LV13762B - Enzyme catalyzed de-acylation of chlorinated sugar derivatives - Google Patents

Abstract

A process of production of trichlorogalactosucrose is described in which deacylation of sucrose-6-ester is achieved by subjecting the reaction mixture after chlorination, neutralization and adjustment of pH to 6.5 to 7 to deacylation by using a lipase or a protease, in a free or in an immobilized form.

Description

CATALYTIC DEACILATION OF CHLORINATED SUGAR DERIVATIVES

TECHNICAL FIELD

This invention relates to a novel process and a new strategy for the preparation of 1 -6-dichloro-16-DIDEOXY-p-fructofuranosyl-4-chloro-4-deoxy-galactopyranoside (TGS), including the enzymatic deacylation of 6-O-protected TGS. chlorination reactions.

PRE-CONDITIONS OF THE INVENTION

Strategies for prototype methods for the preparation of 4, T, 6'-trichloro-galactosucrose (TGS) mainly involve the chlorination of sucrose-6-ester using Vilsmeier Haack reagent derived from chlorinated sucrose-6-ester to form 6-acetyl 4, T, 6'-trichloro-galactosucrose using various chlorinating reagents such as phosphorus oxychloride, oxalyl chloride, phosphorus pentachloride, etc., and a tertiary amide such as dimethylformamide (DMF). The reaction mass after the above chlorination reaction is neutralized to pH 7.0-7.5 using appropriate calcium, sodium and the like. alkaline hydroxides. The pH of the neutralized mass is then raised to 9.5 or higher to de-esterify / deacylate 6-acetyl 4,1 ', 6' trichlorgalactosucrose to form 4, T, 6 'trichlorgalactosucrose using calcium, sodium, potassium and the like. alkaline hydroxides. This alkaline deesterification / deacylation involves exposure of the reagent to high pH values in the alkali range, which results in the degradation of a significant amount of DMF, which is an expensive investment adversely affecting its recovery after the reaction.

The reaction mixture of the prototype process during deacylation is also exposed to high temperatures, which results in the degradation of the TGS product itself.

Therefore, there is a need for a deacylation method that will not obey

DMF for degradation effects.

A method has been developed to achieve enzymatic deacylation at pH that does not subject to DMF degradation.

DETAILED DESCRIPTION OF THE INVENTION

Enzymatic deacylation was described by Palmer.c. (1995) U.S. Pat. No. 5445951 for the preparation of partially acylated derivatives of sucrose by enzyme-catalyzed deacylation of sucrose esters from a sucrose ester selected from the group consisting of sucrose octaacylate, sucrose heptaacylate and sucrose hexacylate in anhydrous organic medium, together with an enzyme or enzyme catalysed ester deacylation to obtain a partially acylated sucrose derivative having a free hydroxyl group at the preselected position (s) and resulting in the recovery of a partially deacylated sucrose derivative.

There are no other reports of enzymatic deacylation of sucrose ester or its derivative / product.

This invention relates to the enzymatic deacylation of 6-0-protected TGS obtained after the chlorination reaction during the preparation of the artificial sweetener TGS. The formulation of the chlorination reaction mixture that may be subjected to the process effect described in this invention includes, but is not limited to, the process stream obtained after mixing the sucrose-6-ester with the chlorinating agent described by Mufti et al. (1983)

U.S. Pat. 4380476, Walkup, etc. (1990 No. 4980463), Jenner et al.

(1982) U.S. Pat. 4,362,869, Tulley et al. (1989) U.S. Pat

No. 4,801,700, Rathbone et al. (1989) U.S. Pat. 4,826,962,

Bornemann et al. (1992) U.S. Pat. 5,141,860, Navia et al. (1996) U.S. Pat. U.S. Patent No. 5,498,709 to Simpson (1989). No. 4,889,928 to Navia (1990), U.S. Pat. 4,950,746 to Neiditch et al. (1991) U.S. Pat. 5,023,329 to Walkup et al. (1992) 5,089,608 to Dordick et al. (1992) U.S. Pat. 5,128,248 to Khan et al. (1995) U.S. Pat. 5,440,026 to Palmer et al. (1995) U.S. Pat. 5,445,951, Sankey et al. (1995) U.S. Pat

No. 5,449,772, Sankey et al. (1995) U.S. Pat. 5,470,969, Navia et al.

(1996) U.S. Pat. 5,498,709, Navia et al. (1996) U.S. Pat. 5,530,106.

Enzymatic deacylation is carried out in the process stream obtained as described above after neutralization of the chlorinated reaction mass with or without isolation of the intermediate from 6-O-protected TGS. The solvent tertiary amide present in the neutralized reaction mass is not degraded by the enzymatic reaction and therefore provides improved recovery of the above solvent.

In the present invention, the chlorinated reaction mass is neutralized with a suitable base after the chlorination reaction. When the pH that is controlled during neutralization is below 6.0, the resulting TGS compound has an intact protected group at position 6. The position 6 unblocking is performed with or without isolation of the above compound. The following various references also emphasize that the unblocking may be carried out with or without a tertiary amide and another solvent under alkaline conditions.

-i

The present invention describes deacylation at position 6 using an enzymatic process wherein the enzyme selectively removes a protected group in the presence or absence of a tertiary amide, including DMF, used in the chlorination reaction.

The process of this invention also works well for deacylation of formulations that are not the result of a chlorination reaction, such as a simple solution of pure TGS6-ester.

Enzyme-catalyzed deacylation is well known, lipase and proteolytic enzymes carry out deacylation and acylation reactions under mild reaction conditions and have been extensively described by Soedjak HS, Spradlin JE (1994).

Biocatalysis 11: 241-248; Therisod M, Klibanov AM (1986) J. Am. Chem. Soc. 108: 5638-5640; B. Cambou and A.M. Klibanov, J. Am. Chem. SOC., 106, 2687 (1984); For Whip S Bisht, Pure & Appl. Cbem., Vol. 68, No. 3, p. 749-752, 1996; F.J. Plou1; _, M.A. Crucesl, Biotechnology Letters 21:

635-639, 1999.

In the present invention, after neutralizing the reaction mass, the pH is adjusted to 6.5 using an appropriate base. The lipase enzyme is added to the reaction mass with slow stirring at room temperature. The amount of enzyme added to the reaction mass varies from 10% to 40% (w / v) depending on enzyme activity and reaction conditions. The content of the tertiary amide in the neutralized reaction mass is about 10% to 40%. The reaction mixture is stirred continuously for 10 to 60 hours, preferably 16 to 20 hours. The conversion of 6-O- protected TGS to TGS is controlled by TLC. After complete deacylation reaction, the mixture is taken for affinity chromatography on TGS. Dedicated

The TGS is then crystallized using suitable techniques.

The use of lipase or proteolytic enzymes to deactivate 6-0-protected TGS to TGS may be in their natural form or in immobilized form. When an immobilized enzyme is used, the enzyme is filtered after the deacylation is complete. This recovered enzyme can then be reused. In addition, the immobilized enzyme can be loaded onto the column, passed through the reaction mass and the deacylation of the 6-0-protected TGS in situ. These enzymes can be immobilized on or on synthetic polymer supports, such as, but not limited to, polyacrylic, polystyrene, polyacrylamide or nylon-based supports; or on semi-synthetic or natural organic supports similar to those based on polysaccharides, such as, but not limited to, cellulose, starch, dextran, agarose, chitosan, chitin, etc., or inorganic supports such as carbon, silica, zirconium, aluminum, zirconium phosphate, etc.

The source of the lipase enzyme may be of animal, plant or microbial origin, preferably microbial or bacterial origin, such as Bacillus thermocatenulatusis, Pseudomonas aeruginosa and the like. of fungal origin such as Penicillium Roquefortii, Asperigillus niger, Asperigillus oryzae,

Rhizopus niveus, Candida rugosa, Rhizomucor miheii, Candida antartctica

etc.

During the above-mentioned process of the invention, the TGS product is not subjected to any drastic pH or temperature conditions, as is the case with conventional deacylation processes using acid or alkali. Product

losses in most cases are less than other forms of deacylation processes.

During the above-mentioned process of the invention, the tertiary amide is not subjected to any drastic pH or temperature conditions, as in the case of conventional deacylation processes using acid or alkali. Therefore, there is no degradation of the third amide. Therefore, the recovery efficiency of the tertiary amide increases to a very high degree.

The following are examples illustrating the scope of the present invention without limiting the scope of the invention in any way. The reagents used, the proportion of reagents, the range of reaction conditions described, the enzymes used and the like are illustrative only and the scope extends to analogous reagents, reaction conditions and reactions of a similar nature. In general, any equivalent alternative obvious to a person skilled in the art for the production of chlorinated sucrose is within the scope of this specification. Accordingly, the mention of acetate encompasses any equivalent ester group which may perform the same function in the context of the present invention, and the use of the enzyme should include any alternative capable of providing the enzyme described herein or analogous operation under analogous reaction conditions. Several adaptations of other formulations can be easily predicted by the prototype competitor and are also within the scope of this specification. The singular mention is constructed to include its plural, unless the context permits, i.e., the use of an organic solvent for extraction involves the use of one or more organic solvents, sequentially or in combination.

EXAMPLE 1

Chlorination of sucrose-6-acetate

1280 ml of dimethylformamide are added to reaction flask I and cooled to 0-5 ° C. This is followed by slow addition of 635 g (5.4 moles) of phosphorus pentachloride with stirring and maintaining the reaction mass at a temperature below 30 ° C. The mass is further cooled to below 0 ° C and sucrose-6-acetate is slowly added together with

DMF at 0-5 ° C. The reaction mass is then heated to 80 ° C and held for 1 hour, but then heated to 100 ° C and held for 6 hours, and finally held at 110-115 ° C for 2-3 hours. The progress of the reaction is controlled by HPLC analysis.

The reaction mixture is then cooled to -5 to -8 ° C and 20% sodium hydroxide solution is added slowly to maintain the pH at 5.5-6.5. The yield of sucrose using this method was 55.4%.

EXAMPLE 2

Enzymatic deacetylation of 6-O-acetyl TGS using lipase enzyme

A 1.51 reaction mass containing 15 g of 6-O-acetylated TGS prepared as described in Example 1 was neutralized using 50% calcium hydroxide solution at pH 7.5. The neutralized reaction mass was diluted with water to 6 I. The DMF content was 33% of the neutralized mass. 84 g of lipase enzyme isolated from Aspergillus oryzae ATCC 26850; NCIM 1212 was added to the reaction mixture with stirring at ambient temperature. The reaction was continued for several hours and the formation of TGS and disappearance of 6-O-acetylated TGS was controlled by TLC. After 42 hours, up to 98.4% deacetylation was achieved.

After deacetylation, the mass was removed for TGS removal using appropriate methods.

EXAMPLE 3

Enzymatic deacetylation of 6-0-acetyl TGS using lipase enzyme on Eudragit RL100

In the experiment, 2.5 L reaction masses containing 80 g of 6-O-acetylated TGS were neutralized using 50% calcium hydroxide solution to pH 5.5. The neutralized reaction mass was diluted to 6 L with water. The DMF content was 33% of the neutralized mass. To the reaction mixture was added 120 g of immobilized lipase to Eudragit RL100 with continuous mixing

25 ° C to 30 ° C, which is usually ambient temperature.

The reaction was continued for several hours and the formation of TGS and disappearance of 6-acetylated TGS was controlled by TLC. After 24 hours, up to 98.3% deacetylation was achieved.

The mass was then filtered and taken for TGS removal. The enzyme obtained in the filter sediment was washed with water and stored for reuse.

EXAMPLE 4

Enzymatic deacetylation of 6-O-acetyl TGS using a lipase enzyme on Eudragit RL 100 packed in a column

In the experiment, 12 g of the immobilized enzyme was packed with 2 cm diameter and

8 cm high glass column. The inlet of the column was connected to the peristaltic pump feed and the outlet was connected to a flask containing 500 ml of neutralized mass containing 5 g of 6-O-acetyl-TGS. The peristaltic pump was also connected to a neutralized mass. The neutralized mass was circulated at a flow rate of 5 mL / min. through the immobilized lipase layer for 6 hours.

TLC was performed every hour to see the degree of deacetylation that occurred in the flask. After 6 hours, deacetylation above 98% was observed.

Upon completion of deacetylation of 6-O-acetyl-TGS, the enzyme immobilized on TGS was washed with deionized water and stored under 10% acetone in water for further use.

EXAMPLE 5

Enzymatic deacetylation of 6-O-acetyl TGS using alkalase proteolytic enzyme

A 1.0 L neutralized mass after chlorination containing 10 g of 6-O-acetylated TGS was removed for enzymatic reaction. The neutralized reaction mass was dissolved in 3.0 L of water. 200 ml alcalase 2.4 L enzyme from novozyme obtained from B. lichenformis was added to the reaction mixture with continuous stirring at 25-30 ° C. The reaction was continued for several hours and the formation of TGS and disappearance of 6-O-acetylated TGS was controlled by TLC. After 36 hours, up to 96.4% deacetylation was achieved. After deacetylation, the mass was removed for TGS isolation using appropriate methods.

Claims (6)

A process for the preparation of a chlorinated sucrose compound, wherein the chlorinated 6-protected sucrose derivative in the solution was dea protected by the action of an enzyme capable of separating 5 protecting groups. The method of claim 1, wherein: a. the aforementioned 6-0-protected sucrose containing one or more sucrose-6-acetate, sucrose-6-benzoate, sucrose-6-propionate, sucrose-6-laurate, sucrose-610 glutarate, sucrose-6-palmitate and like, b. the aforesaid solution comprising a solution of pure chlorinated 6-O-protected sucrose, or (ii) a process stream obtained by the process of obtaining a chlorinated sucrose compound. The method of claim 2, wherein: 15th century the flow of the aforesaid process comprising one or more processes for the production of chlorinated sucrose, including the chlorination process for sucrose or the chlorination process for 6-O-protected sucrose, and b.The aforementioned chlorinated sucrose compound contains one 20 or more chlorinated sucrose, including trichlorgalactosucrose, dichlorgalactosucrose, tetrachloro-galactosucrose, and the like. The process of claim 3, wherein the above chlorination process comprises the reaction of a sucrose derivative with one or more chlorinating agents in one or more processes, 25 including: • i n a. 6-0- protected sucrose dissolved in pyridine with sulfur chloride, or b. Reaction of 6-O-protected sucrose with thionyl chloride in the presence of triphenylphosphine and 1,1,2-trichloroethane, or 5 c. 6-0- protected sucrose reaction including sucrose-6 ester with Vilsmeier reagent of general formula [HCIC = N.sup. + R.sub.2] Cl.sup.- or [HPOCI.sub.2.OCsup + = N.sup + .R.sub.2] Cl.sup.- where R represents an alkyl group, preferably a methyl or ethyl group . The method of claim 4, wherein the activity of the enzyme capable of deprotecting is derivatized from a lipase or protease enzyme. The method of claim 5, wherein the above lipase or protease is a free or immobilized enzyme. The method of claim 6, comprising the steps of: a.chlorination of sucrose-6-acetate in (i) solution or (ii) process stream obtained by the chlorinated sucrose process with a chlorinating reagent selected from the group consisting of Vilsmeier reagent, sulfur chloride or thionyl chloride, B) adjusting the pH of the process stream of step (a) of this invention to about pH 6.5 to about 7, c.6-0- deacitylation of the protected TGS formed in the process stream of step (a) by (i) contacting the same with free or immobilized lipase or free or immobilized protease, 25 preferably with shaking in a reaction container or by recirculation through a column of enzyme packed in a column, preferably about 25 to At an ambient temperature of 30 ° C for a sufficient period of time to achieve the maximum possible deacetylation above 95%, d. Separating the TGS from one or more undesirable components of the process stream of step (c) of this claim.