Anti-microbial activities of ionic liquids

Abstract

Ionic liquids (ILs) are shown to display anti-microbial activity with the activities being greatly affected by alkyl chain lengths. Shorter substituents on the cation result in a lack of activity against cocci, rods and fungi. ILs containing 10, 11, 12 and 14 carbon atoms in an alkoxy group show very high anti-microbial activities. The use of microorganisms in the IL require consideration of their minimum inhibitory concentration (MIC) values.

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Green Context

As often is the case with more interesting new areas of technology a high level of research activity leads to spin-off benefits outside of the main focus of effort. The dramatic growth in interest is the use of ionic liquids as alternative ‘greener’ solvents has led to such added benefits with somewhat unexpected applications being reported in synthesis and in biotechnology. Here we can read about their use as anti-microbial agents. The ILs studied are shown to be active against cocci, rods and fungi. The research reveals a relationship between the structure of the cation and the anti-microbial activities.

JHC

Introduction

Ionic liquids (ILs), a class of neoteric solvent, have attracted increasing interest over recent years as excellent alternatives to organic solvents in homogeneous and biphasic processes. These solvents can be used as media for a wide range of chemical reactions which have been the subject of several excellent reviews.^1–7 ILs are non-volatile, non-flammable, have low melting points, high thermal stability in a wide temperature range and relatively low viscosity. They can be used as replacements for selected organic solvents and have shown promise toward important applications such as synthesis,^1–10 separation and extraction processes.^11–14 Investigation of this group of compounds as solvents is in its very early stages. Before the full potential of these ILs are realized, much more information about them as solvent systems must be obtained. The polarity,^15–17 physical^18–20 and thermodynamic^21,22 properties of ILs have been investigated. ILs have been proposed as green solvents for organic synthesis due to the ease with which they can be recycled and reused whilst offering inert, dipolar media compatible with much of conventional chemistry.¹

One of the most exciting recent developments is the use of enzymes and other types of biotransformations in ILs. Reactions have been carried out in a biphasic H₂O–IL system^23,24 and show higher final yields; also an anhydrous system has been reported.^25–32 The lipase reaction in ILs can be scaled up without major difficulty.²⁷ The enzyme suspended in the IL could be reused three times with less than 10% loss of activity per cycle and the enantioselectivity was not influenced.²⁶ The enzymes are stable in solvents such as 3-butyl-1-methylimidazolium hexafluorophosphate or tetrafluroborate. Based on these initial studies the use of enzymes in ionic liquids would appear to open up a new field of non-aqueous enzymonology. The most important finding of the preliminary reports is the fact that many enzymes retain their activity in ILs. In the literature there are examples of the use of a whole-cell biotransformation in an IL for the bacterium Rhodococcus R312 that contains the nitrile hydratase enzyme²³ or Baker’s yeast.³³ The studies of ABE fermentation with 3-octyl-1-methylimidazolium hexafluorophosphate present at saturation level suggest that the IL suppresses biological activity in the system.³⁴

In this paper we report our results of the investigation of anti-microbial activities of ILs.

Results and discussion

A series of 3-alkoxymethyl-1-methylimidazolium salts of [Cl]⁻ (1), [BF₄]⁻ (2) and [PF₆]⁻ (3) were prepared (Table 1). All chlorides synthesized were very hygroscopic. The [BF₄]⁻ and [PF₆]⁻ salts were obtained by simple metathesis reactions from the corresponding chlorides using aqueous KBF₄ or KPF₆. The [BF₄]⁻ salts have a greater miscibility with water than the corresponding [PF₆]⁻ salts. The water miscibility of 3-alkoxymethyl-1-methylimidazolium [BF₄]⁻ and [PF₆]⁻ depends on the length of the alkoxymethyl chain. The water soluble ones are those salts with short chains (propoxymethyl, butoxymethyl and pentyloxymethyl). Water was present in all salts of the type we studied even after a moderate drying procedure (drying at 70 °C for 4 h on a vacuum line). The water content in the dried ILs depends on the length of the alkyl chain and was found to be between 2200 and 400 ppm.¹⁹ All the salts prepared are air-stable under ambient conditions and may be handled under normal laboratory conditions. Molecular states of water in ILs have been investigated based on the 1-alkyl-3-methylimidazolium salts.³⁵ It has been shown that in these ILs water moleculas absorbed from the air are present mostly in the ‘free’ state, bound via H-bonding with [PF₆]⁻ and [BF₄]⁻ with the concentration of dissolved water in the range 0.2–1.0 mol L⁻¹. It has been concluded that most of the water molecules at these concentrations exist as 1∶2 H-bonded complexes: anion⋯HOH⋯anion.

Table 1 3-Alkoxymethyl-1-methylimidazolium salts 1–3

Chlorides			Tetrafluoroborates			Hexafluorophosphates
	R	Mp/°C		R	Mp/°C		R	mp/°C
Grease = heavy oil.
1a	C3H7	Oil	2a	C3H7	Liquid	3a	C3H7	Liquid
1b	C4H9	Oil	2b	C4H9	Liquid	3b	C4H9	Liquid
1c	C5H11	Oil	2c	C5H11	Liquid	3c	C5H11	Liquid
1d	C6H13	Grease	2d	C6H13	Liquid	3d	C6H13	Liquid
1e	C7H15	Grease	2e	C7H15	Liquid	3e	C7H15	37–38
1f	C8H17	Grease	2f	C8H17	Liquid	3f	C8H17	48–50
1g	C9H19	Grease	2g	C9H19	Liquid	3g	C9H19	47–49
1h	C10H21	Grease	2h	C10H21	56–57	3h	C10H21	46–47
1i	C11H23	66–68	2i	C11H23	61–62	3i	C11H23	52–53
1j	C12H25	67–70	2j	C12H25	62–64	3j	C12H25	61–63
1k	C14H29	70–73	2k	C14H29	65–67	3k	C14H29	67–69
1l	C16H33	73–75	2l	C16H33	68–69	3l	C16H33	71–73

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All synthesized imidazolium salts were tested for anti-microbial activity against cocci, rods and fungi. Minimum inhibitory concentration (MIC) values and minimum bactericidal or fungicidal concentration (MBC) values are given in Tables 2–4. Also the MIC and MBC values of benzalkonium chloride (BAC) were determined and shown in Table 2. The calculated average MIC and MBC values for cocci, rods and fungi are presented in Figs. 1–3 as a relationship between the alkyl chain length and anti-microbial activity. As shown by the results, the salts studied are active against cocci, rods and fungi. Some of them exhibited strong activity and wide anti-bacterial action. Their activities are greatly affected by the alkyl chain length in the alkoxymethyl substituent but do not depend on the type of anion. The MIC values of imidazolium chlorides indicate the anti-microbial activities for the same imidazolium salts with [BF₄]⁻ or [PF₆]⁻. For the studied salts the same correlation is observed for the MIC and MBC values. The most active salts against cocci and rods have an alkoxy group which contains 10, 11, 12 or 14 carbon atoms. The curves in Figs. 1 and 2 demonstrate the optimum anti-microbial efficiency, the most effective group being dodecyloxymethyl. Activity against fungi is significantly different with the curves in Fig. 3 indicating that no optimum activity was clear with the salts under study here. Though the observed increase of activity with number of carbon atoms in the alkoxy group will not be unlimited the hydrophobic chain has the important function of adsorbing onto the surface of the microbial cell.

Table 2 MIC^a and MBC^a values of 3-alkoxymethyl-1-methylimidazolium chlorides (1a–l) and of BAC

		Chloride
Strain		1a–1c	1d	1e	1f	1g	1h	1i	1j	1k	1l	BAC^b
a In μM, the number of microorganisms in 1 mL range from 10^4 to 10^5.b BAC, benzalkonium chloride.c No data available (opacity of solution).
Cocci
M. luteus	MIC	>8600	4300	2030	960	230	108	52	25	—^c	—^c	7
	MBC	>8600	4300	4060	3800	910	217	207	49	91	336	11
S. epidermidis	MIC	>8600	4300	1000	480	110	54	52	25	—^c	—^c	3
	MBC	>8600	4300	2030	1900	1820	867	103	49	91	1340	3
S. aureus	MIC	>8600	4300	500	480	228	54	52	25	—^c	—^c	7
	MBC	>8600	8600	1000	960	1820	867	826	99	91	168	7
S. aureus MRSA	MIC	>8600	8600	4060	3800	1820	433	207	99	—^c	—^c	7
	MBC	>8600	8600	8100	7680	3640	867	413	395	363	671	11
E. hirae	MIC	>8600	8600	8100	1900	228	108	103	99	—^c	—^c	11
	MBC	>8600	8600	8100	7680	3640	433	207	197	91	168	22
Rods
E. coli	MIC	>8600	8600	8100	3800	1820	433	413	99	181	671	7
	MBC	>8600	8600	8100	7680	1820	867	413	395	363	2680	11
P. vulgaris	MIC	>8600	8600	4060	3800	1820	217	207	197	181	336	22
	MBC	>8600	8600	8100	3800	1820	867	413	197	363	336	22
K. pneumoniae	MIC	>8600	8600	8100	3800	1820	433	207	197	181	168	11
	MBC	>8600	8600	8100	7680	3640	1733	413	395	181	336	11
P. aeruginosa	MIC	>8600	8600	4060	3800	1820	867	826	395	726	2680	54
	MBC	>8600	8600	8100	7680	3640	1733	1653	790	5800	>5370	205
Fungi
C. albicans	MIC	>8600	8600	4060	1900	1820	433	413	197	91	84	7
	MBC	>8600	8600	8100	3800	3640	867	413	395	181	84	11
R. rubra	MIC	>8600	8600	2030	1900	910	217	103	99	45	42	11
	MBC	>8600	8600	4060	3800	3640	867	207	197	91	42	11

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Table 3 MIC^a and MBC^a values of 3-alkoxymethyl-1-methylimidazolium tetrafluoroborates (2a–2k)

		Tetraflouroborates
Strain		2a–c	2d	2e	2f	2g	2h	2i	2j	2k
a In μM, the number of microorganisms in 1 mL range from 10^4 to 10^5.b No data available (opacity of solution)
Cocci
M. luteus	MIC	>7000	3500	3360	800	192	46	44	21	—^b
	MBC	>7000	7000	3360	1600	1540	368	88	21	39
S. epidermidis	MIC	>7000	3500	1680	400	192	46	44	21	—^b
	MBC	>7000	7000	3360	800	1540	368	177	85	158
S. aureus	MIC	>7000	7000	840	800	384	92	22	21	—^b
	MBC	>7000	7000	1680	3200	1540	736	353	85	79
S. aureus MRSA	MIC	>7000	7000	3360	3200	1540	368	177	85	—^b
	MBC	>7000	7000	6700	6400	3070	736	353	680	632
E. hirae	MIC	>7000	3500	3360	1600	384	92	88	85	—^b
	MBC	>7000	7000	6700	6400	3070	1471	177	340	158
Rods
E. coli	MIC	>7000	7000	6700	3200	1540	368	177	170	316
	MBC	>7000	7000	6700	6400	3070	1471	177	170	158
P. vulgaris	MIC	>7000	7000	6700	3200	1540	184	177	170	158
	MBC	>7000	7000	6700	3200	3070	736	353	170	316
K. pneumoniae	MIC	>7000	7000	6700	1600	3070	368	177	170	158
	MBC	>7000	7000	6700	6400	3070	736	353	340	158
P. aeruginosa	MIC	>7000	7000	6700	3200	1540	1471	707	340	632
	MBC	>7000	7000	6700	6400	3070	1471	1413	680	1260
Fungi
C. albicans	MIC	>7000	7000	3360	1600	770	736	707	340	158
	MBC	>7000	7000	6700	3200	1540	1471	1413	340	158
R. rubra	MIC	>7000	7000	3360	1600	192	184	177	21	20
	MBC	>7000	7000	6700	3200	1540	736	177	85	39

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Table 4 MIC^a and MBC^a values of 1-alkoxymethyl-3-methylimidazolium hexafluorophosphates (3a–3k)

		Hexafluorophosphates
Strain		3a–c	3d	3e	3f	3g	3h	3i	3j	3k
a In μM, the number of microorganisms in 1 mL range from 10^4 to 10^5.b No data available (opacity of solution)
Cocci
M. luteus	MIC	>5850	2900	2800	1350	330	160	152	37	—^b
	MBC	>5850	5850	5600	2700	1300	630	152	73	138
S. epidermidis	MIC	>5850	2900	1400	680	330	80	76	18	—^b
	MBC	>5850	5850	5600	2700	1300	1250	303	147	138
S. aureus	MIC	>5850	2900	2800	1350	650	160	19	18	—^b
	MBC	>5850	5850	5600	2700	2600	1250	607	73	69
S. aureus MRSA	MIC	>5850	5850	2800	2700	1300	630	607	73	—^b
	MBC	>5850	5850	5600	5400	2600	2500	1214	587	551
E. hirae	MIC	>5850	2900	2800	2700	330	630	152	37	—^b
	MBC	>5850	5850	5600	5400	2600	2500	607	587	275
Rods
E. coli	MIC	>5850	5850	5600	2700	1300	1250	607	73	138
	MBC	>5850	5850	5600	5400	2600	1250	607	293	275
P. vulgaris	MIC	>5850	5850	5600	2700	1300	314	303	147	138
	MBC	>5850	5850	5600	5400	2600	2500	1214	293	551
K. pneumoniae	MIC	>5850	5850	5600	2700	2600	630	152	147	138
	MBC	>5850	5850	5600	5400	2600	2500	1214	147	138
P. aeruginosa	MIC	>5850	5850	5600	2700	2600	1250	1214	587	1100
	MBC	>5850	5850	5600	5400	2600	2500	1214	587	2200
Fungi
C. albicans	MIC	>5850	5850	2800	1350	1300	630	607	587	138
	MBC	>5850	5850	5600	2700	2600	1250	1214	587	275
R. rubra	MIC	>5850	5850	2800	1350	650	314	152	37	17
	MBC	>5850	5850	5600	2700	2600	630	303	587	138

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Fig. 1 Mean MIC and MBC values for cocci.

Fig. 2 Mean MIC and MBC values for rods.

Fig. 3 Mean MIC and MBC values for fungi.

The activity of salts 1j, 2j, and 3j approach the activity of commercially available benzalkonium chloride (BAC in which R represents a mixture of alkyls from C₈H₁₇ to C₁₈H₃₇). The ILs with short substituents (propoxymethyl, butoxymethyl and pentyloxymethyl) are not active against bacteria and fungi.

In summary we found that ILs showed anti-microbial activities. There is a relationship between the structure of the cation and the anti-microbial activities. These salts with short substituents are not active against bacteria and fungi. The results indicate that salts containing 10, 11, 12 and 14 carbon atoms in the alkoxy group show very high anti-microbial activities. The use of microorganisms in ILs requires consideration of their MIC values.

Experimental

3-Alkoxymethyl-1-methylimidazolium chlorides (1), tetrafluoroborates (2) and heksafluorophosphates (3) were prepared according to the published method.³⁶

Anti-microbial actibity

Anti-microbial activity was determined by the tube dilution method. A series of imidazolium salt dilutions were prepared on Müller–Hinton broth medium (bacteria) or Sabouraud broth medium (fungi). A suspension of the microorganisms, prepared from 24 h cultures of bacteria in the Müller–Hinton broth medium and from 48 h cultures in the Sabouraud agar medium for fungi at a concentration of 106 cfu mL⁻¹, were added to each dilution in a 1∶1 ratio. Growth (or lack thereof) of the microorganisms was determined visually after incubation for 24 h at 37 °C (bacteria) or 48 h at 28–30 °C (fungi). The lowest concentration at which there was no visible growth (turbidity) was taken as the MIC. Then from each tube, one loopful was cultured on an agar medium with inactivates (0.3% lecithin, 3% polysorbate 80 and 0.1% cysteine L) and incubated for 48 h at 37 °C (bacteria) or for 5 days at 28–30 °C (fungi). The lowest concentration of the salt supporting no colony formation was defined as the MBC.

Microorganisms used: eleven standard strains representative of cocci; Micrococcus luteus ATCC 9341, Straphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 6538, Staphylococcus aureus MRSA, Enterococcus hirae, rods; Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 15442, fungi; Candida albicans ATCC 10231, Rhodotorula rubra (Demml 1889, Lodder 1934). Standard strains were supplied by the National Collection of Type Cultures (NCTC) London and American Type Culture Collection (ATCC). The R. rubra was taken from the Department of Pharmaceutical Bacteriology, K. Marcinkowski University of Medical Sciences, Poznan.

Acknowledgments

This investigation received financial support from the Polish Committee of Scientific Research Grant KBN No 4 T09B 008 22.

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