Oxygen therapy for terpenes

An innovative approach to the oxidation of terpenes could open up new ways of making perfumes, flavourings, and pharmaceuticals, according to chemists in the UK.

Researchers at Oxford University have devised a new way to selectively oxidize terpenes, a class of common starting materials for the flavour and fragrance industries. The products are formed stereoselectively, which should mean their further functionalization could take place with only one of the possible isomers forming for each terpene reaction.

Reactions such as the functionalization of allylic as well as non-activated C-H bonds in a parent terpene should not be possible as well as the introduction of hydroxyl groups into specific sites in the terpene skeleton. The technique relies on nature's own catalysts - enzymes - and produces high yields of relatively pure compounds.

Luet-Lok Wong

The enzyme on which the Oxford team, led by Luet-Lok Wong of the Inorganic Chemistry Laboratory, has focused is the haem-containing metabolic enzyme, cytochrome P450_cam from pseudomonas putida. This enzyme oxidizes camphor to 5-exo-hydroxycamphor. Yet others can catalyze the oxidation of unactivated carbon-hydrogen bonds forming alcohol groups, they can also catalyze oxygen atom transfer to heteroatoms, the epoxidation of alkenes and aromatic hydrocarbons, and several other reactions. There are different plant P450 enzymes which oxidize limonene to different products, e.g., carveol, isopiperitenol, and perrilyl alcohol. "The advantage of using the microbial P450_cam enzyme is that it is easier to produce in Escherichia coli and much faster than the plant enzymes so if it can be engineered to oxidize the terpenes stereoselectively it will be easier to produce more product," team member Stephen Bell told us.

The mutant P450s are produced by the enzymologist's best friend the microbe Escherichia coli, which acts simply as a host DNA system to allow the new versions of P450 to be expressed in usable quantities by a fermentation process. The P450 electron transfer proteins can be expressed in the same microbe as the P450 to yield an efficient in vivo hydroxylation system (Protein Engineering, 2001, 14, 797-802).

Earlier this year Wong's team engineered P450 to allow them to oxidize the simple gaseous alkanes butane and propane to butan-2-ol and propan-2-ol, respectively. They persuaded the enzyme to act on these starting materials by incorporating bulky amino acid substitutions into the active site of the enzyme using genetic engineering, this improves the enzyme-substrate fit (Chem Commun, 2002, 5, 490).

Based on the preliminary work, the team has now obtained crystal structures of the terpenes bound in a mutant P450 and used this structure to make further mutations to enhance the selectivity of product formation. For limonene (the smell of oranges and lemons depending on the enantiomer to hand) three products (+)-trans-isopiperitenol, (+)trans-carveol, and the trans-limonene epoxide are possible. Another mutant can convert camphor to 5-exo-hydroxycamphor, (+)-alpha-pinene to (+)-verbenol, and valencene to nootkatone (Chem. Commun., 2001, (7), 635).

The team is also now working on Isis Innovation, Oxford University's technology transfer company, is actively seeking partners for the licensing and commercial development of the promising technology.