Efficient quenching of a guanidinium-containing fluorescence sensor

Article abstract of DOI:10.1002/cphc.201000943

Quenching: A novel water-soluble guanidinium-containing phenylene-ethynylene-based fluorescence sensor (see picture) is reported with a two-fold improvement in sensitivity to electron-deficient quenchers compared to an equivalent alkoxy-substituted phenyl

Full text of DOI:10.1002/cphc.201000943

DOI: 10.1002/cphc.201000943
Efficient Quenching of a Guanidinium-Containing Fluorescence Sensor
Wayne N. George,[a] Mark Giles,[b] Iain McCulloch,[a] Joachim H. G. Steinke,[a] and John C. deMello*[a]
Conjugated polymers (CPs) have attracted considerable inter-
est owing to their applications in optoelectronic devices such
as light-emitting diodes (LEDs),[1] solar cells,[2] and thin-film
transistors (TFTs).[3] In recent years there has been increasing in-
terest in exploiting CPs as chemical and biological sensors[4–7]
due to the strong fluorescence quenching they exhibit in the
presence of electron deficient species, and their consequent
ability to detect trace levels of biological analytes at mM con-
centrations and below.[5,8,9]
vantage of enabling a wide palette of existing conjugated
polymers to be derivatised with ionic groups but results in
poor electronic connectivity between the p-electron system
and the complexation site, potentially reducing the quenching
effect (especially at low quencher concentrations).
Subsequent attempts to develop improved fluorescence
sensors have involved perturbing the electron density along
the conjugated chain by introducing electron-donating[21] or
electron-withdrawing[22] substituents. The inclusion of such
groups can enhance electron/energy transfer to a complexed
electron-deficient quencher, but their chemical incorporation
into the sensor typically requires multistep synthetic routes
that significantly limit the scope for further functionalisation of
the sensors.[13,14]
The efficiency of the quenching process can be quantified
by the Stern–Volmer equation [Eq. (1)] which relates the emis-
sion intensity I to the quencher concentration [Q]:
I0
I ¼
ð1Þ
1 þ kSV½Qꢀ
There are potential advantages, in terms of faster electron or
energy transfer and improved sensitivity, to incorporating the
quenching unit directly into the p system itself but, surprising-
ly, such an approach does not appear to have been reported
in the literature due perhaps to the level of synthetic challenge
involved. Herein we report proof-of-principle investigations
into a novel small molecule sensor PE-1 (Figure 1), in which a
guanidinium unit is incorporated into the conjugated back-
bone to impart both water solubility and molecular recogni-
tion properties, whilst also maximising electronic connectivity
between the fluorophore and a complexed quencher mole-
cule. The behaviour of PE-1 is compared with that of the struc-
turally related side-chain electrolyte PE-2, analogues of which
have been widely studied in the literature.
where I0 is the emission intensity in the absence of quencher,
and the Stern–Volmer constant kSV is the effective association
constant for the complex formed between the fluorophore
and the analyte.[10,11] One of the most widely studied families
of fluorescence sensors is that of conjugated polyelectrolytes
[CPEs], in which ionically functionalised side chains impart
water solubility and provide binding sites for complexation
with electron deficient quencher groups. CPEs—and in particu-
lar poly(phenylene ethynylenes) [PPEs]—have previously been
shown to be highly effective materials for sensing trace quanti-
ties of biological materials,[12–15] exhibiting kSV values that are
typically 3–6 orders of magnitude higher than those of their
smaller model analogues.[16] The enhanced sensitivity arises
from the tendency of CPEs to form loosely aggregated assem-
blies in solution, which allows excitons to migrate effectively
along and between chains to quenching sites where they
decay non-radiatively by an energy or electron transfer pro-
cess.[8] Since a single quencher can deactivate the entire as-
sembly, extremely high kSV values can be attained.[16–19]
The guanidinium unit is able to form strong ion pairs with
oxo-anions such as carboxylates, sulfates and phosphates due
CPE sensors studied to date have been based on side-chain
polyelectrolytes, in which the ionisable groups are typically at-
tached to alkoxy side-chains and hence are physically isolated
from the conjugated backbone.[14,20] This approach has the ad-
[a] Dr. W. N. George, Prof. I. McCulloch, Dr. J. H. G. Steinke, Dr. J. C. deMello
Department of Chemistry
Imperial College London, South Kensington Campus
London, SW7 2AZ (UK)
Fax:(+44)2075945801
E-mail: j.demello@imperial.ac.uk
[b] Dr. M. Giles
Smart Holograms
291 Cambridge Science Park
Milton Road, Cambridge, CB4 0WF (UK)
Figure 1. Chemical structures of the two phenylene-ethynylene (PE) based
sensors used herein. PE-1 incorporates a guanidinium unit into the conju-
gated backbone for solubility and complexation with electron-deficient
quenchers, whereas PE-2 uses ionisable side chains.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cphc.201000943.
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765

to its planar Y-shaped configuration and its high pKa of ~12
which ensures protonation over a wide pH range.[23] We rea-
soned that connecting the guanidinium motif directly to the
chromophore would lead to an enhanced level of quenching
relative to PE-2 due to the direct incorporation of the com-
plexation site into the p-electron system.
The principal steps in the synthesis of PE-1 are shown in
Scheme 1 (see also the Supporting Information). To incorporate
the guanidinium unit into PE-1, amine 1 was treated with 1,3-
bis-(tert-butoxycarbonyl)-2-methyl-2-thiopseudo-urea
2
and
mercury(II) chloride in DMF following literature procedures,
Figure 2. Normalised absorption and emission spectra in water (pH 7) for
8 mm PE-1 and PE-2. For PE-1 the absorption and emission peaks are at 326
and 376 nm, respectively, whereas for PE-2 they are at 351 and 391 nm.
quinone-2,6-disulfonic acid disodium salt (AQS)—a widely used
electron deficient quencher molecule. Strong fluorescence
quenching was observed for both sensors, with a slight broad-
ening of the emission spectra and an associated loss of vibron-
ic structure at higher quencher concentrations. Stern–Volmer
plots are shown as insets to the main diagrams, with the mark-
ers denoting experimental data points and the solid line indi-
cating the optimal fit to the Stern–Volmer relation as described
Scheme 1. Synthesis of PE-1. Conditions: i) Et3N, HgCl2, 2, DMF, 258C, 50 h,
68%; ii) K2CO3, MeOH/THF, 258C, 4 h, 71%; iii) Pd(PPh3)4, CuII, iPr2NH, 4-iodo-
toluene, THF, 258C, 24 h, 91%; iv) TFA in DCM, 258C, 1 h, 59%.
which yielded the guanidinylated derivative 3.[24,25] Selective
deprotection of the TMS groups produced the corresponding
bisalkyne; cross-coupling and cleavage of the Boc groups from
4 gave PE-1 as a light-yellow crystalline solid in 11% overall
yield (7 steps). In addition to providing ready access to PE-1,
this concise synthetic route has the advantage of generating
the orthogonally protected bisalkyne monomer 3 which can
be partnered with a wide range of substituted diiodo aryl
monomers by palladium cross-coupling chemistry, providing a
facile pathway to the synthesis of multiple PPE architectures.
The synthesis of PE-2 has been described previously.[12]
Figure 2 shows the absorption and emission spectra for PE-1
and PE-2 in water. PE-1 shows a pronounced blue-shift in both
absorption and emission relative to PE-2. This differs from the
usual situation for amine-containing PPEs, in which the absorp-
tion and emission bands are usually red-shifted relative to the
alkoxy derivatives due to the electron-rich nature of the
chain.[21] The shorter wavelength emission from PE-1 suggests
the overall effect of the guanidinium unit is to reduce the elec-
tron density along the PE backbone, consistent with its proton-
ated (and hence electron-withdrawing) state. This is supported
Figure 3. 8 mm emission spectra for a) PE-1 and b) PE-2 at various concentra-
tions of the quencher 9,10-anthraquinone-2,6-disulfonic acid disodium salt
(AQS) The insets show Stern–Volmer plots using data extracted from the
spectra. The intensities in the Stern–Volmer plots are determined from the
area beneath the corresponding spectra, and are normalised with respect to
the measured emission intensity in the absence of AQS. The markers denote
experimental data lines and the solid-lines indicate the optimal fit to the
Stern–Volmer relation as outlined in the ESI.
1
by the H NMR data which shows a downfield shift of 0.3 ppm
for the proton ortho to the guanidinium motif compared to
the equivalent proton in PE-2.
The emission spectra of 8 mm PE-1 and PE-2 in water are
shown in Figure 3 for varying concentrations of 9,10-anthra-
766
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ChemPhysChem 2011, 12, 765 – 768

in the ESI. Both sensors exhibited strong quenching in the
presence of AQS: PE-1 had a Stern–Volmer coefficient of 8.9ꢁ
104 mꢁ1, compared to 4.6ꢁ104 mꢁ1 for PE-2—the higher value
for PE-1 being consistent with the direct incorporation of the
complexation site into the p system.
To verify the generality of the enhanced quenching effect
observed with PE-1, measurements were also made using a
chemically unrelated phosphate-based quencher 4-nitrophenyl
phosphate, bis(cyclohexyl-ammonium) salt hydrate (NPP),
which has previously been found to be an excellent emission
quencher for PPE-based systems.[12] The emission spectra of
8 mm PE-1 and PE-2 in water are shown in Figure 4 for varying
concentrations of NPP. Like AQS, NPP induces strong quench-
ing of PE-1 and PE-2 (with only a weak influence on spectral
shape), resulting in kSV values of 3.1ꢁ104 and 1.4ꢁ104 mꢁ1, re-
spectively.
Figure 5. Stern–Volmer constants for 8 mm PE-1 and PE-2 in pure water
(pH 7.0) under the influence of the electron-deficient quenchers AQS and
NPP. Also see Table 1.
Table 1. Stern–Volmer constants for 8 mm PE-1 and PE-2 in pure water
(pH 7.0) under the influence of the electron-deficient quenchers AQS and
NPP.
Sensor
Quencher
kSV [mꢁ1
]
Error [mꢁ1
]
PE-1
PE-1
PE-2
PE-2
AQS
NPP
AQS
NPP
8.9ꢁ104
3.1ꢁ104
4.6ꢁ104
1.4ꢁ104
2.0ꢁ103
1.0ꢁ103
1.3ꢁ103
9.6ꢁ101
be partially offset by the less basic nature of sulfate, which re-
sults in weaker individual bond strengths[23]).
To further compare the behaviour of PE-1 and PE-2, their in-
teraction with simple monovalent ions was investigated by
performing additional quenching measurements with the
(non-electron-deficient) molecules disodium hydrogen phos-
phate (DHP) and sodium acetate. PE-1 underwent unexpected-
ly strong emission quenching in the presence of DHP, yielding
a Stern–Volmer coefficient of 8.2ꢁ103 mꢁ1 at low DHP concen-
trations (<0.25 mm), see the Supporting Information.1 Minimal
quenching was observed in the case of sodium acetate, with a
kSV value of <5mꢁ1 being observed, indicating a clear selectivi-
ty towards phosphates. PE-2 showed no quenching with either
DHP or sodium acetate. The comparative response of PE-1 and
PE-2 to DHP and (electron-deficient) NPP is shown in Figure 6
and Table 2.
Although PE-1 was designed to interact strongly with oxo-
anions, the high degree of quenching observed with DHP is
unexpected since neither electron nor energy transfer is ex-
pected to occur to DHP. One possible explanation for the
quenching effect is the formation of non-emissive aggregates
in the presence of DHP. The Y-shaped configuration of the gua-
nidinium unit gives rise to two geometric binding modes for
oxo-anions, meaning tetrahedral phosphates like DHP can bind
to two guanidinium units at once.[23] This behaviour was ex-
ploited by Nishizawa et al. to develop a pyrene-based guanidi-
nium sensor that self-assembled in the presence of pyrophos-
phate anions inducing a spectral change that they attributed
to excimer emission from the newly p-stacked assembly.[29] In
the present case, it may be that DHP promotes dimer (or
Figure 4. 8 mm emission spectra for a) PE-1 and b) PE-2 at various concentra-
tions of the quencher 4-nitrophenyl phosphate, bis(cyclohexyl-ammonium)
salt hydrate (NPP). The insets show Stern–Volmer plots using data extracted
from the spectra.
For both AQS and NPP the quenching efficiency of the gua-
nidinium-containing PE-1 was approximately twice that of the
bis-ammonium derivative PE-2 (see Figure 5 and Table 1), con-
sistent with the incorporation of the ionic recognition unit into
the conjugated backbone and its strong tendency for ion-pair
formation with oxo-anions, leading to faster consequent elec-
tron transfer to the quencher.[26–28] The stronger quenching by
AQS is presumably a consequence of there being two sulfate
binding groups for complexation with guanidinium compared
to just one phosphate group in NPP (although this factor may
1
Note, the quenching rate diminished at higher DHP concentrations, causing a
deviation from the Stern–Volmer relation.
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767

Acknowledgements
We are grateful to the Engineering and Physical Sciences Re-
search Council and Merck Chemicals Ltd., Chilworth Science Park
(Dr. Steve Tierney) for financial support.
Keywords: conjugated
polymers
·
fluorescence
·
polyelectrolytes · quenching · sensors
Figure 6. Quenching results for 8 mm PE-1 and PE-2 in pure water (pH 7.0)
under the influence of the traditional electron-deficient quencher NPP and
the oxo-anion disodium hydrogen phosphate (DHP). Also see Table 2. The
guanidinium-containing PE-1 undergoes strong quenching in the presence
of phosphate ions even with non-electron-deficient molecules such as DHP.
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]
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]
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PE-1
PE-2
PE-2
NPP
DHP
NPP
DHP
3.1ꢁ104
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larger aggregate) formation, leading to a loss of emission by
p–p stacking. Whatever the cause, though, it is clear that PE-1
is an effective sensor for phosphate ions.
In conclusion, we report a novel fluorescent oligo(phenylene
ethynylene) PE-1, which contains a guanidinium group in
direct electronic contact with the conjugated backbone and
shows a two-fold gain in sensitivity compared to the structural-
ly related molecule PE-2. The results reported herein indicate
that the usual strategy of maximising the number of binding
sites along a conjugated segment is just one factor for achiev-
ing high sensitivity, and that the electronic connectivity be-
tween the chromophore unit and the quencher plays a critical
role too. Specific recognition groups such as guanidinium and
isothiouronium units offer interesting alternatives to the com-
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Future work will focus on the synthesis of polymeric ana-
logues of PE-1, with a view to signficantly amplifying the fluo-
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shira–Hagihara conditions to polymerise the bisalkyne mono-
mer derived from 3 with 1,4-diiodobenzene, yielded a polymer
that was highly aggregated in solution. Whilst it was non-emis-
sive in unbuffered aqueous conditions, the addition of a small
quantity (0.3 wt%) of the non-ionic surfactant Triton X-100 re-
sulted in strong solution-phase emission that is typical of dia-
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will yield less strongly aggregrated polymers and so can act as
efficient fluorescence sensors. The results of this work will be
reported in due course.
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Received: November 11, 2010
Published online on February 15, 2011
768
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ChemPhysChem 2011, 12, 765 – 768