Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

l ‐Asparaginase (l ‐Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l ‐glutamine (l ‐Gln) and l ‐asparagine (l ‐Asn). To study the cytotoxic effect and the secondary complications of l ‐Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l ‐Asnase for the hydrolysis of l ‐Gln, employing the enzyme‐gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)‐LIF was established for the separation of l ‐amino acids (l ‐Gln and l ‐glutamic acid) and studying the hydrolysis of l ‐Gln by using l ‐Asnase enzyme reactor. Then, using l ‐Gln as target analyte, the enzyme kinetics of l ‐Asnase in free solution, enzyme‐gold nanoparticle conjugates (E‐GNP), and the enzyme‐gold nanoparticle conjugates immobilized in capillary (E‐GNP‐C) were investigated in detail with the proposed MCE‐LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l ‐Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E‐GNP‐C enzyme reactor exhibited the best performance for the hydrolysis of l ‐Gln.     

氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,本文对水解前驱体3-氨基丙基三甲氧基硅烷进行氨基酸分子改性。具体过程包括N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再和3-氨基丙基三甲氧基硅烷反应生成N-（3-三甲氧基硅基）丙基-N′-Fmoc-L-缬氨酰胺后。然后去除Fmoc,得到N-（3-三甲氧基硅基）丙基-L-缬氨酰胺作为氨基酸修饰的硅源前驱体。通过IR、MS、1H-NMR等分析测试手段对合成得到的各个化合物的结构进行了表征。利用正硅酸甲酯（TMOS）和N-（3-三甲氧基硅基）丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶过程来包埋了胰蛋白酶,研究得到最适的固定化条件为,N-（3-三甲氧基硅基）丙基-L-缬氨酰胺的含量为15mol%。在该条件下,固定化胰蛋白酶活力的绝对值是199U,游离酶的酶活力的绝对值是103U, 四甲氧基硅烷直接包埋的固定化酶活力的活性是38 U。在该条件下,杂化硅源得到的固定化酶的活性是以四甲氧基硅烷水解前驱体的固定化酶活性的5倍,杂化硅源固定化胰蛋白酶的最相比游离酶,酶的最高活力提高的几乎2倍。这些结果表明氨基酸分子对水解前驱体修饰以后,水解产生的固定化载体具有良好的生物相容性。通过改性载体制备的固定化酶,对甲醇变性剂的稳定性,对酸碱的抵抗性及热稳定性也有明显地提高。     

Asymmetric β‐Methylation of l‐ and d‐α‐Amino Acids by a Self‐Contained Enzyme Cascade

This report describes a modular enzyme‐catalyzed cascade reaction that transforms l ‐ or d ‐α‐amino acids to β‐methyl‐α‐amino acids. In this process an α‐amino acid transaminase, an α‐keto acid methyltransferase, and a halide methyltransferase cooperate in two orthogonal reaction cycles that mediate product formation and regeneration of the cofactor pyridoxal‐5′‐phosphate and the co‐substrate S‐adenosylmethionine. The only stoichiometric reagents consumed in this process are the unprotected l ‐ or d ‐α‐amino acid and methyl iodide.     

The effect of solvation processes on amino acid‐ and peptide‐silica stationary phases

The surface excess adsorption isotherms of water, acetonitrile, and methanol from binary hydro‐organic mobile phases were investigated on nine home‐made stationary phases with chemically bonded amino acids, dipeptides, and tripeptides using the dynamic minor disturbance method. The stationary phases were modified by the following amino acids: glycine, alanine, phenylalanine, leucine, and aspartic acid. We investigated the influence of the type of immobilized amino acids, in particular their different side chains, on the solvent adsorption. The interpretation of solvation phenomena shows significant accumulation of investigated solvents on the adsorbent surface according to their hydrophilic or hydrophobic properties. Moreover, the accumulated amount was dependent on the length and type of amino acid sequences bonded to the silica surface. Stationary phases with bonded amino acids and peptides show stronger water and acetonitrile adsorption in contrast to the stationary phase modified with aminopropyl groups—a support for the synthesis. The comparison of water and acetonitrile adsorption as well as a data obtained with the two‐site adsorption model reveal and confirm the heterogeneity of chemically bonded phases. As a consequence of performed investigations, the classification of tested stationary phases for the potential usage in particular high‐performance liquid chromatography mode was also accomplished.     

The immobilized porcine pancreatic exopeptidases and its application in casein hydrolysates debittering

The practical application of exopeptidase has been limited by the high cost of the enzymes resulting from the low content of individual exopeptidase in the raw material. This can be overcome by the use of a combination of all the exopeptidases in the same enzyme source, as well as the use of the enzyme immobilization technology. A porcine pancreatic exopeptidase mixture was prepared by the ammonium sulfate precipitation at 35% saturation of the autolyzed pancreatic juice. The enzyme preparation was immobilized on thin shrimp chitin film by crosslinking with glutaraldehyde. The immobilized porcine pancreatic exopeptidases (IPPE) was effective in releasing the free amino acids from peptides. Of these amino acids, the concentrations of arginine, lysine, histidine, tyrosine, phenylalanine, leucine, and glutamine were increased much more than those of other amino acids. This indicated that both the porcine pancreatic exopeptidases preparation and the IPPE contained carboxypeptidase A, B, and aminopeptidase. The IPPE was also efficient in the decrease of the hydrophobicity of protein hydrolysates demonstrated by hydrophobic Chromatographic analysis. This led to the application of the immobilized exopeptidases in protein hydrolysate debittering. The IPPE was able to remove the bitterness of the tryptic/chymotryptic casein hydrolysates.     

Adding a Functional Handle to Nature′s Building Blocks: The Asymmetric Synthesis of β‐Hydroxy‐α‐Amino Acids

β‐Hydroxy‐α‐amino acids are not only used by synthetic chemists but are also found in natural products, many of which show anti‐microbial or anti‐cancer properties. Over the past 30 years, chemists have searched for many asymmetric routes to these useful building blocks. Initial attempts to synthesize these compounds utilized chiral auxiliaries and the reactions of glycine equivalents with aldehydes to form two stereocenters in one step. Other methods with the formation of specific intermediates or that were aimed at a specific amino acid have also been investigated. Asymmetric hydrogenation by dynamic kinetic resolution has emerged as a high‐yielding method for the synthesis of an array of modified amino acids with good stereoselectivity. More recently, amino‐acid functionalization and multicomponent reactions have increased the atom economy and simplified many long and difficult routes. In this Focus Review, many of the elegant syntheses of these compounds are explored. The applications of β‐hydroxy‐α‐amino acids in natural‐product synthesis are also mentioned.     

Recent Advances in Rapid Synthesis of Non-proteinogenic Amino Acids from Proteinogenic Amino Acids Derivatives via Direct Photo-Mediated C–H Functionalization

Non-proteinogenic amino acids have attracted tremendous interest for their essential applications in the realm of biology and chemistry. Recently, rising C–H functionalization has been considered an alternative powerful method for the direct synthesis of non-proteinogenic amino acids. Meanwhile, photochemistry has become popular for its predominant advantages of mild conditions and conservation of energy. Therefore, C–H functionalization and photochemistry have been merged to synthesize diverse non-proteinogenic amino acids in a mild and environmentally friendly way. In this review, the recent developments in the photo-mediated C–H functionalization of proteinogenic amino acids derivatives for the rapid synthesis of versatile non-proteinogenic amino acids are presented. Moreover, postulated mechanisms are also described wherever needed.     

Integrative Chemical Biology Approaches for Identification and Characterization of “Erasers” for Fatty‐Acid‐Acylated Lysine Residues within Proteins

Acylation of proteins with fatty acids is important for the regulation of membrane association, trafficking, subcellular localization, and activity of many cellular proteins. While significant progress has been made in our understanding of the two major forms of protein acylation with fatty acids, N‐myristoylation and S‐palmitoylation, studies of the acylation of lysine residues, within proteins, with fatty acids have lagged behind. Demonstrated here is the use of integrative chemical biology approaches to examine human sirtuins as de‐fatty‐acid acylases in vitro and in cells. Photo‐crosslinking chemistry is used to investigate enzymes which recognize fatty‐acid acylated lysine. Human Sirt2 was identified as a robust lysine de‐fatty‐acid acylase in vitro. The results also show that Sirt2 can regulate the acylation of lysine residues, of proteins, with fatty acids within cells.     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Immobilization of endo-1,4-β-xylanase on polysulfone acrylate membranes: Synthesis and characterization

We report on a new enzyme/support system to immobilize proteins such as enzymes through a covalent bond on polysulfone membranes. In the present case the enzyme endo-1,4-β-xylanase (E.C.3.2.1.8) is attached to polysulfone previously derivatized by introducing an acrylate group. Membranes are properly prepared from this polysulfone acrylate. Afterwards the enzyme is immobilized though the amino groups of side chains of the amino acids of the enzyme and the acrylate group of the derivatized polysulfone. Such immobilization of the enzyme is confirmed by microelemental analysis as well as by amino acid analysis by HPLC. Moreover, the enzymatic activity of the membranes was evaluated and compared with that corresponding to the free enzyme. Certain physical parameters (asymmetry, irregularity, pore size and surface roughness) of the corresponding enzymatic membranes were obtained from SEM and AFM image interpretation.     

蛋白质功能化新策略:嵌入非天然氨基酸

天然蛋白质由20种天然氨基酸组成,这些蛋白质的构筑基元包含功能基团:羧基、氨基、巯基、硫醚、羟基、碱性胺、烷基和芳基。然而,这些有限的功能基团却不足以完成生物体内所有的生物学功能。为了更好地让生命的体现者--蛋白质完成更加精确和多样的生物学功能,自然界会对蛋白质进行翻译后的修饰,包括:磷酸化,甲基化,乙酰化或者羟基化,甚至在某些情况下,进化出一种新型的翻译机制以便插入硒代半胱氨酸或者吡咯霉素。受此启发,生物化学家发展出各种生物或化学方法来改变或插入新的蛋白质构筑基元,使天然蛋白质完成其相应的生物学功能或者使其具有某些特殊的性质,甚至是创造一种新酶。该文将简单介绍这些蛋白质修饰策略以及该领域的最新进展。     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Enantio‐ and Chemoselective Differentiation of Protected α‐Amino Acids and β‐Homoamino Acids with a Single Copper(II) Host

The association between an achiral copper(II)‐containing host 1 and chiral carboxylates has been expanded beyond previous studies to new chiral carboxylate guests, both α‐amino acids and β‐homoamino acids. The observed exciton‐coupled circular dichroism (ECCD) signals for the enantiomers of each carboxylate were equal and opposite, and these signals differed in size and shape between the individual amino acids. Linear discriminant analysis (LDA) was applied as a statistical analysis technique to differentiate the amino acids, both enantioselectively and chemoselectively, giving the absolute configuration and identity of the amino acid. The identity of each of the α‐amino acids and β‐homoamino acids were determined independently by LDA, and then the two were considered together. Each of these analyses showed good differentiation of the amino acid guests with the use of only one host molecule.     

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

Boosting the Peroxidase‐like Activity of Cobalt Ions by Amino Acid‐based Biological Species and Its Applications

Enzyme mimics have been widely used as alternatives to natural enzymes, owing to their high stability and low cost. However, the activity and atom economy of enzyme mimics still need to be improved. Herein, we report the boosting effects of amino acids, peptides and proteins on the peroxidase‐like activity of Co²⁺. Among 20 amino acids, tryptophan (Trp) enhanced the activity of Co²⁺ approximately 8 times and was identified as the best stimulator. The study revealed the synergy of amino acids‐based species and HCO₃^? for efficient catalysis. Co²⁺ is proposed to bind simultaneously to HCO₃^? and Trp, and to form a ternary catalyst which facilitates the generation of reactive oxygen species. Based on the selective boosting by Trp, a simple and low‐cost Co²⁺ sensor with high sensitivity was developed, which showed a linear range of 10–300 μM and a limit of detection of 0.4 μM for Co²⁺.     

[80]Fullerene–amino acid interactions: Theoretical insights

This work reports the interactions of a C₈₀ fullerene species with the 20 naturally occurring amino acids. As a result of the analysis of multiple configurations, we have determined that the most stable C₈₀ complexes form with thiol containing amino acids. The stabilities of the resulting complexes have been classified according to biochemical classifications that are used to develop trends among the calculated data. The computed trends in the dissociation energies are related to the backbone structure of the corresponding amino acids. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

从磷酰化氨基酸到丝组二肽

Phosphorus is one of the important elements for life, which participates in various biochemical processes. L-Amino acids are the basic structural units of proteins. N-phosphorylation of amino acids makes them be activated and become one kind of "micro activating enzyme", which possesses a variety of biochemical reaction activities, such as peptide formation. Based on the peptide formation reaction of N-phosphoryl amino acids, L-seryl-L-histidine dipeptide (Seryl-histidine dipeptide) is produced and found that it is the smallest functional peptide with a variety of biological enzyme activities. Seryl-histidine dipeptide could be regarded as the original evolution prototype of modern hydrolytic enzyme. This paper reviews the research process from N-phosphoryl amino acids to the discovery of seryl-histidine dipeptide in detail. There are pain, confusion and joy, which fully embodies the charm of scientific exploration.     

七氟丁酰氨基酸正丁酯的GC/MS分析

本文报道了19种蛋白质氨基酸的七氟丁酰正丁酯衍生物的GC/MS分析方法;优选了衍生反应条件;建立了GC分离、定量分析方法,最小检测量为0.1ng;研究了衍生物的EI质谱数据,确定了可作为定性依据的特征离子;测定了儿童用复方氨基酸注射液及儿童血清样品,得到了满意的结果。     

Synthesis and Spectroscopy of Novel‐α‐Pyrazolylglycine Derivatives

The synthesis of α‐pyrazolylglycine derivatives(7a‐d) with different substituents, starting from glycine have been pre pared. The spectroscopy of intermediate compounds and the final amino acids have been discussed.