Enzymatic Resolution of Ethyl 3-Hydroxy-3-Phenylpropanoate and Analogs using Hydrolases

Este trabalho contribui com o estudo da relação substrato-modelo em reações de hidrólise de ésteres β-hidroxilados secundários e terciários. Foram utilizados um β-hidroxiéster secundário e quatro β-hidroxiésteres terciários, com as enzimas PCL, PLE, CRL e AOP. O melhor resultado foi obtido quando efetuou-se a reação enzimática do éster secundário 3-hidroxi-3-fenilpropanoato de etila (1a) com a PCL (50% de conversão, éster (R)-1a recuperado com 98% e.e., e o ácido 1 obtido em 93% e.e. Por outro lado, a PLE apresentou o melhor resultado para os ésteres terciários 3-hidroxi-3-fenilbutanoato de etila (2a) e 3-cicloexil-3-hidroxi-3-fenilpropanoato de etila (3a), apesar da baixa seletividade. PLE e CRL também foram avaliadas na resolução enzimática de 2-(1-hidroxicicloexil)-butanoato de etila (4a) e 2-(1-hidroxiciclopentil)-butanoato de etila (5a), entretanto, não apresentaram seletividade.


Introduction
Enzyme catalysis has been one of the most useful methods for the preparation of enantiomerically pure compounds.Numerous studies have indicated the application of enzymes to prepare synthons for use in asymmetric synthesis and many reviews on this subject have been published recently 1 .Enzymes such as Pig liver esterase (PLE), Pseudomonas cepacia lipase (PCL), Candida rugosa lipase (CRL), Burkholderia cepacia lipase (BCL), and Aspergillus oryzae protease (AOP) have been used.Enzymes can be employed in the resolution of alcohols and esters and many examples have been reported in the literature 1 .The enzymatic hydrolysis of ethyl 3-hydroxy-3-phenylpropanoate (1a) or other ester derivatives to the 3-hydroxy-3-phenylpropanoic acid (1) have interested several research groups.Acid 1 is an important intermediate in the synthesis of tomoxetine (I) and fluoxetine (II) hydrochlorides, widely used as antidepressants 2 (Figure 1).To this end, PLE 3 has been applied and more recently Pseudomonas sp 2 (Reaction 1).Penicillin G Amidohydrolase 4 (PGA) and lipase A 4b were also used in the hydrolysis of 1a O-acetyl derivative.
These results prompted us to develop a complementary study on the resolution of 1a (prepared as stated in the literature by a classical Aldol reaction) 5 , using the lipases from Pseudomonas cepacia and Candida rugosa, the protease from Aspergilus oryzae, as well as the esterase from Pig liver at pH 8 in a phosphate buffer solution to evaluate the best hydrolysis conditions.

Experimental
The solvents and reagents used were supplied by E. Merck or Aldrich Co. and THF was purified through a process of distillation over LiAlH 4 , according to standard laboratory techniques.Melting points were determined on a Fischer-Johns apparatus.Flash chromatography was performed using Kieselgel 60 (230-400 mesh, E. Merck).IR spectra were recorded on films or KBr pellets with a Perkin Elmer 1420 spectrometer. 1H MNR and 13 C NMR spectra were obtained in CDCl 3 solutions (Me 4 Si as internal standard) with a Varian Unity Plus 300 instrument.Whenever necessary, the e.e. was observed using tris-[3-heptafluoropropylhydroxymethylene)-(+)-camphorato] europium (III) derivative (Eu(hfc) 3 ).Optical rotations were measured in EtOH or CHCl 3 solutions with a Polartronic NH8 Schmidt/Haensch polarimeter at room temperature.The mass spectra were obtained with a VG Autoespec.Pig liver esterase (PLE, E3128, 300 units/mg protein, 15 mg protein/mL suspension), Pseudomonas cepacia lipase (PCL, L9156, 90 units/g solid), Candida rugosa lipase (CRL, type VII, L1754, 835 units/mg solid) and Aspergillus oryzae protease (AOP, type XXIII, P4032, 3,6 units/mg solid) were purchased from Sigma Co.
Preparation of racemic β-hydroxy esters 1a, 2a, 3a, 4a and 5a 5 General procedure: Under N 2 atmosphere, n-butyllithium (24 mmol) was added to a solution of diisopropylamine (3.7 mL, 26.4 mmol) and THF (15 mL) at -78 o C. The solution was stirred for 15 min.The corresponding ethyl ester (22 mmol) was added dropwise to a solution of LDA and the reaction was conducted under stirring for 45 min.After that, benzaldehyde or ketone was added dropwise.The mixture was stirred for 6 h at -78 o C, and then treated with NH 4 Cl saturated solution and extracted with ethyl acetate.The organic layer was washed with water, saturated with NaCl, and dried over MgSO 4 .The solvent was evaporated and the crude product was purified through chromatography on silica gel (n-hexane:ethyl acetate, 95:5).

Results and Discussion
The hydrolysis of ester 1a using PCL, PLE, CRL and AOP enzymes were initially studied and the results are presented in Table 1.
The results observed in this work were similar to those obtained by Boaz 2 and Santaniello 3 , where the Pseudomonas sp lipase was more active than PLE esterase, in the enzymatic ester 1a hydrolysis.These differences in the activities between these enzymes have been discussed elsewhere 1g .
Figure 3 shows the reaction curve for the ester hydrolysis, which demonstrates that the conversion rate decreased drastically after 50% of conversion.Our results showed that the ester hydrolysis could be carried out in a single step without successive reactions, as already mentioned.Similar conclusions were reached for the enzymatic acetylation of the 3-hydroxy-4-alkoxy methylbutanoate, as reported recently by Wunsche et al 7 .
The results obtained from the hydrolysis of ester 1a using PLE at pH 8 (entry 7, Table 1) were similar to those reported by Santaniello et al 3 .However, at pH 8 a faster hydrolysis reaction was achieved compared with the data reported by Santaniello at pH 7 (40 min versus 4h).The hydrolysis of 1a using AOP was comparable with PLE results, with a significant enhancement in the e.e. towards the recovered ester and the obtained acid (entry 10, Table 1).Compared with the other enzymes, CRL showed little enantioselectivity for the hydrolysis of 1a and a rather extended time of reaction (entry 8, Table 1).Esters 2a and 3a were hydrolyzed with PLE, PCL and CRL, under the same reaction conditions employed for ester 1a.
PLE at pH 8 provided the best results (Table 2).
When PLE was used in the hydrolysis of 2a and 3a, the recovered esters showed poor selectivity (entry 1 and 4, Table 2).(S)-acid 2 presented a modest e.e. (50%), and acid 3 was racemic.DMSO was used as a cosolvent and the temperature was lowered in order to improve the results for the hydrolysis of 2a.Notwithstanding these modifications the results remained unsatisfactory (entry 2 and 3, Table 2).
CRL (150 mg enzyme and pH 7) promoted the hydrolysis of 2a.However, the products obtained were racemic.After 7 days at room temperature and a conversion of 32%, the yields of recovered ester 2a and acid 2 were 62% and 32%, respectively.Esters 2a and 3a were inert to PCL catalyzed hydrolysis under the same conditions of the 1a hydrolysis.
From the results presented in Tables 1 and 2, a decrease of the enantiomeric excess and an increase in the reaction time may be observed when an hydrogen on C-3 is replaced by methyl and cyclohexyl groups (entry 7, Table 1; entry 1 and 4, Table 2), showing that for larger groups at C-3 the enzyme was not selective.There are useful models for acylated secondary alcohols and α-substituted esters that predict which enantiomer will be faster hydrolyzed 1g,k,n,o,p .The importance of the groups at C-3 concerning enzyme active site are discussed in some papers 1o , and using these models we can infer that bulkier groups at C-3 decrease the enzyme selectivity.The results corroborate the above PLE and CRL were also used in the hydrolysis of the β-hydroxy esters 4a and 5a.Although these enzymes promoted hydrolyses, only racemic esters and corresponding racemic acids were obtained under these conditions.When PLE (50 units) was employed for the ester hydrolysis of 4a at pH 8 and at room temperature, the yield of recovered ester 4a was 42% and for the corresponding acid was 30% after 9 days at a conversion of 37%.When CRL (150 mg) was used, the reaction time for ester 4a was 5 days at a conversion of 14% (the yield of recovered ester 4a was 11%) and 2 h for ester 5a at a conversion of 15% (the yield of recovered ester 5a was 55%), at pH 7 at room temperature.These results showed that the presence of five and six member rings reduce the enzyme discrimination in these substrates, as observed for 2a and 3a.
The results presented in this paper have added new data to the previously developed works [2][3][4] and expand the scope of the estimation of the enzymes active sites through molecular modeling studies 1p .The hydrolyses were carried out under different reaction conditions and other enzymes were employed.Four different β-hydroxy esters were also analyzed and the results obtained demonstrated the importance of the group size present at the β carbon during the reaction, as above mentioned.Results confirm that PCL is a better enzyme to hydrolyze secondary β-hydroxy esters than PLE, CRL and AOP.On the other hand PLE, though depicting poor selectivity is more substrate specific.Experiments are in progress in our laboratory to improve the reactions conversions and selectivities.