Self-calibrating viscosity probes: Design and subcellular localization

Article abstract of DOI:10.1016/j.bmc.2012.05.026

We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through resonance energy transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm.

Full text of DOI:10.1016/j.bmc.2012.05.026

Bioorganic & Medicinal Chemistry 20 (2012) 4443–4450
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Self-calibrating viscosity probes: Design and subcellular localization
Marianna Dakanali a, , Thai H. Do a, Austin Horn b, Akaraphon Chongchivivat a, Tuptim Jarusreni a,
⇑
a,
Darcy Lichlyter b, Gianni Guizzunti c, Mark A. Haidekker b, , Emmanuel A. Theodorakis
⇑
⇑
a Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, MC: 0358, La Jolla, CA 92093-0358, USA
b Faculty of Engineering, University of Georgia, Athens, GA 30602, USA
c Department of Cell Biology and Infection, Membrane Traffic and Pathogenesis Unit, Pasteur Institute, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 March 2012
Revised 8 May 2012
Accepted 12 May 2012
Available online 19 May 2012
We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in
which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity
sensor). Characterization of their fluorescence properties was made with separate excitation of the units
and through resonance energy transfer from the reference to the sensor dye. We have modified the linker
and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering
their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside
the cell and represents a suitable probe for viscosity measurements in the cytoplasm.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescence
Ratiometric dye
Molecular rotor
Subcellular viscosity
1. Introduction
used in biological systems. Although, different from each other,
they both rely on the diffusivity of specific fluorophores in order
The cell biomechanics are primarily determined by the cyto-
skeleton, the cell membrane and the cytoplasm among which the
latter two possess viscoelastic properties that change in various
states of diseases. It is well documented that changes in cell mem-
brane viscosity can affect the activity of membrane-bound pro-
teins,1 which in turn can lead to disorders both at the cellular2
and organismal level.3 For instance, cardiovascular diseases,4 cell
malignancy,5 Alzheimer’s disease,6 diabetes,7 hypertension8 and
aging9 are some examples of disorders associated with changes
in cell membrane viscosity. On the other hand, the effects of vari-
ations in cytoplasmic viscosity have not been widely investigated,
perhaps due to the relatively difficult measurement methods avail-
able. Magnetic microparticles have been used to measure cytoplas-
mic viscosity;10 however, this method demands the use of very
expensive equipment, has limited temporal resolution and suffers
from interactions between particles and cellular environment.10a
General methods for measuring bulk viscosity are the cone-and-
plate viscometers and the capillary viscometers,11 but their use is
limited due to the large sample size needed and the low temporal
resolution.11b Viscosity in microenvironments, such as in cells, can
be measured with the use of fluorescence-based methods.12,13 In
addition, techniques such as fluorescence anisotropy (FA)14 and
fluorescence recovery after photobleaching (FRAP)15 are commonly
to report information about the viscosity of the environment. FA
shows better spatial and temporal resolution than FRAP, yet local
viscosity can only approximately be computed and even minor
misalignments of the polarizers can cause major measurement
errors during FA-based studies.16
Recently, efforts have been focused on the use of environment-
sensitive fluorescent probes for measuring viscosity.13,16,17
Extended discussion and examples on fluorophores are presented
in several recent reviews.17 These probes, referred to as molecular
rotors, form twisted intermolecular charge transfer (TICT)
complexes in the excited state producing a fluorescence quantum
yield that is dependent of the surrounding environment.18 Com-
mon to their chemical structure is a motif composed of an electron
donor group in
p-conjugation with an electron acceptor. Upon
photoexcitation, these probes can relax via two competing path-
ways that include: (a) fluorescence emission; or (b) mechanical
non-radiative deexcitation that proceeds via bond rotations be-
tween the donor and the acceptor. Viscous environments delay
the mechanical deexcitation resulting in the increase of fluores-
cence emission. On the other hand, in media of low viscosity, the
relaxation occurs primarily through mechanical motion. Modifica-
tions in any of the individual components of molecular rotors,
namely the donor, the acceptor or the
p-conjugation system, can
affect their fluorescence profile.19
The Förster-Hoffmann equation20 describes a power law rela-
tionship between the quantum yield UF of a single emission molec-
ular rotor and the solvent viscosity (g), Eq. (1), where, C is a dye-
⇑
Corresponding authors. Tel.: +1 858 822 0456; fax: +1 858 822 0386.
E-mail address: etheodor@ucsd.edu (E.A. Theodorakis).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.026

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M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
dependent constant and x is a constant related to dye-solvent
interactions:18a
self-calibrating measurement of the solvent viscosity. The structure
of compound 1 highlights this design, in which a coumarin donor
(primary, viscosity insensitive fluorophore) is covalently attached
to an amino thiophene rotor (secondary, viscosity sensitive fluoro-
phore) via a polymethylene linker.
For the design of more hydrophilic ratiometric dyes we chose to
substitute the polymethylene linker with a triethylene glycol moi-
ety. The 7-methoxycoumarin-3-carboxylic acid was used as the
primary fluorophore and was covalently attached to either a thio-
phene- or an aniline-based molecular rotor. For the more lipophilic
ratiometric dye, we attached the same coumarin donor to an ami-
no-thiophene rotor via a polymethylene linker of five carbons.21c,23
To further increase the overall lipophilicity we substituted the
piperidine motif of the rotor with a more fatty dihexylamine
subunit.
/F ¼ C ꢀ gx
ð1Þ
The emission intensity of the rotor (IEM) is proportionally re-
lated to the quantum yield (UF), the excitation intensity (IEX), the
dye concentration (c) and instrument gain factors (G).
IEM ¼ ðG ꢀ c ꢀ IEXÞ/F
ð2Þ
These above equations illustrate the drawbacks in the use of
molecular rotors for viscosity studies; namely this method is sen-
sitive to changes of the fluid optical properties and to the dye con-
centration. These drawbacks can be overcome by the use of a dual
dye, composed of an internal reference dye bound to a molecular
rotor. In this case, a second, viscosity-independent emission IREF
becomes available that is proportional to the same factors apart
from the reference quantum yield UREF, which is viscosity indepen-
dent. The ratio of the rotor and reference emissions serves as the
internal calibration emission and can be simplified to Eq. (3):
2.2. Synthesis of the hydrophilic linker
The synthesis of the hydrophilic linker 7 is illustrated in Scheme
1. Commercially available 2-[2-(2-chloro-ethoxy)ethoxy]ethanol
(2) was converted to azide 3 upon treatment with sodium azide
in DMF.24 Reduction of 3 using Pd/C as catalyst formed amine
4,25 which was subsequently protected to yield 526 in 85% yield
after two steps. Esterification of alcohol 5 with cyanoacetic acid
(6) produced the hydrophilic linker 7 in 78% overall yield.
IEM
ðG ꢀ c ꢀ IEXÞ ꢀ /F
C
/
REF
¼
¼
gx
ð3Þ
IREF ðG ꢀ c ꢀ IEXÞ ꢀ /REF
We have explored the above principle for the design of self-cal-
ibrating dyes.21 Here we expand this design to the synthesis and
study of self-calibrating dyes with different solubility profiles for
potential applications in cell imaging. We envisioned that, by vir-
tue of their solubility profiles, these new compounds could be
localized in different parts of cells allowing studies either in the
cytoplasm (hydrophilic dye) or the cell membrane (hydrophobic
dye). In a similar fashion, those dyes could also be used in aqueous
solutions and oils, respectively, for bulk viscosity measurements.
2. Results and discussion
Scheme 1. Reagents and conditions: (a) 1.0 equiv 2, 1.0 equiv NaN3, DMF, 18 h,
90 °C, 82%; (b) 0.05 equiv Pd/C, methanol, 18 h, 25 °C, 95%; (c) 1.0 equiv 4, 2.0 equiv
Boc2O, 2.0 equiv Et3N, methanol, 18 h, reflux, 90%; (d) 1.0 equiv 5, 1.0 equiv 6,
1.1 equiv EDC, 1.1 equiv HOBT, CH2Cl2, 18 h, 25 °C, 78%.
2.1. Design criteria of a ratiometric self-calibrating dye
According to the general design of ratiometric dyes, illustrated in
Figure 1, two fluorophores, one acting as an internal reference and
the other being a molecular rotor should be covalently linked. These
two fluorophores should form a resonance energy transfer (RET)
pair which requires that: (a) the emission spectrum of the primary
fluorophore should have a significant overlap with the excitation
spectrum of the secondary fluorophore and (b) that the two fluoro-
phores are kept within a distance approximately equal to the Förster
radius.12,22 As implied by Eqs. (1)–(3), the primary fluorophore
should be a viscosity insensitive dye (reference dye) with a high
but constant fluorescence emission quantum yield and should suf-
ficiently excite the molecular rotor (viscosity sensitive dye). Follow-
ing excitation of the reference dye, the ratio of the emission A to
emission B would then produce a concentration-independent
2.3. Synthesis of ratiometric dyes
The synthesis of the hydrophilic ratiometric dye containing a
thiophene-based rotor is highlighted in Scheme 2. Knövenagel con-
densation of 827 with b-cyanoester 7 gave compound 9 in good
yield as a single stereoisomer (E).19 Deprotection of the primary
amine 9 by treatment with trifluoroacetic acid, followed by cou-
pling with N-succinimidyl-7-methoxycoumarin-3-carboxylate
(11), yielded ratiometric dye 12 in 83% yield.
Scheme 2. Reagents and conditions: (a) 1.0 equiv 7, 1.5 equiv 8, 0.1 equiv DBU,
18 h, 25 °C, 62%; (b) 1.0 equiv 9, TFA/CH2Cl2, 30 min, 25 °C; (c) 1.0 equiv 11,
1.1 equiv 10, 2.0 equiv DIPEA, 0.1 equiv DMAP, CH2Cl2, 18 h, 25 °C, 83% (over two
steps).
Figure 1. General design of ratiometric self-calibrating viscosity sensors.

M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
4445
fluorophore and the molecular rotor, respectively. The emission
spectra of the rotor motif, when directly excited, are shown in Fig-
ure 2b, e and h for dyes 12, 16 and 24, respectively. Figure 2c, f and
i represent the viscosity sensitivity profiles of the ratiometric dyes.
Table
1 summarizes the spectroscopic data for all new
compounds including, for comparison, those of compound 1.
Methoxycoumarin (donor) was exited near 355 nm and emitted
at around 400 nm. The molecular rotor unit, either when excited
through RET or excited directly, emits at around 490 nm for the
thiophene rotors 12 and 24 and at around 485 nm for the aniline
rotor 16 which is in accordance to previously reported data.
Since the Eq. (2) is logarithmic, data points of intensity versus
viscosity in a double logarithmic plot would lie on a straight line.
Extrapolation of this line for log(viscosity) = 0 (i.e., the y-intercept)
would then give the intensity of the dye in photon counts per sec-
ond at a theoretical viscosity of 1 mPaꢀsec. It therefore gives infor-
mation about the relative brightness of the dye, and it also
Scheme 3. Reagents and conditions: (a) 1.0 equiv 7, 1.5 equiv 13, 0.1 equiv DBU,
18 h, 25 °C, 67%; (b) 1.0 equiv 14, TFA/CH2Cl2, 30 min, 25 °C; (c) 1.0 equiv 15,
1.1 equiv 11, 2.0 equiv DIPEA, 0.1 equiv DMAP, CH2Cl2, 18 h, 25 °C, 81% (over two
steps).
combines the proportionality constants G, C, IEX and c from Eq.
18a,19
(2).
The term viscosity sensitivity refers to the exponent x
in Eqs. (1) and (3) and shows how much the rotor intensity in-
creases as the viscosity increases. The exponent x can be obtained
from the slope of the regression line in the double logarithmic plot.
Moreover, the ratiometric intensity, shown in Table 1 (column 12),
represents the ratio of rotor intensity over the donor intensity
extrapolated at a theoretical viscosity of 1 mPaꢀsec. Qualitatively,
this indicates the efficiency of the RET between the primary and
the secondary fluorophore.
In all cases the fluorescence of the coumarin donor was viscos-
ity-independent. This can be evidenced by (a) the almost identical
maximum intensity of the donor; and (b) the power law slope of
the donor that remains almost zero. Small variations in the emis-
sion intensity, especially for compound 16 (Figure 2a), may be
due to its limited solubility in higher viscosity solutions. Further-
more, upon direct excitation of the rotor, the y-intercept was higher
than when excited through RET (Figure 2a/b, d/e and g/h). The low-
er rotor emission intensity through RET can be explained by the
losses in the donor and by the donor emission itself, which both
take away from the resonant energy that arrives at the acceptor.
These findings are in accordance with previously reported data. 21c
Comparison of hydrophilic dyes 12 and 16, containing a thio-
phene- and an aniline-rotor, respectively, indicates that 12 is
brighter than 16 when excited directly (see Table 1, column 11).
Scheme 4. Reagents and conditions: (a) 1.0 equiv 17, 3.0 equiv dihexylamine, H2O,
18 h, 100 °C, 83%; (b) 1.0 equiv 19, 1.6 equiv 20, 1.1 equiv EDC, 1.1 equiv HOBT,
CH2Cl2, 18 h, 25 °C, 73%; (c) 1.2 equiv 18, 1.0 equiv 21, 1.1 equiv DBU, THF, 18 h,
25 °C, 70%; (d) 1.0 equiv 22, TFA/CH2Cl2, 30 min, 25 °C; (e) 1.0 equiv 23, 0.95 equiv
11, 2.0 equiv DIPEA, 0.1 equiv DMAP, CH2Cl2, 2 h, 25 °C, 84% (over two steps).
In a similar manner, the ratiometric dye 16 was synthesized, as
depicted in Scheme 3. Knövenagel condensation of the commer-
cially available N,N-dimethylamino benzaldehyde (13) with 7 gave
rise to carbamate 14 as the E isomer.19 Deprotection of the primary
amine of 14 and coupling with 11 produced the aniline containing
dye 16 in 81% yield.
The synthesis of the more lipophilic dye 24 containing the long
alkyl amine substituents is highlighted in Scheme 4. Commercially
available aldehyde 17 was converted to the dihexylamine deriva-
tive 18 in 83% yield.28 Knövenagel condensation of 18 with b-
cyanoester 2121c gave compound 22 in very good yield as a single
stereoisomer.19 Deprotection of the primary amine 22 by treat-
ment with trifluoroacetic acid, followed by coupling with N-succin-
imidyl-7-methoxycoumarin-3-carboxylate (11), yielded dye 24 in
84% yield.
This is due to the
p-system of 12 that is more electronically rich
than that of 16.19 However, upon RET excitation, the brightness
(y-intercept) of 16 is slightly higher than that of 12 (see Table 1,
column 9). This can be explained by considering the more efficient
excitation of the aniline fluorophore from the coumarin. Specifi-
cally, for 12 the kmax of the coumarin emission is 404 nm while
the kmax for the thiophene excitation is 470 nm. On the other hand,
in 16 the excitation maximum for the aniline dye is 436 nm allow-
ing a more efficient RET. In the case of dye 16, a better ratiometric
intensity is also observed (see Table 1, column 12). Finally, the ani-
line-based rotor 16, is more viscosity sensitive than the thiophene
rotor 12 both when excited through RET or directly.
Comparison of the thiophene-based dyes 12, 24 and 1 shows
that they have almost the same sensitivity when excited through
RET (Table 1, column 8). The above observation suggests that alter-
ations of the linker between the two fluorophores and/or the sub-
stitution of the nitrogen donor do not affect the viscosity
sensitivity of the dyes. In turn, this observation allows optimiza-
tion of the physical properties of the probes, such as solubility
and hydrophilicity, for a selected application. In all cases, whether
the dyes are excited through RET or directly at the rotor, compound
24 shows higher brightness than 12 and 1. This can be explained
due to the size of the nitrogen substituents that block the rotation
2.4. Fluorescence properties of ratiometric dyes
The fluorescence viscosity studies were performed from 4.15
to 414.41 mPaꢀsec. The emission spectra of dyes 12, 16 and 24
recorded either via RET or direct excitation of the rotor are pre-
sented in Figure 2. As expected, all compounds exhibited dual
emission.21c The fluorescence spectra of the dyes when excited
via RET are depicted in Figures 2a, d and g; the peaks with emission
maxima at around 400 and 490 nm correspond to the coumarin

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M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
Figure 2. Fluorescence emission spectra and viscosity sensitivity plots of hydrophilic ratiometric dyes 12 (Figs. a, b and c), 16 (Figs. d, e and f) and 24 (Figs. g, h and i) in
ethylene glycol:glycerol (or MeOH) mixtures (viscosity is recorded in mPaꢀsec). Fig. a, d and g: emission spectra via RET; Figs. b, e and h: emission spectra under direct
excitation of the rotor motif; Figs. c, f and i: viscosity sensitivity plots.
Table 1
Fluorescent properties and viscosity sensitivity of the dyes
Cmp
Donor
Rotor
Ratiometric
intensity
Exc k
(nm)
Em k
(nm)
Power-law
slope of donor
y-Intercept
(ꢁ106 cps)
Exc k
(nm)
Em k
(nm)
Viscosity
sensitivity
(donor exc)
y-Intercept
(donor exc)
(ꢁ106 cps)
Viscosity
sensitivity
(rotor exc)
y-Intercept
(rotor exc)
(ꢁ106 cps)
IEm/Iref
(donor exc)
12
16
24
1
355
350
354
354
400
400
401
402
ꢂ0.018
ꢂ0.0059
0.0013
0.0055
9.08
6.84
6.41
6.86
467
430
471
467
492
485
491
494
0.24
0.31
0.23
0.23
0.91
0.94
1.53
0.81
0.23
0.35
0.32
0.24
1.86
1.12
2.51
1.98
0.10
0.14
0.24
0.12
of the N-C bond and enhance the deexcitation through fluores-
cence. This event is pronounced in more viscous environments.19
different. The more hydrophilic compound 12 shows a relatively
homogeneous distribution across the cell and absence of local con-
centrations that would be indicative of strong protein binding.
Based on this, we conclude that 12 dissolves in the cytoplasm
and remains in the aqueous phase. As such, 12 could serve as a
self-calibrating dye suitable for viscosity measurements in the
cytoplasm.
2.5. Localization of self-calibrating dyes in cells
As illustrated in Figures 3 and 4, both compounds 12 and 24 can
cross the cell membrane, but their localization pattern is distinctly
Figure 3. Representative He-La cells stained with 12. The dye exhibits a fairly homogeneous distribution inside the cell. The intensity profile along the dashed line A–A
further illustrates the homogeneous dye distribution.

M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
4447
Figure 4. Representative T-24 cells dual-labeled with 24 (green) and the membrane dye DiI-C18 (red). Dye 24 exhibits a more complex staining pattern with a clear
preference for intracellular compartments. Magnified sections and the intensity profiles along the dashed lines A–A and B–B show that a moderate correlation between the
locales of DiI and 24 exists.
Conversely, 24 displays a non-uniform staining pattern in the
cell. Analysis of emission intensity shows that 24 emits both from
the cell membrane and from compartments inside the cell, most
likely the cytoskeleton. Co-localization with the known membrane
dye 1,10-dioctadecyl-3,3,30,30-tetramethylindo carbocyanine (DiI)
shows elevated fluorescence of 24 at locations preferred by DiI,
that is, the cell membrane, but 24 exhibits even higher fluores-
cence from compartments inside the cell. It is known that a molec-
ular rotor increases its fluorescence when it binds to a protein.29
This behavior can explain the relatively high emission intensity
from compartments inside the cell compared to emission from
the cell membrane. Due to its relatively low localization specificity,
24 needs to be used in conjunction with a confocal microscope
when it is intended as a membrane viscosity probe. Inside the cell,
it can be used to study protein conformation, although the exact
target proteins are subjects for further studies. The advantage of
the ratiometric dye 24 compared to pure intensity-based studies
with DCVJ29 is that a calibration of the ratiometric intensity can
provide the apparent viscosity of the microenvironment in
mPaꢀsec.23
4. Experimental
4.1. Chemistry
4.1.1. General notes
All reagents were purchased at highest commercial quality and
used without further purification except where noted. Air- and
moisture-sensitive liquids and solutions were transferred via syr-
inge or stainless steel cannula. Organic solutions were concen-
trated by rotary evaporation below 40 °C at approximately
20 mmHg. All non-aqueous reactions were carried out under anhy-
drous conditions. Yields refer to chromatographically and spectro-
scopically (1H NMR, 13C NMR) homogeneous materials, unless
otherwise stated. Reactions were monitored by thin-layer chroma-
tography (TLC) carried out on 0.25 EMD TLC glass silica gel 60 F254
plates and visualized under UV light and/or developed by dipping
in solutions of 10% ethanolic phosphomolybdic acid (PMA) and
applying heat. Dynamic Adsorbents, Inc. silica gel (60, particle size
0.040–0.063 mm) was used for flash chromatography. NMR spec-
tra were recorded on a Varian Mercury 400 MHz instrument and
calibrated using the residual non-deuterated solvent as an internal
reference. The following abbreviations were used to explain the
multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet,
b = broad. High resolution mass spectra (HRMS) were recorded
on a VG 7070 HS mass spectrometer under electron spray ioniza-
tion (ESI) or electron impact (EI) conditions.
3. Conclusions
We present here the design, synthesis and spectroscopic
evaluation of representative self-calibrating dyes. These dyes have
been synthesized by covalently linking a coumarin (reference
fluorophore) with a molecular rotor (viscosity sensor). Modifica-
tions on the linker between the molecular rotor and the coumarin
do not affect significantly the fluorescence profile of these dyes.
This can be further explored for optimization of ratiometric viscos-
ity probes toward specific applications. Subcellular localization
studies of the new dyes show that they can cross the cell mem-
brane and stay in the cytosol. Interestingly, hydrophilic dye 12
shows a homogeneous distribution inside the cell and represents
a suitable probe for viscosity measurements in the cytoplasm. On
the other hand, hydrophobic dye 24 behaves to some extent as a
membrane dye, although it also crosses the cell membrane and
likely binds to intracellular proteins, from where additional fluo-
rescence is seen. Optimization of the chemical motif and fluores-
cence properties of self-calibrating dyes could provide probes for
both microviscosity and bulk viscosity measurements.
4.1.2. 2-[2-(2-Azidoethoxy)ethoxy]ethanol (3)
To a solution of 2-[2-(2-chloroethoxy)ethoxy]ethanol (2) (2 g,
11.86 mmol) in anhydrous DMF (18 mL), sodium azide (0.77 g,
11.86 mmol) potassium carbonate (14.3 g, 103 mmol) were added
and stirred overnight at 90 °C. The mixture was cooled, diluted
with THF (20 mL) and after stirring for 1 h, it was filtered. The solid
was washed with THF and the filtrate and washings were com-
bined and concentrated to afford azide 3 (1.70 g, 82%). Crude com-
pound 3 was used to the next step without further purification. 1H
NMR (400 MHz, CDCl3) d 3.75–3.72 (m, 2H), 3.69–3.66 (m, 6H),
3.62–3.60 (m, 2H), 3.39 (m, 2H); 13C NMR (100 MHz, CDCl3) d
72.3, 70.2, 70.0, 69.6, 61.1, 50.3; HRMS Calcd for C6H13N3O3Na
(M+Na)+ 198.0849. Found 198.0848.

4448
M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
4.1.3. 2-[2-(2-Aminoethoxy)ethoxy]ethanol (4)
added. The reaction was left stirring at room temperature and
monitored by TLC. Upon completion, the reaction was concen-
trated under reduced pressure and purified via flash chromatogra-
phy (30–50% EtOAc–hexanes) to yield 12 (44 mg, 83% yield) as
orange solid. 1H NMR (400 MHz, CDCl3) d 9.05 (bs, 1H), 8.79 (s,
1H), 7.97 (s, 1H), 7.56 (d, 1H, J = 8.7 Hz), 7.39 (bs, 1H), 6.89 (dd,
1H, J = 2.1 Hz, J = 8.7 Hz), 6.81 (d, 1H, J = 2.1 Hz), 6.06 (d, 1H,
J = 4.6 Hz), 4.36 (m, 2H), 3.88 (s, 3H), 3.79 (m, 2H), 3.72–3.63 (m,
8H), 3.41 (m, 4H), 1.67 (bs, 6H); 13C NMR (100 MHz, CDCl3) d
172.3, 169.6, 165.3, 165.0, 162.6, 161.7, 156.8, 148.4, 146.5,
144.2, 131.1, 119.9, 118.5, 114.7, 114.1, 112.5, 105.0, 100.4, 70.9,
70.7, 69.7, 69.3, 64.7, 63.9, 56.2, 51.4, 36.9, 25.2, 23.6; HRMS Calcd
for C30H34N3O8S (M+H)+ 596.2061. Found 596.2060.
The above synthesized azide 3 (1.27 g, 7.25 mmol) was dis-
solved in MeOH (15 mL). Pd/C (0.22 g, 0.03 mmol) was added, the
flask was purged of argon and filled with hydrogen. The solution
was stirred overnight at room temperature. The mixture was fil-
tered through celite and the solvent was evaporated to yield the
pure amino alcohol 4 (1.03 g, 95%). 4: Light yellow oil; 1H NMR
(400 MHz, CDCl3) d 3.73–3.58 (m, 10H), 2.95 (bs, 2H), 2.82–2.79
(m, 2H); 13C NMR (100 MHz, CDCl3) d 72.9, 70.4, 70.3, 61.7, 49.2;
HRMS Calcd for C6H16NO3 (M+H)+ 150.1125. Found 150.1125.
4.1.4. tert-Butyl N-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}
carbamate (5)
Di-tert-butyl dicarbonate (1.75 g, 8.04 mmol) was added to a
solution of amino alcohol 4 (600 mg, 4.02 mmol) in a 9:1 (v/v) mix-
ture of methanol/triethylamine (68 mL). The reaction was left stir-
ring under reflux and upon completion, the solvent was removed
under reduced pressure and the residue extracted with DCM/
water. The organic layer was dried over anhydrous MgSO4 and con-
centrated under reduced pressure to yield 5 (902 mg, 90%). 5: Yel-
low oil; 1H NMR (400 MHz, CDCl3) d 3.77–371 (m, 2H), 3.66–3.55
(m, 8H), 3.49–3.31 (m, 2H), 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 156.2, 79.9, 73.0, 70.6, 70.5, 70.0, 61.9, 40.5, 28.6; HRMS Calcd for
4.1.8. Carbamate 14
To a solution of commercially available compound 13 (94 mg,
0.63 mmol) and 7 (200 mg, 0.63 mmol) in dry THF (2.4 mL), DBU
(0.01 mL, 0.06 mmol) was added and left stirring at room temper-
ature. Upon completion, the solution was concentrated under re-
duced pressure and the product was purified via flash
chromatography (30–50% EtOAc–hexanes) to yield 14 (189 mg,
67% yield) as yellow solid. 1H NMR (400 MHz, CDCl3) d 8.08 (s,
1H), 7.94 (d, 2H, J = 9.1 Hz), 6.70 (d, 2H, J = 9.1 Hz), 5.05 (bs, 1H),
4.43 (m, 2H), 3.81 (m, 2H), 3.71 (m, 2H), 3.63 (m, 2H), 3.55 (m,
2H), 3.32 (m, 2H), 3.12 (s, 6H), 1.43 (s, 9H); 13C NMR (100 MHz,
CDCl3) d 164.5, 156.2, 155.0, 153.9, 134.4, 119.5, 117.6, 111.7,
93.7, 79.3, 71.0, 70.5, 70.5, 69.1, 65.2, 40.6, 40.2, 28.6; HRMS Calcd
for C23H34N3O6 (M+H)+ 448.2442. Found 448.2439.
C
11H24NO5 (M+H)+ 250.1654. Found 150.1649.
4.1.5. b-Cyanoacetate 7
To a round bottom flask containing a solution of the BOC-pro-
tected amino alcohol 5 (750 mg, 3.01 mmol) and cyanoacetic acid
(260 mg, 3.01 mmol) in 6 mL of anhydrous DCM, EDC (470 mg,
3.02 mmol) and HOBT (408 mg, 3.02 mmol) were added. The
formation of the product was monitored by TLC and was com-
pleted after overnight stirring at room temperature. The crude
mixture was concentrated under reduced pressure and the product
was purified via flash chromatography (50–70% EtOAc–hexanes). 7
(743 mg, 78% yield) as light yellow oil; 1H NMR (400 MHz, CDCl3) d
5.00 (bs, 1H), 4.37 (m, 2H), 3.74 (m, 2H), 3.64 (m, 4H), 3.53 (m, 4H),
3.31 (s, 2H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3) d 163.2, 156.3,
113.1, 79.8, 70.8, 70.5, 70.4, 68.7, 66.0, 40.5, 28.6, 24.9; HRMS Calcd
for C14H24N2O6Na (M+Na)+ 339.1527. Found 339.1525.
4.1.9. Ratiometric dye 16
A TFA solution was prepared by combining 5 mL of TFA with
0.1 mL of anisole in 4.9 mL of DCM. 1.1 mL of this solution was
added to 14 (50 mg, 0.11 mmol) and the reaction was left stirring
at room temperature. After 30 min, reaction was completed and
the solution was concentrated, rinsed with toluene (4 ꢁ 10 mL),
concentrated again, and dried under high vacuum to yield com-
pound 15. To a round bottom flask containing 15 in dry DCM
(0.8 mL); DMAP (1 mg, 0.01 mmol), N-succinimidyl-7-methoxy-
coumarin-3-carboxylate (11) (34 mg, 0.10 mmol), and DIPEA
(0.04 mL, 0.22 mmol) were added. The reaction was left stirring
overnight at room temperature. Upon completion, the reaction
was concentrated and purified via flash chromatography (30–50%
EtOAc: hexanes) to yield ratiometric dye 16 (49 mg, 81% yield) as
orange solid. 1H NMR (400 MHz, CDCl3) d 9.06 (bs, 1H), 8.81 (s,
1H), 8.04 (s, 1H), 7.89 (d, 2H, J = 9.0 Hz), 7.56 (d, 1H, J = 8.7 Hz),
6.90 (dd, 1H, J = 2.2 Hz, J = 8.7 Hz), 6.82 (d, 1H, J = 2.2 Hz), 6.66 (d,
2H, J = 9.0 Hz), 4.43 (m, 2H), 3.89 (s, 3H), 3.84 (m, 2H), 3.74 (m,
2H), 3.70–3.65 (m, 6H), 3.09 (s, 6H); 13C NMR (100 MHz, CDCl3) d
165.0, 164.5, 162.6, 161.8, 156.9, 154.9, 153.8, 148.5, 134.3,
131.1, 119.5, 117.6, 114.9, 114.2, 112.6, 111.7, 100.4, 93.9, 71.0,
70.8, 69.8, 69.2, 65.3, 56.2, 40.2, 39.9; HRMS Calcd for C29H31N3O8-
Na (M+Na)+ 572.2003. Found 572.2005.
4.1.6. Carbamate 9
To a round bottom flask, compounds 7 (200 mg, 0.63 mmol) and
8 (122 mg, 0.63 mmol) were dissolved in dry THF (5 mL). To that,
DBU (0.01 mL, 0.06 mmol) was added and left stirring at room tem-
perature. Upon completion, the crude solution was concentrated
under reduced pressure and purified via flash chromatography
(30–50% EtOAc–hexanes) to yield 9 (193 mg, 62% yield) as yellow
solid. 1H NMR (400 MHz, CDCl3) d 8.00 (s, 1H), 7.41 (bs, 1H), 6.08
(d, 1H, J = 4.6 Hz), 5.10 (bs, 1H), 4.35 (m, 2H), 3.76 (m, 2H), 3.67
(m, 2H), 3.60 (m, 2H), 3.52 (m, 2H), 3.42 (m, 4H), 3.29 (m, 2H),
1.67 (m, 6H), 1.40 (s, 9H); 13C NMR (100 MHz, CDCl3) d 169.7,
165.3, 156.3, 146.7, 144.4, 120.0, 118.7, 105.2, 79.3, 70.9, 70.5,
70.5, 69.3, 64.7, 51.5, 40.6, 29.9, 28.6, 25.3, 23.7; HRMS Calcd for
C
24H36N3O6S (M+H)+ 494.2319. Found 494.2316.
4.1.10. 5-(Dihexylamino)thiophene-2-carbaldehyde (18)
2-bromo-5-formylthiophene (17) (0.4 g, 2.09 mmol), dihexyl-
amine (1.5 mL, 6.28 mmol) and p-toluenesulfonic acid (20 mg,
0.06 mmol) were heated at 100 °C for 24 h. The mixture was cooled
and 5 mL of water were added. After stirring for an additional half
hour, the product was extracted with CH2Cl2. The solution was
dried over MgSO4, concentrated under reduced pressure and puri-
fied via flash chromatography (5–50% EtOAc:hexanes) to yield 18
(518 mg, 83% yield) as oil. 1H NMR (400 MHz, CDCl3) d 9.38 (d,
1H, J = 1.0 Hz), 7.37 (d, 1H, J = 4.3 Hz), 5.83 (dd, 1H, J = 1.0 Hz,
J = 4.3 Hz), 3.25 (m, 4H), 1.58 (m, 4H), 1.24 (bs, 12H), 0.83 (m,
6H); 13C NMR (100 MHz, CDCl3) d 179.5, 167.0, 140.7, 125.1,
4.1.7. Ratiometric dye 12
A TFA solution was prepared by combining 5 mL of TFA with
0.1 mL of anisole in 4.9 mL of DCM. 0.90 mL of this solution was
added to 9 (45 mg, 0.09 mmol) and the reaction was left stirring
at room temperature. After 30 min, reaction was completed and
the solution was concentrated, rinsed with toluene (4 ꢁ 10 mL),
concentrated, and dried under high vacuum to yield 10, which
was used immediately to the next step. To a round bottom flask
containing 10 dissolved in dry DCM (0.6 mL); DMAP (1 mg,
0.009 mmol), N-succinimidyl-7-methoxycoumarin-3-carboxylate
(11) (27 mg, 0.08 mmol), and DIPEA (0.03 mL, 0.17 mmol) were

M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
4449
102.7, 53.8, 31.5, 26.5, 22.6, 14.0; HRMS Calcd for C13H22N2O4Na
(M+Na)+ 293.1472. Found 293.1473.
samples were placed on rotating mixer for a minimum of 1 h be-
fore pouring into cuvettes for scanning. Preliminary fluorescent
scanning was done on each dye dissolved in 414.4 mPaꢀsec viscos-
ity solvent to determine optimal excitation and peak emission and
slit settings for each molecular rotor derivative. All fluorescent
scanning was done in a Fluoromax-3 photon-counting spectropho-
tometer (Jobin-Yvon) with the temperature-controlled turret
(Quantum Northwest) set at room temperature (22 °C). Each sam-
ple was inserted in the turret and allowed to equilibrate for 10 min
before testing. The monochromator slit settings for all calculations
were 5 nm. For each solvent the fluorescent emission in an 11 nm
range around peak emission was averaged and the logarithm of the
average peak intensity was plotted against the logarithm of the vis-
cosity. The slope was obtained for each molecular rotor derivative
by linear regression (Graphpad Prism 4.01, San Diego, CA). The
exponent x of each viscosity gradient was used to evaluate viscos-
ity sensitivity, with a higher value of the exponent x indicating
higher sensitivity. R2 values indicate the linear regression of the
log-transformed data (intensity over viscosity).
4.1.11. Carbamate 22
To a solution of compound 18 (130 mg, 0.44 mmol) and 2121c
(100 mg, 0.37 mmol) in dry THF (2.9 mL), DBU (0.08 mL,
0.53 mmol) was added and left stirring at room temperature. Upon
completion, the solution was concentrated under reduced pressure
and the product was purified via flash chromatography (10–30%
EtOAc–hexanes) to yield 22 (169 mg, 70% yield) as orange solid.
1H NMR (400 MHz, CDCl3) d 7.96 (bs, 1H), 7.35 (bs, 1H), 5.95 (d,
2H, J = 4.4 Hz), 4.61 (bs, 1H), 4.18 (t, 2H, J = 6.6 Hz), 3.35 (m, 4H),
3.09 (m, 2H), 1.73–1.57 (m, 6H), 1.54–1.47 (m, 2H), 1.41 (bs,
11H), 1.28 (m, 12H), 0.87 (m, 6H); 13C NMR (100 MHz, CDCl3) d
168.6, 165.6, 156.1, 146.1, 144.6, 119.3, 118.8, 104.4, 79.1, 65.2,
54.0, 40.5, 31.6, 29.8, 28.5, 27.1, 26.6, 23.4, 22.7, 14.1; HRMS Calcd
for C30H50N3O4S (M+H)+ 570.3336. Found 570.3333.
4.1.12. Ratiometric dye 24
A TFA solution was prepared by combining 5 mL of TFA with
0.1 mL of anisole in 4.9 mL of DCM. 1.1 mL of this solution was
added to 22 (60 mg, 0.11 mmol) and the reaction was left stirring
at room temperature. After 30 min, reaction was completed and
the solution was concentrated, rinsed with toluene (4 ꢁ 10 mL),
concentrated again, and dried under high vacuum to yield com-
pound 23. To a round bottom flask containing 23 in dry DCM
(2.0 mL); DMAP (1 mg, 0.01 mmol), N-succinimidyl-7-methoxy-
coumarin-3-carboxylate (11) (38 mg, 0.12 mmol), and DIPEA
(0.04 mL, 0.25 mmol) were added. The reaction was left stirring
overnight at room temperature. Upon completion, the reaction
was concentrated and purified via flash chromatography (10–30%
EtOAc: hexanes) to yield ratiometric dye 24 (60 mg, 84% yield) as
orange solid. 1H NMR (400 MHz, CDCl3) d 8.83 (s, 1H), 8.78 (s,
1H), 7.99 (bs, 1H), 7.57 (d, 2H, J = 8.7 Hz), 7.40 (bs, 1H), 6.93 (dd,
1H, J = 2.4 Hz, J = 8.7 Hz), 6.86 (d, 1H, J = 2.3 Hz), 5.96 (d, 1H,
J = 4.6 Hz), 4.23 (t, 2H, J = 6.7 Hz), 3.91 (s, 3H), 3.47 (m, 2H), 3.37
(m, 4H), 1.81–1.74 (m, 2H), 1.70–1.62 (m, 8H), 1.54–1.46 (m,
2H), 1.35–1.27 (m, 12H), 0.86 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 168.6, 165.7, 164.9, 162.2, 162.1, 156.8, 148.4, 146.2, 144.6,
131.1, 119.4, 118.8, 115.1, 114.1, 112.7, 104.4, 100.5,65.2, 56.2,
54.1, 39.8, 31.7, 29.9, 29.4, 28.7, 27.2, 26.7, 23.6, 22.8, 14.2; HRMS
Calcd for C36H47N3O6SNa (M+Na)+ 672.3078. Found 672.3082.
4.3. General procedure for cell culture staining and imaging
Immortalized T24 human bladder epithelial cells (HTB-4™
ATCC, Manassas, VA, USA) were grown to confluence on glass cul-
ture slides (Fisher Scientific, Pittsburg, PA, USA). The cells were
pre-stained with an aqueous Vybrant DiI (Invintrogen) solution
as suggested by invitrogen/molecular probes.31 A liposome solu-
tion was prepared. 200
line (Avanti Polar Lipids, Inc., Alabaster AL, USA) in chloroform and
200 l of chloroform (Sigma–Aldrich, St. Louis, MO, USA) were
added to a small glass vial. 50 l of 5 mM dye solution in chloro-
ll of 1,2-dilauroyl-sn-glycero-phosphocho-
l
l
form was added. Chloroform was evaporated while phial was ro-
tated creating a thin layer on the inner walls of the vial. 1 ml of
Ca2+ and Mg2+ free phosphate buffered Saline (PBS, Sigma–Aldrich
St. Louis, MO, USA) solution was added. The solution is treated to 3
cycles of 10 min in a water bath at 37 °C and 5 min in an ice bath.
The solution was then extruded 11 times through a 0.1
lm mem-
brane with the Avanti Mini-Extruder creating 0.1 m liposomes.
l
Liposome solution was added to serum-free McCoy’s 5A modified
media (Sigma Aldrich, St. Louis, MO, USA) at a ratio of 1:9. A
500 ll volume of liposome-media solution is added to the slide
wells and allowed to incubate for 30 min at 37 °C and 5% CO2.
The dye concentration applied to the cells during staining is esti-
4.2. General procedure for the determination of spectral
properties
mated at 25 lM by dilution. Staining solution was removed and
the slides gently washed with serum-free PBS. Images were taken
at 60X magnification using Olympus IX71 microscope with both
WBV (exciter D425/40X, emitter 475lpv2, beamsplitter 450dcxr)
and TRITC (exciter D540/25X emitter D605/55 m beamsplitter
565dclp) cube filters with an Apogee Alta 2000 camera. A compos-
ite image was created from the WBV image (green channel) and
the corresponding TRITC image (red channel) and the image was
reduced in size by binning a 2 ꢁ 2 pixel area. Image processing
and extraction of the intensity profiles were performed with Crys-
Each viscosity sample was mixed according to the volumes
shown in the first column in Table 2 below. Viscosity of each sam-
ple was estimated by the summation of weighted ratio30 of each
solvent’s viscosity at 25 °C from the 92st Edition of the CRC Hand-
book of Chemistry and Physics, 2011–2012. The glycerol (Gly) was
heated to ensure more exact measuring during pipetting. The pre-
stained ethylene glycol (EG) for each sample contained 100 lM of
32
dye resulting in a final concentration of 10
lM for each sample. All
tal Image.
A 20 mM stock solution of compound 12 in DMSO was prepared
and it was then diluted and used at a final concentration of 2 lM in
Table 2
DMEM medium. HeLa cells were maintained at 37 °C in a 5% CO2
incubator in DMEM supplemented with 10% FBS. The day prior to
the experiment the cells were plated on 35 mm Ibidi dishes (Biov-
Solvent composition and viscosity
Pre-stained EG/EG/Gly
or MeOH volumes (mL)
Viscosity
(mPa ꢀsec)
Log
viscosity
alley #81156) at 70% confluency. The probes were added at 2 lM
concentration into DMEM. Control cells were treated with DMSO
at 0.1%. After 1 h incubation, the cells were washed three times
with DMEM and then kept into DMEM supplemented with
25 mM HEPES (pH = 7.4). The cells were then imaged live using a
Zeiss Observer Z1 156 inverted microscope with 63ꢁ objective
controlled by AxioVision software (Zeiss, Thornwood, NY).
0.5/0.5/4.0
0.5/1.5/3.0
0.5/3.5/1.0
0.5/4.5/0.0
0.5/3.5/1.0 (MeOH)
0.5/2.5/2.0 (MeOH)
414.4
183.8
36.2
16.1
8.16
4.15
2.62
2.26
1.56
1.21
0.91
0.62

4450
M. Dakanali et al. / Bioorg. Med. Chem. 20 (2012) 4443–4450
11. (a) International Committee for Standardization in Haematology, J. Clin. Pathol.
Acknowledgments
1984, 37, 1147.; (b) Wang, S.; Boss, A. H.; Kensey, K. R.; Rosenson, R. S. Clin.
Chim. Acta 2003, 332, 79.
Financial support from the National Institutes of Health (1R21
RR025358 and CA 133002) is gratefully acknowledged. We thank
the NSF for instrumentation grants CHE-9709183 and CHE-
0741968. We also thank Dr. A. Mrse and Dr. Y. Su for NMR and
MS assistance, respectively.
12. Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer: New York, NY,
2006.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.026.
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