Facile solid-phase synthesis of sulfated tyrosine-containing peptides:: Total synthesis of human big gastrin-II and cholecystokinin (CCK)-391,2
Facile solid-phase synthesis of sulfated tyrosine-containing peptides:: Total synthesis of human big gastrin-II and cholecystokinin (CCK)-391,2
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DOI：
10.1021/jo000895y
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发表时间：
2001-01-12
影响因子：
3.6
通讯作者：
Inoue, K
中科院分区：
文献类型：
--
作者：
Kitagawa, K;Aida, C;Inoue, K
由于酪氨酸O-硫酸化肽的硫酸酯部分具有固有的酸不稳定性，因此其化学合成仍然是肽化学家的一项艰巨任务。对于基于芴-9-基甲氧羰基（Fmoc）的硫酸化肽固相合成来说，高效的切割/脱保护过程而不损失硫酸根是尚待解决的关键困难。至;克服了困难，TFA介导的酪氨酸O-硫酸酯[Tyr（SO 3 H）]残基和两个保护基，Ser的羟基的Bu-t和Arg的胍基的2，2，4，6，7-五甲基二氢苯并呋喃-5-磺酰基（Pbf）的溶剂分解速率被详细检查。该反应符合一级动力学，具有较大的熵（59.6J（.）K（-1.）mol（-1））和焓（110.5kJ（.）mol（-1））活化。这些值证实了严格溶剂化的Tyr（SO 3 H）残基的反硝化速率具有强烈的温度依赖性。相比之下，S（N）1型脱保护的温度依赖性较小，并在高电离功率的TFA中顺利进行。基于在冷TFA中的脱保护和SN 1-型脱保护之间的大的速率差，开发了用于硫酸化肽的有效脱保护方案。我们用这种有效的脱保护方案合成含Tyr（SO 3 H）的肽的策略如下：（i）使用Fmoc-Tyr（SO 3 Na）-OH作为结构单元，用基于Fmoc的固相化学在2-氯三苯甲基树脂上直接构建硫酸化的肽链;（ii）用90%TFA水溶液在0 ℃下处理被保护的肽-树脂适当的时间以进行切割和脱保护。用此方法合成了人胆囊收缩素（CCK）-12、小胃泌素-Ⅱ（14个残基）和小胃泌素-Ⅱ（17个残基），产率为26-38%，没有任何困难。将此方法进一步应用于人大胃泌素-II（14个残基）、CCK-33和CCK-39的分步合成。尽管经过长时间的酸处理（0 ℃下15-18 h），但硫酸化肽的比例小于15%，并且以约10%的产率获得纯的硫酸化肽。
Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To;overcome:the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue: and two protecting groups, Bu-t for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J(.)K(-1.)mol(-1)) and enthalpy (110.5 kJ(.)mol(-1)) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the S(N)1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i)a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degreesC for an appropriate period of time for the cleavage and deprotection.; Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was-further applied to the stepwise synthesis of human big gastrin-II (14 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degreesC), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.