Patent Description:
There are a number of significant challenges involved in the replacement of petroleum-derived plastic materials with biomass-derived alternatives, such as feedstock sourcing and cost, processing conditions and final material performance.

Carbohydrates, including seaweed-derived polysaccharides such as sodium alginate, agar and carrageenan, are promising biopolymers for the production of novel biomaterials. However, due to the hydrophilic nature of these carbohydrates direct replacement of fossil-derived plastics, which demonstrate highly water resistant properties, is not attainable. One possible route to increase the hydrophobic nature of these biopolymers is the esterification reaction with long chain fatty acid derivatives. This esterification process converts polar hydroxyl groups of the polysaccharide into hydrophobic esters with long alkane chains. While the conversion can be hindered by low reactivity and solubility issues of the carbohydrate starting materials, the formed modified ester derivatives exhibit promising properties for bioplastics applications.

As such, carbohydrate long-chain esters are traditionally manufactured by recurring to acyl chlorides and excess organic bases. Although this route is efficient, said substances are highly toxic to humans and the environment, and represent a significant cost and ultimately a barrier to industrial production and commercialisation.

So far, alternative processes have mainly focused on replacing acyl chlorides with lower-cost fatty acids and esters. However, due to the lower reactivity of these derivatives and the abovementioned intrinsic low reactivity of polysaccharides, the extent of functionalisation is often very limited, resulting in poor product performance preventing commercial exploitation.

<NPL>) describe a microwave assisted synthesis of hydrophobically modified nano-sized particulate esters of agarose and stearic and palmitic acids, using carbodiimide chemistry.

<NPL>) describe a study in which starch materials of different amylose content were reacted with saturated and unsaturated fatty acids of varying chain length from C<NUM> to C<NUM> under homogenous conditions applying the solvent N,N-dimethyl acetamide in combination with LiCl.

<CIT> describes anionic polysaccharides functionalized by at least one hydrophobic acid derivative.

Esterification pathways that are more sustainable and easy to scale are therefore needed, while maintaining a high degree of functionalisation and product performance.

According to a first aspect of the present invention, there is provided a method of preparing a fatty acid ester derivative of polysaccharide derived from seaweed, comprising:.

Reference hereinbelow to "polysaccharide" should be interpreted as meaning "polysaccharide derived from seaweed". The term "Polysaccharide derived from seaweed" includes "macroalgal polysaccharide" and a "phycocolloid". Thus, in one embodiment "polysaccharide derived from seaweed" is macroalgal polysaccharide. In one embodiment "polysaccharide derived from seaweed" is a phycocolloid.

The method of the invention provides the fatty acid derivatives having a high degree of functionalisation, while avoiding the use of fatty acid acyl chlorides. The fatty acid derivatised polysaccharides produced by the method of the invention are hydrophobic and have excellent water barrier properties, as shown in Example <NUM>.

In the method of the invention, the polysaccharide derived from seaweed is derivatised to form a proportion of fatty acid ester moieties. Specifically, a proportion of the hydroxyl groups of the polysaccharide are esterified to form fatty acid ester moieties. The fatty acid derivative is a fatty acid ester, wherein at least a proportion of the hydroxyl groups of the polysaccharide obtained from seaweed have been esterified to form fatty acid esters.

In one embodiment, the method of the invention provides a fatty acid ester derivative wherein at least <NUM>% of the hydroxyl groups of the polysaccharide obtained from seaweed have been esterified to form fatty acid esters, such as at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>%.

The functionalisation yield (corresponding to the % of hydroxyl groups of the polysaccharide derived from seaweed that have been esterified) is determined as set out in the Evaluation Methods.

The at least one polysaccharide derived from seaweed is selected from one or more of: agar (technical or biological grade), agarose, carrageenan, fucoidan, laminarin, or any combination thereof. Preferably the at least one polysaccharide derived from seaweed is selected from agar (technical or biological grade) and/or carrageenan. Preferably the at least one polysaccharide is agar (technical or biological grade).

The fatty acid source comprises a fatty acid. Preferably, the fatty acid source is a fatty acid.

The fatty acid source preferably has a chain length of at least C<NUM>. For the avoidance of doubt, the "chain length" includes the carbon attached to the carbonyl group (e.g. an example of a C<NUM> fatty acid source is lauric acid). In one embodiment, the chain is branched. In another embodiment, the chain is unbranched. Preferably, the chain is unbranched. In one embodiment, the chain contains unsaturation. In another embodiment, the chain is saturated. Preferably, the chain is saturated. Preferably, the fatty acid source has a chain length of no more than C<NUM>. Preferably, the fatty acid source has a chain length of one or more of: C<NUM>, C<NUM>, and/or C<NUM>.

The at least one fatty acid is preferably selected from: octanoic acid, lauric acid, linoleic acid, palmitic acid, stearic acid, myristic acid, or any combination thereof. The at least one fatty acid is preferably selected from: lauric acid, myristic acid, palmitic acid, stearic acid, or any combination thereof. The at least one fatty acid is preferably selected from: palmitic acid or stearic acid, or any combination thereof.

In the method of the invention, typically a suspension of (the at least one) polysaccharide obtained from seaweed in a solvent is prepared, and then the (at least one) fatty acid source is added to the suspension.

In one embodiment, the molar ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide (within the reaction mixture) is at least <NUM>:<NUM>, for example at least <NUM>:<NUM>. In one embodiment, the ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide (within the reaction mixture) is no more than <NUM>:<NUM>.

The at least one fatty acid source is preferably in stoichiometric excess to the polymer repeat unit of the at least one polysaccharide (within the reaction mixture). For example, the molar ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide (within the reaction mixture) is preferably at least <NUM>:<NUM>, preferably at least <NUM>:<NUM>, preferably at least <NUM>:<NUM>, preferably at least <NUM>:<NUM>, for example <NUM>:<NUM>.

Thus, in one embodiment, the molar ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide is in the range of from <NUM>:<NUM> to <NUM>:<NUM>, such as from <NUM>:<NUM> to <NUM>:<NUM>, from <NUM>:<NUM> to <NUM>:<NUM> or from <NUM>:<NUM> to <NUM>:<NUM>. Preferably the molar ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide is in the range of from <NUM>:<NUM> to <NUM>:<NUM>, preferably from <NUM>:<NUM> to <NUM>:<NUM>.

The method may further comprise drying the at least one polysaccharide derived from seaweed prior to functionalisation. The at least one polysaccharide derived from seaweed may be oven dried, for example at <NUM>.

Suitably, the solvent is non aqueous. In the context of the present invention, non aqueous refers to solvent that contains less than <NUM>% (v/v), such as less than <NUM> % (v/v), <NUM>% (v/v), <NUM>% (v/v), <NUM>% (v/v), <NUM>% (v/v), <NUM>% (v/v) or <NUM>% (v/v) of water.

In one embodiment, the solvent is present in the reaction mixture in an amount of <NUM>-<NUM> per gram of polysaccharide derived from seaweed e.g. <NUM>-<NUM> of solvent, <NUM>-<NUM> of solvent or about <NUM> of solvent is used for every gram of polysaccharide derived from seaweed.

In the context of the present invention, the activator is a moiety that is capable of enhancing the electrophilicity of the fatty acid source (in particular the fatty acid) carbonyl group, or it is a moiety that is capable of converting the hydroxyl groups of the polysaccharide into better leaving groups. The exact mechanism of the activator may be a combination of mechanisms, or it may be unknown. For example, it is hypothesized in the literature that using p-toluenesulfonyl chloride as activator may result in sulphonate formation of the fatty acid source (in particular fatty acid) and/or activation of the alcohol groups of the polysaccharide by conversion to sulphonate groups.

The activator is a chemical moiety rather than a biological moiety. For example, the activator is not an enzyme. The activator is not a catalyst for the esterification reaction. In particular, the activator is not a strong acid catalyst or a strong base catalyst. Thus, in one embodiment, the reaction mixture does not contain added strong acid or added strong base.

The activator is selected from one or more of: trifluoromethanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonyl chloride, or any combination thereof. In one embodiment, the activator is preferably selected from one or more of: p-toluenesulfonyl chloride, methanesulfonyl chloride, or any combination thereof. In a preferred embodiment, the activator is p-toluenesulfonyl chloride.

Suitably, the activator is present in the reaction mixture at a proportion of <NUM> - <NUM> molar equivalents vs. the polysaccharide repeat unit, such as <NUM> - <NUM> molar equivalents or <NUM> - <NUM> molar equivalents. In one embodiment, <NUM> - <NUM> molar equivalents of activator (vs. the polysaccharide repeat unit) are present, such as about <NUM> molar equivalents.

In one embodiment, the activator is used together with a base. In one embodiment, the base is a nitrogen-containing (in particular an amine-containing) organic base. Suitably, the base is selected from the group consisting of pyridine, triethylamine, <NUM>-dimethylaminopyridine (DMAP), imidazole and <NUM>-methylimidazole; and mixtures thereof. In one embodiment, the base is selected from the group consisting of pyridine, DMAP, imidazole and <NUM>-methylimidazole; and mixtures thereof. In one embodiment, the base is selected from the group consisting of pyridine, DMAP and imidazole; and mixtures thereof, and in particular is pyridine, imidazole, <NUM>-methylimidazole or a mixture thereof. In one embodiment the base is pyridine. In one embodiment, the base is imidazole and/or <NUM>-methylimidazole. In embodiments where the base is pyridine, imidazole and/or <NUM>-methylimidazole the base may also act as solvent if present at sufficient volume. Suitably, the base is present in the reaction mixture at a proportion of <NUM>-<NUM> molar equivalents vs. the polysaccharide repeat unit, such as <NUM>-<NUM> molar equivalents, <NUM>-<NUM> molar equivalents or about <NUM> molar equivalents.

In one embodiment, the method comprises reacting the polysaccharide with a fatty acid or fatty acid ester; an activator; and a solvent, optionally in the presence of a base.

The reaction mixture is preferably stirred or agitated during the reaction, for example using a mechanical stirrer.

The method preferably further comprises heating the reaction mixture to a temperature of between room temperature and <NUM>. Preferably, the temperature of the reaction mixture is at least <NUM>, for example at least <NUM>. The temperature of the reaction mixture is preferably no more than <NUM>, no more than <NUM>, preferably no more than <NUM>. In one embodiment, the temperature of the reaction mixture is between room temperature and <NUM>, such as between <NUM> to <NUM>, preferably between <NUM> to <NUM>, for example about <NUM>.

In one embodiment, the temperature of the reaction mixture is <NUM>-<NUM>, such as <NUM>-<NUM> or <NUM>-<NUM>.

The method preferably further comprises precipitating the fatty acid ester derivative of a polysaccharide obtained from seaweed. The method preferably further comprises the addition of water and/or ethanol to initiate/cause precipitation of the at least one fatty acid ester derivative of polysaccharides obtained from seaweed. Alternatively, the method preferably further comprises the addition the reaction mixture to water and/or ethanol, to initiate/cause precipitation of the at least one fatty acid ester derivative of polysaccharides obtained from seaweed. In both cases, suitably, the precipitation is initiated/caused by the addition of ethanol.

Preferably, the method further comprises obtaining the fatty acid ester derivative of polysaccharides obtained from seaweed by filtration. Filtration may comprise vacuum filtration.

The precipitated fatty acid ester derivative of at least one polysaccharide obtained from seaweed may be washed, for example with hot ethanol, to remove further impurities.

The precipitated, and optionally washed, fatty acid ester derivative of at least one polysaccharide obtained from seaweed is preferably dried, for example oven dried. The fatty acid ester derivative(s) may for example be dried at a temperature of <NUM>.

The reaction time for the reaction is preferably at least <NUM> minutes. The reaction time for the reaction is preferably no more than <NUM> hours. For example, the reaction time is preferably between <NUM> and <NUM> hours, for example between <NUM> and <NUM> hours, for example about <NUM> hours.

In one embodiment, is provided a method of preparing a fatty acid ester derivative of a polysaccharide derived from seaweed, comprising:.

Suitably, the molar ratio of the at least one fatty acid source to the polymer repeat unit of the at least one polysaccharide is in the range of from <NUM>:<NUM> to <NUM>:<NUM>. Suitably, at least <NUM>% of the hydroxyl groups of the polysaccharide derived from seaweed have been esterified to form fatty acid esters, such as at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>%.

In all of the above embodiments, the imidazole used as solvent may also function as a base.

The extent of esterification of the polysaccharide derived from seaweed corresponds to the percentage of hydroxyl groups of the polysaccharide derived from seaweed that have been esterified according to the method of the invention. The extent of esterification is quantified gravimetrically and is calculated as the ratio between weight gain after reaction and theoretical maximum weight of covalently bound fatty acid groups. This % value is also known as the functionalisation yield.

Infrared (FTIR) spectra were recorded on a Perkin Elmer Frontier FT-IR equipped with a zinc selenide crystal ATR module. Each spectrum was recorded with <NUM> scans between <NUM>,<NUM> and <NUM>-<NUM>, with a resolution of <NUM>-<NUM>. FTIR spectra are plotted as transmittance (%) vs. wavelength (cm-<NUM>). Intensity of a particular signal is reported as % transmittance (%T) of the signal's maximum (minimum transmittance value). In the Examples below where the palmitate derivative of agar is formed, the new ester moieties produce a new ester signal at ca. <NUM>-<NUM>. Absence of this signal generally indicates absence of ester groups; when the signal is observed, transmittance values close to <NUM>% (for example <NUM>% or <NUM>%) generally indicate low concentration of ester groups, while lower values (for example <NUM>% or <NUM>%, corresponding to <NUM>% and <NUM>% absorbance, respectively), generally indicate higher concentration of ester groups.

In order to determine suitable reaction conditions for forming a fatty acid derivatised polysaccharide, agar (<NUM>) was suspended with various palmitic acyl functionalisation agents (<NUM> molar equivalents per repeat unit of the polysaccharide), in pyridine (<NUM>), optionally in the presence of an activator (<NUM> molar equivalents per repeat unit of the polysaccharide). Each reaction mixture was stirred at <NUM> for <NUM> hours under magnetic stirring. The functionalisation yield and FTIR intensity were calculated as set out in Evaluation Methods.

It can be seen that reaction with the acid chloride palmitoyl chloride produced the desired fatty acid derivatised agar ester in a functionalisation yield of <NUM>%, with <NUM>% FTIR ester transmittance. Reaction with methyl palmitate and palmitic anhydride yield an insignificant amount of, or no, fatty acid derivatised ester. Palmitic acid also failed to produce any esterified product, but when combined with the activator TsCl produced the desired fatty acid derivatised ester in a functionalisation yield of <NUM>%, with <NUM>% FTIR ester transmittance. As such, using a fatty acid as functionalisation agent together with the activator TsCl avoids the use acyl chlorides while providing material which is equally good in terms of functionalisation yield and % FTIR ester transmittance.

Pre-dried (<NUM>, overnight) agar (<NUM>) and palmitic acid (<NUM> - <NUM> molar equivalents vs. polymer repeat unit) are suspended in a solvent (selected from DMAc, DMF, pyridine, imidazole and <NUM>-methylimidazole, and combinations thereof)) (<NUM>) into a round bottom flask under magnetic stirring. An activator, p-toluenesulfonyl chloride (<NUM> - <NUM> molar equivalents vs. polymer repeat unit) is added. The mixture is stirred at <NUM> -<NUM> for <NUM> to <NUM> hours under magnetic or mechanical stirring. The mixture is then cooled. Ethanol is added, the solid filtered under reduced pressure and washed repeatedly with hot ethanol to provide a palmitic acid ester agar. Using <NUM> molar equivalents of TsCl and DMAc as solvent, heated at <NUM> for <NUM> hours, the desired palmitate agar ester was produced with a functionalisation yield of <NUM>% and % FTIR ester transmittance of <NUM>% (calculated as set out in the Evaluation Methods).

Pre-dried (<NUM>, overnight) agar (<NUM>) and palmitic acid (<NUM> molar equivalents vs. polymer repeat unit) are suspended in dimethylacetamide (<NUM>) into a round bottom flask under magnetic stirring. An activator, N,N'-dicyclohexylcarbodiimide (<NUM> molar equivalents vs. polymer repeat unit) and optionally a base, <NUM>-dimethylaminopyridine (<NUM> molar equivalents vs. polymer repeat unit) are added. The mixture is stirred at <NUM> - <NUM> for <NUM> to <NUM> hours under magnetic stirring. The mixture is then cooled. Ethanol is added, the solid filtered under reduced pressure and washed repeatedly with hot ethanol to provide a palmitic acid ester agar.

The functionalised polysaccharides were evaluated using FTIR, as set out in Evaluation Methods. <FIG> illustrates the overlapped infrared spectrum of agar, palmitic acid and agar palmitate formed according to the method of Example <NUM>, carried out at <NUM> for <NUM> hours. The new ester signal is clearly visible at ca. <NUM>-<NUM>. The functionalisation yield was determined to be <NUM>%.

Agar was reacted with palmitic acid in the presence of differing types and amounts of activators and bases, over differing time periods. Table <NUM> summarises the reaction conditions, the intensity (as % transmittance) of the signal at <NUM>-<NUM> of the newly formed ester group during the functionalisation reaction, and the functionalisation yield (determined as set out in Evaluation Methods). The first entry in Table <NUM> corresponds to the agar functionalised with palmitic acid as prepared in Example <NUM>.

The Examples in Table <NUM> demonstrate methods for preparing fatty-acid esterified polysaccharides derived from seaweed using differing types and amounts of activators and bases, over differing time periods and at different temperatures.

Agar functionalised with palmitic acid, prepared according to Example <NUM> (Table <NUM>, entry <NUM>), exhibits a hydrophobic nature due to the presence of the long chain hydrocarbon moieties. When agar is placed into a beaker of water it sinks to the bottom and swells, whereas the agar functionalised with palmitic acid swims on the water surface without any absorption of water occurring.

Claim 1:
A method of preparing a fatty acid ester derivative of a polysaccharide derived from seaweed, comprising:
reacting at least one polysaccharide derived from seaweed with a fatty acid source comprising a fatty acid; an activator; and a solvent;
wherein the at least one polysaccharide derived from seaweed is selected from one or more of: agar, agarose, carrageenan, fucoidan, laminarin, or any combination thereof;
wherein the activator is selected from the group consisting of trifluoromethanesulfonyl chloride, p-toluenesulfonyl chloride and methanesulfonyl chloride; or any combination thereof; and
wherein the solvent is imidazole.