CN111748019A - Synthetic method of polypeptide derivative compound - Google Patents

Synthetic method of polypeptide derivative compound Download PDF

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CN111748019A
CN111748019A CN201910249711.5A CN201910249711A CN111748019A CN 111748019 A CN111748019 A CN 111748019A CN 201910249711 A CN201910249711 A CN 201910249711A CN 111748019 A CN111748019 A CN 111748019A
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fmoc
otbu
gly
glu
tbu
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陈友金
尹传龙
宓鹏程
陶安进
袁建成
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Hybio Pharmaceutical Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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Abstract

The invention relates to a method for synthesizing a polypeptide derivative compound, which comprises the following steps: step 1: connecting Fmoc-Gly-OH to the resin to obtain Fmoc-Gly resin; step 2: sequentially coupling amino acids by Fmoc/tBu strategy according to the sequence of the polypeptide derivative compound to obtain polypeptide derivative compound main chain peptide resin; and step 3: removal of Lys26Sequentially coupling Fmoc-AEEA-AEEA-OH and octadienoic acid (OtBu) -Glu-OtBu to obtain polypeptide derivative compound peptide resin; and 4, step 4: cracking the peptide resin of the polypeptide derivative compound and removing side chain protecting groups at the same time to obtain a crude product of the polypeptide derivative compound; optionally, step 5: purifying by RP-HPLC to obtain pure polypeptide derivative compound. The inventionThe method is simple, and has high synthesis efficiency and high purity.

Description

Synthetic method of polypeptide derivative compound
Technical Field
The invention relates to the field of drug synthesis, in particular to a synthesis method of a polypeptide derivative compound.
Background
The preparation prepared from the polypeptide derivative compound is subcutaneously injected once a week and is approved by the FDA in the United states to be marketed in 12-5 months in 2017. Because the fatty chain of the polypeptide derivative compound is longer, the hydrophobicity is increased, and the hydrophilicity is greatly enhanced through short-chain PEG modification. After being modified by PEG, the modified PEG not only can be tightly combined with albumin to cover DPP-4 enzyme hydrolysis sites, but also can reduce renal excretion, prolong the biological half-life and achieve the effect of long circulation.
The chemical name of the polypeptide derivative compound is N6,26-{18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaocta decanoyl}-[8-(2-amino-2-propanoic acid),34-L-arginine]human glucose-like peptide 1(7-37) with molecular formula of C187H291N45O59Molecular weight of 4113.5775 and sequence of H-His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-G ln23-Ala24-Ala25-Lys26(PEG-PEG-γ-Glu-Octadecanoic acid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-OH。
CN101133082A provides two methods of solid phase stepwise synthesis and main chain synthesis first and then liquid phase modification to obtain polypeptide derivative compounds, the first method is easy to generate a large amount of default impurities and is difficult to obtain high-quality products, the second method needs RP-HPLC to purify the main chain first, and the modification reaction is complicated, the cost is high and the yield is low. CN108059666B used a large number of short peptide fragments for solid phase synthesis, which were not commercialized, had high customization cost, and were not suitable for large-scale production.
Disclosure of Invention
The invention adopts Fmoc/tBu solid phase method for synthesis, and uses Fmoc-AEEA-AEEA-OH, octadecenoic acid (OtBu) -Glu-OtBu, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Aib-Glu (OtBu) -OH and other full-protection dipeptide fragments at some special positions respectively, which not only solves the main default impurities in the product pertinently, but also can reduce the production cost, obtain the product with high yield and high quality, and is suitable for large-scale production.
One aspect of the present invention provides a method for synthesizing a polypeptide derivative compound, comprising the steps of:
step 1: connecting Fmoc-Gly-OH to solid-phase synthetic resin to obtain Fmoc-Gly-resin;
step 2: sequentially coupling Fmoc-Arg by Fmoc/tBu strategy according to the sequence of polypeptide derivative compound36(pbf)-OH、Fmoc-Gly35-OH、Fmoc-Arg34(pbf)-OH、Fmoc-Val33-OH、Fmoc-Leu32-OH、Fmoc-Trp31(Boc)-OH、Fmoc-Ala30-OH、Fmoc-Ile29-OH、Fmoc-Phe28-OH、Fmoc-Glu27(OtBu)-OH、Fmoc-Lys26(X)-OH、Fmoc-Ala25-OH、Fmoc-Gln23(Trt)-Ala24-OH、Fmoc-Gly22-OH、Fmoc-Glu21(OtBu)-OH、 Fmoc-Leu20-OH、Fmoc-Tyr19(tBu)-OH、Fmoc-Ser18(tBu)-OH、Fmoc-Ser17(tBu)-OH、 Fmoc-Val16-OH、Fmoc-Asp15(OtBu)-OH、Fmoc-Ser14(tBu)-OH、Fmoc-Thr13(tBu)-OH、 Fmoc-Phe12-OH、Fmoc-Thr11(tBu)-OH、Fmoc-Gly10-OH、Fmoc-Aib8-Glu9(OtBu) -OH and Y-His7(Z) -OH to obtain polypeptide derivative compound main chain peptide resin;
and step 3: removal of Lys26Sequentially coupling Fmoc-AEEA-AEEA-OH and octadienoic acid (OtBu) -Glu-OtBu to obtain polypeptide derivative compound peptide resin;
and 4, step 4: cracking the peptide resin of the polypeptide derivative compound and removing side chain protecting groups at the same time to obtain a crude product of the polypeptide derivative compound;
optionally, step 5: purifying by RP-HPLC to obtain pure polypeptide derivative compound.
In the technical scheme of the invention, the polypeptide derivative compound has a sequence of H-His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-G ln23-Ala24-Ala25-Lys26(PEG-PEG-γ-Glu-Octadecanoic acid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-OH.
The solid phase synthetic resin used in the step 1 is 2-Chlorotrityl resin, Wang resin or RinkAcid resin;
the substitution degree of the resin used in the step 1 is 0.3-0.6 mmol/g, preferably 0.4-0.5 mmol/g;
in the step 1, the substitution degree of Fmoc-Gly-resin is 0.3-0.5 mmol/g, preferably 0.3-0.4 mmol/g;
Fmoc-Gly-OH and Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, (Fmoc-OH, Fmoc-Gly-Ser (Ty) -OH, Fmoc-Gly-Leu, Coupling reagents of Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Aib-Glu (OtBu) -OH and Y-His (Z) -OH are DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
X of Fmoc-Lys (X) -OH in step 1 is an amino protecting group selected from Mmt, ivDDe, DDe, Mtt or Alloc;
in step 1, Y and Z of Y-His (Z) -OH are both amino protecting groups, and Y-His (Z) -OH is selected from Boc-His (Boc) -OH, Boc-His (Trt) -OH or Trt-His (Boc) -OH;
the synthesis method of Octadecanoic acid (OtBu) -Glu-OtBu in the step 3 comprises the following steps:
3-1) mono-tert-butyl octadecanedioate under the action of HONB and DIC to obtain octadienoic acid (OtBu) -ONB;
3-2) reacting H-Glu-OtBu with Octadecanoic acid (OtBu) -ONB in an aqueous alkaline solution to obtain Octadecanoic acid (OtBu) -Glu-OtBu, and purifying by crystallization.
In the step 3, a crystallization solvent of Octadecanoic acid (OtBu) -Glu-OtBu is methanol, acetonitrile, a combination of acetonitrile and water, and a combination of tetrahydrofuran and water, preferably the combination of acetonitrile and water, and the crystallization temperature is 2-8 ℃;
in the step 4, the cracking time of the peptide resin is 2-4 h, preferably 3-4 h;
in the step 4, the cracking temperature is 18-35 ℃, and preferably 25-30 ℃.
The cleavage reagent in step 4 is TFA, phenol and EDT in water, preferably TFA phenol H2O:EDT＝85-90:3-8: 3-8:1-5，v/v。
The chromatographic column condition in the step 5 is two-step purification, and the method specifically comprises the following steps:
the first step of purification conditions: a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica filler as a stationary phase,
the column diameter and length were: 10cm × 25cm. mobile phase: phase A: 0.1% phosphoric acid, adjusting pH to 8.5 with triethylamine; phase B: acetonitrile solution. Flow rate: 190-210 ml/min. Detection wavelength: 280nm. Gradient: the mass percentage concentration of the mobile phase B is as follows: 30-60%, eluting with linear gradient, collecting target peak, concentrating the target peak, and purifying;
and the second step of purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase A: 0.01% phosphoric acid in water; phase B: chromatographically pure acetonitrile solution; flow rate: 190-; detection wavelength: gradient at 280 nm: the mass percentage concentration of the mobile phase B is as follows: 35 to 45 percent.
Advantageous effects
1. Selectively using dipeptide fragment aiming at key problems, and avoiding default impurity Des-Glu in the synthesis process of polypeptide derivative compounds3，Des-Aib2，Des-AEEA，Des-Gln23Des-octadecanedioic acid can not only obtain high-yield and high-quality products, but also ensure the commercialization of raw materials and the reduction of production cost, and is suitable for large-scale production;
2. the octadienoic acid (OtBu) -Glu-OtBu crystallization process ensures high quality and supply of raw materials and is beneficial to commercial production.
Abbreviations and English meanings
Figure RE-GDA0002039795520000031
Figure RE-GDA0002039795520000041
Drawings
FIG. 1 is a mass spectrum of Octadecanoic acid (OtBu) -ONB.
FIG. 2 is a spectrum of Octadecanoic acid (OtBu) -ONB HPLC.
FIG. 3 is a mass spectrum of Octadecanoic acid (OtBu) -Glu-OtBu.
FIG. 4 is an Octadecanoic acid (OtBu) -Glu-OtBu HPLC chromatogram.
FIG. 5 is a mass spectrum of crude polypeptide-derived compound of example 11.
FIG. 6 is an HPLC chromatogram of crude polypeptide derivative compound of example 11.
FIG. 7 is a pure mass spectrum of the polypeptide derivative compound of example 14.
FIG. 8 is an HPLC chromatogram of a pure product of the polypeptide derivative compound of example 14.
Detailed Description
Example 1 Synthesis of Octadecanoic acid (OtBu) -ONB
Figure RE-GDA0002039795520000042
Reacting octadecanedioic acidMono-tert-butyl ester (37.1g, 100mmol) and HONB (2.1.5g, 120mmol) were dissolved in 400ml tetrahydrofuran, DIC (23.2ml, 150mmol) was added under ice salt bath, the reaction was stirred for 2h, ice salt bath was removed, and the reaction was stirred at room temperature for 4 h. Filtration, concentration of the filtrate in vacuo and crystallization of the residue with acetonitrile gave 48.04 white solid, yield: 87.38%, purity: 98.78%, MS: 476.23(M-tBu + H)+)，531.17(M+H+)，554.29(M+Na+)。
Example 2 Synthesis of Octadecanoic acid (OtBu) -Glu-OtBu
H-Glu-OtBu (18.17g, 89.4mmol) and Na2CO3(37.9g, 357.6mmol) was dissolved in 300ml H2Adding Octadecanoid acid (OtBu) -ONB (47.486g, 89.4mmol) solution in THF (300ml) slowly dropwise under stirring at room temperature, continuing to react for 16h after dropwise addition, vacuum concentrating to remove THF, adding 300ml water into water phase, extracting with EA for 2 times, combining organic phases, washing with 1M HCl for 3 times, washing with water for 3 times, washing with saturated salt water for 2 times, and adding anhydrous Na2SO4Drying, concentration in vacuo, and crystallization of the residue with acetonitrile and water at 5 ℃ gave 43.686 as a white solid in yield: 90.32%, purity: 98.55%, MS: 556.12(M + H)+),578.31(M+Na+)。
Example 3: synthesis of Octadecanoic acid (OtBu) -Glu-OtBu
H-Glu-OtBu (2.03g, 10mmol) and Na2CO3(4.24g, 40mmol) was dissolved in 40ml of H2Adding Octadecanoid acid (OtBu) -ONB (5.31g, 10mmol) in THF (40ml) slowly dropwise under stirring at room temperature, continuing to react for 16h after dropwise addition, vacuum concentrating to remove THF, adding 40ml water into water phase, extracting EA for 2 times, combining organic phases, washing with 1M HCl for 3 times, washing with water for 3 times, washing with saturated salt water for 2 times, and adding anhydrous Na2SO4Drying, vacuum concentration, crystallization of the residue with methanol at 5 ℃ gave 2.54g of a white solid in yield: 45.8%, purity: 95.32%, MS: 556.12(M + H +),578.31(M + Na +).
Example 4: synthesis of Octadecanoic acid (OtBu) -Glu-OtBu
H-Glu-OtBu (2.03g, 10mmol) and Na2CO3(4.24g, 40mmol) in 40ml H2Adding Octadecanoid acid (OtBu) -ONB (5.31g, 10mmol) in THF (40ml) slowly dropwise under stirring at room temperature, continuing to react for 16h after dropwise addition, vacuum concentrating to remove THF, adding 40ml water into water phase, extracting EA for 2 times, combining organic phases, washing with 1M HCl for 3 times, washing with water for 3 times, washing with saturated salt water for 2 times, and adding anhydrous Na2SO4Drying, concentration in vacuo, crystallization of the residue with acetonitrile at 5 ℃ gave 2.86g of a white solid in yield: 51.5%, purity: 96.14%, MS: 556.12(M + H +),578.31(M + Na +).
Example 5: synthesis of Octadecanoic acid (OtBu) -Glu-OtBu
H-Glu-OtBu (2.03g, 10mmol) and Na2CO3(4.24g, 40mmol) was dissolved in 40ml of H2Adding Octadecanoid acid (OtBu) -ONB (5.31g, 10mmol) in THF (40ml) slowly dropwise under stirring at room temperature, continuing to react for 16h after dropwise addition, vacuum concentrating to remove THF, adding 40ml water into water phase, extracting EA for 2 times, combining organic phases, washing with 1M HCl for 3 times, washing with water for 3 times, washing with saturated salt water for 2 times, and adding anhydrous Na2SO4Drying, vacuum concentration, crystallization of the residue with tetrahydrofuran and water at 5 ℃ gave 3.24g of a white solid in yield: 58.4%, purity: 96.81%, MS: 556.12(M + H +),578.31(M + Na +).
Example 6 Synthesis of Fmoc-Gly-Wang resin
0.4mmol/g Wang resin (25g, 10mmol) was added to the solid phase reaction column, washed with DMF 2 times, swelled with DMF for 30min, washed with DMF 2 times, and drained.
Dissolving Fmoc-Gly-OH (8.92g, 30mmol) and HOBt (4.86g, 36mmol) in 50ml DMF, adding DIC (4.54g, 36mmol) under ice bath condition, stirring and activating for 5min, putting the activated solution into the above solid phase reaction column filled with Wang resin, adding DMAP (0.37g, 0.3mmol) under nitrogen stirring, reacting for 2h at room temperature, draining the reaction solution, washing DMF for 6 times, washing DCM for 3 times, and Ac with equal molar ratio2Blocking the O/pyridine mixed solution for 6h, draining, washing with DCM 6 times, shrinking methanol 3 times (5min +5min +10min), drying in vacuum, and taking a small sample to measure the substitution Sub ═ 0.371 mmol/g.
Example 7 Synthesis of Fmoc-Gly-CTC resin
Adding 0.5mmol/g 2-CTC resin (20g, 10mmol) into solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, and draining.
Dissolving Fmoc-Gly-OH (8.92g and 30mmol) in 50ml of DMF, adding DIPEA (7.75g and 60mmol) under ice bath conditions, stirring and activating for 5min, putting the activated solution into the solid phase reaction column filled with the 2-CTC resin, stirring and reacting for 2h at room temperature under nitrogen, adding MeOH (6.41g and 0.2mol) into the reaction solution, continuing stirring and reacting for 20min under nitrogen, draining the reaction solution, washing the DMF for 6 times, washing the DCM for 3 times, shrinking the methanol for 3 times (5min +5min +10min), drying in vacuum, and taking a small sample to measure the substitution degree Sub which is 0.412 mmol/g.
Example 8 Synthesis of peptide resin of polypeptide derivative Compound
Adding Fmoc-Gly-Wang resin (13.48g, 5mmol) of 0.371mmol/g into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, deprotecting 20% DBLK for 2 times (10min +15min), washing with DMF for 6 times, and draining for later use.
Fmoc-Arg (pbf) -OH (9.73g, 15mmol) and HOBt (2.43g, 18mmol) were dissolved in DMF (25ml), DIC (2.27g, 18mmol) was added under ice bath conditions, activation was performed for 5min, the activated solution was added to the above solid phase reaction column, the reaction was stirred with nitrogen at room temperature for 2h, the reaction was aspirated off, DMF was washed 3 times, 20% DBLK was deprotected 2 times (5min +7min), and DMF was washed 6 times.
Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Gly-OH, Fmoc-Glu OtBu-OH, (Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Leu, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Aib-Glu (OtBu) -OH and Boc-His (trt) -OH to obtain the polypeptide derivative compound main chain peptide resin.
The backbone peptide resin of the above-mentioned polypeptide derivative compound was washed 6 times with DCM, 2% TFA/3% TIS/DCM desalys26Mmt protection (10min × 12), DCM washing 6 times, DMF washing 3 times, coupling Fmoc-AEEA-AEEA-OH and Octadecanoic acid (OtBu) -Glu-OtBu sequentially, DMF washing 6 times, DCM washing 6 times, methanol shrinkage 3 times (5min +5min +10min), vacuum drying to obtain 38.61g polypeptide derivative compound peptide resin.
Example 9 Synthesis of peptide resin of polypeptide derivative Compound
Adding Fmoc-Gly-Wang resin (13.48g, 5mmol) of 0.371mmol/g into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, deprotecting 20% DBLK for 2 times (10min +15min), washing with DMF for 6 times, and draining for later use.
Fmoc-Arg (pbf) -OH (9.73g, 15mmol) and HOBt (2.43g, 18mmol) were dissolved in DMF (25ml), DIC (2.27g, 18mmol) was added under ice bath conditions, activation was performed for 5min, the activated solution was added to the above solid phase reaction column, the reaction was stirred with nitrogen at room temperature for 2h, the reaction was aspirated off, DMF was washed 3 times, 20% DBLK was deprotected 2 times (5min +7min), and DMF was washed 6 times.
Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Mmt) -OH, Fmoc-Ala-OH, Fmoc-Gln (trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (Asp) (OH), Fmoc-Leu-OH, Fmoc-Lys (Mmt) -OH, Fmoc-Leu-OH, Fmoc, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Aib-OH and Boc-His (trt) -OH to obtain a polypeptide derivative compound backbone peptide resin.
The peptide resin of the main chain of the polypeptide derivative compound is washed 6 times by DCM, Mmt protection of Lys26 is removed by 2% TFA/3% TIS/DCM (10min multiplied by 12), DCM is washed 6 times, DMF is washed 3 times, Fmoc-AEEA-OH, Fmoc-Glu-OtBu and Octadecanoic acid-OtBu are coupled in sequence, DMF is washed 6 times, DCM is washed 6 times, methanol is contracted 3 times (5min +5min +10min), and 38.51g of peptide resin of the polypeptide derivative compound is obtained after vacuum drying.
Example 10 Synthesis of peptide resin of polypeptide derivative Compound
Adding Fmoc-Gly-CTC resin (12.14g, 5mmol) of 0.412mmol/g into a solid phase reaction column, washing with DMF for 2 times, swelling with DMF for 30min, washing with DMF for 2 times, deprotecting 20% DBLK for 2 times (10min +15min), washing with DMF for 6 times, and draining for later use.
Fmoc-Arg (pbf) -OH (9.73g, 15mmol), PyBOP (7.81g, 15mmol) and HOBt (2.43g, 18mmol) were dissolved in DMF (25ml), DIPEA (2.58g, 20mmol) was added under ice bath conditions, activation was performed for 5min, the activated solution was added to the above solid phase reaction column, the reaction was stirred with nitrogen at room temperature for 2h, the reaction solution was withdrawn, DMF was washed 3 times, 20% DBLK was deprotected 2 times (5min +7min), DMF was washed 6 times.
Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Alloc) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Gly-OH, Fmoc-Glu OtBu-OH, (Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, F, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Aib-Glu (OtBu) -OH and Boc-His (Boc) -OH to obtain the polypeptide derivative compound backbone peptide resin.
The peptide resin of the main chain of the polypeptide derivative compound is washed 6 times by DCM, Mmt protection of Lys26 is removed by 2% TFA/3% TIS/DCM (10 min. times.12), DCM is washed 6 times, DMF is washed 3 times, Fmoc-AEEA-OH and Octadecanoic acid (OtBu) -Glu-OtBu are coupled in sequence, DMF is washed 6 times, DCM is washed 6 times, methanol is contracted 3 times (5min +5min +10min), and 34.59g of peptide resin of the polypeptide derivative compound is obtained by vacuum drying.
Example 11 cleavage of peptide resin of polypeptide derivative Compound
38.61g of the peptide resin of the polypeptide derivative compound of example 8 was placed in a 500ml round-bottomed flask, and 400ml of frozen lysate (TFA: Phenol: H) was added for 2 to 4 hours2EDT (87.5: 5:5:2.5, v/v), stirring and reacting at 18 ℃ for 2-4 h, filtering, settling filtrate by using 4L frozen anhydrous ether, centrifugally collecting solid, washing 3 times by using anhydrous ether, drying in vacuum to obtain 19.58g of crude polypeptide derivative compound,yield: 95.2%, purity: 72.66%, MS:4111.846(M + H)+)。
Example 12 cleavage of peptide resin of polypeptide derivative Compound
38.51g of the peptide resin of the polypeptide derivative compound of example 9 was placed in a 500ml round-bottomed flask, and 400ml of frozen lysate (TFA: Phenol: H) was added for 2 to 4 hours2And (2) EDT (87.5: 5:5:2.5, v/v), stirring at 35 ℃ for reaction for 2-4 h, filtering, settling filtrate by using 4L of frozen anhydrous ether, centrifugally collecting solid, washing 3 times by using anhydrous ether, and drying in vacuum to obtain 19.58g of crude polypeptide derivative compound, wherein the yield is as follows: 93.2%, purity: 43.37%, MS:4111.846(M + H)+)。
Example 13 cleavage of peptide resin of polypeptide derivative Compound
34.59g of the peptide resin of the polypeptide derivative compound of example 10 was placed in a 500-ml round-bottomed flask, and 350ml of frozen lysate (TFA: DTT: H) was added for 2 to 4 hours2O ═ 90:5:5, v/v), stirring at 30 ℃ for reaction for 2-4 h, filtering, settling the filtrate with 4L of frozen anhydrous ether, centrifugally collecting the solid, washing with anhydrous ether for 3 times, and drying under vacuum to obtain 17.69g of crude polypeptide derivative compound, with yield: 86.0%, purity: 70.79%, MS:4111.846(M + H)+)。
Example 14: RP-HPLC (reverse phase high Performance liquid chromatography) for preparing pure polypeptide derivative compounds
Taking 19.58g of polypeptide derivative compound, dissolving with 400ml of water, using ammonia water for assisting dissolution, having pH of 8.0-9.0, and performing reversed phase purification after a sample passes through a 0.45 mu m filter membrane.
The first step of purification conditions: a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica filler as a stationary phase,
the column diameter and length were: 10cm × 25cm. mobile phase: phase A: 0.1% phosphoric acid, adjusting pH to 8.5 with triethylamine; phase B: acetonitrile solution. Flow rate: 190-210 ml/min. Detection wavelength: 280nm. Gradient: the mass percentage concentration of the mobile phase B is as follows: 30-60% and gradient treatment time of 60 min. The sample injection amount is 19.58 g;
and (3) purification process: and (4) washing acetonitrile above the chromatographic column, and then loading the sample, wherein the loading amount is the sample solution after dissolution and filtration. Carrying out linear gradient elution, collecting a target peak with the purity of about 90%, and placing the collected peptide solution in a collection bottle for later use;
and the second step of purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase A: 0.01% phosphoric acid in water; phase B: chromatographically pure acetonitrile solution; flow rate: 190-; detection wavelength: gradient at 280 nm: the mass percentage concentration of the mobile phase B is as follows: 35-45%, gradient processing time 45-60 min; the sample amount is a sample solution with the content of 99 percent after the first step of purification and concentration;
and (3) purification process: washing acetonitrile above a chromatographic column, loading the acetonitrile, carrying out linear gradient elution on a sample solution with the loading amount being 90% of the content of the sample solution after the first-step purification and concentration, collecting a target peak with the purity being about 99%, and placing the collected peptide solution in a collection bottle for later use;
desalting: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase A: an aqueous solution; phase B: chromatographically pure acetonitrile solution; flow rate: 190-; detection wavelength: gradient at 280 nm: the mass percentage concentration of the mobile phase B is as follows: 35-45% and gradient treatment time is 45 min;
and (3) purification process: washing acetonitrile above a chromatographic column, loading the acetonitrile, performing linear gradient elution on a sample solution with the loading amount being 99% of the content of the sample solution after the second-step purification and concentration, collecting a target peak, and freeze-drying the collected peptide solution; 6.0 g of refined peptide was obtained, purity: 99.84%, total yield: 29.18%, MS: 2057.209(M + 2H)+)，4111.991(M+H+)。

Claims (10)

1. A method of synthesizing a polypeptide-derived compound, comprising the steps of:
step 1: connecting Fmoc-Gly-OH to solid-phase synthetic resin to obtain Fmoc-Gly-resin;
step 2: Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, (Fmoc-Val-OH, Fmoc-Gly-Il-OH, Fmoc-Gly-Leu-OH, Fmoc-Leu, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Aib-Glu (OtBu) -OH and Y-His (Z) -OH to obtain a polypeptide-derived compound backbone peptide resin;
and step 3: removal of Lys26Sequentially coupling Fmoc-AEEA-AEEA-OH and octadienoic acid (OtBu) -Glu-OtBu to obtain polypeptide derivative compound peptide resin;
and 4, step 4: cracking the peptide resin of the polypeptide derivative compound and removing side chain protecting groups at the same time to obtain a crude product of the polypeptide derivative compound;
optionally, step 5: purifying by RP-HPLC to obtain pure polypeptide derivative compound;
wherein X, Y and Z are independently selected amino protecting groups;
the polypeptide derivative compound has a sequence of H-His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(PEG-PEG-γ-Glu-Octadecanoic acid)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-OH.
2. The method of claim 1, wherein the solid phase synthetic resin used in step 1 is selected from 2-Chlorotritylresin, Wang resin and Rink Acid resin, preferably the solid phase synthetic resin has a degree of substitution of 0.3 to 0.6mmol/g, more preferably 0.4 to 0.5 mmol/g.
3. The method of synthesis according to any one of claims 1-2, X is selected from Mmt, ivDDe, DDe, Mtt or Alloc; Y-His (Z) -OH is selected from Boc-His (Boc) -OH, Boc-His (Trt) -OH or Trt-His (Boc) -OH.
4. The method of synthesizing according to any one of claims 1 to 3, wherein Fmoc-Gly-OH and Fmoc-Arg (pbf) -OH, Fmoc-Gly-OH, Fmoc-Arg (pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (X) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -Ala-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser tBu) -OH, (Fmoc-Gly (tBu) -OH, Fmoc-Gly (tBu) -OH, Fmoc-Leu-Lys (X) -OH, Fmoc, Coupling agents of Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Aib-Glu (OtBu) -OH and Y-His (Z) -OH are DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HAHATU or PyBOP, and C is DIPEA or TMP.
5. The method according to any one of claims 1 to 4, wherein the method for synthesizing Octadecanoic acid (OtBu) -Glu-OtBu in step 3 comprises:
3-1) mono-tert-butyl octadecanedioate under the action of HONB and DIC to obtain octadienoic acid (OtBu) -ONB;
3-2) reacting H-Glu-OtBu with Octadecanoic acid (OtBu) -ONB in an aqueous alkaline solution to obtain Octadecanoic acid (OtBu) -Glu-OtBu, and purifying by crystallization.
6. The synthesis method according to any one of claims 1 to 5, wherein the crystallization solvent of Octadecanoic acid (OtBu) -Glu-OtBu in step 3 is methanol, acetonitrile, a combination of acetonitrile and water, a combination of tetrahydrofuran and water, preferably a combination of acetonitrile and water, and the crystallization temperature is 2 to 8 ℃.
7. The synthesis process according to any one of claims 1 to 6, wherein the cleavage time in step 4 is 2 to 4 hours, preferably 3 to 4 hours.
8. The synthesis process according to any one of claims 1 to 7, the cracking temperature in step 4 being 18 to 35 ℃, preferably 25 to 30 ℃.
9. The method according to any one of claims 1 to 8, wherein the cleavage reagent in step 4 is TFA, an aqueous solution of phenol and EDT, preferably TFA phenol H2O:EDT＝85-90:3-8:3-8:1-5，v/v。
10. The synthesis process according to any one of claims 1 to 9, the column conditions in step 5 being a two-step purification, comprising in particular the following steps:
the first step of purification conditions: a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica filler as a stationary phase,
the column diameter and length were: 10cm × 25cm. mobile phase: phase A: 0.1% phosphoric acid, adjusting pH to 8.5 with triethylamine; phase B: acetonitrile solution. Flow rate: 190-210 ml/min. Detection wavelength: 280nm. Gradient: the mass percentage concentration of the mobile phase B is as follows: 30-60%, eluting with linear gradient, collecting target peak, concentrating the target peak, and purifying;
and the second step of purification conditions: a chromatographic column: the chromatographic column using octadecylsilane chemically bonded silica as a stationary phase has the following diameter and length: 10cm × 25cm, mobile phase A: 0.01% phosphoric acid in water; phase B: chromatographically pure acetonitrile solution; flow rate: 190-; detection wavelength: gradient at 280 nm: the mass percentage concentration of the mobile phase B is as follows: 35 to 45 percent.
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