Stereoselective enzymatic synthesis of monoglucosyl-myo-inositols with in vivo anti-inflammatory activity

Article abstract of DOI:10.1016/j.tetasy.2009.12.009

The monoglucosyl-inositols α-d-glucopyranosyl-(1→4)-4d-myo-inositol 3 and α-d-glucopyranosyl-(1→1)-1d-myo-inositol 4 were synthesized by a combined enzymatic transglucosylation and hydrolysis strategy, using cyclodextrin glucosyl transferase (CGTase) from Thermoanaerobacter sp., followed by hydrolysis with Aspergillus niger glucoamylase. The glucosides were separated by preparative HPLC and fully characterized by extensive 1D and 2D NMR studies. The structure of the regioisomer 4 was confirmed by X-ray crystallography of its perbenzoylated derivative 4a. Both isomers demonstrated in vivo anti-inflammatory activity at comparative levels to corticosterone on mouse ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and in rat hind paw oedema induced by carrageenan.

Full text of DOI:10.1016/j.tetasy.2009.12.009

Tetrahedron: Asymmetry 21 (2010) 43–50
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Stereoselective enzymatic synthesis of monoglucosyl-myo-inositols
with in vivo anti-inflammatory activity
Alfonso Miranda-Molina a, Agustín López-Munguía b, María Luisa San Román a, Jaime Escalante a,
a,
Marco Antonio Leyva c, Ana María Puebla d, Edmundo Castillo b, , Laura Álvarez
*
*
a Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos. Av. Universidad 1001, Col. Chamilpa, C.P. 62209, Cuernavaca-Mor., Mexico
b Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, C.P. 62209, Cuernavaca-Mor., Mexico
c Centro De Investigación y Estudios Avanzados, Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, C.P. 07000, México D.F., Mexico
d Centro Universitario de Ciencias Exactas e Ingeniería, Departamento de Farmacobiología, Universidad de Guadalajara, Blvd. Marcelino García Barragán 1421, esq. Calzada
Olímpica, 44430, Guadalajarara, Jalisco, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The monoglucosyl-inositols
(1?1)-1D-myo-inositol 4 were synthesized by a combined enzymatic transglucosylation and hydrolysis
a-D-glucopyranosyl-(1?4)-4D-myo-inositol 3 and a-D-glucopyranosyl-
Received 4 November 2009
Accepted 21 December 2009
Available online 18 January 2010
strategy, using cyclodextrin glucosyl transferase (CGTase) from Thermoanaerobacter sp., followed by
hydrolysis with Aspergillus niger glucoamylase. The glucosides were separated by preparative HPLC and
fully characterized by extensive 1D and 2D NMR studies. The structure of the regioisomer 4 was con-
firmed by X-ray crystallography of its perbenzoylated derivative 4a. Both isomers demonstrated
in vivo anti-inflammatory activity at comparative levels to corticosterone on mouse ear oedema induced
by 12-O-tetradecanoylphorbol-13-acetate (TPA) and in rat hind paw oedema induced by carrageenan.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and more complex myo-inositol phospho-glycans (IPG), which
have been demonstrated in numerous in vitro studies to exert par-
Inositols, the nine isomeric forms of hexahydroxycyclohexane,
are a group of small and chemically very stable polar molecules
bearing versatile biological properties. Six stereoisomers, myo-,
tial insulin-mimetic activity on glucose and lipid metabolism in
insulin-sensitive cells (adipocytes, cardiomyocytes and dia-
phragms).10–12 Recently, two series of new myo-inositol-derived
glycolipid analogues, named lanceolitols A1–A7 and B1–B7, were
isolated from the leaves of the Mexican medicinal plant Solanum
lanceolatum. These compounds showed important in vivo anti-
inflammatory activity, through inhibition of phospholipase A2
and cyclooxygenase-2 (COX-2), as demonstrated by in vitro
experiments.13
The physiological roles of myo-inositol and its glycosylated
derivatives, as well as their potential pharmaceutical applications
have focused interest in the regio- and stereoselective syntheses
of these compounds. Consequently, the synthesis of partially pro-
tected inositols with free hydroxyl groups at specific positions
has been the subject of intensive research.14–20 However, due to
the similar reactivity of the multiple inositol hydroxyl groups,
the controlled glycosylation remains a challenge in organic chem-
istry.2 In this context, considering that one of the most interesting
properties of enzymes is their regio- and stereoselectivity, a bio-
catalytic approach represents an interesting alternative to cope
with this limitation. In particular, the regio-selective glycosylation
of a wide variety of chemical compounds has been achieved by the
proper application of enzymes such as glycosyltransferases and
glycosidases using several glycosyl donors through different
scyllo-, neo-,
D-chiro-, epi- and muco-inositol, have been recognized
to occur in nature, while the remaining three,
L-chiro-, allo- and cis-
inositol are unnatural synthetic isomers.1–4 Among them, myo-ino-
sitol is the most widely distributed form in microorganisms, plants,
as well as in mammalian cells, where it plays very important phys-
iological roles, such as a growth factor and a precursor of phospha-
tidylinositol an anti-fatty liver agent.5 Particular attention has been
devoted to glycosylated forms of myo-inositol due to their implica-
tion in various signal transduction pathways. Examples include
inositol-containing monosaccharides such as galactinol (
topyranosyl-(1?1)-1 -myo-inositol) which acts as a galactosyl
donor in the synthesis of glycosinolates;6,7 mycothiol (1
-myo-
inosityl2-(N-acethylcystein)amino-2-deoxy- -glucopyranoside),
a functional analogue of glutathione;8 disaccharides such as
a-D-galac-
D
D
a-D
a-D-
mannopyranosyl-(1?4)-a-D-glucuronopyranosyl-(1?2)-myo-ino-
sitol which has effects on the amino acid metabolism of plants;9
* Corresponding authors. Tel./fax: +52 777 3 29 79 97 (L.A.); tel.: +52 55 56 22 76
09; fax: +52 777 17 23 88 (E.C.).
E-mail addresses: edmundo@ibt.unam.mx (E. Castillo), lalvarez@uaem.mx
(L. Álvarez).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.12.009

44
A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
glycosylation strategies.21–31 More specifically, the glycosylation of
various inositols has been reported. The first successful enzymatic
were analyzed by TLC and/or HPLC in a search for glycosylated
myo-inositols (data not shown).
galactosylation of 1
using a b-galactosidase from Bacillus circulans resulting in the syn-
D
-chiro-inositol and 1
D
-pinitol was achieved
From these experiments, it became clear that myo-inositol is
not easily recognized as an acceptor by most glycosidases, and
although a large range of reaction conditions was explored; only
hydrolysis and transglycosylation products (oligosaccharides)
thesis of mono- and digalactosylated inositols in yields of up to
45%.32 In a later report, the formation of 5-O- and 1-O-b-
D-glucopyr-
anosyl-myo-inositols and 5-O-b-
D-galactopyranosyl-myo-inositol
were obtained from enzymes such as a-amylase from Aspergillus
using growing cultures and enzyme extracts of Sporobolomyces sing-
oryzae, levansucrase from Bacillus subtillis and b-galactosidases
from Escherichia coli, A. oryzae and Kluyveromyces lactis. Neverthe-
less, glycosylated myo-inositol products were observed with
Thermotoga maritima b-glucosidase and Thermoanaerobacter sp.
CGTase. Figure 1 shows the TLC analysis of the reaction products
obtained when T. maritima b-glucosidase (1a) and Thermoanaerob-
acter sp. CGTase (1b) were employed. In both cases, the reaction
products were compared to controls obtained with the enzyme
and the glycosyl donor in the absence of myo-inositol (lanes 1a–
7a and lanes 1c–3c). It is interesting to observe that in reactions
carried out in the presence of myo-inositol, a series of products cor-
responding to mono- and/or poly-glycosylated myo-inositols were
formed (highlighted spots in Fig. 1). In order to reduce the number
of poly-glycosylated myo-inositol products, a glucoamylase diges-
tion treatment of the reaction products obtained with CGTase
was carried out (Scheme 1). It was found that this enzyme is capa-
ble not only of transforming the residual dextrins to glucose,
through the hydrolysis of glucose units linked in the non-reducing
end of the chain, but also to hydrolyze the poly-glucosylated myo-
inositol forms to the simplest monoglucosylated forms. The reac-
tion products obtained after glucoamylase digestion were analyzed
by analytical HPLC and purified by preparative HPLC.
The modification of CGTase specificity by the presence of myo-
inositol in the enzymatic reaction is illustrated in Figure 2, where
in HPLC chromatograms, the products of CGTase activity when b-
cyclodextrin is used as a substrate (Fig. 2a) are compared to those
obtained in the same reaction conditions but in the presence of
myo-inositol as acceptor (Fig. 2b). Finally, the glycosylated myo-
inositols 3 and 4 and glucose 1, obtained after glucoamylase diges-
tion of the reaction products (both glycosylated myo-inositols as
well as the maltooligosaccharides) are shown in Figure 2c.
ularis was described.33 On the other hand, a wide variety of inositols
such as 1
D
-chiro-inositol, 1
D
-pinitol, 1D-3-O-allyl-4-O-methyl-chiro-
inositol,
1D
-3,4-di-O-methyl-chiro-inositol, 1L
-chiro-inositol and
myo-inositol were successfully galactosylated with a thermophilic
b-galactosidase isolated from Thermoanaerobacter sp. strain TP6-B1
using p-nitrophenyl b-D-galactopyranoside as donor, with yields
ranging from 46% to 64%.34 Inositol glucosylation has also been
achieved with enzymes other than glycosidases; this is the case of
kojibiose phosphorylase which was used as a biocatalyst for the syn-
thesis of four glucosyl-myo-inositols.35
A successful approach to the synthesis of glucosylated myo-
inositols was also reported by Sato et al., who obtained two
monoglucosyl myo-inositols trough a transglucosylation reaction
with CGTase from Bacillus obhensis.35 The monoglycosylated prod-
ucts were effective when applied as a prebiotic on intestinal
Bifidobacterium.5
CGTase (EC 2.4.1.19) is an important industrial enzyme which
catalyzes the transformation of starch and linear maltodextrin sub-
strates to produce cyclodextrins (CDs) through intramolecular
transglycosylation. CDs are cyclic ring structures consisting of 6,
7 or 8 glucose residues joined by
-, b- and
-CDs, respectively.36,37 CGTases use the non-reducing
end of a bound oligosaccharide as an acceptor molecule for cyclic
-(1?4)-linked oligosaccharide formation.38 Additionally, these
a-(1?4) linkages, designated as
a
c
a
enzymes also catalyze three intermolecular transglycosylations:
hydrolysis of starch to produce linear dextrins, a coupling reaction
between cyclodextrins and/or linear dextrin to produce glucosides,
and disproportionation, a type of activity in which linear oligosac-
charides are recombined through transglucosylation modifying
their size.39
Acceptors that have been successfully subjected to glycosyla-
tion by CGTases include sugar alcohols such as sorbitol;40 sugars,
including glucose, xylose, sorbose, sucrose, maltose, lactose;27,38
glycosides such as rutin, stevioside, rebaudioside A, capsaicin, 8-
From this analysis, the profile of transfer products obtained
after 24 h of reaction with CGTase and myo-inositol before gluco-
amylase digestion were identified as: G1-myo-inositols 3 and 4,
G2-myo-inositols
5 and 6, G3- myo-inositols
7 and 8 and
nordihydrocapsaicin, methyl-
a
-
D
-glucopyranoside, methyl-b-
D
-
G4-myo-inositol 9, which correspond to one or several glucose
molecules transferred to myo-inositol.35,5 After quantification of
the products it was found that 59.8% of the initial myo-inositol
was glucosylated; 32% yield of the products were found as
G1-myo-inositols 3 and 4 in a 73:27 ratio corresponding to
308 mM and 114 mM, respectively, 18.1% yield as G2-myo-inosi-
tols, 7.6% yield as G3-myo-inositols and 2.2% yield as G4-myo-ino-
sitol. After 1H NMR analysis, compounds 3 and 4 were confirmed as
monoglycosylated myo-inositol products.
These results confirm that among all the enzymatic reactions
tested the combined enzymatic transglucosylation using CGTase
from Thermoanaerobacter sp. followed by A. niger glucoamylase
digestion result in the synthesis of monoglucosyl-myo-inositols.
glucopyranoside, phenyl- -glucopyranoside, phenyl-b-D
a
-D
-gluco-
pyranoside, sucrose laurate and alkyl glycosides; flavonoides such
as hesperidin, neohesperidin, naringin41 and ascorbic acid.42
The aim of the present work was to synthesize glucosyl myo-
inositol analogues of the lanceolitols13 through an enzymatic regio
and stereoselective approach and to explore their anti-inflamma-
tory properties. We report in detail the synthesis, purification
and characterization of two monoglucosyl-inositols obtained
through a glucosylation strategy using a b-cyclodextrin as a gluco-
syl donor and CGTase from Thermoanaerobacter sp. as biocatalyst,
followed by the selective hydrolysis of the poly-glucosylated prod-
ucts with Aspergillus niger glucoamylase. The in vivo anti-inflam-
matory properties of the purified monoglucosyl-inositols are also
reported.
2.2. Structural elucidation of the monoglycosylated myo-
inositols 3 and 4
2. Results and discussion
It has been reported that biological properties of glycosylated
molecules rely not only on the number, type, sequence and glyco-
sidic linkage of the glycosyl moieties, but also on the absolute config-
uration of the oligosaccharide.42 Therefore, the detailed elucidation
of the structural features of the oligosaccharide results are essential
to fully understand their biological properties. Hence, complete
structure elucidation of the monoglucosyl-myo-inositols 3 and 4
2.1. Enzymatic synthesis of glycosylated myo-inositol
Initially, the screening of various enzymes capable of perform-
ing the glycosylation of myo-inositol in the presence of different
glycosyl donors was carried out. In all cases, the reaction products

A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
45
Figure 1. (a) Thin-layer chromatogram of the reaction products from cellobiose (donor) and myo-inositol (acceptor). Lines 1a and 1b show profiles before adding
b-glucosidase; 2a–7a and 2b–7b profiles at different reaction times; G, glucose, C, cellobiose. (b) Thin-layer chromatogram of the reaction products from b-CD (donor) and
myo-inositol (acceptor). Lines 1c and 1d show profiles before adding CGTase; lines 2c and 2d show profiles after adding CGTase at 24 h of reaction and lines 3c and 3d show
profiles after 48 h of reaction.
OH
G1-myo-inositols 3 and 4 + G2-myo-inositols 5 and 6 +
HO
Thermoanaerobacter sp. CGTase
HO
HO
OH
G3-myo-inositols 7 and 8 + G4-myo-inositol 9
Aspergillus niger glucoamylase
OH
+
β-CD
OH
OH
HO
4'
6'
4
2
O
3
5'
HO
HO
HO
OH
HO
O
1'
HO
OH
4
+
6
2'
2
1
6'
4'
3
3'
5
5'
OH
1'
OH
O
HO
O
OH
OH
6
1
5
3'
HO
OH
2'
OH
3
4
Scheme 1. Synthetic strategy using myo-inositol, b-cyclodextrin as a glucosyl donor and CGTase from Thermoanaerobacter sp., followed by the selective hydrolysis of the
poly-glucosylated products with Aspergillus niger glucoamylase as biocatalysts.
was determined by an extensive nuclear magnetic resonance (NMR)
study. Indeed, full proton and carbon assignments were carried out
using 1D (1H, 13C) and 2D (gCOSY, TOCSY, HETCOR, NOESY and
gHMBC) NMR experiments and HRFAB mass spectrometry analysis.
HRFAB mass spectrometry analysis of compounds 3 and 4 gave
a peak at m/z 343.1249 for [M+H]+ corresponding to the molecular
formula C12H23O11. The 13C NMR spectrum of the more polar com-
pound 4 (Rt = 14.59 min) contained 12 signals. In the 1H NMR spec-

46
A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
Figure 2. (a) HPLC profile of a carbohydrate mixture after 24 h of reaction with CGTase using b-cyclodextrin (b-CD) in the absence of myo-inositol (b) HPLC profile of a
carbohydrate mixture obtained by the transglycosylation of CGTase using b-cyclodextrin (b-CD) after 24 h of reaction in presence of myo-inositol. (c) HPLC profile of a
carbohydrate mixture obtained by glucoamylase digestion of the mixture of oligoglucosyl myo-inositols after 24 h. Retention times (min) are shown at the top of each peak.
trum, only one anomeric proton was observed at d 5.15 (1H, d,
J = 4.4 Hz) and HETCOR showed its attachment to the anomeric
carbon at d 98.0. This served as the starting point for tracing con-
nectivities within the sugar spin system by gCOSY and TOCSY
experiments. Once the 1H signals of the glucopyranose residue
have been assigned, the six remaining 1H resonances were as-
signed to the myo-inositol ring, and connectivities were delineated
from 1H–1H COSY and TOCSY experiments with the characteristic
signal of H-2 at d 4.19 (1H, dd, J2,1 = 2.0; J2,3 = 2.4 Hz) as the starting
point. The carbon to which each hydrogen was attached was de-
fined from HETCOR. The most downfield of these carbons
(d 79.86) in the cyclitol ring was the point of linkage to the glucose.
Coupling constants (J1,2 = 2.4 Hz; J1,6 = 10.0 Hz) observed for its
hydrogen (d 3.57, H-1) and those observed for the hydrogen as-
tol leads to the configurational
numbering gives the substituted myo-inositol an
D
-prefix, and the clockwise
L
-prefix.2,43,44
Therefore, substitution either side of the plane of symmetry (which
bisects C-2 and C-5), such as at C-1 or C-3, results in two different
compounds.
The absolute configuration of the myo-inositol ring was deter-
mined by an X-ray crystallographic study of the nonabenzoyl
derivative 4a. Figure 3a shows a perspective drawing of the
X-ray structure of 4a, and Figure 3b shows another perspective
drawing in which, in order to gain clarity, the benzoyl groups were
omitted. In this last figure it is observed the 1
myo-inositol moiety in 4a. Therefore, compound 4 can be formu-
lated as -glucopyranosyl-(1?1)-1 -myo-inositol.
D-configuration of the
a-
D
D
This compound was obtained by a transglucosylation reaction
signed to H-2 at
d
4.19 and H-3 at
d
3.50 (J3,2 = 2.4 Hz;
of Kojibiose phosphorylase using myo-inositol as an acceptor and
J3,4 = 10.0 Hz) confirmed assignment of H-1 as an equatorial hydro-
gen with adjacent axial and equatorial hydrogens. The remaining
assignments for the myo-inositol ring were straightforward. Full
assignments of the proton and carbon resonances were secured
from the TOCSY, COSY, NOESY and gHMBC data. Based on the
gHMBC correlations, the glucose moiety was linked to C-1 of the
cyclitol ring on the basis of long-range correlation between C-1
(d 79.86) of the myo-inositol and H-10 of glucose at d 5.15. Similar
analysis by NOESY established that the glucose residue was
b-D
-glucose 1-phosphate as a donor.28 However, the 13C NMR data
of compound 4 obtained in this work, were very different with
those described, and unfortunately, the physical data of the saccha-
ride were not described, and we were not able to find a structural
relationship between them.
A detailed analysis of the unassigned NMR correlations in the
2D NMR spectra revealed that the second product 3 was
a-D-gluco-
pyranosyl-(1?4)-4 -myo-inositol, and comparison with com-
D
pound 4 showed that they differ only in the linkage site of the
glucose to the myo-inositol ring. Instead, compound 3 is attached
to C-4. The presence of the glucopyranosyl unit was deduced from
the 1H NMR anomeric proton signal at d 5.10 (d, J = 4.0 Hz), which
was compatible with the anomeric carbon signal at d 99.62.
Also, in this case, the proton-coupling network within the pseu-
do-disaccharide residue was established using a combination of
1H–1H gCOSY, NOESY, TOCSY, HETCOR and gHMBC experiments.
Based on the gHMBC correlations, the glucose moiety was linked
to C-4 of the cyclitol ring on the basis of long-range correlation be-
a
-linked to O-1 of myo-inositol because of the correlation between
H-10 of glucose at d 5.15 and H-1 of the cyclitol at d 3.57.
It should be noted that myo-inositol is a meso-isomer with five
equatorial hydroxyl groups and an axial hydroxyl group. The car-
bon bearing the axial hydroxyl group is designated as C-2, and
the other ring carbons can be numbered from C-1 to C-6, starting
from a C-1 atom and proceeding around the ring in clockwise or
counterclockwise fashion. According to convention, a counter-
clockwise numbering in an asymmetrically substituted myo-inosi-

A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
47
Figure 3. (a) A stereoscopic view of compound 4a. (b) Perspective drawing in which, in order to gain clarity, the benzoyl groups were omitted.
tween C-4 (d 80.92) of myo-inositol and the H-10 of glucose moiety
at d 5.10. Moreover, NOESY experiment established that the glu-
of ear oedema (80%) at a dose of 1.5 mg/kg of body weight, than
corticosterone (52.5%) at a dose of 10 mg/kg of body weight;
whereas -glucopyranosyl-(1?1)-1 -myo-inositol 4 was nearly
cose residue was
a
-linked to O-4 of myo-inositol because a corre-
a-
D
D
lation between H-10 of glucose at d 5.10 and H-40 of the cyclitol
at d 3.52 was observed. This compound was previously obtained
by Sato et al.35 by using a transglucosylation reaction of CGTase
from B. obhensis, myo-inositol as an acceptor and b-cyclodextrin
as donor. The physical and NMR data of compound 3 and its nano-
benzoyl derivative 3a were very similar with those described,35,45
indicating that these compounds have the same configuration.
equipotent (40 % of inhibition) to corticosterone at the same dose.
In the carrageenan-induced oedema test, both compounds 3 and 4
showed an inhibition of paw oedema of 76% and 80%, respectively
at a dose of 1.5 mg/kg, comparable to the reference drug (79%) at a
dose of 10 mg/kg. The present data clearly showed that compounds
3 and 4 exhibit important anti-inflammatory activity on inhibiting
oedema formation after carrageenan subplantar injection, and TPA
administration in the ear of mice. Further studies must be con-
ducted in order to clarify the exact anti-inflammatory mechanism
of these compounds.
Therefore compound 3 can be formulated as
(1?4)-4 -myo-inositol.
a-D-glucopyranosyl-
D
In addition, it was noteworthy the downfield shift of 0.17 ppm
displayed by H-50 in the 1H NMR spectra of the regioisomer 3, in
comparison with H-50 in compound 4. In order to understand this
downfield shift, we performed a theoretical study of the two pos-
sible diasteromeric products that could be obtained by glucosyla-
tion at the enantiotopic 4- and 6-positions of myo-inositol. For
this, we employed PM3 calculations at the semi empiric level, with
correction of the H–H interactions, with the SPARTAN programme.
These calculations predicted considerably shorter interatomic dis-
4. Conclusion
The coupling reaction between b-CD and myo-inositol via an
enzymatic glycosylation with CGTase from Thermoanaerobacter
sp. and selective hydrolysis of the poly-glucosylated products with
glucoamylase from A. niger, provides a versatile method to produce
monoglucosyl-myo-inositols in moderate yield (32%). The products
tance between H-50 and OH-C-60 for
D
-myo-inositol (2.588 Å) than
-isomer (2.629 Å). This result shows that the cyclitol ring has
-configuration.
obtained were identified as
a-
D-glucopyranosyl-(1?4)-4D-myo-
the
the
L
inositol 3 and -glucopyranosyl-(1?1)-1D
a
-
D
-myo-inositol 4. In
D
addition, this study was able to demonstrate that CGTase from
Thermoanaerobacter sp. stereoselectively glycosylate the (1S)- and
(4S)-positions of myo-inositol.
3. Biological activity
These regioisomeric
markedly inhibited inflammation induced in mice. This is the first
report about the anti-inflammatory properties of -glucosyl myo-
a-D-glucopyranosyl myo-inositols 3 and 4,
Taking into consideration that the natural lanceolitols (for
example: -myo-inositol-2-O-dodecanoyl-1-O-b- -glucopyrano-
D
D
a
side), isolated previously from S. lanceolatum, showed important
anti-inflammatory activity,13 we carried out a preliminary study
exploring the anti-inflammatory properties of the regioisomeric
analogues 3 and 4. For this purpose both compounds were tested
in vivo for their ability to reduce the inflammatory response in
the TPA-induced ear oedema and carrageenan-induced paw oede-
ma of mice, and the inhibitory activity was compared with that of
corticosterone (Cort), a commercially available steroidal (SAI) anti-
inflammatory drug. Taking the anti-inflammatory activity after
inositols, which allow us to consider them as a useful tool for
obtaining new analgesic and anti-inflammatory agents.
5. Experimental
5.1. General
Melting points were determined on a Fisher Johns melting point
apparatus and were not corrected. HRFABMS spectra in a matrix of
m-nitrobenzyl alcohol were recorded on a JEOL JMX-AX 505 HA
2.5 h as criteria for comparison, it can be concluded that
a-D-gluco-
mass spectrometer. Optical rotations were measured on
a
pyranosyl-(1?4)-4 -myo-inositol 3 displayed a higher inhibition
D

48
A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
Perkin–Elmer 241MC polarimeter (10 cm, 1 mL cell). The benzoyl-
ation reactions were made in a CEM microwave apparatus. X-ray
crystallographic data were acquired in an Enraf–Nonius–Bruker
l
Bondapak™ NH2 10
l
m, 125 Å, 7.8 ꢁ 300 mm (waters); eluent
CH3CN–H2O (80:20 v/v); flow rate, 7.0 mL/min; column tempera-
ture, 35 °C; RI detector (waters).
Kappa CCD equipment; method used for solve the structure: soft-
46
ware XM SHELXTL
.
NMR spectra were recorded on a Varian Unity
5.5.1. a-D-Glucopyranosyl-(1?4)-4D-myo-inositol 3
NMR Spectrometer at 400 MHz for 1H NMR, 1H-1H gCOSY, NOESY,
gHMBC, HETCOR, HSQC and 1H-1H TOCSY and 100 MHz for 13C
NMR using D2O for compounds 3 and 4 and CDCl3 for compounds
3a and 4a as solvent. NMR experiments are referenced to H2O and
TMS and chemical shifts are reported in ppm (d).
White amorphous powder, mp 153–155 °C (decomposed),
½
a 2D0
ꢂ
¼ þ66:5 (c 0.1, H2O). 1H NMR (400 MHz, D2O): d = 5.10 (d,
1H, J1 ,2 = 4.0 Hz, H-10), 3.83 (dd, 1H, J2,1 = 2.8, J2,3 = 3.2 Hz, H-2),
0
0
3.78 (ddd, 1H, J5 ,6a = 2.8, J5 ,6b = 4.8, J5 ,4 = 10, H-50), 3.61 (m, 1H,
H-6a0), 3.55 (dd, 1H, J6,1 = 8.8, J6,5 = 10.0 Hz, H-6 or H-4), 3.54 (m,
1H, H-6b0), 3.52 (dd, 1H, J4,3 = 9.2, J4,5 = 10.0 Hz, H-4 or H-6), 3.42
0
0
0
0
0
0
T. maritime b-glucosidase was obtained using a similar proce-
-amylase AmyA from the same strain.47 CGTase
(dd, 1H, J3,2 = 3.2, J3,4 = 9.8 Hz, H-1 or H3), 3.40 (dd, 1H, J3 ,4 = 9.6,
0
0
dure reported for
a
from Thermoanaerobacter sp. (ToruzymeÒ 3.0 L, a liquid enzyme
preparation) was obtained from Novozyme. Myo-inositol was ob-
tained from Sigma. b-cyclodextrin was obtained from American
Maize-Products Company. Glucoamylase from A. niger used for
digestion of oligoglucosyl-inositols was obtained by ANZECOÒ.
J3 ,2 = 10.0 Hz, H-30), 3.34 (dd, 1H, J2 ,1 = 4.0, J2 ,3 = 9.6 Hz, H-20),
3.30 (dd, 1H, J1,2 = 2.8, J1,6 = 10.4 Hz, H-3 or H1), 3.25 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
J5,4 = 8.8, J5,6 = 10.0 Hz, H-5), 3.21 (dd, 1H, J4 ,3 = 9.2, J4 ,5 = 10.0 Hz,
H-40). 13C NMR (100 MHz, D2O): d = 99.62, 80.92, d 74.85, 73.21,
72.69, 72.48, 72.18, 71.99, 71.23, 69.99, 68.68, 60.70. HRFABMS
(positive mode), calcd for C12H23O11: 343.3759, found: 343.1249.
5.2. Thin-layer chromatography
5.5.2.
a-D-Glucopyranosyl-(1?1)-1D-myo-inositol 4
The TLC was performed on a Silica Gel 60 (Merck) pre-coated
plate with a solvent system (v/v) of n-butanol/ethanol/water
(3:5:2). The spots of sugars were detected by spraying with a-nap-
thol solution, followed by heating at 120 °C for 10 min.
White amorphous powder, mp 155–157 °C (decomposed),
½
a 2D0
ꢂ
¼ þ85:8 (c 0.1, H2O). 1H NMR (400 MHz, D2O): d = 5.15 (d,
1H, J1 ,2 = 4.4 Hz, H-10), 4.19 (dd, 1H, J2,1 = 2.0, J2,3 = 2.4 Hz, H-2),
3.82 (m, 1H, H-60b), 3.80 (dd, 1H, J6,1 = 9.2, J6,5 = 10.0 Hz, H-6 or
H4), 3.76 (dd, 1H, J4,3 = 8.8, J4,5 = 10.0 Hz, H-4 or H6), 3.75 (dd,
0
0
1H, J3 ,2 = 8.8, J3 ,4 = 10.0 Hz, H-30), 3.72 (m, 1H, H-60a), 3.61 (m,
0
0
0
0
5.3. Transglucosylation reaction with b-glucosidase from T.
maritima
1H, H-50), 3.57 (dd, 1H, J1,2 = 2.4, J1,6 = 10.0 Hz, H-1or H-3), 3.52
(dd, 1H, J2 ,1 = 4.4, J2 ,3 = 10.0 Hz, H-20), 3.50 (dd, 1H, J3,2 = 2.4,
0
0
0
0
0
0
0
0
Solutions of myo-inositol 1.0 M and 1.0 M of cellobiose were
prepared in 100 mM K2HPO4 buffer at pH 6.0 and b-glucosidase
from T. maritima was added at a final concentration of 5 units/mL.
The transglucosylation reaction was performed at a final volume
of 3 mL, at 70 °C for 48 h. The reaction mixture was stored at
ꢀ18 °C until subsequent analyzes and compared to blank experi-
ments obtained with the enzyme in absence of myo-inositol.
J3,4 = 10.0 Hz, H-3 or H-1), 3.38 (dd, 1H, J4 ,3 = 9.6, J4 ,5 = 10.0 Hz,
H-40), 3.28 (dd, 1H, J5,4 = 9.2, J5,6 = 10.0 Hz, H-5). 13C NMR
(100 MHz, D2O): d = 100.81, 79.86, 74.30, 73.15, 72.63, 72.42,
72.21, 72.05, 72.05, 71.39, 69.93, 60.92. HRFABMS (positive mode),
calcd for C12H23O11: 343.3759, found: 343.1249.
5.6. General benzoylation procedure
5.4. Transglucosylation reaction with CGTase from Thermoanae-
robacter sp.
Benzoyl chloride was added dropwise to a 10 mL glass micro-
wave reaction vessel at room temperature containing a stirred
solution of 3 or 4 in pyridine. The reaction vessel was sealed with
a cap and then placed into the microwave cavity. The microwave
was programmed to heat the reaction mixture to the desired tem-
perature. After the reaction was completed, the vessel was cooled
below 50 °C using a flow of compressed air. The 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 by column
chromatography (hexane-ethyl acetate, 90:10?50:50) on silica gel
to give the corresponding perbenzoylated derivatives.
Solutions of myo-inositol 1.07 M and 0.17 M of b-CD were pre-
pared in 50 mM Na2HPO4 buffer at pH 6.0 and CGTase enzyme
from Thermoanaerobacter sp. was added at a final concentration
of 8.6 units/mL. The transglucosylation reaction was performed
at a final volume of 3 mL, at 50 °C for 24 h. The reaction mixture
was immersed in boiling water for 10 min to inactivate the enzyme
and was stored at -18 °C until subsequent analyzes and compared
to blank experiments obtained with the enzyme in the absence of
myo-inositol.
5.5. Glucoamylase digestion of incubated broth and purification
of monoglucosyl-myo-inositols
5.6.1.
(1,2,3,5,6-penta-O-benzoyl)-myo-inositol 3a
Prepared from (0.0236 g, 0.069 mmol), benzoyl chloride
a-D-(2,3,4,6-Tetra-O-benzoyl)-glucopyranosyl-(1?4)-4D-
3
Anincubatedbroth(3 mL)of CGTasewasdigestedwith258 U/mL
of glucoamylase at 50 °C, in buffer (pH 6.0) Na2HPO4 for 24 h. The
reaction was stopped by boiling for 10 min and was stored at
ꢀ18 °C until subsequent high-performance chromatographic
separation.
Formation of oligoglucosyl-inositols and monoglucosyl-inosi-
tols was monitored by high-performance liquid chromatography
(HPLC). The measurement conditions were as follows: Colum Pre-
(0.172 mL, 1.48 mmol) and pyridine (0.26 mL). The reaction was
heated at 90 °C with 50 W and 13 psi for 30 min to afford com-
pound 3a (in 75 % yield) as a white solid after purification; mp
139–141 °C, ½a 2D0
ꢂ
¼ þ72:95 (c 1.05, CHCl3). 1H NMR (400 MHz,
CDCl3): d = 8.3–7.0 (m, 45H, ArH), 6.29 (t, 1H, J2,1 = 2.8,
J2,3 = 2.8 Hz, H-2), 6.24 (t, 1H, J6,1 = 10.4, J6,5 = 10.4 Hz, H-6 or H4),
6.02 (dd, 1H, J5,4 = 9.6, J5,6 = 10.0 Hz, H-5), 5.98 (dd, 1H, J3,2 = 3.2,
0
0
0
0
J3,4 = 10.0 Hz, H-3 or H-1), 5.93 (dd, 1H, J3 ,2 = 10.4, J3 ,4 = 10.0 Hz,
vail Carbohydrate ES 5
(68:32 v/v); flow rate, 1.0 mL/min; colum temperature, 32 °C; RI
detector (Waters). The injection vol. was 10 L.
l
4.6 mm ꢁ 250 mm; eluent, CH3CN–H2O
H-30), 5.82 (dd, 1H, J1,2 = 3.2, J1,6 = 10.4 Hz, H-1 or H-3), 5.73 (d,
1H, J1 ,2 = 3.6 Hz, H-10), 5.59 (dd, 1H, J4 ,3 = 10.0, J4 ,5’ = 10.4 Hz, H-
0
0
0
0
0
40), 5.22 (dd, 1H, J2 ,1 = 3.6, J2 ,3 = 10.4 Hz, H-20), 5.01 (dd, 1H,
J4,3 = 9.6, J4,5 = 10.0 Hz, H-4 or H6), 4.35 (m, 1H, H-60a), 4.35 (m,
H-50), 3.68 (br d, H-60b). 13C NMR (100 MHz, CDCl3): d = 166.14,
165.67, 165.52, 165.43, 165.36, 165.09, 164.73, 134.05, 133.74,
0
0
0
0
l
After glucoamylase digestion treatment of the reaction products
obtained with CGTase, compounds 3 and 4 were separated by pre-
parative HPLC. The HPLC conditions were as follows: Column

A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
49
133.36, 133.26, 133.0, 130.12, 129.99, 129.83, 129.80, 129.68,
Acknowledgements
129.62, 129.11, 128.95, 128.66, 128.48, 128.40, 128.29, 128.19,
128.14, 96.81, 73.83, 73.12, 70.84, 70.63, 70.23, 70.13, 69.65,
69.49, 68.52, 68.18, 61.87. HRFABMS (positive mode), calcd for
C75H58O20: 1279.2510, found: 1279.3827.
The authors wish to acknowledge PAPIIT (project: IN226706-
3) and CONACyT (projects: 56431 and CB 82851) for the finan-
cial support. We also thank Fernando González Munoz and M.
C. María Elena Rodríguez Alegría, both from IBt-UNAM, for HPLC
analysis and for the production of T. maritima b-glucosidase,
respectively. Alfonso Miranda thanks CONACyT for Ph.D. fellow-
ship 189120.
5.6.2.
(2,3,4,5,6-penta-O-benzoyl)-myo-inositol 4a
Prepared from (0.0364 g, 0.12 mmol), benzoyl chloride
a-D-(2,3,4,6-Tetra-O-benzoyl)-glucopyranosyl-(1?1)-1D-
4
(0.27 mL, 2.32 mmol) and pyridine (0.4 mL). The reaction was
heated at 90 °C with 50 W and 13 psi for 30 min, to afford com-
pound 4a (77.85% yield) as a white solid after purification, crystal-
References
lized from acetone/methanol (1:3), mp 115–118 °C, ½a D20
¼ þ30:5
ꢂ
1. Michell, H. R. Nat. Rev. Mol. Cell Biol. 2008, 9, 151.
2. Sureshan, K. M.; Shashidhar, M. S.; Praveen, T.; Das, T. Chem. Rev. 2003, 103,
4477.
3. Loewus, F. A.; Murthy, P. P. N. Plant. Sci. 2000, 150, 1.
4. Michell, H. R. Biochem. Soc. Symp. 2007, 74, 223.
5. Sato, M.; Matsuo, T.; Orita, N.; Yagi, Y. Biotechnol. Lett. 1991, 13, 69.
6. Kabat, E. A.; MacDonald, D. L.; Ballou, C. E.; Fischer, H. O. L. J. Am. Chem. Soc.
1953, 75, 4507.
7. Noguchi, K.; Okuyama, K.; Ohno, S.; Hidano, T.; Wakiuchi, N.; Tarui, T.; Tamaki,
H.; Kishihara, S.; Fujii, S. Carbohydr. Res. 2000, 328, 241.
8. Newton, G. L.; Fahey, R. C. Arch. Microbiol. 2002, 178, 388.
9. Smith, C. K.; Hewage, C. M.; Fry, S. C.; Sadler, I. H. Phytochemistry 1999, 52,
387.
10. Müller, G.; Wied, S.; Crecelius, A.; Kessler, A.; Eckel J. Endocrinol. 1997, 138,
3459.
11. Müller, G.; Wied, S.; Piossek, C.; Bauer, A.; Bauer, J.; Frick, W. Mol. Med. 1998, 4,
299.
12. Frick, W.; Bauer, A.; Bauer, J.; Wied, S.; Müller, G. Biochem. J. 1998, 336, 163.
13. Salgado, H. Y.; Ramirez, M. L. G.; Vazquez, L.; Rios, M. Y.; Alvarez, L. J. Nat. Prod.
2005, 68, 1031.
14. Koto, S.; Hirooka, M.; Yoshida, T.; Takenaka, K.; Asai, Ch.; Nagamitsu, T.;
Sakuma, H.; Sakurai, M.; Masuzawa, S.; Komiya, M.; Sato, T.; Zen, S.; Yago, K.;
Tomonaga, F. Bull. Chem. Soc. Jap. 2000, 73, 2521.
15. Daniellou, R.; Palmer, D. R. J. Carbohydr. Res. 2006, 341, 2145.
16. Cid, M. B.; Francisco, A.; Lomas, M. M. Carbohydr. Res. 2004, 339, 2303.
17. Kornienko, A.; Marnera, G.; d’Alarcao, M. Carbohydr. Res. 1998, 310, 141.
18. Marnera, G.; d’Alarcao, M. Carbohydr. Res. 2006, 341, 1105.
19. Zapata, A.; León, Y.; Mato, J. M.; Nieto, I. V.; Penadés, S.; Lomas, M. M.
Carbohydr. Res. 1994, 264, 21.
20. Jaworek, C. H.; Iacobucci, S.; Calias, P.; d’Alarcao, M. Carbohydr. Res. 2001, 331,
375.
21. Romero, G.; Lutrell, I.; Rogol, A.; Zeller, K.; Hewlett, E.; Larner, J. Science 1988,
240, 509.
22. Larner, J.; Price, J. D.; Heimark, D.; Smith, L.; Rule, G.; Piccariello, T.; Fonteles, M.
C.; Pontes, C.; Vale, D.; Huang, L. J. Med. Chem. 2003, 46, 3283.
23. Weijers, C. A. G. M.; Franssen, M. C. R.; Visser, G. M. Biotechnol. Adv. 2008, 26,
436.
24. Field, M. C. Glycobiology 1997, 7, 161.
25. Lomas, M. M.; Mosquera, F. M.; Khiar, N. Eur. J. Org. Chem. 2000, 23,
1539.
(c 2.58, CHCl3). 1H NMR (400 MHz, CDCl3): d = 8.5–7.0 (m, 45H,
ArH), 6.36 (dd, 1H, J2,1 = 2.8, J2,3 = 3.2 Hz, H-2), 6.32 (t, 1H,
J6,1 = 10.4, J6,5 = 10.4 Hz, H-6 or H4), 6.22 (dd, 1H, J4,3 = 10.0,
0
0
0
0
J4,5 = 10.4 Hz, H-4 or H6), 5.97 (dd, 1H, J3 ,2 = 9.6, J3 ,4 = 10.0 Hz,
H-30), 5.82 (dd, 1H, J5,4 = 10.0, J5,6 = 10.4 Hz, H-5), 5.73 (dd, 1H,
0
0
J3,2 = 3.2, J3,4 = 10.6 Hz, H-3 or H-1), 5.66 (dd, 1H, J4 ,3 = 9.6,
J4 ,5 = 10.0 Hz, H-40), 5.59 (d, 1H, J1 ,2 = 4.0 Hz, H-10), 5.22 (dd, 1H,
0
0
0
0
J2 ,1 = 3.6, J2 ,3 = 10.4 Hz, H-20), 4.78 (ddd, J5 ,6 a = 3.2, J5 ,6 b = 4.8,
0
0
0
0
0
0
0
0
J5 ,4 = 8.6, H-50), 4.67 (dd, 1H, J1,2 = 3.2, J1,6 = 8.4 Hz, H-1 or H-3),
0
0
4.64 (m, 1H, H-60a), 4.54 (dd, J6 b,5 = 4.0, J6 b,6 a = 12.8, H-6b0). 13C
NMR (100 MHz, CDCl3): d = 166.34, 166.31, 165.64, 165.55,
165.33, 165.26, 165.17, 165.12, 164.80, 133.85, 133.55, 133.39,
133.26, 133.21, 133.14, 133.09, 132.92, 130.42, 130.06, 130.01,
129.98, 129.92, 129.81, 129.62, 129.40, 129.28, 129.08, 129.0,
128.84, 128.72, 128.61, 128.51, 128.45, 128.29, 128.20, 128.02,
98.00, 74.13, 72.02, 71.31, 71.02, 70.78, 70.78, 70.20, 70.05,
69.23, 62.81. HRFABMS, calcd for C75H58O20: 1279.2510, found:
1279.4065. X-ray crystallographic structure in Figure 3.48
0
0
0
0
5.7. Biological evaluation
5.7.1. Experimental animals
Adult male BALB/c mice with a body weight ranging from 20 to
22 g were used. All animals had free access to food and water and
were kept on a 12/12 h light-dark cycle.
5.7.2. TPA-induced mouse ear oedema
Oedema was induced by topical application of 2.5
(12-O-tetradecanoylphorbol-13-acetate) dissolved in 20
tone to the right ear of ten mice. Solutions of compounds 3 and 4
(1.5 mg/kg) were dissolved in 20 L of physiological solution and
lg of TPA
lL ace-
26. Larner, J.; Huang, L. C.; Schwartz, C. F. W.; Oswald, A. S.; Shen, T.-Y.; Kinter, M.;
Tang, G.; Zeller, K. Biochem. Biophys. Res. Commun. 1988, 151, 1416.
27. Shibuya, T.; Miwa, Y.; Nakano, M.; Yamauchi, T.; Chaen, H.; Sakai, S.; Kurimoto,
M. Biosci. Biotechnol. Biochem. 2003, 57, 56.
28. Yamamoto, T.; Watanabe, H.; Nishimoto, T.; Aga, H.; Kubota, M.; Chaen, H.;
Fukuda, S. J. Biosci. Bioeng. 2006, 101, 427.
29. Kurashima, K.; Fujii, M.; Ida, Y.; Akita, H. J. Mol. Catal. B Enzym. 2003, 26, 87.
30. Sauerbrei, B.; Thiem, J. Tetrahedron Lett. 1992, 33, 201.
31. Bridiau, N.; Taboubi, S.; Marzouki, N.; Legoy, M. D.; Maugard, T. Biotechnol.
Prog. 2006, 22, 326.
l
were applied 30 min before TPA administration. A group of five
mice were treated with the standard drug corticosterone (10 mg/kg)
as reference. Left ears of five mice (control) were treated with
20
lL acetone only. The ear swelling was measured with a micro-
metre before and at 0.5, 1, 2 and 2.5 h after TPA application. The
oedema was expressed as the increase in thickness in lm.
32. Hart, J.; Falshaw, A.; Farkas, E.; Thiem, J. Synlett 2001, 3, 329.
33. Gorin, P. A. J.; Horitsu, K.; Spencer, J. F. T. Can. J. Chem. 1965, 43, 2259.
34. Hart, J. B.; Kröger, L.; Falshaw, A.; Falshaw, R.; Farkas, E.; Thiem, J.; Win, A. L.
Carbohydr. Res. 2004, 339, 1857.
35. Sato, M.; Nakamura, K.; Nagano, H.; Yagi, Y.; Koizumi, K. Biotechnol. Lett. 1992,
14, 659.
36. Blackwood, A. D.; Bucke, C. Enzyme Microbial Technol. 2000, 27, 704.
37. Zain, W. S. W., Md.; Illias, R., Md.; Salleh, M., Md.; Hassan, O.; Rahman, R. A.;
Hamid, A. A. Biochem. Eng. 2007, 33, 26.
38. Monthieu, C.; Gubert, A.; Taravel, F. R.; Nardin, R.; Combes, D. Biocatal.
Biotransfor. 2003, 21, 7.
5.7.3. Carrageenan-induced mouse paw oedema
Oedema was induced in the right hind paw by subplantar injec-
tion of carrageenan (0.1% w/v in physiological solution, 20 L) of
five mice. Compounds 3 and 4, dissolved in physiological solution
were administered ip at a dose of 1.5 mg/kg, 30 min before carra-
geenan injection. A group received the reference drug corticoste-
rone (10 mg/kg, po). The left paw volumes were measured with a
micrometre at zero time prior to carrageenan and at 0.5, 1, 2 and
2.5 h after inflammation induction. The oedema was expressed as
the increase in thickness.
l
39. Van Der Veen, B. A.; Bart, A.; Alebeek, V.; Gert-Jan, W. M.; Uitdehaag, J. C. M.;
Dijkstra, B. W.; Dijkhuizen, L. Eur. J. Biochem. 2000, 267, 658.
40. Park, K. H.; Kim, M. J.; Lee, H. S.; Han, N. S.; Kim, D.; Robyt, J. F. Carbohydr. Res.
1998, 313, 235.
41. Svensson, D.; Ulvenlund, S.; Adlercreutz, P. Biotechnol. Bioeng. 2009.
doi:10.1002/bit.22472.
42. Park, D. C.; Kim, T. K.; Lee, Y. H. Enzyme Microb. Technol. 1998, 22, 217.
43. Irvine, R. F.; Schell, M. J. Nat. Rev. Mol. Cell Biol. 2001, 2, 327.
44. Agranoff, B. W. J. Biol. Chem. 2009, 284, 21121.
5.7.4. Statistical analysis
Data are expressed as a mean SEM. Data were analyzed by using
Dunnett’s test post Anova test. P values <0.05 were considered to
be significant.

50
A. Miranda-Molina et al. / Tetrahedron: Asymmetry 21 (2010) 43–50
45. Watanabe, Y.; Nakamoto, Ch.; Ozaki, S.; Sato, M.; Koizumi, K. J. Carbohydr.
Chem. 1993, 12, 685.
46. Sheldrick, G. M. Acta Cryst. 2008, A64, 112.
47. Damián-Almazo, J. Y.; Moreno, A.; López-Munguía, A.; Soberón, X.; González-
Muñoz, F.; Saab-Rincón, G. Appl. Environ. Microbiol. 2008, 74, 5168.
48. Crystallographic data (excluding structure factors) for 4a have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 737220. Copies of the data can be obtained, free
of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax:
+44(0) 336033 or e-mail: deposit@ccdc.cm.ac.uk].