Carnitine alkyl ester bromides as novel biosourced ionic liquids, cationic hydrotropes and surfactants

Abstract

Hypothesis

In contrast to anionic and nonionic amphiphilic substances, bio-based cationic ones are very rare. Cationic amphiphiles are mostly based on quaternary ammonium, pyridinium or imidazolium groups that are either badly biodegradable or have toxic residues even after degradation. In the search for green alternatives to cationic hydrotropes and amphiphiles, natural l-carnitine could be a promising candidate for a cationic headgroup.

Experiments

By esterification of carnitine in one step and with low cost, cationic molecules with alkyl chain length of n = 2–14 could be obtained. Their thermal properties, aggregation behaviour and cytotoxicity were determined. Hydrophobic compounds were solubilized in their aqueous solutions and the PIT-slope method was applied to determine a relative hydrophilicity.

Findings

It was found that some pure carnitine ester bromides were liquid at room temperature and thus can be classified as ionic liquids. They are highly water-soluble, and in aqueous solutions, they showed hydrotrope or surfactant behaviour depending on their alkyl chain length. Their high hydrotropic efficiency was demonstrated by solubilizing Disperse Red 13, while also biomolecules, like vanillin, could be dissolved in reasonable amounts. In all tests, they performed at least as good as the tested reference substances, while showing similar cytotoxicity towards human skin keratinocytes, thus demonstrating their potential as green functional amphiphilic molecules of positive charge.

Introduction

Carnitine, also called 3-hydroxy-4-N-trimethylaminobutyrate, is a naturally sourced quaternary ammonium compound [1]. It occurs in different biological materials, like animal tissue, plants, fruits and microorganisms, whereas mammal muscles are the richest source. In the organism, it can be partly bio-synthesized from the lysine and methionine amino acids. It is needed for fatty acid transportation through mitochondrial membranes [2], [3]. As a chiral compound, l-carnitine is the biologically active form. It is commercially available as a nutritional supplement and supposed to enhance fat burning. Carnitine is a zwitterion in its neutral state and combines the functional groups of choline (hydroxyl group) and betaine (carboxylic group). While choline and its derivatives are well known in the field of green alternative solvents, surfactants and catalysis [4], [5], [6], [7], [8], carnitine has only been scarcely studied in this context up to now. This quaternary ammonium, which is authorized in food, pharmaceutics and cosmetics, is therefore a relevant candidate for the design of novel bio-based, biocompatible and non-toxic amphiphiles [9].

A lot of literature can be found on acylcarnitine compounds regarding their biological functions [10], [11], [12], [13]. Most reports are on their formation in the organism during fatty acid transport through the inner mitochondrial membrane. Only a few studies dealing with the surfactant properties of long-chain acylcarnitine compounds have been published [14], [15], [16]. Goñi et al. have investigated the surfactant properties of palmitoylcarnitine, a zwitterionic amphiphile used as a biological detergent [17]. In contrast, literature on linear carnitine amphiphiles formed by modification of the carboxyl group is rather rare. De Maria et al. and Cipollone et al. have shown the formation of cubic, hexagonal and lamellar phases as well as multilamellar vesicles by different diesters of carnitine depending on the alkyl substituents [18], [19]. A detailed study of the self-assembly of carnitine dodecyl amide (i.e. 3-(dodecylcarbamoyl-2-hydroxypropyl)-trimethylammonium) has been published by Patra et al. [20] who described the spontaneous formation of cationic vesicles in water. Another paper of this group deals with the interactions between the cationic carnitine hexadecyl amide surfactant with the protein pepsin [21]. Apart from these studies, carnitine has been rarely used for the design of amphiphilic molecules. In the present work, we have thus been interested in bromide salts of carnitine alkyl esters (Fig. 1) with alkyl chain lengths ranging from C₂ to C₁₄.

Depending on the chain length, different properties and behaviours were observed, some of them typical of hydrotropes and others of surfactants. Analogous to the critical micellar concentration (CMC) that characterizes surfactants, there is a certain hydrotrope concentration beyond which the solubilization power of hydrophobic compounds significantly increases. This concentration is often called the minimum hydrotrope concentration (MHC) [22].

Beside this, some ionic hydrotropes can also exhibit ionic liquid properties as reported by several authors. For instance, Claudio et al. reported the hydrotropic activity of several ionic liquids based on imidazolium, pyridinium, piperidinium, quaternary ammonium and phosphonium compounds as well as their ability to solubilize some antioxidant biomolecules. In this way, they established a connection between the concept of ionic liquids and the hydrotropic solubility phenomenon [23]. However, it should be noted that none of these positively charged headgroups are biobased.

In the present study, a series of alkyl carnitine ester bromides from C₂ to C₁₄ has been prepared in a simple one-step synthesis (Fig. 2). Since esters are relatively easy to cleave, the (bio-) degradation products of them will mainly consist of products found in nature and as thus should be more easily biodegradable.

The pure compounds were characterized for their thermal properties. Their aqueous phase behaviour has been investigated by surface tension measurements and dynamic light scattering. The ability of the short-chain derivatives to solubilize the Disperse Red 13 hydrophobic dye was studied in order to examine their hydrotropic action and the solubility of vanillin was determined to demonstrate their usefulness in application. For such a purpose, the cytotoxicity of the newly developed compounds has also been determined and compared to two conventionally used cationic surfactants (DTAB, CTAB), one typical hydrotrope (SXS) and one ionic liquid ([C₁C₄Im]Br). Finally, the carnitine ester compounds were classified according to their hydrophobic/lipophilic behaviour using the PIT-slope method [24] which is more powerful and more robust than the well-known HLB of Griffin [25]. The results are discussed in terms of properties related to chain length and potential applications of these novel carnitine-based compounds.