Enzymatic reduction of acetoacetate

Abstract

The invention relates to an enzymatic reduction of polyalcohols comprising at least two acetoacetate groups.

Description

[0001] Enzymatic reduction of acetoacetate

[0002] Technical Field

[0003] The invention relates to an enzymatic reduction of polyalcohols comprising at least two acetoacetate groups (also referred to as p-keto esters).

[0004] Technological Background

[0005] Acetoacetylated polyalcohols and p-hydroxyl butyric acid esters (also referred to as “P-hydroxy- butyrate”; BHB) of polyalcohols (also referred to as “polyalcohol-based p-hydroxybuterate”) prepared therefrom are valuable compounds with a versatile utilization for example as parenteral nutrients or for the treatment of certain diseases, such as migraine, Parkinson's, Alzheimer’s, and any other diseases related to the keton metabolism of the brain.

[0006] US 2019 / 117612 A1 pertains to the field of migraine headaches and the management of the symp- tomology thereof using 3-hydroxybutyrate glycerides.

[0007] US 2018 / 193300 A1 pertains to a method of treatment of mild to moderate non-penetrating closed traumatic brain injury and mild to moderate traumatic brain injury due to surgical intervention using 3-hydroxybutyate glycerides.

[0008] Hence, ketone bodies like acetoacetate (AA) and p-hydroxybutyrate (BHB) are believed to have a positive impact on brain health and may alleviate brain related disease and the associated symptoms. Therefore, it can be beneficial to supplement AA and / or BHB to improve health of a subject such as brain health.

[0009] In the human body, AA and BHB are in a natural equilibrium wherein the interconversion of AA to BHB and BHB to AA is catalysed by the enzyme p-hydroxybutyrate dehydrogenase involving nicotinamide adenine dinucleotide (NAD) (cf. for example H. Kolb et al. “Ketone bodies: from enemy to friend and guardian angel”, BMC Med., 2021 , 19(1), 313). The naturally occurring p-hydroxybutyrate form is the enantiopure R-p-hydroxybutyrate, which is the actual active form. However, in respects to potential diabetes treatment there are beneficial effects of the S-enantiomeric form described (A. Buga et. al. “Fasting and diurnal blood ketonemia and glycemia responses to a six-week, energy- controlled ketogenic diet, supplemented with racemic R / S-BHB salts" Clinical Nutrition, Volume 54, 2023, P227-287). Therefore it is highly desirable to have synthetic methods in place, to selectively synthesis the R-enantiomer or the S-enantiomer, either in an enantiopure or enantioenriched version.

[0010] In view of the natural equilibrium between AA and BHB in the human body, it can be desirable to supplement AA and BHB at the same time in order to maintain and respect the natural physiological equilibrium between BHB and AA when supplementing ketone bodies.

[0011] In general, molecules comprising AA units are accessible via e.g. reacting diketene with an organic polyol or a p-hydroxyl butyric acid ester of an organic polyol (WO 2023 / 094654). The respective AA units can be reduced in order to obtain BHB units. However, reduction conditions are often accompanied by applying heavy metal catalysts. It is particularly difficult to synthesize chiral BHB units. Such reaction often require expensive homogenous chiral catalysts (based on heavy metal) and high pressures, wherein the obtained product may contain traces of the applied catalyst. However, impurities such as the heavy metals like ruthenium must be reduced to low levels for food or pharma products.

[0012] The use of highly expensive chiral catalysts should also be avoided for economic reasons. To reduce the amount of used catalyst, the hydrogen pressure and / or the temperature needs to be increased, which increases the risk to obtain undesired side product. In addition, increasing the hydrogen pressure requires special high-pressure equipment, which is not only expensive but also less user-friendly.

[0013] Against this background, there is an ongoing need for excellent processes for the synthesis of polyalcohol esters comprising BHB. In particular, there is a need for mild and / or efficient processes for the synthesis of polyalcohol esters comprising BHB that suitably are applicable under industrial scale. Further, there is a need for processes for the synthesis of polyalcohol esters comprising BHB in an enantiomeric excess such as in R-configuration. Further, there is a need for processes for the synthesis of said polyalcohol esters comprising BHB, wherein normal reactors can be applied and / or wherein undesired side products are reduced. Summary of the invention

[0014] In a first aspect, the present invention provides a process of manufacturing a polyalcohol-based p- hydroxybuterate, the process comprising the step of reducing a polyalcohol comprising at least two acetoacetate groups with an enzyme in the presence of a hydrogen source. More specifically the process affords an enantiomeric pure product, which is selectively the R- or the S- enantiomer or and enantiomeric enriched mixture with an uneven ratio between R- / S-enantiomeric forms.

[0015] According to a second aspect, the present invention relates to a composition comprising a polyalcohol-based p-hydroxybuterate derived from an enzymatic reduction of a polyalcohol comprising at least two acetoacetate groups, wherein all of the p-hydroxyl butyric acid ester groups of the polyalcohol-based P-hydroxybuterate are in R-configuration or wherein all of the p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in S-configuration.

[0016] It has surprisingly been found that the inventive process solves at least one of the above needs and that the present invention provides inter alia an efficient and mild process of manufacturing polyalcohol esters comprising BHB. The obtained product does not need to be excessively purified since the reduction does not need the presence of heavy metal-based catalysts such as palladium- or ruthenium-based catalysts.

[0017] Detailed description of the invention

[0018] In the following, the invention will be explained in more detail.

[0019] Definitions

[0020] In order for the present invention to be readily understood, several definitions of terms used in the course of the invention are set forth below.

[0021] According to the present invention, the term “linear or branched C2-12 alkyl” refers to a straight- chained or branched saturated hydrocarbon group having 2 to 12 carbon atoms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms including methyl, ethyl, propyl, 1 -methylethyl, butyl, 1- methylpropyl, 2-methylpropyl, 1 ,1 -dimethylethyl, pentyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 1 ,1-dimethylpropyl, 1 ,2-dimethylpropyl, hexyl, 1 -methylpentyl, 2- methylpentyl, 3-methylpentyl, 4-methylpentyl, 1 , 1 -dimethylbutyl, 1 ,2-dimethylbutyl, 1 ,3-dimethyl- butyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1 -ethylbutyl, 2-ethylbutyl, 1 ,1 ,2-trime- thyl propyl, 1 ,2,2-trimethylpropyl, 1 -ethyl-1 -methylpropyl and 1-ethyl-2-methylpropyl.

[0022] According to the present invention, the term “C3-8 cycloalkyl” refers to a monocyclic saturated hydrocarbon group having 3 to 8 carbon ring members, such as 2, 3, 4, 5, 6, 7, or 8 carbon ring members, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0023] It is to be understood that the linear or branched C2-12 alkyl and C3-8 cycloalkyl may optionally be further substituted. Exemplary substituents include hydroxy, linear or branched C1-12 alkyl, Cs s cycloalkyl, a carboxy group, halogen, and phenyl.

[0024] According to the present invention, the term “organic polyol” refers to a linear, branched, or cyclic organic compound with 2 to 18 carbon atoms having at least two hydroxyl groups, preferably at least three hydroxyl groups. As such, the organic polyol may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 carbon atoms. In one embodiment, no more than one hydroxyl group is connected to one carbon atom. In one embodiment, the organic polyol contains only carbon, hydrogen, and oxygen atoms.

[0025] According to the present invention, the term “at least three hydroxyl groups” means that the respective compound has three or more hydroxyl groups. In one embodiment, “at least three hydroxyl groups” includes 3 to 18 hydroxyl groups such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 hydroxyl groups. In one embodiment, “at least three hydroxyl groups” includes 3 to 12 hydroxyl groups such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 hydroxyl groups. In one embodiment, “at least three hydroxyl groups” includes 3 to 9 hydroxyl groups such as 3, 4, 5, 6, 7, 8, or 9 hydroxyl groups. In one embodiment, “at least three hydroxyl groups” includes 3 to 6 hydroxyl groups such as 3, 4, 5, or 6 hydroxyl groups. Equally, the term “at least two hydroxyl groups” means that the respective compound has two or more hydroxyl groups.

[0026] It is to be understood that if not explicitly stated otherwise, all stereoisomers, conformations and configurations are encompassed by compounds and functional groups which can be present as different stereoisomers or in different conformations and configurations. For example, the term “inositol” is to be understood as to include all stereoisomers and conformations such as myo-, scyllo-, muco-, D-chiro-, neo-inositol, L-chiro-, allo-, epi-, and c / s-inositol. For example, the term “hexanetriol” is to be understood as to include all hexane isomers including three hydroxyl groups such as 1 ,1 ,1-hexanetriol, 1 ,1 ,2-hexanetriol, 1 ,2,2-hexanetriol, 1 ,2,3-hexanetriol, 1 ,2,4-hexanetriol, 1 ,2,5- hexanetriol, 1 ,2,6-hexanetriol, 1 ,3,5-hexanetriol, 1 ,3,6-hexanetriol, 2,3,4-hexanetriol, 2,3,5-hex- anetriol etc.

[0027] It is to be understand that denotes the bond of the respective moiety to the remainder of the molecule.

[0028] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of’, both meanings being specifically intended, and hence individually disclosed, embodiments according to the present invention.

[0029] As used herein, the articles “a” and “an” preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore, “a” or “an” is to be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0030] As used herein, the term “about” modifying the quantity of a substance, ingredient, component, or parameter employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures, e.g., liquid handling procedures used for making concentrates or solutions. Furthermore, variation can occur from inadvertent error in measuring procedures, differences in the manufacture, source, or purity of the ingredients employed to carry out the methods, and the like. In one embodiment, the term “about” means within 10% of the reported numerical value. In a more specific embodiment, the term “about” means within 5% or within 2% of the reported numerical value.

[0031] As indicated above, the present invention relates in one aspect to a process of manufacturing a polyalcohol-based p-hydroxybuterate, the process comprising the step of reducing a polyalcohol comprising at least two acetoacetate groups with an enzyme in the presence of a hydrogen source.

[0032] In the following, particular embodiments of the present invention such as moieties and reaction conditions are described in further details. It is to be understood that each embodiment is relevant on its own as well as in combination with other embodiments.

[0033] In one embodiment, the polyalcohol comprising at least two acetoacetate groups is derived from an organic polyol comprising at least 2 hydroxyl groups such as at least 3 hydroxyl groups. In this connection, it is to be understood that in a polyalcohol comprising at least two acetoacetate groups that is derived from an organic polyol comprising at least 2 hydroxyl groups, at least two of said at least 2 hydroxyl groups are substituted by the respective acetoacetate group.

[0034] In one embodiment, the organic polyol has from 2 to 10 hydroxyl groups. Preferably, the organic polyol has from 3 to 8 hydroxyl groups, such as from 3 to 7 hydroxyl groups, from 3 to 6 hydroxyl groups, from 3 to 5 hydroxyl groups, or from 3 to 4 hydroxyl groups.

[0035] In one embodiment, polyalcohol comprising at least two acetoacetate groups comprises from 2 to 10 acetoacetate groups, preferably from 3 to 10 acetoacetate groups, more preferably from 3 to 8 acetoacetate groups, such as from 3 to 7 acetoacetate groups, from 3 to 6 acetoacetate groups, from 3 to 5 acetoacetate groups, or from 3 to 4 acetoacetate groups.

[0036] In one embodiment, the polyalcohol-based p-hydroxybuterate is a compound of formula 1

[0037] 1 wherein

[0038] A is derived from an organic polyol with at least 2 hydroxyl groups, x is at least 1 , y is 0 or at least 1 , x + y is from 2 to the number of hydroxyl groups of the initial organic polyol A, n is 0 or 1 ; and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2

[0039] In one embodiment n is 0 such as all n are 0.

[0040] In another embodiment n is 1 such as all n are 1 . In one embodiment, the compound of formula 1 is a compound of formula 1-2

[0041] 1-2 and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2-2,

[0042] 2-2.

[0043] In one embodiment, the compound of formula 1 is a compound of formula 1-3

[0044] 1-3 and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2-2,

[0045] 2-2.

[0046] In one embodiment, the compound of formula 1 is a compound of formula 1-3a

[0047] 1-3a.

[0048] In another embodiment, the compound of formula 1 is a compound of formula 1-3b

[0049] 1-3b. In one embodiment, the compound of formula 1 is a compound of formula 1-4

[0050] 1-4 wherein A is derived from an organic polyol with at least 2 hydroxyl groups, x is at least 1 , y is 0 or at least 1 , x + y is from 2 to the number of hydroxyl groups of the initial organic polyol A, and n is 0 or 1 ; and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2-4

[0051] 2-4.

[0052] In one embodiment, the compound of formula 1-4 is a compound of formula 1-5 and the compound of formula 2-4 is a compound of formula 2-5

[0053] 2-5. In one embodiment, the compound of formula 1-5 is a compound of formula 1-5a

[0054] 1-5a.

[0055] In one embodiment, the compound of formula 1-5 is a compound of formula 1-5b

[0056] 1-5b.

[0057] In one embodiment, the compound of formula 1-5 is a compound of formula 1-5aa

[0058] 1-5aa and the compound of formula 2-5 is preferably a compound of formula 2-5aa

[0059] 2-5aa.

[0060] In one embodiment, the compound of formula 1-5 is a compound of formula 1-5bb

[0061] 1-5bb and the compound of formula 2-5 is preferably a compound of formula 2-5bb

[0062] 2-5bb

[0063] In one embodiment, the organic polyol has from 2 to 10 hydroxyl groups such as from 3 to 10 hydroxyl groups. Preferably, the organic polyol has from 3 to 8 hydroxyl groups, such as from 3 to 7 hydroxyl groups, from 3 to 6 hydroxyl groups, from 3 to 5 hydroxyl groups, or from 3 to 4 hydroxyl groups, or from 4 to 7 hydroxyl groups, or from 4 to 6 hydroxyl groups.

[0064] In one embodiment, x is at least 2, at least 3, or at least 4. In one embodiment, x is 1 , 2, 3, 4, 5, or 6. Preferably, x is 2, 3, or 4.

[0065] In one embodiment, y is 0, at least 1 , at least 2, at least 3, or at least 4. In a preferred embodiment, y is 0.

[0066] In one embodiment, x is 1 and y is 3. In one embodiment, x is 2 and y is 2. In one embodiment, x is 3 and y is 1 . In one embodiment, x is 4 and y is 0.

[0067] In one embodiment, x + y is from 2 to 10, such as from 3 to 10, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5 or from 3 to 4. Accordingly, x + y may be 3, 4, 5, 6, 7, 8, 9, or 10, preferably 3, 4, 5, or 6.

[0068] In one embodiment, x + y is equal to the number of hydroxyl groups of the initial polyol A. In one embodiment, x + y is less than the number of hydroxyl groups of the initial polyol A.

[0069] In one embodiment, the organic polyol is a linear, branched, or cyclic organic compound with 2 to 18 carbon atoms having at least two hydroxyl groups, preferably at least three hydroxyl groups.

[0070] In one embodiment, the organic polyol is selected from a linear or branched C2-12 alkyl substituted with at least 2 hydroxyl groups, preferably at least 3 hydroxyl groups, or a C3-8 cycloalkyl substituted with at least 2 hydroxyl groups, preferably at least 3 hydroxyl groups.

[0071] Preferably, the linear or branched C2-12 alkyl substituted with at least 2 hydroxyl groups, preferably at least 3 hydroxyl groups, is selected from the group consisting of glycerol, trimethylolpropane, butanetriol, 2-methyl-propanetriol, pentanetriol, 3-methyl-pentanetriol, hexanetriol, pentaerythritol, butanetetrol, pentanetetrol, hexanetetrol, hexanepentol, and combinations thereof. Preferably, the C3-8 cycloalkyl substituted with at least 2 hydroxyl groups, preferably at least 3 hydroxyl groups, is selected from the group consisting of cyclopentanetriol, cyclohexanetriol, cyclopentanetetrol, cyclohexanetetrol, and combinations thereof.

[0072] In one embodiment, the organic polyol is selected from the group consisting of monosaccharides, sugar alcohols, and sugar acids.

[0073] Monosaccharides generally have the chemical formula CnH2nOn. Monosaccharides can be classified by the number x of carbon atoms they contain (CH2O)X: trioses (x=3), tetroses (x=4), pentoses (x=5), hexoses (x=6) and heptoses (x=7).

[0074] In one embodiment, the monosaccharide is selected from tetroses, pentoses, hexoses, heptoses, and combinations thereof. Preferably, the monosaccharide is selected from aldotetroses, ketotetroses, aldopentoses, ketopentoses, aldohexosen, ketohexoses, aldoheptoses, ketoheptoses, and combinations thereof.

[0075] In one embodiment, the monosaccharide is selected from the group consisting of erythrose, thre- ose, erythrulose, ribose, arabinose, xylose, lyxose, desoxyribose, ketopentose, ribulose, xylulose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, n-acetyl-d-glucosamin, glucosa- min, N-acetyl-D-galactosamin, fucose, rhamnose, chinovose, fructose, 2-desoxy-D-glucose, fluordesoxyglucose, 6-desoxyfructose, 1 ,6-dichlorfructose, 3,6-anhydrogalactose, 1-O-methylgalac- tose, 1-O-methyl-D-glucose, 1-O-methyl-D-fructose, 3-O-methyl-D-fructose, 6-O-methyl-D-galac- tose, sedoheptulose, mannoheptulose, L-glycero-D-manno-heptose, and combinations thereof.

[0076] Sugar alcohols (also called polyhydric alcohols, polyalcohols, alditols or glycitols) are organic compounds, typically derived from sugars, containing one hydroxyl group (-OH) attached to each carbon atom.

[0077] In one embodiment, the sugar alcohol is selected from the group consisting of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, and combinations thereof.

[0078] A sugar acid is generally a monosaccharide with a carboxyl group at one end or both ends of the carbon chain. Main classes of sugar acids include aldonic acids, ulosonic acids, uronic acids, and aldaric acids. In aldonic acids, the aldehyde group (-CHO) located at the initial end (position 1) of an aldose is oxidized. In ulosonic acids, the -CH2(OH) group at the initial end of a 2-ketose is oxidized yielding an a-ketoacid. In uronic acids, the -CH2(OH) group at the terminal end of an aldose or ketose is oxidized. In aldaric acids, both ends (-CHO and -CH2(OH)) of an aldose are oxidized. In one embodiment, the sugar acid is selected from aldonic acids, ulosonic acids, uronic acids, al- daric acids, and combinations thereof. Preferably, the sugar acid is selected from the group consisting of xylonic acid, gluconic acid, ascorbic acid, neuraminic acid, ketodeoxyoctonic acid, glucuronic acid, galacturonic acid, iduronic acid, mucic acid, saccharic acid, and combinations thereof. In one embodiment, the organic polyol is selected from the group consisting of glycerol, sorbitol, xylitol, mannitol, erythritol, maltitol, glucose, glucitol, ribulose, pentaerythritol, trimethylolpropane, and combinations thereof.

[0079] In one embodiment, the compound of formula 1 is selected from the group consisting of

[0080] In one embodiment, the compound of formula 1 is selected from the group consisting of

[0081] In one embodiment, the compound of formula 2 may be butane-1 ,2,3,4-tetrayl tetrakis(3-oxobuta- noate). In one embodiment, the p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxy- buterate are in form of a predominantly in S-configuration or predominantly in R-configuration. In one preferred embodiment, all p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hy- droxybuterate are in R-configuration. In another preferred embodiment, all p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in S-configuration.

[0082] In one embodiment, the p-hydroxyl butyric acid ester units of the polyalcohol-based p-hydroxybuter- ate are in form of a predominantly in S-configuration or predominantly in R-configuration. In one preferred embodiment, all p-hydroxyl butyric acid ester units of the polyalcohol-based p-hydroxy- buterate are in R-configuration. In another preferred embodiment, all p-hydroxyl butyric acid ester units of the polyalcohol-based p-hydroxybuterate are in S-configuration.

[0083] As used herein, the term “ -hydroxyl butyric acid ester units” is directed to the structures

[0084] In one embodiment, in the compound according to formula 1 , all p-hydroxyl butyric acid ester groups, preferably all p-hydroxyl butyric acid ester units, are either D-configured or L-configured. In another embodiment, all p-hydroxyl butyric acid ester groups, preferably all p-hydroxyl butyric acid ester units, are present in the compound according to formula 1 as a non-racemic mixture of D- and L- configurations.

[0085] In one embodiment, the compound according to formula 1 contains more D-configured p-hydroxyl butyric acid ester units than L-configured p-hydroxyl butyric acid ester units. Preferably all p-hy- droxyl butyric acid ester groups, preferably all p-hydroxyl butyric acid ester units, are in D-configura- tion.

[0086] In one embodiment, in the compound according to formula 1 , all p-hydroxyl butyric acid ester groups, preferably all p-hydroxyl butyric acid ester units, are either R-configured or S-configured. In another embodiment, all p-hydroxyl butyric acid ester groups, preferably all p-hydroxyl butyric acid ester units, are present in the compound according to formula 1 as a non-racemic mixture of R- and S- configurations. In one embodiment, the compound according to formula 1 contains more R-configured p-hydroxyl butyric acid ester units than S-configured p-hydroxyl butyric acid ester units. Preferably all p-hy- droxyl butyric acid ester groups, preferably all -hydroxyl butyric acid ester units, are in R-configura- tion.

[0087] In one embodiment, the process is free of metal-based catalysts. In this connection, Ni-based catalysts, Pd-based catalysts, Pt-based catalysts, Ru-based catalysts, Co-based catalysts, Ir-based catalysts, or Rh-based catalysts may be named.

[0088] In one embodiment, the process is free of ruthenium-based catalysts.

[0089] In one embodiment, the process is free of palladium-based catalysts.

[0090] In one embodiment, the process is free of chiral ligands selected from the group consisting of 2,2'- bis(diphenylphosphino)-1 ,1 '-binaphthyl (BINAP), 1 ,1 '-Bi-2-naphthol (BINOL), 2,3-O-isopropylidene- 2,3-dihydroxy-1 ,4-bis(diphenylphosphino)butane (DIOP), 2,2’,5,5'-tetramethyl-4,4'-bis-(diphe- nylphoshino)-3,3'-bithiophene (tetraMe-BITlOP), Bis(diphenylphosphino)-7,8-dihydro-6H- dibenzo[f,h][1 ,5]dioxonin (C3-TunePhos), 4,4'-Bis(bis(3,5-dimethylphenyl)phosphino)-2,2',6,6'-tetra- methoxy-3,3'-bipyridine (Xyl-p-PHOS), (6,6'-Dimethoxybiphenyl-2,2’-diyl)-bis-(diphenylphosphin) (MeO-BIPHEP), and 1 ,2-Bis[(2-methoxyphenyl)phenylphosphino]ethane (DIPAMP).

[0091] In one embodiment, the process is conducted in aqueous medium such as water. In this connection, the starting material (e.g. the polyalcohol comprising at least two acetoacetate groups) may be dissolved in a suitable solvent such as dimethyl sulfoxide as necessary.

[0092] In one embodiment, the process is conducted in a two-phase system, wherein one phase is an aqueous medium and the other phase is hydrophobic medium. The hydrophobic medium may be selected from the group consisting of n-butyl acetate, toluene, n-octane, and combinations thereof.

[0093] In one embodiment, the process is conducted at a pressure of about 0.1 to about 2.0 bar, preferably of about 0.2 to about 1 .7 bar or of about 0.5 to about 1 .5 bar, and in particular of about 0.8 to about 1.2 bar. In one embodiment, the process is conducted at a temperature of about 0 to 45 °C, preferably of about 20 to about 45 °C, more preferably of about 28 to about 43 °C, still more preferably of about 30 to about 40 °C, and in particular of about 30 to about 35 °C.

[0094] Any suitable hydrogen source may be applied.

[0095] In one embodiment, the hydrogen source is hydrogen gas.

[0096] In one embodiment, the hydrogen source is a monohydric alcohol. Suitable monohydric alcohols are ethanol, propanol, butanol, and pentanol. Monohydric, secondary alcohols are preferred. Preferably, isopropanol is applied. In this connection, the process suitably comprises an alcohol dehydrogenase enzyme that converts the hydroxyl group of said alcohol to the respective keto group. For example, phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) as disclosed in Itoh et al. (Efficient synthesis of optically pure alcohols by asymmetric hydrogen-trans- fer biocatalysis: application of engineered enzymes in a 2-propanol-water medium, Appl Microbiol Biotechnol (2012) 93:1075-1085) may be suitable.

[0097] In one embodiment, the hydrogen source is a monosaccharide. Suitable, the monosaccharide is an aldohexose such as (DZL)-allose, (D / L)-glucose, (DZL)-mannose, (DZL)-galactose, or the like. In this connection, the process suitably comprises the respective dehydrogenase enzyme. For example, D-glucose dehydrogenase (GDH) as disclosed in Zhu et al. (A recombinant ketoreductase tool-box. Assessing the substrate selectivity and stereoselectivity toward the reduction of p-ketoesters, Tetrahedron (2006) 62:901-905) may be suitable.

[0098] In one embodiment, the hydrogen source is isopropanol, glucose, or sodium formate, preferably isopropanol or glucose, and in particular isopropanol.

[0099] In one embodiment, the enzyme is selected from the group consisting of alcohol dehydrogenase enzyme, ketoreductase enzyme, and mixtures thereof.

[0100] The enzyme can be applied in purified form, in the form of a cell lysate, or via an organism, such as Escherichia coli, which expresses the same.

[0101] Suitable alcohol dehydrogenase enzymes may be selected from the list consisting of phenylacetaldehyde reductase (PAR), Leifsonia alcohol dehydrogenase (LSADH), and mixtures thereof.

[0102] Alcohol dehydrogenase enzymes are suitably accessible via Escherichia coli. In one embodiment, the enzyme is a ketoreductase enzyme and the process comprises an oxidore- ductase enzyme. Said oxidoreductase enzyme is suitably a D-glucose dehydrogenase (GDH), whereas the hydrogen source is suitably D-glucose.

[0103] In a preferred embodiment, the process comprises nicotinamide adenine dinucleotide (NAD+).

[0104] In a preferred embodiment, the process comprises nicotinamide adenine dinucleotide phosphate (NADP+).

[0105] Preferably, the nicotinamide adenine dinucleotide, the nicotinamide adenine dinucleotide phosphate, or a mixture thereof is present in the process in a concentration of about 0.001 to about 100 mM, preferably of about 0.01 to about 10 mM, more preferably of about 0.1 to 5 mM or of about 0.5 to about 3 mM.

[0106] In one embodiment, the process comprises formate dehydrogenases.

[0107] In one embodiment, the process is conducted for about 5 to about 40 hours, preferably for about 10 to about 30 hours, still more preferably for about 12 to about 25 hours, and in particular for about 15 to about 20 hours.

[0108] In one embodiment, the hydrogen source is adjusted as necessary depending on the desired reduction grade of the product, i.e. depending on how many BHB groups are desired.

[0109] In one embodiment, the molar amount of hydrogen source in the process is of about 1 to about 500 equivalents, preferably of about 1 to about 100 equivalents, more preferably of about 1 to about 10 equivalents, and in particular of about 1 to about 2 equivalents or of about 1 to about 1 .05 equivalents, of the total molar equivalents of acetoacetate groups of the polyalcohol comprising at least two acetoacetate groups.

[0110] In another embodiment, the molar amount of hydrogen source in the process is of about 0.1 to about 1 equivalents, preferably of about 0.2 to about 0.9 equivalents, more preferably of about 0.3 to about 0.7 equivalents, and in particular of about 0.4 to about 0.6 equivalents, of the total molar equivalents of acetoacetate groups of the polyalcohol comprising at least two acetoacetate groups.

[0111] In one embodiment, the step of reducing is performed in the presence of a buffer. In one embodiment, the buffer is selected form the group consisting of phosphate buffer such as K2HPO4 / KH2PO4, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hy- droxymethyl)aminomethane (TRIS) buffer, 3-(N-morpholino)propanesulfonic acid (MOPS), ethylenediaminetetraacetic acid (EDTA), and combinations thereof.

[0112] In one embodiment, the buffer is present in a concentration of about 0.1 mM to about 1 ,000 mM, preferably of about 1 mM to about 500 mM, more preferably of about 10 mM to about 300 mM. and in particular of about 20 mM to about 250 mM.

[0113] In one embodiment, the process is conducted at a pH of about 4 to about 9, preferably about 5 to about 8, and in particular about 6 to about 7.

[0114] In one embodiment, the process further comprises additives such as magnesium sulfate.

[0115] As aforementioned above, the invention further relates in a second aspect to a composition comprising a polyalcohol-based p-hydroxybuterate derived from an enzymatic reduction of a polyalcohol comprising at least two acetoacetate groups, wherein all of the p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in R-configuration or wherein all of the p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in S-configuration.

[0116] Particular embodiments (e.g. regarding the polyalcohol) are already above-outlined in connection with the inventive process and shall hold for the composition, as well. In the following, particular embodiments of the composition are described in further detail. It is to be understood that each embodiment is relevant on its own as well as in combination with other embodiments.

[0117] In one embodiment, the polyalcohol-based p-hydroxybuterate derived from an enzymatic reduction of polyalcohol comprising at least two acetoacetate groups is a polyalcohol-based p-hydroxybuter- ate obtained via the process of manufacturing a polyalcohol-based p-hydroxybuterate according to the present invention.

[0118] In one embodiment, the polyalcohol-based p-hydroxybuterate comprises no acetoacetate groups.

[0119] In another embodiment, the polyalcohol-based p-hydroxybuterate comprises the p-hydroxyl butyric acid ester groups and the acetoacetate groups in a molar ratio of about 0.1 :2 to about 2:0.1 , preferably of about 0.5:1 .5 to about 1 .5:0.5, and in particular of about 0.8:1 .2 to about 1 .2:0.8. In one embodiment, the polyalcohol-based P-hydroxybuterate comprises the P-hydroxyl butyric acid ester units and the acetoacetate groups in a molar ratio of about 0.1 :2 to about 2:0.1 , preferably of about 0.5:1 .5 to about 1 .5:0.5, and in particular of about 0.8:1 .2 to about 1 .2:0.8. Without being bound by any theory, the ratio of the p-hydroxyl butyric acid ester groups (or units) and the acetoacetate groups of the polyalcohol-based p-hydroxybuterate can be adjusted as necessary (e.g. via the process according to the present invention).

[0120] In one embodiment, all of the p-hydroxyl butyric acid ester units of the polyalcohol-based p-hydroxy- buterate are in R-configuration or all of the p-hydroxyl butyric acid ester units of the polyalcohol- based p-hydroxybuterate are in S-configuration.

[0121] In one embodiment, the composition further comprises the polyalcohol comprising at least two acetoacetate groups.

[0122] In one embodiment, the amount of the polyalcohol-based p-hydroxybuterate is at least about 20 wt.- %, preferably at least about 30 wt.-%, more preferably at least about 40 wt.-%, even more preferably at least about 50 wt.-%, still more preferably at least about 60 wt.-%, and in particular at least about 70 wt.-% or at least about 80 wt.-% or at least about 85 wt.-%, based on the total amount of the polyalcohol-based p-hydroxybuterate and the polyalcohol comprising at least two acetoacetate groups.

[0123] In one embodiment, the amount of the polyalcohol-based p-hydroxybuterate is in the range of about 20 to about 99 wt.-%, preferably of about 30 to about 98 wt.-%, more preferably of about 40 to about 97 wt.-%, even more preferably of about 50 to about 97 wt.-%, still more preferably of about 60 to about 96 wt.-%, and in particular of about 70 to about 95 wt.-% or of about 80 to about 95 wt.-% or of about 85 to about 95 wt.-%, based on the total amount of the polyalcohol-based p- hydroxybuterate and the polyalcohol comprising at least two acetoacetate groups.

[0124] In one embodiment, the composition further comprises acetone.

[0125] In one embodiment, the amount of the polyalcohol-based p-hydroxybuterate is at least about 20 wt.- %, preferably at least about 30 wt.-%, more preferably at least about 40 wt.-%, even more preferably at least about 50 wt.-%, still more preferably at least about 60 wt.-%, and in particular at least about 70 wt.-% or at least about 80 wt.-%, based on the total amount of the polyalcohol-based p- hydroxybuterate, the polyalcohol comprising at least two acetoacetate groups, and the acetone.

[0126] In one embodiment, the amount of the polyalcohol-based p-hydroxybuterate is in the range of about 20 to about 99 wt.-%, preferably of about 30 to about 98 wt.-%, more preferably of about 40 to about 97 wt.-%, even more preferably of about 50 to about 97 wt.-%, still more preferably of about 60 to about 96 wt.-%, and in particular of about 70 to about 95 wt.-% or of about 80 to about 95 wt.- %, based on the total amount of the polyalcohol-based P-hydroxybuterate, the polyalcohol comprising at least two acetoacetate groups, and the acetone.

[0127] It will be obvious for a person skilled in the art that these embodiments and items only depict examples of a plurality of possibilities. Hence, the embodiments shown here should not be understood to form a limitation of these features and configurations. Any possible combination and configuration of the described features can be chosen according to the scope of the invention. All embodiments and preferred embodiments described herein in connection with one particular aspect of the invention (e.g. the inventive preservative composition) shall likewise apply to all other aspects of the present inventions such as end-use formulations, uses or methods according to the present invention.

[0128] The present invention will be further illustrated by the following examples.

[0129] Examples

[0130] Preparation of the potassium phosphate buffer (250 mM, pH 7):

[0131] K2HPO4 (5.85 g) and KH2PO4 (3.95 g) were mixed in H2O (milliQ, 240.26 g).

[0132] Preparation of the NAD-solution:

[0133] NAD+(27.6 mg), NADP+(29.9 mg) and MgSO4 (6.2 mg) were mixed with the potassium phosphate buffer (36 ml).

[0134] Preparation of the substrate solution:

[0135] Butane-1 ,2,3,4-tetrayl tetrakis(3-oxobutanoate) (1 .84 g) was mixed with dimethyl sulfoxide

[0136] (6.17 ml).

[0137] Procedure for the enzymatic hydrogenation of polyalcohols:

[0138] The alcohol dehydrogenase (ADH, 40.3 mg) was mixed with the NAD-solution (3.6 ml) and the substrate solution (0.4 ml) in a reaction vial. Isopropanol (0.2 ml) was added for cofactor regeneration. The vial was inserted into a heating block and stirred for 18 hours at 32.5 °C. The conversion and enantioselectivity was measured with a chiral GC-method.

[0139] All used alcohol dehydrogenases are from E coli and were purchased from Johnson Matthey.

Claims

Claims1 . A process of manufacturing a polyalcohol-based p-hydroxybuterate, the process comprising the step of reducing a polyalcohol comprising at least two acetoacetate groups with an enzyme in the presence of a hydrogen source.

2. The process according to claim 1 , wherein the polyalcohol-based p-hydroxybuterate is a compound of formula 1whereinA is derived from an organic polyol with at least 2 hydroxyl groups, x is at least 1 , y is 0 or at least 1 , x + y is from 2 to the number of hydroxyl groups of the initial organic polyol A, n is 0 or 1 ; and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 23. The process according to claim 2, wherein the compound of formula 1 is a compound of formula 1-2, preferably a compound of formula 1-3,and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2-2,2-2.

4. The process according to claim 2 or 3, wherein in the compound of formula 1 y is at least 1 .

5. The process according to claim 2 or 3, wherein in the compound of formula 1 y is 0.

6. The process according to claim 1 , wherein the compound of formula 1 is a compound of formula 1-41-4 whereinA is derived from an organic polyol with at least 2 hydroxyl groups, x is at least 1 , y is 0 or at least 1 , x + y is from 2 to the number of hydroxyl groups of the initial organic polyol A, and n is 0 or 1 ; and the polyalcohol comprising at least two acetoacetate groups is a compound of formula 2-42-4.

7. The process according to claim 6, wherein the compound of formula 1-4 is a compound of formula 1-51-5 and the compound of formula 2-4 is a compound of formula 2-52-5.

8. The process according to any one of claims 1 to 7, wherein the organic polyol is selected from the group consisting of a linear or branched C2-12 alkyl substituted with at least 2 hydroxyl groups, a C3-8 cycloalkyl substituted with at least 2 hydroxyl groups, monosaccharides, sugar alcohols, sugar acids, and combinations thereof.

9. The process according to claim 8, wherein the linear or branched C2-12 alkyl substituted with at least 2 hydroxyl groups is selected from the group consisting of glycerol, trimethylolpropane, butanetriol, 2-methyl-propanetriol, pentanetriol, 3-methyl-pentanetriol, hexanetriol, pentaerythritol, butanetetrol, pentanetetrol, hexanetetrol, hexanepentol, and combinations thereof; and / or wherein the C3-8 cycloalkyl substituted with at least 2 hydroxyl groups is selected from the group consisting of cyclopentanetriol, cyclohexanetriol, cyclopentanetetrol, cyclohexanetetrol, and combinations thereof.

10. The process according to claim 8 or 9, wherein the monosaccharide is selected from tetroses, pentoses, hexoses, heptoses, and combinations thereof, preferably wherein the monosaccharide is selected from aldotetroses, ketotetroses, aldopentoses, ketopentoses, aldohexosen, ketohexoses, aldoheptoses, ketoheptoses, and combinations thereof; and / or wherein the monosaccharide is selected from the group consisting of erythrose, threose, erythrulose, ribose, arabinose, xylose, lyxose, desoxyribose, ketopentose, ribulose, xylulose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, n-acetyl-d-glucosamin, glucosamin, N-acetyl-D-galactosamin, fucose, rhamnose, chinovose, fructose, 2-desoxy-D-glucose, fluordesoxyglucose, 6-desoxyfructose, 1 ,6-dichlorfructose, 3,6-anhydrogalactose, 1-O-methylgalactose, 1-0- methyl-D-glucose, 1-O-methyl-D-fructose, 3-O-methyl-D-fructose, 6-O-methyl-D-galactose, sedoheptulose, mannoheptulose, L-glycero-D-manno-heptose, and combinations thereof.11 . The process according to any one of claims 8 to 10, wherein the sugar alcohol is selected from the group consisting of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, and combinations thereof; and / or wherein the sugar acid is selected from the group consisting of xylonic acid, gluconic acid, ascorbic acid, neuraminic acid, ketodeoxyoctonic acid, glucuronic acid, galacturonic acid, iduronic acid, mucic acid, saccharic acid, and combinations thereof.

12. The process according to any one of claims 8 to 11 , wherein the organic polyol is selected from the group consisting of glycerol, sorbitol, xylitol, mannitol, erythritol, maltitol, glucose, glucitol, ribulose, pentaerythritol, trimethylolpropane, and combinations thereof.

13. The process according to any one of claims 1 to 12, wherein the organic polyol has from 2 to 10 hydroxyl groups, preferably from 3 to 10 hydroxyl groups, more preferably from 3 to 8 hydroxyl groups such as from 3 to 7 hydroxyl groups, from 3 to 6 hydroxyl groups, from 3 to 5 hydroxyl groups, or from 3 to 4 hydroxyl groups.

14. The process according to any one of claim 1 to 13, the p-hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in form of predominantly in S-configuration or predominantly in R-configuration, preferably all p-hydroxyl butyric acid ester groups of the polyalcohol- based p-hydroxybuterate are in R-configuration.

15. The process according to any one of claims 1 to 14, wherein the process is conducted at a pressure of about 0.1 to about 2.0 bar, preferably of about 0.2 to about 1 .7 bar or of about 0.5 to about 1 .5 bar, and in particular of about 0.8 to about 1 .2 bar and / or wherein the process is conducted at a temperature of about 0 to about 45 °C, preferably of about 20 to about 45 °C, more preferably of about 28 to about 43 °C, still more preferably of about 30 to about 40 °C, and in particular of about 30 to about 35 °C.

16. The process according to any one of claim 1 to 15, wherein the hydrogen source is isopropanol, glucose, or sodium formate.

17. The process according to any one of claims 1 to 16, wherein the enzyme is selected from the group consisting of alcohol dehydrogenase enzyme, ketoreductase enzyme, and mixtures thereof.

18. The process according to any one of claims 1 to 17, wherein the process comprises nicotin- amide adenine dinucleotide and / or nicotinamide adenine dinucleotide phosphate, preferably in a concentration of about 0.001 to about 100 mM, more preferably of about 0.01 to about 10 mM, still more preferably of about 0.1 to 5 mM or of about 0.5 to about 3 mM.

19. The process according to any one of claims 1 to 17, wherein the step of reducing is performed in the presence of a buffer, preferably selected form the group consisting of phosphate buffer, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)ami- nomethane (TRIS) buffer, 3-(N-morpholino)propanesulfonic acid (MOPS), ethylenediaminetetraacetic acid (EDTA), and combinations thereof.

20. A composition comprising a polyalcohol-based p-hydroxybuterate derived from an enzymatic reduction of a polyalcohol comprising at least two acetoacetate groups, wherein all of the - hydroxyl butyric acid ester groups of the polyalcohol-based p-hydroxybuterate are in R-configuration or wherein all of the p-hydroxyl butyric acid ester groups of the polyalcohol-based P-hydroxybuter- ate are in S-configuration.