A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)-limonene and (+)-valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

SummaryAllylic alcohols are valuable precursors in the synthesis of pharmaceutical intermediates, agrochemicals and natural products. Regioselective oxidation of parental alkenes is a challenging task for chemical catalysts and requires several steps including protection and deprotection. Many cytochrome P450 enzymes are known to catalyse selective allylic hydroxylation under mild conditions. Here, we describe CYP154E1 from Thermobifida fusca YX that enables this type of oxidation. Several acyclic terpenoids were tested as possible substrates for CYP154E1, and the regio- and chemoselectivity of their oxidation was investigated. Using a previously established bioinformatics approach we identified position 286 in the active site of CYP154E1 which is putatively involved in substrate binding and thereby might have an effect on enzyme selectivity. To tune regio- and chemoselectivity of the enzyme three mutants at position 286 were constructed and used for substrate oxidation. All formed products were analysed with GC–MS and identified using chemically synthesised authentic samples and known compounds as references. Best regioselectivity towards geraniol and nerol was observed with the wild type enzyme mainly leading to 8-hydroxy derivatives (8-hydroxygeraniol or 8-hydroxynerol) with high selectivity (100% and 96% respectively). Highest selectivities during the oxidation of geranylacetone and nerylacetone were observed with the following variants: V286F led mainly to 7-hydroxygeranylacetone (60% of the total product) and V286A produced predominantly 12-hydroxynerylacetone (75% of total product). Thus, CYP154E1 and its mutants expand the tool-box for allylic hydroxylation in synthetic chemistry.

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The (E)‐ and (Z)‐terpene‐based aldehydes 6b and 6c with a silyl ether function in the γ‐position were prepared and investigated in boron‐mediated asymmetric Evans aldol reactions. Screening experiments of chiral N‐acylated oxazolidinones 7, which are conveniently accessible from 5‐methyl‐5‐hexenoic acid and Evans oxazolidinone auxiliaries, with various boron triflates and terpenoid neral (Z)‐6a as aldehyde component, provided conditions in which highly selective formation of syn‐aldol adduct 5a occurred and competing C=C double bond isomerization to 10 was completely suppressed. Applying the optimized conditions to O‐silylated aldehydes 6b and 6c and N‐acyloxazolidinone derivative (R)‐7a confirmed the syn‐selectivity and gave the appropriate products syn‐5b,c and syn‐21b,c in good yields. In the case of neral‐derived syn adduct 5a, the configuration of the new stereogenic centers C‐2/C‐3 could be assigned as (2R,3S).