Field of the invention

The invention relates to oligosaccharides composed of alternating glucose and N- acetylglucosamine units, with an unsaturated glucose unit on the non-reducing end and a reduced anomeric end according to the structural formula I: where n = 0 to 10 the production thereof and methods of use thereof. The oligosaccharide having this structure shows an increased resistance to enzymatic degradation and it negatively affects the growth of certain cancer cell lines.

State of the art

Stepwise synthesis of oligosaccharides

The synthesis of penta- and hexasaccharides with alternating units of glucose and N- acetyl glucosamine linked by a 1— >3 bond with a methoxyphenyl group at the reducing end was described in the article (Halkes, K. M. et al.: Carbohydrate Research, 30, 161-174, 1998). Trichloroacetimidate activation catalyzed by boron fluoride or trimethyl silyl trifluoromethanesulphonate was used for coupling of protected carbohydrates in yield 69-81%.

In publication (Lin, Ch. -Ch. et al.: Journal of the Chinese Chemical Society, 47, 921- 928, 2000) the preparation of the disaccharide B-D-Glucopyranosyl( l ^3)- l -thiol-B- glucosamine as a building block for the production of longer oligosaccharides was described. The glucose unit donor is synthesized in 9 steps, the glucosamine acceptor in 5 steps, the coupling of both units and the change of protecting groups proceeds in 3 steps.

One-pot synthesis of a protected disaccharide, tetrasaccharide, and hexasaccharide with alternating glucose and N-acetylglucosamine units linked by a 1— >3 bond was disclosed in article (Huang, L.; Huang, X.: Chemistry - a European Journal, 13, 529-540, 2007). The monosaccharides or disaccharides were attached with a 54-75% yield for glycosylation.

In general, it can be stated that the stepwise synthesis of oligosaccharides is demanding, involves many protection and deprotection steps, working in toxic solvents and has a low yield, especially for longer oligosaccharides. A disadvantage is also the necessary and often difficult control of the stereochemistry of the resulting glycosidic bonds. Another disadvantage can be the use of stringent conditions (strong acidic environment), which can lead to the decomposition of carbohydrate cycles containing a double C=C bond.

Carbohydrates containing multiple bonds

The production of unsaturated esters of uronic acids by means of P-elimination in a basic environment was described (BeMiller, J. N. et al.: Carbohydrate research, 25, 419-428, 1972). In order to avoid undesirable hydrolysis, the elimination was performed with sodium methoxide in methanol. Alginates are depolymerized more slowly than pectins.

By heating of pectin in a weak acidic aqueous solution (pH=6.1), shorter fragments containing a double bond can be obtained (Sajjaanantakul, T. et al.: Journal of Food Science, 54, 5, 1272-1277, 1989). The intensity of the cleavage increases with the extent of esterification of the carboxyl functions by the methyl group. In this work, no more than 2% of glycosidic bonds were cleaved.

The production of a saccharide having a double bond in positions 4 and 5 was described (Mathad, V. T. et al.: Indian Journal of Chemistry, 36B, 808-809, 1997). In the first step, acetylation of hydroxyl groups is performed, followed by oxidation of the position 6 to an aldehyde with a simultaneous formation of a double bond, then the aldehyde is reduced by NaBHj and finally the acetyl groups are removed in a basic environment.

Several methods have been described to introduce a multiple -C=C- bond directly into the saccharide cycle in the polymer chain. One of them is the enzymatic cleavage of polymers using lyases, where a double bond is formed at the non-reducing end of the polymer (Kelly S. J. et al. Glycobiology, 11, 4, 294-304, 2001), in this case hyaluronic acid.

Another method allows to introduce a double bond into the structure of various glycosaminoglycans at positions 4 and 5 along the entire length of the chain, so that even polymers having a higher molecular weight can be effectively modified (Buffa R. et al. CZ305106, WO2014023272A1). These materials showed a selective negative effect on the cancer cells viability. Furthermore, an alkaline cleavage of the chondroitin sulphate benzyl ester in the mixture of DMF/EtOH/EtONa was disclosed (Gao, N. et al.: Carbohydrate Polymers, 127, 427-437, 2015). After de-esterification the resulting oligosaccharides showed better anticoagulant effects than a low-molecular-weight heparin.

In general, it can be stated that unsaturated oligosaccharides can be produced by enzymatic or chemical cleavage. Chemical cleavage is most often performed basicly and requires an activation, for example by converting an alcohol to an aldehyde or a carboxylic acid to its ester. Both oligosaccharides and polysaccharides can be used for cleavage.

Hyaluronic acid

Hyaluronic acid or its sodium salt is an non-sulphated glycosaminoglycan composed of two repeating units of D -glucuronic acid and 7V-acetyl-D-glucosamine (Formula II).

Formula II.

The molecular weight of the native hyaluronic acid can reach up to 5.10 ⁶ g.mol' ¹ . This polysaccharide forms an important part of connective tissues, skin and synovial fluid of joints and plays an important role in a number of biological processes such as hydration, cell differentiation and proteoglycans organization. Hyaluronic acid occurs naturally in biological systems, making it naturally biodegradable and biocompatible. Therefore, it is a suitable substrate for a wide range of biomedical applications.

Hyaluronic acid is degraded in biological conditions by hydrolases and lyases. In the first case, the products retain the structure according to the formula II, only the chain is shortened. When hyaluronic acid is cleaved by lyase, a chain with a double bond is formed at the terminal saccharide at the non-reducing end (Formula III). The biological effects of oligomeric and polymeric hyaluronic acid differ significantly (Russo, R. I. C. et al.: International journal of cancer, 122, 1012-1018, 2008), it also depends on the way of hyaluronan cleavage (Jobe, K. L. et al.: Immunology letters, 89, 99-109, 2003).

Formula III.

Reduction of carboxylic acids

The reduction of aliphatic carboxylic acids, which contain acetyl and other ester groups in their structure, can be accomplished by means out using a dimethyl sulfide/borane complex in tetrahydrofuran (Williams S. J. et al.: Journal of the American Chemical Society, 122, 10, 2223 - 2235, 2000). The reaction proceeds at 20 °C, with a longer reaction time required (up to 72 hours).

The use of boranes generated by the system (NaBHj + H2SO4) for the reduction of aliphatic carboxylic acids containing ether groups was described in the patent (Haidar P. et al.: US250454, 2011). The reaction proceeded for 45 minutes in tetrahydrofuran and diethyl ether in the temperature range from 0 - 35 °C.

The selective reduction of aliphatic carboxylic acids, which contain hydroxyl groups and ethers in their structure, has been disclosed in the article (Murray J. et al.: Chem. Comm., 50, 88, 13608 - 13611, 2014). The reaction proceeds for 12 hours at 0 to 60 °C in an inert atmosphere and the system (NaBHj + I2) in tetrahydrofuran was used as the reducing agent.

An effective reduction of low molecular weight aliphatic and aromatic carboxylic acids to the respective alcohols can be achieved with an excess (3-4 equivalents) of pinacol borane at 20 °C in absence of solvents and catalyst (Harinath A. et al.: Chem. Comm., 10, 55, 1386-9, 2019). However, this approach cannot be applied for molecules containing a large number of hydroxyl groups, which would react rapidly with borane to form hydrogen.

In general, it can be stated that in the reduction of carboxylic acids either directly with boranes, or with boranes complexed with dimethyl sulfide, or with boranes generated in situ, the use of protic solvents is unsuitable due to the rapid reaction of boranes with the hydrogen of the solvent, forming the molecular hydrogen. These reactions proceed efficiently either in aprotic systems (ethers) or in absence of a solvent. The reduction of aliphatic carboxylic acids, which contain ester groups in their structure, can be carried out by a two-step synthesis, where in the first step the carboxyl group is activated by means oxalyl dichloride in N,N-dimethylformamide, tetrahydrofuran, dichloromethane or acetonitrile at -78 to +20 °C , and in the second step the activated carboxyl group is reduced with lithium tri-(tert-butoxy)aluminum hydride in tetrahydrofuran, dichloromethane and acetonitrile at 20 °C for 1.5 hours (Chany A.-C. et al.: Organic and Biomolecular Chemistry, 13, 35, 9190 - 9193, 2015).

The reduction of the hyaluronic acid hexasaccharide modified on the carboxyl to methyl ester was disclosed in the article (Onoera K. et al.: Agricultural and Biological Chem., 27, 2, 143-149, 1963). LiAlEU in tetrahydrofuran was used as the agent.

Even in the reduction of carboxylic acids with aluminum-based hydrides, the use of protic solvents is unsuitable due to the rapid reaction of aluminum hydrides with the hydrogen of the solvent to form molecular hydrogen. Reductions proceed efficiently in aprotic solvents such as tetrahydrofuran, dichloromethane or acetonitrile.

In the article (Montchamp J.-L. et al.: Journal of the American Chemical Society, 114, 12, 4453, 1992) the reduction of aliphatic carboxylic acids, which also contain hydroxyl groups and ethers in their structure, was disclosed, by means of carboxyl activation with triethyl orthoformate and 4-methylmorpholine-N-oxide. The subsequent reduction was carried out using NaBEU with osmium oxide in ethanol and water. Racemization at the alpha carboxyl position was observed as a byproduct.

NaBEU has been used to reduce a wide range of carboxylic acids after in situ activation with benzotriazole-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate. The reaction proceeds rapidly under mild conditions in tetrahydrofuran with the addition of N,N- diisopropyl ethylamine to form high-yield alcohols (McGeary R. P.: Tetrahedron Letters, 39, 3319-3322, 1998). Reduction of the conjugate acid, in this case cinnamic acid, results in an 18% reduction of the double bond.

The carboxyl groups of the tetrasaccharide esterified to methyl esters, which also contain unprotected hydroxyl groups, can be reduced with NaBEU in methanol. Reduction to the corresponding alcohol proceeds for 1 hour at 20 °C. (DAcquarica I. et al.: Tetrahedron, 58, 51, 10127-10136, 2002).

A similar approach was described in the article (Stolz F. et al.: European Journal of Organic Chemistry, 15, 3304-3312, 2004), where aliphatic carboxylic acids also containing ether groups were reduced by NaBEU after carboxyl activation with 1,1 '-carbonyl diimidazole. The reaction was carried out in an ethanol, dichloromethane, N,N-dimethylformamide system. A disaccharide with free hydroxyl groups and with cholesterol linked to the reducing end by an O-glycosidic bond was reduced at carboxyl following its esterification with diazomethane in methanol. NaBHj was used as the reagent and the reaction proceeded for 2 hours in methanol at ambient temperature (Yoshikawa K. et al.: Chemical and Pharmaceutical Bulletin, 46, 7, 1102-1107, 1998).

A mixture of hyaluronic acid oligosaccharides was esterified by a reaction with diazomethane and subsequently reduced by means of 0.2-20 equivalents of NaBFB in water at 25 °C. The obtained material showed a degree of substitution of 5-50% (Christner, J. E. et al.: Biochemistry Journal, 167, 711-716, 1977).

Reduction of carboxylic acid methyl esters in methanol by means of NaBEU occurs at lower temperatures if catalytic sodium methanolate is added to the system (Prasanth, C. P. et al.: Journal of Organic Chemistry, 2018, 83, 1431-1440). In this way, decomposition of the agent due to the acidity of the solvent is prevented.

In general, it can be stated that in the reduction of carboxylic acids with boron-based hydrides, activation of the carboxyl to its more reactive (electrophilic) derivative is necessary. Activation usually takes place in aprotic conditions, and the subsequent reduction with NaBEU usually takes place in mixtures with protic solvents such as methanol, ethanol or water. Compounds containing hydroxyl and amide groups were also used as reduced substrates.

Enzymatic reductions

Aliphatic carboxylic acids containing ethers and acetamides can be reduced in water using an enzyme from Gloeosporium olivarum at 27 °C. However, the reaction time of 768 hours is extremely long, while racemization of the alpha position of the reduced carbonyl was also observed (TsudaY. et al.: Chemical and Pharmaceutical Bulletin, 33, 5, 1955-1960, 1985).

Another enzyme capable of reducing aliphatic carboxylic acids containing ether groups was isolated from the fungus Glomerella cingulata. This enzyme works at 27 °C in water, the reaction time is up to 624 hours, and racemization of the alpha position of the carbonyl was also observed (Tsuda Y. et al.: Agricultural and Biological Chemistry, 48, 5, 1373-1374, 1984).

The use of an enzyme from the organism Glomerella cingulata is also described, when it is used with the addition of KH2PO4, MgSCU, peptone and sucrose, the reaction time is only 24 hours in water at 27 °C (Tsuda Y. : Chemical and pharmaceutical bulletin, 35, 6, 2554-2557, 1987). In general, it can be stated that the reduction can be carried out even under mild conditions in water using an enzyme, but it is necessary to take into account a longer reaction time and also the racemization of the alpha carbonyl position.

Reduction of the -C=C- bond of unsaturated esters of carboxylic acids

The reduction of the conjugated double bond was described in the publication (Falorni, M. et al.: Tetrahedron Letters, 1999, 40, 4395-4396). In this case, the activation of the unsaturated acid by cyanuric chloride takes place in the presence of N-methylmorpholine. Reduction of the intermediate with sodium borohydride was carried out in water. The obtained material showed 20% saturated bonds. The reason for the reduction of the C=C bond is the basic environment of the reduction.

An even higher proportion of the reduced double bond was reported in the reduction of cinnamic acid pentafluorophenyl ester in the publication (Papavassilopoulou, E. et al.: Tetrahedron Letters, 2007, 48, 8323-8325), in total, 25% of double bond was reduced.

The enantioselective reduction of the -C=C- bond of an unsaturated ester using sodium borohydride with the addition of cobalt chloride and an azabi soxazoline ligand in an ethanol/diglyme mixture was described in the publication (Geiger, C. et al.: Adv. Synth. Catal. 2005, 347, 249-254). In this case, there is no reduction of the carboxyl group. Reaction yields exceeded 80%.

Reduction of the -C=C- bond of highly electron-deficient alkenes using 3 -butyl- 1- methylimidazolinium borohydride in a mixture of acetonitrile/water (6: 1) was described in the publication (Wang, J. et al.: Tetrahedron Letters, 2008, 49, 6518-6520). Anionic metathesis with sodium borohydride is used to regenerate the agent. The reduction proceeds with a high yield of 75-92%.

In general, it can be stated that the reduction of the conjugated double bond can proceed under the conditions used for the reduction of esters. In some cases, this reaction may be done on purpose, but often it is not desirable.

In general, it can be stated that oligosaccharides according to the structural formula I have not been disclosed yet. Only glycosides with a double bond in positions 4 and 5 of the glucose cycle were mentioned, as well as polysaccharides having a -C=C- double bond in positions 4 and 5 of the glucosamine cycle, and oligosaccharides having a -C=C- double bond in positions 4 and 5 of the terminal uronic cycle where a carboxyl group is always present. Reduced saturated oligosaccharides of hyaluronic acid were also mentioned, but they do not contain a double bond and their degree of substitution does not exceed 50%. It is surprising that unsaturated hyaluronic acid esters can be prepared by cleavage of the polymer in dimethyl sulfoxide in the presence of nitrogenous bases, since hyaluronic acid oligosaccharides in a basic environment undergo decomposition at an elevated temperature and cleave N-acetylglucosamine from the reducing end (Muckenschnabel, I. et al.: Cancer Letters, 1998, 131, 13-20). When using a solvent mixture of DMSO/water (Example 8) or DMSO/methanol (Example 9), the reaction is significantly slowed down. It can therefore be concluded that the use of pure DMSO is crucial for polymer cleavage.

Furthermore, it is surprising that the reduction of hyaluronic acid esters by sodium borohydride proceeds under mild conditions in a protic medium (water or methanol), since this agent is generally not used in a protic medium (Smith, M. B. MARCH’S ADVANCED ORGANIC CHEMISTRY REACTIONS, MECHANISMS, AND STRUCTURE, 7th ed.; Wiley: USA, 2013, page 1500) due to its low reactivity and decomposition to form hydrogen and borate esters.

Furthermore, it is surprising that under these conditions virtually quantitative conversion was achieved using a small excess (4 equivalents) of sodium borohydride versus a 50% degree of substitution (Christner, J.E. et al.: Biochemistry Journal, 167, 711-716, 1997) using a significantly larger amount of agent (20 equivalents) for the reduction of hyaluronic acid methyl ester.

Furthermore, it is surprising that there is no reduction of the conjugated double bond (Falorni, M. et al.: Tetrahedron Letters, 1999, 40, 4395-4396, McGeary R. P.: Tetrahedron Letters, 39, 3319-3322, 1998).

Summary of the invention

The invention relates to unsaturated oligosaccharides composed of alternating glucose and N-acetylglucosamine units, with an unsaturated glucose unit at the non-reducing end and a reduced anomeric end, according to the structural formula I: - where n = 0 to 10 units.

The oligosaccharide thus modified shows an increased resistance to enzymatic degradation and negatively affects the growth of certain cancer cell lines.

Furthermore, the invention relates to a method of production of the oligosaccharide according to the structural formula I, where in the first step the carboxyl group of hyaluronic acid is alkylated to an ester, in the second step the ester is cleaved to form a double bond in positions 4 and 5 of the cycle, and in the third step the ester groups and the terminal anomeric center are reduced. The oligosaccharide according to the structural formula I can also be prepared by another method according to the invention, where, in the first step, hyaluronic acid is enzymatically cleaved using a lyase to an unsaturated oligosaccharide with a double bond in positions 4 and 5 of the cycle; in the second step, the unsaturated oligosaccharide is alkylated to an ester, and in the third step the ester groups and the terminal anomeric center are reduced to form a primary alcohol.

The following two options can therefore be used for the oligosaccharide preparation:

Option 1 :

The starting material is hyaluronic acid having a molecular weight of up to 2,000 kg. mol' ¹ , where in the first step it is alkylated on the carboxyl group to form an alkyl ester of polymeric hyaluronan, in the second step selective chemical cleavage of the alkylated polysaccharide is carried out to form an unsaturated oligosaccharide and in the third step the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.

Option 2:

The starting material is hyaluronic acid having a molecular weight of up to 2,000 kg.mol-1, where in the first step it is enzymatically cleaved to form unsaturated oligosaccharides, in the second step it is alkylated on the carboxyl group to form alkyl esters of oligosaccharides, and in the third step the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.

These two options are discussed in more detail below:

Option 1 :

In the first step, the carboxyl group of polymeric hyaluronic acid is alkylated using an alkylating agent, for example benzyl bromide, dimethyl sulphate or ethyl iodide, in dimethyl sulfoxide in the presence of a base, for example diisopropyl ethylamine or triethylamine, to form an alkyl ester. This step proceeds preferably for 20 to 120 hours at a temperature in the range from 20 to 25 °C. The molar amount of the base is preferably in the range from 1.7 to 6 equivalents and the molar amount of the alkylating agent is preferably in the range from 1.5 to 5 equivalents relative to the hyaluronic acid disaccharide. The initial hyaluronic acid can have a molecular weight in the range from 10 to 2,000 kg. mol' ¹ . The concentration of hyaluronic acid in DMSO is preferably in the range from 0.3 to 10% by weight.

In the second step, a selective chemical cleavage of the solution of esterified hyaluronic acid in DMSO proceeds at a temperature in the range from 20 to 80 °C, preferably with heating, for 1 to 116 hours, in the presence of a base, for example tri ethylamine, N-methylmorpholine, or diisopropylethylamine, preferably in the range from 2 to 20 equivalents relative to the hyaluronic acid disaccharide.

In the third step, the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols. Preferably, to the solution of esterified oligosaccharide of hyaluronic acid in water, in methanol, in DMSO or in a methanol/pyridine mixture, sodium borohydride (NaBPU) is added in an amount of 1 to 20 equivalents relative to the hyaluronic acid disaccharide and the mixture is stirred for 1 to 140 hours at a temperature in the range from 0 to 70 °C.

If necessary, the native hyaluronic acid is converted to a DMSO-soluble form before the first step, alkylation, for example by conversion to an acidic form by an acidification step with catex, or by conversion of the hyaluronic acid to a salt with organic counterions (e.g., tetrabutyl ammonium).

NaBH ₄

RX base Solvent T DMSO

Scheme I - Option 1 where R is Ci - C7 - linear or a branched or aromatic hydrocarbon and X is a halide or sulphate residue. Option 2:

In the first step, the polymeric hyaluronic acid is enzymatically cleaved by the enzyme hyaluronate lyase in water at 36 to 38 °C for 16 to 92 hours to form an unsaturated oligosaccharide. The starting hyaluronic acid can have a molecular weight in the range from 100 to 2,000 kg. mol' ¹ . The lyase is preferably selected from the group comprising Streptococcus pneumoniae hyaluronan lyase (SpHyl) and Streptococcus pyogenes hyaluronan lyase (Hylpl) and the lyase activity is preferably in the range from 1.632 to 1.75 lU/mL. The cleavage can be complete (down to di saccharides), preferably using SpHyl, or partial (a mixture of oligosaccharides).

In the second step, the carboxyl groups of oligomeric hyaluronic acid are alkylated using an alkylating agent, for example dimethyl sulphate, ethyl iodide or benzyl bromide, in dimethylsulfoxide in the presence of a base, for example triethylamine or diisopropylethylamine, to form an alkyl ester. This step proceeds preferably for 5 to 120 hours at a temperature in the range from 20 to 25 °C, where the molar amount of the alkylating agent is preferably in the range from 1.2 to 3 equivalents relative to the hyaluronic acid disaccharide and the molar amount of the base is preferably 1.2 to 3 molar equivalent relative to the hyaluronic acid disaccharide. The concentration of the hyaluronic acid oligomer solution in DMSO is preferably in the range from 5 to 30% by weight.

In the third step, the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols. Preferably, to the solution of esterified oligosaccharide of hyaluronic acid in water, in methanol, in DMSO or in a methanol/pyridine mixture, sodium borohydride (NaBHj) is added in an amount of 1 to 20 equivalents relative to the disaccharide of hyaluronic acid and the mixture is stirred for 1 to 140 hours at a temperature in the range from 0 to 70 °C.

Scheme II - Option 2 where R is Ci - C7 - linear or a branched or aromatic hydrocarbon and X is a halide or sulphate residue.

Furthermore, the invention relates to the use of oligosaccharides composed of alternating glucose and N-acetyl glucosamine units, with an unsaturated glucose unit at the nonreducing end and a reduced anomeric end according to the structural formula (I). These materials negatively affect the growth of certain cancer cell lines, while not having a negative effect on the growth of normal cells. Furthermore, they show a high resistance to degradation by certain enzymes, which can increase the efficiency of their transport and prolong the duration of their effect. Therefore, they can be used in anti-cancer materials and compositions, for example, but not limited to, in the form of a solution for intravenous administration (by injection or infusion), or in the form of a solid tablet for oral administration. The unsaturated oligosaccharide according to the invention can therefore be used for the preparation of materials and compositions with an anti-cancer effect, preferably with an anti-cancer effect against colorectal cancer in humans.

Description of the drawings

Fig. 1 - Comparison of the rate of enzymatic degradation of the solution of the material prepared according to Example 36 (full line) and the native oligomer of hyaluronic acid (dotted line) after application of enzyme Streptoccus pneumoniae hyaluronate lyase (SpHyl). Fig. 2 - Comparison of the rate of enzymatic degradation of the solution of the material prepared according to Example 36 (full line)) and the native oligomer of hyaluronic acid (dotted line) after application of bovine testicular hyaluronate hydrolase enzyme (BTH).

Fig. 3 - The effect of the materials prepared according to Examples 31, 33, 35 and 36 on fibroblasts NHDF viability.

Fig. 4 - The effect of the materials prepared according to Examples 31, 33, 35 and 36 on HT- 29 viability.

Fig. 5 - Comparison of the effect of native unsaturated HA oligosaccharides and the materials prepared according to Examples 31, 33, 35 and 36 on HT-29 viability.

Examples of embodiments

Ac = acetyl

DMSO = dimethyl sulfoxide

DS = degree of substitution = 100% * (molar amount of modified oligosaccharide unit) / (molar amount of all oligosaccharide units)

HA = hyaluronic acid

MeOH = methanol

Mw = molecular weight

NMR analysis = (700 MHz, D2O/DMSO-de, 8 ppm)

Hylpl = Streptococcus pyogenes hyaluronate lyase

SpHyl = Streptococcus pneumoniae hyaluronate lyase

DMEM = Dulbecco's modified Eagle's medium

FBS = fetal bovine serum

IP A = isopropyl alcohol

As used herein, the term equivalent (eq.) refers, unless otherwise indicated, to the repeating unit of the relevant polysaccharide or oligosaccharide, for example hyaluronic acid disaccharide. The percentages are given as percentages by weight unless otherwise stated. The molecular weight of the initial polysaccharides is weight average molecular weight, determined using the SECMALLS method.

The value of average Mw was determined by comparing the integral of the double bond signal at 6.2 ppm and the integral of the acetyl group signals at 2.0 ppm according to the formula Mw= 0.407* [I2.o]/(3*[l6.2]) kg.mol’ ¹ . Example 1

Alkylation of hyaluronic acid with ethyl iodide

The hyaluronic acid solution (5.9 g, Mw=10 kg.moT ¹ ) in demineralized water (150 mL) was cooled to 0 °C and stirred for 2 hours with catex (6 g) in H ⁺ cycle. Then, the catex was filtered off and the solution was lyophilized. The lyophilizate was dissolved in dry DMSO (60 mL) under a nitrogen atmosphere. Then triethylamine (4.8 mL, 2.2 eq.) and ethyl iodide (2.44 mL, 2 eq.) were added and the reaction mixture was stirred for 20 hours at 20 °C. The product was then precipitated by the addition of ethyl acetate (180 mL), washed and air dried. The product was analyzed using 'H NMR (DMSO-d ₆ ) 6 4.25-4.05 (m, 2H, CH ₂ ); 1.75 (s, 3H, Ac); 1.23 (t, J= 7.0 Hz, 3H, CH ₃ ). DS=100%.

Example 2

Alkylation of hyaluronic acid with dimethyl sulphate

The hyaluronic acid solution (1 g, Mw=2000 kg. mol' ¹ ) in demineralized water (200 mL) was cooled to 0 °C and stirred for 2 hours with catex (2 g) in H ⁺ cycle. Then, the catex was filtered off and the solution was lyophilized. The lyophilizate was dissolved in dry DMSO (300 mL) under a nitrogen atmosphere. Then diisopropylethylamine (1.5 mL, 1.7 eq.) and dimethyl sulphate (0.375 mL, 1.5 eq.) were added and the reaction mixture was stirred 120 hours at 25 °C. The product was then precipitated by the addition of water (200 mL), washed and air dried. The product was analyzed using 'H NMR (DMSO-de) 8 3.75 (s, 3H, CH ₂ ); 1.80 (s, 3H, Ac). DS=98%.

Example 3

Alkylation of hyaluronic acid with benzyl bromide

The hyaluronic acid solution (1 g, Mw=400 kg.mol' ¹ ) in demineralized water (100 mL) was cooled to 0 °C and stirred for 2 hours with catex (1 g) in H ⁺ cycle. Then, the catex was filtered off and the solution was lyophilized. The lyophilizate was dissolved in dry DMSO (10 mL) under a nitrogen atmosphere. Then diisopropyl ethylamine (2.77 mL, 6 eq.) and benzyl bromide (1.57 mL, 5 eq.) were added and the reaction mixture was stirred for 90 hours at 20 °C. The product was then precipitated by the addition of ethyl acetate (40 mL), washed with water and isopropyl alcohol and air dried. The product was analyzed using 'H NMR (DMSO-de) 6 7.51- 7.25 (m, 5H, Ar-H); 5.23 (d, J = 12.55 Hz, 1H, ArCH ₂ ); 5.12 (d, J = 12.55 Hz, 1H, ArCH ₂ ); 1.75 (s, 3H, Ac). DS=100%. Example 4

Alkylation of hyaluronic acid with ethyl iodide

The hyaluronic acid solution (5.6 g, Mw=130 kg.moT ¹ ) in demineralized water (160 mL) was cooled to 0 °C and stirred for 2 hours with catex (6 g) in H ⁺ cycle. Then, the catex was filtered off and the solution was lyophilized. The lyophilizate was dissolved in dry DMSO (60 mL) under a nitrogen atmosphere. Then diisopropyl ethylamine (5.7 mL, 2.3 eq.) and ethyl iodide (2.4 mL, 2.1 eq.) were added and the reaction mixture was stirred for 90 hours at 20 °C. The product was then precipitated by the addition of ethyl acetate (240 mL), washed and air dried. The product was analyzed using ^T H NMR (DMSO-de) 6 4.25-4.05 (m, 2H, CEL); 1.75 (s, 3H, Ac); 1.23 (t, J = 7.0 Hz, 3H, CH ₃ ). DS=100%.

Example 5

Chemical cleavage of hyaluronic acid ethyl esters

To the solution of alkylated HA prepared according to Example 1 (0.16 g) in dry DMSO (7 mL), triethylamine (1.14 mL, 20 eq.) was added and the reaction mixture was stirred at 20 °C for 16 hours. The product was then precipitated by an addition of ethyl acetate (28 mL), washed and air dried. The product was analyzed using NMR (DMSO-de): 5.92 (d, J= 3.9 Hz, 1H, H- C=C); 4.25-4.05 (m, 2H, CH ₂ ); 1.75 (s, 3H, Ac); 1.23 (t, J = 7.0 Hz, 3H, CH ₃ ). Average Mw= 6.12 kg.moT ¹ .

Example 6

Chemical cleavage of hyaluronic acid methyl esters

To the solution of alkylated HA prepared according to Example 2 (0.5 g) in dry DMSO (5 mL), triethylamine (0.35 mL, 2 eq.) was added and the reaction mixture was heated to 80 °C for 1 hour. The product was then precipitated by an addition of ethyl acetate (20 mL), washed and air dried. The product was analyzed using 'H NMR (DMSO-de) 8 5.95 (d, J= 3.9 Hz, 1H, H- C=C); 3.86 (s, 3H, CH ₃ ); 1.75 (s, 3H, Ac). Average Mw= 16.8 kg.mol’ ¹ .

Example 7

Chemical cleavage of hyaluronic acid benzyl esters

To the solution of alkylated HA prepared according to Example 3 (1 g) in dry DMSO (10 mL), triethylamine (1 mL, 3.4 eq.) was added and the reaction mixture was stirred at 50 °C for 72 hours. The product was then precipitated by an addition of ethyl acetate (30 mL), washed and air dried. The product was analyzed using NMR (DMSO-de): 7.55-7.30 (m, 5H, Ar-H); 6.02 (d, J = 3.9 Hz, 1H, HOC); 5.26 - 5.28 (m, 2H, ArCH ₂ ); 1.75 (s, 3H, Ac). Average Mw= 2 kg.mol' ¹ .

Example 8

Chemical cleavage of hyaluronic acid ethyl esters in DMSO/water mixture

To the solution of alkylated HA prepared according to Example 4 (0.5 g) in DMSO/water mixture (4: 1, 5 mL), triethylamine (0.5 mL, 3 eq.) was added and the reaction mixture was heated to 50 °C for 72 hours. The product was then precipitated by an addition of ethyl acetate (20 mL), washed and air dried. The product was analyzed using 'H NMR (DMSO-de) 6 5.92 (d, J= 3.9 Hz, 1H, HOC); 4.25-4.05 (m, 2H, CH ₂ ); 1.75 (s, 3H, Ac); 1.23 (t, J = 7.0 Hz, 3H, CH3). Average Mw= 9.26 kg.mol' ¹ .

Example 9

Chemical cleavage of hyaluronic acid ethyl esters in a mixture of DMSO/methanol

To the solution of alkylated HA prepared according to Example 4 (0.2 g) in DMSO/methanol mixture (4: 1, 2 mL), triethylamine (0.2 mL, 3 eq.) was added and the reaction mixture was heated to 50 °C for 72 hours. The product was then precipitated by an addition of ethyl acetate (20 mL), washed and air dried. The product was analyzed using 'H NMR (DMSO-de) 8 5.92 (d, J= 3.9 Hz, 1H, H-C=C); 4.25-4.05 (m, 2H, CH ₂ ); 1.75 (s, 3H, Ac); 1.23 (t, J = 7.0 Hz, 3H, CH3). Average Mw= 3 kg.mol' ¹ .

Example 10

Chemical cleavage of hyaluronic acid ethyl esters

To the solution of alkylated HA prepared according to Example 4 (1 g) in dry DMSO (10 mL), triethylamine (1 mL, 2.9 eq.) was added and the reaction mixture was heated to 50 °C for 92 hours. The product was then precipitated by an addition of ethyl acetate (100 mL), washed and air dried. The product was analyzed using 'H NMR (D ₂ O) 6 6.25 (d, J = 4.2 Hz, 1H, H-C=C); 5.37-5.25 (m, 2H, CH ₂ ); 2.10-1.98 (m, 3H, Ac); 1.35-1.23 (m, 3H, CH ₃ ). Average Mw= 1.2 kg.mol' ¹ .

Example 11

Chemical cleavage of hyaluronic acid esters with chromatographic separation

To the solution of alkylated HA prepared according to Example 4 (1 g) in dry DMSO (10 mL), triethylamine (1 mL, 2.9 eq.) was added and the reaction mixture was heated to 50 °C for 116 hours. Then acetic acid (0.6 mL) was added and the product was separated on reverse phase HPLC column in water/methanol system. After evaporation, the products were analyzed using 'H NMR (D ₂ O) 8 6.25 (d, J = 4.2 Hz, 1H, H-C=C); 5.37-5.25 (m, 2H, CH ₂ ); 2.10-1.98 (m, 3H, Ac); 1.35-1.23 (m, 3H, CH ₃ ).

Example 12

Chemical cleavage of hyaluronic acid esters by means of A-methylmorpholine

To the solution of alkylated HA prepared according to Example 4 (0.2 g) in dry DMSO (2 mL), A-methylmorpholine was added (0.35 mL, 6.5 eq.) and the reaction mixture was heated to 50 °C for 112 hours. The product was then precipitated by an addition of ethyl acetate (20 mL), washed and air dried. The product was analyzed using 'H NMR (D ₂ O) 6 6.25 (d, J = 4.2 Hz, 1H, H-C=C); 5.37-5.25 (m, 2H, CH ₂ ); 2.10-1.98 (m, 3H, Ac); 1.35-1.23 (m, 3H, CH ₃ ). Average Mw= 5.5 kg.mol' ¹ .

Example 13

Chemical cleavage of hyaluronic acid esters by means of diisopropylethylamine

To the solution of alkylated HA prepared according to Example 4 (0.12 g) in dry DMSO (7 mL), diisopropyl ethylamine (0.2 mL, 3.8 eq.) was added and the reaction mixture was heated to 50 °C for 3 hours. The product was then precipitated by an addition of ethyl acetate (20 mL), washed and air dried. The product was analyzed using 'H NMR (D ₂ O) 6 6.25 (d, J = 4.2 Hz, 1H, H-C=C); 5.37-5.25 (m, 2H, CH ₂ ); 2.10-1.98 (m, 3H, Ac); 1.35-1.23 (m, 3H, CH ₃ ). Average Mw= 4 kg.mol' ¹ .

Example 14

Enzymatic degradation of hyaluronic acid by lyase

To the solution of native HA (5 g, 100 kg.mol-1) in demineralized water (250 mL), a solution of enzyme Streptococcus pneumoniae hyaluronate lyase (0.25 mL, 408 IU, 50% v/v glycerol, 25 mM phosphate buffer (NaxHyPCU), 250 mM NaCl, pH=8) was added. The solution was stirred at 36 °C overnight (16 hours). Then it was filtered through a 0.2 mm nylon filter and the filter was washed with demineralized water (50 mL). The solution was concentrated to the volume of 100 ml and then it was lyophilized. The resulting product was analyzed using 'H NMR (D2O) 8 5.89 - 5.85 (m, 1H, H-C=C); 2.07 (s, 3H, Ac). Example 15

Enzymatic degradation of hyaluronic acid by lyase

To the solution of native HA (1 g, 2,000 kg. mol' ¹ ) in acetate buffer (200 mL, 50 mM CH ₃ COONa, 50 mMNaCl, pH=6), Hylpl enzyme (350 U) was added. The solution was stirred at 38 °C for 92 hours. Then the sample was boiled (100 °C), cooled, supplemented with demineralized water to a volume of 1 1 and ultrafiltered. The solution thus prepared was partially evaporated and lyophilized. The product was analyzed using 'H NMR (D2O) 6 5.86 (d, J= 4.0 Hz, 1H, H-C=C); 3.39 - 3.33 (m, 3H, H-2); 2. 10 - 2.00 (m, 12H, Ac). Average Mw= 1.6 kg.moT ¹ .

Example 16

Alkylation of hyaluronic acid disaccharide with ethyl iodide

Unsaturated disaccharide, prepared according to Example 14 (0.4 g), was dissolved in dry DMSO (1 mL). Then diisopropylethylamine (0.35 mL, 2 eq.) and ethyl iodide (0.1 mL, 1.2 eq.) were added and the reaction mixture was stirred at temperature 20 °C for 60 hours. Then acetic acid (0.1 mL) was added and the reaction mixture was separated on a C18 column in water/methanol mixture. The product was analyzed using 'H NMR (D2O) 6 6.27-6.25 (m, H- C=C, 1H); 4.33-4.27 (m, 2H, CH ₂ ); 2.08 (2xs, 3H, Ac); 1.32, 1.31 (d x t, J = 7.1 Hz, 3H, CH ₃ ). DS=100%

Example 17

Alkylation of hyaluronic acid disaccharides with ethyl iodide

Unsaturated disaccharide, prepared according to Example 14 (0.4 g), was dissolved in dry DMSO (1 mL). Then triethylamine (0.27 mL, 2 eq.) and ethyl iodide (0.24 mL, 3 eq.) were added and the reaction mixture was stirred at temperature 25 °C for 20 hours. Then acetic acid (0.1 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol. The product was analyzed using ¹ H NMR (D2O) 66.27-6.25 (m, H-C=C, 1H); 4.33-4.27 (m, 2H, CH ₂ ); 2.08 (2xs, 3H, Ac); 1.32, 1.31 (d x t, J = 7.1 Hz, 3H, CH ₃ ). DS=100%.

Example 18

Alkylation of hyaluronic acid disaccharides with benzyl bromide.

Unsaturated disaccharide, prepared according to Example 14 (0.8 g) was dissolved in dry DMSO (2 mL). Then diisopropylethylamine (0.7 mL, 2 eq.) and benzyl bromide (0.47 mL, 2 eq.) were added and the reaction mixture was stirred at temperature 20 °C for 16 hours. Then acetic acid (0.2 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol. The product was analyzed using 'H NMR (D2O) 6 7.50-7.40 (m, 5H, Ar); 6.28-6.26 (m, 1H, H-C=C); 5.31 (s, 2H, ArCH ₂ ); 2.07, 2.06 (2 x s, 3H, Ac). DS=100%. Yield 0.76 g.

Example 19

Alkylation of hyaluronic acid oligosaccharides with benzyl bromide.

A mixture of unsaturated oligosaccharides prepared according to Example 15 (0.1 g) was dissolved in dry DMSO (1 mL). Then diisopropyl ethylamine (0.083 mL, 2 eq.) and benzyl bromide (0.06 mL, 2 eq.) were added and the reaction mixture was stirred at temperature 25 °C for 120 hours. Then acetic acid (0.05 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol.

The product was analyzed using ^T H NMR (D ₂ O) 6 7.60-7.37 (m, 20H, Ar); 6.28 (d, J= 4.3 Hz, 1H, H-C=C); 5.45-5.23 (m, 8H, ArCH ₂ ); 2.10-1.94 (m, 12H, Ac). DS=93%. Yield 0.07 g.

Example 20

Alkylation of a hyaluronic acid disaccharide with dimethyl sulphate.

Unsaturated disaccharide, prepared according to Example 14 (0.2 g) was dissolved in dry DMSO (1 mL). Then diisopropylethylamine (0.2 mL, 2 eq.) and dimethyl sulphate (0.1 mL, 2 eq.) were added and the reaction mixture was stirred at 20 °C for 5 hours. Then acetic acid (0.05 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol. The product was analyzed using ¹ H NMR (D ₂ O) 66.27-6.24 (m, 1H, H-C=C); 3.84 (2 x s, 3H, OCH ₃ ); 2.07, 2.06 (2 x s, 3H, Ac). DS=100%. Yield 0.21 g.

Example 21

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (130 mg) prepared according to Example 10 was dissolved in DMSO (5 mL). Then NaBHj (130 mg, 10.5 eq.) was added and the reaction mixture was heated to 70 °C for 140 hours. Then acetic acid (5 mL, 20%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2-propanol (20 mL) and ethyl acetate (30 mL), washed and air dried.

The product was analyzed using 'H NMR (D ₂ O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=89%. Example 22

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (252 mg) prepared according to Example 10 was dissolved in DMSO (5 mL). Then NaBEU (243 mg, 10 eq.) was added and the reaction mixture was heated to 50 °C for 140 hours. Then acetic acid (7.5 mL, 20%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2- propanol (30 mL), washed and air dried.

The product was analyzed using ^T H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 5H, H-2); 2.09-2.03 (7 x _s , 21H, Ac). DS=71%.

Example 23

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (101 mg) prepared according to Example 10 was dissolved in H2O (1 mL). Then NaBHj (22 mg, 2 eq.) was added and the reaction mixture was stirred at 25 °C for 19 hours. Then acetic acid (0.08 mL, 99%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2- propanol (10 mL), washed and air dried. The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=62%.

Example 24

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (11 mg) prepared according to Example 10 was dissolved in H2O (0.7 mL). Then NaBHj (20 mg, 20 eq.) was added and the reaction mixture was stirred at 0 °C for 2 hours. Then acetic acid (0.02 mL, 99%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2- propanol (1 mL), washed and air dried.

The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=89%. Example 25

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (12 mg) prepared according to Example 10 was suspended in MeOH-d4 (0.7 mL). Then NaBEU (13 mg, 12 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.01 mL, 99%) was added. The precipitate was filtered and air dried. The product was analyzed using ^T H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H- 2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=100%.

Example 26

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (101 mg) prepared according to Example 10 was suspended in MeOH (2 mL). The solution was cooled to 0 °C, then NaBHj (94 mg, 10 eq.) was added and the reaction mixture was stirred at 0 °C for 1 hour and then at 25 °C for 15 hours. Then acetic acid (0.1 mL, 99%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2-propanol (10 mL), washed and air dried. The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=40%.

Example 27

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (11 mg) prepared according to Example 10 was suspended in dry MeOH (0.7 mL). Then NaBHj (11 mg, 10 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.01 mL, 99%) was added. The precipitate was filtered and air dried.

The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=51%.

Example 28

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (98 mg) prepared according to Example 10 was suspended in MeOH (2 mL) and pyridine (0.2 mL). Then NaBHj (97 mg, 10 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.1 mL, 99%) was added and the reaction mixture was stirred for 30 minutes. The precipitate was filtered and air dried.

The product was analyzed using ^T H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=98%.

Example 29

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (19 mg) prepared according to Example 10 was suspended in MeOH (0.7 mL) and pyridine (0.004 mL). Then NaBHj (1.89 mg, 1 eq.) was added and the reaction mixture was stirred at 25 °C for 20 hours. Then acetic acid (0.1 mL, 99%) and MeOH (0.5 mL) were added. The precipitate was filtered and air dried.

The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=9%.

Example 30

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (10 mg) prepared according to Example 10 was suspended in MeOH (0.7 mL) and pyridine (0.008 mL). Further NaBHj (4 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 18 hours. Then acetic acid (0.01 mL, 99%) and MeOH (0.5 mL) were added. The precipitate was filtered and air dried. The product was analyzed using 'H NMR (D ₂ O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H- C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=97%.

Example 31

Reduction of hyaluronic acid oligomer esters

A mixture of alkylated oligosaccharides (200 mg) prepared according to Example 10 was suspended in MeOH (2 mL) and pyridine (0.16 mL). Then NaBHj (86 mg, 5 eq.) was added and the reaction mixture was stirred at 0 °C for 20 minutes and then at 25 °C for 18 hours. Then acetic acid (0.125 mL, 99%) and MeOH (0.5 mL) were added. The precipitate was filtered and air dried. The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 3H, H-2); 2.09-2.03 (5 x _s , 15H, Ac). DS=98%.

Example 32

Reduction of esters of the isolated hyaluronic acid disaccharide

The isolated alkylated disaccharide (103 mg) prepared according to Example 16 was suspended in MeOH (2 mL) and pyridine (0.08 mL). Then NaBHj (38 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2- propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.

The product was analyzed using ¹ H NMR (D20) 6= 5.10 (d, J = 4.08 Hz, 1H, H-C=C); 2.06 (s, 3H, Ac). DS=95%.

Example 33

Reduction of esters of isolated hyaluronic acid tetrasaccharide

The isolated alkylated tetrasaccharide (203 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBHj (76 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.

The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 2.09-2.03 (2 x _s , 6H, Ac). DS=100%.

Example 34

Reduction of esters of isolated hyaluronic acid hexasaccharide

The isolated alkylated hexasaccharide (204 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBHj (81 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.

The product was analyzed using 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 1H, H-2); 2.09-2.03 (3 x s, 9H, Ac). DS=100%. Example 35

Reduction of esters of isolated hyaluronic acid octasaccharide

The isolated alkylated octasaccharide (203 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBEU (76 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried. The product was analyzed using ^T H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 2H, H- 2); 2.09-2.03 (5 x _s , 12H, Ac). DS=100%.

Example 36

Reduction of a mixture of esters of higher hyaluronic acid saccharides

The mixture of higher saccharide esters (200 mg, 10-20 saccharides) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBHj (76 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried. The product was analyzed using NMR, 'H NMR (D2O) 6 5.13 (d, J = 4.7 Hz, 1H, H-l); 5.09 (d, J = 4.0 Hz, 1H, H-C=C); 3.38 (dd, J = 9.4 Hz, 8.0 Hz, 1H, H-2); 3.32 (dd, J = 9.1 Hz, 8.0 Hz, 4H, H-2); 2.09-2.03 (m, 18H, Ac). DS=100%.

Example 37

Reduction of benzyl ester of isolated hyaluronic acid disaccharide

The benzylated disaccharide (59 mg) prepared according to Example 18 was dissolved in MeOH (1 mL) and pyridine (0.04 mL). Then NaBHj (18 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.04 mL, 99%) and 2-propanol (4 mL) were added. The precipitate was centrifuged, washed and air dried.

The product was analyzed using ¹ H NMR (D20) 6= 5.10 (d, J = 4.08 Hz, 1H, H-C=C); 2.06 (s, 3H, Ac). Conversion 95%.

Example 38

Reduction of methyl ester of isolated hyaluronic acid disaccharide

The methylated disaccharide (56 mg) prepared according to Example 20 was dissolved in MeOH (1 mL) and pyridine (0.04 mL). Then NaBHj (19 mg, 3.5 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.04 mL, 99%) and 2- propanol (4 mL) were added. The precipitate was centrifuged, washed and air dried.

The product was analyzed using ¹ H NMR (D20) 6= 5.10 (d, J = 4.08 Hz, 1H, H-C=C); 2.06 (s, 3H, Ac). Conversion = 89%.

Example 39

Comparison of enzymatic degradation of reduced HA oligomer prepared according to Example 36 and native HA10 oligomer.

8 mg of the native or the reduced oligosaccharide of HA prepared according to Example 36 were dissolved in 0.8 ml of solution containing 0.01 mol.L' ¹ acetate buffer and 0.01 mol.L' ¹ NaCl in D2O. Then acetic acid was added to reach the pH 5.3. To this mixture, 800 units of BTH enzyme were added and the mixture was stirred for 24 hours at 37 °C. The final solution was then cooled to 20 °C and measured by NMR. The average molecular weight of the oligosaccharide was calculated based on the ratio of the hydrogen signals integral of anomeric end at 5.16 ppm and hydrogen integral of -CH3 group at 2.0 ppm.

Example 40

Comparison of enzymatic degradation of the reduced HA oligomer prepared according to Example 36 and native HA10 oligomer.

8 mg of the native or of the reduced oligosaccharide of HA prepared according to Example 36 were dissolved in 0.5 ml of D2O and then a solution of the enzyme Streptococcus pneumonia hyaluronate lyase was added (0.005 mL, 8 IU, 50% v/v glycerol, 25 mM phosphate buffer (NaxHyPCU), 250 mM NaCl, pH=8). The solution was stirred for 24 hours at 37 °C overnight (16 hours). The final solution was then cooled to 20 °C, diluted with D2O (0.2 mL) and measured by NMR. The extent of cleavage was determined from the hydrogen signals integral arising from the double bond at 5.06 ppm and hydrogen integrals of -CH3 group at 2.0 ppm.

Example 41

Testing of the effect of the reduced hyaluronic acid oligosaccharide prepared according to Examples 31, 33, 35, 36 on NHDF fibroblasts viability.

The human fibroblasts NHDF were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). The cytotoxicity was measured via MTT test. After reaching 80% confluence, the cells were passaged and plated in a 96-well plate in density 5,000 cells per well. After incubation until the second day, the culture medium was replaced for the tested samples (CTRL = culture medium). After 24, 48 and 72 hours of cells treatment, 20 ul MTT (5 mg/mL) were added, followed by incubation for 2.5 hours. Finally, the culture medium with the samples was tapped from the plate and the lysis solution was added (IPA: DMSO (1: 1), Triton X-100 (10%). After cell lysis, the absorbance was measured on a spectrometer Ensight (Perkin Elmer) at wavelength 570 nm, (690 nm background).

Graph The change in NHDF viability (Fig. 3) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.

Example 42

Testing the effect of the reduced hyaluronic acid oligosaccharides prepared according to Examples 31, 33, 35, 36 on the HT-29 viability

Cells of human colorectal carcinoma HT-29 were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). The cytotoxicity was measured via MTT test. After reaching 80% confluence, the cells were passaged and plated in a 96-well plate in density 3,000 cells per well. After incubation until the second day, the culture medium was replaced for the tested samples (CTRL = culture medium). After 24, 48 and 72 hours of cells treatment, 20 ul MTT (5 mg/mL) was added, followed by incubation for 2.5 hours. Finally, the culture medium with the samples was tapped from the plate and a lysis solution was added (IPA: DMSO (1 : 1), Triton X-100 (10%). After cell lysis, the absorbance was measured on a spectrometer Ensight (Perkin Elmer) at wavelength 570 nm, (690 nm background).

Graph The change in HT-29 viability (Fig. 4) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.

Example 43

Comparison of the effect of native unsaturated hyaluronic acid oligosaccharides and derivatives prepared according to Examples 31, 33, 35 and 36 on HT-29 viability

Cells of human colorectal carcinoma HT-29 were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). The cytotoxicity was measured via an MTT test. After reaching 80% confluence, the cells were passaged and plated in a 96-well plate in density 3,000 cells per well. After incubation until the second day, the culture medium was replaced for the tested samples (CTRL = culture medium). After 72 hours of cells treatment, 20 ul MTT (5 mg/mL) was added, followed by incubation for 2.5 hours. Finally, the culture medium with the samples was tapped from the plate and a lysis solution was added (IPA: DMSO (1 : 1), Triton X-100 (10%). After cell lysis, the absorbance was measured on a spectrometer Ensight (Perkin Elmer) at wavelength 570 nm, (690 nm background).

Graph The change in HT-29 viability (Fig. 5) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.