Morpholine‐Facilitated Enrichment‐Triggered Delivery of Carbon Monoxide to Lysosome: A Feasibility Study

Targeted delivery of carbon monoxide (CO) prodrugs holds important therapeutic potential for various applications. Along this line, we developed an enrichment-triggered release (ETR) approach for activating 2-component (a diene and a dienophile) CO prodrugs upon enrichment in the mitochondrion, giving a "one stone, two birds" approach. Herein, we aim to broaden the scope of application to targeted delivery to the lysosome. We tethered a CO prodrug pair, a diene and a dienophile, with morpholine, a lysosomal targeting moiety. Several analogs were synthesized to tune the second-order rate constants (k₂) to a desirable range. We chose two pairs of the prodrugs with different second-order rate constants (0.087 and 0.21 M^-1 s^-1) to further study their enrichment and CO release ability. For one pair, LC-MS experiments revealed > 13-fold enrichment of the morpholine-conjugated CO prodrug pair compared to non-targeted controls in HeLa cells. Fluorescence studies demonstrated the same enrichment and co-localization of LysoTracker. For the second pair, conjugation with morpholine did not lead to improved enrichment in the lysosome. This study represents the first demonstration of lysosome-targeted delivery of CO. However, our findings also note the nonuniversal nature for a morpholine moiety to lead to lysosomal enrichment. The modest magnitude of enrichment also means that this method may only be applicable for targeted delivery of a highly potent drug.