13C-NMR quantification of proton exchange at LewisX hydroxyl groups in water

Article abstract of DOI:10.1039/c1cc13310a

NMR-based analysis of glycans by directly observing hydroxyl protons has been difficult because of their inherently fast exchange with water. We observed hydroxyl proton exchanges in a LewisX-LewisX interaction by using deuterium isotope shifts on ¹³

Full text of DOI:10.1039/c1cc13310a

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13C-NMR quantification of proton exchange at LewisX hydroxyl groups
in waterw
Shinya Hanashima,a Koichi Katobc and Yoshiki Yamaguchi*a
Received 3rd June 2011, Accepted 19th August 2011
DOI: 10.1039/c1cc13310a
NMR-based analysis of glycans by directly observing hydroxyl
protons has been difficult because of their inherently fast
exchange with water. We observed hydroxyl proton exchanges
in a LewisX–LewisX interaction by using deuterium isotope
shifts on 13C-NMR. This strategy is suitable for analyzing weak
interactions by identifying involved protons.
Protein–carbohydrate and carbohydrate–carbohydrate inter-
actions underlie phenomena such as cell adhesion, signal
transduction, protein folding, and immunological responses.1
The glycans participating in such important biological events
use their hydroxyl functionalities to form dynamic hydrogen-
bonding networks that determine binding rates and strength to
Fig. 1 13C-NMR isotope shifts for analyzing the proton exchange
rate of sugar hydroxyl groups. kex, exchange rates of hydroxyl protons.
The chemical shift difference is B0.15 ppm.
cognate partners. Hydrogen bonding influences the rate of
exchange of the hydroxyl protons, as does the surrounding
environment, and nearby electronegativities. Intermolecular
hydrogen-bond bridges are a major force in carbohydrate–
protein and carbohydrate–carbohydrate interactions.2
Deuterium secondary isotope shifts on 13C-chemical shifts
(deuterium isotope shifts) have been observed at the geminal
13C-signal of exchangeable hydroxyl protons (Fig. 1). Pioneering
work by Pfeffer et al. showed the deuterium-induced 13C
isotope shifts on a series of mono- and disaccharides using a
dual coaxial NMR tube.11 In the presence of H2O and D2O,
the line shape of the 13C–OH/D signal is highly dependent on the
H/D exchange rate. In a very slow H/D exchange environment,
deuterium induced 13C differential isotope shifts (DIS) are
observed based on corresponding isotopomers, 13C–OH and
13C–OD, with a 0.09–0.15 ppm difference, whereas in the case
of fast exchange, the equilibrium makes each isotopomer
indistinguishable and gives a singlet signal at an averaged
chemical shift. These properties have allowed the 13C-NMR
signal shape to be used as a good indicator of low energy barrier
hydrogen bonds.12–14 Recently, deuterium isotope shifts have
been applied to proteins to analyze the proton exchange rate at
backbone amides,15 as well as side-chain phenolic protons of
tyrosine and sulfhydryl protons of cysteine.16,17
13C-NMR has great potential because of its wide chemical shift
range and sharp signals, and is now being applied in structure
determination and dynamics of protein–ligand binding, especially
since recent advances that augment sensitivity through using a
cryogenic probe and the dynamic nuclear polarization technique.18
However, there have been few applications of deuterium
isotope shifts in the structural analysis of carbohydrates, and
those were often performed with aprotic solvents.19–21
Protein NMR spectroscopy has been developed on the basis
of observation of exchangeable backbone amides.3 The exchange
rate of the amide proton is well suited to the NMR time scale,
and this parameter has provided key information in structural and
dynamical analyses, particularly in recent NMR techniques.4,5
If the exchange of hydroxyl protons on glycans were equally
amenable to measurement, it would be a significant advance in
the structural analysis of glycans. However, the fast exchange
nature of sugar hydroxyl protons has made it difficult to
observe directly the proton NMR signals.6 To attain this in
aqueous media, NMR spectroscopy currently requires either
low temperature with co-existing organic solvents or utilization
of a capillary NMR tube.7–9 Another problem is that aprotic
organic solvents greatly influence proton exchange rates, and
the presence of an organic solvent can abolish the binding of
glycan to a cognate protein.10
a Structural Glycobiology Team, RIKEN Advanced Science Institute,
2-1 Hirosawa, Wako-shi, 351-0198 Saitama, Japan.
E-mail: yyoshiki@riken.jp; Fax: +81 48-467-9620;
Tel: +81 48-467-9619
b Graduate School of Pharmaceutical Sciences, Nagoya City
University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
c Okazaki Institute for Integrative Bioscience and Institute for
Molecular Science, National Institutes of Natural Sciences,
5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13310a
c
10800 Chem. Commun., 2011, 47, 10800–10802
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Here we demonstrate a NMR strategy using deuterium
isotope shifts for analysis of LewisX–LewisX interactions under
100% water conditions, similar to the biological environment.
We focused on fast exchange O–H groups of the LewisX glycan
because they are expected to be directly involved in stabilizing
tertiary structures and interactions with other molecules by
hydrogen bonding and/or coordinating the metallic ions.
LewisX 1, a trisaccharide of galactose (Gal), fucose (Fuc)
and N-acetylglucosamine (GlcNAc) often at the termini of
glycolipids and glycoproteins, is involved in initial cell–cell
interactions by forming a LewisX–Ca2+ complex.22 Even
though the cis- and trans-binding manner of LewisX has been
investigated using such techniques as mass spectrometry and
NMR,8,23–25 there is no consensus about the spatial atomic-
arrangements of the LewisX–Ca2+ complex in water, and
progress has been hampered by its weak binding (Kd = several
hundred mM to several M) and dynamic nature.26
We initially investigated the effect of Ca2+ on the LewisX–
LewisX interaction by performing titration experiments,
monitored by 1H–13C HSQC chemical shift perturbations
(Fig. S1, ESIw). During titration of CaCl2, chemical shift
changes were observed in all the HSQC correlation signals.
However, the pattern of the changes was not specific for
LewisX 1 because comparable perturbations were observed
with b-(OMe)-galactoside 2 and a-(OMe)-fucoside 3 (Fig. S2
and S3, ESIw). We concluded that the results indicated non-
specific binding and did not necessarily apply to the specific
groups involved in the LewisX–LewisX interaction.
Fig. 2 13C-NMR spectra of 40 mM LewisX with expanded secondary
carbon area. LewisX at 5 1C (a); LewisX with 1.0 M CaCl2 at 5 1C (b);
LewisX at À10 1C (c); LewisX with 1.0 M CaCl2 at À10 1C (d).
In contrast, we obtained the data suggestive of a LewisX–
LewisX interaction when we analyzed the interaction by
deuterium isotope shifts through evaluation of proton exchange
rates of the sugar hydroxyl groups.
splitting patterns because of the g-shifts (0.005–0.02 ppm)
arising from vicinal slow-exchanging protons at hydroxyl
functionalities. Again, this time under slow H/D exchange
conditions, the sugar hydroxyl protons attached at the carbons
giving DIS signals must be involved in the hydrogen bond
13C-NMR spectra were collected at temperatures from
À10 1C to 20 1C in 5 1C increments. The synthetic LewisX
trisaccharide 1 (40 mM) was dissolved in H2O/D2O = 50 : 50
solution with 1.0 M CaCl2 (pH 6.0), and representative 13C-
NMR spectra at 5 1C are shown in Fig. 2b and Fig. S4b (ESIw).
DIS based on the b-shifts of 13C-signals originating from
FucC2 (0.08 ppm), FucC4 (0.08 ppm), GalC2 (0.07 ppm),
GalC4 (0.11 ppm), GalC6 (0.08 ppm) and GlcNAcC2 (0.09 ppm)
were observed. In contrast, 13C-NMR spectra collected without
CaCl2 provided sets of DIS signals originating from GalC4
(0.11 ppm) and GlcNAcC2 (0.09 ppm) (Fig. 2a, Fig. S4a, ESIw).
Thus C2 and C6 of Gal, and C2 and C4 of Fuc provide DIS
only in the presence of Ca2+. b-(OMe)-galactoside 2 and
a-(OMe)-fucoside 3 were also analyzed under the same experi-
mental conditions and no significant Ca2+ effect was observed
(Fig. S6, ESIw). These results suggest that Ca2+ participates in
the LewisX trisaccharide complex formation, but has no effect on
the monosaccharide components of LewisX. Furthermore, it is
evident that all of the C–OH residues that exhibit DIS signals are
directly or indirectly involved in the LewisX–LewisX interactions.
To analyze proton-exchange under more restricted exchange
conditions, we collected 13C-NMR spectra at À10 1C using a
3 mm NMR tube.27,28 Now DIS were additionally observed
from GalC3 (0.09 ppm), FucC3 (0.07 ppm) and GlcNAcC6
(0.11 ppm), and only in the presence of 1.0 M CaCl2 (Fig. 2c
and d, Fig. S4c and S4d, ESIw). Furthermore, 13C-signals
originating from GalC2, GalC3 and FucC3 show complicated
network and/or co-ordination to Ca2+
.
The proton exchange rates of the hydroxyl groups, which
express the isotope-shifts, are estimated by applying each
deuterium isotope shift to Gutowski’s equation (Table 1 and
ESIw). The coalescence temperature of the 13C-signals originating
from GalC2, GalC6, FucC2, FucC3 and FucC4 is elevated by
more than 10 K in the presence of 1.0 M CaCl2. Stabilization of
these hydroxyl protons suggests that they play a significant role in
LewisX–Ca2+ complex formation due to their formation of inter-
and intra-molecular hydrogen-bond networks with neighboring
hydroxyl groups or through coordination with Ca2+. In addition,
GalC4 shows a partial increase in proton exchange, which might
be due to disruption of their hydrogen bonding network.
Those hydroxyl protons with significantly slow exchange
rates in the presence of Ca2+ are summarized in Fig. 3 by
showing their position on the chemical structure and tertiary
structure of LewisX.29 The slowly exchanging protons are
mostly clustered on Fuc and Gal residues, and they should play
key roles in Ca2+ binding and/or intermolecular hydrogen-
bond formations. Ca2+ belongs to Group 2 alkaline earth
metals and has significant Lewis acidity.30 The characteristic
Lewis acidity of Ca2+ can induce further electronegativity of
the coordinating oxygen, which, in our experiments, we detect
through the slow exchange rate of the hydroxyl protons. Geyer
c
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Table 1 The coalescence temperatures and proton exchange rates of
the hydroxyl proton signals
exchange rates. This simple 13C-NMR strategy is suitable for
analyzing extremely weak interactions including those between
carbohydrates, and identifies the hydroxyl protons involved in
hydrogen bonding and metal coordination.
CaCl2 (1.0 M)
No CaCl2
Tc/K
kex/sÀ1
Tc/K
kex/sÀ1
Residues
We thank Drs F. Hayashi, M. Yoshida, and C. Kurosaki
(RIKEN) for technical support of high-field NMR spectra.
We thank Dr Y. Ito (RIKEN) for advice on the synthesis. We
also thank Dr T. Suetake for the initial 13C-NMR study. This
work was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan.
Gal 2
Gal 3
Gal 4
Gal 6
Fuc 2
Fuc 3
Fuc 4
GlcNAc 6
283
263
298
278
283
273
283
268
25
27
37
32
33
23
36
39
273
o263
4298
o263
273
30
n.d.
n.d.
n.d.
42
n.d.
45
o263
273
263
32
Notes and references
13C-signals were collected from 263 K to 298 K in 5 K increments. Tc:
coalescence temperatures, kex: proton exchange rates at coalescence
temperatures calculated using Gutowski’s equation, with an overall
error of 10%. n.d.: not determined.
1 H. J. Gabius, H. C. Siebert, S. Andre, J. Jime
H. Rudiger, ChemBioChem, 2004, 5, 740–764.
2 I. Bucior andM. M. Burger, Curr. Opin. Struct. Biol., 2004, 14, 631–637.
3 A. Bax and S. Grzesiek, Acc. Chem. Res., 1993, 26, 131–138.
4 S. Grzesiek and A. Bax, J. Biomol. NMR, 1993, 3, 627–638.
´
nez-Barbero and
5 G. Wagner and K. Wuthrich, J. Mol. Biol., 1982, 160, 343–361.
¨
6 A. K. Mittermaier and L. E. Kay, Trends Biochem. Sci., 2009, 34,
601–611.
7 L. Poppe and H. van Halbeek, Nat. Struct. Biol., 1994, 1, 215–216.
8 B. Henry, H. Desvaux, M. Pristchepa, P. Berthault, Y. M. Zhang,
J. M. Mallet, J. Esnault andP. Sinay, Carbohydr. Res., 1999, 315, 48–62.
9 H. C. Siebert, M. Frank, C.-W. von der Lieth, J. Jime
´
nez-Barbero and
nez-
H. J. Gabius, in NMR Spectroscopy of Glycoconjugates, ed. J. Jime
´
Barbero and T. Peters, WILEY-VCH, Weinheim, 2003, pp. 39–57.
10 H. C. Siebert, S. Andre, J. L. Asensio, F. J. Canada, X. Dong,
J. F. Espinosa, M. Frank, M. Gilleron, H. Kaltner, T. Kozar,
´
N. V. Bovin, C. W. von Der Lieth, J. F. Vliegenthart, J. Jimenez-
Barbero and H. J. Gabius, ChemBioChem, 2000, 1, 181–195.
11 P. E. Pfeffer, K. M. Valentine and F. W. Parrish, J. Am. Chem.
Soc., 1979, 101, 1265–1274.
12 T. W. Marshall, Mol. Phys., 1961, 4, 61–63.
Fig. 3 The hydroxyl protons whose exchange rate slows in the
presence of Ca2+ are indicated in gray circles (A) as well as large
white balls (B) on the crystal structure of the LewisX trisaccharide.29
and coworkers analyzed a cis-interaction mode of LewisX using
a methylene-bridged LewisX dimer (LewisXa–CH2–LewisXb)
attached at the 6-position of GlcNAc, and their model study
suggested that Ca2+ is pentavalently co-ordinated at O2,3 of
Gala, O6 of Galb, and O2,3 of Fucb.31 These residues are
included in our analyses. The slow proton exchange rates of
GalC2,3,6 and FucC2,3 hydroxyl groups, which we observed
in the presence of Ca2+, would be at least partially due to
co-ordination with Ca2+. Further, our data indicate that
Ca2+ slows the proton-exchange of the hydroxyl groups of
GlcNAcC6 and FucC4. They might also coordinate the Ca2+
in water since the large ionic radius of this metal ion allows for
octavalent coordination,32 or they could form intermolecular
hydrogen-bonds with another LewisX molecule or with the
surrounding water. The hydrogen bonds produced upon an
addition of Ca2+ would be in an intermolecular manner
because no significant structural change has been observed
upon addition of Ca2+. This information will be of great value
in elucidating the interaction mode of LewisX glycans, which
cannot be easily provided by other conventional NMR methods
e.g. in a typical chemical shift perturbation experiment.
We describe a 13C-NMR technique that analyzes LewisX–
LewisX interactions through measurement of proton-exchange
rates under physiological conditions. The technique is able to
quantify the proton exchange of hydroxyl groups on glycans in
100% water, in the ten to hundred milliseconds range. It
revealed that Ca2+ slows the rates of H/D exchange of several
hydroxyl protons of LewisX. These groups must be involved
in the LewisX–LewisX interaction. Our approach not only
observes hydroxyl protons but also precisely evaluates their
dynamics through measurement of the small difference in their
13 T. Dziembowska, P. E. Hansen and Z. Rozwadowski, Prog. Nucl.
Magn. Reson. Spectrosc., 2004, 45, 1–29.
14 S. N. Smirnov, N. S. Golubev, G. S. Denisov, H. Benedict,
P. Schah-Mohammedi and H.-H. Limbach, J. Am. Chem. Soc.,
1996, 118, 4094–4101.
15 K. Uchida, J. L. Markley and M. Kainosho, Biochemistry, 2005,
44, 11811–11820.
16 M. Takeda, J. Jee, T. Terauchi and M. Kainosho, J. Am. Chem.
Soc., 2010, 132, 6254–6260.
17 M. Takeda, J. Jee, A. M. Ono, T. Terauchi and M. Kainosho,
J. Am. Chem. Soc., 2009, 131, 18556–18562.
18 J. H. Ardenkjaer-Larsen, B. Fridlund, A. Gram, G. Hansson,
L. Hansson, M. H. Lerche, R. Servin, M. Thaning and
K. Golman, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 10158–10163.
19 J. Reuben, J. Am. Chem. Soc., 1984, 106, 6180–6186.
20 J. C. Christofides and D. B. Davies, J. Am. Chem. Soc., 1983, 105,
5099–5105.
21 K. Bock and R. U. Lemieux, Carbohydr. Res., 1982, 100, 63–74.
22 I. Eggens, B. Fenderson, T. Toyokuni, B. Dean, M. Stroud and
S. Hakomori, J. Biol. Chem., 1989, 264, 9476–9484.
23 G. Siuzdak, Y. Ichikawa, T. J. Caulfield, B. Munoz, C. H. Wong
and K. C. Nicolaou, J. Am. Chem. Soc., 1993, 115, 2877–2881.
24 C. Gege, A. Geyer and R. R. Schmidt, Eur. J. Org. Chem., 2002,
2475–2485.
25 G. Nodet, L. Poggi, D. Abergel, C. Gourmala, D. X. Dong,
Y. M. Zhang, J. M. Mallet and G. Bodenhausen, J. Am. Chem.
Soc., 2007, 129, 9080–9085.
26 A. Geyer, C. Gege and R. R. Schmidt, Angew. Chem., Int. Ed.,
1999, 38, 1466–1468.
27 J. L. Mills and T. Szyperski, J. Biomol. NMR, 2002, 23, 63–67.
28 J. J. Skalicky, J. L. Mills, S. Sharma and T. Szyperski, J. Am.
Chem. Soc., 2001, 123, 388–397.
29 S. Perez, N. Mouhous-Riou, N. E. Nifant’ev, Y. E. Tsvetkov,
B. Bachet and A. Imberty, Glycobiology, 1996, 6, 537–542.
30 S. Kobayashi and Y. Yamashita, Acc. Chem. Res., 2011, 44, 58–71.
31 A. Geyer, C. Gege and R. R. Schmidt, Angew. Chem., Int. Ed.,
2000, 39, 3245–3249.
32 W. J. Cook and C. E. Bugg, Carbohydr. Res., 1973, 31, 265–275.
c
10802 Chem. Commun., 2011, 47, 10800–10802
This journal is The Royal Society of Chemistry 2011