Construction of a library of rhodol fluorophores for developing new fluorescent probes

Article abstract of DOI:10.1021/ol902706b

[Chemical equation presented] A highly efficient and concise synthetic scheme for rhodol fluorophores is developed with palladium-catalyzed amination reaction as the key step. This new synthetic route is utilized to construct a rhodol library, potentially useful for the design of novel fluorescent probes.

Full text of DOI:10.1021/ol902706b

ORGANIC
LETTERS
2010
Vol. 12, No. 3
496-499
Construction of a Library of Rhodol
Fluorophores for Developing New
Fluorescent Probes
Tao Peng and Dan Yang*
Morningside Laboratory for Chemical Biology and Department of Chemistry, The
UniVersity of Hong Kong, Pokfulam Road, Hong Kong, People’s Republic of China
yangdan@hku.hk
Received November 23, 2009
ABSTRACT
A highly efficient and concise synthetic scheme for rhodol fluorophores is developed with palladium-catalyzed amination reaction as the key
step. This new synthetic route is utilized to construct a rhodol library, potentially useful for the design of novel fluorescent probes.
Fluorescence techniques have been widely used and are still
enjoying ever-increasing interest from chemistry to many
areas of biology due to high sensitivity, simplicity, fast
response, a wealth of molecular information, and capability
of spatial imaging.1 However, the feasibility of using
fluorescence techniques for a particular application is often
limited by the availability of appropriate fluorescent mol-
ecules. Although there have been some reports about
theoretical approaches for the rational design of fluorescent
probes,2 these are still far from enough. Combinatorial
construction of libraries of fluorescent probe candidates has
been demonstrated to be a very powerful and promising
approach3 with some impressive discoveries of novel fluo-
rescent probes.4
As the hybrid structure of fluorescein and rhodamine,
rhodol fluorophores,5 also named “Rhodafluor”,6 are interest-
ing candidates for fluorescent probes since they inherit all
the excellent photophysical properties from fluorescein and
rhodamine, such as high extinction coefficients, quantum
yields, photostability, and solubility in a variety of solvents,
yet low pH-dependence.5c More interestingly, the spectral
characteristics of rhodol fluorophores, such as fluorescence
emission maximum and quantum yields, are quite dependent
on the substitution patterns of the nitrogen atom in a similar
manner to rhodamine.7
Unfortunately, despite their excellent photophysical prop-
erties there are actually limited examples about the applica-
(1) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer US: New York, 2006.
(3) (a) Kauffman, S.; Ellington, A. D. Curr. Opin. Chem. Biol. 1999, 3,
256–259. (b) Finney, N. S. Curr. Opin. Chem. Biol. 2006, 10, 238–245.
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129, 4510–4511.
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A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
97, 1515–1566. (b) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron
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2536. (d) Miura, T.; Urano, Y.; Tanaka, K.; Nagano, T.; Ohkubo, K.;
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Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126,
3357–3367.
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10.1021/ol902706b  2010 American Chemical Society
Published on Web 01/12/2010

tions of rhodol fluorophores,5b,6,8 probably due to the lack
of a convenient synthetic method. Here, we report a highly
efficient and concise synthetic scheme for constructing a
library of rhodol fluorophores to explore their applications.
and employing a much weaker base cesium carbonate,11 the
detriflation side reaction could be suppressed and the desired
product 3a was isolated in 96% yield. This condition was
thus adopted for the coupling reactions with other amines
or amides.
Scheme 1. Divergent Synthsis of Rhodol Fluorophores
Scheme 2. Construction of the Rhodol Library
As presented in Scheme 1, the synthesis of rhodol
fluorophores started from the commercially available fluo-
rescein or 2′,7′-dichlorofluorescein, which was first mono-
protected with a methoxymethyl (MOM) group and then
triflated to provide the intermediate fluorescein triflate 2a or
2b. The triflate 2a or 2b was next coupled with different
amines under the catalysis of palladium-phosphine complex,
widely known as Buchwald-Hartwig amination reaction.9
Finally, the MOM group was easily removed under acidic
condition to afford rhodol fluorophores.
The key amination conditions were optimized for the
reaction of 2a with secondary amine morpholine (data not
shown). Under the typical reaction conditions with
Pd(OAc)2-BINAP complex (2 and 3 mol %, respectively)
as the catalyst and sodium tert-butoxide as the base in toluene
at elevated temperature (80 °C),10 the desired product 3a
was obtained, although the coupling efficency was low.
Competitive detriflation was found to be the major side
reaction to reduce the yield of the coupling reaction. By
increasing the amounts of Pd(OAc)2 to 10 mol % and BINAP
to 15 mol %, raising the reaction temperature to 100 °C,
a Obtained from hydrolysis of coupling product between 2a and
benzophenone imine (see the Supporting Information).
As shown in Scheme 2, the fluorescein triflates 2a and 2b
reacted with various amines or amides (HNR1R2), including
aliphatic amines, aromatic amines, cyclic amines, acyclic
amines, and amides, yielding the corresponding O-substituted
rhodol derivatives 3a-r. The amination reactions with most
amines having high boiling points proceeded smoothly under
standard conditions, while the couplings with amines of low
boiling points, such as monoallylamine, dimethylamine, and
diethylamine, provided much lower yields and required
(7) Haugland, R. P. The Handbook: A Guide to Fluorescent Probes and
Labeling Technologies, 10th ed.; Molecular Probes: Eugene, OR, 2005
.
(8) (a) Smith, G. A.; Metcalfe, J. C.; Clarke, S. D. J. Chem. Soc., Perkin
Trans. 2 1993, 1195–1204. (b) Clark, M. A.; Duffy, K.; Tibrewala, J.;
Lippard, S. J. Org. Lett. 2003, 5, 2051–2054. (c) Dickinson, B. C.; Chang,
C. J. J. Am. Chem. Soc. 2008, 130, 9638–9639
.
(9) (a) Hartwig, J. F. Synlett 1997, 4, 329–340. (b) Yang, B. H.;
Buchwald, S. L. J. Organomet. Chem. 1999, 576, 125–146.
(10) (a) Wolfe, J. P.; Buchwald, S. L. J. Org. Chem. 1997, 62, 1264–
1267. (b) Louie, J.; Driver, M. S.; Hamann, B. C.; Hartwig, J. F. J. Org.
Chem. 1997, 62, 1268–1273.
(11) Åhman, J.; Buchwald, S. L. Tetrahedron Lett. 1997, 38, 6363–
6366.
Org. Lett., Vol. 12, No. 3, 2010
497

excess amounts of amines (e.g., 10 equiv) to make the
reactions complete. For the coupling reactions with amides
(3n and 3o), xantphos turned out to be a more effective ligand
than BINAP,12 and dioxane was found to be a better solvent
than toluene due to the poor solubility of amides in toluene.
The O-substituted rhodol derivatives 3a-r were then
treated with TFA in CH2Cl2 to provide the corresponding
rhodols 4a-r in nearly quantitative yields. The resulting
crude rhodols exhibit excellent purities as shown by NMR
and HPLC analysis (see Table S1 in the Supporting Informa-
tion).
emission wavelengths. In general, quantum yields of rhodol
fluorophores decrease with increasing carbon number and
bulk of the substituents. This phenomenon could be explained
by the formation of twisted intramolecular charge transfer
(TICT) states, which result from steric factors of the nitrogen
atoms and compete with fluorescence generation by the
nonradiative decay of excited states.1,5c,13 The rhodol fluo-
rophores with an amido group substituted on the nitrogen
(e.g., 4n and 4o) or with a phenyl ring attached on nitrogen
(e.g., 4l, 4m, and 4r) show dramatically decreased fluores-
cence quantum yields. The diminished fluorescence of
N-amido and N-phenyl rhodols could also be attributed to
the easy formation of TICT states. Moreover, substitution
on the oxygen atom of the other side significantly decreases
the quantum yields of the fluorophores (e.g., 3d, 3h, and
3o).
The photophysical properties of the fluorophores were
characterized in 50 mM aqueous phosphate buffer at pH 8.0,
and the results are summarized in Table 1.
This library with a series of rhodol fluorophores is
obviously very useful for the design and discovery of novel
fluorescent probes based on the rhodol core structure. The
strong fluorescence of N-monosubstituted rhodols (e.g.,
4h-k) suggests their potential applications as fluorescent
labels or sensors for biologically related species after some
modification on the aliphatic chain. Compound 5 bearing an
alkyne group on the N-substitutent was thus synthesized
(Scheme 3). This strongly fluorescent compound could be
Table 1. Photophysical Properties of Rhodol Fluorophoresa
b
compd
λmax (nm)
εmax
λem (nm)
Φc
4a
4b
4c
4d
4e
4f
4g
4h
4i
522
523
519
518
522
525
525
507
508
507
503
513
61 000
62 500
63 000
64 000
61 000
59 000
63 000
61 000
60 000
61 500
71 000
55 000
57 000
28 500
30 100
∼70 000
71 000
62 000
553
555
550
544
549
552
553
531
533
534
529
n.d.d
n.d.d
524
525
516
533
n.d.d
n.d.d
532
n.d.d
516
0.13
0.10
0.12
0.20
0.15
0.10
0.08
0.94
0.93
0.92
0.95
n.d.d
n.d.d
0.19
0.16
0.98
0.67
<0.0001
n.d.d
0.35
n.d.d
0.85
4j
4k
4l
Scheme 3. Synthesis of a Fluorescent Label Molecule 5
4m
4n
4o
4p
4q
4r
3d
3h
3o
3p
517
462/490
462/490
493
512
520
297
494
293
477
43 000
58 000
a Fluorescence excitation and emission spectra were recorded in 50 mM
phosphate buffer at pH 8.0 containing <0.1% CH3CN as a cosolvent. b Unit:
M-1 cm-1 c Determined with rhodamine 6G (Φ ) 0.76 in water) as
.
reference. d Fluorescence too weak to be detected.
Depending on different substituents on the nitrogen atom,
the maximal absorption wavelengths (λmax) of investigated
fluorophores in aqueous solution vary from approximately
470 to 530 nm, while the maximal fluorescence emission
wavelengths (λem) vary from approximately 510 to 560 nm,
with extinction coefficients (εmax) generally greater than
50 000 M-1 cm-1. Rhodols with only one or no substituent
on the nitrogen (e.g., 4h-k, and 4p) are strongly fluorescent,
with quantum yields (Φ) approaching unity in phosphate
buffer. The fluorophores with two substitutes (e.g., 4a-g)
on the nitrogen, however, generally display much weaker
fluorescence, albeit longer absorption and fluorescence
conjugated with an azide molecule under the catalysis of
Cu(I) to form a triazole linkage,14 and therefore effectively
avoid the isomer problem in commonly used fluorescent
labels such as 5(6)-carboxyfluorescein and 5(6)-carbox-
yrhodamine.7 The difference in quantum yields between
N-amido rhodol (e.g., 4n) and N-free rhodol (e.g., 4p)
(13) (a) Jones, G.; Jackson, W. R.; Halpern, A. M. Chem. Phys. Lett.
1980, 72, 391–395. (b) Taneja, L.; Sharma, A. K.; Singh, R. D. J. Lumin.
1995, 63, 203–214
.
(14) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004–2021. (b) Moses, J. E.; Moorhouse, A. D. Chem. Soc.
ReV. 2007, 36, 1249–1262.
(12) Yin, J.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 6043–6048.
498
Org. Lett., Vol. 12, No. 3, 2010

implicates rhodol-based fluorogenic probes for detecting
peptidase activity, which transforms an amido group to an
amino group. Similarly, conversion of the O-substituted
rhodol (e.g., 3d and 3h) to O-free rhodol (e.g., 4d and 4h)
also induces a change in fluorescence intensity, indicating
fluorogenic probes for dealkylating enzymes, such as gly-
cosidases.
In summary, a highly efficient and concise synthetic route
for rhodol fluorophores has been developed to construct a
library of rhodols. This library has been demonstrated to be
potentially very useful for the design of new fluorescent
probes. The extensive use of this library for developing novel
fluorescent probes for biological applications will be reported
in due course.
Acknowledgment. This work was supported by the
University of Hong Kong, the Hong Kong Research Grants
Council (HKU7060/05P), and Morningside Foundation.
Supporting Information Available: All experimental
procedures, characterization of new compounds, and pho-
tophysical characterization of fluorophores. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL902706B
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