# Patent

## Title
Method of preparation of an oxidized derivative of hyaluronic acid and a method of modification thereof

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase of PCT/CZ2010/000129, filed Dec. 10, 2010, which claims priority to Czech Republic application No. PV 2009-835, filed Dec. 11, 2009, the disclosures of which are hereby incorporated by reference herein. This application is also related to PCT/CZ2010/000128, filed Dec. 10, 2010.
FIELD OF THE INVENTION
The present invention relates to a new method of preparation of a hyaluronic acid derivative containing an aldehydic group —CH═O instead of the primary hydroxyl group —CH2—OH. The oxidation can be performed in protic solvents by use of a system of 4-substituted-TEMPO (2,2,6,6-tetramethyl-piperidinyloxyl)/co-oxidant:




wherein R is hydrogen or any substitute and the co-oxidant is sodium hypochlorite (NaClO) or trichloroisocyanuric acid (TCC).

BACKGROUND OF THE INVENTION
The hyaluronic acid is an important polysaccharide, composed of two repeating units of β-(1,3)-D-glucuronic acid and β-(1,4)-N-acetyl-D-glucosamine. The molecular weight, depending on the method of isolation and on the source material, is within the range from 5·104 to 5·106 g·mol−1. The hyaluronic acid or its sodium salt hyaluronan is an essential component of connective tissues, synovial fluid of joints, and plays an important role in biological processes, such as hydratation, organization of proteoglycans, cell differentiation, proliferation and angiogenesis. The hyaluronic acid is a considerably hydrophilic polysaccharide and is soluble in water in the form of a salt in the whole range of pH.




Oxidation of the Hyaluronic Acid
Oxidation of polysaccharides is a process in which the oxidation degree of the polysaccharide functional groups is changing. Most frequently, carboxylic acids or aldehydes are formed, which can dramatically change the properties of the polysaccharide. In most cases, the reaction is performed by use of agents containing atoms in higher oxidation degrees.
The method of selective oxidation of saccharides on the primary hydroxyl group, described in Angelino, European Journal of Organic Chemistry 2006, 19, 4323-4326, the system of 2,2,6,6-tetramethyl-1-piperidinyloxyl radical TEMPO/TCC in DMF at the temperature of 0° C. was used, with the corresponding aldehyde as the main product.
TEMPO is a relatively stable radical which can exist in reaction in three redox forms. Only the oxidized form of TEMPO is effective for alcohols or geminal diols oxidation.




A catalytic amount of TEMPO is added to the reaction and therefore, it is necessary to use a co-oxidant which restores the presence of the TEMPO oxidized form.
2,2,6,6-tetramethyl-1-piperidinyloxyl radical (TEMPO)- and NaOCl-mediated oxidation of the primary hydroxyl group of hyaluronan to a carboxylic acid was performed at pH 10.2 and at the temperature of 0° C. (Scheme 2) (Carbohydr Res 2000, 327 (4), 455-61).




Analogous to other polysaccharides, a high regioselectivity and slight degradation of the polymer were observed. An increase of the concentration of the salt (NaBr, NaCl, Na2SO4) in the solution caused a decrease in the oxidation rate.

Oxidation of hyaluronan by use of TEMPO/NaClO system was described in the patent application WO 02/18448 A2. The authors also dealt with interactions of percarboxylated polysaccharides, while forming biological complexes.
The rate of oxidation of HA and other polysaccharides by use of sodium periodate was studied by Scott et al. (Scheme 3) (Histochemie 1969, 19 (2), 155-61).




The factors such as the chain length, substitution, polymer configuration and temperature were studied and quantified. The use of NaIO4 for an oxidation of hyaluronan was also disclosed in the U.S. Pat. No. 6,683,064 and U.S. Pat. No. 6,953,784.

Model reactions of HA analogues with low molecular weight in a physiological buffer were studied (Carbohydr Res 1999, 321, (3-4), 228-34). Oxidation products of the glucuronic and glucosamine parts were identified by GC-MS analysis. The results also suggest that the oxidation is performed primarily on the glucuronic part, while the meso-tartaric acid is the main product and may be used as a biomarker of the hyaluronan oxidation.
Use of an Oxidized HA in Cross-Linking Reactions
The use of an oxidized HA for the preparation of cross-linked hydrogels was described by Weng et al. (Scheme 4), J Biomed Mater Res A 2008, 85 (2), 352-65. Two precursors were used in this case: a partially oxidized hyaluronan and gelatin:




The physico-chemical properties of the resulting hydrogels have been elucidated by instrumental analyses FT-IR, SEM (scanning electron Microscopy) and rheometry. Increasing the oxidation degree of the hyaluronan lead to a corresponding increase of hydrogels compatibility and decrease of water absorption capacity. Dermal fibroblasts were used to study the cell-hydrogel interactions. Both the hydrogels and their degradation products are biocompatible, as proved by the long-term cell viability assay. When cultured with cells, the hydrogel underwent a degradation within 4 weeks, with an obvious loss of cohesiveness. The good biocompatibility and biodegradability was further demonstrated in mice subdermal implantations. Lastly, in vitro and in vivo depositions of extracellular matrix in hydrogels were demonstrated by SEM analysis.

The method of preparation of cross-linked HA from an oxidized hyaluronan and gelatin by a water-in-oil-emulsion method, where a 3-dimensional hydrogel is formed in the absence of any external cross-linker, was described in the publication of Weng et al., Biomaterials 2008, 29, (31), 4149-56. In this work, incorporation of model drugs into the hydrogel structure (encapsulation) and their releasing through macrophages were studied by HPLC.
The preparation of elastic hydro gels by coupling the HA oxidized to HA-aldehyde by means of sodium periodate and the HA modified with adipic acid dihydrazide, was described by Sahiner et al., (Scheme 5), J. Biomater. Sci. Polym. Ed 2008, 19 (2), 223-43.




The resulting derivatives did not have any observable effect on the proliferation of the cultured fibroblasts, as shown by a MTT assay.

SUMMARY OF THE INVENTION
The present invention relates to an improvement of a method of selective oxidation of the primary hydroxyl group of the hyaluronic acid in the position 6 of the polysaccharide glucosamine part to aldehyde. The new reaction is performed using a system of 2,2,6,6-tetramethyl-1-piperidinyloxyl radical TEMPO/co-oxidant. Approaches published up to date have introduced either an aldehydic group to the position 2 and 3 of the hyaluronan glucuronic part, while opening the saccharide ring (scheme 6, structure 2), or a carboxyl group to the position 6 of the hyaluronan glucosamine part (scheme 6, structure 1).




The method according to the invention is more advantageous in that the respective oxidation product (structure 3, scheme 6) maintains the structure of conjugated saccharide rings. Ring opening in the product oxidized to dialdehyde (structure 2, scheme 6) gives rise to the chain linearity “breakage” and, therefore, a significant change of the polysaccharide 3-dimensional structure compared to the non-modified hyaluronan. Although in the product oxidized to a carboxylic acid (structure 1, scheme 6) the chain linearity “breakage” does not occur, the carboxyl group does not enable such various modification (binding) possibilities like the aldehydic group. As the carboxyl group is a component of the non-modified polysaccharide already, the oxidation to the structure 1 (scheme 6) brings only an increase of the polysaccharide polarity, not a development of new quality utilizable for binding new substitutes.
It is known that an aldehydic group with a bound alkyl group exists in water in the so-called geminal diol form HA-CH(OH)2, which reacts with nucleophiles similarly as aldehydes do. In aqueous solutions, more than 95% of the hyaluronan oxidized in the position 6 of the glucosamine part (product 3, scheme 6) exists in the form of a geminal diol, as demonstrated by NMR spectroscopy.
In method according to the present invention, the hyaluronic acid is dissolved in water, then an oxidizing agent is added and the mixture is stirred at the temperature of −10 to 20° C., preferably at 0° C., for 1 to 150 hours, preferably for 24 hours.
The prepared oxidized hyaluronan can be used for binding compounds containing for example an amino group. The binding can be realized either in an imine form or after the reduction in an amine form (reductive amination) (scheme 7):




Both degrees of this modification are performed in an aqueous solution, the reduction is performed by means of NaBH3CN. Both degrees of the reaction described in scheme 7 can be performed in one step.
The modification of the hyaluronic acid derivative can be performed by a reaction of the oxidized derivative with an amine of the general formula H2N—R or with a hyaluronan substituted by an —R—NH2 group, wherein R is an alkyl, linear or branched chain C1-C30, optionally containing aromatic or heteroaromatic groups. This amine can be an alkylamine, e.g. butylamine or hexanediamine, amino acid, peptide or polysaccharide containing a free amino group. In case of using a diamine or compounds containing three or more amino groups, cross-linked hyaluronan derivatives can be prepared. The cross-linked derivatives can be prepared also by the reaction of an aldehyde with a hyaluronan substituted by an aminoalkyl group HA-alkyl-NH2.


DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
DS=substitution degree=100%*the molar amount of the bound substitute/the molar amount of all the polysaccharide dimers.
The term “equivalent (eq)” as used herein, refers to a hyaluronic acid dimer, unless otherwise indicated. The percentages as used herein mean a percentage by weight, unless otherwise indicated.
The molecular weight of the starting hyaluronan (source: CPN spol. s r.o., Dolní Dobrou{hacek over (c)}, CZ) was determined by the SEC-MALLS assay.
Example 1
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight of NaCl), TEMPO (0.01 eq) and NaHCO3 (30 eq). The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 10% (determined by NMR)
1H NMR (D2O) δ5.26 (s, 1H, polymer-CH(OH)2)
HSQC (D2O) cross signal 5.26 ppm (1H)-90 ppm (13C) (polymer-CH(OH)2)
FT-IR (KBr) 1740 cm−1 —CH═O
Example 2
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (0.5%) (0.1 g, 2000 kDa) (containing 10% by weight of NaCl), TEMPO (0.01 eq) and NaHCO3 (30 eq). The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 10% (determined by NMR, for details see Example 1)
Example 3
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight of NaCl), 4-acetamido-TEMPO (0.01 eq) and NaHCO3 (30 eq). The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 10% (determined by NMR, for details see Example 1)
Example 4
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaCIO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 10% by weight of NaCl), 4-acetamido-TEMPO (0.05 eq) and NaHCO3 (30 eq). The mixture was stirred for 10 hours at the temperature of −10° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 10% (determined by NMR, for details see Example 1)
Example 5
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaClO (0.3 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 20 kDa), 4-acetamido-TEMPO (0.1 eq) and NaHCO3 (30 eq). The mixture was stirred for 1 hour at the temperature of 20° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 5% (determined by NMR, for details see Example 1)
Example 6
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
4-acetamido-TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution of hyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture of N,N-dimethylformamide/water 70/30. The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 5% (determined by NMR, for details see Example 1)
Example 7
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
4-acetamido-TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution of hyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture of N,N-dimethylformamide/water 90/10. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 10% (determined by NMR, for details see Example 1)
Example 8
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
4-acetamido-TEMPO (0.01 eq) and TCC (1 eq) were added to a solution of hyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture of N,N-dimethylformamide/water 40/60. The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 15% (determined by NMR, for details see Example 1)
Example 9
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
4-acetamido-TEMPO (0.01 eq) and TCC (1 eq) were added to a solution of hyaluronic acid (0.5%) (0.1 g, 2000 kDa) in the mixture of N,N-dimethylformamide/water 40/60. The mixture was stirred for 24 hours at the temperature of −5° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 15% (determined by NMR, for details see Example 1)
Example 10
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
TEMPO (0.01 eq) and TCC (0.2 eq) were added to a solution of hyaluronic acid (1%) (0.1 g, 20 kDa) in the mixture of N,N-dimethylformamide/water 70/30. The mixture was stirred for 1 hour at the temperature of 20° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 5% (determined by NMR, for details see Example 1)
Example 11
Oxidation of the Hyaluronic Acid by the TEMPO/TCC System
TEMPO (0.01 eq) and TCC (0.2 eq) were added to a hyaluronic acid solution (1%) (0.1 g, 20 kDa) in the mixture of DMF/water 70/30. The mixture was stirred for 100 hours at the temperature of −5° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS (determined by NMR, for details see Example 1)
Example 12
Oxidation of the Hyaluronic Acid by the TEMPO/NaBrO System
An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 20 kDa) (containing 5% by weight of NaBr), 4-acetamido-TEMPO (0.5 eq) and NaHCO3 (30 eq). The mixture was stirred for 24 hours at the temperature of 0° C. The solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaClO, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 14% (determined by NMR, for details see Example 1)
Example 13
Oxidation of the Hyaluronic Acid by the TEMPO/NaClO System
An aqueous solution of NaClO (0.5 eq) was gradually added to an aqueous solution of hyaluronan (1%) (0.1 g, 200 kDa) (containing 10% by weight of NaBr), TEMPO (0.5 eq) and NaHCO3 (5 eq). The mixture was stirred for 24 hours at the temperature of 0° C. The solution was then diluted to 0.2% with distilled water and dialyzed against mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 18% (determined by NMR, for details see Example 1)
Example 14
Reaction of the Oxidized Hyaluronan with an Amine
Butylamine (0.4 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then precipitated with a double amount of acetone and 0.1 ml of a saturated aqueous solution of NaCl, filtrated and dried in vacuum. The resulting yellow material was then analyzed.
UV-VIS 328 nm, n→π*transition —CH═N—
Example 15
Reaction of Oxidized Hyaluronan with Butylamine and Subsequent Reduction
Butylamine (0.4 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN in 0.5 ml of water (3 eq). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 15% (determined by NMR)
1H NMR (D2O) δ3.05 (m, 2H, polymer-CH2—NH—CH2—), 1.60 (m, 2H, polymer-CH2—NH—CH2—CH2—), 1.35 (m, 2H, polymer-CH2—NH—CH2—CH2—CH2—), 0.85 (m, 3H, —CH2—CH3)
DOSY NMR (D2O) log D (0.85 ppm, —CH2—CH3) ̃−10.3 m2/s

    
    
        log D (1.35 ppm, polymer-CH2—NH—CH2—CH2—CH2—) ̃−10.3 m2/s
        log D (1.60 ppm, polymer-CH2—NH—CH2—CH2—) ̃−10.3 m2/s
        log D (3.05 ppm, polymer-CH2—NH—CH2—) ̃−10.3 m2/s
        log D (2.03 ppm, CH3—CO—NH-polymer) ̃−10.3 m2/s
        log D (H2O) ̃−8.6 m2/s
    
    


Example 16
Reaction of the Oxidized Hyaluronan with a Diamine and the Subsequent Reduction
Hexanediamine (0.4 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN (3 eq) in 0.5 ml of water. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 16% (determined by NMR)
1H NMR (D2O) δ3.12 (m, 2H, polymer-CH2—NH—CH2—), 3.02 (m, 2H, —CH2—NH2), 1.7 (m, 4H, —NH—CH2—CH2—CH2—CH2—CH2—), 1.45 (m, 411, —NH—CH2—CH2—CH2—CH2—CH2—)
DOSY (D2O) log D (1.45 ppm, —NH—CH2—CH2—CH2—CH2—CH2—) ̃−10.5 m2/s

    
    
        log D (1.7 ppm, —NH—CH2—CH2—CH2—CH2—CH2—) ̃−10.5 m2/s
        log D (3.02 ppm, —CH2—NH2) ̃−10.5 m2/s
        log D (2.03 ppm, CH3—CO—NH-polymer) ̃−10.5 m2/s
        log D (H2O) ̃−8.7 m2/s
    
    


Example 17
Reaction of the Oxidized Hyaluronan with an Amine-Hyaluronan
A 1% aqueous solution of hyaluronan derivative substituted by hexanediamine (1 eq, DS=16%, Example 16) was added to an aqueous solution of oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13) at the temperature of 20° C. The resulting insoluble compact gummy precipitate, obtained after several minutes, was mechanically ground to small pieces, filtrated and dried under reduced pressure.
FT-IR (KBr) 1700 cm−1 
Example 18
Reductive Amination of the Oxidized Hyaluronan with Lysine
Lysine (0.3 eq) was added to an aqueous solution of oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN (3 eq) in 0.5 ml of water. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 14% (determined by NMR)
1H, HSQC, DOSY NMR (2% NaOD/D2O): δ1.33 (m, 2H, —CH—CH2—CH2—), 1.48 (m, 2H, —CH—CH2—CH2—CH2—), 1.55 (m, 1H, —CH—CHH—), 1.63 (m, 1H, —CH—CHH—), 2.62 (m, 2H, —CH—CH2—CH2—CH2—CH2—), 2.65 (m, 1H, polymer-CHH—NH—), 2.99 (m, 1H, polymer-CHH—NH—), 3.16 (m, 1H, —CH—CH2—).
Example 19
Reductive Amination of the Oxidized Hyaluronan with Lysine
Lysine (20 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN (10 eq) in 0.5 ml of water. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 17% (determined by NMR, Example 10)
Example 20
Reductive Amination of the Oxidized Hyaluronan with Serine
Serine (0.3 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 1 min at the temperature of 20° C. The solution was then mixed with NaBH3CN (3 eq) in 0.5 ml of water.
The mixture was stirred for 24 hours at the temperature of 20° C. The solution was diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 14% (determined by NMR)
1H, HSQC, DOSY NMR (2% NaOD/D2O): δ2.74 (m, 1H, polymer-CHH—NH—), 3.08 (m, 1H, polymer-CHH—NH—), 3.21 (m, 1H, —CH—CH2—OH), 3.72 (m, 1H, —CH—CHH—OH), 3.78 (m, 1H, —CH—CHH—OH).
Example 21
Reductive Amination of the Oxidized Hyaluronan with Arginine
Arginine (0.3 eq) was added to an aqueous solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13). The mixture was stirred for 100 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN (3 eq) in 0.5 ml of water. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was evaporated and analyzed.
DS 11% (determined by NMR)
1H, HSQC, DOSY NMR (2% NaOD/D2O): δ1.61 (m, 2H, —CH—CH2—CH2—), 1.63 (m, 1H, —CH—CHH—), 1.70 (m, 1H, —CH—CHH—), 2.65 (m, 1H, polymer-CHH—NH—), 3.01 (m, 1H, polymer-CHH—NH—), 3.13 (m, 1H, —CH—CH2—), 3.21 (m, 2H, —CH—CH2—CH2—CH2—).
Example 22
Reductive Amination of the Oxidized Hyaluronan with Pentapeptide PAL-KTTKS (palmytoyl-Lys-Thr-Thr-Lys-Ser)
1% solution of substituted pentapeptide PAL-KTTKS (0.05 eq) in isopropyl alcohol was added to a solution of the oxidized hyaluronic acid (1%) (0.1 g, oxidation degree DS=18%, Example 13) in the system of water/isopropanol 2/1. The mixture was stirred for 24 hours at the temperature of 20° C. The solution was then mixed with NaBH3CN in 0.5 ml of water (3 eq). The mixture was stirred for 24 hours at the temperature of 20° C. The resulting solution was diluted to 0.1% and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO3) 3×5 liters (once per day) and against distilled water 7×5 liters (twice per day). The resulting solution was then evaporated and analyzed.
DS 9% (determined by NMR)
1H, HSQC, DOSP NMR (2% NaOD/D2O): δ1.61 (m, 2H, —CH—CH2—CH2—), 1.63 (m, 1H, —CH—CHH—), 1.70 (m, 1H, —CH—CHH—), 2.65 (m, 1H, polymer-CHH—NH—), 3.01 (m, 1H, polymer-CHH—NH—), 3.13 (m, 1H, —CH—CH2—), 3.21 (m, 2H, —CH—CH2—CH2—CH2—).

## Claims

1. A method of preparation of an oxidized derivative of hyaluronic acid 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.

2. The method of preparation according to claim 1, wherein the co-oxidant includes sodium hypochlorite or TCC (trichlorocyanuric acid).

3. The method of preparation according to claim 1, wherein the hyaluronic acid is in the form of a free acid or a salt.

4. The method of preparation according to claim 1, wherein the hyaluronic acid molecular weight is within the range from 1·104 to 5·106 g·mol−1.

5. The method of preparation 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.

6. The method of preparation according to claim 5, wherein the concentration of the aqueous salt solutions is 0 to 15% by weight.

7. The method of preparation 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.

8. The method of preparation 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.

9. The method of preparation 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).

10. A method of modification of the hyaluronic acid derivative prepared by the method of claim 1 comprising:
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

11. The method of modification according to claim 10, wherein the oxidized hyaluronic acid derivative reacts with the amino acid.

12. The method of modification according to claim 10, wherein the oxidized hyaluronic acid derivative reacts with the peptide.

13. The method of modification according to claim 10, wherein the oxidized hyaluronic acid derivative reacts with the polysaccharide containing a free amino group.

14. The method of modification according to claim 10, 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.

15. The method of modification according to claim 10, 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.

16. The method of modification according to claim 15, 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

17. The method of modification according to claim 16, 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.

18. The method of modification according to claim 15, wherein the organic solvent is selected from the group comprising water-miscible alcohols, and water-miscible polar aprotic solvents.

19. The method of modification according to claim 15, wherein the amount of water is at least 50% (v/v) with respect to the volume of the whole solution.