CN114014925B - Somaltulipide main peptide chain and preparation method thereof - Google Patents

Somaltulipide main peptide chain and preparation method thereof Download PDF

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CN114014925B
CN114014925B CN202111640670.6A CN202111640670A CN114014925B CN 114014925 B CN114014925 B CN 114014925B CN 202111640670 A CN202111640670 A CN 202111640670A CN 114014925 B CN114014925 B CN 114014925B
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silica gel
polypeptide
fmoc
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peptide chain
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CN114014925A (en
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刘慧敏
张启鹏
魏祝宇
潘海良
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Zhejiang Paitide Biological Co ltd Nanjing Branch
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Zhejiang Pai Peptide Biology Co ltd Nanjing Branch
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    • C—CHEMISTRY; METALLURGY
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Abstract

The invention provides a somaglutide main peptide chain and a preparation method thereof, belonging to the technical field of polypeptide synthesis and specifically comprising the following steps: the self-made polypeptide condensing agent and organic base are used as a condensation coupling system, and amino acid is subjected to condensation coupling according to the polypeptide sequence of the main peptide chain of the soxhlet peptide, so that the coupling efficiency is improved, the generation of impurities is reduced, the purity and yield of crude peptide are improved, the synthesis cost is reduced, the purification difficulty is reduced, and the purification yield is improved; in the purification process of the somaglutide main peptide chain reversed-phase high performance liquid chromatography, 1-pyridine-2-yl-1, 4-butanediamine and the silica gel chromatographic filler modified by epoxy siloxane are used as fillers, which is beneficial to improving the purification efficiency.

Description

Somaltulipide main peptide chain and preparation method thereof
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a somaglutide main peptide chain and a preparation method thereof.
Background
The somaglutide is a new long-acting GLP-1 receptor agonist, and clinical application proves that the somaglutide can effectively control blood sugar by combining different oral hypoglycemic drugs and has obvious weight-losing efficacy. Structurally, the somaglutide is obtained by substituting Aib for Ala at position 8, Arg for Lys at position 34, and attaching short-chain PEG, glutamic acid, octadecanoic acid fatty chain and other side chains to Lys at position 26 on a human glucagon-like peptide-1 chain, so that the somaglutide has greatly improved hydrophilicity and enhanced binding force with albumin. After being modified by PEG, the PEG can be tightly combined with albumin to cover DPP-4 enzyme hydrolysis sites, and is also helpful to reduce renal excretion, prolong the biological half-life and achieve the effect of long circulation.
The most effective synthesis method of the polypeptide drug is a solid-phase synthesis method at present, namely, starting from the carbon end of the polypeptide, connecting the first amino acid to the resin, then removing the Fmoc protecting group at the N end, then coupling the next amino acid, and cracking the polypeptide from the resin until the coupling of the last N-end amino acid is completed, and simultaneously removing all side chain protecting groups to obtain a complete polypeptide. In the solid-phase synthesis process of the somaglutide, a large amount of hydrophobic amino acids exist between the His at the 7 th position and the Lys at the 26 th position, so that hydrogen bonds between peptide chains are stable, serious beta folding is generated, acting force between the peptide chains is enhanced, resin polycondensation is caused, the difficulty of amino acid coupling is increased, and the yield and the purity of the somaglutide are greatly reduced. The polypeptide condensing agent is an important factor influencing the solid-phase synthesis yield and purity of the somaltulipide, and the currently commonly used condensing agents mainly comprise three types, namely carbodiimide, urea positive ion type and phosphine positive ion type. The carbodiimide condensing agent has low condensation activity, and the generated by-product is not easy to remove, which can influence the purity of the synthesized polypeptide; the condensation agent of urea cation type and phosphine cation type has high condensation activity, but the recovery rate is low, and the condensation agent can not be recovered for continuous utilization.
Therefore, it is necessary to develop a new polypeptide condensing agent in order to obtain a somaglutide main peptide chain having high purity and high yield and a preparation method thereof.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a soxhlet peptide main peptide chain, which adopts a polypeptide condensation coupling system to carry out coupling condensation of amino acid, is beneficial to improving coupling efficiency, reducing the generation of impurities, improving the purity and yield of crude soxhlet peptide of the soxhlet peptide main peptide chain, reducing synthesis cost, reducing purification difficulty and greatly improving purification efficiency.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention discloses a preparation method of a soxhlet peptide main peptide chain, which comprises the following steps:
activating a resin solid phase carrier, and coupling Fmoc-Gly-OH to the resin solid phase carrier by adopting a condensation coupling system to obtain Fmoc-Gly-resin;
step two, performing amino acid activation coupling by adopting a condensation coupling system from the C end to the N end according to the polypeptide sequence of the Somallu peptide main peptide chain by a solid-phase synthesis method, and sequentially coupling Arg, Gly, Arg, Val, Leu, Trp, Ala, Ile, Phe and Glu to obtain first polypeptide resin;
step three, adopting a condensation coupling system to carry out activation coupling of amino acid, and coupling Lys to obtain second polypeptide resin;
step four, adopting a condensation coupling system to carry out activation coupling on amino acid, sequentially coupling Ala, Ala, Gln, Gly, Glu, Leu, Tyr, Ser, Val, Asp, Ser, Thr, Phe, Thr, Gly, Glu, Aib and His to obtain the somalufide main peptide chain resin;
step five, cutting by using a cracking agent, and removing resin and side chain protecting groups to obtain crude somaglutide main peptide chain peptide;
and step six, obtaining the somaglutide main peptide chain by reversed phase high performance liquid chromatography purification, anion exchange chromatography salt conversion and freeze drying.
Preferably, in the first to fourth steps, the condensation coupling system is a mixture of a polypeptide condensation agent and an organic base, the mixing molar ratio is 1: 1-1.6, and the organic base is selected from DIEA or TRIS.
Preferably, the polypeptide condensing agent has a structural formula shown in formula (1):
Figure 917553DEST_PATH_IMAGE001
(1)。
the invention provides a polypeptide condensing agent with a structural formula shown in the formula, wherein a benzotriazine structure exists in the structure, and a piperidine group and other groups are matched, so that the polypeptide condensing agent has higher condensation activity at normal temperature, and can reduce side reactions in the polypeptide condensation process, thereby reducing racemization byproducts and improving the purity and yield of crude peptides; the polypeptide condensing agent also contains hydrophobic structures such as ether bonds, aliphatic hydrocarbon and the like, has low water solubility, is convenient to separate from a solution, is beneficial to improving the subsequent purification efficiency, and can be recycled.
The polypeptide condensing agent is prepared by the following method: 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine reacts with epoxypropane to obtain a product A, and then the product A reacts with 2-chloro-1- (2-vinyl-1-piperidyl) ethanone to obtain the polypeptide condensing agent.
In a specific embodiment of the present invention, the polypeptide condensing agent is prepared by the following method:
i) adding 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine into THF, stirring and dissolving, adding epoxypropane and tetramethylammonium hydroxide, stirring and reacting at 50-80 ℃ for 2-5 h, and washing a product with methanol to obtain a product A;
ii) dissolving 2-chloro-1- (2-vinyl-1-piperidyl) ethanone in DCM, adding the product A, adding triethylamine under a stirring state, continuing stirring for reacting for 3-5 h after dropwise adding, filtering, washing a filter cake with methanol, collecting the filter cake, and drying to obtain the polypeptide condensing agent.
Preferably, in the step i), the molar ratio of the 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine to the propylene oxide is 1: 1.1-1.5.
Preferably, in the step ii), the molar ratio of the product A to the 2-chloro-1- (2-vinyl-1-piperidyl) ethanone is 1: 1.1-1.3.
Preferably, in the step ii), the addition amount of triethylamine is 0.1-0.4 times of the molar mass of the 2-chloro-1- (2-vinyl-1-piperidyl) ethanone.
According to the method, 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine is used as a main active structure of the polypeptide condensing agent, and the polypeptide condensing agent shown in the formula (1) is prepared by sequentially reacting with epoxypropane and 2-chloro-1- (2-vinyl-1-piperidyl) ethanone through the steps.
Preferably, in the first to fourth steps, the molar ratio of the amino acid to the condensation coupling system is 1: 2.5-3.
Preferably, in the first step, the resin solid phase carrier is any one of Wang resin, 4-Methoxyytrityl-Cl resin and 2-Cl-trity-Cl resin.
Preferably, in the first step, the degree of substitution of Fmoc-Gly-resin is 0.2-0.45 mmol/L.
Preferably, in the first step, the Fmoc-Gly-resin is prepared by the following method:
the resin was fully swollen in DCM and the solvent was drained; adding Fmoc-Gly-OH into a DCM solution, stirring and dissolving, cooling to 0-10 ℃, adding a condensation coupling system, stirring and activating for 10-30 min, and then adding into resin, stirring and reacting for 4-6 h; pumping out the solvent, adding acetic anhydride, 4-aminopyridine and DCM, carrying out end capping for 1-2.5 h, alternately washing DCM and methanol, and carrying out vacuum drying to obtain Fmoc-Gly-resin; wherein the addition amount of Fmoc-Gly-OH is 0.9-1.4 times of the molar mass of the resin; the addition amount of the condensation coupling system is 2-3 times of the molar mass of the resin; the addition amount of the acetic anhydride is 0.5-0.8 time of the molar mass of the resin; the addition amount of the 4-aminopyridine is 0.4-0.8 time of the molar mass of the resin.
Preferably, in the second step, the coupled amino acids are: 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.
Preferably, in the third step, Lys is R1-Lys (Boc) -OMe.HCl as starting material, R1Selected from H or Fmoc.
Preferably, in the fourth step, the coupled amino acids are sequentially applied as follows: 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 (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、Fmoc-His(R2) -OH; the R is2Selected from Boc or Trt.
Preferably, in the fifth step, the cracking agent is prepared by mixing 90-93 wt% of trifluoroacetic acid, 2-3 wt% of thioanisole, 1-2 wt% of triisopropylsilane, 1-2 wt% of 1, 2-ethanedithiol and the balance of deionized water.
Preferably, in the sixth step, the purification conditions of the reversed-phase high performance liquid chromatography are as follows:
chromatographic packing: the modified silica gel chromatographic packing is filled with 20-100 mL of filler;
mobile phase: the phase A is a trifluoroacetic acid aqueous solution with the concentration of 0.05-0.5 vol%, and the phase B is acetonitrile;
detection wavelength: 210-230 nm;
flow rate: 50-100 mL/min;
elution conditions: mixed liquid of phase A and phase B, wherein the volume percentage of phase B is increased from 20% to 50% in 40 min.
As a preferred scheme, the modified silica gel chromatographic packing is prepared by reacting porous silica gel activated by hydrochloric acid solution with an epoxy silane coupling agent and then reacting with 1-pyridine-2-yl-1, 4-butanediamine.
As a preferred scheme, the preparation method of the modified silica gel chromatographic packing specifically comprises the following steps:
1) soaking and activating C18 silica gel for 2-5 h by using a hydrochloric acid aqueous solution, then washing the activated C18 silica gel to be neutral by using deionized water, and finally washing the activated C18 silica gel by using acetone and drying the acetone to obtain activated silica gel; exposing silicon hydroxyl groups on the surface;
2) dissolving activated silica gel in toluene, adding epoxy siloxane, reacting overnight at 80-100 ℃, cleaning with hot water, and drying to obtain epoxidized modified silica gel;
3) and dissolving the epoxidized modified silica gel in toluene, adding 1-pyridine-2-yl-1, 4-butanediamine at 80-100 ℃, stirring for reacting overnight, washing a product with hot water, and drying to obtain the modified silica gel chromatographic filler.
Preferably, in the step 2), the epoxysiloxane is 2- (3, 4-epoxycyclohexyl) methyldiethoxysilane, 2- (3, 4-epoxycyclohexyl) methyldiethoxysilane or (3-epoxypropoxypropyl) dimethylethoxysilane; the addition amount of the epoxy siloxane is 0.8-2 times of the mass of the active silica gel.
Preferably, in the step 3), the addition amount of the 1-pyridin-2-yl-1, 4-butanediamine is 0.2 to 0.35 times the mass of the epoxidized modified silica gel.
According to the method, C18 silica gel is used as a raw material, epoxy groups are bonded to the surface of porous silica gel through the reaction of silicon hydroxyl on the surface of the silica gel and epoxy siloxane, then 1-pyridine-2-yl-1, 4-butanediamine is reacted with epoxy modified silica gel to prepare the modified silica gel chromatographic packing, the number of the silicon hydroxyl on the surface of the silica gel microspheres is reduced, the non-specific adsorption of polypeptides on the surface of the packing is reduced through two-step modification treatment of the epoxy siloxane and the 1-pyridine-2-yl-1, 4-butanediamine, hydrophilic silicon oxygen groups and the like are introduced, and hydrophobic aliphatic hydrocarbon chains are introduced, so that the modified silica gel chromatographic packing has a good separation effect due to the cooperation of charge repulsion force and hydrophobicity, is beneficial to elution of non-target substances, and the purification efficiency of the polypeptides is improved.
Preferably, in the sixth step, the anion exchange chromatography desalting conditions are as follows:
chromatographic packing: DEAE high-flow rate agarose microspheres, wherein the filling volume is 20-100 mL;
mobile phase: 0.1-2 vol% of acetic acid and 0.05-0.5 vol% of ammonium acetate;
measuring the wavelength: 210-230 nm;
flow rate: 50-100 mL/min;
elution conditions: 100% acetic acid/ammonium acetate mixed solution.
The invention also discloses application of the polypeptide condensing agent in improving the purity and yield of crude peptide in preparation of the somaglutide main peptide chain.
The invention also discloses application of the modified silica gel chromatographic packing in improving the separation and purification efficiency of crude peptide of the soxhlet polypeptide main peptide chain.
The invention takes the self-made polypeptide condensing agent and the organic base as a condensation coupling system, and carries out condensation coupling on the amino acid according to the polypeptide sequence of the main peptide chain of the Somaltulip, thereby having the following beneficial effects:
1) when condensing and coupling Lys, H-His (Boc) -OH or Fmoc-His (Boc) -OH is used as a raw material, so that the defects that Lys (alloc) and Lys (MMt) are expensive and a side chain removal process is difficult are overcome;
2) when amino acid is subjected to condensation coupling, a polypeptide condensing agent and organic base are used as a condensation coupling system, so that the coupling efficiency is improved, the generation of impurities is reduced, the purity and yield of crude peptide are improved, and the synthesis cost is reduced;
3) the 1-pyridine-2-yl-1, 4-butanediamine and epoxysiloxane modified silica gel are used as chromatographic packing to carry out reversed-phase high performance liquid chromatography purification on the crude peptide of the somaltulipide main peptide chain, the hydrophilic action of the modified silica gel reduces the adsorption of the polypeptide, and the charge repulsion acting force is matched with the hydrophobicity to improve the purification efficiency of the polypeptide.
Drawings
FIG. 1 is a scheme for synthesis of a polypeptide condensing agent in example 1;
FIG. 2 is an HPLC chromatogram of the main peptide chain of somaglutide in example 3;
FIG. 3 is a FTIR plot of the modified silica gel chromatography packing of example 2; in the figure, a represents C18 silica gel chromatographic packing, and b represents modified silica gel chromatographic packing;
fig. 4 shows the purity and yield of crude peptide of the somaltulin main peptide chain; in the figure, B represents the yield, C represents the purity;
fig. 5 is the purification yield of the somaglutide main peptide chain.
Detailed Description
The following examples are presented to further illustrate the essence of the present invention, and it should be noted that these examples are only intended to specifically describe the present invention and should not be construed as limiting the present invention.
The related terms of the invention explain:
THF: tetrahydrofuran;
DCM: dichloromethane;
fmoc: 9-fluorenylmethoxycarbonyl;
gly: glycine;
arg: arginine;
val: valine;
leu: leucine;
trp: tryptophan;
ala: alanine;
ile: isoleucine;
phe: phenylalanine;
glu: glutamic acid;
gln: (ii) glutamine;
tyr: tyrosine;
ser: serine;
asp: aspartic acid;
thr: threonine;
aib: 2-methylalanine;
his: (ii) histidine;
DIEA: n, N-diisopropylethylamine;
TRIS: tris (2-aminoethyl) amine;
pbf: 2,2,4,6, 7-pentamethylbenzofuran-5-sulfonyl;
boc: a tert-butoxycarbonyl group;
OtBu: a tert-butoxy group;
trt: a trityl group;
tBu: a tertiary butyl group;
HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate;
DEPBT: 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4-one.
Example 1:
this example provides a method for producing a polypeptide condensing agent (the synthetic scheme is shown in fig. 1), comprising:
i) adding 16.3 g of 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine into 100 mL of THF, stirring for dissolving, adding 7.5 g of propylene oxide and 0.8 g of tetramethylammonium hydroxide, stirring for reacting for 4 hours at 60 ℃, and washing a product with methanol to obtain a product A;
ii)24.3 g of 2-chloro-1- (2-vinyl-1-piperidyl) ethanone is dissolved in 100 mL of DCM, the product A is added, 2 g of triethylamine is added under the stirring state, the mixture is continuously stirred and reacts for 4 hours after the dropwise addition, the filtration is carried out, a filter cake is washed by methanol, the filter cake is collected and dried, and the polypeptide condensing agent is obtained.
And (3) carrying out nuclear magnetic hydrogen spectrum test on the obtained polypeptide condensing agent, and observing a nuclear magnetic hydrogen spectrum after the test to show that 7.40-8.05 ppm is the chemical shift of hydrogen on a benzene ring, 3.40 ppm and 3.65 ppm are the chemical shifts of methylene hydrogen and methine hydrogen after the ring opening of propylene oxide, 1.28 ppm is the chemical shift of methyl hydrogen after the ring opening of propylene oxide, and 3.35-1.60 ppm is the chemical shift of hydrogen on a piperidine ring, thereby indicating that the polypeptide condensing agent is successfully prepared.
Example 2:
the embodiment provides a preparation method of a modified silica gel chromatographic packing, which comprises the following steps:
soaking and activating C18 silica gel with 0.1 mol/L hydrochloric acid aqueous solution for 3.5 h, then washing with deionized water to neutrality, finally washing with acetone and drying to obtain activated silica gel; dissolving 1 g of activated silica gel in 10 mL of toluene, adding 1.4 g of 2- (3, 4-epoxycyclohexane) methyldiethoxysilane, reacting at 90 ℃ overnight, cleaning with hot water, and drying to obtain epoxidized modified silica gel; dissolving 1 g of epoxidized modified silica gel in 10 mL of toluene, adding 0.3 g of 1-pyridin-2-yl-1, 4-butanediamine at 90 ℃, stirring for reaction overnight, washing a product with hot water, and drying to obtain the modified silica gel chromatographic filler.
Example 3:
the present embodiment provides a method for preparing a somaglutide main peptide chain, comprising:
step one, preparing Fmoc-Gly-resin:
adding 10 g Wang resin (0.5 mmol/g) into 50 mL DCM to swell for 1 h, and draining the solvent; adding 15 g of Fmoc-Gly-OH into 200 mL of DCM, stirring for dissolving, cooling to below 10 ℃, adding 11.6 g of condensation coupling system, stirring for activating for 20 min, then adding the resin, and stirring for reacting for 5 h; after the reaction is finished, the solvent is drained, 1.2 g of acetic anhydride, 1.13 mL of 4-aminopyridine and 100 mL of DCM are added for end capping for 1.5 h, the product DCM and methanol are alternately washed, and vacuum drying is carried out, so as to obtain Fmoc-Gly-resin; the substitution degree is 0.38 mmol/g by detecting with an ultraviolet spectrophotometer method;
step two, preparing a first polypeptide resin:
adding the Fmoc-Gly-resin obtained in the step one into 100 mL of DCM for swelling for 40 min, draining the solution, adding 100 mL of 20 vol% piperidine/DCM solution, reacting for 20 min at room temperature, and draining the solution; adding 100 mL of 20 vol% piperidine/DCM solution again, reacting for 20 min at room temperature, draining the solution, and washing with DCM, namely finishing Fmoc removal;
adding 17.7 g of Fmoc-Arg (Pbf) -OH into 500 mL of DCM for dissolution, adding 11.6 g of condensation coupling system at 0 ℃ for activation for 10 min, adding the activated reaction liquid into resin, reacting for 2 h at 25 ℃, draining the solution, and washing the resin with DCM for 5 times to obtain Fmoc-Arg (Pbf) -Gly-resin;
repeating the Fmoc-removing and amino acid coupling steps, sequentially coupling 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 and Fmoc-Glu (OtBu) -OH to obtain first resin;
step three, preparing a second polypeptide resin:
coupling H-Lys (Boc) -OMe.HCl according to the Fmoc removal and amino acid coupling steps in the second step to obtain a second resin;
step four, preparing the somaltulin main peptide chain resin:
coupling 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 and Fmoc-Val-OH in sequence according to the Fmoc removal and amino acid coupling steps in the second step, 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, Fmoc-His (Trt) -OH to prepare the soxhlet peptide main peptide chain resin;
step five, preparing the crude peptide of the somaltulin main peptide chain:
adding 2000 mL of lysate into the soxhlet peptide main peptide chain resin, performing pyrolysis for 3 h at room temperature, performing suction filtration, adding the filtrate into methyl tert-butyl ether for precipitation, centrifuging the obtained solid, washing the solid to be neutral by using the methyl tert-butyl ether, and drying the solid to obtain crude soxhlet peptide main peptide chain; the cracking agent is formed by mixing 92.5 wt% of trifluoroacetic acid, 2.5 wt% of thioanisole, 1.5 wt% of triisopropyl silane, 1.5 wt% of 1, 2-ethanedithiol and 2wt% of deionized water;
step six, purifying the somaltulin main peptide chain:
sample treatment: dissolving the crude soxhlet peptide main peptide chain peptide in 0.5 vol% acetonitrile water solution, filtering with a 0.25 mu m filter membrane after complete dissolution, and collecting the filtered crude soxhlet peptide water solution for later use;
purifying by reversed phase high performance liquid chromatography: the chromatographic packing is the modified silica gel chromatographic packing obtained in the example 2, the packing volume is 60 mL, the mobile phase is an aqueous solution of phase A which is 0.1 vol% trifluoroacetic acid, and the phase B is acetonitrile; the detection wavelength is 220 nm; the flow rate is 80 mL/min; loading the modified silica gel chromatographic packing on a column, balancing the column by using a mobile phase A, loading the sample for elution, wherein an eluent is a mixed solution of a phase A and a phase B, the volume percentage of the phase B is increased from 10% to 35% within 45 min, then keeping the proportion of 35% unchanged, and collecting a purified solution at 65 min;
anion exchange chromatography desalination: the chromatographic packing is DEAE high-flow rate agarose microspheres, the packing volume is 60 mL, the mobile phase is a mixed solution containing 0.5 vol% of acetic acid and 0.2 vol% of ammonium acetate, the measurement wavelength is 220 nm, the flow rate is 80 mL/min, the DEAE high-flow rate agarose microspheres are loaded on a column, the column is balanced by the mobile phase, the sample is loaded for elution, the eluent is a mixed solution of 100% of acetic acid and ammonium acetate, the mixture is concentrated and freeze-dried to obtain the pure product of the main peptide chain of the somaglutelin, and the HPLC chromatogram is shown in figure 2.
In this example, the condensation coupling system was DIEA and the polypeptide condensing agent obtained in example 1 in a molar ratio of 1: 1.4.
Example 4:
this example provides another preparation method of a main peptide chain of somagluteptide, which is substantially the same as example 3, except that the condensation coupling system in this example is the polypeptide condensation agent prepared in example 1, DIEA, in a molar ratio of 1: 0.5.
Example 5:
this example provides another method for preparing a main peptide chain of somagluteptide, which is substantially the same as example 3, except that the condensation coupling system in this example is the polypeptide condensation agent prepared in example 1, DIEA, in a mass ratio of 1: 1.
Example 6:
this example provides another preparation method of a main peptide chain of somagluteptide, which is substantially the same as example 3, except that the condensation coupling system in this example is the polypeptide condensation agent prepared in example 1 and DIEA in a mass ratio of 1: 1.6.
Example 7:
this example provides another method for preparing a main peptide chain of somagluteptide, which is substantially the same as example 3, except that the condensation coupling system in this example is the polypeptide condensation agent prepared in example 1 and DIEA in a mass ratio of 1: 2.
Example 8:
this example provides another method for preparing a somaltulip main peptide chain, which is substantially the same as example 3, except that the condensation coupling system in this example is HBTU, DIEA in a mass ratio of 1: 1.4.
Example 9:
this example provides another preparation method of a main peptide chain of somagluteptide, which is substantially the same as example 3, except that the chromatographic packing in the purification process of reverse phase high performance liquid chromatography is epoxidized modified silica gel, which is prepared by the same method as in example 2.
Example 10:
this example provides another method for preparing the main peptide chain of somagluteptide, which is substantially the same as in example 3, except that the chromatographic packing used in the purification by reverse phase high performance liquid chromatography is C18 silica gel.
Test example 1:
condensation activity test of polypeptide condensing agent:
25.2 g of Boc-D-phenylglycine was added to 500 mL of DCM, 14 g L-alanine methyl ester hydrochloride and 15 g of TRIS were added, 60.5 g of the polypeptide condensing agent prepared in example 1 was then added, the mixture was stirred at room temperature for 5 hours to perform condensation, a sample was taken to examine the remaining Boc-D-phenylglycine content by HPLC, and the condensation activity was measured using HBTU and DEPBT, respectively, and the results are shown in Table 1.
TABLE 1 condensation Activity of polypeptide condensing Agents
Figure 241218DEST_PATH_IMAGE002
As can be seen from table 1, compared with HBTU and detbt, the condensation coupling of amino acids by the polypeptide condensing agent prepared by the present invention has less raw material residue, indicating that the polypeptide condensing agent prepared by the present invention has higher condensation activity than HBTU and detbt.
Test example 2:
infrared spectrum characterization of the modified silica gel chromatographic packing:
the chromatographic packings of C18 silica gel and modified silica gel in example 2 were analyzed by IR spectroscopy using a YENSOR-27 model IR spectrometer from Bruker, Germany, and the results are shown in FIG. 3.
As can be seen from an examination of FIG. 3, curve a represents the unmodified C18 silica gel chromatographic packing and curve b represents the modified silica gel chromatographic packing, and as shown in the figure, curve a is at 3420 cm-1The vicinity is strong absorption peak of silicon hydroxyl, 1128 cm-1、810 cm-1The vicinity is a vibration peak of a silicon-oxygen group; comparing the curve a and the curve b at 3420 cm-1The absorption peak of nearby silicon hydroxyl groups is weakened, which indicates that part of the silicon hydroxyl groups are consumed by reaction, and 850 cm-1The characteristic absorption peak of the epoxy group appears nearby, which indicates that 2- (3, 4-epoxy cyclohexyl) methyl diethoxy silane is successfully grafted to silica gel, and the length of the grafting range is 1600-1420 cm-1The characteristic vibration peak of pyridine appears nearby, which shows that 1-pyridine-2-yl-1, 4-butanediamine successfully modifies the silica gel after epoxidation modification.
Test example 3:
yield and purity of crude peptide of the somaltul peptide main chain:
taking the crude peptide of the main peptide chain of the somaglutide prepared in the examples 3-8 as a sample, testing the purity of the crude peptide by using high performance liquid chromatography, and calculating the molar yield according to the molar weight of the reactant and the product, wherein the test result is shown in fig. 4.
Fig. 4 is a result of testing the yield and purity of the crude peptide of the soxhlet polypeptide main peptide chain, wherein B represents the yield, and C represents the purity, as shown in the figure, the blending of the polypeptide condensing agent and the DIEA in a specific mass ratio in the polypeptide synthesis process as a condensation coupling system can make the yield and purity of the crude peptide of the soxhlet polypeptide main peptide chain reach 97.4% and 85.7% at most; compared with the examples 3 to 7, the mass ratio of the polypeptide condensing agent to the DIEA in the condensation coupling system has larger influence on the synthesis yield and the purity of the polypeptide, and when the mass ratio of the polypeptide condensing agent to the DIEA is in the range of 1: 1-1.6, the condensation coupling system has higher condensation activity and can reduce the occurrence of side reactions, thereby reducing racemization byproducts and improving the purity and the yield of the crude peptide; comparing examples 3 and 8, it can be seen that the condensation coupling system composed of the polypeptide condensing agent and the DIEA provided by the present invention has higher condensation activity than the existing condensation coupling systems HBTU and DIEA.
Test example 4:
yield of purified somaltulide main peptide chain:
the purity of the purified soxhlet peptide obtained in examples 3 to 10 was measured by high performance liquid chromatography using the main peptide chain of the purified soxhlet peptide as a sample, and the purification yield was calculated by using the mass before and after purification, and the measurement results are shown in fig. 5.
Fig. 5 shows the yield of the crude soxhlet peptide main peptide chain after purification, wherein the purity of the soxhlet peptide main peptide chain obtained by purifying the crude soxhlet peptide main peptide chain by the purification method of the present invention is higher than 99.7%, the purification yields of examples 3, 5 and 6 are higher than 76.5%, the purification yields are high, and the purification yields of examples 4, 7 and 8 are lower than example 3, which indicates that the polypeptide condensing agent prepared by the present invention has better condensation coupling activity than HBTU and DIEA when blended with DIEA as a condensation coupling system, and can reduce the occurrence of side reactions, thereby reducing racemization byproducts, facilitating the separation and purification of target peptide, byproducts and impurities, and thus improving the purification yield to a certain extent; the purification yields of examples 9 and 10 are significantly lower than those of example 3, and the purification yield of example 10 is the lowest, which indicates that the modified silica gel is more favorable for eluting the non-target substance during the purification process of the high performance liquid chromatography than the epoxidized modified silica gel or the C18 silica gel as the chromatographic packing, thereby improving the purification efficiency of the polypeptide.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims (7)

1. A method for preparing a somaglutide main peptide chain, comprising:
activating a resin solid phase carrier, and coupling Fmoc-Gly-OH to the resin solid phase carrier by adopting a condensation coupling system to obtain Fmoc-Gly-resin;
step two, performing amino acid activation coupling by adopting a condensation coupling system from the C end to the N end according to the polypeptide sequence of the Somallu peptide main peptide chain by a solid-phase synthesis method, and sequentially coupling Arg, Gly, Arg, Val, Leu, Trp, Ala, Ile, Phe and Glu to obtain first polypeptide resin;
step three, adopting a condensation coupling system to carry out activation coupling of amino acid, and coupling Lys to obtain second polypeptide resin;
step four, adopting a condensation coupling system to carry out activation coupling on amino acid, sequentially coupling Ala, Ala, Gln, Gly, Glu, Leu, Tyr, Ser, Val, Asp, Ser, Thr, Phe, Thr, Gly, Glu, Aib and His to obtain the somalufide main peptide chain resin;
step five, cutting by using a cracking agent, and removing resin and side chain protecting groups to obtain crude somaglutide main peptide chain peptide;
sixthly, obtaining the somatodeptide chain by reversed-phase high-performance liquid chromatography purification, anion exchange chromatography salt conversion and freeze drying;
in the first step, the condensation coupling system is a mixture of a polypeptide condensing agent and an organic base, the mixing molar ratio is 1: 1-1.6, and the organic base is selected from DIEA or TRIS;
the structural formula of the polypeptide condensing agent is shown as the formula (1):
Figure DEST_PATH_IMAGE002
(1)；
the polypeptide condensing agent is prepared by the following method:
3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine reacts with epoxypropane to obtain a product A, and then the product A reacts with 2-chloro-1- (2-vinyl-1-piperidyl) ethanone to obtain a polypeptide condensing agent;
in the preparation process of the polypeptide condensing agent, the molar ratio of 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine to propylene oxide is 1: 1.1-1.5; the molar ratio of the product A to the 2-chloro-1- (2-vinyl-1-piperidyl) ethanone is 1: 1.1-1.3;
the addition amount of triethylamine is 0.1-0.4 time of the molar mass of 2-chloro-1- (2-vinyl-1-piperidyl) ethanone.
2. The method according to claim 1, wherein the molar ratio of the amino acid in the first to fourth steps to the condensation coupling system is 1: 2.5-3.
3. The method of claim 1,
lys in step three by R1-Lys (Boc) -OMe.HCl as starting material, R1Selected from H or Fmoc;
his in the fourth step is Fmoc-His (R)2) -OH as starting material, said R2Selected from Boc or Trt.
4. The method of claim 1, wherein the cracking agent in the fifth step is prepared by mixing 90-93 wt% trifluoroacetic acid, 2-3 wt% thioanisole, 1-2 wt% triisopropylsilane, 1-2 wt%1, 2-ethanedithiol, and the balance deionized water.
5. The method as claimed in claim 1, wherein in the purification process of the reversed-phase high performance liquid chromatography, the chromatographic packing is modified silica gel chromatographic packing, and is prepared by activating porous silica gel with hydrochloric acid solution, reacting with epoxy silane coupling agent, and then reacting with 1-pyridin-2-yl-1, 4-butanediamine;
the preparation method of the modified silica gel chromatographic packing specifically comprises the following steps:
1) soaking and activating C18 silica gel for 2-5 h by using a hydrochloric acid aqueous solution, then washing the activated C18 silica gel to be neutral by using deionized water, and finally washing the activated C18 silica gel by using acetone and drying the acetone to obtain activated silica gel;
2) dissolving activated silica gel in toluene, adding epoxy siloxane, reacting overnight at 80-100 ℃, cleaning with hot water, and drying to obtain epoxidized modified silica gel;
3) dissolving epoxy modified silica gel in toluene, adding 1-pyridine-2-yl-1, 4-butanediamine at 80-100 ℃, stirring for reacting overnight, washing a product with hot water, and drying to obtain a modified silica gel chromatographic filler;
in the step 2), the epoxy siloxane is 2- (3, 4-epoxycyclohexyl) methyldiethoxysilane, 2- (3, 4-epoxycyclohexyl) methyldiethoxysilane or (3-epoxypropoxypropyl) dimethylethoxysilane; the addition amount of the epoxy siloxane is 0.8-2 times of the mass of the active silica gel;
in the step 3), the addition amount of the 1-pyridine-2-yl-1, 4-butanediamine is 0.2-0.35 times of the mass of the epoxidized modified silica gel.
6. Use of a polypeptide condensing agent as claimed in claim 1 for increasing the purity and yield of crude peptide in the preparation of a somagluteptide chain.
7. Use of the modified silica gel chromatography packing material of claim 5 for improving the efficiency of separation and purification of crude peptide of the main peptide chain of somagluteptide.
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