Automated prediction of site and sequence of protein modification with ATRP initiators

One of the most straightforward and commonly used chemical modifications of proteins is to react surface amino groups (lysine residues) with activated esters. This chemistry has been used to generate protein-polymer conjugates, many of which are now approved therapeutics. Similar conjugates have also been generated by reacting activated ester atom transfer polymerization initiators with lysine residues to create biomacromolecular initiators for polymerization reactions. The reaction between activated esters and lysine amino groups is rapid and has been consistently described in almost every publication on the topic as a "random reaction". A random reaction implies that every accessible lysine amino group on a protein molecule is equally reactive, and as a result, that the reaction is indiscriminate. Nonetheless, the literature contradicts itself by also suggesting that some lysine amino groups are more reactive than others (as a function of pKa, surface accessibility, temperature, and local environment). If the latter assumption is correct, then the outcome of these reactions cannot be random at all, and we should be able to predict the outcome from the structure of the protein. Predicting the non-random outcome of a reaction between surface lysines and reactive esters could transform the speed at which active bioconjugates can be developed and engineered. Herein, we describe a robust integrated tool that predicts the activated ester reactivity of every lysine in a protein, thereby allowing us to calculate the non-random sequence of reaction as a function of reaction conditions. Specifically, we have predicted the intrinsic reactivity of each lysine in multiple proteins with a bromine-functionalised N-hydroxysuccinimide initiator molecule. We have also shown that the model applied to PEGylation. The rules-based analysis has been coupled together in a single Python program that can bypass tedious trial and error experiments usually needed in protein-polymer conjugate design and synthesis.