Site-Selective C-C Modification of Proteins at Neutral pH Using Organocatalyst-Mediated Cross Aldol Ligations

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing dynamic biological mechanisms. The general use of chemical methods for the functionalisation of proteins remains limited however by the frequent requirement for complicated reaction partners to be present in large excess, and harsh reaction conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH under biocompatible conditions. OPAL enables rapid chemical modification of proteins within an hour using simple aldehyde probes in minimal excess, and is utilised here in the selective affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the b-hydroxy aldehyde product of the OPAL can be functionalised a second time at neutral pH in a subsequent organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the ‘chemical mimicry’ of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.