Fast Microwave-Assisted Resolution of ( ± )-Cyanohydrins Promoted by Lipase from Candida antarctica

Enzymatic kinetic resolution (EKR) of (±)-cyanohydrins was performed by using immobilized lipase from Candida antarctica (CALB) under conventional ordinary conditions (orbital shaking) and under microwave radiation (MW). The use of microwave radiation contributed very expressively on the reduction of the reaction time from 24 to 2 h. Most importantly, high selectivity (up to 92% eep) as well as conversion was achieved under MW radiation (50-56%).


Introduction
][6][7] Microwave has proven to be a very important alternative energy source and has become a usual synthetic tool for chemists.One of the most expressive advantages of the microwave-assisted transformations is centered on the reduction of the reaction time, reproducibility and in many cases, pronounced increase of yields in comparison with the transformations carried out under conventional heating. 3,6,8However, its application for enzymatic-assisted transformations is still almost unexplored. 7,8ecently, our group reported the results concerning the esterification of (±)-mandelonitrile with vinyl acetate as acylating agent, in toluene catalyzed by Candida antarctica lipase B (CALB) under orbital shaking and under microwave radiation. 6Aiming to explore more extensively the scope and limitations of such conditions for enzymatic catalyzed asymmetric transformations, in this paper present our results on the enzymatic kinetic resolution of cyanohydrins by CALB under microwave radiation and conventional conditions.
The enantiomeric excesses (ee) of (R)-alcohols and (S)-acetates were determined by gas chromatography analyses with chiral stationary phase employing the retention times obtained for both enantiomers (±)-2a-g and (±)-3a-g.Reagents and solvents were used as obtained commercially and when necessary were purified and/or dried using procedures described in the literature. 9Column chromatography separations were carried out using silica gel 60 (400-230 mesh) with hexane and ethyl acetate mixtures as eluent.Optical rotations were measured in CHCl 3 , in a JASCO P2000 polarimeter equipped with a 589 nm Na lamp.

Derivatization of unreacted alcohols
The alcohols not esterified by CALB were added to a test tube [2a (0.22 mmol), 2b (0.18 mmol), 2e (0.22 mmol), 2f (0.16 mmol) and 2g (2.19 mmol)] followed by pyridine (6.2 mmol) and acetic anhydride (5.2 mmol) then submitted to magnetic stirring for 24 h.After that, two drops of a 10% HCl solution were added to each test tube.Water (10 mL) was added, followed by extraction with ethyl acetate (3 × 3 mL).The combined organic phases were dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure.Subsequently, samples were diluted in ethyl acetate in vials and analyzed to determine the enantiomeric excesses by GC-FID analysis.

Enzymatic kinetic resolution (EKR) of (±)-cyanohydrins (2a-g) under orbital shaking
The reaction of (±)-2a, under reaction conditions presented in Table 1, resulted in the desired acylated product (S)-3a in good conversion and reasonable selectivity (c = 57%, 87% ee p ) (entry 1, Table 1).Compounds (±)-2b and (±)-2c, both possessing electronwithdrawing substituents at the para position, presented virtually the same behavior with good conversions and high enantioselectivities (96% ee p and 97% ee p , respectively -entries 2 and 3, Table 1).Higher reaction time was necessary to achieve just reasonable conversion (32%) and poor enantioselectivity (28% ee p ) when (±)-cyanohydrin 2d, bearing an electron-donating group at the para position, was submitted to the same reaction conditions (entry 4, Table 1).On the other hand, a p-OMesubstituted substrate, also an electron-rich substituent (entry 5, Table 1), presented similar results to those observed for the examples of entries 2 and 3, containing electron-withdrawing substituents.Compound (±)-2f, the only example bearing a meta-substituent, also presented practically the same conversion (47%) and selectivity (92% ee p ) averages as most compounds discussed before.The only alkyl cyanohydrin 2g submitted to the study presented very poor results, suggesting that the low selectivity is a consequence of the low steric volume of the alkyl chain, which is in accordance with enzymatic resolutions (entry 7, Table 1).
The low conversion observed for cyanohydrin 2d can be attributed to its instability under the reaction conditions.The reaction performance was monitored by GC-MS analysis and beyond the expected unreacted substrate (2d) and the acetylated product 3d, there was also identified the aldehyde precursor of the cyanoydrin, what is a result of the thermic cleavage of the substrate. 6,13he substrates were also submitted to the enzymatic kinetic resolution, under similar reaction condition, instead to the fact that the energy source was supplied by a microwave oven and the results are summarized in Table 2.

Enzymatic kinetic resolution of (±)-cyanohydrins (2a-f) under microwave radiation
It is well known that microwave radiation is frequently associated with improving reaction performances, accelerating organic transformations in comparison with conventional heating, which is due to the higher efficiency of the energy transfer process to the reaction media. 6,13n our experiments, in a general sense, the reaction time under microwave radiation was deeply decreased to just Scheme 1. Syntheses of (±)-cyanohydrins 2a-g and the corresponding acetates 3a-g.
few hours (2-6) resulting in selectivity and conversions of the same average as those of the conventional conditions.In only 2 h cyanohydrin 2a was efficiently converted to the corresponding acetate (S)-3a (52%) in moderate selectivity (79% ee p , entry 1, Table 2).Compounds (±)-2b and (±)-2c yielded the acetylated products in good conversions (c = 53% and 50%, respectively) and selectivities (90% and 92% ee p , respectively) (entries 2 and 3, Table 2).As observed for the study carried out under orbital shaking at 32 o C, the reaction with 2d proceeded poorly presenting very low conversion and optical purity (entry 4, Table 2).

Enzyme recyclability studies
To examine the stability of the enzyme under the microwave-assisted reaction conditions, cyanohydrin 2a was selected as model compound for the recyclability resolution reaction.After each reaction, the catalyst was isolated by filtration, washed with ethyl acetate, dried    at room temperature and submitted to the next reaction cycle.Constant conversion decrease was observed in each cycle as can be visualized in the graph in Figure 1.
Longer reaction times were also necessary to achieve high conversions.Even being compromised, the activity of the enzyme was reasonably maintained, opening opportunities for recyclability of the biocatalyst for preparative purposes, which is quite attractive in the industrial point of view, considering the high price of this catalyst.

Conclusions
In summary, we demonstrated that microwave radiation can be useful also for enzymatic-assisted transformations.Albeit not extraordinary, good conversions and selectivities were achieved and more importantly, the required reaction time was deeply diminished for all studied cases leading to the corresponding desired compounds in reasonable good optical enrichments.The reutilization of the catalyst was possible demonstrating that the activity of the enzyme was not completely lost under the microwave conditions, which is quite attractive considering the price of the catalyst.
a Conversion: c =

Table 3 .
Optical rotations of the cyanohydrins 2a-g and acetates 3a-g obtained by kinetic resolution using CALB