A Facile Resolution of (R, S)-Mandelic Acid Via Lipase-Catalyzed Esterification

The resolution of racemic mixtures to prepare enantiomerically-pure pharmaceuticals and synthetic intermediates is of great commercial relevance, and different strategies are employed including use of lipases. Lipases are now widely used as synthetic tools in organic chemistry because of their broad substrate specificity and high stereoselectivity, even toward unnatural substrates. In particular, besides hydrolysis, lipases can also catalyze several related reactions - esterifications, transesterifications and ammoniolysis.

Enantiomers of mandelic acid and their derivatives are valuable chemicals widely utilized for resolution and synthetic purposes. Typically, the enzymatic resolution of (R, S)-mandelic acid (MA) is achieved via lipase-catalyzed hydrolysis or ammoniolysis of mandelic acid derivatives [Wang et al, 2007], which could be prepared using chemical reactions. Apparently, extra costs, solvents and time are needed in the preparation of mandelic acid derivatives. In order to solve the problem, we proposed a new approach, that is, one-step lipase-catalyzed esterification to resolve MA (Scheme 1).

In our study, Novozym 435 and CALB (lipase from Candida antarctic B) were used to catalyze the esterification of MA and 1-pentanol. Novozym 435 showed higher enantioselectivity toward (R)-MA than free CALB. After an investigation into possible factors, we attributed this phenomenon to changes in the inherent selectivity of the enzyme derived from the immobilization on hydrophobic interface.

Subsequently, measurements of enzyme activity and enantioselectivity at different initial substrates concentrations were carried out. With increasing initial MA concentrations ranging from 0 to 100 mM, both of enzyme activity and enantioselectivity enhanced. According to experimental data, we obtained Michaelis constants K_m(R), K_m(S) for (R)-, (S)-MA and the corresponding kinetic parameters k_cat(R), k_cat(S), respectively. In contrast to the improvement of enzyme activity due to increased MA concentration, the enzyme activity decreased and enantioselectivity had no change when the initial 1-pentanol concentration gradually increased. The phenomenon revealed that 1-pentanol presented a significant inhibition toward the enzyme. Since no changes in enantioselectivity appeared, we could draw a conclusion that the inhibition of 1-pentanol toward lipase was not selective inhibition.

To decrease the inhibition of 1-pentanol, n-hexane was utilized as the solvent due to its low logP. Compared with the reaction in non-solvent system, the conversion (x) and the substrate enantiomeric excess (ee(s)) values both improved with increasing volume of n-hexane. Furthermore, when the volume ratio of 1-pentanol to n-hexane was 1/7, the reaction time decreased from 72 h to 48 h. Through the investigation into solvent logP varying with the ratio of 1-pentanol to n-hexane, we found that the increased enzyme activity not only derived from the decreased inhibition of 1-pentanol, but also resulted from the changes in solvent logP. An ee(s) of 98.7%, which achieved the requirement of the industry, was obtained with 96.7% conversion after 48 h of reaction.

In addition, effects of triethylamine as an additive on the enzyme activity and enantioselectivity were investigated [Tsai et al, 2006]. The experimental results showed that the enzyme activity was inhibited by triethylamine, which differed from previous studies [Fritz Theil, 2000]. The lower enzyme activity induced by addition of triethylamine can be probably due to the change of surface properties of lipase or the formation of acid-base pair, which hindered the formation of acyl-enzyme intermediate.

In conclusion, we employed one-step lipase-catalyzed esterification to resolve MA and demonstrated the feasibility of the new approach. Moreover, effects of initial substrate concentrations, n-hexane amount and triethylamine as an additive on the enzyme activity and enantioselectivity were investigated.

Scheme 1. Resolution of (R, S)-MA via lipase-catalyzed esterification.

This research was supported by the NSF of China (51173128, 31071509, 21206113), the Ministry of Science and Technology of China (Nos. 2012YQ090194, 2013AA102204, 2012BAD29B05), the Program for New Century Excellent Talents in Chinese University (NCET-08-0386), and Beiyang Young Scholar Program (2012).

References

1.       Wang P. Y., Chen T. L., Tsai S. W. and Kroutil W. Hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media: implication for rate-limiting formation or breakdown of tetrahedral intermediates in acylation step, Biotechnology and Bioengineering, 98, 30-38, 2007

2.       Chen C. C., Chen T. L. and Tsai S. W. Altering lipase activity and enantioselectivity in organic media using organo-soluble bases: implication for rate-limiting proton transfer in acylation step, Biotechnology and bioengineering, 94, 201-208, 2006

3.       Fritz T.Enhancement of selectivity and reactivity of lipases by additives, Tetrahedron, 56, 2905-2919, 2000