Enzymatic Synthesis of a CCK-8 Tripeptide Derivative

The enzymatic synthesis of CCK-8 tripeptide derivative Phae-Met-Asp(OMe)-Phe-NH2 is reported.Starting with Phac-Met-OCam,we have successfully synthesized the target tripeptide with three free or immobilized enzymes, α-chymotrypsin,papain and thermolysin in reasonable yields.The key steps in this synthesis were the coupling of Phac-Met-OCam and H-Asp(OMe)2 to from Met-Asp peptide bond catalyzed by α-chymotrypsin and the selective hydrolysis of α-ester of Phac-met-Asp(OMe)2 catalyzed by papain.     

用嗜热菌蛋白酶催化合成天冬甜精并同时进行DL-苯丙氨酸甲酯的光学拆分

以DL-苯丙氨酸甲酯作原料,利用嗜热菌蛋白酶和固定化嗜热菌蛋白酶催化合成了天冬甜精,经拆分得到D-苯丙氨酸甲酯。实验结果表明,不仅天冬甜精产率高,而且D-苯丙氨酸甲酯的回收率好,光学纯度高。     

Studies on enzymic peptide synthesis: I. Synthesis of tryptophane analogue of Leu-enkephalin

A tryptophane analogue of Leu-enkephalin, Tyr-Gly-Gly-Trp-Leu-NH₂, was synthesized by stepwise elongation from C-terminal Leucylamide towards the N-terminal tyrosine using α-chymotrypsin and papain catalysis. The selections of appropriate enzymes and suitable solvents were studied. It was found that an N-blocked amino acid ester served better as a donor than the corresponding free amino acid in the peptide formation, irrespective of whether α-chymotrypsin or papain was used, while, on the other hand, an N-blocked amino acid was needed as the donor in case of thermolysin. In the last step of pentapeptide synthesis catalyzed by α-chymotrypsin, the phase transfer process seems to be preferable to the homogeneous solution method. The results of this work showed some advantages by the use of an immobilized enzyme. The tryptophane analogue of Leu-enkephalin was also synthesized by the conventional organic method. Preliminary in vitro bioassay indicated that the synthetic Try-Gly-Gly-Try-Leu-NH₂ can inhibit the contraction of the guinea Pig's ileum caused by electric stimulation to nearly the same extent as the natural Leu-enkephalin does.     

Model studies on protease-catalysed peptide synthesis using 9-fluorenylmethoxycarbonyl protected amino acid derivatives

Summary Fmoc-Phe-OMe,Fmoc-Ala-OMe andFmoc-Gly-OH were coupled with H-Leu-NH₂ under catalytic action of chymotrypsin, papain and thermolysin, respectively. The influence of different reaction media and several reaction parameters, such as reactants and enzyme concentrations as well as reaction time, on the peptide bond formation was investigated. Abbreviations: IUPAC-IUB rules for peptides are followed, see (1984) Eur. J. Biochem.138: 9. All amino acids except of Gly are ofL-configuration.DMF=N,N-dimethylformamide,DTE=dithioerythritol,EDTA=ethylenediaminetetraacetic acid, disodium salt,N/C=ratio nucleophile component/carboxyl component,-OMe=methyl ester,TLC=thin layer chromatography.     

Identification of novel macrocyclic peptidase substrates via on-bead enzymatic cyclization

Peptidase-catalyzed formation of macrocyclic lactams on solid phase identifies ring systems that are favorably bound in the enzyme active site. We evaluated several cyclic peptide motifs linked by ester bonds between the P2 and P1' or the P1 and P2' side chains. The depsipeptide represented by structure 5 was readily generated by a variety of peptidases from precursor omega-amino acids or omega-amino esters. This strategy for identifying ring systems for potential macrocyclic transition state analogues was demonstrated with the serine peptidases trypsin and chymotrypsin, with the aspartic peptidase pepsin, and with the zinc peptidase thermolysin.     

Enzymatic Synthesis of a CCK-8 Tripeptide Fragment

A process for the synthesis of CCK-8 tripeptide H-Gly-Trp-Met-OH catalyzed by immobilized enzyme was reported. Enzymes were used for the formation of peptide bonds and the removal of protecting group. Starting with phenylacetyl (PhAc) glycin, N-protected dipeptide PhAc-Gly-Trp-OMe was obtained by coupling PhAc-protected glycine carboxamidomethyl ester (OCam) with Trp-OMe catalyzed by immobilized papain in buffered ethyl acetate. Then the condensation between PhAc-Gly-Trp-OMe and Met-OEtoHC1 was carried out by immobilized α-chymotrypsin catalysis in solvent free system. Basic hydrolysis was followed getting PhAc-Gly-Trp-Met-OH. The PhAc-group was removed with penicillin G amidase and H-Gly-Trp-Met-OH was obtained in an overall yield of 43.9%. The reaction conversion of tripeptide in solvent free system was strongly affected by the system of basic salts added. The influence of the support materials used to deposit enzymes and structures of acyl donor and nucleophile on the reaction was also investigated.     

Synthesis of protected peptides from the human IgG1 hinge region on PEG support using disulfide bond synthons and alkaline or enzymatic detachment

Peptides from the hinge region of human IgG1 are potential defined carriers of synthetic immunogens. Their synthesis was carried out on the soluble support—PEG-OMe 2000 using disulfide bond synthons. The loading capacity of the polymer was increased twofold by anchoring the lysine as a simple branching unit. The amino acid leucine was used as an enzymatically cleavable linker. Peptide detachment from the polymer was performed either by classical ester bond saponification or newly by peptide bond cleavage catalyzed by thermolysin in water.     

Evaluation and application of liquid chromatographic columns coated with "intelligent" ligands. (III). Immobilized phospholipid column.

Immobilized enzyme columns have been developed for use as high-performance liquid chromatographic enzyme reactors. Enzyme reactors were prepared by immobilizing trypsin or cytochrome-c on phospholipid columns. Dynamic coating was employed to prepare the reactors by recycling a buffer solution containing trypsin or cytochrome-c through a phospholipid-coated column, on which the enzymes were immobilized by hydrophobic binding. The immobilized trypsin column displayed hydrolytic activity which catalyzed the hydrolysis of L-amino acid esters to amino acid. The immobilized cytochrome-c column exhibited oxidation activity which catalyzed N-demethylation of N,N-dimethylaniline, codeine, and dihydrocodeine in the presence of hydrogen peroxide as an oxygenating agent. The enzyme reaction proceeded rapidly in the column; both product and substrate could be separated and detected simultaneously. The immobilized enzyme columns could be readily regenerated using the original phospholipid column by repeating the dynamic coating. These immobilized enzyme columns could be utilized as enzyme reactors in the high-performance liquid chromatographic mode. Complete hydrolysis of amino acid ester was observed with the trypsin column. Demethylation of codeine and of dihydrocodeine were observed with the cytochrome-c column.     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.     

酶促合成肽的研究 II: 含羧基侧链氨基酸的肽的酶促合成

用嗜热菌酶酶促合成了一系列含门冬氨酸、谷氨酸的肽. 实验证明, 缩合反应中羧基组份中的门冬氨酸β-羧基或谷氨酸γ-羧基毋需保护. 同时也研究了邻近氨基酸残基对缩合反应的影响.     

Immobilization of trypsin on sub-micron skeletal polymer monolith

A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-α-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-α-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30°C, which is comparable to 24 h digestion in solution at 37°C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.     

Immobilization of sunflower trypsin inhibitor (SFTI-1) peptide onto a gold surface and analysis of its interaction with trypsin

Sunflower trypsin inhibitor (SFTI-1) derived peptide having one disulfide bond could be immobilized via a thiol-disulfide exchange reaction onto a gold surface on a quartz crystal microbalance (QCM) chip. This permitted quantitative analysis of the specific interaction with trypsin.     

固定化酶催化合成CCK-8C-端三肽衍生物

报道胆囊收缩素（CCK-8）C-末端片段三肽衍生物外Hpac-Met_Asp(Cam为起始 原料，采用三种固定化酶--α-糜蛋白酶／Celite-545、木瓜蛋白酶／VA-Epoxy、 步酶促反应得到目标三肽化合物．对酶的专一性、酶的固定化、溶剂选择和合成条 件等进行了研究，比较了自由酶法与固定化酶法的结果．     

Penicillin acylase-catalyzed peptide synthesis: a chemo-enzymatic route to stereoisomers of 3,6-diphenylpiperazine-2,5-dione

Chiral dipeptides of phenylglycine were synthesized using immobilized Escherichia coli penicillin acylase. The high selectivity of penicillin acylase for l-amino acids as the nucleophile resulted in the efficient acylation of l-phenylglycine by d-phenylglycine amide at pH 9.7 to give d-phenylglycyl-l-phenylglycine in 69% yield. No isomers or tripeptides were formed. The low enantiospecificity of the enzyme for the acyl donor provided the possibility of preparing the corresponding l,l-dipeptides, starting from l-phenylglycine methyl ester as both donor and acceptor at pH 7.5, resulting in a 63% yield of l-phenylglycyl-l-phenylglycine methyl ester. The product precipitated under the reaction conditions; this effectively prevented the formation of oligomers as well as chemical transformation of the product.The dipeptide esters of phenylglycine easily cyclized to diketopiperazines in aqueous methanol. l-Phenylglycyl-l-phenylglycine methyl ester formed l,l-3,6-diphenylpiperazine-2,5-dione (cis); the achiral trans isomer was obtained from d-phenylglycyl-l-phenylglycine methyl ester.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

固定化胰蛋白酶整体小柱的制备及其用于微量蛋白质的快速酶解

发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20 μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×10⁵个人急性早幼粒白血病（NB4）细胞与人急性T细胞白血病（Jurkat T）细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2489个和2572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。     

固定化木瓜蛋白酶的制备和性质研究

多孔硅球固定化木瓜蛋白酶具有热增活性 .本文在前文研究的基础上 ,用载体交联法制备了甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶 .考察了固定化pH值、戊二醛浓度和给酶量对固定化木瓜蛋白酶活力的影响 .研究了固定化木瓜蛋白酶的性质 ,特别是热稳定性和耐热性 ,并与溶液酶和多孔硅球固定化木瓜蛋白酶进行了比较 .所制得的甲壳胺固定化木瓜蛋白酶和纤维素固定化木瓜蛋白酶的最适反应温度均达到了 80℃ ;90℃温育 1h后固定化酶的活力保持在 95 %以上 ;70℃温育处理 5h和 6h后固定化酶的活力也仍能保持在 90 %以上 .固定化木瓜蛋白酶的热稳定性和耐热性得到了显著提高     

STUDIES ON NATURAL AND MODIFIED PEPTIDE Trichosanthes TRYPSIN INHIBITORS

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C_(18) column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3--4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.     

Studies on natural and modified peptide Trichosanthes trypsin inhibitors

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C18 column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3-4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.     

聚丙烯偕胺肟-聚乙烯醇大孔球状载体的合成及固定化嗜热菌蛋白酶的研究

以线型聚醋酸乙烯酯为致孔剂,由悬浮聚合制得了丙烯腈-醋酸乙烯酯-二乙烯基苯大孔树脂(MR-AV树脂),通过MR-AV树脂的化学改性,合成了聚丙烯偕胺肟-聚乙烯醇大孔球状载体(MR-AVa载体),用FT-IR、SEM、压汞仪和元素分析法研究了树脂及载体的结构与组成,结果表明MR-AVa载体含氮量较高,具有一定的亲水性,且平均孔径大于8,000(?),嗜热菌蛋白酶用戊二醛固定于所得载体之上。以酪蛋白为底物,考察了固定化嗜热菌蛋白酶的催化水解活性及其影响因素,与可溶酶比较,固定化嗜热菌蛋白酶的热稳定性和抗抑制能力显著提高,当载体孔径较大时,所得固定化酶显示出较好的催化活性。