镇痛多肽——内吗啡肽-1的人工合成及活性研究

 用液相合成方法合成了具有镇痛作用的μ阿片受体的内源性配体——内吗啡肽 - 1(endomorphin- 1 ) ,该四肽为 Tyr- Pro- Trp- Phe NH2 .液相合成法是在氨基酸的 N端用 Boc(叔丁氧羰酰基 )作保护基 ,C端用 HOSu(N-羟基琥珀酰亚胺 )活化 ,与未加保护基的氨基酸在碱性条件下接肽 .先分别合成 C端二肽和 N端二肽 ,再缩合为四肽 ,产物的保护基用盐酸脱帽去除 .中间产物用薄层层析和熔点鉴定其纯度 ,最终得到了高纯度的四肽 .小白鼠脑室注射 (i.c.v)测定表明 ,8.2 5nmol剂量给药 ,其镇痛活性为 87% ,明显高于吗啡 (morphine) .     

N-甲基-2-溴代吡啶卤化盐在多肽合成中的应用

本文介绍了N-甲基-2-溴代吡啶卤化盐在多肽合成中应用的研究结果。N-保护氨基酸的三级铵盐在二氯甲烷或二甲基甲酰胺中与N-甲基-2-溴代吡啶卤化盐反应,先形成N-甲基-2-溴代吡啶卤化盐酯的活化中间体,然后在第二分子的三级胺的存在下与氨基酸或肽的酯缩合得到N-保护的肽酯。用该试剂进行缩合反应具有反应速度快、缩合率高、副产物容易去除等优点。我们用这个方法合成了一系列二肽衍生物,并用逐步递增法合成了亮氨酸脑啡肽(五肽)。N-保护的氨基酸活化后与胺基组分缩合成肽酯,其收率一般都在85%左右。     

9-芴甲氧羰基作为N~α-氨基保护基在多肽合成中的应用

<正> 长期以来,多肽合成中的氨基保护基大都是在不同程度的酸性条件下裂解去保护。自从引进能被亲核试剂除去的保护基以来,肽合成的保护战术得到了重要的扩充。本文将讨论的9-芴甲氧羰基就是其中最突出的代表。     

酰氯法合成甘氨酰-甘氨酰-缬氨酸三肽的实验研究

多肽的合成方法很多 ,传统的方法有混合酸酐法、活泼酯法和偶联试剂法 ,本文采用酰氯法 ,以L -缬氨酸为原料 ,经过酰化、氨解形成二肽 ,二肽再经过酰化、氨解最终形成三肽。通过对三肽样品的红外、核磁及高效液相等的鉴定说明该方法实验步骤简单 ,原料易得 ,收率很高 ,很适合于工业化生产 ,目前国内尚无文献报道     

大豆蛋白源性肽调节糖脂代谢机制研究进展

大豆蛋白源性肽是大豆蛋白质经酸法或酶法水解后分离精制而得到的多肽混合物,是植物蛋白源性肽的重要来源,大豆蛋白源性肽通过抑制肠道胆固醇吸收、与胆固醇的相互作用、调节胆固醇代谢相关基因（用于降低胆固醇）、甘油三酯代谢相关基因、二肽基肽酶Ⅳ和葡萄糖代谢相关基因,在降低胆固醇、降低甘油三酯、抗肥胖、抑制脂肪酸合成酶和抗糖尿病等代谢调节方面具有重要作用。综述了大豆蛋白源性肽在体内外改善脂质和糖代谢方面的生理功能,并对大豆蛋白源性肽的前景进行了展望,以期为大豆蛋白源性肽的研究与应用提供参考。     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

RGD肽介导的MR分子探针在体外结直肠癌细胞的显像及对其生物学行为的影响

目的：探讨RGD肽介导的MR分子探针在体外结直肠癌细胞的MRJ显像及对其生物学行为的影响。方法：利用纳米技术构建靶向RGD荧光纳米MR、探针,利用荧光倒置相、差显微镜观察该探针与LOVO细胞结合情况;体外磁共振成像（MRI）显像;利用细胞克隆实验测其增殖活性;流式细胞术检测其细胞周期、凋亡。结果：荧光相差显微镜示该RGD肽介导的MR分子探针能特异性与LOVO细胞结合;体外MRI成像示靶向RGD组T1信号强度高于非靶向组及对照组（P〈0．05）;该探针作用24h后的LOVO细胞增殖活性降低,细胞分裂周期发生变化,阻滞在S＋G2M期的细胞比例上升,细胞凋亡率与其他两组相比有显著增加（P〈0．05）。结论：该RGD肽介导的MR分子探针能与结直肠癌LOVO细胞靶向结合,能增强MR1的显像效果,并对肿瘤细胞具有一定的抑制作用．     

RGD肽介导的MR分子探针在体外结直肠癌细胞的显像及对其生物学行为的影响

目的:探讨RGD肽介导的MR分子探针在体外结直肠癌细胞的MRI显像及对其生物学行为的影响方法:利用纳米技术构建靶向RGD荧光纳米MR探针,利用荧光倒置相差显微镜观察该探针与LOVO细胞结合情况;体外磁共振成像（MRI）显像;利用细胞克隆实验测其增殖活性;流式细胞术检测其细胞周期、凋亡。结果:荧光相差显微镜示该RGD肽介导的MR分子探针能特异性与LOVO细胞结合;体外MRI成像示靶向RGD组T₁信号强度高于非靶向组及对照组（P<0.05）;该探针作用24h后的LOVO细胞增殖活性降低,细胞分裂周期发生变化,阻滞在S+G₂M期的细胞比例上升,细胞凋亡率与其他两组相比有显著增加（P<0.05）结论:该RGD肽介导的MR分子探针能与结直肠癌LOVO细胞靶向结合,能增强MRI的显像效果,并对肿瘤细胞具有一定的抑制作用     

胃泌素类似物的人工合成和泌酸活性研究

本文采用液相合成法合成了九种胃泌素类似物,并用大白鼠胃灌流技术测定其泌酸活性。若以五肽胃泌素(Boc-β-Ala-Trp-Met-Asp-phe-NH_2,Boc-五肽)的泌酸活性为100％,则胃泌其它类似物的活性分别为:Boc-四肽(Boc-Trp-Met-Asp-Phe-NH_2)30.32％,Boc-三肽(Boc-Met-Asp-phe-NH_2)0.13％;Fmoc-五肽(Fmoc-β-Ala-Trp-Met-ASP-Phe-NH_2)171％,Fmoc-四肽(Fmco-Trp-Met-Asp-Phe-NH_2)32.88％,Fmoc-三肽·(Fmoc-Met-Asp-Phe-NH_2)0.17％,五肽·TFA(β-Ala-Trp-Met-Asp-Phe-NH_2·TFA)17.45％,四肽·TFA(Trp-Met-Asp-phe-NH_2·TFA)5.58％。 实验结果显示四肽胃泌素类似物的活性这比三肽的高,表明胃泌素C-端片段中色氨酸残基对泌酸活性十分重要。在胃泌素类似物的N-端导入保护基同样提高它们的生物学活性,而Fmoc-保护基的作用还强于Boc-保护性。推测其主要原因是在N-式加上一个疏水性强的基团后有利于形成一个与受体相结合的活性构象。     

利用响应面法优化Bacillus natto TK-1产脂肽发酵培养基

用响应面法对Bacillus natto TK-1发酵产脂肽的培养基进行了优化。首先用Plackett-Burman法筛选出三个影响较大的重要因素,分别为：蛋白胨、酵母粉、CaCl2 ,然后进行最陡爬坡实验逼近最佳响应面区域,最后通过Box-Behnken设计,利用SAS软件进行回归分析,得到各因素的最佳浓度。在优化培养基条件下,发酵液的溶血圈直径较初始培养基提高了29.3 %,脂肽的产量提高了约30%。     

Novel synthetic RGD analogs incorporating salicylic acid derivatives show antiplatelet activity in vitro

Continuing our investigational efforts for more active GpIIb/IIIa inhibitors we have synthesized four novel RGD (Arg-Gly-Asp) analogs incorporating salicylic acid derivatives at their N-terminal amino group using the solid phase synthesis.The synthesized compounds 5-Cl-2-HO-C₆H₃-CO-Arg-Gly-Asp(OBzl)-NH₂and 5-Br-2-HO-C₆H₃-CO-Arg-Gly-Asp(OBzl)-NH₂ were tested for their inhibitory activity on human platelet aggregationin vitro and found to be potent inhibitors of the platelet aggregation with 75 and 67% inhibitory activity respectively. In order to confirm our results flow cytometry with monoclonal antibodies against GpIb, GpIIb/IIIa, GpIIIa and GMP140receptors was used.     

The synthesis of alternative diketopiperazines as potential RGD mimetics.

Alternative RGD mimetics-with the exception of glycine-c(Arg-Asp) 1, c(Arg-Glu) 2 and c[Arg-Asp(Phe-OH)] 3 were synthesized. The DKPs were prepared on solid phase with orthogonal protection allowing further derivatization in solution. During solution phase cyclization in NH(3)/methanol, the side chain benzyl ester group of H-Arg(Tos)-Asp(OBzl)-OMe and H-Arg(Tos)-Glu(OBzl)-OMe suffer transesterification, while beta-t-butyl or beta-cyclohexyl esters are stable under the same conditions. In spite of the simple structure, all compounds bind selectively to the alpha(v)beta(3) integrin receptor, 3 showing the highest affinity with an IC(50) value of 0.74 microM value. On the other hand only 3 binds with measurable activity to the alpha(IIb)beta(3) receptor (IC(50) 159 microM). The binding affinities seem to be in accordance with the distances between the arginine guanidino and the aspartic acid carboxyl group in extended conformation determined by semiempirical geometry optimization.     

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.     

Novel synthetic RGD analogs incorporating salicylic acid derivatives show antiplatelet activity in vitro

Summary Continuing our investigational efforts for more active GpIIb/IIIa inhibitors we have synthesized four novel RGD (Arg-Gly-Asp) analogs incorporating salicylic acid derivatives at their N-terminal amino group using the solid phase synthesis. The synthesized compounds 5-Cl-2-HO−C₆H₃−CO−Arg-Gly-Asp(OBzl)-NH₂ and 5-Br-2-HO−C₆H₃−CO-Arg-Gly-Asp(OBzl)-NH₂ were tested for their inhibitory activity on human platelet aggregationin vitro and found to be potent inhibitors of the platelet aggregation with 75 and 67% inhibitory activity respectively. In order to confirm our results flow cytometry with monoclonal antibodies against GpIb, GpIIb/IIIa, GpIIIa and GMP140 receptors was used.     

Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Amino acids and peptides. XVIII. Dipeptide formation during the synthesis of Z-Asp(OBzl)-OH

During the benzyloxycarbonylation of H-Asp(OBzl)-OH by the Schotten-Bauman reaction with benzyloxycarbonyl chloride in the presence of NaHCO3 or Na2CO3, besides Z-Asp(OBzl)-OH, Z-Asp(OBzl)-Asp(OBzl)-OH was formed as side product, although the extent of the dipeptide formation differed depending on the base used (10% and 20% respectively). It was found that melting point, rotation value and Rf values upon thin-layer chromatography of Z-Asp(OBzl)-Asp(OBzl)-OH were quite similar to those of Z-Asp(OBzl)-OH.     

Helix-coil stability constants for the naturally occurring amino acids in water. XXIII. Proline parameters from random poly (hydroxybutylglutamine-co-L-proline)

Water-soluble random copolymers containing L-proline and N5-(4-hydroxybutyl)-L-glutamine were synthesized by copolymerization of the tripeptides H-L-Glu(OBzl)-L-Glu(OBzl)-L-Glu(OBzl)-OH and H-L-Glu(OBzl)-L-Pro-L-Glu(OBzl)-OH, using benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate as condensing reagent, and subsequent aminolysis of the Bzl ester groups with 4-amino-1-butanol. These copolymers were found to contain significant amounts of N5-(4-hydroxybutyl)-D-glutamine, thus requiring the synthesis of a binary copolymer containing only D- and L-N5-(4-hydroxybutyl)glutamine residues in order to evaluate the possible effects of the D-residues on the conformational properties of poly(hydroxybutylglutamine-co-L-proline). The different copolymers were fractionated, and their thermally induced helix-coil transition curves were obtained in water at neutral pH. When proper corrections were applied for the helix-destabilizing properties of N5-(4-hydroxybutyl)-D-glutamine, the Zimm-Bragg parameters sigma and s for L-proline could be deduced from the melting curves of poly(hydroxybutylglutamine-co-L-proline). The results indicate that L-proline acts as a very strong helix breaker over the entire temperature range from 0 to 60 degrees C.     

Synthesis of oligophosphoseryl sequences occurring in casein. Identification of beta-elimination during phosphorylation

The protected oligophosphoseryl peptides from bovine caseins, Z-Xxx-(Ser[PO(OPh)2])3-Glu(OBzl)-OBzl for Xxx = Ile, Val, Gly, Leu and Ph = phenyl, were synthesized in high yields by stepwise lengthening using Boc-Ser[PO(OPh)2]-OH as acylating carboxyl component and N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent. The hydrogenolytic deprotection (PtO2) was carried out with the valine derivative and with the tetrapeptide Ser[PO(OPh)2]3-Glu(OBz)-OBzl. Phosphorylation of oligoseryl peptides failed to give the expected products. Large scale phosphorylation of protected serine was carried out in the presence of triethylamine using absolute ether as a solvent. 2,2,2-Trichloroethyl group (Tc) was shown to be a useful phosphorus protecting moiety in phosphopeptide synthesis: Boc-Ser[PO(OTc)2]-OBzl, Z-Ser[PO(OTc)2]-OBzl and Boc-Glu(OBzl)-Ser[PO(OTc)2]-OBzl were synthesized in high yields using bis-(2,2,2-trichloroethyl) phosphochloridate.     

Relationship between enantioselectivity of alternative molecularly imprinted polymeric membranes and species of amino acid residues composing chiral recognition sites

Molecularly imprinted polymeric membranes with tetrapeptide residue H-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-Asp(OcHex)-CH₂- (DDDD) or H-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-Glu(OBzl)-C H₂- (EEEE) were prepared during membrane preparation (casting) processing in the presence of print molecules. The Boc-L-Trp imprinted polymeric membranes thus obtained showed adsorption selectivity toward Ac-L-Trp from its racemic mixtures. From adsorption isotherms of Ac-Trp, the chiral recognition site, that had been formed by the presence of print molecules in the membrane preparation process, exclusively recognized Ac-L-Trp that possessed the same configuration of the print molecule. The affinity constants between chiral recognition sites in the membrane and Ac-L-Trp was determined to be 1.00 × 10⁴ mol^–1 dm³ and 1.08 × 10⁴ mol^–1 dm³ for the DDDD and EEEE membranes, respectively. Enantioselective electrodialysis could be attained by applying an optimum potential difference to give permselectivity, with a value close to its adsorption selectivity.     

Block copolymerization of α-amino acid N-carboxyanhydride initiated by vinyl polymers having a terminal amino group and the characterization of the block copolymers

Poly(methyl methacrylate) and polystyrene having terminal amino groups were synthesized by the radical polymerization of those monomers in the presence of 2-mercaptoethylammonium chloride as a chain-transfer agent. By the terminal group analysis and the molecular weight determination of the polymers, 0.5–1.3 amino groups were found in a chain of poly(methyl methacrylate) and 0.5–2.5 amino groups in a chain of polystyrene. Using these polymers having a terminal amino group as an initiator, the block polymerization of α-amino acid N-carboxyanhydride (NCA) was carried out. In the polymerizations of Glu(OBzl) NCA and Lys(Z) NCA by the poly(methyl methacrylate) initiator, the terminal amino group underwent a nucleophilic addition reaction to NCA and initiated the polymerization, yielding A-B-type block copolymers in a high yield. The same was observed in the polymerizations of Gly(OBzl) NCA and Lys(Z) NCA by the polystyrene initiator. By eliminating the protecting groups of the side chains of the polypeptide segment, the block copolymers poly(methyl methacrylate)-poly(Glu), poly(methyl methacrylate)-poly(Lys), polystyrene-poly(Glu) and polystyrene-poly(Lys) were synthesized with little side reactions. The side chain amino groups of poly(Lys) segment in the poly(methyl methacrylate)-poly(Lys) block copolymers were sulphonated or stearoylated successfully.