Kinetic Resolution of Epimeric Proteins enables Stereoselective Chemical Mutagenesis

15 November 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications (PTMs) and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired L-configuration and a roughly equal amount of the undesired D-isomer. Here we describe a simple remedy for this issue: removal of the D-isomer via proteolysis using a D-stereoselective peptidase, alkaline D-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the D-isomer of epimeric residues within GFP and several sites of histone H3, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications.


Protein chemistry
Protein modification
D-amino acids
Post-translational modifications

Supplementary materials

Supplementary Figures and Methods
Supplementary Figures S1-S16 Experimental procedures Mass spectra


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