Synthesis and characterization of membrane stable bis(arylimino)isoindole dyes and their potential application in nano-biotechnology

Article abstract of DOI:10.1016/j.tetlet.2012.05.128

A synthetic methodology of preparing novel membrane stable, responsive dyes is revealed in this manuscript. 1,3-Bis(arylimino)isoindole dyes were synthesized and their properties to undergo intramolecular hydrogen bonding was studied with fluorescence spectroscopy in varying solvent polarities. Based on the functional moieties, compound that is capable of hydrogen donor and acceptor interactions produces predominant photoexcitation in comparison to the responsive dyes that lack these functionalities. These dyes, by the virtue of the presence of long chain acyl groups could be incorporated stably within the phospholipids membrane of core-shell nanoparticles. Nanoparticle was 'cracked' to release the dye from a hydrophobic to a hydrophilic environment. A significant change in florescence intensity was then observed, indicating the direct change in effect of intramolecular hydrogen bonding based on solvent polarity changes. This unique study provided implications of many further applications toward nanomedicine and nano-biotechnology.

Full text of DOI:10.1016/j.tetlet.2012.05.128

Tetrahedron Letters 53 (2012) 4134–4137
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and characterization of membrane stable bis(arylimino)isoindole
dyes and their potential application in nano-biotechnology
⇑
Benjamin Kim, Ceren Yalaz, Dipanjan Pan
Division of Cardiology, Washington University School of Medicine, 4320 Forest Park Avenue, Saint Louis, MO 63108, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic methodology of preparing novel membrane stable, responsive dyes is revealed in this man-
uscript. 1,3-Bis(arylimino)isoindole dyes were synthesized and their properties to undergo intramolecu-
lar hydrogen bonding was studied with fluorescence spectroscopy in varying solvent polarities. Based on
the functional moieties, compound that is capable of hydrogen donor and acceptor interactions produces
predominant photoexcitation in comparison to the responsive dyes that lack these functionalities. These
dyes, by the virtue of the presence of long chain acyl groups could be incorporated stably within the
phospholipids membrane of core-shell nanoparticles. Nanoparticle was ‘cracked’ to release the dye from
a hydrophobic to a hydrophilic environment. A significant change in florescence intensity was then
observed, indicating the direct change in effect of intramolecular hydrogen bonding based on solvent
polarity changes. This unique study provided implications of many further applications toward nano-
medicine and nano-biotechnology.
Received 3 May 2012
Revised 24 May 2012
Accepted 25 May 2012
Available online 1 June 2012
Keywords:
Fluorescence dye
Bis(imino)isoindole
Responsive dyes
Membrane stable
Nano-biotechnology
Ó 2012 Elsevier Ltd. All rights reserved.
Molecules that are able to undergo intra molecular hydrogen
bonding are significant contributors in diverse applications for
example, high-energy radiation detectors,1–3 ultra violet photosta-
bilizers,4,5 fluorescent probes,6,7 and laser dyes.8,9 These molecules
are well known for their ability to facilitate proton migration from
a donor atom to a proximal acceptor atom usually within a proxim-
ity of <2 Å.10–13 For obvious reasons, they have prominent effects
on molecular structures and properties in terms of lipophilicity,
membrane permeability, water solubility, and rationales for drug
design.14 Many factors can play in affecting the spectrum of a sol-
ute for example, static factors (interactions between the solvent
and the solute), permanent dipoles, dynamic factors (i.e., London
dispersion forces and dielectric relaxation), and specific interac-
tions including hydrogen bonding between the solute and the sol-
vent. Fluorescent probes that undergo intra molecular hydrogen
bonding are found to be highly sensitive to solvent polarity. Subse-
quently, they provide valuable information about the environment
in heterogeneous media.15 Thus changes in solvent composition in-
duce shifts of the absorption and emission peaks of molecules that
have the ability to cause hydrogen bond formation with its sol-
vents.16 Consequently, such fluorescent excitation of the molecules
triggers a shift to higher emission wavelengths with changing sol-
vent polarities which is a result of introducing a specific site for
hydrogen bonding to occur within the solvent media or within
the molecule itself.17 Thus the intra molecular hydrogen bonding
is strongly influenced by the polarity of the solvent or the environ-
ment that it is exposed to. Thereby it regulates the intensities and
shifts the wavelengths of the excited-state intra molecular hydro-
gen bonding emissions.18,19 A probe that could differentiate be-
tween aqueous and non-aqueous environments will be widely
used in applications such as in vitro diagnostics, drug delivery,
clinical pathology, and numerous other nano-biotechnology appli-
cations. Depending on their sensitivity, these molecules may
potentially be used in vitro and in vivo in conjunction with phos-
pholipids coated nanoparticle systems.20–22 We hypothesize that
these probes can be designed based on their ability to form intra
molecular hydrogen bonds (Fig. 1). Alternatively, these probes
may comprise functional moieties that are capable of undergoing
hydrogen bonding with solvents. Toward this aim, we developed
novel molecules as membrane stable, ‘responsive’ dyes centered
on its ability to intuit changes in cellular environments.
Our design of responsive dyes uses 1,3-bis(imino)isoindole as a
central moiety. 1,3-Bis(imino)isoindole derivatives are known to
undergo intra molecular hydrogen bonding through their extended
conjugation by benzannulation of aromatic rings and its pro-
nounced fluorescence excitation properties.23 Platinum coupled
1,3-bis(imino)isoindole derivatives exhibit great photophysical
and photoemission properties.24 In the present work, the design
of the dyes utilized a 1,3-bis(aryl imino)isoindole (aryl: compound
1 = 2-aminopyridine; compound 3 = 4-butyl aniline) moiety and a
strategically placed acyl fatty acid chain (R = –C8H17) to provide
membrane stability (Fig. 1; compounds 1, 3). The aryl amines were
selected based on their potential ability to form intra molecular
⇑
Corresponding author. Tel.: +1 314 454 7674; fax: +1 314 454 7490.
E-mail address: dipanjan@wustl.edu (D. Pan).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.128

B. Kim et al. / Tetrahedron Letters 53 (2012) 4134–4137
4135
(a)
H2N
O
O
N
(c)
O
N
N
N
1
H2N
N
+ mixture of
CN
CN
NH
polymeric side
products (Product
not isolated)
CaCl2
CaCl2
N
1-BuOH, Δ
Microwave 1-3 min
O
2
1
(70%)
(b)
CaCl2
Microwave 1-3 min
H2N
(e)
(d)
CaCl2
1-BuOH, Δ
Product
never isolated
‘Self-assembly’
O
O
N
NH
N
(f)
R
R
OH
O
O
N
N
HO
H
N
H
O
O
3
(81%)
HO
OH
N
N
O
O
OH
b
N
N
N
H
1
R=C8H17
N a
N
a
Moisture
O
N
N
H
N
N
N
H
N
N
Intramolecular
H-Bonding
R
R
R
R
R
R
~20 nm
R
N
N
N
H
O
O
Factors:Solvent
Phospholipids-coated Nanoparticle
N
N
N
N
N
N
H
Provides hydrophobicity/
membrane stability
O
R
(g)
R
= Long chain acyl moiety
O-Phthalimide derivatives
Figure 1. (a and b) Microwave assisted syntheses of 1 and 3 in the presence of CaCl2; (c) 2-aminopyridine, CaCl2, 1-butanol, reflux, 15 h; (d) 4-butyl aniline, CaCl2, 1-butanol,
reflux, 15 h; (e) self-assembly of lipid-nanoparticle and encapsulation of dye 1: preparation of phospholipids thin films composed of egg lecithin PC and 1; resuspension of the
thin film in water (0.2 lM), polysorbate, probe sonication at room temperature, 1 min, dialysis (cellulosic membrane, MWCO 10 K); (f) moisture sensitivity of the responsive
dyes as confirmed by 1H NMR; (g) schematic representation of the proposed responsive dye comprising of moieties for giving membrane stability and probability to undergo
intra molecular H-bonding affected by the solvent polarity.
hydrogen bonds within the molecule itself. We hypothesize that
the benzannulation of pyridyl and isoindole rings in 1 will be influ-
enced by the solvent polarity. A similar compound 3, which lacks
the extended conjugation by benzannulation of pyridyl and isoin-
dole rings, was synthesized as a control compound. In order to
investigate the compounds’ ability to undergo intra molecular
hydrogen bonding in changing environments, dyes will be encap-
sulated within a phospholipids-coated nanoparticle and studied
in vitro.
Our initial attempt to produce 1 and 3 by a solventless micro-
wave assisted method was unsuccessful. Briefly, 3,6-dioctyloxy-
1,2-benzenedicarbonitrile (2) was added to the aryl amine (for
compound 1: 2-aminopyridine; for compound 3: 4-butyl aniline)
and calcium chloride followed by heating the mixture in a domes-
tic microwave for 1–3 min (Fig. 1a and b). The formation of com-
pound 1 was identified by 1H NMR spectroscopy of the crude
mixture. However, we were unsuccessful to isolate the desired
compound from a complex mixture of side products. In case of 3,
the product was never identified by the microwave route. In an
alternative pathway, 1 and 3 were synthesized based on the nucle-
ophilic addition of aryl amines to 1,2-benzenedicarbonitrile to
form 1,3-bis(arylimino)isoindole derivatives (Fig. 1c and d) follow-
ing a modified method.25 Briefly, 3,6-dioctyloxy-1,2-benzenedicar-
bonitrile (2, 0.65 mmol), 2-aminopyridine (1.62 mmol), and 25 mg
of CaCl2 were refluxed in 1-butanol under argon for 15 h to pro-
duce 1 in 70% yield. In a similar fashion, compound 3 was synthe-
sized from 4-butyl-aniline in 81% overall yield. The compounds
were purified by repeated recrystallization to obtain fine needle-
shaped orange crystals and characterized via 1H NMR and Mass
Spectrometric analyses. The synthesized compounds were pre-
served at 4 °C protecting from light and moisture. Interestingly,
upon exposure to moisture, the 1,3-bis(arylimino)isoindole deriva-
tives were found to undergo slow hydrolytic degradation as indi-
cated by the appearance of O-phthalamide derivatives in 1H NMR
studies (Fig. 1f).
To further assess the ability of the dyes for environmentally
sensitive photo-excitability in vitro, a phospholipids-polysorbate
micellar nanoparticle was synthesized. Our design of the nanopar-
ticle involved a co-self assembly of lecithin-PC and polyoxyethyl-
ene (80) sorbitan monooleate in water. Sorbitan monooleate was
strategically chosen as a co-surfactant to divulge stability to the
micelles and to restrict the particle diameter within 20 nm with
low polydispersity. A simple, two step procedure was followed to
incorporate bis(arylimino)isoindole derivatives, which was facili-
tated by the use of the co-surfactant. In a typical procedure, phos-
photidylcholine (100 mg) was weighed in a long neck test tube and
dissolved in 2 mL chloroform. To this, a solution of 1 (5 mg) in chlo-
roform was added. The solvent was evaporated gently to produce
the lipid film and dried for 30 min in vacuum at 40 °C. To this,
nanopure water (0.2 lM, 10 ml) was added with polyoxyethylene
(20) sorbitan monooleate and was probe-sonicated at 25 °C to form
a transparent light yellow suspension. The suspension was dia-
lyzed using 10 kDa MW cut-off cellulosic membrane against infi-
nite sink of nanopure water to remove any unbound dye.

4136
B. Kim et al. / Tetrahedron Letters 53 (2012) 4134–4137
Multiple analytical techniques were employed to characterize
these particles. Dynamic light scattering (DLS) measurements re-
vealed the particle hydrodynamic diameter as 19 7 nm with
low polydispersity (PDI = 0.039). Electrophoretic potential values
(f = À12 7 mV) were measured as negative, which is an indirect
measure of the colloidal stability of the system and further con-
firms a successful encapsulation with phospholipids.
Fluorescence emission spectra of the dyes (ca. 1.8 mM) were
studied in solvents with varying polarity.31 Solvent polarity depen-
dent variations in absorption and emission spectra were observed
for both 1 and 3. However, for 1 a significant change in the fluores-
cence intensity and intrinsic optical properties was observed. As
shown in Figure 2a, the emission from compound 1 is red-shifted
(kem = 356 to 408 nm) with decrease in solvent polarity. In THF,
kmax was observable at 408 nm. This indicated the activation and
presence of an excited state intra molecular hydrogen bond shift
that allowed the photo-excitation to occur. From the fluorescence
experimental data of compound 1, a general trend can be brought
out. As the solvent changes to a more polar environment from
dichloromethane to methanol, the shift in kmax and its emission in-
creases. As shown, compound 1 exhibited the lowest emission kmax
in dichloromethane, and as it shifted its environment toward more
polar, for example methanol and tetrahydrofuran, its emission kmax
increased. However, the effect of tetrahydrofuran and methanol is
similar and thus indicates a threshold for the compound undergo-
ing intra molecular hydrogen bonding according to solvent
changes. The solvent dependence optical property of 1 is expected
and can be attributed to the structural aspects of the amine func-
tionalities. The intra molecular hydrogen bond in 1 is stabilized
through a six-membered ring transition state, which involves
nitrogen Nb to Na to undergo a temporary resonance stabilized
configuration. This further enhances the transition state’s stability
and feasibility. Such effect is enhanced as solvent polarity is in-
creased, enabling the relay of hydrogen ‘hopping’ toward its Na
acceptor site.26 The nitrogen atom from the pyridine ring (Na)
has a higher affinity than the nitrogen atom in the five-membered
ring (Nb) from the 1,3-bis(imino)isoindole due to the trigonal pla-
nar configuration of the sp2 hybridized Na coordination. Such
geometry alignment with the hydrogen from Nb allows it to have
a higher proton affinity, which is also due to the assistance of the
increased electron density of the pyridine ring. Thus the [–Na–
HÁÁÁNb] valence bond (VB) is more favorable than the [–Na ÁÁÁH–
300
250
200
150
100
50
20
15
10
5
450
20
(a)
(b)
Methanol; Toluene; THF;
400
Dichloromethane
15
350
300
10
250
5
200
0
350
500
650
800
150
0
Wavelength (nm)
350
450
550
650
750
850
100
50
0
Wavelength (nm)
Methanol; THF; Butanol;
Isopropano;lDichloromethane
0
350
450
550
650
750
850
350
450
550
650
750
850
Wavelength (nm)
Wavelength (nm)
50
Dyes ‘nestled’ in the hydrophobic
1000
(c)
membrane
Phospholipids
40
(d)
(iii)
800
600
400
200
0
30
20
Hydrophobic
environment
(ii)
10
Nanoparticle
‘cracked’
0
600
700
800
900
Wavelength (nm)
(i)
Dyes
300
400
500
600
700
800
900
exposed to
hydrophilic
environment
Wavelength (nm)
Figure 2. Fluorescence spectroscopic study: compound 1 (a) and compound 3 (b) show variation in their fluorescence properties in solvents of different polarities; (c) a cartoon
illustrating the encapsulation of 1 distributed within the hydrophobic acyl chains of the phospholipids coated nanoparticles; dye molecules are exposed to aqueous
environment once the nanoparticles are ‘cracked’; (d) fluorescence spectrum of dye-loaded nanoparticles showing similar spectral profile as the free drug in non polar solvent
(dichloromethane, (i)); after the nanoparticle cracking (ii–iii), the variation in the fluorescence intensity and emission maxima are predominant. (iii) Corresponds to the
addition of more isopropanol. Solvents were chosen based on their polarity difference. Relative polarity of methanol: 0.762, 1-butanol: 0.586, THF: 0.207, isopropanol: 0.546,
toluene: 0.099, dichloromethane: 0.309.31

B. Kim et al. / Tetrahedron Letters 53 (2012) 4134–4137
4137
Nb] VB, which further assists in the formation of the intra molecu-
lar hydrogen bonding within the neighboring nitrogen moieties to
further drive the compound toward a stable transition state ring
formation. Consequently, the two tautomeric forms become ener-
getically close to each other due to the counterbalance between
the stability gained through the six-membered ring type.27–30 Such
naphthalene ring is also complemented by the conjugated 1,3-
bis(imino)isoindole structure, which further supports the transfer
of electron density around the four adjacent ring systems and thus,
ultimately favors intra molecular hydrogen bond to occur and
exhibits the shift in kmax. Compound 3 was prepared as a control
molecule, which lacks donor or acceptor atom crucial for rendering
potential intra molecular hydrogen bonding capability. Compound
3 (ca. 1.8 mM) in dichloromethane exhibited its absorption maxi-
mum at 348 nm. Although, Figure 2b indicated a change in the
fluorescence intensity with the polarity of the changing solvents,
it is also suggestive that rather than intra molecular hydrogen
bonding occurring within the internal mode of the structure, the
lone hydrogen is forming weak bonds with the solvents. This is
causing a lower shift in wavelength and causing a change in fluo-
rescence properties due to the quick shift in hydrogen position.
The lower shift in kmax could also indicate that the conjugation of
the system just with the solvent interaction is not as prominent
as that of when an intra molecular hydrogen bond occurs forming
a higher level of conjugated ring structures, thus a lower emission
results. Such absence of the capacity to intra molecularly hydrogen
bond with proximal acceptor atoms validates that a shift in hydro-
gen bonds cannot occur without a donor-acceptor interaction, thus
shows minimal variation in shifts in wavelengths with solvent
polarity.
Compound 1 was successfully incorporated within the nanopar-
ticles as described before. (Fig. 2c) The successful encapsulation of
the dye as a component of phospholipid surfactants indicated that
it was membrane stable via hydrophobic interactions of the acyl
chains (–C8H17) on the dye with the internal lipid coating and
was exposed to a hydrophobic environment. In order to expose
the dye into a less hydrophobic and more polar environment, the
nanoparticles were cracked with isopropanol and were transferred
to methanol. Figure 2d exhibits a significant change in the fluores-
cence intensity and properties as the dye gradually encounters a
polar environment with increasing amount of isopropanol. As
shown, the emission wavelength before exposing compound 1 to
a polar environment (methanol) is similar to the emission proper-
ties displayed by 1 alone in dichloromethane. This indicates that
compound 1 is localized stably at the hydrophobic acyl phospho-
lipids chains. Once the dye is exposed to the polar environment,
a significant shift in intensity and excitation maxima was observed.
With the addition of more isopropanol, the effect is more promi-
nent, which altogether suggests a greater degree of intra molecular
hydrogen bonding of the released dye in a polar environment. This
experiment further supports the hypothesis that the ability to form
intra molecular hydrogen bonding is strongly dependent on the
polarity of the solvent, which in turn regulates the intensities
and wavelength shifts.
to form intra molecular hydrogen bonding only showed a variation
presumably due to hydrogen bonding of the compound with the
solvent. Finally, the potential application of these membrane stable
dyes was successfully examined in a phospholipids-based nanopar-
ticle platform, which clearly indicates the implications of many fur-
ther applications toward nanomedicine and nano-biotechnology.
Acknowledgments
This research was supported by Grants from the AHA(0835426N
and 11IRG5690011), NIH (R01CA154737, R01HL094470, R01N
S059302, UO1NS073457). We are thankful to Professor Gregory
M. Lanza and Professor Samuel A. Wickline for their valuable
suggestions.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
05.128.
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In summary, we have successfully designed and synthesized
novel, membrane stable responsive dyes. Fluorescence spectro-
scopic study revealed that the optical properties are highly depen-
dent on the solvent polarity as reflected its ability to cause
photoexcitation and a shift in emission wavelengths. The fluores-
cence properties of the control compound, which lack the ability
31. Reichardt, C. Solvents and Solvent Effects in Organic Chemistry, 3rd ed.; Wiley-
VCH Publishers, 2003.