TETRAHEDRON LETTERS Tetrahedron Letters 42 (2001) 469–471 Pergamon The enantioselectivity of haloalkane dehalogenases Roland J. Pieters, a,b,† Jeffrey H. Lutje Spelberg, a,b Richard M. Kellogg a and Dick B. Janssen b, * a Department of Organic and Molecular Inorganic Chemistry, University of Groningen, Nijenborgh 4, NL -9747 AG Groningen, The Netherlands b Department of Biochemistry, University of Groningen, Nijenborgh 4, NL -9747 AG Groningen, The Netherlands Received 17 July 2000; revised 24 October 2000; accepted 2 November 2000 Abstract —Two haloalkane dehalogenases were tested for their ability to perform kinetic resolutions of a series of racemi substrates and to convert meso substrates enantioselectively. For the kinetic resolutions E -values of up to 9 were measured and in the conversions of the meso substrates, products were obtained with an enantiomeric excess of up to 47%. A kinetic analysi revealed that despite modest overall chiral recognition (expressed as E -values), there are large differences between the K m value ( > 100 fold) of two enantiomeric substrates but that these differences are compensated by correspondingly large differences in k ca © 2001 Elsevier Science Ltd. All rights reserved. Haloalkane dehalogenases are capable of replacing a halide in an organic substrate by a hydroxyl group. 1 These enzymes have been isolated from polluted indus- trial sites from organisms capable of growing on haloalkanes. Such organisms can play an important environmental role in the clean-up of those sites. Haloalkane dehalogenases also hold potential in bio- catalysis, since haloalkanes or haloalcohols are valuable building blocks in organic synthesis, especially when chiral and of high enantiomeric purity. To date no enantioselective conversions with haloalkane dehaloge- nases have been reported. We therefore initiated a search for such reactions using two prominent members of this enzyme family. The first one is a haloalkane dehalogenase designated DhlA, 2 with optimal activity for short-chain haloalkanes and originally obtained from the 1,2-dichloroethane degrading organism Xan - thobacter autotrophicus GJ10. The second enzyme, DhaA, 3 exhibits additional activity for long-chain haloalkanes and was originally obtained from the chlorobutane-degrading Rhodococcus rhodochrous NCIMB 13064. For both of these enzymes the genes have been cloned and they can be obtained in large quantities by efficient overexpression in E. coli. This latter feature makes their practical application feasible. We screened the two enzymes for enantioselectivit with a series of chiral and meso haloalkanes. Com pounds were first identified as substrates by using a assay that quantifies the amount of halide released over time. The degree of chiral recognition in th conversion of the compounds was determined by ana lyzing the reaction mixture by gas chromatography o a chiral column. 5 The enantiodiscrimination was ex pressed in terms of E -values (Table 1). 6 Most com pounds tested were primary halides, although some ha additional secondary halide centers. Secondary alky halides were found to be very poor substrates for DhlA Although DhaA was better at converting secondar halides, 2-bromobutane was converted without observ able chiral selection. Several 1,2 and 1,3 short chain dihaloalkanes wer tested (entries 1–6). They were all converted with littl selectivity by both enzymes. Interestingly, in the conver sion of the 1,2-dihalo compounds by DhlA, not only i the primary halide replaced, but also significan amounts of the secondary halide, although 2-bromobu tane is converted very sluggishly by this enzyme. Wit the 1,3-dihaloalkanes only the primary halogen wa replaced. In all cases E -values were based on the disap pearance of the starting material but in several case additional determinations were based on the e.e. of th product. 6 A small increase in enantioselectivity was see with methyl-3-bromo-2-methyl propionate (entry 7) This compound proved to be a very good substrate fo DhaA and was converted with an E -value of 5. At tempts were made to increase the selectivity by modify ing the structure of the substrate. Changing th * Corresponding author. Tel.: + 31-50-363 4008; fax: + 31-50-363 4165; e-mail: d.b.janssen@chem.rug.nl † Present address: Department of Medicinal Chemistry; Utrecht Insti- tute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, NL-3508 TB Utrecht, The Netherlands. 0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII:S0040-4039(00)01947-X