DK159883B - ENZYMATIC PROCEDURE FOR PREPARING EPOXY SUBSTITUTED ALDOS OR KETOSES - Google Patents

Description

in

DK 159883 B

This invention relates to a particular process for the preparation of known and novel carbohydrates bearing an epoxy group such as a glucosidic aglycone or otherwise specified in a particular process for the preparation of epoxy-substituted aldoses or ketoses having the formula I as set forth in the preamble of claim 1. .

Epoxides are a class of organic compounds which are of great practical importance as intermediates in organic synthesis and as a class of compounds which exhibit many useful properties. As a result, many methods have been devised over the years for the synthesis of epoxides, described in recognized textbooks in organic chemistry, such as e.g. in Comprehensive Organic Chemistry published by Barton and Ollis and published by Pergamon Press, 1984. In principle, two main methods are used for the synthesis of a given desired molecule containing an epoxy group: Either the epoxy group is formed on the basis of appropriate fraction groups found in a precursor for the desired molecule, such as a group containing a carbon-carbon double bond, or the epoxide is introduced into the desired molecule by a condensation reaction in which one of the reactants carries an epoxy group. The present invention is of the latter type.

A major difficulty inherent in organic synthesis of epoxides arises from the reactivity of the epoxy group. Under many different conditions, epoxides will react with solvents and a large number of organic functional groups, so one consequence of which is that introducing an epoxy group into a complex, polyfunctional organic molecule can be an extremely difficult, if not impossible, task.

An example of this kind is related to carbohydrate molecules bearing an epoxy group in the glucosidic aglycon, according to formula I. Due to the presence of many reactive groups in such molecules, the low solubility of the compounds in most organic solvents and the sensitive nature of the molecules is

DK 159883 B

2 it is a difficult and often very slow task to prepare such compounds by organic synthesis. An example described by J.E.G. Barnett and A. Ralph in Carbohydr. Res. 17 (1971), 231, relates to the synthesis of 2,3-epoxy-propyl-g-D-glucopyranoside, formula II.

HO -i

A

) -0 O-CH - CH - CH-, (II) HO 1

OH

A synthesis of carbohydrate molecules upon which an epoxy group is found comprises a fairly wide range of synthesis steps, starting from readily available starting materials.

In recent years there has been a growing interest in the potential of enzymes as organic catalysts. Since these catalysts are unique in many respects, they have many potential uses in the organic synthesis. Eg. For example, the enzymes enable the conducting of organic reactions under extremely mild conditions and are often substrate selective, for example. enables selective conversion of a single group from several chemically related groups into organic molecules or the synthesis of optically pure organic components from racemic starting materials.

A group of enzymes that have attracted some interest as organic catalysts are enzymes capable of cleaving bonds at the C-1 position (aldoses) 30 or the C-2 position (ketoses) of a carbohydrate. Organic chemists have been interested in these enzymes because they allow the execution of certain chemical reactions at the enzymatic cleavage positions.

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The use of β-galactosidases for the synthesis of galactosides illustrates how enzymes capable of cleaving bonds at the C-1 position (aldoses) or the C-2 position (ketoses) of carbohydrates can be used for organic synthesis. In J. Biol. Chem. 248 (1973), 6571-6574, describes T.J. Silhavy et al., How (2R) -glyceryl BD-galactopyranoside (Scheme 1, Formula III) can be synthesized on the basis of lactose and isopropylidene glycerol condensed to the corresponding galactoside (Scheme 1, Formula IV) 10 to effect the 8-galactosidase produced by E. coli and then cleaved to the desired product of formula III by acid catalysis, as per Scheme 1.

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Scheme 1 4 ch3 H2CO-C-CH3

5 I I

GAL-GLU + HC - 0 -3 -: ---.

In β-galactosidase 'h2c-oh ch3 10 I H +

h2co-c-ch3 H

I I

GAL-0-CH2 “CH-0 (IV)

GAL-O-CH 2 -CHOH-CH 2 OH

(III) GAL-GLU denotes lactose and GAL denotes galactose.

Similarly, T. Satoh et al. in Chem.Pharm.Bull 32 (1984), 1183 - 1187, the use of lactase from Coloromyces 20 fragilis for the synthesis of a series of galactosides.

As shown in Scheme 2 below, these chemists make use of aryl glucosides as a starting material for an enzymatic pathway catalyzed exchange reaction for the synthesis of a series of alkyl glucosides. It appears from this publication that in principle the same reaction can be carried out with widely different radicals R.

Scheme 2

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5 HO — i! "0 <r \ Γ 0H 1 /

10 I LACTASE

HO> _0 / \

/ \ ^ OR ^ ArOH

15 h ^ __ X

HO -Γ

OH

Ar = aryl R = alkyl

In the process of the invention, enzymes are used for the synthesis of carbohydrates of formula I which carry at their C-1 position (aldoses) or C-2 position (ketoses) an epoxy group. Thus, it has surprisingly been found that 25 molecules carrying a highly reactive epoxy group can be used in enzymatic reactions and that the epoxy group can be transferred to another organic molecule without disrupting or inactivating the enzyme despite the enzyme containing numerous functional groups known to react with 30 epoxides.

The method according to the invention is characterized by the method of claim 1.

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Thus, if SUGAR is the o-glucose molecule, the enzyme to be used is α-glucosidase, if SUGAR is the β-galactose molecule, it is the enzyme to be used, β-galactosidase, etc. These enzymes not only exhibit a cleavage activity, but also a corresponding transfer activity in connection with the same position as the cleavage position.

Thus, a defined SUGAR radical in the context of this invention can always be associated with an enzyme having a defined activity, as shown in the table set forth below, which is for illustrative purposes only. in some SUGAR radicals, a glycosidase with a relatively nonspecific activity would be able to catalyze the reaction, whereas more specific glycosidases are required in other cases ··.

SUGAR IN Glycosidase Activity Ribose | ribosidase 20 xylose j xylosidase β-galactose j β-galactosidase glucose j glucosidase fructose j fruetosidase

A preferred embodiment of the method 25 according to the invention is characterized by the characterizing part of claim 2. Both β-galactose and β-galactosidase are readily obtainable substances, and the final product is a valuable feedstock for reaction with organic acids.

A preferred embodiment of the method 30 according to the invention is characterized by the characterizing part of claim 3. Both lactose and galactosidase are cheap and readily available.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 4. Both sucrose and glucosidase are cheap and readily available.

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A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 5. In this way, a highly reactive substrate is readily available.

5 A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 6. There are no steric obstacles and the reaction can be carried out smoothly and with high yields.

A preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 7. In this way, all the usual benefits of using an immobilized enzyme are obtained.

A preferred embodiment of the method 15 according to the invention is characterized by the characterizing part of claim 8. When both reactants are heavily soluble in aqueous media, this embodiment is preferred.

A preferred embodiment of the method 20 according to the invention is characterized by the characterizing part of claim 9. When both reactants are heavily soluble in pure aqueous media but readily soluble in a mixture of water and an organic solvent, this embodiment is preferred.

A preferred embodiment of the method according to the invention comprises that it be carried out in a two-phase system. When one reactant is readily soluble in an aqueous medium and the other reactant is readily soluble in an organic solvent, this embodiment is preferred and the reaction takes place in the intermediate phase.

The reactions which can be carried out by the process of the invention can be summarized as indicated below in Scheme 3, wherein X is hydrogen, a carbohydrate residue, alkyl or aryl, all of which may be substituted by hydroxy, mercapto, nitro, alkoxy, carboxy or amino, Z is oxygen or sulfur, Y is alkylene which may be 8

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substituted by hydroxy, mercapto, nitro, alkoxy, carboxy123 or ammo, and R, R and R are the same or different and each represents hydrogen, alkyl or aryl, whereby both of the latter radicals may be substituted by hydroxy, mercapto, nitro, alkoxy, carboxy or amino, wherein the (unsubstituted) alkyl group preferably contains 6 carbon atoms or below 6 carbon atoms, and wherein the (unsubstituted) aryl group preferably contains between 6 and 10 carbon atoms, including.

Preferably, the alkylene group contains 8 carbon atoms or less than 8 carbon atoms, preferably 4 carbon atoms or less than 4 carbon atoms. Examples of preferred carbohydrate residues (X) are monosaccharide residues, e.g. and glucose residue. Preferably, aryl is phenyl or naphthyl which may be substituted or unsubstituted. The substituted phenyl or naphthyl radicals are preferred, e.g. the nitrosubstituted radicals, due to the higher reactivity of compound V.

Scheme 3

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9 ov / \ ~ enzyme SUGAR-O-X + H-Z-Y-C-C-R · ->

I I

5 R1 R3 (V) (VI) / \ 2

SUGAR-Z-Y-C-C-R

I I

R1, R3 (I) 12 3 wherein R, R, R, SUGAR, X, Y and Z are each as defined in the following claim 1.

The enzymes which may be used in the process of the invention are enzymes which can cleave the -OX bond of the compound SUGAR-O-X (V), shown in Scheme 3, cf. claim 1.

The enzymes used in the process of the invention may be in solution or immobilized. The enzymes can also be modified by chemical or genetic methods to optimize their reactivity in the reaction in question.

The process of the invention can be carried out by mixing the acceptor and donor component in the reaction, i.e. the compounds of formulas V and VI, respectively, in water containing the enzyme at room temperature. If desired, the reaction mixture may be heated to accelerate the reaction. Organic solvents can also be added to the reaction mixture to increase the reactants' solubility.

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And the pH of the reaction medium can be adjusted to achieve maximum enzyme activity and / or stability.

The compounds of general formula I which can be prepared by the process of the invention can be used for many purposes. Eg. enables the reactive epoxy group in molecules of general formula I to link them to other molecules. Therefore, carbohydrate carbon moieties can be easily transferred e.g. to a matrix carrying hydroxyl groups, such as Sepharose or cellulose, or carbohydrate residues can be coupled to proteins which thereby become glycoproteins.

A reaction of particular interest which can be carried out using the method of the invention is illustrated in Scheme 4 below. By reaction with the epoxide of formula VIII, a substrate molecule of general formula V as shown can be converted to the epoxidized compound of formula IX which, as shown by reaction with a fatty acid, can further be converted to the monoglyceride of formula X. The compounds of the general formula formula X, which can thus be prepared from compounds of general formula IX prepared by the process of the invention, are surfactants of great utility. Thus, these compounds can be used in food and feed as emulsifying agents, or they can be used to improve many functional properties of food and feed. The reactive compounds of general formula IX may also be coupled to amino acids, e.g. in proteins, thereby altering the functional properties thereof.

Scheme 4

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11 y ° \ SUGAR-O-X + HO-CH2-CH - CH2 -> (V) (VIII)

5 RCOOH

sugar-o-ch2-ch-ch2 -> (IX)

H2C-0-SUGAR

10 HCOH

H2C-OCOR

(X) wherein R represents a carboxylic acid residue, e.g. alkyl, and wherein SUGAR and X are each as defined in the following claim 1.

The process of the invention is illustrated by the following Examples 1 and 2. Examples 3, 4 and 5 illustrate the use of the epoxy compounds prepared by the method of the invention.

EXAMPLE 1

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Preparation of 2,3-epoxypropyl-8-galactopyranoside β-Galactosidase (supplied by Boehringer Mannheim and recovered from E. coli, 830 μΐ, about 50 U, suspended in 5 (NH ^) 2SO ^) was added to a mixture of 5 g (16.6 mmol) of o-nitrophenyl galactopyranoside and 17.5 ml (0.26 mmol) of 2,3-epoxy-1-propanol in 400 ml of buffer (0.07 M phosphate buffer, 10 mM MgCl 2, pH = 7) . The reaction was followed by thin layer chromatography (hereinafter referred to as TLC) and high pressure liquid chromatography (hereinafter referred to as HPLC). After 4 hours, the mixture was extracted with ether (4 x 100 ml) to remove nitrophenol and the aqueous phase was evaporated under reduced pressure. The product was taken up in ethanol and inorganic salts were filtered off. The yield by evaporation was 6.4 g of crude product.

15 This product was subjected to chromatography

SiO 2 and further crystallized from absolute ethanol to give 1.1 g (28%) of the title compound. Melting point: 126 - 127 ° C.

Ή NMR (DMSO-d6) delta; 2.6 (m, 1H), 2.74 (m, 1H), 3.14 (m, 1H), 3.2 - 3.4 (m, 3H), 3.45 - 3.55 (m , 2H), 3.56 - 3.65 (m, 2H), 3.73 (dd, 1H), 4.13 (d, 1H), 4.38 (d, 1H), 4.56 (m, IH), 4.72 (d, 1H), 4.94 (d, 1H).

13 C NMR (DMSO-d 6) delta; 43.8, 50.1, 60.5, 68.2, 69.3, 70.5, 73.4, 75.2, 103.4.

EXAMPLE 2

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Preparation of 2,3-epoxypropyl BD - galactopyranoside β-Galactosidase (supplied by Boehringer Mannheim and recovered from E. coli, 12 mg freeze-dried, about 2400 U) was added to a mixture of 5 g (13.9 mmol ) lactose and 20 ml (0.3 mol) of 2,3-epoxy-1-propanol in 300 ml of buffer (0.07 M phosphate buffer, 10 mM MgCl 2, pH = 7). The reaction mixture was stirred for 15 hours at room temperature and then heated to 80 ° C for 10 minutes. The aqueous phase was evaporated at reduced pressure. The product was taken up in ethanol and filtered from inorganic salts. Evaporation gave the crude product as a yellow syrup. Further purification was performed as in Example 1, and the data was consistent with the above.

EXAMPLE 3 Preparation of 1'-O-tetradecanoyl-3'-OBD-galactopyranosyl-glycerol 2,3-Epoxypropyl-3-D-galactopyranoside (1g, 4.2mmol) was added to myristic acid (1.06g, 1.1 equivalent) at 80 ° C. Then, tetraethylammonium bromide (40 mg, 0.05 20 ml equivalent) was added. The semi-solid mixture was stirred for 3 hours. The product was taken up in diethyl ketone / methanol (hereafter referred to as Et 2 O-MeOH) (50:50) and evaporated on SiC> 2 followed by chromatography to give 1.2 g (61%) of the title compound which was crystallized by acetone.

25 Ή NMR (DMSO-d6) delta; 0.88 (t, 3H), 1.25 (bs, 20H), 1.54 (m, 2H), 2.32 (t, 2H), 3.25 - 3.6 (mm, 6H), 3 , 65 (bs, 1H), 3.72 (dd, 1H), 3.83 (m, 1H), 4.0 (m, 1H), 4.08 (m, 2H), 4.4 (d, 1H), 4.6 (t, 1H), 4.75 (d, 1H), 4.9 (d, 1H), 5.0 (d, 1H).

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13 C NMR (DMSO-d 6) delta; 13/8, 22.0, 24.4, 28.5, 28.6, 28.7, 28.8, 28.9 - 29.0 (4c), 31.2, 33.5, 60.4, 65 , 4, 67.5, 68.1, 70.4, 70.6, 73.3, 104.0, 172.8.

Example 4 5 Preparation of 1'-O-hexadecanoyl-3'-0-3-D-galactopyranosyl glycerol was carried out in an analogous manner as in Example 3 * 1 H NMR (DMSO-d6) delta? 0.86 (t, 3H), 1.25 (bs, 24H), 1.5 (m, 2H), 2.3 (t, 2H), 3.25 - 3.55 (mm, 6H), 3 , 62 (bs, 1H), 3.7 (m, 1H), 3.8 (m, 1H), 3.96 (m, 1H), 4.05 (m, 2H), 4.4 (d, IH), 4.55 (t, 1H), 4.7 (d, 1H), 4.85 (bs, 1H), 4.98 (d, 1H).

13 C NMR (DMSO-d 6) delta; 13.8, 22.0, 24.4, 28.5, 28.7 (2c), 28.9 (2c), 29.0 (5c), 31.2, 33.5, 60.4, 65 , 4, 67.5, 68.1, 70.4, 70.6, 73.4, 75.3, 104.0, 172.8.

Example 5 Preparation of 1'-O-octadecanoyl-3'-O-8-D-galactopyranosyl-glycerol was carried out in an analogous manner as in Example 3 Ή NMR (DMSO-d6) delta; 0.84 (t, 3H), 1.24 (bs, 28H), 1.5 (m, 2H), 2.28 (t, 2H), 3.25 - 3.55 (mm, 6H), 3 , 6 (bs, 1H), 3.68 (m, 1H), 3.8 (m, 1H), 3.95 (m, 1H), 4.05 (m, 2H), 4.35 (d, 1H), 4.55 (t, 1H), 4.7 (d, 1H), 4.85 (d, 1H), 4.95 (d, 1H).

13 C NMR (DMSO-d 6) delta; 13.8, 22.0, 24.4, 28.5, 28.6, 28.7, 28.9, 29.0 (8c), 31.3, 33.5, 60.4, 65.4, 67 , 5, 68.2, 70.5, 70.7, 73.4, 75.3, 104.0, 172.8.

Claims (9)

A process for preparing epoxy-substituted aldoses or ketoses of the general formula I A 2 Process according to claim 1, characterized in that SUGAR is β-galactose and that the enzyme is a β-galactosidase, preferably prepared by E. coli. Process according to claims 1-2, characterized in that the compound of formula V is lactose and that the enzyme is galactosidase. Process according to claims 1-3, characterized in that the compound of formula V is sucrose and that the enzyme is glucosidase. Process according to claims 1-4, characterized in that X is an aryl group having 6-10 carbon atoms. SUGAR-ZYCCR (I) II R1 R3 wherein SUGAR means a galactose, glucose, fructose, xylose or ribose moiety, Z means oxygen or sulfur attached to the terminal anomeric C1 carbon atom (aldoses) or C 2 carbon atom (ketoses) of the sugar moiety, Y means alkylene which may be substituted by hydroxy, mercapto, 12 3 nitro, alkoxy, carboxy or ammo and R, R and R, which are the same or different, each represent hydrogen , alkyl or aryl, where alkyl and aryl may be substituted by hydroxy, mercapto, nitro, alkoxy, carboxy or amino, and where in 12 cases some of the radicals Y, R, R or R are substituted by an OH - or SH group, the radicals in question are preferably protected, characterized by reacting a compound of the general formula V SUGAR-OX (V) wherein SUGAR is as defined above and wherein X is hydrogen, a carbohydrate residue , alkyl or aryl, all of which may be substituted by hydroxy, mercapto, nitro, alkoxy, carboxy or amino, with a compound of the general formula VI / H-Z-Y-C-C-R (VI) I! R1 R3 12 3 5 wherein R, R, R, Y and Z are each as defined above and wherein 12 3 in the case of some of the radicals Y, R, R, R or X is substituted by an OH- or SH- preferably, the radicals in question are protected and the reaction is catalyzed by an enzyme that cleaves the molecular moiety SUGAR-O- at the position indicated by the arrow: SUGAR-O-. Process according to claims 2-5, characterized in that Z is 12 3 oxygen, Y is methylene and R, R or R are each hydrogen. Process according to claims 1-6, characterized in that the enzyme is immobilized. Process according to claims 1-7, characterized in that the reaction is carried out in an organic solvent. DK 159 383 B Process according to claims 1-8, characterized in that the reaction is carried out in a mixture of water and an organic solvent.