# US 9,434,791 B2 · Method of preparation of an oxidized derivative of hyaluronic acid and a method of modification thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,434,791 B2 - Document ID: US9434791B2 - Status: Granted - Kind: B2 - Application number: 13/512,484 - Filed: 2010-12-10 - Published: 2012-10-18 - Granted: 2016-09-06 - Earliest claimed: 2009-12-11 - Assignees: Contipro Pharma a.s. - Applicants: Radovan Buffa; Sofiane Kettou; Lucie Pospisilova; Gloria Huerta-Angeles; Drahomira Chladkova; Vladimir Velebny - Inventors: Radovan Buffa; Sofiane Kettou; Lucie Pospisilova; Gloria Huerta-Angeles; Drahomira Chladkova; Vladimir Velebny - Web page: https://patentgrep.com/patent/US9434791B2 ## CPC - C08B 37/0072: {Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates} - C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21) ## Abstract The invention relates to a new method of preparation of a hyaluronan derivative with an aldehydic group in the position (6) of the polysaccharide glucosamine part. The hyaluronic acid oxidation can be performed by means of TEMPO/NaCIO or TEMPO/TCC systems in a protic environment with or without the presence of anorganic salts. Thus prepared aldehyde can be used for binding amines, diamines, amino acids, peptides and other compounds containing an amino group, e.g. by means of the reductive amination with NaBH3CN in water or in a water-organic solvent system. When a diamine or compounds containing three or more amino groups are used, cross-linked hyaluronan derivatives can be prepared. Cross-linked derivatives can be also prepared by a reaction of the aldehyde with a hyaluronan substituted by an amino-alkyl group HA-alkyl-NH2. ## Claims ### Claim 1 A method of modification of a hyaluronic acid derivative comprising: reacting hyaluronic acid with 4-R-TEMPO and a co-oxidant in a protic solvent, where R is a linear or branched alkyl chain, optionally containing aromatic or heteroaromatic groups, wherein the oxidized derivative of hyaluronic acid is selectively oxidized to an aldehyde at position 6 of the glucosamine portion of the derivative and 4-R-TEMPO is present in an amount within the range from 0.005 to 0.5 molar equivalent with respect to the hyaluronic acid; and reacting the oxidized hyaluronic acid derivative with either a compound of a general formula H2N-R, where the general formula H2N-R defines an amino acid, peptide, or polysaccharide containing a free amino group or where R is a linear or branched C1—C30 alkyl chain, optionally containing aromatic or heteroaromatic groups, thereby defining an amine, or with a hyaluronan, substituted by an -R-NH2 group, where R is a linear or branched C1—C30 alkyl chain, optionally containing aromatic or heteroaromatic groups, to form an imino form of HA of a general formula I ### Claim 2 (depends on 1) The method according to claim 1, wherein the co-oxidant includes sodium hypochlorite or TCC (trichlorocyanuric acid). ### Claim 3 (depends on 1) The method according to claim 1, wherein the hyaluronic acid is in the form of a free acid or a salt. ### Claim 4 (depends on 1) The method according to claim 1, wherein the hyaluronic acid molecular weight is within the range from 1·104 to 5·106 g·mol−1. ### Claim 5 (depends on 1) The method according to claim 1, wherein the protic solvent includes water and aqueous solutions of salts of a general formula MX, where M is an alkali metal and X is selected from the group including HCO3−, CO32−, PO43−, HPO42−, H2PO4− and halogens. ### Claim 6 (depends on 5) The method according to claim 5, wherein the concentration of the aqueous salt solutions is 0 to 15% by weight. ### Claim 7 (depends on 1) The method according to claim 1, wherein reaction of the hyaluronic acid, 4-R-TEMPO and the co-oxidant is performed at the temperature within the range from −10 to 20° C. for 5 minutes to 24 hours. ### Claim 8 (depends on 1) The method according to claim 1, wherein the co-oxidant is present in an amount within the range from 0.05 to 1 molar equivalent with respect to the hyaluronic acid. ### Claim 9 (depends on 1) The method according to claim 1, wherein the solvent is water or a mixture of water and a polar aprotic solvent with the content of water of at least 30% (v/v). ### Claim 10 (depends on 1) The method of modification according to claim 1, wherein the oxidized hyaluronic acid derivative reacts with the amino acid. ### Claim 11 (depends on 1) The method of modification according to claim 1, wherein the oxidized hyaluronic acid derivative reacts with the peptide. ### Claim 12 (depends on 1) The method of modification according to claim 1, wherein the oxidized hyaluronic acid derivative reacts with the polysaccharide containing a free amino group. ### Claim 13 (depends on 1) The method of modification according to claim 1, wherein the oxidized hyaluronic acid derivative reacts with the amine, amino acid, peptide, or polysaccharide and the amount of the amine, amino acid, peptide or polysaccharide is within the range from 0.05 to 10 molar equivalents with respect to the hyaluronic acid derivative. ### Claim 14 (depends on 1) The method of modification according to claim 1, wherein the oxidized hyaluronic acid derivative reacts with the amine, amino acid, peptide, or polysaccharide and the reaction of the oxidized hyaluronic acid derivative with the amine, amino acid, peptide or polysaccharide is performed in water or in a system of water-organic solvent at the temperature within the range from 0 to 80° C. for 1 minute to 24 hours. ### Claim 15 (depends on 14) The method of modification according to claim 14, wherein the oxidized hyaluronic acid derivative reacts with the amine, amino acid, peptide, or polysaccharide and the reaction of the oxidized hyaluronic acid derivative with the amine, amino acid, peptide or polysaccharide is performed in the presence of NaBH3CN as a reduction agent, which is added to the reaction mixture in time 0 to 100 hours after the addition of the amine, amino acid, peptide or polysaccharide, to form an amino form of HA of a general formula II ### Claim 16 (depends on 15) The method of modification according to claim 15, wherein the amount of NaBH3CN as a reduction agent is within the range from 0 to 20 molar equivalents with respect to the molar amount of the aldehyde. ### Claim 17 (depends on 14) The method of modification according to claim 14, wherein the organic solvent is selected from the group comprising water-miscible alcohols, and water-miscible polar aprotic solvents. ### Claim 18 (depends on 14) The method of modification according to claim 14, wherein the amount of water is at least 50% (v/v) with respect to the volume of the whole solution.