Spectral and electrochemical solvatochromic investigations of newly synthesized peptide-based chemosensor bearing azobenzene side chain bio photoswitch

Article abstract of DOI:10.1016/j.dyepig.2021.109348

In the present study, a novel analogue of azobenzene-containing hemorphin-4 has been synthesized and investigated for assessment of spectral, electrochemical, and biological effects. The synthesis was achieved by a modified solid-phase peptide synthesis (SPPS) by Fmoc-strategy. This compound represents a newly synthesized and unstudied peptide-based chemosensor bearing azobenzene side-chain with different spectral and electrochemical properties in the two trans-/cis-states depending on the solvent polarity. Their fluorescence intensity, as well as voltammetric behavior, was found to depend on both the polarity of the solvents and the type of isomers of the azopeptide compound. Both isomer forms have shown pH dependence, as the fluorescence peak and intensity are significantly distinguished in acidic and neutral medium. The electrochemical behavior before and after 90 min UV illumination was investigated using a cyclic voltammetric mode at three-electrode system with HMDE electrode as the working electrode. The novel biopeptide Azo-Tyr-Pro-Trp-Thr-NH₂ was explored in vivo for potential anticonvulsant activity by 6-Hz seizure test and maximal electroshock test (MES) in ICR mice. The cis-Az-H4 isomer showed stronger potency than the trans-Az-H4 against both the 6 Hz-induced psychomotor seizures and tonic-clonic seizures in the maximal electroshock test with 100% protection demonstrated at the lowest dose of 1 μg administered intracerebroventricularly in mice. The effect of 1 and 5 μg cis-Az-H4 and 5 μg trans-Az-H4 was comparable to the positive control valproate in the 6-Hz test. None of the tested isomers displayed neurotoxicity in the rotarod test. Our results suggest that modified biopeptide in the N-terminus of hemorphin-4 with azobenzene deserve further exploration as a promising candidate with both anticonvulsant activity and as a chemosensor for pH determination.

Full text of DOI:10.1016/j.dyepig.2021.109348

Dyes and Pigments 191 (2021) 109348
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Spectral and electrochemical solvatochromic investigations of newly
synthesized peptide-based chemosensor bearing azobenzene side chain
bio photoswitch
,
,
Petar Todorova *, Stela Georgievab, Petia Penevaa d, Jana Tchekalarovac
a Department of Organic Chemistry, University of Chemical Technology and Metallurgy, 1756, Sofia, Bulgaria
b Department of Analytical Chemistry, University of Chemical Technology and Metallurgy, 1756, Sofia, Bulgaria
c Institute of Neurobiology, Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria
d Institute of Mineralogy and Crystallography, Bulgarian Academy of Sciences, Sofia, 1113, Bulgaria
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, a novel analogue of azobenzene-containing hemorphin-4 has been synthesized and inves-
tigated for assessment of spectral, electrochemical, and biological effects. The synthesis was achieved by a
modified solid-phase peptide synthesis (SPPS) by Fmoc-strategy. This compound represents a newly synthesized
and unstudied peptide-based chemosensor bearing azobenzene side-chain with different spectral and electro-
chemical properties in the two trans-/cis-states depending on the solvent polarity. Their fluorescence intensity, as
well as voltammetric behavior, was found to depend on both the polarity of the solvents and the type of isomers
of the azopeptide compound. The influence of the metal ions such as Zn2+, Ni2+, and Cu2+ on the fluorescence
emission intensity of trans- forms in a water solution of the azopeptide has been also studied. Both isomer forms
have shown significant pH dependence, as the fluorescence peak and intensity are significantly distinguished in
acidic and neutral medium. The electrochemical behavior before and after 90 min UV illumination was inves-
tigated using a cyclic voltammetric mode at three-electrode system with HMDE electrode as the working
electrode.
4-Aminoazobenzene
Peptides
Bio photoswitches
Fluorescence spectroscopy
Anticonvulsant activity
Cyclic voltammetry
The novel biopeptide Azo-Tyr-Pro-Trp-Thr-NH2 was explored in vivo for potential anticonvulsant activity by 6-
Hz seizure test and maximal electroshock test (MES) in ICR mice. The cis-Az-H4 isomer showed stronger potency
than the trans-Az-H4 against both the 6 Hz-induced psychomotor seizures and tonic-clonic seizures in the
maximal electroshock test with 100% protection demonstrated at the lowest dose of 1
μ
g administered intra-
cerebroventricularly in mice. The effect of 1 and 5 μg cis-Az-H4 and 5 μg trans-Az-H4 was comparable to the
positive control valproate in the 6-Hz test. None of the tested isomers displayed neurotoxicity in the rotarod test.
Our results suggest that modified biopeptide in the N-terminus of hemorphin-4 with azobenzene deserve further
exploration as a promising candidate with both anticonvulsant activity and as a chemosensor for pH
determination.
1. Introduction
analogues are used as biosensors and other biomedical applications
[1–4].
In recent years, azo-containing peptides are increasingly used due to
their diverse physicochemical and biological properties. Often peptides
containing chromophore units can recognize and respond to external
stimuli. This exogenous influence is usually expressed, such as ligand
binding, change in pH or temperature, or photon absorption can
modulate the biomolecular structure to activate a biochemical signaling
cascade of reactions. Nowadays, azo-containing peptides and their
The photodynamic control of peptide biomolecules can provide an
easy and non-invasive way to study their effect or obtain a specific result
by modulating the activity of the molecule [5,6]. The insertion of azo-
benzenes into peptides to photomodulate their conformational states
was proposed firstly by Murray Goodman [7,8]. Azobenzene and its
derivatives are the most widely used small-molecule chromophores, that
possess interesting features [9]. Among them are photo-recording and
* Corresponding author.
E-mail address: pepi_37@abv.bg (P. Todorov).
https://doi.org/10.1016/j.dyepig.2021.109348
Received 12 January 2021; Received in revised form 26 March 2021; Accepted 26 March 2021
Available online 31 March 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
photo-switching materials [10]. The presence of photo-inducible isom-
erism can be exploited in changing lipid membranes or to modulate the
membrane interaction of peptides [10]. Also, they are applied for
self-assembly processes as they show large changes in the molecule
structure and properties in trans-cis photoisomerization [11–14].
Azobenzene-based biomolecular systems can also be easily manipulated
with acid-base stimuli and photons, which makes them the subject of
extensive research. The photoresponsive behavior is dependent on the
pH and conformation of the azobenzene units. The physicochemical
properties of the system are dependent on the mode of pH reduction and
the isomeric cis/trans composition of the targets [15]. Increasing
attention is being paid to water-soluble azobenzene derivatives syn-
thesized for photo-regulation of functions and properties of bio-
molecules [16,17]. Nevertheless, water-soluble azobenzenes have been
reported rarely and their properties, including the effect of pH and ad-
ditives, are not understood well [18–20]. Azobenzene-containing pep-
tides and photochromic compounds as a whole represent the basic
molecular triggers for very important photo-regulated biological pro-
cesses in living organisms [21,22]. The usage of azobenzene as a
conformational model of peptide switching to control the conforma-
tional preferences of protein fragments by including different types of
chromophores and various modes of their attachment have been
described by Christian Renner and Luis Moroder [23].
azo-peptide decrease the latency for onset of clonic seizures induced by
intravenous pentylenetetrazole infusion test [38]. It should also be
noted that so far, there are no data in the literature for the hemorphins
containing azobenzene residue. Our previous investigation on a variety
of newly synthesized hemorphin analogues that revealed promising
pharmacological effects in mice provoked us further to explore their
properties and future applications [39–43].
In this context, we have motivated our attention on the design and
synthesis of the novel analogue of azobenzene-containing hemorphin-4
investigating its photophysical and electrochemical behavior in the both
trans-/cis-states in different type of solvents as well as in vivo anticon-
vulsant activity of the two isomers. The structure-spectroscopic prop-
erties relationship has been also discussed.
2. Materials and methods
2.1. Synthesis of the peptide (Az-H4)
All reagents and solvents were analytical or HPLC grade and were
bought from Fluka or Merck, and used without further purification. The
protected amino acids and Fmoc (9-fluorenylmethoxycarbonyl)-Rink
Amide MBHA (4-methylbenzhydrylamine) Resin were purchased from
Iris Biotech (Germany). The 3-functional amino acids were embedded as
The active demand of fluorescent materials with excellent properties
and makings has led to the discovery of a newly phenomenon termed as
aggregation-induced emission effect. In other words, organic chromo-
phores emit more efficiently in the aggregate state than in solution [24].
Restriction of intramolecular rotation is the most commonly accepted
mechanism for explaining this behavior [25].
follows: Tyr – as N
α
-Fmoc-Tyr (tBu)-OH, Thr - as N
α-Fmoc-Thr (t-Bu)-
OH, and Trp – as N
α
-Fmoc-Trp (Boc)-OH.
The solid-phase peptide synthesis by Fmoc strategy was used to
obtain a new analogue of hemorphin-4 containing azobenzene moiety.
The Fmoc-Rink-Amide MBHA resin ((loading 0.71 mmol/g resin, cross
linking 1% DVB) was used as solid phase carrier to obtain the C-terminal
amide derivative and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyla-
minium tetrafluoroborate (TBTU) was used as a coupling reagent. The
coupling reactions were performed using for amino acid/TBTU/HOBt/
DIEA/resin a molar ratio of 3/2.9/3/6/1, in a 1:1 mixture of DMF/DCM.
A 20% piperidine solution in N,N-dimethylformamide (DMF) was used
to remove the Fmoc group at every step. After each reaction step, the
resin was washed with DMF (3 × 1 min), isopropyl alcohol (3 × 1 min)
and CH2Cl2 (3 × 1 min). The coupling and deprotection reactions were
checked by the Kaiser test [44,45]. The cleavage of the synthesized
peptide from the resin was done, using a mixture of 95% trifluoroacetic
acid (TFA), 2.5% triisopropylsilan (TIS) and 2.5% water. The peptide
was obtained as a filtrate in TFA and precipitated with cold, dry ether.
The precipitate was filtered, dissolved in water and lyophilized to yield
the compound as a powder. The crude peptide was dissolved in H2O and
acetonitrile was added until complete dissolving was observed. The
peptide was obtained as a white powder with a purity of >97% as
determined by analytical HPLC. The structure was confirmed by
high-resolution electrospray mass spectrometry and NMR spectroscopy.
The purity of the peptide was monitored on a reversed-phase high--
performance liquid chromatography (RP-HPLC), column: Symmetry-
Peptides with various optional building blocks, predictable confor-
mations and high stability, are versatile and can act as tailor-made tar-
geting probes for drug delivery [26]. Peptides with azobenzene moieties
are very valuable as chemosensors because of their higher biological
compatibility and solubility compared to organic dyes as well as stability
compared to proteins in aqueous solutions [27]. The pH plays significant
roles in biochemistry and medicine [28,29]. That’s why methods for
visualizing H+ ions would be powerful tools to examine the concentra-
tion of H+ ion signaling mechanisms in detail in different processes. For
example, a highly pH-sensitive colorimetric chemosensor having a sig-
nificant colour change would be very useful in the field of physiological
fluids [30]. The pH-responsive molecules are based on varied electronic
transfer effects turning in different tautomeric forms in a dependent
manner from pH [20]. Peptide-based chemosensors have their modular
nature, such as water solubility, biocompatibility, and low toxicity,
high-affinity, and specific interactions with a target receptor [31–33].
Moreover, the peptide backbones can be synthesized by changing the
amino acid sequences by the sophisticated solid-phase peptide synthesis
(SPPS) technique and conjugated with different chromophores,
including azobenzene derivatives [27].
Over the last decade, it has become clear that the degradation of
cytosolic proteins can generate peptides that have biological activity.
These include hemoglobin (Hb) peptides [34], which is a major
component of red blood cells. Naturally occurring oxidants, such as
hydrogen peroxide, modify Hb and generate denatured Hb, which is
degraded by proteasomes and oligopeptidase to produce
hemoglobin-active peptides. These are hemopressins and hemorphins,
which can target mood-related receptors, such as cannabinoid and
opioid receptors [35,36]. The first described hemoglobin-derived opioid
peptide was hemorphin-4 (Tyr-Pro-Trp-Thr, β-chain 35-38), which has
been isolated from bovine blood treated with a mixture of gastrointes-
tinal enzymes [37].
Shield™ RP-18, 3.5 μm, (50 × 4.6 mm), flow: 1 mL/min, H2O (0.1%
TFA)/CH3CN (0.1% TFA), gradient 0 → 100% (45 min) and 100% (5
min). The crude peptide was purified using semi-preparative HPLC,
column XBridge™ Prep C18 10 μm (10 × 250 mm), flow: 5 mL/min,
H2O (0.1% TFA)/CH3CN (0.1% TFA), gradient 20 → 100% (50 min).
The analytical data for the synthesized peptide prepared was as
follows: tR 35.47 min, >97% pure, HRMS (ESI) calculated for
C43H47N9O7, [MH+]: 801.8912; found: 802.3760.
The optical rotation in methanol (c = 0.01) at 20 ◦C was ꢀ 14◦.
1H NMR (600 MHz, DMSO‑d6), δ (ppm): 10.61 (s, 1H), 9.10 (s, 1H),
8.07 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 7.4 Hz, 1H), 7.57–7.49 (m, 2H),
7.46 (d, J = 8.9 Hz, 2H), 7.36–7.25 (m, 4H), 7.23–7.18 (m, 1H), 7.07
(dd, J = 10.8, 8.0 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.93 (s, 1H), 6.90 (s,
2H), 6.82–6.65 (m, 2H), 6.45–6.38 (m, 2H), 6.36–6.32 (m, 2H), 4.42 (td,
J = 8.9, 4.5 Hz, 1H), 4.34 (td, J = 7.6, 5.1 Hz, 1H), 4.17–4.11 (m, 1H),
3.92–3.78 (m, 2H), 3.56–3.45 (m, 2H), 3.34 (q, J = 8.5, 7.8 Hz, 2H),
2.96 (dd, J = 14.9, 5.1 Hz, 1H), 2.83 (dd, J = 14.9, 8.4 Hz, 1H),
Recently, a new analogue of hemorphin-5 containing azobenzene
moiety was synthesized and characterized for its photophysical and
electrochemical behavior by our group [38]. The results showed
structure-activity relation to its E→Z photophysical properties activated
by long-wavelength light at 365 nm. It was found that Z-isomer of
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P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
2.63–2.53 (m, 1H), 2.50 (s, 2H), 2.42 (dd, J = 14.0, 9.4 Hz, 1H), 2.27 (m,
2H), 1.54 (m, 2H), 1.04–0.98 (m, 2H), 0.76 (d, J = 6.5 Hz, 3H).
13C NMR (151 MHz, DMSO‑d6), δ (ppm): 172.51, 172.14, 171.77,
170.42, 169.42, 162.79, 156.32, 152.85, 152.18, 143.70, 136.49,
130.80, 130.06, 129.69, 128.05, 127.85, 125.35, 124.04, 122.27,
121.34, 118.80, 118.73, 115.39, 111.72, 110.37, 66.81, 59.99, 58.43,
54.33, 52.74, 47.25, 36.52, 36.26, 31.24, 27.42, 24.74, 20.43.
13C DEPT NMR (151 MHz, DMSO‑d6), δ (ppm): 130.80, 130.06,
129.69, 125.35, 124.03, 122.27, 121.33, 118.80, 118.73, 115.56,
115.38, 112.48, 111.72, 66.81, 59.98, 58.42, 54.33, 52.74, 47.25,
36.52, 36.26, 31.23, 27.42, 24.74, 20.43.
2.3. Electrochemistry
2.3.1. Aparatus
A voltamperometric analyzer (Metrohm-797VA) in connection with
VA-stand 797 (Metrohm) was used with the three-electrode system
consisting of HMDE as a working electrode (areas of 0.030 cm2), Ag|
AgCl|3 M KCl as a reference electrode, and carbon as an auxulary
electrode. Electrochemical data processing were performed by Origin-
Pro 8.0 software.
2.4. Procedures
Synthesis of the N-[4-(2-phenyldiazenyl)phenyl]glycine (3).
The compound (3) was synthesized from 4-aminoazobenzene (1) by
the adapted methodology according to the previously described method
for the synthesis of aminoazobenzene derivatives [14,46].
Measurements were carried out at room temperature (25 ◦C). Azo-
peptide before and after UV illumination at 365 nm was adsorbed on the
electrode from stirred solutions for a 30 s deposition time at ꢀ 1.2V. The
voltamperograms were recorded after 200s purging with argon from 0.3
to ꢀ 1.4 V at a 1.00 V/s scan rate and 0.010 V voltage step in cyclic
voltammetric mode. For an investigation of the ratio of scan rate vs.
current intensity, a concentration from 6.68 × 10ꢀ 5 mol Lꢀ 1 of Az-H4
was used. An aliquot of stock solution of Azo-peptide before and after
90 min UV illumination at 365 nm was placed in a 10 mL-electrode cell
containing 7 mL electrolyte and the signals were recorded.
2.2. Physicochemical characterization
2.2.1. Spectral measurements
A double-beam UV–Vis “Varian-Cary” with 1 cm path length syn-
thetic quartz glass cells spectrophotometer was used for absorption
spectra recording. The fluorescence spectra were recorded on a Perki-
nElmer LS55 spectrophotometer at the same concentrations. The sol-
vents (spectroscopic grade) used in this study were: dimethyl sulfoxide
(DMSO), acetonitrile (AcCN) and phosphatic buffer solution (pH 6.86).
The concentrations of the compounds in the spectral solutions are as
follows: Az-H4: C = 1.04 × 10ꢀ 5 mol Lꢀ 1 (concentration of stock solu-
tion of Az-H4: C = 4.68 × 10ꢀ 3 mol Lꢀ 1 in DMSO); N-[4-(2-phenyl-
diazenyl)phenyl]glycine (Az): C = 1.754 × 10ꢀ 5 mol Lꢀ 1. The effect of
the metal cations as well pH of the aqua solution upon the fluorescence
intensity of both cis(Z)- and trans(E)- isomers was also examined by
preparation of solution with an equal concentration of azo peptide
compound (C = 1.04 × 10ꢀ 5 mol Lꢀ 1) in the presence of certain amounts
of metal solutions as follows: 50.0 µL of stock solution of the metal
cations (concentration of stock solution of: Cu2+ (C = 1.14 × 10-2 mol L-
1); 100.0 µL Zn2+ (C = 1.23 × 10-2 mol L-1); 50.0 µL Ni2+ (C = 1.14 × 10-
2 mol L-1) and HCl, respectively. All used reagent were analytical grade.
The spectrum was recorded in potassium bromide (KBr) pellet with a
Varian 660 FTIR spectrophotometer the spectra in the range 4400–600
cmꢀ 1 using a Fourier Transform Infrared Spectroscopy (FT-IR). The
sample was scanned 256 times with a resolution of 2 cmꢀ 1 and signal-
averaged for each sample in the range of 400–4000 cmꢀ 1. The first
and second derivatives of the absorption spectrum were performed by
Savitsky-Golay with polynomials derivation of convolution coefficients
2 and smoothing factor 40.
2.5. Biology. In vivo experiments
2.5.1. Animals and treatment
ICR male adult mice (25–30 g), delivered from the animal facility of
the Institute of Neurobiology, Bulgarian Academy of Sciences, were
accommodated under standard conditions as follows: room temperature
21 ± 2 ◦C, relative humidity, light/dark (12/12) regime, plexiglass cages
in groups of 3–4 with water and food ad libitum. The peptide analogues
were suspended in saline. All procedures were performed in agreement
with the European Communities Council Directive 2010/63/EU.
2.6. Drugs and dosage
The mice were infused intracerebroventricularly (i.c.v.) (10
ventricle) at doses of 1, 2.5 and 5 g/mouse by means of 28-gauge
stainless steel needle attached to a 10- L Hamilton® syringe as
μL/
μ
μ
described previously [42]. The injection needle was left in place for 2
min to avoid back diffusion of the solution. Ten min following the in-
jection procedure, the test for anticonvulsant activity or neurotoxicity
was evaluated.
The NMR spectra were recorded on a Bruker Avance II + spec-
2.7. Anticonvulsant activity
trometer operating with frequency 600 MHz for 1H and 125 MHz for 13
C
in DMSO‑d6. Chemical shifts δ are reported in ppm, and coupling con-
stants J are reported in Hz.
2.7.1. Maximal electroshock test (MES test)
MES test was performed as described previously [42]. Tonic-clonic
seizures in controls were produced by corneal electrical stimulation
(50 mA, 60 Hz, 0.2 s) via Constant Current Shock Generator. The lack of
hind limb tonic extension of the tested mouse was accepted as a criterion
for anticonvulsant activity of the tested compound. Each mouse was
used only once.
The molecular mass and purity of the compound was confirmed by
high-resolution electrospray mass spectrometry on a Q Exactive high-
resolution mass spectrometer (Thermo Fisher Scientific Inc., USA)
equipped with TurboFlow TM Transcend chromatography system
(Thermo Fisher Scientific Inc., USA) and heated electrospray ionization
(HESI II) source. Data acquisition and processing were done by XCali-
bur® 2.4 software (Thermo Fisher Scientific Inc., USA). The instru-
mental parameters were as follows: Spray Voltage – 4.0 KV, Sheath Gas –
30 AU, Auxiliary Gas – 12 AU, Capillary Temperature – 300 ◦C, Spare
Gas – 3 AU, Heater Temperature – 300 ◦C. Full scan experiments were
carried out in a range of 120–2000 m/z at 140 000 resolution.
Optical rotations were recorded on an MCP200 modular circular
polarimeter (Anton Paar Opto Tec GmbH, Seelze, Germany).
2.8. 6-Hz psychomotor seizure test
The corneal stimulation with 32 mA, 6 Hz for 3 s was applied as
described earlier [42]. The electric stimulus of 32 mA, 6 Hz for 3s was
conducted via corneal electrodes for evaluating the efficacy of the
compounds against psychomotor seizures. The responses in control mice
are characterized by a minimal clonic seizures, stereotyped behaviors,
twitching of the vibrissae or Straub-tail. The criterion for seizure sup-
pression is accepted when the tested mouse resumes normal position 10
s after stimulation.
3

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
Fig. 1. Schematic representation of photoswitchable Azo-Tyr-Pro-Trp-Thr-NH2 (Az-H4) peptide upon long wavelength UV light as trans (E) and cis (Z) isomers. The
azobenzene photoswitch moiety is highlighted as red/blue.
Scheme 1. Synthetic pathway of the azobenzene-containing hemorphin-4 (Az-H4).
4

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
Fig. 2. Zero order (A) and first and second derivative (B) IR spectra of the azopeptide Az-H4 compound; characteristic regions for peptide moiety: Amid A: 4000-
2600 cmꢀ 1, Amid I: 1600-1700 cmꢀ 1, Amid II: 1528-1577 cmꢀ 1 and Amid III at 1221-1331 cmꢀ 1 [55].
2.9. Rotarod test
absolute ethanol (Scheme 1) to give the ethyl (E)-(4-(phenyldiazenyl)
phenyl)glycinate (2). After that, the alkaline hydrolysis of 2 with NaOH
followed by acidification with HCl yielded compound 3 (Scheme 1). The
azo-peptide (Az-H4) was prepared by solid-phase peptide synthesis
(SPPS)-Fmoc (9-fluorenylmethoxy-carbonyl) chemistry using TBTU
(2-(1H-benzo-triazole-1-yl)-1,1,3,3-tetramethyluronium
The neurotoxicity was assessed as described previously in a rotarod
apparatus for mice [42]. The tested mouse was placed on a rotating rod
(3.2 cm in diameter, at a speed 10 rpm). The compound that causes a loss
of coordination of the tested mouse and inability to keep a position on
the rod for at least 1 min from the three possible sessions trials was
accepted as neurotoxic.
tetrafluoro-borate), an efficient peptide coupling reagent (Scheme 1).
The SPPS, based on the reaction of N-[4-(2-phenyldiazenyl)phenyl]
glycine (3) with N-terminal amino group of Tyr-Pro-Trp-Thr tetrapep-
tide directly on the resin. After cleavage of the product from Rink-Amide
MBHA Resin by TFA to give the Az-H4, the azo-peptide was purified
from crude product by semi-preparative HPLC with a C18 column. The
molecular weight was determined, using HRMS (ESI). The structure of
the obtained compound was checked by UV–Vis, IR, NMR (1H, 13C,
DEPT) spectra. The analytical data are presented in the Material and
Methods Section.
2.10. Statistical analysis
Fisher’s exact was used for the analysis of data from MES and 6-Hz
test. Statistical significance was accepted at p < 0.05. The calculations
were carried out with Graph Pad Prizm Version 7.04 (GraphPad Soft-
ware, San Diego, CA, USA.) and Sigmastat (version SigmaStat® 11.0) for
Windows.
3. Results and discussion
3.2. Spectral characterization
3.1. Chemistry
3.2.1. IR spectroscopy
The determination of the type of the secondary peptide structure is
essential for the study of some physicochemical characterizations of the
molecule such as the solubility of the molecule, its passage through
biological membranes as well acid/base (pKa) constant determination
and etc. To check whether the azo component changes the type of
structure of the peptide bone and vice versa whether the peptide influ-
ence the maxima and intensity of the azo component, IR spectra in the
range 4000–350 cmꢀ 1 were recorded. The frequency maxima of the azo
group range from 1500 to 1630 depending on the type of the azo
compound and the functional groups and its participation in different
structural conjugations [52]. In the case of asymmetric azo derivatives,
In view of the above mentioned the main goal in this research is to
synthesis and investigate the spectral, electrochemical and biological
effects of new biopeptide bearing azobenzene to the N-side chain of
hemorphin-4 obtaining azo-peptide Az-H4 (Fig. 1).
For the preparation of Az-H4 firstly it needs to be synthesized N-[4-
(2-phenyldiazenyl)phenyl]glycine (3). Unfortunately, only a few pro-
tocols for obtaining N-[4-(2-phenyldiazenyl)phenyl]glycine have been
reported [14,46]. The compound (3) incorporated in biomolecular sys-
tems are capable of recognizing and responding to external stimuli such
as ligand binding, a change in pH or temperature, or absorbance of a
photon etc. [3]. As a potential fragment for pharmaceutical and many
other research and development applications, finding a general and
improved method to synthesize N-[4-(2-phenyldiazenyl)phenyl]glycine
analogues is essential [2,3,47–49]. The starting compound (3) used to
obtain the desired compound Az-H4 was synthesized from 4-aminoazo-
benzene (1) under the influence of ethyl bromoacetate in the medium of
–
–
the vibration of the –N N- bond in some azo compounds between 1630
and 1575 cmꢀ 1 could not be observed [53,54]. In the mentioned above
one can find the spectral characterization regions for peptide structure
indicated as follows: Amide I, II, and III (Fig. 2). In Fig. 2 are also given
the zero and derivatives (1st and 2nd) spectra of the Az-H4 compound
for more clearly distinguish the absorption maxima. Our data show a
5

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
chain also suppose the possibility of a beta-structure existing and
breaking down of an alfa-helix conformation.
Table 1
Basic photophysical characteristics of the isomers of a compound Az-H4 and Az-
H4(IRr) in solutions with different polarity.
3.2.2. Photophysical properties
Compound/solutions
DMSO
AcCN
H2O
0.1 M HCl
The main absorption and emission characteristics of the synthesized
compound before and after 90 min UV illumination at 365 nm were
studied in solutions with different polarity, increasing in the order:
DMSO < AcCN < H2O and are presented in Table 1. The spectrum in the
visible region were compared with the spectrum of starting azo com-
pound (Figs. 3 and 4).
Az-H4
λ abs., (nm)
410
468
2.01
58
395
457
4.21
62
391
436
3.12
45
513
555
3.27
42
λ emi., (nm)
ε
.104 (L molꢀ 1cmꢀ 1
)
)
Stokes offset (nm)
λ abs., (nm)
Az-H4(IRr)
410
470
2.13
60
395
456
4.56
61
391
437
3.07
46
508
554
3.16
46
λ emi., (nm)
ε
.104 (L molꢀ 1cmꢀ 1
The absorptions spectra are presented in Fig. 3 by means of zero-
order and derivative spectra. Differentiation of the absorption signals
was necessary in order to enhanced resolution and more readily
appreciated more complex spectra. The UV–Vis spectra of Az-H4 and
free from peptide azo component give clearly shaped different intensity
absorbance bands localized at ≈400 nm which could be attributed to
Stokes offset (nm)
broad absorption maximum in the high-frequency region (3600–2860
cmꢀ 1) corresponding to valence vibrations at NH+3 group (νas+
)
NH3
(Fig. 2). The frequency maxima localized at 3302 cmꢀ 1 could be
attributed to N–H stretching vibration (νNH) (Amid A) [43]. Between the
wavenumber values from 1610 to 1663 cmꢀ 1, a high-intensity spectral
–
–N N- group with n→π* energy transition. As the polarity of the solvent
–
increases, a batochromic shift of the signal in the UV spectrum is
observed, showing positive solvatochromism. Compared to the starting
azo compound, it can be seen that the solvatochromic properties of the
hybrid peptide are mainly due to the azo component, as the colour
change of the solution with increasing solvent polarity does not differ
from that reported in the azo compound literature [56,57]. The binding
with peptide moiety increases the absorbance of Az-H4 in all investi-
gated mediums, the most strong is in acetonitrile (Fig. 3). Basically the
band split in two is observed. The peak at 1663 cmꢀ 1 is due to vibration
of
ν
(Amide I) [43] and that at 1610 cmꢀ 1 generally could be
O
–
–
C
–
–
assigned to vibration of –N N- group. The δ vibration of Amide II
NH
band is appeared at about 1530 cmꢀ 1 and fully corresponding to re-
ported in the literature high absorbance maximum of amide spectrum
which proposes β-turn or β-sheet secondary structure of the peptides
[55]. In addition, the characteristic bands of the Amide III region are
clearly visible on the IR spectrum localized at 1236 cmꢀ 1 and 1176 cmꢀ 1
(Fig. 2). The short peptide backbone as well appearing of amino acids
such as Tyr and Trp in the structure of investigated peptides prevent the
helical folding of the peptide and hence the existence of an alpha helix
[55]. Substitution of Pro and presence of azo component on the peptide
–
π→π* transitions of the >C O in the peptide bonds is occurs by UV
–
absorption of the peptide molecule in the range 180–230 nm. Both Trp
and Tyr are primarily responsible for the inherent absorption of the
peptides at higher wavelengths: absorption at 285 nm is dominated by
the π-electron systems of aromatic side-chains of indol of Trp and phenol
Fig. 3. UV-VIS spectra of the Azo-Tyr-Pro-Trp-Thr-NH2 (Az-H4) and N-[4-(2-phenyldiazenyl)phenyl]glycine (Az) in different solvents ((left) and first derivatives
spectrum before and after 90 min UV-illumination with 365 nm (right)).
Fig. 4. Ammonium-azonium tautomerism of the Az-H4 compound in the presence of protonic solvent (0.1 M HCl) [52].
6

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
Fig. 5. Emisson/fluorescence spectra of N-[4-(2-phenyldiazenyl)phenyl]glycine (Az) and Azo-Tyr-Pro-Trp-Thr-NH2 (Az-H4) and after 90 min UV illumination at λ =
365 nm (Ar-H4(IRr) in different solvents; concentration of all analyte solutions: 5.20 × 10ꢀ 5 mol L1.
ring of Tyr [58,59]. The more evident signals, especially in the range of
270–300 nm, could be observed on the derivatives graphics. One can see
that the derivative spectra indicate three bands related to the phenylazo
part, Tyr and Trp. After UV illumination with 365 nm for 90 min, the Tyr
and 508 nm for cis (Z)- form, respectively, and changed the colour of the
solution which indicates the conversion of azo group into a protonic
form and suppose better solvation of each form of the compound in
water solutions [52]. The influence of proton-saturated medium and the
possibility for the intramolecular migration of the proton from one
element to another gives resonance-stable ammonium-azonium
tautomerism of the azopeptide structure and following different colour
of the solution (Fig. 4).
–
and Trp as well for π→π* of –N N- absorption increased due to basically
–
of the conformation changing.
In the presence of hydrochloric acid, the absorption spectrum is
changed (Fig. 3). The addition of hydrochloric acid shifts the maximum
at ≈400 nm of both isomers to longer wavelengths - 513 nm for trans (E)-
The dyes exhibit complex spectral behavior, which strongly depends
Fig. 6. Cyclic voltammograms of Az-H4 and Az-H4
(IRR) in different electrolyte media, scan rate and
concentration using HMDE and Ag/AgCl, KCl as
reference electrode: A) CVs of Az-H4 and Az-H4
(IRr) (in colour) at different concentrations: from
1.67 × 10ꢀ 5 to 6.681 × 10ꢀ 5 mol Lꢀ 1 in pH 6.86
(phosphate buffer solution, 0.1 mol Lꢀ 1), scan rate
1.00 V sꢀ 1; B) CVs of Az-H4 and Az-H4(IRR) (in
colour) at different concentrations: 1.67 × 10ꢀ 5 to
6.681 × 10ꢀ 5 mol Lꢀ 1 in AcCN; C) Cyclic voltam-
mograms of 6.681 × 10ꢀ 5 Az-H4 before and after
(in colour) UV illumination in pH 6.86 (phosphate
buffer solution, 0.1 mol Lꢀ 1) at different scan rate
from 0.2 to 2.0 V sꢀ 1; D) Cyclic voltammograms of
6.681 × 10ꢀ 5 Az-H4 before and after (in colour)
illumination in AcCN at different scan rate from 0.2
to 2.0 V sꢀ 1
.
7

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
with protons such as HCl-solution and are respectively at 395 nm in
H2O, 391 nm in AcCN, 410 nm in DMSO. A bathochromic shift of the
maximum in the order AcCN, H2O < DMSO < HCl as well a hypochromic
effect in the organic solvents when the compound is irradiate are
observed which could be attributed to the relation of intramolecular
interactions and conformational states changed of the compounds. The
type of absorption and emission maxima is also influenced by the pep-
tide sequence associated with the azo component. This statement is
confirmed by the fact that as a result of intramolecular changes of the
compound due to the presence of photosensitive groups of amino acid
fragments displace and mainly increase the absorption and emission
intensity of the azo compound. It is known that the amino acid residues:
Tyr, Phe, and Trp the peptides possess a fluorescence in UV range [38]
and the energy absorbed by Phe and Tyr is often transferred to the Trp,
because of their spectral properties, resonance energy occurs from Phe
to Tyr to Trp [38]. Typically for the investigated compound containing
amino acid sequence -Trp-Pro-Tyr- is that this structural unit suggests a
trans configuration of Tyr and Trp residues around Pro. The excitatio-
n/emission spectra of azopeptide compound with similar peptide bone
has been studied in our previous work where was proved that the indole
from Trp is highly sensitive to polarity, local environment and confor-
mational changes [38]. Moreover, the energy transfer from Tyr to Trp is
very sensitive to the conformational changes because it is realized close
to the trans configuration.
Table 2
Voltamperometric characteristic of Az-H4 before and after (Az-H4 (IRr)) UV
irradiated with λ = 365 nm at concentration 6.681 × 10ꢀ 5 mol Lꢀ 1 in different
electrolyte mеdia at HMDE electrode; heterogen rate constant and diffusion
coefficient of Az-H4 and Az-H4(IRr) at HMDE electrode (with electrode area, A
= 0.030 cm2).
Compound/
-EPc
,
EPa, V
Epc-
Epc/
2
-Ipc
,
Ipa, A
Nature of
ko
sh
electrolyte
V
A
the
×
reduction
process
10ꢀ 4
cm
mV
sꢀ 1
Az-H4/Ph.
buffer
0.547
0.458
18
20
1.02
×
2.16
×
Ra
R
0.692
(pH 6.86)
Az-H4(IRr)/
Ph.buffer
(pH 6.86)
10ꢀ 7
2.73
×
10ꢀ 8
2.07
×
0.573
0.801
0.464
2.01
–
10ꢀ 7
7.16
×
10ꢀ 7
–
10ꢀ 8
3.67
×
Az-H4/
AcCN
0.603
0.603
0.355
0.345
28
25
1.66
×
R
R
1.97
2.14
10ꢀ 7
3.52
×
10ꢀ 7
1.76
×
Az-H4(IRr)/
AcCN
10ꢀ 7
10ꢀ 7
a
–
R-reversible; n-number of electron exchanged of –N N-.
–
The differences between the structure and functional properties of
the azopeptide compound in its exited (S1) and ground (S0) states before
and after UV illumination at 365 nm were assessed by the value of the
Stokes shift in nm. The calculated values of the Stokes offset are also
given in Table 1. As the polarity of the solvents increases, the influence
on the properties of the solvent. Fluorescence spectroscopy was used to
study the solvatochromic behavior of the azopeptide. The spectrum
before and after 90 min UV illumination at 365 nm of Az-H4 in four
solvents with different polarity: DMSO; H2O, HCl and AcCN are given on
Fig. 5. In both cis (Z)- and trans (E)- isomerization the compound has an
intense colour which is changed depending on the polarity of the solvent
from yellow/orange (DMSO, AcCN, H2O) to red (HCl). Their absorption
maxima basically depend on the type of protonic solvent due to the
ongoing tautomerism affecting the azo group in a solution saturated
of the tautomerization in Az-H4 compound shows lower energy of λ
max
and a high molar extinction coefficient, which is an indication of the
course of intramolecular charge transfer during the transition to the
excited state.
From a biological point of view, the interaction of metals with Az-H4
in a medium close to the physiological is important to be studied in order
Fig. 7. (A) Plot of Ipc vs. ˅ (V sꢀ 1) at 6.681 × 10ꢀ 5 mol Lꢀ 1 Az-H4 and (B) Plot of log Ipc vs. C (mol Lꢀ 1) for azo peptide compound before and after 90 min UV
illumination at λ = 365 nm and different solvents: phosphate buffer solution (pH 6.86) and AcCN at HMDE electrode.
8

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
to investigate their influence on the physicochemical properties of the
compound in case of their presence in biological fluids. Therefore, a
qualitative assessment of the effect of metal ions such as Cu2+, Ni2+ and
Zn2+ on the type of fluorescence spectrum was performed. As can be
seen from Fig. 6, the fluorescence spectrum of the solution in the pres-
ence of copper ions is not significantly affected by the presence of the
metal ion. A slight hyperchromic effect is only observed which metal
was added (Fig. 6). In opposite to the mentioned above addition of Ni
and Zn ions, the fluorescent bands of Az-H4 are very low and shifted to
the red region. This leads to the conclusion that these ions affect the
spectral properties of the compound by quenching their fluorescence.
The change in the intensity of the analytical signals can be related to the
performance of complex-forming processes in the solution and deserves
in-depth future research. The photochromic behavior of the Az-H4
shows promising results for a future application for example as a che-
mosensor in photodynamic therapy.
Table 3
The equations of regresion of the function of IPC,A = f (
ν
1/2), mV sꢀ 1) of Az-H4
before and after (Az-H4 (IRr)) UV irradiated with λ = 365 nm at concentration
6.681 × 10ꢀ 5 mol Lꢀ 1 in different electrolyte mеdia and values of diffusion
coefficient; HMDE electrode (with electrode area, A = 0.030 cm2).
Compound/electrolyte
Equation of regresion
R2
D × 10ꢀ 3
cm2 sꢀ 1
Az-H4/Ph.buffer (pH
6.86)
IPC = -8.06.10ꢀ 8 +3.61.10ꢀ 7
×
×
0.991
0.989
0.990
0.995
0.805
1.36
1.36
1.47
(
ν1/2
)
Az-H4(IRr)/Ph.buffer
(pH 6.86)
IPC = -4.19.10ꢀ 7 +1.01.10ꢀ 6
(
ν1/2
)
Az-H4/AcCN
IPC = -1.65.10ꢀ 7+1.02.10ꢀ 6
×
(
ν1/2
)
Az-H4(IRr)/AcCN
IPC = -2.48.10ꢀ 7 +1.17.10ꢀ 6
×
(
ν1/2
)
R-coefficient of regression; n-number of electron number of electrons transferred
in the redox event.
In the next section, we show some structure-active characterization
and in vivo biological experiments related to the trans (E) and cis (Z)
isomers.
ΔEp = Epc-Epc/2 [38] as well the ratio of peak heights: Ipc/Ipa - greater
than one which means that the reduction could be attributed to
reversible; the current increased with the increase in scan rate from 0.2
to 2.00 Vs-1 (Fig. 7) with the cathodic peak potential shifting to the more
negative potentials. The same can be concluded for the behavior of the
electrochemical reduction of the azo group in acetonitrile with small
differences (Fig. 6-B and Fig. 6-D): ΔEp = Epc-Epc/2 = ≈ 25 mV; Ipc/Ipa
greater than one; reversible electrodic process; the current increased
with the increase in scan rate from 0.2 to 2.00 Vs-1 without shifting. That
is meaning the rate of electron transfer between the working electrode
and the solution redox species is lower than the rate of transfer of the
azo-peptide to the electrode space. Different electrochemical behavior of
the azo compound was observed after trans (E)→cis (Z) transformation
of azo-peptide molecule. The cyclic voltammograms of the trans(E)- and
cis(Z)- isomers of Az-H4 on HMDE at different concentration and elec-
trolyte media are shown in Fig. 6-A and B. As a result of the performed
isomerization and the resulting spatial disturbances, the second peak in
the phosphate buffer medium refers to the reduction of the peptide part
disappears as the intensity of the current peak increases, which increases
the sensitivity of the determination (Fig. 6-A). In an acetonitrile me-
dium, a smaller increase in the signal intensity of the cis (Z)- form was
observed compared to the current height in the proton medium without
changing the condition for the signal appearing (at ≈0.550 V). Thus, as a
result of the obtained data can be concluded that both the type of solvent
(electrolyte medium) and the isomeric form of the Az-H4 affect its
electrochemical properties as follows: from completely inactive in the
aprotic DMSO solvent to the appearance of a well-formed azo compo-
nent peak in acetonitrile and a highly intense current signal in phos-
phate buffer with the most pronounced properties in Az-H4(IRr).
Voltammograms shown in Fig. 6-A and Fig. 6-B clearly depict that Ipc
vary linearly with the escalation in concentration at Az-H4 and Az-H4
(IRr) which indicates that radical anion reduction product of the
investigated compound, is adsorbed on the mercury electrode. A
adsorbtion controlled process of the Az-H4(IRr) compound in phos-
phatic buffer solution is proved from potential shifting toward more
negative values with increasing of the azopeptide concentration. On
Fig. 6-B is also showed a proportional increase in the signal with
increasing peptide concentration in AcCN solution without being fol-
lowed by a shift in the potential peak. The regression equations plot of
3.3. Electrochemistry
As for the study of the potential biological action of a compound, it is
important to study electron and proton transfer processes to get a clearer
idea of their structural activity. Proton-coupled electron transfer (PCET)
reactions and the transport of protons are involved in a variety of
biochemical processes, for example, in the respiratory chain, photo-
synthesis and protein functioning [60]. Therefore the electrochemical
parameters as diffusion coefficient and heterogenius rate constant of
Az-H4 and the same compound after 90 min UV illumination at 365 nm
were calculated applying cyclic voltamperometry at HMDE (handing
mercury drop electrode) in solutions with different dielectric constants:
phosphate buffer solution (pH 6.86); DMSO and AcCN. The all voltam-
perograms were recordered in a potential windows from 0.2 to – 1.4 V
(Fig. 6). The voltammetric characterizations are given in Table 2. The
blank voltammogram of the solvent in the potential window of HMDE
strengthened the belief that the oxidation signals are of compounds:
Az-H4 and Az-H4(IRr) only. In the literature, information on the elec-
trochemical behavior of azopeptide compounds and their conforma-
tional states is scarce. In our previous work were discussed the
electrochemical properties of azobenzene-containing VV-hemorphin-5
analogue and confirmed the fact that the E→Z transformation changes
the electrochemical behavior of the compound by altering the reactivity
of the redox-active azo group [38]. The azo center in cis (Z) -isomeric
form surrounded by symmetrically arranged peptide fragments was
spatially inhibited, which is why, together with the bulky molecule it
was a prerequisite for poorer diffusion through the electrode space. To
evaluate the transfer of particles to and from the electrode space of the
–
azo “-N N-“ component in a smaller and mobile azopeptide molecule,
–
the polarograms were recordered in three electrolyte media at a mercury
working electrode. In an approtonic polaric solvent such as DMSO and
analyte concentration from 1.00 to 7.00 × 10ꢀ 5 mol Lꢀ 1 no electro-
chemical electron transfer to the electrode space was observed and the
voltammogram followed the type of the electrolyte signal. Even the
addition of tetra butylammonium persulfate as an electrolyte salt in the
organic solution does not provoke electronic exchange. The presence of
cathodic and anodic signals of the compound was found to be clearly
more favorable for electron exchange in the electrolytes: phosphate
buffer solution (pH 6.86) and acetonitrile. One can see that in the both
phosphatic buffer and AcCN solution a well formed cathodic peak
localized at lower potential is observed which can be attributed to the
I
pc, A vs C, mol Lꢀ 1 are given in Figs. 7 and 8. From the slope of
regression equations obtained using the formula Ipc = nFACoksh (Rein-
muth expression) the heterogeneous electron transfer rate constants
were calculated (Table 2) and the obtained values predict the reduction
process of the azo group to be quasi-reversible [38]. The number of
electron transfer in electrochemical reaction for the reversible systems
Az-H4 and Az-H4(IRr) is equal of 1 and was calculated from the equa-
tion: ΔEp (Epc-Epa = 59/n) mV.
–
reduction of the –N N- group to –NH–NH– (Fig. 6-A, C). A second one
–
peak is appeared at potential ≈ -0.800 V in the protonic solution which
could be related to the peptide fragment (Fig. 6-A) [38,43]. The
reversibility of the first peak in pH 6.86 can be assumed because one can
see a difference from ≈18 mV provoked from peak potential difference:
Scan rate effect was also evaluated at scan rate from 0.2 to 2.00 V sꢀ 1
9

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
600 n3/2AD1/2 Cv1/2 validate at 25 ◦C (Randles-Secik [38]), Ip = current
maximum in Amps; n: number of electrons transferred in the redox
event; A-electrode area in cm2; F: Faraday Constant,C molꢀ 1; C = con-
centration in mol/ml;
ν
= scan rate in Vsꢀ 1; R: Gas constant, Kꢀ 1 molꢀ 1
and temperature T in K. From the obtained values of (D) coefficients of
both Az-H4 and Az-H4(IRr) we can conclude that the change in the
isomeric states does not affect the value of the diffusion coefficient, ie
the movement/diffusion of the molecule from the inside of the solution
to the surface of the electrode space does not depend on its spatial
structure change.
In the next section we show the some in vivo biological experiments
related to the trans (E) and cis (Z) isоmers. The electrochemical tech-
nique allows being used to control of depletion/action of the pharma-
cophore in biological tissue.
3.4. Pharmacology
Screening for anticonvulsant activity and neurotoxicity of novel
analogue of azobenzene-containing hemorphin-4.
The anticonvulsant activity of the novel analogue of azobenzene-
containing hemorphin-4 was evaluated according to the guidelines of
the Antiepileptic Drug Development Program (ADD) of the National
Institutes of Health (USA) [50]. The two tests, 6-Hz and MES were
applied for preliminary screening of anticonvulsant activity in parallel
with neurotoxicity assessment through the test for minimal motor
impairment (rotarod). Valproate (VPA) was used as a positive control in
the 6-Hz test and Phenytoin in the MES test. The results are shown in
Fig. 8 (6-Hz test) and Table 4 (MES test).
The drugs with anticonvulsant activity in the 6-Hz test are suggested
as being effective against drug-resistant epilepsy and potency to modify
sodium channels [51]. The cis-Az-H4 isomer exhibited higher activity
than the trans-Az-H4 against the psychomotor seizures. It demonstrated
100% protection at the lowest dose of 1 μg/mouse used which effect was
comparable to the positive control VPA (Fisher exact test: P < 0.01 vs
control) (Fig. 8). Also, at the highest dose of 5 μg, both the cis-Az-H4 and
trans-Az-H4 showed 100% protection against the 6-Hz psychomotor
seizures (Fisher exact test: P < 0.01 vs control).
Fig. 8. Comparative analysis of the anticonvulsant activities of cis/trans-Az-H4
analogues in the 6-Hz test in mice. Valproate (VPA) is used as a positive control.
Fisher exact test: **P < 0.01 compared to controls.
Like in the 6-Hz test, the cis-Az-H4 exhibited potency to suppress
tonic-clonic seizures in the MES test with 83% protection vs 100 pro-
tection for the positive control Phenytoin (Table 4). Further, higher
doses of this compound showed the decreasing ability of the peptide
analogue to prevent the seizure spread.
Table 4
Anticonvulsant activity of trans Az-H4 and cis Az-H4 in MES test in mice.
Drug
Dose (trans/cis Az-
No. of animals
protected ⁄No. of
animals tested
%
%
4. Conclusion
H4 in
μ
g/mouse)
Protection
Mortality
(Phenytoin in mg/
kg)
In summary, the influence of cis(Z)- and trans(E)- isomers of newly
synthesized biopeptide bearing azobenzene to the N-side chain of
hemorphin-4 from the absorption, fluorescence, and electrochemical
energy have been investigated in different environments. Generally, the
properties of the photoswitchable Az-H4 upon long-wavelength UV light
as trans (E) and cis (Z) isomers depends on the type of solvent and is
observed as a decrease in the intensity of the spectral lines - absorption
and emission and an increase in voltammetric current intensity which
confirms the performance of intramolecular energy transitions mainly
influenced by the transformation of trans into cis isomerization. The
biological properties of the two isomers were also studied. The photo-
physical properties showed structure-activity relation of E→Z activated
by long-wavelength light at 365 nm. The Z-isomer of the azopeptide has
pronounced anticonvulsant activity in the 6 Hz test for psychomotor
seizures where the compound showed potency in the first tested dose
with 83% protection vs 100 protection for the positive control
Phenytoin.
Control
trans Az-
H4
0
1
0/8
2/6
0
67
0
33
2.5
5
5/6
4/6
5/6
4/6
2/6
6/6
83
67
83
67
33
100
16
0
cis Az-H4
Phenytoin
1
16
0
2.5
5
50
0
30
In the rotarod test, the novel analogue of azobenzene-containing hemorphin-4
did not display neurotoxicity at the maximum dose of 5 g/mouse.
μ
(Fig.6-C, Fig. 6-D, Fig. 7). The plot of peak current versus scan rate gave
a straight lines for two isomers of Az-H4, which is expressed for an ideal
reaction of diffusion controlled electrode process for the azo-peptide
compound (Fig.7).
The diffusion coefficients (D, cm2 sꢀ 1) if we assumed that the systems
are reversible are listed in Table 3 and were determined by the equations
Ethical statement
of regresion of the function of IPC,A = f (
ν
1/2), mV sꢀ 1) using: Ip = 268,
All procedures were performed in agreement with the European
10

P. Todorov et al.
Dyes and Pigments 191 (2021) 109348
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was approved by the Institutional Ethics Committee. There are no
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The authors declare that they have no known competing financial
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