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7-24-2020

Application of Enzymes in Regioselective and Stereoselective Organic Reactions

Ruipu Mu Centenary College of Louisiana

Zhaoshuai Wang University of Kentucky, [email protected]

Max C. Wamsley Centenary College of Louisiana

Colbee N. Duke Centenary College of Louisiana

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Part of the Pharmacy and Pharmaceutical Sciences Commons Authors Ruipu Mu, Zhaoshuai Wang, Max C. Wamsley, Colbee N. Duke, Payton H. Lii, Sarah E. Epley, London C. Todd, and Patty J. Roberts

Application of Enzymes in Regioselective and Stereoselective Organic Reactions Notes/Citation Information Published in Catalysts, v. 10, issue 8, 832.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Digital Object Identifier (DOI) https://doi.org/10.3390/catal10080832

This review is available at UKnowledge: https://uknowledge.uky.edu/ps_facpub/147 catalysts

Review Application of Enzymes in Regioselective and Stereoselective Organic Reactions

Ruipu Mu 1,*, Zhaoshuai Wang 2,3,* , Max C. Wamsley 1, Colbee N. Duke 1, Payton H. Lii 1, Sarah E. Epley 1, London C. Todd 1 and Patty J. Roberts 1 1 Department of Chemistry, Centenary College of Louisiana, Shreveport, LA 71104, USA; [email protected] (M.C.W.); [email protected] (C.N.D.); [email protected] (P.H.L.); [email protected] (S.E.E.); [email protected] (L.C.T.); [email protected] (P.J.R.) 2 Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA 3 Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA * Correspondence: [email protected] (R.M.); [email protected] (Z.W.)



Received: 22 June 2020; Accepted: 21 July 2020; Published: 24 July 2020


Abstract: Nowadays, biocatalysts have received much more attention in chemistry regarding their potential to enable high efficiency, high yield, and eco-friendly processes for a myriad of applications. Nature’s vast repository of catalysts has inspired synthetic chemists. Furthermore, the revolutionary technologies in bioengineering have provided the fast discovery and evolution of enzymes that empower chemical synthesis. This article attempts to deliver a comprehensive overview of the last two decades of investigation into enzymatic reactions and highlights the effective performance progress of bio-enzymes exploited in organic synthesis. Based on the types of enzymatic reactions and enzyme commission (E.C.) numbers, the enzymes discussed in the article are classified into oxidoreductases, transferases, hydrolases, and lyases. These applications should provide us with some insight into enzyme design strategies and molecular mechanisms.

Keywords: regioselectivity; stereoselectivity; biocatalyst; enzymatic reactions; protein engineering

1. Introduction

1.1. A Brief History of Enzymes and Their Application Currently, enzymes are applied extensively in many fields, including the food, medical, chemical, and cosmetic industries [1]. Following their discovery in the late 18th century, enzymes were predominantly employed to produce digested food for humans and animals. The concept of catalysis was well defined in the middle of the 19th century when scientists started to study the mechanism of catalysis and the relationship between chemistry and biology. The study of enzymes bloomed in the 20th century. Models resulting from the further study of enzyme kinetics, including the Michaelis-Menten equation, provided more insight into how enzymes work in reactions in vitro and in vivo. Mechanistic studies led to the discovery of the enzyme-substrate intermediate and the first crystal structure of an enzyme (lysozyme), which marked huge milestones in the history of science and technology [1]. At the same time, more and more enzymes were applied in clinical trials, which led to the discovery of many therapeutic enzymes, like collagenases to treat skin ulcers [2] and antibody-drug conjugates to treat cancer [3]. In addition, enzymes started to play an important role in producing organics like polymer materials and inorganics used in treating wastewater in the chemical industry [4]. During this time, enzymes were also widely applied in the cosmetic industry, helping to produce body care products such as skin protective and soothing agents [5].

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Among enzyme applications, utilizing enzymes as a catalyst in an organic reaction to obtain the specific target product is ubiquitous in the chemical industry. The advantages of using enzymes instead of the traditional chemical catalyst include higher stereoselectivity, higher yield, higher efficiency, and less waste [4]. In addition to the advantages of enzyme application, there are several drawbacks of applying enzymes in this industry. Enzymes are sensitive to the temperature and pH of the environment, and their activity is changed drastically with the changes in the parameters. The cost of isolation and preparation of enzymes is high. Furthermore, a lack of long-term operational stability and the difficulty of their recovery or reuse discourage their wide application [6]. To circumvent these issues, three methods of enzyme immobilization have been developed, including binding to a support (carrier), entrapment (encapsulation), and crosslinking [7]. With the increasing number of techniques to solve these problems, the application of enzymes has become an essential part of industrial development and will continue to make significant contributions.

1.2. A Brief Introduction to Regioselective and Stereoselective Reactions A critical advantage related to the three-dimensional structure of enzymes is that they catalyze the reaction in which a target functional group is inserted into a specific position of the reactant. Compared to traditional organic synthesis, the product obtained from an enzymatic reaction usually leans towards a selective structure with a specific configuration [8]. Two main types of selectivity are often observed in enzymatic reactions: regioselectivity and stereoselectivity. Regioselectivity refers to the preference of a functional group to bond to one atom over another atom in the reactant, while stereoselectivity indicates a favoring of one stereoisomer over another stereoisomer. Related to stereoselectivity, “enantioselectivity” is defined as the preference for producing one enantiomer over another. Regioselectivity and stereoselectivity exist ubiquitously in the plant, animal, bacterial, and fungal kingdoms. A known example is melanin, which functions as a pigment and as a radiation protection agent [9]. Tyrosinase catalyzes the hydroxylation of L-tyrosine to regioselectively produce L-DOPA in the melanin biosynthesis pathway [10]. Another important compound existing in humans is norepinephrine (NE), whose main functions are as a hormone and as a neurotransmitter. In its biosynthesis, a dopamine-β-hydroxylase inserts a hydroxyl group to β-carbon on the side chain of dopamine, stereoselectively producing an R-enantiomer [11]. To date, more than half of the small molecule drugs in clinical use are chiral, but the chirality of drug structure was not valued initially [12]. People started to spotlight chiral drugs following the huge thalidomide tragedy that occurred in the 1960s. Thalidomide entered the market in 1957 to treat insomnia and morning sickness as an over-the-counter drug and soon expanded to markets in 46 countries. Four years later, the side effect of severe birth defects was associated with thalidomide. Use of thalidomide by women during pregnancy was linked to the delivery of babies with shortened or flipper-like limbs [13]. More than 10,000 cases were reported, with 40 percent of families losing their babies and those who survived having additional problems such as dysmelia, facial anomalies, and systemic anomalies [14]. Stereochemistry figures into this tragedy because thalidomide sold in the market was a mixture of two stereoisomers, R- and S- forms. R-enantiomer was therapeutically active, while S-enantiomer caused severe side effects. The thalidomide disaster prompted changes to US-FDA regulations that tightened restrictions surrounding the surveillance and approval process, including requiring manufacturers to provide all data on the safety and effectiveness of a drug, including the evaluation of the absolute stereochemistry as part of the approval process [15]. The broad application of enzymes in stereochemistry drives the rapid development of chiral compounds, expanding the drug candidate pool and opportunities to obtain a therapeutically active drug. Herein, we provide a brief overview of the application of enzymes in regioselective and stereoselective reactions, which also involve enantioselective reactions. Catalysts 2020, 10, 832 3 of 25

Catalysts 2020, 10, x FOR PEER REVIEW 3 of 25 2. Examples of Enzyme Application in Regioselective and Stereoselective Reactions 2. Examples of Enzyme Application in Regioselective and Stereoselective Reactions Depending on the types of enzymatic reactions and enzyme commission (E.C.) numbers, Depending on the types of enzymatic reactions and enzyme commission (E.C.) numbers, the the enzymes are classified into different groups. In this review, four main groups of enzymes, enzymes are classified into different groups. In this review, four main groups of enzymes, including including oxidoreductases, transferases, hydrolases, and lyases, will be discussed. oxidoreductases, transferases, hydrolases, and lyases, will be discussed. 2.1. Oxidoreductases E.C.1 2.1. Oxidoreductases E.C.1 2.1.1. Enzymes Applied for Reduction 2.1.1. Enzymes Applied for Reduction Ketoreductases. The enantioselective reduction of ketones to the corresponding alcohols that have significantKetoreductases bioactivity. The has enantioselective attracted more reduction and more ofattention ketones to in the organic corresponding synthesis. alcohols However, that thehave reduction significant of tert-butyl bioactivity and isopropyl has attracted ketones more still and represents more attention a significant in organic challenge synthesis. in both However, enzymatic the andreduction chemical of reductions. tert-butyl and Enzymatic isopropyl protocols, ketones still such represents as utilizing a significant carbonyl reductase challenge extractedin both enzymatic from Sporobolomycesand chemical salmonicolor reductions.(SSCR), Enzymatic proved protocols, to be moresuch easff ectiveutilizing in catalyzingcarbonyl reductase the enantioselective extracted from reductionSporobolomyces of aryl alkyl salmonicolor ketones than(SSCR), traditional proved chemicalto be more formation effective (Scheme in catalyzing1). Notably, the enantioselective the enzyme demonstratedreduction of excellent aryl alkyl enantioselectivity ketones than traditional (>96% ee, chemicalenantiomeric formation excess), (Scheme reducing 1). ketones Notably, with the bulky enzyme alkyldemonstrated groups (tert-butyl, excellent cyclopropyl, enantioselectivity etc.) [16]. (>96% ee, enantiomeric excess), reducing ketones with bulky alkyl groups (tert-butyl, cyclopropyl, etc.) [16].

Scheme 1. The enantioselective reductions of aryl alkyl ketones. Scheme 1. The enantioselective reductions of aryl alkyl ketones. Alpha-hydroxyAlpha-hydroxy ketones ketones were were experimentally experimentally synthesized synthesized usingusing three three different different mechanisms. mechanisms. The Thefirst first method method involved involved reacting aldehydesaldehydes with with thiamine thiamine diphosphate-dependent diphosphate-dependent lyases lyases to catalyzeto catalyze thethe umpolung umpolung carboligation carboligation of aldehydes;of aldehydes; this this allows allows one one aldehyde aldehyde to becometo become an activean active enamine enamine carbanion.carbanion. Another Another aldehyde aldehyde attacks attacks the the carbanion carbanion to formto form an alpha-hydroxyan alpha-hydroxy ketone. ketone. The The second second methodmethod occurred occurred when when a substratea substrate containing containing a a ketoneketone withwith an adjacent adjacent alcohol alcohol group group (either (either R Ror S) or Swas) was reacted reacted using using hydrolases hydrolases via via dynamic dynamic kineti kineticc resolutions. resolutions. The The substrate substrate being being either either (R) (orR) (S) or (allowsS) allows for high for high conversions conversions and enantiomeric and enantiomeric excess excess of the ofdesired the desired alpha-hydroxy alpha-hydroxy ketone. ketone. The third Themethod third method elaborated elaborated on reacting on reacting diketones diketones and andvicinal vicinal diols diols with with whole-cell whole-cell redox redox reactions. reactions. By By employing freefree enzymesenzymesor or whole-cells, whole-cells, Hoyos Hoyos et et al. al were. were able able to to reduce reduce the the diketones diketones and and oxidize oxidize thethe diols diols to produce to produce the the alpha-hydroxy alpha-hydroxy ketones, ketones, which which is racemic is racemic [17 ].[17]. A largeA large number number of reductasesof reductases were were encoded encoded in Saccharomyces in Saccharomyces cerevisiae cerevisiaeyeast yeast genome, genome, many many of of whichwhich have have the abilitythe ability to reduce to reduceα-chloro- α-chloro-β-ketoβ-keto esters. esters. Kaluzna’s Kaluzna’s study study revealed revealed that in that nearly in nearly all the all targetedthe targeted reductases, reductases, more than more one thanα-chloro- one α-chloro-β-hydroxyβ-hydroxy ester diastereomer ester diastereomer was obtained, was obtained, with up with to up 98%toee 98%. The ee only. The deficiency only deficiency in this approach in this isapproach a lack of Dis-specific a lack of reductases D-specific [18 reductases]. Zhu et al. [18]. determined Zhu et al. thedetermined activity and the enantioselectivity activity and enantioselectivity of a carbonyl reductase of a carbonyl from Candida reductase magnoliae from Candida. Using magnoliae NADPH. as Using a cofactor,NADPH the reductaseas a cofactor, catalyzed the reductase the enantioselective catalyzed the reduction enantioselective of a series ofreduction ketones, includingof a series aliphatic, of ketones, aromaticincluding ketones, aliphatic,α, and aromaticβ ketoesters, ketones, to give α, anti-Prelogand β ketoesters, configurated to give alcohols anti-Prelog with configurated high optical purity alcohols (Schemewith 2high). This optical reductase purity with (Scheme a new 2). application This reduct complementedase with a new theapplication known ketoreductase complemented pool the andknown couldketoreductase be employed pool to synthesize and could chiralbe employed alcohols. to This synthesi strategyze chiral also solves alcohols. the This problems strategy in the also chemical solves the synthesis,problems such in asthe the chemical complicated synthesis, process such operation, as the complicated high metal process residue, operation, low enantiomeric high metal purity, residue, andlow productivity enantiomeric [19]. purity, The backbones and productivity of bacterial [19]. aromatic The backbones polyketides of bacterial could be aromatic synthesized polyketides from could be synthesized from acyl-CoA-derived building blocks by polyketide synthases (PKSs). A recent study revealed that some polyketides were attained from nonactate precursors. Exemplarily, the engineered PKSs catalyzed the synthesis of regioselectively modified anthraquinones with

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acyl-CoA-derivedCatalysts 2020, 10, x FOR building PEER REVIEW blocks by polyketide synthases (PKSs). A recent study revealed that some4 of 25 polyketides were attained from nonactate precursors. Exemplarily, the engineered PKSs catalyzed the synthesispredictable of functional regioselectively moiety modified that have anthraquinones improved pharmacological with predictable properties functional over moiety the thatestrogen have improvedreceptor antagonist pharmacological R1128 [20]. properties over the estrogen receptor antagonist R1128 [20].

Scheme 2. Enantioselectivity of carbonyl reductase from Candida magnolia toward the reduction of Scheme 2. Enantioselectivity of carbonyl reductase from Candida magnolia toward the reduction of aryl ketones. aryl ketones. Dehydrogenases.Dehydrogenases. Classen Classen et et al. al. investigated investigated thethe utilizat utilizationion of the of enoate the enoate reductase reductase YqjM YqjM as well as wellas alcohol as alcohol dehydrogenases dehydrogenases and a and glucose a glucose dehydrogenase dehydrogenase to synthesize to synthesize chiral chiralγ-butyrolactones.γ-butyrolactones. Their Theirresults results revealed revealed that the that process the process is scalable, is scalable, with with up upto to90% 90% yields yields and and excellent excellent enantioselectivity enantioselectivity (over 98%98% eeee)[) [21].21]. Furthermore, Furthermore, the the substrate substrate ethyl ethyl 4- 4-oxo-pent-2-enoatesoxo-pent-2-enoates are readily available through simple Wittig-type chemicalchemical reactions.reactions. No Notably,tably, the the enoate enoate reductase reductase YqjM YqjM is strongly substrate-dependent andand hardly hardly predictable, predictable, unlike unlike ADHs ADHs that that follow follow a simple a simple rule of rule prediction of prediction [15,22]. Studies[15,22]. Studies show thatshow the that log the P log of P the of organicthe organic solvent solvent has has a a dramatic dramatic impact impact onon enzyme activity. activity. Exemplarily, alcoholalcohol dehydrogenase ADH-A from Rhodococcus ruber demonstrated robust enzymatic eefficiencyfficiency ( (eeee >99%),>99%), catalyzing catalyzing the thereduction reduction of a large of a variety large variety of ketones of ketonesin micro-aqueous in micro-aqueous media, a media,nonconventional a nonconventional aqueous aqueousorganic solvent organic system solvent (99% system v/v (99%). Thisv/v ).organic This organic media mediashowed showed great greatbiocompatibility, biocompatibility, with 10-fold with 10-fold improvement, improvement, in contrast in contrast to previous to previous observations observations using an usingaqueous an aqueoussolvent [23,24]. solvent Two [23, methods24]. Two were methods used were to synthesize used to synthesize optically pure optically cis- and pure transcis--isomers and trans of-isomers whisky oflactone. whisky The lactone. first method The first employed method employedenzyme-med enzyme-mediatediated reactions reactionsthat involved that involvedeight alcohol eight alcoholdehydrogenases dehydrogenases as biocatalysts as biocatalysts to enantios toelectively enantioselectively oxidize the oxidizeracemic theproducts racemic of erythron- products and of erythronthreo-3-methyloctane-1,4-diols.- and threo-3-methyloctane-1,4-diols. The second Theapproach second approachinvolved involvedbiotransformation, biotransformation, using usingmicroorganisms microorganisms to produce to produce naturally naturally occurring occurring oppo oppositesite isomers isomers of ofthe the whiskey whiskey lactone. lactone. For For this method, the transtrans-isomer of 3-methyl-4-oxooctanoic3-methyl-4-oxooctanoic acid was produced.produced. Boratynski and colleagues were ableable to synthesizeto synthesize enantiomerically enantiomerically enriched en isomersriched ofisomers oak lactones of oak and oppositelactones enantiomerically and opposite enrichedenantiomerically isomers enriched of whisky isomers lactones of whisky based lactones on the method based on of the reaction method used, of reaction and they used, found and they that thefound lactonization that the lactonization of whole-cell of whole-cell microorganisms microorganisms is a significant is a significant alternative alternative to the enzyme-mediated to the enzyme- oxidationmediated oxidation strategy [ 25strategy]. The chemical[25]. The synthesischemical ofsynthesis stereoselective of stereoselective 1,2-diols is 1,2-diols a significant is a significant challenge duechallenge to the due lack to of the a precursorlack of a precursor and toxic and catalyst. toxic Tocatalyst. discover To discover new alcohol new dehydrogenasesalcohol dehydrogenases (ADHs) used(ADHs) to reduceused to 2-hydroxyreduce 2-hydroxy ketones, ketones, Kulig et Kulig al. investigated et al. investigated eight ADHs eight ADHs and found and found that all that of themall of hadthem good had activities. good activities. Among the Among enzymes the tested, enzy alcoholmes dehydrogenasestested, alcohol fromdehydrogenasesThermoanaerobacter from sp.Thermoanaerobacter(ADHT), Lactobacillus sp. (ADHT), brevis Lactobacillus(LBADH), brevis and Ralstonia (LBADH), sp. and(RADH) Ralstonia showed sp. (RADH) high activities showed high and stereoselectivitiesactivities and stereoselectivities in the enzymatic in reactionsthe enzymatic of reducing reactions all reactants of reducing to anti-diols. all reactants In addition, to anti-diols. all three In ADHsaddition, showed all three promising ADHs showed activity promising towards 2-hydroxy activity towards ketones, 2-hydroxy and RADH ketones, was the and most RADH active was ADH the towardsmost active bulky-bulky ADH towards 2-hydroxy bulky-bulky ketones 2-hydroxy [26]. ketones [26]. O’Reilly et al. experimentally experimentally determined determined a mech mechanismanism to regio- regio- and and stereoselectively stereoselectively synthesize 2,5-disubstirutred pyrrolidines from 1,4-diketones1,4-diketones (Scheme 33).). ByBy usingusing aa one-potone-pot ω-transaminase-transaminase and monoamine oxidase from Aspergillus niger , the reaction was accomplished in one step, withoutwithout intermediation due to thethe compatibilitycompatibility ofof thethe enzymes.enzymes. The transaminase mediates the reductive amination of prochiral ketones and favors the productionproduction of chiral amines, while the monoamine oxidase favors the (S)-enantiomer during the conversion of amines to imines.imines. The enzymes do not hinder eacheach other other and and therefore therefore can becan used be inused a two-step, in a two-step, one-potsynthesis one-pot reactionsynthesis of 2,5-disubstitutedreaction of 2,5- pyrrolidines,disubstituted exhibitingpyrrolidines, exceptional exhibiting enantioselectivity exceptional enantioselectivity of greater than 94% of greateree and diastereoselectivity than 94% ee and greaterdiastereoselectivity than 98% de greater[27]. than 98% de [27].

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SchemeScheme 3. A 3. chemoenzymaticA chemoenzymatic approach approach for thethe synthesis synthesis of 2,5-disubstitutedof 2,5-disubstituted pyrrolidines pyrrolidines by employing by employingan ω-transaminase an ω-transaminase (TA) and (TA) a monoamine and a monoamine oxidase oxidase (MAO-N). (MAO-N).

Single-electron-transferSingle-electron-transfer steps steps involve involve alkali alkali metals, metals, radical radical anion anion transition transition states, states, etc., etc.,that thatlie in lie in the centerthe center of Birch of Birch reductions reductions of of aromatic aromatic co compounds.mpounds. Conversely, 2-naphthoyl-coenzyme2-naphthoyl-coenzyme A A (2-NCoA) (2- NCoA)and and 5,6-dihydro-2-NCoA 5,6-dihydro-2-NCoA (5,6-DHNCoA) (5,6-DHNCoA) reductases reductases such such as NCR as NCR and DHNCRand DHNCR reduce reduce two ditwofferent differentnaphthoyl-ring naphthoyl-ring systems systems to tetrahydronaphthoyl-CoA to tetrahydronaphthoyl-CoA by different by different mechanisms mechanisms that are categorized that are as categorizedtwo-electron as two-electron reductions. Both reductions. enzymatic Both reactions enzymatic have demonstrated reactions have high demonstrated stereoselectivity high (30-fold stereoselectivityimprovement) (30-fold in aqueous improvement) solution in at aqueous ambient solution temperature. at ambient Moreover, temperature. these bio-catalysts Moreover, these provide bio-catalystsan enantioselective provide an enantioselective pathway to products pathway that to areproducts not readily that are accessible not readily via accessible Birch reductions via Birch [28]. reductionsThe biosynthesis [28]. The biosynthesis of polyunsaturated of poly fattyunsaturated acids (PUFAs) fatty acid requiress (PUFAs)∆12 and requiresω3 fatty Δ12 acid and desaturases ω3 fatty to acid formdesaturases the second to form and the third second double and bonds third in double the molecule, bonds in respectively, the molecule, as respectively, PUFAs are crucial as PUFAs building are crucialblocks building of membrane blocks glycerolipids of membrane and glycerolipids predecessors and of predecessors signaling molecules of signaling in cells. molecules Sk-FAD2 in and cells.Sk-FAD3 Sk-FAD2 that and are Sk-FAD3∆12 and thatω3 fattyare Δ acid12 and desaturase ω3 fatty genes acid fromdesaturaseSaccharomyces genes from kluyveri Saccharomycesdemonstrated kluyveriexcellent demonstrated substrate excellent specificity substrate and regioselectivity. specificity and Sk-FAD2 regioselectivity. introduced Sk-FAD2 a double introduced bond to C16–20 a doublemonounsaturated bond to C16–20 fatty monounsaturated acids at the ν + fatty3 position, acids at whereas the ν + 3Sk-FAD3 position, selectedwhereas the Sk-FAD3ω3 position selected on C18 the ωand3 position C20. Moreover, on C18 and studies C20. suggestedMoreover, thatstudies both suggested enzymes that showed both littleenzymes to no showed specificity little toward to no the specificitypolarity toward of the the head polarity group of or the the head sn positions group or of the acyl sn groups positions in phospholipids of acyl groups [ 29in ].phospholipids [29]. 2.1.2. Enzymes Applied for Oxidation 2.1.2. Enzymes Applied for Oxidation Cytochrome P450s. Linear alkanes are difficult to hydroxylate by using chemical synthesis due toCytochrome the high carbon–hydrogen P450s. Linear alkanes bond are strength difficul (~97t to hydroxylate kcal/mol) and by high using activation chemical energy.synthesis Therefore, due to thenumerous high carbon–hydrogen hydroxylations bond rely on strength enzymatic (~97 catalysis, kcal/mol) of and which high some activation require energy. the use Therefore, of cytochrome numerousP450s hydroxylations to initiate the electron rely on enzymatic transfer between catalysis, molecules. of which some A common require cytochromethe use of cytochrome P450 enzyme P450sexploited to initiate for the this electron purpose transfer is cytochrome between P450 molecules. BM-3, an A enzyme common that cytochrome uses heme P450 as a cofactorenzyme and exploitedimitates for monooxygenases. this purpose is cytochrome Studies revealed P450 thatBM-3, the an cofactor enzyme increases that uses the heme chemical as a reactioncofactor ratesand and imitatesallows monooxygenases. the enzyme to function Studies revealed efficiently. that Thus, the cofactor monooxygenases increases the are chemical essential toreaction synthetic rates chemistry and allowsto notthe enzyme only increase to function efficiency efficiently. but also Thus, enable monooxygenases a reaction within are essential the constraints to synthetic of a chemistry living system, to notsuch only as increase temperature efficiency or excessive but also amounts enable ofa reaction substrate. within The modificationthe constraints of biocatalystsof a living system, for natural suchsynthetic as temperature use is aor relevantexcessive field amounts in biochemistry of substrate. today. The modification There has beenof biocatalysts much work for done natural on the syntheticmodification use is a of relevant enzymes field that performin biochemistry hydroxylation today.reactions. There has The been engineering much work of these done enzymes on the has modificationbeen investigated of enzymes to modifythat pe aspectsrform hydroxylation such as regioselectivity, reactions. stereoselectivity,The engineering andof these enantioselectivity, enzymes has amongbeen others.investigated Much focusto modify is placed aspects on different such variations as regioselectivity, of the P450 BM-3 stereoselectivity, enzyme. Other enzymesand enantioselectivity,being studied among for their others. hydroxylation Much focus reactions is placed include on different heme-containing variations biocatalysts of the P450 such BM-3 as the enzyme.PikC Other enzyme. enzymes Variations being of studied the P450 for BM-3 their can hydroxylation improve its industrial reactions applicationsinclude heme-containing since it is capable biocatalysts such as the PikC enzyme. Variations of the P450 BM-3 can improve its industrial applications since it is capable of hydroxylating alkanes [30]. A light-activated hybrid P450 BM-3,

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WT/L407C-Ru1, hydroxylates 10-undecenoic acid exclusively at the allylic position. The reaction ofobtains hydroxylating the highly alkanes enantiomerically [30]. A light-activated enriched (R hybrid)-9-hydroxy-10-undecnoic P450 BM-3, WT/L407C-Ru acid in1, hydroxylates85% ee [31]. 10-undecenoicMoreover, cytochrome acid exclusively P450 ΒΜ at-3 the extracted allylic position. from Bacillus The reaction megaterium obtains was the engineered highly enantiomerically by directed enrichedevolution ( Rto)-9-hydroxy-10-undecnoic hydroxylate linear alkanes. acid Variant in 85% 9-10A-A328Vee [31]. Moreover, has the cytochrome ability to P450produce BM-3 S-2-octanol extracted from Bacillusoctane. megateriumThis productwas is engineered obtained in by 40% directed ee, with evolution excellent to hydroxylate total turnovers linear of alkanes. 2000. Another Variant 9-10A-A328Vvariant, 1-12G, has was the made ability from to the produce same Scytochrome-2-octanol fromas P450 octane. BM-3 This via mutagenesis. product is obtained This variant in 40% alsoee, withhydroxylates excellent in total the turnovers 2-position of on 2000. alkanes Another larger variant, than hexane, 1-12G, wasincluding made fromoctane, the nonane, same cytochrome and decane, as P450to produce BM-3 viaR-2-alcohols mutagenesis. in 40%–55% This variant ee [30]. also hydroxylates in the 2-position on alkanes larger than hexane,In another including study, octane, a cytochrome nonane, and P450 decane, BM-3 to producevariant wasR-2-alcohols produced in to 40%–55% aid in theee [production30]. of indirubin,In another an anticancer study, a cytochrome drug. The P450 enzyme BM-3 variantwas engineered was produced using to aidsite-directed in the production saturation of indirubin,mutagenesis. an anticancerThe mutant, drug. D168W, The enzymealtered the was enzyme engineered so that using the site-directedenzyme produced saturation indirubin mutagenesis. (~90%) Theinstead mutant, of indigo D168W, (~85%), altered which the was enzyme synthesized so that the by enzymeits parent produced enzyme. indirubinThis change (~90%) was caused instead by of indigothe shift (~85%), of hydroxylation which was synthesizedC-3 to C-2 [32]. by its Furt parenthermore, enzyme. BM-3 This variant change 9-10A was causedwas engineered by the shift to ofhydroxylate hydroxylation aromatic C-3 tocarboxylic C-2 [32]. acid. Furthermore, This enzyme BM-3 has variant the highest 9-10A efficiency was engineered with propyl to hydroxylate and butyl aromaticesters. When carboxylic variant acid. 9-10A This was enzyme constructed has the with highest the e ffiF87Aciency mutation, with propyl the total and butylturnover esters. numbers When variant(TTN) increased 9-10A was to constructed its highest withamount the F87Aof 1640. mutation, This enzyme the total obtained turnover a propyl numbers mandelate (TTN) increased in 93% ee to. itsWhen highest 9-10A-F87A amount ofis 1640. combined This enzyme with an obtained NADPH a propylregeneration mandelate system, in 93% utilizingee. When NADPH 9-10A-F87A as the is combinedcofactor, the with TTN an NADPHimproves regeneration to over 5800 system, [33]. Regioselectivity utilizing NADPH is ashighly thecofactor, affected theby TTNthe anchoring improves togroup over of 5800 an [enzyme.33]. Regioselectivity This can be demonstrated is highly affected by bythe thebiosynthetic anchoring enzyme, group of P450 an enzyme. monooxygenase This can bePikC demonstratedD50N-RhFRED. by The the natural biosynthetic anchoring enzyme, group P450 desoamine monooxygenase gives a 1:1 PikC mixtureD50N-RhFRED. of methymycin The natural and anchoringneomethymycin, group desoaminebut by using gives a synthetic a 1:1 mixture anchor, of the methymycin ratio can be and altered neomethymycin, [34]. but by using a syntheticAlthough anchor, stereoselective the ratio can epoxidation be altered [34 of]. terminal alkenes has been reported in a few cases, using a chemicalAlthough catalyst stereoselective in the epoxidation epoxidation of simple of terminal linear alkenes terminal has alkenes been reported has not in yet a few been cases, reported. using aCytochrome chemical catalyst P450 BM-3 in the enzymes epoxidation (Scheme of simple 4) paired linear with terminal saturated alkenes mutagenesis has not and yet recombination been reported. Cytochromehave been applied P450 BM-3 to analyze enzymes the (Schemeeffective4 )epoxidation paired with catalysts saturated for mutagenesis linear terminal and recombinationalkenes. In the havepresence been of applied cell lysate to analyzeand alcohol the edehydrogenase,ffective epoxidation various catalysts P450 enzymes for linear could terminal reproduce alkenes. NADPH In the presencecofactor. ofMutagenesis cell lysate andand alcoholscreening dehydrogenase, helped us to achieve various optimal P450 enzymes results. could Exemplarily, reproduce P450 NADPH BM-3 cofactor.from Bacillus Mutagenesis megaterium and was screening utilized helped for the us toenantioselective achieve optimal epoxidation results. Exemplarily, of terminal P450 alkenes BM-3 fromand Bacillusforms (R megaterium)- and (S)-epoxideswas utilized with for desirable the enantioselective catalytic turnovers; epoxidation this ofcatalyst terminal and alkenes the NADPH and forms oxidize (R)- andthe terminal (S)-epoxides alkene with into desirable a terminal catalytic epoxide turnovers; produc thist [35]. catalyst Denard and et theal. experimentally NADPH oxidize determined the terminal a alkeneone-pot into tandem a terminal reaction epoxide to produce product aryl [epoxides35]. Denard from et a al.mixture experimentally of stilbene-derived determined alkenes a one-pot in the tandempresence reaction of ruthenium. to produce This aryl novel epoxides developmen from a mixturet combines of stilbene-derived alkene metathesis alkenes with in the enzymatic presence ofepoxidation. ruthenium. It Thisfavors novel the utilization development of (Z combines)-stilbene alkenesubstrates metathesis and produced with enzymatic an epoxide epoxidation. product with It favorsmoderate the utilizationyields and of good (Z)-stilbene enantioselectivity. substrates and Su producedbstantial anenzyme epoxide and product reaction with engineering moderate yields with andcytochrome good enantioselectivity. P450 BM-3 is currently Substantial under enzyme investig andation reaction to engineeringimprove catalytic with cytochrome activity and P450 increase BM-3 isenzymatic currently underreaction investigation rates [36]. to The improve enantioselecti catalytic activityve total and synthesis increase enzymaticof norditerpenoid reaction ratesalkaloid [36]. Thenigelladine enantioselective A was accomplished total synthesis within of norditerpenoid an expedien alkaloidt 12 steps nigelladine and with Aan was overall accomplished yield of 5%. within The anengineered expedient enzyme 12 steps cytochrome and with an P450 overall BM-3 yield was of used 5%. to The catalyze engineered the chemo- enzyme and cytochrome regioselective P450 allylic BM-3 wasC–H used bond to oxidation catalyze thein the chemo- intermediate and regioselective step of th allylice reaction. C–H bondThe reaction oxidation relied in the on intermediate the asymmetric step ofallylic the reaction.alkylation The that reaction constructed relied onthe the quaternary asymmetric center allylic of alkylation the product, that with constructed prominent the quaternary yield and centerenantioselectivity; of the product, the with cytochrome prominent P450 yield BM-3 and enzy enantioselectivity;me allowed Steven the cytochrome A. Loskot P450 and BM-3coworkers enzyme to allowedsite-select Steven the secondary A. Loskot allylic and coworkersoxidation without to site-select major the intermediate secondary allylicinterference. oxidation Their without results major have intermediaterevealed that interference.bio-transformations Their results have have the revealedpotential thatto circumvent bio-transformations the limitations have the of potentialtraditional to circumventchemical syntheses the limitations [37]. of traditional chemical syntheses [37].

Scheme 4. Oxidation of 1-hexene by P450 BM-3.

Catalysts 2020, 10, x FOR PEER REVIEW 7 of 25 Catalysts 2020, 10, 832x FOR PEER REVIEW 7 of 25

ExperimentationExperimentation withwith intermolecularintermolecular nitrenenitrene trantransfersfer accomplishedaccomplished byby utilizingutilizing thethe cytochromecytochrome P450P450 ExperimentationBM-3BM-3 enzymeenzyme foundfound with inin intermolecular BacillusBacillus megateriummegaterium nitrene waswas transfer performedperformed accomplished byby FarwellFarwell by etet utilizing al.al. TheThe the catalystcatalyst cytochrome allowsallows forforP450 thethe BM-3 intermolecularintermolecular enzyme found insertioninsertion in Bacillus ofof megaterium nitrogen-connitrogen-conwastainingtaining performed functionalfunctional by Farwell groupsgroups et al. Theintointo catalyst thioethersthioethers allows andand for subsequentlysubsequentlythe intermolecular sulfimides.sulfimides. insertion TwoTwo of factors nitrogen-containingfactors thatthat heavilyheavily impaimpa functionalctct thethe groupsreactionreaction into areare thioethers thethe electronicelectronic and properties subsequentlyproperties ofof thethesulfimides. substratessubstrates Two used,used, factors whichwhich that cancan heavily influenceinfluence impact reactivity,reactivity, the reaction andand are thethe the aminoamino electronic acidacid groupsgroups properties attached,attached, of the whichwhich substrates cancan varyvaryused, thethe which raterate can andand influence stereoselectivitystereoselectivity reactivity, ofof andthethe sulfimidationsulfimidation the amino acid reaction.reaction. groups attached,SulfideSulfide substituentssubstituents which can varymaymay thealsoalso rate impactimpact and sulfimidesulfimidestereoselectivity formation,formation, of the asas sulfimidation sulfimidesulfimide sideside reaction. productsproducts Sulfide werewere substituents introduced,introduced, may indicatingindicating also impact thatthat sulfimide thethe nitrenoidnitrenoid formation, maymay onlyonlyas sulfimide permitpermit insertioninsertion side products intointo reactivereactive were introduced, sulfides.sulfides. TheThe indicating orientationorientation that of theof thethe nitrenoid redoxredox statestate may hemeheme only permitgroup,group, insertioninin eithereither thetheinto presencepresence reactive sulfides.oror absenceabsence The ofof orientation thethe nitrenenitrene of sourcesource the redox andand state thethe sulfidesulfide heme group, acceptor,acceptor, in either includingincluding the presence thethe redoxredox or absencestatestate ofof thetheofthe hemeheme nitrene group,group, source helpedhelped and toto the determdeterm sulfideineine acceptor, thethe nitrenenitrene including transfer,transfer, the butbut redox thethe usageusage state of ofof the cytochromecytochrome heme group, P450P450 helped BM-3BM-3 allowsallowsto determine forfor intermolecularintermolecular the nitrene transfer, nitrenenitrene transfertransfer but the in usagein thethe form ofform cytochrome ofof sulfimidationsulfimidation P450 BM-3 [38].[38]. allows for intermolecular nitreneEnzymaticEnzymatic transfer mutation inmutation the form alsoalso of proved sulfimidationproved toto alteralter [ 38 cyclopcyclop]. ropanationropanation reactionsreactions dealingdealing withwith aa heteroatom.heteroatom. CytochromeCytochromeEnzymatic P450P450 mutation BM3BM3 variantsvariants also proved werewere toengineeredengineered alter cyclopropanation toto catalyzecatalyze thethe reactions cyclopropanationcyclopropanation dealing with ofof a heteroatom-heteroatom- heteroatom. bearingbearingCytochrome alkenes.alkenes. P450 BM3CytochromeCytochrome variants were P411P411 engineered hadhad thethe to catalyzecapacapabilitybility the cyclopropanationofof synthesizingsynthesizing ofheteroatom-bearingheteroatom-bearing heteroatom-bearing cyclopropanes.cyclopropanes.alkenes. Cytochrome Furthermore,Furthermore, P411 had evolevol thevedved capability P411P411 providedprovided of synthesizing nitrogen,nitrogen, heteroatom-bearingoxygen,oxygen, andand sulfur-cyclopropanessulfur-cyclopropanes cyclopropanes. withwithFurthermore, highhigh diasteroselectivitiesdiasteroselectivities evolved P411 provided andand enantioselectienantioselecti nitrogen,vities.vities. oxygen, WithWith and certaincertain sulfur-cyclopropanes mutations,mutations, aa singlesingle with parentparent high enzymeenzymediasteroselectivities becamebecame aa selectiveselective and enantioselectivities. catalystcatalyst forfor ciscis andand With transtrans certain heteroatomheteroatom mutations, substitutedsubstituted a single cyclopropanescyclopropanes parent enzyme synthesissynthesis became withwitha selective preferredpreferred catalyst diasteroselectivitiesdiasteroselectivities for cis and trans andandheteroatom enantioselectivitiesenantioselectivities substituted [39].[39]. cyclopropanes synthesis with preferred diasteroselectivitiesHemeproteinHemeprotein biocatalystbiocatalyst and enantioselectivities isis anotheranother typetype [39 ofof]. hyhydroxylase.droxylase. AsAs aa heme-containingheme-containing enzyme,enzyme, SfmDSfmD isis usedusedHemeprotein toto hydroxylatehydroxylate biocatalyst thethe aromaticaromatic is another ringring type ofof tyrosinetyrosine of hydroxylase. inin thethe presencepresence As a heme-containing ofof hydrogenhydrogen peroxideperoxide enzyme, SfmDandand anan is oxidantoxidantused to hydroxylate(Scheme(Scheme 5).5). ItIt the isis aromaticpossiblypossibly ringresponsibleresponsible of tyrosine forfor producing inproducing the presence 3-OH-5-Me-Tyr3-OH-5-Me-Tyr of hydrogen fromfrom peroxide 3-Me-Tyr.3-Me-Tyr. and an ThisThis oxidant hashas significance(Schemesignificance5). It isforfor possibly thethe biosynthesisbiosynthesis responsible for ofof producing tetrahydroisoquinolinetetrahydroisoquinoline 3-OH-5-Me-Tyr fromfamilyfamily 3-Me-Tyr. membersmembers This has[40].[40]. significance AnotherAnother hemeproteinhemeproteinfor the biosynthesis isis wild-typewild-type of tetrahydroisoquinoline RhodothermusRhodothermus marinusmarinus family (Rma)(Rma) members cytochromecytochrome [40]. c. Anotherc. TheThe variant,variant, hemeprotein RmaRma cytochromecytochrome is wild-type cc TQL,RhodothermusTQL, transformstransforms marinus alkenesalkenes(Rma) toto chiralchiral cytochrome 1,2-amino1,2-amino c. Thealcoholsalcohols variant, viavia direct Rmadirect cytochrome amino-hydroxylation.amino-hydroxylation. c TQL, transforms ThisThis enzymeenzyme alkenes isis thermallythermallyto chiral 1,2-amino stablestable andand alcohols demonstrateddemonstrated via direct highhigh amino-hydroxylation. enantioselecenantioselectivity,tivity, withwith This totaltotal enzyme turnoversturnovers is thermally ofof upup toto stable 25002500 and and 90%90%demonstrated eeee underunder optimizedoptimized high enantioselectivity, conditionsconditions [41].[41]. with G.G. total SelloSello turnovers etet al.al. (Scheme(Scheme of up to 6)6) 2500 analyzedanalyzed and 90% variousvariousee under reactionsreactions optimized thatthat wouldwouldconditions turnturn [41 aa]. styrenestyrene G. Sello etsubstratesubstrate al. (Scheme inintoto6 )enantiopureenantiopure analyzed various 1,2-amino1,2-amino reactions alcohoalcoho that wouldl.l. TheThe turnstyrenestyrene a styrene oxidizedoxidized substrate intointo enantiopureenantiopureinto enantiopure phenylphenyl 1,2-amino epoxideepoxide alcohol. viavia anan E.E. The colicoli styrene containingcontaining oxidized monooxygenasemonooxygenase into enantiopure fromfrom phenyl P.P. fluorescensfluorescens epoxide.. TheThe via anepoxideepoxideE. coli thenthencontaining reactedreacted monooxygenase withwith aa nitrogennitrogen from nucleophilenucleophileP. fluorescens toto openopen. The upup epoxide thethe ring,ring, then leavingleaving reacted anan alcoholalcohol with a nitrogengroupgroup andand nucleophile anan amineamine group.group.to open TheirTheir up the workwork ring, leavingindicatedindicated an thatthat alcohol 2-amino,2-amino, group and1-,1-, andand an amine 2-phenyl2-phenyl group. ethanolsethanols Their work werewere indicated producedproduced that withwith 2-amino, highhigh enantiopurities1-,enantiopurities and 2-phenyl andand ethanols inin goodgood were yieldsyields produced [42].[42]. with high enantiopurities and in good yields [42].

SchemeScheme 5.5. BiosyntheticBiosyntheticBiosynthetic pathwaypathway pathway ofof of 3-OH-5-Me-Tyr.3-OH-5-Me-Tyr. 3-OH-5-Me-Tyr.

SchemeScheme 6.6. RegioisomericRegioisomeric additionaddition ofof nitrogennitrogen toto epoxides.epoxides.

A three-enzyme tandem reaction was used to stereoselectively synthesize γ-oxyfunctionalyzed AA three-enzymethree-enzyme tandemtandem reactionreaction waswas usedused toto stereoselectivelystereoselectively synthesizesynthesize γγ-oxyfunctionalyzed-oxyfunctionalyzed amino acids (Scheme7). Combining N-acylamino acid racemase (NAAAR), L-selective aminoacylase aminoamino acidsacids (Scheme(Scheme 7).7). CombiningCombining N-N-acylaminoacylamino acidacid racemaseracemase (NAAAR),(NAAAR), LL-selective-selective aminoacylaseaminoacylase fromfrom GeobacillusGeobacillus thermoglucosidasiusthermoglucosidasius,, andand isoleucineisoleucine dioxygenasedioxygenase fromfrom BacillusBacillus thuringiensisthuringiensis pairedpaired

Catalysts 2020, 10, 832 8 of 25

Catalysts 2020, 10, x FOR PEER REVIEW 8 of 25 from Geobacillus thermoglucosidasius, and isoleucine dioxygenase from Bacillus thuringiensis withpaired dynamic with dynamic kinetic resolution kinetic resolution allows race allowsmic N-acetylmethionine racemic N-acetylmethionine to be converted to to be L converted-methionine- to (LS-methionine-()-sulfoxide, withS)-sulfoxide, a very high with experimental a very high experimentalyield of 97% yieldand 95% of 97% de under and 95% optimizedde under conditions. optimized Theconditions. three-enzyme The three-enzyme cascade reaction cascade results reaction in the resultsseparation in the of separationthe oxyfunctionalization of the oxyfunctionalization step from the dynamicstep from kinetic the dynamic resolution kinetic step, resolution which step,makes which it possible makes it for possible each forenzyme each enzymeto work to at work the atideal the temperatureideal temperature [43]. [43].

Scheme 7. BtDO catalyzes the stereoselective γ-specific-specific oxidation of of aliphatic L-amino acids such as L-methionine (above) and several branched-chainbranched-chain amino acids (below).

A stereoselective, one-pot biocatalytic total sy synthesisnthesis reaction was employed to synthesize bisorbicillinoid natural products and unnatural side-chain analogues by utilizing recombinant oxidoreductase SorbC from thethe sorbicillinsorbicillin biosyntheticbiosynthetic genegene cluster.cluster. Applying Applying an enzymatic oxidative dearomatization or or dimerization cascade cascade allows allows for for the the direct direct preparation of of sorbiquinol, trichodimeral, and disorbicillinol and the synthesissynthesis of bisorbibutenolide. The bio-enzyme SorbC has high stereoselectivity stereoselectivity that that allows allows synthetic synthetic access access for for natural natural products. products. Sib Sib et al. et al.also also found found that thatthe spontaneousthe spontaneous formation formation of bisorbicillinoids of bisorbicillinoids can be can accomplished be accomplished without without biocat biocatalysts,alysts, indicating indicating that thethat natural the natural compounds compounds produced produced can can be beassemble assembledd in in non-enzymatic non-enzymatic natural natural producers. producers. More investigation is in progress to determine thethe fullfull syntheticsynthetic potentialpotential ofof thethe SorbCSorbC enzymeenzyme [[44].44]. In 2016, 2016, Priyanka Priyanka Bajaj and and her her team team (Scheme (Scheme 88)) developed aa newnew strategy toto engineer myoglobin variants that increase the synthesis of 1- 1-carboxyl-2-aryl-cyclopropanescarboxyl-2-aryl-cyclopropanes with trans- ( 1R, 2R ), a product product used in conjunction with its stereo complement of trans- ( 1S, 2S ).). This This method method improved improved the the essential essential asymmetric cyclopropanation cyclopropanation utilized utilized in in drugs drugs (s (suchuch as as Tranylcypromine, Tranylcypromine, Tasimelteon, Tasimelteon, Ticagrelor, Ticagrelor, and a TRPV1 inhibitor), thereby showing that the ad additiveditive mutations of myog myoglobinlobin gave way to the higher control of stereoselectivity in cyclopropanation reactionsreactions [[45].45].

Scheme 8. ActivityActivity and and selectivity selectivity of of representative representative myo myoglobinglobin variants variants for for styrene styrene cyclopropanation cyclopropanation with ethyl-α-diazoacetate (EDA).

2.2. Transferases E.C.2

2.2.1. Enzymes Applied for Alkylation Polymethoxy flavonoids (PMFs) have shown myriad health-supporting applications. However, one of the greatest challenges that limits their use is small production from plant cells. Plant cells

Catalysts 2020, 10, 832 9 of 25

2.2. Transferases E.C.2

2.2.1. Enzymes Applied for Alkylation Polymethoxy flavonoids (PMFs) have shown myriad health-supporting applications. However, Catalysts 2020, 10, x FOR PEER REVIEW 9 of 25 one of the greatest challenges that limits their use is small production from plant cells. Plant cells exploit the methylation methylation of of hydroxyl hydroxyl groups groups on on flav flavonoidsonoids catalyzed catalyzed by by O-me O-methyltransferasesthyltransferases (FOMT) (FOMT) toto produce PMFs. This This cate categorygory of of enzymes has very high substrate specificity specificity and regioselectivity during the synthetic process. Recombinant Recombinant CdFO CdFOMT5MT5 (one (one of of the the genes genes encoding encoding FOMT FOMT from from Citrus depressa)) expressed expressed in in E.E. coli coli cellscells exhibited exhibited a a preference preference for for flav flavonolonol to to other other types types of of flavonoids. flavonoids. Moreover, it converted 3-, 5-, 6-, and 7-hydroxyl groups on flavone flavone to methyl groups under mild conditions. Furthermore, Furthermore, taking taking advantage advantage of of this this engineering engineering strategy strategy of of mutation of CdFOMT5 could provide alternative routes to synthesizing various PMFs [[46].46]. The installation installation of of fluoroalkyl fluoroalkyl gr groupsoups is isan an abiological abiological process process that that could could significantly significantly alter alter the pharmacologicalthe pharmacological properties properties of a molecule. of a molecule. Despite Despite the ubiquity the ubiquity of C–H bonds of C–H in bondscomplex in bioactive complex 3 bioactive molecules, the fluoroalkylation of enantioselective3 C(sp ) H remains a challenge due to molecules, the fluoroalkylation of enantioselective C(sp )−H remains− a challenge due to inherent problemsinherent problems coupled with coupled cross-coupling with cross-coupling pathways pathways of the C- of fluoroalkyl the C- fluoroalkyl bond in bond the transition in the transition metal- catalyzedmetal-catalyzed reaction. reaction. To circumvent To circumvent this thisissue, issue, the theengineered engineered cytochrome cytochrome P450s P450s could could catalyze catalyze the the 3 addition of fluoroalkyl carbene intermediates to C(sp ) H bonds3 (Scheme9). The catalyst demonstrated addition of fluoroalkyl carbene intermediates to− C(sp )−H bonds (Scheme 9). The catalyst excellent selectivity toward α-amino C H bonds, with up to 99% ee. Additionally, the enzyme is demonstrated excellent selectivity toward− α-amino C−H bonds, with up to 99% ee. Additionally, the enzymecapable is of capable introducing of introducing a variety ofa variety fluoroalkyl of fluoroalkyl groups including groups including pentafluoropropyl pentafluoropropyl entity through entity throughthe same the process, same withprocess, great with activity great and activity enantioselectivity. and enantioselectivity. This versatile This enzyme versatile could enzyme potentially could potentiallysynthesize fluorinatedsynthesize fluori bioactivenated compounds bioactive compounds with directed with evolution directed [47 evolution]. [47].

Scheme 9. SubstrateSubstrate scope scope of of P411-PFA-catalyzed P411-PFA-catalyzed C −HH tri trifluoroethylationfluoroethylation reaction. reaction. − Prenyltransferases such such as as FtmPT1 from from Aspergillus fumigatus transfertransfer a a prenyl prenyl moiety regiospecificallyregiospecifically toto thethe C-2 C-2 position position of of brevianamide brevianamide F (cyclo-F (cyclo-L-Trp-L-Trp-L-Pro).L-Pro). Studies Studies have have suggested suggested that thatthe C-2the prenylationC-2 prenylation of various of various tryptophan-containing tryptophan-containing cyclic cyclic dipeptides dipeptides can be can catalyzed be catalyzed by the by same the sameenzyme enzyme in the in presence the presence of dimethylallyl of dimethylallyl diphosphate. diphosphate. However, However, HPLC and HPLC NMR and analyses NMR analyses detected detectedC-3 prenylated C-3 prenylated derivatives derivatives as by-products, as by-products, with yields with of upyields to 19%, of up while to 19%, regularly whileC-2-prenylated regularly C-2- prenylatedindolines were indolines still perceived were still as perceived main products. as main This products. indicates This that indicates prenylation that prenylation catalyzed by catalyzed FtmPT1 bycan FtmPT1 be intermittent, can be intermittent, which increases which the increases challenge th fore challenge purification; for however,purification; it alsohowever, increases it also the increaseschemoenzymatic the chemoenzymatic synthesis structure synthesis diversity structure [48]. diversity [48].

2.2.2. Enzymes Enzymes Applied Applied for for Amination Amination Optically active amines are very important cons constituentstituents for various bioactive compounds, with importantimportant pharmaceutical, agrochemical, agrochemical, or or industri industrialal applications. In In principle, various synthetic routes catalyzed by different different enzymes, including hy hydrolases,drolases, oxidoreductases, or transferases, can be applied for the production of optically active pr primaryimary amines. However, However, amine transaminases offer offer a unique way to obtain pure amine enantiomers directly from prochiralprochiral ketones.ketones. Moreover, these catalysts elucidateelucidate pronounced pronounced enantio- enantio- and and regioselectivity. regioselectivity. Until Until now, now, compared compared to many to ( Smany)-selective (S)- selectiveamines transaminases amines transaminases (S-ATAs), ( onlyS-ATAs), a few only (R)-amine a few transaminases (R)-amine transaminases (R-ATAs) were (R-ATAs) commercially were commerciallyavailable and extensivelyavailable and investigated. extensivelyR investigated.-ATAs such asR-ATAs AspTer such from asAspergillus AspTer from terreus Aspergillus, PenChry terreus from, PenChry from Penicillium, etc., showed excellent conversion rates (>99%) and enantiomeric purities of >99% ee when they were exploited to catalyze the asymmetric synthesis of targeted amines (Scheme 10) [49]. Regioselectivity is a major challenge in amine transformations due to two or more reactive centers in the molecule. Various (S)- and (R)-stereoselective ω-transaminases have provided protecting group-free strategies for the bio-amination of diketone compounds. A number of 1,5- diketones were selected to synthesize the corresponding optically pure amino ketones catalyzed by ω-transaminases (>92% ee). Simon’s study also suggested that the intermediate underwent a

Catalysts 2020, 10, x FOR PEER REVIEW 10 of 25 Catalysts 2020, 10, 832 10 of 25 spontaneous ring-closure reaction (Scheme 11), in which Δ1-piperideines were produced. Diastereoselectively reducing these products yields a second chiral center, which will provide the Penicilliumpossibility, etc.,of making showed chiral excellent 2,6- conversiondisubstituted rates piperidine (>99%) ands. Their enantiomeric method purities is one ofof> 99%the eeshortestwhen theysynthetical were exploited protocols to to catalyze date that the expands asymmetric the tool synthesisbox for ofmore targeted selective amines amine (Scheme transformations 10)[49]. [50].

Catalysts 2020, 10, x FOR PEER REVIEW 10 of 25 spontaneous ring-closure reaction (Scheme 11), in which Δ1-piperideines were produced. Diastereoselectively reducing these products yields a second chiral center, which will provide the possibility of making chiral 2,6-disubstituted piperidines. Their method is one of the shortest synthetical protocols to date that expands the toolbox for more selective amine transformations [50].

SchemeScheme 10.10. Reaction scheme for thethe asymmetricasymmetric synthesissynthesis of chiralchiral ((RR)-amines)-amines withwith thethe ((RR)-amine)-amine transaminasestransaminases (R-ATAs)(R-ATAs) in combinationin combination with the with lactate the dehydrogenase lactate dehydrogenase (LDH)/lactate dehydrogenase(LDH)/lactate (GDH)dehydrogenase system to (GDH) shift the system equilibrium to shift towardsthe equilibrium product towards formation. product formation.

Regioselectivity is a major challenge in amine transformations due to two or more reactive centers in the molecule. Various (S)- and (R)-stereoselective ω-transaminases have provided protecting group-free strategies for the bio-amination of diketone compounds. A number of 1,5-diketones were selected to synthesize the corresponding optically pure amino ketones catalyzed by ω-transaminases (>92% ee). Simon’s study also suggested that the intermediate underwent a spontaneous ring-closure reaction (Scheme 11), in which ∆1-piperideines were produced. Diastereoselectively reducing these products yieldsScheme a second 10. chiralReaction center, scheme which for the will asymmetric provide thesynthesis possibility of chiral of ( makingR)-amines chiral with 2,6-disubstitutedthe (R)-amine piperidines.transaminases Their method(R-ATAs) is one in ofcombination the shortest syntheticalwith the protocolslactate dehydrogenase to date that expands (LDH)/lactate the toolbox for moredehydrogenase selective amine (GDH) transformations system to shift the [50 equilibrium]. towards product formation.

Scheme 11. Regioselective amination of various 1,5-diketones. Compared to inserting oxygen atoms into the inactivated carbon–hydrogen bond, enzyme- catalyzed oxidative amination is more constrained. Where suitable enzymes were absent in nature, synthetic chemists started exploiting evolutionary optimization to engineer natural enzymes. Mutants of P450 enzymes such as P411Bm3-CIS are capable of the amination of 2,4,6-triethylben-zene- 1-sulfonylazide and its analogues, with excellent enantioselectivity of 430 total turnover numbers and 86% ee for benzosultam (Scheme 12) [51]. Chemically, the synthesis of optically pure L-tert-leucine has low yields and enantio-purity. Nevertheless, many enzyme-catalyzed reactions were obstructed, with merely 50% yields. Recent studies show that leucine dehydrogenase identified and cloned from Exiguobacterium sibiricum Scheme 11. Regioselective amination of various 1,5-diketones. (EsLeuDH) has high enantioselectivityScheme 11. Regioselective via the aminationenzymatic of reductive various 1,5-diketones. amination reaction to produce L- Compared to inserting oxygen atoms into the inactivated carbon–hydrogen bond, enzyme- catalyzed oxidative amination is more constrained. Where suitable enzymes were absent in nature, synthetic chemists started exploiting evolutionary optimization to engineer natural enzymes. Mutants of P450 enzymes such as P411Bm3-CIS are capable of the amination of 2,4,6-triethylben-zene- 1-sulfonylazide and its analogues, with excellent enantioselectivity of 430 total turnover numbers and 86% ee for benzosultam (Scheme 12) [51]. Chemically, the synthesis of optically pure L-tert-leucine has low yields and enantio-purity. Nevertheless, many enzyme-catalyzed reactions were obstructed, with merely 50% yields. Recent studies show that leucine dehydrogenase identified and cloned from Exiguobacterium sibiricum (EsLeuDH) has high enantioselectivity via the enzymatic reductive amination reaction to produce L-

Catalysts 2020, 10, 832 11 of 25

CatalystsCompared 2020, 10, x FOR to PEER inserting REVIEW oxygen atoms into the inactivated carbon–hydrogen11 bond, of 25 enzyme-catalyzed oxidative amination is more constrained. Where suitable enzymes were absent tert-leucinein nature, (Scheme synthetic 13). chemists Compared started to other exploiting reported evolutionary LeuDH, the enzyme optimization EsLeuDH to engineerexhibited naturalhigher activityenzymes. at low Mutants temperatures of P450 and enzymes better thermostability such as P411 atBm3 high-CIS temperatures. are capable In of addition, the amination large scale of experimental2,4,6-triethylben-zene-1-sulfonylazide results indicated that the and enzyme its analogues, has the highest with excellent space-time enantioselectivity yield (over 80%) of 430 among total allturnover LeuDHs, numbers with >99% and 86%ee [52].ee for benzosultam (Scheme 12)[51].

Catalysts 2020, 10, x FOR PEER REVIEW 11 of 25

tert-leucine (Scheme 13). Compared to other reported LeuDH, the enzyme EsLeuDH exhibited higher activity at low temperatures and better thermostability at high temperatures. In addition, large scale experimental results indicated that the enzyme has the highest space-time yield (over 80%) among all LeuDHs, withSchemeScheme >99% 12. 12. ee EnantioselectiveEnantioselective [52]. intramolecular intramolecular C–H C–H amination amination catalyzed catalyzed by by P411. P411.

Chemically, the synthesis of optically pure L-tert-leucine has low yields and enantio-purity. Nevertheless, many enzyme-catalyzed reactions were obstructed, with merely 50% yields. Recent studies show that leucine dehydrogenase identified and cloned from Exiguobacterium sibiricum (EsLeuDH) has high enantioselectivity via the enzymatic reductive amination reaction to produce Scheme 13. Substrate specificity of EsLeuDH in reductive amination and oxidative deamination. L-tert-leucine (Scheme 13). Compared to other reported LeuDH, the enzyme EsLeuDH exhibited higher 2.2.3.activity Enzymes at low temperaturesApplied for Glycosylation and better thermostability at high temperatures. In addition, large scale experimental results indicated that the enzyme has the highest space-time yield (over 80%) among all LeuDHs,Chemically, with >Scheme99% to controlee 12.[52 Enantioselective]. the regio- and intramolecular stereoselectivity C–H ofamination glycosidic catalyzed bonds, by the P411. synthesizing of these oligosaccharides involves various protective group manipulations. Moreover, the challenge of large-scale production limits their application in the food and medical industries. Enzymatic glycosylations provide highly expedient transformation approaches in synthetic chemistry and can be easily exploited broadly to the directed alteration of small molecules and polymers via the covalent attachmentSchemeScheme of glycosyl 13.13. Substrate residues specificityspecificity. Sucrose ofof EsLeuDHEsLeuDH phosphorylase inin reductivereductive was aminationamination successfully andand oxidative oxidativeemployed deamination.deamination. in the synthesis of (R)-2-O-α-D-glucopyranosyl glyceric acid amide from single-step diastereoselective glucosylation, utilizing2.2.3.2.2.3. Enzymes sucrose Applied as a glucosyl forfor GlycosylationGlycosylation donor and racemic glyceric acid amide as an acceptor, with d.e. > 83% and high yield (Scheme 14). This protocol could potentially simplify the access to biomimetic Chemically,Chemically, toto controlcontrol thethe regio-regio- andand stereoselectivitystereoselectivity ofof glycosidicglycosidic bonds,bonds, thethe synthesizingsynthesizing ofof glycoside via esterification processes [53]. In another study, Milosavic reported that, when using thesethese oligosaccharidesoligosaccharides involves involves va variousrious protective protective group group manipulations. manipulations. Moreov Moreover,er, the the challenge challenge of sucrose and arbutin, α-glucosidase from Saccharomyces cerevisiae was highly stereospecific for the oflarge-scale large-scale production production limits limits their their application application in in the the food andand medicalmedical industries.industries. EnzymaticEnzymatic formation of 4-hydroxyphenyl-β-isomaltoside in a molar yield of 50% with reference to arbutin glycosylationsglycosylations provideprovide highly highly expedient expedient transformation transformation approaches approaches in in synthetic synthetic chemistry chemistry and and can can be (Scheme 15). Their study also suggested that α-glucosidase can be exploited to glycosylation reaction easilybe easily exploited exploited broadly broadly to to the the directed directed alteration alteration of of small small molecules molecules and and polymers polymers via via thethe covalentcovalent with other bioactive phenolic compounds comprising a hydroquinone moiety [54]. attachmentattachment ofof glycosylglycosyl residues.residues. SucroseSucrose phosphorylasephosphorylase waswas successfullysuccessfully employedemployed inin thethe synthesissynthesis N-acetyl-β-D-galactosamine (GalNAc) and N-acetyl-β-D-glucosamine (GlcNAc) residue ofof ((RR)-2-O-)-2-O-αα--DD-glucopyranosyl-glucopyranosyl glycericglyceric acidacid amideamide from single-step diastereoselective glucosylation, containing oligosaccharides are essential for many biological activities. They exist in the sugar unit utilizingutilizing sucrosesucrose asas aa glucosylglucosyl donordonor andand racemicracemic glycericglyceric acidacid amideamide asas anan acceptor,acceptor, withwithd.e. d.e. >> 83% of blood group P-related glycan, many tumor-related carbohydrate antigens gangliosides, milk andand highhigh yield yield (Scheme (Scheme 14). 14). This This protocol protocol could could potentially potentially simplify simplify the access the to access biomimetic to biomimetic glycoside oligosaccharides, and chitin. Chen’s work showed that a β-N-acetylhexosaminidase, named BbhI, viaglycoside esterification via esterification processes [processes53]. In another [53]. In study,another Milosavic study, Milosavic reported reported that, when that, using when sucrose using isolated from B. bifidum JCM 1254 exhibited excellent efficiency and regioselectivity in andsucrose arbutin, and αarbutin,-glucosidase α-glucosidase from Saccharomyces from Saccharomyces cerevisiae wascerevisiae highly was stereospecific highly stereospecific for the formation for the transglycosylation at 3-OH of galactose (Gal) moieties in lactose (Scheme 16). BbhI has a strict offormation 4-hydroxyphenyl- of 4-hydroxyphenyl-β-isomaltosideβ-isomaltoside in a molar yieldin a molar of 50% yield with of reference 50% with to arbutinreference (Scheme to arbutin 15). regioselectivity of β 1-3 linkage when transferring GalNAc and GlcNAc residues to Gal residue of Their(Scheme study 15). also Their suggested study also that suggestedα-glucosidase that α-glucosidase can be exploited can be to exploited glycosylation to glycosylation reaction with reaction other lactose, with maximal yields of 55.4% and 44.9%, respectively. The enzyme provided a powerful bioactivewith other phenolic bioactive compounds phenolic compounds comprising co a hydroquinonemprising a hydroquinone moiety [54]. moiety [54]. synthetic approach to obtain physiologically active GalNAcβ1-3Lac and GlcNAcβ1-3Lac [55]. N-acetyl-β-D-galactosamine (GalNAc) and N-acetyl-β-D-glucosamine (GlcNAc) residue containing oligosaccharides are essential for many biological activities. They exist in the sugar unit of blood group P-related glycan, many tumor-related carbohydrate antigens gangliosides, milk oligosaccharides, and chitin. Chen’s work showed that a β-N-acetylhexosaminidase, named BbhI, isolated from B. bifidum JCM 1254 exhibited excellent efficiency and regioselectivity in transglycosylation at 3-OH of galactose (Gal) moieties in lactose (Scheme 16). BbhI has a strict regioselectivity of β 1-3 linkage when transferring GalNAc and GlcNAc residues to Gal residue of lactose, with maximal yields of 55.4% and 44.9%, respectively. The enzyme provided a powerful syntheticScheme approach 14. Diastereoselectivityto obtain physiologically of sucrose active phosphoryl GalNAcaseβ 1-3Lacin the glucosylation and GlcNAc ofβ 1-3Lac1,2-diols. [55].

Scheme 14. Diastereoselectivity of sucrose phosphorylase in the glucosylation of 1,2-diols.

Catalysts 2020, 10, x FOR PEER REVIEW 11 of 25

tert-leucine (Scheme 13). Compared to other reported LeuDH, the enzyme EsLeuDH exhibited higher activity at low temperatures and better thermostability at high temperatures. In addition, large scale experimental results indicated that the enzyme has the highest space-time yield (over 80%) among all LeuDHs, with >99% ee [52].

Scheme 12. Enantioselective intramolecular C–H amination catalyzed by P411.

Scheme 13. Substrate specificity of EsLeuDH in reductive amination and oxidative deamination.

2.2.3. Enzymes Applied for Glycosylation Chemically, to control the regio- and stereoselectivity of glycosidic bonds, the synthesizing of these oligosaccharides involves various protective group manipulations. Moreover, the challenge of large-scale production limits their application in the food and medical industries. Enzymatic glycosylations provide highly expedient transformation approaches in synthetic chemistry and can be easily exploited broadly to the directed alteration of small molecules and polymers via the covalent attachment of glycosyl residues. Sucrose phosphorylase was successfully employed in the synthesis of (R)-2-O-α-D-glucopyranosyl glyceric acid amide from single-step diastereoselective glucosylation, utilizing sucrose as a glucosyl donor and racemic glyceric acid amide as an acceptor, with d.e. > 83% and high yield (Scheme 14). This protocol could potentially simplify the access to biomimetic glycoside via esterification processes [53]. In another study, Milosavic reported that, when using sucrose and arbutin, α-glucosidase from Saccharomyces cerevisiae was highly stereospecific for the formation of 4-hydroxyphenyl-β-isomaltoside in a molar yield of 50% with reference to arbutin (Scheme 15). Their study also suggested that α-glucosidase can be exploited to glycosylation reaction with other bioactive phenolic compounds comprising a hydroquinone moiety [54]. N-acetyl-β-D-galactosamine (GalNAc) and N-acetyl-β-D-glucosamine (GlcNAc) residue containing oligosaccharides are essential for many biological activities. They exist in the sugar unit of blood group P-related glycan, many tumor-related carbohydrate antigens gangliosides, milk oligosaccharides, and chitin. Chen’s work showed that a β-N-acetylhexosaminidase, named BbhI, isolated from B. bifidum JCM 1254 exhibited excellent efficiency and regioselectivity in transglycosylation at 3-OH of galactose (Gal) moieties in lactose (Scheme 16). BbhI has a strict regioselectivity of β 1-3 linkage when transferring GalNAc and GlcNAc residues to Gal residue of

Catalystslactose,2020 with, 10 ,maximal 832 yields of 55.4% and 44.9%, respectively. The enzyme provided a powerful12 of 25 synthetic approach to obtain physiologically active GalNAcβ1-3Lac and GlcNAcβ1-3Lac [55].

Catalysts 2020,Scheme Scheme10, x FOR 14.14. PEER Diastereoselectivity REVIEW ofof sucrosesucrose phosphorylasephosphorylase inin thethe glucosylationglucosylation ofof 1,2-diols.1,2-diols. 12 of 25

Catalysts 2020, 10, x FOR PEER REVIEW 12 of 25

Scheme 15. The transglucosylation reaction catalyzed by α-glucosidase was optimized with respect Scheme 15. The transglucosylation reaction catalyzed by α-glucosidase was optimized with respect to to pH, temperature, and time. pH, temperature, and time.

N-acetyl-β-D-galactosamine (GalNAc) and N-acetyl-β-D-glucosamine (GlcNAc) residue containing oligosaccharides are essential for many biological activities. They exist in the sugar unit of blood group P-related glycan, many tumor-related carbohydrate antigens gangliosides, milk oligosaccharides, and chitin. Chen’s work showed that a β-N-acetylhexosaminidase, named BbhI, isolated from B. bifidum JCM 1254 exhibited excellent efficiency and regioselectivity in transglycosylation at 3-OH of galactose (Gal) moieties in lactose (Scheme 16). BbhI has a strict regioselectivity of β 1-3 linkage when transferring Scheme 16. Synthesis of β-GalNAc/GlcNAc-Lactose. GalNAc and GlcNAc residues to Gal residue of lactose, with maximal yields of 55.4% and 44.9%, respectively. The enzyme provided a powerful synthetic approach to obtain physiologically active DespiteScheme great 15. The progress transglucosylation in the enzymatic reaction catalyzed synthesis by αof-glucosidase sialic acid-containing was optimized molecules with respect using GalNAcβ1-3Lac and GlcNAcβ1-3Lac [55]. sialyltransferaseto pH, temperature, from mammalian and time. sources, a major challenge exists in the low expression levels in the processes. A bacterial based enzyme (α2−6-sialyltransferase) extracted from Photobacterium damselae (Pd2, 6ST) [56] could easily circumvent this issue. Pd2, 6ST could identify the terminal galactose (Gal) and N-Acetylgalactosamine (GalNAc) to produce Neu5Acα2−6Gal and Neu5Acα2−6GalNAc, respectively, in the presence of N-acetylneuraminic acid (Neu5Ac). This novel protocol opens a toolbox for the synthesis of disialyl tetrasaccharide epitopes and their derivatives [57]. Scheme 16. Synthesis of β-GalNAc/GlcNAc-Lactose. The Pictet–Spengler reaction is a stepwise reaction in which a β-arylethylamine proceeds in a Scheme 16. Synthesis of β-GalNAc/GlcNAc-Lactose. condensationDespite reaction great progress with a ketone in the or enzymatic aldehyde synthesisto produce of the sialic addition acid-containing product followed molecules by ring using closure.sialyltransferaseDespite To synthesize great from progress benzyl-isoquinoline mammalian in the sources, enzymatic alkaloid a major synthesis, plants challenge ofutilize sialic exists the acid-containing in stereoselective the low expression molecules Pictet–Spengler levels using in the reactionprocesses.sialyltransferase between A bacterial dopamine from basedmammalian and enzyme 4-hydroxyphenylace sources, (α2 6-sialyltransferase) a major taldehydechallenge extractedexistscatalyzed in the from by low norcoclaurinePhotobacterium expression synthaselevels damselae in − (NCS).(Pd2,the processes. Recent 6ST) [56 studies] A could bacterial (Scheme easily based circumvent 17) revealedenzyme this that(α issue.2− 6-sialyltransferase)the Pd2,enzyme 6ST could could catalyzeextracted identify the thefrom manufacturing terminal Photobacterium galactose of artificial,(Gal)damselae and optically (Pd2, N-Acetylgalactosamine 6ST) active [56] tetrahydroisoquinolines could easily (GalNAc) circumvent to produce du thise to issue.its Neu5Ac versatility Pd2,α 26ST 6Galtoward could and aldehydes.identify Neu5Ac theα 2Especially terminal6GalNAc, − − whenrespectively,galactose exploiting (Gal) in theCjNCS presenceand (N-terminallyN-Acetylgalactosamine of N-acetylneuraminic truncated) from(GalNAc) acid (Neu5Ac).Coptis japonicato Thisproduce expressed novel Neu5Ac protocol in Escherichiaα opens2−6Gal a toolbox andcoli, overforNeu5Ac 85.0% the synthesisα molar2−6GalNAc, yields of disialyl respectively,and excellent tetrasaccharide in e.e the. could presence epitopes be achieved of andN-acetylneuraminic their by synthesizing derivatives acid 6,7-dihy-droxy-1-propyl- [57]. (Neu5Ac). This novel 1,2,3,4-tetrahydroisoquinolineprotocolThe opens Pictet–Spengler a toolbox for reaction theand synthesis 6,7-dihydroxy-1-phen-ethyl-1,2,3,4-tetrahydroisoquinoline is a stepwise of disial reactionyl tetrasaccharide in which aepitopesβ-arylethylamine and their derivatives proceeds [58]. in Alessandraa[57]. condensation Bonamore’s reaction work with (Scheme a ketone 18) also or suggests aldehyde that to recombinant produce the (S addition)-NCS produced product in followed E. coli is an efficientThe Pictet–Spengler and stereoselective reaction isenvironmentall a stepwise reactiony friendly in which enzyme a β-arylethylamine that catalyzes proceedstyrosine andin a dopaminecondensation substrates reaction inwith a aone-pot, ketone or simple, aldehyde two-st to produceep process the addition to produce product ( Sfollowed)-norcoclaurine by ring (higenamine),closure. To synthesize with ee of benzyl-isoquinoline 93% and a yield higher alkaloid than, plants 80% inutilize the optimizedthe stereoselective condition. Pictet–Spengler The process allowedreaction the between recycling dopamine of NCS and with 4-hydroxyphenylace good recovery. Theirtaldehyde strategy catalyzed provided by norcoclaurinea novel, efficient, synthase and green(NCS). route Recent for producing studies (Scheme plant-derived 17) revealed metabolites that the such enzyme as benzyl-isoquinoline could catalyze the alkaloids manufacturing [59]. of artificial, optically active tetrahydroisoquinolines due to its versatility toward aldehydes. Especially when exploiting CjNCS (N-terminally truncated) from Coptis japonica expressed in Escherichia coli, over 85.0% molar yields and excellent e.e. could be achieved by synthesizing 6,7-dihy-droxy-1-propyl- 1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-phen-ethyl-1,2,3,4-tetrahydroisoquinoline [58]. Alessandra Bonamore’s work (Scheme 18) also suggests that recombinant (S)-NCS produced in E. coli is an efficient and stereoselective environmentally friendly enzyme that catalyzes tyrosine and dopamine substrates in a one-pot, simple, two-step process to produce (S)-norcoclaurine

(higenamine), with ee of 93% and a yield higher than 80% in the optimized condition. The process allowed the recycling of NCS with good recovery. Their strategy provided a novel, efficient, and green route for producing plant-derived metabolites such as benzyl-isoquinoline alkaloids [59].

Catalysts 2020, 10, x FOR PEER REVIEW 12 of 25

Scheme 15. The transglucosylation reaction catalyzed by α-glucosidase was optimized with respect to pH, temperature, and time.

Scheme 16. Synthesis of β-GalNAc/GlcNAc-Lactose.

Despite great progress in the enzymatic synthesis of sialic acid-containing molecules using sialyltransferase from mammalian sources, a major challenge exists in the low expression levels in the processes. A bacterial based enzyme (α2−6-sialyltransferase) extracted from Photobacterium damselae (Pd2, 6ST) [56] could easily circumvent this issue. Pd2, 6ST could identify the terminal galactose (Gal) and N-Acetylgalactosamine (GalNAc) to produce Neu5Acα2−6Gal and Neu5Acα2−6GalNAc, respectively, in the presence of N-acetylneuraminic acid (Neu5Ac). This novel Catalystsprotocol2020 opens, 10, 832 a toolbox for the synthesis of disialyl tetrasaccharide epitopes and their derivatives13 of 25 [57]. The Pictet–Spengler reaction is a stepwise reaction in which a β-arylethylamine proceeds in a bycondensation ring closure. reaction To synthesizewith a ketone benzyl-isoquinoline or aldehyde to produce alkaloid, the addition plants utilize product the followed stereoselective by ring Pictet–Spenglerclosure. To synthesize reaction benzyl-isoquinoline between dopamine alkaloid and, plants 4-hydroxyphenylacetaldehyde utilize the stereoselective Pictet–Spengler catalyzed by norcoclaurinereaction between synthase dopamine (NCS). and 4-hydroxyphenylace Recent studies (Schemetaldehyde 17) catalyzed revealed by that norcoclaurine the enzyme synthase could catalyze(NCS). Recent the manufacturingstudies (Scheme of 17) artificial, revealed opticallythat the enzyme active tetrahydroisoquinolinescould catalyze the manufacturing due to itsof versatilityartificial, optically toward active aldehydes. tetrahydroisoquinolines Especially when due exploitingto its versatility CjNCS toward (N-terminally aldehydes. truncated)Especially fromwhen Coptisexploiting japonica CjNCSexpressed (N-terminally in Escherichia truncated) coli from, over Coptis 85.0% japonica molar expressed yields andin Escherichia excellent colie.e,. couldover 85.0% be achieved molar yields by and synthesizing excellent e.e 6,7-dihy-droxy-1-propyl-1,2,3,4-tetrahydroisoquinoline. could be achieved by synthesizing 6,7-dihy-droxy-1-propyl- and 6,7-dihydroxy-1-phen-ethyl-1,2,3,4-tetrahydroisoquinoline1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-phen-ethyl-1,2,3,4-tetrahydroisoquinoline [58]. Alessandra Bonamore’s work[58]. (SchemeAlessandra 18) Bonamore’s also suggests work that recombinant(Scheme 18) (alsoS)-NCS suggests produced that recombinant in E. coli is an ( eSffi)-NCScient andproduced stereoselective in E. coli environmentallyis an efficient and friendly stereoselective enzyme that environmentall catalyzes tyrosiney friendly and dopamine enzyme substrates that catalyzes in a one-pot, tyrosine simple, and two-stepdopamine process substrates to produce in a (Sone-pot,)-norcoclaurine simple, (higenamine), two-step process with ee toof 93%produce and a yield(S)-norcoclaurine higher than 80%(higenamine), in the optimized with ee of condition. 93% and a The yield process higher allowed than 80% the in recycling the optimized of NCS condition. with good The recovery. process Theirallowed strategy the recycling provided of aNCS novel, with effi cient,good recovery. and green Their route strategy for producing provided plant-derived a novel, efficient, metabolites and suchgreen as route benzyl-isoquinoline for producing plant-derived alkaloids [59 ].metabolites such as benzyl-isoquinoline alkaloids [59].

Catalysts 2020, 10, x FOR PEER REVIEW 13 of 25

Scheme 17. Enzymatic synthesis of unnatural 1-substituted 1,2,3,4-tetrahydroisoquinolines. Scheme 17. Enzymatic synthesis of unnatural 1-substituted 1,2,3,4-tetrahydroisoquinolines.

H2N OH NH2 O HO COOH OH

H2O NH NaClO HO H phosphater NCS (S) buffer pH 7.0 OH OH Phosphate buffer 0.1 M 1h, 37 °C OH pH 7.0, 0.5 h, 25 °C Scheme 18. TheThe stereospecific stereospecific chemoenzymatic chemoenzymatic synthesis synthesis of ( ofS)-norcoclaurine (S)-norcoclaurine from from tyrosine tyrosine and anddopamine. dopamine. 2.2.4. Enzymes Applied for Halogenation 2.2.4. Enzymes Applied for Halogenation In organic synthesis, the introduction of halogen substituents, especially in mechanistically In organic synthesis, the introduction of halogen substituents, especially in mechanistically less- less-favored positions, remains a challenge. The antibiotic compound pyrroindomycin B was initially favored positions, remains a challenge. The antibiotic compound pyrroindomycin B was initially isolated by Ding’s research group in 1994 [60]. The molecule is composed of a pyrroloindole entity and isolated by Ding’s research group in 1994 [60]. The molecule is composed of a pyrroloindole entity a polyketide macro-ring system with a tetramic acid functional group, covalently appended to each end and a polyketide macro-ring system with a tetramic acid functional group, covalently appended to of an unbranched deoxytrisaccharide. The indole ring moiety of pyrroindomycin B is halogenated at the each end of an unbranched deoxytrisaccharide. The indole ring moiety of pyrroindomycin B is C5 position. As the indole ring is a derivative of tryptophan, typtophan 5-halogenase is responsible for halogenated at the C5 position. As the indole ring is a derivative of tryptophan, typtophan 5- the biosynthesis of pyrroindomycin B in Streptomyces rugosporus LL-42D005. The disruption mutation halogenase is responsible for the biosynthesis of pyrroindomycin B in Streptomyces rugosporus LL- of the tryptophan 5-halogenase gene resulting in the nonhalogenated pyrroindomycin confirmed this 42D005. The disruption mutation of the tryptophan 5-halogenase gene resulting in the theory [61]. nonhalogenated pyrroindomycin confirmed this theory [61]. C-1027 or lidamycin is an antitumor antibiotic comprising a reactive enediyne chromophore and C-1027 or lidamycin is an antitumor antibiotic comprising a reactive enediyne chromophore and an apoprotein unit (CagA). The chromophore readily generates benzenoid diradical intermediate, an apoprotein unit (CagA). The chromophore readily generates benzenoid diradical intermediate, which is capable of separating hydrogen atoms from the DNA backbone in the presence of free which is capable of separating hydrogen atoms from the DNA backbone in the presence of free oxygen. The destruction of DNA is associated well with cytotoxicity. The bioactive chromophore oxygen. The destruction of DNA is associated well with cytotoxicity. The bioactive chromophore has has three chemical subunits that are covalently bonded to the nine-membered enediynes, including three chemical subunits that are covalently bonded to the nine-membered enediynes, including (S)- (S)-3-chloro-4,5-dihydroxy-β-phenylalanine component. This moiety is synthesized from L-α-tyrosine 3-chloro-4,5-dihydroxy-β-phenylalanine component. This moiety is synthesized from L-α-tyrosine catalyzed by six proteins, one of which is SgcC3. The intermediate (S)-β-tyrosyl-S-SgcC2 can be catalyzed by six proteins, one of which is SgcC3. The intermediate (S)-β-tyrosyl-S-SgcC2 can be regioselectively chlorinated by SgcC3 (Scheme 19). The study indicated that the activity of that enzyme is oxygen and FADH2 dependent [62].

Scheme 19. SgcC3-catalyzed halogenation of (S)-β-tyrosyl-S-SgcC2.

Another FAD-dependent halogenase, RebH, was translated from Lechevalieria aerocolonigenes that could halogenate rebeccamycin. This enzyme is competent at overriding the distinct rules of halogenation, which is regioselective. For instance, in comparison to the halogenation of indole or tryptophan at the C-3 or C-2 position correspondingly, RebH would chlorinate or brominate L-

Catalysts 2020, 10, x FOR PEER REVIEW 13 of 25

Scheme 17. Enzymatic synthesis of unnatural 1-substituted 1,2,3,4-tetrahydroisoquinolines.

H2N OH NH2 O HO COOH OH

H2O NH NaClO HO H phosphater NCS (S) buffer pH 7.0 OH OH Phosphate buffer 0.1 M 1h, 37 °C OH pH 7.0, 0.5 h, 25 °C Scheme 18. The stereospecific chemoenzymatic synthesis of (S)-norcoclaurine from tyrosine and dopamine.

2.2.4. Enzymes Applied for Halogenation In organic synthesis, the introduction of halogen substituents, especially in mechanistically less- favored positions, remains a challenge. The antibiotic compound pyrroindomycin B was initially isolated by Ding’s research group in 1994 [60]. The molecule is composed of a pyrroloindole entity and a polyketide macro-ring system with a tetramic acid functional group, covalently appended to each end of an unbranched deoxytrisaccharide. The indole ring moiety of pyrroindomycin B is halogenated at the C5 position. As the indole ring is a derivative of tryptophan, typtophan 5- halogenase is responsible for the biosynthesis of pyrroindomycin B in Streptomyces rugosporus LL- 42D005. The disruption mutation of the tryptophan 5-halogenase gene resulting in the nonhalogenated pyrroindomycin confirmed this theory [61]. C-1027 or lidamycin is an antitumor antibiotic comprising a reactive enediyne chromophore and an apoprotein unit (CagA). The chromophore readily generates benzenoid diradical intermediate, which is capable of separating hydrogen atoms from the DNA backbone in the presence of free oxygen. The destruction of DNA is associated well with cytotoxicity. The bioactive chromophore has threeCatalysts chemical2020, 10, 832subunits that are covalently bonded to the nine-membered enediynes, including14 ( ofS)- 25 3-chloro-4,5-dihydroxy-β-phenylalanine component. This moiety is synthesized from L-α-tyrosine catalyzed by six proteins, one of which is SgcC3. The intermediate (S)-β-tyrosyl-S-SgcC2 can be regioselectivelyregioselectively chlorinatedchlorinated by by SgcC3 SgcC3 (Scheme (Scheme 19 ).19). The The study study indicated indicated that thethat activity the activity of that enzymeof that enzymeis oxygen is andoxygen FADH and2 dependentFADH2 dependent [62]. [62].

SchemeScheme 19. 19. SgcC3-catalyzedSgcC3-catalyzed halogenation halogenation of of ( (SS)-)-ββ-tyrosyl--tyrosyl-SS-SgcC2.-SgcC2.

AnotherAnother FAD-dependent FAD-dependent halogenase halogenase,, RebH, RebH, was was translated from from Lechevalieria aerocolonigenes thatthat could could halogenate halogenate rebeccamycin. rebeccamycin. This This enzyme enzyme is is competent competent at at overriding overriding the the distinct distinct rules rules of of halogenation,Catalystshalogenation, 2020, 10 , whichx which FOR PEER is is regioselective.regioselective. REVIEW For For instance, instance, in incomparison comparison to tothe the halogenation halogenation of indole of indole14 of or 25 tryptophanor tryptophan at the at theC-3C-3 or C-2 or C-2 position position corresp correspondingly,ondingly, RebH RebH would would chlorinate chlorinate or brominate or brominate L- tryptophanL-tryptophan at atthe the C-7 C-7 position, position, which which is is electronic electronicallyally unfavored unfavored (Scheme (Scheme 20), 20), even even in in the the presence presence of ortho/para-directing groups [[63].63].

X H R R H Trp N Trp N OH halogenase R H synthase N RebH + H3N O NaX, pH 7.4 O H3N O H3N O R=F,OH,CH3,NH2 O X=Cl,Br O Scheme 20.20.Combination Combination of tryptophanof tryptophan synthase synthase from Salmonellafrom Salmonella enterica andenterica tryptophan-7-halogenase and tryptophan-7- halogenaseRebH from RebHLechevalieria from Lechevalieria aerocolonigenes aerocolonigenesfor the for regioselectivethe regioselective halogenation halogenation of of substituted tryptophan derivatives.

2.3. Hydrolases Hydrolases E.C.3 Many polyhydroxylated steroids with vicinal di diolsols on the A-ring have remarkable biological activities. Enzyme Enzyme catalysis catalysis has has shown shown a a significant significant impact impact on on the the regio- and stereoselective transformation ofof functionalfunctional groups groups in in steroids. steroids. Silva Silva and and her her colleagues colleagues conducted conducted a systematic a systematic study studyon a series on a ofseries stereoisomeric of stereoisomeric 2, 3- and 2, 3, 3- 4-vicinal and 3, diols4-vicinal with diols selective with transesterificationselective transesterification catalyzed catalyzedby commercially by commercially available lipases available (Scheme lipases 21). (Scheme Their work 21). delineatedTheir work the delineated high regioselectivity the high regioselectivityof enzymatic acylation. of enzymatic The acylation. targeted lipases, The targeted such as lipases, Novozym such 435as Novozym (immobilized 435 lipase(immobilized B from lipaseCandida B from antarctica Candida) and antarcticaC. viscosum) andlipase, C. viscosum were lipase, capable were of discriminating capable of discriminating between A-ring between vicinal A- ringhydroxyl vicinal groups hydroxyl located groups on the located steroids. on Accordingthe steroids. to theirAccording results, to these their lipases results, are these sensitive lipases to are the sensitiveconfiguration to the of configuration diols, affording of diols, highly affording regioselective highly monoesters. regioselective Their monoesters. findings canTheir provide findings useful can providesynthetic useful tools forsynthetic the preparation tools for of the mono-acylated, preparation glycosylated,of mono-acylated, or sulphated glycosylated, steroidal or vicinal sulphated diols steroidaland their vicinal derivatives diols [and64]. their derivatives [64].

Scheme 21. Regioselective enzymatic acylation of vicinal diols of steroids.

The chemical acylation of nucleosides has been reported in a few cases, but the reaction is usually carried out by using protecting groups. Furthermore, the tedious separation processes always result in low yields of the mono-O-acyl derivative. However, 2-methyltetrahydrofuran (MeTHF) is a versatile aprotic solvent. Compared to tetrahydrofuran (THF), MeTHF is an excellent substitute in biotransformation processes and has gained popularity in industrial synthetic applications due to its low environmental impact. In this sense, Yolanda Simeo et al. investigated the benefits of using MeTHF as a solvent in enzyme-catalyzed biotransformation. They applied CAL-B lipase to acylate 1- β-arabinofuranosyl uracil (ara-U), 9-β-arabinofuranosyl adenosine (ara-A, 15), 2′-O-(2- methoxyethyl)-5-methyluridine, adenosine, and uridine with vinyl esters and hexanoic anhydride, with yields of over 90% and exceptional regioselectivity. Both THF and MeTHF behave in a similar way, while MeTHF exhibited better conversions for the acylation of uridine and ara-U [65]. The classical chemical acylation approaches which need a base to catalyze with the acid chloride have their downside due to side isomerization reactions. Díaz-Rodríguez and coworkers utilized biocatalytic methodology to synthesize several O-crotonyl 20-deoxynucleoside derivatives (Scheme 22). All these compounds have crotonyl functional groups present, with biological activities such as anti-tumor or inhabitation of the replication of HIV-1 and HIV-2. Their research group employed lipase B from Candida Antarctica (CAL-B) and the immobilized lipase from Pseudomonas cepacia(PSL-

Catalysts 2020, 10, x FOR PEER REVIEW 14 of 25

tryptophan at the C-7 position, which is electronically unfavored (Scheme 20), even in the presence of ortho/para-directing groups [63].

X H R R H Trp N Trp N OH halogenase R H synthase N RebH + H3N O NaX, pH 7.4 O H3N O H3N O R=F,OH,CH3,NH2 O X=Cl,Br O Scheme 20. Combination of tryptophan synthase from Salmonella enterica and tryptophan-7- halogenase RebH from Lechevalieria aerocolonigenes for the regioselective halogenation of substituted tryptophan derivatives.

2.3. Hydrolases E.C.3 Many polyhydroxylated steroids with vicinal diols on the A-ring have remarkable biological activities. Enzyme catalysis has shown a significant impact on the regio- and stereoselective transformation of functional groups in steroids. Silva and her colleagues conducted a systematic study on a series of stereoisomeric 2, 3- and 3, 4-vicinal diols with selective transesterification catalyzed by commercially available lipases (Scheme 21). Their work delineated the high regioselectivity of enzymatic acylation. The targeted lipases, such as Novozym 435 (immobilized lipase B from Candida antarctica) and C. viscosum lipase, were capable of discriminating between A- ring vicinal hydroxyl groups located on the steroids. According to their results, these lipases are sensitive to the configuration of diols, affording highly regioselective monoesters. Their findings can

Catalystsprovide2020 useful, 10, 832 synthetic tools for the preparation of mono-acylated, glycosylated, or sulphated15 of 25 steroidal vicinal diols and their derivatives [64].

Scheme 21. Regioselective enzymatic acylatio acylationn of vicinal diols of steroids.

The chemical chemical acylation acylation of of nucleosides nucleosides has has been been repo reportedrted in a few in a cases, few cases,but the but reaction the reaction is usually is usuallycarried out carried by using out by protecting using protecting groups. groups. Furthermore, Furthermore, the tedious the tedious separation separation processes processes always always result resultin low in yields low yields of the of themono-O-acyl mono-O-acyl derivative. derivative. However, However, 2-methyltetrahydrofuran 2-methyltetrahydrofuran (MeTHF) (MeTHF) is isa aversatile versatile aprotic aprotic solvent. solvent. Compared Compared to totetrahydrofuran tetrahydrofuran (THF), (THF), MeTHF MeTHF is an is anexcellent excellent substitute substitute in inbiotransformation biotransformation processes processes and and has hasgained gained popularity popularity in industrial in industrial synthetic synthetic applications applications due to due its tolow its environmental low environmental impact. impact. In this Insense, this Yolanda sense, Yolanda Simeo et Simeo al. investigated et al. investigated the benefits the of benefits using ofMeTHF using as MeTHF a solvent as in aenzyme-catalyzed solvent in enzyme-catalyzed biotransformation. biotransformation. They applied CAL-B They lipase applied to acylate CAL-B 1- lipaseβ-arabinofuranosyl to acylate 1- β-arabinofuranosyluracil (ara-U), uracil9-β-arabinofuranosyl (ara-U), 9-β-arabinofuranosyl adenosine (ara-A, adenosine 15), (ara-A, 2′-O-(2- 15), 2methoxyethyl)-5-methyluridine,0-O-(2-methoxyethyl)-5-methyluridine, adenosine, adenosine, and uridine and with uridine vinyl with esters vinyl and estershexanoic and anhydride, hexanoic anhydride,with yields withof over yields 90% of and over exceptional 90% and exceptional regioselectivity. regioselectivity. Both THF Both and THF MeTHF and MeTHFbehave in behave a similar in a similarway, while way, MeTHF while MeTHF exhibited exhibited better conversions better conversions for the foracylation the acylation of uridine of uridine and ara-U and [65]. ara-U [65]. The classical chemical acylation approaches which need a base to catalyze with the acid chloride have their downsidedownside duedue toto sideside isomerizationisomerization reactions.reactions. DDíaz-Rodríguezíaz-Rodríguez and coworkers utilized biocatalytic methodologymethodology to to synthesize synthesize several several O-crotonyl O-crotonyl 20-deoxynucleoside 20-deoxynucleoside derivatives derivatives (Scheme (Scheme 22). CatalystsAll22). theseAll 2020 these compounds, 10 ,compounds x FOR PEER have REVIEW have crotonyl crotonyl functional functional groups groups present, present, with with biological biologicalactivities activities suchsuch15 of as25 anti-tumor oror inhabitationinhabitation of of the the replication replication of HIV-1of HIV-1 and and HIV-2. HIV-2. Their Their research research group group employed employed lipase C)Blipase fromto BCandidaattain from Candida5 Antarctica′-O-acylated Antarctica(CAL-B) nucleoside (CAL-B) and the andand immobilized the 3 ′immobilized-O-crotonylated lipase lipase from analogs, Pseudomonasfrom Pseudomonas respectively, cepacia cepacia(PSL-C) without(PSL- to isomerizationattain 5 -O-acylated [66]. nucleoside and 3 -O-crotonylated analogs, respectively, without isomerization [66]. 0 0

Scheme 22. RegioselectiveRegioselective enzymatic enzymatic acylation acylation on 2’-deoxynucleosides.

KoKołodziejska’słodziejska’s researchresearch group group also also investigated investigated the influencethe influence of the solventof the solvent on enzymatic on enzymatic reactions. reactions.Their study Their revealed study thatrevealed both that the choiceboth the of choi solventce of and solvent the acyland groupthe acyl donor group impact donor theimpact activity, the activity,stability, stability, and selectivity and selectivity in enzyme-catalyzed in enzyme-cat acylation.alyzed acylation. The hydrophobic The hydrophobic organic solvent organic indicated solvent indicated enzymatic enantioselectivity improvement [67,68]. Tert-butyl methyl ether (TBME) provided an excellent reaction environment for the lipase Amano PS from Burkholderia cepacia (BCL). Acylation reaction gave (R)-monoesters with 81%–99% ee enantiomeric purity (Scheme 23), due to the selectivity of enantio-topic hydroxyl groups [69]. Allenes are flexible synthetic precursors of numerous axially chiral compounds, with important industrial applications. Many stereoselective methods have been developed to synthesize these chiral allenes, most of which start from a racemic mixture. Thus, the yield of the allene resolution is always limited. However, exploiting prochiral substrates in enzyme-catalyzed transesterification reactions is a pronounced approach to overcome the limitation of yields. Sapu’s work suggests that porcine pancreatic lipase (PPL) is an excellent biocatalyst for the kinetic resolution of allenols (Scheme 24). The enzyme is also suitable for the transesterification of diols with similar structures. Exemplarily, treating the prochiral phenyl-substituted diol with vinyl butyrate catalyzed by PPL led to monoesters with 95% yield and 98% ee under modified conditions. One can conclude that this powerful chemoenzymatic protocol makes either enantiomer of optical allenyl monoesters accessible [70]. Traditionally, the selective benzoylation of the 5′-hydroxyl group of thymidine requires highly toxic hexamethylphosphoric triamide and produces a by-product, triphenylphosphine oxide, that is difficult to remove [71]. Other alternative routes are either time consuming and/or have moderate yields [72,73]. Enzyme-catalyzed acylation is a substitute for standard synthetic methods that can avoid these complications. Enzyme Candida Antarctica lipase B (CAL-B) proved to have high selectivity over 5′-hydroxyl in the transesterification and to be reclaimed (Scheme 25). The benzoylation protocol catalyzed by CAL-B is a mild and efficient synthetic method for 5′-O-benzoyl- 2′-deoxynucleosides, with excellent yields (over 89%). Easy scalability, minimal environmental impact, and recycling of both enzyme and acylating agent make this pathway very attractive for industrial applications [74].

Catalysts 2020, 10, 832 16 of 25

enzymatic enantioselectivity improvement [67,68]. Tert-butyl methyl ether (TBME) provided an excellent reaction environment for the lipase Amano PS from Burkholderia cepacia (BCL). Acylation reaction gave (R)-monoesters with 81%–99% ee enantiomeric purity (Scheme 23), due to the selectivity

Catalystsof enantio-topic 2020, 10, x FOR hydroxyl PEER REVIEW groups [69]. 16 of 25

Catalysts 2020, 10, x FOR PEER REVIEW 16 of 25

Scheme 23. Acylation of the prochiral 1-(([1,3-dihydroxypropan-2-yl]oxy)methyl)-5- Scheme 23. Acylation of the prochiral 1-(([1,3-dihydroxypropan-2-yl]oxy)methyl)-5-methylpyrimidine- methylpyrimidine-2,4(1H,3H)-dione and the hydrolysis of the corresponding diesters in the presence of 2,4(1H,3H)-dionea lipase under various and conditions. the hydrolysis of the corresponding diesters in the presence of a lipase under various conditions.

Allenes are flexible synthetic precursors of numerous axially chiral compounds, with important industrial applications. Many stereoselective methods have been developed to synthesize these chiral allenes, most of which start from a racemic mixture. Thus, the yield of the allene resolution is always limited. However, exploiting prochiral substrates in enzyme-catalyzed transesterification reactions is a pronounced approach to overcome the limitation of yields. Sapu’s work suggests that porcine pancreatic lipase (PPL) is an excellent biocatalyst for the kinetic resolution of allenols (Scheme 24). The enzyme is also suitable for the transesterification of diols with similar structures. Exemplarily, treating the prochiral phenyl-substituted diol with vinyl butyrate catalyzed by PPL led to Scheme 23. AcylationScheme of 24. theDesymmetrization prochiral of1-(([1,3 allendiols.-dihydroxypropan-2-yl]oxy)methyl)-5- monoestersmethylpyrimidine-2,4(1H,3H)-dione with 95% yield and 98% ee under and the modified hydrolysis conditions. of the corresponding One can conclude diesters in that the thispresence powerful chemoenzymaticof a lipase under protocol various makes conditions. either enantiomer of optical allenyl monoesters accessible [70].

Scheme 25. Regioselective enzymatic procedure for 5’-O-benzoylation of 2’-deoxynucleosides.

Xiao et al. (Scheme 26) developed a new method to prepare enantiopure caffeic acid amides with an asymmetric aminolysis reaction between the cinnamic acid ester and (R, S)-α-phenylethylamine, which is catalyzed by an immobilized lipase (Novozym 435) from Candida antarctica. They optimized the reaction conditions, and underScheme those 24. conditio Desymmetrizationns, high enantioselectivity of allendiols. for the enzymatic reaction was gained, with an enantiomeric excess of 98.5% [75]. Intermolecular aziridination is a highly enantioselective and valuable synthetical organic reaction that has no known natural equivalent. An engineered, genetically encoded enzyme, cytochrome P450, demonstrated high enantioselectivity, with up to 99% ee for intermolecular aziridination, and it can be easily optimized

Scheme 25. Regioselective enzymatic procedure for 5’-O-benzoylation of 2’-deoxynucleosides.

Xiao et al. (Scheme 26) developed a new method to prepare enantiopure caffeic acid amides with an asymmetric aminolysis reaction between the cinnamic acid ester and (R, S)-α-phenylethylamine, which is catalyzed by an immobilized lipase (Novozym 435) from Candida antarctica. They optimized the reaction conditions, and under those conditions, high enantioselectivity for the enzymatic reaction was gained, with an enantiomeric excess of 98.5% [75]. Intermolecular aziridination is a highly enantioselective and valuable synthetical organic reaction that has no known natural equivalent. An engineered, genetically encoded enzyme, cytochrome P450, demonstrated high enantioselectivity, with up to 99% ee for intermolecular aziridination, and it can be easily optimized

Catalysts 2020, 10, x FOR PEER REVIEW 16 of 25

Scheme 23. Acylation of the prochiral 1-(([1,3-dihydroxypropan-2-yl]oxy)methyl)-5- methylpyrimidine-2,4(1H,3H)-dione and the hydrolysis of the corresponding diesters in the presence Catalysts 2020, 10, 832 17 of 25 of a lipase under various conditions.

Traditionally, the selective benzoylation of the 50-hydroxyl group of thymidine requires highly toxic hexamethylphosphoric triamide and produces a by-product, triphenylphosphine oxide, that is difficult to remove [71]. Other alternative routes are either time consuming and/or have moderate yields [72,73]. Enzyme-catalyzed acylation is a substitute for standard synthetic methods that can avoid these complications. Enzyme Candida Antarctica lipase B (CAL-B) proved to have high selectivity over 50-hydroxyl in the transesterification and to be reclaimed (Scheme 25). The benzoylation protocol catalyzed by CAL-B is a mild and efficient synthetic method for 50-O-benzoyl-20-deoxynucleosides, with excellent yields (over 89%). Easy scalability, minimal environmental impact, and recycling of both enzyme and acylating agent makeScheme this 24. pathway Desymmetrization very attractive of allendiols. for industrial applications [74].

Scheme 25. Regioselective enzymatic procedure for 5’-O-benzoylation of 2’-deoxynucleosides. Scheme 25. Regioselective enzymatic procedure for 5’-O-benzoylation of 2’-deoxynucleosides.

XiaoXiao etet al.al. (Scheme(Scheme 2626)) developeddeveloped aa newnew methodmethod toto prepareprepare enantiopureenantiopure cacaffeicffeicacid acidamides amideswith with anan asymmetricasymmetric aminolysisaminolysis reactionreaction betweenbetween thethe cinnamiccinnamic acidacid esterester andand ((RR,, S S)-)-αα-phenylethylamine,-phenylethylamine, whichwhich is is catalyzedcatalyzed by by anan immobilizedimmobilized lipaselipase (Novozym(Novozym 435)435) fromfromCandida Candida antarctica antarctica.. TheyThey optimizedoptimized thethe reaction reaction conditions, conditions, and and under under those those conditions, conditio highns, enantioselectivityhigh enantioselectivity for the enzymaticfor the enzymatic reaction wasreaction gained, was with gained, an enantiomericwith an enantiomeric excess of excess 98.5% of [75 98.5%]. Intermolecular [75]. Intermolecular aziridination aziridination is a highly is a enantioselectivehighly enantioselective and valuable and valu syntheticalable synthetical organic reactionorganic thatreacti hason no that known has naturalno known equivalent. natural equivalent. An engineered, genetically encoded enzyme, cytochrome P450, demonstrated high CatalystsAnCatalysts engineered, 20202020,, 1010,, xx geneticallyFORFOR PEERPEER REVIEWREVIEW encoded enzyme, cytochrome P450, demonstrated high enantioselectivity,1717 ofof 2525 withenantioselectivity, up to 99% ee withfor intermolecular up to 99% ee for aziridination, intermolecular and aziridination, it can be easily and it optimized can be easily (Scheme optimized 27). (SchemeExemplarily,(Scheme 27).27). Exemplarily,Exemplarily, enzyme P-I263F-A328V-L437V, enzymeenzyme P-I263F-A328V-L43P-I263F-A328V-L43 a variant7V,7V, of a P411a variantvariant (A mutation ofof P411P411 (A(A from mutationmutation wild-type fromfrom P450),wild-wild- typeshowedtype P450),P450), significant showedshowed significant improvementssignificant improvimprov in enantioselectivityementsements inin enantioselectivityenantioselectivity (25% ee to (25%(25% 99% eeeeee to)to and 99%99% yield eeee)) andand (1.1% yieldyield to (1.1%(1.1% 55%) tocomparedto 55%)55%) comparedcompared to its precursor, toto itsits precursor,precursor, P411, when P411,P411, catalyzing whenwhen catalyzingcatalyzing aziridination aziridinationaziridination of 4-methyl ofof 4-methyl4-methyl styrene [ styrene76styrene]. [76].[76].

SchemeScheme 26. 26. AsymmetricAsymmetric aminolysis aminolysis of of cinnamic cinnamic acid acid ester ester derivatives. derivatives.

NTsNTs Ts-NTs-N33 UpUp to to 1,000 1,000 TTN TTN Up to 99% ee R P450P450BM3 variantvariant R Up to 99% ee R BM3 R SchemeScheme 27. 27. Enzyme-catalyzedEnzyme-catalyzed olefin olefinolefin aziridination. aziridination.

WombacherWombacher et etet al. al.al. (Scheme (Scheme(Scheme 28 28)28)) discovered discovereddiscovered an anan RNA RNRN enzymeAA enzymeenzyme (ribozyme) (ribozyme)(ribozyme) that that catalyzesthat catalyzescatalyzes a Diels–Alder aa Diels–Diels– AlderreactionAlder reactionreaction between betweenbetween an oligo anan anthracene oligooligo anthraceneanthracene diene and diendien a maleimideee andand aa dienophile,maleimidemaleimide stereoselectivelydienophile,dienophile, stereoselectivelystereoselectively synthesizing synthesizingsynthesizing anan R,R, RR enantiomerenantiomer withwith aa highhigh yieldyield ofof ~90%~90% ((eeee%)%) whenwhen nn equalsequals 66 oror 12.12. TheThe ribozymeribozyme waswas selectedselected fromfrom aa librarylibrary ofof RNARNA conjugatesconjugates generatedgenerated byby 120120 randomizedrandomized nucleotides.nucleotides. TheyThey provedproved thatthat thethe sizesize ofof thethe dienediene determineddetermined thethe stereoselectivitystereoselectivity ofof thethe rereactionaction andand explainedexplained thethe mechanismmechanism inin thethe truetrue catalyticcatalytic rereactionaction throughthrough aa crystalcrystal structurestructure studystudy [8].[8]. TheThe synthesissynthesis strategystrategy ofof 5-hydroxyimino-4,5-dihyd5-hydroxyimino-4,5-dihydrofuransrofurans throughthrough aa lipase-catalyzedlipase-catalyzed reactionreaction betweenbetween ββ-nitrostyrenes-nitrostyrenes andand 1,3-dicarbonyl1,3-dicarbonyl compoundscompounds waswas developeddeveloped byby WuWu (Scheme(Scheme 29)29) andand coworkers.coworkers. TheThe highlighthighlight ofof theirtheir studystudy waswas thatthat aa highhigh stereoselectivitystereoselectivity ((Z/EZ/E upup toto 99:1)99:1) waswas achievedachieved byby screeningscreening aa seriesseries ofof differentdifferent ββ-nitrostyrenes-nitrostyrenes andand lipaseslipases fromfrom differentdifferent organisms.organisms. ComparedCompared withwith thethe reactionreaction usingusing traditionaltraditional catalystcatalyst NEtNEt33 inin methanol,methanol, thethe enzymaticenzymatic reactionreaction producedproduced muchmuch lessless by-productby-product ofof dihydrofuran,dihydrofuran, whichwhich ledled toto thethe highhigh stereoselectivitystereoselectivity ofof thethe ZZ productproduct [77].[77].

SchemeScheme 28.28. FormationFormation ofof carbon–carboncarbon–carbon bondsbonds byby aa DielDiels–Alders–Alder reactionreaction betweenbetween anan anthraceneanthracene dienediene andand aa maleimidemaleimide dienophile.dienophile.

Catalysts 2020, 10, x FOR PEER REVIEW 17 of 25

(Scheme 27). Exemplarily, enzyme P-I263F-A328V-L437V, a variant of P411 (A mutation from wild- type P450), showed significant improvements in enantioselectivity (25% ee to 99% ee) and yield (1.1% to 55%) compared to its precursor, P411, when catalyzing aziridination of 4-methyl styrene [76].

Scheme 26. Asymmetric aminolysis of cinnamic acid ester derivatives.

NTs Ts-N3 Up to 1,000 TTN P450 variant Up to 99% ee R BM3 R Scheme 27. Enzyme-catalyzed olefin aziridination. Catalysts 2020, 10, 832 18 of 25 Wombacher et al. (Scheme 28) discovered an RNA enzyme (ribozyme) that catalyzes a Diels– Alder reaction between an oligo anthracene diene and a maleimide dienophile, stereoselectively ansynthesizingR, R enantiomer an R, R with enantiomer a high yield with of a ~90%high yield (ee%) of when ~90% n ( equalsee%) when 6 or 12.n equals The ribozyme 6 or 12. The was ribozyme selected fromwas selected a library from of RNA a library conjugates of RNA generated conjugates by 120 generated randomized by 120 nucleotides. randomized They nucleotides. proved that They the sizeproved of thethat diene the size determined of the diene the determined stereoselectivity the stereoselectivity of the reaction of and the re explainedaction and the explained mechanism the inmechanism the true in catalytic the true reaction catalytic throughreaction athrough crystal a structurecrystal structure study [study8]. The [8]. The synthesis synthesis strategy strategy of 5-hydroxyimino-4,5-dihydrofuransof 5-hydroxyimino-4,5-dihydrofurans through through a lipase-catalyzed a lipase-catalyzed reaction reaction between betweenβ-nitrostyrenes β-nitrostyrenes and 1,3-dicarbonyland 1,3-dicarbonyl compounds compounds was developedwas developed by Wu by (Scheme Wu (Scheme 29) and 29) coworkers. and coworkers. The highlight The highlight of their of studytheirCatalysts study was 2020 that , was10, x a FORthat high PEER a stereoselectivity high REVIEW stereoselectivity (Z/E up (Z/E to 99:1) up to was 99:1) achieved was achieved by screening by screening a series of a diseriesff18erent of of25 βdifferent-nitrostyrenes β-nitrostyrenes and lipases and from lipases different from organisms. different organisms. Compared Compared with the reaction with the using reaction traditional using catalysttraditional NEt catalyst3 in methanol, NEt3 in the methanol, enzymatic the reaction enzymatic produced reaction much produced less by-product much less of dihydrofuran,by-product of whichdihydrofuran, led to the which high stereoselectivityled to the high stereoselectivity of the Z product of [ 77the]. Z product [77].

Scheme 29. Lipase-catalyzed reaction of nitrostyrene and acetylacetone.

As already delineated, the lipase extracted from Pseudomonas aeruginosa (PAL) has been proven to be effective in catalyzing the stereoselective hydrolytic kinetic resolution of a chiral ester. Manfred T. Reetz and coworkers applied iterative saturation mutagenesis (ISM) to maximize the quality of mutant libraries. They were able to subject ISM to systematic and rigorous comparison with directed evolution methods such as error-prone polymerase chain reaction (epPCR), saturation mutagenesis, and SchemeDNAScheme shuffling 28.28. FormationFormation (Scheme of of carbon–carbon carbon–carbon30). Their results bonds bonds byrevealed aby Diels–Alder a Diel thats–Alder the reaction best reaction mutant between between dramatically an anthracene an anthracene dieneimproved Catalysts 2020, 10, x FOR PEER REVIEW 18 of 25 enantioselectivityanddiene a maleimideand a maleimide without dienophile. dienophile. requiring high screening effort [78]. Enantio-pure ethyl- and methyl-3-hydroxyglutaric monocarboxylic acids have numerous industrial applications. The hydrolysis of prochiral diethyl- and dimethyl-3-hydroxy glutarates is one way to obtain these chiral molecules. Jacobsen’s work has demonstrated that lipase B from Candida Antarctica (CALB) is suitable for catalyzing in both hydrolysis and ammonolysis reactions. Moreover, the CALB enzyme provided high efficiency in the hydrolysis of diethyl-3-hydroxyglutarate, with up to 91% ee (Scheme 31). The hydrolase is reusable, with the preservation of excellent selectivity and activity [79]. Chiral bicyclicScheme diols 29. are Lipase-catalyzedLipase-catalyzed potentially important reaction reaction of syntheticni nitrostyrenetrostyrene intermediates. and acetylacetone. In the past, dynamic kinetic resolution (DKR) has been one of the most extensively used methods of asymmetric synthesizingAs already enantioselective delineated, thethe pure lipaselipase primary extractedextracted amines from from Pseudomonasor Pseudomonas secondary aeruginosa alcohols.aeruginosa Thus,(PAL) (PAL) a has modifiedhas been been proven provenmethod to tobecalled be eff effectiveective enzyme- in in catalyzing catalyzingand ruthenium-catalyzed the the stereoselective stereoselective dynamic hydrolytic hydrolytic kinetic kinetic kinetic asymmetric resolution resolution oftransformation of aa chiralchiral ester.ester. (DYKAT), Manfred T.developed Reetz and by coworkerscoworkers Patrik Krumlinde applied and iterative coworkers, saturation was employed mutagenesis to synthe (ISM) sizeto maximize the diacetat the es quality of bicyclic of mutantdiols, withlibraries. high They They enantioselectivity were were able able to to subject subject (99.9% ISM ISM ee to to). systematic systematicThe DYKAT and and protocolrigorous rigorous comparison comparisongives high with enantio- directed and evolutiondiastereoselectivities methods such (Scheme as error-prone 32). Compared polymerase to synthetic chain reaction chemistry, (epPCR), their worksaturation has illustrated mutagenesis, mutagenesis, that andthe synthesisDNA shuffling shu offfl ingenantioselective (Scheme 3030).). sertraline Their results from revealed racemic that cis/trans the best diol mutant (n = 2) dramatically can be achieved improved with a enantioselectivityhigher yield [80]. without requiringrequiring high screening eeffortffort [[78].78]. Enantio-pure ethyl- and methyl-3-hydroxyglutaric monocarboxylic acids have numerous industrial applications. The hydrolysis of prochiral diethyl- and dimethyl-3-hydroxy glutarates is one way to obtain these chiral molecules. Jacobsen’s work has demonstrated that lipase B from Candida Antarctica (CALB) is suitable for catalyzing in both hydrolysis and ammonolysis reactions. Moreover, the CALB enzyme provided high efficiency in the hydrolysis of diethyl-3-hydroxyglutarate, with up to 91% ee (Scheme 31). The hydrolase is reusable, with the preservation of excellent selectivity and activity [79]. Chiral bicyclic diolsSchemeScheme are 30.30. potentially HydrolyticHydrolytic importantkinetickinetic resolutionresolution synthetic ofof thethe intermediates. chiralchiral esterester rac-1.rac-1. In the past, dynamic kinetic resolution (DKR) has been one of the most extensively used methods of asymmetric synthesizingEnantio-pure enantioselective ethyl- and methyl-3-hydroxyglutaricO pure OHprimaryO aminesCALB or monocarboxylicsecondaryO OH alcohols. acidsO Thus, have numerousa modified industrial method calledapplications. enzyme- The and hydrolysis ruthenium-catalyzed of prochiral diethyl- dynamic and kinetic dimethyl-3-hydroxy asymmetric transformation glutarates is one(DYKAT), way to developed by Patrik KrumlindeR1O and coworkers,OR1 was employedR2 to synthesizeOR the1 diacetates of bicyclic diols, with high enantioselectivity1R1=Et (99.9% ee). The DYKAT aRprotocol2=OH gives high enantio- and 2R=Me bR=NH diastereoselectivities (Scheme 32). 1Compared to synthetic chemistry,2 their2 work has illustrated that the synthesis of enantioselective sertraline from racemic cis/trans diol (n = 2) can be achieved with a Scheme 31. Hydrolysis of prochiral diethyl-3-hydroxyglutarate (1) and dimethyl-3-hydroxyglutarate higher yield [80]. (2) with CALB.

Scheme 30. Hydrolytic kinetic resolution of the chiral ester rac-1.

O OH O CALB O OH O

R1O OR1 R2 OR1

1R1=Et aR2=OH 2R=Me bR=NH 1 2 2 Scheme 31. Hydrolysis of prochiral diethyl-3-hydroxyglutarate (1) and dimethyl-3-hydroxyglutarate (2) with CALB.

Catalysts 2020, 10, x FOR PEER REVIEW 18 of 25

Scheme 29. Lipase-catalyzed reaction of nitrostyrene and acetylacetone.

As already delineated, the lipase extracted from Pseudomonas aeruginosa (PAL) has been proven to be effective in catalyzing the stereoselective hydrolytic kinetic resolution of a chiral ester. Manfred T. Reetz and coworkers applied iterative saturation mutagenesis (ISM) to maximize the quality of mutant libraries. They were able to subject ISM to systematic and rigorous comparison with directed evolution methods such as error-prone polymerase chain reaction (epPCR), saturation mutagenesis, and DNA shuffling (Scheme 30). Their results revealed that the best mutant dramatically improved enantioselectivity without requiring high screening effort [78]. Enantio-pure ethyl- and methyl-3-hydroxyglutaric monocarboxylic acids have numerous industrial applications. The hydrolysis of prochiral diethyl- and dimethyl-3-hydroxy glutarates is one way to obtain these chiral molecules. Jacobsen’s work has demonstrated that lipase B from Candida Antarctica (CALB) is suitable for catalyzing in both hydrolysis and ammonolysis reactions. Moreover, the CALB enzyme provided high efficiency in the hydrolysis of diethyl-3-hydroxyglutarate, with up to 91% ee (Scheme 31). The hydrolase is reusable, with the preservation of excellent selectivity and activity [79]. Chiral bicyclic diols are potentially important synthetic intermediates. In the past, dynamic kinetic resolution (DKR) has been one of the most extensively used methods of asymmetric synthesizing enantioselective pure primary amines or secondary alcohols. Thus, a modified method called enzyme- and ruthenium-catalyzed dynamic kinetic asymmetric transformation (DYKAT), developed by Patrik Krumlinde and coworkers, was employed to synthesize the diacetates of bicyclic diols, with high enantioselectivity (99.9% ee). The DYKAT protocol gives high enantio- and diastereoselectivities (Scheme 32). Compared to synthetic chemistry, their work has illustrated that the synthesis of enantioselective sertraline from racemic cis/trans diol (n = 2) can be achieved with a higher yield [80].

Catalysts 2020, 10, 832 19 of 25 obtain these chiral molecules. Jacobsen’s work has demonstrated that lipase B from Candida Antarctica (CALB) is suitable for catalyzing in both hydrolysis and ammonolysis reactions. Moreover, the CALB enzyme provided high efficiency in the hydrolysis of diethyl-3-hydroxyglutarate, with up to 91% ee (Scheme 31). The hydrolaseScheme is 30. reusable, Hydrolytic with kinetic the preservationresolution of the of excellentchiral ester selectivity rac-1. and activity [79].

O OH O CALB O OH O

R1O OR1 R2 OR1

1R1=Et aR2=OH 2R=Me bR=NH 1 2 2 Scheme 31. Hydrolysis of prochiral diethyl-3-hydroxyglutarate (1) and dimethyl-3-hydroxyglutarate (2) withwith CALB.CALB.

Chiral bicyclic diols are potentially important synthetic intermediates. In the past, dynamic kinetic resolution (DKR) has been one of the most extensively used methods of asymmetric synthesizing enantioselective pure primary amines or secondary alcohols. Thus, a modified method called enzyme- and ruthenium-catalyzed dynamic kinetic asymmetric transformation (DYKAT), developed by Patrik Krumlinde and coworkers, was employed to synthesize the diacetates of bicyclic diols, with high enantioselectivity (99.9% ee). The DYKAT protocol gives high enantio- and diastereoselectivities (Scheme 32). Compared to synthetic chemistry, their work has illustrated that the synthesis of CatalystsenantioselectiveCatalysts 20202020,, 1010,, xx FORFOR sertraline PEERPEER REVIEWREVIEW from racemic cis/trans diol (n = 2) can be achieved with a higher yield1919 of [of80 2525].

SchemeScheme 32. 32. EsterEster cleavage cleavage of of diacetates. diacetates.

TheThe carboxylation carboxylation of of phenol phenol and and styrene styrene derivativesderivatiderivativesves by by carboxylasescarboxylases (Scheme (Scheme 33)3333)) in in carbonatecarbonate bufferbubufferffer isis highlyhighly highly regioselective.regioselective. regioselective. WhileWhile While benzoicbenzoic benzoic acidacid acid carboxylasescarboxylases carboxylases formform form thethe orthoortho the ortho-hydroxybenzoic-hydroxybenzoic-hydroxybenzoic acidacid derivative,acidderivative, derivative, thethe phenolicphenolic the phenolic acidacid carboxylasescarboxylases acid carboxylases actact onon act thethe on beta-carbonbeta-carbon the beta-carbon andand formform and form((EE)-)- cinnamiccinnamic (E)- cinnamic acids,acids, acids, withwith thewiththe aromaticaromatic the aromatic segmentsegment segment remainingremaining remaining completelycompletely completely intactintact intact [81].[81]. [81].

SchemeScheme 33.33.33.Regiocomplementary RegiocomplementaryRegiocomplementary enzymatic enzymaticenzymatic carboxylation carboxylcarboxyl ofationation phenols ofof andphenolsphenols hydroxystyrene andand hydroxystyrenehydroxystyrene derivatives. derivatives.derivatives. 2.4. Lyases E.C.4 2.4.2.4. LyasesLyasesWilliams E.C.4E.C.4 et al. (Scheme 34) applied the evolved tagatose-1,6-bisphosphate (TBP) aldolase to catalyze the production of tagatose 1,6-bisphosphate by using dihydroxyacetone phosphate (DHAP) WilliamsWilliams etet al.al. (Schem(Schemee 34)34) appliedapplied thethe evolvedevolved tagatose-1,6-bisphosphatetagatose-1,6-bisphosphate (TBP)(TBP) aldolasealdolase toto and glyceraldehyde 3-phosphate (G3P) as reactants. In their study, they performed three rounds of catalyzecatalyze thethe productionproduction ofof tagatosetagatose 1,6-bisphosp1,6-bisphosphatehate byby usingusing dihydroxyacetonedihydroxyacetone phosphatephosphate (DHAP)(DHAP) gene mutation, including random mutation and direct mutation on the framework of TBP aldolase andand glyceraldehydeglyceraldehyde 3-phosphate3-phosphate (G3P)(G3P) asas reactants.reactants. InIn theirtheir study,study, theythey performedperformed threethree roundsrounds ofof (agaY), followed by screening ~3000 first-generation variants created from the mutation. The evolved genegene mutation,mutation, includingincluding randomrandom mutationmutation andand diredirectct mutationmutation onon thethe frameworkframework ofof TBPTBP aldolasealdolase third-generation TBP aldolase not only increased the reaction rate 80-fold but also increased the (agaY),(agaY), followedfollowed byby screeningscreening ~3000~3000 first-generationfirst-generation variantsvariants createdcreated fromfrom thethe mutation.mutation. TheThe evolvedevolved stereoselectivity 100-fold [82]. third-generationthird-generation TBPTBP aldolasealdolase notnot onlyonly increasedincreased thethe reactionreaction raterate 80-fold80-fold butbut alsoalso increasedincreased thethe stereoselectivitystereoselectivity 100-fold100-fold [82].[82]. AA newnew approachapproach toto stereoselectivelystereoselectively synthesizingsynthesizing synsyn-- oror antianti-2-hydroxy-2-hydroxy diolsdiols waswas studiedstudied byby HusainHusain andand coworkers.coworkers. InIn thisthis study,study, thethe stereoselectivestereoselective synthesissynthesis ofof ((RR)-2-hydroxy)-2-hydroxy carbonylcarbonyl compoundscompounds catalyzedcatalyzed byby benzaldehydebenzaldehyde lyaselyase fromfrom Pichia.Pichia. GlucozymaGlucozyma (BAL)(BAL) waswas carriedcarried outout asas thethe criticalcritical step,step, inin whichwhich anan aliphaticaliphatic andand anan aromaticaromatic ketoneketone werewere usedused asas reactants.reactants. CorrespondingCorresponding compoundscompounds withwith highhigh opticaloptical puritypurity ((eeee%,%, >97%)>97%) werewere obtainedobtained fromfrom thethe enzymaticenzymatic reaction.reaction. ThisThis newnew approachapproach providesprovides aa newnew solutionsolution toto selectivelyselectively synthesizesynthesize purepure syn-syn- oror antianti-2-hydroxy-2-hydroxy diolsdiols withwith unprotectedunprotected substratesubstrate [83].[83].

SchemeScheme 34.34. StereochemistryStereochemistry ofof thethe reactireactionon catalyzedcatalyzed byby aldolases.aldolases.

WuWu etet al.al. (Scheme(Scheme 35)35) establishedestablished aa pracpracticaltical biocatalyticbiocatalytic methodmethod toto prepareprepare LL-norephedrine,-norephedrine, inin whichwhich anan RR-selective-selective pyruvatepyruvate decarboxylasedecarboxylase fromfrom SaccharomycesSaccharomyces cerevisiaecerevisiae andand anan SS-selective-selective ωω-- transaminasetransaminase fromfrom VibrioVibrio fluvialisfluvialis JS17JS17 werewere tandemlytandemly applied.applied. WiWithth aa molarmolar yieldyield ofof overover 60%,60%, dede (diastereomeric(diastereomeric excess)excess) andand eeee (enantiomeric(enantiomeric excess)excess) valuesvalues ofof greatergreater thanthan 99.5%99.5% werewere achievedachieved throughthrough thethe enzyme-catalyzedenzyme-catalyzed reactionreaction [84].[84]. InIn ththee presencepresence ofof carboncarbon dioxidedioxide asas aa C-1C-1 unit,unit, thethe enzymaticenzymatic carboxylationcarboxylation ofof parapara-hydroxystyrenes-hydroxystyrenes byby phenolicphenolic acidacid decarboxylasesdecarboxylases synthesizessynthesizes intointo ((EE)-cinnamic)-cinnamic acidsacids atat aa conversionconversion raterate ofof upup toto 40%.40%. Wuensch’sWuensch’s studystudy revealedrevealed thatthat thisthis novelnovel enzyme-catalyzedenzyme-catalyzed ββ-carboxylation-carboxylation hashas nono traditionaltraditional chemicalchemical synthesissynthesis counterpartcounterpart [85].[85].

Catalysts 2020, 10, x FOR PEER REVIEW 19 of 25

Scheme 32. Ester cleavage of diacetates.

The carboxylation of phenol and styrene derivatives by carboxylases (Scheme 33) in carbonate buffer is highly regioselective. While benzoic acid carboxylases form the ortho-hydroxybenzoic acid derivative, the phenolic acid carboxylases act on the beta-carbon and form (E)- cinnamic acids, with the aromatic segment remaining completely intact [81].

Scheme 33. Regiocomplementary enzymatic carboxylation of phenols and hydroxystyrene derivatives.

2.4. Lyases E.C.4 Williams et al. (Scheme 34) applied the evolved tagatose-1,6-bisphosphate (TBP) aldolase to catalyze the production of tagatose 1,6-bisphosphate by using dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) as reactants. In their study, they performed three rounds of gene mutation, including random mutation and direct mutation on the framework of TBP aldolase (agaY), followed by screening ~3000 first-generation variants created from the mutation. The evolved third-generation TBP aldolase not only increased the reaction rate 80-fold but also increased the stereoselectivity 100-fold [82]. A new approach to stereoselectively synthesizing syn- or anti-2-hydroxy diols was studied by Husain and coworkers. In this study, the stereoselective synthesis of (R)-2-hydroxy carbonyl compounds catalyzed by benzaldehyde lyase from Pichia. Glucozyma (BAL) was carried out as the critical step, in which an aliphatic and an aromatic ketone were used as reactants. Corresponding compounds with high optical purity (ee%, >97%) were obtained from the enzymatic reaction. This newCatalysts approach2020, 10, 832provides a new solution to selectively synthesize pure syn- or anti-2-hydroxy20 diols of 25 with unprotected substrate [83].

Scheme 34. StereochemistryStereochemistry of of the the reacti reactionon catalyzed by aldolases.

WuA new et al. approach (Scheme to35) stereoselectively established a prac synthesizingtical biocatalyticsyn- method or anti-2-hydroxy to prepare L diols-norephedrine, was studied in whichby Husain an R and-selective coworkers. pyruvate In thisdecarboxylase study, the stereoselectivefrom Saccharomyces synthesis cerevisiae of (R and)-2-hydroxy an S-selective carbonyl ω- transaminasecompounds catalyzed from Vibrio by fluvialis benzaldehyde JS17 were lyase tandemly from Pichia. applied. Glucozyma With a(BAL) molar was yield carried of over out 60%, as the de (diastereomericcritical step, in whichexcess) an and aliphatic ee (enantiomeric and an aromatic excess) ketone values were of greater used as than reactants. 99.5% Correspondingwere achieved throughcompounds the withenzyme-catalyzed high optical purity reaction (ee%, [84].>97%) In werethe presence obtained fromof carbon the enzymatic dioxide as reaction. a C-1 Thisunit, newthe enzymaticapproach providescarboxylation a new of solution para-hydroxystyrenes to selectively synthesizeby phenolic pure acidsyn decarboxylases- or anti-2-hydroxy synthesizes diols withinto (unprotectedE)-cinnamic substrateacids at [a83 conversion]. rate of up to 40%. Wuensch’s study revealed that this novel enzyme-catalyzedWu et al. (Scheme β-carboxylation 35) established has no a traditional practical biocatalytic chemical synthesis method counterpart to prepare L [85].-norephedrine, in which an R-selective pyruvate decarboxylase from Saccharomyces cerevisiae and an S-selective ω-transaminase from Vibrio fluvialis JS17 were tandemly applied. With a molar yield of over 60%, de (diastereomeric excess) and ee (enantiomeric excess) values of greater than 99.5% were achieved through the enzyme-catalyzed reaction [84]. In the presence of carbon dioxide as a C-1 unit, the enzymatic carboxylation of para-hydroxystyrenes by phenolic acid decarboxylases synthesizes into (E)-cinnamic acids at a conversion rate of up to 40%. Wuensch’s study revealed that this novel enzyme-catalyzedCatalysts 2020, 10, x FORβ-carboxylation PEER REVIEW has no traditional chemical synthesis counterpart [85]. 20 of 25

Scheme 35. Biocatalytic preparation of L-norephedrine by ScPDC and VfTA. Scheme 35. Biocatalytic preparation of L-norephedrine by ScPDC and Vf TA.

3.3. Conclusions Nature’sNature’s vastvast repertoirerepertoire hashas inspiredinspired thethe chemistchemist toto exploitexploit biocatalystsbiocatalysts for chemical synthesis. TheyThey alsoalso utilize utilize the the evolution evolution engineering engineering strategy strategy of mutation of mutation and selection and selection to improve to theimprove efficiency, the stability,efficiency, and stability, regio- andand enantioselectivityregio- and enantioselectivity of enzyme of function enzyme for function various for applications. various applications. However, challengesHowever, tochallenges enzymatic to reactionsenzymatic remain. reactions Research remain. should Research focus should on investigating focus on investigating nature’s mechanisms nature’s formechanisms enzyme innovation, for enzyme especially innovation, catalysts especially for non-natural catalysts reactions, for non-natural as well as delineating reactions, the as structurewell as anddelineating molecular the interactionsstructure and of molecular enzymes. interactions All these challengesof enzymes. will All havethese achallenges huge impact will have on modern a huge organicimpact on synthetical modern processes.organic synthetical processes. Undoubtedly,Undoubtedly, thethe extensive extensive literature literature on enzymaticon enzymatic reactions reactions highlights highlights the extraordinary the extraordinary ability ofability the biocatalyst of the biocatalyst to adapt to and adapt acquire and newacquire functions. new functions. Protein engineering Protein engineering and new and analytical new analytical methods havemethods opened have a opened toolbox a for toolbox more sustainable,for more sustainable, selective, selective, and cost-e andfficient cost-efficient chemical reactions.chemical reactions.

Funding:Funding: This work was carried by the LouisianaLouisiana Board of Regents, grant number LEQSF-EPS (2020)-SURE-226, andand thethe CentenaryCentenary CollegeCollege ofof LouisianaLouisiana CharltonCharlton H.H. LyonsLyons SummerSummer ResearchResearch Award.Award. TheThe publicationpublication ofof thisthis articlearticle waswas fundedfunded byby thethe CentenaryCentenary CollegeCollege ofof Louisiana.Louisiana.

Acknowledgments:Acknowledgments: WeWe thankthank JamesJames M.M. ClawsonClawson forfor hishis thoughtfulthoughtful commentscomments onon thethe manuscript. manuscript.

ConflictsConflicts ofof Interest:Interest: TheThe authorsauthors declaredeclare nono conflictconflict ofof interest.interest.

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