The remarkable degree of synthetic selectivity found in Nature is exemplified by the biosynthesis of paralytic shellfish toxins such as saxitoxin. The polycyclic core shared by saxitoxin and its relatives is assembled and subsequently elaborated through the installation of hydroxyl groups with exquisite precision that is not possible to replicate with traditional synthetic methods. Here, we report the identification of the enzymes that carry out a subset of C-H functionalizations involved in paralytic shellfish toxin biosynthesis. We have shown that three Rieske oxygenases mediate hydroxylation reactions with perfect site- and stereoselectivity. Specifically, the Rieske oxygenase SxtT is responsible for selective hydroxylation of a tricyclic precursor to the famous natural product saxitoxin, and a second Rieske oxygenase, GxtA, selectively hydroxylates saxitoxin to access the oxidation pattern present in gonyautoxin natural products. Unexpectedly, a third Rieske oxygenase, SxtH, does not hydroxylate tricyclic intermediates, but rather a linear substrate prior to tricycle formation, rewriting the biosynthetic route to paralytic shellfish toxins. Characterization of SxtT, SxtH, and GxtA is the first demonstration of enzymes carrying out C-H hydroxylation reactions in paralytic shellfish toxin biosynthesis. Additionally, the reactions of these oxygenases with a suite of saxitoxin-related molecules are reported, highlighting the substrate promiscuity of these catalysts and the potential for their application in the synthesis of natural and unnatural saxitoxin congeners.

Secondary metabolites are assembled by enzymes that often perform reactions with high selectivity and specificity. Many of these enzymes also tolerate variations in substrate structure, exhibiting promiscuity that enables various applications of a given biocatalyst. However, initial enzyme characterization studies frequently do not explore beyond the native substrates. This limited assessment of substrate scope contributes to the difficulty of identifying appropriate enzymes for specific synthetic applications. Here, we report the natural function of cyanobacterial SxtG, an amidinotransferase involved in the biosynthesis of paralytic shellfish toxins (PSTs), and demonstrate its ability to modify a breadth of non-native substrates. In addition, we report the first X-ray crystal structure of SxtG, which provides rationale for this enzyme's substrate scope. Taken together, these data confirm the function of SxtG and exemplify its potential utility in biocatalytic synthesis.

We report herein the intramolecular α--alkylation of unsaturated β-ketoesters which gives rise to highly functionalized cyclopentanes. This strategy is characterized by its operational simplicity, mild reaction conditions and the use of scandium(III) triflate as a Lewis acid catalyst. Of interest, cyclopentanes bearing heterocycles, sites for post reaction functionalization and spirocyclic architectures are accessible with this strategy.

Xanthohumol [ (E)-6′-methoxy-3′-(3-methylbuten-2-yl) -2′,4′,4″-trihydroxychalcone], the principal prenylated flavonoid from hops, has a complex bioactivity profile and 13C-labelled isotopomers of this compound are of potential use as molecular probes and as analytical standards to study metabolism and mode-of-action. 1,3-[13C]2-Xanthohumol was prepared by an adaptation of the total synthesis of Khupse and Erhardt in 7 steps and 5.7% overall yield from phloroglucinol by a route incorporating a cascade Claisen-Cope rearrangement to install the 3′-prenyl moiety from a 5′-prenyl aryl ether and an aldol condensation between 1-[13C] -2′,4′-bis(benzyloxymethyloxy) -6′-methoxy-3′-(3-methylbuten-2-yl)acetophenone and 1′- [13C]-4-(methoxymethyloxy) benzaldehyde. The 13C-atom in the methyl ketone was derived from 1-[13C]-acetyl chloride while that in the aryl aldehyde was derived from [13C]-iodomethane. Tri- and penta-13C-labelled xanthohumols were similarly prepared by applying minor modifications to the route.