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Here we report on the synthesis and characterization of three new N-modified analogues of hemorphin-4 with rhodamine B. Modified with chloroacetyl, chloride cotton fabric has been dyed and color coordinates of the obtained textile materials were determined. Antiviral and virucidal activities of both the peptide-rhodamine B compounds and the dyed textile material were studied. Basic physicochemical properties (acid-base behavior, solvent influence, kinetics) related to the elucidation of structural activity of the new modified peptides based on their steric open/closed ring effect were studied. The obtained results lead to the conclusion that in protic solvent with change in pH of the environment, direct control over the dyeing of textiles can be achieved. Both the new hybrid peptide compounds and the modification of functionalized textile materials with these bioactive hemorphins showed virucidal activity against the human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5 (HAdV-5) for different time intervals (30 and 60 min) and the most active compound was Rh-3.
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molecules
Article
Synthesis of New Modified with Rhodamine B Peptides for
Antiviral Protection of Textile Materials
Petar Todorov 1, * , Stela Georgieva 2, Desislava Staneva 3, Petia Peneva 1, Petar Grozdanov 4,
Ivanka Nikolova 4and Ivo Grabchev 5


Citation: Todorov, P.; Georgieva, S.;
Staneva, D.; Peneva, P.; Grozdanov, P.;
Nikolova, I.; Grabchev, I. Synthesis of
New Modified with Rhodamine B
Peptides for Antiviral Protection of
Textile Materials. Molecules 2021,26,
6608. https://doi.org/10.3390/
molecules26216608
Academic Editors: Hiroyuki Konno
and Kenichi Akaji
Received: 15 October 2021
Accepted: 29 October 2021
Published: 31 October 2021
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Attribution (CC BY) license (https://
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4.0/).
1Department of Organic Chemistry, University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria;
petenceto_2@abv.bg
2
Department of Analytical Chemistry, University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria;
st.georgieva@uctm.edu
3
Department of Textile and Leathers, University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria;
grabcheva@mail.bg
4The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria;
grozdanov@microbio.bas.bg (P.G.); inikolova@microbio.bas.bg (I.N.)
5Faculty of Medicine, Sofia University “St. Kl. Ohridski”, 1407 Sofia, Bulgaria; i.grabchev@chem.uni-sofia.bg
*Correspondence: pepi_37@abv.bg; Tel.: +359-2-8163423
Abstract:
Here we report on the synthesis and characterization of three new N-modified analogues of
hemorphin-4 with rhodamine B. Modified with chloroacetyl, chloride cotton fabric has been dyed and
color coordinates of the obtained textile materials were determined. Antiviral and virucidal activities
of both the peptide-rhodamine B compounds and the dyed textile material were studied. Basic
physicochemical properties (acid-base behavior, solvent influence, kinetics) related to the elucidation
of structural activity of the new modified peptides based on their steric open/closed ring effect were
studied. The obtained results lead to the conclusion that in protic solvent with change in pH of
the environment, direct control over the dyeing of textiles can be achieved. Both the new hybrid
peptide compounds and the modification of functionalized textile materials with these bioactive
hemorphins showed virucidal activity against the human respiratory syncytial virus (HRSV-S2) and
human adenovirus serotype 5 (HAdV-5) for different time intervals (30 and 60 min) and the most
active compound was Rh-3.
Keywords:
rhodamine-peptides; hemorphins; cotton fabric; absorbance; emission; virucidal effect;
antiviral activity
1. Introduction
The xanthene dyes are widely used in medicinal and bioorganic chemistry as biolog-
ically active compounds (either alone or conjugated). These compounds possess potent
antiviral activity against a human foreskin fibroblast (vesicular stomatitis virus) and en-
hance the antiviral activity of xanthene derivatives from 8- to 15-fold [
1
]. Rhodamine B
derivatives represent an important tool for studies of more complex biochemical processes
and activities. Such kind of fluorescence-based probes of bioactive molecules possess
desirable features; they have an excellent spectral characteristics and relatively facile syn-
theses [
2
,
3
]. Moreover, rhodamine B and its derivatives do not have a cytotoxic effect [
4
]. It
is inexpensive, resistant under a variety of reaction conditions, can be covalently linked
to bioactive molecules such as peptides, and has suitable spectral properties in terms
of absorption and fluorescence wavelength. Rhodamine B is a lipophilic cation belong-
ing to the family of xanthenes, and its derivatives are widely employed as fluorophore
probes [
3
,
5
]. It is known that they are sensitive as fluorescent turn-on compounds. The
only drawback to the use of rhodamine B is the formation of a spirolactam compound,
which is non-fluorescent and cannot be used for all fluorescent microscopic applications [
6
].
Molecules 2021,26, 6608. https://doi.org/10.3390/molecules26216608 https://www.mdpi.com/journal/molecules
Molecules 2021,26, 6608 2 of 19
Recently, a new class of rhodamine derivatives displaying a broad-spectrum antiviral activ-
ity against different enveloped viruses including an HSV-2 acyclovir resistant strain has
been reported [
7
]. Unfortunately, many viral infections are the cause of death worldwide,
and still now there are no efficient antiviral drugs or vaccines for a large number of viruses,
and this represents a great challenge especially for emerging and re-emerging viral dis-
eases [
7
]. Moreover, the peptides and peptide-conjugated molecules, having a propensity to
interact with membrane interfaces, might exert broad antiviral activity against enveloped
viruses. [8].
The peptides can be useful as substituents of the proteins, especially when site-specific
modification of the required protein is difficult or impractical. Peptides are sufficiently
small molecules which can be easily modified in the laboratory using standard synthetic
protocols and solid-phase peptide (SPPS) methods. The large part of peptides can play role
of ligands, since they contain a significant number of precisely located functional groups
and amino acid residues, which possess high-affinity and specific interactions with a target
receptor. This is usually harder to achieve with small molecules [
9
,
10
]. The peptide scaffolds
allow the introduction of fluorophores in their structure while retaining the biological
activity, which can increase the possibilities for the design of fluorescent sensors and their
applications in biochemistry and medicine [
11
,
12
]. This kind of fluorophore molecule is
incorporated into or appended to a known peptide sequence that has high affinity for
the target protein. The presence of amino acid residues that have both aromatic moiety
and hydrophobic nature, such as Trp, Phe, and Tyr, favor protein binding. Replacement
of this residue with fluorophore provides a significant change in protein binding and a
correspondingly strong enhancement of the fluorescence signal [9].
Hemorphins are endogenous peptides, belonging to the family of atypical opioid
peptides, released during the sequential cleavage of hemoglobin proteins [
13
15
]. Hemor-
phins have been shown to exhibit diverse therapeutic effects in both human and animal
models [
16
18
]. Hemorphin-4 (Tyr-Pro-Trp-Thr) is a member of the hemorphins family,
an endogenous nonclassical opioid peptides derived from hemoglobin. There is evidence
that these kinds of natural or synthetic tetrapeptides exert opioid activities
in vivo
and,
therefore, may play an important physiological role [
19
]. Our previously investigations
have demonstrated that not only the position of modification, but also the nature of the
incorporated group, lead to significant changes in the peptide activity and affinity
[2025]
.
Recently, the rhodamine B-labelled arginine-rich peptide has been designed as the heparin
bioreceptor to construct a highly sensitive and selective fluorescent biosensor for hep-
arin detection [
26
]. However, there are no data in the literature concerning rhodamine
conjugated hemorphin analogues.
A recent study has reported that the hemorphins bind with high affinity to angiotensin-
converting enzyme (ACE) [
27
]. ACE2 is expressed in nearly all human organs in varying
degrees. Both SARS-CoV-2 and SARS-CoV enter host cells via the angiotensin-converting
enzyme 2 (ACE2) receptor [
28
]. Thus, targeting the ACE2 receptor of host cells can block
the entry of the virus into the cell, thereby protecting the host from viral infection and
pandemic disease COVID-19 [29].
Different methods of textile materials functionalization exist in order to achieve
biologically-active effects. Most of these methods/reagents are controversial for humans
and the environment due to inorganic salts, phenols and thiophenols, antibiotics, formalde-
hyde derivatives, etc. which are used during production [
30
]. Therefore, the development
of new methods has to fulfil the requirements of being safe for both human health and the
environment. In conjunction with such functionalization, there is a question concerning
to which extent the normal skin flora of healthy people will be destroyed by action of
an antimicrobial compound [
31
]. Therefore, the increasing tendency of research is seen
where the functionalization is performed by the use of non-toxic, biodegradable, and
environmentally-friendly reagents. Currently, the peptides are the candidate therapeu-
tic agents that offer selectivity and specificity, and low levels of side effects. The great
advantage of peptides against viruses consists in the reduced possibility of developing re-
Molecules 2021,26, 6608 3 of 19
sistance during the treatment [
32
]. Cotton has widespread use in textiles and in healthcare
environments. Peptides can be simply adsorbed on cotton through electrostatic interac-
tions but without covalent bonding they are easily lost. Functionalization of cotton can
impart suitable groups for covalent bonding of different polymers and ensure durability of
modification during use [33].
Herein we report on the synthesis and characterization of new N-modified analogues
of hemorphin-4 with rhodamine B. We have also investigated the modification of the func-
tionalized cotton fabric with the new hybrid peptide compounds. The potential antiviral
and virucidal activities of both peptides and textiles material have also been studied.
2. Results and Discussion
2.1. Chemistry
We have synthesized and characterized new rhodamine B-conjugated hemorphin-4
analogues as a potential sensitive fluorescent probe for color, antiviral, and virucidal ac-
tivity of textile materials. These peptides contain different aliphatic amino acid residue
and differed by the increased number of methylene group (from one to three) between
rhodamine B moiety to the N-side and the amino acid scaffold of natural hemorphin-4. The
aim of this study was to determine evidence of the significance of different amino alkyl
residues of newly synthesized hybrid compounds for their physicochemical properties
and to investigate their structurally-related properties and potential textile applications
using different methods. We have also explored an approach to the structural features
of pH-dependent equilibrium between the spirolactam form and the ring-opened form
of these peptides, the potential antiviral and virucidal activities of both of the new hy-
brid peptide molecules, and the modification of functionalized cotton fabrics with these
bioactive hemorphins.
RhodamineB-Gly-Tyr-Pro-Trp-Thr-NH2(Rh-1), rhodamineB-β-Ala-Tyr-Pro-Trp- Thr-
NH
2
(Rh-2), and rhodamineB-
γ
-Abu-Tyr-Pro-Trp-Thr-NH
2
(Rh-3) were efficiently prepared
via solid-phase peptide synthesis (SPPS) using Fmoc (9-fluorenylmethoxy- carbonyl) chem-
istry. This strategy, based on the reaction between rhodamine-B with the N-terminal
amino group of the hemorphin-4 analogues, was applied directly to the resin. The syn-
thetic route is summarized in Figure 1. In order to achieve peptide bond formation and
to improve the efficiency of peptide synthesis, the organic compounds such as TBTU
(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) and HOBt (hy-
droxybenzotriazole) as coupling reagents, and DIPEA (N,N-diisopropylethyl- amine) as
an organic base were added to reaction media in each step. After cleavage of the product
from Rink-Amide MBHA Resin, the compounds were purified from crude product by
semi-preparative HPLC with a C18 column. The Mass spectra confirmed the spirolactam
form of peptides formation.
The handling of the amino acidic scaffold can be regarded as a potentially powerful
tool in both bioorganic and medicinal chemistry investigations and the development of
new drugs and materials [34].
Molecules 2021,26, 6608 4 of 19
Molecules 2021, 26, x FOR PEER REVIEW 4 of 20
Figure 1. Schematic representation of the solid phase synthesis of the new hybrid peptides.
2.2. Physicochemical Characterizations
Determination of Physicochemical Constants
The values of the dissociation constants of the test compounds were determined us-
ing potentiometric titration. The calculated values are important for the direct application
of the compounds in the process of their bonding to the textile material. It is known that
one of the most important physical and chemical factors of micro-and macromolecules is
the value of the acid dissociation constant (pK), used to determine the type of individual
protonated and unprotonated forms of compounds and to measure the strength of acids
and bases. Basically, the physicochemical properties of the compounds depend on the pH
O
NH
O
NH
O
O
O
O
O
NH
O
NH
2
O
O
20% piperidine in DMF
O
NH
O
NH
O
O
(1) Fmoc-Thr(t-Bu)-OH; TBTU; HOBt; DIPEA; DMF
(2) 20% piperidine /DMF; 20 min.; rt
Thr
(t-Bu)
H
2
N
Repeat steps:
(1) condensation with the next Fmoc-amino acid-OH;
TBTU; HOBt; DIPEA; DMF and
(2) 20% piperi dine /DMF; 20 min.;
rt for the coupling of the next amino acids
SPPS
O
NH
O
NH
O
O
NH
2
O
N
N
O
OH
TBTU; HOBt; DIPEA; DMF
O
NH
O
NH
O
O
O
N N
O
NH
NH
2
O
N N
O
NH
TFA/TIS/H
2
O
Thr
Trp
Pro
Tyr
Aaa
Aaa = Gly/beta-Ala/Gaba
Aaa
Aaa
Tyr
Tyr
Pro
Pro
Trp
Trp
Thr
Thr
Figure 1. Schematic representation of the solid phase synthesis of the new hybrid peptides.
2.2. Physicochemical Characterizations
Determination of Physicochemical Constants
The values of the dissociation constants of the test compounds were determined using
potentiometric titration. The calculated values are important for the direct application of
the compounds in the process of their bonding to the textile material. It is known that one of
the most important physical and chemical factors of micro-and macromolecules is the value
of the acid dissociation constant (pK), used to determine the type of individual protonated
and unprotonated forms of compounds and to measure the strength of acids and bases.
Basically, the physicochemical properties of the compounds depend on the pH of the
medium. This parameter is an important factor in the dyeing process and the interaction of
Molecules 2021,26, 6608 5 of 19
the dye with the textile material. After data processing from the titration, the dissociation
constants of the compounds were determined by the Hassenbach equation [
23
,
24
]. From
Figure 2it can be seen that rhodamines are referred to as diprotic acids, dissociating
gradually. The calculated values of pKa and pI are summarized in Table 1. The values of
the dissociation constants of the three rhodamine derivatives are approximately equal but
can still be arranged in the following sequence: pK
1
(Rh-2) < pK
1
(Rh-1) < pK
1
(Rh-3). Since
the pKa of Rhodamine B defines the equilibrium between the spirocyclic form and the ring-
opened form, it can be reasoned that the pKa values of rhodamines could be modulated by
introducing different amino acid residues of the peptide chain. pH-dependent equilibrium
between the spirolactam form and the ring-opened form of Rh-1, Rh-2, and Rh-3 are shown
in Figure 3. As mentioned, compounds 1 and 2 have a slightly lower value than 3, which
may be due to the closer proximity of the hydroxy group of Tyr to the spirocyclic carbon,
where the steric effect may be more pronounced. The calculated values of the isoelectric
points show that in a weakly acidic medium (pH~4) the compounds will have zero charges
(neutrality of the molecule) and, accordingly, insolubility which was taken into account at
the textile dyeing.
Molecules 2021, 26, x FOR PEER REVIEW 5 of 20
of the medium. This parameter is an important factor in the dyeing process and the inter-
action of the dye with the textile material. After data processing from the titration, the
dissociation constants of the compounds were determined by the Hassenbach equation
[23,24]. From Figure 2 it can be seen that rhodamines are referred to as diprotic acids,
dissociating gradually. The calculated values of pKa and pI are summarized in Table 1.
The values of the dissociation constants of the three rhodamine derivatives are approxi-
mately equal but can still be arranged in the following sequence: pK
1
(Rh-2) < pK
1
(Rh-1) <
pK
1
(Rh-3). Since the pKa of Rhodamine B defines the equilibrium between the spirocyclic
form and the ring-opened form, it can be reasoned that the pKa values of rhodamines
could be modulated by introducing different amino acid residues of the peptide chain.
pH-dependent equilibrium between the spirolactam form and the ring-opened form of
Rh-1, Rh-2, and Rh-3 are shown in Figure 3. As mentioned, compounds 1 and 2 have a
slightly lower value than 3, which may be due to the closer proximity of the hydroxy
group of Tyr to the spirocyclic carbon, where the steric effect may be more pronounced.
The calculated values of the isoelectric points show that in a weakly acidic medium (pH ~
4) the compounds will have zero charges (neutrality of the molecule) and, accordingly,
insolubility which was taken into account at the textile dyeing.
Table 1. Physicochemical characterization of Rh-1, Rh-2, and Rh-3.
Compound pK pI k, s
1
τ
1/2
pK
1
pK
2
Rh-1 2.81 6.6 4.7 4.65 × 10
2
± 0.0046 14.9
Rh-2 2.78 6.38 4.58 5.53x10
2
± 0.0012
12.5
Rh-3 2.86 6.39 4.63 8.12x10
2
± 0.0014
8.54
Figure 2. Plot of pH vs. V
NaOH
and first derivatives graphics of 20 µmoL of Rh-1, Rh-2, and Rh-3,
respectively.
Figure 2.
Plot of pH vs. V
NaOH
and first derivatives graphics of 20
µ
moL of Rh-1, Rh-2, and
Rh-3, respectively.
Table 1. Physicochemical characterization of Rh-1, Rh-2, and Rh-3.
Compound pK pI k, s1τ1/2
pK1pK2
Rh-1 2.81 6.6 4.7 4.65 ×102±0.0046 14.9
Rh-2 2.78 6.38 4.58 5.53 ×102±0.0012 12.5
Rh-3 2.86 6.39 4.63 8.12 ×102±0.0014 8.54
Molecules 2021,26, 6608 6 of 19
Molecules 2021, 26, x FOR PEER REVIEW 6 of 20
Figure 3. pH-dependent equilibrium between the spirolactam form and the ring-opened form of rhodamine B-conjugated
hemorphin-4 analogues.
2.3. Spectral Characterizations
The new compounds were characterized by FT-IR, UV-Vis, and fluorescence spec-
troscopy in two types of solvents (water (polar protic solvent) and triethylamine (polar
aprotic solvent)) in order to investigate changes in the color of the solution directly related
to the structure of the compounds. The spectra related to the absorption of electromag-
netic radiation of the rhodamine derivatives are given in Figure 4. Two well-formed ab-
sorption peaks with different intensity in both used solvents, localized at λmax 230,
278nm and 561nm in the water solutions and λmax 300nm and 565nm of solution of
triethylamine, respectively, can be seen (Figure 4). Basically, theππ transitions of the >
C=O in the peptide bonds is occurred by UV absorption of the peptide molecule in the
range 180 to 230nm. The aromatic side-chains of indole of Trp, phenol rings of Tyr, and
rhodamine are primarily responsible for absorption of the π-electron systems of aromatic
groups in the range of 300nm [35,36]. The absorption bands occurring at this wavelength
are equally intense due to the same number of peptide bonds and the concentration of
analyzed solutions. The studied dependence on the pH of the water medium showed that
the colors of the aqueous solutions remain unchanged from the strongly acidic medium
in which they dissolve to the strongly alkaline one. There is a visible change in the hue of
the solutions for the individual compounds, but the difference in the wavelength at which
the compounds absorb in the visible region is minimal (2–5 nm). The studied dependence
on the pH of the water medium showed that the colors of the aqueous solutions remain
unchanged from the strongly acidic medium to the strongly alkaline one. The absorption
ON N
O
N
H
O
N
H
N
OH
O
HN
OHN
NH
O
NH
2
O
OH
ON N
O
N
H
O
H
N
N
OH
O
H
N
OHN
NH
O
NH
2
O
OH
OH
H
ON N
O
N
O
NH N
OH
O
H
N
O
HN
N
H
O
H
2
N
O
HO
Fluorescence off
Fluorescence on
ON N
O
NO
HN
N
OH
O
H
N
OHN
NH
O
NH
2
O
OH
Fluorescence on
Fluorescence off
Rh-1
Rh-2
ON N
O
N
H
O
H
N
N
OH
O
H
N
OHN
NH
O
NH
2
O
OH
ON N
O
N
O
NH N
OH
O
H
N
O
HN
N
H
O
H
2
N
O
HO
Fluorescence on
Fluorescence off
Rh-3
OH
H
OH
H
Figure 3.
pH-dependent equilibrium between the spirolactam form and the ring-opened form of rhodamine B-conjugated
hemorphin-4 analogues.
2.3. Spectral Characterizations
The new compounds were characterized by FT-IR, UV-Vis, and fluorescence spec-
troscopy in two types of solvents (water (polar protic solvent) and triethylamine (polar
aprotic solvent)) in order to investigate changes in the color of the solution directly related
to the structure of the compounds. The spectra related to the absorption of electromagnetic
radiation of the rhodamine derivatives are given in Figure 4. Two well-formed absorption
peaks with different intensity in both used solvents, localized at
λ
max
230,
278 nm and
561 nm in the water solutions and
λ
max
300 nm and 565 nm of solution of triethylamine,
respectively, can be seen (Figure 4). Basically, the
ππ
transitions of the > C=O in the
peptide bonds is occurred by UV absorption of the peptide molecule in the range 180 to
230 nm. The aromatic side-chains of indole of Trp, phenol rings of Tyr, and rhodamine are
primarily responsible for absorption of the
π
-electron systems of aromatic groups in the
range of
300 nm [
35
,
36
]. The absorption bands occurring at this wavelength are equally
intense due to the same number of peptide bonds and the concentration of analyzed solu-
tions. The studied dependence on the pH of the water medium showed that the colors of
the aqueous solutions remain unchanged from the strongly acidic medium in which they
dissolve to the strongly alkaline one. There is a visible change in the hue of the solutions for
the individual compounds, but the difference in the wavelength at which the compounds
absorb in the visible region is minimal (2–5 nm). The studied dependence on the pH of the
water medium showed that the colors of the aqueous solutions remain unchanged from
Molecules 2021,26, 6608 7 of 19
the strongly acidic medium to the strongly alkaline one. The absorption peak intensity
increased three-fold with the decrease of pH values from 12 to 1 (Figures 4and 5).
Molecules 2021, 26, x FOR PEER REVIEW 7 of 20
peak intensity increased three-fold with the decrease of pH values from 12 to 1 (Figures 4
and 5).
Figure 4. UV-Vis spectrum of Rhodamine derivatives at: (A) equal concentration of the compounds
and different pH of solutions. Absorbance of the solution was measured against water; (B) solutions
of Rh-1, Rh-2 and Rh-3 in triethylamine with equal concentration of the compounds. Absorbance of
the solution was measured against triethylamine.
Figure 5. Uv-Vis spectrum of Rh-1 (4.09 × 105 mol L1) at different pH.
Figure 4.
UV-Vis spectrum of Rhodamine derivatives at: (
A
) equal concentration of the compounds
and different pH of solutions. Absorbance of the solution was measured against water; (
B
) solutions
of Rh-1, Rh-2 and Rh-3 in triethylamine with equal concentration of the compounds. Absorbance of
the solution was measured against triethylamine.
Molecules 2021, 26, x FOR PEER REVIEW 7 of 20
peak intensity increased three-fold with the decrease of pH values from 12 to 1 (Figures 4
and 5).
Figure 4. UV-Vis spectrum of Rhodamine derivatives at: (A) equal concentration of the compounds
and different pH of solutions. Absorbance of the solution was measured against water; (B) solutions
of Rh-1, Rh-2 and Rh-3 in triethylamine with equal concentration of the compounds. Absorbance of
the solution was measured against triethylamine.
Figure 5. Uv-Vis spectrum of Rh-1 (4.09 × 105 mol L1) at different pH.
A similar property of rhodamine-related peptides was observed by Meng-Chan Xia
et al. [37]. Color stability was observed in the aqueous solutions of the compounds, alt-
hough the intensity of the absorption maxima depended on the pH of the medium. The
molar extinction coefficients in the long-wavelength absorption maximum are, respec-
tively, Rh-3 (ε = 1.83 × 104 cm.mol.L1); Rh-1 (ε = 2.04 × 104 cm.mol.L1); and Rh-2 (ε = 2.16
× 104 cm.mol.L1). The behavior of the compounds in an aprotic solution of triethylamine
Figure 5. Uv-Vis spectrum of Rh-1 (4.09 ×105mol L1) at different pH.
A similar property of rhodamine-related peptides was observed by
Meng-Chan Xia et al. [37]
.
Color stability was observed in the aqueous solutions of the compounds, although the intensity
of the absorption maxima depended on the pH of the medium. The molar extinction coefficients
Molecules 2021,26, 6608 8 of 19
in the long-wavelength absorption maximum are, respectively, Rh-3 (
ε= 1.83 ×104cm.mol.L1
);
Rh-1 (
ε
= 2.04
×
10
4
cm.mol.L
1
); and Rh-2 (
ε= 2.16 ×104cm.mol.L1
). The behavior of
the compounds in an aprotic solution of triethylamine is different. For about 6–120 min, a
visible fading of the solution is observed until the complete disappearance of the color. This
is the due to the observed steric effect in the molecules of the compounds associated with
the case of spirolactam ring opening. This provoked our interest in studying, in addition,
the influence of the environment on the structural changes of the compounds. The effect of
the peptide substituent on the ring opening/closing time is clearly visible with studying the
kinetics of the process. For this purpose, the spectra of the solution were taken at the initial
moment, immediately after its preparation and subsequently on every 5–10 min. Figure 6
shows the decrease in the intensity of the absorption peak. The kinetic lines were taken at
the same time interval and by fluorescence (Figure 7). The observed small Stock shift is
typical for rhodamine dyes. It is characteristic and of the newly synthesized rhodamine
B-conjugated hemorphin-4 analogues. However, the increase in the spacer length between
chromophore and peptides leads to a minor enhancement of Stock shift due to the changes
in molecule mobility [5,38].
Molecules 2021, 26, x FOR PEER REVIEW 8 of 20
is different. For about 6–120 min, a visible fading of the solution is observed until the
complete disappearance of the color. This is the due to the observed steric effect in the
molecules of the compounds associated with the case of spirolactam ring opening. This
provoked our interest in studying, in addition, the influence of the environment on the
structural changes of the compounds. The effect of the peptide substituent on the ring
opening/closing time is clearly visible with studying the kinetics of the process. For this
purpose, the spectra of the solution were taken at the initial moment, immediately after
its preparation and subsequently on every 5–10 min. Figure 6 shows the decrease in the
intensity of the absorption peak. The kinetic lines were taken at the same time interval and
by fluorescence (Figure 7). The observed small Stock shift is typical for rhodamine dyes.
It is characteristic and of the newly synthesized rhodamine B-conjugated hemorphin-4
analogues. However, the increase in the spacer length between chromophore and pep-
tides leads to a minor enhancement of Stock shift due to the changes in molecule mobility
[5,38].
Figure 6. Uv-Vis kinetic plot of the first-order reaction between ring-opened and formed of the spirolactam form at equal
concentration of Rh-1, Rh-2, and Rh-3 (at 25 °C) and corresponding fluorescence spectra in aprotic solvent (triethylamine).
Rh-1 λabs (nm): 565, λem (nm): 589, Stokes shift (cm1): 721; Rh-2 λabs (nm): 561, λem (nm): 593, Stokes shift (cm1): 962; Rh-3
λabs (nm): 559, λem (nm): 592, Stokes shift (cm1): 997.
From a thermodynamic point of view, the opening/closing reaction of the spirolac-
tam ring is a first-order reaction [36]. The kinetic law with respect to the exhaustion of one
form relative to the other is ln (C0)/C) = k.t, ie the dependence ln C = f (t, min) is linear.
Since the concentration of the colored form of the compound is proportional to the
Figure 6.
Uv-Vis kinetic plot of the first-order reaction between ring-opened and formed of the spirolactam form at equal
concentration of Rh-1, Rh-2, and Rh-3 (at 25
C) and corresponding fluorescence spectra in aprotic solvent (triethylamine).
Rh-1
λabs
(nm): 565,
λem
(nm): 589, Stokes shift (cm
1
): 721; Rh-2
λabs
(nm): 561,
λem
(nm): 593, Stokes shift (cm
1
): 962;
Rh-3 λabs (nm): 559, λem (nm): 592, Stokes shift (cm1): 997.
Molecules 2021,26, 6608 9 of 19
Figure 7.
Fluorescence kinetic plot of the first-order reaction between ring-opened and formed of the
spirolactam form at equal concentration of Rh-1, Rh-2 and Rh-3 and 25 C.
From a thermodynamic point of view, the opening/closing reaction of the spirolactam
ring is a first-order reaction [
36
]. The kinetic law with respect to the exhaustion of one form
relative to the other is ln (C
0
)/C) = k.t, i.e., the dependence ln C = f (t, min) is linear. Since
the concentration of the colored form of the compound is proportional to the absorption
(from UV-Vis) and the emission intensity (in fluorescence analysis) in this equation, the
concentration can be replaced by the corresponding physical quantity according to the
literature [
39
]. The rate of conversion of the ring-opened form to spirolactam was quantified
by calculating the value of the rate constant of the process and half the time using data
from the emission spectra of the compounds. The rate constant k was calculated from
the slope of plot of the left side of Equation (2) versus time and the obtained value are
summarized in Table 1. The value of the rate constant (k) was used to calculate and the
τ
1/2—half time or this is the time for which the current concentration (C) of the ring-
opened form decreases twice with respect to the initial concentration of (C
0
), i.e., when
C=C0/2
, then t =
τ1/2
. For a first-order reaction, the value was calculated by the equation:
τ1/2
= ln2/k (Table 1) [
39
]. Moreover, Rh-1 had its maximum absorption band at 565 nm
and strongest fluorescence emission at 589 nm in triethylamine solution (Figure 6) and the
slowest spirolactam form formation effect (k = 4.6
×
10
2
s
1
, Table 1) associated with
solution discoloration (
Figure 7
). In the other compounds, the emission intensities and the
value of the rate constant increase in proportion to the increase in the number of methylene
groups in the peptide chain (Figures 6and 7). Stokes shift is an important feature that
shows the differences between the structure of the fluorophore in the ground S
0
state and
in the first excited state S
1
and was also calculated for the rhodamine peptide derivatives
(Figure 6). The Stokes shift of the test compounds are in the range of 721 and 997 cm
1
,
which is consistent with rhodamine derivatives known in the literature [40].
The IR spectrum of studied compounds were recorded in KBr tablet (KBr, cm
1
) and
shown main characteristic bands, localized at as follow: 3356 (N-H stretching vibration
(
νNH
)), 1701 (s)—NCO (amide) stretching and 1678–1722 cm
–1
—a high-intensity peak of
νC=O
; 1511–1528 cm
–1
(
δNH
) (Figure 8). As can be seen, the absorption lines in addition
prove the functional groups belonging to the structure of the compounds (Figure 8).
Molecules 2021,26, 6608 10 of 19
Molecules 2021, 26, x FOR PEER REVIEW 10 of 20
Figure 8. IR-spectra of the rhodamine-peptide derivatives.
2.4. Color Characterisation of Cotton Fabrics
CIELab coordinates were used to distinguish the difference in the color of three cot-
ton fabrics dyed with rhodamine-peptides. Figure 9 shows the change in the value of a*
and b* of pristine cotton fabric and the functionalized with chloroacetyl chloride fabrics
treated with peptides Rh-1, Rh-2 or Rh-3. The untreated cotton fabric has a white color in
daylight with coordinates a* and b*, approximately equal to zero. For the dyed samples,
the color coordinates as an absolute value increase in the order Rh-3 < Rh-1 < Rh-2 and
correspond to a red-blue color. The color difference of the fabric compared with untreated
fabric changes in the same order. This is in agreement with molar absorption coefficient
of the new rhodamine-peptide samples. For all compounds, this coefficient is higher than
10
4
which demonstrate a good coloring ability. A better result has been obtained with Rh-
2.
Figure 9. Comparison of the colour coordinates of cotton fabric (Co) and fabrics dyed with Rh-1, Rh-2, and Rh-3: (A) color
characterization with a* and b* coordinates; (B) color difference DE*.
Figure 10A shows the reflection spectra of the initial cotton fabric and after it dyeing
with Rh-1, Rh-2, and Rh-3. The minimum reflectivity (R%) occurs at about 560 nm, where
rhodamine peptides have the maximum absorption. As the dyes are fluorescent, there is
also a band with a maximum wavelength of 640 nm [41]. Both the minimum and maxi-
mum are more pronounced for samples Rh-1 and Rh-2 compared to those of Rh-3. Figure
10B shows the relation of K/S values with the wavelength. The maximum absorbance for
0.3
0.6
0.9
1239
1512
2976
Rh-2
Transmittance, %
0.3
0.6
0.9
1701
Rh-1
4000 3500 3000 2000 1500 1000 500
0.3
0.6
0.9
Rh-3
W avenumber, cm-1
3356
Figure 8. IR-spectra of the rhodamine-peptide derivatives.
2.4. Color Characterisation of Cotton Fabrics
CIELab coordinates were used to distinguish the difference in the color of three cotton
fabrics dyed with rhodamine-peptides. Figure 9shows the change in the value of a* and b*
of pristine cotton fabric and the functionalized with chloroacetyl chloride fabrics treated
with peptides Rh-1, Rh-2 or Rh-3. The untreated cotton fabric has a white color in daylight
with coordinates a* and b*, approximately equal to zero. For the dyed samples, the color
coordinates as an absolute value increase in the order Rh-3 < Rh-1 < Rh-2 and correspond
to a red-blue color. The color difference of the fabric compared with untreated fabric
changes in the same order. This is in agreement with molar absorption coefficient of the
new rhodamine-peptide samples. For all compounds, this coefficient is higher than 10
4
which demonstrate a good coloring ability. A better result has been obtained with Rh-2.
Molecules 2021, 26, x FOR PEER REVIEW 10 of 20
Figure 8. IR-spectra of the rhodamine-peptide derivatives.
2.4. Color Characterisation of Cotton Fabrics
CIELab coordinates were used to distinguish the difference in the color of three cot-
ton fabrics dyed with rhodamine-peptides. Figure 9 shows the change in the value of a*
and b* of pristine cotton fabric and the functionalized with chloroacetyl chloride fabrics
treated with peptides Rh-1, Rh-2 or Rh-3. The untreated cotton fabric has a white color in
daylight with coordinates a* and b*, approximately equal to zero. For the dyed samples,
the color coordinates as an absolute value increase in the order Rh-3 < Rh-1 < Rh-2 and
correspond to a red-blue color. The color difference of the fabric compared with untreated
fabric changes in the same order. This is in agreement with molar absorption coefficient
of the new rhodamine-peptide samples. For all compounds, this coefficient is higher than
10
4
which demonstrate a good coloring ability. A better result has been obtained with Rh-
2.
Figure 9. Comparison of the colour coordinates of cotton fabric (Co) and fabrics dyed with Rh-1, Rh-2, and Rh-3: (A) color
characterization with a* and b* coordinates; (B) color difference DE*.
Figure 10A shows the reflection spectra of the initial cotton fabric and after it dyeing
with Rh-1, Rh-2, and Rh-3. The minimum reflectivity (R%) occurs at about 560 nm, where
rhodamine peptides have the maximum absorption. As the dyes are fluorescent, there is
also a band with a maximum wavelength of 640 nm [41]. Both the minimum and maxi-
mum are more pronounced for samples Rh-1 and Rh-2 compared to those of Rh-3. Figure
10B shows the relation of K/S values with the wavelength. The maximum absorbance for
0.3
0.6
0.9
1239
1512
2976
Rh-2
Transmittance, %
0.3
0.6
0.9
1701
Rh-1
4000 3500 3000 2000 1500 1000 500
0.3
0.6
0.9
Rh-3
W avenumber, cm-1
3356
Figure 9.
Comparison of the colour coordinates of cotton fabric (Co) and fabrics dyed with Rh-1, Rh-2, and Rh-3: (
A
) color
characterization with a* and b* coordinates; (B) color difference DE*.
Figure 10A shows the reflection spectra of the initial cotton fabric and after it dyeing
with Rh-1, Rh-2, and Rh-3. The minimum reflectivity (R%) occurs at about 560 nm, where
rhodamine peptides have the maximum absorption. As the dyes are fluorescent, there is
also a band with a maximum wavelength of 640 nm [
41
]. Both the minimum and maximum
are more pronounced for samples Rh-1 and Rh-2 compared to those of Rh-3. Figure 10B
shows the relation of K/S values with the wavelength. The maximum absorbance for the
three dyes is 560 nm. The K/S values give information for the dye quantity on the fabric
Molecules 2021,26, 6608 11 of 19
and its behavior on the textile substrate [
42
]. It can be seen that the concentrations of the
fixed dyes under dyeing conditions are different. Figure 10B shows the relation of K/S
values with the wavelength. The maximum absorbance for the three dyes is 560 nm. The
K/S values give information for the dye quantity on the fabric and its behavior on the textile
substrate. It can be seen that the concentrations of the fixed dyes under dyeing conditions
are different. The K/S value is the highest for Rh-2, but the value of Rh-1 is closed to it.
The value of Rh-3 is twice as small as Rh-2. The reason for this can be the difference in
molecular mass of this rhodamine-peptide and its different behavior in solution.
Molecules 2021, 26, x FOR PEER REVIEW 11 of 20
the three dyes is 560 nm. The K/S values give information for the dye quantity on the
fabric and its behavior on the textile substrate [42]. It can be seen that the concentrations
of the fixed dyes under dyeing conditions are different. Figure 10B) shows the relation of
K/S values with the wavelength. The maximum absorbance for the three dyes is 560 nm.
The K/S values give information for the dye quantity on the fabric and its behavior on the
textile substrate. It can be seen that the concentrations of the fixed dyes under dyeing con-
ditions are different. The K/S value is the highest for Rh-2, but the value of Rh-1 is closed
to it. The value of Rh-3 is twice as small as Rh-2. The reason for this can be the difference
in molecular mass of this rhodamine-peptide and its different behavior in solution.
Figure 10. Comparison of cotton fabric (Co) and fabrics dyed with Rh-1, Rh-2, and Rh-3: (A) reflection spectra; (B) K/S
values in function of wavelength.
2.5. Fastness Testing
The stability of the textile material after dyeing with the new antiviral agents was
also studied. In order to evaluate the rebinding of the compounds when washing the dyed
textile material with soap and water, the spectra of the obtained soap solutions were taken
following the washing procedure. When washing the materials with water only, no dif-
ference in the spectrum of the solutions after soaking the materials was observed (Figure
11). Studies have shown that in all compounds immediately after washing with soap, the
color of the fabric is slightly affected and after drying there is a visible fading of the mate-
rials (Figure 11). As can be seen from the figure most strongly absorbs the solution of Rh-
2 which can be attributed to a lower binding affinity with the textile material and lower
resistance to washing in an alkaline environment. However, no difference was observed
in the color of the textiles when soaking the materials in the washing solution (water and
soap) for 1 and 24 h.
400 450 500 550 600 650 700
0
20
40
60
80
100
R (%)
Wavelength (nm)
Co
Rh-1
Rh-2
Rh-3
A)
400 450 500 550 600 650
0
2
4
6
8
10
K/S
Wavelength (nm)
Co
Rh-1
Rh-2
Rh-3
B)
Figure 10.
Comparison of cotton fabric (Co) and fabrics dyed with Rh-1, Rh-2, and Rh-3: (
A
) reflection spectra; (
B
) K/S
values in function of wavelength.
2.5. Fastness Testing
The stability of the textile material after dyeing with the new antiviral agents was
also studied. In order to evaluate the rebinding of the compounds when washing the
dyed textile material with soap and water, the spectra of the obtained soap solutions were
taken following the washing procedure. When washing the materials with water only,
no difference in the spectrum of the solutions after soaking the materials was observed
(Figure 11). Studies have shown that in all compounds immediately after washing with
soap, the color of the fabric is slightly affected and after drying there is a visible fading
of the materials (Figure 11). As can be seen from the figure most strongly absorbs the
solution of Rh-2 which can be attributed to a lower binding affinity with the textile material
and lower resistance to washing in an alkaline environment. However, no difference was
observed in the color of the textiles when soaking the materials in the washing solution
(water and soap) for 1 and 24 h.
2.6. Virological Activity
Antimicrobial peptides are able to inhibit many pathogens, including Gram-negative
and Gram-positive bacteria and fungi. Additionally, some antimicrobial peptides have
been shown to have anticancer or antiviral activity, such as indolicidin that has activity
toward HIV [
43
]. Peptides with antiviral activity against influenza can be divided into three
main groups. First, entry blocker peptides such as a FluPep that interact with influenza
hemagglutinin block its binding to host cells and prevent viral fusion. Second, several
peptides display virucidal activity, disrupting viral envelopes (e.g., Melittin). Finally, a
third set of peptides interacts with the viral polymerase complex and act as viral replication
inhibitors such as PB1 derived peptides [44].
Rhodamine B-conjugated hemorphin-4 analogues demonstrated a virucidal effect
against human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5
(HAdV-5) for different time intervals (30 and 60 min). The most active is Rh-3 peptide
Molecules 2021,26, 6608 12 of 19
analogue, which is an analogue of hemorphin-4 containing a rhodamine B residue at the
N-terminus and a hydrophobic -
γ
-Abu-Tyr-Pro-Trp-Thr-CONH
2
amino acid sequence of
the peptide molecule. The difference between others two compounds is only between
one amino acid residues and in particular this is the methylene group (from one to three).
Compound Rh-3 showed higher virucidal activity against HRSV-S2 at 60 min, unlike
compound Rh-2 which is more active at 30 min. (Table 2). All of the peptides did not show
any virucidal activity against HAdV-5 in both 30- and 60-min intervals. Perhaps this is due
to the structure of HAdV-5 (non enveloped virus) which do not have lipid bilayer envelope,
thus making them more resistant to chemicals.
Molecules 2021, 26, x FOR PEER REVIEW 12 of 20
Figure 11. UV-Vis (zero) (A) and zoomed at 560 nm (B) spectra of soap (SS) and water (WS) solutions
obtained after washing the materials; soap solutions immediately after washing the materials (C);
compare the color of the material before and after washing with soap and water. The photos were
taken after drying the materials (D).
2.6. Virological Activity
Antimicrobial peptides are able to inhibit many pathogens, including Gram-negative
and Gram-positive bacteria and fungi. Additionally, some antimicrobial peptides have
been shown to have anticancer or antiviral activity, such as indolicidin that has activity
toward HIV [43]. Peptides with antiviral activity against influenza can be divided into
three main groups. First, entry blocker peptides such as a FluPep that interact with influ-
enza hemagglutinin block its binding to host cells and prevent viral fusion. Second, sev-
eral peptides display virucidal activity, disrupting viral envelopes (e.g., Melittin). Finally,
a third set of peptides interacts with the viral polymerase complex and act as viral repli-
cation inhibitors such as PB1 derived peptides [44].
Rhodamine B-conjugated hemorphin-4 analogues demonstrated a virucidal effect
against human respiratory syncytial virus (HRSV-S2) and human adenovirus serotype 5
(HAdV-5) for different time intervals (30 and 60 min). The most active is Rh-3 peptide
analogue, which is an analogue of hemorphin-4 containing a rhodamine B residue at the
N-terminus and a hydrophobic -γ-Abu-Tyr-Pro-Trp-Thr-CONH
2
amino acid sequence of
the peptide molecule. The difference between others two compounds is only between one
amino acid residues and in particular this is the methylene group (from one to three).
Compound Rh-3 showed higher virucidal activity against HRSV-S2 at 60 min, unlike com-
pound Rh-2 which is more active at 30 min. (Table 2). All of the peptides did not show
any virucidal activity against HAdV-5 in both 30- and 60-min intervals. Perhaps this is
due to the structure of HAdV-5 (non enveloped virus) which do not have lipid bilayer
envelope, thus making them more resistant to chemicals.
For a more in-depth study of the virucidal effect against both HRSV-S2 and HAdV-
5, cotton fabrics dyed with rhodamine-peptides have been also studied. Compared to rho-
damine B-conjugated peptides, the virucidal effect of the textile materials was lower. (Ta-
ble 3). In this case, the virucidal effect of cotton fabrics is due to their direct contact with
viruses. The good retention of the tested peptides to the fabric surface does not allow their
Figure 11.
UV-Vis (zero) (
A
) and zoomed at 560 nm (
B
) spectra of soap (SS) and water (WS) solutions
obtained after washing the materials; soap solutions immediately after washing the materials (
C
);
compare the color of the material before and after washing with soap and water. The photos were
taken after drying the materials (D).
Table 2.
Virucidal effect of new rhodamine B-conjugated hemorphin-4 analogues against human res-
piratory syncytial virus (HRSV-S2) and Human adenovirus serotype 5 (HAdV-5) after 30 min/60 min.
Virus
log 30 min log 60 min
Rh-1 Rh-2 Rh-3 Rh-1 Rh-2 Rh-3
HRSV-2 0.2 0.4 0.2 1.3 1.3 1.7
HAdV-5 000000
For a more in-depth study of the virucidal effect against both HRSV-S2 and HAdV-
5, cotton fabrics dyed with rhodamine-peptides have been also studied. Compared to
rhodamine B-conjugated peptides, the virucidal effect of the textile materials was lower.
(Table 3). In this case, the virucidal effect of cotton fabrics is due to their direct contact with
viruses. The good retention of the tested peptides to the fabric surface does not allow their
easy release and reaction with the viruses in solution, which explains the low virucidal
activity of the fabrics to the tested viruses.
Molecules 2021,26, 6608 13 of 19
Table 3.
Virucidal effect of new cotton fabrics dyed with rhodamine-peptides against human respiratory syncytial virus
(HRSV-S2) and human adenovirus C serotype 5 (HAdV-5) after 30 min/60 min.
Virus
log 30 min log 60 min
Rh-1-Textile Rh-2-Textile Rh-3-Textile Rh-1-Textile Rh-2-Textile Rh-3-Textile
HRSV-2 0.2 0.2 0.1
HAdV-5 000000
The newly synthesized rhodamine B-conjugated compounds shown weak virucidal
activity. Rh-3 is more potent as opposed to Rh-1 and Rh-2 (see Figures 12 and 13).
Molecules 2021, 26, x FOR PEER REVIEW 13 of 20
easy release and reaction with the viruses in solution, which explains the low virucidal
activity of the fabrics to the tested viruses.
Table 2. Virucidal effect of new rhodamine B-conjugated hemorphin-4 analogues against human
respiratory syncytial virus (HRSV-S2) and Human adenovirus serotype 5 (HAdV-5) after 30 min/60
min.
Virus Δlog 30 min Δlog 60 min
Rh-1 Rh-2 Rh-3 Rh-1 Rh-2 Rh-3
HRSV-2 0.2 0.4 0.2 1.3 1.3 1.7
HAdV-5 0 0 0 0 0 0
Table 3. Virucidal effect of new cotton fabrics dyed with rhodamine-peptides against human res-
piratory syncytial virus (HRSV-S2) and human adenovirus C serotype 5 (HAdV-5) after 30 min/60
min.
Virus Δlog 30 min Δlog 60 min
Rh-1-textile Rh-2-textile Rh-3-textile Rh-1-textile Rh-2-textile Rh-3-textile
HRSV-2 0.2 0.2 0.1
HAdV-5 0 0 0 0 0 0
The newly synthesized rhodamine B-conjugated compounds shown weak virucidal
activity. Rh-3 is more potent as opposed to Rh-1 and Rh-2 (see Figures 12 and 13).
Our experimental data suggest that in the new hybrid peptide compounds contain-
ing a rhodamine B residue, not only the position of the modification but also the nature
and length of the amino acid sequence leads to significant changes in peptide activity and
affinity. The results suggest that incorporation of different amino acids at the N-terminus
of the hemorphin-4 scaffold deserve further evaluation in antiviral and virucidal effects.
(a) (b)
Figure 12. Antiviral activity curve (in red) and cytotoxicity curve (in black) of: (a) Rh-1 and (b) Rh-2.
Figure 12. Antiviral activity curve (in red) and cytotoxicity curve (in black) of: (a) Rh-1 and (b) Rh-2.
Molecules 2021, 26, x FOR PEER REVIEW 14 of 20
Figure 13. Antiviral activity curve (in red) and cytotoxicity curve (in black) activity of Rh-3.
The cytotoxicity data and antiviral activity of the compounds against human respir-
atory syncytial virus (HRSV-S2) and human adenovirus C serotype 5 (HAdV-5) in HEp-2
cell culture are shown in Table 4.
Table 4. Cytotoxicity and antiviral activity of new rhodamine-peptides against human respiratory
syncytial virus (HRSV-S2) and Human adenovirus C serotype 5 (HAdV-5) in HEp-2 cell culture.
Compound
Cytotoxicity Antiviral Activity
CC50 (µM/mL) in
HEp-2 cells
HRSV-S2 HAdV-5
IC50 (µM
/
mL) SI IC50 (µM
/
mL) SI
Rh-1 35 NA - NA -
Rh-2 23 NA - NA -
Rh-3 113 NA - NA -
3. Materials and Methods
3.1. Synthesis of the Peptides (Rh-1, Rh-2, and Rh-3)
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 follows: Tyr—as Nα-Fmoc-Tyr(tBu)-OH, Thr—as Nα-Fmoc-Thr(t-Bu)-OH,
and Trp—as Nα-Fmoc-Trp(Boc)-OH.
The solid-phase peptide synthesis by Fmoc chemistry was used to obtain new rho-
damine B-conjugated hemorphin-4 analogues. The Fmoc-Rink-Amide MBHA resin was
used as solid phase carrier to obtain the C-terminal amide derivatives and 2-(1H-benzotri-
azole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) was used as a coupling
reagent. The coupling reactions were performed using for amino acid/TBTU/HOBt/DI-
PEA/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 al-
cohol (3 × 1 min) and CH2Cl2 (3 × 1 min). The coupling and deprotection reactions were
checked by the Kaiser test [45,46]. The cleavage of the synthesized peptide from the resin
was done, using a mixture of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilan (TIS)
Figure 13. Antiviral activity curve (in red) and cytotoxicity curve (in black) activity of Rh-3.
Our experimental data suggest that in the new hybrid peptide compounds containing
a rhodamine B residue, not only the position of the modification but also the nature and
length of the amino acid sequence leads to significant changes in peptide activity and
affinity. The results suggest that incorporation of different amino acids at the N-terminus
of the hemorphin-4 scaffold deserve further evaluation in antiviral and virucidal effects.
Molecules 2021,26, 6608 14 of 19
The cytotoxicity data and antiviral activity of the compounds against human respira-
tory syncytial virus (HRSV-S2) and human adenovirus C serotype 5 (HAdV-5) in HEp-2
cell culture are shown in Table 4.
Table 4.
Cytotoxicity and antiviral activity of new rhodamine-peptides against human respiratory syncytial virus (HRSV-S2)
and Human adenovirus C serotype 5 (HAdV-5) in HEp-2 cell culture.
Compound
Cytotoxicity Antiviral Activity
CC50 (µM/mL) in HEp-2 Cells HRSV-S2 HAdV-5
IC50 (µM/mL) SI IC50 (µM/mL) SI
Rh-1 35 NA - NA -
Rh-2 23 NA - NA -
Rh-3 113 NA - NA -
3. Materials and Methods
3.1. Synthesis of the Peptides (Rh-1, Rh-2, and Rh-3)
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
follows: Tyr—as N
α
-Fmoc-Tyr(tBu)-OH, Thr—as N
α
-Fmoc-Thr(t-Bu)-OH, and Trp—as
Nα-Fmoc-Trp(Boc)-OH.
The solid-phase peptide synthesis by Fmoc chemistry was used to obtain new rhodamine
B-conjugated hemorphin-4 analogues. The Fmoc-Rink-Amide MBHA resin was used as
solid phase carrier to obtain the C-terminal amide derivatives and 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) was used as a coupling reagent. The
coupling reactions were performed using for amino acid/TBTU/HOBt/DIPEA/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 CH
2
Cl
2
(3
×
1 min). The coupling and deprotection reactions were checked
by the Kaiser test [
45
,
46
]. 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
compounds as a powder. The crude peptides were dissolved in H
2
O and acetonitrile
was added until complete dissolving was observed. The peptides were obtained as white
powders with a purity of >97% as determined by analytical HPLC. The structures were
confirmed by high-resolution electrospray mass spectrometry. The purity of the peptides
was monitored on a reversed-phase high-performance liquid chromatography (RP-HPLC),
column: SymmetryShield
TM
RP-18, 3.5
µ
, (50
×
4.6 mm), flow: 1 mL/min, H
2
O (0.1%
TFA)/CH
3
CN (0.1% TFA), gradient 0
100% (45 min) and 100% (5 min). The crude
peptides were purified using semi-preparative HPLC, column XBridge
TM
Prep C18 10
µ
m
(10
×
250 mm), flow: 5 mL/min, H
2
O (0.1% TFA)/CH
3
CN (0.1% TFA), gradient 20
100%
(50 min). The analytical data for the synthesized peptides (Supplementary Materials)
prepared were as follows: compound Rh-1 t
R
25.90 min, 1045.5062 calculated [M + H
+
],
1046.5051 observed [M + H
+
]; compound Rh-2 t
R
26.60 min, 1059.5218 calculated [M + H
+
],
1060.5421 observed [M + H
+
]; compound Rh-3 t
R
27.20 min, 1073.5375 calculated [M + H
+
],
1074.5581 observed [M + H
+
]. The rhodamine B-conjugated hemorphin-4 analogues were
checked by optical rotation in methanol (c= 0.01) at 20
C, [
α
]
58920
as follows: compound
Rh-1—40, compound Rh-2—34, compound Rh-3—48.
Molecules 2021,26, 6608 15 of 19
3.2. Physicochemical Characterization
3.2.1. Spectral Measurements
“Varian-Cary” Spectrophotometer has been used for UV-vis spectrophotometric mea-
surements. The fluorescence spectra were recorded on a Perkin Elmer LS55 spectrophotome-
ter at the same concentrations. The concentrations of the compounds in the triethylamine
and double distilled water spectral solutions are as follows: Rh-1:
C = 4.09 ×105mol L1
;
Rh-2: C = 3.63
×
10
5
mol L
1
; Rh-3: C = 3.98
×
10
5
mol L
1
. All used reagent were
analytical grade. The kinetic data for the degree of open/closed of spirolactam ring were ob-
tained according to Equations (1) and (2) and the rate constant k was fitted by Equation (2)
(openclose of the ring) taking into account first-order of the reactions [39]:
ln (I0/It) = k.t (1)
lnII0
IIt=k.t (2)
where (I
0
), (I
t
), and (I
) refer to the signal proportional to the initial-, time t-, and final
intensity of emission, respectively.
The IR spectrum was recorded in potassium bromide (KBr) pellet with a Varian 660
FTIR spectrophotometer the spectra in the 4000–500 cm
1
range using a Fourier Transform
Infrared Spectroscopy (FT-IR). The sample was scanned 256 times with a resolution of
2 cm
1
. 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., San Jose, CA, USA) equipped with TurboFlow TM Transcend chro-
matography system (Thermo Fisher Scientific Inc., San Jose, CA, USA) and heated electro-
spray ionization (HESI II) source. Data acquisition and processing were done by XCalibur
®
2.4 software (Thermo Fisher Scientific Inc., San Jose, CA, USA). The instrumental parame-
ters 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/zat 140,000 resolution.
Optical rotations were recorded on an MCP200 modular circular polarimeter (Anton
Paar Opto Tec GmbH, Seelze, Germany).
3.2.2. Potentiometric Titration
A digital pH-meter (Jenway) was used for potentiometric titrations of the com-
pounds in order to determinate pKa and pI values. For each titration, 5.00 mL from
stock water solutions of the rhodamine derivatives were titrated with standardized base
(
0.0112 mol L1
NaOH). Data (volume of titrant vs. pH) were processed by Origin8Pro
software for determination of pKa of the sample.
3.3. Cotton Fabric Modification and Characterization
3.3.1. Functionalization of Cotton Fabric with Chloroacetyl Chloride
Cotton fabric (140 g/m
2
) was modified with 10% (v/v) chloroacetyl chloride (ClCOCH
2
Cl)
in N,N-dimethylformamide at 25
C for 2 h. The solution was of liquor to goods ratio of
20:1. Then the fabric has been removed from the solution, washed thoroughly with water,
and dried in the open air [47].
3.3.2. Dyeing of Functionalized Cotton Fabric with Rh-1, Rh-2, and Rh-3
The dyeing solution of rhodamine-peptide derivatives were prepared by dissolving
each dye in DMF and water (the ratio 1:14.5). The modified with chloroacetyl chloride
cotton fabric was impregnated with relevant dye solution (2.0 owf %) at a liquor to goods
ratio 5:1, and then dried at room temperature for 30 min and at 50
C for 60 min. The
colored cotton fabric has been washed thoroughly with water.
Molecules 2021,26, 6608 16 of 19
3.3.3. Characterization of Cotton Fabrics
The color coordinates (L* a* b*) and the reflectance spectra (R%) of dyed cotton fabrics
have been determined by using Datacolor Spectraflash SF300 spectrophotometer (Datacolor,
NJ, USA) and Micromatch 2000
®
software. The samples were measured under illuminant
D65 using the 10
standard observer. A non-treated cotton fabric has been used for the
color difference quantification [48].
For the assessment of the depth of color and the obtained shade from rhodamine-
peptides the Kubelka–Munk theory and Equation (3) has been applied.
K/S = (1 Rλ)2/2 ·Rλ(3)
where K is the light absorption coefficient, S is the light scattering coefficient, and R
λ
is the
reflectance of a dyed fabric.
3.3.4. Fastness Testing
The dyed fabrics were tested for colorfastness at washed with water and with soap
water solution of the dyed material following standard procedures given in [
49
]. The
UV-Vis spectra recording and monitored the absorption at
λ
= 561 nm corresponding to
the analytical signal of the peptide derivatives on the solutions after washing of the fabric,
immediately and after 1 and 24 h were carried out. The soap solutions were pre-diluted
twice with d.H2O and filtered through a paper filter to measure the absorbed radiation.
3.4. Virology
3.4.1. Cytotoxicity Assay
Inoculation of monolayer cells in 96-well plates (Costar
®
, Corning Inc., Kennebunk,
ME, USA) was performed with 0.1 mL/well-containing concentrations of the compounds
diluted in a maintenance medium. Cells were incubated in a humidified atmosphere
at 37
C and 5% CO
2
for 48 h. After microscopic evaluation, the maintenance medium
containing the test compound was removed, cells were washed, and 0.1 mL of maintenance
medium supplemented with 0.005% neutral red dye was added to each well, and cells
were incubated at 37
C for 3 h. After incubation, the neutral red day was removed, and
cells were washed once with PBS, and 0.15 mL/well desorb solution (1% glacial acetic acid
and 49% ethanol in distilled water) was added. The optical density (OD) of each well was
read at 540 nm in a microplate reader (Biotek Organon, West Chester, PA, USA). The 50%
cytotoxic concentration (CC
50
) was defined as the material concentration that reduced the
cell viability by 50% when compared to untreated control.
3.4.2. Antiviral Activity Assay
Antiviral screening was based on the viral yield reduction technique. Cytopathic
effect (CPE) inhibition test used confluent cell monolayer in 96-well plates infected with
100 CCID
50
in 0.1 mL. After 1 h of virus adsorption, the extract was added in various
concentrations and cells were incubated for 48 h at 37
C. The viable cells were stained
according to the neutral red uptake procedure and the percentage of CPE inhibition
for each concentration of the test sample was calculated using the following formula:
% CPE = [OD
test sample
OD
virus control
]/[OD
toxicity control
OD
virus control
]
×
100, where
OD
test sample
is the mean value of the ODs of the wells inoculated with virus and treated
with the test sample in the respective concentration, OD
virus control
is the mean value of the
ODs of the virus control wells (with no compound in the medium) and OD
toxicity control
is the mean value of the ODs of the wells not inoculated with virus but treated with the
corresponding concentration of the test sample. The 50% inhibitory concentration (IC
50
)
was defined as the concentration of the material that inhibited 50% of viral replication
when compared to the virus control. The selectivity index (SI) was calculated from the ratio
CC50/IC50 .
Molecules 2021,26, 6608 17 of 19
3.4.3. Virucidal Assay
Samples of 1 mL, containing human respiratory syncytial virus (HRSV-S2) (105,3
CCID50) or respectively human adenovirus type 5 (HAdV5) (106,3 CCID50), and tested
compound in its maximum tolerable concentration (MTC) were contacted in a 1:1 ratio
and subsequently stored at room temperature for different time intervals (30 and 60 min).
Then, the residual infectious virus content in each sample was determined by the end-point
dilution method, and lgs as compared to the untreated controls were evaluated.
Our approach to this test for virocidal effect of fabrics dyed with Rh-1, Rh-2 and Rh-3
was to cut identical pieces of the textiles (1 cm
2
) and immerse them in a viral suspension
(100
µ
L) for the respective times (30 and 60 min). We used non dyed textiles of the same
material for the control. The virus suspension was recovered by exhaustion after the
appropriate time has elapsed. The residual infectious virus content was determined by the
end-point dilution method followed by evaluation of
lgs through comparison between
each sample and control.
4. Conclusions
A systematic study of three novel rhodamine-peptide derivatives for textile dyeing has
been presented. A modified with chloroacetyl chloride cotton fabric was used, which facili-
tated the deposition and retention of the new compounds. An intense and even color with
good moisture resistance is obtained. A slight difference in the color characteristics of the
tissues was found by colorimetric analysis. By using combined experimental approaches,
the structural-textile application has been investigated by UV-Vis and fluorometric meth-
ods. Antiviral and virucidal activities of both the peptide-rhodamine B compounds and the
dyed textile material were also studied. The most potent is Rh-3 peptide analogue, which is
an analogue of hemorphin-4 containing a rhodamine B residue at the N-terminus and a hy-
drophobic -
γ
-Abu-Tyr-Pro-Trp-Thr-CONH
2
amino acid sequence of the peptide molecule.
Spectral studies have shown that the color of the aqueous solutions of compounds depends
on their structural behavior and the type of solvent. In a polar protic solvent such as water,
regardless of the pH of the medium, the compounds showed stability of ring-opened form
and fluorescence on of the compounds without being affected by the number of methylene
groups in the structure of the peptide moiety. The steric effect is more pronounced in the
aprotic solvent of the compounds and the effect depends on the type of peptide chain.
These structural characteristics are directly related to the dyeing of textile materials.
Supplementary Materials:
The following are available online, Figure S1. ESI-MS spectrum of Rh-1;
Figure S2. ESI-MS spectrum of Rh-2; Figure S3. ESI-MS spectrum of Rh-3.
Author Contributions:
Conceptualization, P.T., S.G. and D.S.; methodology, P.T., S.G. and D.S.;
software, S.G. and P.T.; formal analysis, P.T., S.G., P.P., D.S., I.G., P.G. and I.N.; investigation, P.T., S.G.,
P.P., D.S., I.G., P.G. and I.N.; writing—original draft preparations, P.T., S.G. and D.S.; writing—review
and editing—P.T., S.G., D.S. and I.G.; visualization, P.T., S.G. and D.S.; project administration, P.T. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Bulgarian National Scientific Fund project K
Π
-06-
Д
K1/11 of
the Ministry of Education and Science, Bulgaria.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Material.
Acknowledgments:
This work was financial supported by the Bulgarian National Scientific Fund
project K
Π
-06-ДK1/11 (title: “Textile materials to prevent the spread of SARS-CoV2 and other
pathogens”) of the Ministry of Education and Science, Bulgaria.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
Molecules 2021,26, 6608 18 of 19
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