Chemoenzymatic Synthesis of trans-β-Aryl-δ-hydroxy-γ-lactones and Enzymatic Kinetic Resolution of Their Racemic Mixtures

Two novel and convenient routes to obtain enantiomerically enriched trans-β-aryl-δ-hydroxy-γ-lactones 5a–d with potential antifeedant and anticancer activity were developed. In the first method starting from corresponding enantiomers of γ,δ-unsaturated esters 4a–d derived from enzymatically resolved allyl alcohols 1a–d, both enantiomers of hydroxylactones 5a–d were synthesized with high enantiomeric excesses (73%–97%). Configurations of the stereogenic centers of the synthesized compounds were assigned based on the mechanism of acidic lactonization of esters 4a–d in the presence of m-chloroperbenzoic acid (m-CPBA). An alternative method for the production of optically active trans-β-aryl-δ-hydroxy-γ-lactones 5a–d was lipase-catalyzed kinetic resolution of their racemic mixtures by transesterification with vinyl propionate as the acyl donor. The most efficient enzyme in the screening procedure was lipase B from Candida antarctica. Its application on a preparative scale after 6 h afforded unreacted (+)-(4S,5R,6S)-hydroxylactones 5a–d and (+)-(4R,5S,6R)-propionates 6a–d, most of them with high enantiomeric excesses (92%–98%). Resolution of lactone 5d with bulky 1,3-benzodioxol ring provided products with significantly lower optical purity (ee = 89% and 84% for hydroxylactone 5d and propionate 6d, respectively). The elaborated methods give access to both enantiomers of trans-β-aryl-δ-hydroxy-γ-lactones 5a–d with the defined absolute configurations of stereogenic centers, which is crucial requirement for the investigations of relationship: spatial structure–biological activity.

Optically active lactones are of high importance due to their application as building blocks in natural therapeutic compounds [19,20] and the well-known relationship between the configuration of stereogenic centers and their biological activity [21,22].Therefore, there is a growing need to develop new methods for the synthesis of enantiomerically enriched lactones.These strategies involve i.a.alkylation of chiral precursors [23,24], catalytic asymmetric hydrogenation of butenolides [25], kinetic resolution of lactone precursors by hydrolysis of ester bond [26,27] and different whole-cell mediated reactions including reduction of unsaturated formyl esters [28], stereoselective reduction of a carbonyl group in γ-acetyl-γ-lactones [29] and enantioselective hydrolysis of nitriles [30].
With regard to these reports, we have taken up research on the synthesis of a series of racemic γ-lactones with various β-phenyl substituents at the lactone ring [31][32][33][34][35].We have also developed convenient chemoenzymatic methods for the synthesis of optically active iodolactones with antiproliferative activity starting from enantiomerically enriched allyl alcohols with 4-arylbut-3-en-2-ol system as chiral precursors [36][37][38].In this paper, we would like to present two novel, alternative methods for the production of optically active trans-β-aryl-δ-hydroxy-γ-lactones.One of these methods involves the application of chiral precursors, and an alternative method is based on lipase-catalyzed kinetic resolution of racemic mixtures in the transesterification process.

Results and Discussion
In our previous paper [33] using methodology reported earlier by Gowri-Shankar [39], we reported the synthesis of racemic trans-δ-hydroxy-γ-lactones 5a-d as the main products of lactonization of corresponding of γ,δ-unsaturated esters 4a-d.Because of their proven biological activities, i.e., antifeedant and antiproliferative, we decided to evaluate methods for the production of these compounds in an optically active form.
The first elaborated method was based on the lactonization of chiral precursors, enantiomerically enriched γ,δ-unsaturated esters 4a-d in the presence of m-CPBA.These precursors are products of the stereoselective Claisen-Johnson rearrangement of enzymatically resolved allyl alcohols 1a-d.During this reaction, a complete transfer of chirality from atom C-2 of alcohols to the benzylic position C-3 of ester is observed.The details of this two-step synthesis involving lipase-catalyzed enzymatic resolution of allyl alcohols 4a,b,d and their orthoester Claisen rearrangement were reported earlier for synthesis of enantiomeric pairs of esters with unsubstituted phenyl ring (4a), p-methylphenyl substituent (4b) and 1,3-benzodioxol ring (4d) [36,38].In order to obtain both enantiomers of ester 4c with p-methoxyphenyl substituent, a kinetic resolution of racemic alcohol 1c via transesterification process catalyzed by lipase B from C. antarctica (CAL-B) was applied (Scheme 1).Reaction was monitored by chiral GC after derivatization of unreacted alcohol into acetate.Standards used in this analysis-racemic acetate 2 and propionate 3-were synthesized by the esterification of rac-1c with acetyl and propionyl chloride, respectively (Section 3.3).reactions including reduction of unsaturated formyl esters [28], stereoselective reduction of a carbonyl group in γ-acetyl-γ-lactones [29] and enantioselective hydrolysis of nitriles [30].
With regard to these reports, we have taken up research on the synthesis of a series of racemic γ-lactones with various β-phenyl substituents at the lactone ring [31][32][33][34][35].We have also developed convenient chemoenzymatic methods for the synthesis of optically active iodolactones with antiproliferative activity starting from enantiomerically enriched allyl alcohols with 4-arylbut-3-en-2ol system as chiral precursors [36][37][38].In this paper, we would like to present two novel, alternative methods for the production of optically active trans-β-aryl-δ-hydroxy-γ-lactones.One of these methods involves the application of chiral precursors, and an alternative method is based on lipase-catalyzed kinetic resolution of racemic mixtures in the transesterification process.

Results and Discussion
In our previous paper [33] using methodology reported earlier by Gowri-Shankar [39], we reported the synthesis of racemic trans-δ-hydroxy-γ-lactones 5a-d as the main products of lactonization of corresponding of γ,δ-unsaturated esters 4a-d.Because of their proven biological activities, i.e., antifeedant and antiproliferative, we decided to evaluate methods for the production of these compounds in an optically active form.
Both enantiomeric forms of four γ,δ-unsaturated esters 4a-d were subjected to lactonization with m-CPBA and catalytic amount of trifluoroacetic acid (Scheme 2).
Both enantiomeric forms of four γ,δ-unsaturated esters 4a-d were subjected to lactonization with m-CPBA and catalytic amount of trifluoroacetic acid (Scheme 2).During the reaction two epoxyesters, threo and erythro are formed and four possible lactones could be the final products: two γ-lactones and two δ-lactones (Figure S1).Unfortunately, similarly to the reaction carried out earlier for racemic compounds [33], δ-lactones were isolated as the unseparable mixture of stereoisomers.cis γ-Lactones formed from erythro epoxyesters were not isolated and the only isomers isolated in pure form were trans γ-lactones 5a-d, which are the products of lactonization of threo epoxyesters.Advantageous formation of trans isomers 5a-d over the cis isomers can be explained by the fact that in threo epoxyesters oxygen of the epoxide ring is less hindered by large aromatic substituent compared with erythro epoxyesters (Scheme 3).During the reaction two epoxyesters, threo and erythro are formed and four possible lactones could be the final products: two γ-lactones and two δ-lactones (Figure S1).Unfortunately, similarly to the reaction carried out earlier for racemic compounds [33], δ-lactones were isolated as the unseparable mixture of stereoisomers.cis γ-Lactones formed from erythro epoxyesters were not isolated and the only isomers isolated in pure form were trans γ-lactones 5a-d, which are the products of lactonization of threo epoxyesters.Advantageous formation of trans isomers 5a-d over the cis isomers can be explained by the fact that in threo epoxyesters oxygen of the epoxide ring is less hindered by large aromatic substituent compared with erythro epoxyesters (Scheme 3).Chiral GC analysis indicated that enantiomeric excesses of lactones 5a-d corresponded with those determined earlier for starting esters 4a-d and ranged from 73% to 90% in the case of hydroxylactones obtained from (S)-esters 4a-d and 82%-97% for their antipodes synthesized from (R)-esters 4a-d.The important challenge was the assignment of the configurations of stereogenic centers at C-4, C-5 and C-6 for newly synthesized enantiomers, which was made based on the mechanism of acidic lactonization (Scheme 3).Monitoring the composition of the reaction mixture did not show the presence of possible intermediate diolester, which excluded the opening of epoxide ring by water and confirmed the mechanism of lactonization proposed by Olejniczak et al. [41].This mechanism involves the protonation of an oxirane ring followed by its nucleophilic opening by the attack of an oxygen from carboethoxy group with the simultaneous formation of hydroxy group.In the next step of reaction, nucleophilic addition of water to carboethoxy group takes place with subsequent release of the ethanol.Considering lactonization of (S)-esters 4a-d (Scheme 3), the configuration R at C-4 of forming trans-(−)-δ-hydroxyγ-lactones 5a-d is the result of the configuration of starting ester, and its apparent change is only the result of different priority of substituents after formation of oxirane ring.The configurations S at C-5 and R at C-6 are the consequence of stereochemical course of reaction in which the carboethoxy group approaches the C-5 atom from the opposite side of the oxirane ring.Thus, the C-O bond of the γ-lactone ring and hydroxy group are oriented antiperiplanary.Similar reasoning let us assign the configuration of (+)-δ-hydroxy-γ-lactones 5a-d formed from (R)-esters 4a-d as 4S,5R and 6S.The presented stereochemical course of reaction was earlier confirmed for the products of lactonization of ethyl Chiral GC analysis indicated that enantiomeric excesses of lactones 5a-d corresponded with those determined earlier for starting esters 4a-d and ranged from 73% to 90% in the case of hydroxylactones obtained from (S)-esters 4a-d and 82%-97% for their antipodes synthesized from (R)-esters 4a-d.The important challenge was the assignment of the configurations of stereogenic centers at C-4, C-5 and C-6 for newly synthesized enantiomers, which was made based on the mechanism of acidic lactonization (Scheme 3).Monitoring the composition of the reaction mixture did not show the presence of possible intermediate diolester, which excluded the opening of epoxide ring by water and confirmed the mechanism of lactonization proposed by Olejniczak et al. [41].This mechanism involves the protonation of an oxirane ring followed by its nucleophilic opening by the attack of an oxygen from carboethoxy group with the simultaneous formation of hydroxy group.In the next step of reaction, nucleophilic addition of water to carboethoxy group takes place with subsequent release of the ethanol.Considering lactonization of (S)-esters 4a-d (Scheme 3), the configuration R at C-4 of forming trans-(−)-δ-hydroxy-γ-lactones 5a-d is the result of the configuration of starting ester, and its apparent change is only the result of different priority of substituents after formation of oxirane ring.The configurations S at C-5 and R at C-6 are the consequence of stereochemical course of reaction in which the carboethoxy group approaches the C-5 atom from the opposite side of the oxirane ring.Thus, the C-O bond of the γ-lactone ring and hydroxy group are oriented antiperiplanary.Similar reasoning let us assign the configuration of (+)-δ-hydroxy-γ-lactones 5a-d formed from (R)-esters 4a-d as 4S,5R and 6S.The presented stereochemical course of reaction was earlier confirmed for the products of lactonization of ethyl esters of 3,7-dimethyl-4,5-epoxyoctanoic acid and its 7-methyl homolog, in which the configurations of stereocenters were determined by CD (Circular Dichroism) measurements [42].
The alternative method for the production of trans-β-aryl-δ-hydroxy-γ-lactones 5a-d in enantiomerically enriched form was kinetic resolution of their racemic forms synthesized previously [33].Due to the large difference in sizes of two substituents at the stereogenic center C-6 joined with a secondary hydroxy group, the hydroxylactones 5a-d seemed to be excellent substrates for the lipase-catalyzed transesterification [43,44], and high enantiomeric excesses of the products were expected.In the screening procedure, racemic trans-β-phenyl-δ-hydroxy-γ-lactone 5a as a model substrate was subjected to the action of four commercially available lipases in the presence of vinyl propionate as the acyl donor (Scheme 4).The reaction was conducted in diisopropyl ether (DIPE) and monitored by chiral gas chromatography with racemic lactones 5a-d [33] and racemic propionates 6a-d (Section 3.3) as the standards.The results are shown in Table 1.
The alternative method for the production of trans-β-aryl-δ-hydroxy-γ-lactones 5a-d in enantiomerically enriched form was kinetic resolution of their racemic forms synthesized previously [33].Due to the large difference in sizes of two substituents at the stereogenic center C-6 joined with a secondary hydroxy group, the hydroxylactones 5a-d seemed to be excellent substrates for the lipasecatalyzed transesterification [43,44], and high enantiomeric excesses of the products were expected.In the screening procedure, racemic trans-β-phenyl-δ-hydroxy-γ-lactone 5a as a model substrate was subjected to the action of four commercially available lipases in the presence of vinyl propionate as the acyl donor (Scheme 4).The reaction was conducted in diisopropyl ether (DIPE) and monitored by chiral gas chromatography with racemic lactones 5a-d [33] and racemic propionates 6a-d (Section 3.3) as the standards.The results are shown in Table 1.The most effective biocatalyst was CAL-B.At 49% conversion after 1 h of the reaction catalyzed by this enzyme, the enantiomeric excesses of the slower reacting (+)-enantiomer of δ-hydroxy-γ-lactone 5a and its propionate (+)-6a were 96% and 99% respectively.Continuing the process, a slight decrease of ee for (+)-propionate 6a was observed, reaching 96% after 6 h, whereas ee of hydroxylactone 5a raised to 99%.Good results were achieved for Lipozyme TL IM (Thermomyces lanuginosus lipase) as well, but the transesterification proceeded at a lower rate.After 6 h, the 53% conversion was observed.During this period, the enantiomeric excess of unreacted (+)-hydroxylactone 5a gradually increased from 12% to 91%, whereas the optical purity of (+)-propionate 6a decreased from 99% to 80%.High  The most effective biocatalyst was CAL-B.At 49% conversion after 1 h of the reaction catalyzed by this enzyme, the enantiomeric excesses of the slower reacting (+)-enantiomer of δ-hydroxy-γ-lactone 5a and its propionate (+)-6a were 96% and 99% respectively.Continuing the process, a slight decrease of ee for (+)-propionate 6a was observed, reaching 96% after 6 h, whereas ee of hydroxylactone 5a raised to 99%.Good results were achieved for Lipozyme TL IM (Thermomyces lanuginosus lipase) as well, but the transesterification proceeded at a lower rate.After 6 h, the 53% conversion was observed.During this period, the enantiomeric excess of unreacted (+)-hydroxylactone 5a gradually increased from 12% to 91%, whereas the optical purity of (+)-propionate 6a decreased from 99% to 80%.High enantioselectivity (E > 200) was found for Amano PS (lipase from Burkholderia cepacia).In this case, high enantiomeric enrichment of (+)-propionate 6a was found (ee = 99%), but the ee of unreacted (+)-hydroxylactone 5a even after 6 h was unsatisfactory (65%).After 6 h of the process catalyzed by CCL low (ee = 55%) and moderate (ee = 71%), enantiomeric purity of substrate 5a and ester 6a, respectively, was observed at the 44% conversion.For all enzymes, prolonging reaction time affected neither conversion nor enantiomeric composition of the products.
Taking into consideration the highest enantiomeric excesses obtained after 6 h for both hydroxylactone 5a and propionate 6a, CAL-B was selected as the biocatalyst for the kinetic resolution of hydroxylactones 5a-d on a preparative scale (Table 2).After 6 h of reaction, the conversion of the substrates in all cases was 50%-52%.The unreacted substrates 5a-d and their propionates 6a-d were separated by column chromatography in yields ranging from 34% to 45%, respectively, which is a very satisfactory result for kinetic resolution.(+)-δ-Hydroxy-γ-lactone 5a with unsubstituted phenyl ring and its (+)-propionate 6a were obtained with a very high enantiomeric excesses, 99% and 92%, respectively.Taking into consideration the increasing size of an aryl substituent at β-position in substrates 5b-d, comparably high enantioselectivity of the kinetic resolution was expected.It was confirmed in the case of (+)-hydroxylactones with p-methylphenyland p-methoxyphenyl ring (5b and 5c) (ee = 99% and 98%, respectively) and their corresponding (+)-propionates 6b and 6c (ee = 91% and 98%, respectively).The presence of additional 1,3-benzodioxol ring condensed with benzene resulted in the lowest effectiveness of kinetic resolution for hydroxylactone 5d, of which (+)-enantiomer was obtained with ee = 89%.Optical purity of its (+)-propionate 6d was comparably low (ee = 84%).A similar effect of the 1,3-benzodioxol ring on the enantioselectivity of lipase-catalyzed transesterification with vinyl propionate was also reported for allyl alcohol 1d [37].It is likely that the steric hindrance in the molecules alcohol 1d and hydroxylactone 5d impedes the binding of these substrates to the active site of the lipase.Comparison of the specific rotation signs of slowly transesterified δ-hydroxy-γ-lactones 5a-d and those found for isomers with defined absolute configurations synthesized earlier from (R)-and (S)-esters 4a-d let us establish undoubtedly the absolute configurations of both products of enzymatic transesterification.Slowly reacting (+)-δ-hydroxy-γ-lactones 5a-d possessed configurations 4S,5R,6S, and opposite configurations 4R,5S,6R were consequently ascribed to (+)-propionates 6a-d.The configuration of these products at C-6 is consistent with that predicted based on the Kazlauskas' rule.In this case, taking into consideration that the order of substituents at stereogenic center C-6 follows the rule: OH > large substituent (β-aryl substituted γ-lactone ring) > medium substituent (methyl group), lipases preferentially catalyze the esterification of isomers with configuration 6R, leaving those with 6S configuration untouched.
The course of reactions was monitored by gas chromatography (GC) on Agilent Technologies (Palo Alto, CA, USA) 6890N with hydrogen as the gas carrier and autosampler instrument using DB-5HT column (polyimide-coated fused silica tubing, 30 m × 0.25 mm × 0.10 µm).The temperature programme was as follows: injector 220 Products of synthesis and enzymatic reactions were separated by preparative column chromatography on silica gel (Kieselgel 60, 230-400 mesh, Merck) using mixtures of various organic solvents as a mobile phase.
The NMR spectra ( 1 H-NMR, 13 C-NMR, HMBC and HMQC) were recorded in a CDCl 3 solution on a Bruker Avance II 600 MHz spectrometer (Bruker, Rheinstetten, Germany).The IR spectra were determined using a Mattson IR 300 Thermo Nicolet spectrophotometer (Mattson, Waltham, MA, USA) using KBr pellets or as neats.High resolution mass spectra (HRMS) were recorded using electron spray ionization (ESI) technique on spectrometer Waters ESI-QTOF Premier XE (Waters Corp., Millford, MA, USA).The optical rotations were measured on a Jasco P-2000-Na digital polarimeter (Easton, PA, USA) with an intelligent Remote Module (iRM) controller.The indexes of refraction were measured on an Abbe refractometer (Carl Zeiss, Jena, Germany).Melting points were determined on Boetius apparatus (Nagema, Germany).

Enzymatic Resolution of Racemic Allyl Alcohol 1c
To the solution of racemic alcohol 1c (5 g, 28 mmol) in DIPE (50 mL), 1 mL of vinyl propionate and lipase B from C. antartica (2 g) were added.The reaction was carried out in the 250 mL round-bottom flask on a magnetic stirrer at room temperature.The reaction was monitored by chiral GC after treating the samples with acetyl chloride to derivatize the unreacted alcohol into corresponding acetate 2. Racemic acetate 2 and propionate 3 were used as the standards.After 4 h of reaction, the enzyme was filtered off and the organic solvent was evaporated in vacuo.Products of enzymatic tranesterification were separated by column chromatography (hexane/acetone, 15:1) and analyzed by chiral GC.Hydrolysis of (+)-(2R,3E)-4-(4 -methoxyphenyl)but-3-en-2-yl propionate.Ester (+)-3 (11 mmol) was hydrolyzed under reflux in 5% ethanolic solution of NaOH (40 mL).When the reaction was finished (3 h, TLC), ethanol was evaporated in vacuo and the residue was diluted with water.Alcohol (+)-1c was extracted with CH 2 Cl 2 (3 × 30 mL) and the organic fractions were pooled, washed with brine until neutral and dried over anhydrous MgSO 4 .After evaporation of solvent in vacuo, pure alcohol (+)-1c was obtained.

Johnson-Claisen Rearrangement of Enantiomerically Enriched Allyl Alcohols (−)-1c and (+)-1c
Mixture of enantiomerically enriched alcohol (−)-1c or (+)-1c (0.012 mol), triethyl orthoacetate (25 mL) and a drop of propionic acid was heated at 138-140 • C under reflux for 24 h with simultaneous removal of ethanol by distillation.The crude product was purified by column chromatography (hexane/acetone, 10:1, v/v).Enantiomerically enriched γ,δ-unsaturated ester (S)-4a-d or (R)-4a-d (0.007 mol) was dissolved in 50 mL of CHCl 3 and m-CPBA (0.008 mol) and a drop of trifluoroacetic acid were added.The reaction mixture was stirred on a magnetic stirrer for 24 h.Then, the crude mixture was diluted with CHCl 3 (100 mL), and successively washed with NaHSO 3 , NaHCO 3 and brine.The combined organic layers were dried over anhydrous MgSO 4 and filtered.The organic solvent was evaporated under vacuo and after column chromatography (silica gel, hexane/isopropanol/acetone/ethyl acetate/methylene chloride/diethyl ether (100:10:0.1:0.1:0.1:0.1, v/v/v/v/v/v) unseparable mixture of γ-hydroxy-δ-lactones and pure δ-hydroxy-γ-lactones 5a-d were isolated.Their spectroscopic data were consistent those reported for their racemic form [33]. To a solution of hydroxylactone 5a in 10 mL of DIPE 5 mg of lipase and 0.1 mL of vinyl propionate was added.The reaction mixture was stirred in a magnetic stirrer in 20 mL-vial at room temperature.At several time intervals, the samples (0.5 mL) were withdrawn from reaction mixture and filtered through Celite 560.The organic solvent was evaporated under vacuo, and the residue was dissolved in acetone (0.2 mL) and analyzed by CGC.The results are shown in Table 2.

Preparative Transesterification
To a solution of β-aryl-δ-hydroxy-γ-lactone 5a-d (0.97 mmol) in 50 mL of DIPE, 100 mg of lipase B from Candida antarctica (CAL-B) and 1 mL of vinyl propionate were added and the mixture was stirred at room temperature.The reaction was monitored by chiral GC and racemic hydroxylactones 5a-d and propionates 6a-d were used as the standards.After 6 h the enzyme was filtered off and organic solvent was evaporated in vacuo.Slowly reacting hydroxylactones 5a-d and propionates 6a-d were