Note: Descriptions are shown in the official language in which they were submitted.
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ENZYME CATALYZED DE-ACYLATION OF CHLORINATED SUGAR
DERIVATIVES
TECHNICAL FIELD
The present invention relates to a novel process and a novel strategy for
production of 1-6-Dichloro-1-6-DIDEOXY-P-Fructofuranasyl-4-chloro-4-
deoxy-galactopyranoside (TGS) involving enzymatic deacylation of the 6-
0-protected TGS obtained after the chlorination reaction.
BACKGROUND OF THE INVENTION
Strategies of prior art methods of production of 4,1', 6'
trichlorogalactosucrose (TGS) predominantly involve chlorination of
sucrose-6-ester by use of Vilsmeier Haack reagent derived from to
chlorinate Sucrose-6-ester, to form 6 acetyl 4,1', 6'trichlorogalactosucrose,
using various chlorinating agents such as phosphorus oxychloride, oxalyl
chloride, phosphorus pentachloride etc, and a tertiary amide such as
dimethylformamide (DMF). After the said chlorination reaction, the
reaction mass is neutralized to pH 7.0 -7.5 using appropriate alkali
hydroxides of calcium, sodium, etc. The pH of the neutralized mass is
then further raised to 9.5 or above to deesterify / deacetylate the 6 acetyl
4,1', 6'trichlorogalactosucrose to form 4,1', 6' trichlorogalactosucrose
using alkali hydroxides of calcium, sodium, potassium, etc. This alkaline
de-esterification / deacylation involves exposure of the reactants to harsh
pH in alkaline range which leads to destruction of a significant quantity of
DMF, which is an expensive input, adversely affecting its recovery after
the reaction.
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In prior art process, the reaction mixture is also exposed during the
process of deacylation to harsh temperatures which lead to destruction of
the product TGS itself.
Hence, there is a need for a method of deacylation which shall not expose
DMF to destruction.
A method has been developed to achieve deacylation enzymatically at a
pH which does not expose DMF to destruction.
DETAILED DESGRIPTI6N OF THE INVENTION
Enzymatic deacylation has been reported by Palmer et al (1995) in a US
patent no. 5445951 for the preparation of partially acylated derivatives of
sucrose by the enzyme catalyzed deacylation of sucrose esters from a
sucrose ester selected from the group consisting of sucrose octaacylate,
sucrose heptaacylate, and sucrose hexaacylate in an anhydrous organic
medium, with an enzyme or combination of enzymes capable of catalyzing
the deacylation of said sucrose ester to produce a partially deacylated
sucrose derivative having free hydroxyl group(s) in pre-selected
position(s), and recovering the, resulting partially deacylated sucrose
derivative.
No other report is known on enzymatic deacylation of a sucrose ester or
its derivative I a precursor.
This invention relates to the enzymatic deacylation of the 6-0-protected
TGS obtained after the chlorination reaction during the preparation of the
artificial sweetener, TGS. Embodiments of chlorination reaction mixture
which can be subjected to the process described in this invention includes,
without being limited to, a process stream obtained after mixing sucrose-6-
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ester with a chlorinating agent described in Mufti et al. (1983) US patent no
4380476, Walkup et al. (1990 No.4980463), Jenner et al. (1982) US patent
no. 4,362,869, Tu11ey et al. (1989) US pat no. 4,801,700, Rathbone et al.
(1989) US pat no. 4,826,962, Bornemann et al. (1992) US pat no.
5,141,860, Navia et al. (1996) US Pat no. 5,498,709, Simpson (1989) US
Pat no. 4,889,928, Navia (1990) US Pat no. 4,950,746, Neiditch et al.
(1991) US Pat no. 5,023,329, Walkup et al. (1992) 5,089,608, Dordick et al.
(1992) US pat no. 5,128,248, Khan et al. (1995) US Pat no. 5,440,026,
Palmer et al. (1995) US Pat no. 5,445,951, Sankey et al. (1995) US Pat no.
5,449,772, Sankey et al. (1995) US Pat no. 5,470,969, Navia et al. (1996)
US Pat no. 5,498,709, Navia et al.(1996) US Pat no. 5,530,106
The enzymatic deacylation is carried out on the process stream obtained in
a way as mentioned above after neutralization of the chlorinated reaction
mass after or without intermediate isolation of the 6-0-protected TGS. The
solvent, tertiary amide present in the neutralized reaction mass doesn't get
decomposed due to the enzymatic reaction and therefore results in
enhanced recovery of the said solvent.
In this invention, the chlorinated reaction mass, after chlorination reaction
is
neutralized with a suitable base. When the pH is controlled during
neutralization below 6.0, the compound TGS formed still has the protected
group intact at the 6th position. The deblocking of the 6th position is
carried
out either with or without the isolation of the said compound. Further
various references also point out that the deblocking can be carried out with
or without the tertiary amide and other solvent and aqueous conditions.
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The present invention describes the deacylation at the 6{h position using an
enzymatic process where in the enzyme selectively removes the protected
group in the presence or absence of the tertiary amide, including DMF
which is used in the chlorination reaction.
The process of this invention also works well for deacylation of
embodiments which are not result of a chlorination reaction, such as a
simple solution of pure TGS-6-ester.
Enzyme catalyzed deacylation is well known and the lipase and proteolytic
enzymes carry out deacylation and acylation reactions under benign
reaction conditions and is widely reported 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); Kirpal S Bisht,Pure & Appi. Cbem., Vol. 68,
No. 3, pp. 749-752, 1996; F.J. Plou1;_, M.A. Cruces1, Biotechnology Letters
21: 635-639, 1999.
In this invention, after the neutralization of the reaction mass, the pH is
adjusted to 6.5 using an appropriate base. The lipase enzyme is then
added to the reaction mass slowly under stirring at room temperature. The
quantity of enzyme added to the reaction mass varies from 10% to 40% w/v
depending on the enzyme activity and reaction conditions. The tertiary
amide content in the neutralized reaction mass is around 10% to 40%. The
reaction mixture is stirred continuously for a period of 10 to 60 hours, more
preferably 16 to 20 hours. The conversion of the 6-0-protected TGS to TGS
is monitored by TLC.- After the complete deacylation, the reaction mixture is
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taken for TGS isolation by affinity chromatography. The isolated TGS is
then crystallized by suitable methods.
The use of the lipase or proteolytic enzymes for deacylation of 6-0-
protected TGS to TGS can be in its native form or in immobilized form.
When the immobilized enzyme is used, the enzyme is filtered off after the
completion of the deacylation. This recovered enzyme can be further re-
used. Also the immobilized enzyme can be packed in a column and the
reaction mass can be passed through the column and in situ deacylation of
the 6-0-protected TGS pan be carried out. These enzymes can be
immobilized in or on synthetic polymeric supports such as, but not limited to
polyacrylic, or polystyrene or polyacrylamide, nylon based supports; or
semisynthetic or natural organic supports like those based on
polysaccharides such as, but not limited to cellulose, starch, dextran,
agarose, chitosan, chitin, etc.; or inorganic supports like those based on
carbon, silica, zirconia, alumina, zirconium phosphate, etc.
The source of the enzyme lipase can be of animal, plant or microbial origin,
more preferably microbial or bacterial origin such as Bacillus
thermocatenulatusis, Pseudomonas aeruginosa, etc., fungal origin such as
Penicillium Roquefortii, Asperigillus niger, Asperigillus oryzae, Rhizopus
niveus, Candida rugosa, Rhizomucor miheii, Candida antartctica, etc.
During the process of the said invention, the TGS product is not exposed to
any harsh pH or temperature conditions as in the case of the conventional
deacylation processes using acid, alkali. The product loss is the most
minimal compared any other form of deacylation process.
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During the process of the said invention, the tertiary amide is not exposed to
any harsh pH or temperature conditions as in the case of the conventional
deacylation processes using acid, alkali. Hence the decomposition of the
tertiary amide doesn't take place at all. Therefore the efficiency of the
recovery of the tertiary amide enhances to a very large extent.
Described in the following are examples, which illustrate working of this
invention without limiting the scope of this invention in any manner.
Reactants, proportion of reactants used, range of reaction conditions
described, enzymes used and the like are only illustrative and the scope
extends to their analogous reactants, reaction conditions and reactions of
analogous generic nature. In general, any equivalent alternative which is
obvious to a person skilled in art of clorinated sucrose production is covered
within the scope of this specification. Thus, mention of an acetate covers
any equivalent ester group which can perform the same function in the
contest of this invention, and use of an enzyme shall cover any alternative
capable of providing the action or analogous action of the enzyme described
herein under analogous reaction conditions. Several other adaptations of
the embodiments will be easily anticipated by those skilled in this art and
they are also included within the scope of this specification. Mention in
singular is construed to cover its plural also, unless the context does not
permit so, viz: use of "an organic solvent" for extraction covers use of one
or
more of an organic solvent in succession or in a combination as a mixture.
E7(AMPLE 1
Chlorination of sucrose 6-acetate
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In a 5L reaction flask1280ml of Dimethylformamide was added and cooled
to 0 - 5 C. This was followed by addition of 635g of Phosphorous
pentachloride (5.4 moles) slowly under stirring, maintaining the temperature
of the reaction mass below 30 C. The mass is further cooled to below 0 C
and the sucose-6-acetate in DMF is added slowly at 0-5 C. Then the
reaction mass is heated to 80 C and held for 1 hour, further heated to
100 C and held for 6 hours and finally at 110 -115 C and held for 2 - 3
hours. The progress of the reaction is monitored by HPLC analysis.
Then the reaction mixture is cooled to -5 to - 8 C and a 20% solution of
Sodium hydroxide is slowly added so as to bring the pH of the mass to 5.5 -
6.5. The yield obtained by this method was 55.4%of the sucrose input.
EXAMPLE 2
Enzymatic deacetylation of 6-0-acetyf TGS by' lipase enzyme
The Reaction mass, 1.5 L, containing 15g of 6-0-acetylated TGS and
prepared as described in Example 1 was neutralized using 50% calcium
hydroxide slurry up to pH 7.5. The neutralized reaction mass was diluted to
6L using water. The DMF content was 33% in the neutralized mass. 84g of
lipase enzyme isolated from Aspergillus oryzae ATCC 26850; NCIM 1212
was added to the reaction mixture with continuous stirring at ambient
temperature. The reaction was continued for several hours and formation of
TGS and disappearance of 6-0-acetylated TGS was monitored by TLC. At
the end of 42 hours, deacylation upto 98.4 % was achieved.
After the deacylation, the mass was taken for isolation of TGS by suitable
methods.
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EXAMPLE 3
Enzymatic deacetylation of 6-0-acetyl TGS by immobilized lipase
enzyme on Eudragit RL100
In an experiment, 2.5 L of reaction mass containing 80g of 6-0-acetylated
TGS was neutralized using 50% calcium hydroxide slurry up to pH 5.5. The
neutralized reaction mass was diluted to 6L using water. The DMF content
was 33% in the neutralized mass. 120g of immobilized lipase on Eudragit
RL100 was added to the reactiori mixture with continuous stirring at 25 C -
30 C temperature, which is usually the ambient temperature. The reaction
was continued for several hours and formation of TGS and disappearance
of 6-0-acetylated TGS was monitored by TLC. At the end of 24 hours,
deacylation up to 98.3% was achieved.
The mass was then filtered and taken for TGS isolation. The enzyme
obtained in filter cake was washed with water and stored for reuse
EXAMPLE 4
Enzymatic deacetylation of 6-0-acetyl TGS by lipase enzyme
immobilized on Eudragit RL 100, packed in a column
In an experiment, 12g of immobilized enzyme was packed on to a 2cm
diameter and height 8cm glass column. The column inlet was connected to
the delivery of a peristaltic pump and the outlet was connected to a flask
containing 500 ml of neutralized mass which had 5 g of 6-0-acetyl-. The
inlet of the peristaltic pump was also connected to the neutralized mass.
The neutralized mass was circulated at 5 ml/min flow rate through the bed
of immobilized lipase, for 6 hours.
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The TLC was carried out every one hour to see the extent of deacetylation
taking place in the flask. After 6 hours, deacetylation above 98% was
observed.
After completion of deacetylation of 6-0-acetyl-TGS to TGS, the bed of
immobilized enzyme was washed with de-ionized water and was stored
under 10% Acetone in water until further use.
EXAMPLE 5
Enzymatic deacetylation of 6-0-acety9 TGS by Alcalase - a proteolytic
enzyme
1.0 L of neutralized mass after chlorination, containing lOg of 6-0-
acetylated TGS was taken up for the enzymatic reaction. The neutralized
reaction mass was diluted to 3.0L using water. 200 ml of Alcialase 2.4L
enzyme obtained commercially from Novozymes derived from B.
lichenformis was added to the reaction mixture with continuous stirring at
25-30 G temperature. The reaction was continued for several hours and
formation of TGS and disappearance of 6-0-acetylated TGS was monitored
by TLC. At the end of 36 hours, deacylation upto 96.4 % was achieved.
After the deacylation, the mass was taken for isolation of TGS by suitable
methods.
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