EP4698567A1

EP4698567A1 - Hydrogels containing covalently linked cofactors and methods for preparing the same

Info

Publication number: EP4698567A1
Application number: EP24725052.5A
Authority: EP
Inventors: Oliver Plettenburg; Christin AHLBRECHT
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH; Leibniz Universitaet Hannover
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH; Leibniz Universitaet Hannover
Priority date: 2023-04-19
Filing date: 2024-04-19
Publication date: 2026-02-25
Also published as: WO2024218289A1

Abstract

The invention provides a method of preparing a biocompatible hydrogel, a hydrogel obtainable by said method and a biocompatible hydrogel. The present invention further provides a cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a 1,3- dipolar cycloaddition. Additionally, the present invention provides a composition comprising any of said hydrogels according to the invention. The invention further provides the use of any of said hydrogels for non-covalent immobilization of at least one enzyme(s) in the hydrogel, or as a catalyst, or in a biosensor. Additionally, the present invention provides a kit comprising the hydrogel according to the present invention.

Description

HYDROGELS CONTAINING COVALENTLY LINKED COFACTORS AND METHODS FOR PREPARING THE SAME

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention provides a method of preparing a biocompatible hydrogel providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, and a hydrogel obtainable by said method. The present invention further provides a biocompatible hydrogel comprising a crosslinked polymer comprising at least one cofactor(s). Additionally, the present invention is directed to a cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition, and a composition comprising the hydrogel according to the present invention. In a further aspect, the present invention is directed to the use of any of said hydrogels according to the present invention for non-covalent immobilization of at least one enzyme(s) in the hydrogel, or the use of any of said hydrogels as a catalyst, or the use of any of said hydrogels in a biosensor. Additionally, the present invention provides a kit comprising any of said hydrogel(s) according to the present invention.

BACKGROUND ART

[0002] Enzymes in solution usually have a limited lifetime, as they can quickly degrade at ambient temperature. Furthermore, they show limited tolerability towards organic solvents. The purpose of the present invention is inter alia to provide an effective method for enzyme stabilization, inter alia by the use of cofactors which are covalently bound in the hydrogel. Enzyme encapsulation for various purposes usually requires covalent modification, leading to impaired activity. Furthermore, the bonds used for hydrogel formation may frequently not be stable, for example, when using Schiff bases, leading to premature degradation of the gel and subsequent leaching of the payload or may contain toxic and carcinogenic, e.g. hydrazones, functionalities.

[0003] Different methods have been developed to immobilize enzymes in a hydrogel network, e.g. under use of glutaraldehyde. However, for example, BSA mixtures provide limited protection of the enzymes and suffer from short lifetime and toxicity. Other methods of using wired enzymes require chemistry using toxic components and have limited application in implantable devices [1], [0004] Other methods of the prior art use polymers, which are modified with furan/ furan derivatives (methyl furan), reacting with poly(ethylene glycol) [2], Other methods of the prior art uses, for the preparation of hydrogels, the formation of a Schiff base (between amino and aldehyde groups), leading to the formation of an imine linkage [3], For example, Ma et al. uses the formation of a Schiff base (aldehyde and hydrazide groups) for building an injectable hydrogel [4],

[0005] Other possibilities comprise the chemo-specific 'click reaction' by an oxime crosslink. Though the oxime bound is more stable than hydrazine, this has the disadvantage that the hydrogel might be reversible in some biological environments [5],

[0006] Peng et al. instead provides stabilisation of collagen sponges by glutaraldehyde and uses vapour crosslinking [6], Jia et al. [7] teaches to use an enzyme solution and a BSA stabilizer with chitosan in acetic acid, while the final crosslinking of the hydrogel was done with glutaraldehyde vapor.

[0007] However, all these described methods of the prior art have several disadvantages, which makes them not applicable for the encapsulation of enzymes. For example, the formation of a Schiff base has the problem of being reversible and hydrazones are undesired, because of their toxicity. Further, with the hydrogels provided in the prior art, reproducibility is limited, e.g. with regard to layer thickness. Further disadvantages are that coatings with imine/ hydrazone are biodegradable, that products of degraded hydrazones (hydrazine) are toxic, that glutaraldehyde can't be spun, and that the quality control is insufficient, additionally, any vapour process is random and not reproducible [6],

[0008] Further, a major disadvantage of the prior art, for example, with regard to encapsulation of an enzyme within the hydrogel is that crosslinking by glutaraldehyde may result in covalent modifications of amino groups at the enzyme, which will lead to structural and conformational changes of the enzyme, possibly leading to undesired inactivation of the enzyme.

[0009] Also, the gelation process is starting immediately after mixing the individual components, leading to possible incomplete filling of form factors and to inhomogeneous hydrogels.

[0010] Many biosensors like most conventional implantable glucose sensors rely on oxidases as the enzymatic recognition element in the biosensor, because dehydrogenases are less stable and the cofactor is not prosthetically bound.

[0011] Oxidases, however, are oxygen-dependent. In contrast, dehydrogenases are oxygenindependent and may be more sensitive in vivo at low oxygen concentrations.

[0012] For enzymes with non-prosthetically bound cofactors, cofactor lixiviation is a major issue, limiting the number of productive enzymatic reaction cycles and thus enzymatic turnover and lifetime.

[0013] However, presence of the cofactor can also stabilize the enzyme. [0014] Velasco-Lozano et al. [9] reported the reversible adsorption of negatively charged cofactors to cationic beads, which was applied to bioreactors, but loss of cofactor was observed depending on ionic strength, leading to a limited lifetime and cycle times, the cofactor was provided as a substrate to the flow reactor system and later recycled [10], The described system is thus not suitable for biosensor applications.

[0015] Menegatti et al. [11] describe encapsulation of a mixture of non modified pyridoxal phosphate and transaminase in a 8% polyvinal acetate and 2% alginate solution. Crosslinking was induced by adding calcium chloride and phenyl boronic acid. This reversible hydrogel is unsuitable for in vivo applications and leaching of cofactor or enzyme at prolonged time points cannot be excluded.

[0016] Zhang et al. [12] described amide coupling of an N¹-carboxymethyl-NAD⁺ species to amino groups of chitosan and subsequent formation of a hydrogel by glutaraldehyde based crosslinking. As the condensation reaction is not bioorthogonal, modification of the cofactor cannot be excluded, furthermore, the enzyme is covalently linked, as well.

[0017] Yan et al. [13] describe tethering of NAD⁺ and NADP⁺ to carbon nanotubes via formation of a benzoic acid boronic ester of the ribose unit and amide formation of the carboxylic acid and an amino group of the carbon nanotube to create a biofuel cell. Carbon nanotubes were adsorbed on electrode surfaces, cofactor was added and enzyme was immobilized on top of the electrodes via crosslinking by glutaraldehyde. This preparation is not applicable for in vivo sensors, glutaraldehyde treatment can also modify the cofactor and no longevity data is presented.

[0018] The present invention aims at and addresses these above described needs.

SUMMARY OF THE INVENTION

[0019] The above-mentioned problems are solved by the subject-matter as defined in the claims and as defined herein.

[0020] The present invention describes a novel strategy to immobilize enzymes in a biocompatible hydrogel containing covalently bound cofactor(s) or derivatives thereof that does not require a covalent binding of one or more enzyme(s). The hydrogel according to the present invention provides a perfect stabilizing environment for the enzyme, resulting in a better lifetime and enzyme stability. The hydrogel according to the present invention protects the enzyme from biofouling and body immune response.

[0021] In a first aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, c) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; d) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

X is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group Y; and functionalization of the second polysaccharide with one or more linker unit(s) of the structure -A'-Y, when the monomeric repeating unit of the second polysaccharide not comprises a carboxylic acid residue, or functionalization of the second polysaccharide with one or more group(s) of the structure -Y, when the monomeric repeating unit of the second polysaccharide comprises a carboxylic acid residue, wherein A' is a second spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z; e) optionally addition of at least one enzyme(s), f) incubation of the functionalized second polysaccharide with the at least one functionalized cofactor in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hours, to carry out a 1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y, and g) incubation of the functionalized first polysaccharide with the product of step f) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted in step f).

[0022] The present invention provides in one further aspect a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s) and incubation of the at least one enzyme with the at least one functionalized cofactor of step a), such that the at least one enzyme is non-covalently bound to the at least one cofactor, c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z; f) incubation of the functionalized first polysaccharide and the functionalized second polysaccharide of step e) with the at least one enzyme non-covalently bound to the at least one cofactor of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1,3-dipolar cycloaddition between the group Z and a part of the group(s) Y, and a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z.

[0023] In a further aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z; f) incubation of the functionalized second polysaccharide with the at least one functionalized cofactor in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y, and g) incubation of the functionalized first polysaccharide with the at least one enzyme and the product of step f) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted in step f), and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0024] The present invention also provides in a further aspect, a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z; f) incubation of the functionalized first polysaccharide of step e), the functionalized second polysaccharide of step e), the at least one functionalized cofactor of step a) and the at least one enzyme of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y, and to carry out a 1,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z, and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0025] In a further aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor, wherein a part of the cofactor is functionalized with at least one group Z and a part of the cofactor is functionalized with at least one group E, wherein Z and E each independently comprises or is a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z and/or the group E; f) incubation of the functionalized first polysaccharide of step e), the functionalized second polysaccharide of step e), the at least one functionalized cofactor of step a) and the at least one enzyme of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group Z and/or the group E and a part of the group(s) Y, and to carry out a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y, not reacted with Z or E, and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0026] In a further aspect, the present invention is directed to a hydrogel obtainable by a method of preparing a biocompatible hydrogel as described herein.

[0027] In another aspect, the present invention provides a biocompatible hydrogel comprising, a crosslinked polymer comprising:

- at least one cofactor(s),

- a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating units of the first and/ or the second polysaccharide and with n being an integer from 10 to 10000, - at least one first linker unit(s), which link(s) the first polysaccharide with the second polysaccharide, wherein the structure of the at least one first linker unit(s) is -A-Gi-A’-, -A- Gi~ , — A — Gi- , or— Gi- , wherein A is a first spacer unit;

Gi is a first moiety being or comprising a group obtainable or being obtained from a 1,3- dipolar cycloaddition; and

A’ is a second spacer unit; and at least one second linker unit(s), which link(s) the first polysaccharide or the second polysaccharide with the at least one cofactor(s), wherein the structure of the at least one second linker unit(s) is -A-G₂-,-A’-G₂-, or-G₂-; wherein A is the first spacer unit;

A’ is the second spacer unit; and

G₂ is a second moiety being or comprising a group obtainable or being obtained from a 1 ,3- dipolar cycloaddition.

[0028] In a further aspect, the present invention provides a cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition.

[0029] Further is provided by the present invention a composition comprising the hydrogel as described herein.

[0030] The present invention further provides the use of a hydrogel according to any aspect as described herein a) for non-covalent immobilization of one or more enzyme(s) in the hydrogel, or b) as a catalyst, or c) in a biosensor.

[0031] The present invention further provides a kit comprising the composition or the hydrogel according to any aspect of the present invention as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows an exemplary structure of a biocompatible hydrogel with non-covalently immobilized enzyme (E) and covalently attached cofactor (C) according to the present invention.

[0033] Figure 2 shows an exemplary basic principle of the pre-functionalization of the biopolymer strands with cofactors according to the method of the present invention. Figure 2A shows an example of oxanorbornadiene functionalized (marked as triangle) biopolymer strand and azide functionalized (marked as circle) cofactor. The formation of the pre-functionalized biopolymer out of the aforementioned individual components is induced by heat. Figure 2B shows an example of azide functionalized (marked as circle) biopolymer strand and two different functionalized cofactors. The formation of the pre-functionalized biopolymer out of azide functionalized (marked as circle) biopolymer and oxanorbornadiene functionalized (marked as triangle) cofactor is induced by heat (a). The formation of the pre-functionalized biopolymer out of azide functionalized (circle) biopolymer and alkyne functionalized (L) cofactor is induced by [Cu] (b). The formation of the pre-functionalized biopolymer out of azide functionalized (circle) biopolymer and oxanorbornadiene functionalized (marked as triangle) cofactor and alkyne functionalized (L) cofactor is induced by heat and [Cu] (c).

[0034] Figure 3 shows an exemplary basic principle of the gelation to form the biocompatible hydrogel with non-covalently immobilized enzyme and covalently attached cofactor according to the method of the present invention out of azide (marked as circle) and oxanorbornadiene (marked as triangle) functionalized biopolymer strands. Figure 3A shows the aforementioned principle with an azide functionalized (marked as circle) cofactor. A pre-incubation of the enzyme and the azide functionalized (marked as circle) cofactor before mixing with the two biopolymer strands is shown in (a). A pre-functionalized biopolymer strand (marked as square) before mixing with the azide functionalized (marked as circle) second biopolymer strand and enzyme is shown in (b). The mixture of the four aforementioned individual components is shown in (c). Figure 3B shows the aforementioned principle with an alkyne functionalized (L) cofactor. A pre-incubation of the enzyme and the alkyne functionalized (L) cofactor before mixing with the biopolymer strands is shown in (a). A pre-functionalized biopolymer strand (marked as square) before mixing with the oxanorbornadiene functionalized (marked as triangle) second biopolymer strand and enzyme is shown in (b). The mixture of the four aforementioned individual components is shown in (c). The mixture of the four aforementioned individual components and an oxanorbornadiene functionalized cofactor (marked as triangle) is shown in (d). Figure 3C shows the aforementioned principle with an oxanorbornadiene functionalized (marked as triangle) cofactor. A pre-incubation of the enzyme and the oxanorbornadiene functionalized (marked as triangle) cofactor before mixing with the biopolymer strands is shown in (a). A prefunctionalized biopolymer strand (marked as square) before mixing with the oxanorbornadiene functionalized (marked as triangle) second biopolymer strand and enzyme is shown in (b). The mixture of the four aforementioned individual components are shown in (c). The mixture of the four aforementioned individual components and an alkyne functionalized cofactor (L) is shown in (d). [0035] Figure 4 shows the degree of substitution in a pullulan biopolymer with different amounts of introduced carboxymethyl groups. It displays different fingerprint regions in the FT- IR spectrum to characterize the carboxymethylation degree of pullulan (for PCM1 , PCM3 and PCM5). The intensity of the bands between 1600 - 1000 cm'¹ increases with the substitution degree.

[0036] Figure 5 shows the increase of the introduced carboxymethyl-groups in a biopolymer by ¹H-NMR spectroscopy. The intensity of the signals of the anomeric protons of the polysaccharide (marked as square) decreases with higher degrees of modification, while the signals of the polysaccharide backbone broadens (marked as dashed) and the intensity of the signals of the introduced carboxymethyl-group(s) (marked as dotted) increases.

[0037] Figure 6 shows the stepwise modification process of polysaccharides (e.g. pullulan) by ¹H-NMR spectroscopy. It compares the carboxymethylated with the unmodified polysaccharide. In this context, the intensity of the anomeric proton signals decreases (see c and d). The signals of the oxanorbomadiene linker unit introduced by the copper-free cycloaddition into the polysaccharide-chain are visible in the NMR-spectra of the ‘PCM3 linker’ (see a and b).

[0038] Figure 7 shows the direct correlation of the degree of substitution of the polysaccharide with the introduced oxanorbomadiene linker units by ¹H-NMR spectroscopy. Figure 7 c and d show the decrease of the intensity of the anomeric proton signals with increasing degree of substitution. Figure 7 a and b show the increase of the intensity of the introduced linker signals due to the increasing number of carboxymethyl-groups in the polysaccharide.

[0039] Figure 8 shows carboxymethylation reaction, measured by conductive titration. Data is given for a low molecular weight dextran (10 kDa) and molecular weight pullulan (100 kDa).

[0040] Figure 9 shows IR-spectra of five different batches of a five times carboxymethylated pullulan strand (PCM5), which contains an azide linker unit ((-NH-(CH2CH₂O)s-CH2CH₂N3 with s = 3)). Data is given for molecular weight pullulan (100 kDa).

[0041] Figure 10 shows the increase of the intensity of the signal in a ¹⁹F-NMR spectrum of an introduced linker in a pullulan strand due to the increase of the degree of substitution and the number of introduced linker units in relation to an internal standard.

[0042] Figure 11 shows the degree of substitution in a dextran biopolymer (10 kDa) with different amounts of introduced carboxymethyl-groups. It displays different fingerprint regions in the FT-IR spectrum to characterize the different carboxymethylation degree (for CMI , CM3 and CM5). The intensity of the bands between 1600 - 1000 cm'¹ increases with the substitution degree.

[0043] Figure 12 shows the differences between a native lentinan and a carboxymethylated lentinan (CM1). It visualizes different fingerprint regions in the FT-IR spectrum to verify the carboxymethylation degree (lentinan and lentinan CM1). The intensity of the bands between 1600 - 1000 cm'¹ increases with the substitution degree.

[0044] Figure 13 shows the differences between hyaluronan and hyaluronan with linker units (azide unit and an oxanorbornadiene unit). The wavenumbers of the bands in the FT-IR spectra vary between 1600 - 1000 cm'¹ due to the differently employed linker units.

[0045] Figure 14 shows a ¹H-NMR spectrum of a 5-times carboxymethylated pullulan biopolymer with an azide linker unit (-NH-(CH₂)_r-N₃ with r = 6).

[0046] Figure 15 shows a ¹H-NMR spectrum of a 5-times carboxymethylated pullulan with an oxanorbornadiene unit (-NH-(CH₂)_r-Q with r = 6, Q = Q-1 (CF₃) and M/M’ = H).

[0047] Figure 16 shows a ¹H-NMR spectrum of a 5-times carboxymethylated pullulan with an azide unit (-NH-(CH₂CH₂O)_S-CH₂CH₂N₃ with s = 8).

[0048] Figure 17 shows a ¹H-NMR spectrum of hyaluronan (A) in comparison to hyaluronan with an oxanorbornadiene unit (-NH-(CH₂)r-Q with r = 2, Q = Q-1 (CF₃) and M/M' = H) (B), and hyaluronan with an azide unit (-NH-(CH₂CH₂O)_S-CH₂CH₂N₃ with s = 3) (C).

[0049] Figure 18 shows ¹H-NMR spectra of dextran (10 kDa) with different amounts of introduced carboxymethyl groups.

[0050] Figure 19 shows the viscosities of various biopolymers at a shear rate of 100 s'¹. Different polysaccharides are shown in Figure 19A, pullulan with various carboxymethyl groups are shown in Figure 19B.

[0051] Figure 20 shows three different synthesized NAD⁺-derivatives.

[0052] Figure 21 shows the reaction between NAD⁺-azide with an oxanorbornadiene unit to form NAD⁺-triazole. [0053] Figure 22 shows enzyme kinetics of GDH in solution with different NAD⁺-derivatives.

[0054] Figure 23 shows enzyme kinetics of G6PD in solution with different NAD⁺-derivatives.

[0055] Figure 24 shows the reaction between an oxanorbomadiene modified biopolymer strand with NAD⁺-azide to form a pre-functionalized biopolymer strand.

[0056] Figure 25 shows a ¹H-NMR spectrum of a pre-functionalized biopolymer strand. Figure 25A and 25B show different equivalent amount of covalently attached cofactor (NAD⁺) to the biopolymer strand.

[0057] Figure 26 shows enzyme kinetics of G6PD immobilized in hydrogels with native NAD⁺ (in solution) or covalently immobilized NAD⁺.

[0058] Figure 27 shows enzyme kinetics of GDH immobilized in hydrogels with covalently immobilized NAD⁺ on different days.

[0059] Figure 28 shows enzyme kinetics of GDH in solution with native NAD⁺, GDH immobilized in hydrogels with native NAD⁺ and GDH immobilized in hydrogels with covalently immobilized NAD⁺. All samples were incubated in phosphate buffer (pB) for 20 days at 37 degree Celsius.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The invention provides in a first aspect a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, c) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; d) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z; e) optionally addition of at least one enzyme(s), f) incubation of the functionalized second polysaccharide with the at least one functionalized cofactor in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y, and g) incubation of the functionalized first polysaccharide with the product of step f) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted in step f).

[0061] The invention provides in one further aspect a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a click chemistry reaction with a group Y, b) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, c) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; d) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

X is a group comprising or being a functional group suitable for a click chemistry reaction with the group Y; and functionalization of the second polysaccharide with one or more linker unit(s) of the structure -A'-Y, when the monomeric repeating unit of the second polysaccharide not comprises a carboxylic acid residue, or functionalization of the second polysaccharide with one or more group(s) of the structure -Y, when the monomeric repeating unit of the second polysaccharide comprises a carboxylic acid residue, wherein A' is a second spacer unit; and

Y is a group comprising or being a functional group suitable for a click chemistry reaction with the group X and/or the group Z; e) optionally addition of at least one enzyme(s), f) incubation of the functionalized second polysaccharide with the at least one functionalized cofactor in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group Z and a part of the group(s) Y, and g) incubation of the functionalized first polysaccharide with the product of step f) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group X and a part of the group(s) Y not reacted in step f).

[0062] In a further aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s) and incubation of the at least one enzyme with the at least one functionalized cofactor of step a), such that the at least one enzyme is non-covalently bound to the at least one cofactor, c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

[0063] In a further aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a click chemistry reaction with a group Y, b) providing at least one enzyme(s) and incubation of the at least one enzyme with the at least one functionalized cofactor of step a), such that the at least one enzyme is non-covalently bound to the at least one cofactor, c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a click chemistry reaction with the group X and/or the group Z; f) incubation of the functionalized first polysaccharide and the functionalized second polysaccharide of step e) with the at least one enzyme non-covalently bound to the at least one cofactor of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group Z and a part of the group(s) Y, and a click chemistry reaction between the group X and a part of the group(s) Y not reacted with Z.

[0064] The present invention provides in a further aspect a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

[0065] Additionally, the present invention provides in one aspect a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a click chemistry reaction with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a click chemistry reaction with the group X and/or the group Z; f) incubation of the functionalized second polysaccharide with the at least one functionalized cofactor in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group Z and a part of the group(s) Y, and g) incubation of the functionalized first polysaccharide with the at least one enzyme and the product of step f) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group X and a part of the group(s) Y not reacted in step f), and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0066] In another aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

[0067] In another aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a click chemistry reaction with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a click chemistry reaction with the group X and/or the group Z; f) incubation of the functionalized first polysaccharide of step e), the functionalized second polysaccharide of step e), the at least one functionalized cofactor of step a) and the at least one enzyme of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group Z and a part of the group(s) Y, and to carry out a click chemistry reaction between the group X and a part of the group(s) Y not reacted with Z, and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0068] In another aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor, wherein a part of the cofactor is functionalized with at least one group Z and a part of the cofactor is functionalized with at least one group E, wherein Z and E each independently comprises or is a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

[0069] In another aspect, the present invention provides a method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor, wherein a part of the cofactor is functionalized with at least one group Z and a part of the cofactor is functionalized with at least one group E, wherein Z and E each independently comprises or is a functional group suitable for a click chemistry reaction with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

Y is a group comprising or being a functional group suitable for a click chemistry reaction with the group X and/or the group Z and/or the group E; f) incubation of the functionalized first polysaccharide of step e), the functionalized second polysaccharide of step e), the at least one functionalized cofactor of step a) and the at least one enzyme of step b) in an aqueous medium at a temperature being in the range from 5°C to 70°C, preferably in the range from 35°C to 40°C, for at least 1 hour, preferably for 1 to 10 hour(s), to carry out a click chemistry reaction between the group Z and/or the group E and a part of the group(s) Y, and to carry out a click chemistry reaction between the group X and a part of the group(s) Y, not reacted with Z or E, and wherein the at least one enzyme is non-covalently bound to the at least one cofactor.

[0070] The term “the cofactor is functionalized with at least one group Z” in general means that the cofactor can be functionalized with one or more group(s) Z. As illustrative examples, the cofactor may be functionalized with 1 , 2 or 3 group(s) Z. Preferably, the cofactor is functionalized with one group Z. The term “functionalized with one group Z” means that the cofactor is functionalized with exactly one group Z.

[0071] The term “a part of the cofactor” refers to a population of the molecules of the cofactor. Accordingly, the term “a part of the cofactor is functionalized with at least one group Z” means that a population of the molecules of the cofactor is functionalized with at least one group Z. The term “a part of the cofactor is functionalized with at least one group E” means that a population of the molecules of the cofactor is functionalized with at least one group E. In particular, the population of the molecules of the cofactor which is functionalized with the group E is another population of the molecules of the cofactor than the population of molecules of the cofactor which is functionalized with the group Z. Accordingly, the term “a part of the cofactor is functionalized with a least one group Z and a part of the cofactor is functionalized with at least one group E” in particular means that a part of the cofactor is functionalized with at least one group Z and another part of the cofactor is functionalized with at least one group E.

[0072] As used herein and in the context of the present invention, the term “biocompatible” means, especially in connection with a hydrogel, that the respective material being called or assessed as being biocompatible has the quality of not having toxic or injurious effects on biological systems, that it has the ability to perform its desired function without eliciting any undesirable local or systemic effects in the recipient, but generating the most appropriate beneficial response in that specific situation, or the ability to exist in harmony with tissue without causing deleterious changes. Preferable properties of biocompatible materials are reduced inflammation and immunological response and/ or low/ limited fibrotic encapsulation.

[0073] The term “hydrogel”, as used herein and in the context of the present invention, is a term being well known to a person skilled in the art and includes any network of covalently crosslinked polymer chains that are hydrophilic. It usually builds up a three-dimensional solid, consisting of hydrophilic polymer chains, being held together by specific crosslinkers. Because of the inherent crosslinkers, the structural integrity of the hydrogel network does not dissolve in water. Hydrogels are highly absorbent (they can contain over 90% water) natural or synthetic polymeric networks.

[0074] The “first polysaccharide” and the “second polysaccharide” as used within the context of the present invention may be any polysaccharide known to a person skilled in the art. However, it is preferred that the first polysaccharide and/ or the second polysaccharide may be independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof, more preferably from the group consisting of pullulan, alginate, hyaluronan and dextran. In this connection, the respective “monomeric repeating unit” for each of these exemplary examples for the first and/ or second polysaccharide may be defined as follows herein below:

[0075] Pullulan is a polysaccharide polymer consisting of maltotriose units. Three glucose units of maltotriose are connected by an a-1,4-glycosidic bond, whereas consecutive maltotriose units are connected to each other by an a-1,6-glycosidic bond. Pullulan may be produced from starch by the fungus Aureobasidium pullulans. It may be mainly used by the cell to resist desiccation and predation. The presence of this polysaccharide also facilitates diffusion of molecules both into and out of the cell.

[0076] In the context of the present invention, the respective monomeric repeating unit for pullulan has the structure of

, with n being the number of said monomeric repeating unit of pullulan, and with n being an integer from 10 to 10000. In the context of the present invention, the carboxymethylation degree of pullulan is presented by the expression “PCM” followed by a number (e.g. PC 1, PCM3 and PCM5). This number characterizes the respective carboxymethylation degree, meaning the carboxymethyl-groups introduced into pullulan by applying the designated number of repetitive carboxymethylation reaction cycles. As used herein, CM1, CM2, CM3, etc. describe the respective carboxymethylation degree of a polysaccharide in general (without specifically referring to pullulan).

[0077] Alginic acid, also called algin, is a polysaccharide distributed widely in the cell walls of brown algae that is hydrophilic and forms a viscous gum, when being hydrated. Alginic acid is a linear copolymer with homopolymeric blocks of (1 -4)-linked p-D-mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively, are covalently linked together in different sequences or blocks. The monomers may appear in homopolymeric blocks of consecutive G- residues (G-blocks), consecutive M-residues (M-blocks) or alternating M- and G-residues (MG- blocks). With metals, such as sodium and calcium, its salts are known as alginates. In the context of the present invention, the respective monomeric repeating unit for alginate has the structure of , with n and m being the number of the monomeric repeating unit of alginate, and with n and m being each independently from each other an integer in the range from 10 to 10000.

[0078] Hyaluronic acid (abbreviated HA; conjugate base: hyaluronate), also called hyaluronan, is an anionic, non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial and neural tissues. It is unique among glycosaminoglycans in that it is non-sulfated, forms in the plasma membrane instead of the Golgi apparatus and can be very large. Hyaluronic acid is a polymer of disaccharides, themselves composed of D-glucuronic acid and /V-acetyl-D-glucosamine, linked via alternating [3-(1— >4) and p-(1— >3) glycosidic bonds. In the context of the present invention, the respective monomeric repeating unit for hyaluronan has the structure of being the number of said monomeric repeating unit of hyaluronan, and with n being an integer from 10 to 10000. [0079] Dextran is a complex branched glucan (polysaccharide derived from the condensation of glucose). IUPAC defines dextrans as "branched poly-a-D-glucosides of microbial origin having glycosidic bonds predominantly C-1 — > C-6". Dextran chains are of varying lengths (from 3 to 2000 kilodaltons). The polymer main chain consists of a-1 ,6-glycosidic linkages between glucose monomers, with random branches from a-1 ,3-linkages. This characteristic branching distinguishes a dextran from a dextrin, which is a straight chain glucose polymer tethered by a- 1 ,4- or a-1,6-linkages. In the context of the present invention, the respective monomeric repeating unit for dextran has the structure of , with n being the number of said monomeric repeating unit of dextran, and with n being an integer from 10 to 10000.

[0080] The term “functional group” as used in the context of the present invention is well know to the person skilled in the art. Such a functional group is preferably used herein for chemoselective or bioorthogonal modifications. The functional group may be suitable for click chemistry.

[0081] The term “click chemistry” or “click chemistry reaction” refers to a chemical philosophy introduced by Kolb, Finn and Sharpless in 2001 and encompasses a group of powerful linking reactions that are able to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. Click chemistry reactions are typically modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, and/or can be carried out using readily available starting materials and reagents under simple, physiological reaction conditions. In addition, click chemistry reactions preferably use no toxic solvents or use a solvent that is benign or easily removed (preferably water) and/or provides simple product isolation by non-chromatographic methods (crystallization or distillation).

[0082] Click chemistry reactions comprise, e.g., cycloaddition reactions, especially from the 1 ,3- dipolar family, hetero-Diels-Alder reactions; nucleophilic ring-opening reactions, e.g. of strained heterocyclic electrophiles, such as epoxides, aziridines, cyclic sulfates, cyclic sulfamidates, aziridinium ions and episulfonium ions, carbonyl chemistry of the non-aldol type (e.g. the formation of oxime ethers, hydrazones and aromatic heterocycles); and addition to carboncarbon multiple bonds; e.g. oxidation reactions, such as epoxidation, dihydroxylation, aziridination, and nitrosyl and sulfenyl halide additions, but also certain Michael addition reactions. General principles of click chemistry reactions have been described by Kolb, Finn and Sharpless (2001).

[0083] Further, a “group” or “functional group” as used in the context of the present invention may also be a “click chemistry handle”. Such is also known to the person skilled in the art in the context of click chemistry reactions. That term refers to a reactant, or a reactive group, that can partake in a click chemistry reaction. Such a reactant or reactive group is preferably a functional group for chemoselective or bioorthogonal click chemistry reactions. For example, an alkyne, an azide or an oxanorbornadiene, is a click chemistry handle since it can partake in a strain- promoted cycloaddition. In general, click chemistry reactions require at least two molecules comprising click chemistry handle pairs that can react with each other. Such click chemistry handle pairs that are reactive with each other are sometimes referred to as “partner click chemistry handles”. For example, an azide is a partner click chemistry handle to an alkyne or an oxanorbornadiene. In the context of the present invention, the click chemistry handle can preferably be selected from the group consisting of terminal alkyne, azide, strained alkyne, diene, dieneophile, alkoxamine, tetrazine, alkene, cyclooctene, norbornene, tetrazine, nitrone, and cyclooctyne. Other suitable click chemistry handles are readily accessible to the person skilled in the art.

[0084] In the context of conjugation via click chemistry, the conjugation is via a covalent bond formed by the reaction of the click chemistry handles. In certain embodiments, the association is covalent, and the entities are said to be “conjugated” to one another.

[0085] It should be noted that the invention is not limited to the foregoing, exemplary click chemistry handles, and additional click chemistry handles, reactive click chemistry handle pairs, and reaction conditions for such click chemistry handle pairs will be apparent to those of skill in the art.

[0086] The term “1,3-dipolar cycloaddition” is generally known in the art and describes a chemical reaction between a 1,3-dipole and a dipolarophile to form a five-membered ring. Suitable 1 ,3-dipoles and dipolarophiles are also generally known in the art. As a merely illustrative example, a 1 ,3-dipole may be an azide, and a dipolarophile may be an alkyne or an oxanorbornadiene, to generate a 1,2,3-triazole. A 1 ,3-dipolar cycloaddition may be also referred to as “Huisgen cycloaddition”. [0087] The term “spacer unit” as used in the context of the present invention in general means any chemical group which is suitable to provide a distance (or spacing) between the groups to which the spacer unit is bound. Illustrative spacer units, which are suitable for use as spacer units as described herein, such as e.g. a first spacer unit A and/or a second spacer unit A’, may be selected from the group consisting of -(Ci-Cw)alkylene-C(O)-, -(C₃-C₈)carbocyclo-C(O)-, - arylene-C(O)-, -(Ci-Cw)alkylene-arylene-C(O)-, -arylene-(Ci-Cw)alkylene-C(O)-, -(Ci- Cio)alkylene-(C3-C₈)carbocyclo-C(0)-, -(C3-C₈)carbocyclo-(Ci-Ci₀)alkylene-C(O)-, -(C₃- C₈)heterocyclo-C(O)-, -(Ci-Cio)alkylene-(C₃-C₈)heterocyclo-C(0)-, and -(C₃-C₈)heterocyclo-(Ci- Cio)alkylene-C(O)-. Illustrative spacer units, which are suitable for use as spacer units as described herein, such as e.g. a first spacer unit A and/or a second spacer unit A’, may be also selected from the group consisting of -(Ci-Cio)alkylene-, -(C₃-C₈)carbocyclo-, -arylene-, -(Cr C )alkylene-arylene-, -arylene-(C Cio)alkylene-, -(Ci-Ci₀)alkylene-(C₃-C₈)carbocyclo-, -(C₃- C₈)carbocyclo-(Ci-Cio)alkylene-, -(C₃-C₈)heterocyclo-, -(Ci-Ci₀)alkylene-(C₃-C₈)heterocyclo-, and -(C₃-C₈)heterocyclo-(Ci-Cio)alkylene-.

[0088] In one preferred embodiment, the first spacer unit A is -(CH₂)_d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3. It is also preferred for the present invention, that the second spacer unit A’ is -(CH₂)d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

[0089] It is preferred for the methods of the present invention, more preferably for the methods of forming a hydrogel, that the method is carried out in the absence of at least one catalyst(s), preferably in the absence of copper or a copper catalyst. In particular, a click chemistry reaction or a 1 ,3-dipolar cycloaddition may be carried out in the absence of a catalyst, in particular, in the absence of copper or a copper catalyst. In particular, any 1 ,3-dipolar cycloaddition described herein may be carried out in the absence of a catalyst, in particular in the absence of copper or a copper catalyst.

[0090] In one further embodiment, the method of the present invention is carried out in presence of at least one catalyst. In particular, a click chemistry reaction or a 1 ,3-dipolar cycloaddition may be carried out in presence of a catalyst. In particular, any 1 ,3-dipolar cycloaddition described herein may be carried out in presence of a catalyst. It is preferred for this embodiment that the catalyst is a copper catalyst. A suitable copper catalyst for 1 ,3-dipolar cycloadditions, in particular for a 1 ,3-dipolar cycloaddition of an azide with an alkyne, may be, e.g., a copper(l) catalyst, such as e.g. CuCI or CuBr.

[0091] It is preferred for the methods of the present invention in one embodiment that: X is selected from the group consisting of -NH-(CH₂)_r-N₃, -NH-(CH₂CH₂O)_S-CH₂CH₂N₃ and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

Y is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M’ = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Z is selected from the group consisting of -C(O)-(CH₂)_r-N₃, -C(O)-(CH₂CH₂O)s-CH₂CH2-N3 and -C(O)-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15.

More preferably, X is selected from the group consisting of -NH-(CH₂)_r-N₃, -NH-(CH₂CH₂O)_S-CH₂CH₂N₃ and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

Y is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M’ = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; and

Z is selected from the group consisting of -C(O)-(CH₂)_r-N₃, -C(O)-(CH₂CH₂O)_S-CH₂CH₂-N₃ and

-C(O)-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15.

In these embodiments, when Q comprised in the group a 1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y can be a thermoinduced 1 ,3-dipolar cycloaddition, and a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with the group(s) Z can be a thermo-induced 1 ,3-dipolar cycloaddition. Accordingly, in these embodiments, the method can be carried out in the absence of a catalyst, in particular in the absence of copper or a copper catalyst.

[0092] In another embodiment of the methods of the present invention, it is preferred that:

X is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH2O)s-CH₂CH2Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Y is selected from the group consisting of -NH-(CH₂)_r-N₃, -NH-(CH₂CH₂O)_S-CH₂CH₂N3 and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and Z is selected from the group consisting of -C(O)-(CH₂)_r-Q, -C(O)-(CH₂CH₂O)_S-CH₂CH₂Q and -CO-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Q is - ^C=^CH

More preferably, X is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)s-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; and

Y is selected from the group consisting of -NH-(CH₂)_r-N₃, -NH-(CH₂CH₂O)_S-CH₂CH₂N₃ and -NH-(CH₂-CH₂-C(O))_rCH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and

Z is selected from the group consisting of -C(O)-(CH₂)_r-Q, -C(O)-(CH₂CH₂O)_S-CH₂CH₂Q and -CO-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or Q is - ^C=^CH .

In these embodiments, when the Q comprised in the group Z is or

1 ,3-dipolar cycloaddition between the group Z and a part of the group(s) Y can be a thermoinduced 1,3-dipolar cycloaddition, and a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with the group(s) Z can be a thermo-induced 1 ,3-dipolar cycloaddition. Accordingly, in these embodiments, the method can be carried out in the absence of a catalyst, in particular in the absence of copper or a copper catalyst.

In these embodiments, when the Q comprised in the group Z is C^CH _anc| Q comprised in the group -dipolar cycloaddition between the group Z and a part of the group(s) Y can be carried out in the presence of a catalyst, in particular copper or a copper catalyst, and a 1,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with the group(s) Z can be a thermo-induced 1 ,3-dipolar cycloaddition.

[0093] It is preferred for the method of the present invention that the first polysaccharide and/or the second polysaccharide is/are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof. It is more preferred for this embodiment that the first polysaccharide and/or the second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and mixtures thereof. It is even more preferred that the first polysaccharide and the second polysaccharide are pullulan. [0094] The first and/ or second polysaccharide of the method of the present invention may be optionally carboxymethylated, wherein at least one OH-group of the first and/ or second polysaccharide as defined herein above may be carboxymethylated. In one embodiment of the method of the present invention, the carboxymethylation is preferably carried out, when the first and/ or second polysaccharide is/ are pullulan or dextran. In one embodiment of the present invention, the first and/ or second polysaccharide is dextran or pullulan and carboxymethylation of at least one OH-group of dextran or pullulan is carried out according to step c) or step d) of the method of the present invention as described herein.

[0095] It is further preferred for the method of the present invention that in step f) the at least one group Z of the at least one cofactor is reacted in a first 1 ,3-dipolar cycloaddition with a part of the group(s) Y of the functionalized second polysaccharide. It is also preferred for this embodiment that in step g) the part of the group(s) Y not reacted in step f) of the product of step f) is reacted in a second 1 ,3-dipolar cycloaddition with the group X of the functionalized first polysaccharide.

[0096] Functionalization of the first polysaccharide as used in the context of the present invention is with at least one linker unit(s) of the structure -A-X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

X is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group Y.

[0097] Functionalization of the second polysaccharide as used in the context of the present invention is with one or more linker unit(s) of the structure -A'-Y, when the monomeric repeating unit of the second polysaccharide not comprises a carboxylic acid residue, or functionalization of the second polysaccharide with one or more group(s) of the structure -Y, when the monomeric repeating unit of the second polysaccharide comprises a carboxylic acid residue, wherein A' is a second spacer unit; and

Y is a group comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with the group X and/or the group Z.

[0098]°ln one embodiment, in step f) and/or step g), a 1 ,3-dipolar cycloaddition between Z and a part of the group(s) Y is a thermo-induced 1 ,3-dipolar cycloaddition, and a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z is a thermoinduced 1,3-dipolar cycloaddition. In these embodiments, attachment of the cofactor to the second polysaccharide and cross-linking of the first polysaccharide and the second polysaccharide to form a hydrogel can be carried out using thermo-induced 1 ,3-dipolar cycloadditions. Accordingly, in these embodiments, the method can be carried out in the absence of a catalyst, in particular in the absence of copper or a copper catalyst.

[0099] In one embodiment, in step f) and/or step g), a 1 ,3-dipolar cycloaddition between Z and a part of the group(s) Y is a catalyst-induced 1 ,3-dipolar cycloaddition, and a 1 ,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z is a thermoinduced 1,3-dipolar cycloaddition. In these embodiments, attachment of the cofactor to the second polysaccharide can be carried out using a catalyst-induced 1 ,3-dipolar cycloaddition, and cross-linking of the first polysaccharide and the second polysaccharide to form a hydrogel can be carried out using a thermo-induced 1,3-dipolar cycloaddition. The catalyst-induced 1 ,3- dipolar cycloaddition can be carried out in presence of copper, in particular a copper catalyst.

[0100] In one embodiment, the content of N₃, when present, is 0.01-1.5 N₃ per monomeric repeating unit of the first polysaccharide.

[0101] In one embodiment of the present invention, the content of Q, when present, is 0.01-1.5 per monomeric repeating unit of the first and/ or the second polysaccharide.

[0102] As used in the present invention, Q may be used elsewherein herein, this does not mean for the latter mentioned structural formula that both M have to be the same (each H or each Me). Rather, the present invention also comprises that, in one embodiment both M can be H, in one embodiment both M can be Me, and in one embodiment one M can be H and the other M can be Me (independent from the position of M, two possibilities for one M being H and the other M being Me). Thus, in the context of the present invention, the two structural formulas can be used interchangeably herein.

[0103] As used herein and in the context of the present invention, the term “functionalization” or “functionalized” means in general the addition of specific functional groups to afford the compound new, desirable properties, e.g. in the present invention, the addition of a linker unit or linker units as defined above to the existent polysaccharide structure.

[0104] In one embodiment, in step d) of the method of the present invention (enzyme optional) or in step e) of the method of the present invention (enzyme present), -A-X is linked to at least one primary or secondary OH-group of the first polysaccharide, preferably via at least one of C₂, C₃, C₄ or C₆ of the monomeric repeating unit(s) of the first polysaccharide, more preferably via C₆ of the monomeric repeating unit(s) of the first polysaccharide.

[0105] In one embodiment of the present invention, the method of the present invention is without the use of toxic reagents, preferably without the use of glutaraldehyde.

[0106] It is preferred for the method of the present invention that the molecular weight of the unfunctionalized first polysaccharide is in the range from 5 to 2000 kDa. It is also preferred for the methods of the present invention that the molecular weight of the unfunctionalized second polysaccharide is in the range from 5 to 2000 kDa.

[0107] This method of preparing a biocompatible hydrogel according to the present invention may comprise with step f) and/or step g) a step comprising thermo-induced gelation, which e.g. allows formation of specific form factors, e.g. by complete bubble-free filling of a suitably formed mould or by dropping a mixture of the two non-viscous solutions of the individual components into a lipophilic organic medium to form droplets of defined diameter. Formation of a covalent, non-degradable network of a biocompatible hydrogel can be induced by heating to mild temperatures as described above, which is compatible with maintaining enzymatic activity, when an enzyme is encapsulated therein. The method may comprise crosslinking via 1,3- dipolar cycloaddition, thermo-gelation under very mild reaction conditions (e.g. 40°C in aqueous media) with no side reaction and no toxic reagent (e.g. glutaraldehyde). Optimization of pore sizes of the biocompatible hydrogel is possible by feasible adjustment of parameters and adjusting the degree of optional carboxymethylation. If an enzyme is added as described herein, the one or more enzyme(s) will be immobilized in the produced biocompatible hydrogel, wherein the one or more enzyme(s) is/ are then significantly longer stable and active than the free enzyme in solution at ambient or elevated temperature, e.g. body temperature, 37°C. Unstable sensitive enzymes, like glucose-6-phosphate-dehydrogenase or glucose-dehydrogenase, have better life time performances under these conditions. This embodiment is also applicable to other sensitive enzymes. Such a produced biocompatible hydrogel can be stored dry without losing higher amounts of enzyme activity. Further, no or little leaching of enzyme can be achieved. Such hydrogels prepared according to this method of the present invention can be suspended in aqueous or organic solvents, while maintaining enzymatic activity (e.g. acetone). The viscosity of the individual components, as well as of the mixture can be easily adjusted.

[0108] Consequently, the stability of enzymes can be significantly improved by the methods described herein, resulting in longer usable enzymes.

[0109] In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof. In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan and mixtures thereof. In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan and mixtures thereof. In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and mixtures thereof. The polysaccharides pullulan, alginate, hyaluronan, dextran are defined herein above.

[0110] Cellulose is an organic compound with the formula (C₆H₁₀O5)n, a polysaccharide consisting of a linear chain of several hundred to many thousands of |3(1— >4)-linked D-glucose units. Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to form biofilms. In the context of the present invention, the respective monomeric repeating unit for cellulose has the structure of repeating unit of cellulose, and with n being an integer from 10 to 10000.

[0111] Lichenin, also known as lichenan or moss starch, is a complex glucan occurring in certain species of lichens. It is chemically a mixed-linkage glycan, consisting of repeating glucose units linked by P-1 ,3- and p-1 ,4-glycosidic bonds. In the context of the present invention, the respective monomeric repeating unit for lichenin has the structure of being the number of said monomeric repeating unit of lichenin, and with n being an integer from 10 to 10000.

[0112] Lentinan is a polysaccharide isolated from the fruit body of the shiitake mushroom.

Chemically, lentinan is a P-1 ,3 beta-glucan with P-1 ,6 branching. In the context of the present invention, the respective monomeric repeating unit for lentinan has the structure of with n being the number of said monomeric repeating unit of lentinan, and with n being an integer from 10 to 10000.

[0113] In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin and lentinan. In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin and lentinan. In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and lentinan. In a further embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide and the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan and dextran. The respective monomeric repeating unit for each of these polysaccharides is as defined above herein.

[0114] In one embodiment of the method of preparing a biocompatible hydrogel, the first and/ or second polysaccharide is dextran or pullulan and carboxymethylation of at least one OH-group of dextran or pullulan is carried out in the method of the present invention. It is preferred for this embodiment that the carboxymethylation of at least one OH-group of dextran or pullulan is carried out at C₅ of dextran and/ or pullulan. It is preferred for this embodiment that the carboxymethylation of at least one OH-group of dextran or pullulan is carried out at C₂ of dextran and/ or pullulan. It is preferred for this embodiment that the carboxymethylation of at least one OH-group of dextran or pullulan is carried out at C₃ of dextran and/ or pullulan. It is preferred for this embodiment that the carboxymethylation of at least one OH-group of dextran or pullulan is carried out at C₄ of dextran and/ or pullulan.

[0115] In one embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide is functionalized with 0.01-1.5 of -A-X or -X per monomeric repeating unit of the first polysaccharide. In a further preferred embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide is functionalized with 0.05-1.5 of -A-X or -X per monomeric repeating unit of the first polysaccharide. In a further preferred embodiment of the method of preparing a biocompatible hydrogel, the first polysaccharide is functionalized with 0.05-1.0 of -A-X or -X per monomeric repeating unit of the first polysaccharide.

[0116] For the method of preparing a biocompatible hydrogel according to the present invention, it is preferred that the second polysaccharide is functionalized with 0.01-1.5 of -A'-Y or -Y per monomeric repeating unit of the second polysaccharide. In a further preferred embodiment of the method of preparing a biocompatible hydrogel according to the present invention, the second polysaccharide is functionalized with 0.05-1.5 of -A'-Y or -Y per monomeric repeating unit of the second polysaccharide. In a further preferred embodiment of the method of preparing a biocompatible hydrogel according to the present invention, the second polysaccharide is functionalized with 0.05-1.0 of -A'-Y or -Y per monomeric repeating unit of the second polysaccharide.

[0117] The present invention further provides the hydrogel obtainable by any method of preparing a biocompatible hydrogel as described above. It is preferred that said hydrogel comprises one or more encapsulated enzyme(s). It is further preferred that the hydrogel is a swellable or swollen hydrogel matrix.

[0118] In a further aspect, the present invention provides a biocompatible hydrogel comprising, a crosslinked polymer comprising:

- at least one cofactor(s),

- a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating units of the first and/ or the second polysaccharide and with n being an integer from 10 to 10000,

- at least one first linker unit(s), which link(s) the first polysaccharide with the second polysaccharide, wherein the structure of the at least one first linker unit(s) is -A-Gi-A’-, -A- G-i— , — A — Gi- , or— Gi- , wherein A is a first spacer unit;

Gi is a first moiety being or comprising a group obtainable or being obtained from a 1 ,3- dipolar cycloaddition; and

A’ is the second spacer unit; and

[0119] In a further aspect, the present invention provides a biocompatible hydrogel comprising, a crosslinked polymer comprising:

- at least one cofactor(s),

- a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating units of the first and/ or the second polysaccharide and with n being an integer from 10 to 10000, - at least one first linker unit(s), which link(s) the first polysaccharide with the second polysaccharide, wherein the structure of the at least one first linker unit(s) is -A-Gi-A’-, -A- G-i— , — A — Gi- , or— Gi- , wherein A is a first spacer unit;

Gi is a first moiety being or comprising a group obtainable or being obtained from a click chemistry reaction; and

A’ is the second spacer unit; and

G₂ is a second moiety being or comprising a group obtainable or being obtained from a click chemistry reaction.

[0120] In one preferred embodiment of the hydrogel according to the present invention, Gi comprises or consists of the structure -D|-B-D₂-, wherein:

D-i is independently selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

D₂ is independently selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and wherein R' is independently selected from the group consisting of -H, -CF₃, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH₂ and -C(O)-NHMe.

[0121] In one embodiment, A and A' are each independently -(CH₂)_d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3. [0122] It is further preferred for the hydrogel according to the present invention, that G₂ comprises or consists of the structure -D₃-B-, wherein:

D₃ is selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂-, -NH-(CH₂-

CH₂)_S-NH-CO- and -NH-(CH₂-CH₂-C(O))t-CH2-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and wherein R' is independently selected from the group consisting of -CF₃, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH₂ and -C(O)-NHMe.

[0123] The above given definitions for the method of preparing a biocompatible hydrogel also apply to the biocompatible hydrogel, if the same terms are used.

[0124] In one embodiment of the biocompatible hydrogel according to the present invention, the hydrogel is a swellable or swollen hydrogel matrix.

[0125] In one embodiment of the biocompatible hydrogel according to the present invention, the at least one enzyme(s) is/are non-covalently bound in the hydrogel to the at least one cofactor(s). It is also preferred for the biocompatible hydrogel according to the present invention that the at least one enzyme(s) is encapsulated in the hydrogel and that the at least one cofactor(s) is/are covalently bound to the hydrogel. As used herein and in the context of the present invention, the term “non-covalent” means an interaction, which differs from a covalent bond in that it does not involve the sharing of a bond, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule like dipol- dipol or charge-charge interactions. The chemical energy released in the formation of non- covalent interactions is typically in the order of 1-5 kcal/mol. Non-covalent interactions can be classified into different categories, such as electrostatic, TT-effects, van der Waals forces, and hydrophobic effects. Non-covalent interactions are critical in maintaining the three-dimensional structure of large molecules, such as proteins and nucleic acids. In addition, they are also involved in many biological processes in which large molecules bind specifically, but transiently, to one another.

[0126] In one embodiment of the biocompatible hydrogel according to the present invention, the first and/ or second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

[0127] In one further embodiment of the biocompatible hydrogel according to the present invention, the first and/ or second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan and mixtures thereof. In one further embodiment of the biocompatible hydrogel according to the present invention, the first and/ or second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lentinan and mixtures thereof. In one further embodiment of the biocompatible hydrogel according to the present invention, the first and/ or second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and mixtures thereof.

[0128] In one embodiment of the biocompatible hydrogel according to the present invention, the first and/ or second polysaccharide is/ are pullulan.

[0129] In one further preferred embodiment of the biocompatible hydrogel according to the present invention, — A-D-i- comprises or consists of the formula -CH₂-CO-NH-(CH₂-CH₂-O)_n- CH₂-CH₂-, wherein n is preferably from 1 to 5; and/or wherein -A’-D₂- comprises or consists of the formula -CH₂-CO-NH-(CH₂-CH₂-O)_n-CH₂-CH₂-, wherein n is preferably from 1 to 5.

[0130] In one embodiment of the biocompatible hydrogel according to the present invention, the first and/ or the second polysaccharide has/ have a concentration of 5-120 mg/ml, preferably 10-80 mg/ml, more preferably 20-60 mg/ml, with respect to the total hydrogel.

[0131] In one embodiment of the biocompatible hydrogel according to the present invention, the at least one enzyme(s) for non-covalent immobilization is a dehydrogenase. In one embodiment of the biocompatible hydrogel according to the present invention, the at least one enzyme(s) for non-covalent immobilization is an enzyme with EC-number 1.1.1. Those are enzymes which use NAD(+) or NADP(+) as acceptor. In one preferred embodiment of the biocompatible hydrogel according to the present invention, the at least one enzyme(s) is/ are selected from the group consisting of glucose-dehydrogenase, glucose 1 -dehydrogenase, glucose-6-phosphat- dehydrogenase, lactate dehydrogenase, glycerine-aldehyde-3-phosphate-dehydrogenase, indole-3-acetaldehyde reductase, xanthine dehydrogenase, menthol dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, gluconate 5-dehydrogenase, xylulose reductase and sorbitol dehydrogenase. In one more preferred embodiment of the biocompatible hydrogel according to the present invention, the at least one enzyme(s) is/ are glucose-6-phosphate- dehydrogenase (G6PD) or glucose-dehydrogenase (GDH).

[0132] The present invention further provides a cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a 1,3-dipolar cycloaddition. In one embodiment, the cofactor comprises one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition. The present invention also provides a cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a click chemistry reaction. In one embodiment, the cofactor comprises one group Z comprising or being a functional group suitable for a click chemistry reaction. The term “comprises one group Z” means that the cofactor comprises exactly one group Z.

[0133] It is preferred for the cofactor of the present invention that the cofactor is selected from the group consisting of vitamin C, glutathione, coenzyme A, thiamine pyrophosphate, AMP, ADP, ATP, S-adenosyl methionine, biotin, pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺).

[0134] Those cofactor(s) are presented by the following formulas:

Thiamine pyrophosphate

AMP, but also ADP and ATP

S-Adenosyl methionine

Pyridoxal phosphate (PLP) Nicotinamide adenine dinucleotide (NAD⁺)

Nicotinamide adenine dinucleotide phosphate (NADP⁺).

[0135] It is preferred for the cofactor of the present invention that the at least one group(s) Z is/are selected from the group consisting of -C(O)-(CH₂)rQ, -C(O)-(CH2CH2O)s-CH₂CH2Q and -CO-(CH2-CH2-C(O))t-CH2-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Z is selected from the group consisting of -C(O)-(CH₂)_r-N₃, -C(O)-(CH₂CH₂O)_S-CH₂CH₂-N₃ and -C(O)-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15.

[0136] The group Z may be attached to the cofactor via a suitable atom or a suitable functional group of the cofactor. Suitable atoms or functional groups are generally known in the art. Illustrative examples may be, e.g., C, NH (derived, e.g., from an NH₂ group), and O (derived, e.g., from an OH group).

[0137] The present invention further provides a composition comprising the biocompatible hydrogel according to the present invention and as described herein.

[0138] In a further aspect, the present invention provides the use of a hydrogel according to the present invention and as defined herein, a) for non-covalent immobilization of one or more enzyme(s) in the hydrogel, or b) as a catalyst, or c) in a biosensor. As used herein, the term “biosensor” means, unlike biotic sensors or biotests, a self-contained integrated system that provides specific quantitative or semi-quantitative analytical information, consisting of biological recognition element (biochemical receptor or enzyme) and transducers (e.g. an electrode) in direct spatial contact.

[0139] The present invention also provides a kit comprising the composition or the hydrogel according to the present invention and as defined herein.

* * * * *

[0140] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. [0141] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0142] The term "and/ or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0143] The term “less than’’ or in turn “more than” does not include the concrete number.

[0144] For example, “less than 20” means less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. “more than 80 %” means more than or greater than the indicated number of 80 %.

[0145] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including” or sometimes, when used herein, with the term “having”. When used herein, “consisting of" excludes any element, step, or ingredient not specified.

[0146] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0147] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0148] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0149] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0150] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way. EXAMPLES

[0151] Materials and methods:

[0152] Synthesis of oxanorbornadiene linker:

(92 %) furane - ► microwave, 60 °C, 60 min H₂O/THF (7:1 ), rt, 2 d teps ,

Scheme 1 : Synthesis of oxanorbornadiene linker NH₂-(CH₂)_r-Q (with r = 2/3/6, Q = Q-1 (CF₃), M = H or Me and M' = H or Me) as a TFA salt.

[0153] Ethyl 4,4,4-trifluoro-3-oxo-2-(triphenyl-A,⁵-phosphaneylidene)-butanoate (Compound 1)

At 0 °C, (2-ethoxy-2-oxoethyl)triphenylphosphonium bromide (100 g, 0.233 mol) was dissolved in dry THF (400 mL) under argon atmosphere. Triethylamine (65 ml_, 0.47 mol) was slowly added to the reaction mixture over 20 min. Trifluoroacetic anhydride (36 mL, 0.26 mol) was then added dropwise. The reaction was stirred for 24 h while slowly warming to rt. The precipitate was filtered off and washed with cold THF. The solvent of the filtrate was removed on the rotary evaporator and H₂O/THF (3:1, 400 mL) was added to the residue. The precipitated pale yellow solid was then filtered off, dried in vacuo at 40 °C, and recrystallized several times from methanol/H₂O. The product (90.9 g, 0.205 mol, 88 %) was obtained as a crystalline solid.

HR-MS: m/z calculated for C₂₄H₂₀F₃O3PNa⁺ [M+Na]⁺: 467.1000, found (ESI-MS⁺): 467.0997. ¹H-NMR (600 MHz, DMSO): 5 = 7.73-7.59 (m, 15H, CH_(Ph)), 3.63 (q, 2H, CHzCHs, J= 7.1 Hz), 0.69 (t, 3H, CH₂CHS, J= 7.1 Hz) ppm. ¹³C{¹H}-NMR (151 MHz, DMSO) 5 = 172.6 (qd, J = 55.2, 6.4 Hz, COCF₃), 164.5 (d, J = 12.1 Hz, CO₂Et), 132.8 (d, J = 10.1 Hz, CH_(Ph)), 132.7 (d, J = 2.9 Hz, CH_(Ph)), 129.2 (d, J = 12.6 Hz, CH_(Ph)), 123.4 (d, J = 93.2 Hz, C_q(Ph)), 115.8 (qd, J = 14.9, 483.8 Hz, CF₃), 69.3 (d, J = 109.7 Hz, C=P), 59.2 (s, CH₂CH₃), 13.2 (s, CH₂CH₃) ppm.

[0154] Ethyl 4,4,4-trifluorobut-2-ynoate (Compound 2)

Ethyl 4,4,4-trifluoro-3-oxo-2-(triphenyl-2 -phosphaneylidene)butanoate (10 g, 23 mmol) distilled over a pyrolysis apparatus for 4-5 h at 220 °C and 100-160 mbar. The product (3.30 g, 19.9 mmol, 89 %) was obtained as a clear colorless liquid.

¹H-NMR (400 MHz, CDCI₃): 5 = 4.32 (q, J = 7.1 Hz, 2H, CH₂), 1.34 (t, J = 7.2 Hz, 3H, CH₃) ppm. ¹³C{¹H}-NMR (101 MHz, CDCI₃) 5 = 151.0 (q, J = 1.7 Hz, C=O), 113.6 (q, J = 260.1 Hz, CF₃), 75.8 (q, J = 6.5 Hz, CCF₃), 70.1 (q, J = 54.8 Hz, CC=O), 63.7 (s, CH₂CH₃), 14.0 (s, CH₂CH₃) ppm.

[0155] Ethyl 1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.11hepta-2,5-diene-2- carboxylate (Compound 3)

Ethyl 4,4,4-trifluorobut-2-ynoate (3.30 g, 19.9 mmol) was mixed with 2,5-dimethylfuran (3.15 ml_, 29.8 mmol) and heated in a microwave reactor to 60 °C for 60 min. The reaction mixture was concentrated by coevaporation with toluene (3x). The product was obtained as a brown oily residue (2.91 g, 0.11 mol, 56 %).

HR-MS: m/z calculated for Ci₂H₁₃F₃O₃Na⁺: 285.0714 [M+Na]⁺, found (ESI-MS⁺): 285.0718.

¹H-NMR (400 MHz, DMSO): 5 = 7.15 (d, J = 5.0 Hz, 1 H, HC=CH), 7.13 (d, J = 5.1 Hz, 1 H, HC=CH), 4.34-4.19 (m, 2H, CHzCHj), 1.72 (s, 3H, CH₃), 1.64 (s, 3H, CH₃), 1.23 (t, J = 7.1 Hz, 3H, CHzCH^) ppm.

¹³C{¹H]-NMR (101 MHz, DMSO) 8 = 163.1 (q, J = 1.5 Hz, C=O), 155.9 (q, J = 4.9 Hz, CCF₃),

147.5 (q, J = 34.2 Hz, CC=O), 147.5 (d, J = 0.7 Hz, CCH₃), 146.9 (s, CCH₃), 122.0 (q, J =

269.5 Hz, CF₃), 92.3 (d, J = 0.5 Hz, HC=CH), 91.0 (q, J = 1.4 Hz, HC=CH), 61.6 (s, CH₂CH₃), 14.7 (d, J = 1.0 Hz, CH₂CH₃), 14.4 (s, CH₃), 13.7 (s, CH₃).

[0156] 3-(Trifluoromethyl)-7-oxabicvclor2.2.1lhepta-2,5-diene-2-carboxylic acid (Compound 4)

In a round bottom flask, ethyl 4,4,4-trifluoro-3-oxo-2-(triphenyl-X⁵-phosphaneylidene)butanoate (38.8 g, 87.4 mmol) was distilled over a pyrolysis apparatus for 4-5 h at 210 °C and 100-160 mbar. Furan (38.0 mL, 52.4 mmol) was then added to the colorless distillate (ethyl 4,4,4- trifluorobut-2-ynoate) and reacted in a microwave reactor at 60 °C for 60 min. The reaction mixture was concentrated by co-evaporation with toluene (3x) under reduced pressure. The ester could be obtained as an oily residue and was taken up without further purification in H₂O/THF (7:1, 100 mL), mixed with 1 M aq. LiOH solution (100 ml_, 100 mM) and stirred at rt for three days. The aqueous phase was then adjusted to pH 1-2 with 2 M HCI and extracted with diethyl ether (3x). The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The oily residue was dissolved in dichloromethane and petroleum ether was added. The precipitated solid was filtered off and washed with petroleum ether. This afforded the product (6.16 g, 29.9 mmol, 35 % over three steps) as a crystalline solid.

HR-MS: m/z calculated for C₈H₄F₃O₃’ [M-H]’: 205.0113, found (ESI-MS’): 205.0108.

¹H-NMR (600 MHz, DMSO): 8 = 13.75 (bs, 1H, COOH), 7.38 (dd, J = 5.3 Hz, 2.0 Hz, 1H, HC=CH), 7.34 (dd, J = 5.3 Hz, 2.0 Hz, 1 H, HC=CH), 5.86 (t, J = 1.7 Hz, 1 H, HC-O), 5.73 (d, J = 1.3 Hz, 1 H, HC-O) ppm.

¹³C{¹H}-NMR (151 MHz, DMSO) 8 = 163.2 (q, = 1.5 Hz, COOH), 154.3 (q, J = 4.9 Hz, CCF₃), 147.4 (q, J = 36.3 Hz, CC=O), 143.9 (s, HC-O), 143.1 (s, HC-O), 122.0 (q, J = 268.8 Hz, CF₃), 84.8 (s, HC=CH), 83.0 (q, = 2.5 Hz, HC=CH) ppm.

[0157] 1,4-dimethyl-3-(trifluoromethyl)-7-oxabicvclof2.2.11hepta-2,5-diene-2-carboxylic acid (Compound 5)

Ethyl 1,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylate (191 mg, 0.73 mmol) was taken up in H₂O/THF (7:1, 2 mL), mixed with 1 M aq. LiOH solution (2 mL, 2 mM) and stirred at rt for 1.5 days. The aqueous phase was then brought to pH 1-2 with 2 M HCI and extracted with diethyl ether (3x). The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product (127 mg, 0.54 mmol, 75 %) was obtained as a brown oil.

HR-MS: m/z calculated for CI₀H₈F₃O₃ [M-H]’: 233.0426, found (ESI-MS’): 233.0422.

¹H-NMR (400 MHz, DMSO): 8 = 13.93 (bs, 1 H, COOH), 7.12 (d, J = 5.1 Hz, 1 H, HC=CH), 7.10 (d, J = 5.1 Hz, 1 H, HC=CH), 1.71 (s, 3H, CH₃), 1.63 (s, 3H, CH₃) ppm.

¹³C{¹H}-NMR (101 MHz, DMSO) 8 = 164.9 (q, = 1.4 Hz, COOH), 157.1 (q, J = 4.9 Hz, CCF₃), 147.4 (s, HC-O), 146.8 (s, HC-O), 145.1 (q, = 34.0 Hz, CC=O), 122.3 (q, J = 269.2 Hz, CF₃), 92.2 (d, J = 0.6 Hz, HC=CH), 90.7 (q, = 1.4 Hz, HC=CH), 14.82 (s, CH₃), 14.5 (s, CH₃) ppm.

[0158] Tert-butyl (2-(3-(trifluoromethyl)-7-oxabicyclor2.2.11hepta-2,5-diene-2- carboxamido)ethyl)carbamate (Compound 6)

3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (1.18 g, 5.73 mmol) was dissolved in dry dichloromethane (16 mL) under argon atmosphere. After stepwise addition of 4-DMAP (1.4 g, 11 mmol), EDC-HCI (1.6 g, 8.6 mmol), and Boc-ethylenediamine (1.1 mL, 6.9 mmol), the mixture was stirred for 22 h at rt. The reaction mixture was washed with sat. aq. NaCI solution, the organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure. After the crude product was purified by column chromatography (eluent: PE/EtOAc 1 :1), the product was obtained as a white solid (1.01 g, 2.91 mmol, 51 %).

R_f: (PE/EtOAc: 1/1): 0.45.

HR-MS: m/z calculated for Ci₅Hi₉F₃N₂O₄Na⁺: 371.1195 [M+Na]⁺, found (ESI-MS⁺): 371.1189.

¹H-NMR (500 MHz, DMSO): 8 = 8.46 (t, = 5.5 Hz, 1 H, NH-C=O), 7.33 (dd, = 5.3, 2.0 Hz, 1H, HC=CH), 7.28 (dd, = 5.3, 2 Hz, 1 H, HC=CH), 6.83 (t, J =5.7 Hz, 1 H, NH-Boc), 5.79 (t, 1 H, HC- O), 5.67 (d, J = 1.0 Hz, 1 H, HC-O), 3.18-3.15 (m, 2H, CHg-NH), 3.04-3.00 (m, 2H, CH₂-Boc), 1.38 (s, 9H, Boc) ppm.

¹³C{¹H}-NMR (126 MHz, DMSO): 8 = 161.9 (q, J = 1.2 Hz, CONH), 155.8 (q, J = 5,1 Hz, CCF₃), 155.6 (s, NC=O), 143.6 (d, J = 0.7 Hz, HC=CH), 142.7 (s, HC=CH), 141.7 (q, J = 35.7 Hz, CC=O), 122.4 (q, J = 268.2 Hz, CF₃), 85.1 (s, HC-O), 82.4 (q, J = 2.1 Hz, HC-O), 77.7 (s, Cq(Boc)) , 39.1 (s, NHCH₂), 38.9 (s, NHCH₂), 28.2 (s, CH_3(Boc)).

[0159] Tert-butyl (2-(1 ,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclor2.2.1]hepta-2,5-diene-2- carboxamido)ethyl)carbamate (Compound 7)

1 ,4-Dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (0.68 g, 2.90 mmol) was dissolved in dry dichloromethane (12 ml_) under Ar atmosphere. After stepwise addition of 4-DMAP (0.71 g, 5.8 mmol), EDC-HCI (0.83 g, 4.3 mmol), and Boc-ethylenediamine (0.55 mL, 3.5 mmol), the mixture was stirred for 20 h at rt. The reaction mixture was washed with sat. aq. NaCI solution, the organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure. After the crude product was purified by column chromatography (eluent: PE/EtOAc 3:1 ; 1 :1), the product was obtained as oil (0.23 g, 0.60 mmol, 21%).

R_f: (PE/EtOAc: 1/1): 0.63.

HR-MS: m/z calculated for Ci₇H₂₃F₃N₂O₄Na⁺: 399.1508 [M+Na]⁺, found (ESI-MS⁺): 399.1502.

¹H-NMR (400 MHz, DMSO): 8 = 8.56 (t, J = 5.4 Hz, 1 H, NH-CO), 7.06 (d, J = 5.0 Hz, 1H, HC=CH), 7.03 (d, J = 5.0 Hz, 1 H, HC=CH), 6.77 (t, J = 5.5 Hz, 1 H, NH-Boc), 3.27-3.08 (m, 2H, CH2-NH), 3.05-2.93 (m, 2H, CHg-NHBoc), 1.70 (s, 3H, CH₃), 1.55 (s, 3H, CH₃), 1.37 (s, 9H, Boc) ppm.

¹³C{¹H}-NMR (101 MHz, DMSO): 8 = 163.0 (s, CONH), 155.7 (q, J = 5.0 Hz, CCF₃), 155.5 (s, NC=O), 147.3 (s, HC=CH), 146.2 (s, HC=CH), 140.9 (q, = 33.2 Hz, CC=O), 122.6 (q, J = 268.9 Hz, CF₃), 92.3 (s, HC-O), 90.4 (q, J = 1.3 Hz, HC-O), 77.7 (s, C_q(Boc)), 39.2 (s, NHCH₂), 38.5 (s, NHCH₂), 28.2 (s, CH_3(Boc)), 14.9 (s, CH₃), 14.1 (s, CH₃) ppm. [0160] Tert-butyl (3-(3-(trifluoromethyl)-7-oxabicyclor2.2.11hepta-2,5-diene-2- carboxamido)propyl)-X²-azanecarboxylate (Compound 8)

3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (512 mg, 2.49 mmol) was dissolved in dry dichloromethane (6 mL) under Ar atmosphere. After stepwise addition of 4- DMAP (607 mg, 4.97 mmol), EDC-HCI (715 mg, 3.73 mmol), and A/-Boc-1 ,3 diaminopropane (0.52 mL, 3.0 mmol), the mixture was stirred for 22 h at rt. The reaction mixture was washed with sat. aq. NaCI solution, the organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure. After the crude product was purified by column chromatography (eluent: PE/EtOAc 1 :1), the product was obtained as oil (528 mg, 1.46 mmol, 55 %).

R_f: (PE/EtOAc: 3/2): 0.29.

HR-MS: m/z calculated for Ci₆H₂iF₃N₂O₄Na⁺: 385.1351 [M+Na]⁺, found (ESI-MS⁺): 385.1360.

¹H-NMR (400 MHz, DMSO): 8 = 8.43 (t, J = 5.6 Hz, 1H, C=ONH), 7.32 (dd, J = 5.3, 2.0 Hz, 1 H, HC=CH), 7.28 (dd, J = 5.3, 2.0 Hz, 1 H, HC=CH), 6.77 (t, = 5.4 Hz, 1 H, NHBoc), 5.79 (t, J =

1.5 Hz, 1 H, HC-O), 5.65 (s, 1 H, HC-O), 3.17-3.14 (m, 1 H, CONCH₂), 3.13-3.07 (m, 1H, CONCH₂), 2.95-2.91 (m, 2H, CHgNHBoc), 1.57-1.52 (m, 2H, CCH₂C), 1.37 (s, 9H, Boc).

¹³C{¹H}-NMR (101 MHz, DMSO): 8 = 161.8 (s, CONH), 155.7 (q, J = 5.0 Hz, CCF₃), 155.6 (s, NC=O), 143.5 (s, HC=CH), 142.6 (s, HC=CH), 141.2 (q, J = 35.6 Hz, CC=O), 122.5 (q, J = 268.1 Hz, CF₃), 85.2 (s, HC-O), 82.4 (d, J = 2.0 Hz, HC-O), 77.5 (s, C_q(Boc)), 37.4 (s, NHCH₂),

36.5 (s, NHCH₂), 29.2 (s, CCH₂C), 28.2 (s, CH_3(Boc)).

[0161] Tert-butyl (6-(3-(trifluoromethyl)-7-oxabicyclor2.2.1lhepta-2,5-diene-2- carboxamido)hexyl)-X²-azanecarboxylate (Compound 9)

3-(Trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid (0.50 g, 2.4 mmol) was dissolved in dry dichloromethane (10 mL) under Ar atmosphere. After stepwise addition of 4- DMAP (597 mg, 4.88 mmol), EDC-HCI (702 mg, 3.66 mmol), and /V-Boc-1,6-diaminohexane (657 pL, 2.93 mmol), the mixture was stirred for 22 h at rt. The reaction mixture was washed with sat. aq. NaCI solution, the organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure. After the crude product was purified by column chromatography (eluent: PE/EtOAc 7:1 , 3:1 , 1 :1), the product was obtained as oil (610 mg, 1.51 mmol, 62 % yield).

R_f: (PE/EtOAc: 3:2): 0.34.

HR-MS: m/z calculated for Ci₉H₂₇F₃N₂O₄Na⁺: 427.1821 [M+Na]⁺, found (ESI-MS⁺): 427.1830.

¹H-NMR (600 MHz, DMSO): 8 = 8.47 (t, J = 5.6 Hz, CONH), 7.33 (dd, J = 5.3, 2.0 Hz, 1H, HC=CH), 7.28 (dd, J = 5.2, 1.9 Hz, 1 H, HC=CH), 6.75 (t, J = 5.3 Hz, 1 H, NHBoc), 5.79 (t, J =

1.5 Hz, 1H, HC-O), 5.65 (s, 1 H, HC-O), 3.22-3.16 (m, 1 H, CONCH₂), 3.10-3.04 (m, 1 H, CONCH₂), 2.89 (q, J = 6.6 Hz, 2H, CHgNHBoc), 1.45-1.33 (m, 13H, CH₃(BOC), CCH₂C), 1.28-1.22 (m, 4H, CCH₂C) ppm.

¹³C{¹H}-NMR (151 MHz, DMSO): 5 = 161.7 (s, CONH), 155.7 (q, J = 5.1 Hz, CCF₃), 155.5 (s, NC=O), 143.5 (s, HC=CH), 142.5 (s, HC=CH), 140.9 (q, J = 35.6 Hz, CC=O), 122.5 (q, J = 268.1 Hz, CF₃), 85.2 (s, HC-O), 82.4 (d, J = 2.0 Hz, HC-O), 77.2 (s, C_qBoc), 40.0 (s, NHCH₂), 38.5 (s, NHCH₂), 29.4 (s, CCH₂C), 28.7 (s, CCH₂C), 28.2 (s, CH_3(Boc)), 25.9 (s, CCH₂C).

[0162] 2-(3-(Trifluoromethyl)-7-oxabicvclo[2.2.1lhepta-2,5-diene-2-carboxamido)ethan-1- ammonium trifluoroacetate (Compound 10) Te/t-butyl(2-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)ethyl) carbamate (1.00 g, 2.87 mmol) was dissolved in dichloromethane (10 mL). At 0 °C, TFA (8.8 mL, 0.12 mol) was added dropwise. After stirring for 30 min at the same temperature, no starting material could be observed by TLC and the reaction mixture was concentrated by aceotropic distillation with toluene (3x). Subsequently, the brown residue was treated with ethyl acetate and crystallized overnight at 4 °C. The product was obtained after filtration as a crystalline solid (0.98 g, 2.7 mmol, 95 %).

HR-MS: m/z calculated for Ci₀H₁₁F₃N₂O₂Na⁺: 271.0670 [M+H]⁺, found (ESI-MS⁺): 271.0681.

¹H-NMR (400 MHz, DMSO): 8 = 8.64 (t, J =5.6 Hz, 1 H, NH-Boc), 7.94 (s, 3H, NH₃ ⁺), 7.34 (dd, J = 5.2 Hz, 1.9 Hz, 1 H, HC=CH), 7.30 (dd, J = 5.2 Hz, 1.9 Hz, 1 H, HC=CH), 5.81 (t, J = 1.4 Hz, 1 H, HC-O), 5.73 (d, J = 1.2 Hz, 1 H, HC-O), 3.48-3.32 (m, 2H, CH₂-NH), 2.91 (t, J = 6.4 Hz, 2H, CHgNH ) ppm.

¹³C{¹H}-NMR (101 MHz, CDCI₃): 8 = 162.3 (q, J = 1.4 Hz, CONH), 158.3 (q, J = 31.2 Hz, O₂CCF₃), 155.7 (q, J = 5,2 Hz, CCF₃), 143.7 (d, J = 0.8 Hz, HC=CH), 142.8 (q, J = 35.9 Hz, CC=O), 142.8 (s, HC=CH), 122.4 (q, J = 268.3 Hz, CCF₃), 85.2 (s, CH-O), 82.6 (q, J = 2.2 Hz, CH-O), 38.1 (s, CH₂NHCO), 36.5 (s, CH₂NH₃ ⁺) ppm.

[0163] 2-(1,4-dimethyl-3-(trifluoromethyl)-7-oxabicvclo(2.2.11hepta-2,5-diene-2- carboxamido)ethan-1 -ammonium trifluoroacetate (Compound 11)

Tert-butyl (2-(1 ,4-dimethyl-3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2- carboxamido)ethyl)carbamate (88.6 mg, 0.235 mmol) was dissolved in dichloromethane (2 mL). At 0 °C, TFA (0.72 mL, 9.4 mmol) was added dropwise. After stirring for 30 min at the same temperature, no starting material could be observed by TLC and the reaction mixture was concentrated by aceotropic distillation with toluene (3x). The product was obtained as oil (88.9 mg, 0.228 mmol, 97%).

HR-MS: m/z calculated for Ci₂H₁₆F₃N₂O₂Na⁺: 277.1164 [M+H]⁺, found (ESI-MS⁺): 277.1164. ¹H-NMR (400 MHz, DMSO): 8 = 8.74 (t, J = 5.6 Hz, 1 H, NH-Boc), 7.92 (s, 3H, NH₃ ⁺), 7.08 (d, J = 5.0 Hz, 1 H, HC=CH), 7.05 (d, J = 5.1 Hz, 1 H, HC=CH), 3.47-3.32 (m, 2H, CH₂), 2.86 (q, J =

6.1 Hz, 2H, CH₂), 1.70 (s, 3H, CH₃), 1.57 (s, 3H, CH₃) ppm.

¹³C{¹H}-NMR (126 MHz, DMSO): 8 = 163.4 (q, J = 1.2 Hz, CONH), 158.4 (q, J = 5.0 Hz, CCF₃),

158.1 (q, J = 32.4 Hz, O₂CCF₃), 147.3 (s, HC=CH), 146.3 (s, HC=CH), 141.8 (q, J = 33.4 Hz, CC=O), 122.5 (q, J = 269.0 Hz, CCF₃), 92.3 (s, CH-O), 90.5 (q, J = 1.3 Hz, CH-O), 37.9 (s, CH₂NHCO), 36.2 (s, CH₂NH₃ ⁺), 14.9 (s, CH₃), 14.1 (s, CH₃) ppm.

[0164] 3-(3-(Trifluoromethyl)-7-oxabicvclo[2.2.1lhepta-2,5-diene-2-carboxamido)propan-1- ammonium trifluoroacetate (Compound 12)

Tert-butyl (3-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)propyl)-Z²- azanecarboxylate (100 mg, 0.28 mmol) was dissolved in dichloromethane (2 ml_). At 0 °C, TFA (0.85 ml_, 0.011 mol) was added dropwise. After stirring for 30 min at the same temperature, no starting material was observed by TLC and the reaction mixture was concentrated by aceotropic distillation with toluene (3x). The residue was then treated with ethyl acetate and diethyl ether and decanted. The product was obtained as an oil (66 mg, 0.18 mmol, 67%).

HR-MS: m/z calculated for CnH₁₄F₃N₂O₂ ⁺: 263,1007 [M+H]⁺, found (ESI-MS⁺): 263,100.

¹H-NMR (400 MHz, DMSO): 8 = 8.65 (t, J = 5.8 Hz, 1 H, NH_Amid), 7.86 (s, 3H, NH₃ ⁺), 7.33 (dd, J = 5.3, 2.0 Hz, 1 H, HC=CH), 7.29 (dd, J = 5.3, 1.9 Hz, 1 H, HC=CH), 5.81 (t, J = 1.6 Hz, 1 H, HC- O), 5.68 (d, J = 1.3 Hz, 1 H, HC-O), 3.27 (m, 1 H, CH₂-NHCO), 3.17 (m, 1 H, CH₂-NHCO), 2.80 (q, J = 6.9 Hz, 2H, CHgNH ), 1.73 (quin, J = 7.2 Hz, 2H, CH₂CH2CH₂) ppm.

¹³C{¹H}-NMR (101 MHz, CDCI₃): 8 = 162.2 (d, J = 1.3 Hz, CONH), 158.4 (q, J = 31.9 Hz, O₂CCF₃), 155.6 (q, J = 5.1 Hz, CF₃), 143.6 (d, J = 0.7 Hz, HC=CH), 142.7 (s, HC=CH), 141.7 (q, J = 35.7 Hz, CC=O), 122.5 (q, J = 268.2 Hz, CCF₃), 117.0 (q, J = 298.3 Hz, O₂CCF₃), 85.2 (s, CH-O), 82.5 (q, J = 2.1 Hz, CH-O), 36.7 (s, CH₂NHCO), 35.9 (s, CH₂NH₃ ⁺), 27.1 (s, CH₂CH₂CH₂) ppm.

[0165] 6-(3-(Trifluoromethyl)-7-oxabicvclo[2.2.1lhepta-2,5-diene-2-carboxamido)hexan-1- ammonium trifluoroacetate (Compound 13)

Tert-butyl (6-(3-(trifluoromethyl)-7-oxabicyclo[2.2.1]hepta-2,5-diene-2-carboxamido)hexyl)-Z²- azanecarboxylate (205 mg, 0.507 mmol) was dissolved in dichloromethane (4 ml_). At 0 °C, TFA (1.55 ml_, 0.020 mol) was added dropwise. After stirring for 30 min at the same temperature, no starting material was observed by TLC and the reaction mixture was concentrated by aceotropic distillation with toluene (3x). Subsequently, the residue was treated with diethyl ether and petroleum ether and decanted. The product was obtained as an oil (215 mg, 0.051 mmol, quant.).

HR-MS: m/z calculated for CI₄H₁₉F₃N₂O₂ ⁺: 305.1477 [M+H]⁺, found (ESI-MS⁺): 305.1479. ¹H-NMR (500 MHz, DMSO): 8 = 8.51 (t, J = 5.7 Hz, 1 H, NH_Amid), 7.72 (s, 3H, NH₃ ⁺), 7.32 (dd, J = 5.3, 2.0 Hz, 1 H, HC=CH), 7.27 (dd, J = 5.3, 1.9 Hz, 1 H, HC=CH), 5.79 (t, J = 1.6 Hz, HC-O), 5.64 (d, J = 1.3 Hz, HC-O), 3.25-3.18 (m, 1 H, CH₂-NHCO), 3.10-3.03 (m, 1 H, CH₂-NHCO), 2.80-2.73 (m, 2H, CHgNH ), 1.54-1.48 (m, 2H, CH₂), 1.46-1.40 (m, 2H, CH₂), 1.32-1.25 (m, 4H, CH₂) ppm.

¹³C{¹H}-NMR (126 MHz, DMSO): 8 = 161.8 (q, J = 1.3 Hz, CONH), 158.1 (q, J = 33.0 Hz, O₂CCF₃), 155.7 (q, J = 5.1 Hz, CCF₃), 143.6 (d, J = 0.8 Hz, HC=CH), 142.5 (s, HC=CH), 141.0 (q, J = 35.6 Hz, CC=O), 122.5 (q, J = 268.1 Hz, CCF₃), 85.2 (s, CH-O), 82.4 (q, J = 2.1 Hz, CH- O), 38.8 (s, CH₂NHCO), 38.5 (s, CH₂NH₃ ⁺), 28.6 (s, CH₂CH₂CH₂), 26.9 (s, CH₂CH₂CH₂), 25.7 (s, CH₂CH₂CH₂) 25.4 (s, CH₂CH₂CH₂) ppm.

[0166] 1-r(2S,3R,4S,5S)-5-f((r(n(2R,3R,4S,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxy- tetrahvdrofuran-2-yl1methoxy}oxidophosphoryl)oxyl(hvdroxy)phosphoryl}oxy)-methyll-4- n4-azidobutanoyl)oxy1-3-hvdroxytetrahvdrofuran-2-yl1-3-carbamoyl-pyridin-1-ium (Compound 14, 15, 16)

NAD⁺ (194 mg, 0.292 mmol) was dissolved in 1x PBS buffer (4 mL) and the pH was adjusted to pH 10-11 using triethylamine. 2,5-dioxopyrrolidin-1-yl 4-azidobutanoate (132.3 mg, 58.48 mmol) was dissolved in DMF (3 mL) and added to the reaction solution over 2 h. The reaction solution was then added to DMF. After a reaction time of 3.5 h, mono- as well as diacetylated NAD⁺ was detected by LC-MS controls and purification of the reaction solution by column chromatography was performed by HPLC using a C8 column. After lyophilization, monoacetylated NAD⁺ was obtained as a colorless solid (F1: 6.75 mg, 8.72 pmol, 3%; F2: 25.45 mg, 32.86 pmol, 12% and F3: 8.42 mg, 10.9 pmol, 4%). In addition, diacetylated NAD⁺ was isolated as a colorless solid (25.5 mg, 28.8 pmol, 10 %).

HR-MS: HR-ESI-MS: calculated for C₂5H₃₂N₁₀Oi5P₂Na⁺: 797,1422 [M+Na]⁺, found: 797,1431. HR-MS: HR-ESI-MS: calculated for C₂9H₃₇N₁₃Oi6 ₂Na⁺: 908,1854 [M+Na]⁺, found: 908,1854.

[0167] Compound 14

¹H-NMR (600 MHz, D₂O): 8 = 9.47 (s, 1 H, C₂(Pyr)-H), 9.37 (d, J = 6.3 Hz, 1 H C₆(Pyr)-H), 8.98 (dt, J = 8.1 Hz, J = 1.4 Hz, 1 H, C₄(Pyr)-H), 8.64 (s, 1 H, C₈(Ad)-H), 8.45 (s, 1 H, C₂(Ad)-H), 8.34 (dd, J = 8.0 Hz, J = 6.3 Hz, 1 H, C₅(Pyr)-H), 6.51 (d, J = 3.1 Hz, 1 H, Ci(RibPyr)-H), 6.18 (d, J = 5.4 Hz, 1 H, Ci(RibAd)-H), 5.44 (dd, J = 5.3 Hz, J = 3.2 Hz, 1 H, C₃(RibPyr)-H), 4.77 (bs, 2H, C(Rib/AdPyr)-H), 4.62 (dd, J = 5.8 Hz, J = 2.2 Hz, 1 H, C(RibAd)-H), 4.56-4.51 (m, 2H, C(Rib/AdPyr)-H), 4.42 (bs, 1 H, C(Rib/AdPyr)-H), 4.35-4.23 (m, 3H, C(RibPyr)-H, C(RibAd)-H)), 3.43 (t, J = 6.2 Hz, 2H, CHjN₃Linker), 2.68 (t, J = 7.2 Hz, 2H, CH^Ester), 1.96 (t, J = 6.9 Hz, 2H, CHgLinker). ¹³C{¹H}-NMR (151 MHz, D₂0): 8 = 174.5 (Ci-linker), 165.4 (C₇Pyr), 150.2 (C₆Ad), 148.4 (C₇Ad), 146.3 (C₄Pyr), 145.1 (C₄Ad), 142.9 (C₆Pyr), 142.3 (C₂Pyr), 140.4 (CsAd), 133.9 (C₃Pyr), 128.8 (C₅Pyr), 118.5 (C_sAd), 97.7 (CiRibPyr), 87.8 (CiRibAd), 85.7 (d, J_CP = 9.0 Hz, (C₄RibAd/Pyr),

84.1 (d, _Cp = 8.8 Hz, (C₄RibAd/Pyr), 78.5 (C(RibAd/Pyr)), 74.6 (C(RibAdZPyr)), 70.2 (C(RibAdZPyr)), 67.2 (C(RibAd/Pyr)), 65.1 (d, J_CP = 4.7 Hz, (C₅RibAd/Pyr), 63.6 (d, J_CP = 4.4 Hz, (C₅RibAd/Pyr), 50.3 (CN₃-linker), 30.7 (CH₂-linker), 23.4 (CH₂-ester) ppm.

[0168] Compound 15

¹H-NMR (600 MHz, D₂O): 8 = 9.46 (s, 1 H, C₂(Pyr)-H), 9.28 (d, J = 6.4 Hz, 1 H, Cg(Pyr)-H), 9.00 (dt, J = 8.0 Hz, J = 1.4 Hz, 1 H, C₄(Pyr)-H), 8.64 (s, 1 H, C₈(Ad)-H), 8.44 (s, 1 H, C₂(Ad)-H), 8.33 (dd, J = 8.0 Hz, J = 6.3 Hz, 1 H, C₅(Pyr)-H), 6.23 (d, J = 6.6 Hz, 1H, Ci(RibPyr)-H), 6.19 (d, J =

5.4 Hz, 1 H, Cr(RibAd)-H), 5.49 (dd, J = 5.4 Hz, J = 1.5 Hz, 1H, C₃(RibPyr)-H), 4.77 (bs, 3H, C(RibAdZPyr)-H), 4.54 (dd, J = 4.9 Hz, J = 4.1 Hz, 1 H, C₃(RibAd)-H), 4.44-4.41 (m, 2H, C₅(RibPyr)-H, C₄(RibAd)-H), 4.32-4.21 (m, 3H, C₅(RibPyr)-H, C₅(RibAd)-H)), 3.43 (t, J = 6,7 Hz, 2H, CHgNsLinker), 2.64 (t, = 7.2 Hz, 2H, CHgEster), 1.95 (q, = 7.0 Hz, 2H, CHgLinker) ppm.

¹³C{¹H}-NMR (151 MHz, D₂0): 8 = 174.3 (CiLinker), 165.5 (C₇Pyr), 150.3 (C₆Ad), 148.4 (C₇Ad),

146.4 (C₄Ad), 145.3 (C₄Pyr), 142.7 (C₆Pyr), 142.3 (C₂Pyr), 139.9 (CsAd), 134.0 (C₃Pyr), 128.7 (C₅Pyr), 118.6 (CsAd), 99.5 (CiRibPyr), 87.9 (CiRibAd), 85.5 (d, J_CP = 8.5 Hz, (C₄RibPyr), 84.1 (d, _Cp = 8.7 Hz, (C₄RibAd), 76.1 (C₂RibPyr), 74.6 (C₂RibAd), 73.9 (C₃RibPyr), 70.2 (C₃RibAd), 65.1(C₅RibPyr/Ad), 50.3 (CN₃Linker), 30.8 (CH₂Linker), 23.5 (CH₂Ester) ppm.

[0169] Compound 16

¹H-NMR (600 MHz, D₂O): 8 = 9.46 (s, 1 H, C₂(Pyr)-H), 9.30 (d, J = 6.3 Hz, 1 H, Cg(Pyr)-H), 8.96 (dt, J = 8,1 Hz, J = 1.3 Hz, 1 H, C₄(Pyr)-H), 8.67 (s, 1 H, C₈(Ad)-H), 8.45 (s, 1 H, C₂(Ad)-H), 8.32 (dd, J = 8.0 Hz, J = 6.3 Hz, 1 H, C₅(Pyr)-H), 6.22 (d, J = 6.8 Hz, 1 H, C_r(RibAd)-H), 6.20 (d, J = 5.5 Hz, 1 H, Ci(RibPyr)-H), 5.51 (dd, J = 5.4 Hz, J = 2.6 Hz, 1H, C₃(RibAd)-H), 5.03 (dd, J = 6.7 Hz, J = 5.5 Hz, 1H, C₂(RibAd)-H), 4.62-4.58 (m, 3H, C(RibAd/Pyr)-H), 4.50 (dd, J = 5.0 Hz, J = 2.7 Hz, 1 H, C(RibPyr)-H), 4.43-4.40 (m, 1 H, C₅(RibAd/Pyr)-H), 4.29-4.25 (m, 3H, C(RibAdZPyr)-H), 3.44 (t, J = 6.7 Hz, 2H, CHg hLinker), 2.65 (t, J = 7.3 Hz, 2H, CHgEster), 1.97 (q, J - 7.0 Hz, 2H, CHgLinker) ppm.

¹³C{¹H}-NMR (151 MHz, D₂O): 8 = 174.5 (CiLinker), 165.6 (C₇Pyr), 150.2 (CsAd), 148.7 (C₇Ad),

146.1 (C₄Ad), 145.1 (C₄Pyr), 142.6 (C₆Pyr), 142.3 (C₂Pyr), 139.9 (CsAd), 133.9 (C₃Pyr), 128.6 (C₅Pyr), 118.5 (C₆Ad), 100.0 (CiRibPyr), 87.2 (CiRibAd), 82.5 (d, J_CP = 8.7 Hz, (C(RibAdZPyr)), 77.6 (C(RibAdZPyr)), 73.4 (C₃RibAd), 73.3 (C₂RibAd), 70.9 (C(RibAdZPyr)), 65.3 (C₅RibPyrZAd), 64.9 (C₅RibPyrZAd), 50.3 (CN₃Linker), 30.9 (CH₂Linker), 23.6 (CH₂Ester) ppm. [0170] 1-n2S.3R.4S.5S)-5-f((rar(2/?,3/?.4S,5/?)-5-(6-amino-9H-purin-9-yl)-3.4-dihvdroxy- tetrahvdrofuran-2-yl1methoxy}oxidophosphoryl)oxyl(hvdroxy)phosphoryl}oxy)-methyl1-4- H4-(4-[(2-aminoethyl)carbamoyl1-5-(trifluoromethyl)-1F/-1,2,3-triazole-1-yl}butanoyl)oxy1-3- hvdroxytetrahvdrofuran-2-yl1-3-carbamoylpyridin-1-ium (Compound 17)

Monoacylated NAD⁺ (Compound 15) (7.32 mg, 9.45 pmol) was dissolved in 1 mL of D₂O. Oxanorbornadiene linker 5 (3.42 mg, 9.45 pmol) was added and the reaction solution was stirred for 3 h at RT. The reaction solution was then transferred to an NMR tube, incubated at 40 °C for another 3 days, and the conversion was determined by ¹H-NMR spectroscopy. The mixture was then freeze-dried and the product was obtained as a mixture with the starting materials in the form of a colorless solid (8.81 mg mixture, product content by ¹H-NMR spectrum: 4.96 mg, 5.20 pmol, 55 %).

HR-MS: m/z calculated for C3iH₃9N₁₂Oi₆P₂F₃Na⁺: 977.1932 [M+Na]⁺, found (ESI-MS⁺): 977.1932.

¹H-NMR (400 MHz, D₂O): 8 = 9.43 (s, 1 H, C₂(Pyr)-H), 9.29-9.25 (m, 1H, C₆(Pyr)-H), 8.99-8.92 (m, 1 H, C₄(Pyr)-H), 8.62-8.61 (m, 1 H, C₈(Ad)-H), 8.42-8.40 (m, 1H, C₂(Ad)-H), 8.34-8.27 (m, 1H, C₅(Pyr)-H), 6.22-6.14 (m, 2H, Ci(Pyr)-H, Ci(Ad)-H), 4.75-4.70 (m, 4H), 4.63-4.46 (m, 3H), 4.38 (bs, 2H), 4.31-4.22 (m, 3H), 3.77-3.55 (m, 2H, CHgLinker), 3.28-3.17 (m, 2H, CHgLinker), 2.71- 2.59 (m, 2H, CHgEster), 2.34-2.20 (m, 2H, CH₂CH2CH₂) ppm.

[0171] PCM5-triazolo-NAD⁺ (Compound 18)

Monoacylated NAD⁺ (Compound 15) (3.12 mg, 4.15 pmol) was dissolved in 400 pL of D₂O. Oxanorbornadiene functionalized PCM5 (9.20 mg) dissolved in 400 pL D₂O was added and the reaction solution was stirred for 36 h at 37°C (Figure 24). Successful conversion was confirmed by ¹H-NMR spectroscopy (Figure 25). The mixture was then purified by filtration using AMICON filters (cut-off: 3 kDa) and the solution was lyophilized. The product was obtained as a colorless solid (7.75 mg).

[0172] A/-(26-azido-3,6,9,12,15, 18,21 ,24-octaoxahexacosyl)-5-(2-oxohexahydro-1 H- thieno[3,4-onimidazol-4-yl)pentanamide (Compound 19)

To a solution of biotin A/-hydroxysuccinimide ester (100 mg, 0.293 mmol) in DMF (2.5 ml) was added PEG-linker (NH₂-PEG(9)-azide) (122 mg, 0.293 mmol) and the reaction mixture was allowed to stir for 44 h at room temperature. Upon completion the solvent was removed under reduced pressure and the residue was diluted by DCM (1 ml).

By adding diethyl ether a colorless solid precipitate formed, which was filtered off, washed with diethyl ether (3x) and redissolved in dichloromethane. After the solvent was removed under reduced pressure, the product could be obtained as a white solid (0.145 g, 0.218 mmol, 75%). HR-ESI-MS: calculated for C₂₈H₆₂N₆OioSNa⁺: 687.3363 [M+Na]⁺, found: 687.3351. ¹H-NMR (400 MHz, CDCI₃): 8 = 6.57 (t, J = 5.2 Hz, 1H), 5.97 (s, 1H), 5.10 (s, 1H), 4.50 (dd, J = 5.1, 7.6 Hz, 1H), 4.32 (t, J= 5.8 Hz, 1H), 3.67-3.63 (m, 33H), 3.45-3.42 (m, 2H), 3.39 (t, J= 5.1 Hz, 2H), 3.17-3.12 (m, 1H), 2.91 (dd, J = 4.9, 12.8 Hz, 1H), 2.74 (d, J= 12.7 Hz, 1H), 2.26-2.18 (m, 2H), 1.78-1.62 (m, 3H), 1.49-1.41 (m, 2H). rO1731 Table 1: The chemical structures of the compounds mentioned above are presented in this Table.

[0174] General procedures:

[0175] Carboxymethylation of biopolymers:

[0176] The desired biopolymer (e.g. pullulan (25 g)) was dissolved in deionized water (375 mL). Subsequently, 8 M aq. NaOH (125 mL) and chloroacetic acid (50.1 g, 0.53 mol) were added and the reaction mixture was heated to 62°C. After 90 min, the solution was adjusted to pH 6.5 with 6 M aq. HCI and poured into distilled methanol (3 L). The resulting precipitate was filtered off and the residue was dried at 40 °C and 20 mbar. The carboxymethylated biopolymer was obtained as a white solid.

[0177] A higher degree of substitution was achieved by subjecting the obtained product to repetitive carboxymethylation reactions. The number of repetitive cycles is described by using a CM suffix (e.g. CM3 corresponds to 3 repetitive carboxymethylation cycles).

[0178] Two component system (adding linker unit to carboxymethylated biopolymer):

[0179] A desired biopolymer (dextran 250 kDa CM6, 100 mg, 0.50 mmol) was dissolved in 0.025 M MES buffer (50 mL). The desired linker unit NH₂-(CH₂CH2O)_S-CH2CH₂N3 with s = 3 (98.15 pL, 0.50 mmol), EDC-HCI (0.948 g, 4.95 mmol) and NHS (56.93 mg, 0.50 mmol) were added sequentially. The reaction was stirred at RT for 2.5 days and then transferred into a dialysis tube (cut-off: 14 kDa). The latter was layed into a 5 L beaker containing an aqueous deionized NaCI solution and dialyzed for 4 days with decreasing NaCI concentration (day 1 : 20 g/L, day 2: 10 g/L, and day 3-4: 0 g/L). Each day, the aqueous deionized NaCI solution was renewed three times. Subsequently, the dialyzed solution was filtered through absorbent cotton and freeze-dried. The modified biopolymer could be isolated as a cotton wool-like solid (87.80 mg).

[0180] NMR analysis:

[0181] The ¹H and ¹³C and ¹⁹F NMR spectra were measured on Bruker AVANCE-400/500/600 and DPX-200/400 spectrometers at room temperature and in the indicated deuterated solvents. The residual proton signal of the solvent (CDCI₃: 5 (¹H-NMR) = 7.26 ppm, D₂O: 8 (¹H-NMR) = 4.79 ppm und DMSO: 3 (¹H-NMR) = 2.50 ppm) served as a reference and for calibration of the ¹H-NMR spectra. The ¹³C-NMR spectra were recorded broadband decoupled and also calibrated to the solvent signal (CDCI₃: (¹³C-NMR) = 77.2 ppm, and DMSO: (¹³C-NMR) = 39.5 ppm). For the ¹⁹F-NMR, based quantification, trifluoroacetic acid methyl ester was used as an internal standard. Samples in deuterated water were measured using a water suppression method.

[0182] Coupling constants J were expressed in Hz and chemical shifts in ppm. The signal multiplicities were abbreviated as follows for simplicity: singlet (s), duplet (d), triplet (t), quartet (q), and multiplet (m).

[0183] High-resolution mass spectroscopy (HR-MS):

[0184] High-resolution mass spectra were measured on a Water Aquity UPLC system with a QTof Premier detector (ESI and APCI-MS/MS) or on a Water Allicance 2695 with Micromass LCTPremier detector. Samples were dissolved in water, acetonitrile, or methanol and injected using either an HPLC system or a direct inlet. The measured values are given in mass/charge (m/z).

[0185] Column chromatography:

[0186] Manual column chromatography was carried out using overpressure. For this purpose, silica gel (particle size: 40-63 pm, normal phase) from Macherey-Nagel and indicated running medium mixtures (e.g. PE/EtOAc 1 :1) were used. Thin layer chromatography (DC silica gel 60 F254 glass plates) from Merck (pore size: 60 A, layer thickness: 210-270 pm, fluorescent indicator UV254) was used to detect the products.

[0187] Viscosity measurements of polysaccharides:

[0188] The viscosity properties of the polysaccharides were studied using the rheometer MCR302 from Anton Paar. The viscosity measurements were investigated by using a PP20-SN33813 system with a normal force of 0 N, an amplitude y = 0.1 , 100 % log, [slope] = 10 points/decade, a given shear rate (y) from 10 s'¹ to 10,000 s'¹, a temperature of 19.3 °C and a gap size of 1 mm. The sample was prepared as follows: The resulting modified biopolymer chains (e.g. pullulan, dextran, alginate and hyaluronan) with different functional groups (degrees of substitutions are variable) were dissolved in an appropriate solvent (e.g. deionized water, buffer, organic solvent mixtures) and analyzed in the rheometer.

[0189] Determination of enzyme activity (dehydrogenases) via a colorimetric assay

[0190] The resulting modified biopolymer chains (e.g. pullulan, dextran, alginate and hyaluronan) with different functional groups (degrees of substitutions are variable) were dissolved in an appropriate solvent (e.g. deionized water, buffer, organic solvent mixtures) and these two components were mixed with native NAD⁺ (final concentration in well 0.5 mM), NAD⁺- azide or NAD⁺-triazole. To this mixture, a certain concentration of enzyme was added (according to Figure 3). The gelation was conducted in a microtiter plate with a volume of 25 pl by incubation at 37 or 40°C overnight. Afterwards, the hydrogel samples were washed with deionized water by shaking for 30 min at 50 rpm and the excess of deionized water was removed. To all standards, blanks and samples, the specified reaction mixture (see Table 2) was added and the absorbance was measured at X = 450 nm using the Cytation5 from BioTek. The measurement was conducted at a constant temperature of 37°C.

In addition to hydrogel samples, aqueous enzyme solutions were also measured following the same protocol (except for the washing step). A NADH standard curve was generated for each experiment to calculate the amount of NADH converted and to determine enzymatic kinetics.

[0191] Table 2: Composition of the WST-8 assay mix depending on the enzyme of interest. To the 25 pL sample volume, 25 pL reaction mix was added. The NAD⁺ concentration is explicitly final concentration in well will be divided by two (25 pL sample, 25 pL assay reaction mix)

[0192] Fourier-transform infrared spectroscopy:

[0193] Infrared spectra (IR) were recorded on a Shimadzu ATR-FT-IR spectrometer. All biopolymer samples were measured as freeze-dried lyophilizate.

[0194] Conductive titration:

[0195] The degree of substitution of the biopolymers was determined by conductive titration with a Titrol_ine®7000.

[0196] A two component system (crosslinked polysaccharides with three degrees of variability) according to Figure 1 is provided: Two modified biopolymer chains (e.g. pullulan, dextran, lentinan, alginate and hyaluronan) with reactive functional groups (one biopolymer carrying azide residues, the other biopolymer carrying oxanorbornadiene derivatives; degrees of substitutions are variable) form a triazole via thermo-induced copper-free “click-reaction, which is described in detail with concrete examples in the following: [0197] Example 1 : Carboxymethylation of dextran

[0198] Dextran (500 kDa, 10.00 g, 0.06 mol) was dissolved in deionized water (150 mL). Subsequently, 8 M aq. NaOH (50 mL) and chloroacetic acid (20.40 g, 0.22 mol) were added and the reaction mixture was heated to 62°C. After 90 min, the solution was adjusted to pH 6.5 with 6 M aq. HCI and poured into distilled methanol (1 .4 L). The resulting precipitate was filtered off and the residue was dried at 40 °C and 20 mbar. The carboxymethylated biopolymer was obtained as a white solid (12.34 g).

[0199] A higher degree of substitution was achieved by subjecting the obtained product to repetitive carboxymethylation (CM) reactions.

[0200] The following examples were obtained in a similar fashion as described above for Example 1 :

[0201] Table 3: Carboxymethylated biopolymers.

Carboxymethylated Lentinan

Lentinan (400 - 800 kDa) CM1 : scale: 8 g; yield: 3.49 g.

[0202] Table 4: Carboxymethylated pullulan.

[0203] The degree of substitution of the obtained polysaccharides was characterized by FT-IR spectroscopy (see Figure 5), ¹H-NMR spectroscopy (see Figure 6) and conductive titration (see Figure 9).

[0204] Both biopolymer chains are conjugated to functionalized spacers, e g. a PEG-linker unit. The PEG-linker is variable in length and should be short (PEG(3)-PEG(25)). Different PEG- linker units (as indicated in Figure 1) and carboxymethylated biopolymers lead to different pore sizes. The pore size will influence the activity of the enzyme and the diffusion behaviour of the substrate and of other molecules (see Figure 1). In this context, the term “functionalized” refers to spacers carrying either an azide moiety or an oxanorbornadiene derivative Q, like described above (see scheme 1). Conjugation is achieved via amide coupling of carboxy groups of the biopolymer and amine groups in the linker.

[0205] Example 2: Azide functionalization of dextran

[0206] A desired biopolymer (dextran, 250 kDa, CM4, 100 mg, 0.45 mmol) was dissolved in 0.025 M MES buffer (50 mL). The desired linker NH₂-(CH₂CH2O)_S-CH2CH₂N3 with s = 3, 90.12 L, 0.45 mmol, EDC-HCI (0.87 g, 4.54 mmol) and NHS (52.27 mg, 0.45 mmol) were added sequentially. The reaction was stirred at RT for 2.5 days and then transferred into a dialysis tube (cut-off: 14 kDa). The latter was deposited in a 5 L beaker containing an aqueous deionized NaCI solution and dialyzed for 4 days with decreasing NaCI concentration (day 1 : 20 g/L, day 2: 10 g/L, and day 3-4: 0 g/L). Each day, the aqueous deionized NaCI solution was renewed three times. Subsequently, the dialyzed solution was filtered through absorbent cotton and freeze-dried. The modified biopolymer could be isolated as a cotton wool-like solid (95 mg).

[0207] The following examples were obtained as described above:

[0208] Table 5: Introducing an azide unit (-NH-(CH₂CH₂O)_S-CH₂CH₂N₃ with s = 3) to a polysaccharide strand. Alginate (120 - 190 kDa): scale: 200 mg; yield: 172 mg.

Hyaluronan (70 - 80 kDa): scale: 100 mg; yield: 80 mg.

Lentinan CM1 (400 - 800 kDa): scale: 100 mg; yield: 74 mg.

[0209] Table 6: Introducing an azide unit (-NH-(CH2CH₂O)_S-CH₂CH2N3 with s = 3) to pullulan.

Introducing an azide unit (-NH-(CH₂CH2O)_S-CH2CH₂N3 with s = 8) to pullulan.

Pullulan (100 kDa) PCM5: scale: 50 mg; yield: 63 mg.

Introducing an azide unit (-NH-(CH₂)r-N₃with r = 6) to pullulan.

Pullulan (100 kDa) PCM1 : scale: 150 mg; yield: 122 mg.

Pullulan (100 kDa) PCM5: scale: 150 mg; yield: 147 mg.

Alginate (120 - 190 kDa): scale: 200 mg; yield: 89 mg.

[0210] Example 3: Oxanorbornadiene functionalization of dextran

[0211] A desired biopolymer (dextran, 250 kDa, CM4, 100 mg, 0.45 mmol) was dissolved in 0.025 M MES buffer (50 ml_). The desired linker NH₂-(CH₂)_r-Q with r = 2, Q-1 (CF₃) and M/M' = H, 112.73 mg, 0.45 mmol, EDC-HCI (0.87 g, 4.54 mmol) and NHS (52.27 mg, 0.45 mmol) were added sequentially. The reaction was stirred at RT for 2.5 days and then transferred into a dialysis tube (cut-off: 14 kDa). The latter was deposited in a 5 L beaker containing an aqueous deionized NaCI solution and dialyzed for 4 days with decreasing NaCI concentration (day 1 : 20 g/L, day 2: 10 g/L, and day 3-4: 0 g/L). Each day, the aqueous deionized NaCI solution was renewed three times. Subsequently, the dialyzed solution was filtered through absorbent cotton and freeze-dried. The modified biopolymer could be isolated as a cotton wool-like solid (88 mg).

[0212] The following examples were obtained as described above: (02131 Table 7: Introducing an oxanorbomadiene unit (-NH-(CH₂)_r-Q with r = 2, Q-1 (CF₃) and M/M' = H) to a polysaccharide strand.

Alginate (120 - 190 kDa): scale: 200 mg; yield: 234 mg.

Hyaluronan (70 - 80 kDa): scale 100 mg; yield: 99 mg.

Lentinan CM1 (400 - 800 kDa): scale: 100 mg; yield: 65 mg.

(02141 Table 8: Introducing an oxanorbomadiene unit (-NH-(CH₂)_r-Q with r = 2, Q-1 (CF₃) and M/M' = H) to pullulan.

Introducing an oxanorbomadiene unit (-NH-(CH₂)_r-Q with r = 6, Q-1 (CF₃) and M/M' = H) to pullulan.

Pullulan (100 kDa) PCM5: scale: 100 mg; yield: 122 mg.

[0215] The obtained products can be analyzed by ¹H- and ¹⁹F-NMR spectroscopy (see e.g. Figures 6, 7, 10, 13 and 17). Specific IR-spectra of samples from different batches of one derivative are shown in Figure 9.

[0216] Example 4: NAD⁺-functionalization

Functionalized NAD⁺ can be obtained by reaction with a suitably functionalized nhs ester (e.g. azido/alkyne/oxanorbornadiene moiety). Three different NAD⁺-azide derivatives were obtained by reaction with 2,5-dioxopyrrolidin-1-yl 4-azidobutanoate (Figure 20) and separated by HPLC column chromatography. NAD⁺-triazole was obtained by a reaction between NAD⁺-azide with an oxanorbornadiene unit in D₂O at 40°C (Figure 21).

[0217] Example 5: Cofactor attachment to a biopolymer strand

[0218] A pre-functionalization of a biopolymer strand was obtained by a reaction between NAD⁺-azide (Compound 15) with an oxanorbornadiene biopolymer strand (e.g. in this example PCM5) in D₂O at 37°C (Figure 24). By increasing the concentration of NAD⁺ in the reaction, a higher amount of NAD⁺ was covalently attached to the biopolymer strand. This could be detected by ¹H-NMR spectroscopy, comparing the ratio of one NAD⁺ signal (5 = 8.83-8.74 ppm) and furan signals generated in the reaction (8 = 7.45 and 6.39 ppm) (as an example, in A twice the NAD⁺ concentration was added to the reaction compared to B, Figure 25, compound 18).

[0219] Example 6: Bio-orthogonal 1,3-dipolar cycloaddition

[0220] The reaction (see Figure 3) comprises: Crosslinking two biopolymers via bio-orthogonal copper-free “click“-reaction which is a 1 ,3-dipolar cycloaddition. To enable said reaction, the biopolymers of the Examples described above, containing suitable linkers (azide-linked biopolymer and oxanorbomadiene-linked biopolymer, respectively) were mixed. Specific thermal gelation takes place under mild conditions (e.g. < 40°C, in aqueous media) between azide and oxanorbornadiene moieties, only. NAD⁺ derivatives, bearing a suitable azide or oxanorbonadiene moiety can be covalently attached to the formed hydrogel by co-incubation of a solution of the functionalized cofactor with the two individual biopolymers (e.g. Figure 3A (a, c) and Figure 3C (a, c)). Mild reaction conditions are suitable for maintaining activity and integrity of sensitive enzymes. The irreversible network of sugar chains with immobilized enzymes obtained by this method provides high specificity and selectivity and provides a bio- orthogonal material. Further, adjustment of viscosity of the individual biopolymer solutions can be achieved easily, e.g. by choice of biopolymer (Figure 19A), adjusting the degree of functionalization (Figure 19B) or adding water or other suitable co-solvents, which enables optimal processing of the reaction mixture. Additionally, not crosslinked biopolymer and not encapsulated enzyme can be washed away. Gelation takes place after a certain time (of about 1 hour to 10 hours). Processing to different form factors, e.g. by printing or spin coating of the material is possible. Thus, the hydrogel according to the present invention allows to immobilize enzymes in a biocompatible network that does not require a covalent binding of the enzyme (see Figure 3).

[0221] No side reactions occur and no toxic reagents (e.g. glutaraldehyde) are used. Very mild reaction conditions (40°C, in aqueous media) are applied as described above, while gelation time can be controlled by choice of material (substitution degree) and concentration of biopolymer (e.g. 10 - 40 mg/ml). Higher modified biopolymers (PCM5 - PCM9) have a short gelation time (2 - 5 h; 40 mg/ml material). Low modified biopolymers (PCM 1-3) have a gelation time from 3 - 10 h (40 mg/ml material).

[0222] Example 7:

[0223] The biocompatible hydrogel according to the present invention is a modular system, whose synthetic steps can be analyzed using various methods and can be manufactured in a well-defined manner.

[0224] The repetitive carboxymethylation of a polysaccharide can be analyzed by conductive titration (see Figure 8) and the degree of substitution rises with increasing carboxymethylation steps. Furthermore, the increase of substitution can be measured by FT-IR and ¹H-NMR. In the FT-IR spectra specific bands in the fingerprint region become more intense with a higher degree of substitution and an increasing number of functional groups (see Figure 4, Figure 11 and Figure 12). In the ¹H-NMR spectra, the decrease of the intensity of the signal of the anomeric proton was observed with increasing the introduced carboxymethyl-groups (see Figure 5 and Figure 18). Quantification by ¹⁹F-NMR showed the increased intensity of the signal of an introduced linker to differently modified polysaccharides. The number of introduced linker units correlates to the present carboxymethyl-groups in the polysaccharide (see Figure 10). Moreover, five different batches of a five-fold repetitively modified pullulan carrying an azide linker showed similar FT-IR spectra (see Figure 9).

[02251 Example 8:

Biological activity of modified cofactors (NAD⁺ and NAD⁺-triazole) was tested with two dehydrogenases in a WST-8 assay in solution (see Figure 22 and Figure 23). Higher absorbance was recorded for a GDH solution with NAD⁺-triazole compared to using the NAD⁺- azide (Figure 22). With an increase of NAD⁺-triazole concentration, a higher absorbance signal was obtained in G6PD solution. Both lower concentrations of NAD⁺-triazole had higher substrate conversion in G6PD solutions compared to NAD⁺-azide (Figure 23).

[0226] Example 9:

[0227] Enzymes can be immobilized non-covalently in the hydrogel. After complete gelation, non-immobilized enzyme can be removed by washing.

[0228] Preparation: The modified biopolymer chain containing covalently bound cofactor (e.g. in this example pullulan CM5, compound 18) was reacted with an azide containing biopolymer (azide unit -NH-(CH₂CH2O)_S-CH2CH₂N3 with s = 3 connected to a three times carboxymethylated pullulan, PCM3) were dissolved in phosphate buffer pH 7.4. As a control, the non-cofactor bound pullulan biopolymer (-NH-(CH₂)_r-Q with r = 2, Q = Q-1 (CF₃) and M/M' = H connected to carboxymethylated pullulan) was used and the two obtained components were mixed with non-bound native NAD⁺. Other mixtures were prepared in a similar manner (their composition is described in Figure 26 and 28).

[0229] To this mixture, a given concentration of an enzyme (e.g. in this example glucose dehydrogenase (GDH) 1 U/mL or glucose-6-phosphate dehydrogenase (G6PD) 0.5 U/mL final concentration in well) was added. The gelation was conducted in a microtiter plate with a volume of 25 pL (each sample) by incubation at 37 °C over night. Afterwards, the hydrogel samples were washed with deionized water (25 pL) by shaking for 30 min at 50 rpm and the excess of deionized water was removed. To all standards, blanks, and samples the specified reaction mixture (see Table 1) was added and the absorbance was measured. As an additional control, enzyme solution with native NAD⁺ was used.

[0230] The example of immobilized G6PD and GDH was obtained as described above. The reaction mixture was adjusted according to Table 2.

[0231] Results: Enzyme saturation curves were recorded in different modified pullulan hydrogels (e.g. PCM3 and PCM9, meaning pullulan biopolymers obtained by running 3, respectively 9, carboxymethylation cycles). As a control, the enzyme was used in solution (see Figure 26 and Figure 28).

[0232] After incubation over night at 37 °C enzymatic saturation curves of G6PD were recorded in solution with native NAD⁺, in hydrogels (PCM3 and PCM9) with non-covalently native NAD⁺ and in hydrogels (PCM7/5) with covalently immobilized NAD⁺ and results are summarized in Figure 26.

[0233] Long-term data of covalently immobilized NAD⁺ in a PCM5/3 hydrogel with non- covalently immobilized GDH were recorded, as well and the enzymatic activity was plotted on different days (Figure 27).

[0234] Long-term data of GDH in solution with native NAD⁺, GDH immobilized in hydrogels with native NAD⁺ and GDH immobilized in hydrogels with covalently immobilized NAD⁺ were performed and the enzymatic activity was plotted after incubation at 37 degree Celsius for 20 days (Figure 28).

[0235] The invention is further characterized by the following items:

[0236] Items:

1. Method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, c) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; d) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

2. Method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s) and incubation of the at least one enzyme with the at least one functionalized cofactor of step a), such that the at least one enzyme is non-covalently bound to the at least one cofactor, c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

3. Method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

4. Method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor functionalized with at least one group Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

5. Method of preparing a biocompatible hydrogel, comprising the following steps: a) Providing at least one cofactor, wherein a part of the cofactor is functionalized with at least one group Z and a part of the cofactor is functionalized with at least one group E, wherein Z and E each independently comprises or is a functional group suitable for a 1 ,3-dipolar cycloaddition with a group Y, b) providing at least one enzyme(s), c) providing a first polysaccharide and a second polysaccharide, with n being the number of the monomeric repeating unit(s) of the first and/ or the second polysaccharide and with n being an integer in the range from 10 to 10000, d) optionally carboxymethylation of at least one OH-group of at least one monomeric repeating unit(s) of the first and/ or the second polysaccharide; e) functionalization of the first polysaccharide with at least one linker unit(s) of the structure -A- X, when the monomeric repeating unit of the first polysaccharide not comprises a carboxylic acid residue, or functionalization of the first polysaccharide with one or more group(s) of the structure -X, when the monomeric repeating unit of the first polysaccharide comprises a carboxylic acid residue, wherein A is a first spacer unit; and

6. The method according to any one of items 1 to 5, wherein the method is carried out in the absence of at least one catalyst(s), preferably in the absence of copper.

7. The method according to any one of the previous items, wherein the method is carried out in presence of at least one catalyst, preferably wherein the catalyst is a copper catalyst.

8. The method according to any one of the preceding items, wherein the first spacer unit A is -(CH₂)d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

9. The method according to any one of the preceding items, wherein the second spacer unit A’ is -(CH₂)d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

7Q 10. The method according to any one of the preceding items, wherein:

X is selected from the group consisting of -NH-(CH₂)_r-N3, -NH-(CH₂CH₂O)_S-CH₂CH2N3 and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N3, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

Y is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))t-CH₂-CH₂-Q, wherein wherein M, M’ = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Z is selected from the group consisting of -C(O)-(CH₂)_r-N₃, -C(O)-(CH₂CH₂O)_S-CH₂CH₂-N3 and -C(O)-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15.

11. The method according to any one of items 1 to 9, wherein:

X is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M’ = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Y is selected from the group consisting of -NH-(CH₂)r-N₃, -NH-(CH2CH₂O)_S-CH₂CH2N3 and -NH-(CH₂-CH₂-C(O))t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and

Z is selected from the group consisting of -C(O)-(CH₂)_r-Q, -C(O)-(CH₂CH₂O)_S-CH₂CH₂Q and -CO-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Q is - ^C=^CH .

12. The method according to any one of the preceding items, wherein the first polysaccharide and/or the second polysaccharide is/are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

13. The method according to any one of the preceding items, wherein the first polysaccharide and/or the second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and mixtures thereof.

14. The method according to any one of the preceding items, wherein the first polysaccharide and the second polysaccharide are pullulan.

15. The method according to any one of the preceding items, wherein the first and/ or second polysaccharide is dextran or pullulan and carboxymethylation of at least one OH-group of dextran or pullulan is carried out according to step c) of claim 1 or according to step d) of any one of items 2 to 5.

16. The method according to item 1 , wherein in step f) the at least one group Z of the at least one cofactor is reacted in a first 1 ,3-dipolar cycloaddition with a part of the group(s) Y of the functionalized second polysaccharide.

17. The method of item 16, wherein in step g) the part of the group(s) Y not reacted in step f) of the product of step f) is reacted in a second 1 ,3-dipolar cycloaddition with the group X of the functionalized first polysaccharide.

18. The method according to any one of the preceding items, wherein in step d) of item 1 or in step e) of any one of items 2 to 5 the first polysaccharide is functionalized with 0.01-1 .5 of -A-X or -X per monomeric repeating unit of the first polysaccharide.

19. The method according to any one of the preceding items, wherein in step d) of item 1 or in step e) of any one of items 2 to 5 the second polysaccharide is functionalized with 0.01-1 .5 of - A'-Y or -Y per monomeric repeating unit of the second polysaccharide.

20. The method according to any one of the preceding items, wherein d is 1.

21. The method according any one of the previous items, wherein step f) and/or step g) is a thermo-induced cycloaddition reaction between Z and a part of the group(s) Y and/or between X and a part of the groups(s) Y not reacted in step f) for forming a crosslinked polymer.

22. The method according to any one of items 1 to 20, wherein step f) and/or step g) is a catalyst-induced cycloaddition reaction between Z and a part of the group(s) Y and/or between X and a part of the group(s) Y not reacted with Z for forming a crosslinked polymer, preferably wherein the catalyst is a copper catalyst.

23. The method according to any one of the preceding items, wherein the content of N₃, when present, is 0.01-1 .5 N₃ per monomeric repeating unit of the first polysaccharide.

24. The method according to any one of the preceding items, wherein the content of Q, when present, is 0.01-1 .5 per monomeric repeating unit of the first and/ or the second polysaccharide. 25. The method according to any one of the preceding items, wherein in step d) of item 1 or in step e) of any one of items 2 to 5, -A-X is linked to at least one primary or secondary OH-group of the first polysaccharide, preferably via at least one of C₂, C₃, C₄ or C_s of the monomeric repeating unit(s) of the first polysaccharide, more preferably via C₆ of the monomeric repeating unit(s) of the first polysaccharide.

26. The method according to any one of the preceding items, wherein in step d) of item 1 or in step e) of any one of items 2 to 5, -A'-Y is linked to at least one primary or secondary OH-group of the second polysaccharide, preferably via at least one of C₂, C₃, C₄ or C₆ of the monomeric repeating unit(s) of the second polysaccharide, more preferably via C₆ of the monomeric repeating unit(s) of the second polysaccharide.

27. The method according to any one of the preceding items, wherein the method is without the use of toxic reagents, preferably without the use of glutaraldehyde.

28. The method according to any one of the preceding items, wherein the molecular weight of the unfunctionalized first polysaccharide is in the range from 5 to 2000 kDa.

29. The method according to any one of the preceding items, wherein the molecular weight of the unfunctionalized second polysaccharide is in the range from 5 to 2000 kDa.

30. Hydrogel obtainable by a method of any one of the items 1 to 29.

31. The hydrogel according to item 30, wherein the hydrogel comprises at least one encapsulated enzyme(s).

32. The hydrogel according to item 30 or 31 , wherein the hydrogel is a swellable or swollen hydrogel matrix.

33. A biocompatible hydrogel comprising, a crosslinked polymer comprising:

- at least one cofactor(s),

- at least one first linker unit(s), which link(s) the first polysaccharide with the second polysaccharide, wherein the structure of the at least one first linker unit(s) is -A-Gi-A’-, -A- Gi~ , — A’— Gi- , or— Gi- , wherein A is a first spacer unit;

A’ is a second spacer unit; and at least one second linker unit(s), which link(s) the first polysaccharide or the second polysaccharide with the at least one cofactor(s), wherein the structure of the at least one second linker unit(s) is -A-G2- -A-G2-, or-G₂-; wherein A is the first spacer unit;

A’ is the second spacer unit; and

G₂ is a second moiety being or comprising a group obtainable or being obtained from a 1 ,3-dipolar cycloaddition.

34. The hydrogel according to item 33, wherein G1 comprises or consists of the structure -D^ B-D₂-, wherein:

DT is independently selected from the group consisting of -NH-(CH₂)_r, -NH-(CH₂CH₂O)_S-CH₂CH₂- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

35. The hydrogel according to item 33 or item 34, wherein A and A' are each independently -(CH₂)_d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

36. The hydrogel according to any one of items 33 to 35, wherein G₂ comprises or consists of the structure -D₃-B- wherein: D₃ is selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂-, -NH-(CH₂- CH₂)_S-NH-CO- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and wherein R' is independently selected from the group consisting of -CF₃, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH₂ and -C(O)-NHMe.

37. The hydrogel according to any one of items 33 to 36, wherein at least one enzyme(s) is/are non-covalently bound in the hydrogel to the at least one cofactor(s).

38. The hydrogel according to any one of items 33 to 37, wherein the hydrogel is a swellable or swollen hydrogel matrix.

39. The hydrogel according to item 37, wherein the at least one enzyme(s) is encapsulated in the hydrogel and the at least one cofactor(s) is/are covalently bound to the hydrogel.

40. The hydrogel according to any one of items 33 to 39, wherein the first and/ or the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

41. The hydrogel according to any one of items 33 to 40, wherein the first and/ or the second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

42. The hydrogel according to any one of items 33 to 41 , wherein the first polysaccharide and/ or the second polysaccharide is/ are pullulan.

43. The hydrogel according to any one of items 34 to 42, wherein — A-Di- comprises or consists of the formula -CH₂-CO-NH-(CH2-CH2-O)n-CH2-CH₂-, wherein n is preferably from 1 to 5; and/or wherein -A’-D₂- comprises or consists of the formula -CH₂-CO-NH-(CH₂-CH₂-O)_n-CH₂-CH₂-, wherein n is preferably from 1 to 5. 44. The hydrogel according to any one of items 33 to 43, wherein the first and/ or the second polysaccharide has/ have a concentration of 5-120 mg/ml, preferably 10-80 mg/ml, more preferably 20-60 mg/ml, with respect to the total hydrogel.

45. The hydrogel according to item 37 or item 39, wherein the at least one enzyme(s) for non- covalent immobilization is a dehydrogenase or an enzyme with EC-number 1.1.1 , preferably wherein the at least one enzyme(s) is/ are selected from the group consisting of glucosedehydrogenase, glucose 1 -dehydrogenase, glucose-6-phosphat-dehydrogenase, lactate dehydrogenase, glycerine-aldehyde-3-phosphate-dehydrogenase, indole-3-acetaldehyde reductase, xanthine dehydrogenase, menthol dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, gluconate 5-dehydrogenase, xylulose reductase and sorbitol dehydrogenase, more preferably wherein the at least one enzyme(s) is/ are glucose-6-phosphate- dehydrogenase (G6PD) or glucose-dehydrogenase (GDH).

46. The hydrogel according to any one of items 33 to 45, wherein the at least one cofactor(s) is/are selected from the group consisting of vitamin C, glutathione, coenzyme A, thiamine pyrophosphate, AMP, ADP, ATP, S-adenosyl methionine, biotin, pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺).

47. A cofactor comprising at least one group(s) Z comprising or being a functional group suitable for a 1 ,3-dipolar cycloaddition.

48. The cofactor according to item 47, wherein the cofactor is selected from the group consisting of vitamin C, glutathione, coenzyme A, thiamine pyrophosphate, AMP, ADP, ATP, S- adenosyl methionine, biotin, pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺).

49. The cofactor according to item 47 or item 48, wherein the at least one group(s) Z is/are selected from the group consisting of -C(O)-(CH₂)_r-Q, -C(O)-(CH2CH₂O)_S-CH₂CH2Q and -CO-(CH₂-CH₂-C(O))t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

50. A composition comprising the hydrogel according to any one of items 33 to 46.

51. Use of a hydrogel according to any one of items 33 to 46 a) for non-covalent immobilization of at least one enzyme(s) in the hydrogel, or b) as a catalyst, or c) in a biosensor.

52. A kit comprising the hydrogel according to any one of items 33 to 46.

REFERENCES

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Claims

6. The method according to any one of claims 1 to 5, wherein the method is carried out in the absence of at least one catalyst(s), preferably in the absence of a copper catalyst.

7. The method according to any one of the previous claims, wherein the method is carried out in presence of at least one catalyst, preferably wherein the catalyst is a copper catalyst.

8. The method according to any one of the preceding claims, wherein the first spacer unit A is -(CH₂)d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

9. The method according to any one of the preceding claims, wherein the second spacer unit A’ is -(CH₂)_d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

10. The method according to any one of the preceding claims, wherein:

X is selected from the group consisting of -NH-(CH₂)_r-N₃, -NH-(CH₂CH₂O)_S-CH₂CH₂N₃ and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15;

Y is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Q is ^c — ^CH ; and

11 . The method according to any one of claims 1 to 9, wherein: X is selected from the group consisting of -NH-(CH₂)_r-Q, -NH-(CH₂CH₂O)_S-CH₂CH₂Q and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M' = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Q is ^c — ^CH ; and

Y is selected from the group consisting of -NH-(CH₂)r-N₃, -NH-(CH₂CH2O)s-CH2CH₂N3 and -NH-(CH₂-CH₂-C(O))t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and

Q is - C=CH

12. The method according to any one of the preceding claims, wherein the first polysaccharide and/or the second polysaccharide is/are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

13. The method according to any one of the preceding claims, wherein the first polysaccharide and/or the second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran and mixtures thereof.

14. The method according to any one of the preceding claims, wherein the first polysaccharide and the second polysaccharide are pullulan.

15. The method according to any one of the preceding claims, wherein the first and/ or second polysaccharide is dextran or pullulan and carboxymethylation of at least one OH-group of dextran or pullulan is carried out according to step c) of claim 1 or according to step d) of any one of claims 2 to 5.

16. The method according to claim 1 , wherein in step f) the at least one group Z of the at least one cofactor is reacted in a first 1 ,3-dipolar cycloaddition with a part of the group(s) Y of the functionalized second polysaccharide.

17. The method of claim 16, wherein in step g) the part of the group(s) Y not reacted in step f) of the product of step f) is reacted in a second 1 ,3-dipolar cycloaddition with the group X of the functionalized first polysaccharide.

18. The method according to any one of the preceding claims, wherein in step d) of claim 1 or in step e) of any one of claims 2 to 5 the first polysaccharide is functionalized with 0.01-1.5 of -A- X or -X per monomeric repeating unit of the first polysaccharide.

19. The method according to any one of the preceding claims, wherein in step d) of claim 1 or in step e) of any one of claims 2 to 5 the second polysaccharide is functionalized with 0.01-1.5 of -A'-Y or -Y per monomeric repeating unit of the second polysaccharide.

20. The method according to any one of the preceding claims, wherein d is 1.

21. The method according any one of the previous claims, wherein, in step f) and/or step g), the 1 ,3-dipolar cycloaddition between Z and a part of the group(s) Y is a thermo-induced 1 ,3-dipolar cycloaddition, and the 1,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z is a thermo-induced 1,3-dipolar cycloaddition.

22. The method according to any one of claims 1 to 20, wherein, in step f) and/or step g), the 1 ,3-dipolar cycloaddition between Z and a part of the group(s) Y is a catalyst-induced 1,3- dipolar cycloaddition, and the 1,3-dipolar cycloaddition between the group X and a part of the group(s) Y not reacted with Z is a thermo-induced 1 ,3-dipolar cycloaddition, preferably wherein the catalyst is copper or a copper catalyst.

23. The method according to any one of the preceding claims, wherein the content of N₃, when present, is 0.01-1 .5 N₃ per monomeric repeating unit of the first polysaccharide.

24. The method according to any one of the preceding claims, wherein the content of Q, when present, is 0.01-1 .5 per monomeric repeating unit of the first and/ or the second polysaccharide.

25. The method according to any one of the preceding claims, wherein in step d) of claim 1 or in step e) of any one of claims 2 to 5, -A-X is linked to at least one primary or secondary OH- group of the first polysaccharide, preferably via at least one of C₂, C₃, C₄ or C₆ of the monomeric repeating unit(s) of the first polysaccharide, more preferably via C₆ of the monomeric repeating unit(s) of the first polysaccharide.

26. The method according to any one of the preceding claims, wherein in step d) of claim 1 or in step e) of any one of claims 2 to 5, -A'-Y is linked to at least one primary or secondary OH- group of the second polysaccharide, preferably via at least one of C₂, C₃, C₄ or C₆ of the monomeric repeating unit(s) of the second polysaccharide, more preferably via C₆ of the monomeric repeating unit(s) of the second polysaccharide.

27. The method according to any one of the preceding claims, wherein the method is without the use of toxic reagents, preferably without the use of glutaraldehyde.

28. The method according to any one of the preceding claims, wherein the molecular weight of the unfunctionalized first polysaccharide is in the range from 5 to 2000 kDa.

29. The method according to any one of the preceding claims, wherein the molecular weight of the unfunctionalized second polysaccharide is in the range from 5 to 2000 kDa.

30. Hydrogel obtainable by a method of any one of the claims 1 to 29.

31. The hydrogel according to claim 30, wherein the hydrogel comprises at least one encapsulated enzyme(s).

32. The hydrogel according to claim 30 or 31 , wherein the hydrogel is a swellable or swollen hydrogel matrix.

33. A biocompatible hydrogel comprising, a crosslinked polymer comprising:

- at least one cofactor(s),

- at least one first linker unit(s), which link(s) the first polysaccharide with the second polysaccharide, wherein the structure of the at least one first linker unit(s) is -A-Gi-A’-, -A- Gq— , — A — Gi- , or— Gi- , wherein A is a first spacer unit;

A’ is a second spacer unit; and at least one second linker unit(s), which link(s) the first polysaccharide or the second polysaccharide with the at least one cofactor(s), wherein the structure of the at least one second linker unit(s) is -A-G₂-, -A’-G₂- or-G₂-; wherein A is the first spacer unit;

A’ is the second spacer unit; and

34. The hydrogel according to claim 33, wherein G₁ comprises or consists of the structure -D^ B— D₂— , wherein:

D₁ is independently selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15;

D₂ is independently selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH₂- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; and wherein R' is independently selected from the group consisting of -H, -CF₃, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH₂ and -C(O)-NHMe.

35. The hydrogel according to claim 33 or 34, wherein A and A' are each independently -(CH₂)_d-C(O)- or -C(O)-, wherein d is an integer from 1 to 3.

36. The hydrogel according to any one of claims 33 to 35, wherein G₂ comprises or consists of the structure -D₃-B-, wherein:

D₃ is selected from the group consisting of -NH-(CH₂)_r-, -NH-(CH₂CH₂O)_S-CH₂CH2-, -NH-(CH₂- CH₂)_S-NH-CO- and -NH-(CH₂-CH₂-C(O))_t-CH₂-CH₂-, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15; and wherein R' is independently selected from the group consisting of -CF₃, -C(O)-OMe, -C(O)-OEt, -C(O)-OH, -C(O)-NH₂ and -C(O)-NHMe.

37. The hydrogel according to any one of claims 33 to 36, wherein at least one enzyme(s) is/are non-covalently bound in the hydrogel to the at least one cofactor(s).

38. The hydrogel according to any one of claims 33 to 37, wherein the hydrogel is a swellable or swollen hydrogel matrix.

39. The hydrogel according to claim 37, wherein the at least one enzyme(s) is encapsulated in the hydrogel and the at least one cofactor(s) is/are covalently bound to the hydrogel.

40. The hydrogel according to any one of claims 33 to 39, wherein the first and/ or the second polysaccharide are independently from each other selected from the group consisting of pullulan, alginate, cellulose, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

41. The hydrogel according to any one of claims 33 to 40, wherein the first and/ or the second polysaccharide is/ are independently from each other selected from the group consisting of pullulan, alginate, hyaluronan, dextran, lichenin, lentinan and mixtures thereof.

42. The hydrogel according to any one of claims 33 to 41, wherein the first polysaccharide and/ or the second polysaccharide is/ are pullulan.

43. The hydrogel according to any one of claims 34 to 42, wherein — A-D^ comprises or consists of the formula -CH₂-CO-NH-(CH2-CH2-O)n-CH2-CH₂-, wherein n is preferably from 1 to 5; and/or wherein -A’-D₂- comprises or consists of the formula -CH₂-CO-NH-(CH₂-CH₂-O)_n-CH₂-CH₂-, wherein n is preferably from 1 to 5.

44. The hydrogel according to any one of claims 33 to 43, wherein the first and/ or the second polysaccharide has/ have a concentration of 5-120 mg/ml, preferably 10-80 mg/ml, more preferably 20-60 mg/ml, with respect to the total hydrogel.

45. The hydrogel according to claim 37 or 39, wherein the at least one enzyme(s) for non- covalent immobilization is a dehydrogenase or an enzyme with EC-number 1.1.1 , preferably wherein the at least one enzyme(s) is/ are selected from the group consisting of glucosedehydrogenase, glucose 1 -dehydrogenase, glucose-6-phosphat-dehydrogenase, lactate dehydrogenase, glycerine-aldehyde-3-phosphate-dehydrogenase, indole-3-acetaldehyde reductase, xanthine dehydrogenase, menthol dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, gluconate 5-dehydrogenase, xylulose reductase and sorbitol dehydrogenase, more preferably wherein the at least one enzyme(s) is/ are glucose-6-phosphate- dehydrogenase (G6PD) or glucose-dehydrogenase (GDH).

46. The hydrogel according to any one of claims 33 to 45, wherein the at least one cofactor(s) is/are selected from the group consisting of vitamin C, glutathione, coenzyme A, thiamine pyrophosphate, AMP, ADP, ATP, S-adenosyl methionine, biotin, pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺).

48. The cofactor according to claim 47, wherein the cofactor is selected from the group consisting of vitamin C, glutathione, coenzyme A, thiamine pyrophosphate, AMP, ADP, ATP, S- adenosyl methionine, biotin, pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺).

49. The cofactor according to claim 47 or 48, wherein the at least one group(s) Z is/are selected from the group consisting of -C(O)-(CH₂)_r-Q, -C(O)-(CH₂CH₂O)_S-CH₂CH₂Q and -CO-(CH₂-CH₂-C(O))_t-CH₂-CH₂-Q, wherein wherein M, M’ = H or Me, and wherein W = OMe, OEt, OH, NH₂ or NHMe, with r being an integer from 2 to 20, s being an integer from 1 to 15 and t being an integer from 1 to 15; or

Z is selected from the group consisting of -C(O)-(CH₂)_r-N₃, -C(OHCH₂CH₂O)_S-CH₂CH₂-N₃ and -C(O)-(CH₂-CH₂-C(O))_t-CH₂-CH₂-N₃, with r being an integer from 2 to 20, s being an integer from 1 to 15, and t being an integer from 1 to 15.

50. A composition comprising the hydrogel according to any one of claims 33 to 46.

51. Use of a hydrogel according to any one of claims 33 to 46 a) for non-covalent immobilization of at least one enzyme(s) in the hydrogel, or b) as a catalyst, or c) in a biosensor.

52. A kit comprising the hydrogel according to any one of claims 33 to 46.