L-alanine Amide Research Articles

Assessment and application of Marfey's reagent and analogs in enantioseparation: a decade's perspective.

Of the various methods available for high-performance liquid chromatography separation of enantiomers (of e.g. amino acids and amino group containing compounds) by the pre-column derivatization approach, use of Marfey's reagent has been most successful with continued application since its introduction in 1984. The reagent is prepared from difluoro dinitro benzene by nucleophilic substitution of one of its F atoms by l-alanine amide. There is flexibility to prepare several chiral variants (by substituting the F atom with different chiral auxiliaries) and to tailor the hydrophobicity and resolution, ultimately, of the diastereomeric derivatives. The present paper assesses and reviews applications of Marfey's reagent and its chiral variants (i.e. other FDNP reagents) for enantioseparation of certain amino group containing drugs/amino acids, and to provide some case studies on enantiomeric separations that are important for the pharmaceutical industry. Various explanations for separation mechanism and elution order using FDNP reagents are included and the question of the configuration of the corresponding enantiomer using an indirect approach has also been addressed.

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from Citreicella sp. SE45.

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4kDa on SDS-polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including L-alanine amide and L-serine amide with a specific activity of 17.5 and 21.6U/mg, respectively. The K m and V max values for D- and L-ACL were 5.3 and 2.17mM, and 769 and 558μmol/min.mg protein, respectively. Moreover, the turn over number (K cat) and catalytic efficiency (K cat/K m ) of purified ACL racemase from Citreicella sp. SE45 using L-ACL as a substrate were 465S-1 and 214S-1mM-1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.

ChemInform Abstract: Enantioselective 1,3‐Dipolar Cycloaddition of Azomethine Imines with Propioloylpyrazoles Induced by Chiral π—Cation Catalysts.

We developed 1,3-dipolar cycloadditions of azomethine imines with propioloylpyrazoles catalyzed by a chiral copper(II) complex of 3-(2-naphthyl)-l-alanine amide. The asymmetric environment created by intramolecular π-cation interaction and the N-alkyl group of the chiral ligand gives the corresponding adducts in high yields with excellent enantioselectivity. This is the first successful method for the catalytic enantioselective 1,3-dipolar cycloaddition of azomethine imines with internal alkyne derivatives to give fully substituted pyrazolines.

Enantioselective 1,3-dipolar cycloaddition of azomethine imines with propioloylpyrazoles induced by chiral π-cation catalysts.

We developed 1,3-dipolar cycloadditions of azomethine imines with propioloylpyrazoles catalyzed by a chiral copper(II) complex of 3-(2-naphthyl)-l-alanine amide. The asymmetric environment created by intramolecular π-cation interaction and the N-alkyl group of the chiral ligand gives the corresponding adducts in high yields with excellent enantioselectivity. This is the first successful method for the catalytic enantioselective 1,3-dipolar cycloaddition of azomethine imines with internal alkyne derivatives to give fully substituted pyrazolines.

Facile determination of the absolute configurations of α-hydroxy acids by chiral derivatization coupled with liquid chromatography–mass spectrometry analysis

α-Hydroxy acids are commonly encountered in natural bioactive molecules. For convenient determination of the absolute configurations of α-hydroxy acids, a simple method, named the “O-Marfey method” was developed using a chiral derivatization strategy with (1-fluoro-2,4-dinitrophenyl-5)-l-alanine amide (l-FDAA). The liquid chromatography–mass spectrometry (LC–MS) analysis of the derivatives clearly determined the absolute configurations of various α-hydroxy acids based on their elution times and sequences. This new method is operationally simple with short reaction time and sensitive enough for application at a sub-milligram scale without any purification. This method also enables the simultaneous chirality analysis of α-hydroxy acids and α-amino acids. The absolute configuration of a natural depsipeptide bearing an α-hydroxy acid and α-amino acids was successfully determined by our O-Marfey application.

Catalytic Enantioselective 1,3-Dipolar Cycloadditions of Nitrones with Propioloylpyrazoles and Acryloylpyrazoles Induced by Chiral π-Cation Catalysts

A chiral copper(II) complex of 3-(2-naphthyl)-l-alanine amide successfully catalyzes the enantioselective 1,3-dipolar cycloaddition reaction of nitrones with propioloylpyrazole and acryloylpyrazole derivatives. The asymmetric environment created by intramolecular π-cation interaction gives the corresponding adducts in high yields with excellent enantioselectivity. This is the first successful method for the catalytic enantioselective 1,3-dipolar cycloaddition of nitrones with acetylene derivatives. The 1,3-dipolar cycloadducts can be stereoselectively converted to β-lactams via reductive cleavage of the N-O bond using SmI(2).

Model peptide–water complexes in Ar matrix: Complexation induced conformation change and chirality transfer

The hydrogen bonded complexes between water and N-acetyl N′-methyl glycine amide (Ac-Gly-NHMe), as well as between water and N-acetyl N′-methyl l-alanine amide (Ac-L-Ala-NHMe) have been studied by matrix isolation infrared spectroscopy and quantum chemical (B3LYP) calculations. Analysis of the spectra shows that from the β DL/ β L(D) and the γ D=L/ γ L conformers of the monomers mainly β DL/ β L(D) complex with water. In the complexes water binds to both amide groups making water-bridged complexes in which the monomer has a γ D=L/ γ L-like structure. The Ac-L-Ala-NHMe–water complexes have also been studied by matrix isolation vibrational circular dichroism (MI-VCD) spectroscopy as well as by recording the VCD spectra of Ac-L-Ala-NHMe in pure and water-saturated dichloromethane. Transitions of the achiral water molecule, which gained rotational activity through chirality transfer from the model peptide, could be identified in both MI and solution VCD spectra. It is also shown that the signs of these transitions are sensitive to the alignment of the complexed water and can be used for structural identification of the complexes.

Catalytic Enantioselective [2 + 4] and [2 + 2] Cycloaddition Reactions with Propiolamides

We report here the catalytic and highly enantioselective [2 + 4] and [2 + 2] cycloaddition reactions of electron-rich dienes or silyl enol ethers with electron-deficient propiolamide derivatives induced by copper(II).3-(2-naphthyl)-L-alanine amide complex.

Quantification of serine enantiomers in rat brain microdialysate using Marfey's reagent and LC/MS/MS

The ability to selectively measure serine enantiomer concentrations in rat brain microdialysate is essential during drug discovery to study the interaction of d-serine with the N-methyl- d-aspartate (NMDA) subtype of the glutamate receptor. NMDA receptor-stimulating agents, such as d-serine, have been shown to reduce the negative symptoms and cognitive dysfunction in individuals with schizophrenia when added to conventional or atypical antipsychotic drug regimens. In the work presented here, an LC/MS/MS assay was developed and validated to simultaneously measure d-serine and l-serine concentrations in rat brain microdialysate. Reverse phase chromatographic resolution of the enantiomers was obtained through derivatization with 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide (Marfey's reagent). The assay was validated to determine concentrations over the range of 10–7500 ng/mL using electrospray ionization and multiple reaction monitoring (MRM). Both intra- and inter-day precision and accuracy were less than 16.5% (RE) and 7% (CV) for both analytes, respectively, and assay throughput was increased significantly relative to existing methodologies.

Discovery of amino acid amides as new substrates for α-amino-ɛ-caprolactam racemase from Achromobacter obae

Amino acid amide racemizing activity was discovered in α-amino-ɛ-caprolactam (ACL) racemase (EC 5. 1. 1. 15) from Achromobacter obae. The nucleotide sequence of 1305 bp ACL racemase gene was designed for expression in Escherichia coli and synthesized in a reaction with 46 oligonucleotides by assembly PCR technique. The gene was expressed under the control of the lac promoter, and ACL racemase was purified from E. coli JM109/pACL60 harboring the gene for the enzyme. The enzyme catalyzed the racemization of newly discovered substrates such as 2-aminobutyric acid amide, alanine amide, threonine amide, norvaline amide, norleucine amide, leucine amide, methionine amide, serine amide, and phenylalanine amide. The relative activity towards l-2-aminobutyric acid amide was 2.7% that for l-ACL (350 U/mg), followed by l-alanine amide by 2.1%. Kinetic parameters for the two d- and l-alanine-isomers of amino acid amides (2-aminobutyric acid amide and alanine amide) and l-ACL were obtained from Lineweaver–Burk plots. The K cat values for l-ACL, d- and l-2-aminobutyric acid amide, and d- and l-alanine amide were calculated to be 360, 40, 13, 9.8 and 7.2 S −1, respectively. The K m values for l-ACL, d- and l-2-aminobutyric acid amide, and d- and l-alanine amide were shown to be 10.1, 3.5, 1.1, 3.4 and 2.5 mM, respectively. The l-enantiomer of 2-aminobutyric acid amide (22.5 μmol) was completely racemized by ACL racemase (136 μg) in 100 min, and the same amount of l-alanine amide in 120 min. The catalytic efficiency of the newly discovered racemization reaction of 2-aminobutyric acid amide was calculated for be fairly high at about 33% that for l-ACL, while that for alanine amide was about 8.1%. Here, we report that amino acid amides act as new substrates for the ACL racemase.

Purification, characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

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.

Liquid chromatographic enantioseparation of spin-labelled β-amino acids

Direct and indirect high-performance liquid chromatographic (HPLC) methods were developed for the enantioseparation of spin-labelled, cyclic, chiral β-amino acids containing nitroxide free radicals, trans-3-amino-1-oxyl-2,2,5,5-tetramethylpyrrolidine-4-carboxylic acid ( trans-POAC), cis-4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid ( cis-β-TOAC) and their N-Fmoc-protected analogues, synthesized in racemic and enantiomerically pure forms. The direct method involved the use of a Chiralcel OD-RH column, while indirect separation was carried out by application of either 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate or ( S)- N-(4-nitrophenoxycarbonyl)-phenylalanine methoxyethyl ester as chiral derivatizing agent. Use of 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide (Marfey’s reagent) as chiral derivatizing agent failed because of the low of yield of the derivatization reaction. Selection and variation of the mobile phase was restricted by the sensitivity of the spin-labelled amino acids to acidic conditions. Conditions affording the best resolution were found and the differences in separation capability of the methods were noted. The sequence of elution of the enantiomers was determined by different methods and, in the case of the β-TOAC analogues, the absolute configurations of the enantiomers corresponding to each peak were identified.

Design of chiral monochloro- s-triazine reagents for the liquid chromatographic separation of amino acid enantiomers

A series of chiral derivatizing reagents (CDRs) was synthesized by nucleophilic replacement of one chlorine atom in cyanuric chloride (2,4,6-trichloro-1,3,5-triazine; s-triazine) by alkoxy (methoxy, butoxy, 1,1,1-trifluoroethoxy) or aryloxy groups (phenoxy, nitrophenoxy, phenylphenoxy, 4-methylcoumaryloxy), and displacement of a second chlorine by l-alanine amide, l-phenylalanine amide, l-proline tert.-butyl ester, or Boc- l-lysine tert.-butyl ester. Further, CDRs were investigated in which two chlorine atoms in cyanuric chloride were substituted consecutively by l-valine amide and l-phenylalanine amide. The resulting CDRs having a remaining reactive chlorine were tested for their capability of derivatizing dl-amino acids followed by liquid chromatographic separation of the resulting diastereomers.

Ab initio conformational analysis of N-formyl l-alanine amide including electron correlation

The conformational properties of N-formyl l-alanine amide (ALA) were investigated using RMP2/6-311G∗∗ ab initio gradient geometry optimization. One hundred forty four structures of ALA were optimized at 30° grid points in its φ(N–C(α)), ψ(C(α)–C′) conformational space. Using cubic spline functions, the grid structures were then used to construct analytical representations of complete surfaces, in φ,ψ-space, of bond lengths, bond angles, torsional sensitivity and electrostatic atomic charges. Analyses show that, in agreement with previous studies, the right-handed helical conformation, αR, is not a local energy minimum of the potential energy surface of ALA. Comparisons with protein crystallographic data show that the characteristic differences between geometrical trends in dipeptides and proteins, previously found for ab initio dipeptide structures obtained without electron correlation, are also found in the electron-correlated geometries. In contrast to generally accepted features of force fields used in empirical molecular modeling, partial atomic charges obtained by the CHELPG method are found to be not constant, but to vary significantly throughout the φ,ψ-space. By comparing RHF and MP2 structures, the effects of dispersion forces on ALA were studied, revealing molecular contractions for those conformations, in which small adjustments of torsional angles entail large changes in non-bonded distances.

Enantiomeric resolution of amino acids by thin-layer chromatography

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase TLC plate, without using impregnated plates or a chiral mobile phase, is described. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide were spotted on a reversed phase pre-coated TLC plate. Enantiomers of glutamate and aspartate were separated most effectively with solvent consisting of 25% acetonitrile in triethylamine-phosphate buffer (50 mM, pH 5.5) (v/v). Separation of l- and d-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline and alanine were separated with 35% of acetonitrile solvent, and those of methionine, valine, phenylalanine and leucine with 40% of acetonitrile solvent. The possibility of using TLC for quantitative determination of amino acid enantiomers was shown by the quantitative recovery of d- and l-alanine from the TLC plate in the range of 0.56–4.48 nmol.

Ab Initio Study of the Conformational Dependence of the Nonplanarity of the Peptide Group

To study the nonplanarity of peptide bonds, the conformationally dependent variations of the N−C torsional angle, ω2 (Figure 1), of the central peptide group in N-formyl l-alanyl l-alanine amide (ALA-ALA) was investigated using a database of 11 664 RHF/4-21G ab initio gradient optimized structures. The database was generated by optimizing the geometries of ALA-ALA at grid points in its four-dimensional (φ1,ψ1,φ2,ψ2) conformational space (Figure 1) defined by 40° increments along the outer torsions φ1 and ψ2, and by 30° increments along the inner torsions ψ1 and φ2. Using cubic spline functions, the grid structures were then used to construct analytical representations of complete surfaces of ω2 in (φ1,ψ1,φ2,ψ2)-space. Analyses of the conformational surfaces of ω2 reveal that the peptide N−C torsion is a smoothly varying function of associated φ and ψ angles and that, for many conformational regions, deviations from planarity are the rule rather than the exception. Comparisons with protein crystallographic data show that, in contrast to peptide torsional angles calculated for an entire protein, the ω2 angles of smaller model peptides, such as ALA-ALA, cannot be used to model peptide groups in proteins, because of long-range effects present in the latter but not the former. This finding indicates the general difficulty of predicting the exact positions of backbone torsional angles in proteins from smaller model peptides. Furthermore, the results confirm the directional nature of polypeptide chains. That is, conformation transmission effects from neighboring groups differ, depending on whether they are transmitted from right to left or from left to right in the peptide chain.

High-performance liquid chromatographic separation of the enantiomers of unusual α-amino acid analogues

The direct and indirect stereochemical resolution of the enantiomers of ring- and α-methyl-substituted phenylalanines and phenylalanine amides was attempted by high-performance liquid chromatographic methods. The direct separation was carried out on two chiral stationary phases, the crown-ether-based Crownpak CR(+), and the teicoplanin-based Chirobiotic T, while the indirect resolution was performed by applying pre-column derivatization with 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate (GITC) and Nα-(2,4-dinitro-5-fluorophenyl)- l-alanine amide (Marfey’s reagent, FDAA). The Chirobiotic T column was efficient in the separation of ring- and α-methyl-substituted phenylalanine analogues, but was ineffective for the amides of these analogues. The Crownpak CR(+) column separated the ring-substituted phenylalanines and amides, whereas the α-methylated analogues were coeluted. Of the two indirect methods, GITC derivatization seemed more effective than FDAA derivatization.

High-performance liquid chromatographic separation of novel atropic α,α-disubstituted-β-amino acids, either on different β-cyclodextrin-bonded phases or as their 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide derivatives

The high-performance liquid chromatographic enantioresolution of free and N- and/or C-protected derivatives of ( R, S)-2′,1′:1,2;1″,2″:3,4-dinaphthcyclohepta-1,3-diene-6-aminomethyl-6-carboxylic acid (β 2-Bin) by direct and indirect methods is reported. The direct separation was carried out on native and different derivatized β-cyclodextrin-bonded phases. The indirect resolution was achieved by applying pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide. The effects of different parameters such as the mobile phase composition and the structures of the compounds on the enantiomeric resolution are discussed.

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Elucidation of Limitations of Marfey's Method and of Its Separation Mechanism

As the first step in establishing our proposed method, the advanced Marfey's method, which is planned for the simultaneous determination of the absolute configuration of amino acids in a peptide, we applied Marfey's method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and the applicability of the method was confirmed, the (1-fluoro-2,4-dinitrophenyl)-5-l-alaninamide (FDAA) derivative of the l-amino acid was not always eluted prior to its corresponding d-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, its separation mechanism was carefully investigated using UV and NMR spectral techniques. The results suggested that the resulting conformations of the l- and d-amino acid derivatives are stable and that the resolution between the l- and d-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both α-carbons of an amino acid and l-alanine amide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)-type arrangement. Therefore, the l-amino acid derivative is usually eluted from the column before its corresponding d-amino acid derivative in Marfey's method. According to this separation mechanism, the elution order of a desired amino acid can be elucidated from the average retention time of the l- and d-amino acid derivatives, and the dl-serine and -asparagine derivatives are critical for Marfey's method.