16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: potent mechanism-based inhibitors of recombinant ent-kaurene synthase from Arabidopsis thaliana.
The secondary ent-beyeran-16-yl carbocation (7) is a key branch point intermediate in mechanistic schemes to rationalize the cyclic structures of many tetra- and pentacyclic diterpenes, including ent-beyerene, ent-kaurene, ent-trachylobane, and ent-atiserene, presumed precursors to >1000 known diterpenes. To evaluate these mechanistic hypotheses, we synthesized the heterocyclic analogues 16-aza-ent-beyerane (12) and 16-aza-ent-trachylobane (13) by means of Hg(II)- and Pb(IV)-induced cyclizations onto the Delta12 double bonds of tricyclic intermediates bearing carbamoylmethyl and aminomethyl groups at C-8. The 13,16-seco-16-norcarbamate (20a) was obtained from ent-beyeran-16-one oxime (17) by Beckmann fragmentation, hydrolysis, and Curtius rearrangement. The aza analogues inhibited recombinant ent-kaurene synthase from Arabidopsis thaliana (GST-rAtKS) with inhibition constants (IC50 = 1 x 10-7 and 1 x 10-6 M) similar in magnitude to the pseudo-binding constant of the bicyclic ent-copalyl diphosphate substrate (Km = 3 x 10-7 M). Large enhancements of binding affinities (IC50 = 4 x 10-9 and 2 x 10-8 M) were observed in the presence of 1 mM pyrophosphate, which is consistent with a tightly bound ent-beyeranyl+/pyrophosphate- ion pair intermediate in the cyclization-rearrangement catalyzed by this diterpene synthase. The weak inhibition (IC50 = 1 x 10-5 M) exhibited by ent-beyeran-16-exo-yl diphosphate (11) and its failure to undergo bridge rearrangement to kaurene appear to rule out the covalent diphosphate as a free intermediate. 16-Aza-ent-beyerane is proposed as an effective mimic for the ent-beyeran-16-yl carbocation with potential applications as an active site probe for the various ent-diterpene cyclases and as a novel, selective inhibitor of gibberellin biosynthesis in plants.
Roy
Arnab
A
Department of Chemistry University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Roberts
Frank G
FG
Wilderman
P Ross
PR
Zhou
Ke
K
Peters
Reuben J
RJ
Coates
Robert M
RM
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D001372:Aza Compounds / Q000737:chemistry* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry; D013729:Terpenes / Q000737:chemistry
D001372:Aza Compounds; D004791:Enzyme Inhibitors; D010710:Phosphates; D013729:Terpenes; D019883:Alkyl and Aryl Transferases; C105712:ent-kaurene synthetase B