Introduction of isothiuronium surfactant series: Synthesis, structure-dependent aggregation overview and biological activity

Article abstract of DOI:10.1016/j.molliq.2020.114721

For a homologous series of isothiuronium surfactants (S-alkylisothiuronium bromides, C_nSU, where n = 10, 12, 14, 16, 18), a synthesis procedure is described and aggregation properties are comprehensively characterized by a variety of techniques. Krafft temperature and critical micelle concentration (cmc) are obtained by methods of conductometry, tensiometry, spectrophotometry, fluorimetry. Using dynamic light scattering and NMR diffusometry, aggregate sizes in aqueous solutions were determined. The aggregation numbers of C_nSU systems were estimated by alternative methods. An increase in the length of the alkyl tail from 10 to 16 carbon atoms leads to a decrease in cmc from 16 to 0.5 mM with a decrease in aggregation numbers. S-alkylisothiuronium bromides exhibit solubilization capacity toward a hydrophobic dye Orange OT which is 2–3 times higher than that of alkyltrimethylammonium analogues. Improved solubilization characteristics along with antimicrobial properties manifested at the concentrations of low hemolytic activity allow us to recommend these amphiphiles for the fabrication of soft nanocontainers for hydrophobic guests showing their own biological functionality.

Full text of DOI:10.1016/j.molliq.2020.114721

MOLLIQ-114721; No of Pages 11
Journal of Molecular Liquids xxx (2020) xxx
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Introduction of isothiuronium surfactant series: Synthesis,
structure-dependent aggregation overview and biological activity
F.G. Valeeva a, T.R. Karimovaa, R.V. Pavlov a, D.I. Bakhtiyarov b, A.S. Sapunovaa, K.A. Ivshin a, O.N. Kataeva a,
a
a
b
a
a
G.A. Gaynanovaa, , V.V. Syakaev , A.D. Voloshina , I.V. Galkina , Sh.K. Latypov , L.Ya. Zakharova
⁎
a
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS, 8 Arbuzov str., 420088, Kazan, Russian Federation
Kazan Federal University, 18 Kremlyovskaya str., 420008 Kazan, Russian Federation
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2020
Received in revised form 1 November 2020
Accepted 5 November 2020
Available online xxxx
For a homologous series of isothiuronium surfactants (S-alkylisothiuronium bromides, CnSU, where n = 10, 12,
14, 16, 18), a synthesis procedure is described and aggregation properties are comprehensively characterized by
a variety of techniques. Krafft temperature and critical micelle concentration (cmc) are obtained by methods of
conductometry, tensiometry, spectrophotometry, fluorimetry. Using dynamic light scattering and NMR
diffusometry, aggregate sizes in aqueous solutions were determined. The aggregation numbers of CnSU systems
were estimated by alternative methods. An increase in the length of the alkyl tail from 10 to 16 carbon atoms
leads to a decrease in cmc from 16 to 0.5 mM with a decrease in aggregation numbers. S-alkylisothiuronium bro-
mides exhibit solubilization capacity toward a hydrophobic dye Orange OT which is 2–3 times higher than that of
alkyltrimethylammonium analogues. Improved solubilization characteristics along with antimicrobial properties
manifested at the concentrations of low hemolytic activity allow us to recommend these amphiphiles for the fab-
rication of soft nanocontainers for hydrophobic guests showing their own biological functionality.
© 2020 Elsevier B.V. All rights reserved.
Keywords:
Isothiuronium cationic surfactant
Synthesis
Self-assembly
Solubilization
Aggregation number
1. Introduction
[17]. But sometimes such surfactants have a high production cost due
to synthesis aspects. Another trend is application of biodegradable sur-
Surfactants practical applications, such as nanoparticle synthesis,
micellar catalysis, emulsion polymerization, solubilization of biologi-
cally active and medicinal substances, detergent industry and oil pro-
duction, are based on their ability to reduce surface tension, form
nanoscale aggregates and influence wettability [1–8]. Cationic surfac-
tants have a special place among the others [2,9,10], since interest in
them is growing quickly due to applications in cosmetics and
pharmaceutics [11], corrosion inhibition [12] and nanomaterial synthe-
sis [13]. The synthesis of new amphiphilic compounds with the intro-
duction of functional fragments into the head group or the
replacement of a charge-bearing atom is a worldwide practice in the
search for new nanocontainers, nanoreactors, antimicrobial composi-
tions based on surfactants. The bulk of the experimental material in
this direction was obtained by the example of ammonium salts. A new
generation of surfactants should, along with the preserved properties,
have less toxicity to living organisms [14,15]. One of the trends in reduc-
ing toxicity is the transition to gemini surfactants, since they are charac-
terized by very low critical micelle concentration (cmc) values, which
allows working with them in the micro- and millimolar concentration
ranges [16]. They are of increasing popularity in many fields of study
factants [14,18]. Researchers implement biodegradable parts such as
aminoacid, carbamate, amide or ester groups to lower ecological after-
math [19–23].
Compounds with an isothiuronium fragment attracted our atten-
tion, since this functional group has a number of practically useful prop-
erties, including antibacterial [24–26], catalytic [27,28], anti-
tuberculosis [29] activities, and also plays an important role in many
chemical and biological processes, for example, they have been ex-
plored as an agonists of GABA-type receptors [30], short-chain (<C4)
S-alkylisothioureas can be considered to be prodrugs of the aldehyde
dehydrogenase inhibitor cyanamide [31]. It is known that the
isothiuronium group sewn to the surface of halloysite is able to extract
uranium [32] from aqueous solutions, and polystyrene – divinylbenzene
resins functionalized with isothiouronium allows efficient binding of
palladium from acidic solutions [33]. A series of aryl- and alkyl-
substituted isothiuronium salts was synthesized in [34] and it was
shown that alkyl substituents exhibit high anticancer activity and selec-
tivity for leukemia cells.
In the large volume of articles devoted to the synthesis and study of
the properties of isothiuronium salts, only a few describe the properties
of amphiphilic derivatives [26,35–38]. Based on the series of
azobenzene isothiouronium salts of different alkyl chains (propyl,
hexyl and dodecyl), metal complexes were obtained, their surface-
⁎
Corresponding author.
E-mail address: ggulnara@bk.ru (G.A. Gaynanova).
https://doi.org/10.1016/j.molliq.2020.114721
0167-7322/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: F.G. Valeeva, T.R. Karimova, R.V. Pavlov, et al., Introduction of isothiuronium surfactant series: Synthesis, structure-
dependent aggregation overview..., Journal of Molecular Liquids, https://doi.org/10.1016/j.molliq.2020.114721

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
active properties and biological activity toward the pathogenic Gram-
negative bacteria, Gram-positive bacteria, and fungi [26]. The results in-
dicate that the copper complexes of the synthesized azobenzene
isothiouronium salts have a relatively higher biocidal activity than the
parent salts.
The authors of [35] synthesized a number of new cationic surfac-
tants, isothiuroniumethylalkyldimethylammonium bromides, which
contain split hydrophilic isothiuronium head groups covalently at-
tached to tertiary amines with alkyl chain of C8-C12. These surfactants
2. Materials and methods
2.1. Chemicals
Fluorescent probe pyrene (≥99%, Sigma Aldrich), hydrophobic dye
Orange OT (75%, Sigma Aldrich), cetylpyridinium bromide (99%,
AppliChem) were used as received. Purified water (18.2 MΩ cm resis-
tivity at 25 °C) from Direct-Q 5 UV equipment was used for all solution
preparation.
have high cmc values from 300 mM for C8 and up to 10 mM for C12
,
which allows them to compact plasmid DNA. A practically useful prop-
erty of the isothiuronium group is that it can be hydrolyzed to form a
sulfhydryl group at pH above 8.5 and a temperature closer to 333 K.
As a result of hydrolysis, surfactants with a lower cmc value are formed,
and the thiol group can undergo further reaction after surfactant accu-
mulation on the DNA matrix to form a lipid containing a disulfide bond
and two alkyl chains. Template self-assembly of vesicles from a single-
tailed surfactant isothiuroniumethylhexadecyldimethylammonium
bromide followed by dimerization of surfactant molecules was de-
scribed [36]. This leads to additional stability of nanocontainers
under changing environmental conditions (pH, temperature, salt
concentration).
The surface properties of the cationic surfactant dodecyl
isothiouronium bromide in a mixed solvent of water and polyethylene
glycol with different molecular weights (ethylene glycol, triethylene
glycol and polyethylene glycol-600) are described in [37]. By the
methods of tensiometry and conductometry, it was established that
an increase in the molecular weight of glycols leads to an increase in
the cmc value, and an increase in the mass fraction of glycol in the
mixed solution, on the contrary, decreases the cmc value.
2.2. General synthesis procedure of S-n-alkylisothiuronium bromides
Firstly, synthesis of S-alkylisothiuronium salts (dodecyl, hexadecyl
and octadecyl derivatives) was described in [39] without specifying
the structural formula of the obtained salts. Our group planned to syn-
thesize the whole range of compounds starting with a decyl derivative,
the procedure was modified as follows.
A solution of 1.0 g
(0.01314 mol) of thiourea in 25 mL of ethyl alcohol with a small addi-
tion of diethyl ether (9:1) was heated in a water bath until the crystals
of thiourea were completely dissolved. Equimolar amount of alkyl bro-
mide was then added dropwise with stirring. The reaction mixture was
heated in a flask under reflux for an appropriate time, which is listed for
each compound separately, at a temperature of 78 °C. After completion
of the reaction, a white precipitate formed upon cooling the reaction
mixture. The precipitate was dried on a rotary evaporator and recrystal-
lized - dissolved in acetone and precipitated by diethyl ether. The white
precipitate was dried in vacuo. The structures of surfactants were con-
firmed by elemental analysis, IR (Spectrum Two FT-IR spectrometer)
and 1H and 13C NMR spectroscopy. IR and NMR spectra are given in SI
(Figs. S1-S5).
The surface properties of polyvinyl alcohol and three synthesized cat-
ionic surfactants were studied, namely S-dodecyl-, S-tetradecyl-, and S-
hexadecyl isothiuronium bromide surfactant [38]. The cmc value for the
dodecyl derivative is 4.2 mM, for tetradecyl - 1.95 mM, for hexadecyl -
0.58 mM in an individual aqueous solution. The formation of a mixed
polymer compositions with an isothiuronium surfactant at molar ratios
of 1:9, 3:7, 5:5, 7:3, and 9:1 lead to an increase in cmc values. It is impor-
tant to note that all studies were carried out in aqueous solutions at 25°С,
and the cmc values were determined by only one method — the measure-
ment of surface tension by the ring detachment method.
Taking into account all of the above, the aim of this work was to syn-
thesize a number of S-alkylisothiuronium bromides (CnSU) (Fig. 1,
where n = 10, 12, 14, 16, 18) according to the original procedure and
to prove the exact structure of the obtained compounds using the
methods of IR-, 1H and 13C NMR spectroscopy, x-ray structural analysis.
Then planned to study their self-assembly in an aqueous solution by a
complex of physicochemical methods (tensiometry, conductometry,
spectrophotometry, dynamic and electrophoretic light scattering, fluo-
rimetry, NMR) to obtain comprehensive information about
nanocontainers based on relatively new types of surfactants with an as-
sessment of their biological activity as well.
2.3. Characterization of S-n-alkylisothiuronium bromides
2.3.1. S-n-decylisothiuronium bromide (C10SU)
Reflux time: 5 h. Yield: 91%, melt. = 103.6°С. Decomp. 226.7°С. M.w.
279.30.
Elem. anal. Found: % С, 44.23; Н, 8.67; N, 9.30; S, 10.69. C11H25BrN2S
calc. %: С, 44.44; Н, 8.48; N, 9.42; S, 10.79. IR, ν, cm−1: 3300–3000 (NH),
2918–2850 (CH), 1649 (C=N). 1H NMR (CDCl3, 500 MHz, δ, ppm, J, Hz):
0.892 (t, 3JHH = 7.1 Hz, 3H, CH3), 1.20–1.50 (m, 14H, (CH2)7), 1.75 (m,
3
2H, CH2), 3.32 (t, JHH = 7.2 Hz, 2H, CH2S), 7.95 (br, 2H, NH2), 9.26
(br, 2H, NH2). 13C{H} NMR (CDCl3, 126 MHz, δ, ppm): 172.7 (C1), 32.4
(C2), 32.0–22.9 (C4-C9), 28.6 (C3), 14.3 (C10).
2.3.2. S-n-dodecylisothiuronium bromide (C12SU)
Reflux time: 6 h. Yield: 87%, melt. = 109.5°С. Decomp. 225°С. M.w.
327.35.
Elem. anal. Found: % С, 47.79; Н, 8.81; N, 8.31; S, 9.98. C13H29BrN2S
calc. %: С, 47.99; Н, 8.98; N, 8.61; S, 9.86. IR, ν, cm−1: 3300–3000
(NH), 2912–2848 (CH), 1625 (C=N). 1H NMR (CDCl3, 500 MHz):
3
0.892 (t, JHH = 7.1 Hz, 3H, CH3), 1.20–1.50 (m, 18H, (CH2)9), 1.738
Fig. 1. Chemical formulas of compounds used in the work.
2

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
(m, 2H, CH2), 3.294 (t, 3JHH = 7.2 Hz, 2H, CH2S), 7.659 (br, 2H, NH2),
9.265 (br, 2H, NH2). 13C{H} NMR (CDCl3, 126 MHz, δ, ppm): 172.7
(C1), 32.3 (C2), 32.1–22.9 (C4-C11), 28.4 (C3), 14.3 (C12). 15N NMR
(CDCl3, 51 MHz, δ, ppm): 109.1 (N1,N3).
at nitrogen atoms were revealed from difference Fourier map and re-
fined isotropically with geometry constraints. Alkyl group is disordered.
CCDC 2041059 contains the supplementary crystallographic data for
this paper.
Crystal data: formula C16H45ClN12S6, M = 633.45 g/mol, monoclinic,
space group P21/c (No. 14), Z = 4, a = 24.956(2) Å, b = 8.4210(7) Å,
2.3.3. S-n-tetradecylisothiuronium bromide (C14SU)
Reflux time: 7 h. Yield: 92%, melt. = 113.2°С. Decomp. 225°С. M.w.
353.40.
c = 15.7289(13) Å, β = 96.517(4)°, V = 3284.1(5) Å3, ρcalc
=
1.281 g cm−3, μ = 0.526 mm−1, 68,603 reflections collected
(−33 ≤ h ≤ 32, −11 ≤ k ≤ 11, −20 ≤ l ≤ 20), θ range = 2.554° to
28.430°, 8241 independent (Rint = 0.1299) and 4667 observed reflec-
tions [I ≥ 2 σ(I)], 439 refined parameters, R = 0.0589, wR2 = 0.1424,
Elem. anal. Found: % С, 50.78; Н, 9.62; N, 7.61; S, 9.25. C15H33BrN2S
calc. %: С, 50.98; Н, 9.41; N, 7.93; S, 9.07. IR, ν, cm−1: 3300–3000
(NH), 2915–2847 (CH), 1649 (C=N). 1H NMR (CDCl3, 500 MHz): 0.89
(t, 3JHH = 7.1 Hz, 3H, CH3), 1.20–1.50 (m, 14H, (CH2)11), 1.78 (m, 2H,
max.(min.) residual electron density 0.846 (−0.845) e Å−3
.
3
CH2), 3.32 (t, JHH = 7.2 Hz, 2H, CH2S), 7.48 (br, 2H, NH2), 9.30 (br,
2H, NH2). 13C{H} NMR (CDCl3, 126 MHz, δ, ppm): 172.8 (C1), 32.5
(C2), 32.1–22.9 (C4-C13), 28.7 (C3), 14.3 (C14).
2.3.7. Thermogravimetry and differential scanning calorimetry
Melting and decomposition temperatures were studied by simulta-
neous thermogravimetry and differential scanning calorimetry
thermoanalyzer STA 449C Jupiter (Netzsch) coupled with quadrupolar
mass spectrometer QMS 403C Aeolos in the temperature range of
40–500 °C (Fig. S6). The heating rate was linear at 10°С/min. Approxi-
mately 10 mg of the compound was analyzed in an aluminum crucible
in dynamic argon atmosphere with flowrate of 75 mL/min.
2.3.4. S-n-hexadecylisothiuronium bromide (C16SU)
Reflux time: 8 h. Yield: 88%, melt = 114.7°С. Decomp. 222.3°С. M.w.
381.46.
Elem. anal. found: % С, 53.40; Н, 9.49; N, 7.15; S, 8.67. C17H37BrN2S
calc. %: С, 53.53; Н, 9.78; N, 7.34; S, 8.41. IR, ν, cm−1: 3300–3000
(NH), 2915–2848 (CH), 1648 (C=N). 1H NMR (CDCl3, 500 MHz): 0.90
(t, 3JHH = 7.1 Hz, 3H, CH3), 1.20–1.50 (m, 14H, (CH2)13), 1.78 (m, 2H,
2.4. Surface tension measurement
3
CH2), 3.31 (t, JHH = 7.2 Hz, 2H, CH2S), 7.36 (br, 2H, NH2), 9.45 (br,
2H, NH2). 13C{H} NMR (CDCl3, 126 MHz, δ, ppm): 172.9 (C1), 32.5
(C2), 32.1–22.9 (C4-C15), 28.6 (C3), 14.3 (C16).
The surface tension was measured using the Du Nouy ring detach-
ment method on a K6 tensiometer (Kruss, Germany) with use of an op-
timally wettable platinum ring. The measurements were carried out
manually on the surface of 10 mL solutions located in a thermostatically
controlled cell. Surface tension was measured until a constant value was
obtained indicating equilibrium. The concentration dependences of the
surface tension represent the average value of all measurements, and
the error was within 2%. Values of cmc were determined at inflection
points on the dependence of surface tension on the logarithm of surfac-
tant concentration. From the tensiometric data, thermodynamic param-
eters of surfactant interfacial adsorption and micelle formation can be
obtained [42,43]. The surface excess, Γmax, and the surface area per a
molecule, Amin, have been calculated using the Gibbs equation:
2.3.5. S-n-octadecylisothiuronium bromide (C18SU)
Reflux time: 9 h. Melt. 116.3°С. Decomp. 219.7°С. M.w. 409.51.
Elem. anal. found, % С, 53.55; Н, 10.31; N, 6.60; S, 7.70. C19H41BrN2S
calc. %: С, 53.73; Н, 10.09; N, 6.84; S, 7.83. IR (KBr), ν, cm−1: 3300–3000
(NH), 2915–2847 (CH), 1649 (C=N). 1H NMR (CDCl3, 500 MHz): 0.90
(t, 3JHH = 7.1 Hz, 3H, CH3), 1.20–1.50 (m, 30H, (CH2)15), 1.67 (m, 2H,
3
CH2), 3.74 (t, JHH = 7.2 Hz, 2H, CH2S), 7.01 (br, 2H, NH2), 9.72 (br,
2H, NH2). 13C{H} NMR (CDCl3, 126 MHz, δ, ppm): 172.0 (C1), 31.6
(C2), 31.5–22.3 (C4-C17), 28.1 (C3), 13.7 (C18).
2.3.6. X-ray crystallography
We managed to take a single crystal x-ray, but only for S-
decylisothiuronium chloride, which was synthesized analogously to
the bromide using decyl chloride. X-ray structural analysis data of the
isothiuronium salts synthesized is shown in Fig. 2.
Data set for single crystal C16H45ClN12S6 was collected on a Bruker
AXS Kappa APEX II CCD diffractometer with graphite-monochromated
Mo Kα radiation (λ = 0.71073 Å). The structure was solved by direct
methods using APEX3 for data collection, SAINT for data reduction,
SHELXT [40] for structure solution, SHELXL [40] for structure refinement
by full-matrix least-squares against F2, and SADABS [41] for multi-scan
absorption correction. Hydrogen atoms at carbon atoms were placed
into calculated positions and refined as riding atoms. Hydrogen atoms
1
Γmax
¼
lim ðdπ=dlogCC!cmcÞ
2:3nRT
А
min = 1018/(NA × Гmax
)
where R = 8.31 J mol−1 K−1 (gas constant), π is the surface pressure
obtained from the surface tension of water minus the surface tension of
the surfactant solution, and T is the absolute temperature in K. NA is Avo-
gadro number (6.02 × 1023 mol−1). The parameter n represents the
number of species at the interface the concentration of which changes
with surfactant concentration. The constant n takes the value 2 for mo-
nomeric surfactants. С is the surfactant concentration.
The value of the standard free energy of micellization (ΔGm) was cal-
culated according to equation:
ΔGm ¼ ð1 þ βÞRT ln ðcmcÞ
where β is degree of counterions binding, cmc is the concentration cor-
responding to the onset of aggregation, i.e. the molar concentration of
surfactants in monomeric (non-micellized) form.
The standard free energy of adsorption (ΔGad) is correlated with the
standard free energy of micellization by the following equation:
πCMC
ΔGad ¼ ΔGm−
Гmax
Fig. 2. S-decylisothiuronium chloride structure obtained from X-ray data.
3

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
2.5. Electrical conductivity measurement
where I0 and I are the fluorescent intensities of pyrene in the absence
and the presence of cetylpyridinium bromide; Cq, Ccmc, and CCnSU are the
concentrations of the quencher, micelles, and CnSU. Equation was de-
rived under the assumption that the probe and the quencher are en-
tirely present in the micelle phase, their distributions obey the Poisson
statistics, and the probe fluoresces only in the absence of the quencher.
The electrical conductivity (χ, μS·cm−1) was measured on an InoLab
Cond 7110 conductivity meter (WTW, Germany) using a TetraCon sen-
sor. The cmc value was determined by the kink in the graph of the con-
ductivity versus surfactant concentration. The conductivity of water,
which was used to prepare the systems under study, was taken as the
zero surfactant concentration point.
2.10. NMR diffusometry
2.6. Spectrophotometry
NMR diffusometry experiments were performed on a Bruker
AVANCE-500 spectrometer. The spectrometer is equipped with a z-
gradient inverse probehead capable of producing gradients with the
Absorption spectra were recorded on a Specord 250 Plus spectro-
photometer (Analytik Jena, Germany) using 0.5 and 1 cm thick glass cu-
vettes. To study the solubilizing ability toward the Orange OT spectral
probe, a sample of a dye weighing 0.0014 g was added to all the studied
solutions. Then the absorbance was measured at 495 nm. According to
the slope of the linear part of the concentration dependences of the re-
duced optical density at the point of maximum absorption, the solubili-
zation capacity were calculated according to equation:
strength of 50 G cm−1. Experiments were carried out at 40
0.2 °C.
Chemical shifts (δ) were reported relative to the solvent as internal
standard (HDO δ(1H) 4.70 ppm).
The Fourier transform pulsed-gradient spin-echo (FT-PGSE) experi-
ments were performed by BPP-STE-LED (bipolar pulse pair–stimulated
echo-longitudinal eddy current delay) sequence [46]. After Fourier
transformation and baseline correction, the diffusion dimension was
processed with the Bruker TopSpin software package (version 3.2). All
separated peaks were analyzed and the average values were presented
[47,48]. DOSY 1H NMR spectra of C10SU (Fig. S7) and C14SU (Fig. S8) are
given in SI.
S = b/ε,
where b is D/l = f (C) dependency slope, ε – an extinction coefficient
of Orange OT (17,400 M−1 cm−1).
2.7. Dynamic light scattering
Hydrodynamic radius (RH) has been calculated from the self-
diffusion coefficients Ds applying Stokes-Einstein equation: RH = kBT/
6πηDs, where RH is hydrodynamic radius, kB is Boltzmann constant, T
is the temperature (K), η (Pa·s) is dynamic viscosity of the solvent
and Ds is self-diffusion coefficient. The aggregation numbers of CnSU
were estimated from the ratio Nagg = (RHa/RHm)3, where RHa и RHm
are hydrodynamic radii of the aggregate and monomer CnSU molecules,
respectively.
The diameter of the aggregates was determined by dynamic light
scattering on Zetasizer Nano Instrument (Malvern, UK). The source of
laser radiation was a He-Ne gas laser with a power of 4 mW and a wave-
length of 633 nm. The effective hydrodynamic diameter was automati-
cally calculated on the device from the diffusion coefficients according
to the Stokes-Einstein equation for spherical particles. The obtained ex-
perimental data were analyzed using the Malvern DTS program. For
each system, the hydrodynamic diameter was determined as the aver-
age of at least 5 measurements.
2.11. Antimicrobial activity
The antibacterial and antifungal activity of the CnSU were investi-
gated against several pathogenic representative Gram-positive bacteria
Staphylococcus aureus ATCC 209p (Sa), Bacillus cereus ATCC 8035 (Bc);
Gram-negative bacteria Escherichia coli CDC F-50 (Ec), Pseudomonas
aeruginosa ATCC 9027 (Pa); and fungi Aspergillus niger BKMF-1119
(An), Trichophyton mentagrophytes var. gypseum 1773 (Tm), Candida
albicans 855–653 (Ca). The bacteriostatic and fungistatic activity was
studied in Muller Hinton Broth 2 (bacteria 3 × 105 cfu mL−1) and
Sabouraud dextrose broth (fungi 2 × 103 cfu mL−1). Chloramphenicol
and ketoconazole were used as standard drugs. The results were re-
corded every 24 h for 5–14 days. Cultures were incubated at 25–37 °C.
The experiment was repeated three times. The dilutions of the com-
pounds were prepared immediately in nutrient media; 5% DMSO was
added for better solubility and the test strains were not inhibited at
this concentration. The minimum inhibitory concentration (MIC) was
defined as the minimum concentration of a compound that inhibits
the growth of the corresponding test microorganism. The minimum
bactericidal (MBC) and fungicidal (MFC) concentrations were deter-
mined by adding an aliquot of the bacterial (fungal) culture to
Mueller-Hinton or Sabouraud dextrose agar in a 10 cm Petri dish and in-
cubated for 24–48 h at 25–37 °C. MBC and MFC were the minimum con-
centration in which bacterial colonies were not detected, indicating that
bacteria were killed with an efficiency of >99.9% [49].
2.8. The Krafft temperature (TK) measurement
Krafft temperatures were determined using conductometry. Surfac-
tant solutions of certain concentration higher than cmc value but within
the surfactant solubility (30 mM for C12SU, 20 mM for C14SU, 17.5 mM
for C16SU, 10 mM for C18SU) were placed in a freezer at a negative tem-
perature for at least 24 h to reach equilibrium state. Then the tempera-
ture was gradually increased using a thermostatic water bath with
conductivity measurements each minute. Once the turbid solution be-
came clear, a discontinuity in the conductivity versus temperature plot
was taken as the Krafft temperature. The measurements were con-
ducted three times.
2.9. Fluorescence spectroscopy
The fluorescence spectra of pyrene (1·10−6 mol L−1) in amphiphile
solutions were recorded in Hitachi F-7100 spectrofluorimeter (Hitachi,
Japan). Sample excitation was at a wavelength of 335 nm. Emission
spectra were recorded within 350–500 nm range. The thickness of the
cell was 10 × 10 mm. Fluorescence intensities of the first peak at
373 nm (II) and of the third peak at 384 nm (IIII) of pyrene were ob-
tained from the spectra [44]. The data were analyzed using the
Microsoft Excel and Origin Pro 8.5 software.
The aggregation numbers were measured using cetylpyridinium
bromide (1 mM stock solution) as a quencher [45]. The peak at
395 nm was used to calculate the aggregation numbers according to
the equation:
2.12. Hemolytic activity
Series of CnSU surfactants were evaluated in terms of hemolytic ac-
tivity by comparing the optical density of CnSU treated erythrocyte sus-
pension with the optical density of blood at 100% hemolysis [50]. 10%
suspension of human erythrocytes was used as an object of investiga-
tion. An erythrocytic mass was treated with heparin and washed thrice
with physiological saline (p.sal., 0.9% NaCl), centrifuged for 10 min at
I0
I
Nagg
ln
¼
ꢀ Cq
CC −Ccmc
nSU
4

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
800 rpm, and resuspended in p.sal. to make a 10% suspension. The con-
centrations of the surfactants that correspond to the MIC values for the
bacterial test strains were prepared in saline (supplemented with 5%
DMSO), and 450 μL of the compound at the corresponding dilution
was added to 50 μL erythrocyte suspension. The samples were treated
for 1 h at 37 °C and then centrifuged for 10 min at 2000 rpm. Released
hemoglobin was controlled by measuring the optical density of the su-
pernatant on Microplate reader Invitrologic (Russia) at 540 nm. Control
sample with 0% hemolysis was prepared by adding of 50 μL of 10%
erythrocyte suspension to 450 μL of p.sal., and the 100% hemolysis con-
trol sample was prepared by adding of 50 μL of 10% red blood cell sus-
pension to 450 μL of distilled water.
C
16SU : y ¼ 29 þ 1:72x ðlower partÞ; y ¼ −885 þ 24, 6x ðupperÞ
C18SU : y ¼ 106 þ 0:758x ðlower partÞ; y ¼ −1000 þ 22x ðupperÞ
The approach is based on the fact that the electrical conductivity of a
slowly defrosting solution increases with increasing temperature due to
the dissolution of surfactants [51,52]. For C12SU, the exact value of the
Krafft temperature could not be determined, since it turned out to be
less than 10°С. Since the Krafft temperature increases with increasing
length of the surfactant alkyl tail, we hypothesized that C10SU also has
a low TK. For C14SU and C16SU, the Krafft temperature is 36°С and
39°С (Table 1), respectively. It is worth noting that these are quite
high TK values. For example, for the classic series of
alkyltrimethylammonium bromides with tetradecyl and hexadecyl sub-
stituents, the Krafft temperature is 0°С and 25°С, respectively [52]. The
Krafft temperature for C18SU is 51°С (Table 1), which significantly limits
its application from a practical point of view, therefore, further studies
with this representative of the series were not carried out. It is known
that the Krafft temperature is affected by two main factors: the solubil-
ity of surfactant monomers and intermolecular attraction in the hydro-
carbon regions of crystals or in layers of polar groups. The obtained low
3. Results and discussion
3.1. Krafft temperature (TK) of CnSU series
Isothiuronium cationic surfactants starting with C14SU have limited
solubility in water. So, their Krafft temperatures were determined
(Fig. 3) using the conductometry method in order to select the operat-
ing temperature range for self-assembly studies. Linear fitting was ap-
plied to lower and upper parts of the curves and the Krafft
temperature was taken as the intersection point x-axis value (Fig. 3).
The linear fit equations from which the temperature was determined
as the intersection are the following:
TK values for the decyl and dodecyl derivatives indicate the availability
of nitrogen lone pair of the surfactant head group for the formation of
hydrogen bonds, which leads to good solubility of the surfactant.
When passing to higher homologs, an increase in intermolecular inter-
action of hydrophobic fragments in the crystalline state probably occurs,
which is reflected in high TK values.
C14SU : y ¼ 73 þ 2:7x ðlower partÞ; y ¼ −615 þ 22x ðupper partÞ
Next, self-assembly study of a new homologous series of amphi-
philic isothiuronium salts was carried out using a wide range of
physicochemical methods: tensiometry, conductometry, fluorimetry,
spectrophotometry, dynamic light scattering and NMR diffusometry,
each of which provide their own insight into the aggregation behavior
of surfactant molecules. Tensiometric data are used to obtain thermody-
namic parameters of micelle formation and interfacial adsorption. Solu-
bilization data are helpful to assess the ability of the aggregates formed
by the surfactants in study to act as drug nanocarriers. With some con-
siderations, aggregation numbers can also be obtained from solubiliza-
tion data. Fluorimetry data provide details on the polarity inside the
micellar core and aggregation numbers.
3.2. Tensiometry and conductometry data
It was found that the Krafft temperature for C16SU is 39°С, therefore,
in order to conduct a comparative analysis within the homologous se-
ries of surfactants, all the dependences were obtained at 40°С.
Tensiometry and conductometry are traditionally used to determine
the cmc. The tensiometry method is based on the ability of a surfactant
to reduce surface tension. With an increase in surfactant concentration,
surface tension decreases sharply, and after reaching a cmc, it remains
constant. The dependence of the surface tension CnSU as a function of
the concentration of the amphiphilic compound in an aqueous solution
Table 1
Krafft temperature and critical micelle concentrations of CnSU determined by tensiometry
and conductometry.
Surfactants
cmc × 103 (M)
Tensiometry
Krafft point (°С)
Conductometry
C10SU
16
4
1.3
0.5
18
5
1.8
0.45
–
C
C
C
12SU
14SU
16SU
˂ 10
36
39
Fig. 3. The dependence of the electrical conductivity of CnSU aqueous solutions on the
temperature, that was used to estimate the Krafft temperature of surfactants.
5

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
at 40°С is shown in Fig. 4,A: for all surfactants, the surface tension grad-
ually decreases to a plateau with a value of about 30 mN m−1. The cmc
values obtained from the tensiometric dependences are presented in
Table 1. As expected, an increase in the length of the alkyl tail from 10
to 16 carbon atoms leads to a decrease in the cmc values from 16 to
0.5 mM. The logarithm of the obtained cmc values on the number of car-
bon atoms in the alkyl tail is characterized by a linear dependence
(Fig. 5): y = 0.6567–0.25x (r2 = 0.9895).
Using the surface tension isotherms of micellar systems, the thermo-
dynamic characteristics of the surface layer were calculated, the values
of which are summarized in Table 2. The obtained values of surface
area per surfactant molecule (Amin) are in agreement with their struc-
ture and remain at the same level for surfactants with different hydro-
carbon chain lengths. For comparison, the CTAB has an area of about
0.32 nm2. The obtained areas of the order of 1 nm2 may be reasonable,
if we consider that the CTAB head group is sterically hindered at all
the sides except the top, so hydration is occurring axially, which does
not allow for additional water molecules that would increase the head
group equilibrium cross-section area. In the case of CnSU, the area is no-
ticeably larger, firstly, due to the larger structure of the head group, and
secondly, because it does not have hydrophobic methyl groups, which
allows for attachment of water molecules that contribute to the head
group cross section area. The delocalized nature of the CnSU head
groups allows for more hydrogen bonds from all the sides, which in
turn make the equilibrium head group area larger. These assumptions
are not supported by physicochemical methods in the current work,
Fig. 5. The logarithm of cmc and solubilization capacity of CnSU surfactants toward Orange
OT versus the number of carbon atoms, 40°С.
however an x-ray study has previously shown that thiourea group by it-
self can readily form hydrogen bonds [53]. The values of adsorption en-
ergy tend to obtain more negative values with an increase in the
number of hydrophobic carbon atoms in CnSU molecules. This is due
to the fact that longer hydrophobic parts of surfactants represent a
Fig. 4. The obtained plots that were used for determination of cmc values with various methods: (A) tensiometry, (B) conductometry (the inset shows the dependence for C10SU),
(C) Orange OT solubilization (the inset shows the dependence for C10SU), λ = 495 nm, (D) spectrofluorimetry, C(pyrene) = 1 × 10−6 M. All of these measurements were made in
H2O at 40°С.
6

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
Table 2
The solubilization capacity values (the number of moles of dye solu-
bilized per mole of surfactant) are given in Table 3, which are 2–3 times
higher than for alkylammonium analogues [51]. An increase in temper-
ature from 25 to 40°С leads to an increase in solubilization capacity from
0.0072 to 0.0087. These data can also be used to calculate aggregation
numbers by taking into account the extinction coefficient of the dye
and making an assumption that only one hydrophobic molecule is
being solubilized per aggregate [56]. The dependence of the solubiliza-
tion capacity on the number of carbon atoms in the alkyl tail is charac-
terized by a linear dependence (Fig. 5) with a good correlation
coefficient: y = 6.245× - 53.76 (r2 = 0.99968).
Values of surface excess (Гmax), surface area per surfactant molecule (Amin), standard free
energy of micellization (ΔGm) and the standard free energy of interfacial adsorption at the
air/saturated monolayer interface (ΔGad) of isothiuronium surfactants, from tensiometry
data obtained at 40 °C.
Surfactants
Г
max x 106
Amin
-ΔGad
-ΔGm
(mol·m−2
)
(nm2)
(kJ·mol−2
)
(kJ·mol−2
)
C10SU
1.47
2.44
1.87
1.60
1.1
0.9
0.9
1.0
38.9
44.7
44.5
47.7
16.4
26.7
26.4
30.2
C
C
C
12SU
14SU
16SU
3.4. Fluorescence data. DLS data
larger water displacement due to their volume. Therefore, the processes
of micelle formation and adsorption for longer surfactants are more fa-
vorable than for short ones.
The study of the behavior of a series of isothiuronium surfactants in
aqueous solutions was supplemented by the results of the method of
fluorimetry using pyrene. This method is widely used to study the prop-
erties of surfactants, due to its high sensitivity and a very low concentra-
tion of the introduced fluorophore, and it allows to determine the
micropolarity of the medium around the probe, the cmc value and the
aggregation numbers [57,58]. For C10SU and C12SU, which have good
solubility at 25°С, these parameters were determined at two tempera-
tures: 25°С and 40°С. For the other homologs, measurements were
done at 40°С.
Pyrene has a spectrum with 5 characteristic peaks. The first (II) and
third (IIII) peaks are very sensitive to the polarity of the microenviron-
ment, therefore, when the solvent is replaced, their intensities change.
In a polar medium (in water) the II/IIII ratio (polarity parameter) is in
the range of 1.8–2.0, and in a non-polar medium (for example, hexane),
around 0.6. With the addition of amphiphile with an increase in its con-
centration, the overall polarity of the pyrene environment decreases,
due to its localization in the micellar hydrophobic core. Fig. 4,D shows
the dependences of the polarity parameter on the concentration of
CnSU at 40°С, which were plotted from the fluorescence spectra of
pyrene in the presence of increasing concentrations of isothiuronium
surfactants (Fig. S12). From the dependencies shown in Fig. 4D, the
cmc values were determined, which are consistent with the cmc values
obtained by other methods. For C10SU and C12SU, the cmc values deter-
mined at 25°С (Figs. S13, S14) practically do not differ from those ob-
tained at 40°С. This can be explained by the complex dependence of
the cmc on temperature, which can be simultaneously influenced by
several factors that cancel each other out. The cmc values obtained by
fluorimetry are presented in Table 3.
Conductometry also shows aggregation in the systems under study.
It is known that the mobility of individual unassociated ions is signifi-
cantly higher than the mobility of aggregated ions. This phenomenon
is the basis of the conductometric method [54]. Fig. 4,B shows the de-
pendences of the electrical conductivity (χ) of a series of surfactants
under study at 40°С. For each surfactant, the results fit into two straight
lines with different slopes. In the region of low concentration, the
change in electrical conductivity can be attributed to a higher concen-
tration of free cations and anions of CnSU, while the inflection point
can be attributed to the onset of micelle formation (cmc values are pre-
sented in Table 1). After cmc, the change in electrical conductivity has a
lower slope, which can be explained in two ways: (1) by immobilizing
the fraction of counterions around the surface of the micelles, which ef-
fectively reduces the number of free bromide anions; (2) micelles are
less likely to promote charge transfer than free cations of surfactant
monomers due to their lower mobility. The obtained values are in
good agreement with the tensiometric data.
3.3. Solubilization properties of CnSU
The solubilization of hydrophobic compounds in micelles is the most
important property of surfactants, which determines their practical ap-
plication. In addition, solubilization of model spectral probes makes it
possible to determine the cmc values. The method is based on two ob-
servations: (1) at a solution concentration below cmc, the dye is practi-
cally not soluble; (2) when the cmc is reached, the solubility of the dye
increases sharply [55]. The ability of CnSU aggregates to act as
nanocontainers was demonstrated with hydrophobic dye Orange OT
as a probe, which has a characteristic band in the visible region at
495 nm. The absorption spectra of Orange OT in CnSU micellar solution
at 40°С are shown in Fig. S9. The dependences of the reduced optical
density on the surfactant concentration (Fig. 4,C) show points of a
sharp increase in optical density, which can be attributed to cmc points
(Table 3). The cmc determined by the solubilization method for C10SU
and C12SU at 25°С (Figs. S10, S11) practically do not differ from the
cmc at 40°С (Table 3).
At a fixed surfactant concentration (twice the cmc value), aggrega-
tion numbers (Nagg) were determined using cetylpyridinium bromide
as a quencher. The quenching spectra of pyrene fluorescence in the
presence of cetylpyridinium bromide are shown in Figs. S15, S16. Ac-
cording to the obtained spectra, linearization was carried out and the
aggregation numbers were calculated from the slope angles (Fig. 6).
Since two representatives of the series (C10SU and C12SU) are soluble
both at 25°С and 40°С, the aggregation numbers were calculated at
two temperatures.
Table 3
The cmc values and aggregation numbers derived from the fluorescence and the dye solubilization study. Z-Average from DLS.
Surfactants
Т (°С)
cmc × 103 (M)
S × 103
Aggregation number
by dye solubilization
Aggregation number
by fluorescence,
(2 x cmc, M)
Z-Average
(d, nm)
Pyrene
Orange OT
fluorescence
solubilization
C
C
10SU
12SU
25
40
25
40
40
40
16.5
16
3.2
3.1
1.1
0.4
15
16
4.1
4.4
1.5
0.45
7.2
8.7
19
21
34
46
–
110
–
37
23
15
76 (0.033)
106 (0.032)
80 (0.0063)
90 (0.0062)
36 (0.0022)
6 (0.0008)
–
366
–
285
157
135
C
C
14SU
16SU
7

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
diffusion measurements (Fig. 7, Tables S1, S2). As previously shown,
NMR-self-diffusion technique is very sensitive to the formation of
small spherical micelles, including pre-micellar aggregates [51], which
is valuable information in case of different morphology of aggregates as-
sumed. As can be seen in Fig. 7, a sharp inflection point can be observed
in the self-diffusion coefficient plot, which indicates the formation of ag-
gregates with low mobility compared to free surfactant molecules. Ag-
gregate sizes and aggregation number values were calculated as
function of surfactant concentration (Tables S1, S2). It should be noted
that for C14SU, the formation of pre-micellar aggregates, dimers, is
shown at a concentration of 0.75 mM (before the cmc value). Aggregate
sizes for both surfactants studied increase with an increase in surfactant
concentration, with hydrodynamic diameter reaching ca.2 to 6 nm.
Probably this is due to the contribution of small micelle-like particles
that represent the beginning of self-assembly of the systems.
For C14SU, aggregation numbers increase up to 40 (Table S1) at a
concentration of 5 mM (3.8 times greater than the tensiometric cmc).
This is consistent with values found by fluorimetry, provided that no
structural changes occur in this concentration range, and this is not in
essential contradiction with the dye solubilization results. For C10SU,
the aggregation numbers determined by NMR are significantly lower
than those determined by fluorimetry and dye solubilization tech-
niques. This can be due to several reasons, namely, (i) fluorescence
and dye solubilization techniques detect data based on aggregates that
contain probes in their composition, which can significantly alter the
morphological behavior, especially in the lower concentrations range
(ii) high sensitivity of NMR-self-diffusion method to the occurrence of
small micelle-like aggregates and pre-micellar assemblies; (iii)
incompact packing mode of aggregates, which may result in averaging
of self-diffusion coefficients due to exchange of surfactant molecules be-
tween aggregated and monomer state under the conditions of relatively
long NMR-self-diffusion experiments of 50 ms.
Fig. 6. The ratio of fluorescence intensity of pyrene vibronic peak at 395 nm in the absence
and the presence of cetylpyridinium bromide vs quencher concentration, that was used to
obtain aggregation numbers; C(pyrene) = 1 × 10−6 М, H2O. Temperatures are given on
the graph legend. The fluorescence spectra are shown in Figs. S15, S16.
It was established that an increase in the length of the alkyl tail in the
series C10SU - C12SU - C14SU - C16SU leads to a decrease in the aggrega-
tion numbers from 106 to 6 (Table 3). This trend is confirmed by data
obtained from the dye solubilization study given in Table 3 for compar-
ison. The Nagg can be roughly estimated from the absorbance of CnSU so-
lutions saturated with Orange OT (Fig. 4,C) by making the following
assumptions: (a) the solubilization ratio is one micelle per dye mole-
cule; (b) the concentration of detergent not associated into micelles is
constant and equal to the cmc [59]. The decrease in the Nagg with in-
crease in the length of the alkyl tail is nontypical for classical surfactants,
for example, for a series of alkyltrimethylammonium bromides, it has
been shown that the aggregation numbers are 55 (C12TAB), 70
(C14TAB), and 89 (C16TAB) [44]. Aggregation number of alkyl
pyrrolidinium bromides (L-CnPB) obtained from steady-state fluores-
cence quenching was found to be 42, 48, and 53 for L-C12PB, L-C14PB,
and L-C16PB, respectively [60]. However, upon the transition to cationic
surfactants with a more voluminous structure of the head group, the op-
posite tendency in the change of aggregation numbers can be observed.
For example, this was shown for a series of triphenylphosphonium sur-
factants [61] and quaternary ammonium derivatives of quinuclidine
[62]. However, it is assumed that the CN2S core is planar, therefore, it
is not possible to explain the decrease in aggregation numbers from
the point of view of the volume head group.
3.6. Antimicrobial and hemolytic action
The antimicrobial activity of CnSU surfactants was tested against
gram-positive bacteria Staphylococcus aureus ATCC 209p (Sa) and Bacil-
lus cereus ATCC 8035 (Bc); gram-negative bacteria Escherichia coli CDC F-
50 (Ec) and Pseudomonas aeruginosa ATCC 9027 (Pa); fungi Aspergillus
niger BKMF-1119 (An), Trichophyton mentagrophytes var. gypseum 1773
(Tm), and Candida albicans 855–653 (Ca). The minimum inhibitory con-
centrations, the minimal bactericidal concentrations and the minimal
fungicidal concentrations are summarized in Table 4. Bacteriostatic
Therefore, the dynamic light scattering method was additionally
used to determine the hydrodynamic diameter of the aggregates in
aqueous CnSU solutions at a concentration twice as large as cmc. It
was found that for the CnSU series the hydrodynamic diameter averaged
over the number of particles is 50–70 nm (Figs. S17-S20). These values
are not typical for classical micellar aggregates, which suggests the for-
mation of aggregate through another packing model. It is also worth
noting that the hydrodynamic diameter averaged over intensity
(Figs. S17-S20) has a bimodal distribution: 50–70 nm and about
300 nm. That is, the calculated aggregation numbers are effective and
reflect contributions by at least two types of particles. Importantly, the
values of Z-Average (Table 3) decrease with increasing length of the sur-
factant alkyl tail, which indicates the predominance of smaller aggre-
gates and is in good agreement with a decrease in aggregation numbers.
3.5. NMR diffusometry data
To further specify aggregation behavior two representatives of the
homologous series, namely, C10SU and C14SU, were studied by NMR
Fig. 7. The self-diffusion coefficient of cationic surfactants C10SU and C14SU vs. reciprocal
surfactant concentration at 40 °C in D2O.
8

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
Table 4
⁎
Antimicrobial activity of CnSU.
Surfactants
Bacteriostatic and fungistatic activity MIC (μg·mL−1
)
Sa
Bc
Ec
Pa
An
Tm
Ca
C10SU
C12SU
15.6
15.6
15.6
31.3
0.5
1.2
1.2
1.3
2.6
15.6
15.6
15.6
62.5
3.1
1.4
1.3
1.2
5.7
>500
>500
>500
>500
6.3
>500
>500
>500
>500
250
–
>500
>500
>500
>500
62.5
–
>500
>500
>500
>500
31.3
–
62.5
62.5
15.6
31.3
3.1
5.6
5.3
1.4
2.7
C
C
14SU
16SU
⁎⁎
CTAB
Chloramphenicol
Ketoconazole
62.5
–
5.6
62.5
–
5.7
125
–
10
–
4.0
–
–
4.0
0.3
0.3
Bactericidal and fungicidal activity MBC, MFC (μg·mL−1
)
C
C
C
C
10SU
12SU
14SU
16SU
62.5
62.5
62.5
250
50
5.8
5.4
5.8
21
31.3
62.5
62.5
125
2.5
5.3
5.4
10
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
125
62.5
62.5
125
50
11
5.6
5.3
10
⁎⁎
CTAB
>500
⁎
⁎⁎
Average of three values measured; standard deviation (SD).
From [63].
activity of test compounds is more expressed than bactericidal. It can be
seen that all compounds demonstrate antimicrobial activity against test
strains of gram-positive bacteria S. aureus 209p and B. cereus 8035 and
the yeast-like fungus C. albicans 855–653. By bacteriostatic activity
degree of hemolysis of human red blood cells caused by the studied
compounds in the concentration range of 15.6–250 μg·mL−1. The MIC
values of the tested compounds are in this range. It can be seen that
all compounds in the studied concentrations have lower hemolytic ac-
tivity than the antibiotic Gramicidin S, which is known to cause erythro-
cyte hemolysis and therefore cannot be used for intravenous
administration. Yet, due to the sufficiently high antimicrobial activity,
it is widely used in medicine for external treatment [65]. The hemolysis
caused by C10SU and C12SU at a concentration of 15.6 μg·mL−1 does not
exceed 1%, that is, they may be of interest for further research with liv-
ing objects.
C
10SU - C14SU compounds are 4 times superior to the comparison
drug chloramphenicol (bacteriostatic drug) against S. aureus 209p and
B. cereus 8035. C16SU was less active, but also showed antibacterial ac-
tivity at the level of the reference drug. In experiments with gram-
negative bacteria, the tested compounds in the studied concentrations
did not exhibit antimicrobial properties. Surfactants C12SU and C14SU
have a fungicidal effect at a concentration of 62.5 μg·mL−1 toward the
yeast-like fungus C. albicans. Against T. mentagrophytes and A. niger all
compounds were inactive. Compared to the classical cationic surfactant
cetyltrimethylammonium bromide isothiuronium series has been less
active toward all studied bacterial strains, which was assessed previ-
ously in the same workspace as the surfactants in study [63].
Hemolysis with surfactant molecules is a process of great fundamen-
tal and practical importance. Hemolytic activity of surfactants can be
used as a criterion for their toxicological assessment [64]. Thus, in the
summary round of our investigation the hemolytic activity of CnSU com-
pounds was studied. Fig. 8 presents data on the assessment of the
4. Conclusion
A series of surfactants bearing an isothiuronium head group (CnSU,
where n = 10, 12, 14, 16, 18) were synthesized and characterized by
IR, NMR, elemental analysis. For the first time it is shown that such sur-
factants contain a double bond in the head group, which was evident
from x-ray data. Krafft temperature and critical micelle concentration
are obtained by methods of tensiometry, conductometry, spectropho-
tometry, fluorimetry. Physicochemical characterization has shown
that the CnSU surfactants have 2–3 times higher solubilization capacity
than alkyltrimethylammonium analogues. They tend to form aggre-
gates of different sizes, with DLS study testifying the larger size popula-
tion, while NMR diffusometry the contribution of smaller micelle-like
population. The aggregation numbers for CnSU were calculated by fluo-
rimetry, dye solubilization and NMR self-diffusion methods. The transi-
tion from C10SU to C16SU is accompanied by a decrease in aggregation
numbers. CnSU surfactants demonstrate antimicrobial activity against
test strains of gram-positive bacteria S. aureus 209p and B. cereus 8035
and the yeast-like fungus C. albicans 855–653, with no hemolytic activity
observed.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
This work in the part of the study of self-organization, solubilization
and biological activity of isothiuronium surfactants was supported by
Russian Science Foundation (project № 19-73-30012) for V.F.G., P.R.V.,
Fig. 8. Hemolytic activity of CnSU surfactants.
9

F.G. Valeeva, T.R. Karimova, R.V. Pavlov et al.
Journal of Molecular Liquids xxx (2020) xxx
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G.G.A., V.A.D., Z.L.Ya. This work in the part of synthesis of isothiuronium
surfactants was funded by the subsidy allocated to Kazan Federal Uni-
versity for the state assignment in the sphere of scientific activities №
0671-2020-0063 for G.I.V. and B.D.I. X-ray single crystal study and
NMR experiment were supported from the government assignment
for FRC Kazan Scientific Centre of RAS for I.K.A. and K.O.N. The authors
gratefully acknowledge the CSF-SAC FRC KSC RAS for providing neces-
sary facilities to carry out this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.molliq.2020.114721.
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