Figure 1. Enzymatic reactions can be performed as kinetic resolutions yielding at maximum 50% product as shown for a lipase-catalyzed or a transaminase-catalyzed kinetic resolution. In contrast, asymmetric synthesis gives in principle 100% of the desired optically pure product exemplified for a reduction using an alcohol dehydrogenase (ADH/ketoreductase) and the cofactor NADH or by a C C coupling reaction using a hydroxynitrile lyase.

Figure 2. According to the Prelog rule, the size of the substituents R ₁ and R ₂ (here, R ₁ < R ₂) determines whether the carbonyl of a ketone is attacked by the hydride either from the re- or from the si-face. In the example shown above, the (S)-product is formed, if a sequence rule of R ₂ > R ₁ is assumed.

Figure 3. Example for the synthesis of optically active alcohols using an alcohol dehydrogenase. The cofactor NADH is regenerated directly from isopropanol.

Figure 4. Example for the synthesis of optically active 3-oxo-5-hydroxycarboxylic acids using a recombinant alcohol dehydrogenase from Lactobacillus brevis (LBADH) exemplifying the regio- and stereoselectivity of this ADH. The cofactor NADPH is regenerated directly from isopropanol.

Figure 5. Proposed reaction mechanism for LeuDH.

Figure 6. Example for the synthesis of optically active l-amino acids using an amino acid dehydrogenase. The cofactor NADH is regenerated using a formate dehydrogenase from C. boidinii. The equilibrium is shifted toward NADH by using ammonium formate yielding carbon dioxide as by-product.

Figure 7. Overview of reactions catalyzed by monooxygenases.

Figure 8. Reaction cycle of a hydroxylation catalyzed by a P450 monooxygenase.

Figure 9. Principle of the colorimetric pNCA assay allowing the determination of the fatty acid hydroxylating activity of P450 BM3 mutant F87A.

Figure 10. Principle of the assay used to identify P450 _cam variants hydroxylating naphthalene. The resulting naphthols are oxidatively coupled with horseradish peroxidase (HRP) to fluorescent polymers. All reactions occur intracellularly in the recombinant E. coli cell.

Figure 11. Selected examples of products obtained from monooxygenase-catalyzed hydroxylations. Note that most examples represent the use of engineered P450s.

Figure 12. Early example of an enzyme-catalyzed Baeyer–Villiger oxidation.

Figure 13. Proposed mechanism of an enzyme-catalyzed Baeyer–Villiger oxidation.

Figure 14. Selected examples of BVMO-catalyzed conversion of mono- and bicyclic ketones using BVMO from Acinetobacter sp. or camphor-induced P. putida. (a) R = CH ₂CO ₂Et; (b) R = CH ₂OAc; (c) R = CH ₂CH ₂O(CH ₂) ₂OMe.

Figure 15. A BVMO from P. fluorescens converts β-hydroxyketones into the monoacetate of a 1,2-diol as major product and the β-hydroxyacid methyl ester as minor by-product.

Figure 16. Examples of epoxidations and benzylic hydroxylations catalyzed by chloroperoxidase (CPO) from C. fumago.

Figure 17. Catalytic mechanism of stereoselective trans-hydrogenation of CC bond by flavin mononucleotide (FMN) containing old yellow enzyme. The H _R of NAD(P)H is transferred to the β-carbon via FMN _red while a proton from the tyrosine or water is transferred to the α-carbon of the activated alkene, giving a net trans-addition product.

Figure 18. Examples for enoate reductase-catalyzed reductions to yield optically pure products.

Figure 19. OYE1 wild type and W116I form products with opposite stereochemistry from (S)-carvone. With (R)-carvone, both enzymes produced the same diastereomer.

Figure 20. Deracemization of α-MBA using a (S)-selective monoamine oxidase coupled with ammonia–borane reduction of the intermediate imine to form racemic amine. In the first round, 50% (S)-amine is oxidized to the imine yielding after chemical reduction 25% (R)-amine and 25% (S)-amine, the latter being oxidized again. After seven to eight rounds, the racemic starting material is completely converted into optically pure (R)-α-MBA.

Figure 21. Chemoenzymatic synthesis of (+)-crispine and its framework using a deracemization catalyzed by a mutant of MAO-N.

Figure 22. Reaction cycle for asymmetric synthesis of amines with ω-TA. Although all reactions are fully reversible, only simple reaction arrows are shown to indicate the direction of the desired asymmetric synthesis and the chronological order in which the substrates and products (shaded in light gray) have to be bound or released from the enzyme. During the reaction cycle, two forms of the free enzyme exist (E-PLP and E-PMP). Inhibition may be caused by binding of substrates (shaded in dark gray) to the “wrong” free enzyme forming abortive complexes, which results in inhibition of the enzyme.

Figure 23. Strategies for the synthesis of optically active amines using amine transaminases. In a kinetic resolution (a), the amine transaminase converts in the ideal case only one of the amine enantiomers to the corresponding ketone. The remaining enantiomer can be isolated in high optical purity and at a (theoretical) maximum yield of 50%. In an asymmetric synthesis (b), a prostereogenic ketone is aminated enantioselectively, yielding directly the optically active amine at a theoretical yield of 100%. Common cosubstrates for amine transaminases are pyruvate and alanine. As the equilibrium favors ketone formation, high yields in asymmetric synthesis can only be achieved by shifting the equilibrium, for example, by enzymatic removal of the formed coproduct pyruvate. The same principles also apply to α-transaminases useful for the production of α-amino acids.

Figure 24. Asymmetric synthesis of (S)-methoxyisopropylamine.

Figure 25. An engineered (R)-selective amine transaminase is used in a large-scale process for the production of the drug Sitagliptin®.

Figure 26. Mechanism of lipase-catalyzed ester hydrolysis of a butyrate ester. Numbering of amino acid residues is for lipase from C. rugosa (CRL).

Figure 27. Example for a lipase-catalyzed kinetic resolution by hydrolysis. A preparative separation of an ester/alcohol mixture is straightforward by distillation or chromatography.

Figure 28. Example for a lipase-catalyzed kinetic resolution by acylation. The use of the enol ester vinyl acetate ensures an irreversible reaction as the by-product acetaldehyde is generated via a keto–enol tautomerization from the vinyl alcohol.

Figure 29. Some acyl donors used in lipase-catalyzed kinetic resolutions.

Figure 30. An empirical rule (“Kazlauskas rule”) developed for lipase from B. cepacia (BCL) summarizing the enantiopreference for primary and secondary alcohols. The scheme shows the favored enantiomer. For primary alcohols, this rule is only reliable if no oxygen is attached to the stereocenter. Note that BCL shows an opposite enantiopreference for primary alcohols.

Figure 31. Some examples of carboxylic acid resolved using lipase from C. rugosa (CRL). CRL-CLEC is a cross-linked enzyme crystal preparation of CRL. Higher E-values might be due to the removal of interfering isoenzymes during crystallization.

Figure 32. Some examples of secondary alcohols resolved using lipases. In all cases, vinyl acetate served as acyl donor. Note the changes in enantioselectivity with varying substrate structure.

Figure 33. Some examples of primary alcohols resolved using lipase from P. cepacia (PCL). In all cases, vinyl acetate served as acyl donor. Note the changes in enantioselectivity with varying substrate structure and the lower E-values compared to secondary alcohols.

Figure 34. Kinetic resolution of aryl aliphatic tertiary alcohol acetates using a double mutant of esterase BS2 (E188D/M193C) resulted in higher or inverted enantioselectivity.

Figure 35. Lipase-catalyzed kinetic resolution of amines in the BASF process.

Figure 36. Lipase-catalyzed kinetic resolution of a building block for the synthesis of captopril.

Figure 37. Principle of dynamic kinetic resolutions.

Figure 38. Examples of the dynamic kinetic resolution of secondary alcohols using a ruthenium catalyst.

Figure 39. Example of the dynamic kinetic resolution of an allylic alcohol using Pd(0).

Figure 40. Examples of esterase-catalyzed resolutions. AGE, A. globiformis esterase; BCE, Bacillus coagulans esterase; BGE, Burkholderia gladioli esterase; BSE, B. stearothermophilus esterase; PAE, Pseudomonas aeruginosa esterase; PFE, P. fluorescens esterase; PPE, P. putida esterase; RRE, Rh. ruber esterase; SDE, Streptomyces diastatochromogenes esterase.

Figure 41. Recombinant pig liver esterase isoenzymes (1–5) show significantly higher and even inverted enantioselectivity in the kinetic resolution of four acetates of secondary alcohols compared to a crude preparation from Fluka.

Figure 42. Synthesis of amide bonds using proteases and amidases. (a) Thermodynamic control shifts the equilibrium toward synthesis by changing the reaction conditions. For example, organic solvents are added to reduce the concentration of water and to suppress ionization of the starting materials. (b) Kinetic control starts with an activated carboxyl component (e.g., an ester or an amide) and forms an acyl-enzyme intermediate. The acyl-enzyme intermediate then reacts with an amine to form the amide. In a competing side reaction, water may react with the acyl-enzyme intermediate.

Figure 43. Commercial process for the production of aspartame (α-l-aspartyl-l-phenylalanine methyl ester). Thermolysin catalyzes the coupling of an N-Cbz-protected aspartic acid with phenylalanine methyl ester. The product forms an insoluble salt with excess of phenylalanine methyl ester. This precipitation drives this thermodynamically controlled peptide synthesis. The high regioselectivity of thermolysin for the α-carboxylate allows the β-carboxylate in aspartate to be left unprotected. The enantioselectivity of thermolysin allows the use of racemic starting materials.

Figure 44. The penicillin acylase (PGA)-catalyzed coupling of 6-aminopenicillanic acid with (R)-(–)-phenylglycine to produce ampicillin, a β-lactam antibiotic.

Figure 45. Mechanism proposed for epoxide hydrolase from A. radiobacter.

Figure 46. Hydrolysis of epoxides can proceed with retention or inversion of configuration.

Figure 47. Examples of the resolution of epoxides using bacterial epoxide hydrolases.

Figure 48. Examples of the resolution of epoxides using yeast and fungal epoxide hydrolases.

Figure 49. Resolution of styrene oxide using fungal epoxide hydrolases from A. niger or B. sulfurescens or a mixture of both for an enantioconvergent synthesis.

Figure 50. Proposed mechanism for the reversible epoxide ring opening catalyzed by A. radiobacter AD1 haloalcohol dehalogenase. The Arg–Tyr pair is involved in leaving group protonation.

Figure 51. Haloalcohol dehalogenase-catalyzed ring opening of p-nitrostyrene oxide in the presence of azide proceeds with excellent regio- and enantioselectivity and yields an azido alcohol as product.

Figure 52. Hydrolysis of nitriles follows two different pathways.

Figure 53. Proposed mechanism of nitrile hydration catalyzed by a nitrile hydratase involving Fe(III) and PQQ.

Figure 54. Commercial production of acrylamide and nicotinamide using resting cells of Rh. rhodochrous J1.

Figure 55. Examples of the regioselective hydrolyses using nitrile hydratases and nitrilases.

Figure 56. Examples of the synthesis of nonsteroidal anti-inflammatory drugs by hydrolysis of nitriles.

Figure 57. Synthesis of (R)-(–)-mandelic acid by dynamic kinetic resolution using a nitrilase involves in situ recycling of (S)-mandelonitrile by disproportion into benzaldehyde and HCN followed by formation of (R,S)-mandelonitrile.

Figure 58. Desymmetrization of prochiral hydroxyglutaronitrile using engineered nitrilases.

Figure 59. Synthesis of l- or d-amino acids using a combination of hydantoinase and carbamoylase. Complete conversion of racemic d,l-hydantoin can be achieved by racemization at alkaline pH with specific racemases.

Figure 60. Example for the synthesis of optically active cyanohydrins from aldehydes and hydrogen cyanide catalyzed by a hydroxynitrile lyase (HNL).

Figure 61. Examples of building blocks, which can be obtained by chemical synthesis from chiral cyanohydrins. Products with two stereocenters are accessible by these routes.

Figure 62. Suggested reaction mechanism for hydroxynitrile lyase from S. bicolor.

Figure 63. Selected examples of the hydroxynitrile lyase-catalyzed synthesis of chiral cyanohydrins. Pa, HNL from P. amygdalus; Sb, HNL from S. bicolor; Hb, HNL from H. brasiliensis; Me, HNL from M. esculenta.

Figure 64. The principle of transhydrocyanation.

Figure 65. Synthesis of (S)-m-phenoxybenzaldehyde cyanohydrin as an intermediate of pyrethroids using a recombinant HNL from H. brasiliensis.

Figure 66. Mechanism of type I (a) and type II (b) aldolases. The example shows dihydroxyacetone phosphate (DHAP) as nucleophilic ketone.

Figure 67. Aldolase reactions catalyzed by the four stereocomplementary aldolases FruA (fructose-1,6-diphosphate aldolase), FucA (fuculose-1-phosphate aldolase), TagA (tagatose-1,6-diphosphate aldolase), and RhuA (rhamnulose-1-phosphate aldolase).

Figure 68. Selected examples of molecules synthesized with FDP aldolase (ManNAc, N-acetyl-d-mannosamine).

Figure 69. Enzymatic total synthesis of 5-deoxy-5-ethyl-xylulose with in situ formation of dihydroxyacetone phosphate. The key to success was the switch in pH from 4.0 (phytase reaction) to 7.5 (glycerol phosphate oxidase (GPO) and aldolase (FruA) reaction).

Figure 70. In vivo aldol reaction catalyzed by NeuAc aldolase.

Figure 71. Mechanism of the PLP-dependent alanine racemase.

Figure 72. Mechanism of a PLP-independent racemase, that is, glutamate racemase.