Pyrrole‐Assisted and Easy Oxidation of Cyclic α‐Amino Acid‐ Derived Diketopiperazines under Mild Conditions

A new procedure for the aerobic oxidation of α‐amino acids acylated by pyrrole‐carboxylic acid with triplet dioxygen is introduced. The reaction is general for a variety of pyrrole‐amino acid derivatives and represents a very practical and controllable method for the selective preparation of α‐hydroperoxy‐ or α‐hydroxy‐α‐amino acid diketopiperazines with molecular dioxygen. Furthermore, the non‐catalyzed direct oxidation of amino acid derivatives at the α‐position with molecular dioxygen represents a fundamental question.     

Chemoenzymatic synthesis of chiral carboxylic acids via nitriles

BACKGROUND: Enantiomerically pure 1,4‐benzodioxane‐2‐carboxylic acid derivatives are useful building blocks for the synthesis of pharmaceuticals and biologically active compounds whose interaction with their biological target (enzyme, receptor) depends very much on the absolute configuration of the chiral carbon at the 2‐position. The aim of the present work is to investigate the route to racemic nitriles and the subsequent selective enzymatic hydrolysis by nitrilase to optically active 1,4‐benzodioxane‐2‐carboxylic acid and 6‐formyl‐1,4‐benzodioxane‐2‐carboxylic acid. RESULTS: A range of microbial nitrilases from Rhodococcus, Alcaligenes and Pseudomonas strains have been prepared and screened for the desired biotransformations using a chiral high performance liquid chromatography (HPLC) analytical method. The nitrilase from Alcaligenes faecalis ATCC 8750 showed the highest and the nitrilase from Rhodococcus rhodochrous NCIMB 11216 the lowest activity towards 2‐cyano‐6‐formyl‐1,4‐benzodioxane. Lyophilised cells of Rhodococcus R 312 gave the (R)‐1,4‐benzodioxane‐2‐carboxylic acid with high enantioselectivity after 25% conversion. Excellent enantioselectivities for the hydrolysis of both 2‐cyano‐1,4‐benzodioxane as well as 2‐cyano‐6‐formyl‐1,4‐benzodioxane have been achieved and the absolute configuration of 1,4‐benzodioxane‐2‐carboxylic acid was determined to be R by comparison with the specific rotation of commercially available (R)‐1,4‐benzodioxane‐2‐carboxylic acid. CONCLUSIONS: This new nitrilase‐catalysed kinetic resolution of 2‐cyano‐ and 2‐cyano‐6‐formyl‐1,4‐benzodioxane opens a mild route to optically active 1,4‐benzodioxane‐2‐carboxylic acids. As the formyl functional group would be damaged in chemical nitrile hydrolysis, nitrilase‐catalysed hydrolysis solves this synthetic bottleneck and advances nitrilase biocatalytic tools for the preparation of more complex 1,4‐benzodioxane‐2‐carboxylic acids. Copyright © 2007 Society of Chemical Industry     

Catalytic Asymmetric Synthesis of Functionalized α,α‐Disubstituted α‐Amino Acid Derivatives from Racemic Unprotected α‐Amino Acids via in‐situ Generated Azlactones

Masked and activated highly enantioenriched α,α‐disubstituted α‐amino acids with an additional adjacent stereocenter were formed by a tandem reaction involving five steps using racemic unprotected amino acid substrates. Key step is the 1,4‐addition of in‐situ generated azlactones to a broad number of enones. The products of this step‐economic route can, e.g., be useful for a divergent and rapid access to biologically interesting unnatural glutamic acid derivatives.     

光学纯氨基酸的研究进展

氨基酸能参与多种多样的生命活动，此外它还是合成多种药物的重要中间体。大多数氨基酸都有一个或一个以上的手性中心，手性不同其生物活性也不同。因为生命活动对不同的氨基酸会有立体选择性，所以光学纯氨基酸在制药与食品行业有着非常重要的应用。本文综述了近些年国内外光学纯氨基酸的研究进展。     

A highly stable whole‐cell biocatalyst for the enantioselective synthesis of optically active alpha‐hydroxy acids

BACKGROUND: Biocatalysts have gained increasing attention because of their inherent advantages over chemical catalysts. However, the poor operational stability has always prevented their broad application. In this study, (R)‐mandelic acid was chosen as a model compound of alpha‐hydroxy acids. The objective was to obtain a new biocatalyst with desired operational stability for the preparation of (R)‐mandelic acid as well as other optically pure alpha‐hydroxy acids of pharmaceutical importance. RESULTS: Using a two‐step screening strategy, Saccharomyces ellipsoideus GIM2.105 was selected as an effective biocatalyst with high enantioselectivity and remarkable operational stability. After 20 cycles of reuse, whole cells of S. ellipsoideus GIM2.105 maintained its activity, and no obvious decrease in conversion or enantiomeric excess (ee) was observed. Furthermore, effects of various reaction parameters, including pH, temperature, co‐substrate (type, concentration), substrate concentration and reaction time, on the bioreduction were studied. Under optimal conditions, (R)‐mandelic acid and four substituted aromatic (R)‐alpha‐hydroxy acids were prepared in high ee (95–>99%) and good conversion (>90%). CONCLUSION: The high enantioselectivity, remarkable operational stability and mild reaction conditions showed S. ellipsoideus GIM2.105 to be an economical biocatalyst with great industrial application potential for the production of optically active alpha‐hydroxy acids. Copyright © 2009 Society of Chemical Industry     

Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

2,2-二甲基环丙烷甲酸的合成与拆分

S-( )-2,2-二甲基环丙烷甲酸是合成西司他丁(一种肾脱氢二肽酶抑制剂)的关键中间体,今设计了一条新的2,2-二甲基环丙烷甲酸合成路线并改进了拆分工艺,它是以异戊烯酸为原料,经酸的酯化、烯键的环丙烷化、酯水解制得2,2-二甲基环丙烷甲酸,收率为44．1％。其中,环丙烷化反应用锌粉／氯化亚铜-乙酰氯作为催化剂,二溴甲烷作为环丙烷化试剂,这样可以在温和的条件下进行反应,降低成本。此外用L-肉碱草酸盐作为手性拆分试剂,经酰化、成盐、部分结晶、水解得到S-( )-2,2-二甲基环丙烷甲酸．收率为16．7％,手性纯度大于95％．此路线工艺简单、环境友好、成本较低,易于工业化。     

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short‐chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C₂ to C₆. In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole‐cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane‐1,2‐diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)‐1,2‐PDO (1.0 % yield) or 9.6 mm (S)‐1,2‐PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.     

Synthesis of Chiral Amino Acid Anilides by Ligand‐Free Copper‐Catalyzed Selective N‐Arylation of Amino Acid Amides

An atom‐economic, practical and cost‐effective protocol for synthesis of chiral amino acid anilides via ligand‐free copper‐catalyzed selective C N cross coupling of chiral amino acid amides and aryl halides, hetereoaryl halides and a vinyl bromide has been developed. No racemization occurred during the C N coupling. A plausible mechanism is proposed.     

Palladium‐Catalyzed Cyclization Reactions of Acetylene‐Containing Amino Acids

Enantiomerically pure acetylene‐containing α‐amino acids were used as versatile starting materials for the synthesis of a variety of heterocycles via Pd‐mediated cyclization reactions. Depending on the protecting group strategy, both the carboxylate and the amine function of the amino acids could participate in the cyclizations, thus giving rise to oxygen heterocycles (α‐aminolactones) and nitrogen heterocycles (cyclic α‐amino acid derivatives), respectively. Beside the straightforward cyclization, cyclization/cross‐coupling reactions were also successfully carried out to provide the corresponding substituted cyclic amino acid derivatives.     

Influence of Substrate Structure on PGA‐Catalyzed Acylations. Evaluation of Different Approaches for the Enzymatic Synthesis of Cefonicid

The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β‐lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (v_s) and the rate of hydrolysis of the acylating ester (v_h1). 7‐Aminocephalosporanic acid (7‐ACA) and 7‐amino‐3‐(1‐sulfomethyl‐1,2,3,4‐tetrazol‐5‐yl)thiomethyl‐3‐cephem‐4‐carboxylic acid (7‐SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R‐methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7‐SACA. Cefonicid ( 8 ) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7‐ACA, a “one‐pot” chemo‐enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D ‐amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.     

Surface modification of ethylene‐co‐acrylic acid copolymer films: Addition of amide groups by covalently bonded amino acid intermediates

Amide groups were anchored covalently on the surface of ethylene‐co‐acrylic acid (EAA) copolymer film by surface grafting of amino acid intermediates. The process consisted of four steps: conversion of carboxylic acid groups on the EAA surface to acid chloride groups, amino acid attachment, conversion of amino acid carboxyl groups to acid chloride groups, and amidation. All steps were carried out at room temperature. ATR‐FTIR spectroscopy was used to characterize the film after each step and to measure the kinetics of amino acid attachment. Three amino acids were studied: 12‐aminododecanoic acid (12‐ADDA), 5‐aminophthalic acid (5‐APA), and L ‐aspartic acid (AA). The longer‐chain 12‐ADDA compound was selected for its chemical similarity to migratory fatty amides that are commonly used to alter the frictional behavior of polyolefin films. The 5‐APA and AA compounds were selected because each has two carboxylic acid groups that can be converted to amide groups. After amidation, the modified EAA films were characterized by static water contact angle measurements and scanning probe microscopy. Results showed that the 12‐ADDA reacted to the surface much faster than the 5‐APA or AA. Several steps of aggressive rinsing confirmed that the 12‐aminododecanamide was chemically anchored onto the EAA surface. As a result, both hydrophilicity and surface roughness were increased. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 1688–1694, 2004     

Kinetic Resolution of Aliphatic β‐Amino Acid Amides by β‐Aminopeptidases

Access to enantiopure β‐amino acids : β‐Aminopeptidases are hydrolases that possess the unique ability to cleave N‐terminal β‐amino acids from peptides and amides. Hydrolysis of racemic β‐amino acid amides catalyzed by these enzymes displays enantioselectivity with strong preference for substrates with the L ‐configuration, and gives access to various aliphatic β‐amino acids of high enantiopurity.

     

Active‐Site Engineering Expands the Substrate Profile of the Basidiomycete l‐Tryptophan Decarboxylase CsTDC

Aromatic l ‐amino acid decarboxylases (AADCs) catalyze the release of CO₂ from proteinogenic and non‐proteinogenic l ‐amino acid substrates and are involved in pathways that biosynthesize neurotransmitters or bioactive natural products. In contrast to AADCs from animals and plants, fungal AADCs have received very little attention. Here, we report on the in vitro characterization of heterologously produced Ceriporiopsis subvermispora AADC, now referred to as CsTDC, which is the first characterized basidiomycete AADC. This study identified the enzyme as a decarboxylase that is strictly specific for l ‐tryptophan and 5‐hydroxy‐l ‐tryptophan. The tdc gene was subjected to saturation mutagenesis so as to vary the key active site residue, Gly351. Aliphatic amino acid residues, l ‐serine, or l ‐threonine at position 351 added l ‐tyrosine and 3,4‐dihydroxy‐l ‐phenylalanine (l ‐DOPA) decarboxylase activity while retaining stereospecificity and l ‐tryptophan decarboxylase activity.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Combination of a hydroxy‐functional organophosphorus oligomer and a multifunctional carboxylic acid as a flame retardant finishing system for cotton: Part II. Formation of calcium salt during laundering

Multifunctional carboxylic acids, such as 1,2,3,4‐butanetetracarboxylic acid (BTCA), were used to bond a hydroxy‐functional organophosphorus oligomer (FR) to cotton fabric in the presence of a catalyst, such as sodium hypophosphite (NaH₂PO₂). Previously, it was found that the cotton fabric treated with FR and BTCA showed a high level of phosphorus retention after one home laundering cycle. However, the flame retardant properties quickly deteriorated as the number of home laundering cycles was increased. In this research, it was found that the free carboxylic acid groups bound to the cotton fabric form an insoluble calcium salt during home laundering, thus diminishing the flame retardant properties of the treated cotton fabric. It was also found that the free carboxylic acid groups on the treated cotton fabric were esterified by triethanolamine (TEA), and that the formation of calcium salt on the fabric was suppressed by the esterification of the free carboxylic acid groups by TEA. The cotton fabric treated with BTCA and the hydroxy‐functional organophosphorus oligomer significantly improved its flame retardance when a new catalyst system consisting of hypophosphorous acid (H₃PO₂) and TEA was used in the system. Copyright © 2003 John Wiley & Sons, Ltd.     

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.     

Rhodium‐Catalyzed Asymmetric Hydroformylation of 1,1‐Disubstituted Allylphthalimides: A Catalytic Route to β3‐Amino Acids

The high enantioselective rhodium‐catalyzed hydroformylation of 1,1‐disubstituted allylphthalimides has been developed. By employing chiral ligand 1,2‐bis[(2S,5S)‐2,5‐diphenylphospholano]ethane [(S,S)‐Ph‐BPE], a series of β³‐aminoaldehydes can be prepared with up to 95% enantioselectivity. This asymmetric procedure provides an efficient alternative route to prepare chiral β³‐amino acids and alcohols.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

Combination of a hydroxy‐functional organophosphorus oligomer and a multifunctional carboxylic acid as a flame retardant finishing system for cotton: Part I. The chemical reactions

Multifunctional carboxylic acids, such as 1,2,3,4‐butanetetracarboxylic acid (BTCA), have been used as crosslinking agents for cotton cellulose to produce wrinkle‐resistant cotton fabrics and garments. Polycarboxylic acids were used to bond hydroxy‐functional organophosphorus oligomer to cotton, thus imparting durable flame retarding properties to the cotton fabric. This research investigated the chemical reactions between the hydroxy‐functional organophosphorus compound and BTCA on cotton. BTCA crosslinks cotton cellulose through the formation of a 5‐membered cyclic anhydride intermediate and esterification of the anhydride with cellulose. In the presence of the organophosphorus compound, BTCA reacts with both the organophosphorus compound and cellulose, thus functioning as a binder between cotton cellulose and the organophosphorus compound and making the flame retarding system durable to laundering. The cotton fabric treated by the combination of the organophosphorus compound and BTCA demonstrated lower wrinkle resistance and less tensile strength loss than that treated by BTCA alone. The phosphorus retention on the cotton fabric after one home laundering cycle was approximately 70%. Copyright © 2003 John Wiley & Sons, Ltd.