Title of Invention	A PROCESS FOR THE PREPARATION OF NOVEL MACROPOROUS CYCLODEXTRIN BOUND POLYMER SUPPORT
Abstract	A process for the preparation of novel macroporous cyclodextrin bound polymer support A process for the preparation of novel macroporous cyclodextrin bound polymer support by contacting a macroporous polymer containing epoxy groups with the reaction product of monotosyl p-cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a period ranging from 24 to 96 hours in presence of inert gas, washing the product with the aprotic solvent, separating the solids by conventional methods, washing with an alkanol and drying the product by conventional methods to get macroporous polymer support.

Title of Invention

A PROCESS FOR THE PREPARATION OF NOVEL MACROPOROUS CYCLODEXTRIN BOUND POLYMER SUPPORT

Abstract

A process for the preparation of novel macroporous cyclodextrin bound polymer support A process for the preparation of novel macroporous cyclodextrin bound polymer support by contacting a macroporous polymer containing epoxy groups with the reaction product of monotosyl p-cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a period ranging from 24 to 96 hours in presence of inert gas, washing the product with the aprotic solvent, separating the solids by conventional methods, washing with an alkanol and drying the product by conventional methods to get macroporous polymer support.

Full Text	This invention relates to a process for the preparation of novel macroporous cyclodextrin bound polymer support. More particularly, it relates to a process for the preparation of macroporous copolymers with functional groups based on cyclodextrin. These are prepared by the modification of macroporous polymers containing epoxy functional groups. The macroporous polymer bound supports with functional groups prepared by the process of this invention can be used to purify cyclodextrin glycosyl transferase under ambient conditions. The purification of proteins such as cyclodextrin glycosyl transferase can be effected by using conventional techniques such as expended bed column chromatography or packed bed chromatography. Purification of proteins involves a series of steps - such as clarification, concentration and separation. The operational difficulties encountered are high viscosity of the crude fermentation broth, particulate matter fouling of the membranes and loss encountered in each step in handling etc. affects the high cost of process economics. Cononventional purification of proteins involves the use of suitable support as alumina, olymer or membranes. The polymer support use involves the additional step of crosslinking the base polymer with a crosslinking agent such as gluraraldehyde . Thus polyacrylamides had been used for such applications and is also marketed under the trade name Bio-Gel by M/s Bio-Rad Laboratory; (S. Hjertein Arch. Biochem. Biophys. Suppl 147, 1962); the use of Agarose has been described Stellen Hjerten Biochim. Biophys. Acta79,393, 1964; Dextran gels have been used as described by Flodin et.al Gels and their applications in Gel filtration, Thesis, University of Uppasala, 1962; Cellulose and its derivatives have also been reported -U S Patent 4, 055, 510 (1977); German Patents DD 29 5861 (1991) The above polymers suffer from disadvantages such as mechanical stability during the process of operation; resistance to microbial attack; chemical stability under alkaline conditions etc. Additonally, the affinity of the proteins to the support varies and affects the process economics to a great extent. The polymeric supports prepared as per the process of this invention have affinity towards a particular protein and used directly for the purification of the protein. The primary requirements for such applications are - 1. The support material should contain functional groups that have high affinity/ion exchange capacity to selectively bind proteins from mixed stream of crude fermentation broth feed. 2. The support material should have sufficient mechanical strength so that during the processing stage, there is no damage. 3. The support materials have densities high enough to allow stable bed expansion during operation. 4. The matrix material should be stable to variation in operating pH range during use. The main object of the present invention is therefore to provide a process for the preparation of novel macroporous cyclodextrin bound polymer support material with functional groups that are suitable for purification of cyclodextrin glycosyl transferase by conventional purification techniques. Another object of the present invention is to provide a process for the preparation of macroporous polymer bound support with functional groups that has sufficient mechanical strength and resistance to variations in pH. Yet another object of the present invention is to provide a process for the preparation of macroporous polymer matrix with functional groups that can be used repeatedly for purification of cyclodextrin glycosyl transferase without further derivatization using the property of affinity of the proteins to the support. Accordingly, the present invention provides a process for the preparation of novel macroporous cyclodextrin bound polymer support charaterized in that contacting a macroporous polymer containing epoxy groups with the reaction product of monotosyl P-cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a period ranging from 24 to 96 hours in presence of inert gas, washing the product with the aprotic solvent, separating the solids by conventional methods, washing with an alkanol and drying the product -by under reduced pressure at a temperalure range 40-60oC methods to get macroporous polymer support. In an embodiment of the invention, the macroporous polymer containing hydroxyl groups may be a copolymer of allyl glycidyl either-ethylene glycol dimethacrylate prepared as described and claimed in our copcnding application no. 1282/DKL/92, derivatives of polymers synthesized using hydroxy ethyl metiuicrylale derivatives, ion exchange resins with hydroxyl functional groups. In another embodiment of the invention, the inert gas used may be such as nitrogen or argon. In yet another embodiment of the present invention, the diamine used may be such as elhylene diamine, hexamethylene diamine, 1,10-dodecanediamine, 1,4 phenylene diamine, 1,3 phenylene diamine, 1,2 phenylene diamine. In still another embodiment of the present invention, the aprotic solvent used may be such as 1,4-dioxan, dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide. In yet another embodiment of the invention, the alkanol used may be such as isopropanol, methanol, ethanol. The process of the present invention is described with reference to following examples, which are illustrative only and should not be construed to limit the scope of this invention in any manner. EXAMPLE 1 A known process was used to prepare Monotosyl p-cyclodextrin. The procedure followed is given below : P-cyclodextrin 8.0 grams was suspended in 80 ml water and 1 gram sodium hydroxide in 5 ml water was added dropwise to this solution over a period of 5 minutes. The suspension became homogenous and slightly yellowish before the addition was complete. 1.36 grams of para toluene sulphonyl chloride in 6 ml dicholoroethane was then added dropwisc over 10 minutes. After stirring for 2 hours at a temperature of 20°C, the reaction mixture was refrigerated overnight. The resulting white precipitate was recovered and recrystallised from water. 0.6 ml of ethylene diamine was mixed with 20 ml dimethyl formamide and warmed to 70°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated P-cyclodextrin. The reaction mixture was stirred at 70°C for a period of 36 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono {3 amino ethyl amino-6-deoxy (3-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono p amino ethyl amino-6-deoxy p-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 20°C for 96 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 40°C. EXAMPLE 2 1.16 gms of hexaniethylene diamine was mixed with 20 ml dimethyl sulfoxide and warmed to 60°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.15 gram of potassium iodide were added followed by 2 grams of monotosylated ß-cyclodextrin prepared as shown in example 1. The reaction mixture was stirred at 60°C for a period of 40 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß ammo hexa amino-6-dcoxy ß-cyclodcxlrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepired as per our copending application no; 1282/DEL792 was contacted with a solution of 2 grams of mono P amino hexa amino-6-deoxy P-cyclodexlrin in 20 ml dimethyl acctamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 20°C for 96 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with ethanol, followed by drying at 60°C. Example 3 1.1 gms of 1,4-phenylene diamine was mixed with 20 ml dimethyl formamide and warmed to 80°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated p-cyclodextrin. The reaction mixture was stirred at 80°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono P amino phenyl amino-6-deoxy p-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono P amino phenyl amino-6-deoxy P-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with isopropanol, followed by drying at 60°C. EXAMPLE 4 1.1 gins of 1,3-phcnylcnc diamine was mixed with 20 ml dimethyl formamidc and warmed to 50°C. To this mixture, 0.60 gram of dimethyl amino pyridine and 0.15 gram of potassium iodide were added followed by 2 grams of monotosylaled ß cyclodcxlrin. The reaction mixture was stirred at 80°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono (3 amino phenyl amino-6-deoxy ß-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application nek 1282/DHL/92 was.contacted with a solution of 2 grams of mono p amino phenyl amino-6-deoxy ß-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30oC for 50 hours. The reaclunts were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with isopropanol, followed by drying at 60°C. Example 5 1.1 gms of 1,2-phenylene diamine was mixed with 20 ml dimethyl acetamide and warmed to 40°C. To this mixture, 0.57 grain of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylaled ß-cyclodextrin. The reaction mixture was stirred at 40°C for a period of 60 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono (3 amino 1,2-phenylene amino-6-dcoxy ß-cyclodcxtrin. In an inert atmosphere of nitrogen. 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,2-phenylenc amino-6-deoxy P-cyclodextrin in 20 ml dimethyl acetamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 40°C. EXAMPLE 6 1.1 gms of 1,2-phenylene diamine was mixed with 20 ml dimethyl formamide and warmed to 40°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß cyclodextrin. The reaction mixture was stirred at 40°C for a period of 70 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,2-phenylene amino-6-deoxy ß-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,2-phenylene amino-6-deoxy p-cyclodextrin in 20 ml dimethyl formarnide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 60°C. EXAMPLE? 1.1 gms of 1,2-phcnylcne diamine was mixed with 20 ml dimethyl acetamide and warmed to 40°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß- cyclodextrin. The reaction mixture was stirred at 40°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,2-phenylene amino-6-deoxy P-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,2-phenylene amino-6-deoxy ß-cyclodextrin in 20 ml dimethyl acetami.de. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 60°C. EXAMPLE 8 1.45 gins of 1,8-diamino octane was mixed with 20 ml dimethyl sulfoxide and warmed to 30°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß-cyclodextrin. The reaction mixture was stirred at 30°C for a period of 96 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,8-octane amino-6-deoxy ß-cyclodextrin. In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,8-octane amino-6-deoxy ß-cyclodcxtrin in 20 ml dimethyl sulfoxide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 25°C for 96 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 60°C. Advantages of the present invention: 1. The macroporous polymer bound supports with functional groups synthesized by the process of this invention are not biodegradable ensuring enhanced shelf life. 2. The macroporous polymer bound support with functional groups obtained by the process of this invention can be used for the purification of proteins without any derivatization. 3. The process of this invention is amenable for scale-up operation 4. The macroporous polymer bound support with functional groups prepared by the process of this invention have adequate mechanical strength. We claim : 1. A process for the preparation of novel macroporous cyclodextrin bound polymer support which comprises contacting a macroporous polymer containing epoxy groups with the reaction product of monotosyl P- cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a period ranging from 24 to 96 hours in presence of inert gas, washing the product with the aprotic solvent, separating the solids by conventional methods, washing with an alkanol and drying the product by conventional methods to get macroporous polymer support. 2. A process wherein the macroporous polymer containing epoxy groups is a copolymer of allyl glycidyl ether and ethylene glycol dimethacrylate. 3. A process as claimed in claims 1-2 wherein the inert gas used is nitrogen or argon. 4. A process as claimed in claims 1-3 wherein the aprotic solvent used is 1,4- dioxan, dirnethy sulfoxide, dimethyl formamide, dimethyl acetamide. 5. A process as claimed in claims 1-4 wherein the diamine used is ethylene diamine, hexamethylene diamine, 1,10-dodecanediamine, 1,4 phenylene diamine, 1,3 phenylene diamine, 1,2 phenylene diamine. 6. A process as claimed in claims 1-5 wherein the alkanol used is isopropanol, methanol, ethanol. 7. A process for the preparation of novel macroporous cyclodescterin bound polymer support substantially as herein described with reference to the examples.

Full Text

This invention relates to a process for the preparation of novel macroporous cyclodextrin bound polymer support. More particularly, it relates to a process for the preparation of macroporous copolymers with functional groups based on cyclodextrin. These are prepared by the modification of macroporous polymers containing epoxy functional groups. The macroporous polymer bound supports with functional groups prepared by the process of this invention can be used to purify cyclodextrin glycosyl transferase under ambient conditions.
The purification of proteins such as cyclodextrin glycosyl transferase can be effected by using conventional techniques such as expended bed column chromatography or packed bed chromatography.
Purification of proteins involves a series of steps - such as clarification, concentration and separation. The operational difficulties encountered are high viscosity of the crude fermentation broth, particulate matter fouling of the membranes and loss encountered in each step in handling etc. affects the high cost of process economics. Cononventional purification of proteins involves the use of suitable support as alumina, olymer or membranes. The polymer support use involves the additional step of crosslinking the base polymer with a crosslinking agent such as gluraraldehyde . Thus polyacrylamides had been used for such applications and is also marketed under the trade name Bio-Gel by M/s Bio-Rad Laboratory; (S. Hjertein Arch. Biochem. Biophys. Suppl 147, 1962); the use of Agarose has been described Stellen Hjerten Biochim. Biophys. Acta79,393, 1964; Dextran gels have been used
as described by Flodin et.al Gels and their applications in Gel filtration, Thesis, University of Uppasala, 1962; Cellulose and its derivatives have also been reported -U S Patent 4, 055, 510 (1977); German Patents DD 29 5861 (1991)
The above polymers suffer from disadvantages such as mechanical stability during the process of operation; resistance to microbial attack; chemical stability under alkaline conditions etc. Additonally, the affinity of the proteins to the support varies and affects the process economics to a great extent.
The polymeric supports prepared as per the process of this invention have affinity towards a particular protein and used directly for the purification of the protein.
The primary requirements for such applications are -
1. The support material should contain functional groups that have high
affinity/ion exchange capacity to selectively bind proteins from
mixed stream of crude fermentation broth feed.
2. The support material should have sufficient mechanical strength so
that during the processing stage, there is no damage.
3. The support materials have densities high enough to allow stable bed
expansion during operation.
4. The matrix material should be stable to variation in operating pH
range during use.
The main object of the present invention is therefore to provide a process for the
preparation of novel macroporous cyclodextrin bound polymer support material with
functional groups that are suitable for purification of cyclodextrin glycosyl transferase by
conventional purification techniques.
Another object of the present invention is to provide a process for the preparation of
macroporous polymer bound support with functional groups that has sufficient
mechanical strength and resistance to variations in pH.
Yet another object of the present invention is to provide a process for the preparation of
macroporous polymer matrix with functional groups that can be used repeatedly for
purification of cyclodextrin glycosyl transferase without further derivatization using the
property of affinity of the proteins to the support.
Accordingly, the present invention provides a process for the preparation of novel
macroporous cyclodextrin bound polymer support charaterized in that contacting a
macroporous polymer containing epoxy groups with the reaction product of monotosyl P-cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a period ranging from 24 to 96 hours in presence of inert gas, washing the product with the aprotic solvent, separating the solids by conventional methods, washing with an alkanol and drying the product -by

under reduced pressure at a temperalure range 40-60oC methods to get macroporous polymer support.
In an embodiment of the invention, the macroporous polymer containing hydroxyl groups may be a copolymer of allyl glycidyl either-ethylene glycol
dimethacrylate prepared as described and claimed in our copcnding application no. 1282/DKL/92, derivatives of polymers synthesized using hydroxy ethyl metiuicrylale derivatives, ion exchange resins with hydroxyl functional groups.
In another embodiment of the invention, the inert gas used may be such as nitrogen or argon.
In yet another embodiment of the present invention, the diamine used may be such as elhylene diamine, hexamethylene diamine, 1,10-dodecanediamine, 1,4 phenylene diamine, 1,3 phenylene diamine, 1,2 phenylene diamine.
In still another embodiment of the present invention, the aprotic solvent used may be such as 1,4-dioxan, dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide.
In yet another embodiment of the invention, the alkanol used may be such as isopropanol, methanol, ethanol.
The process of the present invention is described with reference to following examples, which are illustrative only and should not be construed to limit the scope of this invention in any manner.
EXAMPLE 1
A known process was used to prepare Monotosyl p-cyclodextrin. The procedure followed is given below :
P-cyclodextrin 8.0 grams was suspended in 80 ml water and 1 gram sodium hydroxide in 5 ml water was added dropwise to this solution over a period of 5
minutes. The suspension became homogenous and slightly yellowish before the addition was complete. 1.36 grams of para toluene sulphonyl chloride in 6 ml dicholoroethane was then added dropwisc over 10 minutes. After stirring for 2 hours at a temperature of 20°C, the reaction mixture was refrigerated overnight. The resulting white precipitate was recovered and recrystallised from water.
0.6 ml of ethylene diamine was mixed with 20 ml dimethyl formamide and warmed to 70°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated P-cyclodextrin. The reaction mixture was stirred at 70°C for a period of 36 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono {3 amino ethyl amino-6-deoxy (3-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono p amino ethyl amino-6-deoxy p-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 20°C for 96 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 40°C.
EXAMPLE 2
1.16 gms of hexaniethylene diamine was mixed with 20 ml dimethyl sulfoxide and warmed to 60°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.15 gram of potassium iodide were added followed by 2 grams of monotosylated ß-cyclodextrin prepared as shown in example 1. The reaction mixture was stirred at
60°C for a period of 40 hours. The reaction mixture was cooled to ambient
temperature and acetone was added to precipitate mono ß ammo hexa amino-6-dcoxy ß-cyclodcxlrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepired as per our copending application no; 1282/DEL792 was contacted with a solution of 2 grams of mono P amino hexa amino-6-deoxy P-cyclodexlrin in 20 ml dimethyl acctamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 20°C for 96 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with ethanol, followed by drying at 60°C.
Example 3
1.1 gms of 1,4-phenylene diamine was mixed with 20 ml dimethyl formamide and warmed to 80°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated p-cyclodextrin. The reaction mixture was stirred at 80°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono P amino phenyl amino-6-deoxy p-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono P amino phenyl amino-6-deoxy P-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with isopropanol, followed by drying at 60°C.
EXAMPLE 4 1.1 gins of 1,3-phcnylcnc diamine was mixed with 20 ml dimethyl formamidc
and warmed to 50°C. To this mixture, 0.60 gram of dimethyl amino pyridine and 0.15 gram of potassium iodide were added followed by 2 grams of monotosylaled ß cyclodcxlrin. The reaction mixture was stirred at 80°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono (3 amino phenyl amino-6-deoxy ß-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application nek 1282/DHL/92 was.contacted with a solution of 2 grams of mono p amino phenyl amino-6-deoxy ß-cyclodextrin in 20 ml dimethyl formamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30oC for 50 hours. The reaclunts were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with isopropanol, followed by drying at 60°C.
Example 5
1.1 gms of 1,2-phenylene diamine was mixed with 20 ml dimethyl acetamide and warmed to 40°C. To this mixture, 0.57 grain of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylaled ß-cyclodextrin. The reaction mixture was stirred at 40°C for a period of 60 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono (3 amino 1,2-phenylene amino-6-dcoxy ß-cyclodcxtrin.
In an inert atmosphere of nitrogen. 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2
grams of mono ß amino 1,2-phenylenc amino-6-deoxy P-cyclodextrin in 20 ml dimethyl acetamide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 40°C.
EXAMPLE 6
1.1 gms of 1,2-phenylene diamine was mixed with 20 ml dimethyl formamide and warmed to 40°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß cyclodextrin. The reaction mixture was stirred at 40°C for a period of 70 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,2-phenylene amino-6-deoxy ß-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,2-phenylene amino-6-deoxy p-cyclodextrin in 20 ml dimethyl formarnide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl formamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 60°C.
EXAMPLE?
1.1 gms of 1,2-phcnylcne diamine was mixed with 20 ml dimethyl acetamide and warmed to 40°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß-
cyclodextrin. The reaction mixture was stirred at 40°C for a period of 30 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,2-phenylene amino-6-deoxy P-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,2-phenylene amino-6-deoxy ß-cyclodextrin in 20 ml dimethyl acetami.de. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 30°C for 50 hours. The reactants were decanted, washed with dimethyl acetamide and the product, cyclodextrin bound polymer support was isolated by filtration, washed with methanol, followed by drying at 60°C.
EXAMPLE 8
1.45 gins of 1,8-diamino octane was mixed with 20 ml dimethyl sulfoxide and warmed to 30°C. To this mixture, 0.57 gram of dimethyl amino pyridine and 0.13 gram of potassium iodide were added followed by 2 grams of monotosylated ß-cyclodextrin. The reaction mixture was stirred at 30°C for a period of 96 hours. The reaction mixture was cooled to ambient temperature and acetone was added to precipitate mono ß amino 1,8-octane amino-6-deoxy ß-cyclodextrin.
In an inert atmosphere of nitrogen, 1 gram of macroporous polymer prepared as per our copending application no. 1282/DEL/92 was contacted with a solution of 2 grams of mono ß amino 1,8-octane amino-6-deoxy ß-cyclodcxtrin in 20 ml dimethyl sulfoxide. The reactants were gently stirred at a rpm of 200 and maintained at a temperature of 25°C for 96 hours. The reactants were decanted, washed with dimethyl sulfoxide and the product, cyclodextrin bound polymer support was isolated by
filtration, washed with methanol, followed by drying at 60°C.
Advantages of the present invention:
1. The macroporous polymer bound supports with functional groups synthesized by
the process of this invention are not biodegradable ensuring enhanced shelf life.
2. The macroporous polymer bound support with functional groups obtained by the
process of this invention can be used for the purification of proteins without any
derivatization.
3. The process of this invention is amenable for scale-up operation
4. The macroporous polymer bound support with functional groups prepared by the
process of this invention have adequate mechanical strength.

We claim :
1. A process for the preparation of novel macroporous cyclodextrin bound
polymer support which comprises contacting a macroporous polymer
containing epoxy groups with the reaction product of monotosyl P-
cyclodextrin and diamine in a aprotic solvent, and stirring the mixture in the
range of 150 to 200 rpm at a temperature in the range of 25 to 80°C, for a
period ranging from 24 to 96 hours in presence of inert gas, washing the
product with the aprotic solvent, separating the solids by conventional
methods, washing with an alkanol and drying the product by conventional
methods to get macroporous polymer support.
2. A process wherein the macroporous polymer containing epoxy groups is a
copolymer of allyl glycidyl ether and ethylene glycol dimethacrylate.
3. A process as claimed in claims 1-2 wherein the inert gas used is nitrogen or
argon.
4. A process as claimed in claims 1-3 wherein the aprotic solvent used is 1,4-
dioxan, dirnethy sulfoxide, dimethyl formamide, dimethyl acetamide.
5. A process as claimed in claims 1-4 wherein the diamine used is ethylene
diamine, hexamethylene diamine, 1,10-dodecanediamine, 1,4 phenylene
diamine, 1,3 phenylene diamine, 1,2 phenylene diamine.
6. A process as claimed in claims 1-5 wherein the alkanol used is isopropanol,
methanol, ethanol.
7. A process for the preparation of novel macroporous cyclodescterin bound polymer support substantially as herein described with reference to the examples.

Documents:

1027-del-2000-abstract.pdf

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Patent Number

232355

Indian Patent Application Number

1027/DEL/2000

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

16-Mar-2009

Date of Filing

17-Nov-2000

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ARIKA KOTHA	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
2	SMITA ATMARAM MULE	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
3	ANANT PATKAR	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
4	SURENDRA PONRATHNAM	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
5	DEBAPRASAD NAYAK	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.
6	CHELANATTU KHIZHAKKE MADATH RAMAN RAJAN	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.

PCT International Classification Number

C08B 37/16

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA