Vitrimer containing a biocatalyst

[0001] This invention relates to vitrimers, processes for making the vitrimers and the use of said vitrimers in various applications.

BACKGROUND

[0002] Classes of polymer materials traditionally included thermosetting polymers, or thermosets, thermoplastics and elastomers.

[0003] Thermosets are formed by curing a thermosetting resin to irreversibly transform the resin into a hardened, insoluble polymer network, characterized by covalent bonds forming between polymer chains. The curing process can be induced by heat, radiation or catalysis. Thermosets are typically moulded prior to curing, as once hardened a thermoset cannot be reheated for remoulding, nor can they easily be machined. The advantages of thermosets include high mechanical strength and high thermal and chemical resistance.

[0004] Thermoplastics are polymers which become pliable and mouldable above a specific temperature and solidify on cooling. The temperature range in which they can be reshaped is typically very narrow, as the viscosity of thermoplastic materials decreases rapidly in the region of the melting points and glass transition temperatures. Thermoplastics suffer from inferior mechanical strength and reduced thermal/chemical resistance compared with thermosets.

[0005] Elastomers are polymers that can be readily stretched to several times their original dimensions, but then return to their original dimensions when the stress or stretching force is released. They are crosslinked polymers, but with a low crosslink density. The polymer chains still have some freedom to move but the crosslinks prevent the deformation from being permanent. An elastomer shows the characteristic elastomeric behaviour above its glass transition temperature. Elastomers typically have low mechanical stiffness and strength and have poor chemical stability and heat resistance.

[0006] More recently, a class of polymers has been developed which combines advantages of both thermosets and thermoplastics. Vitrimers benefit from both the mechanical and solvent- resistant properties of thermosets, and the capacity to be reshaped and/or repaired of thermoplastics. Vitrimers are associative covalent adaptable networks, characterized by covalently bonded polymer networks which change their topology by internal bond-exchange reactions activated, for example, by the input of thermal or light energy. The network is capable of reorganizing above a certain temperature, without altering the number of intramolecular bonds. At, or above this temperature (T _v - topology freezing transition temperature), the vitrimer becomes malleable, making them applicable to a wide range of moulding, reshaping or repairing processes. Unlike thermoplastics, vitrimers’ decrease in viscosity with temperature follows an Arrhenius-law relationship. Below T _v the bond-exchange reactions slow down to the extent that they freeze and vitrimers behave like classical thermosets. The reversible nature of this associative covalent bond-exchange process means that vitrimers are capable of infinitely going from a set, glassy state to a viscoelastic liquid.

[0007] The internal bond-exchange reactions that take place in vitrimers are sped up by the presence of a catalyst. Vitrimers have been reported whereby the bond-exchange reactions occur via transesterification of ester linkages in epoxy-anhydride type vitrimers - obtained from an epoxy thermosetting resin and an anhydride/acid hardener (Montarnal et ai, Science 201 1 , 334, 965-968). The catalysts used in such epoxy-based vitrimers include metal-based catalysts such as zinc acetylacetonate (WO 2012/101078) and titanium bis(3-phenoxy-1 , 2-propane dioxide) (US 2017/0044307) or organic catalysts such as triazabicyclodecene (M. Capelot et ai, ACS Macro Lett. 2012, 1 , 789-792; US 2017/0044305).

[0008] The bond-exchange reactions in vitrimers containing these inorganic or organic catalysts are activated at temperatures typically in excess of 150 °C. The high activation temperatures of these vitrimers limits the range of applications to which they might be suited. For example, they would not be suitable as a coating on temperature-sensitive materials, such as microelectronics or medical prosthetics.

[0009] Lipases are a type of esterase that have been used to carry out enzymatic transesterifications in the production of biodiesel (Zhao et ai, Renewable and Sustainable Energy Reviews 2015, 44, 182-197). This process utilizes immobilized lipases to carry out the transesterification of triglycerides, found in animal or plant oils, with short-chain alcohols. Immobilized lipase has also been used in the esterification of a precursor in the synthesis of a self-healing siloxane-based elastomer (Nasresfahani et ai., Poiym. Chem. 2017, 8, 2942-2952). The lipase is removed from the system via filtration, however, and is not implicated in the healing mechanism of the elastomer.

[0010] The present invention is devised with the foregoing in mind.

SUMMARY OF THE DISCLOSURE

[0011] The present invention provides a vitrimer containing a biomolecule with esterase activity. It has been surprisingly found that biomolecules, such as enzymes, are sufficiently robust to be incorporated into vitrimers. Example vitrimers have been prepared with lipase enzymes and curing of the thermosetting resin at temperatures up to and exceeding 100 °C, which temperatures might be expected to denature the enzyme; surprisingly the enzymes maintained their transesterification activity after curing, even though the curing time and temperature would typically denature these proteins. The incorporation of enzymes into vitrimers, in place of conventionally used catalysts, allows much lower T _v values to be accessed. This invention offers new potential applications whereby the healing vitrimer properties can be reached typically below 140 °C, or even below 100 °C. Due to the non-hazardous and non-toxic nature of the enzyme, the vitrimers of the present invention also present a green alternative to vitrimers incorporating conventional catalysts, as they can be reverted to monomers absent of environmentally harmful pollutants, for instance the presence of metallic catalyst particles, thereby facilitating end-of-life recycling.

[0012] Therefore, in a first aspect, the present invention provides a vitrimer comprising a thermosetting polymer and a biomolecule having esterase activity, wherein the thermosetting polymer comprises ester linkages.

[0013] In a further aspect, the present invention provides a process for forming a vitrimer as described herein, the process comprising:

a) combining a thermosetting resin and a biomolecule having esterase activity, wherein the resin comprises at least one free hydroxyl and/or ester function and/or epoxide function and/or anhydride function;

b) curing of the combination from step a); and optionally

c) cooling the resultant thermoset polymer to ambient temperature.

[0014] In another aspect, the present invention provides the use of a vitrimer as described herein as a healable barrier, preferably for corrosion protection.

[0015] In a further aspect, the present invention provides an object comprising a vitrimer as described herein.

[0016] In a further aspect, the present invention provides the use of an object comprising a vitrimer as described herein, in the automotive, aeronautical, nautical, aerospace, biomedical, sport, construction, petrochemical, chemical engineering, defense, electrical, electrical insulation, electronics, wind power, packaging or printing fields.

[0017] In a further aspect, the present invention provides a kit for producing a vitrimer as described herein, the kit comprising:

a thermosetting resin, said resin comprising at least one free hydroxyl and/or ester function and/or epoxide function and/or anhydride function; and

a biomolecule with esterase activity;

and optionally a hardener.

DEFINITIONS

[0018] The term ‘vitrimer’ refers to a covalently crosslinked polymer that behaves as a conventional thermoset below the glass transition temperature (T _g ), but can deform above a particular temperature, T _v , by bond-exchange reactions. Above T _v the viscosity follows an Arrhenius-law response enabling hot deformation or thermoforming of the vitrimer over a broad temperature range, which is a characteristic of strong glasses. [0019] The term‘thermosetting polymer’ is intended to mean a thermosetting resin which has been crosslinked to have become‘thermoset’.

[0020] The term‘thermosetting resin’ is intended to mean a monomer, oligomer, polymer or macromolecule capable of being crosslinked. In this case, crosslinking relates to the formation of ester linkages within and/or between monomers, oligomers, polymers and/or macromolecules. Preferably, the term ‘thermosetting resin’ refers to a monomer, oligomer, polymer or macromolecule capable of being chemically crosslinked when reacted in the presence of a hardener (also sometimes referred to as a curing agent or crosslinking agent).

[0021] The term‘hardener’ refers to an agent capable of chemically crosslinking a thermosetting resin. Typically, it is a compound bearing multiple functional groups, such as anhydride, acid or hydroxyl moieties, where the functional groups are capable of reacting with reactive functions of the thermosetting resin to provide ester linkages.

[0022] The term‘curing’ is intended to mean the process by which the thermosetting resin is crosslinked to become a thermosetting polymer. The curing process typically involves heating, but other energy inputs such as radiation are possible. The presence of the enzyme significantly lowers the temperature at which curing takes place compared to curing of a conventional thermosetting resin.

[0023] The term‘healing temperature’ refers to a temperature at which the vitrimer becomes malleable and a defect such as a crack in the material can be simply healed or repaired by contacting together of the parts of the material. Therefore, the‘healing’ properties of the vitrimer are observed at a temperature when one or more of the following occurs:

• pieces of the vitrimer weld and/or fuse to one another;

• a crack in the vitrimer is no longer visible;

• the vitrimer becomes soluble in protic solvents, such as ethylene glycol; or

• the viscosity is less than 10 ¹² Pa s.

DETAILED DESCRIPTION

[0024] The present invention provides a vitrimer comprising a thermosetting polymer and a biomolecule having esterase activity, wherein the thermosetting polymer comprises ester linkages.

[0025] The vitrimer of the invention comprises a thermosetting polymer, which comprises ester linkages. The ester linkages refer to covalent ester bonds either found within the polymer chains or preferably crosslinking the polymer chains. In an embodiment, there is provided a vitrimer comprising a thermosetting polymer and a biomolecule having esterase activity, wherein the thermosetting polymer comprises ester crosslinking.

[0026] The vitrimer of the invention also comprises a biomolecule having esterase activity. A biomolecule having esterase activity is intended to mean a biological molecule capable of catalyzing the formation/cleavage of ester bonds. This is typically a protein such as an enzyme, but other biomolecules, such as nucleic acid polymers, may also exhibit esterase activity. In an embodiment, there is provided a vitrimer comprising a thermosetting polymer and a protein having esterase activity, wherein the thermosetting polymer comprises ester linkages. Preferably the protein is an enzyme. More preferably the enzyme is an esterase, most preferably a lipase. In an embodiment, there is provided a vitrimer comprising a thermosetting polymer and an esterase (such as lipase) enzyme, wherein the thermosetting polymer comprises ester linkages. In an embodiment, the biomolecule having esterase activity is an esterase or a phosphatase enzyme. In an embodiment, the biomolecule having esterase activity is selected from trehalose phosphatase, sugar phosphatase, carboxylesterase and feruloyl esterase. In an embodiment, the biomolecule having esterase activity is selected from trehalose phosphatase ( Mycobacterium tuberculosis H36Rv), sugar phosphatase (£. coli), carboxylesterase ( Bacillus subtilis subsp. subtilis 168) and feruloyl esterase ( Acidothermus cellulolyticus CD2).

[0027] Lipase enzymes, both wild-type and engineered, have been isolated from a number of biological sources. In an embodiment, the biomolecule having esterase activity is a lipase isolated from Alcaligenes sp., Achromobacter sp., Aspergillus sp., Aspergillus oryzae, Aspergillus niger, Thermomyces lanuginosus, Candida antarctica, Candida rugosa, Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas cepacia, Pseudomonas stutzeri, Rhizmucor miehei, Rhizopus oryzae, Rhizopus niveus, Mucor javanicus, Penicillium camemberti or Penicillium roqueforti. In an embodiment, the biomolecule having esterase activity is a lipase isolated from Alcaligenes sp., Achromobacter sp., Aspergillus sp., Aspergillus oryzae, Aspergillus niger, Thermomyces lanuginosus, Candida antarctica, Penicillium roqueforti, Pseudomonas fluorescens, Pseudomonas cepacia, Pseudomonas stutzeri or Rhizopus oryzae. In a preferred embodiment, the biomolecule having esterase activity is a lipase isolated from Aspergillus niger, Aspergillus oryzae, Candida antarctica, Penicillium roqueforti, Pseudomonas fluorescens, Pseudomonas stutzeri, or Thermomyces lanuginosus. In a more preferred embodiment, the biomolecule having esterase activity is a lipase isolated from Candida antarctica, Pseudomonas fluorescens or Pseudomonas stutzeri. In an even more preferred embodiment, the biomolecule having esterase activity is a lipase isolated from Pseudomonas fluorescens or Pseudomonas stutzeri. In a most preferred embodiment, the biomolecule having esterase activity is a lipase isolated from Pseudomonas stutzeri.

[0028] Various commercially available lipases may be used in the vitrimers of the present invention. In an embodiment, the biomolecule having esterase activity is a commercial lipase selected from Palatase® 20000 ( Rhizmucor miehei), Resinase® HT ( Aspergillus oryzae), Novozym® 51032 ( Aspergillus sp.), Lipozyme® TL 100 L ( Thermomyces lanuginosus), Lipozyme® CALB L ( Candida antarctica B), Novocor® AD L ( Candida antarctica A), AK (Pseudomonas fluorescens), PSSD ( Pseudomonas cepacia), DF15 ( Rhizopus oryzae), R (Penicillium roqueforti), A12 ( Aspergillus niger), MER ( Rhizopus oryzae), MH10SD ( Mucor javanicus ), AHSD ( Pseudomonas sp.), AYS ( Candida rugosa ), G50 ( Penicillium camemberti), AE07 or Lipase TL ( Pseudomonas stutzeri), AE01 1 or Lipase PL/QL ( Alcaligenes sp.), A1AE05 or Lipase PL/QL ( Alcaligenes sp.), AE04 or Lipase AL (Achromobacter sp.), porcine liver esterase and human/porcine pancreatic lipase. In an embodiment, the biomolecule having esterase activity is a commercial lipase selected from Resinase® HT ( Aspergillus oryzae), Novozym® 51032 ( Aspergillus sp.), Lipozyme® TL 100 L ( Thermomyces lanuginosus), Lipozyme® CALB L ( Candida antarctica B), Novocor® AD L ( Candida antarctica A), AK ( Pseudomonas fluorescens), DF15 ( Rhizopus oryzae), R ( Penicillium roqueforti), A12 ( Aspergillus niger), AE07 or TL (Pseudomonas stutzeri), AE011 or PL/QL ( Alcaligenes sp.), A1AE05 or PL/QL ( Alcaligenes sp.), AE04 and AL (Achromobacter sp.). In an embodiment, the biomolecule having esterase activity is a commercial lipase selected from Novocor® AD L (Candida antarctica A), AK (Pseudomonas fluorescens), Resinase® HT (Aspergillus oryzae), A12 (Aspergillus niger), AE07 or TL (Pseudomonas stutzeri), AE011 or PL/QL (Alcaligenes sp.), A1AE05 or PL/QL (Alcaligenes sp.), AE04 and AL (Achromobacter sp.). In a preferred embodiment, the biomolecule having esterase activity is a commercial lipase selected from AK (Pseudomonas fluorescens), Resinase® HT (Aspergillus oryzae), Lipozyme® CALB L (Candida antarctica B), AE07 or Lipase TL (Pseudomonas stutzeri), Lipozyme® TL 100 L (Thermomyces lanuginosus), R (Penicillium roqueforti ) and A12 (Aspergillus niger). In a more preferred embodiment, the biomolecule having esterase activity is a commercial lipase selected from AK (Pseudomonas fluorescens), Lipozyme® CALB L (Candida antarctica B), and AE07 or Lipase TL (Pseudomonas stutzeri). In an even more preferred embodiment, the biomolecule having esterase activity is a commercial lipase selected from AK (Pseudomonas fluorescens), and AE07 or Lipase TL (Pseudomonas stutzeri).

[0029] In order to modify the properties of the vitrimer, the biomolecule having esterase activity may be present at various concentrations. In an embodiment, the biomolecule is present at a concentration of greater than or equal to 0.1 % wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of greater than or equal to 0.25% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of greater than or equal to 1 % wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of greater than or equal to 2% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of 1-15% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of 1-12% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of 1-7% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of 2-6% wt/wt relative to the thermosetting polymer. In an embodiment, the biomolecule is present at a concentration of 3-5% wt/wt relative to the thermosetting polymer. [0030] The vitrimers of the present invention are deformable or healable at much lower temperatures than conventional catalyst-based vitrimers. In an embodiment, there is provided a vitrimer with a healing temperature of less than 130 °C. In an embodiment, there is provided a vitrimer with a healing temperature of less than 100 °C. In an embodiment, there is provided a vitrimer with a healing temperature of less than 80 °C. In an embodiment, there is provided a vitrimer with a healing temperature of less than 60 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 30-130 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 50-120 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 60-100 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 70-90 °C.

[0031] In another embodiment of the invention, there is provided a vitrimer with a healing temperature of greater than 130 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 130-240 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 130-180 °C. In an embodiment, there is provided a vitrimer with a healing temperature of 130-160 °C.

[0032] When the vitrimers of the present invention are heated above approximately 240 °C, the biomolecule no longer has esterase activity and accordingly the vitrimer is no longer capable of performing as a vitrimer, as it has lost its healing capability. Therefore, in an embodiment, a vitrimer according to the present invention loses its reversible bond-exchange capability if heated at about 240 °C or above. In an embodiment, a vitrimer according to the present invention loses its reversible bond-exchange capability if heated at about 180 °C or above.

[0033] The present invention also provides a process for forming a vitrimer according to the invention, the process comprising:

a) combining a thermosetting resin and a biomolecule having esterase activity, wherein the resin comprises at least one free hydroxyl function and/or ester function and/or epoxide function and/or anhydride function;

b) curing of the combination from step a); and optionally

c) cooling the resultant thermoset polymer to ambient temperature.

[0034] The thermosetting resin provided in step a) contains functional groups that allow ester linkages to be formed during the step b) curing process. The functional groups may react with other functional groups present in the thermosetting resin, or with functional groups present in the optional hardener, to form the ester linkages. Therefore, the thermosetting resin comprises at least one free hydroxyl function and/or ester function and/or epoxide function and/or anhydride function. In an embodiment, the thermosetting resin comprises at least one free hydroxyl function and/or ester function and/or anhydride function and at least one epoxide function.

[0035] In an embodiment, the thermosetting resin is an epoxy resin. Non-glycidyl-type epoxy resins are formed by the peroxidation of the C=C double bonds of a polymer. Glycidyl-type epoxy resins can be represented by general formula (II) shown below and are formed by the condensation of a monomer (I) where X is O, NH or CO2 and R is a divalent aliphatic and/or aromatic moiety, with epichlorohydrin:

[0036] In an embodiment, the thermosetting resin is a glycidyl-type epoxy resin, suitably a glycidyl-type epoxy resin of formula (II), wherein X is O, NH or C(0)0; R is a divalent aliphatic and/or aromatic moiety; and n is the degree of polymerization, suitably n = 0-25. In an embodiment, the thermosetting resin is of formula (II), wherein X is O; R is a divalent aliphatic and/or aromatic moiety; and n = 0—5.

[0037] The skilled person will recognize epoxy resins suitable for the present invention. Suitable epoxy resins include bisphenol A diglycidyl ether (DGEBA), Novolac epoxy resins, bisphenol F diglycidyl ether, trimethylol triglycidyl ether (TMPTGE), ethylene glycol glycidyl ether, bisphenol A polyethylene glycol diglycidyl ether, bisphenol A polypropylene glycol diglycidyl ether, epoxidized polyunsaturated fatty acids, epoxidized vegetable oils (such as epoxidized soybean oil) and epoxidized cycloaliphatic resins sold under the name Araldite® CY179, CY184, MY720 and MY0510, or a mixture of one or more of these epoxy resins. In an embodiment, the thermosetting resin comprises DGEBA. In a preferred embodiment, the thermosetting resin is DGEBA. The structures of DGEBA and the hydrogenated analogue of DGEBA are shown below:

[0038] Preferably, the thermosetting resin is Araldite® LY564 (sold by the company Huntsman) which is a composition containing 60-100% DGEBA and 13-30% butanediol diglycidyl ether.

[0039] In an embodiment, the thermosetting resin is hydrogenated DGEBA. Hydrogenated DGEBA is an alternative to DGEBA, typically giving superior UV resistance or anti-yellowing properties to the resulting vitrimer. In an embodiment, the thermosetting resin is Eponex™ 1510 (supplied by Hexion).

[0040] In an embodiment, the biomolecule having esterase activity provided in step a) is a protein, preferably an enzyme, most preferably a lipase. In an embodiment, the biomolecule having esterase activity provided in step a) is a lipase isolated from a biological source as detailed in paragraph [0027] hereinabove. In an embodiment, the biomolecule having esterase activity provided in step a) is a commercial lipase as detailed in paragraph [0028] hereinabove.

[0041] In a preferred embodiment, step a) of the process further comprises a hardener. Suitable hardeners contain multiple functional groups, such as anhydride, acid or hydroxyl moieties, which are capable of reacting with reactive functions of the thermosetting resin to provide ester linkages. In an embodiment, the hardener is selected from carboxylic acids, carboxylic acid anhydrides, polyfunctional acids, or polyols. Preferably the hardener is a carboxylic acid anhydride or polyfunctional acid. More preferably, the hardener is a carboxylic acid anhydride or sebacic acid. The structures of some of the suitable hardeners are shown below:

1 ,2,3,64et?'ahyd!'oph{ha!tc succinic anhydride sebacic acid anhydride {THPA}

1 ,2,3, 6detrahyd ro-3- methyl· f g,3 _; S4etrahydn>4-meihyl· hexahydro-4-methyi- phthalic anhydride (3MTHPA phthaiic anhydride (4MTHPA) phthaiic anhydride (HMPA)

[0042] In a preferred embodiment the carboxylic acid anhydride is a cyclic anhydride. Cyclic anhydrides include phthalic anhydride, succinic anhydride, glutaric anhydride, tetrahydrophthalic anhydride (THPA), hexahydrophthalic anhydride (HHPA), hexahydro-4-methylphthalic anhydride (HMPA) and methyl-tetrahydrophthalic anhydride (MTHPA). In a preferred embodiment, the hardener is hexahydro-4-methylphthalic anhydride (HMPA) or methyl-tetrahydrophthalic anhydride (MTHPA). Preferably the hardener is MTHPA sold as Aradur® 917 by Huntsman. Preferably the hardener is a liquid at ambient temperature. Polyfunctional acid hardeners include compounds with more than one carboxylic acid moiety, such as sebacic acid. In a preferred embodiment, the hardener is sebacic acid. Suitably the hardener is soluble in the resin.

[0043] In an embodiment, the thermosetting resin comprises an epoxy resin and the hardener comprises a carboxylic acid anhydride or polyfunctional acid. In an embodiment, the thermosetting resin is an epoxy resin and the hardener is a carboxylic acid anhydride or polyfunctional acid. In an embodiment, the thermosetting resin is DGEBA and the hardener is a carboxylic acid anhydride or polyfunctional acid. In an embodiment, the thermosetting resin is DGEBA and the hardener is hexahydro-4-methylphthalic anhydride (HMPA), methyl- tetrahydrophthalic anhydride (MTHPA) or sebacic acid. In an embodiment, no hardener is added in step a). In an embodiment, the thermosetting resin is an epoxy resin and no hardener is added in step a).

[0044] The amount of hardener relative to the amount of thermosetting resin influences the rate of the curing reaction, as well as the amount of crosslinking in the cured polymer. In an embodiment, the hardener is provided at a ratio in the range from 0.3: 1 to 2: 1 molar equivalents relative to the thermosetting resin. In an embodiment, the hardener is provided at a ratio in the range from 0.3: 1 to 1 : 1 molar equivalents relative to the thermosetting resin. In an embodiment, the hardener is provided at a ratio in the range from 0.3: 1 to 0.7: 1 molar equivalents relative to the thermosetting resin. In an embodiment, the hardener is provided at a ratio of 0.5: 1 molar equivalents relative to the thermosetting resin. In an embodiment, the hardener is provided at a ratio in the range from 1 :1 to 2: 1 molar equivalents relative to the thermosetting resin. In an embodiment, the hardener is provided at a ratio in the range from 1.5: 1 to 2:1 molar equivalents relative to the thermosetting resin. In a preferred embodiment, the hardener is provided at a ratio of 1 :1 molar equivalents relative to the thermosetting resin.

[0045] The amount of biomolecule provided in step a) relative to the amount of the thermosetting resin must be sufficient to ensure that bond-exchange reactions of the resultant vitrimer are accessible at temperatures below 130 °C. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 0.1 % wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 0.25% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 0.5% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 1 % wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 2% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration greater than or equal to 3% wt/wt. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration of 1-15% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration of 1-12% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration of 1-7% wt/wt relative to the amount of the thermosetting resin. In an embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration of 3-6% wt/wt relative to the amount of the thermosetting resin. In a preferred embodiment, the biomolecule (such as an enzyme, e.g. lipase) is provided at a concentration of 4.5-5.5% wt/wt relative to the amount of the thermosetting resin.

[0046] The vitrimer formation process may be carried out in the absence of added water. This may be suitable when the biomolecule (such as an enzyme) is provided as a liquid. In an embodiment, step a) further comprises water. Water may be preferably added when the biomolecule (such as an enzyme) is provided as a solid. The presence of water may help to achieve homogenous mixing of the components in step a). In an embodiment, the water is added at greater than 0.5% v/v relative to the total amount of the mixture. In an embodiment, the water is added at greater than 1 % v/v relative to the total amount of the mixture. In an embodiment, the water is added at 1-3% v/v relative to the total amount of the mixture. In an embodiment, the water is added up to 50% w/w relative to the total amount of the mixture. In an embodiment, the water is added up to 25% w/w relative to the total amount of the mixture. In an embodiment, the water is added up to 15% w/w relative to the total amount of the mixture. In a preferred embodiment, water is added at 1-10% w/w relative to the total amount of the mixture. In a preferred embodiment, water is added at 1-5% w/w relative to the total amount of the mixture. In a more preferred embodiment, water is added at 3% w/w relative to the total amount of the mixture.

[0047] In step b) of the vitrimer formation process, the combination of components from step a) is cured to crosslink the thermosetting resin. The curing process can be carried out at any suitable temperature at which crosslinking can occur; at higher temperatures the rate of curing is faster, but it is feasible to cure the vitrimer of the present invention at ambient temperature, albeit very slowly. In an embodiment, the curing in step b) is carried out at 50 to 130 °C. In an embodiment, the curing in step b) is carried out at 60 to 1 10 °C. In an embodiment, the curing in step b) is carried out at 70 to 100 °C. In an embodiment, the curing in step b) is carried out at 80 to 90 °C. In an embodiment, the curing in step b) is carried out at 50 to 70 °C. In an embodiment, the curing in step b) is carried out at 70 to 90 °C.

[0048] It has also been discovered that curing at temperatures above 130 °C may still result in materials still displaying vitrimeric behaviour, but with improved temperature cycling stability. In an alternative embodiment, the curing in step b) is carried out at 130 to 180 °C. In an embodiment, the curing in step b) is carried out at 140 to 170 °C. In an embodiment, the curing in step b) is carried out at 145 to 160 °C.

[0049] The length of time for the curing process will typically depend on the curing temperature to some extent; at higher temperatures curing may progress faster. Nevertheless, curing should typically be carried out for at least 24 hours. In an embodiment, the curing in step b) is carried out for greater than 24 hours, such as greater than 48 hours, or greater than 72 hours. In an embodiment, the curing in step b) is carried out for 1 to 10 days, such as 2 to 8 days, or 3 to 5 days. In an embodiment, the curing in step b) is carried out at 50 to 70 °C for 5 to 10 days. In an embodiment, the curing in step b) is carried out at 70 to 90 °C for 1 to 7 days. In an alternative embodiment, the curing in step b) is carried out at 140 to 170 °C for 16 to 40 hours.

[0050] Vitrimers of the present invention can optionally comprise one or more additional compounds, providing they do not impair the advantageous properties associated with the invention. Examples of such additional compounds are: accelerators, dyes, pigments, fillers, plasticizers, woven or unwoven fibres, lubricants, flame retardants, antioxidants, glass, wood, metals and mixtures thereof.

[0051] The term ‘accelerators’ refers to compounds which are capable of accelerating the vitrimer curing process. Such compounds include additional curing agents chosen from amines (such as dimethylbenzylamine, diethylenetriamine, triethylenetetramine, hexanediamine, phenylenediamine and diaminodiphenylmethane), isocyanates, polymercaptans and dicyanodiamides. In an embodiment, step a) further comprises adding an accelerator. In a preferred embodiment, step a) further comprises adding A/,/\/-dimethylbenzylamine as an accelerator. In a preferred embodiment, step a) further comprises adding 0.1-1.5% wt/wt N,N- dimethylbenzylamine as an accelerator. In alternative embodiment, step a) further comprises adding 0.1-1.5% wt/wt accelerator and the curing in step b) is carried out at 80 to 100 °C for 6 to 24 hours. In a further embodiment, step a) further comprises adding 0.1-1.5% wt/wt N,N- dimethylbenzylamine and the curing in step b) is carried out at 80 to 100 °C for 6 to 24 hours.

[0052] Polyols may also be added to step a) of the process, in order to control the amount of crosslinking in the vitrimer product, which may be beneficial in terms of improved dimensional stability and solvent resistance and an increased heat distortion temperature. An example of a polyol is trimethylolpropane (1 , 1 , 1-tris(hydroxymethyl)propane). In an embodiment, step a) further comprises adding a polyol. In a preferred embodiment, step a) further comprises adding trimethylolpropane. In an embodiment, step a) further comprises adding a polyol and water. In a preferred embodiment, step a) further comprises adding trimethylolpropane and water. In a more preferred embodiment, step a) further comprises adding 0.01 - 1.5 % wt/wt trimethylolpropane and 1-5 % v/v water.

[0053] The term‘dyes’ refers to molecules that are soluble in the component mixture of step a) and absorb light in the visible range. For example, fat-soluble coloured dyes may be added to the formulation, at a concentration of 0.1-5.0% w/w, such as 1-3% w/w (e.g. 2% w/w) with respect to the thermosetting resin.

[0054] The term ‘pigments’ refers to coloured particles that are insoluble in the component mixture of step a). Such pigments include titanium oxide, carbon black, silica, or other mineral pigment; anthraquinones, azo pigments or other organic pigment.

[0055] Fillers that may be used include fillers conventionally used in polymer formulations. Fillers may include nanoparticles, microparticles, silica, clays, talc, calcium carbonate and kaolin. [0056] Among the fibres that can be optionally incorporated in the vitrimers of the present invention are glass, silicon carbide or other inorganic fibres; carbon fibres; polyamide fibres; cellulose-based fibres or other plant or organic fibres.

[0057] In one embodiment, there is provided a vitrimer according to the present invention, comprising a chromophore-labelled biomolecule with esterase activity. This would allow visualisation of the biomolecule within the vitrimer in visible light. In an embodiment, there is provided a vitrimer according to the present invention, comprising a fluorescent or fluorescently- labelled biomolecule with esterase activity. This would allow visualisation of the biomolecule within the vitrimer under fluorescent light. Such fluorescent or fluorescently labelled biomolecules are known in the biological arts and a skilled person will be able to identify suitable biomolecules. In one embodiment, there is provided a vitrimer according to the present invention, comprising a fluorescent or fluorescently labelled esterase enzyme (such as a lipase). An enzyme may be fluorescently labelled with a suitable molecule, such as fluorescein isothiocyanate.

[0058] In a further aspect, the present invention provides a vitrimer obtainable, obtained or directly obtained by a process defined herein.

Applications

[0059] In an aspect of the invention, there is provided an object comprising a vitrimer as defined herein. The term‘object’ refers to a three-dimensional part and includes coatings, films, sheets, powders, granules, fibres, etc. The object may be formed by any technique known for processing thermoplastic and thermosetting resins, such as moulding, casting, continuous moulding, contact moulding (hand lay-up, spray-up), vacuum bagging, filament winding, pultrusion, press and compression moulding, resin injection / resin transfer moulding, resin infusion, prepreg moulding and autoclaving, injection moulding, reaction injection moulding, reinforced reaction injection moulding, structural reaction injection moulding and 3D-printing.

[0060] In a further aspect of the invention, there is provided the use of an object comprising a vitrimer as defined herein, in the automotive, aeronautical, nautical, aerospace, biomedical, sport, construction, petrochemical, chemical engineering, defense, electrical, electrical insulation, electronics, wind power, packaging or printing fields. Examples in the petrochemical field include petrochemical storage and distribution, such as oil pipelines. Examples in the chemical engineering field include reaction vessels. Examples in the defense field include personal armour, such as visors. Examples in the printing field include objects made by 3D-printing of a vitrimer as defined herein. Due to their low healing temperatures and low toxicity, objects comprising vitrimers as defined herein, may find applications in fields previously not suited to vitrimer technology. For example, in the biomedical field, they may find applicability as coatings for medical devices (e.g. stents) and prostheses or scaffolds for tissue regeneration.

[0061] The present invention further provides the use of a vitrimer, as described herein, as a healable barrier or coating. In an embodiment, there is provided the use of a vitrimer, as described herein, as a healable barrier for corrosion protection. The coating of an object with a vitrimer according to the present invention provides a barrier to the deleterious effects of air, moisture, chemicals and abrasion. Should a defect appear in the coating, such as a scratch or crack, then the defect can be readily repaired by heating the coating to a suitable healing temperature. In contrast to previous vitrimers, this healing process can take place at much lower temperatures, e.g. below 130 °C, below 100 °C, such as below 80 °C. For example, defects in vitrimer coatings applied to objects such as bicycles or automobiles may be readily repairable, e.g. by the use of a domestic hairdryer applying heat to the vitrimer defect at approximately 60 °C.

[0062] In an embodiment, there is provided the use of a vitrimer, as described herein, as a healable coating for temperature-sensitive materials, such as microelectronics or medical prosthetics.

[0063] In another embodiment, when a vitrimer according to the present invention comprises a fluorescent or fluorescently-labelled biomolecule with esterase activity, then there is provided the use of such a vitrimer in the structural health monitoring of an object comprising said vitrimer. ‘Structural health monitoring’ refers to the ability to detect, localize and characterize damage or defects. The presence of the fluorescent biomolecule/enzyme within the vitrimer will allow the detection of defects in the object, by monitoring the vitrimer under fluorescent light. By a similar method, it may also be possible to monitor the successful healing of such defects.

Kits

[0064] In a further aspect of the invention, there is provided a kit for producing a vitrimer as defined herein, the kit comprising a thermosetting resin, said resin comprising at least one free hydroxyl and/or ester function and/or epoxide function and/or anhydride function; a biomolecule with esterase activity; and optionally a hardener. Preferably the thermosetting resin is an epoxy resin. Preferably the biomolecule with esterase activity is a protein with esterase activity, such as an esterase enzyme (e.g. a lipase). Preferably the hardener is a carboxylic acid anhydride, or an aliphatic or aromatic diacid (e.g. sebacic acid). More preferably, the thermosetting resin is an epoxy resin and the biomolecule with esterase activity is a lipase.

Healing Process

[0065] The vitrimers obtained as described herein can be deformed, repaired and reshaped at temperatures above the healing temperature of the vitrimer. This is possible due to their slow variation in viscosity over a wide temperature range, meaning they can be worked in a similar manner to strong glasses, unlike conventional thermosets. As a consequence of its properties, the vitrimer can be repaired by simply bringing two parts together to weld or fuse them to one another at a healing temperature, without the need for a mould. An object comprising vitrimer as defined herein can be deformed or reshaped by the application of a mechanical stress at a temperature equal to or greater than the healing temperature of the vitrimer. Examples of mechanical stresses include moulding, extrusion, blowing, injection, blending, stamping, twisting, flexing, tensile stress and shear. More than one type of mechanical stress may be applied to the object simultaneously or successively.

[0066] The increase in temperature necessary to repair or reshape a vitrimer may be provided by any means known in the art, such as an oven, a microwave oven, a flame, an iron, a hot air gun, a heated punch, an ultrasonic bath, electromagnetic fields including light or microwaves, chemical reactions, body heat, electrical heating, etc.

[0067] Therefore, in a further aspect, there is provided a process for healing an object comprising a vitrimer as defined herein, the process comprising subjecting the vitrimer surface to be healed to a temperature equal to or greater than the healing temperature of the vitrimer. In an embodiment, there is provided a process for healing or reshaping an object comprising a vitrimer as defined herein, the process comprising applying a mechanical stress to the object at a temperature equal to or greater than the healing temperature of the vitrimer.

Recycling Processes

[0068] Due to the presence of biomolecules in the vitrimers of the present invention, rather than for example, the toxic metal salts used as catalysts in previous vitrimers, their end-of-life recycling is advantageously facilitated. The vitrimers as defined herein comprising proteins (e.g. enzymes) can be deactivated by heating them to temperatures in excess of approximately 240 °C, thereby denaturing the proteins. Furthermore, the vitrimers may be dissolved in protic solvents (such as ethylene glycol) at temperatures above T _v which allows their removal and recycling.

EXAMPLES

[0069] Embodiments of the invention will now be described, for the purpose of illustration only, with reference to the accompanying figures, in which:

Figure 1 : Epifluorescence image of a vitrimer containing a fluorescently labelled lipase prepared according to example B29. Image width 2.88 mm.

Figure 2: Differential scanning calorimetry (DSC) heat-cool-heat cycle on lipase-containing vitrimer from example F1 showing a single glass transition temperature at 5.9 °C on both cycles. The maximum sample temperature was 100 °C to preserve enzyme activity. The dashed line denotes the glass transition temperature (T _g ). Heating at 10 °C min ^-1 in air.

Figure 3: Modulated DSC on lipase-containing vitrimer from example F1 after heat-cool-heat cycle shown in Figure 2. The dashed line denotes the glass transition temperature (T _g ). The dash- dot line denotes the enzyme degradation temperature. Parameters: Heating from -90 °C to 200 °C at 5 °C min ^-1 in air, modulated by ±1 °C every 60 s. Figure 4: Photographs of enzyme-containing vitrimer from example B18. Left: cracked, cured sample in a polystyrene weighing boat. Right: same sample after subsequent heat treatment of 10 min. at 80 °C.

Figure 5: Photographs showing reprocessing of vitrimer containing 3% AE07 prepared according to example F2; Left: ground vitrimer in mould before heat treatment; Middle: reprocessed vitrimer in the mould after heat treatment at 100 °C and 10 bar for 2.5 h followed by 100 °C and 30 bar for 2.5 h; Right: free-standing reprocessed vitrimer.

Figure 6: Swelling behaviour of various enzyme-containing vitrimer samples after immersion in 1 ,2,4-trichlorobenzene for 1 week at 135 °C.

Figure 7: Arrhenius plot for vitrimers containing 3%, 6% or 15% Resinase® HT prepared according to examples G1-G3 and their respective activation energies.

Figure 8: A) Creep test measurements at various temperatures for vitrimer containing 6% AE07 prepared according to example F3; B) corresponding Arrhenius plots and calculated activation energies.

Figure 9: Creep test measurements at various temperatures for vitrimer containing 3% AE07 prepared according to example F2, cycle 1 (top) and cycle 2 (bottom).

Figure 10: A) Viscosity evolution plots for Example F2 determined from creep test data shown in Figure 10, cycle 1 (left) and cycle 2 (right); B) Arrhenius plots for example F2 determined from creep test data shown in Figure 10, cycle 1 (left) and cycle 2 (right); C) Viscosity evolution plot (left) and Arrhenius plot (right) for reprocessed example F2.

Figure 11 : A) Storage modulus plot and B) Loss modulus plot for example F2 before and after reprocessing.

Figure 12: Tensile test plots for example F2 before and after reprocessing.

Figure 13: MDSC plots for a) vitrimer containing 1.5% AE07 prepared according to example H1 ; b) vitrimer containing 3% AE07 prepared according to example H2; c) vitrimer containing 6% AE07 prepared according to example H3; d) heat-cool-heat DSC plot for vitrimer containing 1.5% AE07 prepared according to example H1 and vitrimer containing 6% AE07 prepared according to example H3.

Figure 14: Creep test measurements at various temperatures for A) vitrimer containing 3% AE07 prepared according to example H2; B) vitrimer containing 6% AE07 prepared according to example H3.

Figure 15: Arrhenius plots generated from creep test data shown in Figure 14 for A) vitrimer containing 3% AE07 prepared according to example H2; B) vitrimer containing 6% AE07 prepared according to example H3. Figure 16: A) Storage Modulus plot; B) Tan delta plot and C) Loss Modulus plot for vitrimer containing 1.5% AE07 prepared according to example H1 and vitrimer containing 6% AE07 prepared according to example H3.

Figure 17: Visible light microscope images of the surface of lipase-containing vitrimer from example B24; A) scratch test - surface after slicing with a scalpel (left) and after heating the scratched surface for 2 min with a domestic hairdryer (right); B) abrasion test - surface as prepared (left), after abrasion (middle) and after heating the abraded surface for 2 min with a domestic hairdryer (right) [all scale bars = 500 mhi].

Figure 18: Attenuated Total Reflectance (ATR)-FTIR spectra of vitrimers L1-L8 containing Lipase AK (left) or Lipase TL (right) with (i) 0 wt% water (dotted lines) or 3 wt% water (solid lines); (ii) 3 wt% lipase (L1 , L2, L5, L6) or 5 wt% lipase (L3, L4, L7, L8).

[0070] Commercial lipases were sourced from the following suppliers and used as supplied: Strem/Novozymes - Palatase® 20000 {Rhizmucor miehei), Resinase® HT ( Aspergillus oryzae), Novozym® 51032 ( Aspergillus sp.), Lipozyme® TL 100 L ( Thermomyces lanuginosus), Lipozyme® CALB L ( Candida antarctica B) and Novocor® AD L ( Candida antarctica A);

Amano Enzymes (via Sigma Aldrich) - AK ( Pseudomonas fluorescens) and PSSD ( Pseudomonas cepacia );

Amano Enzymes - DF15 ( Rhizopus oryzae), R ( Penicillium roqueforti), A12 ( Aspergillus niger), MER ( Rhizopus oryzae), MH10SD (Mucor javanicus), AHSD ( Pseudomonas sp.), AYS ( Candida rugosa) and G50 ( Penicillium camemberti)·,

Mann Associates (MAVMeito Sangyo (MS) - AE07 (MA) or Lipase TL (MS) ( Pseudomonas stutzeri), AE011 (MA) or Lipase PL/QL (MS) ( Alcaligenes sp.), A1AE05 (MA) or Lipase PL/QL (MS) ( Alcaligenes sp.) and AE04 (MA) or Lipase AL (MS) (Achromobacter sp.).

[0071] Araldite® LY 564 (Huntsman, epoxide equivalent weight 165-175 g) and Aradur 917ch (Huntsman, MW 166 g mol ^-1 ) were obtained from Mouldlife. Aradite LY 564 is a low-viscosity epoxy resin based on DGEBA. Aradur 917CH is a liquid mixture of cyclic phthalic anhydrides. The manufacturer’s composition is given as 60-100% 4-MTHPA, 13-30% 3-MTHPA, and 7-13% of THPA and HMPA. Eponex 1510 (Hexion, supplied by Miller-Stephenson, epoxide equivalent weight 205-215 g), is a hydrogenated analogue of DGEBA. DGEBA (epoxide equivalent weight 172-176 g), A/,A/-dimethylbenzylamine (X¾99%, 135 g mol ^-1 ), HMPA (96%, 168 g mol ^-1 ), and 1 ,1 , 1-tris(hydroxymethyl)propane (TMP, 134 g mol ^-1 ) were obtained from Sigma-Aldrich. Sebacic acid (202 g mol ^-1 ) was obtained from TCI UK. All reagents were used without further purification.

[0072] Other enzymes used were trehalose phosphatase ( Mycobacterium tuberculosis H36Rv), sugar phosphatase (E. coli), carboxylesterase ( Bacillus subtilis subsp. subtilis 168) and feruloyl esterase ( Acidothermus cellulolyticus CD2) supplied by Prozomix Ltd as ammonium sulphate precipitates in water after immobilized metal affinity chromatography purification.

[0073] Swelling tests used anhydrous 1 ,2,4-trichlorobenzene (Sigma-Aldrich, >99%). Dull matte, bare, mild steel Q-panels (R23.5, Q-Lab, 89 mm ^c 51 mm ^c 0.81 mm) were used for tests involving corrosion and healing after abrasion and scratching.

Analysis

Calorimetry

[0074] Differential scanning calorimetry (DSC) measurements were carried out with a TA Instruments Q100. A heat-cool-heat cycle consisted of -30°C to 250 °C to -90 °C to 250 °C with heating at 10 °C min ^-1 , and cooling at 5 °C min ^-1 . The glass transition temperature, T _g , was calculated as the midpoint of step in the cooling and second heating profiles. The transition temperature from the first heating profile is convoluted with heat release from additional curing and thermal history.

[0075] Modulated DSC (MDSC) was run between -90 °C to 250 °C at 10 °C min ^-1 with a ±1 °C temperature modulation with a period of 60 s. The MDSC separates the reversible and non- reversible heat flows.

Mechanical Testing

[0076] Dynamic mechanical analysis (DMA) was carried out on a TA Instruments Q800 DMA. Creep tests were used over a temperature range of 100°C to 250°C to characterise the viscous component of the viscoelastic behaviour. Samples were 1 cm diameter ^c 4mm thick. Samples were preloaded at 0.01 N for 5 min, deformed for 45 min at an applied stress of 0.05 MPa on samples, then allowed to recover for 15 min. A compression clamp geometry was used to prevent the samples from yielding when softened at higher temperatures.

[0077] Activation energy values were calculated from DMA creep data. The effective viscosity, /7 _eff , of the vitrimer was calculated based on the gradient of strain versus time. A linear region of plots of In /7 _eff versus 1/7 is indicative of a strong (rather than fragile) glassy network. The slope of this plot is equal to E _n IR, where E _n is the viscous activation energy and R is the gas constant.

[0078] Tensile testing was carried out at room temperature. Samples were either cut into 60mm x 4mm x 4mm strips for tensile testing or moulded into dog-bone test pieces. Samples were tested at a constant extension rate of 10 mm min ^-1 with a gauge length of 25 mm.

Corrosion

[0079] Coated Q-panels were used for corrosion testing and compared to a clean but uncoated Q-panel. Coated panels soaked in covered beakers containing 500 mL of 3.5 wt% sodium chloride. Images were taken at 0, 6, 24 and 168 hours (7 days). Corrosion was qualitatively analysed and observations on the changes of every specimen were recorded. Swelling

[0080] Swelling tests were performed in a silicone oil bath at 135°C on 10mm x 10mm x 4mm samples immersed in 3 ml of TCB. The changes in mass and volume were recorded after 1 week. Dimensional changes were recorded by visible-light microscopy. Images were recorded of the top surface and side of each sample. Two measurements were taken for each dimension on the top surface and three thickness measurements were made on the side to calculate the average dimensions.

General Procedure for preparation of lipase-based epoxy resin vitrimers

[0081] Unless specified otherwise, all percentages are wt/wt relative to the mass of epoxy resin.

[0082] A carboxylic acid anhydride hardener (hexahydro-4-methylphthalic anhydride - HMPA [Sigma] or methyltetrahydrophthalic anhydride - Aradur 917CH) was combined with epoxy resin (Araldite® LY564) in a 0.5: 1 molar ratio of hardener: resin and stirred until homogenous. Water (see Table 1 for volume added relative to the volume of the whole mixture) and lipase biocatalyst were added (see Table 1 for amounts), and the mixture stirred until evenly dispersed. The mixture was then transferred to a polystyrene weighing boat and heated in an oven at a curing temperature, T _c (see Table 1) for a certain length of time (see Table 1). Samples were removed from the oven and cooled to room temperature prior to analysis.

[0083] Vitrimer performance was assessed by taking the cooled vitrimer samples and returning them to the oven at the curing temperature, T _c .

[0084] T _c Hold - if a sample was judged to have held its shape at T _c then it is shown as‘yes’ in the subsequent tables; if a sample melted at T _c , then it is shown as‘no’ in the subsequent tables; and if some pieces of a sample were judged to have held their shape, while some pieces melted at T _c , then it is shown as‘partial’ in the subsequent tables.

[0085] T _c Heal - two or more pieces of sample material were broken and placed on top of each in the oven at T _c . If the sample pieces adhered to each other and could not be pulled apart easily (‘healed’) it is shown as‘yes’ in the subsequent tables; if the sample pieces did not adhere to each other it is shown as‘no’ in the subsequent tables; and if some pieces of a sample were lightly adhered, but could be pulled apart easily, then it is shown as‘partial’ in the subsequent tables. If a sample did not successfully cure under the given conditions, then under T _c Hold/ T _c Heal it is marked as‘-‘ in the subsequent tables.

Table 1

[0086] The following vitrimers summarised in Table 2 were prepared according to the general procedure described above, but with a 0.3:1 molar ratio of hardenerresin. Enzymes were labelled with fluorescein isothiocyanate (FITC) (examples B28 and B29) by preparing 1 g/L of FITC powder in DMSO. 2.23 ml_ of FITC stock was added slowly to 5 g/L DF15 stock in phosphate buffer pH 8. The mixture was covered with light-blocking material and left rocking at room temperature for 3 h. 10 pi of 5 M NH ₄ CI was added per ml_ of reaction mixture; this was left rocking for 1 h at room temperature. The unreacted dye was separated by centrifugal filtration with a Vivaspin 20 10 kDa MWCO over about 20 min. The retentate was washed with 4 ml_ phosphate- buffered saline. The retentate was either freeze dried before use or used without further processing.

Table 2

[0087] The healing behaviour of the lipase-containing vitrimer from example B18 is illustrated in Figure 4. The cured sample was cracked within the polystyrene vessel in which the resin was cured (left panel). The cracked sample was warmed above its T _v for 10 min, after which point the cracks were no longer visible (right panel). The sample still held its original shape despite the heat treatment.

[0088] The following vitrimers summarised in Table 3 were prepared according to the general procedure described above, but with variable molar ratios of hardener: resin as specified in Table 3, with HMPA as the hardener and with curing being carried out for 72 hours at 80 °C.

Table 3

[0089] The following vitrimers in Table 4 were prepared according to the general procedure described above, using lipase AK (5 % w/w with respect to weight of epoxy), 0.5:1 molar ratio of AR917 hardener to Araldite LY564 epoxy resin, and varying the amount of water added to the mixture. The amount of water is shown on a w/w basis corresponding to the mass of water as a percentage of the mass of the entire mixture comprising resin, hardener, enzyme and water. All samples were cured at 80 °C.“Phase separation” refers to the presence of two liquid phases discernible by eye before oven curing.

Table 4

[0090] The following vitrimers in Table 5 were prepared according to the general procedure described above, using lipase AK (in varied amount with respect to weight of epoxy), 0.5:1 molar ratio of hardener to Araldite LY564 epoxy resin, and 1.5% v/v water added to the mixture. All samples were cured at 80 °C.“Homogeneity” refers to the presence of visible inclusions in the complete mixture after oven curing - a homogeneous sample had no visible inclusions. Table 5

Example F1

[0091] Sebacic acid (hardener) and Araldite LY564 epoxy resin were mechanically stirred at 50 °C for 24 h, then 3 % w/w lipase AE07 ( Pseudomonas stutzeri) was stirred in by hand. The mixture was cured for 72 h at 80 °C.

Example F2

[0092] Powdered sebacic acid (hardener) and DGEBA (epoxy resin) in a 1 :1 molar ratio were mechanically stirred at 60 °C for 24 h, then 3 % w/w lipase AE07 ( Pseudomonas stutzeri) was added and the mixture was degassed for 2 hours at 50 °C under vacuum. The mixture was then transferred to an open mould for curing for 72 h at 100 °C.

Example F3

[0093] Example F3 was prepared analogously to example F2, but with 6 % w/w lipase AE07 (Pseudomonas stutzeri).

Example G1

[0094] Powdered sebacic acid and DGEBA (epoxy resin) in a 1 : 1 molar ratio were mechanically stirred at 60 °C for 24 h, then 3 % w/w Resinase® HT ( Aspergillus oryzae) was added and the mixture was degassed for 2 hours at 50 °C under vacuum. The mixture was then transferred to an open mould for curing for 72 h at 100 °C.

Examples G2 & G3

[0095] Examples G2 and G3 were prepared analogously to example G1 , but with 6 % w/w and 15 % w/w Resinase® HT ( Aspergillus oryzae) respectively.

Example H1

[0096] Powdered sebacic acid and DGEBA (epoxy resin) in a 1 : 1 molar ratio were mechanically stirred at 60 °C for 24 h, then 1.5 % w/w lipase AE07 ( Pseudomonas stutzeri) was added and the mixture was degassed for 2 hours at 50 °C under vacuum. The mixture was then transferred to an open mould for curing for 8 h at 145 °C, followed by 8 h at 160 °C and then it was held at 160 °C for 24 h under vacuum. Examples H2 & H3

[0097] Examples H2 and H3 were prepared analogously to example H1 , but with 3 % w/w and 6 % w/w lipase AE07 ( Pseudomonas stutzeri) respectively.

[0098] The following vitrimers in Table 6 were prepared according to the general procedure described above in paragraph [0065], using HMPA as hardener and no added water. The epoxy resin used was either Araldite® LY564 or Eponex™ 1510. All samples were cured at 80 °C for 48 h.

Table 6

[0099] The following vitrimers in Table 7 were prepared according to the general procedure described above in paragraph [0065], using Aradur 917CH as hardener, 5 % w/w lipase AK, 0.5:1 molar ratio of hardener to Araldite LY564 epoxy resin, and 3% v/v water. Varying amounts of trimethylolpropane (TMP) were also added as indicated in Table 7, as % w/w relative to the mass of epoxy resin. All samples were cured at 80 °C for 48 h.

[00100] Analogous vitrimers were prepared to K1-K5 except without any water being added.

These samples did not fully polymerise. Samples prepared without enzyme also did not polymerise.

Table 7

[00101] The following vitrimers in Table 8 were prepared according to the general procedure described above in paragraph [0065], using 1 :1 molar ratio of Aradur 917CH hardener to Araldite LY564 epoxy resin. Varying amounts of lipase AK ( Pseudomonas fluorescens) or Lipase TL ( Pseudomonas stutzeri) and either 0 or 3 wt% water were also added as indicated in Table 8. All samples were cured at 80 °C for 72 h. Table 8 shows the relative cure of the vitrimers in terms of the relative ester (1731 cm ^-1 ) and oxirane (917, 828 cm ^-1 ) band absorptions calculated from the ATR-FTIR spectra of the respective samples (Fig. 18). The formation of ester linkages relative to the amount of oxirane (epoxide) moieties is indicative of the progression of the curing process.

Table 8

[00102] Samples typically had a higher relative ester absorption if they (1) included 5 wt% lipase instead of 3 wt% lipase, (2) included 3 wt% water instead of no water, and (3) used Lipase TL instead of lipase AK.

[00103] The following vitrimers in Table 9 were prepared according to the general procedure described above in paragraph [0065], using molar ratios of Aradur 917CH hardener to Araldite LY564 epoxy resin as shown in the table. Lipase TL ( Pseudomonas stutzeri) and the accelerator A/,/\/-dimethylbenzylamine (DMBA) were also added as indicated in Table 9. All samples were cured at 80-100 °C for 6-24 h.

Table 9

[00104] The following vitrimers in Table 10 were prepared according to the general procedure described above in paragraph [0065], using 1 : 1 molar ratios of Aradur 917CH hardener to Araldite LY564 epoxy resin. Enzymes and water were added as indicated in Table 10. All samples were cured at 80 °C for 75 h.

Table 10

[00105] The results from examples N1 and N4 demonstrate that biomolecules having esterase activity, even if not explicitly recognised as lipases, are still capable of catalysing the ester bond exchange reactions that give the material its vitrimeric properties, in terms of the ability to hold its shape and undergo healing at the curing temperature. Vitrimer Testing

[00106] Example F1 was tested using DSC. Further curing of the resin would manifest itself as an increase in T _g , but no change was observed over two heating cycles (Figure 2). The modulated DSC (MDSC) divides the heat flow required to raise the sample’s temperature into reversible and non-reversible components. The increased exothermic character of the heat flow above ca. 120 °C (Figure 3) may be indicative of enzyme decomposition or residual curing.

[00107] Creep tests were carried out on numerous samples to measure the viscosities at different temperatures. The sample is placed in dynamic mechanical analysis (DMA) apparatus (compression clamps) and a stress (0.5 MPa) is applied for 30 min at different temperatures. The straight slope of the curve (secondary stage of creep) is equal to the stress applied divided by the viscosity. Creep test data are shown in Fig. 8A, Fig. 9 and Fig. 14 for examples F3, F2 and H2 & H3 respectively. From these data, the viscosity of a sample depending on temperature can be calculated, and an Arrhenius plot drawn to determine the activation energy of the sample.

[00108] Fig. 7 shows Arrhenius plots for Examples G1 (3% Resinase® HT), G2 (6% Resinase® HT) and G3 (15% Resinase® HT); activation energies were calculated at 45.7 kJ mol ^-1 , 50.0 kJ mol ^-1 and 51.2 kJ mol ^-1 , respectively.

[00109] Fig. 8 shows the creep test and Arrhenius plot for Example F3 prepared with 6 wt% AE07 lipase. Typically, two phases of creep could be observed during testing of the vitrimer material. The former one appearing at low temperature (typically between 80 °C and 130°C), then a second one appearing at higher temperature (typically 150°C). Once samples were tested at higher temperature, the first stage of creep was not present anymore and vitrimer samples would only creep at temperature higher than 150 °C (Fig. 8A). The Arrhenius plot (Fig. 8B) shows that after a first creep cycle, the creep remains stable at high temperature.

[00110] Fig. 9 and Fig. 10A & B show the creep test, viscosity evolution and Arrhenius plots respectively for Example F2 prepared with 3 wt% AE07 lipase. Two cycles were done to ensure sample stability (cycle 1 and cycle 2). The activation energy (Fig. 10B) was calculated at 52.6 kJ mol ^-1 (cycle 1) and 56.0 kJ mol ^-1 (cycle 2).

[00111] F2 sample was also subjected to reprocessing as follows: example F2 was frozen and then broken into pieces with a hammer. The pieces were frozen again and put in a coffee grinder to obtain a powder. The powder was added to a mould. Compression moulding was realised at 100 °C and 10 bar pressure for 2.5 h. Pressure was increased to 30 bar for a further 2.5 h at 100 °C (Fig. 5 shows photos of the stages of this reprocessing procedure).

[00112] Fig. 10C shows the viscosity evolution and Arrhenius plot for the reprocessed sample. The material had an activation energy of 62.3 kJ mol ^-1 , similar to the original vitrimer, indicating that after reprocessing the material still has the vitrimer properties. Fig. 1 1 shows that the glass transition temperature (via storage modulus and loss modulus) did not change due to reprocessing of the sample. Tensile tests exhibited similar properties between initial and reprocessed sample as shown in Error! Reference source not found.12.

[00113] Swelling tests were carried out to show if the vitrimers prepared from AE07 lipase and Resinase® HT were crosslinked. Linear polymers would be expected to dissolve rather than swell under the test conditions of immersion in 1 ,2,4-trichlorobenzene (TCB) at 135 °C for 1 week. The mass increase was measured to determine the swelling capacity of the network and the results are shown in Fig. 6. Samples with AE07 did not dissolve and swelled. Higher AE07 loadings led to lower swelling than samples with lower AE07 loading. This follows a logical trend as the enzyme should not swell, so with a higher content the polymer network has less“free volume” for solvent to absorb. Similar results were observed for vitrimer samples prepared from Resinase® HT containing 3% and 6% lipase solution, but the sample containing 15% lipase solution partly dissolved when immersed in TCB.

[00114] Scratch tests (Fig. 17A) and abrasion tests (Fig. 17B) demonstrated the healing behaviour of the vitrimers. Heating for 2 min with a domestic hairdryer was sufficient to allow repair of the surface defects.

[00115] Corrosion tests were carried out on steel Q-panels either (i) uncoated; (ii) coated with 40 pm of example M2 vitrimer; or (iii) coated with 40 pm of example M7 vitrimer. None of the panels had degradation or corrosion at the start of the measurement. After 24 h immersion in a 3.5 wt% aqueous NaCI solution, the uncoated control Q-panel showed corrosion around the edges and surface. Similar effects were found on the uncoated regions of all the coated Q-panels, but no signs of degradation were observed within the coated region. After 1 week of immersion of the Q- panels in the solution, the control was heavily corroded. Both coated Q-panels showed similar signs of corrosion on the uncoated surface. There was no rust under the coating nor delamination of the coating.

[00116] Further vitrimer samples were prepared with AE07 lipase and high temperature (145- 160 °C) curing (Examples H1-H3). MDSC and heat-cool-heat (HCH) DSC results are shown in Fig. 13. HCH mode DSC results shows that when samples are cured at higher temperature the samples exhibit more stability to temperature cycling than samples cured at 100 °C. For both example H1 and example H3 the first cycle of heating is the same as the second cycle of DSC heating. MDSC on examples H1 , H2 and H3 (Fig. 13 (a), (b) and (c) respectively) showed that, despite curing at higher temperature, the samples exhibited a non-reversible transition upon curing which could possibly indicate residual stress/thermal history or oxidation.

[00117] Creep tests were performed on examples H2 and H3 at temperatures from 140 °C with 0.1 MPa forced applied for 45 min, followed by 15 min of relaxation at different temperatures (Fig. 14). The creep tests show that the samples relax at high temperature and the relaxation increases as the temperature increases, which is typical of vitrimer polymers. The Arrhenius plots derived from the creep test data (Fig. 15) were used to calculate activation energies of 78.7 kJ mol ^-1 and 44.0 kJ mol ^-1 for examples H2 and H3, respectively. The higher activation energy for the sample with 3 wt% rather than 6 wt% lipase was not unexpected, as the sample was prepared with less catalyst and so needs more thermal energy to trigger the bond exchange reactions.

[00118] DMA experiments were carried out on examples H1 and H3, as shown in Fig. 16. DMA tests were performed from -50°C to 250 °C. There is no phase separation within the samples.

[00119] The results on the high temperature-cured samples are summarised in Table 11.

Table 11

[00120] These results show that even after curing samples at high temperatures exceeding 130 °C, the samples exhibit the same properties as a vitrimer prepared with a classic catalyst (such as metal-based or organic catalysts) in that, for example, T _g does not change and creep persists at high temperature. High temperature usually degrades such traditional catalysts leading to loss of vitrimeric properties. In the case of the enzyme-containing vitrimers of the present invention, high temperature curing brings normal vitrimer behaviour and good stability at higher temperatures. The enzymes’ activity may be preserved by the low water activity within the polymer, preventing hydrolytic degradation of the enzyme, and steric constraints imposed by the polymer, preventing structural degradation of the enzyme.

Summary of Results

[00121] Without being bound by theory, it is postulated that the enzymes AK ( Pseudomonas fluorescens), Resinase® HT ( Aspergillus oryzae), Lipase TL/AE07 ( Pseudomonas stutzeri) and A12 ( Aspergillus niger) consistently showed superior performance (in that they most consistently cross-linked the resin-hardener mixture, and the cured materials healed and held their shape when subjected to the stated tests) due to their improved thermostability at higher temperatures (e.g. in the range 60-90 °C).

[00122] It is also noted that resin-hardener mixtures, which were formulated from cyclic anhydrides and included water, either through the direct addition of water to the mixtures or through the use of lipase formulations that include water, cured more quickly than resin-hardener mixtures without added water.