High-performance liquid chromatographic detection of L- and D-amino acids by use of immobilized enzyme electrodes as detectors

Two enzyme electrodes based on immobilized L- and D-amino acid oxidases give specific responses to L- and D-amino acids, respectively. They are used as amperometric detectors for high-performance liquid chromatography, by splitting the flow after elution from the column and detecting D-isomers in one line, L-isomers in the other. The detection limit is about 2 pmol for some amino acids (methionine, tyrosine, leucine, and phenylalanine). The procedure is useful for the specific detection of L- and D-amino acids without complicated pretreatment. The electrodes retain most of their original activities after repetitive use for one month.     

Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with L- and D-amino acid substitutions

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.     

Application of stepwise discriminant analysis to classify commercial orange juices using chiral micellar electrokinetic chromatography-laser induced fluorescence data of amino acids

The use of chiral amino acids content and stepwise discriminant analysis to classify three types of commercial orange juices (i.e., nectars, orange juices reconstituted from concentrates, and pasteurized orange juices not from concentrates) is presented. Micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) and beta-cyclodextrins are used to determine L- and D-amino acids previously derivatized with fluorescein isothiocyanate (FITC). This chiral MEKC-LIF procedure is easy to implement and provides information about the main amino acids content in orange juices (i.e., L-proline; L-aspartic acid, D-Asp, L-serine, L-asparagine, L-glutamic acid, D-Glu, L-alanine, L-.arginine, D-Arg, and the non-chiral gamma-amino-n-butyric acid (GABA), i.e., gamma-aminobutyric acid). From these results, it is clearly demonstrated that some D-amino acids occur naturally in orange juices. Application of stepwise discriminant analysis to 26 standard samples showed that the amino acids L-Arg, L-Asp and GABA were the most important variables to differentiate the three groups of samples. With these three selected amino acids a 100% correct classification of the samples was obtained either by standard or by leave-one-out cross-validation procedures. These classification functions based on the content in L-Arg, L-Asp and GABA were also applied to nine test samples and provided an adequate classification and/or interesting information on these samples. It is concluded that chiral MEKC-LIF analysis of amino acids and stepwise discriminant analysis can be used as a consistent procedure to classify commercial orange juices providing useful information about their quality and processing. To our knowledge, this is the first report about the combined use of chiral capillary electrophoresis and discriminant techniques to classify foods.     

Synthesis of second-generation sansalvamide A derivatives: novel templates as potential antitumor agents

We report the synthesis of 34 second-generation Sansalvamide A derivatives. San A derivatives have unique anticancer properties and target multiple cancers, including colon, pancreatic, breast, prostate, and melanoma. As novel templates, the derivatives described herein explore the role of stereochemistry, amide bond geometry, transannular hydrogen bonding, and polarity on antitumor potency. Testing the chemotherapeutic activity of these derivatives against multiple cancer cell lines will provide clear structural motifs and identify conformational space that is important for cytotoxicity. The 34 compounds presented are divided into six series, where five series involve the insertion of D-amino acids in conjunction with four structural features at each of the five positions of the macrocycle. The sixth series involves comparison between all L- and all D-amino acid derivatives with N-methyls placed at each position around the macrocyclic core. The four structural features explored in conjunction with D-amino acids include N-methyl amino acids, aromatic amino acids, polar amino acids, and hydrophobic alkyl amino acids.     

固定化D-氨基酸氧化酶催化DL-氨基酸去消旋化制备非天然L-氨基酸

在过氧化氢酶和氧气存在下,固定化D-氨基酸氧化酶(D-AAO)对映选择性催化DL-氨基酸中的D-对映体氧化脱氨为相应酮酸,L-对映体保留.研究了D-AAO的底物特异性并对反应条件进行了优化.结果表明:D-AAO具有较宽的底物谱,能够催化疏水性α-氨基酸的D-对映体氧化脱氨.在最优反应条件下,D-AAO催化DL-2-氨基丁酸、DL-2-氨基戊酸去消旋化,L-2-氨基丁酸、L-2-氨基戊酸的收率分别为48%和47%,ee分别为99.5%和99.8%.进一步地利用Pd-C/HCOONH4催化氧化脱氨过程中产生的亚氨基酸原位还原,有效提高了L-2-氨基丁酸、L-2-氨基戊酸的收率并保持高的光学纯度.     

Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with L- and D-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.     

A sensitive and simple ultra-high-performance-liquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids

D-Amino acids are increasingly being recognized as important signaling molecules in mammals, including humans. D-Serine and D-aspartate are believed to act as signaling molecules in the central nervous system. Interestingly, several other D-amino acids also occur in human plasma, but very little is currently known regarding their function and origin. Abnormal levels of D-amino acids have been implicated in the pathogenesis of different diseases, including schizophrenia and amyotrophic lateral sclerosis (ALS), indicating that D-amino acid levels hold potential as diagnostic markers. Research into the biological functions of D-amino acids is hindered, however, by the lack of sufficiently sensitive, high-throughput analytical methods. In particular, the interference of large amounts of L-amino acids in biological samples and the low concentrations of D-amino acids are challenging. In this paper, we compared 7 different chiral derivatization agents for the analysis of D-amino acids and show that the chiral reagent (S)-NIFE offers outstanding performance in terms of sensitivity and enantioselectivity. An UPLC-MS/MS based method for the quantification of D-amino acids human biological fluids was then developed using (S)-NIFE. Baseline separation (R(s)>2.45) was achieved for the isomers of all 19 chiral proteinogenic amino acids. The limit of detection was <1 nM for all amino acids except d-alanine (1.98 nM), d-methionine (1.18 nM) and d-asparagine (5.15 nM). For measurements in human plasma, cerebrospinal fluid and urine, the accuracy ranged between 85% and 107%. The intra-assay and inter-assay were both <16% RSD for these three different matrices. Importantly, the method does not suffer from spontaneous racemization during sample preparation and derivatization. Using the described method, D-amino acid levels in human cerebrospinal fluid, plasma and urine were measured.     

Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach

D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.     

Synthesis and Molecular Recognition of Novel Cyclic Pseudopeptides Containing L-Glutamic Acid or L-Aspartic Acid Backbones

Novel cyclic pseudopeptides containing L-glutamic acid or L-aspartic acid backbone structures were efficiently synthesized and characterized. Their chiral recognition properties for L- and D-amino acid methyl ester hydrochloride were discussed also.     

An enhanced β turn in water

Aiming to design short linear peptides featuring strong intramolecular hydrogen bonds in water, a series of tetrapeptides based on the sequence Ac-Ala-Pro-Ala-Ala-NH(2) containing all possible combinations of L- and D-amino acids was synthesized. A regiospecific combination of heterochiral residues (DDLL or its mirror image LLDD) can be used to increase turn formation and stability within short peptides in water.     

Determination of enantiomeric purity in biogenic11C-labelled amino acids by aminoacylation of tRNA

In syntheses of naturally occurring amino acids labelled with short-lived radionuclides, such as¹¹C, the determination of the enantiomeric purity presents problems. The aminoacyl coupling of L-amino acids to tRNA followed by a separation with gel filtration of the tRNA-amino acid complex and free amino acid, was here shown to be an adequate method for determining the enantiomeric purity of [methyl-¹¹C]-methionine even in the pmol range. This method has also been used for the determination of the specific radioactivity of the¹¹C-labelled methionine. The method is probably valid for other naturally occurring amino acids and it might also be of interest for enantiomeric separations of L- and D-amino acids of high specific radioactivity.     

Correlation Study on Sweetness of Amino Acid with Different Configurations and Quantum Chemical Parameters

1 INTRODUCTION Since the introduction of QSAR by Hansch and Fujita in 1964, Deutsch and Hansch have quickly used it in the study of nitrophenylamine sweet reagents. They found good correlation between their distribution coefficients in octanol/water system and sweetness degree. Subsequently, they detected that vibration of aroma-substituent compounds has so- mething to do with sweetness. Henceforth, statistic correlations between structure and sweetness ofseries compounds have been inv…     

Chromatographic determination of amino acid enantiomers in beers and raw materials used for their manufacture

Using gas chromatography (GC) on a chiral stationary phase, accompanied by high-performance liquid chromatography, beers and raw materials used for manufacturing (hops, barley grains, malts) were investigated for the pattern and quantities of amino acid enantiomers. Although L-amino acids were most abundant, certain D-amino acids were detected in all beers and most of the raw materials. Highest amounts of D-amino acids were detected in special beers such as Berliner Weisse that underwent bottle-conditioning with lactic cultures, and Belgian fruit beers produced by spontaneous fermentation. It is demonstrated that GC on chiral stationary phases is highly suitable for the quantitative determination of amino acid enantiomers in beers and raw materials used for their manufacture. Quantities, relative amounts and pattern of amino acid enantiomers can serve in particular as chiral markers for the authenticity of special beers.     

Homochirality and life

Before the emergence of life, left-handed amino acids (L-enantiomers) were selected and right-handed amino acids (D-enantiomers) were eliminated on the primal earth. Nevertheless, with the progress of analytical methods, D-amino acids have recently been found in higher order living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids have numerous physiological functions. D-amino acids containing animal peptides are well known as opioid peptides. D-amino acids in protein are related to aging. In this review, we describe the D-amino acids that are present and function as D-amino acid biosystems in our bodies.     

Capillary zone electrophoresis of alpha-helical diastereomeric peptide pairs with anionic ion-pairing reagents

The present study uses an unique capillary electrophoresis (CE) approach, that we have termed ion-interaction capillary zone electrophoresis (II-CZE), for the separation of diastereomeric peptide pairs where a single site in the centre of the non-polar face of an 18-residue amphipathic alpha-helical peptide is substituted by the 19 L- or D-amino acids. Through the addition of perfluorinated acids at very high concentrations (up to 400 mM), such concentration levels not having been used previously in chromatography or CE, to the background electrolyte (pH 2.0), we have been able to achieve baseline resolution of all 19 diastereomeric peptide pairs with an uncoated capillary. Since each diastereomeric peptide pair has the same sequence, identical mass-to-charge ratio and identical intrinsic hydrophobicity, such a separation by CZE has previously been considered theoretically impossible. Excellent resolution was achieved due to maximum advantage being taken of even subtle disruption of peptide structure/conformation (due to the presence of D-amino acids) of the non-polar face of the amphipathic alpha-helix and its interaction with the hydrophobic anionic ion-pairing reagents. In addition, due to the excellent resolution of diastereomeric peptide pairs by this novel CZE approach, we have also been able to separate a mixture of these closely-related alpha-helical peptides.     

Molecular recognition on supramolecular systems (XXXV)-- Synthesis of novel b-cyclodextrin derivative bearing pyridinio group and its chiral discrimination of amino acids

A novel b-cyclodextrin derivative 4 bearing a pyridinio group on the primary side was synthesized by the reaction of 2-aminopyridine with 6-b-cyclodextrin monoaldehyde 3, and its complexation stability constants with several aliphatic amino acids have been determined in phosphate buffer solution ( pH = 7.2, 0.1 mol·L-1) at 25 ℃ by using spectrofluormetric titrations. The stoichiometry is 1︰1 for the inclusion complexation of amino acids with compound 4. Circular dichroism study indicates that the aromatic moiety was embedded shallowly into the cyclodextrin cavity. As a spectral probe, the pyridinio group in the modified cyclodextrin can recognize not only differences of the size and shape of amino acid molecules, but also the L/D-amino acid chiral isomer. As com-pared with mono-[6-(1-pyridinio)-6-deoxy]-b-cyclodextrin 5, compound 4 switched the enantiomer preference for L- to D-isomer, and showed the highest enantioselectivity of 5.4 for D/L-serine. The-se results are discussed from the viewpoints of geometric compensation, induced-fit concept and cooperation of several weak interactions.     

Temperature selectivity effects in reversed-phase liquid chromatography due to conformation differences between helical and non-helical peptides

In order to characterize the effect of temperature on the retention behaviour and selectivity of separation of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four series of peptides, with different peptide conformations, have been studied as a function of temperature (5-80 degrees C). The secondary structure of model peptides was based on either the amphipathic alpha-helical peptide sequence Ac-EAEKAAKEX(D/L)EKAAKEAEK-amide, (position X being in the centre of the hydrophobic face of the alpha-helix), or the random coil peptide sequence Ac-X(D/L)LGAKGAGVG-amide, where position X is substituted by the 19 L- or D-amino acids and glycine. We have shown that the helical peptide analogues exhibited a greater effect of varying temperature on elution behaviour compared to the random coil peptide analogues, due to the unfolding of alpha-helical structure with the increase of temperature during RP-HPLC. In addition, temperature generally produced different effects on the separations of peptides with different L- or D-amino acid substitutions within the groups of helical or non-helical peptides. The results demonstrate that variations in temperature can be used to effect significant changes in selectivity among the peptide analogues despite their very high degree of sequence homology. Our results also suggest that a temperature-based approach to RP-HPLC can be used to distinguish varying amino acid substitutions at the same site of the peptide sequence. We believe that the peptide mixtures presented here provide a good model for studying temperature effects on selectivity due to conformational differences of peptides, both for the rational development of peptide separation optimization protocols and a probe to distinguish between peptide conformations.     

谷胱甘肽与D型氨基酸非共价复合物的质谱

为了研究谷胱甘肽和D型氨基酸的非共价相互作用, 将一定化学剂量比的还原型谷胱甘肽与D-苯丙氨酸、D-组氨酸或D-谷氨酰胺在室温下混合后, 温育1 h, 使反应达到平衡. 电喷雾质谱测量结果表明, 在生理pH条件下, 谷胱甘肽可以和D 型氨基酸经反应生成非共价复合物. 串级质谱中的碰撞诱导解离(CID)以及紫外光谱进一步确认了非共价复合物的生成. 为了避免严重的离子化效率和质谱信号相互抑制作用, 对谷胱甘肽和D-谷氨酰胺的相互作用作了定量的评估. 配制一系列不同初始浓度的谷胱甘肽和D型氨基酸的混合溶液, 并用电喷雾质谱测定混合溶液中不同质点的质谱峰强度, 计算了谷胱甘肽与三个D型氨基酸结合形成的复合物的解离常数. 计算结果表明, 谷胱甘肽可以和D型氨基酸结合形成不同键合强度的非共价复合物, 其稳定性按照D-谷氨酰胺、D-苯丙氨酸、D-组氨酸的次序逐渐增大.     

Enantioselectivity in Ni(II) Schiff-base complexes derived from amino-acids and (S)-o-N-(N-benzylprolyl)aminobenzophenone: molecular structure of several chiral Ni(II) Schiff-base complexes, circular dichroism and molecular mechanics studies

Several Ni(II) complexes derived from (S)-o-N-(N-benzylprolyl)aminobenzophenone ((S)-BBP) and amino acids of general formula [Ni((S)-BBP-L-(or D-)-aa)] were prepared. The crystal and molecular structures of [Ni((S)-BBP-Gly)], [Ni((S)-BBP-L-Ser)] and [Ni((S)-BBP-L-aaIm)](aaIm =L-2-amino-3-(imidazol-1-yl)propanoate were determined by X-ray diffraction analysis. In the three complexes the nickel atoms display a square-planar coordination and the overall structure around the metal indicates that the entire Schiff-base ligands form quite rigid frameworks. Molecular mechanics calculations were carried out for complexes [Ni((S)-BBP-Gly)], [Ni((S)-BBP-Ser)] and [Ni((S)-BBP-aaIm)] containing either the L- or D-amino acid forms, and the factors controlling the stereoselectivity are discussed. Several other [Ni((S)-BBP-L-aa)] complexes are also prepared and their circular dichroism spectra in solution and of the solids dispersed in KBr disks are measured and discussed. In agreement with other studies in solution with similar [Ni((S)-BBP-aa)] complexes, the Cotton effects for the bands with lambda(max) at 520--530 nm are positive when the amino acids have the L-configuration at the alpha-carbon. The same is observed in this work for the solid-state CD spectra of all compounds.