Β-amino ester, poly-β-amino ester compound, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a β-amino ester monomer compound and a preparation method therefor. The main chain of the β-amino ester monomer has a tertiary amine functional group. Further provided are a poly-β-amino ester compound having an accurate molecular weight, and a preparation method therefor. The poly-β-amino ester compound has an adjustable main chain and side chain. By using an exponential iterative growth strategy with orthogonal protecting groups and by means of performing selective TBDPS deprotection and selective benzyl deprotection on the β-amino ester monomer, and then subjecting the two substances obtained after deprotection to esterification coupling, a poly-β-amino ester having a twice-amplified molecular weight is obtained. In the present invention, the exponential iterative growth polymerization method with orthogonal protecting groups is used to realize accurate synthesis of a poly-β-amino ester, and poly-β-amino esters having completely controllable molecular weights, side chains and end groups, and having single-molecular dispersity are thus obtained. Further provided in the present invention is the use of a deprotected poly-β-amino ester compound having functional groups in the preparation of a gene delivery drug.

Description

β-amino ester, poly-β-amino ester compound, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of polymer synthesis, in particular to beta-amino ester, poly-beta-amino ester compounds and preparation methods and applications thereof. Background Art

[0002] The molecular weight, monomer sequence and topological structure of a polymer jointly determine its properties and functions. Polymers with single-molecule divergence have unique properties due to the molecular-level precision of their structure. Poly (β-amino ester) is a class of high-molecular-weight compounds with gene delivery activity. Poly (β-amino ester) can form nanoparticles by electrostatic interaction with negatively charged biomacromolecules such as mRNA and DNA, and is widely used in gene and drug delivery. Traditionally, poly (β-amino ester) can be prepared by the polycondensation reaction between primary amines or di-secondary amines and diacrylate compounds. Its molecular weight distribution is relatively wide, and the obtained product does not have a single, precise molecular weight, which affects the precise regulation of its properties and also limits further research on its pharmacology and toxicology. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a β-amino ester, a poly-β-amino ester compound and a preparation method and application thereof, which are used to solve the technical problem that the molecular weight, side chain and end group cannot be accurately controlled in the synthesis of poly-β-amino ester in the prior art.

[0004] To achieve the above-mentioned and other related purposes, the present invention provides a β-amino ester monomer compound, the chemical structure of which is shown in Formula I:

[0005] Wherein, x=0~16, y=0~10;

[0006] R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0007] The present invention also provides a method for preparing the above-mentioned β-amino ester monomer compound, comprising the following steps: obtaining a compound of formula I by Michael addition reaction of a compound of formula II with benzyl acrylate;

[0008] Wherein, x=0~16; y=0~10;

[0009] R1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0010] The present invention also provides use of the above-mentioned β-amino ester monomer compound or the β-amino ester monomer compound prepared by the above-mentioned preparation method in the preparation of a poly-β-amino ester compound.

[0011] The present invention also provides a poly-β-amino ester compound, which is obtained by polymerization of the β-amino ester monomer compound or the β-amino ester monomer compound prepared by the preparation method described above. The chemical structure of the poly-β-amino ester compound is as shown in Formula A:

[0012] Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1;

[0013] R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0014] The present invention also provides a method for preparing the poly-β-amino ester compound as described above, comprising the following steps:

[0015] 1) selectively removing the TBDPS protecting group from the β-amino ester monomer compound described above or the β-amino ester monomer compound prepared by the preparation method described above to obtain a compound of formula V;

[0016] 2) selectively removing the benzyl protecting group from the β-amino ester monomer compound described above or the β-amino ester monomer compound prepared by the preparation method described above to obtain a compound of formula VI;

[0017] 3) esterifying and coupling the compound of formula V and the compound of formula VI in the presence of a coupling reagent to obtain a dimerized poly (β-amino ester);

[0018] 4) Repeating the TBDPS protection group removal process of step 1) with the dimerized poly-β-amino ester, repeating the benzyl protection group removal process of step 2) with the dimerized poly-β-amino ester, and then repeating the esterification coupling process of step 3) to obtain a tetramerized poly-β-amino ester;

[0019] 5) Repeat the above steps 1) to 3) of removing TBDPS protecting group, removing benzyl protecting group and esterification coupling to obtain a series of tetramer poly β amino esters with a degree of polymerization of 2 m Poly-β-amino ester compounds;

[0020] Where x = 0 to 16; y = 0 to 10; n = 2 m ; m is a positive integer greater than or equal to 1;

[0021] R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0022] The present invention also provides a poly (beta) amino ester compound with a functional group after removing the protecting group, which is obtained by using the poly (beta) amino ester compound of formula A described above through one or more reactions selected from the group consisting of removing the TBDPS protecting group, removing the benzyl protecting group, removing the PG group, and modifying the carboxyl group.

[0023] The chemical structural formula of the poly (beta) amino ester compound with the functional group removed is as shown in Formula B1 or B2:

[0024] Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1;

[0025] R 1’ One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl and guanidinyl.

[0026] R 2 is selected from H or FG; FG is a functional group, and FG is selected from one of polyethylene glycol monomethyl ether, C3-C11 alcohol, C3-C11 guanidine group, C3-C11 dimethylamino group, C3-C11 amino group, C3-C11 4-methylpiperazine or C3-C11 biguanidine group, and the corresponding structural formula is as follows:

[0027] f = 1 to 8; g = 0 to 8, * is the connection site.

[0028] The present invention also provides a method for preparing the poly (beta) amino ester compound having functional groups removed from the protective groups as described above, comprising the following steps:

[0029] If the poly (beta) amino ester compound in claim 4 has R1 Selected from -O-PG group, removing the TBDPS protecting group, removing the PG group, and removing the benzyl protecting group from the poly (beta) amino ester compound as claimed in claim 4 to obtain the poly (beta) amino ester compound having carboxyl modification of formula B1 as described above; and / or,

[0030] If the poly (beta) amino ester compound in claim 4 has R 1 Selected from -O-PG group, the poly (beta) amino ester compound as claimed in claim 4 is subjected to the steps of removing the benzyl protecting group, modifying the carboxyl group, removing the TBDPS protecting group, and removing the PG group to obtain the poly (beta) amino ester compound having carboxyl modification as described above, Formula B1; and / or,

[0031] If the poly (beta) amino ester compound in claim 4 has R 1 One selected from the group consisting of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, removing the TBDPS protecting group and the benzyl protecting group from the poly-β-amino ester compound according to claim 4 to obtain the poly-β-amino ester compound having a carboxyl modification of formula B2 as described above; and / or

[0032] If the poly (beta) amino ester compound in claim 4 has R 1 One selected from the group consisting of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, the poly (beta) amino ester compound as described in claim 4 is subjected to the steps of removing the benzyl protecting group, modifying the carboxyl group, and removing the TBDPS protecting group to obtain the poly (beta) amino ester compound having carboxyl modification as described above, formula B2.

[0033] The present invention also provides a use of the poly-β-amino ester compound as described above, or the poly-β-amino ester compound with functional groups removed from the protective groups as described above, or the poly-β-amino ester compound with functional groups removed from the protective groups prepared by the preparation method as described above in the preparation of gene delivery drugs.

[0034] As described above, the β-amino ester and poly-β-amino ester compounds of the present invention and their preparation methods and applications have the following beneficial effects:

[0035] The β-amino ester of the present invention selects two orthogonal protecting groups, tert-butyldiphenylsilyl (TBDPS) and benzyl (Bn), to protect the hydroxyl group and the carboxyl group respectively. 1 When the group is a hydroxyl group, a third orthogonal protecting group 2-tetrahydropyranyl ether (THP) is introduced to protect the hydroxyl group on the functional side chain (when the R 1When the group is imidazolyl, morpholinyl, 2-tetrahydrofuranyl or 4-methylpiperazinyl, there is no need to introduce a third orthogonal protecting group). Tetrabutylammonium fluoride (TBAF) is used to selectively remove the TBDPS protecting group, and Pd / C and H2 are used to selectively remove the benzyl protecting group. Then, esterification coupling is achieved using efficient coupling reagents EDCI and DPTS. The above experimental path is repeated for polyester compounds with different degrees of polymerization to achieve exponential iterative growth polymerization cycles, and a series of exponential polymerizations with a degree of polymerization of 2 (DP = 2 m )'s precision poly-β-amino ester.

[0036] The present invention also designs a modular end group modification strategy for poly (beta) amino esters. By selectively removing the benzyl group, the carboxylic acid end can be directly used as the end modification group; or a variety of end chains with hydroxyl groups can be modified into the poly (beta) amino ester molecule through esterification coupling to achieve modular modification.

[0037] The present invention adopts an exponential iterative growth polymerization method to achieve the precise synthesis of poly (β-amino esters), and obtains a series of poly (β-amino esters) with completely and precisely controllable molecular weights, side chains, and end groups, and a dispersion at the single-molecule level. Based on the technical route provided by the present invention, the structurally precise poly (β-amino esters) can be used in the fields of gene delivery, drug delivery, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 is a schematic diagram of the synthesis route of exponential iterative growth polymerization in the present invention.

[0039] FIG2 is a size exclusion chromatography SEC curve diagram of compound 7, compound 10 and compound 13.

[0040] FIG3 is a diagram showing the expression of green fluorescent protein after HEK-293T cells were transfected with plasmid DNA (pDNA) encoding EGFP, after the poly (β-amino ester) with functional groups removed from the protective groups of the present invention was transfected with EGFP. DETAILED DESCRIPTION

[0041] The following describes the embodiments of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in this embodiment only illustrate the basic concept of the present invention in a schematic manner, and the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components during actual implementation. During actual implementation, the type, quantity and proportion of each component can be changed at will, and the component layout type may also be more complicated.

[0042] In the present invention:

[0043] The first aspect of the present invention provides a β-amino ester monomer compound, the chemical structure of the β-amino ester monomer compound is shown in Formula I:

[0044] Wherein, x=0~16, y=0~10;

[0045] R 1 One selected from the group consisting of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG groups; wherein the structural formulas corresponding to imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl and di-tert-butyloxycarbonylguanidine are as follows:

[0046] The -O-PG group is a hydroxyl group with a PG protecting group, wherein the PG protecting group is selected from 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl. The corresponding structural formula is as follows:

[0047] Among them, * is the connection site.

[0048] A second aspect of the present invention provides a method for preparing the above-mentioned β-amino ester monomer compound, comprising the following steps: obtaining a compound of formula I by Michael addition reaction of a compound of formula II with benzyl acrylate;

[0049] Where, x=0~16; y=0~10; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0050] In some embodiments of the present invention, the molar ratio of the compound of formula II to benzyl acrylate is 1:1.2-2, for example, 1:1.2-1.4, 1:1.4-1.6, 1:1.6-1.8, or 1:1.8-2.

[0051] In some embodiments of the present invention, the Michael addition reaction of the compound of formula II with benzyl acrylate is carried out in an organic solvent, wherein the organic solvent is selected from one or more of dichloromethane, tetrahydrofuran, N,N-dimethylformamide, and isopropyl alcohol.

[0052] The temperature for the Michael addition reaction of the compound of formula II with benzyl acrylate is 20-50° C., for example, 20-25° C., 25-30° C., 30-35° C., 35-40° C., 40-45° C., or 45-50° C.

[0053] In the preparation method of the β-amino ester monomer compound of the present invention, the compound of formula II is obtained by a nucleophilic substitution reaction between a compound of formula III and a compound of formula IV:

[0054] In some embodiments of the present invention, the molar ratio of the compound of formula III to the compound of formula IV is 1:1.2-1.5, for example, 1:1.2-1.3, 1:1.3-1.4, or 1:1.4-1.5.

[0055] In some embodiments of the present invention, the nucleophilic substitution reaction of the compound of formula III with the compound of formula IV is carried out in the presence of an organic solvent and a base.

[0056] Wherein, the organic solvent is DMF (N,N-dimethylformamide), N,N-dimethylacetamide, acetonitrile, acetone. In a preferred embodiment of the present invention, the organic solvent is DMF.

[0057] The base is DBU (1,8-diazacyclo[5,4,0]undecene-7-ene).

[0058] The temperature of the nucleophilic substitution reaction is 60-95°C, for example, 60-65°C, 65-70°C, 70-75°C, 75-80°C, 80-85°C, 85-90°C or 90-95°C.

[0059] The nucleophilic substitution reaction time is 24-72 hours, for example, 24-28 hours, 28-32 hours, 32-36 hours, 36-40 hours, 40-44 hours, 44-48 hours, 48-52 hours, 52-56 hours, 56-60 hours, 60-64 hours, 64-68 hours or 68-72 hours.

[0060] A third aspect of the present invention provides use of the β-amino ester monomer compound as described above or the β-amino ester monomer compound prepared by the preparation method as described above in the preparation of a poly-β-amino ester compound.

[0061] A fourth aspect of the present invention provides a poly (β-amino ester) compound, which is obtained by polymerization of the β-amino ester monomer compound described above or the β-amino ester monomer compound prepared by the preparation method described above, wherein the chemical structure of the poly (β-amino ester) compound is as shown in Formula A:

[0062] Where x = 0 to 16; y = 0 to 10; n = 2 m, m is a positive integer greater than or equal to 1; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0063] A fifth aspect of the present invention provides a method for preparing the poly-β-amino ester compound as described above, comprising the following steps:

[0064] 1) selectively removing the TBDPS protecting group from the β-amino ester monomer compound described above or the β-amino ester monomer compound prepared by the preparation method described above to obtain a compound of formula V;

[0065] 2) selectively removing the benzyl protecting group from the β-amino ester monomer compound described above or the β-amino ester monomer compound prepared by the preparation method described above to obtain a compound of formula VI;

[0066] 3) esterifying and coupling the compound of formula V and the compound of formula VI in the presence of a coupling reagent to obtain a dimerized poly (β-amino ester);

[0067] 4) Repeating the TBDPS protecting group removal process in step 1) with the dimerized poly-β-amino ester, repeating the benzyl protecting group removal process in step 2) with the dimerized poly-β-amino ester, and then repeating the esterification coupling process in step 3) to obtain a tetramerized poly-β-amino ester;

[0068] 5) Repeat the above steps 1) to 3) of removing the TBDPS protecting group, removing the benzyl protecting group and esterification coupling process on the tetramer poly β-amino ester to obtain a series of polymerization degrees of 2 m Poly-β-amino ester compounds;

[0069] Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

[0070] The synthetic route of the poly (β-amino ester) compound of the present invention is shown in FIG1 .

[0071] The preparation method of the poly-β-amino ester compound of the present invention further includes one or more of the following features:

[0072] a) removing the TBDPS protecting group in step 1) in the presence of tetra-n-butylammonium fluoride;

[0073] b) the debenzylation of the protecting group in step 2) is carried out in the presence of Pd / C and H2;

[0074] c) The coupling reagents in step 3) are 4-(dimethylamino)pyridine p-toluenesulfonate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0075] Among them, 4-(dimethylamino)pyridine p-toluenesulfonate, abbreviated as DPTS, CAS: 91944-64-8.

[0076] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, abbreviation: EDCI; CAS: 25952-53-8.

[0077] In some embodiments of the present invention, the removal of the TBDPS protecting group in step 1) is performed at 0-25°C, for example, 0-5°C, 5-10°C, 10-15°C, 15-20°C or 20-25°C.

[0078] The removal of the TBDPS protecting group in step 1) is carried out in an organic solvent, wherein the organic solvent is anhydrous tetrahydrofuran (THF).

[0079] In the reaction of removing the TBDPS protecting group in step 1), the molar ratio of the β-amino ester compound monomer to tetra-n-butylammonium fluoride is 1:1.2 to 2. For example, 1:1.2 to 1.4, 1:1.4 to 1.6, 1:1.6 to 1.8, or 1:1.8 to 2, etc.

[0080] In some embodiments of the present invention, the removal of the benzyl protecting group in step 2) is carried out in one or more of anhydrous ethyl acetate, anhydrous tetrahydrofuran, anhydrous ethanol or anhydrous methanol.

[0081] In the debenzylation reaction in step 2), the ratio of Pd / C to the β-amino ester compound monomer is 5-10 wt %, for example, 5-6 wt %, 6-7 wt %, 7-8 wt %, 8-9 wt % or 9-10 wt %.

[0082] In some embodiments of the present invention, the esterification coupling reaction in step 3) is carried out in one or more of anhydrous dichloromethane, anhydrous tetrahydrofuran, anhydrous chloroform, and anhydrous acetonitrile.

[0083] In the esterification coupling reaction in step 3), the molar ratio of the compound of formula V, the compound of formula VI, DPTS, and EDCI is 1:(1-1.2):(0.3-0.5):(2-3). For example, 1:(1~1.1):(0.3~0.5):(2~3), 1:(1.1~1.2):(0.3~0.5):(2~3), 1:(1~1.2):(0.3~0.4):(2~3), 1:(1~1.2):(0.4~0.5):(2~3), 1:(1~1.2):(0.3~0.5):(2~2.2), 1:(1~1.2):(0.3~0.5):(2.2~2.4), 1:(1~1.2):(0.3~0.5):(2.4~2.6), 1:(1~1.2):(0.3~0.5):(2.6~2.8) or 1:(1~1.2):(0.3~0.5):(2.8~3).

[0084] A sixth aspect of the present invention provides a poly (beta) amino ester compound having a functional group and a deprotected group, which is obtained by removing the protective group from the poly (beta) amino ester compound of formula A described above and performing one or more reactions selected from the group consisting of a TBDPS protective group removal reaction, a benzyl protective group removal reaction, a PG group removal reaction, and a carboxyl group modification reaction.

[0085] The chemical structural formula of the poly (beta) amino ester compound with the functional group removed is as shown in Formula B1 or B2:

[0086] Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1;

[0087] R 1’ One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl or 4-methylpiperazinyl, methyl, guanidinyl;

[0088] R 2 is selected from H or FG; FG is a functional group, and FG is selected from one of polyethylene glycol monomethyl ether, C3-C11 alcohol, C3-C11 guanidine group, C3-C11 dimethylamino group, C3-C11 amino group, C3-C11 4-methylpiperazine or C3-C11 biguanidine group, and the corresponding structural formula is as follows:

[0089] f = 1 to 8; g = 0 to 8, * is the connection site.

[0090] A seventh aspect of the present invention provides a method for preparing the poly (beta) amino ester compound having functional groups removed from the protective groups as described above, comprising the following steps:

[0091] If the poly (beta) amino ester compound in claim 4 has R1 The poly (beta) amino ester compound according to claim 4 is obtained by removing the TBDPS protecting group, removing the PG group, and removing the benzyl protecting group to obtain the poly (beta) amino ester compound having the functional group (formula B1) without the protecting group selected from the group consisting of -O-PG group; and / or

[0092] If the poly (beta) amino ester compound in claim 4 has R 1 The poly (beta) amino ester compound according to claim 4 is obtained by removing the benzyl protecting group, modifying the carboxyl group, removing the TBDPS protecting group, and removing the PG group to obtain the poly (beta) amino ester compound having the functional group and the formula B1; and / or

[0093] If the poly (beta) amino ester compound in claim 4 has R 1 One selected from the group consisting of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, wherein the poly-β-amino ester compound according to claim 4 is subjected to the step of removing the TBDPS protecting group and removing the benzyl protecting group to obtain the poly-β-amino ester compound having functional groups (Formula B2) without the protecting group; and / or

[0094] If R1 of the poly (beta) amino ester compound in claim 4 is selected from one of imidazole, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, the poly (beta) amino ester compound as claimed in claim 4 is obtained by removing the benzyl protecting group, modifying the carboxyl group, and removing the TBDPS protecting group to obtain the poly (beta) amino ester compound formula B2 with the functional group removed.

[0095] Wherein, the removal of TBDPS protecting group is carried out in the presence of tetra-n-butylammonium fluoride;

[0096] The PG group removal is carried out in the presence of p-toluenesulfonic acid (TsOH);

[0097] The debenzylation of the protecting group is carried out in the presence of Pd / C and H2;

[0098] The modification of the carboxyl group is carried out in the presence of DPTS and EDCI.

[0099] In some embodiments of the present invention, the PG group removal reaction is carried out in isopropyl alcohol (IPA) and / or hexafluoroisopropanol (HFIP).

[0100] In the PG group removal reaction, the molar ratio of the added amount of TsOH to the reaction substrate is 4 to 24:1, for example, 4 to 6:1, 6 to 8:1, 8 to 10:1, 10 to 12:1, 12 to 14:1, 14 to 16:1, 16 to 18:1, 18 to 20:1, 20 to 22:1, or 22 to 24:1.

[0101] In an eighth aspect, the present invention provides a use of the poly (beta) amino ester compound as described above, or the poly (beta) amino ester compound with functional groups removed from the protective group as described above, or the poly (beta) amino ester compound with functional groups removed from the protective group prepared by the preparation method as described above in the preparation of gene delivery drugs.

[0102] The invention of the present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0103] Example 1 Preparation of Compound 1

[0104] 6-Amino-1-hexanol (1.0 eq.) and imidazole (3 eq.) were dissolved in CHCl. ​​Tert-butyldiphenylsilyl chloride (TBDPSCl, 2 eq.) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous NaSO, filtered, concentrated in vacuo, and purified by silica gel column chromatography (CHCl:MeOH = 125:3) to afford a dark yellow oil, compound 1 (yield 76%). NMR results were consistent with those reported in the literature.

[0105] Example 2 Preparation of Compound 2

[0106] Pyridinium p-toluenesulfonate (PPTS, 0.1 eq.) was placed in a round-bottom flask and degassed with N2. CH2Cl2 was then added via syringe. N2 was bubbled continuously into the flask for 15 minutes. 4-Chloro-1-butanol (1.0 eq.) and dihydropyran (1.2 eq.) were then added via syringe. The reaction mixture was stirred at room temperature overnight. The reaction was quenched with brine, washed with water (30 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexane:EtOAc = 10:1). The purified product was a colorless oil, compound 2 (75% yield). NMR results were consistent with those reported in the literature.

[0107] Example 3 Preparation of Compound 3

[0108] In a dry round-bottom flask, compound 1 (1.0 eq.) and compound 2 (1.2 eq.) were dissolved in N,N'-dimethylformamide (DMF). 1,5-diazabicyclo[5.4.0]-5-undecene (DBU, 0.3 M in DMF, 1.5 eq.) was added dropwise. The reaction mixture was stirred at 90°C for 48 hours. The reaction mixture was then concentrated in vacuo and dried in a vacuum oven at 90°C for 24 hours. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH = 200:1, containing 0.5% triethylamine, TEA). The purified product, compound 3, was obtained as a brownish-yellow oil (50% yield). 1 H NMR (400MHz, CDCl3, 298K) δ (ppm) = 7.68-7.63 (m, 4H), 7.44-7.34 (m, 6H), 4.57 (t, 1H), 3.88-3.82 (m, 1H),3.80-3.74(m,1H),3.64(t,2H),3.53-3.47(m,1H),2.62(t,2H),2.55(t,2H),1.92-1.28(m,18H expected,21H integrated),1.04(s,9H). 13 C NMR (100MHz, CDCl3, 298K) δ (ppm) = 135.70, 134.21, 129.66, 127.74, 99.00, 66.75, 63.88, 62.50, 45.12, 41.07,32.52,30.86,30.65,29.80,27.27,27.02,26.58,25.55,25.60,19.76,19.35.HR-MS(ESI):[M+H] + calculated m / z=512.3555,found:512.3558.

[0109] Example 4 Preparation of TBDPS-THP-OBn-Monomer 4

[0110] Compound 3 (1 eq.) and benzyl acrylate (1.2 eq.) were dissolved in CHCl in a dry round-bottom flask and stirred at room temperature overnight. The reaction was then concentrated in vacuo and purified by silica gel column chromatography (hexane:EtOAc=2:1). The purified product was a colorless oil, TBDPS-THP-OBn-monomer 4 (yield 72%). 1H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.68-7.66 (m, 4H), 7.43-7.30 (m, 11H), 5.11 (s, 2H), 4.57 (t, 1H), 3.88-3.84 (m, 1H), 3 .75-3.71(m,1H),3.64(t,2H),3.51-3.47(m,1H),2.81(t,2H),2.50(t,2H),2.43(t,2H),2.38(t,2H),1.85-1.20(m,18H expected,22H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.81, 136.15, 135.71, 134.29, 129.62, 128.66, 128.35, 128.31, 127.70, 98.95, 67.55, 66.32, 64.0 8,62.44,53.99,53.80,49.50,32.74,32.54,30.89,27.75,27.41,27.23,27.01,25.91,25.63,24.01,19.79,19.35.HR-MS(ESI):[M+H] + calculated m / z=674.4236,found:674.4236.

[0111] Example 5 Preparation of OH-THP-OBn-Monomer 5

[0112] TBDPS-THP-OBn-monomer 4 (1 eq.) was dissolved in dry tetrahydrofuran (THF) in a dry round-bottom flask and cooled to 0°C in an ice bath. Tetrabutylammonium fluoride (TBAF, 1 M in THF, 1.2 eq.) was slowly added. The reaction mixture was stirred at 0°C for 4 hours and then concentrated in vacuo. The concentrated material was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), and washed with NaCl (30 mL×3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (developing solvent from hexane:EtOAc=4:1 to pure EtOAc) to obtain a brown oil, OH-THP-OBn-monomer 5 (70% yield). 1H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.38-7.28 (m, 5H), 5.11 (s, 2H), 4.57 (m, 1H), 3.86 (m, 1H), 3.72 (m, 1H),3.62(t,2H),3.49(m,1H),3.37(m,1H),2.79(m,2H),2.47(m,2H),2.40(m,4H),1.88-1.20(m,18H expected,22H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.86, 136.15, 128.67, 128.36, 128.32, 98.98, 67.59, 66.33, 63.02, 62.48,53.81,49.52,32.85,32.58,30.89,27.75,27.23,25.72,25.63,24.00,19.80.HR-MS(ESI):[M+H] + calculated m / z=584.3766,found:584.3752.

[0113] Example 6 Preparation of TBDPS-THP-COOH-Monomer 6

[0114] TBDPS-THP-OBn-monomer 4 is dissolved in the ethyl acetate of drying, and uses N 2 bubbling 20 minutes.Pd / C (10wt%) is joined in the round-bottom flask of oven drying.Then the degassed solution of compound 4 is transferred in the flask that contains Pd / C by syringe.Gained mixture is at room temperature inserted into H 2 balloon, stirred and spent the night, filtered through diatomite, and concentrated in vacuum.Crude product is dried under high vacuum, obtains transparent oily compound 6 (quantitative yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.70-7.31 (m, 10H), 4.54 (d, 1H), 3.84 (t, 1H), 3.77 (dt, 1H), 3.65 (s, 2H), 3.53-3.47 (m,1H),3.41(dt,1H),2.87(t,2H),2.77-2.70(m,2H),2.69-2.63(m,2H),2.49(t,2H),1.86-1.20(m,18H),1.04(s,9H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 174.33, 135.65, 134.10, 129.66, 127.71, 99.31, 66.85, 63.76, 62.84, 60.49, 52. 24,52.04,50.06,32.42,30.86,29.51,27.30,26.98,25.67,25.49,24.92,22.03,19.95,19.32.HR-MS(ESI):[M+H] + calculated m / z=436.3058,found:436.3042.

[0115] Example 7 Preparation of TBDPS-THP-OBn-Dimer 7

[0116] Compound 5 (1 eq.) and compound 6 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. 4-(Dimethylamino)pyridine p-toluenesulfonate (DPTS, 0.3 eq.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2 eq.) were added to the mixed solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=50:3) to obtain a brownish-yellow oil, TBDPS-THP-OBn-dimer 7 (96% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.73-7.29 (m, 14H), 5.11 (s, 2H), 4.57 (td, 2H), 4.03 (t ,2H),3.89-3.82(m,2H),3.76-3.68(m,2H),3.61(t,2H),3.52-3.46(m,2H),3.41-3.34(m 2H),2.84-2.30(m,16H),1.88-1.19(m,36H expected,41H integrated),1.04(s,9H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.11, 172.82, 136.14, 135.70, 134.28, 129. 61,128.65,128.34,128.30,127.69,98.94,67.57,66.30,64.58,64.07,62.43,54 .02,53.96,53.82,49.50,32.75,32.60,32.47,30.89,28.78,27.78,27.75,27.43 ,27.27,27.01,26.03,25.92,25.63,24.06,19.78,19.35.MS(MALDI-TOF):[M+Na] + calculated m / z=1024.5,found:1024.8.

[0117] Example 8 Preparation of OH-THP-OBn-Dimer 8

[0118] TBDPS-THP-OBn-dimer 7 (1 eq.) dissolved in dry THF was added to a dry round-bottom flask and the solution was cooled to 0°C in an ice bath. TBAF (1 M, THF solution, 1.2 eq.) was slowly added. The reaction mixture was stirred at 0°C for 5 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=100:6) to obtain a brown oil, i.e., OH-THP-OBn-dimer 8 (80% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.42-7.29 (m, 5H), 5.10 (s, 2H), 4.56 (td, 2H), 4.03 (t ,2H),3.88-3.81(m,2H),3.76-3.68(m,2H),3.61(t,2H),3.55-3.44(m,2H),3.41-3.33(m 2H),2.85-2.68(m,4H),2.55-2.30(m,12H),1.93-1.17(m,36H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.09, 172.81, 136.12, 128.66, 128.35, 128.32, 98.96, 67.58, 67.55, 66.33, 64.60, 62.92, 62. 46,53.96,53.81,49.52,49.46,32.87,32.52,30.89,28.78,27.74,27.26,26.06,25.76,25.62,23.98,19.79.HR-MS(ESI):[M+2H] 2+ calculated m / z=382.2770,found:382.2765.

[0119] Example 9 Preparation of TBDPS-THP-COOH-Dimer 9

[0120] TBDPS-THP-OBn-dimer 7 (1 eq.) was dissolved in dry ethyl acetate and bubbling with N2 for 20 minutes. Pd / C (10 wt%) was added to an oven-dried round-bottomed flask. The degassed solution of dimer 7 was then transferred to the Pd / C-containing flask via a syringe. The resulting mixture was stirred overnight at room temperature under H2 (1 atm, balloon), filtered through diatomaceous earth, and concentrated in a vacuum. The crude product was dried under high vacuum to obtain transparent, oily TBDPS-THP-COOH-dimer 9 in a quantitative yield. 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.73-7.32 (m, 10H), 4.55 (dt, 2H), 4.05 (t, 2H), 3 .89-3.81(m,,2H),3.80-3.70(m,2H),3.64(t,2H),3.53-3.46(m,2H),3.44-3.34(m 2H),2.90-2.30(m,16H),1.87-1.17(m,36H),1.04(s,9H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 174.08, 173.07, 135.70, 134.28, 129.63, 127.71, 99.34 ,98.99,77.41,77.16,76.91,67.58,66.97,64.26,64.08,62.86,62.49,53.97,53.77,52. 43,52.25,49.93,49.47,32.76,32.45,30.91,29.53,28.62,27.78,27.45,27.22,27.02,2 5.93,25.88,25.63,25.54,25.46,24.00,22.44,19.99,19.81,19.36.HR-MS(ESI):[M+2H] 2+ calculated m / z=456.3124,found:456.3112.

[0121] Example 10 Preparation of TBDPS-THP-OBn-tetramer 10

[0122] OH-THP-OBn dimer 8 (1 eq.) and TBDPS-THP-COOH dimer 9 were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.3 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with brine (30 mL x 3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=50:3) to obtain a dark yellow oil, TBDPS-THP-OBn-tetramer 10 (94% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.70-7.63 (m, 5H), 7.43-7.34 (m, 10H), 5.11 (s, 2H), 4.59-4.55 (m, 4H), 4.09-4.00 (m, 6H), 3.90-3.83 (m, 4H) ,3.77-3.69(m,4H),3.64(t,2H),3.53-3.46(m,4H),3.42-3.34(m,4H), 2.83-2.72(m,8H),2.50-2.35(m,24H),1.87-1.20(m,72H),1.04(s,9H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.06, 172.81, 136.14, 135.69, 134.27, 129.60, 12 8.65,128.34,128.30,127.69,98.94,77.41,77.16,76.91,67.56,66.30,64.58,64.07 ,62.43,53.96,53.81,53.78,49.48,32.74,32.59,32.45,30.89,28.79,27.76,27.42, 27.30,27.26,27.01,26.04,25.91,25.63,24.06,19.78,19.34.MS(MALDI-TOF):[M+Na] + calculated m / z=1679.1,found:1679.1.

[0123] Example 11 Preparation of OH-THP-OBn-tetramer 11

[0124] TBDPS-THP-OBn tetramer 10 (1 eq.) was dissolved in dry THF in an oven-dried round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M in THF, 1.2 eq.) was slowly added. The reaction mixture was stirred at 0°C for 5 hours and then concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), and then washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=50:3) to obtain OH-THP-OBn tetramer 11 as a dark yellow oil (70% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.38-7.30 (m, 5H), 5.11 (s, 2H), 4.60-4.51 (m, 4H), 4.10-3.97 (m, 6H), 3.90-3.81 (m, 4H) ,3.77-3.68(m,4H),3.63(t,2H),3.54-3.44(m,4H),3.44-3.34(m,4H),2.78(t,8H),2.59-2.25(m,24H),1.93-1.17(m,72H expected,88H integrated). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.98, 172.74, 136.07, 128.62, 128.49, 128.31, 1 28.28,126.15,98.93,77.42,77.16,76.91,67.50,66.29,64.58,62.77,62.43,59.02, 53.89,53.76,53.73,53.69,49.44,32.82,32.47,32.39,32.28,30.85,28.74,27.69,2 7.24,27.11,26.00,25.74,25.57,24.20,23.88,19.84,19.76.MS(MALDI-TOF):[M+Na] + calculated m / z=1440.01found:1440.17.

[0125] Example 12 Preparation of TBTPS-THP-COOH-tetramer 12

[0126] TBDPS-THP-OBn tetramer 10 (1 eq.) was dissolved in dry ethyl acetate and bubbling with N for 20 minutes. Pd / C (10 wt%) was added to a dry round-bottomed flask. The degassed solution of 10 was then transferred to the Pd / C flask via a syringe. The resulting mixture was stirred overnight at room temperature under H (1 atm, balloon), filtered through diatomaceous earth, and concentrated in a vacuum. The crude product was dried under high vacuum to obtain a pale yellow oily compound, i.e., TBTPS-THP-COOH-tetramer 12, in a quantitative yield. 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.43-7.28 (m, 5H), 5.10 (s, 2H), 4.63-4.46 (m, 4H), 4.03 (tt, 6H), 3.90-3.78 (m, 4H), 3.77 -3.67(m,4H),3.61(t,2H),3.54-3.43(m,4H),3.44-3.30(m,4H),2.95-2.68(m,8H),2.57-2.27(m,24H),1.93-1.19(m,74H). 13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 174.20, 173.04, 135.69, 134.27, 129.61, 127.69, 99.32, 98.96 ,98.93,77.42,77.16,76.91,67.56,66.97,64.58,64.27,64.07,62.84,62.45,53.92,53.75,52.4 8,52.30,49.95,49.44,32.74,32.43,30.89,29.64,28.79,28.62,27.76,27.44,27.30,27.22,27. 16,27.01,26.04,25.91,25.62,25.53,24.00,22.46,19.97,19.80,19.34.MS(MALDI-TOF):[M+Na] + calculated m / z=1589.08,found:1589.17.

[0127] Example 13 Preparation of TBDPS-THP-OBn-octamer 13

[0128] OH-THP-OBn tetramer 11 (1 eq.) and TBDPS-THP-COOH tetramer 12 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.5 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then washed with brine (30 mL x 3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH = 10:1) to obtain a brownish-yellow oil, TBDPS-THP-OBn-octamer 13 (92% yield). 1H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.69-7.58 (m, 4H), 7.42-7.31 (m, 11H), 5.10 (s, 2H), 4.60-4.53 (m, 8H), 4.04 (t, 14H), 3.90-3.81 (m, 8H),3.76-3.69(m,8H),3.64(t,2H),3.54-3.44(m,8H),3.43-3.33(m,8H),2.82-2.70(m,16H),2.51-2.34(m,48H),1.92-1.17(m,144H expected,168H integrated),1.04(s,9H).13C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.02, 135.69, 134.27, 129.61, 128.65, 128. 34,128.30,127.69,98.95,77.41,77.16,76.91,67.54,66.31,64.60,64.06,62. 45,53.95,53.75,49.46,32.73,32.41,30.89,29.82,28.79,27.75,27.41,27.30 ,27.25,27.01,26.03,25.90,25.63,24.03,19.80,19.35.MS(MALDI-TOF):[M+Na] + calculated m / z=2988.10,found:2989.42.

[0129] From the SCE curves of TBDPS-THP-OBn-dimer 7, TBDPS-THP-OBn-tetramer 10 and TBDPS-THP-OBn-octamer 13 in FIG2 , it can be seen that the preparation method of the present invention can obtain poly-β-amino ester compounds with precise molecular weight.

[0130] Example 14 Preparation of OH-THP-OBn-octamer 14

[0131] TBDPS-THP-OBn octamer 13 (1 eq.) dissolved in dry THF was added to an oven-dried round-bottom flask and the solution was cooled to 0°C in an ice bath. TBAF (1 M in THF, 1.5 eq.) was slowly added. The reaction mixture was stirred at 0°C for 5 hours and then concentrated in vacuo. The residue was dissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=10:1) to give OH-THP-OBn-octamer 14 as a brownish-yellow oil (65% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.42-7.30 (m, 6H), 5.11 (s, 2H), 4.62-4.51 (m, 8H), 4.11-3.96 (m, 14H), 3.91-3.81 (m, 8H), 3 .78-3.69(m,8H),3.62(t,2H),3.54-3.45(m,8H),3.43-3.33(m,8H),2.85-2.70(m,16H),2.5-5.25(m,48H),1.91-1.19(m,144H expected,172H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.00, 136.12, 128.66, 128.35, 128.32, 98.97, 77.41, 77.16, 76.91, 67.54, 66.34, 64.62, 62.8 8,62.47,53.93,53.78,53.73,49.42,32.86,32.33,30.89,28.78,27.73,27.30,27.26,27.15,26.03,25.76,25.62,23.93,19.80.

[0132] Example 15 Preparation of TBTPS-THP-COOH-octamer 15

[0133] TBDPS-THP-OBn octamer 13 (1 eq.) was dissolved in dry ethyl acetate and bubbling with N for 20 minutes. Pd / C (10 wt %) was added to a dry round-bottomed flask. The degassed solution of octamer 13 was then transferred to the Pd / C flask via a syringe. The resulting mixture was stirred overnight under H (1 atm, balloon), filtered through diatomaceous earth, and concentrated in a vacuum. The crude product was dried under high vacuum to obtain a brownish-yellow oily compound, i.e., TBTPS-THP-COOH-octamer 15, in a quantitative yield. 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.76-7.31 (m, 10H), 4.63-4.49 (m, 8H), 4.11-3.99 (m, 14H), 3.89-3.81 (m, 8H), 3.80-3.68 (m ,9H),3.64(t,2H),3.56-3.44(m,8H),3.44-3.32(m,8H),2.90-2.62(m,20H),2.43(tq,44H),1.90-1.16(m,159H),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 174.13, 173.05, 135.70, 134.29, 129.62, 127.70, 99. 32,98.96,77.42,77.16,76.91,67.57,66.99,64.60,64.29,64.08,62.85,62.45,53.96, 53.76,52.53,52.36,49.93,49.47,32.75,32.44,30.91,29.83,29.68,28.81,28.64,27.78,27.44,27.32,27.24,27.02,26.05,25.93,25.64,24.06,22.51,19.98,19.81,19.36.

[0134] Example 16 Preparation of TBDPS-THP-OTg-Dimer 16

[0135] Triethylene glycol monomethyl ether (1.2 eq.) and TBDPS-THP-COOH dimer 9 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.3 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH = 50:3) to afford TBDPS-THP-OTg-dimer 16 as a brownish-yellow oil (92% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.74-7.31 (m, 10H), 4.57 (t, 2H), 4.22 (t, 2H), 4.04 (t, 2H), 3.90-3.82 (m, 2H), 3.79-3.58 (m, 12H expected, 15H integrated),3.57-3.52(m,2H),3.41-3.34(m,5H),2.83-2.71(t,4H),2.56-2.28(m,12H),1.97-1.18(m,36H expected,42H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.90, 135.68, 134.25, 129.60, 127.68, 98.94, 77.41 ,77.16,76.91,72.61,72.05,72.02,70.73,70.70,70.61,70.44,69.26,67.53,64.58,64. 04,63.61,62.44,61.85,59.16,53.93,53.76,49.44,49.33,32.72,32.29,30.87,28.77,2 7.74,27.40,27.27,26.99,26.02,25.89,25.61,23.99,19.78,19.33.HR-MS(ESI):[M+2H] 2+ calculated m / z=529.3596,found:529.3591.

[0136] Example 17 Preparation of TBDPS-THP-Guanidine-Boc-Dimer 17

[0137] Tert-butyloxycarbonyl (Boc)-protected N-(4-hydroxybutyl)guanidine (1 eq.) and TBDPS-THP-COOH-dimer 9 (1 eq.) were dissolved in dry CHCl and cooled to 0°C in an ice bath. DPTS (0.3 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight and washed with brine (30 mL x 3). The combined organic phases were dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl:MeOH = 10:1) to afford TBDPS-THP-Guanidine-Boc-dimer 17 as a brownish-yellow oil (90% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 11.50 (s, 1H), 8.32 (s, 1H), 7.74-7.32 (m, 10H), 4.57 (t, 2H), 4.14-3.96 (m, 4H), 3.92- 3.78(m,2H),3.77-3.68(m,2H),3.64(t,2H),3.54-3.32(m,6H),2.91-2.69(m,4H),2.64-2.20(m,12H),1.97-1.15(m,40H expected,68H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.12, 156.31, 153.48, 135.71, 134.28, 129.63, 127.72, 98.98, 83.26, 79.43, 67.57, 64.07, 62. 46,49.46,40.58,32.75,30.91,28.81,28.45,28.22,27.76,27.31,27.02,26.20,26.05,25.93,25.86,25.64,24.07,19.82,19.36.

[0138] Example 18 Preparation of OH-OH-COOH-Dimer 19

[0139] OH-THP-OBn dimer 8 was dissolved in dry isopropyl alcohol (IPA). To this solution was added p-toluenesulfonic acid (TsOH, 4 eq.). The reaction mixture was stirred at room temperature for 6 h and washed with saturated Na2CO3 solution (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 18 was obtained without further purification.

[0140] Compound 18 is dissolved in dry ethyl acetate, and uses N bubbling 20 minutes.Pd / C (10wt%) is added in dry round-bottom flask.Then with syringe the degassed solution of compound 18 is transferred in the flask containing Pd / C.With the gained mixture under H 2 (1atm, balloon) stirring at room temperature spends the night, passes through diatomite filtration, and vacuum concentration.Crude product is dried under high vacuum to obtain colorless oily product, i.e. OH-OH-COOH-dimer 19 (60% productive rate). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.13-3.99 (m, 2H), 3.72-3.58 (m, 4H), 3.56 (t,2H),2.97(t,2H),2.90-2.67(m,6H),2.57-2.35(m,8H),1.83-1.17(m,24H expected,32H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.84, 172.67, 77.42, 77.16, 76.91, 67.73, 64.52, 62.55, 62.42, 54.48, 52.69, 52.42, 48.81 ,45.11,32.68,32.09,31.49,30.26,30.02,29.83,28.46,27.07,26.72,26.25,25.80,25.50,24.46,22.03,27-22(overlapping peaks).HR-MS(ESI):[M+2H] 2+ calculated m / z=253.1960,found:253.1952.

[0141] Example 19 Preparation of OH-OH-OTg-Dimer 21

[0142] TBDPS-THP-OTg-dimer 16 (1 eq.) was dissolved in dry THF in a dry round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M, THF solution, 2 eq.) was then slowly added. The reaction mixture was stirred at 0°C for 6 hours and concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), then washed with brine (30 mL×3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 20 was obtained without further purification.

[0143] Compound 20 (1 eq.) was dissolved in dry IPA. TsOH (4 eq.) was slowly added to the solution. The reaction mixture was stirred at room temperature for 6 h and washed with saturated NaCO solution (30 mL x 3). The organic phases were combined, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl:MeOH = 25:3) to afford a brownish-yellow oil, OH-OH-COOH-dimer 21 (30% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.23 (t, 2H), 4.05 (t, 2H), 3.71-3.58 (m, 10H), 3 .57-3.51(m,6H),3.37(s,3H),2.82(t,4H),2.54-2.37(m,12H),1.69-1.18(m,24H expected,28H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.72, 172.57, 77.42, 77.16, 76.91, 72.05, 70.73, 70.68, 69.23, 64.74, 63.83, 62.72, 62.54, 59.18, 54 .53,54.46,53.86,50.94,48.65,48.60,32.73,32.35,31.30,31.21,28.69,27.31,27.04,26.39,26.02,25.85,25.51.HR-MS(ESI):[M+2H] 2+ calculated m / z=326.2432,found:326.2425.

[0144] Example 20 Preparation of OH-OH-Guanidine-Dimer 23

[0145] TBDPS-THP-Guanidine-Boc-dimer 17 (1 eq.) was dissolved in dry THF in a dry round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M in THF, 2 eq.) was then slowly added to the flask. The reaction mixture was stirred at 0°C for 6 hours and then concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), and then washed with brine (30 mL×3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give compound 22 without further purification.

[0146] Compound 22 was dissolved in dry IPA, TsOH (8 eq.) was added to the solution, and the reaction mixture was stirred at room temperature for 6 h and washed with saturated Na2CO3 solution (30 mL×3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The material was redissolved in hexafluoroisopropanol (HFIP), and TsOH (6 eq.) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 4 h and washed with saturated Na2CO3 solution (30 mL×3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo; the product was precipitated using anhydrous ether to obtain a brown oil, OH-OH-Guanidine-dimer 23 (yield 30%). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.06 (m, 4H), 3.77-3.41 (m, 6H), 3.23 (s, 2H expected, 1H integrated), 2.91-2.66 (m, 4H), 2.55-2.22 (m, 12H), 1.80-1.21 (m, 28H). 13 C NMR (125MHz, DMSO-d6, 298K) δ (ppm) = 172.23, 63.69, 60.73, 60.28, 53.15, 52.71, 49.08, 40.11, 40.02, 39.95, 39.85, 39.78, 39.69, 3 9.61,39.52,39.35,39.19,39.02,32.61,32.10,29.75,29.52,28.22,26.76,26.51,26.02,25.50,23.30,23.16.HR-MS(ESI):[M+3H] 3+ calculated m / z=206.8315,found:206.8312.

[0147] Example 21 Preparation of TBTPS-THP-OTg-tetramer 24

[0148] Triethylene glycol monomethyl ether (2 eq.) and TBDPS-THP-COOH tetramer 12 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.3 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=10:1) to give TBTPS-THP-OTg-tetramer 24 as a brown oil (83% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.71-7.32 (m, 10H), 4.56 (t, 4H), 4.22 (t, 2H), 4.04 (t, 6H), 3.89-3.82 (m, 4H), 3.77-3. 61(m,15H),3.55(dd,2H),3.52-3.44(m,4H),3.42-3.34(m,7H),2.84-2.71(m,8H),2.54-2.30(m,24H),1.90-1.14(m,72H expected,84H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.00, 135.68, 134.25, 129.60, 127.69, 98.95, 77. 42,77.16,76.91,72.65,72.04,70.69,70.59,70.41,69.26,67.52,64.60,64.04,63.62 ,62.45,61.84,59.15,53.92,53.74,49.41,32.72,32.31,30.87,28.77,27.73,27.40, 27.28,27.11,26.99,26.02,25.89,25.61,23.93,19.78,19.33.MS(MALDI-TOF):[M+Na] + calculated m / z=1713.2,found:1713.1.

[0149] Example 22 Preparation of TBTPS-THP-Guanidine-Boc-tetramer 25

[0150] Tert-Butyloxycarbonyl (Boc)-protected N-(4-hydroxybutyl)guanidine (1 eq.) and TBTPS-THP-COOH-tetramer 12 (1 eq.) were dissolved in dry CHCl and cooled to 0°C in an ice bath. DPTS (0.5 eq.) and EDCI (2 eq.) were added to the solution, and the reaction mixture was stirred at room temperature overnight. The mixture was washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl:MeOH = 25:3) to afford TBTPS-THP-OTg-tetramer 25 as a brownish-yellow oil (80% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 11.50 (s, 1H), 8.33 (s, 1H), 7.75-7.34 (m, 10H), 4.57 (t, 4H), 4.16-3.97 (m, 8H), 3.90-3 .80(m,4H),3.78-3.70(m,4H),3.64(t,3H),3.54-3.34(m,10H),2.90-2.69(m,8H),2.67-2.20(m,24H),1.88-1.20(m,76H expected,98H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.01, 135.70, 134.27, 129.62, 127.70, 98.97,79.42,77.41,77.16,76.91,67.54,64.62,64.06,62.46,53.95,53.7 5,49.44,40.57,32.74,32.36,30.90,28.79,28.44,28.21,27.75,27.42,27.30,27.15,27.01,26.19,26.04,25.91,25.84,25.63,23.97,19.80,19.35.

[0151] Example 23 Preparation of OH-OH-COOH-tetramer 27

[0152] OH-THP-OBn tetramer 11 was dissolved in a dry mixed solvent (IPA:HFIP = 1:1). To this solution was added p-toluenesulfonic acid (TsOH, 12 eq.). The reaction mixture was stirred at room temperature for 6 h and washed with saturated Na2CO3 solution (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 26 was obtained without further purification.

[0153] Compound 26 is dissolved in the ethyl acetate of drying, and uses N bubbling 20 minutes.Pd / C (10wt%) is added in the round-bottom flask of drying, then with syringe the degassed solution of compound 26 is transferred in the flask that contains Pd / C.With the gained mixture at H 2 (1atm, balloon) stirring at room temperature is spent the night, by diatomite filtration, and vacuum concentration.Crude product is dried under high vacuum, obtains brown oily product, i.e. OH-OHCOOH-tetramer 27 (50% productive rate). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.08 (m, 6H), 3.69-3.60 (m, 6H), 3.53-3.53 (m, 4H), 3.00-2.69 (m, 8H expected, 14H integrated), 2.60-2.37 (m, 8H expected, 18H integrated),1.68-1.17(m,48H expected,103H integrated). 13 C NMR has extremely poor signal to noise ratio.HR-MS(ESI):[M+4H] 4+ m / z=248.6934,found:248.6934.

[0154] Example 24 Preparation of OH-OH-OTg-tetramer 29

[0155] TBDPS-THP-OTg-tetramer 24 (1 eq.) was dissolved in dry THF in a dry round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M, THF solution, 2 eq.) was then slowly added. The reaction mixture was stirred at 0°C for 6 hours and concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), then washed with brine (30 mL×3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 28 was obtained without further purification.

[0156] Compound 28 (1 eq.) was dissolved in a dry mixed solvent (IPA: HFIP = 1: 1). TsOH (12 eq.) was slowly added to the solution. The reaction mixture was stirred at room temperature for 6 h and washed with a saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The material was redissolved in a mixed solvent IPA: HFIP = 1: 1, and TsOH (8 eq.) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 5 h and washed with a saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product was precipitated using anhydrous ether to obtain a brown oil, i.e., OH-OH-OTg-tetramer 29 (40% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.23 (t, 2H), 4.11-4.01 (m, 6H), 3.71-3.51 ( m,20H),3.38(s,3H),2.85-2.77(m,8H),2.51-2.38(m,24H),1.75-1.18(m,32H expected,68H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.71,77.41,77.16,76.91,72.06,70.70,64.75,63.84,62.73,62.52,59 .19,54.54,54.45,53.85,52.12,48.69,48.62,32.73,32.36,31.28,28.69,27.30,26.38,25.98,25.85.(with 4 peaks missing in the range between 33 to 24 ppm due to poor signal to noise ratio).HR-MS(ESI):[M+4H] 4+ m / z=285.2169,found:285.2159.

[0157] Example 25 Preparation of OH-OH--Guanidine-tetramer 31

[0158] TBTPS-THP-Guanidine-Boc-tetramer 25 (1 eq.) was dissolved in dry THF in a dry round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M, THF solution, 2 eq.) was then slowly added to the flask. The reaction mixture was stirred at 0°C for 6 hours and then concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), then washed with brine (30 mL×3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 30 was obtained without further purification.

[0159] Compound 30 was dissolved in a dry mixed solvent (IPA:HFIP=3:4). TsOH (12 eq.) was added to the solution. The reaction mixture was stirred at room temperature for 7 hours and washed with saturated Na2CO3 solution (30 mL×3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting material was redissolved in HFIP, and TsOH (10 eq.) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 4 hours and washed with saturated Na2CO3 solution (30 mL×3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product was precipitated using anhydrous ether to obtain a brown oil, i.e., OH-OH-Guanidine-tetramer 31 (yield 30%). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.19-4.00 (m, 8H), 3.69-3.47 (m, 10H), 3.3 3-3.22(m,2H),2.93-2.66(m,8H),2.61-2.61-2.19(m,24H),1.77-1.26(m,36H expected,56H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.77,77.42,77.36,77.16,76.91,64.73,62.56,54.43 ,53.84,48.83,32.71,32.22,31.43,28.68,27.29,26.44,25.98,25.60.HR-MS(ESI):[M+5H] 5+ m / z=221.7752,found:221.7747.

[0160] Example 26 Preparation of TBDPS-THP-OTg-octamer 32

[0161] Triethylene glycol monomethyl ether (3 eq.) and TBDPS-THP-COOH octamer 15 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.6 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2: MeOH = 10: 1) to give a brownish-yellow oil, TBDPS-THP-OTg-octamer 32 (73% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 7.70-7.29 (m, 10H), 4.56 (t, 8H), 4.22 (t, 2H), 4.04 (t, 14H), 3.90-3.81 (m, 8H), 3.76-3.59 (m, 18H expected, 28H integrated),3.56-3.53(m,2H),3.50-3.45(m,8H),3.42-3.34(m,11H),2.93-2.71(m,16H),2.60-2.22(m,48H),1.89-1.14(m,144H),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 173.00, 135.69, 134.26, 129.61, 127.7 0,98.97,77.41,77.16,76.90,72.64,72.06,70.71,70.44,67.53,64.62,6 4.05,62.47,61.87,59.17,53.94,53.72,49.42,32.73,32.33,30.89,28. 79,27.74,27.29,27.13,27.01,26.03,25.90,25.62,23.95,19.80,19.35.

[0162] Example 27 Preparation of TBDPS-THP-Guanidine-Boc-Octamer 33

[0163] Boc-protected N-(4-hydroxybutyl)guanidine and TBDPS-THP-COOH-octamer 15 (1 eq.) were dissolved in dry CH2Cl2 and cooled to 0°C in an ice bath. DPTS (0.5 eq.) and EDCI (2 eq.) were added to the solution. The reaction mixture was stirred at room temperature overnight and washed with brine (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CH2Cl2:MeOH=25:3) to give a brownish-yellow oil, TBDPS-THP-Guanidine-Boc-octamer 33 (88% yield). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 11.50 (s, 1H), 8.39-8.20 (m, 1H), 7.73-7.31 (m, 10H), 4.57 (t, 8H), 4.17-3.94 (m, 16H), 3.90-3.82 (m, 8H), 3.7 7-3.70(m,8H),3.64(t,2H),3.54-3.46(m,8H),3.46-3.43(m,2H),3.42- 3.33(m,8H),2.94-2.66(m,16H),2.63-2.25(s,48H),1.91-1.17(m,148H expected,180H integrated),1.04(s,9H). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.97, 135.71, 129.63, 127.71, 99.00, 77.41, 77.16, 76.91, 67.53, 64.64, 64.06, 62.4 9,53.95,53.73,49.44,32.37,30.91,28.80,28.45,28.23,27.74,27.29,27.03,26.20,26.04,25.91,25.64,23.99,19.83.

[0164] Example 28 Preparation of OH-OH-COOH-octamer 35

[0165] OH-THP-OBn-octamer 14 was dissolved in a dry mixed solvent (IPA:HFIP = 2:3). To this solution was added p-toluenesulfonic acid (TsOH, 16 eq.). The reaction mixture was stirred at room temperature for 7 hours and washed with saturated Na2CO3 solution (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 34 was obtained without further purification.

[0166] The compound 34 that obtains is dissolved in the ethyl acetate of drying, and adds the ethanol of 10v / v%, and uses N Bubble 20 minutes.Pd / C (10wt%) is joined in the round-bottom flask of oven drying.Then with syringe the degassed solution of compound 34 is transferred in the flask that contains Pd / C.With the gained mixture at H (1atm, balloon) stirring at room temperature is spent the night, by diatomite filtration, and vacuum concentration.Crude product is dried under high vacuum, obtains brown oily product, i.e. OH-OH-COOH-octamer 35 (50% productive rate). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.06 (q, 14H), 3.64-3.52 (m, 18H), 2.85-2.76 (m, 16H), 2.62-2.34 (m, 48H), 1.80-1.16 (m, 96H expected, 137H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.71,77.41,77.16,76.91,64.75,62.68,54.42,53 .83,48.77,32.70,32.25,31.41,31.40,29.83,28.68,27.29,27.06,25.98,25.50.(with 4peaks missing due to poor signal to noise ratio).HR-MS(ESI):[M+7H] 7+ m / z=281.6475,found:281.6477.

[0167] Example 29 Preparation of OH-OH-OTg-octamer 37

[0168] TBDPS-THP-OTg-octamer 32 (1 eq.) was dissolved in dry THF in a dry round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M in THF, 2 eq.) was then slowly added. The reaction mixture was stirred at 0°C for 6 hours and concentrated in vacuo. The residue was redissolved in EtOAc, quenched with saturated CaCl2 solution (30 mL), then washed with brine (30 mL×3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to obtain compound 36 without further purification.

[0169] The obtained compound 36 was dissolved in a dry mixed solvent IPA: HFIP = 2: 3. TsOH (16 eq.) was slowly added to the solution. The reaction mixture was stirred at room temperature for 6 h and washed with saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The obtained material was redissolved in a mixed solvent IPA: HFIP = 2: 3, and TsOH (10 eq.) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 7 hours and washed with saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product was precipitated using anhydrous ether to obtain a brown oil, i.e., OH-OH-OTg-octamer 37 (yield 41%). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.26-4.20 (m, 2H), 4.11-4.02 (m, 14H), 3.72-3.59 (m, 12H) ,3.57-3.53(m,16H),3.38(s,3H),2.85-2.77(m,16H),2.51-2.38(m,48H),1.75-1.18(m,96H expected,146H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.60, 77.30, 77.05, 76.80, 64.63, 62.62, 54.35 ,53.73,48.68,43.70,32.63,32.21,31.33,29.73,28.59,27.20,26.28,25.88.(with 9 peaks missing due to poor signal to noise ratio).HR-MS(ESI):[M+6H] 6+ m / z=352.7699,found:352.7698.

[0170] Example 30 Preparation of OH-OH-Guanidine-octamer 39

[0171] TBDPS-THP-Guanidine-Boc-octamer 33 (1 eq.) was dissolved in dry THF in an oven-dried round-bottom flask and cooled to 0°C in an ice bath. TBAF (1 M in THF, 2 eq.) was then slowly added to the flask. The reaction mixture was stirred at 0°C for 6 hours and then concentrated in vacuo. The residue was redissolved in ethyl acetate, quenched with saturated CaCl2 solution (30 mL), then washed with brine (30 mL x 3), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Compound 38 was obtained without further purification.

[0172] The obtained compound 38 was dissolved in a dry mixed solvent IPA: HFIP = 2: 3. TsOH (24 eq.) was added to the solution. The reaction mixture was stirred at room temperature for 7 hours and washed with saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The resulting material was redissolved in HFIP, and TsOH (15 eq.) was subsequently added to the solution. The reaction mixture was stirred at room temperature for 4 hours and washed with saturated Na2CO3 solution (30 mL × 3), the organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product was precipitated using anhydrous ether to obtain a brown oil, i.e., OH-OH-Guanidine-octamer 39 (yield 30%). 1 H NMR (500MHz, CDCl3, 298K) δ (ppm) = 4.05 (t, 16H), 3.63-3.42 (m, 18H), 3.29-3.13 (m, 2H expected, 3H integrated),2.93-2.63(m,16H),2.52-2.31(m,48H),1.75-1.19(m,100H expected,112H integrated). 13 C NMR (125MHz, CDCl3, 298K) δ (ppm) = 172.73, 77.41, 77.16, 76.91, 64.72, 62.68, 54.42, 53.82, 53.66, 51.88 ,48.81,48.65,32.75,32.28,31.47,28.68,27.29,27.10,26.42,25.98,25.79,25.58.HR-MS(ESI):[M+8H] 8+ m / z=260.7044,found:260.7043.

[0173] Performance study: cell transfection experiment

[0174] This experiment demonstrates the transfection of adherent HEK-293T cells with a deprotected poly(β-amino ester) compound carrying functional groups into a plasmid DNA (pDNA) encoding EGFP. Polymer-pDNA nanoparticles were prepared by dissolving the deprotected poly(β-amino ester) compound and pDNA in tetrahydrofuran and 25 mM sodium acetate (NaOAc, pH 5), respectively. The deprotected poly(β-amino ester) compound and pDNA were mixed at the desired weight ratio (20:1 to 180:1, in the order of 20:1, 40:1, 80:1, 100:1, 120:1, 140:1, 160:1, and 180:1) and incubated at 20-25°C for 10 minutes (final pDNA concentration of 20 μg / mL). HEK-293T cells were then plated in 96-well cell culture plates and allowed to adhere overnight. When the cell confluence reaches 70-90%, the prepared polymer-pDNA nanoparticles are used for transfection. Polymer-pDNA nanoparticles containing 200 or 100 ng pDNA are added to 100 μL of serum-free DMEM (dulbecco's modified Eagle medium) or Hanks medium, respectively. After incubation of the nanoparticles for 3-4 hours, the transfection solution is removed from the well plate and replaced with fresh DMEM or Hanks cell culture medium. The expression of enhanced green fluorescent protein (EGFP) is observed 24h and 48h after transfection of HEK-293T cells, and the gene transfection effect is observed by fluorescence microscopy.

[0175] The gene transfection results are as follows:

[0176] Experimental results demonstrated that poly(β-amino ester) compounds with guanidine functional groups exhibited significant transfection efficacy (Figure 3). A tetramerized β-amino ester compound (Compound 31) and an octamerized β-amino ester compound (Compound 39), both of which had been deprotected and now carried guanidine functional groups, demonstrated transfection efficacy in different culture media and at varying polymer-pDNA ratios, demonstrating the compound's gene delivery capabilities.

[0177] In summary, the present invention designs a system for synthesizing poly(β-amino esters) using an exponential iterative growth polymerization method with three orthogonal protecting groups. This system enables selective deprotection and coupling of monomers, dimers, tetramers, octamers, and so on, successfully obtaining precise poly(β-amino ester) molecules of dimers, tetramers, octamers, and so on. Furthermore, a modular end-group modification strategy for poly(β-amino esters) is designed, whereby the carboxylic acid termini are directly used as terminal modification groups by selectively removing benzyl groups. Subsequently, various hydroxyl-bearing terminal chains are modified into the poly(β-amino ester) molecules through esterification and coupling, achieving modular modification. This invention effectively overcomes the shortcomings of the prior art and has high industrial value.

[0178] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A β-amino ester monomer compound, characterized in that The chemical structural formula of the β-amino ester monomer compound is shown in Formula I: Wherein, x=0~16, y=0~10; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

2. The method for preparing a β-amino ester monomer compound according to claim 1, wherein The following steps are involved: The compound of formula I is obtained by Michael addition reaction of the compound of formula II with benzyl acrylate; Wherein, x=0~16; y=0~10; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

3. Use of the β-amino ester monomer compound according to claim 1 or the β-amino ester monomer compound prepared by the preparation method according to claim 2 in the preparation of a poly-β-amino ester compound.

4. A poly (β-amino ester) compound, characterized in that Obtained by polymerization reaction of the β-amino ester monomer compound according to claim 1 or the β-amino ester monomer compound prepared by the preparation method according to claim 2, the chemical structure of the poly-β-amino ester compound is as shown in Formula A: Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1; R1 is selected from one of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG groups; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

5. A method for preparing the poly (beta) amino ester compound according to claim 4, characterized in that: The following steps are involved: 1) selectively removing the TBDPS protecting group from the β-amino ester monomer compound according to claim 1 or the β-amino ester monomer compound prepared by the preparation method according to claim 2 to obtain a compound of formula V; 2) selectively removing the benzyl protecting group from the β-amino ester monomer compound according to claim 1 or the β-amino ester monomer compound prepared by the preparation method according to claim 2 to obtain a compound of formula VI; 3) esterifying and coupling the compound of formula V and the compound of formula VI in the presence of a coupling reagent to obtain a dimerized poly (β-amino ester); 4) Repeating the TBDPS protecting group removal process in step 1) with the dimerized poly-β-amino ester, repeating the benzyl protecting group removal process in step 2) with the dimerized poly-β-amino ester, and then repeating the esterification coupling process in step 3) to obtain a tetramerized poly-β-amino ester; 5) Repeat the above steps 1) to 3) of removing the TBDPS protecting group, removing the benzyl protecting group and esterification coupling process on the tetramer poly β-amino ester to obtain a series of polymerization degrees of 2 m Poly-β-amino ester compounds; Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1; R 1 One selected from imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, di-tert-butyloxycarbonylguanidine or -O-PG group; the -O-PG group is a hydroxyl group with a PG protecting group, and the PG protecting group is selected from one of 2-tetrahydropyranyl, 2-methoxyethoxymethyl or methoxymethyl.

6. The method for preparing the poly (beta) amino ester compound according to claim 5, wherein: Also includes one or more of the following characteristics: a) removing the TBDPS protecting group in step 1) in the presence of tetra-n-butylammonium fluoride; b) the removal of the benzyl protecting group in step 2) is carried out in the presence of Pd / C and H2; c) The coupling reagents in step 3) are 4-(dimethylamino)pyridine p-toluenesulfonate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

7. A poly (β-amino ester) compound with a functional group after removing the protecting group, characterized in that: The poly (beta) amino ester compound according to claim 4 is obtained by one or more reactions selected from the group consisting of TBDPS protective group removal reaction, benzyl protective group removal reaction, PG group removal reaction, and carboxyl group modification reaction; The chemical structural formula of the poly (beta) amino ester compound with the functional group removed is as shown in Formula B1 or B2: Where x = 0 to 16; y = 0 to 10; n = 2 m , m is a positive integer greater than or equal to 1; R 1’ One selected from the group consisting of imidazolyl, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and guanidinyl; R 2 is selected from H or FG; FG is a functional group, and FG is selected from one of polyethylene glycol monomethyl ether, C3-C11 alcohol, C3-C11 guanidine group, C3-C11 dimethylamino group, C3-C11 amino group, C3-C11 4-methylpiperazine or C3-C11 biguanidine group, and the corresponding structural formula is as follows: f = 1 to 8; g = 0 to 8, * is the connection site.

8. A method for preparing a poly (β-amino ester) compound with functional groups after removing the protecting groups as claimed in claim 7, characterized in that: The following steps are involved: If the poly (beta) amino ester compound in claim 4 has R 1 The poly (beta) amino ester compound according to claim 4 is obtained by removing the TBDPS protecting group, removing the PG group, and removing the benzyl protecting group to obtain the poly (beta) amino ester compound according to claim 7 having the functional group and the protective group removed, which is a formula B1; and / or If the poly (beta) amino ester compound in claim 4 has R 1 The poly-β-amino ester compound according to claim 4 is subjected to the steps of removing the benzyl protecting group, modifying the carboxyl group, removing the TBDPS protecting group, and removing the PG group to obtain the poly-β-amino ester compound according to claim 7 having the functional group removed and the functional group Formula B1; and / or, if R1 of the poly-β-amino ester compound according to claim 4 is selected from one of imidazole, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, the poly-β-amino ester compound according to claim 4 is subjected to the steps of removing the TBDPS protecting group and removing the benzyl protecting group to obtain the poly-β-amino ester compound according to claim 7 having the functional group removed and the functional group Formula B2; and / or, If R1 of the poly (beta) amino ester compound in claim 4 is selected from one of imidazole, morpholinyl, 2-tetrahydrofuranyl, 4-methylpiperazinyl, methyl, and di-tert-butyloxycarbonylguanidine, the poly (beta) amino ester compound as claimed in claim 4 is obtained by removing the benzyl protecting group, modifying the carboxyl group, and removing the TBDPS protecting group to obtain the poly (beta) amino ester compound formula B2 with functional groups removed as claimed in claim 7.

9. The method for preparing a poly (beta) amino ester compound having functional groups and removed protecting groups according to claim 8, wherein: The removal of the TBDPS protecting group is carried out in the presence of tetra-n-butylammonium fluoride; And / or, the removal of the PG group is carried out in the presence of p-toluenesulfonic acid; And / or, the debenzylation of the protecting group is carried out in the presence of Pd / C and H2; And / or, the modification of the carboxyl group is carried out in the presence of DPTS and EDCI.

10. Use of the poly (beta) amino ester compound according to claim 4, the poly (beta) amino ester compound having functional groups removed from the protecting groups according to claim 7, or the poly (beta) amino ester compound having functional groups removed from the protecting groups prepared by the method according to any one of claims 8 to 9 in the preparation of a gene delivery drug.

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