A fluorescent chiral chemosensor for the recognition of the two enantiomers of chiral carboxylates

A new 7-nitrobenz-2-oxa-1,3-diazole (NBD)-based fluorescent chiral chemosensor (NBD-1) was prepared and applied to the recognition of the two enantiomers of the tetrabutylammonium salts of N-t-Boc-α-amino acids and chiral carboxylic acids including naproxen. In particular, the chiral recognition by the new fluorescent chiral chemosensor for the two enantiomers of N-t-Boc-threonine (tetrabutylammonium salt) was quite excellent, the Stern-Volmer constant ratio (K(D)/K(L)) for the two enantiomers being as high as 4.89.     

人羧酸酯酶1结构和功能的研究进展

人羧酸酯酶1(Human carboxylesterase 1,HCE1)是丝氨酸水解酶多基因家族的重要成员之一,它是一种参与肝脏外源物质解毒及代谢的肝羧酸酯酶。HCE1还能参与人体内胆固醇酯和游离脂肪酸的运输和代谢过程,与肝细胞肝癌的发生发展密切相关。文中就近十年来人羧酸酯酶1的分子结构、药物代谢、毒物代谢、脂质代谢及早期诊断肝细胞肝癌等功能方面的相关研究进展进行综述。     

Geometric specificity of porcine liver carboxylesterase and its application for the production of cis-arylacrylic esters

Porcine liver carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) hydrolyses trans isomers of three different methyl 3-arylacrylates approximately one order of magnitude faster than the corresponding cis isomers. This phenomenon can be used for preparative production of cis esters from their trans counterparts as exemplified by methyl cinnamate. A solution of commercial, predominantly trans methyl cinnamate was irradiated by ultraviolet light and the resultant mixture of trans and cis esters was passed through a column packed with immobilized esterase. The effluent contained mainly trans cinnamic acid and cis methyl cinnamate. The latter was then extracted with methylene chloride, and the cis ester was isolated by evaporating the solvent. By esterifying the co-produced trans acid, the process can be made continuous.     

Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines

Substrate specificity of the omega-aminotransferase obtained from Vibrio fluvialis (omega-ATVf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. omega-ATVf showed unique substrate specificity toward aromatic amines with a high enantioselectivity (E > 100) for (S)-enantiomers. However, the substrate specificity of this enzyme was much narrower toward aliphatic amines. To overcome the narrow substrate specificity toward aliphatic amines, we redesigned the substrate specificity of omega-ATVf using homology modeling and the substrate structure- activity relationship. The homology model and the substrate structure-activity relationship showed that the active site of omega-ATVf consists of one large substrate-binding site and another small substrate-binding site. The key determinant in the small substrate-binding site was D25, whose role was expected to mask R415 and to generate the electrostatic repulsion with the substrate's alpha-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the alpha-carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase subgroup II enzymes. Among the several amino acid residues in the large substrate-binding site, W57 and W147, with their bulky side chains, were expected to restrict the recognition of aliphatic amines. Two mutant enzymes, W57G and W147G, showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity.     

Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.     

NMR studies of chiral recognition by cyclodextrins

Chiral recognition by cyclodextrins is of considerable importance, especially for pharmaceutical industry, in view of the possible side effects of the second enantiometer of chiral drugs. In general, it manifests itself in all NMR parameters (chemical shifts, coupling constants, NOE and ROE effects, and relaxation rates) on one hand. On the other hand, it allows one to determine the thermodynamic parameters characterizing diastereomeric complexes formed by cyclodextrins with enantiomeric guests. After an introduction and a general discussion of NMR manifestations of chiral recognition by cyclodextrin, the existing literature data on this problem will be discussed herein. Chirality 16:90-105, 2004.     

Probing the substrate recognition mechanism of the human MTH1 protein by nucleotide analogs

To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.     

Discovery of triterpenoids as potent dual inhibitors of pancreatic lipase and human carboxylesterase 1

Pancreatic lipase (PL) is a well-known key target for the prevention and treatment of obesity. Human carboxylesterase 1A (hCES1A) has become an important target for the treatment of hyperlipidaemia. Thus, the discovery of potent dual-target inhibitors based on PL and hCES1A hold great potential for the development of remedies for treating related metabolic diseases. In this study, a series of natural triterpenoids were collected and the inhibitory effects of these triterpenoids on PL and hCES1A were determined using fluorescence-based biochemical assays. It was found that oleanolic acid (OA) and ursolic acid (UA) have the excellent inhibitory effects against PL and hCES1A, and highly selectivity over hCES2A. Subsequently, a number of compounds based on the OA and UA skeletons were synthesised and evaluated. Structure–activity relationship (SAR) analysis of these compounds revealed that the acetyl group at the C-3 site of UA (compound 41) was very essential for both PL and hCES1A inhibition, with IC₅₀ of 0.75 µM and 0.014 µM, respectively. In addition, compound 39 with 2-enol and 3-ketal moiety of OA also has strong inhibitory effects against both PL and hCES1A, with IC₅₀ of 2.13 µM and 0.055 µM, respectively. Furthermore, compound 39 and 41 exhibited good selectivity over other human serine hydrolases including hCES2A, butyrylcholinesterase (BChE) and dipeptidyl peptidase IV (DPP-IV). Inhibitory kinetics and molecular docking studies demonstrated that both compounds 39 and 41 were effective mixed inhibitors of PL, while competitive inhibitors of hCES1A. Further investigations demonstrated that both compounds 39 and 41 could inhibit adipocyte adipogenesis induced by mouse preadipocytes. Collectively, we found two triterpenoid derivatives with strong inhibitory ability on both PL and hCES1A, which can be served as promising lead compounds for the development of more potent dual-target inhibitors targeting on PL and hCES1A.     

Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the V_max value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.     

Synthesis and characterization of novel chiral ionic liquids and investigation of their enantiomeric recognition properties

We report the synthesis and characterization of amino acid ester based chiral ionic liquids, derived from L- and D-alanine tert butyl ester chloride. The synthesis was accomplished via an anion metathesis reaction between commercially available L- and D-alanine tert butyl ester chloride using a variety of counterions such as lithium bis (trifluoromethane) sulfonimide, silver nitrate, silver lactate, and silver tetrafluoroborate. Both enantiomeric forms were obtained as confirmed by bands of opposite sign in the circular dichroism spectra. The L- and D-alanine tert butyl ester bis (trifluoromethane) sulfonimide were obtained as liquids at room temperature and intriguingly exhibited the highest thermal stability (up to 263 degrees C). In addition, the ionic liquids demonstrated enantiomeric recognition ability as evidenced by splitting of racemic Mosher's sodium salt signal using a liquid state (19)F nuclear magnetic resonance (NMR) and fluorescence spectroscopy. The L- and D-alanine tert butyl ester chloride resulted in solid salts with nitrate, lactate, and tetrafluoroborate anions. This illustrates the previously observed tunability of ionic liquid synthesis, resulting in ionic liquids of varying properties as a function of varying the anion.     

The development of biphasic chiral recognition in chiral separation

In chiral separation, enantioseparation factor is an important parameter which influences the resolution of enantiomers. In this current overview, a biphasic chiral recognition method is introduced to the readers. This method can significantly improve the enantioseparation factor in two‐phase solvent through adding lipophilic and hydrophilic chiral selectors which have opposite chiral recognition ability to organic and aqueous phases, respectively. This overview presents the development and applications of biphasic chiral recognition in liquid‐liquid extraction and counter current chromatography. It mainly focuses on the topics of mechanism, advantages and limitations, applications, and key factors of biphasic chiral recognition. In addition, the future outlook on development of biphasic chiral recognition also has been discussed in this overview.     

Steric hindrance influence on the enantiorecognition ability of tyrosine-derived chiral stationary phases

Four chiral stationary phases (CSPs) derived from N-(3,5-dinitrobenzoyl)tyrosine have been synthesized. They differ by the substituent nature (methyl, ethyl, isopropyl, tert-butyl) of the aliphatic amide function. The enantiorecognition ability of these CSPs was evaluated with 10 racemates. For the majority of them, the stereoselectivity increases with the steric hindrance of the substituent. The chiral selector enantiomeric separation on the resulting CSPs has evidenced a reversal of elution order only for CS 4 on CSP 4 (tert-butyl substituent), suggesting a change in its conformation.     

手性源、手性池和手性农药中间体的开发研究

介绍手性源、手性池和手性分子化合物的基本概念;由手性池化合物制备手性衍生物;比较了手性化合物生物加工与化学加工过程的优、缺点,寻求高效、经济和最合理的综合工艺流程。     

Enantiomeric recognition of chiral 3,3-bridged-1,1'-binaphthol dimer toward alpha-phenylethylamine and alpha-amino acid ester

The 1,1'-binaphthol-based dimers with p-phenylenebis(2-ethynyl) spacer, (+)-6 and (+)-2, were synthesized as chiral host compounds. (1)H NMR, UV-vis, and fluorescent titration were used to evaluate the enantiomeric recognition abilities of the chiral host dimers toward the guest amine 7 and alpha-amino acid ester 8. The chiral BINOL-based dimers were found to have good enantiomeric recognition ability. The computer simulation of the host-guest complex molecules was carried out to describe the conformational changes of both naphthyl ring in the molecule of chiral host dimer after complexation with the guest molecule.     

Effect of constituting amino acid residue numbers on molecularly imprinted chiral recognition sites

In the present study, the effect of constituting amino acid residue numbers of oligopeptide derivatives, which are candidate materials to construct molecular recognition sites, on chiral recognition ability was investigated. Chiral recognition sites were formed from oligopeptide derivatives, of which constituting amino acid residue numbers were three to six, by adopting an alternative molecular imprinting. It was made clear that the number four, in other words, the tetrapeptide derivative, is the best candidate material to form a chiral recognition site.     

Enantioseparation of chiral pharmaceuticals by vancomycin‐bonded stationary phase and analysis of chiral recognition mechanism

The drug chirality is attracting increasing attention because of different biological activities, metabolic pathways, and toxicities of chiral enantiomers. The chiral separation has been a great challenge. Optimized high‐performance liquid chromatography (HPLC) methods based on vancomycin chiral stationary phase (CSP) were developed for the enantioseparation of propranolol, atenolol, metoprolol, venlafaxine, fluoxetine, and amlodipine. The retention and enantioseparation properties of these analytes were investigated in the variety of mobile phase additives, flow rate, and column temperature. As a result, the optimal chromatographic condition was achieved using methanol as a main mobile phase with triethylamine (TEA) and glacial acetic acid (HOAc) added as modifiers in a volume ratio of 0.01% at a flow rate of 0.3 mL/minute and at a column temperature of 5°C. The thermodynamic parameters (eg, ΔH, ΔΔH, and ΔΔS) from linear van 't Hoff plots revealed that the retention of investigated pharmaceuticals on vancomycin CSP was an exothermic process. The nonlinear behavior of lnk′ against 1/T for propranolol, atenolol, and metoprolol suggested the presence of multiple binding mechanisms for these analytes on CSP with variation of temperature. The simulated interaction processes between vancomycin and pharmaceutical enantiomers using molecular docking technique and binding energy calculations indicated that the calculated magnitudes of steady combination energy (ΔG) coincided with experimental elution order for most of these enantiomers.     

Structural requirements for the inhibition of human monocyte carboxylesterase by organophosphorus compounds

Human blood monocyte carboxylesterase (CBE) is inhibited by a variety of organophosphorus compounds including arylphosphates and arylphosphites and some alkylphosphites. Triphenyl phosphate and triphenyl phosphite with K_i values of 8 × 10⁻⁹ M and 4.8 × 10⁻⁸ M, respectively, are the most potent inhibitors of this enzyme evaluated by this study. The arylphosphates vary in their capacity to inhibit carboxylesterase activity. Diphenyl phosphate with its strong negative charge is not a potent inhibitor (K_i = 1 × 10⁻⁴ M), whereas if its negative charge is neutralized, as in diphenyl methyl phosphate, its capacity to inhibit carboxylesterase is significantly increased. Compounds with increased bulk, such as trinaphthyl phosphate, only inhibit the enzyme at concentrations of 10⁻⁵ M or greater. Arylphosphites have inhibitory capacities similar to the arylphosphates. Alkylphosphites (tributyl phosphite/triethyl phosphite) inhibit carboxylesterase activity, whereas alkylphosphates (tributyl phosphate/triethyl phosphate) have no inhibitory effect. Arylphosphines and arylphosphine oxides do not inhibit carboxylesterase activity. This study demonstrates that organophosphates and organophosphites are relatively effective inhibitors of human monocyte CBE activity with the exception of the alkylphosphates which have no inhibitory activity. We conclude that molecular bulk and charge have a significant role in determining the potency of organophosphorus inhibitors of monocyte CBE. The observed variations in the degree of esterase inhibition by organophosphorus compounds as well as the differences in the pathological expression of neuropathic disorders associated with such chemicals suggest that different esterase enzymes derived from the family of esterase genes may mediate the different neuropathies observed with organophosphorus exposures. Such data also provide the rationale for the kinetic analyses of esterases and the design of non-toxic organophosphorus compounds with low or no monocyte CBE inhibitory capacity to reduce the potential of these commonly used chemicals for human toxicity.     

Enantioselective separation of chiral arylcarboxylic acids on an immobilized human serum albumin chiral stationary phase

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II. Chirality 9:178–183, 1997. © 1997 Wiley-Liss, Inc.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part II. Optical resolution of racemic (thio)glycidyl esters on modified cellulose chiral stationary phases

Chiral chromatography on cellulose tris(3,5-dimethylphenyl carbamate) (Chiralcel OD) and cellulose tribenzoate (Chiralcel OB) coated stationary phases has been successfully used for the optical resolution of rac-(thio)glycidyl esters (acetate, propionate, butyrate). Glycidyl esters could sufficiently be resolved on the OD column whereas for the thio analogues baseline resolution is obtained on CSP OB using hexane/2-propanol mobile phases. The separation factor (α) and resolution (R_S) depend on column temperature, eluent composition, and flow rate, respectively. Best results were obtained for the butyrates and at low temperatures in general. © 1993 Wiley-Liss, Inc.     

Enantiomeric separation and recognition mechanism of 2-arylpropionic acid derivatives by high-performance liquid chromatography using a chiral column

An optical resolution of the amide derivatives of ibuprofen and the carbamate-alkylester derivatives of the trans-alcohol metabolite of loxoprofen and an analogous compound, CS-670, was studied by chiral high-performance liquid chromatography (HPLC). The chiral columns SUMIPAX OA-4000 and OA-4100 were used to investigate the enantiomeric separation behavior of these derivatives using both reversed and normal mobile phases. A better separation factor (α) of the amide and the carbamate ester derivatives was obtained in the normal mobile phase than in the reversed mobile phase HPLC. In addition, the recognition mechanisms of both amide and carbamate ester enantiomers were investigated by ¹H-nuclear magnetic resonance (NMR). It is suggested that the important driving forces for the enantiomeric separation are the formation of hydrogen bonding and the charge transfer complex between these derivatives and an active site of the chiral stationary phase. © 1995 Wiley-Liss, Inc.