KR20140011098A

KR20140011098A - Core-shell aminopolystyrene resin and process for preparing the same

Info

Publication number: KR20140011098A
Application number: KR1020120077778A
Authority: KR
Inventors: 이윤식; 조홍준; 이상명
Original assignee: 서울대학교산학협력단
Priority date: 2012-07-17
Filing date: 2012-07-17
Publication date: 2014-01-28
Also published as: KR101855531B1

Abstract

The present invention relates to an aminopolystyrene resin having a core-shell structure and a method for producing the same. More specifically, the present invention provides a core-shell structured aminopolystyrene resin; And (i) swelling the aminomethylpolystyrene resin; (ii) using a protecting group to protect the amine groups in the outer portion of the swollen aminomethylpolystyrene resin to form a shell; (iii) acetylating the amine groups of the resin except the shell to form a core; And (iv) removing the protecting groups present in the shell layer of the resin of step (iii), wherein the aminomethylpolystyrene resin has a core-shell structure.

Description

Core-Shell Aminopolystyrene Resin and Process for Preparing the Same}

The present invention relates to an aminopolystyrene (AMPS) resin having a core-shell structure and a method for preparing the same. More specifically, the present invention provides a core-shell structured aminopolystyrene resin; And (i) swelling the aminomethylpolystyrene resin; (ii) using a protecting group to protect the amine groups in the outer portion of the swollen aminomethylpolystyrene resin to form a shell; (iii) acetylating the amine groups of the resin except the shell to form a core; And (iv) removing the protecting groups present in the shell layer of the resin of step (iii), wherein the aminomethylpolystyrene resin has a core-shell structure.

Recently, the importance of hydrophobic peptide synthesis technology has been further emphasized in the identification of the principle of action of neurodegenerative diseases such as Alzheimer's disease, mad cow disease, Parkinson's disease and Huntington's disease and the development of therapeutic agents.

In order to synthesize such hydrophobic peptides, biological methods such as genetic recombination methods and chemical synthesis methods combining amino acids one by one have been widely used. Despite the advantages of genetic recombination, genetic recombination has the disadvantage of requiring complex processes and high production costs.

Chemical synthesis, in particular solid phase synthesis, is simpler and easier to scale up than genetic recombination. However, chemical synthesis is limited in the synthesis of long hydrophobic peptides.

Since the introduction of solid phase peptide synthesis by Bruce Merrifield, various studies have been actively conducted for effective peptide synthesis. There is still much room for improvement in the development of polymer support, which is the core of solid phase synthesis.

In order to increase the compatibility between the peptide and the polymer support in the synthesis of solid phase peptides, various polymer supports modified with a hydrophilic polymer called polyethylene glycol have been developed (TentaGel, CLEAR resin, ChemMatrix resin, etc.). ). The polymer supports thus produced showed good results for peptide synthesis with hydrophobic amino acid sequences and various enzymatic reactions. However, polymer supports using polyethylene glycol are expensive and have complicated manufacturing processes.

As another approach for effective solid phase peptide synthesis, the inventors have developed core-shell resins by the present inventors to overcome the disadvantages of steric hindrances possessed by polymeric supports. In the core-shell resin, the active functional group present in the core portion of the polymer support is inactivated, and the active functional group is used for the shell layer that is the outer portion of the support. The contact between the functional groups of the shell layer and the reactants can be maximized to increase the yield and purity of the peptide synthesis.

As a conventional core-shell resin synthesis method, two-step polymerization method (Tetrahedron Lett. Vol. 41, pp7481-7485, 2000), central crosslinking method (Org. Lett. Vol. 6, pp3273-3276, 2004), partial hydrolysis method (J. Comb. Chem. Vol. 7, pp170-173, 2005) and the like.

However, since the conventional core-shell resin synthesis methods require harsh conditions or undergo complicated manufacturing processes, the core-shell structure is not uniformly produced. Using such heterogeneous core-shell resins, there is a disadvantage in that the yield of peptide products is lowered in the synthesis of solid phase peptides. Accordingly, there is a need for a method for efficiently controlling solid phase peptide synthesis by efficiently controlling a uniform core-shell structure.

The present inventors have completed the present invention in view of the fact that the amine group in the central portion of the aminopolystyrene resin can be inactivated by acetylation and only the amine group in the shell portion can be used for peptide synthesis.

A basic object of the present invention is to provide a core-shell aminomethylpolystyrene resin in which an amine group in the central portion of the aminopolystyrene resin is acetylated in an aminopolystyrene resin.

Still another object of the present invention is to provide a method for preparing a polymer comprising: (i) swelling an aminomethylpolystyrene resin; (ii) using a protecting group to protect the amine groups in the outer portion of the swollen aminomethylpolystyrene resin to form a shell; (iii) acetylating the amine groups of the resin except the shell to form a core; And (iv) removing the protecting group present in the shell layer of the resin of step (iii), to provide a method for preparing an aminomethylpolystyrene resin having a core-shell structure.

The basic object of the present invention can be achieved by providing an aminomethylpolystyrene resin having a core-shell structure in which an amine group in the central portion of the aminopolystyrene resin is acetylated in an aminopolystyrene resin.

In the core-shell structured aminomethylpolystyrene resin of the present invention, only the shell portion is activated. Therefore, when the peptide is synthesized using the core-shell aminomethylpolystyrene resin, since the peptide is generated only in the shell layer of the core-shell aminomethylpolystyrene resin, the synthesis efficiency and yield of the peptide are increased. .

That is, when the amine group is activated in the center of the resin during peptide synthesis, the peptide synthesis proceeds partially even in the center of the resin, and in this case, it is difficult to synthesize the desired peptide with high purity due to the steric hindrance of the center. This not only causes unwanted peptides to be synthesized, but also results in waste of reactants.

In particular, the core-shell aminomethylpolystyrene resin of the present invention is very useful for hydrophobic peptide synthesis. In addition, by using the amino-methyl polystyrene resin of the core-shell structure of the present invention, peptides can be effectively synthesized under mild conditions.

Still another object of the present invention is to provide a method for preparing a polymer comprising: (i) swelling an aminomethylpolystyrene resin; (ii) using a protecting group to protect the amine groups in the outer portion of the swollen aminomethylpolystyrene resin to form a shell; (iii) acetylating the amine groups of the resin except the shell to form a core; And (iv) removing the protecting group present in the shell layer of the resin of step (iii), by providing a method for preparing an aminomethylpolystyrene resin having a core-shell structure.

In step (i) of the process of the present invention, the aminomethylpolystyrene resin is swelled by treating the aminomethylpolystyrene resin with hydrochloric acid and tetrahydrofuran.

In the step (ii), to protect the amine group by combining a protecting group such as Fmoc (fluorenylmethyloxycarbonyl), Boc (butoxycarbonyl), Cbz (carboxybenzyl) or Alloc (allyloxycarbonyl) group to the amine group of the outer portion of the resin Can be.

When the Fmoc group is used as the protecting group, the swelled aminopolystyrene resin, Fmoc-OSu (N- (9-fluorenylmethoxycarbonyloxy) succinimide) and DIPEA (N, N-diisopropylethylamine) may be reacted to bind the Fmoc group to the amine group. .

In addition, when the Boc group is used as the protecting group, the swelled aminopolystyrene resin, di-tert-butyl dicarbonate, and DIPEA may be reacted to bond the Boc group to the amine group.

In addition, when the Cbz group is used as the protecting group, the swollen aminopolystyrene resin, benzyl chloroformate and DIPEA may be reacted to bind the Cbz group to the amine group.

In addition, when the Alloc group is used as the protecting group, the swelled aminopolystyrene resin, allyl chloroformate and DIPEA may be reacted to bind the Alloc group to the amine group.

In the step (ii), it is preferable to use a hydrophobic organic solvent such as DCM, THF, chloroform, toluene and the like. In the reaction of step (ii), the reaction proceeds at the interface between the solvent and the resin due to the polarity difference between the solvent and the resin.

Moreover, the core / shell structure is simply formed under mild conditions while the amine group of the outer AMPS resin of the aminopolystyrene resin is protected by Fmoc group, Boc group, Cbz group or Alloc group.

In addition, the amount of Fmoc-OSu and DIPEA in step (ii); The amount of di-tertiary-butyl dicarbonate and DIPEA; The amount of benzyl chloroformate and DIPEA; Or by varying the amounts of allyl chloroformate and DIPEA, the thickness of the shell of the resulting aminopolystyrene resin changes. Thus, according to the method of the present invention, aminopolystyrene resins having shells of various thicknesses can be prepared.

In step (iii), the aminopolystyrene resin obtained in step (ii) is reacted with acetic anhydride, DIPEA and NMP (1-Methyl-2-pyrrolidinone) to acetylate the amine group present in the center of the aminopolystyrene resin. Can be. In step (iii), NMP or DMF can be used as a solvent.

In the step (iv), if the aminopolystyrene resin obtained in step (iii) in DMF or NMP solvent is reacted with piperidine, the Fmoc group can be removed and the amino-polystyrene resin of the core-shell structure of the present invention is obtained. Can be.

Further, in step (iv), when the aminopolystyrene resin obtained in step (iii) in a DCM solvent is reacted with trifluoroacetic acid (TFA), the Boc group can be removed, and the amino-polystyrene resin of the core-shell structure of the present invention. Can be obtained.

In addition, in step (iv), when the aminopolystyrene resin obtained in step (iii) in DCM solvent is reacted with Pd / C (palladium / charcoal), the Cbz group can be removed and the core-shell structure of the present invention Aminopolystyrene resin can be obtained.

In addition, in step (iv), when the aminopolystyrene resin obtained in step (iii) in the TFA solvent is reacted with HBr, the Alloc group can be removed and the core-shell structured aminopolystyrene resin of the present invention can be obtained. .

The figure below is one embodiment illustrating the method for preparing the aminomethylpolystyrene resin of the core-shell structure of the present invention.

As can be seen in the above figure, the core-shell structure of the aminomethyl polystyrene resin manufacturing method of the present invention is a reaction under very mild conditions.

Using the core-shell aminoaminostyrene resin of the present invention, peptides can be synthesized at low cost under mild conditions. In particular, the core-shell structured aminopolystyrene resin of the present invention is suitable for hydrophobic peptide synthesis.

1 is a confocal fluorescence photograph of (1) aminomethyl polystyrene resin and (2) aminomethyl polystyrene resin of core-shell structure prepared in Example 1 of the present invention.
2 is a confocal fluorescence photograph showing the thickness change of the shell layer of the amino-methyl polystyrene resin of the core-shell structure produced according to the concentration of Fmoc-OSu and DIPEA in Example 2 of the present invention.
Figure 3 is a graph showing the amount of amine group loading (loading) according to the concentration of Fmoc-OSu and DIPEA in Example 2 of the present invention.
Figure 4 shows the results of high performance liquid chromatography analysis of MoPrP 105-125 peptides synthesized using aminopolystyrene resin, ChemMatrix and the core-shell aminomethylpolystyrene resin of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples or drawings. It is to be understood, however, that the following description of the embodiments or drawings is intended to illustrate specific embodiments of the invention and is not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed.

Example 1. Preparation of Core-Shell Resin

Aminomethylpolystyrene (AMPS) resin (10 g, 2.3 mmol / g) was treated with 1 normal hydrochloric acid / tetrahydrofuran (50/50, v / v) at room temperature for 2 hours and then washed with water. After filtration of water, Fmoc-OSu (2.87g, 8.5mmol) and N, N-Diisopropylethylamine ( N, N- Diisopropylethylamine, DIPEA) (1.48mL, 8.5mL) was dissolved and reacted at room temperature for 12 hours. The resin was then washed with dimethylformamide (DMF), DCM and methanol (3 times each 100 mL). In order to eliminate the reactivity of the amine group of the core portion of the resin, the resin was treated with acetic anhydride and DIPEA in DMF for 2 hours at room temperature (acetylation reaction). In order to remove the Fmoc group protecting the amine group of the shell portion, it was treated with 20% piperidine / N-methylpyrrolidinone (1-Methyl-2-pyrrolidinone, NMP) solution for 1 hour. After filtration, the resin was washed with DMF, DCM and methanol (three times 100 mL each) and dried under vacuum. The progress of the reaction was confirmed by Fourier transform infrared spectroscopy (absorption band-amide group: 1652 cm ^-1 ). The loading amount of the active amine group was determined to be 0.67 mmol / g through Fmoc group titration.

Comparative Example 1. Conventional AMPS Of the resin and the core-shell resin of the present invention Confocal Fluorescence Comparison

In order to prove the core-shell structure, two equivalents of AMPS resin, which is widely used in the prior art, two equivalents of 5-fluorescein isothiocyanate (FITC) and four equivalents of DIPEA, which are used as fluorescent materials in the core-shell resin of the present invention, were treated for two hours. After the reaction, the resin was washed with DMF, DCM and methanol, and dried in vacuo, and fluorescent images were taken using a confocal fluorescence microscope (FIG. 1).

Figure 1 (1) is a picture of AMPS resin, Figure 1 (2) is a picture of the core-shell resin of the present invention. Looking through the photos of Figure 1, it can be seen that the core-shell resin of the present invention is clearly a core-shell structure.

Example 2. Preparation of Core-Shell Resin with Adjustable Shell Thickness

Various core-shell resins were prepared in the same manner as in Example 1 by adjusting the concentrations of Fmoc-OSu and DIPEA. In order to visualize the shell of the prepared core-shell resins, FITC was introduced in the same manner as in Comparative Example 1, and fluorescence pictures were taken using a confocal fluorescence microscope (FIG. 2). Figure 3 shows the loading value according to the concentration of Fmoc-OSu and DIPEA. The correlation between the added amount of Fmoc-OSu and DIPEA and the loading amount of the reactive amine group on the resin showed that 82% reacted with the resin and the loading amount of the amine group was proportional (FIG. 3). It can be seen that the thickness of the shell increases as the amount of amine loading increases (FIG. 2), so that the thickness of the shell of the core-shell resin can be easily controlled.

Example 3. Peptides synthesis

9-mer peptide acyl carrier protein 65-74 (ACP 65-74, H-VQAAIDYING-NH ₂ ) after introducing the linker using the core-shell resin prepared in Example 1 ) And a 10-mer peptide, Jung-Redemann 10-mer (JR 10-mer, H-WFTTLISTIM-NH ₂ ), were synthesized, respectively. In the core-shell resin and AMPS, a link amide linker (4-[(2,4-Dimethoxyphenyl) (Fmoc-amino) methyl] phenoxyacetic acid), in which an amine group is protected with Fmoc group, was added with HBTU ( O- (Benzotriazol). -1-yl) -N, N, N ' , N ' -tetramethyl uronium hexafluorophosphate), HOBt (1-Hydroxybenzotriazole), DIPEA was introduced under NMP solvent. Each resin was swelled in NMP solvent in advance, and then the link amide linker (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIPEA (6 equivalents) were added to NMP and reacted for 2 hours. After completion of the reaction was confirmed by Kaiser test, washed in the order of DMF, DCM, methanol and dried in a vacuum oven. In order to remove the Fmoc group protecting the amine group, it was treated with 20% piperidine / NMP solution for 1 hour. For each resin, the loading amount of the amine group was calculated through the Fmoc group titration method (core-shell resin: 0.25, 0.51 mmol / g).

ACP 65-74 was synthesized by solid phase synthesis in an NMP solvent using amino acids having an amine group protected with a Fmoc protecting group and an HBTU coupling agent. 50 mg of the core-shell resin incorporating the link amide linker was previously swollen in NMP solvent. 3 equivalents of Fmoc amino acid, HBTU, HOBt and 6 equivalents of DIPEA were dissolved in NMP to activate carboxylic acid of amino acid in advance, and then added to each resin and reacted at room temperature for 1 hour. The Fmoc protecting group of the amino acid thus introduced was removed by two treatments of 5 minutes and 10 minutes with 20% piperidine / NMP solution. The Fmoc amino acids were repeatedly introduced into each resin in the same manner according to the peptide sequence. After finishing the last amino acid synthesis, the peptide was recovered by treatment with trifluoroacetic acid / triisopropylsilane / water (95: 2.5: 2.5) for 1 hour at room temperature.

JR 10-mer peptides were synthesized in the same manner as in ACP 65-74 peptide synthesis using the respective Fmoc amino acids and coupling reagents. Peptides were recovered from the resin by treating the synthesized resin to the last amino acid with trifluoroacetic acid / thioanizol / 1,2-ethanedithiol / anizol (90: 5: 3: 2) solution for 1 hour.

Comparative Example 2. AMPS Using resin ACP 65-74 and JR 10- mer Peptides Synthetic comparison experiment

As a control of the core-shell resin of the present invention, ACP 65-74 and JR 10-mer peptides were synthesized under the same conditions as in Example 3 using an AMPS resin having no core-shell structure. In the case of AMPS, after the introduction of the link amide linker, the remaining reactive amine group was acetylated by dissolving an excess of acetic anhydride and DIPEA in NMP. The amount of amine group loading of AMPS resin was measured by Fmoc titration (AMPS resin: 0.28, 0.48 mmol / g).

Example 4. Using microwave Peptides synthesis

Mouse prion peptide 105-125 which is a 21-mer peptide containing a large number of hydrophobic amino acid sequences using microwaves in the core-shell resin to which the link amide linker prepared in Examples 1 and 3 was introduced MoPrP 105-125, H-KTNLKHVAGAAAAGAVVGGLG-NH ₂ ) were synthesized. Microwave autosynthesizer was used in the CEM, each reaction was carried out in a nitrogen bubbling environment using a DMF solvent. For peptide synthesis, each amino acid and coupling reagent were Fmoc amino acid (5 equivalents), HBTU (5 equivalents), DIPEA (10 equivalents), and the synthesis was performed by irradiation of microwave at 70 ° C. for 300 seconds (maximum power: 20 W). ). Fmoc protecting groups were removed by two treatments with 20% piperidine / NMP solution. A double Fmoc deprotection process was performed by applying the conditions of 75 ° C., 30 seconds (maximum power: 40W) first, and 70 ° C., 180 seconds (max power: 35W). After the peptide synthesis was completed, the peptide was recovered by treatment with trifluoroacetic acid / triisopropylsilane / water (95: 2.5: 2.5) at room temperature for 1 hour.

Comparative Example 3. AMPS Suzy and ChemMatrix Using resin MoPrP 105-125 Peptides Synthetic comparison experiment

MoPrP 105-125 peptide was synthesized under the same conditions as in Example 4 using AMPS resin and ChemMatrix resin having no core-shell structure as a control of the core-shell resin.

Example 5 Peptides analysis

ACP 65-74 and JR 10-mer peptides synthesized in the previous example were subjected to high performance liquid chromatography (solvent A: distilled water containing 0.1% trifluoroacetic acid; solvent B: acetonitrile containing 0.1% fluoroacetic acid; flow rate 1.0 mL). Gradient conditions for 30 minutes / min, concentration of solvent B is 10-60% and constant flow for 10 minutes with solvent B; column is SPIRIT PEPTIDE 120 C ₁₈ column (5mm, 250mm × 4.6mm); absorption wavelength 230nm and Measured at 260 nm) and MALDI-TOF mass spectrometry. Mass spectrometry of each peptide ACP 65-74: calculated value 1061.6, C ₄₇ H ₇₅ N ₁₃ O ₁₅ , [M + Na] +, found 1084.6; JR 10-mer: calculated value 1210.6, C ₅₈ H ₉₀ N ₁₂ O ₁₄ S, [M + Na] +, confirmed 1233.8, and shows the synthetic yield and purity of the peptide synthesized in the core-shell resin and AMPS resin. We summarized in 1.

Loading value Suzy ACP (65-74) JR 10-mer yield water yield water High loading Core-shell resin Quant. 88% 90% 45% AMPS Resin 60% 28% 83% 35% Low loading Core-shell resin Quant. 88% 99% 54% AMPS Resin 85% 37% 89% 51%

As can be seen from Table 1, the core-shell resin prepared according to the method of the present invention was synthesized with high yields of ACP 65-74 and JR 10-mer peptides compared to the case of using an AMPS resin that is not a core-shell structure. .

In addition, the MoPrP 105-125 peptide synthesized using microwave was analyzed in the same manner. Mass spectrometry results were obtained with MoPrP 105-125: calculated value 1860.1, C ₈₁ H ₁₄₁ N ₂₇ O ₂₃ , [M + H] +, confirmed value 1861.1, and high performance liquid chromatography results are shown in FIG. 4.

In the case of MoPrP 105-125, as shown in Figure 4, when using a different AMPS resin and ChemMatrix resin, a large number of side reactions are not produced hydrophobic amino acid sequences synthesized, while the core-shell prepared according to the method of the present invention In the case of resins, the hydrophobic amino acid sequences are well synthesized, indicating that the synthetic yield is improved.

Claims

The aminomethyl polystyrene resin of the core-shell structure in amino polystyrene resin in which the amine group of the center part of the said amino polystyrene resin is acetylated.

(i) swelling the aminomethylpolystyrene resin;
(ii) using a protecting group to protect the amine groups in the outer portion of the swollen aminomethylpolystyrene resin to form a shell;
(iii) acetylating the amine groups of the resin except the shell to form a core; And
(iv) removing the protecting group present in the shell layer of the resin of step (iii), the core-shell structure aminomethyl polystyrene resin production method.

The method of claim 2, wherein in the step (i), the aminomethylpolystyrene resin is treated with hydrochloric acid and tetrahydrofuran.

The method of claim 2, wherein the protecting group of step (ii) is selected from the group consisting of Fmoc group, Boc group, Cbz group and Alloc group.

5. The method of claim 4, wherein the Fmoc group is bonded by reacting the swollen amino polystyrene resin with Fmoc-OSu and DIPEA. 6.

The method of claim 4, wherein the Boc group is bonded by reacting the swollen amino polystyrene resin with di-tertiary-butyl dicarbonate and DIPEA. 6.

The method of claim 4, wherein the Cbz group is bonded by reacting the swollen aminopolystyrene resin with benzyl chloroformate and DIPEA to form a core-shell structured aminomethylpolystyrene resin.

5. The method of claim 4, wherein the Alloc group is bonded by reacting the swollen aminopolystyrene resin with allyl chloroformate and DIPEA. 6.

The method of producing aminomethylpolystyrene resin having a core-shell structure according to claim 2, wherein acetic anhydride, DIPEA, and NMP are used for the acetylation of step (iii).

The method of claim 2, wherein in step (iv), piperidine is used to remove the Fmoc protecting group.

3. The method of claim 2, wherein the step (iv) removes the Boc protecting group using TFA in the core-shell structure of aminomethylpolystyrene resin.

The method of claim 2, wherein in the step (iv), the Cbz protecting group is removed using Pd / C.

The method of claim 2, wherein the alloc protecting group is removed using HBr in step (iv).