The effect of boronic acid acidity on performance of viologen-based boronic acids in a two-component optical glucose-sensing system

Article abstract of DOI:10.1016/j.tetlet.2007.05.075

A two-component saccharide sensing system using the fluorescent dye, hydroxypyrene trisulfonic acid, combined with a boronic acid functional viologen as a receptor/quencher in pH 7.4 buffer solution has been further investigated. The effect of substituents on the acidity of the boronic acid was measured. The boronic acid pK_a changed in the expected manner when electron donating or withdrawing groups were present. The glucose binding constants were dependent on pK_a, but no simple correlation was observed for the Stern-Volmer quenching constants and the fluorescence signal modulation.

Full text of DOI:10.1016/j.tetlet.2007.05.075

Tetrahedron Letters 48 (2007) 5125–5129
The effect of boronic acid acidity on performance
of viologen-based boronic acids in a two-component
optical glucose-sensing system
Zachary Sharrett, Soya Gamsey, Jonathan Fat, Daniel Cunningham-Bryant,
Ritchie A. Wessling and Bakthan Singaram*
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA
Received 6 April 2007; revised 2 May 2007; accepted 11 May 2007
Available online 18 May 2007
Abstract—A two-component saccharide sensing system using the fluorescent dye, hydroxypyrene trisulfonic acid, combined with a
boronic acid functional viologen as a receptor/quencher in pH 7.4 buffer solution has been further investigated. The effect of sub-
stituents on the acidity of the boronic acid was measured. The boronic acid pKa changed in the expected manner when electron
donating or withdrawing groups were present. The glucose binding constants were dependent on pKa, but no simple correlation
was observed for the Stern–Volmer quenching constants and the fluorescence signal modulation.
Published by Elsevier Ltd.
The detection of glucose using boronic acid-based fluo-
rescent chemosensors has been a major area of research
in recent years.1 This is driven in part by the need for
better techniques for monitoring blood sugar concentra-
tions in diabetics2 and critically ill patients.3 Boronic
acid-based sensors can be utilized for this application
because of their ability to bind reversibly to 1,2- and
1,3-diols in aqueous media.4 In most of these systems,
the boronic acid receptor is attached directly to a fluoro-
phore, and the saccharide recognition event causes a
change in fluorescence emission.5
The existence of complexes has been shown both in solu-
tion6b,e and in the solid state.6h The complex exhibits
diminished fluorescence relative to that of the free dye.
We propose that a signal is generated when the complex
dissociates as a consequence of the reaction of the
quencher component with glucose, resulting in an
increase in fluorescence intensity related to glucose
concentration.
The first step in the dissociation process involves reac-
tion of the boronic acid receptor on the viologen with
glucose. The conversion of the neutral boronic acid (tri-
gonal boron) to a negatively charged boronate ester (tetra-
hedral boron) alters the properties of the viologen
leading to a reduction in quenching efficacy. Though
electronic and steric effects may play a role, at least part
of the process is simple charge neutralization, which
reduces the electrostatic attraction of dye for quencher
and leads to weaker binding and therefore enhanced
fluorescence.
In contrast to systems in which the boronic acid and
fluorophore are incorporated into a single molecule,
the study described in this Letter uses a two-component
system, developed earlier,6 comprising the anionic fluo-
rescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid tri-
sodium salt (HPTS), and a boronic acid-appended
viologen. The latter serves both as a fluorescence
quencher and a glucose receptor.
Though the detailed mechanism of glucose modulation
in the two-component system has not been completely
elucidated, it has been established that the quenching
step involves ground-state charge-transfer complex for-
mation between anionic dye and cationic quencher.
The purpose of the present study was to explore the
effect of boronic acid acidity on quenching and glucose
response. This knowledge should lead to a better
understanding of the sensing mechanism. The system
studied combines benzyl viologens as the quenching
component with HPTS as the fluorescent dye in aqueous
media at physiological pH. The approach taken is to
manipulate the pKa of the boronic acid by adding
*
Corresponding author. E-mail: singaram@chemistry.ucsc.edu
0040-4039/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2007.05.075

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Z. Sharrett et al. / Tetrahedron Letters 48 (2007) 5125–5129
Table 1. pKa values determined by potentiometric titration for the
BBVs (4 mM) in the absence and presence of glucose (0.1 M)
electron donating and withdrawing groups in the para
position and observing how such substituents affect the
quenching ability and glucose response of the viologens.
It has been shown that the pKa values for arylboronic
acids are dependent upon the substitution pattern and
nature of the substituents on the aromatic ring.7
pKa without glucose pKa with 0.1 M glucose DpKa
BBV
F-BBV
OMe-BBV 8.2
7.7
7.5
6.8
6.7
7.4
0.9
0.8
0.8
Three quencher/receptors, N,N0-4,40-bis(benzyl-3-boro-
nic acid)-bipyridinium dibromide (BBV), N,N0-4,40-bis-
(benzyl-5-fluoro-3-boronic acid)-bipyridinium dibromide
(F-BBV), and N,N0-4,40-bis(benzyl-5-methoxy-3-boro-
nic acid)-bipyridinium dibromide (OMe-BBV), were
prepared and used in this study (Fig. 1). The effect of
an electron donating substituent (methoxy) and electron
withdrawing substituent (fluoro) on the pKa of the boro-
nic acid, as well as on the quenching ability and glucose
response of these compounds was investigated. Non-
boronic acid analogs, benzyl viologen (BV), fluoro-
benzyl viologen (F-BV), and methoxy-benzyl viologen
(OMe-BV), were used as controls. The six compounds
were synthesized as outlined in Scheme 1.
tion and are listed in Table 1. As expected, the presence
of an electron-donating methoxy group para to the
boronic acid in OMe-BBV raised the apparent pKa rela-
tive to that of the unsubstituted compound (BBV). Sim-
ilarly, the apparent pKa of F-BBV was lower than that
of the unsubstituted compound, BBV, due to the elec-
tron-withdrawing fluoro group para to the boronic acid.
As observed earlier,6g the value of BBV is substantially
lower than that of phenyl boronic acid (pKa = 8.8) and
this is attributed to the effect of the positively charged
nitrogen atom in the pyridinium ring. The change in
pKa observed by adding a second substituent to the aro-
matic ring is therefore, the net effect of both groups. The
F-atom adds to the electron withdrawing effect of N+;
the OMe-group counteracts it.
The apparent pKa values of the boronic acid-containing
compounds were determined using potentiometric titra-
N
N
F
N
N
F
O
N
N
O
2 Br-
2 Br-
2 Br-
B(OH)2
(HO)2B
B(OH)2
(HO)2B
B(OH)2
(HO)2B
OMe-BBV
BBV
F-BBV
N
N
F
N
N
F
O
N
N
O
2 Br-
2 Br-
2 Br-
BV
F-BV
OMe-BV
Figure 1. Structures of boronic acid-substituted benzyl viologens and benzyl viologens.
X
BBV (X = H, Y = B(OH)2)
BV (X, Y = H)
a
Br
F-BV (X = F, Y = H)
Y
X = F, H
Y = B(OH)2, H
X
X
b, c
a
Br
F-BBV (X = F)
OMe-BBV (X = OMe)
B
B(OH)2
O
O
X = F, OMe
OMe
OMe
c
a
Br
OMe-BV
Scheme 1. Synthesis of viologen-based quenchers. Reagents and conditions: (a) 4,40-dipyridyl, DMF, 60 ꢀC, 48 h; (b) 1,3-propanediol, CaH2, DCE,
88 ꢀC, 1 h; (c) N-bromo succinimide, 2,20-azobisisobutyronitrile, DCE, 88 ꢀC, 3 h.

Z. Sharrett et al. / Tetrahedron Letters 48 (2007) 5125–5129
5127
Table 2. Static (Ks) and dynamic (V) quenching components of
pyranine (4 · 10ꢀ6 M in pH 7.4 phosphate buffer, kex = 460 nm and
8.4
8.2
8
k
em = 510 nm) for the BBV and BV quenchers
Ks (Mꢀ1
)
V (Mꢀ1
)
BBV
7.8 · 103
3.5 · 103
5.8 · 103
1.5 · 104
7.7 · 103
7.9 · 103
3.1 · 103
2.8 · 103
3.2 · 103
2.3 · 103
2.9 · 103
3.3 · 103
7.8
7.6
7.4
F-BBV
OMe-BBV
BV
F-BV
OMe-BV
y = -2.1x + 7.6
R2 = 0.97
-0.3
-0.2
-0.1
0
0.1
σ
Figure 2. Hammet plot for BBVs.
pH 7.4 phosphate buffer) was titrated with increasing
amounts of the viologens, and the fluorescence emission
at 510 nm (kex = 460 nm) was recorded after each addi-
tion. Values for Ks and V, which represent the static and
dynamic quenching constants, respectively, were deter-
mined from these data (Table 2).9
The pKa values of these compounds were also deter-
mined in the presence of glucose (Table 1). Consistent
with typical boronic acids, all of the compounds dis-
played lower pKa values with glucose present, versus
those measured in the absence of glucose.
A comparison of F-BV, OMe-BV, and BV is given in
Figure. 3a.
A Hammett plot was generated using this data (Fig. 2).8
The data in the Hammet plot were fitted with a straight
line with a slope of 2.1. This value is consistent with the
value previously reported for phenyl boronic acids.7a
From the Stern–Volmer plot and the Ks values, it is clear
that each ring substitution decreases the quenching abil-
ity of benzyl viologen to nearly the same extent. One
possible explanation of this diminished quenching is a
weak intramolecular charge-transfer between the substi-
tuent heteroatom and the quaternized pyridinium nitro-
gen (Fig. 4). Because the quenching mechanism depends
on the transfer of an electron from the dye to the violo-
gen quencher in the ground-state complex,6a any change
in the viologen that reduces its electron affinity would
make it a less effective quencher. It is also possible that
the substituents interfere sterically with the complex
formation.
To determine the relative fluorescence quenching ability
of each viologen with HPTS, Stern–Volmer plots were
generated (Fig. 3). A solution of HPTS (4 · 10ꢀ6 M in
4
a
BV
F-BV
3.5
3
OMe-BV
2.5
2
When analyzing the relative quenching characteristics of
the three boronic acid-based viologens, two main factors
have to be considered as noted above. One is the possi-
ble steric implications of the fluoro and methoxy substit-
uents. As was observed with the non-boronic acid
containing viologens (BVs), this could explain why both
OMe-BBV and F-BBV are weaker quenchers than BBV
(Fig. 3b, Table 2). The second factor is the charge-neu-
tralization mechanism. In further support of this mech-
anism of diminished quenching, it should be noted that
F-BBV is the poorest quencher of all. The reason why
the quenching strengths of OMe-BBV and F-BBV differ
is likely due to the electronic effects that the para substi-
tuent exert on the boronic acid. As evidenced by the
apparent pKa value of F-BBV (Table 1), the electron
withdrawing fluoro group causes the boronic acid to
be partially in the anionic boronate form at pH 7.4, thus
decreasing its ability to complex with HPTS. The same
rationale can be used to explain the quenching behavior
1.5
1
0
2
4
6
8
10
12
[Quencher] (M-5)
3
2.8
2.6
2.4
2.2
2
b
BBV
F-BBV
OMe-BBV
1.8
1.6
1.4
1.2
1
0
2
4
6
8
10
12
[Quencher] (M-5)
X
N
X
N
Figure 3. (a) Stern–Volmer plots of HPTS (4 · 10ꢀ6 M) with increas-
ing concentrations of (j) BV, (m) F-BV, and (d) OMe-BV at pH 7.4
k
ex = 460 nm, kem = 510 nm. (b) Stern–Volmer plots of HPTS
(4 · 10ꢀ6 M) with increasing concentrations of (j) BBV, (m) F-BBV,
and (d) OMe-BBV at pH 7.4 kex = 460 nm, kem = 510 nm.
Figure 4. Proposed charge interaction resulting in diminished quench-
ing ability.

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Z. Sharrett et al. / Tetrahedron Letters 48 (2007) 5125–5129
of OMe-BBV, relative to F-BBV. That is, the boronic
acid is mainly in the trigonal neutral state at this pH.
The observation that the quenching by OMe-BBV is
poorer than BBV indicates that other factors are in-
volved which are not forthcoming at present.
Besides binding constants, another metric that can be
used to evaluate these compounds is the degree of fluo-
rescence recovery (F/F0) obtained upon glucose addi-
tion. In this case, HPTS/BBV produced the largest
degree of fluorescence enhancement, followed by
HPTS/F-BBV, then HPTS/OMe-BBV. In spite of a
higher apparent binding constant, F-BBV did not pro-
duce a larger F/F0 value relative to BBV. This could
be the result of F-BBV existing more in the boronate
form due to its lower apparent pKa. As pointed out ear-
lier, this would weaken the F-BBV/HPTS interaction
thereby decreasing the overall modulation.
The use of BBV and HPTS as a viable saccharide sens-
ing system has been previously demonstrated.6 In this
system, the amount of fluorescence signal modulation
is dependant on the amount of saccharide that is titrated
into a solution of HPTS quenched by BBV. When glu-
cose binds to the boronic acid on BBV, the Lewis acidity
of boron is increased, resulting in tetrahedral anionic
glucoboronate formation at pH 7.4. This zwitterionic
form of the BBV molecule is a less effective quencher
and thus an increase in the fluorescence intensity of
HPTS is observed.
The BBV quenchers used in this study, when combined
with the dye HPTS in pH 7.4 buffer, modulated the fluo-
rescence of HPTS when titrated with physiological
amounts (2.5–20 mM) of glucose. This study showed
the expected substituent effect on the pKa of boronic
acid-substituted viologens. A clear correlation was
shown between apparent pKa and the binding constants
of the boronic acids with glucose. The addition of either
an electron withdrawing group or an electron donating
group to the benzyl ring of boronic acid-substituted ben-
zyl viologens also affected the quenching and glucose
sensing behavior of the viologens relative to the unsub-
stituted derivatives; however the relationship is more
complex. The addition of the fluoro group increased
the apparent binding constant but slightly decreased
the fluorescence modulation. The methoxy group unex-
pectedly produced a slight decrease in quenching effi-
cacy, and caused a drop in both the binding constant
and modulation. Further research will be required to
establish the exact relationship between the apparent
pKa of the boronic acids and the performance of the
HPTS/BBV sensing system, though the correlation with
glucose binding is clear.
The apparent binding affinities of the boronic acid-based
viologens for glucose were determined by measuring the
relative fluorescence intensity (F/F0) (kex = 460 nm,
k
em = 510 nm) when increasing amounts of glucose (0–
30 mM) were added to an aqueous solution of HPTS
(4 · 10ꢀ6 M, pH 7.4) and a BBV (1.2 · 10ꢀ4 M). The
binding curves generated from the titration are shown
in Figure 5, and the calculated apparent binding con-
stants (Kb) are listed in Table 3. It is known that the
binding constants of boronic acids with diols increase
with decreasing boronic acid pKa.7 The BBV com-
pounds display a behavior that is consistent with this
trend. The compound with the lowest pKa, F-BBV, pro-
duced the highest binding constant and OMe-BBV,
which has the highest pKa, gave the lowest binding
constant.
1.4
mBBV
1.35
1.3
F-mBBV
OMe-mBBV
Acknowledgments
The authors thank Lacie Hirayama, Boaz Vilozny, Jeff
Gardina, Alexander Schiller, Jeff Suri, and Monica
Lares for their helpful discussions. The authors would
also like to thank the BioSTAR Project and the Indus-
try-University Cooperative Research Program with
GluMetrics, Inc. for their financial support.
1.25
1.2
1.15
1.1
1.05
1
0
5
10
15
20
25
30
References and notes
[Glucose] (mM)
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