DL-amino Acids Research Articles

First detection of free d-amino acids in human nails by combination of derivatization and UPLC-ESI-TOF-MS

The resolution of free DL-amino acids in the human nail was carried out by combination of the R(−)-DBD-PyNCS derivatives and UPLC-ESI-TOF-MS. As we know, for the first time, these five kinds of D-amino acids, were determined from human nail samples. Fifteen kinds of L-amino acids were also recognized from human nails. Application to DL-amino acids in healthy person and diabetes patients nails is also described in this communication.

Preparation of novel chiral stationary phase based on click chemistry for ligand exchange chromatography

Click chemistry was applied to immobilize l-proline derivative onto azide-modified silica gel to give a novel chiral stationary phase (denoted as click-CSP) for ligand exchange chromatography. The developed protocol combines the benefits of operational simplicity, exceptionally mild conditions and high surface loadings. The enantioselectivity α of some dl-amino acids on the click-CSP were found to be in the range from 1.13 to 3.46. The chromatographic resolutions of some dl-amino acids and the stability study firmly illustrate the potential of click chemistry for preparation chiral stationary phase for ligand exchange chromatography.

Highly sensitive detection of chiral amino acids by CE based on on‐line stacking techniques

We report a novel means for chiral separation and stacking of amino acids by MEKC with LED-induced fluorescence detection. Naphthalene-2,3-dicarboxaldehyde, hydroxypropyl-beta-cyclodextrin (Hp-beta-CD), SDS, and poly(ethylene oxide) (PEO) serve as a derivatized agent, chiral selector, pseudostationary phase, and concentrated medium, in sequence. To improve speed, resolution, and stacking efficiency, the analysis of chiral amino acids was performed under discontinuous conditions--the capillary was filled with a solution of 100 mM Tris-borate, 150 mM SDS, and 50 mM Hp-beta-CD, whereas buffer vials contain 20 mM Tris-borate, 150 mM SDS, 50 mM Hp-beta-CD, and 0.5% w/v PEO. A solution of nonionic PEO enters the capillary with the help of EOF during the separation. Through interaction of SDS micelles/Hp-beta-CD and chiral amino acid, the negatively charged complexes migrated into the PEO solution and stacked at the boundary between the sample zone and the PEO solution. Compared with normal sample injection (10 nL sample volume), a several hundred-fold sensitivity improvement for chiral amino acids was obtained under the injection of 270 nL sample volume (30% of the capillary volume). Meanwhile, the LOD at S/N of three for DL-amino acids were in the range of 0.18-0.22 nM. The proposed method has been applied for the determination of DL-leucine in urine and plasma samples.

A chiral stationary phase with special hydrophobic framework for ligand-exchange chromatography

A novel chiral stationary phase (CSP1) for ligand-exchange chromatography (CLEC) was prepared by firstly using dimethylchlorosilane as an endcapping reagent for decreasing residual silanol group on the surface of silica gel, and then introducing l-Pro as a chiral selector and hydrophobic octyl group to the silica gel surface simultaneously. The enantioseparations of 14 dl-amino acids on CSP1 were achieved with the enantioselectivity α ranging from 1.09 to 2.44 and the resolution Rs being between 0.8 and 6.3. The chromatographic performances of CSP1 with the bonded phase (CSP2) prepared using reference method were compared. The results showed that the column efficiency and resolution Rs of chiral stationary phase could be improved by using the above modifying method.

Preparation of new hybrid organic/inorganic polymeric chiral stationary phases for ligand-exchange chromatography

Three new hybrid organic/inorganic polymeric ligand-exchange chiral stationary phases were developed by radical chain transfer reaction and surface grafting on silica gel, and successfully used for the enantioseparations of dl-amino acids and dl-hydroxyl acids. The resolutions were achieved by using water containing 2.0 × 10 −4 mol/L of CuAc 2 as a mobile phase, column temperature of 40 °C, flow rate of 1.0 mL/min and detection at UV 254 nm. The elution order of d-isomer before l-isomer was observed for all dl-amino acids resolved except dl-Pro.

Indirect TLC resolution of amino acid enantiomers after derivatization withMarfey’sreagent and its chiral variants

A simple and rapid method has been established for indirect separation of the optical isomers of seventeen DL amino acids by reversed-phase and normal-phase TLC. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L -alaninamide (FDNP-L -Ala-NH 2 ), 1-fluoro-2,4-dinitrophenyl-5-L -phenylalaninamide (FDNP-L -Phe-NH 2 ), or 1-fluoro-2,4-dinitrophenyl-5-L -valinamide (FDNP-L -Val-NH 2 ) were spotted on precoated plates. Diastereomers of all the DL amino acids were separated most effectively by normal-phase TLC with phenol-water, 3:1 ( v / v ), as mobile phase. In reversed-phase TLC the diastereomers were separated most effectively by use of mobile phases containing acetonitrile and triethylamine-phosphate buffer (50 mM, pH 5.5). The results obtained by use of the classical Marfey’s reagent (FDNP-L -Ala-NH 2 ) were compared with those obtained by use of FDNP-L -Phe-NH 2 and FDNP-L -Val-NH 2 . The effects of buffer concentration, pH, and concentration of organic modifier were studied. This indirect metho...

Biotransformation of D‐methionine into L‐methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).

Molecular Speciation Controlling Stereoselectivity of Additives: Impact on the Habit Modification in α-Glycine Crystals

The demonstration of stereoselective habit modification in molecular crystals using “tailor-made” (or structurally related) additives, over the last two decades, has been a milestone in understanding the phenomenon of molecular recognition at crystal interfaces. The centrosymmetric α-glycine crystal has provided a classic example in earlier studies for elucidating the mechanisms of such stereoselective processes. In these previous studies, with chirally resolved (l or d-amino acids) and racemic α-amino acids (dl-amino acids) as tailor-made additives, habit modification was observed to be along the enantiopolar b-axis of α-glycine crystals. Revisiting this work has, however, revealed additional habit modification along the c-axis of the α-glycine crystal with certain α-amino acids as additives (viz. aspartic acid and glutamic acid). In the presence of l-Asp and l-Glu, the (01̄1) and (01̄1̄)faces were of morphological importance and with the d-amino acids, the (011) and (011̄) faces were well-developed. On the basis of the fact that these amino acids exist in two charged states (zwitterion and anion) and building on the stereoselectivity mechanism, it is surmised that the zwitterionic species interact along the b-axis of the α-glycine crystal and the anions alter their interaction from the crystallographic b-axis to the c-axis, due to electronic repulsive forces acting at the docking sites on the (010) and (01̄0) faces. In this paper, we have used optical microscopy, molecular modeling, and IR spectroscopy to demonstrate and explain the newly observed habit modification.

Synthesis of dl-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive L-amino acids to DL-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of D-amino acids, especially D-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. L-Trp (20 mM) was successfully racemized, and the proportion of D-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial L-Trp.

Dissecting the Pretransitional Conformational Changes in Aminoacylase I Thermal Denaturation

Aminoacylase I (ACYI) catalyzes the stereospecific hydrolysis of L-acylamino acids and is generally assumed to be involved in the final step of the degradation of intracellular N-acetylated proteins. Apart from its crucial functions in intracellular amino acid metabolism, ACYI also has substantial commercial importance for the optical resolution of N-acylated DL-amino acids. As a zinc-dependent enzyme, ACYI is quite stable against heat-induced denaturation and can be regarded as a thermostable enzyme with an optimal temperature for activity of ∼65°C. In this research, the sequential events in ACYI thermal denaturation were investigated by a combination of spectroscopic methods and related resolution-enhancing techniques. Interestingly, the results from fluorescence and infrared (IR) spectroscopy clearly indicated that a pretransitional stage existed at temperatures from 50°C to 66°C. The thermal unfolding of ACYI might be a three-state process involving an aggregation-prone intermediate appearing at ∼68°C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed β-sheet structures and the transformation of about half of the Class I Trp fluorophores to Class II. Our results also suggested that the usage of resolution-enhancing techniques could provide valuable information of the step-wise unfolding of proteins.

Synthesis, potentiometric and antimicrobial activity studies on DL-amino acids-Schiff bases and their complexes

A relationship between antimicrobial activities and the formation constants of amino acid-Schiff bases and their Cu(II) and Ni(II) complexes was studied. For this purpose, a series of Schiff bases were prepared from DL-amino acids (DL-glycine, DL-alanine) and halo aldehydes (5-chloro-2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde). Schiff bases and their Cu(II) and Ni(II) complexes were characterized by the elemental analysis, spectral analysis, magnetic moment (at ca. 25°C), molar conductivity, and thermal analysis data. The complexes were found to have general compositions [ML(H2O)]. The protonation constants of the Schiff bases and stability constants of the complexes were determined potentiometrically in a dioxane-water (1: 1) solution at 25°C and 0.1 M KCl ionic strength. Antimicrobial activities of the Schiff bases and their complexes were estimated for six bacteria, such as Bacillus cereus RSKK 863, Staphylococcus aureus ATCC 259231, Micrococcus luteus NRLL B-4375, Escherichia coli ATCC 11230, Aeromonas hydrophila 106, Pseudomonas aeroginosa ATCC 29212, and the yeast Candida albicans ATCC 10239. The role of halogen substitution on the ligands, effect of the metal ion, and stabilities of the complexes are discussed on antimicrobial activities.

Precolumn diastereomerization and micellar electrokinetic chromatography on a plastic microchip: Rapid chiral analysis of amino acids

Precolumn derivatization and chiral separation of DL-amino acids based on diastereomerization have been performed on an integrated poly(dimethylsiloxane) microchip. Diastereomeric derivatives were formed in a microfabricated precolumn reactor by the reaction of amino acid enantiomers with o-phthaldialdehyde/2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose (OPA/TATG), and separated by MEKC in an achiral environment without chiral selectors in the running buffer. Optimized precolumn reactions and chiral separations of amino acids were achieved within 2.5 min. Resolutions of diastereomers of OPA/TATG-amino acids were in the range of 2.5-6.1 at optimized separation conditions. Simultaneous separation of a mixture of five chiral amino acids was successfully performed in a single run in less than 100 s.

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H4) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H4 chiral stationary phase to study chiral recognition by 18C6H4 of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H4, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H4. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.

Resolution of non-protein amino acids via Carica papaya lipase-catalyzed enantioselective transesterification

Carica papaya lipase-catalyzed transesterification of the 2,2,2-trifluoroethyl esters of N-benzyloxycarbonylated dl-amino acids carrying aliphatic side chains proceeded smoothly and, in almost all the cases, enantiospecifically ( E = >200), affording the l-methyl esters and leaving the d-trifluoroethyl esters intact.

Enantioseparation of dansyl amino acids by a novel permanently positively charged single-isomer cyclodextrin: Mono-6- N-allylammonium-6-deoxy-β-cyclodextrin chloride by capillary electrophoresis

A permanently positively charged single-isomer β-CD, mono-6- N-allylammonium-6-deoxy-β-CD chloride, ALAM-β-CD, has been synthesized and successfully used as chiral selector in capillary electrophoresis for enantioseparation of amino acids and dansyl amino acids. The effects of pH, CD's concentration and capillary length on enantioseparation were studied in order to investigate its potential as chiral selector for acidic racemates. By increasing buffer's pH and CD's concentration, fairly robust separations were achieved. Good separations were also obtained with a shorter capillary. In addition, baseline enantiomeric separations of a mixture of six pairs of dansyl dl-amino acids were achieved under different pH conditions.

Synthesis and Characterization of a New Series of 3‐(4‐Antipyrinyl)‐2‐thiohydantoin Derivatives.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Dynamic Kinetic Resolution of Amino Acid Amide Catalyzed byd-Aminopeptidase and α-Amino-ε-caprolactam Racemase

Amino acid amide racemizing activity was discovered in alpha-amino-epsilon-caprolactam (ACL) racemase (EC 5. 1. 1. 15) from Achromobacter obae. The enzymatic synthesis of d-alanine from l-alanine amide has been demonstrated by use of d-aminopeptidase (DAP; EC 3. 4. 11. 19) from Ochrobactrum anthropi C1-38 and ACL racemase. The conversion of 45 mM l-alanine amide was carried out at 30 degrees C for 7 h; l-alanine amide was completely converted to d-alanine, and no l-alanine was detected. The result of successive enzymatic reaction shows that the combination of ACL racemase and DAP can be applied for dynamic kinetic resolution of dl-amino acid amides to yield d-amino acids.

Synthesis and Characterization of a New Series of 3-(4-Antipyrinyl)-2-thiohydantoin Derivatives

This paper reports the reaction of 4-antipyrinyl isothiocyanate with DL-amino acids affording a new heterocyclic series of 3-(4-antipyrinyl)-2-thiohydantoin derivatives in mild conditions. The reaction proves to be simpleand the experiment is easy to operate with high yields (86-96%).

Marfey?s reagent for chiral amino acid analysis: A review

The present paper describes characteristics and application of Marfey's reagent (MR) including general protocols for synthesis of the reagent and diastereomers along with advantages, disadvantages and the required precautions. Applications, and comparison with other derivatizing agents, for the resolution of complex mixtures of DL-amino acids, amines and non-proteinogenic amino acids, peptides/amino acids from microorganisms, cysteine residues in peptides, and evaluation of racemizing characteristics have been discussed. Separation mechanisms of resolution of amino acid diastereomers and replacement of Ala-NH2 by suitable chiral moieties providing structural analogs and different chiral variants and their application as a derivatizing agent to examine the efficiency, and reactivity of the reagent have been focussed. Use of MR for preparing CSPs for direct enantiomeric resolution has also been included.

In vitro activity and potency of an intravenously injected antimicrobial peptide and its DL amino acid analog in mice infected with bacteria.

We report that intravenous injection (3 mg/kg of body weight twice daily) of a diastereomer (containing 33% D amino acids) of an antimicrobial peptide, K6L9 (LKLLKKLLKKLLKLL-NH2), but not the all-L-amino-acid parental peptide, cures neutropenic mice infected with gentamicin-sensitive Pseudomonas aeruginosa and gentamicin-resistant Acinetobacter baumannii bacteria. Various biophysical experiments suggest a membranolytic-like effect.