A unique carbazole-coumarin fused two-photon platform: development of a robust two-photon fluorescent probe for imaging carbon monoxide in living tissues

Two-photon fluorescent probes are favorable as powerful molecular tools for studies in biology and medicine. To construct two-photon fluorescent probes, it is necessary to have suitable two-photon fluorescent platforms. Herein, we present the rational design, synthesis, and spectral properties of carbazole-coumarin (CC) derivatives, a unique family of two-photon fluorescent dyes. Significantly, the action cross-sections of CC are tunable by modifications at the 4 '-position of the coumarin moiety, implying that CC could be exploited as a novel platform to design two-photon fluorescent probes. Carbon monoxide (CO) plays an important role in many physiological and pathological processes. Although the tracking of CO in living cells has been previously reported, the detection of CO in much thicker biosamples, for instance, living tissues, has not been realized. It is known that two-photon fluorescent probes are favorable for monitoring biomolecules in living tissues. Thus, based on the unique CC platform, we rationally engineered CC-CO as the first two-photon fluorescent CO probe, and we further demonstrated that CC-CO could monitor the changes of CO levels not only in living cells but also in living tissues for the first time, demonstrating the value of our two-photon fluorescent CO probe. The CC platform is complementary to the current two-photon fluorescent platforms and it may be used to develop a wide variety of two-photon fluorescent probes. In addition, we expect that CC-CO and its next generation analogues may be useful for unraveling the functions of CO in complicated living systems.