# Programmable wide-range pH gradients for NMR titrations: application to antibody–drug conjugate linker group modifications

## TL;DR

This paper introduces a method to create pH gradients in NMR tubes for quickly measuring pKa values, which helps in designing better antibody-drug conjugate linkers.

## Contribution

A new NMR method for rapid pKa determination of polyprotic compounds using tunable pH gradients in standard NMR tubes.

## Key findings

- pKa values of polyprotic compounds can be determined in a single 20-minute NMR experiment.
- A strong correlation was found between pKa and the rate of conjugate addition of thiol nucleophiles to vinyl groups.
- The method is suitable for routine use in organic molecule optimization during synthesis.

## Abstract

We generate pH gradients spanning more than six units in standard NMR tubes and determine all the pKa values of polyprotic compounds in single 20 minutes chemical shift imaging (CSI) NMR experiments. The modest demands of our method in terms of sample quantity and preparation time allow it be performed as part of the routine characterisation and optimisation of organic molecules during synthesis campaigns. As proof of concept, we measure the pKa values of a family of vinylpyridines employed as antibody drug conjugate linkers. Our analysis reveals a strong correlation between the experimental aqueous pKa and the rate of conjugate addition of thiol nucleophiles to the vinyl group, representing a powerful predictive method.

Tuneable pH gradients are generated within NMR tubes using buffer components enabling extraction of the pKa of polyprotic molecules by chemical shift imaging. Knowledge of pKa informs the design of vinylpyridine antibody drug conjugate linkers.