Azide-terminated oligomer or polymer

Abstract

The present invention relates to the use of an azide-terminated oligomer or polymer Z, or functional polymers comprising said azide-terminated oligomers or polymers at least partly as side chains, as coating agent.

Description

[0001] Azide-terminated oligomer or polymer

[0002] The present invention relates to the use of an azide-terminated oligomer or polymer or functional polymers comprising said azide-terminated oligomers or polymers at least partly as side chains as coating agent.

[0003] Polyoxazolines, polymers with unique chemical and physical properties, have been a subject of interest for researchers in the field of polymer chemistry. These polymers exhibit characteristics that make them highly suitable for a range of applications, from drug delivery to material science. Central to the production of these polymers is a process known as cationic ring opening polymerization (CROP). This methodology, specifically for the synthesis of oxazolines, has been rigorously investigated and reported in numerous scientific publications over the years. The cationic ring opening polymerization (CROP) of oxazolines to form polyoxazolines is, therefore, a well -described procedure in the literature. However, despite the availability of commercial sources for diversely functionalized polyoxazolines, certain combinations of chain terminations remain mostly inaccessible to date.

[0004] Glassner et al (Polym Int 2018;67:32-45) disclose the growing prominence of poly(2-oxazoline)s (PAOx) across various domains, with a special emphasis on the biomedical field. The review articulates the processes involved in the synthesis of 2-oxazoline monomers and their subsequent cationic ring-opening polymerization. It further explores the intricate properties of these homopolymers, particularly those with diverse side-chain configurations. Covering a wide spectrum of applications, from polymer-drug interactions to wound care, this in-depth review stands as a comprehensive reference for poly(2-oxazoline)s.

[0005] Podevyn et al (European Polymer Journal 120 (2019) 109273) discusses diverse application possibilities of poly(2-alkyl / aryl-2-oxazoline)s (PAOx) due to their extensive chemical adaptability. This study introduces an approach to infuse diverse functional end-groups into PAOx using methyl bromoacetate (MeBrAc) as an initiator. While its initiation and polymerization rates were slower than the popularly used methyl tosylate (MeOTs), MeBrAc produced well-defined polymers with almost complete methyl ester end-group functionality. Subsequent modifications introduced functionalities like hydroxyl groups.

[0006] Stafast et al (European Polymer Journal 184 (2023) 111779) discusses the synthesis of end- functionalized block copolymers of poly(2-oxazoline) (POx) and polyesters. Therefore, telechelic poly(2 -oxazolines) building blocks with one amino and one azide end group are synthesized. The amino terminus is accessible via the use of a protected amine -functionalized initiator. The polymers described in this study are intended for the use in drug delivery, especially hydrophobic drugs. WO2023144228A1 discloses azide-functionalized copolymers as biocompatible polymers. The copolymers are synthesized via cationic ring opening polymerization and comprise two different monomers. However, sodium azide is needed for end functionalization, which can lead to the formation of side products. The introduction of an azide moiety as a terminus for polyoxazolines is generally obtained by employing sodium azide as a terminating agent. However, due to occurrence of competitive termination reactions it is not possible to obtain complete azido-terminated polymers with this synthesis strategy without a high degree of side products.

[0007] The object of the present invention is to provide a coating agent with tailorable-length chain having the possibility of being grafted onto functionalized polymers / surfaces.

[0008] The problem is solved by the use of the azide-terminated oligomers or polymers or functional polymers comprising said azide-terminated oligomers or polymers at least partly as side chains, as coating agent according to claim 1. Further preferred embodiments are subject of dependent claims 2 to 17.

[0009] The present invention relates to the use of oligomer or polymer Z, according to the general structure (I) shown below, or a functional polymer FP comprising a polymer backbone and a plurality of side chains, wherein at least a part of said side chains is oligomer or polymer Z, as a coating agent. It was found that the azide-terminated oligomer or polymer Z according to the present invention can be obtained by a straightforward protocol. Due to the use of a terminating agent (VI) which will be described below in detail, unwanted side reactions can be avoided. Furthermore, the polymers lead to a comparable or better coating performance than their corresponding PEG analogues. Moreover, they provide a good long-term stability at room temperature.

[0010] In the azide-terminated oligomer or polymer Z according to the present invention (Formula I)

[0011] A is a linear or branched Ci to Ci2alkylene,

[0012] Ri and Ri ’ are independently of each other a linear or branched Ci to C6alkyl,

[0013] B and B’ are independently of each other a linear or branched C2to C3alkylene group, ni is an integer of 1 to 1000, n2is an integer of 0 to 100, and the sum of n!+n2is at least 2,

[0014] R2is selected from the group consisting of hydrogen, methyl, ethyl and COR4;

[0015] R3is selected from the group consisting of methyl and ethyl, m is 0 or 1, and if m is 1, compound of formula I has a permanent positive charge,

[0016] R4 is selected from the group consisting of CH3, CF3and phenyl, and if R2is COR4, m is 0, and D is a linear or branched Ci to C]2alkylene.

[0017] Thus, oligomer or polymer Z according to the present invention comprises a terminal primary amine group and a terminal azide group. This allows to obtain a polymer with a tailorable-length chain, while having at the same time the possibility of being grafted onto polymers / surfaces, preferably onto carboxylate -functionalized (such as activated esters) polymers / surfaces via an amide bond formation. The oligomer or polymer according to the present invention offers a unique combination of properties that makes it especially well-suited for biosensing applications, in particular for detecting biological and chemical analytes.

[0018] Within the context of the present invention, a coating agent is a chemical ingredient that is added to coatings or coating formulations to enhance properties such as wetting, protein resistance, dispersion, film formation, surface quality, and appearance.

[0019] Within the context of the present invention “an oligomer” is a molecule that consists of 2 to 10 monomer units, while “a polymer” consists of more than 10 monomer units.

[0020] Within the context of the present invention, surface relates to any interface between the body of an object and its environment. This can be large scale objects such as windows, glass slides, medical or industrial devices etc., but also micro and nanoscale objects such as nanoparticles, small structures on electronic chips, or structures in a microflucidic device.

[0021] One embodiment of the present invention relates to the use of an azide -terminated oligomer Z according to formula (I), wherein the sum of n!+n2is an integer of 2 to 10, as coating agent. Oligomers are especially preferred if thin, uniform coatings with precise control over film thickness and smooth surface properties are required.

[0022] One embodiment of the present invention relates to the use of an azide -terminated polymer Z according to formula (I), wherein the sum of n!+n2is an integer of 11 to 1100, as coating agent. Polymers are especially preferred when a more durable coating is needed, as they form stronger intermolecular interactions due to their longer chains, resulting in higher mechanical strength and stability.

[0023] A further aspect of the present invention relates to the use of oligomer or polymer Z according to the present invention for coating a surface comprising a surface active headgroup, wherein the terminal amine group is intended to irreversibly bind to a substrate. In one aspect of the present invention this concerns coating a surface of a substrate comprising a surface active headgroup, in which the surface active headgroup has a linear or branched, substituted or unsubstituted Ci to Ci2alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group K5 selected from the group of alkoxy silanes, chloro silanes, catechols, nitrocatechols, bromocatechols, chlorocatechols, phosphates, phosphonates, mimosine derivatives, anacheline, gallols, thiols, N-heterocyclic carbenes, perfluorophenyl azides, benzophenon, diaryldiazomethane, aryltrifluoromethyldiazomethane, and organoboron. Such a coating provides free azide groups that can be modified further. Such azide functionalized surfaces can be used as substrates for example for next generation sequencing by functionalizing with single stranded DNA or RNA, functionalized with antibodies or antigens for ELISA applications or biosensing applications in general.

[0024] If the oligomer or polymer according to the present invention is prepared with only one type of monomer unit, n2in formula I is 0 resulting in a polymer of formula IA.

[0025] If the polymer is a copolymer of two different types of monomer units n2is an integer of 1 to 100.

[0026] In polymer of formula (I), and in particular of formula (IA), A is preferably a Ci to C6alkylene, most preferably a propylene, and Ri, Rf, R2, R3, B, B’, n and D are defined as above. The length of A allows for an optimal distance of the terminal group from the polymer chain, which simplifies the reaction with other end groups. Having the terminal group at an optimal distance improves the efficiency of post-polymerization modifications, resulting in more uniform grafting and polymer chain adjustments.

[0027] In polymer of formula I, and in particular of formula (IA), D is preferably a Ci to C6alkylene, preferably ethylene, and A, R R! ’, R2, R3, B, B’, n! and n2are defined as above. As explained below in detail, D is introduced into oligomer or polymer Z as terminating agent of the formula (VI) wherein R2’ is selected from the group consisting of hydrogen, methyl, ethyl; R3’ is selected from the group consisting of hydrogen, methyl and ethyl, and D is a linear or branched Ci to Ci2alkylene, preferably Ci to C6alkylene, and most preferably ethylene.

[0028] In one aspect of the present invention in polymer of formula I, and in particular of formula (IA), A, Ri, Ri’, B, B’, D, n! and n2are defined as above, R2is hydrogen and R3is absent, i.e., in the terminating agent of formula VI, R2’ and R3’ are both hydrogens. In this embodiment, oligomer or polymer Z comprises a secondary amine, which can become protonated at physiological pH.

[0029] In a further aspect of the present invention in polymer of formula I, and in particular of formula (IA), A, Rj , Rj ’ , B, B ’ , D, n! and n2are defined as above, R2is selected from the group consisting of methyl and ethyl and R3is absent, i.e., in the terminating agent of formula VI, R2’ and is methyl or ethyl and R3’ is hydrogen. In this embodiment, oligomer or polymer Z comprises a tertiary amine, which can become protonated at physiological pH.

[0030] In a further aspect of the present invention in polymer of formula I, and in particular of formula (IA), A, R1?Rf . B, B’ D, n! and n2are defined as above, R2is selected from the group consisting of methyl and ethyl and R3is selected from the group consisting of methyl and ethyl, i.e., in the terminating agent of formula VI, R2’ and is methyl or ethyl and R3’ is methyl or ethyl. In this embodiment, oligomer or polymer Z comprises a quaternary ammonium, with a permanent positive charge.

[0031] Preferably, the oligomer or polymer Z according to the present invention is a polymer of formula IA wherein

[0032] A is a linear or branched Ci to C]2alkylene, Ri is a linear or branched Ci to C6alkyl,

[0033] B is a linear or branched Ci to C2alkylene group , ip is an integer of 2 to 1000,

[0034] R2is selected from the group consisting of hydrogen, methyl, ethyl and COR4;

[0035] R3is selected from the group consisting of methyl and ethyl, m is 0 or 1, and if m is 1, compound of formula I has a permanent positive charge,

[0036] R4 is selected from the group consisting of CH3, CF3and phenyl, and if R2 is COR4, m is 0, and D is a linear or branched Ci to C]2alkylene.

[0037] Thus, n2in polymer of formula 1 is 0, i.e. only one type of monomer has been used.

[0038] In a further embodiment of the present invention, oligomer or polymer Z has the general formula (IB) i.e., in compound (I) R2is COR4 R4 is selected from the group consisting of CH3, CF3and phenyl, preferably CH3, and A, R|. B, n! and D are defined as above. The resistance of the resulting amid group to protonation under physiological conditions results in a higher resistance to enzymatic hydrolysis, which increases the stability.

[0039] In polymer of formula I, Ri is selected from the group consisting of methyl, ethyl, propyl and isopropyl, preferably methyl and ethyl and most preferably methyl and A, R2, R3, B, n and D are defined as above.

[0040] Preferably, oligomer or polymer Z of formula IA is selected from the group consisting of compounds 1 to 256, wherein n! is an integer between 2 and 1000 and

[0041] Especially good results can be obtained with compounds 1, 2, 5, 25, 65, 66, 69, 89, 129, 130, 133, 153, 193, 194, 197 and 217.

[0042] Also to be mentioned are reaction intermediates of the formula (X), (XA) and (XB) wherein A, B, B’, D, n1?n2, Ri, Rf, R2and R4 have the same definition as above and P is an amine protecting group. Intermediate X and XA refer to the reaction product obtained after termination of the cationic polymerization reaction, prior to the removal of the protective group from the primary amine, and intermediate XB to the reaction product after termination and acylation, prior to the removal of the protective group from the primary amine.

[0043] One method for preparing oligomer or polymer Z of formula (I) involves a cationic ring polymerization of a monomer unit M selected from the group consisting of 2-alkyl-4,5-dihydro-l,3- oxazole and 2-alkyl-5,6-dihydro-4H-l,3-oxazine, involving an initiator (V) which initiates a cationic ring polymerization and a terminating agent (VI) which terminates the cationic ring polymerization. An initiator (V) is added that forms a cationic species, which starts and propagates the reaction by opening monomer rings and incorporating them into the growing polymer chain. The initiator is the first molecule of the polymer chain, thus the protected primary amine group forms one of the two terminal end groups.

[0044] The initiator has the general formula (V)

[0045] P-N-A-X (V) wherein

[0046] A is a linear or branched Ci to C]2alkylene,

[0047] P is a protecting group for a primary amine group,

[0048] X is a leaving group. The protecting group P for a primary amine group, may be selected from the group consisting of t- Butyloxycarbonyl (Boc), Phthalimide, Benzyl carbamate and p-Methoxybenzyl carbamate (PMB), most preferably t-Butyloxycarbonyl (Boc) and Phthalimide.

[0049] The term "leaving group" refers to an atom or functional group that departs from the initiator, taking with it an electron pair, and consequently leaving behind a positively charged cation, resulting in a stable cationic state. Preferably, the leaving group X is selected from the group consisting of bromo, iodo, mesylate, tosylate, brosylate, nosylate and triflate, most preferably, bromo, iodo, tosylate and triflate.

[0050] The initiator (V) which initiates a cationic ring polymerization may be selected from the group consisting of and n!2is 1 to 12, preferably 1 to 6, and most preferably 2 or 3.

[0051] In one method the termination reagent (VI) that is added to the reaction mixture of the cationic ring polymerization to intentionally quench or stop the polymerization reaction by reacting with the active cationic species, that is, with the living polymer end. Due to its dual functionality, it does not only quench or stop the reaction but also introduces an azide group to the polymer end, thus forming the second of the two terminal end groups.

[0052] The terminating agent has the following formula (VI) wherein

[0053] R2’ is selected from the group consisting of hydrogen, methyl, ethyl;

[0054] R3’ is selected from the group consisting of hydrogen, methyl and ethyl, and D is a linear or branched Ci to Ci2alkylene.

[0055] The terminating agents of formula (VI) have a good solubility in polar solvents such as acetonitrile which facilitates the straightforward reaction of the present invention and avoids unwanted side reactions.

[0056] The terminating agent (VI) which terminates a cationic ring polymerization may be selected from the group consisting of If R2and R3are hydrogen or R2is methyl or ethyl and R3is hydrogen, oligomer or polymer Z of formula I comprises a secondary amine which can become protonated under neutral conditions, such as at physiological pH (around pH 7.4), and is unprotonated under basic conditions. This increases the solubility in water and can increase the adhesion to biological tissues.

[0057] If R2is methyl or ethyl and R3is hydrogen, oligomer or polymer Z of formula I comprises tertiary amine which can become protonated under neutral conditions, such as at physiological pH (around pH 7.4), and is unprotonated under basic conditions. This increases the solubility in water and can increase the adhesion to biological tissues.

[0058] If R2and R3are both methyl and / or ethyl, the final polymer will become a quaternary ammonium with a permanent positive charge which results in interesting anti-bacterial properties. Examples are the inhibition of biofilm development and the electrostatic attachment to bacterial cell walls.

[0059] An oligomer or polymer Z which does not contain a positively charged amine, even under physiological conditions can be obtained by reacting an intermediate XA with an acylating agent and subsequent deprotection.

[0060] Within the context of the present invention an acylating agent is a compound that introduces an acyl group (R4-C=O) into intermediate X forming an amide. Examples are acid chlorides, anhydrides, esters and acid halides. Preferably, the acylating agent is selected from the group consisting of acetyl chloride, trifluoroacetyl chloride and benzoyl chloride.

[0061] Within the context of the present invention “an oligomer” is a molecule that consists of 2 to 10 monomer units, while “a polymer” consists of more than 10 monomer units.

[0062] The deprotection conditions for the primary amine group in Intermediate X are determined by the type of protecting group used. Each protecting group requires specific conditions for efficient deprotection which has to be chosen in such a way that the azido moiety remains intact. For example, the phthalimide protecting group can be removed employing a mild protocol (treatment with methylamine in ethanol at 70°C for 4 hours) which ensures the survival of the azido moiety.

[0063] Within the context of the present invention the term of 2-alkyl-4,5-dihydro- 1,3-oxazole and 2-alkyl- 5,6-dihydro-4H-l,3-oxazine stands for 4,5-dihydro-l,3-oxazoles and 5,6-dihydro-4H-l,3-oxazine having a linear or branched Ci to C6alkyl group in position 2. Preferably, the monomer unit M is selected from the group consisting of 2-methyl-4,5-dihydro-l,3-oxazole, 2-ethyl-4,5-dihydro-l,3- oxazole, 2-n-propyl-4,5-dihydro-l,3-oxazole, 2-iso-propyl-4,5-dihydro-l,3-oxazole, 2-methyl-5,6- dihydro-4H-l,3-oxazine and 2-ethyl-5,6-dihydro-4H-l,3-oxazine, 2-n-propyl-5,6-dihydro-4H-l,3- oxazine, and 2-iso-propyl-5,6-dihydro-4H-l,3-oxazine, or a mixture thereof. Said monomers form a polymer chain that dissolves in water. Therefore, oligomer or polymer Z can serve as alternative to azide functionalized PEG in various biochemical uses. This includes enhancing protein stability in blood, modifying surfaces to decrease fouling and unwanted protein adsorption.

[0064] The azide group of the oligomer or polymer Z according to the present invention can be easily modified, for example via click chemistry to form triazoles, reduced to amines, or by exposure to energy (temperature, light) they can release N2to form highly reactive nitrene intermediates that react nonspecific with neighbouring groups (for example insertion into C-H bonds).

[0065] A further aspect of the present invention relates to the use of a functional polymer FP comprising a polymer backbone and a plurality of side chains, wherein at least a part of said side chains are compounds according to the present invention, as coating agent. For example, EP3191559A1 discloses functional polymers. The contents of EP3191559A1 are hereby incorporated by reference in its entirety. Preferably, the polymer backbone is a polyacrylamide.

[0066] Functional polymer FP has a polymeric backbone that can be easily functionalized, allowing for the seamless incorporation of various functional and binding groups through a stable and reproducible coupling method, all designed for efficiency and precision. This process is uniquely facilitated by using dip-and-rinse aqueous solutions, which naturally assemble into monolayer carpets with a thickness of just a few nanometers. This method is compatible with a wide range of substrates, including metals, glass, ceramics, and plastics. One of the key advantages of this functional polymer FP is that it eliminates the need for curing, making it highly versatile. It can be applied directly onto open glass or metallic chips and can be readily utilized within closed microfluidic channels without any constraints. It can also be used to coat nanoparticles or nanostructured surfaces.

[0067] In one embodiment of the present invention functional polymer FP is a polymer in which all side chains are polymers Z, as described in the present invention. Such functional polymers are highly desirable for biosensing applications. They can be tailored in chain length using click chemistry and grafted onto carboxylate -functionalized polymers via an amide bond formation. It was found that functional polymers comprising oligomer or polymer Z as side chain have an excellent proteinresistance. The term protein-resistance is expressed in % as reduction of protein uptake on a sample coated with the copolymer according to the present invention compared to uncoated reference sample and is higher than 90%. Such a high protein-resistance is a prerequisite for many medical and biomedical applications. Within the context of the present invention the term "non-fouling" refers to a quality of preventing or minimizing fouling. Thus, a non-fouling interfacial biomaterial can be used to reduce attachment of biomaterials to a surface, and to reduce aesthetic and operational consequences of fouling. The term protein-resistance and non-fouling characteristics can be used as synonyms.

[0068] In another embodiment of the present invention functional polymer FP is a polymer that comprises at least two types of side chains, wherein at least one type of side chain is oligomer or polymer Z as described in the present invention. Of course, each type of side chain comprises a plurality of identical side chains.

[0069] As mentioned before, oligomer or polymer Z according to the present invention comprises a primary amine as a terminal linker group which can react with a reactive group on the polymer backbone. Such a reactive group on the polymer backbone is preferably selected from the group consisting of esters, activated esters, chloro, fluoro, acrylate, methacrylate, NHS esters, epoxides, anhydrides, azides, alkynes, and acyltrifluoroborates. Thus, the compound according to the present invention can be introduced into a functional polymer by post-modification of a polymer backbone carrying a reactive group. Preferably, the polymer backbone is a polyacrylamide, and polymer backbone and the side chains are linked by amide bonds.

[0070] In a further aspect of the present invention functional polymer FP comprises at least two different types of side chains, and wherein at least one type of side chain is an oligomer or a polymer according to the present invention.

[0071] Preferably, such a functional polymer comprises the oligomer or polymer according to the present invention, designated as Z, as the first type of side chain and at least one other type of side chain W which is intended to reversibly bind to a substrate or has a coating function, and

[0072] H, is an alkylene or arylene group and [H2]qis an amide, an ester or an ether group and q is either 0 or 1, preferably 0.

[0073] W is selected from the group consisting of a short side chain W1 having a linear or branched, substituted or unsubstituted Ci to Ci2alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional side or end group KI selected from the group consisting of amines, carboxy, polypropylene sulfide), and thioethers, whereby said functional group KI is preferably an end group; a long side chain W2 comprising more than 15 carbon or silicium atoms in the chain being selected from the group consisting of polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, poly dimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly(N-isopropylacrylamide), poly(N- hydroxy ethylacrylamide), poly(2-hydroxyethyl methacrylate), poly(hydroxypropylmethacrylate), poly(methacryloyloxylethyl phosphorylcholine), poly(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragments and poly(sulfobetaine acrylamide), whereby W2 has no further functional end group or side group; a long side chain W3 selected from the group consisting of a polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, poly dimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly(N- isopropylacryl-amide), poly (N-hydroxy ethylacrylamide), poly(2-hydroxyethyl methacrylate), poly-hydroxypropylmeth-acrylate), poly(methacryloyloxylethyl phosphoryl-choline), poly(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragments and poly(sulfobetaine acrylamide), whereby W3 carries at least one functional end or side group K3 selected from the group consisting of amines, carboxy, nitrilotriacetic acid (NTA), biotin, azide, terminal alkene groups, terminal alkine groups, tetrazine whereby said functional group K3 is preferably an end group.

[0074] Preferably, the functional polymer, in addition or alternatively to at least one type of side chain W, may comprise a further type of side chain which is intended to irreversibly bind to a substrate, said side chain Q having a linear or branched, substituted or unsubstituted Ci to C]2alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional side or end group K4 selected from the group of alkoxy silanes, chloro silanes, catechols, nitrocatechols, bromocatechols, chlorocatechols, phosphates, phosphonates, mimosine derivatives (including compounds having an N-(3-hydroxy-4-oxypyridyl residue), anacheline, gallols, thiols, N-heterocyclic carbenes, arylazides, perfluorophenyl azides, benzophenon, diaryldiazomethane, aryltrifluoromethyldiazomethane, and organoboron, whereby said functional group K4 is preferably an end group. Interestingly, arylazides, especially perfluorophenyl azides can be selectively activated by UV or temperature without inactivating the azide groups in the azide-functionalized polymer.

[0075] Within the context of the present invention the term side group means a group of atoms attached to a carbon atom within the side chain and an end group is a group at the end of the side chain, that is its terminal group. For example, side chain W2 can be a polyethylene glycol chain that does not have any other functional groups attached to it, either at its end or along its backbone, whereas side chain W3 can be a polyethylene glycol chain with a terminal carboxy group.

[0076] In preferred embodiments of the present invention the functional polymer FP is selected from polymers comprising the following types of side chains

[0077] - azide-terminated oligomer or polymer Z

[0078] - azide-terminated oligomer or polymer Z and side chain W 1

[0079] - azide-terminated oligomer or polymer Z and side chain W2

[0080] - azide-terminated oligomer or polymer Z and side chain W3

[0081] - azide-terminated oligomer or polymer Z and side chain Q

[0082] - azide-terminated oligomer or polymer Z, side chain W 1 and side chain W2

[0083] - azide-terminated oligomer or polymer Z, side chain W 1 and side chain W3

[0084] - azide-terminated oligomer or polymer Z, side chain W3 and side chain W2

[0085] - azide-terminated oligomer or polymer Z, side chain W 1 and side chain E

[0086] - azide-terminated oligomer or polymer Z, side chain Q and side chain W2

[0087] - azide-terminated oligomer or polymer Z, side chain Q and side chain W3

[0088] - azide-terminated oligomer or polymer Z, side chain Wl, side chain W2 and side chain W3

[0089] - azide-terminated oligomer or polymer Z, side chain Wl, side chain Q and side chain W3

[0090] - azide-terminated oligomer or polymer Z, side chain Wl, side chain W2 and side chain Q

[0091] - azide-terminated oligomer or polymer Z, side chain W2, side chain Q and side chain W3, and - azide-terminated oligomer or polymer Z, side chain Wl, side chain W2, side chain W3 and side chain Q; whereby, the order within the oligomer or polymer chain is not defined by the order of the chains as listed above.

[0092] Preferably, the functional polymer according to the present invention comprises at least 1 to 200 identical side chains per type of side chain.

[0093] Especially preferred side chains Wl are selected from the group consisting of aminobutyl, aminopentyl and aminohexyl, preferably aminohexyl.

[0094] Especially preferred side chains W2 are selected from the group consisting of polyalkyloxazolines, polyalkyloxazines and polyethylene glycol;

[0095] Especially preferred side chains W3 are selected from the group consisting of biotin, NTA, a terminal alkene group and a terminal alkine group.

[0096] Especially preferred side chains Q are selected from the group consisting of alkoxy silanes, chloro silanes, especially aminopropyldimehylethoxysilane, phosphates especially alkoxy-phosphates, phosphonates especially alkoxy-phosphonate and catechols, especially nitrocatechol, arylazides, especially perfluorophenyl azides.

[0097] Preferably, all different types of side chains Q and W comprise a terminal linker group selected from the group consisting of hydroxy, amine and thiol, preferably amine, to link the side chain to the polymer backbone. Thus, for example for aminohexyl side chain N-Boc-l,6-hexanediamine can be used as starting compound, for the 4-(ethyl)-5-nitrobenzene-l,2-diol side chain nitrodopamine can be used as starting compound and for dimethylsiloxane side chain 3-[ethoxybis(methyl)silyl] propylamine can be used as starting compound.

[0098] The coating agent described in the present invention leverages its unique physicochemical properties to effectively prevent or minimize fouling on a variety of surfaces selected from the group consisting of glass, polydimethylsiloxane, A12O3, Ta2O5, ITO, TiO2, ZrO2, Fe2O3, steel, brass, bronze, Au, Pt, polystyrene, COC, polypropylene, polyethylene, ABS, nylon, PLA, PETG, polyurethane, PMMA and PVA. For example, in biosensing applications, the coating agent’s compatibility with these diverse materials ensures a versatile platform for the sensor, enhancing its performance across various environments and conditions. The formulation of the coating is designed to maintain a stable surface free energy, reduce the adhesion of biomolecules, and limit non-specific binding, which are critical factors in improving the sensitivity and specificity of biosensors. In the context of glass and PDMS, the coating can provide enhanced stability and compatibility for biosensors. For glass, which is often utilized in optical sensors, the coating can help maintain clarity while preventing biofouling, ensuring that optical signals remain strong and detectable. For PDMS, the flexibility of the material combined with the coating according to the present invention allows for the creation of robust coated substrates that can be used in various biological assays without losing performance overtime. In case of nanoparticles, the functional polymer FP leads to a shell preventing the particles from non-specific binding and agglomeration, while the azide can be used to selectively functionalize the particles with biomolecules.

[0099] This combination of the above described properties makes the coating agent an excellent choice for protecting surfaces in diverse applications, including environmental monitoring, clinical diagnostics, and other areas where the interaction with biological and chemical analytes must be managed effectively and selectively.

[0100] Experiments

[0101] Synthesis of 2-Azidoethylamine - Terminating agent [I. A. Inverarity, A. N. Hulme, Org

[0102] Biomol Chem 2007, 5, 636.]

[0103] 1) NaN H 0 80°C 18h 2) KOH

[0104] 2-Chloroethylamine hydrochloride (2.00 g, 17.27 mmol) was dissolved in 20 mb water, and sodium azide (3.36 g, 51.70 mmol) was added. The resulting pale orange solution was heated at 80°C and left stirring overnight. Subsequently, potassium hydroxide was added until pH = 11, and the paleyellow solution was extracted with diethyl ether. The combined organic phase was dried over magnesium sulfate, and dried on the rotary evaporator at slightly reduced pressure (40°C, 800 mbar). 2 -Azidoethylamine was obtained as a colorless, volatile oil (736 mg, 8.54 mmol, 49%). 1H NMR (400 MHz, CDC13) 5 = 3.37 (2H, t, J = 5.6 Hz, -CH2-), 2.89 (2H, t, J = 5.6 Hz, -CH2-), 1.52 - 1.42 (2H, m, -NH2) ppm.

[0105] Synthesis of PhthN-C3H6-PMOXA-C2H4-N3

[0106] N-(3-Bromopropyl)phthalimide (320 mg, 1.19 mmol) was dissolved in 20 mb anhydrous acetonitrile, operating under inert atmosphere. Then, freshly distilled 2-methyl-2-oxazoline (5 mb, 59.05 mmol) was added, and the resulting colorless solution was heated to reflux (95 °C bath temperature) for 24 hours. Subsequently, the solution of 2-azidoethylamine (308 mg, 3.58 mmol) was added and refluxed for additional 24 hours. After cooling down to room temperature, the crude reaction mixture was added to 250 mL diethyl ether, and the opaque mixture was cooled down to - 20°C to favor complete precipitation and sedimentation of the abundant white precipitate. The clear supernatant was finally discarded, and after drying under vacuum, the product was recovered as a white powder (5.65 g, 100%). IR (ATR) = 2104 (-N3); 1711 (Phthalimide-C=O); 1626 cm-1 (PMOXA-C=O). 1H NMR (400 MHz, D2O) 5 = 7.90 (m, 4 H, Phthalimide); 7.55; 4.32; 4.19; 4.02

[0107] - 3.91 (m, 4 H, CH2); 3.67 - 3.53 (m, 280 H, PMOXA-CH2); 3.17; 2.98 - 2.64 (m, 2 H, CH2); 2.13

[0108] - 2.06 ppm (m, 210 H, PMOXA-CH3). n21= 70 (1H NMR).

[0109] Deprotection of PhthN-C3H6-PMOXA-C2H4-N3: Synthesis of NH2-C3H6-PMOXA-C2H4-N3

[0110] PhthN-C3H6-PMOXA-C2H4-N3 (2501 mg, 0.55 mmol) and ethanol (100 mL) were stirred to obtain an opaque, off-white mixture, to which methylamine (1.45 mL, 40% w / w in H2O, 16.81 mmol) was added. The mixture was then heated at 70°C for 4 hours. The resulting clear, colorless solution was concentrated under reduced pressure, dissolved in water (30 mL), and dialyzed (MWCO = 3.5 kDa) against water for 24 hours, changing the dialysis medium 3 times. After filtration (0.22 pm PES membrane), the dialyzed solution was lyophilized to obtain a white powder, corresponding to the product NH2-C3H6-PMOXA-C2H4-N3 (1.59 g, 66%). IR (ATR) = 2108 (-N3); 1628 cm-1 (PMOXA-C=O). 1H NMR (400 MHz, D2O) 5 = 7.52; 3.99; 3.67 - 3.53 (m, 280 H, PMOXA-CH2); 3.02 - 2.74 (m, 4 H, CH2); 2.27 - 1.93 ppm (m, 210 H, PMOXA-CH3). GPC (EluentDMAc / 0.05M LiBr; Columns: PSS GRAM, 10pm, Calibration: PMMA): Mn [Da]=6350, Mw[Da]=8790, PDI (=Mw / Mn)=1.39, degree of polymerisation DP(GPC)= 75.

[0111] Synthesis of PAcrAm™-g-(PMOXA-N3,NH2,Si) wherein x is 20, y is 40 and z is 40.

[0112] NH2-C3H6-PMOXA-C2H4-N3 (752 mg, 0.17 mmol) and triethylamine (43 mg, 0.42 mmol) were dissolved in 5 mL of dimethylformamide, and the resulting solution was added to a solution of polyperfluorophenylacrylate, pPFPAc, (201 mg, 0.84 mmol) in 2 mL DMF. The mixture was heated at 50°C and let stir overnight. Subsequently, a solution of N-Boc-l,6-hexanediamine hydrochloride (86 mg, 0.34 mmol) and triethylamine (79 mg, 0.78 mmol) in 1 mL DMF was added, and the mixture was again stirred at 50°C overnight. Finally, a solution of (3-aminopropyl)dimethylethoxysilane (121 mg, 0.75 mmol) and triethylamine (161 mg, 1.59 mmol) in 1 mL DMF was added, and the mixture was again stirred at 50°C overnight. Then, the volatiles were removed by evaporation under reduced pressure, and the crude was redissolved in 18 mL of dichloromethane. Trifluoroacetic acid (3 mL, 39 mmol) was added, and the mixture was stirred overnight at room temperature. After evaporation under reduced pressure, water (20 mL) and 6M NaOH (2 mL, 12 mmol) were added, and the solution was dialyzed (MWCO = 3.5 kDa) against water for 48 hours, changing the dialysis medium 5 times. After filtration (0.22 pm PES membrane), the dialyzed solution was lyophilized to obtain a white powder, corresponding to the product (734 mg, 62%).

[0113] Assembly of PAcr Am™-g-(PMOXA-N3,NH2,Si) on SiO2Surface: Protein Resistance and Azide Functionality Investigation

[0114] The substrates (SiO2-coated silicon wafer chips) were treated with a O2-plasma for 2 minutes, immersed for 0.1 mg / mL solution of PAcrAm™-g-(PMOXA-N3,NH2,Si) in 1 mM HEPES buffer (HEPES 0, pH = 7.4) for 30 minutes, then rinsed with ultrapure water and blow-dried with a filtered nitrogen flow. The polymer-coated chips were then immersed overnight in 10 mM HEPES buffer with 150 mM sodium chloride (HEPES II, pH = 7.4). For the protein resistance test, both PAcrAm™- coated and uncoated chips were covered with full human serum for 30 minutes, then rinsed with HEPES II and ultrapure water, and blow-dried with a filtered nitrogen flow. For the azide functionality test, PAcrAm™-coated chips were immersed in a 0. 1 mM solution of DBCO-Biotin in HEPES II for 1 hour, then rinsed with ultrapure water and blow-dried. Afterwards, the same chips were immersed in a 0. 1 mg / mL solution of Streptavidin for 30 minutes. As a control, chips that were not exposed to DBCO-Biotin were also immersed in Streptavidin.

[0115] Functionality Testing: Synthesis and Analysis of PAcrAm™-g-(PMOXA-N3,NH2,Si)

[0116] The functionality of NH2-C3H6-PMOXA-C2H4-N3, was tested by synthesizing PAcrAm™-g- (PMOXA-N3,NH2,Si) (d = 0.2, 0.4, 0.4), a poly(acrylamide) to which NH2-C3H6-PMOXA-C2H4-N3was grafted, together with amino and silane functionalities. Such polymer was employed to coat a SiO2 surface, and protein resistance and azide functionality were investigated: the coating was found to grant resistance against exposure to human serum (< 0. 1 nm adlayer thickness uptake, as measured by Variable Angle Spectroscopic Ellipsometry), and, after in situ functionalization with Biotin (via copper-free click reaction with DBCO), selective binding to Streptavidin. In particular, a 2.5 nm thick film is formed after adsorption and ageing in Hepes II buffer (see Table 1).

[0117] Table 1: Adlayer thickness (nm) on SiO2surface, measured by Variable Angle Spectroscopic Ellipsometry at various steps: 1) coating with PAcrAm™-g-(PMOXA-N3,NH2,Si), 2) aging overnight in HEPES II buffer, 3) coupling with DBCO-Biotin, 4) exposure to Streptavidin before coupling with DBCO-Biotin (control), 5) exposure to Streptavidin after coupling with DBCO-Biotin, 6) exposure to human serum after Hepes II ageing, 7) exposure to human serum of an uncoated SiO2surface.

[0118] Immersion of such coated samples in Full Human Serum or Streptavidin does not lead to a significant increase in the film thickness, proving resistance to protein adsorption (non-foulingness) (6.). When the surface is first functionalized with DBCO-Biotin and then exposed to Streptavidin, a significant increase of the film thickness (to 4.8 nm) can be observed, showing the selective sensing capability (5.). A graphical representation of the film thickness values at each step can be observed in Figure 1. Figure 1 shows a graphical representation of the adlayer thickness (nm) on SiO2surface, measured by Variable Angle Spectroscopic Ellipsometry at various steps: 1) coating with PAcrAm™-g- (PMOXA-N3,NH2,Si), 2) aging overnight in HEPES II buffer, 3) coupling with DBCO-Biotin, 4) exposure to Streptavidin before coupling with DBCO-Biotin (control), 5) exposure to Streptavidin after coupling with DBCO-Biotin, 6) exposure to human serum, 7) exposure to human serum of an uncoated SiO2surface.

Claims

Claims1. Use of an azide-terminated oligomer or polymer Z of the general formula (I)or a functional polymer FP comprising a polymer backbone and a plurality of side chains, wherein at least a part of said side chains is oligomer or polymer Z, as coating agent, whereinA is a linear or branched Ci to Ci2alkylene,Ri and Rf are independently of each other a linear or branched Ci to C6alkyl,B and B’ are independently of each other a linear or branched C2to C3alkylene group, ly is an integer of 1 to 1000, n2is an integer of 0 to 100, and the sum of n!+n2is at least 2,R2is selected from the group consisting of hydrogen, methyl, ethyl and COR4;R3is selected from the group consisting of methyl and ethyl, m is 0 or 1, and if m is 1, compound of formula I has a permanent positive charge,R4 is selected from the group consisting of CH3, CF3and phenyl, and if R2is COR4, m is 0, and D is a linear or branched Ci to C]2alkylene.

2. Use of an azide-terminated oligomer Z as coating agent according to claim 1, wherein the sum of ni+n2is an integer of 2 to 10.

3. Use of an azide-terminated polymer Z as coating agent according to claim 1, wherein the sum of n!+n2is an integer of 11 to 1100.

4. Use according to any of the preceding claims for coating a surface of a substrate comprising a surface active headgroup, wherein terminal amine group of oligomer or polymer Z is intended to irreversibly bind to a substrate.

5. Use according to claim 4, wherein the surface active headgroup of the surface has a linear or branched, substituted or unsubstituted Ci to Ci2alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group K5selected from the group of alkoxy silanes, chloro silanes, catechols, nitrocatechols, bromocatechols, chlorocatechols, phosphates, phosphonates, mimosine derivatives, anacheline, gallols, thiols, N-heterocyclic carbenes, perfluorophenyl azides, benzophenon, diaryldiazomethane, aryltrifluoromethyldiazomethane, and organoboron.

6. Use of azide-terminated oligomer or polymer Z as coating agent according to any of the preceding claims, wherein R2is hydrogen.

7. Use of azide-terminated oligomer or polymer Z as coating agent according to any of the preceding claims, wherein A is a Ci to C6alkylene, preferably propylene.

8. Use of azide-terminated oligomer or polymer Z as coating agent according to any of the preceding claims, wherein D is a Ci to C6alkylene, preferably ethylene.

9. Use of azide-terminated oligomer or polymer Z as coating agent according to any of the preceding claims, wherein R, and Rf are independently of each other selected from the group consisting of methyl, ethyl, propyl and isopropyl, preferably methyl and ethyl and most preferably methyl.

10. Use of azide-terminated oligomer or polymer Z as coating agent according to any of the preceding claims having the formula IA or IBwherein R4 is selected from the group consisting of CH3, CF3and phenyl, and if R2is COR4, m is 0.

11. Use of azide-terminated oligomer or polymer Z of formula IA as coating agent according to any of the preceding claims selected from the group consisting of12. Use of functional polymer FP according to claim 1 as coating agent.

13. Use of functional polymer FP as coating agent according to claim 12, wherein all side chains are oligomer or polymer Z according to any of claims 1 to 3 and 6 to 11.

14. Use of functional polymer FP as coating agent according to claim 12, wherein said functional polymer FP comprises at least two different types of side chains, and wherein at least one type of side chain is oligomer or polymer Z according to any of claim 1 to 3 and 6 to 11.

15. Use of functional polymer FP as coating agent according to claim 14, wherein said functional polymer FP comprises at least one type of side chain W which is intended to reversible bind to a substrate or has a coating function, and side chain W is selected from the group consisting ofI. a short chain side chain W 1 having a linear or branched, substituted or unsubstituted Cl to C 12 alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group KI selected from the group consisting of amines, carboxy, poly (propylene sulfide), and thioethers;II. a side chain W2 having a long chain W2 comprising more than 15 carbon or silicon atoms in the chain, wherein said long chain W2 is selected from the group consisting of polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, polydimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly(N- isopropylacrylamide), poly (N-hydroxy ethylacrylamide, poly(2 -hydroxyethyl methacrylate), poly-hydroxypropylmethacrylate), poly-(methacryloyloxylethyl phosphorylcholine), poly-(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragements and poly-(sulfobetaineacrylamide), whereby W2 has no functional end group or side group;III. a side chain W3 having a long chain W3 selected from the group consisting of a polydimethylsiloxane, perfluoroethers, perfluoroalkyls, polyisobutene, polyethylene glycol, polydimethylacrylamide, polyvinylpyrrolidone, polyalkyloxazolines, dextran, carboxymethyl dextran, poly( N isopropylacrylamide), poly (N-hydroxy ethylacrylamide, poly(2 -hydroxyethyl methacrylate), poly-hydroxypropylmethacrylate), poly-(methacryloyloxylethyl phosphorylcholine), poly-(sulfobetaine methacrylate), polyalkylene residues having more than 20 carbon atoms, peptide chains, DNA fragments and poly-(sulfobetaine acrylamide) whereby W3 carries at least one functional end or side group K3 selected from the group consisting of amines, carboxy, and nitrilotriacetic acid (NTA), biotin, azide, terminal alkene groups, terminal alkine groups, tetrazine.

16. Use of functional polymer FP as coating agent according to any of claims 14 or 15, wherein said functional polymer FP comprises at least one type of side chain which is intended to irreversibly bind to a substrate, said side chain Q having a linear or branched, substituted or unsubstituted Ci to Ci2alkylene group which optionally comprises heteroatoms selected from the group consisting of oxygen and nitrogen, and which carries at least one functional end or side group K4 selected from the group of alkoxy silanes, chloro silanes, catechols, nitrocatechols, bromocatechols, chlorocatechols, phosphates, phosphonates, mimosine derivatives, anacheline, gallols, thiols, N-heterocyclic carbenes, perfluorophenyl azides, benzophenon, diaryldiazomethane, aryltrifluoromethyldiazomethane, and organoboron.

17. Use according to any of the preceding claims, as coating agent of a surface selected from the group consisting of glass, polydimethylsiloxane, A12O3, Ta2O5, ITO, TiO2, ZrO2, Fe2O3, steel, brass, bronze, Au, Pt, polystyrene, COC, polypropylene, polyethylene, ABS, nylon, PUA, PETG, polyurethane, PMMA, and PVA.

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