Chemically modified dansyl probes: A fluorescent diagnostic for ion and proton detection in solution and in polymers

Article abstract of DOI:10.1021/ol060130y

The fluorescence emission intensity of the dansyl group is significantly diminished upon appending an ethyldimethylamino group to the N1 nitrogen substituent. Addition of acids and metal ions (i.e., Zn²⁺) to solutions of trimethylethylenediamine naphthalene sulfonamide (trinsyl) 2 produces a >25-fold increase in fluorescence intensity. Trinsyl probe 2 has been used as a diagnostic for the diffusion of protons and metal ions in a network polymer as well as an optical reporter for the glass transition temperature.

Full text of DOI:10.1021/ol060130y

ORGANIC
LETTERS
2006
Vol. 8, No. 8
1581-1584
Chemically Modified Dansyl Probes:
A Fluorescent Diagnostic for Ion and
Proton Detection in Solution and in
Polymers
Naphtali A. O’Connor, Steven T. Sakata, Huide Zhu, and Kenneth J. Shea*
Department of Chemistry, UniVersity of California, IrVine, California 92717
kjshea@uci.edu
Received January 16, 2006
ABSTRACT
The fluorescence emission intensity of the dansyl group is significantly diminished upon appending an ethyldimethylamino group to the N1
+
nitrogen substituent. Addition of acids and metal ions (i.e., Zn2 ) to solutions of trimethylethylenediamine naphthalene sulfonamide (trinsyl)
2 produces a >25-fold increase in fluorescence intensity. Trinsyl probe 2 has been used as a diagnostic for the diffusion of protons and metal
ions in a network polymer as well as an optical reporter for the glass transition temperature.
The fluorescence emission of 1,5-dimethylaminonaphthalene
sulfonamide (dansyl) derivatives (i.e., 1) serves as an
important probe in both biological1 and synthetic polymers.2
Dansyl chloride is a widely used derivatization reagent for
end-group analysis of proteins and amino acid detection.
Dansyl derivatives are environmentally sensitive and are
known to exhibit large Stokes shifts along with varying
fluorescence quantum yields due to changes in the local
environment.
emission intensity when compared with the parent dansyl
(1).1a However, upon addition of 1 equiv of acid or 4 equiv
of Zn2+ ion to 2, a >25-fold enhancement of fluorescence
emission intensity is exhibited. The location and substitution
pattern of the aminoethyl group is crucial in producing this
effect. The synthesis of trinsyl 2 is shown in Scheme 1.
Scheme 1. Synthesis of Trinsyl 2
We report that the fluorescence characteristics of this probe
can be profoundly modified by appending an ethyldimethyl-
amino group to the N1 nitrogen substituent. The modified
probe 2 (trinsyl) exhibits significantly reduced fluorescence
(1) (a) Haugland, R. P. Molecular Probes, 6th ed.; Molecular Probes:
Eugene, OR, 1996. (b) Pfleiderer, W.; Matysiak, S.; Bergmann, F.; Schnell,
R. Acta Biochim. Pol. 1996, 43, 37-44. (c) Walker, J. M. Methods Mol.
Biol. 1997, 64, 189-195. (d) Tolosa, L.; Nowaczyk, K.; Lakowicz, J. In
Introduction to Laser Spectroscopy, 2nd ed.; Andrews, D. L., Demidov, A.
A., Eds.; Kluwer Academic/Plenum: New York, 2002; pp 139-170.
(2) (a) Chu, D. Y.; Thomas, J. K. In Photochemistry and Photophysics;
Rabek, J. F., Ed.; CRC: Boca Raton, FL, 1991; Vol. 3. (b) Guillet, J.
Photophysics and Photochemistry; Cambridge University Press: Cambridge,
UK, 1985. (c) Hollaender, A. Surf. Interface Anal. 2004, 36, 1023-1026.
(d) Shea, K. J.; Sasaki, D. Y.; Stoddard, G. J. Macromolecules 1989, 22,
1722-1730.
Plots of the fluorescence emission intensity of a 10-5
solution (CH3CN) of 2 in the presence and absence of CF3-
M
10.1021/ol060130y CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/17/2006

Zn complexed trinsyl 2 in a 1:1 mixture is estimated to be
less than 33%. The binding constant of a 3° aliphatic amine
for a proton is much higher, on the order of 1010 (pKa of
10). In 1:1 mixture of protic acid and aliphatic amine at a
concentration of 10-5 M, approximately 99% of the amine
will be protonated. This agrees with our observations that
approximately 4 equiv of ZnCl2 are needed for complete
fluorescence enhancement versus 1 equiv of CF3CO2H.
Intramolecular quenching of the fluorescence of aromatic
chromophores by amines by photoinduced electron transfer
(PET) is well-precedented5 and has been demonstrated
recently in striking examples of metal-enhanced intensity of
fluorescence emission.6
The fluorescence enhancement accompanying proto-
nation or coordination of metal of the aliphatic amine (N2)
prevents quenching by the pendant amine. Interestingly
the position of this pendant amine was found to be impor-
tant. For example, the fluorescence emission intensity of
Figure 1. Dansyl derivatives and their fluorescence excitation and
emission maxima in acetonitrile (10-5 M).
N,N-dimethylaminoethyl sulfonamide derivative
3 is
essentially identical with that of dansyl benzylamine 1. Upon
addition of 1 equiv of CF3CO2H, the fluorescence emis-
sion intensity decreases 30%. Under similar conditions,
the fluorescence emission intensity of dansyl probe 1
also experiences a slight (3%) decrease. The >25-fold
increase in fluorescene emission intensity of trinsyl probe 2
upon treatment with 1 equiv of CF3CO2H arises from
protonation of the most basic nitrogen (N2), which
removes the amine as a source for intramolecular quenching.
Similar regiochemical effects of PET have been observed
by de Silva et al.7
CO2H are shown in Figure 2a. An analogous plot of
fluorescence intensity as a function of added Zn2+ (CH3-
CN) is shown in Figure 2b. In both cases, added electrophile
Indeed, their explanation for the regiochemical effect on
quenching is consistent with the results of this study. The
donor-acceptor substitution pattern on the naphthalene ring
produces a strong dipole moment (internal charge transfer,
ITC) in the excited state. In models proposed by both de
Silva and Lewis (Figure 3), PET requires overlap of the
ground-state dimethylamino lone pair (N2) with the positive
end of the molecular dipole at N1. This position corresponds
to the LUMO of the excited state. Overlap (and PET) occurs
with facility in 2 but in 3, because of poor overlap of the
N2 lone pair or repulsive interactions of N2 with the induced
electric field of the ITC, PET is not observed.7,8
Figure 2. (a) Emission fluorescence spectra of 10-5 trinsyl 2 in
CH3CN, titrated with CF3CO2H: 0, 0.25, 0.50, and 1 equiv. (b)
Emission fluorescence spectra of 10-5 trinsyl 2 in CH3CN, titrated
with ZnCl2: 0, 1, 2, and 4 equiv (λex ) 332 nm).
Addition of CF3CO2H also produces a blue shift from 339
to 318 nm in the UV spectrum of trinsyl probe 2. This
produces a >25-fold increase in fluorescence emission
intensity. The intensity maximum is achieved at 1 equiv in
the case of protic acid and with 4 equiv in that of Zn2+. The
difference in stoichiometry needed for each electrophile can
be attributed to their respective binding constants to the
dimethylaminoethyl group. The binding constant of ethyl-
enediamine with ZnCl2 is on the order of 105.3 The binding
constant of ZnCl2 for trinsyl 2 is expected to be lower
because of the reduced basicity of N1 due to conjugation
with an electron poor aromatic ring.4
(5) (a) Callan, J. F.; de Silva, A. P.; Ferguson, J.; Huxley, A. J. M.;
O’Brien, A. M. Tetrahedron 2004, 60, 11125-11131. (b) Pischel, U.; Abad,
S.; Miranda, M. A. Chem. Commun. (Cambridge, U.K.) 2003, 1088-1089.
(c) Griesbeck, A. G.; Schieffer, S. Photochem. Photobiol. Sci. 2003, 2, 113-
117. (d) Lewis, F. D.; Wagner-Brennan, J. M.; Denari, J. M. J. Phys. Chem.
A 1998, 102, 519-525. (e) Huston, M. E.; Haider, E. W.; Czarnik, A. W.
J. Am. Chem. Soc. 1988, 110, 4460-4462. (f) de Silva, A. P.; Nimal
Gunaratne, H. Q. J. Chem. Soc., Chem. Commun. 1985, 23, 1669-1670.
(g) Chandross, E. A.; Thomas, H. T. Chem. Phys. Lett. 1971, 9, 393-397.
(6) (a) Lewis, F. D.; Kultgen, S. G. J. Photochem. Photobiol., A 1998,
112, 159-164. (b) Kimura, E.; Koike, T. Chem. Soc. ReV. 1998, 27, 179-
184.
(7) de Silva, A. P.; Nimal Gunaratne, H. Q.; Habib-Jiwan, J.; McCoy,
C. P.; Rice, T. E.; Soumillion, J. Angew. Chem., Int. Ed. Engl. 1995, 34,
1728-1731.
(8) (a) Lewis, F. D.; Li, L.; Kurth, T. L.; Kalgutkar, R. S. J. Am. Chem.
Soc. 2000, 122, 8573-8574. (b) Lewis, F. D.; Ahrens, A.; Kurth, T. L.
Photochem. Photobiol. Sci. 2004, 3, 341-347. (c) Lewis, F. D.; Kalgutkar,
R. S.; Kurth, T. L. J. Phys. Chem. A 2004, 108, 1425-1434.
By using this binding constant and a trinsyl 2 concentration
of 10-5 M, calculation of the equilibrium concentration of
(3) McIntyre, G. H. J.; Block, B. P.; Fernelius, W. C. J. Am. Chem. Soc.
1959, 81, 529-535.
(4) Whidby, J. F.; Leyden, D. E.; Himel, C. M.; Mayer, R. T. J. Phys.
Chem. 1971, 75, 4056-4059.
1582
Org. Lett., Vol. 8, No. 8, 2006

Scheme 2. Synthesis of TMS-Trinsyl 4
Figure 3. Regiochemical effect of pendant amine on intramolecular
quenching of the ICT excited state.
spectral shift may be attributed to the destablilization of the
ICT excited singlet state of the fluorophore by the interaction
of the protonated amine with the positive dipole of the
fluorphore. Similar observations are seen in the UV spectrum
of trinsyl probe 2 when it is titrated with ZnCl2.
and TMS-trinsyl 4 are 9.0 and 12.0, respectively. This
corresponds to a pKa value in aqueous solution for trinsyl 2
The substitution pattern on the aliphatic amine (N2) is also
important. Dansyl derivatives incorporating a simple primary
ethylamine are fluorescent, and protonation of this nitrogen
does not affect fluorescence. Quenching ability is attributed
to the lowered oxidation potential of tertiary amines com-
pared to that of primary amines.9
We attempted the synthesis of a trimethylsilyl-substituted
trinsyl derivative 9 with the goal of obtaining even greater
fluorescence quenching. The C-Si δ orbitals are higher in
energy than C-H or C-C δ orbitals and can interact with
the neighboring nonbonding orbital of the nitrogen.10-12 The
result of this hyperconjugation is a decrease in oxidation
potential9,10,13 and an increase in basicity.14 It was anticipated
this would increase the quenching efficiency of the aliphatic
amine and thus upon protonation effect a larger fluorescence
change.
Attempts at bis alkylation of the primary amine were,
however, unsuccessful; we observed alkylation on both
nitrogens to obtain methylenetrimethylsilyl derivative 10
(Scheme 2). This derivative was carried on to trinsyl
analogue 4 (TMS-trinsyl). The addition of CF3CO2H or
ZnCl2 to TMS-trinsyl 4 had no effect on its fluoresence
emission intensity.
TMS-trinsyl 4 had a similar pH profile to trinsyl 2.
Titration of 10-5 M trinsyl probe 2 and TMS-trinsyl 4 in
aqueous acidic solution with 10 N NaOH is shown in Figure
4. Titrations starting at approximately pH 2 and ending at
pH 12 resulted in a 12.5-fold increase fluorescence intensity
for trinsyl 2 while only a 2-fold increase for TMS-trinsyl 4.
Both exhibit the opposite behavior to dansyl derivative 1,
which has a lowered fluorescence at acidic pH. The pH
values at the midpoints of the titration curve for trinsyl 2
Figure 4. Relative fluorescence intensity (%) vs pH value of 10-5
M trinsyl 2 in aqueous solution and TMS-trinsyl 4. Titration of
TMS-trinsyl 4 was performed in 30% MeOH aqueous solution due
to decreased solubility in H2O. The excitation λ was set at 332 nm
and the fluorescence emission maximum was recorded at each pH
value.
of 9.0, which is quite close to the pKa1 value of tetraethyl-
enediamine (pKa1 ) 9.2 and pKa2 ) 5.2 in water). A pKa
value of 12.0 for TMS-trinsyl 4 also follows our expectations
of an increased basicity. The reduced quenching efficiency
of TMS-trinsyl 4 as compared to that of trinsyl 2 may be a
result of steric hindrance in exciplex formation caused by
the increased size of the trimethylsilyl substituents. It has
been shown that steric bulk can adversely affect the quench-
ing efficiency of amines.15
Trinsyl probe 2 offers a sensitive diagnostic for protons
and metal ions. An example of its utility is given below.
The differential functionality of the dansyl molecule allows
for synthesis of probes that can be covalently incorporated
into synthetic polymers or attached to proteins. Fluorescence
intensity measurements can then be used to monitor proton
(9) Beeson, J. C.; Huston, M. E.; Pollard, D. A.; Venkatachalam, T. K.;
Czarnik, A. W. J. Fluoresc. 1993, 3, 65-68.
(10) Yoshida, J.; Isoe, S. Tetrahedron Lett. 1987, 28, 6621-6624.
(11) Yoshida, J.; Nishiwaki, K. Dalton Trans. 1998, 16, 2589-2596.
(12) Cooper, B. E.; Owen, W. J. J. Organomet. Chem. 1971, 29, 33-
40.
(13) Yoon, U. C. Acc. Chem. Res. 1992, 25, 233-240.
(14) (a) Sommer, L. H.; Rockett, J. J. Am. Chem. Soc. 1951, 73, 5130-
5134. (b) Abel, E. W.; Farrow, G. W. J. Inorg. Nucl. Chem. 1980, 42,
541-545.
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473. (b) Wang, Y. Chem. Phys. Lett. 1985, 116, 286-291.
Org. Lett., Vol. 8, No. 8, 2006
1583

or ion flux.16 We demonstrate this by synthesis and copo-
lymerization of methacrylamide probe 5. To ensure compat-
ibility with free radical polymerization the trifluoroacetic acid
salt 5 was covalently incorporated into a macroporous cross-
linked copolymer by copolymerization with ethyleneglycol
dimethacrylate and methyl methacrylate (10-4:7:3) in aceto-
nitrile. The resulting polymer particles were suspended in
CH2C12 and treated with 4 equiv of diethylamine, which
resulted in a decrease in fluorescence emission intensity.
Addition of 4 equiv of trifluoroacetic acid resulted in
recovery of fluorescence intensity (Figure 5). Similar results
intensity was measured at different temperatures. Figure 6
Figure 6. Fluorescence emission intensity of PMMA doped with
trinsyl 2 as a function of temperature (λem ) 472 nm; λex ) 332
nm).
shows the plot of fluorescence emission intensity versus
temperature. As the temperature increased a decrease in
fluorescence intensity is observed. This is attributed to a
change in free volume of the polymer.19 The excited state
of the probe can return to the ground state by either
fluorescing or internal conversion (dissipation of excess
energy by friction or momentum transfer to the polymer
matrix). As the polymer is heated, its free volume increases
and the probe is more free to rotate and vibrate, causing an
increase in the occurrence of internal conversion. A transition
temperature at approximately 43 °C can be observed in the
plot. This correlates to the glass transition temperature (Tg)
of 42 °C for the material, as measured by DSC.
In summary, we have developed a simple and sensitive
fluorescent probe, trinsyl 2. Unlike dansyl, trinsyl 2 is
sensitive to the presence of Zn2+ ions and protic acids. We
have demonstated its use as a diagnostic of proton and ion
diffusion in solids as well as the determination of Tg. The
application of this probe to the development of sensors for
acidic molecules is currently underway.
Figure 5. Plot of fluorescence emission intensity (λem ) 472 nm;
λex ) 332 nm) vs time for a stirred suspension of macroporous
cross-linked poly(ethylene glycol)dimethacrylate doped with probe
5 in CH2C12. Time A (arrow) corresponds to addition of diethy-
lamine (4:1 amine:probe) and time B (arrow) corresponds to
addition of CF3CO2H (4:1 acid:probe).
are obtained upon addition of Zn2+ ions at the second stage.
These changes in fluorescence intensity were completely
reversible. The rates of recovery and diminution of fluores-
cence emission intensity are related to the rate at which these
reagents diffuse throughout the bulk material.
Fluorescent molecules have been shown to be useful
nondestructive probes for the determination of physical
properties of polymeric materials.17 Blending trinsyl 2 with
commercial PMMA provides a method to determine the Tg
and changes in free volume of the polymer.18
Acknowledgment. Support fron the Joint Institute of
Food Safety and Applied Nutrition is gratefully acknowl-
edged.
Trinsyl molecule 2 and PMMA (Mw ) 3 × 105) were
dissolved in chloroform. After evaporation the solid was
ground into small particles. The fluorescence emission
Supporting Information Available: Experimental details
and characterizations for all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
(16) (a) Klok, H.; Mo¨ller, M. Macromol. Chem. Phys. 1996, 197, 1395-
1409. (b) Shea, K. J.; Stoddard, G. J.; Sasaki, D. Y. Macromolecules 1989,
22, 4303-4308.
OL060130Y
(17) Bosch, P.; Catalina, T. C.; Peinado, C. Chem. Eur. J. 2005, 11,
4314-4325.
(19) Meyer, E. F.; Jamieson, A. M.; Shima, R.; Palmen, J. H. M.; Booij,
H. C.; Maurer, F. H. J. Polymer 1990, 31, 243-247.
(18) Loutfy, R. O. Macromolecules 1981, 14, 270-275.
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Org. Lett., Vol. 8, No. 8, 2006