Methods for the preparation of a polyalkoxylated nucleic acid molecule
A method of obtaining a polyalkoxylated nucleic acid, which can be used to make a modified drug, from a liquid mixture of polyalkoxylated and non-polyalkoxylated nucleic acids is described. The method involves use of a solvent or a mixture of solvents that allow the polyalkoxylated nucleic acids to precipitate out of the solution.
1. A method for obtaining a polyalkoxylated nucleic acid molecule comprising separating said polyalkoxylated nucleic acid molecule from a liquid mixture comprising said polyalkoxylated nucleic acid molecule and a non-polyalkoxylated nucleic acid molecule by precipitating said polyalkoxylated nucleic acid molecule from said mixture,
wherein said mixture comprises a solution comprising a solvent or a mixture of solvents;
wherein said polyalkoxylated nucleic acid molecule comprises a nucleic acid moiety and a polyalkoxylate moiety, and said non-polyalkoxylated nucleic acid molecule comprises a nucleic acid moiety and is lacking a polyalkoxylate moiety;
wherein said polyalkoxylated nucleic acid molecule comprises a plurality of polyalkoxylated nucleic acid molecules comprising a plurality of nucleic acid moieties;
wherein said non-polyalkoxylated nucleic acid molecule comprises a plurality of non-polyalkoxylated nucleic acid molecules comprising a plurality of nucleic acid moieties; and
wherein said precipitated polyalkoxylated nucleic acid molecule is separated from said non-polyalkoxylated nucleic acid molecule by separating liquid from solid.
2. The method according to claim 1 , wherein said solvent comprises water and/or a water miscible organic solvent.
3. The method according to claim 1 , wherein said precipitating comprises a temperature of 50° C. to 30° C.; a temperature of −25° C. to 25° C.; a temperature of −20° C. to 4° C.; a temperature of −20° C.; or a temperature of 4° C.
4. The method according to claim 1 , wherein said precipitating comprises a pH range of 4 to 11; a pH range of 6 to 10; or a pH range of 7 to 9.5.
5. The method according to claim 1 , wherein said precipitating is carried out until 75% to 100% of said polyalkoxylated nucleic acid molecule is precipitated; or until 90% to 100% of said polyalkoxylated nucleic acid molecule is precipitated.
6. The method according to claim 1 , wherein said polyalkoxylate moiety comprises polyethylene glycol, polypropylene glycol, polybutylene glycol or polyglycerol.
7. The method according to claim 1 , wherein said polyalkoxylate moiety comprises a molecular weight of 5,000 Da to 100,000 Da.
8. A method for preparing a polyalkoxylated nucleic acid molecule comprising a nucleic acid moiety and a polyalkoxylate moiety, comprising reacting a nucleic acid molecule with a polyalkoxylate in presence of a quaternary ammonium compound forming said polyalkoxylated nucleic acid molecule, wherein said nucleic acid molecule forms said nucleic acid moiety of said polyalkoxylated nucleic acid molecule; said polyalkoxylate forms said polyalkoxylate moiety of said polyalkoxylated nucleic acid molecule; and said quaternary ammonium compound is dissolved in water, in a water miscible organic solvent, or in a combination thereof, wherein said nucleic acid molecule comprises a reactive group, wherein when there is more than one reactive group, said reactive groups are different, and said polyalkoxylate comprises a reactive group that reacts with said nucleic acid molecule comprising a reactive group of the nucleic acid molecule forming said polyalkoxylated nucleic acid molecule;
wherein said reactive group of said nucleic acid molecule comprises an amine, a thiol, an azide, an alkyne, a carboxylate, a carboxylic acid ester, an aldehyde, an iodoalkyl or a maleimide; and
wherein said reactive group of the polyalkoxylate comprises an amine, a thiol, an azide, an alkyne, a carboxylate, a carboxylic acid ester, an aldehyde, an N-hydroxysuccinimide, an iodoalkyl or a maleimide.
9. The method according to claim 8 , wherein said quaternary ammonium compound comprises tetraalkylammonium chloride, tetraalkylammonium bromide, tetraalkylammonium tetrafluoroborate tetraalkylammonium hexafluorophosphate, tetraalkylammonium hydrogen sulphate or tetraalkylammonium dihydrogen phosphate, wherein alkyl is an alkyl chain of 1 to 18 C atoms.
10. The method according to claim 8 , wherein said water miscible organic solvent comprises dimethyl sulfoxide, diethyl sulfoxide, methyl ethyl sulfoxide, methyl formamide, dimethyl formamide, ethyl formamide, ethyl methyl formamide, diethyl formamide, 2-pyrrolidone, N-methyl-2-pyrrolidone, N-ethyl-2-pyrrolidone, acetonitrile, acetone, ethyl methyl ketone, methyl propyl ketone, diethyl ketone, methyl isopropyl ketone, methyl formate, ethyl formate, propyl formate, isopropyl formate, methyl acetate, ethyl acetate, methyl propanoate, tetrahydrofuran or dioxan.
11. The method according to claim 8 , wherein said reacting comprises 1 to 20 mole or 2 to 10 mole equivalents of said quaternary ammonium compound per nucleotide of said nucleic acid molecule.
12. The method according to claim 8 , wherein said reacting comprises a temperature of 10° C. to 50° C. or a temperature of 20° C. to 40° C.
13. The method according to claim 8 , wherein said reacting comprises a pH range of 4 to 11 or a pH range of 6 to 10.
14. The method according to claim 8 , wherein said polyalkoxylate moiety comprises a molecular weight of 5,000 Da to 100,000 Da.
15. The method according to claim 8 , wherein said polyalkoxylate moiety comprises polyethylene glycol, polypropylene glycol, polybutylene glycol or polyglycerol.
16. The method according to claim 8 , wherein said quaternary ammonium compound comprises tetrabutylammonium bromide.
17. The method according to claim 8 , wherein said reactive group of said nucleic acid molecule comprises an amine and said reactive group of said polyalkoxylate comprises a carboxylic acid ester.