EP3433374A1

EP3433374A1 - Glycosylated mono(2-hydroxyethyl) terephthalic acid and glycosylated bis(2-hydroxyethyl) terephthalic acid

Info

Publication number: EP3433374A1
Application number: EP17713276.8A
Authority: EP
Inventors: Jürgen SEIBEL; Malte TIMM
Original assignee: Julius Maximilians Universitaet Wuerzburg
Current assignee: Julius Maximilians Universitaet Wuerzburg
Priority date: 2016-03-23
Filing date: 2017-03-23
Publication date: 2019-01-30
Also published as: CA3018435A1; JP2019512521A; US11279963B2; JP7297444B2; US20200332334A1; WO2017162825A1

Abstract

The invention relates to a compound, characterized by a mono(2-hydroxyethyl) terephthalic acid (MHET) and a bis(2-hydroxyethyl) terephthalic acid which are chemically bound to a saccharide. The invention further relates to a corresponding compound for the use as a synthetic building block for polymers or fine chemicals.

Description

DESCRIPTION

GLYCOSYLATED MONO (2-HYDROXYETHYL) TEREPHTHALIC ACID AND GLYCOSYLATED BIS (2-HYDROXYETHYL) TEREPHTHALIC ACID

FIELD OF THE INVENTION

The invention relates to a mono (2-hydroxyethyl) terephthalic acid or bis (2-hydroxyethyl) terephthalic acid-containing compound, which serves for use as fine chemicals, active ingredient, or starting material for the Biohybhdpolymere.

STATE OF THE ART

S. Yoshida et. al reported on the Baktehen strain. sakaiensis. This is able to attach to the polyethylene terephthalate (abbreviation PET), a polycondensation produced by the thermoplastic family of polyesters, and reduce this. The enzyme PETase is released from the bacterial strain, it hydrolyzes PET to form mono (2-hydroxyethyl) terephthalic acid (MHET) (Figure 1) (Ref: S. Yoshida et al., Science 351, 1961 (2016);

U. Bornscheuer, Science 351, 1 154 (2016). In a further step, MHET is hydrolyzed to the monomers ethylene glycol and terephthalic acid.

DESCRIPTION OF THE INVENTION

PET serves u.a. the production of plastic bottles (PET bottles), films and textile fibers. Worldwide production is 40 million tons per year. So far, it has been difficult to break down PET into usable substances or to use them for synthesis.

This object is achieved according to the invention by the glycosylation of the substance mono (2-hydroxyethyl) terephthalic acid (MHET) and glycosylation of the substance bis (2-hydroxyethyl) terephthalic acid (BHET).

The present invention has the advantage that it allows Abfallbe. Decomposition products of PET production to produce new chemical compounds. These chemical compounds comprise according to the invention glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET). These chemical compounds can be advantageously used in various ways, for example as fine chemicals, agents or biopolymers. Thus, the invention makes it possible to produce new chemicals from PET waste or degradation products.

Another advantageous aspect of the invention is that the glycosylation of mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid (BHET) as well as the other optional process steps of the invention can be carried out almost completely enzymatically.

In addition, the glycosylated compounds according to the invention have advantageous surface properties. Because of these properties, the compounds according to the invention can be used in a variety of ways, such as cell culture substrates.

In addition, according to the invention, the glycosylation of the compounds of the present invention favors their biodegradability and biocompatibility. For this reason, the compounds of the invention can advantageously be used according to the invention in medical applications, in particular in biomedical applications.

The present invention thus provides the following preferred embodiments:

Preferred embodiments:

1 . A compound comprising glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET).

2. A compound according to embodiment 1, wherein the MHET or BHET and a saccharide are chemically bonded to each other via a glycosidic bond.

A compound comprising mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid chemically bound to a saccharide.

4. A compound according to embodiment 3, wherein the MHET or BHET and the saccharide are chemically bonded to each other via a glycosidic bond.

5. A compound according to any one of embodiments 2 to 4, wherein the saccharide is a monosaccharide or disaccharide.

6. A compound according to embodiment 5, wherein the monosaccharide or disaccharide is selected from a group containing hexoses and pentoses.

7. A compound according to embodiment 5, wherein the monosaccharide or disaccharide is selected from α-glucose, β-glucose, α-fructose, β-fructose, α-galactose and β-galactose, α-mannose and β-mannose, xylose, N Acetylglucosamine, glucosamine and glucuronic acid.

8. A compound according to any one of embodiments 1 to 7, wherein the compound is obtainable by enzymatic glycosylation of MHET or BHET. A compound according to any one of embodiments 1 to 8, wherein the compound is formed by enzymatic glycosylation of MHET or BHET. A compound according to embodiment 8 or 9, wherein the enzymatic glycosylation is catalyzed by a glucosidase, a galactosidase or a fructosidase. A compound according to embodiment 10, wherein the enzymatic glycosylation is catalyzed by a glucosidase. A compound according to embodiment 1 1, wherein the glucosidase is an α-glucosidase or a β-glucosidase. A compound according to any one of embodiments 8 to 12, wherein the MHET or BHET is obtainable by bacterial degradation or enzymatic degradation from polyethylene terephthalate (PET). A compound according to any one of embodiments 8 to 13, wherein the MHET or BHET is formed by bacterial degradation or enzymatic degradation from polyethylene terephthalate (PET). A compound according to embodiment 13 or 14, wherein the enzymatic degradation of PET is catalyzed by a hydrolase. A compound according to embodiment 15, wherein the hydrolase is PETase from Idionella sakaiensis. A compound according to embodiment 15 or 16, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1. A compound according to any one of embodiments 13 to 17, wherein the enzyme for the enzymatic glycosylation of MHET or BHET and the enzyme are used together for the enzymatic degradation of PET. A compound according to embodiment 18, wherein a microorganism harboring the enzyme for enzymatic glycosylation and the enzyme for enzymatic degradation of PET is used. A compound according to any one of embodiments 1 to 19, consisting of MHET or BHET, which is chemically bonded to a saccharide via a glycosidic linkage. A compound according to any one of embodiments 1 to 20, wherein the compound has any of the following structures (a) or (b): (a)

wherein Ri comprises a glycosidic bond-bound saccharide, and R ₂ comprises a glycosidic bond-bound saccharide or H.

A compound according to any one of embodiments 1 to 19, wherein the compound further comprises at least one methacrylic residue. A compound according to embodiment 22, wherein the compound has the structure:

wherein Ri comprises a glycosidic bond-bound saccharide and R2 comprises a methacrylic residue. A compound according to embodiment 23, wherein the compound has the structure:

A compound according to embodiment 23, wherein the compound has the structure:

A compound according to any one of embodiments 1 to 19, wherein the compound further comprises a lipophilic side chain. A compound according to embodiment 27, wherein the compound has the structure:

wherein Ri comprises a glycosidic bond-bound saccharide and R2 comprises a lipophilic side chain, preferably a saturated or unsaturated aliphatic hydrocarbon side chain, or a linker.

A compound according to embodiment 28, wherein R _{2 is a} C ₅ to C 20 saturated or unsaturated aliphatic hydrocarbon side chain, preferably a saturated or unsaturated C ₅ to C 15 aliphatic hydrocarbon side chain, more preferably a Cs to C 12 saturated or unsaturated aliphatic hydrocarbon side chain, more preferably a saturated or unsaturated aliphatic C 10 Hydrocarbon side chain and more preferably a saturated aliphatic C10 hydrocarbon side chain is.

A compound according to embodiment 28 selected from a compound having the following structure (a) to (j):

 where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. A compound according to embodiments 2 to 24, 26 to 29 or 30 (b) - (j), wherein the saccharide chemically bonded to MHET or BHET through a glycosidic linkage is methacrylated. Polymer of a compound according to any one of embodiments 1 to 31. The polymer according to embodiment 32, wherein the polymer is a biohybrid polymer. A process for the preparation of a compound comprising glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET), said process comprising the step of enzymatic glycosylation of mono (2-hydroxyethyl) terephthalic acid (MHET ) or bis (2-hydroxyethyl) terephthalic acid (BHET). The method according to embodiment 34, wherein in the step of enzymatic glycosylation, the MHET or BHET and a saccharide are chemically bonded to each other through a glycosidic bond. A method according to embodiment 35, wherein the saccharide is a monosaccharide or disaccharide. The method according to embodiment 36, wherein the monosaccharide or disaccharide is selected from a group containing hexoses and pentoses. Process according to embodiment 37, wherein the monosaccharide or disaccharide is selected from α-glucose, β-glucose, α-fructose, β-fructose, α-galactose and β-galactose, α-mannose and β-mannose, xylose, N-acetylglucosamine , Glucosamine and glucuronic acid. A method according to any one of embodiments 34 to 38, wherein the enzymatic glycosylation is catalyzed by a glucosidase, a galactosidase or a fructosidase. A method according to embodiment 39, wherein the enzymatic glycosylation is catalyzed by a glucosidase. A method according to embodiment 40, wherein the glucosidase is an α-glucosidase or a β-glucosidase. A method according to any of embodiments 34 to 41, wherein the MHET or BHET is obtainable by bacterial degradation or enzymatic degradation from polyethylene terephthalate (PET). A method according to any of embodiments 34 to 42, wherein the method prior to the enzymatic glycosylation step preferably comprises a step of bacterially degrading or enzymatically degrading polyethylene terephthalate (PET) to MHET or BHET. A process according to embodiment 42 or 43, wherein the enzymatic degradation of PET is catalyzed by a hydrolase. The method according to embodiment 44, wherein the hydrolase is PETase from Idionella sakaiensis. A method according to embodiment 44 or 45, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1. A method according to any one of embodiments 42 to 46, wherein the enzyme for the enzymatic glycosylation of MHET or BHET and the enzyme for the enzymatic degradation of PET are used together. A process according to Embodiment 47, wherein a microorganism harboring the enzyme for enzymatic glycosylation and the enzyme for enzymatic degradation of PET is used. The method according to any of embodiments 34 to 48, wherein in a further step, a methacrylic residue is chemically bonded to the glycosylated MHET or BHET, the methacrylic residue being preferentially bound by enzymatic esterification to the glycosylated MHET or BHET, wherein the enzymatic esterification is preferably catalyzed by a lipase. Process according to embodiment 49, wherein the methacrylic radical is chemically bound to the glycosylated MHET or BHET by addition of vinylmethyl methacrylate. Method according to at least one of embodiments 34 to 48, wherein in a further step a lipophilic side chain, preferably a saturated or unsaturated aliphatic hydrocarbon side chain, is chemically bonded to the glycosylated MHET or BHET. Method according to embodiment 51, wherein the lipophilic side chain, a saturated or unsaturated C ₅ to C 20 hydrocarbon side chain, preferably a saturated or unsaturated C ₅ to C 15 hydrocarbon side chain, more preferably a saturated or unsaturated Cs-C12 hydrocarbon side chain, more preferably a saturated or unsaturated C10 hydrocarbon side chain and particularly preferably a saturated C10 hydrocarbon side chain. A process according to embodiment 52, wherein the lipophilic side chain is a saturated C10 hydrocarbon side chain, process according to any of embodiments 51 to 53, wherein the lipophilic side chain is attached by the addition of decanol. A method according to any one of embodiments 34 to 54, wherein after the step of enzymatic glycosylation, a step of polymerization follows. A polymer obtainable by the process of any one of embodiments 34 to 55. The polymer of embodiment 56, wherein the polymer is a bio-hybrid polymer. Microorganism harboring at least one enzyme for the enzymatic glycosylation of MHET and / or BHET and at least one enzyme for the enzymatic degradation of PET. A microorganism according to embodiment 58, wherein the microorganism is a recombinant microorganism and the enzyme for the enzymatic glycosylation of MHET and / or BHET and / or the enzyme for the enzymatic degradation of PET is a recombinant enzyme. A microorganism according to embodiment 58 or 59, wherein the enzyme for enzymatic glycosylation is a glucosidase, a galactosidase or a fructosidase. The microorganism according to embodiment 60, wherein the enzymatic glycosylation enzyme is a glucosidase. The microorganism according to embodiment 61, wherein the glucosidase is an α-glucosidase or a β-glucosidase. The microorganism according to any one of the embodiments 58 to 62, wherein the enzyme for enzymatic degradation of PET is a hydrolase. A microorganism according to embodiment 63, wherein the hydrolase is PETase from Idionella sakaiensis. The microorganism according to embodiment 63 or 64, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1. A compound resulting from the enzymatic glycosylation of mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid to produce MHET or bis (2-hydroxyethyl) terephthalic acid by bacterial degradation or enzymatic degradation from PET.

A compound characterized by a mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid chemically bound to a saccharide. 68. A compound according to embodiments 66 and 67, wherein the MHET or bis (2-hydroxyethyl) terephthalic acid and the saccharide are chemically bonded to each other via a glycosidic bond. 69. A compound which can be polymerized after glycosylation of mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid.

70. A compound according to embodiment 66 or 67, wherein the saccharide is a monosaccharide or disaccharide.

71. A compound according to embodiment 70, wherein the monosaccharide or disaccharide is selected from a group comprising hexoses and pentoses (α-glucose, β-glucose, α-fructose, β-fructose, α-galactose and β-galactose, α-mannose and β -Mannose, xylose, N-acetylglucosamine,

Glucosamine, glucuronic acid).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Figure: U. Bornscheuer, Science 351, 1 154 (2016); Degradation of PET by /. sakaiensis.

FIG. 2: Exemplary representation of the glucosylation of PET obtained. FIG

MHET through the use of microorganisms.

Fig. 3: A biohybrid polymer of glucosylated MHET.

Fig. 4: Glycosylated MHET serves as an active ingredient, fine chemical or the synthesis of polymers.

Fig. 5: Exemplary structure of a biohybrid polymer of glucosylated MHET or bis (2-hydroxyethyl) terephthalic acid.

Fig. 6: Glycosylated MHET and glycosylated bis (2-hydroxyethyl) terephthalic acid can be linked to methacrylate. For this purpose, methacrylate is enzymatically esterified with one or more alcohol functions of MHET or bis (2-hydroxyethyl) terephthalic acid.

Figure 7: Glycosylated MHET and glycosylated bis (2-hydroxyethyl) terephthalic acid can be linked to methacrylate. For this purpose, methacrylate is enzymatically esterified with one or more alcohol functions of MHET or bis (2-hydroxyethyl) terephthalic acid.

Fig. 8: Synthesized glycosylated BHET methacrylates and glycosylated MHET methacrylates can be prepared by means of a radical initiator, e.g. Polymerize potassium peroxodisulfate.

Figure 9: Synthesized glycosylated BHET methacrylates and glycosylated MHET methacrylates can be prepared with the aid of a radical initiator, e.g. Polymerize potassium peroxodisulfate.

Fig. 10: Synthesized glycosylated BHET methacrylates and glycosylated MHET methacrylates can be prepared with the aid of a radical initiator, e.g. Polymerize potassium peroxodisulfate.

Figure 1: Glycosylated MHET and glycosylated bis (2-hydroxyethyl) terephthalic acid may also be linked to aliphatic alcohols. For example, e.g. Dodecanol enzymatically esterified with ΒΗΕΤ-α-Glc.

Fig. 12: Thiols, amines and other alcohols are also conjugated with glycosylated BHET instead of the decanol.

FIG. 13: Exemplary representation of the structure of α-glycosylated MHET. FIG. Fig. 14: Exemplary representation of the structure of MHET.

FIG. 15: Exemplary representation of the structure of β-glycosylated MHET. FIG.

Fig. 16: ¹ H-NMR spectrum of .beta.-glycosylated MHET.

Fig. 17: ¹³ C-NMR spectrum of .beta.-glycosylated MHET.

FIG. 18: Exemplary representation of the structure of di-1,3-β-glucosylated MHET. FIG.

Fig. 19: Exemplary representation of the structure of β-galactosylated MHET.

Fig. 20: ¹ H-NMR spectrum of β-galactosylated MHET.

Fig. 21: ¹³ C-NMR spectrum of beta-galactosylated MHET.

Fig. 22: Exemplary representation of the structure of α-glucosylated bis (2-hydroxyethyl) terephthalate.

Fig. 23: ¹ H-NMR spectrum of α-glucosylated bis (2-hydroxyethyl) terephthalate. Fig. 24: ¹³ C-NMR spectrum of α-glucosylated bis (2-hydroxyethyl) terephthalate. FIG. 25: Exemplary representation of the structure of di-1,6-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

FIG. 26: ¹ H-NMR spectrum of di-1,6-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

FIG. 27: ¹³ C-NMR spectrum of di-1,6-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

Fig. 28: Exemplary representation of the structure of β-glucosylated bis (2-hydroxyethyl) terephthalate.

Fig. 29: ¹ H-NMR spectrum of β-glucosylated bis (2-hydroxyethyl) terephthalate. Fig. 30: ¹³ C-NMR spectrum of β-glucosylated bis (2-hydroxyethyl) terephthalate. Fig. 31: Exemplary representation of the structure of β-galactosylated bis (2-hydroxyethyl) terephthalate.

Figure 32: ¹ H-NMR spectrum of β-galactosylated bis (2-hydroxyethyl) terephthalate. Fig. 33: ¹³ C-NMR spectrum of β-galactosylated bis (2-hydroxyethyl) terephthalate. FIG. 34: Exemplary representation of the structure of di-β-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

FIG. 35: ¹ H-NMR spectrum of di-β-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

FIG. 36: ¹³ C-NMR spectrum of di-β-glucosylated bis (2-hydroxyethyl) terephthalate. FIG.

Fig. 37: Exemplary representation of the structure of glycosylated MA-BHET-α-Glc. Fig. 38: ¹ H-NMR spectrum of glycosylated MA-BHET-α-Glc.

Fig. 39: ¹³ C-NMR spectrum of glycosylated MA-BHET-α-Glc.

FIG. 40: Exemplary representation of the structure of glycosylated MA ₂ -BHET-α-

Glc.

Fig. 41: ¹ H-NMR spectrum of glycosylated MA ₂ -BHET-a-Glc.

Fig. 42: ¹³ C-NMR spectrum of glycosylated MA ₂ -BHET-a-Glc.

FIG. 43: Exemplary representation of the structure of glycosylated a-Glc-MHET

Decanol.

Figure 44: ¹ H-NMR spectrum of glycosylated α-Glc-MHET decanol.

Figure 45: ¹³ C NMR spectrum of glycosylated α-Glc-MHET decanol.

Fig. 46: Exemplary representation of the structure of α-glucosylated MHET.

Fig. 47: ¹ H-NMR spectrum of α-glucosylated MHET.

Fig. 48: ¹³ C-NMR spectrum of α-glucosylated MHET.

Fig. 49: detection of the polymer; shown is the ¹ H-NMR spectrum of the polymer. Fig. 50: Exemplary ¹³ C NMR spectrum of MHET shown

DETAILED DESCRIPTION OF THE INVENTION

Definitions and general techniques

Unless otherwise indicated, the terms used in the present invention are to be understood as meaning to those skilled in the art.

Terms such as "comprise" or "comprising" are used here in such a way that each occurrence of these terms can optionally be replaced by "consist of" or "consisting of".

Terms such as "contained" or "containing" are used herein in the meaning familiar to those skilled in the art. Optionally, each occurrence of these terms can be replaced by "consist of" or "consisting of".

The term "compound" is used herein as it would be understood by those skilled in the art, ie, meaning in particular "chemical compound". The term "compound comprising glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET)" is to be understood that with the respective mono (2-hydroxyethyl) terephthalic acid (MHET) or Bis (2-hydroxyethyl) terephthalic acid (BHET) is also optionally meant esters, amides, thioesters and ethers of the respective mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid (BHET) are esters, amides, thioesters and ethers of the respective mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid (BHET) not from the term "compound comprising glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET ) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET) ".

A glycosidic bond is the chemical bond between the anomeric carbon atom of a glycan, ie a saccharide, and the hetero or rare carbon atom of an aglycone, a "non-sugar." A glycosidic bond can have both an α-glycosidic bond and a β-glycosidic bond The prefixes α and β define the anomeric configuration.

A glycosidic bond can be formed by enzymatic glycosylation. Enzymatic glycosylation can be catalyzed by a glycosidase, glycosidases belong to the enzyme class of hydrolases and are classified under EC 3.2.1. Glycosidases can reversibly hydrolyze a- or ß-glycosidic bonds. Examples of glycosidases include glucosidases, galactosidases, sialidases, mannosidases, fucosidases, xylosidases, glucuronidase, α-L-arabinofuranosidase, exo-β-glucosaminidase, galacturonase, glucosaminidase, N-acetylglucosaminidase and fructosidases. Specifically, a glycosidase may be an α- or β-glucosidase. Furthermore, a glycosidase may be a β-galactosidase.

Lipophile may in particular mean hydrophobic. Lipophilic side chains include, for example, hydrocarbons, and especially aliphatic hydrocarbons. A hydrocarbon side chain is a side chain consisting of carbon and hydrogen. The hydrocarbon side chain may be saturated or unsaturated. An unsaturated hydrocarbon side chain includes both a mono- and polyunsaturated hydrocarbon side chain. The hydrocarbon side chain may include double bonds and triple bonds between two carbon atoms. Furthermore, if appropriate, individual carbon atoms in the hydrocarbon chain can be replaced by heteroatoms. The hydrocarbon chain may be unbranched or branched. The hydrocarbon chain may be acyclic or cyclic.

In multiple glycosylations, the acceptor is typically first glycosylated. If enough monoglycosylated acceptor is present in the reaction mixture, multiple glycosylations can occur.

As glycosidases for the enzymatic syntheses can be used, for example: α-glucosidase (sucrose isomerase): in suspension of microorganisms (Protaminobacter rubrum Z 12 (CBD 574.77)). It can preferably be used for the monoglucosylation. The optimum pH for the enzymatic reaction is pH = 6; α-glucosidase, GTFR from Streptococcus oralis first transfers glucose to the acceptor. After a prolonged reaction time, another glucose unit is transferred to the glucose of the glucosylated acceptor, as demonstrated, for example, in Example (6). The optimum pH for the enzymatic reaction is pH = 6. GTFR from S. oralis can be used as described in Fujiwara et al. (Fujiwara et al., Infect Immun., 2000; 68 (5): 2475-83 2000). almonds β-glucosidase EC.3.2.1 .21, CAS 9001 -22-3 (BioChemika 49290, WA10531). The optimum pH for the enzymatic reaction is pH = 5.2. Aspergillus oryzae β-galactosidase, CAS 9031-1 1 -2 (Sigma G5160-25KU). The optimum pH for the enzymatic reaction is pH = 5.2. Yeast β-fructosidase (Boehringer Mannheim GmbH, order number 104914). The optimum pH for the enzymatic reaction is pH = 5.2.

For enzymatic esterification or transesterification, the lipase Novozymes 435 can be used. The optimum pH for the enzymatic reaction is pH = 7.5.

General description for the separation of reaction mixtures and isolation of reaction products according to the invention:

All compounds of the invention may be isolated. Accordingly, all processes according to the invention preferably comprise isolating the respective compound.

This can preferably be carried out as follows:

The reaction mixtures are distilled off from solvents or lyophilized. Glycosylated MHET or glycosylated BHET is separated by column chromatography. The chromatography material is Kieselgel 60 (Macherey Nagel, 0.044-0.063 mm). 100 g of silica gel are used per gram of reaction mixture to be separated. The separation vessel are glass columns. Preference is given to using columns with a diameter of 1: 20. The silica gel is used in a solvent mixture of ethyl acetate: isopropanol: water (volume ratio 6: 3: 1), in the case of nonpolar substances, a mixture of ethyl acetate: methanol (volume ratio 12: 1) is used, the mixture is padded and the column is filled. Subsequently, the reaction mixture is dissolved in as little eluent as possible (preferably 1 g in 1 ml) and added to the column. The separation is carried out by elution with the eluent. The eluate is collected in vessels (a 10 mL) and determined by HPLC in which vessels the product is contained. The vessels are combined with product and the solvents are evaporated. A solid is obtained. embodiments

In the invention, MHET or bis (2-hydroxyethyl) terephthalic acid is glycosylated by means of an enzyme and a glycosubstrate. As glycosubstrate, disaccharides (e.g., sucrose, lactose, maltose), oligosaccharides (maltooligosaccharides) or polysaccharides (dextran, fructo-oligosaccharides, chitin, mannan, cellulose) are used. The preparation of glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid can be carried out starting from MHET or bis (2-hydroxyethyl) terephthalic acid. MHET or bis (2-hydroxyethyl) terephthalic acid can be prepared from PET by means of a hydrolase e.g. with the PETase from l.sakaiensis, e.g. having the amino acid sequence shown in SEQ ID NO: 1:

MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVR SFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFV VITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGWSM GGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSAL PIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYS TFACENPNSTRVSDFRTANCS (SEQ ID NO: 1). Both enzymes can be used together. A microorganism harboring both enzymes can also be used for production (see Fig. 2).

The compound according to the invention is characterized by a chemically bound to a saccharide MHET or bis (2-hydroxyethyl) terephthalic acid. Glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid can be used as an active ingredient. Glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid can also be polymerized to biohybrid polymers (see Fig. 3).

More preferably, the MHET or bis (2-hydroxyethyl) terephthalic acid and the saccharide are chemically bonded to each other via a glycosidic bond. A glycosidic bond is the chemical bond between the anomeric carbon atom of a glycan, that is, a saccharide, and the hetero or rare carbon atom of an aglycone, a "non-sugar." Compounds containing a glycosidic bond commonly referred to as glycosides. The hydrolysis of a glycosidic bond in a glycoside is reversibly catalyzed in particular by glucosidases, wherein the glycon, in this case the saccharide, and the aglycone, here MHET or bis (2-hydroxyethyl) terephthalic acid, are liberated while consuming a water molecule.

Preferably, the saccharide is a monosaccharide. Monosaccharides are simple sugars having a backbone of at least three chained carbon atoms which have a carbonyl group (-C = O) and at least one hydroxyl group (-OH).

Glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid serves as fine chemical, active ingredient or starting material for biohybrid polymers (see Fig. 4). Thus, by polycondensation of glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid, a thermoplastic polymer produced from the polyester family can be obtained. Thus, polycondensation of glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid will give rise to a thermoplastic polymer from the polyester family.

Likewise, a polyester can be prepared by transesterification of di- and monoglucosylated bis (2-hydroxyethyl) terephthalic acid with ethanediol. Since it is an equilibrium reaction, split-off ethanediol is distilled off. It comes to the accumulation of glycosylated bis (2-hydroxyethyl) terephthalic acid. Likewise, mono- and diglycosylated bis (2-hydroxyethyl) terephthalic acid is esterified with terephthalic acid directly to a polyester.

As the catalyst, e.g. Antimony (III) oxide can be used.

A biohybrid polymer of glucosylated MHET or bis (2-hydroxyethyl) terephthalic acid may adopt the structure of FIG.

Glucosylated MHET and BHET methacrylates

Similarly, glycosylated MHET and glycosylated bis (2-hydroxyethyl) terephthalic acid can be linked to methacrylate. This is methacrylate enzymatically esterified with one or more alcohol functions of MHET or bis (2-hydroxyethyl) terephthalic acid (FIG. 6, FIG. 7). In the reaction of ΒΗΕΤ-α-Glc with vinylmethyl acrylate at 50 ° C and the lipase Novozymes 435 arises when using the solvent tert-butyl alcohol, the di-esterified product MA ₂ -BHET-a-Glc and the glycosylated ΜΑ-ΒΗΕΤ-α- Glc (Figure 6).

Polymerization of qv cosylated BHET methacrylates and qv cosylated MHET methacrylates

The glycosylated BHET methacrylates and glycosylated MHET methacrylates so synthesized can be purified by means of a radical initiator, e.g. Potassium peroxodisulfate polymerize (Fig. 8, Fig.9, Fig.10).

Likewise, it is also possible to mix various methacrylates in different ratios with each other and to polymerize the mixtures. Thus block polymers can also be formed.

Glucosylated MHET and BHET lipids

Glycosylated MHET and glycosylated bis (2-hydroxyethyl) terephthalic acid can also be linked to aliphatic alcohols. For this purpose, e.g. Decanol enzymatically esterified with ΒΗΕΤ-α-Glc (Fig. 1 1). In the reaction of BHET-α-Glc with decanol at 50 ° C. and the lipase Novozymes 435, tert-butanol preferably forms α-Glc-MHET-decanol when the solvent is used (FIG. 11). Likewise, instead of the decanol, thiols, amines and other alcohols can be conjugated with glycosylated BHET. This allows the introduction of different linkers, which i.a. Contain biotin or can be used for bioorthogonal click reactions for the construction of cell culture scaffolds (FIG. 12).

Most preferably, the monosaccharide is selected from a group containing pentoses and hexoses. hexoses have a carbon backbone with six carbon atoms and basically differ by the nature of the carbonyl function. In a non-terminal carbonyl function (Ri-C (O) -R ₂ ), a keto group, one speaks of ketohexoses. A terminal carbonyl function, an aldehyde group, is aldohexose.

Pentoses (C ₅ H 10 O ₅ ) have a carbon backbone with five carbon atoms.

The hexoses are preferably selected from a group containing α-glucose, β-glucose, α-fructose, β-fructose, α-mannose, β-mannose, α-galactose and β-galactose, N-acetylglucosamine, glucosamine, glucuronic acid , Depending on which saccharide is chemically bound to the MHET or bis (2-hydroxyethyl) terephthalic acid, the nomenclature of the compound differs. In the case of α- or β-glucose chemically bound to the MHET or bis (2-hydroxyethyl) terephthalic acid, the compound is α- or β-glucosylated MHET or bis (2-hydroxyethyl) terephthalic acid. In the case of α- or β-galactose as chemically bound saccharide, this is referred to as α- or β-galactosylated MHET or bis (2-hydroxyethyl) terephthalic acid. If an α- or β-fructose is bound as a saccharide to the MHET, it is α- or β-fructosylated MHET or bis (2-hydroxyethyl) terephthalic acid.

Advantageously, the pentoses are selected from a group containing xylose, arabinose or xylose.

In a particularly advantageous embodiment of the invention, α-glucose is linked via a glycosidic bond with MHET or bis (2-hydroxyethyl) terephthalic acid. The synthesis of the α-glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid takes place with dehydration of α-glucose and MHET or bis (2-hydroxyethyl) terephthalic acid. This produces α-glycosylated MHET or bis (2-hydroxyethyl) terephthalic acid.

The compound α-glycosylated MHET is preferably characterized by the structural formula shown in FIG. In the synthesis of the α-glycosylated MHET, a glycosidic linkage is made between the α-glucose and the MHET. The bonding is particularly advantageously carried out selectively via the hydroxyl group of the MHET bonded to the 1 ^' carbon atom. The oxygen atom bridging the α-glucose with the MHET comes from the MHET. In principle, this linkage-independently of the saccharide used-is preferably carried out selectively via the hydroxyl group of the MHET bound to the 1 ^' carbon atom. The enzyme used is responsible for the selectivity of the bond formation at the 1 ^'carbon atom.

In the following, embodiments of the invention will be explained in more detail by means of production methods of the connection.

EXAMPLES

All following embodiments were carried out experimentally.

(1) Production of MHET

MHET is shown by way of example in FIG. 14.

To a solution of terephthalic acid (2.00 g, 12.04 mmol) in DMF (15 mL) was added NaHCO3 (3.05 g, 36 mmol). The mixture was stirred at 85 ° C for 50 min. Then 2-bromo-1-ethanol (0.75g, 426μΙ, 6mmol) was added and stirring was continued at 85 ° C for 4h. The course of the reaction is monitored by thin-layer chromatography (MeOH / CH ₂ Cl ₂ , 1: 4). Then the reaction mixture is filtered through silica and after concentration of silica (MeOH / CH ₂ Cl ₂ , 1: 4) separated.

For the spectroscopic detection of MHET ¹ H-NMR spectra and ¹³ C-NMR spectra were recorded.

The ¹ H NMR spectrum 1 was recorded at 400 MHz in deuterated dimethyl sulfoxide, the assignment of the signals is shown in Table 1.

Table 1: Signal assignment ¹ H-NMR spectrum of MHET Relative coupling constant δ [ppm] Multiplicity assignment

Integral J [Hz]

8.01 H-3, H-7 2H doublet 8.37

7.93 H-4, H-6 2H doublet 8.37

4.28 H-1 '2H triplet

3.71 H-2 '2H triplet

In Fig. 50, an exemplary ¹³ C-NMR spectrum of MHET is shown. The ¹³ C NMR spectrum 1 1 was also recorded in deuterated dimethyl sulfoxide at 100 MHz. The assignment of the signals is listed in Table 2.

Table 2: Signal assignment of the ¹³ C NMR spectrum of MHET

The chemical shifts of the signals of the H or C atoms and of the integral values (in the case of the H atoms) which can be taken from the tables clearly determine MHET. Likewise, a mass spectrum was measured. This also confirms MHET.

MS (ESI, negative): calculated for Ci ₀ H ₉ O ₅ (MH) ^" 209.045, according to 209.045 (2) Preparation of g-glucosylated MHET

MHET (0.05 mol / L) and sucrose (0.4 mol / L) are dissolved in 0.05 M phosphate buffer (0.05 mol / L, pH = 6) and a suspension of microorganisms (Protaminobacter rubrum Z 12 ( CBS 574.77)). In an alternative embodiment, an α-glucosidase solution (100 U in phosphate buffer) is added. Sucrose consists of aD-glucose and ß-D-fructose, which are linked via an a, ß-1, 2-glycosidic bond. The microorganism Protaminobacter rubrum Z 12 contains an enzyme, an α-glucosidase. It catalyzes the cleavage of the sucrose used in aD-glucose and ß-D-fructose. The aD-glucose chemically binds to the MHET during the reaction.

For this purpose, the reaction mixture is shaken at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. The product α-glycosylated MHET is obtained. The optimal product formation is determined by continuous sampling and thin-layer chromatography. α-glucosylated MHET is exemplified in FIG.

The test results of the embodiment with a suspension of microorganisms (Protaminobacter rubrum Z 12 (CBS 574.77)) are as follows.

The product is confirmed by mass spectrometry (MT203).

MS (ESI, negative): calculated for Ci ₆ H ₁ ₉ Oi ₀ (M-HV 371 .0978, according to 371 .09727

(3) Preparation of β-glucosylated MHET

MHET (0.05 mol / L) and cellobiose (0.4 mol / L) are dissolved in Natnumacetatpuffer (0.05 mol / L, pH = 5.2) or phosphate buffer (0.05 mol / L, pH = 7) and added a β-glucosidase solution (100 U in Natnumacetatpuffer or phosphate buffer). The cellobiose is a disaccharide of two β-1,4-glucosidically linked glucose molecules. The enzyme β-glucosidase allows the degradation of cellobiose to glucose. The glucose chemically binds to the MHET during the reaction.

The reaction mixture is also shaken at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. After column chromatography, β-glucosylated MHET is obtained. β-glucosylated MHET is exemplified in FIG. The experimental results of the sodium acetate buffer embodiment are as follows.

The product is confirmed by mass spectrometry (MT204).

MS (ESI, negative): calculated for Ci ₆ H ₁ ₉ Oi ₀ (M-HV 371 .0978, according to 371 .0972.

Further experimental results are shown in FIGS. 16, 17 and the following tables.

Table 3: Signal assignment ¹ H-NMR spectrum of β-glucosylated MHET

Table 4: Signal assignment ¹³ C-NMR spectrum of β-glucosylated MHET δ [ppm] assignment 167.50 C-1 ", C-8"

134.17 C-2 ", C-5"

130.52 C-3 ", C-4",

130.50 C-6 ", C-7"

104.68 C-1

78.04 C-5

77.99 C-3

75.01 C-2

71.55 C-4

68.62 c-r

65.75 C-2 '

62.71 C-6

Di-1 .3-.beta.-glucosylated MHET

MS (ESI, positive): calculated for C ₂ 2H ₃ oNaOi ₅ (M-HV 557.1482, corresponding to 557.1477) Di-1,3-β-glucosylated MHET is exemplified in Figure 18. Further experimental results are shown in the following tables shown.

Table 5: Signal assignment ¹ H NMR spectrum of di-1,3-β-glucosylated

MHET

Doublet of

3.88 H-6a 1H 11,786.54

doublet

Doublet of

3.69 H-6b 1H 11,865.46

doublet

Doublet of

3.64 H-6 "'b 1H 11,766.16

doublet

3.57 H-3 1H triplet 8.57

H-2, H-2 '",

H-3 '", H-4,

3.45-3.21 7H Multiplett

H-4 '", H-5,

H-5 ' "

Table 6: Signal assignment ¹³ C-NMR spectrum of di-1,3-β-glucosylated

MHET

62.56 C-6, C-6 '

(4) Preparation of β-galactosylated MHET

MHET (0.05 mol / L) and lactose (0.4 mol / L) are dissolved in sodium acetate buffer (0.05 mol / L, pH = 5.2) or phosphate buffer (0.05 mol / L, pH = 7). and added a β-galactosidase solution (100 U in sodium acetate buffer or phosphate buffer). Lactose consists of D-galactose and D-glucose, which are linked by a β-1,4-glycosidic bond. β-galactosidase enzymatically catalyzes the hydrolysis of this bond to produce galactose, which chemically binds to the MHET during the reaction.

The reaction mixture is shaken for this purpose at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. The product β-galactosylated MHET is isolated by column chromatography. β-galactosylated MHET is exemplified in FIG. The experimental results of the sodium acetate buffer embodiment are as follows.

The product is confirmed by mass spectrometry (MT206).

MS (ESI, negative): calculated for C ₆ H1 ₉ Oi ₀ (MH) ^"371 .0978; gem 371 .0975 Further experimental results are shown in Fig 20, Fig 21 and in the following tables.....

Table 7: Signal assignment ¹ H-NMR spectrum of β-galactosylated MHET

4.22 Η-1 'a 1 H Multiplett

3.96 Η-1 'b 1 H multiplet

Doublet of

3.84 H-4 1 H 3.32 0.92

doublet

3.76 H-6a 1 H Multiplett

3.72 H-6b 1 H Multiplett

Doublet of

3.56 H-2 1 H 9.72 7.52

doublet

3.56 - 3.51 H-3 1 H Multiplett

Doublet of

3.48 H-5 1 H 9.72 3.32

doublet

Table 8: Signal assignment ¹³ C-NMR spectrum of β-galactosylated MHET

(5) Preparation of fructosylated MHET

MHET (0.05 mol / L) and sucrose (0.4 mol / L) were dissolved in sodium acetate buffer (0.05 mol / L, pH = 5.2) and a fructosidase solution (100 U in sodium acetate buffer) added. The fructosidase solution catalyzes the Cleavage of the sucrose into aD-glucose and ß-D-fructose enzymatically. The ß-D-fructose binds chemically to the MHET during the reaction.

The reaction mixture is shaken for this purpose also at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped.

(6) Preparation of g-glucosylated bis (2-hydroxyethyl) terephthalate

Bis (2-hydroxyethyl) terephthalate (0.05 mol / L) and sucrose (0.4 mol / L) are dissolved in 0.05 M phosphate buffer (0.05 mol / L, pH = 7) or are in 0, Dissolve 05 M phosphate buffer (0.05 mol / L, pH = 6) and add a suspension of microorganisms (Protaminobacter rubrum Z 12 (CBD 574.77)). In an alternative embodiment, an α-glucosidase solution (100 U in phosphate buffer) is added.

Sucrose consists of α-D-glucose and β-D-fructose, which are linked via an α, β-1,2-glycosidic linkage. The microorganism Protaminobacter rubrum Z 12 contains an enzyme, an α-glucosidase. It catalyses the cleavage of the sucrose used in α-D-glucose and β-D-fructose. The α-D-glucose chemically binds to bis (2-hydroxyethyl) terephthalate during the reaction.

For this purpose, the reaction mixture is shaken at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. The product α-glycosylated bis (2-hydroxyethyl) terephthalate is obtained. The optimal product formation is determined by continuous sampling and thin-layer chromatography.

α-glucosylated bis (2-hydroxyethyl) terephthalate is exemplified in FIG. Experimental results of the embodiment with 0.05 M phosphate buffer (0.05 mol / L, pH = 6) and a suspension of microorganisms (Protaminobacter rubrum Z 12 (CBD 574.77)) are shown in Fig. 23, Fig. 24 and in the following tables shown.

Table 9: Signal assignment ¹ H-NMR spectrum of α-glucosylated bis (2-hydroxyethyl) terephthalate Relative coupling constant δ [ppm] Multiplicity assignment

Integral J [Hz]

H-3 ", H-4",

8.16 4H Singlet

H-6 ", H-7"

4.89 H-1 1H doublet 3.96

4.57-4.53 H-2 '"2H Multiplett

4.41 H-1 '"2H Multiplett

4.09 Η-1'a 1H Multiplett

3.87 H-2 '2H Multiplett

3.88-3.84 Η-1'b 1H Multiplett

3.79 - 3.74 H-6a 1H Multiplett

H-3, H-4,

3.69 - 3.62 3H Multiplett

H-6b

Doublet of

3.41 H-2 1H 9,723.76

doublet

3.30 H-5 1H Multiplett

Table 10: Signal assignment ¹³ C-NMR spectrum of α-glucosylated bis (2-hydroxyethyl) terephthalate

67.96 C-1 '"

67.00 c-r

65.70 C-2 '"

62.59 C-6

61.05 C-2 '

Di-1,6-glucosylated bis (2-hydroxyethyl) terephthalate

Di-a-1, 6-glucosylated bis (2-hydroxyethyl) terephthalate is exemplified in FIG. Further experimental results are shown in FIGS. 26, 27 and in the following tables.

Table n: Signal assignment ¹ H NMR spectrum of di-a-1, 6-glucosylated

Bis (2-hydroxyethyl) terephthalate

doubled for

3.73 H-5 "" 1 H Triplet 9.26

Doublet of

3.21 H-2 "" 1 H 9.06 7.78

doublet

3.31H-5 1H MeOH

Table 12: Signal assignment ¹³ C-NMR spectrum of di-a-1, 6-glucosylated

Bis (2-hydroxyethyl) terephthalate

(7) Preparation of β-glucosylated bis (2-hydroxyethyl) terephthalate

Bis (2-hydroxyethyl) terephthalate (0.05 mol / L) and cellobiose (0.4 mol / L) are dissolved in Natnumacetatpuffer (0.05 mol / L, pH = 5.2) and a ß-glucosidase solution (100 U in Natnumacetatpuffer) was added. The cellobiose is a disaccharide of two β-1,4-glucosidically linked glucose molecules. The enzyme β-glucosidase allows the degradation of cellobiose to glucose. The glucose chemically binds to bis (2-hydroxyethyl) terephthalate during the reaction.

The reaction mixture is also shaken at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. After column chromatography, β-glucosylated bis (2-hydroxyethyl) terephthalate is obtained (see FIG. 28). Further experimental results are shown in FIG. 29, FIG. 30 and in the following tables. Table 13: Signal assignment ¹ H-NMR spectrum of β-glucosylated bis (2-hydroxyethyl) terephthalate

3.30 - 3.28 H-4 1 H Multiplett

Doublet of

3.21 H-2 1 H 9.06 7.78

doublet

Table 14: Signal assignment ¹³ C-NMR spectrum of β-glucosylated bis (2-hydroxyethyl) terephthalate

(8) Preparation of β-qalactosylated bis (2-hydroxyethyl) terephthalate

Bis (2-hydroxyethyl) terephthalate (0.05 mol / L) and lactose (0.4 mol / L) are dissolved in sodium acetate buffer (0.05 mol / L, pH 5.2) or phosphate buffer (0.05 mol / L). pH = 7) and a β-galactosidase solution (100 U in Natnumacetatpuffer or Phosphate buffer). Lactose consists of D-galactose and D-glucose, which are linked via a β-1,4-glycosidic bond. Β-galactosidases enzymatically catalyzes the hydrolysis of this bond to produce galactose, which chemically reacts with bis (2 - hydroxyethyl) terephthalate binds.

The reaction mixture is shaken for this purpose at a temperature of 37 ° C in a water bath. After optimal product formation, the reaction is stopped. The product β-galactosylated bis (2-hydroxyethyl) terephthalate is isolated by column chromatography. β-galactosylated bis (2-hydroxyethyl) terephthalate is exemplified in FIG. 31. Experimental results of the sodium acetate buffer embodiment are shown in Figs. 32, 33 and the following tables.

Table 15: Signal assignment ¹ H-NMR spectrum of β-galactosylated bis (2-hydroxyethyl) terephthalate

doublet

3.55 - 3.52 H-5 1 H Multiplett

Doublet of

3.48 H-3 1 H 9.70 3.34

doublet

Table 16: Signal assignment ¹³ C-NMR spectrum of β-galactosylated bis (2-hydroxyethyl) terephthalate

Disequalactosylated bis (2-hydroxyethyl) terephthalate

MS (ESI, positive): calculated for C ₂ H ₃₄ NaOi ₆ (M-HV 601, 1745, according to 601, 1739)

Di-β-glucosylated bis (2-hydroxyethyl) terephthalate Di-β-glucosylated bis (2-hydroxyethyl) terephthalate is exemplified in FIG.

The product is confirmed by mass spectrometry.

MS (ESI, positive): calculated for C ₂ H ₃ Oi ₆ Na (MH) ⁺ 601 .1745, acc. 601 .1739 Further experimental results are shown in Fig. 35, Fig. 36 and in the following tables.

Table 17: Signal assignment ¹ H-NMR spectrum of bis-glucosylated bis (2-hydroxyethyl) terephthalate

Table 18: Signal assignment ¹³ C-NMR spectrum of bis-glucosylated bis (2-hydroxyethyl) terephthalate δ [ppm] assignment 165.31 C-1 ", C-8"

133.80 C-2 ", C-5"

C-3 ", C-4",

129.77

C-6 ", C-7"

103.30 C-1

77.01 C-5

76.59 C-3

73.33 C-2

70.00 C-4

66.69 c-r

64.97 C-2 '

61 .07 C-6

(9) Preparation of fructosylated BHET

BHET (0.05 mol / L) and sucrose (0.4 mol / L) were dissolved in sodium acetate buffer (0.05 mol / L, pH = 5.2) and a fructosidase solution (100 U in sodium acetate buffer) added. The fructosidase solution catalyzes the cleavage of the sucrose into α-D-glucose and β-D-fructose enzymatically. The ß-D-fructose binds chemically to the BHET during the reaction.

MS (ESI, positive): calculated for Ci ₆ Hi ₉ NaOi ₀ (M-HV 439.1216, according to 439.121 1

(10) Preparation of polyester from g-glucosylated MHET

α-Glucosylated MHET (20 mg) is heated to 250 ° C for 50 sec. There is a gas generation. There remains a brown-white solid, which is partially suspended in water and dissolved. Thin-layer chromatography analysis (MeOH / CH ₂ Cl ₂ ) shows that a high-polymer substance is formed from the suspension and is also UV-active.

(1 1) Preparation of glvcosvlated MA-BHET-a-Glc In 2 ml of tert-butanol, 40 mg of α-glucosylated BHET and 72 μΙ vinylnethacrylate are dissolved. Then 25 mg of Novozymes 435 (immobilized on acrylic resin) are added. At 50 ° C, the suspension is shaken for 18 h. The suspension is then centrifuged, the supernatant decanted and concentrated on a rotary evaporator. The residue is chromatographed on silica gel 60 (Macherey Nagel, 0.044-0.063 mm). (Eluent system: 1 liter of ethyl acetate to 1 volume of methanol). A white solid was obtained.

Glycosylated ΜΑ-ΒΗΕΤ-α-Glc is exemplified in FIG. Further experimental results are shown in FIG. 38, FIG. 39 and in the following tables.

NMR measured in deuterated (CD ₃ ) ₂ SO.

Table 19: Signal assignment ¹ H-NMR spectrum of glycosylated MA-BHET-a-Glc

Doublet of

3.94 Η-1'a 1H 11,545.01

Triplett

Doublet of

3.75 Η-1'b 1H 11,624.59

Triplett

3.60-3.56 H-6a 1H Multiplett

H-6b, H-4, H-

3.47-3.38 3H Multiplett

3

3.23-3.18 H-2 1H Multiplett

3.09 - 3.04 H-5 1H Multiplett

Doublet of

3.41 H-4 "" 1H 9,723.76

doublet

Table 20: Signal Assignment ¹³ C-NMR spectrum of glycosyliertenn MA-BHET- a-Glc

54.86 C-2 '"

54.17 C-1 '"

53.12 C-6

8.90 C-4 ""

(12) Preparation of q-cosylated MA-BHET-g-Glc

In 2 ml of tert-butanol, 40 mg of α-glucosylated BHET and 72 μΙ vinylmethyl acrylate are dissolved. Then, 25 mg of Novozymes 435 immobilized on acrylic resin are added. At 50 ° C, the suspension is shaken for 30 h. The suspension is then centrifuged, the supernatant decanted and concentrated on a rotary evaporator. The residue is chromatographed on silica gel. (Eluent system: 1 liter of ethyl acetate to 1 volume of methanol). A white solid was obtained.

Glycosylated MA ₂ -BET-α-Glc is exemplified in FIG. Further experimental results are shown in FIG. 41, FIG. 42 and in the following tables.

NMR measured in CD ₃ OD.

Table 21: Signal assignment ¹ H-NMR spectrum of glycosylated MA ₂ -BHET-a-Glc

4.62-4.60 Η-1 '"2H Multiplett

4.57-4.55 H-2 '2H Multiplett

4.52-4.50 Η-2 '"2H Multiplett

Doublet of

4.43 H-6a 1H 11,802,12

doublet

Doublet of

4.18 H-6b 1H 11,846.40

doublet

4.08-4.03 Η-1'a 1H Multiplett

3.94-3.83 H-5, Η-1'b 2H Multiplett

Doublet of

3.66 H-3 1H 9,548.98

doublet

Under MeOH

-3.31 H-2 1H

peak

Doublet of

3.41 H-4 1H 9,723.76

doublet

H-4 "" + doublet of

1.90 + 1.89 2H 1.581,02 + 1,501.02

H-4 doublet

Table 22: Signal assignment ¹³ C-NMR spectrum of glycosylated MA ₂ -BET-α-Glc

130.67 C-6 ", C-7"

126.61

C-3 "", C-3

126.38

100.43 C-1

74.91 C-3

73.39 C-4

71.96 C-2

71 .45 C-5

67.19 c-r

65.66 C-2 '

65.19 C-6

64.34 C-1 '"

63.65 C-2 '"

18:42 18:39 C-4 "", C-4

(13) Preparation of qv cosylated a-Glc-MHET decanol

In 2 ml of tert-butanol are added 40 mg of α-glucosylated BHET and 120 μΜ-decanol. Then, 25 mg of Novozymes 435 immobilized on acrylic resin are added. At 50 ° C, the suspension is shaken for 24 h. The suspension is then centrifuged, the supernatant decanted and concentrated on a rotary evaporator. The residue is chromatographed on silica gel. (Eluent system: 1 liter of ethyl acetate to 1 volume of methanol). A white solid was obtained.

Glycosylated α-Glc-MHET-decanol is shown by way of example in FIG. Further experimental results are shown in FIG. 44, FIG. 45 and in the following tables.

NMR measured in CDCl ₃ .

Table 23: Signal Assignment ¹ H-NMR spectrum of glycosylated a-GIC MHET-decanol Relative coupling constant δ [ppm] Multiplicity assignment

Integral J [Hz]

H-3 ", H-4",

8.10 4H Singlet

H-6 ", H-7"

4.96 H-1 1H doublet 3.88

Doublet of

4.61 H-2 "a 1H doublet of 12,186,283.10

doublet

Doublet of

4.52 H-2 "b 1H doublet of 12,176,313.05

doublet

4.34 H-1 '"2H triplet 6.72

Doublet of

4.09 H-1 "a 1H doublet of 11,706,243.04

doublet

Doublet of

3.87 H-1 "b 1H doublet of 11,696,293.09

doublet

Doublet of

3.82 H-6 2H 5,884.00

doublet

3.73 H-3 1H triplet 9,18

Doublet of

3.71 H-2 1H 9,574.04

doublet

3.58 H-4 1H triplet 9.30

3:52

H-5 1H Multiplett

3:49

Doublet of

1.78 H-2 '"2H 14,466.98

Triplett

1:46

H-3 '"2H Multiplett

1:41

1:38

H- (4,5,6,7,8,9) "12H multiplet

1.22 0.88 H-10 '"3H singlet

Table 24: Signal assignment ¹³ C NMR spectrum of glycosylated

MHET-decanol

(14) Alternative preparation of g-glucosylated MHET In 2 ml of 50 mM HEPES buffer pH 7.5, 40 mg of α-glucosylated BHET. Then, 25 mg of Novozymes 435 immobilized on acrylic resin are added. At 50 ° C, the suspension is shaken for 24 h. The pH of 7.5 of the batch is kept constant by adding 1 N NaOH solution. The suspension is then centrifuged, the supernatant decanted and concentrated on a rotary evaporator. The residue is chromatographed on silica gel. (Eluent system: 6 volumes of ethyl acetate to 3 volumes of isopropanol and 1 volume of water). A white solid was obtained.

α-Glucosylated MHET is exemplified in FIG. Further experimental results are shown in Fig. 47, Fig. 48 and in the following tables.

NMR measured in CD ₃ OD.

Table 25: Signal assignment ¹ H-NMR spectrum of α-glucosylated MHET

doublet

Under MeOH

-3.31 H-5 1 H

peak

Table 26: Signal assignment ¹³ C-NMR spectrum of α-glucosylated MHET

(15) Polymerization of MA-BHET-α-Glc

To 2 ml of DMSO are added 1 ml of water and 50 mg of ΜΑ-ΒΗΕΤ-α-Glc and 1 mg of K ₂ OsS2. This solution is sparged with nitrogen for 2 hours. Then the reaction is sealed and shaken at 50 ° C for 12 hours. The solvents were removed by freeze-drying, then a portion of the residue was dissolved overnight in deuterated methanol at 50 ° C and measured ¹ H NMR 400 MHz. Experimental results are shown in FIG. INDUSTRIAL APPLICABILITY

The present invention is industrially applicable in a variety of ways. For example, the further use of degradation products from the hydrolysis of PET in the form of biohybrid polymers is made possible. For example, biohybrid polymers according to the invention have increased biocompatibility, compostability, and / or allow improved adhesion of cultured cells to surfaces.

Claims

claims

A compound according to claim 1, wherein the MHET or BHET and a saccharide are chemically bonded to each other via a glycosidic bond.

3. A compound comprising mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid chemically bound to a saccharide.

4. A compound according to claim 3, wherein the MHET or BHET and the saccharide are chemically bonded to each other via a glycosidic bond.

5. A compound according to any one of claims 2 to 4, wherein the saccharide is a monosaccharide or disaccharide.

A compound according to claim 5, wherein the monosaccharide or disaccharide is selected from a group containing hexoses and pentoses.

A compound according to claim 5, wherein the monosaccharide or disaccharide is selected from α-glucose, β-glucose, α-fructose, β-fructose, α-galactose and β-galactose, α-mannose and β-mannose, xylose, N Acetylglucosamine, glucosamine and glucuronic acid.

8. A compound according to any one of claims 1 to 7, wherein the compound is obtainable by enzymatic glycosylation of MHET or BHET.

9. A compound according to any one of claims 1 to 8, wherein the compound is formed by enzymatic glycosylation of MHET or BHET.

10. A compound according to claim 8 or 9, wherein the enzymatic glycosylation is catalyzed by a glucosidase, a galactosidase or a fructosidase.

1 1. A compound according to claim 10, wherein the enzymatic glycosylation is catalyzed by a glucosidase.

12. A compound according to claim 1 1, wherein the glucosidase is an α-glucosidase or a β-glucosidase.

A compound according to any one of claims 8 to 12, wherein the MHET or BHET is obtainable by bacterial degradation or enzymatic degradation from polyethylene terephthalate (PET).

14. A compound according to any one of claims 8 to 13, wherein the MHET or BHET is formed by bacterial degradation or enzymatic degradation of polyethylene terephthalate (PET).

A compound according to claim 13 or 14, wherein the enzymatic degradation of PET is catalyzed by a hydrolase.

16. A compound according to claim 15, wherein the hydrolase is PETase from Idionella sakaiensis.

17. A compound according to claim 15 or 16, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1.

18. A compound according to any one of claims 13 to 17, wherein the enzyme for the enzymatic glycosylation of MHET or BHET and the enzyme for the enzymatic degradation of PET are used together.

A compound according to claim 18, wherein a microorganism harboring the enzyme for enzymatic glycosylation and enzyme for enzymatic degradation of PET is used.

20. A compound according to any one of claims 1 to 19, consisting of MHET or BHET, which is chemically bonded to a saccharide via a glycosidic bond.

21. A compound according to any one of claims 1 to 20, wherein the compound has any of the following structures (a) or (b):

(A)

22. A compound according to any one of claims 1 to 19, wherein the compound further comprises at least one methacrylic radical.

A compound according to claim 22 wherein the compound has the structure:

wherein R ₁ comprises a glycosidic bond-bound saccharide and R ₂ comprises a methacrylic residue.

A compound according to claim 23, wherein the compound has the structure:

A compound according to claim 23, wherein the compound has the following structure:

A compound according to any one of claims 1 to 19, wherein the compound further comprises a lipophilic side chain.

A compound according to claim 27, wherein the compound has the structure:

wherein Ri comprises a saccharide bound via a glycosidic bond and R ₂ comprises a lipophilic side chain, preferably a saturated or unsaturated aliphatic hydrocarbon side chain, or comprises a linker.

29. A compound according to claim 28, wherein R2 is a saturated or unsaturated aliphatic C ₅ to C 20 hydrocarbon side chain, preferably a saturated or unsaturated aliphatic C ₅ to C 15 hydrocarbon side chain, more preferably a saturated or unsaturated aliphatic Cs to C12 hydrocarbon side chain, more preferably a saturated or unsaturated aliphatic C 10 hydrocarbon side chain and more preferably a saturated aliphatic C10 hydrocarbon side chain.

A compound according to claim 28, selected from a compound having the following structure (a) to (j):

60

where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

31. A compound according to any one of claims 2 to 24, 26 to 29 or 30 (b) - (j), wherein the saccharide chemically bonded to MHET or BHET via a glycosidic linkage is methacrylated.

32. Polymer of a compound according to any one of claims 1 to 31.

33. The polymer of claim 32, wherein the polymer is a biohybrid polymer.

34. A process for preparing a compound comprising glycosylated mono (2-hydroxyethyl) terephthalic acid (MHET) or glycosylated bis (2-hydroxyethyl) terephthalic acid (BHET), which process comprises the step of enzymatically glycosylating mono (2-hydroxyethyl) terephthalic acid (MHET) or bis (2-hydroxyethyl) terephthalic acid (BHET).

35. The method of claim 34, wherein in the step of enzymatic glycosylation, the MHET or BHET and a saccharide are chemically bonded together via a glycosidic linkage.

A method according to claim 35, wherein the saccharide is a monosaccharide or disaccharide.

37. The method according to claim 36, wherein the monosaccharide or disaccharide is selected from a group containing hexoses and pentoses.

A process according to claim 37, wherein the monosaccharide or disaccharide is selected from α-glucose, β-glucose, α-fructose, β-fructose, α-galactose and β-galactose, α-mannose and β-mannose, xylose, N - acetylglucosamine, glucosamine and glucuronic acid.

39. The method according to any one of claims 34 to 38, wherein the enzymatic glycosylation is catalysed by a glucosidase, a galactosidase or a fructosidase.

40. The method of claim 39, wherein the enzymatic glycosylation is catalysed by a glucosidase.

41. The method of claim 40, wherein the glucosidase is an α-glucosidase or a β-glucosidase.

42. The method according to any one of claims 34 to 41, wherein the MHET or BHET is obtainable by bacterial degradation or enzymatic degradation of polyethylene terephthalate (PET).

43. The method according to any one of claims 34 to 42, wherein the method prior to the step of enzymatic glycosylation preferably comprises a step of bacterial degradation or enzymatic degradation of polyethylene terephthalate (PET) to MHET or BHET.

44. The method of claim 42 or 43, wherein the enzymatic degradation of PET is catalyzed by a hydrolase.

45. The method of claim 44, wherein the hydrolase is PETase from Idionella sakaiensis.

46. The method according to claim 44 or 45, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1.

47. The method according to any one of claims 42 to 46, wherein the enzyme for the enzymatic glycosylation of MHET or BHET and the enzyme for the enzymatic degradation of PET are used together.

A method according to claim 47, wherein a microorganism harboring the enzyme for enzymatic glycosylation and the enzyme for enzymatic degradation of PET is used.

49. The method according to any one of claims 34 to 48, wherein in a further step, a methacrylic radical is chemically attached to the glycosylated MHET or BHET is bound, wherein the methacrylic radical is preferably bound by an enzymatic esterification of the glycosylated MHET or BHET chemically, wherein the enzymatic esterification is preferably catalyzed by a lipase.

50. The method of claim 49, wherein the methacrylic radical is chemically bound to the glycosylated MHET or BHET by the addition of vinylmethyl methacrylate.

51. A method according to any one of claims 34 to 48, wherein in a further step, a lipophilic side chain, preferably a saturated or unsaturated aliphatic hydrocarbon side chain, is chemically bonded to the glycosylated MHET or BHET.

The process of claim 51, wherein the lipophilic side chain is a saturated or unsaturated C ₅ to C 20 hydrocarbon side chain, preferably a saturated or unsaturated C ₅ to C 15 hydrocarbon side chain, more preferably a saturated or unsaturated C to C 12 hydrocarbon side chain, more preferably a saturated or unsaturated C ₁₀ hydrocarbon side chain and particularly preferred is a saturated C10 hydrocarbon side chain.

53. The method of claim 52, wherein the lipophilic side chain is a saturated C10 hydrocarbon side chain,

54. The method according to any one of claims 51 to 53, wherein the lipophilic side chain is bound by the addition of decanol.

55. The method according to any one of claims 34 to 54, wherein after the step of enzymatic glycosylation, a step of polymerisation follows.

56. A polymer obtainable by the process according to any one of claims 34 to 55.

57. The polymer of claim 56, wherein the polymer is a biohybrid polymer.

58. A microorganism which houses at least one enzyme for the enzymatic glycosylation of MHET and / or BHET and at least one enzyme for the enzymatic degradation of PET.

59. The microorganism according to claim 58, wherein the microorganism is a recombinant microorganism and the enzyme for enzymatic glycosylation of MHET and / or BHET and / or the enzyme for enzymatic degradation of PET is a recombinant enzyme.

60. The microorganism according to claim 58 or 59, wherein the enzyme for enzymatic glycosylation is a glucosidase, a galactosidase or a fructosidase.

61. The microorganism according to claim 60, wherein the enzymatic glycosylation enzyme is a glucosidase.

62. The microorganism according to claim 61, wherein the glucosidase is an α-glucosidase or a β-glucosidase.

63. The microorganism according to any one of claims 58 to 62, wherein the enzyme for the enzymatic degradation of PET is a hydrolase.

64. The microorganism according to claim 63, wherein the hydrolase is PETase from Idionella sakaiensis.

65. The microorganism according to claim 63 or 64, wherein the hydrolase comprises the amino acid sequence shown in SEQ ID NO: 1.