Diels-Alder ligation of peptides and proteins
Diels-Alder ligation of peptides and proteins
复制标题
DOI：
10.1002/chem.200600148
复制
发表时间：
2006-08-07
影响因子：
4.3
通讯作者：
Waldmann, Herbert
中科院分区：
文献类型：
--
作者：
de Araujo, Aline Dantas;Palomo, Jose M.;Waldmann, Herbert
报道了Diels-Alder环加成反应作为一种在温和条件下进行肽和蛋白质位点特异性化学选择性连接的新方法的发展。配备有2，4-己二烯基酯和N-末端马来酰亚胺的肽在水性介质中反应，以高产率得到环加合物，并且取决于具有高立体选择性的氨基酸序列，除了半胱氨酸SH基团，该转化与所有氨基酸侧链官能团相容。为了连接到蛋白质，通过与生物素化肽形成复合物或通过将含己二烯基酯的标记共价连接到掺入到蛋白质中的赖氨酸侧链，将己二烯基非共价连接到抗生物素蛋白和链霉抗生物素蛋白。通过表达的蛋白质连接，然后通过Ellman试剂保护产生的半胱氨酸SH，实现己二烯基单元到Rab蛋白中的位点特异性连接。该蛋白与不同的马来酰亚胺修饰的肽在温和的条件下反应，高产率地得到全功能的环加合物。结果表明，Diels-Alder连接提供了一个有利的和技术上简单的新的机会，为肽和蛋白质的位点特异性设备与进一步的官能团和标签。它在非常温和的条件下进行，并与蛋白质中发现的大多数官能团相容。它与其他连接方法，特别是表达蛋白连接的组合是可行的。
The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncovalently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible.