The attraction of cationic surfactants for a negatively charged bacteria surface gives them the ability to intercalate into phospholipid membranes, thus causing the leakage of intercellular materials into the environment and cell death (Devínsky et al., 1985; Kopecká-Leitmanová et al., 1989; Mlynarčík et al., 1981). The disruption of membrane, and subsequent solubilization of its interior, plays an essential role in the mode of antimicrobial action of cationic surfactants and firmly depends on the ability of surfactant molecules to form micelles. Whereas micellization properties are linearly related to the length of the alkyl chains of cationic surfactant, the antimicrobial activity also depends on the number of carbon atoms in the long alkyl chain of surfactant molecule (Marek et al., 2018). The optimal antimicrobial activity for gram-positive bacteria is achieved when the carbon chain length is C12–C14, while for gram-negative bacteria, the highest activity is obtained for the chain length of C14–C16 (Feder-Kubis & Tomczuk 2013; Pernak and Skrzypczak, 1996; Thebault et al., 2009). Molecules with n-alkyl chain length below C4 and above C18 are antimicrobially ineffective. In the series of structurally related QASs, the antimicrobial activity increases with the growing chain length until it reaches the maximum. From this point, with a continuous growing chain length, the antimicrobial activity starts to decrease. This phenomenon, called cut-off effect, is typical for many of the biologically active compounds, and it can be caused by many reasons (limited aqueous solubility, kinetic effects, interaction with lipid bilayers or proteins) (Balgavý & Devínsky, 1996).
The alkyl chain length is not the only factor affecting the antimicrobial activity of QASs. The other hydrophobic groups in the molecule of surfactant also influence the aggregation properties, so they can also affect the biological activity. The study of this effect on antimicrobial properties could help in the development of new active QASs (Benkova et al., 2019; Malinak et al., 2014).
In addition, introduction of new structural motives such as heteroatoms or aromatics (Semenov et al., 2011; Pernak et al., 2001) may result in potential antimicrobial agents with higher biological activities, and they could even conquer the growing resistance phenomenon (Jennings 2015). As long as essential oils containing bicyclical camphor or borneol moiety exhibit antibacterial effect (Ruiz-Navajas et al., 2012; Miguel et al., 2011), we decided to design and synthesize QASs bearing hydrophobic bicyclic moiety, hoping that incorporation of two important antimicrobially active structures in one compound will improve their bioactivity. In this study, we have prepared, as illustrated in Fig. 1, three new optically active amphiphilic QASs starting from (1R,3S)-(+)-camphoric acid. The aggregation properties of prepared surfactants were studied by tensiometry and critical micelle concentration (CMC); surface tension at CMC (γCMC) and efficiency of adsorption at the surface (pC20) were calculated for each compound from a break in the plot of logarithm of surfactant's concentration versus surface tension. Their antimicrobial activity was tested against gram-negative bacteria Escherichia coli, gram-positive human pathogenic bacteria Staphylococcus aureus, and human fungal pathogen Candida albicans.
Figure 1
Synthesis of QASs derived from (1R,3S)-(+)-camphoric acid.
EXPERIMENTAL
Materials and methods
All compounds used ((1R,3S)-(+)-camphoric acid, methanesulfonyl chloride (MsCl), triethylamine (TEA), N,N-dimethylethane-1,2-diamine, acetone, dichloromethane (DCM), ethyl acetate, bromoalkanes) are commercially available. DCM was pre-dried over CaCl2 and then distilled from CaH2 under nitrogen atmosphere. 1H and 13C NMR spectra were measured on a Varian Gemini 300 spectrometer at 300 MHz and 75 MHz, respectively. Chemical shifts have been reported in ppm relative to an internal reference (TMS). IR spectra were recorded on NICOLET 6700 FT-IR instrument. Polarimetric measurements were obtained using a Jasco P-1010 polarimeter at 589 nm. Elemental analyses were carried out on a Carlo Erba 1108A instrument. All melting points reported were uncorrected and measured on Kofler hot stage. The surface tension measurements were performed on Krüss processor tensiometer K100 (Wilhelmy plate method). The temperature was kept constant at the desired level using thermostatted (Thermo Haake SC100) water bath. Double-distilled water was used for the preparation of all samples. Measurements of equilibrium surface tension were taken repeatedly until the change in surface tension was less than 0.08 mNm−1. The values of surface tension decrease with increasing concentration and the break point provides the CMC value.
Microbiology
The antimicrobial activity was tested against gram-negative bacteria Escherichia coli CNCTC 377/79, gram-positive bacteria Staphylococcus aureus CNCTC 29/58, and fungi Candida albicans CCM 8186. Solutions of compounds studied were prepared in water (5%). A suspension of the standard microorganism, prepared from 24 h cultures of bacteria in blood agar and from 24 h cultures in the Sabouraud agar for fungi had a concentration of 5 × 107 cfu mL−1 of bacteria and 5 × 105 cfu mL−1 of Candida. The concentration of microorganisms was determined spectrophotometrically at 540 nm and adjusted to absorbance A = 0.35. The microorganism suspension was added to solutions containing the tested compound and to double concentrated peptone broth medium (8%) for bacteria or Sabouraud medium (12%) for Candida. The stock solution of the tested compounds was serially diluted by half. The cultures were done in 96-well microliter plates. The microorganisms were incubated for 24 h at 37°C, and then, from each well, 5 μL of suspension were cultured on blood agar (bacteria) or on Sabouraud agar (fungi). After 24 h at 37°C, the lowest concentration of QAS that prevented colony formation was determined as minimal inhibitory concentration (MIC). Clinically used benzalkonuim bromide (BAB) was used as a standard.
30 g (150 mmol) of (1R,3S)-camphoric acid was dissolved in 750 mL of dry DCM in the argon purged vessel. TEA (62.6 mL, 450 mmol, 3 equiv.) was added to the mixture and stirred to give homogenous solution. Then MsCl (11.6 mL, 150 mmol) was added dropwise for several minutes and allowed to stir overnight. The mixture was transferred to a separatory funnel and washed with 3 M HCl (3 × 100 ml) followed by 100 mL of brine. The organic phase was dried over anhydrous Na2SO4, and the solvents were removed on rotary evaporator to give yellow crystals. Recrystallization from ethyl acetate gave 20.2 g (74%) of white needles. M.p. = 223–225°C; [α]D21= −0.99 (1.0, CHCl3). Spectral data were in agreement with the literature (Eagles & Hitchcock, 2010).
20 g (0.11 mol) of anhydride 3 and 12.1 mL (0.11 mol) of N,N-dimethylethane-1,2-diamine were mixed and gently heated (90–100 °C) until a clear reaction mixture was obtained. The resulting partially reacted mass was heated to a temperature of 180°C and maintained at that temperature for 7 h. Reaction mixture was allowed to cool, and the resulting oil was vacuum distilled. The fraction distilled at 165–168°C / 10 mmHg was identified as a final product which was obtained in 94% yield as colorless oil. Spectral data were in agreement with the literature (Rice & Grogan, 1957).
General procedure for the synthesis of QAS 1
8 mmol of imide 2 was dissolved in 15 mL of acetonitrile. To this solution was added 9.6 mmol (1.2 eqiv.) of bromoalkene, and the reaction mixture was heated at 100°C for 24 h. After the reaction, the mixture was allowed to cool, and the solvent was removed by rotary evaporation. To the resulting mixture, orange oil was added 40 mL of anhydrous benzene, and again, it was evaporated. This procedure was repeated three times. The resulting material, solidified by cooling, was repeatedly crystallized from acetone/hexane mixture, filtered, washed with anhydrous diethyl ether, and dried in vacuum to afford final salts 1. Structural and spectral characterization of prepared QASs are summarized in the Table 1 and Table 2.
Characterization of camphoric acid-derived QAS 1.
Compound
Formula / [α]D21 (conc[g/100 mL]., solvent)
wi(calc.)/%wi(found)/%
Yield
M.p.
C
H
N
%
°C
1a
C26H49BrN2O2/+7.99 (0.85, CHCl3)
62.2662.34
9.859.81
5.585.61
80
82–83.5
1b
C28H53BrN2O2/+6.75 (0.809, CHCl3)
63.5063.26
10.099.96
5.295.15
87
85–85.5
1c
C30H57BrN2O2/+7.2 (c = 0.799, CHCl3)
64.6164.36
10.309.96
5.024.85
84
95–96.8
1d
C32H61BrN2O2/+6,7d (c = 0.80, CHCl3)
65.6265.68
10.5010.45
4.784.82
85
96.8–97.5
Spectroscopic data of camphoric acid-derived QASs 1.
Enantiopure QASs of the compounds in series 1 were synthesized, as illustrated in Fig. 1, starting from (1R,3S)-(+)-camphoric acid. In the first step of the synthesis, (1R,3S)-(+)-camphoric acid was dehydrated using MsCl to yield anhydride 3 (Eagles & Hitchcock, 2010). The prepared anhydride 3 was subsequently heated with N,N-dimethylethane-1,2-diamine (Rice & Grogan, 1957). This nucleophilic attack followed by dehydration at 180°C gives as a product N-substituted imide of camphoric acid 2. The final step of the synthesis was nucleophilic substitution of bromide in bromoalkanes by tertiary amino group of derivative 2. QASs 1 were isolated after several crystallizations from acetone/hexane mixture as white solids in the yields 80–87%. All of the prepared QASs 1 were identified and characterized thoroughly from spectral and analytical data summarized in Table 1 and Table 2.
The antimicrobial activities of synthesized QASs were determined as a MIC, [μmol l−1] against the gram-positive human pathogenic bacteria S. aureus, gram-negative bacteria E. coli, and human fungal pathogen C. albicans, the values for which are given in Table 3. The MIC values were determined as lowest concentration of the salts 1 that completely prevented visible colony formation. All the compounds were dissolved in water for biological evaluation. Clinically used BAB was used as a standard.
Minimal inhibitory concentrations [μmol L−1] of prepared QAS 1.
Compound
S. aureus CNCTC 29/58
E. coli CNCTC 377/79N
C. albicans CCM 8186
1a
6.1
24.3
12.2
1b
0.7
5.8
0.7
1c
0.6
5.5
0.6
1d
324.1
665.8
324.1
BAB
26
260
26
According to the results, it can be observed that all of the synthesized QASs exhibit growth inhibition effect against all three types of microbes, with higher efficiency against S. aureus and C. albicans (Table 3). Gram-negative E. coli was found to be most resistant to the prepared salts among the tested microorganisms. This is presumably due to the cell membrane composition. Gram-negative bacteria contain an outer membrane with an external component that consists mainly of lipopolysaccharides, which acts as a barrier and prevents antimicrobial agents and biocides from entering the cell (Pérez et al., 2009).
The main target site of QASs is the cytoplasmic membrane comprised of a phospholipid bilayer. QASs are able to insert into the phospholipid bilayer, which is accompanied by membrane disorganization and structural and functional changes in the cell membrane, inducing leakage of intracellular components (Gilbert and Moore, 2005). In addition, QASs were also found to inhibit ATP synthesis by neutralizing the proton motive force (PMF) (Denyer & Hugo, 1977). The PMF is initiated by a proton gradient across the cytoplasmic membrane and is responsible for many respiratory and photosynthetic processes, including ATP synthesis. As long as QASs are surface active agents, they can cause denaturation of proteins anchored in the cytoplasmic membrane or dissociation of an enzyme from its prosthetic group. Because this effect was observed at concentrations much higher than lethal ones, the enzyme inhibition is not the primary injury caused by cationic surfactants (Merianos 1991). If we take a deeper look into the mechanism of action of QASs, for the most of them, no specific target site has been recognized. However, it is not excluded that there can exist some target specificities, like DNA binding, as shown by Menzel (2011) and Zhang (2013), because the antimicrobial activity of QASs changes significantly against various types of microorganisms and explanation only by the cationic charge and hydrophobic tail cannot be used.
The antimicrobial activity of surfactants generally depends on the alkyl chain length, although this correlation is not linear. In the series of prepared QASs 1a–1d, maximum antimicrobial activity was observed for compound 1c with 16 carbon atoms in alkyl chain, though salt 1b with 14 carbon atoms in alkyl chain exhibits the similar antimicrobial activity. It is noteworthy that among the salts examined in this study, all of the prepared QASs inhibited the growth of microorganisms at the concentrations lower than clinically used BAB.
QASs 1a–1d exhibit surface activities such as surface tension lowering and micelle formation. The surface properties of prepared QASs were investigated by surface tension measurements. The plots of surface tension against the logarithm of surfactant's concentration are presented in Fig. 2. CMC, surface tension at CMC (γCMC), and efficiency of adsorption at the surface (pC20) (pC20 – the negative log of C20, the surfactant molar concentration required to reduce surface tension by 20 mNm−1) of QASs were calculated from the break in the plots and are shown in Table 4.
Figure 2
Surface tension versus the logarithm of the aqueous molar concentration of 1a–1d at 25°C.
CMC, g(CMC) and pC20 values of prepared QASs calculated from tensiometry measurements.
1a
1b
1c
1d
CMC [molL−1]
5.07×10−3±0.23×10−3
9.13×10−4±0.98×10−4
0.97×10−4±0.05×10−4
8.09×10−6±1.13×10−6
γ(CMC) [mNm−1]
36.36
37.32
38.49
42.51
pC20
3.5
4.1
5.1
5.5
The surfactant concentration required to reduce the surface tension of pure water by 20 mNm−1 was used to compare the efficiency of surfactant (pC20). Higher values indicate that the surfactant adsorbs at the interface more efficiently. The values of pC20 in Table 4 show that with increasing alkyl chain length the pC20 increases. This means that better reduction in surface tension is achieved for surfactants with longer alkyl chain used at a smaller concentration. It is known that by increasing the alkyl chain length of a surfactant the CMC values decrease (Rosen 1989). It can be seen from Fig. 3 that values of the log CMC decrease linearly with number of carbon atoms in the chain from 1a to 1d. This linear relationship between chain length and CMC for homologous series of surfactants is known as Kleven's equation: log CMC = A – Bn, where n is the number of carbon atoms in the long alkyl chain and A and B are constants (Klevens, 1953). For prepared QASs 1, the linear relationship was found to be log CMC = 2.89 – 0.43n. The surface tension at CMC values (γCMC) are in the range of 36.36–42.51 mNm−1.
Figure 3
Variation of log CMC with chain length of 1a–1d at 25°C.
With regard to better understanding of the relation between the antimicrobial activities and micelle forming ability, the MIC values [mmolL−1] of the prepared QASs were correlated with their CMC values [mmolL−1] (Fig. 4). As seen, the CMC curve intersects the MIC curve at the cut-off point (16 carbon atoms chain length) for all the microorganisms tested. In addition, MICs of the prepared cationic surfactants with the alkyl chain length shorter than 16 carbon atoms were found below the CMCs, while above the cut-off point, the MICs appear above the CMC. A similar behavior was observed also in the study of cationic surfactants derived from amino acids (Joondan et al., 2014), alkyl betaines, and N-oxides (Birnie et al. 2000). This observation could be explained according to Joondan et al. that at the concentration under the CMC, the surfactant molecules are soluble; therefore, monomers predominate and participate in the interaction with phospholipid bilayer in the bacterial cell wall. On the contrary, molecules with longer chain length aggregate into the micelles at much lower concentration, thus decreasing the concentration of monomers. To achieve bactericidal effect, the higher concentration of the surfactant is needed (Joondan et al., 2014).
Figure 4
Correlation of CMC [mmolL−1] and MIC [mmolL−1] of QASs 1a–1d for a) S. aureus; b) E. coli; c) C. albicans.
CONCLUSIONS
In summary, we have designed and synthesized a new amphiphilic ammonium salt that could be classified as potential antimicrobials. All of the prepared QASs showed higher antimicrobial activity on gram-positive bacteria and fungi than on gram-negative strain. The maximum antimicrobial activity was observed for compound 1c with 16 carbon atoms in alkyl chain. QASs 1a–1c exhibit higher antimicrobial and antifungal activity than clinically used BAB. Aggregation parameters like CMC, γCMC, pC20 of synthesized cationic surfactants 1a–1d were studied by tensiometry, and based on the correlation between CMC and MIC, we can conclude that monomers of surfactants are mainly responsible for antimicrobial activity.
