构建含三苯基膦的人工酶
Construction of triphenylphosphine-containing artificial enzymes
摘要
随着持续深入的研究，不对称膦催化在过去二十年中取得了显着的进展，使不对称膦催化成为有机化学家最常见和最可靠的工具之一。在生物催化领域，酶通常被认为具有高选择性、绿色高效的特点。然而不对称磷催化涉及的广泛且条件温和的化学反应却极少与生物大分子结合，三取代膦通常作为配体引入蛋白骨架中，但其本身也是一类应用广泛的催化基团。
本研究利用马来酰亚胺与蛋白中的半胱氨酸中的巯基部分偶联形成酰肼，再在脱气条件下将二苯基膦苯甲醛与酰肼在温和的水相条件中形成共价结构，将三苯基膦催化中心引入蛋白疏水空腔中，过量的磷醛通过离心超滤去除。
在HPLC-MS中我们观察到三苯基膦修饰的蛋白质分子量，并对该偶联方案进行优化，最终得到了最优修饰条件，即第一步修饰为pH 7.0，底物当量12，第二步修饰为pH 6.0，底物当量10。
Abstract
With continuous and in-depth research, asymmetric phosphine catalysis has made significant progress in the past two decades, making asymmetric phosphine catalysis one of the most common and reliable tools for organic chemists. In the field of biocatalysis, enzymes are generally considered to be highly selective, green and efficient. However, asymmetric phosphorus catalysis involves a wide range of mild chemical reactions that rarely bind to biological macromolecules, and trisubstituted phosphine is usually introduced into the protein backbone as a ligand, but it is also a widely used catalytic group in its own right.
In this study, maleimide was conjugated to the sulfhydryl moiety of cysteine in the protein to form hydrazide, and then diphenylphosphine benzaldehyde and hydrazide were formed into a covalent structure under degassing conditions, the triphenylphosphine catalytic center was introduced into the protein hydrophobic cavity, and the excess phosphoformaldehyde was removed by centrifugal ultrafiltration.
In HPLC-MS, we observed the molecular weight of the triphenylphosphine-modified protein and optimized the coupling protocol to obtain the optimal modification conditions, i.e., the first step was modified to pH 7.0 with substrate equivalent of 12, and the second step was modified to pH 6.0 with substrate equivalent of 10.关键词
人工酶、合成生物学、生物偶联、磷催化Key words
Artificial enzymes/ synthetic biology/ bioconjugation/ phosphorus catalysis引用本文复制引用
黄薇,周志.构建含三苯基膦的人工酶[EB/OL].(2024-03-06)[2026-04-03].http://www.paper.edu.cn/releasepaper/content/202403-79.学科分类
生物科学研究方法、生物科学研究技术/生物化学/生物工程学
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