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 ...

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E 1 ), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B 6 -dependent enzymes, albeit with two important distinctions. It is a rare example of a B 6 -dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B 6 -dependent enzymes is replaced by histidine at position 220 in E 1 . Since alteration of His220 to a lysine residue may produce a putative progenitor of E 1 , the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E 1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic function and mechanism of E 1 .The C-3 deoxygenation step in the biosynthesis of 3,6-dideoxyhexoses, in which CDP-4-keto-6-deoxy-D-glucose (1) is converted to CDP-4-keto-3,6-dideoxy-D-glucose (2), is catalyzed by CDP-6-deoxy-D-glycero-L-threo-4-hexulose-3-dehydrase (E 1 ) and a reductase (E 3 ). 1-4 E 1 is a dimeric protein containing one pyridoxamine 5′-phosphate (PMP) and a [2Fe-2S] cluster per subunit. 5-7 E 3 belongs to the flavodoxin-NADP + reductase family and contains FAD and a plant-type ferredoxin [2Fe-2S] center in the active site. 8,9 The reaction catalyzed by E 1 and E 3 is initiated by the formation of a Schiff base between PMP and the 4-keto group of 1 (Scheme 1). 5,6 Subsequent abstraction of the pro-S 4′-H of the external Schiff base (3) results in the loss of the 3-OH group and leads to the conjugated intermediate (4). 1-7 A sequential two-electron reduction relayed from NADH via E 3 -bound FAD and the [2Fe-2S] centers of E 1 and E 3 drives the reaction to completion and regenerates the PMP coenzyme. 10,11 A radical intermediate represented by 5 has been established by EPR analysis during turnover. 12,13 The participation of PMP in deoxygenation is unusual, as is the direct

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.

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 elaborate post-PKS modifications.