A rigid lanthanide binding tag for NMR structural analysis of carbohydrates

Article abstract of DOI:10.1039/c1cc11860a

The first synthesis of a carbohydrate molecule covalently bound to a rigid lanthanide binding tag is reported. This derivative has been designed as a new tool to provide long-range restraints for structural elucidation and molecular recognition studies of carbohydrates, thus extending the methodology already developed for proteins.

Full text of DOI:10.1039/c1cc11860a

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COMMUNICATION
A rigid lanthanide binding tag for NMR structural analysis of
carbohydrateswz
c
Alvaro Mallagaray,a Angeles Canales,b Gema Domı
´
Javier Perez-Castells*
´
nguez,a Jesu
´
´
s Jimenez-Barbero* and
a
Received 1st April 2011, Accepted 28th April 2011
DOI: 10.1039/c1cc11860a
The first synthesis of a carbohydrate molecule covalently bound
to a rigid lanthanide binding tag is reported. This derivative has
been designed as a new tool to provide long-range restraints for
structural elucidation and molecular recognition studies of
carbohydrates, thus extending the methodology already developed
for proteins.
fertilization, hormonal activities, cell proliferation, invasion
and attachment of pathogens, inflammation, metastasis, and
organization into specific tissues.7 Therefore, the knowledge
of the 3D structure of carbohydrates and the fine details of
their interaction features is a matter of high interest.8 Despite
the inherent difficulty of NMR studies of carbohydrate
molecules due to overlapping problems, even at high magnetic
fields, a variety of NMR methods are available to unravel the
three-dimensional structure of carbohydrates in their free and
bound states.9 However, in many instances, access to comple-
mentary and easy-to-get information on conformational and
molecular recognition aspects may be of interest. On this basis,
we herein present a novel approach to get structural information
on these molecules by employing paramagnetic tags attached
to sugar moieties.10 As exposed above, PCS and RDC
may give crucial information for spectral assignment, confor-
mational details and molecular recognition features.11
Paramagnetic metal ions cause paramagnetic shifts, enhanced
nuclear relaxation, and molecular alignment in the presence of
magnetic fields.1 In particular, residual dipolar couplings
(RDCs), paramagnetic relaxation enhancement (PRE) and
pseudocontact shifts (PCS) have been applied for structural
biology studies of proteins and protein–ligand complexes,
under different experimental conditions.2 Moreover, lanthanides
are suitable for these studies as they bear varied paramagnetism.3
If coordinated to the biomolecule, detailed information about
the spatial anisotropy of the paramagnetic effects induced by
different lanthanide ions can be obtained.4
To show the feasibility of this idea, we selected a structurally
simple carbohydrate, 1-b-aminochitobiose, and designed a
derivative consisting of a rigid linker attached to the anomeric
amino group, which ended in an EDTA chelating unit. As the
rigid linker, we decided to use a biphenyl unit, as it presents a
length of 7.1 A between the extreme carbons, which will situate
the metal at more than 10 A from the carbohydrate in the final
complex, thus minimizing PREs. The synthesis of this derivative
was planned in a convergent way. 1-b-Aminochitobiose was
obtained by synthesizing heptaacetylated 1-b-aminochitobiose
from chitin followed by deacetylation.12
The metal may be coordinated to the metal binding site of
metalloproteins or chelated to an EDTA moiety which is
attached to a cysteine residue.5 In order to decrease the effect
of the paramagnetic relaxation enhancement (PRE), the metal
can be separated from the biomolecule by a rigid structure. Rigid
positioning of the metal is needed to avoid drastic reduction
of PCS due to motional averaging. Various different linkers
have been designed, and applied to structural and molecular
recognition studies of proteins and their complexes.6
Herein, we show that analogous methods can be extended to
study carbohydrates and their complexes to receptor molecules.
Carbohydrate interactions are involved in a wide variety of
biological cell–cell recognition events such as embryogenesis,
The biphenyl linker 3 was synthesized through the Suzuki
coupling of p-aminoboronate 1 and protected p-iodobenzoic
acid 2. The protecting group selected for the carboxylic acid
was trimethylsilylethyl (TMSE), because this group can be
selectively cleaved in the presence of tert-butylcarboxylates
and is not cleaved under Suzuki conditions. The synthesis
of 1 from p-iodoaniline was carried out with 5 mol% of
PdCl2(dppf) and 3 equiv. of triethylamine for 15 h (80%
yield).13 With this intermediate in hand we carried out the
Suzuki coupling following literature conditions.14 Yield of the
desired biphenyl 3 was 75% thus completing a global yield of
60%. The synthesis of the chelating unit 4 was accomplished
from (S)-2,3-diaminopropionic acid hydrochloride, which was
prepared in the enantiomerically pure way according to Rao’s
method from L-aspartic acid.15 From this acid an exhaustive
a
´
Facultad de Farmacia, Dpto. Quımica, Universidad San Pablo CEU,
Urb. Monteprıncipe, ctra. Boadilla km 5,300 Boadilla del Monte,
´
28668 Madrid, Spain. E-mail: jpercas@ceu.es
b
´
´
Dpto. Quımica Organica I, Facultad de Ciencias Quimıcas,
Universidad Complutense de Madrid, Avd. Complutense s/n, 28040,
Madrid, Spain
c Departamento de Biologı´a Fı´sico-Quı´mica,
´
Centro de Investigaciones Biologicas, CSIC E-28040 Madrid, Spain.
E-mail: jjbarbero@cib.csic.es
w This article is part of the ‘Glycochemistry and glycobiology’
web-theme issue for ChemComm.
z Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc11860a
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7179–7181 7179

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Scheme 1
alkylation with tert-butyl bromoacetate in the presence of
Pseudocontact shifts (PCS) measurements: pseudocontact
N-ethyldiisopropylamine, following Griesinger’s methodology,5
and subsequent hydrolysis of the crude mixture with LiOH
provided the carboxylic acid 4 in 35% yield for the two steps.
Condensation of 4 with compound 3 using HATU as the
condensation reagent and selective hydrolysis of the TMSE
group afforded 5 with a global yield of 70%. Finally,
1-b-aminochitobiose was condensed with 5, under similar
conditions as for previous condensation and upon hydrolysis
of the tert-butylesters the final carbohydrate derivative 6
was obtained. All the condensations and hydrolysis steps
proceeded with high yields (Scheme 1).
shifts arise from dipolar interactions between the unpaired
electrons of a metal ion and the nuclei in the vicinity of this
ion. PCS give rise to large changes in chemical shifts that
decrease with the distance between the metal ion and the
nuclear spin with a 1/r3 dependence. Therefore, PCS provide
long-range structural information. PCS are measured by
comparison of the spectrum of a complex with a diamagnetic
ion (La3+, Lu3+) with a spectrum of a complex with a
paramagnetic ion (Dy3+, Tb3+). Superimposition of the
1H-13C HSQC spectra acquired for the carbohydrate derivative
6 in the presence of La3+ (in red) and Tb3+ (in blue) can be
seen in Fig. 2 (ratio 6 : Tb3+; 2 : 1).
Paramagnetic relaxation enhancements (PREs): 1D proton
NMR spectra were collected for the carbohydrate derivative 6
in the presence of increasing amounts of Tb3+. Addition of the
paramagnetic ion gave rise to line broadening of all the proton
signals of the molecule (Fig. 1). The aromatic signals almost
disappeared when the titration reached the 1 : 1 ratio. Tb3+
broadened the NMR signals of the anomeric proton closer to
the metal (H-1) by ca. 40 Hz while the sugar signal at the
further distance of the metal (H-40) showed a line width
increase of 8 Hz.
Clear shifts were observed due to the paramagnetic ion.
1H NMR pseudocontact shifts assumed values ranging from
ꢀ0.17 to 0.73 ppm. Shifts of ꢀ0.14 and ꢀ0.15 ppm are
detected even for those carbohydrate protons located at higher
distances from the metal, H30 and H40, respectively (Table 1).
It is worth noting that the protons with positive PCS belong to
Fig. 1 Titration 1H NMR study of 6 with Tb3+. Three different
metal : ligand molar ratios are shown, namely 1 : 1, 1 : 2 and 1 : 3.5.
The bottom spectrum was acquired in the presence of La3+
Fig. 2 1H-13C HSQC spectra of 6 acquired under the isotropic
.
conditions (red) and anisotropic conditions (blue).
c
7180 Chem. Commun., 2011, 47, 7179–7181
This journal is The Royal Society of Chemistry 2011

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Table 1 Pseudocontact shifts for 6 in the presence of Tb3+ ions
(2 : 1 molar ratio) at 600 MHz
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ꢀ0.14
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ꢀ0.05
ꢀ0.01
ꢀ0.16
ꢀ0.14
ꢀ0.15
ꢀ0.14
0.15
H60a
H60b
H6a
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H50
H30
H40
CH3 20
CH3 2
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1-b-amino-N-acetylglucosamine unit). In contrast, the protons
of the O-linked acetylglucosamine (with primes) showed
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The synthesis of a sugar molecule containing a rigid
lanthanide binding tag has been achieved. A linker with a
fixed distance and orientation between the metal and the
carbohydrate unit has been employed that should provide
additional parameters for structural, conformational and
molecular recognition studies. PCS and PRE have been
observed, providing structural information for the different
signals. The linker protons are the ones whose signals
disappear from the spectra due to the fast relaxation of the
metal in the paramagnetic complexes. In addition, PCS could
be measured for all the carbohydrate signals. Therefore, this
linker and structural variations thereof could be used for the
study of larger carbohydrates and their interactions with
proteins. Further studies on this topic are currently underway.
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7179–7181 7181