Disulfide linkages of proteins are attractive yet underutilized sites for bioconjugation. Their use is hampered by limitations in existing disulfide bioconjugation chemistry including slow reaction kinetics, moderate yields, and suboptimal conjugate stability. Moreover, an intrinsic limitation of disulfide bioconjugation is that it can be performed only on proteins that either contain native cystine disulfide linkages or have been engineered by introducing two non-native cysteines that form this linkage during protein folding. Introducing two exogenous cysteines into a protein is not ideal as it renders it susceptible to misfolding. Herein, we report the StapleAld technology and combine it with the LAP tag technology (LAP-StapleAld) to overcome these limitations. StapleAld employs 2-formylallyl carboxylates for the rapid and quantitative formation of stable disulfide rebridged adducts appended with aldehyde tags for late-stage labeling. LAP-StapleAld integrates StapleAld with a chemoenzymatic approach to install lipoyl disulfide linkages at any desired site on any protein post-folding circumventing the protein misfolding problem, rendering disulfide bioconjugation universally applicable and location agnostic. Building on model studies on small molecules, we employ these platforms for the rapid, quantitative, and stable labeling of the cyclic peptides somatostatin and vasopressin and proteins including lysozyme, maltose-binding protein, eGFP, BtuF, and the therapeutic antibody, trastuzumab.
Shaikh et al. (Thu,) studied this question.