conifer defense ͉ gibberellic acid ͉ diterpene resin acids ͉ conifer genomics ͉ plant secondary metabolism D iterpene resin acids (DRAs) (Fig. 1A) are important defense compounds of conifers against potential herbivores and pathogens, such as bark beetles and their associated fungi (1-3). DRAs are formed and sequestered as major components of complex oleoresin blends in resin ducts, resin blisters, or resin cells in stems, needles, and roots of most conifers. Biosynthesis of DRAs involves formation of geranylgeranyl diphosphate (GGDP), cyclization of GGDP to a series of diterpene olefins by activity of diterpene synthases (diTPSs), and three subsequent oxidations at carbon 18 (Fig. 1B). A small family of singleproduct or multiproduct diTPSs of DRA biosynthesis has recently been cloned and characterized (4-6). Studies with grand fir (Abies grandis) and lodgepole pine (Pinus contorta) tissue extracts showed that stepwise oxidation of the diterpene olefin abietadiene (1a) to abietic acid (1d) can be achieved by membrane-bound cytochrome P450 monooxygenase (P450) and soluble aldehyde dehydrogenase enzyme activities (Fig. 1B) (7, 8). The general pathway scheme of oxidation of abietadiene to abietic acid in conifer secondary metabolism resembles that of oxidation of ent-kaurene to ent-kaurenoic acid in the biosynthesis of gibberellin phytohormones (9-11). A multifunctional P450 responsible for the three-step oxidation of ent-kaurene to kaurenoic acid has previously been identified by using genetic approaches in Arabidopsis thaliana (10-11). Similarly, a multifunctional P450 also catalyzes the subsequent three-step oxidation from kaurenoic acid to GA 12 in Arabidopsis (12). Cloning and identification of P450s of DRA secondary metabolism have been hampered by difficulties in purifying the corresponding enzymes and by lack of suitable forward genetic tools for conifers. However, large collections of ESTs for loblolly pine (Pinus taeda, Pt) (13) can enable gene discovery and biochemical identification of candidate P450 cDNAs of terpenoid secondary metabolism in conifers. Here, we describe cloning and functional characterization of abietadienol͞abietadienal oxidase (PtAO, CYP720B1), a P450 enzyme that is unusual in that it catalyzes an array of consecutive oxidations of multiple diterpene alcohol and aldehyde intermediates in DRA biosynthesis in loblolly pine. The methyl jasmonate (MJ)-inducible PtAO can account for much of the oxidative diversification of diterpenoid natural product defense compounds in loblolly pine. Materials and MethodsMaterials. Seedlings of loblolly pine and Sitka spruce (Picea sitchensis) were grown to 2-year-old trees as described in ref. 14. Yeast strain YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-⌬63, his3-⌬200, and leu2-⌬1) and yeast dual expression vectors (pESC-Leu and pESC-His) were from Stratagene. Diterpenoid substrates were prepared from the corresponding DRAs (Helix Biotech, Surrey, BC, Canada) as described in Supporting Materials and Methods, which is published as supporting informat...

The antimitotic sponge tripeptide hemiasterlin (1) and a number of structural analogues have been synthesized and evaluated in cell-based assays for both cytotoxic and antimitotic activity in order to explore the SAR for this promising anticancer drug lead. One synthetic analogue, SPA110 (8), showed more potent in vitro cytotoxicty and antimitotic activity than the natural product hemiasterlin (1), and consequently it has been subjected to thorough preclinical evaluation and targeted for clinical evaluation. The details of the synthesis of hemiasterlin (1) and the analogues and a discussion of how their biological activities vary with their structures are presented in this paper.

Crude methanol extracts of the ascidian Didemnum granulatum collected in Brazil showed activity in a new screen for G2 cell cycle checkpoint inhibitors. Bioassay-guided fractionation of the extract yielded the known alkaloids didemnimides A (1) and D (2), the new alkaloid didemnimide E (3), and a new G2 checkpoint inhibitor. Two candidate structures for the inhibitor, named granulatimide (4) and isogranulatimide (5), have been prepared via a short and efficient biomimetic synthesis involving the photolysis of didemnimide A (1). The synthesis revealed that the correct structure for the naturally occurring G2 checkpoint inhibitor is isogranulatimide (5). Granulatimide (4), the other candidate structure, was also found to be a G2 checkpoint inhibitor, and it was subsequently detected in chromatographic fractions associated with purification of D. granulatum alkaloids. Granulatimide (4) and isogranulatimide (5) represent the first examples of a new class of G2 specific cell cycle checkpoint inhibitors and the first ones identified through a rational screening program.

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