On the development of sensor molecules that display FeIII- amplified fluorescence

Article abstract of DOI:10.1021/ja050652t

Incorporation of a tailor-made size-restricted dithia-aza-oxa macrocycle, 1-oxa-4,10-dithia-7-aza-cyclododecane, via a phenyl linker into two fluorescent sensor molecules with electronically decoupled, rigidly fixed, and sterically preoriented architectur

Full text of DOI:10.1021/ja050652t

Published on Web 09/13/2005
On the Development of Sensor Molecules that Display
FeIII-amplified Fluorescence
Julia L. Bricks,*,† Anton Kovalchuk,‡ Christian Trieflinger,§ Marianne Nofz,‡
Michael Bu¨schel,§ Alexei I. Tolmachev,† Jo¨rg Daub,*,§ and Knut Rurack*,‡
Contribution from the Institute of Organic Chemistry, National Academy of Sciences of the
Ukraine, 5 Murmanskaya Street, 02094 KieV, Ukraine, DiV. I.3 and V.4, Federal Institute for
Materials Research and Testing (BAM), Richard-Willsta¨tter-Strasse 11, D-12489 Berlin,
Germany, Institute of Organic Chemistry, UniVersity of Regensburg,
D-93040 Regensburg, Germany
Received February 1, 2005; E-mail: knut.rurack@bam.de; joerg.daub@chemie.uni-regensburg.de; timophei@bricks.kiev.ua
Abstract: Incorporation of a tailor-made size-restricted dithia-aza-oxa macrocycle, 1-oxa-4,10-dithia-7-
aza-cyclododecane, via a phenyl linker into two fluorescent sensor molecules with electronically decoupled,
rigidly fixed, and sterically preoriented architectures, a 1,3,5-triaryl-∆2-pyrazoline and a meso-substituted
boron-dipyrromethene (BDP), yields amplified fluorescence in the red-visible spectral range upon binding
of FeIII ions. The response to FeIII and potentially interfering metal ions is studied in highly polar aprotic
and protic solvents for both probes as well as in neat and buffered aqueous solution for one of the sensor
molecules, the BDP derivative. In organic solvents, the fluorescence of both indicators is quenched by an
intramolecular charge or electron transfer in the excited state and coordination of FeIII leads to a revival of
their fluorescence without pronounced spectral shifts. Most remarkably, the unbound BDP derivative shows
dual emission in water and can be employed for the selective ratiometric signaling of FeIII in buffered aqueous
solutions.
Introduction
constantly growing attention. Examples of specific molecular
fluorosensors for less aminophilic metal ions that are at the
bottom of this order, e.g., FeIII, are still scarce.8-12 This is
surprising as especially FeIII for instance plays a key role in
many biochemical processes at the cellular level.13 Iron is
indispensable for most organisms, and both its deficiency and
overload can induce various disorders14 with iron trafficking,
In recent years, the search for selective and sensitive
fluorescent probes for metal ions has tremendously gained in
importance.1 Besides the development of fluoroionophores for
physiologically relevant main group I and II metal ions,2
significant progress has been made in the design of fluorescent
molecular sensors and switches for heavy and transition metal
ions.3 In particular, thiophilic ions4 such as HgII and AgI as well
as “soft”5 transition metal ions that head the Irving-Williams
order6 of complex stabilities, e.g., CuII and ZnII,7 have received
(8) For reviews on fluorescent iron indicators, see: Espo´sito, B. P.; Epsztejn,
S.; Breuer, W.; Cabantchik, Z. I. Anal. Biochem. 2002, 304, 1-18.
Reynolds, I. J. Ann. N. Y. Acad. Sci. 2004, 1012, 27-36.
(9) For iron-responsive fluorescent probes, see: Weizman, H.; Ardon, O.;
Mester, B.; Libman, J.; Dwir, O.; Hadar, Y.; Chen, Y.; Shanzer, A. J. Am.
Chem. Soc. 1996, 118, 12386-12375. Nudelman, R.; Ardon, O.; Hadar,
Y.; Chen, Y.; Libman, J.; Shanzer, A. J. Med. Chem. 1998, 41, 1671-
1678. Hankovszky, O. H.; Kalai, T.; Hideg, E.; Jeko, J.; Hideg, K. Synth.
Commun. 2001, 31, 975-986. Petrat, F.; Weisheit, D.; Lensen, M.; de
Groot, H.; Sustmann, R.; Rauen, U. Biochem. J. 2002, 362, 137-147. Ma,
Y.; Luo, W.; Quinn, P. J.; Liu, Z.; Hider, R. C. J. Med. Chem. 2004, 47,
6349-6362. Tumambac, G. E.; Rosencrance, C. M.; Wolf, C. Tetrahedron
2004, 60, 11293-11297. Ouchetto, H.; Dias, M.; Mornet, R.; Lesuisse,
E.; Camadro, J. M. Bioorg. Med. Chem. 2005, 13, 1799-1803.
† National Academy of Sciences of the Ukraine.
‡ Federal Institute for Materials Research and Testing (BAM).
§ University of Regensburg.
(1) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515-
1566. Valeur, B.; Leray, I. Coord. Chem. ReV. 2000, 205, 3-40. de Silva,
A. P.; McCaughan, B.; McKinney, B. O. F.; Querol, M. Dalton Trans.
2003, 1902-1913.
(2) Tsien, R. Y. Methods Cell Biol. 1989, 30, 127-156. Brownlee, C. Trends
Cell Biol. 2000, 10, 451-457.
(3) Fabbrizzi, L.; Licchelli, M.; Pallavicini, P.; Sacchi, D.; Taglietti, A. Analyst
1996, 121, 1763-1768. Bargossi, C.; Fiorini, M. C.; Montalti, M.; Prodi,
L.; Zaccheroni, N. Coord. Chem. ReV. 2000, 208, 17-32. Rurack, K.
Spectrochim. Acta, Part A 2001, 57, 2161-2195.
(4) For recent representative examples, see: Nolan, E. M.; Lippard, S. J. J.
Am. Chem. Soc. 2003, 125, 14270-14271. Liu, W.; Jiao, T.; Li, Y.; Liu,
Q.; Tan, M.; Wang, H.; Wang, L. J. Am. Chem. Soc. 2004, 126, 2280-
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R. G. J. Am. Chem. Soc. 1963, 85, 3533-3539.
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C.; Park, W. H. Macromol. Rapid Commun. 2000, 21, 951-955. Grabchev,
I.; Chovelon, J. M.; Bojinov, V. Polym. AdV. Technol. 2004, 15, 382-
386. Sumner, J. P.; Kopelman, R. Analyst 2005, 130, 528-533.
(11) A number of unspecific fluorescent sensor molecules or sensory polymers
that also respond to ionic iron have been reported lately as well, see:
Ramachandram, B.; Samanta, A. Chem. Commun. 1997, 1037-1038.
Ghosh, P.; Bharadwaj, P. K.; Roy, J.; Ghosh, S. J. Am. Chem. Soc. 1997,
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9
13522
J. AM. CHEM. SOC. 2005, 127, 13522-13529
10.1021/ja050652t CCC: $30.25 © 2005 American Chemical Society

Probes Showing FeIII-Enhanced Fluorescence
A R T I C L E S
Figure 2. Ionic radii (Å; for six-coordination) of the metal ions employed
in our studies.21 The radius of the simple all-oxygen 12-crown-4 analogue
of AT212C4 was estimated to 0.6-0.7 Å.22
Figure 1. Absorption spectra of FeCl3 (solid black line), Fe(ClO4)3 (dashed
black line), and the title compounds 2 (blue) and 3 (red) in MeCN. The
spectra of the salts were multiplied by a factor of 5.
Chart 1
storage, and balance being tightly regulated in an organism.15
The lack of suitable fluorescent iron indicators is even more
obvious when judged in terms of application-oriented features.
The receptors employed are often synthetically demanding
analogues of ferrichromes or siderophores and, due to the
paramagnetic nature of ionic iron, the indication is mostly
signaled by fluorescence quenching. To the best of our
knowledge, molecular probes that selectively show FeIII-ampli-
fied fluorescence in protic solvents have not been reported yet.
When aiming at the rational development of specific molec-
ular reporters for target transition metal ions, the choice of the
binding motif is a critical factor. At the cellular level, chemically
closely related metal ions frequently are transported by the same
or very similar proteins, and thus, for instance, ZnII or CuII can
interfere with iron binding and transport.16 Therefore, the
strategy that we followed to reach the goal of selective FeIII-
amplified fluorescence signaling equally relied on careful
considerations of the binding preferences of the biochemically
important transition metal ions and on the choice of a powerful
fluorophore as well as a robust supramolecular architecture of
the probe. On one hand, the latter is essential in the sense that
it should prevent undesired communication between guest and
host during the lifetime of the excited state to avoid unspecific
quenching by electron transfer between paramagnetic ion and
fluorophore; yet the probe’s architecture should facilitate
efficient signaling. On the other hand, aqueous as well as organic
solutions of dissolved iron salts are colored and show substantial
absorption in the UV/near-visible spectral range so that the
chromophore should absorb and emit further to the red (Figure
1).
mixed-donor atom (N,S,O) macrocycles.17 Further functional-
ization of such crowned anilines is usually straightforward and
thus allows facile integration of these simple receptor modules
to a great variety of supramolecular signaling architectures.
Targeting FeIII, the wealth of knowledge on synthetic non-heme
iron enzyme mimics18 suggested that a size-restricted aza-oxa-
thia macrocycle might be a promising candidate to achieve the
required discrimination against other small but more aminophilic
or larger and “softer” transition metal ions (for size relations,
see Figure 2). Accordingly, we synthesized the crowned
benzaldehyde 1, containing the 1-oxa-4,10-dithia-7-aza-cy-
clododecane or AT212C4 macrocycle, and integrated it to two
types of fluoroionophores (2,3) (Chart 1) that have been recently
identified by us as suitable platforms to obtain enhanced
emission output in the desired wavelength region upon binding
to a well-known fluorescence quencher such as HgII.19,20
The selection of the receptor unit was driven by our
experience in the design of tailor-made N-phenyl-substituted
(12) In the past few years, several researchers have also successfully employed
modified fluorescent ligands that either bind to a large number of metal
ions (e.g., 8-hydroxyquinoline) or that are usually used for the indication
of other cations (e.g., calcein) for the fluorometric monitoring of ionic iron,
see: Thomas, F.; Serratrice, G.; Beguin, C.; Aman, E. S.; Pierre, J. L.;
Fontecave, M.; Laulhere, J. P. J. Biol. Chem. 1999, 274, 13375-13383.
Pierre, J. L.; Baret, P.; Serratrice, G. Curr. Med. Chem. 2003, 10, 1077-
1084. Ali, A.; Zhang, Q.; Dai, J.; Huang, X. BioMetals 2003, 16, 285-
293.
(13) Aisen, P.; Wessling-Resnick, M.; Leibold, E. A. Curr. Opin. Chem. Biol.
1999, 3, 200-206. Eisenstein, R. S. Annu. ReV. Nutr. 2000, 20, 627-662.
(14) Andrews, N. C. N. Engl. J. Med. 1999, 341, 1986-1195. Touati, D. Arch.
Biochem. Biophys. 2000, 373, 1-6.
(15) Cairo, G.; Pietrangelo, A. Biochem. J. 2000, 352, 241-250. Beutler, E.;
Felitti, V.; Gelbart, T.; Ho, N. Drug Metab. Dispos. 2001, 29, 495-499.
(16) Askwith, C.; Kaplan, J. Trends Biochem. Sci. 1998, 23, 135-138.
(17) Rurack, K.; Bricks, J. L.; Reck, G.; Radeglia, R.; Resch-Genger, U. J. Phys.
Chem. A 2000, 104, 3087-3109. Rurack, K.; Koval’chuck, A.; Bricks, J.
L.; Slominskii, J. L. J. Am. Chem. Soc. 2001, 123, 6205-6206. Descalzo,
A. B.; Mart´ınez-Ma´n˜ez, R.; Radeglia, R.; Rurack, K.; Soto, J. J. Am. Chem.
Soc. 2003, 125, 3418-3419. Ros-Lis, J. V.; Mart´ınez-Ma´n˜ez, R.; Rurack,
K.; Sanceno´n, F.; Soto, J.; Spieles, M. Inorg. Chem. 2004, 43, 5183-5185.
(18) Mascharak, P. K. Coord. Chem. ReV. 2002, 225, 201-214. Kovacs, J. A.
Chem. ReV. 2004, 104, 825-848.
(19) Rurack, K.; Resch-Genger, U.; Bricks, J. L.; Spieles, M. Chem. Commun.
2000, 2103-2104.
(20) Rurack, K.; Kollmannsberger, M.; Resch-Genger, U.; Daub, J. J. Am. Chem.
Soc. 2000, 122, 968-969.
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J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005 13523

A R T I C L E S
Bricks et al.
Scheme 1. Synthetic Scheme of the Preparation of 1-3a
Figure 3. Absorption and emission spectra of 2 (black) and 2a (red) in
MeCN at 298 K.
Table 1. Selected Spectroscopic Data of 2, Its Complexes, and
2a in Different Solvents at 298 Ka
1
solvent
λ
abs nm
ꢀabs M-1 cm-
λ
em nm
Φf
τf ns
2
2
MeCN
MeOH
MeCN
MeOH
MeCN
MeCN
MeOH
MeCNc
493
502
477
491
478
484
493
714
34,600b
30,900b
38,100
32,800
37,900
36,400b
34,600b
75,000
677
692
662
688
663
670
703
-
0.004
0.002
0.12
0.008
0.14
0.18
0.011
-
0.04
0.02
1.50
0.10
1.72
1.32
0.16
2-FeIII
2-FeIII
2-CuII
2a
2a
2a/FeIII
-
a cdye ) 3 × 10-6 M, λexc ) 480 nm. Counteranion perchlorate. b The
reduction in molar absorptivity is due to the fact that the spectra are broader
in MeOH as compared to those in MeCN. The oscillator strengths, i.e., the
integrals of the longest-wavelength absorption bands, however, are virtually
identical for each dye in both solvents. c FeIII does not trigger Knorr’s
reaction in (neutral) methanol which is conceivable with its reduced
oxidizing power in this solvent and based on the fact that Knorr’s test works
only in the presence of acids in protic solvents.29
a Reagents and conditions: i) NaOiPr/iPrOH; ii) POCl3/DMF; iii)
piperidine/abs. EtOH; iv) phenylhydrazine hydrochloride/AcOH; v) v.i)
CH2Cl2/TFA, v.ii) DDQ, v.iii) BF3‚OEt2, diisopropylethylamine.
Results and Discussion
central five-membered ∆2-pyrazoline ring results in an optimal
preorientation of the three subunits in terms of spectroscopic
recognition features typically displayed by photoinduced electron
transfer (PET) probes.28 Whereas the N(1)-phenyl donor and
the 3-(N-phenyl)naphthalimide acceptor are rather planar and
electronically conjugated, the phenyl-AT212C4 receptor at the
sp3-hybridized C(5) is bent in a pseudospiro-like conformation
(a twist angle of ∼80° between both molecular planes has been
found for similar molecules)27b and cannot directly interact
electronically with the 1,3-chromophore. Moreover for 2, in
particular, the high conjugation of 1- and 3-substituent in
combination with the strong 3-(N-phenyl)naphthalimidyl ac-
ceptor guarantees to obtain broad and well-separated charge-
transfer absorption and emission bands in the red/far-red spectral
region at ca. 500 and 680 nm in highly polar solvents such as
acetonitrile or alcohols (Figure 3, Table 1).19 The composite
nature of the architecture of 2 is further evident from the
absorption spectrum shown in Figure 3. The spectrum is a linear
combination of the transition localized on the 1,3-chromophore
at ca. 500 nm and the characteristic transition localized on the
substituted alkylaminophenyl fragment at ca. 265 nm. Since the
5-anilino receptor is also a strong electron donor, in the unbound
state, a fast excited-state PET process from the 5-fragment to
the 3-acceptor can compete with the CT in the conjugated 1,3-
Synthesis. For the preparation of the phenyl-appended
receptor unit c, we employed modified reactions based on the
chemistry that was utilized to synthesize the parent macrocycle23
as well as several N-alkylated derivatives24 (Scheme 1). Formy-
lation of c then afforded 1.25 The crowned benzaldehyde was
subsequently transformed into the 1,3,5-triaryl-∆2-pyrazoline 2
via the intermediate chalcone e as well as into the meso-
substituted boron-dipyrromethene 3 via a one-pot reaction by
successive use of 2,4-dimethylpyrrole, trifluoroacetic acid
(TFA), dichloro-dicyano-benzoquinone (DDQ), and BF3‚OEt2.
The syntheses of the model compounds 2a and 3a that carry a
phenyl group instead of the anilino crown have been published
previously by us.19,26
Spectroscopic Properties of 2. The 1,3,5-triaryl-∆2-pyrazo-
line27 2 was primarily prepared by us to get a profound picture
of complex formation with FeIII. The 1,3,5-substitution of the
(21) Shannon, R. D. Acta Crystallogr. 1976, 32A, 751-767.
(22) Dalley, N. K. In Synthetic Multidentate Macrocyclic Compounds; Izatt, R.
M., Christensen, J. J., Eds.; Academic Press: New York, 1978; pp 207-
243.
(23) Black, D. S. C.; McLean, I. A. Tetrahedron Lett. 1969, 45, 3961-3964.
(24) Shinkai, S.; Shigematsu, K.; Honda, Y.; Manabe, O. Bull. Chem. Soc. Jpn.
1984, 57, 2879-2884. Youinou, M.-T.; Osborn, J. A.; Collin, J.-P.;
Lagrange, P. Inorg. Chem. 1986, 25, 453-461.
(25) Adapted from Ross, W. C. J. J. Chem. Soc. 1949, 183-191.
(26) Kollmannsberger, M.; Rurack, K.; Resch-Genger, U.; Daub, J. J. Phys.
Chem. A 1998, 102, 10211-10220.
(27) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M. J. Chem. Soc.,
Perkin Trans. 2 1995, 685-690. (b) Rurack, K.; Bricks, J. L.; Schulz, B.;
Maus, M.; Reck, G.; Resch-Genger, U. J. Phys. Chem. A 2000, 104, 6171-
6188. (c) Fahrni, C. J.; Yang, L.; VanDerveer, D. G. J. Am. Chem. Soc.
2003, 125, 3799-3812.
(28) (a) Bissell, R. A.; de Silva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M.;
Maguire, G. E. M.; Sandanayake, K. R. A. S. Chem. Soc. ReV. 1992, 21,
187-195. (b) Rurack, K.; Resch-Genger, U. Chem. Soc. ReV. 2002, 31,
116-127.
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13524 J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005

Probes Showing FeIII-Enhanced Fluorescence
A R T I C L E S
Figure 4. Absorption and emission spectra of 2 (black), 2-FeIII (green),
and the reaction product of 2a and FeIII in MeCN (blue); arrows indicate
the changes measured for 2 upon addition of FeIII. (Inset) Plot according to
the Method of Continuous Variations,30 indicating the 1:1 stoichiometry of
2-FeIII.
Figure 5. ESR spectra of (a) FeIII in MeCN and FeIII:2 ) 2 (b) and ) 1
(c). The asterisk indicates the resonance of a MgO/Cr3+ standard at g′ )
1.9796.
chromophore. As a consequence, the fluorescence of 2 is
drastically quenched as compared to that of model dye 2a that
lacks an electron-rich 5-substitutent, with the band position
remaining virtually unchanged (Figure 3, Table 1). For instance,
based on the data given in Table 1 and a formalism described
in ref 27b, the rate constant of the nonadiabatic intramolecular
electron-transfer process is determined to kET ) 3.05 × 1010
ns-1 in acetonitrile. In protic solvents such as methanol, not
only is the fluorescence of 2 strongly quenched, but the emission
of 2a is also considerably reduced (Table 1). These findings
are obviously related to previous observations by us that
∆2-pyrazolines with such strong 3-acceptors tend to suffer from
fluorescence quenching due to hydrogen-bonding interactions
with solvent molecules in alcohols.27b
Complexation Features of 2. Employing 2 and 2a allowed
us to follow the complexation characteristics of the phenyl-
AT212C4 moiety in more detail. First, binding of the target ion
at the crown engages the lone electron pair of the receptor’s
anilino nitrogen atom in coordination and thus leads to a
decrease of the characteristic absorption band at 265 nm for 2
(Figure 4). Second, the electron-donating potential of the
5-receptor is drastically reduced in its bound state, leading to a
suppression of the PET process and amplified fluorescence with
only minor spectral shifts (Figure 4). The inset of Figure 4
indicates a 1:1 stoichiometry, and formation of the strong
complex (log K > 5.5) is immediate. As a cross-check, the UV/
vis spectra of reference compound 2a, carrying an unsubstituted
phenyl group at the 5-position, were followed upon addition of
FeIII in acetonitrile. Already at equimolar ratios, the color of
the solution is deep blue (Figure 4), conceivable with a redox
reaction which is known as Knorr’s Pyrazoline Test.29 The
product of this test is a planar bis(1-[3-aryl-∆2-pyrazolinyl])-
biphenyl dication with extended electron delocalization in the
entire chromophore,29c exemplified by the intense near-IR
absorption. ESR measurements provided further insight into the
coordination mode (Figure 5). Whereas Fe(ClO4)3 dissolved in
acetonitrile is largely ESR-silent under the experimental condi-
tions used here, gradual formation of 2-FeIII leads to the
Figure 6. Fluorescence enhancement factors for 2 in the presence of various
transition metal ions in MeCN (black; equimolar amounts of metal ions)
and MeOH (white; 5-fold excess of metal ions). The relative fluorescence
yield of 2a vs 2 in both solvents is included on the far right for comparison,
see Table 1 for actual fluorescence quantum yield data.
appearance of a narrow resonance at g′ ) 2.0037 (∆Bpp ) 1
mT), suggesting low-spin complexation in analogy to tetra (or
penta-)-dentate FeIII complexes with mixed donor-atom ligation
- 18
and one (or two) weakly bound sixth ligand(s), e.g., ClO4
.
2 displays favorable selectivity features already in acetonitrile.
Whereas an equimolar amount of FeIII induces a ca. 30-fold
increase in fluorescence, NiII, CoII, and CdII are entirely silent,
ZnII and FeII lead to a 1.5-fold and HgII and AgI to a 2-fold
enhancement. Only CuII yields equally amplified emission in
MeCN (Figure 6). Upon taking a step toward protic solvents,
the spectroscopic characteristics of the host-guest reaction
between 2 and FeIII remain virtually identical. Binding of the
cation leads to increased emission due to the formation of a
1:1 complex with log K ) 4.9. The reduced fluorescence
enhancement factor found in methanol as compared to the
aprotic solvent is in accordance with the considerably low
fluorescence quantum yield of 2a in methanol and the mecha-
nistic implications described above (Figure 6, Table 1). Fur-
thermore, in methanol, no enhanced fluorescence could be
(29) (a) Knorr, L.; Laubmann, H. Chem. Ber. 1888, 21, 1205-1212. (b) Knorr,
L. Chem. Ber. 1893, 26, 100-103. (c) Pragst, F.; Vieth, B. Z. Phys. Chem.
1976, 275, 849-867.
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J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005 13525

A R T I C L E S
Bricks et al.
Table 2. Spectroscopic Data of 3 and 3a in Different Solvents at 298 Ka
1
solventb
λ
abs nm
fwhmc nm
ꢀmax M-1 cm-
λ
em (LE/CT)d nm
fwhmc nm
Φfe
ΦfCT
/
ΦfLE
τf ns
3
MeCN
MeOH
H2O
MOPS
Tris/HCl
MeCN
MeOH
497
497
505
506
508
497
498
23
22
59
59
61
21
19
79,200
78,700
34,800
36,700
33,100
83,100
80,600
508/-
25
26
43/123
52/121
49/124
25
0.0003
0.0008
0.022
0.026
0.027
0.60
-
-
<0.003
<0.003
510/-
508/635
508/634
509/632
505/-
21
0.30/1.94f
11.3
10.0
-
0.18/3.03f
0.17/3.09f
3.17
3ag
508/-
23
0.65
-
3.21
a cdye) 1 × 10-6 M, λexc ) 470 nm for steady-state and 480 nm for time-resolved fluorescence measurements. b MOPS (3-[N-morpholino]propane sulfonic
acid) buffer, adjusted to pH 5.1; Tris (tris-(hydroxymethyl) amino methane)/HCl buffer, adjusted to 5.8. c Full width at half-maximum. d First maximum
corresponds to the typical BDP-localized band, second one to the charge-transfer band. e Overall fluorescence quantum yield. f Major decay components in
the blue () LE) and red () CT) part of the emission spectrum; for a detailed description of such dual emission features, see refs 26,33. g 3a is only very
weakly soluble in water and shows strong tendencies of aggregation already at lower micromolar concentrations.
of 3 at e.g. 508 nm in MeCN is strongly quenched due to an
ultrafast charge-transfer (CT) process from the electron-rich
8-anilino receptor moiety to the electron-accepting BDP (Table
2). In highly polar solvents, the CT process occurs almost
quantitatively, and due to the forbidden nature of CT emission
from perpendicularly oriented ensembles,32 only a weak BDP-
localized (or locally excited, LE) emission and no CT fluores-
cence can be found.20,26 For instance, the fluorescence quantum
yield of 3 in solvents such as MeCN and MeOH is ca. 10-4
,
orders of magnitude lower than that of the 8-phenyl analogue
3a (Φf ≈ 0.6).26 Although strongly quenched, the spectral shape
in fluorescence of 3 is again virtually identical to that of 3a,
reflecting the decoupled architecture of the sensor molecule.
Addition of FeIII to 3 in MeCN accordingly leads to a drastic
increase in LE fluorescence with typical PET features such as
a virtually unchanged band position (Figure 8) and mono-
exponential decay kinetics close to that of the model dye, τf )
2.95 vs 3.17 ns for 3-FeIII vs 3a (Table 3). In this solvent, the
ion selectivity is similar to that of 2 (Figure 9), with CuII leading
also to strongly enhanced fluorescence (τf[3-CuII] ) 2.79 ns).
Emerging to MeOH, the spectral and switching features of 3 in
the presence of FeIII are preserved (τf[3-FeIII] ) 2.71 ns; Figure
8). However, CuII is now less strongly bound, yielding only a
slight fluorescence enhancement and a biexponential decay with
two shorter lifetimes (Figure 9, Table 3), indicative of the
formation of two weak complexes of different conformation
related to the endo/exo isomerization of N-substituted aza
crowns.34 Such a behavior has been found by us before for some
of the complexes of alkali- and alkaline-earth metal ions and a
BDP dye carrying a N-phenyl-monoaza-tetraoxa-15-crown-5
unit at the 8-position.26 The behavior is based on the fact that
the excited state CT process in 8-anilino-substituted BDPs is
exceptionally sensitive to changes in the electron-donating ability
of the unit in the meso-position, also exemplified by the
remarkably high FEF that can be obtained upon cation binding
in dyes such as 3.20,26 As in the case of 2, all the other potential
competitors remain silent in MeOH (Figure 9). The complex
formation features of 3 and FeIII in both organic solvents reflect
the results found for 2 and the target metal ion, i.e. complexation
is immediate with log K > 5.5 in MeCN and 4.8 in MeOH and
1:1 stoichiometries (Figure 8).
Figure 7. Absorption spectra of 3 in MeCN (black), MeOH (red), 0.01 M
MOPS-buffered (green), and 0.01 M Tris/HCl-buffered (blue) aqueous
solution. The absorption spectrum of 3a in MeCN (dotted line) is included
for comparison.
detected for CuII, and all the other metal ions also do not show
any competition (Figure 6).
These results distinguish the AT212C4 macrocycle as a
suitable receptor for FeIII and suggest that sensor molecules with
electronically decoupled, rigidly fixed, and sterically preoriented
architectures are largely immune against the inherently quench-
ing nature of paramagnetic metal ions. However, as the solubility
of 2 is very limited in aqueous media and as these ∆2-
pyrazolines principally suffer from fluorescence quenching in
alcohols, another type of decoupled, rigid, and preoriented probe
scaffold was chosen to obtain an FeIII-selective indicator that
operates in aqueous solutions, the meso-substituted boron-
dipyrromethene (BDP) platform.20,26,28b,31
Spectroscopic and Complexation Properties of 3 in Non-
aqueous Solvents. Figure 7 reveals that in 3, due to methyl
group substitution at the 1,7-positions of the BDP core, the
crowned aniline in the 8- or meso-position is also electronically
decoupled from the main fluorophore, resulting in a linear
combination of the absorption bands as described above for 2.
The spectral absorption features of 3 are thus very similar to
those of 3a and typical for such BDP dyes, e.g., λabs ) 497
nm, ꢀ ≈ 79,000 M-1 cm-1 in organic solvents (Table 2).
Moreover, in the unbound state, the typical BDP fluorescence
(32) Rettig, W. Top. Curr. Chem. 1994, 169, 253-299.
(33) Schuddeboom, W.; Jonker, S. A.; Warman, J. M.; Leinhos, U.; Ku¨hnle,
W.; Zachariasse, K. A. J. Phys. Chem. 1992, 96, 10809-10819. Leinhos,
U.; Ku¨hnle, W.; Zachariasse, K. A. J. Phys. Chem. 1991, 95, 5, 2013-
2021.
(30) Vosburgh, W. C.; Cooper, G. R. J. Am. Chem. Soc. 1941, 63, 437-442.
(31) Kollmannsberger, M.; Gareis, T.; Heinl, S.; Breu, J.; Daub, J. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 1333-1335. Gareis, T.; Huber, C.; Wolfbeis, O.
S.; Daub, J. Chem. Commun. 1997, 1717-1718.
(34) Gokel, G. W.; Echegoyen, L.; Kim, M. S.; Eyring, E. M.; Petrucci, S.
Biophys. Chem. 1987, 26, 225-233. Echegoyen, L.; Gokel, G. W.; Kim,
M. S.; Eyring, E. M.; Petrucci, S. J. Phys. Chem. 1987, 91, 3854-3862.
9
13526 J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005

Probes Showing FeIII-Enhanced Fluorescence
A R T I C L E S
Figure 8. Fluorescence titration spectra of 3 with FeIII in MeCN (A) and MeOH (B). (Inset) Plot according to the Method of Continuous Variations,30
indicating the 1:1 stoichiometry of 3-FeIII.
Table 3. Spectroscopic Data of the Complexes of 3 in Different
Solvents at 298 Ka
λabs fwhmc
ꢀmax
M-1 cm-
λem fwhmc
τf
ns
1
solventb
nm
nm
nm
nm
FEFLE d
3-FeIII MeCN
3-FeIII MeOH
3-FeIII H2O
498
498
506
508
22
22
62
63
65
22
22
80,300
80,200
31,800
33,400
30,600
80,500
78,700
512
513
510
512
511
512
510
27
27
31
31
30
27
26
>500 2.95
>500 2.71
14 3.48
14 3.40
15 3.54
3-FeIII MOPS
3-FeIII Tris/HCl 510
3-CuII MeCN
3-CuII MeOH
500
497
>500 2.79
∼10 0.24, 1.25e
a-c See Table 2. d Cation-induced fluorescence enhancement factor for
the BDP (LE) emission, for respective data of unbound 3, see Table 2.
e Biexponential decay of the LE band, most probably due to the formation
of two complexes with different conformation as has been observed before
for various main group metal ions and another meso-substituted BDP probe
in ref 26. The relative amplitudes are arel[0.24] ) 0.83 and arel[1.25] )
0.17.
Figure 10. Fluorescence spectra of 3 (black) and 3-FeIII (red) in 0.01 M
MOPS buffer. The arrows indicate the changes upon complexation. The
ratiometric signal F634/F511 of a typical titration is included as the left inset
(r2 ) 0.994), a plot according to the Method of Continuous Variations30 in
water as the right inset. Note that Continuous Variations plots tend to flatten
as the complex stability decreases; see, for example a recent discussion in
ref 43.
the BDP-localized LE band and the charge transfer (CT) band
are found at 508 ( 1 and 634 ( 2 nm, respectively, with overall
Φf ) 0.025 ( 0.003. The ratio of Φf[CT]/Φf[LE] is 21 in neat
water and 11 ( 1 in the buffers. Apparently, water is also
strongly solvating the receptor unit so that high quenching rates
as in MeCN or MeOH (Φf ≈ 10-4) cannot be achieved.
Nonetheless, the interaction of the solvent with the anilino
nitrogen is weak enough to generate the red-shifted CT emission,
suggesting that 3 might be suitable for analytically advantageous
ratiometric sensing.35 Indeed, titration with FeIII results in a
displacement of water molecules from the receptor, entailing
an increase of the typical BDP emission and a concomitant
decrease of the CT band (Figure 10). Besides these favorable
ratiometric signaling features, 3 indicates FeIII by a strong
enhancement of BDP fluorescence. Concerning selectivity and
interference in aqueous solution, 3 displays negligible response
to the cations listed before, including CuII (Figure 9). Com-
plexation of FeIII by 3 is again immediate in neat water as well
as the buffered solutions, and the complexes of 1:1 stoichiometry
possess log K ) 4.2, 4.0, and 4.1 in H2O, 0.01 M MOPS, and
0.01 M Tris/HCl buffer, respectively.
Figure 9. Enhancement factors for the LE fluorescence of 3 in the presence
of the biochemically important transition metal ions of the first row in MeCN
(black), MeOH (white), and water (blue). The response toward the metal
ions in the buffered solutions is similar to that in water; see also Table 3
for 3-FeIII.
Spectroscopic and Complexation Properties of 3 in Aque-
ous Solution. In neat as well as buffered (0.01 M MOPS or
Tris/HCl) aqueous solution, the absorption features of 3 are
slightly changed. The intensity of the molecular C-C frame
vibration of 1300 cm-1, which is typical for BDPs, is enhanced,
and the spectra are thus slightly broadened (Figure 7). However,
although the molar absorptivity at the maximum is reduced in
aqueous solution, the integral absorption of the typical lowest-
energy transition localized on the BDP fragment is virtually
unchanged (Table 2). Most importantly, 3 shows dual emission
in all of these aqueous solutions (Figure 10). The maxima of
Conclusion
In this contribution, we have introduced a receptor unit that
allows selective detection of FeIII in various media. By integra-
(35) Silver, R. B. Methods Cell Biol. 1998, 56, 237-251. Dustin, L. B. Clin.
Appl. Immunol. ReV. 2000, 1, 5-15.
9
J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005 13527

A R T I C L E S
Bricks et al.
recrystallized. Then, a solution of 0.3 mmol e and 0.6 mmol phenyl-
hydrazine hydrochloride in 5 mL of acetic acid was heated for 10 h at
100 °C. The resulting red reaction mixture was poured into 50 mL of
ice water and neutralized with 10% aqueous sodium carbonate. The
solid precipitate was separated, washed with water, and purified by
column chromatography (silica/CHCl3). After evaporation of the
solvent, 2 was crystallized from toluene. Dark-red crystals, mp 253-
256 °C, yield 35%. 1H NMR (CDCl3) δ (ppm): 2.782-2.811 (CH2N,
t, J ) 4 Hz, 4H); 2.845-2.887 (S-CH2-CH2-O, t, J ) 6.5 Hz, 4H);
3.355-3.433 (CH2CdN, dd, 1H); 3.724-3.754 (S-CH2-CH2-N,
OCH2, m, 8H); 4.020-4.118 (CH2CdN, dd, 1H); 5.335-5.397
(ArCHN, dd, 1H); 6.875-10.061 (arom. H, 19H). 13C NMR (CDCl3)
δ (ppm): 30.520, 32.288 (CH2-S); 45.126 (CH2 pyrazoline); 63.109
(ArCHN); 73.455 (CH2-O); 113.954; 120.250; 121.594; 122,820;
126.210; 127. 098; 127.773; 128.324; 128.684; 128.895; 129.017;
129.164; 129.340; 129.617; 131.029; 131.543; 134.885; 135.432;
135.650; 143.677; 145.310 (arom. C); 164.020, 164.328 (CdO amide).
Anal. Calcd for C41H38N4O3S2: C 70.46, H 5.48, N 8.02; found C 70.33,
H 5.44, N 8.12.
tion of the unit to two decoupled and rigid molecular architec-
tures, we obtained, to our knowledge, the first examples of
fluorescent probes that selectively show FeIII-amplified emission.
One of the sensor molecules even allows for ratiometric
signaling in aqueous solution with both bands well within the
visible range of the spectrum. Although the complex stability
constants of the couple AT212C4/FeIII are only moderate in neat
and buffered aqueous solutions, the exceptional sensitivity of
the signaling reaction in the boron-dipyrromethene-type probe
3 still allows detection of the target cation in the lower
micromolar range. On one hand, such a strategy might be
valuable in designing probes that only indicate the “free” or
labile ionic form of transition metals in a sample or environ-
ment,36 without liberating the cation from naturally occurring
biotic or abiotic complexes, removing it from a system or
saturating the molecular sensor by irreversible complex forma-
tion. On the other hand, emerging from the present structure to
lariat-type macrocyclic receptors that are equipped with one or
two pendant arms to satisfy the need of the ion for six-
coordination should lead better to related systems with increased
complex stabilities. However, concerning the latter step of
modification, it should be kept in mind that increasing denticity
of the chelating moieties of iron-sensitive probes might result
in drawbacks in selectivity.37 In summary, we believe that the
studies presented here will stimulate further research in the area
of fluorescent sensor molecules for other largely neglected metal
ions such as CoII, FeII, or MnII.
1,3,5,7-Tetramethyl-8-[4-(1-oxa-4,10-dithia-7-aza-cyclododec-7-
yl)-phenyl]-4H-3a,4a-diaza-4-difluorobora-s-indacene (3). The reac-
tion was carried out under nitrogen atmosphere. 0.100 g (0.32 mmol)
of 1 were reacted at room temperature with 0.1 mL (0.096 g, 1.0 mmol)
of 2,4-dimethylpyrrole in 200 mL of CH2Cl2 in the presence of three
drops of trifluoroacetic acid (TFA). After complete conversion of the
benzaldehyde (TLC-control), 0.073 g (0.32 mmol) of dichloro-dicyano-
benzoquinone (DDQ) were added. After stirring for 15 min, 3 mL of
both diisopropyl-ethylamine and BF3‚OEt2 were added. After quenching
the reaction with water and extracting with dichloromethane, the organic
layer was dried over sodium sulfate, and the solvent was removed in
vacuo. Column chromatography on silica using CH2Cl2 as solvent
yielded 0.028 g (0.053 mmol) of 3. Orange plates, mp 218-219 °C,
Experimental Section
1
Materials. All the solvents employed were of UV-spectroscopic
grade and purchased from Aldrich. Metal perchlorates and FeCl3
purchased from Merck, Acros, and Aldrich were of highest purity
available and dried as described previously. Caution: Perchlorate salts
present a potenital explosion hazard and should be handled with care
and possibly only in small quantities. The spectroscopic response of 2
and 3 in the presence of FeIII was independent of the counteranion used,
i.e., perchlorate or chloride.
Synthesis. General Methods. The chemical structures of the
synthesized compounds were confirmed by elemental analysis, 1H
NMR, 13C NMR, and MS, and their purity was checked by reversed
phase HPLC (HPLC set up from Merck-Hitachi; RP18 column;
acetonitrile/water ) 75/25 as eluent) employing UV detection (UV
detector from Knaur; fixed wavelength at 310 nm). NMR spectra were
obtained with a 500 MHz NMR spectrometer Varian Unityplus 500. The
mass spectra were recorded on a Finnigan MAT 95 spectrometer with
an ESI-II/APCI-Source for electrospray ionization and the base peaks
[M + Na]+ were determined.
4-(1-Oxa-4,10-dithia-7-aza-cyclododec-7-yl)-benzaldehyde (1). As
shown in Scheme 1, condensation of a and bis-(2-mercaptoethyl)ether
in the presence of NaOiPr/iPrOH yielded c,23,24 that was then formy-
lated25 to give 1 in 41% yield.
1-Phenyl-3-(N-phenyl-1,8-naphthalimid-4-yl)-5-[4-(1-oxa-4,10-
dithia-7-aza-cyclododec-7-yl)-phenyl]-∆2-pyrazoline (2). For e, a
mixture of 4-acetylnaphthalic acid phenylamide38 d (1 mmol), 1 and
piperidine was refluxed in absolute ethanol for 8-10 h. After cooling,
a solid precipitate was separated, washed with ethanol, purified by
column chromatography (silica, dichloromethane, chloroform), and
yield 17%. H NMR (CDCl3) δ (ppm): 1.50 (γ CH3, s, 6H), 2.54 (R
CH3, s, 6H,), 2.82-2.85 (CH2, m, 4H), 2.91-2.96 (CH2, m, 4H), 3.76-
3.82 (CH2, m, 8H), 5.97 (pyrrole-H, s, 2H), 6.70-7.04 (arom. H, m,
4H). 13C NMR (CDCl3) δ (ppm): 14.6 (+), 14.9 (+), 30.6 (-), 32.4
(-), 50.2 (-), 73.7 (-), 111.4 (+), 120.9 (+), 121.9 (q), 129.1 (+),
132.2 (q), 143.1 (q), 143.2 (q), 148.2 (q), 154.7 (q). MS (EI, 70 eV):
m/z (%) ) 529 (100) [M+]. Anal. Calcd for C25H34BF2N3O1S2: C 61.24,
H 6.47, N 7.93; found C 61.13, H 6.40, N 7.89. FT-IR (KBr) ν˜ )
2963, 2930, 2868, 1541, 1476, 1316, 1187, 1155, 1074, 978, 825 cm-1
.
Optical Spectroscopic Procedures. UV/vis spectra were recorded
on a Bruins Instruments Omega 10 spectrophotometer, and for the
steady-state fluorescence spectra, a Spectronics Instruments 8100
spectrofluorometer was employed. Titrations were performed on a
Perkin-Elmer LS50B spectrofluorometer. All measurements were
carried out at 298 ( 1 K and with upper micro- or millimolar stock
solutions in MeCN. Molar extinction coefficients were determined from
N ) 8 individual samples. For the fluorescence experiments, only dilute
solutions with an optical density (OD) below 0.1 at the absorption
maximum were used, and the fluorescence measurements were
performed with a 90° standard geometry and polarizers set at 54.7°
(emission) and 0° (excitation). The fluorescence quantum yields (Φf)
were determined relative to fluorescein 27 in 0.1 N NaOH (Φf ) 0.90
( 0.03)39 and DCM in methanol (Φf ) 0.43 ( 0.08).40 All the
fluorescence spectra were corrected for the spectral response of the
detection system (calibrated quartz halogen lamp placed inside an
integrating sphere; Gigahertz-Optik) and for the spectral irradiance of
the excitation channel (calibrated silicon diode mounted at a sphere
port; Gigahertz-Optik). The uncertainties of the fluorescence quantum
yields were determined to (5% (for Φf > 0.2), (10% (for 0.2 > Φf
> 0.02), (20% (for 0.02 > Φf > 5 × 10-3), and (30% (for 5 × 10-3
> Φf), respectively.
(36) Pietrangelo, A. J. Hepatol. 2000, 32, 862-864.
(37) Esposito, B. P.; Breuer, W.; Cabantchik, Z. I. Biochem. Soc. Trans. 2002,
30, 729-732.
(38) Adapted from Krasovitskii, B. M.; Afanasiadi, L. M. PreparatiVnaya
Khimiya Organicheskikh LuminoforoV; Folio: Kharkov, 1997; p 186. The
last step in the synthesis of d had to be carried out with propionic acid to
allow for higher reaction temperatures.
(39) Olmsted, J., III. J. Phys. Chem. 1979, 83, 2581-2584.
(40) Drake, J. M.; Lesiecki, M. L.; Camaioni, D. M. Chem. Phys. Lett. 1985,
113, 530-534.
9
13528 J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005

Probes Showing FeIII-Enhanced Fluorescence
A R T I C L E S
Fluorescence lifetimes (τf) were measured with a unique laser
impulse fluorometer with picosecond time resolution described by us
in an earlier publication41 and modified according to a description in
ref 42. Either the second harmonic output of the Ti:sapphire laser or
the frequency quadrupled idler of the OPA were used for excitation.
The fluorescence was collected at right angles (polarizer set at 54.7°;
monochromator with spectral bandwidths of 4, 8, and 16 nm), and the
fluorescence decays were recorded by the single-photon timing method.
While realizing typical instrumental response functions of 25-30 ps
(full width at half-maximum), the time division was 5.2 ps channel-1
and the experimental accuracy amounted to (3 ps, respectively. The
laser beam was attenuated using a double prism attenuator from LTB,
and typical excitation energies were in the nanowatt to microwatt range
(average laser power). The fluorescence lifetime profiles were analyzed
with a PC using the software package Global Unlimited v2.2 (Labora-
the calculated absorbance Aλ′ is plotted vs the molar fraction x of one
reactand. The absorbances measured for the pure solutions of metal
salt (subscript M) and probe (subscript P) are used to calculate Aλ′
according to eq 1. In the experiment, solutions of different molar ratios
are prepared from equimolar solutions of M and P and the actual
absorbance Aλ is measured. Then, Yλ is calculated (eq 2), and when
plotting Yλ vs xM, a maximum at 0.5 corresponds to a complex of 1:1
stoichiometry, respectively (see inset of Figure 4).
A′λ ) xMꢀ(λ)McMd + xPꢀ(λ)PcPd
Yλ ) Aλ - A′λ
(1)
(2)
with cM0 ) cP0 and xM + xP ) 1; d is the optical path length.
Alternatively, continuous variations diagrams can be constructed from
fluorescence data in a similar fashion (see inset of Figure 8).
tory for Fluorescence Dynamics, University of Illinois). The goodness
ESR Spectroscopy. ESR spectra were recorded with an ERS300
spectrometer (Zentrum fu¨r Wissenschaftlichen Gera¨tebau, Berlin) at
X-band frequencies with 20 mW microwave power at 300 K,
respectively. Measurements were performed in narrow silica capillaries
(“Q-band tubes”). For the complexation experiments, 2 and Fe(ClO4)3
of highest purity available were dissolved in acetonitrile at 1.2 × 10-4
M (concentration is limited by the solubility of the probe), and the
respective metal-to-probe ratios were accordingly adjusted by mixing
of the metal solution with either 2 in MeCN or pure MeCN.
2
of the fit of the single decays as judged by reduced chi-squared (øR
)
and the autocorrelation function C(j) of the residuals was always below
øR2 < 1.2. For 3 in aqueous media, decays were recorded at 10 different
wavelengths over the entire emission spectrum and analyzed by global
analysis. Such a global analysis of decays recorded at different emission
wavelengths implies that the decay times of the species are linked while
the program varies the preexponential factors and lifetimes until the
changes in the error surface (ø2 surface) are minimal, i.e., convergence
is reached. The fitting results are judged for every single decay (local
øR2) and for all the decays (global øR2), respectively. The errors for all
Acknowledgment. Financial support by the Deutsche Fors-
chungsgemeinschaft DFG, the Studienstiftung des Deutschen
Volkes, and BAM’s Ph.D. Program is gratefully acknowledged.
The work is part of the Graduate College “Sensory Photo-
receptors in Natural and Artificial Systems” (DFG, GRK 640,
University of Regensburg). We thank M. Spieles (BAM) for
experimental help.
2
the global analytical results were below a global øR ) 1.2.
Method of Continuous Variations.30 For a continuous variations
diagram, the difference Yλ between the measured absorbance Aλ and
(41) Resch, U.; Rurack, K. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 3105, 96-
103.
(42) Shen, Z.; Ro¨hr, H.; Rurack, K.; Uno, H.; Spieles, M.; Schulz, B.; Reck,
G.; Ono, N. Chem. Eur. J. 2004, 10, 4853-4871.
(43) Sayao, A.; Boccio, M.; Asuero, A. G. Int. J. Pharm. 2005, 295, 29-34.
JA050652T
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J. AM. CHEM. SOC. VOL. 127, NO. 39, 2005 13529