Biocatalytic Approach for the Synthesis of Enantiopure Acebutolol as a β1‐Selective Blocker

A new chemoenzymatic route is reported to synthesize acebutolol, a selective β₁ adrenergic receptor blocking agent in enantiopure (R and S) forms. The enzymatic kinetic resolution strategy was used to synthesize enantiopure intermediates (R)‐ and (S)‐N‐(3‐acetyl‐4‐(3‐chloro‐2‐hydroxypropoxy)phenyl)butyramide from the corresponding racemic alcohols. The results showed that out of eleven commercially available lipase preparations, two enzyme preparations (Lipase A, Candida antarctica, CLEA [CAL CLEA] and Candida rugosa lipase, 62316 [CRL 62316]) act in enantioselective manner. Under optimized conditions the enantiomeric excess of both (R)‐ and (S)‐N‐(3‐acetyl‐4‐(3‐chloro‐2‐hydroxypropoxy)phenyl)butyramide were 99.9 and 96.8%, respectively. N‐alkylation of both the (R) and (S) intermediates with isopropylamine gave enantiomerically pure (R and S)‐ acebutolol with a yield 68 and 72%, respectively. This study suggests a high yielding, easy and environmentally green approach to synthesize enantiopure acebutolol. Chirality 27:382–391, 2015. © 2015 Wiley Periodicals, Inc.     

Bromolactamization: Key Step in the Stereoselective Synthesis of Enantiomerically Pure,cis‐Configured Perhydropyrroloquinoxalines

Compounds based on the pyrroloquinoxaline system can interact with serotonin 5‐HT₃, cannabinoid CB₁, and μ‐opioid receptors. Herein, a chiral pool synthesis of diastereomerically and enantiomerically pure bromolactam (S,R,R,R)‐ 14A is presented. Introduction of the cyclohexenyl ring at the N‐atom of (S)‐proline derivatives 8 or methyl (S)‐pyroglutamate ( 12 ) led to the N‐cyclohexenyl substituted pyrrolidine derivatives 4 and 13 , respectively. All attempts to cyclize the (S)‐proline derivatives 4 with a basic pyrrolidine N‐atom via [3 + 2] cycloaddition, aziridination, or bromolactamization failed. Fast aromatization occurred during treatment of cyclohexenamines under halolactamization conditions. In contrast, reaction of a 1:1 mixture of diastereomeric pyroglutamates (S,R)‐ 13bA and (S,S)‐ 13bB with LiO^tBu and NBS provided the tricyclic bromolactam (S,R,R,R)‐ 14A with high diastereoselectivity from (S,R)‐ 13bA , but did not transform the diastereomer (S,S)‐ 13bB . The different behavior of the diastereomeric pyroglutamates (S,R)‐ 13bA and (S,S)‐ 13bB is explained by different energetically favored conformations. Chirality 26:793–800, 2014. © 2014 Wiley Periodicals, Inc.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

Synthesis and nootropic activity of some 2,3-dihydro-1H-isoindol-1-one derivatives structurally related with piracetam

Three 2,3‐dihydro‐1H‐isoindol‐1‐ones structurally related with piracetam (=2‐oxopyrrolidine‐1‐acetamide) have been synthesized and tested for their nootropic effects in the passive avoidance test in mice. Compounds (RS)‐ 2 , (R,R)‐ 3 , and (R,S)‐ 3 were obtained in good yields in only two steps starting from methyl dl ‐phthaloylalanine. Compound (RS)‐ 2 exhibited nootropic activity at lower doses than piracetam, used as reference drug, but it showed lower efficacy. Whereas diastereoisomers (R,R)‐ 3 and (R,S)‐ 3 were as potent as piracetam to revert amnesia induced by scopolamine, (R,S)‐ 3 showed lower efficacy than (R,R)‐ 3 . Only (R,R)‐ 3 showed myorelaxant effect at doses of 10 and 30 mg/kg; other compounds did not exhibit any anticonvulsant, sedative, myorelaxant, or impaired motor‐coordination effect in mice. These synthesized 2,3‐dihydro‐1H‐isoindol‐1‐one derivatives constitute a new kind of nootropic compounds.     

Amino acids as chiral derivatizing agents for antiproliferative substituted N‐benzyl isoindolinones

The absolute configurations of the diastereomers of novel amino acid ester derivatives of 2,3‐substituted isoindolinones, which are known as apoptosis activators due to their ability to inhibit the MDM2‐p53 PPI, were assigned using NMR and computational methods. Procedures for diastereomer separation and determining the absolute configuration were developed to perform the study. The high significance of N‐benzyl fragment for the determination of the diastereomer absolute configuration by NMR methods was established; it is determined by a number of factors inherent in this fragment and the structural features of the studied substrates. Analysis of the individual isomer activity showed that the target inhibitory effect of S‐ and R‐isoindolinone L‐valinates differs by less than 20%. It can be explained by the presence of a flexible linker between the isoindolinone core and amino acid fragment, which provides the optimal arrangement of the molecule in the hydrophobic cavity of MDM2 for both isomers.     

Supramolecular chirality transfer in a stereodynamic catalysts

We present rhodium catalysts that contain stereodynamic axially chiral biphenol‐derived phosphinite ligands modified with non‐stereoselective amides for non‐covalent interactions. A chirality transfer was achieved with (R)‐ or (S)‐acetylphenylalanine methyl amide, and the interaction mechanism was investigated by NMR measurements. These interactions at the non‐stereoselective interaction sites and the formation of supramolecular complexes result in an enrichment of either the (R_ax)‐ or (S_ax) enantiomer of the tropos catalysts, which in turn provide the (R)‐ or (S)‐acetylphenylalanine methyl ester in the hydrogenation of (Z)‐methyl‐α‐acetamidocinnamate.     

Diastereomeric Resolution of Racemic o‐Chloromandelic Acid

The separation of rac‐o‐chloromandelic acid 1 with enantiopure aryloxypropylamine via diastereomeric salt formation was investigated. (R)‐o‐chloromandelic acid (R)‐ 1 , a key intermediate for the antithrombotic agent clopidogrel, was obtained in 65% yield and 98% ee by Dutch resolution of rac‐ 1 with (S)‐2‐hydroxyl‐3‐(p‐chlorophenoxy) propylamine (S)‐ 5 as resolving agent and (S)‐2‐hydroxyl‐3‐(o‐nitrophenoxy) propylamine (S)‐ 4 as nucleation inhibitor. Chirality 24:1013–1017, 2012. © 2012 Wiley Periodicals, Inc.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

6‐[2‐Phosphonomethoxy)Alkoxy]‐2,4‐Diaminopyrimidines: A New Class of Acyclic Pyrimidine Nucleoside Phosphonates with Antiviral Activity

Acyclic nucleoside phosphonate derivatives containing a pyrimidine base preferably bearing amino groups at C‐2 and C‐4 (DAPym), and linked at the C‐6 position to (S)‐[3‐hydroxy‐2‐(phosphonomethoxy)propoxy] (HPMPO), 2‐(phosphonomethoxy) ethoxy (PMEO) or (R)‐[2‐(phosphonomethoxy)propoxy] (PMPO), display an antiviral sensitivity spectrum that closely mimic that of the parental (S)‐HPMP‐, PME‐ and (R)‐PMP‐purine derivatives. Several PMEO‐DAPym derivatives proved as potent as PMEA (adefovir) and (R)‐PMPA (tenofovir) in inhibiting Moloney murine sarcoma virus (MSV)‐induced tumor formation in newborn NMRI mice. The HPMPO‐, PMEO‐ and PMPO‐DAPym derivatives represent a novel well‐defined subclass among the acyclic nucleoside phosphonates endowed with potent and selective antiviral activity.     

Enantioselective pharmacokinetics of (R)‐ and (S)‐ketamine after a 5‐day infusion in patients with complex regional pain syndrome

Introduction: This study determined the pharmacokinetics and pharmacodynamics of (R)‐ and (S)‐ketamine and (R)‐ and (S)‐norketamine following a 5‐day moderate dose, as a continuous (R,S)‐ketamine infusion in complex regional pain syndrome (CRPS) patients. Materials and methods: Ketamine was titrated to 10–40 mg/h and maintained for 5 days. (R)‐ and (S)‐Ketamine and (R)‐ and (S)‐norketamine pharmacokinetic and pharmacodynamic studies were performed. Blood samples were obtained on Day 1 preinfusion, and at 60–90, 120–150, 180–210, and 240–300 min after the start of the infusion, on Days 2, 3, 4, 5, and on Day 5 at 60 min after the end of infusion. The plasma concentrations of (R)‐ and (S)‐ketamine and (R)‐ and (S)‐norketamine were determined using enantioselective liquid chromatography–mass spectrometry. Results: Ketamine and norketamine levels stabilized 5 h after the start of the infusion. (R)‐Ketamine clearance was significantly lower resulting in higher steady‐state plasma concentrations than (S)‐ketamine. The first‐order elimination for (S)‐norketamine was significantly greater than that of (R)‐enantiomer. When comparing the pharmacokinetic parameters of the patients who responded to ketamine treatment with those who did not, no differences were observed in ketamine clearance and the first‐order elimination of norketamine. Conclusion: The results indicate that (R)‐ and (S)‐ketamine and (R)‐ and (S)‐norketamine plasma concentrations do not explain the antinociceptive activity of the drug in patients suffering from CRPS. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Chiral syntheses of methyl (R)‐2‐Sulfanylcarboxylic esters and acids with optical purity determination using HPLC

Accessible chiral syntheses of 3 types of (R)‐2‐sulfanylcarboxylic esters and acids were performed: (R)‐2‐sulfanylpropanoic (thiolactic) ester (53%, 98%ee) and acid (39%, 96%ee), (R)‐2‐sulfanylsucciinic diester (59%, 96%ee), and (R)‐2‐mandelic ester (78%, 90%ee) and acid (59%, 96%ee). The present practical and robust method involves (i) clean S_N2 displacement of methanesulfonates of (S)‐2‐hydroxyesters by using commercially available AcSK with tris(2‐[2‐methoxyethoxy])ethylamine and (ii) sufficiently mild deacetylation. The optical purity was determined by the corresponding (2R,5R)‐trans‐thiazolidin‐4‐one and (2S,5R)‐cis‐thiazolidin‐4‐one derivatives based on accurate high‐performance liquid chromatography analysis with high‐resolution efficiency. Compared with the reported method utilizing AcSCs (generated from AcSH and CsCO₃), the present method has several advantages, that is, the use of odorless AcCOSK reagent, reasonable reaction velocity, isolation procedure, and accurate, reliable optical purity determination. The use of accessible AcSK has advantages because of easy‐to‐handle odorless and hygroscopic solid that can be used in a bench‐top procedure. The Ti(OiPr)₄ catalyst promoted smooth trans‐cyclo‐condensation to afford (2R,5R)‐trans‐thiazolidin‐4‐one formation of (R)‐2‐sulfanylcarboxylic esters with available N‐(benzylidene)methylamine under neutral conditions without any racemization, whereas (2S,5R)‐cis‐thiazollidin‐4‐ones were obtained via cis‐cyclo‐condensation and no catalysts. Direct high‐performance liquid chromatography analysis of methyl (R)‐mandelate was also performed; however, the resolution efficiency was inferior to that of the thaizolidin‐4‐one derivatizations.     

Enantioselective recognition of carboxylic acids by novel fluorescent triazine‐based thiazoles

Hydrogen bonding and π‐π interactions take special part in the enantioselectivity task. In this regard, because of having both hydrogen acceptor and hydrogen donor groups, melamine derivatives become more of an issue for enantioselectivity. In the light of such information, triazine‐based chiral, fluorescence active novel thiazole derivatives L1 and L2 were designed and synthesized from (S)‐(?)‐2‐amino‐1‐butanol and (1S,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol. The structural establishment of these compounds was made by spectroscopic methods such as FTIR, ¹H, and ¹³C NMR. While the solution of these compounds in DMSO did not show any fluorescence emission, it was observed that the emission increased 44‐fold for L1 and 55‐fold for L2 in 95% water, similar to the aggregation‐induced emission (AIE) characterized compounds. In this regard, enantioselective capabilities of these compounds against carboxylic acids were tested, and in experiments carried out at a ratio of 40/60 DMSO/H₂O, it was determined that R‐2ClMA increased the fluorescence emission of L1 chiral receptor by 2.59 times compared to S‐isomer.     

Absolute configurations and CD spectra of axially chiral biphenyls prepared in a facile manner by crystallization‐induced configuration transformation

Axially chiral biphenyls such as (M,S)‐ 3k have been conveniently obtained by crystallization of their diastereomeric mixtures, which were synthesized from racemic 4,4′‐dimethoxy‐5,6,5′,6′‐bis(methylenedioxy)‐2‐carboxylester‐2′‐carboxyl‐biphenyls 4 and chiral amino alcohols (R)‐alaninol, (S)‐alaninol, (S)‐valinol, and (S)‐phenylalaninol. A crystallization‐induced configuration transformation of the biphenyls was thus achieved. It was found that amide formation of an (S)‐valinol or (S)‐phenylalaninol at the 2′‐position of the biphenyl usually induced the deposition of crystals with the (M)‐configuration from ethanol in yields higher than 50%. The absolute configurations (ACs) of two crystalline biphenyls have been determined by X‐ray crystallographic analysis. The ACs of nine biphenyls have been assigned based on their CD spectra. Further, stability investigation of these axially chiral biphenyls revealed that the ACs could revert upon redissolution. The energy barrier to epimerization between (P,R)‐ 3b and (M,R)‐ 3b was measured as ΔG^# = 21.45 kcal/mol and the half‐life in ethanol at 301 K was 17.1 h. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Enantioselective induction of cyclophosphamide metabolism by phenytoin

The objective of this study was to investigate the effect of phenytoin (PHE) on cyclophosphamide (CP) disposition. CP was administered to 6 adult patients in a preparative regimen for bone marrow transplantation consisting of busulfan and CP. Three of the patients received PHE and the other 3 “control” patients received diazepam (DZP) as anti‐epileptic prophylactic treatment. Plasma samples were collected at intervals up to 24 h after CP administration. The plasma concentrations of (R)‐ and (S)‐CP and their respective N‐dechloroethylated metabolites, (R)‐ and (S)‐DCE‐CP were simultaneously fitted using an enantiospecific 2‐compartment pharmacokinetic (PK) model with Bayesian control estimation. DZP had no significant effect on the metabolism of CP and any of its PK parameters. PHE, however, increased significantly the formation of (S)‐DCE‐CP while having no effect on the formation of (R)‐DCE‐CP. These results suggest that different enzymes are responsible for the formation of (S)‐DCE‐CP from (S)‐CP and (R)‐DCE‐CP from (R)‐CP. Additionally, assuming that PHE does not affect the passive renal elimination of (R)‐ and (S)‐CP, this analysis suggests that the clearance of both (R)‐ and (S)‐CP to 4‐hydroxy‐CP (the activation pathway) is increased by PHE. Chirality 11:569–574, 1999. © 1999 Wiley‐Liss, Inc.     

Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step

A thermally stable esterase (SNSM‐87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)‐2‐hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure–enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)‐2‐chloromandelates as the model substrates, an expanded Michaelis–Menten mechanism for the rate‐limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast‐reacting (S)‐2‐chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow‐reacting (R)‐2‐chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl‐enzyme intermediates is rate‐limiting. However, the rate‐limiting step shifts to the formation of tetrahedral intermediates for the (S)‐2‐chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate. Biotechnol. Bioeng. 2007; 98: 30–38. © 2007 Wiley Periodicals, Inc.     

A Simple Method for Resolution of Endo‐/Exo‐Monoesters of Trans‐Norborn‐5‐Ene‐2,3‐Dicarboxylic Acids Into Their Enantiomers

Separation of optical isomers obtainable from trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid methyl and tert‐butyl monoesters was performed by crystallization of the respective salts prepared with (R)‐ and (S)‐1‐phenylethylamine. Starting from racemic endo‐monomethyl ester of trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid, prepared by partial hydrolysis of the cyclopentadiene‐dimethyl fumarate adduct, the corresponding (2R,3R)‐endo‐monoester was isolated in 97% enantiomeric excess (ee) yield after seven repeated crystallizations from tetrachloromethane. Starting from exo‐mono‐tert‐butyl ester of the same acid, prepared by alcoholysis of the cyclopentadiene‐maleic anhydride adduct followed by isomerization, (2R,3R)‐exo‐monoester was isolated in >98% ee yield after four repeated crystallizations from ethanol. Crystallization of the acids from the mother liquor containing (S)‐1‐phenylethylamine yielded products with inverse stereochemical configuration. Chirality 27:151–155, 2015. © 2014 Wiley Periodicals, Inc.     

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2‐bromo phenyl acetic acid esters

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S‐enantioselectivity (E‐value = 5) during the hydrolytic kinetic resolution of 2‐bromo‐phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R‐selective enzyme (E‐value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low‐hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site‐directed mutagenesis. This work led to the isolation of one double substituted variant (D97A‐V232F), which displays a total preference for the R‐enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5‐fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R‐ and S‐enantiomers in the wild‐type enzyme and the D97A‐V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R‐enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity. Biotechnol. Bioeng. 2010;106: 852–859. © 2010 Wiley Periodicals, Inc.     

Enantioselective synthesis and teratogenicity of propylisopropyl acetamide,a CNS‐active chiral amide analogue of valproic acid

Propylisopropyl acetamide (PID), an amide analogue of the major antiepileptic drug valproic acid (VPA), possesses favorable anticonvulsant and CNS properties. PID contains one chiral carbon atom and therefore exists in two enantiomeric forms. The purpose of this work was to synthesize the two PID enantiomers and evaluate their enantiospecific teratogenicity. Enantioselective synthesis of PID enantiomers was achieved by coupling valeroyl chloride with optically pure (4S)‐ and (4R)‐benzyl‐2‐oxazolidinone chiral auxiliaries. The two oxazolidinone enolates were alkylated with isopropyl triflate, hydrolyzed, and amidated to yield (2R)‐ and (2S)‐PID. These two PID enantiomers were obtained with excellent enantiomeric purity, exceeding 99.4%. Unlike VPA, both (2R)‐ and (2S)‐PID failed to exert teratogenic effects in NMRI mice following a single 3 mmol/kg subcutaneous injection. From this study we can conclude that individual PID enantiomers do not demonstrate stereoselective teratogenicity in NMRI mice. Due to its better anticonvulsant activity than VPA and lack of teratogenicity, PID (in a stereospecific or racemic form) has the potential to become a new antiepileptic and CNS drug. Chirality 11:645–650, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis of enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate and endo‐2‐norbornyl‐N‐n‐butylcarbamate for stereoselective inhibition of acetylcholinesterase

The acetylcholinesterase inhibition by enantiomers of exo‐ and endo‐2‐norbornyl‐N‐n‐butylcarbamates shows high stereoselelectivity. For the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐exo‐2‐norbornyl‐N‐n‐butylcarbamates, the R‐enantiomer is more potent than the S‐enantiomer. But, for the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates, the S‐enantiomer is more potent than the R‐enantiomer. Optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates are synthesized from condensations of optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norborneols with n‐butyl isocyanate, respectively. Optically pure norborneols are obtained from kinetic resolutions of their racemic esters by lipase catalysis in organic solvent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.