Synthesis of sucrose laurate using a new alkaline protease

Article abstract of DOI:10.1016/S0957-4166(03)00086-7

Sucrose laurate esters were synthesized from sucrose and vinyl laurate in organic solvents using an alkaline protease from a new alkalophilic strain, Bacillus pseudofirmus AL-89. Maximum synthetic activity was observed in the presence of 7.5% v/v water and in the pH range of 7-10. With protease AL-89 esterification occurred predominantly at the 2-O-position while subtilisin A-catalyzed monoester formation predominantly at the 1′-O position. In the absence of enzyme, buffer salts catalyzed non-specific reactions, resulting in the formation of a number of esters. Non-specific catalysis was also observed upon inhibition of the enzyme using a serine protease inhibitor or upon deactivation of the enzyme at pH above 10.

Full text of DOI:10.1016/S0957-4166(03)00086-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 667–673
Synthesis of sucrose laurate using a new alkaline protease
Ninfa Rangel Pedersen, Reinhard Wimmer, Rune Matthiesen, Lars Haastrup Pedersen* and
Amare Gessesse
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, 9000 Aalborg, Denmark
Received 17 October 2002; accepted 21 January 2003
Abstract—Sucrose laurate esters were synthesized from sucrose and vinyl laurate in organic solvents using an alkaline protease
from a new alkalophilic strain, Bacillus pseudofirmus AL-89. Maximum synthetic activity was observed in the presence of 7.5% v/v
water and in the pH range of 7–10. With protease AL-89 esterification occurred predominantly at the 2-O-position while subtilisin A-
catalyzed monoester formation predominantly at the 1%-O position. In the absence of enzyme, buffer salts catalyzed non-specific
reactions, resulting in the formation of a number of esters. Non-specific catalysis was also observed upon inhibition of the enzyme
using a serine protease inhibitor or upon deactivation of the enzyme at pH above 10. © 2003 Elsevier Science Ltd. All rights
reserved.
1. Introduction
catalyze reactions by a similar mechanism.9 To date
only a handful of commercially available lipases and
serine proteases have been used for the synthesis of
sugar esters.10 The serine protease, subtilisin is known
to synthesize sucrose esters predominantly at the 1%-O-
position in DMF and pyridine11–13 and the metallo-
protease, thermolysin has recently been shown to
catalyze the acylation of sucrose at the 2-O-position in
DMSO.14 Thus, enzymes from different sources have
different properties, including stability and selectivity in
the presence of chemical denaturants. This shows the
need to search for new and better biocatalysts that can
withstand the reaction conditions required for efficient
industrial productions. Herein, we report on the use of
an alkaline protease from Bacillus pseudofirmus AL-89,
recently isolated from an alkaline soda lake in
Ethiopia15 for sucrose ester synthesis in organic
solvents.
Sugar esters find important applications in a variety of
industrial processes including their use as biodegradable
surfactants in the food and cosmetic industry, as antitu-
moral agents and plant growth inhibitors.1,2 Currently
sucrose esters are synthesized non-enzymatically at high
temperatures, resulting in low specificity and side reac-
tions with the formation of colored derivatives.3 In
recent years, hydrolytic enzymes have received increas-
ing attention for specific and regioselective acylation of
carbohydrates in organic solvents.4–7 In addition to
their selectivity, enzyme-catalyzed reactions are advan-
tageous as they can be performed under milder condi-
tions than non-enzymatic reactions, potentially
lowering the cost of ester production.
The two main problems encountered with the enzy-
matic acylation of carbohydrates in organic solvents are
the stability of the biocatalyst and the solubility of the
sugar substrate.6,7 While better sugar solubility is
obtained in water miscible organic solvents, such as
dimethylsulfoxide (DMSO) and dimethylformamide
(DMF), most enzymes are denatured in the presence of
these polar solvents.7,8
2. Results
2.1. Effect of water
Protease AL-89 catalyzed the synthesis of sucrose esters
using vinyl laurate as the acyl donor in DMF. No ester
synthesis was detected in the absence of water showing
that the enzyme needed to be hydrated to catalyze the
reaction. Maximum synthetic activity was observed in
the presence of 7.5% v/v water (Fig. 1). Following the
Lipases and serine proteases are commonly used for the
synthesis of sugar fatty acid esters and the enzymes
* Corresponding author. Tel.: +45 9635 8497; fax: +45 98141808;
e-mail: lhp@bio.auc.dk
0957-4166/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00086-7

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N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
Figure 1. Effect of water on sucrose laurate ester synthesis
catalyzed by protease AL-89 in DMF. The yield of ester was
calculated on the basis of the carbohydrate substrate.
progress of the reaction in the presence of 10% v/v
water by TLC, showed that ester formation reached a
maximum at 12 h and decreased markedly at 24 h,
resulting in a reduced yield of the ester.
Figure 2. Effect of sodium carbonate concentration (pH 10)
on the non-enzymatic synthesis of sucrose laurate esters in
DMF. Na2CO3 was deposited on Celite. TLC analysis of
samples incubated with 1.0 M (lane 1), 0.5 M (lane 2) and 0.1
M (lane 3) solutions of sodium carbonate for 6 h. Lane 4
shows a sample incubated with 10 mM sodium carbonate for
29 h, showing complete conversion. 100 mg Celite containing
Na2CO3 buffer was incubated with 150 mM sucrose and 0.5
M vinyl laurate at 45°C Lane 5 shows a sample incubated
with protease AL-89, adjusted to pH 10 with 10 mM sodium
carbonate, and 7.5%, v/v water for 24 h at 45°C. The mobility
of sucrose and sucrose laurate monoesters are indicated to the
right.
2.2. Effect of buffer salts and immobilization matrices
With lauric acid as the acyl donor neither subtilisin A
nor protease AL-89 catalyzed the synthesis of sucrose
ester. However, both enzymes catalyzed the formation
of sucrose esters when using vinyl laurate as the acyl
donor. In all cases a reaction mixture without any
enzyme added was used as a control to determine the
extent of non-enzymatic catalysis. Sucrose ester forma-
tion was repeatedly observed in the control reaction
mixtures. However, when vinyl laurate and sucrose in
the appropriate solvent were mixed in the absence of
buffer salts or Celite no ester formation was observed
even after prolonged incubation. This showed that
buffer salts in combination with the immobilization
matrix catalyze the reaction between vinyl laurate and
sucrose. To investigate the role of immobilization
matrices on ester synthesis, in addition to Celite, three
other matrices (silica gel, Amberlite XAD-7 and
Accurel) were treated with 10 mM sodium carbonate
buffer and used in the reaction. Sucrose laurate ester
formation was observed with all the matrices tested.
Given the wide variation in chemical composition of
these matrices, the similarity in the rate of ester synthe-
sis indicates that the buffer salts play a major role in
catalyzing the reaction. The rate of ester synthesis
increased on increasing the buffer salt concentration
from 10 mM to 1 M (Fig. 2) further indicating that the
buffer salts play an important role in the non-enzymatic
formation of these esters. Similarly, when immobilized
subtilisin A adjusted to pH 7.8 with 10 mM sodium
phosphate buffer was used instead of protease AL-89,
only monoesters were formed in the presence of
enzyme, while a mixture of esters varying in the degree
of substitution (DS) was formed in the absence of
enzyme (Fig. 3B).
Figure 3. Enzymatic and non-enzymatic catalyzed sucrose
ester synthesis in DMF–pyridine 1:1 v/v. Enzymes pH-
adjusted and immobilized on Celite. TLC analysis of reac-
tions: (A) 100 mg immobilized enzyme or Celite containing
buffer was incubated with 150 mM sucrose and 0.5 M vinyl
laurate at 45°C for 24 h Ester formation with PMSF inhibited
(lane 1) or uninhibited (lane 2) protease AL-89, pH 10. (B)
Ester formation in the presence (lane 1) or absence (lane 2) of
subtilisin A, pH 7.8.

N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
669
It is interesting to note that while buffers deposited on
Celite or other matrices catalyzed the synthesis of a
mixture of sucrose laurate esters (Fig. 2 and Fig. 3B),
only monoesters were synthesized in the presence of
protease AL-89 and subtilisin A (Fig. 3A and 3B).
When PMSF (a serine protease inhibitor) inactivated
enzyme was used in the reaction a mixture of esters
similar to that obtained from the non-enzymatic reac-
tion was formed (Fig. 3A). In addition, when the
enzyme was replaced with BSA a similar product mix-
ture was obtained.
of sucrose laurate monoester as the sodium adduct
(Fig. 4A). The reaction with enzyme adjusted at pH 11
and 12 gave four peaks corresponding to the calculated
mass of the sodium adducts of the mono-, di-, tri and
tetrasubstituted esters (Fig. 4B). At pH 11 and 12 no
proteolytic activity in aqueous solution was detected,
indicating that the enzyme was deactivated at these
high pH values. The substitution pattern was identical
to that observed with buffer salt deposited on immobi-
lization matrix suggesting that the ester synthesis was
catalyzed non-enzymatically.
2.3. Effect of solvents
The monoesters synthesized by protease AL-89 were
analyzed by NMR (Table 2). In the range of pH 7–10,
between 50 and 60% of the esters was at the 2-O-posi-
tion and 5–25% were substituted at the 1%-O-position.
Small amounts of other esters were also detected. A
similar reaction carried out using subtilisin A adjusted
in the pH range of 6–9 gave 60–75% of the 1%-
monoester. HPLC analysis revealed no hydrolysis of
the acyl donor occurred with either protease.
With AL-89 adjusted at pH 10 and a water content of
7.5% v/v ester synthesis was observed in pyridine,
DMF, and a 1:1 v/v mixture of DMF–pyridine and
DMF–DMSO. No ester formation was detected in
THF, acetone, dioxane, acetonitrile, or a 1:1 v/v mix-
ture of the above solvents with DMF (Table 1).
2.4. The effect of pH
2.5. Hydrolysis of esters by protease AL-89
Using non-immobilized enzyme, protease AL-89
adjusted to pH 6 prior to lyophilization failed to cata-
lyze ester synthesis while raising the pH from 7 to 10 in
1:1 v/v DMF:pyridine, ester formation was observed.
Mass spectrometry analyses of the reaction products
showed one peak corresponding to the calculated mass
To investigate if sucrose laurate esters of DS 2-4 were
hydrolyzed selectively by protease AL-89, resulting in
the accumulation of sucrose monoesters, a mixture
of non-enzymatically synthesized esters (Fig. 2) was
incubated with AL-89 in increasing amounts of water.
Table 1. Effect of solvents on protease AL-89 catalyzed sucrose laurate ester formation
Solvent
Sucrose laurate (mM)
Solvent
Sucrose laurate (mM)
Solvent
Sucrose laurate (mM)
Pyridine
DMF
DMF–pyridine 80
DMF–DMSO 85
15
72
Acetone
Acetonitrile
dioxane
THF
0
0
0
0
DMSO
0
0
0
0
DMF–acetone
DMF–acetonitrile
DMF–dioxane
The reaction mixture (total volume 1 ml) in the respective solvents or a 1:1 mixture of solvents was composed of 7.5% v/v water, 10 mg of pH
10 adjusted lyophilized enzyme, 150 mM sucrose, and 0.5 M vinyl laurate. The total amount of ester formed after 24 h incubation was quantified
by measuring the amount of unreacted vinyl laurate.
Figure 4. Mass spectra of sucrose laurate esters. Sucrose esters obtained with protease AL-89 adjusted to pH 10 (A) and pH 12
(B). Masses of sodium adducts of mono-, di-, tri- and tetralaurate esters shown on labelled peaks. The unlabelled peaks were also
present in the calibration spectra.

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N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
Table 2. The effect of pH on the position of acylation of sucrose catalysed by protease AL-89 and subtilisin A
Protease
pH
1%O-Lauroyl ester
2-O-Lauroyl ester
6-O-Lauroyl ester
6%-O-Lauroyl ester
Unidentifiable compounds
(%)
(%)
(%)
(%)
AL-89
AL-89
AL-89
AL-89
Subtilisin A
Subtilisin A
Subtilisin A
7.0
8.0
9.0
10.0
6.0
7.8
N.D.
5–10
15–20
20–25
60
ꢀ60
55–60
40–50
ꢀ50
N.D.
N.D.
5
N.D.
3–5
N.D.
N.D.
N.D.
N.D.
ꢀ20
10–15
15
D
D
D
N.D.
N.D.
N.D.
N.D.
5–10
ꢀ15
ꢀ20
10–15
15
75
65
9.0
Immobilized enzyme (100 mg) in DMF:pyridine, 1:1 v/v was incubated with 150 mM sucrose and 0.5 M vinyl laurate at 45°C for 24 h. The
position of acylation was determined by NMR analysis. D: detected, N.D.: not detected.
After 24 h of incubation at 45°C the reaction mixture
was analyzed by TLC. At water content of up to 7.5%
v/v no hydrolysis of the sucrose esters was detected. At
water contents of 10 and 20% v/v hydrolysis of sucrose
esters was evident, with the concomitant production of
sucrose. However, no accumulation of monoesters was
observed.
In the presence of active enzyme (protease AL-89 or
subtilisin A) only monoesters were synthesized. How-
ever, not all sucrose was converted during 24 h. With
protease AL-89 sucrose esters predominantly substi-
tuted at the 2-O-position were obtained. In contrast,
with subtilisin the monoesters substituted primarily at
the 1%-O-position were obtained. Incubation of the non-
enzymatically synthesized heterogeneous mixture of
esters with protease AL-89 at a water content of 0%–
7.5% v/v did not generate any breakdown products,
demonstrating that appearance of only monoesters in
the enzyme catalyzed reaction was not due to hydroly-
sis of sucrose esters of DS 2-4. Ester hydrolysis
occurred at water contents of 10% and 20% v/v, but no
selectivity towards hydrolysis of esters of DS 2-4 was
observed. Furthermore, at a water content of 7.5% and
below, no hydrolysis of the acyl donor was observed.
With an initial molar ratio of 3.3 equivalents of acyl
donor to acyl acceptor, the absence of higher esters in
the protease-catalyzed process was not due to limited
availability of the acyl donor.
3. Discussion
Protease AL-89 adjusted at pH 10 catalyzed the synthe-
sis of sucrose laurate esters using vinyl laurate as the
acyl donor in three hydrophilic solvents. Ester synthesis
was observed only when water was present in the
reaction medium. In organic solvents the enzyme con-
formation is believed to be very rigid, leading to signifi-
cant reduction in activity.16 Hydration is thought to
reduce the rigidity of the enzyme by forming multiple
hydrogen bonds with the amino acid residues, thus
increasing the conformational flexibility of the
enzyme.16,17 However, increasing the concentration of
water past the optimum resulted in a significant reduc-
tion in ester yield, indicating an increase in the rate of
hydrolysis.
When the enzyme was deactivated using an active site
specific serine protease inhibitor (PMSF) and used in
the reaction, a mixture of esters similar to that formed
in the non-enzymatic reaction was obtained. Unreacted
sucrose and a lower yield of higher esters than the yield
of monoesters was observed after 24 h incubation,
showing that the non-enzymatically catalyzed forma-
tion of higher esters occurred more slowly than the
enzyme catalyzed formation of monoesters. In the pres-
ence of pH adjusted, active enzyme both enzymatic and
non-enzymatic catalysis would be expected to occur
simultaneously, resulting in a mixture of mono- and
higher esters at a thermodynamically controlled stage
of the process. Therefore we concluded that the regiose-
lective synthesis of monoester in the presence of active
enzyme was enzymatically catalyzed and that at reac-
tion time of 24 h the ester synthesis seems to be kinetic
controlled.
In contrast to lipases, proteases do not catalyze the
esterification reaction between native fatty acids and
sucrose, but both of these serine hydrolases readily
catalyze a transesterification reaction with vinyl esters
as acyl donors.11,18 The use of such substrates for ester
synthesis results in the liberation of enols, which rapidly
tautomerize to the corresponding aldehyde or ketone,19
driving the reaction forwards and avoiding the need
for continuous removal of the water liberated, which
would otherwise be necessary in an esterification reac-
tion.
In the absence of enzyme, but in the presence of the
buffer salts or bases added for pH adjustment, the
formation of esters also occurred and within 24 h
complete conversion of sucrose to esters of DS 1-4 was
observed. The non-regioselective synthesis of sucrose
esters of varying DS catalyzed by 0.1 M phosphate
buffer has been reported previously.20
Previous studies showed that the solvent composition in
the reaction medium could significantly affect the rate
of enzyme-catalyzed reactions in organic media.6–8 In
our study, of the 13 different solvents and solvent
mixtures tested, sucrose ester synthesis was detected in
three of the solvents. The solvent plays two important

N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
671
roles, determining the solubility of the sugar and alter-
ing the stability of the enzyme. Thus, it is essential to
select the best solvent for high sugar solubility whilst
maintaining enzyme activity so as to increase the yield
of esters synthesized in organic solvents.
was from Akzo Nobel, Obernburg, Germany. Silica gel
60 and thin-layer chromatography (TLC) plates were
from Merck (Darmstadt, Germany). Lauric acid and
vinyl laurate were from Fluka (Buchs, Switzerland).
The organic solvents and vinyl laurate were stored over
molecular sieves.
The pH at which the enzymes are treated prior to
lyophilization is also very important for synthetic activ-
ity in organic solvents.16 Protease AL-89 adjusted in the
pH range of 7-10 catalyzed the synthesis of only
monoesters of sucrose. When the pH was raised to
11–12 the enzyme was deactivated and a mixture of
esters similar to that obtained from the buffer-catalyzed
reaction was formed. Protease AL-89 is an alkaline
serine protease with maximum stability around pH 10
in aqueous medium. It becomes unstable at pH values
above 10.5.15 Thus, the esters synthesized at pH 11 and
12 must result from non-enzymatic catalysis as the
enzyme is inactive at this high pH.
4.1.2. Enzyme sources. Subtilisin A was a kind gift from
Novozymes, Denmark. Protease AL-89 was produced
in the laboratory using B. pseudofirmus AL-89. The
medium used for enzyme production was composed of
(g/l) peptone, 5.0; yeast extract, 2.0; casein 5.0;
K2HPO4, 1.0; magnesium sulfate, 0.2; calcium chloride,
0.1; glucose, 5.0 and Na2CO3, 10.0. Sodium carbonate
was autoclaved separately and added to the rest of the
medium after cooling. 500 ml of the medium in 2 l
flasks was inoculated with 40 ml of pre-culture grown
overnight and incubated 48 h at 37°C in a shaking
incubator. The culture was centrifuged at 3000×g for 20
min and the cell free culture supernatant used as the
enzyme source. To concentrate the enzyme ammonium
sulfate was added to the cell free culture supernatant to
75% saturation followed by centrifugation at 6000×g.
The precipitate was dissolved in water and dialyzed
against three changes of Mili Q water and lyophilized.
The monoester formed in the pH range of 7–10 by
protease AL-89 was predominantly (50–60%) 2-O-lau-
royl sucrose, showing that the enzyme has selectivity
towards reaction at the 2-O-position. In contrast, 60–
75% of the monoesters obtained using subtilisin A in
the pH range 6–9 were at the 1%-O position. The identity
of the ester products was confirmed by NMR analysis
and were shown to be identical to those reported by
Potier et al.11
4.1.3. Preparation of the enzyme catalyst. For sucrose
ester synthesis, the enzyme was used as an immobilized
protein or as a lyophilized protein powder directly into
the reaction medium. Enzyme immobilization was done
by adsorption or deposition. For deposition, 30 mg of
protein was dissolved in 10 mM Na2CO3 buffer (pH 10)
and mixed with 1 g of acid washed Celite followed by
vacuum drying for 3 h. As a control the same amount
of buffer without any protein added was mixed with
Celite and dried under vacuum. In addition, 1 ml of
different molarities (1.0, 0.5, 0.1 and 0.01 M) of
Na2CO3 buffer, pH 10 was each deposited on 1 g
Celite, dried under vacuum and used as control.
Subtilisin has been shown to synthesize esters predomi-
nantly at the 1%-O-position in DMF and pyridine.11–13
Thus, protease AL-89 differed from subtilisin with
respect to regioselectivity. A recent investigation has
shown that another serine protease (Proleather) also
catalyses acylation of sucrose at the 2-O-position in
DMF using divinyl adipate as the acyl donor.5 These
results show that the regioselectivity of different serine
proteases is not the same.
In some cases the regioselectivity of an enzyme has
been shown to be affected by the solvent. For example,
the alkaline protease from Streptomyces spp. catalyzed
the synthesis of the 6-O-galactose ester in DMF, but in
DMF:DMSO 4:1 v/v the 2-O-galactose ester was
formed.21
Alternatively the pH of the enzyme in aqueous solution
was adjusted to 10 with either 0.1 M KOH or 0.1 M
NaOH and then deposited on Celite and vacuum dried
for 3 h. A corresponding control without enzyme was
made with water containing the same volume of 0.1 M
KOH or 0.1 M NaOH. The enzyme preparations were
stored in a dessicator at 4°C.
In conclusion, protease AL-89 adjusted at pH between
7 and 10 catalyzed the acylation of sucrose in a transes-
terification reaction with vinyl laurate. The enzyme
differed from subtilisin in that it shows specificity for
reaction at the 2-O position of sucrose in
DMF:pyridine (1:1, v/v).
Immobilization by adsorption was carried out using a
modification of the method of Ulijin et al.22 Enzyme (10
mg) dissolved in 2.5 ml of 10 mM sodium carbonate
buffer, pH 10 was added to 100 mg Celite, Amberlite
XAD-7 or Accurel EP100 and gently shaken at ambient
temperature until no proteolytic activity detected in the
liquid phase. The immobilized enzyme preparation was
washed several times with DMF–pyridine (1:1 v/v).
Celite and Accurel EP100 treated in a similar way
without the addition of enzyme were used as controls.
The immobilized enzyme and controls were used imme-
diately in an ester synthesis reaction as described below.
4. Experimental
4.1. Materials and methods
4.1.1. Materials. Celite, silica gel, Amberlite XAD 7,
,
sucrose, molecular sieves (3 A, 8–12 mesh), phenyl
methylsulfonyl fluoride (PMSF) and organic solvents
For the preparation of lyophilized powders the enzyme
were from Sigma, St. Louis, MO, USA. Accurel EP100
(protease AL-89 and subtilisin A) was first dialyzed

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N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
against water, followed by adjustment of the pH to the
desired values (from pH 6–12) and lyophilized. The
pH-adjusted enzyme powder was directly used in the
reaction medium.
4.2.2. Thin-layer chromatography (TLC). For TLC anal-
ysis, 2 ml of the reaction mixture was applied to silica
gel 60 plates (0.1 mm) and chloroform/methanol/acetic
acid/water 70:20:8:2 v/v was used as mobile phase.7
Unused substrate and esters were detected by spraying
with methanol/sulfuric acid (1:1 v/v) and heating at
140°C for 10 min.
4.1.4. Ester synthesis. The reaction mixture in total a
volume of 1 ml, contained 150 mM sucrose, 0.5 M vinyl
laurate, immobilized enzyme (100 mg) or lyophilized
enzyme (10 mg) and appropriate amount of water or
for control reactions, 100 mg of Celite buffer control
with or without water. The different solvents tested
were DMF, DMSO, pyridine, acetone, acetonitrile,
dioxane or a 1:1 v/v mixture of these solvents with
DMF. The reaction mixture was incubated at 45°C
with constant stirring at 250 rpm. After 24 h the
reaction was terminated by removing the immobilized
enzyme by filtration through glass wool or by centrifug-
ing to remove the lyophilized enzyme powder. Samples
from the above reaction mixture were then analyzed
qualitatively using TLC, or quantified using HPLC by
measuring the unused vinyl laurate and/or sucrose. The
esters were further characterized by mass spectrometry
and NMR.
4.2.3. Purification of sucrose esters. After removal of the
enzyme by filtration or centrifugation, the reaction
mixture was mixed with three volumes of hexane,
resulting in the precipitation of the unreacted sucrose
and sucrose laurate esters. After washing with n-hexane
three times to remove residual vinyl laurate, the precip-
itate was dissolved in methanol and subjected to
preparative TLC (0.5 mm TLC plates). The sucrose
ester band was collected and eluted from a column with
ethyl acetate/methanol/water (17:4:1 v/v/v).24 Ester
fractions were pooled, dried under vacuum and ana-
lyzed using MS and NMR.
4.2.4. High-pressure liquid chromatography (HPLC).
Analysis was carried out by reversed-phase HPLC
using a system equipped with a Waters-Physics pump, a
Waters Nucleosil 100-C18 column (250×4.6 mm) and a
refractive index detector (Knauer, Germany) For analy-
sis, methanol/water 7:3 v/v or methanol/water 85:15
v/v16 was used as the mobile phase at a flow rate of 1
ml/min at 40°C. The degree of conversion was calcu-
lated from the concentrations of the substrate (sucrose
and/or vinyl laurate).
4.1.5. Hydrolysis of esters using protease AL-89. Sucrose
laurate esters of up to four substitutions were synthe-
sized in DMF using sodium carbonate deposited onto
Celite. After complete conversion, sodium carbonate
Celite control was separated from the reaction medium
by centrifugation and DMF was evaporated on a vac-
uum drier leaving behind the esters. These esters were
dissolved in DMF:pyridine, 1:1 v/v and 10 mg of
protease AL-89 was added with or without the addition
of water and incubated at 45°C with constant stirring at
250 rpm. After 24 h the reaction mixture was analyzed
by TLC.
4.2.5. Mass spectrometry (MS). After 24 h reaction, the
catalyst was removed from the reaction mixture as
described earlier. To 20 ml of the reaction mixture 1.5
ml methanol was added. From this mixture 2 ml were
analyzed by matrix-assisted laser desorption ionization
time of flight (MALDI-TOF) MS on a Bruker Reflex
III, using dried droplet and CCA (a-cyano-4-hydroxy-
cinnamic acid) as matrix. The spectra where calibrated
using Angiotensin II (1046.5423 Da, Sigma), Bombesin
(1619.8229 Da, Fluka), Adrenocorticotropic hormone
fragments 18–39 (2465.1989 Da, Sigma), and Somato-
statin 28 (3149.4783 Da, Sigma).
4.1.6. Enzyme inhibition. To the enzyme (subtilisin A or
AL-89, 10 mg) dissolved in MiliQ water adjusted to pH
7.5 or 10, with 0.1 M KOH, was added 35 ml of a 235
mM stock solution of phenyl methyl sulfonyl fluoride
(PMSF) in methanol. After reacting for 30 min with
occasional shaking, remaining proteolytic activity was
determined and the mixture was freeze dried. Inhibited
freeze-dried enzyme (subtilisin A or AL-89) was incu-
bated with sucrose and vinyl laurate in DMF:pyridine
1:1 v/v in a similar manner to the uninhibited enzymes.
4.2.6. NMR. NMR studies were carried out on a
BRUKER DRX600 spectrometer equipped with a
TXI(H/C/N)xyz-grad probe. Solutions of the samples
in pyridine-d5 were prepared and analyzed by means of
2QF-COSY, TOCSY (120 ms mixing time) and 13C–
1H–HSQC spectra.
4.2. Analytical methods
4.2.1. Proteolytic and protein assays. Proteolytic activity
was assayed using casein as substrate at pH 10 as
described earlier.23
Acknowledgements
One unit (U) of protease activity was defined as the
amount of enzyme, which resulted in the release of 1 mg
of amino acid (equivalent to tyrosine) per minute.
Protein concentration was determined by the Bio-Rad
Bradford assay using bovine serum albumin as a stan-
dard. Protease activity of subtilisin A used was 71.84
U/mg protein. Protease activity of AL89 used was 19.12
U/mg protein.
The present work was financed by Aalborg University.
The authors wish to thank Professor P. Halling (Uni-
versity of Strathclyde, Glasgow, UK) for valuable sug-
gestions and Associate professor Dr. M. Wiebe
(Aalborg University, Denmark) for valuable sugges-
tions and critical reading of the manuscript.

N. R. Pedersen et al. / Tetrahedron: Asymmetry 14 (2003) 667–673
673
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