4 Enzymatic Synthesis of Unnatural Novel Compounds on the Basis of Structure Function Analysis of Biosynthetic Enzymes
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1. A C_<35> polyprene in which a farnesyl C_<15> unit is connected in a head-to-head fashion to a geranylgeranyl C_<20> unit was enzymatically converted to an unnatural hexacyclic polyprenoid by squalene: hopene cyclase from Alicyclobacillus acidocaldarius. The cyclization of the C_<35> polyprene was initiated by a proton attack on the terminal double bond of the C_<15> unit, and proceeded without rearrangement of carbon and hydrogen. The substrate should be folded in chair-chair-chair-chair-boat-boat conformation in order to achieve the stereochemistry of the cyclization product. 2. Substrate specificities of chalcone synthase (CHS) were investigated using analogs of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, CHS from Scutellaria baicalensis afforded an unnatural C_6-C_5 aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the substrates. In contrast, succinyl-CoA was not accepted as a substrate. Next, both the starter and the extension unit were simultaneously replaced with non-physiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA, CHS afforded a novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
