Subtilisin-catalyzed esterification of di- and oligosaccharides containing a D-fructose moiety

Article abstract of DOI:10.1016/S0008-6215(98)00320-6

Several di- and oligosaccharides containing a D-fructose moiety have been acylated by protease subtilisin in anhydrous dimethylformamide in the presence of the activated ester trifluoroethyl butanoate. Under the reaction conditions used, all the substrates were converted into the corresponding monobutanoates in ca. 50% isolated yields. Structural determination of the products by ¹³C NMR indicated a strong preference of subtilisin towards the regioselective esterification of the primary hydroxyls of the fructose moiety and, specifically, of the C-1 OH, as already observed with sucrose. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(98)00320-6

Carbohydrate Research 314 (1998) 259–266
Subtilisin-catalyzed esterification of di- and
oligosaccharides containing a
D
-fructose moiety
a,
a
a
b
Sergio Riva *, Micol Nonini , Gianluca Ottolina , Bruno Danieli
a
Istituto di Biocatalisi e Riconoscimento Molecolare, C.N.R., 6ia Mario Bianco 9, I-20131 Milan, Italy
Dipartimento di Chimica Organica e Industriale, Centro C.N.R.. di Studio sulle Sostanze Organiche Naturali,
ia Venezian 21, I-20133 Milan, Italy
Received 5 September 1998; accepted 2 November 1998
b
6
Abstract
Several di- and oligosaccharides containing a
D-fructose moiety have been acylated by protease subtilisin in
anhydrous dimethylformamide in the presence of the activated ester trifluoroethyl butanoate. Under the reaction
conditions used, all the substrates were converted into the corresponding monobutanoates in ca. 50% isolated yields.
13
Structural determination of the products by C NMR indicated a strong preference of subtilisin towards the
regioselective esterification of the primary hydroxyls of the fructose moiety and, specifically, of the C-1 OH, as
already observed with sucrose. © 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Subtilisin; Regioselectivity; Transesterification; Fructose; Lactulose; Disaccharide acylation
1
. Introduction
The upgrading of agriculture by-products is
one of the key issues of the food industry. In
some instances, raw materials are enriched in
specific molecules that can be potentially ex-
ploited as chemical commodities [1].
that is, either into its ‘first generation’ deriva-
tives such as lactitol (2), lactobionic acid (3)
and lactulose (4) or into novel lactose-based
materials, i.e., surfactants and polymers [2].
Lactose (1) is one of these molecules since,
throughout the processing of milk, it accumu-
lates in very large amounts (estimated as 1.2
million tons worldwide annually) from the
dairy by-product cheese whey. Research is in
progress to develop biocatalytic processes of
industrial relevance that might convert lactose
into more valuable products;
In general, sugar-based surfactants can be ob-
tained by selective esterification catalyzed by
lipases or proteases in organic solvents [3],
while esterification with acrylate derivatives
can allow the isolation of sugar-based
monomers suitable for chemical polymeriza-
tion [4]. As recently reviewed, most of the
research performed in this area has been
devoted to the modification of monosaccha-
rides or of the non-reducing disaccharide su-
*
Corresponding author. Fax: +39-02-285-00036.
E-mail address: rivas@ico.mi.cnr.it (S. Riva)
0008-6215/98/$ - see front matter © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(98)00320-6

260
S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
crose (5), and to the investigation of the per-
formances of different lipases and proteases
with these compounds [5,6]. Specifically, su-
crose-based polymers and surfactants have
been synthesized and characterized [7,8], while
to the knowledge of the authors, lactose has
never been considered as an additional freely
available disaccharidic starting material for
the production of such materials.
selective acylation of sucrose to give 1%-O-bu-
tanoyl sucrose (5a). Compounds 4 and 5 have
several structural differences: the other
monosaccharidic moiety is galactose in 4,
whereas it is glucose in 5; the glycosidic bond
is b in 4 and a in 5; the fructose hydroxyl
implicated in the glycosydic bond is the C-4
hydroxyl in 4 and the C-2 hydroxyl in 5.
Therefore, we decided to extend our investiga-
tion on subtilisin regioselectivity to other di-
This paper presents the preliminary results
obtained in the acylation of lactose and of its
derivatives catalyzed by the protease subtilisin
and oligosaccharides containing a
D
-fructose
moiety. To our knowledge, not only is this
information not already available in the litera-
ture, but more generally, very few reports
have been published on the selective esterifica-
tion of commercially available di- and
oligosaccharides catalyzed by lipases and/or
proteases [12–14], despite the fact that these
compounds can be useful building blocks for
the preparation of more complex oligosaccha-
rides.
The commercially available compounds that
have been considered in this investigation are
shown in Fig. 2. Specifically, compounds 6–8
are a homogeneous set of fructose-containing
(
EC 3.4.21.62), as well as an application of
this methodology to several di- and oligosac-
charides containing a -fructose moiety.
D
2
. Results and discussion
Our investigation on the enzymatic esterifi-
cation of lactose 1 and of its derivatives 2–4
started by using the activated ester trifl-
uoroethyl butanoate (TFEB) and the protease
subtilisin suspended in anhydrous dimethyl-
formamide, according to the protocol re-
ported in the first paper published on the
biocatalytic regioselective acylation of di- and
oligosaccharides [9]. As previously reported
disaccharides in which a
D-glucosyl moiety is
a-linked to the fructose C-4 hydroxyl in mal-
tulose (6), C-6 hydroxyl in palatinose (7) and
C-3 hydroxyl in turanose (8). Additionally, we
have considered the disaccharide leucrose (9),
[
9], subtilisin-catalyzed acylation of lactose
was mainly directed to the C-6 primary hy-
droxyl group of the galactosyl moiety. Under
the same conditions, lactitol (2) and lacto-
bionic acid (3) gave a mixture of monoesters,
1
3
as deduced by C NMR spectroscopy. On the
other hand, the acylation of lactulose (4) was
quite regioselective, affording a single mo-
noester in 48% isolated yield (Fig. 1). The
structure of 1-O-butanoyl-lactulose (4a) for
this derivative was suggested by the analysis
1
3
of its C NMR spectrum, which indicated a
small downfield shift of the fructosyl C-1 sig-
nal and a concomitant upfield shift of the
neighboring fructosyl C-2 signal [10]. More-
over, the product structure was confirmed by
submitting 4a to the catalytic action of a
b-galactosidase, which split it into two
monosaccharides, identified as
D
-galactose
and 1-O-butanoyl-
D
-fructose by comparison
Fig. 1. Acylation of lactulose (4) and sucrose (5) catalyzed by
subtilisin in anhydrous dimethylformamide. Arrows indicate
the acylation site. Numbers indicate the percentage of regiose-
lectivity.
with authentic samples [11].
This result was quite peculiar and matched
the previously reported [9] subtilisin-catalyzed

S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
261
Fig. 2. Di- and oligosaccharides acylated by subtilisin in anhydrous dimethylformamide. Arrows indicate the acylation site.
Numbers indicate the percentage of regioselectivity.
in which the fructosyl moiety is forced into a
chair conformation due to the fact that -glu-
can be considered either as a C-3% hydroxyl-
glucosylated sucrose or as a C-2 hydroxyl-glu-
cosylated turanose, was checked.
D
cose is a-linked to the fructose C-5 hydroxyl.
Moreover, the tri- and tetrasaccharides
raffinose (10) and stachyose (11), which can be
considered as sucrose molecules substituted at
C-6 hydroxyl groups, were investigated. Fi-
nally, the trisaccharide melezitose (12), which
All these compounds were acylated by sub-
tilisin following the usual protocol, and the
corresponding monoesters were isolated by
flash chromatography and characterized by
1
3
C NMR spectroscopy using the well-estab-

262
S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
lished acylation-induced shift methodology [10]
following the attribution of the signals of the
carbons of the starting sugars as reported in
refs. [15–19]. The results (Tables 1 and 2),
clearly indicate that the fructose moiety is
indeed highly preferred by subtilisin in the
esterification reaction. Some of the sugars con-
sidered (i.e., maltulose 6 and the tetrasaccha-
ride stachyose 11, besides the already
mentioned lactulose, 4, and sucrose, 5) were
regioselectively acylated at the fructose C-1
hydroxyl, giving the corresponding monoester
in ca. 50% isolated yield. Compounds 7, 9 and
As it is obvious that the spatial orientation
of the hydroxyl groups plays an essential role
in the regioselective acylation of sugars by
hydrolases, molecular modeling has been used
recently to investigate a plausible rationaliza-
tion of the observed selectivity [20–22]. Spe-
cifically, concerning sucrose, it has been
shown [20] that the glucosyl moiety is exposed
to a favorable interaction with a polar solvent
(
such as pyridine or dimethylformamide)
when the fructosyl C-1 hydroxyl is acylated by
subtilisin BPN%. On the other hand, acylation
at the glucosyl C-6 hydroxyl would require
that the entire sucrose molecule must be
buried within the enzyme binding pocket, giv-
ing a sterically less favorable spatial orienta-
tion of the complex enzyme–substrate. The
same approach also explains the results ob-
tained with raffinose (10) and stachyose (11),
as these compounds can be considered as su-
crose derivatives modified at their glucosyl
C-6 hydroxyl. The acylation of melezitose (12)
and turanose (8) at their fructosyl C-6 hy-
droxyl, could also be explained by docking
these two molecules into subtilisin active site
according to the procedure suggested by ref.
[20].
1
0 gave more complex mixtures of monoesters,
in which, however, the corresponding 1-O-acyl
derivatives were the most abundant products.
At variance with turanose (8) and melezitose
(
12), the preferred acylation site was still the
fructose moiety, but the C-6 hydroxyl was
esterified.
The similar selectivity observed with palati-
nose (7) and leucrose (9) (:65–70% acylation
at fructose C-1 OH and :30–35% acylation at
glucose C-6 OH) rules out the possibility that
a lower regioselectivity is determined by the
absence of a fructopyranosyl ring structure (as
in 7) or of a fructofuranosyl structure (as in 9).
On the other hand, the excellent results ob-
tained with the non-reducing sugars sucrose (5),
stachyose (11) and to a lesser extent, raffinose
Recently, Sheldon and co-workers have re-
ported the results obtained in the acylation of
disaccharides by action of Candida antarctica
lipase [12]. As it is shown in Fig. 3, the
(
10), exclude the need for an open fructosyl
chain to get better enzyme selectivity.
Table 1
a
Subtilisin-catalyzed acylation of fructose-containing di- and oligosaccharides
Substrate (mg) Temperature (°C) Reaction time (h) Monoester(s) isolated yield [mg (%)]
Acylated OH
4
6
7
(684)
(342)
(342)
45
45
45
15
15
9
390 (48)
182 (44)
217 (53)
Fructosyl 1-OH
Fructosyl 1-OH
Fructosyl 1-OH (:70%)
and
b
Glucosyl 6-OH (:30%)
Fructosyl 6-OH (:60%)
Fructosyl 1-OH (:66%)
c
8
9
(342)
(342)
45
45
24
15
227 (55)
243 (59)
b
and Glucosyl 6-OH (:34%)
Fructosyl 1-OH
1
0 (504)
30
24
247 (43)
34 (6)
Galactosyl 6-OH
1
1
1 (333)
2 (504)
30
30
24
24
181 (49)
287 (50)
Fructosyl 1-OH
Fructosyl 6-OH (:45%)
c
a
See Section 3.
b
c
As a mixture.
Main product in mixture with other unidentified monoesters.

S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
263
Table 2
a
Physical data for acylated sugars
a
Substrate
Product structure
Eluent
Rf
[h]D (H 0, c 0.5)
Elemental analysis
Calcd for
2
Found
4
6
4a
6a
A
A
0.24
0.34
−23.2
C H O
28 12
16
C, 46.60; H, 6.80
C H O
C, 46.48; H, 6.75
C, 46.58; H, 6.84
47.2
16
28 12
C, 46.60; H, 6.80
b
7
8
9
Mixture of monoesters
Mixture of monoesters
Mixture of monoesters
10a
A
A
A
B
0.29
0.32
0.25
0.23
n.d.
n.d.
n.d.
1
0
102.2
C H O
22
38 17
C, 45.99; H, 6.62
C H O
C, 45.87; H, 6.75
C, 46.12; H, 6.71
C, 45.46; H, 6.48
1
0b
B
C
B
0.39
0.31
0.35
87.4
114.4
n.d.
22
38 17
C, 45.99; H, 6.62
C H O
1
1
2
11a
28
48 22
C, 45,65; H, 6.52
1
Mixture of monoesters
a
A: 16:4:1 EtOAc:MeOH:H O; B: 85:30:10 EtOAc:MeOH:H O; C: 5:5:1 EtOAc:MeOH:H O.
n.d., not determined.
2
2
2
b
esterification of the fructose-containing disac-
charides 5, 6 and 7 catalyzed by this lipase
was mainly directed to the C-6 hydroxyl of the
non-reducing glucose unit, even though the
selectivity was not very high. Compared with
the data reported by Sheldon’s group, our
results offer an additional example of comple-
mentary regioselective acylation by action of
different hydrolases on the same molecule
for solutions in D O or Me SO-d . Optical
2
2
6
rotations were measured using a Perkin–
Elmer 141 polarimeter. TLC were performed
on E. Merck Silica Gel 60 F254 plates, and
[
23,24].
According to the initial goal of this investi-
gation, we will exploit the information ob-
tained in the regioselective modification of 1
and 4 to get new sugar-based surfactants and
polymers.
3
. Experimental
General methods.—Subtilisin Carlsberg
(
protease from Bacillus licheniformis) and b-
galactosidase from Aspergillus oryzae were
from Sigma. The sugars lactose (1), lactitol
(
2), lactobionic acid (3), maltulose (6), palati-
nose (7), turanose (8), raffinose (10), stachyose
11) and melezitose (12) were from Aldrich,
(
Fig. 3. Disaccharides acylated by Novozym 435 (lipase from
Candida antarctica) in tert-butanol. Arrows indicate the acyl-
ation site. Numbers indicate the percentage of regioselectivity
(see ref. [12]).
while lactulose (4) and leucrose (9) were from
Fluka. NMR spectra (see Tables 3–5) were
recorded with a Bruker AC 300 spectrometer

Table 3
13
C NMR data for compounds 4a, 6a, 7a and 7b
a
a
b
b
1
-O-Butanoyl-lactulose (4a)
1-O-Butanoyl-maltulose (6a)
1-O-Butanoyl-palatinose (7a)
6%-O-butanoyl-palatinose (7b)
b-P
b-F
a-F
b-P
b-F
a-F
a-F b-F
a-F b-F
Galactosyl
C-1
C-2
C-3
C-4
Glucosyl
Fructosyl
Butanoyl
Glucosyl
Fructosyl
Butanoyl
Glucosyl
Fructosyl
Butanoyl
101.1
71.0
72.8
68.9
75.9
61.4
103.1
71.0
72.8
68.9
75.9
61.4
103.7
71.0
72.8
68.9
75.9
61.4
103.3
74.6
75.6
72.5
75.1
63.4
101.2
74.0
75.6
72.3
75.1
63.3
100.4
74.0
75.6
72.3
75.1
63.3
99.1
99.1
72.1
73.6
70.4
72.6
61.2
72.1
73.6
70.3
69.7
62.7
/
C-5
C-6
61.0
Fructosyl
C-1
C-2
C-3
C-4
65.6
97.3
66.7
77.3
66.8
63.5
65.6
101.2
75.6
84.0
80.4
62.6
64.6
104.8
81.4
85.2
81.2
62.6
68.1
100.0
70.1
80.6
71.7
66.5
67.1
103.2
78.9
83.7
83.0
65.3
67.1
106.8
83.7
84.4
84.1
64.2
64.7
103.4
82.4
77.5
79.6
68.4
64.2
100.5
75.3
76.7
80.0
68.4
63.8
104.2
83.0
77.1
79.5
68.4
63.2
102.4
75.7
75.8
79.9
68.4
c
c
c
c
314
C-5
C-6
(
1
9
9
8
)
259
Butanoyl
CꢀO
C-2
C-3
C-4
176.6
35.9
18.2
13.2
176.8
35.8
18.1
13.2
172.4
35.6
17.9
13.2
172.4
35.6
17.9
13.2
266
a
Solvent D O.
2
b
Solvent: Me SO-d .
2
6
c
Assignments may be reversed.

S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
265
Table 4
13
C NMR data for compounds 8a, 9a and 9b
a
b
b
6
-O-Butanoyl-turanose (8a)
a-F
1-O-Butanoyl-leucrose (9a)
b-P
6%-O-Butanoyl-leucrose (9b)
b-F
b-P
Glucosyl
C-1
C-2
C-3
C-4
Glucosyl
101.4
72.6
Glucosyl
101.3
72.4
97.8
72.0
73.5
70.2
75.6
61.2
99.2
72.2
73.5
70.2
75.3
61.2
73.7
69.8
72.6
60.9
73.2
70.0
70.1
63.6
C-5
C-6
Fructosyl
C-1
C-2
C-3
C-4
Fructosyl
64.5
Fructosyl
64.4
61.9
105.0
85.4
73.2
80.8
65.2
63.6
102.5
80.8
73.2
79.0
65.2
96.8
68.4
70.1
79.9
97.9
68.8
70.3
79.9
C-5
C-6
62.1
62.0
Butanoyl
CꢀO
C-2
C-3
C-4
Butanoyl
172.7
35.3
Butanoyl
172.7
35.4
177.5
36.5
18.8
13.7
17.9
13.5
17.9
13.5
a
Solvent D O.
2
b
Solvent Me SO-d .
2
6
Table 5
13
C NMR data for compounds 10a, 10b, 11a and 12a
Compound
C-1
C-2
69.3
71.7
103.3
36.5
C-3
C-4
C-5
C-6
a
a
b
a-Galactopyranosyl-(1“6)-
a-glucopyranosyl-(1“2)-
b-fructofuranose
10a
10b
11a
99.3
93.4
63.4
70.3
73.5
77.5
18.8
70.1
70.3
74.3
13.7
71.8
72.3
82.3
62.0
66.8
63.0
butanoate
176.9
a-Galactopyranosyl-(1“6)-
a-glucopyranosyl-(1“2)-
b-fructofuranose
99.1
93.4
62.4
69.1
71.7
104.6
36.5
70.1
73.5
77.2
18.8
70.1
70.3
74.9
13.7
69.6
72.3
82.3
64.9
66.8
63.0
butanoate
177.3
c
c
c
c
a-Galactopyranosyl-(1“6)-
a-galactopyranosyl-(1“6)-
a-glucopyranosyl-(1“2)-
b-fructofuranoside
99.0
98.9
92.1
62.4
172.2
68.6
68.6
69.4
69.8
72.8
76.7
18.0
69.0
69.0
70.2
73.6
13.4
71.2
68.8
71.2
82.7
60.6
66.6
66.6
62.2
c
71.4
102.3
35.5
butanoate
b
a-Glucopyranosyl-(1“3)-
b-fructofuranosyl-(2“1)-
a-glucopyranoside
butanoate
12a
98.2
61.7
92.0
72.1
104.8
71.8
73.6
80.9
73.3
17.9
70.2
73.4
70.2
13.5
72.6
79.5
72.7
60.8
65.8
60.8
172.8
35.3
a
Solvent D O.
2
b
Solvent Me SO-d .
2
6
c
Assignments may be reversed.

266
S. Ri6a et al. / Carbohydrate Research 314 (1998) 259–266
visualized by spraying with a molybdate
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(
buffer pH 4.5 (10 mL). b-Galactosidase from
[
Aspergillus oryzae (10 mg, :900 units) was
added and the solution was left at rt for 6 h
.
(
TLC, 80:20:5 AcOEt:MeOH:H O). Water
2
was lyophilized and the residue was purified
by flash chromatography to give 80 mg (79%
[
[
[
[
[
[
[
[
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4
isolated yield) of 1-O-butanoyl- -fructose
D
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(
identified by comparison with an authentic
sample [11]).
2
779–2781.
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245–3252.
4
Acknowledgements
3
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The authors would like to thank the Eu-
ropean Union for financial support (EC
FAIR Contract 96-1048: Enzymatic Lactose
Valorization).