Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

Review Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

Ana Caroline Lustosa de Melo Carvalho 1, Thiago de Sousa Fonseca 1, Marcos Carlos de Mattos 1,*, Maria da Conceição Ferreira de Oliveira 1, Telma Leda Gomes de Lemos 1, Francesco Molinari 2, Diego Romano 2 and Immacolata Serra 2

Received: 28 October 2015; Accepted: 04 November 2015; Published: 11 December 2015 Academic Editor: Vladimír Kˇren

1 Laboratório de Biotecnologia e Síntese Orgânica (LABS), Department of Organic and Inorganic Chemistry, Federal University of Ceará, Campus do Pici, Postal box 6044, 60455-970 Fortaleza, Ceará, Brazil; [email protected] (A.C.L.M.C.); [email protected] (T.S.F.); [email protected] (M.C.F.O.); [email protected] (T.L.G.L.) 2 Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, via Mangiagalli 25, 20133 Milan, Italy; [email protected] (F.M.); [email protected] (D.R.); [email protected] (I.S.) * Correspondance: [email protected]; Tel.: +55-85-3366-9144; Fax: +55-85-3366-9978

Abstract: Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modiﬁed. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase.

Keywords: biocatalysis; lipases; kinetic resolution; enantioselectivity; pharmaceuticals

1. Introduction Among the applications of lipases, the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates using these enzymes is a subject of continuing interest. It is noteworthy that most of the drugs are chiral, and it is important to know which stereoisomer has the desired biological activity. Thus, only the active stereoisomer is administered, thereby preventing the patient receiving a dose of stereoisomer with unnecessary activity. Such practice avoids unnecessary consumption of the substance and minimizes side effects. Biocatalysis offers an alternative approach to conventional chemical processes for the production of enantiomerically pure chiral drugs. Lipases are highlighted as being among the most frequently used enzymes for the production of drugs and their intermediates, a subject covered in an elegant review in 2006 [1]. The use of lipases is mainly due to the characteristics of the regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modiﬁed [1]. Some reviews on the preparation of APIs via biocatalysis were also published, but involved various types of enzymes and not only lipases [2–6].

Int. J. Mol. Sci. 2015, 16, 29682–29716; doi:10.3390/ijms161226191 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2015, 16, page–page

On this topic, we will present representative examples of recent reports in the literature (2007 to mid-2015) concerning the lipase-catalyzed preparation of APIs and their intermediates through hydrolytic (Section 2) and esterification (Section 3) approaches. Additionally, examples involving both approachesInt. J. Mol. Sci. as2015 complementary, 16, 29682–29716 methods are discussed (Section 4).

2. Hydrolytic Approach On this topic, we will present representative examples of recent reports in the literature (2007 Hydrolysisto mid-2015) of racemic concerning or theprochiral lipase-catalyzed esters catalyzed preparation by of lipases APIs and for their preparing intermediates an enantiomerically through hydrolytic (Section2) and esteriﬁcation (Section3) approaches. Additionally, examples involving pure drug intermediate is a well-established method in organic chemistry; nevertheless, new both approaches as complementary methods are discussed (Section4). applications, new enzymes, and improved techniques for bettering the efficiency of already known lipase-catalyzed2. Hydrolytic hydrolysis Approach are continuously proposed. Hydrolysis of racemic or prochiral esters catalyzed by lipases for preparing an enantiomerically 2.1. Key Intermediatespure drug intermediate of Paclitaxel is aSide well-established Chain method in organic chemistry; nevertheless, new applications, new enzymes, and improved techniques for bettering the efﬁciency of already known An interestinglipase-catalyzed example hydrolysis of are the continuously application proposed. of lipases for preparing a series of useful chiral intermediates was proposed for the chemoenzymatic synthesis of analogues of Paclitaxel (trade 2.1. Key Intermediates of Paclitaxel Side Chain names: Taxol or Onxal). This compound is a polyoxygenated diterpenoid isolated from the bark of Taxus brevifoliaAn, which interesting has example been extensively of the application used of in lipases anti-cancer for preparing therapy. a series A number of useful of chiral compounds intermediates was proposed for the chemoenzymatic synthesis of analogues of Paclitaxel (trade from the names:class of Taxol arylazetidiones, or Onxal). This precursors compound is of a polyoxygenatedthe amino acid diterpenoid side chain isolated of Paclitaxel, from the bark as well as a new generationof Taxus brevifoliaof taxanoides, which has side been chains, extensively were used prepared in anti-cancer in enantiomerically therapy. A number ofpure compounds form by kinetic resolution,from via the hydrolysis, class of arylazetidiones, of the corresponding precursors of the esters amino in acid the side presence chain of Paclitaxel,of lipases. as A well screening as a was performednew with generation 14 lipases, of taxanoides and four side of chains, them were (PS prepared(Burkholderia in enantiomerically cepacia), PS-C pure (B. form cepacia by kinetic immobilized resolution, via hydrolysis, of the corresponding esters in the presence of lipases. A screening was on ceramics),performed AS (Aspergillus with 14 lipases, niger and), and four ofABL them (Arthrobacter (PS (Burkholderia sp.)) cepacia showed), PS-C positive (B. cepacia results.immobilized Among these lipases, Arthrobacteron ceramics), sp. AS (ABL) (Aspergillus was niger the), only and ABLone (thatArthrobacter hydrolyzsp.))ed showed all tested positive substrates. results. Among ABL was used on the resolutionthese lipases, ofArthrobacter four arylazetidionessp. (ABL) was thederivatives only one that (Scheme hydrolyzed 1) allin testedbuffer substrates. solution ABL pH was 7.0, in the presence usedof co-solvents on the resolution such of as four acetonitrile arylazetidiones (MeCN), derivatives dimethylformamide (Scheme1) in buffer solution(DMF) pHor 7.0,dimethylsulfoxide in the presence of co-solvents such as acetonitrile (MeCN), dimethylformamide (DMF) or dimethylsulfoxide (DMSO), and resulted in enantiomeric excess of both alcohols and acetates of >99% and conversions (DMSO), and resulted in enantiomeric excess of both alcohols and acetates of >99% and conversions close to 50%close [7]. to 50% [7].

Scheme 1.Scheme Kinetic 1. Kineticenzymatic enzymatic hydrolysis hydrolysis of of racemic racemic arylazetidiones,arylazetidiones, precursors precursors of amino of acidamino side acid side chain of Paclitaxelchain of Paclitaxel [7]. [7].

The compoundThe compound (2R,3S (2)-3-phenylisoserineR,3S)-3-phenylisoserine is isalso also a akey key interm intermediateediate ofof thethe Paclitaxel Paclitaxel side side chain, chain, and it has been already produced by biocatalytical methods [8]. The racemate of and it hasethyl been 3-amino-3-phenyl-2-hydroxy-propionate already produced by biocatalytical (3-phenylisoserine methods [8]. ethyl The ester) racemate was synthesized of ethyl and 3-amino-3- phenyl-2-hydroxy-propionateresolved via lipase-mediated (3-phenylisoserine kinetic hydrolysis. Variousethyl lipasesester) werewas screened synthesized and the bestand resultresolved via lipase-mediated(c 50%, E kinetic> 200) was hydrolysis. obtained with Various lipase from lipasesB. cepacia wereimmobilized screened on and diatomaceous the best result earth (PS (c IM).50%, E > 200) ˝ was obtainedAfter optimizing with lipase the reaction from conditions B. cepacia (diisopropyl immobilized ether (DIPE) on withdiatomaceo 0.5 eq. H2Ous as solvent,earth (PS 50 C),IM). After 3 h, the (2R,3S)-3-amino-3-phenyl-2-hydroxy-propionate was obtained with 100% ee, c 50% and optimizing the reaction conditions (diisopropyl ether (DIPE) with 0.5 eq. H2O as solvent, 50 °C), 3 h, E > 200. Subsequent hydrolysis of this latter compound led to (2R,3S)-3-phenylisoserine (Scheme2a). the (2R,3SOne)-3-amino-3-phenyl-2-hydroxy-propionate advantage of this strategy is that it was not necessary was to obtained protect the with amino 100% group ee [9,]. c 50% and E > 200. Subsequent hydrolysis of this latter compound led to (2R,3S)-3-phenylisoserine (Scheme 2a). One advantage of this strategy is that it was not necessary to protect the amino group [9]. Another strategy to produce the (2R,3S)-3-phenylisoserine involved the enantioselective ring-cleavage of (3R*,4S*)-β-lactam (R=H) and its 3-acetoxy derivative (R=Ac) using CAL-B (lipase B from Candida antarctica produced by the submerged fermentation of a genetically modified Aspergillus oryzae and absorbed on a macroporous resin) as an enzyme29683 at 60 °C (Scheme 2b). The kinetic hydrolyses were performed on gram-scale (1.0 g of substrate) after optimizing the reaction conditions for each starting material. The enantioselective ring-cleavage of the (3R*,4S*)-β-lactams (R=H) in tert-butylmethyl ether (TBME) (with 0.5 eq. of water) yielded, after 18 h, the (3S,4R)-β-lactam (>99% ee, 48% yield) and (2R,3S)-3-phenylisoserine (98% ee, 48% yield). These two compounds were also formed with high 2 Int. J. Mol. Sci. 2015, 16, 29682–29716

Another strategy to produce the (2R,3S)-3-phenylisoserine involved the enantioselective ring-cleavage of (3R*,4S*)-β-lactam (R=H) and its 3-acetoxy derivative (R=Ac) using CAL-B (lipase B from Candida antarctica produced by the submerged fermentation of a genetically modiﬁed Aspergillus oryzae and absorbed on a macroporous resin) as an enzyme at 60 ˝C (Scheme2b). The kinetic hydrolyses were performed on gram-scale (1.0 g of substrate) after optimizing the reaction conditions for each starting material. The enantioselective ring-cleavage of the (3R*,4S*)-β-lactams (R=H) in tert-butylmethyl ether (TBME) (with 0.5 eq. of water) yielded, after 18 h, the (3S,4R)-β-lactam Int. J. Mol. Sci. 2015, 16, page–page (>99% ee, 48% yield) and (2R,3S)-3-phenylisoserine (98% ee, 48% yield). These two compounds were also formed with high enantioselectivity (>98% ee) and good yields (43% and 49%, respectively) enantioselectivitywhen the reaction (>98% wasee) performedand good with yields the (3(43%R*,4S *)-3-acetoxy-and 49%, βrespectively)-lactam (R=Ac). when In this the case, reaction the was performedsolvent with was the DIPE(3R*,4 (1.0S*)-3-acetoxy- eq. H2O) and,β-lactam after 50 (R=Ac). h, the starting In this material case, the was solvent 100% converted was DIPE into (1.0 the eq. H2O) and, afterenantiopure 50 h, the starting products material (>98% eewas)[10 100%]. converted into the enantiopure products (>98% ee) [10].

Scheme Scheme2. Synthesis 2. Synthesis of (2R,3 ofS (2)-3-phenylisoserine,R,3S)-3-phenylisoserine, key key intermediates intermediates ofof PaclitaxelPaclitaxel side side chain, chain, via enzymaticvia hydrolysis enzymatic of hydrolysis the (a) racemic of the ethyl (a) racemic3-amino-3-phenyl-2-hydroxypropionate ethyl 3-amino-3-phenyl-2-hydroxypropionate [7] or (b) β-lactams [7] or [10]. (b) β-lactams [10]. 2.2. Key Intermediate of Crizotinib 2.2. Key Intermediate of Crizotinib An extensiveAn extensive screening screening among among commercial commercial lipases lipases and and esterases esterases was was carried carried out by out researchers by researchers at Agouronat Agouron Pharmaceuticals Pharmaceuticals for forthe the preparation preparation of of (SS)-1-(2,6-dichloro-3-ﬂuorophenyl)ethanol)-1-(2,6-dichloro-3-fluorophenyl)ethanol from from its correspondingits corresponding racemic racemic ester ester(Scheme (Scheme 3);3 this); this chir chiralal intermediate intermediate cancan be be linked linked with with an an ether ether bond to differentbond 2-aminopyridine to different 2-aminopyridine compounds compounds that that po potentlytently inhibit auto-phosphorylation auto-phosphorylation of human of human heptocyteheptocyte growth growth factor factorreceptor. receptor. In addition, In addition, it is itan is intermediate an intermediate on on the the preparation preparation ofof thethe potent potent antitumor compound Crizotinib [11]. Highly enantioselective hydrolysis (E > 100) was antitumor compound Crizotinib [11]. Highly enantioselective hydrolysis (E > 100) was observed with observed with different commercial lipases (CAL-B and Rhizopus delemar lipase, among the others), differentand commercial provided thelipases (S)-ester (CAL-B and (R and)-alcohol Rhizopus with eedelemarranging lipase, from 80% among to 97%. the Then,others), these and two provided the (S)-estercompounds and (R were)-alcohol not separated, with ee ranging and the mixture from 80% was subjectedto 97%. Then, to mesylation these two reaction compounds (conversion were not separated,of and the ( Rthe)-alcohol mixture into was its mesylsubjected derivative), to mesylation followed reaction by treatment (conversion with potassium of the acetate (R)-alcohol to into its mesylexclusively derivative), yield followed the (S)-ester. by treatment Hydrolysis with of potassium this compound acetate furnished to exclusively the desired yield product the (S)-ester. (S)-1-(2,6-dichloro-3-ﬂuorophenyl)ethanol in high yield [12]. Hydrolysis of this compound furnished the desired product (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in high yield [12].

29684

Scheme 3. Synthesis of (S)-1-(2,6-dichloro-3 fluorophenyl)ethanol via kinetic enzymatic hydrolysis of the corresponding racemic ester [12].

3 Int. J. Mol. Sci. 2015, 16, page–page

enantioselectivity (>98% ee) and good yields (43% and 49%, respectively) when the reaction was performed with the (3R*,4S*)-3-acetoxy-β-lactam (R=Ac). In this case, the solvent was DIPE (1.0 eq. H2O) and, after 50 h, the starting material was 100% converted into the enantiopure products (>98% ee) [10].

Scheme 2. Synthesis of (2R,3S)-3-phenylisoserine, key intermediates of Paclitaxel side chain, via enzymatic hydrolysis of the (a) racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate [7] or (b) β-lactams [10].

2.2. Key Intermediate of Crizotinib An extensive screening among commercial lipases and esterases was carried out by researchers at Agouron Pharmaceuticals for the preparation of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol from its corresponding racemic ester (Scheme 3); this chiral intermediate can be linked with an ether bond to different 2-aminopyridine compounds that potently inhibit auto-phosphorylation of human heptocyte growth factor receptor. In addition, it is an intermediate on the preparation of the potent antitumor compound Crizotinib [11]. Highly enantioselective hydrolysis (E > 100) was observed with different commercial lipases (CAL-B and Rhizopus delemar lipase, among the others), and provided the (S)-ester and (R)-alcohol with ee ranging from 80% to 97%. Then, these two compounds were not separated, and the mixture was subjected to mesylation reaction (conversion of the (R)-alcohol into its mesyl derivative), followed by treatment with potassium acetate to exclusively yield the (S)-ester. HydrolysisInt. J. Mol. Sci. of2015 this, 16 compound, 29682–29716 furnished the desired product (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol in high yield [12].

Int. J. Mol. Sci. 2015, 16, page–page

SchemeScheme 3. 3. SynthesisSynthesis of of ( (SS)-1-(2,6-dichloro-3)-1-(2,6-dichloro-3 fluorophenyl)ethanol ﬂuorophenyl)ethanol via via kinetic kinetic enzymatic enzymatic hydrolysis hydrolysis of of 2.3. Pregabalin and Analogues thethe corresponding corresponding racemic racemic ester [12]. [12].

Pregabalin ((S)-3-(aminomethyl)-5-methylhexanoic acid, trade name Lyrica®) is a drug used in 2.3. Pregabalin and Analogues anticonvulsant therapy. Pregabalin and its analogues were obtained by hydrolysis of γ-nitro esters, followed byPregabalin hydrogenation ((S)-3-(aminomethyl)-5-methylhexanoic of the nitro group on3 Raney acid, nickel. trade Hydrolysis name Lyrica rof) isthe a γ drug-nitro used esters can γ be effectivelyin anticonvulsant catalyzed therapy. by lipase Pregabalin Novozym and its435, analogues as seen were in Scheme obtained 4a, by hydrolysisaffording of (S)--nitroγ-nitro esters, acids with followed by hydrogenation of the nitro group on Raney nickel. Hydrolysis of the γ-nitro esters can be moderateeffectively conversion catalyzed (18%–24%) by lipase Novozymand ee 435,ranging as seen between in Scheme 87%4a,affording and 94% ( S )-γ[13].-nitro Additionally, acids 2-carboxyethyl-3-cyano-5-methylhexanoicwith moderate conversion (18%–24%) and acidee ranging ethyl between ester 87% (CNDE) and 94% [was13]. Additionally,enantioselectively hydrolyzed2-carboxyethyl-3-cyano-5-methylhexanoic as the key step in the preparation acid of ethyl Pregabalin ester (CNDE) (Scheme was enantioselectively 4b). This biotransformation hydrolyzed was achievedas by the keyusing step Thermomyces in the preparation lanuginosus of Pregabalin lipase (Scheme (TLL),4b). Thisthe biotransformation enzyme contained was achieved in commercial r Lipolase®by usingand LipozymeThermomyces TL lanuginosus IM®. Recombinantlipase (TLL), the lipase enzyme from contained T. lanuginosus in commercial DSM Lipolase 10635 showed and Lipozyme TL IMr. Recombinant lipase from T. lanuginosus DSM 10635 showed excellent excellent(S ()-enantioselectivityS)-enantioselectivity to CNDE to CNDE (E > 200), (E but > with200), low but catalytic with low activity. catalytic The enzyme activity. was evolvedThe enzyme by was evolved proteinby protein engineering, engineering, allowing the preparationallowing of (3theS)-2-carboxyethyl-3-cyano-5-methylhexanoic preparation of (3S)-2-carboxyethyl-3-cyano-5- acid methylhexanoicwith 42% acid conversion with 42% and 98%conversionee, starting and from 98% 255 ee g/L, starting of substrate from [14 255]. g/L of substrate [14].

Scheme Scheme4. Synthesis 4. Synthesis of Pregabalin of Pregabalin intermediates intermediates through through kinetic kinetic enzymatic enzymatic hydrolysis hydrolysis of (a) rac-γ-nitro of (a) rac-γ- nitro estersesters [13] [13 ]and and ( b)) racrac-2-carboxyethyl-3-cyano-5-methylhexanoic-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl acid ester [ethyl14]. ester [14].

29685

Scheme 5. Kinetic enzymatic hydrolysis of racemic methyl-1,4-benzodioxan-2-carboxylate, affording intermediates used in the synthesis of (S)-Prosimpal, (S)-Piperoxan, (S,S)-Dibozane and (S)-Doxazosin [15].

2.4. Prosimpal, Piperoxan, Dibozane and Doxazosin The drugs (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane are α-adrenergic receptor antagonists, and (S)-Doxazosin is a drug used in the treatment of hypertension. All (S)-isomers are more effective than the corresponding (R)-isomers. The aforementioned drugs were synthesized by hydrolytic kinetic resolution of the racemic intermediate methyl-1,4-benzodioxan-2-carboxylate in the presence of lipase from whole cells of wild species of Arthrobacter (ABL), as seen in Scheme 5. The

4 Int. J. Mol. Sci. 2015, 16, page–page

2.3. Pregabalin and Analogues

Pregabalin ((S)-3-(aminomethyl)-5-methylhexanoic acid, trade name Lyrica®) is a drug used in anticonvulsant therapy. Pregabalin and its analogues were obtained by hydrolysis of γ-nitro esters, followed by hydrogenation of the nitro group on Raney nickel. Hydrolysis of the γ-nitro esters can be effectively catalyzed by lipase Novozym 435, as seen in Scheme 4a, affording (S)-γ-nitro acids with moderate conversion (18%–24%) and ee ranging between 87% and 94% [13]. Additionally, 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) was enantioselectively hydrolyzed as the key step in the preparation of Pregabalin (Scheme 4b). This biotransformation was achieved by using Thermomyces lanuginosus lipase (TLL), the enzyme contained in commercial Lipolase® and Lipozyme TL IM®. Recombinant lipase from T. lanuginosus DSM 10635 showed excellent (S)-enantioselectivity to CNDE (E > 200), but with low catalytic activity. The enzyme was evolved by protein engineering, allowing the preparation of (3S)-2-carboxyethyl-3-cyano-5- methylhexanoic acid with 42% conversion and 98% ee, starting from 255 g/L of substrate [14].

Int.Scheme J. Mol. Sci.4. Synthesis2015, 16, 29682–29716 of Pregabalin intermediates through kinetic enzymatic hydrolysis of (a) rac-γ- nitro esters [13] and (b) rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester [14].

SchemeScheme 5. Kinetic 5. Kineticenzymatic enzymatic hydrolysis hydrolysis of racemic of racemicmethyl-1,4-benzodioxan-2-carboxylate, methyl-1,4-benzodioxan-2-carboxylate, affording intermediatesaffording used intermediates in the synthesis used in of the (S)-Prosimpal, synthesis of ( (SS)-Piperoxan,)-Prosimpal, ( (SS,)-Piperoxan,S)-Dibozane (andS,S)-Dibozane (S)-Doxazosin and [15]. (S)-Doxazosin [15]. 2.4. Prosimpal, Piperoxan, Dibozane and Doxazosin 2.4. Prosimpal, Piperoxan, Dibozane and Doxazosin The drugs (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane are α-adrenergic receptor α antagonists,The and drugs (S (S)-Doxazosin)-Prosimpal, (isS)-Piperoxan a drug used and in (S the,S)-Dibozane treatment are of -adrenergichypertension. receptor All antagonists,(S)-isomers are and (S)-Doxazosin is a drug used in the treatment of hypertension. All (S)-isomers are more effective more effective than the corresponding (R)-isomers. The aforementioned drugs were synthesized by than the corresponding (R)-isomers. The aforementioned drugs were synthesized by hydrolytic hydrolytic kinetic resolution of the racemic intermediate methyl-1,4-benzodioxan-2-carboxylate in kinetic resolution of the racemic intermediate methyl-1,4-benzodioxan-2-carboxylate in the presence the presenceof lipase of from lipase whole from cells whole of wild cells species of wild of Arthrobacter species of Arthrobacter(ABL), as seen (ABL), in Scheme as seen5. The in Scheme reactions 5. The were performed in two different conditions: (i) 0.1 M phosphate buffer with 20% DIPE as a co-solvent and (ii) 0.1 M phosphate buffer with 20% of n-butanol4 as a co-solvent. In the ﬁrst reaction condition, after 2 h of reaction, the corresponding (S)-carboxylic acid (79% ee) and the (R)-methyl ester (>99% ee) were obtained with c 55% and E-value of 33. In the second reaction condition, after 4 h, (S)-carboxylic acid (>99% ee) and (R)-methyl ester (73% ee) were obtained with c 42% and E > 200. The syntheses of the pharmaceuticals (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane were performed by using the (R)-methyl ester (>99% ee) obtained from the ﬁrst reaction condition, while the (S)-carboxylic acid (>99% ee) obtained in the second condition was used in the synthesis of (S)-Doxazosin [15].

2.5. Key Intermediate of Ezetimibe Ezetimibe is a drug used in the reduction of cholesterol and blood lipids. The synthesis of this drug requires the enantiopure 3-[5-(4-ﬂuorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1, 3-oxazolidin-2-one ((S)-FOP alcohol) as a key intermediate. Kinetic resolution of the diastereoisomeric mixture of FOP acetates was assessed using several commercial lipases; the most efﬁcient lipase was from Candida rugosa (CRL) and the best reaction conditions were buffer solution (pH 7.0) containing 30% of DIPE as a co-solvent, at 40 ˝C, and an enzyme:substrate (w/w) ratio of 2.5:1. In such conditions, after reaching 50% conversion, it was possible to obtain (S)-FOP acetate with diastereomeric excess (de) 98.5% (Scheme6). The same results were obtained when the method was applied to scale-up, starting from 1 g of the diastereoisomeric mixture of FOP acetates [16].

29686 Int. J. Mol. Sci. 2015, 16, page–page reactions were performed in two different conditions: (i) 0.1 M phosphate buffer with 20% DIPE as a co-solvent and (ii) 0.1 M phosphate buffer with 20% of n-butanol as a co-solvent. In the first reaction condition, after 2 h of reaction, the corresponding (S)-carboxylic acid (79% ee) and the (R)-methyl ester (>99% ee) were obtained with c 55% and E-value of 33. In the second reaction condition, after 4 h, (S)-carboxylic acid (>99% ee) and (R)-methyl ester (73% ee) were obtained with c 42% and E > 200. The syntheses of the pharmaceuticals (S)-Prosimpal, (S)-Piperoxan and (S,S)-Dibozane were performed by using the (R)-methyl ester (>99% ee) obtained from the first reaction condition, while the (S)-carboxylic acid (>99% ee) obtained in the second condition was used in the synthesis of (S)-Doxazosin [15].

2.5. Key Intermediate of Ezetimibe Ezetimibe is a drug used in the reduction of cholesterol and blood lipids. The synthesis of this drug requires the enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3- oxazolidin-2-one ((S)-FOP alcohol) as a key intermediate. Kinetic resolution of the diastereoisomeric mixture of FOP acetates was assessed using several commercial lipases; the most efficient lipase was from Candida rugosa (CRL) and the best reaction conditions were buffer solution (pH 7.0) containing 30% of DIPE as a co-solvent, at 40 °C, and an enzyme:substrate (w/w) ratio of 2.5:1. In such conditions, after reaching 50% conversion, it was possible to obtain (S)-FOP acetate with diastereomeric excess Int.(de) J. 98.5% Mol. Sci. (Scheme2015, 16, 29682–29716 6). The same results were obtained when the method was applied to scale-up, starting from 1 g of the diastereoisomeric mixture of FOP acetates [16].

Scheme 6. Kinetic enzymatic hydrolysis of diastereoisomeric mixture of FOP acetates to produce the (S)-FOP alcoholalcohol usedused inin thethe synthesissynthesis ofof thethe drugdrug EzetimibeEzetimibe [[16].16].

2.6. Key Intermediate of Hepatitis C Virus Protease Inhibitor 2.6. Key Intermediate of Hepatitis C Virus Protease Inhibitor The chiral compound trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate The chiral compound trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate is an important intermediate for the synthesis of certain Hepatitis C virus protease inhibitors. Initially, is an important intermediate for the synthesis of certain Hepatitis C virus protease inhibitors. rac-dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate was submitted to a kinetic resolution in the Initially, rac-dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate was submitted to a kinetic resolution presence of various hydrolytic enzymes including lipases (lipase OF from Candida rugosa; lipase from in the presence of various hydrolytic enzymes including lipases (lipase OF from Candida rugosa; lipase Mucor miehei; lipase OF from C. rugosa (formerly C. cylindracea); and lipase N from Rhizopus niveus). from Mucor miehei; lipase OF from C. rugosa (formerly C. cylindracea); and lipase N from Rhizopus niveus). The reactions were performed in phosphate buffer, pH 7.0, at 28 °C. As results, the remaining dialkyl The reactions were performed in phosphate buffer, pH 7.0, at 28 ˝C. As results, the remaining dialkyl (2R)-2-vinylcyclopropane-1,1-dicarboxylate and the hydrolysis product, the 1-alcoxycarbonyl-2- (2R)-2-vinylcyclopropane-1,1-dicarboxylate and the hydrolysis product, the 1-alcoxycarbonyl- vinylcyclopropane-1-carboxylic acid, were obtained with diastereomeric excess values (de) >90% in 2-vinylcyclopropane-1-carboxylic acid, were obtained with diastereomeric excess values (de) >90% favor of the cis-isomer (ester/vinyl group), and enantiomeric excess values (ee) ranging from 70% to in favor of the cis-isomer (ester/vinyl group), and enantiomeric excess values (ee) ranging from 70% >99% in favor of the cis-(1S,2S) enantiomer (Scheme 7). After a few chemical steps, it was possible to to >99% in favor of the cis-(1S,2S) enantiomer (Scheme7). After a few chemical steps, it was possible obtain the key chiral intermediate for the synthesis of the virus protease inhibitors of Hepatitis C, to obtain the key chiral intermediate for the synthesis of the virus protease inhibitors of Hepatitis C, trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate [17]. trans-alkyl (1R,2S)-1-(tert-butoxycarbonylamino)-2-vinylcyclopropanecarboxylate [17]. Int. J. Mol. Sci. 2015, 16, page–page

Scheme 7. Kinetic enzymatic hydrolysis hydrolysis of dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate to produce the cis-(1S,2S)-1-alcoxycarbonyl-2-vinylcyclopropane-1-ca)-1-alcoxycarbonyl-2-vinylcyclopropane-1-carboxylicrboxylic acid used to obtain the key intermediate in the synthesissynthesis of the virus protease inhibitorsinhibitors ofof HepatitisHepatitis CC [[17].17].

2.7. Naproxen In the the last last few few years, years, a anumber number of of examples examples ha haveve been been reported reported concerning concerning the the application application of immobilizedof immobilized lipases lipases for achieving for achievinghighly efficient highly stereoselective efﬁcient hydrolysis stereoselective of esters hydrolysis of pharmaceutical of esters interest. of A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)- pharmaceutical interest. Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)-Naproxen belongs to a class of non-steroidal anti-inflammatory drugs and their activity is 28-fold (S)-Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis (S)-Naproxen belongs to a class of non-steroidal anti-inﬂammatory drugs and their activity is 28-fold of racemic Naproxen methyl ester. The kinetic resolution was carried out in the presence of lipase higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis of from Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained racemic Naproxen methyl ester. The kinetic resolution was carried out in the presence of lipase from at 45 °C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained at and a substrate concentration of 10 mg/mL. Under these conditions, a conversion of 49% and an 45 ˝C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL and E-value of 174.2 were reached (Scheme 8) [18]. The same biotransformation was improved by using nanoparticles as additives for the encapsulation of the enzyme via the sol–gel method. The encapsulated lipase showed outstanding 29687 enantioselectivity, with an E-value ranging from 265 to 371 and >98% ee depending on the sol–gel encapsulation process employed [19].

Scheme 8. Synthesis of (S)-Naproxen through kinetic enzymatic hydrolysis of the racemic Naproxen methyl ester [18].

2.8. Key Intermediate of Prostaglandins, Prostacyclins and Thromboxane The compound (1S,4R)-4-hydroxycyclopent-2-enyl acetate is an important intermediate in the synthesis of cyclopentenoid molecules with important biological activity, such as prostaglandins, prostacyclins and thromboxane. Enzymatic hydrolysis of meso-cyclopent-2-en-1,4-diacetate may give access to (1S,4R)-4-hydroxycyclopent-2-enyl acetate, taking advantage of the so-called meso-trick. Lipases from Pseudomonas fluorescens (PFL) and Candida rugosa (CRL) were immobilized by physical adsorption or by chemical functionalization on core-shell superparamagnetic nanoparticles, and their performances were compared with the ones of the free enzymes. The biotransformations were performed in a two-liquid-phase system composed with 80% hexane and 20% water under mild end-over-end rotation. CRL was poorly enantioselective, while free and immobilized PFL afforded enantiopure (1S,4R)-4-hydroxycyclopent-2-enyl acetate (Scheme 9). The re-use of the differently nanoparticle-immobilized PFL showed that the activity of physically adsorbed PFL decreased more than 50% after two cycles, whereas the chemically immobilized enzyme was much more stable [20].

6 Int. J. Mol. Sci. 2015, 16, page–page

Scheme 7. Kinetic enzymatic hydrolysis of dialkyl 2-vinyl-1,1-cyclopropanedicarboxylate to produce the cis-(1S,2S)-1-alcoxycarbonyl-2-vinylcyclopropane-1-carboxylic acid used to obtain the key intermediate in the synthesis of the virus protease inhibitors of Hepatitis C [17].

2.7. Naproxen In the last few years, a number of examples have been reported concerning the application of immobilized lipases for achieving highly efficient stereoselective hydrolysis of esters of pharmaceutical interest. A classic example of the use of immobilized lipase in hydrolysis is the obtainment of (S)- Naproxen ((S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid) from racemic Naproxen methyl ester. (S)-Naproxen belongs to a class of non-steroidal anti-inflammatory drugs and their activity is 28-fold higher than the corresponding (R)-enantiomer. This drug was prepared from enzymatic hydrolysis ofInt. racemic J. Mol. Sci. Naproxen2015, 16, 29682–29716 methyl ester. The kinetic resolution was carried out in the presence of lipase from Candida rugosa immobilized on Amberlite XAD7 (CRL type VII). The best results were obtained at 45 °C in an aqueous phase/isooctane biphasic batch system, at pH 6.0, a lipase load of 800 U/mL anda substrate a substrate concentration concentration of 10 mg/mL.of 10 mg/mL. Under Under these conditions,these conditions, a conversion a conversion of 49% of and 49% an Eand-value an Eof-value 174.2 wereof 174.2 reached were reached (Scheme (Scheme8)[18]. 8) [18]. The same biotransformation was improved by using nanoparticles as additives for the encapsulation of the enzyme via the sol–gel method.method. The The encapsulated lipase showed outstanding enantioselectivity, with an E-value-value rangingranging fromfrom 265265 toto 371371 andand >98%>98% ee depending on the sol–gel encapsulation processprocess employed [[19].19].

Scheme 8. Synthesis of (S)-Naproxen through kinetic enzymatic hydrolysis of the racemic Naproxen Scheme 8. Synthesis of (S)-Naproxen through kinetic enzymatic hydrolysis of the racemic Naproxen methyl ester [18]. methyl ester [18]. 2.8. Key Intermediate of Prostaglandins, Prostacyclins and Thromboxane 2.8. Key Intermediate of Prostaglandins, Prostacyclins and Thromboxane The compound (1S,4R)-4-hydroxycyclopent-2-enyl acetate is an important intermediate in the The compound (1S,4R)-4-hydroxycyclopent-2-enyl acetate is an important intermediate in the synthesis of cyclopentenoid molecules with important biological activity, such as prostaglandins, synthesis of cyclopentenoid molecules with important biological activity, such as prostaglandins, prostacyclins and thromboxane. Enzymatic hydrolysis of meso-cyclopent-2-en-1,4-diacetate may give prostacyclins and thromboxane. Enzymatic hydrolysis of meso-cyclopent-2-en-1,4-diacetate may give access to (1S,4R)-4-hydroxycyclopent-2-enyl acetate, taking advantage of the so-called meso-trick. access to (1S,4R)-4-hydroxycyclopent-2-enyl acetate, taking advantage of the so-called meso-trick. Lipases from Pseudomonas fluorescens (PFL) and Candida rugosa (CRL) were immobilized by physical Lipases from Pseudomonas ﬂuorescens (PFL) and Candida rugosa (CRL) were immobilized by physical adsorption or by chemical functionalization on core-shell superparamagnetic nanoparticles, and their adsorption or by chemical functionalization on core-shell superparamagnetic nanoparticles, and their performances were compared with the ones of the free enzymes. The biotransformations were performances were compared with the ones of the free enzymes. The biotransformations were performed in a two-liquid-phase system composed with 80% hexane and 20% water under mild performed in a two-liquid-phase system composed with 80% hexane and 20% water under mild end-over-end rotation. CRL was poorly enantioselective, while free and immobilized PFL afforded end-over-end rotation. CRL was poorly enantioselective, while free and immobilized PFL afforded enantiopure (1S,4R)-4-hydroxycyclopent-2-enyl acetate (Scheme 9). The re-use of the differently enantiopure (1S,4R)-4-hydroxycyclopent-2-enyl acetate (Scheme9). The re-use of the differently nanoparticle-immobilized PFL showed that the activity of physically adsorbed PFL decreased more nanoparticle-immobilized PFL showed that the activity of physically adsorbed PFL decreased more than 50% after two cycles, whereas the chemically immobilized enzyme was much more stable [20]. than 50% after two cycles, whereas the chemically immobilized enzyme was much more stable [20]. Int. J. Mol. Sci. 2015, 16, page–page

Scheme 9. Desymmetrization of meso-cyclopent-2-en-1,4-diacetate by enzymatic hydrolysis [20]. Scheme 9. Desymmetrization of meso-cyclopent-2-en-1,4-diacetate by enzymatic hydrolysis [20].

2.9. Ketoprofen 2.9. Ketoprofen Only the (S)-enantiomer of Ketoprofen (2-(3-benzoylphenyl)propionic acid) is therapeutically Only the (S)-enantiomer of Ketoprofen (2-(3-benzoylphenyl)propionic acid) is therapeutically relevant as a nonsteroidal anti-inflammatory drug (NSAID). Kinetic resolution of racemic Ketoprofen relevant as a nonsteroidal anti-inﬂammatory drug (NSAID). Kinetic resolution of racemic Ketoprofen vinyl ester was obtained by enzymatic hydrolysis using a lipase from Aspergillus terreus immobilized vinyl ester was obtained by enzymatic hydrolysis using a lipase from Aspergillus terreus immobilized on modified alginate and cyclodextrin hollow spheres [21]. Under optimized conditions (enzyme on modiﬁed alginate and cyclodextrin hollow spheres [21]. Under optimized conditions (enzyme immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 °C), the immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 ˝C), biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at 46% the biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with low 46% conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table 1. Notably, immobilized low enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table1. Notably, immobilized A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free enzyme cannot be recycled. enzyme cannot be recycled. A new approach to resolve racemic Ketoprofen vinyl ester was developed after comparison of the performances of different lipases (from Mucor javanicus, Rhizomucor miehei, Candida rugosa and Pseudomonas cepacia) employed both as free enzymes29688 or immobilized in micro-emulsion-based organogels (MBGs) [22]. The reactions were conducted in the presence of DIPE at 30 °C. The bioconversion with free lipases generally provided low conversions (maximum 12%) compared with the immobilized lipases (maximum yield 49%). Immobilized Mucor javanicus lipase (MJL) exhibited the highest enantioselectivity (E > 200), whereas immobilized Rhizomucor miehei lipase (RML) displayed a moderate E-value (35), but proved to be tolerant to various organic solvents such as DIPE, TBME, tetrahydrofuran (THF), 2-MeTHF, 1,4-dioxane, acetone and MeCN. Moreover, immobilized RML was very thermostable up to 50 °C. It is noteworthy that immobilized RML and MJL showed high activity over 30 cycles and maintained the same initial enantioselectivity. RML immobilized in micro- emulsion-based organogels was employed for a 5-g-scale kinetic resolution of Ketoprofen vinyl ester, furnishing the desired product (91% ee) in 47% yield after 72 h (Scheme 10b and Table 1). The authors highlight the advantages of biotransformations catalyzed by immobilized enzymes in the form of MBGs; these advantages include an improved enzymatic efficiency (conversion and enantioselectivity), excellent reusability, low enzyme loading, and enhanced resistance to organic solvents. Long-term resistance to high concentrations of organic solvents is a crucial feature in lipase-catalyzed reactions, especially when working with poorly water-soluble substrates. Studies on the effect of interfacial composition on lipase activity in two-liquid phase systems revealed that substrate inaccessibility is a major reason for the low activity of lipase in the absence of organic solvents [23].

Scheme 10. Kinetic enzymatic hydrolysis of racemic Ketoprofen vinyl ester, a member of the class of nonsteroidal anti-inflammatory drugs (NSAIDs), using lipases immobilized on (a) Alg-g-PEG/α-CD [21] or in (b) micro-emulsion-based organogels (MBGs) [22].

7 Int. J. Mol. Sci. 2015, 16, page–page

Scheme 9. Desymmetrization of meso-cyclopent-2-en-1,4-diacetate by enzymatic hydrolysis [20].

2.9. Ketoprofen Only the (S)-enantiomer of Ketoprofen (2-(3-benzoylphenyl)propionic acid) is therapeutically relevant as a nonsteroidal anti-inflammatory drug (NSAID). Kinetic resolution of racemic Ketoprofen vinyl ester was obtained by enzymatic hydrolysis using a lipase from Aspergillus terreus immobilized on modified alginate and cyclodextrin hollow spheres [21]. Under optimized conditions (enzyme immobilized on Alg-g-PEG/α-CD hollow spheres used in acetone/water 80/20, pH 7.4, at 30 °C), the biotransformation was enantioselective (E-value of 129), furnishing (R)-Ketoprofen (96% ee at 46% conversion). Under the same conditions, free A. terreus lipase gave only 16% conversion with low enantioselectivity (E-value of 11.4), as seen in Scheme 10a and Table 1. Notably, immobilized Int. J. Mol. Sci. 2015, 16, 29682–29716 A. terreus lipase was continuously used up to 20 cycles with minimal loss of activity, whereas the free enzyme cannot be recycled. A Anew new approach approach to toresolve resolve racemic racemic Ketoprofen Ketoprofen vinyl vinyl ester ester was was developed developed after after comparison comparison of theof performances the performances of different of different lipases lipases (from (from MucorMucor javanicus javanicus, Rhizomucor, Rhizomucor miehei miehei, Candida, Candida rugosa rugosa and Pseudomonasand Pseudomonas cepacia) cepaciaemployed) employed both as free both enzymes as free enzymes or immobilized or immobilized in micro-emulsion-based in micro-emulsion-based organogels ˝ (MBGs)organogels [22]. The (MBGs) reactions [22]. were The conducted reactions were in the conducted presence inof DIPE the presence at 30 °C. of The DIPE bioconversion at 30 C. The with freebioconversion lipases generally with free provided lipases generallylow conversions provided (maximum low conversions 12%) (maximumcompared 12%)with comparedthe immobilized with lipasesthe immobilized (maximum lipases yield (maximum49%). Immobilized yield 49%). Mucor Immobilized javanicusMucor lipase javanicus (MJL)lipase exhibited (MJL) the exhibited highest enantioselectivitythe highest enantioselectivity (E > 200), whereas (E > 200), immobilized whereas immobilizedRhizomucor mieheiRhizomucor lipase miehei (RML)lipase displayed (RML) a moderatedisplayed E-value a moderate (35), Ebut-value proved (35), butto be proved tolerant to be to tolerant various to organic various organicsolvents solvents such as such DIPE, as DIPE, TBME, TBME, tetrahydrofuran (THF), 2-MeTHF, 1,4-dioxane, acetone and MeCN. Moreover, immobilized tetrahydrofuran (THF), 2-MeTHF, 1,4-dioxane, acetone and MeCN. Moreover, immobilized RML RML was very thermostable up to 50 ˝C. It is noteworthy that immobilized RML and MJL showed was very thermostable up to 50 °C. It is noteworthy that immobilized RML and MJL showed high high activity over 30 cycles and maintained the same initial enantioselectivity. RML immobilized activity over 30 cycles and maintained the same initial enantioselectivity. RML immobilized in micro- in micro-emulsion-based organogels was employed for a 5-g-scale kinetic resolution of Ketoprofen emulsion-based organogels was employed for a 5-g-scale kinetic resolution of Ketoprofen vinyl ester, vinyl ester, furnishing the desired product (91% ee) in 47% yield after 72 h (Scheme 10b and Table1). furnishing the desired product (91% ee) in 47% yield after 72 h (Scheme 10b and Table 1). The authors The authors highlight the advantages of biotransformations catalyzed by immobilized enzymes in highlightthe form the of advantages MBGs; these of advantages biotransformations include an catalyzed improved by enzymatic immobilized efﬁciency enzymes (conversion in the form and of MBGs;enantioselectivity), these advantages excellent reusability,include an low improv enzymeed loading, enzymatic and enhancedefficiency resistance (conversion to organic and enantioselectivity),solvents. Long-term excellent resistance reusability, to high low concentrations enzyme loading, of organic and solventsenhanced is aresistance crucial feature to organic in solvents.lipase-catalyzed Long-term reactions,resistance to especially high concentrations when working of organic with poorlysolvents water-soluble is a crucial feature substrates. in lipase-catalyzed Studies reactions,on the effect especially of interfacial when compositionworking with on poorly lipase activitywater-soluble in two-liquid substrates. phase Studies systems on revealed the effect that of interfacialsubstrate composition inaccessibility on is alipase major activity reason forin thetwo-liquid low activity phase of lipasesystems in therevealed absence that of organicsubstrate inaccessibilitysolvents [23]. is a major reason for the low activity of lipase in the absence of organic solvents [23].

Scheme 10. Kinetic enzymatic hydrolysis of racemic Ketoprofen vinyl ester, a member of the class of Scheme 10. Kinetic enzymatic hydrolysis of racemic Ketoprofen vinyl ester, a member of the class nonsteroidalof nonsteroidal anti-inflammatory anti-inﬂammatory drugs drugs (NSAIDs), (NSAIDs), using usinglipases lipases immobilized immobilized on (a) onAlg- (ag)-PEG/ Alg-gα-PEG/-CD [21] or αin-CD (b) [micro-emulsion-based21] or in (b) micro-emulsion-based organogels organogels (MBGs) [22]. (MBGs) [22].

Table 1. Enzymes and conditions used on the kinetic enzymatic hydrolysis of rac-Ketoprofen vinyl ester by methods a and b.

Method Enzyme Conditions Reference lipase from A. terreus immobilized on acetone/water (80/20, v/v), a 7 [21] Alg-g-PEG/α-CD, hollow spheres pH 7.4, 30 ˝C, 4 days b MJL MBGs or RML MBGs DIPE, 30 ˝C, 72 h [22]

29689 Int. J. Mol. Sci. 2015, 16, page–page

Table 1. Enzymes and conditions used on the kinetic enzymatic hydrolysis of rac-Ketoprofen vinyl ester by methods a and b.

Method Enzyme Conditions Reference lipase from A. terreus immobilized on acetone/water (80/20, v/v), a [21] Alg-g-PEG/α-CD, hollow spheres pH 7.4, 30 °C, 4 days b MJL MBGs or RML MBGs DIPE, 30 °C, 72 h [22]

2.10. Dihydropyridine Derivatives The 1,4-Dihydropyridines (1,4-DHPs) are recognized as pharmacophores widely used in clinics for the treatment of cardiovascular diseases. A series of racemic 1,4-DHPs were prepared through several chemical steps including a multicomponent Hantzsch process and a Vilsmeir-Haack reaction. These 1,4-DHPs were subjected to enzymatic kinetic resolution via hydrolysis in the presence of lipases in water saturated with organic solvent [24]. Reactions were carried out until conversions next to 50% and several lipases (such as from Candida antarctica A (CAL-A, NZL-101), porcine pancreatic lipaseInt. J. Mol. (PPL), Sci. 2015 from, 16, C. 29682–29716 antarctica B (CAL-B, Novozym 435) and from C. rugosa (CRL, type VII)) were assayed. The best results were obtained in the presence of CRL or CAL-B depending on the aryl group and the organic solvent. For reaction systems containing CRL and EtOAc as a solvent, the highest enantioselectivity values (E)) werewere obtainedobtained withwith 1,4-DHPs1,4-DHPs containingcontaining arylaryl groupsgroups asas 2-NO2-NO22–C–C66HH44, naphtyl and 2-Cl–5-NO2–C6HH33 withwith EE-values-values of of 103, 103, 170 170 and and >200, >200, respectively. respectively. The The eeee values of the remaining ester were in the range of 84% to >99% while the ee of the carboxylic acid product ranged from 94% to 98%. It It is is noteworthy noteworthy that that some some of of the the carboxylic carboxylic acid acid products products had the enantiomeric excess increased by crystallization.crystallization. Applying Applying this this methodology, methodology, eeee valuesvalues of the carboxylic acid products containing aryl groups ( (i.e.i.e.,, 3-NO 3-NO22–C–C66HH4;4 Ph;; Ph; and and 4-NO 4-NO2–C2–C6H64H) were4) were enhanced enhanced from from 88% 88% to 97%;to 97%; 60% 60% to 92%; to 92%; and and 69% 69% to 95%, to 95%, respectively. respectively. In the In thepresence presence of CAL-B of CAL-B and and EtOAc EtOAc as a assolvent, a solvent, the bestthe bestresult result was wasobtained obtained with witha 1,4-DHP-containing a 1,4-DHP-containing aryl group aryl groupsuch as such 3-CH as3O–C 3-CH6H3O–C4 with6H an4 withE-value an ofE-value 63, c 34%, of 63, 49% c 34%, ee to 49% the remainingee to the remaining ester and ester 95% andee to 95%the carboxylicee to the carboxylic acid product. acid In product. order to In obtain order theto obtain remaining the remainingsubstrate with substrate high ee with, hydrolysis high ee, of hydrolysis 1,4-DHPs of containing 1,4-DHPs aryl containing groups such aryl as groups 4-Br–C such6H4 andas 4-Br–C 3-CH63O–CH4 and6H4 3-CHwas carried3O–C6H out4 was with carried CAL-B out and with TBME CAL-B as a andsolvent TBME affording as a solvent 57% and affording 54% molar 57% conversion,and 54% molar respectively. conversion, In this respectively. case, remaining In this substrates case, remaining (aryl = 4-Br–C substrates6H4) and (aryl (aryl = 4-Br–C= 3-CH63O–CH4)6 andH4) were(aryl =obtained 3-CH3O–C with6H high4) were ee, 97% obtained and 94%, with respectively high ee, 97% (Scheme and 94%, 11). respectively (Scheme 11).

Scheme 11. KineticKinetic enzymatic enzymatic hydrolysis hydrolysis of racemic 1,4- 1,4-DHPsDHPs to obtain the chiral pharmacophores used in clinics for the treatment of cardiovascular diseases [[24].24].

3. Esterification Approach 3. Esterification Approach As known to most of the researchers working in this area, lipases can efficiently catalyze the As known to most of the researchers working in this area, lipases can efficiently catalyze stereoselective acylation of alcohols when the reaction is performed under low water activity the stereoselective acylation of alcohols when the reaction is performed under low water activity conditions. This opportunity can be exploited for achieving the highly regio- or stereoselective conditions. This opportunity can be exploited for achieving the highly regio- or stereoselective preparation of esters with pharmaceutical relevance under mild and controlled conditions. Examples preparation of esters with pharmaceutical relevance under mild and controlled conditions. Examples span from structurally simple drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to span from structurally simple drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to multifunctional complex molecules, such as immunosuppressive agents (i.e., Rapamycin or multifunctional complex molecules, such as immunosuppressive agents (i.e., Rapamycin or Pimecrolimus), Pimecrolimus), where regioselectivity is a major issue. where regioselectivity is a major issue.

3.1. Rapamycin 8 A brilliant example of regioselectivity observed in lipase-catalyzed acylation is the synthesis of the proline analogue of Rapamycin dihydroxyesters, where the chemoenzymatic approach allowed the preparation of the desired molecule in only two steps [25]. Identiﬁcation of a suitable activated ester of the 2,2-bis(hydroxymethyl) propionic acid side chain was crucial for performing the lipase-catalyzed acylation; vinyl esters provided the highest activity and the best yield, allowing for preparative synthesis. Immobilized lipase PS-C “Amano” II was the best biocatalyst, displaying optimal activity in anhydrous TBME (Scheme 12).

29690 Int. J. Mol. Sci. 2015, 16, page–page

3.1. Rapamycin A brilliant example of regioselectivity observed in lipase-catalyzed acylation is the synthesis of the proline analogue of Rapamycin dihydroxyesters, where the chemoenzymatic approach allowed the preparation of the desired molecule in only two steps [25]. Identification of a suitable activated ester of theInt. J. 2,2-bis(hydroxymethyl) Mol. Sci. 2015, 16, page–page propionic acid side chain was crucial for performing the lipase-catalyzed acylation; vinyl esters provided the highest activity and the best yield, allowing for preparative synthesis.3.1. ImmobilizedRapamycin lipase PS-C “Amano” II was the best biocatalyst, displaying optimal activity in anhydrousA brilliant TBME example(Scheme of 12). regioselectivity observed in lipase-catalyzed acylation is the synthesis of the proline analogue of Rapamycin dihydroxyesters, where the chemoenzymatic approach allowed the preparation of the desired molecule in only two steps [25]. Identification of a suitable activated ester of the 2,2-bis(hydroxymethyl) propionic acid side chain was crucial for performing the lipase-catalyzed acylation; vinyl esters provided the highest activity and the best yield, allowing for preparative

synthesis.Int. J. Mol. Immobilized Sci. 2015, 16, 29682–29716 lipase PS-C “Amano” II was the best biocatalyst, displaying optimal activity in anhydrous TBME (Scheme 12).

Scheme 12. Chemoenzymatic synthesis of proline analogue of Rapamycin dihydroxyester [25].

3.2. Pimecrolimus

Pimecrolimus (32-epi-chloro-derivative of Ascomycyn) is a macrolide which has anti-inflammatory, Scheme 12. Chemoenzymatic synthesis of proline analogue of Rapamycin dihydroxyester [25]. antiproliferative andScheme immunosuppres 12. Chemoenzymaticsive synthesis properties, of proline analogue used ofin Rapamycin the topical dihydroxyester treatment [25]. of inflammatory ® skin diseases.3.2. Pimecrolimus3.2. PimecrolimusThis substance is present as an active ingredient in the drug marketed as Elidel . The first chemoenzymaticPimecrolimusPimecrolimus (32-synthesis (32-epi-chloro-derivativeepi-chloro-derivative of Pimecrolimus of of Ascomycyn) Ascomycyn) was isisperformed aa macrolide macrolide which whichfrom has hasAscomycin, anti-inflammatory, anti-inflammatory, a substance isolatedantiproliferative fromantiproliferative the fermentation and and immunosuppres immunosuppressive broth ofsive Streptomyces properties, properties, usedused hygroscopicus in in the the topical topical. treatment Ascomycin treatment of inflammatoryof inflammatoryhas two hydroxyl groups skinattached skindiseases. diseases. to This secondary Thissubstance substance carbons,is present is present asone asan aninactive activethe ingredient cyclohexane ingredient in in the the drugmoiety drug marketed marketed (C-32) as and ElidelElidel rthe®.. The other in macrolactamfirst Thechemoenzymatic cycle first chemoenzymatic (C-24). Insynthesis this synthesis synthesis, of Pimecrolimus of Pimecrolimus it was wasnecessary was performed performed to acetylate fromfrom Ascomycin, Ascomycin, the hydroxyl asubstance a substance specifically isolated from the fermentation broth of Streptomyces hygroscopicus. Ascomycin has two hydroxyl locatedisolated in cyclohexane from the moiety.fermentation The brothstrategy of Streptomyces was based hygroscopicus on the regioselectivity. Ascomycin hasof lipasetwo hydroxyl from Candida groupsgroups attached attached to tosecondary secondary carbons, carbons, one one in in the cyclohexanecyclohexane moiety moiety (C-32) (C-32) and and the the other other in in antarctica Bmacrolactam (CAL-B, Novozym cycle (C-24). 435) In this with synthesis, regioselective it was necessary acylation to acetylate of the the hydroxyl hydroxyl specifically group in question, macrolactam cycle (C-24). In this synthesis, it was necessary to acetylate the hydroxyl specifically located in cyclohexane moiety. The strategy was based on the regioselectivity of lipase from in the presencelocated in cyclohexaneof vinyl acetate moiety. and The toluene,strategy was leading based onto the regioselectivity corresponding of lipaseacetate from with Candida 94% yield Candida antarctica B (CAL-B, Novozym 435) with regioselective acylation of the hydroxyl group (Scheme 13). Then, the 32-monoacetate derivative was subjected to silylation with trimethylsilyl antarcticain question, B (CAL-B, in the Novozym presence 435) of vinyl with acetateregioselective and toluene, acylation leading of the to thehydroxyl corresponding group in acetate question, trifluoromethanesulfonatein thewith presence 94% yield of vinyl (Scheme (TBMSOTf), acetate 13). and Then, leadingtoluene, the 32-monoacetate letoading a 24-silyloxy-32-monoacetate to the derivative corresponding was subjected acetate derivative. towith silylation 94% yield The latter was subjected(Schemewith 13). trimethylsilylto aThen, regioselective the trifluoromethanesulfonate 32-monoacetate alcoholysis derivative (TBMSOTf),(n-octanol) was subjected leading in the to to apresence silylation 24-silyloxy-32-monoacetate ofwith a trimethylsilylCAL-B-producing 24-silyloxy-32-hydroxytrifluoromethanesulfonatederivative. The derivative. latter was (TBMSOTf), subjected After the leading to a steps regioselective to of a 24-silyloxy-32-monoacetate introducing alcoholysis the (n-octanol) chlorine inderivative. atom the presence at position The of latter 32 with was asubjected CAL-B-producing to a regioselective 24-silyloxy-32-hydroxy alcoholysis derivative. (n-octanol) After in thethe steps presence of introducing of a CAL-B-producing the chlorine polymer-bound triphenyl phosphine and deprotection of the silyl group at position 24, Pimecrolimus 24-silyloxy-32-hydroxyatom at position 32 with derivative. polymer-bound After the triphenyl steps of phosphine introducing and the deprotection chlorine atom of the at silyl position group 32 at with was obtainedpolymer-boundposition with 24, an Pimecrolimus triphenyl overall phosphineyield was of obtained 29% and [26,27].withdeprotection an overall of yield the ofsilyl 29% group [26,27 at]. position 24, Pimecrolimus was obtained with an overall yield of 29% [26,27].

Scheme 13. Synthesis of Pimecrolimus via regioselective enzymatic acylation of Ascomycin [26,27].

Scheme 13.Scheme Synthesis 13. Synthesis of Pimecrolimus of Pimecrolimus via via regioselecti regioselective9 ve enzymatic enzymatic acylation acylation of Ascomycin of Ascomycin [26,27]. [26,27]. 9

29691 Int. J. Mol. Sci. 2015, 16, page–page Int. J. Mol. Sci. 2015, 16, 29682–29716 3.3. LoxoprofenInt. J. Mol. Sci. 2015, 16, page–page

3.3.A considerable Loxoprofen number of biocatalytic routes have been developed so far for the production of (S)-profens; nevertheless, more efficient and sustainable processes are needed due to the commercial A considerable number of biocatalytic routes havehave been developed so far for the production of importance of these drugs. Loxoprofen, an (S)-2-(4-(R)-2-oxo–cyclopentylmethyl)phenyl propionic (S)-profens; nevertheless, more efficientefficient and sustainablesustainable processes are needed due to the commercial acid, is a NSAI drug belonging to the group of propionic acid derivatives with anti-inflammatory, importance of thesethese drugs.drugs. Loxoprofen, Loxoprofen, an an ( (SS)-2-(4-()-2-(4-(R)-2-oxo–cyclopentylmethyl)phenyl)-2-oxo–cyclopentylmethyl)phenyl propionic analgesicacid, is and a NSAI antipyretic drug belonging activities. to Inthe fact, group group Loxoprof of of propionic propionicen is acid acida pro-drug derivatives derivatives because with with anti-inflammatory, anti-inflammatory,its active form is the correspondinganalgesic and trans antipyretic antipyretic-alcohol activities. activities.metabolite. In In fact, The fact, Loxoprof Loxoprofenpreparationen is a is ofpro-drug a pro-drugLoxoprofen because because involved its active its active formthe formenzymatic is the is kineticthecorresponding resolution corresponding oftrans thetrans-alcohol racemic-alcohol metabolite. alcohol metabolite. 2-( Thep-{[( The ppreparation-methoxy preparation phenyl)methoxy]methyl}phenyl)propanolof of Loxoprofen Loxoprofen involved involved the the enzymatic via transesterificationkinetic resolution of the reaction racemic in alcohol the 2-(presence p-{[(-{[(p-methoxy-methoxy of lipase phenyl)methoxy]methyl}phenyl)propanol phenyl)methoxy]methyl}phenyl)propanol from Burkholderia cepacia (lipase-PS), molecularvia transesterificationtransesterification sieves 4 Å, in DIPE reaction reaction as ina thesolventin presencethe presenceand of vinyl lipase of acetate fromlipaseBurkholderia asfrom an acylBurkholderia cepaciadonor(lipase-PS), (Scheme cepacia (lipase-PS),14). molecular After 12 h, it wassievesmolecular possible 4 Å, sieves in to DIPE obtain 4 Å, as in a DIPEthe solvent corresponding as a and solvent vinyl and acetate (vinylS)-acetate as acetate an acyl in as donor98%an acyl enantiomeric (Scheme donor (Scheme 14). Afterexcess 14). 12 After h,(ee it) 12 wasand h, the possible to obtain the corresponding (S)-acetate in 98% enantiomeric excess (ee) and the (R)-alcohol (R)-alcoholit was possiblein 94% toee . obtainThe synthesis the corresponding of Loxoprofen (S)-acetate followed in 98% with enantiomeric (S)-acetate excess having (ee) andthe desiredthe in 94% ee. The synthesis of Loxoprofen followed with (S)-acetate having the desired configuration of configuration(R)-alcohol of in the 94% drug ee. The[28]. synthesis of Loxoprofen followed with (S)-acetate having the desired theconfiguration drug [28]. of the drug [28].

SchemeScheme 14. 14.Enzymatic Enzymatic kinetic kinetic resolutionresolution ofof the racemicracemic alcoholalcohol 2-( 2-(p-{[(p-{[(p-methoxyp-methoxy phenyl) phenyl) Scheme 14. Enzymatic kinetic resolution of the racemic alcohol 2-(p-{[(p-methoxy phenyl) methoxy]methyl}phenyl)propanolmethoxy]methyl}phenyl)propanol to to produce produce the the ( (SS)-acetate)-acetate usedused in in the the synthesis synthesis of theof the drug drug Loxoprofen Loxoprofen [28]. [28]. methoxy]methyl}phenyl)propanol to produce the (S)-acetate used in the synthesis of the drug Loxoprofen [28]. 3.4. Flurbiprofen3.4. Flurbiprofen (S)-Flurbiprofen is a NSAI drug, whereas its enantiomer was found to inhibit tumor growth in 3.4.(S)-Flurbiprofen Flurbiprofen is a NSAI drug, whereas its enantiomer was found to inhibit tumor growth in animal models. The kinetic resolution of racemic Flurbiprofen was performed using dry mycelia of animalAspergillus models.(S)-Flurbiprofen oryzae The , kineticMIM is as a NSAIresolutionbiocatalysts drug, of whereas[29]. racemic After its optimizing Flurbiprofen enantiomer the was reactionwas found performe conditions to inhibitd using tumor(organic dry growth solvent, mycelia in of Aspergillusanimaltype of oryzae models.alcohol, MIM and The astemperature), kinetic biocatalysts resolution ( R[29].)-Flurbiprofen of After racemic optimizing Flurbiprofen ester was the obtained wasreaction performed in conditions varied using conversion (organic dry mycelia andsolvent, type ofenantioselectivityofAspergillus alcohol and oryzae (62%–92%temperature),, MIM as ee biocatalysts), as ( Rseen)-Flurbiprofen in [Scheme29]. After 15. ester optimizingLater was on, obtainedthe the kinetic reaction in resolution varied conditions conversionof the (organic same and enantioselectivitysolvent,racemic typedrug ofwas (62%–92% alcohol performed and ee temperature),), in as flow-reactor, seen in (SchemeR)-Flurbiprofen which 15. is consideredLater ester on, was thean obtained interestingkinetic inresolution variedapproach conversion of for the the same racemicanddevelopment drug enantioselectivity was of performed the lipase-catalyzed (62%–92% in flow-reactor,ee), preparation as seen inwhich Scheme of enantiopure is considered15. Later molecules on, an the interesting kinetic[30,31]. resolution It approachis also worth of thefor the same racemic drug was performed in flow-reactor, which is considered an interesting approach for developmentmentioning of thatthe lipase-catalyzedthese automated systemspreparation help ofcontinuous enantiopure bioprocesses, molecules provide [30,31]. easy It is reaction also worth the development of the lipase-catalyzed preparation of enantiopure molecules [30,31]. It is also mentioningoptimization that (includingthese automated parameters systems such as helpresidence continuous time), and bioprocesses, allow for multistep provide reactions easy reactionand worth mentioning that these automated systems help continuous bioprocesses, provide easy reaction optimizationinline recovery (including and purificationparameters ofsuch the as products residence [32,33]. time), Thus, and allowas a prooffor multistep of concept, reactions direct and optimizationesterification (includingin organic parameterssolvent using such commerc as residenceial enzyme time), (Novozym and allow 435) for [30] multistep and the reactions whole inline recovery and purification of the products [32,33]. Thus, as a proof of concept, direct andmicroorganism inline recovery (A. oryzae and, purification MIM) [31] were of the compared products with [32,33 the]. classical Thus, as batch a proof method. of concept, The protocol direct esterification in organic solvent using commercial enzyme (Novozym 435) [30] and the whole esterificationinflow reactor inshowed organic a significant solvent using reduction commercial of the reaction enzyme time (Novozym (from 6 h 435)to 15–60 [30 ]min) and and the yielded whole microorganismmicroorganismboth the (S)-Flurbiprofen (A. (A.oryzae oryzae, MIM), and MIM) ( R[31])-Flurbiprofen [31 ]were were compared compared butyl withesterwith thewith the classical classical≥90% ee batch (chemical batch method. method. purity The >98%)The protocol protocol by inflowinflowmodulating reactor reactor showed the showed reaction a significant a significantconditions reduction reduction(temperature of of the and reactionreaction residence time time time), (from (from as 6 hseen6 toh 15–60to in 15–60 Scheme min) min) and15. and yielded yielded bothboth the ( theS)-Flurbiprofen (S)-Flurbiprofen and and (R (R)-Flurbiprofen)-Flurbiprofen butylbutyl esterester with withě 90%≥90%ee ee(chemical (chemical purity purity >98%) >98%) by by modulatingmodulating the thereaction reaction conditions conditions (temperature (temperature andand residenceresidence time), time), as as seen seen in Schemein Scheme 15. 15.

Scheme 15. Enzymatic kinetic resolution of racemic Flurbiprofen, a non-steroidal anti-inflammatory drug, using classical batch method [29] and inflow reactors [30,31].

3.5.Scheme IbuprofenScheme 15. Enzymatic 15. Enzymatic kinetic kinetic resolution resolution of of racemic racemic Flurbiprofen, a a non-steroidal non-steroidal anti-inﬂammatory anti-inflammatory drug,drug, using using classical classical batch batch method method [29] [29 ]and and inflow inﬂow reactorsreactors [ 30[30,31].,31]. Ibuprofen is another NSAI agent with anti-inflammatory activity and wide commercial interest. 3.5. IbuprofenA racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was performedIbuprofen viais another a glutaraldehyde NSAI agent or withN-3-(3-dimethylaminopropyl)- anti-inflammatory activityN´-ethylcarbodiimide and wide commercial (EDC)/ interest.N- 29692 A racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF 10 and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was performed via a glutaraldehyde or N-3-(3-dimethylaminopropyl)-N´-ethylcarbodiimide (EDC)/N-

10 Int. J. Mol. Sci. 2015, 16, 29682–29716

3.5. Ibuprofen Ibuprofen is another NSAI agent with anti-inﬂammatory activity and wide commercial interest. A racemic mixture of Ibuprofen was resolved in the presence of commercially available lipases OF and CRL type VII from Candida rugosa immobilized onto magnetic beads. Immobilization was Int. J. Mol. Sci. 2015, 16, page–page performedInt. J. Mol. Sci. 2015 via, 16 a, glutaraldehydepage–page or N-3-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC)/ hydroxysulfosuccinimideN-hydroxysulfosuccinimide sodium sodium salt salt (sulfo-NHS) (sulfo-NHS) cross-linking cross-linking reaction. reaction. The The immobilized immobilized enzymes werehydroxysulfosuccinimide evaluated for the preparation sodium salt of Ibuprofen (sulfo-NHS) propyl cross-linking ester. All reaction. reactions The provided immobilized the ( S )-esterenzymes as thewere product, product, evaluated and and for the the the best best preparation result result (E (E-value-value of Ibuprofen of of 19, 19, 83% 83%propyl ee eeand andester. c 42%) c 42%)All was reactions was achieved achieved provided in the in thepresencethe presence(S)-ester of the as of immobilizedthe immobilizedproduct, andlipase lipasethe from best from resultC. rugosaC. rugosa(E-value OF,OF, as of as well19, well 83% as as inee in theand the presencec presence 42%) was of of achievedadditives additives in such the presence as NaNa22SO of4 and the molecularimmobilized sieves lipase 4 Å from (Scheme C. rugosa 1616).). The OF, study as well of reuseas in demonstratedthe presence of continued additives stability such as and Na2 catalytic SO4 and activitymolecular after sieves ﬁvefive 4 reaction Å (Scheme cycles 16). [[34].34 The]. study of reuse demonstrated continued stability and catalytic activity after five reaction cycles [34].

Scheme 16. Kinetic enzymatic esterification of rac-Ibuprofen, a nonsteroidal drug with anti-inflammatory Scheme 16. Kinetic enzymatic esteriﬁcation of rac-Ibuprofen, a nonsteroidal drug with anti-inﬂammatory activityScheme [34].16. Kinetic enzymatic esterification of rac-Ibuprofen, a nonsteroidal drug with anti-inflammatory activity [34]. activity [34]. 3.6. Ketorolac 3.6. Ketorolac Ketorolac (rac-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid) is a potent NSAIKetorolac drug belonging (rac-5-benzoyl-1,2-dihydro-3 to the class of the heterocyclicH-pyrrolo[1,2- acetica aacid]pyrrole-1-carboxylic]pyrrole-1-carboxylic derivatives, and it acid)acid) is widely isis a used potent as anNSAI analgesic. drug belongingbelonging The microwave toto thethe class class (MW)-assisted of of the the heterocyclic heterocyclic kinetic acetic aceticresolution acid acid derivatives, ofderivatives, this compound and and it isit widelyiswas widely investigated used used as anas usinganalgesic.an analgesic. different The The microwave immobilized microwave (MW)-assisted lipases, (MW)-assisted such kinetic as kineticNovozym resolution resolution 435, of thisLipozyme of compound this compound TL IM, was Lipozyme investigated was investigated RM using IM, lipasedifferentusing differentAmano immobilized immobilizedAS, and lipases, lipase lipases, such AYS as suchAmano Novozym as Novozym [35]. 435, Among Lipozyme 435, Lipozymethem, TL Novozym IM, TL Lipozyme IM, Lipozyme435 RMcatalyzed IM, RM lipase IM,the enantioselectiveAmanolipase Amano AS, and AS,acylation lipase and AYS oflipase Amano rac-Ketorolac AYS [35 ].Amano Among in 3 h, [35]. at them, 50 Among°C Novozym and 300 them, rpm, 435 Novozym catalyzedwith c 50% the435 and enantioselective catalyzedhigh ee values the ˝ (>99%)acylationenantioselective for of bothrac-Ketorolac the acylation (R)-ester inof 3andrac h,-Ketorolac atremaining 50 C and in ( S3 300)-acid h, at rpm, 50 (Scheme °C with and c 17). 50%300 Inrpm, and addition, highwithee c the50%values reaction and (>99%) high was ee for valuesfound both tothe(>99%) follow (R)-ester for the both andPing-Pong the remaining (R)-ester bi-bi (and Smechanism,)-acid remaining (Scheme and (S )-acid 17to). be In inhibited(Scheme addition, 17).by the nIn-octanol. reactionaddition, was the foundreaction to was follow found the Ping-Pongto follow the bi-bi Ping-Pong mechanism, bi-bi and mechanism, to be inhibited and to by ben inhibited-octanol. by n-octanol.

Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory drug [35]. Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory drug [35]. Scheme 17. Enzymatic kinetic resolution of rac-Ketorolac, a potent nonsteroidal anti-inflammatory 3.7. Chloramphenicol and Thiamphenicol drug [35]. 3.7. Chloramphenicol and Thiamphenicol Another pharmacological field where the use of lipases has been particularly active in the recent past3.7. ChloramphenicolAnotheris the elaboration pharmacological and Thiamphenicolof antibiotics. field where The the compound use of lipas (1Res,2 hasR)-( been−)-Chloramphenicol particularly active is inan the effective recent antibioticpast is the against elaboration a wide of rangeantibiotics. of Gram-positive The compound and (1Gram-negativeR,2R)-(−)-Chloramphenicol bacteria. This is drugan effective can be Another pharmacological field where the use of lipases has been particularly active in the recent administeredantibiotic against in capsule a wide or rangeliquid offorms, Gram-positive and it has aand bitter Gram-negative taste. One way bacteria. to overcome This thisdrug problem can be past is the elaboration of antibiotics. The compound (1R,2R)-(´)-Chloramphenicol is an effective isadministered to use esters in of capsule chloramphenicol, or liquid forms, which and are it morehas a palatable.bitter taste. Several One way chloramphenicol to overcome this esters problem were antibiotic against a wide range of Gram-positive and Gram-negative bacteria. This drug can be preparedis to use esters regioselectively of chloramphenicol, in the presence which areof lipases.more palatable. Then (− )-ChloramphenicolSeveral chloramphenicol was subjected esters were to administered in capsule or liquid forms, and it has a bitter taste. One way to overcome this problem esterificationprepared regioselectively reaction in the in presencethe presence of a seriesof lipases. of vinyl Then esters (−)-Chloramphenicol containing alkyl chainswas subjected of varying to is to use esters of chloramphenicol, which are more palatable. Several chloramphenicol esters were sizesesterification (one to 15reaction carbon in atoms), the presence as well ofas ain series the presence of vinyl ofesters a number containing of lipases. alkyl Thechains more of efficientvarying prepared regioselectively in the presence of lipases. Then (´)-Chloramphenicol was subjected to lipasessizes (one were to from15 carbon Candida atoms), antarctica as well B (CAL-B,as in the Novozym presence of435) a number and two of different lipases. preparationsThe more efficient from esterification reaction in the presence of a series of vinyl esters containing alkyl chains of varying Pseudomonaslipases were cepaciafrom Candida, one from antarctica Amano B (PSL-C (CAL-B, Amano, Novozym also known435) and as twoBurkholderia different cepacia preparations) and another from sizes (one to 15 carbon atoms), as well as in the presence of a number of lipases. The more efficient fromPseudomonas Sigma-Aldrich cepacia, ®one (PSLC-I). from Amano With (PSL-CCAL-B, Amano, the best also reaction known conditions as Burkholderia were cepacia in the) andpresence another of lipases were from Candida antarctica B (CAL-B, Novozym 435) and two different preparations from MeCNfrom Sigma-Aldrich as a solvent at® 20 (PSLC-I). °C; with WithPSL-C CAL-B, I and PSL-C the best Amano, reaction the ideal conditions solvent werewas 1,4-dioxane in the presence at 30 °C. of Pseudomonas cepacia, one from Amano (PSL-C Amano, also known as Burkholderia cepacia) and another Generally,MeCN as a monoesters solvent at 20 of °C;chloramphenicol with PSL-C I and acylated PSL-C on Amano, the primary the ideal hydroxyl solvent we wasre obtained 1,4-dioxane with at c 30>99% °C. (isolatedGenerally, yields monoesters between of 75% chloramphenicol and 91%) for reactionacylated times on the between primary 3 hydroxyland 10 h (Schemewere obtained 18 and with Table c >99% 2). It is(isolated noteworthy yields betweenthat the 75%study and of 91%) the forreuse reaction of29693 CA timesL-B between in esterification 3 and 10 hwith (Scheme vinyl 18 palmitateand Table 2).was It performed.is noteworthy As thata result, the itstudy was ofobserved the reuse that of afte CAr L-B3 h ofin reaction,esterification there withwas completevinyl palmitate conversion was duringperformed. 10 cycles As a result,of reuse it wasof the observed enzyme that [36]. afte Ther 3 sameh of reaction, strategy therewas usedwas completeon the synthesis conversion of Thiamphenicolduring 10 cycles esters of reuse from of(1 Rthe,2R enzyme)-(−)-Thiamphenicol, [36]. The same an antibioticstrategy was analogue used onof chloramphenicolthe synthesis of withThiamphenicol veterinary estersapplications. from (1 TheR,2 Resters)-(−)-Thiamphenicol, were regioselectively an antibiotic prepared analogue via acylation of chloramphenicol reaction in the presencewith veterinary of lipases applications. and vinyl esters The esterswith variable were regioselectively lengths. The most prepared effective via lipase acylation was CAL-B reaction in MeCN, in the presence of lipases and vinyl esters with variable lengths. The most effective lipase was CAL-B in MeCN, 11 11 Int. J. Mol. Sci. 2015, 16, 29682–29716 from Sigma-Aldrichr (PSLC-I). With CAL-B, the best reaction conditions were in the presence of MeCN as a solvent at 20 ˝C; with PSL-C I and PSL-C Amano, the ideal solvent was 1,4-dioxane at 30 ˝C. Generally, monoesters of chloramphenicol acylated on the primary hydroxyl were obtained with c >99% (isolated yields between 75% and 91%) for reaction times between 3 and 10 h (Scheme 18 and Table2). It is noteworthy that the study of the reuse of CAL-B in esterification with vinyl palmitate was performed. As a result, it was observed that after 3 h of reaction, there was complete conversion during 10 cycles of reuse of the enzyme [36]. The same strategy was used on the synthesis of Thiamphenicol esters from (1R,2R)-(´)-Thiamphenicol, an antibiotic analogue of chloramphenicol with veterinary applications. The esters were regioselectively prepared via acylation reaction in the Int. J. Mol. Sci. 2015, 16, page–page presence of lipases and vinyl esters with variable lengths. The most effective lipase was CAL-B in ˝ atMeCN, 20 °C. at 20ThiamphenicolC. Thiamphenicol esters esters(acylated (acylated at the at theprimary primary hydroxyl hydroxyl group) group) were were regioselectively produced with c >99% and isolated yields between 94% and 98%, as seen seen in in Scheme Scheme 1818 andand Table Table 22.. A study of the reuse of lipase from C. antarctica type B (CAL-B, Novozym 435) was conducted for the acylation of (´−)-Thiamphenicol)-Thiamphenicol with with vinyl vinyl decanoate. decanoate. The The corresponding corresponding 3’-monoester 3’-monoester was obtained in 96% yield and the enzyme was reused fivefive timestimes withoutwithout anyany lossloss ofof thethe activityactivity [[37].37].

Scheme 18. Regioselective enzymatic acylation of (−)-Chloramphenicol [36] and (−)-Thiamphenicol [37]. Scheme 18. Regioselective enzymatic acylation of (´)-Chloramphenicol [36] and (´)-Thiamphenicol [37].

Table 2. Optimized conditions for the regioselective enzymatic acylation of (−)-Chloramphenicol and Table 2. Optimized conditions for the regioselective enzymatic acylation of (´)-Chloramphenicol and (−)-Thiamphenicol. (´)-Thiamphenicol. Compound Enzyme Conditions 3‘-Monoester Yield (%) Ref. CompoundCAL-B Enzyme MeCN, Conditions 20 °C 3‘-Monoester Yield (%) Ref. (−)-Chloramphenicol 75–91 36 PSL-C I or PSL-CCAL-B Amano 1,4-dioxane,MeCN, 2030˝ °CC (´)-Chloramphenicol ˝ 75–91 [36] (−)-Thiamphenicol PSL-CCAL-B I or PSL-C Amano 1,4-dioxane, MeCN, 20 °C 30 C 94–98 37 (´)-Thiamphenicol CAL-B MeCN, 20 ˝C 94–98 [37] 3.8. Key Intermediates of Modified Cephalosporins 3.8. Key Intermediates of Modified Cephalosporins Some modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9- ylethoxy)carbonyl]-Some modifiedL-leucine Cephalosporins tert-butyl ester and have the a chiral anti-inflammatory fragment resulting agent from Nthe-[(9 incorporationH-fluoren-9- ofylethoxy)carbonyl]- 1-(9H-fluoren-9-yl)ethanolL-leucine tertin -butyltheir structures. ester have The a chiral first fragment chemoenzymatic resulting synthesis from the incorporationof this chiral fragmentof 1-(9H-fluoren-9-yl)ethanol was performed in the in presence their structures. of lipases. The Initially, first chemoenzymatic racemic 1-(9H-fluoren-9-yl)ethanol synthesis of this chiral was chemicallyfragment was obtained, performed and then in theit was presence submitted of lipases. to a kinetic Initially, resolution racemic via acet 1-(9ylationH-fluoren-9-yl)ethanol reaction. Fifteen commercialwas chemically lipases obtained, were andevaluated then it by was studying submitted parameters to a kinetic considered resolution important via acetylation in the reaction. kinetic resolutionFifteen commercial process, such lipases as the were substrate/lipase evaluated by ratio studying and solvent. parameters The best considered results importantwere obtained in the in thekinetic kinetic resolution resolution process, with an such enzymatic as the aggregate substrate/lipase of lipase ratio from and Candida solvent. antarctica The A best (CAL-A results CLEA, were Amanoobtained lipase in the A). kinetic It is worth resolution noting with that andepending enzymatic on aggregatethe reaction of conditions, lipase from bothCandida (S)- and antarctica (R)-1- (9AH (CAL-A-fluoren-9-yl) CLEA, ethanol Amano (obtained lipase A). from It is worththe hydrol notingysis that of dependingthe corresponding on the reaction acetate) conditions, could be preparedboth (S)- with and >99% (R)-1-(9 ee.H In-fluoren-9-yl) the first condition ethanol (i), (obtainedafter 24 h of from reaction, the hydrolysis with an enzyme/substrate of the corresponding (w/w) ratioacetate) of 10% could and be TBME prepared as a with solvent, >99% it eewas. In possible the first to condition obtain the (i), (S after)-alcohol 24 h and of reaction, (R)-acetate with with an enantiomericenzyme/substrate excess (w (/eew) )>99% ratio and of 10% 93%, and respectively TBME as a ( solvent,E-value itof was145, possible c 52%), as to seen obtain in theScheme (S)-alcohol 19. In theand second (R)-acetate condition with enantiomeric (ii), a dramatic excess decrease (ee) >99% in the and reaction 93%, respectively rate was observed (E-value with of 145, a decreasing c 52%), as enzyme/substrateseen in Scheme 19 .ratio In the to second1.25%. conditionAfter nine (ii), days, a dramatic the (S)-alcohol decrease (68% in the ee) reaction and (R)-acetate rate was (>99% observed ee) werewith aobtained decreasing with enzyme/substrate c 41% and E > 200 ratio[38]. to 1.25%. After nine days, the (S)-alcohol (68% ee) and (R)-acetate (>99% ee) were obtained with c 41% and E > 200 [38].

29694

Scheme 19. Kinetic enzymatic acylation of rac-1-(9H-fluoren-9-yl)ethanol, affording intermediates in the synthesis of modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9- ylethoxy)carbonyl]-L-leucine tert-butyl ester [38].

3.9. Key Intermediate of Duloxetin BASF AG patented the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2- yl)propan-1-ol, a key intermediate in the synthesis of the serotonin-norepinephrine reuptake 12 Int. J. Mol. Sci. 2015, 16, page–page at 20 °C. Thiamphenicol esters (acylated at the primary hydroxyl group) were regioselectively produced with c >99% and isolated yields between 94% and 98%, as seen in Scheme 18 and Table 2. A study of the reuse of lipase from C. antarctica type B (CAL-B, Novozym 435) was conducted for the acylation of (−)-Thiamphenicol with vinyl decanoate. The corresponding 3’-monoester was obtained in 96% yield and the enzyme was reused five times without any loss of the activity [37].

Scheme 18. Regioselective enzymatic acylation of (−)-Chloramphenicol [36] and (−)-Thiamphenicol [37].

Table 2. Optimized conditions for the regioselective enzymatic acylation of (−)-Chloramphenicol and (−)-Thiamphenicol.

Compound Enzyme Conditions 3‘-Monoester Yield (%) Ref. CAL-B MeCN, 20 °C (−)-Chloramphenicol 75–91 36 PSL-C I or PSL-C Amano 1,4-dioxane, 30 °C (−)-Thiamphenicol CAL-B MeCN, 20 °C 94–98 37

3.8. Key Intermediates of Modified Cephalosporins Some modified Cephalosporins and the anti-inflammatory agent N-[(9H-fluoren-9- ylethoxy)carbonyl]-L-leucine tert-butyl ester have a chiral fragment resulting from the incorporation of 1-(9H-fluoren-9-yl)ethanol in their structures. The first chemoenzymatic synthesis of this chiral fragment was performed in the presence of lipases. Initially, racemic 1-(9H-fluoren-9-yl)ethanol was chemically obtained, and then it was submitted to a kinetic resolution via acetylation reaction. Fifteen commercial lipases were evaluated by studying parameters considered important in the kinetic resolution process, such as the substrate/lipase ratio and solvent. The best results were obtained in the kinetic resolution with an enzymatic aggregate of lipase from Candida antarctica A (CAL-A CLEA, Amano lipase A). It is worth noting that depending on the reaction conditions, both (S)- and (R)-1- (9H-fluoren-9-yl) ethanol (obtained from the hydrolysis of the corresponding acetate) could be prepared with >99% ee. In the first condition (i), after 24 h of reaction, with an enzyme/substrate (w/w) ratio of 10% and TBME as a solvent, it was possible to obtain the (S)-alcohol and (R)-acetate with enantiomeric excess (ee) >99% and 93%, respectively (E-value of 145, c 52%), as seen in Scheme 19. In the second condition (ii), a dramatic decrease in the reaction rate was observed with a decreasing Int.enzyme/substrate J. Mol. Sci. 2015, 16 ,ratio 29682–29716 to 1.25%. After nine days, the (S)-alcohol (68% ee) and (R)-acetate (>99% ee) were obtained with c 41% and E > 200 [38].

Scheme 19. KineticKinetic enzymatic enzymatic acylation acylation of of racrac-1-(9-1-(9HH-fluoren-9-yl)ethanol,-fluoren-9-yl)ethanol, affording intermediatesintermediates inin thethe synthesissynthesis ofof modifiedmodified CephalosporinsCephalosporins andand the anti-inflammatoryanti-inflammatory agentagent N-[(9H-fluoren-9--fluoren-9- ylethoxy)carbonyl]-L-leucine terttert-butyl-butyl esterester [[38].38].

3.9. Key Intermediate of Duloxetin 3.9. Key Intermediate of Duloxetin BASF AG patented the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2- BASF AG patented the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2- Int.yl)propan-1-ol, J. Mol. Sci. 2015 , 16a , page–pagekey intermediate in the synthesis of the serotonin-norepinephrine reuptake yl)propan-1-ol,Int. J. Mol. Sci. 2015, a 16 key, page–page intermediate in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetineinhibitor Duloxetine ((+)-(S)-N -methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine12 oxalate) [39 oxalate)]. Chemical [39]. reductioninhibitorChemical reductionDuloxetine of 3-chloro-1-(thiophen-2-yl)propan-1-one of 3-chloro-1-(thiophen-2-yl((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine)propan-1-one gives gives access access to theto the corresponding corresponding oxalate)rac rac-alcohol,-alcohol, [39]. whichChemical waswas reduction enantioselectively enantioselectively of 3-chloro-1-(thiophen-2-yl acylated acylated with with succinic)propan-1-one succinic anhydride anhydride gives using access lipase using to the from lipase correspondingBurkholderia from Burkholderia plantariirac-alcohol,or fromplantariiwhichPseudomonas was or from enantioselectively Pseudomonassp., furnishing sp., acylated thefurnishing corresponding with the succinic corresponding (R)-semiester, anhydride (R)-semiester, leaving using thelipase unreactedleaving from the Burkholderia ( Sunreacted)-alcohol. The(plantariiS)-alcohol. reaction or from The between Pseudomonasreaction the between (S)-alcohol sp., furnishing the and (S)-alcohol methylamine the corresponding and methylamine afforded (R the)-semiester, enantiomericallyafforded leavingthe enantiomerically the pure unreacted desired productpure(S)-alcohol. desired (Scheme The product reaction20). (Scheme between 20). the (S)-alcohol and methylamine afforded the enantiomerically pure desired product (Scheme 20).

Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the Schemechemoenzymatic 20. Kinetic synthesis enzymatic of (acylationS)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol a key intermediate used in the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, a key intermediate inchemoenzymatic the synthesis of synthesis the serotonin-norepi of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol,nephrine reuptake inhibitor Duloxetine [39]. a key intermediate in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetine [39]. in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetine [39]. 3.10. Nebracetam 3.10. Nebracetam Nebracetam, a nootropic drug from the racetam family, is used as an anti-depressant (M1 acetylcholineNebracetam, receptor a nootropic agonist). drug The from first asymmetric the racetam synthesis family, isof used (S)-Nebracetam as an anti-depressant was efficiently (M1 acetylcholineconducted from receptor receptor the agonist). intermediateagonist). The The ﬁrst (5first asymmetricS)-1-benzyl-5-hydroxy-1,5-dih asymmetric synthesis synthesis of (S)-Nebracetam of (Sydropyrrol-2-one)-Nebracetam was efﬁciently was which efficiently conducted was fromobtainedconducted the by intermediate kineticfrom theresolution intermediate (5S)-1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one of the racemic (5S)-1-benzyl-5-hydroxy-1,5-dih hydroxylactam (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one).ydropyrrol-2-one which was obtainedwhich was by kineticTheobtained acetylation resolution by kinetic was ofresolution performed the racemic of thein hydroxylactam theracemic presence hydrox of (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). ylactamlipase from (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). Burkholderia cepacia (lipase PS-D), vinyl The acetylationTheacetate acetylation as an was acyl was performed donor, performed 1,4-dioxane in thein the presence as presence the organic of of lipase lipase solvent from from atBurkholderia roomBurkholderia temperature cepacia cepacia (lipaseand(lipase 48 h PS-D),PS-D), of reaction vinyl acetate(Scheme as 21). an Accordingly,acyl donor, 1,4-dioxane the corresponding as the organic (R)-acetate solvent and at ( S room)-alcohol temperature were obtained and 48 with h of excellent reaction (Schemeenantioselectivity 2121).). Accordingly, Accordingly, and conversion the corresponding (>99% ee, c 49% (R)-acetate and E > and 200). (S )-alcoholThen, after were several obtained chemical with steps, excellent the enantioselectivity(5S)-alcohol was converted and conversion conversion into (S (>99%)-Nebracetam (>99% eeee, ,c c49% 49% [40]. and and E E> 200).> 200). Then, Then, after after several several chemical chemical steps, steps, the the(5S)-alcohol (5S)-alcohol was was converted converted into into (S)-Nebracetam (S)-Nebracetam [40]. [40 ].

Scheme 21. Kinetic resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxy- 1,5-dihydropyrrol-2-oneScheme 21. Kinetic resolution used ofin the racemicsynthesis hydroxylactam of the drug (S )-Nebracetamto produce the [40]. (5S)-1-benzyl-5-hydroxy- Scheme 21. Kinetic resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxy-1, 1,5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40]. 5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40]. 3.11. Naphthofurandione derivative 3.11. Naphthofurandione derivative The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with antitumorThe naphthofurandione activity, is a secondary (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3- metabolite isolated from the bark ofb ]furan-4,9-dione,Tabebuia impetiginosa with antitumor(Bignoniaceae). activity, Naphthofurandione is a secondary wasmetabolite prepared29695 isolated in racemic from form the throughbark of severalTabebuia chemical impetiginosa steps from(Bignoniaceae). commercially Naphthofurandione available 1,5-dihydroxynaphthalene. was prepared in racemic Then, form the through rac-naphthofurandione several chemical stepswas subjectedfrom commercially to enzymatic available kinetic resolution1,5-dihydroxynaphthalene. via acetylation reaction Then, (Scheme the rac 22).-naphthofurandione Among the evaluated was lipases,subjected Pseudomonas to enzymatic cepacia kinetic, (PSL-CI, resolution 3 h of viareaction) acetylation and lipase reaction from (Scheme Candida antarctica22). Among B, (Novozym the evaluated 435, lipases,2 h of reaction) Pseudomonas were cepacia the most, (PSL-CI, efficient. 3 h of The reaction) best reactionand lipase conditions from Candida were antarctica vinyl acetate B, (Novozym as the acyl435, donor,2 h of reaction) THF as the were solvent, the most 250 rpm, efficient. at 30 The°C, leadingbest reaction to (S)-alcohol conditions and were the corresponding vinyl acetate as(R )-acetatethe acyl donor,with 99% THF ee asfor the both solvent, and E 250> 200 rpm, [41]. at 30 °C, leading to (S)-alcohol and the corresponding (R)-acetate with 99% ee for both and E > 200 [41].

Scheme 22. Synthesis of the naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3- b]furan- Scheme4,9-dione 22. via Synthesis kinetic enzymatic of the naphthofurandione acylation of the corresponding (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3- racemic alcohol [41]. b]furan- 4,9-dione via kinetic enzymatic acylation of the corresponding13 racemic alcohol [41]. 13 Int. J. Mol. Sci. 2015, 16, page–page inhibitor Duloxetine ((+)-(S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)propylamine oxalate) [39]. Chemical reduction of 3-chloro-1-(thiophen-2-yl)propan-1-one gives access to the corresponding rac-alcohol, which was enantioselectively acylated with succinic anhydride using lipase from Burkholderia plantarii or from Pseudomonas sp., furnishing the corresponding (R)-semiester, leaving the unreacted (S)-alcohol. The reaction between the (S)-alcohol and methylamine afforded the enantiomerically pure desired product (Scheme 20).

Scheme 20. Kinetic enzymatic acylation of the rac-3-chloro-1-(thiophen-2-yl)propan-1-ol used in the chemoenzymatic synthesis of (S)-3-(methylamino)-1-(thiophen-2-yl)propan-1-ol, a key intermediate in the synthesis of the serotonin-norepinephrine reuptake inhibitor Duloxetine [39].

3.10. Nebracetam Nebracetam, a nootropic drug from the racetam family, is used as an anti-depressant (M1 acetylcholine receptor agonist). The first asymmetric synthesis of (S)-Nebracetam was efficiently conducted from the intermediate (5S)-1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one which was obtained by kinetic resolution of the racemic hydroxylactam (1-benzyl-5-hydroxy-1,5-dihydropyrrol-2-one). The acetylation was performed in the presence of lipase from Burkholderia cepacia (lipase PS-D), vinyl acetate as an acyl donor, 1,4-dioxane as the organic solvent at room temperature and 48 h of reaction (Scheme 21). Accordingly, the corresponding (R)-acetate and (S)-alcohol were obtained with excellent enantioselectivity and conversion (>99% ee, c 49% and E > 200). Then, after several chemical steps, the (5S)-alcohol was converted into (S)-Nebracetam [40].

Int. J. Mol.Scheme Sci. 2015 21. Kinetic, 16, 29682–29716 resolution of the racemic hydroxylactam to produce the (5S)-1-benzyl-5-hydroxy- 1,5-dihydropyrrol-2-one used in the synthesis of the drug (S)-Nebracetam [40]. 3.11. Naphthofurandione Derivative 3.11. Naphthofurandione derivative The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with The naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione, with antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa antitumor activity, is a secondary metabolite isolated from the bark of Tabebuia impetiginosa (Bignoniaceae). Naphthofurandione was prepared in racemic form through several chemical steps (Bignoniaceae). Naphthofurandione was prepared in racemic form through several chemical steps from commercially available 1,5-dihydroxynaphthalene. Then, the rac-naphthofurandione was from commercially available 1,5-dihydroxynaphthalene. Then, the rac-naphthofurandione was subjected to enzymatic kinetic resolution via acetylation reaction (Scheme 22). Among the evaluated subjected to enzymatic kinetic resolution via acetylation reaction (Scheme 22). Among the evaluated lipases, Pseudomonas cepacia, (PSL-CI, 3 h of reaction) and lipase from Candida antarctica B, (Novozym lipases, Pseudomonas cepacia, (PSL-CI, 3 h of reaction) and lipase from Candida antarctica B, (Novozym 435, 435, 2 h of reaction) were the most efﬁcient. The best reaction conditions were vinyl acetate as the acyl 2 h of reaction) were the most efficient. The best reaction conditions were vinyl acetate as the acyl donor, THF as the solvent, 250 rpm, at 30 ˝C, leading to (S)-alcohol and the corresponding (R)-acetate donor, THF as the solvent, 250 rpm, at 30 °C, leading to (S)-alcohol and the corresponding (R)-acetate with 99% ee for both and E > 200 [41]. with 99% ee for both and E > 200 [41].

Scheme 22. Synthesis of the naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan- Scheme 22. Synthesis of the naphthofurandione (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b] 4,9-dione via kinetic enzymatic acylation of the corresponding racemic alcohol [41]. furan-4,9-dione via kinetic enzymatic acylation of the corresponding racemic alcohol [41]. Int. J. Mol. Sci. 2015, 16, page–page 13 3.12. GABOB GABOB and and carnitine Carnitine The compoundscompounds (R )-(Rγ)--Amino-γ-Amino-β-hydroxybutyricβ-hydroxybutyric acid acid (GABOB) (GABOB) and ( Rand)-carnitine (R)-carnitine are two importantare two importantbioactive moleculesbioactive molecules that play that an importantplay an import roleant in mammalianrole in mammalian systems. systems. The keyThe stepkey step of the of thechemoenzymatic chemoenzymatic syntheses syntheses of of these these two two compounds compounds involved involved the the lipase-mediatedlipase-mediated resolution of thethe racemic 3-hydroxy-4-tosyloxybu 3-hydroxy-4-tosyloxybutanenitriletanenitrile inin organicorganic solventssolvents (Scheme(Scheme 2323).). Lipases Lipases (free (free or or immobilizedimmobilized forms) forms) from from PseudomonasPseudomonas cepacia cepacia (PS),(PS), P.P. fluorescens ﬂuorescens (AK),(AK), CandidaCandida rugosa rugosa (CRL(CRL AYS), AYS), C. antarcticaC. antarctica (CAL-B)(CAL-B) and Mucor and Mucor miehei miehei(Lipozyme)(Lipozyme) were used were as biocatalysts, used as biocatalysts, and yielded and the yielded (S)-alcohol the and(S)-alcohol (R)-acetate and in (R high)-acetate enantioselectivity in high enantioselectivity (>99% ee). After (>99% someee chemical). After some transformations, chemical transformations, the (R)-acetate isomerthe (R)-acetate was converted isomer wasinto converted(R)-GABOB into and (R ()-GABOBR)-carnitine and [42]. (R)-carnitine [42].

Scheme 23. Kinetic resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile to produce (R)-GABOB Scheme 23. Kinetic resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile to produce and (R)-carnitine [42]. (R)-GABOB and (R)-carnitine [42].

3.13. Quinolone Derivatives 3.13. Quinolone Derivatives The quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one The quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)- was chemically synthesized and subsequently subjected to enzymatic kinetic resolution via acylation one was chemically synthesized and subsequently subjected to enzymatic kinetic resolution via reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica B (CAL-B), acylation reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h of reaction, it was B (CAL-B), and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; (S)-quinolone was obtained of reaction, it was possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well as the corresponding (R)-acetate (S)-quinolone was obtained in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well showed significant cytotoxic activity against human neuroblastoma tumor cells (SK-N-SH) and lung cells as the corresponding (R)-acetate showed signiﬁcant cytotoxic activity against human neuroblastoma (A549) compared to the control doxorubicin [43]. tumor cells (SK-N-SH) and lung cells (A549) compared to the control doxorubicin [43].

29696

Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1- yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43].

3.14. Key Intermediate of Mevinic Acid Analogues Analogues of mevinic acid can be obtained from (S)-1-(1-naphthyl)ethanol. A racemic mixture of this compound was resolved in the presence of lipases using microwave. Several parameters were studied and the best reaction conditions were Novozym 435 as a biocatalyst, vinyl acetate as an acyl donor, n-heptane as a solvent, a stirring speed of 400 rpm, temperature at 60 °C and 30 mg of enzyme loading. In such conditions, after 3 h of reaction, the (S)-1-(1-naphthyl)ethanol was obtained with c 48%, 90.0% ee and E > 200 (Scheme 25). The study of reuse of the enzyme was performed with a slight reduction of conversion from 48% to 45% after being used three times. It is noteworthy that the results from the conventional heating (c 39%, 64% ee and E-value of 164 after 5 h of reaction) were inferior to those obtained under microwave conditions [44].

14 Int. J. Mol. Sci. 2015, 16, page–page

3.12. GABOB and carnitine The compounds (R)-γ-Amino-β-hydroxybutyric acid (GABOB) and (R)-carnitine are two important bioactive molecules that play an important role in mammalian systems. The key step of the chemoenzymatic syntheses of these two compounds involved the lipase-mediated resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile in organic solvents (Scheme 23). Lipases (free or immobilized forms) from Pseudomonas cepacia (PS), P. fluorescens (AK), Candida rugosa (CRL AYS), C. antarctica (CAL-B) and Mucor miehei (Lipozyme) were used as biocatalysts, and yielded the (S)-alcohol and (R)-acetate in high enantioselectivity (>99% ee). After some chemical transformations, the (R)-acetate isomer was converted into (R)-GABOB and (R)-carnitine [42].

Scheme 23. Kinetic resolution of the racemic 3-hydroxy-4-tosyloxybutanenitrile to produce (R)-GABOB and (R)-carnitine [42].

3.13. Quinolone Derivatives The quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one was chemically synthesized and subsequently subjected to enzymatic kinetic resolution via acylation reaction (Scheme 24). The best results were obtained using lipase from Candida antarctica B (CAL-B), and vinyl acetate as a both solvent and acyl donor at room temperature. After 48 h of reaction, it was possible to obtain the corresponding (R)-acetate in 36% yield and 96% ee; (S)-quinolone was obtained in 41% yield and 98% ee, c 50% and E > 200. The (S)-quinolone as well as the corresponding (R)-acetate Int.showed J. Mol. significant Sci. 2015, 16 ,cytotoxic 29682–29716 activity against human neuroblastoma tumor cells (SK-N-SH) and lung cells (A549) compared to the control doxorubicin [43].

Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazin-1- Scheme 24. Kinetic enzymatic acylation of the racemic quinolone 2-[2-hydroxy-3-(4-phenylpiperazin- yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43]. 1-yl)propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one [43].

3.14. Key Intermediate of Mevinic Acid Analogues 3.14. Key Intermediate of Mevinic Acid Analogues Analogues of mevinic acid can be obtained from (S)-1-(1-naphthyl)ethanol. A racemic mixture Analogues of mevinic acid can be obtained from (S)-1-(1-naphthyl)ethanol. A racemic mixture of this compound was resolved in the presence of lipases using microwave. Several parameters were of this compound was resolved in the presence of lipases using microwave. Several parameters were studied and the best reaction conditions were Novozym 435 as a biocatalyst, vinyl acetate as an acyl studied and the best reaction conditions were Novozym 435 as a biocatalyst, vinyl acetate as an acyl donor, n-heptane as a solvent, a stirring speed of 400 rpm, temperature at 60 °C and 30 mg of enzyme donor, n-heptane as a solvent, a stirring speed of 400 rpm, temperature at 60 ˝C and 30 mg of enzyme loading. In such conditions, after 3 h of reaction, the (S)-1-(1-naphthyl)ethanol was obtained with loading. In such conditions, after 3 h of reaction, the (S)-1-(1-naphthyl)ethanol was obtained with c 48%, 90.0% ee and E > 200 (Scheme 25). The study of reuse of the enzyme was performed with a c 48%, 90.0% ee and E > 200 (Scheme 25). The study of reuse of the enzyme was performed with a slight reduction of conversion from 48% to 45% after being used three times. It is noteworthy that the slight reduction of conversion from 48% to 45% after being used three times. It is noteworthy that results from the conventional heating (c 39%, 64% ee and E-value of 164 after 5 h of reaction) were the results from the conventional heating (c 39%, 64% ee and E-value of 164 after 5 h of reaction) were inferior to those obtained under microwave conditions [44]. inferior to those obtained under microwave conditions [44]. Int. J. Mol. Sci. 2015, 16, page–page

14 Scheme 25. 25. KineticKinetic enzymatic enzymatic acylation acylation of rac-1-(1-naphthyl)ethanol of rac-1-(1-naphthyl)ethanol to produce to producethe (S)-1-(1- the (naphthyl)ethanol,S)-1-(1-naphthyl)ethanol, an intermed an intermediateiate in the synthesis in the synthesis of mevinic of mevinic acid analogues acid analogues [44]. [44].

3.15. Key Intermediate of Tetrahydrofuran DerivativesDerivatives Compounds (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine showed Compounds (+)- and (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine neuroprotective, antidepressant and antiepileptic activities. Such compounds were prepared by showed neuroprotective, antidepressant and antiepileptic activities. Such compounds were prepared chemoenzymatic synthesis starting from 5,5-diphenyltetrahydrofuran-2(3H)-one. The key step for by chemoenzymatic synthesis starting from 5,5-diphenyltetrahydrofuran-2(3H)-one. The key step introducing chirality in the target molecule was the desymmetrization of the intermediate for introducing chirality in the target molecule was the desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol, using vinyl acetate as an acylating agent in the 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol, using vinyl acetate as an acylating agent in the presence presence of lipase from Burkholderia cepacia (Amano lipase PS30), to obtain the (+)-4-hydroxy-2- of lipase from Burkholderia cepacia (Amano lipase PS30), to obtain the (+)-4-hydroxy-2-(hydroxymethyl)-4, (hydroxymethyl)-4,4-diphenylbutyl acetate (Scheme 26). The latter compound was subjected to two 4-diphenylbutyl acetate (Scheme 26). The latter compound was subjected to two different synthetic different synthetic routes. The first (a) involved a sequence of reactions including tosylation, and routes. The first (a) involved a sequence of reactions including tosylation, and hydrolysis and reaction hydrolysis and reaction with 2,6-lutidine in the presence of trifluoromethane sulfonic anhydride to with 2,6-lutidine in the presence of trifluoromethane sulfonic anhydride to afford the (+) afford the (+) 1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. In the second 1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. In the second synthetic route (b), synthetic route (b), the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was subjected to a step of subjected to a step of protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), followed by followed by hydrolysis in the presence of LiOH/H2O, tosylation reaction and deprotection with hydrolysis in the presence of LiOH/H2O, tosylation reaction and deprotection with tetrabutylammonium tetrabutylammonium fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic sulfonic sulfonic anydride led to the (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should anydride led to the (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should be mentioned that the absolute configurations of the products were not reported by the authors [45]. be mentioned that the absolute configurations of the products were not reported by the authors [45].

29697

Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol to produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the synthesis of (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine [45].

3.16. Key Intermediates of β-Amino Alcohols β-Amino alcohols are part of the structure of numerous active pharmaceuticals such as Vernakalant and Indinavir. The cis- and trans-2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol were subjected to kinetic resolution in the presence of lipases. The best reaction conditions were obtained in the presence of vinyl acetate and TBME. For both trans-2-phthalimidocyclopentanol and trans-2- phthalimidocyclohexanol the most efficient lipase was from Pseudomonas cepacia (PS IM), as seen in Table 3, leading to (1R,2R)-acetates and (1S,2S)-alcohols with >99% ee, c 50% and E > 200. In this case, the biocatalyst maintained its activity and enantioselectivity after five reaction cycles. For the cis-2- phthalimidocyclopentanol, four lipases were efficient: lipase A from Candida antarctica (CAL-A), lipase from Rhizomucor miehei (RM IM), lipase from P. fluorescens (AK) and P. cepacia lipase (PS IM), as seen in Table 3. In all cases (1S,2R)-cis-alcohol and (1R,2S)-cis-acetate with >99% ee, c 50%, and E > 200 were obtained. For the cis-2-phthalimidocyclohexanol the only effective lipase was P. cepacia (PS IM), Table 3, with a reaction time of 72 h, at 45 °C. In such conditions, (1R,2S)-cis-acetate and (1S,2R)-cis- alcohol were obtained with >99% ee, c 50% and E > 200 (Scheme 27) [46].

15 Int. J. Mol. Sci. 2015, 16, page–page

Scheme 25. Kinetic enzymatic acylation of rac-1-(1-naphthyl)ethanol to produce the (S)-1-(1- naphthyl)ethanol, an intermediate in the synthesis of mevinic acid analogues [44].

3.15. Key Intermediate of Tetrahydrofuran Derivatives Compounds (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine showed neuroprotective, antidepressant and antiepileptic activities. Such compounds were prepared by chemoenzymatic synthesis starting from 5,5-diphenyltetrahydrofuran-2(3H)-one. The key step for introducing chirality in the target molecule was the desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol, using vinyl acetate as an acylating agent in the presence of lipase from Burkholderia cepacia (Amano lipase PS30), to obtain the (+)-4-hydroxy-2- (hydroxymethyl)-4,4-diphenylbutyl acetate (Scheme 26). The latter compound was subjected to two different synthetic routes. The first (a) involved a sequence of reactions including tosylation, and hydrolysis and reaction with 2,6-lutidine in the presence of trifluoromethane sulfonic anhydride to afford the (+) 1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. In the second synthetic route (b), the intermediate (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate was subjected to a step of protecting one of the hydroxyl groups with tert-butyldimethylsilyl chlroride (TBSCl), followed by hydrolysis in the presence of LiOH/H2O, tosylation reaction and deprotection with tetrabutylammonium fluoride (TBAF). Finally, a reaction with 2,6-lutidine in the presence of trifluoroacetic sulfonicInt. J. Mol. anydride Sci. 2015, 16led, 29682–29716 to the (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine. It should be mentioned that the absolute configurations of the products were not reported by the authors [45].

Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol to Scheme 26. Desymmetrization of the intermediate 3-(hydroxymethyl)-1,1-diphenylbutane-1,4-diol produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the to produce (+)-4-hydroxy-2-(hydroxymethyl)-4,4-diphenylbutyl acetate, a key intermediate in the synthesis of (+)- and (−)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine [45]. synthesis of (+)- and (´)-1-(5,5-diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine [45]. 3.16. Key Intermediates of β-Amino Alcohols 3.16. Key Intermediates of β-Amino Alcohols β-Amino alcohols are part of the structure of numerous active pharmaceuticals such as Vernakalant β-Amino alcohols are part of the structure of numerous active pharmaceuticals such as Vernakalant and Indinavir. The cis- and trans-2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol were and Indinavir. The cis- and trans-2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol were subjected to kinetic resolution in the presence of lipases. The best reaction conditions were obtained subjected to kinetic resolution in the presence of lipases. The best reaction conditions were in the presence of vinyl acetate and TBME. For both trans-2-phthalimidocyclopentanol and trans-2- obtained in the presence of vinyl acetate and TBME. For both trans-2-phthalimidocyclopentanol and phthalimidocyclohexanol the most efficient lipase was from Pseudomonas cepacia (PS IM), as seen in trans-2-phthalimidocyclohexanol the most efficient lipase was from Pseudomonas cepacia (PS IM), as Table 3, leading to (1R,2R)-acetates and (1S,2S)-alcohols with >99% ee, c 50% and E > 200. In this case, seen in Table3, leading to (1 R,2R)-acetates and (1S,2S)-alcohols with >99% ee, c 50% and E > 200. the biocatalyst maintained its activity and enantioselectivity after five reaction cycles. For the cis-2- In this case, the biocatalyst maintained its activity and enantioselectivity after five reaction cycles. phthalimidocyclopentanol, four lipases were efficient: lipase A from Candida antarctica (CAL-A), For the cis-2-phthalimidocyclopentanol, four lipases were efficient: lipase A from Candida antarctica lipase from Rhizomucor miehei (RM IM), lipase from P. fluorescens (AK) and P. cepacia lipase (PS IM), (CAL-A), lipase from Rhizomucor miehei (RM IM), lipase from P. fluorescens (AK) and P. cepacia as seen in Table 3. In all cases (1S,2R)-cis-alcohol and (1R,2S)-cis-acetate with >99% ee, c 50%, and E > lipase (PS IM), as seen in Table3. In all cases (1 S,2R)-cis-alcohol and (1R,2S)-cis-acetate with >99% ee, 200 were obtained. For the cis-2-phthalimidocyclohexanol the only effective lipase was P. cepacia (PS IM), c 50%, and E > 200 were obtained. For the cis-2-phthalimidocyclohexanol the only effective lipase was Table 3, with a reaction time of 72 h, at 45 °C. In such conditions, (1R,2S)-cis-acetate and (1S,2R)-cis- P. cepacia (PS IM), Table3, with a reaction time of 72 h, at 45 ˝C. In such conditions, (1R,2S)-cis-acetate alcohol were obtained with >99% ee, c 50% and E > 200 (Scheme 27) [46]. Int. J. andMol. Sci. (1S ,22015R)-,cis 16-alcohol, page–page were obtained with >99% ee, c 50% and E > 200 (Scheme 27)[46]. 15

SchemeScheme 27. 27.KineticKinetic enzymatic enzymatic acylation acylation of of racemic racemic transtrans-- andand ciscis-diastereoisomers-diastereoisomers of of 2-phthalimidocyclopentanol2-phthalimidocyclopentanol and and 2-phthalimidocyclohexanol 2-phthalimidocyclohexanol toto produceproduce intermediates intermediates in thein the synthesissynthesis of β of-aminoβ-amino alcohols alcohols [46]. [46 ].

TableTable 3. Efficient 3. Efﬁcient lipases lipases for for the the kinetic kinetic enzymatic enzymatic acylation of of racemic racemictrans trans- and- andcis -diastereoisomerscis-diastereoisomers of 2-phthalimidocyclopentanolof 2-phthalimidocyclopentanol and and 2-phthalimidocyclohexanol 2-phthalimidocyclohexanol [46 [46].].

CompoundCompound LipaseLipase trans-2-phthalimidocyclopentanoltrans-2-phthalimidocyclopentanol P. cepaciaP. cepacia(PS IM) (PS IM) C. antarctica (CAL-A), R. miehei (RM IM), P. ﬂuorescens (AK) cis-2-phthalimidocyclopentanol C. antarctica (CAL-A), R. miehei (RM IM), P. fluorescens cis-2-phthalimidocyclopentanol and P. cepacia (PS IM) trans-2-phthalimidocyclohexanol (AK)P. cepacia and(PS P. IM)cepacia (PS IM) trans-2-phthalimidocyclohexanolcis-2-phthalimidocyclohexanol P. cepaciaP. cepacia(PS IM) (PS IM) cis-2-phthalimidocyclohexanol P. cepacia (PS IM)

3.17. Key Intermediates of Iminocyclitols

Enantiomerically pure 1,3-propanediols 29698are important intermediates in the synthesis of iminocyclitols (glycosidases inhibitors used as anti-diabetic drugs). The desymmetrization of carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) via acetylation reaction in THF mediated by crude pig pancreatic lipase (PPL, L3126, type II, Steapsin) (i) or purified PPL (L0382, type VI-S) (ii) was reported [47]. When the reaction was carried out in vinyl acetate as both a solvent and acyl donor, in the presence of crude PPL (i), only the (2R)-monoacetate was formed in 98% ee. On the other hand, only the (2S)-monoacetate (100% ee) was formed when purified PPL (ii) catalyzed the desymmetrization (Scheme 28).

Scheme 28. Kinetic enzymatic acylation of racemic carboxybenzyl-2-amino-1,3-propanediol (Cbz- serinol) to produce intermediates in the synthesis of iminocyclitols [47].

3.18. N-Acetyl Phenylalanine and Analogues Phenylalanine and analogues are important building blocks in the preparation of complex structures of interest to the medicinal chemistry area. N-Acetyl phenylalanine and a series of analogues were obtained in enantiomerically pure form by interesterification reaction in the presence of lipase from Rhizomucor miehei (RML). Ethyl acetamidocianoacetate was alkylated in the presence of various alkyl halides in phase transfer catalysis (PTC), followed by acidic hydrolysis, esterification and N-acetylation leading to N-acetyl-phenylalanine methyl and allyl esters derivatives. These derivatives were submitted to an enzymatic kinetic resolution via interesterification reaction. After screening with various commercial lipases, it was found that only RML was able to promote the kinetic resolution (Scheme 29). Several parameters were studied and the best reaction conditions were obtained in the presence of butyl butyrate as an interesterification agent, with an enzyme:substrate

16 Int. J. Mol. Sci. 2015, 16, page–page

Scheme 27. Kinetic enzymatic acylation of racemic trans- and cis-diastereoisomers of 2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol to produce intermediates in the synthesis of β-amino alcohols [46].

Table 3. Efficient lipases for the kinetic enzymatic acylation of racemic trans- and cis-diastereoisomers of 2-phthalimidocyclopentanol and 2-phthalimidocyclohexanol [46].

Compound Lipase trans-2-phthalimidocyclopentanol P. cepacia (PS IM) C. antarctica (CAL-A), R. miehei (RM IM), P. fluorescens cis-2-phthalimidocyclopentanol (AK) and P. cepacia (PS IM) trans-2-phthalimidocyclohexanol P. cepacia (PS IM) cis-2-phthalimidocyclohexanol P. cepacia (PS IM)

Int. J. Mol. Sci. 2015, 16, 29682–29716 3.17. Key Intermediates of Iminocyclitols Enantiomerically3.17. Key Intermediates pure of Iminocyclitols1,3-propanediols are important intermediates in the synthesis of iminocyclitolsEnantiomerically (glycosidases pure inhibitors 1,3-propanediols used as are anti-diabetic important intermediates drugs). The in thedesymmetrization synthesis of of carboxybenzyl-2-amino-1,3-propanedioliminocyclitols (glycosidases inhibitors (Cbz-serinol) used as anti-diabetic via acetylation drugs). Thereaction desymmetrization in THF mediated of by crude pigcarboxybenzyl-2-amino-1,3-propanediol pancreatic lipase (PPL, L3126, type (Cbz-serinol) II, Steapsin) via acetylation (i) or purified reaction PPL in THF (L0382, mediated type by VI-S) (ii) crude pig pancreatic lipase (PPL, L3126, type II, Steapsin) (i) or puriﬁed PPL (L0382, type VI-S) was reported [47]. When the reaction was carried out in vinyl acetate as both a solvent and acyl donor, (ii) was reported [47]. When the reaction was carried out in vinyl acetate as both a solvent and acyl in the presencedonor, in of the crude presence PPL of (i), crude only PPL the (i), (2 onlyR)-monoacetate the (2R)-monoacetate was formed was formed in 98% in ee 98%. Onee. the On other the hand, only theother (2S hand,)-monoacetate only the (2S )-monoacetate(100% ee) (100%was eeformed) was formed when when purified puriﬁed PPLPPL (ii) catalyzed(ii) catalyzed the the desymmetrizationdesymmetrization (Scheme (Scheme 28). 28 ).

Scheme Scheme28. Kinetic 28. Kinetic enzymatic enzymatic acylation acylation of of racemic racemic carboxybenzyl-2-amino-1,3-propanediol carboxybenzyl-2-amino-1,3-propanediol (Cbz-serinol) (Cbz- serinol) to produceproduce intermediates intermediates in the in synthesis the synthesis of iminocyclitols of iminocyclitols [47]. [47].

3.18. N-Acetyl3.18. N-Acetyl Phenylalanine Phenylalanine and andAnalogues Analogues Phenylalanine and analogues are important building blocks in the preparation of complex Phenylalaninestructures of interestand analogues to the medicinal are important chemistry area.buildingN-Acetyl blocks phenylalanine in the preparation and a series of of complex structuresanalogues of interest were obtainedto the inmedicinal enantiomerically chemistry pure form area. by interesterificationN-Acetyl phenylalanine reaction in the and presence a series of analoguesof lipasewere fromobtainedRhizomucor in enantiomerically miehei (RML). Ethyl pure acetamidocianoacetate form by interesterification was alkylated reaction in the presence in the of presence of lipasevarious from Rhizomucor alkyl halides inmiehei phase (RML). transfer Ethyl catalysis acetamidocianoacetate (PTC), followed by acidic was hydrolysis, alkylated esterification in the presence and N-acetylation leading to N-acetyl-phenylalanine methyl and allyl esters derivatives. These of various alkyl halides in phase transfer catalysis (PTC), followed by acidic hydrolysis, esterification derivatives were submitted to an enzymatic kinetic resolution via interesterification reaction. After and N-acetylationscreening with leading various to commercial N-acetyl-phenylalanine lipases, it was found methyl that only and RML allyl was esters able to promotederivatives. the These derivativesInt. J. Mol.kinetic Sci. were 2015 resolution , submitted16, page–page (Scheme to 29 an). Severalenzymatic parameters kinetic were resolution studied and via the interesterification best reaction conditions reaction. were After screeningobtained with various in the presence commercial of butyl butyratelipases, asit anwas interesterification found that only agent, RML with anwas enzyme:substrate able to promote the ˝ kineticratio resolutionofratio 2:1 of(w 2:1/w (),(Schemew at/w 30), at °C, 30 29). andC, Several and MeCN MeCN parameters as as a asolvent. solvent. we Mostre studied derivatives derivatives and (S-configuration) the(S-configuration) best reaction were conditions obtainedwere obtained were with >99% ee and E > 200 [48]. obtainedwith >99% in the ee and presence E > 200 of [48]. butyl butyrate as an interesterification agent, with an enzyme:substrate

SchemeScheme 29. Kinetic 29. Kinetic enzymatic enzymatic resolution resolution of of racemic racemic NN-acetyl-acetyl phenylalanine phenylalanin ande analoguesand analogues via via interesterificationinteresteriﬁcation reaction reaction [48]. [48 ].

3.19. (S)-1-(2-Furyl)ethanol3.19. (S)-1-(2-Furyl)ethanol The (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various The (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various natural products such as ﬂavonoids, polyketide antibiotics and carbohydrate derivatives. This naturalintermediate products wassuch obtained as flavon by lipase-catalyzedoids, polyketide kinetic antibiotics resolution ofand racemic carbohydrate 1-(2-furyl)ethanol. derivatives. After This intermediate was obtained by lipase-catalyzed kinetic resolution of racemic 1-(2-furyl)ethanol. After commercial lipases screening in n-heptane as a solvent and 2 h of reaction time, the lipase from 29699 Candida antarctica B (Novozym 435) was the most efficient enzyme, leading to higher conversion (c 47%) than those observed with the other evaluated enzymes, RM IM Lipozyme (c 1.8%) and Lipozyme TL IM (c 1.5%). Various parameters were studied before establishing the best reaction conditions (vinyl acetate as the acyl donor, n-heptane as the solvent, a stirring speed of 300 rpm, 5 mg of enzyme loading at 60 °C). In these conditions, the (S)-1-(2-furyl)ethanol was obtained with c 47% and 89% ee (Scheme 30). The study of the reuse of the biocatalyst was also performed, and it was shown that it can be recycled three times with a small decrease in conversion from 47.0% to 44.5% [49].

Scheme 30. Kinetic enzymatic acylation of the rac-1-(2-furyl)ethanol to produce the (S)-alcohol, an important building block for the synthesis of various natural products [49].

3.20. Ascorbyl Ester Derivatives Ascorbic acid (Asc) is a very important natural antioxidant, but with limited industrial application because of its hydrophilic nature. Alternatively, ascorbyl esters are considered antioxidants to be used in hydrophobic formulations [50]. A series of lipophilic esters derived from ascorbic acid was prepared by using lipase from Staphylococcus xylosus immobilized on silica aerogel. The regioselective enzymatic esterification of Asc with seven carboxylic acids (acetic acid and six fatty acids) was performed in MeCN and 2-methyl-2-propanol (co-solvent) for 72 h. Yields varied with the length of the fatty acid chain, and the best result was obtained on the preparation of the short chain derivative ascorbyl acetate (82.6 % yield), as seen in Scheme 31. All Asc derivatives were submitted to some bioassays (antibacterial, antioxidant, and antileishmanial), and the results suggested them as candidates to be used in the cosmetic and pharmaceutical industries [51].

Scheme 31. Kinetic enzymatic acylation of the ascorbic acid (Asc) to produce ascorbyl esters derivatives [51]. 17 Int. J. Mol. Sci. 2015, 16, page–page ratio of 2:1 (w/w), at 30 °C, and MeCN as a solvent. Most derivatives (S-configuration) were obtained Int. J. Mol. Sci. 2015, 16, page–page with >99% ee and E > 200 [48]. ratio of 2:1 (w/w), at 30 °C, and MeCN as a solvent. Most derivatives (S-configuration) were obtained with >99% ee and E > 200 [48].

Scheme 29. Kinetic enzymatic resolution of racemic N-acetyl phenylalanine and analogues via interesterification reaction [48]. Scheme 29. Kinetic enzymatic resolution of racemic N-acetyl phenylalanine and analogues via 3.19. (S)-1-(2-Furyl)ethanolinteresterification reaction [48].

3.19.The (S)-1-(2-Furyl)ethanol (S)-1-(2-furyl)ethanol is used as an important building block for the synthesis of various natural products such as flavonoids, polyketide antibiotics and carbohydrate derivatives. This intermediateInt.The J. Mol. (S Sci. was)-1-(2-furyl)ethanol2015 obtained, 16, 29682–29716 by lipase-catalyzed is used as an important kinetic resolution building ofblock racemic for the 1-(2-furyl)ethanol. synthesis of various After commercialnatural products lipases suchscreening as flavon in n-heptaneoids, polyketide as a solvent antibiotics and 2and h of carbohydrate reaction time, derivatives. the lipase This from intermediate was obtained by lipase-catalyzed kinetic resolution of racemic 1-(2-furyl)ethanol. After Candidacommercial antarctica lipases B (Novozym screening 435) in wasn-heptane the most as aefficient solvent enzy andme, 2 h leading of reaction to higher time, theconversion lipase from (c 47%) commercial lipases screening in n-heptane as a solvent and 2 h of reaction time, the lipase from than Candidathose observed antarctica withB (Novozym the other 435) evaluated was the enzy mostmes, efﬁcient RM IM enzyme, Lipozyme leading (c 1.8%) to higher and conversion Lipozyme TL Candida antarctica B (Novozym 435) was the most efficient enzyme, leading to higher conversion (c 47%) IM (c(c 1.5%). 47%) thanVarious those parameters observed withwere the studied other before evaluated establishing enzymes, the RM best IM Lipozymereaction conditions (c 1.8%) and (vinyl than those observed with the other evaluated enzymes, RM IM Lipozyme (c 1.8%) and Lipozyme TL acetateLipozyme as the acyl TL donor, IM (c 1.5%).n-heptane Various as the parameters solvent, a werestirring studied speed before of 300 establishing rpm, 5 mg of the enzyme best reaction loading at IM (c 1.5%). Various parameters were studied before establishing the best reaction conditions (vinyl 60 °C).conditions In these (vinylconditions, acetate the as ( theS)-1-(2-furyl)ethanol acyl donor, n-heptane was asobtained the solvent, with ac stirring47% and speed 89% ofee 300(Scheme rpm, 30). acetate5 mg as of the enzyme acyl donor, loading n-heptane at 60 ˝C). as In the these solvent, conditions, a stirring the speed (S)-1-(2-furyl)ethanol of 300 rpm, 5 mg wasof enzyme obtained loading with at The study of the reuse of the biocatalyst was also performed, and it was shown that it can be recycled 60 c°C). 47% In and these 89% conditions,ee (Scheme the 30 ().S)-1-(2-furyl)ethanol The study of the reuse was obtained of the biocatalyst with c 47% was and also 89% performed, ee (Scheme and 30). three times with a small decrease in conversion from 47.0% to 44.5% [49]. Theit wasstudy shown of the that reuse it canof the be biocatalyst recycled three was times also pe withrformed, a small and decrease it was in shown conversion that it from can be 47.0% recycled to three44.5% times [49 ].with a small decrease in conversion from 47.0% to 44.5% [49].

Scheme 30. Kinetic enzymatic acylation of the rac-1-(2-furyl)ethanol to produce the (S)-alcohol, an importantSchemeScheme building30. 30. KineticKinetic block enzymatic enzymatic for the acylationsynthesis acylation of of of thevarious the racrac-1-(2-furyl)ethanol -1-(2-furyl)ethanolnatural products to [49]. to produce produce the the (S (S)-alcohol,)-alcohol, an importantan important building building block block for the for thesynthesis synthesis of various of various natural natural products products [49]. [49]. 3.20. Ascorbyl Ester Derivatives 3.20.3.20. Ascorbyl Ascorbyl Ester Ester Derivatives Derivatives Ascorbic acid (Asc) is a very important natural antioxidant, but with limited industrial applicationAscorbicAscorbic because acid acid (Asc)(Asc)of its is is ahydrophilic verya very important important nature. natural natu antioxidant,Alternatively,ral antioxidant, but with ascorbyl limitedbut with industrialesters limited are application industrialconsidered antioxidantsapplicationbecause ofto because itsbe hydrophilicused ofin hydrophobicits nature.hydrophilic Alternatively, formulations nature. Alternatively, ascorbyl [50]. A estersseries ascorbyl areof lipophilic considered esters esters antioxidantsare derivedconsidered to from be used in hydrophobic formulations [50]. A series of lipophilic esters derived from ascorbic ascorbicantioxidants acid was to beprepared used in byhydrophobic using lipase formulations from Staphylococcus [50]. A series xylosus of lipophilic immobilized esters on derived silica aerogel. from ascorbicacid was acid prepared was prepared by using by lipaseusing fromlipaseStaphylococcus from Staphylococcus xylosus xylosusimmobilized immobilized on silica on aerogel. silica aerogel. The The regioselective enzymatic esterification of Asc with seven carboxylic acids (acetic acid and six fatty Theregioselective regioselective enzymatic enzymatic esteriﬁcation esterification of Ascof Asc with with seven seven carboxylic carboxylic acids acids (acetic (acetic acid acid and and six six fatty fatty acids) was performed in MeCN and 2-methyl-2-propanol (co-solvent) for 72 h. Yields varied with the acids)acids) was was performed performed in in MeCN MeCN and and 2-methyl-2-propanol 2-methyl-2-propanol (co-solvent)(co-solvent) for for 72 72 h. h. Yields Yields varied varied with with the the length of the fatty acid chain, and the best result was obtained on the preparation of the short chain lengthlength of ofthe the fatty fatty acid acid chain, chain, an andd the the best best result result was obtainedobtained onon the the preparation preparation of of the the short short chain chain derivative ascorbyl acetate (82.6 % yield), as seen in Scheme 31. All Asc derivatives were submitted derivativederivative ascorbyl ascorbyl acetate acetate (82.6 (82.6 % % yield), yield), as seen inin SchemeScheme 31 31.. AllAll Asc Asc derivatives derivatives were were submitted submitted to some bioassays (antibacterial, antioxidant, and antileishmanial), and the results suggested them as to tosome some bioassays bioassays (antibacterial, (antibacterial, antioxidant, antioxidant, and antileishmanial), and and the the results results suggested suggested them them as as candidates to be used in the cosmetic and pharmaceutical industries [51]. candidatescandidates to tobe be used used in in the the cosmetic cosmetic and and pharmaceutical pharmaceutical industries [51].[51].

SchemeSchemeScheme 31. 31. Kinetic Kinetic 31. enzymaticKinetic enzymatic enzymatic acylation acylation acylationof of the the ascorbic ascorbic of the acidacid ascorbic (Asc) to acid produce (Asc) ascorbyl ascorbyl to produce esters esters derivatives ascorbyl derivatives esters [51]. [51]. derivatives [51]. 1717

29700 Int. J. Mol. Sci. 2015, 16, page–page

3.21. Key Intermediates of Stagonolide E, Pyrenophorol and Decarestrictine L Enantiomerically pure hept-6-ene-2,5-diol derivatives were prepared by lipase-catalyzed acetylation of the corresponding racemates, and used as key intermediates in the synthesis of the bioactive compounds Stagonolide E, Pyrenophorol and Decarestrictine L (Scheme 32), all of them isolated from filamentous fungi. Racemic 6-methyl-5-hepten-2-ol was resolved by acetylation with Novozym 435, vinyl acetate in hexane at room temperature, and yielded both the (R)-acetate and (S)-alcohol with 98% ee (E > 195) after 50% conversion. The (R)-alcohol was also produced by treatment of (R)-acetate with LiAlHInt. J.4 Mol.. Then, Sci. 2015 both, 16, 29682–29716 the (R)- and (S)-alcohols were submitted, individually, to some chemical steps that included protection of the hydroxyl group with tert-butyldiphenylsilyl (TBDPS), ozonolysiswere and submitted, reaction individually, of the obtained to some aldehy chemicaldes steps with that vinylmagnesium included protection bromide, of the hydroxyl providing the allylic alcoholsgroup with (3RStert,6-butyldiphenylsilylR) and (3RS,6S). (TBDPS), A similar ozonolysis protocol and was reaction used of in the the obtained enzyme-catalyzed aldehydes with acylation of these vinylmagnesiumallylic alcohols bromide, (Scheme providing 32). In this the allyliccase, alcoholsboth key (3 RSintermediates,6R) and (3RS,6 (3S).S,6 AR similar)-acetate protocol and (3R,6S)- alcohol waswere used obtained in the enzyme-catalyzed with high enantioselectivity acylation of these allylic(E > alcohols195), c (Scheme50%. The 32). Inenantiomerically this case, both pure (3R,6S)-alcoholkey intermediates was used (3 asS,6 Ran)-acetate intermediate and (3R,6 inS)-alcohol the synthesis were obtained of Stagonolide with high E. enantioselectivity The chemoenzymatic (E > 195), c 50%. The enantiomerically pure (3R,6S)-alcohol was used as an intermediate in the synthesissynthesis of Pyrenophorol of Stagonolide and E. TheDecarestrictine chemoenzymatic L involved synthesis ofthe Pyrenophorol enantiomericaly and Decarestrictine pure allylic L (3S,6R)- acetate asinvolved key intermediate the enantiomericaly (Scheme pure 32) allylic [52]. (3S ,6R)-acetate as key intermediate (Scheme 32)[52].

Scheme Scheme32. Kinetic 32. Kinetic enzymatic enzymatic acylation acylation of of racrac-hept-6-ene-2,5-diol-hept-6-ene-2,5-diol derivatives derivatives to produce to produce key key intermediatesintermediates in the insynthesis the synthesis of the of the Stagonolide Stagonolide E,E, PyrenophorolPyrenophor andol and Decarestrictine Decarestrictine L [52]. L [52].

3.22. Key3.22. Intermediates Key Intermediates of Macrolide of Macrolide Antibiotic Antibiotic (− (´)-A26771B)-A26771B The chemoenzymatic synthesis of the macrolide antibiotic (´)-A26771B involved the lipase-catalyzed Theacylation chemoenzymatic for resolving synthesis both a methylcarbinolof the macrolide and antibiotic an allylic ( alcohol−)-A26771B in order involved to establish the lipase-catalyzed the two acylationstereogenic for resolving centers both of the a target methylcarbinol molecule. A seriesand ofan commercial allylic alcohol lipases in were order investigated to establish on the the two stereogenicacetylation centers of theof therac-methylcarbinol target molecule. (tridec-12-en-2-ol) A series of with commercial vinyl acetate lipases in hexane were orDIPE investigated at 25 ˝C. on the acetylationThe of best the result rac-methylcarbinol was achieved when (tridec-12-en-2-ol) Novozym 435 (lipase with B vinyl from Candida acetate antarctica in hexaneimmobilized or DIPE at 25 °C. on acrylic resin) was used as the biocatalyst and DIPE as the solvent. This condition yielded The best result was achieved when Novozym 435 (lipase B from Candida antarctica immobilized on (S)-methylcarbinol (96% ee) and the corresponding (R)-acetate (92% ee) after 2 h (c 51%), as seen in acrylic resin)Scheme 33wasa. Theused same as condition the biocatalyst was used for and the acetylation DIPE as of thetherac solvent.-allylic alcohol This (Schemecondition 33b), yielded (S)-methylcarbinolwhich produced (96% (3R ,13ee)R and)-alcohol the (98%correspondingee) and (3S,13 (RR)-acetate)-acetate (95% (92%ee) after ee) 6after h (c 50%)2 h (c [53 51%),]. as seen in Scheme 33a. The same condition was used for the acetylation of the rac-allylic alcohol (Scheme 33b), which produced (3R,13R)-alcohol (98% ee) and (3S,13R)-acetate (95% ee) after 6 h (c 50%) [53].

2970118 Int. J. Mol. Sci. 2015, 16, 29682–29716 Int. J. Mol. Sci. 2015, 16, page–page Int. J. Mol. Sci. 2015, 16, page–page

Scheme 33. Kinetic enzymatic acylation of the (a) racemic tridec-12-en-2-ol and (b) allylic alcohol, Scheme 33. KineticKinetic enzymatic enzymatic acylation acylation of the ( a)) racemic tridec-12-en-2-ol and (b) allylic alcohol, affording key intermediates in the synthesis of the macrolide antibiotic (−)-A26771B [53]. affording keykey intermediatesintermediates inin thethe synthesissynthesis ofof thethe macrolidemacrolide antibioticantibiotic ((´−)-A26771B)-A26771B [53]. [53]. 3.23. Key Intermediate of Vitamin E Acetate 3.23. Key Intermediate of Vitamin E Acetate Lipase-catalyzed regioselective transesterification between trimethylhydroquinone diacetate Lipase-catalyzed regioselective transesterificationtransesterification between trimethylhydroquinone diacetate (TMHQ-DA) and n-butanol was applied on the preparation of the trimethylhydroquinone-1- (TMHQ-DA) and andn -butanoln-butanol was was applied applied on the on preparation the preparation of the trimethylhydroquinone-1-monoacetate of the trimethylhydroquinone-1- monoacetate (TMHQ-1-MA) derivative, which is an important aromatic intermediate used in the (TMHQ-1-MA)monoacetate (TMHQ-1-MA) derivative, which derivative, is an importantwhich is an aromatic important intermediate aromatic intermediate used in the synthesisused in the of synthesis of Vitamin E acetate (commercialized form of Vitamin E), as seen in Scheme 34. Seven Vitaminsynthesis E of acetate Vitamin (commercialized E acetate (commercialized form of Vitamin form E), of as Vitamin seen in E), Scheme as seen 34. in Seven Scheme commercially 34. Seven commercially available lipases were screened, and the highest yield (99.1%) of the TMHQ-1-MA was availablecommercially lipases available were screened,lipases were and screened, the highest and yieldthe highest (99.1%) yield of the(99.1%) TMHQ-1-MA of the TMHQ-1-MA was obtained was obtained using Lipozyme RM IM (lipase from Rhizomucor miehei). No activity was observed when usingobtained Lipozyme using Lipozyme RM IM (lipaseRM IM from (lipaseRhizomucor from Rhizomucor miehei). miehei No activity). No activity was observed was observed when usingwhen using lipase A (from Aspergillus niger) as an enzyme. Except for Lipozyme 435 (CAL-B, lipase B from lipaseusing lipase A (from A (fromAspergillus Aspergillus niger )niger as an) as enzyme. an enzyme. Except Except for for Lipozyme Lipozyme 435 435 (CAL-B, (CAL-B, lipase lipase BB from Candida antarctica immobilized on macroporous polyacrylate resin), all active enzymes showed 100% Candida antarctica immobilized onon macroporousmacroporous polyacrylate resin), all active enzymes showed 100% regioselectivity on the formation of TMHQ-1-MA. Several reaction parameters were investigated and regioselectivity on the formation of TMHQ-1-MA. SeveralSeveral reaction parameters were investigated and the optimum conditions for the Lipozyme RM IM catalyzed regioselective transesterification were: the optimumoptimum conditionsconditions forfor thethe LipozymeLipozyme RMRM IMIM catalyzedcatalyzed regioselectiveregioselective transesterificationtransesterification were:were: TMHQ-DA:n-butanol ratio of 1:1, 200 rpm, 50 °C,˝ and TBME:n-hexane (3:7) as a solvent. In such TMHQ-DA:nn-butanol-butanol ratio ratio of of 1:1, 1:1, 200 200 rpm, rpm, 50 50 °C,C, and and TBME: TBME:n-hexane-hexane (3:7) as aa solvent.solvent. In such conditions, the enzyme was active even after 20 cycles. A Ping-Pong bi-bi mechanism with n-butanol conditions, the enzyme was active even after 20 cycles. A Ping-Pong bi-bi mechanism with n-butanol inhibition was proposed based on the initial rate data and concentration profiles [54]. inhibition waswas proposedproposed basedbased onon thethe initialinitial raterate datadata andand concentrationconcentration profilesprofiles [[54].54].

Scheme 34. Lipase-catalyzed regioselective transesterification between TMHQ-DA and n-butanol on Scheme 34. Lipase-catalyzed regioselective transesterification between TMHQ-DA and n-butanol on Schemethe preparation 34. Lipase-catalyzed of TMHQ-1-MA, regioselective an intermediate transesteriﬁcation in the synthesis between of Vitamin TMHQ-DA E acetate and [54].n-butanol on the preparation ofof TMHQ-1-MA,TMHQ-1-MA, anan intermediateintermediate inin thethe synthesissynthesis ofof VitaminVitamin EE acetateacetate [[54].54]. 3.24. Rasagiline Mesylate 3.24. Rasagiline Mesylate 3.24. Rasagiline Mesylate A straightforward chemoenzymatic synthesis of Rasagiline mesylate has been developed A straightforward chemoenzymatic synthesis of Rasagiline mesylate has been developed (SchemeA straightforward 35). This drug is chemoenzymatic used in monotherapy synthesis of Parkinson of Rasagiline patients mesylate at an early has beenstage, developedand as an (Scheme 35). This drug is used in monotherapy of Parkinson patients at an early stage, and as an (Schemeadjunct to35 moderate). This drug the advanced is used in stage monotherapy of the disease. of Parkinson The synthesis patients began at an with early the stage, chemical and adjunct to moderate the advanced stage of the disease. The synthesis began with the chemical asreduction an adjunct of indanone to moderate to give the rac-indanol advanced in 86% stage yield. of the Then, disease. rac-indanol The was synthesis subjected began to enzymatic with the reduction of indanone to give rac-indanol in 86% yield. Then, rac-indanol was subjected to enzymatic chemicalkinetic resolution reduction using of indanonethe lipase from to give Thermomycesrac-indanol lanuginosus in 86% yield. (TLL) Then,immobilizedrac-indanol on immobead-150 was subjected as kinetic resolution using the lipase from Thermomyces lanuginosus (TLL) immobilized on immobead-150 as toa biocatalyst, enzymatic hexane kinetic resolutionas an organic using solvent, the lipase vinyl fromacetateThermomyces as an acyl donor, lanuginosus at 35 (TLL)°C and immobilized with 15 min onof a biocatalyst, hexane as an organic solvent, vinyl acetate as an acyl donor, at 35 °C and with 15 min of immobead-150reaction time. In as this a biocatalyst, case, (R)-indanyl hexane acetate as an organicand (S)-indanol solvent, vinylwere obtained acetate as with an acyl >99% donor, ee, c 50% at 35 and˝C reaction time. In this case, (R)-indanyl acetate and (S)-indanol were obtained with >99% ee, c 50% and andE > with200. 15Then, min the ofreaction (S)-indanol time. was In this subjected case, (R )-indanylto a sequence acetate of andchemical (S)-indanol steps, were which obtained included with a E > 200. Then, the (S)-indanol was subjected to a sequence of chemical steps, which included a Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, finally, the Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, finally, the reaction with methanesulfonic acid, which afforded Rasagiline mesylate. Immobilized lipase from reaction with methanesulfonic acid, which afforded29702 Rasagiline mesylate. Immobilized lipase from 19 19 Int. J. Mol. Sci. 2015, 16, 29682–29716

>99% ee, c 50% and E > 200. Then, the (S)-indanol was subjected to a sequence of chemical steps, which included a Mitsunobu reaction, a Staudinger reaction, the introduction of the propargyl group and, Int. J. Mol. Sci. 2015, 16, page–page ﬁnally,Int. J. Mol. the Sci. reaction2015, 16, page–page with methanesulfonic acid, which afforded Rasagiline mesylate. Immobilized lipaseT. lanuginosus from T. lanuginosus was foundwas to found be an to beefficient an efﬁcient biocatalyst biocatalyst to produce to produce (S ()-indanolS)-indanol with with high ˝ enantioselectivityT. lanuginosus was (>99% found eeee,, EE to>> 200)be 200) inan in hexane,efficient hexane, at at35biocatalyst 35°C andC and 15 to min. 15 produce min. Additionally, Additionally, (S)-indanol this enzyme thiswith enzyme highwas wasenantioselectivityreused reused 10 times, 10 times, (>99%maintaining maintaining ee, E > 200)both both inthe hexane, the activity activity at 35and and °C selectivity and selectivity 15 min. unchanged. unchanged. Additionally, This This this preparation preparation enzyme was of Rasagilinereused 10 mesylatetimes, maintaining can be considered both the an activityenvironmen environmentally andtally selectivity sustainable unchanged. strategy, This since preparation it involved theof useRasagiline of a biocatalyst mesylate can that be is considered commercially an environmen available, low-cost,tally sustainable stable, reusable strategy, for since multiple it involved reaction the cyclesuse of anda biocatalyst highly enantioselectiveenan thattioselective is commercially [[55,56].55,56]. available, low-cost, stable, reusable for multiple reaction cycles and highly enantioselective [55,56].

Scheme 35. Enzymatic kinetic resolution rac-indanol in the chemoenzymatic synthesis of Rasagiline Scheme 35. Enzymatic kinetic resolution rac-indanol in the chemoenzymatic synthesis of Rasagiline mesylate [[55,56].55,56]. mesylate [55,56].

Scheme 36. Enzymatic kinetic resolution of rac-N-Boc-HMTHQ to prepare the (R)- and (S)-3-methyl- Scheme 36. Enzymatic kinetic resolution of rac-N-Boc-HMTHQ to prepare the (R)- and (S)-3-methyl- Scheme1,2,3,4-tetrahydroquinoline, 36. Enzymatic kinetic chir resolutional intermediates of rac-N -Boc-HMTHQfor the preparation to prepare of the the antithrombotic (R)- and (S)-3-methyl-1, (21R)- and 1,2,3,4-tetrahydroquinoline, chiral intermediates for the preparation of the antithrombotic (21R)- and 2,3,4-tetrahydroquinoline,(21S)-Argatroban [57]. chiral intermediates for the preparation of the antithrombotic (21R)- and (21S)-Argatroban [57]. (21S)-Argatroban [57]. 3.25. Argatroban 3.25. Argatroban 3.25.Argatroban Argatroban is an inhibitor of thrombim, the protease that plays a key role in blood coagulation Argatroban is an inhibitor of thrombim, the protease that plays a key role in blood coagulation and fibrinolysis.Argatroban isThe an diastereoisomeric inhibitor of thrombim, mixture the of protease epimers that ((21 playsR)- and a key (21 roleS)-epimers) in blood is coagulation used as an and fibrinolysis. The diastereoisomeric mixture of epimers ((21R)- and (21S)-epimers) is used as an andantithrombotic ﬁbrinolysis. drug, The diastereoisomericbut the (21S)-isomer mixture is twice of as epimers potent as ((21 21RR)-. The and chiral (21S)-epimers) intermediates is used (R)- as and an antithrombotic drug, but the (21S)-isomer is twice as potent as 21R. The chiral intermediates (R)- and antithrombotic(S)-3-methyl-1,2,3,4-tetrahydroquinoline drug, but the (21S)-isomer are used is twice for the as potentpreparation as 21 Rof. the The antithrombotic chiral intermediates (21R)- (S)-3-methyl-1,2,3,4-tetrahydroquinoline are used for the preparation of the antithrombotic (21R)- (andR)- and(21S ()-Argatroban.S)-3-methyl-1,2,3,4-tetrahydroquinoline These intermediates were are prepar useded for using the preparation3-quinoline ofcarboxylic the antithrombotic acid as a and (21S)-Argatroban. These intermediates were prepared using 3-quinoline carboxylic acid as a (21startingR)- and material. (21S)-Argatroban. After some These chemical intermediates steps, the were rac-3-(1’-hydroxymethyl)-1- prepared using 3-quinolinetert-butyloxycarbonyl- carboxylic acid as a starting material. After some chemical steps, the rac-3-(1’-hydroxymethyl)-1-tert-butyloxycarbonyl- starting1,2,3,4-tetrahydroquinoline material. After some (rac chemical-N-Boc-HMTHQ) steps, the rac was-3-(1’-hydroxymethyl)-1- obtained, and subjectedtert to-butyloxycarbonyl-1, kinetic resolution in 1,2,3,4-tetrahydroquinoline (rac-N-Boc-HMTHQ) was obtained, and subjected to kinetic resolution in 2,3,4-tetrahydroquinolinethe presence of lipase from (rac Pseudomonas-N-Boc-HMTHQ) fluorescens was (PFL), obtained, with andtoluene subjected as a solvent to kinetic and resolutionvinyl acetate in the presence of lipase from Pseudomonas fluorescens (PFL), with toluene as a solvent and vinyl acetate theas an presence acyl donor. of lipase After fromthis procedure,Pseudomonas the ﬂuorescens (S)-ester (>99%(PFL), ee with for c toluene 30%–40%) as aand solvent the (R and)-alcohol vinyl (81% acetate ee as an acyl donor. After this procedure, the (S)-ester (>99% ee for c 30%–40%) and the (R)-alcohol (81% ee asfor anc 55–65%) acyl donor. were After obtained. this procedure, For obtaining the (theS)-ester enantiomerically (>99% ee for enriched c 30%–40%) (R)-alcohol and the (>99% (R)-alcohol ee), a for c 55–65%) were obtained. For obtaining the enantiomerically enriched (R)-alcohol (>99% ee), a (81%secondee kineticfor c resolution 55–65%) were was performed obtained. unde For obtainingr the aforementioned the enantiomerically conditions enrichedusing the (R)-alcohol second kinetic resolution was performed under the aforementioned conditions using the (R)-alcohol (>99%(81% eeee). ),In athis second case, kineticthe reaction resolution was stopped was performed at a conversion under ranging the aforementioned from 30% to 45% conditions (Scheme using 36). (81% ee). In this case, the reaction was stopped at a conversion ranging from 30% to 45% (Scheme 36). theThe ((RS)-alcohol)-acetate (>99% (81% ee ee).) was In this converted case, the into reaction the corresponding was stopped (S at)-alcohol a conversion by enzymatic ranging hydrolysis from 30% Thein the (S )-acetatepresence (>99% of PFL. ee) Subsequently,was converted bothinto the(S)- corresponding and (R)-alcohol (S )-alcoholwere subjected by enzymatic to a sequence hydrolysis of inchemical the presence steps which of PFL. included Subsequently, tosylation, both reaction (S)- and with (R lithium)-alcohol aluminum were subjected hydride to and a sequence removal of 29703 chemicalthe Boc protecting steps which group, included leading tosylation, to enantiomerically reaction with lithiumpure intermediates aluminum hydride (S)- and and (R )-3-methyl-removal of the1,2,3,4-tetrahydroquinoline Boc protecting group, leading[57]. to enantiomerically pure intermediates (S)- and (R)-3-methyl- 1,2,3,4-tetrahydroquinoline [57].

20 20 Int. J. Mol. Sci. 2015, 16, 29682–29716 to 45% (Scheme 36). The (S)-acetate (>99% ee) was converted into the corresponding (S)-alcohol by enzymatic hydrolysis in the presence of PFL. Subsequently, both (S)- and (R)-alcohol were subjected to a sequence of chemical steps which included tosylation, reaction with lithium aluminum hydride and removal of the Boc protecting group, leading to enantiomerically pure intermediates (S)- and (Int.R)-3-methyl-1,2,3,4-tetrahydroquinoline J. Mol. Sci. 2015, 16, page–page [57].

3.26. Key Intermediate of the Paclitaxel Side Chain The synthesis synthesis of of a akey key intermediate intermediate for for the the Paclitaxel Paclitaxel side side chain, chain, (2R,3 (2S)-3-phenylisoserine,R,3S)-3-phenylisoserine, was wasperformed performed by kinetic by kinetic enzymatic enzymatic resolution resolution of of racemic racemic NN-hydroxymethylated-hydroxymethylated cis-3-acetoxy-4- phenylazetidin-2-one ((racrac-azetidin-2-one-azetidin-2-one derivative),derivative), viavia acylationacylation reactionreaction (Scheme(Scheme 3737).). Various parameters were evaluatedevaluated such as lipases from Burkholderia cepacia (PS IM), Candida antarctica A (CAL-A), Pseudomonas ﬂuorescensfluorescens (lipase(lipase AK) and C. rugosa (lipase AY); acylacyl donorsdonors suchsuch as vinyl ˝ acetate, vinyl butyrate and 2,2,2-triﬂuoroethyl2,2,2-trifluoroethyl butyrate;butyrate; temperature (4,(4, 25 and 50 °C)C) and organic solvent (DIPE, (DIPE, toluene, toluene, TBME TBME and and 2-MeTHF). 2-MeTHF). The The best best results results were were obtained obtained in the in presence the presence of lipase of ˝ lipasePS IM, PSDIPE IM, as DIPEa solvent, as a vinyl solvent, butyrate vinyl as butyrate an acyl donor, as an at acyl 25 °C. donor, The reaction at 25 C. was The conducted reaction with was conducteda higher amount with a of higher substrate amount (100 of mg) substrate and, after (100 20 mg) min, and, the after conversion 20 min, thereached conversion 50%, with reached the 50%,formation with of the the formation diester (3 ofR,4 theS) with diester >99% (3R ee,4 Sand) with the >99%remainingee and substrate the remaining (3S,4R) substratewith 98% (3eeS, ,4andR) withE > 200, 98% withee, traces and E of> a 200, byproduct with traces from of the a migration byproduct of from the acyl the migrationgroup. Both of the the (3 acylR,4S group.)-diester Both and the (3remainingR,4S)-diester (3 andS,4R the)-substrate remaining were (3S ,4Rsubj)-substrateected to were acid subjected hydrolysis to acid leading hydrolysis to leading(2R,3S)-3- to (2phenylisoserineR,3S)-3-phenylisoserine hydrochloride hydrochloride with >99% with ee >99%(key eeintermediate(key intermediate for the for Paclitaxel the Paclitaxel side chain) side chain) and and(2S,3 (2RS)-3-phenylisoserine,3R)-3-phenylisoserine hydrochloride hydrochloride with with 98% 98%ee [58].ee [ 58].

Scheme 37. Enzymatic kinetic resolution of racemic N-hydroxymethylated cis-3-acetoxy-4- Scheme 37. Enzymatic kinetic resolution of racemic N-hydroxymethylated cis-3-acetoxy-4- phenylazetidin-2-one (rac-azetidin-2-one derivative), affording the key intermediate for the Paclitaxel phenylazetidin-2-one (rac-azetidin-2-one derivative), affording the key intermediate for the Paclitaxel side chain, (2R,3S)-3-phenylisoserine [58]. side chain, (2R,3S)-3-phenylisoserine [58].

3.27. Piperidine Derivative 3.27. Piperidine Derivative The chiral compound (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine is a novel triple- The chiral compound (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine is a novel reuptake inhibitor (inhibiting the reuptake of serotonin, norepinephrine and dopamine), which was triple-reuptake inhibitor (inhibiting the reuptake of serotonin, norepinephrine and dopamine), created as a candidate for a novel antidepressant agent. Among the strategies to synthesize the which was created as a candidate for a novel antidepressant agent. Among the strategies to synthesize aforementioned compound, it is possible to highlight the enzymatic kinetic resolution of racemic tert- the aforementioned compound, it is possible to highlight the enzymatic kinetic resolution of butyl 4-(1-(3,4-dichlorophenyl)-2-hydroxyethyl)piperidine-1-carboxylate (rac-alcohol) via acetylation racemic tert-butyl 4-(1-(3,4-dichlorophenyl)-2-hydroxyethyl)piperidine-1-carboxylate (rac-alcohol) in the presence of lipases [59]. A preliminary enzyme screening was conducted using 38 lipases, via acetylation in the presence of lipases [59]. A preliminary enzyme screening was conducted using wherein the lipase from Pseudomonas sp. immobilized on diatomite (Amano Enzyme Inc.) gave the 38 lipases, wherein the lipase from Pseudomonas sp. immobilized on diatomite (Amano Enzyme Inc.) best E-value (E 243). Several solvents were evaluated such as DIPE, THF, acetone, MeCN, DMF and TBME. In this case, DIPE showed the best performance leading to an E-value of 525. Thus, the kinetic resolution was performed in the optimized conditions29704 using lipase PS IM (from Pseudomonas sp.), vinyl acetate as an acyl donor and DIPE as a solvent, at 35 °C. After the reaction reached 48% conversion, the product (S)-ester was obtained with 96% ee and (R)-alcohol with >99% ee, and E > 200 (Scheme 38). Then, after some chemical steps, the (S)-ester was converted into the triple-reuptake inhibitor (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine with 96% ee. It is worth mentioning that the reaction was performed on a 10 g scale, and after 40 h it was necessary to add

21 Int. J. Mol. Sci. 2015, 16, 29682–29716 gave the best E-value (E 243). Several solvents were evaluated such as DIPE, THF, acetone, MeCN, DMF and TBME. In this case, DIPE showed the best performance leading to an E-value of 525. Thus, the kinetic resolution was performed in the optimized conditions using lipase PS IM (from Pseudomonas sp.), vinyl acetate as an acyl donor and DIPE as a solvent, at 35 ˝C. After the reaction reached 48% conversion, the product (S)-ester was obtained with 96% ee and (R)-alcohol with >99% ee, and E > 200 (Scheme 38). Then, after some chemical steps, the (S)-ester was converted into the triple-reuptake inhibitor (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine with 96% ee. Int. J. Mol. Sci. 2015, 16, page–page It is worth mentioning that the reaction was performed on a 10 g scale, and after 40 h it was necessary toadditional add additional lipase lipaseand vinyl and vinylacetate. acetate. After After over over 24 h 24 of h ofagitation, agitation, the the kinetic kinetic resolution resolution gave gave an excellent enantioselectivity.enantioselectivity.

Scheme 38.38. EnzymaticEnzymatic kinetic kinetic resolution resolution of of racemic racemic terttert-butyl-butyl 4-(1-(3,4-dichlorophenyl)-2- hydroxyethyl)piperidine-1-carboxylate ( racrac-alcohol),-alcohol), affording affording the the novel novel triple triple reuptake reuptake inhibitor inhibitor (S)- (4-(1-(3,4-dichlorophenyl)-2-mS)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidineethoxyethyl)piperidine [59]. [59 ].

3.28. Tetrahydroquinolinol and Tetrahydrobenzoazepinol Derivatives 3.28. Tetrahydroquinolinol and Tetrahydrobenzoazepinol Derivatives Tetrahydroquinolin-4-ols and tetrahydro-1H-benzo[b]azepin-5-ols, in chiral form, are building Tetrahydroquinolin-4-ols and tetrahydro-1H-benzo[b]azepin-5-ols, in chiral form, are building blocks for drug candidates due to their promising biological activities. A series of racemic blocks for drug candidates due to their promising biological activities. A series of racemic N-protected N-protected tetrahydroquinolin-4-ols were chemically prepared from 1,2,3,4-tetrahydroquinoline. tetrahydroquinolin-4-ols were chemically prepared from 1,2,3,4-tetrahydroquinoline. Subsequently, Subsequently, the rac-N-Boc-tetrahydroquinolin-4-ol (R1 = H; R2 = Boc) was subjected to kinetic the rac-N-Boc-tetrahydroquinolin-4-ol (R1 = H; R2 = Boc) was subjected to kinetic resolution in the resolution in the presence of lipases, using TBME as a solvent and vinyl acetate as an acyl donor, at presence of lipases, using TBME as a solvent and vinyl acetate as an acyl donor, at 30 ˝C and for 30 °C and for 8 h (n = 1, Scheme 39 and Table 4). Among the evaluated lipases (Amano lipase Type VII, 8 h (n = 1, Scheme 39 and Table4). Among the evaluated lipases (Amano lipase Type VII, CAL-A, CAL-A, Amano lipase A, Novozym 435, lipase RM IM and lipase TL IM), Novozym 435 lipase Amano lipase A, Novozym 435, lipase RM IM and lipase TL IM), Novozym 435 lipase provided provided the highest enantioselectivity for the (S)-alcohol (98% ee) and the (R)-acetate (97% ee), c 50% the highest enantioselectivity for the (S)-alcohol (98% ee) and the (R)-acetate (97% ee), c 50% and and E > 200. Then, under the same conditions, various acyl donors were tested (vinyl chloroacetate, E > 200. Then, under the same conditions, various acyl donors were tested (vinyl chloroacetate, divinyl heptanedioate, vinyl hexanoate, vinyl undecanoate and vinyl 2,2-dimethylpropionate). In this divinyl heptanedioate, vinyl hexanoate, vinyl undecanoate and vinyl 2,2-dimethylpropionate). In this case, vinyl chloroacetate was the most effective acyl donor, producing both (R)-ester and (S)-alcohol case, vinyl chloroacetate was the most effective acyl donor, producing both (R)-ester and (S)-alcohol with >99% ee, c 50% and E > 200. Besides TBME, other solvents (DIPE, MeCN, toluene and n-hexane) with >99% ee, c 50% and E > 200. Besides TBME, other solvents (DIPE, MeCN, toluene and n-hexane) were evaluated, but TBME proved to be more efficient in the kinetic resolution. After optimizing the were evaluated, but TBME proved to be more efﬁcient in the kinetic resolution. After optimizing the reaction conditions (Novozym 435, TBME as a solvent, vinyl chloroacetate as an acyl donor, 30 °C, 8 h), reaction conditions (Novozym 435, TBME as a solvent, vinyl chloroacetate as an acyl donor, 30 ˝C, the kinetic resolution was extended to the other racemic N-protected tetrahydroquinolin-4-ols 8 h), the kinetic resolution was extended to the other racemic N-protected tetrahydroquinolin-4-ols containing substituent groups at the aromatic ring (C-6) and different N-protecting groups (Table 4). containing substituent groups at the aromatic ring (C-6) and different N-protecting groups (Table4). In general, the kinetic resolutions provided the (R)-esters ranging from 37% to 49% yields, 92% to In general, the kinetic resolutions provided the (R)-esters ranging from 37% to 49% yields, 92% to >99% ee, and the (S)-alcohols ranging from 29% to 48% yields, 94% to >99% ee, with E-values ranging >99% ee, and the (S)-alcohols ranging from 29% to 48% yields, 94% to >99% ee, with E-values ranging from 126 to >200. It is worth noting that the formation of any desired products was not observed when the substrate had the benzyl group linked to the nitrogen atom (n = 1; R1 = H; R2 = Bn), as seen in Scheme 39 and Table 4. In this same study, th29705e authors conducted the kinetic resolution of racemic N-protected tetrahydro-1H-benzo[b]azepin-5-ols (R = Bz, 2-furoyl and Cbz), (n = 2, Scheme 39 and Table 4). As result, the kinetic resolutions produced the corresponding (R)-esters and (S)-alcohols in excellent yields (up to 44%) and enantioselectivity (ee up to 98% and E > 200) [60].

22 Int. J. Mol. Sci. 2015, 16, 29682–29716 from 126 to >200. It is worth noting that the formation of any desired products was not observed when the substrate had the benzyl group linked to the nitrogen atom (n = 1; R1 = H; R2 = Bn), as seen in Scheme 39 and Table4. In this same study, the authors conducted the kinetic resolution of racemic N-protected tetrahydro-1H-benzo[b]azepin-5-ols (R = Bz, 2-furoyl and Cbz), (n = 2, Scheme 39 and Table4). As result, the kinetic resolutions produced the corresponding ( R)-esters and (S)-alcohols in excellentInt. J. Mol. Sci. yields 2015, (up16, page–page to 44%) and enantioselectivity (ee up to 98% and E > 200) [60].

Scheme 39.39. Enzymatickinetic kinetic resolution resolution of of the the racemic racemic compounds compoundsN-protected N-protected tetrahydroquinolin-4-ols tetrahydroquinolin- (4-olsn = 1)(n and= 1) Nand-protected N-protected tetrahydro-1 tetrahydro-1H-benzo[H-benzo[b]azepin-5-olsb]azepin-5-ols (n = ( 2)n = to 2) produce to produce building building blocks blocks for drugfor drug candidates candidates [60]. [60].

Table 4.4. Substrates used for the enzymatic kinetic re resolutionsolution to produce tetrahydroquinolinoltetrahydroquinolinol and tetrahydrobenzoazepinol derivativesderivatives [[60].60].

Substrates n R1 R2 Substrates n R1 R2 H Boc H Boc ClCl BocBoc BrBr BocBoc rac-N-protect tetrahydroquinolin-4-ols 1 rac-N-protect tetrahydroquinolin-4-ols 1 OMe Ac OMe Ac H CO2Ph HH CO Cbz2Ph HH Cbz Bz rac-N-protect tetrahydro-1H-benzo[b]azepin-5-ols 2 H 2-furoyl HH CbzBz rac-N-protect tetrahydro-1H-benzo[b]azepin-5-ols 2 H 2-furoyl 3.29. Aminohydroxypiperidine Derivatives H Cbz

3.29. TheAminohydroxypiperidinetrans-3-amino-4-hydroxypiperidine Derivatives moiety is present in the structures of some bioactive compounds, such as an inhibitor of non-receptor tyrosine kinase (Scheme 40) that is used for the treatment of auto-immuneThe trans-3-amino-4-hydroxypiperidine disorders. Enantiopure, orthogonally moiety protectedis present (3inR ,4theR)-3-amino-4-hydroxypiperidine structures of some bioactive acetatecompounds, was obtainedsuch as an by inhibitor lipase-mediated of non-receptor kinetic tyrosine acylation kinase of the (Scheme corresponding 40) that racemate.is used for Thethe racemictreatmenttrans of -Nauto-immune-benzyl-3-(diallylamino)-4-hydroxypiperidine disorders. Enantiopure, orthogonally (trans- hydroxypiperidineprotected (3R,4R)-3-amino-4- derivative) washydroxypiperidine obtained by the acetate regioselective was obtained opening by lipase- of themediated epoxide at kinetic the rac acylation-1-benzyl-3,4-epoxypiperidine of the corresponding (racemate.rac-epoxide) The with racemic diallylamine. trans-N-benzyl-3-(diallylamino)-4-hydroxypiperidine Later, it was subjected to the enzymatic transesterification (trans- hydroxypiperidine reaction withderivative) vinyl acetatewas obtained (5 eq.) asby the the acyl regioselective donor, lipases opening from Candida of the antarcticaepoxide typeat the A (lipaserac-1-benzyl-3,4- NZL-101) andepoxypiperidineC. antarctica (Brac (Novozym-epoxide) 435),with Burkholderiadiallylamine. cepacia Later,(PSL it was IM), subjectedPseudomonas to fluorescensthe enzymatic(AK) andtransesterificationThermomyces lanuginosusreaction with(TL vinyl IM), acetate in TBME (5 eq.) as as the the solvent, acyl donor, and atlipases 30 ˝C. from Although Candida all antarctica lipases catalyzedtype A (lipase the NZL-101) acylation ofand the C. antarctica substrate B with (Novozym the same 435), stereochemical Burkholderia cepacia preference, (PSL IM), (3R ,4PseudomonasR)-product andfluorescens (3S,4S (AK))-substrate, and Thermomyces the conversions lanuginosus were moderate(TL IM), in (11%–31%). TBME as the Thus, solvent, the and influence at 30 °C. of Although different reactionall lipases parameters catalyzed the (organic acylation solvent, of the temperature, substrate with and the triethylamine same stereochemical as additive) preference, was studied (3R,4R in)- orderproduct to improveand (3S,4 theS)-substrate, reaction rate the using conversions the lipases were CAL-A modera andte CAL-B. (11%–31%). The best Thus, result the was influence obtained of different reaction parameters (organic solvent, temperature, and triethylamine as additive)˝ was by using CAL-B, TBME/Et3N (10:1) as the solvent, three days of reaction time, and at 45 C. In this case,studied the in kineticorder to resolution improve the yielded reaction the rate (3R using,4R)-product the lipases and CAL-A (3S,4S and)-substrate CAL-B. withThe best enantiomeric result was excessobtained of by >99% using and CAL-B, 81%, respectively,TBME/Et3N (10:1) c 45% as and the Esolvent,> 200 three (Scheme days 40 of). reaction It should time, be mentionedand at 45 °C. that In this case, the kinetic resolution yielded the (3R,4R)-product and (3S,4S)-substrate with enantiomeric excess of >99% and 81%, respectively, c 45% and E > 200 (Scheme 40). It should be mentioned that 29706 when the reaction was performed without triethylamine as an additive, a significant decrease in the reaction rate was observed (from three to seven days) [61].

23 Int. J. Mol. Sci. 2015, 16, 29682–29716 when the reaction was performed without triethylamine as an additive, a signiﬁcant decrease in the

Int.reaction J. Mol. Sci. rate 2015 was, 16 observed, page–page (from three to seven days) [61].

Scheme 40. EnzymaticEnzymatic kinetic kinetic resolution resolution of of the the racemic racemic transtrans-hydroxypiperidine-hydroxypiperidine derivative derivative to produce the orthogonally protected (3R,4R)-3-amino-4-hydroxypiperidine, which is present in the structure of an inhibitor of non-receptornon-receptor tyrosinetyrosine kinasekinase [[61].61].

3.30. Azole Derivatives The azole subunit is part ofof thethe structurestructure of of a a nu numbermber of of drugs drugs such such as as Econazole, Econazole, Fl Fluconazole,uconazole, Ketoconalole, Miconazole, Voriconazole andand Ravuconazole.Ravuconazole. It is noteworthy that cycloalkyl azoles have been described as potent antileishmanial agents. A new family of racemic trans- and cis-azole derivatives was prepared and submitted to a kinetic resolution through transesterification transesterification in the presence of of lipases lipases (Scheme (Scheme 41). 41). The The kineti kineticc resolution resolution was was conducted conducted with with the racemic the racemic trans-trans3-(1-3-H- imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol(1H-imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol (rac-trans (rac-trans-3-imidazol-3-imidazol THN) THN) in the in presence the presence of vinyl of acetatevinyl acetate as the asacyl the donor acyl donorand THF and as THF the as solven the solventt (Scheme (Scheme 41a and 41a Table and Table5). Several5). Several lipases lipases were evaluated,were evaluated, such as such lipases as lipasesfrom Candida from Candidaantarcticaantarctica B (CAL-B,B immobilized (CAL-B, immobilized by adsorption by adsorptionin Lewatit E), in fromLewatit C. E),antarctica from C. A antarctica (CAL-A),A (CAL-A),from C. rugosa from C. (CRL), rugosa lipase(CRL), AK lipase from AK Pseudomonas from Pseudomonas fluorescens fluorescens, from, P.from cepaciaP. cepacia (PSL),(PSL), from from RhizomucorRhizomucor miehei miehei (RML),(RML), porcine porcine pancreatic pancreatic lipase lipase (PPL), (PPL), and and from Thermomyces lanuginosus (TLL). Among them, only PSL produced an appreciable degree of activity with good good selectivity. selectivity. Subsequently, Subsequently, several several commercially commercially available available types types of PSL of PSL were were evaluated, evaluated, and PSL-Cand PSL-C I (immobilized I (immobilized onto a ceramic onto a carrier) ceramic was carrier) the mo wasst efficient the most (with efficient a ratio of (with 1:1 lipase/substrate a ratio of 1:1 inlipase/substrate weight), producing in weight), (after 48 producing h at 30 °C) (after trans 48-(2 hR,3 atR)-acetate 30 ˝C) trans in 95%-(2R ee,3 Rand)-acetate trans-(2 inS 95%,3S)-alcoholee and withtrans -(283%S,3 eeS)-alcohol, c 46%, E-value with 83% of 102.ee, cPSL-C 46%, EI -valuewas also of the 102. most PSL-C efficient I was in also the thekinetic most resolution efficient inof the ciskinetic-isomer. resolution After 24 of h the at cis45-isomer. °C, using After the ratio 24 h at2:1 45 of˝ C,lipase:substrate using the ratio in 2:1 weight, of lipase:substrate the cis-(2R,3 inS)-acetate weight, (95%the cis ee-(2) andR,3S cis)-acetate-(2S,3R)-alcohol (95% ee) (71% and cisee)-(2 wereS,3R obtained)-alcohol with (71% c ee43%) were and obtainedan E-value with of 83 c (Scheme 43% and 41a an andE-value Table of 835). (Scheme The strategy 41a and Tableused 5).to The prepare strategy the used chiral to prepare imidazole the chiral derivatives imidazole was derivatives used to enantioselectivelywas used to enantioselectively obtain the trans obtain- and the cis-triazoltrans- and compoundscis-triazol (Scheme compounds 41b and (Scheme Table 415).b Again, and Table PSL-C5). IAgain, was the PSL-C most efficient I was the enzyme most efficientto promot enzymee the kinetic to promote resolution the of kinetic both racemic resolution trans of- and both cis racemic-3-(1H- 1,2,4-triazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-olstrans- and cis-3-(1H-1,2,4-triazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols (rac-trans- and (racrac--cistrans-3-triazol- and rac THN).-cis-3-triazol The experimentsTHN). The experiments were performed were performedusing THF usingas the THF solvent as the and solvent vinyl andacetate vinyl as acetatethe acyl as donor, the acyl at donor, 30 °C andat 30 48˝ Ch. andThe 48reaction h. The with reaction the rac with-trans the- isomerrac-trans provided- isomer the provided trans-(2R the,3Rtrans)-acetate-(2R,3 withR)-acetate 99% ee withand trans99% -(2ee Sand,3S)-alcoholtrans-(2S ,3withS)-alcohol 93% ee, withc 48% 93% andee E, > c 200. 48% In and theE case> 200. of the In rac the-cis case-isomer, of the it racwas-cis obtained-isomer, fromit was the obtained cis-(2R,3 fromS)-acetate the ciswith-(2R >99%,3S)-acetate ee and withcis-(2 >99%S,3R)-alcoholee and withcis-(2 S29%,3R )-alcoholee, c 23% withand E 29% > 200ee, (Scheme 41b and Table 5) [62]. Tetrahydronaphthylazoles are known to exhibit antileishmanial properties, which has inspired the authors [62] to resolve the racemate of trans- and cis-1-(1H- imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols29707 (rac-trans- and rac-cis-1-imidazol THN) using the same aforementioned enzymatic methodology. The kinetic resolutions of these isomers were conducted in THF as a solvent and vinyl acetate as an acyl donor, at 30 °C and for 48 h. The lipases CAL-B and PSL-C I were tested and the latter led to the best results. Thus, for the rac-trans-isomer, 24 Int. J. Mol. Sci. 2015, 16, 29682–29716 c 23% and E > 200 (Scheme 41b and Table5)[62]. Tetrahydronaphthylazoles are known to exhibit antileishmanial properties, which has inspired the authors [62] to resolve the racemate of trans- and cis-1-(1H-imidazol-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ols (rac-trans- and rac-cis-1-imidazol THN) using the same aforementioned enzymatic methodology. The kinetic resolutions of these isomers were conducted in THF as a solvent and vinyl acetate as an acyl donor, at 30 ˝C and for 48 h. The lipases CAL-B and PSL-C I were tested and the latter led to the best results. Thus, for the Int. J. Mol. Sci. 2015, 16, page–page rac-trans-isomer, both the trans-(1R,2R)-acetate in 96% ee and trans-(1S,2S)-alcohol with 85% ee (cboth 43% the and trans an-(1ER-value,2R)-acetate of 133) in were96% ee obtained. and trans-(1 InS the,2S)-alcohol case of thewithrac 85%-cis -isomer,ee (c 43% the and compounds an E-value cisof -(1133)S,2 wereR)-acetate obtained. and cis In-(1 theR,2 caseS)-alcohol of the withrac-cis 98%-isomer,ee (c 50% the andcompoundsE > 200) werecis-(1 obtainedS,2R)-acetate (Scheme and 41cisc- and(1R,2 TableS)-alcohol5). with 98% ee (c 50% and E > 200) were obtained (Scheme 41c and Table 5).

Scheme 41. Enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a), racemic Scheme 41. Enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a); racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62]. trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c)[62].

Table 5. Results from the enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol THN (a), Table 5. Results from the enzymatic kinetic resolution of the racemic trans- and cis-3-imidazol racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62]. THN (a), racemic trans- and cis-3-triazol THN (b) and racemic trans- and cis-1-imidazol THN (c) [62].

eeP (%) eeS (%) SchemeScheme 41 41 SubstrateSubstrate c (%) c ee(%)(%) Acetate ee (%) AlcoholE E P Acetate AlcoholS trans-3-imidazol THN 46 95 83 102 a trans-3-imidazol THN 46 95 83 102 a rac-cis-3-imidazol THN 43 95 71 83 rac-trans-3-triazolrac-cis-3-imidazol THN THN 48 43 99 95 71 93 83 >200 b rac-cis-3-triazolrac-trans THN-3-triazol THN 23 48 >99 99 93 29 >200 >200 rac-trans-1-imidazol THN 43 96 85 133 c b rac-cis-1-imidazolrac-cis-3-triazol THN THN 50 23 98 >99 29 98 >200 >200 rac-trans-1-imidazol THN 43 96 85 133 c 3.31. Benzoin Derivative rac-cis-1-imidazol THN 50 98 98 >200 Chiral α-hydroxy ketones are important building blocks in the syntheses of several biologically 3.31. Benzoin Derivative active compounds such as pharmaceuticals, agrochemicals and pheromones. Aiming to obtain the (S)-benzoinChiral α butyrate,-hydroxy a studyketones for are the important dynamic enzymaticbuilding blocks kinetic in resolutionthe syntheses (DKR) of several of racemic biologically benzoin viaactive acylation compounds was developed such as pharmaceuticals, [63]; an efﬁcient agrochemicals DKR is needed and for competingpheromones. with Aiming other biocatalyticalto obtain the approaches,(S)-benzoin butyrate, such as thea study enantioselective for the dynamic reduction enzyma oftic benzyl kinetic ketones, resolution which (DKR) has of been racemic reported benzoin as avia useful acylation technique was developed for preparing [63]; an (S)-benzoins efficient DKR [64 ,is65 needed]. The for reactions competing were with conducted other biocatalytical in batch or continuousapproaches, mode such usingas the lipaseenantiosel fromectivePseudomonas reduction stutzeri of benzyl(lipase ketone TL) immobilizeds, which has been on Accurel reported carrier as a useful technique for preparing (S)-benzoins [64,65]. The reactions were conducted in batch or continuous mode using lipase from Pseudomonas29708 stutzeri (lipase TL) immobilized on Accurel carrier MP1001 [63]. Various solvents were evaluated in relation to TL lipase activity such as toluene, 2-MeTHF, 1,3-dioxolane, cyclopentyl methyl ether (CPME), as well as some solvents classified as deep eutectic solvents (DESs). At the same time, a study was conducted to verify the influence of the amount of water in the solvents on the enzyme activity. As result, dry CPME was the most efficient solvent for the catalytic activity of the lipase TL as well as for catalyst activity of Zr-TUD-1 (a three- dimensional mesoporous silicate containing zirconium), with this latter used for in situ racemization of the (R)-benzoin. The DKR of rac-benzoin, in batch mode, was carried out at 50 °C in the presence of 25 Int. J. Mol. Sci. 2015, 16, 29682–29716

MP1001 [63]. Various solvents were evaluated in relation to TL lipase activity such as toluene, 2-MeTHF, 1,3-dioxolane, cyclopentyl methyl ether (CPME), as well as some solvents classified as deep eutectic solvents (DESs). At the same time, a study was conducted to verify the influence of the amount of water in the solvents on the enzyme activity. As result, dry CPME was the most Int. J. Mol. efficientSci. 2015, solvent 16, page–page for the catalytic activity of the lipase TL as well as for catalyst activity of Zr-TUD-1 (a three-dimensional mesoporous silicate containing zirconium), with this latter used for in situ vinyl butyrateracemization (6 eq.) of as the the (R)-benzoin. acyl donor, The DKRdried of CPMErac-benzoin, as the in batch solvent, mode, Zr-TUD-1 was carried as out the at 50catalyst˝C and immobilizedin the TL presence lipase of vinylas the butyrate enzyme. (6 eq.) After as the 5 acylh, ( donor,S)-benzoin dried CPME butyrate as the (c solvent, 98.2% Zr-TUD-1 and 99% as ee) was obtained.the Subsequently, catalyst and immobilized the DKR TLof lipaserac-benzoin as the enzyme. was investigated After 5 h, (S)-benzoin in a continuous butyrate (cflow 98.2% system and in the presence99% of eevinyl) was butyrate obtained. Subsequently,(3 eq.), TL lipa the DKRse, and of rac Zr-TUD-1-benzoin was in investigated CPME. After in a continuous25 h, the flow(S)-benzoin system in the presence of vinyl butyrate (3 eq.), TL lipase, and Zr-TUD-1 in CPME. After 25 h, the butyrate( Swas)-benzoin obtained butyrate with was obtaineda maximum with a maximumconversion conversion of 40%, of 40%,decreasing decreasing to to 11% 11% atat 76 76 h. h. The enantiomericThe enantiomeric excess values excess ( valuesee) of ( eethe) of the(S)-benzoin (S)-benzoin butyrate butyrate werewereě98% ≥98% throughout throughout the process the process (Scheme(Scheme 42) [63]. 42 )[63].

Scheme Scheme42. Dynamic 42. Dynamic enzymatic enzymatic kinetic kinetic resolution resolution (DKR) of ofrac rac-benzoin-benzoin in conventional in conventional batch or batch or continuouscontinuous flow [63]. ﬂow [63].

The DKRThe in DKR the inpresence the presence of lipase of lipase is isan an important important approachapproach for for obtaining obtaining enantiomerically enantiomerically pure pure intermediatesintermediates for the for synthesis the synthesis of biol of biologicallyogically active active compoundscompounds with with both bothee and ee conversion and conversion up to up to 100%. Recently, an elegant review covering this subject was published [66]. 100%. Recently, an elegant review covering this subject was published [66]. 4. Complementary Approaches: Hydrolysis and Esteriﬁcation 4. Complementary Approaches: Hydrolysis and Esterification 4.1. Acetonide Derivatives 4.1. AcetonideThe Derivatives compound ((R)-2,2,4-trimethyl-1,3-dioxolan-4-yl)-methanol ((R)-acetonide alcohol), a chiral intermediate used in the synthesis of some bioactive compounds, was prepared in high ee by the Theenzymatic compound resolution ((R)-2,2,4-trimethyl-1,3-dioxolan-4-yl)-methanol of its racemate form (Scheme 43a). In this case, ((R both)-acetonide succinic anhydridealcohol), a chiral intermediateand vinyl used laurate in the were synthesis used as of acyl some donors, bioactive and Amano compounds, PS-D lipase was was prepared the selected in enzyme.high ee by the enzymaticThe resolution (S)-acetonide of alcoholits racemate was selectively form (Scheme acylated 43a). (with TBMEIn this as case, the solvent)both succinic and yielded anhydride the and (R)-acetonide semiester (with succinic anhydride as the acyl donor) and (R)-acetonide ester (with vinyl laurate were used as acyl donors, and Amano PS-D lipase was the selected enzyme. The (S)-acetonide vinyl laurate as the acyl donor), besides the remaining (R)-acetonide alcohol (Scheme 43a). The alcohol was(R)-acetonide selectively alcohol acylated was also (with obtained TBME by as kinetic the solvent) resolution and of theyielded corresponding the (R)-acetonide racemic ester semiester (with succinicvia hydrolysis anhydride using as CAL-B the acyl (Novozym donor) 435) and as ( theR)-acetonide lipase, and tester-BuOH:H (with2O (9:1)vinyl as laurate the solvent as the acyl donor), besides(Scheme the43b) remaining [67]. (R)-acetonide alcohol (Scheme 43a). The (R)-acetonide alcohol was also obtained by kinetic resolution of the corresponding racemic ester via hydrolysis using CAL-B (Novozym 435) as the lipase, and t-BuOH:H2O (9:1) as the solvent (Scheme 43b) [67].

29709

Scheme 43. Syntheses of (R)-acetonide derivatives by enantiocomplementary kinetic resolution via acylation of the racemic rac-acetonide alcohol (a) and via hydrolysis of (R)-acetonide ester (b) [67].

26 Int. J. Mol. Sci. 2015, 16, page–page vinyl butyrate (6 eq.) as the acyl donor, dried CPME as the solvent, Zr-TUD-1 as the catalyst and immobilized TL lipase as the enzyme. After 5 h, (S)-benzoin butyrate (c 98.2% and 99% ee) was obtained. Subsequently, the DKR of rac-benzoin was investigated in a continuous flow system in the presence of vinyl butyrate (3 eq.), TL lipase, and Zr-TUD-1 in CPME. After 25 h, the (S)-benzoin butyrate was obtained with a maximum conversion of 40%, decreasing to 11% at 76 h. The enantiomeric excess values (ee) of the (S)-benzoin butyrate were ≥98% throughout the process (Scheme 42) [63].

Scheme 42. Dynamic enzymatic kinetic resolution (DKR) of rac-benzoin in conventional batch or continuous flow [63].

The DKR in the presence of lipase is an important approach for obtaining enantiomerically pure intermediates for the synthesis of biologically active compounds with both ee and conversion up to 100%. Recently, an elegant review covering this subject was published [66].

4. Complementary Approaches: Hydrolysis and Esterification

4.1. Acetonide Derivatives The compound ((R)-2,2,4-trimethyl-1,3-dioxolan-4-yl)-methanol ((R)-acetonide alcohol), a chiral intermediate used in the synthesis of some bioactive compounds, was prepared in high ee by the enzymatic resolution of its racemate form (Scheme 43a). In this case, both succinic anhydride and vinyl laurate were used as acyl donors, and Amano PS-D lipase was the selected enzyme. The (S)-acetonide alcohol was selectively acylated (with TBME as the solvent) and yielded the (R)-acetonide semiester (with succinic anhydride as the acyl donor) and (R)-acetonide ester (with vinyl laurate as the acyl donor), besides the remaining (R)-acetonide alcohol (Scheme 43a). The (R)-acetonide alcohol was also Int.obtained J. Mol. Sci.by2015 kinetic, 16, 29682–29716 resolution of the corresponding racemic ester via hydrolysis using CAL-B (Novozym 435) as the lipase, and t-BuOH:H2O (9:1) as the solvent (Scheme 43b) [67].

Scheme 43. Syntheses of (R)-acetonide derivatives by enantiocomplementary kinetic resolution via Scheme 43. Syntheses of (R)-acetonide derivatives by enantiocomplementary kinetic resolution via acylation of the racemic rac-acetonide alcohol (a) and via hydrolysis of (R)-acetonide ester (b) [67]. Int. J. Mol.acylation Sci. 2015 of,the 16, page–page racemic rac -acetonide alcohol (a) and via hydrolysis of (R)-acetonide ester (b)[67].

4.2. Vitamin D Analogues 26 Vitamin DD playsplays aa vital vital role role in in maintaining maintaining calcium calcium levels levels within within the the normal normal range. range. Analogues Analogues of vitaminof vitamin D have D have been been prepared prepared in order in toorder obtain to compoundsobtain compounds with calcemic with activity.calcemic Such activity. analogues Such wereanalogues obtained were by obtained separation by separa of a mixturetion of ofa mixture epimers of (at epimers C-24 containing (at C-24 containing an OH group) an OH using group) two approaches,using two approaches, (a) enzymatic (a) kineticenzymatic resolution kinetic via resoluti esterificationon via oresterification (b) solvolysis or of(b) the solvolysis corresponding of the esters,corresponding and the epimeresters, ofand interest the epimer (with biologicalof interest activity) (with biological was the one activity) with S wasconfiguration. the one with In the S firstconfiguration. approach (routeIn the a),first the approach esterifications (route were a), the carried esterifications out using were an acylating carried agentout using (especially an acylating vinyl acetate),agent (especially organic solvent vinyl (especiallyacetate), organic hexane orsolvent DIPE) and(especially lipases fromhexaneAlcalagenes or DIPE)sp. orandPseudomonas lipases fromsp. (inAlcalagenes free or immobilizedsp. or Pseudomonas form). sp. The (in reactions free or immobilized were monitored form). by The HPLC reactions to ensure were diastereomeric monitored by excessHPLC amountsto ensure indiastereomeric the range of 80%–95%.excess amounts In this in case, the therange desired of 80%–95%. epimer isIn thethis remaining case, the desired alcohol (epimerS configuration is the remaining at C-24), asalcohol seen in(S Scheme configuration 44a. In theat C-24), second as approach seen in Scheme (route b), 44a. esterified In the vitaminsecond Dapproach analogues (route have b), undergone esterified vitamin alcoholysis D analogues or hydrolysis have inundergone the presence alcoholysis of the same or hydrolysis lipases used in the in routepresence a. These of the reactionssame lipases gave used a diastereomeric in route a. These excess reactions in the gave range a ofdiastereomeric 80%–95% (Scheme excess 44 inb) the and range the desiredof 80%–95% epimer (Scheme was the 44b) non-hydrolyzed and the desire (dS )-acetateepimer was [68 ].the non-hydrolyzed (S)-acetate [68].

Scheme 44. Synthesis of vitamin D analogues by enzymatic kinetic resolution via esterification (a) or Scheme 44. Synthesis of vitamin D analogues by enzymatic kinetic resolution via esteriﬁcation (a) or (b) solvolysis of the corresponding esters [68]. (b) solvolysis of the corresponding esters [68].

4.3. Arylalkylcarbinol Derivatives 4.3. Arylalkylcarbinol Derivatives Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in the Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important the complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important intermediates for the syntheses of several compounds with industrial application. This enzyme was intermediates for the syntheses of several compounds with industrial application. This enzyme was used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected to and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee (n = 2)), as seen in Scheme 45a. The complementary process was conducted using CAL-B on the catalyzed acylation of the rac-arylalkylcarbinols, producing29710 the (S)-alcohols and (R)-acetates in high enantioselectivities (route b). Subsequently, these mixtures were submitted to a Mitsunobu reaction and provided only the (R)-acetates (99% ee (n = 1 or 2)), as seen in Scheme 45b [69].

Scheme 45. Complementary enzymatic kinetic resolution of rac-arylalkylcarbinyl acetates or rac- arylalkylcarbinols combined with Mitsunobu reaction on the preparation of (S)- and (R)- arylalkylcarbinyl acetates via hydrolysis (a) or acylation (b), respectively [69].

27 Int. J. Mol. Sci. 2015, 16, page–page

4.2. Vitamin D Analogues Vitamin D plays a vital role in maintaining calcium levels within the normal range. Analogues of vitamin D have been prepared in order to obtain compounds with calcemic activity. Such analogues were obtained by separation of a mixture of epimers (at C-24 containing an OH group) using two approaches, (a) enzymatic kinetic resolution via esterification or (b) solvolysis of the corresponding esters, and the epimer of interest (with biological activity) was the one with S configuration. In the first approach (route a), the esterifications were carried out using an acylating agent (especially vinyl acetate), organic solvent (especially hexane or DIPE) and lipases from Alcalagenes sp. or Pseudomonas sp. (in free or immobilized form). The reactions were monitored by HPLC to ensure diastereomeric excess amounts in the range of 80%–95%. In this case, the desired epimer is the remaining alcohol (S configuration at C-24), as seen in Scheme 44a. In the second approach (route b), esterified vitamin D analogues have undergone alcoholysis or hydrolysis in the presence of the same lipases used in route a. These reactions gave a diastereomeric excess in the range of 80%–95% (Scheme 44b) and the desired epimer was the non-hydrolyzed (S)-acetate [68].

Scheme 44. Synthesis of vitamin D analogues by enzymatic kinetic resolution via esterification (a) or (b) solvolysis of the corresponding esters [68].

4.3. Arylalkylcarbinol Derivatives Candida antarctica lipase B (CAL-B, Novozym 435) was investigated as a biocatalyst in the complementary preparation of (S)- and (R)-arylalkylcarbinol acetates, which are important intermediates for the syntheses of several compounds with industrial application. This enzyme was used in the hydrolysis of racemic arylalkylcarbinyl acetates (route a) and produced both (R)-alcohols Int. J. Mol. Sci. 2015, 16, 29682–29716 and (S)-acetates in high enantioselectivities. Then, the mixture of these compounds was subjected to Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee (n = 2)), to Mitsunobu reaction to yield the (S)-acetates as the only products (94% ee (n = 1) and 99% ee as seen in(n =Scheme 2)), as seen 45a. in The Scheme complementary 45a. The complementary process was process conducted was conducted using using CAL-B CAL-B on onthe the catalyzed acylationcatalyzed of the acylation rac-arylalkylcarbinols, of the rac-arylalkylcarbinols, producing producing the the(S ()-alcoholsS)-alcohols andand (R )-acetates(R)-acetates in high in high enantioselectivitiesenantioselectivities (route (route b). Su b).bsequently, Subsequently, these these mixtures were were submitted submitted to a Mitsunobuto a Mitsunobu reaction reaction and providedand provided only the only (R the)-acetates (R)-acetates (99% (99% eeee (n(n == 1 1 or 2)),2)), as as seen seen in Schemein Scheme 45b [ 6945b]. [69].

Scheme Scheme45. Complementary 45. Complementary enzymatic enzymatic kinetic kinetic resolution resolution of rac of -arylalkylcarbinylrac-arylalkylcarbinyl acetates acetates or rac- arylalkylcarbinolsor rac-arylalkylcarbinols combined combinedwith Mitsun withobu Mitsunobu reaction reaction on onthe the preparation preparation ofof (S()-S)- and and (R)- arylalkylcarbinyl(R)-arylalkylcarbinyl acetates acetatesvia hydrolysis via hydrolysis (a) or (a acylation) or acylation (b (),b ),respectively respectively [ 69[69].].

4.4. Key Intermediate of Levofloxacin 27 The benzoxazine moiety is part of the structures of a series of molecules with biological activities such as antibacterial, anticancer, antifungal and antimicrobial. A number of 1,4-benzoxazine derivatives (with or without substituents at the aromatic ring) were prepared by a combination of chemical steps and lipase-mediated kinetic resolution [70]. The enzymatic step was conducted with rac-1-(2-nitrophenoxy)propan-2-ols (rac-alcohols), via acylation (route a), and with the corresponding rac-acetates, via hydrolysis (route b), as seen in Scheme 46. These two approaches are considered complementary lipase-catalyzed processes. The process of kinetic resolution via acetylation was first evaluated using the rac-1-(2-nitrophenoxy)propan-2-ol as a substrate, in the presence of vinyl acetate as an acyl donor, TBME as a solvent, at 30 ˝C and with 5 h of reaction time. After screening lipases from Candida antarctica (CAL-A), from Pseudomonascepacia (PSL C-I), from C. antarctica B (CAL-B) and from Rhizomucor miehei in immobilized form (RML IM), it was found that the latter (RM IM) was the most efficient. In the aforementioned conditions, the (R)-acetate (94% ee) and the remaining (S)-alcohol (89% ee) were obtained with c 48%. The study was extended to the rac-alcohols with substituents at the aromatic ring (4-F, 4-OMe and 5-Me), leading to the (R)-acetates (93%–95% ee) and the remaining (S)-alcohols (89%–94% ee) with c 48%–50% and E-values ranging from 103 to >200 (Scheme 46a and Table6). By a sequence of reactions, the (R)-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine derivatives ((R)-3-methyl-1,4-benzoxazines) were synthesized from the (S)-alcohols. The second approach (route b) was related to the resolution of the corresponding rac-acetates via hydrolysis reaction. In this case, the reactions were carried out in the presence of RML IM, at 30 ˝C, using TBME and water (5 eq.) as solvents. After 52 h, the (R)-alcohols (96% to >99% ee) and the remaining (S)-acetates (91%–97% ee) were obtained with c 48%–50% and E > 200 (Scheme 46b and Table6). Due to the successful strategies for obtaining the chiral 1,4-benzoxazines, the authors performed the production of the (S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine, precursor of the potent antibacterial Levofloxacin (Scheme 46c). Then, the kinetic resolution of the rac-1-(2,3-difluoro-6-nitrophenoxy)propan-2-yl acetate was performed, via hydrolysis in the

29711 Int. J. Mol. Sci. 2015, 16, page–page

4.4. Key Intermediate of Levofloxacin The benzoxazine moiety is part of the structures of a series of molecules with biological activities such as antibacterial, anticancer, antifungal and antimicrobial. A number of 1,4-benzoxazine derivatives (with or without substituents at the aromatic ring) were prepared by a combination of chemical steps and lipase-mediated kinetic resolution [70]. The enzymatic step was conducted with rac-1-(2-nitrophenoxy)propan-2-ols (rac-alcohols), via acylation (route a), and with the corresponding rac-acetates, via hydrolysis (route b), as seen in Scheme 46. These two approaches are considered complementary lipase-catalyzed processes. The process of kinetic resolution via acetylation was first evaluated using the rac-1-(2-nitrophenoxy)propan-2-ol as a substrate, in the presence of vinyl acetate as an acyl donor, TBME as a solvent, at 30 °C and with 5 h of reaction time. After screening lipases from Candida antarctica (CAL-A), from Pseudomonas cepacia (PSL C-I), from C. antarctica B (CAL-B) and from Rhizomucor miehei in immobilized form (RML IM), it was found that the latter (RM IM) was the most efficient. In the aforementioned conditions, the (R)-acetate (94% ee) and the remaining (S)-alcohol (89% ee) were obtained with c 48%. The study was extended to the rac-alcohols with substituents at the aromatic ring (4-F, 4-OMe and 5-Me), leading to the (R)-acetates (93%–95% ee) and the remaining (S)-alcohols (89%–94% ee) with c 48%–50% and E-values ranging from 103 to >200 (Scheme 46a and Table 6). By a sequence of reactions, the (R)-3-methyl-3,4-dihydro-2H- benzo[b][1,4]oxazine derivatives ((R)-3-methyl-1,4-benzoxazines) were synthesized from the (S)-alcohols. The second approach (route b) was related to the resolution of the corresponding rac- acetates via hydrolysis reaction. In this case, the reactions were carried out in the presence of RML IM, at 30 °C, using TBME and water (5 eq.) as solvents. After 52 h, the (R)-alcohols (96% to >99% ee) and the remaining (S)-acetates (91%–97% ee) were obtained with c 48%–50% and E > 200 (Scheme 46b and Table 6). Due to the successful strategies for obtaining the chiral 1,4-benzoxazines, the authors performedInt. J. Mol.the Sci.production2015, 16, 29682–29716 of the (S)-7,8-difluoro-3-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine, precursor of the potent antibacterial Levofloxacin (Scheme 46c). Then, the kinetic resolution of the rac-1-(2,3-difluoro-6-nitrophenoxy)propan-2-yl acetate was performed, via hydrolysis in the presence presence of lipase RM IM, leading to the (R)-alcohol (>99% ee) and the remaining (S)-acetate of lipase(84% RMee IM,) with leading c 46%. to Subsequently, the (R)-alcohol after several (>99% chemical ee) and steps, the theremaining (R)-alcohol (S was)-acetate converted (84% into ee) with c 46%. Subsequently,the (S)-7,8-diﬂuoro-3-methyl-3,4-dihydro-2 after several chemical Hsteps,-benzo[ theb][1,4]oxazine (R)-alcohol (36% was yield, converted >99% ee) Levoﬂoxacin into the (S)-7,8- difluoro-3-methyl-3,4-dihydro-2precursor [70]. H-benzo[b][1,4]oxazine (36% yield, >99% ee) Levofloxacin precursor [70].

Scheme Scheme46. Enzymatic 46. Enzymatic kinetic kinetic resolution resolution of racemic of racemic alcohols alcohols 1-(2-nitrophenoxy)propan-2-ols 1-(2-nitrophenoxy)propan-2-ols via acetylationvia acetylation(a) or via (ahydrolysis) or via hydrolysis (b) of the (b) ofcorresponding the corresponding racemic racemic acetates. acetates. Production Production ofof (S)-7,8- difluoro-3-methyl-3,4-dihydro-2(S)-7,8-diﬂuoro-3-methyl-3,4-dihydro-2H-benzo[Hb-benzo[][1,4]oxazineb][1,4]oxazine precursor precursor ofof thethe antibacterial antibacterial drug drug c LevofloxacinLevoﬂoxacin (c) [70]. ( )[ 70].

Table 6. Results from the enzymatic kinetic28 resolution of racemic alcohols 1-(2-nitrophenoxy) propan-2-ols via acetylation (a) or via hydrolysis (b) of the corresponding racemic acetates [70].

Acetates Alcohols Scheme 46 c (%) E ee (%) Yield (%) ee (%) Yield (%) a 93–95 45–47 89–94 44–48 48–50 103–>200 b 91–97 41–48 96–>99 44–47 48–50 >200

5. Conclusions Recent examples of the use of lipases in the preparation of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates were reviewed, conﬁrming the importance of these enzymes in obtaining compounds with high added value. Most reported biocatalytic processes refer to kinetic resolutions of racemic substrates, which occur under mild conditions with a high degree of regio- or enantioselectivity. Among the examples presented, the action of lipases can be seen in a wide range of substrates with varied structures such as aromatic, heteroaromatic (containing atoms of nitrogen, sulfur, chlorine, etc.), as well as aliphatic with either open or cyclic chain (branched or not). The substances produced by the action of lipases, in the examples herein, are pharmaceutical intermediates or drugs, which makes the biocatalytic process of great interest in

29712 Int. J. Mol. Sci. 2015, 16, 29682–29716 the pharmaceutical industry. Additionally, it was also possible to confirm the versatility of lipases for acting in either aqueous medium or organic solvents for the hydrolysis of esters, the acylation of alcohols or the esterification of carboxylic acid, allowing the production of the desired stereoisomer. It highlights the operational simplicity, as it is not necessary to add cofactors in the reaction system, and the ease of finding a wide range of commercially available and relatively low-cost lipases. The use of immobilized lipase often allows the reuse of the enzymatic system for several cycles, making the process more effective and economically viable. In summary, it is clear that lipases will continue to be an excellent alternative for obtaining biologically active compounds.

Acknowledgments: The authors thank the Brazilian funding agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing the Special Visiting Researcher fellowship (process 400171/2014-7) under the Brazilian Scientific Program “Ciência sem Fronteira”. Author Contributions: Marcos Carlos de Mattos supervised the manuscript preparation. Marcos Carlos de Mattos, Maria da Conceição Ferreira de Oliveira, Telma Leda Gomes de Lemos, Francesco Molinari, Diego Romano and Immacolata Serra reviewed the literature and co-wrote the manuscript. Ana Caroline Lustosa de Melo Carvalho and Thiago de Sousa Fonseca prepared all illustrations. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Gotor-Fernandez, V.; Brieva, R.; Gotor, V. Lipases: Useful biocatalysts for the preparation of pharmaceuticals. J. Mol. Catal. B. 2006, 40, 111–120. [CrossRef] 2. Tao, J.; Zhao, L.; Ran, N. Recent advances in developing chemoenzymatic processes for active pharmaceutical ingredients. Org. Process Res. Dev. 2007, 11, 259–267. 3. Pollard, D.J.; Woodley, J.M. Biocatalysis for pharmaceutical intermediates: The future is now. Trends Biotechnol. 2007, 25, 66–73. [CrossRef][PubMed] 4. Patel, R.N. Synthesis of chiral pharmaceutical intermediates by Biocatalysis. Coord. Chem. Rev. 2008, 252, 659–701. [CrossRef] 5. Tao, J.; Xu, J.-H. Biocatalysis in development of green pharmaceutical processes. Curr. Opin. Chem. Biol. 2009, 13, 43–50. [CrossRef][PubMed] 6. Patel, R.N. Biocatalysis: Synthesis of key intermediates for development of pharmaceuticals. ACS Catal. 2011, 1, 1056–1074. [CrossRef] 7. Anand, N.; Kapoor, M.; Ahmad, K.; Koul, S.; Parshad, R.; Manhas, K.S.; Sharma, L.R.; Qazi, G.N.; Taneja, S.C. Arthrobacter sp.: A lipase of choice for the kinetic resolution of racemic arylazetidinone precursors of taxanoid side chains. Tetrahedron 2007, 18, 1059–1069. [CrossRef] 8. Rimoldi, M.; Pellizzoni, G.; Facchetti, F.E.; Molinari, F.; Zerla, D.S.; Gandolﬁ, R. Chemo- and biocatalytic strategies to obtain phenylisoserine, lateral chain of taxol by asymmetric reduction. Tetrahedron 2011, 22, 2110–2116. [CrossRef] 9. Forró, E.; Fülöp, F. A new enzymatic strategy for the preparation of (2R,3S)-3-phenylisoserine: A key intermediate for the Taxol side chain. Tetrahedron 2010, 21, 637–639. [CrossRef] 10. Forró, E.; Fülöp, F. New enzymatic two-step cascade reaction for the preparation of a key intermediate for the taxol side-chain. Eur. J. Org. Chem. 2010, 16, 3074–3079. [CrossRef] 11. Cui, J.J.; Tran-Dubé, M.; Shen, H.; Nambu, M.; Kung, P.-P.; Pairish, M.; Jia, L.; Meng, J.; Funk, L.; Botrous, I.; et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J. Med. Chem. 2011, 54, 6342–6363. [CrossRef][PubMed] 12. Kung, P.-P.; Martinez, C.; Tao, J. Enantioselective Biotransformation for Preparation of Protein Tyrosine Kinase Inhibitor Intermediates. US 7465842 B2, 16 December 2008. 13. Felluga, F.; Pitacco, G.; Valentin, E.; Venneri, C.D. A facile chemoenzymatic approach to chiral non-racemic β-alkyl-γ-amino acids and 2-alkylsuccinic acids. A concise synthesis of (S)-(+)-Pregabalin. Tetrahedron 2008, 19, 945–955. [CrossRef] 14. Li, X.-J.; Zheng, R.-C.; Ma, H.-Y.; Zheng, Y.-G. Engineering of Thermomyces lanuginosus lipase Lip: Creation of novel biocatalyst for efﬁcient biosynthesis of chiral intermediate of Pregablin. Appl. Microbiol. Biotechnol. 2014, 98, 2473–2483. [CrossRef][PubMed]

29713 Int. J. Mol. Sci. 2015, 16, 29682–29716

15. Rouf, A.; Gupta, P.; Aga, M.A.; Kumar, B.; Chaubey, A.; Parshad, R.; Taneja, S.C. Chemoenzymatic synthesis of piperoxan, prosympal, dibozane, and doxazosin. Tetrahedron 2012, 23, 1615–1623. [CrossRef] 16. Singh, A.; Goel, Y.; Rai, A.K.; Banerjee, U.C. Lipase catalyzed kinetic resolution for the production of (S)-3-[5-(4-fluoro-phenyl)-5-hydroxy-pentanoyl]-4-phenyl-oxazolidin-2-one: An intermediate for the synthesis of ezetimibe. J. Mol. Catal. B 2013, 85–86, 99–104. [CrossRef] 17. Goswami, A.; Guo, Z.; Qiu, Y. Process for Resolving cyclopropyl diesters. EP2817412 A1, 31 December 2014. 18. Takaç, S.; Bakkal, M. Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production. Pro. Biochem. 2007, 42, 1021–1027. [CrossRef] 19. Sayin, S.; Akoz, E.; Yilmaz, M. Enhanced catalysis and enantioselective resolution of racemic naproxen methyl ester by lipase encapsulated within iron oxide nanoparticles coated with calix[8]arene valeric acid complexes. Org. Biomol. Chem. 2014, 12, 6634–6642. [CrossRef][PubMed] 20. Sharifabad, M.E.; Hodgson, B.; Jellite, M.; Mercer, T.; Sen, T. Enzyme immobilised novel core–shell superparamagnetic nanocomposites for enantioselective formation of 4-(R)-hydroxycyclopent-2- en-1-(S)-acetate. Chem. Commun. 2014, 50, 11185–11187. [CrossRef][PubMed] 21. Hu, C.; Wang, N.; Zhang, W.; Zhang, S.; Meng, Y.; Yu, X. Immobilization of Aspergillus terreus lipase in self-assembled hollow nanospheres for enantioselective hydrolysis of ketoprofen vinyl ester. J. Biotechnol. 2015, 194, 12–18. [CrossRef][PubMed] 22. Zhang, W.-W.; Jia, J.-Q.; Wang, N.; Hu, C.-L.; Yang, S.-Y.; Yu, X.-Q. Improved activity of lipase immobilized in microemulsion-based organogels for (R,S)-ketoprofen ester resolution: Long-term stability and reusability. Biotechnol. Rep. 2015, 7, 1–8. [CrossRef] 23. Reis, P.; Holmberg, K.; Watzke, H.; Leser, M.E.; Miller, R. Lipases at interfaces: A review. Adv. Colloid Interface Sci. 2009, 147–148, 237–250. [CrossRef][PubMed] 24. Torres, S.Y.; Verdecia, Y.; Rebolledo, F. Chemoenzymatic approach to optically active 1,4-dihydropyridine derivatives. Tetrahedron 2015, 71, 3976–3984. [CrossRef] 25. Gu, J.; Rupeen, M.E.; Raveendranath, P.; Chew, W.; Shaw, C.C. Proline cci-779 (Proline-Rapamycin 42-Ester with 2,2-Bis(hydroxymethyl) Propionic Acid) and Two-Step Enzymatic Synthesis of Proline cci-779 and cci-779 Using Microbial Lipase. EP1751168A1, 14 February 2007. 26. Ferraboschi, P.; Colombo, D.; de Mieri, M.; Grisenti, P. First chemoenzymatic synthesis of immunomodulating macrolactam pimecrolimus. Tetrahedron Lett. 2009, 50, 4384–4388. [CrossRef] 27. Grisenti, P.; Reza, E.S.; Verza, E. A Chemo-Enzymatic Approach to the Synthesis of Primecrolimus. EP2432791B1, 8 July 2015. 28. Bhuniya, R.; Nanda, S. Asymmetric synthesis of the active form of loxoprofen and its analogue. Tetrahedron 2011, 22, 1125–1132. [CrossRef] 29. Spizzo, P.; Basso, A.; Ebert, C.; Gardossi, L.; Ferrario, V.; Romano, D.; Molinari, F. Resolution of (R,S)-flurbiprofen catalysed by dry mycelia in organic solvente. Tetrahedron 2007, 63, 11005–11010. [CrossRef] 30. Tamborini, L.; Romano, D.; Pinto, A.; Bertolani, A.; Molinari, F.; Conti, P. An efficient method for the lipase-catalysed resolution and in-line purification of racemic flurbiprofen in a continuous-flow reactor. J. Mol. Catal. B 2012, 84, 78–82. [CrossRef] 31. Tamborini, L.; Romano, D.; Pinto, A.; Contente, M.; Iannuzzi, M.C.; Conti, P.; Molinari, F. Biotransformation with whole microbial systems in a continuous flow reactor: Resolution of (RS)-flurbiprofen using Aspergillus oryzae by direct esterification with ethanol in organic solvent. Tetrahedron Lett. 2013, 54, 6090–6093. [CrossRef] 32. Pastre, J.C.; Browne, D.L.; Ley, S.V. Flow chemistry syntheses of natural products. Chem. Soc. Rev. 2013, 42, 8849–8869. [CrossRef][PubMed] 33. Itabaiana, I.; Miranda, L.S.M.; Souza, R.O.M.A. Towards a continuous flow environment for lipase-catalyzed reactions. J. Mol. Catal. B 2013, 85–86, 1–9. [CrossRef] 34. Marszałł, M.P.; Siódmiak, T. Immobilization of Candida rugosa lipase onto magnetic beads for kinetic resolution of (R,S)-ibuprofen. Catal. Commun. 2012, 24, 80–84. [CrossRef] 35. Shinde, S.D.; Yadav, G.D. Insight into microwave assisted immobilized Candida Antarctica lipase B catalyzed kinetic resolution of R,S-(˘)-ketorolac. Process Biochem. 2015, 50, 230–236. [CrossRef]

29714 Int. J. Mol. Sci. 2015, 16, 29682–29716

36. Bizerra, A.M.C.; Montenegro, T.G.C.; Lemos, T.L.G.; de Oliveira, M.C.F.; de Mattos, M.C.; Lavandera, I.; Gotor-Fernandez, V.; Gonzalo, G.; Gotor, V. Enzymatic regioselective production of chloramphenicol esters. Tetrahedron 2011, 67, 2858–2862. [CrossRef] 37. Da Silva, M.R.; Montenegro, T.G.C.; de Mattos, M.C.; de Oliveira, M.C.F.; de Lemos, T.L.G.; de Gonzalo, G.; Lavandera, I.; Gotor-Fernandez, V.; Gotor, V. Regioselective preparation of thiamphenicol esters through lipase-catalyzed processes. J. Braz. Chem. Soc. 2014, 25, 987–994. [CrossRef] 38. Borowiecki, P.; Balter, S.; Justyniak, I.; Ochal, Z. First chemoenzymatic synthesis of (R)- and (S)-1-(9H-fluoren-9-yl)ethanol. Tetrahedron 2013, 24, 1120–1126. [CrossRef] 39. Stürmer, R. Method for producing enantiomer-pure aminoalcohols. US 7,435,835 B2, 14 October 2008. 40. Yamashita, S.; Mase, N.; Takabe, K. Chemoenzymatic total synthesis and determination of the absolute configuration of (S)-nebracetam. Tetrahedron 2008, 19, 2115–2118. [CrossRef] 41. Araujo, D.M.F.; Vieira, G.A.B.; Mattos, M.C.; Lemos, T.L.G.; Oliveira, M.C.F.; Melo, V.M.M.; Gonzalo, G.; Gotor-Fernandez, V.; Gotor, V. Chemoenzymatic preparation of a biologically active naphthoquinone from Tabebuia impetiginosa using lipases or alcohol dehydrogenases. J. Mol. Catal. B 2009, 61, 279–283. [CrossRef] 42. Kamal, A.; Khanna, G.; Krishnaji, T.; Ramu, R. Chemoenzymatic Process for the Stereoselective Preparation of (R)-γ-Amino-β-hydroxybutyric Acid or (R)-Carnitine from 3,4-Dihydroxybutanenitrile. US 7816119B2, 19 October 2010. 43. Nagarapu, L.; Gaikwad, H.K.; Bantu, R.; Manikonda, S.R. Chemoenzymatic synthesis with lipase catalyzed resolution and evaluation of antitumor activity of (R/S)-2-[2-hydroxy-3-(4-phenylpiperazin-1-yl) propyl]-1H-pyrrolo[3,4-b]quinolin-3(2H)-one. Eur. J. Med. Chem. 2011, 46, 2152–2156. [CrossRef][PubMed] 44. Yadav, G.D.; Devendran, S. Lipase catalyzed kinetic resolution of (˘)-1-(1-naphthyl) ethanol under microwave irradiation. J. Mol. Catal. B 2012, 81, 58–65. [CrossRef] 45. Wamvakides, A.; Moutsos, V.; Schmitt, M. Synthesis of (+) and (–) 1-(5,5-Diphenyltetrahydrofuran-3-yl)-N, N-dimethylmethanamine, (+) and (–) 1-(2,2-Diphenyltetrahydrofuran-3-yl)-N,N-dimethylmethanamine and (+) and (–) 1-(2,2-Diphenyltetrahydrofuran-3-yl)-N-methylmethanamine. WO2013008044 A1, 17 January 2013. 46. González-Sabín, J.; Ríos-Lombardía, N.; Gotor, V.; Morís, F. Enzymatic transesterification of pharmacologically interesting β-aminocycloalkanol precursors. Tetrahedron 2013, 24, 1421–1425. [CrossRef] 47. Jacobsen, E.E.; Lie, A.; Frigstad, M.M.H.; el-Behairy, M.F.; Ljones, T.; Wohlgemuth, R.; Anthonsen, T. Desymmetrization of cbz-serinol catalyzed by crude pig pancreatic lipase reveals action of lipases with opposite enantioselectivity. J. Mol. Catal. B 2013, 85–86, 134–139. [CrossRef] 48. Da Silva, M.R.; de Mattos, M.C.; de Oliveira, M.C.F.; de Lemos, T.L.G.; Ricardo, N.M.P.S.; de Gonzalo, G.; Lavandera, I.; Gotor-Fernández, V.; Gotor, V. Asymmetric chemoenzymatic synthesis of N-acetyl-α-amino esters based on lipase-catalyzed kinetic resolutions through interesterification reactions. Tetrahedron 2014, 70, 2264–2271. [CrossRef] 49. Devendran, S.; Yadav, G.D. Lipase-catalyzed kinetic resolution of (˘)-1-(2-furyl) ethanol in nonaqueous media. Chirality 2014, 26, 286–292. [CrossRef][PubMed] 50. Karmee, S.K. The synthesis, properties, and applications of ascorbyl esters. Lipid. Technol. 2011, 23, 227–229. [CrossRef] 51. Kharrat, N.; Aissa, I.; Sghaier, M.; Bouaziz, M.; Sellami, M.; Laouini, D.; Gargouri, Y. Lipophilization of ascorbic acid: A monolayer study and biological and antileishmanial activities. J. Agric. Food Chem. 2014, 62, 9118–9127. 52. Chatterjee, S.; Ghadigaonkar, S.; Sur, P.; Sharma, A.; Chattopadhyay, S. A chemoenzymatic synthesis of hept-6-ene-2,5-diol stereomers: Application to asymmetric synthesis of decarestrictine L, pyrenophorol, and stagonolide E. J. Org. Chem. 2014, 79, 8067–8076. [CrossRef][PubMed] 53. Chatterjee, S.; Sharma, A.; Chattopadhyay, S. Chemoenzymatic synthesis of macrolide antibiotic (´)-A26771B. RSC Adv. 2014, 4, 42697–42705. [CrossRef] 54. Wei, C.; Fu, X.-F.; Wang, Z.; Yu, X.-J.; Zhang, Y.-J.; Zheng, J.-Y. Efficient synthesis of vitamin E intermediate by lipase-catalyzed regioselective transesterification. J. Mol. Catal. B 2014, 106, 90–94. [CrossRef] 55. Fonseca, T.S.; Silva, M.R.; Oliveira, M.C.F.; Lemos, T.L.G.; Marques, R.A.; Mattos, M.C. Chemoenzymatic synthesis of rasagiline mesylate using lipases. Appl. Catal. A 2015, 492, 76–82. [CrossRef]

29715 Int. J. Mol. Sci. 2015, 16, 29682–29716

56. Carvalho, A.C.L.M.; Araujo, D.M.F.; Gonçalves, L.R.B.; Mattos, M.C.; Silva, M.R.; Oliveira, M.C.F.; Marques, R.A.; Lemos, T.L.G.; Fonseca, T.S.; Oliveira, U.M.F. Desenvolvimento de um Processo Biocatalítico para a Produção do (S)-Indanol, Precursor do Fármaco Mesilato de Rasagilina. BR102013024675–1A2, 15 September 2015. 57. Grisenti, P. Biocatalyzed Synthesis of the Optically Pure (R) and (S) 3-Methyl-1,2,3,4-tetrahydroquinoline and Their Use as Chiral Synthons for the Preparation of the Antithrombotic (21R)- and (21S)-Argatroban. WO2015004015 A1, 15 January 2015. 58. Forró, E.; Galla, Z.; Nádasdia, Z.; Árva, J.; Fülöp, F. Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration. J. Mol. Catal. B 2015, 116, 101–105. [CrossRef] 59. Yamashita, M.; Taya, N.; Nishitani, M.; Oda, K.; Kawamoto, T.; Kimura, E.; Ishichi, Y.; Terauchi, J.; Yamano, T. Preparation of (S)-4-(1-(3,4-dichlorophenyl)-2-methoxyethyl)piperidine. Tetrahedron 2015, 26, 935–942. [CrossRef] 60. Zhou, X.; Zheng, D.; Cui, B.; Han, W.; Chen, Y. Novozym 435 lipase mediated enantioselective kinetic resolution: A facile method for the synthesis of chiral tetrahydroquinolin-4-ol and tetrahydro1H-benzo[b]azepin-5-ol derivatives. Tetrahedron 2015, 71, 4738–4744. [CrossRef] 61. Villa-Barro, A.; Gotor, V.; Brieva, R. Highly selective chemoenzymatic synthesis of enantiopure orthogonally protected trans-3-amino-4-hydroxypiperidines. Tetrahedron 2015, 71, 6907–6912. [CrossRef] 62. Méndez-Sánchez, D.; Ríos-Lombardía, N.; Gotor, V.; Gotor-Fernández, V. Asymmetric synthesis of azolium-based 1,2,3,4-tetrahydronaphthalen-2-ols through lipase-catalyzed resolutions. Tetrahedron 2015, 26, 760–767. [CrossRef] 63. Petrenz, A.; de Maria, P.D.; Ramanathan, A.; Hanefeld, U.; Ansorge-Schumacher, M.B.; Kara, S. Medium and reaction engineering for the establishment of a chemo-enzymatic dynamic kinetic resolution of rac-benzoin in batch and continuous mode. J. Mol. Catal. B 2015, 114, 42–49. [CrossRef] 64. Hoyos, P.; Sansottera, G.; Fernández, M.; Molinari, F.; Sinisterra, J.V.; Alcántara, A.R. Enantioselective monoreduction of different 1,2-diaryl-1,2-diketones catalysed by lyophilised whole cells from Pichia glucozyma. Tetrahedron 2008, 64, 7929–7936. [CrossRef] 65. Fragnelli, M.C.; Hoyos, P.; Romano, D.; Gandolfi, R.; Alcántara, A.R.; Molinari, F. Enantioselective reduction and deracemisation using the non-conventional yeast Pichia glucozyma in water-organic solvent biphasic systems: Preparation of (S)-1,2-diaryl-2-hydroxyethanones (benzoins). Tetrahedron 2012, 68, 523–528. [CrossRef] 66. De Miranda, A.S.; Miranda, L.S.M.; de Souza, R.O.M.A. Lipases: Valuable catalysts for dynamic kinetic resolutions. Biotechnol. Adv. 2015, 33, 372–393. [CrossRef][PubMed] 67. Ainge, D.; Gnad, F.; Sinclair, R.; Vaz, L.M.; Wells, A. Use of Intermediates (R)-2,2,4-Trimethyl-l, 3-dioxolane-4-yl) methanol (a), 3-Fluoro-4-nitro-phenol (b) and 1-(4-Chloro-benzyl)-piperidin-4-ylamine (c). WO2009035407 A1, 19 March 2009. 68. Shapiro, E.; Fishman, A.; Effenberger, R.; Maymon, A.; Schwartz, A. Selective Enzymatic Esterification and Solvolysis of Epimeric Vitamin D Analog and Separation of the Epimers. US 8129173 B2, 6 March 2012. 69. Bouzemi, N.; Grib, I.; Houiene, Z.; Aribi-Zouioueche, L. Enantiocomplementary preparation of (S)- and (R)-arylalkylcarbinols by lipase-catalysed resolution and Mitsunobu inversion: Impact of lipase amount. Catalysts 2014, 4, 215–225. [CrossRef] 70. López-Iglesias, M.; Busto, E.; Gotor, V.; Gotor-Fernández, V. Chemoenzymatic asymmetric synthesis of 1,4-benzoxazine derivatives: Application in the synthesis of a levofloxacin precursor. J. Org. Chem. 2015, 80, 3815–3824. [CrossRef][PubMed]

© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

29716