Abstract:
Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.
摘要:
蛋白质由于其独特的功能和在细胞和非生物应用中的广泛用途而代表强大的生物大分子。非规范氨基酸(ncAA)的遗传编码促进了这些已经强大的蛋白质的功能多样化。具体来说,ncAA已被证明为通过缀合反应生物正交引入新的功能提供了独特的功能柄。在本文中,我们研究了单个ncAA作为产生多价生物缀合物的手柄的能力,以将两种或更多种额外的组分引入蛋白质。产生多价共轭物。为了实现这一目标,对-溴丙炔氧基苯丙氨酸(pBrPrF)被遗传编码到GFP和泛素模型蛋白中以用作缀合柄。然后优化了涉及铜辅助环加成反应以及随后的Sonogashira交叉偶联的顺序生物缀合序列。通过这些反应将两个另外的分子与模型蛋白连接产生所需的多价生物缀合物。这种使用单个ncAA的多米诺骨牌方法在治疗和诊断两者中具有过多的应用,因为可以以高度受控的方式将多个独特的部分引入蛋白质中。