WO2020199461A1 - Method for synthesizing polypeptide-derived compound - Google Patents

Abstract

Disclosed is a method for synthesizing a polypeptide-derived compound, comprising the following steps: step 1: attaching Fmoc-Gly-OH to a resin to obtain a Fmoc-Gly resin; step 2: in accordance with a sequence of a peptide-derived compound, sequentially coupling amino acids by a Fmoc/tBu scheme to obtain a peptide resin with a main chain of the polypeptide-derived compound; step 3: removing a protecting group on Lys ²⁶ and sequentially coupling Fmoc-AEEA-AEEA-OH and Octadecanoicacid(OtBu)-Glu-OtBu to obtain a peptide resin with the polypeptide-derived compound; step 4: cleaving the peptide resin with the polypeptide-derived compound and simultaneously removing a side chain protecting group to obtain a crude peptide-derived compound; and selectively, step 5: purifying by RP-HPLC to obtain a pure peptide-derived compound. The method is simple and has high synthesis yield as well as high purity.

Description

A synthetic method of peptide derivative compound Technical field

The invention relates to the field of drug synthesis, in particular to a method for synthesizing polypeptide derivative compounds.

Background technique

The preparation prepared from the polypeptide derivative compound is subcutaneously injected once a week, and was approved for marketing by the US FDA on December 5, 2017. Since the fatty chain of the polypeptide derivative compound is longer, the hydrophobicity is increased, and the hydrophilicity is greatly enhanced after the short-chain PEG modification. After PEG modification, it can not only bind tightly to albumin, mask the DPP-4 enzymatic hydrolysis site, but also reduce renal excretion, prolong the biological half-life, and achieve the effect of long circulation.

The chemical name of the above peptide derivative compound is N ^6,26 -{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 glucagon-like peptide 1(7-37), molecular formula is C ₁₈₇ H ₂₉₁ N ₄₅ O ₅₉ , molecular weight is 4113.5775, The sequence is H-His ⁷ -Aib ⁸ -Glu ⁹ -Gly ¹⁰ -Thr ¹¹ -Phe ¹² -Thr ¹³ -Ser ¹⁴ -Asp ¹⁵ -Val ¹⁶ -Ser ¹⁷ -Ser ¹⁸ -Tyr ¹⁹ -Leu ²⁰ -Glu ²¹ -Gly ²² -G ln ²³ -Ala ²⁴ -Ala ²⁵ -Lys ²⁶ (PEG-PEG-γ-Glu-Octadecanoic acid)-Glu ²⁷ -Phe ²⁸ -Ile ²⁹ -Ala ³⁰ -Trp ³¹ -Leu ³² -Val ³³ -Arg ³⁴ -Gly ³⁵ -Arg ³⁶ -Gly ³⁷ -OH.

CN101133082A provides two methods of solid-phase gradual synthesis and first solid-phase synthesis of the backbone and then liquid-phase modification to obtain polypeptide derivative compounds. The first method is prone to produce a large amount of default impurities and it is difficult to obtain high-quality products. The second method requires The main chain is purified by RP-HPLC first, and the modification reaction is complicated, the cost is high, and the yield is low. CN108059666B uses a large number of short peptide fragments for solid-phase synthesis. These fragments have not been commercialized, and the cost of customization is high, which is not suitable for large-scale production.

Summary of the invention

The present invention adopts Fmoc/tBu solid-phase synthesis method, and uses Fmoc-AEEA-AEEA-OH, Octadecanoic acid(OtBu)-Glu-OtBu, Fmoc-Gln(Trt)-Ala-OH and Fmoc-Aib in some special positions -Glu(OtBu)-OH and other fully protected dipeptide fragments, which not only specifically solve the main default impurities in the product, but also reduce production costs, obtain high-yield and high-quality products, and are suitable for large-scale production.

One aspect of the present invention provides a method for synthesizing a polypeptide derivative compound, which includes the following steps:

Step 1: Connect Fmoc-Gly-OH to the solid phase synthetic resin to obtain Fmoc-Gly-resin;

Step 2: According to the sequence of the peptide derivative compound, Fmoc-Arg ³⁶ (pbf)-OH, Fmoc-Gly ³⁵ -OH, Fmoc-Arg ³⁴ (pbf)-OH, Fmoc-Val ³³ -OH are sequentially coupled using Fmoc/tBu strategy , Fmoc-Leu ³² -OH, Fmoc-Trp ³¹ (Boc)-OH, Fmoc-Ala ³⁰ -OH, Fmoc-Ile ²⁹ -OH, Fmoc-Phe ²⁸ -OH, Fmoc-Glu ²⁷ (OtBu)-OH, Fmoc -Lys ²⁶ (X)-OH, Fmoc-Ala ²⁵ -OH, Fmoc-Gln ²³ (Trt)-Ala ²⁴ -OH, Fmoc-Gly ²² -OH, Fmoc-Glu ²¹ (OtBu)-OH, Fmoc-Leu ²⁰ -OH, Fmoc-Tyr ¹⁹ (tBu)-OH, Fmoc-Ser ¹⁸ (tBu)-OH, Fmoc-Ser ¹⁷ (tBu)-OH, Fmoc-Val ¹⁶ -OH, Fmoc-Asp ¹⁵ (OtBu)-OH, Fmoc-Ser ¹⁴ (tBu)-OH, Fmoc-Thr ¹³ (tBu)-OH, Fmoc-Phe ¹² -OH, Fmoc-Thr ¹¹ (tBu)-OH, Fmoc-Gly ¹⁰ -OH, Fmoc-Aib ⁸ -Glu ⁹ (OtBu)-OH and Y-His ⁷ (Z)-OH to obtain the peptide resin of the peptide derivative compound backbone;

Step 3: Remove the protective group on Lys ²⁶ , and couple Fmoc-AEEA-AEEA-OH and Octadecanoic acid(OtBu)-Glu-OtBu in sequence to obtain peptide resin of peptide derivative compound;

Step 4: Cleavage the peptide resin of the polypeptide derivative compound and simultaneously remove the side chain protecting groups to obtain the crude polypeptide derivative compound;

Optionally, step 5: Purify by RP-HPLC to obtain a pure polypeptide derivative compound.

In the technical scheme of the present invention, the polypeptide derivative compound has a sequence of H-His ⁷ -Aib ⁸ -Glu ⁹ -Gly ¹⁰ -Thr ¹¹ -Phe ¹² -Thr ¹³ -Ser ¹⁴ -Asp ¹⁵ -Val ¹⁶ -Ser ¹⁷ -Ser ¹⁸ -Tyr ¹⁹ -Leu ²⁰ -Glu ²¹ -Gly ²² -G ln ²³ -Ala ²⁴ -Ala ²⁵ -Lys ²⁶ (PEG-PEG-γ-Glu-Octadecanoic acid)-Glu ²⁷ -Phe ²⁸ -Ile ²⁹ -Ala ³⁰ -Trp ³¹ -Leu ³² -Val ³³ -Arg ³⁴ -Gly ³⁵ -Arg ³⁶ -Gly ³⁷ -OH compound.

The solid phase synthetic resin used in step 1 is 2-Chlorotrityl resin, Wang resin or Rink Acid resin;

The resin substitution degree used in step 1 is 0.3-0.6 mmol/g, preferably 0.4-0.5 mmol/g;

The degree of substitution of Fmoc-Gly-resin in step 1 is 0.3-0.5 mmol/g, preferably 0.3-0.4 mmol/g;

Connect Fmoc-Gly-OH and Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc in step 1 and step 2. -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-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc- The coupling agent of Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Aib-Glu(OtBu)-OH and Y-His(Z)-OH is DIC+A or B+A+C, where A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.

In step 1, X of Fmoc-Lys(X)-OH is an ε amino protecting group, which is 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 step 3 is:

3-1) Octadecanoic acid (OtBu)-ONB is obtained under the action of HONB and DIC;

3-2) H-Glu-OtBu and Octadecanoic acid (OtBu)-ONB are reacted in an alkaline aqueous solution to obtain Octadecanoic acid (OtBu)-Glu-OtBu, which is purified by crystallization.

In step 3, the crystallization solvent of Octadecanoic acid (OtBu)-Glu-OtBu 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 2-8°C;

The cleavage time of the peptide resin in step 4 is 2 to 4 hours, preferably 3 to 4 hours;

The pyrolysis temperature in step 4 is 18 to 35°C, preferably 25 to 30°C.

The lysis reagent in step 4 is an aqueous solution of TFA, phenol and EDT, preferably TFA:phenol:H ₂ O:EDT=85-90:3-8:3-8:1-5, v/v.

The column conditions in step 5 are two-step purification, which specifically includes the following steps:

Purification conditions for the first step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica filler as the stationary phase,

The diameter and length of the column are: 10cm×25cm. Mobile phase: Phase A: 0.1% phosphoric acid, adjusted to pH 8.5 with triethylamine; Phase B: Acetonitrile solution. Flow rate: 190-210ml/min. Detection wavelength: 280nm. Gradient: The mass percentage concentration of mobile phase B: 30-60%, linear gradient elution, collect the target peak, and then concentrate the target peak for the second step of purification;

Purification conditions for the second step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica gel as the stationary phase, the diameter and length of the column: 10cm×25cm, mobile phase A: 0.01% phosphoric acid aqueous solution; Phase B: chromatographic purity Acetonitrile solution; flow rate: 190-210ml/min; detection wavelength: 280nm. Gradient: mass percentage concentration of mobile phase B: 35-45%.

Beneficial effect

1. Selective use of dipeptide fragments for key issues, avoiding the default impurities Des-Glu ³ , Des-Aib ² , Des-AEEA, Des-Gln ²³ and Des-octadecanedioic acid during the synthesis of peptide derivative compounds , Not only can obtain high-yield and high-quality products, but also ensure the commercialization of raw materials and reduce production costs, which is suitable for large-scale production;

2. Octadecanoic acid (OtBu)-Glu-OtBu crystallization process ensures the high quality and supply of raw materials, which is conducive to commercial production.

Abbreviations and English meanings

Abbreviations and English meaning Boc Tert-Butoxycarbonyl TFA Trifluoroacetate EDT 1,2-ethanedithiol tBu Tert-butyl Trt Trityl

HOBt 1-Hydroxybenzotriazole HONb N-hydroxy-5-norbornene-2,3-dicarboximide DIC N,N'-Diisopropylcarbodiimide Na ₂ CO ₃ Sodium carbonate Na ₂ SO ₄ Sodium sulfate THF Tetrahydrofuran DCM Dichloromethane EA Ethyl acetate DMF N,N-Dimethylformamide 20% DBLK 20% piperidine/N,N-dimethylformamide MeOH Methanol Aib 2-aminoisobutyric acid Octadecanoic acid Octadecanedioic acid Octadecanoic acid(OtBu) Octadecanedioic acid mono-tert-butyl ester .

Description of the drawings

Figure 1 shows the mass spectrum of Octadecanoic acid (OtBu)-ONB.

Figure 2 shows the HPLC spectrum of Octadecanoic acid (OtBu)-ONB.

Figure 3 shows the mass spectrum of Octadecanoic acid (OtBu)-Glu-OtBu.

Figure 4 shows the HPLC spectrum of Octadecanoic acid (OtBu)-Glu-OtBu.

Figure 5 is a crude quality profile of the polypeptide derivative compound of Example 11.

Figure 6 is the HPLC spectrum of the crude polypeptide derivative compound of Example 11.

Fig. 7 is a pure quality spectrum of the polypeptide derivative compound of Example 14.

Figure 8 is the HPLC spectrum of the pure polypeptide derivative compound of Example 14.

detailed description

Example 1: Synthesis of Octadecanoic acid (OtBu)-ONB

Dissolve mono-tert-butyl octadecanedioic acid (37.1g, 100mmol) and HONB (2.1.5g, 120mmol) in 400ml of tetrahydrofuran, add DIC (23.2ml, 150mmol) under an ice-salt bath, stir and react for 2h, then remove In an ice salt bath, the reaction was stirred at room temperature for 4h. Filtration, the filtrate was concentrated in vacuo, the residue was crystallized with acetonitrile to obtain 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

Dissolve H-Glu-OtBu (18.17g, 89.4mmol) and Na ₂ CO ₃ (37.9g, 357.6mmol) in 300ml H ₂ O, add Octadecanoic acid(OtBu)-ONB (47.486g, 89.4mmol) in THF (300ml) solution. After the addition is complete, continue the reaction for 16 hours. The THF is concentrated in vacuo, 300ml of water is added to the aqueous phase, and EA is extracted twice, the organic phases are combined, washed with 1M HCl 3 times, and washed 3 times with water , Washed with saturated brine twice, dried with anhydrous Na ₂ SO ₄ , concentrated in vacuo, and the residue was crystallized with acetonitrile and water at a crystallization temperature of 5 degrees Celsius to obtain 43.686 white solid, yield: 90.32%, purity: 98.55%, MS: 556.12 (M+H ⁺ ), 578.31 (M+Na ⁺ ).

Example 3: Synthesis of Octadecanoic acid(OtBu)-Glu-OtBu

Dissolve H-Glu-OtBu (2.03g, 10mmol) and Na ₂ CO ₃ (4.24g, 40mmol) in 40ml H ₂ O, add Octadecanoic acid(OtBu)-ONB (5.31g, 10mmol) slowly while stirring at room temperature THF (40ml) solution, after the dropwise addition, continue to react for 16h, concentrate to remove THF, add 40ml of water to the aqueous phase, extract 2 times with EA, combine the organic phases, wash 3 times with 1M HCl, wash 3 times with water, saturated salt Washed twice with water, dried with anhydrous Na ₂ SO ₄ and concentrated in vacuo. The residue was crystallized with methanol at a crystallization temperature of 5 degrees Celsius to obtain 2.54 g of white solid. Yield: 45.8%, purity: 95.32%, MS: 556.12 (M+ H+), 578.31 (M+Na+).

Example 4: Synthesis of Octadecanoic acid(OtBu)-Glu-OtBu

Dissolve H-Glu-OtBu (2.03g, 10mmol) and Na ₂ CO ₃ (4.24g, 40mmol) in 40ml H ₂ O, add Octadecanoic acid(OtBu)-ONB (5.31g, 10mmol) slowly while stirring at room temperature THF (40ml) solution, after the dropwise addition, continue to react for 16h, concentrate to remove THF, add 40ml of water to the aqueous phase, extract 2 times with EA, combine the organic phases, wash 3 times with 1M HCl, wash 3 times with water, saturated salt Washed twice with water, dried with anhydrous Na ₂ SO ₄ and concentrated in vacuo. The residue was crystallized with acetonitrile at a crystallization temperature of 5 degrees Celsius to obtain 2.86 g of a white solid. Yield: 51.5%, purity: 96.14%, MS: 556.12 (M+ H+), 578.31 (M+Na+).

Example 5: Synthesis of Octadecanoic acid(OtBu)-Glu-OtBu

Dissolve H-Glu-OtBu (2.03g, 10mmol) and Na ₂ CO ₃ (4.24g, 40mmol) in 40ml H ₂ O, add Octadecanoic acid(OtBu)-ONB (5.31g, 10mmol) slowly while stirring at room temperature THF (40ml) solution, after the dropwise addition, continue to react for 16h, concentrate to remove THF, add 40ml of water to the aqueous phase, extract 2 times with EA, combine the organic phases, wash 3 times with 1M HCl, wash 3 times with water, saturated salt Washed twice with water, dried with anhydrous Na ₂ SO ₄ and concentrated in vacuo. The residue was crystallized with tetrahydrofuran and water at a crystallization temperature of 5 degrees Celsius to obtain 3.24 g of white solid. 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 twice with DMF, swelled with DMF for 30min, washed twice with DMF, and drained.

Dissolve Fmoc-Gly-OH (8.92g, 30mmol) and HOBt (4.86g, 36mmol) in 50ml DMF, add DIC (4.54g, 36mmol) under ice bath conditions, stir and activate for 5min, and put the activation solution into the above Solid phase reaction column of Wang resin, add DMAP (0.37g, 0.3mmol) under nitrogen stirring, react at room temperature for 2h, drain the reaction solution, wash 6 times with DMF, wash 3 times with DCM, and mix with Ac ₂ O/pyridine in equal molar ratio The solution was sealed for 6h, drained, washed with DCM 6 times, methanol was contracted 3 times (5min+5min+10min), dried in vacuum, and a small sample was taken to measure the degree of substitution Sub=0.371mmol/g.

Example 7: Synthesis of Fmoc-Gly-CTC resin

0.5mmol/g 2-CTC resin (20g, 10mmol) was added to the solid phase reaction column, DMF washed twice, DMF swelled for 30 minutes, DMF washed twice, and drained.

Dissolve Fmoc-Gly-OH (8.92g, 30mmol) in 50ml DMF, add DIPEA (7.75g, 60mmol) under ice bath conditions, stir and activate for 5 minutes, and put the activation solution into the above solid phase reaction with 2-CTC resin Column, room temperature nitrogen stirred and reacted for 2h, MeOH (6.41g, 0.2mol) was added to the reaction solution, and the reaction was continued to stir for 20min with nitrogen. 5min+10min), vacuum drying, take a small sample to measure the degree of substitution Sub=0.412mmol/g.

Example 8: Synthesis of peptide resin of peptide derivative compound

Add 0.371mmol/g Fmoc-Gly-Wang resin (13.48g, 5mmol) to the solid phase reaction column, wash 2 times with DMF, swell with DMF 30min, wash 2 times with DMF, and deprotect 2 times with 20% DBLK (10min+15min) , DMF wash 6 times, drain and set aside.

Dissolve Fmoc-Arg(pbf)-OH (9.73g, 15mmol) and HOBt (2.43g, 18mmol) in DMF (25ml), add DIC (2.27g, 18mmol) under ice-bath conditions, activate for 5min, change the activation solution It was added to the solid phase reaction column, stirred with nitrogen at room temperature for 2 hours, the reaction solution was removed, DMF washed 3 times, 20% DBLK deprotected 2 times (5min+7min), DMF washed 6 times.

According to the sequence of the peptide derivative compound, 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-Ser(tBu)-OH, 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 Boc-His(trt)-OH to obtain the peptide resin of the main chain of the peptide derivative compound.

Wash the main chain peptide resin of the above polypeptide derivative compound with DCM 6 times, 2%TFA/3%TIS/DCM to remove Lys ²⁶ Mmt protection (10min×12), DCM wash 6 times, DMF wash 3 times, and then couple Fmoc -AEEA-AEEA-OH and Octadecanoic acid (OtBu)-Glu-OtBu, DMF wash 6 times, DCM wash 6 times, methanol shrinkage 3 times (5min+5min+10min), vacuum drying to obtain 38.61g peptide derivative of peptide resin.

Example 9: Synthesis of peptide resin of peptide derivative compound

Add 0.371mmol/g Fmoc-Gly-Wang resin (13.48g, 5mmol) to the solid phase reaction column, wash 2 times with DMF, swell with DMF 30min, wash 2 times with DMF, and deprotect 2 times with 20% DBLK (10min+15min) , DMF wash 6 times, drain and set aside.

Dissolve Fmoc-Arg(pbf)-OH (9.73g, 15mmol) and HOBt (2.43g, 18mmol) in DMF (25ml), add DIC (2.27g, 18mmol) under ice-bath conditions, activate for 5min, change the activation solution It was added to the solid phase reaction column, stirred with nitrogen at room temperature for 2 hours, the reaction solution was removed, DMF washed 3 times, 20% DBLK deprotected 2 times (5min+7min), DMF washed 6 times.

According to the sequence of the peptide derivative compound, 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-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-Ser(tBu) -OH, 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-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-His(trt)-OH to obtain peptide resins for the main chain of peptide derivatives.

Wash the main chain peptide resin of the above polypeptide derivative compound with DCM 6 times, 2%TFA/3%TIS/DCM to remove Lys26 Mmt protection (10min×12), wash 6 times with DCM, wash 3 times with DMF, and then couple Fmoc- AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu and Octadecanoic acid-OtBu, DMF washing 6 times, DCM washing 6 times, methanol shrinkage 3 times (5min+5min+10min), vacuum drying to obtain 38.51g peptide derivative Compound peptide resin.

Example 10: Synthesis of peptide resin of peptide derivative compound

Add 0.412mmol/g Fmoc-Gly-CTC resin (12.14g, 5mmol) to the solid phase reaction column, wash 2 times with DMF, swell with DMF 30min, wash 2 times with DMF, and deprotect 2 times with 20% DBLK (10min+15min) , DMF wash 6 times, drain and set aside.

Dissolve Fmoc-Arg(pbf)-OH (9.73g, 15mmol), PyBOP (7.81g, 15mmol) and HOBt (2.43g, 18mmol) in DMF (25ml), add DIPEA (2.58g, 20mmol) under ice bath conditions ), activate for 5min, add the activation solution to the above solid phase reaction column, bubbling nitrogen at room temperature and stirring for 2h, extract the reaction solution, DMF wash 3 times, 20% DBLK deprotection 2 times (5min+7min), DMF wash 6 Times.

According to the sequence of the peptide derivative compound, 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-Ser(tBu)-OH, 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 Boc-His(Boc)-OH to obtain the peptide resin of the peptide derivative compound backbone.

Wash the main chain peptide resin of the above polypeptide derivative compound with DCM 6 times, 2%TFA/3%TIS/DCM to remove Lys26 Mmt protection (10min×12), wash 6 times with DCM, wash 3 times with DMF, and then couple Fmoc- AEEA-AEEA-OH and Octadecanoic acid (OtBu)-Glu-OtBu were washed with DMF 6 times, DCM washed 6 times, methanol contracted 3 times (5min+5min+10min), and vacuum dried to obtain 34.59g of peptide resin derived from peptides.

Example 11: Cleavage of peptide resins of peptide derivatives

The 38.61g polypeptide derivative compound peptide resin of Example 8 was added to a 500ml round-bottomed flask, and 400ml of frozen lysis solution (TFA:Phenol:H ₂ O:EDT=87.5:5:5:2.5, v/ v) Stir the reaction at 18℃ for 2～4h, filter, settle the filtrate with 4L frozen anhydrous ether, collect the solid by centrifugation, wash 3 times with anhydrous ether, and dry in vacuum to obtain 19.58g crude peptide derivative compound, yield: 95.2% , Purity: 72.66%, MS: 4111.846 (M+H ⁺ ).

Example 12: Cleavage of peptide resins of peptide derivatives

Add 38.51g of the polypeptide derivative compound peptide resin of Example 9 to a 500ml round bottom flask, and add 400ml of frozen lysis solution (TFA:Phenol:H ₂ O:EDT=87.5:5:5:2.5, v/ v) Stir the reaction at 35℃ for 2～4h, filter, settle the filtrate with 4L ice-free anhydrous ether, collect the solid by centrifugation, wash 3 times with anhydrous ether, and dry in vacuum to obtain 19.58g crude peptide derivative compound, yield: 93.2% , Purity: 43.37%, MS: 4111.846 (M+H ⁺ ).

Example 13: Cleavage of peptide resins of peptide derivatives

Add 34.59g of the polypeptide derivative compound peptide resin of Example 10 to a 500ml round bottom flask, add 350ml of frozen lysis solution (TFA:DTT:H ₂ O=90:5:5, v/v) for 2 to 4 hours, 30 The reaction was stirred at ℃ for 2～4h, filtered, the filtrate was settled with 4L of ice-free anhydrous ether, the solid was collected by centrifugation, washed with anhydrous ether 3 times, and vacuum dried to obtain 17.69g of crude peptide derivative compound, yield: 86.0%, purity: 70.79 %, MS: 4111.846 (M+H ⁺ ).

Example 14: Preparation of pure polypeptide derivative compounds by RP-HPLC

Take 19.58 g of the peptide derivative compound, dissolve it in 400 ml of water, and aid the dissolution with ammonia water, pH 8.0-9.0, the sample is filtered through a 0.45 μm membrane, and then purified by reverse phase.

Purification conditions for the first step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica filler as the stationary phase,

The diameter and length of the column are: 10cm×25cm. Mobile phase: Phase A: 0.1% phosphoric acid, adjusted to pH 8.5 with triethylamine; Phase B: Acetonitrile solution. Flow rate: 190-210ml/min. Detection wavelength: 280nm. Gradient: The mass percentage concentration of mobile phase B: 30-60%, and the gradient processing time is 60 min. The injection volume is 19.58g;

Purification process: Rinse the acetonitrile above the chromatographic column and load the sample. The sample load is the sample solution after dissolution and filtration. Linear gradient elution, collect the target peak, the purity is about 90%, put the collected peptide solution in the collection bottle for use;

Purification conditions for the second step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica gel as the stationary phase, the diameter and length of the column: 10cm×25cm, mobile phase A: 0.01% phosphoric acid aqueous solution; Phase B: chromatographic purity Acetonitrile solution; Flow rate: 190-210ml/min; Detection wavelength: 280nm. Gradient: Mass percentage concentration of mobile phase B: 35-45%, gradient processing time 45-60min; The injection volume is the content after the first purification and concentration 99% sample solution;

Purification process: Rinse the acetonitrile above the chromatographic column and load the sample. The sample amount is the 90% sample solution after purification and concentration in the first step. Linear gradient elution is used to collect the target peak with a purity of about 99% and it will be collected. Place the peptide solution in the collection bottle for use;

Desalination: Chromatographic column: Column with octadecylsilane bonded silica gel as stationary phase, column diameter and length: 10cm×25cm, mobile phase A: aqueous solution; phase B: chromatographic pure acetonitrile solution; flow rate: 190-210ml /min; Detection wavelength: 280nm. Gradient: Mass percentage concentration of mobile phase B: 35-45%, gradient processing time 45min;

Purification process: Rinse the acetonitrile above the chromatographic column and load the sample. The sample amount is 99% of the sample solution after purification and concentration in the second step, linear gradient elution, collect the target peak, and freeze-dry the collected peptide solution; Obtain 6.0 g of refined peptide, purity: 99.84%, total yield: 29.18%, MS: 2057.209 (M+2H ⁺ ), 4111.991 (M+H ⁺ ).

Claims (10)

A method for synthesizing polypeptide derivative compounds, which includes the following steps: Step 1: Connect Fmoc-Gly-OH to the solid phase synthetic resin to obtain Fmoc-Gly-resin; Step 2: According to the sequence of the peptide derivative compound, Fmoc-Arg(pbf)-OH, Fmoc-Gly-OH, Fmoc-Arg(pbf)-OH, Fmoc-Val-OH, Fmoc-Leu are sequentially coupled with Fmoc/tBu strategy -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-Ser(tBu)-OH, 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 to obtain peptide resins of peptide derivatives; Step 3: Remove the protective group on Lys ²⁶ , and couple Fmoc-AEEA-AEEA-OH and Octadecanoic acid(OtBu)-Glu-OtBu in sequence to obtain peptide resin of peptide derivative compound; Step 4: Cleavage the peptide resin of the polypeptide derivative compound and simultaneously remove the side chain protecting groups to obtain the crude polypeptide derivative compound; Optionally, Step 5: Purify by RP-HPLC to obtain a pure product of the polypeptide derivative compound; Wherein X, Y and Z are independently selected amino protecting groups; The polypeptide derivative compound has a sequence of H-His ⁷ -Aib ⁸ -Glu ⁹ -Gly ¹⁰ -Thr ¹¹ -Phe ¹² -Thr ¹³ -Ser ¹⁴ -Asp ¹⁵ -Val ¹⁶ -Ser ¹⁷ -Ser ¹⁸ -Tyr ^19- Leu ²⁰ -Glu ²¹ -Gly ²² -Gln ²³ -Ala ²⁴ -Ala ²⁵ -Lys ²⁶ (PEG-PEG-γ-Glu-Octadecanoic acid)-Glu ²⁷ -Phe ²⁸ -Ile ²⁹ -Ala ³⁰ -Trp ³¹ -Leu ³² -Val ³³ -Arg ³⁴ -Gly ³⁵ -Arg ³⁶ -Gly ³⁷ -OH compound. The synthesis method according to claim 1, wherein the solid phase synthetic resin used in step 1 is selected from 2-Chlorotrityl resin, Wang resin or Rink Acid resin. Preferably, the solid phase synthetic resin substitution degree is 0.3-0.6 mmol/g, More preferably, it is 0.4 to 0.5 mmol/g. The synthesis method 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. The synthesis method 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-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc- Coupling of Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Aib-Glu(OtBu)-OH and Y-His(Z)-OH The agent is DIC+A or B+A+C, where A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP. The synthesis method according to any one of claims 1-4, the synthesis method of Octadecanoic acid (OtBu)-Glu-OtBu in step 3 is: 3-1) Octadecanoic acid (OtBu)-ONB is obtained under the action of HONB and DIC; 3-2) H-Glu-OtBu and Octadecanoic acid (OtBu)-ONB are reacted in an alkaline aqueous solution to obtain Octadecanoic acid (OtBu)-Glu-OtBu, which is purified by crystallization. The synthesis method according to any one of claims 1 to 5, in step 3, the crystallization solvent of Octadecanoic acid (OtBu)-Glu-OtBu is methanol, acetonitrile, a combination of acetonitrile and water, a combination of tetrahydrofuran and water, preferably acetonitrile When combined with water, the crystallization temperature is 2～8℃. According to the synthesis method of any one of claims 1-6, the lysis time in step 4 is 2 to 4 hours, preferably 3 to 4 hours. The synthesis method according to any one of claims 1-7, the cracking temperature in step 4 is 18-35°C, preferably 25-30°C. The synthesis method according to any one of claims 1-8, the lysis reagent in step 4 is an aqueous solution of TFA, phenol and EDT, preferably TFA:phenol:H ₂ O:EDT=85-90:3-8: 3-8:1-5, v/v. According to the synthesis method of any one of claims 1-9, the chromatographic column conditions in step 5 are two-step purification, which specifically includes the following steps: Purification conditions for the first step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica filler as the stationary phase, The diameter and length of the column are: 10cm×25cm. Mobile phase: Phase A: 0.1% phosphoric acid, pH 8.5 adjusted with triethylamine; Phase B: Acetonitrile solution, flow rate: 190-210ml/min, detection wavelength: 280nm, gradient: The mass percentage concentration of mobile phase B: 30-60%, linear gradient elution, collect the target peak, the target peak is concentrated and then purified in the second step; Purification conditions for the second step: Chromatographic column: Chromatographic column with octadecylsilane bonded silica gel as the stationary phase, the diameter and length of the column: 10cm×25cm, mobile phase A: 0.01% phosphoric acid aqueous solution; Phase B: chromatographic purity Acetonitrile solution; flow rate: 190-210ml/min; detection wavelength: 280nm. Gradient: mass percentage concentration of mobile phase B: 35-45%.

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