Free and immobilized lecitase ultra as the biocatalyst in the kinetic resolution of (E)‐4‐arylbut‐3‐en‐2‐yl esters

Article abstract of DOI:10.3390/molecules25051067

The influence of buffer type, co‐solvent type, and acyl chain length was investigated for the enantioselective hydrolysis of racemic 4‐arylbut‐3‐en‐2‐yl esters using Lecitase Ultra (LU). Immobilized preparations of the Lecitase Ultra enzyme had

Full text of DOI:10.3390/molecules25051067

Article
Free and Immobilized Lecitase™ Ultra
as the Biocatalyst in the Kinetic Resolution of
(E)‐4‐Arylbut‐3‐en‐2‐yl Esters
1,
1
2
2
Aleksandra Leśniarek *, Anna Chojnacka , Radosław Drozd , Magdalena Szymańska and
Witold Gładkowski *
1,
1
Department of Chemistry, Wroclaw University of Environmental and Life Sciences, Norwida 25,
50‐375 Wroclaw, Poland; anna.chojnacka@upwr.edu.pl
Department of Microbiology and Biotechnology, Faculty of Biotechnology and Animal Husbandry, West
2
Pomeranian University of Technology, Szczecin, 45 Piastów Avenue, 71‐311 Szczecin, Poland;
rdrozd@zut.edu.pl (R.D.); magdalena.szymanska@zut.edu.pl (M.S.)
* Correspondence: witold.gladkowski@upwr.edu.pl (W.G.); aleksandrajurabio@gmail.com (A.L.);
Tel.: +48‐713205154
Received: 12 February 2020; Accepted: 25 February 2020; Published: 27 February 2020
Abstract: The influence of buffer type, co‐solvent type, and acyl chain length was investigated for
™
the enantioselective hydrolysis of racemic 4‐arylbut‐3‐en‐2‐yl esters using Lecitase Ultra (LU).
™
Immobilized preparations of the Lecitase Ultra enzyme had significantly higher activity and
enantioselectivity than the free enzyme, particularly for 4‐phenylbut‐3‐en‐2‐yl butyrate as the
substrate. Moreover, the kinetic resolution with the immobilized enzyme was achieved in a much
™
shorter time (24–48 h). Lecitase Ultra, immobilized on cyanogen bromide‐activated agarose, was
particularly effective, producing, after 24 h of reaction time in phosphate buffer (pH 7.2) with
acetone as co‐solvent, both (R)‐alcohols and unreacted (S)‐esters with good to excellent
enantiomeric excesses (ee 90–99%). These conditions and enzyme were also suitable for the kinetic
separation of racemic (E)‐4‐phenylbut‐3‐en‐2‐yl butyrate analogs containing methyl substituents
on the benzene ring (4b,4c), but they did not show any enantioselectivity toward
(E)‐4‐(4’‐methoxyphenyl)but‐3‐en‐2‐yl butyrate (4d).
™
Keywords: (E)‐4‐arylbut‐3‐en‐2‐ols; enantioselective hydrolysis; kinetic resolution; Lecitase Ultra;
immobilization; cyanogen bromide‐activated agarose
1. Introduction
™
Lecitase Ultra (LU) is a commercially available chimeric enzyme that is the product of the
fusion of lipase genes from Thermomyces lanuginosus and phospholipase A1 genes from Fusarium
oxysporum [1]. This enzyme combines the stability of lipase and activity of phospholipase A1 and was
initially designed for the degumming of plant oils during rafination [2–6]. In this process,
™
phospholipids contained in the oil phase are hydrolyzed by Lecitase Ultra at the sn‐1 position to
lysophospholipids, which migrate to the aqueous phase. Since then, this fundamental reaction
™
catalyzed by Lecitase Ultra has also found other food and none‐food applications i.a. hydrolysis of
soy phosphatidylcholine to produce lysophosphatidylcholine [7,8] and α‐glycerophosphocholine
[9,10], modification of flour in bread making [11–13], and rafination of oils used in the production of
™
biodiesel [14,15]. Except for hydrolytic activity, Lecitase Ultra is also able to catalyze esterification,
acidolysis, and alcoholysis, which has found application in the production of oils enriched with
omega‐3 fatty acids [16] as well as in the production of diglycerides [17–20] and structured
Molecules 2020, 25, 1067; doi:10.3390/molecules25051067
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1067
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™
phospholipids [21–27]. Due to its wide enzymatic activity, the application of Lecitase Ultra has
been further extended to the production of flavor low‐chain esters i.a. methyl benzoate and methyl
butanoate [28] and regioselective hydrolysis of peracetylated mono‐ and disaccharides to produce
key intermediates in the preparation of different glycoderivatives such as oligosaccharides,
glycolipids, or glycopeptides [29].
One of the most popular uses of lipases in organic chemistry is the kinetic resolution of racemic
™
mixtures [30–32]. Lecitase Ultra has been also employed for this goal and there are few reports
™
concerning the application of Lecitase Ultra‐catalyzed enantioselective hydrolysis of esters for the
production of some important chiral drug intermediates. These applications covered the resolution
of esters of 2‐hydroxyacids with a phenyl ring including mandelates [33–38], resolution of glycidate
esters [35,39,40], and N‐acetyl‐α‐amino acid methyl esters [35]. Another example of an application in
™
asymmetric synthesis was Lecitase Ultra‐catalyzed regioselective hydrolysis of prochiral dimethyl
3‐phenylglutarate [41].
Immobilization of enzymes on different supports is a useful tool to increase their stability,
activity, and selectivity under operational conditions [42–44]. Due to a simpler separation of the
biocatalyst from the reaction environment, such preparations may be reused in many catalytic cycles
without losing their catalytic properties, which increases the economic efficiency of the industrial
process [45]. However, in many cases, it is also related with a decrease in enzyme activity caused by
™
various types of distortion, diffusion, and steric problems [46]. As Lecitase Ultra is commercially
available only in its free form as the water solution, many attempts have been made to prepare an
immobilized form of this enzyme for different purposes, for example, by entrapment in gelatin
hydrogel [47] or in different calcium alginates [48], immobilization on the derivatives of cellulose
[49,50], polystyrene resin [51], styrene‐divinylbenzene beads [37,52], magnetic nanoparticles [53], or
by encapsulation in AOT(bis(2‐ethylhexyl) sulfosuccinate sodium salt)/isooctane reverse micelles
™
[54]. In the processes of the kinetic resolution of racemates catalyzed by immobilized Lecitase Ultra,
the best results were obtained using gelatin [35,39], various derivatives of agarose [33,36,38,41,55],
hexyl‐ and butyl‐toyopearl [34], or epoxy activated polymer Dilbead VWR coated with
polyethylenimine (PEI) and crosslinked with glutaraldehyde [40].
™
In previous investigations, kinetic resolutions of racemic esters through their Lecitase
Ultra‐catalyzed enantioselective hydrolysis has been limited mainly to the esters of chiral carboxylic
acids [34,35,39,41]. However, since a large variety of chiral esters possess the stereogenic center in
the nucleophilic part of molecule, to extend the substrate scope and increase the usability of this
™
enzyme in the asymmetric synthesis, we employed Lecitase Ultra for the biocatalytic resolution of
racemic 4‐arylbut‐3‐en‐2‐ol esters. In our working group, these alcohols are the subject of special
interest as chiral precursors of the optically active lactones with antiproliferative activity [56,57].
Likewise, they have been used in the production of chiral drugs [58,59].
™
In our previous paper, we reported the preliminary results of the free Lecitase Ultra‐mediated
kinetic resolution of model racemic esters of (E)‐4‐phenylbut‐3‐en‐2‐ol through their enantioselective
hydrolysis, achieving moderate to good enantiopurity of the products [60]. Here, we present the
results of our further investigations in this field, aiming to increase the process efficacy through
optimization of some reaction conditions (pH of the reaction medium, addition of organic
co‐solvent) as well as by enzyme immobilization. To determine the substrate specificity and extend
™
the application of Lecitase Ultra, the hydrolysis of some analogs of (E)‐4‐phenylbut‐3‐en‐3‐ol esters
differing in aromatic fragment of the molecule was also studied.
2. Results and Discussion
Eleven known (2a–d, 3a–d and 4a,b,d) and one new (4c) racemic allyl esters were obtained in
high yields (96–98%) by esterification of corresponding known [61–63] (E)‐4‐arylbut‐3‐en‐2‐ols
(1a–d) with appropriate acid chlorides, according to the standard procedure (Scheme 1).

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Scheme 1. General scheme for the synthesis of racemic esters 2a–d, 3a–d and 4a–d.
Studies toward the kinetic resolution of the prepared compounds were started for the free
™
Lecitase Ultra (LU)‐catalyzed hydrolysis of model 4‐phenylbut‐3‐en‐2‐ol esters (2a–4a) to
investigate the influence of the length of an acyl chain, type of co‐solvent, and type of buffer on the
process stereoselectivity (Table 1).
Table 1. Free Lecitase™ Ultra‐catalyzed hydrolysis of racemic esters 2a–4a.
O
O
O
R
OH
R
O
free Lecitase Ultra
buffer
+
(R)-1a
rac-2a-4a
(S)-2a-4a
ees
[%]
25
52
77
50
70
39
28
68
39
30
22
69
eep
[%]
29
71
88
49
84
>99
29
80
Entry
Substrate
R
Co‐solvent
Buffer
t [h]
c [%]1
E2
1
2
3
4
5
6
7
8
9
2a
3a
4a
2a
3a
4a
2a
3a
4a
2a
3a
4a
Me
Et
Pr
Me
Et
Pr
Me
Et
Pr
Me
Et
Pr
acetone
acetone
acetone
acetone
acetone
acetone
DMF
DMF
DMF
DMSO
DMSO
Tris/HCl3
Tris/HCl
Tris/HCl
120
120
168
168
144
96
168
144
96
46
42
47
51
45
28
49
46
52
42
27
53
2
10
36
5
24
>200
2
18
23
3
phosphate4
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
phosphate
88
41
59
87
10
11
168
144
96
5
30
12
DMSO
2
1
Conversion, c = ees/(ees + eep). The enantiomeric ratio calculated at the highest conversion rate
according to the following equation: E = ln[(1 − ees)/(1 + (ees/eep)]/ln[(1 + ees)/(1 + (ees/eep)]; ees =
enantiomeric excess of unreacted ester, eep = enantiomeric excess of alcohol. 3 pH 8.2. 4 pH 7.2.
In previous studies, the enantioselective hydrolysis of carboxylic acid esters catalyzed by LU
was carried out in a phosphate buffer [33] or Tris‐HCl buffer [35], therefore in our first experiments,
we employed these two reaction media. (E)‐4‐Phenylbut‐3‐en‐2‐ol esters with different acyl chain
lengths (2a–4a) were subjected to hydrolysis at room temperature, with acetone as a co‐solvent. In all
cases, a higher enantioselectivity of hydrolysis was noticed in the reactions carried out in the
phosphate buffer (Entries 4–6). Higher enantiomeric excess of alcohol 1a and a shorter reaction time
necessary to achieve the best resolution was observed with the elongation of the acyl chain. The best

Molecules 2020, 25, 1067
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results were obtained for butyrate 4a; in this case, the highest enantioselectivity (E > 200) was
observed after 96 h of reaction, resulting in the optically pure product (Entry 6). The positive effect of
the acyl chain length of hydrolyzed esters 2a–4a on their kinetic resolution was a constant tendency,
observed also in experiments carried out in Tris‐HCl (Entries 1–3) as well as using other co‐solvents
™
(Entries 7–9 and 10–12). These investigations confirm the high specificity of Lecitase Ultra toward
™
esters of butyric acid, which was earlier found by Mishra et al. [35]. In their experiments, Lecitase
Ultra did not accept glycerol triacetate as the substrate but exhibited hydrolytic activity toward
glycerol tributyrate.
The influence of the co‐solvent on the kinetic resolution of esters 2a–4a was determined for the
reactions carried out in the phosphate buffer. For each of the substrates, the results of the reactions
with acetone as the co‐solvent were compared with those carried out with the addition of
dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). In all cases, the highest
enantioselectivity was observed when the co‐solvent was acetone (Entries 4–6). In the hydrolysis of
propionate 3a with the addition of acetone (Entry 5) and DMF (Entry 8), the enantiomeric excesses of
both unreacted ester and the produced alcohol were comparable, while the addition of DMSO was
the least effective (Entry 11). In the case of the hydrolysis of acetate 2a, the highest enantiomeric
purity of both products was observed for the reaction with the addition of acetone (Entry 4), while
the addition of DMSO resulted in slightly better results than the addition of DMF (Entries 7 and 10).
Significant impact of the co‐solvent on the enantioselectivity of the reaction was observed for the
hydrolysis of butyrate 4a. In this case, a pure enantiomer of alcohol 1a was produced only in the
presence of acetone (Entry 6); the addition of DMF (Entry 9) and DMSO (Entry 12) resulted in a
decrease of ee for alcohol 1a to 87–88%. On the other hand, the highest ee of unreacted butyrate 4a
(69%) was noticed using DMSO as the co‐solvent (Entry 12).
™
In subsequent experiments, free Lecitase Ultra was applied to the hydrolysis of esters of
(E)‐4‐phenylbut‐3‐en‐2‐ol analogues differing in the structure of the aromatic fragment (Table 2).
Table 2. Hydrolysis of allyl esters with different phenyl ring substituents 2b–d, 3b–d, and 4b–d
catalyzed by free Lecitase™ Ultra.
Entry
Substrate
2b
Ar
R
t [h]
c [%]1
ees [%]
eep [%]
E2
1
Me
72
43
9
12
1
2
3
4
5
6
7
3b
4b
2c
3c
4c
2d
Et
Pr
96
120
144
144
96
30
30
41
45
32
59
28
37
42
65
36
0
64
85
61
80
77
0
6
18
6
Me
Et
18
11
0
Pr
Me
6
8
3d
4d
Et
Pr
6
56
65
0
0
0
0
0
0
9
96
1
2
Conversion, c = ees/(ees + eep). The enantiomeric ratio calculated at the highest conversion rate
according to the following equation: E = ln[(1 − ees)/(1 + (ees/eep)]/ln[(1 + ees)/(1 + (ees/eep)]; ees =
enantiomeric excess of unreacted ester, eep = enantiomeric excess of alcohol.

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Based on the previous results of the kinetic resolution of esters 2a–4a, the procedure using
phosphate buffer as the reaction medium and the addition of acetone as a co‐solvent was applied.
Regardless of the acyl chain length, esters of (E)‐4‐(4′‐methoxyphenyl)but‐3‐en‐2‐ol (2d–4d) were
hydrolyzed with no enantioselectivity (Entries 7–9), and for esters containing methyl groups (2b–4b,
2c–4c) lower enantioselectivities of the reactions were observed (E ≤ 18) compared to
(E)‐4‐phenylbut‐3‐en‐2‐yl esters 2a–4a (Table 1, entries 4–6). The beneficial effect of the elongation of
the acyl chain on the results of kinetic resolution was confirmed only in the group of esters with the
p‐methyl substituted phenyl ring (2b–4b). Better resolution was observed for compounds with a
2,5‐dimethylsubstituted phenyl ring than for those with the p‐methylphenyl substituent, which was
particularly noticeable in the case of acetates and propionates.
The positive effect of immobilization on the activity and enantioselectivity of enzymatic
reactions is known from the literature [42]. In the case of LU, this relationship was confirmed in the
hydrolysis of α‐hydroxy acids esters. The highest increase of enantioselectivity, from E = 4 for the
free enzyme to E = 19.5 for the enzyme immobilized in gelatin was observed for the hydrolysis of
ethyl 2‐hydroxy‐4‐oxo‐4‐phenylbutyrate, resulting in optically pure unreacted ester at 60–65%
conversion [35]. Taking this into consideration, in our further studies, immobilized preparations of
™
Lecitase Ultra were used as biocatalysts of the kinetic resolution of esters of alcohols with a
4‐arylbut‐3‐en‐2‐ol system. For this purpose, the commercial enzyme was immobilized on four
™
carriers: calcium alginate; Supelite DAX‐8 acrylic resin; cyanogen bromide‐activated agarose; and
modified bacterial cellulose. The activity of immobilized preparations (Table 3) was measured using
p‐nitrophenyl palmitate as a substrate [64]. The highest, comparable activity was determined for the
preparations obtained after immobilization on cyanogen bromide‐activated agarose (LU‐CNBr) and
acrylic resin (LU‐DAX). Preparation LU‐CNBr was previously used in the production of optically
pure (S)‐3‐methyl phenylglutarate through the asymmetric hydrolysis of dimethyl
3‐phenylglutarate (48 h, 80% yield) [41].
Table 3. Activity of Lecitase™ Ultra (LU) preparations.
Activity
[U/ g of support]
Entry
Carrier
Biocatalyst
1
2
3
Supelite™ DAX‐8
Sodium Alginate
Modified Bacterial Celulose
Cyanogen Bromide‐Activated
Agarose
LU‐DAX
LU‐ALG
LU‐MBC
0.06
0.04
0.045
4
LU‐CNBr
0.06
(E)‐4‐Phenylbut‐3‐en‐2‐yl acetate (2a), (E)‐4‐phenylbut‐3‐en‐2‐yl propionate (3a), and
(E)‐4‐phenylbut‐3‐en‐2‐yl butyrate (4a) were subjected to kinetic resolution with immobilized
preparations of LU and the results were compared with those obtained previously for the free
enzyme (Figures 1–3).
One can see that the use of LU‐CNBr and LU‐MBC significantly shortened the time needed to
achieve the kinetic resolution of (E)‐4‐phenylbut‐3‐en‐2‐yl acetate (2a) up to 24 h and 48 h,
respectively (Figure 1).

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Figure 1. The effect of enzyme immobilization on the conversion, enantioselectivity, and
enantiopurity of the products of Lecitase™ Ultra‐catalyzed hydrolysis of racemic acetate 2a (reaction
scheme presented above)
Likewise, the use of these preparations also had a positive effect on the parameters of kinetic
resolution. The most spectacular improvement in the enantioselectivity of reaction (E > 200) was
achieved using the LU‐MBC preparation (Figure 1). In this case, very high enantiomeric excesses of
alcohol 1a and unreacted acetate 2a were achieved of 97% and 96%, respectively. A highly
enantioselective (E = 99) was also the reaction catalyzed by the LU‐CNBr, in this case, after 24 h, the
conversion exceeded 50%, which resulted in a lower ee of alcohol 1a (90%), but excellent ee of acetate
2a (99%). At a shorter reaction time (20 h, data not shown), when the conversion was close to 50%,
the ee of alcohol 1a was higher, but a lower ee of acetate 2a was observed. The process of
™
immobilization of the enzyme on Supelite DAX‐8 acrylic resin and in calcium alginate did not bring
the expected results. For the reaction catalyzed by LU‐DAX, a slight increase of enantioselectivity
was observed, affording alcohol 1a with 76% ee. However, the only 20% conversion degree achieved
in this reaction resulted in significantly lower enantiomeric excess of unreacted ester 2a compared to
the reaction catalyzed by free LU. Very low enantioselectivity of hydrolysis was observed in the case
™
using Lecitase Ultra Alginate (LU‐ALG).
Preparations LU‐CNBr and LU‐MBC were also the most efficient biocatalysts for the hydrolysis
of propionate 3a (Figure 2).

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Figure 2. The effect of enzyme immobilization on the conversion, enantioselectivity, and
enantiopurity of the products of Lecitase™ Ultra‐catalyzed hydrolysis of racemic propionate 3a
(reaction scheme presented above).
The highest increase in the enantioselectivity of the reaction (E = 170) compared to the reaction
catalyzed by the free enzyme was observed for LU‐CNBr. Using this preparation allowed us to
obtain enantiomerically pure propionate 3a and enantiomerically enriched alcohol 1a (ee 94%) after
24 h. The LU‐MBC preparation hydrolyzed propionate 3a with an enantioselectivity comparable to
that observed for the free LU‐catalyzed reaction, but at a much higher rate. Accordingly, the
enantiomers of the substrate were resolved in a significantly shorter time (24 h) and a 53%
conversion of alcohol 1a and unreacted ester 3a was obtained with enantiomeric excesses of 79% and
90%, respectively. In the processes catalyzed by LU‐DAX and LU‐ALG, both the enantioselectivity of
the reaction and the optical purities of unreacted propionate 3a and alcohol 1a were lower than those
observed in free LU‐catalyzed hydrolysis.
Particularly noteworthy are the results of the hydrolysis of (E)‐4‐phenylbut‐3‐en‐2‐yl butyrate
(4a) (Figure 3).

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Figure 3. The effect of enzyme immobilization on the conversion, enantioselectivity, and
enantiopurity of the products of Lecitase™ Ultra‐catalyzed hydrolysis of racemic butyrate 4a
(reaction scheme presented above)
For each preparation, the enzyme immobilization significantly increased its hydrolytic activity
toward the substrate. Using preparations LU‐CNBr, LU‐DAX, and LU‐ALG, kinetic resolution was
achieved just after 24 h, affording alcohol 1a with ee in the range of 94–97% and unreacted ester 4a
with 95–96% ee. A lower enantiomeric excess of alcohol 1a (ee 83%) was observed only when
LU‐MBC preparation was used; in this case, after 24 h, the conversion exceeded 50% and it was
possible to obtain optically pure unreacted butyrate 4a.
Enantiomeric purities of the products of the kinetic resolution of racemic
™
(E)‐4‐phenylbut‐3‐en‐2‐yl esters through Lecitase Ultra‐catalyzed hydrolysis were comparable or
higher than those obtained previously involving commercially available lipase preparations. Among
the different lipases tested by Ghanem et al. [65], the most effective biocatalyst of the kinetic
resolution of acetate 2a was lipase B from Candida antarctica (CALB). Optically pure alcohol 1a and
unreacted acetate 2a with only 80% ee were obtained after 24 h of reaction in the phosphate buffer
(pH 6.0) with the addition of toluene as the co‐solvent. Similarly, in our earlier work, CALB was
used to resolve racemic acetate 2a and propionate 3a in the reaction conditions analogous to those
used in this study. A short time was needed to achieve kinetic resolution (6 h and 2 h, respectively),
but significantly lower enantiomeric excess of unreacted ester was observed (80–85%) [60]. In other
work, Thalén et al. [66] used CALB in the hydrolysis of enantiomerically enriched
(S,E)‐4‐phenylbut‐3‐en‐2‐yl butyrate (ee 95%) to obtain optically pure butyrate 4a after 2 h of reaction
carried out in a phosphate buffer (pH 7.2), but the reaction was carried out at high temperature (60 °C).
Taking into consideration that the most effective biocatalyst in the kinetic resolution of all
4‐phenylbut‐3‐en‐2‐yl esters 2a–4a was LU‐CNBr, it was decided to extend its scope of application to
the kinetic resolution of butyrates 4b, 4c, and 4d containing the substituted benzene ring (Table 4),
which have been hydrolyzed previously with free enzyme (Table 2).

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Table 4. Hydrolysis of butyrates 4b–d with different substituents on a benzene ring catalyzed by
Lecitase™ Ultra immobilized on cyanogen bromide‐activated agarose (LU‐CNBr).
Entry
Substrate
4b
Ar
t [h]
c [%]1
ees [%]
eep [%]
E2
1
24
48
89
98
>200
2
3
4c
48
72
50
56
99
0
99
0
>200
0
4d
1
2
Conversion, c = ees/(ees + eep). The enantiomeric ratio calculated at the highest conversion rate
according to the following equation: E = ln[(1 − ees)/(1 + (ees/eep)]/ln[(1 + ees)/(1 + (ees/eep)]; ees =
enantiomeric excess of unreacted ester, eep = enantiomeric excess of alcohol.
The preparation was an effective biocatalyst for the enantioselective hydrolysis of butyrate with
the p‐methylphenyl (4b) and 2,5‐dimethylphenyl (4c) substituent. Significantly higher reaction rates
and enantioselectivities were observed compared to the reactions catalyzed by free LU.
Enantiomerically enriched alcohols (98% ee for 1b and 99% ee for 1c) and unreacted esters (89% ee for
4b and 99% ee for 4c) were obtained after 24 h or 48 h. On the other hand, the use of LU‐CNBr did not
affect the enantioselectivity of the hydrolysis of butyrate with the p‐methoxyphenyl substituent (4d)
and, similar to the reaction catalyzed by the free enzyme, both the alcohol 1d and unreacted ester 4d
were racemic mixtures.
The aim of our last experiment was to determine the minimal enzyme dosage required for the
effective kinetic resolution of the tested substrate in a 24 h reaction (Figure 4). The substrate of this
reaction was (E)‐4‐phenylbut‐3‐en‐2‐yl acetate (2a) and hydrolysis was carried out using 100 mg of
the starting ester. Regardless of an enzyme dosage, the enantioselectivity of reaction was
consistently high (E > 200), but significant decrease of the conversion and the optical purity of
unreacted ester 2a was observed when the enzyme dosage was lower than 0.03 U.
Figure 4. The effect of the dose of Lecitase Ultra immobilized on cyanogen bromide‐activated
agarose (LU‐CNBr) on the conversion and enantiopurity of the products after 24 h of hydrolysis of
racemic acetate 2a in phosphate buffer (pH 7.2) and acetone as the co‐solvent.

Molecules 2020, 25, 1067
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™
In order to prove the enantiopreference of Lecitase Ultra toward 4‐arylbut‐3‐en‐2‐yl esters,
alcohols and unreacted esters obtained after hydrolysis of butyrates 4a–4c by LU‐CNBr were
separated using preparative TLC and the configurations of known isolated isomers were
unambiguously confirmed by comparing the signs of their specific rotation with the data in the
™
literature (see Materials and Methods, paragraph 3.9.3). Similar to most lipases, Lecitase Ultra
shows enantiopreference according to Kazlauskas’ rule, hydrolyzing (R)‐enantiomers of tested
substrates with a higher reaction rate, which results in the production of (R)‐alcohols and
(S)‐enantiomers of unreacted esters.
3. Materials and Methods
3.1. Enzyme and Chemicals
™
−1
Lecitase Ultra (LU, >10 000 U × g ) was obtained from Sigma‐Aldrich (St. Louis, MO, USA).
Racemic alcohols: (E)‐4‐phenylbut‐3‐en‐2‐ol (1a), (E)‐4‐(4′‐methylphenyl)but‐3‐en‐2‐ol (1b),
(E)‐4‐(2′,5′‐dimethylphenyl)but‐3‐en‐2‐ol (1c), and (E)‐4‐(4′‐methoxyphenyl)but‐3‐en‐2‐ol (1d) were
synthesized according to the procedures described earlier [61–63]. Acetyl chloride (≥99%), propionyl
chloride (98%), butyryl chloride (≥99%), sodium alginate, calcium chloride (≥96%), p‐nitrophenyl
palmitate (p‐NPP), p‐nitrophenol (p‐NP), and polyethyleneimine (PEI) were purchased from
®
Sigma‐Aldrich (St. Louis, MO, USA). Cyanogen bromide‐activated agarose (Sepharose 4B) was
™
purchased from GE Healthcare Bio‐Sciences AB (Uppsala, Sweden). Supelite DAX‐8 was
purchased from Supelco Analytical (Bellefonte, PA, USA). Sodium alginate was purchased from
Sigma‐Aldrich. Other chemicals were of analytical grade.
3.2. Analysis
Racemic esters 2a–d, 3a–d, and 4a–d were analyzed on an Agilent Technologies 6890N gas
chromatograph (Santa Clara, CA, USA) equipped with hydrogen as the gas carrier, autosampler,
split injection (50:1), and a flame ionization detector (FID) detector. Enantiomeric purity of the
compounds was determined using a CP(Chrompack)‐Chiral‐Dex CB column (25 m × 0.25 mm × 0.25
m, Varian, Palo Alto, CA, USA). The temperature program for the products of hydrolysis of acetates
2a–d and propionates 3a–d are as follows: injector 280 °C, detector (FID) 280 °C, column 80–130 °C
−1
−1
(0.5 °C × min ), 130–200 °C (30 °C min ), 200 °C (2 min); for the products of hydrolysis of butyrate
−1
−1
4a: injector 280 °C, detector (FID) 250 °C, column 80–120 °C (1 °C min ), 120–140 °C (0.5 °C min );
for the products of hydrolysis of butyrate 4b: injector 230 °C, detector (FID) 250 °C, column 40–160
−1
−1
°C (0.5 °C min ), 160–190 °C (5 °C min ); for the products of hydrolysis of butyrate 4c and 4d:
−1
−1
injector 280 °C, detector (FID) 250 °C, column 40–140 °C (0.3 °C min ), 140–180 °C (1 °C min ).
Analytical thin layer chromatography (TLC) was carried out on silica gel coated aluminum plates
(DC‐Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany). Compounds were visualized by
spraying the plates with solution of 1% Ce(SO4)2 and 2% H3[P(Mo 3O10)4] in 10% H2SO4. Silica gel
(Kieselgel 60, 230–400 mesh, Merck) was used as a stationary phase in the chromatographic
purification of synthesized esters 2a–d, 3a–d, and 4a–d. Products of kinetic resolution were
™
separated on preparative TLC silica gel glass plates (Uniplate UV254, layer thickness 1000 μm, 20 cm
× 20 cm, Analtech, Newar, DE, USA). The optical rotations were measured on a Jasco P‐2000‐Na
digital polarimeter (Easton, PA, USA) with an intelligent Remote Module (iRM) controller. The
1
13
NMR spectra ( H NMR, C NMR, HMBC, and HMQC) were recorded on a Bruker Avance II 600
MHz spectrometer (Bruker, Rheinstetten, Germany). Samples were dissolved in CDCl3 and chemical
shifts were referenced to residual solvent signals (δH = 7.26, δC = 77.0). The IR spectra were recorded
on a Mattson IR 300 Thermo Nicolet spectrophotometer (Mattson, Waltham, MA, USA). High
resolution mass spectrum (HRMS) of 4‐(2′,5′‐dimethylphenyl)but‐3‐en‐2‐yl butyrate (4c) was
recorded on a Waters ESI‐QTOF Premier XE spectrometer (Waters Corp., Millford, MA, USA) using
the electron spray ionization (ESI) technique. The index of refraction was measured on an Abbe
refractometer (Carl Zeiss, Jena, Germany).

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3.3. Synthesis of Racemic Esters 2a–d, 3a–d, and 4a–d—General Procedure
Alcohol 1a–d (14 mmol) was dissolved in 100 mL of dry diethyl ether and 10 mL of pyridine.
The mixture was stirred in an ice bath and 19 mmol of corresponding acylating agent (acetyl
chloride, propionyl chloride, or butyryl chloride) was added dropwise. The reaction was continued
at room temperature until the alcohol reacted completely (24 h, TLC). The reaction mixture was
acidified with 1M HCl, the product was extracted with diethyl ether (3 × 40 mL) and separated by
silica gel column chromatography (hexane:acetone, 10:1) to afford pure ester.
(E)‐4‐phenylbut‐3‐en‐2‐yl acetate (2a): Yield 96% (2.7 g), spectroscopic data consistent with
those reported in the literature [67].
(E)‐4‐(4′‐methylphenyl)but‐3‐en‐2‐yl acetate (2b): Yield 98% (2.6 g), spectroscopic data
consistent with those reported in the literature [68].
(E)‐4‐(2′,5′‐dimethylphenyl)but‐3‐en‐2‐yl acetate (2c): Yield 97% (2.8 g), spectroscopic data
consistent with those reported in the literature [63].
(E)‐4‐(4’‐methoxyphenyl)but‐3‐en‐2‐yl acetate (2d): Yield 98% (2.6 g), spectroscopic data
consistent with those reported in the literature [68].
(E)‐4‐phenylbut‐3‐en‐2‐yl propionate (3a): Yield 96% (2.9 g), spectroscopic data consistent with
those reported in the literature [69].
(E)‐4‐(4′‐methylphenyl)but‐3‐en‐2‐yl propionate (3b): Yield 97% (2.9 g), spectroscopic data
consistent with those reported in the literature [69].
(E)‐4‐(2’,5’‐dimethylphenyl)but‐3‐en‐2‐yl propionate (3c): Yield 98% (3.0 g), spectroscopic data
consistent with those reported in the literature [63].
(E)‐4‐(4’‐methoxyphenyl)but‐3‐en‐2‐yl propionate (3d): Yield 97% (2.8 g), spectroscopic data
consistent with those reported in the literature [70].
(E)‐4‐phenylbut‐3‐en‐2‐yl butyrate (4a): Yield 96% (2.7 g), spectroscopic data consistent with
those reported in the literature [71].
(E)‐4‐(4′‐methylphenyl)but‐3‐en‐2‐yl butyrate (4b): Yield 98% (2.9 g), spectroscopic data
consistent with those reported in the literature [71].
(E)‐4‐(2,5‐dimethylphenyl)but‐3‐en‐2‐yl butyrate (4c): Yield 97% (3.1 g), colorless liquid, n 2D0
=
1
1.5175; H NMR, δ: 0.96 (t, J = 7.8 Hz, 3H, CH3‐8), 1.42 (d, J = 6.6 Hz, 3H, CH3‐1), 1.68 (sextet, J = 7.8
Hz, 2H, CH2‐7), 2.30, 2.31 (two s, 6H, CH3‐9 and CH3‐10), 2.29–2.32 (m, 2H, CH2‐6), 5.55 (m, 1H, H‐2),
6.06 (dd, J = 15.6 and 6.6 Hz, 1H, H‐3), 6.79 (d, J = 15.6 Hz, 1H, H‐4), 6.98 (m, 1H, H‐4′), 7.03 (d, J = 7.8
13
Hz, 1H, H‐3′), 7.25 (s, 1H, H‐6′); C NMR, δ: 13.6 (C‐8), 18.5 (C‐7), 19.2 (C‐9), 20.9 (C‐10), 20.5 (C‐1),
36.6 (C‐6), 70.9 (C‐2), 126.2 (C‐6′), 128.5 (C‐4′), 129.4 (C‐4), 130.0 (C‐3), 130.2 (C‐3′), 132.6 (C‐2′), 135.2,
‐1
(C‐1′), 135.4 (C‐5′), 172.9 (C‐5); IR (cm ): 1735 (s), 1457 (m), 1182 (s), 1040 (s), 966 (s), 808 (m); HRMS:
+
calcd for C16H22O2 [M + Na] : 269.1517, found 269.1518.
(E)‐4‐(4’‐methoxyphenyl)but‐3‐en‐2‐yl butyrate (4d): Yield 98% (3.0 g), spectroscopic data
consistent with those reported in the literature [71].
3.4. Determination of Enzyme Activity
™
The enzymatic activity of the free and immobilized preparations of Lecitase Ultra was
determined according to the procedure described by Pencreac’h and Baratti [72] with some
modifications, based on the amount of p‐nitrophenol released during the hydrolysis of
p‐nitrophenyl palmitate. The reaction mixture consisted of 75 μL of 1 mM p‐NPP, 5.75 mL of 10 mM
Tris/HCl buffer (pH 8.0), and 50 μL of free lipase solution or 30 mg of immobilized lipase. The
mixture was kept at 37 °C for 130 min and the reaction was terminated by the addition of cold
ethanol. The absorbance of released p‐nitrophenol was measured on
a
Cintra 101
spectrophotometer, GBC Scientific Equipment at 410 nm. One international unit of p‐NPP activity (1
U) was defined as the amount of enzyme necessary to produce 1 μmol of p‐NP per minute under the
conditions described above.

Molecules 2020, 25, 1067
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™
3.5. Immobilization of Lecitase Ultra by Entrapment in Calcium Alginate
Procedure of immobilization described by Blandino et al. was used with some modifications
™
[73]. Lecitase Ultra (2.2 mL) was added to 10 mL of 6% of sodium alginate solution and stirred for 1
h on a magnetic stirrer. The mixture was dropped using a syringe into 500ꢀmL of 0.3ꢀM CaCl2.
Formed beads of immobilized preparation were left for 1 h in the solution. Finally, the preparation
was filtered, washed with distilled water to remove the residual CaCl2, and freeze‐dried for 24 h
™
using an LYO GT2‐Basic lyophilizer at 10 mbar. As a result, 0.55 g of Lecitase Ultra immobilized in
calcium alginate (LU‐CA) was obtained and stored at 4 °C until use.
™
™
3.6. Immobilization of Lecitase Ultra by Adsorption on Polyacrylic Resin (Supelite DAX‐8)
Immobilization of the enzyme was carried out using the method described by Egger et al. [74]
™
with some modifications. Polyacrylic resin Supelite DAX‐8 (20 g) was washed with Tris‐HCl buffer
and distilled water (4 × 20 mL). The carrier was left at room temperature for 24 h, and 1 g of
pretreated support was placed in a twisted 20 mL vial containing 10 mL of Tris‐HCl buffer and 4 mL
™
of Lecitase Ultra. The mixture was shaken on a laboratory end‐over‐end shaker for 4 days.
Immobilized enzyme was filtered, washed with Tris‐HCl buffer (2 × 10 mL) and freeze‐dried as
™
™
described in paragraph 3.5 (24 h). Finally, 0.8 g of Lecitase Ultra immobilized on Supelite DAX‐8
(LU‐DAX) was obtained and stored at 4 °C until use.
™
3.7. Immobilization of Lecitase Ultra by Covalent Bonds on Cyanogen Bromide‐Activated Agarose (CNBr)
Immobilization of the enzyme was carried out according to the modified protocol reported by
Dos Santos [36]. Cyanogen bromide‐activated agarose (3 g) was successively washed on a Schott G3
funnel with 600 mL of cold 1 mM HCl in several portions for 30 min and then with 50 mL of
™
coupling buffer (0.1M NaHCO3, 0.5 M NaCl, pH 8.3). Commercial Lecitase Ultra (2 mL) in a 250 mL
Erlenmayer flask was diluted in 12 mL of coupling buffer and 3 g of prepared wet carrier was added.
The mixture was shaken on a rotary shaker for 1 h at room temperature, filtered on a Schott G3
funnel, and rinsed with 50 mL of coupling buffer. The immobilized enzyme was transferred back to
the Erlenmayer flask and incubated with 50 mL of 1 M ethanolamine (pH 8.0) at room temperature
for 2 h, filtered on a Schott G3 funnel, then washed with acetate buffer (pH 4.0, 4 × 10 mL) and finally
™
with 50 mL of coupling buffer. After freeze‐drying (24 h), 2.8 g of Lecitase Ultra immobilized on
cyanogen bromide‐activated agarose (LU‐CNBr) was obtained and stored at 4 °C until use.
™
3.8. Immobilization of Lecitase Ultra by Covalent Bonds on Modified Bacterial Cellulose (MBC)
Bacterial cellulose spheres produced by shaking cultures of Komagataeibacter xylinus and
modified with polyethyleneimine and ferromagnetic particles were used as a carrier for enzyme
™
immobilization. The detailed procedure of the carrier preparation as well as Lecitase Ultra
immobilization has been described earlier [50]. Briefly, prior to immobilization, the carrier was
activated with 1% glutaraldehyde solution in 100 mM phosphate buffer (pH 7.0) by shaking at a
roller shaker. After activation, the modified bacterial cellulose (MBC) was collected using a magnetic
separator and rinsed with phosphate buffer to remove an excess of glutaraldehyde. Next, 10 mL of
enzyme solution was added to 5 mL of activated carrier and the mixture was incubated at 4 °C at a
roller shaker. After 24 h, the immobilized enzyme was collected using a magnetic separator and the
supernatant was removed. The preparation was washed with phosphate buffer and incubated at 4
™
°C for 1 h with NaBH4 solution in the phosphate buffer. Finally, 4 g of Lecitase Ultra immobilized
on modified bacterial cellulose (LU‐MBC) was collected using a magnetic separator and flushed
successively with phosphate buffer containing 100 mM NaCl, 0.25% Triton‐X100, and phosphate
buffer. The preparation was freeze‐dried for 24 h and stored at 4 °C until use.

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™
3.9. Hydrolysis of Esters 2a–d, 3a–d and 4a–d Catalyzed by free or Immobilized Lecitase Ultra
3.9.1. General Procedure
™
A total of 0.06 U of free enzyme Lecitase Ultra (2 mL) or 0.06 U of immobilized preparation of
this enzyme (1.33 g LU‐MBC or 1.15 g LU‐DAX‐8, or 1.56 g LU‐CA or 1.05 g LU‐CNBr) and 0.2 g of
ester dissolved in 0.5 mL of organic solvent (acetone, DMF or DMSO) were placed in 10 mL screw
cap glass vials containing 3.5 mL of the phosphate buffer (pH 7.2) or Tris/HCl buffer (pH 8.2). Vials
were shaken at 750 rpm on a magnetic stirrer at 20 °C. Samples (0.6 mL) of the reaction mixture were
taken at different time intervals and extracted with diethyl ether (2 × 2 mL). In the case of the
reaction with the immobilized enzyme, the extracts were filtrated through diatomaceous earth
(Celite 560) to completely remove an immobilized biocatalyst. The extracts were dried and solvent
was removed by evaporation in vacuo. Before chiral gas chromatography (CGC) samples taken from
the reaction mixture were treated with propionyl or acetyl chloride to derivatize inseparable
enantiomers of alcohols 1a–d into the corresponding esters as described earlier [60].
3.9.2. The Effect of Dosage of Lecitase Ultra immobilized Cyanogen Bromide‐Activated Agarose
(LU‐CNBr)
Different dosages of LU‐CNBr and (E)‐4‐phenylbut‐3‐en‐2‐yl acetate 2a (0.2g) in 0.5 mL of
acetone were placed in 10 mL screw cap glass vials containing 3.5 mL of the phosphate buffer (pH
7.2). Vials were shaken at 750 rpm on magnetic stirrer for 24 h. Samples (0.6 mL) from the reaction
mixture were taken after 24 h, filtrated through Celite 560, and prepared for chiral GC as described
in paragraph 3.9.1.
3.9.3. Isolation of Products Obtained by Hydrolysis of Esters 4a–4c—General Procedure
Hydrolysis of butyrate 4a–4c (0.2 g) in the phosphate buffer was carried out as described in
paragraph 3.9.2 using LU‐CNBr (0.06 U). After 24 h of reaction, the products were extracted with
ether diethyl (2 × 5 mL) and the combined organic layers were filtered through Celite 560, washing
the adsorbent with 8 mL of diethyl ether. The extract was dried over anhydrous magnesium sulfate,
the solvent was evaporated in vacuo, and the mixture of products was separated by preparative TLC
(hexane: acetone, 10:1).
After hydrolysis of ester 4a (0.2 g, 0.9 mmol) the following products were obtained:
(S,E)‐4‐phenylbut‐3‐en‐2‐yl butyrate ((S)‐4a): yield 45% (0.09 g), ee 96%, [] 2D0 = −51.3 (c 0.36;
CHCl3); lit. [71]: [] 2D0 = −111.3 (c 1.0; CHCl3, ee 97%).
(R,E)‐4‐phenylbut‐3‐en‐2‐ol ((R)‐1a): yield 47% (0.063 g), ee 97%, [] 2D0 = +19.6 (c 1.35; CH2Cl2); lit.
[65]: [] 2D0 = +19.9 (c 1.6; CH2Cl2, ee 96%)
After hydrolysis of ester 4b (0.2 g, 0.8 mmol) the following products were isolated:
(S,E)‐4‐(4′‐methylphenyl)but‐3‐en‐2‐yl butyrate ((S)‐4b): yield 45% (0.089 g), ee 89%, [] 2D0 = −108.1
(c 0.39; CHCl3), lit. [71]: [] 2D0 = −115.9 (c 1.0; CHCl3, ee 98%)
(R,E)‐ 4‐(4′‐methylphenyl)but‐3‐en‐2‐ol ((R)‐1b): yield 47% (0.066 g), ee 98%, [] 2D0 = +22.7 (c 1.22;
CH2Cl2), lit [69]: [] 2D0 = +22.5 (c 1.5; CH2Cl2, ee 96%)
Hydrolysis of ester 4c (0.2 g, 0.8 mmol) afforded the following products:
(S,E)‐4‐(2′,5′‐dimethylphenyl)but‐3‐en‐2‐yl butyrate ((S)‐4c): yield 48% (0.096 g), ee 99%, [] 2D0 = −46.2
(c 0.38; CH2Cl2).

Molecules 2020, 25, 1067
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(R,E)‐4‐(2′,5′‐dimethylphenyl)but‐3‐en‐2‐ol ((R)‐1c): yield 43% (0.062 g), ee 99%, [] 2D0 = +16.5 (c 0.37;
CH2Cl2); lit. [63]: [] 2D0 = +16.2 (c 1.7; CH2Cl2, ee 98%).
4. Conclusions
™
Results of this survey allowed for the extension of the scope of application for enzyme Lecitase
Ultra in the asymmetric synthesis. This biocatalyst proved to be an attractive alternative to the
commonly used commercial lipase preparations in the kinetic resolution of 4‐arylbut‐3‐en‐2‐yl esters
through their enantioselective hydrolysis. In the reactions catalyzed by the free enzyme, very good
kinetic resolution was obtained in reactions carried out in the phosphate buffer, with the addition of
acetone as a co‐solvent. The enantioselectivity of hydrolysis increased along with the acyl chain
length. The significant increase in the optical purity of unreacted substrates and products (up to
90–99%) as well as the reduction of a reaction time necessary for optimal kinetic resolution was
achieved using immobilized preparations (up to 24–48 h). Particularly effective was the enzyme
immobilized on cyanogen bromide‐activated agarose (LU‐CNBr). This preparation also catalyzed
effective kinetic resolution of substrates containing methyl substituents on phenyl ring (4b,4c),
however, it did not show any enantioselectivity toward 4‐(4’‐methoxyphenyl)but‐3‐en‐2‐yl butyrate
(4d).
™
Further research will be aimed at the extension of the scope of the applicability of Lecitase
Ultra not only in the hydrolysis of structurally diverse esters, but also in the kinetic resolution of
alcohols through their transesterification.
Author Contributions: Conceptualization, A.L. and W.G.; Investigation, A.L., R.D., M.S.; Validation, W.G.;
Writing—original draft, A.L.; Writing—Review & Editing, A.L., W.G., and A.C. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was financed by the statutory activities of the Department of Chemistry, Wrocław
University of Environmental and Life Sciences. Article Processing Charge (APC) was financed under the
Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of
2% of the subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained
in 2019.
Conflicts of Interest: The authors declare no conflicts of interest.
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