- Email: [email protected]
7.1 Introduction and General Concepts
7.1 Introduction and General Concepts NJ Turner, University of Manchester, Manchester, UK r 2012 Elsevier Ltd. All rights reserved.
7.1.1 7.1.2 7.1.3 7.1.4 References
Biocatalysis in Organic Synthesis The Current Biocatalysis Toolbox New Synthetic Transformations using Biocatalysts Future Trends
Glossary API Active Pharmaceutical Ingredient (API). An API is the substance of a pharmaceutical drug that is biologically active. Biocatalyst A collective term used to describe either an enzyme or whole cell used as a catalyst for a synthetic transformation. Chimeric enzyme Fusion proteins or chimeric enzymes are proteins created through the joining of two or more genes which originally coded for separate proteins. Deracemization Deracemization is a term used to describe a process in which a racemate is converted into a nonracemic product in 100% theoretical yield without intermediate separation of materials.
7.1.1
1 2 3 4 5
Directed evolution Enhancement of a characteristic of an enzyme (e.g., enantioselectivity, substrate specificity, and thermostability) by iterative rounds of random mutagenesis coupled with either screening or selection. Synthetic biology Synthetic biology is a new area of biological research that combines science and engineering. Synthetic biology involves the design and construction of new biological functions and systems not found in nature. Water activity (aW) Simply stated, it is a measure of the energy status of the water in a system. It is defined as the vapor pressure of a liquid divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.
Biocatalysis in Organic Synthesis
The origins of biocatalysis as a technology for producing chemical products can be traced back many centuries, largely as a result of the development of fermentation-based processes for the baking and brewing industries.1 However, the emergence of biocatalysis as a tool for synthetic organic chemistry is by comparison – a relatively recent development. In the late 1970s and early 1980s, research groups in both academe and industry began to explore the use of enzymes and whole cells for the production of chiral building blocks for organic synthesis.2 Much of this early work was focused on either hydrolytic enzymes (e.g., lipases, proteases, and acylases) or redox enzymes (e.g., alcohol dehydrogenases and lyophilized yeasts) simply because these biocatalysts were readily available and also relatively easy for synthetic organic chemists to use in the laboratory. As the range of enzymes began to increase, and hence their application became more widespread, landmark reviews and seminal textbooks3 appeared dealing with some of the general concepts and principles governing the application of biocatalysis in organic synthesis. During the 1990s and into the new millennium, the field of biocatalysis really accelerated apace, largely fuelled by the development of some key enabling technologies which transformed the way in which new enzymes could be discovered and optimized for practical applications. Firstly, high-throughput DNA sequencing enabled whole microbial genomes to be sequenced and annotated on a scale previously unimaginable, providing a panoply of potentially new enzymes for applications in biocatalysis.4 Secondly, coupled with advances in screening technology,5 these new gene sequences could be translated into biocatalysts that provided new starting points for synthetic transformations. Thirdly, the ability to engineer proteins and enzymes by applying technologies such as laboratory evolution of enzymes6 meant that one was no longer restricted to use wild-type enzymes found in Nature. Enzymes, and hence biocatalysts, could now be engineered to be ‘fit-for-purpose’ by improving characteristics such as substrate acceptance, enantioselectivity, and stability.7 This combination of enzyme discovery, reaction screening and protein engineering has proven to be a very powerful trinity of interlinked technologies and has rapidly accelerated to a rate at which new biocatalytic reactions can be initially identified and subsequently optimized for practical applications in industry. In addition to discovering new biocatalysts, substantial advances have also been made in our understanding of how to use enzymes and whole cells on large-scale, an issue which is clearly of importance to those in industry wishing to use biocatalysts.8 Much fundamental work has been carried out on reaction process engineering, enzyme and whole cell immobilization and also the regeneration of cofactors in order to develop cost-effective processes for manufacturing chemicals using enzymes and whole cells. Many examples now exist of biocatalytic processes that have been successfully transferred from the laboratory to plant scale.9 The use of biocatalysts, both as enzymes and also engineered whole cells, in the manufacture of chemicals offers major advantages in terms of enhanced reaction selectivity, cost of raw materials, lower energy costs, safety, and importantly sustainability.
Comprehensive Chirality, Volume 7
http://dx.doi.org/10.1016/B978-0-08-095167-6.00701-1
1
2
Introduction and General Concepts
Biocatalysis is increasingly applied in the development of second generation manufacturing processes, particularly in the pharmaceutical industry for API production. Although a current limitation is the time taken to develop biocatalytic processes, this situation will undoubtedly change in the future resulting in more applications of biocatalysis for first generation processes. This Volume of Comprehensive Chirality deals with the application of enzymes and whole cells, in the form of biocatalysts, for stereoselective synthesis. Each of the major classes of biocatalytic reactions are dealt with and grouped under the general themes: (1) hydrolysis/reverse hydrolysis, (2) reduction, (3) oxidation, and (4) C–X bond formation. The Volume concludes with a series of special topics, namely, Synthesis of Carbohydrates; Enzyme Promiscuity; Multi-Enzyme Reactions; Emerging Reactions; and Hybrid Enzymes.
7.1.2
The Current Biocatalysis Toolbox
Nature has evolved a wide range of different enzymes to catalyze all of the essential reactions in primary and secondary metabolism within a cell. An analysis of these enzymes from the perspective of a synthetic organic chemist reveals that certain reaction classes are very well represented and indeed may be grouped into four basic types of synthetic transformation as shown in Figure 1. Many of these biocatalysts have been used on a practical scale to access specific target molecules and today one is able to
Hydrolysis/reverse hydrolysis O
O lipase/esterase
OR1
R
R
O R
RCN
R
O amidase/protease
NHR1
R
+ R1NH2
OH
Reduction
OH/NH2
dehalogenase
Hal
R
O
nitrilase/ nitrile hydratase
+ R1OH
OH
OH
R
Oxidation OH
O R
R1
ketoreductase
R1
R amino acid dehydrogenase
O R
P450
OH
R
NH2
CO 2H
R
X
enoate reductase R
lyase
R1
R
OH/NH2
CO2H R
R
S
R
X
R
C−X Bond Formation O R
O
OH aldolase
+ OH
R
OPi
OH
O R
CN
+
O R
OH
lyase R
Figure 1 Synthetic transformations catalyzed by enzymes.
CN
NH2
transaminase R1
O
R
R1
OPi
R1 O
haloperoxidase
R
R1
O/NH
oxidase
R1
O S
Introduction and General Concepts
3
follow Organic Synthesis style protocols in order to be able to repeat literature preparations before developing new applications.10,11 Particularly prevalent in the biocatalysis toolbox are a wide range of enzymes that are able to catalyze different hydrolytic reactions, especially those involving acyl group transfer (e.g., hydrolysis of esters and amides). This group includes lipases, esterases, acylases, amidases, and proteases. Other types of hydrolytic enzyme include those that are involved in the hydrolysis of nitriles (nitrilases and nitrile hydratases), epoxides (epoxide hydrolases), and carbon–halogen bonds (dehalogenases). Hydrolytic enzymes are in general stable and can often be used above ambient temperature to increase reaction rate and also facilitate solubilization of the substrate. A number of hydrolytic enzymes (e.g., lipases, proteases) are produced commercially on a large scale for applications in the food processing and detergent industries and hence are commercially available at relatively low cost. Hydrolytic enzymes, particularly lipases, are also tolerant of high concentrations of organic solvent which has led to their use under conditions of low-water activity (aW). In this environment, these enzymes can be used to catalyze reverse-hydrolysis reactions since lowering the water activity of the system disfavors hydrolysis and consequently favors the synthesis of esters or amides. In addition to hydrolysis, the three other main types of transformation catalyzed by enzymes, which are of interest to synthetic chemists, are reduction, oxidation, and C–X bond formation. Enzyme-catalyzed reduction of ketones to enantiomerically pure chiral secondary alcohols has become a powerful and increasingly used method. Many ketoreductases, carbonyl reductases, and alcohol dehydrogenases are now commercially available with broad and overlapping substrate range. Methods for recycling both NADH and NADPH have been developed which are efficient and allow these reactions to be applied on a commercial scale. Attention is increasingly being directed toward other types of biocatalysts that mediate reduction processes, including those involved in reductive amination (amino acid dehydrogenases) and also the enantioselective reduction of activated CQC bonds (enoate reductases). Oxidation using biocatalysis is a very diverse field with many types of different transformations known including hydroxylation, sulfoxidation, epoxidation, Baeyer–Villiger oxidation, and the oxidation of alcohols and amines to aldehydes and imines, respectively. Biocatalytic oxidation reactions frequently require cofactors (e.g., NADH, NADPH) and other auxiliary proteins to assist with electron transfer and hence are often used as whole cell systems. Enzyme-catalyzed oxidation represents both a challenge and opportunity for biocatalysis and may provide an alternative to the use of metal catalyzed oxidants. The final group of enzymes include those that catalyze C–X bond formation including carbon–carbon (lyases, aldolases, and decarboxylases) and carbon–nitrogen (transaminases and ammonia lyases) bonds. Clearly many other enzymes are known, and exist, which catalyze reactions that fall outside of the five types of process described above. Most of the biocatalysts that find application today originate from primary metabolic pathways. If one looks into secondary metabolism, that is, those enzymes associated with the biosynthesis of structurally complex natural products, then one finds a Pandora’s box of potential biocatalysts that might one day be used for synthetic transformations.
7.1.3
New Synthetic Transformations using Biocatalysts
In order to continue to further expand the ‘biocatalysis toolbox,’ research groups are investigating a wider range of enzymes for their potential to be applied as biocatalysts. Figure 2 provides an overview of reaction types that during the next 5–10 years are likely to move from the academic laboratory to application in an industrial context. For example, flavin dependent halogenases are known to catalyze both the chlorination and bromination of electron-rich heteroaromatic substrates such as tryptophan and indole.12 These enzymes utilize Cl and Br as the halide source which are oxidized by the enzyme to Cl þ and Br þ , respectively. Halogenases with complementary regioselectivity have been reported allowing access to differentially halogenated products. A fluorinase from Streptomyces cattaleya has been described which catalyzes the conversion of S-adenosylmethionine to 5-fluororibose using fluoride ion.13 5-Fluororibose then serves as a building block for other fluorinated metabolites found in Nature. It now remains to be seen whether other related fluorinases will be discovered in order to broaden the range of fluorinated products which can be accessed. Racemases are known which catalyze the interconversion of enantiomers of both free a-amino acids and also N-acylated derivatives. Alanine racemases are pyridoxal phosphate (PLP) dependent enzymes and generally possess fairly narrow substrate specificity although a broad spectrum amino acid racemase from a Pseudomonas sp. has been reported.14 Glutamate and aspartate catalyze racemization of amino acids via a different mechanism employing a metal ion and two lysine residues to catalyze racemization via an intermediate stabilized carbanion. The enzyme N-acetyl amino acid racemase from Amycolatopsis sp. belongs to the enolase superfamily and accepts both N-acetyl and N-succinyl amino acids as substrates. Racemases can potentially be combined with other enantioselective biocatalysts to develop efficient dynamic kinetic resolution (DKR) processes.15 Ammonia lyases catalyze the reversible addition of ammonia to cinnamic acid and derivatives. For example, phenylalanine ammonia lyase (PAL) converts cinnamic acid to L-phenylalanine in high enantiomeric excess (ee).16 This transformation constitutes a ‘reverse’ Michael addition and is a consequence of the mechanism of the reaction which involves participation by an electrophilic active-site cofactor. Aminomutases are closely related enzymes which catalyze the interconversion of a- and b-amino acids, for example, a-Phe and b-Phe. A challenge in using these enzymes is to find conditions under which either a- or b-amino acids might be preferentially stabilized in solution. Although the reduction of imino acids to amino acids is well documented (amino acid dehydrogenase), the corresponding reduction of imines to amines using imine reductases is much less described. Recently a Japanese group has reported the conversion of 2-methylpyrroline to 2-methylpyrrolidine using various Streptomyces sp. These microorganisms were found to be effective imine-reducing strains with both high (R)- and (S)-selectivity yielding the
4
Introduction and General Concepts
Halogenase
Fluorinase Cl/Br
Met
halogenase N H
N H
Racemase
HO
R
CO2H
Imine reductase
Ad
O HO
OH
ammonia lyase
R
CO2H
OH
NO2
CH3
N H
R1
CO2H
Sulfatase OSO3−
OPO32−
kinase phosphatase
NHOH/NH2
nitroreductase
CH3
Phosphatase/Kinase OH
NH2 R
Nitroreductase
imine reductase
R
F fluorinase
NH2
racemase
CO2H
N
Ad
Ammonia lyase
NH2 R
O
R
R1
R
R1
OH
sulfatase R
R1
Figure 2 New synthetic transformations for biocatalysis.
product in good yield and high ee.17 Nitroreductases are NADPH dependent enzymes that are able to catalyze a wide range of redox processes including the reduction of nitroarenes to the corresponding hydroxylamines and anilines. Salmonella typhimurium (NRSal) has recently been investigated for the reduction of a,b-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics.18 Finally, there is interest in using both kinases and phosphatases to catalyze phosphorylation/dephosphosphorylation of chiral building blocks.19 Kinases are well known in the field of carbohydrate enzymology and can be used to regioselectively phosphorylate hexoses using ATP as the phosphoryl donor. Methods for recycling ADP back to ATP have also been developed. Finally, both retaining and inverting sulfatases have been reported which can be used to produce enantiomerically pure secondary alcohols from the corresponding racemic sulphate esters.20 The continued development of biocatalysis as a tool for organic synthesis will depend critically on the rate at which new reaction classes are discovered and developed for practical purposes.
7.1.4
Future Trends
The period from 2000–2011 has witnessed an enormous increase in the application of biocatalysis for the synthesis of a broad range of chemical-based products. There is a now growing trend amongst the chemical industry to develop sustainable manufacturing processes for chemical production including pharmaceutical building blocks, agrochemicals, nutraceuticals, polymers, personal health-care products, and more recently fuel molecules. Indeed, it is estimated that at least 20% of global chemicals (B2290 billion US$ today) could be produced by biotechnological means in 2020. Biocatalysis has the potential to be applied to an increasingly diverse range of products that are traditionally produced from raw materials derived from oil, coal, and gas.21 Currently there are a number of exciting new areas of research and development which are likely to further increase the impact of biocatalysis in the future. A major theme is the idea of telescoping two or more biocatalytic steps in order to carry out a series of sequential or concurrent reactions in a single flask. In some cases it is possible to fuse, at the genetic level, two or more genes resulting in chimeric enzymes possessing dual catalytic function. For example, an aldolase has been fused with a kinase22 and a Baeyer–Villiger monooxygenase has been fused with phosphite dehydrogenase to provide an enzyme that is able to recycle NADPH as well as oxidize ketones to lactones.23 New ways of combining bio- and chemo-catalysts are being sought in order to carry out multistep transformations, particularly in the areas of deracemization and dynamic kinetic resolution. An area of great promise is that of Synthetic Biology wherein multiple genes encoding for entire biosynthetic pathways could be coexpressed in a single cell enabling the conversion of cheap renewable starting materials to high-value products ranging from antimalarial drugs to jet fuel.24
Introduction and General Concepts
References 1. Roberts, S. M.; Turner, N. J.; Willetts, A. J.; Turner, M. K. Introduction to Biocatalysis Using Enzymes and Microorganisms; Cambridge University Press: UK, 1995, pp 1–31. 2. Porter, R.; Clark, S., Eds. Enzymes in Organic Synthesis, Ciba Foundation Symposium 111, Pitman: London, 1985. 3. Kieslich, K. Microbial Transformations of Non-Steroidal Cyclic Compounds; Wiley: Chichester, UK, 1976, 2 pp; Suckling, C. J., Ed. Enzyme Chemistry: Impact and Applications; Chapman and Hall: London, 1985; Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Pergamon: UK, 1994; Faber, K. Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin, 2011. 4. See: http://en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes 5. Reymond, J. L., Ed. Enzyme Assays; Wiley-VCH: Weinheim, 2006. 6. Brakmann, S., Johnsson, K., Eds. Directed Molecular Evolution of Proteins; Wiley-VCH: Weinheim, 2002. 7. Turner, N. J. Nat. Chem. Biol. 2009, 5, 567. 8. Tufvesson, P.; Lima-Ramos, J.; Nordblad, M.; Woodley, J. M. Org. Process Res. Dev. 2011, 15, 266. 9. Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, 2nd ed.; Wiley-VCH: Weinheim, 2006. 10. Grogan, G. Practical Biotransformations: A Beginner’s Guide; Wiley: UK, 2009. 11. Whittall, J., Sutton, P. W., Eds. Practical Methods for Biocatalysis and Biotransformations; Wiley: UK, 2010. 12. Lang, A.; Polnick, S.; Nicke, T.; et al. Angew. Chem. Int. Ed. 2011, 50, 2951. 13. Deng, H.; Cross, S. M.; McGlinchey, R. P.; Hamilton, J. T. G.; O’Hagan, D. Chem. Biol. 2008, 15, 1268. 14. Ikeda, H.; Yoshiyuki, Y.; Shin-ichi, H.; Yugasaki, M.; Soda, K. US Patent No. 7,666,653 82, 2010. 15. May, O.; Verseck, S.; Bommarius, A.; Drauz, K.-H. Org. Proc. Res. Dev. 2002, 6, 452. 16. Turner, N. J. Curr. Opin. Chem. Biol. 2011, 15, 234. 17. Mitsukura, K.; Suzuki, M.; Tada, K.; Yoshida, T.; Nagasawa, T. Org. Biomol. Chem. 2010, 8, 4533. 18. Yanto, Y.; Hall, M.; Bommarius, A. S. Org. Biomol. Chem. 2010, 8, 1826. 19. van Herk, T.; Hartog, A. F.; van der Burg, A. M.; Wever, R. Adv. Synth. Catal. 2005, 347, 1155. 20. Wallner, S. R.; Bauer, M.; Wuerdemann, C.; et al. Angew. Chem. Int. Ed. 2005, 44, 6381. 21. Meyer, H.-P.; Turner, N. J. Min. Rev. Org. Chem. 2009, 6, 300. 22. Iturrate, L.; Sanchez-Moreno, I.; Oroz-Guinea, I.; Perez-Gil, J.; Garcia-Junceda, E. Chem. Eur. J. 2010, 16, 4018. 23. Torres Pazmin˜o, D. E.; Snajdrova, R.; Baas, J. B.; et al. Angew. Chem. Int. Ed. 2008, 47, 2275. 24. Keasling, J. BioSocieties 2009, 4, 275.
5
7.1.1 7.1.2 7.1.3 7.1.4 References
Biocatalysis in Organic Synthesis The Current Biocatalysis Toolbox New Synthetic Transformations using Biocatalysts Future Trends
Glossary API Active Pharmaceutical Ingredient (API). An API is the substance of a pharmaceutical drug that is biologically active. Biocatalyst A collective term used to describe either an enzyme or whole cell used as a catalyst for a synthetic transformation. Chimeric enzyme Fusion proteins or chimeric enzymes are proteins created through the joining of two or more genes which originally coded for separate proteins. Deracemization Deracemization is a term used to describe a process in which a racemate is converted into a nonracemic product in 100% theoretical yield without intermediate separation of materials.
7.1.1
1 2 3 4 5
Directed evolution Enhancement of a characteristic of an enzyme (e.g., enantioselectivity, substrate specificity, and thermostability) by iterative rounds of random mutagenesis coupled with either screening or selection. Synthetic biology Synthetic biology is a new area of biological research that combines science and engineering. Synthetic biology involves the design and construction of new biological functions and systems not found in nature. Water activity (aW) Simply stated, it is a measure of the energy status of the water in a system. It is defined as the vapor pressure of a liquid divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.
Biocatalysis in Organic Synthesis
The origins of biocatalysis as a technology for producing chemical products can be traced back many centuries, largely as a result of the development of fermentation-based processes for the baking and brewing industries.1 However, the emergence of biocatalysis as a tool for synthetic organic chemistry is by comparison – a relatively recent development. In the late 1970s and early 1980s, research groups in both academe and industry began to explore the use of enzymes and whole cells for the production of chiral building blocks for organic synthesis.2 Much of this early work was focused on either hydrolytic enzymes (e.g., lipases, proteases, and acylases) or redox enzymes (e.g., alcohol dehydrogenases and lyophilized yeasts) simply because these biocatalysts were readily available and also relatively easy for synthetic organic chemists to use in the laboratory. As the range of enzymes began to increase, and hence their application became more widespread, landmark reviews and seminal textbooks3 appeared dealing with some of the general concepts and principles governing the application of biocatalysis in organic synthesis. During the 1990s and into the new millennium, the field of biocatalysis really accelerated apace, largely fuelled by the development of some key enabling technologies which transformed the way in which new enzymes could be discovered and optimized for practical applications. Firstly, high-throughput DNA sequencing enabled whole microbial genomes to be sequenced and annotated on a scale previously unimaginable, providing a panoply of potentially new enzymes for applications in biocatalysis.4 Secondly, coupled with advances in screening technology,5 these new gene sequences could be translated into biocatalysts that provided new starting points for synthetic transformations. Thirdly, the ability to engineer proteins and enzymes by applying technologies such as laboratory evolution of enzymes6 meant that one was no longer restricted to use wild-type enzymes found in Nature. Enzymes, and hence biocatalysts, could now be engineered to be ‘fit-for-purpose’ by improving characteristics such as substrate acceptance, enantioselectivity, and stability.7 This combination of enzyme discovery, reaction screening and protein engineering has proven to be a very powerful trinity of interlinked technologies and has rapidly accelerated to a rate at which new biocatalytic reactions can be initially identified and subsequently optimized for practical applications in industry. In addition to discovering new biocatalysts, substantial advances have also been made in our understanding of how to use enzymes and whole cells on large-scale, an issue which is clearly of importance to those in industry wishing to use biocatalysts.8 Much fundamental work has been carried out on reaction process engineering, enzyme and whole cell immobilization and also the regeneration of cofactors in order to develop cost-effective processes for manufacturing chemicals using enzymes and whole cells. Many examples now exist of biocatalytic processes that have been successfully transferred from the laboratory to plant scale.9 The use of biocatalysts, both as enzymes and also engineered whole cells, in the manufacture of chemicals offers major advantages in terms of enhanced reaction selectivity, cost of raw materials, lower energy costs, safety, and importantly sustainability.
Comprehensive Chirality, Volume 7
http://dx.doi.org/10.1016/B978-0-08-095167-6.00701-1
1
2
Introduction and General Concepts
Biocatalysis is increasingly applied in the development of second generation manufacturing processes, particularly in the pharmaceutical industry for API production. Although a current limitation is the time taken to develop biocatalytic processes, this situation will undoubtedly change in the future resulting in more applications of biocatalysis for first generation processes. This Volume of Comprehensive Chirality deals with the application of enzymes and whole cells, in the form of biocatalysts, for stereoselective synthesis. Each of the major classes of biocatalytic reactions are dealt with and grouped under the general themes: (1) hydrolysis/reverse hydrolysis, (2) reduction, (3) oxidation, and (4) C–X bond formation. The Volume concludes with a series of special topics, namely, Synthesis of Carbohydrates; Enzyme Promiscuity; Multi-Enzyme Reactions; Emerging Reactions; and Hybrid Enzymes.
7.1.2
The Current Biocatalysis Toolbox
Nature has evolved a wide range of different enzymes to catalyze all of the essential reactions in primary and secondary metabolism within a cell. An analysis of these enzymes from the perspective of a synthetic organic chemist reveals that certain reaction classes are very well represented and indeed may be grouped into four basic types of synthetic transformation as shown in Figure 1. Many of these biocatalysts have been used on a practical scale to access specific target molecules and today one is able to
Hydrolysis/reverse hydrolysis O
O lipase/esterase
OR1
R
R
O R
RCN
R
O amidase/protease
NHR1
R
+ R1NH2
OH
Reduction
OH/NH2
dehalogenase
Hal
R
O
nitrilase/ nitrile hydratase
+ R1OH
OH
OH
R
Oxidation OH
O R
R1
ketoreductase
R1
R amino acid dehydrogenase
O R
P450
OH
R
NH2
CO 2H
R
X
enoate reductase R
lyase
R1
R
OH/NH2
CO2H R
R
S
R
X
R
C−X Bond Formation O R
O
OH aldolase
+ OH
R
OPi
OH
O R
CN
+
O R
OH
lyase R
Figure 1 Synthetic transformations catalyzed by enzymes.
CN
NH2
transaminase R1
O
R
R1
OPi
R1 O
haloperoxidase
R
R1
O/NH
oxidase
R1
O S
Introduction and General Concepts
3
follow Organic Synthesis style protocols in order to be able to repeat literature preparations before developing new applications.10,11 Particularly prevalent in the biocatalysis toolbox are a wide range of enzymes that are able to catalyze different hydrolytic reactions, especially those involving acyl group transfer (e.g., hydrolysis of esters and amides). This group includes lipases, esterases, acylases, amidases, and proteases. Other types of hydrolytic enzyme include those that are involved in the hydrolysis of nitriles (nitrilases and nitrile hydratases), epoxides (epoxide hydrolases), and carbon–halogen bonds (dehalogenases). Hydrolytic enzymes are in general stable and can often be used above ambient temperature to increase reaction rate and also facilitate solubilization of the substrate. A number of hydrolytic enzymes (e.g., lipases, proteases) are produced commercially on a large scale for applications in the food processing and detergent industries and hence are commercially available at relatively low cost. Hydrolytic enzymes, particularly lipases, are also tolerant of high concentrations of organic solvent which has led to their use under conditions of low-water activity (aW). In this environment, these enzymes can be used to catalyze reverse-hydrolysis reactions since lowering the water activity of the system disfavors hydrolysis and consequently favors the synthesis of esters or amides. In addition to hydrolysis, the three other main types of transformation catalyzed by enzymes, which are of interest to synthetic chemists, are reduction, oxidation, and C–X bond formation. Enzyme-catalyzed reduction of ketones to enantiomerically pure chiral secondary alcohols has become a powerful and increasingly used method. Many ketoreductases, carbonyl reductases, and alcohol dehydrogenases are now commercially available with broad and overlapping substrate range. Methods for recycling both NADH and NADPH have been developed which are efficient and allow these reactions to be applied on a commercial scale. Attention is increasingly being directed toward other types of biocatalysts that mediate reduction processes, including those involved in reductive amination (amino acid dehydrogenases) and also the enantioselective reduction of activated CQC bonds (enoate reductases). Oxidation using biocatalysis is a very diverse field with many types of different transformations known including hydroxylation, sulfoxidation, epoxidation, Baeyer–Villiger oxidation, and the oxidation of alcohols and amines to aldehydes and imines, respectively. Biocatalytic oxidation reactions frequently require cofactors (e.g., NADH, NADPH) and other auxiliary proteins to assist with electron transfer and hence are often used as whole cell systems. Enzyme-catalyzed oxidation represents both a challenge and opportunity for biocatalysis and may provide an alternative to the use of metal catalyzed oxidants. The final group of enzymes include those that catalyze C–X bond formation including carbon–carbon (lyases, aldolases, and decarboxylases) and carbon–nitrogen (transaminases and ammonia lyases) bonds. Clearly many other enzymes are known, and exist, which catalyze reactions that fall outside of the five types of process described above. Most of the biocatalysts that find application today originate from primary metabolic pathways. If one looks into secondary metabolism, that is, those enzymes associated with the biosynthesis of structurally complex natural products, then one finds a Pandora’s box of potential biocatalysts that might one day be used for synthetic transformations.
7.1.3
New Synthetic Transformations using Biocatalysts
In order to continue to further expand the ‘biocatalysis toolbox,’ research groups are investigating a wider range of enzymes for their potential to be applied as biocatalysts. Figure 2 provides an overview of reaction types that during the next 5–10 years are likely to move from the academic laboratory to application in an industrial context. For example, flavin dependent halogenases are known to catalyze both the chlorination and bromination of electron-rich heteroaromatic substrates such as tryptophan and indole.12 These enzymes utilize Cl and Br as the halide source which are oxidized by the enzyme to Cl þ and Br þ , respectively. Halogenases with complementary regioselectivity have been reported allowing access to differentially halogenated products. A fluorinase from Streptomyces cattaleya has been described which catalyzes the conversion of S-adenosylmethionine to 5-fluororibose using fluoride ion.13 5-Fluororibose then serves as a building block for other fluorinated metabolites found in Nature. It now remains to be seen whether other related fluorinases will be discovered in order to broaden the range of fluorinated products which can be accessed. Racemases are known which catalyze the interconversion of enantiomers of both free a-amino acids and also N-acylated derivatives. Alanine racemases are pyridoxal phosphate (PLP) dependent enzymes and generally possess fairly narrow substrate specificity although a broad spectrum amino acid racemase from a Pseudomonas sp. has been reported.14 Glutamate and aspartate catalyze racemization of amino acids via a different mechanism employing a metal ion and two lysine residues to catalyze racemization via an intermediate stabilized carbanion. The enzyme N-acetyl amino acid racemase from Amycolatopsis sp. belongs to the enolase superfamily and accepts both N-acetyl and N-succinyl amino acids as substrates. Racemases can potentially be combined with other enantioselective biocatalysts to develop efficient dynamic kinetic resolution (DKR) processes.15 Ammonia lyases catalyze the reversible addition of ammonia to cinnamic acid and derivatives. For example, phenylalanine ammonia lyase (PAL) converts cinnamic acid to L-phenylalanine in high enantiomeric excess (ee).16 This transformation constitutes a ‘reverse’ Michael addition and is a consequence of the mechanism of the reaction which involves participation by an electrophilic active-site cofactor. Aminomutases are closely related enzymes which catalyze the interconversion of a- and b-amino acids, for example, a-Phe and b-Phe. A challenge in using these enzymes is to find conditions under which either a- or b-amino acids might be preferentially stabilized in solution. Although the reduction of imino acids to amino acids is well documented (amino acid dehydrogenase), the corresponding reduction of imines to amines using imine reductases is much less described. Recently a Japanese group has reported the conversion of 2-methylpyrroline to 2-methylpyrrolidine using various Streptomyces sp. These microorganisms were found to be effective imine-reducing strains with both high (R)- and (S)-selectivity yielding the
4
Introduction and General Concepts
Halogenase
Fluorinase Cl/Br
Met
halogenase N H
N H
Racemase
HO
R
CO2H
Imine reductase
Ad
O HO
OH
ammonia lyase
R
CO2H
OH
NO2
CH3
N H
R1
CO2H
Sulfatase OSO3−
OPO32−
kinase phosphatase
NHOH/NH2
nitroreductase
CH3
Phosphatase/Kinase OH
NH2 R
Nitroreductase
imine reductase
R
F fluorinase
NH2
racemase
CO2H
N
Ad
Ammonia lyase
NH2 R
O
R
R1
R
R1
OH
sulfatase R
R1
Figure 2 New synthetic transformations for biocatalysis.
product in good yield and high ee.17 Nitroreductases are NADPH dependent enzymes that are able to catalyze a wide range of redox processes including the reduction of nitroarenes to the corresponding hydroxylamines and anilines. Salmonella typhimurium (NRSal) has recently been investigated for the reduction of a,b-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics.18 Finally, there is interest in using both kinases and phosphatases to catalyze phosphorylation/dephosphosphorylation of chiral building blocks.19 Kinases are well known in the field of carbohydrate enzymology and can be used to regioselectively phosphorylate hexoses using ATP as the phosphoryl donor. Methods for recycling ADP back to ATP have also been developed. Finally, both retaining and inverting sulfatases have been reported which can be used to produce enantiomerically pure secondary alcohols from the corresponding racemic sulphate esters.20 The continued development of biocatalysis as a tool for organic synthesis will depend critically on the rate at which new reaction classes are discovered and developed for practical purposes.
7.1.4
Future Trends
The period from 2000–2011 has witnessed an enormous increase in the application of biocatalysis for the synthesis of a broad range of chemical-based products. There is a now growing trend amongst the chemical industry to develop sustainable manufacturing processes for chemical production including pharmaceutical building blocks, agrochemicals, nutraceuticals, polymers, personal health-care products, and more recently fuel molecules. Indeed, it is estimated that at least 20% of global chemicals (B2290 billion US$ today) could be produced by biotechnological means in 2020. Biocatalysis has the potential to be applied to an increasingly diverse range of products that are traditionally produced from raw materials derived from oil, coal, and gas.21 Currently there are a number of exciting new areas of research and development which are likely to further increase the impact of biocatalysis in the future. A major theme is the idea of telescoping two or more biocatalytic steps in order to carry out a series of sequential or concurrent reactions in a single flask. In some cases it is possible to fuse, at the genetic level, two or more genes resulting in chimeric enzymes possessing dual catalytic function. For example, an aldolase has been fused with a kinase22 and a Baeyer–Villiger monooxygenase has been fused with phosphite dehydrogenase to provide an enzyme that is able to recycle NADPH as well as oxidize ketones to lactones.23 New ways of combining bio- and chemo-catalysts are being sought in order to carry out multistep transformations, particularly in the areas of deracemization and dynamic kinetic resolution. An area of great promise is that of Synthetic Biology wherein multiple genes encoding for entire biosynthetic pathways could be coexpressed in a single cell enabling the conversion of cheap renewable starting materials to high-value products ranging from antimalarial drugs to jet fuel.24
Introduction and General Concepts
References 1. Roberts, S. M.; Turner, N. J.; Willetts, A. J.; Turner, M. K. Introduction to Biocatalysis Using Enzymes and Microorganisms; Cambridge University Press: UK, 1995, pp 1–31. 2. Porter, R.; Clark, S., Eds. Enzymes in Organic Synthesis, Ciba Foundation Symposium 111, Pitman: London, 1985. 3. Kieslich, K. Microbial Transformations of Non-Steroidal Cyclic Compounds; Wiley: Chichester, UK, 1976, 2 pp; Suckling, C. J., Ed. Enzyme Chemistry: Impact and Applications; Chapman and Hall: London, 1985; Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Pergamon: UK, 1994; Faber, K. Biotransformations in Organic Chemistry, 6th ed.; Springer: Berlin, 2011. 4. See: http://en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes 5. Reymond, J. L., Ed. Enzyme Assays; Wiley-VCH: Weinheim, 2006. 6. Brakmann, S., Johnsson, K., Eds. Directed Molecular Evolution of Proteins; Wiley-VCH: Weinheim, 2002. 7. Turner, N. J. Nat. Chem. Biol. 2009, 5, 567. 8. Tufvesson, P.; Lima-Ramos, J.; Nordblad, M.; Woodley, J. M. Org. Process Res. Dev. 2011, 15, 266. 9. Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, 2nd ed.; Wiley-VCH: Weinheim, 2006. 10. Grogan, G. Practical Biotransformations: A Beginner’s Guide; Wiley: UK, 2009. 11. Whittall, J., Sutton, P. W., Eds. Practical Methods for Biocatalysis and Biotransformations; Wiley: UK, 2010. 12. Lang, A.; Polnick, S.; Nicke, T.; et al. Angew. Chem. Int. Ed. 2011, 50, 2951. 13. Deng, H.; Cross, S. M.; McGlinchey, R. P.; Hamilton, J. T. G.; O’Hagan, D. Chem. Biol. 2008, 15, 1268. 14. Ikeda, H.; Yoshiyuki, Y.; Shin-ichi, H.; Yugasaki, M.; Soda, K. US Patent No. 7,666,653 82, 2010. 15. May, O.; Verseck, S.; Bommarius, A.; Drauz, K.-H. Org. Proc. Res. Dev. 2002, 6, 452. 16. Turner, N. J. Curr. Opin. Chem. Biol. 2011, 15, 234. 17. Mitsukura, K.; Suzuki, M.; Tada, K.; Yoshida, T.; Nagasawa, T. Org. Biomol. Chem. 2010, 8, 4533. 18. Yanto, Y.; Hall, M.; Bommarius, A. S. Org. Biomol. Chem. 2010, 8, 1826. 19. van Herk, T.; Hartog, A. F.; van der Burg, A. M.; Wever, R. Adv. Synth. Catal. 2005, 347, 1155. 20. Wallner, S. R.; Bauer, M.; Wuerdemann, C.; et al. Angew. Chem. Int. Ed. 2005, 44, 6381. 21. Meyer, H.-P.; Turner, N. J. Min. Rev. Org. Chem. 2009, 6, 300. 22. Iturrate, L.; Sanchez-Moreno, I.; Oroz-Guinea, I.; Perez-Gil, J.; Garcia-Junceda, E. Chem. Eur. J. 2010, 16, 4018. 23. Torres Pazmin˜o, D. E.; Snajdrova, R.; Baas, J. B.; et al. Angew. Chem. Int. Ed. 2008, 47, 2275. 24. Keasling, J. BioSocieties 2009, 4, 275.
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