A fluorescein-derived anthocyanidin-inspired pH sensor

Article abstract of DOI:10.1016/j.tetlet.2009.03.223

A new multicolor pH-dependent fluorophore was synthesized via Pd-mediated cross-coupling chemistry of the mono-triflate of fluorescein with p-hydroxyphenylboronic acid. The novel indicator, named anthofluorescein, is highly sensitive to pH changes between

Full text of DOI:10.1016/j.tetlet.2009.03.223

Tetrahedron Letters 50 (2009) 3713–3715
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A fluorescein-derived anthocyanidin-inspired pH sensor
*
Asier Unciti-Broceta, M. Rahimi Yusop , Patricia R. Richardson , Jeffrey G. A. Walton, Mark Bradley
School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A new multicolor pH-dependent fluorophore was synthesized via Pd-mediated cross-coupling chemistry
of the mono-triflate of fluorescein with p-hydroxyphenylboronic acid. The novel indicator, named antho-
fluorescein, is highly sensitive to pH changes between 7 and 10 and displays a green to red fluorescence
shift, making it a valuable candidate for biological studies.
Received 19 January 2009
Revised 26 March 2009
Accepted 31 March 2009
Available online 17 April 2009
Ó 2009 Elsevier Ltd. All rights reserved.
R3'
R3'
The direct visualization of general biochemical events, specific
molecular targets, or defined biochemical processes are indispens-
able for both in vitro1–4 and, increasingly, in vivo applications.3–6
These applications, because of the remarkable advances in imaging
techniques, have begun to use a multitude of different fluorescent
labels, probes, and sensors in multi-channel multiplex studies7–9
which include the real-time analysis of cells and whole organ-
isms.10–13
O
OH
R5'
-H
HO
O
HO
O
R5'
R3
R3
+H
R5
R5
Anthocyanidins, 1
Due to the particular needs of each and every study, fluorescent
dyes and probes with a myriad of defined optical, chemical, and
biological properties are in high demand. Much effort has been fo-
cused on the development of optical pH chemosensors,14–18 with
specific attention paid to highly sensitive indicators within the
physiological pH range.19,20 In this area fluorescein and its deriva-
tives are perhaps the most widely used fluorescent pH probes,21,22
due in part to their excellent spectral and physical properties.
Anthocyanidins are the chromophores of a well-known family
of natural dyes and are characterized by their broad spectral win-
dow, which is controlled by the various substituents on the benzo-
pyrylium core and the pH.23 Inspired by the general structures of
the anthocyanidins 1, the work described herein focuses on the
modification of fluorescein 2 via substitution of one of its phenolic
groups with p-hydroxyphenyl (see Fig. 1), a feature found in many
anthocyanidins.23 Due to its extended conjugation, compound 3
(which has been named anthofluorescein) was expected to display
optical properties similar to both fluorescein and the anthocyani-
dins, but without the chemical instability of the latter.23,24
To assess the usefulness of the sensor prior to synthesis, the po-
tential bathochromic shift in the excitation/emission wavelength
of the different prototropic forms of derivative 3 was calculated
via a series of configuration interaction singles, CIS/6-31g(d,p) cal-
culations. These predicted that the 400-oxo dianion form (D-I in the
Supplementary data) would display a maximum absorbance at
O
HO
O
O
HO
O
COOH
COOH
Fluorescein, 2
Anthofluorescein, 3
Figure 1. Structures of anthocyanidins, fluorescein (quinoid form), and anthoflu-
orescein (quinoid form). Similarities in the structures are highlighted in blue.
530 nm and emission at 570 nm, the emission being red shifted
by over 50 nm relative to fluorescein (see Supplementary data).
In contrast, the 30-oxo dianion form (D-II in the Supplementary
data) should keep the original spectroscopic properties of fluores-
cein, with absorption maximum at 480 nm and emission maxi-
mum at 513 nm. These dianionic forms were predicted to have
significant oscillator strengths. Due to the different pKa values of
the two phenolic OH groups, we envisaged that we would observe
a dual-color performance tuned by the pH of the solution.
Compound 3 was synthesized via sequential microwave-as-
sisted mono-triflation of fluorescein 2 using one equivalent of N-
phenylbis(trifluoromethanesulfonimide)25 (a mild triflation re-
agent) followed by Suzuki aryl palladium-catalyzed cross-coupling
of 4 with p-hydroxyphenylboronic acid (see Scheme 1) to give rise
to anthofluorescein 3. Heterogeneous catalysis using polystyrene
resin captured palladium was successful but lower yields were ob-
tained in this case (41%).26
* Corresponding author. Tel.: +44 131 650 4820; fax: +44 131 777 0334.
E-mail address: mark.bradley@ed.ac.uk (Mark Bradley).
These authors contributed equally to this work.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.223

3714
A. Unciti-Broceta et al. / Tetrahedron Letters 50 (2009) 3713–3715
OH
OAc
O
OTf
O
O
OH
O
O
O
AcO
O
(a)
(b)
(c)
O
COOH
COOH
COOH
O
Fluorescein, 2
4
Anthofluorescein, 3
5
Scheme 1. Reagents and conditions: (a) PhN(Tf)2, K2CO3, DMF, lx 80 °C, 20 min, 61%; (b) p-hydroxyphenylboronic acid, Pd(OAc)2, K2CO3, PPh3, H2O:dioxane (1:9), lx 120 °C,
30 min, 78%; (c) Ac2O:Pyr (1:1), 16 h, 87%.
A study of the absorptivity and the fluorescent properties of the
novel dye confirmed that the optical properties of anthofluorescein
were quite different from those of fluorescein, with both the
absorption and emission spectra highly influenced by pH (as is also
observed for the anthocyanidins). Spectrophotometric analysis of
due to a decrease in the rate of internal conversion mediated by
the p-hydroxyphenyl group torsional motion, similar to that as-
cribed to the diethylamino torsional motion in Rhodamine B.28 It
would be expected that the changes in emission intensity and
quantum yield would be mirrored by changes in fluorescence life-
time and, as such, the molecule could find application as a, poten-
tially, very sensitive probe of local temperature and viscosity in
fluorescence lifetime imaging microscopy.
A non-fluorescent derivative of anthofluorescein 3, diacetylated
derivative 5, was synthesized (see Scheme 1) in order to enhance
the cellular penetrability of anthofluorescein and to reduce extra-
cellular background. Compound 5 was incubated with HeLa cells
for 2 h and then imaged using a 488/20 excitation filter (in the ab-
sence of an emission filter). As expected, intracellular deacetylation
led to the formation of anthofluorescein 3, which was identified by
fluorescently yellow cells (Fig. 4), highlighting the viability of the
cells. Due to the higher viscosity of the cell cytoplasm (expected
to be more akin to glycerol than water)29 the anthofluorescein
dye was strongly emissive within the cells, confirming the previous
viscosity observations.
anthofluorescein solutions (50 lM) at distinct pH values (Fig. 2)
showed that absorbance increased with pH, with the largest im-
pact being observed between pH 7 and 10 (see Fig. 2 for the appar-
ent colors of the solution), with a maximum absorptivity at pH 11
(e
= 25,000 Mꢀ1 cmꢀ1). Absorptivity greatly decayed at pH 13, indi-
cating a large change in the prototropic composition of the
solution.
We attribute the changes seen in the fluorescence emission
spectra between pH 7 and pH 13 (see Fig. 3) as being predomi-
nantly due to the prototropic equilibrium existing between the
two dianionic forms of the molecule (for a more detailed explana-
tion see Supplementary data). The experimental quantum yield27
of the dye at pH 8, where maximal emission was observed, was
very low (0.02), which in practice would limit applicability for cell
assays. However, further analysis indicated that the quantum yield
was highly influenced by the viscosity of the solution, with the
quantum yield rising to 0.3 at pH 8 in 20% glycerol, presumably
In conclusion a new pH-sensitive dye synthesized from fluores-
cein using a straightforward microwave-assisted two-step proce-
Figure 2. (A) Absorption spectra of 50 lM solutions of anthofluorescein 3 at pH 6–13. (B) Images of the dye solutions at various pH values.
Figure 3. Emission spectra of anthofluorescein 3, with excitation at 450 nm: (A) pH 6–9 and (B) pH 9–13.

A. Unciti-Broceta et al. / Tetrahedron Letters 50 (2009) 3713–3715
3715
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.03.223.
References and notes
1. Johnsson, N.; Johnsson, K. ACS Chem. Biol. 2007, 2, 31–38.
2. Unciti-Broceta, A.; Díaz-Mochón, J.; Mizomoto, H.; Bradley, M. J. Comb. Chem.
2008, 10, 179–184.
3. Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008, 4, 168–175.
4. Giepmans, B. N.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312,
217–224.
5. Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123–128.
6. Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. Rev. Mol. Cell Biol. 2002, 3,
906–918.
7. Díaz-Mochón, J. J.; Tourniaire, G.; Bradley, M. Chem. Soc. Rev. 2007, 36, 449–457.
8. Pernagallo, S.; Unciti-Broceta, A.; Díaz-Mochón, J. J.; Bradley, M. Biomed. Mater.
2008, 3, Art. No. 034112 (6pp).
9. Unciti-Broceta, A.; Diezmann, F.; Ou-Yang, C. Y.; Fara, M. A.; Bradley, M. Bioorg.
Med. Chem. 2009, 17, 959–966.
10. Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155.
11. Hell, S. W. Nat. Biotechnol. 2003, 11, 1347–1355.
12. Weissleder, R. Science 2006, 312, 1168–1171.
13. Alexander, L.; Dhaliwal, K.; Simpson, J.; Bradley, M. Chem. Commun. 2008,
3507–3509.
Figure 4. (A) Intracellular deacetylation of compound 5 to give anthofluorescein 3.
HeLa cells were incubated for 2 h with a 25 M solution of diacetylated anthoflu-
orescein 5. (B) Brightfield. (C) Fluorescent image with 488 nm excitation and no
l
emission filter.
14. Charier, S.; Ruel, O.; Baudin, J.-B.; Alcor, D.; Allemand, J.-F.; Meglio, A.; Jullien, L.
Angew. Chem., Int. Ed. 2004, 43, 4785–4788.
15. Wong, L. S.; Birembaut, F.; Bocklesby, W. S.; Frey, J. G.; Bradley, M. Anal. Chem.
2005, 77, 2247–2251.
16. Cho, J. K.; Wong, L. S.; Dean, T. W.; Ichihara, O.; Muller, C.; Bradley, M. Chem.
Commun. 2004, 1470–1471.
17. Cho, J. K.; White, P. D.; Klute, W.; Dean, T. W.; Bradley, M. J. Comb. Chem. 2003,
5, 632–636.
18. Wong, L. S.; Brocklesby, W. S.; Bradley, M. Sens. Actuators B-Chem. 2005, 107,
957–962.
19. Bradley, M.; Alexander, L.; Duncan, K.; Chennaoui, M.; Jones, A. C.; Sanchez-
Martin, R. M. Bioorg. Med. Chem. Lett. 2008, 18, 313–317.
20. Vasylevska, A. S.; Karasyov, A. A.; Borisov, S. M.; Krause, C. Anal. Bioanal. Chem.
2007, 387, 2131–2141.
21. Lavis, L. D.; Rutkoski, T. J.; Raines, R. T. Anal. Chem. 2007, 79, 6775–6782.
22. Smith, J. P.; Drewes, L. R. J. Biol. Chem. 2006, 281, 2053–2060.
23. Castañeda-Ovando, A.; Pacheco-Hernández, M. L.; Páez-Hernández, M. E.;
Rodríguez, J. A.; Galán-Vidal, C. A. Food Chem. 2009, 113, 859–871.
24. Jurd, L.; Geissman, T. A. J. Org. Chem. 1963, 28, 2394–2397.
25. Miller, E. W.; Albers, A. E.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc.
2005, 127, 16652–16659.
dure consisting of mono-triflation and subsequent Suzuki aryl
cross-coupling with p-hydroxyphenylboronic acid has been devel-
oped. The new dye, named anthofluorescein, was characterized by
a highly sensitive absorption and fluorescence emission particu-
larly in the pH range from 7 to 10. A diacetylated derivative of
anthofluorescein successfully labeled living HeLa cells, which indi-
cates that it might become a valuable tool for cell labeling and via-
bility studies and, with the assistance of advanced microscopy
techniques, for uses such as a ratiometric reporter of pH, viscosity,
and/or temperature. Finally, the synthetic method provides a facile
route to the development of new multicolor fluorescence dyes with
red-shifted absorption and emission, which are in high demand as
fluorescence probes.
Acknowledgments
26. Cho, J. K.; Najman, R.; Dean, T. W.; Ichihara, O.; Muller, C.; Bradley, M. J. Am.
Chem. Soc. 2006, 128, 6276–6277.
27. Williams, A. T. R.; Winfield, S. A.; Miller, J. N. Analyst 1983, 108, 1067.
28. Lopez Arbeloa, I.; Rohatgi-Mukherjee, K. K. Chem. Phys. Lett. 1986, 129, 607–
614.
We thank the MRC, the Government of Malaysia (MRY) and
edikt2 (PRR) for funding. Computational resources were provided
by the EaStCHEM research computing facility.
29. Daniels, B. R.; Masi, B. C.; Wirtz, D. Biophys. J. 2006, 90, 4712–4719.