Copolymers based on poly(isobutylene-alt-maleic anhydride) for solubilization, isolation and molecular tagging of membrane proteins in aqueous media

Abstract

The present application relates to a specifically defined copolymer, a process for its preparation, a complex containing the copolymer, the use of the copolymer for solubilizing and stabilizing membrane proteins, and a kit comprising the polymer according to the application and for this use.

Description

Technical Field

[0001] This invention relates to specifically defined copolymers, methods for their preparation, complexes containing the copolymers, the use of the copolymers in dissolving and stabilizing membrane proteins, and kits comprising the polymers described herein for use in this purpose. Background Technology

[0002] Membrane proteins are of paramount importance in biomedical research. Approximately one-third of the human genome encodes membrane proteins, which are deeply involved in cell communication, intermembranous transport, and enzymatic reactions, thus serving as potential drug targets. The isolation and stabilization of membrane proteins are challenging due to their natural interaction with the alkyl chains of cell membrane lipids and their helical and folded structures that insert into hydrophobic membranes. Amphiphilic detergents are required to disrupt the cell membrane, followed by mimicking it. Typically, the surface of membrane proteins is characterized by hydrophobic amino acids localized in the regions inserted into the cell membrane, while hydrophilic amino acids are located in the peripheral portions of the protein. This Janus-like structure is the root cause of the reduced solubility and consequently decreased stability of membrane proteins in aqueous media.

[0003] However, the function of cell membrane proteins typically depends on their native lipid environment, which is disrupted by dissolution with detergents. Furthermore, the structural stability of membrane proteins is compromised after dissolution with common detergents such as sodium dodecyl sulfate (SDS) or dodecyl-β-maltodextrin (DDM), as they tend to aggregate, especially during high-level expression and purification.

[0004] Commercially available detergents (such as SDS) can dissolve membranes and target membrane proteins in high yields [1]. However, detergent dissolution of membrane proteins is often accompanied by denaturation and inactivation. Furthermore, detergent concentrations must always be kept above the critical micelle concentration (CMC). Detergent concentrations within the CMC range can adversely affect membrane protein function because they are difficult to mimic the lipids of the cell membrane. In addition, the presence of detergent in the membrane protein solution can adversely affect further analysis or applications of the membrane protein (e.g., cryotransmission electron spectroscopy or crystallization). Below the CMC, the solubility of the membrane protein cannot be ensured because the hydrophobic plaques of the transmembrane portion of the protein are not completely covered by the detergent.

[0005] [ODDS] Classical amphiphilic polymers, known as amphiphilic polymers (amphipols), are used to overcome the aforementioned challenges because they are able to stabilize dissolved membrane proteins. Amphiphilic polymers bind directly to the equally hydrophobic portion of the protein's transmembrane region via their hydrophobic portion. This makes the use of detergents redundant, as the dissolution and stabilization of membrane proteins can be performed in a unique, single-step process. The amphiphilic polymer prototype A8-35, as described in WO1998027434A1, consists of a polyacrylic acid backbone grafted with octylamine (approximately 25%), isopropylamine (approximately 40%), and approximately 35% remaining carboxylic acid groups. However, the use of A8-35 typically requires detergent-based membrane protein dissolution, which can lead to loss of protein function because the amphiphilic polymer surrounding the membrane protein does not mimic the unique native environment of the cell membrane surrounding the protein.

[0006] One class of novel amphiphilic polymers that have been published and patented are SMA (styrene-maleic acid, WO2006129127, WO2011004158), CyclApol (US20220119558A1), and AASTY (poly(acrylic acid-co-styrene)) [2-4]. The stabilization of these novel polymers is based on their ability to mimic the native environment of membrane proteins by forming small complexes known as nanodisks [2, 5].

[0007] Cyclic nanodisc complexes consist of one or more amphiphilic polymer molecules surrounding a small piece of cell membrane, including membrane proteins [6, 7]. Furthermore, SMA, CyclApol, and AASTY allow for the dissolution and stabilization of membrane proteins without the use of detergents. However, these polymers have limitations in terms of flexibility in controlling polymer length, polydispersity, monomer sequence, altering the hydrophobic or hydrophilic side chains, and the ability to further functionalize the polymer chains using fluorophores or biomolecular tags without affecting the polymerization efficiency itself. The dissolution efficiency and the stability of the resulting membrane protein-containing nanodiscs depend heavily on the properties of the polymer used. Polymers such as SMA, CyclApol, and AASTY are stabilized by the electrostatic repulsion of negatively charged polymer chains. The charge is generated by carboxyl groups, which are achieved in the polymer by copolymerizing monomers such as acrylic acid or maleic acid with styrene in the case of SMA and AASTY [3, 8-10]. These polymers that form nanodisks are at risk of aggregation because they are highly sensitive to the presence of divalent ions, low pH values ​​(<6.5), and high ionic strength due to the negative charge masked by the stabilizing carboxylic acid groups. In contrast, polymers with highly charged polar groups (such as phosphates, sulfates, and quaternary ammoniums) and flexible nonpolar side chains (as described in this invention) can improve the dissolution efficiency and stability of nanodisks

[11] . In addition to the aforementioned drawbacks of copolymers currently used for membrane protein dissolution, the new polymer backbone allows for the realization of functions such as fluorophores or biomolecular tags within the polymer chain, which has been demonstrated in various fields

[12] . Furthermore, the new backbone exhibits different backbone flexibility, which can strongly affect the migration of copolymers within the membrane during dissolution and may limit downstream applications.

[0008] Polymer length, polydispersity, and homogeneous sequence of comonomers (such as acrylic acid and styrene) are key factors in dissolution itself or in applications such as cryo-transmission electron microscopy

[13] . Cryo-transmission electron microscopy (Cryo-TEM) typically requires highly pure and homogeneous samples to obtain high-resolution structures. For detergent-dissolved membrane proteins, the heterogeneity of detergent micelles is known to be a key factor in determining the success of Cryo-TEM work

[14] . For polymer-stabilized membrane proteins, polymer nanodisc regions represent one of the regions with the highest structural heterogeneity, which can hinder successful structure determination. Therefore, controlling the flexibility and length of the polymer backbone can significantly affect the resolution and feasibility of Cryo-TEM projects. A common problem in Cryo-TEM sample preparation is that proteins tend to adhere to the harsh environment of the interface between the sample and air / vacuum, leading to preferential orientation or denaturation of proteins. One approach to address this problem is to use a mesh with a support layer that can specifically recognize target proteins by His tags or other affinity markers, thereby keeping them away from the air-water interface. [WO2020041202A1, US20210041388A1,

[15] ] Adding affinity markers (e.g., biotin) to copolymers will create a universal affinity-based tool for Cryo-TEM work on membrane proteins, offering two key advantages: (i) it will keep proteins away from the air-water interface, and (ii) it should impart the correct orientation distribution to the sample, regardless of the protein's identity. Therefore, it is extremely important to find new polymer backbones with different combinations of hydrophilic and hydrophobic side chains to enable protein structure analysis of membrane proteins using Cryo-TEM. Summary of the Invention

[0009] To overcome the shortcomings of current copolymers used for membrane dissolution, the present invention aims to provide a copolymer having a novel polymer backbone that is adaptable to both hydrophilic and hydrophobic side chains, stable under high ionic strength, and resistant to divalent ions (e.g., Ca2+). 2+ (>5 mM), can be chemically modified, can efficiently dissolve membrane proteins, and can efficiently stabilize membrane proteins.

[0010] This has already been achieved through the subject matter of the independent claims. Preferred embodiments are defined in the dependent claims.

[0011] According to the present invention, a copolymer is provided comprising repeating units of formulas (1) and (1') and / or repeating units of formulas (2) and (2'),

[0012] Equation (1),

[0013] Equation (1´),

[0014] Equation (2),

[0015] Equation (2´),

[0016] In the formula,

[0017] R1 and R3 are independently selected from the following groups:

[0018] (C3-C10) cycloalkyl or (C3-C10) heterocycloalkyl, which is unsubstituted or substituted by one or more free radicals, wherein the free radicals are selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, and straight-chain or branched (C1-C8) alkynyl.

[0019] (C3-C10) cycloalkenyl or (C3-C10) (hetero)cycloalkenyl, which are polyunsaturated or monounsaturated, unsubstituted or substituted by one or more free radicals, wherein the free radicals are selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, straight-chain or branched (C1-C8) alkynyl;

[0020] A polyunsaturated or monounsaturated polycyclic group that is unsubstituted or substituted by one or more free radicals, wherein the free radicals are selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, straight-chain or branched (C1-C8) alkynyl.

[0021] Furthermore, the side-chain hydrophilic group may include one or more of the following: hydroxyl, amino, ether, carboxyl, carboxylate, phosphate, phosphonate, phoscholine, carboxylate ether, carboxylate ester, phosphate ester, amide, phosphonamide, ammonium, or their respective salts. The side-chain hydrophilic group may be positively charged, negatively charged, amphoteric, or neutral.

[0022] Positively charged hydrophilic groups may include, but are not limited to, ammonium cations (e.g., alkylammonium cations, such as monoalkylammonium cations, dialkylammonium cations, trialkylammonium cations, or tetraalkylammonium cations). Negatively charged hydrophilic groups may include, but are not limited to, sulfate groups, carboxyl groups, or phosphate groups;

[0023] Straight-chain alkyl primary amines, or straight-chain alkyl monomethylated, dimethylated or trimethylated amines, wherein in the case of trimethylated amines, a positively charged quaternary amine is present. In embodiments with zwitterionic hydrophilic side chains, straight-chain alkyl amines (e.g., dimethylaminopropylamine) may be further sulfonated using, for example, 1,3-propanesulfonyl lactone, further carboxylated using, for example, halo-(C2-C7) alkyl acids (e.g., chloroacetic acid), or phosphorylated.

[0024] Polyol molecules, such as tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol or 2-amino-2-deoxy-D-glucanol;

[0025] Amine-functionalized linear or cyclic monosaccharides, disaccharides, or polysaccharides, such as N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxygalactose, 2-amino-2-deoxymannose, 2-amino-2-deoxyribose, 2-amino-2-deoxyarabinose, and 2-amino-2-deoxyxylose;

[0026] Amine-functionalized linear or branched polyethylene oxide molecules, such as 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol, wherein the number of ethoxy units can vary between 1 and 15. The terminal functional group can be changed and can be hydroxyl, methoxy, carboxyl, amino, thio, or other groups;

[0027] Choline-containing molecules, such as (2-aminoethyl)trimethylammonium chloride and N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride,

[0028] In addition to the above, R3 is H or an alkali metal ion;

[0029] X is NH, O, or S, and

[0030] x and y are greater than 0, that is, in the copolymer described in this invention, both units with subscripts x and y are present.

[0031] The dashed lines in all structural formulas of this application do not represent chemical bonds, but rather indicate relationships between the corresponding repeating units, particularly their coexistence in any kind of polymer, especially as random copolymers or block copolymers. In the art to which this is addressed, it is common practice to describe such relationships within polymers using dashed lines (see, for example, US2022 / 0119558 A1 and US 11,092,605 B2).

[0032] The sum of x and y can be 100% of the copolymer. Furthermore, the copolymer described in this invention can be a random copolymer or an alternating copolymer.

[0033] If necessary, the negative charge of the carboxyl group can be balanced by cations.

[0034] The copolymers described in this invention are based on maleic acid-isobutylene copolymers. The starting maleic acid-isobutylene copolymers are commercially available in the form of anhydrides. The maleic anhydride copolymer (the starting material) can react with an amine; this produces an acid amide (R-CO-NH-R') with an amine on one side, and an acid R-COOH (where R: polymer group, R': hydrocarbon group on the amine) on the other side.

[0035] Heating these compounds can lead to ring closure to form maleimide. However, ring closure can be achieved using different techniques based on condensation and dehydration, such as DCC HOBt-catalyzed ring closure.

[0036] The copolymers described in this invention can be zwitterionic compounds as exemplified by formulas (5) and (5'):

[0037] Equation (5)

[0038] Equation (5´)

[0039] According to the present invention, a method for preparing the above-mentioned copolymer is also provided, wherein the anhydride of the maleic acid-isobutylene copolymer reacts with the amine of R6 and the R5 functional group to provide copolymers of formulas (5) and (5'). The amine of R6 and the additional R5 functional group are defined as follows:

[0040] R5 is a branched or straight-chain C2-C7 alkyl group;

[0041] R6 is a branched or straight-chain C1-C6 alkyl group with a terminal functional group containing a negative charge (such as a carboxyl group, phosphate group, or sulfonate group).

[0042] R7 and R8 are each independently branched or straight-chain C1-C4 alkyl groups;

[0043] The copolymers described in this invention (exemplified as maleic amides in Formulas 5 and 5') can be converted into maleimide forms as shown in Formula 6.

[0044] According to this disclosure, the amide is formed on one of the two carbonyl carbons, while the carboxylic acid is formed on the other carbonyl carbon.

[0045] In one embodiment, the copolymer of the present invention may be represented by the following formulas (3) and (3') or the following formulas (4) and (4'):

[0046] Equation (3)

[0047] Formula (3´)

[0048] Equation (4)

[0049] Equation (4´)

[0050] In the formula:

[0051] R1, R3, and X are defined as above. That is, the compounds of formula (3) and formula (4) respectively contain the compounds of formula (1) and formula (2) as a part.

[0052] R2 is derived from biotags, fluorophores, peptides, biotin, and functional groups used in click chemistry, and R4 is independently selected from groups defined for R3.

[0053] In Equations 3-4, the letters AD represent the relative number of statistically distributed units in the copolymer and determine the molecular weight of the copolymer, which can range from 2,000 to 24,000 Daltons. The sum of AD can be 100% and is equal to the number of maleic anhydride or isobutylene units in the initial PIMA polymer. Functionalization can range from 0% to 100% relative to the corresponding maleic anhydride units, and typically A + B + C + D = 100% of the total functionalized and unfunctionalized maleic anhydride groups in the polymer.

[0054] Additional functional groups can be provided to the copolymers via formulas 3-4, such as fluorophores, tags (His, Rho, FLAG), biotin, functional groups for click chemistry (e.g., azides and alkynes), and others. These compounds can be attached to acid anhydrides as amine-functionalized molecules. Theoretically, this can be done with amides of formula (1) or with ring closures of formula (2).

[0055] In one embodiment of the copolymer described in this invention, R1 and R3 are independently selected from cycloalkyl groups, such as cyclooctyl, ethylcyclohexyl, and methylcyclohexyl. Surprisingly, cycloalkyl compounds have been found to dissolve membrane proteins, while straight-chain alkyl compounds have not. Therefore, the copolymer described in this invention is unexpectedly particularly suitable for dissolving and stabilizing hydrophobic proteins, membrane proteins, and GPCRs.

[0056] According to the present invention, a method for preparing the above-mentioned copolymers is also provided, wherein the anhydride of the maleic acid-isobutylene copolymer reacts with the amines of R1 and R3 to introduce groups R2 and R4 to provide copolymers of formulas (1), (1') and (3), (3'), which can then be dehydrated to generate copolymers of formulas (2), (2') and (4), (4'). The amines of R1 and R3 can be represented by R1-NH2 and R3-NH2, wherein R1 and R3 are as defined above, and wherein atomic N is introduced into the copolymers of the present invention through these amines.

[0057] In a further embodiment, a complex is provided comprising a copolymer as defined above and a hydrophobic protein, membrane protein, and / or G protein-coupled receptor (GPCR). The complex may also comprise lipids. The membrane protein may be selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transport proteins such as ABC transporters, ion channel proteins, aquaporins, membrane-based ATPases, and SLC transporters.

[0058] According to the present invention, a method for obtaining the above-described complex is further provided, the method comprising contacting the copolymer as defined above with a hydrophobic protein, a membrane protein, a G protein-coupled receptor, and optionally a lipid.

[0059] The copolymers described in this invention can be used for the dissolution, stabilization, and / or purification of membrane proteins. Specifically, they can be dissolved, stabilized, and / or purified from their native membrane environment. In one embodiment, the membrane proteins are selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transport proteins such as ABC transporters, ion channel proteins, aquaporins (water channel proteins), membrane-based ATPases, and SLC transporters.

[0060] Furthermore, the copolymers described in this invention can be used in cryo-electron microscopy, particularly for stabilizing the position and orientation of membrane proteins.

[0061] Furthermore, according to the present invention, a complex is provided comprising the copolymer described in this invention (e.g., in the zwitterionic form described above), lipids, and biomolecules. This complex can be used for cell-free protein expression.

[0062] The present invention also provides a kit comprising the copolymer described herein and optional instructions for use, particularly instructions for the above-described uses. Detailed Implementation

[0063] The invention will be described in more detail below.

[0064] According to the present invention, a copolymer based on poly(isobutylene-alternating-maleic anhydride) having the general formula (6) is provided:

[0065] Equation (6)

[0066] Two segments, each bearing groups R1 and R2, are randomly distributed along the length of the polymer chain.

[0067] In the above formula (6), X or Y is an initiator fragment derived from a free radical initiator molecule, an isobutylene monomer, a functional group (such as a hydroxyl group, a carboxylic acid group, etc.) or a hydrogen atom.

[0068] The term "functional group" refers to any functional group known in the field of organic chemistry.

[0069] The letters AD represent the relative number of statistically distributed units in the polymer and determine the polymer's molecular weight, which can range from 2,000 to 24,000 Daltons. The sum of AD is 100% and equal to the number of maleic anhydride or isobutylene units in the initial PIMA polymer. Functionalization can range from 0% to 100% relative to the corresponding maleic anhydride units, and typically A + B + C + D = 100% of the total functionalized and unfunctionalized maleic anhydride groups in the polymer.

[0070] R1 to R4 are the hydrophilic or hydrophobic portions of the polymer. R1-R4 can be derived from the following substituent groups bonded to amino, thio, or hydroxyl groups:

[0071] Unsubstituted or substituted by one or more free radicals (C3-C10) cycloalkyl or (C3-C10) (hetero)cycloalkyl, wherein the free radical is selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, and straight-chain or branched (C1-C8) ynyl; polyunsaturated or monounsaturated, unsubstituted or substituted by one or more free radicals (C3-C10) alkyl, straight-chain or branched (C1-C8) alkenyl, or straight-chain or branched (C1-C8) ynyl. The free radicals are: polyunsaturated or monounsaturated, unsubstituted or substituted with one or more free radicals, wherein the free radicals are selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, straight-chain or branched (C1-C8) ynyl; and phenyl groups that are unsubstituted or substituted with one or more free radicals, wherein the free radicals are selected from straight-chain, cyclic or branched (C1-C8) alkyl, straight-chain or branched (C1-C8) alkenyl, straight-chain or branched (C1-C8) ynyl.

[0072] In addition, the side chain hydrophilic group may include one or more of the following: hydroxyl, amino, ether, carboxylic acid, carboxylate, phosphate, phosphonate, phosphocholine, choline, carboxylic acid ether, carboxylic acid ester, phosphate ester, amide, phosphonamide, ammonium or their respective salts; the side chain hydrophilic group may be positively charged, negatively charged, amphoteric or neutral.

[0073] Positively charged hydrophilic groups may include, but are not limited to, ammonium cations (e.g., alkylammonium cations, such as monoalkylammonium cations, dialkylammonium cations, trialkylammonium cations, or tetraalkylammonium cations, such as choline and its derivatives). Negatively charged hydrophilic groups may include, but are not limited to, sulfate groups, carboxyl groups, or phosphate groups;

[0074] Straight-chain alkyl primary amines, or monomethylated, dimethylated, or trimethylated amines. In the case of trimethylated amines, a positively charged quaternary amine is present. In embodiments with zwitterionic hydrophilic side chains, the straight-chain alkylamine (e.g., dimethylaminopropylamine) is further sulfonated (e.g., using 1,3-propanesulfonyl lactone), but may also contain carboxyl or phosphate moieties.

[0075] Polyol molecules, such as tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol, or 2-amino-2-deoxy-D-glucol.

[0076] Amine-functionalized linear or cyclic monosaccharides, disaccharides, or polysaccharides, such as N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxygalactose, 2-amino-2-deoxymannose, 2-amino-2-deoxyribose, 2-amino-2-deoxyarabinose, and 2-amino-2-deoxyxylose.

[0077] Amine-functionalized linear or branched polyethylene oxide molecules, such as 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol, wherein the number of ethoxy units can vary between 1 and 15. The terminal functional group can be changed and can be hydroxyl, methoxy, carboxyl, amino, thio, or other groups.

[0078] Choline-containing molecules, such as (2-aminoethyl)trimethylammonium chloride and N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride,

[0079] Amino-containing biotags (e.g., biotin-PEG4-amino), fluorophores (e.g., fluoresceinamine), or peptides (e.g., Rho tags).

[0080] Specifically, R3 and R4 can be hydroxyl groups or alkali metal ions ionically bonded to oxygen (e.g., Li). + Na + K + ).

[0081] definition

[0082] The term "amphiphilic polymer" refers to a vinyl polymer that can dissolve membrane proteins and stabilize them in solution in their natural form.

[0083] The term "nanoplasm" refers to a nanoscale complex formed from amphiphilic polymers and lipids that can stabilize membrane proteins.

[0084] In relation to this invention, the term "PIMA" refers to a polymer backbone based on the polymerization of isobutylene and maleic anhydride monomers.

[0085] The term "membrane protein" includes proteins that contain hydrophobic transmembrane domains or are bound to the membrane through at least one hydrophobic domain. Membrane proteins can be monomers or oligomers and can bind to cofactors.

[0086] In the context of this invention, the term "grafting" describes the functionalization of maleic anhydride units within a polymer chain by other molecules containing amino, thio, or hydroxyl groups.

[0087] The prefix "sulfo-" refers to polymers grafted with varying percentages of zwitterionic side chains to enhance the solubility of the polymer and thereby improve the dissolution and stabilization efficiency in membrane protein dissolution, processing, and downstream processes.

[0088] The prefix "glyco-" refers to polymers grafted with varying percentages of meglumine side chains to reduce the net charge of the polymer and enhance the resulting dissolution and stabilization efficiency in membrane protein dissolution, processing, and downstream processes.

[0089] The term "affinity chromatography" is a method for separating biomolecules from a mixture based on highly specific macromolecular binding interactions between biomolecules and another substance. Compared to other chromatographic methods, affinity chromatography is extremely useful due to its high selectivity and high resolution of separation. Examples of affinity chromatography include the purification of His or Rho-tagged proteins. These proteins have a polyHis (e.g., his5 or his10) or a TETSQVAPA amino acid sequence known as the Rho-Tag at the C-terminus or N-terminus, allowing purification from the mixture by selectively binding to Ni-NTA agarose (for His-tagged proteins) or anti-Rho-1D4 antibody agarose (for Rho-tagged proteins). Protocols for these purification procedures can be found on the Cube Biotech website and in the literature by Hochuli et al. and Corin et al.

[0090] This invention uses poly(isobutylene-alternating-maleic anhydride) copolymer (PIMA) to synthesize amphiphilic polymers.

[0091] PIMA used in this invention is commercially available (e.g., Thermo Scientific, Sigma Aldrich, 26426-80-2). The molecular weight of PIMA can be in the range of 2-20 kDa, but is not limited to that range.

[0092] The basic copolymers of anhydrides can also be selected from the following group: poly(maleic anhydride), poly(isobutylene-alternating-maleic anhydride), poly(maleic anhydride-alternating-1-octadecene), poly(maleic anhydride-alternating-1-tetradecene), poly(ethylene-alternating-maleic anhydride), polyethylene-grafted-maleic anhydride, polyisoprene-grafted-maleic anhydride, polypropylene-grafted-maleic anhydride, poly(styrene-co-maleic anhydride), and poly(methyl vinyl ether-alternating-maleic anhydride).

[0093] To achieve the amphiphilic properties of the copolymers described in this invention, the copolymers can be grafted using various techniques, including but not limited to forming amides by combining a target containing a primary amine or thiol with the maleic anhydride moiety in the polymer chain via a ring-opening reaction. The grafting rate of the copolymer can be controlled by an excess of amine. The ring-open state is characterized by the formation of both an amide and a carboxyl group. This carboxyl group can be used to form maleimides via a thermally induced ring-closing reaction, which helps to minimize the negative charge in the copolymer.

[0094] The grafting rate of the base polymer having both hydrophilic and hydrophobic molecules can be in the range of >1%, particularly between 1-100%, and preferably between 40-60%. This grafting rate refers to the amount of maleic anhydride in the polymer backbone that reacts with molecules containing amino, sulfide, or hydroxyl groups to form amides or esters. After the grafting reaction, if the amide and carboxyl groups are not cyclized, 100% grafting will result in equal amounts of amide and carboxyl groups in the final copolymer. If the amide and carboxyl groups from the pre-maleic anhydride are cyclized, maleimide is formed via a condensation reaction.

[0095] The polar molecule used for grafting can be a straight-chain alkyl (primary or monomethylated, dimethylated or trimethylated) amine. In the case of trimethylated amines, a positively charged quaternary amine exists.

[0096] In the case of polyols, tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol, or 2-amino-2-deoxy-D-glucanol and similar substances may be used.

[0097] In the case of amine-functionalized linear or cyclic monosaccharides, disaccharides, or polysaccharides, substances such as N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxygalactose, 2-amino-2-deoxymannose, 2-amino-2-deoxyribose, 2-amino-2-deoxyarabinose, and 2-amino-2-deoxyxylose can be used.

[0098] In the case of amine-functionalized linear or branched polyethylene oxides, ethanol such as 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol can be used. The number of ethoxy units can be varied between 1 and 15. The terminal functional group can be changed and can be hydroxyl, methoxy, carboxyl, amino, thio, or other groups.

[0099] In the case of amine-functionalized choline, (2-aminoethyl)trimethylammonium chloride or N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride can be used.

[0100] In embodiments with zwitterionic side chains, linear alkylamines (e.g., dimethylaminopropylamine) may be further sulfonated (e.g., using 1,3-propanesulfonyl lactone). Amino-containing biotags (e.g., biotin-PEG4-amino), fluorophores (e.g., fluoresceinamine), or peptides (e.g., Rho tags) may also be grafted onto the copolymer to achieve intrinsic functionalization in the polymer, such as target specificity, binding to affinity resins, or labeling with chromophores for spectroscopic detection. Polyol molecules, such as tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol, or 2-amino-2-deoxy-D-glucanol, may also be used.

[0101] The novel amphiphilic copolymers, both functionalized and unfunctionalized, can be used for the dissolution and stabilization of transmembrane proteins and membrane-associated proteins.

[0102] The copolymers described in this invention can be used for labeling nanodisk complexes for use in biomolecular research, diagnostic applications, and medical product development.

[0103] Compared with existing polymers and copolymers, the copolymers of the present invention have at least one of the following advantages:

[0104] It has enhanced solubility for membrane proteins expressed in prokaryotic and eukaryotic cells and / or native proteins expressed in the cell membrane.

[0105] Compared to commercially available products, the membrane protein nanodisc assemblies formed exhibit enhanced stability in terms of high ionic strength and high cation concentration, during dissolution and purification processes, and in applications involving novel amphiphilic polymers and cell membrane fragments.

[0106] Due to the improved dissolution efficiency, the polymer described in this invention can be used at a lower concentration, thereby enabling the stabilized membrane proteins to have higher purity and functionality.

[0107] Novel copolymers can be functionalized using molecules (e.g., fluorophores, biotin derivatives, etc.) for protein purification and labeling.

[0108] The chemically modifiable backbone exhibits molecular flexibility unlike other copolymers on the market (such as SMALP) and can achieve site-specific modification to locate dissolved target proteins in the cryo-TEM grid, thereby enhancing the orientation and quality of the obtained protein structure through cryo-TEM.

[0109] The dissolution, stabilization, and purification of membrane proteins from their native membrane environment depends on a number of parameters. Most of these parameters can be optimized during the purification process for greater efficiency. These parameters include buffer conditions (e.g., salt, pH), polymer selection, protein-to-solvent ratio, temperature, and time. First, cell lysis and centrifugation are performed, for example, using the following parameters: adding a protease inhibitor (PI) to the buffer and readjusting the pH, then lysing the cells (e.g., sonication, Freund's crusher), centrifuging at 9,000 rcf for 30 minutes at 4°C, discarding the precipitate (cell debris), collecting the supernatant, centrifuging the supernatant at 100,000 rcf for 1 hour at 4°C, discarding the supernatant, and homogenizing the precipitate. Then, the membrane protein is dissolved: the polymer forms synthetic nanodisks around the protein, thereby maintaining the native phospholipid environment and preserving the protein's native properties and resulting functional characteristics in a convenient one-step process (dissolution and stabilization). On the other hand, detergents form micelles around the hydrophobic band, thereby removing the surrounding lipids. For native conditions, it is necessary to preserve the unique lipid environment.

[0110] In one embodiment, the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transport proteins such as ABC transporters, ion channel proteins, aquaporins (water channel proteins), membrane-based ATPases, and SLC transporters. That is, as the starting material for the method described in this invention, a solution of a free polymer is used, derived from the dissolution, stabilization, and purification of the aforementioned membrane proteins from their natural environment using a polymer.

[0111] The amphiphilic copolymers described in this invention can be used for the dissolution and stabilization of membrane proteins for biotechnological and pharmaceutical applications. Therefore, it is also possible to use the amphiphilic polymers described in this invention as reagents in kits and diagnostic kits (e.g., lateral flow chromatography assays) that contain at least a portion of the invention. One application of the copolymers described in this invention is the dissolution and stabilization of membrane proteins in solution with or without detergent pretreatment. This means that they are capable of stabilizing fully functional or nonfunctional but still immunogenic membrane proteins in solution and preventing their aggregation or precipitation during dissolution and handling. Therefore, this invention also relates to the formation of water-soluble complexes composed of one or more amphiphilic polymer molecules, artificial or natural lipids derived from cell membranes, and transmembrane proteins or membrane-associated proteins.

[0112] Examples of the uses of the copolymer described in this invention are as follows:

[0113] One or more recombinant membrane proteins or membrane-associated proteins are expressed at high density in prokaryotic or eukaryotic cells and are located in or on the cell membrane, and potentially in inclusion bodies.

[0114] The dissolution step was performed using whole-cell suspension, supernatant of cell lysate, or precipitate obtained by centrifugation of supernatant derived from cell lysate.

[0115] The protein solution or precipitate was added directly to the polymer solution to a final polymer concentration of up to 5 wt%, and incubated with stirring for up to 24 hours.

[0116] Dissolution efficiency can be determined using standard biomolecular methods (e.g., SDS-PAGE, Western blotting).

[0117] Centrifugation can separate undissolved proteins and debris from dissolved proteins located in the formed nanodisc complex.

[0118] In diagnostics, biological components such as DNA, RNA, proteins, and metabolites are examined qualitatively and quantitatively. This provides information about disease, genetic susceptibility, or health status. Diagnostic tests can be performed by medical professionals or by individuals.

[0119] The copolymers described in this invention can be used to dissolve and stabilize membrane proteins, preferably in their native lipid environment, to maintain their activity. These stabilized membrane proteins can be used to detect interactions. The interactions between the copolymer-stabilized membrane proteins and their interacting partners can be detected by various analytical methods, including but not limited to.

[0120] Examples of optical detection include SPR (surface plasmon resonance), RM (resonance mirror), GCI (grating-coupled interferometry), ELISA (enzyme-linked immunosorbent assay) such as direct ELISA, sandwich ELISA, competitive ELISA or reverse ELISA, and LFA (lateral flow chromatography assay).

[0121] The interactions between copolymer-stabilized membrane proteins and their interacting partners can be detected using various analytical methods, including but not limited to.

[0122] Furthermore, the copolymer can lyse eukaryotic cells and tissues very rapidly (within seconds to minutes) at low concentrations (0.01% to 5%) without mechanical assistance. Due to this mild lysis capability, it allows users to obtain nucleic acids with low fragmentation and soluble proteins and membrane proteins in their native state. The polymers of the present invention are particularly suitable for diagnostic tests, especially those based on DNA, RNA, soluble proteins, and membrane proteins.

[0123] The copolymers described in this invention, or complexes of the copolymers with lipids, drugs, or biomolecules, can be used for diagnostic or therapeutic applications. Example

[0124] The invention is described below by way of examples. It is explicitly stated that these examples should not be construed as limiting the invention.

[0125] The attached diagram shows the following:

[0126] Figure 1 ATR-FTIR spectra of PIMA and Cubipolnh (acid anhydride).

[0127] Figure 2 ATR-FTIR spectra of Cubipolnh (acid anhydride) and hydrolyzed Cubipol.

[0128] Figure 3 ATR-FTIR spectra of Cubipolnh (acid anhydride) and functionalized Cubipol-glycerol (Cubipol-Glycerol).

[0129] Figure 4 ATR-FTIR spectra of Cubipolnh (acid anhydride) and functionalized glycosyl-Cubipol (Glyco-Cubipol).

[0130] Figure 5 ATR-FTIR spectra of Cubipolnh (acid anhydride) and functionalized sulfonyl-Cubipol (Sulfo-Cubipol).

[0131] Figure 6 ATR-FTIR spectra of Cubipolnh (acid anhydride) and functionalized Cubipol Amin.

[0132] Figure 7 ATR-FTIR spectra of Cubipolnh (acid anhydride) and functionalized Cubipol PEG.

[0133] Figure 8 UV-Vis extinction spectra of Cubipol, Cubipol-glycerol, sulfonyl-Cubipol, glycosyl-Cubipol, Cubipol PEG, and Cubipol Amin. Polymer solutions at a concentration of 25 mg / mL were measured in 150 mM NaCl and 20 mM HEPES buffer (pH 7.5).

[0134] Figure 9 Cubipol's tolerance to divalent ions (top) and different pH values ​​(bottom) was screened. A white gel precipitate indicates polymer instability.

[0135] Figure 10 Screening for the tolerance of Cubipol-glycerol to divalent ions (top) and different pH values ​​(bottom). A polymer precipitate as a white gel indicates polymer instability.

[0136] Figure 11 Glyco Cubipol's tolerance to divalent ions (top) and different pH values ​​(bottom) was screened. A white gel precipitate indicates polymer instability.

[0137] Figure 12 Sulfo Cubipol's tolerance to divalent ions (top) and different pH values ​​(bottom) was screened. A white gel precipitate indicates polymer instability.

[0138] Figure 13 Cubipol Amin was screened for tolerance to divalent ions (top) and different pH values ​​(bottom). A white gel precipitate indicates polymer instability.

[0139] Figure 14 Cubipol PEG was screened for tolerance to divalent ions (top) and different pH values ​​(bottom). A polymer precipitate as a white gel indicates polymer instability.

[0140] Figure 15HPLC chromatograms of Cubipol and Cubipol Fluoresceine. Excitation was performed at 490 nm, followed by detection of the fluorescence signal at 520 nm. The Cubipol-based polymer showed no fluorescence, while Cubipol Fluoresceine showed strong fluorescence. Measurements were performed using an Agilent 1260 Infinity II system equipped with a Plgel 3 µM MIXED E column and a mobile phase of DMF containing 0.5 mg / mL LiBr, at 60°C and a flow rate of 1 mL / min. The injection volume was 50 µL (0.1 mg / mL polymer).

[0141] Figure 16 FPLC chromatogram of human G6PC stabilized by Cubipol fluorescein and purified by affinity resin (Rho1D4 MagBeads, Cube Biotech GmbH). An Agilent 1260 Infinity II system equipped with a Superose 6 5 / 150 GL column was used, with a mobile phase of 150 mM NaCl and 20 mM HEPES (pH 7.5) at a flow rate of 0.5 ml / min at room temperature. After elution from the purification resin, 50 µl of protein eluent was injected. Excitation was performed at 490 nm, followed by detection at 520 nm. The chromatogram shows an empty volume peak (5.3 min), nanodiscs (6–11 min), and excess fluorescent polymer (11–19 min).

[0142] Figure 17 FPLC chromatograms of Cubipol Biotin (0.65 mg / ml), streptavidin (1.5 mg / ml), and a mixture of Cubipol Biotin (0.65 mg / ml) and streptavidin (1.5 mg / ml) were obtained. An Agilent 1260 Infinity II system equipped with a Superdex 75 10 / 300 column was used, with a mobile phase of 150 mM NaCl and 20 mM HEPES (pH 7.5) at room temperature and a flow rate of 0.5 ml / min. The refractive index was monitored over time. The chromatograms show the Cubipol Biotin peak (22 min), the streptavidin peak (17.8 min), and the shifted streptavidin peak after conjugation of the four Cubipol Biotin polymers (19.2 min).

[0143] Figure 18Coomassie Brilliant Blue SDS-PAGE of UCP1, a human membrane protein purified with affinity resin, tagged with Rho1D4, and stabilized by Cubipol, Sulfo Cubipol, GlycoCubipol, Cubipol PEG, Cubipol Amin, Cubipol Biotin, or Cubipol Biotin. Based on the sequence, the expected size of UCP1 is 34.17 kDa.

[0144] Figure 19 SDS-PAGE proteoblotting of the human membrane protein UCP1, purified with affinity resin, tagged with Rho1D4, and stabilized by Cubipol, Sulfo Cubipol, GlycoCubipol, Cubipol PEG, Cubipol Amin, Cubipol Biotin, or Cubipol Biotin. Based on the sequence, the expected size of UCP1 is 34.17 kDa.

[0145] Figure 20 Hydrodynamic radius of copolymer DMPC nanodisks determined by DLS.

[0146] Synthesis of PIMA-C8C0

[0147] Under vigorous stirring, 3.084 g of PIMA (20.02 mmol MAnh monomer) was suspended in 50 ml of THF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 2.037 g of cyclooctylamine (16.02 mmol) dissolved in 5 ml of THF was slowly added dropwise, considering the hydrolyzed form of PIMA maleic anhydride and the total amount of available carboxyl groups. The suspension was placed in an oil bath and heated at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently forming a clear solution. The THF was then evaporated under reduced pressure. The pale yellow solid polymer was dissolved at 5 wt.% in 1 M NaOH and then hydrolyzed by reflux at 100°C. The hydrolytic and ring-opening forms of the amphiphilic PIMA-C8C0 copolymer were precipitated with 1 M HCl and washed three times with double-distilled water. The polymer was then obtained after freeze-drying and lyophilization.

[0148]

[0149] Open-loop PIMA-C8C0

[0150] To obtain the ring-closed form of the amphiphilic PIMA polymer, DMF was used instead of THF as the solvent in the reaction. After reacting 1.019 g (8.008 mmol) of C8CO with PIMA to obtain a 40% grafted ring-closed polymer form, an oil bubbler was placed in the flask and the mixture was heated at 120°C for 24 hours. This converts the amide form of PIMA-C8CO to the maleimide form by eliminating H2O. Hydrolysis and purification were performed as described above.

[0151] Synthesis of sulfonyl-PIMA-C8C0

[0152] Under vigorous stirring, 3.084 g of PIMA (20.02 mmol MAnh monomer) was suspended in 50 ml of THF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 2.037 g of cyclooctylamine (16.02 mmol) dissolved in 5 ml of THF was slowly added dropwise, taking into account the hydrolytic form of PIMA maleic anhydride and the total amount of available carboxyl groups. The flask was sealed with a Schlenk adapter, and the suspension was heated in an oil bath at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently becoming a clear solution. To introduce the quaternary ammonium moiety, 0.613 g (6 mmol) of dimethylaminopropylamine dissolved in 5 ml of THF was added to the PIMA-C8C0 THF solution under vigorous stirring at room temperature. The flask was sealed with a Schlenk adapter, and the suspension was heated in an oil bath at 60°C for 24 hours. Then, to introduce the sulfonate moiety, 1 g (8 mmol) of 1,3-propanesulfonyl lactone dissolved in 5 ml of THF was added to a THF solution of PIMA-C8C0 at room temperature with vigorous stirring. The flask was sealed with a Schlenk adapter, and the suspension was heated in an oil bath at 60°C for 24 hours. The THF was then removed under reduced pressure, and the solid product was resuspended in 100 ml of trichloromethane, filtered, and dried under vacuum for purification. The solid product was dissolved in 20 ml of NaOH (1 M) and dialyzed against H₂O, followed by lyophilization to obtain a pale yellow solid amphiphilic copolymer grafted with 40 mol% hydrophobic, 50 mol% carboxyl, and 10 mol% zwitterionic moieties.

[0153]

[0154] Open-ring sulfonyl-PIMA-C8C0

[0155] The proportions of the final hydrophobic, carboxyl, and zwitterionic moieties in the polymer chain can be adjusted by changing their respective molar amounts. To obtain the closed-ring form of the polymer, DMF was used instead of THF in the reaction, and after the introduction of dimethylaminopropylamine, the flask was sealed with an oil bubbler, and the solution was then heated at 120°C for 24 hours, which would generate the maleimide form in the absence of H2O.

[0156] Synthesis of PIMA precursor polymers with labels or tags

[0157] Under vigorous stirring, an argon atmosphere, at room temperature, and in the dark, 3.084 g of PIMA (20.02 mmol MAnh monomer) was dissolved in 10 ml of DMF in a 100 ml round-bottom flask. A solution of 178 mg of 5-aminofluorescein in 5 ml of DMF was added dropwise. The flask was sealed with a Schlenk adapter, and the suspension was heated in an oil bath at 60°C for 24 hours. The DMF was then removed using a rotary evaporator under reduced pressure at 75°C. The solid product was dissolved in 15 ml of DMF, and the procedure was repeated. Successful integration of one fluorescein unit in each chain can be monitored using HPLC. For the integration of the hydrophobic portion, the synthesis of PIMA-C80 described above can be applied.

[0158]

[0159] PIMA-fluorescein was used as a fluorescent precursor polymer in the synthesis of PIMA-C8C0-fluorescein.

[0160] A wide variety of functions can be achieved on the final copolymer backbone by changing the molar amount and / or replacing the 5-aminofluorescein with a primary amine-containing biomolecular tag, other fluorophores, or molecules.

[0161] Synthesis of PIMA-C8C0-Tris or PIMA-C8C0-glycerol

[0162] Under vigorous stirring, 3.084 g of PIMA (20.02 mmol MAnh monomer) was suspended in 50 ml of THF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 1.273 g of cyclooctylamine (10.01 mmol) dissolved in 5 ml of THF was slowly added dropwise, considering the hydrolyzed form of PIMA maleic anhydride and the total amount of available carboxyl groups. The suspension was placed in an oil bath and heated at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently becoming a clear solution. The reaction of the residual maleic anhydride monomer in the polymer backbone could be achieved by adding three molar amounts of polyol amine. For the ring-opening form PIMA-C8C0-Tris, 3.6 g of tris(hydroxymethyl)aminomethane dissolved in 5 ml of THF was added dropwise. For PIMA-C8C0-glycerol, 4.6 g of 2-amino-1,3-propanediol dissolved in 5 ml of THF was added dropwise. The solution was placed in an oil bath and heated at 60°C for 24 hours. The THF was then evaporated under reduced pressure. The pale yellow solid polymer was dissolved at 5 wt.% in 1 M NaOH and then hydrolyzed by reflux at 100°C. The hydrolyzed and ring-opened amphiphilic PIMA-C8C0-Tris copolymer was dialyzed against double-distilled water. After freeze-drying, the polymer was obtained.

[0163]

[0164] PIMA-C8C0-Tris in open-loop state

[0165]

[0166] PIMA-C8C0-glycerol in open-ring state

[0167] Synthesis of glycosyl-PIMA-C8C0

[0168] Under vigorous stirring, 8.0 g of PIMA (51.93 mmol MAnh monomer) was suspended in 40 ml of DMF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF was slowly added dropwise, considering the hydrolyzed form of PIMA maleic anhydride and the total amount of available carboxyl groups. The suspension was placed in an oil bath and heated at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently becoming a clear solution. The anhydride form of the grafted polymer was precipitated in 400 ml of diethyl ether and dried under vacuum. 10 g of the dried solid polymer was dissolved in 100 ml of dry methanol at 65°C along with 6.75 g of N-methyl-D-glucamine. After dissolution, a solution of 0.651 mg sodium in 25 ml methanol was added dropwise under argon atmosphere and stirring. After incubation for 24 hours and the addition of 100 ml ethanol, the final glycol-PIMA-C8CO was separated by rotary evaporation of the organic solvent. For purification, the crude product was dissolved in pure water and dialyzed against water. After freeze-drying, the final polymer was obtained.

[0169]

[0170] Open-ring glycosyl-PIMA-C8C0

[0171] Synthesis of PIMA-C8C0-PEG

[0172] Under vigorous stirring, 8.0 g of PIMA (51.93 mmol MAnh monomer) was suspended in 40 ml of DMF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF was slowly added dropwise, considering the hydrolyzed form of PIMA maleic anhydride and the total amount of available carboxyl groups. The suspension was placed in an oil bath and heated at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently becoming a clear solution. The anhydride form of the grafted polymer was precipitated in 400 ml of diethyl ether and dried under vacuum. 10 g of the dried solid polymer was dissolved in 20 ml of DMF at 65°C. After dissolution, a solution of 5.34 g PEG4-Amin (27.6 mmol) in 20 ml DMF was added dropwise under argon atmosphere and stirring. After incubation for 24 hours, the final PIMA-C8C0-PEG was isolated. For purification, the crude product was dissolved in pure water and dialyzed against water. The final polymer was obtained after freeze-drying and lyophilization. As previously described, the closed-ring derivative can be synthesized by condensation of the ring-opening form.

[0173]

[0174] Open-ring PIMA-C8C0-PEG

[0175] Synthesis of PIMA-C8C0-Amin

[0176] Under vigorous stirring, 8.0 g of PIMA (51.93 mmol MAnh monomer) was suspended in 40 ml of DMF in a 100 ml round-bottom flask. The suspension was covered with argon, and varying amounts of cyclooctylamine diluted in THF were added. To synthesize 40% grafted PIMA-C8C0, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF was slowly added dropwise, considering the hydrolyzed form of PIMA maleic anhydride and the total amount of available carboxyl groups. The suspension was placed in an oil bath and heated at 60°C for 24 hours. After the reaction, the suspended PIMA was grafted with the C8C0 monomer and dissolved, subsequently becoming a clear solution. The anhydride form of the grafted polymer was precipitated in 400 ml of diethyl ether and dried under vacuum. 10 g of the dried solid polymer was dissolved in 20 ml of DMF at 65°C. After dissolution, a solution of 5.0 g (2-aminoethyl)trimethylammonium chloride-hydrochloride (5.71 mmol) in 10 ml DMF and 5 ml H2O was added dropwise under argon atmosphere and stirring. After incubation for 24 hours, the final PIMA-C8C0-Amin was isolated. For purification, the crude product was dissolved in pure water and dialyzed against water. The final polymer was obtained after freeze-drying and lyophilization. As previously described, the closed-ring derivative can be synthesized by condensation of the ring-opening form.

[0177]

[0178] Open-loop PIMA-C8C0-Amin

[0179] Dissolution and purification of membrane protein G6PC with his or rho tags

[0180] Dynamic light scattering

[0181] Dynamic light scattering experiments were performed using the Nanotemper Prometheus Panter and its accompanying software. Each sample was loaded into a 10 µl glass capillary. Each measurement consisted of 10 scans at 25°C, each lasting 5 seconds. The attenuator position and laser power were automatically optimized for dimensional determination.

[0182] Preparation of DMPC nanodisks

[0183] DMPC was suspended at 1.25 mg / ml in 5 w / v% copolymer solutions containing 150 mM NaCl and 20 mM HEPES buffer (pH 7.5), respectively, and stirred at room temperature for 4 hours. The solutions were centrifuged at 10,000 rcf for 30 minutes prior to analysis.

[0184] pH and divalent ion stability determination

[0185] Copolymer solutions (2.5 w / v%) containing 150 mM NaCl, 20 mM HEPES, and 0-150 mM MgCl2 or CaCl2 or pH range 3-10 were prepared.

[0186] Expression of rho-tagged membrane proteins G6PC and UCP1

[0187] The preparation, expression, and purification of G6PC and UCP1 were described in the literature (M. Jastroch, V. Hirschberg and M. Klingenspor, 2012, Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artifacts and reveals no contribution to basal proton leakage, BBA, 1817 (9), 1660-1670, doi.org / 10.1016 / j.bbabio.2012.05.014).

[0188] The dissolution, stabilization, and purification of membrane proteins from their natural membrane environment depends on many parameters. Most of these parameters can be optimized during the purification process for greater efficiency. These parameters include: buffer conditions (e.g., salt, pH), polymer selection, protein-to-solvent ratio, temperature, and time.

[0189] Cell lysis and centrifugation:

[0190] Add a protease inhibitor (PI) to the buffer and readjust the pH, then lyse the cells (e.g., sonication, Freund's crusher). Centrifuge at 9,000 rcf for 30 minutes at 4°C, discard the pellet (cell debris), and collect the supernatant. Centrifuge the supernatant at 100,000 rcf for 1 hour at 4°C, discard the supernatant, and homogenize the pellet.

[0191] Dissolution of membrane proteins:

[0192] Polymers form synthetic nanodisks around proteins, thereby maintaining the natural phospholipid environment and preserving the protein's native properties and resulting functional characteristics in a convenient one-step process (dissolution and stabilization). Conversely, detergents form micelles around the hydrophobic band, thereby removing the surrounding lipids. For natural conditions, it is necessary to preserve the unique lipid environment.

[0193] If the solubility is low, it is recommended to change the screening parameters to improve the overall yield of soluble protein. The standard protocol is described below:

[0194] Add the solvent (copolymer) to the protein solution. The ideal concentration may vary, but a good starting point is:

[0195] 0.1-2.5 w / v% copolymer (e.g., PIMA-based amphiphilic copolymer). Dissolve by stirring at 4°C for 3 to 24 hours.

[0196] Higher temperatures can be selected for optimization. Centrifuge at 100,000 rcf for 1 hour at 4°C.

[0197] Discard the precipitate containing cell debris and collect the supernatant.

[0198] The dissolved membrane proteins (supernatant) located in polymer nanodisks were used for affinity chromatography to separate membrane protein copolymer complexes tagged with his or rho from the mixture using commercially available agarose products.

[0199] Dissolved membrane proteins were visualized using SDS-PAGE, Western blotting, and Rho antibody / HRP staining for chemiluminescence detection.

[0200] result :

[0201] The functionalization of the base polymer PIMA using the modifications described in this invention can be monitored using standard analytical techniques such as UV-Vis electronic absorption spectroscopy, high-performance liquid chromatography (HPLC) with different online detectors, ATR-FTIR spectroscopy, and dynamic light scattering (DLS) to characterize the intrinsic physicochemical properties of the copolymer and its ability to dissolve lipids and proteins.

[0202] Figure 1 This demonstrates the process by which unfunctionalized polyisobutylene-maleic anhydride (PIMA) backbone polymers react with cyclooctylamine to transform into Cubipol polymers in the anhydride state. For all FTIR spectra ( Figure 1-7 Characteristic vibrational modes can be detected in all of them. After maleic anhydride functionalization, approximately 1770 cm⁻¹ -1 Approximately 1850 cm -1 The characteristic C=O carbonyl stretching band at this location weakens or disappears completely upon hydrolysis, while the band at approximately 1720 cm⁻¹... -1 The stretching vibration of carboxylic acid C=O at 1660 cm⁻¹ -1 The stretching vibration of the amide C=O at 1550 cm⁻¹ -1 The amide NH vibration also appears. Furthermore, for Sulfo Cubipol, the characteristic S=O stretching band appears in the 1100-1250 cm⁻¹ region. -1 This indicates successful functionalization. For Cubipol PEG, it can be achieved at 1100 cm⁻¹. -1 COC deformation vibration was detected at the location.

[0203] The different chemical properties of the copolymers described in this invention can be associated with two characteristics. First, the hydrophobicity derived from the aliphatic backbone and its hydrophobic modification. In this embodiment, the hydrophobic modification is cyclooctylamide. Both the backbone and the modification are hydrophobic elements that determine the solubility and can be altered by the modification rate of the backbone. Second, the hydrophilicity derived from the hydrophilic modification of the backbone. This hydrophilic modification determines the stability of the polymer in solution, as well as the stability of the nanodisks formed with or without stable proteins. Many modification methods exist; as examples, modifications and results of PIMA-C8C0 (Cubipol), PIMA-C8C0-glycerol (Cubipol-glycerol), glycosyl-PIMA-C8C0 (glycosyl-Cubipol), PIMA-C8C0-PEG (Cubipol PEG), PIMA-C8C0-Amin (Cubipol Amin), and sulfonyl-PIMA-C8C0 (sulfonyl-Cubipol) are described. Most importantly, resistance to monovalent and divalent ions (e.g., H+) is observed. + Mg 2+ Ca 2+ The ability to precipitate was determined through precipitation experiments. The results are shown in Table 1 and... Figure 9-14 In the case of polymer precipitation after adjusting the pH to 3-10 or adding the appropriate amount of divalent ions to achieve the target concentration, the polymer was found to be unstable under the selected conditions.

[0204] Table 1:

[0205] A property overview of Cubipol, Cubipol-glycerol, sulfonyl-Cubipol, glycosyl-Cubipol, Cubipol PEG, and CubipolAmin. The stability of the polymers at a concentration of 25 mg / ml was tested in aqueous solutions containing 150 mM NaCl, 20 mM HEPES buffer, and different concentrations of calcium chloride and magnesium chloride (pH 7.5). For pH stability determinations, the pH of the polymer solutions was adjusted accordingly.

[0206]

[0207] Copolymers used to stabilize membrane proteins should exhibit minimum absorption above 260 nm so that protein concentration can be determined by UV-Vis absorption. Specifically, tryptophan, an amino acid that absorbs light at 280 nm, is quantified. Figure 8 The UV-Vis absorption spectra of the exemplary copolymers described in this invention are shown. The absorption of a 25 mg / ml copolymer solution is <0.1 au, which allows for the quantification of protein yield after protein purification and removal of excess copolymer.

[0208] The copolymer technology described in this invention allows for the functionalization of different copolymers using biomolecular tags and fluorophores, as previously mentioned. Figure 15 The HPLC chromatograms of Cubipol and Cubipol fluorescein are shown. A fluorescence signal was detected at 520 nm after excitation at 490 nm. The chromatograms show that the Cubipol polymer has no nonspecific fluorescence, while Cubipol fluorescein exhibits a strong fluorescence signal with a peak at approximately 7 minutes. Figure 16 FPLC chromatograms of human G6PC membrane protein stabilized in Cubipol fluorescein and purified with Rho1D4 Magbeads are shown. The chromatograms show a fluorescent empty volume peak of the insufficiently stabilized protein at 5.3 min, a fluorescent nanodisc peak containing the membrane protein G6PC between 6 and 11 min, and an excess of copolymer between 11 and 18 min.

[0209] Through Figure 17 FPLC analysis of Cubipol biotin exemplifies the attachment of biomolecular tags to the copolymer backbone. Figure 17 The changes in refractive index signal after size exclusion chromatography of samples containing Cubipol biotin, streptavidin, and mixtures of both are shown. Four molar amounts of streptavidin were incubated with one molar amount of Cubipol biotin to ensure streptavidin saturation. Successful binding of Cubipol biotin was observed as the peak shifted from approximately 22 min (Cubipol biotin) to 19.5 min (Cubipol biotin + streptavidin). Streptavidin itself showed a weak refractive index signal at approximately 18 min.

[0210] To evaluate the lipid-dissolving ability of the polymers described in this invention, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) was used as an exemplary lipid for its evaluation. Dynamic light scattering (DLS) was used as the analytical technique to characterize the hydrodynamic dimensions of the formed synthetic copolymer nanodiscs. The formed lipid copolymer nanodiscs exhibited hydrodynamic radii ranging from 2.5 nm to 12 nm. However, it is important to note that the DLS data did not reveal the physical dimensions of the formed copolymer nanodiscs, but rather the hydrodynamic dimensions including the bound ions. Therefore, this size is correlated with the actual charge of the copolymer backbone. Sulfo Cubipol exhibits the highest number of charges per copolymer chain, resulting in the highest number of bound ions and thus a larger hydrodynamic radius.

[0211] The ability to dissolve membrane proteins was demonstrated using the human membrane protein UCP1, which is tagged with Rho1D4 and overexpressed in HEK cell cultures. Dissolution and purification were performed according to the previously described protocol. Following affinity resin purification and elution, the eluent samples were analyzed using SDS-PAGE, Western blotting, and DLS. Figure 18 The gel stained with Coomassie Brilliant Blue was displayed, with the target protein UCP1 located at approximately 35 kDa, consistent with the expected size of 34.2 kDa. The identity of the band at approximately 35 kDa was further confirmed by Western blotting with anti-Rho1D4 antibody, followed by secondary HRP antibody labeling and chemiluminescence detection. Furthermore, the cumulative radius of the copolymer nanodiscs containing UCP1 was determined by DLS, indicating that the copolymer nanodiscs are stable. A low PDI indicates that the stable membrane protein did not aggregate in the different copolymers.

[0212] Table 2: Cumulative radius and cumulative PDI of protein UCP1 stabilized in different Cubipol derivatives by dynamic light scattering analysis.

[0213]

[0214] The multimodal copolymer platform technology described in this invention can efficiently stabilize different membrane proteins, thereby producing copolymer nanodisks with tunable chemical properties of the copolymer bands—a unique feature. Furthermore, the copolymers described in this invention can be functionalized using biomolecular tags, fluorophores, or, in some embodiments, lipids or bioactive molecules, enabling their application in life sciences or biomedical fields.

[0215] The above description is provided for illustrative purposes and is not intended to be exhaustive or to limit the invention to the specific forms disclosed. Those skilled in the art will recognize that various modifications and adaptations can be made based on the provided disclosure.

[0216] The terminology used in this specification has been chosen primarily for clarity and guidance purposes and is not necessarily intended to define the precise scope of the invention. Therefore, the scope should not be determined by this detailed description but by the claims associated with this application.

[0217] Therefore, the embodiments described herein are examples of the invention and not limitations, as defined in the following claims.

[0218] Furthermore, throughout this specification and the appended claims, unless otherwise expressly stated, the term "comprising" and its variations (such as "comprising (singular)" or "containing") shall be understood to indicate the inclusion of the specified element or feature without excluding others.

[0219] References:

[0220] 1.Rabilloud, T., Membrane proteins and proteomics: love is possible,but so difficult. Electrophoresis, 2009. 30 Suppl 1: p. S174-80.

[0221] 2.Marconnet, A., et al., Solubilization and stabilization of membraneproteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules,2020: p. 3459-3467.

[0222] 3.Smith, A.A.A., et al., Lipid Nanodiscs via Ordered Copolymers.Chem, 2020. 6(10): p. 2782-2795.

[0099]

[0223] 4.Darr, J.M., et al., The styrene-maleic acid copolymer: a versatiletool in membrane research. Eur Biophys J, 2016. 45(1): p. 3-21.

[0224] 5.Marconnet, A., et al., influence of Hydrophobic Groups Attached toAmphipathic Polymers on the Solubilization of Membrane Proteins along withTheir Lipids SI. 2022.

[0225] 6.Zoonens, M. and J.L. Popot, Amphipols for each season. J MembrBiol, 2014, 247(9-10): p. 759-96.

[0226] 7.Le Bon, C., et al., Folding and stabilizing membrane proteins inamphipol AB-35. Methods, 2018. 147: p. 95-105.

[0227] 8.Smith, A.A.A., et al., Controlling Styrene Maleic Acid LipidParticles through RAFT. Biomacromolecules, 2017. 18(11): p. 3706-3713.

[0228] 9.Autzen, H.E., D. Julius, and Y. Cheng, Membrane mimetic systems inCryoEM: keeping membrane proteins in their native environment. Curr OpinStruct Biol, 2019. 58: p. 259268.

[0229] 10.Timcenko, M., A.A.A. Autzen, and H.E. Autzen, Characterization ofDivalent Cation lnteractions with AASTY Nanodiscs. ACS Applied PolymerMaterials, 2022. 4(2): p. 1071-1083.

[0230] 11.Glueck, D., et al., Electroneutral Polymer Nanodiscs Enablelnterference-Free Probing of Membrane Proteins in a Lipid-BilayerEnvironment. Small, 2022: p. e2202492.

[0231] 12.Lin, C.A., et al., Design of an amphiphilic polymer fornanoparticle coating and functionalization. Small, 2008. 4(3): p. 334-41.

[0232] 13.Mueller, S., et al., The bigger picture: global analysis ofsolubilization performance of classical detergents versus new syntheticpolymers utilizing shotgun proteomics. 2023.

[0233] 14.Yuan, Y., et al., Cryo-EM structure of human glucose transporterGLUT4. Nat Commun, 2022. 13(1): p. 2671.

[0234] 15.Cookis, T., et al., Streptavidin-Affinity Grid Fabrication forCryo-Electron Microscopy Sample Preparation. J Vis Exp, 2023(202).

Claims

1. A copolymer comprising repeating units of formula (1) and / or formula (2) and / or formula (1') and formula (2'): Equation (1), Equation (2), Equation (1´), Equation (2´), In the formula, R1 and R3 are independently selected from the following groups: (C3-C10) cycloalkyl or (C3-C10) (hetero)cycloalkyl, unsubstituted or substituted by one or more free radicals, wherein the free radicals are selected from straight-chain (C1-C8), cyclic or branched (C3-C8) alkyl, straight-chain (C1-C8) or branched (C3-C8) alkenyl, and straight-chain (C1-C8) or branched (C3-C8) alkynyl. Polyunsaturated or monounsaturated, unsubstituted or substituted by one or more free radicals, comprising (C3-C10) cycloalkenyl or (C3-C10) (hetero)cycloalkenyl groups, wherein the free radicals are selected from straight-chain (C1-C8), cyclic or branched (C3-C8) alkyl, straight-chain (C1-C8) or branched (C3-C8) alkenyl, and straight-chain (C1-C8) or branched (C3-C8) alkynyl groups. Polyunsaturated or monounsaturated, unsubstituted or substituted with one or more free radicals, wherein the free radicals are selected from straight-chain (C1-C8), cyclic or branched (C3-C8) alkyl, straight-chain (C1-C8) or branched (C3-C8) alkenyl, and straight-chain (C1-C8) or branched (C3-C8) alkynyl. A phenyl group that is unsubstituted or substituted by one or more free radicals, wherein the free radicals are selected from straight-chain (C1-C8), cyclic or branched (C3-C8) alkyl, straight-chain (C1-C8) or branched (C3-C8) alkenyl, and straight-chain (C1-C8) or branched (C3-C8) alkynyl. Straight-chain alkyl primary amines, or monomethylated, dimethylated, or trimethylated amines; in the case of trimethylated amines, a positively charged quaternary ammonium is present; in the case of zwitterionic hydrophilic side chains, straight-chain alkyl amines can be further sulfonated. Polyol groups, such as tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol, or 2-amino-2-deoxy-D-glucol, Amine-functionalized linear or cyclic monosaccharides, disaccharides, or polysaccharides, such as N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N-methyl-D-glucamine, 2-amino-2-deoxyglucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxygalactose, 2-amino-2-deoxymannose, 2-amino-2-deoxyribose, 2-amino-2-deoxyarabinose, 2-amino-2-deoxyxylose. Amine-functionalized linear or branched polyoxyethylene groups, such as 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol. Choline-like groups, such as (2-aminoethyl)trimethylammonium chloride and N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride, In addition to the above, R3 is H or an alkali metal ion, and X is NH, O, or S. y and x are greater than 0.

2. The copolymer according to claim 1, wherein, The copolymer has the following formula (3) or the following formula (4), and / or formula (3') and formula (4'): Equation (3) Equation (4) Formula (3´) Equation (4´) In the formula, R1, R3, and X are defined as described in claim 1. R2 is derived from biotags, fluorophores, peptides, biotin, and functional groups used in click chemistry, and R4 is independently selected from groups defined for R3.

3. The copolymer according to claim 1 or 2, wherein, R1 and R3 are independently selected from cycloalkyl groups, especially cyclooctyl groups.

4. The copolymer according to any one of the preceding claims, wherein, The copolymer is an amphoteric compound as illustrated in formula (5) or formula (5'), or a maleimide compound obtained by cyclization of a compound of formula (5) or formula (5'): Equation (5) Equation (5´) In the formula, R5 is a branched C3-C7 or straight-chain C1-C7 alkyl group; R6 is a branched C3-C6 or straight-chain C1-C6 alkyl group with a terminal functional group, wherein the terminal functional group contains a negatively charged moiety such as a carboxylic acid group, a phosphate group, or a sulfonate group. R7 and R8 are each independently a branched C3-C4 or straight-chain C1-C4 alkyl group.

5. The copolymer according to any one of the preceding claims, wherein, The copolymer is a compound grafted with anamined linear or cyclic monosaccharide, disaccharide, or polysaccharide, such as N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-mannose, 2-amino-2-deoxyribose, 2-amino-2-deoxyarabinose, 2-amino-2-deoxyxylose, as exemplified by maleimide compounds of formula (6) or formula (6') or obtained by cyclization of a compound of formula (6) or formula (6'): Equation (6) Equation (6´).

6. The copolymer according to any one of the preceding claims, wherein, The copolymer is a compound grafted with an amination polyol, such as tris(hydroxymethyl)aminomethane, 1-amino-2,3-propanediol, 2-amino-2-methyl-1,3-propanediol, 1-amino-2,3-butanediol, 3-amino-1,2-propanediol, 1-amino-1,2-ethylenediol, or 2-amino-2-deoxy-D-glucanol, as exemplified by maleimide compounds of formula (7), formula (7'), or obtained by cyclization of a compound of formula (7) or formula (7'): Equation (7) Equation (7´).

7. A method for preparing the copolymer according to any one of claims 1 to 6, wherein, The anhydride of the maleic acid-isobutylene copolymer is reacted with the amines of R1 and R3, and groups R2 and R4 are introduced to provide copolymers of formula (1), formula (1'), formula (3) and formula (3'), and then the copolymers of formula (1), formula (1'), formula (3) and formula (3') can be reacted to obtain copolymers of formula (2), formula (2'), formula (4) and formula (4').

8. A complex comprising a copolymer as defined in any one of claims 1 to 6 and a hydrophobic protein, membrane protein and / or G protein-coupled receptor (GPCR).

9. The complex according to claim 8, wherein, The complex also contains lipids.

10. The complex according to claim 8 or 9, wherein, The membrane proteins are selected from the following group: membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transport proteins such as ABC transporters, ion channel proteins, aquaporins, membrane-based ATPases, and SLC transporters.

11. A method for obtaining the complex of any one of claims 8 to 10, the method comprising contacting the copolymer as defined in any one of claims 1 to 6 with a hydrophobic protein, a membrane protein, a G protein-coupled receptor, and optionally a lipid.

12. Use of the copolymer of any one of claims 1 to 6 for dissolving, stabilizing and / or purifying membrane proteins.

13. The use according to claim 12, wherein, The solubility, stabilization, and / or purification are provided from the natural membrane environment.

14. The use according to claim 12, wherein, The solubility, stabilization, and / or purification are provided using a cell-free expression system.

15. The use according to claim 12, 13, or 14, wherein, The membrane proteins are selected from the following group: membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transport proteins such as ABC transporters, ion channel proteins, aquaporins, membrane-based ATPases, and SLC transporters.

16. Use of the copolymer of any one of claims 1 to 6 for cryo-electron microscopy.

17. A complex comprising the copolymer of any one of claims 1 to 6, a lipid, and optionally a biomolecule.

18. A kit comprising the polymer of any one of claims 1 to 6 and optional instructions for use.

19. Use of the copolymer of any one of claims 1 to 6 or a complex of said copolymer with lipids, pharmaceuticals or biomolecules for diagnostic or therapeutic applications.

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