Applications of transketolases in organic synthesis

527

Applications of transketolases in organic synthesis Nicholas J Turner The enzyme transketolase has been employed as a catalyst for asymmetric carbon-carbon bond formation in the synthesis of biologically important molecules. A number of important parameters have been addressed including substrate specificity, over-expression of the protein in suitable host systems, scale-up of the reaction and use of transketolase in multi-enzyme experiments. X-ray structural studies have been used to probe the origin of the asymmetry of the carbon-carbon bond-forming process.

sources, including spinach [2]. Most of the recent work has been carried out on the Escherichia coil [3-5] and S. cerevisiae [6] enzymes, the genes for which have been over-expressed, thereby making available greater quantities of enzyme for biophysical studies and applications in organic synthesis. [n vivo the enzyme catalyses the reversible transfer of a two carbon ketol unit from D-xylulose 5-phosphate (1; Figure la) to D-ribose 5-phosphate (2) to generate D-scdulose 7-phosphate (3) and D-glyceraldchyde 3-phosphate (4). Although this type of transformation has been exploited in a synthetic manner, the reaction can be made more generally useful by using hydroxypyruvic acid (HPA; 5) as the ketol donor (Figure lb). T h e use of HPA renders the reaction irreversible by virtue of the concomitant release of carbon dioxide as a by-product. T K is dependent for activity upon divalent metal cations (usually Mg z+) and thiamine pyrophosphate (TPP).

Addresses Department of Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JJ, UK; e-maiL [email protected] Current Opinion in Biotechnology 2000, 11:527-531

0958-1669/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations FBP fructose 1,6-bis-phosphate HPA hydroxypyruvic acid TK transketolase TPP thiamine pyrophosphate

TK-catalysed carbon-carbon bond formation is both stereospecific, in that the new chiral centre formed in the product has the (S)-configuration (the same configuration as C-3 of the natural donor substrates), and stereoselective, in that the enzyme has a preference for cz-hydroxyaldehydes with (R)-configuration at C-2. Thus, TK-mediated condensation of an (z-substituted aldehyde with HPA produces enantiomerically pure chiral triols (6) with the [3S,4R] or D-threo stereochemistry. T h e specificity of C - C bond formation is near total with T K from yeast and E. coli, producing an (S)-chiral centre, though with spinach T K some loss of enantiospecificity has been observed with certain substrates, notably cz-unsubstituted aldehydes. Non-phosphorylated, as well as phosphorylated, substrates are accepted by the enzyme; the best substrates for T K are

Introduction

Transketolase (TK; EC 2.2.1.1) occupies a pivotal place in metabolic regulation, providing a link between the glycolytic (degradative) and pentose phosphate pathways. T h e enzyme has a controlling role in the supply of ribose units for nucleoside biosynthesis, and (in microorganisms) in the supply of erythrose-4-phosphate into the shikimate pathway for aromatic amino acid biosynthesis. Transkctolase was first identified by Racker eta]. [1] in 1953 in the yeast Saccharomyces cerevisiae and subsequently located in other Figure 1 (a)

OH

OH

OH I O

TK

OH

OH I O

OH +

OH 0 D-Ribose 5-P2

OH

OH

D-Xylulose 5-P 1

2-O3PO= P

O

Mg 2+, TPP

(b)

OH

O D-Glyceraldehyde 3-P 4

D-Sedoheptulose7-P 3

TK -O2 c

OH

~x

0

~

HO

O

R OH

HPA 5

CO2

H_O OH

+

R O

6 Current Opinion in Biotechnology

TK-catalysed reactions (a) in vivo and (b) in vitro using HPA as the ketol donor. P, phosphate.

528 Chemicalbiotechnology

Figure 2 H

OEt

~ ' L "

Epoxide hydrolase

OEt

H

OEt

OH

0~[.. ~

(+/-)-7

+

OEt

Combined use of an epoxide hydrolase and S. cerevisiae TK to synthesise 4-deoxy D-fructose 6-phosphate. ee, enantiomeric excess.

OEt

HO-v~OEt

(S)-7 >98% e.e. i. K2HPO4 ii. H+, H20

OH 2-03P0 ~

0

(S)-8

OH OH

2-/

OH 0

OH OH OH 2"03P0~/'~, ~ 0 4-Deoxy-D-fructose 6-phosphate 10

7110 OH

Alcohol dehydrogenase~ ,- ) H NADH NAD÷

\)

002+ H20 "

OH HC02-

CurrentOpinionin Biotechnology

(z-hydroxyaldehydes of (R)-configuration, though (~-oxoand (z-unsubstituted aldehydes are also accepted. T h e stereochemistry of product formation with T K is the same as that obtained with fructose 1,6-bis-phosphate (FBP) aldolase and it is therefore instructive to compare the relative merits of the two enzymes [7]. Both enzymes accept a wide range of aldehydes as substrates, besides the natural ones. T K adds a two-carbon unit, rather than the three-carbon unit added by FBP aldolase. T K also produces unphosphorylated products, which makes the course of reaction easier to follow, simplifies product isolation and obviates the need for subsequent removal of phosphate from the product. T h e selectivity of T K for (R)-(x-hydroxyaldehydes means that the enzyme can be employed in the kinetic resolution of racemic substrates, forming a single condensation product, whereas FBP aldolase would tend to produce diastereomeric mixtures of products. T K therefore appears to possess a number of advantages as a useful catalyst for asymmetric C - C bond formation. It does, however, share a number of the disadvantages of FBP aldolase: availability of the catalyst and expense of the necessary co-substrate (HPA). Any attempt to develop T K as a viable process catalyst must therefore address these factors. T K is a member of a much larger family of TPP-dependent enzymes (e.g. pyruvate decarboxylase, dihydroxyacetone synthase, 1-deoxyxylulose-5-phosphate synthase), some others of which have also found application as catalysts in organic synthesis. This review focuses specifically on T K and deals principally with the literature published in the past 3-4 years. T h e literature up to 1997 has already been

reviewed [8-9]. For a comparative discussion of other TPPdependent enzymes related to T K the reader is referred to an excellent recent review [10].

Recent applications of transketolase in organic synthesis There is currently great interest in the use of biocatalysis for preparing flavour and fragrance components because of the desire to produce 'natural' molecules that can command a premium price as food additives. T K from spinach has been employed in a chemoenzymatic synthesis of 6-deoxy-L-sorbose, which is a known precursor of furaneol, a compound with caramel-like flavour [11]. In this synthesis, the hydroxypyruvate was prepared from L-serine by the use of serine glyoxylate aminotransferase, also isolated from spinach. T h e required aldehyde, 4-deoxy-L-threose, was obtained by microbial isomerisation of 4-deoxy-L-erythrulose using intact cells of Co~ynebacterium equi or Serratia liquefaciens. Finally, T K was employed to couple 4-deoxy-L-threose with HPA, yielding 6-deoxy-L-sorbose in up to 35% yield. Two key enzyme-catalysed reactions have been combined to great effect in the synthesis of the unnatural sugar 4-deoxy-D-fructose 6-phosphate (10; Figure 2) [12"]. Firstly, (R,S)-l,l-diethoxy-3,4-epoxybutanc (7) is resolved by treatment with an epoxide hydrolase from Aspergillus niger to give the recovered (S)-epoxide 7 in 30% yield and >98% cnantiomeric excess. T h e epoxide is converted to the (S)-aldehyde 8 by treatment with inorganic phosphate followed by acidic hydrolysis. T h e aldehyde 8 was then reacted with L-erythrulose (9) in the presence of S. cerevisiae T K to generate 4-deoxy-D-fructose 6-phosphate

Applications of transketolases in organic synthesis Turner 529

Figure 3

OPO32/ O OH s~~OPO3 HO

OH

O 2-O3PO~ Fructose 1,6-bisOH phosphatealdolase 1L 2~. TPI OH 2-OsPO~l

12

a-Xylulose5-phosphate 1

13

OH O 2-O3PO-@[~

TK

OH OH

O

002

O -O2C,~

OH CurrentOpinionin Biotechnology

One-pot aldolase/TK-catalysedsynthesisof D-xylulose5-phosphate.TPI, triosephosphateisomerase.

(10) in 52% yield. In order to drive the reaction to completion, the liberated glycolaldehyde 11 was reduced in situ with yeast alcohol dehydrogenase and catalytic NADH, the latter being recycled using the conventional formate/formate dehydrogenase system. It is noteworthy that this synthesis uses an cz-unsubstituted aldehyde in the TK-catalysed step. A second example of a multi-enzyme approach is shown in Figure 3 [13°]. In a one-pot procedure, fructose 1,6-hisphosphate aldolase initially catalyses the retro-aldol cleavage of D-fructose 1,6-his-phosphate to give a mixture of dihydroxyacetone phosphate (13) and D-glyceraldehyde 3-phosphate (4), which can be equilibrated by the use of triosephosphate isomerase (TPI). D-glyceraldehyde 3-phosphate (4) then couples with HPA (5) in a reaction mediated by E. coil T K to give D-xylulose 5-phosphate (1) in an overall yield of 82% on a gram scale.

E. coli TK-mediated coupling of racemic 3-0-benzylglyceraldehyde (14; Figure 4) with HPA has been carried out on a multi-gram scale to provide triol 15 as a convenient starting material for synthesis [14]. Subsequently, the triol 15 has been used in the preparation of the novel N-hydroxypyrrolidine 20, which has been designed to mimic the presumed oxonium ion intermediate and thereby act as a potential glycosidase inhibitor (Figure 4) [15°]. T h e synthesis proceeds via the protected oxime 16, which after conversion to the aldehyde 17 is cyclised to the 1,2-oxazine 18. This oxazine then undergoes an unusual ring contraction to form the protected N-hydroxypyrrolidine 19, which upon deprotection with HF produces 20. As part of their studies into the biosynthetic origin of the carbohydrate-based natural products acarbose and validamycin, Floss and co-workers [16] required access to t3C/NC/3H-labelled D-sedohepmlose 7-phosphate and D-ido-heptulose 7-phosphate. D-[5-14C, 5-3H]ribose

5-phosphate served as a key aldehyde acceptor for the TK-catalysed reactions, thereby providing a route to these labelled compounds. Of especial interest is the use of c-[3-tSC]serine as the precursor of 3-13C-HPA via the action of alanine racemase and D-amino acid oxidase/catalase. Various methods have been reported for the preparation of aldehyde substrates for TK. A procedure for converting enantiomerically pure (R)4z-hydroxycarboxylic acids to the corresponding (R)-o~-hydroxyaldehydes has been shown to proceed with no loss of optical purity [17]. Analogues of L-threose and D-erythrose have been prepared in which the C-2 position has been modified (i.e. 2-O-methyl, 2-deoxy) [18]. Such aldehydes have been shown to be substrates for T K and used to make some novel heptulose analogues [19]. Finally, Li and Frost [20] have spent a number of years developing multi-enzyme-based routes that are able to convert cheap, renewable carbon sources (e.g. glucose) to high-value fine chemicals, such as adipic acid, quinic acid, and 3-dehydroshikimic acid. In order to be able to manipulate the glycolytic and pentose phosphate pathway in a synthetically useful way, it is essential to ensure that the key enzymes are adequately expressed. One such recombinant strain, E. coli KLS/pKL4.124A, which overexpresses T K and feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, synthesizes higher concentrations and yields of 3-dehydroshikimic acid when the glucose/xylose/arabinose mixture is used as the carbon source relative to when either xylose or glucose is used as a carbon source. T h e y conclude that this high-yielding synthesis of 3-dehydroshikimic acid from the glucose/xylose/arabinose mixture carries significant ramifications that are relevant to the employment of corn fibre, which has a similar mixture of carbohydrate components, in the microbial synthesis of value-added chemicals [20].

530 Chemicalbiotechnology

Figure 4

HO

HO BnOv~l

TK >

BnO

o

0

i. TBSOTf, EtaN ii. NH20H TBSO N~OTBS iii. TBSOTf, EtaN OH ,. B n O ~ O T B S OTBS

OH 15

-02C~/OH

14

0

16

i. H2/Pd ii. NaOCI, TEMPO TBSO

DTBS

NaCNBH3 ,I

N~OTBS I OH 19

O!BS T B S O ~

TBSO

N~OTBS

OTBS (EtO)aCH'pTsOH i ~ ' ~

o

EtO/¢"-O~N

OTBS

OTBS

18

17

HF

~

HO

OH HO,,..~..~ ..,,OH

.OH

I

OH

OH

20

%O/k,~/OH

Oxonium ion intermediate in glycosidase-catalysed hydrolysis of glycosides

+ Current Opinion in Biotechnology

Synthesis of an N-hydroxypyrrolidineusing E. coil TK in the key C-C bond-forming step. Et, ethyl; Bn, benzyl;TBS, tert-butyldimethylsilyl; Tf, triflate; Ts, tosyl; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy.

D e v e l o p m e n t of large-scale processes Several groups have investigated the various parameters that are critical for developing a large-scale process employing T K in the key step. T h e group of Wandrey [21] have specialised in the development of membrane reactor systems for biocatalytic processes, in which the enzyme (and co-factor where appropriate) is located in a membrane, thereby improving biocatalyst stability- and also facilitating downstream purification. T h e y have applied this methodology to the TK-catalysed synthesis of erythrulose using a continuous process [21]. T h e y note that the starting aldehyde, glycolaldehyde, causes deactivation of the enzyme, thereby placing a limit on the concentration of starting material that can be used. Overall they were able to achieve a productivity level, as measured by spacetime-yield, of 45 g L -1 d q. T h e group of Woodley and Lilly at University College London has also reported on several aspects of scale-up. Their approach to the problem of product (etythrulose) inhibition is to develop methods for in situ product removal (ISPR) [22]. After considering a number of possible methods (e.g. ion-exchange, complex formation, physical absorption), they found that the use of phenylboronic acid to specifically form the phenylboronate derivative of the vic-diol in products of type 6 (Figure 1) offered the most promise. By using an immobilised phenylboronate resin (Affigel 601) they were able to remove L-erythrulose

together with some glycolaldehyde (21% on a molar basis) from a mixture containing these two components and hydroxypyruvic acid, which did not bind [23]. T h e y have also examined other facets of the biotransformation, including the effect of immobilising T K from E. coil on two commercially available supports (Eupergit-C and Amberlite XAD-7) has been reported. Using the TK-catalysed synthesis of L-erythrulose from glycolaldehyde and HPA as a model system, the authors report improvements of 80-100-fold in stability [24].

Structural and m u t a g e n e s i s studies T K is a dimeric protein composed of two identical subunits of -70 kDa each. The X-ray structures of TKs from both E. coli [2S] and S. cerevisiae [26,27] have been solved. Since the initial structure determination, Schneider and co-workers [28,29] have reported a series of studies in which they have systematically mutated key residues at the active site of T K in order to shed light on the catalytic mechanism and also explain the origin of the stereospecificity of the C-C bond forming process. Recently, this group has shown that the conserved residue Asp477, which forms a hydrogen bond with the C-2 hydroxyl group of the aldehyde substrate, is an important residue involved in determining the enantioselectivity of the reaction [30]. It is known that in the wild-type enzyme, the kcat/K M values are 103-104 lower for 2-deoxyaldehydes and also L-configured substrates. For the Asp477--+Ala mutant, the kcaJKM values for D-(x-hydroxyaldehydes are

Applications of transketolases in organic synthesis Turner

lowered by 103, whereas the kcat/KM values for the L- or 2-deoxy aldehydes are similar to the wild-type enzyme. T h e results imply that Asp477 plays a role in binding the aldehyde substrate and controlling the enantioselectivity.

Conclusions During the past five years there has been substantial progress in developing T K as a catalyst for asymmetric C42 bond synthesis. T h e enzyme has been over-expressed and is now truly available in substantial quantities if required. Much effort has gone into defining the problems associated with operating the reaction at scale and in certain cases generic solutions have been identified. T h e range of potential targets accessible via TK-catalysed reactions has been expanded by widening the pool of successful substrates and also exploring further synthetic applications of the products. Finally, our understanding of the mechanism of action of transketolase has been transformed by X-ray and mutagenesis studies leading to the possibility of redesigning the enzyme in a directed manner to improve certain desired characteristics.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • " of outstanding interest 1.

RackerE, de la Haba G, Leder IG: Thiamine pyrophosphate, a coenzyme of transketolase. J Am Chem Soc 1953, 75:1010-1011.

2.

VillafrancaJ, Axelrod B: Heptulose synthesis from nonphosphorylated aldoses and ketoses by spinach transketolase. J Biol Chem 1971,246:3126-3131.

3.

4.

Hobbs GR, Lilly MD, Turner NJ, Ward JM, Willetts A.I, Woodley JM: Enzyme-catalysed carbon-carbon bond formation: use of transketolase from Escherichia coll. J Chem Soc Perkin Trans 1 1993:165-166. FrenchC, Ward JM: Improved production and stability of Escherichia coil recombinants expressing transketolase for large scale biotransformation. Bioteehnol Lett 1995, 17:247-252.

5.

Sprenger GA, Schorken U, Sprenger G, Sahm H: Transketolase-A of

531

reaction is of significance because it utilises an aldehyde substrate lacking a C-2 hydroxyl group. 13. Zimmermann FT, Schneider A, Schorken U, Sprenger GA, • FessnerW-D: Efficient multi-enzymatic synthesis of D-xylulose 5-phosphate. Tetrahedron - Asymmetry 1999, 9:1643-1646. One pot synthesis of D-xylulose 5-phosphate by using combination of aldolase and transketolase enzymes. 14. Morris KG, Smith MEB, Turner NJ, Humphrey AJ: Transketolase from Escherichia coil: a practical procedure for using the biocatalyst for asymmetric carbon-carbon bond synthesis. Tetrahedron - Asymmetry 1996, 7:2185-2188. 15. Humphrey A J, Parsons SF, Smith MEB, Turner NJ: Synthesis of a • novel N-hydroxypyrrolidine using enzyme catalysed asymmetric carbon-carbon bond synthesis. Tetrahedron Lett 2000, 41:4481-4485. This paper demonstrates that transketolase-catalysed reactions can be used to generate sufficient quantities of material to undertake multi-step synthesis of natural and unnatural products. 16. Lee S, Kirschning A, Muller M, Way C, Floss HG: Enzymatic synthesis of [7-C-14, 7-H-3]- and [1 -C-13]sedoheptulose 7-phosphate and [1-C-13]ido-heptulose 7-phosphate. J Mol Cat B - Enzymatic 1999, 6:369-377. 17. Humphrey A J, Turner NJ, McCague R, Taylor SJC: Synthesis of enantiomerically pure alpha-hydroxyaldehydes from the corresponding alpha-hydroxycarboxylic acids: novel substrates for Escherichia coli transketolase. J Chem Soc Chem Commun 1995:2475-2476. 18. Andre C, Bolte J, DemuynckC: Syntheses of L-threose and #erythrose analogues modified at position 2. Tetrahedron - Asymmetry 1998, 9:1359-1367. 19. Andre C, Guerard C, Hecquet L, Demuynck C, Bolte J: Modified t-threose and D-erythrose as substrates of transketolase and fructose-1,6-bisphosphate aldolase. Application to the synthesis of new heptulose analogues. J Me/Cat B - Enzymatic 1998, 5:459-466. 20. Li K, Frost JW: Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of D-xylose, L-arabinose, and D-glucose carbon sources. Biotechno/Prog 1999, 15:876-683. 21. Bongs J, Hahn D, Schorken U, Sprenger GA, Wandrey C: Continuous production of erythrulose using transketolase in a membrane reactor. Biotechnol Lett 1997, 19:213-215. 22. Lye GJ, Woodley JM: Application of in situ product-removal techniques to biocatalytic processes. Trends Biotechnol 1999, 17:395-402.

Escherichia coil K12 - purification and properties of the enzyme from recombinant strains. Eur J Biochem 1995, 230:525-532.

23. Chauhan RP, Powell LW, Woodley JM: Boron based separations for in situ recovery of L-erythrulose from transketolase-catalyzed condensation. Biotechno/Bioeng 1997, 56:345-351.

6.

WiknerC, MeshalkinaL, Nilsson U, Nikkola M, Lindqvist Y, Sundstrom M, Schneider G: Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. J BiD/Chem 1994, 269:32144-32150.

24. Brocklebank S, Woodley JM, Lilly MD: Immobilised transketolase for carbon-carbon bond synthesis: biocatalyst stability. J Me/ Cat B - Enzymatic 1999, 7:223-231.

7.

Andre C, DemuynckC, Gefflaut T, Guerard C, Hecquet L, Lemaire M, Bolte J: Fructose-l,6-bis-phosphate aldolase and transketolase: complementary tools for the de nero syntheses of monosaccharides and analogues. J Me/Cat B - Enzymatic 1998, 5:113-118.

25. Littlechild J, Turner NJ, Hobbs G, Lilly M, Rawas A, Watson H: Crystallisation and preliminary X-ray crystallographic data with Escherichia coil transketolase. Acta Crystallogr D - BiD/Crysta//ogr 1995, 51:1074-1076.

8.

TakayamaS, McGarvey GJ, Wong C-H: Microbial aldolases and transketolases: new biocatalytic approaches to simple and complex sugars. Annu Rev Microbic/1997, 51:285-310.

26. Lindqvist Y, Schneider G, Ermler U, Sundstrom M: 3-Dimensional structure of transketolase, a thiamine diphosphate-dependent enzyme, at 2.5 A resolution. E M B O J 1992, 11:2373-2379.

FessnerW-D: Enzyme mediated C-C bond formation. Curt Qpin

27. Nikkela M, Lindqvist Y, Schneider G: Refined structure of transketolase from Saccharomyces cerevisiae at 2 ~. resolution. J Me/BiD/1994, 238:387-404.

9.

Chem BiD/1998, 2:85-97.

10. Sprenger GA, Pehl M: Synthetic potential of thiamin diphosphatedependent enzymes. J M e / C a t B - Enzymatic 1998, 3:145-159. 11. Hecquet L, Bolte J, Demuynck C: Enzymatic synthesis of 'naturallabeled' 6-deoxy-L-sorbose, precursor of an important food flavor. Tetrahedron 1996, 52:8223-8232. 12. Uerard C, Alphand V, Archelas A, Demuynck C, Hecquet L, • • FurstossR, 8olte J: Transketolase mediated synthesis of 4-deoxy-Dfructose 6-phosphate by epoxide hydrolase-catalysed resolution of 1,1-diethoxy-3,4-epoxybutane. Eur J Org Chem 1999:3399-3402. The authors demonstrate the effective combination of two different enzymes, namely epoxide hydrolase and transketolase. The S. cerevisiae transketolase

28. Wikner C, Nilsson U, Meshalkina L, Udekwu C, Lindqvist Y, Schneider G: Identification of catalytically important residues in yeast transketolase. Biochemistry 1997, 36:15643-15649. 29. Nilsson U, Meshalkina L, Lindqvist Y, Schneider G: Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis. J Biol Chem 1997, 272:1864-1869. 30. Nilsson U, Hecquet L, Gefflaut T, Guerard C, Schneider G: Asp(477) is a determinant of the enantioselectivity in yeast transketolase. FEBS Lett 1998,424:49-52.