Aggregation Behavior of Carbamate-Functionalized Monocationic Morpholinium Surfactants in Aqueous Media

Alkyl isocyanates (98%), Orange OT (OOT, 75%), pyrene (≥99%) were purchased from Sigma-Aldrich (USA). 4-Methylmorpholine (99%) and 1,4-diazabicyclo[2.2.2]octane (DABCO, 97%) were purchased from Acros Organics (USA). Cetylpyridinium bromide (CPB, 99%) was purchased from AppliChem (Germany). Acetonitrile (Ecos-1, Russia) and diethyl ether (Kuzbassorghim, Russia) were purified by standard procedures before use. Aniline dye crystal violet (CV, 98%, BLDpharm, China), fluorescent dyes propidium iodide (PI, ≥94%, Sigma-Aldrich, USA), and 3′,3′-dipropylthiadicarbocyanine (DiSC3(5), 98%, BLDpharm, China) were used as received.

General procedure for synthesis of 4-[2-(alkylcarbamoyloxy)ethyl]-4-methylmorpholinium bromides. 4-(2-Hydroxyethyl)-4-methylmorpholinium bromide was synthesized according to previous reports with some modifications by the reaction of 4-methylmorpholine (1 equiv.) with an excess of 2-bromoethanol (1.2 equiv.) in 50 mL of dry acetonitrile [44, 45]. For the synthesis of CnMB-carb, alkyl isocyanate (1.1 equiv.) was added to a solution of 4-(2-hydroxyethyl)-4-methylmorpholinium bromide (1 equiv.), DABCO (0.1 equiv.) and dry acetonitrile (30 mL). The reaction mixture was stirred for 16 h under heating. The resulting precipitate was filtered, washed twice with diethyl ether and dried on a water bath (313 K) under vacuum (15 mmHg). The structures of the compounds were characterized by IR, 1H NMR spectroscopy, mass spectrometry, and elemental analysis.

4-Methyl-4-(2-(octylcarbamoyloxy)ethyl)morpholinium bromide (C8MB-carb). Yield 93.6%, white solid, mp 56–59°С. IR spectrum (KBr), ν, cm–1: 3417, 2957, 2927, 2857, 1712, 1639, 1539, 1467, 1256, 1127, 1062, 951, 885, 774, 722, 623. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 0.88 m [3H, NH(CH2)7CH3], 1.28 m [10H, NH(CH2)2(CH2)5CH3], 1.51 m [2H, NHCH2CH2(CH2)5CH3], 3.14 t [2H, NHCH2(CH2)6CH3, 3JНH 7.3], 3.62 s (3H, N+CH3), 3.85 m (4H, N+CH2CH2OCH2CH2N+), 4.08 m (4H, N+CH2CH2OCH2CH2N+), 4.23 m (2H, N+CH2CH2OCO), 4.62 m (2H, N+CH2CH2OCO). Mass spectrum (ESI), m/z: 301.12 [M]+ (calcd for C16H33BrN2O3: 381.36). Found, %: C 50.31; H 8.76; Br 20.88; N 7.30. C16H33BrN2O3. Calculated, %: C 50.39; H 8.72; Br 20.95; N 7.35.

4-[2-(Decylcarbamoyloxy)ethyl]-4-methylmorpholinium bromide (C10MB-carb). Yield 98%, white solid, mp 82–85°С. IR spectrum, (KBr), ν, cm–1: 3434, 3275, 2954, 2922, 2851, 1720, 1702, 1614, 1542, 1468, 1378, 1305, 1261, 1128, 1104, 1067, 1043, 1021, 989, 951, 918, 883, 779, 721, 619. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 0.88 m [3H, NH(CH2)9CH3], 1.26 m [14H, NH(CH2)2(CH2)7CH3], 1.52 m [2H, NHCH2CH2(CH2)7CH3], 3.14 t [2H, NHCH2(CH2)8CH3, 3JНH 7.1], 3.61 s (3H, N+CH3), 3.84 m (4H, N+CH2CH2OCH2CH2N+), 4.09 m (4H, N+CH2CH2OCH2CH2N+), 4.22 m (2H, N+CH2CH2OCO), 4.63 m (2H, N+CH2CH2OCO). Mass spectrum (ESI), m/z: 329.35 [M]+ (calcd for C18H37BrN2O3: 409.41). Found, %: C 52.85; H 9.05; Br 19.44; N 6.85. C18H37BrN2O3. Calculated, %: C 52.81; H 9.11; Br 19.52; N 6.84.

4-[2-(Dodecylcarbamoyloxy)ethyl]-4-methylmorpholinium bromide (C12MB-carb). Yield 85.3%, white solid, mp 98–101°С. IR spectrum (KBr), ν, cm–1: 3339, 3279, 2953, 2919, 2849, 1721, 1701, 1614, 1576, 1548, 1468, 1379, 1304, 1268, 1250, 1141, 1128, 1104, 1068, 1041, 1022, 986, 951, 920, 883, 779, 722, 623. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 0.88 t [3H, NH(CH2)11CH3, 3JНH 6.8], 1.25–1.30 m [18H, NH(CH2)2(CH2)9CH3], 1.51 m [2H, NHCH2CH2(CH2)9CH3], 3.11–3.16 m [2H, NHCH2(CH2)10CH3, 2H], 3.64 s (3H, N+CH3), 3.87 m (4H, N+CH2CH2OCH2CH2N+), 4.07 m (4H, N+CH2CH2OCH2CH2N+), 4.27 m (2H, N+CH2CH2OCO), 4.62 m (2H, N+CH2CH2OCO). Mass spectrum (ESI), m/z: 357.42 [M]+ (calcd for C20H41BrN2O3: 437.46). Found, %: C 54.82; H 9.42; Br 18.35; N 6.33. C20H41BrN2O3. Calculated, %: C 54.91; H 9.45; Br 18.27; N 6.40.

4-Methyl-4-[2-(tetradecylcarbamoyloxy)ethyl]morpholinium bromide (C14MB-carb). Yield 92%, white solid, mp 88–91°С. IR spectrum (KBr), ν, cm–1: 3275, 3021, 2917, 2850, 1721, 1701, 1616, 1549, 1468, 1379, 1351, 1304, 1260, 1128, 1104, 1068, 1036, 1022, 989, 951, 919, 883, 779, 722, 646. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 0.88 t [3H, NH(CH2)13CH3, 3JНH 6.8], 1.25–1.28 m [22H, NH(CH2)2(CH2)11CH3], 1.51 m [2H, NHCH2CH2(CH2)11CH3], 3.13 q [2H, NHCH2(CH2)12CH3, 3JНH 6.8], 3.63 s (3H, N+CH3), 3.83–3.90 m (4H, N+CH2CH2OCH2CH2N+), 4.07 m (4H, N+CH2CH2OCH2CH2N+), 4.26 m (2H, N+CH2CH2OCO), 4.62 m (2H, N+CH2CH2OCO). Mass spectrum (ESI), m/z: 385.45 [M]+ (calcd for C22H45BrN2O3: 465.52). Found, %: C 56.68; H 9.80; Br 17.09; N 6.00. C22H45BrN2O3. Calculated, %: C 56.76; H 9.74; Br 17.16; N 6.02.

4-[2-(Hexadecylcarbamoyloxy)ethyl]-4-methylmorpholinium bromide (C16MB-carb). Yield 95%, white solid, mp 93–96°С. IR spectrum (KBr), ν, cm–1: 3403, 3276, 3020, 2917, 2850, 1720, 1700, 1547, 1469, 1378, 1304, 1264, 1251, 1141, 1128, 1104, 1068, 1037, 987, 952, 918, 883, 779, 721, 626. 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 0.88 t [3H, NH(CH2)15CH3, 3JНH 6.8], 1.25–1.30 m [26H, NH(CH2)2(CH2)13CH3], 1.51 m [2H, NHCH2CH2(CH2)13CH3], 3.13 q [2H, NHCH2(CH2)14CH3, 3JНH 6.8], 3.64 s (3H, N+CH3), 3.83–3.91 m (4H, N+CH2CH2OCH2CH2N+), 4.07 m (4H, N+CH2CH2OCH2CH2N+), 4.26 m (2H, N+CH2CH2OCO), 4.62 m (2H, N+CH2CH2OCO). Mass spectrum (ESI), m/z: 413.31 [M]+ (calcd for C24H49BrN2O3: 493.57). Found, %: C 58.42; H 9.98; Br 16.10; N 5.72. C24H49BrN2O3. Calculated, %: C 58.40; H 10.01; Br 16.19; N 5.68.

Tensiometry. A series of 10 mL solutions of CnMB-сarb with varying concentrations were prepared using ultrapure water (Simplicity®UV, Millipore SAS, France) and automatic pipettes (Eppendorf, Germany) to determine the aggregation thresholds. The surface tension of each solution at 298 K was determined using platinum ring (Du Noüy method). A K6 tensiometer (KRŰSS, Germany) was calibrated with ultrapure water before each experiment to obtain the fitting coefficient. The measurement error of surface tension did not exceed 2%. After measurements, surface tension isotherms were plotted against surfactant concentration, and the cmc values were determined from the inflection points. The first-degree polynomial equation, i.e., linear equation, was used to determine the slope of changes in surface tension against log c to determine the thermodynamic parameters of adsorption and micellization [the maximum adsorption (Γmax) and the minimum surface area per surfactant molecule (Amin)] using the Eqs. (1), (2):

$$} = - }\mathop \limits_} \to }} \left( \over }} \right),$$

((1))

where R is the gas constant, n = 2 for monocationic surfactants, σ is equal to the difference between the surface tensions of ultrapure water and the amphiphile solution, Т is the absolute temperature (K).

$$} = ^}} \over }}}}},$$

((2))

where NА is the Avogadro number.

When calculating the values of Γmax, the main contribution to the accuracy of the calculations is made by the accuracy of determining the slope of the isotherm premicellar section. To minimize the error, the isotherms were obtained three times for each homologue, with average value taken into account.

Conductometry. The conductivity of a series of 10 mL CnMB-carb solutions was determined using the InoLab Cond 7110 system (WTW, Germany) with an error of no more than 0.5%. Measurements were conducted at 298, 308, 318, and 328 K using a circulatory thermostat LT-TWC/7 (LabTex, China). The conductometer was calibrated every 6 months using a potassium chloride solution (0.01 mol/L), and the conductivity of ultrapure water was measured before each experiment.

After the measurements, temperature dependencies of the specific conductivity against the CnMB-carb concentration were plotted, and the cmc values were determined from the inflection points. The values of the degree of counterion binding (β) were determined based on the slope of the dependence of specific conductivity on surfactant concentration before (S1) and after (S2) cmc (Tables S1 and S2, see Supporting Information) using the following equation:

$$\beta = 1 - } \over }}.$$

((3))

Additionally, the enthalpy (ΔHmic), entropy (ΔSmic), and Gibbs free energy (ΔGmic) of micellization were calculated using the following equations:

$$\Delta }}} = - R\left[ \right)\left( }} \over }} \right) - \ln }\left( \over }} \right)} \right],$$

((4))

$$\Delta }}} = \left( \right)RT\ln }}_}}},$$

((5))

$$\Delta }}} = \Delta }}} - T\Delta }}},$$

((6))

$$\Delta }}} = }}} - \Delta }}}} \over T},$$

((7))

where cmccond is the value of the critical micelle concentration determined by the conductometry. Based on the work of Zana [29], the use this equation for ΔGmic calculation is possible for surfactants with cmc values below than 10 mM.

The Gibbs free energy of adsorption (ΔGad) was calculated using the following equation:

$$\Delta }}} = \Delta }}} - }}}} \over }}},$$

((8))

where πcmc is the surface pressure equal to the surface tension of water and the surface tension of the solution at the cmc.

Spectrophotometry. The solubilizing capacity of CnMB-сarb toward hydrophobic dye Orange OT (OOT) was determined on Specord 250 Plus spectrophotometer (Analytik Jena AG, Germany). A series of 3 mL solutions with an excess of dye were kept for 48 h to ensure complete transition of the probe into the hydrophobic core, while the unsolubilized dye was filtered using 450 nm Millex®-HV PVDF Membrane syringe filter units (Merck Millipore, USA) before measurement. From the obtained absorption spectra of the micellar solutions of OOT, the dependence of the reduced optical density of the dye at the absorption maximum (495 nm) on the surfactant concentration was plotted. The error of the obtained cmc values did not exceed 4%. To calculate the solubilization capacity (S), the region of sharp increase in optical density was approximated using a linear equation (9):

where b is the slope of the dependence (above cmc), ε is the OOT extinction coefficient (17400 M–1 cm–1).

Fluorimetry. The cmc values were determined using the fluorescent probe pyrene. The probe was added to a surfactant solution at concentrations of 1 μM, 30–40 min before measurement. The experiment was carried out on an F-7100 fluorimeter (Hitachi, Japan) using 10 × 10 mm quartz cuvettes. For micellar solutions with pyrene, the following settings were used: excitation wavelength is 335 nm, emission range is 350–500 nm, measurement speed is 1200 nm/min, excitation and emission slits are 5 and 2.5 nm, respectively. From the fluorescence spectra of pyrene, the dependence of the polarity index (I1/I3) on the surfactant concentration was plotted. The error of the obtained cmc values was no more than 2%.

Electrophoretic light scattering. The zeta potential for C16MB-carb was determined on a Malvern ZetaSizer Nano (Malvern Instruments, UK) using a DTS1070 U-shaped zeta cuvette (Malvern, USA). The device is equipped with a helium-neon laser with a wavelength of 633 nm, a power of 10 kW and a light scattering angle of 173°. The zeta potential was determined by converting the electrophoretic mobility of the particles using the Smoluchowski equation (10):

$$\zeta = \over \varepsilon },$$

((10))

where ζ is the zeta potential, ε is the dielectric constant, η is the dynamic viscosity of the solution, μ is the electrophoretic mobility of particles.

Biodegradability assay. The biodegradability of the surfactant series was evaluated in the presence of microorganisms sourced from municipal wastewater treatment sludge using the “closed bottle test” [46]. Mineral salts and the surfactants (at a concentration of 10 mg/L) were added to aerated deionized water. The filtrate of inoculum was added in an amount of 5 mL per liter of solution. The resulting solution was incubated in closed bottles in the dark at 293±1 K, and the concentration of dissolved oxygen, which changes during biodegradation (the initial oxygen concentration in the nutrient medium was 9.02 g/L), was determined using an Oxi 7310 oximeter (WTW, Germany). The analysis was carried out for 28 days. A sodium acetate (G-Biosciences, USA) with inoculum and a blank experiment without surfactants were used as a control. The degree of biodegradation was determined by the following equation:

$$} = }\;}\;}\;\left( }} \right)}\;}\;\left( }} \right)} \over }}} \times 100.$$

((11))

The theoretical oxygen demand (ThOD) was calculated using the standard equations (for sodium acetate ThOD = 1.07 g/L) [46].

Antimicrobial activity. The antimicrobial activity of the surfactants was investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC). The following strains of pathogenic microorganisms were selected for the experiment: gram-positive bacteria Staphylococcus aureus ATCC 209p (Sa), including methicillin-resistant strains of S. aureus MRSA-1 and MRSA-2, Bacillus cereus ATCC 8035 (Bc), and Enterococcus faecalis ATCC 29212 (Ef), gram-negative bacteria Escherichia coli CDC F-50 (Ec) and Pseudomonas aeruginosa ATCC 9027 (Pa), as well as fungi Candida albicans ATCC 10231 (Ca) (State Collection of Pathogenic Microorganisms and Cell Cultures “GKPM-Obolensk”). Bacteria and fungi were cultivated at 3.0 × 105 and 2 × 103 CFU/mL in Mueller–Hinton and Sabouraud broth at 310 and 298 K, respectively. Subsequently, the surfactant solutions were added and incubated with the bacteria. Data were collected every 24 h for 5 days, and the experiment was replicated three times. Chloramphenicol, norfloxacin, and ketoconazole were used as a reference drugs.

Mechanism of antimicrobial activity. The experiments were carried out using Staphylococcus aureus ATCC 209p strain in three directions: (i) determination of the bacterial cell wall damage degree using the CV dye; (ii) assessment of bacterial membrane permeability using the high molecular weight PI dye; (iii) the evaluation of bacterial membrane potential change (∆Ψ) using DiSC3(5). Bacterial cells were cultured overnight in Muller–Hinton broth, centrifuged, washed twice with 0.01 M phosphate buffer solution (PBS) and then again centrifugation at 5000 rpm for 10 min. After that, cells were resuspended in the PBS to obtain 2 × 108 CFU per mL. Then CnMB-сarb aqueous solutions were added to the resulting inoculum in a 1 : 1 ratio and incubated for 30 min at 310 K, followed by the addition of 0.001% CV (i), 1.5 μM PI (ii), and 1 μM DiSC3(5) (iii) and incubated in darkness. The optical density of CV was measured at 540 nm using InVitroLogic microplate reader (LLC “Medico-Biological Union,” Russia). Fluorescence intensity of PI and DiSC3(5) was measured using F-7100 fluorimeter (Hitachi, Japan) at λex of 544 nm and 622 nm, respectively.

Data processing and statistical analysis. The data was analyzed using Microsoft Excel 2016® (version 2403) and OriginPro 8.5 (version 9.8.0.200) softwares. Statistical analysis was performed using the Mann-Whitney test (p < 0.05). Tabular and graphical data contain mean values and standard deviation (SD).

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