Design and Synthesis of Thioglycosylated Monolignol Dual Probes for Bioimaging of Lignin Biosynthesis

Lignin biosynthesis is a critical process that underpins plant structural integrity and defenses. Central to this pathway are monolignol glucosides (MLGs), whose role as intermediates remains debated. To elucidate MLGs' involvement, we developed thioglycosylated monolignol probes compatible with click chemistry for in situ visualization of lignin biosynthesis. Using a highly selective Buchwald-Hartwig-Migita cross-coupling approach, these probes incorporate glycosyl thiols into MLGs, creating stable thioacetal bonds to enhance both metabolic stability and tracking precision. The unique chemistry of these probes allows for incorporation within the lignification pathway, enabling specific visualization of MLG involvement in lignin formation. The probes are compatible with bioorthogonal chemistry labeling and confocal microscopy, allowing detailed tracking of MLG transport, storage, and incorporation into cell walls. Our findings provide new insights into lignification dynamics, underscoring the metabolic roles of MLGs and demonstrating their potential as metabolic intermediates in lignin polymerization. This approach offers a novel chemical biology toolset to dissect plant cell wall biosynthesis and will help elucidatethe molecular roles of MLGs in the context of plant biochemistry and resilience.