Thioconjugates of oligosaccharides, method of their preparation and use, composition

Abstract

The invention relates to oligosaccharides composed of alternating glucuronic and N-acetylglucosamine units, with a β-D-glucuronic, β-D-galacturonic or β-L-altruronic unit at the non-reducing end with a substituent attached by a sulfide bond according to the structural formula (I), (II) or (III), their preparation and method of use. The invention also relates to a pharmaceutical composition comprising the oligosaccharide according to the structural formula (I), (II) or (III). Formulae for abstract (formulae I, II and III) wherein n = 0 to 8; X is O or N-H; Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7; R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally with content of N, S, Cl or O atoms.

Description

[0001] Thioconjugates of oligosaccharides, method of their preparation and use, composition

[0002] Technical field The invention relates to oligosaccharides composed of alternating glucuronic and N- acetylglucosamine units, with a P-D-galacturonic, P-D-glucuronic or P-L-altruronic unit at the non-reducing end with a substituent attached by a sulfide bond according to the structural formulas (I, II a III), wherein n = 0 to 8;

[0003] X is O or N-H;

[0004] Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7; R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally with content of N, S, Cl or O atoms;

[0005] Furthermore, the invention relates to a method for preparing compounds of general formula I, II and III and a method of use.

[0006] The invention also relates to pharmaceutical compositions containing a compound of general formula I, II and III.

[0007] State of the art

[0008] Hyaluronic acid

[0009] Hyaluronic acid or its sodium salt is a non-sulfated glycosaminoglycan composed of two repeating units of D -glucuronic acid and 7V-acetyl-D-glucosamine.

[0010] The molecular weight of native hyaluronic acid can reach up to 5.106g.mol'1. This polysaccharide forms a significant component 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 proteoglycan organization. Hyaluronic acid occurs naturally in biological systems, therefore it is naturally biodegradable and biocompatible. Therefore, it is a suitable substrate for a wide range of biomedical applications.

[0011] Hyaluronic acid is degraded under biological conditions by hydrolases and lyases. In the first case, the products maintain the structure according to formula II, only the chain is shortened. When hyaluronic acid is cleaved by a lyase, a chain with a double bond is formed at the terminal saccharide at the non-reducing end. An alternative method of cleavage to form a double bond is heating the hyaluronic acid ester in the presence of a base in a non-aqueous solvent (Klejch, T. et al.: Carbohydrate Polymers, 336, 122129, 2024).

[0012] Modification of the non-reducing end of hyaluronic acid oligosaccharides

[0013] In the article (Ruhela, D et al.: Bioconjugate Chemistry, 17, 1360-1363, 2006), the ozonation of the double bond at the non-reducing end of the unsaturated HA oligosaccharide was described. The resulting aldehyde was coupled by reductive amination to biotin, proteins, other polymers, or dipalmitoyl phosphatidylethanolamine. The disadvantage of this approach is the opening of the terminal saccharide ring and its decrease of stability.

[0014] In the article (Yamaguchi, M. et al.: Journal of Carbohydrate Chemistry, 29, 315-331, 2010), the mercurati on-oxidation of an unsaturated bond at the non-reducing end of HA di saccharide to form a saturated disaccharide was described. Products of the same structure can be prepared using hydrolytic enzymes and without the need to use highly toxic mercury.

[0015] In the article (Wei, G.H. et al.: Tetrahedron Letters, 50, 5920-5922, 2009), chemical synthesis of F-4-GlcA beta(l -> 3)GlcNAc-UDP as a substrate or inhibitor for hyaluronan synthase has been described. However, the subsequent reaction with the enzyme is not disclosed in this article.

[0016] In the article (Zhang, X. et al.: Journal of Organic Chemistry, 82, 9910-9915, 2017), the synthesis of 4-azido-2-deoxy-2-N-acetylglukosamine activated by UDP group for polymeration reactions with HAS enzymes was described. The azinated monosaccharide terminates the reaction sequence and can be used for click reactions with azides.

[0017] In the article (Wu, Z. L. et al.: Glycobiology, 31, 1435-1443, 2021), the modification of the non-reducing end of glycosaminoglycans by the addition of azido glucosamine or galactosamine was described. This reaction is catalyzed by the enzyme HAS. The products were linked to fluorophores using a click reaction and subsequently used as a substrate for endoglycosidases testing. This procedure was even patented (US10890586B2, 2021).

[0018] In the article (Weigel, P. H. et al.: Glycobiology, 27, 536-554, 2017), the use of unsaturated chitin oligosaccharides with an activating UDP group at the reducing end as primers for a HAS-catalyzed reaction has been described. In this way, a chitin-HA copolymer can be prepared. In general, it can be stated that the modification of the non-reducing end of hyaluronic acid oligosaccharides is poorly described. Chemical modifications of the double bond or enzymatic reaction with modified UDP-activated monosaccharides are used. Reactions with thiols are not described.

[0019] Thiooligosaccharides of HA and other GAGs

[0020] In the article (Rye, C. S. et al.: Carbohydrate Research, 339, 699-703, 2004), the synthesis of a sulfide-bridged chondroitin disaccharide by a multistep synthesis using protecting groups was described. This substance was considered as a potential inhibitor of the lyase from Flavobacterium heparinum. but it is a slow substrate.

[0021] In the article (Cao, H. et al.: Tetrahedron Letters, 46, 4337-4340, 2005), the preparation of a sulfur-bridged heparan sulfate trisaccharide as a heparanase inhibitor has been disclosed. This synthesis utilizes classical saccharide synthesis with extensive use of protecting groups.

[0022] In the next article (Teki, D. S. E. K. et al.: Organic Chemistry Frontiers, 6, 2718-2725, 2019), the preparation of disaccharides GlcA-S-Glc is described. These oligosaccharides were attached to maltotriose using a click reaction. Again, classical saccharide chemistry is used for the synthesis.

[0023] In the following article (Teki, D. S. E. K. Organic & Biomolecular Chemistry, 20, 3528- 3534, 2022), the synthesis of a monosulfated heparan disaccharide bridged by a sulfide bridge was described using saccharide chemistry, again with extensive use of protecting groups.

[0024] In the article (Cristofalo, A. E. et al. : Journal of Organic Chemistry, 85, 306-317, 2020), the synthesis of sulfur-bridged basic units of hyaluronan and keratan has been disclosed (P-S-GlcA(l— >3)P-GlcNAc and P-S-GalA( l ^3)P-GlcNAc). The synthesis is again multi-step with the use of protecting groups. In the following article (Cristofalo, A. E. et al.: Organic & Biomolecular Chemistry, 19, 6455-6467, 2021), the use of this disaccharide for the preparation of a glycocluster was described. Furthermore, the publication (Cristofalo, A. E. et al.: Molecules, 26, 180, 2021) described the synthesis of HA disaccharide epimer (P-S- GlcA(l — >3)P-AllNAc), linked by a sulfur bridge, also using classical saccharide chemistry.

[0025] In the patent publication (CN110790845 A, 2019), the preparation of heparosan or a similar glycosaminoglycan with a sulfur bond GlcA-S-NAcGlc was described. The product was synthesized from UDP-activated saccharides under enzymatic catalysis by PmHAS. The chain length is controlled by successive additions of activated saccharides and isolation from the mixture. The activated saccharides were prepared in a multistep process using the described methods (Schultz, V. L. et al.: Journal Of Organic Chemistry, 82, 2243-2248, 2017). Antitumor, anticoagulant and anti-inflammatory activity of these substances is claimed, as well as their use as carriers for targeted drugs. This method requires complex preparation of building blocks, activation with UDP and a suitable enzyme.

[0026] In the article (He, P. et al.: Nature Communications, 13, 7438, 2022), the preparation of sulfur analogues of heparosan (structures (-4-GlcA-l-P-S-4-GlcNAc-l-a-)nand S-4-GlcNAc- l-a-(-4-GlcA-l-P-O-4-GlcNAc-l-a-)n) and hyaluronic acid with a thiol group in position 4 of N-acetylglucosamine (structures (-4-GlcA-l-P-O-3-GlcNAc(4S)-l-P-)nand GlcNAc(4S)-l- beta-(-4-GlcA-l -P-O-3 -GlcNAc-l-P-)n) was described. These substances were prepared from UDP activated monosaccharides using the appropriate synthases. The reaction with sulfur analogues proceeds significantly slower than with oxygen analogues, a molecular weight of approximately 18 kDa was achieved. This process was also patented (US2021230313A1, 2021).

[0027] In general, the synthesis of sulfide-bridged glycosaminoglycan dimers using classical saccharide chemistry has been described, and the enzymatic synthesis of longer sulfide-bridged chains from UDP-activated monosaccharides has been described. The starting materials for these syntheses are prepared by multistep reactions with extensive use of protecting groups.

[0028] Radical addition to the -C=C- double bond

[0029] In the article (Dondoni, A. et al.: Chemistry A European Journal, 15, 43, 11444-11449, 2009), the radical thiolation of an allyl group attached to the 1 -position of a protected galactoside was described. The reactions were carried out in DMF and in DMF / water or DMSO / water mixtures. DPAP was used as the photoinitiator. A large excess of thiol (3 equivalents) was used, otherwise the yield would be reduced. In this case, the double bond is not sterically hindered, which significantly improves its reactivity.

[0030] In the publication (Cao, J. et al.: New Journal of Chemistry, 41, 8546-8553, 2017), the photochemical addition of thiol to acrylic acid has been described. The reaction occurs without a solvent. The reaction is again facilitated by the good accessibility of the double bond on acrylic acid.

[0031] In the article (Staderini, S. et al.: Tetrahedron Letters, 53, 702-704, 2012), the synthesis of thiodisaccharides was described by the photochemical addition of thiol glycosides to glycals with an unsubstituted double bond. The reactions were carried out with a large excess of 6 equivalents of thiol and with long irradiation (1 hour). When the amount of thiol was reduced or the irradiation was shortened, the yield decreased. In these reactions, low stereoselectivity was achieved, with a mixture of axial and equatorial products in a ratio of approximately 1 : 1. To improve stereoselectivity, it was necessary for the substituent in position 3 to be in the axial position.

[0032] In the article (Lazar, L. et al.: Organic Letters, 14, 17, 4650-4653, 2012), the photochemical addition of thiols to endocyclic double bonds of enoses with an acetoxy group in position 2 has been described. Additions to enose with an unsaturated double bond in position 2-3 have also been described. Reactions without deoxygenation gave lower yields. The reactions are carried out with repeated addition of photoinitiator and irradiation. Even with prolonged irradiation, full conversion was not achieved.

[0033] In the publication (Lazar, L. et al.: Organic and Biomolecular Chemistry, 11, 5339, 2013), radical additions of thiols to unsaturated bonds of glycals protected by acetyl or benzyl groups are described. Reactions in methanol, DMF and water: DMF mixtures are mentioned. Changing the solvent did not improve the course of the reaction. The reactions were again carried out with 3-4 photoinitiator additions, followed by 15 minutes of irradiation. Full conversion was not achieved, in some cases the yields were low. Higher yields are provided by substrates with exoglycosidic double bond.

[0034] In the following publication (Lazar, L. et al.: New Journal of Chemistry, 41, 1284, 2017), the addition of thiols to a protected unsaturated saccharide with a carboxamide, methoxycarbonyl and nitrile group in position 1 is described. These substrates should behave as captodative radicals and their reaction should theoretically be facilitated due to the higher stability of the intermediate radical. In practice, these substrates react worse. According to the authors, the reverse step, elimination of the thiol radical, may be preferred. Full conversion was not achieved, even in the case of prolonging the irradiation time, increasing the excess of thiol or photoinitiator. The use of a polar protic solvent did not lead to a noticeable increase in conversion. Furthermore, by-products are formed by reactions with the solvent.

[0035] In the article (Lazar, L. et al.: Carbohydrate Research, 470, 8-12, 2018), the photoinitiated reaction of acetylated glucal with 1.3 equivalents of protected 4-thioglucose was described. The reaction was carried out under very mild conditions (- 80 °C, 75 min) under irradiation with Xmax=365 nm in the presence of the photoinitiator 2,2-dimethoxy-2- phenylacetophenone (DPAP) in a yield of 53 %. The resulting disaccharide can be further transformed and used for the preparation of the tetrasaccharide.

[0036] In the article (Ma, S.-H. et al.: RSC Advances, 11, 18409, 2021) the addition of thiols to the 4-position of a protected 4, 5 -unsaturated uronate was described. The radical reaction had a low yield due to the electron-withdrawing substituent on the unsaturated bond. 5 equivalents of the photoinitiator DPAP and 3 equivalents of benzyl mercaptan were added to the unsaturated uronate solution and the mixture was irradiated for 1 hour with radiation at a wavelength of 365 nm. The addition of reagents and irradiation were performed 4 times in total. The yields were around 30 %. The addition of a protic solvent reduces the reaction yield.

[0037] In general, it can be stated that radical addition occurs more easily on less hindered substrates. Addition to unsaturated saccharides is accompanied by elimination of the thiol radical and requires repeated addition of photoinitiator and cooling to very low temperatures (- 80 °C). Furthermore, protected oligosaccharides were used exclusively. The yields of reactions of hindered substrates are generally rather low.

[0038] Ionic addition to unsaturated oligosaccharides.

[0039] In the article (Lutolf, M. P. et al.: Bioconjugate Chemistry, 12, 1051-1056, 2001), the effect of charge on the addition of charged cysteine-containing peptides to alkenes with a TI- acceptor group was investigated. It was found that the concentration of thiolate anions has the greatest influence on the reaction, and that this effect dominates over other parameters.

[0040] Authors of the article (Gennari, A. et al.: Tetrahedron, 76, 47, 131637, 2020) investigated the effect of conditions on the addition of thiol to various alkenes with a 7t-acceptor group. They found that the course of the reaction depends only on the PKA of the thiol, not on its structural parameters such as size or polarity.

[0041] In the article (Schuster, M. C. et al.: Organic Letters, 5, 1407-1410, 2003), the addition of various thiols to the unsaturated bond of shikimic acid ester was described. The reaction was carried out in alcohols under the catalysis of strongly basic alcoholates. The addition occurs exclusively to the axial position of the double bond.

[0042] In the article (Wang, Z. et al.: Chemical Communications, 50, 7004, 2014), the Michael addition of a thiol to the non-reducing end of an unsaturated benzylated heparin was described. The reaction takes place in formamide under catalysis by boric acid at 50 °C. The authors did not determine the configuration at the terminal saccharide. In another publication (Przybylski, C. et al.: Chemical Communications, 55, 4182-4185, 2019), an identical procedure was used, this time with 2-mercaptoethanol.

[0043] In the aforementioned article (Ma, S.-H. et al.: RSC Advances, 11, 18409, 2021), the addition of thiols to the 4-position of a protected 4, 5 -unsaturated uronate was described. The ionic addition was carried out at 60 and 80 °C for 18 hours with 10 equivalents of thiol and 1 equivalent of nitrogenous base. The reactions were carried out in acetonitrile, THF, toluene and CHCh. The highest yield achieved was 86 % in acetonitrile. The products were formed in D- galacto and L-altro configurations in various ratios. The D-galacto product predominated in most cases. The reaction with thiosaccharides or cysteine derivatives occurs only in trace amounts.

[0044] In general, ionic additions of thiols to unsaturated saccharides or similar substances have been carried out in non-aqueous solvents at elevated temperatures and catalyzed by strong bases. These conditions are not suitable for HA oligosaccharides, as there is cleavage of N- acetyl glucosamine from the reducing end (Muckenschnabel, I. et al. : Cancer Letters, 1998, 131, 13-20). Furthermore, only addition to the axial position of the oligosaccharide was observed, which corresponds to products in the D-galacto or L-altro configuration. Addition was unsuccessful with some thiols, for example cysteine.

[0045] General summary

[0046] In general, it can be stated that oligosaccharides according to structural formula I have not been described yet. Basic disaccharides of glycosaminoglycans with a sulfide bridge prepared using classical saccharide chemistry with the use of protecting groups have been described. Similar oligosaccharides based on the heparosan structure, prepared using transglycosylation enzymes, have also been described. Thiol adducts to the non-reducing end of benzylated heparin have also been described, but without determining the configuration. Modification of the non-reducing end of HA oligosaccharide by addition of a thiol has not been described.

[0047] It is surprising that the radical addition of thiol proceeds with high conversion even on the unsaturated HA oligosaccharide with an electron-withdrawing substituent, because according to the literature, these systems are less reactive (Lazar, L. et al.: New Journal of Chemistry, 41, 1284, 2017). When adding thiol to a similar system, the conversion was impaired by the addition of methanol as a polar protic solvent (Ma, S.-H. et al. : RSC Advances, 11, 18409, 2021), therefore it is surprising that the addition to the HA oligosaccharide proceeds well even in a protic environment. In the literature (Lazar, L. et al.: Organic Letters, 14, 17, 4650-4653, 2012; Borbas, A.: Chemistry - A European Journal, 26, 6090-6101, 2020), the necessity of multiple additions of photoinitiator and thiol for high conversion is often described, therefore it is surprising that full conversion is achieved even without repeated additions. Furthermore, for photochemical reactions on unsaturated saccharides it is important to carry out the reaction at low temperature, (Borbas, A.: Chemistry - A European Journal, 26, 6090- 6101, 2020), therefore it is surprising that addition to HA oligosaccharides occurs even at normal temperatures (-10 to 30 °C).

[0048] Ionic addition to similar unsaturated oligosaccharides is described only upon heating with strong bases in organic solvents (Schuster, M. C. et al.: Organic Letters, 5, 1407-1410, 2003, Ma, S.-H. et al.: RSC Advances, 11, 18409, 2021; Wang, Z. et al.: Chemical Communications, 50, 7004, 2014), therefore it is surprising that addition to the HA oligosaccharide occurs under very mild conditions in an aqueous environment.

[0049] Furthermore, it is surprising that ionic addition does not occur on amides (Comparative Examples 17, 18, 23, 24, 51, 52), although they are generally suitable substrates for binding thiols (-SH) or thiolates (-S') (Gennari, A. et al.: Tetrahedron, 76, 47, 131637, 2020).

[0050] It is also surprising that the addition also occurs with cysteine, because on a similar system, the addition of cysteine practically did not occur (Ma, S.-H. et al.: RSC Advances, 11, 18409, 2021). Furthermore, it is surprising that the mixture of ionic addition products also contains the D-gluco isomer, since only products in the L-altro and D-galacto configurations are described in the literature (Ma, S.-H. et al.: RSC Advances, 11, 18409, 2021; Przybylski, C. et al.: Chemical Communications, 55, 4182-4185, 2019). According to the literature, the main structural parameter affecting the reaction rate should be the PKA value of the thiol (Lutolf, M. P. et al.: Bioconjugate Chemistry, 12, 1051-1056, 2001; Gennari, A. et al.: Tetrahedron, 76, 47, 131637, 2020), therefore, it is surprising that the product distribution is strongly dependent on the thiol structure, where thiols with a hydroxyl or amino group in the beta position to the thiol function also yield the D-gluco product. Conversely, if this group is blocked, for example by an acetyl group, the D-gluco product is not formed.

[0051] The advantage of the solution described in this invention is that the structure of the anomeric end of the oligosaccharide is preserved unchanged. This end is the most reactive site from a general chemical point of view, which can be important for interactions with receptors, or it can be used for subsequent reversible or irreversible binding of a wide range of nucleophiles such as amino compounds (CZ305153, 2015; WO 2008 / 014787; WO 2008 / 115799; WO 2007 / 101243; US 2012 / 0277416).

[0052] Summary of the invention

[0053] The invention relates to oligosaccharides composed of alternating glucuronic and N- acetylglucosamine units, with a P-D-galacturonic, P-D-glucuronic or P-L-altruronic unit at the non-reducing end with a substituent attached by a sulfide bond according to the structural formulae (I, II a III), wherein n = 0 to 8;

[0054] X is O or N-H;

[0055] Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7;

[0056] R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally containing N, S, Cl or O atoms. Preferably, the substituent R is selected from the group comprising amino acids, peptides, monosaccharides or oligosaccharides. In one preferred embodiment, the substituent R is an aliphatic linear chain C4-C8 containing a free terminal group -SH and optionally one or more N, S, or O atoms. In another preferred embodiment, the substituent R comprises the structure of a pharmaceutically active substance. Furthermore, the invention relates to a method for preparing an oligosaccharide according to structural formula I, II or III, where the following 4 options can be used:

[0057] Option 1 : The input material is an unsaturated oligosaccharide of hyaluronic acid with a length of 2 to 18 saccharides with carboxyl groups in the form of ester, amide, acid or its salt, which is irradiated in the presence of a photoinitiator and a thiol.

[0058] Option 2:

[0059] The input material is an unsaturated oligosaccharide of hyaluronic acid with a length of 2 to 18 saccharides with carboxyl groups in the form of an ester, which reacts with a thiol in solution.

[0060] Option 3 :

[0061] The input material is an unsaturated hyaluronic acid oligosaccharide of 2 to 18 saccharides in length with carboxyl groups converted to an ester, which in the first step reacts with a thiol in solution, and in the second step the ester groups of the oligosaccharide are hydrolyzed to form carboxyls.

[0062] Option 4:

[0063] The material prepared according to Options 1-3, which has a free thiol group, is reacted in the next step with an acrylic acid ester or amide.

[0064] These 4 options are described in more detail below:

[0065] Option 1 :

[0066] The starting unsaturated oligosaccharide of hyaluronic acid or its derivative in the form of an ester or amide is dissolved at a concentration of 0.05 to 0.68 mol.L'1in water or in a mixture of water and an organic solvent, for example DMF, DMSO, THF, acetone, acetonitrile, methanol or 2-propanol, with the amount of organic solvent being 0 - 60 vol %, then a thiol is added in an amount of 0.36 to 2.08 molar equivalents relative to the oligosaccharide, and a photoinitiator, for example DPAP, TPO, Irgacure 2959, Irgacure 1173 or LAP, in an amount of 0.1 to 0.36 molar equivalents relative to the oligosaccharide in 1 to 3 doses, and the resulting mixture is irradiated with light of wavelength 254, 365 or 455 nm at a temperature of -10 to 30 °C for 5 to 240 minutes to form a modified oligosaccharide or a mixture of modified and unmodified oligosaccharide. The products can be separated by precipitation or chromatography.

[0067] Scheme 1 - Option 1 wherein n = 0 to 8;

[0068] X is O or N-H;

[0069] Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7;

[0070] R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally containing N, S, Cl or O atoms. The thiol is preferably selected from the group comprising cysteamine, dithiothreitol, DODT, glutathione, L-cysteine, N-acetyl-L-cysteine methyl ester, N-acetyl-L-cysteine, 2-mercaptoethanol, N-acetyl-L-cysteine 2-mercaptoethylamide, 3- mercaptopropionic acid, meso-2, 3 -dimercaptosuccinic acid, 1-thio-B-D-glucose, thioglycolic acid, HA thiol tetrasaccharide, HA thiol hexasaccharide, HA thiol octasaccharide, HA thiol decasaccharide.

[0071] Option 2:

[0072] The starting unsaturated oligosaccharide of hyaluronic acid in the form of an ester is dissolved at a concentration of 0.02 to 0.6 mol.L'1in water or a mixture of water and an organic solvent, for example acetonitrile or 2-propanol, with the amount of organic solvent being 0 - 25 vol %, then a thiol is added in an amount of 2 to 8 molar equivalents relative to the oligosaccharide, the pH of the solution is adjusted to 6.02 to 9.1 using a base, for example LiOH, NaOH, NaOD, NazCOs, NaHCOs, K2CO3, KHCO3 or triethylamine, and the resulting mixture is stirred at a temperature of -20 to 50 °C for 1-72 hours to form a modified oligosaccharide or a mixture of modified and unmodified oligosaccharide. The products can be separated by precipitation or chromatography.

[0073] Scheme 2 - Option 2 wherein n = 0 to 8;

[0074] Y is aliphatic, aromatic, linear, branched or cyclic chain C2-C7;

[0075] R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally containing N, S, Cl or O atoms. The thiol is preferably selected from the group comprising cysteamine, D-cysteine, 2-(diethylamino)ethanethiol, dithiothreitol, DODT, thioglycolic acid ethyl ester, glutathione, L-cysteine, L-cysteine methyl ester, N-acetyl-L-cysteine methyl ester, 2-mercaptoethanol, N-acetyl-L-cysteine 2-mercaptoethyl amide, meso-2, 3 -dimercaptosuccinic acid, N-acetyl-L-cysteine, thioglycolic acid, thiocholine.

[0076] The resulting modified oligosaccharide or the mixture of modified and unmodified oligosaccharide can undergo hydrolysis of ester groups to form carboxyl groups - see Option 3.

[0077] Option 3 :

[0078] The starting unsaturated oligosaccharide of hyaluronic acid in the form of an ester is dissolved at a concentration of 0.02 to 0.6 mol.L'1in water or a mixture of water and an organic solvent, for example acetonitrile or 2-propanol, with the amount of organic solvent being 0 - 25 vol %, then a thiol is added in an amount of 2 to 8 molar equivalents relative to the oligosaccharide, the pH of the solution is adjusted to 6.02 to 9.1 and the resulting mixture is stirred at a temperature of -20 to 50 °C for 1-72 hours. The pH is then adjusted to 8.0 to 9.0, for example using LiOH, NaOH, NaOD, ISfeCCh, NaHCCh, K2CO3, KHCO3 or triethylamine, and the resulting mixture is stirred at a temperature of 20 - 50 °C for 24-120 hours. The products can be separated by precipitation or chromatography.

[0079] Scheme 3 - Option 3 wherein n = 0 to 8;

[0080] X is aliphatic, aromatic, linear, branched or cyclic chain C2-C7;

[0081] Y is H, Na or K; R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally containing N, S, Cl or O atoms.

[0082] Option 4

[0083] The initial modified oligosaccharide of hyaluronic acid containing in its structure a -SH group, prepared according to any of options 1, 2 or 3, is dissolved in water and added to an ester or amide of acrylic acid optionally with a covalently bound active substance, the ester or amide being dissolved in water or an organic solvent selected from the group comprising methanol, ethanol, 2-propanol, dimethyl sulfoxide, dimethyl formamide, acetonitrile or tetrahydrofuran, in an amount of 1-3 molar equivalents relative to the oligosaccharide, the resulting amount of organic solvent is 0 - 25 vol %, the pH of the solution is adjusted to 7 to 8 and the reaction mixture is then stirred at a temperature of 25 °C for 1-72 hours. The products can be separated by precipitation or chromatography.

[0084] Scheme 4 - Option 4 wherein n = 0 to 8;

[0085] A is O or N-H;

[0086] B contains a fragment of the active substance;

[0087] X is O or N-H;

[0088] Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7;

[0089] R is aliphatic linear chain C4-C8, optionally containing N, S, or O atoms.

[0090] The invention further relates to a method for preparing a modified oligosaccharide according to structural formulae (I, II and III), where R contains the structure of a pharmaceutically active substance, wherein first an acryloyl derivative of a pharmaceutically active substance according to the formula (VI) is prepared wherein A is O, N-H or NH-CH2CH2-O and B is a pharmaceutically active substance; further, a modified oligosaccharide according to the structural formulae (I, II and III) is prepared, where R is an aliphatic linear chain C4-C8 containing a free terminal group -SH and optionally one or more N, S, or O atoms, by the method according to any of options 1 to 3. Subsequently, the resulting oligosaccharide is dissolved in water and added to the derivative according to structural formula (VI) dissolved in water, a water-miscible organic solvent or a mixture thereof, wherein the organic solvent is selected from the group comprising methanol, ethanol, 2-propanol, dimethyl sulfoxide, dimethyl formamide, acetonitrile, tetrahydrofuran, or a mixture thereof, where the amount of the derivative according to formula (VI) is 1-3 molar equivalents relative to the oligosaccharide and the resulting amount of organic solvent is 0-25 vol %. Then, the pH of the solution is adjusted to 7 to 8 and the reaction mixture is then stirred at a temperature of 25 °C for 1-72 hours.

[0091] The pharmaceutically active substance is preferably selected from the group comprising substances with anti-inflammatory, antibacterial, antiviral or anticancer effects, for example acetaminophen, 5-aminosalicylic acid, diclofenac, ibuprofen, indomethacin, ketoprofen, naproxen, ciprofloxacin, metronidazole, acyclovir, darunavir, didanosine, ganciclovir or ribavirin, daunorubicin, doxorubicin, lenalidomide, mercaptopurine.

[0092] A modified oligosaccharide with a bound pharmaceutically active substance can be used, for example, in the treatment of inflammatory diseases, bacterial diseases, viral diseases, cancer, chronic intestinal inflammation, Crohn's disease or ulcerative colitis.

[0093] The materials prepared according to present invention do not show cytotoxic effects on 3T3, NHDF, HaCaT, HT-29 and AD-MSC cells at concentrations 10-1,000 pg / mL (Examples 130, 131, 132, 133, 134).

[0094] The materials prepared according to the present invention contain a hyaluronic acid fragment and optionally a fragment of an active substance, both of which may exhibit biological activity. The materials may also be used as intermediates for further syntheses. Option 4 also includes the attachment of active medicinal substances, such as diclofenac, to the oligosaccharide and its use as a carrier. The ester or amide bond with the active substance may be hydrolyzed in the body (Shandbag, V.R. et al.: Journal of Pharmaceutical Sciences, 81, 2, 149-154, 1992), alternatively, conjugates may show activity even without prior hydrolysis (Galanakis, D. et al: Bioorganic & Medicinal Chemistry Letters, 14, 3639-3643, 2004). Other possibilities include the attachment of a wide range of compounds to the electrophilic anomeric end of the saccharide, which allows reversible and irreversible binding of various nucleophiles, such as amines, oxyamines, or hydrazides. Furthermore, the present invention provides pharmaceutical compositions with antiinflammatory, antibacterial, antiviral or anticancer effects.

[0095] The compositions in solution form contain:

[0096] 0.1 - 5 wt. % of modified oligosaccharide with a bound pharmaceutically active substance,

[0097] 0.1 - 1.0 wt. % of sodium or potassium chloride,

[0098] 94 - 99.8 wt. % of water.

[0099] In a preferred embodiment, the pharmaceutical compositions in solution form contain:

[0100] 0.1 - 5 wt. % of modified oligosaccharide with a bound pharmaceutically active substance,

[0101] 0.1 - 1.0 wt. % of sodium or potassium chloride,

[0102] 0.1 - 1.0 wt. % of sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate or mixtures thereof, and / or 0.001 - 5 % of other additives selected from the group comprising calcium chloride, glycerol, hyaluronic acid oligosaccharide or its derivative, hyaluronic acid or its derivative, chondroitin sulfate,

[0103] 88 - 99.699 wt. % of water.

[0104] Solid compositions in the form of a film or lyophilisate contain:

[0105] 1 - 10 wt. % of modified oligosaccharide as defined in claim 4,

[0106] 75 - 98.9 wt. % of one or more carrier polymers selected from the group comprising hyaluronic acid or its derivative, chondroitin sulfate,

[0107] 0.1 - 15 wt. % of water.

[0108] In a preferred embodiment, the solid compositions contain:

[0109] - 1 - 10 wt. % of modified oligosaccharide,

[0110] 70 - 98.8 wt. % of one or more carrier polymers selected from the group comprising hyaluronic acid or its derivative, chondroitin sulfate.

[0111] 0.1 - 5 wt. % of hyaluronic acid oligosaccharide or its derivative, sodium or potassium chloride, calcium chloride, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate or mixtures thereof,

[0112] 0.1 - 15 wt. % of water.

[0113] The term hyaluronic acid includes a polymer composed of alternating D-glucuronic and D-N-acetylglucosamine units, linked by P-( 1 — 3) and P-(l — 4) glycosidic bonds, with a weight average molecular weight of 10-2,000 kDa.

[0114] The term hyaluronic acid derivative includes: hyaluronic acid oxidized at the 6-position of N-acetyl glucosamine to an aldehyde; hyaluronic acid oxidized at the 6-position of N- acetylglucosamine to an aldehyde to which an amino group-containing substance, e.g. benzylamine, cysteamine, amino acid or peptide, is attached via an imine bond; hyaluronic acid with a carboxylic acid, e.g. acetic, hexanoic, lauric or oleic acid, attached to the 6-position of N-acetylglucosamine via an ester bond; hyaluronic acid reduced at the 6-position of glucuronic acid to an alcohol; hyaluronic acid with an amino compound, e.g. benzylamine, cysteamine, amino acid or peptide, attached to the 6-position of glucuronic acid via an amide bond.

[0115] The term hyaluronic acid oligosaccharide includes an oligomer composed of alternating D-glucuronic and D-N-acetylglucosamine units, linked by -(l— >3) and 0-(l— >4) glycosidic bonds, with a weight average molecular weight of 400-10,000 Da.

[0116] The term hyaluronic acid oligosaccharide derivative includes: hyaluronic acid oligosaccharide with a double bond at position 4,5 of the terminal glucuronic acid; hyaluronic acid oligosaccharide with an amino compound, e.g., benzylamine, cysteamine, amino acid or peptide, attached to the reducing end of the oligosaccharide via an imine or amine bond; modified oligosaccharide prepared by the present invention.

[0117] The term "structure of a pharmaceutically active substance" or "fragment of a pharmaceutically active substance" means that the structure contains a part of the pharmaceutically active substance, formed, for example, by removing a hydrogen or hydroxyl group. The fragment or structure can be converted back to the starting pharmaceutically active substance by a suitable reaction, for example, hydrolysis.

[0118] Conversion can be reduced by reducing the excess of thiol and shortening the reaction time. In some cases, higher conversion can be achieved by changing the reaction conditions (for example, extending the reaction time, more equivalents of thiol, adjusting the pH). Furthermore, the material can be separated (for example, chromatographically), thereby achieving higher purity.

[0119] Description of the drawings

[0120] Fig. 1 - The effect of materials prepared according to Examples 71, 77, 98, 102, 103, 105, 106, and 107 on 3T3 cell viability.

[0121] Fig. 2 - The effect of materials prepared according to Examples 71, 77, 98, 102, 103, 105, 106, and 107 on HaCaT cell viability.

[0122] Fig. 3 - The effect of materials prepared according to Examples 71, 77, 98, 102, 103, 105, 106, and 107 on HT-29 cell viability. Fig. 4 - The effect of materials prepared according to Examples 71, 77, 98, 102, 103, 105, 106, and 107 on NHDF cell viability.

[0123] Fig. 5 - The effect of materials prepared according to Examples 71, 77, 98, 102, 103, 106, and 107 on AD-MSC cell viability.

[0124] Examples of embodiments

[0125] CDI = 1,1'- carbonyl diimidazole

[0126] DCM = dichloromethane

[0127] DMEM = Dulbecco's Modified Eagle's Medium

[0128] DMF = dimethyl formamide

[0129] DMSO = dimethyl sulfoxide

[0130] DODT = 2,2-(ethylenedioxy)diethanethiol

[0131] DPAP = 2,2-dimethoxy-2-phenylacetophenone

[0132] DTT = dithiothreitol

[0133] FBS = fetal bovine serum

[0134] HA = hyaluronic acid

[0135] IPA = 2-propanol

[0136] Irgacure 1173 - 2-hydroxy-2-methyl-l-phenylpropan-l-one

[0137] Irgacure 2959 = 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone

[0138] LAP = lithium phenyl-2,4,6-trimethylbenzoyl phosphinate

[0139] MeOH = methanol

[0140] MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0141] Mw = molecular weight

[0142] NMR analysis = (700 MHz, D2O, 5 ppm)

[0143] TEA = triethylamine

[0144] THF = tetrahydrofuran

[0145] TPO = diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

[0146] Percentages are given as mole percentages unless otherwise stated.

[0147] The Conversion value was determined by comparing the sum of the integrals of the double bond signals at 6.2 ppm and 5.8 ppm with the integral of the acetyl group signals at 2.0 ppm according to the formula a = 100% x (1 - 3 x [I6 2+ Is.s b.o]).

[0148] The identity of the isomers at the non-reducing end of the oligosaccharide was determined by the presence of signals of anomeric hydrogens on the saccharide in question as follows: L-altro 4.86 and 4.83 (2 x d, J= 2.75 Hz); D-gluco 4.57 and 4.52 (2 x d, J= 7.87 Hz); D-galacto 4.46 a 4.42 (2 x d, J = 7.80 Hz). The distribution of isomers was determined from the values of the integrals of the acetyl signals according to the following formula xiSOmer = 100% x ([IiSomer] / ([lAit] + [loai] + [IGIU]); wherein Xisomer is the distribution of the given isomer, lisomer is the integral of the signal belonging to the given isomer, [IAH] is the value of the signal integral at 2.04 and belongs to the L-altro isomer, [loai] is the value of the integral of the signal at 2.02 and belongs to the D-galacto isomer, [IGIU] is the value of the signal integral at 2.01 and belongs to the D-gluco isomer.

[0149] The designation D-galacto corresponds to structure I, D-gluco corresponds to structure II, L-altro corresponds to structure III.

[0150] Preparation of the starting materials

[0151] Example 1

[0152] Preparation of HA unsaturated disaccharide ethyl ester

[0153] HA unsaturated disaccharide (804 mg, 2 mmol) was dissolved in DMSO (2 mL). Then DIPEA (0.7 mL, 4 mmol) and ethyl iodide (0.32 mL, 4 mmol) were added and the reaction mixture was stirred at room temperature for 16 hours. Then the reaction mixture was acidified with acetic acid (0.2 mL), diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured. 'H NMR 5 6.25 (1H, C=C-H), 4.30 (2H, COOCH2), 2.08 (3H, COCH3), 1.32 (3H, CH3).

[0154] Example 2

[0155] Preparation of HA unsaturated decasaccharide ethyl ester

[0156] HA unsaturated decasaccharide (800 mg, 0.4 mmol) was dissolved in DMSO (2 mL). Then DIPEA (0.7 mL, 4 mmol) and ethyl iodide (0.32 mL, 4 mmol) were added and the reaction mixture was stirred at room temperature for 44 hours. Then the reaction mixture was acidified with acetic acid (0.2 mL), diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0157] 'H NMR 5 6.25 (1H, C=C-H), 4.30 (10H, COOCH2), 2.08 (3H, COCH3), 2.02 (12H, COCH3), 1.32 (15H, CH3).

[0158] Example 3

[0159] Preparation of HA unsaturated octadecasaccharide ethyl ester HA unsaturated octadecasaccharide (360 mg, 0.1 mmol) was dissolved in DMSO (1 mL). Then DIPEA (0.35 mL, 2 mmol) and ethyl iodide (0.16 mL, 2 mmol) were added and the reaction mixture was stirred at room temperature for 120 hours. Then the reaction mixture was acidified with acetic acid (0.2 mL), diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0160] 'H NMR 5 6.25 (1H, C=C-H), 4.30 (18H, COOCH2), 2.08 (3H, COCH3), 2.02 (24H, COCH3), 1.32 (27H, CH3).

[0161] Example 4

[0162] Preparation of HA unsaturated disaccharide benzyl ester

[0163] HA unsaturated disaccharide (804 mg, 2 mmol) was dissolved in DMSO (2 mL). Then DIPEA (0.7 mL, 4 mmol) and benzyl bromide (0.47 mL, 4 mmol) were added and the reaction mixture was stirred at room temperature for 16 hours. Then the reaction mixture was acidified with acetic acid (0.2 mL), diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0164] XH NMR 5 7.45 (5H, Ar-H), 6.25 (1H, C=C-H), 5.30 (2H, ArCH2), 2.07 (3H, COCH3).

[0165] Example 5

[0166] Preparation of HA unsaturated disaccharide benzyl amide

[0167] HA unsaturated disaccharide (200 mg, 0.5 mmol) and benzylamine (0.28 mL, 2.6 mmol) were dissolved in distilled water (1.8 mL) and the pH was adjusted to 7.6 by means of 5M HC1. Then DMTMM (144 mg, 0.52 mmol) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 120 hours. Then the reaction mixture was filtered and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0168] XH NMR 5 7.41 (2H, Ar-H), 7.36 (2H, Ar-H), 6.06 (1H, C=C-H), 4.51 (2H, ArCH2), 2.07 (3H, COCH3).

[0169] Example 6

[0170] Preparation of HA unsaturated disaccharide butyl ester

[0171] HA unsaturated disaccharide (200 mg, 0.5 mmol) and butylamine (0.26 mL, 2.6 mmol) were dissolved in distilled water (1.8 mL) and the pH was adjusted to 7.6 by means of 5M HC1. Then DMTMM (144 mg, 0.52 mmol) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 120 hours. Then the reaction mixture was filtered and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions andTH NMR was measured.

[0172] XH NMR 5 6.00 (1H, C=C-H), 3.29 (2H, COOCH2), 2.09 (3H, COCH3), 1.18 (3H, CH3).

[0173] Example 7

[0174] Preparation of HA unsaturated disaccharide nitrobenzyl ester

[0175] HA unsaturated disaccharide (400 mg, 1 mmol) was dissolved in DMSO (1 mL). Then DIPEA (0.35 mL, 2 mmol) and 4-nitrobenzyl bromide (430 mg, 2 mmol) were added and the reaction mixture was stirred at room temperature for 16 hours. Then the reaction mixture was acidified with acetic acid (0.1 mL), diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0176] 'H NMR 5 8.27 (2H, Ar-H), 7.67 (2H, Ar-H), 6.31 (1H, C=C-H), 5.41 (2H, ArCH2), 2.05 (3H, COCH3).

[0177] Example 8

[0178] Preparation of 2-chloro-l,3-dimethylimidazolinium chloride (DMC) l,3-Dimethyl-2-imidazolidinone (5.28 g, 46 mmol) was dissolved in toluene (20 mL) and then oxalyl chloride was added dropwise (5 mL, 58 mmol). The reaction mixture was heated for 6 hours at 60 °C, then heating was stopped, the reaction mixture was filtered through a frit. The precipitate was washed with hexane (20 mL), dried in a desiccator and analyzed by 'H NMR. Conversion >98 %, yield 79 %.

[0179] XH NMR 5 4.01 (4H, CH2), 3.18 (6H, CH3).

[0180] Example 9

[0181] Preparation of HA tetrasaccharide thiol

[0182] HA saturated tetrasaccharide (1.0 g, 1.22 mmol) and DMC (2.1 g, 12.2 mmol), prepared according to Example 8, were dissolved in a mixture of water (45 mL) and acetonitrile (20 mL) and cooled to 0 °C. DIPEA (6.4 mL, 36.6 mmol) was added to the reaction mixture and the reaction mixture was stirred for 5 minutes at 0 °C and then for 85 minutes at room temperature. Thiobenzoic acid (2.5 g, 18.3 mmol) in acetonitrile (15 mL) was then added and the reaction mixture was stirred for an additional 15 minutes. The reaction was terminated by addition of water (45 mL) and extraction with chloroform (3x30 mL). The aqueous phase was evaporated, the residue was dissolved in distilled water and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated, dissolved in methanol (6 mL) and slowly added to a mixture of IPA (20 mL) and MeONa (2.2 mL, 25% solution in MeOH). The solid was centrifuged, air-dried and analyzed by 'H NMR.

[0183] XH NMR 5 4.71 (1H, SCH), 2.03 (6H, COCH3).

[0184] Example 10

[0185] Preparation of HA hexasaccharide thiol

[0186] HA saturated hexasaccharide (1.5 g, 1.22 mmol) and DMC (2,1 g, 12,2 mmol), prepared according to Example 8, were dissolved in a mixture of water (45 mL) and acetonitrile (20 mL) and cooled to 0 °C. DIPEA (6.4 mL, 36.6 mmol) was added to the reaction mixture and the reaction mixture was stirred for 5 minutes at 0 °C and then for 85 minutes at room temperature. Thiobenzoic acid (2.5 g, 18.3 mmol) in acetonitrile (15 mL) was then added and the reaction mixture was stirred for further 15 min. The reaction was quenched by addition of water (45 mL) and extraction with chloroform (3^30 mL). The aqueous phase was evaporated, the residue was dissolved in distilled water and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated, dissolved in methanol (9 mL) and slowly added to a mixture of IPA (20 mL) and MeONa (2.2 mL, 25% solution in MeOH). The solid was centrifuged, air-dried and analyzed by 'H NMR.

[0187] XH NMR 5 4.71 (1H, SCH), 2.03 (9H, COCH3).

[0188] Example 11

[0189] Preparation of HA octasaccharide thiol

[0190] HA saturated octasaccharide (2.0 g, 1.22 mmol) and DMC (2,1 g, 12,2 mmol), prepared according to Example 8, were dissolved in a mixture of water (45 mL) and acetonitrile (20 mL) and cooled to 0 °C. DIPEA (6.4 mL, 36.6 mmol) was added to the reaction mixture and the reaction mixture was stirred for 5 minutes at 0 °C and then for 85 minutes at room temperature. Thiobenzoic acid (2.5 g, 18.3 mmol) in acetonitrile (15 mL) was then added and the reaction mixture was stirred for further 15 min. The reaction was quenched by addition of water (45 mL) and extraction with chloroform (3x30 mL). The aqueous phase was evaporated, the residue was dissolved in distilled water and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated, dissolved in methanol (9 mL) and slowly added to a mixture of IPA (20 mL) and MeONa (2.2 mL, 25% solution in MeOH). The solid was centrifuged, air-dried and analyzed by 'H NMR.

[0191] 'H NMR 5 4.71 (1H, SCH), 2.03 (12H, COCH3). Example 12

[0192] Preparation of HA decasaccharide thiol

[0193] HA saturated hexasaccharide (2.5 g, 1.22 mmol) and DMC (2,1 g, 12,2 mmol), prepared according to Example 8, were dissolved in a mixture of water (45 mL) and acetonitrile (20 mL) and cooled to 0 °C. DIPEA (6.4 mL, 36.6 mmol) was added to the reaction mixture and the reaction mixture was stirred for 5 minutes at 0 °C and then for 85 minutes at room temperature. Thiobenzoic acid (2.5 g, 18.3 mmol) in acetonitrile (15 mL) was then added and the reaction mixture was stirred for further 15 min. The reaction was quenched by addition of water (45 mL) and extraction with chloroform (3^30 mL). The aqueous phase was evaporated, the residue was dissolved in distilled water and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated, dissolved in methanol (9 mL) and slowly added to a mixture of IPA (20 mL) and MeONa (2.2 mL, 25% solution in MeOH). The solid was centrifuged, air-dried and analyzed by 'H NMR.

[0194] XH NMR 5 4.71 (1H, SCH), 2.03 (15H, COCH3).

[0195] Example 13

[0196] Preparation of HA decasaccharide with cysteine at the reducing end

[0197] HA saturated decasaccharide (0.5 g, 0.25 mmol) and L-cysteine hydrochloride (9.5 mg, 0.060 mmol; 240 mg, 1.5 mmol) were dissolved in water (10 mL). The solution was acidified with acetic acid (0.07 mL, 1.25 mmol) and the reaction mixture was stirred at 25 °C for 72 hours. The reaction mixture was then neutralized with NaHCCL, precipitated with IPA (50 mL) and reprecipitated twice with water and IPA. The material thus obtained was dried in vacuum and redissolved in water (10 mL) together with NaBHsCN (5 eq.). The reaction mixture was stirred at 25 °C for 48 hours and then precipitated with IPA (50 mL). The solid was dried in vacuum and analyzed by1H NMR.

[0198] 'H NMR 5 2.03 (15H, COCH3).

[0199] Ionic reactions

[0200] Example 14

[0201] Ionic addition of cysteamine to HA unsaturated disaccharide ethyl ester

[0202] HA unsaturated disaccharide ethyl ester (4.9 mg, 0.012 mmol), prepared according to Example 1, and cysteamine hydrochloride (6.8 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.25 with 0.1 M Na2CC>3 and the reaction mixture was left to stand at room temperature for 20 hours. Then1H NMR was measured and the isomer distribution was determined.

[0203] Conversion 79 %. Distribution 3 % D-galacto (I), 16 % D-gluco (II), 81 % L-altro (III).

[0204] Example 15

[0205] Ionic addition of cysteamine to HA unsaturated disaccharide benzyl ester

[0206] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and cysteamine hydrochloride (6.8 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.25 and the reaction mixture was left to stand at room temperature for 20 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0207] Conversion 95 %. Distribution 5 % D-galacto (I), 14 % D-gluco (II), 81 % L-altro (III).

[0208] Example 16

[0209] Ionic addition of cysteamine to HA unsaturated disaccharide benzyl ester

[0210] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example

[0211] 4, and cysteamine hydrochloride (17 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 6.36. The sample was left to stand at room temperature for 16 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0212] Conversion 95 %. Distribution 7 % D-galacto (I), 16 % D-gluco (II), 77 % L-altro (III).

[0213] Comparative Example 17

[0214] Ionic addition of cysteamine to HA unsaturated disaccharide benzyl amide

[0215] HA unsaturated disaccharide benzyl amide (14 mg, 0.03 mmol), prepared according to Example

[0216] 5, and cysteamine hydrochloride (17 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 6.80. The sample was left to stand at room temperature for 24 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0217] Conversion <2%.

[0218] Comparative Example 18

[0219] Ionic addition of cysteamine to HA unsaturated disaccharide butyl amide HA unsaturated disaccharide butyl amide (14 mg, 0.03 mmol), prepared according to Example 6, and cysteamine hydrochloride (17 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.18. The sample was left to stand at room temperature. After 64 hours, 'H NMR was measured and the isomer distribution was determined.

[0220] Conversion <2%.

[0221] Example 19

[0222] Ionic addition of D-cysteine to HA unsaturated disaccharide benzyl ester

[0223] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and D-cysteine hydrochloride (26 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 6.74. The sample was left to stand at room temperature for 20 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0224] Conversion 88 %. Distribution <2% D-galacto (I), 25 % D-gluco (II), 75 % L-altro (III).

[0225] Example 20

[0226] Ionic addition of HA decasaccharide with cysteine on its reducing end to HA unsaturated disaccharide benzyl ester

[0227] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and material prepared according to Example 13 (132 mg, 0.06 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.05 and the mixture was left to stand at room temperature for 48 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0228] Conversion 28 %. Distribution 3% D-galacto (I), 22 % D-gluco (II), 75 % L-altro (III).

[0229] Example 21

[0230] Ionic addition of 2-(diethylamino) ethanthiole to HA unsaturated disaccharide benzyl ester

[0231] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and 2-(diethylamino)ethanthiole hydrochloride (25 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 6.5 by means of 0.1 M NaHCOs and the mixture was left to stand at room temperature for 20 hours. Then 'H NMR was measured and the isomer distribution was determined. Conversion >98%. Distribution 3% D-galacto (I), 23 % D-gluco (II), 74 % L-altro (III).

[0232] Example 22

[0233] Ionic addition of dithiothreitole to HA unsaturated disaccharide benzyl ester

[0234] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example

[0235] 4, and dithiothreitole (23 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH of the mixture was adjusted to 8.02 by means of 0.1 M KHCO3. The sample was left to stand at room temperature and after 22 hours, the 'H NMR was measured and the isomer distribution was determined.

[0236] Conversion 98%. Distribution 24% D-galacto (I), 9 % D-gluco (II), 67 % L-altro (III).

[0237] Comparative Example 23

[0238] Ionic addition of dithiothreitole to HA unsaturated disaccharide benzyl amide

[0239] HA unsaturated disaccharide benzyl amide (14 mg, 0.03 mmol), prepared according to Example

[0240] 5, and dithiothreitole (23 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH of the mixture was adjusted to 7.99. The sample was left to stand at room temperature and after 120 hours, the 'H NMR was measured and the isomer distribution was determined. Conversion <2%.

[0241] Comparative Example 24

[0242] Ionic addition of dithiothreitole to HA unsaturated disaccharide butyl amide

[0243] HA unsaturated disaccharide butyl amide (14 mg, 0.03 mmol), prepared according to Example

[0244] 6, and dithiothreitole (23 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH of the mixture was adjusted to 7.25. The sample was left to stand at room temperature and after 64 hours, the1H NMR was measured and the isomer distribution was determined. Conversion <2%.

[0245] Example 25

[0246] Ionic addition of DODT to HA unsaturated disaccharide benzyl ester

[0247] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and DODT (27 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH was adjusted to 8.94 by means of 0.1 M NaOD. The sample was left to stand at room temperature and after 17 hours, the 'H NMR was measured and the isomer distribution was determined. Conversion 95%. Distribution 55% D-galacto (I), <2%. D-gluco (II), 45 % L-altro (III). Example 26

[0248] Ionic addition of ethyl thioglycolate to HA unsaturated disaccharide benzyl ester

[0249] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and ethyl thioglycolate (18 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH was adjusted to 7.05 and the mixture was left to stand at room temperature for 20 hours. Then1H NMR was measured and the isomer distribution was determined.

[0250] Conversion 36%. Distribution 63% D-galacto (I), <2%. D-gluco (II), 37 % L-altro (III).

[0251] Example 27

[0252] Ionic addition of glutathione to HA unsaturated disaccharide benzyl ester

[0253] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and glutathione (46 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH was adjusted to 8.14. The sample was left to stand at room temperature and after 21 hours, the1H NMR was measured and the isomer distribution was determined.

[0254] Conversion 92%. Distribution 51% D-galacto (I), <2%. D-gluco (II), 49 % L-altro (III).

[0255] Example 28

[0256] Ionic addition of cysteine to HA unsaturated disaccharide ethyl ester

[0257] HA unsaturated disaccharide ethyl ester (4.9 mg, 0.012 mmol), prepared according to Example 1, and L-cysteine hydrochloride (9.5 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH was adjusted to 7.05 and the mixture was left to stand at room temperature for 20 hours. Then1H NMR was measured and the isomer distribution was determined.

[0258] Conversion 75%. Distribution 4% D-galacto (I), 23 %. D-gluco (II), 73 % L-altro (III).

[0259] Example 29

[0260] Ionic addition of cysteine to HA unsaturated disaccharide ethyl ester in PBS

[0261] HA unsaturated disaccharide ethyl ester (4.9 mg, 0.012 mmol), prepared according to Example 1, and L-cysteine hydrochloride (6.1 mg, 0.039 mmol) were dissolved in 100 mM phosphate buffer in deuterated water (0.6 mL, pH = 7.25). The reaction mixture was left to stand at room temperature for 72 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0262] Conversion 87%. Distribution 6% D-galacto (I), 25 %. D-gluco (II), 69 % L-altro (III). Example 30

[0263] Ionic addition of cysteine to HA unsaturated decasaccharide ethyl ester

[0264] HA unsaturated decasaccharide ethyl ester (5.4 mg, 0.0027 mmol), prepared according to Example 2, and L-cysteine hydrochloride (5.0 mg, 0.032 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.89 and the reaction mixture was left to stand at room temperature for 24 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0265] Conversion 89%. Distribution 5% D-galacto (I), 20 %. D-gluco (II), 75 % L-altro (III).

[0266] Example 31

[0267] Ionic addition of cysteine to HA unsaturated octadecasaccharide ethyl ester

[0268] HA unsaturated octadecasaccharide ethyl ester (5.4 mg, 0.0027 mmol), prepared according to Example 3, and L-cysteine hydrochloride (5.0 mg, 0.032 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.45 and the reaction mixture was left to stand at room temperature for 57 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0269] Conversion 42%. Distribution 5% D-galacto (I), 21 %. D-gluco (II), 74 % L-altro (III).

[0270] Example 32

[0271] Ionic addition of cysteine to HA unsaturated disaccharide benzyl ester

[0272] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and L-cysteine hydrochloride (9.5 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.05 and the reaction mixture was left to stand at room temperature for 20 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0273] Conversion 93%. Distribution 5% D-galacto (I), 24 %. D-gluco (II), 71 % L-altro (III).

[0274] Example 33

[0275] Ionic addition of cysteine to HA unsaturated disaccharide benzyl ester

[0276] HA unsaturated disaccharide benzyl ester (100 mg, 0.21 mmol), prepared according to Example 4, and L-cysteine hydrochloride (168 mg, 1.07 mmol) were dissolved in water (3 mL). The pH of the mixture was adjusted to 7.0 by means of 0.1 M NaOH and the reaction mixture was stirred at room temperature for 23 hours. The reaction mixture was acidified with acetic acid (0.2 mL) and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated and analyzed by 'H NMR.

[0277] Conversion >98%. Distribution 10% D-galacto (I), 29 %. D-gluco (II), 61 % L-altro (III).

[0278] Example 34

[0279] Ionic addition of cysteine to HA unsaturated disaccharide benzyl ester

[0280] HA unsaturated disaccharide benzyl ester (27 mg, 0.06 mmol), prepared according to Example 4, and L-cysteine hydrochloride (43 mg, 0.27 mmol) were dissolved in water (0.75 mL). The pH of the mixture was adjusted to 6.82 by means of 0.1 M K2CO3 and the reaction mixture was stirred at room temperature for 22 hours. The reaction mixture was acidified with acetic acid (0.2 mL) and separated by reverse phase chromatography with a water / methanol gradient. The product-containing fractions were evaporated and analyzed by 'H NMR.

[0281] Conversion >98%. Distribution 11% D-galacto (I), 27 %. D-gluco (II), 62 % L-altro (III).

[0282] Example 35

[0283] Ionic addition of cysteine to the HA unsaturated disaccharide benzyl ester followed by hydrolysis

[0284] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and L-cysteine hydrochloride (9.5 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.05 and the reaction mixture was allowed to stand at room temperature for 20 hours. The pH was then adjusted to 9.00 and the reaction mixture was left to stand at room temperature for 60 hours. 'H NMR was then measured and the isomer distribution was determined.

[0285] Conversion 93 %. Distribution 5 % D-galacto (I), 24 % D-gluco (II), 71 % L-altro (III). <5 % of ester is present in the sample.

[0286] Example 36

[0287] Ionic addition of cysteine to the HA unsaturated disaccharide benzyl ester in PBS

[0288] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and L-cysteine hydrochloride (6.1 mg, 0.039 mmol) were dissolved in 100 mM phosphate buffer in deuterated water (0.6 mL, pH = 7.22). The reaction mixture was left to stand at room temperature for 48 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0289] Conversion 93 %. Distribution 4 % D-galacto (I), 24 % D-gluco (II), 72 % L-altro (III). Example 37

[0290] Ionic addition of cysteine to the HA unsaturated disaccharide 4-nitrobenzyl ester followed by hydrolysis

[0291] HA unsaturated disaccharide 4-nitrobenzyl ester (14 mg, 0.03 mmol), prepared according to Example 7, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.08 and the mixture was left to stand at room temperature for 68 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0292] Conversion 95 %. Distribution <2 % D-galacto (I), 20 % D-gluco (II), 80 % L-altro (III).

[0293] <5 % ester is present in the sample.

[0294] Example 38

[0295] Ionic addition of N-acetyl cysteine mercaptoethylamide to HA unsaturated disaccharide benzyl ester

[0296] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and N-acetyl-L-cysteine 2-mercaptoethylamide (33 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.8 and the mixture was allowed to stand at room temperature for 16 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0297] Conversion 90 %. Distribution 73 % D-galacto (I), <2 % D-gluco (II), 27 % L-altro (III).

[0298] Example 39

[0299] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0300] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.8 mL). The pH of the mixture was adjusted to 6.02 and the mixture was allowed to stand at room temperature for 18 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0301] Conversion 94 %. Distribution <2% D-galacto (I), 21% D-gluco (II), 79% L-altro (III).

[0302] Example 40

[0303] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0304] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.8 mL). The pH of the mixture was adjusted to 8.54 and the mixture was allowed to stand at room temperature for 1 hour. Then1H NMR was measured and the isomer distribution was determined.

[0305] Conversion 95 %. Distribution 11% D-galacto (I), 20% D-gluco (II), 69% L-altro (III).

[0306] Example 41

[0307] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0308] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.8 mL). The pH of the mixture was adjusted to 7.05 and the mixture was allowed to stand at room temperature for 24 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0309] Conversion 95 %. Distribution 4% D-galacto (I), 20% D-gluco (II), 76% L-altro (III).

[0310] Example 42

[0311] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0312] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in a mixture of deuterated water (0.6 mL) and CD3CN (0.2 mL). The pH of the mixture was adjusted to 7.05 and the mixture was allowed to stand at room temperature for 24 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0313] Conversion 95 %. Distribution 3% D-galacto (I), 24% D-gluco (II), 73% L-altro (III).

[0314] Example 43

[0315] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0316] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.8 mL). The pH of the mixture was adjusted to 7.05 and the mixture was allowed to stand at -20 °C for 72 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0317] Conversion >98%. Distribution 9% D-galacto (I), 17% D-gluco (II), 74% L-altro (III). Example 44

[0318] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0319] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.8 mL). The pH of the mixture was adjusted to 7.09 and the mixture was stirred at 50 °C for 2 hours. Then1H NMR was measured and the isomer distribution was determined. Conversion 97 %. Distribution 5% D-galacto (I), 20% D-gluco (II), 75% L-altro (III).

[0320] Example 45

[0321] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0322] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in a mixture of deuterated water (0.6 mL) and deuterated IPA (0.2 mL). The pH of the mixture was adjusted to 7.05 and the mixture was allowed to stand at room temperature for 21 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0323] Conversion >98%. Distribution 3% D-galacto (I), 20% D-gluco (II), 77% L-altro (III).

[0324] Example 46

[0325] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0326] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.12 by means of tri ethyl amine and the mixture was allowed to stand at room temperature for 28 hours. Then1H NMR was measured and the isomer distribution was determined.

[0327] Conversion >98%. Distribution 4% D-galacto (I), 25% D-gluco (II), 71% L-altro (III).

[0328] Example 47

[0329] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0330] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 7.05 by means of lithium hydroxide and the mixture was allowed to stand at room temperature for 21 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0331] Conversion >98%. Distribution 3% D-galacto (I), 21% D-gluco (II), 76% L-altro (III).

[0332] Example 48

[0333] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0334] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (42 mg, 0.24 mmol) were dissolved in deuterated water (0.6 mL) and the pH was adjusted to 6.49. The sample was allowed to stand at room temperature and after 16 hours, 'H NMR was measured and the isomer distribution was determined.

[0335] Conversion 96 %. Distribution <2% D-galacto (I), 21% D-gluco (II), 78% L-altro (III).

[0336] Example 49

[0337] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0338] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (26 mg, 0.15 mmol) were dissolved in deuterated water (0.05 mL). The pH of the mixture was adjusted to 6.8 and the mixture was allowed to stand at room temperature for 1 hour. Then the solution was diluted with D2O (0,6 mL), 'H NMR was measured and the isomer distribution was determined.

[0339] Conversion 85 %. Distribution 10% D-galacto (I), 16% D-gluco (II), 74% L-altro (III).

[0340] Example 50

[0341] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0342] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and L-cysteine methyl ester hydrochloride (11 mg, 0.06 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 6.8 and the mixture was allowed to stand at room temperature for 16 hours. Then1H NMR was measured and the isomer distribution was determined.

[0343] Conversion 88 %. Distribution 2% D-galacto (I), 23% D-gluco (II), 75% L-altro (III). Comparative Example 51

[0344] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide benzyl amide

[0345] HA unsaturated disaccharide benzyl amide (14 mg, 0.03 mmol), prepared according to Example

[0346] 5, and L-cysteine methyl ester hydrochloride (24 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH of the mixture was adjusted to 6.04. The sample was allowed to stand at room temperature and after 24 hours, 'H NMR was measured and the isomer distribution was determined.

[0347] Conversion <2%.

[0348] Comparative Example 52

[0349] Ionic addition of cysteine methyl ester to HA unsaturated disaccharide butyl amide

[0350] HA unsaturated disaccharide butyl amide (14 mg, 0.03 mmol), prepared according to Example

[0351] 6, and L-cysteine methyl ester hydrochloride (24 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH of the mixture was adjusted to 6.66. The sample was allowed to stand at room temperature and after 64 hours, 'H NMR was measured and the isomer distribution was determined.

[0352] Conversion <2%.

[0353] Example 53

[0354] Ionic addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide ethyl ester in PBS

[0355] HA unsaturated disaccharide ethyl ester (4.9 mg, 0.012 mmol), prepared according to Example 1, and N-acetyl-L-cysteine methyl ester (10.06 mg, 0.060 mmol) were dissolved in 100 mM phosphate buffer in deuterated water (0.6 mL, pH = 7.3). The reaction mixture was allowed to stand at room temperature for 72 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0356] Conversion 51 %. Distribution 71% D-galacto (I), <2% D-gluco (II), 29% L-altro (III).

[0357] Example 54

[0358] Ionic addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide benzyl ester in PBS

[0359] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and N-acetyl-L-cysteine methyl ester (10.06 mg, 0.060 mmol) were dissolved in 100 mM phosphate buffer in deuterated water (0.6 mL, pH = 7.28). The reaction mixture was allowed to stand at room temperature for 72 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0360] Conversion 76 %. Distribution 60% D-galacto (I), <2% D-gluco (II), 40% L-altro (III).

[0361] Example 55

[0362] Ionic addition of 2-mercaptoethanol to HA unsaturated disaccharide benzyl ester

[0363] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and 2-mercaptoethanol (12 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH was adjusted to 8.04. The sample was allowed to stand at room temperature and after 17 hours, 'H NMR was measured and the isomer distribution was determined.

[0364] Conversion 66 %. Distribution 24% D-galacto (I), 7% D-gluco (II), 69% L-altro (III).

[0365] Example 56

[0366] Ionic addition of meso-2,3-dimercaptosuccinic acid to HA unsaturated disaccharide benzyl ester

[0367] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and meso-2,3-dimercaptosuccinic acid (27 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL) and the pH was adjusted to 8.74. The sample was allowed to stand at room temperature and after 50 hours, 'H NMR was measured and the isomer distribution was determined.

[0368] Conversion 47 %. Distribution >96% D-galacto (I), <2% D-gluco (II), <2% L-altro (III).

[0369] Example 57

[0370] Ionic addition of N-acetylcysteine to HA unsaturated disaccharide benzyl ester

[0371] HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, and N-acetyl-L-cysteine (24 mg, 0.15 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 9.0 and the mixture was allowed to stand at room temperature for 16 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0372] Conversion 90 %. Distribution 84% D-galacto (I), <2% D-gluco (II), 16% L-altro (III). Example 58

[0373] Ionic addition of sodium thioglycolate to HA unsaturated disaccharide ethyl ester

[0374] HA unsaturated disaccharide ethyl ester (4.9 mg, 0.012 mmol), prepared according to Example 1, and sodium thioglycolate (6.8 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 9.1 and the mixture was allowed to stand at room temperature for 20 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0375] Conversion 55 %. Distribution 77% D-galacto (I), <2% D-gluco (II), 23% L-altro (III).

[0376] Example 59

[0377] Ionic addition of sodium thioglycolate to HA unsaturated disaccharide benzyl ester

[0378] HA unsaturated disaccharide benzyl ester (6.1 mg, 0.012 mmol), prepared according to Example 4, and sodium thioglycolate (6.8 mg, 0.060 mmol) were dissolved in deuterated water (0.6 mL). The pH of the mixture was adjusted to 9.1 and the mixture was allowed to stand at room temperature for 20 hours. Then1H NMR was measured and the isomer distribution was determined.

[0379] Conversion 60 %. Distribution 4% D-galacto (I), 23% D-gluco (II), 73% L-altro (III).

[0380] Example 60

[0381] Ionic addition of thiocholine to HA unsaturated disaccharide benzyl ester

[0382] Acetylcholine iodide (43 mg, 15 mmol) was dissolved in deuterated water (0.6 mL) and alkalized with 20% NaOD (0.06 mL). Then the pH was adjusted to 7.32, and HA unsaturated disaccharide benzyl ester (14 mg, 0.03 mmol), prepared according to Example 4, was added. The reaction mixture was allowed to stand at room temperature for 66 hours. Then 'H NMR was measured and the isomer distribution was determined.

[0383] Conversion 95 %. Distribution 84% D-galacto (I), <2% D-gluco (II), 16% L-altro (III).

[0384] Radical reaction

[0385] Example 61

[0386] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0387] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and IPA (1 mL, 1 : 1). The reaction mixture was cooled to -10 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0388] Conversion >98 %. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0389] Example 62

[0390] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0391] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and IP A (1 mL, 1 : 1). The reaction mixture was heated to 30 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0392] Conversion 94 %. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0393] Example 63

[0394] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0395] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and IPA (0.2 mL, 1 : 1). The reaction mixture was cooled to -10 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0396] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0397] Example 64

[0398] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0399] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and IPA (0.2 mL, 1 : 1). The reaction mixture was tempered to 30 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0400] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0401] Example 65

[0402] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0403] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and DMF (0.2 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0404] Conversion 98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0405] Example 66

[0406] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0407] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and acetone (0.2 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0408] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0409] Example 67

[0410] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0411] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and acetonitrile (0.2 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0412] Conversion 97%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0413] Example 68

[0414] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0415] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and DMSO (0.2 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0416] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0417] Example 69

[0418] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0419] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and THF (0.2 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0420] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0421] Example 70

[0422] Photochemical addition of cysteamine to HA unsaturated disaccharide

[0423] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and cysteamine hydrochloride (11.4 mg, 0.10 mmol) were dissolved in a mixture of water and DMF (1 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 5 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0424] Conversion 94%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0425] Example 71

[0426] Photochemical addition of cysteamine to HA unsaturated octasaccharide

[0427] HA unsaturated octasaccharide (200 mg, 0.132 mmol), photoinitiator LAP (13 mg, 0.044 mmol), and cysteamine hydrochloride (39 mg, 0.343 mmol) were dissolved in a mixture of water and DMF (0.65 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 120 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier. 'H NMR was measured and the isomer distribution was determined.

[0428] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0429] Example 72

[0430] Photochemical addition of dithiothreitol to HA unsaturated octasaccharide

[0431] HA unsaturated octasaccharide (400 mg, 0.25 mmol), photoinitiator Irgacure 2959 (15 mg, 0.075 mmol), and dithiothreitol (154 mg, 1 mmol) were dissolved in a mixture of water and DMF (5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 30 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier. 'H NMR was measured and the isomer distribution was determined.

[0432] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0433] Example 73

[0434] Photochemical addition of DTT to HA unsaturated disaccharide benzyl ester

[0435] HA unsaturated disaccharide benzyl ester (12 mg, 0.025 mmol), prepared according to Example 4, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and dithiothreitol (15 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 30 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0436] Conversion 87%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0437] Example 74

[0438] Photochemical addition of DTT to HA unsaturated disaccharide benzyl amide

[0439] HA unsaturated disaccharide benzyl amide (12 mg, 0.025 mmol), prepared according to Example 5, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and dithiothreitol (15 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 5 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0440] Conversion 95%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0441] Example 75

[0442] Photochemical addition of DTT to HA unsaturated disaccharide butyl amide

[0443] HA unsaturated disaccharide butyl amide (12 mg, 0.025 mmol), prepared according to Example 6, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and dithiothreitol (15 mg, 0.1 mmol) were dissolved in a mixture of water / IPA (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 5 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0444] Conversion 87%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0445] Example 76

[0446] Photochemical addition of DODT to HA unsaturated disaccharide

[0447] HA unsaturated disaccharide (100 mg, 0.25 mmol), photoinitiator Irgacure 2959 (16 mg, 0.08 mmol), and DODT (91 mg, 0.5 mmol) were dissolved in a mixture of water and IPA (1 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 60 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0448] Conversion 96%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0449] Example 77

[0450] Photochemical addition of L-cysteine to HA unsaturated octasaccharide

[0451] HA unsaturated octasaccharide (200 mg, 0.132 mmol), photoinitiator LAP (13 mg, 0.044 mmol), and L-cysteine hydrochloride (54 mg, 0.343 mmol) were dissolved in a mixture of water and DMF (0.65 mL, 1 :1). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 120 minutes. The reaction mixture was precipitated by means of IP A and the precipitate was dried in a drier. ThenTH NMR was measured and the isomer distribution was determined. Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0452] Example 78

[0453] Photochemical addition of glutathione to HA unsaturated disaccharide

[0454] HA unsaturated disaccharide (40 mg, 0.1 mmol), photoinitiator LAP (4 mg, 0.014 mmol), and glutathione (61 mg, 0.2 mmol) were dissolved in a mixture of water and DMF (1 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated at 365 nm for 60 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier. Then1H NMR was measured and the isomer distribution was determined.

[0455] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0456] Example 79

[0457] Photochemical addition of cysteine to HA unsaturated disaccharide benzyl ester

[0458] HA unsaturated disaccharide benzyl ester (12 mg, 0.025 mmol), prepared according to Example 4, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and L-cysteine hydrochloride (16 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with UV light at 365 nm for 60 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0459] Conversion 50%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0460] Example 80

[0461] Photochemical addition of cysteine to HA unsaturated disaccharide benzyl amide

[0462] HA unsaturated disaccharide benzyl amide (12 mg, 0.025 mmol), prepared according to Example 5, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and L-cysteine hydrochloride (16 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with UV light at 365 nm for 30 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0463] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0464] Example 81

[0465] Photochemical addition of cysteine to HA unsaturated disaccharide butyl amide

[0466] HA unsaturated disaccharide butyl amide (12 mg, 0.025 mmol), prepared according to Example 6, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and L-cysteine hydrochloride (16 mg, 0.1 mmol) were dissolved in a mixture of water / IPA (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with UV light at 365 nm for 30 minutes. Then 'H NMR was measured and the isomer distribution was determined.

[0467] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0468] Example 82

[0469] Photochemical addition of N-acetylcysteine mercaptoethylamide to HA unsaturated disaccharide

[0470] HA unsaturated disaccharide (10 mg, 0.025 mmol), photoinitiator LAP (2.1 mg, 0.007 mmol), and N-acetylcysteine mercaptoethylamide (11 mg, 0.05 mmol) were dissolved in a water. The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 120 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0471] Conversion 95%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0472] Example 83

[0473] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide

[0474] HA unsaturated disaccharide (12 mg, 0.029 mmol) and N-acetyl-L-cysteine methyl ester (9.13 mg, 0.052 mmol) were dissolved in a mixture of DMF and D2O (0.05 mL, 1 : 1). Photoinitiator TPO (1 mg, 0.0029 mmol) in DMF (0.005 mL) was added to the reaction mixture and the reaction mixture was irradiated on ice with a UV lamp for 45 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0475] Conversion 73%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0476] Example 84

[0477] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide ethyl ester

[0478] HA unsaturated disaccharide ethyl ester (10 mg, 0.025 mmol), prepared according to Example 1, and N-acetyl-L-cysteine methyl ester (9.3 mg, 0.052 mmol) were dissolved in a mixture of DMF and D2O (0.05 mL, 1 : 1). Photoinitiator TPO (1 mg, 0.0029 mmol) in DMF (0.005 mL) was added to the reaction mixture and the reaction mixture was irradiated on ice with a UV lamp for 45 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0479] Conversion 45%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III). Example 85

[0480] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 254 nm

[0481] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator Irgacure 2959 (1.2 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 254 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0482] Conversion 39%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0483] Example 86

[0484] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 254 nm

[0485] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator Irgacure 1173 (1 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 254 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0486] Conversion 54%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0487] Example 87

[0488] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 254 nm

[0489] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator DP AP (1.6 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 254 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0490] Conversion 62%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0491] Example 88

[0492] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 365 nm

[0493] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator DP AP (1.6 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0494] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0495] Example 89

[0496] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 365 nm

[0497] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator IC1173 (1 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0498] Conversion 46%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0499] Example 90

[0500] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide at 365 nm

[0501] HA unsaturated disaccharide (8 mg, 0.02 mmol), photoinitiator DP AP (1.6 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 5:3). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0502] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0503] Example 91

[0504] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide

[0505] HA unsaturated disaccharide (10 mg, 0.025 mmol), photoinitiator LAP (2.1 mg, 0.007 mmol), and N-acetyl-L-cysteine methyl ester (9.6 mg, 0.054 mmol) were dissolved in a water (0.06 mL). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 120 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0506] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III). Example 92

[0507] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide ethyl ester at 365 nm

[0508] HA unsaturated disaccharide ethyl ester (8 mg, 0.02 mmol), prepared according to Example 1, photoinitiator DP AP (1.6 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 5:3). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0509] Conversion 73%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0510] Example 93

[0511] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide benzyl ester at 365 nm

[0512] HA unsaturated disaccharide benzyl ester (9 mg, 0.02 mmol), prepared according to Example 4, photoinitiator DPAP (1.6 mg, 0.0062 mmol), and N-acetyl-L-cysteine methyl ester (7 mg, 0.039 mmol) were dissolved in a mixture of DMF and H2O (0.05 mL, 5:3). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 90 minutes. The reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0513] Conversion 89%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0514] Example 94

[0515] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide benzyl ester

[0516] HA unsaturated disaccharide benzyl ester (12 mg, 0.025 mmol), prepared according to Example 4, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and N-acetyl-L-cysteine methyl ester (18 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 30 minutes. Then the reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0517] Conversion 67%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III). Example 95

[0518] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide benzyl amide

[0519] HA unsaturated disaccharide benzyl amide (12 mg, 0.025 mmol), prepared according to Example 5, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and N-acetyl-L-cysteine methyl ester (18 mg, 0.1 mmol) were dissolved in a mixture of water / DMF (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 30 minutes. Then the reaction mixture was diluted with deuterated water (0.6 mL),1H NMR was measured and the isomer distribution was determined.

[0520] Conversion 96%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0521] Example 96

[0522] Photochemical addition of N-acetyl-L-cysteine methyl ester to HA unsaturated disaccharide butyl amide

[0523] HA unsaturated disaccharide butyl amide (12 mg, 0.025 mmol), prepared according to Example 6, photoinitiator Irgacure 2959 (1.5 mg, 0.0075 mmol), and N-acetyl-L-cysteine methyl ester (18 mg, 0.1 mmol) were dissolved in a mixture of water / IPA (0.5 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 30 minutes. Then the reaction mixture was diluted with deuterated water (0.6 mL), 'H NMR was measured and the isomer distribution was determined.

[0524] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0525] Example 97

[0526] Photochemical addition of N-acetyl-L-cysteine to HA unsaturated disaccharide

[0527] HA unsaturated disaccharide (100 mg, 0.25 mmol), photoinitiator LAP (23 mg, 0.08 mmol), and N-acetyl-L-cysteine (81 mg, 0.5 mmol) were dissolved in a mixture of water and DMF (1 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated at 455 nm for 60 minutes. The reaction mixture was precipitated by means of IP A and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0528] Conversion 88%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0529] Example 98

[0530] Photochemical addition of mercaptoethanol to HA unsaturated octasaccharide

[0531] HA unsaturated octasaccharide (200 mg, 0.132 mmol), photoinitiator LAP (13 mg, 0.044 mmol), and 2-mercaptoethanol (27 mg, 0.343 mmol) were dissolved in a mixture of water and DMF (0.65 mL, 1 : 1). The reaction mixture was irradiated on ice at 365 nm for 120 minutes. The reaction mixture was precipitated by means of IP A and the precipitate was dried in a drier.TH NMR was measured and the isomer distribution was determined.

[0532] Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0533] Example 99

[0534] Photochemical addition of 3-mercaptopropionic acid to HA unsaturated disaccharide

[0535] HA unsaturated disaccharide (20 mg, 0.05 mmol), photoinitiator Irgacure 2959 (3.2 mg, 0.016 mmol), and 3-mercaptopropionic acid (11 mg, 0.10 mmol) were dissolved in a mixture of water and DMF (1 mL, 1 :1). The reaction mixture was cooled to 0 °C and irradiated with a UV lamp at 365 nm for 15 minutes. The reaction mixture was diluted with deuterated water (0.06 mL),1H NMR was measured and the isomer distribution was determined.

[0536] Conversion 84%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0537] Example 100

[0538] Photoaddition of meso-2,3-dimercaptosuccinic acid to HA unsaturated disaccharide

[0539] HA unsaturated disaccharide (40 mg, 0.1 mmol), photoinitiator LAP (8 mg, 0.028 mmol), and meso-2, 3 -mercaptosuccinic acid (18 mg, 0.1 mmol) were dissolved in a mixture of water and DMF (1 mL, 1 : 1). The reaction mixture was cooled to 0 °C and irradiated at 455 nm for 60 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0540] Conversion 58%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0541] Example 101

[0542] Photochemical addition of 1 -thioglucose to HA unsaturated disaccharide

[0543] HA unsaturated disaccharide (10 mg, 0.025 mmol) and P-D-thioglucose (11.8 mg, 0.054 mmol) were dissolved in a mixture of DMF and D2O (0.05 mL, 1 : 1) and acidified with CD3COOD (0.005 mL). Photoinitiator Irgacure 2959 (0.62 mg, 0.0028 mmol) in DMF (0.005 mL) were added to the reaction mixture and the reaction mixture was irradiated on ice with a UV lamp for 45 minutes. The reaction mixture was diluted with D2O (0.6 mL) and 'H NMR was measured and the isomer distribution was determined.

[0544] Conversion 76%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0545] Example 102

[0546] Photochemical addition of sodium thioglycolate to HA unsaturated octasaccharide HA unsaturated octasaccharide (200 mg, 0.132 mmol), photoinitiator LAP (13 mg, 0.044 mmol), and sodium thioglycolate (39 mg, 0.343 mmol) were dissolved in a mixture of water and DMF (0.65 mL, 1 : 1). The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 120 minutes. The reaction mixture was precipitated by means of IP A and the precipitate was dried in a drier. 'H NMR was measured and the isomer distribution was determined. Conversion >98%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0547] Example 103

[0548] Photochemical addition of HA tetrasaccharide thiol to HA unsaturated disaccharide

[0549] HA unsaturated disaccharide (47 mg, 0.116 mmol), photoinitiator LAP (3.4 mg, 0.0116 mmol) and HA tetrasaccharide thiol (50 mg, 0.058 mmol), prepared according to Example 9, were dissolved in a mixture of DMF and H2O (0.16 mL, 1 : 1) and acidified with CH3COOH (0.025 mL). The reaction mixture was cooled to 0 °C and irradiated at 365 nm for 30 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0550] Conversion 67%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0551] Example 104

[0552] Photochemical addition of HA hexasaccharide thiol to HA unsaturated disaccharide

[0553] HA unsaturated disaccharide (118 mg, 0.295 mmol), photoinitiator LAP (15 mg, 0.0045 mmol) and HA hexasaccharide thiol (190 mg, 0.155 mmol), prepared according to Example 10, were dissolved in a mixture of DMF and H2O (0.6 mL, 1 :1) and acidified with CH3COOH (0.12 mL) to pH = 6.17. The reaction mixture was irradiated on ice with a UV lamp at 365 nm for 240 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0554] Conversion 56%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0555] Example 105

[0556] Photochemical addition of HA hexasaccharide thiol to HA unsaturated disaccharide

[0557] HA unsaturated disaccharide (34 mg, 0.082 mmol), photoinitiator LAP (2.62 mg, 0.0082 mmol) and HA hexasaccharide thiol (49 mg, 0.041 mmol), prepared according to Example 10, were dissolved in a mixture of DMF and H2O (0.16 mL, 1 : 1) and acidified with CH3COOH (0.016 mL). The reaction mixture was cooled to 0 °C and irradiated at 365 nm for 120 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0558] Conversion 64%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III). Example 106

[0559] Photochemical addition of HA octasaccharide thiol to HA unsaturated disaccharide

[0560] HA unsaturated disaccharide (112 mg, 0.280 mmol), photoinitiator Irgacure 2959 (14 mg, 0.063 mmol) and HA octasaccharide thiol (182 mg, 0.113 mmol), prepared according to Example 11, were dissolved in a mixture of DMF and H2O (0.6 mL, 1 : 1) and acidified with CH3COOH (0.105 mL) to pH = 5.3. The reaction mixture was irradiated on ice with a UV lamp for 90 minutes. The reaction mixture was precipitated by adding IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0561] Conversion 60%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0562] Example 107

[0563] Photochemical addition of HA decasaccharide thiol to HA unsaturated disaccharide

[0564] HA unsaturated disaccharide (80 mg, 0.2 mmol), photoinitiator LAP (6.73mg, 0.023 mmol) and HA decasaccharide thiol (200 mg, 0.1 mmol), prepared according to Example 12, were dissolved in a mixture of IPA and H2O (1 mL, 1 : 1) and acidified with CH3COOH (0.04 mL). The reaction mixture was cooled to -12 °C and irradiated at 365 nm for 240 minutes. The reaction mixture was precipitated by means of IPA and the precipitate was dried in a drier.1H NMR was measured and the isomer distribution was determined.

[0565] Conversion 54%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0566] Example 108

[0567] Photochemical addition of HA decasaccharide thiol to HA unsaturated disaccharide

[0568] HA unsaturated disaccharide (17 mg, 0.042 mmol), photoinitiator Irgacure 2959 (1.6 mg, 0.007 mmol) and HA decasaccharide thiol (31 mg, 0.015 mmol), prepared according to Example 12, were dissolved in a mixture of DMF and H2O (0.08 mL, 1 : 1) and acidified with CH3COOH (0.015 mL) to pH = 5.30. The reaction mixture was irradiated on ice with a UV lamp for 90 minutes. The reaction mixture was precipitated by adding IPA and the precipitate was dried in a drier. 'H NMR was measured and the isomer distribution was determined.

[0569] Conversion 50%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0570] Example 109

[0571] Photochemical addition of HA decasaccharide thiol to HA unsaturated decasaccharide

[0572] HA unsaturated decaaccharide (10 mg, 0.005 mmol), photoinitiator Irgacure 2959 (1.5 mg, 0.0054 mmol) and HA decasaccharide thiol (9 mg, 0.006 mmol), prepared according to Example 12, were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1) and acidified with CH3COOH (0.002 mL) to pH = 5.8. The reaction mixture was irradiated on ice with a UV lamp for 90 minutes. The reaction mixture was precipitated by adding IPA and the precipitate was dried in a drier. 1H NMR was measured and the isomer distribution was determined.

[0573] Conversion 42%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0574] Example 110

[0575] Photochemical addition of HA decasaccharide thiol to HA unsaturated tetrasaccharide

[0576] HA unsaturated tetrasaccharide (14 mg, 0.005 mmol), photoinitiator Irgacure 2959 (1.5 mg, 0.0054 mmol) and HA decasaccharide thiol (9 mg, 0.006 mmol), prepared according to Example 12, were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1) and acidified with CH3COOH (0.002 mL) to pH = 5.4. The reaction mixture was irradiated on ice with a UV lamp for 90 minutes. The reaction mixture was precipitated by adding IPA and the precipitate was dried in a drier. 1H NMR was measured and the isomer distribution was determined.

[0577] Conversion 18%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0578] Example 111

[0579] Photochemical addition of HA decasaccharide thiol to HA unsaturated octadecasaccharide

[0580] HA unsaturated octadecasaccharide (18 mg, 0.005 mmol), photoinitiator Irgacure 2959 (1.5 mg, 0.0054 mmol) and HA decasaccharide thiol (9 mg, 0.006 mmol), prepared according to Example 12, were dissolved in a mixture of DMF and H2O (0.05 mL, 1 : 1) and acidified with CH3COOH (0.002 mL) to pH = 5.5. The reaction mixture was irradiated on ice with a UV lamp for 90 minutes. The reaction mixture was precipitated by adding IPA and the precipitate was dried in a drier. 1H NMR was measured and the isomer distribution was determined.

[0581] Conversion 4%. Distribution >96 % D-galacto (I), <2 % D-gluco (II), <2 % L-altro (III).

[0582] Conjugates with API

[0583] Example 112

[0584] Preparation of a thioconjugate of HA octasaccharide with acetaminophen

[0585] Acetaminophen (30 mg, 0.2 mmol) and DIPEA (0.14 mL, 0.8 mmol) were suspended in dry DCM (1 mL). The reaction mixture was cooled to 0 °C and then acryloyl chloride (0.065 mL, 0.8 mmol) was added. The reaction mixture was warmed to RT and stirred for 20 hours. The reaction mixture was diluted with DCM (3 mL) and washed with IM HC1 (2 mL), IM NaHCCh (2 mL) and water (2x1 mL) and dried over MgSCL. Then the DCM was evaporated and the residue was stirred in water (1.5 mL), the conjugate of HA octasaccharide and DTT, prepared according to Example 72, was added (176 mg, 0.1 mmol), the mixture was acidified to pH=7.41 and the reaction mixture was stirred for 24 hours at 30 °C. The reaction mixture was then diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0586] ^HNMR S dJS (1H, Gal-1), 4.26 (1H, Gal-5), 3.01 (2H, SCH2), 2.82 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C7IHIO3N5049S2= 936.8 [M - 2H]2', found 936.5 [M - 2H]2'.

[0587] Example 113

[0588] Preparation of thioconjugate of HA octasaccharide with 5-aminosalicylic acid 5-Aminosalicylic acid (31 mg, 0.2 mmol) and DIPEA (0.14 mL, 0.8 mmol) were suspended in dry DCM (1 mL). The reaction mixture was cooled to 0 °C and then acryloyl chloride (0.065 mL, 0.8 mmol) was added. The reaction mixture was warmed to RT and stirred for 20 hours. The reaction mixture was diluted with DCM (3 mL) and washed with IM HC1 (2 mL), IM NaHCCL (2 mL) and water (2x1 mL) and dried over MgSCU. The DCM was then evaporated and the residue was dissolved in water (1.5 mL) and basified with 20% NaOH (0.052 mL). The mixture was stirred until dissolved and then acidified with IM HC1 to pH = 8.0. The conjugate of HA octasaccharide and DTT, prepared according to Example 72 (176 mg, 0.1 mmol), was added to the reaction mixture, and the reaction mixture was stirred for 24 hours at 30 °C. The reaction mixture was then diluted with water and separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR and LC-MS were measured.

[0589] ^NMR S d JS (1H, Gal-1), 4.26 (1H, Gal-5), 2.96 (2H, SCH2), 2.76 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C7OHIOIN505OS2= 937.8 [M - 2H]2', found 937.7 [M - 2H]2'.

[0590] Example 114

[0591] Preparation of thioconjugate of HA octasaccharide with diclofenac

[0592] Diclofenac (318 mg, 1 mmol) was dissolved in water (10 mL), acidified with IM HC1 (10 mL), extracted with ethyl acetate (3 x 10 mL), dried over MgSCU and evaporated.

[0593] The residue was stirred under nitrogen in DCM (5 mL) and cooled to 0 °C. CDI (180 mg, mmol) was then added and the whole mixture was stirred for 1 hour. Ethanolamine (0.151 mL, 2.5 mmol) in DCM (5 mL) was then added, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (10 mL) and water (2 x 10 mL), dried over MgSCU and evaporated. The residue was dissolved in a mixture of DCM (5 mL) and TEA (101 mg, 1 mmol). The mixture was then cooled to 0 °C and acryloyl chloride (90 mg, 1 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (5 mL), IM NaHCOs (5 mL) and water (2 x 5 mL), dried over MgSCU and evaporated.

[0594] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of the conjugate of HA octasaccharide and DTT, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.5 and stirred at 20 °C for 72 hours. The reaction mixture was then diluted with water (8 mL) and washed with chloroform (3 x 5 mL). The aqueous phase was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and the1H NMR LC-MS was measured.

[0595] ^NMR S d JS (1H, Gal-1), 4.26 (1H, Gal-5), 2.61 (2H, SCH2), 2.47 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C79HIIOC12N6049S2= 1030.3 [M - 2H]2', found 1030.3 [M - 2H]2'.

[0596] Example 115

[0597] Preparation of thioconjugate of HA octasaccharide with ibuprofen

[0598] Ibuprofen (206 mg, 1 mmol) was stirred under nitrogen in DCM (5 mL) and cooled to 0 °C. CDI (180 mg, mmol) was then added and the whole mixture was stirred for 1 hour. Ethanolamine (0.151 mL, 2.5 mmol) in DCM (5 mL) was then added, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (10 mL) and water (2 x 10 mL), dried over MgSCU and evaporated.

[0599] The residue was dissolved in a mixture of DCM (5 mL) and TEA (101 mg, 1 mmol). The mixture was then cooled to 0 °C and acryloyl chloride (90 mg, 1 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (5 mL), IM NaHCOs (5 mL) and water (2 x 5 mL), dried over MgSCU and evaporated.

[0600] The residue was dissolved in methanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.5 and stirred at 20 °C for 48 hours. The reaction mixture was then diluted with water (8 mL) and washed with chloroform (3 x 5 mL). The aqueous phase was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0601] ^HNMR S dJS (1H, Gal-1), 4.26 (1H, Gal-5), 2.63 (2H, SCH2), 2.48 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C sH NsO^Si = 985.8 [M - 2H]2', found 985.9 [M - 2H]2'.

[0602] Example 116

[0603] Preparation of thioconjugate of HA octasaccharide with indomethacin

[0604] Indomethacin (358 mg, 1 mmol) was stirred under nitrogen in DCM (5 mL) and cooled to 0 °C. CDI (180 mg, mmol) was then added and the whole mixture was stirred for 1 hour. Ethanolamine (0.151 mL, 2.5 mmol) in DCM (5 mL) was then added, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (10 mL) and water (2 x 10 mL), dried over MgSCU and evaporated.

[0605] The residue was dissolved in a mixture of DCM (5 mL) and TEA (101 mg, 1 mmol). The mixture was then cooled to 0 °C and acryloyl chloride (90 mg, 1 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (5 mL), IM NaHCOs (5 mL) and water (2 x 5 mL), dried over MgSCU and evaporated.

[0606] The residue was dissolved in tetrahydrofuran (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.5 and stirred at 20 °C for 70 hours. The reaction mixture was then diluted with water (8 mL) and washed with chloroform (3 x 5 mL). The aqueous phase was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0607] ^NMR S d JS (1H, Gal-1), 4.26 (1H, Gal-5), 2.60 (2H, SCH2), 2.48 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C84Hii5ClN6O5iS2= 1061.3 [M - 2H]2', found 1061.5 [M - 2H]2'.

[0608] Example 117

[0609] Preparation of thioconjugate of HA octasaccharide with ketoprofen

[0610] Ketoprofen (254 mg, 1 mmol) was stirred under nitrogen in DCM (5 mL) and cooled to 0 °C. CDI (180 mg, mmol) was then added and the whole mixture was stirred for 1 hour. Ethanolamine (0.151 mL, 2.5 mmol) in DCM (5 mL) was then added, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (10 mL) and water (2 x 10 mL), dried over MgSC and evaporated.

[0611] The residue was dissolved in a mixture of DCM (5 mL) and TEA (101 mg, 1 mmol). The mixture was then cooled to 0 °C and acryloyl chloride (90 mg, 1 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (5 mL), IM NaHCOs (5 mL) and water (2 x 5 mL), dried over MgSCU and evaporated.

[0612] The residue was dissolved in dimethyl sulfoxide (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.5 and stirred at 20 °C for 72 hours. The reaction mixture was then diluted with water (8 mL) and washed with chloroform (3 x 5 mL). The aqueous phase was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured. 'H NMR 5 4.35 (1H, Gal-1), 4.27 (1H, Gal-5), 2.61 (2H, SCH2), 2.48 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C81H113N5O50S2 = 1009.8 [M - 2H]2', found 1010.0 [M - 2H]2'.

[0613] Example 118

[0614] Preparation of thioconjugate of HA octasaccharide with naproxen

[0615] Naproxen (230 mg, 1 mmol) was stirred under nitrogen in DCM (5 mL) and cooled to 0 °C. CDI (180 mg, mmol) was then added and the whole mixture was stirred for 1 hour. Ethanolamine (0.151 mL, 2.5 mmol) in DCM (5 mL) was then added, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (10 mL) and water (2 x 10 mL), dried over MgSCU and evaporated.

[0616] The residue was dissolved in a mixture of DCM (5 mL) and TEA (101 mg, 1 mmol). The mixture was then cooled to 0 °C and acryloyl chloride (90 mg, 1 mmol) was added. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was washed with IM HC1 (5 mL), IM NaHCCf (5 mL) and water (2 x 5 mL), dried over MgSCU and evaporated.

[0617] The residue was dissolved in acetonitrile (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.5 and stirred at 20 °C for 3 days. Then the reaction mixture was diluted with water (8 mL) and washed with chloroform (3 x 5 mL). The aqueous phase was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.XHNMR 54.36 (1H, Gal-1), 4.26 (1H, Gal-5), 2.61 (2H, SCH2), 2.49 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C79Hii3N505oS2= 997.8 [M - 2H]2', found 997.7 [M - 2H]2'.

[0618] Example 119

[0619] Preparation of thioconjugate of HA octasaccharide with cirpofloxacin

[0620] Ciprofloxacin (165 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCU and evaporated.

[0621] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0622] XH NMR 54.34 (1H, Gal-1), 4.28 (1H, Gal-5), 2.72 (2H, SCH2), 2.64 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C8OHII2N705OS2= 1026.8 [M - 2H]2', found 1027.1 [M - 2H]2'.

[0623] Example 120

[0624] Preparation of thioconjugate of HA octasaccharide with metronidazol

[0625] Metronidazol (85 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCL (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0626] The residue was dissolved in ethanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR and LC-MS was measured.

[0627] XH NMR 54.34 (1H, Gal-1), 4.28 (1H, Gal-5), 2.75 (2H, SCH2), 2.68 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C69HIO3N705OS2= 946.8 [M - 2H]2', found 947.3 [M - 2H]2'.

[0628] Example 121

[0629] Preparation of thioconjugate of HA octasaccharide with aciclovir

[0630] Aciclovir (112 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCU and evaporated.

[0631] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0632] XH NMR 54.34 (1H, Gal-1), 4.28 (1H, Gal-5), 2.76 (2H, SCH2), 2.68 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C7iHio5N905oS2= 973.8 [M - 2H]2', found 973.9 [M - 2H]2'.

[0633] Example 122

[0634] Preparation of thioconjugate of HA octasaccharide with darunavir

[0635] Darunavir (273 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCL (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCU and evaporated.

[0636] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0637] ^HNMR S dJd (1H, Gal-1), 4.28 (1H, Gal-5), 2.75 (2H, SCH2), 2.68 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C90H131N7O54S3 = 1134.8 [M - 2H]2', found 1134.8 [M - 2H]2'.

[0638] Example 123

[0639] Preparation of thioconjugate of HA octasaccharide with didanosine

[0640] Didanosine (118 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCU and evaporated.

[0641] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0642] ^NMR S d Jd (1H, Gal-1), 4.28 (1H, Gal-5), 2.75 (2H, SCH2), 2.68 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C73HIO6N805OS2= 979.3 [M - 2H]2', found 979.6 [M - 2H]2'.

[0643] Example 124

[0644] Preparation of thioconjugate of HA octasaccharide with ganciclovir

[0645] Ganciclovir (122 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCL (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0646] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0647] ^HNMR S dJd (1H, Gal-1), 4.28 (1H, Gal-5), 2.74 (2H, SCH2), 2.68 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C72HIO7N705IS2= 988.8 [M - 2H]2', found 989.1 [M - 2H]2'.

[0648] Example 125

[0649] Preparation of thioconjugate of HA octasaccharide with ribavirin

[0650] Ribavirin (122 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0651] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0652] ^NMR S d Jd (1H, Gal-1), 4.28 (1H, Gal-5), 2.73 (2H, SCH2), 2.67 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C7iHio6N8052S2= 983.3 [M - 2H]2', found 983.2 [M - 2H]2'.

[0653] Example 126

[0654] Preparation of thioconjugate of HA octasaccharide with daunorubicin

[0655] Daunorubicin (263 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCL (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0656] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0657] ^HNMR S dJd (1H, Gal-1), 4.28 (1H, Gal-5), 2.72 (2H, SCH2), 2.61 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C9OHI23N5057S2= 1124.8 [M - 2H]2', found 1124.7 [M

[0658] - 2H]2'.

[0659] Example 127

[0660] Preparation of thioconjugate of HA octasaccharide with doxorubicin

[0661] Doxorubicin (272 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0662] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0663] ^NMR S d Jd (1H, Gal-1), 4.28 (1H, Gal-5), 2.72 (2H, SCH2), 2.61 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C9OHI23N505SS2= 1132.8 [M - 2H]2', found 1132.6 [M

[0664] - 2H]2'.

[0665] Example 128

[0666] Preparation of thioconjugate of HA octasaccharide with lenalidomide

[0667] Lenalidomide (130 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCL (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCh and evaporated.

[0668] The residue was dissolved in dimethyl formamide (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and1H NMR was measured.

[0669] XH NMR 54.34 (1H, Gal-1), 4.28 (1H, Gal-5), 2.73 (2H, SCH2), 2.60 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C76HIO7N705OS2= 990.8 [M - 2H]2', found 990.8 [M - 2H]2'.

[0670] Example 129

[0671] Preparation of thioconjugate of HA octasaccharide with mercaptopurine

[0672] Mercaptopurine (76 mg, 0.5 mmol) was stirred in a mixture of chloroform (2.5 mL) and TEA (0.15 mL, 1.08 mmol). Then acryloyl chloride (90 mg, 1.0 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding IM NaHCCE (2.5 mL). The organic phase was washed with water (2 x 5 mL), dried over MgSCU and evaporated.

[0673] The residue was dissolved in 2-propanol (1 mL) and mixed with an aqueous solution (2 mL) of HA octasaccharide and DTT conjugate, prepared according to Example 72 (440 mg, 0.25 mmol). The reaction mixture was alkalized to pH=7.0 and stirred at 25 °C for 24 hours. Then the reaction mixture was separated by reverse phase chromatography with a water / methanol gradient. The solvents were evaporated from the product-containing fractions and 'H NMR was measured.

[0674] 1HNMR 54.35 (1H, Gal-1), 4.26 (1H, Gal-5), 2.96 (2H, SCH2), 2.76 (2H, COCH2), 2.03 (12H, COCH3). MS (ESI-) m / z: calculated for C68H98N8O47S3 = 937.2 [M - 2H]2', found 937.0 [M - 2H]2'.

[0675] Biological tests

[0676] Example 130

[0677] Testing the effect of oligosaccharide thioconjugates prepared according to Examples 71, 77, 98, 102, 103, 105, 106, 107 on 3T3 viability

[0678] Mouse embryonic cells 3T3 were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). Cytotoxicity was measured via MTT assay. After reaching 80% confluence, cells were seeded into a 96-well plate at a density of 3,000 cells per well. After incubation until the next day, the culture medium was replaced with the tested samples (control = culture medium). After 24, 48 and 72 hours of cell exposure, 20 pL of MTT (5 mg / mL) were added and incubated for 2.5 hours. Finally, the culture medium with samples was tapped from the plate and lysis solution (IPA: DMSO (1 : 1), Triton X-100 (10%)) was added. After cell lysis, absorbance was measured on an Ensight spectrometer (Perkin Elmer) at a wavelength of 570 nm (690 nm background).

[0679] Graph 1 shows the change in cell viability relative to the control at a given time - i.e., the unaffected control corresponds to the value of 0; if the value is positive or negative up to -20%, it is interpreted as meaning that the substance has no cytotoxic effect.

[0680] Example 131

[0681] Testing the effect of oligosaccharide thioconjugates prepared according to Examples 71, 77, 98, 102, 103, 105, 106, 107 on HaCaT viability

[0682] Human keratinocytes HaCaT were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). Cytotoxicity was measured via MTT assay. After reaching 80% confluence, cells were seeded into a 96-well plate at a density of 3,000 cells per well. After incubation until the next day, the culture medium was replaced with the tested samples (control = culture medium). After 24, 48 and 72 hours of cell exposure, 20 pL of MTT (5 mg / mL) were added and incubated for 2.5 hours. Finally, the culture medium with samples was tapped from the plate and lysis solution (IPA: DMSO (1 : 1), Triton X-100 (10%)) was added. After cell lysis, absorbance was measured on an Ensight spectrometer (Perkin Elmer) at a wavelength of 570 nm (690 nm background). Graph 2 shows the change in cell viability relative to the control at a given time - i.e., the unaffected control corresponds to the value of 0; if the value is positive or negative up to -20 %, it is interpreted as meaning that the substance has no cytotoxic effect

[0683] Example 132

[0684] Testing the effect of oligosaccharide thioconjugates prepared according to Examples 71, 77, 98, 102, 103, 105, 106, 107 on HT-29 viability

[0685] Human colorectal cancer cells HT-29 were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). Cytotoxicity was measured via MTT assay. After reaching 80% confluence, cells were seeded into a 96-well plate at a density of 3,000 cells per well. After incubation until the next day, the culture medium was replaced with the tested samples (control = culture medium). After 24, 48 and 72 hours of cell exposure, 20 pL of MTT (5 mg / mL) was added and incubated for 2.5 hours. Finally, the culture medium with samples was tapped from the plate and lysis solution (IPA: DMSO (1 :1), Triton X-100 (10%)) was added. After cell lysis, absorbance was measured on an Ensight spectrometer (Perkin Elmer) at a wavelength of 570 nm (690 nm background). Graph 3 shows the change in cell viability relative to the control at a given time - i.e., the unaffected control corresponds to the value of 0; if the value is positive or negative up to -20%, it is interpreted as meaning that the substance has no cytotoxic effect.

[0686] Example 133

[0687] Testing the effect of oligosaccharide thioconjugates prepared according to Examples 71, 77, 98, 102, 103, 105, 106, 107 on the viability of NHDF fibroblasts.

[0688] Human fibroblasts NHDF were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). Cytotoxicity was measured via MTT assay. After reaching 80% confluence, cells were plated in a 96-well plate at a density of 5,000 cells per well. After incubation until the next day, the culture medium was replaced with the tested samples (control = culture medium). After 24, 48 and 72 hours of cell exposure, 20 pL of MTT (5 mg / mL) was added and incubated for 2.5 hours. Finally, the culture medium with samples was tapped from the plate and lysis solution (IPA: DMSO (1 : 1), Triton X-100 (10%)) was added. After cell lysis, absorbance was measured on an Ensight spectrometer (Perkin Elmer) at a wavelength of 570 nm (690 nm background).

[0689] Graph 4 shows the change in cell viability relative to the control at a given time - i.e., the unaffected control corresponds to the value of 0; if the value is positive or negative up to -20%, it is interpreted as meaning that the substance has no cytotoxic effect.

[0690] Example 134

[0691] Testing the effect of oligosaccharide thioconjugates prepared according to Examples 71, 77, 98, 102, 103, 106, 107 on the viability of AD-MSC cells.

[0692] Stem cells from canine adipose tissue AD-MSC were cultured in DMEM medium containing 10% FBS (5% CO2, 37 °C). Cytotoxicity was measured via MTT assay. After reaching 80% confluence, cells were plated in a 96-well plate at a density of 5,000 cells per well. After incubation until the next day, the culture medium was replaced with the tested samples (control = culture medium). After 24, 48 and 72 hours of cell exposure, 20 pL of MTT (5 mg / mL) was added and incubated for 2.5 hours. Finally, the culture medium with samples was tapped from the plate and lysis solution (IPA: DMSO (1 : 1), Triton X-100 (10%)) was added. After cell lysis, absorbance was measured on an Ensight spectrometer (Perkin Elmer) at a wavelength of 570 nm (690 nm background).

[0693] Graph 5 shows the change in cell viability relative to the control at a given time - i.e., the unaffected control corresponds to the value of 0; if the value is positive or negative up to -20%, it is interpreted as meaning that the substance has no cytotoxic effect. Preparation of compositions

[0694] Example 135

[0695] Preparation of a solution composition

[0696] 500 mg of modified oligosaccharide, prepared according to Example 114, 10 mg of sodium hydrogen phosphate and 0.1 mg of calcium chloride were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0697] Example 136

[0698] Preparation of a solution composition

[0699] 500 mg of modified oligosaccharide, prepared according to Example 114, 100 mg of sodium dihydrogen phosphate and 10 mg of hyaluronic acid (Mw = 10 kDa) were dissolved in 10 ml of 0.1 % NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0700] Example 137

[0701] Preparation of a solution composition

[0702] 500 mg of modified oligosaccharide, prepared according to Example 120, 10 mg of sodium phosphate and 0.1 mg of glycerol were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0703] Example 138

[0704] Preparation of a solution composition

[0705] 500 mg of modified oligosaccharide, prepared according to Example 120, 10 mg of sodium phosphate and 500 mg of modified oligosaccharide, prepared according to Example 77, were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter. Example 139

[0706] Preparation of a solution composition

[0707] 500 mg of modified oligosaccharide, prepared according to Example 121 were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0708] Example 140

[0709] Preparation of a solution composition

[0710] 500 mg of modified oligosaccharide, prepared according to Example 121, 10 mg of sodium dihydrogen phosphate and 500 mg of modified oligosaccharide, prepared according to Example 72, were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0711] Example 141

[0712] Preparation of a solution composition

[0713] 500 mg of modified oligosaccharide, prepared according to Example 129, and 10 mg of chondroitin sulfate were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0714] Example 142

[0715] Preparation of a solution composition

[0716] 10 mg of modified oligosaccharide, prepared according to Example 129, and 500 mg of modified oligosaccharide, prepared according to Example 107, were dissolved in 10 ml of 0.9% NaCl solution in water. The resulting solution was then filtered under sterile conditions through a 0.1 to 0.2 pm nitrocellulose filter.

[0717] Example 143

[0718] Preparation of a film

[0719] 50 mg of modified oligosaccharide, prepared according to Example 114, and 350 mg of hyaluronic acid (Mw = 500 kDa) and 25 mg of calcium dihydrogen phosphate were dissolved in 40 ml of demineralized water. The resulting solution was poured on a Petri dish and slowly evaporated until formation of a film. Example 144

[0720] Preparation of a lyophilizate

[0721] 10 mg of modified oligosaccharide, prepared according to Example 114, 988 mg of hyaluronic acid (Mw = 10 kDa) and 1 mg of NaCl were dissolved in 100 ml of demineralized water. The resulting solution was frozen at -20 °C for 16 hours and lyophilized for 48 hours.

[0722] Example 145

[0723] Preparation of a lyophilizate

[0724] 100 mg of modified oligosaccharide, prepared according to Example 114, 50 mg of modified oligosaccharide, prepared according to Example 105, 750 mg of hyaluronic acid (Mw = 2000 kDa) and 50 mg of chondroitin sulfate were dissolved in 100 ml of demineralized water. The resulting solution was frozen at -78 °C for 6 hours and lyophilized for 72 hours.

Claims

CLAIMS1. Modified oligosaccharide according to the structural formula (A)wherein n = 0 to 8;X is O or N-H;Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7;R is aliphatic, aromatic, heteroaromatic, linear, branched or cyclic chain C2-C70, optionally containing N, S, Cl or O atoms, wherein in the case the modified oligosaccharide according to the structural formula(A) stereometrically corresponds to the formula (I)then X = O or NH, and wherein in the case the modified oligosaccharide according to the structural formula (A) stereometrically corresponds to the formula (II) or (III)then X = O.The modified oligosaccharide according to claim 1, characterized in that the substituent R is selected from the group comprising amino acids, peptides, monosaccharides or oligosaccharides.

3. The modified oligosaccharide according to claim 1, characterized in that the substituent R is an aliphatic linear chain C4-C8 containing a free terminal group -SH and optionally one or more N, S, or O atoms.

4. The modified oligosaccharide according to claim 1, characterized in that the substituent R contains a structure of a pharmaceutically active substance.

5. A method for preparing the modified oligosaccharide defined in claims 1 to 4, characterized in that an unsaturated oligosaccharide of hyaluronic acid according to the structural formula IVwherein n = 0 to 8;X is O or N-H;Y is H, Na, K or aliphatic, aromatic, linear, branched or cyclic chain C2-C7; is dissolved in a concentration of 0.05 to 0.68 mol.L'1in water or in a mixture of water and an organic solvent, wherein the organic solvent is selected from the group comprising dimethyl formamide, dimethyl sulfoxide, 2-propanol, methanol, tetrahydrofuran, acetone, acetonitrile or a mixture thereof, with the addition of a thiol, in an amount of 0.36 to 2.08 molar equivalents relative to the oligosaccharide, and aphotoinitiator selected from the group comprising LAP, Irgacure 2959, DPAP, TPO or Irgacure 1173, in an amount of 0.1 to 0.36 molar equivalents relative to the oligosaccharide, after which the mixture is irradiated with a light source of wavelength 254, 365 or 455 nm at a temperature of -10 to 30 °C for a period of 5 to 240 minutes to form the modified oligosaccharide or a mixture of modified and unmodified oligosaccharide.

6. The method of preparation according to claim 5, characterized in that the thiol is selected from the group comprising cysteamine, dithiothreitol, DODT, glutathione, L- cysteine, N-acetyl-L-cysteine methyl ester, N-acetyl-L-cysteine, 2-mercaptoethanol, N-acetyl-L-cysteine 2-mercaptoethylamide, 3 -mercaptopropionic acid, meso-2,3- dimercaptosuccinic acid, 1-thio-B-D-glucose, thioglycolic acid, HA thiol tetrasaccharide, HA thiol hexasaccharide, HA thiol octasaccharide, HA thiol decasaccharide.

7. A method for preparing the modified oligosaccharide defined in the claims 1 to 4, characterized in that an ester of unsaturated oligosaccharide of hyaluronic acid according to the structural formula V,wherein n = 0 to 8 and Y is aliphatic, aromatic, linear, branched or cyclic chain C2-C7; is dissolved in water or in a mixture of water and an organic solvent selected from the group comprising acetonitrile and 2-propanol, with the amount of organic solvent being 0 - 25 %vol., at a concentration of 0.02 to 0.6 mol.L’1, in the presence of a thiol in an amount of 2 to 10 molar equivalents relative to the oligosaccharide, the pH of the solution is adjusted to 6.02 to 9.1 and the resulting mixture is stirred at a temperature of -20 to 50 °C for 1-72 hours to form the modified oligosaccharide or a mixture of modified and unmodified oligosaccharide.

8. The method of preparation according to claim 7, characterized in that the thiol is selected from the group comprising cysteamine, D-cysteine, 2-(diethylamino)ethanethiol, dithiothreitol, DODT, thioglycolic acid ethyl ester,glutathione, L-cysteine, L-cysteine methyl ester, N-acetyl-L-cysteine methyl ester, 2- mercaptoethanol, N-acetyl-L-cysteine 2-mercaptoethyl amide, meso-2,3- dimercaptosuccinic acid, N-acetyl-L-cysteine, thioglycolic acid, thiocholine.

9. The method of preparation according to claim 7 or 8, characterized in that the resulting modified oligosaccharide or the mixture of modified and unmodified oligosaccharide is subjected to hydrolysis of ester groups to form carboxyl groups.

10. A method for preparing the modified oligosaccharide defined in claim 4, characterized in that first an acryloyl derivative of a pharmaceutically active substance according to the formula (VI) is preparedwherein A is O, N-H or NH-CH2CH2-O and B is a pharmaceutically active substance; further, the modified oligosaccharide as defined in claim 3 is prepared by the method defined in any one of claims 5 - 9, after which the resulting oligosaccharide is dissolved in water and added to the derivative according to the structural formula (VI) dissolved in water, a water-miscible organic solvent or a mixture thereof, wherein the organic solvent is selected from the group comprising methanol, ethanol, 2-propanol, dimethyl sulfoxide, dimethyl formamide, acetonitrile, tetrahydrofuran, or a mixture thereof, wherein the amount of the derivative according to the formula (VI) is 1 - 3 molar equivalents relative to the oligosaccharide and the resulting amount of organic solvent is 0 - 25 vol %, the pH of the solution is adjusted to 7 to 8, the reaction mixture is then stirred at a temperature of 25 °C for 1-72 hours.

11. The method of preparation according to claim 10, characterized in that the pharmaceutically active substance is selected from the group comprising substances with anti-inflammatory, antibacterial, antiviral or anticancer effects.

12. The method of preparation according to claim 11, characterized in that the active substance is selected from the group comprising acetaminophen, 5-aminosalicylic acid, diclofenac, ibuprofen, indomethacin, ketoprofen, naproxen, ciprofloxacin, metronidazole, aciclovir, darunavir, didanosine, ganciclovir or ribavirin, daunorubicin, doxorubicin, lenalidomide, mercaptopurine.

13. Use of the modified oligosaccharide defined in the claims 1 to 3 as a precursor for the preparation of other derivatives.

14. Use of the modified oligosaccharide as defined in the claims 1 to 3 as a precursor for the preparation of the derivative defined in claim 4.

15. Use of the modified oligosaccharide defined in claim 4 for the production of pharmaceutical compositions.

16. The modified oligosaccharide as defined in the claim 4 for use in the treatment of inflammatory diseases, bacterial diseases, viral diseases, cancer, chronic intestinal inflammation, Crohn's disease or ulcerative colitis.

17. A pharmaceutical composition, characterized in that it contains the modified oligosaccharide as defined in claim 4, and is in the form of a solution, film or lyophilizate.

18. The pharmaceutical composition according to claim 17 for use in the treatment of inflammatory diseases, bacterial diseases, viral diseases, cancer, chronic intestinal inflammation, Crohn's disease or ulcerative colitis.

19. The pharmaceutical composition according to claim 17 or 18, characterized in that it is in the form of a solution and contains:- 0.1 - 5 wt. % of the modified oligosaccharide defined in claim 4,- 0.1 - 1.0 wt. % of sodium or potassium chloride,- 94 - 99.8 wt. % of water.

20. The pharmaceutical composition according to claim 19, characterized in that it contains:- 0.1 - 5 wt. % of the modified oligosaccharide defined in claim 4,- 0.1 - 1.0 wt. % of sodium or potassium chloride,- 0.1 - 1.0 wt. % of sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate or a mixture thereof, and / or 0.001 - 5 % of other additives selected from the group comprising calcium chloride, glycerol, hyaluronic acid oligosaccharide or its derivative, hyaluronic acid or its derivative, chondroitin sulfate,- 88 - 99.699 wt. % of water.

21. The pharmaceutical composition according to claim 17 or 18, characterized in that it is in the form of a film or lyophilisate and that it contains:- 1 - 10 wt. % of the modified oligosaccharide defined in claim 4,- 75 - 98.9 wt. % of one or more carrier polymers selected from the group comprising hyaluronic acid or its derivative, chondroitin sulfate,- 0.1 - 15 wt. % of water.

22. The pharmaceutical composition according to claim 21, characterized in that it contains:- 1 - 10 wt. % of the modified oligosaccharide defined in claim 4,- 70 - 98.8 wt. % of one or more carrier polymers selected from the group comprising hyaluronic acid or its derivative, chondroitin sulfate,- 0.1 - 5 wt. % of hyaluronic acid oligosaccharide or its derivative, sodium or potassium chloride, calcium chloride, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate or a mixture thereof,- 0.1 - 15 wt. % of water.

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