Studies on α-Nucleophiles for Efficient Protein Labeling

Abstract

Specific and efficient incorporation of covalent tags into a protein is of great significance in chemical biology. One common strategy involves targeting electrophilic functional groups in proteins with specific nucleophiles harboring fluorophores or affinity tags. We designed some novel nucleophiles with neighboring groups for intramolecular catalytic effects for more efficient bioconjugation under mild conditions in aqueous buffers. Our nucleophiles are based on hydrazine and aminooxy groups, which are known for their enhanced nucleophilicity. Chapter 1 describes our investigation on these nucleophiles for reactions with acyl phosphates. Phosphoaspartate (pAsp), an acyl phosphate, is a key element in two-component systems (TCSs), which are crucial in bacterial virulence, survival, and pathogenicity. Fast and specific labeling of pAsp-containing proteins will enable facile detection, isolation, and identification of these modified proteins from cell lysates, and it will lay the foundation for the further studies on TCSs, as well as the development of novel antibacterial strategies. We tested a number of nucleophiles on various conditions using a model acyl phosphate and the results are described herein. Chapter 2 discusses our progress on the utilization of novel alpha-nucleophiles for bioconjugation to aldehydes/ketones and thioesters. Aldehyde and ketone handles are commonly used for bioconjugation via the formation of hydrazones and oximes, but their slow kinetics with nucleophiles in neutral conditions hampered their widespread applications. Recently, it was found that aniline catalyst can accelerate these reactions, but very high concentration (>100 mM) of these catalysts are required for practically useful reaction rates. To address this issue, we aimed at the development of novel nucleophiles for fast bioconjugation without the need of catalysts. Moreover, our nucleophiles can yield cyclic stable products with aldehydes. So further studies on our nucleophiles can lead to superior nucleophiles for aldehyde/ketone bioconjugation. Thioesters are another group of electrophiles utilized for protein bioconjugation. Hydrazinylation and aminoxylation of thioesters have been reported to site-specifically label proteins. However, these reactions are very slow in neutral conditions and large excess (>100 mM) of nucleophiles are typically used. We tested our nucleophiles to thioesters, and our novel alpha-nucleophiles might turn out be a solution to this issue.