(582dr) 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

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