Two Approaches for CAL-B-Catalyzed Enantioselective Deacylation of a Set of α-Phenyl Ethyl Esters: Organic Solvent with Sodium Carbonate and Micro-aqueous Medium

Article abstract of DOI:10.1007/s10562-020-03525-0

Herein, we report an efficient enantioselective cleavage of the acyl- moiety of a set of α- phenyl ethyl esters with different chain-lengths catalyzed by lipase B from Candida antarctica (CAL-B) by comparing two reactional approaches: anhydrous media with sodium carbonates and micro-aqueous medium. The deacylation is performed in organic solvent, in the presence of Na₂CO₃ in the first case, and by addition of a drop of phosphate buffer solution pH 7 in the second. The results show the high efficiency of the deacylation in the presence of the sodium carbonate for the enzymatic resolution of all the esters and that in term of reactivity (31% ≤ conv ≤ 50%) and selectivity (E > 200). While, during the hydrolysis in micro-aqueous media, the conversion is strongly affected by the length of the acyl-chain side, the conversion decreases from conv = 50% with the 1-phenylethyl acetate 1a to conv = 19% with 1-phenyethyl dodecanoate 6a, and this, even if the selectivity remains high (E > 89). In both conditions, the lipase CAL-B shows a high enantioselectivities in favor of (R)-1-phenyl ethanol enantiomer (conv > 45%, E > 200) but the reactivity is modulated by the form and the size of the acyl-chain side. Graphic Abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s10562-020-03525-0

Catalysis Letters
https://doi.org/10.1007/s10562-020-03525-0
Two Approaches for CAL‑B‑Catalyzed Enantioselective Deacylation
of a Set of α‑Phenyl Ethyl Esters: Organic Solvent with Sodium
Carbonate and Micro‑aqueous Medium
Samra Razi1 · Saoussen Zeror1 · Mounia Merabet‑Khelassi1 · Emilie Kolodziej2 · Martial Tofano2
Louisa Aribi‑Zouioueche1
·
Received: 16 October 2020 / Accepted: 26 December 2020
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Herein, we report an efcient enantioselective cleavage of the acyl- moiety of a set of α- phenyl ethyl esters with diferent
chain-lengths catalyzed by lipase B from Candida antarctica (CAL-B) by comparing two reactional approaches: anhydrous
media with sodium carbonates and micro-aqueous medium. The deacylation is performed in organic solvent, in the presence
of Na2CO3 in the frst case, and by addition of a drop of phosphate bufer solution pH 7 in the second. The results show the
high efciency of the deacylation in the presence of the sodium carbonate for the enzymatic resolution of all the esters and
that in term of reactivity (31%≤conv≤50%) and selectivity (E>200). While, during the hydrolysis in micro-aqueous media,
the conversion is strongly afected by the length of the acyl-chain side, the conversion decreases from conv=50% with the
1-phenylethyl acetate 1a to conv=19% with 1-phenyethyl dodecanoate 6a, and this, even if the selectivity remains high
(E>89). In both conditions, the lipase CAL-B shows a high enantioselectivities in favor of (R)-1-phenyl ethanol enantiomer
(conv>45%, E>200) but the reactivity is modulated by the form and the size of the acyl-chain side.
Graphic Abstract
Keywords Kinetic resolution · CAL-B · Deacylation · Na2CO3 · α-Phenyl ethyl esters · Flavoring agent · Anhydrous
medium · Micro-aqueous medium
Supplementary Information The online version contains
1 Introduction
supplementary material available at https://doi.org/10.1007/s1056
2-020-03525-0.
An impressive range of industrial scale biocatalytic appli-
cations keeps developing [1, 2]. These tools are highly ef-
cient and commercially available with high selectivities
and clean reactions [3]. Enzymatic hydrolysis resolution is
one of the most important, fundamental and practical reac-
tions to have access to optically enriched compounds which
can be transformed with various functionalities such as
* Saoussen Zeror
saoussenzeror@gmail.com
1
Ecocompatible Asymmetric Catalysis Laboratory, Badji
Mokhtar Annaba-University, B.P 12, 23000 Annaba, Algeria
2
Equipe de Catalyse Moléculaire-ICMMO Bât 420, Université
Paris-Sud, Paris Saclay, 91405 Orsay Cedex, France
Vol.:(0123456789)
1 3

S. Razi et al.
pharmaceuticals, agrochemicals, and natural products [4–9].
Serine hydrolases (EC.3.1.1.3) are considered as promising
catalysts due to their advantages, such as mild and environ-
mentally benign reaction conditions not requiring cofactors
and especially remarkable chemo-, regio-, stereoselectivities
[10–14]. Immobilized lipases are excellent biocatalysts for
the enzymatic synthesis of short- and medium-chain fatty
esters used as food favor compounds. The properties of fa-
vor esters make possible a great variety of applications in the
food sector in many beverages, candies, jellies, jams, wines
and dairy and also in the cosmetic industry, as fragrances
in perfumes, deodorants, creams and soaps and favors in
lip cosmetics [15]. Among the employed lipases, Candida
antarctica fraction B, under an immobilized form, is one of
the most popular it is a very robust biocatalyst in various
non-conventional media, with high catalytic efciency in
kinetic resolutions. The exclusive properties of immobilized
CAL-B such as facile recovery, thermostability, environmen-
tal harmlessness, are making it attractive for wide applica-
tions in pharmaceuticals, food technology, organic synthesis,
paper industry and laundry [4, 6, 7, 9, 16, 17].
amino alcohols [20], the enantio- and the diastereoselective
deacylation of cis/trans -2-aryl-1-cyclohexanol derivatives
[21] (Scheme 1). In all these previous works, the leaving
group was acyl moiety (O-Acetyl).
In the continuity of our work, the main objective focused
the efect of the carbon chain length of the leaving group,
on both reactivity and selectivity, in the CAL-B-catalyzed
deacylation of a set of α-phenyl ethyl esters in organic
medium, without external water, in the presence of the car-
bonate sodium. The major advantage of this approach is to
limit the constraints of the aqueous medium including slow
solubility of the substrate and the liquid–liquid extraction
step. So, the study has been extended to examine the hydrol-
ysis reaction in the presence of large amount of external
water, catalyzed by CAL-B and without carbonate salts. The
idea was to compare between two environments of hydroly-
sis with CAL-B lipase, one with large amount of external
water and the other in micro- aqueous media (Scheme 2,
Path A and B).
In our previous work, Candida antarctica lipase (CAL-B)
was successfully used for resolving a set of acetates via alka-
line enzymatic hydrolysis in the presence of the carbonate
salts [18]. The main objective of this process was to circum-
vent some of disadvantages of the conventional methods for
enzymatic hydrolysis, such as the perturbation on the lipase’s
enantioselectivity due to the co-solvent nature as well as the
difculty of controlling the pH value of the aqueous solution
during the hydrolysis procedure. The application of these
alkaline hydrolysis by means of CAL-B and sodium carbon-
ate in organic media gave an excellent resolutions which
have been successfully applied to the deracemization process
[19]. Moreover, this procedure has been successfully applied
to deacylate numerous substrates with a high stereoselectiv-
ity such the chemoselective deacylation of N,O-protected
2 Experimental Section
2.1 Chemicals and Materials
All reagents and solvents were of analytical grade and were
purchased from Sigma-Aldrich. The Candida antarctica
lipase fraction B immobilized on acrylic resin, recombi-
nant, expressed in Aspergillus niger purchased from Sigma-
Aldrich with specifc activity up to 5000 U/g, was used
without any pre-treatment. The monitoring of the reactions
was conducted using TLC on Silica gel 60F254 plates type
MERCK 5179, 250 mesh. The separation of the resulting
alcohols and the remaining acetates was performed by col-
umn chromatography using Silica gel 60 Å, 70–230 mesh
63–200 µm.
[18-19]
[20]
[21]
Scheme 1 CAL-B catalyzed O-deacylation of aryl-ethyl acetates in the presence of Na2CO3
1 3

Two Approaches for CAL‑B‑Catalyzed Enantioselective Deacylation of a Set of α‑Phenyl Ethyl…
O
O
O
R
OH
(S)
Path A- CAL-B/Na2CO3
Path B- CAL-B/H2O
O
R
+
(R)
This work
(R, S)
1a: R= -CH3; 2a: R= -CH2-CH3; 3a: R= -(CH2)2-CH3; 4a: R= -iPr
5a: R= -(CH2)8-CH3; 6a: R= -(CH2)10-CH3 ; 7a: R= -Ph
Scheme 2 Investigated reactions
chromatography on silica gel (petroleum ether/ethyl acetate,
8:2) to aford the corresponding esters. All spectroscopic anal-
ysis were detailed in the supplementary data.
2.2 Instrumentations
The spectroscopic characterisation was performed with
Brüker spectrometers (300 MHz for 1H, 75 MHz for 13C).
Chemical shifts were reported in δ ppm from tetramethyl-
silane with the solvent resonance as internal standard for
1H NMR and chloroform-d (delta 77.0) for 13C NMR. The
enantiomeric excesses of alcohol and esters were measured
by a chiral stationary phase HPLC: Chiralcel-ODH column
(4.6 × 250 mm) column, by a chiral stationary phase GC:
column Astec® CHIRALDEX®B-PM. Retention times are
reported in minutes.
2.5 General Procedure for the Chemical Preparation
of [5a‑6a]
To 1 mmol of 1-phenyl ethanol dissolved in 5 mL of diethyl-
ether, 1.2 eq. of the appropriate chloride acid was added fol-
lowed by the addition of 0.1 mmol 4-dimethylaminopyridine
(DMAP). The suspension was stirred at room temperature. The
reaction was monitored by TLC, after total consumption of
the alcohol, the solvent was removed in vacuo, and the crude
reaction mixture was purifed by column chromatography on
silica gel (petroleum ether/ethyl acetate, 8:2) to aford the cor-
responding esters. All spectroscopic analysis were detailed in
the supplementary data.
2.3 General Procedure for the Chemical Preparation
of 1a and 4a
The racemic esters (1a and 4a) were obtained by standard
classical chemical acetylation of 1-phenylethanol, accord-
ing to the following procedure: to 1 equivalent of racemic
1-phenylethanol, 1.2 equivalent of triethylamine and 0.1
equivalent of 4-dimethylaminopyridine (DMAP) dissolved
in 4 mL of ether, 1.5 equivalent acetic anhydride or iso-
butyric anhydride were added slowly. The evolution of the
reactions was monitored by TLC. The esters are obtained
pure after standard work up, in good yields. All spectro-
scopic analysis were detailed in the supplementary data.
2.6 General Procedure for the Deacylation
of Racemic Esters [1a‑7a] in the Presence
of Na2CO3
To 1 mmol of the racemic acetate dissolved in of organic sol-
vent which was used directly from the bottle P.A quality (for
analysis), without distillation, 1 mmol of sodium carbonate,
previously dried over night, and an appropriate amount of the
lipase, are added. The suspension was stirred at 40 °C for the
indicated time. The reaction mixture is fltered and concen-
trated in vacuo. The remaining acetate and the obtained alco-
hol were separated by chromatography on silica gel (petroleum
ether/ethyl acetate: 80/20) and analyzed by chiral GC.
2.4 General Procedure for the Chemical Preparation
of [2a‑3a‑7a]
2.7 General Procedure for the Conventional
Hydrolysis of Racemic Esters [1a‑7a] in Biphasic
Media
To 1 mmol of 1-phenyl ethanol dissolved in 10 mL of chlo-
roform, 2 eq. of the appropriate carboxylic acid was added
followed by the addition of 1.1 mmol N, N’-dicyclohexyl-
carbodiimide (DCC). The reaction mixture was stirred at
room temperature for 5 h. The solvent was removed in vacuo,
and the crude reaction mixture was purified by column
1 mmol of racemic esters [1a–7a] were dissolved in 1 mL
of organic solvent and added to the appropriate volume of
1 3

S. Razi et al.
phosphate bufer pH 7. An appropriate amount of CAL-B
(UA=5000 U/g) was then added. The suspension was stirred
at room temperature for two days. The reaction mixture was
fltered on celite and concentrated in vacuo. The furnished
ester and the remaining alcohol were separated by fash
chromatography on silica gel (petroleum ether/ethyl acetate:
80/20) and analyzed by chiral HPLC or GC.
hexane/iPrOH: 95/5, flow 1 mL/min): tR = 3.81 min,
tS =4.23 min.
2.8.5 (R,S)‑1‑phenylethyl isobutyrate (4a)
C12H16O2.Crude oil. Yield = 80%. Rf = 0.80. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel ADH,
hexane/iPrOH: 95/5, flow 1 ml/min): tR = 3.67 min,
tS =4.02 min.
2.8 Chiral GC Analysis and/or Chiral HPLC Analysis
2.8.6 (R,S)‑1‑phenylethyl decanoate (5a)
Retention times are reported in minutes. The conditions
for the analysis of (R) alcohol 1 and acetates (S)-1a–7a are
reported below:
C18H28O2.Crude oil. Yield = 74%. Rf = 0.84. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel ADH,
hexane/iPrOH: 95/5, flow 1 mL/min): tR = 3.49 min,
tS =3.87 min.
2.8.1 (R,S)‑1‑Phenylethanol (1)
HPLC (Chiralcel IB, hexane/iPrOH: 95/5, fow 0.5 mL/min):
2.8.7 (R,S)‑1‑phenylethyl dodecanoate (6a)
tR =14.22 min, tS =15.71 min.
C20H32O2. Crude oil. Yield=82%. Rf =0.85. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel ADH,
hexane/iPrOH: 95/5, flow 1 mL/min): tR = 3.39 min,
tS =3.70 min.
2.8.2 (R,S)‑1‑Phenylethyl acetate (1a)
C10H12O2. Crude oil. Yield=78%. Rf =0.65. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel OD-H,
hexane/iPrOH: 95/5, flow 0.5 ml/min):tR = 9.25 min,
tS =9.70 min.
2.8.8 (R,S)‑1‑phenylethyl benzoate (7a)
C15H14O2. Crude oil. Yield=75%. Rf =0.73. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel OD-H,
hexane/iPrOH: 95/5, flow 0.5 mL/min): t1 = 9.37 min,
t2 =9.99 min.
2.8.3 (R,S)‑1‑Phenylethyl benzoate (2a)
C15H14O2. Crude oil. Yield=75%. Rf =0.73. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel OD-H,
hexane/iPrOH: 95/5, flow 0.5 mL/min): t1 = 9.37 min,
t2 =9.99 min.
3 Results and Discussion
2.8.4 (R,S)‑1‑Phenylethyl butyrate (3a)
We have selected the rac-α-phenyl ethyl esters [1a–7a]
with different chain (lengths and congestion) to examine
enzymatic hydrolysis resolution of esters (Scheme 3).
The low molecular weight esters represent an important
C11H14O2. Crude oil. Yield=82%. Rf =0.75. Eluent (v,v):
hexane/ethyl acetate (80/20). HPLC (Chiralcel ADH,
Scheme 3 Rac-α-phenyl ethyl esters [1a–7a]
1 3

Two Approaches for CAL‑B‑Catalyzed Enantioselective Deacylation of a Set of α‑Phenyl Ethyl…
class of fragrances, consisting of compounds derived
from short chain acids, such as acetates, propionates and
butyrates, which are often responsible for fruity aroma
[22]. Moreover, the odour intensity of esters decreases
with the increase of molecular weight; however, the fla-
vour industry needs esters with strong odours and high
molecular weight to prolong the duration of the odour of
flavourings [23]. Our work could be offer an interesting
approach in this area. The enzymatic deacylation of the
esters [1a–7a] experiments were performed in the pres-
ence of Candida antarctica lipase fraction B immobilized
on acrylic resin (CAL-B) at different proportion. For the
reaction of CAL-B-catalyzed kinetic resolution of α- phe-
nyl alkyl esters in the presence of Na2CO3 in non-aqueous
media, we have applied the operating protocol previously
described [18]. Regarding the CAL-B-catalyzed kinetic
resolution of rac-α-phenyl ethyl esters, the conventional
hydrolysis in buffered medium has been studied, from
biphasic medium to micro-aqueous medium, under vari-
ous experimental conditions on 1-phenylethyl acetate (1a)
as study model.
3.1 CAL‑B Catalyzed Kinetic Resolution of α‑Phenyl
Ethyl Esters in the Presence of Na2CO3
in Non‑aqueous Media
We investigated the hydrolysis of phenyl ethyl esters [1a–7a]
to examine the infuence of the acyl part in the hydrolysis in
the presence of Na2CO3. The enzymatic deacylation reac-
tions were carried out on equimolecular mixture of the race-
mic esters [1a–7a] and sodium carbonate in 2 mL of toluene
to which is added 40 mg of CAL-B. The mixture is stirred
three days at 40 °C without addition of any external amount
of water (Scheme 2) [18]. Both enantiomers of the remained
esters and furnished alcohols were recovered after fltration
of the lipase and evaporation in vaccuo. Their enantiomeric
excesses were evaluated by chiral chromatography (GC or
HPLC), and the results are summarized in Table 1.
The result from Table 1 shows that the activity and selec-
tivity of lipase in this basic hydrolysis of racemic esters are
related to the efect of the migrating group structure. The
nature of acyl moiety is of great importance. We observe
that the acyl chain length did not afect the selectivity of
the lipase CAL-B which remains optimal (E>> 200). The
CAL-B exhibits R-enantiopreference [25–27], according to
Table 1 CAL-B Catalyzed kinetic resolution of rac-α-phenyl ethyl esters in non-aqueous media
Entry
Substratea
ees (%)d (S)
eep (%)d (R)
conv (%)e
Ee
1
2
3
4
5
6
7
1a
2a
3a
4a
5a
6a
7a
>99
93
98
44
92
80
1
>99
>99
>99
>99
>99
>99
74
50
48.4
50
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
7
31
48.2
44.7
1
9
10
8(b)
9(c)
8
8
84
74
12
7
a1mmol of racemic ester, 1 mmol of Na2CO3, 40 mg of CAL-B in 2 mL of Toluene at 40 °C
b2 mL of hexane
c2 mL of TBME
dEnantiomeric excess of recovered alcohols and remainingesters are measured by chiral GC or HPLC
econv=eeS/eeP +ees; Selectivity: E=Ln [(1−C) (1−ee(S))]/Ln [(1−C) (1+ee(S))][24]
1 3

S. Razi et al.
the Kazlauskas rules, where the R-enantiomer reacts faster
than the S-enantiomer during the enzymatic kinetic resolu-
tion of secondary alcohols [28].
water until micro-aqueous media. The reactivity and selec-
tivity of the lipase CAL-B as catalyst was studied on deacyla-
tion of 1-phenylethyl acetate 1a as study model. A series of
experiments of enzymatic deacylation reactions were car-
ried out on 1 mol of rac-phenylethyl acetate (1a) diluted in
2 mL of organic co-solvent in the presence several propor-
tion of phosphate bufer solution (pH 7) with the appropri-
ate amount of lipase. The reaction mixtures were stirred for
48 h at room temperature. Both enantiomers of the remained
esters and furnished alcohols were recovered after fltration
of the lipase and liquid–liquid extraction. The course and
selectivity of the enzymatic hydrolysis were quantifed by
chiral chromatography, and the results are summarized in
Table 2
High enantioselectivities were observed with aliphatic
acyl chain side (ee>99%), but reactivities varies between
good to high depending of their bulkiness (31≤conv≤50).
The deacylation of 1-phenylethyl butyrate 3a was achieved
with conv = 31% compared to the 1-phenylethyl propion-
ate 2a and the 1-phenylethyl acetate 1a (entry 4 vs entries
3–2). Good results were obtained for the deacylation of fatty
esters 5a–6a with conversion varied 44.7<conv<48.2 and
excellent selectivity E>200 (entries 5–6). Whilst, using a
benzoate as acyl side 7a, the conversion and the selectivity
drops sharply in the three tested solvents, the best, conv=9%
and E = 12 was noted in TBME (Entry 8 vs Entries 7, 9).
This result suggests that the deleterious impact on the con-
formational changes on lipase due to a aryl group impact
more strongly than the efect of chain portion with isopropyl
group 4a [29].
The results from Table 2 indicate the high enantioselectiv-
ity of the lipase CAL-B under all the experimental conditions
applied (E>> 200). According to previous experimental
conditions for the conventional hydrolysis [30], an impor-
tant infuence of CAL-B loading on the lipase reactivity is
observed. The frst test is carried out in an ethyl ether/water
mixture (2 mL/12 mL) and with 150 mg of CAL-B (entry
1). More eco-friendly reaction conditions [31–33] were
then introduced, such decreasing to the half, of both phos-
phate bufer solution also the CAL-B amount with the use
of tertiobutylmethyl ether (TBME) as alternative to of the
diethylether in term of environmental exigencies [34, 35].
A similar result was reached and an ideal EKR is achieved:
the conversion value of 50%, the (R)-alcohols are obtained
with excellent enantiomeric excesses, ees = eep = 99% and
E>200 (entries 1 and 2). No disturb on both reactivity and
selectivity was recorded by more reducing the amount of
CAL-B to lower values of 12.5 mg per 1 mmole of substrate
(entries 6–7).The decreasing of the proportion of the bufer
solution/co-solvent (v/v) from (5/1) to (0.02/1) only a slight
drop of the conversion from 50 to 45% (entry 5 vs 9). No
reactivity without external source of water was observed
(entry 10). So, to ensure the hydrolysis reaction, it’s suf-
cient to use micro-aqueous system under the optimum elabo-
rated conditions and a single drop of water was essential to
catalytic activity. To study the scope of the CAL-B catalyzed
hydrolysis in micro-aqueous media with the lipase CAL B,
we extended the methodology using our optimized condi-
tions to other substrates. These experimental conditions have
been performed on deacylation of various rac-α-phenyl ethyl
esters [1a–7a] in micro-aqueous media with the lipase CAL
B. The obtained results have been compared to those given
by in non-aqueous media in presence of the carbonate salts.
The obtained results are detailed in Table 3.
The obtained results are in according of previous results
of enzymatic hydrolysis in biphasic medium, which is
explained by the shape of the active site of the lipase CAL-
B. It is more adapted to accept linear substituent, such as
decanoate or laurate, which can more easily penetrate into
it [30].
In the previous investigation, the basic non-conventional
deacylation catalyzed by CAL-B on the aryl ethyl acetates,
had not shown signifcant efect of the aryl moiety, on both
the reactivity and the selectivity, while here the structure
of the acyl side moiety have a signifcant efect on catalytic
performance of the lipase CAL-B.
The aryl substituent as a leaving group shows a drastic
impact on the reactivity and the selectivity of the CAL-B dur-
ing the deacylation with sodium carbonate. It is not the case
with the aliphatic substituent’s, which seem the best candi-
dates for this pathway. This easy non-conventional meth-
odology, assisted by sodium carbonates has been applied
without any external water addition shows ideal for the
cleavage of the aliphatic chain acyl moieties, especially for
the deacylation of fatty esters with high enantioselectivities.
3.2 CAL‑B Catalyzed Kinetic Resolution
of rac‑α‑phenyl Ethyl Esters: From Biphasic
to Micro‑aqueous Media
The objective of this study was to examine the enzymatic
hydrolysis operated in micro-aqueous media. Traditional
enzymatic hydrolysis of racemic esters was mostly operated
within a large amount of external water (phosphate bufer
solution pH 7). To fnd the optimum conditions, the catalytic
amount of the biocatalysts, was frst examined. Then, the
enzymatic hydrolysis have realized under diferent condi-
tions of aqueous medium, from large amount of external
The enzymatic hydrolysis resolution in micro-aqueous
media is carried out with an optimal amount of lipase CAL-
B, the conditions of 12.5 mg of lipase CAL-B in TBME/
H2O (1/0.02) have been selected for the kinetic resolution of
rac-α- phenyl alkyl esters [1a–7a] in micro-aqueous media.
1 3

Two Approaches for CAL‑B‑Catalyzed Enantioselective Deacylation of a Set of α‑Phenyl Ethyl…
Table 2 CAL-B catalyzed kinetic resolution of rac-1a: from biphasic to micro-aqueous media
Entrya
CAL-B
(mg)
Org. Solv./Bufer phosphate
solution (v/v) mL
ees (%)b
eep (%)b (R)
conv (%)c
Ec
(S)
1
2
3
4
5
6
7
8
9
150
75
50
50
25
12.5
12.5
12.5
12.5
12.5
Et2O/H2O (2/12)
Et2O/H2O (2/12)
Et2O/H2O (2/12)
TBME/H2O (1/5)
TBME/H2O (1/5)
TBME/H2O (1/5)
TBME/H2O (1/1)
TBME/H2O (1/0.5)
TBME/H2O (1/0.02)
TBME/H2O (1/0)
>99
>99
>99
>99
>99
>99
90.3
88
>99
>99
>99
>99
>99
>99
>99
>99
>99
–
50
50
50
50
50
50
47.7
47
45
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
>> 200
80
–
10
NR
Org. Solv. organic solvent, NR no reaction
a1 mmol of rac-1a, an appropriate amount of CAL-B, in mixture of organic solvent and phosphate bufer, stirred room temperature for 48 h
bEnantiomeric excess of recovered alcohols and remaining acetates are measured by chiral GC
cConversion: conv=eeS/eeP +ees; Selectivity: E=Ln [(1−C) (1−ee(S))]/Ln [(1−C) (1+ee(S))] [24]
The data of Table 3 show a high selectivity of CAL-B
lipase (E>89) under the selected conditions for the reaction
in the micro-aqueous system although the reactivity is sig-
nifcantly destabilized. The esters [2a–6a] were hydrolyzed
with conversions that vary according on the length of the
acyl moiety. The best reactivities were achieved with the
1-phenyl ethyl acetate 1a and the 1-phenyl ethyl propionate
2a (entries 1 and 2). It has been observed that when a migrat-
ing group chain has more than 2 carbon atoms in length, the
reactivity decreases. In the hydrolysis of 3a, the selectivity
obtained is E=89 for a conversion of conv=24.2% (entry
3). A very similar efect was observed in the hydrolysis of
4a which the butyl group is replaced by the more bulky iso-
butyl group (entries 3 and 4). The hydrolysis of the fatty
esters 5a and 6a was done with more moderate conversions
(18% ≤ conv ≤ 22%). This reveal that the micro-aqueous
conditions are not really efcient for the long chain esters
despite an excellent enantioselectivity factor (E>> 200). No
hydrolysis of the 1-phenylethyl benzoate 7a.
reactivity and selectivity for the rac-α- phenyl ethyl esters,
especially for the fatty esters, molecules of high interest
in several domains such: pharmaceutics and cosmetics
[36]. The path B method, using the traditional hydrolysis
in micro-aqueous system (Scheme 1 path B) showed once
again the limits of enzymatic hydrolysis in biphasic media
especially the problems of solubility of the substrates and
the difculty of controlling the pH value of the aqueous solu-
tion during the hydrolysis procedure [37].
4 Conclusion
Deacylation of a set of α-phenyl ethyl esters with dif-
ferent chain-lengths [1a–7a] via Candida antarctica
lipase B as catalyst in diferent conditions was studied:
in organic solvent with sodium carbonate and in micro-
aqueous medium. The best results recorded shown are
signifcantly related to the nature of the leaving group
structure. During the alkaline hydrolysis, high selectivity
were observed with aliphatic acyl chain side (E>> 200),
The comparison between two deacylations approaches
reveals the efciency of the path A, the protocol involving
sodium carbonate in organic solvent (Scheme 1), in terms of
1 3

S. Razi et al.
Table 3 CAL-B catalyzed kinetic resolution of rac-α-phenyl alkyl esters in micro-aqueous media
Entry
Substratea
ees (%)b(S)
eep (%)b (R)
conv (%)c
Ec
1
2
3
4
5
6
7
1a
2a
3a
4a
5a
6a
7a
80
87
31
29
22
23
–
>99
>99
97
>99
98
45
46
24.2
22
18
>> 200
>> 200
89
>> 200
>> 200
>> 200
–
98
19
NRd
a1 mmol of rac-ester, 12.5 mg of CAL-B, in TBME/H2O (1/0.02) mL, stirred room temperature for 48 h
bEnantiomeric excess of recovered alcohols and remaining acetates are measured by chiral GC or HPLC
cConversion: conv=eeS/eeP +ees; Selectivity [24]: E=Ln [(1−C) (1−ee(S))]/Ln [(1−C) (1+ee(S))]
dNR no reaction
Acknowledgements Algerian Ministry of Higher Education and Sci-
entifc Research (MESRS, FNR 2000) are gratefully acknowledged for
fnancial support of this work.
but, reactivity varied between good to high in function of
their bulkiness (31 ≤ conv ≤ 50), the acyl chain length of
the esters [1a–6a] has afects slightly lipase reactivity. The
obtained results show the efectiveness of the deacylation
in the presence of the sodium carbonate for the enzymatic
resolution, especially of the fatty esters. For the conven-
tional hydrolysis in biphasic media, we have optimized
the minimum quantity of necessary water, and shown the
limits of micro-aqueous conditions for the hydrolysis of
esters. Unfortunately, except for 1a and 2a, the conver-
sions obtained with other esters [3a-6a] are very moderate
(18%≤conv≤22%). So, the alkaline enzymatic hydrolysis
path stills the best alternative for the cleavage of aliphatic
long chain acyl esters, which can be very precious for the
enantioselective deacylation of fatty esters. Contrariwise,
the micro-aqueous conditions are less adapted and only
compatible with short chain esters. A novel hydrolysis
process in toluene with sodium carbonate catalyzed by
CAL-B has been successfully extended to a set of α-phenyl
ethyl esters with diferent chain-lengths which are valuable
commodity chemicals widely found in agrochemicals and
pharmaceuticals.
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