WO2025210045A1

WO2025210045A1 - Method to prepare polyalkyleneimines

Info

Publication number: WO2025210045A1
Application number: PCT/EP2025/058899
Authority: WO
Inventors: Richard Hoogenboom; Emiel PATTYN; Ine MERTENS
Original assignee: Universiteit Gent
Current assignee: Universiteit Gent
Priority date: 2024-04-02
Filing date: 2025-04-02
Publication date: 2025-10-09
Anticipated expiration: 2026-10-02

Abstract

The present invention generally relates to a method for preparing polyalkyleneimines, and to polyalkyleneimines obtainable by the method. The present invention further relates to particular polyalkyleneimines, and to the use of the polyalkyleneimines in human or veterinary medicine.

Description

METHOD TO PREPARE POLYALKYLENEIMINES

FIELD OF THE INVENTION

BACKGROUND TO THE INVENTION

Polyethyleneimine (PEI) is a versatile polymer known for its wide range of applications in various fields, including chemistry, materials science, and biotechnology. It is characterized by its repeating unit composed of ethyleneimine, giving it unique properties such as high reactivity, ionizability and excellent film-forming capabilities.

There are two primary forms of polyethyleneimine: branched polyethyleneimine and linear polyethyleneimine. Branched PEI, with its highly branched structure, has limited use in medical and in vivo applications due to its broad molar mass distribution and cytotoxicity, related to the presence of tertiary amines in the polymer structure and high molar mass fractions. On the other hand, linear PEI, being absent of tertiary amines, exhibits a much lower cytotoxicity and can be prepared with narrow molar mass distribution, enhancing its application in biomedical applications.

Linear polyethyleneimine (L-PEI) has been widely studied for its potential in gene delivery applications. Gene delivery is the process of introducing foreign genetic material into target cells for various purposes, such as gene therapy, genetic engineering, or basic research. PEI's ability to condense and protect nucleic acids, such as DNA or RNA, and facilitate their entry into cells has made it an attractive candidate for gene delivery.

Branched PEI can be prepared by the ring opening polymerization of aziridine, while linear PEI can be prepared by post-modification of poly(2-oxazolines). In particular, linear, well-defined PEI has been prepared by the hydrolysis of poly(2-ethyl-2-oxazoline). A disadvantage of the hydrolysis of poly(2-alkyl-2-oxazoline)s, i.e. A/-acyl polyethyleneimines, are the harsh acidic conditions (6M aq. HCI, reflux) needed to hydrolyze the amide group. These harsh acidic conditions prevent the presence of other acid-sensitive groups present in the starting poly(2- alkyl-2-oxazoline).

More recently, Hoogenboom et al. have reported the acidic hydrolysis of poly(2-alkyl-2- oxazoline)s under milder conditions by using 3M HCI at 100 °C in an ethanol-water solvent mixture. They also found that specific block copoly(2-oxazolines) could be preferentially hydrolyzed, albeit not selective, based on the difference in hydrolysis kinetics of the corresponding poly(2-oxazoline) homopolymers. Specifically, a block copolymer of 2-methyl-2- oxazoline (MeOx) units and 2-phenyl-2-oxazoline (PhOx) units could be hydrolyzed with a conversion of roughly 60 %, wherein 95 % of the PMeOx block was hydrolyzed, while only 10 % of the PPhOx block was hydrolyzed. Similar observations were reported by Guegan et al. for the hydrolysis of a block copolymer of 2-methyl-2-oxazoline (MeOx) units and 2-isopropyl-2- oxazoline (iPrOx) units. Because these methods rely on the inherent difference in hydrolysis kinetics of the corresponding poly(2-oxazoline) homopolymers related to their solvation, they do not allow to retain the more reactive, and more hydrophilic 2-oxazoline unit (e.g. MeOx) in the hydrolyzed polymer. Moreover, the inherent difference in hydrolysis kinetics may allow for a preference in hydrolysis of one 2-oxazoline unit relative to another one, but the less reactive 2- oxazoline unit will still be hydrolyzed to some extent. Moreover, these methods do not allow the preparation of L-PEI copolymers with a hydrophilic poly(2-oxazoline) block as the preferential hydrolysis largely results from insolubility of one of the blocks in the aqueous hydrolysis medium.

As there is growing interest in block copolymers having multiple hydrophilic blocks or block copolymers having both hydrophobic blocks and hydrophilic blocks, having numerous applications, such as stabilization of nanoparticles, there is a need for methods that allow preparing tailored (block) copolymers with a high degree of selectivity.

It is therefore an object of the present invention to provide an improved method of preparing polyalkyleneimines compared to the methods known in the state of the art, or at least provide one or more alternative methods.

SUMMARY OF THE INVENTION

According to first aspect, the present invention provides a method to modify a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, comprising the steps of: a) providing a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, wherein the polyalkyleneimine comprises first alkyleneimine structural units represented by formula (I) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , Rr, R2, R2', R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

R is selected from -C(O)SR_a, -C(O)S(O)R_a, and -C(O)S(O)2R_a; and R_a is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl; and b) reacting the polymer provided in step a) with one or more alkoxides in a solvent.

In an embodiment, the present invention provides the method as defined herein, wherein the polymer comprising a polyalkyleneimine provided in step a) is the polyalkyleneimine, in particular the linear polyalkyleneimine.

In a further embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII) or (VII’) wherein n is an integer from 2 to 1000; m is 0, or an integer selected from 1 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1 , each instance of Rr, each instance of R2, each instance of R2 , each instance of R3, and each instance of R3' is independently selected from -H, and -Ci ealkyl; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of X2 is independently selected from a direct bond, -CH2-, and -CR6R6 CH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -C1- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; each instance of Re is independently selected from -H, -Ci-i2alkyl, -Ci-i2alkenyl, -C(O)R_c, and -C(O)NRdRd'; each instance of R_c, and each instance of Rd is independently selected from -Ci-i2alkyl, -C2- i2alkenyl, and -Ar2; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of Rd' is independently selected from -H, -Ci-i2alkyl, -C2-i2alkenyl, and -Ars; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally and independently substituted; and each instance of An , each instance of Ar2, and each instance of Ars is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

In yet a further embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (Vila) or (Vila’) wherein n is an integer from 2 to 1000; m is an integer selected from 1 to 1000; each instance of Xi and each instance of X2 is independently selected from a direct bond, - CH2-, and -(CH₂)₂-; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted;

Rs is -C(O)R_c; and

Re is selected from -Ci-i2alkyl, in particular from -Ci-ealkyl, more in particular from -Ci-4alkyl.

In yet a further embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is a gradient copolymer.

In yet a further embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is a block copolymer. In yet a further embodiment, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O, wherein Rb is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

In yet a further embodiment, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is a tertiary alkyl group, in particular wherein Rb is selected from tert-butyl, tert-pentyl, and tert-hexyl.

In yet a further embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises A/-alkoxy- or aryloxycarbonyl alkyleneimine structural units.

In yet a further embodiment, the present invention provides the method as defined herein, further comprising a step c), wherein at least part of the A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units are removed by reaction with a chemical reagent.

In yet a further embodiment, the present invention provides the method as defined herein, further comprising a step c), wherein at least part of the A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units are removed by acidic hydrolysis.

According to a further aspect, the present invention provides a polymer, in particular a polyalkyleneimine, obtainable by the method as defined herein.

According to yet a further aspect, the present invention provides a polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, wherein the polyalkyleneimine is a gradient copolymer or a block copolymer represented by formula (VIII) or wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1, each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci-ealkyl ; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of X2 is independently selected from a direct bond, -CH2-, and -CR6R6CH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -Ci- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; and

Re is selected from -Ci-i2alkyl, in particular from -Ci-ealkyl, more in particular from -Ci-4alkyl, even more in particular from -CH3, and -CH2CH3.

In an embodiment, the present invention provides the polymer, in particular the polyalkyleneimine as defined herein, wherein the polyalkyleneimine is a gradient copolymer or a block copolymer represented by formula (Villa) or (Villa’) wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi and each instance of X2 is independently selected from a direct bond, -

CH2-, and -(CH2)2-; and

The present invention further provides a delivery system comprising a polymer, as defined herein, and a therapeutic agent that is non-covalently bound to said polymer, in particular wherein the therapeutic agent is a pharmaceutically active agent or a biomolecule, such as a gene, a peptide, a protein or a nucleic acid.

The present invention further provides a polymer, in particular a polyalkyleneimine, as defined herein, or a delivery system, as defined herein, for use in human or veterinary medicine. BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description provided with the drawings makes apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Figure 1A, also abbreviated as FIG. 1A, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 1 , after each of General Procedures A and C, respectively.

Figure 1 B, also abbreviated as FIG. 1 B, shows superimposed FT-IR spectra of the polymers prepared according to Example 1 , after each of General Procedures A and C, respectively.

Figure 2, also abbreviated as FIG. 2, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 2, after each of General Procedures A, B and C, respectively.

Figure 3, also abbreviated as FIG. 3, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 2 after General Procedure C, and after acidic hydrolysis of said polymer, according to Example 3, respectively.

Figure 4, also abbreviated as FIG. 4, shows a SEC spectrum of the polymer prepared according to Example 4.

Figure 5, also abbreviated as FIG. 5, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 5, after each of General Procedures A, B and C, respectively.

Figure 6, also abbreviated as FIG. 6, shows an ¹ H NMR spectrum of the polymer prepared according to Example 6, after General Procedure A.

Figure 7, also abbreviated as FIG. 7, shows an ¹ H NMR spectrum of the polymer prepared according to Example 6, after General Procedure A, B and C and acidic hydrolysis.

Figure 8, also abbreviated as FIG. 8, shows an ¹ H NMR spectrum of the polymer prepared according to Comparative example A, after General Procedure A, dissolution in water and stirring with 1 equivalent of trifluoroacetic acid overnight at room temperature. Figure 9, also abbreviated as FIG. 9, shows a SEC overlay of the polymers prepared according to Comparative example A, after General Procedure A, and after further dissolution in water and stirring with 1 equivalent of trifluoroacetic acid overnight at room temperature, respectively.

Figure 10, also abbreviated as FIG. 10, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 8, after each of General Procedures A and B, respectively.

Figure 11 , also abbreviated as FIG. 11 , shows a SEC spectrum of the polymer prepared according to Example 9.

Figure 12, also abbreviated as FIG. 12, shows superimposed ¹H NMR spectra of the polymers prepared according to Example 1 1 , after each of General Procedures A and B, respectively.

Figure 13, also abbreviated as FIG. 13, shows a SEC overlay of the polymers prepared according to Example 7 (PEtSOx) and Example 8 (PiPrSOx).

Figure 14, also abbreviated as FIG. 14, shows a SEC overlay of the polymers prepared according to Example 10 (PEtSOzi) and Example 1 1 (PiPrSOzi).

Figure 15, also abbreviated as FIG. 15, shows a SEC spectrum of the polymer prepared according to Example 12.

Figure 16A, also abbreviated as FIG. 16A, shows an ¹H NMR spectrum of the polymer prepared according to Example 13.

Figure 16B, also abbreviated as FIG. 16B, shows a SEC spectrum of the polymer prepared according to Example 13.

Figure 17A, also abbreviated as FIG. 17A, shows superimposed ¹H NMR spectra of the polymer prepared according to Example 14 (PEtOx-b-PiPrSOx), as well as the polymerization solution before polymerization.

Figure 17B, also abbreviated as FIG. 17B, shows a SEC overlay of the polymer prepared according to Example 14.

Figure 18A, also abbreviated as FIG. 18A, shows superimposed ¹H NMR spectra of the polymer prepared according to Example 15 (PEtOx-b-PiPrSOzi), as well as the polymerization solution before polymerization. Figure 18B, also abbreviated as FIG. 18B, shows a SEC overlay of the polymer prepared according to Example 15.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

When describing the compounds of the present invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise:

The term "alkyl" by itself or as part of another substituent refers to a fully saturated hydrocarbon of Formula CxHzx+i wherein x is a number greater than or equal to 1 . Generally, alkyl groups of this invention comprise from 1 to 20 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, Ci-4alkyl means an alkyl of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, butyl, and its isomers (e.g. n-butyl, i-butyl and t- butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers, undecyl and its isomers, dodecyl and its isomers, tridecyl and its isomers, tetradecyl and its isomers, pentadecyl and its isomers, hexadecyl and its isomers, heptadecyl and its isomers, octadecyl and its isomers, nonadecyl and its isomers, eicosanyl and its isomers. The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3, or 4 substituents) at any available point of attachment.

Whenever the term “substituted” is used in the present invention, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using “substituted” is replaced with a selection from the indicated group, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.

Where groups may be optionally substituted, such groups may be substituted once or more, and preferably once, twice or thrice. Non-limiting examples of such substituents are selected from halogen (-halo), hydroxy (-OH), oxo (=0), nitro (-NO2), amino (-NR’R”), cyano (-CN), alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy or aryloxy (-OR’”), aryl, heteroaryl, carbonyl (-C(O)R^iv), carboxyl (-COOH), ester or alkoxycarbonyl (-C(O)OR^V), ester or alkylcarbonyloxy (-OC(O)R^vi), amido or aminocarbonyl (-NR’C(O)), amido or carbonylamino (-C(O)NR’), heterocyclyl, carbonyl, acyl, arylcarbonyl, thio (-SH), alkylthio (-SR^vi), and the like.

The term "alkenyl" or “alkene”, as used herein, unless otherwise indicated, means straight-chain, cyclic, or branched-chain hydrocarbon radicals containing at least one carbon-carbon double bond. Examples of alkenyl radicals include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, hexadienyl, be it in the terminal or internal positions and the like. Generally alkenyl or alkene moieties of the present invention comprise from 2 to 20 C atoms. An optionally substituted alkenyl refers to an alkenyl having optionally one or more substituents (for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term "alkynyl", as used herein, unless otherwise indicated, means straight-chain or branched-chain hydrocarbon radicals containing at least one carbon-carbon triple bond. Examples of alkynyl radicals include ethynyl, propynyl, butynyl, pentynyl, hexynyl, hexadiynyl, be it in the terminal or internal positions, and the like. An optionally substituted alkynyl refers to an alkynyl having optionally one or more substituents (for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl.

The term “cycloalkyl” by itself or as part of another substituent is a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 , 2, or 3 cyclic structure. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups containing 1 to 3 rings, including monocyclic, bicyclic, or polycyclic alkyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 15 atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantanyl and cyclodecyl with cyclopropyl being particularly preferred. An “optionally substituted cycloalkyl” refers to a cycloalkyl having optionally one or more substituents (for example 1 to 3 substituents, for example 1 , 2, 3 or 4 substituents), selected from those defined above for substituted alkyl.

Where alkyl groups as defined are divalent, i.e., with two single bonds for attachment to two other groups, they are termed "alkylene" groups. Non-limiting examples of alkylene groups includes methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1 ,2-dimethylethylene, pentamethylene and hexamethylene. Similarly, where alkenyl groups as defined above and alkynyl groups as defined above, respectively, are divalent radicals having single bonds for attachment to two other groups, they are termed "alkenylene" and "alkynylene" respectively. In the context of the present invention, the alkyl(ene), alkenyl(ene) and alkynyl(ene) moieties as defined herein may also further comprise one or more heteroatoms, such as selected from N, S or O, in that for example a carbon atom in an alkyl, alkene or alkyne chain is replaced by a heteroatom. When two or more C atoms are replaced by heteroatoms, the heteroatoms may be adjacent or separated, as long as it results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent. An example of a stable combination of two adjacent heteroatoms is a disulfide (-S-S-) group. Where a carbon atom in an alkyl(ene), alkenyl(ene) or alkynyl(ene) chain is replaced by an N atom, the N atom may be N or NH depending on the number of bonds connected to said C atom.

The term “alkoxy" or “alkyloxy” as used herein refers to a radical having the Formula -OR’” wherein R’” is alkyl, alkenyl, or alkynyl. Non-limiting examples of suitable alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy. The term “aryloxy" as used herein refers to a radical having the Formula -OR’” wherein R’” is aryl.

Where the oxygen atom in an alkoxy group is substituted with sulfur, the resultant radical is referred to as alkylthio or arylthio, such as methylthio, ethylthio, phenylthio, and the like.

The term "carbonyl" by itself or as part of another substituent refers to the group -C(O)R^iv, wherein R^iv is a hydrogen atom (i.e. an aldehyde), or alkyl, alkenyl, alkynyl or aryl (i.e. a ketone).

The term "carboxy" or “carboxyl” or “hydroxycarbonyl” by itself or as part of another substituent refers to the group -COOH, -C(O)OH, or -CO2H.

The term "alkoxycarbonyl" by itself or as part of another substituent refers to a carboxy group linked to an alkyl radical i.e. to form -C(O)OR^V, wherein R^v is alkyl, alkenyl, alkynyl or aryl.

The term “alkylcarbonyloxy” by itself or as part of another substituent refers to a -OC(O)R^vi wherein R^vi is alkyl, alkenyl, alkynyl or aryl.

The term "direct bond" as used herein, refers to a chemical linkage directly connecting two or more specified moieties, without the presence of any intervening elements or groups.

The term "heterocycle" as used herein by itself or as part of another group refers to nonaromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atomcontaining ring. Each ring of the heterocyclic group containing a heteroatom may have 1 , 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms. An optionally substituted heterocyclic refers to a heterocyclic having optionally one or more substituents (for example 1 to 4 substituents, or for example 1 , 2, 3 or 4), selected from those defined above for substituted alkyl. Non-limiting examples of heterocycle comprise: piperidinyl, azepanyl, morpholinyl.

The term “aryl" (herein also referred to as aromatic heterocycle, and represented by -Ar or -aryl) as used herein refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthalene or anthracene) or linked covalently, typically containing 6 to 10 atoms; wherein at least one ring is aromatic. The aromatic ring may optionally include one to three additional rings (either cycloalkyl, heterocyclyl, or heteroaryl) fused thereto. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein. Non-limiting examples of aryl comprise phenyl.

The aryl ring or heterocycle as defined herein can optionally be substituted by one or more substituents (for example 1 to 5 substituents, for example 1 , 2, 3 or 4) at any available point of attachment. Where a carbon atom in an aryl group is replaced with a heteroatom, the resultant ring is referred to herein as a heteroaryl ring.

The term “heteroaryl” as used herein by itself or as part of another group refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 3 rings which are fused together or linked covalently, typically containing 5 to 8 atoms; at least one of which is aromatic in which one or more carbon atoms in one or more of these rings can be replaced by oxygen, nitrogen or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of such heteroaryl, include piridinyl, azepinyl.

An “optionally substituted heteroaryl” refers to a heteroaryl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted aryl.

As used herein the terms such as “alkyl, aryl, or cycloalkyl, each being optionally substituted with” or “alkyl, aryl, or cycloalkyl, optionally substituted with” refers to optionally substituted alkyl, optionally substituted aryl and optionally substituted cycloalkyl.

As already mentioned herein before, in a first aspect, the present invention provides a method to modify a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, the method comprising the steps of: a) providing a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, wherein the polyalkyleneimine comprises first alkyleneimine structural units represented by formula (I) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R is selected from -C(O)SR_a, -C(O)S(O)R_a, and -C(O)S(O)2R_a; and

R_a is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl; and b) reacting the polymer provided in step a) with one or more alkoxides in a solvent.

It is an advantage of the method of the present invention that it provides a polyalkyleneimine polymer allowing removal of the substituents on the amine group of the alkyleneimine structural units under less harsh conditions and/or more selectively, compared to methods known in the art, such as the acidic or alkaline hydrolysis of alkylcarbonyl or arylcarbonyl substituents. It is a further advantage of the method of the present invention that it allows obtaining a polyalkyleneimine polymer having alkoxycarbonyl or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units, which may not be accessible via methods known in the art, such as the cationic ring opening polymerization of 2-alkoxy-2-oxazolines reported by Miyamoto et al. Yet a further advantage of the method of the present invention is that it allows to prepare gradient copolymers or block copolymers with a high selectivity, comprising unsubstituted alkyleneimine structural units and alkylcarbonyl substituted alkyleneimine structural units, wherein the alkyl group is methyl or ethyl, which cannot be obtained by the processes known in the art.

In the context of the present invention, the term “polyalkyleneimine” refers to any polymer substantially consisting of substituted or unsubstituted alkyleneimine structural units, wherein each of the alkyleneimine structural units consists of an amine group and an alkylene spacer. In the case of unsubstituted alkyleneimine structural units, the amine group bears a hydrogen atom, while in the case of substituted alkyleneimine structural units, the amine group bears any group other than hydrogen, such as alkyl, alkenyl, alkynyl, aryl, alkylcarbonyl, alkoxycarbonyl, alkylthiocarbonyl, alkylsulfinylcarbonyl, alkylsulfonylcarbonyl, and alkylaminocarbonyl. In the context of the present invention, the term “linear polyalkyleneimine” may refer to a polyalkyleneimine, as defined herein, wherein each alkyleneimine structural unit - except for the structural units at each end of the polymer chain - is directly connected to one further alkyleneimine structural unit on the N-terminal of the structural unit and directly connected to one further alkyleneimine structural unit on the C-terminal of the structural unit. Scheme 1 provides a representation of a number of structural units of a polyalkyleneimine, in particular a linear polyalkyleneimine, as defined herein, wherein A represents any alkylene, and R represents hydrogen or any group other than hydrogen, such as alkyl, alkenyl, alkynyl, aryl, alkylcarbonyl, alkoxycarbonyl, alkylthiocarbonyl, alkylsulfinylcarbonyl, alkylsulfonylcarbonyl, and alkylaminocarbonyl.

Scheme 1

In the context of the present invention, “any polymer substantially consisting of substituted or unsubstituted alkyleneimine structural units” refers to any polymer comprising at least 90 % of substituted or unsubstituted alkyleneimine structural units, relative to the total number of structural units, preferably at least 95 %, more preferably at least 99 %, and preferably is a polymer 100 % consisting of substituted or unsubstituted alkyleneimine structural units.

The term “polyalkyleneimine”, as used herein, therefore includes unsubstituted homopolymers such as polyethyleneimine, polypropyleneimine, polybutyleneimine, polypentyleneimine, and the like. Consequently, the term “linear polyalkyleneimine”, as used herein, includes unsubstituted linear homopolymers such as linear polyethyleneimine, linear polypropyleneimine, linear polybutyleneimine, linear polypentyleneimine, and the like.

The term “polyalkyleneimine”, in particular the term “linear polyalkyleneimine”, as used herein, also includes substituted homopolymers such as poly(2-alkyl-2-oxazoline)s, poly(2-aryl-2- oxazoline)s, poly(2-alkoxy-2-oxazoline)s, poly(2-aryloxy-2-oxazoline)s, poly(2-alkylthio-2- oxazoline)s, poly(2-alkylsulfinyl-2-oxazoline)s, poly(2-alkylsulfonyl-2-oxazoline)s, poly(2- arylthio-2-oxazoline)s, poly(2-arylsulfinyl-2-oxazoline)s, poly(2-arylsulfonyl-2-oxazoline)s, poly(2-alkylamino-2-oxazoline)s, poly(2-arylamino-2-oxazoline)s, and their longer-chain counterparts, such as poly(2-alkyl-2-oxazine)s, and poly(2-alkyl-2-oxazepine)s.

The term “polyalkyleneimine”, in particular the term “linear polyalkyleneimine”, as used herein, also includes any random, alternating, statistical, gradient, or block copolymer combining two or more different alkyleneimine structural units.

In the context of the present invention, the term “different alkyleneimine structural units” may refer to alkyleneimine structural units having a different length of alkylene group, to alkyleneimine structural units having different groups on the amine group, and to any combination thereof. Preferably, the term “different alkyleneimine structural units” refers to alkyleneimine structural units having different groups on the amine group.

In the context of the present invention, the term “a polymer comprising a polyalkyleneimine” refers to any polymer comprising a polyalkyleneimine as defined herein. Said polymer may include 1 or more polyalkyleneimines, such as 1 , 2, 3, or more polyalkyleneimines. Said polymer may also be a star-shaped polymer, having multiple polyalkyleneimines arising from a single junction point. Said polymer may also be a graft copolymer having one or polyalkyleneimines grafted on a polymer. Said polymer may also be a cyclic polymer, such as a cyclic polyalkyleneimine.

In the context of the present invention, the term "gradient copolymer" herein refers to a statistical polymer that exhibits a gradual change in monomeric composition along the chain. This arrangement is different from random copolymers, which maintain a constant average composition along the chain, and block copolymers, which change abruptly along the chain. The statistical copolymers of the present invention can suitably be characterized by comparing the composition of structural units of different fragments of the copolymer. To this end the copolymer is divided in 3 equal fragments, i.e. a fragment adjacent to the initiator residue I (initiating fragment), a fragment adjacent to the terminating residue T (terminating fragment) and a central fragment that separates the initiating fragment and the terminating fragment. In case the total number of structural units is not a multiple of 3, the size of the central fragment is chosen such that the initiating fragment and the terminating fragment are of equal size. In the following table a few arithmetic examples are provided to illustrate this further.

While in random copolymers the composition of structural units of the initiating fragment and the terminating fragment are quite similar, in the case of gradient copolymers, the composition of structural units of these fragments are dissimilar.

In preferred embodiments of the invention, the polyalkyleneimine comprised in the polymer is a linear polyalkyleneimine. In preferred embodiments of the invention, the polymer comprising a polyalkyleneimine is the polyalkyleneimine, in particular a linear polyalkyleneimine.

In the context of the present invention, the terms “first alkyleneimine structural unit(s)” and “first structural unit(s)” are used synonymously.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises substituted first alkyleneimine structural units, wherein for each structural unit, the substituent on the amine group is independently selected from any substituted or unsubstituted alkyl-, alkenyl- or arylthiocarbonyl group, from any substituted or unsubstituted alkyl-, alkenyl- or arylsulfinylcarbonyl group, or from any substituted or unsubstituted alkyl-, alkenyl- or arylsulfonylcarbonyl group.

The alkyl-, alkenyl- or arylthiocarbonyl substituted alkyleneimine structural units may be represented by formula (II) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , Ri', R2, R2', R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

R_a is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

The alkyl-, alkenyl- or arylsulfinylcarbonyl substituted alkyleneimine structural units may be represented by formula (III) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , Rr, R2, R2’, R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

The alkyl-, alkenyl- or arylsulfonylcarbonyl substituted alkyleneimine structural units may be represented by formula (IV) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , R , R2, R2', R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises first structural units represented by formula (I), wherein each instance of X is independently selected from a direct bond, -CH2-, and -(CH2)2-.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises first alkyleneimine structural units represented by formula (la) wherein

X is selected from a direct bond, -CH2-, and -(CH2)z-;

R is selected from -C(O)SR_a, -C(O)S(O)R_a, and -C(O)S(O)2R_a; and

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a), comprises structural units represented by formula (I) or (la), wherein R_a is selected from -Ci-i2alkyl, -C2-i2alkenyl, -C2-i2alkynyl, and -Ar; wherein each of said -Ci-i2alkyl, -C2-i2alkenyl, and -C2-i2alkynyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted with one or more substituents selected from ethers, thioethers, esters, and amides.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a), comprises structural units represented by formula (I) or (la), wherein R_a is selected from -Ci-i2alkyl, -C2-i2alkenyl, -C2-i2alkynyl, and -Ar; wherein each of said -Ci-i2alkyl, -C2-i2alkenyl, and -C2-i2alkynyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted with one or more substituents selected from -OCi-i2alkyl, -SCi-i2alkyl, -C(O)OCi-i2alkyl, -OC(O)Ci-i2alkyl, - C(O)NHCi-i₂alkyl, and -NHC(O)Ci-i₂alkyl.

In embodiments of the invention, each instance of R_a is independently selected from -Ci ealkyl, in particular from -Ci-4alkyl. In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a), comprises structural units represented by formula (I) or (la), wherein R_a is selected from -Ci ealkyl , in particular from -Ci-4alkyl .

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises first structural units represented by formula (I) or (la), wherein each instance of R is -C(O)SR_a; and wherein X and R_a are as defined in any one of the embodiments as described herein.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises first structural units represented by formula (I), wherein each instance of R is C(O)S(O)2R_a; and wherein X and R_a are as defined in any one of the embodiments as described herein.

In step b) of the method of the invention, the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is reacted with an alkoxide in a solvent.

In the context of the present invention, the term “alkoxide” refers to a conjugate base of an alcohol that comprises an organic group bonded to a negatively charged oxygen atom and may be represented by Rb-O-. The alkoxide may be provided as the conjugate base directly, such as the sodium or potassium salt of the corresponding alcohol, or it may be prepared in situ by providing the alcohol and a strong base, such as sodium hydroxide or potassium hydroxide.

In embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, and wherein the alkoxide is provided as the conjugate base of the alcohol Rb-OH, such as the sodium or potassium salt, represented by Rb-O ⁺Na or Rb-O ⁺K, respectively.

In embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, and wherein the alkoxide is prepared in situ in step b), by providing the alcohol Rb-OH and a strong base, such as sodium hydroxide or potassium hydroxide.

Reaction of the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, provided in step a) with the alkoxide in step b) comprises reaction of at least part of the first structural units represented by formula (I), and as defined herein, with the alkoxide. This reaction leads to modification of the A/-thio-, sulfinyl- and/or sulfonylcarbonyl groups of said first structural units, thereby forming the corresponding A/-alkoxy- or aryloxycarbonyl alkyleneimine structural units.

The method as defined herein is not limited to any particular alkoxide. Rb may therefore be selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl. The alkoxide of step b) may also be a mixture of two or more different alkoxides. The two or more different alkoxides may be represented by Rb-O-, Rb -O-, and so forth, wherein R , Rb", and so forth are each independently selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

In embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

In the context of the current invention, the term “at least part of” refers to a definite, non-zero, portion of the item it refers to. The term “at least part of” may therefore refer to about 1 %, 2 %, 3 %, 5 %, 10 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 %, 70 %, 75 %, 80 %, 85 %, or substantially all, such as about 90 %, 95 %, 99 %, and even about 100 %, of the item it refers to.

The polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) therefore comprises A/-alkoxy- or aryloxycarbonyl alkyleneimine structural units.

The A/-alkoxy- or aryloxycarbonyl structural units may be represented by formula (V) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , Rr, R2, R2’, R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H; and

Rb is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

In embodiments, the present invention provides the method as defined herein, wherein the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises structural units represented by formula (V), wherein each instance of X is independently selected from a direct bond, -CH2-, and -(CH2)2-. In embodiments, the present invention provides the method as defined herein, wherein the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises structural units represented by formula (Va) wherein

X is selected from a direct bond, -CH2-, and -(CH2)z-; and

The solvent used in step b) of the method as described herein, can be any suitable solvent capable of dissolving the polymer provided in step a) and the alkoxide. It was found that tetrahydrofuran (THF) may be particularly suited to be used as the solvent in step b) of the method as described herein.

The method according to the present invention may further comprise a step c), wherein at least part of the A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units of the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, obtained in step b), are removed by reaction with a chemical reagent. When the A/-alkoxy- or aryloxycarbonyl substituents are removed from the amine in the alkyleneimine structural units, they provide the corresponding unsubstituted amine groups, i.e. -NH- groups, in the respective structural units.

In embodiments, the present invention provides the method as defined herein, wherein the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises A/-alkoxy- or aryloxycarbonyl alkyleneimine structural units, the method further comprising a step c), wherein at least part of the A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units are removed by reaction with a chemical reagent.

When the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, provided in step a) consists of first structural units, as defined herein, the method allows to obtain unsubstituted homopolymers such as polyethyleneimine, polypropyleneimine, polybutyleneimine, polypentyleneimine, and the like, after step b) and c).

When the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, provided in step a) consists of first structural units and second structural units, as defined herein, the method allows to obtain copolymers having unsubstituted and substituted alkyleneimine structural units, after step b) and c).

When the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, provided in step a) consists of a block copolymer of first structural units and second structural units, as defined herein, the method allows to obtain copolymers having at least one block of unsubstituted alkyleneimine structural units and at least one block of substituted alkyleneimine structural units, after step b) and c).

In particular, the method according to the invention allows to prepare block copolymers having at least one block of unsubstituted alkyleneimine structural units and at least one block of substituted alkyleneimine structural units, wherein the at least one block of unsubstituted alkyleneimine structural units is essentially free of substituted alkyleneimine structural units.

In particular, the method according to the invention allows to prepare block copolymers having at least one block of unsubstituted alkyleneimine structural units and at least one block of substituted alkyleneimine structural units, wherein the at least one block of substituted alkyleneimine structural units is essentially free of unsubstituted alkyleneimine structural units.

In the context of the present invention, “essentially free of” refers to the presence of at most 5 % of the item it refers to, preferably at most 3 %, more preferably at most 2 %, even more preferably at most 1 %, most preferably there is no presence of the item it refers to.

The A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units may for instance be removed by acidic or alkaline hydrolysis. The A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units may also be removed by hydrogenolysis. The A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units may also be removed by reaction with an amine reagent, such as piperidine, or piperazine.

The terms “acid hydrolysis” and “acidic hydrolysis” are generally known terms and refer to the chemical reaction in which a compound is broken down into its constituent molecules through the use of an acid (catalyst) and water. In the context of the present invention, these terms may in particular refer to the removal of A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units by reaction with an acid in the presence of water. The terms “base hydrolysis” and “alkaline hydrolysis” are generally known terms and refer to the chemical reaction in which a compound is broken down into its constituent molecules through the use of a base (catalyst) and water. In the context of the present invention, these terms may in particular refer to the removal of A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units by reaction with a base in the presence of water.

The term “hydrogenolysis” is a generally known term and refers to a chemical reaction in which a compound is broken down into its constituent molecules through the addition of hydrogen atoms. This process often involves the cleavage of chemical bonds with the concurrent addition of hydrogen in the presence of a metal catalyst. In the context of the present invention, this term may in particular refer to the removal of A/-alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units by reaction with Hz in the presence of a catalyst.

It was found that particular alkoxides used in step b), thereby providing the corresponding N- alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units, may allow for even milder removal of the A/-alkoxy- or aryloxycarbonyl substituents.

In embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is selected from -Ci-izalkyl, -Cz-izalkenyl, and -Ar; wherein each of said -Ci-izalkyl, and -Cz-izalkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally and independently substituted with one or more substituents selected from -halo, -Ci-izalkyl, -Ci- izalkenyl, -Ci-izalkynyl, -OCi-izalkyl, -SCi-izalkyl, -C(O)OCi-izalkyl, -OC(O)Ci-izalkyl, - C(O)NHCi-izalkyl, and -NHC(O)Ci-i₂alkyl.

In particular, it was found that an alkoxide derived from a tertiary alcohol, such as 2-methyl-2- propanol (tert-butanol), 2-methyl-2-butanol (tert-pentanol), 2-methyl-2-pentanol or 3-methyl-3- hexanol (tert-hexanol), may allow for even milder removal of the corresponding A/-tert- alkoxycarbonyl substituents by acidic hydrolysis, such as acidic hydrolysis at a temperature below 50 °C, preferably below 40 °C, more preferably below 30 °C, such as at about room temperature. In preferred embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is a tertiary alkyl group, in particular wherein Rb is selected from tert-butyl, tert-pentyl, and tert-hexyl.

It was also found, in particular, that an alkoxide derived from an arylmethylene alcohol, such as a substituted or unsubsituted benzyl alcohol, may allow for even easier removal of the corresponding A/-tert-alkoxycarbonyl substituents by hydrogenolysis. In particular embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O^_, wherein Rb is selected from -CH2-A , wherein AR is a 5- to 10-membered aromatic cycle optionally and independently comprising one or more heteroatoms selected from O, N and S and/or optionally substituted with substituents independently selected from -halo, - Ci-izalkyl, -Ci-i2alkenyl, -Ci-i2alkynyl, -OCi-i2alkyl, -SCi-i2alkyl, -C(O)OCi-i2alkyl, -OC(O)Ci- i₂alkyl, -C(O)NHCi-i₂alkyl, and -NHC(O)Ci-i₂alkyl.

It was further found, in particular, that an alkoxide derived from 9-fluorenemethanol may allow for even easier removal by reaction with an amine reagent. In particular embodiments, the present invention provides the method as defined herein, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is -CH2-9-fluorenyl.

In embodiments, the present invention provides the method as defined herein, wherein the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises structural units represented by formula (V) or (Va), wherein Rb is as defined in any one of the embodiments as described herein.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) can be prepared by cationic ring-opening polymerization (CROP) of cyclic iminoethers. Cationic ring-opening polymerization (CROP) of cyclic iminoethers, such as 2-oxazolines, 2-oxazines, and 2-oxazepines is known, and provides the corresponding polalkyleneimines, such as poly(2-oxazoline)s, poly(2-oxazine)s, and poly(2- oxazepine)s, respectively. Scheme 2 provides a graphic representation of the cationic ringopening polymerization (CROP) of cyclic iminoethers, wherein A represent a -Ci-i2alkylene-, R’ represent a side group, Z represents a residue from the initiator of the polymerization, and T represents a terminating residue resulting from the nucleophile used to terminate the polymerization.

Scheme 2

Any suitable initiator for cationic ring-opening polymerization may be used to prepare the polyalkyleneimine comprising structural units represented by formula (I) or (la), and as defined herein, including alkyl-, benzyl-, and allylhalides, and alkyl-, benzyl-, and allylsulfonates. Commonly used initiators are methyl tosylate, methyl iodide and methyl triflate, leading to a residue Z being -CH3. The initiator can also be an oligomer or polymer which is capable of initiating the cationic ring-opening polymerization, such as a polyethylene glycol (PEG) polymer, wherein the OH-end group is functionalised with a good leaving group, such as an iodo or p- nitrophenylsulfonyl group. In this case, Z would be a PEG oligomer or polymer residue.

Any suitable terminating agent for cationic ring-opening polymerization may be used to prepare the polyalkyleneimine comprising structural units represented by formula (I) or (la), and as defined herein, including carboxylates, thiolates, amines, hydroxide, and azide. A commonly used terminating agent used is hydroxide (OHj, leading to a T residue being -OH. Another commonly used terminating agent used is a piperidine, leading to a T residue being piperidinyl. Another commonly used terminating agent used is sodium azide, leading to a T residue being - N3. Depending on the terminating agent used, the polyalkyleneimine comprising structural units represented by formula (I), and as defined herein, and/or the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprising structural units represented by formula (II), and as defined herein, may be coupled with a further oligomer or polymer.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) can be prepared by cationic ring-opening polymerization (CROP) of cyclic iminoethers, wherein at least part of the cyclic iminoethers bears an alkyl-, alkenyl- or arylthiocarbonyl side group, such as represented by formula (VI) wherein

A is selected from -Ci-salkylene-; and

Examples of suitable cyclic iminoethers bearing an alkyl-, alkenyl- or arylthiocarbonyl side group include the following cyclic iminoethers, as represented by formula (Via), (Via’), (Via”), (Va’”), (Vlb), (Vlb’), (Vlb”), (Vlb’”), (Vlb^iv) , (Vlb^v), (Vic), (Vic’), (Vic”), (Vic’”), (Vlc^iv), or (Vlc^v)

wherein R_a is as defined in any one of the embodiments described herein.

In an embodiment, the present invention provides the method as defined herein, comprising the further step of preparing the polyalkyleneimine of step a) by cationic ring-opening polymerization of cyclic iminoethers, wherein at least part of the cyclic iminoethers is represented by formula

(VI) wherein

A is selected from -Ci-salkylene-; and

R_a is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl. In a particular embodiment, the present invention provides the method as defined herein, comprising the further step of preparing the polyalkyleneimine of step a) by cationic ring-opening polymerization of cyclic iminoethers, wherein at least part of the cyclic iminoethers is represented by formula (VI) wherein

A is selected from -Ci-salkylene-; and

Ra is selected from -Ci -1 zalkyl , in particular from branched -C3-1 salkyl , more in particular from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2-ethylbutyl, 2,3-dimethylbutyl, isoheptyl, 2- methylhexyl, 3-methylhexyl, isooctyl, 2-ethylhexyl, isodecyl, 2-propylheptyl, isododecyl, and 2-butyloctyl.

The alkyl-, alkenyl- or arylthiocarbonyl substituents on the amine group present in the resulting polyalkyleneimine may be oxidized, such as partially or substantially, to the corresponding alkyl- , alkenyl- or arylsulinfylcarbonyl substituents and/or the corresponding alkyl-, alkenyl- or arylsulonfylcarbonyl substituents. Oxidation is for instance known to readily proceed with mCPBA as reported by Swager et al. It was found that alkyl-, alkenyl- or arylsulonfylcarbonyl substituents allow reaction with the alkoxide in step b) at a lower temperature.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) may therefore comprise first alkylene structural units according to formula (II), (III), or (IV), as defined herein, or any combination of two or more thereof.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) may also comprise second alkyleneimine structural units other than first alkyleneimine structural units, such as represented by formula (I), or as represented by formula (II), (III) or (IV), and as defined herein. The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) may comprise second alkyleneimine structural units wherein the amine group bears any group other than alkyl-, alkenyl-, or arylthiocarbonyl, alkyl-, alkenyl-, or arylsulfinylcarbonyl, or alkyl-, alkenyl-, or arylsulfonylcarbonyl, such as hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylcarbonyl, alkoxycarbonyl, and alkylaminocarbonyl.

The presence of second structural units may be the result of the use of cyclic iminoether monomers other than bearing an alkyl-, alkenyl- or arylthiocarbonyl side group, such as represented by formula (VI), and as defined herein. The presence of second structural units may also or alternatively be the result of further modification of substituents on the amine groups present in the polyalkyleneimine after polymerization.

In the context of the present invention, the terms “second alkyleneimine structural unit(s)” and “second structural unit(s)” are used synonymously.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII) or (VII’) wherein n is an integer from 2 to 1000; m is 0, or an integer selected from 1 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1 , each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci-ealkyl ; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of X2 is independently selected from a direct bond, -CH2-, and -CR6R6 CH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -Ci-ealkyl ; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; each instance of Re is independently selected from -H, -Ci-i2alkyl, -Ci-i2alkenyl, -C(O)R_c, and -C(O)NRdRd'; each instance of R_c, and each instance of Rd is independently selected from -Ci-i2alkyl, -C2- izalkenyl, and -Arz; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of Rd' is independently selected from -H, -Ci-i2alkyl, -C2-i2alkenyl, and -Ars; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally and independently substituted; and each instance of An , each instance of Ar2, and each instance of Ars is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

In this embodiment, the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) comprises from 2 to 1000 first structural units as defined in any one of the embodiments as described herein. Said 2 to 1000 first structural units form a first oligomer or polymer part.

In this embodiment, the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) may further comprise 1 to 1000 second structural units as defined in any of the embodiments described herein. Said 1 to 1000 second structural units, if present, form a second oligomer or polymer part.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a), may contain any suitable number of first structural units as defined herein, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, and even 1000 first structural units. In useful embodiments, the present invention provides the method as defined herein, wherein n is an integer from 2 to 1000, preferably from 2 to 500, more preferably from 50 to 500, even more preferably from 100 to 500, yet even more preferably n is an integer from 200 to 500. In further useful embodiments, the present invention provides the method as defined herein, wherein n is an integer from 2 to 1000, preferably from 25 to 700, more preferably from 50 to 500, even more preferably from 150 to 400, yet even more preferably n is an integer from 200 to 300.

The polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a), may further contain any suitable number of second structural units as defined herein, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, and even 1000 second structural units. In useful embodiments, the present invention provides the method as defined herein, wherein m is 0. In useful embodiments, the present invention provides the method as defined herein, wherein m is an integer from 1 to 1000, preferably from 1 to 500, more preferably from 50 to 500, even more preferably from 100 to 500, yet even more preferably m is an integer from 200 to 500. In further useful embodiments, the present invention provides the method as defined herein, wherein m is an integer from 2 to 1000, preferably from 25 to 700, more preferably from 50 to 500, even more preferably from 150 to 400, yet even more preferably n is an integer from 200 to 300.

In the context of the present invention, the terms “degree of polymerization” or “DP” refer to the number-averaged degree of polymerization. It can be calculated using the equation: M_n/Mo, where M_n is the number-averaged molecular weight of the polymer and Mo is the molecular weight of the structural unit.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII) or (VII’), wherein each instance of Xi and each instance of X2 is independently selected from a direct bond, -CH2-, and -(CH₂)₂-.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (Vila) or (Vila’) wherein n is an integer from 2 to 1000; m is 0, or an integer selected from 1 to 1000; each instance of Xi and each instance of X2 is independently selected from a direct bond, - CH2-, and -(CH₂)₂-; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of Rs is independently selected from -H, -Ci-i2alkyl, -Ci-i2alkenyl, -C(O)R_c, and -C(O)NRdRd'; each instance of R_c, and each instance of Rd is independently selected from -Ci-i2alkyl, -C2- izalkenyl, and -Arz; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of Rd' is independently selected from -H, -Ci-i2alkyl, -C2-i2alkenyl, and -Ars; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally and independently substituted; and each instance of An , each instance of Ar2, and each instance of Ars is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

While in the formulae described herein, such as formula (VII), (VII’), (Vila) or (Vila’), the first oligomer or polymer part and the second oligomer or polymer part may be represented as blocks, the invention is not limited thereto. In other words, first structural units, which may be the same or different, and second structural units, if present, which may be the same or different, may be present as any random, alternating, statistical, gradient, or block copolymer.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine is represented by formula (VII), (VII’), (Vila) or (Vila’), wherein R_c is independently selected from -Ci-i2alkyl , in particular from -Ci ealkyl , more in particular from -Ci- 4alkyl.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII), (VII’), (Vila) or (Vila’), and comprises first structural units and second structural units, wherein the first structural units and the second structural units are present as a block copolymer.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII), (VII’), (Vila) or (Vila’), and comprises first structural units and second structural units, wherein the first and second structural units are present as a gradient copolymer.

In these embodiments, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is a block copolymer or a gradient copolymer, n is preferably an integer from 10 to 1000, more preferably from 20 to 500, even more preferably from 30 to 500, yet even more preferably from 50 to 500, yet even more preferably n is an integer from 100 to 500. Similarly, m is preferably an integer from 10 to 1000, more preferably from 20 to 500, even more preferably from 30 to 500, yet even more preferably from 50 to 500, yet even more preferably m is an integer from 100 to 500.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII), (VII’), (Vila) or (Vila’), and comprises first structural units and second structural units, wherein the first structural units and the second structural units are present as a gradient copolymer, wherein the gradient copolymer consists of an initiating fragment, a central fragment, and a terminating fragment, wherein the ratio of first and second structural units in the initiating fragment is at least 2 times higher or lower than the same ratio in the terminating fragment of the copolymer. Preferably, the ratio of first and second structural units in the initiating fragment is at least 3 times higher or lower than the same ratio in the terminating fragment of the copolymer, more preferably at least 5 times, even more preferably at most 20 times higher or lower.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII), (VII’), (Vila) or (Vila’), wherein each instance of R? is independently selected from -C(O)S-Ci-6alkyl, -C(O)S(O)-Ci- ealkyl, and -C(O)S(O)2-Ci-6alkyl; in particular wherein each instance of R? is independently selected from -C(O)S-Ci-4alkyl, -C(O)S(O)-Ci-4alkyl, and -C(O)S(O)2-Ci-4alkyl.

In embodiments, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (VII), (VII’), (Vila) or (Vila’), wherein Xi and X2 are the same, in essence wherein Xi and X2 are both a direct bond, -(CH2)-, or -(CH₂)₂-.

In a particular embodiment, the present invention provides the method as defined herein, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (Vila) or (Vila’) wherein

Rs is -C(O)R_c; and

Re is independently selected from -Ci-i2alkyl, in particular from -Ci-ealkyl, more in particular from -Ci-4alkyl; and

Xi , X2, R7, Ra, n, and m are as defined in any one of the embodiments described herein.

In a further aspect, the present invention provides the polymers, in particular the polyalkyleneimines, obtainable by the method, as defined herein.

In a further aspect, the present invention provides a polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, wherein the polyalkyleneimine is a gradient copolymer or a block copolymer represented by formula (VIII) or (VIII’) wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1 , each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci-ealkyl ; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of X2 is independently selected from a direct bond, -CH2-, and -CR6R6 CH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -C1- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H;

Rc is selected from -Ci -12alkyl , -C2-i2alkenyl, and -Ar2; wherein each of said -Ci -12alkyl , and - C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; and each instance of Ar2 is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is represented by formula (VIII) or (VIII’), and wherein each instance of Xi and each instance of X2 is independently selected from a direct bond, -CH2-, and -(CH2)2-. In preferred embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, wherein the polyalkyleneimine is a gradient copolymer or a block copolymer represented by formula (Villa) or (Villa’) wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi and each instance of X₂ is independently selected from a direct bond, - CH2-, and -(CH₂)₂-;

Rc is selected from -Ci -1 salkyl , -C2-i2alkenyl, and -Ar₂; wherein each of said -Ci -1 salkyl , and - C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; and each instance of Ar₂ is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, having a dispersity of at most 1 .9, as determined by size exclusion chromatography, preferably at most 1 .7, more preferably at most 1 .6, even more preferably at most 1 .5, yet even more preferably from 1 .0 to 1 .4.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein R_c is selected from -Ci-i2alkyl, in particular from -Ci ealkyl, more in particular from -Ci-4alkyl.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein n is an integer from 10 to 1000, preferably from 20 to 500, more preferably from 30 to 500, even more preferably from 50 to 500, yet even more preferably n is an integer from 100 to 500.

In particular embodiments, the present invention provides the polyalkyleneimine as defined herein, and represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein n is an integer from 100 to 400, preferably from 150 to 350, more preferably n is an integer from 200 to 300.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein m is an integer from 10 to 1000, preferably from 20 to 500, more preferably from 30 to 500, even more preferably from 50 to 500, yet even more preferably n is an integer from 100 to 500.

In particular embodiments, the present invention provides the polyalkyleneimine as defined herein, and represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein m is an integer from 10 to 200, preferably from 15 to 150, more preferably m is an integer from 20 to 100, even more preferably from 25 to 50.

In specific embodiments, the present invention provides the polyalkyleneimine as defined herein, and represented by formula (VIII), (VIII’), (Villa) or (Villa’), wherein n is an integer from 100 to 400, preferably from 150 to 350, more preferably n is an integer from 200 to 300; and wherein m is an integer from 10 to 200, preferably from 15 to 150, more preferably m is an integer from 20 to 100, even more preferably from 25 to 50.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein R_c is -CH3 or -CH2CH3.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is a gradient copolymer.

In particular, when the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, is a gradient copolymer, the gradient copolymer consists of an initiating fragment, a central fragment, and a terminating fragment, wherein the ratio of unsubstituted alkyleneimine structural units (-NH-) and substituted alkyleneimine structural units (-NC(O)Rc-) in the initiating fragment is at least 2 times higher or lower than the same ratio in the terminating fragment of the copolymer. Preferably, the ratio of unsubstituted and substituted alkyleneimine structural units in the initiating fragment is at least 3 times higher or lower than the same ratio in the terminating fragment of the copolymer, more preferably at least 5 times, even more preferably at most 20 times higher or lower.

In embodiments, the present invention provides the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, wherein the polyalkyleneimine is a block copolymer.

In particular, when the polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, as defined herein, is a block copolymer, it then has at least one block of unsubstituted alkyleneimine structural units (-NH-) and at least one block of substituted alkyleneimine structural units (-NC(O)Rc-), wherein the at least one block of unsubstituted alkyleneimine structural units is essentially free of substituted alkyleneimine structural units and/or wherein the at least one block of substituted alkyleneimine structural units is essentially free of unsubstituted alkyleneimine structural units.

The block copolymer as described herein can have multiple blocks of unsubstituted alkyleneimine structural units (-NH-) and/or multiple blocks of substituted alkyleneimine structural units (-NC(O)Rc-). Preferably, when there are multiple blocks of unsubstituted alkyleneimine structural units (-NH-), then all of them are essentially free of substituted alkyleneimine structural units (-NC(O)Rc-). Preferably, when there are multiple blocks of substituted alkyleneimine structural units (-NC(O)Rc-), then all of them are essentially free of unsubstituted alkyleneimine structural units (-NH-).

While in the formulae described herein, such as formula (VII), (VII’), (Vila), (Vila’), (VIII), (VIII’), (Villa) or (Villa’), the polyalkyleneimine is represented without an initiating residue and a terminating residue, the invention is not intended to be limited thereto. Either one of formula (VII), (VII’), (Vila), (Vila’), (VIII), (VIII’), (Villa) or (Villa’) may therefore be replaced by formula wherein Z and T are defined as described hereinbefore.

In a further aspect, the present invention provides a delivery system comprising a polymer, in particular a copolymer, as defined herein, and a therapeutic agent that is non-covalently bound to said polymer. The therapeutic agent can be a pharmaceutically active agent or a biomolecule, such as a gene, a peptide, a protein or a nucleic acid.

A “pharmaceutically active agent” in the context of the invention may include chemotherapeutic agents, hydrophobic drugs, and/or small molecule drugs.

A “nucleic acid” in the context of the invention may include deoxyribonucleic acid, ribonucleic acid, recombinantly produced and chemically synthesized molecules. In particular a nucleic acid may include DNA, genomic DNA, cDNA, RNA, tRNA, mRNA, small interfering RNA (siRNA), micro RNA (miRNA), agRNA, smRNA, antisense oligonucleotides, ribozymes, plasmids, immune stimulating nucleic acids, antisense nucleic acids, antagomirs (anti-miRs), miRs, supermiRs, U1 adaptors, and aptamers.

A nucleic acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic acid can be employed for introduction into, i.e. transfection of cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and/or polyadenylation.

In the context of the present invention, the term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a p- D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. Nucleic acids may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial or analogs of naturally-occurring RNA.

Preferably, the therapeutic agent has a charge at a predetermined pH in the range of 6.5 to 8, and the polymer, in particular the copolymer, as defined herein, has an opposite charge than the therapeutic agent at a predetermined pH, thereby enabling an electrostatic bond being formed between the therapeutic agent and the (co)polymer at the predetermined pH. Preferably, the therapeutic agent has a negative charge at the aforementioned predetermined pH, whereas the (co)polymer has a positive charge at the same predetermined pH.

It was found that non-covalent binding of the therapeutic agent to the polymer, in particular the copolymer, as defined herein, may enable efficient transfer of the therapeutic agent into cells with low cytotoxicity.

In an embodiment, the present invention provides the delivery system, as defined herein, wherein the therapeutic agent is selected from a gene, DNA, RNA, siRNA, miRNA, isRNA, agRNA and smRNA.

The present invention also provides the polymers, in particular the polyalkyleneimines, and the delivery systems, as defined herein, for use in human or veterinary medicine. The use of the polymers, in particular the polyalkyleneimines, and the delivery systems, according to this invention for human or veterinary medicine is also intended. In addition, the invention provides a method for the prophylaxis and treatment of human and veterinary disorders, by administering the polymers, in particular the polyalkyleneimines, and the delivery systems, as defined herein, to a subject in need thereof. The invention also provides the polymers, in particular the polyalkyleneimines, and the delivery systems, for use in the preparation of a medicament.

The delivery system, as defined herein, may be suitably used in therapeutic treatment, said treatment preferably comprising parenteral administration of the delivery system.

According to yet a further aspect, the present invention provides a method for preparing a polyalkyleneimine, the method comprising the step of performing cationic ring-opening polymerization of cyclic iminoethers, wherein at least part of the cyclic iminoethers is represented by formula (VI) wherein

A is selected from -Ci-salkylene-; and

Ra is selected from any substituted or unsubstituted branched alkyl.

It was found that the cationic ring-opening polymerization of cyclic iminoethers having more sterically demanding side chains, such as isopropylthio side chains (Ra = iPr), may lead to polyalkyleneimines with a higher average molecular weight and/or a lower dispersity. Without willing to be bound to be bound by any theory, it is believed that the bulkier side chains more efficiently suppress chain transfer side reaction during cationic ring-opening polymerization, compared to an ethyl side group.

The method for preparing a polyalkyleneimine as defined herein is not limited to particular alkylthio side chains, as long as the alkyl group is more sterically demanding than an ethyl group.

In an embodiment, the present invention provides the method for preparing a polyalkyleneimine as defined herein, wherein at least part of the cyclic iminoethers is represented by formula (VI) wherein

A is selected from -Ci-salkylene-; and

R_a is selected from any branched -Cs-izalkyl.

A is selected from -Ci-salkylene-; and R_a is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2-ethylbutyl, 2,3-dimethylbutyl, isoheptyl, 2-methylhexyl, 3-methylhexyl, isooctyl, 2-ethylhexyl, isodecyl, 2-propylheptyl, isododecyl, and 2-butyloctyl.

In a particular embodiment, the present invention provides the method for preparing a polyalkyleneimine as defined herein, wherein at least part of the cyclic iminoethers is represented by formula (Via), (Via’), (Via”), (Va’”), (Vlb), (Vlb’), (Vlb”), (Vlb’”), (Vlb^iv), (Vlb^v), (Vic), (Vic’), (Vic”), (Vic’”), (Vlc^iv), or (Vlc^v) wherein R_a is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2-ethylbutyl, 2,3- dimethylbutyl, isoheptyl, 2-methylhexyl, 3-methylhexyl, isooctyl, 2-ethylhexyl, isodecyl, 2- propylheptyl, isododecyl, and 2-butyloctyl.

In yet a further aspect, the present invention provides a polyalkyleneimine bearing alkylthiocarbonyl side groups, and represented by formula (IX) wherein n is an integer from 2 to 1000, preferably from 25 to 700, more preferably from 50 to 500, even more preferably from 150 to 400, yet even more preferably from 200 to 300;

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1 , Ri', R2, R2’, R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

R_a is selected from any substituted or unsubstituted branched alkyl.

It was found that the presence of the isopropyl side-chain substituent (iPrSOx and iPrSOzi) enables the preparation of much more defined higher molar mass polymers via cationic ringopening polymerization compared to polymerization of the monomers with ethyl side-chains (EtSOx and EtSOzi). This is illustrated by the size exclusion chromatography traces of the obtained polymers when aiming for a degree of polymerization of 250 (Fig. 13 and Fig. 14). Especially for therapeutic applications, having access to defined polymers with narrow molar mass distribution is advantageous for regulatory purposes, while for gene delivery polyalkyleneimines with higher molar mass prove to be more effective.

Preferably, R_a is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tertpentyl, isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2-ethylbutyl, 2,3- dimethylbutyl, isoheptyl, 2-methylhexyl, 3-methylhexyl, isooctyl, 2-ethylhexyl, isodecyl, 2- propylheptyl, isododecyl, and 2-butyloctyl.

The compounds of the present invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention.

EXAMPLES Materials

All the chemicals were obtained from commercial sources and used as received, unless stated otherwise. All solvents used, such as methanol, THF, DCM, acetone, hexane, diethyl ether and ethanol were purchased from Chem-Lab. Acetonitrile was dried in a solvent purification system (Pure Solv EN, Innovative Technology) before use as a polymerization solvent. Triethylamine (99 %), carbon disulfide (99.9 %), hydrogen peroxide (30 % w/v), potassium hydroxide and sodium ethoxide (21 % in ethanol) were purchased from Fisher Scientific®. The deuterated solvents were obtained from Eurisotop. 2-Ethyl-2-oxazoline (EtOx) was kindly donated by Polymer Chemistry Innovations and distilled over BaO before use. 4-Amino-1 -butanol was purchased form Aaron Chemicals. All other chemicals, including 3-aminopropan-1 -ol, magnesium sulfate, iodoethane (copper stabilized), 2-iodopropane (copper stabilized), 1 -iodo-

2.2-dimethylpropane, trifluoroacetic acid, 1 ,1 '-carbonyldiimidazole, methyl p-toluenesulfonate, cesium carbonate, meta-chloroperoxybenzoic acid, Lawesson reagent, methyl trifluoromethanesulfonate (TfOMe), sodium and benzophenone were purchased from Sigma- Aldrich. Methyl p-toluenesulfonate (TsOMe) was distilled over barium before use.

All manipulations concerning the preparation of polymerization mixtures until capping of the microwave vails were carried out in a VIGOR Sci-Lab SG 1200/750 Glovebox System with a water concentration of < 0.1 ppm. For the polymerizations, a Biotage Initiator Microwave System with Robot Sixty autosampler was used. During polymerization the microwave synthesizer operated at a constant set temperature of 140 °C, which was monitored by an IR-sensor. Some reactions were monitored via thin layer chromatography (TLC), using SIL G25 UV254 TLC plates with a silica layer of 0.25 mm thickness. TLC plates were developed using a potassium permanganate solution (potassium permanganate (3 g) + potassium carbonate (20 g) + 5% aqueous, NaOH (5 ml) + water (300 ml)).

2-(Ethylthio)-4,5-dihydrooxazole (EtSOx) was prepared according to a known procedure (Swager et al. 2019).

1 .3-Oxazinane-2-thione was prepared according to a known procedure (Li, G. et al. 1997).

2-(Ethylthio)-5,6-dihydro-4H-1 ,3-oxazine (EtSOzi) was prepared according to the following procedure: 1 ,3-Oxazinane-2-thione (20 g, 176.6 mmol, 1 eq) was dissolved in 600 ml ethanol in a flask. Subsequently sodium ethoxide (12.2 w/w% in ethanol, 100ml 1 .05 eq) and iodoethane (265.0 mmol, 21 .3 ml, 1 .5 eq) were added to the reaction mixture. The flask was shielded from light and left to stir for one hour at room temperature. Next, the reaction mixture was refluxed for one hour. Afterwards, the solution was concentrated under reduced pressure. Saturated NaHCOs solution was added and then the mixture was extracted three times with DCM. The organic phases were combined, washed two times with brine and dried over MgSO4. Following, DCM was evaporated to give a light orange-brown oil. The obtained crude was distilled (0.2 mbar, 55 °C) two times, first over BaO and ninhydrin and secondly over metallic sodium and benzophenone. In the end pure 2-ethylthio-2-oxazine was obtained as a colorless liquid. (15 g, 58.3 %) The product was stored under Argon. ¹HNMR (300 MHz, CDCh): 6 = 4.21 (2H, t, OCH2), 3.44 (2H, t, NCH2), 2.81 (2H, q, CH3CH2), 1 .92 (2H, m, CH2CH2CH2), 1 .26 (3H, t, CH3CH2).

¹³CNMR (300 MHz, CDCIs): 5 = 157.20 (NCO), 66.66 (OCH2), 43.40 (NCH2), 24.90 (CH3CH2), 22.31 (CH2CH2CH2), 14.67 (CH3CH2). FT-IR: v (cm ¹) = 2931 -2861 (m), 1639 (s), 1472-1439 (m),1330 (s), 1 1 16 (s). HRMS: m/z = 146.0639 (M+H⁺).

1 .3-Oxazepan-2-one was prepared according to the following procedure: To a solution of 4- amino-1 -butanol (26.7g, 0.3 mol, 1 .0 eq) in anhydrous THF 1 .2L was 1 ,1 ’-carbonyldiimidazole (53.4 g, 0.33 mol, 1 .1 eq) added under argon atmosphere. The resulting mixture was stirred for 2h at room temperature and consequently refluxed for 3 days. The solvent was evaporated in vacuo and dichloromethane 350 ml was added. The mixture was moved into a separation funnel and washed with 1 M aqueous hydrochloric acid solution (3x 40ml) and brine (1 x 80ml). The organic phase was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The crude yellow oil was purified by column chromatography (eluent: Ethyl acetate ; Rf = 0.3) yielding a crystalline white solid (10.5g, 30% yield). ¹H NMR (300 MHz, CDCh) 6 5.51 (1 H, s (br.), NH), 4.13 (2H, m, -CH2-O), 3.17 ( 2H, m, -CH2-N), 1 .89 (2H, m, CH2-CH2-O), 1 .74 (2H, m, CH2-CH2-N). ¹³C NMR (300 MHz, CDCh) 5 162.1 (Cq=O), 70.39 (CH2-O) , 42.75 (CH2-N), 29.40 5(CH₂-CH₂-O), 27.27 (CH2-CH2-N). HRMS: m/z = 1 16,16 (M+H⁺).

1 .3-Oxazepane-2-thione was prepared according to the following procedure: To a solution of

1 .3-oxazepan-2-one (10.5g, 0.091 mol, 1 eq) in 2L DCM was Lawesson’s reagent (18.45g, 0.045 mol, 0.5eq) added. The suspension was stirred at room temperature for 5 days. Upon completion the solvent was removed in vacuo and the crude oil was purified by column chromatography (eluent: 70% Hexane 30% Ethyl acetate; rf = 0.14) yielding a crystalline white solid (7.5g, 63% yield) .¹ H NMR (300 MHz, CDCh) 6 7.84 (1 H,s (br), NH), 4.38 - 4.19 (2H, m, CH2-O), 3.47 - 3.18 (2H, m, CH2-N), 2.01 - 1 .92 (2H, m, CH2-CH2-O). 1 .79-1 .70 (2H, m, CH2-CH2-N). ¹³C NMR (300 MHz, CDCh) 5197.86 (Cq=S), 74.43 (CH2-O), 46.38 (CH2-N), 29.03 (CH2-CH2-O), 25.82 (CH2- CH2-N). HRMS: m/z = 132,01 (M+H⁺).

2-(Ethylthio)-4,5,6,7-tetrahydro-1 ,3-oxazepine (EtSOpi) was prepared according to the following procedure: To a solution of 1 ,3-Oxazepane-2-thione (5.6g, 0.042mol, 1 eq) in 1 .1 L acetone was CS2CO3 (13.77g, 0.042mol, 1 eq) added. The flask was covered with aluminum foil and ethyl iodide (6.55g, 0.042mol, 1 eq) was added dropwise to the suspension. The mixture was refluxed for 4 days. Thereafter the reaction mixture was filtered over celite and the solvent was removed in vacuo. The mixture was redissolved in DCM and filtered again over celite. The solvent was removed in vacuo and the obtained crude product was purified twice with a static vacuum distillation (40°C; 1 mbar). The first distillation was done over barium oxide, the second distillation over a benzophenone ketyl/sodium dispersion to remove traces of diacetone alcohol and to completely dry the product. The product (5.68g, 0.036mol, 85% yield) was stored under argon. ¹H NMR (300 MHz, CDCI₃) 6 4.08 - 3.81 (2H, m, CH2-O), 3.65 - 3.36 ( 2H, m, CH2-N), 2.78 (2H, dd, J = 14.9, 7.4 Hz, S-CH2-CH3), 1 .99 - 1 .79 (2H, m, CH2-CH2-O). 1 .74 - 1 .59 (2H, m, CH2-CH2-N), 1 .27 ( 3H, td, J = 7.4, 0.6 Hz, S-CH2-CH3). ¹³C NMR (300 MHz, CDCI3) 6 162.64 (N=Cq), 71 .87 (CH2-O), 50.19 (CH2-N) 30.59 (CH2-CH2-O), 26.71 (CH3-CH2-S), 26.64 (CH₂- CH2-N) 14.33 (CH3-CH2-S). HRMS: m/z = 160,09 (M+H⁺).

2-(lsopropylthio)-4,5-dihydrooxazole (iPrSOx) was prepared according to the following procedure: To a solution of oxazolidine-2-thione (8.5 g, 82.4 mmol, 1 eq.) in 100 mL acetone, CS2CO3 (26.86 g, 82.4 mmol, 1 eq.) was added. The flask was covered with aluminium foil and 2-iodopropane (13.98 g, 82.4 mmol, 1 eq.) was added dropwise to the suspension. The mixture was refluxed for 1 day. Thereafter the reaction mixture was filtered over celite and the solvent was removed in vacuo. The mixture was redissolved in DCM and filtered again over celite. The solvent was removed in vacuo and the obtained crude product was purified twice with a static vacuum distillation (57°C; 10 mbar). The first distillation was done over barium oxide, the second distillation over a benzophenone ketyl/sodium dispersion to remove traces of diacetone alcohol and to completely dry the product. The product (7.85g, 66% yield) was stored under argon. ¹H NMR (400 MHz, CDCI3) 6 4.30 (t, J = 9.1 Hz, 2H, O-CHg ), 3.87 (t, J = 9.2 Hz, 2H, N-CHg). 3.63 (h, J = 6.8 Hz, 1 H, S-CH), 1 .37 (d, J = 6.8 Hz, 6H, S-CH-fCHg ). ¹³C NMR (101 MHz, CDCI3) 6 165.80 (N=Cq), 68.61 (CH2-O), 54.93 (CH2-N), 37.42 (S-CH), 23.42 (S-CH-(CH₃)₂). FT-IR: v (cm ¹) = 2968-2864 (m), 1606 (s), 1 130 (s)

2-(lsopropylthio)-5,6-dihydro-4H-1 ,3-oxazine (iPrSOzi) was prepared according to the following procedure: To a solution of 1 ,3-oxazine-2-thione (2 g, 17.1 mmol, 1 eq.) in 30 mL acetone, CS2CO3 (5.57 g, 17.1 mmol, 1 eq.) was added. The flask was covered with aluminium foil and 2- iodopropane (1 eq, 17.1 mmol, 2.9 g) was added dropwise to the suspension. The mixture was refluxed for 1 day. Thereafter the reaction mixture was filtered over celite and the solvent was removed in vacuo. The mixture was redissolved in DCM and filtered again over celite. The solvent was removed in vacuo and the obtained crude product was purified twice with a static vacuum distillation (50°C; 1 mbar). The first distillation was done over barium oxide, the second distillation over a benzophenone ketyl/sodium dispersion to remove traces of diacetone alcohol and to completely dry the product. The product (1 .4g, 55% yield) was stored under argon. ¹H NMR (400 MHz, CDCI3) 6 4.21 (t, J = 5.4 Hz, 2H, O-CHg). 3.57 - 3.41 (m, 3H, N-CHg + S-CH), 1 .93 (p, J = 5.8 Hz, 2H, CH2-CH2-CH2), 1 .30 (d, J = 6.9, 6H, S-CH-(CH3)₂). ¹³C NMR (101 MHz, CDCI3) 6 157.64 (N=Cq), 66.75 (CH2-O), 43.75 (CH2-N), 35.79 (S-CH), 23.26 (S-CH-(CH₃)₂), 22.59 (CH2-CH2-CH2). FT-IR: v (cm ¹) = 2968-2864 (m), 1636 (s), 1 1 14 (s) 2-(neopentylthio)-5,6-dihydro-4H-1 ,3-oxazine (neoPnSOzi) was prepared according to the following procedure: To a solution of 1 ,3-oxazine-2-thione (2.37 g, 20.2 mmol, 1 eq.) in 30 mL acetonitrile, CS2CO3 (6.59 g, 20.2 mmol, 1 eq.) was added. The flask was covered with aluminium foil and 1 -iodo-2,2-dimethylpropane (4 g, 20.2 mmol, 1 eq.) was added to the suspension. The mixture was refluxed for 5 days. Thereafter the reaction mixture was filtered over celite and the solvent was removed in vacuo. The mixture was redissolved in acetonitrile and carefully decanted separating the solution from the solid CS2CO3. The solvent was removed in vacuo and the obtained crude product was purified with a static vacuum distillation over BaO. The product (2.2 g, 1 1 .76 mmol, 58%) was stored in the glove box under nitrogen atmosphere. ¹H NMR (400 MHz, CDCI3) 6 4.21 (t, J = 5.4 Hz, 2H, O-CH₂). 3.43 (t, J = 6.0 Hz, 2H, N-CH₂). 2.83 (s, 2H, S-CH2). 1 .94 - 1 ,89(m, 2H), 0.98 (s, 9H). FT-IR: v (cm ¹) = 2954-2862 (m), 1642 (s), 1474-1464 (m), 1363 (s), 1330 (s), 1226 (s), 1 1 14 (s), 1050 (s), 802 (s). GCMS: m/z = 187.05

Analysis

Proton Nuclear Magnetic Resonance (¹HNMR) spectra were recorded on a Bruker 300 or 400 MHz NMR-spectrometer. All chemical shifts (5) are given relative to the solvent peak of the deuterated solvent used to record the spectra.

Hydrolysis in general, and where applicable, selectivity of the hydrolysis, was determined using quantitative ¹H NMR spectroscopy, with PEtOx as internal standard.

Fourier-transform infrared spectroscopy (FT-IR) were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer and were reported in wavenumber (cm^-1).

High resolution mass spectrometry (HRMS) measurements were performed on an Agilent high 1 100 HPLC with quaternary pump and UV-DAD detection, coupled to an Agilent 6220A TOFMSD with ESI/APCI (multimode) ionization source. A methanol/5 mM NH4OAc in H2O (50:50 v:v) mobile phase was used at a flow rate of 300 pl/min.

Size exclusion chromatography in dimethylacetamide (SEC DMA) was performed on an Agilent 1260-series HPLC system equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a thermostatted column compartment (TCC) at 50 °C equipped with two Plgel 5 pm mixed-D columns and a precolumn in series, a 1260 diode array detector (DAD) and a 1260 refractive index detector (RID). The used eluent was DMA containing 50mM of LiCI at a flow rate of 0.500 ml/min. The spectra were analyzed using the Agilent Chemstation software with the GPC add-on. Molar mass values and £> values were calculated against PMMA standards from PSS.

General procedure A - Cationic ring-opening polymerization A solution containing a total monomer concentration of 1 M was prepared by mixing cyclic iminoether monomer(s), initiator (methyl tosylate (TsOMe)) and solvent (dry acetonitrile (MeCN)) in a Biotage vial containing a stir bar. The mixture was heated up to 140°C in a microwave, and upon completion, the polymer was terminated with the addition of a solution of KOH in MeOH (1 M, 2.5 eq. relative to TsOMe). Purification of the polymer was done through precipitation in cold hexane, diethyl ether or a mixture of the two, followed by overnight under vacuum.

General procedure B - Oxidation

The polymer was dissolved in dichloromethane (DCM) and cooled in an ice bath. A solution of meta-chloroperoxybenzoic acid (mCPBA, 2.1 eq.) in DCM was prepared and added dropwise to the polymer solution, after which the mixture was stirred overnight at room temperature. The polymer was purified by precipitating in cold hexane, diethyl ether or a mixture of the two, and dried overnight under vacuum.

General procedure C - Reaction with the alkoxide

The polymer was dissolved in THF. A solution of a potassium or sodium alkoxide (2.1 eq.) in THF was prepared and added to the polymer solution, after which the mixture was stirred overnight at room temperature or reflux. Purification of the polymer was done by precipitating in cold hexane, diethyl ether, or a mixture of the two, followed by dialysis against water. Subsequently, the polymer was recovered by lyophilization.

Example 1

A polymer was prepared according to General procedure A, using 2-(ethylthio)-5,6-dihydro-4H- 1 ,3-oxazine (EtSOzi) (1 .9 ml, 0.015 mol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 50. This was achieved by adding (0.045 mL, 0.0003 mol, 0.02 eq.) of TsOMe as an initiator. The polymer was precipitated in a cold mixture of diethyl ether and hexane in a 1 :1 ratio. Subsequently, the obtained polymer was reacted with sodium methoxide at reflux according to General procedure C, thereby providing a polymer having methoxycarbonyl substituents on the amine groups of the polyalkyleneimine. The following scheme provides a representation of the reaction according to this example.

The ¹H NMR spectra (both recorded in chloroform-d), and the FT-IR spectra, of the polymers obtained after General Procedure A and General Procedure C, were superimposed, as shown in Figure 1A and Figure 1 B, respectively. The top spectrum corresponds to poly(2-ethylthio-2- oxazine) (PEtsOzi) and the bottom spectrum depicts poly(2-methoxy-2-oxazine). The modification may be supported by the disappearance of ethylthio group peaks in the ¹H NMR spectra (FIG. 1 A), shifting of the backbone and the appearance of the 2-methoxy peak at 3.66 ppm.

Further confirmation of the modification may be provided by the carbonyl of PEt-S-Ozi (top) in the FT-IR spectrum (FIG. 1 B), having a wavenumber of 1635 cm ¹ corresponding to the thiocarbamate. After the reaction with sodium methoxide (bottom) a carbamate is formed for which the carbonyl shows a characteristic wavenumber at 1688 cm ¹.

Example 2

A polymer was prepared according to General Procedure A, utilizing 2-(ethylthio)-4,5- dihydrooxazole (EtSOx) (0.083 ml, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 100. This was achieved by adding (0.001 mL, 0.007 mmol, 0.01 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether. Subsequently, oxidation was carried out following General Procedure B, and finally General Procedure C was employed, utilizing potassium tert-butoxide as the alkoxide at room temperature. The following scheme provides a representation of the reaction according to this example.

The ¹H NMR spectra after each of General Procedures A, B, and C were superimposed, resulting in Figure 2. The top spectrum (recorded in chloroform-d) corresponds to poly(2- ethylthio-2-oxazoline) (PEtSOx), the middle spectrum (recorded in chloroform-d) represents oxidized PEtSOx, and the bottom spectrum (recorded in deuterium oxide (D2O)) depicts poly(2- tert-butoxy-2-oxazoline). Confirmation of removal of the sulfonyl group and introduction of a tertbutoxy group, leading to a tert-butoxycarbonyl (BOC) group, onto the polymer may be found by the presence of a peak at 1 .48 ppm in the bottom spectrum.

Example 3

The polyalkyleneime polymer prepared according to Example 2 (poly(2-tert-butoxy-2-oxazoline)) was dissolved in water and 1 equivalent of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, leading to the removal of the BOC group. Finally, all volatiles were removed in vacuo, thereby providing a polyethyleneimine (PEI) homopolymer. The following scheme provides a representation of the reaction according to this example.

The ¹H NMR spectrum in D2O of the polymer before (top) and after (bottom) acidic hydrolysis were superimposed and are shown in Figure 3. Confirmation of removal of the BOC group may be found by the absence of the peak at 1 .48 ppm in the bottom spectrum. Example 4

A polymer was prepared according to General Procedure A, utilizing 2-(ethylthio)-4,5- dihydrooxazole (EtSOx) (0.03 ml, 0.251 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 103. This was achieved by adding (0.0003 mL, 0.0024 mmol, 0.01 eq) of TfOMe as an initiator. SEC analysis was performed on PEtSOx prepared according to this example. The SEC is shown in Figure 4.

It appeared that the polymerization with TfOMe as the initiator reduces tailing and leads to a relatively low dispersity of 1 .26.

Example 5

A gradient copolymer was prepared according to General Procedure A, utilizing EtSOx (0.043 ml, 0.35 mmol) and EtOx (0.031 mol, 0.35 mmol) as comonomers for statistical copolymerization, with the aim of achieving a degree of polymerization (DP) of 100. This was achieved by adding (0.001 mL, 0.007 mmol, 0.01 eq) of TsOMe as an initiator. By following the polymerization kinetics it was observed that the EtOx monomers were incorporated faster than the EtSOx monomers, leading to reactivity ratios of rEtox = 1 .65 and rEtsox = 0.45 confirming the formation of a gradient copolymer. The obtained gradient copolymer was precipitated in cold diethyl ether. Subsequently, oxidation was carried out according to General Procedure B. Following this, General Procedure C was applied, utilizing potassium tert-butoxide as the alkoxide at room temperature. The following scheme provides a representation of the reaction according to this example. The ¹H NMR spectra after each of General Procedures A, B, and C were superimposed, resulting in Figure 5. The top spectrum, recorded in chloroform-d, corresponds to the PEtOx- PEtsOx copolymer; the middle spectrum, also recorded in chloroform-d, represents the PEtOx- oxidized PEtsOx copolymer; and the bottom spectrum, recorded in D2O, depicts the PEtOx- poly(2-tert-butoxy-2-oxazoline) copolymer. Figure 5 illustrates the ability to selectively transform the sulfonyl groups into alkoxy groups, while preserving the amide groups originating from PEtOx. Due to the limited stability of poly(2-tert-butoxy-2-oxazine), only a small peak can be observed at 1 .48 ppm. A larger peak at 2.80 ppm is observed, which can be attributed to the free base polyethylenimine (PEI), resulting from the removal of the BOC group.

Example 6

A gradient copolymer was prepared according to Example 5, followed by dissolution in water and stirring with 1 equivalent of trifluoroacetic acid was added overnight at room temperature, leading to the removal of the BOC group. Finally, all volatiles were removed in vacuo, thereby providing a PEtOx-PEI gradient copolymer. The following scheme provides a representation of the reaction according to this example.

Confirmation of the selectivity of this transformation may be shown by comparing the ¹ H NMR spectra of the copolymer after General Procedure A (Figure 6) and after acidic hydrolysis (Figure 7). By examining the integration of the polymer backbone (3.5 ppm region) and side chains related to PEtOx (2.4 ppm and 1 .1 ppm) in both figures, it can be shown that the amide side chains remain unchanged, as the integration values before and after the reaction are identical. The peaks (2.8 ppm and 1 .2 ppm) associated with the sulfonyl side chain, on the other hand, are completely absent after the reaction. Comparative Example A

A polymer was prepared according to General Procedure A, utilizing EtOx (0.063 ml, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 100. This was achieved by adding (0.001 mL, 0.007 mmol, 0.01 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether, followed by dissolution in water and stirring with 1 equivalent of trifluoroacetic acid was added overnight at room temperature. The following scheme provides a representation of the absence of a reaction according to this example.

The ¹ H spectrum obtained after stirring with 1 eq. of TFA at room temperature overnight is depicted in Figure 8, illustrating that the amide side groups remain completely unreacted, as evidenced by the integration showing the correct ratios. Furthermore, Figure 9, which shows the SEC overlay of the polymer before (solid line) and after (dashed line) stirring with 1 eq. of TFA at room temperature overnight, confirms this observation, as no shift between the curves can be observed.

Example 7

A polymer was prepared according to General Procedure A, utilizing 2-(ethylthio)-4,5- dihydrooxazole (EtSOx) (0.083 mL, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.00042 mL, 0.0028 mmol, 0.004 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether.

Example 8

A polymer was prepared according to General Procedure A, utilizing 2-(isopropylthio)-4,5- dihydrooxazole (iPrSOx) (0.092 mL, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.00042mL, 0.0028 mmol, 0.004 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether. The resulting poly(2-isopropylthio-2-oxazoline) (PiPrSOx) was characterized by ¹H NMR spectroscopy, which shows a broad peak at 3.48 ppm, corresponding to the polymer backbone; a sharp peak at 1 .33 ppm attributed to the methyl protons of the isopropyl group; while the methine proton signal overlapped with the polymer backbone peaks and could not be distinctly observed. The integration of the peaks matched the expected values, confirming the successful formation of PiPrSOx.

Subsequently, oxidation was carried out according to General Procedure B. The oxidation is clearly observed by examining the changes in chemical shifts in the ¹H NMR spectra of PiPrSOx before and after oxidation. In the side chain, the methylene protons next to the sulphur atom shifts from 3.5 to 3.8 ppm after oxidation. Additionally, the protons on the polymer backbone are also affected by oxidation. The methylene protons, which appear as a single peak before oxidation, split into two peaks after oxidation, with one peak shifting further downfield. These results confirm the successful oxidation of PiPrSOx. The ¹H NMR spectra after each of General Procedures A, and B were superimposed, resulting in Figure 10. The top spectrum (recorded in chloroform-d) corresponds to poly(2-isopropylthio-2-oxazoline) (PiPrSOx), while the bottom spectrum (recorded in chloroform-d) represents oxidized PiPrSOx.

The following scheme provides a representation of the reaction according to this example.

SEC analysis was performed on PiPrSOx prepared according to this example, and compared with PEtSOx prepared according to Example 6, both with a targeted DP of 250. The SEC of both polymers are shown in Figure 13. It appeared that chain transfer becomes predominant in the polymerization of EtSOx when targeting this higher DP of 250, resulting in significant tailing and a high dispersity of 1 .8. In contrast, the polymerization of PiPrSOx with DP 250 showed minimal tailing and maintained a relatively low dispersity of 1 .4.

Example 9

A polymer was prepared according to General Procedure A, utilizing 2-(isopropylthio)-4,5- dihydrooxazole (iPrSOx) (0.0282 mL, 0.22 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.0001 mL, 0.00088 mmol, 0.004 eq) of TfOMe as an initiator. SEC analysis was performed on PiPrSOx prepared according to this example. The SEC is shown in Figure 1 1 . It appeared that the polymerization with TfOMe as the initiator reduces the tailing and maintains a relatively low dispersity of 1 .25.

Example 10

A polymer was prepared according to General Procedure A, utilizing 2-(ethylthio)-5,6-dihydro- 4H-1 ,3-oxazine (EtSOzi) (0.092 mL, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.0004 mL, 0.0028 mmol, 0.004 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether.

Example 11

A polymer was prepared according to General Procedure A, utilizing 2-(isopropylthio)-5,6- dihydro-4H-1 ,3-oxazine (iPrSOzi) (0.101 ml, 0.7 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.0004 mL, 0.0028 mmol, 0.004 eq) of TsOMe as an initiator. The polymer was precipitated in cold diethyl ether. The resulting poly(2-isopropylthio-2-oxazine) (PiPrSOzi) was characterized by ¹H NMR spectroscopy, displaying two broad characteristic peaks related to the polymer backbone: a signal at 1 .85 ppm corresponding to the methylene protons in the middle of the polymer backbone (-N-CH₂-CH₂-CH₂-), and a signal at 3.32 ppm corresponding to the methylene protons adjacent to the nitrogen atoms in the PiPrSOzi backbone (-N-CH₂-CH₂-CH₂-). Additionally, two sharp peaks related to the side chain protons were observed: a peak at 1 .33 ppm corresponding to the methyl protons of the isopropyl group, and another peak at 3.58 ppm corresponding to the methine proton. The integration of the peaks matched the expected values, confirming the successful formation of PiPrSOzi.

Subsequently, oxidation was carried out according to General Procedure B. The oxidation is clearly observed by examining the changes in chemical shifts in the ¹H NMR spectra of PiPrSOzi before and after oxidation. The ¹H NMR spectra after each of General Procedures A, and B were superimposed, resulting in Figure 12. The top spectrum (recorded in chloroform-d) corresponds to poly(2-isopropylthio-2-oxazine) (PiPrSOzi), while the bottom spectrum (recorded in chloroform-d) represents oxidized PiPrSOzi.

SEC analysis was performed on PiPrSOzi prepared according to this example, and compared with PEtSOzi prepared according to Example 8, both with a targeted DP of 250. The SEC of both polymers are shown in Figure 14. It appeared that chain transfer in iPrSOzi is significantly reduced compared to EtSOzi, as indicated by the much lower dispersity of 1 .3 for PiPrOzi compared to a dispersity of 1 .9 for PEtSOzi.

Example 12

A polymer was prepared according to General Procedure A, utilizing 2-(ethylthio)-5,6-dihydro- 4H-1 ,3-oxazine iPrSOzi (0.031 mL, 0.22 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 250. This was achieved by adding (0.0001 mL, 0.00088 mmol, 0.004 eq) of TfOMe as an initiator. SEC analysis was performed on PiPrSOzi prepared according to this example. The SEC is shown in Figure 15. It appeared that the polymerization with TfOMe as the initiator reduces the tailing and maintains a relatively low dispersity of 1 .38.

Example 13

A polymer was prepared according to General Procedure A, utilizing 2-(neopentylthio)-5,6- dihydro-4H-1 ,3-oxazine (neoPnSOzi) (0.0425 mL, 0.25 mmol) as the monomer, with the aim of achieving a degree of polymerization (DP) of 106. This was achieved by adding (0.00036 mL, 0.0024 mmol, 0.0095 eq) of TsOMe as an initiator. The polymer was precipitated in cold hexane. The resulting poly(2-neopentylthio-2-oxazine) (PneoPnSOzi) was characterized by ¹H NMR spectroscopy, displaying two broad characteristic peaks related to the polymer backbone: a signal at 1 .88 ppm corresponding to the methylene protons in the middle of the polymer backbone (-N-CH7-CH7-CH7-), and a signal at 3.39 ppm corresponding to the methylene protons adjacent to the nitrogen atoms in the PneoPnSOzi backbone (-N-CH7-CH7-CH7-). Additionally, two sharp peaks related to the side chain protons were observed: a peak at 0.96 ppm corresponding to the methyl protons of the neopentyl group, and another peak at 2.90 ppm corresponding to the methylene protons of the neopentyl group. The integration of the peaks matched the expected values, confirming the successful formation of PneoPnSOzi. The ¹H NMR in chloroform-d is shown in Figure 16A.

SEC analysis was performed on PneoPnSOzi prepared according to this example. The SEC spectrum of the polymer is shown in Figure 16B. The polymer has a dispersity of 1 .3.

Example 14

A block copolymer was prepared according to General Procedure A, utilizing iPrSOx (0.057 mL, 0.445 mmol) as the monomer with the aim of achieving degree of polymerization (DP) 250. This was achieved by adding living PEtOx (0.33 mg, 0.0018 mmol, 0.004 eq) with degree of polymerization (DP) 50 as a macroinitiator. Dry acetonitrile was used to dilute the mixture until a monomer concentration of 2M was achieved. The following scheme provides a representation of the reaction according to this example.

The ¹H NMR spectra before and after the polymerization of the iPrSOx monomer were superimposed resulting in Figure 17A. Both spectra were recorded in chloroform-d. The bottom spectrum represents the polymerization solution before polymerization and the top spectrum represents the PEtOx-b-PiPrSOx copolymer. Both spectra are displaying the characteristic peaks of PEtOx backbone (-N-CH?-CH?-) at 3.43 ppm. Additionally, two peaks related to the side chain protons of PEtOx were observed: a peak at 2.27-2.37 ppm corresponding to the methylene protons (-O=C-CH2-) and another peak at 1 .09 ppm corresponding to the methyl protons (-CH2-CH3) of the ethyl group. Similarly, the bottom spectrum is displaying the characteristic peaks of the iPrSOx monomer with two peaks for the methylene protons (-O-CH2- ) at 4.30 ppm and (-N-CH2-) at 3.87 ppm of the ring, one peak at 3.63 ppm for the methine proton (S-CH) and one peak at 1 .37 ppm for the methyl protons (S-CH-(CH3)2). In addition, the characteristic peaks of the PiPrSOx polymer are included at the top spectrum. The methylene (- N-CH2-CH2-) and methine (S-CH) protons are overlapping with the backbone protons of PEtOx at 3.43 ppm and a sharper peak at 1 .29 ppm corresponds to the methyl protons (S-CH-(CHs)2) of the isopropyl group. The integration of the peaks matched the expected values, confirming the successful formation of PEtOx-b-PiPrSOx copolymer.

SEC analysis was performed on PEtOx-b-PiPrSOx prepared according to this example. The SEC overlay of the copolymer is shown in Figure 17B, confirming the successful chain extension of the iPrSOx from the PEtOx macroinitiator. Peak a corresponds to the PEtOx polymer with a dispersity of 1 .1 1 and peak b corresponds to the PEtOx-b-PiPrSOx with a dispersity of 1 .64.

Example 15

A block copolymer was prepared according to General Procedure A, utilizing iPrSOzi (0.062 mL, 0.4416 mmol) as the monomer with the aim of achieving degree of polymerization (DP) 250. This was achieved by adding living PEtOx (0.329 mg, 0.00177 mmol, 0.004 eq) with degree of polymerization (DP) 50 as a macroinitiator. Dry acetonitrile was used to dilute the mixture until a monomer concentration of 2M was achieved. The following scheme provides a representation of the reaction according to this example.

The ¹H NMR spectra before and after the polymerization of the iPrSOzi monomer were superimposed resulting in Figure 18A. Both spectra were recorded in chloroform-d. The bottom spectrum represents the polymerization solution before polymerization and the top spectrum represents the PEtOx-b-PiPrSOzi copolymer. Both spectra are displaying the characteristic peaks of PEtOx backbone (-N-CH7-CH7-) at 3.33 ppm. Additionally, two peaks related to the side chain protons of PEtOx were observed: a peak at 2.30-2.40 ppm corresponding to the methylene protons (-O=C-CH2-) and another peak at 1 .12 ppm corresponding to the methyl protons (-CH2-CH3) of the ethyl group. Similarly, the bottom spectrum is displaying the characteristic peaks of the iPrSOzi monomer with two peaks for the methylene protons (-O-CH2- ) at 4.23 ppm and (-N-CH2-) at 3.44 ppm of the ring and one peak at 3.49 ppm for the methine proton (S-CH), one peak at 1 .37 ppm for the methyl protons (S-CH-(CH3)2) and one peak at 1 .91 -1 .94 ppm for the methylene protons (-CH2-CH2-CH2-). In addition, the top spectrum is displaying two broad characteristic peaks related to the PiPrSOzi backbone: a signal at 1 .86 ppm corresponding to the methylene protons in the middle of the polymer backbone (-N-CH₂- CH7-CH7-), and a signal at 3.33 ppm corresponding to the methylene protons adjacent to the nitrogen atoms in the PiPrSOzi backbone (-N-CH7-CH7-CH7-). Additionally, two sharp peaks related to the side chain protons were observed: a peak at 1 .33 ppm corresponding to the methyl protons (S-CH-(CHS)2) of the isopropyl group, and another peak at 3.57-3.61 ppm corresponding to the methine proton (S-CH) of the isopropyl group.

SEC analysis was performed on PEtOx-b-PiPrSOzi prepared according to this example. The SEC overlay of the copolymer is shown in Figure 18B confirming the successful chain extension of the iPrSOzi from the PEtOx macroinitiator. Peak a corresponds to the PEtOx polymer with a dispersity of 1 .10 and peak b corresponds to the PEtOx-b-PiPrSOzi with a dispersity of 1 .78.

REFERENCES van Kuringen, H. P. C.; de la Rosa, V. R.; Fijten, M. W. M.; Heuts, J. P. A.; Hoogenboom*, R.; Enhanced Selectivity for the Hydrolysis of Block Copoly(2-oxazoline)s in Ethanol-Water Resulting in Linear Polyethylene imine) Copolymers Macromol. Rapid Common. 2012, 33, 827-832.

Delecourt, G.; Piet, L.; Bennevault, V.; Guegan*, P. Synthesis of Double Hydrophilic Block Copolymers Poly(2-oxazoline-b-ethylenimine) in a Two-Step Procedure ACS Appl. Polym. Mater. 2020, 2, 7, 2696-2705.

Miyamoto*, M.; Aoi, K.; Morimoto, M.; Chujo, Y.; Saegusa, T. Ring-Opening Isomerization Polymerization of Cyclic Iminocarbonates Macromolecules 1992, 25, 5878-5885.

Wu, Y. C. M.; Swager*, T. M. Living Polymerization of 2-Ethylthio-2-Oxazoline and Postpolymerization Diversification. J Am Chem Soc 2019, 141 (32), 12498-12501 .

Li, G.; Ohtani, T. A New Method for the Synthesis of 5- and 6-Membered 2-Thioxo-1 ,3-0, N- Heterocycles. Heterocycles 1997, 45 (12), 2471-2474.

Claims

1. A method to modify a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, said method comprising the steps of: a) providing a polymer comprising a polyalkyleneimine, in particular a linear polyalkyleneimine, wherein the polyalkyleneimine comprises first alkyleneimine structural units represented by formula (I) wherein

X is selected from a direct bond, -CH2-, and -CR3R3CH2-;

R1, Rr, R2, R2', R3, and R3' are each independently selected from -H, and -Ci ealkyl; when X is a direct bond, then R2 and R2' are both -H;

R is selected from -C(O)SR_a, -C(O)S(O)R_a, and -C(O)S(O)2R_a; and

2. Method as claimed in claim 1 , wherein the polymer comprising a polyalkyleneimine provided in step a) is the polyalkyleneimine, in particular the linear polyalkyleneimine.

3. Method as claimed in claim 1 or 2, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by form wherein n is an integer from 2 to 1000; m is 0, or an integer selected from 1 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1, each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci ealkyl; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of X2 is independently selected from a direct bond, -CH2-, and -CR6R6CH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -C1- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; each instance of Re is independently selected from -H, -Ci-i2alkyl, -Ci-i2alkenyl, -C(O)R_c, and -C(O)NRdRd'; each instance of R_c, and each instance of Rd is independently selected from -Ci-i2alkyl, -C2- i2alkenyl, and -Ar₂; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted; each instance of Rd' is independently selected from -H, -Ci-i2alkyl, -C2-i2alkenyl, and -Ars; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally and independently substituted; and each instance of An , each instance of Ar₂, and each instance of Ars is independently a 5- to 10-membered aromatic cycle optionally comprising one or more heteroatoms selected from N, O and S and/or is optionally and independently substituted.

4. Method as claimed in any one of claims 1 to 3, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is represented by formula (Vila) or (Vila’) wherein n is an integer from 2 to 1000; m is an integer selected from 1 to 1000; each instance of Xi and each instance of X₂ is independently selected from a direct bond, - CH2-, and -(CH₂)₂-; each instance of R7 is independently selected from -C(O)SR_a, -C(O)S(O)R_a, and - C(O)S(O)₂R_a; each instance of R_a is independently selected from -Ci-i2alkyl, -C2-i2alkenyl, and -An ; wherein each of said -Ci-i2alkyl, and -C2-i2alkenyl optionally and independently comprises one or more heteroatoms selected from O, N and S and/or is optionally substituted;

Rs is -C(O)R_c; and

Re is selected from -Ci-i2alkyl, in particular from -Ci salkyl, more in particular from -Ci-4alkyl.

5. Method as claimed in any one of claims 1 to 4, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, provided in step a) is a gradient copolymer, or a block copolymer.

6. Method as claimed in any one of claims 1 to 5, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, any substituted or unsubstituted alkenyl, and any substituted or unsubstituted aryl.

7. Method as claimed in any one of claims 1 to 6, wherein the alkoxide in step b) is represented by Rb-O-, wherein Rb is a tertiary alkyl group, in particular wherein Rb is selected from tert-butyl, tert-pentyl, and tert-hexyl.

8. Method as claimed in any one of claims 1 to 7, wherein the polyalkyleneimine comprised in the polymer, in particular wherein the polymer is the polyalkyleneimine, obtained in step b) comprises A/-alkoxy- or aryloxycarbonyl alkyleneimine structural units.

9. Method as claimed in claim 8, further comprising a step c), wherein at least part of the N- alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units are removed by reaction with a chemical reagent.

10. Method as claimed in claim 8, further comprising a step c), wherein at least part of the N- alkoxy- or aryloxycarbonyl substituents on the amine group of the alkyleneimine structural units are removed by acidic hydrolysis.

1 1 . A polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, wherein the polyalkyleneimine is a block copolymer represented by formula wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1 , each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci ealkyl; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of X2 is independently selected from a direct bond, -CH2-, and -CReReCH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -C1- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; and

12. A polymer comprising a polyalkyleneimine, in particular wherein the polymer is the polyalkyleneimine, wherein the polyalkyleneimine is a gradient copolymer represented by form wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi is independently selected from a direct bond, -CH2-, and -CR3R3CH2-; each instance of R1 , each instance of Rr, each instance of R2, each instance of R2', each instance of R3, and each instance of R3' is independently selected from -H, and -Ci ealkyl; when for a given structural unit Xi is a direct bond, then R2 and R2' are both -H; each instance of X2 is independently selected from a direct bond, -CH2-, and -CReReCH2-; each instance of R4, each instance of R4', each instance of R5, each instance of R5', each instance of Re, and each instance of Re' is independently selected from -H, and -C1- ealkyl; when for a given structural unit X2 is a direct bond, then R5 and R5' are both -H; and

Re is selected from -Ci-i2alkyl, in particular from -Ci ealkyl, more in particular from -Ci-4alkyl, even more in particular from -CH3, and -CH2CH3; wherein the gradient copolymer consists of an initiating fragment, a central fragment, and a terminating fragment, wherein the ratio of first and second structural units in the initiating fragment is at least 2 times higher or lower than the same ratio in the terminating fragment of the copolymer.

13. Polymer as claimed in claim 1 1 or 12, wherein the polyalkyleneimine is represented by formula (Villa) or (Villa’) wherein n is an integer from 10 to 1000; m is an integer selected from 10 to 1000; each instance of Xi and each instance of X2 is independently selected from a direct bond, - CH2-, and -(CH2)2-; and

Re is selected from -Ci-i2alkyl, in particular from -Ci ealkyl, more in particular from -Ci-4alkyl, even more in particular from -CH3, and -CH2CH3.

14. A delivery system comprising a polymer as defined in any one of claims 1 1 to 13, and a therapeutic agent that is non-covalently bound to said polymer, in particular wherein the therapeutic agent is a pharmaceutically active agent or a biomolecule, such as a gene, a peptide, a protein or a nucleic acid.

15. Polymer, in particular a polyalkyleneimine, as claimed in any one of claims 1 1 to 13, or a delivery system as claimed in claim 14, for use in human or veterinary medicine.