WO1994012651A1

WO1994012651A1 - Enzymatic synthesis of sugar alcohol esters

Info

Publication number: WO1994012651A1
Application number: PCT/DK1993/000396
Authority: WO
Inventors: Martin Barfoed; Jan Peter Skagerlind
Original assignee: Novo Nordisk A/S
Priority date: 1992-12-01
Filing date: 1993-12-01
Publication date: 1994-06-09
Also published as: AU5561494A; DK144292D0

Abstract

A method of preparing a compound of the general formula (I): R1-COO-X-O-R2, wherein R1-CO is the acyl group of a carboxylic acid which may be saturated or unsaturated, O-X-O is a saccharide moiety or a sugar alcohol moiety corresponding to a saccharide or a sugar alcohol of the general formula X(OH)2, and R<2> is hydrogen, acetyl, or an alkyl group.

Description

Enzymatic synthesis of sugar alcohol esters.

FIELD OF THE INVENTION

The present invention relates to a method for enzyme-catalyzed preparation of certain saccharide esters, alkyl glycoside esters, and sugar alcohol esters. Such esters are useful as surfactants.

BACKGROUND OF THE INVENTION

Surfactants constitute an extremely important class of industrial chemicals and have a wide variety of uses, for instance as detergents for washing purposes, as emulsifiers in food products and other products and as active ingredients in various personal care products such as shampoos, soaps or creams.

At the molecular level, surfactants are substances which are characterized by the presence of hydrophobic and hydrophilic regions within each individual surfactant molecule and which owe their ability to reduce surface tension to this particular structure. The combination of hydrophobic and hydrophilic regions within the same molecule may be obtained in many different ways, for instance by combining hydrophilic moieties such as sulphonic acid residues, quaternized ammonium moieties or glycerol moieties with hydrophobic moieties such as alkyl chains as is the case with the linear alkyl surfactants, the quarternized alkyl amines or the monoglycerides, respectively. When designing a surfactant molecule, the detailed molecular architecture of the compound is a major concern, care being taken to achieve a proper balance between the hydrophobic and the hydrophilic regions of the molecule as well as to achieve a favourable spatial arrangement of these individual regions of the molecule. However, also the possibility of producing the product by a high-yielding pro cess and on the basis of inexpensive and readily available raw materials is carefully considered. Finally, the environmental issues related to the eventual loading of the surfactant into the environment are matters of major concern. As a result of these considerations, much effort has been devoted to attempts to produce surfactants based on sugars and fatty acids, e.g. sugar esters. Such substances were expected to exhibit surfactant properties due to the hydrophilic properties of their sugar moiety and the hydrophobic properties of their fatty acid residue. The balance between the hydrophobic and the hydrophilic properties of such molecules can be adjusted by separate modification of the sugar moiety and/or the fatty acid moiety. Such surfactants could be produced under mild conditions from very inexpensive starting materials and, being prepared from and degradable into naturally occurring components, they would not constitute an environmental hazard.

One traditional method of preparing sugar esters, including glycoside esters, has been by transesterification. Thus, US 3,597,417 discloses the preparation of alkyl monoglycoside esters by transesterification in a two-step process by reacting a glycoside with a short-chain ester and subsequently with a fatty acid ester. Another method is disclosed in US 2,759,922 which relates to a process for producing esterified glycosides, e.g. methyl glycoside, by reacting the glycoside with a fatty acid at a temperature of 160 - 300°C.

In spite of the strong interest in producing sugar esters of fatty acids, it has been found rather difficult to produce these esters by conventional synthesis procedures. This may partly be explained as being due to the presence of several chemically similar groups in the sugar molecules which may therefore be esterified at many different positions and to varying degrees when exposed to esterification reagents. Therefore, reaction mixtures resulting from attempts to prepare sugar esters by traditional chemical synthesis are inhomogeneous in that they are composed of mixtures of compounds different in their degree of esterification and in the position of the acyl groups on the sugar moiety. This may cause differences in the surfactant properties of the compounds. As, additionally, the preparation of sugar esters by conventional chemical synthesis has been found to be rather cost-intensive, the currently available sugar esters prepared by these methods have found limited application only. In view of the difficulties encountered in the production of sugar esters by chemical synthesis and in view or the attractiveness of these compounds as surfactants, alternative methods have been suggested for the production of esterified sugars. Thus, one interesting method involves the use of enzymes which are known to be highly regioselective and enan- tioselective so that they may be employed for the selective esterification of one or more hydroxy groups on the sugar molecules. Such enzymatic processes may exploit cheap starting materials which means that the resulting sugar esters are inexpensive even though they are of a high quality.

The attempts to develop efficient enzymatic syntheses of sugar esters have so far been of limited success. Thus, Sweers and Wong (J. Am. Chem. Soc., 108 (1986), 6421 - 2) briefly discuss the regioselective esterification of sugars, e.g. methyl glycoside, with pentanoic acid in the presence of a Candida cylindracea lipase and report that the yield of this process was very low (2 - 3%). Similarly, US 4,614,718 discloses the preparation of sugar or sugar alcohol esters by reacting the sugar or sugar alcohol with a higher fatty acid in finely divided or emulsified form in the presence of a lipase until an equilibrium is reached. A large amount of water is used as solvent and as a result of this, the equilibrium of the reaction cannot be shifted towards a favourable yield of the product. Furthermore, the reaction is slow even when large amounts of the enzyme are employed.

One reason why poor yields are obtained and/or long reaction times are required in the known enzymatic processes is the considerable difference in polarity between the sugar compo- nent and the fatty acid component which makes it difficult to find a solvent in which both are soluble. When using water as a solvent as taught in US 4,614,718, the fatty acid is not dissolved. This results in an inefficient reaction and a low utilization of the fatty acid reagent. Few solvents are available which will dissolve both sugars and fatty acids and such solvents (e.g. dimethylformamide) will generally inactivate the enzyme. Also, in most cases such solvents are toxic, constituting an environmental hazard.

Japanese patent application having publication No. 62-195,292 discloses a method of preparing sugar esters or sugar alcohol esters by reacting a sugar or a sugar alcohol with a fatty acid in an aqueous medium in the presence of a lipase after which the water is gradually removed and incubation is continued. Japanese patent application having publication No. 62-289,190 discloses a method of preparing sugar esters or sugar alcohol esters by mixing a sugar ester or a sugar alcohol, a fatty acid and a lipase and adding only a minor amount of water to the reaction mixture. Japanese patent application having publication No. 63-112,993 discloses a method of preparing sugar esters or sugar alcohol esters by reacting an acetylated sugar or an acetylated sugar alcohol with a fatty acid in an organic solvent in the presence of a lipase.

A method of providing alkylglycoside esters with fatty acids by enzyme catalyzed acylation of the corresponding alkyl- glycosides with the pertinent fatty acids or lower alkyl- esters thereof is described in WO 89/01480 and in WO 90/09451. All embodiments illustrated in these two documents use an immobilized enzyme catalyst. The reaction times are very long, often several days. An object of the present invention is to provide a fast process for the production of saccharide esters, alkylglyco- side esters, and sugar alcohol esters with carboxylic acids in high yield and purity from inexpensive starting materials by enzymatic catalysis without the use of toxic solvents.

SUMMARY OF THE INVENTION

Accordingly, in its broadest aspect the present invention relates to a method of preparing a compound of the general formula (I)

R¹-COO-X-O-R² (I) wherein R¹-CO is the acyl group of a carboxylic acid which may be saturated or unsaturated, O-X-O is a saccharide moiety or a sugar alcohol moiety corresponding to a saccharide or a sugar alcohol of the general formula X(OH)₂, and R² is hydrogen or a lower alkyl group, the method comprising reacting a saccharide or a glycoside or a sugar alcohol of the general formula (II)

HO-X-OR² (Il) wherein O-X-O and R² are as defined above, with a carboxylic acid of the general formula (III)

R¹-COOH (III) wherein R¹-CO is as defined above, in the presence of a detergent, a soluble enzyme catalyst, and water at a temperature at which the carboxylic acid is liquid to form the desired product which is subsequently isolated by methods known per se. In a first preferred embodiment of the invention, hexanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, heptanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, octanoic acid is used as the carboxylic acid of the general formula (III). In a further preferred embodiment of the invention, nonanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, decanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, undecanoic acid is used as the carboxylic acid of the general formula (lll).

In a further preferred embodiment of the invention, dodecanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, tetradecanoic acid is used as the carboxylic acid of the general formula (III). In a further preferred embodiment of the invention, hexadecanoic acid is used as the carboxylic acid of the general formula (III). In a further preferred embodiment of the invention, octadecanoic acid is used as the carboxylic acid of the general formula (lll).

In a further preferred embodiment of the invention, eicosanoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, docosanoic acid is used as the carboxylic acid of the general formula (III). In a further preferred embodiment of the invention, 9-octadecenoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, 9,12- octadecadienoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, 9,12,15- octadecatrienoic acid is used as the carboxylic acid of the general formula (III).

In a further preferred embodiment of the invention, coconut fatty acid, i.e. a mixture of fatty acids as found in coconuts, is used as the carboxylic acid component of the general formula (III).

In a further preferred embodiment of the invention, tallow fatty acid, i.e. a mixture of fatty acids as found in tallow, is used as the carboxylic acid component of the general formula (III).

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is a monosaccharide. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is an aldohexose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is glucose. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is galactose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is mannose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is a ketohexose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is fructose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is an aldopentose. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is ribose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is arabinose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is xylose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is an oligosaccharide.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is a disaccharide. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is sucrose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is lactose. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is maltose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is cellobiose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is isomaltose.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is a sugar alcohol.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is xylitol. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is ribitol.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is glucitol.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is mannitol.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is inositol.

In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is galactitol. In a further preferred embodiment of the invention, the compound of the general formula X(OH)₂ is allitol.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is an alkyl group having from 1 to 6 carbon atoms.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is a methyl group.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is an ethyl group. In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is a propyl group.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is an isopropyl group.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is a butyl group.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is an isobutyl group.

In a further preferred embodiment of the invention, the R² of formula (I) and formula (II) is an acetyl group. In a further preferred embodiment of the invention, the detergent used is a salt of the acid R¹-COOH.

In a further preferred embodiment of the invention, the salt of the acid R¹-COOH used as detergent has a monovalent cation. In a further preferred embodiment of the invention, the detergent used is the sodium salt of the acid R¹-COOH. In a further preferred embodiment of the invention, the detergent used is the potassium salt of the acid R¹-COOH.

In a further preferred embodiment of the invention, the detergent used is an ammonium salt of the acid R¹-COOH, the ammonium ion optionally being substituted with one or more groups which can be any combination of alkyl groups, aryl groups, or aralkyl groups.

In a further preferred embodiment of the invention, the detergent used has the general formula (I) as described above.

In a further preferred embodiment of the invention, the enzyme catalyst is dissolved in the reaction mixture.

In a further preferred embodiment of the invention, the enzyme catalyst is a hydrolase. In a further preferred embodiment of the invention, the enzyme catalyst is a lipase.

In a further preferred embodiment of the invention, the enzyme catalyst is an esterase.

In a further preferred embodiment of the invention, the enzyme catalyst is a lipase produced by species of Mucor, Humicola, Pseudomonas, or Candida.

In a further preferred embodiment of the invention, the enzyme catalyst is a lipase produced by Candida antarctica, DSM 3855, DSM 3908 or DSM 3909, Pseudomonas cephacia, DSM 3959, Humicola lanuαinosa, DSM 3819 or DSM 4109, Humicola brevispora, DSM 4110, Humicola brevis var. thermoidea, DSM 4111, or Humicola insolens, DSM 1800. In a further preferred embodiment of the invention, the enzyme catalyst is a lipase produced by species of Hyphozyma.

In a further preferred embodiment of the invention, the enzyme catalyst is a lipase produced by Hypozvma sp. LF132, CBS 648.91.

In a further preferred embodiment of the invention, the water contents in the reaction mixture is in the range of from 0.1 to 50 per cent by weight, more preferred from 0.1 to 10 per cent by weight, most preferred from 0.1 to 5 per cent by weight.

DETAILED DESCRIPTION OF THE INVENTION

Compared to the processes disclosed in WO 89/01480 and in WO 90/09451 for enzymatically preparing sugar esters or sugar alcohol esters, the reaction times required to prepare sugar esters or alkyl glycoside esters of formula (I) by the present process are significantly lower. The present process therefore represents an important economic advantage. Furthermore, it results in a high yield of regiospecifically esterified monoesters (e.g. the 6-0 monoesters) when the starting materials of formula (II) are alkyl glycosides. The use of sugars as starting materials may lead to the formation of a mixture of mono-, di-, tri-, etc. esters. The production of monoesters of the alkylglycosides of formula (I) in a high yield is particularly desirable as these compounds have been found to be very useful detergents, as demonstrated e.g. in WO 89/01480 and in WO 90/09451.

When the saccharide corresponding to the starting material of formula (II) is a pentose or a hexose, it is preferably in cyclic (furanose or pyranose) form. According to the method of the invention, the carboxylic acid of formula (III) used as starting material is most often used in an amount which is equimolar with or in excess of the amount of the saccharide or glycoside of formula (II). The amount of detergent added to the reaction mixture is usually in the range of from 0.05% to 25% more preferred from 0.05% to 5% by weight of the carboxylic acid. When the detergent used is a salt of the carboxylic acid of formula (III) the carboxylic acid salt may be provided in situ by the addition of an equivalent amount of a suitable metal salt or amine salt (e.g. the hydroxide or the carbonate) to the reaction mixture to which initially has been added an excess of the carboxylic acid which corresponds to the desired amount of the salt to be formed in situ. The ratio between the amount of the carboxylic acid of formula (III) and the corresponding salt used as detergent can be adjusted during the process by audition of an appropriate acid stronger than the acid of formula (III) or of an appropriate base e.g. the hydroxide or the carbonate of the desired cation. Also, if desired, the water initially added to the reaction mixture may comprise a buffer.

By the method according to the invention, the reaction of the saccharide or glycoside of the formula (II) with the carboxylic acid of the formula (III) proceeds in a microemulsion comprising these starting materials, an enzyme and a detergent. In order to ensure a satisfactory reactivity of the enzyme at least a minor amount of water must also be present. If larger amounts of water is present, the reaction mixture may appear as two separate phases of which one is the microemulsion and the other one is a water phase. Microemulsions are systems which i.a. are characterized by being monophasic, transparent, isotropic, and - contrary to disperse emulsions - they are also kinetically and thermodynamically stable. These last-mentioned stability properties implies that microemulsions form spontaneously when the components are mixed. When the reaction is carried out as preferred using a dissolved enzyme in a microemulsion, there is a very intimate contact between the enzyme and the substrate and thus a very high rate of reaction can be achieved. Accordingly, preferred compounds of the formula (I) prepared by the process of the invention may be selected from the group consisting of glucose octanoate, glucose nonanoate, glucose decanoate, glucose dodecanoate, glucose tetradecanoa- te, glucose hexadecanoate, glucose octadecanoate, glucose eicosanoate, glucose docosanoate, glucose cis-9-octadecenoate, glucose cis,cis-9,12-octadecadienoate and glucose cis,cis,cis-9,12,15-octadecatrienoate.

Other preferred compounds of the formula (I) prepared by the process of the invention may be selected from the group consisting of ethyl 6-O-octanoylglucoside, ethyl 6-O-nonanoyl- glucoside, ethyl 6-O-decanolyglucoside, ethyl 6-O-dodecano- ylglucoside, ethyl 6-O-tetradecanoylglucoside, ethyl 6-O-hex- adecanoylglucoside, ethyl 6-O-octadecanoylglucoside, ethyl 6- O-eicosanoylglucoside, ethyl 6-O-docosanoylglucoside, ethyl 6-O-cis-9-octadecenoylglucoside, ethyl 6-O-cis,cis-9,12- octadecadienoylglucoside and ethyl 6-O-cis,cis,cis-9,12,15- octadecatrienoylglucoside.

Other preferred compounds of the formula (I) prepared by the process of the invention may be selected from the group consisting of propyl 6-O-octanoylglucoside, propyl 6-O-nonanoylglucoside, propyl 6-O-decanolyglucoside, propyl 6-O-dodecanoylglucoside, propyl 6-O-tetradecanoylglucoside, propyl 6-O- hexadecanoylglucoside, propyl 6-O-octadecanoylglucoside, propyl 6-O-eicosanoylglucoside, propyl 6-0-docosanoylgluco- side, propyl 6-O-cis-9-octadecenoylglucoside, propyl 6-O- cis,cis-9,12-octadecadienoylglucoside and propyl 6-O- cis,cis,cis-9,12,15-octadecatrienoylglucoside. Further preferred compounds of formula (I) prepared by the process of the invention may be selected from the group consisting of methyl 6-O-octanoylglucoside, methyl 6-O-nonanoyl- glucoside, methyl 6-O-decanolyglucoside, methyl 6-O-dodeca- noylglucoside, methyl 6-O-tetradecanoylglucoside, methyl 6-0- hexadecanoylglucoside, methyl 6-O-octadecanoylglucoside, methyl 6-O-eicosanoylglucoside, methyl 6-O-docosanoylglucoside, methyl 6-O-cis-9-octadecenoylglucoside, methyl 6-O- cis,cis-9,12-octadecadienoylglucoside and methyl 6-O- cis,cis,cis-9,12,15-octadecatrienoylglucoside.

Still further preferred compounds of formula (I) prepared by the process of the invention may be selected from the group consisting of the esters corresponding to the esters described in the four preceding paragraphs, but derived from galactose, from fructose or from mannose instead of glucose.

Enzymes which may be useful as catalysts in the process of the invention are those which catalyze the hydrolysis of ester bonds, i.e. hydrolases. Such enzymes may be lipases or esterases, in particular lipases which may be defined as enzymes catalyzing reactions involving ester bonds, e.g. hydrolysis, synthesis and/or exchange of ester bonds. Lipases which may be employed in the present process may be pancreatic lipase or microbial lipases produced, for instance, by strains of Aspergillus, Enterobacterium, Chromobacterium, Geotricium or Penicillium. Preferred lipases for use according to the invention are those produced by species of Mucor (e.g. Lipozyme™), Humicola, Pseudomonas or Candida.

Particularly preferred lipases are those produced by the following strains of microorganisms, all of which have been deposited in Deutsche Sammlung von Mikroorganismen (except Hyphozyma sp. LF132 which is deposited in Centraalbureau voor Schimmelcultures) in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Candida antarctica, deposited on 29 September 1986, with the number DSM 3855, and on 8 December 1986, with the numbers DSM 3908 and DSM 3909.

Pseudomonas cephacia, deposited on 30 January 1987, with the number 3959.

Humicola lanuginosa, deposited on 13 August 1986 and 4 May, with the deposit numbers 3819 and 4109, respectively.

Humicola brevispora, deposited on 4 May 1987, with the deposit number DSM 4110.

Humicola brevis var. thermoidea, deposited on 4 May 1987, with the deposit number DSM 4111.

Humicola insolens, deposited on 1 October 1981, with the deposit number DSM 1800.

Hyphozyma sp. LF132, deposited on 12 November 1991, with the deposit number CBS 648.91.

Currently preferred lipases are those produced by Candida antarctica, DSM 3855, DSM 3908 and DSM 3909. These enzymes may be produced by the process disclosed in WO 88/02775. Briefly, the Candida strains in question are cultivated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen sources as well as essential minerals, trace elements etc., the medium being composed according to established practice in the art. After cultivation, liquid enzyme concentrates may be prepared by removing in- soluble materials, e.g. by filtration or centrifugation, after which the broth is concentrated by evaporation or reverse osmosis.

Additional lipases may be obtained from the following strains which are publicly available without restriction from the Centraalbureau voor Schimmelculturen (CBS), American Type Culture Collection (ATCC), Agricultural Research Culture Collection (NRRL) and Institute of Fermentation, Osaka (IFO) with the following deposit numbers: Candida antarctica, CBS 5955, ATCC 34888, NRRL Y-8295, CBS 6678, ATCC 28323, CBS 6821 and NRRL Y-7954; Candida tsukubaensis, CBS 6389, ATCC 24555 and NRRL Y-7795; Candida auriculariae, CBS 6379, ATTC 24121 and IFO 1580; Candida humicola, CBS 571, ATCC 14438, IFO 0760, CBS 2041, ATCC 9949, NRRL Y-1266, IFO 0753 and IFO 1527; and Candida foliorum, CBS 5234 and ATCC 18820. It is known to produce lipase by recombinant DNA techniques, cf. for instance European patent application having publication No. 238,023 or European patent application having publication No. 305,216. Recombinant lipases may also be employed for the present purpose. When employed in the method of the present invention, the enzyme is in a soluble state. The amount of enzyme used relative to the other components of the reaction mixture may vary within wide limits. Usually, however, 1 to 200 Lipase Units, preferably 1 to 100 Lipase Units are used per gram of reaction mixture. One Lipase Unit (LU) is the amount of enzyme which liberates one micromole of butyric acid per minute from tributyrin as a substrate at 30°C in a pH-stat at pH 7 (a detailed description of the assay (AF 95) is available from Novo Nordisk A/S upon request). The lower limit of the temperature range within which the reaction is carried out is selected so as to ensure that the reaction mixture is liquid and it thus depends on the melting point of the specific carboxylic acid of formula (III) employed. The upper limit of the range is selected so as to ensure that the heat does not inactivate the enzyme employed.

During the reaction or when the reaction is complete, the water phase - if any - is separated from the microemulsion which contains most or all of the desired product. The product may in some cases be a solid which can be separated from the excess of carboxylic acid by filtration. An adjustment of the temperature may help to break the microemulsion. Excess carboxylic acid adhering to the filter cake can be removed by short path distillation or by other methods known per se. When the product is isolated in this manner, most of the enzyme will be in the filtrate and there is a possibility that it can be reused. In other cases, the product can be isolated or further purified by extraction or by distillation, e.g. by short path distillation.

When the saccharide X(OH)₂ corresponding to the starting material of formula (II) is a monosaccharide, it can be a pentose or hexose, but preferably it is a hexose. Out of economical considerations, the monohexose is preferably glucose, galactose or fructose, i.e. when the starting material is a glycoside, it is preferably a glucoside, a galactoside or a fructoside. The starting material of formula (II) may be in the furanose or in the pyranose form as indicated above. Due to the ease of preparation, the most accessible of the isomers is preferred, e.g. a glucopyranoside, a galactopyranoside or a fructofuranoside.

When the starting material of formula (II) is an alkylglycoside corresponding to a monohexose, the ester bond linking the acyl group to the hexose moiety of the product is preferably attached in the 6-position of the hexose moiety.

The present invention is further illustrated in the following example which is not in any way intended to be limiting to the scope of the invention for which protection is sought.

EXAMPLES General procedures

HPLC analysis was performed on a Shimadzu LC-4A instrument (refractive index detector) equipped with a Merck LiChrosorb NH₂-column. 96% ethanol was used as eluent. EXAMPLE 1

Preparation of ethyl 6-O-dodecanoyl-D-glucopyranoside

Dodecanoic acid (118 g, 0.59 mol) was melted and heated with stirring to 85°C. Potassium dodecanoate (6.4 g, 0.027 mol) was dissolved in the melt which was subsequently cooled to 50°C. Then, crude ethyl-D-glucopyranoside (122 g, 0.59 mol, obtained as described in WO 89/01480) and Candida antarctica component B lipase (obtainable from Novo Nordisk A/S, Bagsvaerd, Denmark, 25000 lipase units (LU), 256 LU/mg) were dissolved in water (83 ml). The aqueous solution was heated to 50°C and added to the above-mentioned melt. The resulting reaction mixture which comprised two clear phases was stirred at 50°C for 15 minutes after which the stirring was stopped. When the phases had separated a sample of the top phase was withdrawn for analysis by HPLC. By comparison with an authentic sample obtained as described in WO 89/01480 it was found that the conversion in the reaction mixture after 15 minutes was 16%.

EXAMPLE 2

Preparation of ethyl 6-O-decanoyl-D-glucopyranoside

Experiment A:

Ethyl-D-glucopyranoside (4 g, 19.2 mmol) and decanoic acid (4 g, 23.2 mmol) were heated together to 50°C under mechanical stirring. Sodium decanoate (1 g, 5.1 mmol) was added and the reaction was then started by adding Candida antarctica component B lipase (obtainable from Novo Nordisk A/S, Bagsvaerd, Denmark, 1 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50°C was maintained for 44 hours, samples for analysis (HPLC) being withdrawn after 1, 3.5, 20 and 44 hours. The results (% conversion of ethyl-D-glucopyranoside to the monoester, ethyl 6-O-decanoyl-D-glucopyranoside) are given in Table 1. Experiment B:

This experiment was performed similarly to Experiment A except that the reaction was carried out in vacuum (0.2 bar) in order to remove as much as possible of the water formed during the reaction. The results (% conversion of ethyl-Dglucopyranoside to the monoester, ethyl 6-O-decanoyl-D-glucopyranoside) are given in Table 1.

† This sample further contained 3% of a mixture of the diester and the triester.

The experiments demonstrate the advantage of reducing the amount of water present in the reaction mixture.

EXAMPLE 3

Preparation of xylitol decanoate

Xylitol (0.5 g, 3.3 mmol) and decanoic acid (4 g, 23.2 mmol) were heated together to 50 °C under mechanical stirring. Sodium decanoate (1 g, 5.1 mmol) was added and the reaction was then started by adding Candida antarctica component B lipase (0.5 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50°C was maintained for 20 hours. At this point HPLC analysis indicated 80% conversion of the sugar to a mixture of the monoester (39%), the diester (11%), and the triester (30%), respectively. EXAMPLE 4

Preparation of sorbitol decanoate

Sorbitol (0.5 g, 2.7 mmol) and decanoic acid (4 g, 23.2 mmol) were heated together to 50°C under mechanical stirring. Sodium decanoate (1 g, 5.1 mmol) was added and the reaction was then started by adding Candida antarctica component B lipase (0.5 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50°C was maintained for 20 hours. At this point HPLC analysis indicated 20% conversion of the sugar.

EXAMPLE 5

Preparation of mannose decanoate

Mannose (0.5 g, 2.8 mmol) and decanoic acid (4 g, 23.2 mmol) were heated together to 50°C under mechanical stirring. Sodium decanoate (1 g, 5.1 mmol) was added and the reaction was then started by adding Candida antarctica component B lipase (0.5 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50°C was maintained for 20 hours. At this point HPLC analysis indicated 22% conversion of the sugar.

EXAMPLE 6

Preparation of ethyl 6-O-decanoyl-D-glucopyranoside

Ethyl-D-glucopyranoside (4 g, 19.2 mmol) and decanoic acid (5.4 g, 31.3 mmol) were heated together to 50°C under mecha- nical stirring. 6-O-decanoyl-D-glucopyranoside (0.28 g, 0.8 mmol, obtained as described in WO 89/01480) was added and the reaction was started by adding Candida antarctica component B lipase (0.33 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50°C in vacuum (0.2 bar) was maintained for 24 hours. At this point HPLC analysis of a sample indicated 40% conversion of the ethyl-D-glucopyranoside to ethyl 6-O-decanoyl-D-glucopyranoside.

The structure of ethyl 6-O-decanoyl-D-glucopyranoside was confirmed by NMR spectroscopy using a 400 MHz Bruker apparatus. The ¹H-NMR spectroscopic data for a sample of the product is given below. In comparison with the NMR spectra of decanoic acid and ethyl-D-glucopyranoside there are new peaks at 4.2 and 4.3 which can be ascribed to an ester function at the 6-position of the sugar moiety. ¹H-NMR (CDCl₃), ppm: 0.9, 1.2, 1.6, 2.2, 3.3, 3.5, 3.7, 3.9, 4.1, 4.2, 4.3, 4.8.

EXAMPLE 7

Preparation of ethyl 6-O-decanoyl-D-glucopyranoside

Ethyl-D-glucopyranoside (4 g, 19.2 mmol) and decanoic acid (4 g, 23.2 mmol) were heated together to 50 °C under mechanical stirring. 6-O-decanoyl-D-glucopyranoside (0.82 g, 2.3 mmol, obtained as described in WO 89/01480) was added and the reaction was started by adding Candida antarctica component B lipase (1.9 ml of an aqueous solution containing 5000 LU/ml). Stirring and heating to 50 °C in vacuum (0.2 bar) was main- tained for 24 hours. At this point HPLC analysis of a sample indicated 80% conversion of the ethyl-D-glucopyranoside to ethyl 6-O-decanoyl-D-glucopyranoside.

The structure of the product was confirmed by NMR spectroscopy as described in Example 6.

Claims

1. A method of preparing a compound of the general formula (I)

R¹-COO-X-O-R² (I) wherein R¹-CO is the acyl group of a carboxylic acid which may be saturated or unsaturated, O-X-O is a saccharide moiety or sugar alcohol moiety corresponding to a saccharide or a sugar alcohol of the general formula X(OH)₂, and R² is hydrogen, acetyl, or an alkyl group, characterized in that a saccharide or a glycoside or a sugar alcohol of the general formula (II)

HO-X-OR² (II) wherein O-X-O and R² are as defined above, a carboxylic acid of the general formula (III) R¹-COOH (III) wherein R¹-CO is as defined above, a detergent, a soluble enzyme catalyst, and water are mixed and allowed to react at a temperature at which the carboxylic acid is liquid to form the desired product which is subsequently isolated by methods known per se.

2. A method according to claim 1, characterized in that the carboxylic acid of the general formula R¹-COOH is selected from the group consisting of hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, 9-octadecenoic acid, 9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid, coconut oil fatty acid and tallow fatty acid.

3. A method according to claim 1 or 2, characterized in that the compound of the general formula X(OH)₂ is a monosaccharide.

4. A method according to claim 3, characterized in that the monosaccharide of the general formula X(OH)₂ is an aldohexose.

5. A method according to claim 4, characterized in that the aldohexose is selected from the group comprising glucose, galactose, and mannose.

6. A method according to claim 3, characterized in that the monosaccharide of the general formula X(OH)₂ is a ketohexose.

7. A method according to claim 6, characterized in that the ketohexose is fructose.

8. A method according to claim 3, characterized in that the compound of the general formula X(OH)₂ is an aldopentose.

9. A method according to claim 8, characterized in that the aldopentose is selected from the group comprising ribose, arabinose, and xylose.

10. A method according to claim 1 or 2, characterized in that the compound of the general formula X(OH)₂ is an oligosaccharide.

11. A method according to claim 10, characterized in that the oligosaccharide of the general formula X(OH)₂ is a disaccharide.

12. A method according to claim 11, characterized in that the disaccharide is selected from the group comprising sucrose, lactose, maltose, cellobiose, and isomaltose.

13. A method according to claim 1 or 2, characterized in that the compound of the general formula X(OH)₂ is a sugar alcohol.

14. A method according to claim 13, characterized in that the sugar alcohol of the general formula X(OH)₂ is sorbitol.

15. A method according to any one of the preceding claims, characterized in that R² is an alkyl group having from 1 to 6 carbon atoms.

16. A method according to claim 15, characterized in that R² is methyl, ethyl, propyl, isopropyl, butyl, or isobutyl.

17. A method according to any one of the claims 1 to 14, characterized in that R² is an acetyl group.

18. A method according to any one of the preceding claims, characterized in that the detergent used is a salt of the acid R¹-COOH.

19. A method according to claim 18, characterized in that the salt of the acid R¹-COOH has a monovalent cation.

20. A method according to claim 19, characterized in that the salt of the acid R¹-COOH is the sodium salt or the potassium salt.

21. A method according to claim 19, characterized in that the salt of the acid R¹-COOH is a salt with an ammonium ion which is optionally substituted with one or more groups which can be alkyl groups, aryl groups, or aralkyl groups.

22. A method according to any one of the claims 1 to 17, characterized in that the detergent used is a compound of the general formula (I) as defined in claim 1.

23. A method according to any one of the preceding claims, characterized in that the enzyme catalyst is a hydrolase.

24. A method according to claim 23, characterized in that the hydrolase is a lipase or an esterase.

25. A method according to claim 24, characterized in that the lipase is one produced by species of Mucor, Humicola, Pseudomonas or Candida.

26. A method according to claim 25, characterized in that the lipase is one produced by Candida antarctica, DSM 3855, DSM 3908 or DSM 3909, Pseudomonas cephacia, DSM 3959, Humicola lanuqinosa, DSM 3819 or DSM 4109, Humicola brevispora, DSM 4110, Humicola brevis var. thermoidea, DSM 4111, or Humicola insolens, DSM 1800.

27. A method according to claim 24, characterized in that the lipase is one produced by species of Hyphozyma.

28. A method according to claim 25, characterized in that the lipase is one produced by Hypozyma sp. LF132, CBS 648.91.

29. A method according to any one of the preceding claims, characterized in that the water contents in the reaction mixture is in the range of from 0.1 to 50, preferably from 0.1 to 10, most preferred from 0.1 to 5 per cent by weight.

30. Any novel feature or combinatino of features as herein described.