Csingle bondC bond formation using ThDP-dependent lyases

In memory of Ayhan S. Demir, a pioneer in the field of enzymatic benzoin condensation.
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The present review summarizes recent achievements in enzymatic thiamine catalysis during the past three years. With well-established enzymes such as BAL, PDC and TK new reactions have been identified and respective variants were prepared, which enable access to stereoisomeric products. Further we highlight recent progress with ‘new’ ThDP-dependent enzymes like MenD and PigD, which catalyze the Stetter-like 1,4 addition of aldehydes and YerE, which is the first known ThDP-dependent enzyme accepting ketones as acceptors.

Introduction

Thiamine diphosphate (ThDP)-dependent enzymes are exceedingly multifunctional biocatalysts and catalyze a broad range of reactions. Most importantly, they are involved in the making and breaking of Csingle bondC bonds. Any enzymatic reaction involving a Csingle bondC bond which links two vicinal carbonyl groups or the carbinol and carbonyl groups of a 2-hydroxy ketone is almost certain to be ThDP-dependent. This article deals with some recent developments in enzymatic asymmetric thiamine catalysis.

Several comprehensive reviews have been published recently. Brovetto et al. summarized synthetic applications of the thiamine diphosphate (ThDP)-dependent enzymes pyruvate decarboxylase (PDC), benzoylformate decarboxylase (BFD), phenylpyruvate decarboxylase (PhPDC) and benzaldehyde lyase (BAL) [1••]. Herein, new syntheses with these enzymes and transformations catalyzed by additional enzymes will be presented. Domínguez de Maria et al. described selected enzymatic and organocatalytic benzoin-type and Stetter-type reactions [2, 3•]. Pohl et al. gave a comprehensive review with respect to enzymatic acyloin and benzoin condensation [4••], and several review articles dealing with ThDP catalysis were published in a special issue of the FEBS Journal [5].

BAL, which is the Turkish word for honey, was pioneered as a catalyst in organic synthesis by the recently deceased Ayhan Demir. He introduced the use of DMSO as a cosolvent and thus could identify a broad range of reactions catalyzed by BAL. More recently, he and his coworkers published the enantioselective self-condensation and cross-condensation of acetaldehyde derivatives. In addition to the already known aliphatic compounds acetaldehyde, monomethoxyacetaldehyde and dimethoxyacetaldehyde, benzyloxy acetaldehyde was successfully applied as a substrate [6]. Depending on the cosubstrate and the reaction conditions benzyloxyacetaldehyde functioned either as the donor or the acceptor or both (Figure 1a–c). BAL also catalyzes the direct amidation of aldehydes with nitroso compounds [7]. Moreover, Demir and coworkers performed reaction engineering, for example, they immobilized BAL on supermagnetic solid support and applied this resin in catalytic asymmetric Csingle bondC bond formation [8]. Many other groups have applied BAL for different purposes, which have been summarized in the above mentioned review articles. Active-site engineering of BAL and BFD have been performed among others by McLeish and coworkers [9•, 10•].

PDC and BFD are well-established ThDP-dependent enzymes in asymmetric syntheses. Whereas only the BFD (from Pseudomonas putida) was found to be appropriate for carboligation reactions, several PDCs from Saccharomyces cerevisiae, Zymomonas mobilis and Acetobacter pasteurianus have been studied and optimized concerning their carboligation activity [11]. Protein engineering has been used successfully to enhance stability and/or efficiency of the catalysts and to optimize and to alter enantioselectivity. An impressive example was given by Tittmann et al., who designed the Glu473Gln variant of PDC from Zymomonas mobilis (ZmPDC). Whereas the variant showed, as expected, a very low residual decarboxylase activity of 0.1%, the carboligase activity was up to 20-fold higher for the variant resulting in highly enantio-enriched (>98% ee) (R)-phenylacetylcarbinol in up to 98% yield (Figure 1d) [12••].

On the basis of the crystal structure of PDC from Acetobacter pasteurianus (ApPDC) Rother et al. designed the first (S)-selective PDC-variant by site-directed modification of the glutamic acid residue at position 469 to glycine. It is important to note that Glu469 in ApPDC is equivalent to Glu473 in ZmPDC. Both glutamate residues are located in a structural element called S-pocket, which was found to be largest in ApPDC. The Glu469Gly mutation opened this S-pocket in ApPDC and generated a variant which provides access to (S)-phenylacetylcarbinol and derivatives thereof (61–89% ee) through enzymatic Csingle bondC bond formation for the first time (Figure 1e) [13••].

McLeish et al. characterized a PhPDC from Saccharomyces cerevisiae [14]. PhPDC has been used before by others in vitro and in vivo for different Csingle bondC bond formations.

Transketolase (TK), either from Escherichia coli or yeast, has been used for synthetic purposes in the past; TK transfers a glycolaldehyde group from a donor compound (naturally the best donor is xylulose-5-phosphate, X5P) to α-(R)-hydroxyaldehydes as acceptors to form products with a 3S,4R stereoconfiguration (Figure 2a). β-Hydroxypyruvate (HP) can substitute for many reactions of microbial transketolases with the advantage that the reactions are drawn to the product side as carbon dioxide is being formed [15]. The field was recently reviewed with an emphasis on improving TK-based formation of X5P which is a rare and expensive donor compound for TK [16]. Yeast TK was used for the preparative scale synthesis of the rare d-sedoheptulose-7-phosphate (an intermediate of the pentose phosphate pathway) by reacting ribose-5-phosphate (R5P) and HP. This yielded the desired product in 81% overall yield in a multigram scale [17]. Halogenated pyruvates as fluoro-derivatives or bromo-derivatives were shown as donor substrates for the yeast TK at least as being able to form the respective thiamine adducts [18].

A general assay method allows screening in a microtitre plate format with the pH indicator, phenol red. The assay is based on the TK donor lithium-HP which releases LiHCO3 upon reaction with various aldehyde acceptors. In a low-buffered medium (2 mM of triethanolamine at pH 7.5), this can be monitored by an increase in pH. Even very low catalytic activities could be determined precisely [19••].

The general understanding of TK activity depends upon structural insights. The E. coli transketolase TktA was analyzed with X-ray crystallography in covalent complex with X5P [20••]. X5P is covalently bound to ThDP in a tetrahedral shape (sp3-hybridized) where the C2 atom of X5P is connected to the sp2-hybridized C2 of the aromatic thiazolium ring. Importantly, this bond is distorted out-of-plane for about 25–30° which implies considerable strain in the intermediate [20••]. This strain could be relieved by elimination of the glyceraldehyde-3-phosphate and formation of the planar and unstrained dihydroxyethyl-ThDP enamine intermediate. This reactive enamine could then covalently add to the C1 aldo function of R5P and eventually lead to the liberation of the C7 keto compound, sedoheptulose-7-phosphate.

The covalent X5P-ThDP intermediate in the active site was shown to be surrounded by amino acid side chains whereof the Asp469 residue is especially close to the C2 atom of both, the keto group of donor compounds and the hydroxyl group of α-hydroxyaldehyde acceptor compounds. Asp469 also governs the stereoselectivity of yeast TK [21]. Now, in an extension of earlier findings with mutants of the E. coli TktA enzyme [22] or the yeast TKL1 enzyme [21], the group of Hailes has shown that mutants of E. coli TktA at position Asp469 (especially Asp469Glu) can be successfully used for improved utilization of unphosphorylated acceptors (e.g. glycolaldehyde) or of compounds which are not hydroxylated in the C2 position (e.g. acetaldehyde or propanal) [23, 24]. These findings were recently corroborated by work of the groups of Hecquet and Fessner [19••]. These mutant enzymes also accept other linear [19••, 23, 24] or cyclic aliphatic aldehydes lacking a hydroxyl group in C2 [24].

Additionally, the Asp469Glu mutant displayed improved or even excellent ee values (over the wild type TktA) for the products [19••, 23, 24]. Moreover, a His26Tyr mutant showed similarly improved activity with nonphosphorylated acceptors (e.g. propanal) and an altered stereospecifity (R) of the products [23, 24]. The groups of Fessner and Hecquet, however, could not confirm the improved activity with the same TktA enzyme variant, but agree on the inverted enantioselectivity of the 4-deoxyketose products [19••]. An Asp469Thr single or Asp469Thr/Arg358Pro double mutant showed activity towards aromatic aldehydes as 3-formyl or 4-formyl-benzoic acid (67% and 30% conversion to products, respectively), compounds which are not accepted by wild type TktA (Figure 2c) [25].

Two conserved residues (Arg358/Arg 520 in TktA, Arg359 and Arg528 in TKL1) are involved in binding of the phosphoryl group of acceptor compounds [20••, 21, 22]. TK mutants with alterations of these arginine residues had been shown to have weakened binding to phosphorylated acceptor compounds and reduced activities [21, 22] while almost pertaining wild type catalytic efficiencies (kcat/KM) for unphosphorylated acceptors [22]. On the basis of the model with E4P bound to the yeast TK [21] saturation mutagenesis was performed with the E. coli tktA gene targeting for residues of the active site. From the mutant libraries, some showed improved activities with non-phosphorylated (e.g. propanal) or aliphatic aldehyde substrates (Figure 2b). Also, mutations leading to improved or reversed stereoselectivities of TktA have been detected (Ref. in [23]). The yeast TK was similarly improved for the acceptance of non-phosphorylated acceptors; for the improved enzymatic reaction of Li-HP and glycolaldehyde, either various Arg526 (equivalent to Arg528 in Ref. [21]) mutants or double mutants with exchanges in Arg526 plus substitutions at Ser525, Arg357 (eq. to Arg359), or Ile189 were found to be most active [26].

Combinations of some single mutations (improved activity with glycolaldehyde or propanal), however, did not lead to the expected combined improvements of TK activity. Some of these double mutants even showed a loss of activity with certain substrates or a loss of enzyme solubility. A ‘readaptation’ was necessary and was successfully accomplished in the case of propanal as acceptor by additional mutations in the network of TK. The best TktA double variant was Asp469Ser/Arg520Gln with a more than fivefold improved kcat/KM value [27].

Finally, cascade reactions with TK and omega-transaminases have led to the preparative-scale biocatalytic synthesis of chiral aminodiols (e.g. (2S,3S)-2-aminopentane-1,3-diol). Herein, TK reactions provide hydroxyketones which are then reductively aminated to the respective chiral aminodiol products [28]. A novel two-component transketolase (NapB/NapD) was recently proposed to transfer a C2-glycolaldehyde unit from X5P to the lipoyl group of a pyruvate dehydrogenase-like complex in the biosynthesis of a non-ribosomal peptide assembly line during the synthesis of the natural antitumour compound, naphthyridinomycin [29].

Biosynthetic studies have been demonstrated to be a valuable source for the identification of novel enzymes catalyzing carboligation. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) is the first enzyme in the mevalonate-independent MEP pathway to isoprenoids. Brammer and Meyers successfully applied DXPS as a catalyst in vitro (Figure 2d) [30]. They showed that pyruvate can act as both donor and acceptor substrate.

Lehwald et al. presented the first example of an enzymatic asymmetric intermolecular crossed aldehyde–ketone carboligation via an Umpolung reaction, which was based on previous biosynthetic studies by Liu and coworkers. The ThDP-dependent flavoenzyme YerE catalyzes the decarboxylation of pyruvate and the transfer of the activated acetaldehyde to aldehydes and ketones (Figure 3a,b) [31••]. Many non-physiological carbonyl compounds are accepted as acceptor substrates by YerE and several of the enzymatic products were prepared on a preparative scale. The broad acceptor substrate range of the enzyme includes also 1,2-diketones (Figure 3c). Such 1,2-diketones were applied by Giovannini et al. in similar transformations using Bacillus licheniformis crude cell extract [32, 33]. Although the enzymes have not been identified yet, it could well be that they are ThDP-dependent. Using hexane-3,4-dione as a substrate, the resulting product with B. licheniformis crude cell extract (Figure 3d) is the (+)-enantiomer (in CH2Cl2) [32], whereas YerE gives the (−)-product (Lehwald, Müller, unpublished data).

In contrast to this, the ThDP-dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) catalyzes the hydrolysis of the title compound (Figure 3e) [34]. Although the structure of this enzyme from a dinitrifying Azoarcus strain has been solved [35], its mechanism and the reason(s) for the different behaviour compared to YerE (Csingle bondC bond cleavage versus ligation) are not known.

In addition to the above described various 1,2-additions of carbonyl compounds, the enzyme PigD was found to catalyze a 1,4-addition. This enzymatic Stetter reaction (conjugate addition of umpoled carbonyl species) was developed starting from a biosynthetic hypothesis, too. Williamson et al. postulated the first enzymatic step in the biosynthesis of prodigiosin in Serratia marcescens as being catalyzed by ThDP-dependent PigD [36]. Dresen et al. successfully demonstrated selective PigD-catalyzed 1,4-addition by using ketones (Figure 4a) as acceptor substrates instead of aldehydes (Figure 4b). Aliphatic, aromatic and heterocyclic α,β-unsaturated ketones selectively reacted with pyruvate (or 2-oxobutanoate) in the presence of PigD to give 1,4-adducts [37••]. The products were usually obtained with good to high enantiomeric excess; however, product yields on a preparative scale were not yet satisfactory.

Also MenD from E. coli is known to catalyze the 1,4-addition of a ThDP adduct of α-ketoglutarate (α-KG) to a α,β-unsaturated carboxylate; this reaction, therefore, constitutes a further Stetter-type reaction. The physiological 1,4-addition of α-KG to isochorismate catalyzed by MenD was enlarged to 2,3-dihydroxy-2,3-dihydro benzoate as a substrate (Figure 4c) [38]. Addition of α-KG to a broad range of aldehydes gave 2-hydroxy ketones with high enantiomeric excess (>94% ee for aromatic aldehydes as substrates, Figure 4d). Hence, a wide variety of new chiral building blocks are available through effective asymmetric enzymatic synthesis with MenD.

Most recently, an interesting decarboxylation-rearrangement has been elucidated by Beigi et al., who demonstrated that the reaction of α-KG and acetaldehyde catalyzed by MenD gave solely the expected, yet racemic, 2-hydroxy-3-oxo-product (Figure 5a), whereas the reaction with pyruvate instead of acetaldehyde resulted in the selective formation of the racemic 2-oxo-3-hydroxy-product (Figure 5b) [39]. Further experiments with other ThDP-dependent enzymes verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations. SucA, the ThDP-dependent E1 subunit of the α-KG dehydrogenase complex from E. coli, catalyzes the formation of acetoin using either pyruvate or acetaldehyde as sole substrate. Incubation of pyruvate with SucA led, via acetolactate, to almost racemic acetoin (8% ee), but the ee increased to 90% when (R)-acetoin was prepared using acetaldehyde as a substrate (Figure 5c). In contrast, CDH showed no formation of acetolactate as an intermediate. Accordingly, highly enantio-enriched (S)-acetoin (up to 90% ee) was directly obtained using pyruvate as the sole substrate (Figure 5d). These results, in combination with the already detailed investigations of the mechanism of acetohydroxy acid synthases [40], pave the way for the regioselective and enantioselective syntheses of short-chain acyloins.

Many other ThDP-dependent enzymes, such as different decarboxylases, have been applied in asymmetric Csingle bondC bond formation. For a more comprehensive overview see the above mentioned reviews. Other ThDP-dependent enzymes, such as phosphono pyruvate decarboxylase [41], phosphoketolase [42, 43] or clavulanic acid synthase [44], might be suitable candidates for this purpose as well.

Section snippets

Recently developed methods

Pleiss and coworkers designed the Thiamine diphosphate-dependent Enzyme Engineering Database (TEED), which serves as a navigation and analysis tool for members of this enzyme family [45]. The annotation of the conserved domains enables comparison and analysis of these domains between different subfamilies. The online accessible version of the database is provided at http://www.teed.uni-stuttgart.de. The same group recently published a new numbering system for this enzyme family [46]. Based

Conclusion and perspective

ThDP-dependent enzymes have been established as broadly applicable catalysts in asymmetric synthesis. Starting from the 1930s and up to several years ago, they have been applied predominantly for the synthesis of acyloins. Meanwhile, both enantiomers of many regioisomeric acyloins can be synthesized using wild type or variant enzymes.

Additional transformations, such as the Stetter reaction or the cross-benzoin condensation of aldehydes and ketones, have been introduced to enzyme catalysis. A

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

Financial support by the DFG in the scope of FOR 1296 and the research training group BioNoCo (GK1166) is gratefully acknowledged.

References (60)

  • M. Brovetto et al.

    Csingle bondC bond-forming lyases in organic synthesis

    Chem Res

    (2011)
  • P. Domínguez de Maria et al.

    Umpolung catalysis in benzoin-type and Stetter-type reactions: from enzymatic performances to bio-mimetic organocatalytic concepts

    Curr Org Chem

    (2011)
  • P. Hoyos et al.

    Biocatalytic strategies for the asymmetric synthesis of α-hydroxy ketones

    Acc Chem Res

    (2010)
  • M. Pohl et al.

    Enzymatic acyloin and benzoin condensations

  • M. Müller et al.

    Thiamin diphosphate in biological chemistry: exploitation of diverse thiamin diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis

    FEBS J

    (2009)
    K. Agyei-Owusu et al.

    Thiamin diphosphate in biological chemistry: analogues of thiamin diphosphate in studies of enzymes and riboswitches

    FEBS J

    (2009)
    L. Bettendorff et al.

    Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors

    FEBS J

    (2009)
  • P. Ayhan et al.

    Benzaldehyde lyase catalyzed enantioselective self and cross condensation reactions of acetaldehyde derivatives

    Org Biomol Chem

    (2011)
  • P. Ayhan et al.

    Benzaldehyde lyase-catalyzed direct amidation of aldehydes with nitroso compounds

    Adv Synth Catal

    (2011)
  • S.B. Sopaci et al.

    Carboligation reactions with benzaldehyde lyase immobilized on superparamagnetic solid support

    Org Biomol Chem

    (2009)
  • G.S. Brandt et al.

    Active-site engineering of benzaldehyde lyase shows that a point mutation can confer both new reactivity and susceptibility to mechanism-based inhibition

    J Am Chem Soc

    (2010)
  • A. Yep et al.

    Engineering the substrate binding site of benzoylformate decarboxylase

    Biochemistry

    (2009)
  • D. Gocke et al.

    Comparative characterisation of thiamin diphosphate-dependent decarboxylases

    J Mol Catal B: Enzym

    (2009)
  • D. Meyer et al.

    Conversion of pyruvate decarboxylase into an enantioselective carboligase with biosynthetic potential

    J Am Chem Soc

    (2011)
  • D. Rother et al.

    S-selective mixed carboligation by structure-based design of the pyruvate decarboxylase from Acetobacter pasteurianus

    ChemCatChem

    (2011)
  • M.M. Kneen et al.

    Characterization of a thiamine diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

    FEBS J

    (2011)
  • U. Schörken et al.

    Thiamin-dependent enzymes as catalysts in chemoenzymatic syntheses

    Biochim Biophys Acta

    (1998)
  • R. Wohlgemuth

    C-2-ketol elongation by transketolase-catalyzed asymmetric synthesis

    J Mol Catal B: Enzym

    (2009)
  • F. Charmantray et al.

    Preparative scale enzymatic synthesis of d-sedoheptulose-7-phosphate from β-hydroxypyruvate and d-ribose-5-phosphate

    J Mol Catal B: Enzym

    (2009)
  • O.A. Esakova et al.

    Halogenated pyruvate derivatives as substrates of transketolase from Saccharomyces cerevisiae

    Biochemistry (Moscow)

    (2009)
  • P.A. Asztalos et al.

    Strain and near attack conformers in enzymic thiamin catalysis: X-ray crystallographic snapshots of bacterial transketolase in covalent complex with donor ketoses xylulose 5-phosphate and fructose 6-phosphate, and in noncovalent complex with acceptor aldose ribose 5-phosphate

    Biochemistry

    (2007)
  • U. Nilsson et al.

    Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis

    J Biol Chem

    (1997)
  • Cited by (0)

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