CN114106080A - Polypeptide fluorogenic substrate method for preparing ubiquitin-like probe - Google Patents

Abstract

The invention provides a method for synthesizing functional polypeptide. The method comprises the following steps: subjecting the carbon-terminal amino acid of the crude peptide to a condensation reaction with Gly-AMC to obtain the polypeptide, the condensation reaction being carried out under activation of a condensing agent comprising at least one selected from HCTU, DIC, HOBT, PyAOP, PyBOP, HBTU, HATU and HOAT. The method is simple and convenient to operate, and the prepared polypeptide fluorogenic substrate is high in yield, high in purity and low in cost, and is beneficial to commercialization of a fluorogenic reagent.

Description

Polypeptide fluorogenic substrate method for preparing ubiquitin-like probe

Technical Field

The invention relates to the technical field of biology, in particular to a polypeptide fluorogenic substrate method for preparing a ubiquitin-like probe.

Background

Cbz-RLRGG-AMC is a fluorogenic substrate for a polypeptide fragment. The main body of the protein translation modification ubiquitin C-terminal sequence is composed of 5 amino acid residues Arg-Leu-Arg-Gly-Gly. The naked amino group at the N terminal of the sequence is selectively protected by a Cbz (benzyloxycarbonyl) protecting group, and the C terminal is connected with an AMC (7-amino-4-methylcoumarin) fluorescent molecule. When encountering deubiquitinating hydrolase such as isopeptidase (IPAseT) and other ubiquitin C terminal hydrolase (UCHs), the substrate can simulate wild ubiquitin chains to generate amido bond hydrolytic cleavage at terminal glycine, thereby releasing AMC molecules to obviously enhance the fluorescence emitted by the AMC molecules, and accurately determining hydrolysis efficiency and enzyme activity through the fluorescence. Ubiquitin Cbz-RLRGG-AMC is used as a fluorescence detection reagent of deubiquitinase, and the catalytic hydrolysis efficiency (Kcat/Km) of deubiquitinase can reach 95M-1 s-1. Based on the fluorescence characteristic of Cbz-RLRGG-AMC, the polypeptide fluorescent substrate is used as a high-throughput screening tool and widely applied to development of deubiquitinase inhibitors and screening of small molecules and polypeptide drugs of targeted deubiquitinase.

Disclosure of Invention

The invention provides a method for synthesizing polypeptide. According to an embodiment of the invention, the method comprises: subjecting the carbon-terminal amino acid of the crude peptide to a condensation reaction with Gly-AMC to obtain the polypeptide, the condensation reaction being carried out under the activation of a condensing agent comprising a compound selected from HCTU (6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole), PyAOP (3H-1,2, 3-triazolo [4,5-b ] pyridin-3-yloxy) tris-1-pyrrolidinylphosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU (benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate), HATU (2- (7-azobenzotriazol) -N, N, n ', N' -tetramethyluronium hexafluorophosphate) and HOAT (1-hydroxy-7-azobenzotriazol). The method provided by the embodiment of the invention is simple and convenient to operate, and the prepared polypeptide fluorogenic substrate is high in yield, high in purity, low in cost and beneficial to commercialization of a fluorogenic reagent.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the condensing agent is HCTU, PyAOP or HATU and HOAT. The inventors found that the condensing agent is HCTU, PyAOP or HATU and HOAT, and the yield of the polypeptide is further improved.

According to an embodiment of the invention, the condensing agent is HATU and HOAT, the ratio of the amounts of the substances of HATU and HOAT being 1: 1. The inventor finds that when the condensing agent is a combination of HATU and HOAT, the amount ratio of the two substances is 1:1, the yield of the polypeptide is remarkably improved.

According to an embodiment of the invention, the condensing agent is PyBOP, PyAOP, HCTU, HATU and HOAT or HBTU and HOBT, the condensation reaction further being carried out with addition of DIEA (N, N-diisopropylethylamine).

According to an embodiment of the invention, the condensation agent is HOBT and the condensation reaction is further carried out with the addition of DIC (N, N' -diisopropylcarbodiimide).

According to an embodiment of the present invention, the crude peptide is synthesized by Fmoc solid phase synthesis.

According to an embodiment of the invention, the crude peptide has the amino acid sequence of RLRGG, which is obtained by: the method comprises the steps of using chlorine resin as a solid phase carrier, activating by a condensing agent, coupling crude peptides to be synthesized to the chlorine resin one by one according to the sequence of amino acids from C end to N end, protecting the last amino acid by Cbz, and obtaining the crude peptides after full-protection cutting.

According to the examples of the present invention, the coupling of the crude peptide to be synthesized onto the chlorine resin one by one in the order of amino acids from the C-terminus to the N-terminus was carried out at a temperature of 30 to 32 ℃ under activation of HCTU.

According to the present example, the Fmoc removal reaction was performed using a DMF solution containing 20% piperidine.

According to an embodiment of the present invention, the protection of the last amino acid with Cbz is performed by placing the chlorine resin to which RLRGG is attached in a mixed solution of DIEA, CbzCl and DMF in a volume ratio of 1:1: 8.

According to an embodiment of the present invention, the full protection cleavage is performed by placing the chlorine resin to which RLRGG is attached in a mixed solution of 1,1, 1,3,3, 3-hexafluoro-2-propanol and DCM in a volume ratio of 1,1, 1,3,3, 3-hexafluoro-2-propanol to DCM of 1: 4.

According to an embodiment of the present invention, coupling the crude peptide to be synthesized to the chlorine resin one by one in the order of amino acids from the C-terminus to the N-terminus comprises:

(1) carrying out first contact on Fmoc-Gly-OH dissolved by DMF and DIEA in a polypeptide synthesis tube at 30-32 ℃ so as to couple Gly to the chlorine resin;

(2) contacting a chlorine resin coupled with Gly with DMF solution containing 20% piperidine so as to remove Fmoc;

(3) carrying out second contact on Fmoc-Arg-OH dissolved by DMF and DIEA in a polypeptide synthesis tube at the temperature of 30-32 ℃, wherein the second contact is carried out under the activation of HCTU so as to couple Arg to the N end of Gly;

(4) contacting a chlorine resin coupled with Arg-Gly with a DMF solution containing 20% piperidine to remove Fmoc;

(5) carrying out third contact on Fmoc-Leu-OH dissolved by DMF and DIEA at 30-32 ℃ in a polypeptide synthesis tube, wherein the third contact is carried out under the activation of HCTU so as to couple Leu to the N terminal of Arg;

(6) contacting the chlorine resin coupled with Leu-Arg-Gly with DMF solution containing 20% piperidine to remove Fmoc;

(5) carrying out fourth contact on Fmoc-Arg-OH dissolved by DMF and DIEA at 30-32 ℃ in a polypeptide synthesis tube, wherein the fourth contact is carried out under the activation of HCTU so as to couple Arg to the N end of Leu;

(6) the chlorine resin coupled with Arg-Leu-Arg-Gly was contacted with DMF solution containing 20% piperidine to remove Fmoc.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a chromatogram of a synthetic polypeptide according to an embodiment of the invention;

FIG. 2 is a mass spectrum of a synthetic polypeptide according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

The technical problem to be solved by the invention is to provide a polypeptide with good stability and high purity.

In order to solve the technical problems, the invention adopts the following technical scheme: the amino acid sequence of the polypeptide is shown as Cbz-RLRGG-AMC, and the invention also provides a synthetic method of the polypeptide, which comprises the following steps:

the Fmoc solid-phase synthesis method is adopted, chlorine resin is used as a solid-phase carrier, the solid-phase carrier is activated by a condensing agent, amino acids are coupled to the resin one by one from the C end to the N end according to the sequence shown, the last amino acid needs to be protected by Cbz, after full protection cutting, crude peptide reacts with Gly-AMC, and the polypeptide is obtained after treatment by cutting fluid.

Furthermore, the resin is chlorine resin, and the resin is low in cost.

Further, the condensing agent is selected from one or any several of DIC, HATU, HBTU, PyAOP, PyBOP and HCTU. The term "plurality" as used herein means two or more.

Further, the synthesis temperature was 31.5 ℃. In the practice of the present invention, the inventors have found that reaction conditions applicable to all amino acids are used at this temperature.

Further, the reaction conditions for Cbz are DIEA: CbzCl: DMF 1:1: 8;

further, the reagent for removing the Fmoc protecting group was a DMF solution containing 20% piperidine.

Further, the cutting fluid is trifluoroacetic acid, phenol, water, TIPS (triisopropylsilane);

further, Gly-AMC is carried out in the presence of at least one of condensing agents HCTU, HOBT, PyAOP, PyBOP, HBTU, HATU and HOAT, when the condensing agent is PyBOP, PyAOP, HCTU, HATU and HOAT or HBTU and HOBT, the condensation reaction is further added with DIEA, when the condensing agent is HOBT, the condensation reaction is further added with DIC;

the invention has the beneficial effects that:

the polypeptide has the advantages of high stability and high purity of the synthetic method, and the method is simple and convenient to operate and high in yield.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

EXAMPLE 1 preparation of the polypeptide

(1) Swelling resin: weighing 222mg of chlorine resin (0.45g/mmol), putting the chlorine resin into a solid-phase synthesis tube, adding DCM and DMF with the same volume, and standing for 30 min;

(2) cleaning: sequentially washing with DMF, DCM and DMF for 3 times respectively, and draining off solvent;

(3) first amino acid condensation: the Fmoc-Gly-OH amino acid was dissolved in 3mL of DMF in a molar amount of 4 times that of the resin, and then 15 equivalents of DIEA was added to the solution, and the mixture was uniformly mixed and transferred to a peptide synthesis tube. The polypeptide synthesis tube was transferred to a constant temperature shaker and shaken overnight at room temperature (31.5 ℃).

(2) Removing Fmoc protecting groups: the peptide synthesis tube was filled with 20% (20% piperidine solution: 100mL piperidine was measured in graduated cylinders and 500mL DMF was added to the graduated cylinders) piperidine solution in DMF to flood the resin, transferred to a shaker and shaken for 5min, the solvent was drained and washed 3 times with DMF, DCM, DMF in sequence. The resin was again flooded with 20% piperidine in DMF and the shaking time was extended to 10 min. The solvent was drained and washed 3 times with DMF/DCM/DMF in sequence.

(3) Condensation of the next amino acid: 4 equivalents of Fmoc-Arg (Pbf, 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl) -OH amino acid and 4 equivalents of condensing agent (HCTU) relative to the resin were dissolved in DMF, 8 equivalents of DIEA was added thereto, and the mixture was uniformly mixed and transferred to a polypeptide synthesis tube. The polypeptide synthesis tube was transferred to a constant temperature shaker and shaken at room temperature for 50 min.

(4) Removing Fmoc protection: and (3) repeating the operation of the step (2).

(5) Coupling of a third amino acid: dissolving Fmoc-Leu-OH amino acid with 4 times of equivalent and condensing agent (HCTU) with 4 times of equivalent by using DMF, adding DIEA with 8 times of equivalent, uniformly mixing, and transferring to a polypeptide synthesis tube. The polypeptide synthesis tube was transferred to a constant temperature shaker and shaken at room temperature for 50 min.

(6) And (5) repeating the step (4) and the step (5) until the synthesis of the last amino acid is completed.

(7) Protection of the polypeptide terminal Cbz:

7-1) removing the Fmoc protecting group of the last arginine according to step 2);

7-2) according to the ratio DIEA: CbzCl: preparing 10mL of solution with DMF (1: 1: 8), adding 5mL of solution into a synthesis tube, transferring the polypeptide synthesis tube to a constant temperature shaking table, oscillating at room temperature for 20min, and repeating the previous step;

(8) full protection cleavage of fragments

1,1, 1,3,3, 3-hexafluoro-2-propanol: preparing 5mL of solution of DCM (1: 4), pouring the solution into a synthesis tube, transferring the polypeptide synthesis tube to a constant temperature shaking table, shaking at room temperature for 60min, collecting the cutting fluid into a 50mL EP tube, precipitating the crude peptide by using glacial ethyl ether, centrifuging, collecting, washing the crude peptide by using the glacial ethyl ether for 2 times, and drying in the air.

(9) Reaction of fragment Gly-AMC

The crude peptide obtained from the above reaction was put into a 50mL centrifuge tube on average, magnetons were added, 29mg Gly-AMC and PyBOP 104mg were weighed and dissolved in 3mL DMF, 66.5. mu.L DIEA was added, mixed well and added to the centrifuge tube, and the centrifuge tube was put into an oil bath pan (37 ℃ C., 200 rpm) for reaction overnight.

(10) Cutting into large pieces

10-1) spin-drying the reaction solution by using a rotary evaporator, washing the reaction solution for three times by using diethyl ether, and airing the reaction solution;

10-2) 500mg of phenol; TIPS 250 μ L; h₂O is 500 mu L; using trifluoroacetic acid to fix the volume to 10 mL;

10-3) transferring the cleavage reagent of 10-2) to a polypeptide synthesis tube for reaction for 3 h.

(11) Drying and washing: collecting the cutting fluid in a 50mL EP tube, concentrating the cutting fluid by bubbling nitrogen, precipitating the polypeptide by using glacial ethyl ether, centrifugally collecting, washing the polypeptide by using the glacial ethyl ether for 2 times, and drying in the air.

(12) Polypeptide chromatographic analysis and separation: the polypeptide was analyzed for correctness by HPLC and mass spectrometry. After verification, the correct product was isolated and lyophilized as shown in fig. 1 and 2.

Example 2 reaction condition screening of fragment Gly-AMC

Taking the crude peptide obtained in the reaction (8) and putting the crude peptide into 6 50mL centrifuge tubes (the labels are 1-6, and the sequence corresponds to the following condensing agent respectively), adding magnetons, weighing 29mg of Gly-AMC and adding the 6 parts into the centrifuge tubes, weighing PyBOP 57mg, PyAOP 57mg, HCTU 78.5mg, HATU 72mg/HOAT 25mg, HBTU 78mg/HOBT 25mg and HOBT 25mg into the centrifuge tubes respectively, dissolving the mixture with 3mL of DMF, adding DIEA of 66.5 mu L into the centrifuge tubes of No. 1-5 and No. 6 respectively, adding DIC of 31 mu L into the centrifuge tubes of No. 6, fully mixing the mixture and adding the centrifuge tubes into an oil bath pot (the temperature is 37 ℃ and the rotation speed is 200) for reaction overnight.

After reaction (10), fragment cutting, (11), blow-drying and washing, and (12), polypeptide chromatography analysis and separation, 3.6mg of fluorescent polypeptide is obtained in tube 1 (yield 8.6%), 7mg of fluorescent polypeptide is obtained in tube 2 (yield 16.6%), 7.2mg of fluorescent polypeptide is obtained in tube 3 (yield 17.1%), 9.3mg of fluorescent polypeptide is obtained in tube 4 (yield 22.1%), 5.6mg of fluorescent polypeptide is obtained in tube 5 (yield 13.3%), and 5.8mg of fluorescent polypeptide is obtained in tube 6 (yield 8.6%), wherein the yield is calculated according to the following formula: mass of fluorescent polypeptide actually obtained/mass of fluorescent polypeptide theoretically obtained.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (9)

1. A method for synthesizing a polypeptide, comprising subjecting a carbon-terminal amino acid of a crude peptide to condensation reaction with Gly-AMC to obtain the polypeptide, wherein the condensation reaction is performed under activation of a condensing agent comprising at least one selected from the group consisting of HCTU, HOBT, PyAOP, PyBOP, HBTU, HATU, and HOAT.

2. The method of claim 1, wherein the condensing agent is HCTU, PyAOP, or HATU and HOAT.

3. The method of claim 1, wherein the condensing agent is HATU and HOAT in a mass ratio of 1: 1;

optionally, the condensing agent is PyBOP, PyAOP, HCTU, HATU and HOAT or HBTU and HOBT, the condensation reaction further being carried out with DIEA added;

optionally, the condensing agent is HOBT, and the condensation reaction is further carried out with the addition of DIC.

4. The method of claim 1, wherein the crude peptide is synthesized by Fmoc solid phase synthesis.

5. The method according to claim 4, wherein the crude peptide has the amino acid sequence of RLRGG, and is obtained by:

the method comprises the steps of using chlorine resin as a solid phase carrier, activating by a condensing agent, coupling crude peptides to be synthesized to the chlorine resin one by one according to the sequence of amino acids from C end to N end, protecting the last amino acid by Cbz, and obtaining the crude peptides after full-protection cutting.

6. The process according to claim 5, wherein the coupling of the crude peptide to be synthesized onto the chlorine resin one by one in the order of amino acids from the C-terminus to the N-terminus is carried out at a temperature of 30 to 32 ℃ under activation of HCTU;

optionally, Fmoc removal was performed using 20% piperidine in DMF.

7. The method of claim 5, wherein the last amino acid is protected with Cbz by placing the chlorine resin with RLRGG attached in a mixed solution of DIEA, CbzCl and DMF at a volume ratio of 1:1: 8.

8. The method of claim 5, wherein the full protection cleavage is performed by placing the chlorine resin with RLRGG attached in a mixed solution of 1,1, 1,3,3, 3-hexafluoro-2-propanol and DCM with a volume ratio of 1,1, 1,3,3, 3-hexafluoro-2-propanol to DCM of 1: 4.

9. The method of claim 6, wherein coupling the crude peptide to be synthesized to the chlorine resin one by one in the order of amino acids from the C-terminus to the N-terminus comprises:

(1) carrying out first contact on Fmoc-Gly-OH dissolved by DMF and DIEA in a polypeptide synthesis tube at 30-32 ℃ so as to couple Gly to the chlorine resin;

(2) contacting a chlorine resin coupled with Gly with DMF solution containing 20% piperidine so as to remove Fmoc;

(3) carrying out second contact on Fmoc-Arg-OH dissolved by DMF and DIEA in a polypeptide synthesis tube at the temperature of 30-32 ℃, wherein the second contact is carried out under the activation of HCTU so as to couple Arg to the N end of Gly;

(4) contacting a chlorine resin coupled with Arg-Gly with a DMF solution containing 20% piperidine to remove Fmoc;

(5) carrying out third contact on Fmoc-Leu-OH dissolved by DMF and DIEA at 30-32 ℃ in a polypeptide synthesis tube, wherein the third contact is carried out under the activation of HCTU so as to couple Leu to the N terminal of Arg;

(6) contacting the chlorine resin coupled with Leu-Arg-Gly with DMF solution containing 20% piperidine to remove Fmoc;

(5) carrying out fourth contact on Fmoc-Arg-OH dissolved by DMF and DIEA at 30-32 ℃ in a polypeptide synthesis tube, wherein the fourth contact is carried out under the activation of HCTU so as to couple Arg to the N end of Leu;

(6) the chlorine resin coupled with Arg-Leu-Arg-Gly was contacted with DMF solution containing 20% piperidine to remove Fmoc.

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