The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.

中文翻译：

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收支平衡：通过原位硫酯化和天然化学连接，微波加速合成富含环状和二硫化物的蛋白质。

环肽合成方法的发展是由环肽药物支架的发现推动的，例如植物来源的环肽、向日葵胰蛋白酶抑制剂 1 (SFTI-1) 和环化芋螺毒素的开发。目前，N 端半胱氨酸和 C 端硫酯基团之间的天然化学连接反应仍然是获得头对尾环化肽的最可靠方法。Boc SPPS 可有效生成肽基硫酯。然而，使用 Fmoc SPPS 生成它们是具有挑战性的，因为硫酯接头在去保护过程中对重复的哌啶暴露不稳定。在此，我们描述了一种基于 Fmoc 的协议，用于合成适用于微波辅助固相肽合成的环肽。该协议依赖于接头 Di-Fmoc-3,4-二氨基苯甲酸，我们证明了使用 Gly、Ser、Arg 和 Ile 作为 C 端氨基酸（使用 HBTU 和 HATU 作为偶联试剂）。合成后，一个N-酰基脲部分在肽的C-末端产生；然后将树脂结合的酰基脲肽脱保护并从树脂上裂解下来。完全脱保护的肽在水性缓冲液中进行硫解，原位生成硫酯。最终，头对尾环化肽是通过天然化学连接获得的。两种天然存在的环肽，原型莫比乌斯环肽 kalata B1 和 SFTI-1 被有效合成，使用优化的协议避免了二氨基接头处的潜在分支。此外，我们通过连接 50 个残基长植物防御素的两个截短片段，证明了使用该方法合成长且具有合成挑战性的线性序列的可能性。