Spinosyns are glycosylated polyketide-derived macrolides possessing a perhydro-as-indacene core that is presumably formed via a series of intramolecular cross-bridging reactions. The unusual structure of the spinosyn aglycone suggests an intriguing biosynthetic pathway for its formation, which is expected to be initiated by the oxidation of the 15-OH group of the mature polyketide precursor and may involve a "Diels-Alder" type [4+2] cycloaddition reaction. Three possible routes, which differ in the order of oxidation and cyclization events, can be envisioned for the biosynthesis of the core structure. Sequence analysis of the spinosyn biosynthetic gene cluster led to the speculation of spnJ as the possible oxidase gene. To explore the early stage of intramolecular ring formation, we cloned and expressed the spnJ gene and purified the SpnJ protein which shows the characteristics of flavoproteins. Two possible substrates for SpnJ, the linear mature polyketide precursor and the corresponding cyclized macrolactone, were also synthesized. TLC and HPLC analysis of the incubation mixture of these compounds with SpnJ revealed that only the synthesized macrolactone could be converted to the corresponding ketone. This result clearly indicated that macrolactone formation proceeds 15-OH oxidation since the linear polyketide is not a substrate for SpnJ. In summary, the experiments described herein detail a convergent synthesis of spinosyn macrolactone and validate the catalytic function of SpnJ as a flavin-dependent oxidase. More significantly, we have established the spinosyn macrolactone as the immediate precursor of the tricyclic nucleus of spinosyns.Spinosyns A (1) and D (2) are polyketide-derived macrolides produced by Saccharopolyspora spinosa. 1 The combination of 1 and 2 serves as the active ingredient in a commercial insecticide, Naturalyte. This mixture exhibits excellent insecticidal activity with low mammalian toxicity and little detrimental environmental effects. 2 Structurally, the Spinosyns consist of a 22-membered macrolactone ring fused to a perhydro-as-indacene core scaffold. In addition, they are glycosylated with tri-O-methylrhamnose and forosamine at C-9 and C-17, respectively. The aglycone portion of Spinosyns is unusual among polyketide-derived secondary metabolites due to the presence of three intramolecular carbon-carbon bonds that constitute the as-indacene skeleton. The unusual nature of the Spinosyn aglycone suggests an intriguing biosynthetic pathway which has generated much recent attention. 3,4The Spinosyn (spn) biosynthetic gene cluster was cloned from S. spinosa. 3 Sequence analysis coupled with gene disruption experiments suggested that macrolactone 4, which is derived from the linear polyketide precursor 3, is a likely intermediate to form the as-indacene aglycone (9), 3,4 and the proteins encoded by spnF, spnJ, spnL, spnM could be involved in the cross- If 4 is indeed a precursor of the polycyclic aglycone and the cross-bridging reactions are all post-cyclization events, oxidation ...

Tyrosine hydroxylase (TyrH) is a mononuclear, non-heme iron monooxygenase that catalyzes the pterin-dependent hydroxylation of tyrosine to dihydroxyphenylalanine. When 4-methylphenylalanine is used as a substrate for TyrH, 4-hydroxymethylphenylalanine is one of the amino acid products. To examine the mechanism of benzylic hydroxylation, the products and their isotopic compositions were determined with 4-methylphenylalanines containing a mono-, di-, or trideuterated methyl group as substrates. Intrinsic primary and secondary deuterium isotope effects for benzylic hydroxylation of 9.6 +/- 0.9 and 1.21 +/- 0.08, respectively, were derived from the data. The magnitudes of these isotope effects are consistent with quantum mechanical tunneling of the hydrogen. The similarity of the effects to those seen for benzylic hydroxylation by other enzymes supports a mechanism where a high valence iron-oxo species, Fe(IV)=O, is the hydroxylating intermediate.

[reaction: see text]. A new one-pot method using glycosyl phosphites for the synthesis of 2-deoxy-beta-oligosaccharides is described.

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Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more involved post-PKS modifications.