oNBS-SPPS: A nem method for solid-phase peptide synthesis
oNBS-SPPS: A nem method for solid-phase peptide synthesis
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DOI：
10.1021/ja974252k
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发表时间：
1998-03-25
影响因子：
15
通讯作者：
Scanlan, TS
中科院分区：
文献类型：
--
作者：
Miller, SC;Scanlan, TS
自从固相肽合成（SPPS）出现以来，1许多不同的保护基策略被用于肽的侧链和氨基末端。 Boc 保护基方案 2 临时 Boc 氨基保护基对三氟乙酸不稳定，侧链保护可被无水 HF 裂解，该方案仍在广泛使用，但在许多实验室已被 Fmoc-SPPS 取代。 3 Fmoc-SPPS 的优点包括试剂更温和、能够通过 UV 监测 Fmoc 脱保护，以及通常所需肽的产量更高。然而，它比 Boc-SPPS 贵得多。在这里，我们介绍了一种基于新的 R-氨基保护基的 SPPS 方法，该方法保留了与 Fmoc-SPPS 的兼容性，并提供了一些额外的优点。 Fukuyama 等人首次描述的邻硝基苯磺酰基 (oNBS) 4 提供了许多支持其用作临时氨基保护基团的示例性特征：(1) oNBS 保护的肽脱保护会释放黄色发色团，从而可以简单地目视确认脱保护，(2) 与 Fmoc-SPPS 不同，oNBS-SPPS（方案 1）允许对在肽合成过程中，oNBS 保护的氮，5 (3) oNBS 氨基酸氯化物可用于与固相支持物上的极度受阻胺偶联，其中类似的 Fmoc 氨基酸氯化物无法很好地偶联，并且 (4) 合成 oNBS 氨基酸所需的试剂 2-硝基苯磺酰氯是可商购的，并且比 Fmoc-Cl 便宜得多。最近，我们报道了一种在固相支持物上选择性甲基化肽的方法。 5 我们的方案需要对支持物结合的 Fmoc 保护肽进行脱保护，并随后用 oNBS 基团对其进行重新保护。如果感兴趣的氨基酸最初作为 oNBS 氨基酸掺入，则可以消除这两个步骤。我们使用 Schotten-Baumann 程序合成了许多 oNBS 氨基酸，并发现它们的偶联可以使用标准 Fmoc 偶联程序干净地进行。例如，在 0.4 M NMM/DMF 中，用肽偶联剂 HBTU 将 oNBS-Leu 和 oNBS-Arg (Pmc) 分别与树脂结合肽 LRN 和 GAP 偶联 20 分钟，得到 oNBS-LLRN 和 oNBS-RGAP，并且通过 HPLC 显示没有杂质。随后 Pd (0) 催化的 oNBS-LLRN 烯丙基化实现了 98% 的产率，7 并且 oNBS-RGAP 的甲基化几乎是定量的。 8 按照报道进行脱保护，5 以高产率得到相应的 N-烷基化肽。然后可以使用 oNBS 或 Fmoc 氨基酸继续进行肽合成。我们感兴趣的是确定 oNBS 氨基酸衍生物是否可以像 Fmoc 氨基酸一样用于非烷基化肽序列的一般合成。因此，我们尝试使用 oNBS 氨基酸在 0.05 mmol 规模上自动固相合成凝血酶受体激动剂肽酰胺 SFLLRN。 9 使用 2-巯基乙醇对 oNBS 保护的氨基酸衍生物进行脱保护，对于 N-烷基化衍生物具有选择性，5 但苯硫酚很容易裂解未烷基化的 oNBS 衍生物。因此，我们最初在 oNBS-SPPS 方面的尝试使用了 5% 苯硫酚的 DMF 溶液以及多种不同的可溶性碱，代替了用于 Fmoc 脱保护的 20% 哌啶/DMF 溶液。苯硫酚和碱的溶液特别容易被空气氧化，因此，所有操作均在惰性气氛下进行。通过对释放的黄色发色团进行简单的目视检查即可轻松脱保护。使用...
Since the advent of solid-phase peptide synthesis (SPPS), 1 many different protecting group strategies have been employed for the side chains and amino termini of peptides. The Boc protecting group scheme, 2 where the temporary Boc amino protecting group is labile to trifluoroacetic acid and the side chain protection is cleaved by anhydrous HF, is still in wide use but has been replaced in many laboratories by Fmoc-SPPS. 3 Advantages of Fmoc-SPPS include milder reagents, ability to monitor Fmoc deprotection by UV, and in general, higher yields of desired peptide. However, it is considerably more expensive than Boc-SPPS. Here, we introduce a method for SPPS based on a new R-amino protecting group that retains compatibility with Fmoc-SPPS and offers several additional advantages. The o-nitrobenzenesulfonyl group (oNBS), first described by Fukuyama et al., 4 offers a number of exemplary features that support its use as a temporary amino protecting group:(1) deprotection of oNBS-protected peptides releases a yellow chromophore which allows simple visual confirmation of deprotection,(2) unlike Fmoc-SPPS, oNBS-SPPS (Scheme 1) allows optional and selectiVe N-methylation of the oNBS-protected nitrogen during peptide synthesis, 5 (3) oNBS amino acid chlorides can be used to couple to extremely hindered amines on a solid support where the analogous Fmoc amino acid chlorides fail to couple well, and (4) the reagent necessary to synthesize oNBS amino acids, 2-nitrobenzenesulfonyl chloride, is commercially available and considerably cheaper than Fmoc-Cl. Recently, we reported a method for the selective methylation of peptides on solid support. 5 Our scheme required the deprotection of a support-bound Fmoc-protected peptide and its subsequent reprotection with the oNBS group. These two steps could be eliminated if the amino acid of interest was originally incorporated as an oNBS amino acid. We have synthesized a number of oNBS amino acids using the Schotten-Baumann procedure6 and have found their coupling to proceed cleanly using standard Fmoc coupling procedures. For example, the coupling of oNBS-Leu and oNBS-Arg (Pmc), respectively, to the resinbound peptides LRN and GAP with the peptide coupling agentHBTU for 20 min in 0.4 M NMM/DMF gave oNBS-LLRN and oNBS-RGAP and showed no impurities by HPLC. Subsequent Pd (0)-catalyzed allylation of oNBS-LLRN was achieved in 98% yield, 7 and methylation of oNBS-RGAP was virtually quantitative. 8 Deprotection was carried out as reported, 5 giving the respective N-alkylated peptides in high yields. Peptide synthesis could then be continued using either oNBS or Fmoc amino acids. We were interested in determining whether oNBS amino acid derivatives could be used, like Fmoc amino acids, in the general synthesis of unalkylated peptide sequences. Thus, we attempted the automated solid-phase synthesis of the thrombin-receptor agonist peptide amide, SFLLRN, on 0.05 mmol scale using oNBS amino acids. 9 Deprotection of oNBS-protected amino acid derivatives with 2-mercaptoethanol is selective for the N-alkylated derivatives, 5 but thiophenol readily cleaves unalkylated oNBS derivatives. Thus, our original attempts at oNBS-SPPS utilized a 5% solution of thiophenol in DMF with a number of different soluble bases in place of the 20% piperidine/DMF solution used for Fmoc deprotection. Solutions of thiophenol and base are particularly prone to air oxidation, and thus, all manipulations were performed under inert atmosphere. Deprotection was easily followed by simple visual inspection of the released yellow chromophore. Use of …