Heterotelechelic compounds

Abstract

The present invention relates to biocompatible polymers suitable for drug and gene delivery, and methods for producing the same. The present invention also enables the creation of compounds with diverse structures by a simple method. The present invention relates to polycyclic iminoether compounds, such as poly(2-oxazoline), having different functional groups at both ends, for example, a compound having a pentafluorophenyl group at one end and an azide group at the other end, derivatives thereof, and methods for producing the same.

Description

[Technical Field]

[0001] The present invention relates to heterotelechelic poly(2-oxazoline) derivative compounds. [Background technology]

[0002] Polymeric materials are being developed as medical materials, including biomaterials that come into direct contact with living organisms. To be used as biomaterials, they must meet various requirements, including functionality, physical properties, non-toxicity, stealth, and biocompatibility. Applications of PEG as such polymers have been attempted, including functional microparticles, functional surfaces, and functional drug carriers. Patent Document 1 discloses a polymer composition for forming a biosensor surface (S) using surface plasmon resonance (SPR), which has a polyethylene glycol segment bearing a mercapto group at one end and a functional group or ligand at the other end. Patent Document 2 also discloses a heterotelechelic block copolymer, which is combined with a hydrophobic polymer and has different functional groups at both ends and polyethylene oxide as a hydrophilic polymer, and can be used as a material for direct application to living organisms. However, PEG has drawbacks, such as the detection of anti-PEG antibodies in a certain percentage of subjects who have not previously been treated with PEG formulations, and the fact that the polyether structure of PEG is easily oxidized and degraded. Therefore, the development of polymeric materials other than PEG is anticipated. Therefore, there is hope for the development of medical materials based on poly(2-oxazoline) (POx) derivative compounds as new, more stable polymeric materials that maintain the above-mentioned stealth and biocompatibility requirements (Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2001 / 086301 [Patent Document 2] WO1996 / 033233 [Non-patent literature]

[0004] [Non-Patent Document 1] Macromol.Rapid Commun.33(19), 2012. [Non-patent document 2] J Mater Sci: Mater Med.25(5):1211-1225. [Non-patent document 3] Polym.Chem., 2021,12, 6392-6403. Summary of the Invention [Problem to be solved by the invention]

[0005] Although research into poly(2-oxazoline) (POx) derivative compounds has been conducted (Non-Patent Document 3), it has not yet been possible to control the reactions at both ends of POx to create a variety of compounds.

[0006] Therefore, the present disclosure provides a technology for controlling the reactivity of both ends of POx to create a wide variety of compounds. Specifically, the present inventors provide compounds and derivatives that can freely introduce desired functional groups into both ends by utilizing the difference in reactivity between the two ends of the polymer, as well as methods for producing them.

[0007] We investigated how to initiate 2-oxazoline polymerization using pentafluorobenzyl bromide or tosylate as starting materials under different experimental conditions (reaction time, temperature, solvent). We also succeeded in selectively initiating the 2-oxazolinium chain end with various nucleophiles, including N-, O-, and S-nucleophiles, without reacting the para-fluoro group. Furthermore, we investigated the derivatization of heterotelechelic compounds via para-fluoro substitution, and obtained novel heterotelechelic compounds, as illustrated in Figure 2.

[0008] As polymeric materials that can be used as biomaterials, the development of novel polymeric materials with low immunogenicity and high stability that can be converted by precisely controlling the functional groups introduced at the terminals is expected. The compounds of the present disclosure are useful for the development of such polymeric materials, although they are not limited thereto. [Means for solving the problem]

[0009] The present disclosure provides compounds of formula (I) and methods for making same.

[0010] As a result of extensive research, the present inventors have found that various functional groups can be suitably derived from a polymerized poly(2-oxazoline) compound having a pentafluoro group at one end by adjusting the reaction conditions, and have thus completed the present invention. That is, one aspect of the present invention is as follows.

[0011] [1] Formula (I): [ka] wherein X is a halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -C≡CR 2 , -NHOR 2 , -ONR 2 R 3 , optionally substituted heteroaryl, a group capable of forming a lipid-based nanoparticle (e.g., a form including a lipid nanoparticle (LNP), a liposome, or a lipoplex), or a micelle containing a lipid-solubilizing group such as a polycationic group, a polyanionic group, or a group derived from a fatty acid, preferably F, -SR 2 , -OR 2 , -CN, -NHR 2 , -N3, [ka] and; Y is SR 2 , -OR 2 (However, when X is F, it is not -OH), -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3 or optionally substituted heteroaryl, preferably -SR 2 , -OR 2 (However, when X is F, it is not -OH), -NHR 2 , -NR 2 R 3 , -N3 or [ka] and more preferably, -SR 2 , -OR 2 (However, if X is F, it is not -OH), -NHR 2 ,or [ka] and; where Repeating Unit [ka] are independent in each occurrence and may be the same or different; R 1 is selected from the group consisting of an optionally substituted alkyl group having 1 to 40 carbon atoms, an optionally substituted alkenyl group having 2 to 40 carbon atoms, an optionally substituted alkynyl group having 2 to 40 carbon atoms, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R 2 and R 3are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl, preferably —CHCH(OR C )CH2OR C and; R A , R AA and R AAA are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R A and R AA may be linked together to form a ring, e.g., a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam; R A , R AA and / or R AAA may further be linked to a functional molecule such as a label, or a biofunctional molecule including a protein, nucleic acid, etc.; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, lipid-soluble groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, and optionally substituted alkynyl groups having 2 to 20 carbon atoms, and oxygen protecting groups such as acyl groups such as acetyl, and ether groups such as methoxymethyl; The alkyl, alkenyl and alkynyl are straight, branched or cyclic chains; m is an integer from 1 to 3; and n is an integer between 1 and 2000. or a pharmaceutically acceptable salt thereof. [2] X is halogen, -SR 2 , -OR 2 , -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -NHOR 2 , -ONR 2 R 3 , optionally substituted heteroaryl; Y is SR 2 , -OR 2 , -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3 or [ka] and; More preferably -SR 2 , -OR 2 , -NHR 2 ,or [ka] and; R 1is selected from the group consisting of an optionally substituted alkyl group having 1 to 40 carbon atoms, an optionally substituted alkenyl group having 2 to 40 carbon atoms, an optionally substituted alkynyl group having 2 to 40 carbon atoms, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl, preferably -CHCH(OR C )CH2OR C and R A , and R AA are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R A and R AA may be linked together to form a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R A and / or R AAmay further be linked to a label or a biofunctional molecule such as a protein, nucleic acid, etc.; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, lipid-solubilizing groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, and optionally substituted alkynyl groups having 2 to 20 carbon atoms, and oxygen protecting groups; The alkyl, alkenyl, and alkynyl are straight or branched chain; The alkyl, alkenyl and alkynyl substituents are, independently of one another, and without limitation, ester groups, amino groups, azide groups, and n is an integer between 2 and 1000; [1] The compound or a pharmaceutically acceptable salt thereof according to [1]. [3] X is F, -SR 2 , -OR 2 , -CN, -NHR 2 , -N3, or [ka] and; Y is -SR 2 , -OR 2 , -NHR 2 , -N3, or [ka] and; R 1 is selected from the group consisting of methyl, ethyl, propyl, butyl, -CH2CH2COOCH3, -CH2CH2CH2COOCH3, -CH2CH2CH2CH2COOCH3, butenyl, butynyl, and -CH2OCH3; R 2 represents hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , a sugar derivative; R A and R AA are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R A and R AA may be linked together to form a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R A and / or R AA may be further linked to a functional molecule such as a label, or a biofunctional molecule such as a protein, nucleic acid, etc.; R B is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, and an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer between 2 and 2000; [1] The compound or a pharmaceutically acceptable salt thereof according to [1]. [4] Y is [ka] and R A and / or R AA may be linked to a label or a functional molecule selected from the group consisting of biofunctional molecules including proteins and nucleic acids; The compound according to [1]. [5] X is -OR 2 or -SR 2 and; Y is [ka] and; R 2 is -CH2CH(OR C )CH2OR C and R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, and an optionally substituted alkenyl group having 2 to 20 carbon atoms; The compound according to [1]. [6] X is -OR 2 or -SR 2 and; R 2 is -CH2CH(OR C )CH2OR C and R C is selected from the group consisting of optionally substituted alkyl groups having 1 to 20 carbon atoms and optionally substituted alkenyl groups having 2 to 20 carbon atoms; The compound according to [1]. [7] X is -OR 2 , -SR 2 , or -NHR 2 and; R 2 is -CH2CH(OR C )CH2OR C and R C is hydrogen; The compound according to [1]. [8] X is -OR 2 , -SR 2 , or -NHR 2 and; R 2 is -CH2CH(OR C )CH2OR C and R C is an oxygen protecting group; The compound according to [1]. [9] X is a group capable of forming lipid-based nanoparticles (e.g., lipid nanoparticles (LNPs), liposomes, or lipoplexes) or micelles; The compound according to [1].

[10] The compound according to [1], wherein the sugar derivative is a glucose derivative.

[11] A composition comprising the compound according to any one of [1] to

[10] .

[12] A composition comprising the compound according to any one of [1] to

[10] , wherein the composition is a vaccine.

[13] A composition comprising the compound according to any one of [1] to

[10] , wherein the composition is used as a diagnostic probe.

[14] Formula (II) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR2 , and -N3, -NHOR 2 , and -ONR 2 R 3 selected from the group consisting of: R 2 and R 3 are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , and sugar derivatives, or R 5 Ga-NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl, preferably -CHCH(OR C )CH2OR C and; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; The alkyl and alkenyl are straight chain; m is an integer from 1 to 3; and n is an integer between 2 and 2000. A compound represented by the formula:

[15] R5 -OR 2 , -SR 2 , or --NHR, R 2 is -CH2CH(OR C )CH2OR C and R C is selected from the group consisting of optionally substituted alkyl groups having 1 to 20 carbon atoms and optionally substituted alkenyl groups having 2 to 20 carbon atoms; The compound according to

[14] .

[16] R 5 -OR 2 , -SR 2 , or -NHR; R 2 is -CH2CH(OR C )CH2OR C and R C is H; The compound according to

[14] .

[17] R 5 -OR 2 , -SR 2 , or -NHR 2 and; R 2 is -CH2CH(OR C )CH2OR C and R C is an oxygen protecting group; The compound described in

[14] .

[18] Formula (III) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5is halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -NHOR 2 , - and -ONR 2 R 3 selected from the group consisting of; R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; The alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. A method for producing a compound represented by the formula: It includes the following steps; (a) Formula (IV) [ka] [In the formula, L is a leaving group.] with a compound of formula (V) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl. in the presence of a base to form a compound of formula (II) [ka] to obtain a compound of the formula (b) reacting a compound of formula (II) with an azide reagent, A method for preparing a compound of formula (III).

[19] The leaving group is selected from the group consisting of halogen, OTf, ONs and OTs;

[18] A method for producing a compound of formula (III) according to

[18] .

[20] The method for producing the compound of formula (III) according to

[18] , wherein the base is selected from the group consisting of Et3N, DBU, NMP, TBD, KOH, iPr2NEt, and t-BuOK.

[21] Azide reagents include NaN3 and TMSN. 3、 A method for producing a compound of formula (III) according to

[18] , wherein the compound is selected from the group consisting of TsN3, tetrabutylammonium azide, and diphenylphosphoryl azide.

[22] Formula (III) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 is halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , and -N3, -NHOR 2 , -ONR 2 R 3 selected from the group consisting of: R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl; R Bis selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. A method for producing a compound represented by the formula: It includes the following steps; Formula (IIIa) [ka] [In the formula, R 4 is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; and n is an integer from 2 to 2000. The compound represented by formula (III) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 is halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R3 , -NHNHR 2 , -N=NR 2 , and -N3, -NHOR 2 , and -ONR 2 R 3 selected from the group consisting of: R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. with a nucleophile to obtain a compound represented by the formula (III).

[23] The method for producing a compound of formula (III) according to

[22] , wherein the nucleophile is an alcohol, phenol, carboxylic acid, amine, azide, thiol, cyanide, or alkyne type nucleophile.

[24] Formula (VI) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 is halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -NHOR 2 , and -ONR 2 R 3 selected from the group consisting of: R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl; R Bis selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 100. A method for producing a compound represented by the formula: Formula (IIa) [ka] [In the formula, R 4 is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; and n is an integer between 2 and 2000. A method for producing a compound represented by formula (IV) above, comprising reacting a compound represented by formula (IV) with a nucleophile to obtain a compound represented by formula (VI).

[25] Formula (Ia) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 -SR 2 , -OR 2 , -CN, -NHR 2 , -NR2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -NHOR 2 , and -ONR 2 R 3 selected from the group consisting of: R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl, preferably —CHCH(OR C )CH2OR C and; R A and R AA are each independently selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R A and R AA may be linked together to form a ring, such as a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R A and / or R AAmay further be linked to a functional molecule such as a label, a biofunctional molecule such as a protein, a nucleic acid, etc.; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, lipid-solubilizing groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, and optionally substituted alkynyl groups having 2 to 20 carbon atoms, or oxygen protecting groups including acyl groups such as acetyl, and ether groups such as methoxymethyl; The alkyl, alkenyl, and alkynyl are straight, branched, or cyclic chains; and n is an integer between 1 and 2000. A method for producing a compound represented by the formula: Formula (III) [ka] [In the formula, R 4 is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl; R 5 is halogen, -SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , and -N3, -NHOR 2 , -ONR 2 R 3 selected from the group consisting of: R2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , -CH2CH(OR C )CH2OR C , and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl; R B is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms; R C is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; The alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. The compound represented by formula (VII) [ka] [R A and R AA is as above] By reacting with a compound represented by A method for producing a compound represented by formula (Ia), which provides a compound represented by formula (Ia).

[26] The method for producing a compound represented by formula (Ia) according to

[25] , wherein the reaction is carried out using a metal catalyst such as a Cu catalyst.

[27] The method for producing the compound represented by formula (Ia) according to

[25] , wherein the reaction is carried out without using a metal catalyst.

[28] The method for producing a compound represented by formula (Ia) according to

[25] , wherein the reaction is carried out in water. [Effects of the Invention]

[0012] The compounds of the present invention are useful as polymeric materials for, but not limited to, drug therapy, gene therapy, laboratory diagnostics, regenerative medicine, and pharmaceutical components such as excipients, parts of protein conjugates, protein delivery, etc. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows an outline of the pentafluorobenzyl poly(2-substituted-2-oxazoline) compound of the present invention. [Figure 2] FIG. 2 shows an outline of the heterocyclic benzyl poly(2-substituted-2-oxazoline) compound of the present invention. [Figure 3] FIG. 3 shows the time course of the polymerization reaction of ethyloxazoline with pentafluorobenzyl bromide as the starting material. [Figure 4] FIG. 4 shows the spectrum of the polymerized 2-ethylpropionic acid oxazoline monomer using pentafluorobenzyl bromide as the starting material and terminated with sodium azide. [Figure 5] Figure 5 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with sodium azide. [Figure 6]Figure 6 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 7] FIG. 7 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 3,3-diethoxypropylamine. [Figure 8] Figure 8 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with phenol. [Figure 9] FIG. 9 shows the spectrum obtained when 2-ethyloxazoline monomer was polymerized using pentafluorobenzyl bromide as a starting material and the reaction was terminated with acetic acid. [Figure 10] FIG. 10 shows the time course of the reaction of 2-ethyl-2-oxazoline with piperidine. [Figure 11] FIG. 11 shows the spectrum of a polymerization reaction starting with 2-ethyloxazoline monomer and pentafluorobenzyl tosylate, terminated with sodium azide. [Figure 12] FIG. 12 shows the spectrum obtained when pentafluorobenzyl tosylate was used as a starting material to polymerize 2-ethyloxazoline monomer, and the reaction was terminated with 2-Boc-aminoethanethiol. [Figure 13] FIG. 13 shows the 1H NMR of PFP-PEtOx when prepared in chlorobenzene and selectively terminated with tert-butyl N-(2-mercaptoethyl)carbamate. [Figure 14] Figure 14 shows A) the retention time shift of extended polymers upon block copolymer synthesis, and B) the H NMR of PFP-PEtOx prepared in chlorobenzene and selectively terminated with tert-butyl N-(2-mercaptoethyl)carbamate. [Figure 15] FIG. 15 shows the measurement results for C18-PMeOx liposomes. [Figure 16]FIG. 16 shows the molecular weight distribution of C14-PEG, C14-POx, and C18-POx, as well as firefly luciferase expression in Balb / C mice 4 hours after intravenous administration of C14-PEG, C14-POx, and C18-POx, respectively. [Figure 17] Figure 17 shows the results of in vitro assays evaluating the quantification of firefly luciferase expression levels in Balb / C mice 4 and 24 hours after intravenous administration of C14-PEG, C14-POx, and C18-POx, respectively, and in major organs 24 hours after intravenous administration. [Figure 18] Figure 18 shows the results of immunizing mice with a C14-POxLNP preparation containing mRNA encoding the COVID-19 spike protein. [Figure 19] FIG. 19 shows the H NMR of PFP-PEtOx-N3. [Figure 20] FIG. 20 shows the 19F NMR and mass spectra of the polymerized 2-ethyloxazoline monomer using pentafluorobenzyl bromide as the starting material, and the reaction terminated with sodium azide. [Figure 21] FIG. 21 shows the 19F NMR and mass spectra of the polymerized 2-methyloxazoline monomer using pentafluorobenzyl bromide as the starting material and terminated with sodium azide. [Figure 22] FIG. 22 shows the 19F NMR and mass spectra of the polymerized 2-propyloxazoline monomer using pentafluorobenzyl bromide as the starting material and terminated with sodium azide. [Figure 23] FIG. 23 shows the 19F NMR and mass spectra of the polymerized 2-methoxycarboxyethyl-2-oxazoline monomer using pentafluorobenzyl bromide as the starting material, and the reaction terminated with sodium azide. [Figure 24]FIG. 24 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 25] Figure 25 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 2-hydroxyethanethiol. [Figure 26] Figure 26 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 2-aminoethanethiol. [Figure 27] FIG. 27 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 3-mercaptopropionic acid. [Figure 28] Figure 28 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 2-aminoethanethiol. [Figure 29] FIG. 29 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 30] FIG. 30 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 31] Figure 31 shows the 19F NMR and mass spectra of the thioglycosidic reaction at the para-fluorine position of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide. [Figure 32] FIG. 32 shows the GPC results of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide modified with cysteamine. [Figure 33]FIG. 33 shows the 19 F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzyl poly(2-methylpropionate-2-oxazoline) azide with 2-aminoethanethiol hydrochloride. [Figure 34] FIG. 34 shows a comparison of parafluoroamine substitution in DMF with varying ethanolamine equivalents. [Figure 35] FIG. 35 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with hydrazine. [Figure 36] Figure 36 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 2-aminoethanol. [Figure 37] FIG. 37 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with piperidine. [Figure 38] Figure 38 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with ethylenediamine. [Figure 39] FIG. 39 shows the GPC result of ethylenediamine-modified pentafluorobenzyl poly(2-ethyl-2-oxazoline) azide. [Figure 40] Figure 40 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with ethylenediamine. [Figure 41] Figure 41 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-methyl-2-oxazoline) azide with ethylenediamine. [Figure 42] FIG. 42 shows the 1H NMR and mass spectra of p-(3-thiopropane-1,2-diyl difatty acid ester) tetrafluorobenzyl poly(2-methyl-2-oxazoline) azide. [Figure 43]Figure 43 shows the results of the toxicity studies. [Figure 44] FIG. 44 shows the spectrum of the polymerization of 2-ethyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 45] Figure 45 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with sodium azide. [Figure 46] Figure 46 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 47] Figure 47 shows the 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 48] Figure 48 shows 19F NMR and mass spectra of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with various amines. [Figure 49] FIG. 49 shows the mass spectrum of the aminated compound shown in FIG. [Figure 50] Figure 50 is a mass spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with phenol. [Figure 51] FIG. 51 shows the 19F NMR spectrum of pentafluorobenzyl poly(2-ethyl-2-oxazoline) when the reaction was terminated with piperidine. [Figure 52] FIG. 52 shows the 19F NMR spectrum of pentafluorobenzyl poly(2-ethyl-2-oxazoline) when the reaction was terminated with piperidine. [Figure 53] FIG. 53 shows the 19F NMR spectrum and mass spectrum of pentafluorobenzyl poly(2-ethyl-2-oxazoline) when the reaction was terminated with piperidine. [Figure 54]FIG. 54 shows the 19F NMR spectrum of pentafluorobenzyl poly(2-ethyl-2-oxazoline) quenched with 2-(Boc-amino)ethanethiol. [Figure 55] FIG. 55 shows the 19F NMR spectrum of pentafluorobenzyl poly(2-ethyl-2-oxazoline) when the reaction was quenched with acetic acid. [Figure 56] Figure 56 shows the liposome size and fluorescence intensity of compounds for nanomedicine applications. [Figure 57] FIG. 57 shows firefly luciferase expression in Balb / C mice intravenously injected with LMP 4 hours after injection. [Figure 58] Figure 58 shows an example of an application of nanomedicine. [Figure 59] Figure 59 shows an example of application to a COVID-19 vaccine. [Figure 60] Figure 60 is a mass spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 61] FIG. 61 shows the 19F NMR spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 62] FIG. 62 shows the 19F NMR spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with thioglycerol. [Figure 63] FIG. 63 shows the 19F NMR spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with 2-aminoethanethiol hydrochloride. [Figure 64] Figure 64 shows the 19F NMR spectrum and mass spectrum of the reaction between the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide and 2,3,4,6-tetra-o-acetyl-1-thioglucose. [Figure 65]FIG. 65 shows the spectrum of the polymerization of 2-methylpropionic acid-2-oxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 66] FIG. 66 shows the 1H NMR spectrum of the polymerization of 2-methylpropionate-2-oxazoline monomer using pentafluorobenzyl bromide as the starting material and terminating the reaction with sodium azide. [Figure 67] Figure 67 is a mass spectrum of the reaction of the para-fluorine of pentafluorobenzyl poly(2-methylpropionate-2-oxazoline) azide with 2-aminoethanethiol hydrochloride. [Figure 68] Figure 68 is a mass spectrum of the reaction of the para-fluorine of pentafluorobenzyl poly(2-methylpropionate-2-oxazoline) azide with 2-aminoethanethiol hydrochloride. [Figure 69] Figure 68 is a 19F NMR spectrum of the reaction of the para-fluorine of pentafluorobenzyl poly(2-methylpropionate-2-oxazoline) azide with 2-aminoethanethiol hydrochloride. [Figure 70] FIG. 70 shows the spectrum of the polymerization of 2-methyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination of the reaction with sodium azide. [Figure 71] FIG. 71 shows the spectrum of the polymerization of 2-methyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 72] FIG. 72 is an NMR spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-methyl-2-oxazoline) azide with 1-thioglycerol. [Figure 73] FIG. 73 is a mass spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-methyl-2-oxazoline) azide with 3-mercaptopropionic acid. [Figure 74]FIG. 74 shows the spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-methyl-2-oxazoline) azide with 3-mercaptopropionic acid. [Figure 75] FIG. 75 shows the mass spectrum of the purified PMeOx-lipid conjugate. [Figure 76] FIG. 76 shows the spectrum of the reaction of the para-fluorine of pentafluorobenzylpoly(2-ethyl-2-oxazoline) azide with ethylenediamine. [Figure 77] FIG. 77 shows the spectrum of the polymerization of 2-propyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 78] FIG. 78 shows the spectrum of the polymerization of 2-propyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 79] FIG. 79 shows the spectrum of the polymerization of 2-methoxymethyloxazoline monomer using pentafluorobenzyl bromide as the starting material, followed by termination with sodium azide. [Figure 80] Figure 80 shows 19F NMR spectra of the termination reaction of 2 equivalents of methyl 3-mercaptopropionate with various bases in MeCN. [Figure 81] Figure 81 is a 19F NMR spectrum of the termination reaction of two equivalents of boc-aminoethanethiol with triethylamine in MeCN. [Figure 82] Figure 82 shows the 19F NMR and mass spectra of dioleic acid-TFP-PEtOx-N3. [Figure 83] Figure 83 is the H NMR spectrum of dioleic acid-TFP-PEtOx-N3 in dmso-d6. [Figure 84] Figure 84 shows the 19F NMR and mass spectrum. [Figure 85] Figure 85 shows the 19F NMR and mass spectra of dioleic acid-TFP-PMeOx-N3. DETAILED DESCRIPTION OF THE INVENTION

[0014] In this specification, halogen is any of F, Cl, Br and I, and preferably F and Cl.

[0015] As used herein, alkyl refers to a straight or branched monovalent hydrocarbon chain containing only carbon and hydrogen, the carbon-carbon bonds consisting only of single bonds, and the hydrocarbon chain is preferably straight. As used herein, alkenyl refers to a straight or branched monovalent hydrocarbon chain containing only carbon and hydrogen and having at least one carbon-carbon double bond at any position, including, but not limited to, vinyl, allyl, 1-propenyl, isopropenyl, butenyl, decenyl, and the like. As used herein, alkynyl refers to a straight or branched monovalent hydrocarbon chain containing only carbon and hydrogen and having at least one carbon-carbon triple bond at any position, including, but not limited to, ethynyl, propynyl, butynyl, and the like.

[0016] As used herein, carbocyclyl refers to a cyclic structure containing only carbon atoms in the ring, which may be fully saturated or partially saturated, including, but not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. As used herein, heterocyclyl refers to a cyclic structure containing a heteroatom in the ring, which may be fully saturated or partially saturated. Examples include, but are not limited to, pyrrolyl, piperidinyl, morpholinyl, thiophenyl, pyridinyl, and piperazinyl.

[0017] In this specification, examples of the substituents in alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl include halogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, a hydroxy group, an ether group having 1 to 3 carbon atoms, a thioether group having 1 to 3 carbon atoms, an ester group having 1 to 3 carbon atoms in the alkyl group, an amino group, an arbitrary substituent, for example, a polymerizable moiety, a formyl group, an acetal group (such as -CH(OC2H5)2), a hemiacetal group, an amino group optionally substituted with a carboxyl group, an arbitrary substituent, for example, C 1-3 An oxycarbonyl group optionally substituted with alkyl or the like, an optional substituent, for example, C 1-3 -Alkyl, C 1-3 - a carbamate group optionally substituted with haloalkyl, etc. (such as -NHCOOtBu), and any substituent, such as C 1-3 -Alkyl, C 1-3 -Amido groups optionally substituted with haloalkyl (such as -NHCOCF3), but are not limited to these. As used herein, oxygen protecting groups include, but are not limited to, acetyl, methoxymethyl, benzyl, benzoyl, and the like. Oxygen protecting groups are also well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, fifth edition, John Wiley & Sons, 2014.

[0018] In one embodiment of the present invention, X is a halogen, —SR 2 , -OR 2 , -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, -C≡CR 2 , NHOR 2 , -ONR 2 R3 , optionally substituted heteroaryl, preferably F, -SR 2 , -OR 2 , -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N3. In one embodiment of the present invention, examples of substituents for the optionally substituted heteroaryl include, but are not limited to, halogen, methyl, ethyl, methoxy, methylamino, dimethylamino, carboxy, and the like.

[0019] In one embodiment of the present invention, Y is SR 2 , -OR 2 (However, when X is F, it is not -OH), -CN, -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N=NR 2 , -N3, or optionally substituted heteroaryl, preferably -SR 2 , -OR 2 (However, if X is F, it is not -OH), -NHR 2 , -NR 2 R 3 , -NHNHR 2 , -N3, or [ka] and; More preferably, -SR 2 , -OR 2 (However, if X is F, it is not -OH), -NHR 2 , -NR 2 R 3 , -N3, or [ka] and most preferably -SR 2 , -OR 2(However, if X is F, it is not -OH), -NHR 2 , -NR 2 R 3 , -N3. In one embodiment of the invention, Y is not OH. In one preferred embodiment of the invention, when X is F, Y is not -OH.

[0020] In one embodiment of the present invention, R 1 is selected from the group consisting of an optionally substituted alkyl group having 1 to 40 carbon atoms, preferably 1 to 20 carbon atoms, and more preferably 1 to 18 carbon atoms, an optionally substituted alkenyl group having 2 to 40 carbon atoms, preferably 2 to 20 carbon atoms, and more preferably 2 to 18 carbon atoms, an optionally substituted alkynyl group having 2 to 40 carbon atoms, preferably 2 to 20 carbon atoms, and more preferably 2 to 18 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl. In one embodiment of the present invention, R 1 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, -CH2COOCH3, -CH2CH2COOCH3, -CH2CH2CH2COOCH3, alcohols and their acetates, for example, -CH2OH, -CH2CH2OH, -CH2OCOCH3, -CH2CH2OCOCH3. In one embodiment of the present invention, R 2 and R 3 are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R B , -C(=O)OR B , -C(=O)N(R B )2, -S(=O)R B , -S(=O)2R B , CH2CH(OR C)CH2OR C and sugar derivatives, or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl, preferably —CHCH(OR C )CH2OR C and preferably an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted aryl, or —C(═O)R B , a sugar derivative, and a repeating unit (a portion having a polymer structure), or -NR 2 R 3 In the case of R 2 and R 3 may be linked together to form an optionally substituted heterocyclyl. In one embodiment of the present invention, R A , R AA and R AAA are each independently selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R A and R AA may be linked together to form a ring, such as a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R A , R AA and / or R AAA may further be conjugated to a functional molecule such as a label, or a biofunctional molecule such as a protein, nucleic acid, etc.; R Bis selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms, preferably an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and more preferably an optionally substituted alkyl group having 1 to 6 carbon atoms; R C is selected from the group consisting of lipid-soluble groups such as hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, as well as oxygen-protecting groups including acyl groups such as acetyl and ether groups such as methoxymethyl, and is preferably hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms. In one embodiment of the present invention, R 4 is methyl, ethyl, propyl, butyl, pentyl, -CH2COOCH3, -CH2CH2COOCH3, -CH2CH2CH2COOCH3, alcohols and acetates thereof, for example -CH2OH, -CH2CH2OH, -CH2OCOCH3, -CH2CH2OCOCH3, preferably methyl, ethyl, propyl, -CH2CH2COOCH3. In one aspect of the present invention, In one embodiment of the present invention, each repeat unit includes, but is not limited to: [ka] etc. wherein o is 1 to 2000, and preferably n is 30 to 300; and In the compounds of the present invention, *1 is the ω-terminal side, and *2 is the α-terminal side.

[0021] The polycationic group in this specification is not particularly limited, but examples thereof include quaternary ammonium salts, polyamines such as polylysine and polyornithine, and the like. The polyanionic group in this specification is not particularly limited, but examples thereof include polyaspartates and polyglutamates.

[0022] The biofunctional molecule in this specification is not particularly limited, but examples thereof include functional molecules including proteins, nucleic acids, and the like. The functional molecules herein are not particularly limited, but include labels, antibodies, antibody fragments, peptides, nucleic acids, aptamers, lipids, carbohydrates, and the like. The lipid-based nanoparticles herein include, but are not limited to, formulations such as lipid nanoparticles (LNPs), liposomes, or lipoplexes. In the copolymers herein, each repeat unit is independent at each occurrence and may be the same or different. Copolymers herein include, but are not limited to, block copolymers, statistical copolymers, and the like. The label used herein is not particularly limited, and may include, for example, a detectable moiety or marker for detection based on fluorescence emission, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, absorbance wavelength, absorbance, Raman, Stokes shift, light scattering, molecular weight, redox, magnetic, radiofrequency, enzymatic reaction, or a combination thereof. The label may also be, for example, a fluorescent label, a colorimetric label, an enzyme label, a redox label, a radioactive label, a Raman tag, a mass tag, an isotope, a magnetic particle, a microparticle, or a nanoparticle. In certain embodiments, the label is preferably a fluorophore. The label used in the present invention may be used in vivo or in vitro.

[0023] In this specification, PFP refers to pentafluorobenzyl, and PEtOx refers to poly(2-ethyl-2-oxazoline). Thus, for example, "PFP-PEtOx45-N3" refers to pentafluorobenzyl poly(2-ethyl-2-oxazoline) azide, which is a compound formed by polymerizing 45 units of (2-ethyl-2-oxazoline), and "PFP-PMeOx50-N3" refers to pentafluorobenzyl poly(2(2-methyl-2-oxazoline)) azide, which is a compound formed by polymerizing 50 units of (2-methyl-2-oxazoline). As used herein, the "α-terminus" refers to the left-hand terminus in FIG. 2, i.e., the Y moiety linked to PFP, and the "ω-terminus" refers to the right-hand terminus in FIG. 2, i.e., the X moiety. As used herein, a leaving group is a group that dissociates during a reaction, and includes, but is not limited to, halogens, OTf, ONs, and OTs. In the polymer compounds described in this specification, the numerical values ​​indicating the degree of polymerization do not indicate only the exact numerical values ​​of the degree of polymerization, but are numerical values ​​within an error range of 10%.

[0024] In one embodiment of the present invention, compounds of formula (I) include, for example, the following compounds: [ka] [ka] [ka] [ka] Here, n is 1 to 2000, preferably n is 2 to 1000, more preferably 3 to 500, and most preferably 30 to 150; o is 1 to 2000, preferably n is 1 to 2000, more preferably 3 to 500, and most preferably 30 to 150. In one embodiment of the present invention, compounds of formula (I) include, for example, the following compounds: [ka] [ka] [ka] where o is between 50 and 200. [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, compounds of formula (IIIa) include, for example, the following compounds: [ka] In one aspect of the invention, the copolymer comprises, for example: [ka] where o is between 50 and 200.

[0025] Preferred nucleophiles used in the 2-oxazoline polymerization of the present invention during the ω-terminal termination reaction include, but are not limited to, azide reagents, thiols, amines, and carboxylic acids, as well as their salts, such as NaN. The solvent used during this ω-terminal termination reaction is also not limited to, but is preferably chlorobenzene, acetonitrile, dimethylacetamide, CHCl, N-methylpyrrolidone (NMP), sulfolane, nitrobenzene, benzonitrile, and ethyl acetate. The ω-terminal termination reaction may be carried out under neat conditions. The reaction temperature for the ω-terminal termination reaction is −70 to 140°C, preferably 0 to 50°C, and more preferably 0 to 30°C. A base can be used in the ω-terminal termination reaction, including, but not limited to, triethylamine (TEA), diisopropylethylamine (DIPEA), NaCO, and DBU. Preferred nucleophiles used in the α-terminal fluorination reaction of the present invention include, but are not limited to, azidation reagents, thiols, amines, carboxylic acids, alcohols, phenols, hydrazines, hydrazides, thiocarboxylic acids, such as NaN3, and phenol. The solvent used in this α-terminal reaction is also not limited to, but preferably includes DMF, NMP, acetonitrile (ACN), water, alcohols, DMSO, sulfane, etc. The reaction temperature in the α-terminal fluorination reaction is 0 to 100°C, preferably 20 to 80°C, and more preferably 20 to 70°C. Any base can also be used in the α-terminal fluorination reaction, including, but not limited to, triethylamine (TEA), diisopropylethylamine (DIPEA), potassium t-butoxide (KOtBu), diazabicycloundecene (DBU), pyrimido[1,2-a]pyrimidine (TBD), potassium hydroxide (KOH), sodium carbonate (Na2CO3), potassium carbonate (Na2CO3), etc.

[0026] In the present invention, all reactions described herein can be carried out under appropriate conditions. In the present invention, the reactions are carried out from the viewpoint of selectively carrying out polymerization termination reaction and fluorine substitution at the ω-terminus. When a base is used, it is preferably a weak base, preferably triethylamine or diisopropylethylamine, and more preferably non-base. To carry out the reaction selectively, an appropriate combination of solvent, temperature, and base should be selected during the ω-terminus termination reaction. Selective Termination The termination step in CROP of 2-oxazolines typically requires the addition of excess N-, O-, or S-nucleophiles to ensure quantitative reaction and avoid undesired side reactions, such as over-alkylation in the case of amines. Therefore, we investigated the possibility of selectively terminating the 2-oxazolinium chain end with different N-, O-, or S-nucleophiles under different experimental conditions (reaction time, temperature, and solvent) to terminate pentafluorobenzyl bromide / tosylate-initiated 2-oxazoline polymerizations. As a result, we obtained heterotelechelic POxs bearing a PFP moiety at the α-terminus and an azide, amine, ester, or thioether group at the ω-terminus. As seen in Figure 3 (patent), the reaction time of the azide anion and the 2-oxazolinium POx must be carefully controlled to ensure selectivity in the termination reaction at room temperature. Under these conditions, acetate anions can also be utilized as nucleophiles to obtain the desired heterotelechelic POx (Figure 9). On the other hand, when amines (Figure 10) or aliphatic thiols (Figures XX2-XX3) were used as nucleophiles to react with 2-oxazolinium in the presence of PFP moieties, no selectivity was observed under the conditions applied. When piperidine was used as a model amine, a mixture of products was obtained when the reaction was carried out in acetonitrile. When a thiol was used as the nucleophile and ACN was used as the solvent, screening revealed that a similar situation occurred when a mild base was used to deprotonate the thiol. Even with careful optimization of the reaction temperature and reaction time, it was proven that selective reaction could not be achieved in polar solvents such as ACN under these conditions. To promote selective termination of 2-oxazolinium in the presence of PFP moieties, we investigated the use of chlorobenzene as a solvent to make the reaction with 2-oxazolinium energetically favorable. Under these conditions and the applied reaction time, selective termination was obtained for amines and aliphatic thiols, as seen in Figures 11-13. Preferred combinations of reaction conditions are not particularly limited, but the following can be exemplified. Acetonitrile, 70°C, no base NMP, 20°C, no base Chlorobenzene, room temperature, no base Chlorobenzene, 70°C, no base Chlorobenzene, 70°C, no base Chlorobenzene, room temperature, no base, Acetonitrile, 20°C, no base, Acetonitrile, 20°C, base Chlorobenzene, 20°C, base.

[0027] Preferred embodiments of the present invention will be described below, but these are provided solely for the purpose of understanding the present disclosure, and it should not be understood that the scope of the present disclosure is limited to the following descriptions. [Example]

[0028] Materials and Methods All chemicals were used as received unless otherwise specified. Pentafluorobenzyl bromide (TCI, 99%), pentafluorobenzyl tosylate (TCI >98%), chlorobenzene (Wako 99%), acetonitrile (Merck, DNA synthesis grade), 2-ethyl-2-oxazoline (TCI >98%), 2-methyl-2-oxazoline (TCI, >98%), 2-propyl-2-oxazoline (TCI >98%), CaH2 (Sigma-Aldrich), NaOH (Wako), piperidine (Sigma-Aldrich 99%, double-distilled), ethylenediamine (TCI), aminoethanol (TCI), phenol (Sigma, ≥99%), 1-thio-β-D-glucose tetraacetate (Cayman Chemical). Chemical), 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), potassium tert-butoxide, triethylamine (TCI), tosyl isocyanate (Sigma), dimethylformamide (DMF), diethyl ether (Nacalai), N-methylpyrrolidone (NMP) (Wako, overdried), sulfuric acid (Wako, 98%), magnesium sulfate (Sigma-Aldrich), dinitrofluorobenzene (TCI), cysteamine hydrochloride (TCI), triazabicyclodecene (TBD, 98%, TCI), BaO (90%, Acros Organics), ninhydrin (Sigma, ACS Reagent), sodium carbonate (Sigma-Aldrich), ethyl acetate (Nacalai), and sodium azide (≥99.5%, Sigma). Deuterated solvents were purchased from Cambridge Isotope Laboratories. CleanCap(R) 5moU FLuc mRNA (Trilink), DSPC and D-Lin-MC3-DMA (MedChemExpress), cholesterol (Sigma), DMG-PEG2000 (Avanti), DiD (Invitrogen), DOPC (NOF).

[0029] Sodium azide was dried at 180 °C before use. Sodium carbonate was ground with a pestle and mortar and then dried at 180 °C. 2-Methoxycarbonylethyl-2-oxazoline (C2MestOx) was synthesized according to a previously published protocol and further purified by fractional distillation over BaO and ninhydrin, and isolated as a white crystalline solid. Pentafluorobenzyl bromide was distilled under reduced pressure and stored at -30 °C.

[0030] The purification of acetonitrile deviated from the literature because distillation over CaH2 or acetonitrile obtained from a solvent purification system revealed residual amine impurities. This was confirmed by Sanger's reagent. DNA synthesis-grade acetonitrile was transferred to a distillation apparatus equipped with a fractional distillation column (40 cm), and the solution was refluxed over tosyl isocyanate (1 mL / 100 mL acetonitrile) under an inert atmosphere for 2 hours to remove nucleophilic impurities. Purified acetonitrile was then obtained by fractional distillation. Purity was confirmed by the absence of color after adding 5 μL of Sanger's reagent to a 1 mL aliquot. Similar quality acetonitrile was also obtained by distillation over barium oxide and ninhydrin.

[0031] The purification of chlorobenzene followed the method of Monnery et al. (Angew. Chem. Int. Ed. 2018, 130 (47)). Briefly, chlorobenzene was purified in 500 mL batches by washing with 50 mL of 98% sulfuric acid until no discoloration occurred after overnight standing (four washes), followed by three washes with 50 mL each of water, saturated aqueous sodium carbonate, and water. The chlorobenzene was then dried over anhydrous magnesium sulfate (pre-dried at 180 °C). The chlorobenzene was then transferred to a distillation apparatus equipped with a fractional column (40 cm), and the solution was refluxed over tosyl isocyanate (1 mL / 100 mL chlorobenzene) under an inert atmosphere for 2 h to remove nucleophilic impurities. Finally, purified chlorobenzene was obtained by fractional distillation.

[0032] Purification of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, and 2-propyl-2-oxazoline. The 2-oxazoline monomer was transferred to a distillation apparatus equipped with a fractional distillation column (40 cm), and the solution was refluxed over CaH2 under an inert atmosphere. After H2 gas evolution ceased (determined by manometer, ±2 h), the monomer was fractionally distilled, discarding the first 10% and collecting the majority in a Schlenk flask equipped with a three-way tap and stir bar. Next, another distillation was performed using a similar setup, adding 1 mL of distilled MeOT to the monomer. The monomer was fractionally vacuum distilled, and heating was carefully maintained below 80 °C to prevent excessive polymerization.

[0033] DMF used for NCA polymerization was distilled over tosyl isocyanate (1 mL / 100 mL) and stored at −30°C over molecular sieves and isocyanate beads (Biotage). Cation exchange chromatography was performed on CM50 Sephadex.

[0034] Refractive index (RI)-2031 detector and TSKgel H HR Size exclusion chromatography (SEC) was performed using a Jasco HLC-8220 system with a combination of columns (G4000 and G3000) at 40 °C with LiCl (10 mM) dissolved in DMF as the eluent (flow rate: 0.8 mL min ). -1 SEC analysis was performed using JASCO ChromNav software, with known M as a calibrant. w PEG standard samples (Agilent Technologies, Santa Clara, CA) were used. NMR measurements were performed on a 400 MHz JEOL ECS 400 (JEOL, Tokyo, Japan) instrument. 1 H chemical shifts are relative to tetramethylsilane. 19Chemical shifts of F are reported relative to the instrument's internal calibration. MALDI-TOF-MS measurements were performed on a Bruker ultrafleXtreme, Bruker Daltonics, Bremen, Germany. Analyte solutions were prepared by mixing 10 μL of a 20 mg / mL solution of α-cyano-4-hydroxycinnamic acid (Sigma Aldrich) with 4 μL of a 4 mg / mL polymer solution and 2 μL of a 2 mg / mL solution of NaTFA (Sigma Aldrich). THF was typically used as the solvent, but MeOH was used for PMeOx samples. For samples containing tert-butyl carbamate groups, trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile was used as the matrix. The analyte solution was then spotted onto a 96-well stainless steel sample stage using the dried droplet method. The analyte solution was spotted at least twice onto the same well.

[0035] Example 1-1 Step 1: Representative polymerization of 2-oxazoline monomers initiated with pentafluorobenzyl bromide An oven-dried (180 °C) Schlenk flask equipped with a stir bar and a three-neck tap was cooled under reduced pressure (0.5 mbar) and then filled with dry argon. This vacuum-argon cycle was repeated two more times. Next, acetonitrile (9 mL) and EtOx (6 mL, 60 mmol) were added to the Schlenk flask to obtain a 4 M monomer solution. Next, an appropriate amount of pentafluorobenzyl bromide was added to the flask to obtain a solution with the desired [monomer]:[initiator] ratio. The mixture was then heated to a temperature ranging from 40 to 80 °C, typically 70 °C. 1 The reaction was carried out until 90% conversion was achieved as determined by H NMR, and the polymer was then terminated with an appropriate nucleophile as described elsewhere.

[0036] Example 1-2 Step 2: Representative polymerization of 2-oxazoline monomers initiated with pentafluorobenzyl tosylate An oven-dried (180 °C) Schlenk flask equipped with a stir bar and a three-neck tap was cooled under reduced pressure (0.5 mbar) and then filled with dry argon. This vacuum-argon cycle was repeated two more times. Next, chlorobenzene (9 mL) and EtOx (6 mL, 60 mmol) were added to the Schlenk flask to obtain a 4 M monomer solution. Next, in a glove bag under an inert atmosphere, an appropriate amount of pentafluorobenzyl tosylate was added to the flask to obtain a solution with the desired [monomer]:[initiator] ratio. This mixture was then heated to a temperature ranging from 40 to 80 °C, typically 70 °C. 1 The reaction was carried out until 90% conversion was achieved as determined by H NMR, and the polymer was then terminated with an appropriate nucleophile as described elsewhere.

[0037] Example 2-1 Step 3: Azide termination of the polymer prepared in Step 1 Sodium azide (10 molar equivalents relative to the initiator) was added to the colorless polymer mixture, and the reaction mixture was stirred at room temperature for 16 hours. Next, 10 mL of ethyl acetate was added, and the insoluble sodium azide was filtered through a PTFE syringe filter with a pore size of 0.44 μm. The filtrate was precipitated with a 10-fold excess of ether. This precipitation procedure was repeated once. Next, the ether was decanted, and most of the remaining ether was evaporated, followed by the addition of hot water. Finally, the polymer was isolated as a white powder after lyophilization.

[0038] Example 2-2 Step 4: Azide termination of the polymer prepared in Step 2 Tetrabutylammonium azide (2 molar equivalents relative to the initiator) was added to the colorless polymer mixture, and the reaction mixture was stirred at room temperature for 16 hours. Next, 10 mL of ethyl acetate was added, and the polymer was precipitated in a 10-fold excess of ether. The precipitation procedure was repeated once. The ether was then decanted, and most of the remaining ether was evaporated, followed by the addition of hot water. Finally, the polymer was isolated as a white powder after lyophilization.

[0039] Example 2-3 Step 5: Amine termination of the polymer prepared in Step 2 Piperidine (10 molar equivalents relative to the initiator) was added to the colorless polymer mixture, and the reaction mixture was stirred at room temperature for 24 hours. Then, 10 mL of ethyl acetate was added. The polymer was then precipitated in a 20-fold excess of ether. After dissolving the polymer in ethyl acetate, the precipitation procedure was repeated once. The ether was then decanted, and most of the remaining ether was evaporated, followed by the addition of hot water. Finally, the polymer was isolated as a white powder after lyophilization.

[0040] Examples 2-4 Step 6: Thiol-terminated polymer prepared in step 2 Thiol and triethylamine (2 and 2.5 molar equivalents relative to the initiator, respectively) were added to the colorless polymer mixture, and the reaction mixture was stirred at room temperature for 10 hours. Next, 10 mL of ethyl acetate was added, and the polymer was precipitated in a 10-fold excess of ether. After dissolving the polymer in ethyl acetate, the precipitation procedure was repeated once. Next, the ether was decanted, and most of the remaining ether was evaporated, followed by the addition of hot water. Finally, the polymer was isolated as a white powder after lyophilization.

[0041] Examples 2-5 Step 7: Carboxylic acid end of the polymer prepared in Step 1 Acetic acid and triethylamine (2 and 2.4 molar equivalents relative to the initiator, respectively) were added to the colorless polymer mixture, and the reaction mixture was stirred at room temperature for 16 hours. Then, 10 mL of ethyl acetate was added. The polymer was then precipitated in a 20-fold excess of ether. After dissolving the polymer in ethyl acetate, the precipitation procedure was repeated once. The ether was then decanted, and most of the remaining ether was evaporated, followed by the addition of hot water. Finally, the polymer was isolated as a white powder after lyophilization.

[0042] Example 3-1 Step 8: Parafluoroazide substitution In other words, PFP-PEtOx 450.2 g of -N3 (0.05 mmol of PFP group) was dissolved in 2 mL of N,N-dimethylformamide (DMF), and 2.5 equivalents of sodium azide (8.2 mg, 0.125 mmol) relative to the PFP group were added. The mixture was heated at 80 °C for 2 h. The reaction mixture was then cooled, and the polymer was precipitated in a 10-fold excess of diethyl ether. The polymer was then dissolved in 2 mL of ethyl acetate and filtered through a 0.2 μm syringe filter to remove excess sodium azide. The resulting filtrate was precipitated again with 20 mL of diethyl ether. The supernatant was decanted, and the polymer was dissolved in water and lyophilized.

[0043] Example 3-2 Step 9: Parafluorothiol substitution Example: In short, PFP-PEtOx 45 0.2 g of -N3 (0.05 mmol of PFP group) was dissolved in 2 mL of N,N-dimethylformamide (DMF) with either 5 equivalents of thiol and 6 equivalents of triethylamine, 5 equivalents of thiol and 4 equivalents of KOtBu, or 2 equivalents of thiol and 1.8 equivalents of DBU relative to the PFP-moiety. When triethylamine was used as the base, the mixture was heated at 60 °C for at least 16 h; when other bases were used, the mixture was stirred at room temperature for 1 h. After the reaction was complete, the polymer was precipitated with a 10-fold excess of diethyl ether. The supernatant was decanted, and the polymer was dissolved in water and lyophilized.

[0044] Example 3-3 Step 10: Parafluoroamine substitution Example: In short, PFP-PEtOx 45 While maintaining an argon atmosphere, 0.2 g of -N3 (0.05 mmol of PFP group) was dissolved in 2 mL of NMP along with 7 equivalents of either amine relative to the PFP group. When ethylenediamine was selected as the amine, 15 equivalents were used to prevent polymer coupling reactions. After the reaction was complete, the polymer was precipitated in a 10-fold excess of diethyl ether. The supernatant was decanted, and the polymer was dissolved in water and lyophilized.

[0045] Examples 3-4 Step 11: POx-lipid synthesis Example: In brief, 1-thioglycerol-TFP-PEtOx 45 0.2 g of -N3 (0.10 mmol hydroxyl group, 1 equiv.) was dissolved in 2 mL of DCM and 5 equiv. of fatty acid, 5 equiv. of diisopropylcarbarodiimide, and 0.05 equiv. of DMAP were added. The reaction mixture was stirred at room temperature for at least 16 h, and the polymer was isolated by precipitation in a 10-fold excess of ether. The polymer was then redissolved in DCM and passed through a neutral aluminum oxide plug to remove excess fatty acid. The polymer solution was then added to benzene and subsequently lyophilized to yield a white powder. For the synthesis of PMeOx-lipid, after lyophilization in benzene, the polymer was dispersed in water. The solution was then added to AMICON diafiltration tubing with a 10 kDa molecular weight cutoff and centrifuged to remove proton-initiated impurities. This centrifugation diafiltration was repeated three times with each 10-fold dilution of the remaining solution.

[0046] Examples 3-5 Step 12: Parafluoroalkoxide substitution Example: In short, PFP-PEtOx 45 While maintaining an argon atmosphere, 0.2 g of -N3 (0.05 mmol of PFP group) was dissolved in 2 mL of NMP along with 3 equivalents of phenol and 3 equivalents of TBD relative to the PFP group. The reaction mixture was then stirred at room temperature for 24 hours. After the reaction was complete, the polymer was precipitated in a 10-fold excess of diethyl ether. The supernatant was decanted, and the polymer was dissolved in water and lyophilized.

[0047] Example 4 Preparation and characterization of POX liposomes POX-lipid-containing liposomes were prepared using the thin film hydration method. 67Briefly, DOPC, cholesterol, C18-MeOX, or C18-PtOX were dissolved separately in chloroform and mixed at a molar ratio of 62.5:32.5:5. The chloroform was evaporated, and the lipid film was hydrated with 10 mM HEPES buffer (pH 7.4) at 37 °C to a final total lipid concentration of 5 mM. Liposomes were then extruded multiple times using a 200 nm and 100 nm NanoSizer MINI Extruder (T&T Scientific). Particle size was measured using dynamic light scattering (DLS) with a diode laser (λ = 532 nm) at a scattering angle of 173° (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). For blood circulation studies, a DiD fluorescent probe was added to the lipid mixture to a final concentration of 0.03 mM in the liposome suspension. DiD-liposomes were diluted 10-fold with PBS, and 200 μL of the diluted solution was injected into the tail vein of Balb / C mice.

[0048] Example 5 IVCLSM investigation The blood circulation of POX-lipid-containing liposomes was measured using intravital confocal laser scanning microscopy (IVCLSM). A system equipped with a 20x objective, a 640 nm diode laser, and a 700 / 75 nm bandpass emission filter was used. The pinhole was set to capture 10 μm-diameter optical slices. Five-week-old female Balb / C mice (The Jackson Laboratory Japan, Inc.) were placed on the microscope stage, and DiD signals in the earlobe dermal blood vessels were observed immediately after tail vein injection of liposomes. All animal experimental protocols followed the guidelines of the Innovation Center for NanoMedicine (iCONM), Kawasaki City Industrial Promotion Foundation. Fluorescence images were recorded every 4 seconds for 3 minutes after injection, and snapshots were taken every minute thereafter. Fluorescence intensity was calculated as the intensity of the vein region of interest (ROI) minus the background. Relative fluorescence intensity was calculated as the ratio of the fluorescence intensity value at each time point to the maximum intensity value during the measurement. Elimination and distribution half-lives were calculated by compartmental analysis using Prism GraphPad software.

[0049] Example 6 mRNA synthesis The DNA encoding the full-length SARS-CoV-2 spike protein was cloned into the pSP73 plasmid vector (Genscript Japan, Tokyo, Japan) with a BsmBI cleavage site added at the 3' end. This plasmid was amplified in Escherichia coli DH5α competent cells (Takara Bio Inc., Otsu, Japan) and then extracted and purified using the Nucleobond xtra maxi plus EF kit (Takara, Japan). The plasmid was linearized and fragmented by incubation with BsmBI overnight at 55°C. The desired DNA fragment was separated by gel electrophoresis and extracted using a gel extraction kit (Qiagen, Hilden, Germany). The extracted DNA was further treated with T4 DNA polymerase (Takara Bio Inc., Otsu, Japan) to obtain blunt-ended DNA. Finally, the mMESSAGE mMACHINE TMIn vitro transcription was performed using a kit (Thermo Fisher Scientific). The reaction proceeded for 14 hours at 37°C, and the transcribed mRNA was purified using the RNeasy mini-Kit (Qiagen, Hilden, Germany). The quality of the mRNA was checked using a Bioanalyzer (Agilent Technology, CA, USA).

[0050] Example 7 Preparation and characterization of mRNA-LNPs Ionizable lipid mRNA-LNPs were prepared by microfluidic mixing. One volume of ethanol containing cholesterol, DSPC, D-Lin-MC3-DMA, and polymer-bound lipids was mixed with three volumes of 50 mM sodium citrate buffer (pH = 3) containing mRNA at a flow rate of 12 mL / min in an Ignite microfluidic mixer (Precision NanoSystems Inc., Vancouver, BC, Canada). The product was then diluted 40-fold with PBS and concentrated using a 30 kD Amicon centrifugal filter (Millipore, MA, USA) to remove residual ethanol. Quant-it TM Encapsulation efficiency was measured using a RiboGreen RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and particle size was measured as described above. Example 8 IVIS research Fluc mRNA-LNPs formulated with either conventional PEG-lipids or novel POX-lipids were intravenously injected into mice (Balb / C, female, 5 weeks old) at a dose of 5 μg mRNA per mouse. Four and 24 hours after injection, 200 μL of 15 mg / mL luciferin substrate (Promega) was intraperitoneally injected into the mice 10 minutes before imaging. The mice were then placed on the temperature-controlled stage of an in vivo imaging system (IVIS, PerkinElmer) equipped with O2 and isoflurane for anesthesia. The image exposure time was set to 10 seconds, and total luminescence flux was measured by gating an abdominal ROI to quantify protein expression in the liver. After 24 hours of whole-body imaging, the mice were sacrificed and immersed in luciferin in a Petri dish. The vital organs were then harvested and imaged. Each organ was gated in a separate ROI and used to calculate protein expression distribution.

[0051] Example 9 immunological research Female Balb / C mice (5 weeks old) were injected with 5 μg of spiked mRNA-LNP into the thigh muscle, followed by a 5 μg booster injection 3 weeks later. Two weeks after the final injection, blood was collected into heparinized tubes, and the mice were euthanized and their spleens were harvested for humoral and cellular immune quantification. Plasma was obtained by spinning at 2000 × g for 10 minutes in a refrigerated centrifuge. Anti-spike IgG in plasma was quantified using enzyme-linked immunosorbent assay (ELISA). First, SARS-CoV-2 spike S1+S2 recombinant protein (SinoBiological) was mounted on clear, flat-bottom Immuno Nonsterile 96-well plates (Thermo) by overnight incubation at 4°C. After washing away excess protein, 50 μL of diluted plasma sample was added to each well and incubated overnight at 4°C. Next, goat anti-mouse IgG-HRP (R&D Systems, 1:8000 dilution) was added to each well after washing and further incubated at 23°C for 2 hours. Finally, 100 μL / well of HRP substrate was added and incubated at 23°C for 30 minutes, protected from light. The reaction was stopped by adding 2 M sulfuric acid, and the absorbance at 492 nm was recorded using a plate reader (Tecan, Switzerland). To quantify spike protein-specific IFNγ-producing T cells in the spleens of immunized mice, spleens were first disrupted using a steel grid mesh in the presence of 5 mL of RPMI-1640 medium containing 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 50 μM mercaptoethanol, and 1% penicillin / streptomycin. The resulting suspension was passed through a 40 μm nylon mesh (Cell strainer, Falcon) to obtain a single-cell suspension. Spleen cells were plated at 2.5 × 10 in an anti-IFNγ ELISpot plate. 5 Plates were seeded at a density of 1000 cells / well and stimulated by the addition of 0.025 μg / well of the total spike epitope mixture (JPT Peptide Technologies). Plates were incubated overnight at 37°C and 5% CO2, washed, and processed according to the manufacturer's protocol. Developed spots were counted using an ELISpot plate reader (AID GmbH, Germany).

[0052] Example 10 Toxicity testing Plasma biochemical markers were assessed 4 and 24 hours after intravenous injection of 5 μg Fluc mRNA-LNP. Samples were measured at Oriental Yeast Co., Ltd. (Tokyo, Japan) using a Hitachi High-Technologies Model 7180 automated analyzer. A JCA-BM6050 automated analyzer (JEOL, Tokyo, Japan) was also used. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels were measured using the methods recommended by the Japan Society of Clinical Chemistry (JSCC). Creatinine (CRE) was measured by the creatininase-HMMPS method, uric acid (UA) by the uricase-HMMPS method, and creatinine amidohydrolase-creatinine amidinohydrolase-SOX-POD enzymatic method. Blood urea nitrogen (BUN) was measured by the urease-GLDH enzymatic method. Uric acid (UA) levels were measured enzymatically.

[0053] Example 11 statistical significance Statistical significance between two groups was analyzed using an unpaired two-tailed Student's t-test. Comparisons with untreated samples were performed using unrepeated ANOVA followed by Dunette's test. Statistically significant differences were considered p<0.05.

[0054] Results and Discussion Polymers bearing pentafluorophenyl (PFP) groups, such as poly(pentafluorostyrene) and poly(pentafluorobenzyl acrylate), have been shown to exhibit reactivity toward a wide range of substrates and react with various N-, O-, and S-nucleophiles with high efficiency in a single reaction pathway, i.e., parafluorosubstitution. This chemistry therefore offers an attractive approach for α-end group diversification of 2-oxazolines, provided that the PFP group does not react with the N-, O-, and S-nucleophiles used in the termination step of cationic ring-opening polymerization (CROP). However, comprehensive studies on the termination of 2-oxazolinium species under polymerization-relevant conditions have not been described, and it should be noted that the relative reactivities of PFP and 2-oxazolinium groups likely depend on the nucleophile employed. A further complication is that the termination step of CROP typically requires the addition of an excess of N-, O-, or S-nucleophile to ensure quantitative reaction and to avoid undesired side reactions, such as over-alkylation in the case of amines. So far, only hydroxide species, which can be applied in equimolar ratio with 2-oxazolinium species, have been employed as terminators for PEtOx initiated with pentafluorobenzyl bromide. 19 F NMR did not confirm the selectivity.

[0055] Here, we investigated the termination reaction of 2-oxazoline polymerization initiated with pentafluorobenzyl bromide / tosylate under different experimental conditions (reaction time, temperature, and solvent), allowing selective termination of the 2-oxazolinium chain ends living with different N-, O-, and S-nucleophiles, to obtain heterotelechelic POx bearing a PFP group at the α-terminus and an azide, amine, ester, or thioether group at the ω-terminus, as shown in Figure 1. Furthermore, we also describe how derivatization of these heterotelechelics by para-fluoro substitution can yield new heterotelechelic combinations, as shown in Figure 2.

[0056] Selective azide termination First, because both the 2-oxazolinium and PFP moieties have been shown to react efficiently with azide anions upon gentle heating in dipolar aprotic solvents, providing an ideal starting point for investigating the differential reactivity of both electrophiles, we investigated the selective termination of live polymer chains with sodium azide. To this end, the polymerization of 2-ethyl-2-oxazoline (EtOx) was initiated with pentafluorobenzyl bromide in acetonitrile (MeCN) and allowed to proceed at 70 °C until monomer conversion reached 90% or higher. Next, 10 molar equivalents of sodium azide relative to the initiator were added to the polymerization mixture, and the mixture was left stirring at room temperature, periodically sampling for analysis. 19 The conversion of the PFP moiety was monitored by F NMR. As can be seen in Figure 3, after 16 hours, the NMR showed a characteristic F peak of the unreacted PFP moiety. 19Note that the F signal is observed, and that on the NMR time scale, the signal splits due to the slow rotation of the amide bond, and as the reaction time increases, a peak corresponding to tetrafluorophenyl azide appears. Nevertheless, these results demonstrate that by controlling the reaction time, it is possible to isolate products with unreacted PFP moieties. This was also confirmed by isolating polymers of different degrees of polymerization (DP) after 16 hours of stirring in the presence of NaN3, as seen in Figure 4, for poly(2-ethyl-2-oxazoline) (PEtOx) prepared at [M] / [I] = 50. Figure 4A shows that the resulting polymer has a narrow molecular weight distribution of D = 1.04, which is consistent with the dispersity value expected for similar ring-opening polymerizations. Figure 4B shows that the terminal PFP groups remain unreacted. This is further supported by MALDI-TOF-MS measurements in Figures 4C-D, which show a monomodal mass distribution and a close match between the experimental and simulated spectra. Because the terminal azide end group is prone to fragmentation in MALDI-TOF-MS, N2 exclusion generates molecular ions with a difference of -23 Da and -28 Da compared to the intact molecular ion, but the latter was not observed. Furthermore, the absence of H-initiating species or other products in MALDI-TOF-MS indicates high end group fidelity in the synthesized PEtOx. The presence of pentafluorobenzyl groups is confirmed by the characteristic benzyl signal appearing at 4.55-4.75 ppm. 1This was further confirmed by H NMR (Figure 19), which showed that the relative integration ratio of the polymer backbone CH3 and pentafluorobenzyl protons (135 / 3 = 45) exactly matched the DP of the most intense signal observed by MALDI-TOF-MS (m = 4682.12 Da), again confirming the absence of side reactions and the high purity of the resulting product. Similarly, this kinetic control could also be applied to the heterotelechelic synthesis of EtOx with higher DP, as well as 2-methyl-2-oxazoline (MeOx), 2-propyl-2-oxazoline (PrOx), and 2-methoxycarbonylethyl-2-oxazoline (C2MestOx), as can be seen in Figures 20-23. However, for MeOx, the relative integration ratio (135 / 3 = 45) of the polymer backbone CH3 and pentafluorobenzyl protons exactly matched the DP of the most intense signal observed by MALDI-TOF-MS (m = 4682.12 Da). 1 The presence of H-initiated species was observed in H NMR, which is believed to be due to the inherently high chain transfer constant of this monomer.

[0057] Parafluoroazide substitution With these heterotelechelic polymers in hand, we next investigated para-fluoro substitution reactions on these substrates. First, to further verify the purity of the polymers synthesized in the previous section, we performed azido-fluoro substitution. Here, we adjusted the conditions reported by Noy et al. (Macromolecules 2019, 52(8), 3083-3091.) to obtain PEtOx with two azide end groups, namely, alkyl azide and tetrafluorophenyl azide. DMF-GPC (Figure 5A) showed a molecular weight distribution similar to that of the starting material, and no chain coupling reactions were observed. Furthermore, 19 In F NMR, 19Only two F clusters were clearly observed, indicating that the para-fluorine was indeed consumed singly, resulting in the formation of a para-substituted tetrafluorophenyl azide, as evidenced by the difference in chemical shifts of the meta fluorines (Figure 5B). Finally, MALDI-TOF-MS further supported product formation, as peaks corresponding to the expected fragmentation products were present in the experimental spectrum (Figure 5C-D). However, it should be noted that additional product peaks were present that could not be identified. These peaks are likely due to photochemical transformation of the para-substituted tetrafluorophenyl azide induced by laser irradiation under the experimental conditions, resulting in the generation of a highly reactive nitrenium ion capable of reacting with the matrix. Nevertheless, these results further confirm that selective azide termination is possible and demonstrate the potential for the formation of a highly reactive para-substituted tetrafluorophenyl azide, which could be utilized in photochemical transformations and azide-alkyne cycloaddition reactions.

[0058] Parafluorothiol substitution Next, we focused on para-fluorothiol substitution, as it is possible to couple a wide variety of commercially available thiols under relatively mild conditions. This reaction is usually base-catalyzed, and the resulting thiolate attacks the para-position of the PFP moiety. First, we investigated the use of triethylamine as a base in the coupling reaction of aliphatic thiols. 1-Thioglycerol was chosen as a model compound because the resulting product could be used as a precursor to lipid conjugates. When triethylamine was used as the base, a significant excess of base and thiol was required (6 and 5 equivalents relative to PFP), and the reaction required heating at 60 °C for 16 h in DMF. Nevertheless, MALDI-TOF-MS and 1 H and 19The reaction was successful, with characteristic product peaks confirmed by F NMR. As shown in Figures 6A–D, there was no significant change in the molecular weight distribution other than a shortened retention time in GPC. Despite this success, attempts were made to reduce the reaction temperature and time by using KOtBu as a base (Table 1). Under these conditions, effective conversion occurred within 1 h at room temperature using 5 equivalents of thiol and 4 equivalents of base. Unfortunately, attempts to reduce the molar excess of base and thiol failed, as the reaction did not reach complete conversion even after 4 days. Therefore, the use of DBU as a base was explored, and complete conversion of the PFP moiety was achieved within 1 h at room temperature with only 1.35 equivalents of DBU and 1.5 equivalents of thiol. Nevertheless, screening of various thiols revealed that 2 equivalents of thiol and 1.8 equivalents of DBU exhibited optimal conditions for a wide variety of thiols, including mercaptoethanol, cysteamine, mercaptopropionic acid, and 1-thioglucosetetraacetic acid (Figures 24–31). It is noteworthy that thiolates can also reduce alkyl azides. However, under the reaction conditions employed, no reduction to the amine was observed by ion exchange chromatography (Figure 32-1). This suggests that reduction is kinetically irrelevant under these conditions. However, the cysteamine-modified polymer exhibited a retention profile indicative of cationic charge on the polymer, confirming the success of the conversion (Figure 32-2). Furthermore, this conversion was also applicable to P(C2MestOx), demonstrating orthogonality to the ester functionality (Figure 33).

[0059] [Table 1]

[0060] Parafluoroamine Substitute Having demonstrated the success of thiol and azide modification, we next explored para-fluorosubstitution with various amines. Initially, we employed the conditions proposed by Noy et al., but these conditions revealed significant by-product formation. Screening experiments suggested that the by-products were formed as a result of the thermal and base-induced composition of DMF, whereby in situ generated N,N-dimethylamine effectively competed with the target amine (Figure 34). Therefore, N-methylpyrrolidone (NMP) was selected as a more suitable solvent, allowing for the efficient introduction of a wide variety of amines, including piperidine, ethanolamine, ethylenediamine, 3-aminopropionaldehyde diethyl acetal, and hydrazine. Figure 7 shows the properties of a representative 3-aminopropionaldehyde diethyl acetal-substituted polymer, and other examples are shown in Figures 35–41.

[0061] Parafluoroalkoxide substitution Finally, we explored alkoxides as the final substrate for para-fluoro substitution. Here, phenol was chosen as a model compound and the corresponding alkoxide was generated by in situ deprotonation using triazabicyclodecene (TBD) as a base. The reaction was shown to proceed in NMP at room temperature for 24 hours with a moderate stoichiometric excess. As is evident from the data in Figure 8, DMF-GPC still showed the characteristic narrow molecular weight distribution. 19 The conversion proceeded smoothly without any significant side reactions, as F NMR showed the expected signals corresponding to ortho- and meta-fluorine. Finally, MALDI-TOF-MS spectra provided conclusive evidence that the desired compound was obtained, further proving the absence of side reactions.

[0062] Selective Carboxylic Acid Termination In addition to azide anion as a terminating agent for CROP of 2-oxazolines, we also investigated the use of deprotonated carboxylic acids to obtain polymers with esters at the ω-terminus. The desired polymers could be isolated simply by adding 2 equivalents of acetic acid and 2.4 equivalents of triethylamine relative to the initiator to the polymerization mixture in MeCN and stirring the mixture for 16 hours. As shown in Figure 9, polymers with well-defined molecular weight distributions were obtained. Meanwhile, 19 F NMR showed the presence of the characteristic PFP fluorine, demonstrating that the PFP moiety did not undergo base-catalyzed substitution reactions. Furthermore, MALDI-TOF-MS spectra showed a narrow molecular weight distribution, whereby the observed masses corresponded closely to the theoretical exact masses of the expected products with sodium ions. Furthermore, the masses observed by MALDI-TOF-MS were 1 This corresponds well with the average molecular weight calculated from H NMR (Figure 42-1).

[0063] Selective amine and thiol termination As shown above, the PFP moiety exhibits high reactivity toward thiols, amines, and alkoxides. When selective termination with thiols and amines was attempted in a polymerization initiated with pentafluorobenzyl bromide in MeCN, the high reactivity of the PFP moiety proved problematic. In the case of amines, piperidine was chosen as a model compound. Its high nucleophilicity and low steric hindrance rapidly terminate the polymerization, whereas other amines have been reported to require significantly longer reaction times for complete termination. Therefore, we evaluated selectivity after 24 hours of polymerization using a 10-fold molar excess of piperidine, a representative example of a less reactive amine. However, under these conditions, we found that more than 30% of the PFP moieties had already reacted with piperidine (Figure 10).

[0064] Similarly, selective thiol termination was problematic. Experiments showed that cooling the reaction mixture resulted in very little conversion of the PFP group within 3 hours at 0 °C, and already after 4 hours. 19 Significant conversion was observed by F NMR. However, when the polymer was isolated after 3 hours,1 H NMR revealed incomplete termination of the model thiol, N-(2-mercaptoethyl) tert-butylcarbamate. These results indicated that both the 2-oxazolinium and PFP moieties have similar reactivity toward these nucleophiles under these conditions.

[0065] To promote selective reaction with the 2-oxazolinium species, we investigated the use of nonpolar chlorobenzene as a polymerization solvent. We hypothesized that the formation of a charged Meisenheimer intermediate in para-fluoro substitution is energetically unfavorable, while the reactivity of the 2-oxazolinium species increases due to reduced solvation by nonpolar chlorobenzene (PhCl). This hypothesis was experimentally confirmed by conducting both the polymerization and termination steps in chlorobenzene. As a result, the isolated products for piperidine and tert-butyl N-(2-mercaptoethyl)carbamate termination yielded the desired product with an intact PFP moiety, as confirmed by the characterization data displayed in Figures 11-14. Note that the molecular weight distribution observed by DMF-GPC is slightly broader for the polymer prepared in PhCl than for the polymer prepared in MeCN. Furthermore, the MALDI-TOF-MS spectrum observed in Figure 13 indicates the presence of distinct molecular ions, which we attribute to fragmentation of the Boc-group under MALDI-TOF-MS conditions. The Boc group has been reported to be unstable under MALDI-TOF conditions, regardless of the acidity of the matrix (in this case trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile), resulting in a population that closely resembles a potassium adduct, despite the addition of sodium trifluoroacetate to the analyte solution. Although an exact assignment of the MALDI-TOF-MS spectrum is not possible, the fit of the simulation with potassium adducts suggests the presence of a Boc group, as reported for other polymers, which is 1 This was also confirmed by 1 H NMR.

[0066] Heterotelechelic PFP-POx derivatives and their application to nanomedicine Next, we explored the developed methodology for the synthesis of heterotelechelic POx suitable for nanomedicine applications. First, we synthesized α-amine and ω-azide heterotelechelic POx by selective azide termination of CROP followed by para-fluoro substitution with excess ethylenediamine. These polymers were then used as macroinitiators for the ring-opening polymerization of N-carboxyanhydrides to yield N3-POx-polypeptide block copolymers. Similar N3-PEG-polypeptide block copolymers have shown promise in preclinical and clinical studies, suggesting that these polymers may be suitable for nanomedicine. To this end, we investigated the synthesis of POX-polypeptide block copolymers using trifluoroacetyl (TFA) L-onithine carboxyanhydride as the monomer. This is because, after TFA-deprotection, polycationic blocks are obtained, suitable for condensation with polyanionic biopolymers such as DNA and RNA. Characterization data from chain extension experiments are shown in Figure 14. Figure 14A shows that the chain extension experiment was successful, as the resulting block copolymers showed narrow molecular weight distributions by GPC and no detectable macroinitiators remained, demonstrating the suitability of the developed methodology for this purpose. Furthermore, the polymerization was well controlled, indicating that the tetrafluoroaniline group does not initiate NCA polymerization. Figure 14B confirms this, as the relative ratios of protons in each block correspond well to the initiator:monomer ratio employed.

[0067] In addition to the synthesis of block copolymers, we also explored the synthesis of lipid-polymer conjugates using 1-thioglycerol-functionalized PFP-POx-N3 as a precursor. This precursor polymer could be efficiently utilized for the synthesis of various lipid-conjugates by simple Steglich esterification with the corresponding fatty acids. Three lipid conjugates were synthesized using oleic acid (C18) and myristic acid (C14) as lipid tails, yielding C18-PEtOx-N3, C14-PEtOx-N3, and C18-PMeOx-N3 (characterization data for C14-PEtOx-N3 are shown in Figure 42-2 as a representative example). These lipids were then formulated into liposomes with a 62.5 / 32.5 / 5 molar ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) / cholesterol / dioleic acid-POx by thin-film hydration followed by solution extrusion. This method yielded well-defined liposomes with hydrodynamic radii of 126 nm for the C18-PEtOx formulation and 141 nm for the C18-PMeOx formulation (Figure 16). Next, we incorporated the lipophilic dioctadecyl-3,3,3,3 tetramethylindodicarbocyanine dye (DiD) into the formulation to prepare fluorescently labeled liposomes. This was done to evaluate the effect of the tetrafluorophenyl (TFP) linker on the in vivo circulation of the resulting liposomes using intravital confocal laser scanning microscopy. This is because the presence of this hydrophobic linker may affect factors such as lipid-membrane fluidity or induce nonspecific protein adsorption. However, the blood circulation profiles of the resulting lipids suggested that TFP had a negligible effect on the pharmacokinetics of the liposomes in mice. It is noteworthy that the previously reported method relied on periodic blood sampling, which may have overlooked the relatively short α-phase of C18-PEtOx.

[0068] While the above results indicate that the TFP linker does not significantly affect the blood circulation of liposomes, the presence of a hydrophobic linker may affect the dynamic exchange of lipoproteins and lipid formulations in the blood. It has been reported that in lipid nanoparticle (LNP) formulations containing mRNA, the length of the lipid tail of the PEG-lipid significantly affects the protein expression and knockdown of the encapsulated cargo. To evaluate the effect of the TFP linker, we prepared three LNP formulations. One contained DMG-PEG as the polymer lipid and served as a positive control, which was capable of eliciting efficient hepatic protein expression. The other contained C14-PEtOx and C18-PEtOx. The negative control was expected to achieve little hepatic protein expression due to limited desorption into the LNPs. We used these lipids to encapsulate mRNA encoding firefly luciferase by fluid mixing, and the resulting LNPs showed a narrow size distribution by DLS (Figure 16). Furthermore, all formulations showed comparable encapsulation efficiencies of 77–82% as assessed by the RiboGreen assay, demonstrating the similar applicability of the three lipids tested for mRNA formulation. Next, the LNPs were intravenously injected into the tail vein of 5-week-old female Balb / C mice (n=3), and protein expression was assessed 4 and 24 hours later by intraperitoneal injection of luciferin. After intraperitoneal administration of luciferin, bioluminescence was measured using an in vivo imaging system. The mice were then sacrificed and protein expression was measured in different organs in vitro to determine biodistribution (Figures 16–17). Overall, the synthesized C14-PEtOx lipids performed similarly to the positive control, indicating that the TFP linker does not significantly affect the desorption of the polymer-lipid conjugate from the LNP. Similarly, protein expression in different organs was comparable to that of the positive control. This is in contrast to the negative control, which showed negligible protein expression compared to the positive control due to inefficient desorption of the polymer-lipid conjugate, resulting in low hepatic uptake and protein expression.Furthermore, all formulations showed negligible toxicity as assessed by measuring various toxicity markers in the blood compared to PBS-injected mice (Figure 43).

[0069] Because the newly developed POx-lipid showed excellent potential for mRNA delivery, we evaluated the potential of the C14-PEtOx formulation by administering mRNA encoding the COVID-19 spike protein and monitored cellular immunity and antigen-specific antibody production. Here, mice were injected intramuscularly in the thigh with 5 μg of mRNA (prime) and then administered a second dose (boost) three weeks later. Two weeks after the last dose, blood was collected into heparinized tubes, and the mice were sacrificed to harvest spleens for quantification of humoral and cellular immunity, respectively. As shown in Figure 18, the C14-PEtOx formulation was able to induce both cellular immunity and antibody production compared to unvaccinated mice, demonstrating the potential of POx-lipid in LNP-vaccination technology.

[0070] conclusion In conclusion, we investigated a modular synthetic approach to facilitate the facile diversification of the α-terminus of POx. In this study, we demonstrated that para-fluorosubstitution is suitable for the structural diversification of poly(2-oxazoline)s, successfully introducing various O-, N-, and S-nucleophiles to the α-terminus. This was achieved by carefully adjusting the reaction conditions, first enabling the selective reaction of the 2-oxazolinium species with O-, N-, and S-nucleophiles in the CROP termination step, followed by derivatization of the pentafluorobenzyl group. Thus, para-fluorosubstitution enables the synthesis of a wide range of telechelic POx in a single post-polymerization step. Next, we demonstrated the utility of this approach by synthesizing POx-lipid conjugates and a POx-based macroinitiator for NCA polymerization to yield POx-polypeptide block copolymers. Given the importance of PEG-polypeptide systems in nanomedicine, the newly synthesized system should ideally be suitable for the development of drug delivery systems. Next, we evaluated the in vivo circulation and in vivo mRNA expression of POx-based liposomes and lipid nanoparticles, respectively. The circulation of POx-based liposomes using the novel tetrafluorobenzyl linker was in good agreement with previous reports. Furthermore, the mRNA expression of POx-LNPs was comparable to that of commercially available PEG-based formulations, and the formulations showed no significant toxicological markers compared with PBS. These results suggest that the tetrafluorophenyl linker has little significant effect on the in vivo performance of POx-based lipid systems.

[0071] Precise control of the end groups of biocompatible polymers has proven key to enabling polymer-based therapeutics and nanomedicines. However, end-group diversification can be a synthetically exhaustive process, especially for polymers prepared via ionic polymerization mechanisms, due to the limited functional group tolerance associated with the polymerization mechanism. In this contribution, we present a one-step post-polymerization modification approach to easily diversify the end groups of poly(2-oxazoline)s (POx) with a wide range of nucleophiles. More specifically, by adjusting reaction parameters, we established a difference in reactivity between the pentafluorophenyl and living 2-oxazolinium chain ends, enabling the selective introduction of nucleophiles via CROP termination followed by nucleophilic parafluorosubstitution, facilitating end-group diversification of POx in a single post-polymerization modification step. Therefore, this work presents an attractive synthetic route to facilitate POx synthesis. The value of this approach is demonstrated through the synthesis of well-defined lipid-polymer conjugates and block copolymers of POx and polypeptides, both of which are suitable for drug and gene delivery. Finally, the application of lipid-POx conjugates to the formulation and delivery of mRNA-lipid nanoparticles was investigated, highlighting the value of POx as a biocompatible polymer platform.

[0072] Precise control and modulation of polymer termini has proven crucial for the biomedical application of polymers, perhaps best exemplified by PEGylation. PEGylation utilizes polymer termini to synthesize well-defined nanoassemblies and for the bioconjugation of proteins, nucleic acids, and targeting ligands. Therefore, the conceptually simple conjugation of non-immunogenic, biocompatible (i.e., "stealth") polymers represents a broadly attractive approach for modulating the biodistribution and circulation of therapeutic agents. Another class of polymers suitable for this purpose is poly(2-oxazoline) and its structural relatives, namely, poly(cyclic iminoethers), which are characterized by a high degree of structural diversity due to the tunability of the ring size and substituents of the cyclic iminoether monomers. In addition to monomer design, numerous efficient post-polymerization modification chemistries are available, allowing further tailoring of polymer architecture to the needs of specific applications, such as tissue engineering and drug / protein / gene delivery. Although structural variation along the polymer backbone offers ample opportunities in biomedicine, facile end-group diversification remains a key feature in bioconjugation and the synthesis of advanced drug delivery systems.

[0073] In this regard, considerable efforts have demonstrated the feasibility of introducing many functionalities to the POx end groups, but the process of end-group diversification has generally been synthetically exhaustive. This is primarily due to the diversification of the α-terminus. Generally, functional groups with the preferred tosylate or triflate groups are commercially available in limited quantities, likely due to poor storage stability, and therefore require in-house synthesis and thorough purification. Furthermore, the steric and electronic factors of the polymerization initiator play an important role in rapidly initiating the polymerization reaction. This is because, in addition to evaluating the fidelity of the end group, accurate studies of the polymerization kinetics for different 2-oxazoline monomers are necessary. Given these constraints, a strategy focusing on post-polymerization diversification of a single end group is synthetically attractive, enabling diverse yet highly reproducible syntheses. To date, only a handful of functional groups (azides, alkynes, esters, and alkenes) can be introduced directly to the α-terminus, i.e., without protecting groups, to facilitate end-group diversification through post-polymerization modification. However, these functional groups are not suitable for introducing relatively simple molecules with diverse functionalities. Although highly efficient, the azide-alkyne cycloaddition reaction is synthetically unattractive for this purpose. Finally, although alkenes can be modified by thermal or photoinitiated thiol-ene chemistry, the radical nature of this process is not ideal for end-group diversification of POx. Other functional groups requiring the use of protecting groups (e.g., aldehydes, ketones, carboxylates, amines, alcohols, or maleimides) require a deprotection step before subsequent reactions, thereby necessitating additional purification, as incomplete deprotection and / or incomplete reaction compromise the purity of the heterotelechelic polymer. Finally, the substrates mentioned above have a relatively narrow substrate scope, making it difficult to determine the quantitative conversion of each synthetic step.

[0074] In contrast to the challenging α-terminus, ω-terminus end-group diversification is straightforward, and electrophilic 2-oxazoliniums can be ring-opened with a wide variety of commercially available O-, N-, or S-based nucleophiles. Inspired by the simplicity and broad reactivity of nucleophilic termination reactions, we attempted to initiate cationic ring-opening polymerization using electrophilic moieties susceptible to nucleophilic substitution across a similar substrate range. In this study, we demonstrated that the reactivity difference between the intact 2-oxazolinium ω-terminus and the electrophilic pentafluorophenyl α-terminus can be established toward various nucleophiles by adjusting the reaction parameters. The intact pentafluorophenyl moiety underwent selective para-fluorosubstitution with O-, N-, and S-nucleophiles, facilitating end-group diversification. Modification of this moiety is, in principle, orthogonal to several functional groups incorporated along the polymer chain. Therefore, this approach is useful for the synthesis of drug and gene delivery vehicles, as demonstrated through the synthesis of well-defined lipid-POx conjugates and POx-polypeptide block copolymers. Finally, we report the first application of lipid-POx conjugates to the formulation and delivery of mRNA-lipid nanoparticles, highlighting POx as a promising candidate for gene delivery applications. [Industrial Applicability]

[0075] The polymers of the present invention can be used in medical materials, such as vaccines, drug delivery formulations, gene delivery formulations, protein conjugates, inactive pharmaceutical ingredients, excipients, and surface coatings.

Claims

1. Formula (I): 【Chemical 1】 [wherein X is a halogen, —SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ , -C≡C-R ２ , -NHOR ２ , -ONR ２ R ３ , optionally substituted heteroaryl, a group capable of forming a lipid based nanoparticle (e.g., a form including a lipid nanoparticle (LNP), a liposome or a lipoplex), or a micelle containing a lipid-solubilizing group such as a polycationic group, a polyanionic group or a group derived from a fatty acid, preferably F, -SR ２ , -OR ２ , -CN, -NHR ２ , -N ３ , 【Chemistry 2】 and Y is SR ２ , -OR ２ (However, when X is F, it is not —OH), —CN, —NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ or optionally substituted heteroaryl, preferably -SR ２ , -OR ２ (However, when X is F, it is not —OH), —NHR ２ , -NR ２ R ３ , -N ３ or 【Chemistry 3】 and more preferably, -SR ２ , -OR ２ (However, when X is F, it is not —OH), —NHR ２ ,or 【Chemistry 4】 and where Repeating Unit 【Chemistry 5】 is independent at each occurrence and may be the same or different; R １ is selected from the group consisting of an optionally substituted alkyl group having 1 to 40 carbon atoms, an optionally substituted alkenyl group having 2 to 40 carbon atoms, an optionally substituted alkynyl group having 2 to 40 carbon atoms, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , a sugar derivative, and a repeating unit or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl, preferably —CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ａ , R ＡＡ and R ＡＡＡ are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R Ａ and R ＡＡ may be linked together to form a ring, such as a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam; R Ａ , R ＡＡ and / or R ＡＡＡ may further be linked to a functional molecule such as a label, or a biofunctional molecule including a protein, nucleic acid, etc.; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, lipid-solubilizing groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, optionally substituted alkynyl groups having 2 to 20 carbon atoms, and oxygen protecting groups such as acyl groups such as acetyl, and ether groups such as methoxymethyl; The alkyl, alkenyl and alkynyl are straight, branched or cyclic chains; m is an integer from 1 to 3; and n is an integer from 1 to 2000. or a pharmaceutically acceptable salt thereof.

2. X is halogen, -SR ２ , -OR ２ , -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ , -NHOR ２ , -ONR ２ R ３ or optionally substituted heteroaryl; Y is SR ２ , -OR ２ , -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ ,or 【Chemistry 6】 and More preferably -SR ２ , -OR ２ , -NHR ２ ,or 【Chemistry 7】 and R １ is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , sugar derivatives, and repeat units, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl, preferably —CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ａ , and R ＡＡ are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R Ａ and R ＡＡ may be linked together to form a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam; R Ａ and / or R ＡＡ may further be linked to a label or a biofunctional molecule such as a protein, nucleic acid, etc.; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, lipid-solubilizing groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, and optionally substituted alkynyl groups having 2 to 20 carbon atoms, and oxygen protecting groups; The alkyl, alkenyl, and alkynyl are straight or branched chain; The alkyl, alkenyl and alkynyl substituents are, independently of one another, and without limitation, ester groups, amino groups, azide groups, and n is an integer from 2 to 1000; 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. X is F, -SR ２ , -OR ２ , -CN, -NHR ２ , -N ３ ,or 【Chemistry 8】 and Y is -SR ２ , -OR ２ , -NHR ２ , -N ３ ,or 【Chemistry 9】 and R １ is methyl, ethyl, propyl, butyl, -CH ２ CH ２ COOCH ３ , -CH ２ CH ２ CH ２ COOCH ３ , -CH ２ CH ２ CH ２ CH ２ COOCH ３ , butenyl, butynyl, and —CH ２ OCH ３ selected from the group consisting of: R ２ represents hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, —C(═O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , sugar derivatives, and repeating units thereof; R Ａ and R ＡＡ are each independently hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R Ａ and R ＡＡ may be linked together to form a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R Ａ and / or R ＡＡ may be further linked to a functional molecule such as a label, or a biofunctional molecule such as a protein, nucleic acid, etc.; R Ｂ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 20 carbon atoms, and optionally substituted alkenyl groups having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000; 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. Y is, 【Chemistry 10】 and R Ａ and / or R ＡＡ may be linked to a label or a functional molecule selected from the group consisting of biofunctional molecules including proteins and nucleic acids; The compound of claim 1.

5. X is -OR ２ or -SR ２ and Y is 【Chemistry 11】 and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 20 carbon atoms, and optionally substituted alkenyl groups having 2 to 20 carbon atoms; The compound of claim 1.

6. X is -OR ２ or -SR ２ and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is selected from the group consisting of optionally substituted alkyl groups having 1 to 20 carbon atoms and optionally substituted alkenyl groups having 2 to 20 carbon atoms; The compound of claim 1.

7. X is -OR ２ , -SR ２ , or -NHR ２ and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is hydrogen; The compound of claim 1.

8. X is -OR ２ , -SR ２ , or -NHR ２ and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is an oxygen protecting group; The compound of claim 1.

9. X is a group capable of forming a lipid-based nanoparticle (e.g., a lipid nanoparticle (LNP), liposome, or lipoplex-containing form) liposome or micelle; The compound of claim 1.

10. The compound of claim 1 , wherein the sugar derivative is a glucose derivative.

11. A composition comprising a compound according to any one of claims 1 to 10.

12. A composition comprising a compound according to any one of claims 1 to 10, wherein the composition is a vaccine.

13. A composition comprising the compound of any one of claims 1 to 10, wherein said composition is used as a diagnostic probe.

14. Formula (II) 【Chemistry 12】 [wherein the repeating unit 【Chemistry 13】 is independent at each occurrence and may be the same or different; R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , and -N ３ , -NHOR ２ , and -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, and an optionally substituted heteroaryl, and -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and a sugar derivative, or R ５ Ga-NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl, preferably —CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight chain; m is an integer from 1 to 3; and and n is an integer from 2 to 2000. A compound represented by the formula:

15. R ５ is -OR ２ , -SR ２ or --NHR, R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is selected from the group consisting of optionally substituted alkyl groups having 1 to 20 carbon atoms or optionally substituted alkenyl groups having 2 to 20 carbon atoms; 15. The compound of claim 14.

16. R ５ is -OR ２ , -SR ２ , or -NHR ２ and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is H; 15. The compound of claim 14.

17. R ５ is -OR ２ , -SR ２ , or -NHR ２ and R ２ is -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ｃ is an oxygen protecting group; 15. The compound of claim 14.

18. Formula (III) 【Chemistry 14】 wherein each occurrence of the repeating unit is independent and may be the same or different; R ４ is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl group, and an optionally substituted heteroaryl group; R ５ is a halogen, -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ , -NHOR ２ , - and -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; The alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. A method for producing a compound represented by the formula: It includes the following steps: (a) Formula (IV) 【Chemistry 15】 wherein L is a leaving group. with a compound of formula (V) 【Chemistry 16】 [In the formula, R ４ is selected from the group consisting of an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted aryl, and an optionally substituted heteroaryl. in the presence of a base to form a compound of formula (II) 【Chemistry 17】 to obtain a compound of the formula (b) A method of preparing a compound of formula (III) by reacting a compound of formula (II) with an azide reagent.

19. the leaving group is selected from the group consisting of halogen, OTf, ONs and OTs; 19. A method for preparing a compound of formula (III) according to claim 18.

20. The base is Et ３ N, DBU, NMP, TBD, KOH, iPr ２ 19. A method for preparing a compound of formula (III) according to claim 18, wherein the compound is selected from the group consisting of NEt, and t-BuOK.

21. The azide reagent is NaN ３ , TMSN ３、 TsN ３ 19. A method for preparing a compound of formula (III) according to claim 18, wherein the azide is selected from the group consisting of tetrabutylammonium azide, tetrabutylammonium azide, and diphenylphosphoryl azide.

22. Formula (III) 【Chemistry 18】 wherein each occurrence of the repeating unit is independent and may be the same or different; R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is a halogen, -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , and -N ３ , -NHOR ２ , -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. A method for producing a compound represented by the formula: It includes the following steps: Formula (IIIa) 【Chemistry 19】 [In the formula, R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; and n is an integer from 2 to 2000. The compound represented by formula (III) 【Chemistry 20】 [In the formula, R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is a halogen, -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , and -N ３ , -NHOR ２ , and -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ , each independently selected from hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, and an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. with a nucleophile to give a compound of the formula:

23. 23. The method of making a compound of formula (III) according to claim 22, wherein the nucleophile is an alcohol, phenol, carboxylic acid, amine, azide, thiol, cyanide, or alkyne type nucleophile.

24. Formula (VI) 【Chemical 21】 wherein each occurrence of the repeating unit is independent and may be the same or different; R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is a halogen, -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ , -NHOR ２ , and -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and and n is an integer from 2 to 100. A method for producing a compound represented by the formula: Formula (IIa) 【Chemical 22】 [In the formula, R ４ , optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; and n is an integer from 2 to 2000. A method for producing a compound represented by formula (IV), comprising reacting a compound represented by formula (IV) with a nucleophile to obtain a compound represented by formula (VI).

25. Formula (Ia) 【Chemical 23】 wherein each occurrence of the repeating unit is independent and may be the same or different; R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , -N ３ , -NHOR ２ , and -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl, preferably —CH ２ CH (OR Ｃ ) CH ２ OR Ｃ and R Ａ and R ＡＡ are each independently selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted alkenyl group having 2 to 20 carbon atoms, and an optionally substituted alkynyl group having 2 to 20 carbon atoms, or R Ａ and R ＡＡ may be linked together to form a ring, such as a carbocycle, heterocycle, aryl, heterocyclyl, lactone, or lactam, and R Ａ and / or R ＡＡ may further be linked to a functional molecule such as a label, a biofunctional molecule such as a protein, a nucleic acid, etc.; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, lipid-solubilizing groups such as optionally substituted alkyl groups having 1 to 20 carbon atoms, optionally substituted alkenyl groups having 2 to 20 carbon atoms, and optionally substituted alkynyl groups having 2 to 20 carbon atoms, or oxygen protecting groups such as acyl groups such as acetyl, and ether groups such as methoxymethyl; The alkyl, alkenyl, and alkynyl are straight, branched, or cyclic chains; and n is an integer from 1 to 2000. A method for producing a compound represented by the formula: Formula (III) 【Chemistry 24】 wherein each occurrence of the repeating unit is independent and may be the same or different; R ４ is selected from the group consisting of optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms, optionally substituted aryl, and optionally substituted heteroaryl; R ５ is a halogen, -SR ２ , -OR ２ , -CN, -NHR ２ , -NR ２ R ３ , -NHNHR ２ , -N=NR ２ , and -N ３ , -NHOR ２ , -ONR ２ R ３ selected from the group consisting of: R ２ and R ３ are each independently hydrogen, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkenyl group having 2 to 6 carbon atoms, an optionally substituted alkynyl group having 2 to 6 carbon atoms, an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl, -C(=O)R Ｂ , -C(=O)OR Ｂ , -C(=O)N(R Ｂ ) ２ , -S(=O)R Ｂ , -S(=O) ２ R Ｂ , -CH ２ CH (OR Ｃ ) CH ２ OR Ｃ , and sugar derivatives, or -NR ２ R ３ In the case of ２ and R ３ may be linked together to form an optionally substituted heterocyclyl; R Ｂ is selected from the group consisting of hydrogen, optionally substituted alkyl groups having 1 to 6 carbon atoms, optionally substituted alkenyl groups having 2 to 6 carbon atoms, and optionally substituted alkynyl groups having 2 to 6 carbon atoms; R Ｃ is selected from the group consisting of hydrogen, an optionally substituted alkyl group having 1 to 20 carbon atoms, or an optionally substituted alkenyl group having 2 to 20 carbon atoms; said alkyl and alkenyl are straight-chain; and n is an integer from 2 to 2000. The compound represented by formula (VII) 【Chemistry 25】 [R Ａ , R ＡＡ is as above] By reacting with a compound represented by A method for producing a compound represented by formula (Ia), which provides a compound represented by formula (Ia).

26. 26. The method for producing a compound of formula (Ia) according to claim 25, wherein the reaction is carried out using a metal catalyst, such as a Cu catalyst.

27. 26. The method for preparing a compound of formula (Ia) according to claim 25, wherein the reaction is carried out without the use of a metal catalyst.

28. 26. The method for preparing a compound of formula (Ia) according to claim 25, wherein the reaction is carried out in water.

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