METHOD FOR PREPARING SURFACTANT COMPOSITIONS COMPRISING ALKYL LIDURONAMIDES D-GLUCURONAMIDES AND L-RHAMNOSIDES FROM ULVANS

Abstract

The present invention relates to a novel process for preparing surfactant compositions based on alkyl L-iduronamides, alkyl L-rhamnosides and alkyl D-glucuronamides, to the compositions obtained via said process and to the uses thereof.

Claims

1) Process for preparing a composition comprising a mixture of alkyl D-glucuronamides (I) in pyranoside form of formula (Ia) and in furanoside form of formula (Ib), of alkyl L-iduronamide of formula (II) and of alkyl L-rhamnoside of formula (III): ##STR00004## in which R.sub.1 is a linear or branched, saturated or unsaturated alkyl chain of 2 to 22 carbon atoms; R.sub.2 is a hydrogen, R.sub.1, a linear or branched, saturated or unsaturated alkyl chain of 2 to 22 carbon atoms including an amine end function, and characterized in that said process comprises: a) a step of butanolysis reaction and of Fischer glycosylation starting with ulvans and/or green algae; b) a step of aminolysis reaction on the reaction medium obtained from step a), in the presence of a linear or branched, saturated or unsaturated amine of formula RNH.sub.2 in which R is composed of from 2 to 22, preferably from 8 to 18, preferentially from 12 to 18 carbon atoms.

2) Process according to claim 1, in which said process comprises a step a) of neutralizing the reaction medium obtained from step a) before step b).

3) Process according to claim 1, in which step a) is performed in the presence (i) of water and/or of an ionic solvent and/or of a eutectic solvent, (ii) of a linear or branched, saturated or unsaturated alcohol of formula ROH, containing from 1 to 4 carbon atoms, and (iii) of an acid catalyst.

4) Process according to claim 3, in which the acid catalyst is chosen from the group consisting of: hydrochloric acid, sulfuric acid, an alkyl sulfuric acid, a sulfonic acid, an alkylsulfonic acid or an alkyl sulfosuccinate, perhalohydric acids, metals, oxides thereof or salts thereof such as the halides thereof.

5) Process according to claim 4, in which the acid catalyst is methanesulfonic acid.

6) Process according to claim 1, in which the alcohol ROH is n-butanol.

7) Process according to claim 1, in which step b) is performed in the presence of a fatty amine chosen from the group consisting of dodecylamine and oleylamine.

8) Process according to claim 1, said process also comprising: c) a step of trans-glycosylation of the reaction medium obtained from step b) or of at least one of the isolated derivatives thereof, with a linear or branched, saturated or unsaturated alcohol of formula R OH containing from 5 to 22 carbon atoms; and d) optionally a step of neutralizing the reaction medium obtained from step c) in the presence of water and of a base M(OH)x in which M is an alkali metal or alkaline-earth metal, and x is the valency.

9) Process according to claim 8, in which the alcohol R OH is chosen from the group consisting of dodecanol and oleyl alcohol.

10) Process according to claim 8, in which the trans-glycosylation step c) is performed at 70 C. under reduced pressure so as to recycle the alcohol ROH.

11) Composition obtained via a process according to claim 1.

12) Composition according to claim 11, in which said composition is an oil-in-water or water-in-oil emulsion.

13) Composition according to claim 11 configured as a surfactant.

14) Composition according to claim 13, in which the surfactant is chosen from the group consisting of solubilizers, hydrotropes, wetting agents, foaming agents, emulsion-forming agents, emulsifiers and/or detergents.

15) Composition according to claim 11, for use as an antibacterial and/or antifungal agent.

16) Surfactant comprising a composition according to claim 11.

17) Antibacterial and/or antifungal agent comprising a composition according to claim 11.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0046] FIG. 1 represents the measurement of the emulsifying power of the surfactant composition UlvC.sub.4N.sub.12 (A) W/O emulsion and (B) O/W emulsion, in comparison with commercial references Montanov and Xyliance.

DETAILED DESCRIPTION AND EXAMPLES

Example 1: Process for Obtaining Compositions Based on Alkyl L-Iduronamides, Alkyl D-Glucuronamides and Alkyl L-Rhamnosides from Ulvans

[0047] Preparation of the Starting Materials:

[0048] The process for extracting the ulvans involves treating green algae with 0.5 M hydrochloric acid solution (pH=2) heated for 2 hours at 60 C. After centrifugation (removal of the insoluble residues), the supernatant containing the ulvans is purified (removal of the polyphenolic contaminants) by precipitation using ethanol (2.5 to 3 times the volume of the aqueous solution containing the ulvans) and the precipitated ulvans are then neutralized with aqueous 0.1 M NaOH solution and the solution is lyophilized to give the ulvans in the form of sodium salts (white solid). By way of example, the chemical composition of the ulvan extracted from the species Ulva linza is characterized by a molar mass of 565 100 g.Math.mol.sup.1, a sulfate content of 17.1% (barium sulfate turbidimetric method and the following sugar composition (HPLC study after methanolysis in 2M HCl for 4 hours): rhamnose=26.2%; glucuronic acid=11.5%; iduronic acid=3.5%; xylose=5.8% and glucose=1.2%.

##STR00002##

[0049] 1) Butanolysis and Fischer Glycosylation Reaction

[0050] 2 g of sodium ulvan extracted from Ulva linza, molecular mass=565 100 g/mol (11.4 mmol, 1 eq) were mixed with 3 mL of distilled water and 1.85 mL of methanesulfonic acid (28.51 mmol, 2.5 eq). 156 mL (150 eq) of butanol were added to the ulvan solution with stirring. The medium was stirred at the reflux temperature of butanol (130-135 C.) for 24 hours. The waters added for the dissolution of the polysaccharide and those formed during the reaction were removed in Dean-Stark apparatus filled with butanol, via water-butanol azeotropic distillation. Since water is denser than butanol, it moves to the bottom of the Dean-Stark apparatus and a few ml of butanol pass into the flask to conserve the initial volume. After 24 hours, thin-layer chromatography (95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH) and proton and carbon NMR were performed for the reaction medium to ensure that the expected product had indeed been synthesized.

[0051] 2) Aminolysis Reaction (without Prior Neutralization of the Reaction Medium Before the Aminolysis Reaction)

[0052] The temperature of the medium was lowered to 60 C., followed by addition of 3 molar equivalents of dodecylamine C.sub.12H.sub.25NH.sub.2 (34.21 mmol, 7.86 g) required to increase the pH to 8.5. After stirring for 30 minutes at 65 C. under a reduced pressure of 150 mbar, the butanol was evaporated off by reducing the pressure from 150 mbar to 6 mbar over a period of 1 hour. The medium was left under a reduced pressure of 6 mbar and at 65 C. for 1 hour 30 minutes to ensure the evaporation of the traces of butanol that were formed.

[0053] The residue obtained was taken up in diethyl ether and then filtered through a sinter and rinsed several times with diethyl ether to remove the salts and the unreacted starting sugar. The filtrate (containing butyl rhamnoside and dodecyl glucuronamide and iduronamide) is concentrated under vacuum to give a dark brown oil.

[0054] After optional chromatography of the oil obtained on a column of silica gel (80 g, using 95/5 v/v CH.sub.2Cl.sub.2/CH.sub.3OH as eluent), the presence of 0.76 g (3.45 mmol, 31%, C.sub.10H.sub.20O.sub.5, 220.27 g/mol) of n-butyl -L-rhamnopyranoside (RhamOC.sub.4) was identified, along with the presence of 0.73 g (1.75 mmol, 16% yield) of a mixture of four isomeric forms of chemical formula C.sub.22H.sub.43NO.sub.6 and of molar mass=417.59 g/mol, monosaccharide surfactant composition named UlvC.sub.4N.sub.12. In the case of the D-glucuronic acid surfactant derivatives present in ulvan, 1D and 2D NMR experiments showed the presence of two isomers, a (H-1: 4.92 ppm, J.sub.1.2=3.8 Hz, C-1: 98.62 ppm) and (H-1: 4.37 ppm, J.sub.1.2=7.8 Hz, C-1: 102.89 ppm) in pyranose form, and an furanose form (H-1: 4.98 ppm, C-1: 108.67 ppm). The L-iduronic acid present in ulvan appears to lead to an pyranose form (H-1: 4.95, J.sub.1.2=0.9 Hz, C-1: 108.15).

[0055] After having determined the chemical shift of the H-1 anomeric proton of each of the isomers and of the anomers thereof, the proportion of each of the four forms present in the UlvC.sub.4N.sub.12 mixture was evaluated from the .sup.1H NMR spectrum by integration of the signals relating to the anomeric proton of each of the four forms obtained. The surfactant composition UlvC.sub.4N.sub.12 is then formed from n-(12-dodecyl)-n-butyl -D-glucurofuranosiduronamide (47%), n-(12-dodecyl)-n-butyl -D-glucuropyranosiduronamide (26%), n-(12-dodecyl)-n-butyl -D-glucuropyranosiduronamide (7%), n-(12-dodecyl)-n-butyl -L-iduronopyranosiduronamide (20%). The proportions of the furanose form () and of the pyranose forms ( and ) in the UlvC.sub.4N.sub.12 mixtures made it possible to evaluate a pyranose/furanose ratio. The value of the pyranose/furanose ratio is of the order of 1.12 for the UlvC.sub.4N.sub.12 mixture indicating that the pyranose forms ( and ) of n-dodecyl n-butyl D-glucuronamide and n-dodecyl n-butyl L-iduronamide are predominant relative to the furanose form of n-dodecyl n-butyl D-glucuronamide.

[0056] On account of the different polarities, it was possible to separate by column chromatography on silica gel (eluent: 95/5 dichloromethane/methanol) the uronamide compositions (UlvC.sub.4N.sub.12) from the more polar n-butyl L-rhamnoside compound (RhamOC.sub.4).

[0057] The molar mass of the n-butyl -L-rhamnopyranoside compound (220.27 g/mol evaluated by mass spectrometry) and the absence of an absorption band characteristic of sulfate functions in its infrared spectrum (at 1260 cm.sup.1) showed that the sulfate group initially present on the rhamnose unit of ulvan is released under the effect of the acidic conditions (pH=1.5) during the first step of the process (butanolysis and/or hydrolysis, glycosylation, esterification).

[0058] 3) Trans-Glycosylation Reaction Starting with Butyl L-Rhamnoside Isolated During the Separation by Column Chromatography on Silica Gel

##STR00003##

[0059] The n-butyl -L-rhamnopyranoside (0.5 g, 2.27 mmol, 1 eq.), separated from the surfactant composition UlvC.sub.4N.sub.12 by column chromatography on silica gel, was taken up in dodecanol (15 eq.) in the presence of one equivalent of MSA (2.27 mmol, 148 L). The trans-glycosylation was then performed for 3 hours at 65 C. under reduced pressure (6 mbar) in sufficiently dilute medium so as to avoid the degradation of the butyl rhamnoside. At the end of the reaction, the reaction medium was allowed to cool and was then neutralized with an NaOH solution (0.1 M).

[0060] The difference in polarity between the compound n-dodecyl -L-rhamnopyranoside, having a hydrophobic chain containing 12 carbon atoms, and n-butyl -L-rhamnopyranoside made it possible to separate these two compounds by column chromatography on silica gel, using a 95/5 dichloromethane/ethanol mixture as eluent. The trans-glycosylation yield (=64%, 0.48 g) was evaluated from the molar masses of n-butyl -L-rhamnopyranoside (220.27 g.Math.mol.sup.1) and n-dodecyl -L-rhamnopyranoside (C.sub.18H.sub.36O.sub.5, 332.48 g.Math.mol.sup.1).

[0061] The 1D and 2D NMR results confirm the structure of n-dodecyl L-rhamnoside. The proton NMR spectrum showed the presence of a dodecyl chain in the anomeric position (doublet of triplets at 3.38 and 3.65 ppm corresponding to the protons of the 0-CH.sub.2 function bonded in the anomeric position on rhamnose (OCH.sub.2: 67.89 ppm). The doublet at 4.75 ppm for the anomeric proton H-1 corresponds to the anomer of dodecyl L-rhamnoside 2=2.1 Hz). The anomeric carbon C-1 of this compound RhamOC.sub.12 gives a signal at 99.77 ppm. Furthermore, the HMBC 2D NMR spectrum showed a correlation between the anomeric proton H-1 (4.75 ppm) and the carbon ones of the OCH.sub.2 function bonded in the anomeric position on rhamnose (67.89 ppm).

Example 2: Measurement of the Interface Tensions of the Surfactant Compositions Based on Alkyl L-Iduronamides and Alkyl D-Glucuronamides from Ulvans

[0062] The interface properties of the surfactant composition UlvC.sub.4N.sub.12 were evaluated by measuring the oil-water interface tensions. The surfactants were dissolved in sunflower oil at concentrations ranging from 0.12 to 0.46 g/L. In order to promote the solubility of the surfactants in the oil, the solutions were left in an ultrasonic bath for 10 minutes at 50 C. The interface tension measurements were taken between Milli-Q water and the solutions of sample in oil.

[0063] The tensions at the interface between the oil and the water were measured at 25 C. with a ring tensiometer (Krss, K 100C model). The ring used was horizontally-suspended calibrated iridium-treated platinum. Before each measurement, the ring was cleaned meticulously and flame-dried. The sample goblet is a cylindrical glass container placed in a heat-regulated chamber.

[0064] The interface tension between the sunflower oil (Carrefour brand) and water at 25 C. ranged between 24.71 and 25.04 mN/m.

[0065] For each concentration of the surfactant composition, the machine initially measured the surface tension of sunflower oil containing the surfactant (low-density liquid) and then the surface tension of water (high-density liquid). Finally, the oil was added delicately to the water, while avoiding the formation of bubbles, and the machine began measuring the interface tension between the sunflower oil and the water (average of 10 measurements).

[0066] Interface tensions of the surfactant composition UlvC.sub.4N.sub.12 (mN/m)]

TABLE-US-00002 UlvC.sub.4N.sub.12 0.12 g .Math. L.sup.1 17.45 0.25 g .Math. L.sup.1 13.89 0.46 g .Math. L.sup.1 10.32

[0067] The surfactant composition UlvC.sub.4N.sub.12 is capable of reducing the interface tension to a value of 10.32 mN/m for a concentration of 0.46 g/L to give the composition emulsifying power.

Example 3: Measurement of the Emulsifying Power of Surfactant Compositions Based on Alkyl L-Iduronamides and Alkyl D-Glucuronamides from Ulvans

[0068] The stability of the oil-in-water (O/W) and water-in-oil (W/O) emulsions formed from the surfactant composition UlvC.sub.4N.sub.12 was studied in comparison with that of commercial alkylpolyglycosides: Montanov 82 from SEPPIC and Xyliance from Soliance/ARD.

[0069] The stability of the two types of emulsion, O/W and W/O, was evaluated considering the two water/oil ratios 75/25 and 25/75, respectively, in round-bottomed graduated tubes, 0.5% of the surfactant product is introduced (20 mg). The sunflower oil was introduced (1 or 3 mL) and the surfactants were then dissolved in an ultrasonic bath for 10 minutes at 50 C. After dissolution of the emulsifier, ultrapure water was added (1 or 3 mL).

[0070] The two phases were then emulsified using an Ultra-Turrax IKA T18 Basic homogenizer for 10 minutes at 11 000 rpm. The emulsion was placed in a bath thermostatically regulated at 20 C.

[0071] The evolution of the emulsion and its gradual demixing was observed for a few hours to several weeks.

[0072] FIG. 1 shows the results of analysis of the emulsifying power of the compositions of the disclosure.

[0073] The surfactant composition UlvC.sub.4N.sub.12 derived from dodecylamine gave an O/W emulsion characterized by high stability ranging from several weeks to several months. Furthermore, the W/O emulsion formed by the product UlvC.sub.4N.sub.12 is very stable.

[0074] These experiments made it possible to demonstrate the good stability of the emulsions formed by the monosaccharide surfactant composition UlvC.sub.4N.sub.12. Specifically, this surfactant composition has better emulsifying properties than those of Montanov and Xyliance, since it makes it possible to form emulsions (W/O and O/W) that are very stable ranging from several weeks to several months, which is not the case for those obtained with the commercial references.

TABLE-US-00003 Type of emulsion W/O emulsion O/W emulsion UlvC.sub.4N.sub.12* +++ +++ Montanov* + Xyliance* [0075] Evaluation of the stability of the emulsion, from a few hours to a few months, in demixing as a function of the type of the emulsion.
*Time for total of the emulsion: <12 hours; +<24 hours; ++=7 days; +++>1 month.

Example 4: Antibacterial Activity of Surfactant Compositions Based on Alkyl L-Iduronamides, Alkyl D-Glucuronamides and Alkyl L-Rhamnosides from Ulvans

[0076] Two protocols were used. The first (protocol A) was applied to n-butyl L-rhamnoside (RhamOC.sub.4) isolated by chromatography on silica gel. The second (protocol B) was followed to test the activity of the surfactant composition UlvC.sub.4N.sub.12 derived from dodecylamine.

Protocol A: Method of Diffusion on Agar in Petri Dishes

[0077] 1) Preparation of the Culture Medium:

[0078] The culture medium used was a mixture of 21 g/L of Muller-Hinton broth and 10 g/L of agar in water. This mixture was stirred and then left to boil. Next, a step of autoclaving of this mixture for 30 minutes was necessary in order to sterilize it before any manipulation. This culture medium was poured, while hot, into Petri dishes and then left to cool.

[0079] 2) Preparation of the Test RhamOC.sub.4:

[0080] 5 milligrams of RhamOC.sub.4 were dissolved in 1 mL of DMSO. Twofold serial dilution with DMSO was then performed starting with the stock solution, so as to obtain the concentrations 2.5 g.Math.L.sup.1, 1.25 g.Math.L.sup.1, 0.625 g.Math.L.sup.1 and 0.3125 g.Math.L.sup.1.

[0081] 3) Preparation of the Bacterial and Fungal Suspensions:

[0082] The bacterial strains used were Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecium and Staphylococcus aureus, in addition to the fungal strain Candida albicans. 10.sup.6 bacteria were collected and then transferred into a 0.9% NaCl solution. Each Petri dish, containing the Muller-Hinton medium, was inoculated with a different bacterial suspension.

[0083] 4) Protocol:

[0084] After having left the bacterial suspensions to dry on the agar, 10 L of the test solution (RhamOC.sub.4), at various concentrations, were deposited on the surface of the agar inoculated with the bacterial suspension. 10 L of DMSO were placed in each Petri dish as negative control.

[0085] The positive controls used were discs soaked with ampicillin for Escherichia coli and Enterococcus faecium, ceftazidim discs for Pseudomonas aeruginosa and vancomycin discs for Staphylococcus aureus.

[0086] After drying, the Petri dishes were finally incubated at 37 C. in an oven for 24 hours. The antibacterial activity was evaluated by measuring the clarification zone in mm around the place of deposition of the various concentrations of the test RhamOC.sub.4 solution.

Results:

[0087]

TABLE-US-00004 Concentration P. C. (mg .Math. mL.sup.1) aeruginosa E. coli E. faecium albicans S. aureus RhamOC.sub.4 5 4 9 6 18 10.5 2.5 0 4 4 14 5 1.25 0 0 0 5 5 0.625 0 0 0 0 0 0.3125 0 0 0 0 0 Positive Ceftazidim Ampicillin Ampicillin Vancomycin control (28 mm) (22 mm) (26 mm) (19 mm)

[0088] The rhamnoside RhamOC.sub.4 showed a very good capacity to inhibit the growth of the gram-positive bacterium Staphylococcus aureus and the yeast Candida albicans. Its power against Enterococcus faecium (6 mm at 5 mg.Math.mL.sup.1) was mediocre. Furthermore, the rhamnoside RhamOC.sub.4 showed inhibitory activity on the gram-negative bacterium Escherichia coli at concentrations of 2.5 and 5 mg.Math.mL.sup.1 with poor inhibitory power on the growth of Pseudomonas aeruginosa.

Protocol B: Method for Evaluating the Number of Live Bacteria

[0089] The antibacterial and antifungal activities of the surfactant composition UlvC.sub.4N.sub.12 were evaluated. In this context, the capacity of this monosaccharide surfactant composition to kill bacteria was studied by counting the number of live bacteria on Muller-Hinton agar.

[0090] 1) Preparation of the Bacterial and Fungal Inoculum

[0091] The inoculum was prepared at a turbidity equivalent to 0.5 MacFarland (Biomrieux France), and then diluted to 1/100 (10.sup.6 CFU/ml)

[0092] From this inoculum, a series of dilutions 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 was produced.

[0093] 100 l of each dilution were plated (counting method) onto the surface of a Muller-Hinton agar (determination of the number of bacteria as CFU/ml in the inoculum N).

[0094] 2) Preparation of the Test Surfactants

[0095] A stock solution was prepared for the surfactant composition UlvC.sub.4N.sub.12 (203 mg.Math.mL.sup.1). A series of twofold dilutions with DMSO was prepared in Muller-Hinton broth, the final dilution being 1/128.

[0096] 3) Protocol

[0097] 1 ml of bacterial inoculum was added to each tube of the surfactant dilutions. After incubation for 24 hours at 36 C. 100 l of each clear tube were plated onto the surface of a Muller-Hinton agar followed by incubation for 24 hours at 37 C.

[0098] The number of live bacteria: N0=n10 CFU/ml (n=number of colonies) was determined.

[0099] The percentage of live bacteria was calculated: N0/N100.

Results:

[0100] The minimum concentration for 100% inhibition of Enterococcus faecium and Candida albicans was of the order of 1.58 mg.Math.mL.sup.1 for the monosaccharide surfactant composition based on D-glucuronic acid and L-iduronic acid.

[0101] As regards the two gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, very high concentrations of UlvC.sub.4N.sub.12 were required to inhibit these two bacteria to 100%, showing that UlvC.sub.4N.sub.12 (25.375 mg.Math.mL.sup.1) has poor antibacterial power against these two types of bacteria.

1These studies of the antibacterial power (Protocols A and B) clearly showed that the gram-positive bacteria Enterococcus faecium and Staphylococcus aureus and also the yeast Candida albicans were more sensitive to the rhamnoside RhamOC.sub.4 and to the amide surfactant composition UlvC.sub.4N.sub.12 than the gram-negative bacteria. Specifically, the gram-positive bacteria are characterized by the presence of a very thick layer of peptidoglycan in their cell membrane, in contrast with that of the gram-negative bacteria. The hydrogen bonds between the cell wall of the gram-positive bacteria and the hydrophilic part of the surfactants are then stronger than in the case of the gram-negative bacteria. The hydrophobic carbon chain, the hydrophilic heads of which are anchored in the thick peptidoglycan membrane, could thus interact with the lipid membrane of the gram-positive bacterium, thus promoting its deformation and thereafter the bacterial cell death (Reis et al., J. Brazilian Chem. Soc., 19 (6), 1065-1072,2008) [12].

TABLE-US-00005 Name of the Name of the surfactant bacterium Results UlvC.sub.4N.sub.12 19 P. aeruginosa 101.5 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria 203 mg/mL 50.75 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria 25.375 mg .Math. mL.sup.1 => inhibition of 99.9% of the bacteria E. coli 101.5 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria 50.75 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria 25.375 mg .Math. mL.sup.1 => inhibition of 99.99% of the bacteria E. faecium 1.58 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria C. albicans 1.58 mg .Math. mL.sup.1 => inhibition of 100% of the bacteria