具有合成核酸活性的多肽 Ⅰ.C-端去四肽和去六肽核糖核酸酶A及其水解和合成活性

核糖核酸酶A(RNase A)的结构与功能的关系虽属已知,但大都限于水解作用方面,而关于合成作用方面的报道则颇少.根据前人用抑制剂法所得羧甲基化核糖核酸酶A的衍生物(CM-RNase A),和用合成法所得分子较简单的RNase A的类似物的研究结果,似乎RNaseA分子中的His_(119)与Lys_(41)残基与酶的合成活性无关.这些衍生物和类似物的合成活性都比它们的水解活性高,但不论它们的合成或水解活性都很显著地低于天然酶的活     

修饰核糖核酸酶A的酶促合成模型研究

为研究Leu¹²¹-核糖核酸酶A(Leu¹²¹-RNaseA)的半合成制备了C-端去HAsp Ala SerValOH四肽核糖核酸酶A即RNase A(1—120),用固相合成法合成了C-端四肽的类似物HLeu Ala Ser Val OH及C¹⁴标记Leu(记为Leu^*)的四肽HLeu^* Ala Ser Val OH。在设计的小肤模型中,以Boc Val Phe OH和HLeu Ala OMe为底物,在含50%乙二醇,pH 4.50的醋酸缓冲溶液中,在胃蛋白酶的作用下成功地得到了四肤Boc Val Phe Leu Ala OMe,并研究了温度对宵蛋白酶酶促反应的影响。在RNase A(1-120)与H Leu Ala Ser Val OH四肤的胃蛋白酶酶促反应的初步研究中,用HPL C检测反应出现一个新峰。此峰的保留时间与预期产物的保留时间相一致。同位素标记酶促合成试验在高压纸电泳上出现一个新的同位素点,估计为预期的产物。本文还对酶促合成的一些问题进行了讨论。     

蜂毒肽C末端片段的反序肽的抗菌活性和溶血活性

设计并合成了具有不同碱性氨基酸残基数和不同疏水性片段链长的基于Mel(12~26)的系列反序肽类似物.结果表明,反序肽的正电荷和疏水性对于抑菌活性都很重要,N端至少保留3个碱性氨基酸(正电荷>4)和C端的疏水性片段的链长至少为8个氨基酸残基的类似物具有较高的抑菌活性,具有较大的抑菌活性的最小反序肽类似物为具有11个氨基酸残基的RetroMel(13~23).这些反序肽的溶血活性都很小.     

半胱氨酸肽片段连接法合成ω-芋螺毒素MVIIA

ω-芋螺毒素MVIIA是已上市的镇痛药Ziconotide的有效成分.采用标准Fmoc保护策略在聚苯乙烯树脂上合成ω-MVIIA比较困难,是固相合成中的"困难肽".本研究将ω-MVIIA分为N-端15肽硫酯和C-端10肽两个片段采用标准Fmoc保护策略分别合成,再通过半胱氨酸肽片段连接得到全长的ω-芋螺毒素MVIIA肽链.该方法提高了合成ω-芋螺毒素MVIIA产率.该研究为"困难肽"的合成提供了较好的参考方法.     

芹菜素衍生物的合成及抗肿瘤活性研究

以芹菜素为起始原料,在C-5、C-6、C-7、C-4′位置上引入甲基、苄基、乙酰基、对甲苯磺酰基、三异丙基硅烷基等多种单元结构基团,合成了16个芹菜素衍生物,采用CCK-8的方法考察了所合成化合物对人肝癌细胞(Hep3β)、人结肠癌细胞(LOVO)、人肺癌细胞(A549)的抑制作用。结果显示,经过化学修饰后的部分化合物比芹菜素有更强的抗肿瘤活性,其中化合物13对人肺癌细胞(A549)的抑制率超过了顺铂,值得进一步探讨和研究。     

胆固醇修饰的非天然HR序列肽类HIV-1融合抑制剂的合成及活性初筛

将胆固醇分子通过1个半胱氨酸侧链硫醚键和1个β-丙氨酸连接臂引入到所设计的非天然HR序列抗HIV融合活性多肽的C端和N端,合成了与天然C肽序列同源性很低的非天然序列的类肽抗HIV融合分子,以考察胆固醇修饰对非天然HR序列活性的影响,探讨克服耐药性的新思路.生物活性评价结果表明,胆固醇与HR肽C端结合物抑制HIV融合活性显著提高,而连接到N端的序列则完全失去了抗病毒活性,表明所设计的非天然序列确实具有与天然序列类似的作用机制.     

海地瓜蛋白水解物中ACE抑制肽的分离纯化及合成

采用Sephadex G-25凝胶柱层析、SP Sephadex C-25 阳离子交换层析和反相高效液相色谱等方法对海地瓜水解产物进行分离纯化, 得到了一种新的强活性ACE抑制肽, 其氨基酸序列为MEGAQEAQGD, IC₅₀值为15.9 μmol/L. 采用逐步缩合和片段缩合的方法对该抑制肽进行了设计合成. 合成肽的纯度为99.72%, 分子量与序列结构均与理论值相符. 研究发现, 抑制肽与胃蛋白酶和糜蛋白酶水解反应后, 活性增强了3.5倍. 动物实验结果表明, 剂量为3 μmol/kg的抑制肽对大鼠自发性高血压具有明显的降压效果.     

胰高血糖素样肽-1类似物的合成及其生物学活性研究

通过改变胰高血糖素样肽-1 (GLP-1)易被酶水解部位, 采用Fmoc/t-Bu正交保护固相合成策略, 并运用微波照射促进高效快速地合成了抗二肽基肽酶IV (DPP IV)的GLP-1类似物, 最后经过反相制备高效液相系统纯化得到目标多肽纯品. 生物学活性研究结果显示, 改造后的GLP-1类似物能有效抵抗DPP IV的水解, 并且有很好的降血糖活性. 所合成的GLP-1及其类似物的分子量和纯度均经过电喷雾质谱和高效液相确证.     

新蛙皮活性肽-绿臭蛙激肽的合成及其活性检定

用液相片断缩合法首次合成了由中国绿臭蛙(Rana margaratae)的蛙皮中分离的速激肽类新活性肽-绿臭蛙激肽(Ranamargarin):Asp-Asp-Ala-Ser-Asp-Arg-Ala-Lys-Lys-Phe-Tyr-Gly-Leu-Met-NH_2。合成的十四肽酰胺在RP-HPLC上显示与天然肽相同的保留时间,与天然肽合并后为单一峰,从而进一步肯定了此新活性肽的结构。根据不同生物标本检定的结果,提示此肽对SP-P型速激肽受体有较高的选择性。     

光学活性双氨基酸的合成

光学活性的双氨基酸作为一类非天然的氨基酸合成子, 在医药、农药和肽类合成中有着重要的作用. 评述了近年来关于光学活性双氨基酸合成的进展.     

应用^3^1P NMR研究RNase A水解核糖核酸的机制

核糖核酸酶A(RNase A)水解核糖核酸的机制前人已有研究.Markham等和Brown等根据水解过程中有2′,3′-环核苷酸的生成提出了两步机制,即磷酰基转移和水解开环.Witzel等用紫外差值(ΔA_(286))光谱和pH-stat(pH恒定器)技术进行动力学研究,所得的结果支持了上述机制.Williams用同样的方法进行研究,提出除了两步机制外,还有另一途径,即二核苷(3′→5′)单磷酸二酯(A)不经过2′,3′-环核苷酸(B)而直接转化为3′-核苷酸     

Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications

Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).     

含新型亲核体大环多胺锌(II)配合物对水解酶的模拟研究

本文探讨苯酚功能化的大环多胺配体6-(2'-羟基-3', 5'-二溴)-苄基-1, 4, 8, 11-四氮杂环十四烷(L)锌(II)配合物作为水解酶模拟物催化4-硝基苯酚醋酸酯(NA)水解的动力学。研究表明催化水解速率对NA及配合物浓度皆呈一级反应。水解速率遵循速率方程V=(kcat[Zn]+kOH[OH^-]+k0)[NA]。其二级反应速率常数kcat在一定范围内随着pH值的增加而增加, kcat的最大值(k)和kOH分别为0.12, 8.55mol^-^1.L.s^-^1。k0为NA的溶剂解速率常数, 其值为1.122×10^-^5s^-^1(298K, I=0.10,0.02mol.L^-^1tris缓冲溶液)。kcat值较以前报道的配合物更大, 催化活性更高。显示配位酚羟基可作为一种新型亲核体有效地催化NA的水解。配合物对NA水解的催化作用受酸碱平衡控制。根据实验结果提出了催化反应的机理。     

Surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) brushes as templates for enzyme immobilization

We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.     

Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.     

Determination of the herbicide diclofop in human urine

A simple and sensitive method for the gas chromatographic determination of diclofop residues in human urine is described. Recoveries of diclofop, as its methyl ester, from fortified urine were greater than 85% at 100, 50, 10 and 1 μg kg⁻¹, and were similar with and without the inclusion of a hydrolytic step in the analytical method. However, a hydrolytic step was necessary for analysis of 24-h urine samples collected from a male applicator following a single exposure to diclofop-methyl during application to wheat using a tractor-pulled sprayer. Diclofop residues determined with hydrolysis were approximately double those without hydrolysis, suggesting that a significant portion of diclofop was excreted in the conjugated form.     

Glycosylated constituents of iris fulva and Iris brevicaulis

The major constituents of leaf extracts of Iris fulva KER GAWL. comprised a known flavone C-glycoside, 5,4'-dihydroxy-7-methoxyflavone-6-C-(6?-O-(E)-p-coumaroyl-β-glucopyranosyl)(1?→2″)-β-glucopyranoside (1) and the new monoterpene glycoside, linalyl-6'-O-(3″-hydroxy-3″-methylglutaroyl)-β-D-glucopyranoside (2), both of which were prominent components of Iris brevicaulis RAF. leaf extracts. The structure of a new polyacylated sucrose derivative (3a) obtained from the rhizomes of I. fulva was elucidated as 3-O-(E)-p-coumaroyl-β-D-fructofuranosyl-(2?1')-[2″,4″,6″-tri-O-acetyl-β-D-glucopyranosyl-(1″→3')-(2',6'-di-O-acetyl-4'-O-(E)-p-coumaroyl-α-D-glucopyranoside)]. Selective hydrolysis of the 4″-O-acetyl moiety of the terminal β-glucopyranosyl residue of 3a occurred after several hours in solution giving 3-O-(E)-p-coumaroyl-β-D-fructofuranosyl-(2?1')-[2″,6″-di-O-acetyl-β-D-glucopyranosyl-(1″→3')-(2',6'-di-O-acetyl-4'-O-(E)-p-coumaroyl-α-D-glucopyranoside)] (3b), which subsequently underwent further deacetylation.     

2,6-二甲基-3,5-二氯-4-吡啶酚糖苷的合成

在相转移催化条件下, 使 a-D-乙酰基化溴代的葡萄糖、半乳糖、葡萄糖醛酸甲酯1a, 1b, 1c分别与2,6-二甲基-3,5-二氯-4-吡啶酚(俗称氯吡醇, 氯羟吡啶)作用, 合成了氯吡醇的糖苷: 1-O-(2',6'-二甲基-3',5'-二氯-4'-吡啶基)-2,3,4,6-四-O-乙酰基-β-D-葡萄吡喃糖苷(2a), 1-O-(2',6'-二甲基-3',5'-二氯-4'-吡啶基)-2,3,4,6-四-O-乙酰基β-D-半乳吡喃糖苷(2b), 1-O-(2'6'-二甲基-3',5'-二氯-4'-吡啶基)-2,3,4-三-O-乙酰基-β-D-葡萄吡喃糖醛酸甲酯(2c)。2a, 2b, 2c分别在甲醇中氨解, 相应得到: 1-O-(2', 6'-二甲基-3',5'-二氯-4'-吡啶基)-β-D-葡萄吡喃糖苷(3a), 1-O(2',6'-二甲基-3',5'-二氯-4'-吡啶基)-β-D-半乳吡喃糖苷(3b),1-O-(2', 6'-二甲基-3',5'-二氯-(4'-吡啶基)-β-D-葡萄吡喃糖醛酸酰胺(3c)。2c用CH~3ONa/CH~3OH处理, 得到1-O-(2',6'-二甲基-3',5'-二氯-4'-吡啶基)-β-D-葡萄吡喃糖醛酸甲酯(3d)。     

络天青S—CPB—Brij35胶束增溶吸光光度法测人发中微量铜

在阳离子表面活性剂ＣＰＢ和非离子表面活性剂Ｂｒｉｊ３５存在下,ｐＨ７．５的弱碱性介质中,二价铜离子与络天青Ｓ形成灵敏度高的１：２络合物。应用吸光度法测定人发中微量铜,铜含量在０．０２～２．０ｍｇ·Ｌ＾－１遵守比耳定律,方法检出限为限为０．０１ｍｇ·Ｌ＾－１,平均回收主继１００．５％±０．２１％,ＲＳＤ为０．２１％。此法用于人发中微量铜的测定。结果满意。     

几个烯胺的邻硝基苯甲酰化和在酸水解中发生的重排

为了了解芳酰基化的效率和双酰基衍生物的形成, 对几个典型醛和酮的吗啉烯胺进行了邻硝基苯甲酰化的探索。丁醛和异丁醛吗啉烯胺的酰化正常, 水解分别生成55%的2-(邻硝基苯甲酰基)丁醛(2)和40%的2-(邻硝基苯甲酰基)异丁醛(3)。3-戊酮和1, 3-二甲氧基丙酮的吗啉烯胺在酰化和水解时分别生成37%和14.8%相应的β-二酮的烯醇邻硝基苯甲酸酯(4)和(5)。4和5的酸水解发生意外的1, 5-和1,3-酰基转位, 分别生成3, 5-二甲基-2, 6-双(邻硝基苯基)吡喃-4-酮(6)和2-甲氧基-2-甲氧乙酰基-1, 3-双(邻硝基苯基)-1, 3-丙二酮(7)。环戊酮和环已酮吗啉烯胺的酰化分别产生9%和34%的双酰基烯胺(9a)和(9b),逐次酸水解得到2-(邻硝基苯甲酰基)环戊酮(11a)和-环已酮(11b)。在总酰化产物的直接水解中, 11a的产率可以达到81%, 而11b只有35%。对双酰化进行了一些讨论。