环状、支链或侧链修饰肽的固相合成通常涉及引入带有临时侧链保护基团的残基,该临时侧链保护基团经历选择性的树脂上去除。具体而言,N α -Fmoc-N ε -(4-甲基三苯基甲基) (Mtt)-保护的赖氨酸及其较短的类似物是可商购的,并在此背景下广泛使用。然而,在tert存在下快速可靠地在树脂上去除 Mtt 基团的方法需要-丁氧羰基(Boc)基团。当前常用的条件包括二氯甲烷中低浓度 (1-3%) 的三氟乙酸 (TFA),尽管需要对每个特定应用进行调整以避免 Boc 基团过早去除或从接头上裂解。因此,进行了几种脱保护条件的头对头比较。所选酸代表从 TFA 到三氟乙醇的广泛酸度。此外,在类似条件下研究了N- (4-甲氧基三苯甲基) (Mmt) 和O-三苯甲基 (丝氨酸上)的树脂上去除。为 Mtt 脱保护确定的最温和条件包括用 30% 六氟异丙醇 (HFIP) 或 30% 全氟叔丁醇 [(CF 3) 3 COH] 在二氯甲烷中(分别为 3 × 5 或 3 × 15 分钟),而在 CH 2 Cl 2中的 30% HFIP、30% (CF 3 ) 3 COH 或 10% AcOH–20% 三氟乙醇 (TFE) ( 3 × 5 分钟)以及 5% 三氯乙酸的 CH 2 Cl 2溶液(3 × 2 分钟)能够去除 Mmt。添加/不添加 2% 三异丙基硅烷的 1% TFA 处理(3 × 5 分钟),以及用 30% (CF 3 ) 3 COH 延长处理时间(5 × 15 分钟),导致 O-Trt 基团选择性脱保护一个丝氨酸残基。在所有情况下,序列还含有 N-Boc 或 O- t Bu 保护基团,它们不受 30% HFIP 或 30% (CF 3) 3 COH 即使经过 4 小时的延长反应时间。最后,涉及 HFIP 或 (CF 3 ) 3 COH 的优化条件证明也适用于较长树脂结合肽的选择性脱保护 [即,Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile- Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] 以及几乎完全脱保护 Dab(Mtt) 残基。
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Perfluoro-tert-butanol for selective on-resin detritylation: a mild alternative to traditionally used methods
Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1–3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH–20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.