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Enzyme catalysed deracemisation and dynamic kinetic resolution reactions
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Nicholas J Turner New catalysts and reaction conditions have been developed for the dynamic kinetic resolution or deracemisation of racemic mixtures of chiral compounds. Specific functional groups that lend themselves particularly well to this approach include chiral secondary alcohols, a-amino acids, amines and carboxylic acids. A general theme of these processes is the combination of an enantioselective enzyme with a chemical reagent, the latter being used either to racemise the unreactive enantiomer or alternatively recycle an intermediate in the deracemisation process. In some examples of dynamic kinetic resolution, a second enzyme (racemase) is used to interconvert the enantiomers of the starting material. Addresses School of Chemistry, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, UK e-mail: [email protected]
Current Opinion in Chemical Biology 2004, 8:114–119 This review comes from a themed issue on Biocatalysis and biotransformation Edited by Bernhard Hauer and Stanley M Roberts 1367-5931/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.02.001
Abbreviations CAL-B Candida antarctica lipase B DKR dynamic kinetic resolution ee enantiomeric excess
Introduction The synthesis of enantiomerically pure chiral compounds via cost-effective methods has become an important goal in the fine chemical and pharmaceutical industries. Although resolution-based strategies (e.g. kinetic resolution using enzymes or separation of diastereoisomers via crystallization of salts) are versatile, and can often be used to rapidly prepare the initial samples of a required chiral intermediate, the inherent limitation of a maximum 50% yield increasingly militates against their ultimate use in manufacturing processes. Attention therefore has turned to the development of asymmetric processes, or their equivalent, in which either achiral starting materials are converted to chiral non-racemic products or, alternatively, racemic mixtures are converted to enantiomerically pure compounds in yields approaching 100%. This review addresses the latter theme and summarizes recent literature, mostly dealing with literature published during Current Opinion in Chemical Biology 2004, 8:114–119
2003, covering the use of enzymes or whole cells for either the dynamic kinetic resolution (DKR) or deracemisation of racemic chiral compounds. DKR involves the combination of an enantioselective transformation with an in situ racemisation process such that, in principle, both enantiomers of the starting material can be converted to the product in high yield and enantiomeric excess (ee). The racemisation step can be either enzyme (e.g. racemase) or non-enzyme (e.g. transition metal) catalysed (Figure 1a). By contrast, the term deracemisation covers reactions in which two enantiomers are interconverted by a stereoinversion process such that a racemate can be transformed to a non-racemic mixture without any net change in the composition of the molecule. Deracemisation reactions tend to involve redox processes, for example the interconversion of chiral secondary alcohols via the ketone or alternatively the interconversion of amino acids/amines via the corresponding imine (Figure 1b). For coverage of earlier literature relating to enzyme mediated DKR and deracemisation reactions the reader is referred to a series of excellent reviews [1,2,3–6,7].
Dynamic kinetic resolution Non-enzyme catalysed racemisation
The group of Kanerva et al. has reported the synthesis of a series of novel (R)-5-phenylfuranyl-2-yl cyanomethylbutanoates by Pseudomonas cepacia lipase catalysed DKR of the corresponding cyanohydrins [8]. The use of a basic resin (amberlite OH ) was found to facilitate not only formation and racemisation of the cyanohydrin but also decomposition of the cyanide donor, acetone cyanohydrin. Yields of up to 94% were achieved with ee of 96%. A series of N-acylhemiaminals have been subjected to DKR using various lipases (e.g. lipase AK, lipase PS, lipase QL) in hexane at 60–70 8C with isopropenyl acetate as the acylating agent. In general, the reactions proceeded in high yield with ee >99%. The authors recommend the use of lipase QL for larger-scale applications (Figure 2) [9]. There has been considerable interest recently in the DKR of secondary alcohols by combining enantioselective lipases with transition-metal-based racemisation catalysts. The groups of Ba¨ckvall and Kim have continued to expand the scope of this reaction; in particular, Kim has recently shown that (S)- as well as (R)-configured alcohols can now be prepared by the use of a commercially available (S)-selective subtilisin (from Bacillus licheniformis) as the enantioselective acylating catalyst [10]. The www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 115
Figure 1
(b)
(a)
+ NH2
kcat(R) (R )-substrate
(R )-product
CO2–
R
D-amino
acid oxidase
(R )
NH
krac
[H]
(S )-substrate
R
+ NH2
kcat(S) (S )-product R
CO2–
CO2–
(S ) Current Opinion in Chemical Biology
DKR and deracemisation processes. (a) Dynamic kinetic resolution (DKR) by combining an enantioselective transformation with an in situ racemisation step. (b) Deracemisation of racemic a-amino acids by combining an enantioselective amine oxidase with a non-selective chemical reducing agent.
reactions were carried out in THF as solvent using trifluoroethyl butyrate as acyl donor and an aminocyclopentadienylruthenium complex as the racemising catalyst. In addition, it was found that pre-treating the subtilisin with a non-ionic surfactant (e.g. Brij 56) significantly improved the activity of the biocatalyst (ca. 4000-fold) under the reaction conditions (Figure 3). Backvall has reported the DKR of both a- and b-hydroxyalkane phosphonates using either Candida antarctica lipase B (CAL-B) or Pseudomonas cepacia lipase (PS-C) using an alternative ruthenium complex with p-chlorophenoxy acetate as acyl donor [11]. Interestingly, the DSM group has recently described details of the development of a large-scale process for the DKR of alcohols using various lipases in combination with a range of ruthenium catalysts [12]. By improvement of the process, reactions can be carried at concentrations up to 1 M with lower catalyst loadings. They have also described a process for the preparation of (R)-3,5-bistrifluoromethyl-phenylethan-1-ol using [RuCl2(p-cymene)]2 as the racemisation catalyst combination with CAL-B as the acylating catalyst [13].
Although not yet evaluated in the presence of lipase catalysts, two groups have reported novel rutheniumbased catalysts for the mild racemisation of chiral secondary alcohols. Ikariya et al. [14] prepared a ternary catalyst system composed of [Z5-C5(CH3)5]Ru complexes bearing tertiary-phosphine–primary-amine chelate ligands. Racemisation of a series of secondary alcohols was achieved in only a few hours at 30 8C using toluene as solvent. By contrast, Wuyts et al. [15] have developed a heterogeneous catalyst based upon Ru(III) immobilised on calcium hydroxyapatite. Both the preparation of the hydroxyapatite support and the ruthenium content upon immobilisation were optimised to ensure maximum catalytic performance. The catalyst was able to efficiently racemise both benzylic and aliphatic secondary alcohols. As an alternative to metal-based racemisation catalysts, Wuyts et al. [16] have shown that acid zeolites (e.g. HBeta) are also efficient heterogeneous catalysts for alcohol racemisation. Using a biphasic organic–aqueous system, they carried out the DKR of (R/S)-1-phenylethanol using a range of acyl donors (e.g. vinyl acetate, vinyl octanoate) with Novozym 435 (CAL-B). Under optimised conditions
Figure 2 OH NR O (S )
O
OH
NHR
NR
O
OAc
Lipase isopropenyl acetate
NR Hexane, 60–70 oC O
O (R )
(R ) Current Opinion in Chemical Biology
DKR of hemiaminals. www.sciencedirect.com
Current Opinion in Chemical Biology 2004, 8:114–119
116 Biocatalysis and biotransformation
Figure 3
OH Ph
OH
Ru-catalyst
Me
Ph
4 mol%
(R )
Ph
Me (S )
Ru-catalyst =
Me (S )
Ph Ph
OCOPr
Subtilisin C3H7CO2CH2CF3
Yield = 97% ee = >99%
NHPri
Ph Ru Ph OC CO Cl
Current Opinion in Chemical Biology
DKR of secondary alcohols using subtilisin to yield (S)-configured products.
(16 equivalents of acyl donor), deracemisation was complete in 8 h (90% yield, 99% ee). The corresponding racemisation of chiral amines is a much more difficult process and to date there have been no successful reports of true DKR reactions in which the racemisation catalysts and enzyme are combined in a onepot process. For example, although it is well known that certain lipases (e.g. CAL-B) are able to catalyse enantioselective acylation of a wide range of racemic chiral primary amines, the conditions required for racemisation are typically high temperature (e.g. 150 8C) [17] often in the presence of a supported dehydrogenation catalyst containing copper and zinc oxide as active components [18]. Although the use of a transition metal (ruthenium) catalyst enables a lower operating temperature of 110 8C, these conditions are still incompatible with the use of an enzyme catalyst. In this case, the enzymatic acylation, using CAL-B, and racemisation reaction were carried out as separate steps and after two cycles the products were obtained in 66–69% yield and >98 ee [19]. Recent work by Backvall et al. has suggested that the rate-limiting step in the racemisation of amines by a (cyclopentadienone)ruthenium complex is b-hydride elimination from the amine followed by proton transfer [20].
a L-carbamoylase, a hydantoin racemase and a hydantoinase, and (ii) the DKR of N-acetyl amino acids using an acylase in combination with an N-acetyl amino acid racemase from Amycalotopsis orientalis subsp. lurida [21].
Deracemisation reactions Deracemisation of secondary alcohols
It is well known that various microbial systems are able to deracemise racemic secondary alcohols via a process that generally involves two different alcohol dehydrogenases with complementary enantiospecificity. In this context, Porto et al. have shown that various fungi, including Aspergillus terreus CCT 3320 and A. terreus CCT 4083, are able to deracemise ortho- and meta-fluorophenyl-1ethanol in good yields and high ee [22]. Likewise, Demir et al. have reported the deracemisation of racemic benzoin using Rhizopus oryzae ATCC 9363. Interestingly, they were able to use the pH of the medium to control the absolute configuration of the enantiomer produced. Thus, at pH 7.5–8.5 the (R)-enantiomer was obtained in 73–76% yield and 97% ee whereas at pH 4-5 the (S)-enantiomer was produced (71% yield; 85% ee) (Figure 4) [23]. Chadha et al. have reported the deracemisation of a-hydroxyesters using whole cells of Candida parapsilosis. For example, Figure 4
Enzyme-catalysed racemisation
The use of an enzyme, rather than a transition metal catalyst, represents an attractive option for combined DKR reactions in view of the likely mild conditions associated with enzyme-catalysed racemisation processes. Racemases belong to the group of enzymes EC 5.1.X.X and contain notable members such as mandelate racemase and various amino acid racemases. A recent review provides an excellent overview of the various types of racemases and their applications in synthetic DKR reactions [2]. The Degussa group have recently described their successful commercialisation of two DKR-based processes that employ racemases, namely (i) the DKR of 5-substitued hydantoins using whole cells coexpressing Current Opinion in Chemical Biology 2004, 8:114–119
O
OH R. oryzae pH = 7.5–8.5 O
R. oryzae pH = 4.0–5.0 O
R. oryzae pH = 7.5–8.5 OH
OH Current Opinion in Chemical Biology
Deracemisation of benzoin using Rhizopus oryzae. www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 117
Figure 5
CH3 CO2H
X
CH3
Acyl-CoA synthetase Hydrolase
COSCoA
X
Net reaction deracemization
2-ArylpropionylCoA epimerase CH3
CH3 X
CO2H
X
Hydrolase
COSCoA
X
Current Opinion in Chemical Biology
Possible mechanism for deracemisation of arylpropionic acids.
racemic ethyl 2-hydroxy-4-phenylbutanoic acid yielded the (S)-enatiomer in 85–90% yield and >99% ee. Analogous deracemisations of racemic ethyl/methyl esters of mandelic acid similarly yielded the (S)-products in high ee [24]. Deracemisation of carboxylic acids
Ohta et al. have reported some elegant studies into the microbial deracemisation of 2-aryl- and 2-aryloxypropanoic acids using growing cells of Nocardia diaphanozonaria JCM 3208. In both cases, the (R)-enantiomer was preferentially obtained from the racemate. Although the reaction was found to be very sensitive to the structure of the substrate, in optimal cases (e.g. for 2-(4-chlorophenoxy)propanoic acid) yields of up to 95% were obtained with an associated ee of 97% [25]. Recently this group has reported a series of studies to elucidate the underlying mechanism of the deracemisation reaction. Labelling studies, together with the use of specific inhibitors, suggest the participation of three enzymes, namely an acylCoA synthetase, an arylpropionyl-CoA epimerase and a hydrolase (Figure 5) [26]. Figure 6
NH2 Ph
Pig kidney D-AAO NH3:BH3
CO2H Me (2R,3R)-3
78% yield 99% ee & de
Snake venom L-AAO NH3:BH3
NH2 Ph
CO2H Me (2S, 3R)-3
82% yield 99% ee & de Current Opinion in Chemical Biology
Stereoinversion of b-substituted a-amino acids using amino acid oxidases (AAO) with ammonia-borane. de, diastereomeric excess. www.sciencedirect.com
Deracemisation of amino acids and amines
Turner et al. have continued to expand the scope of their process for the deracemisation of a-amino acids and amines using a cyclic oxidation–reduction sequence [27]. The key to this approach is to combine a highly enantioselective amino acid or amine oxidase with a nonselective chemical reducing agent (e.g. amine–borane complex, Pd/C-ammonium formate), which reduces the intermediate imine, thereby effecting the stereoinversion of one enantiomer to its antipode. Recent developments include the stereoinversion of b- and g-substituted aamino acids in which diastereomers, rather than enantiomers, are interconverted (Figure 6) [28]. Furthermore, by devising a powerful, high-throughput screening method for selecting active variants, this group has recently use directed evolution to identify an amine oxidase possessing both broad substrate specificity and high enantioselectivity that should have wide application in deracemisation reactions [29].
Conclusions The past year (2003) has witnessed significant developments in the efficiency and scope of application of DKR and deracemisation reactions. The DKR of secondary alcohols, using lipases in combination with transition metal (ruthenium) complexes has been further developed to encompass a greater range of substrates and, significantly, can now be applied to the preparation of (S)configured alcohols. New catalysts for racemisation have been discovered, and of particular interest is the demonstration that this process can be scaled-up and applied to the commercial manufacture of specific target compounds. However, the corresponding DKR of amines is not yet a practical option as a result of the harsh conditions required to racemise chiral amines. Undoubtedly, the next 12 months will witness many new publications in this area. Deracemisation processes are Current Opinion in Chemical Biology 2004, 8:114–119
118 Biocatalysis and biotransformation
also starting to make an impact and now extend from the microbial deracemisation of secondary alcohols and achiral carboxylic acids to reactions involving oxidase/ reducing agent combinations for preparing single enantiomer amino acids and amines in high yield. Deracemisation is an inherently attractive option in view of the simplicity of the system (i.e. avoidance of protecting groups, lack of product inhibition) and it will be interesting to see how well these processes compete with alternative approaches based upon DKR. Finally, there is clearly considerable scope for identifying and evolving a wider range of racemase enzymes to complement the transition-metal-based racemisation catalysts. There is no doubt that the increased availability of microbial genome sequences, coupled with advances in high-throughput screening technologies and directed evolution [30], will provide not only new enzymes with the desired substrate range and selectivity, but also process compatible biocatalysts that are better able to operate under the conditions of the reaction. The latter aspect is particularly relevant to those reactions in which enzymes are combined with chemical reagents or transition metal catalysts as outlined above in this review.
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.
Pellisier H: Dynamic kinetic resolution. Tetrahedron 2003, 59:8291-8327.
2.
Schnell B, Faber K, Kroutil W: Enzymatic racemisation and its application to synthetic biotransformations. Adv Synth Catal 2003, 345:653-666. An excellent comprehensive review summarizing the current literature of racemase enzymes and their application in DKR reactions. 3.
Pamies O, Backvall J-E: Combination of enzymes and metal catalysts. A powerful approach in asymmetric catalysis. Chem Rev 2003, 103:3247-3261.
4.
Pamies O, Backvall J-E: Combined metal catalysis and biocatalysis for an efficient deracemization process. Curr Opin Biotechnol 2003, 14:407-413.
5.
Turner NJ: Controlling chirality. Curr Opin Biotechnol 2003, 14:401-406.
6.
Kim M-J, Ahn Y, Park J: Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis. Curr Opin Biotechnol 2002, 13:578-587.
7.
Faber K: Non-sequential processes for the transformation of a racemate into a single stereoisomeric product: proposal for stereochemical classification. Chemistry 2001, 7:5004-5010. A useful review clarifying the use of terminology to describe enzymecatalysed processes, including dynamic kinetic resolution and deracemisation. 8.
Paizs C, Ta¨ htinen P, Lundell K, Poppe L, Irimie F-D, Kanerva LT: Preparation of novel phenylfuran-based cyanohydrin esters: lipase-catalysed kinetic and dynamic resolution. Tetrahedron Asymmetry 2003, 14:1895-1904.
9.
Sharfuddin M, Narumi A, Iwai Y, Keiko M, Yamada S, Kakuchi T, Kaga H: Lipase-catalyzed dynamic resolution of hemiaminals. Tetrahedron Asymmetry 2003, 14:1581-1585.
10. Kim M-J, Chung YI, Choi YK, Lee HK, Kim D, Park J: (S)-Selective dynamic kinetic resolution of secondary alcohols by the combination of subtilisin and an Current Opinion in Chemical Biology 2004, 8:114–119
aminocyclopentadienylruthenium complex as the catalysts. J Am Chem Soc 2003, 125:11494-11495. Significant extension of the application of DKR of secondary alcohols using hydrolases in combination with transition metal based racemisation catalysts. The use of subtilisin allows access to (S)-configured alcohols for the first time. 11. Pa`mies O, Ba¨ ckvall J-E: An efficient route to chiral a- and bhydroxyalkanephosphonates. J Org Chem 2003, 68:4815-4818. 12. Verzijl GKM, De Vries JG, Broxterman QB: Process for the preparation of enantiomerically enriched esters and alcohols. PCT Int Appl 2001, WO 2001090396 A1 20011129. Demonstration that DKR of secondary alcohols can be carried out at large scale as a result of optimisation of the process with respect to substrate concentration, catalyst loading and recycle and other key parameters. 13. Broxterman QB, Verzijl GKM: Process for the synthesis of (R)-1(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol and esters thereof by dynamic kinetic resolution. PCT Int Appl 2003, WO 2003043575 A2 20030530. 14. Ito M, Osaku A, Kitahara S, Hirakawa M, Ikariya T: Rapid racemization of chiral non-racemic sec-alcohols catalyzed by [g5-C5(CH3)5]Ru complexes bearing tertiary phosphine-primary amine chelate ligands. Tetrahedron Lett 2003, 44:7521-7523. 15. Wuyts S, De Vos DE, Verpoort F, Depla D, De Gryse R, Jacobs PA: A heterogeneous Ru-hydroxyapatite catalyst for mild racemization of alcohols. J Catal 2003, 219:417-424. 16. Wuyts S, De Temmerman K, De Vos D, Jacobs PA: A zeoliteenzyme combination for biphasic dynamic kinetic resolution of benzylic alcohols. Chem Commun 2003, 2003:1928-1929. 17. Skupinska KA, McEachern EJ, Baird IR, Skerlj RT, Bridger GJ: Enzymatic resolution of bicyclic 1-heteroarylamines using Candida antarctica lipase B. J Org Chem 2003, 68:3546-3551. 18. Funke F, Liang S, Kramer A, Stuermer R, Hoehn A: Racemization of optically active amines. Eur Pat Appl 2002, EP1215197 A2 20020619. 19. Pa`mies O, E´ ll AH, Samec JSM, Hermanns N, Ba¨ ckvall JE: An efficient and mild-ruthenium-catalyzed racemization of amines: application to the synthesis of enantiomerically pure amines. Tetrahedron Lett 2002, 43:4699-4702. Reports the use of a ruthenium catalyst for racemisation of amines and, although in situ DKR is not possible at this stage, this paper suggests that further improvements in terms of lower temperatures for racemisation may soon be achievable. 20. E´ ll AH, Johnson JB, Ba¨ ckvall JE: Mechanism of rutheniumcatalyzed hydrogen transfer reactions. Evidence for a stepwise transfer of CH and NH hydrogens from an amine to a (cyclopentadienone)ruthenium complex. Chem Commun 2003, 2003:1652-1653. 21. May O, Verseck S, Bommarius A, Drauz K: Development of dynamic kinetic resolution processes for biocatalytic production of natural and non-natural L-amino acids. Org Proc Res Dev 2002, 6:452-457. Informative review detailing the development and subsequent commercialisation of two DKR processes that involve racemase enzymes. 22. Comasseto JV, Omori A´ T, Andrade LH, Porto ALM: Bioreduction of fluoroacetophenones by the fungi Aspergillus terreus and Rhizopus oryzae. Tetrahedron Asymmetry 2003, 14:711-715. 23. Demir AS, Hamamci H, Sesenoglu O, Neslihanoglu R, Asikoglu B, Capanoglu D: Fungal deracemization of benzoin. Tetrahedron Lett 2002, 43:6447-6449. 24. Chadha A, Baskar B: Biocatalytic deracemisation of a-hydroxy esters: high yield preparation of (S)-ethyl 2-hydroxy-4phenylbutanoate from the racemate. Tetrahedron Asymmetry 2002, 13:1461-1464. 25. Kato D, Mitsuda S, Ohta H: Microbial deracemization of a-substituted carboxylic acids. Org Lett 2002, 4:371-373. 26. Kato D, Mitsuda S, Ohta H: Microbial deracemization of a-substituted carboxylic acids: substrate specificity and mechanistic investigation. J Org Chem 2003, 68:7234-7242. Detailed and revealing study concerning the microbial deracemisation of a-substituted carboxylic acids. Evidence presented suggests that www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 119
deracemisation occurs via enzyme-catalysed formation, epimerisation and subsequent hydrolysis of coenzyme A esters. 27. Alexeeva M, Carr R, Turner NJ: Directed evolution of enzymes: new biocatalysts for asymmetric synthesis. Org Biomol Chem 2003, 1:4133-4137. 28. Enright A, Alexandre F-R, Roff G, Fotheringham IG, Dawson MJ, Turner NJ: Stereoinversion of b- and c-substituted-a-amino acids using a chemoenzymatic oxidation-reduction procedure. Chem Commun 2003, 2003:2636-2637.
www.sciencedirect.com
29. Carr R, Alexeeva M, Enright A, Eve TSC, Dawson MJ, Turner NJ: Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantioselectivity. Angew Chem Int Ed Engl 2003, 42:4807-4810. Use of random mutagenesis coupled with high-throughput screening to identify a variant amine oxidase that has significantly broadened substrate specificity, while retaining high enantioselectivity, compared with the wild-type enzyme. 30. Turner NJ: Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol 2003, 21:474-478.
Current Opinion in Chemical Biology 2004, 8:114–119
Current Opinion in Chemical Biology 2004, 8:114–119 This review comes from a themed issue on Biocatalysis and biotransformation Edited by Bernhard Hauer and Stanley M Roberts 1367-5931/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.02.001
Abbreviations CAL-B Candida antarctica lipase B DKR dynamic kinetic resolution ee enantiomeric excess
Introduction The synthesis of enantiomerically pure chiral compounds via cost-effective methods has become an important goal in the fine chemical and pharmaceutical industries. Although resolution-based strategies (e.g. kinetic resolution using enzymes or separation of diastereoisomers via crystallization of salts) are versatile, and can often be used to rapidly prepare the initial samples of a required chiral intermediate, the inherent limitation of a maximum 50% yield increasingly militates against their ultimate use in manufacturing processes. Attention therefore has turned to the development of asymmetric processes, or their equivalent, in which either achiral starting materials are converted to chiral non-racemic products or, alternatively, racemic mixtures are converted to enantiomerically pure compounds in yields approaching 100%. This review addresses the latter theme and summarizes recent literature, mostly dealing with literature published during Current Opinion in Chemical Biology 2004, 8:114–119
2003, covering the use of enzymes or whole cells for either the dynamic kinetic resolution (DKR) or deracemisation of racemic chiral compounds. DKR involves the combination of an enantioselective transformation with an in situ racemisation process such that, in principle, both enantiomers of the starting material can be converted to the product in high yield and enantiomeric excess (ee). The racemisation step can be either enzyme (e.g. racemase) or non-enzyme (e.g. transition metal) catalysed (Figure 1a). By contrast, the term deracemisation covers reactions in which two enantiomers are interconverted by a stereoinversion process such that a racemate can be transformed to a non-racemic mixture without any net change in the composition of the molecule. Deracemisation reactions tend to involve redox processes, for example the interconversion of chiral secondary alcohols via the ketone or alternatively the interconversion of amino acids/amines via the corresponding imine (Figure 1b). For coverage of earlier literature relating to enzyme mediated DKR and deracemisation reactions the reader is referred to a series of excellent reviews [1,2,3–6,7].
Dynamic kinetic resolution Non-enzyme catalysed racemisation
The group of Kanerva et al. has reported the synthesis of a series of novel (R)-5-phenylfuranyl-2-yl cyanomethylbutanoates by Pseudomonas cepacia lipase catalysed DKR of the corresponding cyanohydrins [8]. The use of a basic resin (amberlite OH ) was found to facilitate not only formation and racemisation of the cyanohydrin but also decomposition of the cyanide donor, acetone cyanohydrin. Yields of up to 94% were achieved with ee of 96%. A series of N-acylhemiaminals have been subjected to DKR using various lipases (e.g. lipase AK, lipase PS, lipase QL) in hexane at 60–70 8C with isopropenyl acetate as the acylating agent. In general, the reactions proceeded in high yield with ee >99%. The authors recommend the use of lipase QL for larger-scale applications (Figure 2) [9]. There has been considerable interest recently in the DKR of secondary alcohols by combining enantioselective lipases with transition-metal-based racemisation catalysts. The groups of Ba¨ckvall and Kim have continued to expand the scope of this reaction; in particular, Kim has recently shown that (S)- as well as (R)-configured alcohols can now be prepared by the use of a commercially available (S)-selective subtilisin (from Bacillus licheniformis) as the enantioselective acylating catalyst [10]. The www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 115
Figure 1
(b)
(a)
+ NH2
kcat(R) (R )-substrate
(R )-product
CO2–
R
D-amino
acid oxidase
(R )
NH
krac
[H]
(S )-substrate
R
+ NH2
kcat(S) (S )-product R
CO2–
CO2–
(S ) Current Opinion in Chemical Biology
DKR and deracemisation processes. (a) Dynamic kinetic resolution (DKR) by combining an enantioselective transformation with an in situ racemisation step. (b) Deracemisation of racemic a-amino acids by combining an enantioselective amine oxidase with a non-selective chemical reducing agent.
reactions were carried out in THF as solvent using trifluoroethyl butyrate as acyl donor and an aminocyclopentadienylruthenium complex as the racemising catalyst. In addition, it was found that pre-treating the subtilisin with a non-ionic surfactant (e.g. Brij 56) significantly improved the activity of the biocatalyst (ca. 4000-fold) under the reaction conditions (Figure 3). Backvall has reported the DKR of both a- and b-hydroxyalkane phosphonates using either Candida antarctica lipase B (CAL-B) or Pseudomonas cepacia lipase (PS-C) using an alternative ruthenium complex with p-chlorophenoxy acetate as acyl donor [11]. Interestingly, the DSM group has recently described details of the development of a large-scale process for the DKR of alcohols using various lipases in combination with a range of ruthenium catalysts [12]. By improvement of the process, reactions can be carried at concentrations up to 1 M with lower catalyst loadings. They have also described a process for the preparation of (R)-3,5-bistrifluoromethyl-phenylethan-1-ol using [RuCl2(p-cymene)]2 as the racemisation catalyst combination with CAL-B as the acylating catalyst [13].
Although not yet evaluated in the presence of lipase catalysts, two groups have reported novel rutheniumbased catalysts for the mild racemisation of chiral secondary alcohols. Ikariya et al. [14] prepared a ternary catalyst system composed of [Z5-C5(CH3)5]Ru complexes bearing tertiary-phosphine–primary-amine chelate ligands. Racemisation of a series of secondary alcohols was achieved in only a few hours at 30 8C using toluene as solvent. By contrast, Wuyts et al. [15] have developed a heterogeneous catalyst based upon Ru(III) immobilised on calcium hydroxyapatite. Both the preparation of the hydroxyapatite support and the ruthenium content upon immobilisation were optimised to ensure maximum catalytic performance. The catalyst was able to efficiently racemise both benzylic and aliphatic secondary alcohols. As an alternative to metal-based racemisation catalysts, Wuyts et al. [16] have shown that acid zeolites (e.g. HBeta) are also efficient heterogeneous catalysts for alcohol racemisation. Using a biphasic organic–aqueous system, they carried out the DKR of (R/S)-1-phenylethanol using a range of acyl donors (e.g. vinyl acetate, vinyl octanoate) with Novozym 435 (CAL-B). Under optimised conditions
Figure 2 OH NR O (S )
O
OH
NHR
NR
O
OAc
Lipase isopropenyl acetate
NR Hexane, 60–70 oC O
O (R )
(R ) Current Opinion in Chemical Biology
DKR of hemiaminals. www.sciencedirect.com
Current Opinion in Chemical Biology 2004, 8:114–119
116 Biocatalysis and biotransformation
Figure 3
OH Ph
OH
Ru-catalyst
Me
Ph
4 mol%
(R )
Ph
Me (S )
Ru-catalyst =
Me (S )
Ph Ph
OCOPr
Subtilisin C3H7CO2CH2CF3
Yield = 97% ee = >99%
NHPri
Ph Ru Ph OC CO Cl
Current Opinion in Chemical Biology
DKR of secondary alcohols using subtilisin to yield (S)-configured products.
(16 equivalents of acyl donor), deracemisation was complete in 8 h (90% yield, 99% ee). The corresponding racemisation of chiral amines is a much more difficult process and to date there have been no successful reports of true DKR reactions in which the racemisation catalysts and enzyme are combined in a onepot process. For example, although it is well known that certain lipases (e.g. CAL-B) are able to catalyse enantioselective acylation of a wide range of racemic chiral primary amines, the conditions required for racemisation are typically high temperature (e.g. 150 8C) [17] often in the presence of a supported dehydrogenation catalyst containing copper and zinc oxide as active components [18]. Although the use of a transition metal (ruthenium) catalyst enables a lower operating temperature of 110 8C, these conditions are still incompatible with the use of an enzyme catalyst. In this case, the enzymatic acylation, using CAL-B, and racemisation reaction were carried out as separate steps and after two cycles the products were obtained in 66–69% yield and >98 ee [19]. Recent work by Backvall et al. has suggested that the rate-limiting step in the racemisation of amines by a (cyclopentadienone)ruthenium complex is b-hydride elimination from the amine followed by proton transfer [20].
a L-carbamoylase, a hydantoin racemase and a hydantoinase, and (ii) the DKR of N-acetyl amino acids using an acylase in combination with an N-acetyl amino acid racemase from Amycalotopsis orientalis subsp. lurida [21].
Deracemisation reactions Deracemisation of secondary alcohols
It is well known that various microbial systems are able to deracemise racemic secondary alcohols via a process that generally involves two different alcohol dehydrogenases with complementary enantiospecificity. In this context, Porto et al. have shown that various fungi, including Aspergillus terreus CCT 3320 and A. terreus CCT 4083, are able to deracemise ortho- and meta-fluorophenyl-1ethanol in good yields and high ee [22]. Likewise, Demir et al. have reported the deracemisation of racemic benzoin using Rhizopus oryzae ATCC 9363. Interestingly, they were able to use the pH of the medium to control the absolute configuration of the enantiomer produced. Thus, at pH 7.5–8.5 the (R)-enantiomer was obtained in 73–76% yield and 97% ee whereas at pH 4-5 the (S)-enantiomer was produced (71% yield; 85% ee) (Figure 4) [23]. Chadha et al. have reported the deracemisation of a-hydroxyesters using whole cells of Candida parapsilosis. For example, Figure 4
Enzyme-catalysed racemisation
The use of an enzyme, rather than a transition metal catalyst, represents an attractive option for combined DKR reactions in view of the likely mild conditions associated with enzyme-catalysed racemisation processes. Racemases belong to the group of enzymes EC 5.1.X.X and contain notable members such as mandelate racemase and various amino acid racemases. A recent review provides an excellent overview of the various types of racemases and their applications in synthetic DKR reactions [2]. The Degussa group have recently described their successful commercialisation of two DKR-based processes that employ racemases, namely (i) the DKR of 5-substitued hydantoins using whole cells coexpressing Current Opinion in Chemical Biology 2004, 8:114–119
O
OH R. oryzae pH = 7.5–8.5 O
R. oryzae pH = 4.0–5.0 O
R. oryzae pH = 7.5–8.5 OH
OH Current Opinion in Chemical Biology
Deracemisation of benzoin using Rhizopus oryzae. www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 117
Figure 5
CH3 CO2H
X
CH3
Acyl-CoA synthetase Hydrolase
COSCoA
X
Net reaction deracemization
2-ArylpropionylCoA epimerase CH3
CH3 X
CO2H
X
Hydrolase
COSCoA
X
Current Opinion in Chemical Biology
Possible mechanism for deracemisation of arylpropionic acids.
racemic ethyl 2-hydroxy-4-phenylbutanoic acid yielded the (S)-enatiomer in 85–90% yield and >99% ee. Analogous deracemisations of racemic ethyl/methyl esters of mandelic acid similarly yielded the (S)-products in high ee [24]. Deracemisation of carboxylic acids
Ohta et al. have reported some elegant studies into the microbial deracemisation of 2-aryl- and 2-aryloxypropanoic acids using growing cells of Nocardia diaphanozonaria JCM 3208. In both cases, the (R)-enantiomer was preferentially obtained from the racemate. Although the reaction was found to be very sensitive to the structure of the substrate, in optimal cases (e.g. for 2-(4-chlorophenoxy)propanoic acid) yields of up to 95% were obtained with an associated ee of 97% [25]. Recently this group has reported a series of studies to elucidate the underlying mechanism of the deracemisation reaction. Labelling studies, together with the use of specific inhibitors, suggest the participation of three enzymes, namely an acylCoA synthetase, an arylpropionyl-CoA epimerase and a hydrolase (Figure 5) [26]. Figure 6
NH2 Ph
Pig kidney D-AAO NH3:BH3
CO2H Me (2R,3R)-3
78% yield 99% ee & de
Snake venom L-AAO NH3:BH3
NH2 Ph
CO2H Me (2S, 3R)-3
82% yield 99% ee & de Current Opinion in Chemical Biology
Stereoinversion of b-substituted a-amino acids using amino acid oxidases (AAO) with ammonia-borane. de, diastereomeric excess. www.sciencedirect.com
Deracemisation of amino acids and amines
Turner et al. have continued to expand the scope of their process for the deracemisation of a-amino acids and amines using a cyclic oxidation–reduction sequence [27]. The key to this approach is to combine a highly enantioselective amino acid or amine oxidase with a nonselective chemical reducing agent (e.g. amine–borane complex, Pd/C-ammonium formate), which reduces the intermediate imine, thereby effecting the stereoinversion of one enantiomer to its antipode. Recent developments include the stereoinversion of b- and g-substituted aamino acids in which diastereomers, rather than enantiomers, are interconverted (Figure 6) [28]. Furthermore, by devising a powerful, high-throughput screening method for selecting active variants, this group has recently use directed evolution to identify an amine oxidase possessing both broad substrate specificity and high enantioselectivity that should have wide application in deracemisation reactions [29].
Conclusions The past year (2003) has witnessed significant developments in the efficiency and scope of application of DKR and deracemisation reactions. The DKR of secondary alcohols, using lipases in combination with transition metal (ruthenium) complexes has been further developed to encompass a greater range of substrates and, significantly, can now be applied to the preparation of (S)configured alcohols. New catalysts for racemisation have been discovered, and of particular interest is the demonstration that this process can be scaled-up and applied to the commercial manufacture of specific target compounds. However, the corresponding DKR of amines is not yet a practical option as a result of the harsh conditions required to racemise chiral amines. Undoubtedly, the next 12 months will witness many new publications in this area. Deracemisation processes are Current Opinion in Chemical Biology 2004, 8:114–119
118 Biocatalysis and biotransformation
also starting to make an impact and now extend from the microbial deracemisation of secondary alcohols and achiral carboxylic acids to reactions involving oxidase/ reducing agent combinations for preparing single enantiomer amino acids and amines in high yield. Deracemisation is an inherently attractive option in view of the simplicity of the system (i.e. avoidance of protecting groups, lack of product inhibition) and it will be interesting to see how well these processes compete with alternative approaches based upon DKR. Finally, there is clearly considerable scope for identifying and evolving a wider range of racemase enzymes to complement the transition-metal-based racemisation catalysts. There is no doubt that the increased availability of microbial genome sequences, coupled with advances in high-throughput screening technologies and directed evolution [30], will provide not only new enzymes with the desired substrate range and selectivity, but also process compatible biocatalysts that are better able to operate under the conditions of the reaction. The latter aspect is particularly relevant to those reactions in which enzymes are combined with chemical reagents or transition metal catalysts as outlined above in this review.
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.
Pellisier H: Dynamic kinetic resolution. Tetrahedron 2003, 59:8291-8327.
2.
Schnell B, Faber K, Kroutil W: Enzymatic racemisation and its application to synthetic biotransformations. Adv Synth Catal 2003, 345:653-666. An excellent comprehensive review summarizing the current literature of racemase enzymes and their application in DKR reactions. 3.
Pamies O, Backvall J-E: Combination of enzymes and metal catalysts. A powerful approach in asymmetric catalysis. Chem Rev 2003, 103:3247-3261.
4.
Pamies O, Backvall J-E: Combined metal catalysis and biocatalysis for an efficient deracemization process. Curr Opin Biotechnol 2003, 14:407-413.
5.
Turner NJ: Controlling chirality. Curr Opin Biotechnol 2003, 14:401-406.
6.
Kim M-J, Ahn Y, Park J: Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis. Curr Opin Biotechnol 2002, 13:578-587.
7.
Faber K: Non-sequential processes for the transformation of a racemate into a single stereoisomeric product: proposal for stereochemical classification. Chemistry 2001, 7:5004-5010. A useful review clarifying the use of terminology to describe enzymecatalysed processes, including dynamic kinetic resolution and deracemisation. 8.
Paizs C, Ta¨ htinen P, Lundell K, Poppe L, Irimie F-D, Kanerva LT: Preparation of novel phenylfuran-based cyanohydrin esters: lipase-catalysed kinetic and dynamic resolution. Tetrahedron Asymmetry 2003, 14:1895-1904.
9.
Sharfuddin M, Narumi A, Iwai Y, Keiko M, Yamada S, Kakuchi T, Kaga H: Lipase-catalyzed dynamic resolution of hemiaminals. Tetrahedron Asymmetry 2003, 14:1581-1585.
10. Kim M-J, Chung YI, Choi YK, Lee HK, Kim D, Park J: (S)-Selective dynamic kinetic resolution of secondary alcohols by the combination of subtilisin and an Current Opinion in Chemical Biology 2004, 8:114–119
aminocyclopentadienylruthenium complex as the catalysts. J Am Chem Soc 2003, 125:11494-11495. Significant extension of the application of DKR of secondary alcohols using hydrolases in combination with transition metal based racemisation catalysts. The use of subtilisin allows access to (S)-configured alcohols for the first time. 11. Pa`mies O, Ba¨ ckvall J-E: An efficient route to chiral a- and bhydroxyalkanephosphonates. J Org Chem 2003, 68:4815-4818. 12. Verzijl GKM, De Vries JG, Broxterman QB: Process for the preparation of enantiomerically enriched esters and alcohols. PCT Int Appl 2001, WO 2001090396 A1 20011129. Demonstration that DKR of secondary alcohols can be carried out at large scale as a result of optimisation of the process with respect to substrate concentration, catalyst loading and recycle and other key parameters. 13. Broxterman QB, Verzijl GKM: Process for the synthesis of (R)-1(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol and esters thereof by dynamic kinetic resolution. PCT Int Appl 2003, WO 2003043575 A2 20030530. 14. Ito M, Osaku A, Kitahara S, Hirakawa M, Ikariya T: Rapid racemization of chiral non-racemic sec-alcohols catalyzed by [g5-C5(CH3)5]Ru complexes bearing tertiary phosphine-primary amine chelate ligands. Tetrahedron Lett 2003, 44:7521-7523. 15. Wuyts S, De Vos DE, Verpoort F, Depla D, De Gryse R, Jacobs PA: A heterogeneous Ru-hydroxyapatite catalyst for mild racemization of alcohols. J Catal 2003, 219:417-424. 16. Wuyts S, De Temmerman K, De Vos D, Jacobs PA: A zeoliteenzyme combination for biphasic dynamic kinetic resolution of benzylic alcohols. Chem Commun 2003, 2003:1928-1929. 17. Skupinska KA, McEachern EJ, Baird IR, Skerlj RT, Bridger GJ: Enzymatic resolution of bicyclic 1-heteroarylamines using Candida antarctica lipase B. J Org Chem 2003, 68:3546-3551. 18. Funke F, Liang S, Kramer A, Stuermer R, Hoehn A: Racemization of optically active amines. Eur Pat Appl 2002, EP1215197 A2 20020619. 19. Pa`mies O, E´ ll AH, Samec JSM, Hermanns N, Ba¨ ckvall JE: An efficient and mild-ruthenium-catalyzed racemization of amines: application to the synthesis of enantiomerically pure amines. Tetrahedron Lett 2002, 43:4699-4702. Reports the use of a ruthenium catalyst for racemisation of amines and, although in situ DKR is not possible at this stage, this paper suggests that further improvements in terms of lower temperatures for racemisation may soon be achievable. 20. E´ ll AH, Johnson JB, Ba¨ ckvall JE: Mechanism of rutheniumcatalyzed hydrogen transfer reactions. Evidence for a stepwise transfer of CH and NH hydrogens from an amine to a (cyclopentadienone)ruthenium complex. Chem Commun 2003, 2003:1652-1653. 21. May O, Verseck S, Bommarius A, Drauz K: Development of dynamic kinetic resolution processes for biocatalytic production of natural and non-natural L-amino acids. Org Proc Res Dev 2002, 6:452-457. Informative review detailing the development and subsequent commercialisation of two DKR processes that involve racemase enzymes. 22. Comasseto JV, Omori A´ T, Andrade LH, Porto ALM: Bioreduction of fluoroacetophenones by the fungi Aspergillus terreus and Rhizopus oryzae. Tetrahedron Asymmetry 2003, 14:711-715. 23. Demir AS, Hamamci H, Sesenoglu O, Neslihanoglu R, Asikoglu B, Capanoglu D: Fungal deracemization of benzoin. Tetrahedron Lett 2002, 43:6447-6449. 24. Chadha A, Baskar B: Biocatalytic deracemisation of a-hydroxy esters: high yield preparation of (S)-ethyl 2-hydroxy-4phenylbutanoate from the racemate. Tetrahedron Asymmetry 2002, 13:1461-1464. 25. Kato D, Mitsuda S, Ohta H: Microbial deracemization of a-substituted carboxylic acids. Org Lett 2002, 4:371-373. 26. Kato D, Mitsuda S, Ohta H: Microbial deracemization of a-substituted carboxylic acids: substrate specificity and mechanistic investigation. J Org Chem 2003, 68:7234-7242. Detailed and revealing study concerning the microbial deracemisation of a-substituted carboxylic acids. Evidence presented suggests that www.sciencedirect.com
Enzyme catalysed deracemisation and dynamic kinetic resolution reactions Turner 119
deracemisation occurs via enzyme-catalysed formation, epimerisation and subsequent hydrolysis of coenzyme A esters. 27. Alexeeva M, Carr R, Turner NJ: Directed evolution of enzymes: new biocatalysts for asymmetric synthesis. Org Biomol Chem 2003, 1:4133-4137. 28. Enright A, Alexandre F-R, Roff G, Fotheringham IG, Dawson MJ, Turner NJ: Stereoinversion of b- and c-substituted-a-amino acids using a chemoenzymatic oxidation-reduction procedure. Chem Commun 2003, 2003:2636-2637.
www.sciencedirect.com
29. Carr R, Alexeeva M, Enright A, Eve TSC, Dawson MJ, Turner NJ: Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantioselectivity. Angew Chem Int Ed Engl 2003, 42:4807-4810. Use of random mutagenesis coupled with high-throughput screening to identify a variant amine oxidase that has significantly broadened substrate specificity, while retaining high enantioselectivity, compared with the wild-type enzyme. 30. Turner NJ: Directed evolution of enzymes for applied biocatalysis. Trends Biotechnol 2003, 21:474-478.
Current Opinion in Chemical Biology 2004, 8:114–119