Turn-on fluorescence detection of cyanide in water: Activation of latent fluorophores through remote hydrogen bonds that mimic peptide β-turn motif

Article abstract of DOI:10.1021/ja907056m

A molecular probe was prepared that selectively responds to cyanide in aqueous solutions by fluorescence enhancement. Using the peptide β-turn as a structural template, we designed a series of diphenylacetylene derivatives in which the π-conjugated backbo

Full text of DOI:10.1021/ja907056m

Published on Web 10/15/2009
Turn-On Fluorescence Detection of Cyanide in Water:
Activation of Latent Fluorophores through Remote Hydrogen
Bonds That Mimic Peptide ꢀ-Turn Motif
Junyong Jo and Dongwhan Lee*
Department of Chemistry, Indiana UniVersity, 800 East Kirkwood AVenue,
Bloomington, Indiana 47405
Received August 20, 2009; E-mail: dongwhan@indiana.edu
Abstract: A molecular probe was prepared that selectively responds to cyanide in aqueous solutions
by fluorescence enhancement. Using the peptide ꢀ-turn as a structural template, we designed a series
of diphenylacetylene derivatives in which the π-conjugated backbone was functionalized with an
aldehyde group to render the molecule nonfluorescent. The N-H · · · O hydrogen bond across the 2,2′-
functionalized diphenylacetylene turn motif activates the carbonyl group toward nucleophilic attack,
and chemical transformation of this internal quencher site by reaction with CN- elicits a rapid (k ) 72
M-1 s-1) enhancement in the emission at λmax ) 375 nm. Tethering of an ammonium group to the
hydrogen bond donor fragment significantly increased both the response kinetics and the intensity of
the fluorescence signal. In addition to providing electrostatic attraction toward the CN- ion, this positively
+
charged R-NH3 fragment can engage in a secondary hydrogen bond to facilitate the formation of the
cyanohydrin adduct responsible for the signaling event. The structurally optimized molecular probe 3
responds exclusively to µM-level cyanide in neutral aqueous solutions, with no interference from other
common anions including F- and AcO-.
metal centers6-8 or demetalation of preassembled complexes9-13
to elicit detectable changes in absorption or emission spectra.
Introduction
Cyanide is a toxic anion that binds heme cofactors to inhibit
the terminal respiratory chain enzyme cytochrome c oxidase.1
Cellular hypoxia resulting from such an event often leads to
fatal consequences. Uptake of CN- also raises the concentration
of intracellular Ca2+ to trigger a cascade of enzymatic events
to increase the level of reactive oxygen species (ROS) and
inhibit antioxidant defense systems.2,3 The widespread use of
cyanide in industrial settings (1.5 million tons per year)1 and
potential threats for terrorism continue to engender significant
research efforts directed toward its detection under biologically
relevant conditions.
Cyanide ion can function as a Lewis base or a nucleophile.
Accordingly, detection schemes that capitalize on either property
have been pursued using a diverse array of structural platforms.
For example, a direct coordination of CN- to electron-deficient
trivalent boron perturbs the [p,π*]-conjugation4 or internal
charge transfer (ICT) pathways,5 and thereby modifies the
optical properties of the cyanoboryl adduct. The strong donor
ability of cyanide is also exploited for coordination to transition
Alternatively, nucleophilic attack of CN- on activated carbonyl
groups14-20 or heterocyclic ring systems21-24 can irreversibly
modify the probe molecule with concomitant changes in optical
properties.25
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Scheme 1. Reactivity-Based Detection of CN- by Nucleophilic
Attack on Hydrogen-Bonded Carbonyl Groups
Figure 1. Chemical structures of peptide ꢀ-turn (1), its synthetic mimic
(2), and cyanide probe 3 built on 2,2′-functionalized diphenylacetylene
template. Solid-state structures generated using CSD codes ADUCEH (for
1) and WUTVOW (for 2) are shown for comparison, in which side chains
and N/O substituents have been removed for clarity. Hydrogen bonds are
denoted with dotted lines.
Despite the increasing number of proof-of-principle molecular
prototypes, however, few cyanide detection systems operate in
genuine aqueous environment with turn-on response.5,10,17
Taking inspirations from naturally occurring ꢀ-turn motif 126-28
and its structural mimic 2,29-32 we have designed a diphenyl-
acetylene-based cyanide probe 3 (Figure 1), which (i) operates
in water at pH ) 6-8, (ii) responds by fluorescence enhance-
ment with fast response kinetics (k ) 72 M-1 s-1) and µM-
level detection limit, and (iii) displays high selectivity without
interference from other common anions including fluoride and
acetate. Comparative studies on a series of structural analogues
provided significant insights into the structural and functional
role of intramolecular hydrogen bonds in mechanism-based
cyanide probes, which constitute the main topic of this paper.
Background: Hydrogen Bonds for Anion Detection. Con-
verging arrays of N-H or O-H hydrogen bond donor (HBD)
groups have been studied extensively for anion sensing.33-44
In these elaborate receptor molecules, multiple X-H· · ·Y-
(X-H ) HBD group; Y- ) anion) contacts are achieved by
positioning of X-H groups as part of the macrocyclic backbone
or on the concave side of crescent-shaped hosts. In addition to
functioning as the recognition sites for Lewis basic anions, HBD
groups have also been exploited in a growing number of
reactivity-based CN- indicators,25 in which covalent modifica-
tion of the reporter molecule is facilitated by HBD functioning
as an internal activating group.
As shown in Scheme 1, nucleophilic attack by CN- on the
carbonyl group of a latent chromophore A converts it to the
cyanohydrin adduct B having different absorption/emission
properties. The efficiency of this reactivity-based detection
scheme can significantly be improved by positioning a HBD
group to stabilize the developing negative charge on the carbonyl
oxygen atom. In addition to facilitating the functionally requisite
chemical transformation, the resulting X-H· · ·O- motif in B
rigidifies the molecule by restricting internal torsional motions,45
which can further enhance the emission quantum yield.46,47
The feasibility of this reactivity-based detection scheme has
been demonstrated by a number of fluorogenic molecular probes
that respond to CN- (Scheme 1). Either the N-H· · ·O hydrogen
bond in I16,19,20 and II15a,c,d,48 or the O-H· · ·O hydrogen bond
in III17,24 facilitates the formation of cyanohydrin from nu-
cleophilic attack by CN-, a process that is often assisted by the
electron-withdrawing trifluoromethyl substituent on the carbonyl
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Turn-On Fluorescence Detection of Cyanide in Water
A R T I C L E S
Scheme 2. Cartoon-Type Representation of the
Fluorophore-Quencher-Activator Triad Organized through
Intramolecular Hydrogen Bonds, and Chemical Structures of 3-5
Scheme 3. Synthetic Routes to 3-5a
carbon.19,20,15a,c,d,48 Formation of such cyanide adduct entails
either diminution () turn-off) or, more desirably, enhancement
() turn-on) in the emission intensity of the reporter group.
Among these hydrogen-bonding assisted cyanide-detecting
probes, only one system operates in genuine aqueous environ-
ment with turn-on fluorescence response.17
a
i
Reagents and conditions: (a) HCCSiMe3, Pd(PPh3)2Cl2, CuI, Pr2NH,
THF, r.t., 82%. (b) LiAlH4, THF, 0 °C, 78%. (c) TBAF, THF/H2O, 50 °C,
>99%. (d) EDC, Boc-Gly, CH2Cl2, 40 °C, 83%. (e) 2-Iodobenzaldehyde,
Pd(PPh3)2Cl2, CuI, iPr2NH, THF, r.t. 92% for 4; 78% for 5. (f) HCl, CH2Cl2,
0 °C, >99%.
Results and Discussion
Design and Synthesis. As shown in Figure 1, an appropriately
functionalized diphenylacetylene 2 can restrict C-C bond
rotation along the molecular axis. A robust ꢀ-turn mimic49-56
based on this structural motif was pioneered by Kemp29 and
subsequently exploited as a conformational lock to assist
structural folding and rigidification of phenyleneethylene-based
oligomers.30-32 We envisioned that a simple aldehyde group
could be placed within a similar structural context to furnish 3,
in which the 2,2′-functionalized diphenylacetylene backbone
functions as a π-extended fluorogenic group (Scheme 2). As in
typical aromatic carbonyls, a rapid intersystem crossing from
[n,π*] singlet-state to [π,π*] triplet-state would render the
molecule nonfluorescent,57,58 but chemical transformation of the
carbonyl group to cyanohydrin (Scheme 1) should suppress the
internal quenching process and restore the intrinsic fluorescence
associated with diphenylacetylene. We anticipated that such
fluorescence “turn-on” scheme could be exploited for detection
of CN-. In addition, comparative studies on a series of structural
analogues 3-5 (Scheme 2), which share a common π-conju-
gated backbone but differ in the number of N-H· · ·O hydrogen
bonds and the overall charge, would provide significant insights
into the structure-function relationships.
Our synthesis commenced with installation of a protected
acetylene fragment onto a known ester 6 (Scheme 3). The ester
functionality in the coupling product 7 was reduced to an alcohol
group to enhance water solubility (of the products 3 and 4). A
standard EDC-mediated amide coupling with Boc-protected
glycine converted 9 to 10, which was subjected to a Sonogash-
ira-Hagihara coupling with 2-iodobenzaldehyde to furnish 4.
Subsequent deprotection of the Boc group provided 3 as a pale
yellow solid material in an overall 48% yield in six steps from
known materials. Compound 5, lacking the hydrogen-bonding
donor motif, was also prepared in a straightforward manner
(Scheme 3).
Solution Structures Probed by NMR Spectroscopy. An
important consideration in the design of 3 is distal hydrogen
bonding between the aldehyde carbonyl and the N-H groups
across the “turn” motif (Figure 1 and Scheme 2). Experimental
evidence for this functionally required (Scheme 1) noncovalent
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1
interaction was obtained by solution H NMR spectroscopic
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studies. In DMSO-d6 at 298 K, 3 (4.5 mM) displays downfield
proton resonances at 10.44, 10.16, and 8.40 ppm, which are
assigned to the aldehyde C-H, amide N-H, and ammonium
N-H protons, respectively (Figure 2a). Notably, the aniline
amide N-H resonance of 3 at 10.16 ppm is significantly (∆δ
≈ 1 ppm) downfield-shifted relative to that of 10 (Scheme 3)
at 9.13 ppm (Figure 2c), which implicates deshielding from
hydrogen-bonding interaction with the carbonyl group across
the turn motif.59-61
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A R T I C L E S
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are brought in close proximity through the N-H· · ·OdC
contact.29 A geometry-optimized model of 3, generated by
density functional theory (DFT) calculations with the B3LYP
functional and the 6-31G** basis set (Figure 3b,c),62 is also
consistent with this observation. The presence of similar
bifurcated hydrogen bonds59-61 in 4 (Scheme 2) was evidenced
by significantly downfield-shifted N-H proton resonances at
9.38 ppm (for aniline amide) and 5.50 ppm (for Boc-amide) in
CDCl3, relative to the corresponding signals at 8.65 ppm and
5.19 ppm determined for 10. In DMSO-d6, however, these values
change to 9.43 ppm and 5.28 ppm for 4 (Figure 2b) and 9.13
ppm and 5.22 ppm for 10 (Figure 2c). This trend presumably
reflects weaker intramolecular hydrogen bond donor ability of
the bulky Boc-amide N-H group compared with the aniline
amide N-H group and, therefore, less pronounced spectral shift
(∆δ) in hydrogen-bonding solvent DMSO.
Figure 2. Partial 1H NMR spectra of (a) 3, (b) 4, and (c) 10 in DMSO-d6.
Sample concentrations ) 4.5 mM; T ) 298 K. The symbols 0, 9, O, and
b indicate the resonances of the aldehyde C-H, aniline amide N-H,
ammonium N-H, and Boc amide N-H proton, respectively.
The strength of the intramolecular hydrogen-bonding network
1
in 3 (Scheme 2) was assessed by concentration-dependent H
NMR studies. The N-H proton resonances of 3 in DMSO-d6
remain essentially invariant within the concentration range of
0.2-6.0 mM but, beyond this point, display systematic down-
field shifts with increasing concentration (Figure S1). This
concentration-dependent changes in chemical shifts of both
amide N-H proton (from 10.05 to 10.16 ppm) and ammonium
N-H proton (from 8.22 to 8.40 ppm) can best be explained by
the formation of intermolecular hydrogen bonds, which becomes
competitive with intramolecular hydrogen bonds only at high
concentrations. Apparently, a combination of (i) positive charge,
(ii) less steric constraints, and (iii) multiple N-H donor units
are responsible for the strong propensity of 3 to engage in
hydrogen bonds, the functional relevance of which was probed
further in the reaction with CN-.
1
Reaction with Cyanide in Water. With solution H NMR
evidence obtained for the intramolecular hydrogen bonding and
its stability in polar solvent environment (ET(30) ) 45.1 for
DMSO),63,64 we initiated investigating the reactivity of 3 toward
CN-. Specifically, we wished to establish that (i) N-H· · ·OdC
hydrogen bonds across the ꢀ-turn mimic can facilitate nucleo-
philic attack on the carbonyl group by CN-, (ii) this chemical
transformation on the side chain can restore the inherent
fluorescence of the π-conjugated molecular backbone, and (iii)
such reactivity-based turn-on detection can be implemented in
an aqueous environment at physiologically relevant pH.
Preliminary studies established a linear correlation between
the absorbance and concentration of 3 up to 70 µM in H2O
(Figure S2), indicating good water solubility and no interchro-
mophore interactions in solution within the concentration range
of 0-70 µM. Accordingly, all UV-vis (with [3] ) 50 µM)
and fluorescence (with [3] ) 5.0 µM) measurements were
conducted for aqueous solution samples at pH ) 7.0 (HEPES,
10 mM). As shown in Figure 4, 3 displays a strong electronic
transition at λmax,abs ) 350 nm (ε ) 1.21 × 104 M-1 cm-1) but
is only weakly fluorescent with emission quantum yield of ΦF
) 1.3% when excited at λexc ) 270 nm. Addition of NaCN
Figure 3. (a) Partial 2D-ROESY spectrum of 3 (4.5 mM) in DMSO-d6
solution at T ) 298 K. (b) Capped-stick representation of the DFT (B3LYP/
6-31G**) geometry optimized model of 3, and (c) a close-up view showing
hydrogen bonds (dotted red lines) and ROE contacts (double-headed arrows).
The protons contributing to the cross-peaks denoted in (a) are labeled with
the corresponding symbols. Selected interatomic distances: N1 · · · O1 )
3.110 Å; N2· · ·O1 ) 2.613 Å.
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In support of this notion, the 2D-ROESY 1H NMR spectrum
(Figure 3a) taken in DMSO-d6 revealed correlation between the
amide N-H, ammonium N-H, and aldehyde C-H protons that
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Turn-On Fluorescence Detection of Cyanide in Water
A R T I C L E S
Scheme 4. Detection of Cyanide by 3 through the Formation of
Fluorescent Cyanohydrin Adduct 3a
Figure 4. (a) Electronic absorption spectra of 3 () 50 µM) prior to (dotted
line) and after (solid line) addition of NaCN (30 equiv). (b) Fluorescence
response of 3 () 5.0 µM; λexc ) 270 nm) toward increasing amount of
NaCN, with spectral traces corresponding to [NaCN] ) 0, 0.2, 0.5, 0.7,
0.9, 1.2, and 1.5 mM from bottom to top. All measurements were conducted
in water at pH ) 7.0 (HEPES, 10 mM).
Scheme 5. Mechanism of Fluorescence Turn-On Detection of CN-
by Quencher-Modified Quantum Dot (Top) or by
Quencher-Modified Fluorescent Dye Molecule, Such as 3 (Bottom)
1
Figure 5. Partial H NMR spectra of (a) 3 in DMSO-d6, (b) 3 + 5 equiv
NaCN in DMSO-d6/D2O (5:2, v/v), and (c) 3 + 5 equiv (n-Bu4N)CN in
DMSO-d6; [3] ) 4.5 mM, T ) 298 K. The symbols 0, 9, O, and 4 indicate
the resonances of the aldehyde C-H, aniline amide N-H, ammonium N-H,
and benzylic C-H proton, respectively. The lines indicate some corre-
sponding signals. In (b), the amide N-H proton resonance disappears as a
result of H/D exchange with D2O.
(Scheme 4).65 Stabilization of the developing negative charge
in 3a by multiple N-H groups should facilitate the forward
step in this inherently reversible reaction.
(300 equiv) to the aqueous solution of 3, however, elicited a
significant (>7-fold) enhancement in the emission (ΦF ) 10%)
at λmax,em ) 375 nm (Figure 4b), with only a slight decrease in
absorption intensity (Figure 4a).
An efficient chemical transformation of the quencher group
by CN- restores the fluorescence of the π-conjugated molecular
backbone of 3 functioning as a reporter group. A conceptual
linkage can be drawn to cyanide detection schemes implemented
with fluorescent quantum dots (QDs),10,13 in which the reporter
remains nonluminescent in the presence of paramagetic quencher
Cu2+ ion. Coordination of CN- dissociates the metal ion from
the QD surface and thereby elicits a turn-on response (Scheme
5).
The reaction between 3 and cyanide was further analyzed by
a continuous variation method () Job’s plot; Figure S3) to
determine a 1:1 reaction stoichiometry. A LC-MS analysis of
the reaction mixture of 3 and NaCN in H2O at pH ) 7.0
(HEPES, 10 mM; Figure S4) revealed two major components,
which were identified as 3 (calculated for [3]+, 309.1239; found,
309.1249) and its cyanide adduct (calculated for [3 + CN +
H]+, 336.1348; found, 336.1352) based on high-resolution MS
analysis (Figures S5 and S6). Addition of NaCN (5 equiv) in
D2O to a DMSO-d6 solution (4.5 mM) of 3 resulted in the
disappearance of the aldehyde proton signal at 10.44 ppm with
concomitant development of a new resonance at 6.00 ppm,
which was assigned to a benzylic proton (Figure 5b). Addition
of cyanide (5 equiv) as a tetrabutylammonium salt resulted in
a similar spectral change, with the benzylic proton of the reaction
product appearing at 6.50 ppm with complete disappearance of
the aldehyde signal of the starting material (Figure 5c). Notably,
the amide N-H proton resonance of the reaction product at
10.49 ppm (Figure 5c) is significantly downfield shifted from
that (10.16 ppm) of 3, which implicates a stronger deshielding
from the newly developing hydrogen bond in the cyanide adduct
of 3.
Kinetics of CN- Signaling and Structure-Property
Relationships. Detection of cyanide ion through the reaction
shown in Scheme 4 critically relies on the efficiency of
nucleophilic attack of cyanide on the carbonyl group of 3. Under
pseudo-first-order conditions ([NaCN]0 > 100[3]), an exponential
increase in the emission intensity () ∆I375nm; λexc ) 270 nm)
was observed from the reaction between 3 and cyanide in H2O
(pH ) 7.0; HEPES, 10 mM) at 298 K (Figure 6a). A linear
dependence of the pseudo-first-order rate constant k′ () k[CN-]0;
eq 1) on [CN-]0 (Figures 6b and S7) indicates a rate-limiting
C-C bond formation by nucleophilic attack of CN- on 3 with
the second-order rate constant of k ) 72 M-1 s-1
.
∆I
I
) 1 - e-k′t
(1)
The functional role of intramolecular hydrogen bonds in this
bimolecular reaction was probed further by using the compounds
The spectroscopic and mass-spectrometric data described
above are fully consistent with direct nucleophilic attack by CN-
on the aldehyde group of 3 to form a cyanohydrin adduct 3a
(65) In aqueous solution, an equilibrium between 3a and its N-protonated
form might also be established.
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A R T I C L E S
Jo and Lee
than 4 toward CN-, with second-order rate constants of 91 M-1
s-1 (for 3) and 13 M-1 s-1 (for 4) determined at 288 K (Figures
S8 and S9). The markedly (7-fold) enhanced reactivity of 3
relative to 4 arises apparently from charge-assisted hydrogen
bonds involving a tethered ammonium group that not only
provides electrostatic attraction for the incoming CN- anion,
but also helps stabilize the developing negative charge in the
transition-state leading to the product 3a.67-69 In addition to
this charge effect, the slower response kinetics of 4 toward CN-
might have its origin in the steric constraints imposed by the
bulky Boc substituent on the terminal amine group.
Internal bond rotations facilitate nonradiative decay of dye
molecules in the excited-states.46,47 Binding-induced restriction
of such torsional motions results in fluorescence enhancement.
Signal transduction mechanisms capitalizing on structural ri-
gidification have previously been implemented with synthetic
receptor molecules built on fluorogenic π-systems.70-74 In a
similar manner, the tight hydrogen-bonding network in 3a
should provide additional structural rigidity to the fluorogenic
π-backbone and give rise to a larger net turn-on response than
the corresponding cyanohydrin adduct of 4 (Figure 7). In support
of this notion, the emission quantum yield of the control
compound 12,75,76 having a π-conjugated backbone identical
to that of 3a but lacking the conformational lock provided by
the hydrogen bonds, is only 1.8% in H2O.77 This value is
significantly lower than that (ΦF ) 10%) of 3a.
Figure 6. (a) Time-dependent changes in the fluorescence intensity at λ
) 375 nm (λexc ) 270 nm) observed from the reaction between 3 (5.0 µM)
and NaCN (0.74 mM) in H2O (pH ) 7.0; HEPES, 10 mM) at T ) 298 K.
The gray curve overlaid on the experimental data points is theoretical fit
generated using k′ ) 5.8 × 10-2 s-1. (b) A plot of k′ () k[CN-]0) vs [CN-]0
to derive the second-order rate constant k ) 72 M-1 s-1
.
Practical Considerations for Cyanide Detection: pH
Window, Sensitivity, and Selectivity. Reactivity-based detection
of cyanide ion by 3 can be operated in a wide pH range of
6-8, with consistently large fluorescence enhancement (Figure
8a,b).78 The detection limit at pH ) 7.0 was estimated by a
(67) When the Taft equation68 was used, the pKa of the ammonium
functionality in 3 is estimated to be 8.0, which is close to the value
pKa ) 7.96, experimentally determined for the HCl salt of N-
glycylaniline.69 The CdO· · ·H-N hydrogen bond in 3 should further
enhance the basicity of the terminal amino group. Even a conservative
estimation of pKa ) 8.0 suggests that 90% of 3 should exist as the
protonated form in water at pH ) 7.0.
(68) Perrin, D. D.; Dempsey, B.; Serjeant, E. P. pKa Prediction for Organic
Acids and Bases; Chapman and Hall: Cambridge, U.K., 1981.
(69) Peters, F. B.; Johnson, H. W., Jr. J. Org. Chem. 1975, 40, 1517–
1519.
Figure 7. Changes in the emission spectra of 5.0 µM solution samples of
(a) 3, (b) 4, and (c) 5, prior to (blue) and after (red) addition of NaCN (300
equiv) in MeCN. λexc ) 270 nm; T ) 298 K.
(70) Sandanayake, K. R. A. S.; Nakashima, K.; Shinkai, S. J. Chem. Soc.,
Chem. Commun. 1994, 1621–1622.
(71) (a) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2001, 123,
1260–1261. (b) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc.
2002, 124, 1178–1179.
3-5 (Scheme 2). Due to the limited solubility of 4 and 5 in
H2O, all comparative reactivity and kinetic studies were
conducted in MeCN. As shown in Figure 7, the efficiency of
cyanide detection, as judged by ∆I375nm upon introduction of
the same amount of NaCN to the solution samples (5.0 µM) of
the probes, follows the trend of 3 > 4 . 5. The largest
fluorescence enhancement was observed for 3 having charge-
assisted hydrogen bond, whereas 5 having a simple non-
hydrogen-bonded aldehyde group shows essentially no reactivity
toward CN-.66
(72) Yamada, K.; Nomura, Y.; Citterio, D.; Iwasawa, N.; Suzuki, K. J. Am.
Chem. Soc. 2005, 127, 6956–6957.
(73) Jiang, X.; Vieweger, M. C.; Bollinger, J. C.; Dragnea, B.; Lee, D.
Org. Lett. 2007, 9, 3579–3582.
(74) Opsitnick, E.; Lee, D. Chem.sEur. J. 2007, 13, 7040–7049.
(75) Matsunaga, N.; Kaku, T.; Itoh, F.; Tanaka, T.; Hara, T.; Miki, H.;
Iwasaki, M.; Aono, T.; Yamaoka, M.; Kusaka, M.; Tasaka, A. Bioorg.
Med. Chem. 2004, 12, 2251–2273.
(76) 1H NMR (400 MHz, CDCl3, 298 K): δ 7.52-7.55 (m, 3H), 7.46-
7.48 (m, 1H), 7.34-7.37 (m, 5H). 13C NMR (100 MHz, CDCl3, 298
K): δ 141.2, 131.8, 130.9, 130.2, 128.7, 128.5, 128.5, 126.9, 123.7,
123.3, 89.7, 89.3, 65.1.
Subsequent kinetic studies revealed that, in addition to the
larger signal response (Figure 7a,b), 3 reacts about 7 times faster
(77) Fluorescence quantum yield of diphenylacetylene is only 0.34% at
298 K. See: Ferrante, C.; Kensy, U.; Dick, B. J. Phys. Chem. 1993,
97, 13457-13463.
(66) 1H NMR spectra of 5 (4.5 mM) in DMSO-d6 taken prior to and after
treatment of NaCN (5 equiv) are essentially identical, which further
confirms the lack of reactivity in the absence of intramolecular
hydrogen bonds.
(78) For a wider pH window, a phosphate buffer system was used instead
-
of HEPES. As shown in Figure 8c,d, 3 does not respond to H2PO4
in water.
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Turn-On Fluorescence Detection of Cyanide in Water
A R T I C L E S
restrictions on the structure and number of hydrogen-bonding
donor groups that activate the carbonyl group for the reactivity-
based detection of cyanide. In addition, an effective structural
“decoupling” of the fluorophore, activator, and receptor com-
ponent (Scheme 2) should facilitate structure optimization of
this proof-of-principle probe through a highly modular synthetic
route (Scheme 3). The logical next step is improving the
sensitivity and optical properties (i.e., longer wavelength emis-
sion and higher quantum yield) of this first-generation molecular
prototype, which is the topic of ongoing research in our
laboratory.
Experimental Section
General Considerations. All reagents were obtained from
commercial suppliers and used as received unless otherwise noted.
The solvents MeCN, THF, and dichloromethane were saturated with
nitrogen and purified by passage through activated Al2O3 columns
under nitrogen (Innovative Technology SPS 400). All water used
in this study was purified by an E-pure water filtration system
(Barnstead Thermolyne Co.). The compounds methyl 4-amino-3-
iodobenzoate (6),80 2-iodobenzaldehyde,81 3-(tert-butoxycarbonyl-
amino)propanoic acid (Boc-Gly-OH),82 and (3-ethynylphenyl)-
methanol (11)83 were prepared according to literature procedures.
The synthesis of (3-(2-phenylethynyl)phenyl)methanol (12) has
previously been reported.72 All air-sensitive manipulations were
carried out under nitrogen atmosphere by standard Schlenk-line
techniques.
Figure 8. (a) Emission spectra of 3 (5.0 µM) in H2O at various pH values
(5.7-8.0, phosphate, 0.2 M), and (b) fluorescence enhancement after
addition of NaCN (1.5 mM). (c) Fluorescence response of 3 (5.0 µM) to
various anions in water at pH ) 7.0 (HEPES, 10 mM). The signal intensity
(I) at λ ) 375 nm is normalized with that of the untreated probe (I0). The
bars represent the emission () I/I0) of 3 in the presence of 300 equiv of the
anion of interest: 1, CN-; 2, F-; 3, Cl-; 4, Br-; 5, I-; 6, HSO4-; 7, N3-; 8,
-
HCO3-; 9, PF6-; 10, H2PO4-; 11, OH-; 12, ClO4-; 13, AcO-; 14, NO3
;
15, SCN-, all delivered as sodium salts. (c) The selectivity of 3 for cyanide
in the presence of other anions. The light gray bars represent the emission
of 3 in the presence of 300 equiv of the anion of interest. The black bars
indicate the change in the emission that occurs upon subsequent addition
of 300 equiv of cyanide to the solution containing 3 and the anion of interest.
λexc ) 270 nm; T ) 298 K.
Physical Measurements. 1H and 13C NMR spectra were recorded
on a 300 MHz Varian Gemini 2000 or a 400 MHz Varian Inova
NMR Spectrometer. Chemical shifts were reported versus tetra-
methylsilane and referenced to the residual solvent peaks. High
resolution chemical ionization (CI; using CH4 as CI reagent) and
electrospray ionization (ESI) mass spectra were obtained on a
Thermo Electron Corporation MAT 95XP-Trap. High resolution
GC-MS (CI, using CH4 as CI reagent) was obtained on a Thermo
Electron Corporation MAT 95XP-Trap. FT-IR spectra were re-
corded on a Nicolet Avatar 360 FT-IR spectrometer with EZ
OMNIC ESP software. UV-vis spectra were recorded on a Varian
Cary 5000 UV-vis-NIR spectrophotometer. Fluorescence spectra
were recorded on a Photon Technology International QM-4-CW
Spectrofluorometer with FeliX32 software.
Fluorescence Quantum Yield Measurements. Quantum yields
were determined by standard methods,84 using p-terphenyl (ΦF )
0.93 in cyclohexane solution)85,86 as a standard. The sample
absorbance was maintained less than 0.1 to minimize internal
absorption and corrections were made to account for the differences
in solvent refractive indexes.
plot of ∆I375nm versus cyanide concentration, which shows a
nice linear correlation down to the value of 2.5 µM (Figure
S10). This value is comparable to the WHO guideline of 0.07
mg/L () 2.7 µM) cyanide in drinking water.79 Besides sensitiv-
ity, selectivity is another important criteria in cyanide detection.
As noted in previous studies, competitive binding44 or nucleo-
philic attack15a,b,d by fluoride or acetate ion compromised the
practical utility of certain cyanide sensory systems. As shown
in Figure 8c, among 15 different anions screened, including F-,
Cl-, Br-, I-, HSO4 , N3 , HCO3 , PF6 , H2PO4 , OH-, ClO4 ,
-
-
-
-
-
-
-
AcO-, NO3 , and SCN-, 3 responds exclusively to cyanide in
water at pH ) 7.0 (HEPES, 10 mM). This excellent selectivity
was further highlighted by competition experiments (Figure 8d),
in which a consistent turn-on fluorescence response (>5-fold)
was observed upon addition of cyanide to sample solutions of
3 containing equal concentrations of potentially competing
anions.
Reactivity Studies. Stock solutions of 3-5 in DMSO (2-7 mM)
were diluted with either buffered H2O (pH ) 7.0, 10 mM HEPES)
or MeCN to prepare sample solutions (5-50 µM). Anion stock
solutions (150 mM) of CN-, F-, Cl-, Br-, I-, HSO4-, N3-, HCO3
,
-
Summary and Outlook
PF6-, H2PO4-, OH-, ClO4-, AcO-, NO3-, and SCN- were prepared
by dissolving the corresponding sodium salt in buffered water (pH
) 7.0, 10 mM HEPES). To minimize dilution effects, 3-40 µL
aliquots of the anion stock solution, corresponding to 30-400 equiv
of the substrate, were delivered using microsyringes into a 3.0 mL
sample solution placed in a thermostatted Peltier cuvette holder.
A mechanism-based turn-on fluorescence probe was prepared
that responds exclusively to cyanide in water at pH ) 6-8. In
our structure design, a 2,2′-functionalized diphenylacetylene
motif was employed to introduce multiple (and positively
charged) N-H groups, which converge at the carbonyl group
and activate it for cyanide capture. The functional relevance of
such charge-assisted bifurcated hydrogen bonding was estab-
lished through comparative studies on a set of structural
analogues built on the common π-conjugated molecular scaffold.
Unlike existing CN- probes that have a single O-H/N-H
group installed R or ꢀ to the carbonyl group (Scheme 1), the
use of synthetic ꢀ-turn mimic here significantly alleviates the
(80) Finaru, A.; Berthault, A.; Besson, T.; Guillaumet, G.; Berteina-Raboin,
S. Tetrahedron Lett. 2002, 43, 787–790.
(81) Kurono, N.; Honda, E.; Komatsu, F.; Orito, K.; Tokuda, M. Tetra-
hedron 2004, 60, 1791–1801.
(82) Fiakpui, C. Y.; Knaus, E. E. Can. J. Chem. 1987, 65, 1158–1161.
(83) Zhang, Q.; Takacs, J. M. Org. Lett. 2008, 10, 545–548.
(84) Williams, A. T. R.; Winfield, S. A.; Miller, J. N. Analyst 1983, 108,
1067–1071.
(85) Pavlopoulos, T. G.; Hammond, P. R. J. Am. Chem. Soc. 1974, 96,
6568–6579.
(86) Berlman, I. B. Handbook of fluorescence spectra of aromatic mol-
ecules; Academic Press: New York, 1971.
(79) Guidelines for drinking-water quality; 3rd ed.; World Health Organiza-
tion: Geneva, 2004. Also available at http://www.who.int/water_sani-
tation_health/dwq/guidelines/en/.
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A R T I C L E S
Jo and Lee
Kinetic Measurements. Solution samples (5.0 µM) of 3 and 4
were prepared by diluting the DMSO stock solutions (2-7 mM)
with either buffered water (pH ) 7.0, 10 mM HEPES) or MeCN.
Time-dependent changes in the fluorescence intensity were moni-
tored at 375 nm (λexc ) 270 nm) at 288 K (for measurements in
MeCN) or 298 K (for measurements in water) with constant stirring.
The ∆I versus t kinetic traces were fitted by nonlinear regression
method (OriginPro 8) using eq 1, in which the parameters k′ ()
k[CN-]0) and I () intensity at t f ∞) were allowed to vary.
Methyl 4-Amino-3-((trimethylsilyl)ethynyl)benzoate (7). An
oven-dried round-bottom flask was loaded with 6 (1.14 g, 4.12
mmol), Pd(PPh3)2Cl2 (78 mg, 0.11 mmol), and CuI (70 mg, 0.37
mmol). The headspace was purged with nitrogen and a premixed
was added slowly Boc-Gly-OH79 (0.161 g, 0.848 mmol). After
stirring for 2 h at 40 °C, the reaction mixture was diluted with
EtOAc (100 mL), washed with satd aq solution (3 × 25 mL) of
NaHCO3, dried over anhyd MgSO4, and filtered. Volatile fractions
were removed under reduced pressure and the residual material was
purified by flash column chromatography on SiO2 (CH2Cl2/EtOAc
) 3:1, v/v) to isolate 10 as a pale yellow solid (0.835 g, 83%). 1H
NMR (300 MHz, CDCl3, 298 K): δ 8.65 (br s, 1H), 8.38-8.41 (d,
J ) 8.5 Hz, 1H), 7.47-7.48 (d, J ) 1.8 Hz, 1H), 7.33-7.36 (dd,
J ) 8.5, 1.8 Hz, 1H), 5.19 (br s, 1H), 4.63 (s, 2H), 3.97-3.98 (d,
2H), 3.49 (s, 1H), 1.82 (br s, 1H), 1.47 (s, 9H). 13C NMR (75 MHz,
CDCl3, 298 K): δ 168.1, 156.1, 138.2, 136.6, 130.6, 128.9, 119.3,
111.2, 84.6, 80.7, 78.9, 64.2, 45.4, 28.5. FT-IR (thin film on NaCl,
cm-1): 3349, 2978, 1684, 1590, 1521, 1418, 1368, 1251, 1164,
1050, 831, 660. HRMS (CI) calcd for C16H20N2O4 [M]+, 304.1423;
found, 304.1417.
i
solvent (100 mL, Pr2NH/THF ) 1:2, v/v) was delivered under
nitrogen to the reaction vessel. A portion of trimethylsilylacetylene
() TMSA; 1.0 mL, 7.1 mmol) was added, and the mixture was
stirred for 2 h at r.t. The crude reaction mixture was diluted with
EtOAc (100 mL) and the organic layer was washed with an aq
solution of EDTA (1 M, 3 × 5 mL) and satd aq solution of NaHCO3
(3 × 20 mL), dried over anhyd MgSO4, and filtered. Volatile
fractions were removed under reduced pressure and the residual
material was purified by flash column chromatography on SiO2
(hexanes/EtOAc ) 4:1, v/v) to furnish 6 as a pale yellow solid
(0.835 g, 82%). 1H NMR (300 MHz, CDCl3, 298 K): δ 8.01 (d, J
) 1.6 Hz, 1H), 7.76-7.80 (dd, J ) 8.5, 1.6 Hz, 1H), 6.64-6.66
tert-Butyl-2-(2-((2-formylphenyl)ethynyl)-4-(hydroxy-methyl)-
phenylamino)-2-oxoethylcarbamate (4). This compound was
prepared using 10 (0.454 g, 1.49 mmol), 2-iodobenzaldehyde (0.438
g, 1.89 mmol), Pd(PPh3)2Cl2 (54 mg, 0.077 mmol), and CuI (28
mg, 0.15 mmol) in a manner similar to that described for 7. After
stirring for 6.5 h at r.t., the reaction mixture was diluted with EtOAc
(100 mL), washed with aq solution (1 M) of EDTA (3 × 5 mL)
and satd aq solution (3 × 5 mL) of NaHCO3, dried over anhyd
MgSO4, and filtered. Volatile fractions were removed under reduced
pressure and the residual material was purified by flash column
chromatography on SiO2 (CH2Cl2/EtOAc ) 3:1, v/v) to furnish 4
as a yellow solid (0.560 g, 92%). 1H NMR (300 MHz, CDCl3, 298
K): δ 10.10 (s, 1H), 9.38 (br s, 1H), 8.40-8.43 (d, J ) 8.5 Hz,
1H), 7.76-7.79 (d, J ) 7.3 Hz, 1H), 7.49-7.64 (m, 4H), 7.26-7.30
(d, J ) 8.7 Hz, 1H), 5.50 (br s, 1H), 4.61 (s, 2H), 4.24-4.25 (d,
2H), 2.61 (br s, 1H), 1.40 (s, 9H). 13C NMR (75 MHz, CDCl3, 298
K): δ 191.8, 168.7, 156.2, 139.3, 136.4, 135.3, 134.0, 133.9, 130.7,
129.2, 128.7, 122.5, 119.8, 116.3, 111.7, 94.0, 90.9, 80.0, 64.3,
45.0, 28.4. FT-IR (thin film on NaCl, cm-1): 3319, 2977, 2929,
1643, 1589, 1522, 1291, 1165, 1052, 762. HRMS (CI) calcd for
C23H24N2O5 [M]+, 408.1685; found, 408.1670.
(d, J ) 8.5 Hz, 1H), 4.67 (br s, 2H), 3.84 (s, 3H), 0.26 (s, 9H). 13
C
NMR (75 MHz, CDCl3, 298 K): δ 166.6, 152.0, 134.7, 131.7, 119.3,
113.3, 107.1, 100.7, 100.6, 51.8, 0.2. FT-IR (thin film on NaCl,
cm-1): 3481, 3356, 3209, 2955, 2147, 1704, 1627, 1439, 1340,
1324, 1247, 843, 761. HRMS (CI) calcd for C13H17NO2Si [M]+,
247.1029; found, 247.1016.
(4-Amino-3-((trimethylsilyl)ethynyl)phenyl)methanol (8). To
a stirred solution of 7 (0.493 g, 1.99 mmol) in THF (20 mL) at 0
°C was added portionwise LiAlH4 (0.075 g, 1.98 mmol), and the
headspace was sparged with nitrogen. After stirring for 2 h at 0
°C, the reaction was quenched with H2O (0.3 mL) and an aq solution
of NaOH (15%, w/w, 0.1 mL). The organic layer was separated,
dried over anhyd MgSO4, and filtered. Volatile fractions were
removed under reduced pressure and the residual material was
purified by flash column chromatography on SiO2 (hexanes/EtOAc
2-(2-((2-Formylphenyl)ethynyl)-4-(hydroxymethyl)-phenyl-
amino)-2-oxoethanaminium chloride (3). A stream of HCl gas,
generated by using a concd H2SO4 (15 mL) trap, was bubbled
through a stirred CH2Cl2 solution (50 mL) of 4 (0.0436 g, 0.121
mmol) at 0 °C. After stirring for 5 min, the crude product was
isolated by filtration and washed with CH2Cl2 (200 mL) to isolate
1
) 3:1, v/v) to furnish 8 as a pale yellow solid (0.341 g, 78%). H
NMR (300 MHz, CDCl3, 298 K): δ 7.28-7.29 (d, J ) 1.8 Hz,
1H), 7.09-7.13 (dd, J ) 8.2, 1.8 Hz, 1H), 6.65-6.67 (d, J ) 8.2
Hz, 1H), 4.49 (s, 2H), 4.25 (br s, 2H), 1.70 (br s, 1H), 0.26 (s,
9H). 13C NMR (75 MHz, CDCl3, 298 K): δ 147.9, 131.5, 130.4,
129.5, 114.5, 107.9, 101.7, 100.0, 65.0, 0.3. FT-IR (thin film on
NaCl, cm-1): 3352, 2923, 2853, 1687, 1591, 1530, 1422, 1274,
1267, 750, 668. HRMS (CI) calcd for C12H17NOSi [M]+, 219.1079;
found, 219.1068.
1
3 as a pale yellow solid (0.0419 g, 99%). H NMR (300 MHz,
DMSO-d6, 298 K): δ 10.44 (s, 1H), 10.16 (s, 1H), 8.40 (br s, 3H),
7.96-7.98 (d, J ) 7.7 Hz, 1H), 7.85-7.87 (m, 2H), 7.76-7.79
(m, 1H), 7.63-7.67 (m, 2H), 7.41-7.43 (d, J ) 8.5 Hz, 1H), 4.51
(s, 2H), 3.96 (s, 2H). 13C NMR (75 MHz, DMSO-d6, 298 K): δ
192.1, 165.4, 139.6, 137.0, 135.3, 134.3, 133.7, 130.4, 129.6, 129.4,
128.3, 124.1, 122.9, 114.3, 91.7, 91.4, 61.9, 40.9. FT-IR (thin film
on NaCl, cm-1): 3275, 2975, 2861, 2682, 2601, 2206, 1967, 1684,
1460, 1365, 1290, 1198, 1065, 911, 766, 658. HRMS (ESI) calcd
for C18H17N2O3 [M - Cl]+, 309.1239; found, 309.1249.
(4-Amino-3-ethynylphenyl)methanol (9). To a stirred solution
of 8 (0.257 g, 1.17 mmol) in THF (5 mL) was added water (0.1
mL) and a THF solution of TBAF (2.0 mL, 1.0 M). The reaction
was heated at 50 °C for 2.5 h and quenched by adding satd aq
solution (25 mL) of NaHCO3. The organic layer was separated,
dried over anhyd MgSO4, and filtered. Volatile fractions were
removed under reduced pressure and the residual material was
purified by flash column chromatography on SiO2 (CH2Cl2/EtOAc
) 10:1, v/v) to furnish 9 as a pale yellow oil (0.173 g, >99%). 1H
NMR (300 MHz, CDCl3, 298 K): δ 7.32 (d, J ) 1.6 Hz, 1H),
7.14-7.18 (dd, J ) 8.2, 1.6 Hz, 1H), 6.68-6.70 (d, J ) 8.2 Hz,
2-((3-(Hydroxymethyl)phenyl)ethynyl)benzaldehyde (5). This
compound was prepared using 11 (0.338 g, 2.56 mmol), 2-iodo-
benzaldehyde (0.596 g, 2.57 mmol), Pd(PPh3)2Cl2 (83 mg, 0.12
mmol), and CuI (41 mg, 0.22 mmol) in a manner similar to that
described for 7. After stirring for 1.5 h at r.t., the reaction mixture
was diluted with EtOAc (100 mL), washed with aq solution (1 M)
of EDTA (3 × 25 mL) and satd aq solution (3 × 25 mL) of
NaHCO3, dried over anhyd MgSO4, and filtered. Volatile fractions
were removed under reduced pressure and the residual material was
purified by flash column chromatography on SiO2 (CH2Cl2/EtOAc
1H), 4.52 (s, 2H), 4.27 (br s, 2H), 3.38 (s, 1H), 1.58 (br s, 1H). 13
C
NMR (75 MHz, CDCl3, 298 K): δ 148.3, 131.9, 130.5, 129.8, 114.6,
106.7, 82.7, 80.5, 65.1. FT-IR (thin film on NaCl, cm-1): 3393,
3313, 2955, 2879, 2548, 1617, 1577, 1423, 1259, 1020, 833, 750,
668. HRMS (CI) calcd for C9H9NO [M]+, 147.0684; found,
147.0679.
1
) 10:1, v/v) to furnish 5 as a yellow solid (0.470 g, 78%). H
NMR (400 MHz, CDCl3, 298 K): δ 10.58 (s, 1H), 7.89-7.91 (d,
J ) 7.6 Hz, 1H), 7.53-7.60 (m, 3H), 7.40-7.45 (m, 2H),
7.31-7.35 (m, 2H), 4.67 (s, 2H), 2.74 (br s, 1H). 13C NMR (75
MHz, CDCl3, 298 K): δ 191.9, 141.5, 135.8, 133.9, 133.3, 130.8,
130.0, 128.7, 128.7, 127.6, 127.4, 126.8, 122.5, 96.3, 85.0, 64.6.
tert-Butyl-2-(2-ethynyl-4-(hydroxymethyl)phenylamino)-2-
oxoethylcarbamate (10). To a stirred CH2Cl2 solution (10 mL) of
9 (0.106 g, 0.717 mmol) and EDC (0.151 g, 0.790 mmol) at 40 °C
9
16290 J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009

Turn-On Fluorescence Detection of Cyanide in Water
A R T I C L E S
FT-IR (thin film on NaCl, cm-1): 3377, 2849, 1693, 1593, 1489,
1268, 1192, 1033, 761, 690, 638. HRMS (CI) calcd for C16H12O2
[M]+, 236.0837; found, 236.0842.
sions and Soumya Ghosh of the Baik group at Indiana University
for assistance in DFT studies.
Supporting Information Available: Additional spectroscopic
and kinetic data. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by the U.S. Army
Research Office (W911NF-07-1-0533) and the National Science
Foundation (CAREER CHE 0547251). D.L. is an Alfred P. Sloan
Research Fellow. We thank Dr. Ho Yong Lee for helpful discus-
JA907056M
9
J. AM. CHEM. SOC. VOL. 131, NO. 44, 2009 16291