The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular and scalable synthesis of sulfonolipid IOR-1A (six steps, 27% overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta . The synthesis features a decarboxylative cross-coupling reaction of a sulfonic acid-containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR-1A derivatives, including fluorescent and photoaffinity-based probes, allowed quantification of IOR-1A, localization studies within S. rosetta cells and evaluation of structure-activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta . These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta .