Direct observation of fluorescent complex formation of acridinium-anilide-thiophene triad with poly-L-glutamic acid

Article abstract of DOI:10.1016/j.jphotochem.2016.11.016

Conformational changes and self-assembly process of polypeptides and proteins are important for biological processes. In order to investigate such changes, we prepared a fluorescent probe, thiophene-phenylanilide-acridinium triad, and investigated its pho

Full text of DOI:10.1016/j.jphotochem.2016.11.016

Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Direct observation of fluorescent complex formation of
acridinium-anilide-thiophene triad with poly- -glutamic acid
L
a,
a
b
Jingqiu Hu *, Monica Joshi , Michael Elioff
a
Department of Chemistry, West Chester University, West Chester, PA 19383, USA
Department of Chemistry, Millersville University, Millersville, PA 17551, USA
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 5 September 2016
Received in revised form 17 October 2016
Accepted 11 November 2016
Available online 18 November 2016
Conformational changes and self-assembly process of polypeptides and proteins are important for
biological processes. In order to investigate such changes, we prepared a fluorescent probe, thiophene-
phenylanilide-acridinium triad, and investigated its photophysical properties in aqueous solutions of
poly-
solution due to fast intramolecular electron transfer. In the acidic solution of PGA, the charge shift state
CSH) emission near 560 nm is significantly enhanced. The intensity of the CSH emission depends on the
L-glutamic acid (PGA) at various pH levels. The triad displayed extremely weak emission in aqueous
(
concentration, size, and conformation of PGA. Fluorescent microscope images reveal the size and shape of
the super molecular structures and crystals formed by PGA-triad complexes in acidic conditions.
©
2016 Elsevier B.V. All rights reserved.
1. Introduction
polypeptide chain length [21] and solvent properties such as pH
14,15], temperature [18,21–23], and the type of interacting ions in
[
Non-covalent interactions between small organic molecules (or
ions) and polyelectrolytes have received substantial attention due
to their applications in energy conversion, fluorescent sensor
design, and drug delivery [1–8]. One well-studied polypeptide
the solution [24]. It is well known that as pH is decreased from
basic to acidic, PGA undergoes coil-to-helix transition. The
neutralization of charges in PGA in acidic condition can also
facilitate the formation of diverse aggregates and ordered super-
molecular structures [20,23]. However, the use of fluorescence
microscopy to directly measure the size and shape of PGA
aggregates has not been reported.
electrolyte is poly-
naturally occurring
through the peptide bonds. The carboxyl group in each repeating
unit of -glutamic acid is negatively charged at neutral pH. This
L-glutamic acid (PGA), which is composed of
L-glutamic acid residues linked together
L
The binding of a few organic dyes to poly-L-glutamic acid has
charge not only causes the polymer to be water soluble but also
provides functionality for fluorescence tagging or electrostatic
attachment by small counter ions including some drugs. Because it
has a pH-controllable conformation, PGA has been frequently used
as a carrier for drugs such as antibiotics and antitumor agents [9–
been studied as a model system for ligand-protein interaction
[5,25–27]. The aromatic compounds studied include porphyrin
derivatives [25], Thioflavin T [5,20], and acridine orange [26,27].
However, the fluorescent properties of organic dyes that bind to
PGA were not investigated in detail. This article describes an
acridinium-anilide-thiophene triad (Compound 1) whose fluores-
cent emission is sensitive to the conformational change and self-
assembly behavior of polypeptides. The probe is based on
suppressed back-electron transfer process when the rotation
within the molecule is restricted as the dye binds to PGA. In the
triad design, an acridinium ion is the light absorber and electron
acceptor, an anilide group is the chemical linker and electron
donor, and thiophene is the terminal electron donor. With a
positive charge and the hydrophobic nature of aromatic acridinium
ring, the triad might be expected to show binding affinity toward
the hydrophobic domains of polyacids. The effects of pH, ion
strength, the polymer residue/dye concentration (R/D) ratio, and
12].
The conformational change of PGA in aqueous solutions has
been extensively investigated as a model system for protein folding
and unfolding [13–19]. Various spectroscopic methods including
vibrational circular dichroism (VCD) [13–17], two-dimensional
Raman analysis [19], Fourier transform infrared (FTIR) [20], and
near infrared absorption (NIR) have been applied in conforma-
tional studies of PGA. The conformation of PGA is affected by the
*
Corresponding author.
E-mail address: jhu@wcupa.edu (J. Hu).
http://dx.doi.org/10.1016/j.jphotochem.2016.11.016
010-6030/© 2016 Elsevier B.V. All rights reserved.
1

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J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
polymer molecular weight on the self-assembly of compound 1 to
PGA were studied. The emission maxima and quantum yield of the
triad are sensitive to the coil-to-helix conformational change of the
PGA. Furthermore, the emission is strongly enhanced when the
PGA aggregates at low pH. Based on fluorescence microscope
images, we describe a model involving the aggregation of helix
coils of PGA in acidic solutions, which forms hydrophobic domains
for ligand complexation and significantly affects photo-induced
electron/hole transfer properties.
A stock solution of polypeptides was prepared by dissolving
8.4 mg PGA in 2 ml of Millipore water (28 mM as a concentration of
monomers). A small aliquot of the PGA stock solution was added to
2.5 ml aqueous solution of 1. The pH of 1/PGA solution was adjusted
by adding dilute HCl (0.1 M aqueous solution) and NaOH (0.1 M
aqueous solution) where necessary. In order to observe fluorescent
crystals formed by 1/PGA complex under various conditions, we
placed 10 ml of 1/PGA solution ([1] = 8.4 mM, [PGA residue] = 1.1
mM) on glass slides and allowed the water evaporate at room
temperature. Fluorescent microscope images were collected using
an Olympus BX43F polarized light microscope with a Lumen
Dynamics X-Cite 120Q epifluorescence source. The filter used was
FITC: 475–490 nm for excitation and 505–535 nm for emission.
2. Experimental
2.1. Materials
The thiophene-phenylanilide-acridinium triad (compound 1)
2.3. Computational studies
was prepared by the coupling reaction between thiophene
carbonyl chloride and the 4-aminophenyl-10-methylacridinium
salt. The preparation of anilide derivative of acridinium has been
reported previously [28,29]. Sodium salts of poly-L-glutamic acid
were purchased from Sigma Aldrich with average molecular
In order to have a more complete understanding of the
electronic and nuclear geometries of compound 1, computations
were performed using the Becke Three-parameter Lee-Yang-Parr
(B3LYP) density functional theory at the 6–31 + G* level using
Spartan ’14 modeling software from Wavefunction, Inc. All
calculations were performed on single molecules. Calculations
were executed on a Dell Optiplex 7010 computer with 8.00 GB RAM
and a 64-bit Windows 7 Professional operating system with an
Intel Core i5-3550 CPU operating at 3.30 GHz. The radii of the
terthiophene, anilide, and acridinium moieties were determined
from calculated equilibrium geometries and used to ascertain the
Gibbs Energy of solvation, as discussed in the following section.
weights of 75 kDa (MW: 50–100 kDa) and 32.5 kDa (MW: 15–
50 kDa). These PGA salts were used directly without purification.
2.1.1. 2-Thiophene carbonyl chloride
The grey powder of thiophene carboxylic acid (4.1 g, 32 mmol)
and 30 ml of thionyl chloride were placed in a flask equipped with a
condenser and a stirring bar. The reaction mixture was refluxed for
1
h. Most of the thionyl chloride was removed by distillation. The
remainder of the solvent was removed by rotary evaporation.
About 4 ml of a dark brown liquid was obtained. The product was
used in the next step without further purification.
3. Results and discussion
3
.1. Photophysical properties in organic solvents
2.1.2. Thiophene-phenylanilide-acridinium triad
The purple solution of 4-aminophenyl-10-methylacridinium
The absorption spectra observed for compound 1 was
hexafluorophosphate (300 mg, 0.70 mmol) in 6 ml CH
slowly added to the brown solution of 2-thiophenecarbonyl
chloride (100 mg, 0.70 mmol) in 2 ml of CH CN and 0.2 ml of
3
CN was
characterized by the principal transitions that are typical of the
acridinium chromophore (Fig. 1, bands at 361 nm and 428–462 nm
with extinction coefficients of approximately 20,000 and
9000 M cm , respectively). The longer wavelength absorption
at around 450 nm resembles that of 9-{4[(benzoylamino)phenyl]}-
10-methylacridinium ion, an acridinium-anilide dyad, in which the
direct links to the ring lead to significant donor-acceptor coupling
in the 9-position. This band is assigned to the charge shift state
(CSH) absorption [28].
As the polarity of the solvent decreases, the CSH absorption
associated with direct excitation to the intermediate radical pair
(Scheme 1) undergoes a red shift. For example, the CSH band shifts
from 436 nm in acetonitrile to 459 nm in methylene chloride
(Fig. 1a). These spectral shifts remain consistent with our former
observation that the less polar CSH state is relatively more stable
than the polar (charge localized) ground state in non-polar
solvents [28–30].
3
ꢁ1
ꢁ1
pyridine. The brown solution of the acid chloride turned to a light
yellow color and eventually turned reddish. An orange precipitate
formed at the bottom of, and along the wall of, the flask. The
reaction was left at room temperature overnight. The orange
residue was filtered and recrystallized from ethanol to yield 90 mg
ꢀ
1
of an orange powder (30%). MP: 198–201 C. H NMR (400 Hz,
DMSO-d ): ppm 10.723 (s, 1H), 8.845 (d, J = 11.5 Hz, 2H), 8.453 (t,
J = 10.0, 9.5 Hz, 2H), 8.180 (s, 2H), 8.163 (s, 1H), 8.048 (d, J = 10.5 Hz,
H), 7.946 (t, J = 9.5, 9.0 Hz, 3H), 7.600 (d, J = 8.0 Hz, 2H), 7.268 (m,
6
d
2
13
J = 5.0, 5.0 Hz,1H), 4.920 (s, 3H). C NMR: (DMSO-d
61.25, 141.96, 141.47, 140.35, 139.10 (2C), 133.17, 131.54 (2C),
30.55, 130.48 (2C), 128.93, 128.61 (2C), 126.33, 120.69 (2C), 119.91
2C), 100.18, 60.33. HRMS (EI, 70 eV) m/z 396.1334, reported
6
) d ppm 171.13,
1
1
(
+
+
+
composition:
25 2 25 2
C H21OSN . ([M + 1] , calcd. for C H20OSN ,
3
95.1218).
While the isolated acridinium ion, 10-methyl-acridinium,
fluoresces strongly with a quantum yield of 1.0 at 495 nm [29],
compound 1 displays no appreciable emission in polar organic
solutions, such as acetonitrile and acetone. The fluorescence
emission of the acridinium ion in compound 1 is fully quenched.
Since both thiophene and anilide were reported to donate
electrons to acridinium ion [28–30], the complete quenching of
fluorescence is due to fast multistep electron transfer from
thiophene and anilide to acridinium ions. In less polar or nonpolar
organic solvents, such as methylene chloride and toluene, the CSH
2.2. Methods and procedures
Measurements of pH were made using a VWR Symphony SB70P
pH meter. UV–vis absorption spectra were recorded using a Cary
00 UV–vis spectrophotometer. Steady state emission and
quantum yield were measured using Shimadzu RF-6000
3
a
fluorophotometer. Except where indicated, measurements were
made at room temperature (25 C) using 1-cm quartz cells.
ꢀ
Fluorescence quantum yields were determined by comparing
the spectrally corrected emission intensity of the sample to that of
a fluorescence standard, phenylacridinium. Circular Dichroism
band is observed at longer wavelength (
Fig. 1b). Furthermore, compound 1 displayed a higher quantum
yield in less polar solvents = 0.0024 in methylene chloride, and
= 0.0065 in toluene). The physical properties of 1 in various
media are summarized in Table 1. An acridnium-anilide dyad, the
lmax = 567–580 nm,
f
(
CD) spectra of the polypeptides were collected with an AVIV
f
Associates Model 60 DS spectrometer.

J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
61
Scheme 1. Energy diagram showing electron/hole transfer steps for 1 along with
structure of 1.
G CSH = F [E D /D ꢁ E A/Aꢁꢂ] ꢁ E00
0
0
+
ꢂ
ꢂ
0
D
S
+ DG + W
where F is the Faraday constant, which is unity when the energy is
0
ꢁ.
0
+.
.
in electon-volts. E A/A and E
D
/D are the standard electrode
0
ꢁ.
potentials of the reduction of the acceptor (acridinium: E A/A = ꢁ
0
+.
D
0
.62 V vs SCE) [27] and the oxidation of the donor (E
/D = 2.0 for
0
+.
thiophene [33], and E
D
/D = 1.86 for the anilide group) [28]. The
energy of the zero to zero transition of acridinium ion, E00, is 2.7 eV
28]. The Coulomb correction term, W, is always zero for charge
shift process [28]. The Born correction term, , is associated with
[
DG
S
the solvation energies and given by the following equation for
monovalent cations:
ꢂ
ꢀ
ꢁ
ꢀ
ꢁꢃ
e2N
1
1
1
1
A
D
G ¼
ꢁ 1 ꢁ r
ꢁ 1
S
8pe
0
r
T
e
T
acr
e
acr
where e is the charge on an electron, N
the permittivity in vacuo, r and racr are the radii of the terthiophene
and acridinium moieties, and and acr are the relative permittivity
A e0
is Avogadro’s number, is
T
e
T
e
of the terthiophene and acridinium moieties, which are taken to be
2.3 when benzene is the solvent. We assume that the dielectric
constants of dilute solutions are approximately equal to the
Fig. 1. Absorption (a) and emission (b) spectra observed for 1 in various solvents
[1] = 8.0 M; ex = 360 nm).
(
m
l
dielectric constant of the solvent, thus
below, the solvation energy calculated using density functional
e
ꢃ
e
acr
ꢃ
eT
. As described
9
-{4[(benzoylamino)phenyl]}-10-methylacridinium ion, was used
as reference compound in which anilide is the sole electron donor
28]. A phenyl group, which does not act as an electron donor, was
theory (DFT) is
S
DG = –0.01 eV. This yields a value for the driving
ꢀ
force of
D
G
CSH2 = –0.09 eV. Using the same approximation, we can
[
determine that the driving force of electron transfer between
anilide and acridnium is
Our calculations suggest a negative Gibbs free energy for both
CSH process, indicating that both charge shift process are
thermodynamically favored. However, the energy of charge shift
ꢀ
G
introduced as the amide terminal group for structural similarity.
Therefore, the difference between the fluorescent parameters of
compound 1 and the dyad should reflect the electron transfer
process due to the presence of the thiophene group. The spectral
features of 1 compare favorably to that of the acridinium-anilide
dyad, (
emission band around 570 nm is assigned to the radiative decay of
the CSH state (CSH ) formed by the electron transfer from anilide
to the acridinium moiety [28]. Note that the CSH state quantum
D
CSH1 = ꢁ0.23 eV.
state CSH
the two charge shift processes occur simultaneously, resulting in
smaller yield of CSH . In summary, the attachment of the thiophene
2 1
is above the CSH state. Therefore it is more likely that
lCSH = 566 nm, fCSH = 0.10 in methylene chloride [28]); this
1
1
groups to the acridinium-anilide base structure introduces a
dramatic reduction in fluorescence yield for 1, indicating a second
charge shift process in 1 involving transfer of holes to the more
remote thiophene. An energy diagram that depicts the possible
pathway of radiative and non-radiative decay in 1 is shown in
Scheme 1.
1
yield of the triad (compound 1) is only 2.4% of that of the dyad. This
is not unexpected because in the triad there is an additional charge
shift process (CSH
thiophene. There are two possible competing pathways that
may result in a smaller quantum yield of CSH . One involves the
CSH process occurring after the formation of CSH and competing
with the radiative decay process of CSH . Another involves the
CSH occurring simultaneously and thus competing with the
formation process of CSH
To determine which pathway is dominant, we calculated the
driving force for the photo-induced electron transfer using the
Rehm-Weller equation [31,32].
2
) involving the second electron donor,
1
2
1
3
.2. Computational studies of ground state structure
1
2
Single-molecule computations were performed on compound 1
1
.
using the B3LYP density functional theory at the 6–31 + G* level
using Spartan ’14. Fig. 2a shows the calculated structure for the
molecule. In the energy-optimized structure, the acridinium
moiety assumes a flat geometry, as do the benzanilide and

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J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
Table 1
a
The photophysical properties of 1 in various solvents .
solvent
l
(
I
e
I
l
II
e
II
lem (nm)
F
f
nm) (M 1cmꢁ1
ꢁ
)
(nm) (M cm
ꢁ1
ꢁ1
)
4
H
CH
CH
2
O
361
23000
21700
21600
10000
17000
428
436
459
462
436
9800
9500
9300
7300
8000
505
–
5.0 ꢄ10ꢁ
ꢁ
3
4
3
CN
Cl
361
361
369
362
<5.0 ꢄ10
ꢁ
2
2
566
577
565
2.4 ꢄ10
6.5 ꢄ10
2.2 ꢄ10
ꢁ
3
Toluene
b
ꢁ2
PGA
a
l
ex = 360 nm.
b
[
PGA residue]/[dye] = 260, pH = 4.7.
thiophene functional groups. The dihedral angle between the
ꢀ
acridinium and benzene ring is approximately 58 . The benzene
and thiophene rings are very nearly in the same plane. The center
to center distance between the acridinium and benzanilide moiety
is 4.3 Å. The distance between the center of the acridinium system
and the thiophene ring is 10.6 Å. The radii of the terthiophene,
anilide, and acridinium moieties are 2.0 Å, 2.2 Å, and 3.1 Å,
respectively, and are used to determine
equation.
S
DG in the Rehm-Weller
Fig. 3. Absorption spectra observed for 1 in PGA solution: R/D = 130, in pH 7.3, 4.7,
.8, and 2.8 aqueous solutions. The R/D = 0 curve (solid line) is included for
comparison. ([1] = 8.4 M; ex = 360 nm).
3
Fig. 2b shows the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO). It is
noteworthy that the HOMO has most of its density located on
the thiophene and benzanilide portions of the molecule, whereas
the LUMO has most of its density located on the acridinium ion.
This evidence supports the theory of intramolecular electron
transfer from the thiophene and anilide moieties to the acridinium
ions. The calculated energy gap between HOMO and LUMO is
m
l
3
3
.3. Photophysical properties of compound 1 upon binding to PGA
.3.1. Absorption spectra
The absorption spectra observed for 1 in aqueous solution
shows small changes on addition of the polypeptide, PGA (Fig. 3).
At pH 7.3, the addition of PGA resulted in only a slight decrease in
intensity of the 361 nm absorption band. At pH 4.7, the addition of
PGA caused a slight decrease of the absorbance at 361 nm, and a
red shift of the CSH band from 428 nm to 436 nm. However, at pH
values below 4.0, the CSH band shifted back to 429 nm.
3
98 kJ/mol, which corresponds to an absorption wavelength of
approximately 300 nm. This slight underestimate of the wave-
length of maximum absorption, which was experimentally
determined to be 361 nm in water and organic solvents, is not
unexpected as the calculations were performed on the gas-phase
ion.
3
.3.2. pH effect on CSH
The interaction between 1 and PGA is strongly affected by the
pH of the solution as indicated by the wavelength and intensity of
1
emission band
Fig. 2. Calculated ground-state geometry (a) of compound 1 using B3LYP at the 6–31 + G* level and screen shot of highest occupied molecular orbital (b) and lowest
unoccupied molecular orbital (c) of compound 1.

J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
63
Fig. 4. pH dependence observed for CSH emission for 1 in aqueous PGA (R/D = 130;
[1] = 8.4 M; ex = 360 nm).
(
m
l
1 1
the CSH band emission (Fig. 4). Starting at pH 6.6, the CSH
emission of 1 with PGA is observed, at around 575 nm with R/D
ratio of 130. The R/D ratio is defined as the ratio of molar
concentration of PGA residues to dye molecules. Compared to the
1
absence of the CSH band emission for aqueous solution, these
results provide evidence for the binding of 1 to PGA at high pH
through mechanisms that involve electrostatic interaction and
possibly hydrophobic or hydrogen-bonding contact. The weak
1
intensity of the CSH emission band indicates that the biopolymer
electrostatic binding does not bring about a large deceleration of
non-radiative decay (i. e., back-electron transfer) for the triad.
Fig. 5. Absorption (a) and emission (b) spectra observed for 1 in the presence of
PGA, pH adjusted to 4.8 with dilute HCl solution. ([1] = 8.4 mM; lex = 360 nm).
However, as pH was decreased, the CSH
significantly, reaching a maximum at pH 4.7, where the coil to
-helical secondary structure transition is complete (Fig. 4). With
1
emission of 1 increased
a
other conditions remaining the same, the intensity of 1 in acidic
condition at pH 4.7 is 30 times higher than that in pH 7.3 solution.
Lowering the pH to 4.5 caused blue shift of the emission band and
decreased intensity.
that the self-assembly of the triads begins at low molar excess of
residues as the peptides enter the coil-to-helix transition.
Furthermore, we see that the higher the peptide concentration,
the more ordered the system and the higher the energy of the CSH
1
Higher levels of polypeptide aggregation were observed at still
lower pH for 1/PGA complexes. At a pH of 3.0, solutions of the long
chain PGA/dye complex became turbid due to the aggregation
induced by the intermolecular hydrophobic interaction between
segments of the PGA backbone (note the decrease of emission
intensity, Fig. 4). A decrease of the pH to 2.5 led to destabilization of
the biopolymer complex and precipitation from solution. These
results indicate the formation of triad/polypeptide aggregates. It
band. Along with the very modest change in absorption features,
was also observed that as pH decreases from 6.6 to 2.8, the CSH
band blue-shifted from 575 nm to 537 nm. The pH dependence of
the CSH emission implies the influence of not only the secondary
structure (coil to helix transition) but also the tertiary structure
size, shape, hydrophobic domain) of PGA on dye binding and
1
1
(
orientation, and on the rate of the competing non-radiative decay
process.
3.3.3. R/D effect on dye binding at fixed pH
For aqueous solutions at a pH of 4.8, the addition of PGA, with R/
D values ranging from 60 to 590, brought about small blue shift
572 nm–565 nm) of the CSH emission band and increased the
intensity by more than 30-fold (Fig. 5). These results demonstrate
(
1
Fig. 6. Benesi Hildebrand plot. Dependence of CSH emission for 1 on PGA
concentration; pH = 4.8, [1] = 8.4 mM, lex = 360 nm.

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J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
the increased intensity of CSH
1
fluorescence indicates that the
competing nonradiative decay processes are slowed for the triad/
peptide complex. This is probably due to the docking of the triad
molecules onto the PGA scaffold. Subsequently, the restriction of
the fast rotations about the sigma bonds within the dye molecule
prohibits it from achieving the geometry favorable for back-
electron transfer. The binding constant of 1 to PGA residues is
ꢁ
1
calculated to be 233 M from the Benesi-Hildebrand plot (Fig. 6).
3
.3.4. Test of influences on PGA binding: electrostatic vs hydrophobic
interaction
To further investigate the binding forces between acridinium
conjugates and PGA, the effect of ionic strength and the
introduction of an organic solvent were studied. The addition of
salt is expected to decrease electrostatic interaction between
charged peptides and acridinium cations, while the addition of
organic solvent should weaken the hydrophobic contacts. Upon
addition of NaCl solution (up to 0.2 M) to 1/PGA aqueous solutions
at pH 4.7, the emission intensity decreased (Fig. 7b) by more than
8
0%. Furthermore, the absorbance at 361 nm recovered to near that
of the free dye, while the CSH band blue shifted back to 429 nm
Fig. 7a). This result implies the electrostatic interaction between
(
the acridinium cation and the deprotonated side chain carboxylate
Fig. 8. Effect of ethanol on the absorption (a) and emission (b) spectra observed for
in PGA (R/D = 260, pH = 4.8). ([1] = 8.4 M; ex = 360 nm).
1
m
l
group of PGA plays an important role in forming 1/PGA complex.
Upon addition of ethanol to 1/PGA complexes at pH 4.7, the
intensity of CSH emission decreased by more than half with 20%
ethanol in the solution (Fig. 8). This result indicates that ethanol
Fig. 7. Salt effect on the absorption (a) and emission (b) spectra observed for 1 in
PGA (R/D = 260, pH 4.7 adjusted with dilute HCl solution), ([1] = 8.4
m
M;
l
ex = 360
Fig. 9. Dependence of the emission of 1/PGA complexes (R/D = 260) on molecular
nm).
weight at pH 4.8 ([1] = 8.4 mM; lex = 360 nm).

J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
65
inhibits the domain structure important for binding
1
by
3.4. Viscosity effect
disrupting the hydrophobic interaction. This suggests that the
PGA binding behavior for the triad is due to the sequestration of the
hydrophobic acridinium groups in non-polar regions of a highly
folded peptide structure.
In order to investigate the effect of restricted rotation on the
fluorescent emission of 1, a series of solutions of 1 in mixture of
glycerol and water were prepared. As the glycerol concentration
increases, the solution becomes more viscous and the CSH
emission at 540 nm increased significantly (Q. Y. = 0.0065 in pure
3.3.5. Molecular weight effect on the binding
ꢀ
The PGA samples used in the binding experiments have an
glycerol at 22.5 C). Additionally, a solution of 1 in pure glycerol
ꢀ
average molecular weight of 75 kDa and contain about 500
glutamic acid monomers. To test the effect of PGA molecular
weight on aggregation and dye binding, a PGA with different
molecular weights (average molecular weight 32.5 kDa) was
applied in the study, while the pH and R/D ratio were kept
constant. Results showed that CSH emission in 1 was significantly
weaker in low molecular weight PGA (Fig. 9) . The higher molecular
weight PGA is much more effective in binding, leading to stronger
emission. This result suggests that the longer peptide chains are
able to provide a protected non-polar environment in which the
dye can self-assemble. As discussed below, the overriding factor
may have more to do with the ability of the larger biopolymer to
create domains via aggregation.
was heated to 65 C to ensure thorough mixing. As the solution
cooled to room temperature, the bulk viscosity increased, and the
CSH emission at 540 nm increased (Fig. 10). The increase of
viscosity during the cooling process restricts the bond rotation in
the triad, thereby deactivating the non-radiative decay (back
electron transfer) pathway that competes with the radiative decay
leading to CSH
confirms our proposed mechanism of enhanced CSH
1
emission. The viscosity-sensitive behavior of 1
emission
1
through suppressed non-radiative decay process as 1 binds to PGA.
In summary, we note that fluorescence parameters of 1 are
strongly influenced by size, concentration, and conformation of
PGA in the solution. The complex formation of triad/PGA is evident
in emission data for all combinations of stoichiometry (R/D = 60–
5
90) at pH range 2.7–7.3, but the extent of the effect of
complexation with the peptide follows a discernible trend that
reflects changes in PGA structure. The binding of triad to PGA is
sensitive to the addition of both salt and an organic solvent,
indicating that both electrostatic and hydrophobic interactions are
important driving force for the complex formation.
3
.5. CD spectra
The CD spectra, shown in Fig. 11, of PGA confirm the coil to helix
transition when pH is changed from neutral to acidic. At pH 6.0,
PGA has random coil conformation and the spectrum displays
characteristic negative at 197 nm and slight positive at 217 nm. At
pH 4.0 and pH 3.0, CD signal shows the characteristic ellipticity of
a-helices at 208 nm and 222 nm [34]. The helical content of PGA
was calculated using Mean Residue Ellipticity (MRE).
observed CD ðmdegÞ
MRE½ ꢅ ¼
u
l
Cnl ꢄ 10
where C is the molar concentration of PGA, n is the average number
of glutamic acid residues per PGA chain, and l is the path length of
the light through the cuvette. The product of C and n gives the
Fig. 10. Emission spectra of 1 in glycerol/water mixture (a), and emission spectra of
ꢀ
1
in 100% glycerol (b) as the solution cooled from 65 C to room temperature.
Fig. 11. CD spectra of PGA at pH 6.0, pH 4.0, and pH 3.0 respectively. [Glu
(
[1] = 8.4
m
M;
l
ex = 360 nm).
residue] = 100 mM, pH was adjusted by 5% TFA.

6
6
J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
ꢀ
Fig. 12. Fluorescence micsroscope image of 1/PGA solution at pH 4.0 (a and b), pH 3.0 (c), and pH 3.0 after heated to 80 C for 1 h and cooled to room temperature (d).
molar concentration of glutamic acid residue. In our experiment,
the PGA residue concentration is 0.100 mM, and the path length of
the cuvette is 1 cm.
globular proteins [38]. Using an average weight of 75,000 g/mol,
we can determine an approximate volume for PGA molecules of
90.7 nm . From this volume, the diameter of a single PGA molecule
3
The helical content was calculated using the following equation
34]:
is estimated to be 5.6 nm. By comparing this to the size of the 1/
PGA aggregates, we estimate the number of PGA molecules in each
aggregate to be 250–400 in one dimension.
At pH 3.0, the solution quickly turned turbid and PGA forms
larger aggregates that are about 20 to 100 mm long. The majority of
[
½
u
ꢅ
ꢁ 3000
2
22
%
a
ꢁ helix ¼
ꢁ
36000 ꢁ 3000
where [
ꢁ
u
]
222nm is the MRE [
u
] at 222 nm, and is equal to
29400 cm dmol at a pH of 4.0. The helix content of PGA at
pH 4.0 is calculated to be 83.1%. The [ 208nm ratio from
the aggregates shape like rods, with some irregularly shaped
superstructure (Fig. 12c). After being heated at 80 C for 1 h, the pH
3.0 PGA solution turned clear and the emission of compound 1 was
ꢀ
2
ꢁ1
ꢀ
u
]
222nm/[[
u
]
the CD spectrum at pH 4.0 is 1.36, indicating the formation of a
coiled coil by multiple helices [35].
extremely weak and featureless. As the solution cools to room
temperature, the CSH emission recovered (Fig. 13) while the
1
3.6. Fluorescence microscope images of 1/PGA complex and crystals
Under a fluorescence microscope, we observed large triad/PGA
aggregates (Fig.12) in acidic aqueous solutions. At pH 4.0, although
the freshly made solution appears to be clear to the naked eye,
fluorescence microscope images show that PGA forms globular
aggregates of various sizes with diameters ranging from 1.4
mm to
2
.5 m (Fig. 12a, b). After one week, a yellow precipitate was
m
observed in the sample solution. A quick shake re-dissolved the
solid and the solution turned clear again. The fluorescent emission
of the one week-old sample was comparable to the fresh sample,
indicating the formation of large aggregates is reversible at pH 4.0.
Our CD spectra predict that PGA consisted of folded
a-helices
connected by free chains. Theoretical study predicted that PGA
molecules assume a near-spherical shape at pH 4.5 [36,37]. We
therefore treated PGA as a globular protein and estimated the
volume of individual peptide molecules as follows:
3
24
3
3
23
V = 0.73 cm /g ꢄ 10 Å /cm ꢄ MWg/mol / 6.02 ꢄ10 molecule/
3
mol = 1.21 ꢄ MW Å /molecule
where MW is the molecular weight of the peptide, and 0.73 is the
average of experimentally determined densities for soluble,
Fig. 13. Time profile of fluorescence emission at 540 nm of compound 1/PGA
complex as temperature changes from 60 C to R. T. over a period of 40 min.
ꢀ
(
[1] = 8.4 mM; lex = 360 nm, R/D = 140).

J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
67
Fig.14. Fluorescence microscope images of 1/PGA crystals formed at various pH 4.0 (a), pH 6.5 (b), pH 4.7 (c), pH 3.4 (d), and bright field images of 1/PGA crystals at pH 4.7 (e)
and pH 3.4 (f).
solution turned turbid again. Under the fluorescence microscope,
long thread-like superstructure formed with the length of several
hundred micrometers and thickness of 3.7 mm (Fig. 12d). A closer
suggesting the crystal formation is unique to the 1/PGA complex.
The size- and shape-dependence of 1/PGA crystals on pH can be
understood by considering the conformational change of PGA as
pH varies. The pKa value of glutamic acid side chain carboxyl group
is 4.25. At pH 6.5, about 2 units above the pKa value, more than 99%
of the side chain is protonated. The PGA molecules assumes an
extended coil structure, which can be brought close to each other
by acridinium cations in 1, forming large rod-like long crystals as
water evaporates. As pH decreases, the side chains become
protonated and the peptide assumes a multi-helical structure,
which is more packed than the random coil structure. They
therefore form smaller crystals as water slowly evaporates.
Meanwhile, as pH decreases, the solubility of PGA decreases. This
means the crystallization process started with more nucleation
which results in smaller crystals in more acidic conditions.
The peptide concentration plays a critical role in crystal
formation. When PGA residue concentration in the 1/PGA solution
was 0.55 mM (R/D = 65), no crystals were observed. When PGA
residue concentration is 1.1 mM, crystals were observed in acidic
conditions. At that concentration, the R/D ratio of glutamic acid
monomer to dye is 130:1. According to the average molecular
weight (75 kDa) of the PGA, the average number of monomers is
500 per peptide molecule. Taken together, these ratios suggest that
look at the structure reveals the inter-chain aggregation of fibril-
like long peptide chains. The image appears to be the brightest at
where the triad is incorporated into the inter-chain aggregation. It
ꢀ
has been reported that after incubation at 65 C at pH 4.1, PGA with
lower molecular weight (MW 15–50 kDa) forms Amyloid-like
fibrils of approximately 200 nm in thickness and 1.6 mm in length
observed under TEM [20,21]. Our result is the first to observe much
larger fibrils like superstructure directly under fluorescence
microscope.
Furthermore, we observed fluorescent crystals formed by 1/
PGA complex under various conditions (Fig.14). The size and shape
of the crystals are affected by the pH of the solution. At pH 6.5, long,
rod-shaped crystals with bright edges were observed under a
fluorescence microscope. The length varies from 45
and the width is 17 m. At pH 4.0, cross-shaped crystals formed
having edge-to-edge distances in the range of 14 m–80 m.
mm to 260 mm,
m
m
m
Those crystals appeared to be hollow in the middle and bright on
the edges. At pH 3.0, even smaller crystals with edge-to-edge
distances of 12–29
mm were observed. Bright field images show no
crystal formation in pure PGA solution or pure dye solution,

6
8
J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
results [42,43]. The fluorescence microscope images provide
evidence for the hypothesis that helical chains develop a tertiary
structure involving multiple helices in acidic condition and
undergo intermolecular aggregation to assume a compact inter-
woven coil conformation with a hydrophobic domain, which is
well suited to accommodate acridinium chromophores. The
process can be viewed simply as: random coil ! single helices !
multiple helices ! interchain aggregation forming hydrophobic
domain (Scheme 2).
Another well-studied polymer, Poly (methylacrylic acid)
(
PMAA), which assumes a compact coil conformation in acidic
solution albeit with little aggregation [46], showed a similar effect
on the CSH emission of 1, which implies a similar hydrophobic
domain forming tendency for the PGA and PMAA structures.
Those results taken together support a model where the high
molecular weight PGA aggregate to form a globular-like quaternary
structure at acidic pH that is well suited for accommodating
hydrophobic organic chromophores and donor-acceptor arrays. At
pH 4.0, the PGA forms multiple helices and slowly aggregates to
form a folded compact coil structure. The bound acridinium-
anilide-thiophene molecules are surrounded by helices of PGA in
the solutions and emit strongly at around 560 nm.
Scheme 2. Conformation change of PGA and complex formation of compound 1
with PGA aggregates.
the molar ratio of dye molecules to PGA molecules in the crystal is
approximately 4:1. The positively charged acridinium ions can bind
to the side chain carboxylate groups of a small number of residues
to bring the peptide chains together and promote the crystal
formation process of peptides. The detailed crystal structure of the
4
. Conclusions
1
/PGA complex is a subject for future studies.
The helix-coil transition for PGA has been a subject of intense
study for decades as a model for protein folding and unfolding [39–
1]. It has been widely thought that there are four regions of
structural distinction for dilute solutions of PGA in water: the
charged random coil important at higher pH (e.g., 8.0), a transition
region (HC: helix to coil) important at lower pH (e.g. pH 5.7), a
region in which the peptide is nearly completely helical (H) at pH
In summary, we report a new acridinium-based dye that forms
fluorescent complexes with PGA. In this molecule, the local excited
state emission of the acridinium chromophore is fully quenched
due to ultrafast photo-induced electron transfer in all solvents. The
charge shift band (CSH) emission resulting from the first electron
transfer step from anilide to acridinium strongly depends on the
solvent polarity and the microenvironment. <H2>In nonpolar
organic solvents, such as methylene chloride and toluene, the weak
CSH state emission was observed. When the data were compared
with the CSH emission for the anilide-acridinium diad, it was
evident that there is a second electron transfer step between
thiophene and acridinium ions.
4
4
.9, and finally at pH 3.0 and lower, a region in which the peptide is
aggregated into complexes of varying size [42]. Indeed it has been
known for some years that the helix-coil transition is concentra-
tion dependent and that PGA solutions precipitate on standing at
lower acidic pH.
More recent reports are consistent with the model in which the
helical form of PGA is in an aggregated structure at equilibrium. A
light scattering study documented the growth of PGA (708-mer)
aggregates at pH < 4.7 [43]. Theoretically treatment predicted that
the helices overlap in a side by side fashion in the PGA aggregates
Upon binding of the triads to PGA in water, the CSH state
emission largely depends on the size of the polypeptide, the R/D
ratio, and the pH of the solution. In the acidic PGA solutions, the
back electron/hole transfer step is considerately retarded and the
1
CSH state emission increased by 30-fold. Fluorescence micro-
[
41]. More convincing support for the helix aggregate comes from
scope images show that high molecular weight PGA in acidic
solutions undergoes intermolecular aggregation to form a slightly
elongated globular shape. The size and nature of the micro-
domains formed in this aggregate may be similar to that formed in
PMAA in acidic condition. This research reports a new fluorescent
dye that can be used for peptide imaging with potential
applications in the field of protein folding and cell imaging. This
level of self-assembly for 1 and PGA, coupled with control on
charge transport properties is unusual, and worthy of further study
to understand the detailed structural requirements for such a
system at a more refined level.
CD measurements coupled with NMR investigation of the process
of chain interaction [44]. Kallenbach in the latter study showed
that distinct amide (non-randomized amide proton bands) could
be distinguished for acidic solutions (8 mM residue concentration,
pH 4.0) for oligomers as small as pentamer [44]. FPLC chromatog-
raphy showed that the 5-mer oligomer consisted of aggregates of
about 10,000 MW (about 13 chains of
a-helix). The precise
structure of the aggregated helical form of the acid form of PGA
was not reported, but it was thought that amphipathic interactions
lead to coiled coils, probably assisted by inter-chain hydrogen
bonding of side chain ꢁꢁ CO
2
H groups [44].
An alternate antiparallel coiled-coil model of PGA has been
proposed according to CD data taken during the multiple-step
folding of PGA [45]. The 4.4 kDa PGA at pH 3.3 showed an
Acknowledgement
“
overshoot” with decreased helix content on equilibrium over
The authors would like to thank Prof. Guilford Jones of Boston
University for supplying the materials for triad preparation and
helpful suggestions.
time. A model with a single to multiple helixes equilibrium was
proposed, where the long peptides assumed many conformations
containing two or more helices, perhaps in helix–turn–helix or
antiparallel coiled-coil motifs. It should be noted that this model
concerns PGA at low concentrations where it is believed, but not
confirmed, that aggregation is not important.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
Our fluorescent microscope data are consistent with the
aggregate model supported by the light scattering and viscosity
the
online
version,
at
http://dx.doi.org/10.1016/j.
jphotochem.2016.11.016.

J. Hu et al. / Journal of Photochemistry and Photobiology A: Chemistry 335 (2017) 59–69
69
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