Conjugated polymer-based fluorescence turn-on sensor for nitric oxide

Article abstract of DOI:10.1021/ol0513903

(Chemical Equation Presented) A turn-on fluorescent sensor for NO (g) in solution was synthesized using a bipyridyl-substituted poly(p-phenylene vinylene) derivative (CP1) as the sensory scaffold. The action of NO (g) upon the CP1-Cu(II) complex reduces it to the CP1-Cu(I) complex with a concomitant 2.8-fold increase in emission intensity. The reagent is selective for NO (g) versus other biological reactive nitrogen species, except for nitroxyl, and has a detection sensitivity limit of 6.3 nM.

Full text of DOI:10.1021/ol0513903

ORGANIC
LETTERS
2005
Vol. 7, No. 16
3573-3575
Conjugated Polymer-Based
Fluorescence Turn-On Sensor for Nitric
Oxide
Rhett C. Smith, Andrew G. Tennyson, Mi Hee Lim, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
lippard@mit.edu
Received June 14, 2005
ABSTRACT
A turn-on fluorescent sensor for NO (g) in solution was synthesized using a bipyridyl-substituted poly(p-phenylene vinylene) derivative (CP1)
as the sensory scaffold. The action of NO (g) upon the CP1 Cu(II) complex reduces it to the CP1 Cu(I) complex with a concomitant 2.8-fold
−
−
increase in emission intensity. The reagent is selective for NO (g) versus other biological reactive nitrogen species, except for nitroxyl, and
has a detection sensitivity limit of 6.3 nM.
The discovery that nitric oxide (NO) is the endothelium-
derived relaxing factor astonished the scientific community.1
Since then, an even wider range of biological roles for NO
have been elucidated.2 We are interested in NO as a possible
signaling agent in the central nervous system,3 where it is
proposed to mediate synaptic plasticity.4 The biological
chemistry of NO has inspired researchers to devise tech-
niques for its bioimaging,5 of which turn-on fluorescence is
especially attractive because of the demonstrated success of
this strategy in real-time monitoring of other cellular signals.6
Neurological research in particular has benefited from the
development of small-molecule fluorescent sensors specific
for Ca2+ 6b,7 or Zn2+.8 Here we report a turn-on sensor for
NO with nM sensitivity employing a π-conjugated polymer
(CP) as the fluorescent reporter.
The enhanced sensitivity of CPs vs small molecule-based
sensors, together with their structural and optoelectronic
tunability, renders them intriguing scaffolds for the design
and construction of detection systems.9 We therefore inves-
tigated this class of materials for imaging NO by turn-on
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10.1021/ol0513903 CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/02/2005

fluorescence, a resistivity-based CP sensor for NO having
been previously described.10 The successful realization of
CPs for fluorescence-based detection of NO, in the form of
a CP-copper complex that exhibits a turn-on response to
NO in solution, represents an advantageous new strategy with
many possible applications.
Previously, we reported transition metal-based NO sensors
that involved displacement of a fluorescent ligand from a
mono- or dimetallic center with attendant turn-on of fluo-
rescence, one of which is reversible (Scheme 1a, L )
Figure 1. Structure of CP1.
that only 0.5 equiv of Cu(I) are necessary to attain maximum
quenching of polymer luminescence, as independently ob-
served in the present study (Supporting Information).
CP1 is a bright red-orange solid (λmax ) 462 nm) with
strong fluorescence emission centered at 542 nm (Φ ) 0.30).
Upon addition of 1 equiv of Cu(OTf)2 to a solution of CP1,
the integrated fluorescence intensity was quenched 4-fold,
whereas addition of 1 equiv of [Cu(NCMe)4][BF4] decreased
the fluorescence by ∼30% (Figure 2).15 Adding >0.5 equiv
of Cu(II) afforded little additional emission quenching.
Scheme 1. Strategies for Fluorescent Detection of Nitric
Oxide
fluorescent ligand).11 More recent work unveiled a related
approach,12 involving NO-induced copper redox chemistry,13
with net conversion of fluorophore-labeled, paramagnetic
(quenched) Cu(II) complexes to a diamagnetic (fluorescent)
Cu(I) state (Scheme 1b). This mechanism has been consid-
ered as a possibility for colorimetric NO sensing.14
In further pursuit of the latter strategy, we prepared a series
of CPs integrating copper-binding units at defined intervals
and screened their relative fluorescence intensities in both
CP-Cu(I) and CP-Cu(II) forms, anticipating turn-on NO
detection by the process depicted in Scheme 1b.15 A poly-
(p-phenylene vinylene) (PPV) derivative incorporating pe-
riodic bipyridyl units along the main chain displayed the most
suitable properties for further investigation (CP1, Figure 1,
Hx ) n-hexyl). A structurally related CP is reportedly
quenched in the presence of Cu(II), whereas only moderate
quenching occurs with added Cu(I).16 A careful study of the
structural and photophysical characteristics of poly(p-
phenylene ethynylene)/bipyridyl-Cu(I) complexes has re-
cently been undertaken.17 This work indicates that stable
complexes exist with two bipyridyl units per Cu(I), indicating
Figure 2. Emission spectra of CP1 (black), CP1-Cu(I) (green),
CP1-Cu(II) (red), and CP1-Cu(II) immediately following addition
of 300 equiv of NO (g). All measurements were made in 4:1 CH2-
Cl2/EtOH.
Introduction of 300 equiv of NO (g) to the CP1-Cu(II)
complex rapidly (<1 min) increased the integrated emission
by 2.8-fold, producing a fluorescence spectrum similar to
that of the CP1-Cu(I) complex (Figure 2). NO (g) did not
alter the fluorescence of CP1 in the absence of Cu(II).
In the proposed mechanism, Scheme 1b, protons are
formed that could affect the fluorescence of CP1. Protons
alone, however, decrease the emission from bipyridyl-
PPVs.18 Using a handheld UV lamp, a decrease in emission
was qualitatively confirmed in the current case following
addition of 3 µL of glacial acetic acid to 5 mL of a 2 µM
solution of CP1 in 4:1 CH2Cl2/EtOH.
(10) Shioya, T.; Swager, T. M. Chem. Commun. 2002, 1364-1365.
(11) (a) Franz, K. J.; Singh, N.; Lippard, S. J. Angew. Chem., Int. Ed.
2000, 39, 2120-2122. (b) Franz, K. J.; Singh, N.; Spingler, B.; Lippard, S.
J. Inorg. Chem. 2000, 39, 4081-4092. (c) Hilderbrand, S. A.; Lim, M. H.;
Lippard, S. J. J. Am. Chem. Soc. 2004, 126, 4872-4878. (d) Lim, M. H.;
Lippard, S. J. Inorg. Chem. 2004, 43, 6366-6370.
(12) Lim, M. H.; Lippard, S. J. 2005, submitted for publication.
(13) Tran, D.; Skelton, B. W.; White, A. H.; Laverman, L. E.; Ford, P.
C. Inorg. Chem. 1998, 37, 2505-2511.
(14) Dacres, H.; Narayanaswamy, R. Sens. Act. B 2003, 90, 222-229.
(15) Details of the full set of polymers examined will be reported
elsewhere.
(16) Wang, B.; Wasielewski, M. R. J. Am. Chem. Soc. 1997, 119, 12-
21.
(17) Kokil, A.; Yao, P.; Weder, C. Macromolecules 2005, 38, 3800-
3807.
(18) Eichen, Y.; Nakhmanovich, G.; Gorelik, V.; Epshtein, O.; Poplawski,
J. M.; Ehrenfreund, E. J. Am. Chem. Soc. 1998, 120, 10463-10470.
3574
Org. Lett., Vol. 7, No. 16, 2005

Following initial trials with NO (g), we examined the
selectivity of the sensor for nitric oxide vs other biologically
relevant reactive nitrogen species (RNS). In parallel with
ongoing work in our laboratory,19 we evaluated a nitrosothiol
(SNAP, S-nitroso-N-acetylpenicillamine), a nitroxyl (HNO)
donor (Angeli’s salt, Na2N2O3),20 and a nitrosonium (NO+)
source (NOBF4) for their ability to alter the emission spectra
of CP1, CP1-Cu(I), or CP1-Cu(II). These donors were
selected on the basis of their commercial availability in high
purity and well-studied kinetics of RNS formation.22 None
of the donors (50 equiv of SNAP, 50 equiv of NOBF4, or
16 equiv of Angeli’s salt, Na2N2O3) induced a change in the
emission spectra of CP1 or CP1-Cu(I). When 50 equiv of
SNAP was added to CP1-Cu(II), a 1.5-fold turn-on of
fluorescence occurred slowly over 2 h, in accord with the
known ability of cupric ion to catalyze the release of NO
from nitrosothiols.22e The same effect was observed upon
reversing the order of Cu(II) and SNAP addition, in which
case the expected 4-fold quenching occurred immediately
upon Cu(II) addition, followed by a slow turn-on. This
control indicates that the presence of excess SNAP does not
interfere with Cu(II) binding by CP1. A full 24 h was re-
quired following addition of SNAP before a turn-on response
(2.1-fold) similar to that evoked by NO (g) was attained.
Because of the significantly longer time for SNAP (24 h) vs
NO (<1 min) to elicit a fluorescence response, nitrosothiols
should not be considered as seriously interfering analytes.
The addition of 50 equiv of NOBF4 did not affect the
fluorescence of solutions of CP1 or of preformed CP1-
Cu(I) or CP1-Cu(II). When Cu(II) was added to a solution
of CP1 containing 50 equiv of NOBF4, the emission
spectrum was the same as that of CP1-Cu(II). In the
presence of EtOH, NOBF4 will form EtONO. The solution
resulting from addition of NOBF4 to CP1-Cu(I) was
therefore expected to contain the same species present in
the reaction of CP1-Cu(II) with NO. These two solutions
exhibited identical emission spectra.
ing a nitrogen-purged CP1-Cu(II) solution slightly de-
creased the integrated fluorescence (∼6%). This response is
presumably due the ability of O2 to serve as a collisional
quencher and is within the error of detection.
The sensitivity of the CP1-Cu(II) complex to NO was
evaluated by addition of progressively lower concentrations
of SNAP to a 630 nM solution of the sensor. On the basis
of multiple measurements versus an emission standard,
NIST-issued quinine sulfate dihydrate, we determined a 10%
increase in integrated emission to be the lowest quantifiable
change discernible at our instrumental detection limit. By
using this method, we computed a sensitivity of about 6.3
nM for the CP1-Cu(II) system.
Although previous work has provided strong evidence for
the mechanism presented in Scheme 1b,13 we carried out a
number of additional checks to confirm its validity in the
present context. The strongest supporting evidence is the near
perfect match of emission spectra derived from CP1-Cu-
(I), CP1-Cu(I)/NO+, and CP1-Cu(II)/NO. Exposure of
CP1-Cu(II) to NO (g) in the absence of EtOH, which is
required to form RONO in the proposed mechanism, did not
produce an increase in fluorescence. Finally, the expected
diminution of the EPR signal of CP1-Cu(II) occurred upon
addition of 1 equiv of NO, confirming reduction to Cu(I).
When 1 equiv of Cu(OTf)2 was added to a solution of CP1
(0.5 equiv bind) followed by 1 equiv of NO (g), the
integrated signal decreased by 45%, indicating that only CP1-
coordinated Cu(II) is reduced to Cu(I). No bands attributable
to stable copper nitrosyls were observed by IR spectroscopy.
Despite the strong evidence and precedence for the
proposed mechanism, we note that, in addition to the d9 f
d10 transformation that occurs upon reduction, some degree
of ligand rearrangement or other structural alteration, to
which CP emission is very sensitive,17,21 could play a role
in the observed sensory response. Work in progress with
small-molecule model compounds will elucidate such pos-
sibilities.
Although no response was elicited by nitroxyl with CP1-
Cu(I), it is noteworthy that an immediate 2.8-fold increase
occurred upon reaction of CP1-Cu(II) with 50 equiv of
nitroxyl, formed from decomposition of Angeli’s salt. The
spectrum produced was nearly identical to that exhibited by
CP1-Cu(I), suggesting that nitroxyl may reduce Cu(II) to
Cu(I).
The system reported here represents an early manifestation
of a new strategy for the fluorescent detection of NO. To
the best of our knowledge, it also signifies the first
fluorescence-based sensor for NO employing a conjugated
polymer scaffold.10 Studies have commenced to identify
additional transition metal-conjugated polymer complexes for
improved sensory response, devise specificity for NO over
nitroxyl, and prepare highly fluorescent water-soluble deriva-
tives for biological imaging of nitric oxide.21
The effect of O2 was also investigated as another possible
interfering biological species. Aeration of a cuvette contain-
(19) Tennyson, A. G.; Lippard, S. J. Manuscript in preparation.
(20) Bartberger, M. D.; Lin, W.; Ford, E.; Miranda, K. M.; Switzer, C.;
Fukuto, J. M.; Farmer, P. J.; Wink, P. A.; Houk, K. N. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 10958-10963.
(21) (a) Wang, D.; Gong, X.; Heeger, P. S.; Rininsland, F.; Bazan, G.
C.; Heeger, A. J. AdV. Funct. Mater. 2003, 13, 463-467. (b) Kuroda, K.;
Swager, T. M. Chem. Commun 2003, 26-27. (c) Xu, Q.-H.; Gaylord, B.
S.; Wang, S.; Bazan, G. C.; Moses, D.; Heeger, A. J. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 11634-11639.
(22) (a) Bonner, F. T.; Ravid, B. Inorg. Chem. 1975, 14, 558-563. (b)
Bazylinski, D. A.; Hollocher, T. C. Inorg. Chem. 1985, 24, 4285-4288.
(c) Singh, R. J.; Hogg, N.; Joseph, J.; Kalyanaraman, B. J. Biol. Chem.
1996, 271, 18596-18603. (d) Williams, D. L. H. Met. Enzymol. 1996, 268,
299-308. (e) Smith, J. N.; Dasgupta, T. P. Nitric Oxide 2000, 4, 57-66.
(f) Chen, Y.; Irie, Y.; Keung, W. M.; Maret, W. Biochem. 2002, 41, 8360-
8367.
Acknowledgment. This work was supported by NSF
Grant CHE-0234951. R.C.S. and A.G.T. thank the NIH and
NSF for Fellowships. The MIT DCIF NMR spectrometer
was funded through NSF Grants CHE-9808061 and DBI-
9729592.
Supporting Information Available: Experimental de-
tails, absorption and emission spectra, EPR spectra, and
details of sensitivity and selectivity measurements. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0513903
Org. Lett., Vol. 7, No. 16, 2005
3575