Synthetic chemists routinely employ inorganic and organometallic catalysts to mediate desired chemical transformations. An emerging area of catalysis is the creation of recombinant microbes to catalyze multistep transformations whereby the genes encoding the requisite enzymes are recruited from different microbes, plants, and animals. The result is an intact microbial catalyst that transforms simple carbohydrate starting materials into a desired chemical product. Our microbial synthesis of 1,2,4-butanetriol enantiomers provides an example of the creation and use of recombinant microbes as synthetic catalysts.

Scheme 1

Nitration of racemic D,L-1,2,4-butanetriol affords D,L-1,2,4-butanetriol trinitrate (Scheme 1). Relative to nitroglycerin, which has been used in industrial and military energetic materials since Nobel's original dynamite formulations, D,L-1,2,4-butanetriol trinitrate is a less hazardous energetic material. D,L-1,2,4-Butanetriol is commercially synthesized using NaBH4 reduction of esterified malic acid (Scheme 1). Such a stoichiometric reduction contributes significantly to the expense of D,L-1,2,4-butanetriol, which precludes widespread substitution of D,L-1,2,4-butanetriol trinitrate as a replacement for nitroglycerin. Catalytic hydrogenation of malic acid over Ru on C is another option (Scheme 1). Unfortunately, such hydrogenations need to be run at 5,000 psi and at elevated temperatures. Byproducts formed during hydrogenation of malic acid complicate purification of product D,L-1,2,4-butanetriol.

Our strategy for the synthesis of 1,2,4-butanetriol entailed recruiting genes that encode enzymes from Pseudomonas fragi, Pseudomonas putida, and Escherichia coli to create two biosynthetic pathways that do not exist in nature (Scheme 2).1 One of these created pathways enables D-xylose to be converted into D-1,2,4-butanetriol (Scheme 2). Another created pathway enables L-arabinose to be converted into L-1,2,4-butanetriol (Scheme 2). With the created biosynthetic pathways, the high H2 pressures and elevated temperatures required for hydrogenation of malic acid are avoided. Byproduct formation is also minimized. D-Xylose and L-arabinose starting materials are abundantly available in corn fiber and sugar beet pulp.

Scheme 2

a) D-xylose dehydrogenase (P. fragi); (a') L-arabinose dehydrogenase (P. fragi); (b) D-xylonate dehydratase (E. coli); (b') L-arabinonate dehydratase (P. fragi); (c) benzoylformate decarboxylase (P. putida); (d) dehydrogenase (E. coli).

Aside from the impact on energetic material manufacture, the pathways created for the biosynthesis of D-1,2,4-butanetriol and L-1,2,4-butanetriol provide access to a valuable group of chiral synthons. (S)-3-Hydroxy-gamma-butyrolactone, which is used in the commercial manufacture of Astra Zeneca's cholesterol-lowering drug Crestor, is derived from D-3,4-dihydroxybutanoic acid. One of two patented routes to the cholesterol-lowering drug Zetia also employs 3-hydroxy-gamma-butyrolactone as a chiral synthon. Zetia is jointly marketed by Merck and Schering-Plough. Microbial synthesis of D-1,2,4-butanetriol and L-1,2,4-butanetriol provides a unique example of dual use (defense/pharmaceutical) synthetic methodology.

1Niu, W.; Molefe, M. N.; Frost, J. W. J. Am. Chem. Soc. 2003, 125, 12998.