Poly(ethylene glycol) having C1 to C3 alkyloxymethyl side chains, its bioconjugate, process for its preparation, and its use.

Abstract

The present invention relates to a polyether polymer represented by the following formula [I]: -(CH2CHR-O-)m - [I], wherein 10 to 90% of the residues R are hydrogen, 10% to 90% of the residues R are selected, independently of one another, from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, 1 to 100% of the residues R are methoxymethyl, up to 50% of the residues R may be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, m is in the range of 10 to 1000, and the dispersity is less than or equal to 1.15. The present invention further relates to processes for their preparation, to their conjugates, and to their use.

Description

[Technical Field]

[0001] The present invention relates to a polyether polymer represented by the following formula [I]. -(CH2CHR-O-) m - [I] In the formula, 10 to 90% of residue R are hydrogen, and 10 to 90% of residue R are independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; 1 to 100% of residue R are methoxymethyl; up to 50% of residue R can be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; m is in the range of 10 to 1000, and the degree of dispersion is 1.15 or less.

[0002] The present invention further relates to processes for the preparation thereof, their conjugates, and their uses. [Background technology]

[0003] Aliphatic polyethers poly(ethylene glycol) (PEG) are among the most widely used polymers in pharmaceuticals, medicine, and drug delivery. PEGulation, or attachment of PEG to peptide drugs, was introduced in the 1970s and has since led to numerous highly efficient drugs. PEGulation is a form of conjugation, i.e., the attachment of a polymer to a molecule via covalent bonds. The main effect of PEG attachment is the so-called "stealth effect," which prevents close approach and recognition by the immune system and subsequent binding and removal by the reticuloendothelial system (RES) in the liver. More simply, when attached to the surface of a drug delivery nanocarrier, PEG attachment also prevents protein opsonization. The inhibition of protein adsorption by PEG is further enhanced by the realization of rules for protein-repellent surfaces derived by Whitesides (Non-Patent Literature 1). PEGylation is also used for "stealth liposomes" with long circulation times, low molecular weight drugs, and lipid nanoparticles. Such structures are used for mRNA transport and delivery. Here, each PEG-based lipid plays a crucial role in COVID-19 pandemic vaccination, enabling mRNA transport to cells and avoiding unwanted degradation processes. Recently approved mRNA vaccines from Moderna Inc. and Pfizer Inc.-BioNTech SE require PEGylated lipids to ensure the stability of mRNA in lipid-based nanoparticles. PEGylation also prevents the coalescence of liposomes and similar structures in aqueous systems. Typically, molecular weights in the range of 2 to 40 kg / mol are preferred for the PEGylation of drugs or nanocarriers. In total, PEG has become the standard for water-soluble medical polymers, specifically for a wide range of medical and pharmaceutical uses in the treatment of cancer and chronic diseases, and more recently, for RNA delivery platforms. The effectiveness of PEG can be attributed to several factors. These include its sufficient quality and broad molar mass marketability from the anionic ring-opening polymerization of ethylene oxide, as well as its extremely low toxicity and high biocompatibility. Furthermore, PEG can be prepared with a narrow molecular weight distribution.

[0004] However, a growing number of studies summarize concerns about the presence of so-called anti-PEG antibodies (APAs) in a large and constantly growing portion of the population. Such antibodies can lead to accelerated clearance of PEGylated drugs from the bloodstream, i.e., loss of the stealth effect crucial for therapeutic response, allergic reactions, and in extreme and rare cases, even potentially fatal anaphylactic shock. This undesirable effect is observed with both PEGylated peptide drugs, PEGylated liposomes, and other PEGylated nanocarriers and PEGylated small molecule drugs. Anti-PEG antibodies also pose a problem with PEGylated lipids in RNA transport lipid nanoparticles, for example, in the context of COVID-19 vaccines. PEG antibodies are often mentioned as a motivation to seek alternatives to PEG, in the context of potential PEG substitutes. Roughly speaking, about 10% of the population experiences allergic reactions when treated with PEGylated formulations. Recently, the nature of binding between PEG and anti-PEG antibodies has been studied by Lai et al. (Non-Patent Literature 2). They concluded that PEG binding is due to the ring-open structure of PEG captured by APA. By counting the number of monomer repeats of the PEG polymer interacting with the inner and outer paratopes of Fab, they found that the size of the PEG antigen epitope is ~700 g / mol, corresponding to 16 monomer subunits. Therefore, at least 16 ethylene glycol units are required for binding by APA. Other studies suggest that shorter, regular chain segments of PEG and the methoxy-terminal groups of the mPEG (α-methoxypoly(ethylene glycol)) structure also play an important role in recognition. Accordingly, Patent Literature 1 discloses a method for preparing PEG conjugates based on modification of the terminal groups of the PEG chain. Variations of PEG terminal groups are disclosed, aiming, for example, to minimize the interaction of the methoxy-terminal groups with anti-PEG antibodies. The authors disclose a conjugate in which all distal polyalkylene glycol termini of the pure conjugate have hydroxyl groups, and the conjugate exhibits reduced antigenicity compared to the mPEG-protein conjugate.Antibodies induced by PEG-OH have similar affinity for both mPEG and PEG-OH, and antibodies induced by mPEG recognize mPEG more effectively than PEG-OH (Non-Patent Literature 3; Non-Patent Literature 4). These results were obtained using competitive ELISA, suggesting that anti-PEG antibodies induced by PEG-OH proteins are oriented towards the PEG backbone (backbone-specific), while antibodies induced by mPEG protein conjugates are methoxy group-specific. It can be concluded that at least 4-5 to 16 regularly arranged segments of ethylene glycol units are required to achieve recognition and immunogenic reaction by anti-PEG antibodies. Recent clinical practice has demonstrated that the presence of antibodies against PEG makes it possible to predict PEG-asparagase allergic reactions and readmission failure, highlighting the clinical importance of anti-PEG antibodies in the success of leukemia treatment (Non-Patent Literature 5).

[0005] Three strategies have been developed for modifying the PEG structure to counteract undesirable APA interactions in PEGylated therapeutics. Replacing the methoxy-terminal group of mPEG with a hydroxyl group resulted in a significant decrease in affinity for anti-PEG antibodies, as demonstrated by competitive ELISA testing. However, for large-scale commercial applications, the presence of a terminal hydroxyl group would require a protected functional initiator, complicating existing synthetic protocols. A second, more recent strategy is disclosed in Patent Document 2, which relies on a PEG-bottlebrush structure, i.e., a small PEG structure attached to a poly(hydroxyethyl methacrylate) backbone. The inventors claim that this type of PEG architecture minimizes anti-PEG antigenicity while preserving the desired stealth properties for surface coating. However, the resulting PEG-grafted copolymers exhibit a fairly broad, multimodal molecular weight distribution, which represents a major obstacle to approval for medical applications and bioconjugates similar to PEGylation with peptides or lipids. A narrower distribution (Mw / Mn < 1.15) was only achievable by performing costly and time-consuming preparative size exclusion chromatography (SEC).

[0006] Another strategy involves replacing PEG with linear polyether polyglycerol to increase the circulating time of liposomes in the bloodstream and avoid accelerated blood clearance (see Non-Patent Literature 6). However, the use of polyglycerol also results in the introduction of numerous hydroxyl functional groups into the liposomes. This is in contrast to Whitesides' aforementioned rules and leads to complex, nonspecific recognition of peptides in the bloodstream. Therefore, targeted use of conjugated drugs or liposomes is not possible with polyglycerol.

[0007] PEGylated therapeutics are often used for chronic or, in many cases, otherwise fatal diseases, such as cancer, and therefore, in many cases, repeated doses must be administered over long periods. PEG is a non-biodegradable polymer and must be excreted from the body by the kidneys. Studies of nephrofiltration of PEGylated drugs have demonstrated undesirable accumulation of PEG in the kidneys and associated pathological changes in the renal system (see, for example, Non-Patent Document 7). Thus, the tendency of polyethylene to crystallize can lead to deposition in the kidneys. Renal excretion also limits the molecular weight of PEG for medical therapeutics to the renal cutoff size, which is approximately 50 to 60 kDa for globular proteins.

[0008] In 2014, the inventors reported on the copolymerization of ethylene oxide with glycidyl methyl ether (GME) using a monomer activation method and the use of tetraoctylammonium bromide and trisisobutylaluminum as initiators (see Non-Patent Literature 8). A fairly broad molecular weight distribution was obtained (dispersion in the range of 1.21 to 1.5). These are clearly not suitable for medical applications. As discussed at that time, conventional anionic ring-opening methods have only been reported to achieve molecular weights up to 3000 g / mol and high dispersion for the polymerization of GME. Previous studies on homopolymerization from GME to poly(glycidyl methyl ether) (PGME) have generally stated that polymerization is difficult and that molecular weight control cannot be achieved by anionic ring-opening polymerization used in medical PEG (see Non-Patent Literature 9). In all available reports on GME polymerization to date, either a phosphazene base was used as a catalyst, or an aluminum compound had to be added in the manner of "monomer-activated polymerization." The same applies to the reaction published by Labbe et al. using monomer-activated ring-opening polymerization for the synthesis of polyglycidyl ethers (Non-Patent Document 10). This technique is subject to the common side reactions shown in Figure 11. Unwanted migration of hydride leads to further initiation species, thus limiting the required high end-group fidelity. In addition, the occurrence of isopropyl initiation by monomer-activated ring-opening polymerization has also been reported in the literature, for example, Non-Patent Documents 11 and 12. None of these polymers or copolymers are suitable for medical applications for several reasons: (i) trace amounts of phosphazene bases are toxic and cannot be removed; (ii) the degree of dispersion exceeds 1.1, which is contraindicated in most medical applications; and (iii) the end group fidelity of the polymers derived from the method is insufficient for attachment to peptides, drugs, or lipids by bioconjugate. Therefore, neither PGME homopolymers nor GME copolymers have been considered for PEGylation applications to date.

[0009] The formation and retention of well-defined polymer end groups (end group fidelity) is a crucial tool for chain elongation and all post-polymerization reactions. End group fidelity is also a critical factor in the use of polymers in bioconjugates for pharmaceutical applications, where biomolecules are linked by end groups. Polymers with low end group fidelity contain large amounts of unbound polymers that cannot be removed from the mixture without excessive expense. The resulting conjugates lack the required purity. Therefore, while loss of end groups is undesirable, it cannot be completely avoided. End group fidelity varies considerably depending on the reaction conditions of polymer preparation. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 8,129,330 [Patent Document 2] U.S. Patent Application Publication No. 2019 / 0015520 [Non-patent literature]

[0011] [Non-Patent Document 1] Yarovsky et al., "Quantitative design rules for protein-resistant surface coatings using machine learning," Scientific Reports, 2019, Vol. 9, p. 265. [Non-Patent Document 2] Lai et al., "Structure of an anti-PEG antibody reveals an open ring that captures highly flexible PEG polymers," Communications Chemistry, 2020, Vol. 3, p. 124. [Non-Patent Document 3] Sherman et al., "Role of the methoxy group in immune responses to mPEG-protein conjugates," Bioconjugate Chemistry, 2012, Vol. 23(3), pp. 485-499. [Non-Patent Document 4] Sherman et al., "Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies induced by methoxyPEG-proteins," Molecular Immunology, 2014, Vol. 57(2), pp. 236-246. [Non-Patent Document 5] Liu et al., "Antibodies Predict Pegasparagase Allergic Reactions and Failure of Rechallenge," Journal of Clinical Oncology, 2019, Vol. 37, p. 2051. [Non-Patent Document 6] Abu Lila et al., "Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration," International Journal of Pharmaceutics, Vol. 456 (2013), pp. 235-242. [Non-Patent Document 7] Bendele, A.; Seely, J.; Richey, C.; Sennello, G.; Shopp, G., "Short Communication: Renal Tubular Vacuolation in Animals Treated with Polyethylene-Glycol-Conjugated Proteins," Toxicological Sciences, 1998, Vol. 42, pp. 152-157. [Non-Patent Document 8] Frey et al., "A Challenging Comonomer Pair: Copolymerization of Ethylene Oxide and Glycidyl Methyl Ether to Thermoresponsive Polyethers," Macromolecules, 2014, Vol. 47, pp. 5492-5500. [Non-Patent Document 9] Frey et al., Biomacromolecules, 2014, Vol. 15, pp. 1935-1954. [Non-Patent Document 10] Macromolecular Symposia, 2007, Vol. 249-250, pp. 392-397. [Non-Patent Document 11] Billouard, C.; Carlotti, S.; Desbois, P.; Deffieux, A., "'Controlled' High-Speed ​​Anionic Polymerization of Propylene Oxide Initiated by Alkali Metal Alkoxide / Trialkylaluminum Systems," Macromolecules, 2004, Vol. 37(11), pp. 4038-4043. DOI: 10.1021 / ma035768t [Non-Patent Document 12] Herzberger, J.; Niederer, K.; Pohlit, H.; Seiwert, J.; Worm, M.; Wurm, FR.; Frey, H., "Polymerization of Ethylene Oxide, Propylene Oxide, and Other Alkylene Oxides: Synthesis, Novel Polymer Architectures, and Bioconjugation," Chemical Reviews, 2016, Vol. 116(4), pp. 2170-2243. DOI: 10.1021 / acs.chemrev.5b00441 [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide an alternative polymer that can replace PEG in its intended use and that can mitigate at least some of the aforementioned drawbacks of PEG. The alternative polymer should be particularly suitable for replacing PEG in pharmaceutical applications. Furthermore, the object is to provide a polymer with low dispersibility. A further object is to provide a polymer with high end-group fidelity. It is also desirable to provide a process that allows for the preparation of such polymers in a simple and commercially viable manner. The alternative polymer should have low affinity for anti-PEG antibodies (APAs). A further goal is to provide a polymer with low immunogenicity. [Means for solving the problem]

[0013] The present invention relates to poly(ethylene glycol) having C1 to C3 alkyloxymethyl side chains. The present invention relates to a polyether polymer represented by the following formula [I]: -(CH2CHR-O-) m - [I] In the formula, 10 to 90% of residue R are hydrogen, and 10 to 90% of residue R are independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; 1 to 100% of residue R are methoxymethyl; up to 50% of residue R may be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; however, if at least one residue R is selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, then at least one residue R is hydrogen; m is in the range of 10 to 1000, and the degree of dispersion measured by size exclusion chromatography in DMF is 1.15 or less, according to calibration according to the PEG standard. In this specification, the percentage of residue R is based on the sum of all residue R, i.e., m. These novel polyether polymers are a new type of material that behaves very differently from PEG in terms of affinity for anti-PEG antibodies (APAs) and have low immunogenicity. They can also have structures ranging from low crystallinity to amorphous. However, the polyether polymers of the present invention behave very similarly to PEG in terms of various characteristics important for pharmaceutical applications, including water solubility, hydration, cell viability, and biocompatibility.

[0014] The key to the reduced interaction with APA lies in the use of substituents on the PEG backbone. The increased spatial requirements of the PEG copolymer interfere with or make impossible interaction with anti-PEG antibodies that follow a specific "lock and key principle." In addition to the steric impact, the random or statistical distribution and / or random or statistical steric orientation of substituents on the polymer backbone further makes the generation and formation of specific anti-polymer antibodies impossible.

[0015] The key to the similarity in characteristics between PEG and the polymer of the present invention lies in the use of alkoxymethyl side chains. This maintains water solubility and other properties, as seen in PEG. The repeating group of this polymer, where R is a methoxymethyl group, i.e., (-CH2-CH(-CH2-OCH3)-O)-, has the same molecular formula C4H8O2 as the two repeating units of PEG (-CH2-CH2-O)2-, and is therefore a structural isomer of the two main chain repeating units of PEG. It contains the same number of atoms and very similar functional groups. This leads to similar properties, particularly similar water solubility, which is important for applications in biological systems. The absence of reactive functional groups on the side chains, such as hydroxyl and amino, reduces undesirable interactions with biological systems.

[0016] Ethoxymethyl, n-propoxymethyl, and isopropoxymethyl can be used as residues R to further adjust the properties of the polymer, such as its solubility, interaction with lipids, and properties for passage through biological membranes. However, these residues result in reduced water solubility. Such a selection of substituents also leads to deviations from the behavior of PEG with respect to other properties. For this reason, the amount of repeating units having residues selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl is limited to 50% of all residues R of the polymer of formula (I). Furthermore, in embodiments of the present invention in which at least 10% of the polymer residues R are selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, at least 10% are hydrogen, and more preferably at least 25% are hydrogen. In a more preferred embodiment, up to 20% of residue R may be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, and even more preferably, up to 50% of residue R may be selected from this group. These residues are not required in some of the pharmaceutical applications of the polymer of the present invention. For this reason, polymers of the present invention in which R is selected from the group consisting of hydrogen and methoxymethyl are preferred. Clearly, the ethylene oxide repeating units of the polymer of the present invention also have properties similar to PEG. Therefore, this selection provides a polymer whose behavior is closest to that of PEG.

[0017] Higher-order congeners of methoxymethyl, i.e., where R is C4 alkoxymethyl and higher-order, lead to increasing low water solubility and other undesirable properties, and therefore should generally be avoided. However, they can be used in small amounts when required, based on the sum of all residues R selected from the group consisting of hydrogen, methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; i.e., based on m, preferably in amounts of 10% or less, more preferably 3% or less, and most preferably 1% or less. If the polyether of the present invention contains higher-order congeners of methoxymethyl, the same conditions regarding the presence of hydrogen apply in the same preferred amounts where R is C4 alkoxymethyl or higher-order. Various other comonomers can also be used, if necessary, in small amounts, i.e., preferably less than 3 wt%, more preferably less than 1 wt%, and most preferably less than 0.1 wt%, based on the weight-average molecular weight of the polymer of formula [I]. These can be used, for example, to introduce functional groups or side chains onto a polymer. However, polymers of the present invention containing only the repeating units of formula (I) are typically best suited for use in pharmaceutical applications.

[0018] As previously mentioned, ethylene oxide repeating units and glycidyl methyl ether repeating units, i.e., repeating units where R is methoxymethyl, provide polymers with very similar properties. Due to these very similar properties, the ethylene oxide repeating units on the PEG backbone can be completely replaced by repeating units substituted with methoxymethyl. That is, 100% of residue R can be selected from methoxymethyl groups, which constitute a homopolymer of glycidyl methyl ether. However, this is undesirable. When at least some of residue R are selected to be hydrogen, the properties of the polymer of the present invention are still closer to those of PEG. Furthermore, compared to the homopolymer of glycidyl methyl ether, the immunogenicity of the copolymer will be reduced. This is because they differ not only in the orientation of their side groups but also in the side groups themselves. Therefore, a copolymer is preferred in the present invention. The greatest difference between side groups, and therefore the lowest immunogenicity, is provided by polymers in which some of the repeating units lack side groups, i.e., R is hydrogen.

[0019] This will ensure lower immunogenicity. Preferably, 10 to 90% of residue R is hydrogen, and 10 to 90% of residue R is independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl. Even more preferably, 20 to 80% of residue R is hydrogen, and 20 to 80% of residue R is independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl. Polymers of the present invention in which approximately 50% of all ethylene oxide repeating units are substituted with alkylglycidyl ethers offer the lowest immunogenicity. For this reason, polymers in which 30 to 70% of residue R is hydrogen, and 30 to 70% of residue R is independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, are preferred, and more preferably 40 to 60%. The polyether polymer of the present invention, in which the polyether is a poly(glycidylmethyl ether-co-ethylene oxide) copolymer, i.e., the residue R is selected from the group consisting of hydrogen and methoxymethyl, is particularly useful in pharmaceutical applications. The same preferred range applies to embodiments in which R is selected from the group consisting of hydrogen and methoxymethyl, i.e., the polyether is a poly(glycidylmethyl ether-co-ethylene oxide) copolymer.

[0020] The polyether polymer of the present invention is particularly preferred in which 30 to 70% of residue R is hydrogen and the remaining residue R is methoxymethyl. These copolymers containing repeating units where R is hydrogen combine most of the aforementioned advantageous properties. They have low immunogenicity, good water solubility, low crystallinity, and otherwise are very similar to PEG.

[0021] A further aspect of the polymers of the present invention is that, while retaining the same properties, the degree of crystallinity, i.e., the degree of crystallization, is reduced compared to PEG. Some polymers have an amorphous structure depending on their specific composition. Statistical copolymers of EO with GME or other glycidyl ethers can reduce or completely eliminate the crystallization of PEG. The degree of crystallinity of PEG and the associated melting point (Table 5) play an important role in various pharmaceutical applications, such as drug suppositories or solid dispersions. In other applications, such as highly hydrophilic and biocompatible polyurethane soft foams used in wound dressings, crystallization is undesirable, and completely amorphous PEG is required. When more than 25% of glycidyl ether monomers are incorporated due to the random incorporation of side chains, the copolymers of the present invention make it possible to obtain a completely amorphous PEG copolymer structure. This feature is applicable in biomaterials based on triblock and multiblock structures, for example, combining polyethers with biodegradable polylactide blocks.

[0022] m is in the range of 10 to 1000; preferably m is in the range of 20 to 900, more preferably in the range of 30 to 800. Most preferably m is in the range of 30 to 700. These ranges correspond to the polymer weight of PEG that is most suitable for medical applications and used in existing medical applications of PEG. Furthermore, the polymer of the present invention preferably has a molecular weight M determined by MALDI-TOF-MS in the range of 500 to 50,000 g / mol, more preferably in the range of 1,000 to 50,000 g / mol, most preferably in the range of 1,000 to 30,000 g / mol. n These have. These scopes apply to all embodiments of the present invention.

[0023] The copolymers of the present invention may be random copolymers or statistical copolymers. Since they do not provide a blueprint for the immune system regarding antibodies, such copolymers offer the lowest immunogenicity. They are inherently resistant to immune responses and therefore represent preferred embodiments of the present invention. These polymers can be prepared by the processes of the present invention discussed below.

[0024] In one embodiment of the present invention, the polymer of the present invention may be a block copolymer or may have a block-like structure or a tapered or gradient structure. Methods for preparing such polymers are known to those skilled in the art. In such embodiments, it is preferable that more than 5% of the polymer's polymer content contains blocks having more than 15 ethylene oxide repeating units, and more preferably, more than 5% of the polymer's polymer content contains blocks having more than 8 ethylene oxide repeating units. This reduces immunogenicity.

[0025] As discussed above, the similarity to PEG is an important feature of the polymer of the present invention. This is particularly true of the end groups of the polymer. Processing of PEG for use in the pharmaceutical field typically involves modification of the end groups. For this purpose, any end group known to be used in PEG may also be used in the present polymer. This includes not only the use of all known end groups for PEG, but also their modifications, including the types of modifications and the processes of modifications. Where necessary, in order to allow the polymer to function similarly to or actually identically to PEG, the polymer may be provided with ethylene oxide repeating units at either end or both ends of the polymer, as represented by formulas [Ia] to [Ic]. These are preferred embodiments of the present invention. -(CH2CH2-O-) a -(CH2CHR-O-) m - [Ia] -(CH2CHR-O-) m -(CH2CH2-O-) a - [Ib] -(CH2CH2-O-) a-(CH2CHR-O-) m -(CH2CH2-O-) a - [Ic]

[0026] In these formulas, the partial formula -(CH2CHR-O-) m - represents the polymer of the present invention described in this specification, and a can be any number, but a number in the range of 1 to 10 is preferred, and more preferably a number in the range of 1 to 3. The polymers according to formulas [Ia] to [Ic] allow for modification of the ends of the polymer as modified in the process known for PEG, especially as modified in current industrial processes. This allows for a smooth transition from PEG to the polymers of the present invention in an established process. As will be discussed below, such materials can be prepared without much additional effort.

[0027] A further embodiment of the present invention is a polyether polymer represented by any of the following formulas [II] and [IIa] to [IIc]: X-(CH2CHR-O-) m -CH2CHR-Y [II] X-(CH2CH2-O-) a -(CH2CHR-O-) m -CH2CHR-Y [IIa] X-(CH2CHR-O-) m -(CH2CH2-O-) a -CH2CH2-Y [IIb] X-(CH2CH2-O-) a -(CH2CHR-O-) m -(CH2CH2-O-) a -CH2CH2-Y [IIc] In these formulas, m is from 19 to 999, R is as defined in formula [I], and a is as defined in any of formulas [Ia] to [Ic]. X is a terminal group derived from the initiator of the reaction and is an α-terminal group, and Y is an ω-terminal group.

[0028] X or Y or both may be selected from any of the end groups known to be used in PEG. The same end groups may also be provided for polymers of formulas [Ia] to [Ic].

[0029] X and Y may independently or together contain one or more functional groups selected from the group consisting of: acetals (dialkoxy), aldehydes (formyl), amides (carboxamide), azides, carbonates (alkoxycarbonyl) (oxy), carboxyls (carboxy), carboxylic acid anhydrides, esters (alkoxycarbonyl), ethers, halos, haloformyl (carbonohaloridoyl), hemiacetals (alkoxyol), hemiketals (alkoxyol), hydroxyls, imides (imido), imines (imino), ketals (dialkoxy), ketones (oil), orthoesters (trialkoxy), primary, secondary, and tertiary amino groups, primary, secondary, and tertiary alkoxy groups, sulfhydryl (sulfanyl, HS-), thioethers, and combinations thereof. In preferred embodiments, X or Y may be selected from the group consisting of alkyl, hydrogen, hydroxy, alkoxy, sulfanyl, phthalimide, amide, amine, and combinations thereof. X or Y or both are of the formula R-CH, where R is a linear, branched, or cyclic alkyl or phenyl, and n is equal to 1 to 20. n It may be a selected primary alkoxy group of -O-. Preferably, X or Y, or both, are selected from the group consisting of methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, octoxy, nonoxy, decanoxy, 3-ethyl-butoxy, 2,3-dialkoxypropoxy, and combinations thereof. X or Y, or both, may also be alkoxy residues of the following alcohol: [ka] In the formula, R is hydrogen, or a linear, branched, or cyclic alkyl or phenyl compound. X or Y or both are in the formula: [ka] It may be an alkoxy residue of a secondary alcohol selected from any of the following. In the formula, R and R' are independently hydrogen, or linear, branched, or cyclic alkyl or phenyl groups. Among these, secondary alkoxy groups selected from the group consisting of 2-propoxy, 2-butoxy, 2-pentoxy, 3-pentoxy, 2-hexoxy, 3-hexoxy, 2-heptoxy, 3-heptoxy, 4-heptoxy, and cyclohexyloxy are preferred.

[0030] X or Y, or both, may also be selected from primary alkenyloxy groups that can be linear, branched, or cyclic. X or Y, or both, may also be alkoxy residues of the following alcohol: [ka] In the formula, R is hydrogen, or a linear, branched, or cyclic alkyl or phenyl group. Among these, a primary alkenyloxy group selected from the group consisting of 2-propenoxy(allyloxy), 3-butenoxy, 2-butenoxy, 3-methyl-2-butenoxy, 4-pentenoxy, 3-pentenoxy, 2-pentenoxy, and 3,5-hexadienoxy is preferred.

[0031] X or Y or both may be further selected from secondary alkenyloxy groups that may be branched or cyclic. X or Y or both may be alkoxy residues of any of the following formulas: [ka] In the formula, R and R' are independently hydrogen, or a linear, branched, or cyclic alkyl or phenyl group. The secondary alkenyloxy group may be any of the following: 4-pentene-2-oxy, 3-pentene-2-oxy, 5-hexene-2-oxy, 4-hexene-2-oxy, 3-hexene-2-oxy, 4,6-heptadiene-2-oxy, or 1,4-pentadiene-3-oxy.

[0032] X or Y, or both, may be further selected from aryloxy and heteroaryloxy groups. A benzyloxy group is particularly preferred. X or Y, or both, may be aryloxy or benzyloxy residues of any of the following alcohols: [ka] In the formula, R, R'', R''', R'''', R''''', and R'''''' are independently hydrogen, or a linear, branched, or cyclic alkyl or phenyl. X or Y or both are preferably selected from the group consisting of benzyloxy, phenyloxy, and naphthyloxy.

[0033] X or Y, or both, may be a multifunctional alkoxy group. That is, multiple ether bonds may be present at the α or ω positions of multiple polymers of the polymer of the present invention according to formula [II] or [IIa] to [IIc]. Thus, any suitable polyol can be selected. X or Y, or both, may be selected from the group consisting of polyethers of carbohydrates, particularly ribose, polyethers of 1,1,1-trimethylolpropane, polyethers of glycerol, and polyethers of sugars or hydride sugars.

[0034] Furthermore, Y may be selected from the group consisting of acrylamide, acrylate, aldehyde, alkyne, amine, aminooxy, azide, benzotriazole carbonate, carboxylic acid, chloroformate, cyanuric acid chloride, dithioester, epoxide, fluorescein, hydrazide, imidazoyl formate, iminoester, isocyanate, maleimide, mesylate, methacrylate, NHS ester, nitrobenzoate, nitrophenyl carbonate, succinimidyl active ester, succinimidyl carbonate, N-succinimidyl carbonate, succinimidyl succinate, thiocarbonate, thiol, thiol carboxylic acid, tosylate, triflate, vinyl sulfone, and xanthate.

[0035] It is particularly preferable that X is selected from the group consisting of hydrogen, alkyl, hydroxy, sulfanyl, C1-C10 alkoxy, C1-C10 thioalkoxy, amino, amino-C1-C10 alkoxy, dibenzylamino-C1-C10 alkoxy, amide, N heterocyclic carbene, and N heterocyclic olefin. It is also particularly preferable that Y is selected from the group consisting of hydrogen, hydroxy, alkoxy, -CH2-C(=O)-R (where R is as defined above), -CHO, alkylcarbonyloxy, alkoxycarbonyloxy, amine, alkylamine, carboxamide, azide, halogen, sulfanyl, thioalkoxy, and sulfonate. In addition, any alkoxy group (rest) defined herein for residue X may be a corresponding thioalkoxy residue, where a sulfur atom is present instead of an oxygen atom.

[0036] The polymer of the present invention is most preferably one in which X and / or Y are selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces, and these are directly bonded to the polymer by covalent bonds or by spacers. Nanocarriers usable in the present invention are selected from the group consisting of, for example, carbon nanotubes (CNTs), dendrimers, gold nanorods, lipid nanoparticles (nanoparticels), liposomes, micelles, nanocrystals, niosomes, polymer nanoparticles (PNPs), solid lipid nanoparticles (SLNs), and virus-based nanoparticles (VNPs).

[0037] The polymers of the present invention have a dispersion degree of 1.15 or less. The dispersion degree of the present invention was determined using size exclusion chromatography (SEC) in DMF, calibrated according to PEG standards, as described in more detail in the examples. They are suitable for applications requiring low dispersion, such as pharmaceutical, medical, and biological applications. Polymer dispersion is of great importance in pharmaceutical, medical, and biological applications because polymers with different molecular weights can behave differently in biological systems. These may include differences in water solubility, membrane permeability, crystallinity, and renal clearance. Since uniform behavior of all molecules is desired in medical applications, low dispersion is often required. Therefore, polymers of the present invention having a dispersion degree of 1.10 or less are most preferred. This applies to all embodiments of the present invention. Most medical and pharmaceutical applications require a dispersion degree of 1.10 or less. Polymers of the present invention having a dispersion degree lower than 1.08 are most preferred.

[0038] A preferred embodiment of the present invention is a polyether polymer represented by the following formula [II]: X-(CH2CHR-O-) m CH2CHR-Y [II] In the formula, residues R are independently selected from the group consisting of hydrogen, methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; 1 to 100% of residues R are methoxymethyl; up to 50% of residues R may be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; however, if at least one residue R is selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl, then at least one residue R is hydrogen; X consists of hydrogen, alkyl, hydroxy, sulfanyl, C1-C10 alkoxy, C1-C10 thioalkoxy, amino, amino-C1-C10 alkoxy, dibenzylamino-C1-C10 alkoxy, amide, N heterocyclic carbene, and N heterocyclic olefin. Selected from the group; Y is selected from the group consisting of hydrogen, hydroxyl, alkoxy, -CH2-C(=O)-R, -CHO, alkylcarbonyloxy, alkoxycarbonyloxy, amine, alkylamine, carboxamide, azide, halogen, sulfanyl, thioalkoxy, N-succinimidyl carbonate, and sulfonate; X and / or Y may also be selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces, characterized in that they are directly bonded to the polymer by covalent bonds or by spacers, m is in the range of 19 to 999, and the degree of dispersion is 1.15 or less. N-succinimidyl carbonate is particularly preferred.

[0039] A particularly preferred embodiment of the present invention is the polyether polymer of the present invention in which X is an alkoxy and Y is a hydroxyl group. More preferably, X is a C1-C10 alkoxy group and Y is a hydroxyl group, and even more preferably, X is a C1-C3 linear alkoxy group and Y is a hydroxyl group. These embodiments are similar to commercially available PEG derivatives. Most preferably, X is methoxy and Y is OH, which is similar to mPEG.

[0040] As discussed regarding dispersion, unified behavior of all molecules is preferred in drugs and excipients used in pharmaceutical and medical applications. The end group fidelity of the polymers of the present invention is determined by MALDI-TOF-MS or 1 The end group fidelity can be determined by known methods, specifically by a combination of 1H-NMR and MALDI-TOF-MS. The polymer of the present invention preferably has an end group fidelity of at least 95%, more preferably at least 98%, with respect to group X. In further embodiments, the polymer of the present invention preferably has an end group fidelity of at least 95%, more preferably at least 98%, with respect to group Y. A polyether polymer of the present invention having an end group fidelity of at least 95%, most preferably at least 98%, with respect to groups X and Y is even more preferred. A polyether polymer of the present invention having an end group fidelity of at least 95% with respect to groups X and Y and a dispersion of 1.10 or less is particularly preferred, and a polyether polymer of the present invention having an end group fidelity of at least 98% with respect to groups X and Y and a dispersion of 1.10 or less is most preferred.

[0041] In another embodiment of the present invention, the polyether polymer is a polyether polymer represented by the following formula [II]: X-(CH2CHR-O-) m CH2CHR-Y [II] In the formula, R has the same meaning as referred to in the other embodiments of [I] and [II], and in the formula, X is selected from the group consisting of the group X referred to in the other embodiments of [I] and [II], or hydrogen or X'. In the formula, X' is selected from the following group: [ka] In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl, preferably, R'' is alkyl, that is [ka] Or alkenil, that is [ka] Or aryl, that is [ka] Or alkoxymethyl, i.e. [ka] Or alkenyloxymethyl, i.e. [ka] Or aryloxymethyl, i.e. [ka] Or aminomethyl, that is [ka] And in the formula, p = 1, 2, 3, 4, or 5, k = 5 to 500, preferably 5 to 250, most preferably 10 to 200; and [ka] In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl, preferably, R'' is alkyl, that is [ka] Or alkenil, that is [ka] Or aryl, that is [ka] Or alkoxymethyl, i.e. [ka] Or alkenyloxymethyl, i.e. [ka] Or aryloxymethyl, i.e. [ka] Or aminomethyl, that is [ka] And in the formula, p = 1, 2, 3, 4, or 5, k = 5 to 500; preferably 5 to 250, most preferably 10 to 200; and [ka] In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl, preferably, R'' is alkyl, that is [ka] Or alkenil, that is [ka] Or aryl, that is [ka] Or alkoxymethyl, i.e. [ka] Or alkenyloxymethyl, i.e. [ka] Or aryloxymethyl, i.e. [ka] Or aminomethyl, that is [ka] And in the formula, q = 3, 4, or 5, k = 5 to 500, preferably 5 to 250, most preferably 10 to 200; and [ka] In the formula, k = 5 to 500, preferably 5 to 250, and most preferably 10 to 200; In the formula, Y is ZW, meaning it is composed of substituents Z and W linked by covalent bonds. During the ceremony, Z is -O-R'-O-, W is -R-CHCH2-(O-CHRCH2-) m -V is; In the equation, V is either X or X'; In the formula, R' is selected from the following group: [ka] During the ceremony, R” = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl, preferably, R'' is alkyl, that is [ka] Or alkenil, that is [ka] Or aryl, that is [ka] Or alkoxymethyl, i.e. [ka] Or alkenyloxymethyl, i.e. [ka] Or aryloxymethyl, i.e. [ka] Or aminomethyl, that is [ka] And in the formula, s = 0 to 20, t = 0 to 20, Further R' is as follows: [ka]

[0042] In another embodiment, block copolymers of P(EG-co-GME) and poly(L-lactic acid)(PLLA) having -O-CH3, -O-Bz, or -OH as functional groups at the ends of the polymer chain, namely diblock polymers MeO-P(EG-co-GME)-b-PLLA-OH, BzO-P(EG-co-GME)-b-PLLA-OH, or HO-P(EG-co-GME)-b-PLLA-OH, and HO-P(EG-co-GME)-b-PLLA-OH, as well as triblock polymers, such as HO-PLLA-bP(EG-co-GME)-b-PLLA-OH, are preferred.

[0043] In all embodiments of the present invention, m is in the range of 9 to 999, more preferably in the range of 30 to 800. Most preferably, m is in the range of 30 to 700. These ranges correspond to the polymer weight of PEG that is most suitable for medical applications and used in existing medical applications of PEG. Furthermore, the polymer of the present invention preferably has a molecular weight M determined by MALDI-TOF in the range of 500 to 50,000 g / mol, more preferably in the range of 1,000 to 50,000 g / mol, most preferably in the range of 1,000 to 30,000 g / mol. n These have. These scopes apply to all embodiments of the present invention.

[0044] Calibrated according to PEG standards, the dispersion of all compounds of the present invention, as measured by size exclusion chromatography in DMF, is 1.15 or less. The end-group fidelity of the polymer is at least 95% for groups X and Y, and the dispersion is 1.10 or less. Most preferably, the end-group fidelity of the polymer is at least 98% for groups X and Y, and the dispersion is 1.10 or less.

[0045] As described above, the polymers of the present invention have properties similar to PEG, particularly in biological systems. Therefore, these polymers can substitute for PEG in most of their applications. The polymers of the present invention are specifically designed for use in the pharmaceutical and medical fields. They can also be used in veterinary and other biological applications for the same benefits. In these fields, the polymers of the present invention can achieve the same functions as PEG, but may have reduced immunogenicity and antigenicity. Due to their irregular structure, they do not induce a strong immune response. The polymers of the present invention show a particularly low response to anti-PEG antibodies, as demonstrated by ELISA testing. Conjugates used to mask drugs and other pharmaceutically active compounds typically contain PEG in addition to a physiologically active compound or adjuvant. The polymers of the present invention can be used as a substitute for PEG in any of these applications. At the same time, the immune response induced by this polymer is weak or absent, thereby resolving one of the main problems with the use of PEG.

[0046] Therefore, a further embodiment of the present invention is a conjugate comprising a polyether polymer and a substrate according to any of the prior claims. The conjugate of the present invention may hereafter also be referred to as a polyether-conjugated substrate or a polymer-conjugated substrate. In the conjugate of the present invention, the polymer of the present invention is directly or indirectly covalently bonded to the substrate. The polymer of the present invention may be bonded to the substrate by spacers constituting indirect bonds. The substrate may be selected from the group consisting of pharmaceutically active compounds, adjuvants, surfaces, and interfaces. The pharmaceutically active compound is preferably selected from the group consisting of low molecular weight drugs, peptides, polypeptides, proteins, glycoproteins, polynucleotides, and polysaccharides. The adjuvant is preferably a vesicle or carrier, which is preferably used to support a pharmaceutically active compound. The adjuvant is preferably selected from the group consisting of nanocarriers, liposome structures, lipid structures, and liposomes. Nanocarriers usable in the present invention are selected from the group consisting of, for example, carbon nanotubes (CNTs), dendrimers, gold nanorods, lipid nanoparticles, liposomes, micelles, nanocrystals, niosomes, polymer nanoparticles (PNPs), solid lipid nanoparticles (SLNs), and virus-based nanoparticles (VNPs). In more preferred embodiments, the substrate is selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, proteins, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces. In these embodiments, the substrate may be directly bonded to the polymer by covalent bonds or by spacers. The conjugate of the present invention with bovine serum albumin is particularly preferred.

[0047] The polymer of the present invention can be conjugated to a substrate directly by covalent bonds or by any type of spacer. Preferably, the hydroxyl residue at the ω terminus of the polymer of the present invention is used to conjugate the substrate or spacer to the polymer. This is the most economical method because the polymer of the present invention, in which X is an inert group and Y is an OH group, is the easiest to prepare (see Examples). Conjugation of substrates to the OH group at the ω terminus of PEG is well established. The same process can be used for the polymer of the present invention. However, the substrate can also be conjugated to the α terminus of the polymer. The polymer of the present invention can also be conjugated to more than one substrate. This can be done by using the α and ω terms of the polymer, and by using functional end groups of the polymer or spacer that can conjugate to multiple substrates. This can also be achieved by using a comonomer having a functional group to which the substrate can be conjugated. The bond between the polymer of the present invention and the substrate can be prepared by any means currently used to conjugate PEG to a substrate. It is also possible to bind multiple polymers of the present invention to a single substrate. This is particularly useful for all types of vesicles, including liposomes.

[0048] Typical examples of PEG conjugates are the lipid nanoparticles essential to the COVID-19 vaccines of BioNTech SE, Pfizer Inc., and Moderna Inc. These contain lipid PEG conjugates, where lipid nanoparticles (LNPs) are used to encapsulate and protect the pharmaceutically active substance mRNA. The PEG in these conjugates can be replaced by the polymer of the present invention, thereby preserving the effectiveness of the PEG polymer in the conjugate while simultaneously reducing the immune response to the conjugate. A further vaccine of this type is the Curevac NV COVID-19 vaccine. Further drugs containing PEG conjugates in which PEG can be replaced by this polymer are listed below.

[0049] [Table 1A] [Table 1B] [Table 1C]

[0050] In another embodiment of the present invention, the present invention relates to a process for preparing polyether polymers by anionic ring-opening copolymerization (AROP), comprising the following steps: - Anion An - Providing, - expression [ka] Add at least one monomer of the formula, where R is defined as described for either formula [I] or [II], - Allow polymerization to proceed at temperatures ranging from -10 to 90°C. The monomer contains less than 1 wt% epichlorohydrin.

[0051] In the process of the present invention, the anion An - The anion must be suitable for anionic ring-opening copolymerization (AROP). Those skilled in the art can easily select from known anions for this purpose. - Anions include alkyl anions, hydrides, and OH groups. - alkoxy - SH - , thioalkoxy -The group can be selected from amine anions, amide anions, imid anions, and combinations thereof. Alkyl anions and hydride anions can be provided in the form of metallic alkyl or metallic hydride compounds. Alkoxy anions and thioalkoxy anions may be the corresponding anions having the same structure as any of the alkoxy group, aryloxy group, and aralkoxy group as defined herein with respect to residue X. However, preferably, alkoxy anions and thioalkoxy anions are not tertiary alkoxy anions. Imid anions are preferably phthalimido anions. Polymers of formulas [Ia], [Ic], [IIa], and [IIc] are firstly composed of the anion An of the corresponding amount of ethylene oxide. - It can be prepared by adding to and allowing polymerization to proceed at a temperature in the range of -10 to 90°C until all of the ethylene oxide is consumed. Thereafter, formula [ka] The steps are to add at least one monomer and to allow polymerization to proceed at a temperature in the range of -10 to 90°C. In this manner, the repeating units adjacent to the α-terminus of the polymer of the present invention are provided with the exact same structure as PEG. Polymers of formulas [Ia], [Ic], [IIa], and [IIc] are of formula X-(CH2CH2-O-) a - It can also be prepared by the use of the anion, where a is as defined for any of formulas [Ia], [Ic], [IIa], and [IIc], and X is any residue of X as defined herein. X' is any residue of X' as defined herein. This is a preferred method. X is an alkyl, alkoxy, dialkylamine anion, or (RCO)2N - The equation is X-(CH2CH2-O-) a - The use of such an anion is particularly preferred. MeOCH2CH2O - MeO(CH2CH2O)2 - , benzyl OCH2CH2O -, BzO(CH2CH2O)2 - (Bz)2N-CH2CH2O - (Bz)2N-(CH2CH2O)2 - phthalimide-CH2CH2O - phthalimide-(CH2CH2O)2 - It is even more preferable to use an anion selected from the group consisting of the following, where Me is methyl and Bz is benzyl. MeO(CH2CH2O)2 - , BzOCH2CH2O - , and (Bz)2N-CH2CH2O - Most preferable.

[0052] In addition, to synthesize a bifunctional initiator α,ω-OH-P(EG-co-GME) copolymer that enables the synthesis of ABA triblock copolymers, P(EG-co-GME) having two hydroxyl ends is used as a polymer initiator for ring-opening polymerization of cyclic polyesters, cyclic diesters, or cyclic monoesters, such as L-lactide, D-lactide, mesolactide, cyclic carbonates, such as trimethylene carbonate (TMC), 2,2-dimethyl-trimethylene carbonate (DTC), 5-butyl-1,3-dioxan-2-one, and 1,3-dioxepant-2-one; glycolides, other cyclic esters, and lactams, and N-carboxyanhydrides. In one embodiment of the present invention, the reaction is carried out using L-lactide, for example, enabling the synthesis of PLLA-bP(EG-co-GME)-b-PLLA, where PLLA is [ka] This means that in the formula, k = 5 to 500, preferably 5 to 250, and most preferably 10 to 200.

[0053] Polymerization is catalyzed by Lewis acids, such as triethylaluminum (AlEt3) and bis-(2-ethylhexanoate)tin(II) (Sn(Oct)2), or by organic bases, such as DBU (1,8-diazabicyclo[5.4.0]undeca-7-ene) and N-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene. The procedures described are not limited to triblock copolymers. Diblock copolymers (P(EG-co-GME)-b-PLLA) can be obtained by using a monofunctional P(EG-co-GME) polymer initiator.

[0054] In these embodiments, An - is alkoxy - , thioalkoxy - The group may be selected from the , and amine anions.

[0055] Furthermore, in the final step of preparation, a small amount of pure EO may be added to ensure that the ω-terminus primary hydroxyl terminal group of the polymer can undergo all established coupling strategies with the therapeutic entity. This leads to the polymers of the present invention having formulas [Ib], [Ic], [IIb], and [IIc].

[0056] An - The counterion for the anion is preferably Na + , K + , and Cs + Selected from the group consisting of: Anion An - It can be provided in an inert solvent.

[0057] Anionic ring-opening polymerization of epoxides can be carried out in aprotic solvents such as DMSO or other polar aprotic solvents, such as tetrahydrofuran (THF), methyl-THF, cyclopentyl methyl ether (CPME), benzene, toluene, xylene, HMPA, dioxane, and diglyme. Surprisingly, it has been found that random copolymers can be synthesized using the aprotic solvent DMSO, while statistical copolymers can be synthesized using a polar aprotic solvent, such as toluene. This can be seen in Figures 9, 10, 14, and 15.

[0058] The solvent is preferably an aprotic solvent, most preferably dimethyl sulfoxide (DMSO). Furthermore, the reaction is preferably carried out in the same solvent.

[0059] Anionic ring-opening polymerization using alkylglycidyl ethers is more prone to chain transfer reactions, as follows: [ka]

[0060] These side reactions increase with temperature. Therefore, high molecular weight polymers can only be obtained when the reaction temperature is not above 90°C. Chain transfer reactions result in a broad distribution of molecular weight and thus high dispersibility. Therefore, lower polymerization temperatures also provide polymers of the present invention having lower dispersibility. On the other hand, alkyl glycidyl ethers are as reactive as ethylene oxide in anionic ring-opening polymerization. For this reason, polymerization can be carried out at temperatures as low as -10°C. Preferably, polymerization is carried out at temperatures in the range of 10 to 70°C, and more preferably in the range of 10 to 60°C.

[0061] A further problem with anionic ring-opening polymerization is that commercially available alkyl glycidyl ethers are prepared from epichlorohydrin. Some of the epichlorohydrin remains in the alkyl glycidyl ether. The presence of the alkyl glycidyl ether leads to the termination reaction as follows: [ka]

[0062] This, in turn, leads to a low molecular weight and high dispersibility of the product. Therefore, the polymer of the present invention is accessible only by reaction temperatures from -10 to 90°C and by the use of an alkylglycidyl ether containing less than 1 wt% epichlorohydrin. Preferably, the alkylglycidyl ether contains less than 0.5 wt% epichlorohydrin, more preferably less than 0.1 wt%. Most preferably, the alkylglycidyl ether is epichlorohydrin-free. Since glycidyl methyl ether and epichlorohydrin have similar boiling points, for example, epichlorohydrin is 117.9°C and glycidyl methyl ether is 110°C, it is not possible to remove epichlorohydrin from glycidyl methyl ether to the extent required. A procedure for preparing an epichlorohydrin-free alkylglycidyl ether is provided in Example 1 of the present invention.

[0063] Furthermore, high temperatures are the starting anion An - Alternatively, it can also lead to side reactions of group X of the living polymer. Therefore, limiting the temperature within the aforementioned range also improves the end group fidelity at the α-terminus of the polymer of the present invention, allowing for an end group fidelity of 95% or more of the X residue. Other known methods for polymerization of alkylglycidyl ethers do not provide the low degree of dispersibility required for this polymer. Furthermore, they do not allow for the same high end group fidelity.

[0064] To provide the random or statistical copolymers of the present invention, it is necessary to add at least two different types of monomers to the anion. The reactivity ratios of both alkylglycidyl ethers and ethylene oxides used are in the range of 0.7 to 1.3 or 0.6 to 1.7. For glycidyl methyl ether and ethylene oxide, the reactivity ratio is in the range of 0.98 to 1.02 in polymerization in DMSO. This yields a nearly ideally random copolymer. Copolymers of other forms, namely block structures, block-like structures, or tapered or gradient structures, can be prepared according to known methods. Block copolymers can be readily prepared by subsequent addition of different monomers to a living polymer. Therefore, the process of the present invention is preferred in which the step of adding at least one monomer and the step of allowing polymerization to proceed are repeated at least once, thereby using at least one monomer different from the first time.

[0065] A further embodiment of the present invention is the use of the polyether polymer of the present invention for the preparation of polymer conjugates with substrates. The substrate is as defined herein with respect to the conjugate of the present invention. The substrate is preferably a bioactive compound. There is a well-developed chemistry for the preparation of PEG conjugates, which is known to those skilled in the art. The same chemistry can be used for the preparation of the conjugate of the present invention. Specifically, the use of the present invention is preferred for the preparation of conjugated lipids for use in vaccines, based on lipid nanoparticles. These nanoparticles are preferably nanoparticles used against COVID-19. Conjugate of the polymer of the present invention with bovine serum albumin (BSA) is preferred. Conjugate with the polymer of the present invention in which the y group of the polymer is an N-succinimidyl carbonate group is particularly preferred. A conjugate of the polymer in which bovine serum albumin is conjugated to the polymer of the present invention by such group Y is even more preferred. Conjugation of bovine serum albumin with α-BzO-ω-N-succinimidyl carbonate-P(EG-co-GME) is most preferred.

[0066] Additional embodiments of the present invention relate to the preparation of the conjugate of the present invention. As discussed above, the conjugate can be prepared by any known process for the preparation of PEG conjugates. In addition, the present invention provides a process in which a process for the preparation of the polymer of the present invention is carried out first, and a substrate is added therefrom to a living polymer, the substrate having functional groups that react with the living polymer to form a conjugate. [Brief explanation of the drawing]

[0067] [Figure 1] Figure 1 shows the results of ELISA tests for three polymers of the present invention and mPEG. [Figure 2] ~ [Figure 4] These figures show the SEC trace of the polymer of the present invention synthesized in DMSO, and Figure 4 also shows the SEC trace of a commercially available mPEG. [Figure 5] Figure 5 shows the 1H-NMR spectrum of α-BzO-P(EG0.51-co-GME0.49). [Figure 6] Figure 6 shows the MALDI-TOF spectrum of α-BzO-P(EG0.51-co-GME0.49). [Figure 7] Figure 7 shows the cloud point of α-BzO-P(EG0.51-co-GME0.49). [Figure 8] Figure 8 shows the MALDI-TOF spectrum of α-MeO-P(PEG0.88-b-PGME0.22), i.e., the block copolymer of the present invention. [Figure 9] Figure 9 shows the decreasing 1H-NMR signal of the monomer during the preparation of the polymer of the present invention. [Figure 10] Figure 10 shows the results of a study on the relative reactivity of ethylene oxide and glycidyl methyl ether in anionic ring-opening polymerization. [Figure 11]Figure 11 shows the mechanism and side reactions of monomer-activated ring-opening polymerization using triisobutylaluminum, which leads to low terminal fidelity. In the formula, a) is epoxide activation; b) is "ate" complex formation; c) is initiation and growth; d) is termination; and e) is a side reaction. [Figure 12] Figure 12 shows the SEC traces of EG-GME copolymers synthesized in toluene at different temperatures. [Figure 13] Figure 13 shows the MALDI-TOF-MS results of BnO-P(EG-co-GME) synthesized in toluene at 25°C. [Figure 14] Figure 14 shows the stacked NMR spectra of copolymerization of EO and GME in toluene-d8 at different times of copolymerization. [Figure 15] Figure 15 shows the composition profile based on the obtained reactivity ratio. [Figure 16] Figure 16 shows the turbidity measurement of P(EG-co-GME) with different mol% GME at a constant concentration. [Figure 17] Figure 17 shows the synthesis of P(EG-co-GME) with different terminal groups. [Figure 18] Figure 18 shows the SEC traces of EO-GME copolymers with different terminal groups. [Figure 19] Figure 19 shows the SEC traces of α,ω-OH-P(EG-co-GME) and two PLLA-bP(EG-co-GME)-b-PLLA molecules. [Figure 20] Figure 20 shows the 1H-NMR spectrum of PLLA-bP(EG-co-GME)-b-PLLA. [Figure 21] Figure 21 shows the 1H-NMR-DOSY spectrum of PLLA-bP(EG-co-GME)-b-PLLA. [Modes for carrying out the invention] [Examples]

[0068] reagent: Unless otherwise noted, chemicals were purchased from TCI Europe NV, Acros Organics BVBA, Carl Roth GmbH, Sigma-Aldrich Co. LLC, and Honeywell Research Chemicals. Ethylene oxide was obtained from Air Liquide SA. Deuterated solvents were purchased from Deutero GmbH. THF was flushed with basic aluminum oxide before use. Glycidyl methyl ether was dried with CaH2 and cryotransferred before polymerization.

[0069] measurement: 1 H and 13 ¹³C-NMR spectra were recorded at 300 MHz using a Bruker Avance III-HD300 spectrometer, with the residual proton signal of the deuterated solvent used as an internal reference. 1 In the case of H-DOSY-NMR, the spectrum was recorded at 400 MHz using a Bruker Avance III-HD400 spectrometer, with the residual proton signal of the deuterated solvent used as an internal reference.

[0070] M of all samples w M n , and the degree of dispersion (M w / M n The PDI (Plant Diagram) was determined from the corresponding size exclusion chromatogram (calibrated according to the refractive index (RI) detector, DMF, and PEG standards).

[0071] Size exclusion chromatography (SEC) was performed at 50°C using a poly(2-hydroxyethyl methacrylate) (PHEMA) 300 / 100 / 40 column with dimethylformamide (DMF with 1 g / L LiBr) as the mobile phase (flow rate 1 mL / min). The polymer concentration was 1 mg / mL. Calibration was performed using a poly(ethylene glycol) standard (from Polymer Standards Service GmbH, Mainz, Germany).

[0072] Differential scanning calorimeter (DSC) measurements were performed on a PerkinElmer DSC8500 at a heating rate of 10 K / min in the temperature range of -100 to 100 °C. The temperature history of the samples was eliminated by two cooling and two heating cycles. For each sample, the glass transition temperature and melting temperature were obtained from the curve of the second heating cycle.

[0073] MALDI-ToF-MS measurements were performed using a Bruker autoflex-maX-MALDI-TOF / TOF system. Potassium trifluoroacetic acid and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) were used as the ionized salt and matrix, respectively.

[0074] Example 1: Synthesis of glycidyl methyl ether (GME) [ka]

[0075] a) Allyl methyl ether (10.0 g, 139 mmol) was dissolved in 274 mL of dichloromethane (DCM), and m-chloroperbenzoic acid (m-CPBA, 70%, 37.6 g, 153 mmol (based on m-CPBA)) was added to the solution. After stirring overnight, the solution was filtered and slowly concentrated under reduced pressure. Crude glycidyl methyl ether was separated from solid impurities by cryotransfer. Slow vacuum evaporation of residual dichloromethane gave pure glycidyl methyl ether as a colorless liquid; yield 41%.

[0076] b) 1-Chloro-3-methoxypropan-2-ol (3.00 g, 2.56 mL, 24.1 mmol) and anhydrous sodium sulfate (1.02 g, 7.23 mmol) were added to a flask equipped with a magnetic stirrer and cooled by a water bath. Grinding sodium hydroxide (1.25 g, 31.3 mmol) was added under stirring. After a complete reaction (TLC control), the crude product was cryotransferred from the reaction flask under vacuum and dried over CaH2 under cooling. After additional cryotransfer, GME was obtained as a colorless liquid in 93% yield.

[0077] Example 2: General procedure for preparing random copolymers α-BzO-P(EG 0.51 -co-GME 0.49 ) synthesis Ph-CH2-O-CH2-CH2-O-[CH2-CH2-O-] 0.51 -[CH2CH(CH2-O-CH3)-O-] 0.49 H Cesium hydroxide monohydrate (28.0 mg, 118 μmol) was dissolved in a THF-water mixture and transferred to a reaction flask equipped with a Teflon® stopcock and septum. 2-Benzyloxyethanol (20.0 mg, 18.7 μL, 131 μmol) was dissolved in benzene and added to the reaction flask. After slow evaporation of the solvent, the resulting solid was dried overnight under high vacuum at 60°C. The residue was dissolved in DMSO (5 mL) and the solution was cooled to -78°C. GME (367 mg, 374 μL, 4.16 mmol) was added by syringe, and EO (183 mg, 189 μL, 4.16 mmol) was cryotransferred from a graduated ampoule to the reaction flask. The solution was stirred under vacuum at room temperature for 48 hours. Subsequently, the solution was added to an excess of chloroform, and the organic phase was extracted with water (three times) and brine, dried with MgSO4, and filtered. After evaporation of the solvent and high-vacuum drying, P(EG) was used. 0.51 -co-GME 0.49 The product was obtained as a viscous liquid in quantitative yield. Product data can be found in Tables 2 and 3, item e.

[0078] Example 3: Synthesis of mPEG-b-PGME Me-O-[CH2-CH2-O-] 0.78 -[CH2CH(CH2-O-CH3)-O-] 0.22 H Potassium tert-butoxide (13.0 mg, 118 μmol) was dissolved in a tetrahydrofuran (THF)-water mixture and transferred to a reaction flask equipped with a Teflon stopcock and septum. Methoxypoly(ethylene glycol) (mPEG, M n2 kg / mol (250 mg, 125 μmol) was dissolved in benzene and added to the reaction flask. After slow evaporation, the residue was dried overnight under high vacuum at 80°C. The residue was dissolved in dimethyl sulfoxide (DMSO, 5 mL), and the solution was cooled to -78°C. Glycidyl methyl ether (253 mg, 258 μL, 2.88 mmol) was cryotransferred into the reaction flask. The solution was heated to 55°C and stirred under vacuum for 24 hours. Thereafter, the solution was poured into excess chloroform, and the organic phase was extracted with water (3 times) and brine, dried with MgSO4, and filtered. After precipitation with ice-cold diethyl ether, mPEG-b-PGME4k was obtained as a colorless solid; quantitative yield. Tables 2 and 3, item l, show the data for this polymer.

[0079] Example 4: Synthesis of α-BzO-ω-N-succinimidyl carbonate-P(EG-co-GME) [ka]

[0080] N,N'-disuccinimidyl carbonate (3.5 mg, 13.8 μmol) was added over 18 hours at room temperature to a stirred solution of α-BzO-P(EG-co-GME) (50.0 mg, 4.6 μmol) from Example 2 in dried CH3CN (1 ml). The reaction mixture was dissolved in dichloromethane, extracted with saturated NaHCO3, water, and brine, dried with MgSO4, and filtered. The solvent was removed under reduced pressure, and the polymer was vacuum-dried. The product was obtained as a white solid; quantitative yield.

[0081] Example 5: Conjugate of α-BzO-ω-N-succinimidyl carbonate-P (EG-co-GME) to bovine serum albumin (BSA). [ka]

[0082] Bovine serum albumin (5 mg, 0.07 μmol) and α-BzO-ω-N-succinimidyl carbonate-P (EG-co-GME) (16.9 mg, 2.6 μmol) from Example 4 were stirred in phosphate-buffered saline (PBS buffer) for 1 hour. Unreacted polymers were removed by dialyzing in deionized water using a 25 kDa membrane filter. The conjugates were then dried by freeze-drying to obtain a quantitative yield. In the preparation of this conjugate, the outer lysine groups (30-35) were targeted for conjugation.

[0083] Example 6: Competitive enzyme-coupled immunosorbent assay (ELISA) The interactions of items c, d, and e of copolymer samples were evaluated using a competitive PEG-ELISA kit with mouse monoclonal horseradish peroxidase conjugate anti-PEG antibody (HRP anti-PEG) (Life Diagnostics Inc., Westchester, PA, USA). Sample sizes ranged from 0 to 4600 μg / ml. -1 Samples of concentrations within the specified range were prepared in dilution buffer. In addition, 5000 g·mol -1 mPEG with a molecular weight of was used as an internal standard for comparison. 50 μl of each prepared sample was dispensed into a PEG-precoated 96-well plate, and 50 μl of HRP-anti-PEG was added to each well. The solution was incubated at 25°C for 1 hour using a microplate shaker, and then washed six times with 400 μl of wash buffer per well. After removing residual droplets, 100 μl of 3,3',5,5'-tetramethylbiphenyl-4,4'-diamine was added to each well, and the solution was mixed on a microplate shaker for 20 min. The reaction was stopped by adding 100 μl of stop solution, and the absorbance at 450 nm was read within 5 minutes.

[0084] Analysis of ELISA data: Determined absorbance values ​​were normalized to visualize the percentage of maximum binding. Sample concentration was log 10The function was converted to a function of the form. The sigmoid fit was calculated using the following equation, where a is the upper bound, b is the lower bound, c is the inflection point, and d is the Hill slope. y = a + (ba) / 1 + 10 (c-x)*d

[0085] Example 7: Kinetics of the synthesis of α-BzO-P(EG-co-GME) The synthesis of α-BzO-P(EG-co-GME) was performed online in situ in fully deuterated DMSO. 1 The process was repeated for 1H-NMR kinetic measurements. 95 mg glycidyl methyl ether (95 μl, 1.1 μmol) and 32 mg ethylene oxide (33 μl, 7.1 μmol), i.e., 60% glycidyl methyl ether and 40% ethylene oxide, were dissolved in fully deuterated DMSO. The consumption of ethylene oxide and glycidyl methyl ether over time was observed. 1 Measurements were performed by 1H-NMR spectroscopy. The proton signal of ethylene oxide at approximately 2.6 ppm was used as a measure of ethylene oxide concentration. For glycidyl methyl ether, the proton signal of the oxirane ring, which has absorption at approximately 2.75 and 3.1 ppm, was used. Figure 9 shows the selected results of the two monomers over time. 1 This shows a decrease in the H-NMR signal. Figure 10 shows monomer consumption M as a percentage of the total conversion rate. x,t / M x,t=0 The plot is shown. As can be seen, ethylene oxide and glycidyl methyl ether are consumed at exactly the same rate. This data indicates that the copolymer of the present invention is an almost ideal random copolymer.

[0086] Furthermore, at that place 1 ¹H-NMR kinetics were performed to investigate the effect of changing the solvent from DMSO to toluene on the copolymerization kinetics, as can be seen in Figure 14. In addition, based on the decrease in monomer signal during copolymerization, r EO =0.61 and r GMEA copolymerization parameter of 1.65 was obtained, showing a somewhat preferred incorporation of GME repeat units at the start of copolymerization due to the higher reactivity of GME compared to EO, leading to the statistical copolymer as can be seen from Figure 15.

[0087] Example 8: Synthesis of mP(EG 0.66 -co-GME 0.34 )(1) mP(EG 0.66 -co-GME 0.34 )(1): Diethylene glycol monomethyl ether (50.8 mg, 420 μmol) was dissolved in benzene (5 mL) and transferred by syringe under a steady vacuum to a flame-dried flask. Potassium tert-butoxide (42.7 mg, 380 μmol) was dissolved in stabilizer-free THF (3 mL) and a small amount of Millipore water and transferred to the flask. High vacuum was applied to the flask and the solvent was removed under high vacuum. The supplied initiator salt was dried at 60 °C overnight under high vacuum and finally dissolved in dry DMSO (5 mL) under a steady vacuum. After the supplied solution was frozen at -90 °C, GME (1.03 g, 11.6 mmol) was added to the flask by syringe. EO (1.04 g, 1.00 mL, 23.6 mmol) was added to the flask by cryotransfer before removing the cooling bath and the reaction mixture was allowed to warm to room temperature. The reaction was allowed to stir at room temperature for 72 h. Subsequently, a mixture of 2 M HCl (500 μL) and MeOH (500 μL) was added to stop the polymerization.

[0088] The reaction mixture was added to chloroform (20 mL) and washed three times with Millipore water (10 mL) and once with brine. Before removing the solvent under vacuum, the organic phase was dried over MgSO4. The supplied polymer was dialyzed against MeOH (MWCO of 2 kDa) for 24 h and freeze-dried overnight under high vacuum to give 1.82 g (86%) of the polymer (1) of Figure 17 as a pale yellow viscous liquid. The product data can be found in Tables 2 and 3, item n.

[0089] Furthermore, α-BzO-P(EG-co-GME) with varying amounts of monomer was prepared using the same procedure, with a polymerization temperature of 55°C and benzyloxyethanol as the initiator instead of diethylene glycol monomethyl ether (items c and d in Tables 2 and 3).

[0090] Example 9: mP(EG) according to Figure 17 0.66 -co-GME 0.34 Synthesis of )-Ms(2) mP(EG 0.66 -co-GME 0.34 )-Ms(2):polymer (1) (600 mg, 120 μmol) was dissolved in benzene (5 mL) and added to a flame-dried flask under argon flow. Benzene was slowly removed under high vacuum. The polymer was heated to 60°C and dried under high vacuum overnight. Under argon flow, the dried polymer was dissolved in dry DCM (10 mL) and triethylamine (97.1 mg, 960 μmol) and methanesulfonyl chloride (110 mg, 960 μmol) were added under ice cooling. The mixture was allowed to be stirred under argon at room temperature for 72 hours. The mixture was then washed three times with Millipore water and once with brine. The organic phase was dried with MgSO4 and the solvent was removed under high vacuum to give 427 mg (70%) of mesylated polymer (2) as a dark yellow viscous liquid. Product data can be found in Tables 2 and 3, item o.

[0091] Example 10: mP(EG) according to Figure 17 0.66 -co-GME 0.34 )-N3(3) synthesis Polymer (2) (400 mg, 80.0 μg) was dissolved in benzene (5 mL) and added to a flame-dried flask under an argon flow. The benzene was removed under high vacuum. The polymer was further heated to 60 °C and dried overnight under high vacuum. Subsequently, the dried polymer was dissolved in dry DMF (8 mL), and sodium azide (41.6 mg, 640 μmol) was added as a suspension in dry DMF (2 mL) under an argon flow. The reaction mixture was stirred at 65 °C for 72 h. The solvent was removed under high vacuum, and the residue was suspended in DCM (10 mL) and treated in an ultrasonic bath for 20 min. The remaining residue was filtered off, and the filtrate was washed three times with Millipore water and once with brine. The organic phase was dried over MgSO4, and the solvent was removed to give 284 mg (71%) of polymer (3) as a dark yellow viscous liquid. The product data can be found in Tables 2 and 3, entry p.

[0092] Example 11: mP(EG 0.66 -co-GME 0.34 )-NH2(4) Polymer (3) (180 mg, 40.0 μg) was dissolved in EtOH (10 mL). A catalytic amount of Pd / C 10 wt% (13.8 mg, 10.0 μmol of Pd) was added to the solution, and a balloon filled with excess H2 gas was connected to the flask. Vacuum was applied to the flask until the solvent began to evaporate, and the flask was then flushed with argon. This process was repeated two more times. Subsequently, vacuum was applied until the solvent began to evaporate, and the flask was flushed with H2 gas. The last step was repeated two more times. The reaction was allowed to stir under 1 atm of H2 gas for 48 h. After 24 h, the H2 balloon was refilled to ensure saturation of the atmosphere with H2. After 48 h, the balloon was removed, the catalyst was filtered off through zeolite, and the solvent was removed under high vacuum to give 142 mg (79%) of amino-terminated polymer (4). The product data can be found in Tables 2 and 3, entry q.

[0093] Example 12: Synthesis of α,ω-OH-P(EG-co-GME) 2,2'-((propane-2,2-diylbis(4,1-phenylene))bis(oxy))bis(ethane-1-ol)(BHEPP) (123 mg, 389 μmol) was dissolved in benzene (5 mL) and transferred by syringe to a flame-dried flask under argon. Potassium tert-butoxide (43.6 mg, 389 μmol) was dissolved in stabilizer-free THF (3 mL) and a small amount of Millipore water and transferred to the flask. High vacuum was applied to the flask and the solvent was removed under high vacuum. The resulting initiator salt was dried overnight at 70°C under high vacuum and finally dissolved in dry DMSO (5 mL) under steady vacuum. After freezing the resulting solution at -78°C, GME (873 mg, 890 μL, 9.91 mmol) was added to the flask by syringe. EO (1.46 g, 1.50 mL, 33.1 mmol) was added to the flask by cryotransfer before removing the cooling bath, allowing the reaction mixture to warm to room temperature. The reaction was allowed to proceed with stirring at 55°C for 22 hours. After venting the flask, Dowex® (30 mg) and water (4 mL) were added to the solution. After dialysis with water and freeze-drying, the polymer was obtained as a viscous liquid (1.36 g, 57%). Product data can be found in Tables 2 and 3, item r.

[0094] Example 13: PLLA 69 -bP(EG-co-GME)-b-PLLA 70 synthesis In a glove box, dried α,ω-OH-P(EG-co-GME) (400 mg, 66.7 μmol) and L-lactide (577 mg, 4.00 mmol) were dissolved in dried DCM (3 mL). DBU (6.08 mg, 40.0 μmol) dissolved in DCM (70.0 μL) was added, and the resulting solution was stirred for 30 min. After adding benzoic acid (24.4 mg, 200 μmol) in DCM (3 mL), the crude polymer was precipitated with cold diethyl ether. The final step was repeated twice. The block copolymer was obtained as a colorless solid (79%) after lyophilization from benzene.

[0095] Example 14: α-BzO-P(EG) in toluene 0.50 -co-GME 0.50 ) synthesis With all other copolymerization conditions except temperature and solvent kept constant compared to Example 8, only the effects of temperature and solvent on copolymerization were investigated. In the temperature range investigated (25-50°C), GME-EO copolymerization in toluene yields a well-defined copolymer with a low dispersion degree <1.10 and a unimodal, narrow molecular weight distribution, as can be seen in Figure 12.

[0096] As can be seen in Figure 13, α-BzO-P(EG 0.50 -co-GME 0.50 MALDI-ToF-MS in toluene (at 25°C) is theoretical and experimentally effective. n The values ​​show a clear overlap (2 kg·mol). -1 ), we have revealed exceptionally high terminal group fidelity (>99%) from copolymerization in DMSO.

[0097] The product data can be found in Tables 2 and 3, items s through u.

[0098] Analysis of the results Tables 2, 3, 4, and 5, and Figures 1 through 21, show the properties of the prepared polymers. These will be discussed below.

[0099] [Table 2]

[0100] [Table 3]

[0101] [Table 4]

[0102] Table 4 shows the temperature characteristics of each compound. Polymers were prepared according to Example 8, using varying amounts of monomer and benzyloxyethanol as an initiator instead of diethylene glycol monomethyl ether. The cloud points of random copolymers of EO and GME were investigated by turbidimeter measurements of copolymers containing a high amount (≧59 mol%) of GME. The cloud point was defined as a 50% transmittance. Copolymers initiated by benzyloxyethanol synthesized by anionic ring-opening polymerization (AROP) in DMSO were used for the measurements.

[0103] The cloud point of the copolymer decreases with increasing amounts of GME on the polymer backbone. Despite the decrease in the cloud point due to increasing mol% of GME, water solubility under physiological conditions is guaranteed for all copolymers.

[0104] immunogenicity As can be seen from Table 3, items c through e and l have approximately the same molecular weight, i.e., approximately 4 to 5 kg / mol M n The present invention shows copolymers comprising ethylene oxide repeating units and glycidyl methyl ether repeating units. The amount of glycidyl methyl ether repeating units increases from 16% to 49% from c to e (see Table 2, items c to e and l). Figure 1 shows the ELISA test results for polymers c to e and commercially available mPEG. It shows the normalized absorption function at a wavelength of 450 nm against the log10 function of the polymer concentration in nanograms per ml, thus illustrating the anti-PEG antibody interaction depending on the polymer concentration examined. The ELISA data shows that a strong effect can be observed with increasing concentrations of alkyl side chains (it is important to note that the x-axis has a logarithmic scale). With increasing amounts of GME incorporated into the polyether structure, significantly higher polymer concentrations are required to observe the interaction between the copolymer and APA. Notably, at 49 mol% GME, there is no interaction between APA and copolymer. This means that each copolymer cannot be detected by APA, i.e., it is not immunogenic.

[0105] degree of dispersion As can be seen from Table 3, items c through e and l, all polymers of the present invention have a very low degree of dispersion, i.e., 1.10 or lower. For visualization, the SEC traces of these polymers are shown in Figures 2 through 4 and 12. In addition, Figure 4 shows the SEC trace of mPEG. It is clear that the present invention provides polymers with a very narrow molecular weight distribution.

[0106] Preparation and Purity of the Polymer of the Present Invention Figure 5 shows α-BzO-P(EG 0.51 -co-GME 0.49 ), that is, the polymer of Example 2 1 The 1H-NMR spectrum is shown (item e in Tables 2 and 3). The product is obtained as in Example 2 without further purification. As can be seen, this method provides not only polymers with a narrow molecular weight distribution, but also very pure polymers without the need for cumbersome purification steps. This is an important feature for use in pharmaceutical applications.

[0107] The synthesis of bifunctionally initiated α,ω-OH-P(EG-co-GME) copolymers enables the synthesis of ABA triblock copolymers. In this case, P(EG-co-GME) with two hydroxyl ends (Example 12) was used as a polymer initiator for ring-opening polymerization of L-lactide, enabling the synthesis of PLLA-bP(EG-co-GME)-b-PLLA. In the formula, PLLA is [ka] Polymerization is catalyzed by the organic base DBU (1,8-diazabicyclo[5.4.0]undeca-7-ene).

[0108] Figure 19 shows the polymer initiator α,ω-OH-P(EG-co-GME) (polymer of Example 12), and two PLLA-bP(EG-co-GME)-b-PLLA(PLLA) having different degrees of polymerization (k=46, 47 and 69, 70). 46 -bP(EG-co-GME)-b-PLLA47 The image shows the overlap of SEC traces of the polymer in Example 13, confirming successful chain elongation from the polymer initiator.

[0109] Figure 20 shows the 1H-NMR spectrum (sprectum) of Example 13, which confirms the successful polymerization of L-lactide.

[0110] Figure 21 confirms that the blocks on the copolymer are linked by covalent bonds. 1 This shows H-DOSY-NMR.

[0111] End-head fidelity As can be seen from Table 3, the end group fidelity of all polymers in the present invention is close to 100%. Figure 6 shows α-BzO-P(EG) having potassium and sodium cations. 0.51 -co-GME 0.49 The MALDI-TOF mass spectrum of the polymer in Example 2 (item e in Tables 2 and 3) is shown. The only peak visible here is that of the product molecule. There are no peaks from polymers with different end groups. This indicates that the end group fidelity of the polymer of the present invention is very high. In addition, the spectrum confirms a narrow molecular weight distribution.

[0112] soluble Figure 7 shows α-BzO-P(EG 0.51 -co-GME 0.49) That is, the cloud point measurement of the polymer of Example 2 (Item e in Tables 2 and 3) is shown. The cloud point determines the temperature at which immiscibility in water / aqueous solution is observed upon heating. As an example, for a copolymer with 49% GME, the cloud point can be observed at \(95^{\circ}C\), indicating excellent water solubility of the P(EG-co-GME) copolymer. The homopolymer PEG shows a cloud point of ca.\(100^{\circ}C\). All other copolymers of the present invention having 26 - 49% GME are between PEG and this copolymer and show cloud points of \(96 - 100^{\circ}C\). For copolymers with 25 mol% and lower GME incorporation, the cloud point was not detected in water at temperatures in the range of 0 to \(99^{\circ}C\). Therefore, GME copolymerization has little effect on water solubility, which is a characteristic of the similar structures of polyethylene glycol and methyl ethyl ether. This is an important characteristic of the polymers of the present invention. Because it shows that they can replace PEG in an aqueous system without causing phase separation or even precipitation problems.

[0113] Comparison of the dispersity of the end - group fidelity of the polymers of the present invention with that of mPEG Figure 8 shows the overlap of the MALDI - TOF mass spectra of commercially available mPEG and the block copolymer of the present invention (the polymer of Example 3 (Item l in Tables 2 and 3)). As can be seen, a distinct shift between the high - molecular - weight initiator mPEG and Item l is observed, indicating successful block copolymer synthesis. The absence of the high - molecular - weight initiator in the MALDI - ToF - MS of Item l further proves quantitative block copolymer formation. This is further proven by the distance of the signal of \(44 g\cdot mol\) -1 depending on the signal distance.

[0114] Degree of crystallinity Table 5 shows some temperature characteristics of the polymers in Tables 2 and 3:

[0115]

Table 5

[0116] As can be seen, the crystallinity of the polymer of the present invention is low and can be completely interrupted by the incorporation of GME monomers in the polymer structure. This prevents, for example, the accumulation of the polymer in the kidney or liver. [Appendix 1] A polyether polymer represented by the following formula [I]: -(CH2CHR-O-) m - [I] In the formula, - 10 to 90% of the residue R is hydrogen, and 10% to 90% of the residue R is independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; - 1 to 100% of the residue R is methoxymethyl; - Up to 50% of the residue R can be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; - And - m is in the range of 10 to 1000, A polyether polymer characterized in that the dispersity measured by size exclusion chromatography in DMF is 1.15 or less according to calibration based on the PEG standard. [Appendix 2] The polyether polymer according to Appendix 1, characterized in that the polyether is a poly(glycidyl methyl ether-co-ethylene oxide) copolymer. [Appendix 3] The polyether polymer according to any one of Appendices 1 and 2, characterized in that 30 to 70% of the residue R is hydrogen and the remaining residue R is methoxymethyl. [Appendix 4] The polyether polymer according to any one of Appendices 1 to 3, characterized in that the polymer is a random copolymer or a statistical copolymer. [Appendix 5] A polyether polymer according to any one of Appendices 1 to 4, represented by the following formula [II]: X-(CH2CHR-O-) m CH2CHR-Y [II] In the formula, X is selected from the group consisting of hydrogen, alkyl, hydroxy, sulfanyl, C1-C10 alkoxy, C1-C10 thioalkoxy, amino, amino-C1-C10 alkoxy, dibenzylamino-C1-C10 alkoxy, amide, N heterocyclic carbene, and N heterocyclic olefin; Y is selected from the group consisting of hydrogen, hydroxyl, alkoxy, -CH2-C(=O)-R, -CHO, alkylcarbonyloxy, alkoxycarbonyloxy, amine, C1-C10 alkylamine, carboxamide, azide, halogen, sulfanyl, thioalkoxy, N-succinimidyl carbonate, and sulfonate; X and / or Y may also be selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, proteins, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces, which are directly bonded to the polymer by covalent bonds or by spacers. m is in the range of 9 to 999. A polyether polymer characterized by having a dispersion degree of 1.15 or less, as measured by size exclusion chromatography in DMF, according to calibration using PEG standards. [Note 6] The polyether polymer described in Appendix 5, characterized in that X is an alkoxy and Y is a hydroxyl group. [Note 7] The polyether polymer described in Appendix 5, characterized in that X and Y are each hydroxyl groups. [Note 8] The polyether polymer described in appendices 1 to 4, represented by the following formula [II]: X-(CH2CHR-O-) m CH2CHR-Y [II] In the formula, X is selected from the group consisting of hydrogen or X', In the formula, X' is selected from the following group: [ka] In the formula, R” = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl, p = 1, 2, 3, 4, or 5, k = 5 to 500;

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

1. A polyether polymer represented by the following formula [I]: -(CH ２ CHR-O-) ｍ - [I] During the ceremony, - 10 to 90% of residue R are hydrogen, and the remaining residue R is independently selected from the group consisting of methoxymethyl, ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; - At least 1% of residue R is methoxymethyl; - Up to 50% of residue R can be selected from the group consisting of ethoxymethyl, n-propoxymethyl, and isopropoxymethyl; - and -m is in the range of 10 to 1000. A polyether polymer characterized by having a dispersion degree of 1.15 or less, as measured by size exclusion chromatography in DMF, based on calibration using PEG standards.

2. The polyether polymer according to claim 1, characterized in that the polyether is a poly(glycidyl methyl ether-co-ethylene oxide) copolymer.

3. The polyether polymer according to claim 1, characterized in that 30 to 70% of residue R is hydrogen and the remaining residue R is methoxymethyl.

4. The polyether polymer according to any one of claims 1 to 3, characterized in that the polymer is a random copolymer or a statistical copolymer.

5. A polyether polymer according to any one of claims 1 to 3, represented by the following formula [II]: X-(CH ２ CHR-O-) ｍ CH ２ CHR-Y [II] In the formula, X is selected from the group consisting of hydrogen, alkyl, hydroxy, sulfanyl, C1-C10 alkoxy, C1-C10 thioalkoxy, amino, amino-C1-C10 alkoxy, dibenzylamino-C1-C10 alkoxy, amide, N heterocyclic carbene, and N heterocyclic olefin; Y is hydrogen, hydroxyl, alkoxy, and R is as defined above - CH ２ Selected from the group consisting of -C(=O)-R, -CHO, alkylcarbonyloxy, alkoxycarbonyloxy, amine, C1-C10 alkylamine, carboxamide, azide, halogen, sulfanyl, thioalkoxy, N-succinimidyl carbonate, and sulfonate; X and / or Y may also be selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, proteins, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces, which are directly bonded to the polymer by covalent bonds or by spacers. m is in the range of 9 to 999. A polyether polymer characterized by having a dispersion degree of 1.15 or less, as measured by size exclusion chromatography in DMF, based on calibration using PEG standards.

6. The polyether polymer according to claim 5, characterized in that X is an alkoxy and Y is a hydroxyl group.

7. The polyether polymer according to claim 5, characterized in that X and Y are each hydroxyl.

8. A polyether polymer according to any one of claims 1 to 3, represented by the following formula [II]: X-(CH ２ CHR-O-) ｍ CH ２ CHR-Y [II] In the formula, X is selected from the group consisting of hydrogen or X', In the formula, X' is selected from the following group: 【Chemistry 1】 In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl. p = 1, 2, 3, 4, or 5, k = 5 to 500; 【Chemistry 2】 In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl. p = 1, 2, 3, 4, or 5, k = 5 to 500; 【Transformation 3】 In the formula, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl. q = 3, 4, or 5, k = 5 to 500; and 【Chemistry 4】 In the formula, k = 5 to 500; In the formula, Y is Z-W, meaning it is composed of substituents Z and W linked by a covalent bond. During the ceremony, Z is -O-R'-O-, W is -R-CHCH ２ -(O-CHRCH2-) ｍ -V is; In the equation, V is either X or X'; In the formula, R' is selected from the following group: 【Transformation 5】 During the ceremony, R'' = H, or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl, and thioalkoxymethyl. s = 0 to 20, t = 0 to 20; Further R's are selected from the following: 【Transformation 6】 During the ceremony, m is in the range of 9 to 999. A polyether polymer characterized by having a dispersion degree of 1.15 or less, as measured by size exclusion chromatography in DMF, based on calibration using PEG standards.

9. The polyether polymer according to claim 5, characterized in that the end group fidelity of the polymer is at least 95% with respect to group X and / or group Y.

10. A conjugate characterized by comprising a polyether polymer and a substrate as described in any one of Claims 1 to 3.

11. The conjugate according to claim 10, characterized in that the substrate is selected from the group consisting of low molecular weight drugs, nanocarriers, liposome structures, peptides, polypeptides, proteins, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces, and interfaces.

12. A process for preparing a polyether polymer according to any one of claims 1 to 3, comprising the following steps: - Anion An － Providing, - expression 【Transformation 7】 Add at least one monomer, wherein R is as described in any one of claims 1 to 3. - Allow polymerization to proceed at temperatures in the range of -10 to 90°C. A process for preparing polyether polymers, characterized by containing less than 1 wt% epichlorohydrin as the monomer.

13. The process according to claim 12, characterized in that the steps of adding at least one monomer and allowing polymerization to proceed are repeated at least once, thereby using at least one monomer different from the first time.

14. An － The counterion for the anion is Na ＋ _K ＋ , and Cs ＋ The process according to claim 12, characterized in that it is selected from the group consisting of the following.

15. Use of the polyether polymer according to any one of claims 1 to 3 for the preparation of polymer conjugates with bioactive compounds.

16. Specifically, the use according to claim 15 for the preparation of conjugated lipids for use in vaccines, characterized in that these nanoparticles are based on lipid nanoparticles, and these nanoparticles are preferably nanoparticles used against COVID-19.

Images