Creation of superior carboxyfluorescein dyes by blocking donor-excited photoinduced electron transfer

Article abstract of DOI:10.1021/ol0623926

(Chemical Equation Presented) Carboxyfluoresceins are widely utilized as fluorescence labeling reagents, but we recently found that their emission intensity is markedly decreased after esterification. On the basis of our hypothesis that the fluorescence d

Full text of DOI:10.1021/ol0623926

ORGANIC
LETTERS
2006
Vol. 8, No. 26
5963-5966
Creation of Superior Carboxyfluorescein
Dyes by Blocking Donor-Excited
Photoinduced Electron Transfer
|
Tomoko Mineno,†,§ Tasuku Ueno,† Yasuteru Urano,†,‡ Hirotatsu Kojima,†, and
|
Tetsuo Nagano*,†,
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, and CREST and PRESTO, JST Agency,
4-8-1 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
tlong@mol.f.u-tokyo.ac.jp
Received September 28, 2006
ABSTRACT
Carboxyfluoresceins are widely utilized as fluorescence labeling reagents, but we recently found that their emission intensity is markedly
decreased after esterification. On the basis of our hypothesis that the fluorescence decrease is due to a donor-excited photoinduced electron
transfer (d-PeT) process, we have developed novel carboxyfluorescein derivatives in which the d-PeT process is hampered, and the emission
intensity is not decreased upon esterification. These novel dye derivatives display high quantum yields and are expected to be useful as
labeling agents.
In the field of chemical biology, it is often required to detect
biological substances, such as proteins, DNAs, and sugars,
with high sensitivity and selectivity. Fluorescence labeling
reagents enable the detection of the target biological mol-
ecules through covalent binding.1-4 Organic compound based
fluorescent dyes are widely used because of their small
molecular weight and convenience in use, and the specific
detection is based on their characteristic excitation and
emission properties. Fluorescein5-9 has a relatively high
(2) Alexiev, U.; Marti, T.; Heyn, M. P.; Khorana, H. G.; Scherrer, P.
Biochemistry 1994, 33, 13693-13699.
† The University of Tokyo.
§ Current address: Laboratory of Organic Chemistry, Faculty of Phar-
maceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082,
Japan.
(3) Maeda, H.; Ishida, N.; Kawauchi, H.; Tuzimura, K. J. Biochem. 1969,
65, 777-783.
(4) Hegyvary, C.; Jorgensen, P. L. J. Biol. Chem. 1981, 256, 6294-
6303.
(5) Baeyer, A. Ber. Dtsch. Chem. Ges. 1871, 4, 555-558.
(6) Orndorff, W. R.; Hemmer, A. J. J. Am. Chem. Soc. 1927, 49, 1272-
1280.
‡ PRESTO, JST Agency.
| CREST, JST Agency.
(1) Scherrer, P.; Alexiev, U.; Marti, T.; Khorana, H. G.; Heyn, M. P.
Biochemistry 1994, 33, 13684-13692.
10.1021/ol0623926 CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/29/2006

fluorescence quantum yield (φfl) of 0.85 with the absorption
maximum (Absmax) of 492 nm and the emission maximum
(Emmax) of 511 nm. Prerequisites for fluorescence labeling
reagents are to bear an intensely emitting fluorophore and a
reactive functional group to bind to the target molecule. From
these viewpoints, carboxyfluoresceins with fluorescence
properties similar to fluorescein are very useful as fluores-
cence labeling reagents and have indeed been widely utilized
for biological applications,10-15 although in most cases a
mixture of 5- and 6-carboxylate isomers is used due to the
difficulty of separation.16,17
Recently, we found that the emission intensity of car-
boxyfluoresceins declines markedly upon esterification of
the carboxyl functional groups. For example, t-butyl esteri-
fication of 6-carboxyfluorescein (6-CF) decreased the φfl
value from 0.915 to 0.378 at pH 7.4, and the corresponding
change for 5-carboxyfluorescein (5-CF) was from 0.834 to
0.694 (Figure 1). This suppression of fluorescence emission
of the φfl values (Figure 1). This hypothesis is also supported
by our previous findings on modulation of fluorescence
properties via the d-PeT process: the φfl value of tricar-
boxyfluorescein (0.817) is far higher than that of its trimethyl
ester derivative (0.001).21 We therefore aimed to design and
synthesize novel fluorescence labeling reagents in which the
d-PeT process is hampered and the emission intensity is
maintained even after labeling events.
The process of d-PeT involves transfer of one electron
from the excited xanthene unit to the lowest unoccupied
molecular orbital (LUMO) of the electron-deficient benzene
unit, leading to fluorescence quenching (Figure 2A). Thus,
Figure 2. Electron flow involved in d-PeT and molecular orbital
diagrams. (A) Fluorescence emission is quenched by d-PeT. (B)
Fluorescence emission is restored by preventing d-PeT.
elevation of the LUMO level by raising the electron density
of the benzene unit should prevent the d-PeT process and
result in a high quantum yield of the compound (Figure 2B).
The ease of electron transfer from the electron donor to
the acceptor is predicted by the Rehm-Weller equation21,22
and is correlated with the LUMO energy level.
Figure 1. Fluorescence quantum yields (φfl) and absorption/
emission maxima (nm) of carboxyfluoresceins and their t-butyl
esters in 0.1 M sodium phosphate buffer (pH 7.4).
The LUMO levels of the acceptor units of 6-CFtBu and
5-CFtBu, terephthalic acid mono t-butyl ester (1) and
isophthalic acid mono t-butyl ester (2), were obtained by
means of B3LYP/6-31G calculations. The small quantum
yields of 6-CFtBu and 5-CFtBu can be explained by the low
LUMO levels of their acceptor units 1 and 2, as shown in
Table 1, and this is also supported by our recent findings
regarding fluorescence quenching owing to the electron-
deficient benzene moiety of the acceptor unit.21 The LUMO
levels in Table 1 were calculated for the neutral forms, as
the electron deficiency of the benzoate derivatives of 6-CF
may be rationally explained by the occurrence of a donor-
excited photoinduced electron transfer (d-PeT) process from
the xanthene unit to the benzene unit18-20 because there is
no major shift in Absmax/Emmax accompanying the reduction
(7) Weber, G.; Teale, F. W. J. Trans. Faraday Soc. 1958, 54, 640-648.
(8) Jiao, G.-S.; Han, J. W.; Burgess, K. J. Org. Chem. 2003, 68, 8264-
8267.
(9) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano,
T. J. Am. Chem. Soc. 2005, 127, 4888-4894.
(10) Tobey, S. L.; Anslyn, E. V. Org. Lett. 2003, 5, 2029-2031.
(11) Paradiso, A. M.; Tsien, R. Y.; Machen, T. E. Proc. Natl. Acad. Sci.
U.S.A. 1984, 81, 7436-7440.
(12) Souto, A. A.; Acun˜a, A. U.; Andreu, J. M.; Barasoain, I.; Abal,
M.; Amat-Guerri, F. Angew. Chem., Int. Ed. Engl. 1995, 34, 2710-2712.
(13) Setsukinai, K.; Urano, Y.; Kakinuma, K.; Majima, H. J. J. Biol.
Chem. 2003, 278, 3170-3175.
(14) Takakusa, H.; Kikuchi, K.; Urano, Y.; Sakamoto, S.; Yamaguchi,
K.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 1653-1657.
(15) Kojima, H.; Nanatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.;
Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453.
(16) Rossi, F. M.; Kao, J. P. Y. Bioconjugate Chem. 1997, 8, 495-497.
(17) Ueno, Y.; Guan-Sheng, J.; Kevin, B. Synthesis 2004, 2591-2593.
(18) Miura, T.; Urano, Y.; Tanaka, K.; Nagano, T.; Ohkubo, K.;
Fukuzumi, S. J. Am. Chem. Soc. 2003, 125, 8666-8671.
(19) Kavarnos, G. J.; Turro, N. J. Chem. ReV. 1986, 86, 401-449.
(20) Zhang, H.; Zhou, Y.; Zhang, M.; Shen, T.; Li, Y.; Zhu, D. J. Phys.
Chem. B 2002, 106, 9597-9603.
(21) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.;
Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc.
2004, 126, 14079-14085.
(22) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259-271.
5964
Org. Lett., Vol. 8, No. 26, 2006

mo-4-methylbenzoic acid (11) was converted to the t-butyl
ester 12 (Scheme S1, provided in the Supporting Informa-
tion), which was treated with 3,6-bis(tert-butyldimethyl-
silyloxy)-9H-xanthen-9-one24,25 in the presence of t-BuLi,
followed by deprotection of the dimethylsilyloxy groups and
condensation in 2 N HCl solution. Cleavage of the t-butyl
group of the resultant compound 13 with TFA/CH2Cl2 (1:1)
furnished the free carboxyl acid 7. Scheme S2 (provided in
the Supporting Information) shows the preparation of
compound 8,23 and Scheme S3 (provided in the Supporting
Information) shows that of compound 9. Both of them were
synthesized in good yields using steps and reagents similar
to those in Scheme S1. After Sandmeyer-like reaction from
20 to 21, compound 21 had to be protected as the pyrrolidine
amide 22 for the succeeding reactions (Scheme S4, provided
in the Supporting Information). The amide intermediate 23
was deprotected with 1 N NaOH to yield the carboxylic acid
10, which was then finally transformed to the corresponding
t-butyl ester 24. The identities of the four compounds and
their t-butyl esters were confirmed by spectrometric char-
acterization in advance of further analysis of the fluorescence
properties.
Table 1. LUMO Energy Levels of the Electron-Acceptor
Portions by B3LYP/6-31G Calculations
and 5-CF could not be calcualted. We also measured the
reduction potential of terephthalic acid mono t-butyl ester
(1), the benzene moiety of the less fluorescent derivative
6-CFtBu, and found that the value was in the range
appropriate for d-PeT (Figure S1, provided in the Supporting
Information), as discussed in our previous paper.21 Thus, both
lines of evidence indicate that the small quantum yield of
6-CFtBu can be explained in terms of the low LUMO level
of the acceptor unit 1.
We have recently discovered that replacement of the
carboxyl group at the 2 position of fluorescein with other
groups does not diminish the intensity of fluorescence. The
compounds bearing a methyl or methoxy group at the 2
position, termed 2-Me TokyoGreen and 2-OMe TokyoGreen,
respectively, exhibit φfl values as high as that of fluores-
cein.9,23 We thus conducted B3LYP/6-31G calculations of
the t-butyl esters 3-6, possessing methyl or methoxy groups
in place of carboxylates. Introduction of electron-donating
methyl and methoxy groups appeared to result in higher
LUMO values of 3-6, as predicted. Considering the xan-
thene unit as the electron donor and the benzene unit as the
electron acceptor, we have newly designed carboxyfluores-
cein dyes bearing methyl and methoxy substituents, 7-10
(Figure 3). On the basis of our hypothesis that a high LUMO
The fluorescence of the dyes 7-10 and their t-butyl esters
13, 16, 19, and 24 and the amide 23 (Figure 4) was
Figure 4. Structures of the t-butyl esters 13, 16, 19, and 24 and
the amide 23.
characterized. In view of the equilibrium constant of the
xanthene moiety (pKa ) 6.2), the spectra were recorded in
sodium phosphate buffer of pH 7.4. The Absmax, the
fluorescence Emmax excited at 490 nm, and the φfl values
are summarized in Table 2. All the spectra displayed Absmax
values of around 490 nm, and the newly synthesized t-butyl
esters were found to possess high quantum yields, as
predicted. Among them, compound 13, the t-butyl ester of
5-carboxy-2-Me TokyoGreen (7), displayed the most promi-
nent fluorescence intensity in contrast to the t-butyl ester of
Figure 3. Structures of the proposed carboxyfluorescein dyes,
7-10.
level suppresses d-PeT, these carboxyfluorescein compounds
7-10 were expected to display higher φfl values than 5-CFt-
Bu and 6-CFtBu after esterification.
The synthesis of the designed compounds began with
bromomethylbenzoic and bromomethoxybenzoic acids. 3-Bro-
(23) Mottram, L. F.; Boonyarattanakalin, S.; Kovel, R. E.; Peterson, B.
R. Org. Lett. 2006, 8, 581-584.
(24) Grover, P. K.; Shah, G. D.; Shah, R. C. J. Chem. Soc. 1955, 3982-
3985.
(25) Kenmoku, S.; Urano, Y.; Kanda, K.; Kojima, H.; Kikuchi, K.;
Nagano, T. Tetrahedron 2004, 60, 11067-11073.
Org. Lett., Vol. 8, No. 26, 2006
5965

Table 2. Fluorescence Properties of the Newly Synthesized
Carboxyfluorescein Dyes and Their t-Butyl Esters in 0.1 M
Sodium Phosphate Buffer (pH 7.4)
pH 7.4
a
Absmax
Emmax
φfl valueb
6-CF
492
492
494
494
493
493
495
496
494
495
497
497
498
515
519
518
525
512
514
518
523
514
517
519
521
527
0.915
0.834
0.378
0.694
0.873
0.830
0.753
0.776
0.877
0.771
0.842
0.740
0.658
5-CF
6-CFtBu
5-CFtBu
7
8
9
10
13
16
19
23
24
a Excited at 490 nm. b Obtained by calculation, based on fluorescein as
the standard (φfl ) 0.850).
6-CF, as graphically shown in Figure 5. Compounds 13 and
19, whose carboxyl esters are para to the methyl or methoxy
group, exhibited remarkable recovery of the quantum yields
(φfl ) 0.877 for 13 and φfl ) 0.842 for 19), compared with
6-CFtBu (φfl ) 0.378) (Table 2). The above-mentioned para-
substituted compounds 13 and 19 have higher values of
quantum yield than the meta-positioned esters 16 and 24. A
plausible explanation is that the electron-donating character
of the para-situated methyl and methoxy groups is greater
than that of meta-situated ones owing to resonance effects,
and this is supported by the higher LUMO values of the para-
substituted acceptors (Table 1). The amide derivative 23 also
showed a high quantum yield (φfl ) 0.740).
On the basis of the rationale of the donor-derived
fluorescence quenching mechanism, we have designed and
synthesized novel carboxyfluorescein dyes in which the
d-PeT process is hampered and full fluorescence is main-
tained, taking t-butyl esters as a model. Replacement of the
carboxylate with electron-donating methyl or methoxy sub-
stituents successfully blocked the d-PeT process and provided
the fully emitting esters 13 and 19. These novel carboxy-
fluorescein dyes should be useful for more sensitive fluo-
rescence labeling, especially for hydroxyl-containing com-
pounds such as steroids, than is possible with currently
available labeling reagents. Further, the synthetic route
presented herein enabled us to acquire a single isomer of
each derivative, whereas the known method generates a
mixture of 5-CF and 6-CF. These novel carboxyfluorescein
dyes and the rationale introduced here provide a solid
foundation for the development of superior fluorescence
labeling agents for chemical-biological research.
Figure 5. Fluorescence emission spectra (excited at 490 nm) in
0.1 M sodium phosphate buffer (pH 7.4). (A) 6-CF and its t-butyl
ester, 6-CFtBu. (B) Compound 7 and its t-butyl ester, compound
13.
Acknowledgment. This study was supported by a grant
for the Advanced and Innovational Research Program in Life
Sciences from the Ministry of Education, Culture, Sports,
Science and Technology of the Japanese Government and
by a grant from Hoansha Foundation.
Supporting Information Available: Supporting experi-
mental details for the preparation and characterization of
compounds 1, 7-10, 12, 13, 15, 16, 18, 19, and 21-24 are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0623926
5966
Org. Lett., Vol. 8, No. 26, 2006