Development of Arginine Based Monopeptides as Cationic Surfactants from Pure Amino Acid

Amino acid-based surfactants constitute a class of cationic surfactants with excellent surface p roperties, wide biological activ ity, low potential toxicity. Essential structural factors for their antimicrobial activity include both the length of the fatty residue and the presence of the protonated guanidine function. These features and the use of natural raw materials such as Arginine and fatty acids for their synthesis, make them interesting candidates as preservatives and antiseptics in pharmaceutical, food and cosmetic fo rmulat ions. The present work describes the synthesis (at lab scale), the physicochemical properties of long chain Nα-acyl arginine ethyl esters with a chain of 8 and 9 carbon atoms. These compounds have been prepared by condensation of L-arg inine ethyl ester dihydrochloride with corresponding acyl chlorides in the presence of base. The basic charastristic properties such as physical appearance, structural identificat ion by IR/HNMR, water solubility at different pH and surface tension studies of the synthesized compounds are discussed. The Antimicrobial properties of the compounds of invention are studied by determin ing their Minimum inhibitory concentration for different microorganisms.


Introduction
Surfactants are widely used in both consumer and industrial application, but they can adversely affect the environment. Therefore there is a gro wing demand for mild, biodegradable and nontoxic products that are made fro m natural raw materials. These surfactants can be obtained fro m mo lecules that mimic natural amph iphilic structures. The association of a polar amino acid (hydrophilic mo iety) and a non-polar long chain compound (hydrophobic moiety) to built amphiphilic structures allow obtaining molecules with a h igh surface activity.
Long chain N α -acyl amino acids have been studied by many groups with the aim to obtain mild surfactants for preservative applications [1,2]. Cation ic surfactants derived fro m the condensation of fatty acids and esterified dibasic amino acids, such as fro m lauric acid and arg in ine, in particu lar the Et ester of the lau ramide of the argin ine monohydrochloride (LA E), may be used for the p rotection against the growth of the microorganisms. The cation ic surfactants of this type are also effect ive against virus infections. Addition of LA E to cultures of Herpes virus type 1 Vaccinia virus and bovine parainfluen zae 3 virus leads to nearly co mplete reduction of the virus organisms in these cultures, such effects being observed after 5 and 60 min [3]. Sugimoto and Toyoshima [4] have studied N α -Cocoyl-L-Arg inine ethyl ester, DL-Pyrog lutamic acid salt as an inactivator of Hepatit is B surface antigen. Lourdes et al [5], have synthesized a novel family of dicat ionic arginine -monoglyceride surfactants. Co mpounds having alkyl chain C10-C14 attached to the glycerol through esters and a dicationic polar head fro m the Arginine. An Article by Infante et al [6] studied the synthesis and properties of N α -Lauroyl-L-Arginine dipeptides as methyl esters (cationic surfactants) and free α-carboxy lic acids (amphoteric surfactants). They found properties of these compounds are comparable to the cationic mono mer derivatives methyl ester of N α -Lauroyl-L-Arg inine and of the amphoteric mono mer derivative N α -Lauroyl-L-Argin ine reported earlier. Seguer et al [7] also reported synthesis and physicochemical properties of N α -acyl-L-argin ine dipeptides with a chain length between 8-16 carbon atoms. Fro m the studies the author has summarized that the introduction of an appropriate long chain (14 carbon atoms) to the amino function of a mixture of amino acids yields an interesting mu ltifunctional co mpound to be applied as soft preservative peptide surfactant in cosmet ics, food and dermo -pharmaceu tical formu lations.
Based on the studies done so far, it is clear that Essential structural factors for their antimicrobial activity include both the length of the fatty residue and the presence of the protonated guanidine function. These features and the use of natural raw materials such as Arginine and fatty acids for their synthesis, make them interesting candidates as preservatives and antiseptics in pharmaceutical, food and cosmetic formu lations.
We were particu larly interested in preparation o f N α -acyl argin ine esters (C8-C16), which will be used as microbicide s in future. Microbicides are the group of pharmaco logic agents and chemical substances that are capable of killing certain microorganis m that commonly cause human infection by bacteria, fungi and viruses like HIV. In this paper we report the synthesis of N α -Octanoyl and N α -Nonanoyl-L-Agin ine ethyl ester by acylat ion of a basic amino acid with a positively charged side chain (arginine) and Octanoyl/Nonanoyl chlorides. The co mpounds are tested as antimicrobial agent against Gram-positive and Gram-Negative Bacteria.

Chemistry
With the aim to systematically study the influence of the alky l chain length on the properties of long chain N α -acyl arginine ethyl ester salts, we synthesized at mult igram scale two pure homo logues with alkyl chain of 8 (CA E) and 9 (NA E) carbon atoms (Fig. 1). The fatty residue, the guanidine basic residue and the protection of the carboxyl group in the molecu le in the mo lecule of the arginine are essential structural factors for antimicrobial activ ity. The role o f the alkyl chain length in the water solubility, surface activity, crit ical micellar concentration and antimicrobial activity of these compounds was examined in order to improve our understanding of the structure/properties relationship.  [8,9] synthesized N α -lauroyl argin ine methyl ester Hydrochloride (LAM), in gel emulsions of the system water/C 14 E 4 /decane and compared with those obtained in conventional dimethylformamide (DMF) media and found that LAM formation is faster in gel emu lsions at short times. Clapes et al [10] have published a review wherein enzy mat ic synthesis, physicochemical and biological properties as well as the potential uses of A mino-acid based surfactants are described. Rondel et al [11] have described the acylation of amino acids by acid chlorides with fro m 8-12 carbon atoms in alkaline aqueous mediu m fo llo wing Schotten-Baumann reaction.
We have used an easy and very efficient procedure for the synthesis of the compounds of our interest. It consisted of three steps using L-Arginine as starting material. (1) Preparation of Fatty acid chloride (2) Synthesis of L-Arginine ethyl ester dihydrochloride by esterification process. (3) Synthesis of N α -acyl arginine ethyl ester by acylation of α-A mino group of L-Arg inine ethyl ester dihydrochloride with the corresponding long chain acid chloride.

General Reagents and Synthetic Method
L-Arginine was purchased from Ajinomoto Co., Octanoic acid, Nonanoic acid and Sodium Dodecyl sulfate (SDS) were received fro m Sig ma-A ldrich. LA E.HCl (N α -Lauroyl arginine ethyl ester Hydrochloride) was supplied by local supplier. So lvents used were of analytical grade or h igher purity and supplied by Sigma-Aldrich. The homogeneity of compounds was checked by thin-layer chro matography on alu min iu m p lates (Kieselgel G, Merck}. The solvent systems were (A) chlo roform/ methanol/acetic acid (8.5:10:5 }; and (B) chloroform/ methanol (7:3). Ninhydrin developer solution was used for qualitative analysis of free amino groups.
Nuclear Magnetic Resonance ( 1 H NM R) and all the NMR measurements were performed with Bruker, Avance 300 spectrometer model at 300M Hz in a 5mm d irect probe (BBO BB-1H) using CDCl 3 as a solvent. Surface Tension was measured using Stalagmo meter. Mass Spectroscopy with fast atom bo mbard ment (FAB) was carried out with VG-QUATTRO fro m Fisons Instrument.

Preparation of Fatty Aci d Chl ori des
In a four necked round bottom flask equipped with stirrer and thermo pocket charge 0.1mole of Fatty Acid at room temperature and heat to 40℃., then add 0.15mo le of Thionyl Chloride under Nitrogen atmosphere over a period of 1 hour and heat the reaction mixture to reflu x for 1 ½ hour to obtain crude Acid Chloride.

Preparation of L-Arginine ethyl ester dihydrochlori de
In a 250ml round bottom flask charge Ethyl alcohol followed by the addition of L-Arginine free base. Stir vigorously to get a clear solution. Charge via addition funnel conc.HCl o f 37 % strength slowly controlling exotherm. Apply heat and reflu x for 4-5 hours. Cool to 60 degree C and add more conc. HCl and reflu x further till the complet ion of the reaction with simu ltaneous removal of ethanol. Apply vacuum and distill residual alcohol. Cool the residual mass to get crude L-Argin ine ethyl ester dihydrochloride.

Preparation of Nα-Acyl L-Arginine ethyl ester compounds by Schotten B aumann reaction
The crude reaction product obtained in the first step is dissolved in water and the pH of the solution is brought to a specific pH value 5.5-7 by the addit ion of aqueous sodium hydroxide. The pH of the reaction is carefully kept constant at this value until co mplet ion of the reaction. To this solution, add 0.96 equivalent of Octanoyl/Nonanoyl chloride drop-wise, whereby the temperature of the mixtu re is kept at a temperature of 10-15 ℃ by means of an appropriate cooling bath containing ethylene glycol. After complet ion of the reaction, the stirring is maintained for a further two hours, after which the pH of the solution is adjusted to a final value of 5.5-7 with hydrochloric acid or sodium hydro xide. Finally, the crude reaction product is obtained either by filt ration or by distillat ion. Compound

Analytical Methods
The characteristic propert ies of the co mpounds synthesize d are summarized in Table 1. Their mass spectra showed only one mass peak corresponding to the molecular ion. The complet ion of reaction was monitored by TLC. Likewise, the 1 H NM R spectra of all co mpounds were in accordance with their proposed structure. The water solubility of CA E and NA E was partially studied at different pH values at a constant concentration of 1% (w/v) and at roo m temperature. CA E is clear in the range of pH 2-11.5, whereas solution of NAE is clear at pH 6-11, but insoluble at pH ≤4. This solubility data was compared with LA E solubility, which is showing clear solubility in the pH range 1-7.3 and insoluble at pH > 8 and ≤0.5 (Tab le 2). These results appear to indicate that the insolubility increases with increase in chain length (Hydrophobic Character) of the co mpound.

Method for Surface Tension
A tensiometer (Stalag mo meter) with a W ilhelmy p late was used for surface tension measurements (γ). Water /surfactant solutions of different concentrations were prepared and allowed to equilibrate at 25℃ between 4 and 10 hr.

Method for crit ical M icellar Concentration (CM C)
The critical micellar concentration (CM C) was determine d fro m the break point of the surface tension /concentration curves.

Antimicrobial Activity
Antimicrobial act ivity is tested by determin ing the Minimu m Inhibitory concentration (MIC) of the compound of invention. MIC is defined as the lowest concentration of antimicrobial agent that inhibits the development of visib le micro -organism gro wth after incubation at 32℃ for 48 hrs and fungal growth at 25℃ for 4 days by Broth Dilution method. Samp le was prepared by simp ly diluting 1ml o f the 1% solution in DMSO with 9ml of broth Tryptic Soy Broth (1000pp m solution). This stock solution did not precipitate. Fro m the above stock solution 1ml was added to each of 12 consecutive sterile 13mm tubes containing 1ml TSB. Each tube is vortexed and asceptic transfer to give the below ppm. Each cu lture is grown in TSB>24hrs<48h rs at 32℃. The culture is diluted to 10,000 cfu/ ml and 10 μl of this is added to each tube. Negative controls (NC) TSB confirm sterility of the TSB, Positive controls (PC) for each culture confirm organism capable of gro wth in the TSB.

Results and Discussions
Single chain Arg inine based surfactants have CMC values ca 1-10mM and these values decrease linearly as the number of methylene groups in the alky l chain increases [12]. The high CM C values observed for the CAE are due to the fact that it has a less hydrophobic chain than that of LA E and NA E. In fact, a longer, mo re hydrophobic chain results in lo wer surface tension. The surface Tension and CMC values of CAE and NA E are co mpared against commercially availab le LA E and SDS ( Table 3). The study indicates that both the values of surface tension and CMC are co mparable and sometimes even lower than that of SDS. They therefore have interesting surface-active properties. In general, the antimicrobial activity o f the surfactants depends on the alkyl chain length and the antimicrobial effect took place at concentrations of surfactant below the cmc. This suggests, in a good agreement with our results, that the antimicrobial activ ity is due to the individual mo lecules and not to the aggregates. For Medical applications, the use of cationic surfactants with low antimicrobial activity would be necessary based on their biodegradability and toxicity data.
Further work is in progress in order to understand the influence of effect of chain length of fatty acid residue on the physicochemical properties and antimicrobial activ ities, to be able to assess the area of applicat ion of these materials.