Accelerated chemical synthesis of peptides and small proteins
Accelerated chemical synthesis of peptides and small proteins
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DOI：
10.1073/pnas.96.4.1181
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发表时间：
1999-02-16
影响因子：
11.1
通讯作者：
Alewood, PF
中科院分区：
文献类型：
--
作者：
Miranda, LP;Alewood, PF
多肽和小蛋白的化学合成是重组蛋白过表达的有力补充策略，广泛应用于结构生物学、免疫学、蛋白质工程和生物医学研究。尽管在肽链组装、侧链保护和合成后分析的保真度方面有了很大的改进，但获得含有50个以上残基的多肽的一个限制因素仍然是链组装所需的时间。这项工作的最终目标是建立高效的化学程序，实现大约每小时10-15个残基的链组装速度，从而支持长多肽和蛋白质的快速化学合成，包括细胞因子、生长因子、蛋白质结构域和小型酶。在这里，我们报告BOC化学使用O-(7-氮杂苯并-三氮唑-1-基)-N，N，N‘，N’-四甲基氟磷酸(HATU)/二甲基亚砜作为偶联剂，每隔5分钟加入一个受保护的氨基酸残基作为偶联剂，以产生高质量的多肽。这种快速的偶联化学已被成功地证明通过合成几个小到中型的多肽，包括HIV-1蛋白酶的“困难的”C-末端序列(残基81-99)；酰基载体蛋白的片段65-74；强有力的神经元尼古丁受体拮抗剂螺杆菌毒素PnIA(A10L)；以及促炎的趋化蛋白CP10，一种88个残基的蛋白质，通过天然的化学连接，这种方法的好处包括增强识别和表征“难偶联”的能力，快速获得用于生物学和结构活性研究的多肽，以及加速合成定制的大肽片段(
The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes, Here we report Boc chemistry that employs O-(7-azabenzo-triazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the "difficult" C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the proinflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation, The benefits of this approach include enhanced ability to identify and characterize "difficult couplings," rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (