Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides
Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides
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DOI：
10.1034/j.1399-3011.2002.02838.x
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发表时间：
2002-11-01
期刊：
影响因子：
--
通讯作者：
Barany, G
中科院分区：
文献类型：
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作者：
Angell, YM;Alsina, J;Barany, G
本研究详细介绍了一系列条件，以确保半胱氨酸在自动化或手动固相多肽合成过程中以最小的外消旋“安全”掺入。我们实验室的早期研究[han等人]。(1997)J.Org.化学。62,4307-4312]显示了几种常见的偶联方法，包括那些利用原位活化剂的方法，例如N-[(dimethylamino)-1H-1，2，3-triazolo[4，5-b]pyridin-1-ylmethylene]-N-methylmethanaminium六氟磷酸盐N-氧化物(HATU)、N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium六氟磷酸盐N-氧化物(HBTU)和(benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium六氟磷酸盐(BOP)[都在N-甲基吗啉或N的存在下，N-二异丙基乙胺(DIEA)作为叔胺碱)，会导致不可接受的半胱氨酸外消旋程度(即5-33%)。在三肽模型H-Gly-Cys-Phe-NH2和九肽二氢催产素上，推荐了以下方法：O-五氟苯酯(O-PFP)在DMF中；O-PFP酯/1-羟基苯并三氮唑(HOBt)在DMF中；N，N‘-二异丙基碳二亚胺(DIPCDI)/HOBt在DMF中；HBTU/HOBt/2，4，6-三甲基吡啶(TIMP)在DMF中(所有情况下的预活化时间均为3.5-7.0min)；HBTU/HOBt/TMP在CH2Cl2/DMF(1：1)中，无预激活。事实上，在我们的实验室中，上述几种方法现在被用于自动合成58个残基的蛋白质牛胰蛋白酶抑制物(BPTI)的类似物。此外，几种高度受阻的碱，如2，6-二甲基吡啶(Lutidine)2-3-5-四甲基吡啶(TEMP)，八氢吖啶(OHA)和2，6-二叔丁基-4(二甲氨基)吡啶(DB[DMAP])可以用来代替通常的DIEA或NMM，以减少半胱氨酸的外消旋，即使在原位偶联方案中也是如此。
This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH2, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DIME; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TIMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH2Cl2/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine) 2 3 5 6-tetramethylpyridine (TEMP), octahydroacricline (OHA), and 2,6-di-tert-butyl-4(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.