Novel Chemoselective Linkage Chemistry toward Controlled Loading of Ligands to Proteins through In Situ Real-Time Quantification of Conjugate Formation

‘Linkage chemistry', which encompasses the science of chemical attachment of a ligand molecule to a carrier moiety, plays a crucial role in a wide range of biochemical and biophysical disciplines. In particular, the production of synthetic vaccines, where quality assurance criteria are an essential part of the approvals procedure for development of medicines, is reliant upon reproducible linkage chemistries. Herein, we describe novel 2-hydroxybenzaldehyde-based quaternary amine containing chemoselective linkers that provide a simple and robust linkage process that overcomes the deficiencies present in state-of-the-art linkage chemistries. The 2-hydroxybenzaldehyde groups undergo a pH-dependent absorbance change that enabled its nondestructive quantification, even when covalently attached to a wide range of proteins. Additionally, formation of a hydrazone bond between the benzaldehyde group and a range of ligand hydrazides resulted in a second reversible absorbance change enabling the forward (ligand loading) and reverse (ligand release for analysis) reactions of ligand-loaded proteins to be monitored in situ and quantified in real time. Incorporation of the quaternary amine moiety into our improved linkage chemistries was found to increase the relative solubility of protein conjugates and enabled significantly higher loading of proteins with linker and subsequent ligands, while retaining aqueous solubility, when compared to standard methods.