CN113527517A

CN113527517A - Method for synthesizing U. hensis probe

Info

Publication number: CN113527517A
Application number: CN202110806725.XA
Authority: CN
Inventors: 李佳斌; 陈聪; 卢成飘; 梁军; 许国强
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-10-22

Abstract

The invention belongs to the technical field of protein chemical synthesis, and particularly relates to a synthesis method of a kosher probe, which comprises the following steps: respectively preparing a JUSU N-terminal hydrazide fragment and a JUSU C-terminal hydrazide fragment by adopting a polypeptide solid phase synthesis technology; coupling the JUSU N-terminal hydrazide fragment and the JUSU C-terminal hydrazide fragment by adopting polypeptide hydrazide connection reaction to obtain the JUSU hydrazide; and carrying out in-situ activation and ammonolysis reaction on the utahenoside to obtain the utahenoside probe. The method adopts total synthesis to prepare the Ustilagin hydrazide, obtains the Ustilagin probe by a one-pot method of in-situ oxidation and ammonolysis of the hydrazide, and has the advantages of high universality, simple operation, high synthesis efficiency, easy functional modification, large-scale preparation and the like.

Description

Method for synthesizing U. hensis probe

Technical Field

The invention belongs to the technical field of protein chemical synthesis, and particularly relates to a synthesis method of a kosher probe, wherein the kosher probe can be used for covalent crosslinking to remove a modification enzyme.

Background

Utilin (UFM1) is a conserved ubiquitin-like protein in eukaryotes, and contains 83 amino acids. Through the cascade reaction catalyzed by the Usherein related enzymes E1, E2 and E3, the C-terminal glycine of the Usherein can form an amido bond (also called isopeptide bond) with the lysine side chain of the protein substrate, so as to form Usherein modification and further regulate and control the structure and the function of the substrate protein. Similar to other post-translational modifications, deuterogamy is also a dynamically reversible process, the isopeptide bond of which can be specifically hydrolyzed by deuterogamy enzyme.

Research shows that the taurochrombosis participates in cell processes such as DNA damage modification, gene transcription, endoplasmic reticulum homeostasis, autophagy and apoptosis, and the functional disorder is proved to be closely related to tumors, schizophrenia, ischemic heart disease, diabetes and the like. In recent years, a plurality of kosher protein substrates (such as p53, histone and the like) are identified successively, and the number of the kosher protein substrates tends to increase year by year, but the corresponding enzyme catalytic system is single, and only one human kosher enzyme with activity exists if the human kosher enzyme is known at present. The diversification of the types and the quantity of the jewish-like enzyme substrates and the singleness of the jewish-like enzyme indicate that the knowledge of the jewish-like enzyme modification system still has a great gap, for example, whether other unknown jewish-like enzyme exists, the selective recognition function between the enzyme and different substrates, the enzyme catalysis mechanism of the jewish-like enzyme modification process and other basic problems still need to be answered.

An active probe of a specific targeting enzyme active center is considered to be an effective molecular tool for researching the jewish-sumatrise, and consists of three parts, namely a purification label, jewish-sumatrise and an active group, on the molecular structure, the principle is that an electrophilic group is assembled at the C end (near an isopeptide bond) of the jewish-sumatrise molecule, the jewish-sumatrise is taken as an identification unit to interact with the corresponding jewish-sumatrise, at the moment, the de-modification enzyme does not hydrolyze an amido bond, and cysteine of the active center and the electrophilic group can generate addition or substitution reaction, so that a covalent cross-linking compound of a substrate-enzyme is formed, and the aim of covalently capturing protease is fulfilled. The probe has various application values, including 1) enriching and purifying from cell lysate and finding novel de-kosher enzyme by combining technologies such as proteomics and the like; 2) analyzing the active abnormal de-modified enzyme in the disease cell to find a potential drug target; 3) monitoring the activity and selectivity of enzyme inhibitors for potential drug molecules; 4) the catalytic conformation of the de-modified enzyme is captured and stabilized to facilitate structural resolution of the substrate-enzyme complex.

The tag-UFM1-PA of C-terminal propargylamine modified Usher probe is proved to have good cross-linking activity with de-Usher enzyme, thus having wide application prospect, but the C-terminal propargylamine modified Usher probe is difficult to prepare by protein recombinant expression or enzyme method, and the existing established full synthesis or semi-synthesis method has the defects of low synthesis efficiency or poor universality and the like. Therefore, the development of a simpler and efficient chemical synthesis method of the jewish enzyme probe is beneficial to promoting the research on a mechanism related to the jewish enzyme, and provides necessary probe molecules for the identification of targets related to the jewish enzyme modification and the development of an inhibitor.

Disclosure of Invention

The invention aims to provide a kosher probe (His)₆UFM1-PA), has the characteristics of high universality, easy functional modification, simple operation, mass preparation and the like.

According to the technical scheme of the invention, in the synthesis method of the U. jequiriti probe, the U. jequiriti probe is His₆UFM1-PA (amino acid sequence shown in SEQ ID No. 1), comprising the steps of,

step 1: respectively preparing a JUSU N-terminal hydrazide fragment and a JUSU C-terminal hydrazide fragment by adopting a polypeptide solid phase synthesis technology;

step 2: coupling the JUSU N-terminal hydrazide fragment and the JUSU C-terminal hydrazide fragment by adopting a polypeptide hydrazide connection technology to obtain the JUSU hydrazide;

and step 3: and carrying out in-situ activation and ammonolysis reaction on the utahenoside to obtain the utahenoside probe.

Further, the N-terminal hydrazide fragment of the utahenisin is His₆-UFM1[S²-A⁴⁵]-NHNH₂The U. jelisol C-terminal hydrazide fragment is UFM1[ C⁴⁶-V⁸²]-NHNH₂。

Further, the specific operation of step 1 is as follows:

1a, mixing solid-phase synthetic resin with Fmoc-NHNH₂Coupling to obtain hydrazine resin;

1b, removing Fmoc protective groups from the hydrazine resin, and performing extension coupling of polypeptide chains in a solution containing Fmoc-amino acid and a condensing agent to obtain a resin connected with the polypeptide;

1c, cracking the resin connected with the polypeptide to remove the resin and amino acid side chain protecting groups to obtain a polypeptide solution;

1d, concentrating the polypeptide solution, precipitating with glacial ethyl ether and purifying by high performance liquid chromatography to obtain the hydrazide fragment His at the N end of the Usherbet₆-UFM1[S²-A⁴⁵]-NHNH₂And Uxisin C-terminal hydrazide fragment UFM1[ C⁴⁶-V⁸²]-NHNH₂。

Step 1, the N-terminal fragment His of Usherein₆-UFM1[S²-A⁴⁵]-NHNH₂In the synthesis of (3), a tag of 6 consecutive histidines is modified at the N terminal, and the method can be used for enrichment and purification based on nickel ions.

Further, in the step 1b, the condensing agent is one or more of common polypeptide solid phase synthesis condensing agents such as HCTU (6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium hexafluorophosphate), HBTU (benzotriazol-N, N '-tetramethyluronium hexafluorophosphate) and HATU (2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate).

Specifically, step 1 may include the following operations:

1a, preparation of hydrazine resin: weighing 2-Chlorotrityl Chloride resin, placing the resin in a polypeptide synthesis tube, washing once in a standard way, and draining; adding the mixture in a volume ratio of 1:1, swelling the mixture of DMF and DCM (N, N-dimethylformamide/dichloromethane) for 25-35min, and draining; Fmoc-NHNH was added₂(4 equivalents) and a DMF solution of DIEA (N, N-diisopropylethylamine, 10 equivalents) are reacted for 4 to 6 hours with shaking at 37 ℃, and then the reaction solution is drained; adding methanol, shaking at room temperature for 10-15min to block unreacted functional groups, and pumping to dry; washing DMF and DCM for 3-6 times respectively, and pumping to dryness;

1b, weighing the hydrazine resin (0.1mmol) prepared in the step 1a, adding 20% piperidine DMF solution (v/v) to remove Fmoc protective groups, carrying out shake reaction at room temperature for 5-8min, washing twice with DMF, and pumping to dry; adding 20% piperidine DMF solution, shake reacting at room temperature for 10-15min, washing with standard solution once (DMF 3 times, DCM 3 times, DMF 3 times), and draining;

standard peptide chain extension, wherein a condensation reaction system is DMF solution of Fmoc-amino acid (4 equivalents), HCTU (3.8 equivalents) and DIEA (8 equivalents), coupling is carried out for 40-50min at 30 ℃, and standard washing is carried out for 1 time after the reaction is finished; adding 20% piperidine for reaction twice (5min and 10min), washing for 1 time, and performing the next condensation cycle of amino acid;

1c, cleavage of the polypeptide from the resin and deprotection of the polypeptide: adding 20% piperidine for reaction twice (5min and 10min), and washing for 1 time; washing the resin with DCM for several times, and draining; standard cleavage reagents (TFA/Thioanisole/Phenol/EDT/H) were added₂The volume ratio of O is 85/5/5/2.5/2.5), and the reaction is carried out for 2 to 3 hours at room temperature; concentrating the polypeptide solution to about 2mL by using a nitrogen bubbling method; adding glacial ethyl ether for precipitation, centrifuging for 5min, removing supernatant, and retaining precipitate; washing the crude peptide twice with glacial ethyl ether, precipitating, standing and airing to obtain crude peptide;

1d, separation and purification: dissolving crude peptide with acetonitrile-water mixed solution, purifying polypeptide with semi-preparative reverse phase high performance liquid chromatography (C18 or C4 column), collecting target component, freeze drying at low temperature to obtain target polypeptide (His)₆-UFM1[S²-A⁴⁵]-NHNH₂And UFM1[ C⁴⁶-V⁸²]-NHNH₂) And sealing and storing at low temperature for later use.

Further, the specific operation of step 2 is as follows:

2a, adding the hydrazide fragment His at the N end of the utahenisin obtained in the step 1₆-UFM1[S²-A⁴⁵]-NHNH₂Dissolving in oxidizing buffer solution, adding oxidant under ice salt bath for reaction, and sequentially adding thiol and Ushinsu C-terminal hydrazide fragment UFM1[ C⁴⁶-V⁸²]-NHNH₂And carrying out reaction respectively;

2b, adding a reducing agent, and purifying to obtain His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂；

2c, His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Dissolving in desulfurization buffer solution, adding initiator and free radical trapping agent, adjusting pH to 7.0-8.0, performing desulfurization reaction, and purifying to obtain the Ustilagin hydrazide His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂。

Further, the oxidation buffer is a mixed solution of guanidine hydrochloride and disodium hydrogen phosphate at pH 3.0-4.0.

Further, in the step 2a, the oxidizing agent is a sodium nitrite aqueous solution, and the mercaptan is a 4-mercaptophenylacetic acid solution.

Further, in the step 2b, the reducing agent is tris (2-carboxyethyl) phosphine solution.

Further, in step 2c, the radical initiator is 2,2' -azabicyclo (2-imidazoline) (VA-044), the capture agent is reduced glutathione or tert-butyl mercaptan, and the desulfurization buffer is a mixture of guanidine hydrochloride, disodium hydrogen phosphate, and TCEP (tris (2-carboxyethyl) phosphine) at pH 7.0.

Specifically, the 4-mercaptophenylacetic acid solution is prepared by dissolving 0.2M 4-mercaptophenylacetic acid in a connection buffer solution; the tris (2-carboxyethyl) phosphine solution is prepared by dissolving 0.1M tris (2-carboxyethyl) phosphine in a connection buffer; the ligation buffer was a mixed solution of guanidine hydrochloride and disodium hydrogen phosphate at pH 7.0.

Specifically, step 2 may include the following operations:

2a, weighing 1 equivalent of His₆-UFM1[S²-A⁴⁵]-NHNH₂Dissolving in oxidation buffer solution (6M guanidine hydrochloride, 0.2M sodium dihydrogen phosphate, pH 3.0), cooling in ice salt bath (about-12 deg.C) for 10-15min, adding 10 equivalents of sodium nitrite water solution (0.2M), stirring in ice salt bath for 20-30 min; directly adding 60 equivalents of 4-mercaptophenylacetic acid solution (4-mercaptophenylacetic acid dissolved in 6M guanidine hydrochloride, 0.2M monobasic sodium phosphate buffer, concentration 0.2M); after stirring for several minutes, 1 equivalent of UFM1[ C ] was added⁴⁶-V⁸²]-NHNH₂Adjusting the pH value to 6.6 by using 2M sodium hydroxide solution, reacting at room temperature for about 2 hours, and monitoring the reaction by using a high performance liquid chromatography in the process;

2b, adding an equal volume of TCEP solution after the reaction is finishedStirring the solution (0.1M solution of ligation buffer) at room temperature for 8-15 min; purifying with high performance liquid chromatography, collecting target components, confirming with mass spectrum, and freeze drying at low temperature to obtain target product His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Sealing and storing for later use;

2c, free radical desulfurization: weighing a certain amount of His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Adding desulfurization buffer solution (6.0M guanidine hydrochloride, 0.2M disodium hydrogen phosphate, 1.0M TCEP, pH 7.0) to dissolve the lyophilized solid, adding 25 equivalents of VA-044 and 50 equivalents of reduced glutathione, adjusting the pH to 7.5, and reacting at 37 ℃ overnight; purifying by high performance liquid chromatography, collecting target components, confirming by mass spectrometry, and freeze-drying at low temperature to obtain target product-Ushinsu hydrazide His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂And sealing and storing for later use.

Further, the specific operation of step 3 is as follows:

subjecting the Ushinsu hydrazide His obtained in the step 2₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂Dissolving in an oxidation buffer solution, adding an oxidant under an ice salt bath for reaction, sequentially adding thiol and propargylamine for reaction respectively, and preparing the Usher probe.

Step 3 is an in-situ one-pot reaction, the intermediate does not need to be purified, and a urea probe His is adopted₆The C terminal of UFM1-PA is N- (2-propynyl) amide structure.

Further, the oxidant is sodium nitrite aqueous solution, and the catalyst is 4-mercaptophenylacetic acid solution (which is prepared as above).

Specifically, step 3 may include the following operations: weighing a certain amount of the utahensu hydrazide, dissolving the utahensu hydrazide in an oxidation buffer solution, cooling the utahensu hydrazide in a salt bath (about-12 ℃) for 8-15min, adding 10 equivalents of sodium nitrite aqueous solution (0.2M), and stirring the utahensu hydrazide in the salt bath for reaction for 20-30 min; directly adding 60 equivalents of 4-mercaptophenylacetic acid solution, and stirring for reaction for 8-15 min; adding propargylamine directly, adjusting the pH value to 9.0, and reacting at room temperature for 8-15 min; high efficiency after reactionPurifying by liquid chromatography, collecting components confirmed by mass spectrum, and freeze drying at low temperature to obtain Ushernin probe His₆-UFM1-PA。

Compared with the prior art, the technical scheme of the invention has the following advantages:

the method adopts the total synthesis to prepare the U. jeldahl hydrazide, and obtains the U. jeldahl probe through the one-pot method of in-situ oxidation and ammonolysis of the hydrazide, compared with the strategy 1 (a total synthesis method with two segment connections, wherein the segment connections adopt thioester mediated standard natural chemical connection, and the coupling of the C end of the U. jeldahl and propargylamine adopts the liquid phase condensation reaction of the total protection peptide), the synthesis operation is simple, the efficiency is high, the preparation can be carried out in large quantities, and the same strategy can be expanded to other C end functionalized modification; compared with the strategy 2 (a semisynthesis method of recombinant expression, namely expression preparation of a mutant UFM1G83C with Usherein C-terminal glycine mutated into cysteine, and small molecules activate the C-terminal cysteine and then carry out ammonolysis to obtain UFM1-PA), the method breaks through the limitation of recombinant expression to 20 natural amino acids, has high universality and wide application prospect, and is easy to functionally modify (such as Biotin and fluorescent molecule modification).

In conclusion, the method has the advantages of high universality, simplicity in operation, high synthesis efficiency, easiness in functional modification, capability of large-scale preparation and the like.

Drawings

FIG. 1 shows that the invention provides synthetic Usin probe (His)₆UFM 1-PA).

FIG. 2 is a schematic diagram of the application of the kosher probe according to the present invention.

FIG. 3 shows the N-terminal fragment His of Usherein₆-UFM1[S²-A⁴⁵]-NHNH₂The high performance liquid chromatogram of (1).

FIG. 4 shows the N-terminal fragment His of Usherein₆-UFM1[S²-A⁴⁵]-NHNH₂ESI-MS mass spectrum of (E).

FIG. 5 shows Uighurin C-terminal fragment UFM1[ C ]⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1).

FIG. 6 shows Uighurin C-terminal fragment UFM1[ C ]⁴⁶-V⁸²]-NHNH₂ESI-MS mass spectrum of (E).

FIG. 7 is a drawing showingLigation product His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1).

FIG. 8 shows the ligation product His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂ESI-MS mass spectrum of (E).

FIG. 9 shows desulfurization product-Ushinsu hydrazide His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1).

FIG. 10 shows desulfurization product-Ushinsu hydrazide His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂ESI-MS mass spectrum of (E).

FIG. 11 shows the Usherin probe His₆-high performance liquid chromatography of UFM 1-PA.

FIG. 12 shows the Usherin probe His₆ESI-MS mass spectrum of UFM 1-PA.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Weighing 1.5g of 2-Chlorotrityl Chloride resin, placing the resin in a polypeptide synthesis tube, washing once in a standard way, and draining; adding 38mL of DMF/DCM mixed solvent with the volume ratio of 1:1 for swelling for 30min, and pumping to dry; Fmoc-NHNH was added₂(4 equiv.) DIEA (10 equiv.) in DMF (25mL) was reacted at 37 ℃ with shaking for 5h, and then drained; adding 1.2mL of methanol, shaking at room temperature for 10min to block unreacted functional groups, and pumping to dry; washing DMF and DCM for many times respectively, and draining the prepared hydrazine resin for later use;

weighing a certain amount of hydrazine resin (0.1mmol), adding 2mL of 20% piperidine DMF solution (v/v) to remove Fmoc protective groups, oscillating and reacting for 5min at room temperature, washing twice with DMF, and draining; adding 2mL of 20% piperidine solution in DMF again, performing shake reaction at room temperature for 10min, performing standard washing once (3 times of DMF washing, 3 times of DCM washing and 3 times of DMF washing), and draining;

standard peptide chain extension, wherein a condensation reaction system is 3mL of DMF solution of Fmoc-amino acid (4 equivalents), HCTU (3.8 equivalents) and DIEA (8 equivalents), coupling is carried out for 45min at 30 ℃, and standard washing is carried out for 1 time after the reaction is finished; adding 2mL of 20% piperidine for reaction twice (5min and 10min), washing for 1 time in a standard manner, and performing condensation circulation of the next amino acid;

cleavage of the polypeptide from the resin and deprotection of the polypeptide: after the peptide chain extension is finished, 2mL of 20% piperidine is added into the reaction tube to react twice (5min and 10min), and the reaction tube is washed for 1 time in a standard way; washing the resin with DCM for several times, and draining; 10mL of cleavage reagent (TFA/Thioanisole/Phenol/EDT/H) was added₂The volume ratio of O is 85/5/5/2.5/2.5), and the reaction is carried out for 2 hours at room temperature; concentrating the polypeptide solution to about 2mL by using a nitrogen bubbling method; adding 40-45mL of glacial ethyl ether for precipitation, centrifuging for 5min, removing supernatant, and retaining the precipitate; washing the crude peptide twice with glacial ethyl ether, precipitating, standing and airing to obtain crude peptide;

separating and purifying crude peptide: dissolving crude peptide with acetonitrile-water mixed solution, purifying polypeptide with semi-preparative reverse phase high performance liquid chromatography (C18 or C4 column), collecting target component, freeze drying at low temperature to obtain target polypeptide (His)₆-UFM1[S²-A⁴⁵]-NHNH₂And UFM1[ C⁴⁶-V⁸²]-NHNH₂) And sealing and storing at low temperature for later use.

As shown in FIG. 3, His₆-UFM1[S²-A⁴⁵]-NHNH₂The high performance liquid chromatogram of (1). Chromatographic separation conditions: c18 column, 250 × 4.6mm, 5 μm particle size, linear gradient 20-80% acetonitrile 30min, flow rate 1.0mL/min, λ 214 nm.

As shown in FIG. 4, His₆-UFM1[S²-A⁴⁵]-NHNH₂ESI-MS mass spectrum of (E). Theoretical molecular weight: 5577.41, actual determination of molecular weight: 5576.55.

UFM1[ C ] as shown in FIG. 5⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1). Chromatographic separation conditions: c18 column, 250 × 4.6mm, 5 μm particle size, linear gradient 20% -80% acetonitrile 30min, flow rate 1.0mL/min, λ ═ 214 nm.

UFM1[ C ] as shown in FIG. 6⁴⁶-V⁸²]-NHNH₂ESI-MS Mass SpectrometryFigure (a). Theoretical molecular weight: 4062.63, actual determination of molecular weight: 4061.50.

16.6mg of His were weighed₆-UFM1[S²-A⁴⁵]-NHNH₂Dissolving in oxidation buffer solution (6M guanidine hydrochloride, 0.2M disodium hydrogen phosphate, pH 3.0), cooling in ice salt bath (about-12 deg.C) for 10min, adding 100 μ L sodium nitrite water solution, stirring in ice salt bath, and reacting for 25 min; 400 μ L of a 0.2M solution of 4-mercaptophenylacetic acid was added directly; after stirring for several minutes 11.0mg of UFM1[ C ] were added⁴⁶-V⁸²]-NHNH₂Adjusting the pH value to 6.6 by using 2M sodium hydroxide solution, reacting at room temperature for about 2 hours, and detecting the reaction by using analytical high performance liquid chromatography in the process; after the reaction is finished, adding an equal volume of TCEP solution (0.1M of connection buffer solution), and stirring at room temperature for 10 min; purifying with high performance liquid chromatography, collecting target components, confirming with mass spectrum, and freeze drying at low temperature to obtain target product His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Sealing and storing for later use;

as shown in FIG. 7, His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1). Chromatographic separation conditions: c18 column, 250 × 4.6mm, 5 μm particle size, linear gradient 20% -80% acetonitrile 30min, flow rate 1.0mL/min, λ ═ 214 nm.

As shown in FIG. 8, His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂ESI-MS mass spectrum of (E). Theoretical molecular weight: 9608.02, actual determination of molecular weight: 9607.20.

and (3) free radical desulfurization: weighing 8.1mg of His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Adding 0.9mL of desulfurization buffer (6.0M guanidine hydrochloride, 0.2M disodium hydrogen phosphate, 1.0M TCEP, pH 7.0) to dissolve the lyophilized solid, adding 6.6mg of VA-044 and 12.5mg of reduced glutathione, adjusting the pH to 7.5, and reacting at 37 ℃ overnight; purifying with high performance liquid chromatography, collecting target components, confirming with mass spectrum, lyophilizing at low temperature to obtain His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂Sealing ofAnd (5) storing for later use.

As shown in FIG. 9, His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂The high performance liquid chromatogram of (1). Chromatographic separation conditions: c18 column, 250 × 4.6mm, 5 μm particle size, linear gradient 20% -60% acetonitrile 30min, flow rate 1.0mL/min, λ ═ 214 nm.

As shown in FIG. 10, His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂ESI-MS mass spectrum of (E). Theoretical molecular weight: 9575.96, actual determination of molecular weight: 9574.80.

in-situ oxidation ammonolysis: weighing 5.3mg of the utahensu hydrazide, dissolving in an oxidation buffer solution, cooling in a salt bath (about-12 ℃) for 10min, adding 20 microliters of sodium nitrite aqueous solution (0.2M), and stirring in the salt bath for reaction for 25 min; directly adding 200 microliter of 4-mercaptophenylacetic acid solution, and stirring for reaction for 10 min; adding propargylamine to adjust the pH value to 9.0, and reacting for 10min at room temperature; purifying with high performance liquid chromatography after reaction, collecting mass spectrum identified component, and freeze drying at low temperature to obtain Ushernin probe His₆-UFM1-PA。

As shown in FIG. 11, His₆-high performance liquid chromatography of UFM 1-PA. Chromatographic separation conditions: c18 column, 250 × 4.6mm, 5 μm particle size, linear gradient 20% -80% acetonitrile 30min, flow rate 1.0mL/min, λ ═ 214 nm.

As shown in FIG. 12, His₆ESI-MS mass spectrum of UFM 1-PA. Theoretical molecular weight: 9598.60, actual determination of molecular weight: 9597.60.

in conclusion, the invention provides a method for simply obtaining the Usherein probe His₆A synthetic method of UFM 1-PA.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

<110> Suzhou university

<120> synthetic method of Usherin probe

<130> 23

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 87

<212> PRT

<213> (Artificial Synthesis)

<400> 1

His His His His His His Ser Lys Val Ser Phe Lys Ile Thr Leu Thr

1 5 10 15

Ser Asp Pro Arg Leu Pro Tyr Lys Val Leu Ser Val Pro Glu Ser Thr

20 25 30

Pro Phe Thr Ala Val Leu Lys Phe Ala Ala Glu Glu Phe Lys Val Pro

35 40 45

Ala Ala Thr Ser Ala Ile Ile Thr Asn Asp Gly Ile Gly Ile Asn Pro

50 55 60

Ala Gln Thr Ala Gly Asn Val Phe Leu Lys His Gly Ser Glu Leu Arg

65 70 75 80

Ile Ile Pro Arg Asp Arg Val

85

Claims

1. UsuThe synthesis method of the probe is characterized in that the Usherein probe is His₆UFM1-PA comprising the steps of,

2. The method for synthesizing the euhesin probe as claimed in claim 1, wherein the euhesin N-terminal hydrazide fragment is His₆-UFM1[S²-A⁴⁵]-NHNH₂The U. jelisol C-terminal hydrazide fragment is UFM1[ C⁴⁶-V⁸²]-NHNH₂。

3. The method for synthesizing the jewish essence probe as claimed in claim 1 or 2, wherein the specific operation of step 1 is as follows:

1d, concentrating and purifying the polypeptide solution to obtain the hydrazide fragment His at the N end of the Usherbet₆-UFM1[S²-A⁴⁵]-NHNH₂And Uxisin C-terminal hydrazide fragment UFM1[ C⁴⁶-V⁸²]-NHNH₂。

4. The method of claim 3, wherein in step 1b, the condensing agent is one or more of HCTU, HBTU and HATU.

5. The method for synthesizing the jewish essence probe as claimed in claim 1 or 2, wherein the specific operation of step 2 is as follows:

2a, adding the hydrazide fragment His at the N end of the utahenisin obtained in the step 1₆-UFM1[S²-A⁴⁵]-NHNH₂Dissolving in oxidizing buffer solution, adding oxidant under ice salt bath for reaction, and sequentially adding thiol and Ushinsu C-terminal hydrazide fragment UFM1[ C⁴⁶-V⁸²]-NHNH₂And reacting respectively;

2b, adding a reducing agent, and reacting to prepare His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂；

2c, His₆-UFM1[S²-A⁴⁵-C⁴⁶-V⁸²]-NHNH₂Dissolving in desulfurization buffer solution, adding free radical initiator and trapping agent, adjusting pH to 7.0-8.0, and performing desulfurization reaction to obtain the Ushernin hydrazide His₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂。

6. The method as claimed in claim 5, wherein in step 2a, the oxidizing agent is an aqueous solution of sodium nitrite and the thiol is a solution of 4-mercaptophenylacetic acid.

7. The method as claimed in claim 5, wherein in step 2b, the reducing agent is tris (2-carboxyethyl) phosphine solution.

8. The method as claimed in claim 5, wherein in step 2c, the radical initiator is 2,2' -azabicyclo (2-imidazoline), and the capture agent is reduced glutathione and/or tert-butyl mercaptan.

9. The method for synthesizing the jewish essence probe as claimed in claim 1 or 2, wherein the specific operation of step 3 is as follows:

subjecting the Ushinsu hydrazide His obtained in the step 2₆-UFM1[S²-A⁴⁵-A⁴⁶-V⁸²]-NHNH₂Dissolving in an oxidation buffer solution, adding an oxidant under an ice salt bath for reaction, sequentially adding thiol and propargylamine, and reacting respectively to obtain the jewish probe.

10. The method for synthesizing the kosher probe as claimed in claim 9, wherein the oxidizing agent is an aqueous solution of sodium nitrite and the thiol is a solution of 4-mercaptophenylacetic acid.