Biotechnological Methods of Sulfoxidation: Yesterday, Today, Tomorrow

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Review Biotechnological Methods of Sulfoxidation: Yesterday, Today, Tomorrow

Wanda M ˛aczka,Katarzyna Wi ´nska* and Małgorzata Grabarczyk

Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland; [email protected] (W.M.); [email protected] (M.G.) * Correspondence: [email protected]; Tel.: +48-71-320-5213

Received: 31 October 2018; Accepted: 25 November 2018; Published: 5 December 2018

Abstract: The production of chiral sulphoxides is an important part of the chemical industry since they have been used not only as pharmaceuticals and pesticides, but also as catalysts or functional materials. The main purpose of this review is to present biotechnological methods for the oxidation of sulfides. The work consists of two parts. In the first part, examples of biosyntransformation of prochiral sulfides using whole cells of bacteria and fungi are discussed. They have more historical significance due to the low predictability of positive results in relation to the workload. In the second part, the main enzymes responsible for sulfoxidation have been characterized such as chloroperoxidase, dioxygenases, cytochrome flavin-dependent monooxygenases, and P450 monooxygenases. Particular emphasis has been placed on the huge variety of cytochrome P450 monooxygenases, and flavin-dependent monooxygenases, which allows for pure sulfoxides enantiomers effectively to be obtained. In the summary, further directions of research on the optimization of enzymatic sulfoxidation are indicated.

Keywords: sulfoxides; biotransformation; enzymatic oxidation

1. Introduction Sulfur is an element that is about one percent of the dry mass of the human body because many sulfur-organic compounds are involved in metabolism. They are not only amino acids (cysteine, methionine) and their derivatives, but also glutathione and sulfolipids. Important enzyme cofactors also contain sulfur such as biotin, thioredoxin, lipoic acid, and coenzyme A [1]. Strong poisons can be found among the sulfur-containing compounds. For example, the consumption of Amanita mushrooms can cause fatal human poisoning and the toxin responsible for this effect is amanitin, which is an R-sulfoxide [2]. The organo-sulfur compounds are also an important group of secondary plant metabolites. They were isolated, among others, from onions, radishes, and cress. The characteristic smell and healing properties of plants of the genus Allium are attributed to sulfur-containing compounds, including chiral sulfoxides such as a compound characteristic of garlic: alliin (3-(2-propenylsulﬁnyl)-L-alanine) [3]. Since many compounds containing sulfur in their structure perform important biological functions, an attempt was made to develop methods for the synthesis of sulfur-based compounds, among which, the largest group are sulfoxides. It is noteworthy that the sulfur atom is a stereogenic center if it is linked to two different alkyl or aryl substituents, as well as to an oxygen atom and a lone electron pair. However, the bond between sulfur and oxygen is indirect between the coordination bond and the polarized double bond [4]. It does not form a typical π bond like between carbon and oxygen. It is possible to obtain stable enatiomers of sulfoxides because the lone electron pair is not inverted. The value of energy needed to change the conﬁguration on sulfur is in the range of 35–43 kcal mol−1

Catalysts 2018, 8, 624; doi:10.3390/catal8120624 www.mdpi.com/journal/catalysts Catalysts 2018, 8, x FOR PEER REVIEW 2 of 27 Catalysts 2018, 8, 624 2 of 27 of 35–43 kcal mol−1 for a typical sulfoxide [1,5]. The first examples of optically active sulfoxides were described in 1926, and the first general method for the synthesis of enantiomeric sulfoxides was reportedfor a typical in 1962 sulfoxide [6]. [1,5]. The first examples of optically active sulfoxides were described in 1926, andSince the first then, general the chemistry method forof chiral the synthesis sulfoxides of enantiomerichas been very sulfoxides popular am wasong reported researchers in 1962 because [6]. these Sincecompounds then, thehave chemistry found many of chiral applications sulfoxides [7–10]. has been They very are popularused as amongchiral ligands researchers in enantioselectivebecause these compounds catalysis. They have foundhave also many found applications wide applications [7–10]. They as are chiral used asauxiliaries chiral ligands and intermediatesin enantioselective in asymmetric catalysis. synthesis. They have High also asymmetric found wide induction applications was observed as chiral when auxiliaries using chiral and sulfinyl.intermediates In addition, in asymmetric several drugs synthesis. containing High asymmetricchirotopic sulfur induction in sulfoxide was observed are used when in using the pharmaceuticalchiral sulfinyl. industry, In addition, such several as esomeprazole drugs containing or modafinil chirotopic [11]. sulfur in sulfoxide are used in the pharmaceuticalDue to their industry, importance, such a as significant esomeprazole effort or ha modafinils been made [11]. to develop efficient methods for their preparation.Due to their importance,There are three a significant main ways effort to obtain has been optically made active to develop sulfoxides: efficient methods for their preparation. There are three main ways to obtain optically active sulfoxides: • nucleophilic substitution of a chiral precursor; •• asymmetricnucleophilic sulphoxidation substitution of of a chiralprochiral precursor; sulfides; •• kineticasymmetric separation sulphoxidation of racemic ofsulfoxides. prochiral sulfides; • Inkinetic this work, separation we will of focus racemic onsulfoxides. discussing the biotechnological methods of oxidation of sulfides becauseIn thisbiocatalysis work, we is willundoubtedly focus on discussing a method theof gentle biotechnological and environmentally methods of friendly oxidation synthesis of sulfides of sulfoxidesbecause biocatalysis [12]. is undoubtedly a method of gentle and environmentally friendly synthesis of sulfoxides [12]. 2. Biotransformations in Cultures of Microorganisms 2. BiotransformationsThe use of whole cells in Cultures of bacteria, of Microorganismsfungi, and yeast in the asymmetric oxidation of sulfides is an excellentThe alternative use of whole to cellsreactions of bacteria, catalyzed fungi, using and pure yeast enzymes. in the asymmetric Such biotransformations oxidation of sulfides are much is an cheaperexcellent and alternative more convenient to reactions than catalyzed enzymatic using reaction pures. enzymes. In addition, Such the biotransformations costly co-factors, are such much as nicotinamidecheaper and adenine more convenient dinucleotd thane/nicotinamide enzymatic reactions.adenine dinucleo In addition,tide phosphate the costly (NADH/NADPH), co-factors, such as arenicotinamide avoided. The adenine costs dinucleotde/nicotinamide associated with the purifi adeninecation dinucleotide of enzymes phosphate also disappear. (NADH/NADPH), It is also extremelyare avoided. important The costs that associated some withmicroorganism the purifications can of simulate enzymes alsothe disappear.metabolism It isof alsomammalian extremely cytochromeimportant thatP450 some monooxygenases microorganisms (CYPs) can simulate[13–17]. the metabolism of mammalian cytochrome P450 monooxygenases (CYPs) [13–17]. 2.1. Fungus and Yeast 2.1. Fungus and Yeast The reactions of asymmetric sulfoxidation with fungi and yeasts are carried out successfully in the secondThe reactions half of the of last asymmetric century [18] sulfoxidation. In 1982, Holland with fungi and and Carter yeasts published are carried a paper out successfullyin which they in presentedthe second the half results of the of last the centurybiotransformation [18]. In 1982, of Hollandaryl-substituted and Carter phenyl published and benzyl-methyl a paper in which sulfides they usingpresented the Mortierella the results isabellina of the biotransformation strain. These compounds of aryl-substituted have been phenyl oxidized and benzyl-methylto sulfoxides sulfidesduring enzymaticusing the oxidationMortierella with isabellina oxygenstrain. from Thesethe atmosphe compoundsre. In havethe studies, been oxidized the authors to sulfoxides focused mainly during onenzymatic determining oxidation the rate with and oxygen mechanism from the of atmosphere. enzymatic Inoxidation the studies, [19]. the In authors subsequent focused studies, mainly the on authorsdetermining confirmed the rate that and benzylic mechanism hydroxylation of enzymatic and oxidation sulfoxidation [19]. Inreactions subsequent can be studies, catalyzed the authors using theconfirmed same enzyme that benzylic in M. hydroxylation isabellina. This and enzyme sulfoxidation has the reactions characteristics can be catalyzed of cytochrome using the P450 same monooxygenaseenzyme in M. isabellina [20]. . This enzyme has the characteristics of cytochrome P450 monooxygenase [20]. FurtherFurther research research by by the the team team led led by by Holland Holland show showeded that that the the phenyl-alkyl phenyl-alkylatedated sulfoxides sulfoxides of of the the (S(S) )configuration configuration are are the the main main biotransformation biotransformation prod productsucts of of the the respective respective sulfides sulfides catalyzed catalyzed using using thethe strain strain HelminthosporiumHelminthosporium sp. NRRL 4671 [[21].21]. SubsequentSubsequent studiesstudies conducted conducted under under the the guidance guidance of ofHolland Holland showed showed that that similar similar sulfides sulfides were were also also oxidized oxidized using using the the strain strainM. M. isabellina isabellinaATCC ATCC 4261 4261 3 3(Scheme (Scheme1 ).1). However, However, the the formation formation of of R-isomers was preferred here here [22,23]. [22,23]. Further Further research research has has suggestedsuggested that that both both strains strains of Helminthosporium andand M.M. isabellinaisabellinause use other other enzymes enzymes for for the the oxidation oxidation of ofsulfides sulfides [24 [24].].

SchemeScheme 1. 1. SulfoxidationSulfoxidation of of sulfides sulﬁdes catalyzed catalyzed by by HelminthosporiumHelminthosporium sp.sp. and and M.M. isabellina. isabellina .

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Also,Also, the the white-rot white-rot BasidiomycetesBasidiomycetes catalyzedcatalyzed oxidation oxidation of of aromatic aromatic pro-chiral pro-chiral sulfides sulﬁdes into into sulfoxidessulfoxides with with good good enantioselectivity andand yield.yield. TheThe reactionsreactionswere were carried carried out out using using whole whole cells cells of ofTrametes Trametes rigida rigida, Trametes, Trametes versicolor versicolor,, Trametes Trametes villosa villosa,, PycnoporusPycnoporus sanguineus ,, TrichaptumTrichaptum byssogenum byssogenum, , andand IrpexIrpex lacteus lacteus. .These These microorganisms microorganisms also also produced produced a a smal smalll amount amount of of sulfones. sulfones. In In the the oxidation oxidation reactionsreactions of of all all the the alkyl alkyl aryl aryl sulfoxides, sulfoxides, formation formation of ( ofS)-enantiomers (S)-enantiomers was was preferred preferred (Scheme (Scheme 2). All2). strainsAll strains of Basidiomycetes of Basidiomycetes gavegave (S)-(phenylpropyl)sulfoxide (S)-(phenylpropyl)sulfoxide with with high high enantiomeric enantiomeric excess excess (ee (ee ≥≥ 99%)99%) fromfrom the the (phenylpropyl)sulfide (phenylpropyl)sulﬁde [25]. [25].

SchemeScheme 2. 2. SulfoxidationSulfoxidation of of alkyl alkyl aryl aryl sulfides sulﬁdes catalyzed catalyzed by by the the white-rot white-rot Basidiomycetes.Basidiomycetes .

InIn 2007, 2007, Pinedo-Rivilla andand coworkers coworkers [ 26[26]] presented presented the the results results of biotransformationof biotransformation of four of four alkyl alkylaryl andaryl alkyl and sulfides alkyl sulfides catalyzed catalyzed by Eutypa by lata Eutypa, Botrytis lata, cinerea Botrytis, and Trichodermacinerea, and viride Trichoderma. Reactions viride were. Reactionscarried out were both carried in resting out both and shakenin resting cultures. and shak It wasen cultures. observed It thatwas theobserved substrates that containingthe substrates the containingaromatic ring the werearomatic mainly ring oxidized. were mainly The alkyl oxidized. sulfides The were alkyl transformed sulfides were with transformed low yields, with mainly low to yields,sulfones mainly or treated to sulfones as a carbon or tr source.eated Theas a bestcarbon result source. for the The oxidation best result of thioanisole for the wasoxidation obtained of thioanisoleduring the was transformation obtained during in aresting the transformation culture of T. in viride a resting.(R)-methylphenyl culture of T. viride sulfoxide. (R)-methylphenyl was obtained sulfoxidewith 70% was ee but obtained with low with yield 70% (10%). ee but With with greater low yiel efficiency,d (10%). this With sulfoxide greater was efficiency, obtained this in a sulfoxide culture of wasB. cinerea obtained(50% in ee, a culture 32% yield). of B. Incinerea the reactions, (50% ee, the32% formation yield). In of the the reactions, corresponding the formation sulfone, of which the correspondingformed during sulfone, the further which oxidation formed of during sulfoxides, the further was observed. oxidationR -sulfoxideof sulfoxides, is obtained was observed. in both Rreactions-sulfoxide catalyzed is obtained using in bothE. lata reactionsand T.viride catalyzed. Oxidation using E. in lata a T. and viride T. shakingviride. Oxidation culture gives in a T. the viride best shakingenantiomeric culture excess gives inthe good best yieldenantiomeric (ee > 95%, excess 60%). in The good optical yield purity(ee > 95%, of this 60%). sulfoxide The optical is extremely purity ofhigh, this suggesting sulfoxide thatis extremely this substrate high, is suggesting well suited that to the this enzymatic substrate system is well in whichsuited theto the oxidation enzymatic takes systemplace. Thisin which result the indicates oxidation the takes potential place. use This of T. result viride indicatesin the production the potential of this use chiral of T. sulphoxide viride in the in productionpreparative of scale. this chiral sulphoxide in preparative scale. AspergillusAspergillus strainsstrains are are also also capable capable of enantioselecti of enantioselectiveve oxidation oxidation of sulfides of sulfides (Scheme (Scheme 3). Two3). sulfidesTwo sulfides were wereselected selected for the for tests: the tests:cyclohexyl cyclohexyl methyl methyl and thioanisole. and thioanisole. All tested All tested strains strains showed showed the abilitythe ability to oxidize to oxidize both both alkyl alkyl sulfides sulfides and andthioanisole. thioanisole.

SchemeScheme 3. 3. SulfoxidationSulfoxidation of alkyl of alkyl aryl aryl andand alkyl alkyl alkyl alkyl sulfides sulﬁdes catalyzed catalyzed by Aspergillus by Aspergillus japonicus japonicus ICFC 744/11.ICFC 744/11.

The formation of R-enantiomers was preferred and full chemoselectivity was confirmed The formation of R-enantiomers was preferred and full chemoselectivity was confirmed because no sulfones were detected. During the research, it was noticed that the addition of because no sulfones were detected. During the research, it was noticed that the addition of isopropyl isopropyl alcohol affects both chemo- and enantioselectivity of the process. Among the tested alcohol affects both chemo- and enantioselectivity of the process. Among the tested strains of strains of Aspergillus japonicus ICFC 744/11 biotransformed the alkyl sulfide with the best yield Aspergillus japonicus ICFC 744/11 biotransformed the alkyl sulfide with the best yield and R stereoselectivityand stereoselectivity (100% (100% reaction, reaction, ee > 99% ee > ( 99%R)) [27]. ( )) [27]. ResearchResearch on on the the oxidation oxidation of of sulfides sulfides allowed allowed to to think think that that the the fungal fungal enzyme enzyme system system can can be be usedused to to simultaneously simultaneously transform transform two two functional functional groups. groups. Therefore, Therefore, research research has has been been carried carried out outon Helminthosporium M. isabellina theon theuse useof Helminthosporium of sp. sp.NRRL NRRL 4671 4671 and and M. isabellina ATCCATCC 42613 42613 for for bioconversion bioconversion of of substratessubstrates that that contain contain both both carbonyl carbonyl and and sulfide sulfide functional functional groups groups (Scheme (Scheme 44).).

Catalysts 2018, 8, x FOR PEER REVIEW 4 of 27

Scheme 4. Biotransformation of ketosulfides catalyzed by Helminthosporium sp. NRRL 4671 and M. isabellina ATCC 42613.

These reactionsCatalysts 2018 allowed, 8, 624 to obtain pure isomers containing two new chiral centers4 of 27 [28]. As in previous studiesCatalysts 2018on , 8sulfide, x FOR PEER oxidation, REVIEW Helminthosporium and M. isabellina strains4 of 27catalyzed the formation of sulfoxides of opposite configuration. The Helminthosporium sp. catalyzed the formation of hydroxy-sulfoxide with high optical purity if the carbonyl group had a methyl substituent (Table 1). Sulfoxide alcohols from phenylcarbonyl-containing substrates were prepared via a reaction with the M. isabellina strain.SchemeScheme 4. 4. BiotransformationBiotransformation ofof ketosulfides ketosulfides catalyzed catalyzed byby HelminthosporiumHelminthosporium sp. sp.NRRL NRRL 4671 4671 and andM. isabellinaM. isabellina ATCCATCC 42613. 42613. Table 1. Biotransformation of the substituted sulfides catalyzed by Helminthosporium sp. and M. TheseThese reactions reactions allowed allowed to toobtain obtain pure pure isomers isomers containing containing two new two chiral new chiralcenters centers [28]. As [ 28in]. isabellina.previous As in previous studies studies on sulfide on sulfide oxidation, oxidation, HelminthosporiumHelminthosporium andand M.M. isabellina isabellina strainsstrains catalyzed catalyzed the the formationformation of of sulfoxides sulfoxides of opposite configuration.configuration. TheTheHelminthosporium Helminthosporiumsp. sp. catalyzed catalyzed the the formation formation of Biocatalyst ofhydroxy-sulfoxide hydroxy-sulfoxide with with high high optical optical purity purity if if the the carbonyl carbonyl group group had had a methyla methyl substituent substituent (Table (Table1). Substrate1).Sulfoxide Sulfoxide alcohols alcohols from from phenylcarbonyl-containing phenylcarbonyl-containingHelminthosporium substrates sp. substrates were were prepared prepared via via a reaction aM. reaction isabellina with with the theM. M. isabellina isabellinastrain. strain. Yield Stereoselectivity Yield Stereoselectivity R1 = H, R2 = CHTable3 1. Biotransformation 90% of the substituted de sulfides 89% catalyzed by Helminthosporium 23% sp. and de 46% Table 1. Biotransformation of the substituted sulfides catalyzed by Helminthosporium sp. and M. M. isabellina. R1 = OCH3, R2isabellina = CH3. 70% de 85% 69% de 52% R1 = Br, R2 = CH3 30% de 86% Biocatalyst 64% de 50% Biocatalyst R1 = OCH3, R2 = PhSubstrate Substrate 6% HelminthosporiumHelminthosporium de <5% sp.sp. M.isabellina 48%M. isabellina de 40% R1 = H, R2 = CH3 66% YieldYield Stereoselectivity Stereoselectivity de 92% YieldYield 48% Stereoselectivity Stereoselectivity de 40% R1 = H, R2 = CH3 90% de 89% 23% de 46% R1 = OCH3, R2 = CH3R 1 = H, R2 = CH 72%3 90% de de 95% 89% 23% 42% de 46% de 85% R1 = OCHR 3=, R OCH2 = CH,R3 = CH 70% 70% de de 85% 85% 69% 69% de 52% de 52% 1 2 3 1 3 2 3 R = Br, R = CH1 2 3 72% de 90% 37% de 40% R = Br, RR1 = CH Br, R 2 = CH3 30% 30% de de 86% 86% 64% 64% de 50% de 50% R1 = OCH3, RR2 1= = PhOCHR 13,= R OCH2 = Ph3,R 2 = 16% Ph 6% 6% de de de <5%26% <5% 48% 48% 69% de 40% de 40% de 54% R1 = H, R2 = CH H, R3 = CH 66% 66% de de 92% 92% 48% 48% de 40% de 40% R1 = Br, R2 =Ph 1 2 3 No products 25% de 28% R1 = OCHR13=, R OCH2 = CH3,R3 2 = CH3 72% 72% de de 95% 95% 42% 42% de 85% de 85% R1 = Br, RR12 = CH Br, R3 2 = CH3 de72% – 72% diastereoisomeric de de 90% 90% excess. 37% 37% de 40% de 40% R = OCH ,R = Ph 16% de 26% 69% de 54% R1 = OCH13, R2 = Ph3 2 16% de 26% 69% de 54% R1 = Br, R2 =Ph No products 25% de 28% R1 = Br, R2 =Ph No products 25% de 28% Another research topic carried out bydede—diastereoisomeric –the diastereoisomeric Holland team excess.excess. was the oxidation of sulfur amino acids. The protected amino acids were biotransformed using Beauveria species strains. Only methionine Another research topic carried out by the Holland team was the oxidation of sulfur amino acids. derivatives wereAnother biotransformed research topic with carried good out by yiel the Hollandd and teamhigh was enantiomeric the oxidation of excess. sulfur amino A strain acids. of Beauveria TheThe protected protected amino amino acids acids were were biotransformed using using Beauveria species strains. Only Only methionine methionine caledonica ATCCderivativesderivatives 64970 were were oxidized biotransformed biotransformed N-phthaloyl-L-methionine with with good good yield yield and high and enantiomeric high to enantiomeric sulfoxide excess. excess. A(S strainSSC) of Awith Beauveria strain de of 90% (Figure Beauveria caledonica ATCC 64970 oxidized N-phthaloyl-L-methionine to sulfoxide (S S ) with de 1). N-phthaloyl-D-methioninecaledonica ATCC 64970 wasoxidized converted N-phthaloyl-L-methionine to (SSRC) sulfoxides to sulfoxide with (SSS Ca) lowerwith de Syield90%C (Figure but equally high 90% (Figure1). N-phthaloyl-D-methionine was converted to (S R ) sulfoxides with a lower yield but de = 92% [29].1). No N-phthaloyl-D-methionine such good results was were converted observed to (SSR forC) sulfoxides cysteineS withC derivatives. a lower yield but equally high deequally = 92% high [29]. deNo = such 92% good [29]. Noresults such were good observed results were for cysteine observed derivatives. for cysteine derivatives.

Figure 1. The structures of products obtained in the oxidation of sulfur amino acids. Figure 1. The structures of products obtained in the oxidation of sulfur amino acids. FigureHowever, 1. The instructures the oxidation of productsreaction of obtainedN-Boc derivative in the ofoxidation l-4-S-morpholine-2-carboxylic of sulfur amino acids. acid, very highHowever, conversion in the (above oxidation 90%) reaction was observed of N-Boc wi derivativeth a high diasteroisomeric of l-4-S-morpholine-2-carboxylic excess (above 95%) acid, at However,thevery insame high the time. conversion oxidation It is worth (above reaction noticing 90%) was thatof observed Nstrains-Boc with ofderivative aB. high caledonica diasteroisomeric ofand l-4- B. bassianaS-morpholine-2-carboxylic excess [30] (above oxidized 95%) at the the acid, substratesame time. to Itcompound, is worth noticing which thatis the strains intermediate of B. caledonica compoundand B.in bassianathe synthesis[30] oxidized of chondrine the substrate (Scheme to very high conversion5).compound, (above which is the90%) intermediate was observed compound wi inth the a synthesis high diasteroisomeric of chondrine (Scheme 5excess). (above 95%) at the same time. It is worth noticing that strains of B. caledonica and B. bassiana [30] oxidized the substrate to compound, which is the intermediate compound in the synthesis of chondrine (Scheme 5).

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Scheme 5. Synthesis of chondrine. SchemeScheme 5. 5. SynthesisSynthesis of of chondrine. chondrine. In order to obtain the active form of rabeprazole, the authors [31] carried out screening tests on In order to obtain the active form of rabeprazole, the authors [31] carried out 650 In ordermicroorganisms. to obtain the activeThe form mostof rabeprazol effectivee, the authorsbiocatalyst [31] carried for out screeningoxidation tests onof screening tests on 650 microorganisms. The most effective biocatalyst for oxidation of 6502-[4-(3-methoxypropoxy)-3-methylpyridin-2-yl microorganisms. The most ]-methylthiobenzimidazoleeffective biocatalyst for (PTBI)oxidation is theof 2-[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]-methylthiobenzimidazole (PTBI) is the 2-[4-(3-methoxypropoxy)-3-methylpyridin-2-ylCunninghamella echinulata MK40 strain (Scheme]-methylthiobenzimidazole 6). After 48 h of transformation, (PTBI) theis authorsthe Cunninghamella echinulata MK40 strain (Scheme6). After 48 h of transformation, the authors Cunninghamellaobserved a 92% echinulataconversion MK40 without strain forming (Scheme the sulf 6).one After as an 48 undesired h of transformation, product. The mainthe authorsproduct observed a 92% conversion without forming the sulfone as an undesired product. The main product observed a 92% conversion without forming the sulfone as an undesired product. The main product waswas the the S-sulfurS-sulfur enantiomer. enantiomer. During During the the st study,udy, it was foundfound thatthat the the addition addition of of glucose glucose affected affected the the wasbiotransformationbiotransformation the S-sulfur enantiomer. efficiency. efﬁciency. During On On this this the basis, basis, study, it it was it was found found thatthat that conversionconversion the addition of of PTBI PTBI of toglucose to rabeprazole rabeprazole affected may may the biotransformationinvolveinvolve the the regeneration regeneration efficiency. of of NADH NADHOn this or or basis, NADPH NADPH it was in foundC.C. echinulataechinulata that conversionMK40 MK40 supported supported of PTBI by byto glucose. rabeprazoleglucose. may involveAlso, Also,the regenerationother other CunninghamellaCunninghamella of NADHstrains orstrains NADPH tested testedwere in C. were justechinulata as just capable MK40as ofcapableconverting supported of PTBI convertingby glucose. to rabeprazole. PTBI to rabeprazole.TheAlso, efﬁciency other The of efficiencyCunninghamella the reactions of catalyzedthe reactionsstrains by C.tested catalyzed echinulata were IAMby just C. 6210 echinulataas andcapableC. blakesleeanaIAM of 6210 converting andstrains C. IAMblakesleeana PTBI 6219 to rabeprazole.strainswere IAM comparable 6219The efficiencywere to those comparable ofofC. the echinulata reactions to thoseMK40 catalyzedof (4C. mMechinulata andby C. 3.9 MK40echinulata mM, respectively, (4 mM IAM and 6210 after 3.9 and mM, 48 h).C. respectively, Theblakesleeana lower strainsafteryield 48 IAM ofh). biotransformation The6219 lower were yieldcomparable by of strainbiotransformation to of thoseC. echinulata of C. echinulatabyIAM strain 6209 MK40of after C. 48echinulata (4 h mM was observed,and IAM 3.9 6209 mM, because after respectively, 0.848 mMh was afterobserved,rabeprazole 48 h). because The was lower formed.0.8 yieldmM rabeprazole of biotransformation was formed. by strain of C. echinulata IAM 6209 after 48 h was observed, because 0.8 mM rabeprazole was formed.

SchemeScheme 6. 6. SulfoxidationSulfoxidation of of PTBI PTBI catalyzed bybyCunninghamella Cunninghamellasp. sp. Scheme 6. Sulfoxidation of PTBI catalyzed by Cunninghamella sp. AsAs well, well, sulfuric sulfuric precursors precursors omeprazole omeprazole and lansoprazole (Figure(Figure2) 2) were were selected selected for for biotransformation. These compounds, like rabeprazole, are proton pump inhibitors. In reactions biotransformation.As well, sulfuric These precursors compound omeprazoles, like rabeprazole, and lansoprazole are proton (Figurepump inhibitors.2) were selectedIn reactions for catalyzed using C. echinulata MK40, omeprazole and lansoprazole were also obtained, but in low biotransformation.catalyzed using C. Theseechinulata compound MK40, s,omeprazole like rabeprazole, and lansoprazole are proton werepump also inhibitors. obtained, In but reactions in low yield. It is surprising that other substrates, such as DL-methionine, DL-ethionine, DL-lanthionine, catalyzedyield. It is using surprising C. echinulata that other MK40, substrates, omeprazole such andas DL-methionine, lansoprazole were DL-ethionine, also obtained, DL-lanthionine, but in low 4-methylthiophenol, thioanisole, and 4-chlorophenylmethyl sulﬁde were not oxidized to the yield.4-methylthiophenol, It is surprising thatthioanisole, other substrates, and 4-chloroph such asenylmethyl DL-methionine, sulfide DL-ethionine, were not oxidizedDL-lanthionine, to the corresponding sulfoxides. 4-methylthiophenol,corresponding sulfoxides. thioanisole, and 4-chlorophenylmethyl sulfide were not oxidized to the corresponding sulfoxides.

Catalysts 2018, 8, 624 6 of 27 Catalysts 2018, 8, x FOR PEER REVIEW 6 of 27

FigureFigure 2. 2. TheThe structures structures of of omeprazole omeprazole and and lansoprazole. lansoprazole. 2.2. Bacteria 2.2. Bacteria The catalytic bio-oxidation reactions can also be catalyzed using bacterial strains. Previous The catalytic bio-oxidation reactions can also be catalyzed using bacterial strains. Previous research [32,33] has shown that this process occurs in bacteria, as in the case of fungi, and can be research [32,33] has shown that this process occurs in bacteria, as in the case of fungi, and can be catalyzed using P450 or dibenzothiophene monooxygenases [34]. catalyzed using P450 or dibenzothiophene monooxygenases [34]. Research carried out by the Ohta team [35,36] in the 1980s proved the usefulness of bacteria of the Research carried out by the Ohta team [35,36] in the 1980s proved the usefulness of bacteria of genus Rhodococcus for the oxidation of alkylaryl sulfides. For biotransformation, the authors used the the genus Rhodococcus for the oxidation of alkylaryl sulfides. For biotransformation, the authors used Rhodococcus equi strain. As a result of the reaction, a high enantiomeric excess (ee 97–100%) of alkyl the Rhodococcus equi strain. As a result of the reaction, a high enantiomeric excess (ee 97–100%) of aryl sulfoxides from sulfide was obtained. The authors also observed the formation of sulfones. alkyl aryl sulfoxides from sulfide was obtained. The authors also observed the formation of sulfones. One of the most commonly selected bacterial strains for the biotransformation of sulfur-containing One of the most commonly selected bacterial strains for the biotransformation of compounds is the Rhodococcus erythropolis, which has been extensively tested for its ability to oxidize sulfur-containing compounds is the Rhodococcus erythropolis, which has been extensively tested for its sulfur-containing compounds. This strain is also used in the biocatalytic desulfurization of fossil ability to oxidize sulfur-containing compounds. This strain is also used in the biocatalytic fuels [37]. The dibenzothiophene desulfurization (dsz) operon from R. erythropolis IGTS8 encodes desulfurization of fossil fuels [37]. The dibenzothiophene desulfurization (dsz) operon from R. three proteins, DscC, DscA, and DsaB. Two of the enzymes are cytoplasmic monooxygenases erythropolis IGTS8 encodes three proteins, DscC, DscA, and DsaB. Two of the enzymes are (DszA and DszC), while the third (DszB) is a desulfinase [33]. The specific role in sulfur oxidation has cytoplasmic monooxygenases (DszA and DszC), while the third (DszB) is a desulfinase [33]. The monooxygenase DscC, which participates in conversion of sulfide to sulfoxide and thence to sulfone. specific role in sulfur oxidation has monooxygenase DscC, which participates in conversion of In 2003, Holland published a study [38] on the oxidation of many sulfur derivatives using the strain sulfide to sulfoxide and thence to sulfone. In 2003, Holland published a study [38] on the oxidation R. erythropolis, in which the enzymes DscA and DscB were lacking, while there were different DscC of many sulfur derivatives using the strain R. erythropolis, in which the enzymes DscA and DscB enzymes. The first group were methyl phenyl sulfides, which were biotransformed mainly to the were lacking, while there were different DscC enzymes. The first group were methyl phenyl isomer with the R configuration of a sulfur asymmetric center (Table2). Sulfoxides having a substitution sulfides, which were biotransformed mainly to the isomer with the R configuration of a sulfur in the para position were obtained with a higher enantiomeric excess. The enantioselectivity of the asymmetric center (Table 2). Sulfoxides having a substitution in the para position were obtained with process was also influenced by the group that substituted the aromatic ring. Higher optical purity has a higher enantiomeric excess. The enantioselectivity of the process was also influenced by the group products with higher electronic withdrawing groups (e.g. p-CN, and p-NO ). that substituted the aromatic ring. Higher optical purity has products2 with higher electronic withdrawing groups (e.g. p-CN, and p-NO2). Table 2. Sulfoxidation of the substituted methyl aryl sulfides catalyzed by R. erythropolis.

TableSubstrate 2. Sulfoxidation of the Yield substituted (%) methyl Sulfoxide aryl sulfides Conﬁguration catalyzed by R. erythropolis ee (%) .

C6H5SCHSubstrate3 67 Yield (%) Sulfoxide Configuration rac ee (%) 2 p-CH3C6H4SCH3 72 R 62 C6H5SCH3 67 rac 2 m-CH3C6H4SCH3 60 R 10 p-CH3C6H4SCH3 72 R 62 o-CH3C6H4SCH3 95 rac 4 p-FC6mH-CH4SCH3C3 6H4SCH3 52 60 RR 10 63 p-ClC6oH-CH4SCH3C36H4SCH3 70 95 racR 4 72 p-BrC H SCH 65 R 76 6 p4-FC6H3 4SCH3 52 R 63 p-NO2C6H4SCH3 83 R >98 p-ClC6H4SCH3 70 R 72 p-CNC6H4SCH3 43 R 85 p-BrC6H4SCH3 65 R 76 p-NO2C6H4SCH3 83 R >98 The second group contained substituted benzyl sulﬁdes (Table3). The authors investigated p-CNC6H4SCH3 43 R 85 the effect of a substituent in the aromatic ring during oxidation. The best optical purity of sulfoxidesThe second was obtained group contained with para -substitutedsubstituted benzyl polynuclear sulfides groups (Table of 3). substrates The authors (p-F, pinvestigated-Cl, p-Br, p-NO the2 , effectp-CN). of Largea substituent alkyl or in aryl the substituents, aromatic ring such during as C oxidation.6H5CH2S(alkyl)C The best6 opticalH13 or ppurity-t-C4H of9C sulfoxides6H4CH2SCH was3 , C H C H SCH , and C H C H SCH , caused a signiﬁcant decrease in enantiomeric excess [38]. obtained6 5 2 with4 para3 -substituted6 5 3 polynuclear6 3 groups of substrates (p-F, p-Cl, p-Br, p-NO2, p-CN). Large alkyl or aryl substituents, such as C6H5CH2S(alkyl)C6H13 or p-t-C4H9C6H4CH2SCH3, C6H5C2H4SCH3, and C6H5C3H6SCH3, caused a significant decrease in enantiomeric excess [38].

Catalysts 2018, 8, 624 7 of 27

Table 3. Sulfoxidation of the substituted benzyl sulﬁdes catalyzed using R. erythropolis.

Substrate Yield (%) Sulfoxide Conﬁguration ee (%)

C6H5CH2SCH3 73 R 26 C6H5CH2SC3H7 58 R 66 C6H5C2H4SCH3 73 R 14 C6H5C3H6SCH3 81 rac 3 C6H5CH2SC6H5 65 R >98 C6H5CH2SC3H6 C6H5 54 R 58 C6H5CH2SC2H4 C6H5 50 R 78 p-CH3OC6H4CH2SCH3 51 R 27 m-CH3OC6H4CH2SCH3 54 rac 4 o-CH3OC6H4CH2SCH3 82 R 44 p-NO2C6H4CH2SCH3 58 R 76 m-NO2C6H4CH2SCH3 15 R 52 o-NO2C6H4CH2SCH3 41 R 70 p-FC6H4CH2SCH3 61 R 62 p-ClC6H4CH2SCH3 61 R 65 p-BrC6H4CH2SCH3 60 R 76 p-CNC6H4CH2SCH3 47 R 72 p-CH3 C6H4CH2SC6H5 62 R 82 p-CH3CONHC6H4CH2SCH3 63 R 39 C6H5SCH2CN 50 S 13 p-CH3OC6H4SCH2CN 91 R 88 p-BrC6H4SCH2CN 92 R 94

The tests were also subjected to sulfur derivatives of amino acids (Table4). In the oxidation reaction of sulﬁdes to sulfoxides, the R conﬁguration was preferred regardless of the amino acid stereochemistry. Similar stereochemistry was observed during the oxidation of methionine and cysteine derivatives by chloroperoxidase [39,40].

Table 4. Sulfoxidation of amino acids catalyzed using R. erythropolis.

Substrate Yield (%) Sulfoxide Conﬁguration de (%) Methyl ester of N-MOC-S-methyl-L-Cys 12 R 34 Propyl ester N-MOC-S-methyl-L-Cys 22 R 52 Methyl ester of N-MOC-L-Met 54 R 83 Propyl ester of N-MOC-L-Met 56 R 90 Pentyl ester of N-MOC-L-Met 59 R 93 Heptyl ester of N-MOC-L-Met 35 R >95 Methyl ester of N-MOC-D-Met 53 R >95 Ethyl ester of N-t-Boc-D-Met 69 R >95 Pentyl ester of N-t-Boc-D-Met 17 R >95

Ai-Tao and coworkers [41] presented an uncommon perception of sulfoxidation reaction. Formerly, sulfone formation was an undesirable reaction, whereas in this case the reaction was used to obtain an optically pure sulfoxide with the S conﬁguration as a result of deracemization (Scheme7). Whole cells of Rhodococcus sp. ECU0066 were used for asymmetric oxidation of racemic sulfoxides, which were excellent substrates for biotransformation due to their lower biotoxicity Catalystscompared 2018,to 8, x sulﬁdes. FOR PEER REVIEW 8 of 27

SchemeScheme 7. 7. SulfoxidationSulfoxidation of of aryl aryl alkil alkil sulfides sulﬁdes catalyzed catalyzed using using RhodococcusRhodococcus sp.sp.

(S)-Phenylmethylsulfoxide was obtained at higher concentrations and good enantiomeric excess (37.8 mM and 93.7% ee (S)) if the substrate was in the form of racemic sulfoxide. In comparison, asymmetric oxidation of phenylmethyl sulfide gave the product at a lower yield and enantiomeric excess (10 mM and 80% ee (S)). Rhodococcus was also characterized by a fairly good activity (yield, 22.7–43.2%, in 1–8 h) and enantioselectivity (ee (S) > 99.0%) to para substituted (methyl and chloro) phenylmethylsulfoxides and phenylethyl sulfonate. He and colleagues realized very good results in the oxidation of phenylmethyl sulfide [42], where biotransformation reactions catalyzed using Rhodococcus sp. CCZU10-1 were carried out in a two-phase n-octane/water system: 55.3 mM (S)-phenyl-methylsulfoxide with an enantiomeric excess >99.9% was conveniently obtained. Pseudomonas bacteria also convert sulfides into chiral sulfoxides. An example is the strain of P. monteilii CCTCC M2013683, which catalyzed the oxidation reaction of a series of aryl alkyl sulfides to sulfoxides with a yield of 54–99% and a good enantiomeric excess (63–99%). The cultivation was carried out in M9 medium with toluene as the carbon source. Oxidation reactions were carried out in resting cultures. The obtained results showed that the aromatic ring of substrates with the substituted group may influence the activity and enantioselectivity of the oxidation reaction with P. monteilii CCTCC M2013683 [43]. In turn, the use of biotransformation cultures of the genus Streptomyces and Bacillus cereus allowed for the formation of both enantiomers of cyclohexylmethyl sulfoxide, where S. badius, S. hiroshimensis, S. phaerocromogenes S. flaviviseus, and B. cereus strains produced significant amounts (>25%) of sulfoxides. The first two strains gave a racemic mixture. The other three performed the sulfoxidation reaction with different enantioselectivity. S. hiroshimensis ATCC 27429 and S. flavogriseus ATCC 33331 oxidized to the R- and S-sulfoxide species without generating sulfone. The reaction catalyzed using S. phaeochromogenes NCIMB 11741 was very interesting as there was the synchronized reaction of two enzymes that simultaneously oxidized the sulfide to the corresponding enantiomers of sulfoxide. During the first six hours of biotransformation, mainly S-sulfoxide with high enantiomeric excess (ee > 99%) was formed. After this time, the oxidation of cyclohexylmethyl sulfide to the R-isomer was also observed. Ultimately after 48 h, 55% R-cyclohexylmethyl sulfoxide with 55% ee was obtained [30]. One of the methods of optimizing biotransformation conditions carried out by other cells may be their immobilization. The use of Gordonia terrae IEGM 136 cells, which was immobilized in a cryogel of polyvinyl alcohol, significantly reduced the oxidation time of methylphenyl sulfide to (R)-sulfoxide from 144 to 24 h. The immobilized Gordonia cells were resistant to high (up to 1.5 g/L) sulfide concentrations and catalyzed a complete bioconversion to (R)-sulfoxide (95% ee) within 72 h. The immobilized biocatalyst also showed high activity against other sulfides giving the right products from 77–95% ee [44].

3. Enzymatic Sulfoxidation

3.1. Chloroperoxidase Fungal chloroperoxidases is one of the classes of enzymes that produce natural organochlorides in the soil. The first enzyme in this class was chloroperoxidase (CPO, EC 1.11.1.10), which was isolated from the marine fungus Caldariomyces fumago and it was first described in 1959. This enzyme is a highly glycosylated heme-dependent haloperoxidase with a molecular mass of 42 kDa. In vivo, it catalyzes the oxidative chlorination of 1,3-cyclopentadione by generating in situ hypochlorite via chloride ion oxidation. In the presence of free chloride and hydrogen peroxide at an acidic pH in the

Catalysts 2018, 8, 624 8 of 27

(S)-Phenylmethylsulfoxide was obtained at higher concentrations and good enantiomeric excess (37.8 mM and 93.7% ee (S)) if the substrate was in the form of racemic sulfoxide. In comparison, asymmetric oxidation of phenylmethyl sulfide gave the product at a lower yield and enantiomeric excess (10 mM and 80% ee (S)). Rhodococcus was also characterized by a fairly good activity (yield, 22.7–43.2%, in 1–8 h) and enantioselectivity (ee (S) > 99.0%) to para substituted (methyl and chloro) phenylmethylsulfoxides and phenylethyl sulfonate. He and colleagues realized very good results in the oxidation of phenylmethyl sulfide [42], where biotransformation reactions catalyzed using Rhodococcus sp. CCZU10-1 were carried out in a two-phase n-octane/water system: 55.3 mM (S)-phenyl-methylsulfoxide with an enantiomeric excess >99.9% was conveniently obtained. Pseudomonas bacteria also convert sulfides into chiral sulfoxides. An example is the strain of P. monteilii CCTCC M2013683, which catalyzed the oxidation reaction of a series of aryl alkyl sulfides to sulfoxides with a yield of 54–99% and a good enantiomeric excess (63–99%). The cultivation was carried out in M9 medium with toluene as the carbon source. Oxidation reactions were carried out in resting cultures. The obtained results showed that the aromatic ring of substrates with the substituted group may influence the activity and enantioselectivity of the oxidation reaction with P. monteilii CCTCC M2013683 [43]. In turn, the use of biotransformation cultures of the genus Streptomyces and Bacillus cereus allowed for the formation of both enantiomers of cyclohexylmethyl sulfoxide, where S. badius, S. hiroshimensis, S. phaerocromogenes S. flaviviseus, and B. cereus strains produced significant amounts (>25%) of sulfoxides. The first two strains gave a racemic mixture. The other three performed the sulfoxidation reaction with different enantioselectivity. S. hiroshimensis ATCC 27429 and S. flavogriseus ATCC 33331 oxidized to the R- and S-sulfoxide species without generating sulfone. The reaction catalyzed using S. phaeochromogenes NCIMB 11741 was very interesting as there was the synchronized reaction of two enzymes that simultaneously oxidized the sulfide to the corresponding enantiomers of sulfoxide. During the first six hours of biotransformation, mainly S-sulfoxide with high enantiomeric excess (ee > 99%) was formed. After this time, the oxidation of cyclohexylmethyl sulfide to the R-isomer was also observed. Ultimately after 48 h, 55% R-cyclohexylmethyl sulfoxide with 55% ee was obtained [30]. One of the methods of optimizing biotransformation conditions carried out by other cells may be their immobilization. The use of Gordonia terrae IEGM 136 cells, which was immobilized in a cryogel of polyvinyl alcohol, significantly reduced the oxidation time of methylphenyl sulfide to (R)-sulfoxide from 144 to 24 h. The immobilized Gordonia cells were resistant to high (up to 1.5 g/L) sulfide concentrations and catalyzed a complete bioconversion to (R)-sulfoxide (95% ee) within 72 h. The immobilized biocatalyst also showed high activity against other sulfides giving the right products from 77–95% ee [44].

3. Enzymatic Sulfoxidation

3.1. Chloroperoxidase Fungal chloroperoxidases is one of the classes of enzymes that produce natural organochlorides in the soil. The first enzyme in this class was chloroperoxidase (CPO, EC 1.11.1.10), which was isolated from the marine fungus Caldariomyces fumago and it was first described in 1959. This enzyme is a highly glycosylated heme-dependent haloperoxidase with a molecular mass of 42 kDa. In vivo, it catalyzes the oxidative chlorination of 1,3-cyclopentadione by generating in situ hypochlorite via chloride ion oxidation. In the presence of free chloride and hydrogen peroxide at an acidic pH in the range of 2.75–4.50, the CPO easily chlorinates compounds with a variety of structures, such as antipirin, barbituric acid, catechin, phenols, and many others [40]. While CPO usually catalyzes the chlorination reaction in the presence of free chlorine and low pH, it can also act as an oxidizing enzyme with cytochrome P450-like activity at higher pH values (pH > 5.0), especially in the absence of chlorides [45]. In the case of an oxidation reaction, the oxygen is transferred directly to the sulfur Catalysts 2018, 8, x FOR PEER REVIEW 9 of 27 range of 2.75–4.50, the CPO easily chlorinates compounds with a variety of structures, such as antipirin, barbituric acid, catechin, phenols, and many others [40]. While CPO usually catalyzes the chlorination reaction in the presence of free chlorine and low pH, it can also act as an oxidizing enzyme with cytochrome P450-like activity at higher pH values (pH > 5.0), especially in the absence of chlorides [45]. In the case of an oxidation reaction, the oxygen is transferred directly to the sulfur from the heme group of CPO [46]. Chloroperoxidase catalyzes many oxidation reactions such as olefin epoxidation, sulfoxidation, hydroxylations, oxidation of indoles, and oxidation of primary alcohols to aldehydes [47]. Activated forms of oxygen, such as hydrogen peroxide or hydroxyl radicals, can cause the suicide inactivation of CPO via oxidation of the heme group. One strategy to circumvent this problem is to ensure that the steady-state hydrogen peroxide concentration is kept as low as possible by controlling Catalystsits addition2018, 8, 624 to the reaction environment. This step is crucial for9 ofenzyme 27 activity, because concentration of H2O2 even as low as 30 μM results in complete deactivation of CPO [42]. Additionally, hydrogenfrom the heme peroxide group of CPO alone [46]. Chloroperoxidase can cause oxidation catalyzes many of oxidation more reactions sensitive such assubstrates. olefin To solve this problem, anepoxidation, electroenzymatic sulfoxidation, hydroxylations, method with oxidation the ofparticipation indoles, and oxidation of CPO of primary was alcohols developed. to A carbon aldehydes [47]. cathode was used,Activated which formsreduced of oxygen, dissolved such as hydrogen oxygen peroxide to hydrogen or hydroxyl radicals, peroxide, can cause which the suicide in the presence of CPO from C. fumagoinactivation, can of CPOenantiomerycally via oxidation of the oxidize heme group. sulfides One strategy to sulfoxides. to circumvent This this problemmethod was used to is to ensure that the steady-state hydrogen peroxide concentration is kept as low as possible by react with thioanisole,controlling itswhich addition was to the oxidized reaction environment. to form This a sulfoxide step is crucial with for enzyme very activity, high becauseoptical purity (ee = 99%). Another concentrationway was ofto H use2O2 even the as cascade low as 30 µ Mreaction results in completewith Pd deactivation (0) for ofoxidation CPO [42]. Additionally, of the same substrate, which catalyzedhydrogen the formation peroxide alone of can the cause hydrogen oxidation of pe moreroxide sensitive necessary substrates. for To solve the thisoperation problem, of CPO. The an electroenzymatic method with the participation of CPO was developed. A carbon cathode was next method wasused, the which use reduced of supe dissolvedrcritical oxygen carbon to hydrogen dioxide peroxide, (scCO which2) as in thea medium presence of for CPO H from2O2 production in C. fumago, can enantiomerycally oxidize sulfides to sulfoxides. This method was used to react with situ with H2 and O2 using a Pd catalyst. The system gave thioanisole sulfoxide with a 34% yield and thioanisole, which was oxidized to form a sulfoxide with very high optical purity (ee = 99%). Another 94% ee [48]. way was to use the cascade reaction with Pd (0) for oxidation of the same substrate, which catalyzed Chloroperoxidasethe formation is of also the hydrogen sensitive peroxide to necessary a number for the of operation factors of CPO. such The nextas temperature, method was the pH, organic solvents, and highuse of salt supercritical concentrations. carbon dioxide This (scCO 2proble) as a mediumm can for be H2O solved2 production via in immobilization. situ with H2 and O2 Therefore, in using a Pd catalyst. The system gave thioanisole sulfoxide with a 34% yield and 94% ee [48]. the oxidation reactionChloroperoxidase of dibenzothiophene is also sensitive to (DBT), a number classified of factors such as aspolycyclic temperature, aromatic pH, organic hydrocarbons (PAHs) [49], tosolvents, the corresponding and high salt concentrations. sulfoxide This was problem carried can beout solved using via immobilization.CPO from C. Therefore, fumago immobilized in the oxidation reaction of dibenzothiophene (DBT), classified as polycyclic aromatic hydrocarbons on (1D)-γ-Al2O(PAHs)3 nanorods [49], to the (Scheme corresponding 8). sulfoxideThe enzymatic was carried outreaction using CPO was from initiatedC. fumago immobilized via the addition on of 1 mM hydrogen peroxide(1D)-γ -Alto2 Oa 3mixturenanorods (Schemecontaining8). The enzymatic1.0 × 10− reaction7 mol wasof DBT initiated and via 1.3 the addition× 10−12 ofmol 1 mM of CPO in 1 mL. hydrogen peroxide to a mixture containing 1.0 × 10−7 mol of DBT and 1.3 × 10−12 mol of CPO in After 120 min, 1the mL. reaction After 120 min, yield the reaction was 42.2% yield was [46]. 42.2% [46].

Scheme 8. Sulfoxidation of DBT catalyzed using CPO from Caldariomyces fumago. Scheme 8. Sulfoxidation of DBT catalyzed using CPO from Caldariomyces fumago. Modafinil is a very effective pharmaceutical for the treatment of excessive drowsiness caused by sleep disorders, narcolepsy, and obstructive sleep apnea. Compared to racemic modafinil, Modafinilthe is Ra-enantiomer very effective of this drug pharmaceutical has a long half-life fo duer tothe its excretiontreatment and slowof excessive metabolism, whichdrowsiness caused by sleep disorders,results innarcolepsy, a longer time ofand exposure obstructive to the drug sleep and consequently apnea. Compared a longer duration to ofracemic its action. modafinil, the R-enantiomer Forof this reason, drug it has is important a long to develophalf-life a method due to for obtainingits excret pureion modafinil and slowR-enantiomer. metabolism, which This was possible via enantioselective sulfoxidation of 2-(diphenylmethylthio)acetamide using results in a longerchloroperoxidase time of exposure (Scheme9). Only to the the usedrug of ionic and liquids consequently (ILs) in the form a oflonger quaternary duration ammonium of its action. For this reason, it issalts important or polyhydric to compoundsdevelop introduceda method into for the obtaining reaction environment pure modafinil allowed for the R increase-enantiomer. in This was its efficiency to 40.8%. The enantiomeric excess of 97.3% (R) was obtained at pH 5.5 in the presence of possible via [EMIM]enantioselective [Br] (v ILs/v 10% buffer)sulfoxidation and enzyme concentration of 2-(diphenylmethylthio)acetamide 0.013 mM. The use of an ionic liquid using chloroperoxidasecaused (Scheme the unveiling 9). of theOnly heme, the making use the of CPO io activenic centerliquids more (ILs) accessible in tothe the substrate.form of quaternary ammonium saltsIn addition, or polyhydric ILs improved compounds the affinity and specificityintroduced of CPO into [50]. the reaction environment allowed for the increase in its efficiency to 40.8%. The enantiomeric excess of 97.3% (R) was obtained at pH 5.5 in the presence of [EMIM] [Br] (v ILs/v 10% buffer) and enzyme concentration 0.013 mM. The use of an ionic liquid caused the unveiling of the heme, making the CPO active center more accessible to the substrate. In addition, ILs improved the affinity and specificity of CPO [50].

Catalysts 2018, 8, 624 10 of 27 Catalysts 2018, 8, x FOR PEER REVIEW 10 of 27

Scheme 9. Sulfoxidation of 2-(diphenylmethylthio) acetamide catalyzed by CPO.

The optimal use of chloroperoxidase from C. fumago inin sulfoxidationsulfoxidation dependsdepends onon thethe methodmethod of adding an an oxidizer oxidizer such such as as hydrogen hydrogen peroxide. peroxide. Due Due to the to thehigh high sensitivity sensitivity of the of enzyme the enzyme to H2O to2 Hconcentrations2O2 concentrations in the inthioanisole the thioanisole oxidation oxidation reaction, reaction, glucose glucose oxidase oxidase (GOX; (GOX;EC 1.1.3.4) EC 1.1.3.4) from fromAspergillusAspergillus niger nigerwas wasused used to togenerate generate hydrogen hydrogen peroxide peroxide in in situ. situ. Maximum Maximum yields yields and enantioselectivity ofof biotransformationbiotransformation were were observed observed at at high high chloroperoxidase chloroperoxidase reaction reaction rates, rates, but but not atnot low at low hydrogen hydrogen peroxide peroxide formation formation rates, rates, as would as would be expected be expected based based on the on CPO the CPO instability instability at high at hydrogenhigh hydrogen peroxide peroxide concentrations. concentrations. It has beenIt has observed been observed that glucose that glucose oxidase oxidase catalyzing catalyzing the oxygen the sulfoxylationoxygen sulfoxylation gave racemic gave racemic sulfoxide, sulfoxide, which iswhich an unexpected is an unexpected and innovative and innovative reaction. reaction. It has It been has attributedbeen attributed to the to ﬂavonoid the flavonoid hydroperoxide hydroperoxide oxidation oxidation resulting resulting from thefrom reaction the reaction of the of free the ﬂavin free cofactorflavin cofactor associated associated with glucose with oxidaseglucose andoxidase dioxygen. and dioxygen. The speed The of this speed side of reaction this side depends reaction on thedepends amount on ofthe co-solvent amount of in theco-solvent system, in and the the system, enantiomeric and the excess enantiomeric of the oxidation excess of product the oxidation can be improvedproduct can by be lowering improved the by concentration lowering the of concentration the co-solvent of [ 47the]. co-solvent [47].

3.2. Horseradish Peroxidase Horseradish peroxidaseperoxidase (HRP, (HRP, EC EC 1.11.1.7) 1.11.1.7) catalyzes catalyzes the oxidation the oxidation of various of substratesvarious substrates including sulfidesincluding by sulfides hydrogen by peroxide. hydrogen The peroxide. most abundant The most is isoenzymeabundant is C, isoenzyme which is a singleC, which polypeptide is a single of 308polypeptide amino acid of 308 residues. amino The acid sequence residues. of The this sequence enzyme wasof this determined enzyme was in 1976 determined [51]. in 1976 [51]. Initial studiesstudies using using horseradish horseradish peroxidase peroxidase in asymmetric in asymmetric sulfide oxidation sulfide were oxidation disappointing. were Bydisappointing. contrast, when By contrast, in sequence when ofin sequence HRP phenylalanine of HRP phenylalanine was replaced was withreplaced leucine, with thisleucine, change this significantlychange significantly improved improved enzyme enzyme yield to yield obtain to (obtainS) alkyl (S aryl) alkyl sulfoxides aryl sulfoxides with high with optical high optical purity (eepurity > 90%). (ee > In 90%). turn, In substitution turn, substitution of the same of the amino same acid amino with acid threonine with inthreonine HRP resulted in HRP in resulted a decrease in of a enantioselectivitydecrease of enantioselectivity [48]. [48]. 3.3. Dioxygenase 3.3. Dioxygenase Dioxygenases are a heterogeneous group of enzymes that are capable of introducing two Dioxygenases are a heterogeneous group of enzymes that are capable of introducing two oxygen atoms into the double bond. These enzymes are involved in the aerobic degradation of oxygen atoms into the double bond. These enzymes are involved in the aerobic degradation of aromatic compounds and they are mainly catalyzed for the cleavage of aromatic ring and the aromatic compounds and they are mainly catalyzed for the cleavage of aromatic ring and the cis-dihydroxylation of arenes [52]. cis-dihydroxylation of arenes.[52] 3.3.1. Toluene Dioxygenase 3.3.1. Toluene Dioxygenase Toluene dioxygenase (TDO, EC 1.14.12.11) from Pseudomonas putida F1 was able to oxidise thioanisoleToluene mainly dioxygenase to the (TDO, (R)-sulfoxide EC 1.14.12.11) (ee > 98%) from [ 53Pseudomonas,54]. It is putida worth F1 emphasizing was able to that oxidise this biotransformationthioanisole mainly wasto the carried (R)-sulfoxide out in higher (ee scale> 98%) (bioreactor [53,54]. It 8 L,is 7.8worth g of emphasizing substrate, glucose that this for thebiotransformation regeneration of was cofactors), carried obtaining out in higher a 90% scale substrate (bioreactor conversion. 8 L, 7.8 g of substrate, glucose for the regenerationThis enzyme of cofactors), was also obta expressedining a in 90%P. putida substrateUV4 conversion. and a biotransformation of benzo[b]thiophene (B[b]T)This (Figure enzyme3), and was its also 2-methyl expressed and 3-methylin P. putida derivatives UV4 and werea biotransformation performed. These of compoundsbenzo[b]thiophene can be found(B[b]T) in (Figure the environment, 3), and its 2-methyl e.g., from and petroleum, 3-methyl coal,derivatives and products were performed. of its combustion. These compounds In the case can of sulfoxidationbe found in the by environment, TDO in addition e.g., from to the petroleum, formation coal, of theand desiredproducts sulfoxides, of its combustion. the formation In the case of a varietyof sulfoxidation of other productsby TDO in has addition been also to observed,the format e.g.,ion of the the formation desired sulfoxides, of di- and monohydroxylsthe formation of or a dimersvariety [of52 ,other55]. products has been also observed, e.g., the formation of di- and monohydroxyls or dimers [52,55].

Catalysts 2018, 8, 624 11 of 27 Catalysts 2018, 8, x FOR PEER REVIEW 11 of 27

Figure 3. The structure of benzo[b]thiophenes (B[(B[b]T).]T).

Although thethe resulting resulting benzo[ benzo[b]thiophenb]thiophen sulfoxide sulfoxide was enantiomerically was enantiomerically enriched (predominantly enriched in(predominantly the R-enantiomer), in the itR quickly-enantiomer), racemized it quickly within racemized a few hours within and a was few thermallyhours and unstable, was thermally either dimerizedunstable, either or disproportionate dimerized or disproportionate over several days. over several The methyl days. derivativesThe methyl werederivatives more stablewere more and thereforestable andR -sulfoxidetherefore withR-sulfoxide ee > 99% with was ee obtained > 99% from was 2-methylbenzo[obtained fromb ]thiophene2-methylbenzo[ [52,55b].]thiophene [52,55]. 3.3.2. Naphthalene Dioxygenase 3.3.2.For Naphthalene the biotransformation Dioxygenase of benzo[b]thiophene and its 2-methyl or 3-methyl derivatives in additionFor the to TDO, biotransformation naphthalene dioxygenaseof benzo[b]thiophene (NDO, EC and 1.14.12.12.) its 2-methyl was used,or 3-methyl which wasderivatives expressed in inadditionPseudomonas to TDO, putida naphthaleneNCIMB dioxygenase 8859 (NDO2 ),(NDO,P. putida ECmutant 1.14.12.12.) 9816/11 was used, expressing which NDO was expressed1, and E. coli in F352V expressing NDO . Pseudomonas putida NCIMB3 8859 (NDO2), P. putida mutant 9816/11 expressing NDO1, and E. coli As with TDO, many different products were also made from benzo[b]thiophen and its derivatives, F352V expressing NDO3. whichAs were with more TDO, stable. many NDO different1,3 produced products sulfoxides were inalso 7–40% made of thefrom yield, benzo[b]thiophen while NDO2 provided and its a relative yield of 90%. In the case of NDO , 2-methylbenzo[b]thiophene sulfoxide was obtained with derivatives, which were more stable. NDO2 1,3 produced sulfoxides in 7–40% of the yield, while NDO2 the isolated yield of 26% and ee = 56%, but with the S-configuration of sulfoxide [52,55]. provided a relative yield of 90%. In the case of NDO2, 2-methylbenzo[b]thiophene sulfoxide was obtained with the isolated yield of 26% and ee = 56%, but with the S-configuration of sulfoxide 3.4. Monooxygenases [52,55]. 3.4.1. Cytochrome P450 Monooxygenases 3.4. Monooxygenases Cytochrome P450 monooxygenases (P450, CYP) are hemoproteins found in viruses, archaea, bacteria,3.4.1. Cytochrome protists, fungi, P450 plants,Monooxygenases and animals. They play an important role in the oxyfunctionalization of many structurally and physiologically important chemical compounds, including fatty acids, steroids, or vitamins.Cytochrome What P450 is important monooxygenases is that they (P450, also participate CYP) are inhemoproteins the detoxification found process in viruses, of xenobiotics, archaea, andbacteria, in this protists, case, some fungi, enzymes plants, and can catalyzeanimals. sulfoxidationThey play an [important56]. role in the oxyfunctionalization of manyThe sulfoxidationstructurally and reaction physiologically can be catalyzed import usingant chemical CYP116B compounds, monooxygenases including classified fatty as acids, class VIIsteroids, [57]. or These vitamins. are self-contained What is important enzymes is that because they also a single participate polypeptide in the detoxification chain has three process distinct of functionalxenobiotics, parts: and in the this flavine case, mononucleotidesome enzymes can (FMN) catalyze binding sulfoxidation domain, the[56]. NADPH binding domain, and theThe [2Fe-2S] sulfoxidation ferredoxin reaction domain. can be Therefore, catalyzed the us redoxing CYP116B partners monooxygenase transfer electronss classified from the reducedas class NADPH,VII [57]. These through are the self-contained specific domains enzymes of FMN becaus ande [2Fe-2S], a single to polypeptide their acceptor, chain the hemehas three domain distinct [58]. Theyfunctional show parts: preference the flavine for more mononucleotide structurally (FMN) differentiated binding substratesdomain, the from NADPH the most binding well-known domain, CYP102A1and the [2Fe-2S] monooxygenase ferredoxin (P450-BM3domain. Therefore, from class the VIII). redox To combinepartners thistransfer catalytic electrons self-sufficiency from the withreduced thermal NADPH, stability, through new P450the specific enzymes domains derived of from FMN thermophilic and [2Fe-2S], microorganisms to their acceptor, have the recently heme beendomain characterized. [58]. They show The P450-TT preference monooxygenase for more stru fromcturallyThermus differentiated thermophilus substrateshas been from shown the to most have exceptionalwell-known thermostability CYP102A1 monooxygena within all multiple-domainse (P450-BM3 enzymesfrom class and VIII) has. aTo half-life combine greater this than catalytic 9 h at 50self-sufficiency◦C[56]. with thermal stability, new P450 enzymes derived from thermophilic microorganisms have recently been characterized. The P450-TT monooxygenase from Thermus An example member of the CYP116B subfamily is P450RhF monooxygenase (CYP116B2), which hasthermophilus been isolated has been from shownRhodococcus to havesp. exceptional NCIMB 9784. thermostability This enzyme within is able all to multiple-domain catalyze various reactions,enzymes and including has a half-life O-dealkylation, greater than aromatic 9 h at hydroxylation, 50 °C [56]. epoxidation of olefins, and asymmetric sulfoxylationAn example of a numbermember of of substituted the CYP116B aromatic subfamily compounds. is P450RhF The monooxygenase natural substrates (CYP116B2), for CYP116B2 which are unknown;has been isolated however, from the incomparably Rhodococcus highsp. NCIMB activity exhibited9784. This by enzyme the enzyme is able against to catalyze sulfides suggestsvarious reactions, including O-dealkylation, aromatic hydroxylation, epoxidation of olefins, and asymmetric that its natural function may involve the detoxification of related compounds via sulfoxidation [59]. sulfoxylation of a number of substituted aromatic compounds. The natural substrates for CYP116B2 The pharmacokinetics of omeprazole is associated with phenotypic P450 2C19 polymorphism are unknown; however, the incomparably high activity exhibited by the enzyme against sulfides in humans. An easily commercially available racemic mixture of omeprazole was used as a proton suggests that its natural function may involve the detoxification of related compounds via pump inhibitor in the treatment of gastric acid related diseases. However, it is noteworthy that the sulfoxidation [59]. omeprazole S-enantiomer esomeprazole was developed as an enantiomerically pure drug [60]. The pharmacokinetics of omeprazole is associated with phenotypic P450 2C19 polymorphism in humans. An easily commercially available racemic mixture of omeprazole was used as a proton

Catalysts 2018, 8, x FOR PEER REVIEW 12 of 27 pump inhibitor in the treatment of gastric acid related diseases. However, it is noteworthy that the Catalysts 2018, 8, 624 12 of 27 omeprazole S-enantiomer esomeprazole was developed as an enantiomerically pure drug [60]. High catalytic activity was observed for recombinant human CYP2C19 in 5-hydroxylation of R-omeprazole,High catalytic CYP2C19 activity of cynomolgus was observed in forhydroxylation recombinant of humanboth R- CYP2C19and S-omeprazole in 5-hydroxylation isomers, and of RCYP2C19-omeprazole, of marmosets CYP2C19 of in cynomolgus 5-hydroxylation in hydroxylation of S-omeprazole. of both R -On and Sthe-omeprazole other hand, isomers, human, and cynomolgus,CYP2C19 of marmosets and marmoset in 5-hydroxylation P4503A ofpreferentiallyS-omeprazole. mediated On the other S-omeprazole hand, human, sulfoxidation. cynomolgus, Correlationsand marmoset and P4503A kinetic preferentially analysis have mediated shown aS high-omeprazole affinity of sulfoxidation. polymorphic Correlations CYP2C19 of and macaques kinetic withanalysis marmoset have shown microsomal a high affinity enzymes of polymorphic in relation to CYP2C19 5-hydroxylation of macaques of R with- and marmoset S-omeprazole, microsomal and highenzymes yield in relationof cynomolgus to 5-hydroxylation P4503A4 of Rand- and micrS-omeprazole,osomal CYP2C19 and high marmosets yield of cynomolgus for omeprazole P4503A4 5-hydroxylationand microsomal CYP2C19and sulfoxidation marmosets [60]. for omeprazole 5-hydroxylation and sulfoxidation [60]. Recombinant E. coli cells (P450pyrI83H-GDH)(P450pyrI83H-GDH) co-expressing three-component P450pyrI83H monooxygenase and glucose dehydrogenase (GDH) (GDH) were were used for sulfoxidation of a number of compounds such as thioanisole, 4-fluorothioanisole, 4-fluorothioanisole, ethyl phenyl sulfide, sulfide, methyl p-tolyl sulfide, sulfide, and 4-methoxythioanisole. High High RR-enantioselectivity-enantioselectivity was was observed. observed. Due Due to to the the high high toxicity toxicity of of both substrates and biotransformation products, the phosphate buffer an andd ionic liquid two-phase system was used toto solvesolve thisthis problem.problem. [P6,6,6,14] [NTf [NTf2]2] showed excellent biocompatibility during the growth of E. coli cells (P450pyrI83H-GDH) and and the the ability ability to to protect substrate toxicity against against cells, cells, being the ideal co-solvent in a two-phase KP/IL buffe bufferr system system for for efficient efficient processes processes of of biooxidation. biooxidation. The applied system contributed to the growth of both productivity and enantioselectivity of transformation. In In such such a a two-phase two-phase system, system, >99 >99%% of of the the substrate substrate and and 35–60% of the products remained inin thethe IL,IL, whichwhich significantly significantly reduced reduced the the toxicity toxicity of of the the substrate substrate and and partially partially reduced reduced the thetoxicity toxicity and and inhibition inhibition of the of productthe product [61]. [61]. Not onlyonly thethe involvement involvement of of CYP CYP in thein the metabolism metabolism of drugs of drugs must must be carefully be carefully studied; studied; it is equally it is equallyimportant important to discover to discover the contribution the contribution of liver enzymes of liver in enzymes the detoxification in the detoxification of pesticides. of Benfuracarb pesticides. (FigureBenfuracarb4) is one(Figure of the 4) carbamateis one of the pesticides. carbamate This pesticides. compound This iscompound a systemic is and a systemic contact and insecticide contact insecticideused to control used insectto control pests insect in various pests crops.in various Benfuracarb crops. Benfuracarb and its main and metabolite, its main carbofuran,metabolite, carbofuran,have been shown have been to cause shown neurotoxic to cause effects.neurotoxic In addition, effects. In they addition, can cross they the can placental cross the barrier placental and barrierhave a significantand have a impact significant on the impact maternal-placental-fetal on the maternal-placental-fetal unit. It is therefore unit. It a is threat therefore to humans, a threat and to humans,therefore and its metabolism therefore its in metabolism liver microsomes in liver was microsomes examined. was examined.

Figure 4. The structure of Benfuracarb.

Benfuracarb was activated via the carbamfuran metabolic pathway, pathway, and in this case, the CYP3A subfamily turned out to be the most active enzymesenzymes (mainly CYP3A4). Only CYP2C9 and CYP2C19 participated in the sulfoxidation of benfuracarb. It It is important to notice that the formation of toxic products of the carbofuran metabolic pathway is faster thanthan sulfoxidationsulfoxidation [[62].62]. Chlorpromazine (Figure(Figure5) belongs5) belongs to phenothiazine to phenothiazine neuroleptics neuroleptics and it is aand potent it dopaminergicis a potent D receptor antagonist that is responsible for antipsychotic activity of this drug. Chlorpromazine is a dopaminergic2 D2 receptor antagonist that is responsible for antipsychotic activity of this drug. α Chlorpromazineblocker of 1 adrenergic is a blocker receptors of α1 andadrenergic muscarinic receptors M1 receptors and muscarinic that may M1 be associatedreceptors that with may certain be associatedside effects, with such certain as anticholinergic side effects, symptoms, such as anticholinergic hypotension, and symptoms, sedation. hypotension, During phase and I of sedation. this drug Duringmetabolism phase in theI of liver, this itdrug undergoes metabolism a number in ofthe transformations liver, it undergoes catalyzed a number via cytochrome of transformations P450 (CYP) catalyzedmonooxygenase-associated via cytochrome P450 isotactic (CYP) compounds monooxygena suchse-associated as 5-sulfoxidation, isotactic mono- compoundsN-demethylation, such as 5-sulfoxidation,and di-N-demethylation. mono-N-demethylation, In the studies describedand di-N-demethylation. by Wójcikowski In [63 ],the selective studies CYP described inhibitors, by Wójcikowskihuman CYP [63], isoforms selective (Supersomes CYP inhibitors, 1A2, 2A6,human 2B6, CYP 2C8, isoforms 2C9, 2C19,(Supersomes 2D6, 2E1, 1A2, 3A4), 2A6, and2B6, liver2C8, 2C9,microsomes 2C19, 2D6, from 2E1, different 3A4), and donors liver weremicrosomes used in from the experiments different donorsin vitro were. The used authors in the experiments suggest that CYP1A2 is the only CYP isoform responsible for mono-N-demethylation and di-N-demethylation of chlorpromazine (100%), and it is also the main isoform that catalyzes 5-sulfoxidation (64%) at the Catalysts 2018,, 8,, xx FORFOR PEERPEER REVIEWREVIEW 13 of 27 Catalysts 2018, 8, 624 13 of 27 in vitro. The authors suggest that CYP1A2 is the only CYP isoform responsible for mono-N-demethylation and di-N-demethylation of chlorpromazine (100%), and it is also the main isoformtherapeutic that concentration catalyzes 5-sulfoxidation of this drug (64%) (10 mM). at the In addition,therapeutic 5-sulfoxidation concentration of of chlorpromazine this drug (10 mM). is also In addition,carried out 5-sulfoxidation by CYP3A4 (34%) of chlorpromazine [63]. is also carried out by CYP3A4 (34%) [63].

Figure 5. TheThe structures structures of Chlorpromazine Chlorpromazine,, Levomepromazine, and Prothipendyl.

The same researchers also became interested in metabolism of of another another drug drug from from this this group, group, namely levomepromazine (methotrimeprazine) (Figur (Figuree 55),), whichwhich isis usedused toto treattreat schizophreniaschizophrenia andand schizoaffective disorder. This drug has also found widespread use in symptom control in palliativepalliative care as an antipsychotic, anxiolytic, antimimetic, andand sedative agent.agent. It is also used as a pre-operative sedative, in the ﬁnalfinal controlcontrol ofof pain,pain, andand post-operativepost-operative analgesia.analgesia. Its Its long long half-life allows for administration once once a a day, day, and and its its cost cost is is also also beneficial. beneﬁcial. This This compound compound is a is moderate a moderate dopamine dopamine D2 receptorD2 receptor antagonist antagonist responsible responsible for for its itsantipsychotic antipsychotic activity, activity, as as well well as as α1-adrenoceptor and M1-muscarinic receptors, which which are are associated with with some some side side effects effects of this drug (hypotension, sedation, and anticholinergic symptoms)symptoms) suchsuch asas thethe chlorpromazinechlorpromazine mentionedmentioned aboveabove [[64].64]. For the biotransformation of this drug in the liver, CYP3A4 is mainly responsible because it performs levomepromazinelevomepromazine 5-sulfoxylation 5-sulfoxylation (72%) (72%) and andN-demethylation N-demethylation (78%) (78%) at the at therapeuticthe therapeutic drug drugconcentration concentration (10 mM). (10 mM). CYP1A2 CYP1A2 contributes contributes less to less levomepromazine to levomepromazine 5-sulfoxide 5-sulfoxide (20%). (20%). The role The of roleCYP2A6, of CYP2A6, CYP2B6, CYP2B6, CYP2C8, CYP2C8, CYP2C9, CYP2C9, CYP2C19, CYP2C19, CYP2D6, CYP2D6, and and CYP2E1 CYP2E1 in in the the catalysis catalysis ofof the above-mentioned reactions is negligible (0.1–8%). The The results results obtained indicate that in case of both chlorpromazine andand levomepromazine levomepromazine provide provide possible possible pharmacokinetic pharmacokinetic interactions interactions between between these thesedrugs drugs and CYP3A4 and CYP3A4 substrates substrates (e.g., testosterone, (e.g., testoste tricyclicrone, antidepressants,tricyclic antidepressants, and macrolide and antibioticsmacrolide antibioticscalcium channel calcium blockers), channel andblockers), inhibitors and (e.g.,inhibitors erythromycin, (e.g., erythromycin, ketoconazole, ketoconazole, and selective and serotonin selective serotoninreuptake inhibitorreuptake (SSRI)), inhibitor or (SSRI)), inducers or (e.g., indu carbamazepine,cers (e.g., carbamazepine, rifampicin) rifampicin) [64]. [64]. ® CYP3A4 is also responsibleresponsible forfor thethe sulfoxidationsulfoxidation ofof ProthipendilProthipendil (Dominal(Dominal®), which which is a neuroleptic drug and tricyclic azapenothiazine derivative. Prothipendil (Figure 5 5)) isis usedused inin patientspatients with psychomotor andand anxietyanxiety disordersdisorders [[65].65]. Thioridazine (Figure(Figure6 )6) is is widely widely used used as anas antipsychotican antipsychotic drug. drug. The mainThe main reactions reactions that take that place take placein the humanin the liverhuman are liver mono-2-sulfoxidation are mono-2-sulfoxidation to mesoridazine to mesoridazine and di-2-sulfoxidation and di-2-sulfoxidation to sulforidazine. to sulforidazine.First of all, these First reactions of all, can thes bee attributed reactions to can CYP2D6. be attributed Both products to CYP2D6. of sulfoxidation Both products also display of sulfoxidationantipsychotic activity.also display The interestantipsychotic of thioridazine activity. usage The interest has increased of thioridazine again because usage it canhas potentially increased againserve asbecause an antimicrobial it can potentially for the controlserve as of an multidrug-resistant antimicrobial for strains,the control including of multidrug-resistant the treatment of strains,drug-resistant including tuberculosis the treatment [66]. of drug-resistant tuberculosis [66].

Figure 6. The structure of thioridazine.

Catalysts 2018, 8, 624 14 of 27

In the genome of Streptomyces platensis DSM 40041, 21 cyp genes were identiﬁed. One of these P450 enzymes was classiﬁed as CYP107L and its ability to biotransform drugs compared to human metabolism was established. The conversion of thioridazine with CYP107L reached 63% after 20 h, compared to 35% with CYP2D6. CYP2D6 provided 33% of the mono-sulfoxidation product and only 1% of the di-sulfoxidation product after 20 h. CYP107L, on the other hand, provided 26% of monosulfoxidation and an additional 6% of disulfoxidation [66].

3.4.2. Flavin-Dependent Monooxygenases Metabolism of xenobiotics in humans and other mammals often begins with oxidation. Most reactions in phase I metabolism are catalyzed via CYP450s, which are discussed in the previous section. However, beyond CYP450s, recent studies indicated that flavin-containing monooxygenases (FMOs) also play a key role in the biotransformation of xenobiotics, including drugs and natural compounds. The human proteome contains five FMO isoforms (FMO1-FMO5). All these enzymes have expression patterns and the role in human metabolism is dependent on the tissue. The major organs among which they are present, consist of the liver, kidneys, small intestine, lungs, and brain. Human FMO (hFMO) and their mammalian orthologs are usually associated with cell membranes and often thermolability, which seems to be the main reason for their problematic isolation from tissue and ineffective production in the form of a recombinant protein. FMO has been shown to be involved in the oxidation of heteroatom-containing compounds such as sulfides. Unlike CYP450, which contain heme, FMO uses flavin for oxidation, which also translates into a different mechanism of catalyzed reactions. For this reason, to distinguish whether a compound is metabolized via FMO or CYP450, specific inhibitors are often used. FMO enzymes require NADPH to reduce flavin adenine dinucleotide (FAD). The reduced flavin then reacts with molecular oxygen to produce a reactive 4α-hydroperoxiflavin. This reactive intermediate from flavin is capable of carrying out various oxidation reactions, e.g., sulfoxylation. The wider use of mammals’ FMO in biocatalysis is difficult and little investigated due to their typical binding to membranes and thermal instability; although, some researchers have presented the involvement of these mammalian enzymes in specific chiral reactions including sulfoxidation. For example, purified FMO2 from a rabbit’s lung managed to catalyze stereoselective oxidation of methyl p-tolyl sulfide with an enantiomeric excess of 96% for (R)-sulfoxide [67,68]. Another example is Sulindac, which contains a chiral sulfoxide moiety. This nonsteroidal anti-inflammatory drug is clinically administered as a racemic mixture. Sulindac sulfoxide is reduced in vivo to sulindac sulfide, a pharmacologically active metabolite [69]. In the serum and urine of volunteers, a clear stereoselective enrichment of (R)-sulindac sulfoxide was detected. Hamman and colleagues showed that purified FMO1 from pig liver, FMO2 from rabbit lung, and human FMO3 were able to stereoselectively oxidize sulindac sulfide to the R-sulfoxide isomer. Using human FMO3, the (R)-sulindac sulfoxide was obtained with an ee of 87%. The remaining FMO were even more stereoselective in the formation of this sulfoxide because in case of FMO1 and FMO2 observed ee of 98 and 99%, respectively [68,69]. In turn, albendazole (Figure7) and fenbendazole sulfoxides, benzimidazole derivatives with anthelmintic activity, were obtained using hepatic microsomes of sheep and cattle liver [68,70]. The authors suggest that the sulfoxidation of albendazole led to enantiomerically pure sulfoxide. Another research group [71] demonstrated that the resulting FMO1 mutants could also oxidize insecticide fenthion with excellent selectivity to (+)-sulfoxide [68]. The ability to catalyze sulfoxidation in the case of FMO5 was also investigated. This FMO, which naturally shows good expression in the small intestine, kidneys, and lungs, accounts for over 50% of all FMO transcripts in the human liver. A moderate conversion (20%) of thioanisole forming the corresponding sulfoxide was observed. The enzyme was also able to oxidize S-methyl-esonarimod and fenthion [72]. Catalysts 2018, 8, 624 15 of 27 Catalysts 2018, 8, x FOR PEER REVIEW 15 of 27

Figure 7. The structures of Albendazole,Albendazole, Fenbandazole, and Fenthion.

ItThe was ability noted to that catalyze Baeyer-Villiger sulfoxidation Monooxygenases in the case (BVMOs)of FMO5 from was manyalso investigated. microbes are sequentiallyThis FMO, associatedwhich naturally with humanshows good FMO expression and exhibit in similar the small effects; intestine, hence, kidneys, they can and be usedlungs, as accounts a tool to for prepare over metabolites50% of all FMO in studies transcripts on the in metabolism the human ofliver. xenobiotics A moderate [73]. conversion (20%) of thioanisole forming the corresponding sulfoxide was observed. The enzyme was also able to oxidize 3.4.3. Baeyer-Villiger Monooxygenases (BVMOs) S-methyl-esonarimod and fenthion [72]. Baeyer-VilligerIt was noted monooxygenasesthat Baeyer-Villiger (BVMOs) Monooxygenases are ﬂavoprotein, (BVMOs which are) from dependent many on microbes nicotinamide are cofactorssequentially and associated capable of with mainly human carrying FMO out and the exhibit oxidation similar of carbonyl effects; hence, compounds they can (Baeyer-Villiger be used as a oxidation)tool to prepare and alsometabolites oxidation in studies of various on heteroatoms,the metabolism including of xenobiotics sulfur [73]. [52]. Many different BVMOs are capable of catalyzing sulfoxidation, which will be discussed later in this chapter. These are, for3.4.3. example: Baeyer-Villiger Monooxygenases (BVMOs)

• cyclohexanoneBaeyer-Villiger monooxygenasemonooxygenases (CHMO, (BVMOs) EC 1.14.13.22) are flavoprotein, which are dependent on •nicotinamidephenylacetone cofactors monooxygenase and capable (PAMO,of mainly EC ca 1.14.13.92)rrying out the oxidation of carbonyl compounds •(Baeyer-Villiger4-hydroxyacetophenone oxidation) and monooxygenase also oxidation (HAPMO, of various EC heteroatoms, 1.14.13.84) including sulfur [52]. Many different BVMOs are capable of catalyzing sulfoxidation, which will be discussed later in this • styrene monooxygenase (SMO). chapter. These are, for example: BVMO from Aspergillus flavus NRRL3357 readily oxidizes thioanisole with both sulfoxide • cyclohexanoneAFL706 monooxygenase (CHMO, EC 1.14.13.22) and excessively oxidized sulfone being formed. The sulfone product is about 60% of the total reacted • phenylacetone monooxygenase (PAMO, EC 1.14.13.92) substrate after 12 h. Researchers found that it is difficult to determine the similarity of this enzyme • 4-hydroxyacetophenone monooxygenase (HAPMO, EC 1.14.13.84) with other known BVMOs, finding only ≈38% sequence similarity with CHMO [74]. • styrene monooxygenase (SMO). Ac Other BVMOs used for the oxidation of thioanisole were BVMOBo from Bradyrhizobium oligotrophicum and BVMOBVMOAmAFL706from fromAeromicrobium Aspergillus flavus marinum NRRL3357, which were readily expressed oxidizes in thioanisoleE. coli BL21 with (DE3). both Evolutionary sulfoxide dependenceand excessively and oxidized sequence sulfone analysis being revealed formed. that The both sulfone BVMOs product belong is to about the BVMO 60% of type the total I family reacted and thesubstrate EtaA monooxygenaseafter 12 h. Researchers mono-oxide found subtype. that it is Thedifficult sequence to determine identity isthe 48% similarity between of BVMO this enzymeBo and BVMOwith otherAm. known Both BVMO BVMOs, showed finding sequence only ≈38% similarity sequence with similarity EtaA from withM. CHMO tuberculosisAc [74].and with EthA fromOtherP. putida BVMOsKT2440. used The identityfor the ofoxidation the BVMO ofBo thioanisoleand BVMO Amweresequences BVMOBo with from EthA Bradyrhizobium was 47% and 54%,oligotrophicum respectively. and BVMOAm from Aeromicrobium marinum, which were expressed in E. coli BL21 (DE3).Both Evolutionary BVMOs are dependence active, not only and for sequence ketones, anal but alsoysis forrevealed various that thioether both BVMOs substrates. belong Among to the studiedBVMO type substrates, I family BVMO and theBo andEtaA BVMO monooxygenaseAm showed mono-oxide the highest subtype. activity inThe relation sequence to thioanisole identity is (0.11748% between and 0.025 BVMO U/mgBo and protein). BVMOAm Importantly,. Both BVMO no showed sulfone sequence formation similarity was observed. with EtaA In addition,from M. thetuberculosis BVMOs usedand with exhibited EthA pronounced from P. putida activity KT2440. and excellent The identity stereoselectivity of the BVMO in relationBo and to BVMO prochiralAm prazolesequences thioethers, with EthA such was as 47% omeprazoles, and 54%, ilaprazoles,respectively. rabeprazoles, pantoprazoles, and lansoprazoles, whichBoth is a BVMOs unique featureare active, in this not BVMO only for family ketones, [75]. but also for various thioether substrates. Among the studiedThe recombinant substrates, BVMO4 BVMOBo from andDietzia BVMOsp.Am D5showed was expressed the highest in activityE. coli BL21-CodonPlus in relation to thioanisole (DE3)-RP. Protein(0.117 and expression 0.025 U/mg was inducedprotein). inImportantly,E. coli with no 1 mM sulfone isopropyl formation b-D-1-thiogalactopyranoside was observed. In addition, (IPTG). the TheBVMOs soluble used part exhibited of the BVMO4 pronounced was obtained activity viaand centrifugation excellent stereoselectivity of the lysed cells in relation [76]. to prochiral prazole thioethers, such as omeprazoles, ilaprazoles, rabeprazoles, pantoprazoles, and lansoprazoles, which is a unique feature in this BVMO family [75].

Catalysts 2018, 8, x FOR PEER REVIEW 16 of 27

The recombinant BVMO4 from Dietzia sp. D5 was expressed in E. coli BL21-CodonPlus (DE3)-RP. Protein expression was induced in E. coli with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG). The soluble part of the BVMO4 was obtained via centrifugation of the lysed cells [76]. BVMO4 from Dietzia sp. D5 has been tested for the oxidation of several sulfur-containing compounds. This enzyme exhibits a preference for unsubstituted aromatic sulfides, but also tolerates small substituents in the para (methyl, fluoro) or meta (chloro) positions. It is noteworthy that the presence of one additional methyl group in the substrate, i.e., ethyl phenyl sulfide, significantly increased the stereospecificity of the oxidation (ee > 99% R) compared to thioanisole (ee = 50% R), while the reaction rate was comparable [76].

3.4.4. Cyclohexanone Monooxygenase

CyclohexanoneCatalysts 2018 monooxygenase, 8, 624 (CHMO, EC 1.14.13.22) from Acinetobacter 16calcoaceticus of 27 NCIB 9871 is currently one of the best-studied Baeyer-Villiger monooxygenases [74,77]. As a member of BVMO type I, CHMOBVMO4 is from dependentDietzia sp. D5on has FAD been and tested NADPH for the oxidation as cofactors of several sulfur-containing[78]. Under physiological compounds. This enzyme exhibits a preference for unsubstituted aromatic sulfides, but also tolerates conditions, CHMOsmall substituents catalyzes in the the parakey(methyl, step in fluoro) the orbiodmetaegradation(chloro) positions. of cyclohexanol, It is noteworthy that i.e., the the conversion of a cyclic ketonepresence into of one a lactone, additional methylthat can group then in the be substrate, hydrolyzed i.e., ethyl phenylto aliphatic sulfide, significantlyacid [68,79]. The first increased the stereospecificity of the oxidation (ee > 99% R) compared to thioanisole (ee = 50% R), CHMO was isolatedwhile the reactionfrom Acinetobacter rate was comparable spp. [76 ].NCIMB 9871 in 1975 [80]. Although typical substrates are four- to eight-membered cyclic ketones, this enzyme is characterized by a broad spectrum of 3.4.4. Cyclohexanone Monooxygenase acceptable substrates and high enantio- and regioselectivity. These features enable its use in Cyclohexanone monooxygenase (CHMO, EC 1.14.13.22) from Acinetobacter calcoaceticus NCIB 9871 industrial productionis currently routes one of the [77]. best-studied Baeyer-Villiger monooxygenases [74,77]. As a member of BVMO Cyclohexanonetype I, CHMO monooxygenase is dependent on FAD not and only NADPH catalyze as cofactorss the [78 ].oxidation Under physiological of Baeyer-Villiger conditions, but also sulfoxidation. CHMOColonna catalyzes et al. the [81] key step(1996) in the showed biodegradation that of CHMO cyclohexanol, from i.e., Acinetobacter the conversion of calcoaceticus a cyclic NCIMB ketone into a lactone, that can then be hydrolyzed to aliphatic acid [68,79]. The first CHMO was was able to oxidizeisolated alkyl from Acinetobacter aryl sulfide,spp. disulphate, NCIMB 9871 in dialkyl 1975 [80]. sulfide, Although typicalcyclic substrates and acyclic are four- 1,3-di-thioacetals, to and 1,3-oxathioacetalseight-membered to the cyclic corresponding ketones, this enzyme mono-sulfoxides, is characterized by a with broad spectrumhigh ee. of The acceptable researchers were substrates and high enantio- and regioselectivity. These features enable its use in industrial production interested in routesthe [synthesis77]. of various sulfoxides, not only because of participation of those compounds in manyCyclohexanone biological monooxygenase activities, but not onlyalso catalyzes because the of oxidation their pharmaceutical of Baeyer-Villiger but importance. also The methodsulfoxidation. of directed Colonna evolution et al. [81] (1996) was showed used thatto generate CHMO from mutantsAcinetobacter of calcoaceticusCHMO fromNCIMB Acinobacter sp. was able to oxidize alkyl aryl sulfide, disulphate, dialkyl sulfide, cyclic and acyclic 1,3-di-thioacetals, NCIMB 9871. andIn this 1,3-oxathioacetals way M.T. to Reetz the corresponding and coworkers mono-sulfoxides, [82] wanted with high to ee. obtain The researchers variants were of CHMO that catalyzed sulfoxidationinterested in the of synthesismethyl- ofp- variousmethylbenzyl sulfoxides, not thioether only because S of or participation R selectively, of those compounds and at the same time, limit the furtherin many oxidation biological activities,of sulfide but also to becausesulfone. of their Finally, pharmaceutical the mutant importance. with six mutations, namely The method of directed evolution was used to generate mutants of CHMO from Acinobacter sp. Q92R, Y132C, NCIMBP169L, 9871. F246N, In this V361A, way M.T. Reetzand andT415A, coworkers allowed [82] wanted the toresearchers obtain variants to of obtain CHMO thatS-sulfoxide with an ee value of catalyzed99.8% and sulfoxidation the amount of methyl- ofp -methylbenzylundesired sulfone thioether S belowor R selectively, 5%. and at the same time, limit the further oxidation of sulfide to sulfone. Finally, the mutant with six mutations, namely Q92R, Also, in Y132C,the sulfoxidation P169L, F246N, V361A, reaction, and T415A, CHMO allowedAc theaccepts researchers a towide obtain rangeS-sulfoxide of withsubstrates an ee including 3-(methylthio)anilinevalue of 99.8% (3.0 and mM) the amountor albendazole of undesired sulfone(Figures below 7 5%.and 8). Using the reduced form of NADPH, phosphate dehydrogenaseAlso, in the sulfoxidation was used reaction, to regenera CHMOAc acceptste coenzymes. a wide range After of substrates 135 includingmin of incubation, 3-(methylthio)aniline (3.0 mM) or albendazole (Figures7 and8). Using the reduced form of (+)-(R)-3-(methylthio)anilineNADPH, phosphate dehydrogenasesulfoxide with was used ee to = regenerate 71% was coenzymes. obtained. After 135 In min turn, of incubation, the R-sulfoxide of albendazole was(+)-( Robtain)-3-(methylthio)anilineed with an sulfoxideee of 32%. with eeIt =is 71% worth was obtained.noting Inthat turn, both the RCHMO-sulfoxideAc of and BVMOMt1 albendazole was obtained with an ee of 32%. It is worth noting that both CHMOAc and BVMOMt1 formed the sameformed product the same as product human as human FMO FMO [73]. [73].

Figure 8. The structure of 3-(methylthio)aniline. Figure 8. The structure of 3-(methylthio)aniline. To solve the problems of stability of this enzyme, a number of CHMO mutants were generated. To solve theParticularly problems noteworthy of stability is the cyclohexanone of this enzy monooxygenaseme, a number mutant (CHMO-QM), of CHMO which mutants contained were generated. four distinct mutations C376L, M400I, T415C, and A463C. This resulted in increased long-term stability Particularly noteworthyand higher activity is at highthe temperaturescyclohexanone compared tomonooxygenase wild CHMO [77]. mutant (CHMO-QM), which contained four distinct mutations C376L, M400I, T415C, and A463C. This resulted in increased long-term stability and higher activity at high temperatures compared to wild CHMO [77].

Catalysts 2018, 8, 624 17 of 27

BVMOs from Rhodoccocus jostii (BVMORj4 and BVMORj24) were subsequently used to oxidize 3-(methylthio)aniline. A substrate at 3.0 mM was incubated with cell extracts supplemented with phosphate, NADPH, and phosphate dehydrogenase to regenerate the reduced coenzyme. A conversion of 72% was achieved with ee > 99.5% of S-(−) after 135 min. Unfortunately, the formation of sulfone in an amount not exceeding 2% was also observed. This enzyme was also able to oxidize albendazole with a conversion of 55% and ee = 95% (S)[73]. In the oxidation of 3-(methylthio)aniline using BVMO from the fungus Myceliophthora thermophila (BVMOMt1), a conversion of substrate of 97% and ee = 88% (R)-(+) was observed, thus the opposite enantiomer of 3-(methylthio)aniline sulfoxide was obtained when BVMORj4 was used for this reaction. Albendazole was even more oxidized (25%) with the predominance of R-enantiomer. It is noteworthy that this enzyme formed the same product as mammalian FMOs [73].

3.4.5. Phenylacetone Monooxygenase Phenylacetone monooxygenase (PAMO, EC 1.14.13.92) from thermophilic bacteria Thermobifida fusca is a monomeric BVMO type I capable of catalyzing inter alia enantioselective sulfoxylation. It deserves special attention due to its good thermostability and tolerance to solvents. For this reason, it is attractive for practical use [12,83]. However, PAMO has a somewhat narrow range of substrates favoring relatively simple aromatic compounds, such as benzyl alkyl sulfides. PAMO was not suitable for the synthesis of alkyl heteroaryl sulfoxides because of its generally low activity and/or selectivity. In addition to aromatic substrates, PAMO can also moderately convert non-aromatic substrates [12]. Rodrigues et al. 2012 [84] attempted to use a regeneration system in the reaction oxidation of thioanisole catalyzed using PAMO at pH 9.0. The highest conversion was observed when glucose/GDH as a recycling system NADPH (c = 95%) was used; however, the obtained (S)-methylphenyl sulfoxide has a slightly lower optical purity (ee = 35%) than for the oxidation of ketones. When condensed CRE2-PAMO was used, 60% of (S)-methylphenyl sulfoxide was obtained. For the three enzymes, sulfoxide was obtained with an optical purity of about 40%. Variability of selectivity depending on the cofactor regeneration system is explained by the allosteric effect on enzyme behavior. The use of the addition of organic solvents to the reaction mixture allows the range of substrates to be extended by working with hydrophobic substrates, and they are used to develop solvent engineering techniques to modify the biocatalytic properties of this BVMO. The use of PAMO with the addition of small amounts of short-chain alcohols, such as methanol or ethanol, or a water-soluble polymer, such as PEG 4000, resulted in a significant improvement in enantioselectivity in the oxidation of prochiral sulfides. Using the I-TASSER methodology with matrix 1 W4X, it was found that PAMO from T. fusca has 86% homology with BVMO4 from Dietzia sp. D5. The difference is mainly due to the longer sequence in BVMO4 at the N-terminus compared to PAMO. The variable regions that correspond to amino acid residues 228–241, 319–334, 448–475, and 548–571 are part of the loop, and thus, can be flexible, which is consistent with the data found in the X-ray structures. The remaining part of the protein shows high homology, especially the binding sites of the cofactor, as well as the active site of the enzyme, i.e., the key sites for its catalytic activity [85]. The methionine 446 in PAMO has recently been replaced with glycine, after comparing the PAMO structure with the homologous cyclopentanone monoxygenase model (CPMO, EC 1.14.13.16). The resulting M446G PAMO protein retains the thermostability of PAMO but has an altered substrate-binding pocket, which explains the essential changes in substrate specificity and enantioselectivity in the sulfoxidation of sulfides. It has been found that this biocatalyst is active in relation to many aromatic sulfides for which the wild-type PAMO has no activity [83]. Catalysts 2018, 8, 624 18 of 27

The presence of various hydrophilic organic solvents has led to an increase in the enzymatic activity of the M446G mutant of PAMO when it is used in enantioselective sulfoxidations. For example, cyclohexyl propyl sulfide was not converted by M446G PAMO in an aqueous medium, but reacted in the presence of hydrophilic solvents. In the case of wild-type PAMOs, short-chain alcohols led to reduced conversion and increase selectivity, while the presence of low (5–10% by volume) hydrophilic solvents and PEG in M446G catalyzed reactions led to increased sulfides conversion; meanwhile, the selectivity of the biocatalyst remained unchanged or slightly improved. This effect can be explained by the better solubility of the substrate and improved stability of the enzyme in sulfoxidation carried out in the presence of solvents [83]. As result of directed evolution of PAMO, several enzyme variants with reversed enantioselectivity have been obtained, which were used in asymmetric sulfoxidation of several structurally different thioethers. Whereas using wild PAMO leads to obtaining S-sulfoxide with an ee value of 90%, the evolved mutant of PAMO with a four-point mutation, namely 167Q, P440F, A442N, L443I, was R-selective (ee = 95%) [86]. Pyridine methyl sulfides containing a nitrogen atom in the 2-, 3-, or 4-position were oxidized by the use of PAMO and its mutant M446G (M-PAMO). A second enzyme, glucose-6-phosphate dehydrogenase, was used to regenerate NADPH. Reactions were carried out in a Tris-HCl 50 mM buffer (pH = 9.0) at 30 ◦C for 24 h [87]. When wild-type PAMO and its mutant M446G were used for the oxidation of these substrates, only (R)-3-(methylsulfinyl)pyridine was acquired while the other compounds were not reactive. In sulfoxidation which was carried out in the presence of M-PAMO led to the formation of (R)-3-(methylsulfinyl)pyridine with 54% conversion and 88% of ee was observed, whereas PAMO showed lower activity (c = 26%) and selectivity (ee = 39%). M-PAMO was also able to transform the corresponding sulfide to (R)-2-(ethylsulfinyl)pyridine with a conversion of 13% and ee of 76% after 24 h. It is important to mention that both 3-(methylsulfinyl)pyridine enantiomers can be obtained with high enantiomeric excess using different BVMO because PAMO has the preference to form (R)-sulfoxide, while HAPMO prefers the formation of (S)-sulfoxide [87]. The 2-furfuryl methyl sulfide was also transformed. The (S)-sulfoxide from this compound can be obtained with a high conversion and an ee of 81% using the wild-type PAMO. The application of M-PAMO led to lower ee values. Regarding the oxidation of 2-(methylthio)thiophene, PAMO showed lower activity and selectivity than HAPMO in the preparation of the opposite enantiomer, (R)-sulfoxide (c = 22% and ee = 34%). The M446G mutant catalyzed sulfoxidation with even lower activity. Sulfoxidation of cyclohexyl alkyl sulfides was also carried out using PAMO, which gave (S)-sulfoxide after 24 h with a 96% conversion and moderate optical purity (ee = 35%); meanwhile, this biocatalyst was not able to oxidize the ethyl derivative. Reactions that were carried out in the presence of M-PAMO led to (S)-cyclohexylmethyl sulfoxide with a moderate conversion and enantiomeric excess, whereas (S)-cyclohexylethyl sulfoxide was obtained after 24 h with a conversion of 19% and an ee of 31%. As expected from previous results, the M446G mutant was able to catalyze cyclohexylethyl sulfide oxidation for which the wild type of PAMO was not active due to the change in the size of the active site of the enzyme [87]. For both PAMO enzymes, wild and M-PAMO, the optimal pH for sulfoxidation activity ranges from 9.0 to 10.0. Regarding the enzymatic selectivity, (S)-cyclohexyl methyl sulfoxide was gathered at the highest enantiomeric excess at pH 10.5 (ee = 43%), while pH 9.0 was more suitable for M-PAMO (ee = 49%). Further increase in pH led to a gradual loss of enantioselectivity. This observation differs from that of previous work, which presented that this biocatalyst is able to catalyze Baeyer-Villiger oxidation with high conversion to 1-isochromanone synthesis at pH 10.5. PAMO and its mutant M446G were able to carry out moderate oxidation of alkyl butyl sulfides (c = 34–45%, ee = 36–65%) [87]. Catalysts 2018, 8, 624 19 of 27

3.4.6. 4-Hydroxyacetophenone Monooxygenase 4-Hydroxyacetophenone monooxygenase (HAPMO, EC 1.14.13.84) is a BVMO isolated from Pseudomonas fluorescens ACB. The primary reaction catalyzed by this enzyme is NADPH-dependent oxidation of 4-hydroxyacetophenone to 4-hydroxyphenylacetate. This enzyme is a 145 kDa homodimer and each subunit contains a non-covalently bound FAD cofactor [88]. In addition to Baeyer-Villiger oxidation, the enzyme is also used in the oxidation of a range of aromatic sulfides including thioanisols. A second enzyme reaction catalyzed via glucose-6-phosphate dehydrogenase (G6PDH) was used in the regeneration of NADPH. The reactions were carried out in a Tris-HCl buffer (50 mM, pH 9.0) at 25 ◦C[88]. In general, the phenyl sulfides appear to be the best substrates for this enzyme, giving (S)-sulfoxides with high optical purity. Low enantiomeric excess was obtained using benzyl sulfides and the enantioselectivity inversion from S to R was observed for alkyl chains longer than ethyl. The inversion of the preferred enantioselectivity was also found when the sulfur atom was further away from the aromatic ring. In the case of para-substituted phenylmethyl sulfides, the enzyme showed high selectivity for electron donor groups, while strongly accepting electron groups showed a negative effect on the selectivity and efficiency of the reaction. Interestingly, in this study, the position of the substituent did not affect selectivity [88]. Pyridine methyl sulfides containing the nitrogen atom in the 2-, 3-, or 4-position were also oxidized using HAPMO. A second enzyme, glucose-6-phosphate dehydrogenase (G6PDH), was used to regenerate NADPH. Reactions were carried out in a Tris-HCl 50mM buffer (pH = 9.0) at 20 ◦C for 24 h [87]. HAPMO was capable of catalyzing the enzymatic sulfoxidation of the three derivatives with moderate to high conversion and excellent enantiomeric excess. In a study by Rioz-Martinez [87], the best substrate for HAPMO was 2-(methylthio)pyridine because (S)-sulfoxide with complete conversion was achieved. The use of substrates containing a nitrogen atom further away from the methylthio group contributed to lower conversions. Thus, (S)-3-(methylsulfinyl)pyridine was obtained with a conversion of 95%, and the least-oxidized 4-(methylthio)pyridine (c = 63%); however, it yielded the opposite enantiomer because (R)-sulfoxide was obtained. It is important to underline that both 3-(methylsulfinyl) pyridine enantiomers can be obtained with high enantiomeric excess using different biocatalysts, because HAPMO has the preference to form (S)-sulfoxide while PAMO both wild type and mutant M446G prefer formation (R)-sulfoxide. HAPMO was also adept at catalyzing the sulfoxidation of ethyl, n-propyl, and allyl sulfide derivatives of 2-pyridine, and high enantioselectivity was observed. Enantiomerically pure (S)-2-(ethylsulfinyl)pyridine was synthesized with complete conversion after 24 h. When the alkyl chain length was extended to the propyl group, (S)-sulfoxide was achieved in low yield (c = 11%). This tendency was previously described for this biocatalyst in the oxidation of thioanisole derivatives [87]. The heteroaryl substrate, 2-furfuryl methyl sulfide, was also transformed to give the (R)-sulfoxide enantiomer with a conversion of 69% and ee ≥ 99%. Upon oxidation of 2-(methylthio)thiophene, HAPMO was able to transform into enantiomerically pure (S)-sulfoxide with good conversion (c = 71%) after 24 h [87]. Sulfoxidation of cyclohexyl alkyl sulfides was also carried out. In this case, HAPMO proved to be a great catalyst because the enantiomerically pure (S)-sulfoxides were obtained in the reaction of the oxidation of cyclohexyl methyl sulfide and cyclohexyl ethyl sulfide. HAPMO also efficiently oxidized tetrahydrothiophene and tetrahydro-2H-thiopyran (close to 80%). Interestingly, both compounds were not transformed by other BVMO PAMOs. HAPMO also oxidizes aliphatic sulfides. For example, n-butyl methyl and n-butyl ethyl sulfoxides were obtained in the enantiomerically pure form with complete conversion after 24 h of reaction [87]. Catalysts 2018, 8, 624 20 of 27

3.4.7. Styrene Monooxygenase Styrene monooxygenase (SMO) is a flavin-dependent enzyme from group E of BVMOs. This enzyme was first described in Pseudomonas sp. It allows for the degradation of styrene, which is a source of coal and energy for this microorganism. SMO catalyzing the conversion of styrene to (S)-styrene oxide is responsible for adapting this microorganism to an environment contaminated with this hydrocarbon [89]. SMOs are known for their ability to convert a huge variety of substrates, often with high enantioselectivity. It is worth noting that while epoxidation is limited to the production of the (S)-enantiomer, in the case of sulfoxidation, the production of S- or R-sulfoxides depends on the used SMOs and structure of the substrate. However, there is still the possibility of optimization because the enantiomeric excess of the (R)-sulfoxides obtained by these monooxygenases is often not high. SMO usually creates a two-component system. Monooxygenase (StyA, StyA1, EC 1.14.14.11) requires a second component for its activity, oxidoreductase (StyB; EC 1.5.1.36), which produces the reduced FAD necessary for the activity of the first component [90,91]. Some of these reductases exist as self-contained natural fusion proteins (or two domain proteins, StyA2B, E2 type of SMO) composed of the domain of oxygenase (A2) and reductase (B). This prototype was first isolated from Rhodococcus opacus 1CP (RoStyA2B). Most of the described monooxygenases (StyA, StyA1) belong to the pathways of degradation and detoxification of aromatic compounds. The studies found that the organization of the gene cluster is heterogeneous, in particular with regard to regulatory and transport mechanisms. In sulfoxidation reactions, the E1-type is characterized by high activity and low enantioselectivity, while the E2-type has low activity and high enantioselectivity [90]. Monooxygenase VpStyA1 and VpStyA2B isolated from Variovorax paradoxus EPS (accession numbers: ADU39063 and ADU39062) are two-component SMOs of type 2. They are the first example of StyA2B isolated from β-proteobacteria. These enzymes are proteins with two domains as an NADH: FAD-oxidoreductase (VpStyA2B). Searching databases for styA2B-like genes/proteins similar to V. paradoxus EPS indicated that these enzymes occur mainly among Actinobacteria (e.g., Amycolatopsis, Arthrobacter, Gordonia, Mycobacterium, Nocardia, Paenarthrobacter, Paeniglutamicibacter, Pseudarthrobacter, Sciscionella, Sinomonas, and Streptomyces). Variovorax belongs to β-proteobacteria and not to Actinobacteria. The closest homologs of two Variovorax domain proteins are in Delftia, although there is no natural variant of the fusion. The similarity of the VpStyA1 and VpStyA2B sequences (74% identity of more than 404 amino acids) between the monooxygenase domains (A1 and A2) was significantly higher than among other StyAl/StyA2B systems. Due to the high sequence similarity between monooxygenase subunits, Variovorax-like enzymes form a separate branch within the phylogenetic tree and results to date suggest a convergent evolution of the StyA1/StyA2B systems [90]. The ability of VpStyA1 and VpStyA2B to transform five aryl alkyl analogues that are analogues of styrene was studied. The reaction time was 2h and the substrate concentration was 2 mM. The degree of substrate conversion for VpStyA1 ranged from 32% to 95%, and the enantiomeric excess ee = 97–99% (S). The most active VpStyA1 was 95% against benzyl methyl sulfide (BMS). It is worth noting that the enzyme oxidized sulfides more efficiently as it transformed styrene; it was 3.3 to 22 times more active in relation to sulfides than styrene. VpStyA2B was definitely less active in relation to these substrates because the conversion was 6–13% [90]. The maximum of specific activity on BMS was determined to be 3.113 U mg−1 for VpStyAl and 1.616 U mg−1 for VpStyA2B. This is around 25 to 26 times faster than the specific activity for styrene. Thus, the two-component monooxygenase from the V. paradoxus EPS strain has a preference for this sulfide. It is worth noting that high sulfide activity is comparable to other SMOs known from the literature, but the high selectivity giving almost pure (S)-enantiomers is outstanding [90]. Catalysts 2018, 8, 624 21 of 27

In addition, the potential linker region was identified and mutated with a protein similar to the StyA2B region. This linker region has been shown to influence both the reductase domain (B) and monooxygenase (A2). However further structural and kinetic studies are necessary to determine the effect of the linker on individual VpStyA2B domains and related monooxygenase. So far, the results indicate that this monooxygenase from Variovorax paradoxus EPS strain is a suitable candidate for structural studies as well as for the development of an enantioselective catalyst for the sulfoxidation reaction [90]. Another interesting styrene monooxygenases are PaSMO and MlSMO. PaSMO is an SMO isolated from Paraglaciecola agarilytica NO2 (previously Glaciecola agarilytica), an agar-digesting marine bacterium isolated from marine sediment, and MlSMO is an SMO isolated from Marinobacterium litorale DSM 23545, which is example of a mesophile bacteria isolated from surface seawater [92]. The MlSMO and PaSMO amino acid sequences have been aligned with other previously known SMOs. Studies have shown the presence of conservative sequence motifs typical of the mechanistic involvement of flavins and NAD(P)H. It is worth emphasizing that both MlSMO and PaSMO have a functionally conserved Thr47 residue, which is important for catalytic activity in StyA. The FAD-binding fingerprints GXGXXG, GG, and DX6G were found in both SMOs. The GXGXXG sequence (residues 9–14) is the fingerprint sequence that is characteristic for FAD- and NAD(P)H-dependent oxidoreductases and plays an important role in the FAD binding proteins. The second FAD-binding motif contains the sequence GDX6P (residues 294–302 in MlSMO, residues 292–300 in PaSMO), where the highly conserved Asp moiety connectswith the O3’ of the ribose moiety of FAD. The DG sequence is highly conserved among all flavoproteins with hydroxylase activity but only Gly is conservative in StyA (MlSMO residue 171, PaSMO residue 169) [92]. Both SMOs were expressed in E. coli BL21 (DE3). The biotransformation reactions were carried out in a two-phase system containing 10% (v/v) addition of cyclohexane to a phosphate buffer pH = 8 and they were catalyzed using whole E. coli BL21 cells (DE3) in which MlSMO or PaSMO were expressed. In general, MlSMO consistently showed higher activity than PaSMO under the same reaction conditions. The sulfoxidation reaction of thioanisole proceeded with poor enantioselectivity (ee = 19–42% (S)) [92]. An interesting bacterium is the Gram-positive actinomycete Rhodococcus opacus 1CP, which contains three different SMOs. In the work of Riedel [93], it was found that in addition to the styA1 and styA2B gene, the third SMO gene is located within the styABCD cluster, completely separated from styA1 and styA2B. The newly discovered FAD StyB reductase from the R. opacus 1CP strain is highly active with NADH as an electron donor. However, compared to StyB homologues, the binding affinity to NADH and FAD is rather low. StyB forms a homodimer as described for other StyB enzymes, but appears to be rather unstable, which limits its further characteristics [93]. In the studies, phenyl vinyl sulfide was selected as a substrate to provide sulfoxidation as well as an epoxidation site. It was found that all three SMO isoforms convert this substrate and only sulfoxidation was determined. The activities are comparable to styrene epoxidation. It is worth noting that StyA2B showed a 2.4-fold higher sulfoxidase as epoxidase activity. It was found that all three SMO isoforms catalyze sulfoxidation in an enantioselective manner. StyA1 is distinguished by the production of the vinyl-p-sulfoxide S-enantiomer with more than 99% ee [93]. In E. coli Bl21 (DE3) cells, SMO from P. putida CA-3 was expressed. Nine different sulfur-containing compounds were biotransformed. Thioanisole was consumed at a rate of 83.3 µmol/min g dry weight of cells, which resulted mainly in the formation of R-sulfoxide of thioanisole with the ee value of 45%. The consumption rate of 2-bromo-, 2-chloro-, and 2-methylothioanisole was 2-fold lower than in the case of thioanisole. Astonishingly, the oxidation product of 2-methylthioanisole had the opposite configuration (S) to the configuration of the other 2-substituted thioanisole derivatives and had a higher ee value of 84%. The rate of the 4-substituted thioanisols sulfoxidation was higher than the corresponding 2-substituted substrates, but the ee values of the products were consistently lower (10–23%). The rate of formation of benzo[b]thiophene sulphoxide and 2-methylbenzo[b]thiophene was Catalysts 2018, 8, 624 22 of 27 about 10 times lower than in the case of thioanisole. The ee values of benzo[b]thiophene sulfoxide could not be determined due to the rapidly racemizing product under the reaction conditions. E. coli cells expressing the modified SMO (SMOeng R3-11) oxidized the 2-substituted thioanisole 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidized benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster than the wild-type of enzyme. The stereospecificity of the modified SMO has not changed compared to the wild-type enzyme. Using X-ray crystallography for P. putida S12 SMO analysis, it was determined that the entry of substrates into the active site of SMO is limited by the bottleneck of the binding cavity formed by the side chains of the carboxylamide groups Val-211 and Asn-46 [55].

4. Conclusions It seems that despite the intensive development of research on sulfoxidation, it will not be possible to find the optimal biocatalyst for a new sulfide molecule for a long time. In this regard, one should not forget about the tedious basic research involving classic biotransformations using whole cultures of microorganisms, especially those coming from extreme environments. Despite the development of genetic methods, there are no effective and rapid screening tools so far. This problem may arise from the fact that the sulfoxidation reaction may catalyze many different enzymes, as discussed in this work. Two streams of research can be distinguished in current research. On the one hand, research using molecular biology tools on the structure of enzymes, especially the construction of their active center. Unfortunately, so far, there are no quick and effective methods to find the optimal mutant for a given sulfoxidation reaction. The second stream of research includes the optimization of reaction conditions via immobilization or the use of various regeneration methods or the purpose of improving substrate access for the enzyme via the use of added solvents, including ionic liquids.

Author Contributions: W.M. and K.W. writing—original draft preparation, M.G. writing—review. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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