Markus Schober

Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives

The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the β-carbon atom of styrenes forming (E)-cinnamic acids.

Inverting hydrolases and their use in enantioconvergent biotransformations

Highlights • Enantioconvergent processes overcome the 50%-yield limits of kinetic resolution.• Inverting enzymes are key catalysts for enantioconvergent processes.• Enzyme engineering provided improved variants of inverting enzymes.

A Stereoselective Inverting sec-Alkylsulfatase for the Deracemization of sec-Alcohols

A metallo-β-lactamase-type alkylsulfatase was found to catalyze the enantioselective hydrolysis of sec-alkylsulfates with strict inversion of configuration. This catalytic event, which does not have an analog in chemocatalysis, yields homochiral (S)-configurated alcohols and nonreacted sulfate esters. The latter could be converted into (S)-sec-alcohols as the sole product in up to >99% ee via a chemoenzymatic deracemization protocol on a preparative scale.

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, 9] 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: kurt.faber@uni-graz.at Homepage: http://biocatalysis.uni-graz.at/

Microbial alkyl- and aryl-sulfatases: mechanism, occurrence, screening and stereoselectivities

This review gives an overview on the occurrence of sulfatases in Prokaryota, Eukaryota and Archaea. The mechanism of enzymes acting with retention or inversion of configuration during sulfate ester hydrolysis is discussed taking two complementary examples. Methods for the discovery of novel alkyl sulfatases are described by way of sequence-based search and enzyme induction. A comprehensive list of organisms with their respective substrate scope regarding prim- and sec-alkyl sulfate esters allows to assess the capabilities and limitations of various biocatalysts employed as whole cell systems or as purified enzymes with respect to their activities and enantioselectivities. Methods for immobilization and selectivity enhancement by addition of metal ions or organic (co)solvents are summarised.

Structure and mechanism of an inverting alkylsulfatase from Pseudomonas sp. DSM6611 specific for secondary alkyl sulfates

A highly enantioselective and stereoselective secondary alkylsulfatase from Pseudomonas sp. DSM6611 (Pisa1) was heterologously expressed in Escherichia coli BL21, and purified to homogeneity for kinetic and structural studies. Structure determination of Pisa1 by X‐ray crystallography showed that the protein belongs to the family of metallo‐β‐lactamases with a conserved binuclear Zn2+ cluster in the active site. In contrast to a closely related alkylsulfatase from Pseudomonas aeruginosa (SdsA1), Pisa1 showed a preference for secondary rather than primary alkyl sulfates, and enantioselectively hydrolyzed the (R)‐enantiomer of rac‐2‐octyl sulfate, yielding (S)‐2‐octanol with inversion of absolute configuration as a result of C–O bond cleavage. In order to elucidate the mechanism of inverting sulfate ester hydrolysis, for which no counterpart in chemical catalysis exists, we designed variants of Pisa1 guided by three‐dimensional structure and docking experiments. In the course of these studies, we identified an invariant histidine (His317) near the sulfate‐binding site as the general acid for crucial protonation of the sulfate leaving group. Additionally, amino acid replacements in the alkyl chain‐binding pocket generated an enzyme variant that lost its stereoselectivity towards rac‐2‐octyl sulfate. These findings are discussed in light of the potential use of this enzyme family for applications in biocatalysis.

Enzymatic Aerobic Alkene Cleavage Catalyzed by a Mn3+‐Dependent Proteinase A Homologue

Mild, selective, safe, and environmentally benign oxidation methods have gained increased importance. However, the chemical cleavage of alkenes to give aldehydes or ketones is still preferentially achieved by ozonization or with stoichiometric amounts of metal salts. Alternative methods include those employing hypervalent iodine or N-hydroxyphthalimide in the presence of molecular oxygen. Although various enzymes have been described for alkene cleavage, only a few are applied for non-natural substrates. For instance, a crude preparation of the white-rot basidiomycete Trametes hirsuta FCC047 has been employed on a preparative scale to cleave a C=C double bond adjacent to a phenyl moiety with molecular oxygen, thereby leading to benzaldehyde derivatives (Scheme 1). For molecules bearing at least two alkene groups, the preparation is able to distinguish between different C=C double bonds within the same molecule. Studies of the mechanism of alkene cleavage revealed a novel mechanism involving radical intermediates and incorporating oxygen atoms of two molecules of molecular oxygen. However, the exact type of enzyme and the nature of the involved cofactor remained unknown. We report here the identification of this enzyme and the unexpected metal dependence. Freeze-dried cells of the fungus showed an activity of 1 U mg 1 for the cleavage of t-anethole 1 a (Scheme 1). Initial attempts to purify the enzyme from T. hirsuta were unsuccessful. After two purification steps (hydrophobic interaction chromatography followed by ion exchange chromatography), no activity remained, even for the very well accepted substrate 1 a. Inductively coupled plasma mass spectrometry (ICP/MS) analysis of the active protein fraction after one purification step (hydrophobic interaction, phenyl Sepharose CL-4B) revealed the presence of three possible metal cofactors : Cu, Mn or Mo. Unfortunately, addition of common metal salts of Cu , Cu , or Mn + (as well as of various Mo salts or cofactors) did not restore enzyme activity. Fe + and Fe + were also tested for enzyme activation, but did not give positive results. Serendipitously, assaying the enzyme in the presence of Mn and hydrogen peroxide resulted in increased activity, which was subsequently attributed to the in situ formation of manganese in oxidation state three. Indeed, alkene-cleaving activity was restored by addition of Mn + salts (e.g. , Mn acetate, 0.4 mm). Fresh Mn acetate on its own did not induce alkene cleaving activity under the assay conditions. Alkene-cleaving enzymes described so far depend on either Fe + or Cu + ; some also show activity in the presence of Mn , Ni + , or Co . Other than in photosystem II, Mn is only found in a few types of enzymes, such as manganese superoxide dismutases (SODs), catalases, pseudocatalases, and ribonucleotide reductases. SODs contain one Mn ion per subunit and catalyze the decomposition of O2 , whereas catalases/ pseudocatalases have two manganese ions in their centers and Scheme 1. Alkene cleavage catalyzed by the fungus Trametes hirsuta FCC047 at the expense of molecular oxygen as the sole oxidant. a) Trametes hirsuta crude extract, buffer (pH 6), 21 8C.

Asymmetric Synthesis of β-Substituted α-Methylenebutyro- lactones via TRIP-Catalyzed Allylation: Mechanistic Studies and Application to the Synthesis of (S)-(−)-Hydroxymatairesinol

Asymmetric allylation of (hetero)aromatic aldehydes by a zinc(II)-allylbutyrolactone species catalyzed by a chiral BINOL-type phosphoric acid gave β-substituted α-methylenebutyrolactones in 68 to >99% ee and 52–91% isolated yield. DFT studies on the intermediate Zn2+-complex – crucial for chiral induction – suggest a six-membered ring intermediate, which allows the phosphoric acid moiety to activate the aldehyde. The methodology was applied to the synthesis of the antitumour natural product (S)-(−)-hydroxymatairesinol.

Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis of Benzylic sec-Sulfate Esters

The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in 18O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting SN2-type nucleophilic attack of [OH–] at the benzylic carbon represents the major pathway, whereas (ii) SN1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining SN2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents.