New brightly coloured, water soluble, core-substituted naphthalene diimides for biophysical applications

Article abstract of DOI:10.1016/j.dyepig.2014.07.018

Symmetrically and dissymmetrically core-substituted amino naphthalene diimides (cNDIs) with water-soluble substituents have been synthesised. The compounds display characteristic steady-state optical properties absorbing and emitting light in the visible part of the electromagnetic spectrum. Time-resolved fluorescence studies reveal long emission lifetimes of ～10 ns in chloroform, ～8 ns in methanol and ～4 ns in water. Suitability for use in biophysical applications is demonstrated by encapsulation into reverse micelles and liposomes. Fluorescence lifetime is shown to indicate cavity size in the reverse micelles and fluorescence correlation spectroscopy measurements of cNDI loaded liposomes successfully determined diffusion coefficients and hydrodynamic radii of the liposomes.

Full text of DOI:10.1016/j.dyepig.2014.07.018

Dyes and Pigments 112 (2015) 290e297
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
New brightly coloured, water soluble, core-substituted naphthalene
diimides for biophysical applications
Heather F. Higginbotham, Subashani Maniam, Steven J. Langford, Toby D.M. Bell*
School of Chemistry, Monash University, Wellington Road, Clayton, Victoria 3800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Symmetrically and dissymmetrically core-substituted amino naphthalene diimides (cNDIs) with water-
soluble substituents have been synthesised. The compounds display characteristic steady-state optical
properties absorbing and emitting light in the visible part of the electromagnetic spectrum. Time-
resolved fluorescence studies reveal long emission lifetimes of ~10 ns in chloroform, ~8 ns in meth-
anol and ~4 ns in water. Suitability for use in biophysical applications is demonstrated by encapsulation
into reverse micelles and liposomes. Fluorescence lifetime is shown to indicate cavity size in the reverse
micelles and fluorescence correlation spectroscopy measurements of cNDI loaded liposomes successfully
determined diffusion coefficients and hydrodynamic radii of the liposomes.
Received 22 April 2014
Received in revised form
26 June 2014
Accepted 16 July 2014
Available online 30 July 2014
Keywords:
Naphthalene diimide
Red emission
© 2014 Elsevier Ltd. All rights reserved.
Water soluble
Time-resolved fluorescence
Reverse micelle
Fluorescence correlation spectroscopy
1. Introduction
Substitution of NDIs on the core has seen the recent develop-
ment of a whole new sub-class of NDIs, the core-substituted
Since the development of the first synthetic dyes in the mid-
19th century, coloured molecules have found applications ranging
from clothing to cellular imaging. The search for new dye com-
pounds with improved and unusual properties is on-going and new
applications for existing dyes are continually being found. Naph-
thalene diimides (NDIs) are a well-known class of functional mol-
ecules which typically absorb light in the UV region of the
electromagnetic spectrum and are thus uncoloured. NDIs have,
however, attracted the attention of researchers for many years who
have sought to take advantage of the many useful properties of
NDIs such as their electron deficient nature, rigid structure, air
stability and solubility in a range of organic solvents. Significant
recent interest in NDIs has been in the drive to create new and
interesting molecules [1]. These aspects have led to NDIs being
used in a variety of applications including in molecular pulleys and
levers in rotaxanes and pseudo-rotaxanes [2], as molecular recog-
nition indicators through pieanion interactions [3], and in porous
metal organic frameworks [4]. The high electron affinity of NDIs has
also been taken advantage of in the design of donoreacceptor
complexes [5,6] and organic semiconductors [7].
naphthalene diimides (cNDIs). Many of these molecules are
intensely coloured with dramatically altered optical properties
with respect to the parent NDI and are being pursued as novel and
versatile fluorescent chromophores. One highly favourable aspect
of cNDIs is that optical properties such as absorption and emission
wavelengths can often be ‘tuned’ through the visible and near
infrared regions of the electromagnetic spectrum by the nature and
particularly, the number, of electron donating groups on the core
[8e10]. One of the most common type of cNDIs, are the substituted
amino naphthalene diimides (SANDIs), which have been investi-
gated for use in fluorescence applications such as cationic and
anionic sensors [11e15], as indicators for G-quaduplex in DNA
[16e18], and in single molecule studies [19]. SANDIs have been
identified as ideal small molecules for such applications due to their
facile functionalisation, creating intramolecular electron poor and
rich domains required for rapid regulation of fluorescence output.
SANDIs are also bright, displaying high emission quantum yields
and good photostability, even at the level of single molecules.
The small size of SANDIs makes them ideal candidates for water
solubilisation creating further potential for use as biologically
relevant materials. Emission in the red region of the electromag-
netic spectrum [9] provides a further advantage by contrasting
auto-fluorescence and diminishing light reabsorption from other
biological materials. Additionally, SANDIs have already been shown
* Corresponding author. Tel.: þ61 3 9905 4566; fax: þ61 3 9905 4597.
E-mail address: toby.bell@monash.edu (T.D.M. Bell).
http://dx.doi.org/10.1016/j.dyepig.2014.07.018
0143-7208/© 2014 Elsevier Ltd. All rights reserved.

H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
291
[10] to have synthetic flexibility in both the core and the imide
positions making the synthesis of a diverse range of hydrophilic
structures possible. Non-core substituted naphthalene diimides
have already been shown to be water soluble through the substi-
tution of hydrophilic groups at the imide positions [20e22]. These
have included the use of imide substituted amino acids as sol-
ubilising units for self assembly [23], and in combinatorial libraries
[5]. SANDIs have also been rendered water soluble through qua-
ternary ammonium salts and used for the detection of singlet ox-
ygen [24], while very recently, a series of phenolate functionalised,
conjugated cNDIs has been described and characterised [25].
In pursuit of the goal of water soluble, red emitting dyes, a series
of SANDI molecules has been synthesised (Scheme 1) and charac-
terised. The SANDIs were synthesised from the common di-bromo
starting material (1), with imidation at the anhydride positions
occurring via a substitution reaction under acidic conditions to
allow for selective substitution in the imides without further sub-
stitution on the core [9]. This imidation reaction was followed by
nucleophilic aromatic substitution to synthesise compounds 5e7. A
one-pot method was utilised to synthesise 8 with the choice of
groups being made on the grounds that they were structurally
straight forward and readily available. The photophysical proper-
ties, including fluorescence lifetimes, of these compounds were
then determined in a range of solvents including water for com-
pounds 6e8. SANDIs 7 and 8 were then further examined in AOT
reverse micelles and 7 in liposomes to investigate their potential
and suitability for use in biophysical applications.
chloride, triphenylphosphine and sodium bis(2-ethylhexyl) sul-
fosuccinate (AOT) were purchased from Aldrich and used without
further purification. Sodium azide was purchased from Ajax
chemicals. Chloroform (CHCl3, Merck spectrophotometric grade),
methanol (MeOH, Merck GR grade) and water (Millipore) were
used as received. 1H and 13C nuclear magnetic resonance (NMR)
spectra were recorded using a Bruker DRX 400 MHz Spectrometer
(1H taken at 400 MHz and 13C taken at 100 MHz) or Bruker DRX
600 MHz Spectrometer (13C taken at 125 MHz), using deuterated
chloroform (CDCl3) unless otherwise stated. The chemical shifts
(
d
) were calibrated with reference to the solvent peak in the
spectrum. For 1H NMR spectra, each resonance was assigned ac-
cording to the following convention: chemical shift ( ), measured
d
in parts per million (ppm), multiplicity, coupling constant (J),
measured in Hz, number of protons and assignment. Multiplicities
are given as (s) singlet, (d) doublet, (t) triplet, (q) quartet, (p)
pentet or (m) multiplet. The 13C NMR spectra were assigned a
chemical shift (d) measured in parts per million. High Resolution-
Electrospray Ionisation Mass Spectrometry (HR-ESI) was
completed on an Agilent Technologies 6220 Accurate- Mass TOF-
time of flight instrument. Absorbance spectra were run on a Cary
100 Bio UVevisible spectrophotometer (Agilent technologies) and
steady state fluorescence measurements were recorded on a
Varian model Cary Eclipse fluorescence spectrophotometer (Agi-
lent technologies) and corrected for detector efficiency. Fluores-
cence quantum yields were determined using Rhodamine 101
(
Ff ¼ 1.0 in ethanol with 0.01% HCl) [26] as the standard. Solu-
tions were prepared in thoroughly cleaned 1.0 cm quartz cuvettes.
For quantum yield measurements absorbance maxima were less
than 0.10 in order to reduce inner filter effects and samples were
deoxygenated by bubbling with N2 for 20 min immediately prior
to measurement. Emission spectra were taken under identical
conditions and the areas under the curve integrated in order to
determine fluorescence quantum yields. Differences in the frac-
tion of light absorbed and refractive index were accounted for
using appropriate corrections.
2. Experimental
2.1. General
1,4,5,8-naphthalene tetracarboxylic dianhydride was pur-
chased from Tokyo Chemical Industries and used without further
purification. 2-(2-aminoethoxy)ethanol (98%), tri(ethylene glycol)
monomethyl ether, 1-octylamine (99%), 4-methylbenzene sulfonyl
Scheme 1. Two step, sequential imidation and core substitution reaction scheme for the synthesis of SANDIs 5e8.

292
H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
2.2. Fluorescence lifetimes
Hydrodynamic radii (rH) of the molecules were estimated from
the diffusion coefficients using the StokeseEinstein equation.
Fluorescence decay histograms were obtained using Time
Correlated Single Photon Counting (TCSPC) with excitation from a
picosecond pulsed supercontinuum fibre laser (Fianium, SC 400-
4-pp) creating of pulses ~50 ps at 5 or 10 MHz repetition rates
across the visible and near IR. Excitation wavelength selection
was achieved using 10 nm bandpass filters (Chroma) and a
700 nm shortpass filter. Emission from the sample was collected
at 90ꢀ to the excitation beam and passed through a mono-
kBT
6phD
rH
¼
(3)
where kB is the Boltzmann constant, T is the temperature in Kelvin,
is the viscosity of water (0.001002 Pa$s at 20 ꢀC).
h
2.4. Synthesis of cNDI dyes
chromator (CVI, dk480) and focused onto
a fast response
avalanche photodiode detector (APD, Id-Quantique, Id-100).
Photon emission times were recorded by a photon counting
module (Picoquant, PicoHarp 300) with the start signal provided
by a fast photodiode triggering from the excitation laser and stop
signal from the APD detector. An instrument response function
(IRF) recorded from a scattering solution (dilute milk powder in
water) had a full width half maximum of ~90e100 ps [15]. Prior to
measurement all samples were degassed by bubbling with ni-
trogen gas for 20 min. Fluorescence decay times were obtained by
2.4.1. (((4,9-Dibromo-1,3,6,8-tetraoxo-1,3,6,8-tetrahydrobenzo
[lmn][3,8]phenanthroline-2,7-diyl)bis(ethane-2,1-diyl))bis(oxy))
bis(ethane-2,1-diyl) diacetate (3)
2-(2-Aminoethoxy)ethanol (0.22 g, 2.2 mmol) was added to a
suspension of 2,6-dibromo-1,4,5,8-naphthalene tetracarboxylic
dianhydride (1) (0.23 g, 0.53 mmol) in 60 mL of glacial acetic acid
and heated to 120 ꢀC for 4 h. The reaction mixture was then cooled
to room temperature and added to water (200 mL), filtered and
washed with water to obtain a light brown precipitate. This crude
product was then purified using column chromatography (20%
acetone in DCM) yielding 0.12 g (33%), Mp. 165e168 ꢀC, of (3) as a
pale yellow solid.
fitting the data with
a single exponential decay function
convolved with the IRF using an iterative least-squares routine
based on the LevenbergeMarquardt algorithm. Goodness of fit
c
2) and by inspection
1H NMR (400 MHz, CDCl3):
d 8.99 (s, 2H, ArH), 4.47 (t, J 6.0 Hz,
was judged by the chi-squared parameter (
of the residuals.
4H, NeCH2), 4.18 (dd, J 4.8 Hz, J 6.0 Hz, 4H,OeCH2), 3.85 (t, J 6.0 Hz,
4H, CH2), 3.74 (dd, J 4.8 Hz, J 4.8 Hz, 4H, OeCH2), 2.00 (s, 6H, CH3).
13C NMR (100 MHz, CDCl3)
d
171.0, 161.0160.9, 139.3, 128.6, 128.0,
2.3. Fluorescence lifetime correlation spectroscopy
125.5, 124.3, 68.8, 67.7, 63.8, 40.1, 21.0. HR-MS (ESI, þve): m/z
observed [M
þ
Na]þ 704.9694, calculated C26H24Br2N2O10Na
Excitation was provided by the supercontinuum fibre laser
described above with the laser light directed into the back aperture
of an inverted microscope (Olympus, IX71) and focussed by a 100X
1.2 NA water immersion objective with a correction collar for cor-
recting refractive index mismatch. Emission from the sample was
passed through a dichroic filter and long pass filter to reject scat-
tered excitation light and focused onto an avalanche photodiode
[M þ Na]þ 704.9695.
2.4.2. 4,9-Dibromo-2,7-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)
benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (4)
2-(2-(2-Methoxyethoxy)ethoxy)ethan-1-amine
(0.39
g,
2.4 mmol) was added to a suspension of 2,6-dibromo-1,4,5,8-
naphthalene tetracarboxylic diimide (1) (0.25 g, 0.59 mmol) in
50 mL of glacial acetic acid and heated to 120 ꢀC for 4 h. The re-
action mixture was then cooled to room temperature and added to
water (100 mL), filtered and washed with water to obtain a yellow/
brown precipitate. This crude product was then purified using
column chromatography (5% acetone in DCM) yielding 0.15 g (35%),
Mp. 147e149 ꢀC, of (4) as a bright yellow powder.
detector (APD, PicoQuant GmbH,
t-SPAD). Time tagged/time
resolved (TTTR) data were recorded using the photon counting
module above allowing lifetime and intensity auto-correlation data
to be recorded simultaneously. The small confocal volume was
created by placing a 30 mm pinhole before the APD. The confocal
volume (height (zo) and waist (wo)) were determined using
Rhodamine B in water at 20 ꢀC [27] (SI Figs S7 and S8). Autocor-
relation curves were analysed using Picoquant SymPhoTime soft-
ware with diffusion coefficients determined via the following
model equation (equation (1)) which contains terms accounting for
3 dimensional Brownian diffusion and the formation of triplet
states.
1H NMR (400 MHz, CDCl3):
d 8.98 (s, 2H, ArH), 4.46 (t, J 5.8 Hz,
4H, NeCH2), 3.86 (t, J 5.8 Hz, 4H, OeCH2), 3.71e3.69 (m, 4H,
OeCH2), 3.62e3.57 (m, 8H, OeCH2), 3.48e3.46 (m, 4H, OeCH2),
3.32 (s, 6H, CH3). 13C NMR (100 MHz, CDCl3)
d 161.0, 160.9, 139.2,
128.5, 128.0, 125.5, 124.3, 72.0, 70.8, 70.7, 70.3, 67.8, 59.2, 40.2. Mass
spectrum HR-MS (ESI, þve): m/z observed [M þ Na]þ 737.0318,
calculated C28H32Br2N2O10Na [M þ Na]þ 737.0321.
!
t
T:eꢁ
1
1
t
triplet
1
N:23
ꢀ
ꢁ
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
GðtÞ ¼ 1 þ
1 þ
ꢀ
ꢁ
2
1 ꢁ T
2
t
tD
1 þ
u0
z0
t
tD
1 þ
2.4.3. 4,9-Bis((2-(2-hydroxyethoxy)ethyl)amino)-2,7-dioctylbenzo
[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (5)
(1)
2-2(Aminoethoxy)ethanol (32 mg, 0.31 mmol) was added to (2)
(50 mg, 0.077 mmol) in 10 mL of CHCl3 and heated at 60 ꢀC for 24 h.
The reaction mixture was then cooled to room temperature and the
solvent evaporated to produce a dark blue precipitate. This crude
product was then purified using column chromatography in DCM
yielding 23 mg (42%) of (5) as a dark blue semi-solid.
N is the number of molecules in the confocal volume,
t
is the lag
time and tD is the diffusional (residence) time of the molecules. u0
and z0 are the height and waist of the confocal volume, respectively,
T is the proportion of molecules residing in the triplet state and
ttriplet is the triplet state lifetime.
From the diffusional time tD, determined in equation (1), the
diffusion coefficient D can be obtained from the following equation.
1H NMR (400 MHz)
d 9.66 (t, J 5.2 Hz, 2H, NH), 8.22 (s, 2H, ArH),
4.16 (dd, J 7.6 Hz, 7.6 Hz, 4H, NeCH2), 3.87e3.81 (m, 4H, NHeCH2),
3.80e3.74 (m, 4H, OeCH2), 3.73e3.68 (m, 8H, OeCH2), 2.78 (t, J
5.8 Hz, 2H, OH), 1.74e1.67 (m, 4H, CH2), 1.42e1.32 (m, 20H, CH2),
u02
4D
0.88 (t, J 7.2 Hz, 6H, CH3). 13C NMR (100 MHz)
d
166.7, 163.8, 126.2,
tD
¼
(2)
121.7, 119.3, 73.2, 70.3, 62.3, 43.3, 41.0, 32.3, 29.8, 29.7, 28.6, 27.7,

H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
293
23.1, 14.6. Mass spectrum HR-MS (ESI, ꢁve): m/z observed [MꢁH]ꢁ
695.4035, calculated C38H55N4O8 [MꢁH]ꢁ 695.4020.
purified using column chromatography (1% MeOH in ethyl acetate)
yielding 64 mg (52%) of (8) as a dark blue semi-solid.
1H NMR (400 MHz)
d 9.52 (t, J 5.2 Hz, 2H, NH). 8.17 (s, 2H, ArH),
4.43 (t, J 6.4 Hz, 2H, NH), 3.86e3.80 (m, 8H, OeCH2), 3.79e3.59 (m,
30H, OeCH2), 3.56e3.54 (m, 4H, OeCH2), 3.37 (s, 3H, OeCH2), 3.33
2.4.4. (((4,9-Bis((2-(2-hydroxyethoxy)ethyl)amino)-1,3,6,8-
tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]phenanthroline-2,7-diyl)
bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) diacetate (6)
2-(2-Aminoethoxy)ethanol (31 mg, 0.29 mmol) was added to (3)
(50 mg, 0.073 mmol) in 10 mL of CHCl3 and heated to 60 ꢀC for 24 h.
The reaction mixture was then cooled to room temperature and the
solvent evaporated to produce a dark blue precipitate. This crude
product was then purified using column chromatography (2%
MeOH in DCM) yielding 16 mg (30%) of (6) as a dark blue semi-
solid.
(s, 3H, CH3). 13C NMR (100 MHz)
d166.1, 163.2, 149.3, 125.9, 121.5,
118.6, 102.3, 72.0, 70.8, 70.7, 70.4, 69.7, 68.0, 59.1, 43.1. Mass spec-
trum HR-MS (ESI, þve): m/z observed [M þ Na]þ 903.4213, calcu-
lated C42H64N4O16Na [M þ Na]þ 903.4215.
3. Results and discussion
3.1. Steady state spectroscopy analysis
1H NMR (400 MHz)
d 9.62 (t, J 5.2 Hz, 2H, NH), 8.20 (s, 2H, ArH),
4.43 (t, J 6.0 Hz, 4H, NeCH2), 4.20 (dd, J 4.4 Hz, J 6.0 Hz, 4H, NeCH2),
3.87e3.79 (m, 12H, OeCH2), 3.75e3.67 (m, 12H, OeCH2), 2.81 (br
UV-visible and fluorescence spectra of compounds 5e8 were
recorded in CHCl3 and MeOH and 6e8 also measured in water. By
way of example, these spectra for 7 are shown in Fig 1. Spectra for
compounds 5, 6 and 8 are in the SI (Fig S1). Absorption is charac-
teristic of di-core-substituted NDIs [19] with the NDI core
m, 2H, OH) 1.99 (s, 6H, CH3). 13C NMR (100 MHz)
d 171.2, 166.2,
163.2, 149.4, 125.7, 121.3, 118.9, 102.1, 72.9, 69.8, 68.7, 68.0, 63.8,
62.1, 42.9, 39.2, 21.0 Mass spectrum HR-MS (ESI, þve): m/z observed
[M þ H]þ 733.2922, calculated C34H45N4O4 [M þ H]þ 733.2932.
absorbing due to a
pe
p* transition in the near UV at around
360 nm and a broad transition with a shoulder due to vibronic
structure in the red part of spectrum. This transition arises from the
substitutions on the core and bestows an intense blue colour to
solutions of these compounds. In a given solvent, the spectra of all
compounds are very similar. Spectra are slightly blue-shifted in
MeOH compared to CHCl3 and there is a small red shift in water
(Table 1). There is also considerable broadening of the absorption
for 6e8 in water. This is likely due to aggregation indicating that
solubility in water for these compounds is not complete. The molar
absorptivities of all four compounds were found to be
~11,500 Mꢁ1 cmꢁ1 in CHCl3 (see SI, Fig S2)).
Fluorescence spectra show good mirror-image properties with a
vibronic shoulder at lower energy to the main emission. Stokes
shifts are ~770 cmꢁ1 in CHCl3, 950 cmꢁ1 in MeOH and 850 cmꢁ1 in
water. As 6e8 were placed in increasingly polar solvents there is a
reduction in intensity in the fluorescence emission intensity. This is
attributed to increased non-radiative relaxation and, in water, the
formation of non-fluorescing aggregates. Emission maxima for 5e8
are similar in CHCl3 and MeOH, and there is a bathochromic shift in
wavelength in water for 6e8 of ~150 cmꢁ1. The quantum yields
(Table 1) of all four compounds in CHCl3 are typical of di-
alkylamino substituted cNDIs [9,10,19] and are marginally greater
than 0.5. These values decrease when placed in MeOH
2.4.5. 4,9-Bis((2-(2-hydroxyethoxy)ethyl)amino)-2,7-bis(2-(2-(2-
methoxyethoxy)ethoxy)ethyl)benzo[lmn][3,8]phenanthroline-
1,3,6,8(2H,7H)-tetraone (7)
2-(2-Aminoethoxy)ethanol (21 mg, 0.20 mmol) was added to (4)
(35 mg, 0.049 mmol) in 10 mL of CHCl3 and heated to 60 ꢀC for 24 h.
The reaction mixture was then cooled to room temperature and the
solvent evaporated to produce a dark blue precipitate. This crude
product was then purified using column chromatography (2%
MeOH in DCM) yielding 22 mg (60%) of (7) as a dark blue semi-
solid.
1H NMR (400 MHz)
d 9.64 (t, J 5.2 Hz, 2H, NH), 8.22 (s, 2H, ArH),
4.43 (t, J 6.0 Hz, 4H, NeCH2), 3.87e3.78 (m, 12H, OeCH2), 3.74e3.58
(m, 20H, OeCH2) 3.48e3.46 (m, 4H, OeCH2), 3.33 (s, 6H, CH3). 13C
NMR (100 MHz)
d 166.3, 163.4, 149.5, 125.8, 119.0, 102.3, 72.9, 72.1,
70.8, 70.6, 70.3, 69.9, 68.1, 62.1, 59.1, 43.0 Mass spectrum HR-MS
(ESI, þve): m/z observed [M
þ
H]þ 765.3550, calculated
C
36H53N4O14 [M þ H]þ 765.3558.
2.4.6. 2,7-Bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-4,9-bis((2-(2-
(2-methoxyethoxy)ethoxy)ethyl)amino)benzo[lmn][3,8]
phenanthroline-1,3,6,8(2H,7H)-tetraone (8)
2-(2-(2-Methoxyethoxy)ethoxy)ethan-1-amine
(0.19
g,
1.1 mmol) was added to a suspension of (1) (60 mg, 0.14 mmol) in
10 mL of CHCl3 and heated at 60 ꢀC overnight. The reaction mixture
was then cooled to room temperature and the solvent evaporated
to produce a dark blue precipitate. This crude product was then
(Ff ~ 0.33e0.43) and are strongly reduced in water (Ff ~ 0.04)
suggesting the presence of non-fluorescent aggregates (see below).
Excitation spectra (see SI, Fig S3) have similar spectral shapes to
absorption spectra in CHCl3 and MeOH and are spectrally narrower
Fig. 1. a) UVevisible absorption spectra and b) fluorescence emission spectra (lex ¼ 590 nm) of 2 ꢂ 10ꢁ5 M solutions of 7 in chloroform (green), methanol (purple) and water (blue).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

294
H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
Table 1
Absorption and emission maxima, quantum yields (Ff) and fluorescence lifetimes (tf) of compounds 5e8 in chloroform, methanol and water.
Compound
labs max (nm)
lem max (nm)
CHCl3/MeOH/H2O
Фf
tf (ns)
(5)
(6)
(7)
(8)
609/604/e
640/642/e
0.58 0.04/0.43 0.01/e
9.7/7.8/e
610/604/622
610/604/621
610/605/623
640/641/657
640/641/658
640/642/655
0.59 0.02/0.37 0.02/0.030a
0.56 0.02/0.37 0.06/0.031a
0.56 0.02/0.33 0.01/0.039a
9.6/8.0/4.4
9.6/8.2/4.3
10.4/8.4/4.4
a
Standard errors for
Ф
f in CHCl3 and MeOH are 1 standard deviation.
Ф
f in water varies with concentration. The values here are at 1 ꢂ 10ꢁ5 M. See the SI, Table S1 for
Фf at
different concentrations for compound 8.
than the absorption spectra in water and similar to absorption
spectra in the other solvents suggesting that emission originates
from non-aggregated species.
contributing to the total fluorescence quenching observed.
Furthermore, the single exponential decay profiles of 6e8 in water
and invariant lifetime of 8 over the concentration range studied,
show that the aggregates are not contributing emission as distinctly
separate fluorescent species and that the emission in water can be
attributed to fully solvated individual molecules. Finally, it is worth
noting that the fluorescence lifetimes of 6e8 in water compare well
to known water-soluble dyes being similar to fluorescein de-
rivatives [28] and relatively long compared the rhodamines [29], for
example.
3.2. Time correlated single photon counting analysis
Fluorescence lifetimes of 5e8 were measured in CHCl3, MeOH
and water using the method of Time Correlated Single Photon
Counting (TCSPC). By way of example, decay histograms and fitted
functions for 7 are shown in Fig. 2 (see SI Fig S4 for 5, 6 and 8). All
compounds displayed single exponential decay kinetics in all sol-
vents investigated. Fitted functions matched the data well with c2
3.3. Reverse micelle analysis
goodness-of-fit parameter values in the range 1.0 < c
2 < 1.2. There
is little variation in lifetime for each solvent with values in CHCl3
~10 ns, in MeOH ~8 ns and in water ~4.4 ns (Table 1). This similarity
indicates that decay from the excited state occurs via the same
mechanism in all compounds regardless of the alteration of side
arm derivatives on the core. The reduction of fluorescence lifetime
in MeOH corresponds with the reduction in quantum yield indi-
cating that this is due to increases in the rate constants of non-
radiative decay pathways. In water, fluorescence lifetimes and
quantum yields are further reduced indicating non-radiative
pathways are more competitive again compared to radiative
decay. The quantum yield, however, is quenched more than is
accounted for by the reduction in lifetime indicating that there is a
further mechanism reducing steady state emission. Given the
broadening of the absorption spectra for 6-8 in water, this can be
attributed to static quenching due to the formation of non-
fluorescent aggregates.
Reverse micelles are small, spherical aggregates formed from
water in oil microemulsions [30]. Reverse micelles have been
used as mimics of biological systems to provide information on
the potential photophysical behaviour of a molecule if it were to
be confined in an environment such as a cell [31e33]. The for-
mation of reverse micelles using sodium bis(2-ethylhexyl)sulfo-
succinate (AOT) as the formation surfactant produces micelles of
precise sizes. When water is confined inside the micelle cavity, it
first forms strong interactions with the ionic sulfosuccinate head
group, creating a more structured water system. As more water is
incorporated into the micelle and the micelle size increases, the
sulfosuccinate anion becomes fully solvated and the remaining
water creates a free molecule pool with properties approaching
those of bulk water [30,32,34]. The size of the micelle is defined
by wo, which is the concentration ratio of AOT against H2O. wo is
a direct measure of the hydrodynamic radius of reverse micelles
and therefore allows careful control of micelle local
environment.
Dye loaded AOT reverse micelles were made according to
literature methods [35,36] of sizes ranging from wo ¼ 1 to wo ¼ 30
with water and hexane as the formulation solvents (see SI Table S2
for preparation details). Both 7 and 8 were investigated in a range
of these reverse micelles. Fig. 3 (a) shows fluorescence spectra of 8
confined in varying sizes of AOT reverse micelles. Fluorescence
spectra of 8 in CHCl3, water and hexane were also taken at iden-
tical concentrations for comparison. The emission spectrum of 8 in
hexane shows a blue-shifted spectrum of very low intensity (pink
spectrum in Fig. 3(a)) consistent with its very low solubility. The
emission spectrum of 8 in water shows the similar quenching and
a bathochromic shift as seen in Fig. 1(b). However once 1 ꢂ 10ꢁ5 M
of 8 was loaded into the reverse micelles, the emission intensity of
8 was restored up to 80% in the smallest wo ¼ 1 micelles compared
to CHCl3 (Fig. 3(a)). There is also a restoration of the vibronic
structure lost in the water spectrum. The intensity of emission
decreases with a bathochromic shift as the reverse micelles
become larger and 8 is further affected by the increasing amount
of water inside the micelle. Similar results were obtained for 7 (see
SI, Fig S5).
Examination of the concentration dependence of quantum yield
and lifetime was performed for 8 in water it was found that the
lifetime remains constant over the range of 2.5 ꢂ 10ꢁ7e5 ꢂ 10ꢁ5 M
whereas the quantum yield steadily decreases as [8] increase (see
SI, Table S1). This behaviour provides further evidence for aggregate
formation at higher concentrations which lead to static quenching
Fig. 2. TCSPC fluorescence decay profiles of 7 and fitted functions (black lines) in CHCl3
(green), methanol (purple) and water (blue). The instrument response function is
shown in red. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
The intensity increase inside the micelles compared to that of
bulk water provides evidence that the cNDIs do not aggregate

H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
295
Fig. 3. a) Fluorescence spectra (lex ¼ 590 nm) of 1 ꢂ 10ꢁ5 M compound 8 in chloroform (green), reverse micelle wo ¼ 1 (purple), wo ¼ 3 (red), wo ¼ 5 (orange), wo ¼ 10 (yellow),
wo ¼ 15 (grey), wo ¼ 30 (dark grey), water (blue) and hexane (pink) b) Fluorescence decay profiles of 8 and fitted functions (black lines) in reverse micelles wo ¼ 1 (grey), wo ¼ 5
(dark grey) and wo ¼ 30 (light grey). The instrument response function is in red. c) Fluorescence lifetime of 1 ꢂ 10ꢁ5 M compound 8 inside reverse micelle versus wo with
exponential decay fitting function. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2
Fluorescence lifetime tf and
wavelength maxima (
of bulk water and as the micelles become larger, this energy dif-
ference becomes smaller, however E of the largest micelle does
DE) for 8 between the reverse micelles to that
D
E of 8 inside AOT reverse micelles.
a
wo
rH (nm)
D
E (cmꢁ1
)
tf (ns)
D
not reach that of bulk water. This may be due to the interior envi-
ronment of the reverse micelles not fully approximate that of bulk
water, even in large reverse micelles.
1
3
5
10
15
30
H2O
1.68
2.03
2.38
3.25
4.13
6.75
e
506
407
382
284
189
141
e
9.9
9.0
8.6
7.4
6.9
6.7
4.4
Fluorescence lifetimes of 8 inside reverse micelles were also
recorded using TCSPC for reverse micelles from wo ¼ 1 to wo ¼ 30
and were found to be single exponential and strongly dependent on
micelle size (Fig. 3(b) and SI Fig S6). In the smallest micelle (wo ¼ 1)
8 was found to have a lifetime of 9.9 ns consistent with 8 residing in
an environment of similar polarity to that of CHCl3 (Table 2). As the
reverse micelles become larger (determined by the hydrodynamic
radius of the micelle, Rh, seen in Table 2) the lifetimes are shown to
decrease to 6.7 ns for reverse micelles of wo ¼ 30. The wo value,
plotted against the fluorescence lifetime can be fit to a simple
exponential decay function (Fig. 3(c)). This is a purely empirical
relationship between the fluorescence lifetime and environment
polarity which may be useful in approximating the hydrophilic
environment inside biological systems. The data and exponential fit
imply that even at ‘infinite’ size, the value of the lifetime and in-
tensity do not reduce to bulk water values again suggesting the
interior environment does not fully approach that of bulk water
even in large reverse micelles.
a
D
E ¼ E (AOT) e E (H2O).
inside AOT reverse micelles at this concentration as the relative
quantum yield (area under the emission spectrum) tracks with the
fluorescence lifetime meaning only dynamic quenching is affecting
the intensity profile of the micelles seen in Fig. 3(a). It has recently
been established that the pH of the interior of AOT reverse micelles
is ~5e5.5 and that these reverse micelles have some buffering ca-
pacity to maintain this pH [37]. This mildly acidic environment is
not expected to affect the photophysics of the cNDI molecules. We
have previously shown that a related cNDI had no change in optical
properties on addition of strong acid to a solution of CHCl3 [38] and
the absorption spectrum of 8, does not show any noticeable change
over a range of pH 3e11. Table 2 shows the change in the
Fig. 4. Autocorrelation functions (black) and fitted functions (red) of a) 6 in water, b) 7 in water and c) 8 in water. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

296
H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
Table 3
triplet states to the ACF curves (see SI Table S3) was on a micro-
second timescale indicating that triplets only contribute to the
ACF at lag times shorter than ~10 ms.
Diffusional parameters of compounds 6e8 in water and 7 in DMPC and DMPC/Chol
liposomes.
Compound
D
tf (ns)
Concentration
(nM)
Radius
(nm)
To test these compounds in a more biologically relevant sce-
nario, compound 7 was loaded into 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC) and DMPC:cholesterol (DMPC/Chol)
(4:1) liposomes. DMPC and DMPC/Chol were chosen to encapsu-
late 7 as they are both known synthetic mimics for the phos-
photidylcholine lipid found in nature and have been extensively
researched as artificial plasma membranes [43,44]. Autocorrela-
tion functions of 7 incorporated into DMPC and DMPC/Chol li-
posomes were recorded using FLCS and compared to that of the
free 7 (Fig. 5). Fig. 4(b) shows the autocorrelation function of the
(m )
m2 sꢁ1
(6)
(7)
(8)
457
400
387
1.20
1.79
4.3
4.4
4.6
4.4
4.6
11.9
19.3
5.4
0.217
8.6
0.47
0.54
0.55
179
120
DMPC (7) liposome
DMPC/Chol (7) liposome
3.4. Fluorescence correlation spectroscopy analysis
free 7 from which a diffusion coefficient of 400
m
m2 sꢁ1 is
Water solubility at millimolar concentrations allowed for
further characterisation of cNDIs 6e8 using Fluorescence Corre-
lation Spectroscopy (FCS). FCS (and variations thereof such as
lifetime (FLCS) and cross correlation (FCCS) versions) has become
a leading research method, particularly in biophysical applica-
tions and has been used, for example, in the determination of
diffusion coefficients, flow rates [39], molecular aggregation [40],
and molecular weights. The technique has also been used for
research in cell receptor recognition and antibody and virus
binding [41] and in a recent example, to measure diffusion of
transcription factors in complex biological systems such as em-
bryonic stem cells and live mouse embryos [42]. To investigate the
potential for these compounds to be employed in such applica-
tions, fluorescence lifetime correlation spectroscopy (FLCS) was
performed to generate auto-correlation function (ACF) curves and
TCSPC fluorescence decay histograms in water (Fig. 4). Analysis of
the autocorrelation curves using equation (1) indicated that only
one diffusional component was required for satisfactory fitting.
The diffusion coefficients calculated from the auto-correlation
data using equation (2) were consistent with diffusion by small
molecules indicating that at the concentrations used for the FLCS
measurements (~5e20 nM), there are no larger fluorescent spe-
cies detected. The diffusion behaviour and molecular weight
follow the expected trend with the largest molecule 8 residing in
the confocal volume the longest and thus returning the smallest
determined (Table 3). The autocorrelation functions of 7 loaded
into liposomes display much longer lag times but can still be well
fitted with the single component diffusion model given by
equation (1) (Table S4). This suggests that the diffusing species is
large, spherical in shape and has a narrow size distribution within
the sample. The diffusion coefficients determined are 1.20 and
1.79 m
m2 sꢁ1 for the DMPC and DMPC/Chol liposomes respectively
(Table 3) and using equation (3) and assuming a spherical shape,
the liposomes sizes are in the range of 200e400 nm in diameter.
Photon counting of 7 in both liposome systems produce single
exponential decays with fluorescence lifetimes essentially the
same as that of free 7 in water. These lifetimes show that
encapsulation inside large liposomal structures does not signifi-
cantly affect the general photophysics of the dyes. This suggests
that the dye is found in the large water filled liposomal cavity and
does not interact strongly with the interior wall. These results
therefore support the use of cNDIs to probe more complex bio-
logical systems in the future.
4. Conclusion
A series of cNDIs with water-soluble substituents has been
synthesised and show some solubility in water with some ag-
gregation also occurring. The compounds display optical prop-
erties characteristic of cNDIs with strong absorption and
emission of light in the red part of the spectrum. Time-resolved
fluorescence studies show that the compounds have emission
lifetimes of ~10 ns in CHCl3, ~8 ns in MeOH and ~4 ns in water.
The compounds are shown to have potential for use in biophys-
ical applications by encapsulation into AOT reverse micelles and
liposomes. Fluorescence lifetime is shown to indicate cavity size
diffusion coefficient (D ¼ 387
6 displayed the shortest lag times and the largest diffusion coef-
m2 ꢁ1) (Table 3). The fluorescence lifetimes
m
m2 sꢁ1), and the smallest molecule
ficient (D ¼ 457
m
s
recorded in the confocal volume were comparable to those ach-
ieved using standard photon counting techniques. The fitting
function used to model the ACF curves also contained a term to
account for triplet state formation. In all cases, the contribution of
Fig. 5. Autocorrelation function (black) and fit (red) of a) 7 loaded into DMPC liposomes and b) 7 loaded into DMPC/Chol (4:1) liposomes. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

H.F. Higginbotham et al. / Dyes and Pigments 112 (2015) 290e297
297
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Appendix A. Supplementary data
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