schultz, J., and Cameron, B.F., eds) Vol. 4, pp. 251-274, Academic Press, New York) presented a preliminary report that the flavoenzyme monooxygenase orcinol hydroxylase shows mixed type 4R, 4S stereospecificity with respect to dihydronicotinamide oxidation when resorcinol and m-cresol were used as substrate analogs. With the natural substrate orcinol, 4R chirality was maintained. In kinetic isotope experiments reported here, we demonstrate in fact that orcinol hydroxylase maintains 4R stereospecificity with respect to dihydronicotinamide oxidation with all three substrates, orcinol, resorcinol, and m-cresol. Deuterium and tritium kinetic isotope effects were detected under Vmax conditions with (4R)-[4-2H]-, and (4R)-[4-3H]NADH for all three substrates. No isotope effect was observed with (4S)-[4-2H]NADH and tritium labilization from assays with (4S)-[4-3H]-NADH was negligible in all cases.     

Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.     

Molecular recognition between oligopeptides and nucleic acids. Sequence specific binding of (4S)-(+)- and (4R)-(-)-dihydrokikumycin B to DNA deduced from 1H NMR, footprinting studies and thermodynamic data

The sequence specific binding of the antibiotic (4S)-(+)-dihydrokikumycin B and its (4R)-(-) enantiomer, [(S)-1 and (R)-1, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)-1 and (R)-1] bind to AT-rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrkikumycins with d-[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5'-A5T6T7-3' reading from left to right. From quantitative 1D-NOE studies, the AH2(5)-ligand H7 distance of complex A [(S)-1 plus decamer (which is bound more strongly)] and complex B [(R)-1 and decamer] are estimated to be 3.8 +/- 0.3 A and 4.9 +/- 0.4 A, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding free energies (delta G degrees) of (S)-1 and (R)-1 to poly d(AT).poly d(AT) at 25 degrees C are -31.8 and -29.3 kJ mol-1, respectively while the corresponding binding enthalpies (delta H degrees) are -11.3 and -0.8 kJ mol-1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.     

Optical resolution of 1-(1-naphthyl)ethylamine by its dicarboxylic acid derivatives: Structural features of the oxalic acid derivative diastereomeric salt pair

Optical resolution methods were established for racemic 1-(1-naphthyl) ethylamine. The resolving agents were synthesized by N-derivatizing (R)-1-(1-naphthyl) ethylamine with dicarboxylic acids. Oxalic, malonic, and succinic acid derivatives were found to be suitable resolving agents. These resolutions are parallel to a series of optical resolutions of 1-phenylethylamine which had been previously performed by our research group using similar derivative resolving agents (Balint et al., Tetrahedron: Asymmetry 2001;12:1511-1518.) The comparison of the results of the enantiomer separations is performed. The diastereomeric salts formed with (R)-N-[1-(1-naphthyl)ethyl]oxalamic acid were investigated by single crystal X-ray diffraction. The crystal structures were compared with the previously published structures of the diastereomers of the phenyl-substituted analogue, namely (R)- and (S)-1-phenylethylammonium (R)-N-(1-phenylethyl)oxalamates (Balint et al., Tetrahedron: Asymmetry 2001;12:1511-1518).     

Extent of enthalpy-entropy compensation in protein-ligand interactions

The extent of enthalpy-entropy compensation in protein-ligand interactions has long been disputed because negatively correlated enthalpy (ΔH) and entropy (TΔS) changes can arise from constraints imposed by experimental and analytical procedures as well as through a physical compensation mechanism. To distinguish these possibilities, we have created quantitative models of the effects of experimental constraints on isothermal titration calorimetry (ITC) measurements. These constraints are found to obscure any compensation that may be present in common data representations and regression analyses (e.g., in ΔH vs. -TΔS plots). However, transforming the thermodynamic data into ΔΔ-plots of the differences between all pairs of ligands that bind each protein diminishes the influence of experimental constraints and representational bias. Statistical analysis of data from 32 diverse proteins shows a significant and widespread tendency to compensation. ΔΔH versus ΔΔG plots reveal a wide variation in the extent of compensation for different ligand modifications. While strong compensation (ΔΔH and -TΔΔS opposed and differing by < 20% in magnitude) is observed for 22% of modifications (twice that expected without compensation), 15% of modifications result in reinforcement (ΔΔH and -TΔΔS of the same sign). Because both enthalpy and entropy changes arise from changes to the distribution of energy states on binding, there is a general theoretical expectation of compensated behavior. However, prior theoretical studies have focussed on explaining a stronger tendency to compensation than actually found here. These results, showing strong but imperfect compensation, will act as a benchmark for future theoretical models of the thermodynamic consequences of ligand modification.     

Kinetic resolutions of indan derivatives using bacteria

Racemic indan derivatives have been resolved by the hydrolysis of amide bonds using Corynebacterium ammoniagenes IFO12612 to produce (S)-amine and (R)-amides. In the kinetic resolution of 1 (N-12-(6-methoxy-indan-1-yl)ethyl]acetamide), it was possible to run the reaction to 44% conversion on a 10-g scale, obtaining (S)-amine 4 ((S)-2-(6-methoxy-indan-1-yl)ethylamine) at >99% enantiomeric excess (ee) and (R)-1 at 98% ee.     

Lipase-catalyzed kinetic resolution of (+/-)-cis-flavan-4-ol and its acetate: synthesis of chiral 3-hydroxyflavanones

Lipase-catalyzed kinetic resolution of (+/-)-cis-flavan-4-ol and its acetate led to enantiomerically enriched flavan-4-ol and its acetate. These chiral compounds were converted to (2R, 3R)- and (25, 3S)-3-hydroxyflavanones.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.