Site specific labeling methods have significant potential to enhance the properties of antibody conjugates. While studied extensively in the context of antibody-drug conjugates (ADCs), few studies have examined the impact of homogenous labeling on the properties of antibody-fluorophore conjugates (AFCs). We report the application of pentafluorophenyl (PFP) esters, which had previously been shown to be reasonably selective for K188 of the kappa light chain of human IGG antibodies, toward producing AFCs. We show that simple replacement of N-hydroxy succinimide (NHS) with PFP dramatically increases the light-chain specificity of near-infrared (NIR) AFCs. Comparing the properties of AFCs labeled using NHS and PFP-activated esters reveals that the latter exhibits reduced aggregation and improved brightness, both in vitro and in vivo. Overall, the use of PFP esters provides a remarkably simple approach to provide selectively labeled antibodies with improved properties.

Heptamethine cyanines are broadly used for a range of near-infrared imaging applications. As with many fluorophores, these molecules are prone to forming nonemissive aggregates upon biomolecule conjugation. Prior work has focused on persulfonation strategies, which only partially address these issues. Here, we report a new set of peripheral substituents, short polyethylene glycol chains on the indolenine nitrogens and a substituted alkyl ether at the C4′ position, that provide exceptionally aggregation-resistant fluorophores. These symmetrical molecules are net-neutral, can be prepared in a concise sequence, and exhibit no evidence of H-aggregation even at high labeling density when appended to monoclonal antibodies or virus-like particles. The resulting fluorophore–biomolecule conjugates exhibit exceptionally bright in vitro and in vivo signals when compared to a conventional persulfonated heptamethine cyanine. Overall, these efforts provide a new class of heptamethine cyanines with significant utility for complex labeling applications.