Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo

Abstract

Increasing evidence has highlighted the endogenous production of formaldehyde (FA) in a variety of fundamental biological processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiological and pathological relevance and functions of FA, fluorescent probes for FA imaging in live biological samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theoretical calculations were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-determining step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative β-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aqueous solutions and live cells. Among these probes, the near-infrared probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chemical tools for fluorescence imaging and biological investigation of FA in living systems.