Multi-substrate screening for lipase-catalyzed resolution of arylalkylethanols with succinic anhydride as acylating agent

Article abstract of DOI:10.1016/j.molcatb.2010.05.016

CAL-B lipase-catalyzed resolution of a number of arylalkylethanols using succinic anhydride as acylating agent has been performed to evaluate the corresponding enantioselectivity factors. Then "one-pot multi-substrate screening" reactions involving two-and four-substrate mixtures were carried out and evaluation of the enantioselectivity factors for each alcohol was undertook by a single-run analysis. It was concluded that the alcohols in the mixture behave independently, validating the "one-pot multisubstrate screening" for a rapid evaluation of the enantioselectivity of the kinetic resolution process for each individual substrate.

Full text of DOI:10.1016/j.molcatb.2010.05.016

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Multi-substrate screening for lipase-catalyzed resolution of arylalkylethanols
with succinic anhydride as acylating agent
Hanane Debbeche , Martial Toffano , Jean-Claude Fiaud , Louisa Aribi-Zouiouechea
a
b
b,∗
a
Groupe de Synthèse Asymétrique et Biocatalyse, Université d’Annaba, 23000 Algeria
Laboratoire de Catalyse Moléculaire, ICMMO, Université de Paris-Sud, 91405 Orsay cedex, France
b
a r t i c l e i n f o
a b s t r a c t
Article history:
CAL-B lipase-catalyzed resolution of a number of arylalkylethanols using succinic anhydride as acylat-
ing agent has been performed to evaluate the corresponding enantioselectivity factors. Then “one-pot
multi-substrate screening” reactions involving two- and four-substrate mixtures were carried out and
evaluation of the enantioselectivity factors for each alcohol was undertook by a single-run analysis. It
was concluded that the alcohols in the mixture behave independently, validating the “one-pot multi-
substrate screening” for a rapid evaluation of the enantioselectivity of the kinetic resolution process for
each individual substrate.
Received 22 February 2010
Received in revised form 12 May 2010
Accepted 31 May 2010
Available online 8 June 2010
Keywords:
Secondary alcohol
Kinetic resolution
Cyclic anhydride
Multi-substrate screening
Lipase-catalysis
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
and ruthenium-catalyzed reduction of ketones by hydride transfer
8].
To date and to the best of our knowledge, only one exam-
[
The “one-pot multi-substrate screening” [1] has been success-
fully used for exploration of catalytic activities and enantioselec-
tivities of a given catalyst by performing reaction on a mixture of
achiral substrates. Since it is of importance to realize the evaluation
of the performance of an enzyme towards several substrates in the
shortest time, the multi-substrate screening methodology appears
particularly suited. The method requires that the ee’s of each unre-
acted substrate (or product) should be measured in a mixture. The
best way is to measure the ee’s by a single run on chiral GLC or HPLC,
with baseline separation for peaks of all compounds. However, the
values measured from multi-substrate reaction are valid for reac-
tions performed onto individual substrates only if these substrates
behave independently in the mixture.
ple of application of enzyme-catalyzed multi-substrate screening
methodology has been reported, dealing with the enzyme-
catalyzed hydrolysis of monoesters [9].
These methods allow the evaluation of the stereoselectivities
of reactions performed onto a mixture of substrates, to get the
stereoselectivity expected on individual substrates.
However, the validation of the methods requires that each sub-
strate reacts in a mixture with the same enantioselectivity as
individually under the same conditions, i.e. that the substrates
behave independently.
This point needs to be addressed since it has been reported that
the stereoselectivity of a lipase-catalyzed acylation of a chiral alco-
hol may be affected by the presence of another substrate, especially
when this latter is of similar structure [10].
In order to have a look to this latter point, we determined the
E-value for lipase-catalyzed acylation of each individual secondary
alcohol, with succinic anhydride as the acylating agent. We then
compared with the values obtained in two- and four-substrate acy-
lation reactions performed under the same conditions. Reactions
were usually performed twice in order to check their reproducibil-
ity.
Most examples of application of such a methodology involve
metal- or organo-catalyzed enantioselective reactions performed
onto achiral substrates: oxazaborolidine-catalyzed borane reduc-
tion of prochiral ketones [2], diethylzinc addition to aldehydes
[
3], rhodium-catalyzed hydroformylation of olefins [4], nickel-
catalyzed hydrosilylation of ketones [5], copper-catalyzed conju-
gate addition of diethylzinc to cycloalkenones [6], nitroalkenes [7]
This work reports our investigation of lipase-catalyzed acylation
of several racemic arylalkylethanols with cyclic, achiral, chiral or
prochiral anhydrides. Use of cyclic anhydrides as acylating agents
has been shown to offer some advantages over the use of vinyl
∗ Corresponding author at: Institut de Chimie Moléculaire et des Matériaux
d’Orsay, Université Paris-Sud, Centre d’Orsay, 91405 Orsay, France.
Fax: +33 1 69 15 46 80.
E-mail address: jean-claude.fiaud@u-psud.fr (J.-C. Fiaud).
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.05.016

3
20
H. Debbeche et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
esters [11]. The product obtained from acylation is a hemiester
which is easily separated from the unreacted alcohol by extraction
with an aqueous sodium bicarbonate solution, avoiding the use of a
chromatographic separation. Furthermore the hemiester liberates
the enantiomeric alcohol through saponification. The enantiomeric
purities of both the unreacted alcohol and the produced one were
measured by chiral HPLC. Conditions for HPLC analysis were set
up to record baseline separations of enantiomers of the mixture
of alcohols. Conversions were calculated from these values. Evalu-
ation of the E-values for each individual substrate would then be
compared with the values recorded for reactions performed onto
two- and four-substrate mixtures. This would indicate whether
the substrates behave independently, and, accordingly, which sub-
strates show high reactivity and selectivity.
(3) 1-(6-Methoxy-2-naphthyl)ethanol (3): hexane/i-PrOH, 90/10;
ꢀ = 254 nm; 1 ml/min; tR(3) (S) = 8.70 min, tR(3) (R) = 11.85 min.
(4) 1-(2-Naphthyl)ethanol (4): hexane/EtOH, 98/2; ꢀ = 254 nm;
0.8 ml/min; tR(4) (S) = 28.2 min, tR(4) (R) = 30.3 min. [(S,S)-ulmo-
column: hexane/i-PrOH, 95/5; ꢀ = 254 nm; 0.5 ml/min; tR(4)
(S) = 12.6 min, tR(4) (R) = 14.5 min].
(5) 1-Acenaphthenol (5): hexane/i-PrOH, 90/10; ꢀ = 254 nm;
0.5 ml/min; tR(5) (S) = 16.0 min, tR(5) (R) = 17.0 min.
(6) 1-(1-Naphthyl)ethanol (6): hexane/i-PrOH, 90/10; ꢀ = 254 nm;
0.9 ml/min; tR(6) (S) = 10.5 min, tR(6) (R) = 16.6 min.
(7) 1-Phenylethanol (7): hexane/i-PrOH, 95/5; ꢀ = 254 nm;
1 ml/min; tR(7) (S) = 8.37 min, tR(7) (R) = 9.67 min.
(8) 1-(4-Methoxyphenyl)ethanol (8): hexane/i-PrOH, 95/5;
ꢀ = 254 nm; 0.8 ml/min; tR(8) (S) = 13.5 min, tR(8) (R) = 15.1 min.
The use of cyclic anhydride in this multi-substrate screening
methodology is of interest: (i) the work-up is easily carried out,
since it does not require a chromatographic separation; (ii) a single
set up ofanalysishas to be done, since boththe unreacted substrates
and the products have the same structure (arylalkylethanols); (iii)
the accurate measurement (chiral HPLC) of ee’s of these alcohols
allows to calculate the conversion, and hence the E factor for each
substrate. We made the assumption that the reactions are irre-
versible and hence the E-values remained constant throughout the
reaction [12].
2
.4. HPLC analysis of the alcohols in two-substrate mixture
(
(
(
(
(
(
(
(
(
1) Alcohols [(1) + (6)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 15.3 min, tR(1) (R) = 17.0 min; tR(6)
S) = 26.9 min, tR(6) (R) = 46.3 min.
2) Alcohols [(6) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(6) (S) = 27.3 min, tR(6) (R) = 46.7 min; tR(8)
S) = 20.0 min, tR(8) (R) = 23.8 min.
3) Alcohols [(1) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 15.7 min, tR(1) (R) = 17.7 min; tR(8)
S) = 20.7 min, tR(8) (R) = 24.5 min.
4) Alcohols [(2) + (3)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(2) (S) = 13.5 min, tR(2) (R) = 15.0 min; tR(3)
S) = 31.1 min, tR(3) (R) = 52.2 min.
5) Alcohols [(2) + (5)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(2) (S) = 13.7 min, tR(2) (R) = 15.2 min; tR(5)
S) = 28.7 min, tR(5) (R) = 32.9 min.
6) Alcohols [(7) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(7) (S) = 13.7 min, tR(7) (R) = 17.1 min; tR(8)
S) = 19.9 min, tR(8) (R) = 21.9 min.
7) Alcohols [(1) + (7)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 14.8 min, tR(1) (R) = 16.5 min; tR(7)
S) = 13.6 min, tR(7) (R) = 17.7 min.
8) Alcohols [(1) + (3)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 14.9 min, tR(1) (R) = 16.9 min; tR(3)
S) = 28.9 min, tR(3) (R) = 40.6 min.
9) Alcohols [(3) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(3) (S) = 31.3 min, tR(3) (R) = 54.1 min; tR(8)
S) = 21.2 min, tR(8) (R) = 23.4 min.
10) Alcohols [(2) + (7)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(2) (S) = 19.2 min, tR(2) (R) = 21.9 min; tR(7)
S) = 16.4 min, tR(7) (R) = 17.1 min.
0
(
0
(
The commercially available Candida antarctica lipase B (CAL B)
was chosen for this study, since it is one of the most used for kinetic
resolution of alcohols. Moreover, we showed that it performed bet-
ter than Pseudomonas fluorescens lipase (PFL) [13].
0
(
0
(
2
. Experimental
2.1. Materials and methods
0
(
Substrates 1–8 and anhydrides 9–12 were commercially avail-
able and purchased from Acros Organics (1, 3, 9), Sigma–Aldrich (2, 5,
1, 12), Fluka (4), Fluka Chemika (6), Janssen (7), Janssen Chemika (10)
0
(
1
and Alfa Aesar (8). They were used as received. Lipase acrylic resin
from C. antarctica (Novozym, L4777, 3 units/mg) was purchased
from Sigma–Aldrich.
Reactions were monitored by TLC and the enantiomeric excesses
of the alcohols were determined by chiral HPLC on Chiralcel® OD-H
column (25 cm × 4.6 mm), with UV-detection at 254 nm.
0
(
0
(
0
(
2.2. General procedure for kinetic resolution
(
The mixture of equimolecular amounts of the racemic alcohol
0
(
(
1 mmol) and anhydride (1 mmol) in diethyl ether (5 ml) and the
lipase (150 mg) was stirred for 24 h at room temperature. After fil-
tration of the enzyme, the ethereal solution was extracted with an
aqueous solution of 2 M Na CO3 (2× 2 ml). The combined aqueous
2
extracts were washed with ethyl ether (5 ml) and the combined
2.5. HPLC analysis of the alcohols in four-substrate mixture
organic phases were dried (MgSO ) and evaporated to recover the
4
unreacted alcohol. To the combined aqueous phases was added an
aqueous NaOH solution (5 ml, 10%). After 2 h the aqueous phase was
extracted with ethyl ether (2× 5 ml), the combined ethereal phases
(
1) Alcohols [(1) + (3) + (6) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 16.3 min, tR(1) (R) = 18.4 min; tR(3)
0
(
(
S) = 34.6 min, tR(3) (R) = 55.9 min; tR(6) (S) = 31.5 min, tR(6)
R) = 50.4 min; tR(8) (S) = 22.3 min, tR(8) (R) = 24.6 min.
were dried (MgSO ) then evaporated to give the alcohol which has
4
reacted in the acylation process.
(
2) Alcohols [(1) + (4) + (7) + (8)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
.8 ml/min; tR(1) (S) = 15.9 min, tR(1) (R) = 18.0 min; tR(4)
(S) = 29.7 min, tR(4) (R) = 33.4 min; tR(7) (S) = 14.3 min, tR(7)
R) = 19.0 min; tR(8) (S) = 21.0 min, tR(8) (R) = 22.9 min.
0
2.3. HPLC analysis of racemic alcohols 1–8
(
(
1) 1-Indanol (1): hexane/i-PrOH, 98/2; ꢀ = 254 nm; 0.8 ml/min;
tR(1) (S) = 20.7 min, tR(1) (R) = 23.9 min.
2) 1,2,3,4-Tetrahydro-1-naphthol (2): hexane/EtOH, 98/2;
(3) Alcohols [(2) + (3) + (5) + (7)]: hexane/EtOH, 98/2; ꢀ = 254 nm;
0.8 ml/min; tR(2) (S) = 10.5 min, tR(2) (R) = 11.2 min; tR(3)
(S) = 21.0 min, tR(3) (R) = 33.7 min; tR(5) (S) = 23.4 min, tR(5)
(R) = 25.8 min; tR(7) (S) = 11.1 min, tR(7) (R) = 14.0 min.
(
ꢀ
= 254 nm; 0.8 ml/min; tR(2) (S) = 9.45 min, tR(2) (R) = 10.1 min.

H. Debbeche et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
321
Table 1
a
CAL-B-catalyzed acylation of alcohols 1–8 by cyclic anhydrides : conversions, yields, enantiomeric excesses and E-values.
Entry
Substrate
Anhydrideb
Substrate
eeS (%)c
Product
eeP (%)c
c (%)e
Ee
Yield (%)d
Yield (%)d
1
2
3
4
SA
GA
MeSA
MeGA
>99
47
75
41
54
52
52
>99
99
>99
85
26
15
24
16
50
32
43
26
>200
>200
>200
f
1
2
3
4
5
6
7
8
30
17
5
6
7
8
SA
GA
MeSA
MeGA
94
24
3
51
71
66
56
>99
>99
76
26
7
3
49
20
4
>200
>200
10
f
f
f
f
f
2
97
2
2
70
9
SA
GA
MeSA
MeGA
74
7
2
52
78
85
82
>99
74
93
32
5
2
43
9
2
>200
8
30
1
1
1
0
1
2
6
98
3
6
>100
1
1
1
1
3
4
5
6
SA
GA
MeSA
MeGA
88
31.5
5
41
58
78
83
>99
61
95
30
24
1
47
34
3
>200
6
40
9
87
5
9
15
1
1
1
2
7
8
9
0
SA
GA
MeSA
MeGA
5
<1
1
56
84
78
87
36
Nd
44
Nd
18
1
1
12
–
2
3
–
4
–
0.2
3
–
2
2
2
2
1
2
3
4
SA
GA
MeSA
MeGA
5
2
1
82
76
88
–
91
71
28
–
5
1
1
–
5
3
2
–
23
7
3
<1
–
2
2
2
2
5
6
7
8
SA
GA
MeSA
MeGA
98
29
11
16
37
55
45
47
99
35
8
3
50
23
9
>200
>200
>99
>99
99
f
>200
7
14
>200
2
3
3
3
9
0
1
2
SA
GA
MeSA
MeGA
97
42
16
23
45
69
82
46
97
97
98
98
30
18
12
14
50
30
14
19
>200
100
f
>100
>100
a
b
c
d
e
f
Reactions were performed onto 1 mmol of alcohol and 1 equiv. of anhydride in diethyl ether (5 ml) in the presence of 150 mg lipase for 24 h.
SA: succinic anhydride; GA: glutaric anhydride; MeSA: 2-methylsuccinic anhydride; MeGA: 3-methylglutaric anhydride.
ees and eep were measured by chiral HPLC on Chiralcel OD-H column.
®
Isolated yield.
Conversion c and enantioselectivity E were calculated from relationships: c = eeS/(eeS + eeP); E = ln [1 − c(1 + eeP)]/ln [1 − c (1 − eeP)] [14].
Calculated with the assumption that the stereoselectivity of attack of the alcohol onto the diastereomeric acylenzymes solely arises from asymmetric induction of the
enzyme chirality.
Fig. 1. Arylalkyl alcohols to be resolved and cyclic anhydrides used as acylating agents.

3
22
H. Debbeche et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
Table 2
Acylation of a two-substrate mixture of alcohols: conversions, yields, enantiomeric excesses and E-values.
Entry
Alcohola
Unreacted alcohol
eeS d (%)
Recovered alcohol
eePd (%)
c (%)e
Ee
Yieldc (%)
Yieldc (%)
1
2
3
4
5
6
7
8
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
>99
94
74
88
5
5
98
97
41
51
52
42
56
82
37
45
>99
>99
>99
>99
36
91
99
97
26
26
32
25
18
5
50
49
43
47
12
5
>200
>200
>200
>200
3
23
>200
>200
35
30
50
50
9
(1) + (6)
(6) + (8)
(1) + (8)
(2) + (3)
(2) + (5)
(7) + (8)
(1) + (7)
(1) + (3)
(3) + (8)
(2) + (7)
(7) + (8)
[7 + Enz] + 8
(1)
6)
>99
1
>99
72
50
2
>200
7
(
1
1
1
1
1
1
1
1
1
1
2
2
0
(6)
8)
4
88
62
95
6
48
5
110
(
1
(1)
8)
80
56
97
95
45
37
160
70
(
2
(2)
3)
75
68
>99
99
43
41
>200
>200
(
3
(2)
5)
41
11
95
70
30
14
60
7
(
4
(7)
8)
17
23
>99
98
15
19
>200
120
(
5
(1)
7)
36
14
96
89
27
14
70
20
(
6
(1)
3)
93
20
89
>99
51
17
60
>200
(
7
(3)
8)
89
84
89
>99
50
46
50
>200
(
8
(2)
7)
6
21
69
85
8
20
7
16
(
9
(7)
8)
17
23
>99
98
15
19
>200
120
(
0b
1b
(7)
8)
59
74
75
74
44
50
15
15
(
c
[8 + Enz ] + 7
(7)
8)
60
76
95
91
39
46
70
50
(
a
b
c
Entries 1–8: alcohol (1 mmol), SA (1 equiv.), lipase (150 mg), diethyl ether (5 ml), 24 h; entries 9–21: under the same conditions but with 0.5 mmol for each alcohol.
The first alcohol and the enzyme were stirred for 10 min before the addition of the second alcohol.
Isolated yield.
d
e
®
ees and eep were measured by chiral HPLC on Chiralcel OD-H column;.
Conversion c and enantioselectivity E were calculated from relationships: c = eeS/(eeS + eeP); E = ln [1 − c (1 + eeP)]/ln [1 − c (1 − eeP)] [14].
3
. Results and discussion
Acylation of the enzyme by MeSa could result in the forma-
tion of regio- and diastereoisomeric acylenzymes. However, the
E-value was estimated on the assumption that the stereoselec-
tion solely arose from the enzyme chirality. Since conversions of
most of the alcohol substrates, with the exception of 1, were low,
results recorded from acylation with MeSA-11 will not be further
discussed.
Low values for activity and enantioselectivity were recorded for
alcohols 5 and 6 whatever the acylating agent was.
On the basis of these results, succinic anhydride was then
selected to carry out the multi-substrate assays.
3.1. CAL-B-catalyzed acylation of benzylic-type alcohols
The kinetic resolution of arylalkyl alcohols 1–8 was carried out
with four different cyclic anhydrides 9–12 under CAL-B catalysis
Fig. 1).
Each alcohol was subjected to acylation according to the
(
reported procedure, in diethyl ether as the solvent. Enantiomeric
excesses of the unreacted alcohol and the alcohol obtained from the
hemiester produced were measured by chiral HPLC. The conversion
was calculated from these values, together with the enantioselec-
tivity factor E. Table 1
With succinic anhydride as the acylating agent, the conversion
was over 43% and the E-value > 200, except for 1-acenaphthenol 5
and 1-(1-naphthyl)-ethanol 6, which show both low reactivity and
enantioselectivity. Glutaric anhydride provided less satisfactory
results, although enantioselectivity remained high for substrates
3.2. CAL-B-catalyzed acylation of a two-substrate mixture of
alcohols
In order to check whether the acylation of both substrates pro-
ceeded independently, we ran two-substrate experiments under
analogous conditions than those established in single-substrate
assays. A number of two-substrate mixtures were subjected to
CAL-B-catalyzed acylation with succinic anhydride under the same
experimental procedure as the one used in single-substrate assay.
1
, 2, 7 and 8. Methylglutaric anhydride afforded high enantioselec-
tivities for the same substrates as glutaric anhydride did (with the
exception of 1 and 4), albeit with lower activities.

H. Debbeche et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
323
Table 3
CAL-B-catalyzed acylation of a four-substrate mixture of alcohols.
Entry
Alcohola
Unreacted alcohol
eeS (%)c
Recovered alcohol
eeP (%)c
c (%)
Ec
Yield (%)
Yield (%)
1
2
3
4
5
6
7
8
9
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
>99
94
74
88
5
41
51
52
42
56
82
37
45
>99
>99
>99
>99
36
91
99
97
>99
99
26
26
32
30
18
5
50
49
43
47
12
5
50
50
50
25
2
50
48
20
42
41
42
26
3
>200
>200
>200
>200
3
5
20
98
96
>99
33
1
>99
93
25
69
67
13
34
3
35
30
>200
>200
>200
>200
5
>200
>200
>200
66
>200
>200
90
20
>200
(1 + 3 + 6 + 8)
(1)
(3)
(6)
(8)
61
>99
>99
99
94
98
>99
79
90
1
0
1
(1 + 4 + 7 + 8)b
(2 + 3 + 5 + 7)
(1)
(4)
(7)
(8)
1
(2)
(3)
(5)
(7)
37
99
27
Conversion c and enantioselectivity E were calculated from relationships: c = eeS/(eeS + eeP); E =conversion c and enantioselectivity E were calculated from relationships:
c = eeS/(eeS + eeP); E = ln [1 − c (1 + eeP)]/ln [1 − c (1 − eeP)] [14]. ln [1 − c (1 + eeP)]/ln [1 − c (1 − eeP)] [14].
a
Reactions were performed onto 0.5 mmol of each alcohol in the presence of 150 mg lipase/mmol in diethylether for 24 h.
b
Reaction carried out with 75 mg lipase.
c
®
ees and eep were measured by chiral HPLC on Chiralcel OD-H column.
The results collected in Table 2 show the following main features:
for runs involving a “reactive” substrate and a “poorly reactive”
one, results – in terms of reactivity and selectivity – are not so
affected by the presence of the poor substrate and are closed to
those obtained in single-substrate runs. Hence the alcohols appear
to react independently.
However, for reaction involving a 2-“good”-substrate mixture,
the conversion appears sometimes depleted although the enan-
tioselectivity is hardly affected (run 14, 7 and 8; run 15, 1 and
The hemiester produced from reaction of one alcohol could be
an acylating agent to provide new acylenzymes which would be
deacylated by the same or other alcohol, with different stereoselec-
tivities than when reacting onto the primary acylenzyme, affording
diesters. This could explain the altered enantioselectivity recorded.
However, we did not detect significant amounts of diesters in the
reaction product.
In most cases, the multi-substrate screening would afford stere-
oselectivity figures generally lower or similar to those shown for
reactions performed with individual substrates.
3).
We may tentatively offer an explanation for the depleted con-
version observed when using mixtures of several – however active
substrates compared to the rates observed for individual sub-
strates.
As mentioned in previous reports, acylation of the enzyme
by an acylating agent of structure similar to the nucleophile
involved in the deacylation process would result in an imprint-
ing process which would favor the following reaction of the
nucleophile. Conversely, it may disfavor the reaction of other
nucleophiles.
3
.3. CAL-B-catalyzed acylation of a four-substrate mixture of
–
alcohols
Some experiments have been conducted with a four-substrate
mixture (Table 3). The results show that enantioselectivities are not
greatly affected, although for some combinations the conversion of
some alcohol may be reduced, as for 3 in the mixture (1 + 3 + 6 + 8)
or (2 + 3 + 5 + 7) and 4 in the mixture (1 + 4 + 7 + 8).
So that for multi-alcohol reactions, we may tentatively sug-
gest that each alcohol would imprint, in the deacylation process
a number of enzyme sites favoring the further reactions of this
alcohol with these sites. This would result in “specialization” of
the sites of an enzyme towards one particular alcohol of the mix-
ture. Hence, in addition of being in lower molecular ratio, each
alcohol would have access to a limited number of sites with
a good reactivity, resulting in a lower overall rate of the pro-
cess.
4
. Conclusion
As a conclusion, the use of a multi-substrate procedure in the
CAL-B-catalyzed acylation by succinic anhydride allows an eval-
uation of the enantioselectivity displayed by the enzyme for the
single substrate. The evaluation may however be underestimated.
The reactivity of some substrates might be affected. The order
of addition of the alcohols and enzyme may also influence both
parameters.
The order of introduction of the reagents (enzyme added to a
mixture of the 2 alcohols) has some influence on the reactivity and
selectivity.
For the mixture of substrates 7 and 8, the enzyme added to
the mixture of alcohols resulted in a reduction of the reactivity
although the enantioselectivities for both substrates were pre-
served. However, addition of the enzyme to the first alcohol before
addition of the second substrate restored the reactivity, albeit with
depletion of the enantioselectivity.
Acknowledgements
We acknowledge the Algerian Ministry of Education and Scien-
tific Research and the French Foreign Ministry for a fellowship for
Hanane Debbeche. We thank Tassili 06 MDU 674 and FNR 2000 and
PNR for financial support.

3
24
H. Debbeche et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 319–324
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