Ionic liquid based support for manufacture of peptides
11192917 · 2021-12-07
Assignee
Inventors
Cpc classification
International classification
Abstract
The present invention relates to an ionic liquid based support of Formula-I: wherein: X.sup.+ is a heteroatom containing cationic part; W is a halogen containing polymeric solid support; n is an integer in the range of 2 to 8; Y is a hydrophobic anion; R is selected from CO—Z or Z; Z is selected from the group consisting of —Cl, —Br, —OH, —O-Alkyl and combinations thereof. The present invention also relates to a process for preparation of said ionic liquid based support used for di, oligo or polypeptide manufacture.
Claims
1. An ionic liquid based support of Formula I: ##STR00003## wherein: X.sup.+ is diethanolamine; W is chloromethyl polystyrene; n is 5; Y.sup.− is hexafluorophosphate; and R is CO—Z wherein Z is OH.
2. The ionic liquid based support as claimed in claim 1, wherein the ionic liquid based support has a mesh size in the range of 10 to 70.
Description
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
(1) The above and other features, aspects, and advantages of the subject matter will be better understood with regard to the following description and accompanying drawings where:
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
(4) The present invention relates to a class of functionalized ionic liquid used as supports as well as solvents for di, oligo or polypeptide manufacture. The present invention also relates to a process for preparation of said functionalized ionic liquids.
(5) In one embodiment of the invention, there is provided an ionic liquid based support of Formula-I;
(6) ##STR00002## wherein: X.sup.+ is a heteroatom containing cationic part; W is a halogen containing polymeric solid support; n is an integer in the range of 2 to 8; Y.sup.− is a hydrophobic anion; R is selected from CO—Z or Z; Z is selected from the group consisting of —Cl, —Br, —OH, —O-Alkyl and combinations thereof.
(7) In one embodiment of the invention, there is provided an ionic liquid based support wherein R is CO—Z. In another embodiment of the invention, there is provided an ionic liquid based support wherein R is Z.
(8) In one embodiment of the invention, there is provided an ionic liquid based support has a mesh size in the range of 10 to 70.
(9) In one embodiment of the invention, the heteroatom containing cationic part is selected from the group consisting of imidazole, triethylamine, diethanolamine, diethyl amine, pyridine, triethanolamine, triethylphosphine and combinations thereof.
(10) In one embodiment of the invention, the halogen containing polymeric solid support is selected from the group consisting of chloromethyl polystyrene, hydroxyethylpolystyrene, divinyl benzene crosslinked chloromethyl polystyrene and combinations thereof.
(11) In one embodiment of the invention, the hydrophobic anion is selected from the group consisting of methane sulfonate, triflate, hexafluorophosphate, trifluoromethanesulfonate, bistrifluoromethane sulfanimide, tetrafluroborate and combinations thereof.
(12) In one embodiment of the invention, the ionic liquid based support comprises ionic solvent. In another embodiment of the invention, ionic solvent is selected from the group consisting of triethylamine, diethanolamine, triethanolamine, diethyl amine, imidazole, triethylphosphine based functionalized ionic liquid.
(13) In one embodiment of the invention, there is provided a method for the preparation of the ionic liquid based support, comprising the steps: i) attaching a heteroatom containing cationic part (X.sup.+) of an ionic liquid with a halogen containing polymeric solid support in an organic solvent at temperature in the range of 40° C. to 110° C. to obtain a solid product; ii) washing the solid product with another organic solvent to obtain a washed solid product; iii) subjecting the washed solid product to anion metathesis using an anionic source selected from the group consisting of methane sulfonate, triflate, hexafluorophosphate, trifluoromethanesulfonate, bistrifluoromethane sulfanimide, and tetrafluroborate or combinations thereof in a solvent under stirring conditions at a temperature ranging from 20° C. to 60° C. to obtain an anion metathesis product; iv) drying the metathesis product at a temperature ranging from 60° C. to 120° C. under reduced pressure in between 60 mm Hg to 20 mm of Hg to obtain ionic liquid based support.
(14) In one embodiment of the invention, the heteroatom containing cationic part (X.sup.+) is attached with halogen containing polymeric solid support in an organic solvent selected from a group of toluene, acetonitrile, ethanol, and acetone or combinations thereof.
(15) In one embodiment of the invention, the heteroatom containing cationic part (X.sup.+) is attached with halogen containing polymeric solid support in an organic solvent at a temperature preferably in the range of 60° C. to 110° C.
(16) In one embodiment of the invention, the side chain containing functional group is attached to the cationic part of the ionic liquid based support in an organic solvent selected from a group of toluene, acetonitrile, ethanol, acetone and combinations thereof.
(17) In one embodiment of the invention, the side chain containing functional group is attached to the cationic part of the ionic liquid based support in an organic solvent at temperature preferably in the range of 0° C. to 50° C.
(18) In one embodiment of the invention, the solid product is washed with organic solvent selected from the group consisting of diethyl ether, acetone, ethanol, toluene and combinations thereof.
(19) In one embodiment of the invention, the washed solid product is subjected to anion metathesis using an anionic source selected from the group consisting of methane sulfonate, triflate, hexafluorophosphate, trifluoromethanesulfonate, bistrifluoromethane sulfanimide, and tetrafluroborate or combinations thereof in a solvent selected from the group consisting of dichloromethane, water, ethanol and combinations thereof.
(20) In one embodiment of the invention, the washed solid product is subjected to anion metathesis using an anionic source in a solvent at a temperature in the range of 35° C. to 60° C.
(21) In one embodiment of the invention, the metathesis product was dried under reduced pressure of less than 20 mm of Hg.
(22) The present invention is further described in terms of N-terminal peptide synthesis approach and C-terminal peptide synthesis approach.
(23) The structure of the ionic liquid based solid support used for the N-terminal approach has several functionalized part as shown in
(24) The structure of the ionic liquid based solid support used for the C-terminal approach has several functionalized part as shown in
(25) The polymeric part in the ionic liquid based support material was of mesh size in between 10 to 70, preferably in between 15 to 60 and before attaching the ionic liquid the polymeric part may contain halogen atom loading of less than 18.05 mmol per g, preferably 10.50 mmol per g, more preferably 5.50 mmol per g was used.
(26) In the present invention the ionic liquid based solid support for the N-terminal approach method was synthesized by attaching the heteroatom containing cationic part of the ionic liquid with the solid support by removal of the halogen pan efficiently under vigorous magnetic stirring in a solvent selected from a group of toluene, acetonitrile, ethyl acetate, water, ethanol and combinations thereof at a temperature in between 40° C. to 110° C., preferably in the range of 60° C. to 110° C., more preferably in between 75° C. to 90° C. for about 8 to 36 h, preferably in between 12 to 30 h, more preferably in between 18 to 24 h. The obtained solid was filtered and washed with an organic solvent selected from a group of acetone, ethanol, methanol, and combinations thereof. Further the side chain containing functionality of the ionic liquid was attached to the obtained solid in an organic solvent selected from a group of toluene, acetonitrile, ethanol, acetone and combinations thereof at temperature in the range of 0° C. to 110° C., preferably in the range of 0° C. to 50° C. The resultant solid product was further washed using common organic solvents like diethyl ether, acetone, ethanol, toluene and combinations thereof. The anion metathesis was carried out using the anionic source anionic source selected from the group consisting of methane sulfonate, triflate, hexafluorophosphate, trifluoromethanesulfonate, bistrifluoromethane sulfanimide, and tetrafluroborate or combinations thereof, containing species in a solvent selected from a group of dichloromethane, water, ethanol and combinations thereof under stirring conditions at a temperature ranging from room temperature to 60° C., specifically in the range of 35° C. to 60° C.
(27) In the present invention the ionic liquid based solid support for the C-terminal approach method was synthesized by attaching the heteroatom containing cationic part of the ionic liquid with the solid support by removal of the halogen part efficiently under vigorous magnetic stirring in a solvent selected from a group of toluene, acetonitrile, ethyl acetate, water, ethanol and combinations thereof at a temperature in between 40° C. to 110° C., preferably in the range of 60° C. to 110° C., more preferably in between 75° C. to 90° C. for about 8 to 36 h, preferably in between 12 to 30 h, more preferably in between 18 to 24 h. The obtained solid was filtered and washed with an organic solvent selected from a group of acetone, ethanol, methanol, and combinations thereof. Further the side chain containing functionality of the ionic liquid was attached to the obtained solid in an organic solvent selected from a group of toluene, acetonitrile, ethanol, acetone and combinations thereof at temperature in the range of 0° C. to 110° C., preferably in the range of 50° C. to 110° C. The resultant solid product was further washed using common organic solvents like diethyl ether, acetone, ethanol, toluene and combinations thereof. The anion metathesis was carried out using an anionic source containing in a solvent selected from a group of dichloromethane, water, ethanol and combinations thereof under stirring conditions at a temperature ranging from room temperature to 60° C., specifically in the range of 35° C. to 60° C.
(28) In the present invention, the ionic solvent was synthesized by attaching the heteroatom containing cationic part of the ionic liquid with the side chain containing functionality in an organic solvent selected from a group of toluene, acetonitrile, ethanol, acetone and combinations thereof at temperature in the range of 0° C. to 110° C., preferably in the range of 80° C. to 110° C. The resultant solid product was further washed using common organic solvents like diethyl ether, acetone, ethanol, toluene and combinations thereof. The anion metathesis was carried out using an anionic source containing species in a solvent selected from a group of dichloromethane, water, ethanol and combinations thereof under stirring conditions at a temperature ranging from room temperature to 60° C., specifically in the range of 35° C. to 60° C. Finally, the ionic liquid was dried at 120° C. under reduced pressure of around 10 mm of Hg.
(29) When the above-mentioned ionic liquid was used as the active support in combination with another ionic liquid to be used as a solvent for the reaction, can be the same ionic liquid as the support or a different ionic liquid which was easy to separate from the system. The combination of the ionic liquids is such that the interaction between them do not interfere their individual function in the reaction.
(30) Ionic liquid used as the solvent for the reaction must possess low to moderate viscosity to facilitate the fast reaction kinetics. The use of more than 20% water by volume of the reagents was not sufficient to stop the puckering effects of longer peptide chain which retards the kinetics of the reaction. The water content of the reaction was controlled by reducing the pressure in the range of 10 to 120 mm of Hg, preferably in the range of 20 to 90 mm of Hg, more preferably in the range of 30 to 60 mm of Hg.
(31) In an embodiment of the present disclosure, there is provided an ionic liquid based support in combination with an ionic solvent for the manufacture of di, oligo or polypeptides, wherein the C-terminal or the N-terminal of the amino acids or peptides to be attached to the chain is blocked. After addition of each blocked amino acid or peptides to the growing peptide chain the blocking functionalities were removed following the conventional literature.
(32) In an embodiment of the present disclosure, there is provided an ionic liquid based support in combination with an ionic solvent for the manufacture of di, oligo or polypeptides, wherein the used blocked amino acids or peptide combinations were selected from a group of Glycine (Gly), Leucine (Leu), Phenyl alanine (Phe-Ala), Lysine (Lys), Tryptophan (Trp), Aspartic acid (Asp) and analogues as well as oligomers and polymerized products.
(33) The di, oligo or polypeptides manufactured using these ionic liquid based solid supports in combination with an ionic solvent can be used as artificial sweeteners, food and food additives, pharmaceutical products, useful chemical reagents, etc.
(34) The present invention is more particularly described in the following examples, that are intended as illustrations only, since numerous modifications and variations within the scope of the present invention will be apparent to those skilled in the art. Unless otherwise noted, all parts, percentages and ratios reported in the following examples are on a weight basis and all reagents used in the examples were obtained or are available from the chemical suppliers.
EXAMPLES
(35) The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the claimed subject matter.
Example 1
(36) Preparation of Functionalized Ionic Liquid for N-Terminal Approach
(37) In a typical ionic liquid preparation procedure. Equimolar amount of an amine was mixed with chloromethyl polystyrene under vigorous stirring at a temperature range of 60° C. to 110° C., preferably in the range of 80° C. to 100° C. for 12 to 24 h in an organic solvent like acetonitrile. After completion of reaction the solid mass was washed with acetone three times and dried in air at 60° C. for 4 to 6 h. An equimolar amount of the solid was reacted with 3-chloropropyl chloroformate at 0° C. in acetone for 2 h. The obtained solid mass was collected by decanting acetone and finally equimolar amount of Lithium bistrifluoromethane sulfanimide was added and stirred vigorously for 2 h to obtain the ionic liquid based solid support.
(38) After 34 h of reaction the yield of the ionic liquid was obtained to be 89% by relative molar percentage with respect to the amount of amine added initially.
Example 2
(39) Preparation of Functionalized Ionic Liquid for C-Terminal Approach
(40) In a typical ionic liquid preparation procedure, Equimolar amount of an amine was mixed with Chloromethyl polystyrene under vigorous stirring at a temperature range of 60° C. to 110° C., preferably in the range of 80° C. to 100° C. for 12 to 24 h in an organic solvent like acetonitrile. After completion of reaction the solid mass was washed with acetone three times and dried in air at 60° C. for 4 to 6 h. An equimolar amount of the solid was reacted with 2-bromoethanol at 110° C. in toluene for 24 h. The obtained solid mass was collected by decanting toluene and finally equimolar amount of Lithium bistrifluoromethane sulfanimide was added and stirred vigorously for 2 h to obtain the ionic liquid based solid support.
(41) After 54 h of reaction the yield of the ionic liquid was obtained to be 82% by relative molar percentage with respect to the amount of amine added initially.
Example 3
(42) Preparation of the Ionic Liquid Used as the Solvent
(43) In a typical ionic liquid preparation procedure, an amine was mixed with 1-chlorobutane stirring in the range of 80° C. to 110° C., preferably in the range of 100° C. to 110° C. for 12 to 24 h in toluene. After completion of the reaction the obtained mass was washed three times with diethyl ether. Then the ionic liquid was charged with equimolar amount of Potassium tetrafluoroborate in water for 2 h and then extracted using dichloromethane. Finally, the ionic liquid was dried under vacuum (120° C., 0.01 Torr).
Example 4
(44) Dipeptide Manufacture Via N-Terminal Approach
(45) The reaction mixture containing L-Phenyl alanine methyl ester and ionic liquid based solid support at 1.1:1 mole ratio was charged in a peptide vessel glass reactor in the ionic liquid solvent under reduced pressure at room temperature for 2-4 h. At the end of the reaction, the solvent along with excess amino acid was first separated from the solid mass and then separated from the amino acids by extraction using dichloromethane. The ester linkage was deprotected by treating with trifluoroacetic acid and then with triethylamine. Similarly, other C-terminal blocked Glycinyl methyl ester was attached to the chain and the ester part was removed to obtain a dipeptide, L-Phe-ala-Gly, attached to the ionic liquid based solid support. Finally, the formed dipeptide can be detached from the ionic liquid support by treating with small amount of NaOH in THF and water mixture followed by acidification at pH 5.0. The products were qualitatively as well as quantitatively analyzed using a .sup.1H NMR and HPLC analysis respectively.
Example 5
(46) Dipeptide Manufacture Via C-Terminal Approach
(47) The reaction mixture containing Boc-Leucine and ionic liquid based solid support at 1.1:1 mole ratio was charged in a peptide vessel glass reactor in the solvent ionic liquid under reduced pressure at room temperature for 2 to 6 h. At the end of the reaction, the solvent along with excess amino acid was first separated from the solid mass and then separated from the amino acids by extraction using dichloromethane. The Boc groups were eliminated from the Leucine moiety by treating with small amount of 1:1 v/v mixture trifluoroacetic acid in dichloromethane followed by acidification at pH 5. Similarly other N-terminal blocked Fmoc-Leucine was attached to the chain and the Fmoc part was removed by treating with piperidine in dichloromethane for 2 h to obtain a dipeptide attached to the ionic liquid based solid support. Finally, the formed peptide can be detached from the ionic liquid support by treating with trifluoroacetic acid and then with triethylamine. The products were qualitatively as well as quantitatively analyzed using a .sup.1H NMR and HPLC analysis respectively.
Example 6
(48) Recycling of the Ionic Liquid Support
(49) The functionalized ionic liquid used in the process was separated from the reaction mixture after use by washing with a solvent like water and solubilizing the subsidiary materials using an organic solvent selected from a group of dichloromethane, toluene, diethyl ether, acetone, etc. The ionic liquid was further dried under normal or reduced pressure at 120° C.
Example 7
(50) Recycling of the Ionic Liquid Solvent
(51) The ionic liquid used as the solvent in the process was separated from the reaction mixture after use by solubilizing the subsidiary materials using an organic solvent selected from a group of dichloromethane, toluene, diethyl ether, etc. The ionic liquid was further dried under reduced pressure at 120° C.
(52) The present invention is not limited to the embodiments discussed herein and can be embodied by various modifications within the scope of the following claims. It should be recognized that the preferred embodiments described above are exemplary only. Certain modifications and improvements will occur to the person skilled in the art upon a reading of forgoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.