A highly sensitive fluorescent viscosity sensor

Article abstract of DOI:10.1016/j.bmcl.2012.07.101

Variation at the 3' position of fluorescein via Suzuki-Miyaura cross-coupling with aryl and heteroaryl moieties gave a family of anthofluoresceins whose spectroscopic properties were studied. The 1-methylindole derivative gave the highest quantum yield and was observed to behave as a molecular rotor, displaying marked variations in fluorescent intensities with viscosity and offering possible application in cellular sensing and fluorescent polarisation assays.

Full text of DOI:10.1016/j.bmcl.2012.07.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5780–5783
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A highly sensitive fluorescent viscosity sensor
Rahimi M. Yusop a, , Asier Unciti-Broceta b, Mark Bradley c
⇑
a School of Chemical Sciences and Food Technology, Faculty of Science and Technology, 43600 Universiti Kebangsaan Malaysia, Selangor, Malaysia
b Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, UK
c 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:
Variation at the 3’ position of fluorescein via Suzuki–Miyaura cross-coupling with aryl and heteroaryl
moieties gave a family of anthofluoresceins whose spectroscopic properties were studied. The 1-methyl-
indole derivative gave the highest quantum yield and was observed to behave as a molecular rotor, dis-
playing marked variations in fluorescent intensities with viscosity and offering possible application in
cellular sensing and fluorescent polarisation assays.
Received 2 May 2012
Revised 17 July 2012
Accepted 31 July 2012
Available online 4 August 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Anthofluorescein
Viscosity sensor
Suzuki cross-coupling
Palladium catalysis
Fluorescein
Fluorophores are a central component of many laboratory tech-
niques, with applications ranging from labels for antibodies and
protease substrates to fluorescent sensors for metal ions and reac-
tive oxygen species.1–3 Typically, organic dyes are rapidly respon-
sive and sensitive and are the tools of choice for labelling a wide
range of biomolecules and probing biochemical processes.1–4 Ever
increasingly sophisticated in vitro and in vivo studies demand ever
more complex fluorescent reporters, such as probes for specific
intracellular processes,4,5 cellular tracking,6,7 pH measurments,8–10
ion sensing,11–13 and in cellulo synthesis.14,15 As such the demands
and expectations placed on specific dyes and chemosensors contin-
ues to increase. This includes growing interests in the development
of environmentally-sensitive dyes; so-called molecular rotors;
whose changes in fluorescence can be ‘translated’ into viscosity
data.16–18 Such a phenomenon can also be applied in place of tech-
niques such as fluorescence lifetime imaging and fluorescent
polarisation.19,20
Xanthene dyes (with fluorescein, 1, the parent of the family,
Fig. 1) remain the most broadly utilized family of dyes.1,2,5,8–16
These versatile dyes can be readily functionalized to allow biomol-
ecule labeling,21 while the introduction of electron-withdrawing/
donating groups on the xanthene core has been used to modify
the phenol’s pKa, thereby tuning its deprotonation to physiologi-
cally relevant pH’s.22–25
sensitivity to pH changes between 7 and 10 (ranging from
460–530 nm to 530–580 nm respectively).26 These optical proper-
ties arise due to a combination of factors (see Fig. 1). Firstly tauto-
mer II contains a 4-oxo-2,5-cyclohexadien-1-ylidene group that
expands the dye’s p-electron system (conjugation expander), lead-
ing to a red-shifted Ex/Em spectra relative to fluorescein; secondly,
the p-hydroxyphenyl group of tautomer I has freedom to rotate,
forming a twisted intramolecular charge transfer (TICT) state upon
photoexcitation and thus exhibiting two competing relaxation
pathways fluorescence emission and non-radiative de-excitation
by internal rotation. Consequently, the ratio of these tautomers
and their non-ionic forms in different solvents and pH’s governs
the overall optical properties of anthofluorescein resulting in a
multi-functional and highly-sensitive probe.
Encouraged by the unique optical properties of anthofluoresce-
in and the potential to generate a sensitive viscosity sensor, a
20-member library of anthofluorescein derivatives was synthe-
sized in order to study the influence of aryl and heteroaryl moieties
decorated with electron-donating/withdrawing groups on the sys-
tem’s fluorescent properties.
A variety of anthofluorescein derivatives were synthesised by
mono-triflation of fluorescein, 1, with N,N-bis(trifluoromethylsul-
fonyl)aniline followed by cross-coupling with a series of aryl and
heteroaryl boronic acids containing electron donating/withdraw-
ing groups and/or rings. The synthetic procedure is outlined in
Scheme 1 (with yields between 63 and 88%) with the list of probes
synthesized given in Table 1.
We recently developed anthofluorescein, 2, a fluorescein-de-
rived dye whose absorption and emission spectra display high
The optical properties of the new derivatives demonstrated a
broad and slightly red-shifted emission compared to unmodified
⇑
Corresponding author. Tel.: +60 3 8921 5448; fax: +60 3 8921 5410.
E-mail address: rahimi@ukm.my (R.M. Yusop).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.101

R. M. Yusop et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5780–5783
OH
5781
O
O
COOH
Fluorescein, 1
Molecular
Rotor
Conjugation
Expander
O
O
O
O
O
O
CO2
CO2
Tautomer I
Anthofluorescein, 2
Tautomer II
Figure 1. Structures of fluorescein, 1, and two tautomeric forms of the deprotonated anthofluorescein, 2.
(HO)2B Ar
PhN(Tf)2, K2CO3
DMF, μω 80°C
O
O
Ar
O
O
OH
O
O
OTf
4a-v
COOH
COOH
COOH
Pd(OAc)2, PPh3
Dioxane/H2O (10:1)
μω
K2CO3,
120°C
5a-v
1
3
Scheme 1. Suzuki cross-coupling between fluorescein-monotriflate (1) and boronic acids (4a–v).
fluorescein. Compounds containing electron rich heterocycles (5a–
i) typically gave quantum yields ( ) in methanol ranging from 0.40
to 0.56, superior to anthofluorescein (0.33). On the contrary, com-
pounds containing substituted phenyl derivatives produced dyes
minimal changes in polarity.17,27 As observed in Figure 2, fluores-
cent analysis showed that fluorescent intensity was highly influ-
enced by the viscosity of the environment: the higher the
viscosity of the mixture the greater the fluorescence emission.
U
with lower
U
than anthofluorescein, with some dyes containing
Plots of log I5e against the log
g as shown in Figure 3, give a lin-
electron withdrawing groups falling below 0.2 (5q, 5r and 5t).
According to the Föster–Hoffmann equation, there is a direct
relationship between viscosity and quantum yield (see equation
ear relationship as predicted.27–29
Incubation of 5e with HeLa cells showed that the dye was rap-
idly up taken and was clearly visible (results not shown). This is
supposed to be due to the high viscosity of the cellular cytoplasm
which in terms of viscosity is more akin to glycerol than water29
confirming the previous viscosity observations. To facilitate future
measurements of intracellular viscosity, it would be desirable that
the probe would be retained inside the cells after fixation. To verify
this, cells were incubated with compound 5e for 2 h, fixed with
paraformaldehyde, washed with PBS and imaged by fluorescent
microscopy, showing clear fluorescence signal coming from the
cells while eliminating all extracellular background (Fig. 4).
In conclusion a series of anthofluorescein derivatives were syn-
thesised via Suzuki cross-coupling chemistry and their spectro-
scopic properties studied. Emission spectra showed all derivatives
had a broad and slightly red-shift emission compared with fluores-
cein. Dye 5e containing a 1-methyl-1H-indol-5-yl moiety showed
significant variations of its fluorescent properties with viscosity,
which followed the Förster–Hoffmann equation. These results
strongly suggest 5e behaves as a molecular rotor where its emission
intensity is highly influenced by the environment viscosity. Preli-
minary in vitro assays showed 5e was cell permeable and success-
fully labelled HeLa cells. This probe could be used as a micro-
viscosity sensor, making it a valuable tool for biological studies
including a new angle on fluorescent polarisation type assays
where binding events cause changes in rotational rates.
below, where
U is the quantum yield; g is the viscosity, C is a sys-
tem-dependant constant; x is a dye dependant-constant).27
log ¼ C þ x log
While for most small molecule dyes viscosity changes lead to
small variations of , these variations are highly prominent in
U
g
U
the case of fluorescent molecular rotors.16,17,27 Due to their inher-
ent potential to dissipate energy by intramolecular rotation, photo-
activated molecules may relax by non-radiative decay processes.
Viscous environments reduce the loss of energy via this mecha-
nism, thus increasing the relative proportion of relaxation via the
fluorescent excited state via photon emission.17
From the library generated, the probe with a 1-methylindole
moiety (compound 5e) had the highest quantum yield (0.56) in
methanol and maximum emission at 537 nm. To investigate which
members of the library were more sensitive to viscosity changes,
we determined their corresponding
cous solvent) and glycerol (highly viscous solvent) (see Table 1).
This study showed that compound 5e showed the highest dynamic
range, with
Thus the optical properties of 5e were measured in mixtures of
ethylene glycol and glycerol; a pair of miscible solvents which al-
low the formation of solvent mixtures of different viscosity with
U in ethylene glycol (low vis-
U = 0.20 in ethylene glycol and U = 0.67 in glycerol.

5782
R. M. Yusop et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5780–5783
Table 1
The anthofluorescein derivatives synthesised and their fluorescent properties (20 lM in methanol, ethylene glycol and glycerol)
Ar
O
O
COOH
5a-v
Probe Ar
Ex/Em (nm)
U
In
U
In
U
In
Probe Ar
Ex/Em (nm)
U
In
U
In
U In
methanol ethylene
glycol
glycerol
methanol ethylene
glycol
glycerol
OH
2
478/538
482/528
0.33
0.49
0.19
0.29
0.40
0.45
5l
478/533
470/532
0.22
0.23
0.24
0.20
0.35
0.26
O
O
O
O
5a
5m
O
N
5b
5c
5d
480/532
480/536
480/531
0.51
0.50
0.47
0.33
0.28
0.25
0.50
0.49
0.54
5n
5o
5p
476/532
475/530
478/529
0.26
0.30
0.27
0.23
0.28
0.21
0.27
0.30
0.27
N
H
O
O
S
S
5e
480/537
0.56
0.20
0.67
5q
470/535
0.13
0.16
0.24
O
5f
482/533
477/528
0.45
0.46
0.29
0.33
0.41
0.36
5r
5s
480/534
482/531
0.18
0.21
0.20
0.18
0.20
0.25
O
NH2
N
5g
Cl
5h
5i
480/536
479/538
470/535
477/525
0.42
0.40
0.30
0.32
0.27
0.29
0.28
0.26
0.40
0.37
0.32
0.28
5t
480/533
480/532
0.19
0.29
0.20
0.22
0.23
0.27
CN
NO2
COOH
5u
CF3
5j
5v
480/530
0.29
0.19
0.32
5k
COOH
η
Figure 2. Effect of solvent viscosity on the fluorescent intensity of 5e. Fluorescence
excitation wavelength = 480 nm. Viscosity values of ethylene glycol and glycerol
mixtures (as reported by Haidekker et al.25) are given between brackets. (Temper-
ature at 25 °C, concentration at 945, 374, 109, 36 and 13.5 mPa s).
Figure 3. Plots of log I5e vs log
g
g. I5e = emission intensity of 5e at 537 nm.
= viscosity values (mPa s) of ethylene glycol/glycerol mixtures.25

R. M. Yusop et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5780–5783
5783
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Figure 4. HeLa cells after incubation with a 5 lM solution of 5e for 2 h at 37 °C. The
cells were fixed and washed twice with PBS. HeLa cells were imaged using a ꢀ20
objective (Leica fluorescence microscope) under brightlight (left) fluorescent image
(right) with 488 nm excitation and no emission filter. Scale bar, 100 lm.
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This work was supported financially by the Government of
Malaysia grant no. FRGS/1/2012/ST01/UKM/02/1, GGPM-2012-
010 (RMY) and the EPSRC. AUB thank the MRC for funding. We
are grateful to Patricia Richardson for her helpful advice regarding
the fluorescent measurement.
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Supplementary data
Supplementary data (general methods, experimental procedures
and characterization) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.101.
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