Development of a fluorescent probe library enabling efficient screening of tumour-imaging probes based on discovery of biomarker enzymatic activities.

Fluorescent probes that can selectively detect tumour lesions have great potential for fluorescence imaging-guided surgery. Here, we established a library-based approach for efficient screening of probes for tumour-selective imaging based on discovery of biomarker enzymes. We constructed a combinatorial fluorescent probe library for aminopeptidases and proteases, which is composed of 380 probes with various substrate moieties. Using this probe library, we performed lysate-based in vitro screening and/or direct imaging-based ex vivo screening of freshly resected clinical specimens from lung or gastric cancer patients, and found promising probes for tumour-selective visualization. Further, we identified two target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues. This library-based approach is expected to be an efficient tool to develop tumour-imaging probes and to discover new biomarker enzyme activities for various tumours and other diseases.

Design of spontaneously blinking fluorophores for live-cell super-resolution imaging based on quantum-chemical calculations.

Spontaneously blinking fluorophores are powerful tools for live-cell super-resolution imaging under physiological conditions. Here we show that quantum-chemical calculations can predict key parameters for fluorophore design. We applied this methodology to develop a spontaneously blinking fluorophore with yellow fluorescence for super-resolution imaging of microtubules in living cells.

Molecular design strategy of fluorogenic probes based on quantum chemical prediction of intramolecular spirocyclization.

Fluorogenic probes are essential tools for real-time visualization of dynamic intracellular processes in living cells, but so far, their design has been largely dependent on trial-and-error methods. Here we propose a quantum chemical calculation-based method for rational prediction of the fluorescence properties of hydroxymethyl rhodamine (HMR)-based fluorogenic probes. Our computational analysis of the intramolecular spirocyclization reaction, which switches the fluorescence properties of HMR derivatives, reveals that consideration of the explicit water molecules is essential for accurate estimation of the free energy difference between the open (fluorescent) and closed (non-fluorescent) forms. We show that this approach can predict the open-closed equilibrium (pKcycl values) of unknown HMR derivatives in aqueous media. We validate this pKcycl prediction methodology by designing red and yellow fluorogenic peptidase probes that are highly activated by γ-glutamyltranspeptidase, without the need for prior synthesis of multiple candidates.