Discrimination of Saccharides by a Simple Array

Article abstract of DOI:10.1002/chem.201700831

We report the development of a two-component probe system as fluorescence turn-on assay of simple saccharides. The quenching of an anionic conjugated water-soluble polymer by a cationic quencher has been reported previously. Three different boronic acid f

Full text of DOI:10.1002/chem.201700831

A Journal of
Accepted Article
Title: Discrimination of Saccharides by a Simple Array
Authors: Uwe Heiko Bunz, Max Bojanowski, Markus Bender, and Kai
Seehafer
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700831
Link to VoR: http://dx.doi.org/10.1002/chem.201700831
Supported by

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
Discrimination of Saccharides by a Simple Array
N. Maximilian Bojanowski,[a] Markus Bender,[a] Kai Seehafer[a] and Uwe H. F. Bunz[a,b]
*
Abstract: We report the development of a two component probe
system as fluorescence turn-on assay of simple saccharides.
The quenching of an anionic conjugated water-soluble polymer
by a cationic quencher has been reported previously. Three
different boronic acid functionalized benzyl viologens and three
conjugated polymers of the poly(aryleneethynylene) type form
nine non-fluorescent complexes. This small library discriminates
nine different simple saccharides in aqueous solutions by
fluorescence turn-on in a displacememt assay. The saccharides
can be discriminated and identified with this simple system.
to give a non-fluorescent construct (Figures 1 and 2). Upon
binding to fructose, galactose or glucose, the quencher is
liberated from the anionic polyelectrolyte and the polymer
solution experiences a turn-on in fluorescence. While
sugars and saccharides are neutral, the corresponding
boron-based chelates probably pick up water and turn into
a negatively charged borate complex, counteracting the
electrostatic interaction between the paraquat and the
anionic polymer. Scheme
mechanism.
1
shows the simplified
Introduction
We have prepared a small self-assembled library consisting
of nine elements, consisting of pairwise combinations of
three boronic acid quenchers and three conjugated
polymers. The library discriminates simple saccharides in
aqueous solution. Boronic acids,[1] “artificial lectins”, are mo-
lecules that bind to saccharides, forming a boronic acid
acetal (Scheme 1).[ 2 ] This binding is attractive for the
detection and potentially also quantitation of sugars[3,4,5,6]: a
significant number of glucose-sensors have successfully
employed boronic acids. Such a task is obviously important
both in biological systems, medicinal applications[7,8] but
Scheme 1. Equilibria between free boronic acid and sac-
charide (diol).
more so in quality control of food stuff and beverages,
[ 9 , 10 ]
including wine and fruit juices.
While in biomedical
applications the monitoring of glucose level is still
interesting, issues in quality control of beverages allow and
require potentially other approaches to discriminate
saccharides in a specific environment. One example should
be mentioned to explain our interest in discriminating
saccharides. The continued demand for premium quality
honey puts a strain on many bee colonies due to new,
highly epidemic mites. Such a situation creates a lure to
add artificial sugar-syrups to honey. Such artificial sugar
syrups typically have a sugar composition that is different
from that found in natural honey. Therefore, discrimination
of simple sugars is a fundamental and attractive, but
immediately application-relevant, yet difficult task, due to
their chemical similarity.
Schanze et al. synthesized paraquat-based boronic acids.[ 1]
They created a probe system in which poly(arylene-
ethynylene) (PAE 1) is combined with the quencher p-BV2+
[a]
§
Prof. Dr. U. Bunz, M. Bojanowski, M. Bender, K. Seehafer
Organisch-Chemisches Institut
Ruprecht-Karls-Universität Heidelberg
Im Neuenheimer Feld 270, 69120 Heidelberg, FRG
E-mail: uwe.bunz@oci.uni-heidelberg.de
Both authors contributed equally to this work
Scheme 2. Turn-on sensing mechanism employed here.
Supporting information for this article is given via a link at the end of
the document.
Schanze et al. observed that the sugars bind differently to
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
saccharides this intensity increase should be proportional to
the quantity of added analyte.
the PAE-viologen complexes and noted that fructose was
the most successful saccharide in displacing the viologen
from the PAEs. Strong binding of fructose to boronic acids
is due to the presence of a five-membered syn-diol, long
known and discussed by Peters in a recent review.[4] We
thought that the system boronic acid-viologen/PAEs was
attractive-not only to determine but perhaps also to
discriminate different sugars. This type of approach has
also recently been employed by different groups[3c,d]
employing either boronic acid treated filter papers in a dye
displacement assay or, more powerful, viologen-substituted
cationic phenylboronic acids that form electrostatic
complexes with pyrene sulfonic acids. In the latter case,
upon decomplexation by polyols (“sugar alcohols”)
differential fluorescence turn on is observed, and PCA or
LDA discriminate the observed patterns. This method is
fairly sensitive for the sugar alcohols and allows their
discrimination down to 0.4 mM, but the authors have not
attempted to discriminate regular mono- and disaccharides.
Figure 2. Selected conjugated polymers (PAE 1 – PAE 3)[14-1516] and discarded polymers
(PAE 4 – PAE 6).[17,18]
Results and Discussion
Table 1: Proposed sensor system and formed complexes.
We first investigated the binding of the three boronic acid
substituted viologens (o-, m-,p-BV2+) to the PAEs (PAE 1-3),
employing the Stern-Volmer formalisms. Figures 1 and 2 display
the chemical structures of the quenchers and the conjugated
polymers, all of which are literature known, except for PAE 5.
complex
p-BV2+
m-BV2+
o-BV2+
PAE 1
P1
PAE 2
P2
PAE 3
P3
P4
P5
P6
P7
P8
P9
Determination of binding constants for the viologens with PAEs.
As our proposed assay is a dye displacement, we extract
the quencher-dye binding constant forming P1-9 from the
Stern-Volmer quenching constants (Figure 3). The binding
+
of PAEs to p-BV2 is by far the strongest, while for m-BV2+
and o-BV2+ the binding to the polymers is a factor of 5 or so
lower. Also, PAE 1 binds strongest to the quencher, while
the two other PAEs show a lesser affinity to the quenchers.
The signalling mechanism is then the sugar-promoted
decomposition of these complexes, freeing the anionic
polymers under strong fluorescence turn-on. We expect a
useful response range of the constructs with the
saccharides. We would expect that the weakest bound
complexes will give the greatest fluorescence turn-on
responses.
Figure 1. Chemical structures of reported glucose-selective benzyl viologens
(BV2+).[11- 13]
12
Schanze`s scheme consists of a cationic viologen and an
anionic PAE to create a neutral complex with quenched
fluorescence (neutral PAEs show no evidence of complex
formation). We expanded this concept and investigated
Discrimination and binding of sugars.
Figure 4 shows the fluorescence response patterns of all
complexes P1-9 towards different sugars at a sugar
concentration of 100 mM. As expected, the weaker bound
complexes, particularly P7, show larger turn-on efficiencies,
yet, the overall patterns are different for each sugar.
three water-soluble and highly fluorescent PAEs (PAE 1-3
)
that were left after screening of PAE 1-6 PAE 4 were
.
-6
discarded as their fluorescence is too weak. Titration curves
were analyzed and PAE 1-3 show a continuous increase in
quenching upon adding p-BV2+. A continuous increase in
the fluorescence intensity suggests that upon adding of
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
our system should be perfect for their investigation; our assay is
not sufficiently sensitive to determine glucose levels in blood (4-
8
mM). The most useful results are obtained at a
concentration of the conjugated polymer of 2.5 M, the
paraquats at 25 M and the saccharides at concentrations


around 70-100 mM. With the results shown in Figure 4 we
performed statistical analysis to discriminate.
Figure 3 Stern-Volmer constants of PAE 1-3 upon binding to p-, m- and o-
BV2+
.
Previous to that (Figure 5) we screened for the complexes
P1-3 the concentration range of the saccharides (glucose,
fructose, galactose and sucrose) that would give the best
responses; we found that 100 mM of sugar is optimal, even
though 25 mM solutions could also be investigated. At
higher concentrations, the solutions became too syrupy to
allow precise analysis. Also, at around 200 mM of the
saccharides we observe saturation and discrimination
becomes less effective (Figure 5b).
Figure 5. a) Fluorescence response pattern ((I-I0)/I0) obtained with P1, P2 and
P3 (2.5 µM PAE and 25 µM p-BV2+, buffered at pH 7) for four different
saccharides (fructose, galactose, glucose and sucrose) at five different
concentrations (25 – 200 mM). Each value is the average of five independent
measurements with standard error. b) Detailed concentration course of the
fluorescence response ((I-I0)/I0) of P1 applied to glucose, fructose and
sucrose.
Figure 4. Fluorescence-response pattern ((I-I0)/I0) with P1-9 (2.5 µM PAE 1-3
and 25 µM o-, m- and p-BV2+, buffered at pH 7) treated with sugars (100 mM).
Each value is the average of five independent measurements with standard
error.
Mathematical processing and interpretation of the turn-on data
We first analyzed responses of the complexes P1-P3 (Figure
6a); some of the saccharides are immediately discriminated, viz.
fructose, maltose, and sucrose. There is a second, unresolved
A 0.1 M solution of sucrose contains around 34 g sugar/L, while
typical soft drinks like cola-types have a sugar content of around
110 g/L as a mix of sucrose, glucose and fructose. Similar
amounts of sugars are hidden in energy drinks, suggesting that
cluster of lactose, raffinose and glucose, and a third cluster with
partial resolution, which contains mannose, galactose and
xylose. While xylose is discriminated from galactose, the data for
mannose overlap with those for both of the other sugars. If we
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
Figure 6. a) Canonical score plot for the first two factors of simplified fluorescence response patterns obtained with an array of P1-3 (2.5 μM PAE 1-3 and 25 µM
p-BV2+ buffered at pH 7) treated with nine different sugars. b) Canonical score plot for the first two factors of simplified fluorescence response patterns obtained
with an array of P1-6 (2.5 μM PAE 1-3 and 25 µM p-BV2+ und m-BV2+ buffered at pH 7) treated with nine different sugars.
increase the library to include P1-P6, the resolution increases
somewhat and the group galactose, mannose and xylose is
better resolved (Figure 6b).
The concentration dependence for the first three elements P1-3
of the turn-on fluorescence pattern (Figure 5), shown in a
standard plot, determines quantitatively the concentration of one
saccharides, similar to experiments published by Schanze et al.
And we can easily quantify solutions of glucose, fructose or
sucrose.
Upon going to the full array P1-P9, the resolution of our
assay increases again and now, to our surprise, even A7
(lactose) and A8 (raffinose) are resolved, as they are most
similar in structure. This is not gleaned from the first two
factors, but if score 1 and score 3 are plotted, the
discrimination is clear (inset Figure 7).
Table 2. Discrimination of Blind Samples of Different Sugars.
An important question is what and if the two axes depicted in the
LDA plots are correlated with any physical property. As usual,
score 1 is connected to the change in emission intensity, here
emission turn on. While in comparison to water all of the added
sugar solutions increase the fluorescence, there is a significant
differentiation between the sugars. Fructose, due to the
presence of the furanose form, featuring a cis-diol, in equilibrium
with its pyranose form, is the sugar giving the highest turn-on.
This tight binding to boronic acids is literature known and
discussed.4 On the other hand, sucrose, without any cis-diol
present is the weakest binding sugar. Table 2 shows the
identification of blind samples. With exception of mannose and
sucrose we could classify all of the tested sugars correctly. Even
lactose and raffinose, similar in their structure and hardly
separated using Score 1 and 2 in a canonical plot, are
discriminated.
Number of
samples
Correctly
identified
Accuracy [%]
Fructose
Galactose
Glucose
Lactose
Maltose
Mannose
Raffinose
Sucrose
Xylose
5
5
5
4
100
80
5
5
100
60
5
3
5
5
100
60
5
3
5
4
80
5
5
100
100
87
5
5
Total
45
39
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
Experimental Section
We prepared the poly(para-aryleneethynylene)s PAE1–PAE6
through standard Sonogashira reactions (see the Supporting
Information). The benzyl viologens (BV2+) were prepared
according to literature. Analytical data, UV-measurements and
canonical score plots are detailed in the Supporting Information.
References
[1]
[2]
G. Springsteen, B. Wang, Tetrahedron 2002, 58, 5291–5300.
a) T. D. James, K. R. A. S. Sandanayake, S. Shinkai, Angew.
Chemie Int. Ed. 1996, 35, 1910–1922; b) K. Lacina, P. Skládal,
T. D. James, Chemistry Central J. 2014, 8, 60.
[3]
a) J. A. Peters, Coord. Chem. Rev. 2014, 268, 1–22; b) H. S.
Mader, O. S. Wolfbeis, Microchim. Acta 2008, 162, 1-34; c) X.
Bi, D. Li, Z. Liu, Anal. Chem. 2015, 87, 4442-4447; d) A.
Resendez, P. Panescu, R. Zuniga, I. Banda, J. Joseph, D. L.
Webb, B. Singaram, Anal. Chem. 2016, 88, 5444-5452.
[4] C. Tan, M. R. Pinto, K. S. Schanze, Chem. Commun. 2002, 38,
446–447.
[5]
a) X. Wu, Z. Li, X.-X. Chen, J. S. Fossey, T. D. James, Y.-B.
Jiang, Chem. Soc. Rev. 2013, 42, 7965–8178; b) G. Vancoillie,
R. Hoogenboom, Sensors 2016, 16, 1736.
Figure 7. Canonical score plot for the first two factors of simplified
fluorescence response patterns obtained with an array of P1-9 (2.5 μM PAE 1-
3 and 25 µM p-BV2+, m-BV2+ and o-BV2+ buffered at pH 7) treated with nine
different sugars and the first and third factors factors of simplified fluorescence
response patterns (inset, A7 and A8 are only represented).
[6]
M. A. Saeed, H. T. M. Le, O. Š. Miljanić, Acc. Chem. Res.
2014, 47, 2074–2083
[7] G. J. Worsley, G. A. Tourniaire, K. E. S. Medlock, F. K. Sartain,
H. E. Harmer, M. Thatcher, A. M. Horgan, J. Pritchard, J.
Diabetes Sci. Technol. 2008, 2, 213–220.
But fundamentally, the discrimination of tightly related
analytes by such ad-hoc sensor arrays is encouraging and
attractive in its simplicity. The lack of sample preparation
and the use of a simple plate reader do not hurt either to
further investigate these attractive systems.
[8] S. G. Elci, D. F. Moyano, S. Rana, G. Y. Tonga, R. L. Phillips,
U. H. F. Bunz, V. M. Rotello, Chem. Sci. 2013, 4, 2076-2080.
[9] J. Han, M. Bender, K. Seehafer, U. H. F. Bunz, Angew. Chemie
Int. Ed. 2016, 55, 7689–7692.
[10] J. Han, B. Wang, M. Bender, K. Seehafer, U. H. F. Bunz,
Analyst 2017, 142, 537-543.
Conclusions
[11] D. B. Cordes, S. Gamsey, Z. Sharrett, A. Miller, P. Thoniyot, R.
We have expanded Schanze’s elegant saccharide assay,
employing a focused nine-element library, consisting of
three anionic conjugated polymers and three different
boronic-acid substituted, cationic paraquat-based quencher
molecules. Their combination allows for a fluorophore
displacement assay, in which the differential fluorescence
turn-on of the conjugated polymers upon release from the
quencher gives a unique pattern that identifies and
discriminates the investigated sugars. This power of
discrimination is surprising for such a simple, focused
system, based only on the formation of fairly similar
adducts. And while the sensitivity of this assay is not as
great as the dye displacement assay published by
Singaram et al. for sugar polyols, the flexibility in both the
viologen but also the conjugated polymer in our case
should make our system attractive where limit of detection
and sensitivity do not play a critical role. Therefore, in the
future, this approach will be exploited to determine sugar
concentrations in beverages and discriminate types of
honey.
A. Wessling, B. Singaram, Langmuir 2005, 21, 6540–6547.
[12] S. Gamsey, N. A. Baxter, Z. Sharrett, D. B. Cordes, M. M.
Olmstead, R. A. Wessling, B. Singaram, Tetrahedron 2006, 62,
6321–6331.
[13] J. N. Camara, J. T. Suri, F. E. Cappuccio, R. A. Wessling, B.
Singaram, Tetrahedron Lett. 2002, 43, 1139–1141.
[14] J. Lim, O. Š. Miljanić, Chem. Commun. 2012, 48, 10301–
10303.
[15] I. B. Kim, R. Phillips, U. H. F. Bunz, Macromolecules 2007, 40,
5290–5293.
[16] M. Bender, K. Seehafer, M. Findt, U. H. F. Bunz, RSC Adv.
2015, 5, 96189–96193.
[17] K. Haskins-Glusac, M. R. Pinto, C. Tan, K. S. Schanze, J. Am.
Chem. Soc. 2004, 126, 14964–14971.
[18] J. Han, M. Bender, S. Hahn, K. Seehafer, U. H. F. Bunz, Chem.
- Eur. J. 2016, 22, 3230–3233.
Acknowledgements
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
We thank Heidelberg University for generous support.
.
Keywords: sugars • sensor field • sensor array • conjugated
polymer • discrimination
This article is protected by copyright. All rights reserved.

10.1002/chem.201700831
Chemistry - A European Journal
FULL PAPER
FULL PAPER
A poly(aryleneethynylene) tongue that
discriminates different saccharides
works through complex formation with
different boronic acid substituted violo-
gens.
Page No. – Page No.
Title:
Discrimination
of Saccharides by a
Simple Sensor Array
Maximilan Bojanowski, Markus
Bender, Kai Seehafer, Uwe H. F.
Bunz*
1 G. Springsteen and B. Wang, Tetrahedron, 2002, 58, 5291–5300.
2 T. D. James, K. R. A. S. Sandanayake and S. Shinkai, Angew. Chemie Int. Ed. English, 1996, 35, 1910–1922.
3 J. A. Peters, Coord. Chem. Rev., 2014, 268, 1–22.
4 J. Lim and O. Š. Miljanić, Chem. Commun. (Camb)., 2012, 48, 10301–10303
5 X. Wu, Z. Li, X.-X. Chen, J. S. Fossey, T. D. James and Y.-B. Jiang, Chem. Soc. Rev, 2013, 42, 7965–8178.
6 M. A. Saeed, H. T. M. Le and O. Š. Miljanić, Acc. Chem. Res., 2014, 47, 2074–2083
7 G. J. Worsley, G. A. Tourniaire, K. E. S. Medlock, F. K. Sartain, H. E. Harmer, M. Thatcher, A. M. Horgan and J. Pritchard, J. Diabetes Sci. Technol., 2008, 2, 213–220.
8 S. G. Elci, D. F. Moyano, S. Rana, G. Y. Tonga, R. L. Phillips, U. H. F. Bunz, V. M. Rotello, Chem. Sci., 2013, 4, 2076-2080.
9 J. Han, M. Bender, K. Seehafer and U. H. F. Bunz, Angew. Chemie Int. Ed., 2016, 55, 7689–7692.
10 J. Han, B. Wang, M. Bender, K. Seehafer and U. H. F. Bunz, Analyst, 2017, 142, 537-543.
11 D. B. Cordes, S. Gamsey, Z. Sharrett, A. Miller, P. Thoniyot, R. A. Wessling and B. Singaram, Langmuir, 2005, 21, 6540–6547.
12 S. Gamsey, N. A. Baxter, Z. Sharrett, D. B. Cordes, M. M. Olmstead, R. A. Wessling and B. Singaram, Tetrahedron, 2006, 62, 6321–6331.
13 J. N. Camara, J. T. Suri, F. E. Cappuccio, R. A. Wessling and B. Singaram, Tetrahedron Lett., 2002, 43, 1139–1141.
14 C. Tan, M. R. Pinto and K. S. Schanze, Chem. Commun., 2002, 446–447.
15 I. B. Kim, R. Phillips and U. H. F. Bunz, Macromolecules, 2007, 40, 5290–5293.
16 M. Bender, K. Seehafer, M. Findt and U. H. F. Bunz, RSC Adv., 2015, 5, 96189–96193.
17 K. Haskins-Glusac, M. R. Pinto, C. Tan and K. S. Schanze, J. Am. Chem. Soc., 2004, 126, 14964–14971.
18 J. Han, M. Bender, S. Hahn, K. Seehafer and U. H. F. Bunz, Chem. - Eur. J., 2016, 22, 3230–3233.
This article is protected by copyright. All rights reserved.