Available online at www.sciencedirect.com Novel carbon–carbon bond formations for biocatalysis Verena Resch, Joerg H Schrittwieser, Elina Siirola and Wolfgang Kroutil Carbon–carbon bond formation is the key transformation in biosynthesis by forming a C–C bond between dopa- organic synthesis to set up the carbon backbone of organic mine and 4-hydroxyphenylacetaldehyde to yield molecules. However, only a limited number of enzymatic C–C (S)-norcoclaurine (Figure 1a). A recombinant norco- bond forming reactions have been applied in biocatalytic claurine synthase originating from the plant Thalictrum organic synthesis. Recently, further name reactions have been flavum (meadow rue) [9,10] was used to prepare (S)- accomplished for the first time employing enzymes on a norcoclaurine starting from cheap tyrosine and dopa- preparative scale, for instance the Stetter and Pictet–Spengler mine as substrates in a one-pot, two-step process [11 ]. reaction or oxidative C–C bond formation. Furthermore, novel Tyrosine was first chemically decarboxylated by enzymatic C–C bond forming reactions have been identified stoichiometric amounts of sodium hypochlorite to gen- like benzylation of aromatics, intermolecular Diels-Alder or erate the aldehyde species (4-hydroxyphenylacetade- reductive coupling of carbon monoxide. hyde), followed by the addition of the enzyme and dopamine substrate. The optimised process Address afforded (S)-norcoclaurine (e.e. 93%) in 81% yield and Department of Chemistry, Organic and Bioorganic Chemistry, University allowed the recycling of the enzyme. of Graz, Heinrichstrasse 28, A-8010 Graz, Austria Another Pictet-Spenglerase is strictosidine synthase (EC Corresponding author: Kroutil, Wolfgang ([email protected]) 4.3.3.2), which triggers in nature the formation of stricto- sidine from tryptamine and secologanin (Figure 1b). The Current Opinion in Biotechnology 2011, 22:793–799 recombinant enzyme from Catharanthus roseus was inves- tigated concerning the acceptance of non-natural sub- This review comes from a themed issue on Chemical biotechnology strates [12] and a strictosidine synthase from Ophiorrhiza Edited by Guo-Qiang Chen and Romas Kazlauskas pumila was shown to accept a range of simple achiral aldehydes and substituted tryptamines to form highly Available online 25th February 2011 enantioenriched (e.e. >98%) tetrahydro-b-carbolines 0958-1669/$ – see front matter [13 ] (Figure 1b). # 2011 Elsevier Ltd. All rights reserved. Thiamine diphosphate-dependent enzymes [7] were recently DOI 10.1016/j.copbio.2011.02.002 shown to catalyse besides the well-known formation of a- hydroxy ketones via 1,2-addition also a 1,4-addition Introduction when employing a,b-unsaturated ketones as substrates Enzymatic carbon–carbon bond forming reactions [1] [14 ] (Figure 1c). This remarkable new development catalysed by aldolases, transketolases [2–5], hydroxynitrile allows exploiting the decarboxylation and umpolung of lyases [1,6] and thiamin diphosphate (ThDP)-depending pyruvate to perform the so-called Stetter reaction giving a-hydroxy ketone forming enzymes [1,7] are well estab- access to 1,4-bifunctional molecules. This 1,4-addition lished for synthetic purposes. This review focuses on was catalysed by the enzyme PigD from Serratia marces- C–C bond formation by enzymes, which are less estab- cens, which is in contrast to other ThDP enzymes, for lished for biocatalysis, which have gained increased sig- example, the enzyme YerE, catalysing 1,2-addition. YerE nificance recently or which have been reported for the on the other hand was successfully employed for the first time. The review covers the period of approximately carboligation of ketones with pyruvate as reagent to form the last two years. Subdivisions have been made according enantioselectively tertiary alcohols with a a-acetyl to the type of enzyme (lyase, oxidoreductase and trans- moiety [15 ]. ferase) and enzymes with promiscuous activity. Most examples belong to the group of lyases. Crotonases. Enzymes of the crotonase superfamily catalyse a wide variety of reaction types including alkene Lyases hydration and isomerisation, coenzyme A ester hydrolysis Pictet-Spenglerases. The group of ‘Pictet-Spenglerases’ and C–C bond cleavage. Two members of the super- [8] encompasses various enzymes such as norcoclaurine family have been reported to catalyse C–C bond for- synthase and strictosidine synthase. The general reac- mation [16], whereby in both cases the substrate bears tion is the condensation of an aryl ethylamine with a coenzyme A ester moiety. Carboxymethylproline an aldehyde to form a six-membered N-heterocycle synthase CarB from Pectobacterium carotovorum activates (Figure 1a,b). Norcoclaurine synthase (EC 4.2.1.78) malonyl CoA derivatives via decarboxylation; the variant catalyses the first step in benzylisoquinoline alkaloid CarB His229Ala and its homologue ThnE from Strepto- www.sciencedirect.com Current Opinion in Biotechnology 2011, 22:793–799 794 Chemical biotechnology Figure 1 Biocatalytic C–C bond formation catalysed by lyases, such as (a) norcoclaurine synthase, (b) strictosidine synthase transforming non-natural substrates, (c) PigD, a ThDP-dependent enzyme, (d) CarB, a member of the crotonase superfamily, (e) tyrosine phenol lyase, (f) halohydrin dehalogenase, (g) computationally de novo designed Diels-Alderase. Current Opinion in Biotechnology 2011, 22:793–799 www.sciencedirect.com Novel carboncarbon bond formations Resch et al. 795 myces cattleya [17] have been applied to convert amino acid a His-tag and expressed in Escherichia coli. Fifty of the aldehydes and malonyl CoA derivatives into 5-mem- expressed enzymes were soluble and therefore chosen for bered, 6-membered and 7-membered N-heterocycles purification. Out of these 50 enzymes, two enzymes were (Figure 1d) [18 ]. The products were converted further found to be active and mutations led to a 100-fold increase to bicyclic b-lactam derivatives by carbapenam synthe- of the catalytic activity. The substrate spectrum of two tase CarA from P. carotovorum. variants was also tested using different dienophiles. Tyrosine phenol lyase (EC 4.1.99.2), a pyridoxal 5-phos- Oxidoreductases phate-dependent enzyme, catalyses in vivo the revers- Oxidoreductases are enzymes less commonly employed ible b-elimination reaction of L-tyrosine leading for C–C bond formation, except for laccases and per- to phenol, ammonium ion and pyruvate. Exploiting oxidases; however, none of these two enzymes controls the reverse reaction, non-natural amino acids can be the actual C–C bond formation reaction; they mediate prepared from substituted phenols, pyruvate and just the formation of a reactive species. Nevertheless, ammonium (Figure 1e). Since the wild-type enzyme novel redox enzymes like the berberine bridge enzyme from Citrobacter freundii did not accept most investi- (BBE; vide infra) were exploited for synthetic purposes gated o-substituted phenols, variants were designed and novel redox reactions for C–C bond formations based on the crystal structure of the enzyme. The were identified. best-identified variant M379V allowed the synthesis of non-natural tyrosine derivatives possessing a chloro, The BBE is involved in the biosynthesis of benzophenan- 0 methoxy or methyl substituent in position 3 of tyrosine thridine alkaloids in plants from the poppy family. It within one step [19 ]. The obtained tyrosine derivatives catalyses an intramolecular oxidative C–C bond formation are building blocks for bioactive compounds or bio- between a phenol moiety and an N-methyl group at the markers. expense of molecular oxygen. The most thoroughly stu- died enzyme is BBE from Eschscholzia californica (califor- Halohydrin dehalogenases catalyse besides the ring-clo- nia poppy) which has been obtained in substantial sure of vicinal halohydrins to the corresponding epox- amounts by overexpression in Pichia pastoris. Its X-ray ides also the nucleophilic ring-opening of epoxides with crystal structure has been determined and the catalytic a broad range of nucleophiles [20]. In case cyanide is mechanism has been studied [25]. Recently, BBE has used for the ring-opening of an epoxide, a new C–C been employed for the preparation of novel optically pure bond is formed. Recently, a multi-enzymatic synthesis (R)-benzylisoquinolines and (S)-berbine derivatives 1 for the manufacture of atorvastatin (Lipitor ), a cho- (Figure 2a) [26 ]. Starting from a racemic mixture, lesterol-lowering drug, has been developed (Scheme 1f) exclusively the (S)-enantiomer was transformed via C– [21,22]: After asymmetric reduction of ethyl 4-chloroa- C bond formation leading to a kinetic resolution with cetoacetate by an alcohol dehydrogenase (ADH), the perfect E-value (E > 200). The reaction could success- obtained halohydrin was converted into the epoxide and fully be performed on a 500 mg scale at a substrate further into the corresponding hydroxynitrile by a halo- concentration of 20 g/L. Reactions were carried out in a hydrin dehalogenase. Under optimised conditions, (R)- toluene/buffer biphasic system to solubilise the substrates ethyl 4-cyano-3-hydroxybutyrate could be obtained in and O2 was required as the stoichiometric oxidant. 95% isolated yield and e.e. > 99.9% with a space-time À1 À1 yield of 480 g L day . In a similar approach, a one- Laccases are multi-copper containing enzymes that cata- pot cascade was investigated, whereby by choosing the lyse the oxidation of various O-substituted and N-sub- appropriate ADH, either enantiomer of various b-hydro-