Intermediacy of eudesmane cation during catalysis by aristolochene synthase.
Aristolochene synthase from Penicillium roqueforti (PR-AS) catalyzes the formation of the bicyclic sesquiterpene (+)-aristolochene (5) from farnesyl diphosphate (1, FDP) in two mechanistically distinct cyclization reactions. The first reaction transforms farnesyl diphosphate to the uncharged intermediate (S)-(-)-germacrene A (3) through a macrocyclization process that links C1 and C10 upon magnesium ion-assisted diphosphate ester activation. In the second reaction mediated by PR-AS, a protonation induced cyclization has been suggested to generate the highly reactive trans-fused eudesmane cation 4 as a consequence of the precise folding of the enzyme-bound germacrene A intermediate. This contribution describes the use of the transition state analogue inhibitor 4-aza-eudesm-11-ene to explore the intermediacy of cation 4 as an on-path intermediate in the biosynthesis of aristolochene. 4-Aza-eudesm-11-ene as the hydrochloride salt 6 was stereospecifically synthesized in seven steps and 37% overall yield starting from chiral enamine 9. The synthetic sequence featured a highly regio- and stereoselective deracemization reaction of 9 that gave rise to the corresponding Michael adduct in >95% diastereomeric excess as evidenced by optical rotation and NMR measurements. 6 acts as a potent competitive inhibitor of PR-AS (K(i) = 0.35 +/- 0.12 microM) independent of the presence of diphosphate (K(i) = 0.24 +/- 0.09 microM). The failure of exogenous PP(i) to enhance the binding affinity of 6 for PR-AS could be interpreted against an eudesmyl cation/diphosphate anion pair mechanism as the enzymatic strategy to stabilize the highly reactive eudesmane cation 4. In addition, these observations seem to rule out simple favorable electrostatic and/or hydrogen bonding interactions between the active site anchored diphosphate ion and the ammonium ion 6 as the binding mode. Ammonium ion 6 seems to act as a genuine mimic of eudesmane cation (4) that most likely binds the active site of PR-AS in a productive conformation resembling that adapted by 4 during the PR-AS-catalyzed synthesis of 5.
Faraldos
Juan A
JA
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, United Kingdom.
Kariuki
Benson
B
Allemann
Rudolf K
RK
D001665:Binding Sites; D002384:Catalysis; D002412:Cations / Q000737:chemistry*; D047248:Crown Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D003500:Cyclization; D007535:Isomerases / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D010407:Penicillium / Q000737:chemistry*; D012717:Sesquiterpenes / Q000737:chemistry*; D045787:Sesquiterpenes, Eudesmane / Q000737:chemistry*; D045788:Sesquiterpenes, Germacrane / Q000138:chemical synthesis* / Q000737:chemistry*; D013237:Stereoisomerism
D002412:Cations; D047248:Crown Compounds; D012717:Sesquiterpenes; D045787:Sesquiterpenes, Eudesmane; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; C015872:eudesmane; D007535:Isomerases; C060050:aristolochene synthase