Abstract
<jats:p> Tuberculosis (TB) remains the world's leading infectious cause of death. Trehalose-based fluorogenic probes have emerged as powerful tools for labeling and studying mycobacteria, including <jats:italic>Mycobacterium tuberculosis</jats:italic> (Mtb), the causative agent of tuberculosis. However, existing probes occupy a limited spectral range and require compromise between brightness, specificity, and functional readouts. Here, we report the design and characterization of two trehalose conjugates derived from Janelia Fluor® dyes, JF <jats:sub>635</jats:sub> -Tre and JF <jats:sub>646</jats:sub> -Tre, which extend the trehalose-based platform into the far-red region. Following NHS ester-mediated synthesis, the trehalose-conjugated analogs displayed strong far-red fluorescence, with excitation/emission maxima at 638/654 nm and 648/663 nm for JF <jats:sub>635</jats:sub> -Tre and JF <jats:sub>646</jats:sub> -Tre, respectively. Both probes exhibited concentration- and time-dependent labeling of <jats:italic>Mycobacterium smegmatis</jats:italic> (Msmeg) and Mtb with minimal background fluorescence from the corresponding unconjugated dyes. Furthermore, we observed reduced labeling in heat-killed cells compared to live Msmeg, particularly for JF <jats:sub>646</jats:sub> -Tre, consistent with sensitivity to metabolic activity. Both JF-Tre derivatives produced significant cellular labeling and JF <jats:sub>635</jats:sub> -Tre distinguished untreated from INH-treated samples in drug susceptible Mtb, demonstrating the potential of the JF-Tre probes to report on INH susceptibility and resistance. Together, these findings expand the toolkit of trehalose-based probes and highlight how fluorophore identity influences probe performance in mycobacterial fluorescence imaging and drug susceptibility testing. </jats:p>