2.1. Copolymerization of Maleic Anhydride with 4-Methyl-1-pentene
Alternating copolymers of maleic anhydride with alkenes were obtained by free-radical copolymerization in solution in the presence of radical initiators [
41,
42,
43,
44,
45]. However, copolymers which contain an excess of olefin have also been described in the literature [
46,
47], but as a general rule, alternating copolymers are formed, in particular, when an excess of anhydride was used.
The alternating copolymer of 4-methyl-1-pentene with maleic anhydride (MSA) was synthesized accordingly via free-radical polymerization. The copolymerization was carried out under homogenous conditions in anhydrous 2-butanone (MEK) at 80 °C in the presence of benzoyl peroxide (BPO) as an initiator.
Scheme 2 depicts the copolymerization reaction of 4-methyl-1-pentene with maleic anhydride.
An excess of maleic anhydride was used to ensure the equimolar composition of the resulting copolymer. The product was separated from the reaction mixture by precipitation in a Et2O:MeOH (4:1, vol:vol) mixture. For its molecular weight determination by gel permeation chromatography (GPC), the succinic anhydride units of the copolymer were methanolized at room temperature. The methanolysis was necessary because maleic anhydride copolymer adsorbed on the inline filter, impeding any measurement. GPC measurement of the methanolyzed copolymer was performed in THF with PMMA standards and the determined values were: Mn = 5800 and Mw/Mn = 1.67.
The composition of the obtained polymer could not be determined by
1H-NMR because of overlapping signals of both monomers.
Figure 1a depicts a typical
1H-NMR-spectrum of a P[MP-alt-MSA] copolymer. The range of overlapping signals between 1 and 4 ppm precludes the calculation of the polymer composition for the non-methanolyzed copolymers (MSA protons 3.25 ppm) as well as methanolyzed products (MSA protons 2.8 ppm) [
47].
Treatment of the copolymer with benzyl alcohol in MEK results in the formation of monobenzyl esters. This method allows determining the content of protons of succinic acid benzyl ester since the benzyl group’s NMR signals were well separated from the other polymer peaks.
Figure 1b depicts the
1H-NMR spectrum of a P[MP-alt-MSA] copolymer after reaction with benzyl alcohol. The aromatic protons as well as the benzylic protons are well separated from the signals of the polymer backbone and the alkyl side groups. The monomer composition was calculated from the integrated signal intensity of the benzylic protons and that of the alkyl signals between 0.8 and 5.2 ppm according to Equation (1):
A—integration value of benzylic proton signal (σ = 4.90–5.30 ppm),
m—number of benzylic protons = 2,
B—integration value of polymer backbone and side chains proton signal (σ = 0.70–4.2),
n—number of protons in signal mentioned in B = 14.
This method was developed for determination of the MSA content in terpolymers, and it was proved that, without a catalyst, only one carboxyl group was esterified [
48]. It is very useful and precise in the range of error of the used spectroscopic method. The 46 ± 5% of calculated content of succinic anhydride in the copolymer is reasonable and allowed us to assume that an alternating copolymer was obtained. The composition of the copolymer was also determined by elemental analysis. The obtained results correspond well to the composition calculated for alternating copolymer (see
Table 1).
2.5. Thermal Properties
Figure 6 depicts the TGA thermograms of copolymer C3 and its quaternized derivatives C4, C5, and C6. The nonquaternized copolymer C3 (curve 1) is the thermally most stable polymer and shows only slight weight loss below 200 °C. The highest weight loss is observed above 300 °C. Any modification of C3 by quaternization causes a decrease in thermal stability of the polymer due to Hofmann elimination of the ammonium groups [
61]. The Hofmann elimination occurs when quaternary ammonium salts are exposed to high temperatures and the reaction yields an alkene and a tertiary amine and a low-molecular-weight compound specific for the counterion (e.g., water, HCl, HI, etc.).
The quaternization with methyl iodide causes the C4 (curve 2) to decompose above 150 °C via a three-stage thermal degradation. The first stage starts at 150 °C and ends at 200 °C with a weight loss of 32% corresponding to the loss of HI (31 wt %). Even partial quaternization with long alkyl-chain iodide versus methyl iodide increased the thermal stability, as observed in curves 3 and 4 (C5 and C6) in
Figure 6However, such derivatives also show faster thermal degradation than nonquaternized C3. In all cases, the investigated copolymers showed a certain weight loss at relatively low temperatures around 100 °C, most probably caused by a loss of adsorbed water because of the hygroscopic nature of salts.
DSC measurement of dodecyl-iodide-quaternized copolymer is typical for all ammonium copolymers based on the C3 copolymer in the temperature range of −50 to 200 °C. Each copolymer showed two thermal transitions: one at 17 °C and a second one in the region of 80–105 °C (see
Table 4). The temperature of the second transition seems to depend on the type of quaternizing agent and is about 20 °C higher for copolymers quaternized with dodecyl iodide. There is a correlation with the TGA data where the presence of dodecyl iodide increased the thermal stability. Since the copolymers undergo decomposition in this range of temperatures, reverse heating does not reproduce the curves. Only the first transition is fully reproducible. The fact that the presence of longer alkyl chain gives a higher
Tg value is not in line with the expectations. It is known that polymers which contain longer alkyl side chains exhibit lower glass transition temperatures than the shorter ones due to the plastifying effect. Although there has been no melting temperature observed, this phenomenon can be assigned to the formation of ordered structures on the micro or even nano scale. These types of crystalline structures usually do not give any measurable thermal response and further investigation is required. Detailed values of the thermal transition of the investigated copolymers are summarized in
Table 4.
2.6. Investigation of Antimicrobial Properties of the Cationic Copolymer
In the World Health Organization’s (WHO) “global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics” of February 2017, experts agreed on grouping pathogens according to the species and the type of antibiotic resistance and classified the results in three priority tiers: critical, high, and medium.
Our selected bacteria belong to genera that were grouped into the priorities critical and high, i.e., here, the prevalence of resistance against antibiotic treatments is extremely high. With our research field, to prepare antimicrobially functional polymers, we would like to contribute to the reduction of the spread of these bacteria.
Amphiphilic polymers with quaternary ammonium groups are known to have antimicrobial properties [
20]. Thus, the water soluble C3, C4, and C5 were tested for their antimicrobial efficacy.
In order to find the MIC of the copolymers, bacterial growth of strains belonging to clinically relevant genera was monitored in the presence of all the copolymers. The tests were performed in microwell plates and the proliferation potential of the bacteria was monitored at 37 °C by measuring the optical density at 612 nm for 20 h using a microwell plate incubator/reader in comparison to a reference without the respective polymer. The MIC values are summarized in
Table 5.
The novelty of this paper is (i) the polymer backbone based on maleic anhydride and 4-methyl-1-pentene and (ii) the fact that the results obtained for E. coli and S. aureus were not as expected based on a great amount of papers showing that amphiphilic polymers with different backbones and manifold kinds of cationic and hydrophobic residues are more active against Gram-positive bacteria (lower MIC values) than against Gram-negative bacteria.
The polymers are more effective by a factor of 5–10 against the Gram-negative
E. coli than against the Gram-positive
S. aureus. This selectivity, although with a lower factor, was also found in [
66], who prepared lysine and arginine mimicking amino and guanidine propyl methacrylamide copolymers; in the latter, the amine-containing copolymers were best.
Compared to polymer C4 (modified with methyl iodide), polymers C3 (nonquaternized) and C5 (modified with both iodides, methyl and dodecyl iodide) are more active against the Gram-negative bacteria
E. coli and
P. aeruginosa, whereas, in the case of
S. aureus, C5 was the most active polymer compared to C3 and C4, with the nonquaternized C3 being the least active in the latter case. Previous studies in our group, although with a different polymer backbone, have shown that the best results against
S. aureus were obtained with the cationic residue directly linked to the aliphatic residue without a spacer in between [
67], and that longer alkyl chains showed best efficacy [
68] and, thus, confirm these results. Regarding the solubility properties (
Table 3), C3 and C5 show similar solubilities in the different solvents compared to C4. The limited solubility of the latter seems to restrict the effect against
E. coli,
P. aeruginosa, and
S. aureus since the hydrophilic–lipophilic balance is decisive for the efficacy of the respective polymer.
Against S. epidermidis, all three polymers are equally active in a comparable range, such as C3 and C5 against E. coli.
Comparing the MIC for the Gram-negative bacteria gave 5- (C3) to 20-fold (C5) higher values for
P. aeruginosa than for
E. coli, meaning that
E. coli is 5–20 times better inhibited compared to
P. aeruginosa. Gram-negative bacteria are known to actively secrete outer membrane vesicles (OMVs) from the outer membrane (OM) [
69]. OMV production is correlated with an increased rate of survival upon antimicrobial peptide treatment [
70] In
Pseudomonas putida, OMVs are generated, e.g., as a response to stress caused by cationic surfactants which can contribute to OMV biogenesis, through a physical mechanism by induction of the curvature of the membrane [
71]. Although OMV production is common in many bacteria, the extent and mechanism of OMV production is species specific, and thus, the higher MIC values for
P. aeruginosa might be due to the level of OMV production, since environmental stresses result in increased OMV formation by
P. aeruginosa [
72].
For the Gram-negative bacteria
E. coli and
P. aeruginosa and for the Gram-positive
S. aureus, C5 quaternized with the long alkyl chain, i.e., the repeat unit structure with the hydrophobic moiety being directly accompanied by the charged moiety, exhibits a higher efficacy compared to C4 quaternized with methyl iodide (this was also confirmed for functionalized polymers with polglycidol backbone [
73]).
C5 exhibits a more ordered structure due to phase separation and orientation of the hydrophobic C
12H
25 chains to hydrophobic domains (see also the discussion on the thermal properties in
Section 2.5).
Whereas polymers C3 and C4 led to an agglutination of human RBC at all concentrations tested (10–1000 µg/mL), C5 did not agglutinate RBC but showed lysis of 50% of the RBC relative to the positive control (HC50) at a concentration of 60 µg/mL. The higher value of HC50 compared to the MIC100 against E. coli (10 μg/mL) proved that polymer C5 has a selectivity to differentiate between mammalian cells and bacterial cell walls. However, since the values are overall in the same order of magnitude, the selectivity is low.
Since the investigated antimicrobial copolymers were designed to mimic peptides, a comparison with a reference compound is needed. The type A lantibiotics, e.g., Pep5 or Nisin, are in general of linear conformation and all the Nisin type peptides are positively charged [
33]. Combination of the cationic nature and the presence of leucine makes Nisin a good reference.
Nisin is a 34-residue-long peptide which is predominantly active against Gram-positive bacteria (Nisin is also active against Gram-negative bacteria but only after a pretreatment) [
74]. It is generally accepted that the bacterial plasma membrane is the target for Nisin, and that Nisin kills the cells by pore formation and inhibition of peptidoglycan synthesis. The pore formation causes collapse of vital ion gradients, resulting in cell death [
75]. In this study, it was shown that Nisin, compared to the copolymer modified with both iodides, is highly active against Gram-positive bacteria, and, as expected, 30 to more than 60 times less active against Gram-negative bacteria. Moreover, comparing the MIC of Nisin against the Gram-positive bacteria on the molar basis instead on the weight basis, the difference between the effect of Nisin and the copolymer is significantly lower.
This gap indicates that the activity of the peptides is determined not only by the amphiphilic nature but most probably the secondary peptide structure plays also a substantial role.