WO2016020534A1

WO2016020534A1 - Atom transfer radical polymerization of monomers with nitrogen-containing hetercyclic aromatic functional groups utilizing biocatalysts

Info

Publication number: WO2016020534A1
Application number: PCT/EP2015/068277
Authority: WO
Inventors: Nico Bruns; Csaba FODOR
Original assignee: Adolphe Merkle Institute University of Fribourg
Current assignee: Adolphe Merkle Institute University of Fribourg
Priority date: 2014-08-07
Filing date: 2015-08-07
Publication date: 2016-02-11
Anticipated expiration: 2017-02-07

Abstract

Methods for polymerizing vinyl monomers having nitrogen-containing heterocyclic aromatic functional groups, such as N-heterocyclic aromatic functional groups, by atom transfer radical polymerization (ATRP). Various biocatalysts in the form of enzymes are utilized to catalyze the reaction. Polymers having a halogen-containing terminal group, controlled molecular weight and relatively low dispersity indices are disclosed, with the polymers having many applications.

Description

ATOM TRANSFER RADICAL POLYMERIZATION OF MONOMERS WITH NITROGEN- CONTAINING HETEROCYCLIC AROMATIC FUNCTIONAL GROUPS UTILIZING

BIOCATALYSTS

FIELD OF THE INVENTION

[0001 ] The present invention relates to methods for polymerizing monomers having nitrogen-containing heterocyclic functional groups, such as N-heterocyclic aromatic functional groups, by atom transfer radical polymerization (ATRP). Various biocatalysts in the form of enzymes are utilized to catalyze the reaction. Polymers having a halogen- containing terminal group, controlled molecular weight and relatively low dispersity indices are disclosed, with the polymers having many applications.

BACKGROUND OF THE INVENTION

[0002] Derivatives of the heterocyclic aromatic compound imidazole have an extraordinarily importance in nature, since the aromatic ring is a structural element of the amino acid histidine, the hormone histamine, and other naturally occurring molecules. Moreover the imidazole moiety is also presented in many biological active pharmaceuticals and agrochemicals. The imidazole ring is a hydrogen bond acceptor and it can be protonated to form a cationic species. These features allow for a multitude of non-covalent interactions with other molecules. Not surprisingly, the imidazole ring has been recognized as a versatile functional group for the preparation of functional macromolecules. Polymers that feature imidazole rings have attracted great interest for biomedical applications, as these rings offer good biocompatibility while at the same time they are able to complex DNA and RNA for gene delivery. Moreover, such polymers are also used in technical applications, e.g. as dye-transfer inhibitors for washing formulations, or in their alkylated form, as polymeric ionic liquids, see Fodor, Cs.; Domjan, A.; Ivan, B. Polym. Chem. 2013, 4, 3714- 3724 and Fodor, Cs.; Bozi, J.; Blazso, M.; Ivan, B. Macromolecules 2012, 45, 8953-8960. The most widely used monomer that features an imidazole side chain is N-vinylimidazole. Homo- and copolymers of this monomer are used as washing formulation ingredients, as pharmaceutical formulation ingredients, in cosmetic and hair care products, and as filtration materials to remove metal ions from alcoholic beverages like wine. Moreover, poly(vinylimidazoles) show potential in applications that range from non-toxic gene delivery vectors, metal ion imprinted matrices, proton conducting fuel cells materials, metal ion complexing or carbon dioxide absorbing membranes, catalysts and catalyst supports, microgels, or biomaterials with various pharmacological activities. Many of these applications would benefit from poly(N-vinylimidazoles) with a predetermined molecular weight and a narrowly dispersed molecular weight distribution. Such well-defined polymers allow to conclusively study their structure-property relationships and consequently to tailor their properties. For example, the length of polycationic polymers can define its drug delivery efficacy and its toxicity. However, N-vinylimidazole is commonly polymerized by free radical polymerization which yields broadly distributed polymers and does not allow controlling the molecular weight and architecture of the polymers due to the occurrence of termination reactions. Moreover, free radical polymerization most often results in macromolecules with high molecular weight and short chains are difficult to obtain. Reversible-deactivation radical polymerizations (controlled/living" radical polymerizations; CRP), on the other hand, suppress termination reactions through a dynamic equilibrium between propagating (living) and nonpropagating (dormant) polymer chains. Thus, methods like atom transfer radical polymerization (ATRP), see K. Matyaszewski; L. Bombalski; W. Jakubowski; K. Min; J. Spanswick; N. Tsavresky WO Patent 2005/087819 A1 Atom transfer radical polymerization process K. Matyaszewski; W. Jakubowski; K. Min; J. Spanswick WO Patent 2007/025310 A1 Polymerization process with catalyst reactivation, S. Balk; G. Lohden; E. Osthaus WO Patent 2008/017523 A1 Process for preparating halogen-free ATRP products, Tsarevsky, N. V.; Matyjaszewski, K. Chem. Rev. 2007, 107, 2270-2299, di Lena, F.; Matyjaszewski, K. Prog. Polym. Sci. 2010, 35, 959-1021 , Matyjaszewski, K. Macromolecules 2012, 45, 4015- 403, nitroxide-mediated polymerization (NMP), see Hawker C.J.; Bosman A.W.; Harth E. Chem. Rev. 2001 , 101, 3661 -3688 and reversible addition-fragmentation chain transfer polymerization (RAFT), see Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffrey, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L; Moad, G.; Rizzardo, E.; Than, S. H. Macromolecules 1998, 31, 5559-5562, Moad, G.; Chiefari, J.; Chong, Y. K.; Krstina, J.; Mayadunne, R. T. A.; Postma, A., Rizzaro, E., Thang, S. H. Polym. Int. 2000, 49, 993-1001 enable the synthesis of well-defined functional polymers with a low dispersity index (D) and also allow tuning of important key macromolecular parameters, such as molecular weight and the functional group at the chain end, see Braunecker, W. A.; Matyjaszewski, K. Prog. Polym. Sci. 2007, 32, 93-146. ATRP continues to be the most widely used CRP method. One reason for its popularity is that it yields halogen-terminated polymer chains. Bromine or chloride chain ends are versatile functional groups that allow to initiate another polymerization or that can be converted into a variety of other functionalities, such as azides for click chemistry and hydroxyl groups. On the other hand, halogenated chain ends are stable and biocompatible and do not interfere with the application of ATRP-derived polymers in biomedical or engineering applications.

[0003] Unfortunately, N-vinyl monomers like N-vinylimidazole or N-vinylpyrrolidone are difficult to polymerize by CRP methods. The propagating radicals are highly reactive because they are not stabilized by resonance. They tend to undergo chain termination and chain transfer reactions. Nevertheless, recent advances in RAFT, see Ge, Z.; Xie, D.; Chen, D.; Jiang, X.; Zhang, Y.; Liu, H.; Liu, S. Macromolecules 2007, 40, 3538-35469, Mori, H.; Yahagi, M.; Endo, T.: Macromolecules 2009, 42, 8082-8092., Skouta, Ft.; Wei, S.; Breslow, Ft.: J. Am. C em. Soc. 2009, 131, 15604-15605. Skouta, Ft.; Wei, S.; Breslow, Ft.: J. Am. Chem. Soc. 2009, 131, 15604-15605., Allen, M. H.; Hemp, S. T.; Smith, A. E.; Long, T. E. Macromolecules 2012, 45, 3669-3676., Allen, M. H.; Hemp, S. T.; Thang, M.; Thang, M.; Smith, A. E.; Moore, R. B.; Long, T. E. Polym. Chem. 2013, 4, 2333-2341 . NMP, see Green, M., D.; Long, T. E. Polym. Prep. 2009, 50(2), 434-435 and cobalt-mediated controlled radical polymerization, see Detremleur, C; Debuigne, A.; Hurtgen, M.; Jerome, C. Macromolecules, 201 1 , 44, 6397-6404 enabled the polymerization of N-vinylimidazole with good control over the polymer's molecular weight. However, all reported attempts to polymerize this monomer with ATRP in a controlled way failed. They either gave none or very small conversions so that the polymers could not be analyzed, see Jana, S.; Vasantha, V. A.; Stubbs, L. P.; Parthiban, A.; Vancso, J. G. Polym. Sci. Parf.A Polym. Chem. 2013, 51, 3260-3273 or the polymers were grown from surfaces and not further characterized with respect to their molecular weight, their chain end functionalization and their molecular structure, see Li, J.; Han, H.; Wang, Q.; Liu, X.; Jiang, S. J. Sep. Sci. 2010, 33, 2804-2810, and Jiang, S.; Sun, M.; Liu, X.; Qiu, H. CN Patent 102101043 B Method for preparing polyvinyl imidazole type silica gel filler. The reason for the failure of ATRP to polymerize N-vinylimidazole and other regioisomeric vinylimidazoles is that the monomers and the resulting polymers complex metal ions, see Green, M. D.; Allen, M. H.; Dennis, J. M.; Cruze, D. S.; Gao, R.; Winey, K. I.; Long, T. E. Eur. Polym. J. 201 1 , 47, 486-496, Allen, M. H.; Hemp, S. T.; Smith, A. E.; Long, T. E. Macromolecules 2012, 45, 3669-3676, and used as metal removing and/or binding polymer membranes, see Bessbousse, H.; Rhlalou, T.; Verchere, J. F.; Lebrun, L. J. Chem. Eng. 2010, 164, 37-48., Ajji, Z.; AN, A. M. J. Hazard. Mater. 2010, 173, 71 -74., Ozmen, F.; Kavlaki, P. A.; Giiven, O. J. Appl. Polym. Sci. 201 1 , 1 19, 613-619, and therefore strip metal ions from the transition metal complexes that are conventionally used as ATRP catalysts. Even if ATRP would yield well-defined poly(vinylimidazoles), the ATRP catalysts would be difficult to remove quantitatively from the polymers due to the polymer's capability to bind metal ions. Residual metal traces can cause unwanted coloration of the polymer product, inhibit its use in biomedical applications due to the toxicity of transition metal ions and deteriorate the material properties when used in electronic, energy, or filtration applications.

[0004] ATRP catalyzed by metallo-enzymes, see Hollmann, F.; Arends, I. W. C. E. Polymers 2012, 4, 759-793 could circumvent these problems, due to the fact that these proteins strongly bind their transition metal ion cofactors within their three-dimensional structure. Metalloproteins with various metal centers, such as horseradish peroxidase (HRP), see Sigg, S. J.; Seidi, F.; Renggli, K.; Silva, T. B.; Kali, G.; Bruns, N. Macromol. Rapid Commun. 201 1 , 32, 1710-1715, Ng, Y-H.; di Lena, F.; Chai, C. L. L. Chem. Commun. 201 1 , 47, 6464-6466; hemoglobin (Hb), see Silva, T. B.; Spulber, M.; Kocik, M. K.; Seidi, F.; Charan, H.; Rother, M.; Sigg, S. J.; Renggli, K.; Kali, G.; Bruns, N. Biomacromolecules, 2013, 14, 2703-2712 ; catalase, see Ng, Y-H.; di Lena, F.; Chai, C. L. L. Polym. Chem. 201 1 , 2, 589-594 and laccase (Lac), see Ng, Y-H.; di Lena, F.; Chai, C. L. L. Chem. Commun. 201 1 , 47, 6464-6466, Ng, Y-H.; di Lena, F.; Chai, C. L. L. Polym. Chem. 201 1 , 2, 589-594 were recently discovered to display catalytic activity under ATRP conditions. These "ATRPases" have been used as catalysts for the polymerizations of acrylamide, see Macromol. Rapid Commun. 201 1 , 32, 1710-1715. Biomacromolecules, 2013, 14, 2703-2712, and Yamashita, K.; Yamamoto, K.; Kadokawa, J-i. Polymer 2013, 54, 1775-1778; acrylate, see Chem. Commun. 201 1 , 47, 6464-6466, Polym. Chem. 201 1 , 2, 589-594 Polym. Chem. 201 1 , Simakova, A.; Mackenzie, M.; Averick, S. E.; Park, S.; Matyjaszewski, K. Angew. Chem. Int. Ed. 2013, 52, 1 -5 and methacrylate monomers. However, other monomers were not yet explored.

SUMMARY OF THE INVENTION

In view of the above, a problem of the present invention was to develop polymers and methods for producing polymers derived from monomers having nitrogen-containing heterocyclic aromatic functional groups that have controlled molecular weight and/or relatively low dispersity (D). The average molecular weight of the polymer made by the enzymatic ATRP can be predetermined by the applied monomer and initiator ratio (degree of polymerization, DP_n = d[M]/[l]₀) while maintaining relatively narrow molecular weight distribution. The use of enzymes as catalysts under ATRP conditions provide a high degree of control over molecular weight of the polymers, especially at lower number average molecular weight (<20 000 g mol^"1 , or preferably <10 000 g mol^"1) often with a low D generally less than 1 .6 and preferably between 1 .0 and 1 .5.

[0005] Yet another problem of the present invention was to develop polymers and methods for producing the polymers that include a halogen-containing end group and repeating units derived from monomers having nitrogen-containing heterocyclic, such as N- heterocyclic aromatic functional groups, wherein the halogen-containing end group can be utilized to initiate a polymerization reaction or can be converted into a variety of other functionality, or does not interfere with the application of the polymer in a particular application.

[0006] These problems and others are solved by the methods and polymers of the present invention. The polymers of the invention include polymers derived from nitrogen- containing, e.g. N-heterocyclic aromatic functional groups, wherein halogen-terminated poly(vinylimidazoles) are produced in one embodiment. The polymers can be produced with a controlled or predetermined molecular weight due to utilization of the ATRP process. [0007] Still another object of the present invention is to provide polymers that are substantially metal free. For example, in one embodiment, the polymers, for example poly(N-vinylimidazole), have a metal (for example copper, iron and zinc) content below 20 ppm, preferably below 10 ppm.

[0008] Another object of the present invention is to provide a process for enzyme- catalyzed ATRP of monomers comprising nitrogen-containing aromatic functional groups such as N-heterocyclic functional groups, aromatic functional groups in some embodiments.

[0009] Yet another object of the present invention is to provide method for polymerization of monomers comprising the nitrogen containing aromatic heterocyclic functional groups utilizing biocatalysts comprising an enzyme, in particular a metal-ion containing enzyme.

[0010] Another object of the present invention is to provide a polymerization method for monomers comprising nitrogen-containing heterocyclic aromatic functional groups, wherein polymerization is initiated with a compound including a transferrable halogen atom.

[0011 ] A further object of the present invention is to provide halogen-terminated polymers derived from monomers with nitrogen-containing aromatic functional groups, e.g. N-heterocyclic aromatic functional groups such as N-vinylimidazoles suitable for use in one or more of the following applications, bio-medical applications, washing formulations, pharmaceutical formulations, filtration applications, non-toxic gene delivery vectors, metal ion imprinted matrices, proton conducting fuel cell materials, metal ion complexing or carbon dioxide absorbing membranes, anti-fouling coatings, catalysts and catalyst supports or microgels.

[0012] Accordingly, in one aspect of the invention, a process for polymerizing at least vinyl monomers having nitrogen-containing heterocyclic aromatic functional groups is disclosed, comprising the steps of polymerizing via ATRP at least the vinyl monomers, that are the same or different, with nitrogen-containing heterocyclic aromatic functional groups in the presence of an enzyme catalyst and an initiator.

[0013] In another aspect of the invention, a (co)polymer derived from enzyme catalyzation is disclosed comprising repeat units derived at least from vinyl monomers, that are the same or different, having nitrogen-containing heterocyclic aromatic functional groups; and an end group comprising a halogen atom.

[0014] In a further aspect of the invention, copolymers derived from enzyme catalyzation are disclosed comprising repeat units derived from vinyl monomers, having nitrogen- containing heterocyclic aromatic functional groups, and vinyl monomers with non-aromatic heterocyclic functional groups, for example Vlm-co-vinylpyrrolidone copolymers. [0015] In another aspect of the invention block copolymers derived from enzyme catalyzation are disclosed comprising at least one block of repeat units derived from vinyl monomers, having nitrogen-containing heterocyclic aromatic functional groups, and at least one second block of two or more same or different repeat units such as ethylene glycol, an ether group, (meth)acrylate group wherein the methyl group is present or absent, or ester group. PEG-block-PVIm copolymer is an example of block copolymer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention will be better understood and other features and advantages will become apparent by reading the detailed description of the invention, taken together with the drawings, wherein:

[0017] Figure 1 illustrates the reaction scheme of Lac-catalyzed ATRP of N- vinylimidazole.

[0018] Figure 2 illustrates first order kinetic plot (a), and molecular weights, D values vs. percent monomer conversion (b) of the Lac-catalyzed ATRP polymerization of Vim, at room temperature (ratio of HEBIB/Vlm/NaAsc/Lac 1 :50:0.2:0.005).

[0019] Figure 3 illustrates ¹ H NMR spectrum of the Vim monomer and of purified poly(N- vinylimidazole) (PVIm) polymer synthesized by Lac-catalyzed ATRP of Vim.

[0020] Figure 4 illustrates MALDI-ToF MS spectrum of the purified poly(N- vinylimidazole) (PVIm) polymer, prepared by Lac-catalyzed ATRP of Vim.

DETAILED DESCRIPTION OF THE INVENTION

[0021 ] Atom transfer radical polymerization of monomers comprising nitrogen- containing heterocyclic aromatic functional groups is described herein. The monomers are polymerized in the presence of an atom transfer radical polymerization initiator and an enzyme, in particular a metal-ion-containing enzyme catalyst. The polymerization process may additionally utilize one or more of aqueous solutions with and without organic (co)solvents and a reducing agent.

[0022] Initiator

[0023] The initiator that can be utilized in the polymerization process of the present invention may be any compound and has one or more atoms or atomic groups that are radically transferrable under the polymerization conditions and can initiate chain growth. Depending on the initiator structure utilized and the number of halogen atoms, the architecture of the prepared polymers can be varied from linear, for example when using alkyl halides with a single halogen atom or two halogen atoms, to star-like or brush-like when using multiple halogen atoms in the initiator. The initiators include compounds with a transferrable halogen that can initiate chain growth in one embodiment. A variety of initiators, such as alkyl halides, have been utilized in the art to perform atom transfer radical polymerization. Many different types of halogenated compounds are potential initiators. The initiators may comprise two or more radically transferrable atoms or groups or be a polymer or other compound comprising a radically transferrable atom or group attached to the polymer or compound.

[0024] Examples of suitable initiators (described by the Y— (X)_n formula) include, but are not limited to:

R¹ R²R³C— X

R¹C(=0)—X

R¹ R²R³Si— X

R¹ R²N— X

R¹ N— X₂

(R¹0)_nP(0)_m-X₃-_n and

(R¹)(R² 0)P(0)_m-X,

[0025] where X is selected from the group consisting of CI, Br, I, OR⁴, [where R⁴ is an alkyl group with 1 to 20 carbon atoms, where each hydrogen atom can independently be replaced by a halide, preferably chloride or fluoride, an alkenyl with 2 to 20 carbon atoms, preferably vinyl, an alkynyl with 2 to 10 carbon atoms, preferably acetylenyl or phenyl, which can be substituted with 1 to 5 halogen atoms or alkyl groups with 1 to 4 carbon atoms, or aralkyl (aryl-substituted alkyl in which the aryl group is phenyl or substituted phenyl and the alkyl group is an alkyl with 1 to 6 carbon atoms, such as benzyl, for example)], SR⁵, SeR⁵, OC(=0)R⁵, OP(=0)R⁵, OP(=0) (OR⁵)₂, OP(=0)OR⁵, O— N(R⁵)₂, S— C(=S)N(R⁵)₂, CN, NC, SCN, CNS, OCN, CNO and N₃, where R⁵ means an alkyl group or a linear or branched alkyl group with 1 to 20, preferably 1 to 10 carbon atoms, where two R⁵ groups, is present, together can form a 5, 6 or 7-member heterocyclic ring; and R¹ , R² and R³ are independently chosen from the group consisting of hydrogen, halogens, alkyl groups with 1 to 20, preferably 1 to 10 and especially preferably 1 to 6 carbon atoms, cycloalkyl groups with 3 to 8 carbon atoms, R^6* ₃Si, C(=Y^*)R^7*, C(=Y^*)NR^8*R^7*, where Y^*, R^6*, R^7*, R^8* and R^9* are independently chosen from an alkyl group with 1 to 20 carbon atoms, hydrogen or R⁸ and R⁹ together can form an alkylene group with 2 to 7, preferably 2 to 5 carbon atoms or they form a 3 to 8 member, preferably 3 to 6 member ring, COCI, OH, (preferably one of the residues R¹ , R² and R³ is OH), CN, alkenyl or alkynyl groups with 2 to 20 carbon atoms, preferably 2 to 6 carbon atoms and especially preferably allyl or vinyl, oxiranyl, glycidyl, alkylene or alkenylene groups with 2 to 6 carbon atoms, which are substituted with oxiranyl or glycidyl, aryl, heterocyclyl, aralkyl, aralkenyl (aryl-substituted alkenyl, where aryl is defined as above and alkenyl is vinyl, which is substituted with one or two d to C₆ alkyl groups and/or halogen atoms, preferably with chlorine), alkyl groups with 1 to 6 carbon atoms, in which one up to all of the hydrogen atoms, preferably one, is/are substituted by halogen (preferably fluorine or chlorine, if one or more hydrogen atoms are replaced, and preferably fluorine, chlorine or bromine, if one hydrogen atom is replaced), alkyl groups with 1 to 6 carbon atoms, which with 1 to 3 substituents (preferably 1 ) are chosen from the group consisting of Ci-C₄ alkoxy, aryl, heterocyclyl, C(=Y^*)R^7*, (where Ft^7* is defined as above), C(=Y^*)NR^8*R^9* (where R^8* and R^9* are defined as above), oxiranyl and glycidyl (preferably not more than 2 of the residues R¹ , R² and R³ are hydrogen, especially preferably a maximum of one of the residues R¹ , R² and R³ is hydrogen);

n is 0 or 1 ; and m=0, 1 or 2.

[0026] Poly(ethylene glycol) methyl ether 2-bromoisobutyrate is another suitable initiator.

[0027] Among the especially preferred initiators are benzyl halides like p- chloromethylstyrene, oc-dichloroxylene, α,α-diochloroxylene, α,α-dibromoxylene and hexakis (oc-bromomethyl)benzene, benzyl chloride, benzyl bromide, 1 -bromo-1 -phenylethane and 1 - chloro-1 -phenylethane; carboxylic acids derivatives that are halogenated in a position, such as propyl 2-bromopropionate, methyl 2-chloropropionate, ethyl 2-chloropropionate, methyl 2- bromopropionate, ethyl 2-bromoisobutyrate, 2-hydroxyethyl-2-bromosiobutyrate; tosyl halides such as p-toluenesulfonyl chloride; alkyl halides like tetrachloromethane, tribromomethane, 1 -vinylethyl chloride, 1 -vinylethyl bromide; poly(ethylene glycol) methyl ether 2-bromoisobutyrate and halogen derivatives of phosphoric acid esters like dimethylphosphoric chloride.

[0028] As known in the art, alkyl halides used as initiators may also contain various functional groups. Some advantages of using functional initiators in the synthesis of polymers of the present invention include, direct functionalization, no post-polymerization modification requirements, oc-telechelic polymers can be produced and multiple applicable functionalities.

[0029] Various suitable initiators are known in the art and/or commercially available from sources such as Sigma-Aldrich.

[0030] The initiator is used in a concentration that ranges generally from about 1 to about 100 mmol/L, desirably from about 10 to about 70 mmol/L, and preferably from about 20 to about 40 mmol/L of halide terminated initiators, such as 2-hydroxyethyl-2- bromosiobutyrate.

[0031 ] Monomers

[0032] The polymerization process and thus the polymers of the invention comprise monomers having nitrogen-containing heterocyclic aromatic functional groups. N- heterocyclic aromatic functional group containing monomers are utilized in some embodiments. Preferably the monomers are vinyl group-containing monomers including the indicated functionality.

[0033] Examples of suitable vinyl-containing monomers having nitrogen containing heterocyclic aromatic functional groups include, but are not limited to, 1 -vinylimidazole, 2- vinylimidazole, 4-vinylimidazole, 1 -vinylpyridine, 2-vinylpyridine, 3-vinylpyridine, 2-methyl-5- vinylpyridine, 3-ethyl-4-vinylpyridine, 2,3-dimethyl-5-vinylpyridine, 2-vinylpyrimidine, 5- vinylpyrimidine, 1 -vinylpiperidine, 2-vinylpiperidine, 4-vinylpiperidine, 9-vinylcarbazole, 3- vinylcarbazole, 4-vinylcarbazole, 2-methyl-1 -vinylimidazole, vinylthiazoles and hydrogenated vinylthiazoles, 1 ,2,3-vinyltriazole, 1 ,2,4-vinyltriazole, 5-vinyltetrazole, vinyloxazoles and hydrogenated vinyloxazoles, derivatives thereof and combinations thereof.

[0034] The vinyl monomers with a nitrogen-containing heterocyclic aromatic functional group and one or more, different monomer(s) can be utilized to form copolymers. Any other monomers compatible with the ATRP polymerization process can be utilized. As such, many different unique copolymers can be created and can be arranged in any form, such as random, alternating, graft or block. Examples of suitable comonomers include, but are not limited to, vinyl monomers with non-aromatic heterocyclic functional groups (such as N-vinyl- 2-pyrrolidone, 2-vinylpyrrolidone, N-vinylpyrrolidine, 3-vinylpyrrolidine, N-vinylcaprolactam, N-vinylbutyrolactam), methacrylates and acrylates (such as hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methycrylate, hydroxypropyl acrylate, butyl methacrylate, butyl acrylate, propyl methacylate, propyl acrylate, methyl methacrylate, methyl acrylate, lauryl methacrylate, lauryl acrylate, methacrylic acid, acrylic acid, dimethylaminoethyl methacylate, dimethylaminoethyl acylate, polyethyleneglycol acrylate, polyethylene glycol methacrylate, oligoethyleneglycol methacrylate, oligoethyleneglycol acrylate), methacrylamides and acrylamides (such as acrylamide, methacrylamide, N- isopropylacrylamide, N-(2-hydroxypropyl) methacrylamide, N-(2-hydroxypropyl) acrylamide), styrene-based monomers (such as styrene, sodium 4-styrene sulfonate), and monomers containing proton donors (such as diethyl- or diisopropyl-p-viniybenzyl phosphonate). The conversion of halogen-terminated chain ends of these homo and copolymers into polymerizable functional groups such as acrylates or methacrylates allows to form cross- linked polymer (co)networks.

[0035] Catalyst

[0036] The catalyst employed in the present invention comprises an enzyme, more particularly a metal-ion containing enzyme. In various embodiments the catalysts can be one or more natural enzymes, genetically engineered variants and proteins that have been chemically modified, e.g. by PEGylation, the conjugation with other polymers, or the substitution of the natural metal with a non-natural metal ion cofactor. The metal-ion containing enzymes have unexpectedly been found to be stable under reaction conditions and provide the reaction with well controlled ATRP. In addition, enzyme catalysts are environmentally friendly alternatives to the commonly utilized catalysts as the catalysts are biocompatible, biodegradable and work under mild conditions, for example ambient pressure and temperature.

[0037] Examples of suitable enzyme catalysts of the present invention include, but are not limited to Cu-containing enzymes and proteins such as laccase (Lac), tyrosinase (TYR), hemocyanin (He), cytochrome oxidase (COX), superoxide dismutase (SOD), plastocyanin (PCY), and ceruloplasmin (CP), enzymes containing heme, such as hemoglobin (Hb), horseradish peroxidase (HRP), soybean peroxidase (SBP), myoglobin (Mb), cytochromes (Cyt), catalase (Cat) and chloroperoxidase (CPO), as well as the Mn-containing enzyme arginase (ARG), V-containing enzymes vanadium-haloperoxidase (V-HPO), vanadium- chloroperoxidase (V-CIPO), vanadium-bromoperoxidase (VBrPO), and the Ni-containing enzyme urease. In one embodiment laccase is a preferred enzyme catalyst.

[0038] The enzyme catalyst is used in a concentration that ranges generally about 0.001 to about 1 mmol/L, desirably from about 0.05 to about 0.5 mmol/L, and preferably from about 0.1 to about 0.3 mmol/L of metal ion containing enzymes, such as laccase.

[0039] Aqueous Solutions

[0040] In various embodiments an aqueous solution is utilized as a medium for polymerization of the indicated monomers. The pH range of the solution depends on the enzyme catalyst utilized.

[0041 ] Examples of suitable compounds utilized to form the acidic and alkaline buffer solutions include, but are not limited to, sodium acetate, sodium citrate, sodium phosphate, sodium bicarbonate, Tris buffers, Good's buffers, or mixtures of buffer solutions, such as phosphate/citrate buffer. The pH of the buffer solution ranges generally from about 3 to about 10, desirably from 4 to about 7 and is preferably about 4.0 for Laccase, 4.0 for hemoglobin (Hb) and 6.0 or 7.0 for horseradish peroxidase (HRP). Beside the aqueous solutions purified, deionized water can be used. Another possibility is the addition of cosolvents, including, but not limited to, tetrahydrofuran or simple or higher alcohols. The cosolvent content ranges generally from about 0 to about 90 %, desirably from 10 to about 30 %, depending on the used catalytic system.

[0042] Reducing Agents

[0043] Reducing agents of the present invention are utilized to reduce the transition metal ion bearing enzymes in the oxidation state. The reducing agents include, but are not limited to inorganic or organic compounds with reducing capability. Examples of suitable reducing agents include, but are not limited to ascorbic acid, sodium-L ascorbate, sodium dithionite hydrazine, hydrazine hydrate, hydrazine derivatives, stannous compounds, mercaptoethanol, acetylacetonate, reducing sugars, and monosaccharides. The reducing agents may also preferably be soluble in the polymerization medium, or at least in one phase of the polymerization medium.

[0044] Polymerization Reaction Conditions

[0045] Advantageously, the polymerization reactions of the present invention can be carried out at a normal pressure and at room temperature. That said, the reaction can be performed at a reduced pressure or elevated pressure, if desired. Furthermore, if desired, the polymerization temperature can range generally from about -20 to about 100°C, desirably from about 20 to about 50°C and preferably from about 20 to about 40°C, depending on the activity and stability of the particular catalytic system at the polymerization temperature. The desired pH of the polymerization media can range generally from about 1 to about 14, desirably from about 3 to about 9 and preferably from about 3.5 to about 4.5 (when Lac and/or Hb is utilized), depending on the activity and stability of the particular catalytic system at the polymerization media.

[0046] In order to form a desired polymer, any desired components of the reaction are combined in an inert atmosphere; preferably, the inert atmosphere is made up of inert gases selected from the group consisting of argon, nitrogen, helium and mixtures thereof in one embodiment. Although an inert atmosphere is preferred, the reaction can also be performed in air. In one preferred embodiment, the initiator and any monomers to be polymerized are combined in a suitable vessel. Any desired buffer solution and/or (co)solvent can also be added to the vessel. Desired quantity of enzyme catalysts are dissolved in desired buffer solution and/or (co)solvent. In various embodiments, the reaction mixture and catalyst- containing solution can be deoxygenated by bubbling an inert gas, e.g. argon, therethrough for a suitable period of time or instead of the described method a freeze-thaw method, that is freeze-evacuate-flush with inert gas-thaw cycle can be used. The desired amount of the catalyst containing solution is transferred into the reaction vessel comprising the initiator and monomers. To start the polymerization, reducing agent dissolved in desired buffer solution and/or (co)solvent and deoxygenated by different methods is added to the polymerization media in the main reaction vessel. In a preferred embodiment, the reaction mixture is stirred during the polymerization process. The length of a polymerization process can vary depending upon the compounds utilized to perform the same.

[0047] Once polymerization has been completed to a desired degree or conversion, the resulting mixture can be exposed to air and further processed as desired. From the polymerization mixture, a desired amount of sample can be removed and diluted with desired amount of non-deoxygenated D₂0 (or other suitable deuterated solvents) after exposed to air, and filtered over neutral aluminum oxide. For preparative purposes, the polymer can be purified e.g. by precipitation. For analytical purposes, the filtrate can be centrifuged and analyzed by ¹ H NMR to determine the conversion and molecular weight of the produced polymer. The NMR sample can be recovered and prepared for gel permeation chromatography (GPC) measurements by evaporating the solvents over the polymer sample to yield a solid, which can be redissolved in a suitable GPC eluent such as dimethylformamide (DMF) containing 0.05 M lithium bromide. The samples can be filtered through a syringe filter (PTFE syringe filter, pore size: 0.20 μηι) and can be analyzed by GPC.

[0048] The molecular weight and the dispersity index (D) of the polymers were determined by GPC using PSS GRAM columns (Mixed bed PSS GRAM analytical linear 10 μηι (300 mm x 8.0 mm) and PSS GRAM analytical precolumn 10 μηι (50 mm x 8.0 mm), separation range from 500 to 10⁶ g mol^"1) thermostated to 60 °C in DMF containing 0.05 M lithium bromide solvent with a flow rate of 1 .0 ml_ min^"1 and a refractive index detector. The molecular weight determination of a polymer is based on poly(methylacrylate) (PMMA) standards (PSS Polymer Standards Service). As described hereinabove, (co)polymers can be produced having a number average molecular weight of less than 20,000 g/mol or preferably less than the 10,000 g/mol. (Co)polymers can be produced having a low dispersity of generally less than 1 .6 and preferably between 1 .0 and 1 .5. As indicated herein, advantageously, substantially metal free or metal free polymers can be produced.

[0049] The living nature of the polymerization is shown by the linear increase of molecular weight with conversion, relatively low dispersity and a first order kinetic. Terminal ends of polymer chains formed include a halogen group that is derived from the initiator and can be utilized for further reaction, if desired. The halogen-terminated nitrogen-containing heterocyclic group containing vinyl polymers open possibilities for the preparation of block copolymers or of polymers with a functional end group that is highly beneficial, for example, as surfactants with dye transfer inhibition properties or as micellar/vesicular drug/gene- delivery systems.

[0050] Examples

[0051 ] Materials

[0052] /V-Vinylimidazole (Vim,≥ 99%) and N-vinylpyrrolidine (NVP,≥ 99%) were distilled under reduced pressure at 72°C and 68 °C, respectively, and kept under argon until used. Laccase from Tramtes versicolor (10 units/mg), Horseradish peroxidase (HRP, 300 units/mg solid), Hemoglobin from bovine blood (Hb), 2-hydroxyethyl-2-bromoisobutyrate (HEBIB, 95%), poly(ethylene glycol) methyl ether 2-bromoisobutyrate (average M_n 2000 g mol^"1 ; PEGBIB), sodium L-ascorbate (crystalline NaAsc, ≥ 98%), sodium acetate (anhydrous, ≥ 99%), and acetic acid (puriss.) were purchased from Sigma-Aldrich, and used as received. Laccase was also obtained from Amano and from Novozymes. Deuterium oxide (D, 99.9%) was obtained from Cambridge Isotope laboratories and dimethyl sulfoxide-d₆ (- 100%, 99.96 atom% D) was purchased from Sigma-Aldrich. Argon (99.998%, Carbagas) was used without further purification.

[0053] Vim polymerization with Lac

[0054] A reaction scheme is shown in Figure 1 . In a typical reaction, HEBIB (59.1 mg, 0.28 mmol) and Vim (1 .32 g, 14.0 mmol) were weighed in a round-bottom flask and dissolved in 6.1 mL sodium acetate buffer (100 mM, pH 4.0) in argon atmosphere. Stock solutions of Lac and NaAsc were prepared by dissolving Lac (184.8 mg, 2.8 μηιοΙ) in 1 .0 mL acetate buffer and NaAsc (22.2 mg, 0.12 mmol) in 1 .0 mL acetate buffer, respectively. The reaction mixture and the stock solutions were deoxygenated by bubbling argon for ca. 30 min. 0.5 mL of the enzyme solution was transferred into the initiator/monomer flask using an argon purged syringe. The polymerization was started by adding 0.5 mL of the reducing agent solution to the reaction mixture with an argon-purged syringe. The molar ratio of the reactants in the reaction mixture was 1 :50:0.2:0.005 (HEBIB:Vlm:NaAsc:Lac). The reaction was stirred at room temperature. 1 mL samples were removed from the polymerization mixture for analytical purposes at periodic time intervals. The samples were exposed to air, diluted with 1 .5 mL non-deoxygenated D₂0 and filtered over a small plug of neutral aluminum oxide. The filtrate was analyzed by ¹ H NMR to determine the conversion. The NMR sample was recovered and prepared for GPC measurements by evaporating the solvents over the polymer sample to yield a solid that was redissolved in the GPC eluent (dimethylformamide (DMF) containing 0.05 M lithium bromide), filtered through a syringe filter (Chromafil O-20/15 MS, PTFE syringe filter, pore size: 0.20 μηι, filter diameter: 15 mm) and analyzed by gel permeation chromatography (GPC). For ¹ H-NMFt, MALDI ToF MS and ICP-OES analysis of the synthesized PVIm a reaction was stopped after 7 h and filtered over a small plug of neutral aluminum oxide. The solution was centrifuged and dried under vacuum. The product was dissolved in methanol. The polymer was purified by precipitation in acetone and dried under vacuum.

[0055] Vim polymerization with HRP

[0056] In a typical reaction, HEBIB (8.4 mg, 0.04 mmol) and Vim (188.2 mg, 2.0 mmol) were weighed in a round-bottom flask and dissolved in 0.213 mL sodium acetate buffer (100 mM, pH 7.0) in argon atmosphere. Stock solutions of HRP and NaAsc were prepared by dissolving HRP (3.52 mg, 0.08 mol) in 0.8 mL acetate buffer and NaAsc (15.9 mg, 0.08 mmol) in 0.8 mL acetate buffer, respectively. The reaction mixture and the stock solutions were deoxygenated by bubbling argon for ca. 30 min. 0.4 mL of the enzyme solution was transferred into the initiator/monomer flask using an argon purged syringe. The polymerization was started by adding 0.4 mL of the reducing agent solution to the reaction mixture with an argon-purged syringe. The molar ratio of the reactants in the reaction mixture was 1 :50:1 :0.001 (HEBIB:Vlm:NaAsc:HRP). The reaction was stirred at room temperature. 1 .0 mL sample was removed from the polymerization mixture for analytical purposes, exposed to air, diluted with 1 .5 mL non-deoxygenated D₂0 and filtered over a small plug of neutral aluminum oxide. The filtrates were analyzed by ¹ H NMR to determine the conversion.

[0057] Vim polymerization with Hb

[0058] In a typical reaction, HEBIB (25.3 mg, 0.12 mmol) and Vim (0.57 g, 6.0 mmol) were weighed in a round-bottom flask and dissolved in 1 .039 mL sodium acetate buffer (100 mM, pH 4.0) in argon atmosphere. Stock solutions of Hb and NaAsc were prepared by dissolving Hb (77.4 mg, 1 .2 μηιοΙ) in 2.0 mL acetate buffer and NaAsc (9.5 mg, 0.05 mmol) in 2.0 mL acetate buffer, respectively. The reaction mixture and the stock solutions were deoxygenated by bubbling argon for ca. 30 min. 1 .0 mL of the enzyme solution was transferred into the initiator/monomer flask using an argon purged syringe. The polymerization was started by adding 1 .0 mL of the reducing agent solution to the reaction mixture with an argon-purged syringe. The molar ratio of the reactants in the reaction mixture was 1 :50:0.2:0.005 (HEBIB:Vlm:NaAsc:Hb). The reaction was stirred at room temperature. The polymerization was stopped after 24h and 1 mL sample were removed from the polymerization mixture for analytical purposes, exposed to air, diluted with 1 .5 mL non-deoxygenated D₂0 and filtered over a small plug of neutral aluminum oxide. The filtrate was analyzed by ¹ H NMR to determine the conversion. The PVIm polymerization was filtered over a plug of neutral aluminum oxide. The solution was centrifuged, dried under vacuum, and dissolved in methanol. The polymer was purified by precipitation in acetone and dried under vacuum and analyzed by ¹ H NMR.

[0059] Copolymerization of VIM and NVP with Lac

[0060] In a typical reaction, HEBIB (59.1 mg, 0.28 mmol), Vim (1 .98 g, 21 mmol) and NVP (0.78 g, 7.0 mmol) were weighed in a round-bottom flask and dissolved in 4.7 mL sodium acetate buffer (100 mM, pH 4.0) in argon atmosphere. Stock solutions of Lac and NaAsc were prepared by dissolving Lac (462 mg) in 2.5 mL acetate buffer and NaAsc (1 1 1 mg) in 5.0 mL acetate buffer, respectively. The reaction mixture and the stock solutions were deoxygenated by bubbling argon for ca. 30 min. 0.5 mL of the enzyme solution was transferred into the initiator/monomer flask using an argon purged syringe. The polymerization was started by adding 0.5 mL of the reducing agent solution to the reaction mixture with an argon-purged syringe. The molar ratio of the reactants in the reaction mixture was 1 :100:0.2:0.005 (HEBIB:monomer:NaAsc:Lac; monomer: 75 % Vim and 25 % NVP). The reaction was stirred at room temperature and the copolymerization was stopped after 24 h. The reaction mixture was filtered over a plug of neutral aluminum oxide. The solution was centrifuged, dried under vacuum, and dissolved in methanol. The PVIm-co-NVP copolymer was purified by precipitation in acetone, dried under vacuum and analyzed by ¹ H NMR. Copolymerizations were also carried out with Vlm:NVP ratios of 25:75 and 50:50 according to the protocol described above.

[0061 ] Block copolymer synthesis with Lac

[0062] In a typical reaction, Vim (1 .04 g, 1 1 .0 mmol), NaAsc (3.6 mg, 0.04 mmol) and Lac (1 .06 mL of a 100 mg mL^"1 solution, resulting in 106.0 mg, 1 .1 μηιοΙ) were weighted in a round-bottom flask and dissolved in 3 mL sodium acetate buffer (100 mM, pH 4.0) under argon atmosphere. A stock solution of PEGBIB (883 mg, 0.44 mmol) was prepared in 2 mL acetate buffer. The reaction mixture and the stock solutions were deoxygenated by bubbling argon for ca. 30 min. The reaction mixture was kept at 40°C in an oil bath. 1 mL of the initiator solution was transferred into the monomer/reducing agent/catalyst flask using an argon purged syringe, to start the polymerization. The molar ratio of the reactants in the reaction mixture was 1 :50:0.2:0.005 (PEGBIB:Vlm:NaAsc:Lac). The reaction was stirred at 40°C. After 24 h, the solution was exposed to air. A sample of 0.5 mL was diluted with 1 .5 mL non-deoxygenated D₂0 and filtered over a small plug of neutral aluminum oxide. The filtrate was analyzed by ¹ H NMR to determine the conversion and M_n. Characterization methods

[0063] NMR spectra were recorded at room temperature on a Bruker Advance III 300 MHz (¹ H: 300 MHz; ¹³C: 75.46 MHz) spectrometer using deuterated solvents. Chemical shifts (δ) are reported in ppm, whereas the chemical shifts are calibrated to the solvent residual peaks. The chemical composition and the purity of the compounds were determined using D₂0 and DMSO-d₆ as solvents. Conversion of Vim was calculated from phase and baseline-corrected spectra using the integrals of the signal at 7.77 ppm, which corresponds to one proton of the residual monomer, and the signals between 2.30 and 1 .70 ppm, which correspond to two backbone protons of the polymer. The number average molecular weight (M_n) was calculated from the integrals of the peak at between 1 .8 and 2.0 ppm (6H of initiator in the polymer) and the signals between 2.30 and 1 .70 ppm (2H of polymer). The molecular weight and the dispersity (D) of polymers was determined by GPC using PSS GRAM columns (Mixed bed PSS GRAM analytical linear 10 μηι (300 mm x 8.0 mm), separation range from 500 to 10⁶ g mol^"1) thermostated to 60 °C in DMF containing 0.05 M lithium bromide solvent with a flow rate of 1 .0 mL min^"1 and a refractive index detector.The molecular weight determination were based on poly(methyl methacrylate) (PMMA) standards. Mass analysis was carried out on a Bruker Utraflextreme Matrix Assisted Laser Desorption/lonization Time of Flight (MALDI-ToF) mass spectrometer. The instrument was working in positive ionization method in linear and reflecting mode for all samples. Samples for MALDI-ToF MS measurements were prepared using a-cyano-4- hydroxycinnamic acid (HCCA) as matrix and potassium- and sodium chloride salts. Data was processed with Bruker Daltonicsflex Analysis software. Number average molecular weight and weight average molecular weight were calculated from Mn =^∑^^Mi and Mw =∑^{i NiMi} where M, is the molecular weight of the chain and N, is the number of chains of

∑_i N_iM_i ^a

that molecular weight.

[0064] Isothermal dehydrobromination measurements were carried out to confirm the presence of bromine in purified PVIm. To this end, the evolution of free hydrogen bromide (HBr) from PVIm was monitored with a Metrohm PVC Thermomat 763 instrument. The solvent was 1 ,2,4-trichlorobenzene (20 mL), the degradation temperature was 200 °C and the oxygen flow rate was constant 3.8 L h^"1 . The amount of formed HBr was purged with the oxygen, dissolved in 60 mL distilled water and then detected with a conductometer. A calibration curve was measured without oxygen flow, with acid solutions of known concentration.

[0065] An Perkin Elmer Optima 7000 inductively coupled plasma atomic absorption spectrometer (ICP-OES) was used to measure the concentration of the residual Cu-, Zn-, and Fe ions (at 224.700, 238.204, 206.200 nm respectively). Moreover, the presence of bromine ions in the polymer samples (at 700.570 nm) was confirmed with this method. To calibrate the method individual 1000 mg/L aqueous stock solutions of Cu²⁺ and Fe²⁺ and a 10 mg/L aqueous stock solution of Zn²⁺ were used. Calibration solutions containing 0.1 , 0.25, 0.5, 1 .0 and 5.0 mg/L were prepared in the same aqueous media. The background emission of pure water was compensated by the preparation of the sample and calibration solutions in the same media. Three parallel measurements were carried out.

[0066] Results and discussion

[0067] Lac are copper-containing enzymes with a molecular weight between 50 and 130 kDa. They are produced by higher fungi and plants, moreover they can be found in several bacteria and insects. The active site of the glycoprotein contains four copper ions, a mononuclear "blue" copper ion (Type 1 ) and a three-nuclear copper cluster (Type 2 and Type 3 Cu pair), classified by UV-w^'s and electron paramagnetic resonance (EPR) spectroscopy. [Morozova, O. V.; Shumakovich, G. P.; Gorbacheva, M. A.; Shleev, S. V.; Yarapolov, A. I. Biochemistry (Moscow) 2007, 72, 1 136-1 150] The copper-containing Lac enzymes are able to catalyze the oxidation of several organic substrates. Laccases are one of the few oxidoreductases that are industrially used, e.g. for the biocatalytic delignification of pulp in the paper industry, for textile dye decolorization in the textile industry, in bioremediation for toxic environmental pollutant waste transformation, inactivation and detoxification, in analytical or synthesis purposes, as well as in medical care applications in the pharmaceutical sector or beverage and food treatment in the food industry.[Xu, F. Industrial Biotechnol. 2005, 1, 38-50; Schauer F, Borriss R. (2004) Biocatalysis and biotransformation. In: Tkacz J. S., Lange L, editors. Advances in fungal biotechnology for industry, agriculture, and medicine. Kluver Academic; 2004 pp. 237-75.] It has been reported that, instead of inhibiting the activity of laccase, vinyl imidazole activates these enzymes.

[0068] Lac is able to catalyze the polymerization of Vim in aqueous solution under ARGET ATRP conditions. For the polymerization the initiator 2-hydroxyethyl-2- bromoisobutyrate (HEBIB) and the monomer Vim were dissolved in aqueous sodium acetate buffer (pH 4) under argon atmosphere. Solutions of Lac and sodium-L-ascorbate (NaAsc) were added, and the polymerization was carried out under argon atmosphere and continuous stirring at room temperature. The formation of poly(/V-vinylimidazole) (PVIm) polymer was proven by ¹ H NMR and GPC. No PVIm polymer formation occurred in the absence of enzyme, initiator or reducing agent, respectively.

[0069] Enzyme-catalyzed Vim polymerization with an initial ratio of HEBIB/Vlm/ascorbate/Lac 1 :50:0.2:0.005 in pH 4 sodium acetate buffer was followed for 24 h to demonstrate that the biocatalytic PVIm polymerization follows the activation/deactivation mechanism of the ATRP process (Figure 2). In sight of the determined macromolecular parameters, the kinetic of the living/controlled polymerization follows a first order kinetic (t ~ ln([M]₀/[M],)) from starting the polymerization to 8 h polymerization time. The living nature of the polymerization is proven by the linear increase of molecular weight with conversion, relative low D and a first order kinetic.

[0070] In addition, a purified PVIm polymer was analyzed by ¹ H NMR and MALDI-ToF MS to identify the end groups of the polymer chains. The presence of the methyl groups that originate from the initiator, as well as bromine termination was verified as shown in Figure 3. In this ¹ H NMR spectrum, the signals corresponding to the backbone protons and the initiator fragments in the polymer structure appear between 1 .5 and 4.0 ppm, while the signals between 6.5 and 7.2 ppm belong to the three protons in the imidazole ring. The the initiator signals are between 1 .8 and 2.0 ppm.

[0071 ] The MALDI-ToF MS spectrum of PVIm polymer ranging from 1000 to 8000 is shown in Figure 3. The series of the main peaks are separated by an interval corresponding to Vim repeating unit (94.1 mass unit). Moreover, bromine chain ends were detected in the mass spectra. The following characteristics of the polymer were calculated from this mass spectrum: M_n = 4740 g mol^"1 , M_w = 51 10 g mol^"1 and D = 1 .08. This low D is a further proof for the living/controlled character of the enzymatically catalyzed polymerization.

[0072] The presence of bromine end groups in PVIm was further confirmed with isothermal dehydrobromination measurements.

[0073] The metal content in the formed polymer was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The examined metals were copper, iron and zinc. No significant amount of these metal ions were found bound to the polymers that were prepared by enzymatic ATRP. For example, the copper concentration that remained in poly(N-vinylimidazole) after a few simple purification steps of was found to be as low as 1 .4 ppm, and the iron and zinc concentrations were below the detection limit of the method (ppm by mass of polymer).

[0074] The activity of Lac enzymes is dependent on several parameters, such as the temperature and the pH of the media, [see: Han, M-J.; Choi, H-T.; Song, H-G. J. Microbiol. 2005, 43(6), 555-560.] According to these findings the influence of temperature on the polymerization was studied. The reaction temperature was varied between 5 (ice water) and 40 °C. At all temperatures formation of PVIm was observed. Higher temperature accelerated the polymerization but decreased the degree of control of the polymerization. An optimal temperature for VIM conversion and degree of control of PVIM formation was room temperature.

[0075] The pH of the polymerization media for Lac was varied between 3 and 6. At all pH formation of PVIm was observed. An optimal pH for high conversions was pH 4.0.

[0076] Random copolymers of VIM and NVP were synthesized by Lac catalyzed ATRP, using HEBIB as initiator. Formation of copolymers was confirmed by ¹ H NMR. A Vlm:NVP feed-ratio of 75:25 resulted in a copolymer with M_n = 3580 g mol^"1 , M_w = 5240 g mol^"1 and D = 1 .46 (as determined by GPC).

[0077] In order to show that enzyme-catalyzed polymerizations allow synthesizing block copolymers, VIM was polymerized by Lac using the macromolecular initiator PEGBIB. After 24 h reaction time a conversion of 92 % was achieved. ¹ H NMR revealed the following characteristics of the PEG-b-PVIM block copolymer: PEG block: degree of polymerization (DP) = 47, M_n = 2180 g mol^"1 ; PVIm block: DP = 51 , M_n = 4740 g mol^"1 ; whole polymer: M_n = 6920 g mol^"1. While in accordance with the patent statutes the best mode and preferred embodiment have been set forth, the scope of the invention is not limited thereof, but rather by the scope of the attached claims.

Claims

Claims:

1 . A process for polymerizing at least vinyl monomers having nitrogen-containing heterocyclic aromatic functional groups, comprising the step of: polymerizing via ATRP at least the vinyl monomers, that are the same or different, with nitrogen-containing heterocyclic aromatic functional groups in the presence of an enzyme catalyst and an initiator.

2. The process according to claim 2, wherein the monomers comprise at least N- vinylimidazole.

3. The process according to claim 3, wherein the enzyme catalyst comprises laccase, hemoglobin and/or horseradish peroxidase.

4. The process according to claim 1 , further including the steps of polymerizing the monomers in the presence of a reducing agent.

5. The process according to claim 1 , wherein the enzyme catalyst is one or more of Cu-containing enzyme laccase, iron-containing enzymes hemoglobin (Hb), horseradish peroxidase (HRP), soybean peroxidase (SBP), chloroperoxidase, myoglobin, enzymes containing heme, Mn-containing enzymes, V-containing enzymes and Ni-containing enzymes.

6. The process according to claim 1 , wherein the monomers include one or more of 1 -vinylimidazole, 2-vinylimidazole, 4-vinylimidazole, 1 -vinylpyridine, 2-vinylpyridine, 3- vinylpyridine, 2-methyl-5-vinylpyridine, 3-ethyl-4-vinylpyridine, 2,3-dimethyl-5-vinylpyridine, 2-vinylpyrimidine, 5-vinylpyrimidine, 1 -vinylpiperidine, 2-vinylpiperidine, 4-vinylpiperidine, 9- vinylcarbazole, 3-vinylcarbazole, 4-vinylcarbazole, 2-methyl-1 -vinylimidazole, vinylthiazoles and hydrogenated vinylthiazoles, 1 ,2,3-vinyltriazole, 1 ,2,4-vinyltriazole, 5-vinyltetrazole, vinyloxazoles and hydrogenated vinyloxazoles, derivatives thereof and combinations thereof.

7. The process according to claim 1 , further including the step of forming a (co)polymer having a number average molecular weight of less than 20,000 g mol^"1.

8. The process according to claim 1 , further including the step of forming a

(co)polymer having a dispersity of less than 1 .6.

9. The process according to claim 8, wherein the dispersity is between 1 .0 and 1 .5, and wherein the (co)polymer formed has a number average molecular weight of less than 10,000 g mol^"1.

10. A (co)polymer derived from enzyme catalyzation, comprising: repeat units derived at least from vinyl monomers, that are the same or different, having nitrogen- containing heterocyclic aromatic functional groups; and an end group comprising a halogen atom.

1 1 . The (co)polymer according to claim 10, wherein the vinyl monomers have nitrogen-containing heterocyclic aromatic functional groups.

12. The (co)polymer according to claim 1 1 , wherein the vinyl monomers comprise N- vinylimidazole.

13. The (co)polymer according to claim 10, wherein the polymer is metal free with a Cu, Fe and Zn content of less than or equal to 20 ppm.

14. The (co)polymer according to claim 10, wherein the vinyl monomers include one or more of 1 -vinylimidazole, 2-vinylimidazole, 4-vinylimidazole, 1 -vinylpyridine, 2- vinylpyridine, 3-vinylpyridine, 2-methyl-5-vinylpyridine, 3-ethyl-4-vinylpyridine, 2,3-dimethyl- 5-vinylpyridine, 2-vinylpyrimidine, 5-vinylpyrimidine, 1 -vinylpiperidine, 2-vinylpiperidine, 4- vinylpiperidine, 9-vinylcarbazole, 3-vinylcarbazole, 4-vinylcarbazole, 2-methyl-1 - vinylimidazole, vinylthiazoles and hydrogenated vinylthiazoles, 1 ,2,3-vinyltriazole, 1 ,2,4- vinyltriazole, 5-vinyltetrazole, vinyloxazoles and hydrogenated vinyloxazoles, or derivatives thereof.

15. The (co)polymer according to claim 10, wherein the (co)polymer has a number average molecular weight of less than 20,000 g mol^"1.

16. The (co)polymer according to claim 10, wherein the (co)polymer has a dispersity of less than 1 .6.

17. The (co)polymer according to claim 16, wherein the dispersity is between 1 .0 and 1 .5, and wherein the (co)polymer number average molecular weight is less than 10,000 g mol^"1.

18. The (co)polymer according to claim 10, wherein the (co)polymer includes at least one monomer not having nitrogen-containing heterocyclic aromatic functional groups, (meth)acrylate, (meth)acrylamide, a styrene-based monomer, or a monomer containing a proton donor or a combination thereof.

19. The (co)polymer according to claim 18, wherein one or more of the following monomers are present N-vinyl-2-pyrrolidone, 2-vinylpyrrolidone, N-vinylpyrrolidine, 3- vinylpyrrolidine, N-vinylcaprolactam, N-vinylbutyrolactam, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methycrylate, hydroxypropyl acrylate, butyl methacrylate, butyl acrylate, propyl methacylate, propyl acrylate, methyl methacrylate, methyl acrylate, lauryl methacrylate, lauryl acrylate, methacrylic acid, acrylic acid, dimethylaminoethyl methacylate, dimethylaminoethyl acylate, polyethyleneglycol acrylate, polyethylene glycol methacrylate, oligoethyleneglycol methacrylate, oligoethyleneglycol acrylate, acrylamide, methacrylamide, N-isopropylacrylamide, N-(2-hydroxypropyl, styrene, sodium 4-styrene sulfonate, or diethyl- or diisopropyl-p-viniybenzyl phosphonate.

20. The (co)polymer according to claim 10, wherein the copolymer comprises two or more blocks including at least one block derived from the repeat units derived at least from the vinyl monomers, that are the same or different, having nitrogen-containing heterocyclic aromatic functional groups.