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Cyclization of Pregaliellalactone in the Fungus Galiella rufa

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COMMUNICATIONS2158 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1433-7851/02/4112-2158 $ 20.00+.50/0 Angew. Chem. Int. Ed. 2002, 41,No.12Cyclization of ()-Pregaliellalactonein the Fungus Galiella rufaMartin Johansson, B‰rbel Kˆpcke, Heidrun Anke, andOlov Sterner*The Diels ± Alder pericyclic [42] cycloaddition between adiene and a dienophile is one of the most powerful carbon ±carbon bond forming reactions in organic chemistry,[1]and ithas been utilized in the total syntheses of many naturalproducts.[2]However, Nature does not appear to haveappreciated the reaction to the same extent as syntheticchemists: only few examples of enzyme-catalyzed Diels ±Alder reactions in biosynthetic pathways have been report-ed.[3]Some secondary metabolites may be Diels ± Alderadducts although it has been difficult to determine whetherthey were formed through an actual [42] cycloaddition orthrough an ionic or stepwise process.[4]In reaction pathwayswhich comprise a Diels ± Alder reaction the question whetherthe reaction is spontaneous or catalyzed, for example by aDiels-Alderase, still remains. The problems to demonstratethe action of a Diels-Alderase in a biosynthetic pathway areassociated with the difficulties to establish such a pathwayunambiguously and with the low availability of biosyntheticprecursors for testing.So far, only two enzyme-catalyzed biosynthetic Diels ±Alder reactions have been reported: Oikawa et al. showedin several experiments that cell-free extracts from the fungusAlternaria solani catalyze the conversion of the linear trieneprecursor prosolanapyrone III to the bicyclic product solana-pyrone A,[5]but they did not characterize the enzyme.Recently, the group of Vederas showed that isolated lova-statin nonaketide synthase, LNKS, involved in the biosyn-thesis of the fungal metabolite lovastatin catalyzes the exo-selective cyclization of a hexaketide triene.[6]The remarkableeffect of the enzyme was not that it increased the reactionrate, but that it stabilized the exo transition state giving theopposite diastereomer compared to the spontaneous cycliza-tion. The exo isomer could not be formed by chemicalmethods.()-Galiellalactone (3) is a fungal metabolite isolated fromthe ascomycetes Galiella rufa (strains A75-86 and A111-95) asa highly selective and potent inhibitor of interleukin-6 (IL-6)signalling in HepG2 cells.[7],[8]Biosynthetic studies and thestructure of the co-metabolites ()-1 and ()-2[9]suggestedthat 3 is formed in vivo through an intramolecular Diels ±Alder reaction.[10],[11]When ()-pregaliellalactone (1)or()-desoxygaliellalactone (2) are fed to the mycelium of thefungus Galiella rufa they are converted to ( )-galiellalactone(3),[12]showing that, in vivo, the conversion of 1 to 3 takesOOOHHHOOOH HHO123place in two discrete steps. Here we wish to discuss the firststep, the cyclization, which, to our knowledge, is the firstexample of an intramolecular Diels ± Alder reaction withinverse electron demand in a polyketide biosynthetic pathwaythat shows a biological rate enhancement.Initial experiments showed that the cyclization of ()-1 to()-2 can proceed spontaneously. The reaction was found tobe very sluggish in organic solvents at room temperature.When ()-1 was left in a chloroform solution, the cyclizedproduct ()-2 could only be detected after weeks. When ()-1in toluene was heated in a sealed tube at 140 8C the conversionto ()-2 was complete after 5 h [Eq. (1)]. As it is known thatthe rate of Diels ± Alder reactions can increase in aqueousmedia due to the hydrophobic effect,[13]reaction (1) was alsoperformed in water: compound 1 cyclized at room temper-ature cleanly to 2, with a half-life of 1 of 65 h. When thecyclization was conducted in a 4.86m LiCl solution the ratewas further increased (approximately twice) due to the™salting-out∫ effect.[13]Spontaneous cyclization yielded onlyone isomer–natural ()-desoxygaliellalactone (2) with allbridgehead hydrogen atoms on the same side of the ringsystem. No product from an exo transition state was everobserved, and the reaction is completely diastereoselective.Taken together, the absence of by-products and the rateenhancement in water as well as the effect of high concen-trations of salt show that the cyclization of ()-1 to ()-2 is infact a Diels ± Alder reaction.A series of experiments in which natural ()-1 was fed tothe mycelium of Galiella rufa were conducted. The fungus wasgrown in a normal growth medium for 10 ± 14 days, and whenthe secondary metabolism started, the mycelium was filteredoff and washed thoroughly with saline to remove the growthmedium. The washed mycelium was re-dispersed in saline and()-1 dissolved in MeOH (0.1%) was added, whereafter themycelium was incubated at 248C. Compared with an abioticcontrol experiment (only 1 in saline), a rate enhancement inthe production of the Diels ± Alder adduct was observed inthe presence of living mycelia (k 0.0015 min1), and theconcentration of 1 was halved in only 9 h with the conditionsused in this investigation (both experiments are summarizedin Figure 1). The rate of the spontaneous abiotic reaction in[*] Prof. Dr. O. Sterner, Dr. M. JohanssonDepartment of Organic and Bioorganic ChemistryLund UniversityP.O.Box 124, 221 00 Lund (Sweden)Fax : ( 46)46-222-8209E-mail: [email protected]. B. Kˆpcke, Prof. Dr. H. AnkeInstitut f¸r Biotechnologie und Wirkstoffforschung (IBWF)Erwin-Schroedinger-Strasse 56, 67663 Kaiserslautern (Germany)COMMUNICATIONSAngew. Chem. Int. Ed. 2002, 41, No. 12 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1433-7851/02/4112-2159 $ 20.00+.50/0 2159Figure 1. Comparison of the spontaneous cyclization of ()-1 (&)to()-2(&) in abiotic saline at 248C with the cyclization of ()-1 (*)to()-2 ()and the formation of ()-3 (*)at248C in the presence of active myceliafrom Galiella rufa (2 gL1mycelium dry weight) in saline.saline (k 0.00018 min1) is approximately 8 times lower.When the mycelium was autoclaved prior to the experimentthe rate enhancement was lost completely, showing that nounspecific interaction of the mycelium is affecting thecyclization and thus suggesting that the cyclization is cata-lyzed by a macromolecule with a well-defined structure. Inaddition, the catalyzed cyclization is highly specific. TheOHHHOOO45cyclization of the nonnatural enantiomer ()-pregaliellalac-tone (4)to5 was not


Cyclization of Pregaliellalactone in the Fungus Galiella rufa

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