Field of Invention

The present invention relates to methods for cleaving (e.g. saponifying and/or hydrolysing) a lipid ester so as to form a fatty acid (or salt thereof). The present invention also relates to products (such as fatty acids and esters thereof; and alcohols derived from the lipid ester) produced by the method of the invention.

Background of the Invention

Lipids may be formed by plants and animals to create membranes and to chemically store energy reserves. A lipid may be created by the esterification of a fatty acid carboxylic acid with an alcohol (such as a polyol, such as glycerol or a glycerol derivative), and hence may be referred to as a lipid ester. Energy storage in the form of lipids is generally associated with the formation of triglycerides, which phase separate more effectively from the aqueous phase than those lipids that contain a water-soluble head group such as phosphoglycerides, the latter lipids being more often associated with biological membrane (lipid bilayer) formation.

The esterification reaction is only slightly endergonic. For example, the dehydration reaction forming ethyl acetate from the condensation of ethyl alcohol and acetic acid at standard state conditions (25 C and 1 bar) has a small positive free energy of reaction (AG° _r = 10.45 kJ mol ¹ ). Phase separation of the resulting lipid esters from the aqueous cellular environment drives the equilibrium towards lipid formation.

CH3C00H(i)+ CzHeOji) /IG° _r ~ +10.45 kJ/mol

The equilibrium constant (K _eq ) is directly related to the Gibb's free energy of the reaction ( lG _r ) . where, C, = the concentration of product i in solution. = the concentration of reactant j in solution.

Vi = stoichiometric coefficient associated with that component in the reaction.

R = gas law constant. T = absolute reaction temperature.

For the ethyl acetate example above, K _sq = 0.015. This implies the ester concentration is directly proportional to the acid and alcohol concentrations and inversely proportional to the water concentration:

> 0.015 C _c tha _nO l acetate ethyl acetate — '-'watei'

For the reverse cleavage reaction (e.g., saponification and/or hydrolysis of the lipid), the same equilibrium constant applies. If we assume that the C _et hanoi = C _ac etate, then:

Cacetate Cethanoi VO.015 C _athyl acetate water

This equation implies that the cleavage (e.g. saponification and/or hydrolysis) of esters to their constitutive fatty acids and alcohols in solution requires high concentrations of both the ester (lipid), water, or both. Increasing the ester and water concentrations to drive the cleavage reaction to the free acid and alcohols is an example of the 'principle of mass action' effect on chemical equilibria.

The alternative to drive this reaction is to selectively remove either the alcohol or the fatty acid from the reaction mixture as it is formed. In the manufacture of soaps from fats, caustic (sodium hydroxide) is added to the aqueous solution both to catalyze the reaction and to cause the free fatty acids formed to precipitate from the aqueous solution as their corresponding sodium salts. The rate of this reaction increases with temperature [Vidal et al. (2018)]. Whale oil was once produced by boiling whale blubber (mostly triglyceride lipids), skimming the resulting fatty acid oils produced by the hydrolysis reaction from the surface of the pot as they accumulated [Tower (1881)]. In this case the fatty acids were removed from the aqueous lipid solution by phase separation. However, this process was slow due to the hydrolysis reaction only occurring on the outside surface of the chunks of whale blubber. The whale blubber itself forming its own water-excluding phase.

While it is possible to drive the final equilibrium yield of fatty acid oils from their lipid ester forms by mass action, the rate at which the cleavage reaction occurs is a critical consideration for the economics of the overall process. Cleavage is thought to occur by nucleophilic substitution of the carbonyl of the ester by the water or its hydroxide. The extra proton of water transferring to form the neutral alcohol from the liberated alkoxide. This can be catalyzed by acid or base.

In the presence of a basic salt (such as an alkoxide or hydroxide, and a cation) which is solubilized in a reaction solvent, ester cleavage will typically occur under equilibrium conditions via nucleophilic substitution. When the basic salt is a hydroxide, the cleavage will generate a carboxylate anion. Depending on the solubility of the cleavage product, the product may stay in the same phase, or may phase separate. The equilibrium may shift to favour the products, depending on whether phase separation occurs or not.

The pH of the aqueous solution determines the concentration of protons and hydroxyl ions and the resulting rate of the reaction [Murthy et al. (2012) and Yates and McClelland (1967)] at any given temperature. Independent of the catalysis mechanism, the reaction rate also increases with temperature [Vidal (2018)].

While the cleavage rate at any pH increases with temperature, many fatty acids are not thermally stable and degrade at higher temperatures. In particular, polyunsaturated fatty acids, such as omega-3 and omega-6 fatty acids and lipid-soluble materials such as antho- and zeo-xanthins spontaneously decompose at temperatures above 45 °C. These compounds are essential nutrients commercially extracted from algae, fish, krill, and mussels and used as dietary supplements.

Furthermore, the analysis of the fatty acid composition of lipids requires that they be cleaved. There are several disease states (e.g., Gaucher disease, Tay-Sachs disease, and Tangier's Disease) presenting with specific lipid dysfunctions before clinically-observable symptoms present that could be diagnosed from analysis of the fatty acid or glyceride compositions of lipids post-cleavage [Yamashita and Matsuzawa (2019)]. The fatty acid compositions of membrane lipids also change with growth temperature of coldblooded organisms [Marr and Ingraham (1962)], allowing the monitoring of the growth conditions under which the organism was produced, or localization of its point of harvest. Similarly, the composition of the head groups of membrane glycerides can vary both by organism [Steger et al (2003)] and growth condition of that organism [Hood et al. (1986)] and once shed of the complexity presented by variable fatty acid compositions as lipids can be used for diagnostic purposes.

Much like whale oil (saponified whale blubber) was used in the 19 ^th Century for lighting, lubrication, and its heating value, before the discovery of fossil fuels, natural lipids are also saponified then esterified with a simple alcohol to render them liquid at cold temperatures for the production of biodiesel [Kwangdinata et al. (2014)]. This process was important in order to remove the glycerides that contained N, P, and S atoms in their head groups that both fouled the air and provided a nutrient source for bacteria and fungi to grow on the oil, reducing its shelf life. It is an object of the invention to provide a method for cleaving (e.g. saponifying and/or hydrolysing) a lipid ester so as to form a fatty acid (or salt thereof) that substantially avoids one or more of the drawbacks of traditional cleavage techniques, including thermal degradation of one or more of the resultant fatty acids.

A second object of the invention is to drive the reaction by chelation of the fatty carboxylate. For example, by forming a contact ion pair with an anion exchange resin. Alternatively to release a cation from a cation exchange resin that will cause the fatty acid cation salt to precipitate or phase separate from the lipid soluble phase to a lipid insoluble phase.

A third object of the invention is to separate the glycerol from the free fatty acid by the selective chelation of the fatty acid to a resin by displacement chromatography.

Alternatively, it is an object of the invention to at least provide the public with a useful choice.

Summary of the Invention

In a first aspect the invention provides a method of cleaving a lipid ester so as to form a fatty acid (or salt thereof), the method comprising the steps of: i) dissolving a lipid ester in a solvent (such as an aprotic solvent) so as to form a solution;

II) in the presence of water, contacting the solution with a functionalized solid phase material so that the lipid ester is cleaved so as to form a fatty acid or derivative thereof (such as an ester or contact ion pair) bound to the functionalized solid phase material and an alcohol derived from the lipid ester; ill) separating at least a portion of the solution from the fatty acid or derivative thereof (such as an ester or contact ion pair) bound to the functionalized solid phase material; and iv) displacing the fatty acid or derivative thereof from the functionalized solid phase material.

In a second aspect the invention provides a fatty acid or alcohol derived from the lipid ester produced by the method of the invention.

It has now been found that cleaving lipid ester functionality, by using a functionalized solid phase surface, provides a high yield of fatty acids by largely avoiding degrading conditions. The solid phase surfaces can include weak or strong anion exchange resins and/or nucleophilic functional group(s). Advantageously, the step of separating at least a portion of the solution from the fatty acid or derivative thereof (such as an ester) bound to the functionalized solid phase material allows for the facile separation and removal of components of the lipid ester such as glycerol.

Anion exchange resins may include an anion exchange functional group, such as: tertiary amino functional group (such as diethylaminoethyl (DEAE)); quaternary ammonium functional group; phosphonium functional group; sulfonium functional group; oxonium functional group; metal-cation based anion exchange functional group.

Examples of nucleophilic functional groups are alcohol and thiol groups (e.g., phenolic, nitro-substituted phenolic polymer surfaces, or cysteine polyamides). Without wishing to be bound by theory, it is believed that these solid phase surfaces sequester the carboxylic acids released from the esters, effectively removing them from the liquid phase onto the solid phase, thus driving the reactions further to completion, and so provide a high yield. The residual alcohols can be removed by filtration before the fatty acids are displaced from the solid phase, thus also separating the glyceride and fatty acid components of the lipid, largely preventing lipid reformation. The process can be conducted in organic solvents that promote the solubility of the esters, particularly where the ester carboxylate is composed of long-chain fatty acids.

In some examples the process can be conducted in a batch process where the solid phase is mixed with the lipid solution, and the glyceride alcohols removed by filtration post-reaction. The solid phase may be optionally rinsed. The fatty acids may be subsequently released from the solid phase into a displacing solution and recovered separately by filtration. In a preferred embodiment the solid phase is packed into a chromatography column and the process is run as a displacement chromatography process.

The process can be used for biodiesel production, the recovery of food oils (including polyunsaturated fatty acids used as dietary supplements), and/or to prepare lipid samples for the analysis of the glyceride composition.

Brief Description of the Drawings

Figure 1. Solid phase cleavage process utilizing either strong or weak anion exchange resins.

Figure 2. Solid phase cleavage process utilizing an alcohol functional surface. Figure 3. Illustration of the basic steps in a displacement chromatography for ester cleavage by the solid support in the chromatography column. This process results in the separation of the ester alcohol and carboxylate moieties. In this figure the ester is a lipid, the ester alcohol is a glyceride, and the ester carboxylate is a fatty acid.

Figure 4. Gas chromatogram of the fatty acids from duck fat samples. Sample 1 corresponds to a sample of duck fat dissolved in chloroform (Example 1). Samples 2 and 3 correspond to a replicate samples of the cleaved duck fat eluted from the Dianion WA30 ion exchange resin with 8% methanolic toluene methylation buffer (Example 5) after the overnight cleavage reaction.

Detailed Description of the Invention

In a preferred embodiment of the present invention anion exchange resins are used as the solid phase (Figure 1). The anion exchange solid surface functional group is optionally charged with hydroxyl ions in a basic solution. Alternatively, a hydroxide base can be added to the ester solution to catalyze the reaction. Equilibration of the hydroxyl ion saturated solid phase with a lipid solution lowers the effective pH of the solution, catalyzing the ester cleavage. At any solution pH above the pK _a of the fatty acids and below that of the pK _a of the anion exchange solid surface function group, the resulting fatty acids are ionized and freely ion exchange with other resin-bound hydroxyl ions freeing additional hydroxyl groups into the lipid solution, and removing the free fatty acids from the solution phase to the solid resin surface driving the reaction equilibrium. The alkoxide ion produced from the ester cleavage consumes the proton liberated from ionization of the fatty acid to become neutralized, so the glycerides do not bind to the solid phase. The effective solution pH is thus effectively maintained between the pK _a of the ester carboxylate moiety (> 4) and the pK _a of the ester alcohol moiety (< 11). Since the ester alcohol is neutralized, it fails to bind to the anion exchange resin and stays in solution. Since the ester carboxylates are ionized at the solution pH, these bind to the anion exchange resin to maintain electroneutrality. An advantage of this process is that it can be conducted in an organic solvent solution in which the esters are more soluble than in an aqueous solution (i.e., minimal to no water is needed to accomplish the cleavage). Furthermore it can be conducted without a solvent on liquid esters. Ion separation and kinetics are improved by using organic solvents with higher polarity or dielectric constants, but that maintain good solubility of the lipids and fatty acids.

In one embodiment, suitable solid phases include crosslinked anion exchange resins, such as aminated styrene-divinylbenzene resin (e.g., Diaion WA30, Mitsubishi) or tetraakylammoniated polystyrenic resin (e.g., Amberlite IRA-900). In another embodiment, suitable solid phases include silicate or aluminate particle surfaces functionalized with pendant amine, alkylammonium, or tetraalkylphosphonium groups. The positively ionized or ionizable groups are covalently attached to the surface by an organic linker of variable length. Furthermore, the solid surfaces may be porous. The solid surfaces may be formed into beads or presented as porous monoliths.

In the case of strong anion exchange surfaces the surface is first treated with a hydroxide base in an aqueous solution (e.g., 1-10 M sodium hydroxide) or in an organic solvent in which the hydroxide salt is soluble (e.g., tetrabutylphosphonium or tetabutylammonium hydroxide in toluene). This treatment displaces any anions ionically bound to the fixed cations on the surface and charges the surface with hydroxide ions. The surface is then rinsed to remove the excess hydroxide and its counterion salt. The hydroxy-charged resin is ready to use.

In the case of weak ion exchange surfaces, the surface may be first treated with a dilute aqueous acid solution in water with a pH below that of the pK _a of the ionizable cation fixed to the surface. This will ionize the fixed surface charges and through mass action substitute hydroxyl ions at most of the fixed cation sites. The surface can then be rinsed with deionized water to displace the remaining acid cations by mass action. The surface may be optionally rinsed with a water-miscible, aprotic, organic solvent to remove the residual moisture. The resulting hydroxyl-charged surface is ready to use for ester cleavage.

The hydroxyl ion-charged anion exchange surface is then exposed to a solution containing the ester.

The ester may be dissolved in any deionized solvent in which it is soluble. Optionally, the ester need not be diluted, if presented to the solid surface in a liquid form. Esters formed of longer chain, more hydrophobic, carboxylic acids typically require water-imiscible organic solvents, such as toluene or chloroform, to remain soluble. Shorter chain esters (e.g., ethylacetate, soluble to 8.3% in water) may be sufficiently soluble in water to allow water to be used for the ester cleavage reaction. In one embodiment the ester-containing solution is contacted with the solid phase in a batch reaction. Mixing may be applied to speed diffusion to the surface where the reaction occurs. In a preferred embodiment the solid surface is contained in a chromatography column and the solution containing the ester is passed slowly through the chromatography column. In this preferred embodiment the reaction is complete when the original ester begins to elute from the back end of the column.

In both embodiments the ester carboxylate anion formed can subsequently be displaced from the solid phase with a displacing agent. In one embodiment this is accomplished by adding a hydroxide salt solution to (e.g., NaOH, Mg(OH) ₂ , or tetraalkylphosphonium hydroxide), which displaces the fatty acid from the resin surface by mass action to form a soluble fatty acid salt in the solution. In another embodiment the pH of the displacing solution is raised above the pK _a of the positively-charged weak anions fixed to the solid surface, liberating the bound fatty acid carboxylates as salts of the conjugate hydroxide counterion. In a preferred embodiment, the pH of the eluting solution is lowered below that of the pK _a of the fatty acid to neutralize the fatty acids to force their displacement from the solid surface by the conjugate acid counterion, such as: Cl’, SO ₄ ² ’, PO ₄ ³ ’, or acetate. The ionically-bound ester carboxylate moieties are effectively displaced from the solid phase by the negatively-charged counter ion of the protic acid used. After elution of the ester carboxylates, the solid phase optionally can be recharged into the hydroxyl ion-charged form as described above and reused.

In another embodiment, a solid phase including one or more nucleophilic functional groups can be used. Examples of nucleophilic functional groups are alcohol and thiol groups.

For example, an alcohol-functional solid phase can be used wherein the pK _a of the alcohol group fixed to the resin is lower than that of the lipid glyceride alcohols (i.e., < 10). Suitable examples for R in Figure 2 include aromatic or electron-withdrawing functionalized aromatic rings, such as phenolate and nitrophenolate moieties. The solid phase fixed alcohol functional group performs a transesterification reaction with the esters displacing original alcohol moiety (pKa > 10) of the ester and forming a resinbound ester with the carboxylate moiety. This reaction is kinetically faster when the fixed alcohol on the solid phase is first ionized under basic conditions to create an alcohol anion salt such as sodium or tetraalkylphosphonium phenoxide. In another embodiment the alcohol-functional solid phase is contacted with the ester solution. A transesterification reaction naturally occurs, which can be catalyzed under either acidic or basic conditions. By creating the alcohol anion salt before ester addition, no additional catalyst is necessary. The carboxylate moiety of the ester will end up covalently attached through an ester linkage to the alcohol functional group of the solid support.

When the reaction is complete, the reaction solution, containing the original ester alcohol group can be separated from the solid phase containing the covalently-bound carboxylates to remove the unbound ester alcohols. The solid phase can optionally, be rinsed.

In one embodiment the solid phase can then be treated with a hydroxide base solution with a pH above the pK _a of the fixed alcohol functional group. This cleaves the solid phase-bound fatty acid esters regenerating the free alcohol anion salt. The carboxylates form salts with the counterion of the hydroxide solution. The carboxylate salts can be recovered by separating the ester carboxylate salts from the solid surface. In another embodiment the solid phase can be treated with an acid solution with a pH below the pK _a of the ester carboxylates. This catalyzes the cleavage of the esters regenerating the free alcohol. The carboxylates are released from the surface as neutral carboxylic acids in solution and can be separated from the solid phase.

By way of another example, the solid phase is composed of thiol functional groups. Both aromatic and aliphatic thiols have pK _a 's below those of aliphatic alcohols and will form thioesters with the ester carboxylates. In one embodiment the fixed thiol group can be ionized before reaction with the ester by incubating the solid phase with a hydroxide base prior to exposure. In one embodiment the solid phase can be exposed to an acidic solution of the ester. In another embodiment the solid phase can be exposed to a basic solution containing the ester. Both the acidic and basic solutions catalyzing the transesterification reaction.

In all the functional solid phase embodiments the carboxylate moiety of the ester is retained on the solid surface. The alcoholic moiety of the ester remains in the solution so that it can be separated from the carboxylate moiety. The carboxylate moiety is then released from the solid phase into solution and the solution containing the dissolved carboxylate moiety separated from the solid phase. The solid phase can optionally be regenerated and re-used.

In one embodiment the process can be conducted in a batch process where the resin is mixed with the lipid solution. In another embodiment the solid phase is packed into a chromatography column and the process is run as a chromatography process. Displacement chromatography [Gu (2012)] is the preferred embodiment as this uses the least reagents and produces the most concentrated form of both the ester alcohol and carboxylate (Figure 3).

In one embodiment, the lipids can be dissolved in a substantially organic phase with sufficient small amount of water present in this phase to drive the reaction. If necessary, water can be drawn into the organic phase through the addition of a polar, jointly-miscible, aprototic, cosolvent (e.g., acetonitrile).

In another embodiment a biphasic water in oil emulsion can be used such that water reacted in the continuous organic phase is replenished from the included aqueous phase. In another embodiment, the water can be suspended in the pores of the solid phase resin with a pure oil phase contacted with the outer surface of the resin. In another embodiment a pure organic solvent can be used. Said organic solvents chosen such that they do not form anions that will complete with the carboxylates for the solid phase.

One or more embodiments of the invention will be described below by way of example only, and without intending to be limiting. Examples

Example 1

A sample of 5 g of commercial duck fat was dissolved in 50 mL of chloroform. A 0.1 mL sample of this lipid solution was taken and diluted into 1 mL of a 30% methanol/70% toluene mixture. A 0.25 mL sample of this dilution was then transferred into 1.13 mL of methylation buffer (Example 2). The sample was capped, mixed, and incubated for 16 hours at 45 °C in a heating block. The samples were centrifuged to remove any particulates transferred to a glass gas chromatography vial and capped with a rubber gasket.

Example 2

An 8% methanolic HCI solution was prepared by mixing 362.5 mL of methanol (Sigma, technical grade) with 100 mL of 37% HCI in water (Sigma) to make 462.5 mL in a glass bottle. Methylation buffer was prepared by mixing 230 mL of this 8% methanolic HCI solution with 600 mL of methanol and 300 mL of toluene (Sigma).

Example 3

A 10 g sample of a weak anion exchange resin (Diaion WA90) was added to a 25 mL solution of 1.0 M NaOH and equilibrated overnight to charge the resin with hydroxide ions. The charged resin was then rinsed with deionized water and dried.

Example 4

The dried and hydroxyl-charged Diaion WA90 resin (Example 3) was added to a 50 mL polypropylene centrifuge tube. The 25 mL sample of duck fat in chloroform (Example 1) was added to the tube along with 2.5 mL of deionized water, which created a second phase. The tube was sealed and rotated overnight end-over-end on a rotary mixer at room temperature.

Example 5

After the solid phase cleavage reaction (Example 4) was allowed to progress overnight, the solution was filtered off the resin. The resin was rinsed with a 70% toluene/30% methanol solution to remove any residual lipids and residual glyceride alcohols. The fatty acid carboxylates were then eluted by adding 25 mL of the methanolic HCI toluene solution (Example 2), incubating for 15 min, and separating the methanolic HCI toluene solution from the resin by filtration. The filtrate was collected and heated at 45 °C overnight [method of Ichihara and Fukubayashi (2010)] to convert the fatty acids to their corresponding methyl esters.

Example 6

The methylated fatty acids obtained from Examples 1 and 5 were analyzed on a Shimadzu GC-2014 gas chromatograph equipped with a 30 m Restek Stabiwax capillary column (0.25 mm ID, 0.25 micron film thickness) using a flame ionization detector (FID). The injection volume was 1 pL using a split injector at 220 °C with a split ratio of 1:186 using hydrogen at 50 cm/s as the carrier gas. The FID was operated at 250 °C. The column was operated with a temperature gradient consisting of 160 °C for 1 min. The temperature was increased to 185 °C at 5 °C/min then increased to 240 °C at 8 °C/min. Finally, the column was held at 240 °C for 10 min before returning to the starting temperature. Since the dilution factors were the same for each sample, the relative abundances of the fatty acid methyl ester peaks provide a direct measure of the completeness of the cleavage reaction. The gas chromatograms nearly overlap, suggesting nearly 100% cleavage efficiency and fatty acid capture on the solid phase.

As used herein, "salt" refers to a compound prepared by the reaction of an organic acid (typically a carboxylic acid herein) with a pharmaceutically acceptable mineral or organic base; as used herein, "salt" also includes hydrates and solvates of salts made in accordance with this invention. Exemplary mineral or organic acids or bases are as listed in Tables 1-8 in Handbook of Pharmaceutical Salts, P.H.

Stahl and C.G. Wermuth (eds.), VHCA, Zurich 2002, pp. 334-345. In particular, salts include, but are not limited to salts formed from a carboxylate and a Group I alkali or Group II alkaline earth metal cation, such as potassium, sodium, lithium, magnesium, calcium.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to".

The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

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