Method for analysis of different oligosacchiride structures

Article abstract of DOI:10.1007/s10895-012-1102-9

In this study, an improved, rapid, high yield synthesis of N,N'-4,4'-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) is described. The obtained o-BVV is applied in a two-component saccharide sensing system (complex) where it serves as a fluoresc

Full text of DOI:10.1007/s10895-012-1102-9

J Fluoresc
DOI 10.1007/s10895-012-1102-9
ORIGINAL PAPER
Method for Analysis of Different Oligosacchiride Structures
Elena Kostadinova & Pavlina Dolashka &
Stefka Kaloyanova & Ludmyla Velkova &
Todor Deligeorgiev & Wolfgang Voelter & Ivan Petkov
Received: 24 January 2012 /Accepted: 27 June 2012
#
Springer Science+Business Media, LLC 2012
. .
Keywords Cyclodextrins Hemocyanins Fluorescence
Abstract In this study, an improved, rapid, high yield syn-
thesis of N,N’-4,4’-bis(benzyl-2-boronic acid)-bipyridinium
dibromide (o-BBV) is described. The obtained o-BVV is
applied in a two-component saccharide sensing system
.
spectroscopy Rapana venosa
(
complex) where it serves as a fluorescence quencher and
Introduction
a saccharide receptor. This system was applied to different
natural oligosaccharides isolated from molluscan Rapana
venosa (RvH1-a) and arthropodan Carcinus aestuarii
The immune system is one of the most commonly studied in
which glycans and glycoproteins play an important physio-
logical role. Therefore, saccharide sensing is a current topic
of interest in molecular recognition chemistry in determin-
ing saccharide concentrations in physiological fluids and
clarifying the functions of glycoproteins at biomembranes
(
CaeH) hemocyanins (Hcs) and cyclodextrins (CDs). The
carbohydrate contents of both Hcs were calculated in our
previous work to be 1,6 % and 7 % for CaeH and RvH1-a,
respectively. We propose that the difference in fluorescence
increase of the native CaeH and RvH1-a when titrating them
with the complex is due to the fact that the carbohydrate
content of CaeH is lower and the carbohydrate chains are
buried in between the structural subunits of the native mol-
ecule, while the glycans of the functional unit RvH1-a are
exposed on the surface of the molecule leading to a 4-fold
fluorescence’s intensity change.
[
1, 2]. There are several methods to identify oligosaccharide
structures, as well as glycosylation patterns on cells, tissues
and body fluids [3–5]. Although several types of saccharide
receptors applying different recognition mechanisms are
available, the most well-understood receptors are based on
arylboronic acids [5–10] because of their favourable stabil-
ity in water, and easy detectable UV-VIS and fluorescence
signals. In contrast to boronic acid-based saccharide recog-
nition systems consisting of a single detector, we provide in
this communication a two-component sensing system [11]
comprising an anionic fluorescent dye (1) and a boronic
acid-appended cationic viologen (2) (Fig. 1) that serves as
fluorescence quencher and saccharide receptor [12–15].
Cyclodextrins (CDs) are a family of cyclic oligosacchar-
ides, composed of α-1,4-linked glucopyranose subunits
[16–18]. CDs are produced from starch by enzymatic deg-
radation. α-Cyclodextrin (α-CD), β-cyclodextrin (β-CD)
and γ-cyclodextrin (γ-CDs) are the most common CD spe-
cies, referred to as first generation parent CDs (six, seven
and eight glucosyl units, respectively). Generally, CDs can
form host-guest complexes with a large variety of solid,
liquid, and gaseous organic compounds by a molecular
inclusion phenomenon. This inner inclusion exerts a pro-
found effect on the physicochemical properties of the guest
:
:
:
E. Kostadinova S. Kaloyanova T. Deligeorgiev I. Petkov
The Sofia University, Faculty of chemistry,
1
James Bourchier Blvd.,
164 Sofia, Bulgaria
1
:
P. Dolashka (*) L. Velkova
Institute of Organic Chemistry, Bulgarian Academy of Sciences,
G. Bonchev 9,
1
113 Sofia, Bulgaria
e-mail: pda54@abv.bg
P. Dolashka
e-mail: dolashka@orgchm.bas.bg
W. Voelter
Interfacultary Institute of Biochemistry, University of Tubingen,
Hoppe-Seyler-Strasse 4,
7
2076 Tubingen, Germany

J Fluoresc
Fig. 1 Proposed mechanism of
saccharide detection:
saccharide-induced dissociation
of ground-state complex results
in fluorescence increase
OH
OH
B
O
B
OH
O
O S
OH
N
N
3
O S
OH
3
N
N
saccharide
+
H O
2
O S
SO3
3
O S
3
SO3
OH
OH
B
O
O
B
OH
1
2
Weak Fluorescence
Strong Fluorescence
molecules as they are temporarily locked or caged within the
host cavity, giving rise to beneficial modifications on the
guest molecule’s properties (solubility, reactivity, volatility)
well as for facilitating studies on the effects of glycosyla-
tion. Therefore, we represent herein the application of a
complex between an anionic fluorescent dye and a boronic
acid-appended cationic viologen, serving as a fluorescence
quencher and a saccharide receptor.
[
19]. Therefore, the native CD modifications are effective
templates for generating wide ranges of molecular hosts [20]
and are employed as carriers for biologically active substan-
ces [21], enzyme models [22], separating agents [23], cata-
lysts [24], mass transfer promoters [25], additives in
perfumes, cosmetics, aliments or food [26], environmental
protection agents [27], or sensors for organic molecules
A literature survey revealed two main synthetic proce-
dures to obtain N,N’-4,4’-bis(benzyl-2-boronic acid)-bipyr-
idinium dibromide, which differ in used solvents and
reaction temperatures. The described methods for quaterni-
zation of 4,4′-bipyridine with o-bromomethylphenylboronic
acid were carried out in different solvents—in methanol at
room temperature for 15 h [40] or in DMF by heating to 70 °C
for 48 h [41]. The most frequently described and cited
method applies stirring for 15 h at room temperature in an
inert atmosphere, followed by a multistep and laborious
isolation procedure [40]. In order to shorten and improve
the synthetic procedure as well as to avoid the use of a
solvent, we simply melted the starting materials and so the
reaction temperature was determined by the melting points
of the starting compounds. It was found that if the tem-
perature of the reaction mixture is below 100 °C, this
does not allow obtaining of a homogenous melt at low
reaction rate. Heating of the reaction mixture to 100–110 °C
for 20 min (Fig. 2) was found to be optimal, giving a final
product in high purity and excellent yield by TLC monitoring.
[
28].
Hemocyanins (Hcs) are high molecular mass oxygen-
transporting proteins, freely dissolved in the hemolymph
of several arthropods and molluscs. Arthropodan Hcs occur
as hexamers or multiples of hexamers (up to 8×6-mers) of
approximately 75 kDa subunits, each containing a Cu(I) pair
able to reversibly bind dioxygen [29, 30]. Molluscan Hcs, in
contrast, are decamers or di-decamers of approximately
3
50–450 kDa subunits, and the (di)decameric structure has
the shape of a hollow cylinder [31, 32]. Each structural
subunit is organized by seven or eight globular functional
units (FUs) of approximately 50 kDa. These FUs contain
one dioxygen-binding Cu(I) pair and are assigned by the
letters a–g (h) starting from the N-terminus [31–34]. Beside
the quaternary structure, there are also large differences in
the carbohydrate content and monosaccharide composition
of Hcs from arthropods and molluscs. The carbohydrate con-
tent of arthropodan Hcs is relatively low (0.1–2 %, w/w) [35],
while the molluscan Hcs usually have a higher carbo-
hydrate content (2–9 %, w/w) and may contain unusual
monosaccharides [36–39].
1
The structure was confirmed by elemental analysis and H
NMR spectroscopy.
The absorbance spectrum of pyrinine shows only one
maximum at 432 nm and two maxima were identified at
440 nm and 470 nm after titration with o-BBV (Fig. 3). For
our fluorescence spectroscopic measurements, a solution of
−6
pyranine (1.33×10 ) in 50 mM phosphate buffer, pH 7.5,
was titrated with increasing amounts of the o-BBV and a
decrease (4-fold) in fluorescence emission of pyranine
Results and Discussion
(
λem0508 nm, λ 0460 nm) was observed (Fig. 4). The
ex
New methods for analyzing glycans and providing informa-
tion about the glycosylation patterns on cells, tissues and in
body fluids, are needed for diagnostics and treatment, as
fluorescence quantum yield (Φ) of pyranine in our instru-
mental setup in aqueous solution has a value of 0.89. After
titration of pyranine with o-BBV, a complex formation has

J Fluoresc
Br
Br
N
N
CH3
CH Br
N
N
Br2
2
CCl4
o
B(OH)2
B(OH)2 100-110 C
20 min
B(OH)2
(HO)2B
1
2
3
Fig. 2 Synthesis of o-bromomethylphenylboronic acid (2) and N,N’-bis-(benzyl-2-boronic acid)-[4,4′]bipyridinium dibromide (o-BBV, 3)
occurred, which cause reduction of the pyranine’s quantum
yield up to 0.18.
similar behaviour towards the used complex (Fig. 5, insert).
This lack of significant changes in the fluorescence intensity
could be partially explained with the structure of these cyclic
In the proposed mechanism in Fig. 1, the electrostatic
association of the pyranine and the quencher results in
ground-state complex formation, facilitating electron trans-
fer from the dye to the viologen, which leads to a decrease in
fluorescence intensity [42, 43]. When different saccharides
are added to the system, formation of two anionic boronate
esters effectively neutralize the dicationic viologen, thus
greatly diminishing its quenching efficiency, and an increase
in the fluorescence intensity of the dye is observed. Fluo-
rescence modulation is therefore directly correlated with
saccharide concentration. Verification of this method
includes titration of the above mentioned complex with
three types of cyclodextrins, namely α-CD, β-CD and
acetyl- β-CD, as well as two different types of hemocyanins
from molluscs and arthropods. All of the above mentioned
components contain carbohydrates with different structures.
The experiment was started with titration of the simplest
samples, namely α-CD, β-CD and acetyl-β-CD with the
above described complex and the effect was estimated by
fluorescence spectroscopy. Тhe interaction of the complex
with the hydroxyl groups in α-CD is represented by the
change of fluorescence intensity (arbitrary units, a.u.) as it is
shown on Fig. 5. Increase in the concentration of the titration
agent does not lead to a significant change in the fluorescence
intensity (12 a.u.) of the pyranine and the quantum yield has
slightly increased from 0.18 to 0.21. The same results were
observed for the other two cyclodehtrins, β-CD and acetyl-β-
CD (results are not shown). All three tested cyclodextrins have
carbohydrates (Fig. 6).
4
As a consequence of the C conformation of the gluco-
1
pyranose units, all secondary hydroxyl groups are situated
on one of the two edges of the ring, whereas all the primary
ones are placed on the other edge. The ring, in reality, is a
conical cylinder, which is frequently characterized as a
Fig. 4 a Fluorescence emission spectra of pyranine (1.33×10− M in
6
5
0 mM phosphate buffer, pH 7.5, λex0460 nm and λem0508 nm) with
−
5
increasing concentrations (0.0–1,67×10 M) of o-BBV; b Relation-
ship between the fluorescence intensity and the concentration of the
titration agents
−5
Fig. 3 UV–absorption spectra of pyranine (1×10 M) (- - - - - - -) and
pyranine (1×10− M) with o-BBV (3×10 M) (—)
5
−4

J Fluoresc
doughnut or wreath-shaped truncated cone. The cavity is
lined by the hydrogen atoms and the glycosidic oxygen
bridges, respectively. The C-2-OH group of one glucopyr-
anoside unit can form a hydrogen bond with the C-3-OH
group of the adjacent glucopyranose unit. In the CD mole-
cule, a complete secondary belt is formed by these hydrogen
bonds; therefore the β-CD forms a rather rigid structure.
This intramolecular hydrogen bond formation is probably
the explanation for the observation of very low fluorescence
effect of all CDs. Moreover, the number of free hydroxyl
groups is limited effectively neutralizing the dicationic viol-
ogen, resulting in very low fluorescence intensity of the
pyranine.
Our new carbohydrate determination method was tested
with glycoproteins, hemocyanins, with different amount and
structure of the glycans. Molluscan and arthropodan hemo-
cyanins are giant oxygen-transporting glycoproteins which
have the same function, but quite different structure
[
32–34]. Arthropodan Hcs exhibit unusual assemblies of
up to 48 structural subunits with molecular masses of
5 kDa. Hemocyanin from crab Carcinus aestuarii (CaeH)
7
is constituted of three major and two minor structural sub-
units with molecular masses of 75 kDa [35, 43]. The glyco-
sylation analyses of Carcinus hemocyanin were reported
which showed that the carbohydrate of native CaeH repre-
sents only 1.6 % of protein mass [35]. Therefore, this
hemocyanin was chosen to test the utility and reliability of
our new carbohydrate determination method.
A change in the fluorescence intensity with only 30 a.u.
and the quantum yield with 0.064 during titration of the
complex with different concentrations of CaeH is shown on
Fig. 7. These data are comparable with those of the cyclo-
dextrins’ fluorescence intensity changes and could be
explained with the oligosaccharide’s structure of CaeH:
Fig. 5 a Fluorescence emission spectra of the complex (pyranine
1.33×10− M) in presence of o-BBV) upon addition of α-CD with
6
(
−5
increasing concentrations (0.0–8×10 M); b Relationship between the
fluorescence intensity and the concentration of the titration agents:
(─•─) α-CD; (─■─) β-CD and (─▲─) acetyl- β-CD
Fig. 6 Common structures of α-CD with 6 Glu, β-CD with 7 Glu and γ-CD with 8 Glu

J Fluoresc
Fig. 8 Fluorescence emission spectra of the complex of pyranine
−
6
(
1.33×10 M) in presence of o-BBV, upon addition of increasing
−
4
concentrations (0.0–1.4×10 M) of RvH1-a
complex pyranine (1.33×10− in 50 mM phosphate buffer,
pH 7.5) and o-BBV, was tested using RvH1-a, a functional
unit of the structural subunit RvH1, the carbohydrate con-
tent of which is known [44]. Several techniques were ap-
plied, including capillary electrophoresis, matrix-assisted
laser desorption ionization MS, and electrospray ionization
MS in combination with glycosidase digestion, to determine
a carbohydrate content for RvH1-a of 7 % and structures for
its N-glycosidically-linked carbohydrates as follows :
6
Glycan 1:
Fig. 7 a Fluorescence emission spectra of the complex (pyranine
1.33×10− M) in presence of o-BBV) upon addition of increasing
6
(
−4
concentrations (0–0.47×10 M) of CaeH; b Relationship between the
fluorescence intensity and the concentration of the titration agents:
CaeH (─♦─) and RvH1-a (─■─)
Glycan 2:
Two short O-glycan sequences with suggested structures (N-
Acetyl-O-NeuAc Gal GalNAc and N-Acetyl-O-di-NeuAc
Based on the models of several FUs from different mol-
luscan Hcs, we suggest that both glycans are exposed on the
surface of the molecule. Thus their hydroxyl groups are not
involved in any interactions, such as van der Waals forces or
hydrogen bonds and as a consequence they can be easily
accessed by the complex. This leads to a 4-fold increase in
fluorescence intensity.
2
3
2
2-
Gal GalNAc ) and one N-glycan sequence (SO Man Glc-
2
2
4
4
NAc ) were identified by MALDI-MS analyses and
3
enzymatic digestions [35]. Carbohydrate groups of these gly-
cans most probably are buried in between the structural sub-
units of the global protein CaeH and interactions through van
der Waals forces or hydrogen bonds may be involved, prevent-
ing the access of the complex to the hydroxyl groups.
In contrast to this result, titration of the functional unit
RvH1-a of the molluscan hemocyanin Rapana venosa with
the complex leads to 4-fold increase in arbitrary units (a.u.)
of fluorescence intensity and 0.46 units in the quantum yield
Conclusion
According to these preliminary results we suggest that our
invention provides a new tool to determine accessible free
hydroxyl groups of carbohydrates attached to glycoproteins,
glycolipids and proteoglycans which are not buried within
the macromolecular structure or fixed by hydrogen bridges.
This kind of information may be of enormous value study-
ing carbohydrate-based interaction between biomolecules or
cells. Studies in our laboratories are under way testing
(
Fig. 8). Hemocyanins of molluscs are high molecular mass
glycoproteins (9,000 kDa) with a complex quaternary struc-
ture organized by ten structural subunits of RvH1 and
RvH2, arranged by 8 FUs [31–33]. The oligosaccharide
structures of both structural subunits and of some FUs are
very well studied [38]. Therefore, the reactivity of the

J Fluoresc
oligosaccharides and glycoproteins of different structure to
develop a more quantitative test system.
analysis calcd for C H B Br N O : C, 49.20; H, 4.13; N,
4.78. Found: C, 48.89; H, 3.92; N, 4.56.
2
4
24
2
2 2 4
Isolation of FU of Hemocyanins
Materials and Methods
The glycosylated functional unit RvH1–a was isolated via
an anion-exchange chromatography column on a FPLC
system, as described by Dolashka et al [34]. Native Hc from
the crab C. aestuarii was prepared from the hemolymph
obtained from the dorsal lacuna of living animals collected
in the lagoon of Venice as reported by Dolashka et al [35].
All starting materials and solvents were commercial prod-
ucts purchased from Sigma-Aldrich (Germany). 2-
Bromomethylphenylboronic acid was synthesized by mod-
ification of the reported procedure [40]. Analytical samples
of the reaction products were obtained by recrystallization in
ethanol. All products were characterized and compared with
1
reported data. H NMR spectra were obtained on a Bruker
Fluorescence Measurements
Advance II 600 MHz instrument in CDCl as solvent. Ele-
3
mental analyses were performed on a Vario III apparatus.
Melting points were determined on a Kofler apparatus and
are uncorrected.
Absorbance spectra were recorded in a Schimadzu spectrom-
−5
eter of (1×10 M) pyranine and after titration of pyranine
−4
with o-BBV (3×10 M). Fluorescence measurements were
carried out on a spectrofluorimeter Jasco FP-6600. The emis-
−6
Synthesis of 2-Bromomethylphenylboronic Acid
sion spectra of pyranine (1.33×10 ) in 50 mM phosphate
buffer, pH 7.5, were measured between 460 and 650 nm, with
excitation wavelength at 460 nm in a 1 cm quartz cuvette upon
addition of samples with different concentrations. For CaeH
and RvH1-a the concentration range was within 0–1×
Synthesis of 2-Bromomethylphenylboronic acid was carried
out as shown in Fig. 2. To a solution of 2-methylphenylboronic
acid (1) (1 g, 0.0073 mol) in CCl (20 ml) was added dropwise
4
−4
−6
a solution of Br (1.2 g, 0.0075 mol) in CCl (3 ml). The
2
4
10 mol/l and for cyclodextrin 0–8×10 mol/l . All studies
reaction mixture was stirred and irradiated with an UV lamp
for 1 h at room temperature and then refluxed for 2 h. The
progress of the reaction was monitored by TLC (Merck F 254
silica gel; dichloromethane). After cooling, the resulting pre-
cipitate was filtered off from the reaction mixture, the filtrate
concentrated under reduced pressure to give a yellowish-
brown oil (2), which became solid after few days of storage
in a refrigerator. The product was used without further purifi-
cation. Yield 82 %. m.p. 156–159 °C (lit. m.p. 158–162 °C)
were carried out under ambient conditions (25°C, in air).
Acknowledgments This work was supported by a research grant by
the Bulgarian National Science Fund TK01-496/2009 and DFG-STE
1
819/5-1/2012 (Germany). P. Dolashka and E. Kostadinova thank to
German Academic Exchange Service (DAAD) for financing this study.
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