Molecular design and biological potential of galacto-type trehalose as a nonnatural ligand of shiga toxins.

Article abstract of DOI:10.1021/ol017135+

Galacto-type trehalose, a "C-4 epimer of trehalose", possesses a stereochemical structure around the alpha(1-1)-linkage analogous to that of the globobiosyl alpha(1-4)-linkage in Gb(2) and Gb(3) ceramides, which are known as the ligands of Shiga toxins produced by pathogenic E. coli. This paper presents evidence supporting the new idea of using a trehalosyl alpha(1-1)-linkage as a substitute for the galactobiosyl alpha(1-4)-linkage.

Full text of DOI:10.1021/ol017135+

ORGANIC
LETTERS
2002
Vol. 4, No. 3
355-357
Molecular Design and Biological
Potential of Galacto-Type Trehalose as a
Nonnatural Ligand of Shiga Toxins
,†
Hirofumi Dohi,† Yoshihiro Nishida,* Yuki Furuta,† Hirotaka Uzawa,‡
Shin-ichiro Yokoyama,§ Saori Ito,§ Hiroshi Mori,§ and Kazukiyo Kobayashi*
,†
Department of Molecular Design and Engineering, Graduate School of Engineering,
Nagoya UniVersity, Chikusa-ku, Nagoya 464-8603, Japan, National Institute of
AdVanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba,
305-8565, Japan, and Department of Public Health Pharmacy,
Gifu Pharmaceutical UniVersity, Mitabora-higashi, Gifu 502-8585, Japan
h996603d@mbox.media.nagoya-u.ac.jp
Received November 27, 2001
ABSTRACT
Galacto-type trehalose, a “C-4 epimer of trehalose”, possesses a stereochemical structure around the r(1−1)-linkage analogous to that of the
globobiosyl r(1−4)-linkage in Gb2 and Gb3 ceramides, which are known as the ligands of Shiga toxins produced by pathogenic E. coli. This
paper presents evidence supporting the new idea of using a trehalosyl r(1−1)-linkage as a substitute for the galactobiosyl r(1−4)-linkage.
Shiga toxins (Stx-I and Stx-II) produced by Escherichia coli
O-157:H-7 and other pathogenic E. coli species are multi-
subunit proteins comprising one toxic A-subunit and five
receptor-binding B-subunits.1 Both Stx-I and Stx-II recognize
a galactobiosyl R(1-4)-linkage in globosyl Gb2 and Gb3
ceramides and induce carbohydrate-mediated internalization
into host cells.2 There have been extensive studies on the
molecular design of artificial Stx ligands that possess simpler
structures and higher activity for blocking the toxin-host
cell adhesion than the natural globosyl ceramides. Most of
the synthetic studies have been focused on the replacement
of the ceramide group with simpler lipids3 or on the
molecular assembly to construct carbohydrate clusters as
glycopolymers, glycodendrimers, and starfish models.4 How-
ever, an effective mimic of the galactobiosyl R(1-4)-linkage
has not yet been found. This is ironic since the synthesis of
many mimics has been hampered by the difficulty of
constructing the R(1-4)-linkage.5 In this communication, we
(3) (a) Debenham, S. D.; Cossrow, J.; Toone, E. J. J. Org. Chem., 1999,
64, 9153-9163. (b) Mylvaganam, M.; Lingwood, C. A. Biochem. Biophys.
Res. Commun. 1999, 257, 391-394. (c) Ling, H.; Boodhoo, A.; Hazes, B.;
Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J.
Biochemistry 1998, 37, 1777-1788.
† Nagoya University.
‡ National Institute of Advanced Industrial Science and Technology.
§ Gifu Pharmaceutical University.
(4) (a) Nishida, Y.; Dohi, H.; Uzawa, H.; Kobayashi, K. Tetrahedron
Lett. 1998, 39, 8681-8684. (b) Matsuoka, K.; Terabatake, M.; Esumi, Y.;
Terunuma, D.; Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839-7842. (c)
Kitov, P. I.; Sadowska, J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.;
Pannu, N. S.; Read, R. J.; Bundle, D. R. Nature 2000, 403, 669-672. (d)
Lundquist, J. J.; Debenham, S. D.; Toone, E. J. J. Org. Chem. 2000, 65,
8245-8250.
(1) (a) Kozlov, Y. V.; Chernaia, M. M.; Fraser, M. E.; James, M. N. J.
Mol. Biol. 1993, 232, 704-706. (b) Picking, W. D.; McCann, J. A.; Nutikka,
A.; Lingwood, C. A. Biochemistry 1999, 38, 7177-7184.
(2) (a) Lingwood, C. A.; Law, H.; Richardson, S.; Petric, M.; Brunton,
J. L.; Grandis, S. D.; Karamali, M. J. Biol. Chem. 1987, 262, 8834-8839.
(b) Lindberg, A. A.; Brown, J. E.; Stro¨mberg, N.; Westling-Ryd, M.; Schultz,
J. E.; Karlsson, K. A. J. Biol. Chem. 1987, 262, 1779-1785. (c) Arab, S.;
Lingwood, C. A. Glycoconjugate J. 1996, 13, 159-166.
(5) Dohi, H.; Nishida, Y.; Tanaka, H.; Kobayashi, K. Synlett 2001, 9,
1446-1448.
10.1021/ol017135+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/05/2002

propose the use of a trehalose R(1-1)-linkage as the
substitute for the galactobiosyl R(1-4)-linkage and demon-
strate the potential of this mimic.
starting from 4,6-mono-O-benzylidene trehalose 39 as sum-
marized in Scheme 1. As a key reference compound to
Trehalose 1a possesses many biological and medicinal
potentials applicable to sugar-based therapeutic reagents.6
This is mainly because the glycoside R(1-1)-linkage is
highly tolerant to both chemical and enzymatic (mammalian
R-glycosidases) degradation compared with glycosyl R(1-
4)-linkages. Here, it can be easily noticed that the trehalose
R(1-1)-linkage has a molecular topology analogous to that
of the galactobiosyl R(1-4)-linkage 2 (Gb2) with binding
activity to Shiga toxins. A critical difference arises from the
configuration at OH-4, which should be axial to be recog-
nized by Shiga toxins. Accordingly, a galacto-trehalose 1b
can be designed as a novel Gb2 mimic and expected to show
a binding affinity to Stxs similar to that of the natural Gb2
ligand as well as the advantageous property mentioned above.
Molecular force field (MM-2) calculation supported the
similarity of the stereochemical environment around the
glycoside linkage between 2 and 1b (Figure 1).
Scheme 1. Synthesis of Galacto-Trehalose Cluster Model 7a
a Reaction conditions: (a) (i) TBDPSCl, Et3N, DMAP, pyridine,
room temperature, 12 h, 91%; (ii) BnBr, NaH, DMF, room
temperature, 12 h, 95%; (iii) BH3-NHMe2, AlCl3, THF,
0 °Cfroom temperature, 2 h, 94%; (b) (i) Tf2O, pyridine, CH2Cl2,
0 °Cfroom temperature, 45 min, quant; (ii) CsOAc, 18-crown-6,
toluene, ultrasound, 40 °C, 2 h, 97%; (c) (i) NaOMe, MeOH, THF,
room temperature, 12 h, quant; (ii) TBAF, THF, room temperature,
24 h, 95%; (iii) 6-azido-1-bromohexane, TBAI, NaH, DMF,
60 °C, 12 h, 92%; (iv) Pd(OH)2/C, H2, THF, MeOH, room
temperature, 1.5 h; (v) acryloyl chloride, Et3N, CH2Cl2, MeOH,
0 °Cfroom temperature, 2 h, 91% (2 steps); (d) 2,2-azobis (2-
amidinopropane) dihydrochloride, H2O, Me2SO, 60 °C, 12 h, 72%.
b Sloping line in the structure represents the random copolymer
comprised of the two constitutional units.
Figure 1. Preferred conformations of galacto R(1-4)-bioside 2
and galacto-type trehalose 1b (1) and superimposed structures (2).
The structures were minimized by MM-2 without optimization of
all OH bonds in the calculation (Insight II/Discover program and
Amber force field); the white and blue lines represent 2 and 1b,
respectively, in panel 2.
In this study, we prepared galacto-type trehalose 67 and
its cluster model 7 in order to verify our expectation.8 The
synthesis was carried out via conventional chemical pathways
investigate the role of the OH-4 configuration, a cluster
model 10 carrying trehalose at the side chain was also
prepared in the same way (6-azidohexylation at O-6, reduc-
tion of the azido group, N-acryloylation, and copolymeriza-
tion with acrylamide in a redox radical manner) (Figure 2).
Acrylamido copolymers 8 and 9 carrying natural Gb2 and
R-D-galactoside clusters, respectively, were prepared in our
previous manner.5,10
(6) Highly effective use of an R-D-mannosyl(1-1)-â-D-galactosyl linkage
as a scaffold constructing a sialyl Lewis X mimic was reported: Hiruma,
K.; Kajimoto, T.; Schumidt, G. W.; Ollmann, I.; Wong, C.-H. J. Am. Chem.
Soc. 1996, 39, 9265-9270.
(7) Compound 6: 1H NMR (δ ppm, 500 MHz, D2O): 5.65-6.24 (dd ×
3, 3H, H-olefin), 5.15 (d, 1H, J1,2 ) 3.4 Hz, GalH-1), 5.14 (d, J1,2 ) 3.1
Hz, GlcH-1), 4.15 (d, J3,4 ) 3.0 Hz, GalH-4), 3.36 (dd, 1H, J2,3 ) 6.7 Hz,
J3,4 ) 9.0 Hz, GlcH-4), 1.40-3.50 (dd × 2 and m × 4, 12H, H-methylene).
(8) Various types of glycosylated polyacrylamides have been reported;
see: (a) Nishimura, S.-I.; Furuike, T.; Matsuoka, K.; Murayama, K.; Nagata,
K.; Kurita, K.; Nishi, N.; Tokura, S. Macromolecules 1994, 27, 4876-
4880. (b) Roy, R.; Tropper, F. D. Glycoconjugate J. 1988, 5, 203-206.
Hemagglutination inhibition assay11 was performed for
trehalose 1a, galacto-trehalose 6, p-N-acrylamidophenyl Gb2,5
(9) Richardson, A. C.; Tarelli, E. J. Chem. Soc. C 1971, 22, 3733-
3735.
356
Org. Lett., Vol. 4, No. 3, 2002

Table 1. Hemagglutination Inhibition Assay and Stx-I
Neutralization Activity Assay
MIC (M)a
neutralization activity
copolymer
Stx-I
Stx-II
against Stx-Ib
7
8
9
6.1 × 10-5
4.1 × 10-5
2.4 × 10-4
1.6 × 10-4
0.88
0.95
0.65
c
1.1 × 10-3 >1.1 × 10-3
10
>5.7 × 10-2 >5.7 × 10-2
a Minimum inhibition concentration.13 b Relative activity to that of Gb3
copolymer. c Not determined.
and 160 µM for Stx-II), the activity was apparently higher
than that of the R-galactoside copolymer 9 (MIC ) 1 mM
for Stx-I and >1 mM for Stx-II). Moreover, the trehalose
copolymer 10 did not show binding activity to Stx-I or Stx-
II. These data are suggestive of the structural characteristics
of the galacto-type trehalose 1b. The axial 4-OH in the
R-galactoside moiety is essential for the Stx binding, and
the R-glucoside moiety in 1b, similarly to the â-galactoside
moiety in 2, plays an important role as a supplementary
binding site.
Stx-I neutralization assay using HeLa cells was performed
for the binding-active copolymers 7-9. Under conditions
in which the Gb3/acrylamide copolymer10 (10 µM) can keep
all cells alive in the presence of Stx-I, galacto-trehalose
copolymer 7 could rescue 88% of the cells. The relative
activity matches well with the preceding hemagglutination
inhibition activity and also supports our expectation that the
galacto-type trehalose 1b serves as a nonnatural carbohydrate
ligand of Shiga toxins.
In conclusion, we have found a potential utility of the
galacto-trehalose R(1-1)-linkage as a promising substitute
for the globosyl R(1-4)-linkage. Though the binding of the
galacto-trehalose to Stx-I and Stx-II, particularly to the latter,
is still weaker than that of the natural Gb2 and Gb3
globosides, galacto-trehalose may integrate the binding
affinity significantly by additionally introducing a â-gluco-
side moiety or other related units. Such efforts are in progress
in our group and will be reported elsewhere. Moreover, the
idea of applying R- (or even â-) (1-1)-linked sugars, instead
of natural and labile glycosyl linkages, will open a promising
pathway leading to sugar-based therapeutic agents.
Figure 2. Structures and properties of glycoconjugate copolymers
8-10.
and p-nitrophenyl R-D-galactoside10 to show that none of
these monomeric glycosides possess notable activity to block
the Stx binding to erythrocytes (MIC > 10 mM). The result
accords well with the report12 that a monomeric methyl
globotriaoside (Gb3) has a very low binding affinity to Stx-I
(Ka < 1.0 × 103 M-1). On the other hand, activity was
observed clearly for copolymers due to a cluster effect (Table
1).13 The galacto-trehalose copolymer 7 showed blocking
activity for both Stx-I (MIC ) ca. 60 µM) and Stx-II (ca.
240 µM). Though the activity was slightly lower than that
of the natural Gb2 copolymer 8 (MIC ) 40 µM for Stx-I
Acknowledgment. Dedicated to Professor Joachim
Thiem of the University of Hamburg on the occasion of
his 60th birthday. We thank the Japan Society for the
Promotion of Science Research for a research fellowship for
young scientists (DC) for H. Dohi.
Supporting Information Available: Experimental pro-
cedures and full characterization for all new compounds
(PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
(10) Dohi, H.; Nishida, Y.; Mizuno, M.; Shinkai, M.; Kobayashi, T.;
Takeda, T.; Uzawa, H.; Kobayashi, K. Bioorg. Med. Chem. 1999, 7, 2053-
2062.
(11) Kawagishi, H.; Yamawaki, M.; Isobe, S.; Usui, T.; Kimura, A.;
Chiba, S. J. Biol. Chem. 1994, 269, 1375-1379.
(12) St. Hilaire, P. M.; Boyd, M. K.; Toone, E. J. Biochemistry 1994,
33, 14452-14463.
(13) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327.
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