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。     

肽的酶促合成及其反应机制

先后以α-胰凝乳蛋白酶和木爪酶作催化剂以逐级缩合方式合成了[色]⁴亮-脑啡肽酰酶(TyGlyGlyTrpLeuNH₂),在合成中,对酶的专一性,有机溶剂的选择和合成条件等进行试探,比较了固相酶促法和液相酶促法,α-胰凝乳蛋白酶和木爪酶连接肽键中,以N-酰基氨基酸酯作为供体比相应的游离酸好,如用嗜热菌蛋白酶只能以N-酰基氨基酸为羧基组份,初步的体外实验说明合成的[色]⁴-亮脑啡肽酰酶与天然亮-脑啡肽有类似的鸦片样生理活性。     

酶促合成肽的研究I.亮-脑啡肽色氨酸类似物的酶促合成

在先后以α-胰凝乳蛋白酶和和木瓜酶作催化剂的作用下,从C端的LeuNH2的氨基开始,逐个与N保护的氨基酸或其衍射生物缩合,合成了亮-脑啡肽色氨酸类似物-TyrGlyGly Trp LeuNH2.对酶的专一性、有机溶剂的选择和合成条件等进行了研究,在Z-TyrOMe和GlyGlyTrp LeuNH2的缩合反应中,比较了相转移法和均相法。上述两种酶的酶促接肽的供体以N-酰基氨基酸酯为好,比相相应的游离酸反应速度快,副反应少,如用嗜热菌蛋白酶,则以N-酰基氨基酸为好,酶促合成与从有机合成所得的产物相同。初步的体外实验表明合成的TyrGlyGlyTrpLeuNH2和天然的亮-脑啡肽对豚鼠回肠的收缩显示相同程度的鸦片样的抑制作用。     

以N-保护消旋氨基酸为羧基组分的酶促合成肽的研究

用不同的氨基保护的消旋氨基酸或酯作为羧基组分,在α-胰凝乳蛋白酶或木瓜蛋白酶的催化下与另一氨基酸的酰胺或酯缩合形成预期的光活性肽产物。以L-构型底物计算肽的产率为55%～83%,化合物的物理学常数与用相应的N-保护-L-氨基酸或酯为羧基组分,分别用化学法或酶促法测定所得结果一致。研究结果表明:利用酶的立体专一性,在形成肽键的过程中进行立体选择可得到光学活性的产物而不需预先拆分;只要溶剂体系中含有酶所需要的微量必需水,酶促反应在有机溶剂中可以顺利地进行。     

酶促合成肽的研究——Ⅰ.亮-脑啡肽色氨酸类似物的酶促合成

在先后以α-胰凝乳蛋白酶和木瓜酶作催化剂的作用下,从C端的LeuNH_2的氨基开始,逐个与N保护的氨基酸或其衍生物缩合,合成了亮-脑啡肽色氨酸类似物—TyrGlyGly Trp LeuNH_2~(**)。对酶的专一性、有机溶剂的选择和合成条件等进行了研究,在Z-TyrOMe和GlyGlyTrp LeuNH_2的缩合反应中,比较了相转移法和均相法。上述两种酶的酶促接肽的供体以N-酰基氨基酸酯为好,比相应的游离酸反应速度快,副反应少。如用嗜热菌蛋白酶,则以N-酰基氨基酸为好。酶促合成与从有机合成所得的产物相同。初步的体外实验表明合成的TyrGlyGlyTrpLeuNH_2和天然的亮-脑啡肽对豚鼠回肠的收缩显示相同程度的鸦片样的抑制作用。     

新型富勒烯α-氨基酸的合成及其纳米颗粒水悬液的制备

通过1,3-偶极环加成反应合成中间体N-取代的3,4-富勒烯吡咯烷, 利用α-氨基与α-羧基均被保护的天冬氨酸或谷氨酸的非α-羧基与中间体N-取代的3,4-富勒烯吡咯烷衍生物的活化羟基进行缩合反应, 产物脱保护后得到了2种新的α-富勒烯氨基酸: 富勒烯天冬氨酸和富勒烯谷氨酸. 采用MALDI-TOF质谱、红外光谱、紫外-可见光谱和1H NMR 等方法对它们进行了结构表征. 采用有机溶剂交换法, 制备富勒烯氨基酸纳米颗粒水悬液, 并进行了电镜和表面zeta-电位分析, 结果表明, 此水悬液体系稳定, 颗粒形态大小均一, 在生物医学领域中具有潜在的应用前景.     

苯并二氮杂酮酸的合成及其在肽模拟中的应用

采用两种方法以靛红酸酐为起始原料,分别与谷氨酸(L-Glu)和亚氨基二乙酸(Ida)反应制备两种含有游离羧基的1,4-苯并二氮杂-2,5-二酮类中间体2a和2b;同时,由2a或2b与氨基组分3c～3f,3h,3j进行缩合制备了含有1,4-苯并二氮杂-2,5-二酮环的肽模拟物4c～4f,4h,4j,5c～5f,5h,5j.另外,讨论了溶剂对微波辅助法合成1,4-苯并二氮杂-2,5-二酮酸的影响.所合成新化合物均经MS,1HNMR证明其结构.     

2-羰基丙酸缩对羧基苯腙的合成

以对氨基苯甲酸为原料,经过重氮化反应和还原反应,合成了对羧基苯肼(2);2与2-羰基丙酸缩合得到新化合物2-羰基丙酸缩对羧基苯腙,其结构经UV,荧光光谱(FS),1H NMR,13C NMR,IR和元素分析表征.     

苯并二氮杂(艹卓)酮酸的合成及其在肽模拟中的应用

采用两种方法以靛红酸酐为起始原料,分别与谷氨酸(L-Glu)和亚氨基二乙酸(Ida)反应制备两种含有游离羧基的1,4-苯并二氮杂(艹卓)-2,5-二酮类中间体2a和2b;同时,由2a或2b与氨基组分3c～3f,3h,3j进行缩合制备了含有1,4-苯并二氮杂(艹卓)-2,5-二酮环的肽模拟物4c～4f,4h,4j,5c～5f,5h,5j.另外,讨论了溶剂对微波辅助法合成1,4-苯并二氮杂(艹卓)-2,5-二酮酸的影响.所合成新化合物均经MS,1H NMR证明其结构.     

接枝树形支化分子双亲性嵌段共聚肽的合成及性能研究

分别以叠氮丙胺和丙炔胺为引发剂,采用氨基酸环内酸酐开环聚合法(NCA-ROP),引发L-谷氨酸-γ-苄酯-N-羧基-环内酸酐和L-谷氨酸-γ-氯乙醇酯-N-羧基-环内酸酐聚合得到链端基为叠氮基的聚(L-谷氨酸-γ-苄酯)(PBLG)和链端基为炔基的聚(L-谷氨酸-γ-氯乙醇酯)(PCELG)均聚物.联合点击化学法(click chemistry)制备了一系列聚(L-谷氨酸-γ-苄酯)-b-聚(L-谷氨酸-γ-氯乙醇酯)(PBLG-b-PCELG),再通过对嵌段共聚物侧基氯进行化学修饰,将二代的甲基烷氧醚类亲水性树形枝化分子(G2)接枝到侧链上,形成一系列树形支化分子接枝聚肽双亲性嵌段共聚物,通过核磁(NMR)、红外光谱(IR)和凝胶渗透色谱(GPC)对其化学结构进行了表征,并采用紫外-可见光谱(UV-Vis)研究了聚合物结构及其溶液浓度对其温敏行为的影响规律.     

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.     

Peptidsynthesen mit immobilisierten Enzymen II. Immobilisiertes Trypsin,Thermolysin und Papain

Model studies were performed on the utility of covalently immobilized trypsin, thermolysin and papain for peptide bond formation. Trypsin and thermolysin catalyzed the formation of peptide bonds with nearly the same efficiency as the soluble proteases and they could be re-used successfully for further coupling experiments. The possibility of using immobilized trypsin and papain for kinetically controlled peptide bond formation was investigated. With the serine type enzyme trypsin excellent product yields were obtained starting with ester carboxyl components and an economical ratio of substrates. Experiments with the thiol protease papain were unsatisfactory because the once formed product is hydrolyzed as fast as the starting ester substrate used.     

The amino- and carboxyl-terminal sequence of bovine rhodopsin

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (similar to 35,000 daltons) is converted to a membrane-bound fragment O' (similar to 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O' are replaced by the membrane-bound fragments F1 (similar to 25,000 daltons), and F2 (similar 9,500 daltons). When 32P-ROS are digested, F2 carries the 32P. Both O' and F1 contain the amino-terminal glycopeptide.     

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。     

Thermolysin catalyses the synthesis of cyclodextrin esters in DMSO

Fatty acid esters of cyclodextrins (CDs) were synthesised in a one-step reaction with native CDs as acyl acceptors and vinyl-activated fatty acid esters as acyl donors. Immobilised preparations of thermolysin, subtilisin, the alkaline protease AL-89 and Candida antarctica lipase B were investigated for their catalytic properties regarding transesterification in solvents of increasing hydrophilicity. The synthesis of cyclodextrin fatty acid esters was proved to be catalysed enzymatically by thermolysin in DMSO. The obtained products were analysed by TLC and their structures characterised by NMR, MS and FTIR spectroscopy. With vinyl decanoate as acyl donor β-CD was esterified at all seven glucose C-2 positions resulting in heptakis(2-O-decanoyl)-β-cyclodextrin as the major product. With vinyl butyrate, substitution occurred at all the C-2 and partially at the C-3 or C-6 positions resulting in an average degree of substitution of nine. Between 20% and 25% (w/w) of the acyl donor was converted to esters in 20 h corresponding to an estimated total conversion of the acyl acceptor in the case of maltosyl-β-CD. In the subtilisin and AL-89 catalysed reactions, product formation was simultaneously catalysed non-enzymatically by inorganic buffer salts in aprotic, hydrophilic solvents and with the lipase no products were formed in any of the solvents investigated.     

吡啶甲酸分子平衡构型的ab initio理论研究

本文用能量梯度法TEXAS从头计算程序,选取4-21G(,N原子含极化d轨道)基组,对α-,β-吡啶甲酸分子的平衡几何构型进行了全优化计算,二套基组的理论优化表明,三个吡啶甲酸分子都具有平面的平衡构型,其计算键长经4-21G 基组的标准经验校正值校正后,所得的理论预测平衡几何构型与X射线单晶衍射实验结果有很好的一致性.对部分键长以及羧基与吡啶华平面的二面角中出现的差异进行了讨论.     

通过Michaelis-Arbuzov重排合成新型

α-溴代-叔丁基甲基酮和3-取代2,2-二甲基环丙烷羧基氯化物与亚磷酸酯反应,生成膦酸酯,并描述了膦酸酯的溴化反应.     

三苄基锡羧酸酯的合成及其结构表征

本文以(三苄基锡)氧化物和相应的羧酸反应, 合成了20个三苄基锡羧酸酯, 由波谱对结构的研究表明, 红外光谱的酰基振动频率与羧基上取代基的空间和电子效应密切相关, 三苄基锡羧酸酯的结构微小变化, 明显地反映了^1^1^9Sn和^1^3CNMR化学位移的差异, ^1^1^9Sn化学位移与三苄基锡取代的苯甲酸酯的对位取代基Hammett常数(σ)之间存在着良好的线性关系: δ~1~1~9~S~n=15.48σ+14.23, n=7, r=0.995, 去烃基化是这一类化合物质谱裂解的特点。     

A silanediol inhibitor of the metalloprotease thermolysin: synthesis and comparison with a phosphinic acid inhibitor

A silanediol inhibitor of the metalloprotease thermolysin was prepared for comparison to a known phosphinic acid inhibitor, providing the first comparison of these second-row element based transition-state analogues. Inhibition of thermolysin by the silanediol (K(i) = 41 nM) was comparable to that of the phosphinic acid (K(i) = 10 nM) even though the silanediol is uncharged and thereby lacks the intrinsic Coulombic attraction of the phosphinate anion to the active-site zinc cation. This silanediol protease inhibitor is the least sterically encumbered example prepared to date and, therefore, the most prone toward polymerization. Hydrolysis of a difluorosilane intermediate to the silanediol leads cleanly to a monomeric product.     

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。