Selective fluorescent detection of flavin adenine dinucleotide in human eosinophils by using bis(Zn2+-dipicolylamine) complex

Article abstract of DOI:10.1021/ja070026r

A novel fluorescent sensor based on the Zn²⁺-DPA complex shows selective detection of flavin adenine dinucleotide (FAD) from among other flavins in water, and it was used to detect FAD in eosinophils by fluorescence microscopy and FACS. Copyrig

Full text of DOI:10.1021/ja070026r

Published on Web 03/24/2007
Selective Fluorescent Detection of Flavin Adenine Dinucleotide in Human
Eosinophils by Using Bis(Zn2+-Dipicolylamine) Complex
Hyun-Woo Rhee,† Ha-Young Choi,‡ Kyungja Han,‡ and Jong-In Hong*,†
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity, Seoul 151-747, Korea,
Department of Laboratory Medicine, Catholic UniVersity Medical College, Seoul, Korea.
Received January 2, 2007; E-mail: jihong@snu.ac.kr
Flavin adenine dinucleotide (FAD) is an important coenzyme in
many biological systems,1 and it is involved in numerous redox
processes of metabolic reactions and biological electron transport.
For example, FAD is an essential component of the electron
transport chain (ETC) of the mitochondria and chlorophylls. It has
been reported that the human white blood cellseosinophilshas a
considerable amount of FAD in its granules,2 and the FAD in
eosinophils plays an important role in redox reactions to generate
hydrogen peroxide or superoxide that can damage a parasitic
organism.2a The essential part of FAD in these biological reactions
is an isoalloxazine ring. This isoalloxazine ring exhibits a strong
fluorescence emission (Q ) 0.26) in the green spectral region
Figure 1. FAD, FMN, and riboflavin.
(excitation, 445 nm; emission, 525 nm).3 The ring is also a part of
flavin mononucleotide (FMN)1d and riboflavin (vitamin B2),1e which
Chart 1. Molecular Probes for FAD
are widely involved in many redox reactions within the cell (Figure
1). Therefore, the flavins (FAD, FMN, and riboflavin) containing
the isoalloxazine nucleus exhibit similar fluorescence emission
spectra in water owing to the presence of the isoalloxazine unit.
Capillary electrophoresis (CE) techniques have been developed
to sensitively detect these flavins.4 However, CE cannot be
applicable to the real-time or in vivo detection of flavins. Hence,
it is necessary to develop a highly selective and sensitive molecular
probe for flavins under physiological conditions.
All the photophysical experiments with flavins were performed
in water (10 mM HEPES, pH 7.4). Changes of FAD fluorescence
emission with 1 are shown in Figure 2A. Fluorescence of FAD
(50 µM) (within a few seconds) increased immediately upon
addition of 1. The fluorescence intensity at 525 nm was increased
approximately 6-fold upon addition of 2 equiv of 1 (Figure 2A),
and the quantum yield of FAD was enhanced by approximately
7-fold from 0.03 to 0.22, which is similar to that of FMN or
riboflavin in water. The enhanced FAD fluorescence could be easily
detected by the naked eye under a UV lamp at 365 nm (Figure
2B).
However, riboflavin (50 µM) shows almost no changes in
fluorescence emission upon addition of 8 equiv of 1. Fluorescence
of FMN (50 µM) that possesses a phosphate group on C5 of the
ribityl chain was slightly quenched upon the addition of 8 equiv of
1 (Figure 4).
There have been many studies that used synthetic molecules for
the molecular recognition of the isoalloxazine ring. Yano5 and
Rotello6 developed synthetic receptors that can recognize the
isoalloxazine ring of flavins based on hydrogen bonding, π-π
stacking, and electrostatic forces in organic solvents. Ko¨nig
employed a method to detect the isoalloxazine ring using Zn2+
-
cyclen-phenothiazine conjugate in water.7 RNA-aptamers were
developed as FAD receptors.8 However, these compounds showed
no or negligible selectivity for riboflavin, FMN, and FAD.
Furthermore, to the best of our knowledge, no study has reported
the development of a small molecular probe that is sensitive and
highly selective, especially for FAD. Here, we report a small
molecular probe that enhances only FAD fluorescence by using
the conformational change of FAD before and after complexation
with a dinuclear Zn2+ complex (1) of 1,3-bis[(bis(2-pyridylmethyl)-
amino)methyl] benzene (see Chart 1).
Fluorescence of FAD showed a slight increase upon addition of
2, which has only one Zn2+-DPA complex. The association constant
of 1 was evaluated to be 1.11 × 105 M-1, but that of 2 was
extremely low and could not be evaluated in water. These results
indicate that the double Zn2+-DPA complexes of 1 were much more
tightly bound to FAD than one Zn2+-DPA complex of 2. Job’s plot
for the binding between 1 and FAD shows a 1:1 binding stoichi-
The intrinsic fluorescence of FAD in an aqueous solution is
considerably lower (Q ) 0.03)3 than that of FMN and riboflavin
(Q ) 0.26)3 because the adenine moiety of FAD is intramolecularly
stacked on the isoalloxazine ring in water, and the fluorescence of
the isoalloxazine ring is almost completely quenched.9
Bis(Zn2+-DPA) (DPA ) dipicolylamine) complexes are known
to be strongly bound to phosphate or a phosphodiester group in
water.10 1 was easily synthesized in two steps with 95% yield (see
the Supporting Information).
1
ometry (see the Supporting Information). H NMR and UV data
indicate that binding of 1 to the diphosphate moiety of FAD forces
the adenine and isoalloxazine rings apart (see the SI).
1 was used as a probe that targeted the FAD present in large
amounts in the eosinophil granules.2 Prior to the addition of 1,
† Seoul National University.
‡ Catholic University Medical College.
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10.1021/ja070026r CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. (A) Fluorescence changes of riboflavin (50 µM) and FMN (50
µM) with addition of 1 (excitation, 445 nm; emission, 525 nm); (B)
fluorescence changes of FAD (50 µM) with 2 (excitation, 445 nm; emission,
525 nm).
from among other flavins in water. The enhancement of fluores-
cence emission of FAD was caused by a change in the intramo-
lecular stacked conformation of FAD in water upon binding of the
phosphodiester group of FAD to 1. This probe was used to detect
FAD in eosinophils by fluorescence microscopy and FACS.
Figure 2. (A) Enhanced fluorescence of FAD (50 µM) upon addition of
1 (excitation, 445 nm; emission, 525 nm); (B) (left) increased FAD (10
µM) fluorescence upon addition of 2 equiv of 1; (right) only FAD
fluorescence (10 µM) under 365 nm UV lamp.
Acknowledgment. This work was supported by a grant from
the KRF (Grant No. KRF-2006-311-C00091). Partial support from
the Seoul R&BD is also acknowledged. H.-W. Rhee is the recipient
of the Seoul Science Fellowship.
Supporting Information Available: Synthesis and results sup-
porting the binding mode between 1 and FAD; complete procedures
of cell study and results with neutrophils. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Bruice, T. C. Acc. Chem. Res. 1980, 13, 256. (b) Ghisla, S.; Massey,
V. Eur. J. Biochem. 1989, 181, 1. (c) Fitzpatrick, P. F. Acc. Chem. Res.
2001, 34, 299. (d) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335. (d)
Walsh, C. Acc. Chem. Res. 1980, 13, 148. (e) Massey, V. Biochem. Soc.
Trans. 2000, 28, 283.
(2) (a) Mayeno, A. N.; Hamann, K. J.; Gleich, G. J. J. Leukocyte Biol. 1992,
51, 172. (b) Weil, G. J.; Chused, T. M. Blood 1981, 57, 1099. (c) Monici,
M.; Pratesi, R.; Bernabei, P. A.; Caporale, R.; Ferrini, P. R.; Croce, A.
C.; Balzarini, P.; Bottiroli, G. J. Photochem. Photobiol. B 1995, 30, 29.
(3) Weber, G. Biochem. J. 1950, 47, 114.
(4) Britz-McKibbin, P.; Markuszewski, M. J.; Iyanagi, T.; Matsuda, K.;
Nishioka, T.; Terabe, S. Anal. Biochem. 2003, 313, 89.
Figure 3. (A) Natural autofluorescence of eosinophils before incubation
with 1; (B) enhanced autofluorescence of eosinophils after 15 min of
incubation with 1 (100 µM) in saline (150 mM NaCl, pH 7.4). The x-axis
in FACS shows the fluorescence intensity of each eosinophil cell and the
y-axis in FACS shows the eosinophil count. After incubation with 1, the
eosinophil distribution shifted to the stronger fluorescence intensity region.
The cells were fixed and permeabilized prior to addition of 1. Argon-ion
laser (488 nm) was used for fluorescence excitation of cell and 530 ((10)
nm emission filter was used in FACS. Scale bar ) 10 µm.
(5) (a) Yano, Y.; Tamura, N.; Mitsui, K.; Nabeshima, T. Chem. Lett. 1989,
1655. (b) Tamura, N.; Kajiki, T.; Nabeshima, T.; Yano, Y. J. Chem. Soc.
Chem. Commun. 1994, 2583 (c) Tamura, N.; Mitsui, K.; Nabeshima, T.;
Yano, Y. J. Chem. Soc., Perkin. Trans. 1994, 2, 2229.
(6) (a) Breinlinger, E.; Niemz, A.; Rotello, V. M. J. Am. Chem. Soc. 1995,
117, 5379. (b) Deans, R.; Cooke, G.; Rotello, V. M. J. Org. Chem. 1997,
62, 836. (c) Breinlinger, E. C.; Keenan, C. J.; Rotello, V. M. J. Am. Chem.
Soc. 1998, 120, 8606. (d) Boal, A. K.; Rotello, V. M. J. Am. Chem. Soc.
1999, 121, 4914.
eosinophils showed weak autofluorescence, and after incubation
with 1 its granules showed bright greenish fluorescence (Figure
3). The enhanced autofluorescence of eosinophils was also detected
by the fluorescence-activated cell sorter (FACS). The increased
autofluorescence of the eosinophils due to addition of 1 can be
used in the flow cytometry for eosinophil count;11 this is crucial to
immunological diagnosis for eosinophilia,11a an abnormal ac-
cumulation of eosinophils in blood or tissue, which occurs in certain
disease states. The control experiment using 2 showed almost no
enhancement of autofluorescence (see the SI). This is in good
agreement with little fluorescence change of FAD upon addition
of 2 as shown in Figure 4.
(7) Konig, B.; Pelka, M.; Zieg, H.; Ritter, T.; Bouas-Laurent, H.; Bonneau,
R.; Desvergne, J. P. J. Am. Chem. Soc. 1999, 121, 1681.
(8) Roychowdhury-Saha, M.; Lato, S. M.; Shank, E. D.; Burke, D. H.
Biochemistry 2002, 41, 2492.
(9) (a) Sarma, R. H.; Dannies, P.; Kaplan, N. O. Biochemistry, 1968, 7, 4359.
(b) Razka, M.; Kaplan, N. O. Proc. Natl. Acad. Sci. U.S.A. 1974, 71,
4546.
(10) (a) Ojida, A.; Inoue, M.; Mito-oka, Y.; Tsutsumi, H.; Sada, K.; Hamachi,
I. J. Am. Soc. Chem. 2006, 128, 2052. (b) Lee, D. H.; Im, J. H.; Son, S.
U.; Chung, Y. K.; Hong, J. I. J. Am. Soc. Chem. 2003, 125, 7752. (c)
Kruppa, M.; Konig, B. Chem. ReV. 2006, 106, 3520.
(11) (a) Rothenberg, M. E. N. Engl. J. Med. 1998, 338, 1592. (b) Weller, P. F.
N. Engl. J. Med. 1991, 324, 1110.
In summary, we have developed a novel fluorescent chemosensor
that uses the Zn2+-DPA complex, which selectively targets FAD
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