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Enzymatic Enantioselective Acylation of Sterically Aromatic Secondary Alcohol.

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Dev. Chem. Eng. Mineral Process. 13(5/6), pp. 605-616, 2005.
Enzymatic Enantioselective Acylation of
Sterically Aromatic Secondary Alcohol
Lee-Suan Chua and Mohamad Roji Sarmidi*
Dept of Bioprocess Engineering, Faculty of Chemical and Natural
Resources Engineering, Universiti Teknologi Malaysia, 81310 UTM
Skudai, Johor, Malaysia.
This study focused on the kinetic resolution of (R,S)-I-phenylethanol using lauric acid
as acyl donor. The enantioselective esterification was catalysed by immobilised
lipases in organic media. From exploratory experiments, several commercial
immobilised lipases were screened for their efficiency in resolving the racemic
alcohol. They were lipases from Pseudomonas cepacia, Candida antarctica and
Candida rugosa (Candida cylindracea) with different immobilisation methods. The
cross-linked enzyme crystal of P. cepacia lipase (ChiroCLEC-PC) and the carrierfured lyophilised C. antarctica lease B (Chirazyme L2, c.$, C3, lyo) showed the
highest pegormance in term of enzyme activity as well as enzyme enantioselectivity.
They were selective towards the R-enantiomer of 1-phenylethanol with enantiomeric
ratio (E) above 200. The presence of S-enantiomers in the racemic alcohol did not
cause inhibition to the resolution. Kinetic studies were carried out by varying the
substrates concentration at the determined reaction conditions. Both enzymes
required threegold molar excess of lauric acid over (R,S)-I-phenylethanol (50 mM)
in order to achieve the highest initial reaction rate. When using the molar excess of
(R,S)-1-phenylethanol, equilibrium conversion dropped due to enzyme deactivation.
Keywordr: Kinetic resolution; immobilised lipase; enantioselectivity; optically active
The current development of biocatalysis is focusing on preparing enantiomerically
pure compounds by utilising the high degree of enzyme selectivity, namely
enantioselectivity. Enzymes are superior to chemicals as catalysts because of high
optical purity of product, mild reaction conditions and environmentally fiiendly
processes. Lipases are widely used enzymes to resolve racemates, they require no
cofactor and are readily available at low cost. They accept a broad structural range of
substrates, while retaining high enantioselectivityfor each.
* Authorfor correspondence (
Lee-Suan Chua and Mohamad Roji Sannidi
In contrast, the chemical method for the preparation of enantiomerically enriched
1-phenylethanol requires a heavy metal catalyst such as lithium aluminium hydride
complexes for the asymmetric reduction of acetophenone [l]. In addition to the
negative impact on the environment, this method is unable to produce
1-phenylethanol with sufficient optical purity (48%) compared to enzymatic
The asymmetric reduction of acetophenone [2] and the enantioselective oxidation
of (S)-1-phenylethanol from its racemate [3] are other examples of microbial methods
for the synthesis of the chiral alcohols. However, the yeast-mediated reduction
required the regeneration of coenzyme NAD(P)H as the reducing reagent.
Furthermore, only about 10% of acetophenone is converted to (S)- 1-phenylethanol.
The stereoselective oxidation produced (R)-1-phenylethanol with high optical purity
(>go%) only after 80 hours of continuous production.
This study investigated the enantioselective acylation of sterically aromatic
secondary alcohol, namely (R,S)-1-phenylethanol with lauric acid in a batch stirredtank reactor. Several studies have been carried out using vinyl ester as acyl donor in
this resolution [4-71. Even though vinyl acetate is more reactive and renders the
reaction irreversible, the liberated acetaldehyde may inactivate the enzyme, especially
lipases fromCandidu rugosa and Geotrichum candidum [8]. The use of lauric acid in
this kinetic resolution could achieve high conversion (48%) as well as high
enantiomeric purity (95% ee,). Water is the only by-product and can be removed by
adding molecular sieve in the reaction mixture.
The reaction followed a Ping-Pong Bi-Bi mechanism with the inhibition of lauric
acid, (R,S)-1-phenylethanol and water [9]. The schematic diagram of the reaction
mechanism is presented in Figure 1. The initial reaction velocity approach was used
to develop the lunetic model of the resolution with steady state assumption in this
The optically active (R)-and (S)-1-phenylethanol are used as chiral building
blocks and synthetic intermediates in the pharmaceutical and fine chemical industries
[lo]. The (R)-1-phenylethanol is widely used as a fragrance in the cosmetics industry
because it contains a mild floral odour [ 111.
F + BR,S= FBR+ Bs
E+BR,s #EBR,s
Figure I . Schematic diagram of Ping-Pong Bi-Bi mechanism with the inhibition of
substrates and product.
Notation: A is lauric acid; B is (R,S)-I-phenylethanol; P is water; Q is
(R)-1-phenylethyl laurate; E is enzyme; F is enzyme complex; subcript R denotes
(R)-1-phenylethanol; subcript S denotes: (S)-1 -phenylethanol.
Enzymatic Enantioselective Acylation of StericaIIy Aromatic Secondary Alcohol
The objective of this study was to investigate the behaviour of immobilised
enzyme lipases for the chiral esterification of I-phenylethanol with lauric acid in
organic media. In the preliminary study, exploratory experiments were carried out to
screen the most suitable enzyme for the chiral esterification. The kinetic studies were
carried out on the selected enzymes by varying the substrates concentration in a batch
stirred-tank reactor. The effect of individual enantiomer on the resolution was also
investigated by carrying out a similar reaction using the enantiomerically pure alcohol
instead of racemic alcohol.
Materials Used and Experimental Methods
(i) Chemicals and enzymes
High-purity grade of substrates, (R,S)-1-phenylethanol and lauric acid were purchased
from Fluka (Switzerland). Isooctane was purchased fiom Merck (Germany). Several
commercial immobilised lipases were obtained from the suppliers listed in Table 1.
(ii) Resolution of (R,S)-I-phenylethanol
Lauric acid (100 mM) and (4s)-1-phenylethanol (50 mM) were dissolved in
isooctane and added into a batch stirred-tank reactor. The mixture (25 ml) was
maintained at 35°C. The enzymes (=15 mg) as listed in Table 1 were added in order to
catalyze the reaction. The solution was continuously stirred to ensure that all enzyme
particles were homogeneously dispersed in the reaction medium. Samples (0.01 ml)
were periodically withdrawn to analyze the time course of the reaction. No reaction
was detected in the absence of the enzyme. Each set of experiments was carried out in
(iii) EjJect of enzyme loading
The enzyme loading of Chirazyme L2, c.-f., C3, lyo and ChiroCLEC-PC was
investigated by varying the enzyme quantity for the reaction described above.
Commercial name
I Microorganism
I Supplier
Lipase PS-C
Pseudornonas cepacia
Ammo Pharmaceuticals Co., Japan
Lipase Sol-Gel-Ak
Candida cylindracea
Fluka Chemie AG, Switzerland
Chirazyme L2, c.-f., C2, lyo
Candida antarctica
Roche Molecular Biochemicals,
Chirazyme L2, c.-f., C3, lyo
Candida antarctica
Roche Molecular Biochemicals,
Candida rugosa
Pseudornonas cepacia
Altus Biologics Inc., USA
Altus Biologics Inc., USA
Lee-Suan Chua and Moharnad Roji Sarmidi
(iv) EHect of lauric acid and (R,S)-1-phenylethanolconcentration
Similar reactions were carried out by varying the concentration of lauric acid
(25-250 mM) at a fixed (R,S)-1-phenylethanol concentration (50 mM). In the
subsequent experiments, the concentration of (R,S)-1-phenylethanol was varied from
25-250 mM and the concentration of lauric acid was fixed at 150mM.
(v) Egect of Single Enantiomer
The single isomer, (R)-1-phenylethanol (25 mM) was esterified by lauric acid
(150 mM) in isooctane (10 ml) at 35°C. Chirazyme L2, c.-f., C3, lyo and ChiroCLECPC were used to catalyse the reaction. Similar reactions were carried out using its
counterpart isomer, (S)- 1-phenylethanol.
(vi) Gas ChromatographyAnalysis
The reaction progress was determined by a Shimadzu GC-17A gas chromatography
equipped with a flame-ionization detector (FID). The column was a chiral Beta-DexTM
120 fused-silica capillary column with dimensions of 0.25 mm ID x 30 mL x 0.25 pm
film hckness (Supelco, USA).
The temperature of injector and detector were maintained at 250°C. The carrier
gas was nitrogen with the total flow rate 104 d m i n at 100 H a . A programmed
temperature for the column was developed. Initially, it was kept at 120°C for
15 minutes. The temperature was then increased from 120°C to 190°C at the rate of
4OoC/min. From 190°C to 220"C, the increased rate of temperature was 2"C/min.
Finally, the column was maintained at this temperature for 15 minutes. The retention
times for (R)-1-pehnylethanol, (S)-1-phenylethanol, lauric acid and (R)-1-phenylethyl
laurate were 12.33, 12.82,25.57 and 44.86 minutes respectively.
(vii) Determination of conversion, enantiomeric excess, enantiomeric ratio
The extent of substrate conversion (G), the optical purity expressed as enantiomeric
excess of substrate (ee,), and the enantiomeric ratio (E) were determined using the
mathematical equations developed by Chen et al. [ 121, as given below:
5 = (1-
+ A s ) x 100%
+ ASi
ees = -x100%
A, +A,
where A is peak area; subscript i is initial time; subscript R denotes
(R)-1-phenylethanol;subscript S denotes (S)-1-phenylethanol.
E v m a t i c EnantioselectiveAcylation of Sterically Aromatic Secondary Alcohol
Results and Discussion
fi) Exproratory experiment of fR,S)-1-phenylethanol resolution
Six commercial immobilised lipases were screened for the kinetic resolution of
(R,S)-1-phenylethanol with lauric acid at 35°C in isooctane. Although the overall
structure and serine triad of lipases are similar, their degrees of substrate specificity
and activity differ widely. All the enzymes except Lipase PS-C and Lipase Sol-GelAk were capable of catalysing the reaction (see Figure 2). All of the lipases show a
preference for the (R)-configuration of 1-phenylethanol. The (R)-1-phenylethanol was
converted into ester while the (S)-1-phenylethanol remained unchanged.
ChiroCLEC-PC exhibited the hlghest performance for the resolution among the
enzymes. The initial rate of reaction was 473.5 f 10 pmol/min and the reaction
reached equilibrium conversion of 45% after 100 minutes reaction. This cross-linking
enzyme is more reactive because it involves protein purification during the formation
of microcrystals
Chirazyme L2, c.-f., C2, lyo and Chazyme L2, c.-f., C3, lyo are lipase B from
Candida antarctica. They are non-covalently bonded to different insoluble organic
polymer carriers. The enzyme with the carrier C3 was more reactive than C2 in the
lunetic resolution of (R,S)-1-phenylethanol,and the initial rate of the former was 1.2
e)? A
Figure 2. Resolution of I-phenylethanol catalysed by immobilised lipases.
Lee-Suan Chua and Mohamad Roji Sannidi
times faster than the latter. This observation can be explained by the difference size of
the enzyme beads. The lipase with the carrier C2 has a larger diameter (0.5 mm),
therefore it has higher internal mass transfer limitation than C3 carrier (0.1 mm). The
reaction progress curves of both enzymes reached equilibrium at 44% conversion
after 250 minutes reaction.
The performance of ChiroCLEC-CR in thls resolution is the fourth after
ChiroCLEC-PC, Chirazyme L2, c.-f., C3, lyo and Chirazyme L2, c.-f., C2, lyo. The
reaction catalysed by ChiroCLEC-CR reached equilibrium only after 300 minutes.
The result indicated that Candida rugosa lipase is capable of discriminating the
racemic alcohol. However, the performance of Candida rugosa lipase was lower than
Pseudomonus cepucia and Candida untarctica lipase B. ChiroCLEC-CR showed
about 45% activity lower than ChiroCLEC-PC in the resolution, even though their
microcrystals are covalently cross-linked by the bifunctional reagent glutralaldehyde
in the similar process. Therefore, the reactivity of Pseudomonas cepacia lipase was
higher than Candida rugosa lipase in the resolution of aromatic secondary alcohol.
Nevertheless, Candida rugosa lipase is a large protein molecule (60 m a ) containing
the binding site which is more accessible to the surface.
Lipase PS-C and Lipase Sol-GeEAk did not catalyse the reaction after stirring for
two hours at 35°C. The resolution of sterically alcohol was not observed in the case of
lipases immobilised on ceramic particles and in Sol-Gel-&. These lipases may
require higher temperature for catalysis, but this is not suitable in the preparative scale
(ii) Eflecr of enqvme loading
Investigation of the effect of enzyme loading is very important to investigate the
enzyme effectiveness in the resolution. This is because enzymes exhibit the highest
activity at the optimal quantity. Hence, the enzyme quantity influences the reaction
rate and the final conversion value.
The results in Figures 3 and 4 show that the initial reaction rates increase with an
increase of enzyme loading. The reaction rate reached a constant plateau value for a
given enzyme loading. This value represents the maximum rate at which the substrate
can be transported from the bulk solution to the enzyme molecules. The optimal
enzyme loading for ChiroCLEC-PC (see Figure 3) and Chirazyme L2, c.-f., C3, lyo
(see Figure 4) are 12.5 f 1 mg and 250 f 5 mg respectively. For those specific
amounts of enzymes, the reactions have the highest initial reaction rate and could
achieve the highest conversion in the shortest time of reaction. The external mass
transfer is not the predominating mechanism at high enzyme loading. The reaction
rate did not increased significantly above the optimal value of enzyme loading.
The result showed that the enzyme loading for Chirazyme L2, c.-f., C3, lyo was
20 times higher than ChiroCLEC-PC. Hence, ChiroCLEC-PC exhibited higher
productivity (12.22 g/mg enzyme) than Chirazyme L2, c.-f, C3, lyo (0.61 g/mg
enzyme). The high productivity of ChiroCLEC-PC can be explained by the high
purity of crystalline enzyme. The cross-linked crystals provide their own support, but
Chirazyme L2 consists mostly of inert carrier material.
Enzymatic Enantioselective Acylation of Sterically Aromatic Secondary Alcohol
6 0.6
Figure 3. Loading of ChiroCLEC-PC in the resolution.
Enzyme loading (mg)
Figure 4. Loading of Chirazyme L2, c.-f. C3, lyo in the resolution.
61 I
Lee-Suan Chua and Mohamad Roji Sarmidi
(iii) Effect of lauric acid Concentration
The initial reaction rates were determined from the initial reaction time of the
progressive curves. The linearity of the curves is consistent with a kinetically
controlled enzymatic reaction. The initial rates of both enzymes, Chirazyme L2,c.-f.,
C3, lyo and ChiroCLEC-PC, are plotted in Figure 5. The cross-linked crystalline
lipase from Pseudomonas cepaciu showed higher activity than lyophilized lipase B
from Candida antarctica in all substrates ratios. They have similar curves which
indicate the concentration of lauric acid, 150 mM is the optimum concentration for
the resolution of (R,S)-1-phenylethanol (50 mM). In accordance with the work of
Okahata et al. [ 131, substrate inhibition was observed when the concentration of lauric
acid was above 150 mM.
An increase in the lauric acid concentration may prevent the depletion of acyl
donor at the enzyme active site. This would reduce the internal diffision if the mass
transfer limitation occurs. Another reason for using an excess of lauric acid is to shift
the reaction towards the synthesis direction, since the reaction is reversible.
Furthermore, the use of excess lauric acid would simplify the kinetic analysis by
reducing the reaction to pseudo-first order.
According to Bakker et al. [8], secondary alcohol is a sluggish reactant, hence an
irreversible reaction is a prerequisite for efficient lunetic resolution. The nonchiral
compound should usually be present in a molar excess over the chiral compound to
secure the complete conversion of the reactive enantiomer.
Figure 5. EHect of lauric acid concentration on the initial reaction rate.
Enzymatic Enantioselective Acylation of Stericallj Aromatic Secondary Alcohol
The initial rate increased with the lauric acid concentration up to a ratio of 1:3.
Further increase of lauric acid concentration did not lead to significant change in the
initial rate values. This is because too much lauric acid in the reaction medium would
cause steric hindrance. The deacylation step of the catalytic mechanism was slower
because of the high concentration of lauric acid around the enzyme molecules. The
active site of the enzyme complex would be less accessible to alcohol molecules.
Therefore, the reaction rate was decreased and a longer reaction time was required in
order to reach equilibrium.
(iv) Effect of (R,S)-I-phenylethanol concentration
The results of alcohol concentration effect on the reaction are presented in Figure 6.
The initial reaction rate increased with an increase of (R,S)-1-phenylethanol
concentration. However, the rate of increase was slow, nearly reaching a plateau value
at (R,S)-1-phenylethanol of 250 mM. The results indicate that the available enzyme
active sites are not sufficient for the increased amount of (R,S)-1-phenylethanol.
Excessive (Ft,S)-1-phenylethanol in the reaction medium would restrict the mobility
of alcohol bound to the enzyme complex.
Another observation from the study was the decrease in fmal conversion value at
high alcohol concentration. Enzyme deactivation might occur because the log P value
(logarithm of the partition coefficient of the organic liquid between n-octanol and
water) for the alcohol is relatively low at 1.62 [3]. It is well established that enzymes
exhlbit optimum activity in organic solvents with the log P values between 2 to 4
4.0 -
(R,S)-1 -phenylethanol (mM)
Figure 6. EjFect of (R,S)-1-phenylethanolconcentration on the initial reaction rate.
Lee-Suan Chua and Mohamad Roji Sannidi
The E values for both catalysed enzymes resolutions are reported as 200 even
though their E values are much greater than 200. An E value greater than 200 cannot
be accurately determined because even a small variation of ees would cause a
significant change in the numerical value of E. Frings et al. [15] also noted that a
precise value makes no sense due to the enormous influence of the smallest deviation
in measurement.
There was a marked decreased in the conversion rate when the reaction reached
40% conversion. This was due to the reversibility of the reaction at high conversion.
The conversion of alcohol did not go beyond 50% indicating that the enzymes are
highly selective toward (R)-1-phenylethanol. The ee, values were 90% for
ChiroCLEC-PC and 95% Chirazyme L2, c.-f., C3, lyo catalysed reactions.
(v) Single enantiomer in chiral esterification
The results obtained show that both enzymes were highly selective toward
(R)- 1-phenylethanol. No reaction was observed for (S)-1-phenylethanol. The
(S)-isomers may fit into the active sites at the nonproductive orientation and therefore
render it non-reactive. Since no chemical bonding was formed between the (S)-isomer
and the enzyme active sites, the (S)-isomer was easily moved out and replaced by
another isomer. The movement of (S)-isomers in and out fiom the active sites has
reduced the chances of the (R)-isomers fitting into the active sites. The final
conversions of the reactions could achieve 48% regardless of the existence of
(S)- 1-phenylethanol. This indicates that (S)-1-phenylethanol caused reversible
inhibition to the enzymes.
The initial velocities of the reaction in the synthesis of (R)-1-phenylethyl laurate
were 34.5 f 3 mh4/min/g enzyme and 817.8 f 11 mM./rnin/g enzyme for Ckazyme
L2, c.-f., C3, lyo and ChiroCLEC-PC respectively. For comparison with the values of
reaction rates in the presence of (S)-1-phenyleythanol, the reaction rate of single
enantiomer was higher than racemic alcohol. It was about two-fold for the Candida
antarctica lipase catalysed reaction, and three-fold for the Pseudomanas cepacia
lipase catalysed reaction.
Furthermore, the rate of racemic alcohol (50 mM) was approximately 62% less
than the rate of the s u m of single enantiomer. Langrand et al. [ 161 explained that the
rates of single enantiomers are equal to the rate of racemic mixture only if the total
concentration is below the apparent Km
The enantioselective esterification of (R,S)-l -phenylethanol had been carried out in a
stirred batch reactor using the commercial immobilised lipases, namely ChiroCLECPC, Chirazyme L2, c.-f., C3, lyo , Chirazyme L2, c.-f., C2, lyo and ChiroCLEC-CR.
ChiroCLEC-PC and Chirazyme L2, c.-f., C3, lyo were the enzymes that showed
good performance for the chiral resolution of (R,S)-1-phenylethanol with
enantiomeric in excess of 90% and 95% substrate respectively. The enantioselectivity
of the enzymes (E > 200) was not affected by substrates concentration. The enzymes
were highly selective toward the (R)-enantiomer of 1-phenylethanol, while the
(S)-enantiomer remained unchanged.
Enzymatic Enantioselective Acylation of Sterically Aromatic Secondary Alcohol
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C3, lyo in the resolution. This result may be due to the great difference in the
concentration of the enzyme active sites which was determined by using
phenylmethanesulfonyl fluoride as an irreversible inhibitor [ 171. The concentration of
the active site of ChiroCLEC-PC was 131.7 mM/g enzyme and 14.3 mM/g enzyme
for Chirazyme L2, c.-f., C3, lyo.
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