Design, Synthesis, and Bioactivation of O‑Glycosylated Prodrugs of the Natural Nitric Oxide Precursor N^ω‑Hydroxy‑l‑arginine

Naturally occurring N^ω-hydroxy-l-arginine (NOHA, 1) is the best substrate of NO synthases (NOS). The development of stable and bioavailable prodrugs would provide a pharmacologically valuable strategy for the treatment of cardiovascular diseases that are associated with endothelial dysfunction. To improve NOHAs druglike properties, we demonstrate that O-substitution by (glycosylic) acetal formation greatly increased the chemical stability of the hydroxyguanidine moiety and provided a nontoxic group that could be easily bioactivated by glycosidases. A straightforward synthetic concept was devised and afforded a series of diversely substituted prodrugs by O-conjugation of the hydroxyguanidine moiety with different monosaccharides. Systematic exploration of their bioactivation profile revealed that glucose-based prodrugs were more efficiently bioactivated than their galactose counterparts. NOS-dependent cytosolic NO release was quantified by automated fluorescence microscopy in a cell-based assay with murine macrophages. Glucose-based prodrugs performed particularly well and delivered cellular NO levels comparable to 1, demonstrating proof-of-concept.