A set of thirty-two natural and ten semisynthetic ecdysteroids was assayed in murine 3T3 cells across ten different ecdysteroid receptor (EcR) ligandbinding domains derived from nine arthropod species in an engineered gene switch format. Among the ecdysteroids tested, the most biologically widespread ecdysteroid, 20-hydroxyecdysone (20E), was moderately and consistently potent across the tested EcRs. The most potent ligand-receptor combination (EC 50 = 0.3 nm) was ponasterone A (PoA) actuating the Nephotettix cincticeps EcR switch. The most robust ligand-receptor combination, as measured by potency and efficacy, was PoA actuating either the Bombyx mori EcR or a 'VY' (E274V ⁄ V390I ⁄ Y410E) mutant of Choristoneura fumiferana EcR. Parallel ecdysteroid structure-activity relationships were observed across species; addition of hydroxyl groups at positions 2, 3, 14, 20 and 22 incrementally enhanced potency, whereas hydroxylation at position 25 retarded potency. Nevertheless, several outlier ligand-EcR combinations, such as cyasterone actuating the VY C. fumiferana EcR mutant and canescensterone activating Bemisia argentifolii EcR, exhibited an inversion of relative potency, and therefore lend themselves to construction of orthogonal duplex gene switches. The potency inversion between these two ligand-receptor pairs can be accounted for by steroid-tail contact residues Tyr411 and Met502 in VY C. fumiferana EcR corresponding to two threonines in B. argentifolii EcR. Another potency inversion was also observed with cyasterone operating on the VY mutant of C. fumiferana EcR and polypodine B activating Aedes aegypti EcR. The ecdysteroid-EcR dataset, generated in a non-natural system, nevertheless invites conjecture regarding relative ecdysteroid potencies, plant species distribution of certain phytoecdysteroids, and the role of phytoecdysteroids as chemodefense against relevant insect herbivores.
Several Anisotome diterpene derivatives were synthesized in an attempt to obtain a crystalline compound for X-ray analysis. Although we were unable to obtain a suitable crystal, the absolute configuration of the irregular diterpene skeleton was determined using two other techniques: a circular dichroism (CD) protocol based on a tetraarylporphyrin molecular tweezer that allowed prediction of the absolute stereochemistry on a microscale level, and a method employing differences in NMR shifts from derivatization of the naturally occurring acid 1 with enantiomers of a phenylglycine methyl ester (PGME) chiral anisotropic reagent. The excellent agreement between the CD and NMR methods led to the assignment of a 2S-absolute configuration for anisotomenoic acid 1.
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