Lipase-catalyzed kinetic resolution of 4-aryl-and 4-heteroarylbut-3-en-2-ols

Several 4-aryl-and 4-heteroarylbut-3-en-2-ones [ 2a-c , bearing 4-phenyl-, 4-(furan-2-yl)- and 4-(1-benzyl-1 H -indol-3yl)-substituents, respectively] were prepared by condensation reaction of acetone with the corresponding aldehydes ( 1a-c ). Reduction of the ketones ( 2a,b ) with NaBH 4 resulted in the formation of the corresponding racemic secondary alcohols ( rac - 3a,b ). On the other hand, reduction of the ketone bearing indole moiety ( 2c ) gave an unstable product. The lipase-catalyzed enantiomer selective acetylation of the alcohols ( rac - 3a,b ) by vinyl acetate has been investigated with a crude lipase from submerged fermentation (SmF) of a thermophilic fungus, with several crude enzyme preparations from solid state fermentation (SSF) of selected mesophilic fungi and with several commercially available lipases. The commercial and SmF lipases and the majority of SSF preparations exhibited high but usual enantiomer selectivities and resulted in the formation of ( R )-acetates [( R )- 4a,b ] according to the Kazlauskas’ rule. Several SSF preparations, however, behaved as selective anti-Kazlauskas catalysts.


Introduction
There is an ever growing demand in the pharmaceutical and fine chemical industry for production of optically active intermediates.In this area, biocatalysis has expanded from a niche technology to a widely used manufacturing method. 1 Biocatalysts − enzymes and whole-cell systems − as a result of their chiral nature are predominantly suited for production of optically pure stereoisomers.Several beneficial characteristics − broad substrate tolerance, no need for cofactors − make some hydrolases as useful biocatalysts for synthetic biotransformations. 2 Among hydrolases, lipases (triacylglycerol carboxyl ester hydrolases, EC 3.1.1.3)proved to be the most versatile biocatalysts in stereoselective biotransformations such as kinetic resolutions, [1][2][3] deracemisations and dynamic kinetic resolutions. 4Lipases are particularly suited for the kinetic resolution of secondary alcohols, since these enzymes exhibit high stability and enantioselectivity in organic solvents, and they are environmentally friendly. 5The commercial availability of a range of lipase preparations at low cost has also widely expanded their use as biocatalysts in organic chemistry.
Several studies on screening some thermophilic filamentous fungi in submerged fermentation (SmF) conditions, 6 and some poorly studied mesophilic fungi in solid state fermentation (SSF) 7 conditions for lipase/esterase activity resulted in biocatalysts exhibiting high enantiomer selectivity in synthetic biotransformations.The recent study of fungal SSF preparations acting on several typical secondary alcohols − such as 1-phenylethanol, 1-cyclohexylethanol and 1-(napth-2-yl)ethanol − as model substrates indicated that SSF biocatalysts with lipase activity are applicable in kinetic resolutions of racemic secondary alcohols. 7As the gently dried SSF preparations do not require costly downstream processes, they can be considered as inexpensive, naturally immobilized biocatalysts.In the enzyme-catalyzed acetylation of the three selected racemic secondary alcohols 7 several of the SSF biocatalysts exhibited high but usual enantioselectivities [formation of the (R)-acetates] according to the so-called "Kazlauskas' rule", 8 whereas one strain (Mucor hiemalis) resulted in a selective "anti-Kazlauskas" biocatalyst by SSF. 7 As the secondary alcohols in this study (with SSF biocatalysts) contained rigid (aromatic) or more flexible (cyclohexane) ring adjacent to the asymmetric carbon centre, 7 one of our aims was to broaden the selection of the substrates for these SSF preparations by studying the kinetic resolution of allylic alcohols containing the rings at more remote position from the asymmetric center.The enantiomerically pure forms of such allylic alcohols (4-aryl-and 4-heteroarylsubstituted but-3-en-2-ols) and acetates are useful synthons which can be transformed to a wide range of more complex molecules. 9Different routes − such as kinetic resolutions by enzymatic acylation in apolar solvents 10,11 or in ionic liquids 12 − leading to both enantiomeric forms of chiral 4-aryl/heteroarylbut-3-en-2-ols have been reported.Other methods − such as ruthenium- 13 or enzyme-catalyzed asymmetric reduction of the corresponding ketones 14 or dynamic kinetic resolution of the racemic secondary alcohols including 4-phenyl-and 4-(furan-2-yl)but-3-en-2ols with lipases 15 − however result in formation of just one particular enantiomeric form.In most cases, however, the availability of both enantiomers of the chiral starting materials in high enantiomer purity is essential.Therefore, we also considered to be important investigating the "anti-Kazlauskas" nature of the hydrolase preparations − which can lead to the opposite enantiomer in dynamic kinetic resolutions as the usual "Kazlauskas" enzymes − with the secondary allylic alcohol type substrates as well.We report here these aspects in kinetic resolution of racemic 4-aryl/heteroarylbut-3-en-2-ols with the best SSF hydrolase preparations, in comparison with commercial lipases.

Results and Discussion
From our previous screens of thermophilic (SmF) 6 and mesophilic filamentous fungi (SSF), 7 several biocatalysts having comparable lipase activities and enantiomer selectivities to the widely used commercially available lipases were available for further screening.Within the strains of the SSF screen, 7 a preparation (Mucor hiemalis) with the less usual anti-Kazlauskas selectivity was also found.Therefore we could study the kinetic resolution of several less characterized racemic allylic alcohols by a range of commercial and in-house prepared enzymes.
Reduction of the ketones proceeded smoothly.In the case of 4-phenylbut-3-en-2-one [(E)and (Z)-2a] the reaction was performed with NaBH 4 in aqueous methanol to yield a hardly separable diastereomeric mixture of the secondary allylic alcohols [(E)-and (Z)-3a].To avoid working with this mixture, pure alcohol (E)-3a was prepared from the commercially available (E)-2a under the same conditions.Reduction of the 4-(furan-2-yl)but-3-en-2-ones [(E)-and (Z)-2b] was also performed with NaBH 4 in aqueous solvent system in the presence of Ba(OH) 2 17 leading to a separable mixture of the secondary allylic alcohols [(E)-and (Z)-3b].Reduction of the indole-containing ketone [(E)-2c] under similar conditions resulted in the formation of a product (detected by TLC) which decomposed under the normal work up conditions.
The majority of the newly tested lipase preparations proved to be quite effective in the enzyme-catalyzed kinetic resolution of racemic (3E)-4-phenyl-but-3-en-2-ol (rac-3a) too.BUTE-3b and SSF-9 biocatalysts were found to be the best (Table 1, entries 8, 9), performing the reaction with even better enantiomer selectivity (E) and productivity than that was observed with the best commercial lipases (Table 1, entries 1, 2).
ARKAT USA, Inc.For details on the biocatalysts and reaction conditions, see the Experimental section.b The conversion and the ee of 4a were determined by GC on Hydrodex-β-PM column.c Enantiomer selectivity (E) was calculated from conversion and ee 4a . 18Due to sensitivity to experimental errors, E values calculated in the 100-300 range are reported as >100, values in the 300-500 range are reported as >200 and values calculated above 500 are given as »200.
These data support the former observations that many preparations of filamentous fungi exhibiting lipase/esterase activity produced by submerged (SmF) 6 or solid state fermentation (SSF) 7 are useful biocatalysts in stereoselective organic reactions.In the kinetic resolution of the allylic alcohol rac-3a, two SSF preparations exhibited better "anti-Kazlauskas" selectivities (E ~ 7, Table 1, entry 13: Mucor hiemalis ATCC 26035, entry 14: Rhizomucor pusillus WFPL 267 A) than the already known anti-Kazlauskas biocatalyst (SSF-63, Mucor hiemalis NRRL 13.009, previously tested on racemic 1-phenylethanol 7 ).Although the selectivities and productivities of these biocatalysts were only moderate, they represent a useful type of complementary selectivity which was precedented in protease-catalyzed reactions so far.15c The enantiomer selective acylation of racemic (3E)-4-(furan-2-yl)but-3-en-2-ol (rac-3b) using lipases (Table 2) has not been characterized in detail, as no data on the lipase-catalyzed kinetic resolution of this substrate was found.However, dynamic kinetic resolution of rac-3b with immobilized P. cepacia lipase exhibiting the usual "Kazlauskas" selectivity is already known. 15For details on the biocatalysts and reaction conditions, see the Experimental section.b The conversion and the ee values of 3b and 4b were determined by GC on Hydrodex-β-PM column.
c Enantiomer selectivity (E) was calculated from conversion and ee 4b 18 (and confirmed by independent calculation from ee 3b and ee 4b 19 ).Due to sensitivity to experimental errors, E values calculated in the 100-300 range are reported as >100, values in the 300-500 range are reported as >200 and values calculated above 500 are given as »200.
Interestingly, replacement of the phenyl (in rac-3a) to a similarly bulky but more polar furan-2-yl moiety (in rac-3b) at position 4 of the racemic secondary allylic alcohol resulted in significant decrease of the enantiomer selectivity (E) of several commercial lipases such as Lipase AK and CaLB (from E > 100 in Table 1, entries 6 and 5 to E = 39 and E = 9 in Table 2, entries 5 and 7; respectively).This drop in enantiomer selectivity (E) in the kinetic resolution of the furyl-substituted alcohol (rac-3b) in comparison with E value for the phenyl-substituted alcohol (rac-3b) was also experienced with the biocatalysts of filamentous fungal origin prepared by us (Table 2, entries 9-13 vs. Table 1, entries 8-12; respectively).The most significant decrease of E has been detected in the reactions catalyzed by BUTE-3b and SSF-9 preparations (from E »200 in Table 1, entries 8 and 9 to E >100 and E >200 in Table 2, entries 10 and 9; respectively).
The fungal SSF preparations exhibiting "anti-Kazlauskas" selectivity with the phenylsubstituted substrate (rac-3a) retained their sense of enantiomeric preference in the biotransformation of the furan-2-yl-substituted alcohol (rac-3b) as well.Thus, they also acted as ARKAT USA, Inc.

Conclusions
It was demonstrated that kinetic resolution of racemic 4-aryl and 4-heteroaryl-substituted but-3en-2-ols (rac-3a,b) can be effectively performed with lipase-catalyzed enantiomer selective acetylation by vinyl acetate.In addition to the commercially available lipases, several crude lipase preparations from submerged (SmF) or solid state (SSF) fermentations of filamentous fungi proved to be useful biocatalysts in this kinetic resolution.Whereas all of the commercial lipases and most of the fungal preparations exhibited high but usual "Kazlauskas-type" enantiomer selectivities with the investigated racemic secondary allylic alcohols (rac-3a,b), several of our SSF preparations turned out to be "anti-Kazlauskas" biocatalysts.