One-Pot Deracemization of sec-Alcohols: Enantioconvergent Enzymatic Hydrolysis of Alkyl Sulfates Using Stereocomplementary Sulfatases**

Given the fact that the theoretically possible number of racemates is larger than that of symmetric prochiral or meso compounds, the development of deracemization methods, which yield a single stereoisomer from a racemate is an important topic. Enantioconvergent processes are based on the transformation of a pair of enantiomers through opposite stereochemical pathways affecting retention and inversion of configuration. Depending on the stereochemical course of enzymatic and chemical reactions, three types of deracemization protocols were recently classified by Feringa et al. Two chemoenzymatic methods start with a biocatalytic kinetic resolution step, which yields a heteroor homochiral 1:1 mixture of the formed product and nonconverted substrate enantiomer. The latter is subjected to a second (nonenzymatic) transformation with retention or inversion of configuration to yield a single stereoisomeric product. Although several one-pot, two-step protocols have been successfully demonstrated, they typically rely on activated species, such as sulfonates, nitrate esters, or Mitsunobu intermediates, and negatively affect the overall atom economy of the process. The most elegant method relies on one (or two) enzyme(s), which mediate the transformation of both enantiomers through stereocomplementary pathways by retention and inversion. Since the requirements of such double selectivities are very difficult to meet, successful examples are rare: This approach has been applied to the hydrolysis of epoxides using two epoxide hydrolases showing opposite enantiopreference or a single enzyme that catalyzes the enantioconvergent hydrolysis of enantiomers with opposite regioselectivity. For enzymes, the ability to act by retention or inversion is a rare feature, which has been found among epoxide hydrolases, dehalogenases, and sulfatases. The latter catalyze the hydrolytic cleavage of (alkyl) sulfate esters by breakage of the S O or the C O bond leading to retention or inversion at the chiral carbon atom, and thus makes them prime candidates for enantioconvergent processes. So far, only a single inverting sec-alkylsulfatase (PISA1) was generated recombinantly and characterized biochemically, thus allowing preparative-scale applications. In combination with acid-catalyzed hydrolysis of the nonreacted substrate enantiomer under retention of configuration a chemoenzymatic two-step deracemization protocol for sec-alcohols was recently developed. However, the method suffers from serious limitations because it requires undesirably large volumes organic solvents and several molar equivalents of a strong acid (typically 2–7 equiv of p-TosOH), which pose the risk of racemization or decomposition to the functionalized substrates, especially when elevated temperatures are required for acidic hydrolysis. Moreover, it is not applicable to retaining sulfatases, because no chemical method for sulfate ester hydrolysis with inversion exists. So far, retaining-sulfatase activity was reported in whole cells of Rhodopirellula baltica DSM 10527, but the corresponding enzymes could not be identified, thus impeding the use of recombinant technology to make the enzyme available for biocatalysis. Furthermore, the retaining sulfatase of Rh. baltica would not be suitable for an enantioconvergent process with PISA1, because both proteins exhibit the same enantiopreference. During our search for a retaining secalkylsulfatase with an enantiopreference opposite to that of PISA1, we discovered that the arylsulfatase from Pseudomonas aeruginosa (PAS) exhibited activity on sec-alkylsulfates. PAS, which has been characterized on a molecular level, showed promiscuous activity on various arylic phosphates and phosphonates. On its standard model substrate (4-nitrophenyl sulfate), PAS exhibited a rate acceleration of kcat/kuncat 2.3 10, and for a less reactive substrate the highest rate enhancement (kcat/kuncat= 2 10 ) of any catalytic reaction known so far has been measured. The stereochemical [*] M. Schober, M. Toesch, Dr. M. Fuchs, Prof. K. Faber Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz Heinrichstrasse 28, 8010 Graz (Austria) E-mail: [email protected] Homepage: http://biocatalysis.uni-graz.at/