Enhanced fluorescence cyanide detection at physiologically lethal levels: Reduced ICT-based signal transduction

Article abstract of DOI:10.1021/ja044421i

Three water-soluble fluorescent probes have been specifically designed to determine free cyanide concentrations up to physiologically lethal levels, >20 μM. The probes have been designed in such a way as to afford many notable sensing features, which render them unique with regard to signal transduction, photophysical characteristics, and their application to physiological cyanide determination and safeguard. The probes are readily able to reversibly bind free aqueous cyanide with dissociation constants around 4 μM³. Subsequent cyanide binding modulates the intramolecular charge transfer within the probes, a change in the electronic properties within the probes, resulting in enhanced fluorescence optical signals as a function of increased solution cyanide concentration. The ground-state chelation with cyanide produces wavelength shifts, which also enable the probes to sense cyanide in both an excitation and emission ratiometric manner, in addition to enhanced fluorescence signaling. This has enabled a generic cyanide sensing platform to be realized that is not dependent on fluorescent probe concentration, probe photodegradation, or fluctuations in the intensity of any employed excitation sources, ideal for remote cyanide sensing applications. Further, the >600 nm fluorescence emission of the probes potentially allows for enhanced fluorescence ratiometric cyanide sensing in the optical window of tissues and blood, facilitating their use for the transdermal monitoring of cyanide for mammalian safeguard or postmortem in fire victims, both areas of active research.

Full text of DOI:10.1021/ja044421i

Published on Web 02/16/2005
Enhanced Fluorescence Cyanide Detection at Physiologically
Lethal Levels: Reduced ICT-Based Signal Transduction
Ramachandram Badugu,† Joseph R. Lakowicz,† and Chris D. Geddes*,†,‡
Contribution from the Center for Fluorescence Spectroscopy, Department of Biochemistry and
Molecular Biology, Medical Biotechnology Center, UniVersity of Maryland School of Medicine,
725 West Lombard Street, Baltimore, Maryland 21201, and Institute of Fluorescence,
Laboratory for AdVanced Fluorescence Spectroscopy, Medical Biotechnology Center, UniVersity
of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, Maryland 21201
Received September 14, 2004; E-mail: chris@cfs.umbi.umd.edu
Abstract: Three water-soluble fluorescent probes have been specifically designed to determine free cyanide
concentrations up to physiologically lethal levels, >20 µM. The probes have been designed in such a way
as to afford many notable sensing features, which render them unique with regard to signal transduction,
photophysical characteristics, and their application to physiological cyanide determination and safeguard.
The probes are readily able to reversibly bind free aqueous cyanide with dissociation constants around 4
µM3. Subsequent cyanide binding modulates the intramolecular charge transfer within the probes, a change
in the electronic properties within the probes, resulting in enhanced fluorescence optical signals as a function
of increased solution cyanide concentration. The ground-state chelation with cyanide produces wavelength
shifts, which also enable the probes to sense cyanide in both an excitation and emission ratiometric manner,
in addition to enhanced fluorescence signaling. This has enabled a generic cyanide sensing platform to be
realized that is not dependent on fluorescent probe concentration, probe photodegradation, or fluctuations
in the intensity of any employed excitation sources, ideal for remote cyanide sensing applications. Further,
the >600 nm fluorescence emission of the probes potentially allows for enhanced fluorescence ratiometric
cyanide sensing in the optical window of tissues and blood, facilitating their use for the transdermal monitoring
of cyanide for mammalian safeguard or postmortem in fire victims, both areas of active research.
Introduction
physiological safeguard would therefore need a cyanide dynamic
range from a few to ∼30 µM free cyanide.
Cyanide has long been known to be a toxic substance.1 The
mechanism of cyanide poisoning is by absorption through the
GI track, skin, and lungs. Cyanide’s toxicity lies in its ability
to inhibit oxygen utilization by cells, binding to the active site
of cytochrome oxidase,2,3 hence, the tissues with the highest
oxygen requirement (brain, heart, and lungs) are the most
affected by acute poisoning.1
While it is difficult to give an exact lethal dosage or exposure
limit, it is estimated that the intravenous dose that is lethal to
50% of the exposed population (LD50) for HCN for an adult is
1.0 mg/Kg, and the estimated lethal dose for liquid contact, that
is, on the skin, is about 100 mg/Kg.1 Recent studies on both
fire victims and survivors have shown that survivors were found
to have <20 µM blood cyanide, while victims were found to
have levels ∼20-30 µM and, in some cases, much higher.4-6
Hence, any cyanide monitoring analytical technique to ensure
While cyanide has earned a well-deserved reputation as the
lethal poison in television murder/mystery, it is somewhat
surprising to learn that the raw material finds common industrial
use in organic chemicals and polymers, such as nitriles, nylon,
and acrylic plastics,7 as well as being a critical reagent in the
gold-extraction process.1,8 Subsequently, there is a widespread
need to develop versatile small-molecule cyanide sensors for
use in a variety of settings.
There have been numerous techniques developed for the
detection and determination of aqueous free cyanide9-13 and
indeed may other inorganic anions. These techniques include
potentiometric, chromatographic, and flow injection to name
just a few.9-13 However, these systems are not simple or cheap
(6) Baud, F. J.; Barriot, P.; Toffis, V. N. Engl. J. Med. 1991, 325, 1761-
1766.
(7) Ullman’s Encyclopedia of Industrial Chemistry, 6th ed.; Wiley-VCH: New
York, 1999.
† University of Maryland School of Medicine.
‡ University of Maryland Biotechnology Institute.
(8) Miller, G. C.; Pritsos, C. A. Cyanide: Social, Industrial, and Economic
Aspects; Proceedings of the TMS Annual Meeting, 2001; pp 73-81.
(9) Giuriati, C.; Cavalli, S.; Gorni, A.; Badocco, D.; Pastore, P. J. Chromatogr.
A 2004, 1023, 105-112.
(1) Baskin, S. I.; Brewer, T. G. Medical Aspects of Chemical and Biological
Warfare; Sidell, F., Takafuji, E. T., Franz, D. R., Eds.; TMM Publica-
tions: Washington, DC, 1997; Chapter 10, pp 271-286 and references
therein.
(10) Beck, H. P.; Zhang, B.; Bordeanu, A. Anal. Lett. 2003, 36, 2211-2228.
(11) Fernandez-Arguelles, M. T.; Costa-Fernandez, J. M. C.; Pereiro, R.; Sanz-
Medel, A. Anal. Chim. Acta 2003, 491, 27-35.
(2) Warburg, O. Hoppe-Seyler’s Z. Physiol. Chem. 1911, 76, 331-346.
(3) Kellin, D. Proc. R. Soc. London, Ser. B: Biol. Sci. 1929, 104, 206-251.
(4) Ishii, A.; Seno, H.; Watanabe-Suzuki, K.; Suzuki, O.; Kumazawa, T. Anal.
Chem. 1998, 70 (22), 4873-4876.
(12) Deep, B.; Balasubramanian, N.; Nagaraja, K. S. Anal. Lett. 2003, 36, 2865-
2874.
(5) Moriva, F.; Hashimoto, Y. J. For. Sci. 2001, 46 (6), 1421-1425.
(13) Favero, J. A. D.; Tubino, M. Anal. Sci. 2003, 19, 1139-1143.
9
10.1021/ja044421i CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 3635-3641
3635

A R T I C L E S
Badugu et al.
and cannot be applied to physiological monitoring or used to
monitor drinking waters alike, many requiring the benefits of
an analytical laboratory.9-13 It is worth noting that one recent
paper, however, has made some progress in this regard by
developing a small-molecule lifetime-based sensor based on a
luminescent Ru(II) metal complex14 that readily chelates
cyanide. While the benefits of lifetime-based sensing are well-
known compared to those of simple absorption or fluorescence-
based measurements, lifetime-based measurements are consid-
erably more complex, expensive, and generally require more
time for data acquisition. One powerful alternative, however,
is a technique based on fluorescence excitation and/or emission
ratiometric sensing.15,16 Similar to lifetime-based sensing, this
approach is impervious to experimental artifacts, such as probe
concentration, excitation source intensity fluctuations, light
scattering, and detector drift, but has the added attraction of
being conceptually simple, with the instrumentation costs being
significantly lower than that for time-resolved lifetime-based
sensing.15,16
Figure 1. Molecular structures of cyanide.
In this article, we describe a very powerful set of new
fluorescent probes, which are readily able to detect cyanide
concentrations in the range of 1-30 µM in the presence of a
high background of potential physiological interferents, such
as aqueous chloride. The reduced intramolecular charge transfer
of the probes upon cyanide chelation provides for the fluores-
cence emission enhancements observed, effectively increasing
the signal-to-noise ratio for sensing, as a function of cyanide
concentration. Subsequently, we have developed the first sensors
for cyanide based on direct and enhanced fluorescence, which
are not prone to biological interferences, such as chloride or
oxygen, which is not likely to be the case for a recently reported
Ru(II)-based sensor.14 In addition, the Ru(II)-based sensor has
the disadvantage of reduced intensity as a function of cyanide,
which is in contrast to our new sensing format. Further, the red
nature of our fluorescence emission is likely to provide for
cyanide sensing in blood or perhaps even transdermally, where
the absorption by hemoglobin and water is minimal.17
Subsequently, we believe we have demonstrated the first
proof-of-concept for enhanced fluorescence wavelength ratio-
metric small-molecule cyanide sensing, which is a platform also
applicable to other anions.
Figure 2. Complexation of DSPBA probes with aqueous free cyanide.
absence of cyanide directly influences the intramolecular charge
transfer (ICT) from the amino group to the electron-deficient
quaternary nitrogen center.15 In the absence of cyanide, ICT is
efficient, with the probe being effectively quenched. However,
in the presence of cyanide, the extent of ICT from the amino
group to the positively charged nitrogen is reduced, facilitated
by an increased electron donation from the cyanide-complexed
boronic acid to the quaternary nitrogen. This reduction in ICT
(effectively unquenching) affords an enhanced and wavelength-
shifted fluorescence as a function of cyanide concentration.
The binding of cyanide to the DSPBA (4-[4-(N,N-dimethyl-
amino)styryl]-1-(x-boronobenzyl)pyridinium bromide (where x
is 2, 3, or 4 for ortho-, meta-, or para, respectively) probes in
the ground state readily leads to changes in the absorption
spectra. These changes are also manifested in the excited state,
where wavelength shifts and intensity changes in the fluores-
cence emission spectra can be readily observed, both the free
and cyanide-bound DSPBA forms being fluorescent when
excited at the isosbestic point, ∼440 nm.
Subsequently, these changes readily allow both an excitation
and emission wavelength ratiometric response to cyanide at
levels of significant physiological importance.1
Results and Discussion
Excitation Wavelength Ratiometric Response to Cyanide.
In the presence of cyanide, the control compound (4-[4-(N,N-
dimethylamino)styryl]-1-benzylpyridinium bromide, DSP), which
does not contain the cyanide chelating group, shows a very slight
response to cyanide (hypsochromic effect), which is attributed
to a trivial cyanide-nitrogen electrostatic interaction19 (Figure3,
top left). However, ortho-, meta-, and para-DSPBA typically
show a hypsochromic shift in the absorption spectra as a function
of cyanide addition (Figure 3). These changes are due to a
reduction in ICT in the conjugated backbone, facilitated by the
electron-donating capability of the R-B-(CN)3 upon cyanide
addition. These wavelength shifts and extinction changes readily
support wavelength ratiometric sensing, as can be seen in Figure
4.
Reduced ICT Resulting in Enhanced Fluorescence Emis-
sion. The signal transduction mechanism employed with the
new probes affords several key probe features, making the
probes unique compared to other cyanide sensing schemes.9-14
The sensing scheme is based on the ability of boronic acid to
complex cyanide18 and its change from being electron deficient
(R-B(OH)2) in the absence of cyanide at physiological pH to
being electron rich (R-B-(CN)3) upon cyanide complexation
(Figures 1 and 2). The modulation in the electron-donating
capabilities of the boronic acid group in the presence and
(14) Anzenbacher, P.; Tyson, D. S.; Jursikova, K.; Castellano, F. N. J. Am.
Chem. Soc. 2002, 124, 6232-6233.
(15) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer/
Academic Plenum Publishers: New York, 1997.
(16) Lakowicz, J. R. Topics in Fluorescence Spectroscopy: Probe Design and
Chemical Sensing; Plenum Press: New York, 1994; Vol. 4.
(17) Richards-Kortum, R.; Sevick-Muraca, E. Annu. ReV. Phys. Chem. 1996,
47, 555-606.
(18) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Anal. Chem. Acta 2004, 522,
9-17.
By plotting the absorption values at 475/375 nm, we can
clearly see that DSP shows little or no response to cyanide,
(19) Geddes, C. D.; Apperson, K.; Karolin, J.; Birch, D. J. S. Anal. Biochem.
2001, 293 (1), 60.
9
3636 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005

Cyanide Detection using Boronic Acid Probes and Reduced ICT
A R T I C L E S
Figure 3. Absorption spectra of DSP in water with increasing cyanide concentrations (top left), and for the ortho-, meta-, and para-DSPBA probes with
increasing cyanide concentrations (top right, bottom left, and bottom right, respectively).
Figure 4 so as to visualize the classic shapes of the binding
isotherms to which eq 1 can readily be applied to determine
the binding/dissociation constants. Interestingly, o-DSPBA
shows a much better response to cyanide than do the other two
isomers. We attribute this to the shorter distance and, therefore,
more effective electrostatic interaction of the positively charged
quaternary nitrogen center with the electron-rich boron atom
upon cyanide complexation.20-22 The cyanide concentrations
of physiological importance can therefore be monitored using
simple absorption-based measurements and ratiometric hypso-
chromic shifts.
For a practical working sensor, which could be potentially
deployed in the field to monitor drinking, stream, or reservoir
waters, etc., it is not practical to determine the cyanide
concentration by using entire absorption spectra. Much better,
however, is an excitation wavelength ratiometric approach which
utilizes two excitation wavelengths and simply ratios the
respective fluorescence intensity bands. This practical approach
requires that fluctuations in excitation photon flux are minimal,
which is easily attainable if one utilizes low power, cheap, light-
emitting diode (LED) and laser diode (LD) sources.23 Such
sources are currently having a major impact in simplifying
remote sensing and indeed in fluorescence spectroscopy as a
whole.15 Using o-DSPBA, we subsequently measured the
intensity at 600 nm as a function of time for both 375 and 475
nm excitation, two wavelengths compatible with current LED
sources15,23 (Figure 5). The emission at 475 nm typically shows
Figure 4. Ratiometric response of the probes in water with increasing
cyanide concentrations.
Figure 5. Fluorescence emission of o-DSPBA in water with time when
excited at 475 and 375 nm.
(20) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Talanta 2005, 65, 762-768.
(21) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Dyes Pigm. 2004, 61, 227-
234.
with o-DSPBA showing a notable change over the range of
physiological importance, that is, up to 30 µM free cyanide.1 It
should be noted that the dynamic response range is typically
2-fold, with the data plotted as the inverse (375/475 nm) in
(22) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. J. Fluoresc. 2003, 13, 371-
374.
(23) Landgraf, S. ReViews in Fluorescence 2004; Geddes, C. D., Lakowicz, J.
R., Eds.; Springer Publishers: New York, 2004.
9
J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005 3637

A R T I C L E S
Badugu et al.
Figure 6. Fluorescence emission spectra of o-DSPBA in water with cyanide for both 375 and 475 nm excitation (left), and the respective ratiometric plot
for the intensity at 600 nm (i.e., 375/475 nm) (right).
a 1% loss in intensity after 1 h. This very slight decrease may
reflect a slight drift in the xenon arc light source of the
fluorometer, as a similar decrease was also observed for the
600 nm emission when excited at 375 nm, noting the y-axis
scale of Figure 5. It is worth noting that the trends in Figure 5
are unlikely to reflect the photostability of DSPBA under these
conditions, as only a small fraction of the 4 × 1 × 1 cm cuvette
was illuminated with moderate intensity (0.5 cm beam diameter),
unperturbed dye readily being able to replenish any photode-
graded DSPBA via rapid diffusion. In any case, our ratiometric
approach removes any issues related to source photon flux drift
on the time scale of data collection.
Figure 6 (left panel) shows the emission spectra of o-DSPBA
in water as a function of cyanide addition when excited at 375
and 475 nm. For both excitation wavelengths, we typically see
a blue-shifted and increased fluorescence emission. This is
consistent with reduced ICT as a function of cyanide binding,
which is described in more detail in the next section. Figure 6
(right panel) shows the subsequent ratiometric response plotted
for fluorescence intensity values from 375/475 nm excitation,
again, plotted to reflect the familiar shape of binding isotherms
and its utility by eq 1, the dynamic sensing range therefore being
>2, that is, when plotted inversely as the intensity from 475/
375 nm excitation. Interestingly, o-DSPBA saturates at about
30 µM free cyanide, a concentration which is considered to be
the lethal free cyanide blood limit.1 In addition, the dynamic
sensing range could be further optimized by choosing other
emission wavelengths; however, we chose the emission maxi-
mum at ∼600 nm since this is optimal with regard to signal
level and would, therefore, be practical for remote sensing
applications.
Figure 7 shows the response of the control compound (DSP)
and the cyanide binding probes (DSPBAs) toward free aqueous
cyanide. DSP typically shows a slight decrease in emission
intensity when excited at 440 nm as a function of cyanide
addition, which is thought to be due to minor collisional
quenching. We subsequently determined the Stern-Volmer
constant25 (KSV) to be 7800 M-1. Importantly, no wavelength
shifts are observed, clearly indicating that for the control
compound, cyanide is not involved in any ICT-based signal
transduction.
Upon cyanide binding to the DSPBA probes, we typically
observe an enhanced and blue-shifted emission spectrum,
indicative of reduced ICT,15 due to the change in electron density
of the boronic acid moiety upon cyanide binding and its
subsequent influence on the charge transfer between the amino
group (donor) and the electron-deficient quaternary nitrogen
center (acceptor) (Figure 7). The response of the ortho probe
is, again, the largest, while beyond the scope of this text, it is
thought to reflect the distance dependence of the through-space
interaction between the electron-deficient nitrogen and the
electron-rich R-B-(CN)3.
We subsequently chose two wavelengths where the intensity
was notable, 600 and 650 nm, and measured the emission
wavelength ratiometric response (Figure 8). As expected, DSP
shows no ratiometric response compared to the classic shapes
of the binding isotherms determined for the DSPBA probes.
Subsequently, from these binding curves and eq 1, we were
able to determine the cyanide dissociation constants to be 4.03,
25.50, and 4.16 µM3 for ortho-, meta-, and para-DSPBA,
respectively.
From a sensing perspective, Figure 8 shows that an ap-
proximately 2-fold dynamic range for cyanide is present,
importantly, using emission wavelengths that provide measur-
able and notable signal levels. Clearly from Figure 8, these
curves could be optimized depending on instrumental parameters
and requirements. The enhanced fluorescence nature of the
signal transduction affords a much better signal-to-noise ratio
for this cyanide-sensing scheme, which is of major significance
compared to others previously published.9-14 Finally, Figure 8
shows that the probes saturate between 20 and 30 µM free
cyanide, a physiological lethal threshold,1 which to the best of
our knowledge, has not been addressed by other small-molecule
sensors.
Emission Wavelength Ratiometric Response to Cyanide.
Both the free (uncomplexed) and bound (complexed) cyanide
forms of the DSPBA probes were found to be fluorescent, which
additionally allows for the possibility of sensing cyanide in an
emission wavelength ratiometric manner, that is, by using one
excitation wavelength and taking the intensity ratio at two
different emission wavelengths. This sensing format is well-
known to have its many advantages,15,24 in particular, the
simplicity and cost of one LED or LD as the excitation source
and two narrow band-pass or interference filters to select the
emission wavelength(s).
(24) Gryczynski, Z.; Gryczynski, I.; Lakowicz, J. R. Methods Enzymol. 2002,
360, 44.
(25) Geddes, C. D. Meas. Sci. Technol. 2001, 12, R53.
9
3638 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005

Cyanide Detection using Boronic Acid Probes and Reduced ICT
A R T I C L E S
Figure 7. Fluorescence spectra of DSP in water with increasing cyanide concentrations (top left), and also for the ortho-, meta-, and para-DSPBA probes
with increasing cyanide concentrations (top right, bottom left, and bottom right, respectively).
for the equilibrium shown in Figure 2, it is difficult to assign
K1, K2, and K3. This is because no additional information is
provided from the ICT-induced optical response, probably
reflecting the fact that further addition of cyanide (i.e., K2 and
K3) does not substantially change the electron density on the
boron atom after the first cyanide complexation (i.e., K1).
In addition, boronic acid-based fluorophores have earned a
well-deserved reputation for sensing diol-containing compounds,
such as monosaccharides,26-31 and both fluoride32 and pH.33
We have found that the binding constants for glucose, fructose,
fluoride, and pH are significantly smaller than those for cyanide,
demonstrating that they are not potential interferents on this
sensing scheme.26-33 In addition, changes in pH in physiologies,
or even drinking waters, are uncommon, and therefore, pH
Figure 8. Emission ratiometric response of the probes in water with
increasing cyanide concentrations.
interference is likely to be minimal also. In this regard, the pKa
of the aromatic amino group of DSPBA is likely to be around
3 and, therefore, not prone to protonation, which could
potentially interfere with the fluorescence signaling mechanism.
Further, given the relatively higher KD values of boronic acid
for fluoride, compared to those for cyanide, and given that the
concentration of fluoride in drinking water fluctuates in the low
Interferences, Scope, and Limitation. As with any sensing
scheme, it is important to determine the degree of interference
from other physiological analytes. We subsequently considered
the response of the probes in the presence of a large excess of
aqueous chloride, which is well-known to collisionally quench
fluorescence15,25 (Figure 9). We can clearly see that the presence
of up to 100 mM NaCl has no interference on the determination
of cyanide up to physiologically lethal levels (Figure 9, bottom
left and right), suggesting that the lifetime of the probe is
relatively short.25 At high cyanide concentrations, where the
physical and therefore optical responses have effectively been
saturated, a slight interference by chloride can be seen. This
reflects the longer lifetime of the DSPBA probes due to reduced
ICT and, therefore, the greater probability of an excited state
chloride collisional encounter.25 It is worth noting that the
fluorophore concentration was 3 µM for these experiments, and
(26) Fang, H.; Kaur, G.; Wang, B. J. Fluoresc. 2004, 14, 481-489.
(27) Kawanishi, T.; Romey, M. A.; Zhu, P. C.; Holody, M. Z.; Shinkai, S. J.
Fluoresc. 2004, 14, 499-512.
(28) Cappuccio, F. E.; Suri, J. T.; Cordes, D. B.; Wessling, R. A.; Singaram B.
J. Fluoresc. 2004, 14, 521-533.
(29) Philips, M. D.; James, T. D. J. Fluoresc. 2004, 14, 549-559.
(30) Cao, H.; Heagy, M. J. Fluoresc. 2004, 14, 569-583.
(31) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. J. Fluoresc. 2004, 14, 617-
633.
(32) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Sens. Actuators, B 2005, 104,
103-110.
(33) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Dyes Pigm. 2004, 61, 227-
234.
(34) Hawkins, R. T.; Snyder, H. R. J. Am. Chem. Soc. 1960, 82, 3863.
9
J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005 3639

A R T I C L E S
Badugu et al.
Figure 9. Absorption and emission spectra of o-DSPBA in water with increasing cyanide concentration and having a 100 mM NaCl background (top), and
the respective excitation and emission ratiometric plots (bottom).
micromolar range,32 these new probes are most unlikely to be
perturbed by fluoride in drinking waters.
affording enhanced fluorescence signals as a function of cyanide
concentration, enabling an enhanced signal-to-noise ratio during
sensing to be achieved. This sensing scheme, while generic to
cyanide by the nature of its specific design and the key
photophysical probe properties, could be applied as a more
general approach for sensing other analytes of interest, such as
fluoride and pH.
Finally, it is worth noting that another key feature of these
cyanide probes is their long wavelength emission, relative to
past boronic acid-based probes.26-31 In fact, a key design feature
of probes for clinical applications is determined, in part, by the
optical properties of water and tissues. The region of low
absorption for tissues and blood is from ∼600 to 900 nm, the
so-called therapeutic range,17 where one minimizes absorption
by hemoglobin and water. Subsequently, these probes were
designed, in part, due to their emission characteristics (>600
nm), making them applicable to either blood sensing/screening
or transdermal cyanide sensing. While we have not tested these
probes in physiological environments to date, the authors are
not aware of any other probes that are capable of transdermally
measuring cyanide levels, never mind using enhanced fluores-
cence-based transduction.
Experimental Section
Methods. All steady-state fluorescence measurements were under-
taken in 4 × 1 × 1 cm fluorometric plastic cuvettes, using a Varian
Cary Eclipse fluorometer, and all absorption measurements were
performed using a Varian UV-vis 50 spectrophotometer.
Data Analysis. Stability (KS) and dissociation (KD) constants were
obtained by fitting the titration curves with cyanide using the relation
Imin + Imax KS[cyanide]
I )
(1)
1 + KS[cyanide]
Conclusions
where Imin and Imax are the initial (no cyanide) and final (plateau)
fluorescence intensities of the titration curves, respectively, where KD
) (1/KS). (KS units are in µM-3 or mol-3 dm9 for CN-, and mM-1 or
mol-1 dm3 for monosaccharides, such as glucose and fructose.18) This
kinetic equation, which is widely used to recover the stability constants
for boronic acid-glucose interactions20-22 has also been utilized here
for cyanide binding with both reliable and reproducible fitting
parameters being obtained. The equation has been used to model the
general cyanide-binding isotherm, and no inference on the values of
K1, K2, or K3 can be made from the data.
We have developed a range of water-soluble fluorescent
probes which are capable of determining cyanide levels up to
physiologically lethal levels in both an excitation and an
emission wavelength ratiometric manner. The probes have a
notable dynamic sensing range and show saturation just after
physiologically lethal levels, making them ideal candidates for
applications of human safeguard. In addition, the emission of
the probes falls within the optical window of blood and tissues,
enabling the possibility for transdermal blood monitoring or even
their use postmortem for the determination of cyanide levels in
fire victims, an area of active research. Finally, the signal
transduction mechanism is one based on reduced charge transfer,
Materials and Preparation of the Cyanide-Sensitive Probes. All
chemicals were purchased from Sigma-Aldrich at the highest purity
available. Bromomethylphenylboronic acids were either purchased from
Combiblocks or prepared from the corresponding methylphenylboronic
9
3640 J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005

Cyanide Detection using Boronic Acid Probes and Reduced ICT
A R T I C L E S
acids using N-bromosuccinimide and a peroxide initiator as described
in the literature.32 The boronic acid containing fluorescent probes (o-,
m-, and p-DSPBA) and a control compound (DSP) were prepared using
the following one-step synthetic procedure, described below for the
control compound DSP. The corresponding o-, m-, or p-boronobenzyl
bromides are employed instead of benzyl bromide to obtain the isomeric
boronic acid derivatives o-, m-, and p-DSPBA, respectively (Figure
1). Equimolar amounts of 4-[4-(N,N-dimethylamino)styryl]pyridine and
benzylbromide were dissolved in 10 mL of dry acetonitrile in a 25 mL
round-bottomed flask equipped with a magnetic stirrer. The reaction
mixture was allowed to stir under an inert atmosphere for 24 h at room
temperature. During this time, a quantitative amount of quaternized
salt was precipitated as a brown-reddish solid. The solid product was
recovered by filtration, washed several times with dry acetonitrile, and
then dried under vacuum for 12 h. The obtained compounds were further
purified for use in the spectral measurements using preparative TLC
(silica gel, 20% methanol in dichloromethane).
13C NMR (DMSO-d6): δ 43.5 (2C), 56.5 (1C), 113.0 (2C), 124.4 (1C),
124.8 (1C), 126.0 (1C), 127.1 (2C), 127.5 (1C), 127.8 (1C), 128.0 (1C),
128.5 (1C), 129.0 (2C), 130.8 (1C), 138.0 (1C), 142.5 (2C), 143.7 (1C),
148.4 (1C). HRMS (FAB+, H2O): m/e calculated 359.1925 (M+),
found 359.1931 (M+).
1
Analytical Data for Compound m-DSPBA. H NMR (D2O): δ
3.1 (s, 6H), 5.9 (s, 2H), 6.8 (d, 2H), 7.2 (d, 1H), 7.5-7.8 (m, 4H),
7.8-8.1 (m, 5H), 8.6 (d, 2H). 13C NMR (DMSO-d6): δ 43.5 (2C),
60.0 (1C), 113.0 (2C), 124.4 (1C), 124.8 (1C), 126.0 (1C), 126.5 (1C),
126.5 (1C), 127.1 (2C), 128.0 (1C), 128.5 (1C), 129.0 (2C), 130.8 (1C),
138.0 (1C), 142.5 (2C), 143.7 (1C), 148.4 (1C). HRMS (FAB+,
H2O): m/e calculated 359.1925 (M+), found 359.1921 (M+).
Analytical Data for Compound p-DSPBA. 1H NMR (D2O): δ 3.1
(s, 6H), 5.7 (s, 2H), 6.8 (d, 2H), 7.1 (d, 1H), 7.5 (d, 2H), 7.7 (d, 2H),
7.9 (d, 3H), 8.0 (d, 2H), 8.8 (d, 2H). 13C NMR (DMSO-d6): δ 43.5
(2C), 59.6 (1C), 113.0 (2C), 124.4 (1C), 124.8 (1C), 126.0 (1C), 127.0
(2C), 127.1 (2C), 128.2 (2C), 129.0 (2C), 130.8 (1C), 138.0 (1C), 142.5
(2C), 143.7 (1C), 148.4 (1C). HRMS (FAB+, H2O): m/e calculated
359.1925 (M+), found 359.1934 (M+).
1
Analytical Data for Compound DSP. H NMR (D2O): δ 3.1 (s,
6H), 5.6 (s, 2H), 6.8 (d, 2H), 7.1 (d, 1H), 7.4-7.6 (m, 5H), 7.7 (d,
2H), 7.9 (d, 1H), 8.0 (d, 2H), 8.7 (d, 2H). 13C NMR (DMSO-d6): δ
43.6 (2C), 59.6 (1C), 113.0 (2C), 124.4 (1C), 124.8 (1C), 125.5 (1C),
127.1 (2C), 128.4 (2C), 129 (2C), 129.2 (2C), 130.8 (1C), 137.7 (1C),
142.5 (2C), 143.7 (1C), 148.4 (1C). HRMS (FAB+, H2O): m/e
calculated 315.1856 (M+), found 315.1861 (M+).
Acknowledgment. This work was supported by the National
Center for Research Resources, RR-08119. Partial salary support
to J.R.L. and C.D.G. from UMBI is also gratefully acknowledged.
Analytical Data for Compound o-DSPBA. 1H NMR (D2O): δ 3.2
(s, 6H), 5.8 (s, 2H), 7.3-7.7 (m, 6H), 7.8-8.2 (m, 6H), 8.8 (d, 2H).
JA044421I
9
J. AM. CHEM. SOC. VOL. 127, NO. 10, 2005 3641