WO2015166038A1

WO2015166038A1 - A synthetic process of a block copolymer and uses thereof

Info

Publication number: WO2015166038A1
Application number: PCT/EP2015/059498
Authority: WO
Inventors: Michael Stolzenburg; Christian Elbek; Søren Junker MENTZEL; Kent Høier NIELSEN
Original assignee: Aquaporin A/S
Priority date: 2014-05-01
Filing date: 2015-04-30
Publication date: 2015-11-05
Also published as: DK178227B1; CN106459410A; TW201602177A; CN106459410B

Abstract

The present invention relates to a process for synthesizing amphiphilic block copolymers. The invention further relates to novel vesicles comprising said block copolymers having the structures according to Formulae (I) and (II) and novel uses of said block copolymers as a matrix forming material for incorporation of transmembrane molecules, such as aquaporins.

Description

A SYNTHETIC PROCESS OF A BLOCK COPOLYMER AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to a novel process foclaimsr synthesizing block copolymers, where said process may be performed in a reaction using a sterically hindered base, optionally using a solvent mixture, and where an increased yield of copolymer having an improved degree of PDMS derivatization is obtained. The present invention further relates to certain di- and tri block copolymers produced in higher degrees of purity, as well as novel di- and triblock copolymer mixtures having a controlled degree of molecular weight dispersity.

BACKGROUND OF THE INVENTION

Nardin et al. (Langmuir 2000, 1035- 1041 ) describes the synthesis of a poly(2-methyloxazoline)- block-poly(dimethylsiloxane)-block-poly(2-methyk)xazoline) (PMOXA-PDMS-P OXA) triblock copolymer carrying polymerizable groups at both chain ends.

Isaacman et al. (2012) describes a modular synthesis of poly(oxazoline)-poly(siloxane)- poly(oxazoline) block copolymers that have been clicked together using the copper catalyzed azide-alkyne cycloaddition reaction.

Chujo et al. (1992) describes the preparation of polyoxazoline-polysiloxane-polyoxazoline block copolymers having the formula

The block copolymers are prepared by reacting amino-terminated telechelic

poly(dimethylsiloxane)s (3) at the end of poly(2-methyl-2-oxazoline)s in CHCb at 60°C in the form of reactive oxazolinium (4) species using three different (4)/(3) reactant ratios: 20.6, 19.6, and 25.2 resulting in correspondingly increasing yields of 13.5, 23.8, and 29.0 %, respectively.

WO 2013/072378 A l (Byk-Chemie GMBH) discloses uses as additives in thermal hardening coating material compositions and molding materials of polysiloxane-polyoxazoline block copolymers having units of the general formulae

wherein n is in the range of 1 to 400 preferably in the range of 5 to 100, and Rl and R2 represent alkyl moieties of various kinds having from 4 to 6 carbon atoms or they represent cyclic alkenyl, aryl, alkylaryl or arylalkyl moieties having up to 12 carbon atoms; m is in the range of 1 to 400 preferably in the range of 5 to 100, and R3 represents alkyl, alkenyl or aryl moieties of various kinds having from 3 to 12 carbon atoms. However, for the syntheses described in the preparation of said copolymers only single component solvents, such as toluol or acetonitrile, are used. For the reactants a large surplus of the polyoxazoline component is needed, cf. the synthesis of Chujo et al. (1992), and where the polyoxazoline-polysiloxane-copolymers are prepared in a one-pot reaction using methyl toluenesulfonate as initiator, PMOXA in excess amounts and as solvent toluene or acetonitrile.

Moreover, the block copolymers of the prior art exhibit a large variation in chain lengths of the blocks and may form different kinds of reaction by-products, in block copolymers polydispersity may be manifested through a molecular weight distribution. It has been shown by Meier et al. (2000) that amphiphilic block copolymers, such as PMOXA-PDMS-PMOXA, when forming biomimetic membranes, e.g. when self assembled into polymersomes, can incorporate

transmembrane proteins in the amphiphilic lipid bilayer like wall (a polymeric bi layer). However, when incorporating transmembrane proteins and peptides in polymersome walls or membranes, the polydispersity of the block copolymer chains may result in a membrane thickness mismatch that could pose a problem for transmembrane protein incorporation. Pata & Dan (2003) found that the mechanism for the inhibition of protein incorporation in polymeric bi layers differs from that of their inclusion in lipid vesicles; because in polymersomes, the equilibrium concentration of transmembrane proteins decreases as a function of the thickness mismatch between the protein and the bilayer core.

SUMMARY OF THE INVENTION

Broadly, the present invention provides methods of preparing block copolymers, such as

PMOXA-PDMS-PMOXA triblock copolymers, having a controlled, well defined and narrow molecular dispersity range making them useful for incorporation of various transmembrane molecules being proteins, such as aquaporin water channels, ion channel proteins, or being transmembrane peptide channels such as gramicidins, and the like according to the preferred incorporation requirements of said molecules. In one aspect, the present invention relates to the use of a triblock copolymer according to Formula I

a diblock copolymer according to Formula II

or a mixture of said block copolymers;

wherein Ri, R₂, Li, L , m, and n are defined as below; as matrix forming material in vesicles being useful for having incorporated transmembrane proteins. In an exemplary embodiment of the invention, the use employs a mixture wherein the triblock copolymer comprises more than about 65 to 70 % (w/w), such as more than about 80 % (w/w). In another exemplary embodiment of the invention said use comprises a mixture wherein the triblock copolymer comprises about 25 to 40 % (w/w) or about one third, and the diblock copolymer comprises about 55 to 70 % (w/w) or about two thirds. A significant feature of the compounds of Formula I and II is that the PMOXA blocks exhibit a retro configuration of the repeat unit compared to PMOXA-PDMS-PMOXA block copolymers used in the relevant prior art cf. Isaacman et al. (2012), and another feature is that the Ri end groups are directly attached to the nitrogen atom of the repeat sequence, whereas end groups in prior art PMOXA-PDMS-PMOXA block copolymers, typically a hydroxyl group, is bound to the ~ CH₂)₂- group of the repeat unit.

The present invention relates to a process for synthesizing a preferably amphiphilic block copolymer having at least one hydrophilic A block polymer and a hydrophobic B block polymer, the process comprising the step of reacting a terminally cationic reactive A block polymer (A⁺) with a terminally di- or mono functionalized B block in a reaction to obtain an A-B or an A-B-A block copolymer wherein said A block is selected from hydrophilic polymeric compounds, such as polyethyleneoxide (PEO/OEG) or polyalkyloxazoline (POXA) polymers, such as PMOXA polymers (poly(2-methyl-oxazoline)) and PEOXA (poly(2-ethyl-oxazoline)), and said B block is selected from hydrophobic polymeric compounds such as polybutadiene or silicone compounds, such as polyorganosiloxanes including polydimethylsiloxane (PDMS), polydiethylsiloxane

(PDES) and polymethylphenylsiloxane (PDPS) polymers. In particular, the present invention relates to a process for synthesizing a block copolymer comprising reacting at least one hydrophilic and terminally cationic reactive polymer, A⁺, with a terminally di- or mono functional ized hydrophobic polymer, B, comprising polydimethylsiloxane (PDMS), to obtain an

A-B block copolymer, an A-B-A block copolymer, or a mixture of said block copolymers;

wherein said reaction is carried out in the presence of a sterically hindered base. In addition, said process preferably takes place in a reaction vessel using a solvent mixture where said mixture comprises a polar organic solvent and an apolar organic solvent where both are able to dissolve the hydrophilic as well as the hydrophobic reactants and reaction products. In said process B could be selected from polydiethylsiloxane (PDES) or polydipropylsiloxane (PDPS). In addition, said process of preparing an amphiphilic block copolymer is terminated by quenching the reaction with water.

In particular, the invention relates to a process for synthesizing a block copolymer, wherein said terminally cationic reactive A block polymer (A⁺) is formed by reacting 2-alkyloxazoline monomers with a nucleophilic reagent, such as having a lower alkyl substituent on the leaving group, e.g. methyl, to produce the desired length of polyalkyloxazoline (POXA⁺) polymer, such as PMOXA polymer (poly(2-methyl-oxazoline)), and reacting said POXA* with said terminally di- or monoamine functional ized B block in the same reaction vessel without the need to exchange solvent (one-pot reaction), thus obtaining the desired A-B or A-B-A block copolymer. In addition, the method of polymerization of the invention can be used to incorporate functional end-groups. Such end-groups are normally added post-polymerization. However, functional end- groups including -NH₂, -OH, -SH, -CHO, -C₂H₄OH, -COCH3, -COOH, methacrylate and epoxides may be introduced in the compounds of the invention via or as the Ri -group in Ts-O-Ri and thus be transferred to the POXA ends during the nucleophilic substitution reaction, cf.

Scheme 1 below. If necessary, the functional end-groups can be protected. The block copolymers prepared using the method of the invention are useful as matrix materials in vesicles having incorporated transmembrane proteins, such as aquaporins including bacterial and yeast aquaporins and aquaporins from higher plants; ion channels including sodium, potassium, chloride, and calcium channels, ligand and voltage gated channels, stretch activated channels and constitute vcly open channels, such as porins; transporters including Na atpase, FOFl atp- synthase, calcium atpases and lithium transporters, taurine transporters and GLUT4.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying examples. However, various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

DETAILED DESCRIPTION OF THE INVENTION

More specifically, the invention relates to a process wherein a mono or diamine end- functionalized silicone polymer, such as of Formula i): X1-L1-PDMS-L2-X2 or Formula ii): Xi-Li- PDMS-L2, wherein Xi and X2 each represents a primary amine group (-NH₂) or one of Xi and X2 represents a -NH₂ group and the other represents a terminal hydrogen on the corresponding L group (i.e. the -NH2 group is absent); Li represents a hydrocarbon chain, such as alkylene, i.a. a^■- (CH )_y- group where y is an integer selected from 1 , 2, 3, and 4; L2 is absent or represents a hydrocarbon chain, such as alkyl, i.a. a {Cl hJy-CIb group where y is an integer selected from 1 , 2, and 3; and where y preferably is the same in Li and L₂; or when one of the Xi and X₂ groups is absent then the Li or the L₂ group which would be connected to said absent Xi or X2 group is also absent, and the number of repeating units of the PDMS is in the range of about 10 to about 100, such as about 35 to about 65, such as about 40.

In one aspect of the process of the invention, i.a. the synthesis of an A-B block copolymer and/or an A-B-A block copolymer, the synthetic route to POXA-HN-(CH₂) PDMS-(CH2)_yH and POXA-HN-(CH₂)_y-PDMS-(CH2)_y-NH-POXA comprises the steps of: a) providing the reactant A+, such as POXA⁺, e.g. through polymerization of the monomer alkyl-2-oxazoline using an initiator such as tosylate, both in a suitable polar organic solvent;

b) reacting the reactant A+, such as POXA^*, with H₂N-(CH₂)y-PDMS-(CH2)y-NH2

the presence of a proton scavenger, preferably a sterically hindered base, in a suitable apolar organic solvent;

c) quenching the polymerization reaction in water.

Steps a), b) and c) may ail take place in one reaction vessel (one pot reaction). In some embodiments of the invention, only steps b) and c) take place in the same reaction vessel.

In a further aspect of the process of the invention formula i) represents H₂N-(CH₂)3-PDMS-

(CH₂)₃-NH₂ which is reacted with a terminally cationic reactive PMOXA⁺ where the number of repeating units of the PMOXA is in the range of about 3 to about 50, such as about 5 to about 20, and the molar ratio of said PMOXA⁺ to the amine groups in the compound i) is about 1 , such as about 1 .1 or such as 0.5, to obtain the desired PMOXA-HN-(CH2)3-PDMS-(CH₂)₃-NH-PMOXA. In a further aspect of the process of the invention formula ii) represents H₂N-(CH₂)3-PDMS- (CH₂)₄H which is reacted with a terminally cationic reactive PMOXA⁺ where the number of repeating units of the PMOXA is in the range of about 3 to about 50, such as about 5 to about 20, and the molar ratio of said PMOXA^* to the amine groups in the compound i) is about 1 , such as about 1.1 or such as 0.5, to obtain the desired PMOXA-HN-(CH₂)₃-PDMS-(CH₂)3-NH-PMOXA.

In the process of the invention the compounds of formulae i) and ii), such as H₂N-(CH2)₃-PDMS- (CH₂)3- H₂ or H₂N-(CH₂)3-PDMS-(CH₂)4H, is preferably dissolved in an organic solvent, such as dichloromethane, chloroform, or the like, and added to the POXA^* reagent, in the reaction vessel, and the A-B or A-B-A reaction may be performed at a temperature of between 20 and 70°C lasting for a period of between 2 hours and up to 30 hours. An example of said coupling reaction is shown in scheme 1 below where a terminal -N¾ group in the presence of a proton scavenger, such as a Hunig's base, e.g. DIPEA, participates in a ring opening of a terminal oxazoline moiety and resulting addition reaction with the release of protons from said -NH₂ group.

Furthermore, prior to reacting terminally cationic reactive POXA with H₂N-(CH₂)3-PDMS- (CH₂)3-NH₂ or H₂N-(CH₂)3-PDMS, the process of the invention may comprise the step of polymerizing 2-alkyl oxazoline monomers, preferably 2-methyl-2-oxazoline, to obtain said cationic reactive POXA^* . In the process of the invention the polymerization of the A block monomer (e.g. 2-methyl-2- oxazoline) is preferably performed with a nucleophilic reagent initiator capable of initiating an

SN2 reaction (also known as bimolecular nucleophilic substitution), such as alkyl p-toluene sulfonate Ri-O-Ts (e.g. methyl tosylate), alkyl trifluoromethane sulfonate (e.g. methyl triflate) or alkyl methanesulfonate (e.g. methyl mesylate), preferably methyl p-toluenesulfonate (methyl tosylate) in a nucleophilic substitution reaction leading to alkyl end capped PMOXA, i.e. methyl end capped PMOXA, cf. Formula II below. When using 2-dimethylammoniumethyl methacrylate tosylate as the initiator the final block copolymer would possess methacrylate end-groups suitable for crosslinking by UV light. The initiator is used at a molar ratio dependent of the desired chain length, and said polymerization of 2-methyl-2-oxazoline monomers is preferably conducted in a solvent such as acetonitrile or DMSO both solvents being compatible with solvents such as dichloromethane or chloroform.

In some embodiments of the invention the mono functionalized B block is a compound of Formula i), i): X 1 -L 1-PDMS-L2-X2, wherein Xi, Li, L₂ and X₂ are as defined below.

In a preferred embodiment of the process of the invention the compound of Formula i) is H₂N- (CH₂)₃-PDMS-(CH₂)3-NH₂, such as a compound, wherein the average number of repeating units of the PDMS is in the range of about 10 to about 100, such as about 25 to about 55, such as about 35. In other preferred embodiments of the invention the compound of Formula ii) is H₂N-(CH₂)3- PDMS-(CH₂)₄H, such as a compound wherein the average number of repeating units of the

PDMS is in the range of about 10 to about 100, such as about 20 to about 30. Examples of amino functionalized PDMS polymers useful in the process of the invention preferably have a number average molecular weight, M_n, of from about 2000 Da to about 4500 Da, e.g. such as about 2500 to about 3000 Da.

In some embodiments of the present invention the polymer, B, is a terminally mono

functionalized block polymer and the block copolymer obtained is predominantly an A-B block copolymer. In some embodiments the polymer, B, is a terminally di functionalized polymer and the block copolymer obtained is a mixture of A-B-A and A-B block copolymers. In some embodiments the polymer, B, is a terminally difunctionalized polymer and the block copolymer obtained is predominantly an A-B-A block copolymer. In some embodiments of the present invention, the process is being conducted in a solvent mixture which dissolves all of the A+, B, A-B, and A-B-A polymers which are present in the reaction mixture. In some embodiments said solvent mixture a comprises

• a polar aprotic solvent selected from acetonitrile or DMSO;

· and an apolar solvent selected from dichloromethane, trichloromethane, or

trichloroethylene.

In a special embodiment, solvent mixture comprises acetonitrile and methylene chloride, preferably in the ratio ranges of from 1 :3 to 3: 1. In some embodiments, the hydrophobic polymer, B, is dissolved in the apolar solvent prior to addition to the reaction pot.

In some embodiments of the process of the present invention, POXA+ is PMOXA+, and the molar ratio of PMOXA+ to functionalized groups is at least 1 : 1, such as equal to or larger than 1.1 : 1 , such as larger than 1.2: 1. In some embodiments, POXA+ is PMOXA+, and the molar ratio of PMOXA+ to functionalized groups is at least 0.5: 1 , such as equal to or larger than 0.55: 1 , such as larger than 0.6: 1.

In some embodiments of the present invention, the process comprises the step prior to reacting the terminally cationic reactive POXA+ with said terminally di- or monoamine functionalized B block, of: polymerization of 2-alkyl oxazoline monomers, such as 2-methyl oxazoline, to obtain said cationic reactive POXA+. In some embodiments the polymerization of 2-alkyl oxazoline monomers, such as 2-methyl oxazoline, is performed with a nucleophilic reagent or initiator, such as methyl p-toluenesulfonate, being capable of initiating an SN2 reaction. In a special

embodiment the polymerization is conducted in a polar aprotic solvent, such as acetonitrile.

The invention further relates to a vesicle comprising a triblock copolymer according to Formula I,

a diblock copolymer according to Formula II,

and a transmembrane molecule selected from the group consisting of aquaporin water channel molecules.

In one embodiment of the above use both Ri groups are the same and selected from straight or branched Ci to C₃ lower alkyl groups, such as methyl and ethyl; Li and L₂ are the same and selected from -{CH₂)₂- and -(CH₂)3-; m is an integer between 30 and 100, such as between 60 and 100; and n is an integer between 7 and 30, such as between 7 and 19, such as 9 to 1 1.

In some embodiments of the above use the triblock copolymer has the structure of Formula III:

The invention further relates to a block copolymer composition comprising a compound according to Formula I and a compound according to Formula II, said composition being prepared according to the process as disclosed herein, and wherein each of said compounds has a degree of PDMS derivatization of more than about 30 to 40 %, such as more than or equal to

50%.

An exemplary synthetic route of preparing an A-B-A type compound of Formula I is shown below in Scheme 1 where Ts-O-Ri represents a tosylate, Ts representing the leaving group, and Ri is a group, such as alkyl (eg. methyl) and H2N-L1-PDMS-L2-NH2 represents the diamine functionalized PDMS, cf. also formula i) above. R_rOTs+

Formu a

H₂N-L_rPDMS-L₂-NH₂

Scheme 1 : Synthesis reaction scheme. Hunig's base is shown here while other sterically hindered bases also may be useful as proton scavengers. Definitions and Terms

The term amine functionalized PDMS as used herein refers to mono- or diamine functionalized polydimethyl siloxane polymers, e.g. such as aminomethyl, aminoethyl, aminopropyl, or aminobutyl terminated polydimethylsi!oxane (CAS: 106214-84-0 ). Example of a diamine functionalized PDMS is the cheap commercial oligomer DMS A21 (bis(3-aminopropyl)- polydimethylsiloxane, calculated Mn app. 6400 Da) from Gelest or polydimethylsiloxane, aminopropyl terminated (Mw 3000.00 g/mol from abcr specialty chemicals, Karlsruhe,

Germany). Or PDMS diamine can be synthesized if needed to tailor the PDMS portion in Mw. Example of synthesis of oligomers with targeted number average molecular weights (Mn) of 1000, 2000, 5000, and 1 1000 g mol is disclosed in Bowens, A.D. Synthesis and Characterization of Poly(siloxane imide) Block Copolymers and End-Functional Polyimides for Interphase Applications (Ph. D. dissertation 1999- 1 1 -29) cf. url>

http://scholar.lib.vt.edu theses/available/etd-120799-131523/ retrieved from the internet on 28-04- 2014. Alternatively as disclosed in Baranauskas, V.V. url>

http://scholar.lib.vt.edu/theses/available/etd-04272005-195048/ retrieved from the internet on 28- 04-2014.

The term "PMOXA" as used herein refers to the polyoxazoline poly(2-methyloxazoline) or poly(2 -methyl -2-oxazoline) being prepared by polymerizing 2-methyloxazoline CAS No. 1 120- 64-5: 2-methyl-2-oxazoline. Other similar oxazolines may be useful in the method of the invention, e.g. oxazoline and 2-ethyl-2-oxazoline.

The term "PDMS" as used herein refers to polydimethylsiloxane. However, in general other polyorganosiloxanes including polydiethylsiloxane (PDES) and polymethylphenylsiloxane (PDPS) polymers may be useful in synthesizing block copolymers according to the methods described herein.

Sterically hindered base

A sterically hindered base is an organic base that has the ability to abstract an acidic hydrogen atom from a compound without otherwise chemically reacting with the compound, that is without displacing a functional group within the compound (i.e. nucle philic substitution). Tertiary amines are good examples of non-nucleophilic bases because they have the ability to abstract an acidic proton from a compound but because of their steric hindrance, they cannot otherwise react with the compound. Other non-limiting illustrative examples of non-nucleophilic bases include lithium diisopropylamide (LDA), lithium 2,2,6,6-tetramethylpiperdine (LTMP), lithium hexamethyldisilazide (LHMDS), and the like.

Specifical examples of sterically hindered bases for use in the present invention include:

N,N-Diisopropylethylamine (DIPEA, or Hiinig's Base), l ,5-Diazabicyclo[4.3.0]non-5-ene (DBN), 1 ,4-Diazabicyclo[2.2.2]octan (TED), tert-butylamine, l,8-diazabicyclo[5.4.0Jundec-7-ene (DBU), l,4-diazabicyclo(2.2.2)octane (DABCO), N,N-dicyclohexylmethylamine,

2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1 ,2,2,6,6-pentamethylpiperidine (PMP), 7- methyl-l,5,7-triazabicyclo(4.4.0)dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine. The abbreviation M_n means number average molecular weight. It means the total weight of polymer divided by the number of polymer molecules. Thus, M_n is the molecular weight weighted according to number fractions.

The abbreviation M_w means weight average molecular weight. The molecular weight weighted according to weight fractions. The term PDI means the polydispersity index. The polydispersity index is calculated as the ratio of Mw to Mn. In some embodiments the PDI of the diblock or triblock copolymer is less than 2, such as less than about 1.9, 1.8, 1.7, 1.6, or 1.5 such as less than about 1.4. In some embodiments of the invention the PDI of the diblock copolymer is less than about 1.8 or about 1.7. In some embodiments of the invention the PDI of the triblock copolymer is less than about 1.6 or about 1.5. In some embodiments the PDI of the functional ized PDMS is less than 1 .5, such as less than 1.4, such as less than 1.3. The term "transmembrane molecule" as used herein means in particular membrane proteins having at least one transmembrane domain such as aquaporin water channels including bacterial aquaporins, yeast aquaporins, plant aquaporins, mammalian aquaporins, and other eukaryotic aquaporins. In addition, "transmembrane molecule" means membrane bound peptides, such as gramicidin peptides, having transmembrane spanning properties as well as other transmembrane molecules having sufficient amphiphilic features to enable transmembrane binding or localization.

The process according to the invention exhibits several advantages compared with the prior art, such as the use of a single solvent mixture that abolishes the need to exchange solvent during the reactions and enables the reaction to be performed in one reaction vessel, i.e. as a one-pot reaction. The use of functional tosylates or triflates for the cationic ring-opening polymerization of 2-methyl- 1 ,3-oxazoline is described in Einzmann & Binder (2001 ), and the polymerization of 2-methyl oxazoline is well described in literature (e.g. Matyjazewski & Hrkach 1992). In addition the following synthesis examples show that:

The process is highly reproducible.

The process is scalable due to the possibility of using only one reaction vessel (one-pot reaction) where the reaction may be carried out using only one or few steps, such as, e.g. two steps,without a need to exchange solvents.

- The POXA polymerization is fast as compared to other types of synthesis described in the literature, such as disclosed by Nardin et al. (Langmuir 2000, 16, 1035-1041).

It is possible to control the polydispersity of the end product through control of the POXA⁺ polymerisation step. It is possible to obtain a consistently low polydispersity matching the lowest found in literature yielding a very defined product.

The synthesis of the invention provides a simple way of generating a relatively well-defined di- or triblock copolymer which would exhibit a lower polydispersity compared to similar block copolymers synthesized according to the prior art.

In some embodiments of the invention, the triblock copolymer is having about 30-70, such as about 35-65, such as about 40-60, such as about 45-55 dimethylsiloxane units and about 10-40, such as about 15-35, such as about 20-30 2-methyloxazoline units in total. In some embodiments of the invention the triblock copolymer is having 49 dimethylsiloxane units and 25 2- methyloxazoline units in total.

In some embodiments of the invention, the dibiock copolymer is having about 30-70, such as about 35-65, such as about 35-55, such as about 35-45 dimethylsiloxane units and about 5-20, such as about 5- 15, such as about 10-15 2-methyloxazoline units in total. In some embodiments of the invention the dibiock copolymer is having 40 dimethylsiloxane units and 12 2- methyloxazoline units in total. EXAMPLES

The present invention is further illustrated by the following examples should not be construed as further limiting the general scope o the invention.

Experimental section

General procedure

The reaction according to the invention for the synthesis of PMOXA_n-PDMS_m-PMOXA_n (poly(2 -methyl oxazolin) block polydimethylsiloxan block poly(2-methyl oxazolin)) in general is as shown in Scheme 1 above. Example 1. Preparation of PMOXA-PDMS-PMOXA/PMOXA-PDMS block copolymers in a two step reaction, cf. Formulae III and IV below:

Prior to reaction the solvent (acetonitrile) and the monomer (2-methyl-2-oxazoline) are both dried, such as using molecular sieve 4. 130 g acetonitrile is charged into a 500 ml glass reactor with stirrer. 50 g 2-methyl-2-oxazoline monomer is added by means of a dried (such as Ar flushed) syringe. The monomer reaction solution is heated to 40 °C. 50 g of initiator solution

(methyl-p-toluenesulfonate in acetonitrile with a concentration of 1.0 mol/L) is added with a dried syringe. The reaction solution is heated to 100 °C. After 3 hours at 100 °C the reaction solution is cooled to room temperature. 1 13 g Dichloromethane is added to the resulting PMOXA+ reaction solution.

10 g Aminopropyl terminated polydimethylsiloxane, symmetric (PDMS difunctional amine, CAS No. 106214-84-0, molecular weight around 3000 kDa, obtained from Gelest DMS A 15), 33 g acetonitrile and 3 g N,N-Diisopropylethylamine (DIPEA) are added to a 250 ml reaction flask. 50 g of the previously prepared PMOXA+ reaction solution are added. The reaction is kept at 60 °C for 64 hours. The copolymerization reaction is quenched in water. The resulting copolymer is purified by ultrafiltration. Derivatization of amines from PDMS: 92%, as determined by NMR. We infer from this finding using binominal distribution modelling that a high percentage of the final copolymer is a triblock copolymer, such as about > 70% or about > 80%, and a low percentage of the final copolymer is a diblock copolymer with a negligible amount of unreacted PDMS.

Example 2. Preparation of PMOXA-PDMS and PMOXA-PDMS-PMOXA block

copolymers according to Formulae III and IV below in a two step reaction:

a) 130 g acetonitrile is charged into a 500 ml glass reactor with stirrer. 50 g 2-methyl-2-oxazoline is added by means of a dried syringe. The monomer reaction solution is heated to 40 °C. 50 g initiator solution (methyl-p-toluenesulfonate in acetonitrile with a concentration of 1.0 mol/L) is added with a dried syringe. The reaction solution is heated to 100 °C. After 3 hours at 100 °C the reaction solution comprising the resulting PMOXA+ polymer is cooled to room temperature, 10 g Aminopropyl terminated polydimethylsiloxane, symmetric (PDMS difunctional amine, CAS No. 106214-84-0, obtained from Gelest DMS A 15), 17 g dichloromethane and 3 g N,N- Diisopropylethylamine are added to a 250 ml reaction flask. 47 g of acetonitrile and 15 g of the previously prepared PMOXA+ reaction solution are added. The reaction is kept at 60 °C for 64 hours. The copolymerization reaction is quenched in water. The resulting copolymer is purified by ultrafiltration. Derivatization of amines from PDMS: 56%, as determined by NMR. We infer from this finding using binominal distribution modelling that about two thirds of the final copolymer is a diblock copolymer and about one third is a triblock copolymer.

Alternatively, b) 130 g acetonitrile is charged into a 500 ml glass reactor with stirrer. 50 g 2-methyl-2-oxazoline is added by means of a dried syringe. The monomer reaction solution is heated to 40 °C. 50 g initiator solution (methyl-p-toluenesulfonate in acetonitrile with a concentration of 1.0 mol/L) is added with a dried syringe. The reaction solution is heated to 100 °C. After 3 hours at 100 °C the reaction solution comprising the resulting PMOXA+ polymer is cooled to room temperature.

10 g Aminopropyl terminated polydimethylsiloxane, symmetric (PDMS di functional amine, CAS NR. 106214-84-0, obtained from Gelest DMS A 15), 17 g dichloromethane and 3 g N,N- Diisopropylethylamine are added to a 250 ml reaction flask. 13 g of acetonitrile and 15 g of the previously prepared PMOXA reaction solution are added. The reaction is kept at 60 °C for 64 hours. The copolymerization reaction is quenched in water. The resulting copolymer is purified by ultrafiltration. Derivatization of amines from PDMS: 53%, as determined by NMR. We infer from this finding using binominal distribution modelling that about two thirds of the final copolymer is a diblock copolymer and about one third is a triblock copolymer. Example 3: Preparation of diblock copolymer PMOXA-PDMS of Formula IV below.

130 g acetonitrile is charged into a 500 ml glass reactor with stirrer. 50 g 2-methyl-2-oxazoline is added by means of a dried syringe. The monomer reaction solution is heated to 40 °C. 50 g initiator solution (methyl-p-toluenesulfonate in acetonitrile with a concentration of 1 ,0 mol/L) is added with a dried syringe. The reaction solution is heated to 100 °C. After 3 hours at 100 °C the reaction solution comprising the resulting PMOXA+ polymer is cooled to room temperature. 10 g Monoaminopropyl terminated polydimethylsiloxane, assymmetric, cf. Formula iia below) where n is approximately 24 (mean value) (PDMS mono functional amine, molecular weight around 2000 kDa, obtained from Gelest MCR A12), 17 g dichloromethane, and 2 g N,N- diisopropylethylamine are added to a 250 ml reaction flask. 8 g of acetonitrile and 21 g of the previously prepared PMOXA+ reaction solution are added. The reaction is kept at 60 °C for 64 hours. The copolymerization reaction is quenched in water. The resulting copolymer is purified by ultrafiltration. Derivatization of amines from PDMS: 50%, as determined by NMR. We infer from this finding using binominal distribution modelling that about two thirds of the final copolymer is a diblock copolymer and about one third is a triblock copolymer.

Formula iia), n « 24 Table I) mol amounts and mol ratios used in the above examples

Table i) clearly shows that the PMOXA reactant is used in approximately equimolar amount relative to the amino functionality.

-40

Formula IV; n ¾ 9 and m « 25

Example 4. Protocol for 1 mg ml proteo-polymersomes, polymer to protein ratio (POPR) of

50

Materials:

Polyoxazoline Based Di- and Triblock Copolymers prepared and purified as described in the examples above, having an average molecular weight of between approximately 1500 (diblock copolymers) and approximately 6000 (triblock copolymers).

Protein: Aquaporin Z (AqpZ), Mw 27233.

Preparation:

1 ) Fill a 50 ml glass evaporation vial with 5ml of a 2 mg/ml stock solution of copolymers (either in pure diblock or pure triblock form or as mixtures of di- and triblock copolymers) in CHCI3. 2) Evaporate the CHCb using a rotation evaporator for at least 2h to complete dryness.

3) Add 3.0 mL of buffer solution (1.3% O.G.; 200mM Sucrose; l OmM Tris pH 8; 50m NaCl) to rehydrate the film obtained in the evaporation vial in step 2.

4) Shake the vial at 200 rpm on a platform shaker (Heidolph orbital platform shaker Unimax 2010 or equivalent) for 3 hours to obtain dissolution of the copolymer.

5) Add 1.55mg μϋ of AqpZ in a protein buffer containing Tris, glucose and OG, and rotate vial over night at 200rpm and 4°C.

6) Add 6.88 ml buffer (lOmM Tris pH 8; 5()mM NaCl) slowly while mixing up and down with pipette.

7) Add 180mg hydrated Biobeads and rotate for 1 h at 200rpm.

8) Add 210mg hydrated Biobeads and rotate for lh at 200 m.

9) Add 240mg hydrated Biobeads and rotate O.N. at 200rpm 4°C.

10) Add 240mg hydrated Biobeads and rotate O.N. at 200rpm 4°C.

1 1 ) The Biobeads with adsorbed OG are then removed by pipetting off the suspension.

12) Extrude the suspension for about 21 times through a 200nm track etched polycarbonate filter using an extruder, such as from at least 1 time and up to about 22 times to obtain a uniform proteopolymersome suspension (vesicles) suspension.

Example 5. Alternative protocol for the synthesis of PMOXA-PDMS-PMOXA triblock copolymer

0.57 g (3mmol) distilled methyltosylate was placed in a 20ml μ-wave vial, 6g anhydrous acetonitrile and 2.8g (33mmol) distilled methyloxazoline were added. The reaction mixture was heated in the μ-wave reactor for 15 min at 140°C. After the reaction 5g dry dicloromethane was added.

In another 20ml μ-wave vial were placed 1.98g (0.68mmol) PDMS(-NH₂)₂ (CAS No. 106214-84- 0, Gelest DMS A15Y02), 5g acetonitrile and 0.38g (2.9mmol) diisopropylethylamine. Added 6.6g of the yellow 'living' PMOx solution (contains 1.33mmol chains).

The mixture was heated in an oil bath for ~80h at 60°C. A syringe with N₂ was inserted to prevent pressure build-up. After 80 hours a sample of the RM was for analysis.

After cooling to room temperature lg AMBERLYST™ A26 OH that was previously washed with acetonitrile (15.20g AMBERLYST™ A26 OH was washed with 5x 50ml acetonitrile, filtered using a folded paper filter and dried on air for 2 hours; obtained 7.08g pink solid) was added and the vial was placed on the shaker for 1 hour. Transferred the liquid to a membrane (Spectra/Por Dialysis Membrane RC MWCO: 1000, Vol/1 ength=4.6ml/cm). This membrane was first placed in water for more than 1 day and washed with water. The membrane was placed in a beaker containing -11 water and stirred overnight. Changed the water and stirred for ~3 hours. The mixture in the membrane was concentrated on the rotavap to 3.45g product.

The resulting triblock copolymer contained 49 dimethylsiloxane units and 25 2-methyloxazoline units in total according to Ή-NMR analysis (data not shown), and the PDI of 1.5 (±0.1) was determined for the blockcopolymer based on GPC measurements (data not shown). The PDI of the Gelest DMI A15 Y02 was determined to 1.2 by GPC (data not shown).

Example 6. Alternative protocol for the synthesis of PDMS-PMOXA diblock copolymer 0.57 g (3mmol) distilled methyltosylate was placed in a 20ml μ-wave vial. Added 6g anhydrous acetonitrile and 2.8g (33mmol) distilled methyloxazoline. The reaction mixture was heated in the μ-wave reactor for 15 min at 140°C.

In another 20ml μ-wave vial were placed 3.83g (1.37mmol) PDMS-NH₂ (Gelest DMS XG-2801 ; monoaminopropyl terminated PDMS; molecular weight around 2500 kDa; viscosity 31.4 cPs), 5g dry DCM and 0.8g (6.2mmol) diisopropylethylamine.

Added 4.5g of the 'living' PMOx solution (contains 1.37mmo! chains). This mixture was heated in the μ-wave reactor for 30' at 140°C. After the heating the reaction mixture had turned orange. Removed some sample for analysis. 1.0g of AMBERLYST™ A26 OH previously washed with acetonitrile.1 and placed the vial on the shaker for 4 hours. Transferred the liquid (a little bit of resin came along) to a membrane (Spectra/Por Dialysis Membrane RC MWCO: 1000,

Vol/1 ength=4.6ml/cm). This membrane was first placed in water and after 1 hour washed with water. The membrane was placed in a beaker containing ~11 water and stirred. After ~3 hours the water was replaced. Stirred over the weekend and changed the water. After 4 hours the milky liquid containing some yellow gels was removed from the membrane, concentrated on the rotavap and dried overnight under vacuum at ~60°C. Obtained 4.58g yellow product.

The prepared di-block copolymer (IV) contained 40 dimethylsiloxane units and 12 2- methyloxazoline units according to Ή-NMR analysis (data not shown). The PDI was 1.7 (±0.1) as calculated for the diblock copolymer based on GPC measurements (data not shown). The PDI of the Gelest DMS XG-2801 was determined to 1.3 by GPC (data not shown).

Example 7. Large scale batch synthesis of PMOXA-PDMS and PMOXA-PDMS-PMOXA block copolymers The large scale syntheses were made according to the steps and principles of Examples 1 -3 using the materials and with the yields as shown in table ii below and with the functionalized PDMS starting materials Gelest DMS A15 Y02 and Gelest DMS XG-2801.

Table ii: Materials used and results obtained. ACN is acetonitril, MOXA is methyloxazoline, MeTs is methyltosylate, ACN is acetonitrile, DCM is dichloromethane, DIPEA is N,N- diisopropylethylamine, and PDMS is the functional ised polydimethylsiloxane.

Workup by continuous diafiltration: The reaction mixture is stripped for methylene chloride on a Rotavapor. The remaining solution is mixed with water and the pH is adjusted to 7 with HQ. The solution is then transferred to a diafiltration unit and filtered at approx. 3.2bar on a 2kDa MWCO hydrosart filter from Sartorious. The retentate is transferred back to the solution and more water is added to keep the volume constant. Conductivity is measured in permeate and retentate and the filtration is stopped when the conductivity of the permeate is constant at around 1 ( S/cm for a polymer solution.

The worked up product can then be freeze dried and analysed for impurities and structure. References:

The references cited herein are expressly incorporated by reference for all purposes in their entirety.

Veena Pata and Nily Dan, Biophysical Journal Volume 85 October 2003 21 1 1-21 18]

Einzmann & Binder, Journal of Polymer Science Part A: Polymer Chemistry, Volume 39, Issue 16, pages 2821-2831 , 15 August 2001.

T. Saegusa, H. Ikeda, H. Fujii; Macromolecules 1972, 5, 359-362; Isomerization Polymerization of 2-Oxazoline IV. Kinetic study of 2-Methyl-2-oxazoline Polymerization.

B. Brissault, C. Guis, H. Cheradame; European Polymer Journal 2002, 38, 219-228; Kinetic study of poly(ethylene oxide-b-2-methyl-2-oxazoline) diblocks synthesis from poly(ethylene oxide) macroinitiators.

R. Hoogenboom, M.W.M. Fijten, U.S.Schubert; Journal of Polymer Science Part A: Polymer Chemistry 2004, 42, 1830-1840; Parallel kinetic Investigation of 2-Oxazoline Polymerizations with Different Initiators as Basis for Designed Copolymer Synthesis.

R. Hoogenboom, M.W.M. Fijten, R.M. Paulus, H.M.L. Thijs, S. Hoeppener, G. Kickelbick, U.S. Schubert; Polymer 2006, 47, 75-84; Accelerated pressure synthesis and characterization of 2- oxazoline block copolymers.

S. Ji, T.T. Hoye, C.W. Macosko: Macromolecules 2005, 38, 4679-4686, Primary Amine (-NH₂) Quantification in Polymers: Functionality by ^I9F NMR Spectroscopy.

Corinne Nardin, Thomas Flirt, Jorg Leukel and Wolfgang Meier, Polymerized ABA triblock Copolymer Vesicles, Langmuir 2000, 16, 1 35 - 1041

Michael J. Isaacman, Kathryn A. Barron and Michael J. Isaacman, Kathryn A. Barron and Luke S. Theogarajan, Clickable Amphiphilic Triblock Copolymers, Polymer Chemistry 2012, 50, 23 19 - 2329.

Corinne Nardin, Sandra Thoeni, Jorg Widmer, Mathias Winterhalter and Wolfgang Meier, Nanoreactors based on (polymerized) ABA-triblock copolymer vesicles, Chem. Commun., 2000, 1433 - 1434.

Matyjaszewski, Krzysztof & Jeffrey S. Hrkach. Cationic ring opening polymerization of oxazolines initiated by trimethylsily! derivatives. Technical Report, Carnegie Mellon

University, Department of Chemistry, May 25, 1992.

W. Meier, C. Nardin, M. Winterhalter, Reconstitution of channel proteins in (polymerised) ABA triblock copolymer membranes. Angew. Chem. Int. Ed. 39(24) (2000) 4599 Mirko Einzmann &Wolfgang H. Binder (2001) Novel functional initiators for oxazoline polymerization. Journal of Polymer Science Part A: Polymer Chemistry, Volume 39, Issue 16, pages 2821-2831 , 15 August 2001

Y. Chujo, E. Ihara & T. Saegusa, Synthesis of Polyoxazoline-polysiloxane block copolymers. Kobunshi ronbunshu, 1992, vol 49, no. 11, pages 943-946.

Claims

CLAIMS:

1. A process for synthesizing a block copolymer comprising reacting at least one hydrophilic and terminally cationic reactive polymer, A⁺, with a terminally di- or mono functionalized hydrophobic polymer, B, comprising polydimethylsiloxane (PDMS), to obtain an A-B block copolymer, an A-B-A block copolymer, or a mixture of said block copolymers; wherein the reaction is carried out in the presence of a sterically hindered base.

2. The process according to claim 1, wherein the sterically hindered base is selected from the group consisting of N,N-Diisopropylethylamine (DIPEA, or Hunig's Base), 1 ,5-

Diazabicyclo[4.3.0]non-5-ene (DBN), 1 ,4-Diazabicyclo[2.2.2]octan (TED), tert-butyl amine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), l,4-diazabieyc!o(2.2.2)octane (DABCO), N,N- dicyclohexylmethylamine, 2,6-di-tert-butyl-4-methylpyridine, quinuclidine, 1 ,2,2,6,6- pentamethylpiperidine (PMP), 7-methyl-l,5,7-triazabicyclo(4.4.())dec-5-ene (MTBD), triphenylphosphine, tri-tert-butylphosphine and tricyclohexylphosphine.

3. The process according to claim 1 or claim 2, wherein the sterically hindered base is selected from the group consisting of DIPEA (Hunig's base), DBN and TED.

4. The process according to any one of claims 1-3, wherein the hydrophilic and terminally cationic reactive polymer, A⁺, comprises POXA⁺ (polyalkyloxazoline), such as PMOXA⁴ (poly(2-methyl-oxazoline)).

5. The process according to any one of claims 1 -4, wherein said hydrophobic polymer B is terminally di- or monofunctionalized with one or two reactive groups being able to perform the coupling reaction with the hydrophilic and terminally cationic reactive polymer A⁺, wherein said reactive group is selected independently from the group of amine, thiol, piperidyl, piperazinyl and acyl.

6. The process according to any one of claims 1-5, wherein said terminally di- or mono functionalized B block is a compound of Formula i)

i): X1-L1-PDMS-L2-X2, wherein Xi and X₂ each represents a primary amine group (-NH₂) or one of Xi and X₂ represents a -NH₂ group and the other represents a terminal hydrogen on the corresponding L group;

Li and L₂ each represents a bond or a hydrocarbon chain, such as alkylene, i.a. a -(CH₂)_y- group where y is an integer selected from 1 , 2, 3, and 4 and where y preferably is the same in Li and L₂; and the average number of repeating units of the PDMS is in the range of about 10 to about 100, such as about 25 to about 55, such as about 35.

7. The process according to any one of claims 1-5, wherein said terminally di- or mono functional ized B block is a compound of Formula ii)

wherein Xi represents a primary amine group (-NH₂);

Li represents a bond or a hydrocarbon chain, such as alkylene, i.a. a -(CH₂)_y- group where y is an integer selected from 1 , 2, 3, and 4;

L₂ is absent or represents a hydrocarbon chain, such as alkyl, i.a. a -(CH2)y-CH₃ group where y is an integer selected from 1 , 2, and 3;

and the average number of repeating units of the PDMS is in the range of about 10 to about 100, such as about 20 to about 30.

8. The process according to any one of claims 1 -7, wherein the polymer, B, is a terminally mono functionalized block polymer and the block copolymer obtained is

predominantly an A-B block copolymer.

9. The process according to any one of claims 1 -8, wherein the polymer, B, is a terminally difunctionalized polymer and the block copolymer obtained is a mixture of A-B-A and A-B block copolymers.

10. The process according to any one of claims 1 -9, said process being conducted in a solvent mixture which dissolves all of the A+, B, A-B, and A-B-A polymers which are present in the reaction mixture.

1 1 . The process according to claim 10, wherein said solvent mixture comprises acetonitrile and methylene chloride, preferably in the ratio ranges of from 1 :3 to 3: 1.

12. The process according to any one of claims 1 -1 1, wherein the hydrophobic polymer, B, is dissolved in the apolar solvent prior to addition to the reaction pot.

13. The process according to any one of claims 1 -1 1 , wherein the process uses a single solvent mixture that abolishes the need to exchange solvent during the reactions and enables the reaction to be performed in one reaction vessel.

14. A vesicle comprising

- a triblock copolymer according to Formula I,

- a diblock copolymer according to Formula II,

- a transmembrane molecule selected from the group consisting of aquaporin water channel molecules;

wherein Ri and R₂ of Formulae I and II

are independently selected from the group consisting of straight or branched Ci to C₆ alkyl, secondary or tertiary amine, -OH, SH, -CHO, -C₂H₄OH, -COCH3, -COOH, methacrylate and epoxides;

R3 is straight or branched lower alkyl; Li and L₂ independently of each other are -(CH₂)^, wherein y is an integer selected from 1 , 2, 3, and 4; or Li is™ CH₂)y-, and L₂ is absent;

m is an integer between 10 and 100; and

n is an integer between 3 and 50;

and optionally in Formula II R₂ may represent hydrogen.

15. The vesicle according to claim 14, wherein the block copolymer is a mixture in which the triblock copolymer comprises between about 25 to 40% (w/w) of the block copolymer and the diblock copolymer comprises between about 55 to 70% (w/w) of the block copolymer.

16. The vesicle according to claim 14 or claim 15, wherein the block copolymer is a mixture in which the triblock copolymer comprises between about one third (w/w) of the block copolymer and the diblock copolymer comprises between about two thirds (w/w) of the block copolymer.

17. A block copolymer composition comprising a compound according to Formula I and a compound according to Formula II, wherein the composition is as obtainable by the process of any one of claims 1 to 13.

18. The block copolymer composition according to claim 17, wherein each of said compounds has a degree of PDMS derivatization of more than about 30 to 40 %, such as more than or equal to 50%.

19. A compound according to Formula I having a polydispersity index (PDI) of less than about 1.7, such as less than about 1.6 or about 1.5.

20. The compound according to claim 19 having 30-70 dimethylsiloxane units and 10- 40 2-methyloxazoline units in total.

21. A compound according to Formula II having a polydispersity index (PDI) of less than about 1.8 or about 1.7.

22. The compound according to claim 21 having 30-70 dimethylsiloxane units and 5- 20 2-methyloxazoline units in total.