Two diasteroisomers of the Penicillium metabolite penicillenol C1 were synthesized for the first time by 3-acylation of an L-threonine-derived tetramic acid with enantiopure 2-methyloct-(6E)-enoic acids. The 5S,6R,9S isomer has NMR spectra and optical rotation identical with those of the natural compound. A bis-azide-tagged penicillenol analogue was also synthesized for photoaffinity labeling of target proteins. The photolysis of the bis-azide in the presence of methanol as a protein-mimicking nucleophile led to reaction only of the aryl azide, while leaving the benzyl azide available for pull-downs or the attachment of fluorescent tracers. As a proof of concept, the distribution of this bis-azide-tagged tetramic acid in living cells was visualized via a Staudinger ligation between the azide tag and a phosphane fluorophore.