Synthesis of enantiopure (r)-3-hydroxybutyric acid salts and methods, compositions and uses related thereto.

Abstract

The invention relates to the field of green chemistry and renewable carbon sources, more in particular to the synthesis of (R)-3-hydroxybutyric acid using renewable carbon feedstock as starting materials. Provided is a method for the manufacture of a (R)-hydroxybutyric acid salt, comprising: (i) subjecting a starting material comprising polyhydroxyalkanoates (PHA) to an extraction / transesterification reaction using isobutanol in the presence of an acid to obtain the isobutyl ester of (R)-hydroxybutyric acid; and (ii) converting the isobutyl ester of (R)-hydroxybutyric acid to the corresponding salt.

Description

[0001]Title: Synthesis of enantiopure (R)-3-hydroxybutyric acid salts and methods, compositions and uses related thereto. The invention relates to the field of green chemistry and renewable carbon sources. More in particular, it relates to the synthesis of (R)-3- hydroxybutyric acid salts for use, among others, as therapeutic agent ornutritional supplement, using renewable carbon feedstock as startingmaterial. (R)-3-hydroxybutyric acid belongs to the group of ketone bodies, which are naturally occurring compounds being formed from fatty acids in the liver. Other examples are acetone and acetoacetic acid. These ketone bodies are intermediates in the citric acid cycle towards energy. Especially (R)-3-hydroxybutyric acid, also referred to as beta-hydroxybutyric acid (BHB) or D-beta-hydroxybutyric acid (D-BHB), has recently gained a lot of attention as it not only improves physical performance but also positively influences cognition. In healthy individuals, BHB consumption as a nutritional supplement provides near term energy for active sports and endurance activities. Cognitively, improved concentration is often attained after exogenous BHB supplementation. Interestingly, this can be especially important for patients suffering from early stage neurodegenerative diseases (Applied Microbiology and Biotechnology (2021) 105:6229–6243). US2021 / 322350 relates to exogenous ketone supplementation and addresses the benefits of in a subject in need thereof through consumption of 10-20 grams or more of exogenous natural D-BHB. Proposed therapeutic applications include the treatment of cancer, kidney disease, cognitive disorders and improved viral resistance. Known methods for the synthesis of (R)-3-hydroxybutyric acid include procedures starting from (m)ethyl acetoacetate (e.g. Biotechnology and Bioprocess Engineering 2015, 20, 324-332 and Angew. Chemie Int. Ed, 2003, 42, 6000-6003), or butyrolactone (e.g., Appl Microbiol Biotechnol, 2014, 98, 621–628) and those involving the fermentation of glucose using recombinant strains (e.g., US 7,262,037 B2 and US 2018 / 0282767 A1). Drawbacks of the first two approaches are that a chiral reduction step (ethyl acetoacetate) or a chiral enzymatic resolution step (butyrolactone) is required to prepare the (R)-enantiomer. Such chiral steps result in high costs for synthesis. Furthermore, both starting materials are prepared from fossil sources, whereas renewable feedstocks for synthesis are generally preferred. Whereas the fermentation of glucose is an alternative strategy, this approach is suffering from low yields, enhanced reaction times and complicated purification steps. It has been recognized by Seebach and co-workers that polyhydroxyalkanoates (PHA’s) are an alternative renewable source for (R)- 3-hydroxybutyric acid and derivatives thereof (e.g., Organic Syntheses 71, 39-47 (1993) ). This class of biopolymers can be prepared by micro- organisms using a wide variety of feedstocks such as carbohydrates, fatty acids (also volatile fatty acids) and even carbon dioxide. The configuration of the monomer in PHA’s is always in the (R)-enantiomer, making it an appropriate and sustainable source for preparation of (R)-3-hydroxybutyric acid. Unfortunately however, standard hydrolysis conditions (acid or basic conditions, high temperatures) have always afforded a mixture of C4- products. Hydrolysis under basic conditions results in beta-elimination and formation of (unwanted) crotonic acid (CA;trans-2-butenoic acid; trans-3- methylacrylic acid). CA is on the list of hazardous substances. Inhalation, ingestion or skin contact with CA may cause severe injury or death. CA was also formed under acidic conditions. Under these conditions, unwanted repolymerization towards oligomers was also observed (Current Opinion in Green and Sustainable Chemistry 2022, 37:100656). In order to prevent the formation of crotonic acid derivatives, an alternative approach was developed which involves the methanolysis of PHA under acidic conditions in mixtures of methanol and hazardous organic solvents (e.g., dichloroethane), resulting in the formation of the methyl ester of (R)-3- hydroxybutyric acid (e.g., Helv. Chimica Acta 1982, 49, 495-503). However, a major drawback of this method is the utilization of toxic chlorinated organic solvents, which is not desirable from both a cost and environmental point of view. An improvement was reported by Parodi et al. (ACS Sustainable Chem. Eng.2021, 9, 12575−12583) involving a single-step catalytic methanolysis using MeOH as a solvent and reactant. The procedure also worked for crude biomass (dried or wet PHA-containing microorganisms) resulting in high / moderate yields of methyl (R)-3- hydroxybutyric acid. Unfortunately, for this approach high temperatures (140 ºC), extended (>24 h) reaction times and costly autogenous pressure conditions are needed to bring the PHA in solution and achieve full depolymerization. The present inventors therefore aimed at providing an improved method for the manufacture of enantiopure (R)-3-hydroxybutyric acid salts from renewable sources, which method is commercially attractive and does not rely on harmful cosolvent(s) and costly equipment, such as pressure reactors. Moreover, the process should yield (R)-3-hydroxybutyric acid salt at a satisfactory yield, while minimizing or even avoiding the formation of unwanted (e.g. toxic) side products, in particular crotonic acid. Surprisingly, it was found that these goals could be met by the use of isobutanol as solvent in the extraction / transesterification of PHA or a PHA-containing biomass. More specifically, it was observed that almost full conversion of PHA to the corresponding isobutyl ester was achieved by dissolving PHA in refluxing isobutanol in the presence of an acid catalyst. This approach allows for short reaction times to achieve full conversions while obviating the need for pressure reactors. Surprisingly, it was observed that the formation of crotonates is almost completely prevented (< 1%) when isobutanol is used as extraction / transesterification solvent. Herewith, the method of the invention is not only attractive from an economic and environmental point of view, but also has important advantages when the product(s) are to be used as nutritional or nutraceutical supplement. Accordingly, in one embodiment the invention provides a method for the manufacture of a (R)-hydroxybutyric acid salt, comprising the steps of: (i) extracting starting material comprising polyhydroxyalkanoate (PHA) containing biomass with isobutanol in the presence of an acid to obtain the isobutyl ester of (R)-hydroxybutyric acid; and (ii) converting the isobutyl ester of (R)-hydroxybutyric acid to the corresponding salt. A method according to the invention is not disclosed or suggested in the art. In fact, according to Seebach and co-workers (vide supra), direct extraction / depolymerization of PHA-containing cells using 1-butanol / H2SO4 was not feasible and the PHA / PHB had to be first extracted from the biomass. This teaches away from the present unexpected observation that a high yield of the isobutanol ester of (R)-3-hydroxybutyric acid can be obtained from PHA-containing biomass using isobutanol as a solvent / reactant. Furthermore, the art is silent about the formation of unwanted crotonic acid and derivatives thereof, let alone that it suggests that CA formation is influenced by the choice of alcoholic solvent. In one aspect, the invention provides a method for the manufacture of a (R)- hydroxybutyric acid salt, comprising: (i) subjecting a starting material comprising polyhydroxyalkanoates (PHA) to an extraction / transesterification reaction using isobutanol in the presence of an acid, e.g. sulfuric or sulfonic acid, to obtain the isobutyl ester of (R)-hydroxybutyric acid; and (ii) converting the isobutyl ester of (R)-hydroxybutyric acid to the corresponding salt. Polyhydroxyalkanoates (PHA) make up a class of very versatile compounds, in which over 100 polymers have been shown to date, differing by the number of carbon atoms in the main chain or the substituent R, according to the formula, as seen in Table 1: Table 1. PHAs Preferred PHA types for use as starting material in a method of the invention include those containing PHB (poly-(3-hydroxybutyrate)). Suitably, the starting material is a PHA-containing biomass. By the term „biomass" is understood hereinafter plant biomass and / or microbial biomass (e.g., bacterial biomass, yeast biomass, fungal biomass, microalgal biomass, archeal biomass). Bio- mass-derived PHA can be formed, for example, via enzymatic polymerization of the monomer units. The biomass can be formed of one or more of a variety of entities. Such entities include, for example, microbial strains for producing PHAs (e.g., Alcaligenes eutrophus / Cupriavidus necator, Alcaligenes latus / Azohydromonas lata, Rhodospirillum rubrum, Halomonas bluephagenesis , Azetobacter , Aeromonas, Caldimonas (e.g., species of Thermodepolymerans), Comamonas, Pseudomonas, Caenibacterium thermophilum, Archaea (e.g., species of Bacillus, Halobacterium, Natronococ- cus, Natronobacterium, Halorubrum, Haloquadratum, Halococcus, Haloterrigena , Natrialba , Haloarcula, and Haloferax), Schlegelella (e.g., species of Thermodepolymerans). Also envisaged is the use of recombinant strains, genetically engineered organisms, preferably containing recombinant plasmids or introduced gene sequences on the chromosome, for producing PHAs (e.g., Escherichia coli species of Pseudomonas, Ralstonia, Klebsiella), yeasts for producing PHAs, and plant systems for producing PHAs. Preferably, the PHA is isolated from plant biomass derived from plants such as sugar cane, switchgrass, soybean, cotton, coconuts, groundnuts, rapeseed, sunflower seed, olive, palm, sesame seed, linseed, castor, safflower seed, tobacco and potato. Also, transgenic plants are used as a source for PHA. Transgenic plant derived PHA polymers or their derivatives can be processed and separated from plant biomass in commercially useful forms. In a specific aspect, the starting material is a PHB- or PHBV-containing biomass. PHBV-containing biomass refers to biological material that includes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a type of biodegradable bioplastic that can be produced by certain bacteria (e.g., Cupriavidus necator, Alcaligenes spp.) under nutrient-limited conditions. Preferably, crude biomass containing PHA (e.g. PHB) is used as feedstock. The C4 chiral hydroxy acid backbone of the most abundant and readily available PHA, poly(3-hydroxybutyrate) (PHB), renders it an attractive circular carbon feedstock for the synthesis of many new polymers or value-added platform molecules such as crotonic acid and (R)-3- hydroxybutyric acid. However, it is also envisaged that PHA containing biomass is first extracted with isobutanol and subsequently hydrolyzed in situ with e.g., sulfuric acid. The isobutanol used for synthesis can easily be recovered by distillation. If needed, also the isobutyl ester of (R)-3-hydroxybutyric acid can be purified by e.g. vacuum distillation. Alternatively, virgin or end-of- life PHA can be utilized as a starting material for subsequent conversion towards (R)-isobutyl esters of hydroxybutyric acid. Figure 1 depicts the exemplary synthesis of magnesium bis (R)-3- hydroxybutyric acid from PHA (as a polymer or included in biomass) according to the invention using isobutanol as extraction / transesterification solvent. A method provided herein comprises subjecting a starting material comprising PHA to an extraction / transesterification reaction using isobutanol in the presence of an acid catalyst to obtain the isobutyl ester of (R)-hydroxybutyric acid. Suitably, this is performed by contacting the starting material with isobutanol under reflux conditions. Exemplary reaction times are 2- 12 hours, preferably 2-5 hours. The extraction / transesterification reaction is preferably performed in a solvent system wherein isobutanol is essentially the sole solvent and no co- solvent(s) is / are added. For example, the solvent system comprises at least 96 wt% isobutanol, preferably at least 97wt%, more preferably at least 98wt%, most preferably at least 99 wt% isobutanol. According to the invention, isobutanol is used in combination with one or more acid(s), for example acid(s) known in the art for acid-catalyzed alcoholysis. These include sulfuric, sulphonic, hydrochloric, formic, acetic, and nitric acids. In one aspect, the acid is selected from the group consisting of Brönsted and Lewis acids (e.g. hydrochloric acid, organic acid (TFA), etc.), more in particular selected from sulfuric acid and sulfonic acid catalysts. Exemplary sulfonic acid catalysts include aromatic sulfonic acids (arenesulfonic acids), preferably toluenesulfonic acid (p-toluenesulfonic acid; TsOH; p-TsOH). The acid catalyst may be used at any effective concentration. In one embodiment, it is present in an amount of 0.5 % v / v to 2 % v / v, preferably about 1 v / v %. In some embodiments, sulfuric acid / sulfonic acid is used at 0.5 % v / v to 2 % v / v, preferably about 1 v / v %. Step (ii) of a method herein disclosed comprises converting the isobutyl ester of (R)-hydroxybutyric acid to the corresponding salt(s). As used herein, the term “salt” or “salts” refer to salts prepared from bases or acids (e.g., pharmaceutically acceptable non‐toxic bases or acids) including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Exemplary preferred embodiments include the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from organic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N, N'‐dibenzylethylene‐diamine, diethylamine, 2‐diethylaminoethanol, 2‐dimethylaminoethanol, ethanolamine, ethylenediamine, N‐ethyl‐morpholine, N‐ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Acids may include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p‐toluenesulfonic acid, and the like. Exemplary embodiments include the citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids. Preferred salts are physiologically acceptable salts. Suitable salts include the salts of mono-valent cations (M+) and bi-valent cations (M2+). Exemplary M+ salts include the salts of sodium and potassium. Exemplary M2+salts include the calcium salt and the magnesium salt. In one aspect, the isobutyl ester of (R)-hydroxybutyric acid is converted to the calcium salt, the sodium salt, the potassium salt, the magnesium salt, or salt mixture thereof. Methods for preparing the corresponding salt are known in the art, which mostly rely on the use of a salts from a base or acid. For example, the isobutylester of (R)-3-hydroxybutyric acid can be hydrolyzed with NaOH or KOH to yield the corresponding sodium or potassium salt. The present inventors observed that hydrolysis of the isobutylester of (R)-3- hydroxybutyric acid with Mg(OH)2 and MgO resulted in high conversions towards the compound aimed for. Accordingly, in a specifically preferred embodiment, step (ii) comprises converting the isobutyl ester of (R)- hydroxybutyric acid to magnesium bis (R)-hydroxybutyric acid((C4H7O3)2Mg) using MgO, more preferably heating the isobutyl ester of (R)-hydroxybutyric acid in an aqueous suspension of MgO. (R)-3-hydroxybutyric acid can easily prepared from the corresponding sodium or calcium salt using Dowex H+ or acidification. The latter (R)-3- hydroxybutyric acid) can also be further converted to commercially available (R)-3-hydroxybutyric acid derivatives such as the commercially available Veech ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate). The present inventors recognized that magnesium bis (R)-3-hydroxybutyric acid is of particular interest as food or dietary supplement or drug because it serves a dual function. On the one hand, as described herein above, it provides the beneficial effect of 3-hydroxybutyrate (physical and cognitive performance). At the same time, it provides a source of exogenous magnesium. Magnesium is present in the human body and plays multiple roles. At the molecular level, magnesium is a cofactor for over 300 enzymes responsible for some of the most important biological activities in mammals, including humans. In living cells, magnesium is involved in the homeostasis of other minerals, such as sodium, potassium and calcium, and the formation, transfer, storage and utilization of adenosine triphosphate (ATP), a principal source of energy in living cells. In the human body, magnesium is involved in the maintenance of normal muscle and nerve function, heart rhythm, bone strength, and immune system health. Magnesium is also involved in the regulation of blood sugar levels and the promotion of normal blood pressure. Despite the physiological role of magnesium in human health, people may not consume enough of the mineral in their diets. Magnesium deficit may lead to or may be associated with many pathological symptoms, such as loss of appetite, nausea, vomiting, fatigue, seizures, abnormal heart rhythms, diabetes, and / or cardiovascular disease, for example. According to several studies, magnesium deficit may lead to or may be associated with attention deficit hyperactivity disorder (ADHD) in children and symptoms associated therewith. Furthermore, there is accumulating evidence that magnesium also positively influences physical and cognitive performance (Tao et al., Alzheimer’s Dement.2022;8:e12250, https: / / doi.org / 10.1002 / trc2.12250). Herewith, ((C4H7O3)2Mg can simultaneously serve as an oral magnesium supplement e.g. for treating magnesium deficiency in a subject in need thereof. Moreover, it was surprisingly found that the oral availability of magnesium when administered in the form of magnesium bis (R)- hydroxybutyric acid is superior to that of other magnesium salts, including magnesium citrate, heretofore considered as an optimal magnesium formulation (see e.g. Rylander, J. of Pharmacy and Nutrition Sciences, 2014, 4, 57-59). The invention therefore also relates to a composition comprising magnesium bis (R)-hydroxybutyric acid for use as pharmaceutical composition, neutraceutical composition or food supplement. In one embodiment, it provides a composition comprising magnesium bis (R)-hydroxybutyric acid for use as (oral) magnesium supplement. In another embodiment, it provides a composition comprising magnesium bis (R)-hydroxybutyric acid for use as exogenous ketone supplement, preferably in exercise or obesity. Still further, it provides a composition comprising magnesium bis (R)- hydroxybutyric acid for use in a method for improving the physical and / or cognitive performance of a subject, in particular a subject suffering from a metabolic or age-related disorder. In one aspect, the subject suffers from magnesium deficiency, mild cognitive impairment, Alzheimer's disease, Huntington's disease, autism, schizophrenia, cognitive decline, depression, dementia, attention deficit hyperactivity disorder (ADHD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, diabetes, hypomagnesemia, glaucoma, migraine, anxiety, mood disorder, or hypertension. The composition may further comprise a pharmaceutically acceptable carrier or excipient, for example a binder, filler, lubricant, dissolution aid, and any combination thereof. Suitable pharmaceutically acceptable excipients include lactose, microcrystalline cellulose, silicon dioxide, titanium dioxide, stearic acid, starch, sodium starch glycolate, povidone, pregelatinized starch, croscarmellose, ethylcellulose, calcium phosphate, talc, sucrose, calcium stearate, hydroxypropylmethylcellulose, shellac, and any combination thereof. The composition may comprise an additional agent, for example a therapeutically active agent, food additive agent, sweetening agent, flavoring agent, coloring agent, filling agent, binding agent, lubricating agent, excipient, preservative, texturing agent, stabilizing agent, manufacturing agent, and any combination thereof. Suitable further active agents include vitamins. The recommended daily allowance of magnesium is 400 - 420 mg for men and 320–360 mg for women. In one embodiment, the present invention provides an oral dosage form comprising 300 mg to 5,0 g of magnesium bis (R)-hydroxybutyric acid. The oral dosage form can be administered in any suitable fashion, e.g. as powder, film coated capsule, (effervescent) tablet, drops, It may also be formulated in the form of liquid, in immediate or sustained release format. In some aspects, the oral dosage form comprises a plurality of beads encapsulated in a capsule. Such format can be used as a sustained release formulation. An exemplary oral formulation containing magnesium (R)-3-hydroxybutyrate may comprise or consist of 25-75 mg magnesium (R)-3-hydroxybutyrate, 200-500 mg Vitamin D3, 30-60 mg Vitamin C, 10-15 mg Vitamin B6, 2-8 mg Phosphatidylserine, 400-600 mg (modified) starch. In one embodiment, the present invention provides a method of enhancing cognitive function. The method comprises administering to a subject an amount of magnesium bis (R)-hydroxybutyric acid effective to achieve a physiological concentration of magnesium at about 0.75 mM or above, wherein said concentration of magnesium is measured under a fasting condition. In some instances, the concentration of magnesium is measured after fasting for at least about twelve hours. In other instances, the physiological concentration is serum concentration, plasma concentration, or cerebrospinal fluid concentration. Also provided is a method where the cognitive function is short-term memory or long-term memory. In some instances, the physiological concentration is maintained for a period of greater than one month. In one embodiment, a method of maintaining cognitive function is provided wherein the method comprises administering to a subject an amount of magnesium bis (R)-hydroxybutyric acid effective to increase a physiological concentration of magnesium by at least about 10% as compared to an initial level of magnesium prior to the administration. In some instances the increase is measured under a fasting condition. In other instances, the physiological concentration is serum concentration, plasma concentration, or cerebrospinal fluid concentration. Also provided is a method of maintaining and / or enhancing cognitive function comprising administering to a subject an amount of magnesium bis (R)-hydroxybutyric acid effective to increase a physiological concentration of magnesium by at least about 10% as compared to an initial level of magnesium prior to said administration. Where desired, the amount of magnesium bis (R)-hydroxybutyric acid is effective to increase a physiological concentration of magnesium by at least about 12%, 14%, 15%, 20%, 25% or more as compared to an initial level of magnesium prior to said administration. The increase in physiological concentration of magnesium can last for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2 years, or even longer. As noted herein, the increase in physiological concentration of magnesium is preferably measured after a fasting condition. The neurological disorders that can be ameliorated by the subject method include dementia, Alzheimer's disease, and depression. In a related but separate embodiment, the present invention provides a method of ameliorating depression by administering to a subject in need for a therapeutic or prophylactic treatment of depression, a composition of magnesium to yield a sustained level of physiological concentration of magnesium of 0.75 mM or above for at least about 15 days, e.g. upon multiple dosages. Preferably, the beneficial effect can last longer than 20 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 1.5 years, 2 years or longer. In another aspect of the invention, a method for therapeutic or prophylactic treatment of a cognitive dysfunction is provided, wherein the method comprises administering to a subject in need of therapeutic or prophylactic treatment of cognitive dysfunction a magnesium bis (R)- hydroxybutyric acid-containing composition to yield a level of physiological concentration of magnesium sustained at the level of 0.75 mM or above for at least about 15 days. In some instances, the magnesium is sustained at the level of 0.75 mM or above for at least about one month or at least about four months. In other instances, magnesium concentration is magnesium plasma concentration measured after fasting for at least about eight hours. In some embodiments, the subject is an adult. In one embodiment, the subject is a patient suffering from or diagnosed with dementia or Alzheimer's disease. In another embodiment, the subject has a magnesium deficiency. Magnesium deficiency, also known as hypomagnesemia, refers to a condition where there is an insufficient level of magnesium in the body. A deficiency can result from various factors, including inadequate dietary intake, gastrointestinal disorders that impair absorption, certain medications, chronic alcoholism, and conditions that increase magnesium excretion such as diabetes and hyperthyroidism. Symptoms of magnesium deficiency can include muscle cramps, fatigue, weakness, irritability, arrhythmias, and in severe cases, seizures and cardiac arrest. Diagnosis is typically made through blood tests measuring serum magnesium levels, though other tests may be used to assess intracellular magnesium levels or overall magnesium status in the body. Normal magnesium levels are between 0.6 and 1.1 mmol / L (1.46–2.68 mg / dL) with levels less than 0.6 mmol / L (1.46 mg / dL) defining hypomagnesemia. However, the specific reference range can vary slightly depending on the laboratory and the measurement techniques used. Treatment usually involves dietary changes, oral magnesium supplements, and in severe cases, intravenous magnesium administration. A composition comprising magnesium bis (R)-hydroxybutyric acid for use as food additive, nutritional or nutraceutical composition is advantageously prepared using a method of the invention which relies on the use of renewable carbon sources and isobutanol as the (sole) extraction and trans- esterification solvent. Accordingly, the invention further relates to a method for the manufacture of a food additive, nutritional, nutraceutical or pharmaceutical composition e.g. for improving the physical and / or cognitive performance of a subject, in particular a subject suffering from a metabolic or age-related disorder, the method comprising the steps of: a) in situ extraction / transesterification of a PHA-containing starting material with isobutanol in the presence of an acid, preferably a sulfuric or sulfonic acid, to obtain the isobutyl ester of (R)-hydroxybutyric acid; b) purifying the isobutyl ester (e.g. distillation); c) converting the purified isobutyl ester of (R)-hydroxybutyric acid to magnesium bis (R)-hydroxybutyric acid ((C4H7O3)2Mg); and d) formulating said magnesium bis (R)-hydroxybutyric acid into a food additive, nutritional or nutraceutical, pharmaceutical composition. Preferably, step c) comprises heating the purified isobutyl ester of (R)- hydroxybutyric acid in an aqueous suspension of MgO to obtain magnesium bis (R)-hydroxybutyric acid ((C4H7O3)2Mg). LEGEND TO THE FIGURES Figure 1: Schematic depiction of the synthesis of magnesium bis (R)-3- hydroxybutyric acid from PHA (as a polymer or included in biomass) using isobutanol. Figure 2: Ratio between oral dosing and IV dosing (∆AUC PO corrected / (∆AUC IV, = bioavailability) in rats for magnesium bis (R)-3- hydroxybutyrate according to the invention (MHB) compared to known supplements magnesium citrate (TMC) and magnesium glycerophosphate (MPG). Example 1: The synthesis of 3-hydroxybutyrate esters by the transesterification of PHB (general procedure). To a suspension of PHB (2.5 g, 28.7 mmol, 1 equiv.) in an alcohol of choice (50 mL,) was added H2SO4 (0.5 mL, 93.3 mmol, 1 V / V%). The reaction was maintained at reflux, overnight,1H-NMR showed the reaction to be complete. The mixture was cooled to room temperature and neutralized with a saturated NaHCO3solution to pH: 7. The layers were separated, after which the organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The resulting product was analyzed by1H-NMR and GC-MS Table 2: Transesterification of PHB with different alcohols Solvent T (ºC) Ester Crotonate formed ester (GC- MS) Isopropanol 82.3 No No 1-Propanol 96.3 Traces No 2-Butanol 99.4 Partly, no < 1 % full conversion Isobutanol 107.8 Yes < 1 % 1-Butanol 117.7 Yes 3.7 % 1-Pentanol 137.5 Yes 7.5% 1-Hexanol 155.8 Yes 12.5% As is shown in Table 2, the depolymerization using higher alcohols (1- pentanol, 1-hexanol) at reflux temperatures suffered from crotonate (ester) formation. Lower molecular weight alcohols (≤ PrOH) did not afford the corresponding esters under reflux conditions, amongst others due to limited solubility of PHA under the conditions used.2-Butanol required much longer reaction times to reach full conversion. The boiling point of isobutanol (108 ºC) or 1-butanol (118 ºC) and the solvent characteristics are also sufficient to extract / depolymerize the PHA directly from the biomass residue (reflux conditions), thereby avoiding the autogenous pressure infrastructure needed for depolymerization using lower molecular weight alcohols (≤ PrOH). Example 2: Synthesis of isobutyl (R)-3-hydroxybutanoate from PHB. To a suspension of PHB (70.1 g, 804 mmol, 1 equiv.) in isobutanol (700 mL, 7.57 mol, 9.5 equiv.) H2SO4 (7 mL, 131 mmol, 1 v / v%) was added. The reaction mixture was maintained at reflux. After stirring for 3 hours,1H- NMR analysis showed the reaction to be complete. The mixture was cooled to room temperature and neutralized with a saturated NaHCO3 solution to pH 7. The layers were separated, after which the organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Isobutyl-3-hydroxybutyrate was obtained as a colorless transparent liquid:1H NMR (60 MHz, CDCl3) δ 4.18 (dd, J = 5.9, 3.1 Hz, 1H), 3.88 (d, J = 6.5 Hz, 2H), 3.00 (d, J = 3.3 Hz, 2H), 2.58 – 2.32 (m, 2H), 2.30 – 1.50 (m, 1H), 1.21 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.5 Hz, 6H).Example 3: Synthesis of magnesium bis (R)-3-hydroxybutyric acidfrom isobutyl (R)-3-hydroxybutanoate. A suspension of MgO (6.70 g, 166 mmol, 1 equiv.) in isobutyl-(R)-3- hydroxybutanoate (25 mL, 165 mmol, 0.99 equiv.) and water (150 mL) was heated to 95-100°C and stirred for 10 hours. The reaction mixture was cooled to room temperature. Remaining MgO was removed by filtration, after which the filtrate was extracted with ethyl acetate. The layers were separated, and the aqueous layer was concentrated in vacuo, and subsequently submitted for lyophilization. Magnesium bis (R)-3- hydroxybutyric acid was isolated as a white crystalline solid:1H NMR (60 MHz, D2O) δ 4.13 (h, J = 6.5 Hz, 1H), 2.33 (dd, J = 6.7, 1.0 Hz, 2H), 1.17 (d, J = 6.2 Hz, 3H). Example 4: Bioavailability of magnesium bis(R)-3-hydroxybutyric acid. This example demonstrates that an oral magnesium supplement according to the invention has a superior bioavailability as compared to known magnesium salt supplements wherein Mg is complexed to citrate. Sprague Dawley rats (N = 3) were exposed in vivo to either magnesium bis (R-3-hydroxybutyrate) or magnesium citrate (magnesium citrate tribasic nonahydrate; Sigma Aldrich) or magnesium glycerophosphate (MGP; Sigma Aldrich) by IV dosing (0.14 mg Mg / rat) and PO dosing (1.4 mg Mg / rat) (identical dosing). As shown in Figure 2, the ratio between oral dosing and IV dosing (∆AUC PO corrected / (∆AUC IV, = bioavailability) is higher for magnesium bis (R-3-hydroxybutyrate) compared to the known Mg-citrate and Mg-glycerophosphate supplements. Example 5: Conversion of isobutyl ester of (R)-hydroxybutyric acid to the corresponding salt(s) (Na, K, Ca, mixtures). This example describes how isobutyl ester of (R)-hydroxybutyric acid can be converted to various different (physiologically acceptable) salts. Sodium 3-hydroxy butyrate (Na3HB). To a solution of NaOH (11.24 g, 280.9 mmol; 0.9 eq.) in demi-water (1 L) was added Isobutyl-3-hydroxybutyrate (50 g, 312.1 mmol, 1.0 eq.). The turbid mixture was stirred at room temperature for two hours. A clear solution was obtained with pH = 7. The mixture was washed with ethyl acetate (2 x 0.5 L) and the aqueous phase was concentrated in vacuo (70 °C) to afford a white solid. The solid was suspended in acetone (100 mL) and subsequently filtered off. The filter dry solids were dried in vacuo to afford the product as a white solid (35.5 g, yield: quant.).1H-NMR (400 MHz, D2O) δ 4.11 (p, J = 6.3 Hz, 1H), 2.46 – 2.18 (m, 2H), 1.16 (d, J = 6.3 Hz, 3H).13C-NMR (101 MHz, D2O) δ 180.38, 65.57, 46.40, 21.62. Potassium 3-hydroxy butyrate (K3HB). To a solution of KOH (85%) (18.5 g, 280.9 mmol, 0.9 eq.) in demi-water (1 L) was added Isobutyl-3-hydroxybutyrate (50.0 g, 312.1 mmol, 1.0 eq.). The turbid mixture was stirred at room temperature for two hours. A clear solution was obtained with pH = 7. The mixture was washed with ethyl acetate (2 x 0.5 L) and the aqueous phase was concentrated in vacuo (70 °C) to afford a white solid. The solid was suspended in acetone (100 mL) and subsequently filtered off. The filter dry solids were dried in vacuo to affordthe product as a white solid (39.4 g, yield: quant.).1H-NMR (400 MHz, D2O)δ 3.97 (p, J = 6.3 Hz, 1H), 2.29 – 2.07 (m, 2H), 1.02 (d, J = 6.3 Hz, 3H).13C- NMR (101 MHz, d2o) δ 180.36, 65.57, 46.41, 21.61. Calcium 3-hydroxy butyrate (Ca(3HB)2). To a suspension of CaO (8.16 g, 140.4 mmol; 0.45 eq.) in demi-water (1 L) was added Isobutyl-3-hydroxybutyrate (50.0 g, 312.1 mmol, 1.0 eq.). The suspension was stirred overnight at room temperature. A slightly turbid solution was obtained with pH = 7. The mixture was washed with ethyl acetate (2 x 0.75L) and the aqueous phase was filtered over a paper filter and concentrated in vacuo (70 °C) to afford a white sticky foam. The product was stripped with isopropyl alcohol (2 x 50 mL) to afford a white foam (26.2 g; yield: 76%).1H-NMR (400 MHz, D2O) δ 3.96 (p, J = 6.3 Hz, 1H), 2.29 –2.07 (m, 2H), 1.01 (d, J = 6.3 Hz, 3H).13C-NMR (101 MHz, D2O) δ 180.54,65.53, 46.27, 21.60. Calcium 3-hydroxy butyrate & Magnesium 3-hydroxy butyrate (2:1) (Ca / Mg(3HB)2). To a suspension of magnesium oxide (0.81 g; 19.97 mmol, 0.16 eq.) and calcium oxide (2.32 g, 39.95 mmol, 0.32 eq.) in demi water (200 mL) wasadded 3HB-iBu-Ester (20.0 g, 124.8 mmol, 1.0 eq.). The white suspensionwas heated to reflux temperature (105 °C) and stirred overnight. To the mixture was added ethyl acetate (100 mL). The layers were separated and the aqueous phase was concentrated in vacuo (65 °C.) to afford a sticky foam. The residue was dissolved in ethanol (abs.100 mL), filtered over a sintered glass filter (P3) and the filtrate was concentrated in vacuo (65 °C) to afford the mixture of the title compounds as white foam (11.4 g; yield: 79%).1H-NMR (400 MHz, D2O) δ 4.18 – 4.04 (m, 1H), 2.48 – 2.12 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H).13C-NMR (101 MHz, D2O) δ 180.36, 65.51, 46.18, 21.60. Example 6: Iso-butyl-3-hydroxybutyrate from microorganisms (3HB iBu-ester). To a suspension of PHB containing microorganisms (15 g; PHB content: 64%) in iso-butanol (75 mL) was added sulfuric acid (1.5 mL, 27.66 mmol, 0.3 eq.). The mixture was heated to reflux temperature (105 °C) and stirred for 16 h. To the black suspension was added Celite (545; 10 g) and the mixture was filtered over a patch of Celite (125 mL filter, 0.5 cm celite). The filtrate was washed with sodium bicarbonate (sat., aq., 1 x 50 mL). The aqueous phase was extracted with Iso-butanol (50 mL), and the combined organics were washed with sodium carbonate(sat., aq., 1 x), brine and concentrated in vacuo (60 °C) to afford a brown oil with a small amount of precipitate. The residue was dissolved in ethyl acetate (20 mL) and filteredover paper filter. The volatiles were removed in vacuo (60 °C) and the crudewas purified by fractional vacuum distillation (2 mbar, 88-96°C), to afford the target compound as a clear oil (7.1 g, yield: 48%).1H-NMR (400 MHz, CDCl3) δ 4.25 – 4.12 (m, 1H), 3.93 – 3.84 (m, 2H), 2.54 – 2.36 (m, 2H), 2.00 –1.85 (m, 1H), 1.22 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.7 Hz, 6H).13C-NMR (101MHz, CDCl3) δ 173.01, 70.76, 64.23, 42.69, 27.63, 22.39, 19.03. Example 7: Magnesium 3-hydroxy butyrate from microorganisms (Mg(3HB)2). To a suspension of magnesium oxide (724 mg, 17.98 mmol, 0.48 eq) in demi water (60 mL) was added 3HB-iBu-ester (prepared from PHB containing microorganisms; see Example 6) (6.0 g; 37.45 mmol, 1.0 eq.) The white suspension was heated to reflux temperature (100 °C) and stirred overnight. An almost clear solution was obtained. The mixture was washed with ethyl acetate (50 mL) and the aqueous phase was concentrated (70 °C) to afford a white foam. The white foam was dissolved in ethanol (abs., 25 mL), filtered over a paper filter and concentrated in vacuo (60 °C) to afford magnesium 3-hydroxy butyrate as white foam (3.07 g; yield: 74%).1H-NMR (400 MHz, D2O) δ 4.15 – 4.02 (m, 1H), 2.44 – 2.19 (m, 2H), 1.13 (d, J = 6.3 Hz, 3H).13C-NMR (101 MHz, D2O) δ 180.36, 65.53, 46.24, 21.58. Example 8: Iso-butyl-3-hydroxybutyrate & Iso-butyl-3- hydroxyvalerate (2:1) (from PHBV containing microorganisms; ) (3HBV iBu-ester). To a solution of PHBV containing microorganisms (47.2 g) in iso-butanol (250 mL) was added sulfuric acid (5 mL, 90.7 mmol). The mixture was heated to reflux temperature (105 °C) and was stirred overnight. A black suspension formed. The mixture was filtered over celite (0.5 cm, 50 mL filter) and the residue was washed with ethyl acetate (25 mL). The filtrate was washed with sodium bicarbonate (sat., aq., 2 x 200 mL) and brine (200 mL). The organic layer was concentrated in vacuo (60 °C) to afford the crude as a brown oil with small amount of solid particles. The crude was dissolved in ethyl acetate (100 mL) and the solids were removed by filtration overpaper filter. The filtrate was concentrated in vacuo (60 °C) to afford a crudemixture of Iso-butyl-3-hydroxybutyrate and Iso-butyl-3-hydroxyvalerate (2:1) (52.8 g). The crude was purified by distillation under reduced pressure (3 mbar, 95 - 99°C) to afford a mixture of Iso-butyl-3-hydroxybutyrate and Iso-butyl-3-hydroxyvalerate (2:1) (37.7 g, 229 mmol).1H-NMR (400 MHz, CDCl3) (iso-butyl-3-hydroxybutyrate): δ 4.23 – 4.12 (m, 1H), 3.88 (d, J = 1.3 Hz, 2H), 3.06 (d, J = 3.7 Hz, 1H), 2.56 – 2.35 (m, 2H), 2.00 – 1.85 (m, 1H),1.22 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.8 Hz, 6H); (Iso-butyl-3-hydroxyvalerate): δ 3.97 – 3.83 (m, 3H), 2.99 (d, J = 4.0 Hz, 1H), 2.56 – 2.35 (m, 2H), 2.00 – 1.85 (m, 1H), 1.60 – 1.42 (m, 2H), 0.96 (d, J = 7.4 Hz, 3H)).13C-NMR (101 MHz, CDCl3) δ 173.19, 173.01, 70.76, 69.33, 64.23, 42.69, 40.81, 29.36, 27.63, 22.39, 19.03, 9.81. Example 9: Magnesium 3-hydroxy butyrate & Magnesium 3- hydroxyvalerate (Mg(3HBV)2) from PHBV containing microorganisms. To a suspension of magnesium oxide (2.58 g, 64.1 mmol, 0.48 eq.) in demi water (150 mL) was added 3HBV iBu-ester (prepared from PHBV containing microorganisms; see Example 8) (21.4 g, 133.3 mmol, 1.0 eq.). The suspension was stirred at reflux temperature (100 °C) overnight. The mixture became an almost clear solution. The solution was allowed to cool to room temperature and was washed with ethyl acetate (3 x 100 mL). The aqueous phase was filtered over a paper filter and concentrated in vacuo (70 °C) to afford a white sticky foam. The product was stripped with ethanol (abs.) (2 x 50 mL) to afford a mixture of magnesium 3-hydroxy butyrate & magnesium 3-hydroxyvalerate (2:1) as a white foam (8.5 g, yield: 57.5%).1H- NMR (400 MHz, D2O)(magnesium 3-hydroxy butyrate): δ 4.15 – 4.03 (m,1H), 2.40 – 2.20 (m, 2H), 1.14 (d, J = 6.3 Hz, 3H); (magnesium 3-hydroxyvalerate): δ 3.93 – 3.80 (m, 1H), 1.61 – 1.31 (m, 2H), 1.11 (d, J = 7.1Hz, 2H), 0.85 (t, J = 7.5 Hz, 3H).13C-NMR (101 MHz, D2O) (magnesium 3-hydroxy butyrate): δ 180.32, 65.52, 46.21, 21.59; (magnesium 3-hydroxyvalerate): δ 180.62, 70.71, 44.24, 28.85, 9.02.

Claims

AMENDED CLAIMS received by the International Bureau on 23 October 2025 (23.10.2025)1. A method for the manufacture of a (R)- 3-hydroxybutyric acid salt, comprising:(i) subjecting a starting material comprising polyhydroxyalkanoates (PHA) to an extraction / transesterification reaction using isobutanol in the presence of an acid to obtain the isobutyl ester of (R) -3 -hydroxybutyric acid; and(ii) converting the isobutyl ester of (R)-3-hydroxybutyric acid to the corresponding salt.

2. The method according to claim 1, wherein the starting material is a PHA-containing biomass.

3. The method according to claim 2, wherein the biomass comprises PHB ((poly(3-hydroxybutyrate)) and / or PHBV ((poly(3-hydroxybutyrate-co-3- hydroxyvalerate)), preferably wherein the starting material is a PHB- containing biomass.

4. The method according to any one of claim 1-3, wherein step (i) comprises contacting the starting material with isobutanol under reflux conditions, preferably for a duration of 2-12 hours, more preferably 2-5 hours.

5. The method according to any one of claims 1-4, wherein isobutanol is the sole solvent in the extraction / transesterification reaction.

6. The method according to any one of the preceding claims, wherein a sulfuric or sulfonic acid is used, preferably a sulfuric acid.

7. The method according to any one of the preceding claims, wherein step (ii) comprises converting the isobutyl ester of (R)-3-hydroxybutyric acid to the corresponding calcium salt, sodium salt, potassium salt, magnesium salt, or salt mixture thereof.

8. The method according to claim 7, comprising converting the isobutyl ester of (R) -3 -hydroxybutyric acid to magnesium bis (R)-3- hydroxybutyric acid ((C4H7O3)2Mg).

9. The method according to claim 8, wherein step (ii) comprises heating the isobutyl ester of (R)-3-hydroxybutyric acid in an aqueous suspension of MgO and / or Mg(0H)2.

10. The method according to claim 8 or 9, further comprising formulating said magnesium bis (R)-3-hydroxybutyric acid into a food additive, nutritional or nutraceutical or pharmaceutical composition.

11. A method for improving the physical and / or cognitive performance of a subject, in particular a subject suffering from a metabolic or age- related disorder, comprising administering magnesium bis (R)-3- hydroxybutyric acid to the subject.

12. A composition comprising magnesium bis (R)-3-hydroxybutyric acid for use in a method for improving the physical and / or cognitive performance of a subject, in particular a subject suffering from a metabolic or age-related disorder.

13. The method according to claim 11 or the composition for use according to claim 12, wherein the subject suffers from magnesium deficiency, mild cognitive impairment, Alzheimer's disease, Huntington's disease, autism, schizophrenia, cognitive decline, depression, dementia, attention deficit hyperactivity disorder (ADHD), amyotrophic lateralsclerosis (ALS), Parkinson's disease, diabetes, hypomagnesemia, glaucoma, migraine, anxiety, mood disorder, or hypertension.

14. A composition comprising magnesium bis (R)-3-hydroxybutyric acid for use as magnesium supplement.

15. A composition comprising magnesium bis (R)-3-hydroxybutyric acid for use as exogenous ketone supplement, preferably in the management of exercise or obesity.

16. A composition comprising magnesium bis (R)-3-hydroxybutyric acid for use in a method to influence metabolism by increasing circulating ketone bodies.

17. Composition for use according to any one of claims 12-16, wherein the composition is a pharmaceutical composition, a neutraceutical composition or a food supplement.

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