FUNGICIDAL AND BACTERICIDAL ACTIVITY OF ALKYL-SUBSTITUTING POLYETHERGUANIDINES

© 2020 M. Ya. Vortman et al.; Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org/ and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 541.49:546.791.6 +546.73

Background. Polyhexamethylene guanidines are widely used as biocides and disinfectants due to the wide range of their antimicrobial activity against Gram-positive and Gram-negative bacteria, viruses, fungi, and molds. The mechanism of biocidal action of polyguanidines is similar to that of quaternary ammonium compounds and has a membrane toxic nature. The advantages of polyhexamethylguanidines salts include their moderate toxicity and a lack of cumulative action against living organisms. The convenience of such polymeric biocides lies in their high water solubility and in the absence of volatility, which allows disinfectant work to be carried out in the presence of people. It is known that the introduction of alkyl radicals into the polymer chain leads to an increase in the bactericidal and fungicidal action of the obtained compounds. In order to enhance these properties, it seems promising to obtain polyetherguanidines with alkyl radicals in their structure. The aim of this work was to study the fungicidal and bactericidal activity of synthesized alkyl-substituted polyetherguanidinium iodides against a number of bacteria and microscopic fungi.
Methods. Bacteria were grown on the meat-peptone agar for 48 hours at 28±2 °C. Test cultures of micromycetes were cultured on agar beer wort (6 ° B), incubated for 14 days at 28±2 °C. Antimicrobial and fungicidal activities of the newly synthesized alkylsub sti tuting polyetherguanidines were determined by the standard disco-diffusion method.

INTRODUCTION
Polyhexamethylene guanidines (PGMG) are widely used as biocides and disinfectants due to the wide range of their antimicrobial activity against Gram-positive and Gram-negative bacteria, viruses, fungi, and molds [5-7, 19, 20]. The mechanism of biocidal action of polyguanidines is similar to that of quaternary ammonium compounds and has a membrane toxic nature [14,17].
The advantages of PGMG salts include their moderate toxicity and the absence of cumulative action against living organisms (hazard class 4 according to GOST 12.1.007-76) [6,10,12]. The convenience of using such polymeric biocides lies in their high solubility in water and in the absence of volatility, which allows for disinfection in the presence of humans [12,14,16,17].
Previously, we investigated the fungicidal activity of guanidine-containing oligoether (GO) against Gram-positive and Gram-negative bacteria and isolates of microscopic fungi that caused damage to rubber materials. GO with terminal guanidinium fragments was obtained on the basis of aromatic oligoepoxide and guanidinium chlo- ride. It was found that guanidine-containing oligoether at a concentration of 3% inhibited growth of bacteria and most of the studied micromycetes [4,8,18]. Fungicidal effect of the functionalized polymer based on the specified oligoetherguanidinium chloride and aliphatic oligooxyethylenediamine, which inhibited growth of most of studied micromycetes isolated from the premises in Kyiv, was also investigated. It is known that the introduction of alkyl radicals into the polymer chain leads to an increased bactericidal and fungicidal effect of the obtained compounds. In order to enhance these properties, it seems promising to obtain polyetherguanidines with alkyl radicals in their composition. The simplest method of introducing an alkyl radical into a polymer chain involves using the most common alkylating agent, methyl iodide. It can be assumed that the introduction of methyl iodide into polyguanidine chain will produce a polymer with a good bactericidal and fungicidal properties.
The aim of this work was to study the fungicidal and bactericidal activities of synthesized alkyl-substituted polyetherguanidinium iodides against a number of bacteria and microscopic fungi. For the synthesis of guanidinium-containing oligoether -GO based on aromatic epoxy oligomer DER-331 MW 365.5 and guanidinium chloride, 10 g of epoxy oligomer was dissolved in 30 ml of dimethylformamide and uploaded to the reactor and a solution of guanidine (3.3 g) was added, which was pre-converted from salt to basic form by alkali. The reaction was carried out for 2-3 h at 50-60 °С. The completeness of the reaction was monitored by IR spectroscopy for the disappearance of epoxy groups absorption at 920 cm -1 . To the solution of the resultant product was added 5 mL hydrochloric acid (37%) and the reaction mixture was mixed to convert oligoether into an acid form, the solvents were separated from the reaction mixture under reduced pressure. The obtained GO was precipitated from dimethylformamide to diethyl ether. To remove solvent residues, the product was kept in vacuum at a pressure of 1 mbar at 80 °C for 12 h. The product yield constituted 95%.

MATERIALS AND METHODS
To obtain polyetherguanidinium chloride (PEG-GC), a mixture of 10 g of GO and 8.2 g of oligooxyethylenediamine with terminal amino groups MW 400 was heated to 80 °C and stirred for 4 h, then the reaction was continued for 4 h at 130-140 °C and 4 h at 180 °C. After cooling the reaction mixture, an amorphous vitreous polymer of PEG-GC was obtained. To purify the product from residual starting reagents, it was dissolved in 40 mL of water and precipitated by adding 20 mL of saturated sodium chloride solution. The purified polymer was separated by decantation, washed with water (20 mL) and dried in vacuum at a pressure of 1 mbar at 80 °C for 24 h. The product represents an amorphous polymer of dark yellow color. The intrinsic viscosity of PEG-GC, determined in 0.1 N aqueous NaCl solution at 25 °C, is 0.065 Dl/g. The obtained polyetherguanidinium chloride was converted from the salt form to basic form by reaction with an equivalent amount of alkali in ethanol. The obtained sodium chloride was removed from the reaction mixture by filtration. The product was dried and reacted with methyl iodide at a molar ratio of PEG: methyl iodide 1 : 2 in methanol at 50 °C. The final product was dried at room temperature and represented an amorphous polymer of dark yellow color. Alkyl-substituted polyetherguanidinium iodides based on oligooxyethylene glieols MW 230 and 2000 were obtained by a similar method.
Cultivation of microorganisms. Bacteria were grown on meat-peptone agar for 48 hours at 28±2 °C. Test cultures of micromycetes were cultured on agar beer wort (6 ° B), incubated for 14 days at 28±2 °C.
The antimicrobial activity of newly synthesized polyetherguanidinium iodides was determined by the standard disco-diffusion method [6]. In our study we invistigated 1 and 3% solutions of polymers in distilled water. The solutions were applied in 0.2 mL on standard paper disks with a diameter of 6 mm and placed on the surface of meatpeptone agar inoculated with the appropriate test culture of bacteria. Incubation was performed for 18 hours at 28±2 °C. Antimicrobial activity was expressed by the diameters (mm) of the zones of growth retardation of microorganisms.
Fungicidal activity of the newly synthesized polyetherguanidinium iodides was determined by the method of diffusion of the investigated polymer solution into agar of nutrient Chapek-Dox medium. Test cultures were taken from the collection of fungal cultures of the Department of Physiology and Systematics of Micromycetes of the Institute of Microbiology and Virology of NAS of Ukraine, isolated from technical, polyethylene and construction materials and stored in the Testing Laboratory of Fungal Resistance and Microbiological Research of Technical and Medical Products and Materials.
A suspension with a concentration of 1×10 6 spores of each species was prepared from a working batch of fungal cultures aged 14 to 28 days. The suspension was prepared as follows: spores from a pure culture tube were transferred to a flask containing 25±5 cm 3 of sterile water. The suspension of spores of each species was mixed, the concentration of spores was calculated using a Goryaev counting chamber. Next, 1 ml of the spore suspension was added to sterile Petri dishes and filled with Chapek-Dox warm medium for preparation of lawns of cultures by a deep method.
After solidification of the medium, holes were made with a sterile drill with a diameter of 5 mm, which filled 0.1 mL of the test drug. Petri dishes were kept in a thermostat at a temperature of (29±2) °C for 14 days.
Evaluation of the sensitivity of cultures isolated from technical and building materials to the studied compounds was carried out by measuring the diameter of the zone of growth retardation of micromycetes: > 25 mm -high; 25-15 mm -average; <15 mmlow; 0 mm -absent.
IR Fourier spectroscopy. The IR spectra of oligomers with Fourier transform were recorded on a TENSOR 37 spectrophotometer in the spectral region of 6000-400 cm -1 in KBr tablets. 1 H NMR spectra were recorded on a Varian VXR-400 MHz instrument (USA) in a CDCI 3 system.
Statistical processing of the results. The experiments were performed in triplicate, the results were expressed using the standard deviation M±n. Data processing was performed using the software package Excel 2016 (MS Office) and Origin 8.5 (MS Office).
Research results. The synthesized alkyl-containing polyetherguanidinium iodides can be represented by the following structural formula: The synthesis of alkyl-containing polyetherguanidinium iodides was carried out in four stages.
The first stage was the synthesis of guanidine-containing oligoether with terminal guanidine moieties by the reaction between aromatic oligoepoxide and guanidine The structure of the obtained oligomer was confirmed by IR ( Table 1) and 1 H NMR spectra (Fig. 1) The second stage is the synthesis of polyetherguanidinium chloride by the reaction between guanidinium-containing oligoether with terminal guanidine fragments and oligooxyethylenediamine of different molecular weight.
In the third stage, the obtained polyetherguanidinium chloride is converted from the salt form to the base form by an equivalent amount of alkali in ethanol.
In the fourth stage, the basic polyetherguanidine is reacted with methyl iodide at a molar ratio of PEG : methyl iodide components of 1:2. The structure of the obtained polyetherguanidinium iodides was confirmed by IR ( Table 2) and 1 H NMR spectra (Fig. 2). The obtained polymers are resinous products of dark yellow color that are soluble in water, ethanol, methanol, methyl ethyl ketone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide and insoluble in diethyl ether, hexane, acetone. Data on the study of bactericidal activity of the obtained polyetherguanidinium iodides in relation to a number of Gram-positive and Gram-negative bacteria are given in Table 3 and Fig. 3  The fungicidal activity of alkyl-containing polyetherguanidinium iodides in relation to different types of microscopic fungi is shown in Table 4 and in Fig. 4-7. Table 4.
Fungicidal activity of alkyl-containing polyetherguanidinium iodides  The fungicidal activity of alkyl-containing polyetherguanidinium iodides at a concentration of 1% was determined with respect to micromicetу isolates, which dominated or often occurred in Kyiv and could pose a significant threat to human health.
According to the obtained data, alkyl-containing polyetherguanidinium iodides at a concentration of 1% showed fungicidal activity against almost all studied isolates after 14 days. With increasing length of the oligoethylene oxide component, the obtained polymers PEG-2 and PEG-3 did not show a fungicidal effect against A. niger, and PEG-3 against the micromycete A. versicolor. If we determine the fungicidal effect as a whole, the polymer PEG-1 showed the highest activity against all studied isolates after 14 days, while the fungicidal effect of PEG-2 and PEG-3 decreased. All synthesized alkyl-containing polyetherguanidinium iodides showed the greatest activity against C. clado sporioides, A. strictum, A. alternata, C. sphaerospermum, P. variotii, S. chartarum. Noteworthy is the selective effect of the obtained polymer PEG-2 on the isolate P. funicu losum (diameter of growth retardation more than 20 mm), as well as of PEG-3 on isolates A. strictum, A. alternata (diameter of growth retardation more than 20 and 21 mm, respectively).

DISCUSSION
The synthesized alkyl-containing polyetherguanidinium iodides showed rather high zones of growth retardation of Gram-negative bacteria E. coli 475 and K. pneumo niae 479 and lower for the Gram-positive bacterium S. aureus 451. It can be assumed that the mechanism of biocidal action of alkyl-containing polyetherguanidinium iodides is similar to that of polyguanidines and has a membrane toxic nature [1,21]. It is known that the biocidal activity of polyguanidines is due to the cooperative interaction of neighboring guanidine groups of the polycation with the microbial cell and is also influenced by the structure of the guanidine group [7,10] in contrast to the cation of quaternary ammonium compounds [13,15] where the positive charge is distributed between three nitrogen atoms. Delocalization of the positive charge softens the action of the biocide and reduces its toxicity.
The fungicidal effect of alkyl-containing polyetherguanidinium iodides is probably due to the presence of guanidine moieties and a diphenylolpropane group. It is known that in the molecules of organic substances under the influence of different atoms or atomic groups present in them there is a redistribution of the electronic density of chemical bonds (positive or negative induction effect) [2,3,9].
The presence of a diphenylolpropane group in the polymer molecule causes a negative induction effect -the substituent reduces the electron density on the carbon atom to which it is attached. In this case, the substituent acquires a partial negative charge (δ-), and the carbon atom acquires a partial positive charge (δ+) [10]. According to the literature, the value (δ+) may be one of the factors that enhances the interaction of fungicidal substances with the cell wall of fungi [19].
The data above show that with increasing length of the oligoethylene oxide component in the molecules of alkyl-containing polyetherguanidinium iodides, the fungicidal activity generally decreases. This is probably due to a decrease in the concentration of guanidine and diphenylolpropane groups in the polymer chain that promote the fungicidal action.