Jung G, Brückner H
Hoppe Seylers Z Physiol Chem. 1981 Mar;362(3):275-89.
The N-terminal sequence H-Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-OH (FIF[1-13]) of human fibroblast interferon HuIFN-beta(Fi) has been synthesized using the solid-phase method. After esterification of N-tert-butyloxycarbonyl-O-benzyl-L-serine cesium salt with chloromethylated polystyrene-1% divinylbenzene (loading 0.25 mmol/g) the tridecapeptide was built up stepwise. Coupling reagents and N-tert-butyloxycarbonylamino acids were used in a six-fold excess. For the second coupling 1-hydroxybenzotriazole was added during carbodiimide and 4-nitrophenyl glutaminate or asparaginate couplings. Side chain functions were masked: O-benzylserine, O-(2,6-dichlorobenzyl)tyrosine and Ng-tosylarginine. After an acetylation step the N-protection was removed by trifluoroacetic acid/dichloromethane 1:1, and for neutralisation triethylamine/-chloroform 1:9 were used, both steps with a prewash. The Ng-tosyltridecapeptide was split-off from the resin by HBr in trifluoroacetic acid and purified by repetitive precipitations. After deprotection of the guanidino group of arginine with sodium in liquid ammonia, the peptide was precipitated from acetic acid/water, chromatographed on Sephadex G-25 coarse in acetic acid/water 1:1 and precipitated from acetic acid/ether and dimethylformamide/acetone. After purification by multiplicative counter-current distribution in butanol/-5% acetic acid/propanol 5:5:1 the tridecapeptide was pure according to chromatographic, electrophoretic, enzymatic and instrumental analyses. The peptide was investigated by circular dichroism in trifluoroethanol and hexafluoroacetonesesquihydrate and 13C-nuclear magnetic resonance, which revealed an alpha-helical conformation. In order to obtain a suitable antigen the tridecapeptide was coupled to poly(L-lysine) (molecular mass 37300) via N,N'-dicyclohexylcarbodiimide followed by dialysis. The resulting poly(L-lysine)-FIF[1-13] conjugate showed a loading of 17.8 mol FIF[1-13] per mol poly(L-lysine).
已采用固相法合成了人成纤维细胞干扰素HuIFN-β(Fi)的N端序列H-Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-OH(FIF[1-13])。用氯甲基化聚苯乙烯-1%二乙烯苯(负载量0.25 mmol/g)将N-叔丁氧羰基-O-苄基-L-丝氨酸铯盐酯化后，逐步合成十三肽。偶联试剂和N-叔丁氧羰基氨基酸使用过量6倍。第二次偶联时，在碳二亚胺以及4-硝基苯基谷氨酸酯或天冬氨酸酯偶联过程中加入1-羟基苯并三唑。侧链官能团被保护：O-苄基丝氨酸、O-(2,6-二氯苄基)酪氨酸和Nε-甲苯磺酰精氨酸。乙酰化步骤后，用三氟乙酸/二氯甲烷1:1除去N-保护基，中和时使用三乙胺/氯仿1:9，两步均进行预洗。用氢溴酸在三氟乙酸中将Nε-甲苯磺酰十三肽从树脂上裂解下来，并通过反复沉淀进行纯化。用钠在液氨中脱去精氨酸胍基的保护基后，将肽从乙酸/水中沉淀出来，在乙酸/水1:1的Sephadex G-25粗柱上进行层析，然后从乙酸/乙醚和二甲基甲酰胺/丙酮中沉淀出来。在丁醇/-5%乙酸/丙醇5:5:1中通过多次逆流分配纯化后，根据色谱、电泳、酶学和仪器分析，该十三肽是纯的。在三氟乙醇和六氟丙酮倍半水合物中通过圆二色性以及通过13C核磁共振对该肽进行了研究，结果显示其具有α-螺旋构象。为了获得合适的抗原，通过N,N'-二环己基碳二亚胺将十三肽与聚(L-赖氨酸)(分子量37300)偶联，随后进行透析。所得的聚(L-赖氨酸)-FIF[1-13]缀合物显示每摩尔聚(L-赖氨酸)负载17.8摩尔FIF[1-13]。
