Modular and scalable biocatalytic tools for practical safety, health and

Biocatalysis and Biotransformation, March
August 2007; 25(2
4): 178
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ORIGINAL ARTICLE

Modular and scalable biocatalytic tools for practical safety, health and environmental improvements in the production of speciality chemicals

ROLAND WOHLGEMUTH Sigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland

Abstract Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with microbubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.

Keywords: Biocatalytic asymmetric Baeyer-Villiger oxidation, biocatalytic asymmetric dihydroxylation, biocatalytic asymmetric condensation

Introduction Methods for the production of chemicals have grown tremendously with the transfer of new scientific discoveries, new practical tools and technology platforms into everyday laboratory use. Key success factors for the growth of both the chemical sciences as well as the chemical industries in the past have been the discovery of novel reactions, process and product innovation, and the availability of inexpensive raw materials. The parallel development of economic growth and globalization, the rapid spread of information technologies, and the increasing spatial and temporal density of working and living areas of a growing world population has led to a vital interest in environment, health and safety (EHS) issues. EHS advantages in production, packaging, transport, storage, application and waste handling are important success factors for speciality chemicals. Adequate conditions for safety, health and environment are prerequisites for the capabilities of working, living and economic growth. A growing world population, distributed over the areas for permanent living and working, requires increasing

use of natural resources limited by space and time. Although the parallel global growth of information technology has led to rapid global spread of news, and has the potential to improve safety, health and environment on a global scale, there is a large gap between effects on a global scale and local action. WHO director-general Lee Jong-Wook stated that ‘over the past 50 years, humans have changed natural ecosystems more rapidly and extensively than in any comparable period in human history’, and has been concerned with the environmental effects on human well-being (Jong-Wook 2005). The challenge for the 21st century is, therefore, to harmonize global economic growth, based on the use of non-renewable natural resources, with the environment, and to develop new product streams based on renewable resources. Early implementation of the best available technology completely eliminating safety and health hazards, incorporating environmental issues, such as the volume of natural resources wasted, harmful substances and the ratio of kilogram total waste to kilogram product (E-factor; Sheldon 2000), will improve delivery time and avoid wasting effort on routes with no long-term

Correspondence: R. Wohlgemuth, Research Specialties, Sigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland. E-mail: [email protected] ISSN 1024-2422 print/ISSN 1029-2446 online # 2007 Informa UK Ltd DOI: 10.1080/10242420701379759

Modular and scalable biocatalytic tools for practical safety future (Butters et al. 2006). The concept of sustainable development has received global attention; a colorful growth of disciplines and many local, sectorial and institutional initiatives have been creating increased awareness. Catalytic process technologies minimize the amount of waste per kilogram of product and improve EHS issues, instead of classic organic syntheses, which require auxiliary reagents in stoichiometric amounts. This minimization of waste has to be balanced with catalyst toxicity and sustainability. Since biocatalysts are easily degradable and non-toxic, the procedures using biocatalytic tools have not only found their way into routine production, but also have the potential to solve EHS issues in the production of new speciality chemicals. Oxidation reactions have been an area where practical EHS issues, like the replacement of oxidants, which are critical to exothermic events, fire and explosion risks, by environmentally compatible and safe reagents, are of prime importance. The development of selective and orthogonal oxidation methodology without the need to introduce additional protection
deprotection loops for other labile functional groups, and with formally direct insertion of oxygen atoms at specific positions in molecules, in a catalytic and asymmetric way, continues to be a synthetic topic. Biocatalysis and biotransformations have made tremendous progress through work on the molecular nature of enzyme action. Haldane (1965) mentioned the importance of enzymes for the discovery of tryptophan, the preparation of maltose, and the classification of complex glucosides. Understanding of the chemistry of living systems and the molecular transformations that occur in them requires knowledge of both the nature of enzymatic catalysis as well as the scope of the biocatalytic reactions (Walsh 1979). Looking at the chemistry
biology interface from both sides, i.e. the use of enzymes in organic chemistry (Drauz & Waldmann 2002; Bommarius & Riebel 2004; Faber 2004; Liese et al. 2006) and the organic chemistry of enzyme-catalyzed reactions (Silverman 2000), has been very fruitful for the advancement of our knowledge of biocatalytic synthesis and degradation of molecules. Further development of this interaction and the application of biocatalysis is the key to sustainable global economic growth with an environmental bonus (selective removal of toxic educts or side-products from the reaction mixture, waste reduction by selective reactions and by avoiding complicated protection
deprotection schemes with side-reactions under non-orthogonal reaction conditions, safety, health and energy improvements by utilizing

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the privileged chiral catalysts from nature and avoiding highly reactive and non-selective auxiliary reagents and catalysts, translation of insight at the microenvironmental level of the biocatalyst into benefits at the macroenvironmental level). Toxic byproducts and waste can thereby be converted into environmentally compatible materials to achieve industrially viable solutions to the challenges mentioned above (Alcalde et al. 2006). The mechanisms biocatalysts use to carry out a desired transformation on only one molecular structure out of a mixture of different compounds and only on one particular atom or functional group within the molecule having several sites has become accessible to time-dependent structural experiments (Schlichting et al. 2000). Beside more service-oriented applications, like decomposition of excess educts and side products or the introduction and removal of protecting groups, modular and scalable asymmetric transformation types are in use for over 100 reactions involving Baeyer-Villiger oxidations, mono- and dihydroxylations, epoxide ring-opening reactions, ester synthesis, cyanohydrin synthesis, ketone reductions, peptide synthesis, carbon
carbon bond formation, nitrile hydrolysis and glycosyl-transfer reactions (Wohlgemuth 2005). The type of products which are produced by biocatalytic processes in target-oriented syntheses are chiral compounds, isomer-free chemicals, isotope-labeled reagents, high-purity reagents, natural compounds, metabolites and specialty biochemicals. Despite their power as a synthetic tool in research, oxidation reactions comprise a small fraction of preparative reactions on an industrial scale, which has been attributed, among other factors, to undesirable waste products and the safety of the processes (Caron et al. 2006). The stabilization of reactants in flammable organic solvents removes oxidants from the system, and is a standard safety measure to prevent fires and explosions. This precaution is absent in oxidation reactions, where oxidants are required and, therefore, all elements necessary for combustion are present in the reactor. The reactive nature of compounds, instabilities of some products and narrow error tolerance, limit the use of oxidation processes despite appropriate safety testing and precautions (Caron et al. 2006). Since about half of the active pharmaceutical ingredients launched over the last 12 years contain at least one element of chirality (Farina et al. 2006), asymmetric oxidation reactions have attracted a lot of interest from both the chemical and biological perspective due to the tremendous challenges in the improvement of selectivity and atom economy, safety, health and environmental aspects of these

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oxidations on a large scale (Katsuki 2001). The field of asymmetric catalysis, in general, has been growing at a phenomenal rate (Jacobsen et al. 1999), and the development of catalytic asymmetric oxidations in particular continues to be of prime interest (Sharpless 2002). The catalytic asymmetric BaeyerVilliger oxidation and catalytic asymmetric dihydroxylation have been chosen as target oxidation reactions for practical safety, health and environment improvements.

anticancer agent mithramycin DK (Gibson et al. 2005). The key process bottlenecks in a preparative biocatalytic Baeyer-Villiger oxidation have been identified as substrate and product inhibition, cell toxicity and substrate solubility (Alphand et al. 2003). A highly efficient methodology of in-situ substrate feeding and in-situ product removal (SFPR process concept) resulted in useful isolated yields of 58
79% for the asymmetric Baeyer-Villiger oxidation of bicyclic ketones (Hilker et al. 2004a) by using glycerol feed control for cofactor regeneration and sintered-metal spargers for microbubble aeration. The first kilogram-scale operation of a biocatalytic Baeyer-Villiger oxidation has been performed on racemic bicyclo[3.2.0]heptenone, which was oxidized in a regiodivergent manner to the corresponding lactones by the SFPR process design. This methodology circumvented the main bottlenecks, such as severe limitation by substrate and/or product solubility, inhibition or cell viability and simplified downstream processing (Hilker et al. 2004b). Moreover, the adsorbent resin is reusable, which considerably lowers the cost of the entire process. Using these experimental conditions, we were able to run a 900 g scale experiment, using a 50 L reactor at a substrate concentration of about 25 g L1 with a volumetric productivity as high as 1.02 g lactones L1 h1 (136 U L 1) and a non-optimized isolated product yield of nearly 60% was obtained (Hilker et al. 2005). This represents the first kilogram-scale BaeyerVilliger biooxidation ever described and opens new perspectives. The rate of biocatalytic Baeyer-Villiger oxidation decreases with cell density in the concentration range of 6
36 g L1, thereby limiting space
time yield significantly. Oxygen supply limitations have been overcome by a new sintered-metal sparger system. A semi-quantitative process model based on

Biocatalytic asymmetric Baeyer-Villiger oxidation tools The classical Baeyer-Villiger oxidation has been of considerable synthetic interest for more than a century in organic chemistry, due to its versatility and to the well-established retention of stereochemistry at the migrating centre (Renz & Meunier 1999). This important reaction of organic chemistry is performed with oxidants that are often intrinsically unstable, toxic or lack functional group selectivity. Shock-sensitive and sacrificial oxidants, like peracids, used in stoichiometric amounts, accumulate considerable amounts of reduced reactant waste. Replacement by safe and environmentally compatible oxidants, like molecular oxygen, is clearly an important and desirable step towards industrially viable process improvement (ten Brink et al. 2004). The challenge of moving from the non-enantioselective and stoichiometric Baeyer-Villiger oxidation to a catalytic asymmetric version has best been achieved by the use of Baeyer-Villiger mono-oxygenases (Figure 1), utilizing molecular oxygen as oxidant to introduce one oxygen atom into the substrate and yielding water as a by-product (Taschner 1988; Mihovilovic et al. 2004). The remarkable selectivity of Baeyer-Villiger mono-oxygenases has been demonstrated with MtmOIV, the key enzyme to convert inactive pre-mithramycin B to the active Asymmetric Synthesis O

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Figure 1. Biocatalytic asymmetric Baeyer-Villiger oxidation types.

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Modular and scalable biocatalytic tools for practical safety the fact that cells prefer endogenous respiration over biocatalytic oxidation helps to identify optimal operating points for this biocatalytic Baeyer-Villiger process (Hilker et al. 2006). Biocatalytic asymmetric dihydroxylation tools The osmium-catalyzed asymmetric dihydroxylation (AD) of carbon
carbon double bonds to vicinal diols, which are important building blocks in enantioselective synthesis, is one of the most efficient catalytic asymmetric transformations to chiral vicinal diols, and reliable, versatile and convenient to use (Kolb et al. 1994). The high cost and toxicity of the metal
ligand system are, however, as in many catalytic asymmetric transformations, bottlenecks for large-scale industrial applications of the AD reaction (Lu et al. 2000). Various approaches have, therefore, focused in recent years on sustainable protocols of the AD reaction, like recycling methods for osmium tetroxide (Ishida et al. 2005; Lee et al. 2006) and the use of more environmentally benign terminal oxidants, like hydrogen peroxide (Jonsson et al. 2003) and molecular oxygen (Do¨bler et al. 2000). Aromatic ring dioxygenases catalyze the addition of molecular oxygen to the aromatic nucleus to form arene cis -dihydrodiols with high regio- and enantioselectivity, which makes these biocatalysts useful for the production of a broad range of chiral synthons (Boyd et al. 2006). As these enzymes are involved in the biodegradation of many chemicals in the environment, it has been interesting to explore what range of functional groups would still be tolerated in the selective asymmetric dihydroxylation of a variety of aromatic nitriles by chlorobenzene-dioxygenase. A range of new 1,2-regiospecific cis -dihydroxylations of aromatic nitriles has thereby been discovered (Figure 2), and the volumetric productivity of the cis-dihydroxylation processes could be increased significantly by use of an efficient and stable recombinant expression system for the biocatalyst (Yildirim et al. 2005). The mild hydrolysis of the corresponding dihydrodiol-nitriles to the dihydroN

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Figure 2. Biocatalytic asymmetric dihydroxylations.

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diol-carboxylic acids has been achieved by the use of nitrilases (Yildirim et al. 2006). Biocatalytic asymmetric condensation tools Our environment contains a large structural variety of carbon-based natural products of prebiotic origin or from micro-organisms, plants and animals reflecting the complex biological functions in the organism and in their interaction with the environment (Barton & Nakanishi 1999). Mankind’s curiosity and practical needs have been at the origin of many natural products whose manufacture has led to major industries. The chemistry and biosynthesis of natural products have made enormous progress with new analytical and synthetic tools, and this has led, in combination with the molecularization of biology, to a renaissance of natural product chemistry. Among the biocatalytic condensation reactions, aldol condensations and ketol transfer reactions have been useful for the synthesis of enantiomerically pure polyhydroxylated compounds (Fessner & Helaine 2001; Drauz & Waldmann 2002), an area which has been of continued interest in organic chemistry for new directions in stereocontrolled synthesis (Mahrwald 2004) since the impressive synthesis of all eight L-hexoses using a reiterative two-carbon extension cycle consisting of four key transformations (Ko et al. 1990). This concept of two-carbon extension cycles is also utilized by acetaldehyde- and pyruvate-dependent aldolases and transketolases in an efficient and environmentally sustainable one-step biocatalytic condensation, which has been observed for the case of the type I aldolase D-2-deoxyribose-5-phosphate aldolase (DERA, E.C. 4.1.2.4) to occur via a covalent Schiff-base intermediate (Heine et al. 2001). The aldolase enzymes and catalytic antibodies are of considerable synthetic utility because of their broad substrate specificity, their large-scale availability and their excellent stereoselectivity under environmentally benign conditions (Machajewski et al. 2000). Dihydroxyacetonephosphate(DHAP)-dependent aldolases have gained much attention for threecarbon extensions with the large-scale availability of DHAP and recombinant aldolase enzymes. These DHAP-dependent aldolase-catalyzed reactions are an important cornerstone of a flexible, reliable and powerful strategy for the rapid and facile synthesis of carbohydrate targets from simple achiral precursors with complete stereocontrol (Henderson et al. 1994). Catalytic and asymmetric condensation reactions forming new C-C or C-hetero-atom bonds are a continuing challenge at the forefront of synthesis. The invention of novel catalysts,

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asymmetric reactions and methodology for the total synthesis of target molecules creates tools of general interest. No matter whether the asymmetric condensation reactions originate from metal-catalyzed (Negishi et al. 2004), organocatalyzed (Enders et al. 2006) or biocatalytic reactions (Menzella et al. 2005), the key question in a total synthesis is what sequence of reactions will finally yield the desired product-in-the-bottle. Ketol-transfer reactions with b-hydroxypyruvate as ketol donor have been very useful, because the subsequent decarboxylation after condensation makes the overall reaction irreversible. The transketolase reaction has been chosen as a model to screen for process conditions in the multi-step synthesis of D-xylulose-5-phosphate (Shaeri et al. 2006). Generalized process development aimed at robust and scalable condensation reactions, including downstream processing, up to the pure product-inthe-bottle are at the heart of biocatalytic condensation reactions. The polyketides are a class of natural products which require challenging chemical syntheses and are, thus, produced in large-scale by biocatalytic routes, involving synthesis from small carbon precursors through the biocatalytic modular polyketide synthases (Tse & Khosla 2005). Triketide lactone scaffolds have been successfully obtained by combining the modules which encode the structures of two-carbon units demonstrating an assembly path of novel multimodule enzymes to produce complex polyketides (Menzella et al. 2005). Macrotetrolide biosynthesis has been shown to involve a novel polyketide synthase (PKS) that lacks an acyl carrier protein, and these macrotetrolide PKS have been proposed to act directly on CoA esters of the carboxylic acid precursors for the biosynthesis of a pair of enantiomeric polyketide intermediates (Kwon et al. 2001). Macrotetrolides are cyclic polyethers, formally composed of four molecules of enantiomeric nonactic acid or its homologues in a chirally alternating ()( )()( )
cyclocondensation (Figure 3). Their discovery started with nonactin (Corbaz et al. 1955), and they exhibit antimicrobial, antitumor, immunosuppressive and chelating activities. The preparation of these macrotetrolides with 16 chiral centers is produced by biocatalytic condensation reactions (Wohlgemuth 2004), under conditions which have been optimized in down-scaled kinetic experiments with MS-analysis. As promising scaffolds for the development of novel drugs with improved or altered activities are often structurally complex, the use of different building blocks from the natural ones, e.g. in non-ribosomally assembled peptides and enzymatic

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Figure 3. Macrotetrolides.

cyclization reactions, are of interest in the synthesis of therapeutically useful macrocyclic peptides (Gru¨newald & Marahiel 2006). The efficient and stereocontrolled one-step enzymatic reaction of squalene or oxidosqualene to tetra- or pentacyclic triterpenes has been of interest for half a century, and recombinant cyclases have enabled mechanistic insights of these elegant biocatalysts (Wendt et al. 2000). Outlook The development of catalytic asymmetric transformations towards sustainable chemistry continues to be of major interest for metal-catalyzed, organocatalyzed and biocatalyzed reaction methodologies in organic chemistry. In order to lead to practical

Modular and scalable biocatalytic tools for practical safety improvements in safety, health and the macroenvironment, it is important to establish criteria for selecting the best reaction methodology and integrate the economic dimensions. As nature has a rich and changing diversity of biological species, the gene pool for enzymes is a huge and unrivalled resource for the discovery of novel functions. As the technologies for the discovery of structure and function differ, the information on gene sequences and genome data, and detailed description of biochemical transformations and pathways is moving towards a multidimensional genome annotation (Reed et al. 2006). The search for novel biocatalysts (Burton et al. 2002) is at the start of a novel biocatalytic process and powerful approaches make use of microbial biodiversity through extensive and persistent screening (Ogawa & Shimizu 2002), by environmental nucleic acid extraction (Cowan et al. 2005; Ferrer et al. 2005; De Lorenzo 2005). Combinatorial and computational methods promise solutions to more complex biocatalyst design challenges including biosynthetic pathways (Arnold 2001). Further development of the reaction and its downstream processing may require improved enzyme properties (Arnold & Georgiou 2003), and strategies for finding the best mutations have been analyzed (Morley & Kazlauskas 2005). One of the central issues in the discovery and development of novel enzyme reactions is the large effort involved. Part of this effort is due to the limitations of classical reactor dimensions, and it is, therefore, of interest to miniaturize chemical and biological processes by microsystem technology. The reaction and assay technology has been miniaturized for a directed evolution experiment on epoxide hydrolases with isolated enzymes, cell lysates and whole-cells (Belder et al. 2006). Another part of the effort is due to the large number of samples, and, here, novel screening and selection methodologies are of much interest (Bornscheuer 2005; Mohn et al. 2006; van Sint Fiet et al. 2006). In summary, there is a rich harvest to be gained by nourishing further development of biocatalytic processes, tools and methodologies for key catalytic asymmetric transformations because solutions to improving atom economy in the microenvironment of biological cells will translate into enormous benefits on the macroenvironmental level. It is, however, also necessary to aim for robust and scalable biocatalytic processes from educt to final product, even at the research level (Wohlgemuth 2005). Sustainability in synthesis from ingredient to final product has to be incorporated at the research level by the concerted efforts of different disciplines, and the timescale from discovery of a novel enzyme

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reaction to large-scale practical application has to be shortened in a global effort. With clear and transparent definition of the interfaces between different scientific and technological areas, and a clear vision and implementation strategy, biocatalytic processes will change the way that chemicals are manufactured in the 21st century and provide a key to the industrial use of bio-technology. Acknowledgements I would like to thank all coworkers and members of the EU-projects on Baeyer-Villiger Oxidation, the Swiss projects on Asymmetric Dihydroxylation and Nitrilases, for their dedicated work and excellent cooperation, and the following organizations for their support: Swiss State Secretariat for Education and Research (SER), the program SPP-Biotech of the Swiss National Science Foundation, Commission on Technology and Innovation (KTI), and the EU Research Directorate General. References Alcalde M, Ferrer M, Plou FJ, Ballesteros A. 2006. Environmental biocatalysis: From remediation with enzymes to novel green processes. Trends Biotechnol 24:281
287. Alphand V, Carrea G, Furstoss R, Woodley JM, Wohlgemuth R. 2003. Towards large-scale synthetic applications of BaeyerVilliger monooxygenases. Trends Biotechnol 21:318
323. Arnold FH. 2001. Combinatorial and computational challenges for biocatalyst design. Nature 409:253
257. Arnold FH, Georgiou G. 2003. Directed enzyme evolution: Screening and selection methods. Totowa: Humana Press. Barton D, Nakanishi K. (editors-in-chief) 1999. Comprehensive natural products chemistry. Vols. 1
9. Amsterdam: Elsevier. Belder D, Ludwig M, Wang L-W, Reetz MT. 2006. Enantioselective catalysis and analysis on a chip. Angew Chem Intl Ed 45:2463
2466. Bommarius AS, Riebel BR. 2004. Biocatalysis. 1st ed. Weinheim: Wiley-VCH. Bornscheuer UT. 2005. Trends and challenges in enzyme technology. Adv Biochem Eng/Biotechnol 100:181
203. Boyd DR, Bugg TDH. 2006. Arene cis -dihydrodiol formation: From biology to application. Org Biomol Chem 4:181
192. Burton SG, Cowan DA, Woodley JM. 2002. The search for the ideal biocatalyst. Nature Biotechnol 20:37
45. Butters M, Catterick D, Craig A, Curzons A, Dale D, Gillmore A, Green SP, Marziano I, Sherlock J-P, White W. 2006. Critical assessment of pharmaceutical processes
a rationale for changing the synthetic route. Chem Rev 106:3002
3027. Caron S, Dugger RW, Ruggeri SG, Ragan JA, Brown Ripin DH. 2006. Large-scale oxidations in the pharmaceutical industry. Chem Rev 106:2943
2989. Corbaz R, Ettlinger L, Ga¨umann E, Keller-Schierlein W, Kradolfer F, Neipp L, Prelog V, Za¨hner H. 1955. Stoffwechselprodukte von Actinomyceten. 3. Mitteilung. Nonactin. Helv Chim Acta 38:1445
1448. Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P. 2005. Metagenomic gene discovery: Past, present and future. Trends Biotechnol 23:321
332. de Lorenzo V. 2005. Problems with metagenomic screening. Nature Biotechnol 23:1045.

184

R. Wohlgemuth

Do¨bler C, Mehltretter GM, Sundermeier U, Beller MJ. 2000. Osmium-catalyzed dihydroxylation of olefins using dioxygen or air as the terminal oxidant. J Am Chem Soc 122:10289
10297. Drauz K, Waldmann H. (editors) 2002. Enzyme catalysis in organic synthesis: A comprehensive handbook, second, completely revised and enlarged edition. Vol. I
III. Weinheim: Wiley-VCH. Enders D, Hu¨ttl MRM, Grondal C, Raabe G. 2006. Control of four stereocenters in a triple cascade organocatalytic reaction. Nature 441:861
863. Faber K. 2004. Biotransformations in organic chemistry. 5th revised and corrected edition. Berlin: Springer. Farina V, Reeves JT, Senanayake CH, Song JJ. 2006. Asymmetric synthesis of active pharmaceutical intermediates. Chem Rev 106:2734
2793. Ferrer M, Martinez-Abarca F, Golyshin PN. 2005. Mining genomes and ‘meta-genomes’ for novel catalysts. Curr Opin Biotechnol 16:588
593. Fessner WD, Helaine V. 2001. Biocatalytic synthesis of hydroxylated natural products using aldolases and related enzymes. Curr Opin Biotechnol 12:574
586. Gibson M, Nur-e-alam M, Lipata F, Oliveira MA, Rohr J. 2005. Characterization of kinetics and products of the Baeyer-Villiger oxygenase MtmOIV, the key enzyme of the biosynthetic pathway toward the natural product anticancer drug mithramycin from Streptomyces argillaceus . J Am Chem Soc 127:17594
17595. Haldane JBS. 1965. Enzymes. 1st MIT Press paperback edition. Cambridge: MIT Press. Gru¨newald J, Marahiel MA. 2006. Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides. Microb Mol Biol Rev 70:121
146. Heine A, DeSantis G, Luz JG, Mitchell M, Wong CH, Wilson IA. 2001. Observation of covalent intermediates in an enzyme mechanism at atomic resolution. Science 294:369
374. Henderson I, Sharpless KB, Wong C-H. 1994. Synthesis of carbohydrates via tandem use of the osmium-catalyzed asymmetric dihydroxylation and enzyme-catalyzed aldol addition reactions. J Am Chem Soc 116:558
561. Hilker I, Alphand V, Wohlgemuth R, Furstoss R. 2004a. Microbial transformations 56. Preparative scale asymmetric BaeyerVilliger oxidation using a highly productive ‘two-in-one’ in situ SFPR concept. Adv Synth Cat 346:203
214. Hilker I, Gutierrez MC, Alphand V, Wohlgemuth R, Furstoss R. 2004b. Microbiological transformations 57. Facile and efficient resin-based in situ SFPR preparative-scale synthesis of an enantiopure unexpected lactone regio-isomer via a BaeyerVilliger oxidation process. Org Lett 6:1955
1958. Hilker I, Wohlgemuth R, Alphand V, Furstoss R. 2005. Microbial transformations 59: First kilogram scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity using a resin-based in situ SFPR strategy. Biotechnol Bioeng 92:702
710. Hilker I, Baldwin C, Alphand V, Furstoss R, Woodley JM, Wohlgemuth R. 2006. On the influence of oxygen and cell concentration in an SFPR whole cell bio-catalytic BaeyerVilliger oxidation process. Biotechnol Bioeng 93:1138
1144. Ishida T, Akiyama R, Kobayashi S. 2005. A novel microencapsulated osmium catalyst using cross-linked polystyrene as an efficient catalyst for asymmetric dihydroxylation of olefins in water. Adv Synth Catal 347:1189
1192. Jacobsen EN, Pfaltz A, Yamamoto H. (editors) 1999. Comprehensive asymmetric catalysis. Berlin: Springer. Jong-Wook L. 2005 Geneva and Bangkok. Available from: http:// www.who.int/WHO-media-release.pdf

Jonsson SY, Adolfsson H, Ba¨ckvall J-E. 2003. MTO and OsO4-an efficient catalytic couple for mild H2O2-based asymmetric dihydroxylation of olefins. Chem Eur J 9:2783
2788. Katsuki T. (editor) 2001. Asymmetric oxidation reactions. Oxford University Press. Ko SY, Lee AWM, Masamune S, Reed LA, Sharpless KB, Walker FJ. 1990. Total synthesis of the L-hexoses. Tetrahedron 46:245
264. Kolb HC, Van Nieuwenhenhze MS, Sharpless KB. 1994. Catalytic asymmetric dihydroxylation. Chem Rev 94:2483
2547. Kwon H-J, Smith WC, Xiang L, Shen B. 2001. Cloning and expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J Am Chem Soc 123:3385
3386. Lee D, Lee H, Kim S, Yeom CE, Moon Kim B. 2006. A novel chemo-entrapment approach for supportless recycling of a catalyst: Catalytic asymmetric dihydroxylation. Adv Synth Cat 348:1021
1024. Liese A, Seelbach K, Wandrey C. (editors) 2006. Industrial biotransformations. 2nd completely revised and extended edition. Weinheim: Wiley-VCH. Lu X, Xu Z, Yang G. 2000. Process development of the Sharpless catalytic asymmetric dihydroxylation reaction to prepare methyl (2R ,3S )-2,3-dihydroxy-3-phenylpropionate. Org Process Res Dev 4:575
576. Machajewski TD, Wong C-H. 2000. The catalytic asymmetric aldol reaction. Angew Chem Intl Ed 39:1352
1375. Mahrwald R. (editor) 2004. Modern aldol reactions. Weinheim: Wiley-VCH. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA, Santi DV. 2005. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nature Biotechnol 23:1171
1176. Mihovilovic MD, Rudroff F, Gro¨tzl B. 2004. Enantioselective Baeyer-Villiger oxidations. Curr Org Chem 8:1057
1069. Mohn WW, Garmedia J, Galvao TC, de Lorenzo V. 2006. Surveying biotransformations with a` la carte genetic traps: Translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ -based phenotypes. Env Microbiol 8:546
555. Morley KL, Kazlauskas RJ. 2005. Improving enzyme properties: When are closer mutations better? Trends Biotechnol 23:231
237. Negishi E, Tan Z, Liang B, Novak T. 2004. An efficient and general route to reduced polypropionates via Zr-catalyzed asymmetric C-C bond formation. PNAS 101:5782
5787. Ogawa J, Shimizu S. 2002. Industrial microbial enzymes: Their discovery by screening and use in large-scale production of useful chemicals in Japan. Curr Opin Biotechnol 13:1
9. Reed JL, Famili I, Thiele I, Palsson BO. 2006. Towards multidimensional genome annotation. Nature Rev Genet 7:130
141. Renz M, Meunier B. 1999. 100 years of Baeyer-Villiger oxidation of ketones. Eur J Org Chem 4:737
750. Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet RM, Ringe D, Petsko GA, Sligar SG. 2000. The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287:1615
1622. Shaeri J, Wohlgemuth R, Woodley JM. 2006. Semiquantitative process screening for the biocatalytic synthesis of D-xylulose5-phosphate. Org Proc Res Dev 10:605
610. Sharpless KB. 2002. Searching for new reactivity. Angew Chem Intl Ed 41:2024
2032.

Modular and scalable biocatalytic tools for practical safety Sheldon RA. 2000. Atom efficiency and catalysis in organic synthesis. Pure Appl Chem 72:1233
1246. Silverman RB. 2000. The organic chemistry of enzyme-catalyzed reactions. San Diego, CA: Academic Press. Taschner MJ, Black DJ. 1988. The enzymatic Baeyer-Villiger oxidation: Enantioselective synthesis of lactones from mesomeric cyclohexanones. J Am Chem Soc 110:6892
6893. ten Brink G-J, Arends IWCE, Sheldon RA. 2004. The BaeyerVilliger reaction: New developments toward greener procedures. Chem Rev 104:4105
4123. Tse LM, Khosla C. 2005. Engineering and overproduction of polyketide natural products. In: Kna¨blein J, editor. Modern biopharmaceuticals. Weinheim: Wiley-VCH. van Sint Fiet S, van Beilen JB, Witholt B. 2006. Selection of biocatalysts for chemical synthesis. PNAS 103:1693
1698. Walsh C. 1979. Enzymatic reaction mechanisms. San Francisco, CA: W.H. Freeman.

185

Wendt KU, Schulz GE, Corey EJ, Liu DR. 2000. Enzyme mechanisms for polycyclic triterpene formation. Angew Chem Intl Ed 39:2812
2833. Wohlgemuth R. 2004. Large-scale applications of biocatalysis in the asymmetric synthesis of laboratory chemicals. In: Blaser HH, Schmidt E, editors. Asymmetric catalysis on an industrial scale. Weinheim: Wiley-VCH. Wohlgemuth R. 2005. Development, production, and application of recombinant yeast biocatalysts in organic synthesis. Chimia 59:735
740. Yildirim S, Franco T, Wohlgemuth R, Kohler HP, Witholt B, Schmid A. 2005. Recombinant chlorobenzene dioxygenase from Pseudomonas sp. P51 : A biocatalyst for regioselective oxidation of aromatic nitriles. Adv Synth Cat 347:1060
1073. Yildirim S, Ruinatscha R, Gross R, Wohlgemuth R, Kohler HP, Witholt B, Schmid A. 2006. Selective hydrolysis of the nitrile group of cis -dihydrodiols from aromatic nitriles. J Mol Cat B: Enzym 38:76
83.