Highly sensitive detection of saccharides under physiological conditions with click synthesized boronic acid-oligomer fluorophores

Article abstract of DOI:10.1039/c0ob01003k

Two phenylboronic acid based saccharide sensors bearing conjugated oligomer fluorophores with linear and cruciform π-frameworks were synthesized in a modular approach utilizing a Cu-catalyzed alkyne azide cycloaddition (click) reaction. The cruciform fluo

Full text of DOI:10.1039/c0ob01003k

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Highly sensitive detection of saccharides under physiological conditions with
click synthesized boronic acid-oligomer fluorophores†
Karimulla Mulla, Prateek Dongare, Ningzhang Zhou, Guang Chen, David W. Thompson and Yuming Zhao*
Received 9th November 2010, Accepted 22nd December 2010
DOI: 10.1039/c0ob01003k
Two phenylboronic acid based saccharide sensors bearing
conjugated oligomer fluorophores with linear and cruciform
p-frameworks were synthesized in a modular approach uti-
lizing a Cu-catalyzed alkyne azide cycloaddition (click) reac-
tion. The cruciform fluorophore showed excellent saccharide
sensing function under physiological conditions in the mM
range, whereas the linear fluorophore gave very limited sens-
ing functions. The different fluorescent sensing behaviours
highlight the important role of oligomer fluorophore in the
development of effective saccharide sensors.
recently designed and synthesized two boronic acid attached
oligomers (5 and 10, Scheme 1) as models for a qualitative
understanding of the basic structure-property relationships for
oligomer-based fluorescent saccharide sensors. Note that the p-
frameworks of the two oligomer fluorophores are designed in
two distinct motifs, cruciform and linear. As such, the effect of
Detection and recognition of saccharides under physiological
conditions and monitoring the concentration of saccharides in vivo
are challenging tasks that require sustained efforts to be dedicated
to development and refinement of new sensor systems. At present,
boronic acid receptors have been extensively used in conjunction
with various fluorogenic groups to form functional fluorescent
sensors for saccharides.1 In this context, the sensor design has
been recently focused on tuning the receptor, donor/ligand, and
linkage groups in order to strengthen boronic acid-saccharide
binding at neutral pH, to enhance photoinduced electron/energy
transfer for effective signaling, and to introduce cooperativity
for binding with specific saccharides.2 On the other hand, the
fluorophore component acting primarily as the “read-out” unit of
the sensor can significantly influence solvation, steric hindrance of
the binding site, and polarity matching with saccharides. Hence,
the fluorophore effect also has key impact on the efficiency and
selectivity of fluorescent sensors.2g
In the current literature, the fluorophores utilized for construct-
ing saccharide sensors are mostly polyaromatic-based fluorogenic
groups such as napthalene, anthracene, and diazobenzene.1,2
Conjugated oligomers and polymers, owing to the excellent con-
trollability and tunability of their optoelectronic properties, have
found wide-ranging applications in fluorescent sensory devices.3
However, fluorescent saccharide sensors based on p-conjugated
oligomer fluorophores have not been investigated, despite their
excellent photophysical properties. To address this issue, we have
Department of Chemistry, Memorial University of Newfoundland, St. John’s,
NL, Canada, A1B 3X7. E-mail: yuming@mun.ca; Fax: +1-709-864-3702;
Tel: +1-709-864-8747
† Electronic supplementary information (ESI) available: Synthetic proce-
dures, spectroscopic characterizations of new compounds, and detailed
data of global fitting analysis. See DOI: 10.1039/c0ob01003k
Scheme 1 Click synthesis of boronic acid appended cruciform and linear
conjugated oligomers.
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Table 1 Stability constants (K) for complexation of cruciform 5 with
various saccharides determined by global spectral fitting
oligomer fluorophore on sensor properties can be readily probed
by way of comparative studies.
The synthesis of the two fluorophores 5 and 10 (henceforth
referred to as cruciform and linear respectively in the following
discussions) was carried out via a modular strategy using a popular
click reaction, namely Cu-catalyzed alkyne azide cycloaddition
(CuAAC),4 as the key synthetic step.
Saccharide
log K1 (M-1)
log K1K2 (M-2)
D-fructose
D-ribose
D-galactose
D-glucose
4.02 0.5
3.24 0.2
3.87 0.2
2.53 0.3
7.27 0.5
6.09 0.1
6.26 0.1
4.21 0.4
As shown in Scheme 1, the synthesis of cruciform 5 began with
a CuAAC reaction between an azido-pendant phenylboronate
25 and an alkynylated phenylene vinylene oligomer 16 in the
presence of CuI and i-Pr2EtN in DMF at 65 ◦C. Compared
with numerous high-yielding CuAAC reactions reported in the
literature, the yield of this click reaction was only at a moderate
level (69%). The reduced efficiency of reaction is likely due to
some side reactions involving the insertion of Cu into the C–
B bonds.7 The boronate groups of intermediate 3 was then
hydrolyzed via a transesterification reaction between boronate
3 and excess o-tolylboronic acid (4) in dioxane/H2O to give
boronic acid-cruciform 5 along with a tolylboronate byproduct
6. It should be noted that compound 6 serves a useful precursor
for the preparation of azido-phenylboronate 2. From a practical
standpoint, this hydrolysis approach can be viewed as synthetically
economical.
In a similar manner, linear oligomer 7 was coupled with azide
2 through a click reaction, yielding boronate intermediate 8 in
55% yield. For the hydrolysis of 8, however, the transesterification
method was problematic due to the formation of some intractable
impurities. To circumvent the purification difficulty, a stepwise
hydrolysis route was then used, in which the boronate was first
converted into trifluoroborate with KHF2 and then hydrolyzed
into boronic acid in the presence of trimethylsilyl chloride
(TMSCl).8 This method led to the desired product 10 in a very
good yield and satisfactory purity.
The modular synthesis of 5 and 10 clearly demonstrates the
power of click chemistry for rapid generation and screening of
boron-based organic functional fluorophores. It is noteworthy that
there has been a surging interest in using click synthesis to prepare
fluorogenic compounds for optical sensing and imaging purposes
in recent years.5,9 Particularly worth mentioning is that Fossey and
James recently devised a “click-fluor”,5,10 the structure of which
comprised of a phenyl group and an o-methylphenylboronic acid
moiety connected through a 1,2,3-triazolyl linker through click
chemistry. The “click-fluor”5 has shown reasonable fluorescent
sensing functions towards saccharides in an alkaline aqueous
medium (pH 8.21) requiring the presence of an organic co-
solvent (52% wt MeOH in H2O), while the concentrations
of saccharides to be detected were in the range of ca. 10-2
to 1 M.
In our work, we tested the fluorescent sensing functions of cru-
ciform 5 and linear 10 for four selected saccharides, D-fructose, D-
ribose, D-galactose, and D-glucose, under physiological conditions.
Despite the limited solubility of fluorophores 5 and 10 in water,
dissolution of them in a neutral (pH 7.41) aqueous potassium
phosphate buffer solution with concentration (10-5 M) suited
for fluorescence spectroscopic measurements was readily attained
with the aid of a minimal amount of DMSO (H2O/DMSO >
99 : 1, v/v). The fluorescence quantum yields (U) of 5 and 10
determined in aqueous phosphate buffer solutions are 7.5% and
6.3% respectively.
For the cruciform sensor 5, significant fluorescence enhance-
ment (turn-on sensing) was observed during the titrations with
saccharides as shown in Fig. 1A–D. Titration of the four
saccharides resulted in similar trends of spectral changes with
increasing titration. The emission spectrum of cruciform 5 is
composed of a peak at 430 nm and a shoulder at 403 nm. The
two bands become more distinctive as the titration progresses,
and the emission profile at the ending point of titration shows
vibronic structures resembling those of the emission spectrum
of cruciform boronate 3, which is indicative of complexation of
boronic acid with saccharides. The affinities of 5 for saccharides
vary to a large extent as manifested by the plots in Fig. 1E, which
show the correlation between fluorescence enhancement (F/F0)
at 430 nm and saccharide concentration. It can be clearly seen
that the selectivity of cruciform 5 for different saccharides is in the
order of: D-fructose > D-ribose ~ D-galactose > D-glucose. Also of
remark in Fig. 1E is the high sensitivity of 5 for D-fructose and D-
ribose in the range of 10-3 to 10-4 M. Such a performance renders
cruciform 5 at least comparable if not superior to those water-
soluble fluorescent sensor systems reported in the recent literature
with high saccharide sensitivity under physiological condition.11
To gain a quantitative understanding of the mechanisms for
complexation between sensor 5 and various saccharides, global
spectral fitting analysis was undertaken using the modified
Marquardt-Levenberg algorithm implemented in the SPECFIT
software package.12 Table 1 lists the stability constants (K)
determined by global spectral fitting.
From Table 1 it can be seen that all the four saccharides bind
to cruciform 5 in a two-step mechanism. The spatial arrangement
of the two boronic acid receptors in 5 does not facilitate any
“pincer-like” binding mode with saccharides. D-Fructose shows
the strongest binding strength with 5 which is about two orders of
magnitude greater than that of D-glucose. The binding constants
for D-ribose and D-galactose are similar and stay in the middle
range among the four saccharides. The quantitative measurements
are in agreement with the trend of saccharide sensitivity revealed
in Fig. 1E.
The complexation of phenylboronic acid with saccharides
(polyols) is reversible and pH dependent,1a,b while the formation
of stable phenylboronic acid and saccharide complex at neutral
pH usually requires stabilization effect by some ligand groups
such as in the case of the widely known “Wulff-type” receptor (o-
dialkylaminomethylphenylboronic acid).13 From the fluorescence
titration experiments, it is evident that the boronic acid groups
in cruciform 5 are effectively bound to saccharides under phys-
iological conditions. This result suggests that the 1,2,3-triazolyl
moiety resulting from the click reaction not only acts as a linker
group, but might play an important role in saccharide binding.
1
To gain a deeper insight into this aspect, H NMR titration of 5
with D-fructose was then performed. For solubility reason, the
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Fig. 1 (A) Fluorescence titration of 5 (11.8 mM) with D-fructose in an aqueous buffer solution (pH 7.41) at 298 3 K (lex = 340 nm). (B) Fluorescence
titration of 5 (13.8 mM) with D-ribose in an aqueous buffer solution (pH 7.41) at 298 3 K (lex = 340 nm). (C) Fluorescence titration of 5 (13.8 mM) with
D-galactose in an aqueous buffer solution (pH 7.41) at 298 3 K (lex = 340 nm). (D) Fluorescence titration of 5 (12.0 mM) with D-glucose in an aqueous
buffer solution (pH 7.41) at 298 3 K (lex = 340 nm). (E) Plots of fluorescence enhancement (F/Fo, l = 430 nm) against saccharide concentrations with
fittings extracted from SPECFIT. (F) Fluorescence titration of 10 (5.8 mM) with D-fructose in a basic aqueous buffer solution (pH 8.21) at 298 3 K (lex
=
340 nm).
experiments were conducted by adding aliquots of D-fructose
dissolved in a phosphate/D2O buffer solution at pD 7.41 to
a DMSO-d6 solution of 5. Detailed NMR titration results are
delineated in Fig. 2A.
Fluorescence titrations of linear sensor 10 with various sac-
charides in neutral aqueous solutions were also carried out using
the same protocol as that for cruciform 5. In a sharp contrast,
the emission spectrum of 10 did show any changes during the
titrations. Increasing the basicity of solution did not improve
the sensing function significantly. For instance, addition of D-
fructose to 10 in a basic buffer solution at pH 8.21 resulted in
only a slight drifting of spectral baseline (Fig. 1F). Given that
boronic acid tends to strongly complex with saccharides under
basic conditions, the poor saccharide sensing function displayed
by sensor 10 can be reasonably ascribed to an inert response
of the linear OPE fluorophore to saccharide binding events.
The marked difference between cruciform 5 and linear 10 in
fluorescent sensing for saccharides thus underscores that the nature
of fluorophore is a determinative factor controlling sensory perfor-
mance. For the remarkable fluorescence transduction properties
exhibited by cruciform 5, neither photoinduced electron transfer
(PeT) nor intramolecular charge transfer (ICT) mechanism offers
meaningful interpretation. Instead, the sensing mechanism can
be rationalized from a standpoint of aggregation-modulated
emission. Recent studies have disclosed that the aggregation state
of OPVs in solution can exert very significant impact on their
excited-state dynamics and emission yields.14 To shed light on
this point, we further analyzed the aggregation behaviour of
cruciform 5 without and with binding to saccharides. Aliquots
taken from the solution prepared for fluorescence titration of 5
with D-fructose were spin-cast on freshly cleaved mica surfaces
for atomic force microscopic (AFM) imaging. The aggregates
Although the change of solvent system from water to DMSO
may lead to considerable variation of binding constants, the
binding motif however should not be changed as much. As
shown in Fig. 2A, the signal of boronic acid protons (Hb)
vanishes gradually with increasing titration of D-fructose, while
the signals of methylene and aromatic protons (Hc and Hd) show
a slight upfield shift by less than 0.1 ppm. These observations
also corroborate the transformation of boronic acid to boronate
ester in binding to saccharides. Of particular note is that the
triazolyl proton (Ha) dramatically shifts to downfield by more than
0.2 ppm. This result indicates that the triazolyl group experiences
a pronounced change of chemical environment in binding with
saccharides, likely induced by solvation or hydrogen bonding
effect. A water insertion binding motif is therefore proposed based
on density functional theory (DFT) calculations. According to the
model system illustrated in Fig. 3, in binding with a 1,2-diol the
triazolyl unit adjacent to the phenylboronic acid group acts as a
ligand (hydrogen bond acceptor) to coordinate with a molecule
of water. This water insertion model is analogous to the binding
motif of the “Wulff-type” boronic acid receptor in aqueous media.
Energetically, it provides stabilization to the boron-diol complex
by DH = -11.2 kcal mol-1, which well accounts for the significant
binding of cruciform 5 with various saccharide in neutral aqueous
solutions.
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Fig. 2 (A) 1H NMR titration of 5 with D-fructose in DMSO-d6 and phosphate buffer solution (pD 7.41) at 298 3 K. (B) AFM image (tapping mode)
of aggregates of 5 on mica. (C) AFM image (tapping mode) of aggregates of 5 and D-fructose on mica.
ciform 5 to suppress fluorescence self-quenching.15 On the other
hand, the possibility that saccharide binding induces more planar
OPV orientation in the aggregates of 5 may also offer a sound
explanation for the observed fluorescence enhancement. The
latter argument is in line with the aggregation-induced emission
(AIE) for some p-conjugated systems.16 Further investigations to
debunk the detailed photophysical mechanism using dynamic light
scattering (DLS) and time-resolved fluorescence spectroscopic
techniques are underway, and the results will be disclosed in due
course.
Fig. 3 DFT (B3LYP/6-31G*) optimized structure for the complex of
o-triazolylmethylphenylboronic acid with ethylene glycol in the presence
of water.
In summary, we have prepared conjugated oligomer-boronic
acid hybrids as fluorescent saccharide sensors using an efficient
and modular synthetic strategy. The cruciform sensor 5 detects
saccharides with high sensitivity under physiological conditions,
which arises from two major factors: (i) the triazole linker acts as
a hydrogen bond donor to stabilize boron-saccharide complex
at neutral pH, and (ii) the OPV fluorophore shows sensitive
fluorescence responses to the aggregation state in solution which
is modulated by saccharide binding. Our study highlights the
importance of tuning and manipulation of the “fluorophore
parameter” to improve the performances of oligomer-based
fluorescent chemo- and bio-sensors.
of cruciform 5 prior to complexation with D-fructose are in a
size range of 60 to 100 nm (see Fig. 2B). After complexation
with D-fructose, much smaller (6–14 nm) aggregates are formed
(Fig. 2C). Clearly, the binding of cruciform 5 with saccharides
results in considerably reduced aggregate sizes in solution, and it
is reasonable to believe that the aggregation effect plays a crucial
role in the fluorescence enhancement observed in the titrations of
cruciform 5 with various saccharides. Two types of rationalization
can be conceived at this juncture. First, the complexation with
saccharides leads to increased de-aggregation/solvation of cru-
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