Development of colloidally stable carbazole-based fluorescent nanoaggregates

Article abstract of DOI:10.1016/j.jphotochem.2017.10.042

Fluorescent nanomaterials require high colloidal stability for effective use in imaging and sensing applications. We herein report the synthesis of carbazole-based organic fluorescent nanoaggregates, and demonstrate the superior colloidal stability of alkyl-substituted dye aggregates over their non-alkylated analogs. The role of alkyl chains in self-assembly and stability of such nanoaggregates is discussed based on both experimental and molecular dynamics simulation data, and spectral characteristics of the precursor dyes and their aggregates are described. The obtained results provide new insights on development of colloidally stable organic fluorescent nanomaterials with low polydispersity.

Full text of DOI:10.1016/j.jphotochem.2017.10.042

Accepted Manuscript
Title: Development of colloidally stable carbazole-based
fluorescent nanoaggregates
Authors: Denis Svechkarev, Alexander Kyrychenko, William
M. Payne, Aaron M. Mohs
PII:
S1010-6030(17)31162-0
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2017.10.042
JPC 10967
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Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
8-8-2017
25-9-2017
21-10-2017
Please cite this article as: Denis Svechkarev, Alexander Kyrychenko, William
M.Payne, Aaron M.Mohs, Development of colloidally stable carbazole-based
fluorescent nanoaggregates, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2017.10.042
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Development of colloidally stable carbazole-based
fluorescent nanoaggregates
Denis Svechkareva, Alexander Kyrychenkob, William M. Paynea, Aaron M. Mohsa,c,d,*
a Department of Pharmaceutical Sciences, University of Nebraska Medical Center,
986858 Nebraska Medical Center, Omaha, NE 68198-6858, United States
b Institute for Chemistry, V. N. Karazin Kharkiv National University,
4 Svobody Square, 61022 Kharkiv, Ukraine
c Department of Biochemistry and Molecular Biology, University of Nebraska Medical
Center, 986858 Nebraska Medical Center, Omaha, NE 68198-6858, United States
d Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center,
986858 Nebraska Medical Center, Omaha, NE 68198-6858, United States
* Corresponding author email: aaron.mohs@unmc.edu.

Graphical Abstract
TOC graphic
Highlights:
 Fluorescent nanoaggregates with improved colloidal stability were obtained
 Alkyl chains contribute to colloidal stability and monodispersity of the aggregates
 DLS and TEM data and MD simulations were used to illustrate aggregation
dynamics
Abstract: Fluorescent nanomaterials require high colloidal stability for effective use in
imaging and sensing applications. We herein report the synthesis of carbazole-based
organic fluorescent nanoaggregates, and demonstrate the superior colloidal stability of
alkyl-substituted dye aggregates over their non-alkylated analogs. The role of alkyl
chains in self-assembly and stability of such nanoaggregates is discussed based on both
experimental and molecular dynamics simulation data, and spectral characteristics of the
precursor dyes and their aggregates are described. The obtained results provide new
insights on development of colloidally stable organic fluorescent nanomaterials with low
polydispersity.

Key words: polydispersity, alkyl chain, dynamic light scattering, fluorescent imaging,
MD simulation.

1. Introduction
In analytical applications, fluorescence provides a number of critical advantages over
absorption: higher sensitivity, better contrast, and superior resolution. This is particularly
true for imaging applications, as well as closely related techniques, such as flow
cytometry. Various fluorescent materials are used for these applications: inorganic
nanocrystals, largely known as quantum dots [1–3], composite materials such as carbon
[4–6] or doped silica [7,8] nanoparticles, and a wide spectrum of organic materials, for
example, small molecules [9,10] and their nanoaggregates [11,12], fluorescent proteins
[13–15], polymers and dendrimers [16], polymeric nanoparticles [17,18] and their
combinations [19,20]. Each type of these materials possesses a unique combination of
properties – brightness, photostability, size, colloidal stability, toxicity, etc. Their
particular advantages and pitfalls discussed elsewhere [21,22].
Variety of fluorescent nanostructures based on organic small molecules are reported to
date. Many of them are described as irregular nanoaggregates (or nanocrystals) with little
control over their shape and morphology due to relatively few approaches to predict the
size and polydispersity during their synthesis. While some organic fluorescent materials
represent nanoparticles of defined shape with controllable size and polydispersity, their
synthesis is usually more complicated [23], and often involve surface modification and/or
encapsulation into polymers [24]. Thus, it is important to identify approaches to improve
control over physico-chemical properties of the resulting fluorescent organic
nanoaggregates.
Studies of complex biological systems present many challenges. When a fluorescent
probe is used for this purpose, it is very important to understand how stable and
reproducible its characteristics are. Since most of the supramolecular systems discussed
above are assembled from small molecules which are not covalently bound to each other,
spontaneous disassembly can potentially occur upon interaction with other components of
the studied complex systems, or further aggregation can lead to sedimentation over time.

Thus, it is important to address the question of stability of fluorescent organic aggregates,
both in colloidal suspensions and upon their interaction with biological targets [25].
Most of fluorescent molecules show dramatic decrease in their emission efficiency in
concentrated solutions or in solid state. This effect, known as aggregation-induced
quenching, is due to reabsorption and energy transfer processes when molecules are close
to each other. The most common and efficient approach to tackle this problem is to
employ the effect of aggregation-induced emission (AIE) and approaches to convert
conventional organic fluorophores into AIEgenic molecules [12]. Several classes of
organic dyes are known that exhibit fairly bright emission both in solutions and in solid
state. The can also be suitable as material for fluorescent nanoaggregates. Particularly,
carbazole has been successfully used as a core for fluorescent materials for a variety of
applications. Its derivatives were used to obtain bright fluorescent aggregates and
nanostructures [26,27] that were subsequently used for sensing and imaging both in vitro
and in vivo [28–30].
In the present report, we explore the influence of the alkyl moieties in the structure of
organic fluorescent dyes on their self-assembly, including the effect on polydispersity and
colloidal stability of the resulting nanoaggregates. We show that the introduction of the
octyl chains into the structure of two organic dyes leads to formation of more
monodisperse and colloidally stable nanoaggregates when compared to their counterparts
with no alkyl chains. We also discuss the spectral characteristics of the most stable
nanoaggregates and their precursor dyes.
2. Materials and Methods
2.1. Materials
Sodium methoxide (anhydrous), copper (I) iodide (98%), potassium carbonate
(anhydrous, 99+%), N,N'-Dicyclohexylcarbodiimide (99%) and silica gel for
chromatography (0.030-0.200 mm, 60A) were purchased from Acros Organics (New

Jersey, USA). 3,6-Dibromocarbazole (99%), 1,4-dibromobenzene (98%), N,N-
dimethylacetamide (anhydrous, 99.8%) and PdCl2(dppf) complex with acetone (13-15%
Pd) were purchased from Alfa Aesar (Ward Hill, USA). Magnesium sulfate (anhydrous)
was purchased from Fisher Chemical (New Jersey, USA). 1-Bromo-4-octylbenzene was
purchased from Maybridge (Loughborough, UK). 5-Carboxythiophene-2-boronic was
purchased from Frontier Scientific (Newark, USA). Hydrochloric acid (certified ACS
plus) was purchased from Fisher Scientific (New Jersey, USA). Sodium sulfate
(anhydrous) was purchased from Fisher BioReagents (New Jersey, USA). N-
Hydroxysuccinimide (98+%) and octylamine (99%) were purchased from Sigma-Aldrich
(St. Louis, USA). Chloroform-d (99.96%) was purchased from Cambridge Isotope
Laboratories, Inc. (Andover, USA). Dimethyl sulfoxide-d6 (99.8%) was purchased from
EMD Millipore (Billerica, USA). Solvents (methanol, DMF, chloroform, petroleum
ether, toluene, DMSO) were all ACS certified or spectroscopic grade, were purchased
from Fisher Chemical (New Jersey, USA) and used without further purification.
2.2. Synthesis
Target compounds III, 3,6-dimethoxy-9-(4-octylphenyl)carbazole, and V, 5-[4-(3,6-
dimethoxycarbazol-9-yl)phenyl]-N-octyl-thiophene-2-carboxamide, were synthesized
following the procedures described elsewhere. Briefly, 3,6-dimethoxycarbazole was
obtained from 3,6-dibromocarbazole [31], which then was reacted with 1-bromo-4-octyl-
benzene to obtain III [32]. The same precursor was also reacted with 1,4-
dibromobenzene [32], and subsequently with 5-boronthiophene-2-carboxylic acid [33]
and octylamine to obtain V (see Figure 1). A more detailed description of the synthetic
procedures is provided in the Supplementary Information.
2.3. Sample preparation
Fluorescent nanoaggregates were obtained using the solvent exchange method. Solutions
of organic dyes in THF were prepared of 4 different concentrations through 3-fold serial

dilutions. A volume containing equal amounts of the dye (50-1350 μL of THF solution)
was the injected into 10 mL of ultrapure water, and the samples were left open overnight
in the desiccator to allow THF evaporate.
2.4. Characterization
Structures of all obtained compounds were confirmed by 1H and 13C NMR spectroscopy
(Bruker Avance-III HD, 500 and 125 MHz respectively). Mass-spectra were acquired on
4800 MALDI TOF (Sciex, Inc.) in SPA/60% acetonitrile/0.1% TFA in reflector positive
mode with Nd:YAG laser (355 nm @ 200 Hz) for a total of 1000 acquisitions for each
sample. Absorption and fluorescence spectra were recorded on a ThermoFisher Evolution
220 UV-vis spectrophotometer and Horiba Jobin Yvon FluoroMax 4 spectrofluorometer,
respectively, in 10 mm quartz cells. Colloidal properties were studied using a Malvern
Zetasizer Nano ZS90 dynamic light scattering instrument. Transmission electron
microscopy imaging was performed using the FEI Tecnai G2 Spirit microscope (2%
aqueous methylamine vanadate, pH 8, was used for negative staining).
2.5. MD simulations
Molecular dynamics simulations were performed using GROMACS 4.6 in combination
with the GROMOS 53a6 force field [34]. The bond length and angle parameters for
compounds II-V were optimized by density functional theory calculations at the
B3LYP/cc-pVDZ level and adopted for the GROMACS force field format. Partial
charges needed for Coulomb interactions were derived from the B3LYP/cc-pVDZ
electron densities by fitting the electrostatic potential to point (ESP) charges. The Simple
Point Charge (SPC) model was used for water [35]. A set of 729 dye molecules was
placed in a rectangular cell of 18.7 nm x 18.7 nm x 18.7 nm, filled with approximately
200000 water molecules. Each system was simulated for 200 ns.
All the MD simulations were carried out at a constant number of particles, constant
pressure of P = 1 atm, and constant temperature T = 298 K (the NPT ensemble). The

reference temperature of 298 K was kept constant using the velocity rescaling weak
coupling scheme [36] with a coupling constant τ = 0.1 ps. The initial atomic velocities
were generated with a Maxwellian distribution at the given absolute temperature.
Periodic boundary conditions were applied to all three directions of the simulated box.
Electrostatic interactions were simulated with the particle mesh Ewald (PME) approach
[37] using the long-range cutoff of 0.8 nm. The cutoff distance of Lennard-Jones
interactions was also equal to 0.8 nm. The MD simulation time step was 2 fs with the
neighbor list updates every 10 fs. All bond lengths were kept constant using the LINCS
routine [38,39]. The MD simulations were carried out using the GROMACS set of
programs, version 4.6 [40]. Molecular graphics and visualization were performed using
VMD 1.8.6 [41]. The analysis was performed using GROMACS tools.
3. Results and Discussion
Target fluorescent dyes were synthesized by substitution of the carbazole in 9 position
with a corresponding aryl bromide via Buchwald-Hartwig reaction [42,43], and
subsequent Suzuki-Miyaura coupling [44] with 5-boronthiopene-2-carboxylic acid
followed by the condensation with octylamine (Figure 1).
[2-column]

Figure 1. Synthetic scheme leading to target compounds III and V.
In order to study colloidal properties of the aggregates of III and V, their solutions in
THF were injected into water as described in the experimental section. Aggregates of
compounds II and IV were also prepared and were used as reference to assess the effect
of the alkyl chain on the colloidal properties and stability of the resulting nanoaggregates.
Data on average particles sizes and size distributions was acquired using DLS with
freshly prepared samples, as well as with the same samples after 2 and 6 weeks of
preparation, to assess their colloidal stability.
Several approaches exist to interpret the primary DLS measurements data. One of them,
known as the method of cumulants [45], uses an expanded expression to fit the original
autocorrelation function that accounts for polydispersity. This method results in two
parameters: the intensity-weighted mean size, often referred to as Z-average size, and the
polydispersity index (PDI). The other approach evaluates the autocorrelation function to
provide the nanoparticle size distribution, which was shown to be much less reliable than
the former [46]. Although discussions still have place in scientific literature regarding the

optimal method to be used to report the DLS data [47,48], we hereby illustrate our results
using both approaches, suggesting that the observed effects are independent on the
method used and that the resulting analyses are in good agreement with each other.
[1.5- or 2-column]
Figure 2. Average aggregate size (top) and polydispersity index (bottom) obtained from
cumulants analysis of the DLS experiment data for the aggregates of compounds II (A),
III (B), IV (C) and V (D) in 0, 2 and 6 weeks after preparation. Each experimental point
is an average of 5 independent measurements, and the error bars represent the standard
deviation.

The aggregates of II are larger in size than those of III, where the bromine atom is
replaced by the octyl hydrocarbon chain (Figure 2A,B). One can also notice that the
average size of the aggregates of II experienced changes with time. First, an increase in
size is observed for almost all the series (except one that was obtained by injecting the
most concentrated organic solution) over the period of 2 weeks, which can be explained
by Ostwald ripening [49] – an effect when smaller aggregates dissolve and redeposit
material onto larger aggregates, making the latter further grow. The same behavior has
been reported for aggregates of the dyes with similar structure [50]. Some precipitation
was observed in the vial with the sample made by injection of the most concentrated
organic solution. Ultimately, precipitation was observed in all 4 samples, explaining the
decrease in size for all samples at the 6 weeks time point. Larger particles sedimented,
and only the smaller ones remained in the suspension and were sampled by DLS. These
observations also explain larger deviations for both the mean sizes and the polydispersity
indices for this time point. At the same time, aggregates of III showed little change in
their mean size with time (the slight increase in size could be also ascribed to Ostwald
ripening). No precipitation was observed for these samples, except for the one obtained
from the most concentrated organic solution after 6 weeks of preparation (note the
increase of polydispersity index deviations for this point on Figure 2B).
In case of the compound IV, bearing a carboxyl group, it is much harder to notice any
trend for the aggregates size in comparison with its analog V, where the carboxyl group is
converted into the octylcarboxamide (Figure 2C,D). The primary reason for this lack of
trend is that the compound IV has noticeably higher polydispersity. This could be
explained by the ability of the molecules of IV to form hydrogen bonds through several
H-bonding centers. Intermolecular hydrogen bonding in such molecules not only allows
formation of dimers, but also chain- and network-like structures [51,52], which may
contribute to the formation of larger aggregates, as well as lead to further association of
smaller ones into larger complex structures. This assumption is supported by
exceptionally high polydispersity indices obtained for aggregates of IV in the experiment.

At the 6-week time point, similar to the aggregates of II, one can notice a decrease in size
due to continuous precipitation. Such size change is much less pronounced for the
samples obtained from the most concentrated organic solution (where precipitation
started almost right after the sample was prepared) and the one obtained from the least
concentrated organic solution (where precipitation was much less active). In contrast,
aggregates of V do not show noticeable change of their average size with time, and
exhibit significantly lower polydispersity indicating higher colloidal stability compared to
IV. No precipitation was observed in either sample of V even several months after
preparation.
In all cases, aggregates obtained upon injection of the most dilute organic solution of the
dyes show higher monodispersity. To keep the overall amount of the dyes the same
across the samples, the more diluted the THF stock solution was, the larger volume of it
had to be injected into water. For the most diluted solutions it led to over 10% (v/v) of
organic phase after injection, which made the dye slightly more soluble. Slow
evaporation of THF allowed for slower nucleation and growth of nanoaggregates, thus
leading to higher monodispersity.
Results obtained from an alternative analysis of the primary DLS data using the size
distribution fitting (see Figure 3, where one of the dilutions for all four compounds was
taken as an example) are in excellent agreement with those of cumulants analysis and
support the above discussion. Considerably wider size distributions of the aggregates of
II and IV demonstrate their higher polydispersity compared to their alkyl-bearing
counterparts III and V. The size distributions change dramatically for the non-alkylated
compounds aggregates as well. As the same time, almost no change is observed for the
aggregates of alkylated derivatives, confirming their superior colloidal stability.
[2-column]

Figure 3. Aggregate size distributions obtained from DLS experiment for the fluorescent
aggregates: A – II, 0.633 mg/mL; B – III, 0.666 μL/mL; C – IV, 0.567 mg/mL; and D –
V, 0.567 mg/mL at 0, 2 and 6 weeks after preparation. Each experimental point is an
average of 5 independent measurements, and the error bars represent the standard
deviation.
Hydrophobic interaction between the alkyl fragments of different molecules can
contribute to their more ordered packing inside the aggregate. This may be one of the
reasons for the observed size and polydispersity difference between alkylated and non-
alkylated analogs. Another potential explanation is that long hydrophobic chains can act
as analogs to capping agents, which are largely used to stabilize the desired size and
produce monodisperse inorganic nanocrystals [53]. For example, oleic acid has been
shown to limit the particle-particle interaction during both nucleation and growth stages
in CdSe quantum dots synthesis, thus leading to slower diffusion-controlled growth of
monodisperse aggregates [54]. Surface passivation by polyvinyl pyrollidone was shown
to be a plausible reason of growth rate decrease, thus leading to formation of more

uniform nanocrystals of ZnO [55,56]. Similarly, thiols have been extensively used as
capping agents in synthesis of various types of nanostructures, such as ZnO nanocrystals
[57], silver [58,59] and gold [60–62] nanoparticles. Improved stability and lower
polydispersity of the aggregates of II and IV obtained by injection of less concentrated
organic dye solutions is an additional indirect evidence supporting this hypothesis.
Although the capping agents are used to reduce the size and size distribution of the
nanoaggregates, their effect on the growth kinetics is very complex, thus not providing
for a general theory at this time [53].
Amphiphilic fluorescent molecules were shown to provide nearly monodisperse and even
size-controlled micelle-like nanoparticles [23]. However, our target compounds are not
quite amphiphilic, particularly due to insufficient hydrophilicity of the methoxy groups
that readily partition into both polar and non-polar phases [63]. Size-concentration
dependence observed for all aggregates also suggests that no micelles are formed by the
described compounds.
The sizes of obtained nanoaggregates depend on the concentration of the organic solution
used to inject into water: using the organic phase with higher dye concentration resulted
in larger aggregate size (Figure 2). At the same time, the final concentration of the
organic dye in water suspension within the series remains the same. Thus, injection of a
more concentrated THF solution into water leads to formation of larger aggregates, which
are also characterized by higher polydispersity. A similar effect was previously reported
in literature [28,64], where the authors showed the dependence of nanoaggregates’
morphology on the concentration of the organic dye in THF that was injected into
deionized water. This effect can be rationally explained by differences in local
concentration of the aggregating dye upon solvent exchange at the early stages of
nucleation and growth [53].
To shed more light on the morphology of our aggregates, TEM imaging was performed
and representative micrographs were examined (see Figure 4). The sizes of aggregates
observed on the micrographs are generally in good accordance with the sizes determined

from the DLS experiments. One can see that the aggregates of II and IV tend to form
larger and more irregular agglomerates (Figure 4A and 4C), showing much more affinity
for further intermolecular interaction and growth of aggregates. On the other hand,
aggregates of the alkyl-substituted analogs III and V appear to be smaller in size and
more regular in shape, supporting the above hypothesis of alkyl groups’ role in
intermolecular interactions on the stage of nanoaggregates growth.
[1-column]
Figure 4. Transmission electron micrographs of the aggregates of compounds II (A), III
(B), IV (C) and V (D). All images are taken at the same magnification (note the scale bar
in section A).
To shed more light onto the dynamics of the aggregation of the studied compounds,
theoretical modeling of this process was performed for all four molecules. Molecular
dynamics simulations snapshots (Figure 5) show the systems before aggregation, during
its early stages (with the time step of 20 ns) and at the late stages (at 100 and 200 ns).

Even at the early times the alkylated derivatives III and V are forming larger well-
defined aggregates. The aggregation behavior appears more evident after 100 ns, when
the aggregation process for them becomes essentially complete (as evidenced by the dye-
water hydrogen bonds dynamics, see Figure S13). In the opposite, non-alkylated analogs
II and IV struggle to form large aggregates even after 100 ns. It is worth to note the
appearance of the formed aggregates: compounds III and V for more uniform and shape-
defined structures, whereas their non-alkylated counterparts self-assemble into more
irregularly shaped particles. These results are in excellent agreement with the TEM data
shown on Figure 4.
[2-column]

Figure 5. Dye aggregation dynamics illustrated by MD simulations snapshots of the
systems consisting of 729 dye molecules solvated by ~2∙105 water molecules (water
molecules are omitted for clarity of representation).
A more detailed analysis of the aggregation driving forces in all cases unveils some
differences in the interactions of alkylated and non-alkylated dyes. Thus, the latters are
predominantly brought together by their general hydrophobicity and the need to minimize
the overall water-accessible surface area. In case of the alkylated dyes, the alkyl chains
play additional role in the aggregate self-assembly. As can be seen from the snapshot of
the aggregate of III at 100 ns (insert in Figure 6), the aggregation starts from formation
of relatively small particles with micelle-like morphology, that further associate to grow
into larger particles. Even in these, however, hydrophobic domains formed by alkyl
moieties can be observed. This effect is less evident for the molecules of V (Figure S14),
which can be explained by less pronounced hydrophobicity of the respective molecular
fragment due to the presence of more polar and hydrogen bonds forming thiophene cycle
and carboxamide group. Similar structural influence on hydrophobicity was demonstrated
earlier for octyl chains adjacent to pyridinium moiety [63].
[1.5- or 1-column]

Figure 6. Snapshot of the system of 729 molecules of III in approximately 2∙105 water
molecules (water molecules are omitted for clarity of representation) at 100 ns of
simulation.
One of the most common uses of the fluorescent nanoaggregates is imaging of biological
samples, particularly cells. Thus, a detailed spectroscopic characterization of the target
compounds III and V and their aggregates in water has been performed to assess their
potential as perspective imaging agents. Absorption and fluorescence spectra of both dyes
were recorded in a series of solvents with different polarity represented by Onsager
polarity function [65], with subsequent modifications: Δf = f(ε) – f(n).
Absorption spectra for both III and V (see Fig. S1 in the Supplementary Information)
show no significant change upon increase of the solvent polarity. The same is observed
for the fluorescence of III (Figure 7A), i.e., it does not exhibit any noticeable
solvatofluorochromism. In contrast, fluorescence of V (Figure 7B) shows high sensitivity
to the solvent polarity – one can notice a significant bathochromic shift of the emission
maximum upon increase of the solvent polarity (Figure 7C). Additionally, the emission
spectrum of V noticeably broadens in more polar solvents.
[1-column]

Figure 7. Intensity-normalized fluorescence spectra of target compounds III (A) and V
(B) in various solvents. Spectral maxima for fluorescence are plotted against solvent
polarity function Δf (C).
Table 1 represents the main spectral characteristics of the title compounds III and V in
various solvents. It can be seen that with increasing polarity of the molecule’s
microenvironment, there is almost no change in the Stokes shift of fluorescence for III,
whereas for V it is quite noticeable. The quantum yields of fluorescence of V are lower in
polar solvents. Hydrogen bonding is another factor that contributed to the emission

efficiency [66]. However, in the aggregated state in water it also exhibits bright
fluorescence.
Table 1. Spectral characteristics of the title compounds in various solvents.
휈̃abs, cm-1 /
λabs, nm
휈̃fluo, cm-1 /
λfluo, nm
훥휈̃St, cm-1 /
ΔλSt, nm
Solvent
Φ[a]
Compound III
22780 / 439
22675 / 441
22270 / 449
22725 / 440
22625 / 442
20575 / 486
Compound V
22625 / 442
20705 / 483
19415 / 515
19840 / 504
19155 / 522
20490 / 488
Toluene
26955 / 371
27322 / 366
27025 / 370
27175 / 368
27250 / 367
26650 / 375
4175 / 68
4647 / 75
4755 / 79
4450 / 72
4625 / 75
6075 / 111
0.25
0.22
0.24
0.34
0.28
0.12
THF
DMSO
Acetone
Acetonitrile
Water
Toluene
THF
26040 / 384
26110 / 383
26180 / 382
26315 / 380
26455 / 378
26740 / 374
3415 / 58
5405 / 100
6765 / 133
6475 / 124
7300 / 144
6250 / 114
0.32
0.36
0.25
0.11
0.13
0.54
DMSO
Acetone
Acetonitrile
Water
[a] perylene solution in ethanol (φ=0.92 [67]) was used as a reference for the
quantum yield of fluorescence.
Carbazole moiety is a well known electron donor [27,68,69]. In the structures of target
compounds, this feature is further enhanced by methoxy substituents in the positions 3
and 6. However, in III a second pole – an electron accepting moiety – is not present, as
the 4-bromophenyl-2 fragment only contributes to electronic delocalization, providing no

means of significant charge transfer within the molecule. In case of V, a thiophene
heterocycle, complemented with the carboxylic group, plays the role of an efficient
electron withdrawing fragment, thus creating necessary conditions for the intramolecular
charge transfer upon excitation. As a result of this process, a high dipole moment of the
excited molecule explains the noticeable solvatofluorochromism observed for V. Solvent
relaxation around the excited molecules leads to broadening of the emission spectrum
bands [70]. Such effects have been described for similar molecules containing methoxy-
substituted diphenylamine as a donor and thiophene [33,50,71] or other heterocycles [72]
as acceptors. These analogs exhibit fairly bright fluorescence in organic solvents, and
somewhat lower emission efficiency for their aggregates in water. However, very few
data about the colloidal properties of these aggregates or their stability has been
published.
Behavior of both target compounds in water-THF solvent mixtures with varying water
content is in good accordance with the spectral properties they show in individual organic
solvents. Thus, for III almost no change in emission maximum position is observed upon
increase of water content up to 60% (Figure 8A). In the system where only 20% of THF
is left, an aggregate starts to form which scatters the excitation and exhibits its own
emission at approximately 475 nm (a small shoulder emerges in that region). In 100%
water (in fact, 1% THF was still present in order to inject the dye into water), a distinct
band of the aggregate emission is observed.
[1-column]

Figure 8. Intensity-normalized fluorescence spectra of target compounds III (A) and V
(B) in water-THF binary solvent systems. Water content is indicated in the legends.
In case of V (Figure 8B), a slight bathochromic shift of the emission maximum is
observed following the increase of water content in the system (and subsequent increase
of the polarity of the dye’s environment). In addition to that, quenching of fluorescence
with increased water content evidences for the hydrogen-bond induced nonradiative
deactivation discussed above (see Figure S16 for original fluorescence spectra). Similarly
to III, the aggregate fluorescence becomes observed when the water content reaches 80%
and remains almost the same in pure water. Unlike III, however, the aggregate of V
exhibits fluorescence at shorter wavelengths than the compound in polar solvents.
Additionally, the emission band noticeably broadens, which may be a consequence of the
compound’s sensitivity to its microenvironment. Similar behavior was observed for a few
other carbazole-based dye aggregates possessing intramolecular charge transfer [26].

Conclusions
We describe the synthesis of two alkyl-substituted fluorescent dyes based on a carbazole
core. Both dyes exhibit bright emission in organic solutions, and are fluorescent in the
condensed state as well as in aggregate form in aqueous solutions. Colloidal properties of
the aggregates are studied using dynamic light scattering and electron microscopy, and
the aggregation process was simulated by means of molecular dynamics. Analogs of the
target compounds having no alkyl substituents are used as comparison systems to
demonstrate the role of the hydrophobic chains in self-assembly and colloidal stability of
obtained aggregates.
The sizes of the fluorescent nanoaggregates are dependent upon the concentration of the
organic solution initially injected into the water phase. Using more concentrated stock
solutions lead to formation of larger aggregates in most cases, with usual sizes of 200-
300 nm and exceeding 1 µm for some samples of IV. For less concentrated THF
solutions injected, aggregates sizes fluctuate around 100 nm for III and V, and are larger
for their respective non-alkylated analogs II and IV.
Introduction of alkyl chains not only leads to formation of more uniform aggregates, but
also significantly enhances their colloidal stability. While precipitation was observed for
the non-alkylated dyes aggregates within days to weeks after formation of the
suspensions, only the sample made with the most concentrated THF solution of III
showed signs of sedimentation 6 weeks after formation. No precipitate was observed in
either sample of V even after 5 months after being prepared.
Molecular dynamics simulations demonstrate the alkylated dyes III and V better ability
to form well-defined aggregates, while those of II and IV tend to be more irregular in
shape and less monodisperse. These results are in excellent accordance with the DLS data
and TEM microscopy images.
Spectral properties of the target dyes III and V reflect their structural features well.
Compound III shows no sensitivity to its microenvironment polarity, whereas the

emission of V undergoes noticeable bathochromic shift upon increase of the solvent
polarity. This shift is due to an intramolecular charge transfer process making V
significantly more polar in its excited state. In binary water-THF solvent systems, III
does not show significant sensitivity to the water content of up to 80%. After this point, it
exhibits a behavior typical for a J-aggregate, where the aggregate’s emission is
bathochromically shifted with respect to the fluorescence of the precursor dye in bulk
solvent. At the same time, the emission of V shifts bathochromically in response to rising
medium polarity due to increasing water content. The aggregate of V exhibits emission at
slightly shorter wavelengths compared to the precursor dye. Such hypsochromic shift and
band broadening have been reported for other carbazole-based fluorescent aggregates
exhibiting intramolecular charge transfer.
The results obtained in this study show that alkyl moieties can play significant role in
formation of more uniform and colloidally stable aggregates of organic fluorescent dyes,
without negatively affecting their fluorescent properties. These results will be useful for
further development of fluorescent nanomaterials for imaging and sensing applications.
Acknowledgements
This work was supported by the National Institutes of Health [grant numbers R21
CA212500 and P30 CA036727] and the Nebraska Research Initiative. Computational
part of this work was completed using Holland Computing Center of the University of
Nebraska, which receives support from the Nebraska Research Initiative.
Conflict of Interests
The authors declare no conflicting financial interests.

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