乙醇系统HPLC分析PTC－氨基酸的条件优化

首次报道用乙醇系统分析ＰＴＣ－氨基酸的新方法中各衍生物获得最佳分离的建立过程。ＰＴＣ－氨基酸衍生后溶于Ａ溶液,然后进样于４μｍＮｏｖａＰａｋＣ１８柱（３．９ｍｍ×１５０ｍｍ）。系统的优化步序包括全面调控流动相的ｐＨ值与ＴＥＡ浓度、乙醇梯度程序、柱温等诸多影响ＨＰＬＣ色谱行为的因素。最适条件为：Ａ溶液含０．１４ＭＧ酸钠、０．７５ｍｌ／ＬＴＥＡ、ＰＨ６．３５;Ｂ溶液为１００％乙醇;柱温３０℃。通过优化的乙醇梯度最终在约４４ｍｉｎ内将１５种ＰＴＣ－氨基酸很好地分离。此法可用于替代代表新科技水平的ＰＴＣ－氨基酸乙腈分析系统。     

氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

Enantioselective Liquid–Liquid Extraction of Zopiclone With Mandelic Acid Ester Derivatives

Enantioselective liquid–liquid extraction of zopiclone was conducted by employing a series of (R)‐mandelic acid esters as chiral extractants. The effects of concentration of extractant, concentration of zopiclone, type of organic solvent, pH value, and temperature on the extraction efficiency were investigated. (R)‐o‐chloromandelic acid propyl ester was demonstrated to be an efficient chiral extractant for zopiclone resolution with a maximum enantioselectivity of 1.6. Chirality 25:952–956, 2013. © 2013 Wiley Periodicals, Inc.     

Unusual solvent effect on protease activity and effective optical resolution of amino acids by hydrolytic reactions in organic solvents

Summary The catalytic activities of -chymotrypsin, subtilisin Carlsberg, and subtilisin BPN' for hydrolysis of amino acid esters in acetonitrile-water were unusually dependent on the solvent composition. The products obtained as precipitates in high concentrations of acetonitrile were L-amino acids of high optical purities, and effective optical resolution of amino acids was achieved.     

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography.

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluene-sulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

Postnatal hypoglycaemia and gluconeogenesis in the newborn rat. Delayed onset of gluconeogenesis in prematurely delivered newborns.

A normal-phase high-pressure liquid-chromatography system was used with amino-propyl-bonded silica as the column packing in order to resolve amino acids, reduced amino acid derivatives and reduced cross-links of collagen. The method utilized a solvent system comprising acetonitrile and 10 mM-potassium phosphate buffer, pH 4.3, as described by Schuster [(1980) Anal. Chem. 52, 617-620]. With modifications to the original gradient elution it was possible to resolve fully the radiolabelled components of reduced collagen in one run of less than 80 min. The method is very sensitive, and small biopsy samples can readily be investigated. Although solute retention times gradually decreased with repeated use, little loss of resolution of the reducible cross-links of collagen occurred during a 30-day trial period.     

Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum

The effects of organic nitrogen on the metabolism of Clostridium acetobutylicum were investigated in batch fermentations. For this study, amino acids were added to a chemically defined medium in groups from the same biosynthetic pathways. In all cases the addition of amino acids shifted the solvent ratio to higher butanol production at the expense of that of acetone (except for the glutamic acid group) and ethanol (except for histidine). Highest biomass production was obtained from media containing aromatic amino acids and histidine (4.57 g · l⁻¹ and 5.4 g · l⁻¹, respectively). However, the solvent production (ca. 20 g · l⁻¹) and the solvent yield (ca. 33%) in both cases, were similar to those obtained from the synthetic medium. Lower values were obtained from fermentations carried out with other families of amino acids. The strongest inhibition of cell growth (1.13 g · l⁻¹) which related to the lowest solvent production (3.15 g · l⁻¹) was observed on a medium complemented with amino acids of the pyruvic acid group. During the second phase of fermentation, amino acids-complemented media caused a less efficient remetabolization of acetic and butyric acids. Highest production of acids was obtained with the aspartic acid group (7.4 g · l⁻¹). These observations suggest that amino acids can be used as a competitive nitrogen source and also modify the level of enzyme activities involved in acid and solvent production.     

Enantioselective resolution of 4‐chloromandelic acid by liquid–liquid extraction using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid

A liquid–liquid extraction resolution of 4‐chloro‐mandelic acid (4‐ClMA) was studied by using 2‐chloro‐N‐carbobenzyloxy‐L‐amino acid (2‐Cl‐Z‐AA) as a chiral extractant. Important factors affecting the extraction efficiency were investigated, including the type of chiral extractant, pH value of aqueous phase, initial concentration of chiral extractant in organic phase, initial concentration of 4‐ClMA in aqueous phase, and resolution temperature. It was observed that the concentration of (R)‐4‐ClMA was much higher than that of (S)‐4‐ClMA in organic phase due to a higher stability of the complex formed between (R)‐4‐ClMA and 2‐Cl‐Z‐AA. A separation factor (α) of 3.05 was obtained at 0.02 mol/L 2‐Cl‐Z‐Valine dissolved in dichloromethane, pH of 2.0, concentration of 4‐ClMA of 0.11 mmol/Land T of 296.7K.     

The effect of peptide length on the cleavage kinetics of 2-chlorotrityl resin-bound ethers.

Different characteristics of cleavage kinetics of resin-bound amino alcohols and their peptide derivatives were observed in acid containing protic and aprotic solvent mixtures. The hydrolysis reactions are hindered by steric crowding around the cleaving C--O bond and accelerated by the special solvation effect of CF(3)CH(2)OH on the peptide chain as well as the increase of the strength and concentration of the acid. In trifluoroacetic acid containing mixtures, trifluoroacetylation of the peptide alcohols was detected. The appearance of O-trifluoroacetyl serine and threonine derivatives is detected in cleavage mixtures containing trifluoroacetic acid in anhydrous solvent.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Influences of salts on high-performance liquid chromatography of leukotrienes

The effects of concentrations and kinds of salts on the resolution of leukotrienes (LT) C4, D4, E4, and B4 were investigated by two kinds of reversed-phase high-performance liquid chromatography columns (muBondapak C18 and Novapak C18). When a mobile phase (acetonitrile/methanol/water) with a lower concentration of acetic acid (0.02-0.1%) at pH 5.6 was used, LTC4 and LTD4 were not eluted from the muBondapak column. On the Novapak column, LTC4 and LTD4 were eluted, but they were poorly resolved. When the concentration of acetic acid in the mobile phase was raised to 1.0% and adjusted to pH 5.6 with ammonium hydroxide or triethylamine, excellent resolution of LTs was obtained. Sodium hydroxide was, to some extent, useful for the pH adjustment of the mobile phase. Sodium chloride could not be substituted for acetic acid-ammonium hydroxide or -triethylamine salt. The resolution of LTC4, LTD4, and LTE4 was affected more strongly than that of LTB4 by changes of concentrations and kinds of salts. When the acetonitrile/methanol/water/acetic acid solvent system adjusted to pH 5.6 with triethylamine was applied to the analysis of the leukotrienes produced from rat peritoneal cells with stimulation of calcium ionophore A23187, de novo-synthesized LTC4, LTD4, LTB4, and isomers were clearly separated. This solvent system may be useful for the investigation of variations in the synthesis of subclasses of LTs with different stimuli and under different circumstances.     

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.     

Gas chromatographic determination of the optical purities of amino acids using N-trifluoroacetyl menthyl esters

Nineteen α-amino acids and one β-amino acid were resolved as their N-TFA menthyl esters by gas-liquid chromatography. Neutral and basic amino acids were well resolved on some conventional packed columns. For the complete resolution of acidic amino acids, a capillary column was used. The results of the applications to some biochemical substances showed that this method is of value for practical usage. The mechanism of the resolution was also investigated and showed that the difference between the derivatives of the enantiomeric pairs in the ability to form intermolecular hydrogen bond contributes essentially to the resolution.     

反相高效液相色谱法测定烟叶中的游离氨基酸

用不同浓度的乙醇溶液提取烟样中的游离氨基酸 ,结果显示 ,存在最佳的乙醇溶液浓度 ,使烟样中被提取的游离氨基酸总量最大 ;对比了活性炭、乙醚、5 %磺基水杨酸、阳离子交换柱的纯化效果 ,发现阳离子交换柱的纯化效果较其它三种试剂要好。在提取和纯化之后 ,采用OPA、FMOC联合在线衍生反相高效液相色谱法测定了烟样中的游离氨基酸 ,该方法使烟样中的氨基酸和亚氨基酸能被同时测定 ,并且分析方法的重现性和回收率均令人满意。最后用该方法对云南B2 F98(上部、橘黄、二等烟叶 ,98年产 )烟叶中的游离氨基酸进行了测定 ,有 15种氨基酸被测出 ,其中Pro含量最高 ,约占总量的 2 5 % ,Thr含量最低 ,约占总量的 1%。     

杜仲中环烯醚萜类化合物的提取工艺研究

本文研究了杜仲中具有生物活性的环烯醚萜类化合物的最佳提取工艺。采用正交设计表L16(4^5),以提取时间、提取温度、提取次数、提取剂A的浓度及料液比为因素进行实验优化。桃叶珊瑚甙的含量由分光光度法测得,京尼平甙酸和京尼平甙的含量通过高效液相色谱法测定。根据试验结果,提取桃叶珊瑚甙的最佳工艺是：70℃下提取三次,每次0．5小时,溶剂A的浓度为80％,料液比为1：12。而京尼平甙和京尼平甙酸的最佳提取工艺为：在70℃下提取一次,提取时间为1小时,溶剂A的浓度为50％,料液比也为1：12。在此工艺条件下,选取了较佳的提取原料。实验表明在实际应用中,应根据提取目标和原材料选择较好的提取工艺。     

The Influence of Measurement Parameters on the Specific Rotation of Amino Acids

The effects of solute and hydrochloric acid concentrations on optical rotation were studied using 20 naturally occuring amino acids.

There appeared to be no common factor among the amino acids as far as the inclination of optical rotation was concerned. Lutz-Jirgenson’s rule could be applied to few amino acids in the cationic form. Therefore, in the determination of the optical rotation, the concentration of the solute, nature of solvent and temperature must be rigorously controlled. The optical conditions of measurement and the specific rotation of 20 amino acids were recommended based on this work.     

High-performance liquid chromatographic enantioseparation of unusual secondary amino acids on a D-penicillamine-based chiral ligand exchange column

The application of a chiral ligand-exchange column (CLEC) for the direct high-performance liquid chromatographic enantioseparation of unusual secondary amino acids using D-penicillamine-Cu(II) complex as chiral selector is reported. The amino acids investigated were pyrrolidine-2-carboxylic acid, piperidine-2-carboxylic acid, piperazine-2-carboxylic acid, morpholine-3-carboxylic acid, and thiomorpholine-3-carboxylic acid analogs. Chromatographic results are given as the retention, separation, and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The elution sequence of the enantiomers was determined and in most cases the S isomer eluted before R.     

Intracellular monosaccharide and amino acid concentrations and activities and the mechanisms of insulin action.

Current amino acid and monosaccharide transport models are based on an assumption which equates the intracellular chemical activity of a solute with its concentration. This assumption was tested for alpha-aminoisobutyric acid and 3-O-methylglucose in a giant cell, the amphibian oocyte, by using recently developed cryomicrodissection and internal reference phase techniques. We found the following. (i) alpha-Aminoisobutyric acid and 3-O-methylglucose activities were much greater in cytoplasm than was suggested by concentration data; i.e., activity coefficients were higher than in ordinary water solutions. This is attributable to the inaccessibility of considerable water as solvent (solute exclusion). (ii) Solute concentrations varied regionally as follows: nucleus > > animal cytoplasm > vegetal cytoplasm. Insulin increased the nucleus/cytoplasm concentration asymmetry, apparently by increasing cytoplasmic solute exclusion. (iii) Nuclear activity coefficients more closely resembled those of ordinary saline solutions so that nucleus/ extracellular concentration ratios reflected transmembrane activity gradients better than did cytoplasm (or whole cell)/extracellular ratios. (iv) Mediated passive alpha-aminoisobutyric acid and 3-O-methylglucose transport were constituent oocyte membrane properties. Membrane active transport was initiated with time (in the presence of substrate) and by insulin. (v) Increased temperature mimicked insulin in enhancing transmembrane alpha-aminoisobutyric acid activity gradients and increasing the nucleus/cytoplasm concentration asymmetry. These results indicated that concentration data are a misleading measure of cellular amino acid and monosaccharide activity; some consequences of this observation were explored. A model is proposed in which cell water has reduced solvent capacity or is compartmentalized (considered less likely) and is susceptible to physiological modulation. The model accounts for many observations in small cells, suggesting generality of the exclusion phenomenon and a previously unrecognized metabolic control mechanism.     

Amino acid composition and level affect Bt protein concentration in Bt cotton

Low insect resistance has been widely observed in Bacillus thuringiensis (Bt) transgenic cotton. The objective of this study was to investigate the effects of amino acid compositions and their concentration on the Bt toxin content in Bt cotton to gain a better physiological understanding of the observed phenomenon. In the 2010 study, the leaf amino acid composition, contents, and Bt protein concentrations were investigated under high temperature (36?°C) and low temperature(18?°C) at both square and boll stage respectively. In 2011, the leaves were sprayed with ethionone sulfoximine (MSO), aminooxyacetic acid (AOA) or combination of MSO and AOA at the two developing stages, and the leaf amino acid composition and Bt protein concentrations were investigated. In 2012, 17 amino acid compositions were used to spray cotton plants at the same two stages, and the Bt protein concentration were measured. In comparison to the control, more amino acids changed and greater concentration changes had been observed under the extreme temperatures at boll stage compared to that at square stage, which were conformity to the changes of the leaf Bt protein concentration. More amino acids reduced and greater reduction was noted with the application of MSO, AOA or combined MSO and AOA at boll period than that at square period, which resulted in the reduction of the leaf Bt toxin concentration. The external uses of Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin enhanced the leaf Bt toxin level significantly at boll stage. However, only Aspartic acid, Glutamic acid, Proline, Methionine, Arginin could increase Bt protein concentration markedly at square period and the bolstered extent were low. Therefore, it was concluded that cotton amino acid composition and concentration was more sensitive to external factors at boll stage. In addition, there are close relationship between leaf Bt toxin content and Aspartic acid, Glutamic acid, Glycine, Proline, Tyrosine, Methionine, Phenylalanine, Histidine and Arginin and their concentrations.