Enantiospecific enzymatic preparation of (2S,3S)-3-alkylaspartic acids of current interest in the synthesis of stereoregular poly[β-(2S,3S)-3-alkylmalic acids] as new optically active functional polyesters

(2S,3S)-3-methyl- and 3-isopropylaspartic acids were synthesized by bioconversion of the corresponding alkylfumarates (mesaconate and 3-isopropylfumarate) using β-methylaspartase from cell-free extracts of Clostridium tetanomorphum. Optically pure (2S,3S)-3-alkylaspartic acids were transformed in several steps to benzyl (3S,4R)-3-alkylmalolactonates without any racemization of the two chiral centers. These optically active α,β-substituted-β-lactones were polymerized by anionic ring opening polymerization yielding optically active semi-crystalline polyesters. ¹³C NMR analysis of poly[benzyl β-3-isopropylmalate] in CDCl₃ has shown that only the iso-type stereosequence is present in the polymer, indicating that the macromolecular chain is constituted by the only units of benzyl β-(2S,3S)-3-isopropylmalate monomer. The polymerization reaction was done without any racemization of the two stereogenic centers as in the case of benzyl (3S,4R)-3-methylmalolactonate. © 1996 Wiley-Liss, Inc.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Chiral derivatization for separation of racemic amino and thiol drugs by liquid chromatography and capillary electrophoresis

Separation of racemic amino drugs (α-methylbenzeneethanamine, 6-amino-2-methyl-2-heptanol and 1-aminoethyl-benzenemethanol) and thiol drugs [N-(2-mercapto-1-oxopropyl) glycine, 2-mercaptopropanoic acid, and N-acetyl-3-mercaptovaline] has been evaluated after derivatization. ortho-Phthalaldehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA) were used with either homochiral thiols (N-acetyl-L-cysteine and N-acetyl-D-penicillamine) or amines [(-)-(1R,2S)-norephedrine, L-phenylalanine, L-tyrosine, and 3-hydroxy-L-tyrosine] as chiral selectors according to the analyte reactive group. The resulting 36 diastereoisomeric derivatives were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Of the CE modes, micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) as surfactant, β-cyclodextrin (β-CD)-modified capillary zone electrophoresis (β-CD-CZE), and β-CD-MEKC were applied. Results highlight respective performance of the reagents and separative techniques. All OPA derivatives of racemic amino drugs were resolved either by MEKC or β-CD-MEKC. In the case of racemic thiol drugs, 10 of the 12 OPA derivatives were resolved in β-CD-CZE. © 1995 Wiley-Liss, Inc.     

The 3-methylaspartase reaction probed using 2H- and 15N-isotope effects for three substrates: a flip from a concerted to a carbocationic amino-enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S.

The mechanisms of the elimination of ammonia from (2S,3S)-3-methylaspartic acid, (2S)-aspartic acid and (2S,3R)-3-methylaspartic acid, catalysed by the enzyme L-threo-3-methylaspartase ammonia-lyase (EC 4.3.1.2) have been probed using 15N-isotope effects. The 15N-isotope effects for V/K for both (2S,3S)-3-methylaspartic acid and aspartic acid are 1.0246 +/- 0.0013 and 1.0390 +/- 0.0031, respectively. The natural substrate, (2S,3S)-3-methylaspartic acid, is eliminated in a concerted fashion such that the C(beta)-H and C(alpha)-N bonds are cleaved in the same transition state. (2S)-Aspartic acid appears to follow the same mechanistic pathway, but deprotonation of the conjugate acid of the base for C-3 is kinetically important and influences the extent of 15N-fractionation. (2S,3R)-3-Methylaspartic acid is deaminated via a stepwise carbocationic mechanism. Here we elaborate on the proposed model for the mechanism of methylaspartase and propose that a change in stereochemistry of the substrate induces a change in the mechanism of ammonia elimination.     

Enantioselective influence of cyclodextrins on cleavage of chiralic esters

Assessing the reactivity of optical antipodes is of central importance in drug research. Using the model of 2-methoxy-2-phenylacetic acid-4-nitrophenylester (MPE), the rate of hydrolysis in the presence of β-cyclodextrin (CD), hydroxyethyl- and hydroxypropyl-β-CD, as well as methyl-β-CD is studied photometrically and by means of HPLC (Chiralcel-OD-R-column). Both β-CD and hydroxyalkylated-β-CD catalyze (?)-(R)-enantiomers to a larger extent than (+)-(S)-enantiomers, resulting in an enrichment of the latter. Methyl-β-CD stabilizes the ester trifold, thus abolishing chiral discrimination. © 1995 Wiley-Liss, Inc.     

Synthesis of enantiopure phthalides including 3-butylphthalide, a fragrance component of celery oil, and determination of their absolute configurations

Enantiopure phthalides 2 and 5-8 were synthesized via enantioresolution of the corresponding alcohols with a chiral auxiliary of camphorsultam dichlorophthalic acid, (1S,2R,4R)-(-)-CSDP acid 3, followed by solvolysis with KOH in MeOH and the catalytic oxidation of chiral glycols with iridium complex 28. The absolute configurations of phthalides 2 and 5-8 were determined by applying the (1)H-NMR anisotropy method of MalphaNP acid (4), 2-methoxy-2-(1-naphthyl)propionic acid, to the chiral synthetic precursory alcohols. In the case of 3-phenylphthalide (R)-(-)-7, the absolute configuration determined by the (1)H-NMR anisotropy method using MalphaNP acid 4 agreed with that by the X-ray crystallographic method. By applying these methods, 3-butylphthalide (S)-(-)-2, a fragrance component of essential oil of celery, has been synthesized in an enantiopure form, and its absolute configuration was unambiguously determined.     

A new model to account for the order in which enantiomers of alkylarylcarbinols elute from a Pirkle chiral HPLC column: preparation, absolute stereochemistry, and chromatographic properties of (+)-1,2-benzocyclononen-3-ol and (+)-1,2-benzocyclodecen-3-ol

Samples enriched in (-)- and (+)-1,2-benzocyclononen-3-ol were prepared by microbially mediated reactions. An enriched sample of (+)-1,2-benzocyclodecen-3-ol was prepared by fractional crystallization of the diastereoisomeric camphanates, followed by hydrolysis. The absolute stereochemistry of both alcohols was established by chemical transformations. The elution order of their enantiomers from a chiral Pirkle HPLC column [(R)-N-(3,5-dinitrobenzoyl)phenyl glycine ionically bound to gamma-aminopropyl silanized silica] was determined. The information in conjunction with other data was used to formulate a rule to predict the configuration of an enantiomer of an alkylarylcarbinol from its elution order from this column.     

Stereoisomeric flavour compounds LXVIII. 2-, 3-, and 4-Alkyl-branched acids,part 2: Chirospecific analysis and sensory evaluation

Enantioselective GC analysis of 4-ethyloctanoic and 4-methylheptanoic acid, using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as the chiral stationary phase, is described and the sensory properties of several 4-alkyl-branched acids, using gas chromatography-olfactometry (GC-O) equipment and octakis(2,3-di-O-methyl-6-tert-butyldimethylsilyl)-γ-cyclodextrin as the stationary phase, are evaluated. The chirospecific analysis of various 2-, 3-, and 4-alkyl-branched acids from commercially available Roman chamomile (Chamaemelum nobile (L.) Allioni), Parmesan cheese, and subcutaneous mutton adipose tissue, using either GC-GC (MDGC) or GC-MS analytical methods, is described. © 1994 Wiley-Liss, Inc.     

(1′R,2′S,5′R)-Menthyl-(5R)-acetoxy-1,3-oxathiolan-(2R)-carboxylate: Synthesis and isomeric purity determination by HPLC

Chemoselective reduction of one isomer of the 1-menthylester of 1,3-oxathiolan-5-one-2-carboxylic acid produces a mixture of four lactol diastereomers from which the title compound was isolated after acylation. The isomeric purity and absolute stereochemistry were determined by spectroscopic methods, chiral HPLC techniques, and conversion to (?)-2′-deoxy-3′-thiacytidine (Lamivudine, 3TC^TM). © 1994 Wiley-Liss, Inc.     

The absolute configuration of benproperinium dihydrogen phosphate, an antitussive drug

The (+)- and (-)-enantiomers of benproperinium dihydrogen phosphate, an antitussive drug, have been assigned the R- and S-configurations, respectively, by syntheses of both enantiomers using (S)-2-hydroxypropanoic acid (L-lactic acid) as chiral synthon. The key intermediate, (S)-1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl p-toluenesulfonate, was subjected to an SN2-type reaction with piperidine furnishing (+)-(R)-benproperinium dihydrogen phosphate. (-)-(S)-Benproperinium dihydrogen phosphate was obtained by submitting the same tosylate to two consecutive SN2-type reactions with Br- and piperidine, respectively, acting as nucleophiles.     

Fenugreekine,a new steroidal sapogenin-peptide ester of Trigonella foenum-graecum

A new C₂₇-steroidal sapogenin-peptide ester, fenugreekine, has been isolated from seeds of Trigonella foenum-graecum. On acid hydrolysis, it afforded diosgenin, yamogenin, (25R)-spirosta-3,5-diene, a mixture of three isomeric (2S,3R,4R-, 2S,3R,4S-, 2S,3S,4R-)-4-hydroxyisoleucine lactones, 4′-hydroxyisoleucyl-4-hydroxyisoleucine lactone, and a C₁₄-dipeptide which was partially characterized. On the basis of this chemical transformation and spectral (UV, IR, PMR, MS) evidence of fenugreekine and its transformation products, the steroidal sapogenin-peptide ester is assigned structure (1). The two dipeptides also have not been encountered before in nature or prepared synthetically. The compound shows a number of interesting pharmacological and virological activities.     

根霉3078的代谢产物的研究

从根霉3078菌丝体的甲醇提取物中分离得到9个化合物,通过波谱分析,鉴定为5α,8α-表二氧-(20S,22E,24R)-麦角甾-6,22-二烯-3β-醇(1)、甘油醇-1-单油酸酯(2)、4-羟基苯乙酮(3)、4-羟基苯乙酸(4)、(20S,22E,24R)-麦角甾-7,22-二烯-3β,5α,6β三醇(5)、(S)-3-羟基-3-苯基丙酸(6)、胸腺嘧啶(7)、尿嘧啶(8)和腺苷(9)。     

NMR investigation of the interaction of (+)- and (−)-flurbiprofen with β-cyclodextrin

The sodium salts of (+)-(S)- and (−)-(R)-2-(2-fluoro-4-biphenylyl)propionic acid (flurbiprofen, FBP) form 1:1 inclusion complexes with β-cyclodextrin (β-CD) having different association constants. Proton selective relaxation rate measurements revealed the existence of superior aggregated forms for both complexes (+)-FBP/β-CD and (−)-FBP/β-CD; information about their stereochemistry has been obtained by 2D ROESY analysis. © 1996 Wiley-Liss, Inc.     

Syntheses and β‐adrenergic binding affinities of (R)‐ and (S)‐fluoronaphthyloxypropanolamines

The β‐adrenergic receptors mediate several physiological processes including heart rate (β₁), bronchodilation (β₂), and lipolysis (β₃). Therefore, selectivity is important for a possible therapeutic agent acting via these receptors. Aryloxypropanolamines are β‐receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring in this class could lead to significant biological effects because of the unique chemical characteristics of fluorine. Because the target compound has a chiral center, we set out to synthesize the two enantiomers so that effects of stereochemistry on biological activity could be evaluated. Syntheses of the enantiomers were performed starting with commercially available fluoronaphthalene and subsequent use of the chiral synthon (2R)‐ or (2S)‐glycidyl 3‐nitrobenzenesulfonate, depending on the desired enantiomer. High‐pressure liquid chromatography (HPLC) methods were used to characterize %ee. Each enantiomer was synthesized. They exhibited nanomolar binding activities on β‐adrenergic receptors. The (S)‐enantiomer was found to be up to 310 times more potent than the (R). It was also found to be about five‐fold more selective for β₂‐ than for β₁‐receptors. The current report demonstrates the importance of stereochemistry for the fluoroaromatic β‐receptor ligands. Chirality 11:144–148, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis of new C3 symmetric amino acid‐ and aminoalcohol‐containing chiral stationary phases and application to HPLC enantioseparations

We recently reported a new C3‐symmetric (R)‐phenylglycinol N‐1,3,5‐benzenetricarboxylic acid‐derived chiral high‐performance liquid chromatography (HPLC) stationary phase (CSP 1) that demonstrated better results as compared to a previously described N‐3,5‐dintrobenzoyl (DNB) (R)‐phenylglycinol‐derived CSP. Over a decade ago, (S)‐leucinol, (R)‐phenylglycine, and (S)‐leucine derivatives were used as the starting materials of 3,5‐DNB‐based Pirkle‐type CSPs for chiral separation. In this study, three new C3‐symmetric CSPs (CSP 2, 3, and 4) were prepared by combining the ideas and results mentioned above. Here we describe the synthetic procedures and applications of the new C3‐symmetric CSPs (CSP 2–CSP 4).     

Biodegradation of the chlorophenoxy herbicide (R)-(+)-mecoprop by Alcaligenes denitrificans

An Alcaligenes denitrificans strain capable of utilizing theherbicide (R)-(+)-2(2-methyl-4-chlorophenoxy)propionicacid (mecoprop) as a sole carbon source was isolated fromsoil and cultured in liquid medium. Crude cell extracts of thebacterium were utilized in spectrophotometric assays toelucidate a biochemical pathway for degradation ofmecoprop. Results indicated a reaction sequence analogousto the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D).GC-MS analysis provided direct evidence for thebiotransformation of mecoprop to the transient metabolite4-chloro-2-methylphenol (MCP). No NADPH-dependentactivity was observed during this reaction. Pyruvate wasverified as the second product derived from the aliphatic sidechain of mecoprop. MCP was subsequently transformed to asubstituted catechol by an NADPH-dependentmonooxygenase. When grown on mecoprop, A.denitrificans was adapted to oxidize catechol and its 4- and3-methylated derivatives indicating the broad substratespecificity of catechol dioxygenase. The microorganism wasdemonstrated to adopt the ortho mechanism of aromaticcleavage which resulted in the formation of2-methyl-4-carboxymethylene but-2-en-4-olide, a reactionintermediate of the -ketoadipate pathway.     

(3R)-2-羧乙基-3-氰基-5-甲基己酸乙酯水解酶产生菌的筛选与产酶条件优化

以2-羧乙基-3-氰基-5-甲基己酸乙酯为唯一碳源,采用手性气相法检测,筛选到一株能产对映选择性水解酶的假单胞菌Pseudomonas CGMCC No.4184.该菌产的水解酶能优先水解R型底物产生(3R)-2-羧乙基-3-氰基-5-甲基己酸,产物对映体过量值达到90%以上.对菌株Pseudomonas CGMCC ...     

Racemization and hydrolysis of tropic acid alkaloids in the presence of cyclodextrins

Because of the constantly increasing demand for optically pure drugs it is of great importance to elucidate factors affecting stereochemistry, in order to provide a stable formulation with a high chiral quality of the desired isomer. Therefore, the effects of cyclodextrins (CyDs) and their alkylated and hydroxyalkylated derivatives on racemization and hydrolysis of (?)-(S)-hyoscyamine and (?)-(S)-scopolamine were examined kinetically and spectroscopically (NMR). Direct methods, based on a chiral and achiral chromatographic phase system, were used to determine their degradation products and enantiomer composition during stability tests. All different CyDs, except α-CyD, retarded racemization and hydrolysis. The inclusion of the drug substances in CyDs inhibits the attack of hydroxyl ions and/or water molecules and thus retards the racemization and hydrolysis. The racemization of the tropic acid alkaloids is dependent on the pH and temperature. NMR studies were used to evidence the formation of a soluble 1:1 complex in aqueous solution. © 1993 Wiley-Liss, Inc.     

(1S,3R)-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Induced Increases in Cyclic AMP Formation in the Neonatal Rat Hippocampus Are Mediated by a Synergistic Interaction Between Phosphoinositide- and Inhibitory Cyclic AMP-Coupled mGluRs

Abstract: A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1 S ,3 R )-1-Aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD), which is an agonist for phosphoinositide-coupled and inhibitory-coupled cAMP-linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five- to 10-fold). However, the agonists 3,5-dihydroxyphenylglycine (DHPG) and (2 R ,4 R )-4-aminopyrrolidine-2,4-dicarboxylate (2 R ,4 R -APDC), which selectively act on phosphoinositide-coupled and inhibitory cAMP-coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype-selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1 S ,3 R -ACPD, were found. Stimulations of cAMP content evoked by 1 S ,3 R -ACPD and combined additions of DHPG plus 2 R ,4 R -APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8- p -sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)-α-methyl-4-carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1 S ,3 R -ACPD-induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).     

生物催化3-(4-氯苯基)-戊二腈去对称性水解合成光学纯巴氯芬的关键前体

研究了利用生物催化剂制备(S)-4-氰基-3-(4-氯苯基)-丁酸.以3-(4-氯苯基)-戊二腈为底物,采用苯酚-次氯酸钠法对实验室保藏的菌株进行筛选,得到一株产物立体选择性较高的菌株赤霉菌Gibberella intermedia WX12,并对其催化特性和发酵条件进行了初步研究.以30 g/L的乳糖和20 g/L的蛋白胨分别为碳、氮源,发酵培养96 h,收集的菌体在50 mmol/L磷酸缓冲液(pH 8.0)中30℃催化反应24 h,将3-(4-氯苯基)-戊二腈转化为4-氰基-3-(4-氯苯基)-丁酸,产率为90％.将产物化学转化为巴氯芬,手性HPLC分析表明水解产物构型是(S),其对映异构体过量值ee＞ 99％.该产物可以用来合成光学纯的(R)-和(S)-巴氯芬.