NOVEL MODIFIED CELLULOSE, METHOD FOR PREPARING THE SAME AND USE THEREOF

Abstract

A modified cellulose, method for preparing the same and use thereof. The structural formulas of the modified cellulose are shown in Formula (I), Formula (II) or Formula (III):

##STR00001## wherein, n is 2-7; the modified cellulose is obtained by substitution. The cellulose is substituted and modified with long-chain compounds containing amino terminal groups, so that the modified cellulose has space formed by long chains and amino terminal groups, which can be used as a matrix in solid-phase peptide synthesis (SPOT), to increase reaction activity and reaction space, reduce the difficulty of peptide synthesis, realize SPOT well, be suitable for synthesis in an automatic synthesizer, have high yield, and have low cost.

Claims

1. A novel modified cellulose, the structural formulas of said modified cellulose are shown in Formula (I), Formula (II) or Formula (III): ##STR00016## wherein, n is 2-7.

2. The modified cellulose according to claim 1, wherein, said cellulose is paper-based cellulose.

3. The modified cellulose according to claim 2, wherein, said paper-based cellulose is derived from cellulose filter papers.

4. The modified cellulose according to claim 1, wherein, n is 3.

5. A method for preparing the modified cellulose according claim 1, comprising the following steps: S1: mixing cellulose and a pyridine solution of p-toluenesulfonyl chloride, oscillating, washing and air-drying; S2: adding long-chain compounds ##STR00017## oscillating until the reaction is complete, washing, air-drying to obtain said modified cellulose of Formula (I).

6. The method for preparing the modified cellulose according to claim 5, further comprising the following steps of S3 and/or S4: S3: adding Ring Amide resin and an activator to said modified cellulose of Formula (I), reacting and then washing to obtain said modified cellulose of Formula (II). S4: adding Wang linker and an activator to said modified cellulose of Formula (I), reacting and then washing to obtain said modified cellulose of Formula (III).

7. The method for preparing the modified cellulose according to claim 5, in step S1, the mass concentration of p-toluenesulfonyl chloride in said pyridine solution of p-toluenesulfonyl chloride is 350-400 g/L.

8. The method for preparing the modified cellulose according to claim 7, said mass concentration of p-toluenesulfonyl chloride is 380 g/L.

9. The method for preparing the modified cellulose according to claim 6, in step S3, said activator is N-hydroxybenzotriazole (HOBt) and N,N-diisopropylcarbodiimide (DIC), or N-hydroxysuccinimide (HOSu) and N,N-diisopropylcarbodiimide (DIC); said reaction temperature is 50-80 C., and said reaction time is 10-40 min; in step S4, said activator is hexamethylphosphoramide (HMPA) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ); said reaction temperature is 18-25 C., and said reaction time is 12-16 h.

10. The method for preparing the modified cellulose according to claim 5, before step S1 said method also comprises the step of waterless treatment of cellulose.

11. The use of the modified cellulose according to claim 1 as a matrix in solid-phase peptide synthesis.

12. A method for preparing the modified cellulose according to claim 2, comprising the following steps: S1: mixing cellulose and a pyridine solution of p-toluenesulfonyl chloride, oscillating, washing and air-drying S2: adding long-chain compounds ##STR00018## oscillating until the reaction is complete, washing, air-drying to obtain said modified cellulose of Formula (I).

13. A method for preparing the modified cellulose according to claim 3, comprising the following steps: S1: mixing cellulose and a pyridine solution of p-toluenesulfonyl chloride, oscillating, washing and air-drying; S2: adding long-chain compounds ##STR00019## oscillating until the reaction is complete, washing, air-drying to obtain said modified cellulose of Formula (I).

14. A method for preparing the modified cellulose according to claim 4, comprising the following steps: S1: mixing cellulose and a pyridine solution of p-toluenesulfonyl chloride, oscillating, washing and air-drying; S2: adding long-chain compounds ##STR00020## oscillating until the reaction is complete, washing, air-drying to obtain said modified cellulose of Formula (I).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0040] FIG. 1: Structure of a glucose unit in paper-based cellulose;

[0041] FIG. 2: Scheme of the modified paper-based cellulose of Example 1 and the preparation process;

[0042] FIG. 3: Scheme of the functional modification of paper-based cellulose and the peptide synthesis;

[0043] FIG. 4: (A) Sequences of synthesized peptides, (B) Intavis peptide synthesizer and UV absorption of the synthesized peptides with the modified paper-based cellulose as a matrix and (C) Western Blotting results;

[0044] FIG. 5: Scheme of the modified paper-based cellulose modified with Rink Amide linker of Example 4 and the preparation process;

[0045] FIG. 6: Determination of the synthesized peptides (Valine-Valine-Valine-Valine-Lysine) by liquid chromatography-mass spectrometry;

[0046] FIG. 7: Scheme of solid-phase peptide synthesis;

[0047] FIG. 8: Determination of the synthesized peptides (Valine-Valine-Valine-Valine-Lysine) by liquid chromatography-mass spectrometry.

EXAMPLES

[0048] Hereinafter, the present invention is further described in detail with reference to the specific embodiments. The scope of the present disclosure is not limited in the following examples. It should be understood that a person skilled in the art can make various changes and modifications to the present disclosure without departing from the spirit and scope of the present disclosure.

[0049] In the following examples, the experimental methods without specifying conditions are usually in accordance with the conventional conditions in the art or the conditions recommended by the manufacturers; the raw materials, reagents, etc., used, unless otherwise specified, are all obtained from commercial approaches such as the conventional market.

Example 1

[0050] The example provided a modified cellulose with the following structure:

##STR00004##

[0051] As shown in FIG. 2, the modified cellulose was prepared by the following method:

[0052] (1) prepare a certain size of lab filtration paper (Whatman 50, and the structure as shown in FIG. 1), place it in a stainless steel container, add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; and then add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, and air-dry;

[0053] (2) place air-dried paper-based cellulose membrane in the stainless steel container, add enough pyridine solution of p-toluenesulfonyl chloride (380 g p-toluenesulfonyl chloride dissolved in 1 L pyridine) to the stainless steel container, oscillate gently at room temperature for 15 minutes, add enough DMF (washing step) to the stainless steel container, oscillate gently for 20 minutes, then pour out the solvent, repeat three times; add enough DCM to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, and air-dry paper-based cellulose;

[0054] (3) place the air-dried paper-based cellulose membrane in the stainless steel container, add enough 4,7,10-trioxa-1,13-tridecanediamine (TTDDA), oscillate gently at room temperature overnight; add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, and repeat three times to obtain the modified cellulose.

Example 2

[0055] The present example provided a modified cellulose with the following structure:

##STR00005##

[0056] The modified cellulose was prepared by the following method:

[0057] (1) prepare a certain size of lab filtration paper (Whatman 50), place it in a stainless steel container, add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; and then add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, and air-dry;

[0058] (2) place air-dried paper-based cellulose membrane in the stainless steel container, add enough pyridine solution of p-toluenesulfonyl chloride (350 g p-toluenesulfonyl chloride dissolved in 1 L pyridine) to the stainless steel container, oscillate gently at room temperature for 15 minutes, add enough DMF (washing step) to the stainless steel container, oscillate gently for 20 minutes, then pour out the solvent, repeat three times; add enough DCM to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, air-dry paper-based cellulose;

[0059] (3) place the air-dried paper-based cellulose membrane in the stainless steel container, add enough bis(3-aminopropoxy)ethane, oscillate gently at room temperature overnight; add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times to obtain the modified cellulose.

Example 3

[0060] The present example provided a modified cellulose with the following structure:

##STR00006##

[0061] The modified cellulose was prepared by the following method:

[0062] (1) prepare a certain size of lab filtration paper (Whatman 50), place it in a stainless steel container, add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; and then add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, and air-dry;

[0063] (2) place air-dried paper-based cellulose membrane in the stainless steel container, add enough pyridine solution of p-toluenesulfonyl chloride (400 g p-toluenesulfonyl chloride dissolved in 1 L pyridine) to the stainless steel container, oscillate gently at room temperature for 15 minutes, add enough DMF (washing step) to the stainless steel container, oscillate gently for 20 minutes, then pour out the solvent, repeat three times; add enough DCM to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times, air-dry paper-based cellulose;

[0064] (3) place the air-dried paper-based cellulose membrane in the stainless steel container, add enough hepta(ethylene glycol)bis(3-aminopropyl), oscillate gently at room temperature overnight; add enough dimethylformamide (DMF) (washing step) to the stainless steel container, oscillate gently for 1 hour, then pour out the solvent, repeat three times; add enough dichloromethane (DCM) to the stainless steel container, oscillate gently for 10 minutes, then pour out the solvent, repeat three times to obtain the modified cellulose.

Example 4

[0065] The example provided a modified cellulose modified with Rink Amide linker, and the structure of the modified cellulose is as follows:

##STR00007##

[0066] The modified cellulose provided in the example was prepared by the following method:

[0067] (1) the modified cellulose obtained according to the method in Example 1, air-dry;

[0068] (2) place the air-dried paper-based cellulose in step (1) in a stainless steel container, add 0.7 mol Rink Amide linker (structure:

##STR00008##

0.7 mol hydroxybenzotriazole (HOBt), 0.7 mol N,N-diisopropyl carboimide (DIC) and solvent DMF, oscillate at 70 C. for 15 minutes, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Example 5

[0069] The example provided a modified cellulose modified with Rink Amide linker, and the structure of the modified cellulose is as follows:

##STR00009##

[0070] The modified cellulose was prepared by the following method:

[0071] (1) the modified cellulose obtained according to the method in Example 2, air-dry;

[0072] (2) place the air-dried paper-based cellulose in step (1) in a stainless steel container, add 0.7 mol Rink Amide linker (structure:

##STR00010##

0.7 mol hydroxybenzotriazole

[0073] (HOBt), 0.7 mol N,N-diisopropyl carboimide (DIC) and solvent DMF, oscillate at 50 C. for 40 minutes, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Example 6

[0074] The example provided a modified cellulose modified with Rink Amide linker, and the structure of the modified cellulose is as follows:

##STR00011##

[0075] The modified cellulose was prepared by the following method:

[0076] (1) the modified cellulose obtained according to the method in Example 3, air-dry;

[0077] (2) place the air-dried paper-based cellulose in step (1) in a stainless steel container, add 0.7 mol Rink Amide linker (structure:

##STR00012##

0.7 mol hydroxybenzotriazole (HOBt), 0.7 mol N,N-diisopropyl carboimide (DIC) and solvent DMF, oscillate at 80 C. for 10 minutes, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Example 7

[0078] The example provided a modified cellulose modified with Wang linker, and the structure of the modified cellulose is as follows:

##STR00013##

[0079] As shown in FIG. 2, the modified cellulose provided in the example was prepared by the following method:

[0080] (1) the modified cellulose obtained according to the method in Example 1, air-dry;

[0081] (2) place the air-dried paper-based cellulose in step (1) in a stainless steel container, add 0.1 mol Wang Linker, 0.11 mmol 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 0.1 mol hexamethylphosphoramide (HMPA) and NMP (N-methylpyrrolidone) solvent, oscillate at 20 C. for 14 hours, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Example 8

[0082] The example provided a modified cellulose modified with Wang linker, and the structure of the modified cellulose is as follows:

##STR00014##

[0083] The modified cellulose was prepared by the following method:

[0084] (1) the modified cellulose obtained according to the method in Example 2, air-dry;

[0085] (2) place the air-dried paper-based cellulose in step (1) in a stainless steel container, add 0.1 mol Wang linker, 0.11 mmol 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 0.1 mol hexamethylphosphoramide (HMPA) and NMP (N-methylpyrrolidone) solvent, oscillate at 20 C. for 14 hours, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Example 9

[0086] The example provided a modified cellulose modified with Wang linker, and the structure of the modified cellulose is as follows:

##STR00015##

[0087] The modified cellulose was prepared by the following method:

[0088] (1) the modified cellulose obtained according to the method in Example 3, air-dry;

[0089] (2) place the air-dried paper-based cellulose in Step (1) in a stainless steel container, add 0.1 mol Wang Linker, 0.11 mmol 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 0.1 mol hexamethylphosphoramide (HMPA) and NMP (N-methylpyrrolidone) solvent, oscillate at 20 C. for 14 hours, then sequentially wash with DMF, ethanol (twice) and DCM, and air-dry to obtain the modified cellulose.

Application Example 1

[0090] Take the modified cellulose provided in Example 1 as an example, which was used as a matrix in solid-phase peptide synthesis, and test the properties of the synthesized products using UV absorption and Western Blotting.

[0091] Peptide synthesizer was used to synthesize peptides in the direction from C-terminal to N-terminal. The schematic process of synthesis is shown in FIG. 3. The structures of the synthesized peptides are shown in FIG. 4 panel (A). Bioactive peptides containing six repeat histidine residues (HHHHHH) were used as a positive control for synthesis and detection. The synthesized peptides had the sequence tyrosine-serine-proline-threonine-serine-proline-serine (YSPTSPS), repeat structure (YSPTSPSYSPTSPS) and serine at positions 2,5,7 substituted with phosphatidylserine(s). FIG. 4 panel (B) is UV absorption of the synthesized peptides, all of which were positive, because the structures of the synthesized peptides were similar, they all absorbed light in the UV range, and there was no significant difference. FIG. 4 panel (C) is Western Blotting test results, because of the different amino acid sequences and biological activities of the peptides, in the Western Blotting tests, there were obvious differences due to the modification with amino acids (phosphatidylserine) at the different positions; even also in the peptides (YsPTsPS) with phosphatidylserine at positions 2 and 5, and their repeat structure (YsPTsPSYsPTsPS). It could be seen from the figures that the modified cellulose provided in the example could be used as a matrix in solid-state peptide synthesis, and the peptides had been successfully synthesized with the productivity and yields of the synthesis, which were suitable for bioactive peptide screening.

Application Example 2

[0092] Take the modified cellulose modified with Rink Amide linker provided in Example 4 as an example, and test the properties.

[0093] Solid-phase peptide synthesis was carried out using the modified cellulose modified with Rink Amide linker as a matrix provided in Example 4, and the synthesized peptides were tested. The steps were as follows: synthesizing by using an automatic synthesizer on Rink Amide linker modified cellulose, then cleaving protective groups and the synthesized peptides from modified cellulose, finally analyzing the synthesized products by LC-mass spectrometry.

[0094] Peptide synthesizer was used to synthesize peptides in the direction from C-terminal to N-terminal. The schematic process of synthesis is shown in FIG. 5. Take the synthesized peptides Valine-Valine-Valine-Valine-Lysine (Ver-Ver-Ver-Ver-Lys) in an automatic synthesizer as an example: firstly, wetting and cleaning modified cellulose modified with Rink Amide linker with DMF and ethanol, removing Fmoc protection by using a pyridine solution, adding C-terminal amino acid Lys in the first step, and then repeating deprotection and synthesis steps until the desired peptides were synthesized; finally, deprotecting by using pyridine. Cleave synthesized peptides (such as Ver-Ver-Ver-Ver-Ver-Lys) from the modified cellulose modified with Rink Amide linker, cleave each SPOT peptides by using 100 L of 90% trifluoroacetic acid, 3% triisopropylsilane, 2% water and 5% dichloromethane, and oscillate gently for 2 hours.

[0095] The synthesized peptides (Valine-Valine-Valine-Valine-Lysine) were determined by liquid chromatography-mass spectrometry (LC-MS). The molecular weight was 541.3. As shown in the LC-MS spectra (FIG. 6), the retention time of the main compound was 8.64 minutes, and the molecular ion peak was 542.4, which was the synthesized targeted peptide. As can be seen from FIG. 6, the peptides (Valine-Valine-Valine-Valine-Lysine) were successfully synthesized, and the modified cellulose had good properties, which could be used as a matrix in solid-phase peptide synthesis, exhibiting good reaction activity and high purity.

Application Example 3

[0096] Take the modified cellulose modified with Wang linker provided in Example 7 as an example, and test the properties.

[0097] Solid-phase peptide synthesis was carried out using the modified cellulose modified with Wang linker as a matrix provided in Example 7, and the synthesized peptides were tested. The steps were as follows:

[0098] The steps were as follows: synthesizing by using an automatic synthesizer on Wang linker modified cellulos, then cleaving protective groups and the synthesized peptides from modified cellulos, then analyzing the synthesized products by LC-mass spectrometry.

[0099] Peptide synthesizer was used to synthesize peptides in the direction from C-terminal to N-terminal. The schematic process of synthesis is shown in FIG. 7. Take the synthesized peptides Valine-Valine-Valine-Valine-Lysine (Ver-Ver-Ver-Ver-Lys) in an automatic synthesizer as an example: firstly, wetting and cleaning modified cellulose modified with Wang linker with DMF and ethanol, removing Fmoc protection by using a pyridine solution, adding C-terminal amino acid Lys in the first step, and then repeating deprotection and synthesis steps until the desired peptides were synthesized; finally, deprotecting by using pyridine. Cleave synthesized peptides (such as Ver-Ver-Ver-Ver-Ver-Lys) from the modified cellulose modified with Wang linker, cleave each SPOT peptide by using 100 L of 90% trifluoroacetic acid, 3% triisopropylsilane, 2% water and 5% dichloromethane, and oscillate gently for 2 hours.

[0100] The synthesized peptides (Valine-Valine-Valine-Valine-Lysine) were determined by liquid chromatography-mass spectrometry (LC-MS). The molecular weight was 542.3. As shown in the LC-MS spectra (FIG. 8), the retention time of the main compound was 10.18 minutes, and the molecular ion peak was 543.39, which was the synthesized targeted peptide. As can be seen from FIG. 8, the peptides (Valine-Valine-Valine-Valine-Lysine) were successfully synthesized, and the modified cellulose had good properties, which could be used as a matrix in solid-phase peptide synthesis, exhibiting good reaction activity and high purity.