Post-translational site-selective protein backbone α-deuteration

Area of Science:

• Chemical Biology
• Protein Chemistry
• Biophysical Chemistry

Background:

• Isotopic replacement is valuable in small molecules but limited in proteins to biosynthesis or labile sites.
• Postbiosynthetic C-H to C-D/H exchange in proteins is needed to probe non-exchangeable sites and reaction mechanisms.
• Current methods for isotopic labeling in proteins are often restricted to specific sites or require complex biosynthetic approaches.

Purpose of the Study:

• To develop a postbiosynthetic chemical method for selective C-H to C-D/H exchange at non-exchangeable protein backbone sites.
• To apply this C-D labeling strategy to investigate the mechanisms of protein bioconjugation reactions.
• To establish a versatile tool for studying protein mechanisms, structure, biotechnology, and medicine.

Main Methods:

• Selective protein deuteration via a Cys to dehydroalanine (Dha) to deutero-Cys conversion pathway.
• Application of the C-D labeling method to intact protein constructs without specialized culture or genetic engineering.
• Integration of experimental analysis with quantum mechanical calculations to elucidate reaction mechanisms.

Main Results:

• Demonstrated selective C-H to C-D/H exchange at a non-exchangeable α-carbon backbone site in proteins.
• Elucidated the mechanism of Cys to Dha conversion, revealing stepwise deprotonations via on-protein carbanions and sulfonium ylides.
• Confirmed a 'carba-Swern' mechanism for the Cys to Dha transformation.

Conclusions:

• The developed chemical method enables postbiosynthetic C-D labeling at previously inaccessible protein backbone sites.
• This strategy provides a powerful tool for mechanistic studies in protein bioconjugation and other chemical transformations.
• The facile application of this technique on intact proteins opens new avenues in protein science, biotechnology, and medicine.