Simultaneous determination of binary diffusion coefficients from multiple response curves by chromatographic measurements

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic D-amino acids in the presence of large amounts of their L-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the D-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of D-Leu were found in all tissues and physiological fluids tested (trace-1.3 nmol/g tissue), and in the urine, the presence of high amounts of D-allo-Ile (D-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. D-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual D-amino acid and the potential need to clarify its unique metabolism in mammals.     

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.     

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.     

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.     

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

Atpresentseparationsandquantitativedeterminationsofaminoacidsbymeansofnewfluorescencereagentsforpre-columnorpost-columnderivatizationinRP-HPLCarestillanactivefiled,developmentshavingbeensummarizedbySnyder'.MostaminoacidsdonotshowUVabsorptionin220-254urn,henceinordertoincreasedetectionsensitivityandimproveselectivity,generallyderivatizationreagentsareemployed.Phenylisothiocyanate(PITC)',OPAand3,5-dinitrobenzoylchloride3arewellknownderivatizationreagefltsforthedeterminationofaminocompounds…     

Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids

Screen-printed three-electrode amperometric sensors incorporating L- and/or D-amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D-amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume.     

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.     

Design,synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4 analogs

A total of 11 novel combretastatin A-4(CA-4) analogs were designed, synthesized, and evaluated for the anti-proliferative effects in tumor cells. The compounds represent four structural classes:(i)hydrogenated derivatives,(ii) ethoxyl derivatives,(iii) amino derivatives and(iv) pro-drugs. Biological evaluations demonstrate that multiple structural features control the biological potency. Three of the compounds, sit-1, sit-2 and sit-3, have potent anti-proliferative activity against multiple cancer cell lines. Their pro-drugs were synthesized to increase water solubility. Structure–activity relationship study and Surflex-Docking were studied in this paper. These results will be useful for the design of new CA-4 analogs that are structurally related to the SAR study.     

Novel enantioselective receptors for N-protected glutamate and aspartate

A series of chiral bisthiourea macrocycles 1-4 have been prepared and their binding properties with various dicarboxylate salts have been examined by using NMR titration and isothermal calorimetry experiments. Macrocycle 1, in particular, favours the 1:1 binding of N-protected L-glutamate and aspartate, but favours 1:2 binding of the corresponding D-amino acids in polar solvents (dimethyl sulfoxide and acetonitrile). The macrocycles, however, do not bind carboxylates at all in the less competitive solvent chloroform. The binding properties of these macrocyles are sensitive to small structural changes as demonstrated by the altered binding properties of macrocycles 2-4 compared with 1.     

Quantitative analysis of 17 amino acids in tobacco leaves using an amino acid analyzer and chemometric resolution

A method was developed for quantifying 17 amino acids in tobacco leaves by using an A300 amino acid analyzer and chemometric resolution. In the method, amino acids were eluted by the buffer solution on an ion‐exchange column. After reacting with ninhydrin, the derivatives of amino acids were detected by ultraviolet detection. Most amino acids are separated by the elution program. However, five peaks of the derivatives are still overlapping. A non‐negative immune algorithm was employed to extract the profiles of the derivatives from the overlapping signals, and then peak areas were adopted for quantitative analysis of the amino acids. The method was validated by the determination of amino acids in tobacco leaves. The relative standard deviations (n = 5) are all less than 2.54% and the recoveries of the spiked samples are in a range of 94.62–108.21%. The feasibility of the method was proved by analyzing the 17 amino acids in 30 tobacco leaf samples.     

Analysis of amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

A gas-liquid chromatographic procedure is described which will separate and quantitate the seventeen amino acids typically found in protein acid hydrolyzates. If present, tryptophan, cysteine and carboxymethylcysteine can also be simultaneously assayed via this method. The amino acids, as their tert.-butyldimethylsilylated derivatives, are readily separable on SE-30 (wall-coated open-tubular) and OV-17 (bonded) capillary columns, as well as on packed gas-liquid chromatographic columns coated with the same liquid phases. Retention times and responses for all amino acids and internal standards are given. Mass spectral analysis of all tert.-butyldimethylsilylated amino acids is presented and displays a characteristic and unique [M- 57] fragment ion for each amino acid which often dominates the mass spectrum. Application of this method is demonstrated using four well characterized proteins.     

新型手性咪唑鎓环番的合成及对氨基酸的对映选择性识别

以L-氨基酸为手性源, 合成了一系列新型手性咪唑鎓环番, 并进行了结构表征. 在碱性条件下, L氨基酸和乙二醛、甲醛缩合生成了(S)-2-(1-咪唑)羧酸钠, 转化为甲酯后与乙二胺进行胺解反应制得开链手性咪唑二酰胺, 然后与二溴化合物在高稀淡技术和无水条件下进行季铵化关环反应, 再进行阴离子交换制得目标分子(4~6). 以手性咪唑鎓环番为主体分子, 研究了对氨基酸及其衍生物的对映选择性识别作用.     

Sulphur in the Strategy of Natural Products Synthesis

Abstract

Syntheses of secondary metabolites of α-and β-mercapto aminoacids are described. The strategy in the first class of compounds consists of using N-hydroxy-α-amino acid derivatives as intermediates and of converting them into α-functionalized, in particular thiol-substituted amino acids; as examples serve syntheses in the gliotoxin and sporidesmin series. In the second class of compounds the cycloaddition products of indole derivatives, having sulfide substituents, and nitroso olefins are transformed into indole alkaloids, in particular tryptathionins, by rearrangement and further reactions. The antitumor agent sparsomycin has been synthesized via an amino sultine and its stereospecific nucleophilic ring opening. Bioassays done with this compound and sixteen of its structural analogs gave insight in the structural features that are required for sparsomycin's antitumor activity.     

Computational design of thermostabilizing D-amino acid substitutions

Judicious incorporation of D-amino acids in engineered proteins confers many advantages such as preventing degradation by endogenous proteases and promoting novel structures and functions not accessible to homochiral polypeptides. Glycine to D-alanine substitutions at the carboxy termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by D-amino acid substitutions. A detailed, molecular understanding of backbone and side chain interactions is important for developing rational, broadly applicable strategies in using D-amino acids to increase protein thermostability. Insight from structural bioinformatics combined with computational protein design can successfully guide the selection of stabilizing D-amino acid mutations. Substituting a key glycine in the Trp-cage miniprotein with D-Gln dramatically stabilizes the fold without altering the protein backbone. Stabilities of individual substitutions can be understood in terms of the balance of intramolecular forces both at the α-helix C-terminus and throughout the protein.     

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…     

The Enantioresolution of Methylthiohydantoin-Amino Acids on R,S-2-hydroxypropyl Derivatized β-Cyclodextrin Bonded Stationary Phase Using the Water-Based Mobile Phase

The enantioresolution of thirteen methylthiohydantoin-amino acids (MTH-amino acids), which are substrates for producing D-amino acids through enzyme-catalyzed hydrolysis, is described on R, S-2-hydroxypropyl derivatized β-cyclodextrin bonded stationary phase (RSP-β-CD CSP) with a water-based mobile phase. The enantioresolution is relatively sensitive to the structural variation of group which is attached to the carbon at position five on methylthiohydantoin moiety and subsequently turns into side-chain group of corresponding D-amino acid produced after hydrolysis. The inclusion complexation is believed to be the mechanism responsible for the observed enantioresolution that cannot be reproduced either on native β-cyclodextrin CSP under the same chromatographic conditions or on both CSPs using the acetonitrile-based mobile phase. Approaches for enantioimprovement include varying the percentage of organic modifier in the mobile phase and using the buffer-typed mobile phase such as triethylammonium acetate (pH 4.1).     

Gas chromatographic separation of protein amino acids in four minutes

A method is presented for analysis of protein amino acids in five minutes, including derivatization. The chemical treatment involves reaction with ethyl chloroformate: if the composition of the reaction medium is optimum this reagent esterifies carboxylic groups simultaneously with derivatization of other reactive groups and the reaction is complete in seconds. The moderately polar silicone OV-1701 was found to be a convenient liquid phase for separation of the derivatives: a five meter capillary column of internal diameter suitable for routine use was able to separate the amino acids in four minutes. Derivatization of arginine was incomplete and it failed to elute from the column.     

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.     

超分子体系中的分子识别研究 5: 单-[6-(1-吡啶)-6-脱氧]-α-和γ-环糊精对氨基酸分子识别的热力学性质

本文用分光光度滴定法测定了单-[6-(1-吡啶)-6-脱氧]-α-和γ-环糊精(1)和(3)与一系列氨基酸在磷酸缓冲溶液中(pH=7.20), 25.0~40.0℃时形成超分子体系的稳定常数, 进而计算了配位焓和配位熵, 并与单-[6-(1-吡啶)-6-脱氧]-β-环糊精(2)的实验结果作了比较。化学计量法表明,所有的氨基酸均与环糊精衍生物形成了1:1的超分子体系。从热力学的观点,讨论了化学修饰环糊精和客体氨基酸的尺寸或形状适合、疏水效应、范德华力和氢键等几种弱相互作用对形成超分子体系的贡献。研究结果发现, 具有正电荷环糊精衍生物的吡啶基, 作为一种分子探针不仅可以识别氨基酸生物分子的尺寸或形状之间的差异, 而且还可以识别L/D-型手性对映体之间的差异, 进一步表明了主-客体间的诱导楔合、几何互补在分子受体选择性键合底物形成超分子体系中的重要作用。     

Enantioselective screen-printed amperometric biosensor for the determination of D-amino acids

D-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of D-amino acids has been constructed by the immobilization of D-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for D-alanine in the concentration range from 5 to 200 microM. Excellent enantioselectivity towards D-amino acids was discovered. During the experiment, D-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.