Iridoids are important natural products and the secoiridois derived thereof are universal precursors of all monoterpene indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. Thus, the precise elucidation of their biosynthesis pathway is a significant step to maximize the biotechnological production of this natural product class. Iridoid synthases (IRIS) belong to the PRISEs among the SDR family of proteins and catalyze the decisive step in the biosynthesis of iridoids, namely the cyclization of 8-oxogeranial to nepetalactol, the common biosynthetic precursor for all iridoids [1]. 8-oxogeranial is not available commercially but can be synthesized chemically in four steps starting from geraniol [2]. We here optimized the synthesis of 8-oxogeranial. This allowed the investigation of the conversion of 8-oxogeranial by various PRISE enzymes. The protocol involved the conversion of geraniol to geranyl acetate (steglich esterification; yield of 99 %). Microwave-assisted oxidation of the allyl group by selenium dioxide and flash column chromatography provided 8-oxogeranyl acetate (yield 42 %). The protecting group was removed by base-catalyzed hydrolysis to afford 8-oxogeraniol (yield 98 %). In the last step, swern oxidation and the subsequent column chromatographic treatment supplied the desired product 8-oxogeranial in a high purity with a yield of 50 %. The substrate was used to characterize recombinant forms of PRISEs from Arabidopsis thaliana, Digitalis lanata and Plantago species. The immediate availability of 8-oxogeranial will now help to elucidate still open questions in PRISE action, such as stereochemistry of bicyclic iridoid scaffold formation and mechanisms of substrate discrimination/ preferences of PRISES.
[1] Lindner, S. et al. 2014. Conversion of substrate analogs suggests a michael cyclization in iridoid biosynthesis. Chemistry and Biology 21(11): p.1452–1456.
[2] Geu-Flores, F. et al. 2012. An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis. Nature 492(7427): p.138–142.