Synthesis and purification of pteroic acid and conjugates thereof
AssigneeEndocyte, Inc.
InventorsLe-Cun Xu, Iontcho Radoslavov Vlahov, Christopher Paul Leamon, Hari Krishna Santhapuram, Chunhong Li
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Abstract
Methods for purifying pteroic acid, analogs of pteroic acid, and derivatives of pteroic acid are described. Methods for synthesizing and purifying conjugates of vitamins, including FITC conjugates of folic acid, folic acid analogs, and derivatives of folic acid and folic acid analogs are also described. Purified forms of pteroic acid, derivatives and analogs of pteroic acid, and conjugates thereof are also described.
Claims21
A method for purifying pteroic acid, a derivative of pteroic acid, an analog of pteroic acid, or a combination thereof, the method comprising the steps of:
- (a) contacting a solution comprising pteroic acid, the derivative of pteroic acid, the analog of pteroic acid, or the combination thereof with an ion exchange chromatographic support;
- (b) eluting a first fraction comprising pteroic acid, the derivative of pteroic acid, or the analog of pteroic acid, with a mobile phase having a pH of about 10 or greater;
- and
- (c) lowering the pH of the first fraction to about 3 or less to form a precipitate of pteroic acid, the derivative of pteroic acid, or the analog of pteroic acid.
A method for purifying pteroic acid, a derivative of pteroic acid, an analog of pteroic acid, or a combination thereof, the method comprising the steps of:
- (a) contacting a solution comprising pteroic acid, the derivative of pteroic acid, the analog of pteroic acid, or the combination thereof with an anion exchange chromatographic support selected from the group consisting of diethylaminoethyl cross-linked dextran, quaternary amine cross-linked dextran, polyethyleneimine cellulose, quaternary amine cellulose, diethylaminoethyl cellulose, and combinations thereof;
- and
- (b) eluting a first fraction comprising pteroic acid, the derivative of pteroic acid, or the analog of pteroic acid.
A method for purifying a conjugate comprising pteroic acid, a derivative thereof, or an analog of pteroic acid, and fluorescein or a derivative of fluorescein, the method comprising the steps of:
- (a) contacting a solution comprising the conjugate with a first reversed phase chromatographic support;
- (b) eluting from the first reversed phase chromatographic support a fraction comprising a phosphate complex of the conjugate with a mobile phase, said mobile phase comprising a phosphate salt and having a pH in the range from about 6 to about 8;
- (c) contacting a solution of the phosphate complex of the conjugate with a second reversed phase chromatographic support;
- and
- (d) eluting from the second reversed phase chromatographic support a fraction comprising the conjugate with a mobile phase comprising water, where the fraction comprising the conjugate is substantially free of phosphate.
Dependencies are inferred from the claim text.