PROJECT SUMMARY Iatrogenic nerve injury represents one of the most feared surgical complications and remains a major morbidity across all surgical specialties. Nerve-sparing radical prostatectomy is a compelling clinical example of significant patient morbidity, where nerve damage is reported in up to 60% of patients resulting in incontinence and impo- tence. Surprisingly, no clinically approved technology can enhance intraoperative nerve visualization, typically performed through neuroanatomical knowledge and conventional white light visualization alone. Development of a near infrared (NIR) fluorophore that specifically highlights nerve tissue in the operating room would have direct clinical translation to nerve sparing prostatectomy through the FDA approved fluorescence channel in the da Vinci surgical robotic system (Firefly, Intuitive Surgical, Inc.), which is used in >80% of prostatectomies per- formed in the United States today. The proposed work will directly address this unmet clinical need. Fluorescence Guided Surgery (FGS) has successfully integrated into clinical medicine with only two FDA-approved NIR fluor- ophores (i.e., indocyanine green [ICG] and methylene blue). FGS systems operate almost exclusively in the NIR (700-900 nm), where tissue chromophore absorbance, autofluorescence and scatter fall to local minima, allowing high contrast and high resolution imaging at up to centimeter depths. All clinical FGS systems have an “800 nm” channel designed to image ICG. To facilitate rapid clinical translation, the overall goal herein is to generate a nerve-specific small molecule fluorophore with spectral properties matched to ICG, enabling both nerve imaging at depth and future clinical translation using existing clinical FGS infrastructure. Design and development of a small molecule nerve-specific fluorophore that can be imaged using FGS systems optimized for ICG has been a significant challenge because these probes need to have a low enough molecular weight to cross the tight blood nerve barrier junction with a sufficient degree of conjugation for NIR excitation and emission. In exciting preliminary work, our team has synthesized first-in-class NIR nerve-specific small molecule fluorophores that can be imaged with standard FGS systems optimized for ICG. Herein, these novel probes will be synthetically tuned and validated for clinical utility through translation to swine and canine models using the da Vinci as well as completion of preclinical pharmacology and toxicology (pharm/tox) studies, enabling a future IND application to the FDA for clinical translation to robotic assisted radical prostatectomies (RARP). This goal will be accom- plished through the following specific aims: Aim 1: Synthetic tuning and characterization of NIR nerve-specific fluorophores for future clinical FGS. Aim 2: Demonstrate compatibility with the da Vinci Firefly and preclinical pharm/tox suitable for clinical translation. Aim 3: Select the optimal 800 nm, ne...