Thermospray liquid chromatography/mass spectrometry study of diastereomeric isoindole derivatives of amino acids and amino acid amides

A thermospray liquid chromatography/mass spectrometry (TSP-LC/MS) method is described for determination of the enantiomeric excess of -amino acids and -amino acid amides as their

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(OPA/NAC) derivatives.

The source temperature is an important factor in optimizing the sensitivity of the TSP-LC/MS analysis, whereas the repeller voltage is of minor importance. On-column mass spectra were acquired for the OPA/NAC derivatives of several -amino acids and -amino acid amides. For the main fragment ions, mass spectra fragmentation pathways are proposed. The applicability of the method is demonstrated by means of the enantiomeric excess determination of valine in a sample from an enzymatic hydrolysis experiment.

Using single ion monitoring, the detection limit of -valine in the presence of excess -valine is 10 pmol. The present TSP-LC/MS method is useful for validating the results obtained from LC/UV or LC/fluorescence methods for the enantiomeric excess determination of -amino acids and -amino acid amides.     

蛋清溶菌酶部分酶学性质及酶活性的影响因素研究

目的探讨将蛋清溶菌酶制成液体制剂的可行性。方法通过改变不同影响因素测定蛋清溶菌酶的活性,观察几种常用辅料对蛋清溶菌酶活性的影响。结果酶活性在pH 6.0~6.5最强,且在pH 5~7范围内较稳定;在25~65℃范围内随着作用温度的升高酶的活性增强,但温度太高则变性失活;Na+、K+对其活性有轻微激活作用,Mn2+、Mg2+对溶菌酶活性无明显影响,Co2+、Ca2+、Cu2+、Fe2+、Zn2+使溶菌酶的活性下降;吐温20、吐温80、甘油溶液对酶活有抑制作用;EDTA-2Na在0.000 5%~0.300 0%浓度范围内对溶菌酶活性具有激活作用,浓度继续增加反而有抑制活性作用。结论初步试验结果表明溶菌酶可以制成合适的液体制剂。     

大海马和刁海龙氨基酸与脂肪酸的组成分析与评价

目的 研究野生大海马、养殖大海马以及刁海龙三者间氨基酸与脂肪酸的组成和含量差异,旨在为野生海马的高效利用及其适宜替代生物的探寻提供依据。方法 氨基酸自动分析仪测定氨基酸含量,气相色谱-质谱联用仪（GC-MS）测定脂肪酸组成与含量。结果 野生与养殖大海马和刁海龙均含有测定的17种氨基酸,含量范围在43.207～74.827 g/100g DW之间,其中养殖大海马的含量最高,刁海龙次之,野生大海马最低;大海马皮膜的氨基酸含量要高于骨粉,而刁海龙则相反;三者限制性氨基酸中均包含亮氨酸,必需氨基酸指数均较高。刁海龙的不饱和脂肪酸含量高于大海马,尤其是EPA和DHA,占总脂肪酸含量的25.41%和32.74%,远高于后者的2.55%和3.87%;其余脂肪酸相近;养殖大海马与野生大海马间的差异不大。结论 野生大海马的皮膜与骨粉中氨基酸和脂肪酸组成与含量相近,可合理开发;养殖大海马在营养评价方面略优于刁海龙,可优先选择作为野生大海马的替代,刁海龙氨基酸与脂肪酸的营养品质也高于野生大海马,尤其是EPA和DHA含量显著高于野生大海马,可合理选择作为替代。     

Evaluation of the impurity profile of amino acids by means of CE

The aim of this study was to develop a general approach for characterizing the impurity profile of amino acids at level 0.1% by means of capillary electrophoresis (CE). Checking a variety of labeling reagents revealed 9-fluorenylmethyl chloroformate to be favorable, due to the high stability of its derivatives and the fact that the reagent peaks do not interfere with the peaks of the impurities. After optimization, the method was sufficiently sensitive to evaluate impurities at a 0.1% level by UV detection. The method was representatively validated for phenylalanine (Phe) with regard to selectivity, precision, linearity and accuracy using model mixtures of potential impurities. The CE analyzes method was applied to Phe samples of different manufacturers and the capabilities of the strategy was also demonstrated by samples of tryptophan and serine.     

电喷雾离子化质谱分析环孢菌素的组成氨基酸

应用电喷雾离子化质谱对9个环孢菌素类化合物强酸水解后的产物进行分析,获得环孢菌素类化合物一些组成氨基酸的特征分子离子峰,特别是1位上特殊的氨基酸-MeBmt,实验结果表明本方法在测定环孢菌素中的MeBmt和其它组成氨基酸方面具有进样量少、快速、准确,无需对照品等优点,可用于环孢菌素类化合物的初步快速鉴别。     

4-Toluenesulfonylureido derivatives of amines, amino acids and dipeptides: a novel class of potential antitumor agents

The screening of a series of arylsulfonylureido derivatives of amines (such as histamine, or dopamine), aliphatic/aromatic amino acids (such as Gly, β-Ala, Val, Lys, Arg, Phe, Tyr, DOPA, etc.) and dipeptides (such as GlyGly, β-AlaHis) led to the identification of three derivatives that possess tumor growth inhibitory properties against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, and breast cancer cell lines in vitro. The new derivatives were prepared by reaction of 4-toluenesulfonyl isocyanate with (protected) amines, amino acids or dipeptides. The mechanism of antitumor action with these new derivatives is not known at the moment but it may imply uncoupling of mitochondria, as for the structurally related diarylsulfonylurea sulofenur, an investigational anticancer agent.     

海南株和北京株白纹伊蚊水解氨基酸的比较

目的：比较海南、北京两地理株白纹伊蚊水解氨基酸含量的差异。方法：对羽化后5天两地理株白纹伊蚊分别用DV－70型紫外分光光度仪和氨基酸自动分析仪6300型测定色氨及其余17种氨基酸含量，并比较两地理株间的差异。结果：海南株与北京株均测出17种氨基酸，其中有8种存在量上的显著差异。结论：不同地理株白纹伊蚊氨基酸含量可有差异，这种差异是否与白纹伊蚊不同地理株对登革病毒易感性的不同有关值得深入研究。     

岗松中氨基酸和维生素E的成分分析

目的分析岗松中氨基酸和维生素E的成分。方法用氨基酸分析仪和液相色谱仪,对岗松中氨基酸和维生素E成分进行分析,并对水解氨基酸和游离氨基酸含量进行比较。结果鉴定出18种氨基酸成分,岗松中水解氨基酸总量为41.8mg.g-1,游离氨基酸总量为0.7089 m.gg-1,维生素E总量为0.1970 m.gg-1。结论岗松中含部分氨基酸和维生素E,氨基酸主要以结合状态存在。     

Design,synthesis, and antimalarial evaluation of thiazole-derived amino acids

A series of thiazole-derived N-Boc amino acids were synthesized and evaluated as targeted potential antimalarials against plasmepsins II enzyme of malaria parasite Plasmodium falciparum. All the compounds showed moderate to good activity. Compounds 3f and 3g were found to have highest the 50% inhibitory concentration (IC₅₀) values (3.45 μM and 4.89 μM, respectively) against Plasmodium falciparum. The compounds docked to the active site of plasmepsin II. Most of the compounds were found to interact with the catalytic amino acids ASP34 and ASP214 of plasmepsin II. A good correlation was observed between binding energy and antiparasitic activity of the thiazole derivatives.     

环丙沙星致痫大鼠脑脊液氨基酸含量的变化

目的观察环丙沙星(CPLX)致痫大鼠脑脊液(CSF)中兴奋性氨基酸[天冬氨酸(Asp)、谷氨酸(Glu)]和抑制性氨基酸[7-氨基丁酸(GABA)、甘氨酸(Gly)]含量的变化,探讨CPLX致痫的发生机制.方法给雄性大鼠静脉注射CPLX 100mg/kg,于用药前,在小脑延髓池抽取CSF作为对照,用药后癞痫发作40min时抽取CSF作为试验样本.CSF中氨基酸的含量用高效液相色谱法测定.结果癫痫发作后40min时,CSF中7-氨基酸(GABA)含量为(1.72士0.94μ)mol/L,明显高于用药前的(0.31士0.23)μmol/L(P＜0.01),而天冬氨酸(Asp)、谷氨酸(Glu)、甘氨酸(Gly)的含量分别为(9.64士2.62)μmol/L、(9.54士8.58)μmol/L、(18.96±2.84)μmol/L,均明显低于用药前的(14.57±5.85)μmol/L、(16.19士7.56)μmol/L、(27.66士8.01)μmol/L(P＜0.05～0.01).结论静注CPLX引起大鼠癫痫发作是由于CPLX明显影响了中枢神经系统内兴奋性氨基酸和抑制性氨基酸的代谢平衡,氨基酸含量的变化并可通过CSF中的氨基酸含量反映出来.     

氨基酸对利用红色糖多孢菌发酵生产红霉素的产量及组分影响研究

研究红色糖多孢菌发酵生产红霉素过程中几种氨基酸对产量及组分的影响。通过摇瓶实验证实了0h时向合成培养基中分别添加数种氨基酸，对红霉素的产量与组分都有着重要的影响。本文重点研究甘氨酸在红色糖多孢菌发酵中对产量和组分的影响，试验结果表明，0h添加0．05％的甘氨酸产量明显提高（72．2％），红霉素A组分相对百分含量提高9．7％，红霉素C组分减少78．7％。通过50L FUS-50（A）发酵罐进行验证，红霉素产量达到8939u／ml，比对照提高12．2％，EMA：EMC从5．1：1提高到8.2：1。     

薯蓣皂苷元A环和F环的结构改造及其抗肿瘤活性初探

目的提高薯蓣皂苷元类衍生物的抗肿瘤活性以及进一步研究其构效关系。方法基于薯蓣皂苷元结构,运用Autodock 4.2进行对接设计,合成了一系列全新A环、F环结构改造的氨基酸酰胺,内酰胺杂环薯蓣皂苷元类衍生物。采用MTT法对人肝癌细胞HepG-2、人恶性黑色素瘤细胞A375和人肺癌细胞A549进行体外抗肿瘤活性实验。结果合成13个新化合物,结构经1HNMR、13CNMR确定,体外抗肿瘤活性结果表明化合物8～13具有一定活性。结论化合物8～13都具有一定的体外抗肿瘤活性,化合物13的药理学活性与阳性对照品紫杉醇活性相近。     

Analysis and biological properties of amino acid derivates formed by Maillard reaction in foods

Maillard reaction products (MRPs), especially early stage MRPs and melanoidins, are currently gaining a lot of attention due to their reported health-promoting properties and their potential to be used as functional food ingredients. It is often not clear which specific biological function is assigned to which MRP, due to the large amount of MRPs formed during the reaction and difficulties in their purification and identification. This paper provides an overview of amino acid derivatives such as Amadori compounds, carboxymethyllysine, pyrraline, cross-linking products and melanoidins, which can be formed by Maillard reaction in foods, their biological properties and the analytical tools commonly employed for their determination.     

Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.     

Profiling analysis of amino acids from hyperlipidaemic rats treated with Gynostemma pentaphyllum and atorvastatin

Context Gynostemma pentaphyllum (Thunb.) Makino has been used in traditional medicine for the treatment of hyperlipidaemic with a long history.

Objective: The objective of this study was to evaluate the influence of Gynostemma pentaphyllum (GP) and atorvastatin on amino acids from the plasma and liver tissue of hyperlipidaemic rats.

Materials and methods The rats were fed a high-fat diet continuously for 11 weeks for the construction of hyperlipidaemic model. The hyperlipidaemic rats were treated with Gynostemma pentaphyllum (120?mg/kg) and atorvastatin (1.8?mg/kg) for 4 weeks, and the rats were intragastric administration one time every day. Chromatographic separation was performed on a Shim-pack XR-ODSIII C18 analytical column (75?mm?×?2.0?mm i.d., 1.6?μm, Shmadazu Corp., Tokyo, Japan). The biomarkers of amino acids were identified by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

Results After feeding with a high-fat diet, the TC and LDL-C values of the hyperlipidaemic mode rats increased dramatically (p?<?0.01). The established method allowed a target analysis of 12 kinds of amino acids. PCA studies showed that the plasma amino acids had not returned to normal after GP treatment, but those had recovered slightly after atorvastatin treatment. GP has almost no impact on the metabolism of amino acids, while atorvastatin can modify the metabolism of amino acids via self-regulatory mechanisms.

Discussion and conclusion UPLC/DAD combined with SCX-SPE can be successfully used for profiling analysis of amino acids. By the comparison of biomarkers following treatment with GP and atorvastatin, the influence of the two drugs on biomarkers is revealed.     

Syntheses and structure-activity relationships of seven manganese-salen derivatives as anti-amyloidogenic and fibril-destabilizing agents against hen egg-white lysozyme aggregation

Accumulation of intra- and/or extracellular misfolded proteins as amyloid fibrils is a key hallmark in more than 20 amyloid-related diseases. In that respect, blocking or reversing amyloid aggregation via the use of small compounds is considered as two useful approaches in hampering the development of these diseases. In this research, we have studied the ability of different manganese-salen derivatives to inhibit amyloid self-assembly as well as to dissolve amyloid aggregates of hen egg-white lysozyme, as an in vitro model system, with the aim of investigating their structure-activity relationships. By coupling several techniques such as thioflavin T and anilinonaphthalene-8-sulfonic acid fluorescence, congo red absorbance, far-UV circular dichroism, and transmission electron microscopy, we demonstrated that all compounds possessed anti-amyloidogenic activities and were capable of dispersing the fibrillar aggregates. In addition, MTT assay of the treated SK-N-MC cells with the preformed fibrils formed in the presence of compounds at a drug-to-protein molar ratio of 5:1, indicated a significant increase in the viability of cells, compared to the fibrils formed in the absence of each of the compounds. Our spectroscopy, electron microscopy, and cellular studies indicated that EUK-15, with a methoxy group at the para position (group R(5)), had higher activity to either inhibit or disrupt the β-sheet structures relative to other compounds. On the basis of these results, it can be concluded that in addition to aromatic rings of each of the derivatives, the type and position of the side group(s) contribute to lower lysozyme fibril accumulation.     

异硫氰酸苯酯柱前衍生化RP-HPLC法测定人血浆中10种氨基酸的浓度

目的:建立一种异硫氰酸苯酯柱前衍生化反相高效液相色谱同时测定人血浆中10种氨基酸的方法。方法:以正亮氨酸为内标物,异硫氰酸苯酯为柱前衍生剂,Venusil-AA柱为分析柱,柱温40℃,采用二元梯度洗脱,254 nm波长处检测。结果:氨基酸血浆浓度在15.6~250μmol/L时,其峰面积与内标物峰面积的比值和氨基酸血浆浓度的线性相关系数,均大于0.99;10种氨基酸的加样回收率在96.3%~103.4%;日间和日内变异系数均在4.8%以下。应用该方法对小儿血浆中氨基酸含量进行了测定,取得了满意的结果。结论:该法简便、稳定,并且分离效果好,可用于人血浆中氨基酸浓度的检测。     

缺氧和/或缺糖对大鼠突触体游离钙浓度和氨基酸释放的影响

目的：研究缺氧和/或缺糖对大鼠突触体游离钙浓度和氨基酸释放的影响。方法：营养液中去除糖和/或氧，建立大鼠脑突触体缺氧、缺糖和缺血模型。检测静息及高钾去极化状态下，各种模型突触体游离钙浓度及兴奋性氨基酸(EAA)和抑制性氨基酸(IAA)释放量的变化。结果：与正常突触体相比，缺氧60min后，静息和KCl除极突触体的游离钙浓度无明显改变，而缺糖导致静息和KCl除极突触体游离钙浓度显著升高。糖和氧同时去除影响更为明显。缺氧和/或缺糖对大鼠突触体氨基酸释放的影响与此相似，缺糖对突触体氨基酸释放的影响比缺氧更明显。其中EAA门冬氨酸和IAA甘氨酸、牛磺酸、γ-氨基丁酸释放量明显增加。而谷氨酸亦有增加趋势。结论：缺糖和缺氧在缺血致脑损伤中的机制可能不同，其中缺糖有更为重要的作用。缺糖和/或缺氧致脑损伤过程中IAA的增加可能是机体的一个自我保护机制。     

Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid

The temporal inter-relationship of various amino acids and monoamine metabolites in rat cerebrospinal fluid was examined after acute administration of milacemide (100, 200 or 400 mg/kg i.p.), a glycine prodrug. Glycine concentrations rose linearly and dose dependently (20–190%) but were only significantly elevated at the higher milacemide dose (200 and 400 mg/kg). In animals given 400 mg/kg, glycine values were still significantly elevated 8 h later. A concomitant increase (20–25%) in serine and taurine and a decrease in alanine cerebrospinal fluid values were observed at the highest milacemide dose. Other amino acids were unaffected. While cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations were unaffected, the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, exhibited a linear dose-dependent reduction. However, only homovanillic acid values were significantly decreased after 400 mg/kg milacemide. Cerebrospinal fluid analysis may be useful as a first screen in ascertaining putative neurochemical changes associated with drug administration.     

红景天苷衍生物的合成及其抗疲劳活性研究

目的 设计并合成系列4-取代的新型红景天苷衍生物并研究其抗疲劳作用。方法 通过五乙酰基葡萄糖与不同的4-取代苄基酪醇,经过糖苷化、脱乙酰基合成红景天苷衍生物;采用小鼠负重游泳为实验模型,通过观察测定小鼠的负重游泳时间研究合成衍生物的抗疲劳作用。结果 合成了10个新型红景天苷衍生物;小鼠负重游泳实验显示：阳性对照组（红景天苷）和3a-1组（苯乙基-β-D-葡萄糖苷）的小鼠游泳时间明显高于空白对照组（P<0.05）,差异有统计学意义;其余组均与空白对照组小鼠负重游泳时间相近,差异无统计学意义。结论 本实验设计的红景天苷衍生物合成方法简便、可行;红景天苷及其衍生物苯环上的4位羟基可能与其抗疲劳活性密切相关。