The efficient synthesis and biological evaluation of both the reported and revised structures of tyroscherin have been achieved. Central to our synthesis is a cross metathesis reaction that generated the trans-olefin regioselectively. This synthetic strategy enabled the facile manipulation of tyroscherin stereochemistry, facilitating the generation of all 16 tyroscherin diastereomers and a photoactivatable tyroscherin-based affinity probe for future mode of action studies.