Occurrence and minimization of cysteine racemization during stepwise solid-phase peptide synthesis
Occurrence and minimization of cysteine racemization during stepwise solid-phase peptide synthesis
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DOI：
10.1021/jo9622744
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发表时间：
1997-06-27
影响因子：
3.6
通讯作者：
Barany, G
中科院分区：
文献类型：
--
作者：
Han, YX;Albericio, F;Barany, G
与肽合成领域的传统智慧相反，N，S保护的半胱氨酸衍生物可以通过广泛使用的试剂和逐步掺入的方案进行大量的外消旋。系统地研究了偶联条件和β-硫醇保护基团S-乙酰氨基甲基、S-三苯甲基(三苯基或三苯基)、S-2，4，6-三甲氧基苄基(Tmob)和S-9H-X-9-基(Xan)对H-Gly-D-Cys-Phe-NH2中H-Gly-L-Cys-Phe-NH2的拆分。例如，用于由膦和铵盐(例如，(benzotriazolyloxy)tris(dimethylamino)phosphonium六氟磷(BOP)、N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium六氟磷(HBTU)、N-[[(dimethylamino)-1H-1，2，3-triazolo[4，5-b]pyridin-1-yl]methylene]-N-methylmethanaminium六氟磷(HATU)和(7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium六氟磷(PYAOP))介导的偶联的标准方案，通常包括5分钟的预活化时间，并在适当的添加剂存在的情况下进行，如1-羟基苯并三氮唑(HOBt)或7-氮杂-1-羟基苯并三唑(HOAT)以及叔胺碱，如N，N-二异丙基乙胺(DIEA)或N-甲基吗啉(NMM)。在此条件下，模型肽的外消旋水平(以D：L肽的比例表示)在5-33%的完全不可接受的范围内。然而，通过避免预激活步骤，这些水平通常降低了6-7倍。减少消旋的其他战略包括改变为较弱的碱，2，4，6-三甲基吡啶(TMP，Coldine)明显好于DIEA或NMM；碱量减少2倍；以及将溶剂从纯N，N-二甲基甲酰胺(DMF)改变为更低极性的CH2Cl2-DMF(1：1)。安全地结合半胱氨酸和最小消旋的耦合方法(
Contrary to the conventional wisdom of the peptide synthesis field, N,S-protected derivatives of cysteine can undergo substantial levels of racemization with widely-used reagents and protocols for stepwise incorporation. A systematic study of this problem has been carried out as a function of coupling conditions and beta-thiol protecting groups, i.e., S-acetamidomethyl (Acm), S-triphenylmethyl (trityl or Trt), S-2,4,6-trimethoxybenzyl (Tmob), and S-9H-xanthen-9-yl (Xan), taking advantage of a convenient and quantitative model system assay involving HPLC resolution of H-Gly-L-Cys-Phe-NH2 from H-Gly-D-Cys-Phe-NH2. For example, standard protocols for couplings mediated by phosphonium and aminium salts, e.g., (benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and (7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), typically involve 5-min preactivation times and are conducted in the presence of suitable additives such as 1-hydroxybenzotriazole (HOBt) or 7-aza-1-hydroxybenzotriazole (HOAt) plus a tertiary amine base such as N,N-diisopropylethylamine (DIEA) or N-methylmorpholine (NMM). Under such conditions, the levels of racemization in the model peptide, expressed as the ratio of D:L peptide formed, were in the entirely unacceptable range of 5-33%. However, these levels were in general reduced by a factor of 6- or 7-fold by avoiding the preactivation step. Additional strategies to reduce racemization involved change to a weaker base, with 2,4,6-trimethylpyridine (TMP, collidine) being substantially better than DIEA or NMM; 2-fold reduction in the amount of base; and change in solvent from neat N,N-dimethylformamide (DMF) to the less polar CH2Cl2-DMF (1:1). Coupling methods for the safe incorporation of cysteine with minimal racemization (