Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter sp. CGTase

Article abstract of DOI:10.1016/j.carres.2012.08.002

Monoglucosylated products of l-chiro-, d-chiro-, muco-, and allo-inositol were synthesized by regioselective α-d-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydrolysis of by products with Aspergillus niger glucoamylase. While the reactions carried out with d-chiro-, muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two products were obtained in the reaction with l-chiro-inositol. Through the structural characterization of the glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation of β-d-glucopyranose and equivalent glucosides.

Full text of DOI:10.1016/j.carres.2012.08.002

Carbohydrate Research 360 (2012) 93–101
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter
sp. CGTase
Alfonso Miranda-Molina a,b, Silvia Marquina-Bahena a, Agustín López-Munguía b, Laura Álvarez a,
,
⇑
Edmundo Castillo b,
⇑
a Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos. Av. Universidad 1001, Col. Chamilpa, C.P. 62209, Cuernavaca, Morelos 62210, México
b Departamento Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Av. Universidad 2001, Col.
Chamilpa, C.P. 62209, Cuernavaca, Morelos 62250, México
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 May 2012
Received in revised form 2 August 2012
Accepted 4 August 2012
Available online 14 August 2012
Monoglucosylated products of L-chiro-, D-chiro-, muco-, and allo-inositol were synthesized by regioselec-
tive
sis of by products with Aspergillus niger glucoamylase. While the reactions carried out with
muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two prod-
ucts were obtained in the reaction with -chiro-inositol. Through the structural characterization of the
a
-
D-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydroly-
D
-chiro-,
L
glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several
inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation
of b-D-glucopyranose and equivalent glucosides.
Keywords:
CGTase
Glucoamylase
Inositols
Ó 2012 Elsevier Ltd. All rights reserved.
a-D-Glucosylation
Regiopecificity
Configuration
1. Introduction
(PIMs), the components of mycobacterial cell walls, have
demonstrated to be immunomodulators.7 More recently, two series
of new myo-inositol-derived glycolipid analogues were isolated
from leaves of the Mexican medicinal plant Solanum lanceolatum,
which demonstrated in vivo important anti-inflammatory activity.8
Although, most of the glycosylated forms of inositols are natu-
rally present in the vegetal kingdom, the low content in plants
has limited their isolation and commercial application.9 In order
to cope with the lack of a convenient source of inositols, several
chemical routes have been described for their synthesis. However,
due to the similar reactivity of the multiple inositol hydroxyl
groups, the regio- and stereo-selective glycosylations of these com-
pounds remain a challenge in organic chemistry.3 During the last
two decades, biocatalysis has become an interesting alternative
in organic synthesis due to the chemo-, regio-, and stereo-selectiv-
ities of enzymes and their ability to carry out reactions in mild con-
ditions.10–17
Inositols (cyclohexanehexols) are sugar-like molecules with
significant chemical and biological properties. Among all possible
inositol stereoisomers, myo-inositol is the most prominent form,
widely occurring in nature. Other naturally occurring isomers are
scyllo-, neo-,
epi-, allo-, -chiro-, and cis-inositol are considered unnatural syn-
D
-chiro-, and muco-inositol.1 The remaining three,
L
thetic inositol isomers.2,3 It has been reported that all inositol ster-
eoisomers may be prepared from myo-inositol by inversion of the
configuration (epimerization) of one or two of its hydroxyl groups.4
In recent years, evidence of the physiological role of inositols and
their glycosylated derivatives in cell functions has considerably
increased the interests in these compounds for pharmaceutical
applications. Indeed, it has been reported that glycosylphosphat-
idylinositols (GPI) contribute to activate innate immune cells or
to exert partial insulin-mimetic activity on glucose and lipid
metabolism in insulin-sensitive cells (adipocytes, cardiomyocytes
and diaphragms).5,6 Moreover, phosphatidylinositol mannosides
Cyclodextrin glucosyl transferases (CGTase) (EC 2.4.1.19) are
hexosyltransferase enzymes, produced extracellularly by a wide
variety of bacteria, particularly Bacillus. The distinctive feature of
these enzymes is the formation of cyclodextrins from starch
through intra-molecular transglycosylation of
D
a-D-(1?4)-linked
⇑
Corresponding authors. Tel./fax: +52 777 329 7997 (L.Á.); tel.: +52 777
56227609 (E.C.).
-glucose oligosaccharides, resulting in cyclization18 Additionally,
these enzymes also catalyze intermolecular transglycosylations
involving disproportionation and hydrolysis of the amylose
E-mail addresses: lalvarez@ciq.uaem.mx (L. Álvarez), edmundo@ibt.unam.mx
(E. Castillo).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.08.002

94
A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
chains.19 An extension of these activities is the transfer of glucose
to a wide variety of acceptors, such as sugars, sugar alcohols,
glycosides, flavonoids, and aglycones, which have been success-
fully subjected to glucosylation by CGTase.20
absence of acceptors (Fig. 1a). All acceptor reaction products ob-
tained from the glycosylation of the five inositol acceptors were
compared after 24 h of reaction ( Fig. 1b–g). In addition, when
b-CD is used as a single substrate (Fig. 1a), only maltooligosaccha-
rides ranging from glucose (Gl) to maltohexose (G6) were
observed.
In a previous work, we reported the enzymatic regioselective
synthesis of
a-
D-glucopyranosyl-(1?4)-4D-myo-inositol (2) and
a
-
D
-glucopyranosyl-(1?1)-1
D
-myo-inositol (3) with anti-inflam-
With the exception of D-pinitol, all inositol were able to act as
matory activity, using the CGTase from Thermoanaerobacter sp.21
The regioselectivity was explained as a consequence of the general
amylase preference of positions C-4 and bC-1 in glucose for trans-
glucosylation.22 In order to extend the scope of the application of
this enzymatic glucosylation-hydrolysis strategy, we then focused
on the regioselective enzymatic synthesis of glucosyl derivatives of
acceptors of glucosyl residues transferred by CGTase. Several of
the observed glucosylation products may correspond to poly-glu-
cosylated inositols, as deduced from their retention times.
Afterward, the hydrolysis of maltooligosaccharides and poly-
glucosylated inositols (Fig. 1) to glucose and single glucosylated
products was carried out with GA. The HPLC profile after hydrolysis
for each inositol is illustrated in Figure 2, where it may be observed
various inositols, such as D-pinitol, L-chiro-inositol, D-chiro-inositol,
muco-inositol, and allo-inositol, with excellent regioselectivities.
Particularly, we focused on the influence of hydroxyl configuration
on enzyme specificity.
that in reactions using L-chiro-inositol (Fig. 2c), D-chiro-inositol
(Fig. 2d), muco-inositol (Fig. 2f), and allo-inositol (Fig. 2g) as accep-
tor substrate, different peaks corresponding to enzymatic products
were observed (10–11, 14, 19, and 24). These products were puri-
fied by preparative HPLC and analyzed by 1D and 2D NMR for
structural characterization. In order to confirm the identity of the
products, benzoylation of the monoglucosylated inositol products
was carried out. As already stated, no glycosylated products were
2. Results and discussion
2.1. Enzymatic glucosylation of L-chiro-, D-chiro-, muco-, and
allo-inositols
observed by TLC and HPLC analysis, in reactions where
was used as acceptor, as illustrated in Figures 1e and 2e.
D-pinitol
An enzymatic process was designed in order to glucosylate five
different inositol stereoisomers: -pinitol, -chiro-inositol, -chiro-
According to these preliminary results,
(1?2)-2 -chiro-inositol (10) and -glucopyranosyl-(1?5)-5
chiro-inositol (11) were synthesized from -chiro-inositol (Scheme
1); -glucopyranosyl-(1?3)-3 -chiro-inositol (14) and -glu-
copyranosyl-(100?40)- -glucopyranosyl-(10?3)-3
-chiro-inositol
(15) were derived from the transglucosylation of -chiro-inositol
(Scheme 2); -glucopyranosyl-(1?1)-muco-inositol (19) was
obtained from the glucosylation of muco-inositol (Scheme 3); and
-glucopyranosyl-(1?5)-allo-inositol (24) and -glucopyrano-
syl-(100?40)-
-glucopyranosyl-(1?5)-allo-inositol (25) derived
from the glucosylation of allo-inositol (Scheme 4).
a-D-glucopyranosyl-
D
L
D
L
a
-D
L-
inositol, muco-inositol, and allo-inositol. Transglucosylation
reactions were carried out with b-cyclodextrin (b-CD) as glucosyl
donor and CGTase from Thermoanaerobacter sp. as biocatalyst.21
The glucosylation reaction was followed by the selective hydrolysis
of the poly-glucosylated products with Aspergillus niger glucoamy-
lase (GA). The products of all reactions were analyzed by TLC (Sup-
plementary data) and high-performance liquid chromatography
(HPLC), as described in Section 4. The HPLC profile of products ob-
tained in the presence of acceptors after CGTase reactions is illus-
trated in Figure 1, including the profile of CGTase products in the
L
a-D
D
a-D
a
-D
D
D
a-D
a-D
a-D
a-D
Figure 1. HPLC product profile obtained in CGTase glucosylation reactions of various inositols using b-CD as glucosyl donor (a) Reaction control in the absence of inositol (b)
myo-inositol (1), (c) -chiro-inositol (9), (d) -chiro-inositol (13), (e) -pinitol (17), (f) muco-inositol (18), and (g) allo-inositol (23). Products are obtained after 24 h of reaction
Retention times (min) are shown for each peak.
L
D
D

A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
95
Figure 2. HPLC product profile obtained after GA hydrolysis of the transglucosylation products using inositols as acceptors as described in Figure 1. (a) Control in the absence
of inositols, (b) myo-inositol,21 (c)
-chiro-inositol, (d) -chiro-inositol, (e) -pinitol, (f) muco-inositol and (g) allo-inositol. Products are obtained after 24 h of reaction.
L
D
D
Retention times (min) are shown at the top of each peak.
OH
Thermoanaerobacter sp. CGTase
8.6 U/mL pH 6,50°C, 24 h
5
1
6
(11)
18.4%
+
+
(10)
21.4%
(12)
11.8%
HO
51.2%
OH
HO
3
4
2
OH
OH
L
-chiro-inositol (9)
Aspergillus niger glucoamylase
+
258 U/mL, 50°C, 24 h
β-CD (0.13 mmol)
HPLC purification
OH
6'
OH
5
4'
HO
1
O
5'
6
HO
HO
OH
O
HO
HO
3
OH
2'
1'
5
+
HO
4
1
OH
2
3'
OH
6'
6
5'
1'
O
O
OH
OH
HO
3
4
2
OH
HO
3'
4'
2'
OH
(11)
12.5%
(10)
36.6%
Scheme 1. Synthetic strategy of L-chiro-inositol glucosylation, using b-cyclodextrin as glucosyl donor and CGTase from Thermoanaerobacter sp., followed by the selective
hydrolysis of the poly-glucosylated products with A. niger glucoamylase.
2.2. Structure characterization of the transfer products
positive mode, while those of compounds 14, 15, and 24, were ob-
tained in the negative mode, and in general, accounted fine for
their molecular formula (see Section 4).
The structures of the transfer products, were elucidated on the
basis of analyses of their spectroscopic data (1H NMR, 13C NMR,
1H–1H COSY, HMQC, HMBC, and HRFABMS), as well as of their
per-benzoylated derivatives.
The molecular ions of monoglucosylated products 10, 11, and
19 were obtained by HRFAB mass spectrometry analysis in the
In the 1H NMR spectra of monoglucosylated products 10, 11,
and 14, only one anomeric proton in the
a orientation was
observed at d 5.07, 5.11, and 5.38, and HSQC showed their attach-
ment to the anomeric carbons at dC 101.4, 101.5, and 99.6,
respectively.

96
A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
OH
3
HO
1
2
HO
β-CD (0.13 mmol)
+
HO
5
6
4
OH
OH
D-chiro-inositol (13)
Thermoanaerobacter sp. CGTase 8.6 U/mL
pH 6,50°C, 24 h
OH
6''
50.2%
HPLC purification
4''
O
5''
HO
OH
6'
HO
2''
1''
3''
OH
4'
O
5'
O
OH
(14)
23.0%
+
(16)
7.7%
+
HO
2'
1'
3
HO
1
OH
3'
2
O
(15)
15.0%
HO
5
6
4
OH
OH
Aspergillus niger glucoamylase 258 U/mL
OH
6'
50°C, 24 h
4'
O
5'
HO
OH
HO
2'
1'
3
HO
1
3'
OH
2
O
HO
5
6
4
OH
(14)
10.1%
OH
Scheme 2. Synthetic strategy using
D-chiro-inositol, b-cyclodextrin as glucosyl donor and CGTase from Thermoanaerobacter sp., followed by the selective hydrolysis of the
poly-glucosylated products with Aspergillus niger glucoamylase.
OH
OH
2
4
3
Thermoanaerobacter sp. CGTase
(21)
5.7%
(22)
2.1%
(20)
14.9%
+
+
+
(19)
34.9%
8.6 U/mL, pH 6,50°C, 24 h
57.8%
OH
HO
6
5
1
OH
OH
muco-inositol (9)
Aspergillus niger glucoamylase 258 U/mL
50°C, 24 h
+
β-CD (0.074 mmol)
HPLC purification
OH
OH
2
4
3
O
HO
6
OH
5
OH
OH
1
6'
5'
1'
O
OH
HO
3'
4'
2'
OH
(19)
58.2%
Scheme 3. Synthetic strategy using muco-inositol, b-cyclodextrin as glucosyl donor and CGTase from Thermoanaerobacter sp., followed by the selective hydrolysis of the poly-
glucosylated products with Aspergillus niger glucoamylase.
The characteristically downfield anomeric carbons and anomer-
ic hydrogens of the -glucopyranosyl residue in each compound,
served as the starting point for the analysis of the 1H–1H COSY
and NOESY experiments for connectivities within the spin system.
The two diasterotopic H-60 hydrogens on the glucosyl residue were
also useful markers, clearly identifiable by a doublet with double
D

A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
97
OH
OH
OH
6
2
1
β-CD (0.074 mmol)
+
4
5
OH
HO
3
HO
allo-inositol (23)
HPLC purification
Thermoanaerobacter sp. CGTase
OH
OH
2
8.6 U/mL, pH 6,50°C, 24 h
32.2%
OH
3
6
1
O
4
5
1'
OH
OH
HO
6'
HO
5'
O
2'
O
4'
HO
4''
1''
(24)
13.9%
+
(26)
6.4%
+
5''
6''
OH
3'
2''
O
HO
(25)
9.8%
3''
HO
HO
Aspergillus niger glucoamylase
OH
OH
2
258 U/mL, 50°C, 24 h
OH
3
6
1
O
4
5
1'
OH
OH
HO
HO
5'
6'
2'
O
4'
3'
HO
HO
(24)
5.6%
Scheme 4. Synthetic strategy using allo-inositol, b-cyclodextrin as glucosyl donor and CGTase from Thermoanaerobacter sp., followed by the selective hydrolysis of the poly-
glucosylated products with Aspergillus niger glucoamylase.
intensity at d 3.65 and at 3.72 in compound 10; at d 3.75 and 3.69
in compound 11; and in d 3.91 and 3.88, in 14, in their 1H NMR
spectrum, and attached to the most upfield carbon at d 61.33,
61.46, and d 60.63, respectively in the 13C spectrum. Once the 1H
signals of the glucopyranose residue have been assigned, the six
remaining 1H resonances, were assigned to each cyclitol ring, and
connectivities were delineated from 1H–1H COSY and TOCSY
experiments with the characteristic signal of the equatorial proton
of each cyclitol as the starting point. The H-1eq and H-6eq signals
at d 5.07 (d, J = 4.0 Hz) of the glucose residue in compound 10, C-5
(d 73.73) of
-chiro-inositol and the H-10 at d 5.11 (d, J = 4.0 Hz) of
L
the glucose residue in compound 11, and C-3 (d 81.23) of D-chiro-
inositol and the H-10 at d 5.38 (d, J = 4.0 Hz) of the glucose residue
in compound 14. It is noteworthy that glucoside 14 is an analogue
of fagopyritol A1 (a-D-galactopyranosyl-(1?3)-3D-chiro-inositol), a
putative insulin mediator, which is useful in the treatment of non-
insulin dependent diabetes mellitus.24,25
The 1H NMR of 15 showed two anomeric signals with the same
relative intensity: H-10 (d, J = 3.5 Hz, d 5.35) and H-100 (d, J = 3.5 Hz,
d 5.43). 13C NMR spectra of 15 in D2O solution also showed two
anomeric signals with the same relative intensity. The peaks at d
99.52 (C-100) and 99.83 (C-10) and two methylene carbons at d
60.40 and 60.34 indicated the presence of two pyranoside type res-
idues. The anomeric proton H-100 showed NOE to H-40 (dd,
J = 9.1 Hz, J = 9.8 Hz, d 3.71), while H-10 of this ring is in close prox-
of the
L
-chiro-inositol residue in compound 10 were observed at d
3.87 (dd, 1H,
4.06 (t, 1H, J1,6 = 3.5 Hz, J1,2 = 3.5 Hz), and
d
J1,6 = 3.5 Hz, J5,6 = 4.0 Hz), respectively; in compound 11, these pro-
tons appeared at d 3.71 (dd, 1H, J1,6 = 2.5 Hz, J1,2 = 3.0 Hz), and 4.08
(d, 1H, J1,6 = 1.5 Hz, J5,6 = 1.5 Hz), respectively. In compound 14, the
equatorial H-1 and H-6 signals of the D-chiro-inositol residue ap-
peared at d 4.08 (dd, 1H, J1,6 = 2.8 Hz, J1,2 = 4.0 Hz), and 4.09 (dd,
1H, J1,6 = 3.2 Hz, J5,6 = 4.0 Hz), respectively.
imity to H-3 (dd, J = 8.4 Hz, J = 9.8 Hz, d 3.73) of the D-chiro-inositol.
Through the HSQC experiment, the proton attached to the most
downfield carbon in each cyclitol ring was determined (10: dC
79.87, 11: dC 73.73, and 14: dC 81.23),23 and correspond to the
point of linkage to glucose. This was confirmed by the interactions
observed between the anomeric hydrogen and this proton in each
compounds in the NOESY experiment (10: dH 5.07?3.66, 11: dH
5.11?3.72, and 14: dH 5.38?3.78). Coupling constants observed
for these hydrogens and those observed for the adjacent ones, con-
firmed assignment of each proton. In addition, in the heteronuclear
multiple bond coherence (HMBC) spectrum, the following 3J corre-
These positions were unambiguously identified by using a gHMBC
experiment. According to these results, the structure for the pseu-
dotrisaccharide may be established as
a-D-glucopyranosyl-
(100?40)- -glucopyranosyl-(10?3)-3
a-D
D
-chiro-inositol (15).
Due to the slow interconversion of the two meso chair forms of
muco-, and allo-inositols,26 broad unresolved 1H NMR in D2O were
obtained for their transfer products 19 and 24, respectively. How-
ever, six well-defined signals for the inositol moiety were obtained
1
in the H NMR of the perbenzoylated product 19a and in the 1H
NMR spectra of 24 ran in Py-d5, indicating unsymmetrical com-
pounds. Full assignments of the proton and carbon resonances on
lations were observed: C-2 (d 79.87) of
L
-chiro-inositol and the H-10

98
A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
19, 19a, and 24 were thus supported from the 2D NMR data. On the
basis of long-range correlation between C-1 (d 75.59) of the muco-
inositol and H-10 of glucose at d 5.72, it was established the struc-
additional inositol acceptors:
muco-inositol, and allo-inositol was demonstrated; it was also
found that no glucosylation products are formed when -pinitol
is used as acceptor. It is noteworthy that these four substrates bear
structural similarity with -glucopyranose, which is a natural
acceptor for CGTase (Fig. 3). Indeed, the transfer of -(1?4)-glu-
cans to the 40-OH of
-glucose is part of the intrinsic mechanism
of CGTases activity, while the transfer of -(1?1)-glucans to the
10-OH of b- -glucose has also been reported.20,21 Observations de-
rived from the structural analysis of -glucose derivatives obtained
in different transglycosylation reactions with CGTase, suggest that
the configuration of the
-glucose 20-OH, 30-OH, and 40-OH groups
is the key structural feature defining specificity.27,28 Moreover,
L
-chiro-inositol,
D-chiro-inositol,
D
ture of compound 19 as a-D-glucopyranosyl-(1?1)-muco-inositol.
Similarly, the long-range correlation between H-10 of the glucose
D
moiety and C-5 or C-4 (d 82.4) of the allo-inositol in the gHMBC
a
spectrum indicated that the glucose residue is
a-linked to OH-5
D
of allo-inositol. Therefore, compound 24 corresponds to
a
-D
-gluco-
a
pyranosyl-(1?5)-allo-inositol.
D
The 1H NMR of 25 showed two anomeric signals with the same
relative intensity: H-10 (d, J = 4.0 Hz, d 5.18) and H-100 (d, J = 4.0 Hz,
d 5.45). The analysis of glucoamylase reaction and the specificity of
CGTase’s, allowed us to conclude that the two glucopyranose rings
D
D
were connected via
that one glucose is also
According to these results, the primary structure proposed for
the diglucosylated allo-inositol is
-glucopyranosyl-(100?40)-
-glucopyranosyl-(1?5)-allo-inositol (25).
a
-(1?4) type linkage like compound 15 and
other enzymes from the a-amylase family are also able to transfer
a-linked to C-5 of the cyclitol residue.
glucose over the b-anomer of the 10-OH of
are depicted in a similar configuration as b-
D
-glucose.22 If inositols
-glucose, it may be ob-
D
a-
D
a-
served that some of the studied inositol hydroxyls have equivalent
D
configurations to that of 10-OH and 40-OH groups of b-
D-glucose in
its 4C1 conformation (Fig. 3) and therefore fulfill the Thermoanae-
robacter sp. CGTase regio- and stereo-selectivity requirements. As
expected, the experimental results showed that inositols are glu-
cosylated by Thermoanaerobacter sp. CGTase on equatorial hydrox-
2.3. Analysis of regiospecificity of CGTase from
Thermoanaerobacter sp.
The acceptor specificity of CGTase from Thermoanaerobacter sp.
in transglucosylation reactions was explored using six different
inositols as acceptors. In a previous work, we had already reported
yls equivalent to 10-OH and/or 40-OH of b-
of myo-inositol, -chiro-inositol,
The glycosylated positions in
5-OH groups in a 76:24 ratio, the minor product being
pyranosyl-(1?5)-5 -chiro-inositol (11). When the configuration
of -chiro-inositol is compared to that of b- -glucopyranose, we
find that the 2-OH, 3-OH, 4-OH, and 5-OH groups of -chiro-inositol
correspond to the 10-OH, 20-OH, 30-OH, and 40-OH groups of a
D
-glucose. This is the case
-chiro-inositol, and muco-inositol.
-chiro-inositol were the 2-OH and
-gluco-
L
D
L
that this enzyme recognizes
ring glucose residues to the 4-OH and 1-OH hydroxyl groups to
yield stereoselectively -glucopyranosyl-(1?4)-4 -myo-inositol
D-myo-inositol as acceptor transfer-
a-D
L
a-
D
D
L
D
(2) and
a
-
D
-glucopyranosyl-(1?1)-1
D
-myo-inositol (3), respec-
L
tively, in a 73:27 ratio.21 In this work, the ability of the CGTase
from Thermoanaerobacter sp. to transfer glucose residues to four
b-
D-glucopyranose. It is reasonable to conclude that the 5-OH
OH
6'
4'
O
75%*
5'
HO
25%*
OH
HO
2'
OH
OH
1
1'
3'
OH
HO
5
β-D
-glucosa
3
1
6
24%
100%
2
HO
HO
76%
OH
HO
HO
3
5
6
OH
2
2
4
4
OH
OH
OH
4
L
-chiro-inositol (9)
HO
OH
73%
3
HO
D-chiro-inositol (13)
27%
OH
HO
6
5
1
OH
OH
D-myo-inositol (1)
OH
OH
2
HO
3
1
2
4
3
H3CO
X
100%
HO
5
6
OH
HO
6
4
OH
5
1
OH
OH
OH
D
-pinitol (17)
muco-inositol (18)
OH
OH
6
OH
5
HO
OH
1
OH
100%
6
2
1
HO
OH
3
4
4
5
OH
2
HO
3
100%
OH
allo-inositol (23b)
OH
allo-inositol (23a)
Conformation detected by NMR
Figure 3. Products from glucosylation of various inositols catalyzed by CGTase from Thermoanaerobacter sp. Percentages represent the proportion of each product formed.
⁄Products of glycosylation catalyzed by Thermotoga maritima -amylase.22
a

A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
99
L
-chiro-inositol, which corresponds to the 40-OH group of
D
-galactose,
D
-ribose,
D
-mannose,
D
-arabinose, and
D
-fructose have
group of
-glucopyranose, is a favorable acceptor site for the transglycosyla-
resulted as poor acceptors for these enzymes.28,31
D
tion of CGTase, according to the described specificity. However, the
main glycosylation took place in the 2-OH group, which
3. Conclusions
corresponds to 10-OH- of b-
D
-glucopyranose, and
In fact, it is important to consider that if the OH groups at 2- and
5-positions of -chiro-inositol were superimposed to the corre-
sponding 10- and 40-OH groups of b-
-glucose, the oxygen in the
D-myo-inositol.
A practical enzymatic method for the regioselective glycosyla-
tion of five inositols is reported. It is proposed that regioselectivity
of CGTase from Thermoanaerobacter sp. is defined by the hydroxyl
configuration of inositols. Indeed, it was demonstrated that inosi-
tols bearing the stereochemical structure corresponding to 10-OH
L
D
ring is replaced by an axial –CH–OH. In the case of myo-inositol
as acceptor, the enzyme showed a better selectivity to the 4-OH
group of
in a 73:3% ratio.21 Thus, the different orientation of the 6-OH group
(axial) of -chiro-inositol with respect to the 3-OH group (equato-
rial) of -myo-inositol may affect the transglycosylation regioselec-
D
-myo-inositol resulting in monoglycosylated products
and/or 40-OH of b-
tol, -chiro-inositol, muco-inositol, and allo-inositol, are recognized
as acceptor for the synthesis of glucosyl-inositols. The reactions
carried out with -chiro-inositol, -chiro-inositol, muco-inositol,
and allo-inositol resulted in the regiospecific formation of glucosy-
lated products. Although, the 3-OH in -pinitol has the same ste-
reochemical structure of the 40-OH of
-glucopyranose, the
presence of a methyl group in the 3-OH did not allow glucosyla-
tion. It was established that the combined enzymatic transglucosy-
lation-hydrolysis strategy here described results in a useful tool for
the simple and rapid access to monoglycosylated inositols.
D-glucose, including myo-inositol, D-chiro-inosi-
L
L
D
L
D
tivity of the enzyme.
Interestingly, when
D
-chiro-inositol was essayed as acceptor,
D
the
not to the 6-OHax. The 3-OHeq is analogous to the 40-OH of
copyranose and -myo-inositol, while the 6-OHax of -chiro-inosi-
tol is not equivalent to the 10-OH of b-
-glucopyranose. -chiro-
Inositol has a conformation in which 3-OH, 4-OH, and 5-OH groups
are all equatorial like in -glucopyranose. Thus, -glucopyrano-
syl-(1?3)-3 -chiro-inositol (14) is the only glucosylated product
of CGTase on -chiro-inositol. The presence of the 6-OHax group
in -chiro-inositol influenced the reaction specificity in such a
way that glucosylation was observed only in the 3-OH, in contrast
to glucosylation in 2-OH and 5-OH observed in -chiro-inositol.
On the other hand, although the 3-OH in -pinitol has the same
orientation of the 40-OH of
-glucopyranose, the presence of a
a
-D
-glucose residue was transferred only to the 3-OHeq, but
D
D
-glu-
D
D
D
D
D
a-D
D
4. Experimental
4.1. General
D
D
L
NMR spectra were acquired either on Varian Unity NMR Spec-
trometers operating at 400 MHz, 500 MHz, and 700 MHz for 1H
and 100 MHz, 126 MHz, and 175 MHz for 13C nuclei, respectively.
Chemical shifts are listed in parts per million (ppm), referenced
to D2O or pyridine-d5 and were made on the basis of 1H–1H gCOSY,
1H–1H TOCSY, NOESY, gHSQC, and gHMBC spectral analyses as re-
quired. NMR experiments performed in CDCl3 are referenced to
Me4Si (0 ppm). HRFABMS spectra in a matrix of m-nitrobenzyl
alcohol or glycerol were recorded on a JEOL JMX-AX 505 HA mass
spectrometer. Melting points were determined using a Fisher Johns
apparatus and were not corrected. All reagents and solvents used
were of analytical grade. Optical rotations were acquired with a
Perkin–Elmer 241MC polarimeter (10 cm, 1 mL cell) at the sodium
D line. The benzoylation reactions were made in a CEM microwave
apparatus.
D
D
methyl group in the 3-OH did not allow glucosylation.
In the case of muco-inositol, there are two possible glycosyla-
tion sites defined by its symmetry plane. The spectroscopic data
indicated that the a-D-glucose residue was attached to one of the
two enantiotopic 1-OH or 5-OH positions of muco-inositol. In this
case, the exact position to which the glucose residue was attached
could not be determined by the NMR data alone. However, from
Thermoanaerobacter sp. CGTase specificity preferences for the 1-
OH and 4-OH positions in D-myo-inositol, it was deduced that the
glucose residue was attached to the 1-OH group of muco-inositol.
It is interesting to note that among the four inositols evaluated,
muco-inositol resulted as the better acceptor (57.8% yield).
From the NMR spectra of monoglucosylated allo-inositol (24), it
was established that glucosylation occurred in the 5-OHax group
Formation of oligoglucosyl-inositols and monoglucosyl-inosi-
tols was monitored by high performance liquid chromatography
(HPLC) using a Waters-Millipore chromatograph with a refraction
index detector (Waters 410) equipped with an automatic injector
model 717 plus. The measurement conditions were as follows: Col-
to give a-D-glucopyranosyl-(1?5)-allo-inositol (24). It is important
to consider that allo-inositol is in fact a meso-compound, which
means that the molecule interconverts the two stereoisomeric
chair forms (23a and 23b, Fig. 3) and averages H-1 and H-4, H-3
and H-2, and H-5 and H-6. Therefore, when the allo-inositol inter-
converts to the chair 23b, this 5-OH group is in an equatorial posi-
tion favoring the glucosylation on this hydroxyl. Indeed, in this
chair conformation the 5-OHeq corresponds to the 40-OH group
umn Prevail Carbohydrate ES
CH3CN–H2O (68–32 v/v); flow rate, 1.0 mL/min; column tempera-
5
l
4.6 mm ꢀ 250 mm; eluent,
ture, 32 °C; RI detector (Waters).
CGTase from Thermoanaerobacter sp. (ToruzymeÒ 3.0 L, a liquid
enzyme preparation) was a kind gift of Novozymes (México).
chiro-Inositol, -chiro-inositol, muco-inositol, allo-inositol, -pinitol,
and -glucose, were obtained from Sigma Aldrich, Inc. (MO, USA).
D-
of
D-glucopyranose.
L
D
It is worth mentioning that glycosylations in non-interconvert-
ible axial hydroxyl groups have been favorably performed only
when using b-galactosidases as catalysts.13,15
D
b-Cyclodextrin was obtained from American Maize-Products Com-
pany. Glucoamylase from Aspergillus niger used for digestion of oli-
goglucosyl-inositols was obtained by ANZECOÒ.
It is known that CGTase catalyzes not only the cyclization reac-
tion forming cyclodextrin from starch but also the transglycosyla-
tion reaction in the presence of suitable acceptors such as
carbohydrate derivatives and hydroxylated compounds. Pyranoses
with similar configuration as glucopyranose are efficient glucosyl
acceptors for CGTases, the transglycosylation reaction occurring
4.2. Transglucosylation of inositols with CGTase from
Thermoanaerobacter sp. followed by glucoamylase digestion
at analogue positions of the 40-OH and/or 10-OH of
Moreover, glucosylation products of -fructose using a CGTase
D
-glucose.28,29
A solution containing 150 mg (0.83 mmol) of inositol (
D-chiro-
inositol or -chiro-inositol) or -pinitol (150 mg, 0.77 mmol) and
L
D
D
150 mg of b-CD (0.13 mmol) was incubated with 8.6 units/mL of
CGTase from Thermoanaerobacter sp. in 1 mL of phosphate buffer
(pH 6.0, 50 mM Na2HPO4) at 50 °C for 24 h. muco- and allo-Inositols
from Bacillus stearothermophilus were determined at the 3-OH
and 1-OH groups, the main product (turanose) resulted from the
glucosylation of D
-fructose at the 3-OH group.30 On the contrary,

100
A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
(84 mg each, 0.46 mmol) were incubated in the same conditions
using 84 mg (0.074 mmol) of b-cyclodextrin. The reaction mixtures
were immersed in boiling water for 10 min to inactivate the en-
zyme and were stored at ꢁ18 °C until subsequent analyses by
TLC and HPLC and compared to blank experiments obtained with
the enzyme in the absence of inositol.
The products obtained after transglucosylation reactions from
inositols were subjected to the action of A. niger glucoamylase.
Reactions were incubated at 50 °C for 24 h with 258 U/mL of the
enzyme, which was directly added to the CGTase reaction medium.
Samples were taken and analyzed by TLC and HPLC. The reaction
was stopped by boiling for 10 min and was stored at ꢁ18 °C until
subsequent high-performance chromatographic separation.
4.2.4.
-chiro-inositol (15)
a-D a-D
-Glucopyranosyl-(100?40)- -glucopyranosyl-(10?3)-
3D
White amorphous powder (15.0% yield); mp 177–180 °C
(decomposed); ½a D20
ꢂ
+103.11 (c 0.15, H2O); 1H NMR (700 MHz,
D2O): d = 5.43 (d, 1H, J1 ,2 = 3.5 Hz, H-100), 5.35 (d, 1H, J1 ,2 = 3.5 Hz,
00 00
0
0
H-10), 4.15 (ddd, 1H, J5 ,6a = 2.3 Hz, J5 ,6b = 4.4 Hz, J4 ,5 = 9.98 Hz, H-
50), 4.05 (t, 1H, J1,6 = 3.5 Hz, J5,6 = 3.5 Hz, H-6), 4.04 (dd, 1H,
0
0
0
0
0
0
J2 ,3 = 8.4 Hz, J3 ,4 = 10.5 Hz, H-30), d = 4.03 (dd, 1H, J1,6 = 2.8 Hz,
0
0
0
0
00
00
00,6a00
J1,2 = 4.2 Hz, H-1), 3.88 (ddd, 1H, J5
= 2.8 Hz, J5 ,6b = 7.88 Hz,
= 11.02 Hz, H-500), 3.87 (dd, 1H, J5 ,6a = 2.8 Hz, J6a ,6b = 11.2 Hz,
00,500
0
0
0
0
J4
H-6ª0), 3.86 (m, 1H, H-6a00), 3.81 (dd, 1H, J3,4 = 9.1 Hz, J4,5 = 10.5 Hz,
H-4), 3.806 (dd, 1H, J4,5 = 3.5 Hz, J5,6 = 10.15 Hz H-5), 3.80 (dd, 1H,
J5 ,6b , = 2.45 Hz, J6a ,6b = 10.85 Hz, H-6b0), 3.75 (dd, 1H, J1,2 = 2.5 Hz,
J2,3 = 9.5 Hz, H-2), 3.73 (dd, 1H, J2,3 = 8.4 Hz, J3,4 = 9.8 Hz, H-3),
0
0
0
0
3.71 (dd, 1H, J3 ,4 = 9.1 Hz, J4 ,5 = 9.8 Hz H-40), 3.68 (t, 1H,
0
0
0
0
4.2.1. a-D-Glucopyranosyl-(1?2)-2L-chiro-inositol (10)
00 00
= 9.8 Hz, J3 ,4 = 9.8 Hz, H-300), 3.63 (dd, 1H, J1 ,2 = 3.85 Hz,
00 00
0
0
White amorphous powder (36.6% yield); mp 158-160 °C
J2
,3
0
(decomposed); ½a D20
ꢂ
+72.90 (c 0.12, H2O); 1H NMR (500 MHz,
J2 ,3 = 10.15 Hz, H-20), 3.60 (dd, 1H, J1
= 4.2 Hz, J2
= 9.8 Hz,
0
00 00
00 00
,2
,3
D2O): d = 5.07 (d, 1H, J1 ,2 = 4.0 Hz, H-10), 4.06 (t, 1H, J1,6 = 3.5 Hz,
J1,2 = 3.5 Hz, H-1), 3.87 (dd, 1H, J1,6 = 3.5 Hz, J5,6 = 4.0 Hz, H-6),
H-200), 3.43 (t, 1H, J4
= 9.8 Hz, J4
= 9.8 Hz H-400); 13C NMR
00 00
00 00
0
0
,3
,5
(175 MHz, D2O): d = 99.83 (C-10), 99.52 (C-100), 80.75 (C-3), 76.66
(C-40), 73.32 (C-6), 72.95 (C-4), 72.81 (C-300), 72.58 (C-2), 71.83
(C-30), 71.67 (C-200), 71.52 (C-20), 71.27 (C-1), 70.38 (C-50), 70.21
3.72 (dd, 1H, J5 ,6b = 2.0 Hz, J6a ,6b = 12.0 Hz, H-6b0), 3.69 (t, 1H,
J3,4 = 10.0 Hz, J4,5 = 10.0 Hz, H-4), 3.66 (m, 1H, H-2), 3.65 (t, 1H,
0
0
0
0
J2 ,3 = 10.0 Hz, J3 ,4 = 10.0 Hz, H-30), 3.65 (dd, 1H, J5 ,
= 2.0 Hz,
(C-5), 69.27 (C-400), 68.95 (C-500), 60.40 (C-600), 60.34 (C-6 );
0
0
0
0
0
0
6a0
J6a ,6b = 12.0 Hz, H-6a0), 3.64 (m, 1H, H-50), 3.63 (m, 1H, H-5), 3.49
HRFABMS (negative mode), calcd for C18H32O16: 503.4381 found:
503.4381.
0
0
0
0
(dd, 1H, J2,3 = 9.0 Hz, J3,4 = 9.5 Hz, H-3), 3.42 (dd, 1H, J1 ,2 = 4.0 Hz,
J2 ,3 = 9.75 Hz, H-20), 3.29 (t, 1H, J3 ,4 = 10.0 Hz, J4 ,5 = 10.0 Hz H-
40); 13C NMR (125 MHz, D2O): d = 101.47 (C-10), 79.87 (C-2),
73.63 (C-5), 73.38 (C-3), 73.10 (C-4), 72.96 (C-30), 72.56 (C-20),
72.43 (C-6), 72.09 (C-1), 70.97 (C-50), 70.34 (C-40), 61.33 (C-60);
0
0
0
0
0
0
4.2.5. a-D-Glucopyranosyl-(1?1)-muco-inositol (19)
White amorphous powder (58.2% yield); mp 158–160 °C
(decomposed); ½a D20
ꢂ
+117.63 (c 0.11, H2O); 1H NMR (500 MHz,
D2O): d = 5.03 (d, 1H, J1 ,2 = 3.5 Hz, H-1 ), 4.02 (dd, 1H,
J1,2 = 5.5 Hz, J2,3 = 6.5 Hz, H-2 or H-4), 3.87 (m, 2H, H-3 and H-4),
0
0
0
HRFABMS (positive mode), calcd for
found: 365.1074.
C
12H22O11Na: 365.2889,
0
3.82 (m, 1H, H-1 or H-5), 3.78 (m, 1H, H-5 ), 3.77 (m, 2H, H-5
0
0
0
0
0
4.2.2.
a
-
D
-Glucopyranosyl-(1?5)-5
L
-chiro-inositol (11)
and H-6), 3.71 (dd, 1H, J5 ,6b , = 2.0 Hz, J6a ,6b = 12.5 Hz, H-6b ), 3.63
0
0
0
0
0
White amorphous powder (12.5% yield); mp 165–167 °C
(dd, 1H, J5 ,6a , = 2.0 Hz, J6a ,6b = 12.0 Hz, H-6a ), 3.62 (dd, 1H,
J2 ,3 = 9.5 Hz, J3 ,4 = 10.0 Hz, H-3 ), 3.42 (dd, 1H, J1 ,2 = 3.5 Hz,
J2 ,3 = 9.75 Hz, H-2 ), 3.29 (t, 1H, J3 ,4 = 9.5 Hz, J4 ,5 = 9.5 Hz, H-4 );
C NMR (125 MHz, D2O): d = 99.27 (C-1 ), 79.62 (C-1 or C-5),
73.77 (C-3 ), 72.94 (C-5 ), 72.94 (C-5 or C-1 and C-6), 72.34 (C-2
and C-3), 70.89 (C-4 or C-2), 70.21 (C-4 ), 69.02 (C-2 or C-4),
61.27 (C-6 ); HRFABMS (positive mode), calcd for C12H22O11Na:
(decomposed); ½a D20
ꢂ
+71.80 (c 0.24, H2O); 1H NMR (500 MHz,
0
0
0
0
0
0
0
D2O): d = 5.11 (d, 1H, J1 ,2 = 4.0 Hz, H-10), 4.08 (d, 1H, J1,6 = 1.5 Hz,
0
0
0
0
0
0
0
0
0
0
13
0
0
0
0
0
J5,6 = 1.5 Hz, H-6), 3.75 (dd, 1H, J5 ,6a = 2.0 Hz, J6a ,6b = 12.0 Hz, H-
6a0), 3.72 (dd, 1H, J5,4 = 9.5 Hz, J5,6 = 3.0 Hz, H-5), 3.72 (dd, 1H,
0
0
0
J2 ,3 = 9.5 Hz, J3 ,4 = 10.0 Hz, H-30), 3.71 (t, 1H, J3,4 = 9.5 Hz,
J4,5 = 9.5 Hz, H-4), 3.71 (dd, 1H, J1,6 = 2.5 Hz, J1,2 = 3.0 Hz, H-1),
0
0
0
0
0
0
0
0
0
0
0
3.69 (dd, 1H, J5 ,6b = 2.0 Hz, J6a ,6b = 12.0, H-6b ), 3.69 (dd, 1H,
J1,2 = 2.5 Hz, J2,3 = 10.0 Hz, H-2), 3.68 (dd, 1H, J2,3 = 9.0 Hz,
365.2889, found: 365.1074.
0
0
0
J3,4 = 10.0 Hz, H-3), 3.66 (m, 1H, H-5 ), 3.46 (dd, 1H, J1 ,2 = 4.0 Hz,
4.2.6. a-D-Glucopyranosyl-(1?5)-allo-inositol (24)
J2 ,3 = 10.0 Hz, H-20), 3.32 (t, 1H, J3 ,4 = 9.5 Hz, J4 ,5 = 9.5 Hz H-40);
13C NMR (125 MHz, D2O): d = 101.53 (C-10), 73.73 (C-5), 73.73 (C-
1, C-2, C-3 and C-50), 73.22 (C-4), 72.96 (C-30), 72.64 (C-20), 72.17
(C-6), 70.46 (C-40), 61.46 (C-60); HRFABMS (positive mode), calcd
for C12H22O11Na: 365.2889, found: 365.1078.
Purified before hydrolysis with glucoamylase. White amor-
0
0
0
0
0
0
phous powder (13.8% yield); mp 156–159 °C (decomposed); ½a D20
ꢂ
+57.58 (c 0.14, H2O); 1H NMR (400 MHz, pyridine-d5): d = 5.71 (d,
0
0
0
0
0
0
0
1H, J1 ,2 = 4.0 Hz, H-1 ), 5.11 (dd, 1H, J2 ,4 = 3.0 Hz, J3 ,4 = 9.0 Hz, H-
0
0
0
0
4), 4.93 (dd, 1H, J2 ,4 = 2.6 Hz, J3 ,4 = 7.8 Hz, H-3), 4.82 (m, 3H, H-
0
0
0
2, H-5 ), 4.72 (br s, H-1), 4.62 (m, 1H, H-5), 4.61 (t, 1H, J2 ,3 = 9.2 Hz,
0
0
0
0
0
4.2.3.
a
-
D
-Glucopyranosyl-(1?3)-3
D
-chiro-inositol (14)
J3 ,4 = 9.2 Hz, H-3 ), 4.54 (br s, 1H, H-6), 4.43 (dd, 1H, J5 ,6b = 2.0 Hz,
0
0
0
0
0
0
0
Purified before hydrolysis with glucoamylase. White amor-
J6a ,6b = 11.6 Hz, H-6b ), 4.36 (dd, 1H, J5 ,6a = 5.0 Hz, J6a ,6b = 11.8 Hz,
phous powder (23.0% yield); mp 145–148 °C (decomposed); ½a D20
ꢂ
H-6a ), 4.25 (dd, 1H, J2 ,4 = 9.2 Hz, J3 ,4 = 9.6 Hz, H-4 ), 4.18 (dd, 1H,
0
0
0
0
0
0
+53.42 (c 0.1, H2O); 1H NMR (400 MHz, D2O): d = 5.38 (d, 1H,
J1 ,2 = 4.0 Hz, J2 ,3 = 9.60 Hz, H-2 ); 13C NMR (175 MHz, pyridine-d5):
0
0
0
0
0
0
0
0
0
0
J1 ,2 = 4.0 Hz, H-1 ), 4.09 (dd, 1H, J1,6 = 3.2 Hz, J5,6 = 4.0 Hz, H-6),
d = 103.52 (C-1 ), 81.18 (C-3), 75.80 (C-1, C-5 and C-3 ), 75.25 (C-2),
0
0
0
0
0
0
0
0
0
0
0
4.08 (ddd, 1H, J5 ,6a = 2.8 Hz, J5 ,6b = 4.8 Hz, J4 ,5 = 10.1 Hz, H-5 ),
74.36 (C-6, C-5 and C-2 ), 72.10 (C-4 ), 70.59 (C-4), 62.89 (C-6 );
HRFABMS (negative mode), calcd for C12H21O11: 341.1078, found:
341.1059.
4.08 (dd, 1H, J1,6 = 2.8 Hz, J1,2 = 4.0 Hz, H-1), 3.91 (dd, 1H,
0
0
0
0
0
J5 ,6b = 2.2 Hz, J6a ,6b = 12.2 Hz, H-6b ), 3.90 (dd, 1H, J3,4 = 10.0 Hz,
J4,5 = 10.4 Hz, H-4), 3.89 (dd, 1H, J1,2 = 2.8 Hz, J2,3 = 9.9 Hz, H-2),
3.88 (dd, 1H, J5 ,6a = 3.2 Hz, J6a ,6b = 13.6 Hz, H-6ª0), 3.80 (dd, 1H,
4.3. General benzoylation procedure
0
0
0
0
J2 ,3 = 9.6 Hz, J3 ,4 = 10.0 Hz, H-30), 3.78 (t, 1H, J2,3 = 9.6 Hz,
J3,4 = 9.6 Hz, H-3), 3.78 (dd, 1H, J5,4 = 2.4 Hz, J5,6 = 9.6 Hz H-5), 3.64
0
0
0
0
Benzoyl chloride was added dropwise to a 10 mL glass micro-
wave reaction vessel at room temperature containing a stirred
solution of 10, 11, 14, or 19 in pyridine. The reaction vessel was
sealed with a cap and then placed into the microwave cavity. The
microwave was programed to heat the reaction mixture to the
desired temperature. After the reaction was completed, the vessel
was cooled below 50 °C using a flow of compressed air. The
(dd, 1H, J1 ,2 = 4.0 Hz, J2 ,3 = 9.6 Hz, H-20), 3.50 (t, 1H, J3 ,4 = 9.6 Hz,
0
0
0
0
0
0
13
J4 ,5 = 9.6 Hz H-40); C NMR (100 MHz, D2O): d = 99.61 (C-10),
81.23 (C-3), 73.16 (C-30 and C-4), 72.14 (C-1), 72.08 (C-6), 71.99
(C-50), 71.52 (C-5), 70.47 (C-2), 69.65 (C-20), 69.23 (C-40), 60.63
(C-60); HRFABMS (negative mode), calcd for C12H21O11Na:
341.1078, found: 341.1063.
0
0

A. Miranda-Molina et al. / Carbohydrate Research 360 (2012) 93–101
101
reaction was quenched with ice (5 mL), washed with water, 1 M
hydrochloric acid, dried (NaSO4), filtered, and extracted with
CH2Cl2 (3 ꢀ 25 mL) and concentrated. The product was purified
and 25 were purified before treatment with glucoamylase. The
HPLC conditions were as follows: Column Bondapak™ NH2
10
l
l
m, 125 Å, 7.8 ꢀ 300 mm (Waters); eluent CH3CN–H2O (80–
by column chromatography (hexane–ethyl acetate, 90:10
?
20 v/v); flow rate, 7.0 mL/min; column temperature, 35 °C; RI
50:50) on silica gel to give the corresponding perbenzoylated
detector (Waters).
derivatives 10a, 11a, 14a, and 19a.
Acknowledgments
4.3.1.
a-D-(2,3,4,6-Tetra-O-benzoyl)-glucopyranosyl-(1?2)-2L-
(1,3,4,5,6-penta-O-benzoyl)-chiro-inositol (10a)
The authors wish to acknowledge PAPIIT (project: IN226706-3)
and CONACyT (projects: 56431, CB 82851 81637) for the financial
support. We also thank F. González from IBt-UNAM for technical
assistance in HPLC analysis, as well as Victoria Labastida and
Gabriela Vargas from CIQ-UAEM, for mass spectroscopy and NMR
analysis respectively. Alfonso Miranda Molina thanks CONACYT
for fellowship 189120 and DGAPA-UNAM for a postdoctoral
fellowship.
Prepared from 10 (0.01 g, 0.033 mmol), benzoyl chloride
(0.2 mL, 1.72 mmol) and pyridine (0.5 mL). The reaction was
heated at 90 °C with 50 W and 13 psi for 1 h to afford compound
10a (in 74.05% yield) as a white solid after purification; mp 115–
118 °C; ½a 2D0
ꢂ
ꢁ18.26 (c 0.55, CHCl3); 1H NMR (400 MHz, CDCl3),
and 13C NMR (100 MHz, CDCl3): See Supplementary data;
HRFABMS (positive mode), calcd for C75H60O20: 1280.2590, found:
1280.3903.
Supplementary data
4.3.2.
a-D-(2,3,4,6-Tetra-O-benzoyl)-glucopyranosyl-(1?5)-5L-
(1,2,3,4,6-penta-O-benzoyl)-chiro-inositol (11a)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.08.
002.
Prepared from 11 (0.0112 g, 0.0370 mmol), benzoyl chloride
(0.2 mL, 1.72 mmol) and pyridine (0.5 mL). The reaction was
heated at 90 °C with 50 W and 13 psi for 1 h to afford compound
11a (in 45% yield) as a white solid after purification; mp 92–
95 °C; ½a 2D0
ꢂ
+3.63 (c 0.43, CHCl3); 1H NMR (700 MHz, CDCl3), and
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75H59O20
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a
-
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heated at 90 °C with 50 W and 13 psi for 1 h to afford compound
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