An Aminocaprolactam Racemase from Ochrobactrum anthropi with Promiscuous Amino Acid Ester Racemase Activity

The kinetic resolution of amino acid esters (AAEs) is a useful synthetic strategy for the preparation of single‐enantiomer amino acids. The development of an enzymatic dynamic kinetic resolution (DKR) process for AAEs, which would give a theoretical yield of 100 % of the enantiopure product, would require an amino acid ester racemase (AAER); however, no such enzyme has been described. We have identified low AAER activity of 15 U mg^?1 in a homologue of a PLP‐dependent α‐amino ?‐caprolactam racemase (ACLR) from Ochrobactrum anthropi. We have determined the structure of this enzyme, OaACLR, to a resolution of 1.87 Å and, by using structure‐guided saturation mutagenesis, in combination with a colorimetric screen for AAER activity, we have identified a mutant, L293C, in which the promiscuous AAER activity of this enzyme towards l ‐phenylalanine methyl ester is improved 3.7‐fold.     

Thermal deactivation and inhibition of D‐Amino acid oxidase in permeabilized cells of the yeast Trigonopsis variabilis

The inhibition of D ‐amino acid oxidase contained in permeabilized cells of the yeast Trigonopsis variabilis by α‐keto acids (pyruvic acid, phenylpyruvic acid and 4‐methylthio‐2‐oxobutanoic acid), products of the transformation of the corresponding D ‐amino acids, was studied. In all cases, inhibition was of the mixed type and significant differences with respect to the inhibition shown by the enzyme from other sources such as pig kidney or the yeast Rhodotorula gracilis were observed. A study was also made of the thermal deactivation of the enzyme contained in permeabilized cells of T variabilis in the temperature range 30–50 °C in sodium phosphate and Tris hydroxylmethyl aminomethane + CaCl₂ buffers. A deactivation mechanism with two steps in series is proposed to account for the variation in activity with time. The results suggest that the enzyme shows greater stability in phosphate buffer, with half‐lives between 7.6 days at 30 °C and 8.6 h at 50 °C. Copyright © 2004 Society of Chemical Industry     

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.

     

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.

     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

α‐Amino acids as initiators of ε‐caprolactone and L,L‐lactide polymerization

Biodegradable polymers/oligomers were successfully synthesized through a ring‐opening polymerization of ε‐caprolactone and L ,L ‐lactide, initiated by L ‐arginine and L ‐citrulline. The α‐amino acid initiators are natural, operationally simple, inexpensive, environmentally friendly and safe for human health. The polymerizations were performed with no solvents and without introducing any metal impurities. The chemical structures of the polymers obtained were elucidated using ¹H NMR, ¹³C NMR and Fourier transform infrared spectroscopies. In addition, incorporation of α‐amino acid molecules into the polymer chain was confirmed using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Due to the significant biological activity of L ‐arginine and L ‐citrulline, these α‐amino acid initiators may open a new route for the synthesis of functional polymers especially for pharmaceutical applications. Copyright © 2011 Society of Chemical Industry     

Reverse Micellar Encapsulation of d‐ and l‐Enantiomers of Some Aromatic α‐Amino Acids and Nucleobases by Glucose‐Derived Non‐ionic Gemini Surfactants in Neat n‐Hexane

Novel carbohydrate‐based non‐ionic gemini surfactants consisting of two sugar head groups, two hydrophobic tails having chain lengths of C12, C14, and C16 and a flexible –(CH₂)₆– spacer were synthesized and investigated for their reverse micellar encapsulation properties. The head groups of the geminis comprise glucose entities (with reducing function blocked in a cyclic acetal group) connected through C‐6 to tertiary amines. These surfactants were explored for reverse micellar encapsulation of d ‐ and l ‐enantiomers of aromatic α‐amino acids viz. histidine (His), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) in neat n‐hexane. Similar studies were carried out for encapsulation of nucleobases viz. adenine (Ade), guanine (Gua), thymine (Thy), cytosine (Cyt) and Uracil (Ura). Reverse micellar studies revealed that aromatic α‐amino acids were encapsulated in the sequence His>Tyr>Phe>Trp. In most cases, a difference in the degree of encapsulation of d ‐ and l ‐enantiomers of aromatic amino acids in reverse micellar phases of gemini amphiphiles in neat n‐hexane, was revealed. For Tyr, l ‐enantiomer was better encapsulated than its antipode, i.e., d ‐enantiomer but for Trp, d ‐enantiomer was better encapsulated then l ‐enantiomer. In the case of nucleobases, Ura was found selectively encapsulated by reverse micelles formed by these new amphiphiles.     

Heterocycles as Nonclassical Bioisosteres of α‐Amino Acids

Bioisosterism of α‐amino acids is often accomplished by replacing the α‐carboxylate with one of the many known carboxylic acid bioisosteres. However, bioisosterism of the whole α‐amino acid moiety is accomplished with heterocyclic bioisosteres that often display an acidic function. In this Minireview, we summarized the reported heterocycles as nonclassical bioisosteres of α‐amino acids, which include quinoxaline‐2,4(1H)‐dione, quinoxaline‐2,3(1H)‐dione and quinolin‐2(1H)‐one, azagrevellin and azepine‐derived structures. The binding mode of the crystalized bioisosteres were compared with those of the crystalized α‐amino acids that bind in the same domain, and where no data on the crystal structure were available, the displacement studies of known orthosteric ligands were used. The reported bioisosteres share the following essential structural features for mimicking α‐amino acids: an aromatic ring system joined to a lactam ring system with an acidic feature next to the lactam carbonyl, where this acidic feature together with the lactam carbonyl can mimic the α‐carboxylate, and the lactam nitrogen together with the aromatic ring system can mimic the α‐ammonium. The majority of these heterocycles can be prepared from three common corresponding starting materials: the corresponding anilines, isatins or anthranilic esters. The data collected here show the potential of this class of bioisosteres in the design of glutamate receptor ligands and beyond.     

Single‐Biocatalyst Synthesis of Enantiopure d‐Arylalanines Exploiting an Engineered d‐Amino Acid Dehydrogenase

A practical and efficient biocatalytic synthesis of aromatic d ‐amino acids has been developed, based on the reductive amination of the corresponding α‐keto acids via a recombinant whole cell system composed of an engineered dehydrogenase and cofactor recycling apparatus. The reaction was shown to give excellent enantioselectivity (≥98%) and good yields at the preparative scale across a broad range of substrates. Additionally, the structure of the variant enzyme was solved to allow rationalisation of the observed reaction rates. The engineered whole cell catalyst was also used to mediate the production of d ‐phenylalanine derivatives from racemic mixtures and cheaper l ‐amino acids by combining it with an enantiocomplementary deaminase.

     

One‐Pot Conversion of Cephalosporin C to 7‐Aminocephalosporanic Acid in the Absence of Hydrogen Peroxide

The main drawback in the production of 7‐aminocephalosporanic acid (7‐ACA) at the industrial level is the inactivation of the enzymes implicated in the process due to the presence of hydrogen peroxide during the reaction. As an alternative, we have developed the conversion of cephalosporin C to 7‐ACA in a single reactor without the presence of hydrogen peroxide during the reaction, achieving more than 80% yield. In order to develop this process, D ‐amino acid oxidase (DAAO) was co‐immobilized with catalase (CAT), which is able to fully eliminate in situ the hydrogen peroxide formed by the neighbouring DAAO molecules. Thus, the product of the reaction is only α‐ketoadipyl‐7‐ACA. This system prevents the inactivation of the oxidase by hydrogen peroxide, solving the main problem of the enzymatic process. Moreover, we have found that α‐ketoadipyl‐7‐ACA is recognized as a substrate by glutaryl acylase (GAC) and hydrolyzed as long as glutaric acid is absent from the reaction medium (because it is able to inhibit the hydrolysis). The low stability of α‐ketoadipyl‐7‐ACA justifies the use of a single reactor, in which glutaryl acylase is already present when this substrate is generated. Thus, the whole process may (and must) be performed in a single step, and in the absence of hydrogen peroxide that could affect the stabilities of the involved enzymes.     

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.     

One‐Pot Substitution Approach for the Syntheses of Nonfunctional and Functional Poly[(amino acid ester)phosphazene] Biomaterials

Biodegradable polyphosphazenes are important class of biomaterials. Their preparation typically requires specialized setup, inert conditions, and cumbersome and multiple processes. This work focuses on the synthesis of both nonfunctional and novel functional poly[(amino acid ester)phosphazene]s using a simplified thermal ring opening polymerization in air, followed by one‐pot ( 1P ) room temperature substitution, also in air. While some hydrolysis was inevitable under such conditions, purified materials with lower polydispersity indices than previously reported and acceptable yields were successfully and reproducibly obtained. The poly[(amino acid ester)phosphazene]s developed in this work are based on l ‐alanine, l ‐phenylalanine, and l ‐methionine with l ‐glutamic acid to render them functionality. Characterization of these synthesized materials demonstrated that the 1P substitution was successful in developing mono‐ and co‐substituted poly[(amino acid ester)phosphazene]s. Cytotoxicity studies on 2D films showed the materials to be compatible with NIH‐3T3 fibroblasts while confocal imaging of cells showed a well‐spread morphology with abundant F‐actin within the cytoskeleton. The l ‐phenylalanine‐based poly[(amino acid ester)phosphazene]s also showed significantly enhanced cell viability over tissue culture polystyrene at days 1 and 3 of cultivation (p < 0.01). Overall, this study has shown that poly[(amino acid ester)phosphazene]s can be obtained with acceptable yields and straightforward reaction conditions, leading to materials suitable for broader biomedical applications.

     

Evolving the N‐Terminal Domain of Pyrrolysyl‐tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids

By evolving the N‐terminal domain of Methanosarcina mazei pyrrolysyl‐tRNA synthetase (PylRS) that directly interacts with tRNA^Pyl, a mutant clone displaying improved amber‐suppression efficiency for the genetic incorporation of N^?‐(tert‐butoxycarbonyl)‐l ‐lysine threefold more than the wild type was identified. The identified mutations were R19H/H29R/T122S. Direct transfer of these mutations to two other PylRS mutants that were previously evolved for the genetic incorporation of N^?‐acetyl‐l ‐lysine and N^?‐(4‐azidobenzoxycarbonyl)‐l ‐δ,?‐dehydrolysine also improved the incorporation efficiency of these two noncanonical amino acids. As the three identified mutations were found in the N‐terminal domain of PylRS that was separated from its catalytic domain for charging tRNA^Pyl with a noncanonical amino acid, they could potentially be introduced to all other PylRS mutants to improve the incorporation efficiency of their corresponding noncanonical amino acids. Therefore, it represents a general strategy to optimize the pyrrolysine incorporation system‐based noncanonical amino‐acid mutagenesis.     

One‐Pot Conversion of L‐Threonine into L‐Homoalanine: Biocatalytic Production of an Unnatural Amino Acid from a Natural One

A novel biocatalytic process for production of L ‐homoalanine from L ‐threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω‐transaminase (ω‐TA). TD catalyzes the dehydration/deamination of L ‐threonine, leading to the generation of 2‐oxobutyrate which is asymmetrically converted to L ‐homoalanine via transamination with benzylamine executed by ω‐TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Escherichia coli and an (S)‐specific ω‐TA from Paracoccus denitrificans. In the coupled reactions, L ‐threonine serves as a precursor of 2‐oxobutyrate for the ω‐TA reaction, eliminating the need for employing the expensive oxo acid as a starting reactant. In contrast to α‐transaminase reactions in which use of amino acids as an exclusive amino donor limits complete conversion, amines are exploited in the ω‐TA reaction and thus maximum conversion could reach 100%. The ω‐TA‐only reaction with 10 mM 2‐oxobutyrate and 20 mM benzylamine resulted in 94% yield of optically pure L ‐homoalanine (ee>99%). However, the ω‐TA‐only reaction did not produce any detectable amount of L ‐homoalanine from 10 mM L ‐threonine and 20 mM benzylamine, whereas the ω‐TA reaction coupled with TD led to 91% conversion of L ‐threonine to L ‐homoalanine.     

Alternative molecularly imprinted membranes from a derivative of natural polymer,cellulose acetate

Molecularly imprinted polymeric membranes were prepared from cellulose acetate (CA), of which acetyl content was 40%, by applying the alternative molecular imprinting technique. The Z‐d ‐Glu imprinted polymeric membranes thus obtained recognized d ‐Glu in preference to l ‐Glu from racemic Glu mixtures and vice versa. The affinity constants between Glu and the chiral recognition site for two kinds of membranes were determined to be 3.1 × 10³ mol⁻¹ dm³ from the adsorption isotherm of d ‐Glu or L ‐Glu in the molecularly imprinted CA membranes. Enantioselective electrodialysis was attained with the present membranes reflecting their adsorption selectivity. d ‐Glu was preferentially permeated through the Z‐d ‐Glu imprinted CA membrane, whereas L ‐Glu was permeated through the Z‐L ‐Glu imprinted CA membrane. The present study suggests that the molecularly imprinted CA membranes are applicable to the optical resolution of racemic amino acids. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 493–499, 1999     

Alanine Scan of the Peptide Antibiotic Feglymycin: Assessment of Amino Acid Side Chains Contributing to Antimicrobial Activity

The antibiotic feglymycin is a linear 13‐mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4‐hydroxyphenylglycine and 3,5‐dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution‐phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di‐ and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D ‐Hpg1, L ‐Hpg5, and L ‐Phe12, the inhibitory activity against MurA and MurC depends mainly on L ‐Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin.     

Molecularly imprinted polymers for the selective recognition of l‐phenylalanine based on 1‐buty‐3‐methylimidazolium ionic liquid

To enhance the affinity of 4‐vinyl pyridine to l ‐phenylalanine (l ‐Phe) and convert the imprinting process from the aqueous phase to the organic phase, an oil‐soluble amino acid ionic liquid was introduced as a template. In this study, 1‐butyl‐3‐methylimidazolium α‐aminohydrocinnamic acid salt was first applied to prepared surface molecularly imprinted polymers (MIPs) in acetonitrile for the selective recognition of l ‐Phe. Fluorescence quenching analysis of the functional monomer on the template was investigated under different conditions to study the imprinting mechanism. Several binding studies, such as the sorption kinetics, sorption thermodynamics, and solid‐phase extraction application, and the chiral resolution of racemic phenylalanine were investigated. The binding isotherms were fitted by nonlinear regression to the Freundlich model to investigate the recognition mechanism. The affinity distribution analysis revealed that polymers imprinted by ionic liquid showed higher homogeneous binding sites than those imprinted by l ‐Phe. The competition tests were conducted by a molecularly imprinting solid‐phase extraction procedure to estimate the selective separation properties of the MIPs for l ‐Phe. The target MIP was shown to be successfully for the separation of l ‐Phe from an amino acid mixture. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42485.     

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.     

Effect of Amino Acids on Micellization,Surface Activity and Micellar Properties of Nonionic Surfactant Hexadecyl Alcohol Ethoxylate (25EO) in Aqueous Solutions

The effects of amino acids (dl ‐glycine, dl ‐alanine, dl ‐phenylalanine, dl ‐serine, l ‐leucine, l ‐aspartic acid, l ‐lysine) on the micellization, surface activity, viscometric and aggregation properties of hexadecyl alcohol polyethoxylate (25EO) in aqueous solution were studied. The critical micelle concentration and free energy of micellization were evaluated and discussed. The surface excess concentration, the minimum area per molecule, the surface pressure at the critical micelle concentration and the standard free energy of adsorption were obtained from surface activity studies. On the basis of the determination of the relative viscosities on concentration, the viscosity B‐coefficients of the Jones–Dole semiempirical equation were calculated for the premicellar and postmicellar regions. The method of fluorescence quenching was used for determination of the micellar aggregation number of hexadecyl alcohol polyethoxylate (25EO) in aqueous solution in the presence of amino acids. It was found that among studied amino acids serine shows behavior that is different from that of the other neutral amino acids.     

Removal of amino acids from water by adsorption on polystyrene resins

The adsorption of eight amino acids, L ‐asparagine, D,L ‐threonine, L ‐lysine, L ‐leucine D,L ‐methionine, L ‐tyrosine, L ‐phenylalanine and D,L ‐tryptophan, on the non‐polar macroporous adsorbents Amberlite XAD‐2 and XAD‐4 (polystyrene–divinylbenzene copolymers) was studied. Equilibrium adsorption experiments were conducted to estimate the types of isotherm and their parameters. The effect the chemical composition and structure of the amino acids on the efficiency of adsorption was evaluated. The influence of pH and ionic strength was also studied. The data of adsorption isotherms of the examined amino acids seemed generally to approach the Freundlich isotherm model. Tryptophan isotherm adsorption data could match in some cases the Langmuir model. The majority of the adsorption isotherms were almost linear. In terms of adsorbed amino acid on both resin surfaces, the amino acids can be ranked thus: D,L ‐tryptophan > L ‐phenylalanine > D,L ‐methionine, L ‐tyrosine > L ‐leucine > L ‐lysine > D,L ‐threonine > L ‐asparagine. In low pH solution, adsorption was generally higher than that at intermediate and high pH values. Generally, as the ionic strength increases, the adsorption of the amino acids increases. © 2001 Society of Chemical Industry