Chemical synthesis method of N-terminal structural domain of TIMP2 protein

Abstract

The invention provides a chemical synthesis method of a TIMP2 protein N-terminal structural domain. An amino acid sequence of an N-terminal structural domain of TIMP2 protein is divided into four segments, the four segments are synthesized by a solid-phase polypeptide synthesis method, full-length linear polypeptide is obtained through natural chemical connection, sulfur removal and sulfydryl removal and acetamino methyl removal, finally impurities in a system are removed, the full-length linear polypeptide is dropwise added into a refolding reaction system, oxidation and refolding reactions are carried out, and the TIMP2 protein is obtained. A target product is obtained. The N-TIMP2 of the N-terminal structural domain of the TIMP2 retains the inhibitory activity of the TIMP2 on the MMP14, the N-TIMP2 obtained through chemical total synthesis can introduce non-natural amino acid to any site in the sequence or perform specific modification or mutation in the synthesis process, an effective tool is provided for researching the specific action mechanism of the N-TIMP2 and the MMP14, and a foundation is further laid for research and development of the MMP14 inhibitor.

Description

Technical Field

[0001] This invention relates to the field of solid-phase synthesis of peptides / proteins, specifically to a chemical synthesis method for the N-terminal domain of the TIMP2 protein. Background Technology

[0002] Since Merrifield proposed solid-phase peptide synthesis (SPPS) in 1963 (a discovery that won the Nobel Prize in Chemistry in 1984), SPPS has become the gold standard for peptide preparation (Merrifield, RB Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963)). The basic strategy involves covalently fixing the C-terminus of the target peptide to an insoluble solid support (such as resin microspheres), and then coupling the protected amino acids one by one in a cyclical "deprotection-coupling" process in a synthesis tube until sequence coupling is complete. Subsequently, the peptide chain is removed from the resin along with the side-chain protecting groups to obtain the target product. Compared to liquid-phase synthesis, SPPS has the following advantages: ① Each step can use excess amino acids and activators to drive the reaction to near completion, improving step yield and overall efficiency; ② The product is always fixed in the resin, and excess reagents and byproducts can be removed by simple filtration and washing after the reaction, making the operation convenient; ③ It has high programmability, which facilitates the precise introduction of non-natural amino acids and the implementation of site-specific modifications (including simulated post-translational modifications), etc. (Merrifield, RB Automated Synthesis of Peptides. Science. 150,178–185 (1965); Kaiser, E., Colescott, RL, Bossinger, CD&Cook, PIColor test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem. 34, 595–598 (1970).; Carpino, LA&Han, GY 9-Fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem.) 37,3404–3409 (1972).

[0003] As peptide chains elongate, long peptides on resin carriers tend to aggregate and form secondary structures, reducing coupling and deprotection efficiency. Consequently, the effective length of traditional SPPS is typically limited to about 50 amino acids. To overcome this bottleneck, Kent proposed "Native Chemical Ligation" (NCL) in 1994: chemically selectively linking peptide fragments with C-terminal thioesters to fragments with N-terminal cysteine ​​residues in an aqueous phase, thereby modularly splicing multiple fragments to synthesize proteins longer than 100 amino acids (Dawson, PE, Muir, TW, Clark-Lewis, I. & Kent, SBH Synthesis of proteins by Native Chemical Ligation. Science. 266, 776–779 (1994)).The development of NCL and its extended methods has significantly expanded the scale and types of chemical protein synthesis, providing efficient and practical routes for obtaining proteins containing non-natural amino acids, site-specific modifications, and isotopic labels (Bode, JW, Fox, RM & Baucom, KD Chemoselective amideligations by decarboxylative condensations of N-alkylhydroxylamines and alpha-ketoacids. AngewChemInt Ed Engl. 45, 1248–1252 (2006).; Lin, S., Mo, Z., Wang, P. & He, C. Oxidation and phenolysis of peptide / protein C-Terminalhydrazides afford salicylaldehyde ester surrogates for chemical protein synthesis. J. Am. Chem. Soc. 145, 16843–16851 (2023).; Liu, H. & Li, X. Serine / Threonine Ligation: origin, mechanistic aspects, and applications. Acc. Chem. Res. 51, 1643–1655 (2018).; Mitchell, NJ et al. Rapid additive-freeSelenocystine–Selenoester peptide ligation. J. Am. Chem. Soc. 137, 14011–14014 (2015).; Liao, P.&He, C. Azole reagents enabled ligation of peptide acylpyrazoles for chemical protein synthesis. Chem. Sci. 15, 7965–7974 (2024).).

[0004] Tissue inhibitors of matrix metalloproteinases (TIMPs) are a family of four endogenous proteins (TIMP1–4) that primarily inhibit the proteolytic activity of matrix metalloproteinases (MMPs) by forming 1:1 complexes with them. MMPs are involved in extracellular matrix (ECM) remodeling and are crucial in tumor cell migration and invasion (The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell 41, 374–403 (2023)). Among them, TIMP2 maintains microenvironment homeostasis by limiting the hydrolysis of surrounding proteins in the ECM and cell surface. More interestingly, TIMP2 possesses dual functions of "inhibition" and "bridging": its N-terminal domain binds to MMP14 (MT1-MMP), and its C-terminal domain binds to pro-MMP2, forming a ternary complex of MMP14–TIMP2–proMMP2 on the cell surface. Subsequently, the adjacent second MMP14 molecule acts as an activating enzyme to cleave proMMP2, releasing active MMP2 (Hernandez-Barrantes, S. et al. Binding of active (57 kDa) membranetype 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation. J Biol Chem. 275, 12080–12089 (2000).). Early development of broad-spectrum MMP inhibitors failed in clinical trials, essentially due to an incomplete understanding of the specific mechanisms of action between TIMP2 and MMP. Therefore, a detailed analysis of the interaction between TIMP2 and MMP14 / MMP2 may provide new insights for the development of specific inhibitors.Previous studies have shown that the N-terminal domain of TIMP2 (N-TIMP2) can retain the inhibitory activity against MMP14 (Fernandez-Catalan, C. et al. Crystal structure of the complex formed by themembrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor. EMBO J. 17, 5238–5248 (1998).; Hayun, H. et al. Bioorthogonal PEGylation prolongs theelimination half-life of N-TIMP2 while retaining MMP inhibition. BioconjugChem. 33, 795–806 (2022)). Therefore, the chemical total synthesis of high-purity N-TIMP2 is not only an ideal model for elucidating its mechanism of action with MMP14, but also lays the foundation for the rational design of MMP14 inhibitors. Summary of the Invention

[0005] The primary objective of this invention is to provide a chemical synthesis method for the N-terminal domain of the TIMP2 protein.

[0006] The segmented, modular N-terminal domain of the TIMP2 protein prepared by the method of this invention is separable and ultimately homogenized after natural chemical linkage and protein refolding.

[0007] The main inventive point of this invention is that it is the first time that N-TIMP has been fully synthesized using the solid-phase peptide synthesis method SPPS and the natural chemical linking reaction NCL.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention provides a chemical synthesis method for the N-terminal domain of the TIMP2 protein, comprising the following steps: (1) The amino acid sequence of the N-terminal domain N-TIMP2 of the TIMP2 protein was divided into fragment 1, fragment 2, fragment 3 and fragment 4; (2) Using the Fmoc solid-phase peptide synthesis method, fragments 1, 2, and 3 with a C-terminus of hydrazide and fragment 4 with a C-terminus of amide were synthesized. Thioester conversion yielded fragment 4 with a C-terminus of amide. Fragments 1 and 3 of mercaptophenylacetic acid (MPAA); (3) The fragments 1 and 3 with C-terminus MPAA, fragment 2 with C-terminus hydrazide and fragment 4 with C-terminus amide were ligated by natural chemical linking, desulfurized and desulfurized, and acetaminomethyl was removed to obtain a full-length linear polypeptide and remove impurities. (4) The full-length linear polypeptide with impurities removed is added dropwise to the refolding reaction system to carry out oxidation and refolding reactions to obtain the target product.

[0010] Furthermore, in step (1), the amino acid sequence of N-TIMP2 is CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSA (32)NDIYGNPIKRIQYEIKQIKM (52)FKGPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKM (96)HITLCDFIVPWDTLSTTQKKSLNHRYQM (124)GCE, as shown in SEQ ID No.1.

[0011] Furthermore, in step (1), fragments 1, 2, 3 and 4 are divided according to the amino acid sequence of the N-terminal domain of the TIMP2 protein, namely amino acids 1-31, 32-71, 72-100 and 101-127.

[0012] Furthermore, the alanine at position 32 in fragment 2 is synthesized as cysteine, which is then desulfurized and desulfurized to form alanine. The methionine at position 52 in fragment 2, position 96 in fragment 3, and position 124 in fragment 4 are replaced by ortholeucine (Nle) during the synthesis process.

[0013] Furthermore, the sequence of the N-TIMP2 polypeptide fragment 1 synthesized during the synthesis process, which has a C-terminus of hydrazide, is as follows: NH2-Cys(Acm)-Ser(R1)-Cys(Acm)-Ser(R1)-Pro-Val-His(R3)-Pro-Gln(R3)-Gln(R3)-Ala-Phe-Cys(Acm)-Asn(R3)-Ala-Asp (R2)-Val-Val-Ile-Arg(R5)-Ala-Lys(R4)-Ala-Val-Ser(R1)-Glu(R2)-Lys(R4)-Glu(R2)-Val-Asp(R2)-Ser(R1)-NHNH2, as SEQ Shown as IDNo.2.

[0014] Furthermore, the sequence of the N-TIMP2 polypeptide fragment 2 synthesized during the synthesis process, which has an acylhydrazine terminal, is as follows: NH2-Cys(R3)-Asn(R3)-Asp(R2)-Ile-Tyr(R1)-Gly-Asn(R3)-Pro-Ile-Lys(R4)-Arg(R5)-Ile-Gln(R3)-Tyr(R1)-Glu(R2)-Ile-Lys(R4)-Gln(R3)-Ile-Lys (R4)-Nle-Phe-Lys(R4)-Gly-Pro-Glu(R2)-Lys(R4)-Asp(R2)-Ile-Glu(R2)-Phe-Ile-Tyr(R1)-Thr(R1)-Ala-Pro-Ser(R1)-Ser(R1)-Ala-Val-NHNH2, as SEQ Shown as ID No.3.

[0015] Furthermore, the sequence of the N-TIMP2 polypeptide fragment 3, which has a C-terminus of hydrazide and is synthesized during the synthesis process, is as follows: NH2-Cys(R3)-Gly-(Val-Ser)-Leu-Asp(R2)-Val-Gly-Gly-Lys(R4)-Lys(R4)-Glu(R2)-Tyr(R1)-Leu-Il e-Ala-Gly-Lys(R4)-Ala-Glu(R2)-Gly-(Asp-Gly)-Lys(R4)-Nle-His(R3)-(Ile-Thr)-Leu-NHNH2, as SEQ Shown as ID No.4.

[0016] Furthermore, the sequence of the N-TIMP2 polypeptide fragment 4 synthesized during the synthesis process, with an amide terminus at the C-terminus, is as follows: NH2-Cys(R3)-Asp(R2)-Phe-Ile-Val-Pro-Trp(R4)-Asp(R2)-Thr(R1)-Lys(R4)-Ser(R1)-Thr(R1)-Thr(R1)-Gln(R3)-L ys(R4)-Lys(R4)-Ser(R1)-Leu-Asn(R3)-His(R3)-Arg(R5)-Tyr(R1)-Gln(R3)-Nle-Gly-Cys(Acm)-Glu(R2)-CONH2, as SEQ Shown as ID No.5.

[0017] Furthermore, in step (2), the Fmoc solid-phase polypeptide synthesis method involves connecting the amino acids sequentially from the C-terminus to the N-terminus through repeated coupling and deprotection steps to obtain an amino acid fragment.

[0018] Furthermore, the specific operation of sequentially condensing Fmoc-protected amino acids from the C-terminus to the N-terminus involves: under the action of a coupling system, first reacting the first amino acid with an acylhydrazine or amide resin that has had Fmoc removed to generate an amino acid. The amino resin is then coupled one by one with other Fmoc-protected amino acids to obtain a linear polypeptide resin; the condensing agent in the coupling system is "HOBT+DIC" or "TBTU+DIEA"; the resin loading is 0.3-0.5 mmol / g.

[0019] Furthermore, in step (2), fragments 1, 2, and 3 with a C-terminus of acylhydrazide and fragment 4 with a C-terminus of amide are synthesized. The amino acid sequence of N-TIMP2 contains isoleucine. The threonine amino acid site is used to form Fmoc isoleucine with the Psi(Me,Me)Pro structure using the threonine amino group and the side chain carboxyl group. The threonine dipeptide Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH is synthesized; for example, when the amino acid sequence of the N-terminal domain of the TIMP2 protein is as shown in the sequence listing, fragment 3 of the N-terminal domain of the TIMP2 protein is prone to aggregation, so when the 98th and 99th amino acid sites are isoleucine (Ile). The problems related to the synthesis of threonine (Thr) amino acids can be solved by using the above-mentioned dipeptides; among them, the pseudoproline structure can be reduced to the native peptide bond by removing the protecting group with trifluoroacetic acid; The amino acid sequence of N-TIMP2 contains aspartic acid. Fmoc aspartic acid with side chain carboxyl and amino protecting groups of OtBu and Dmb, respectively, at the glycine amino acid site. Glycine dipeptide Fmoc Asp(OtBu) (Dmb)Gly OH is used for synthesis; for example, when the amino acid sequence of the N-terminal domain of the TIMP2 protein is as shown in the sequence listing, the aspartic acid (Asp) at amino acid positions 93 and 94. Glycine (Gly) has a specific sequence, and stepwise synthesis is prone to isomerism, which makes it impossible to obtain a homogeneous polypeptide product during final purification. Using the above-mentioned dipeptide for synthesis can solve this problem; among them, the OtBu protecting group and Dmb protecting group can be removed by trifluoroacetic acid. The amino acid sequence of N-TIMP2 contains valine. Fmoc valine, formed by using the serine amino group and the side chain carboxyl group to form the Psi(Me,Me)Pro structure at the serine amino group site. The serine dipeptide Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH is synthesized; for example, when the amino acid sequence of the N-terminal domain of the TIMP2 protein is as shown in the sequence listing, fragment 3 of the N-terminal domain of the TIMP2 protein is prone to aggregation, so when the 74th and 75th amino acid sites are valine (Val). The serine (Ser) amino acid site can be addressed by using the aforementioned dipeptide for synthesis; in particular, the pseudoproline structure can be reduced to its native peptide bond by removing the protecting group with trifluoroacetic acid. The cysteine ​​residue in fragment 1 is protected with Fmoc cysteine, which has an acetaminomethyl side chain protecting group. Cys(Acm) Synthesized using OH; Cysteine ​​residues at non-linking sites in fragment 4 are protected with Fmoc cysteine ​​(Fmoc) whose side chain thiol protecting group is acetaminomethyl. Cys(Acm) Synthesized using OH.

[0020] Furthermore, the structural formula of Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH is shown below: ; The amount of Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH is calculated based on a molar ratio of 1:2~4 for the loading amount of Fmoc amino resin per gram to Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH. Furthermore, Fmoc Asp(OtBu) (Dmb)Gly The structural formula of OH is shown below: ; Fmoc Asp(OtBu) (Dmb)Gly The amount of OH is based on the loading amount per gram of Fmoc amino resin: Fmoc Asp(OtBu) (Dmb)Gly Calculation of molar ratios of OH = 1:2~4; Furthermore, the structural formula of Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH is shown below: ; The amount of Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH is calculated based on a molar ratio of 1:2~4 for the loading amount of Fmoc amino resin per gram to Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH. Furthermore, Fmoc Cys(Acm) The structural formula of OH is shown below: ; Fmoc Cys(Acm) The amount of OH is based on the loading amount per g of Fmoc amino resin: Fmoc Cys(Acm) Calculation of the molar ratio of OH = 1:2~4.

[0021] Further, in step (2), fragments 1, 2, and 3 with C-terminus hydrazide and fragment 4 with C-terminus amide are synthesized. Other sites in the amino acid sequence of N-TIMP2 are synthesized by Fmoc-protected amino acids without side chains, or Fmoc-protected amino acids with side chain protecting groups that can be removed by trifluoroacetic acid, namely R1, R2, R3, R4, or R5. R1 represents tert-butyl, R2 represents tert-butyl ester, R3 represents triphenylmethyl, R4 represents tert-butyloxycarbonyl, and R5 represents 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl.

[0022] Furthermore, when synthesizing Fmoc protected amino acids without side chains, or Fmoc protected amino acids with side chain protecting groups of R1, R2, R3, R4, or R5, the amount used is calculated based on a molar ratio of Fmoc amino resin loading to Fmoc protected amino acid of 1:4; where: R1 represents tert-butyl, R2 represents tert-butyl ester, R3 represents triphenylmethyl, R4 represents tert-butyloxycarbonyl, and R5 represents 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl. The coupling time between the Fmoc protected amino acid without side chains and the Fmoc amino resin is 2-4 h.

[0023] Furthermore, in step (3), during the natural chemical linking and desulfurization process, fragment 1 with C-terminus MPAA and fragment 2 with C-terminus hydrazide are linked in a one-pot linking-desulfurization process in a solution containing imidazole, without the need to add additional thiol reagents.

[0024] Furthermore, in step (4), during the refolding reaction, the six free thiol groups at the six cysteine ​​sites of the linear polypeptide of the N-terminal domain of the TIMP2 protein are oxidized to form three pairs of disulfide bonds: the first and 72nd positions form one pair, the third and 101st positions form one pair, and the 13th and 126th positions form one pair.

[0025] Furthermore, a chemical synthesis method for the N-terminal domain of the TIMP2 protein includes the following steps: (1) Using Fmoc amino resin with a C-terminus of hydrazide, referred to as hydrazide resin, the Fmoc solid-phase peptide synthesis method was adopted. According to the sequences of fragments 1, 2, and 3, Fmoc protected amino acids were sequentially condensed from the C-terminus to the N-terminus. After washing and drying, hydrazide resins loaded with fragments 1, 2, and 3 were obtained. Among them, the cysteine ​​in the sequence of fragment 1 was protected by Fmoc cysteine ​​Fmoc with a side chain thiol protecting group of acetaminomethyl. Cys(Acm) OH is synthesized to protect it from being affected in subsequent desulfurization reactions; (2) Using Fmoc amino resin with an amide terminal at the C-terminus (referred to as amide resin), the Fmoc solid-phase peptide synthesis method was employed. Based on the sequence of fragment 4, Fmoc protected amino acids were sequentially condensed from the C-terminus to the N-terminus. After washing and drying, the amide resin loaded with fragment 4 was obtained. Among them, the cysteine ​​residues at non-linking sites in the sequence of fragment 4 were protected by Fmoc cysteine ​​with acetaminomethyl side chain thiol protecting groups. Cys(Acm) OH is used in the synthesis to ensure that subsequent linking reactions proceed with maximum efficiency; (3) Take the hydrazide resin loaded with fragment 1, add the cleavage reagent to remove the polypeptide chain from the hydrazide resin and remove the remaining side chain protecting groups; after filtration, rotary drying, extraction, centrifugation and freeze drying, the crude product of fragment 1 with hydrazide at the C-terminus is obtained. (4) Take the hydrazide resin loaded with fragment 2, add the cleavage reagent to remove the polypeptide chain from the amide resin and remove the remaining side chain protecting groups; after filtration, rotary drying, extraction, centrifugation and freeze drying, crude fragment 2 is obtained; further separation and purification, freeze drying, to obtain pure fragment 2 with hydrazide at the C-terminus. (5) Take the hydrazide resin loaded with fragment 3, add the cleavage reagent to remove the polypeptide chain from the hydrazide resin and remove the remaining side chain protecting groups; after filtration, rotary drying, extraction, centrifugation and freeze drying, the crude product of fragment 3 with hydrazide at the C-terminus is obtained. (6) Take the amide resin loaded with fragment 4, add the cleavage reagent to remove the polypeptide chain from the amide resin and remove the remaining side chain protecting groups; after filtration, rotary drying, extraction, centrifugation and freeze drying, crude fragment 2 is obtained; further separation and purification, freeze drying, to obtain pure fragment 4 with C-terminus amide; (7) Take the crude fragment 1 with the C-terminus of hydrazide, dissolve it, add acetylacetone, shake well, and then add 4 Mercaptophenylacetic acid (MPAA), first step reaction, addition of tri(2) The second step of the reaction was to convert carboxyethyl phosphine (TCEP) to TCEP, followed by centrifugation, filtration, further separation and purification, and lyophilization to obtain fragment 1 (thioester conversion) with MPAA at the C-terminus. (8) Take the crude fragment 3 with the C-terminus of hydrazide, dissolve it, add acetylacetone, shake well, and then add 4 Mercaptophenylacetic acid (MPAA), first step reaction, addition of tri(2) The second step of the reaction was to convert carboxyethyl phosphine (TCEP) to TCEP, followed by centrifugation, filtration, further separation and purification, and lyophilization to obtain fragment 3 (thioester conversion) with MPAA at the C-terminus. (9) Take fragment 2 with a C-terminus of hydrazide and fragment 1 with a C-terminus of MPAA, dissolve them separately, mix them, and add tri(2) Carboxyethyl)phosphine TCEP undergoes a natural chemical linkage reaction, and then tri(2)phosphine is added to the solution. The fragment 6, which was desulfurized by carboxyethyl phosphine TCEP, azobisisobutyridine imidazoline hydrochloride VA-044, and tert-butylthiol t-BuSH, was centrifuged, filtered, further separated and purified, and lyophilized to obtain a pure fragment with an acylhydrazine at the C-terminus. The free cysteine ​​in the sequence was desulfurized and the thiol group was removed to become alanine. (10) Take fragment 4, which has an amide at the C-terminus, and fragment 3, which has an MPAA at the C-terminus, dissolve them separately, mix them, and add tri(2) Carboxyethylphosphine TCEP was used for a natural chemical linking reaction. Palladium chloride was added to the solution for the first step reaction. Dithiothreitol was added for the second step reaction. After centrifugation and filtration, the fragment 8 with an amide at the C-terminus was obtained. The free cysteine ​​in the sequence was deprotected by acetaminomethyl. (11) Take fragment 6 with the C-terminus of acylhydrazine, dissolve it, add acetylacetone (acac), shake well, and then add 4 The MPAA reaction of mercaptophenylacetic acid was confirmed by reversed-phase high-performance liquid chromatography-mass spectrometry to convert the terminal of the hydrazide to a thioester. Fragment 8, with an amide at the C-terminus, was taken, dissolved, and tri(2)-methyl-2-ethylhexylene (MTJ) was added. Carboxyethylphosphine (TCEP) was used to adjust the pH to 6.5 and then subjected to natural chemical linkage to obtain a crude solution of N-TIMP2 linear peptide, referred to as solution M. (12) Take solution M, add it to an ultrafiltration tube, and ultrafilter the concentrated replacement solution system to obtain N-TIMP2 crude polypeptide solution. Add palladium chloride to N-TIMP2 crude polypeptide solution to carry out the first step reaction, then add dithiothreitol to carry out the second step reaction. Centrifuge, filter, separate and purify to obtain N-TIMP2 polypeptide solution with cysteine ​​deacetaminomethyl protection, which is called solution N. (13) Take solution N, add it to an ultrafiltration tube, and replace the solution system while concentrating it by ultrafiltration to obtain a full-length linear peptide solution of N-TIMP2 without MPAA and TCEP. Slowly add it to the refolding reaction system, stir at room temperature, place it in a 4℃ refrigerator and let it stand overnight (18h) to react. Centrifuge, filter, and further separate and purify to obtain the refolded N-TIMP2.

[0026] Furthermore, in step (9), the amount of tert-butylthiol used is calculated as 5% to 10% of the solution volume; Furthermore, in step (9), the reaction time for the natural chemical linkage reaction is 4 to 6 hours.

[0027] Furthermore, in step (9), the desulfurization reaction takes 20 to 24 hours.

[0028] Furthermore, in step (10), the reaction time for the natural chemical linkage reaction is 4 to 6 hours.

[0029] Furthermore, in step (11), the reaction time of fragment 6, which has an acylhydrazine at the C-terminus, with MAPP is 6 to 10 hours.

[0030] Furthermore, in step (11), the reaction time of the natural chemical linkage reaction is 4 to 6 hours; Furthermore, the volume of the refolding reaction system in step (13) is calculated based on 0.2 to 0.5 mg of linear polypeptide dissolved in 1 mL of refolding buffer solution.

[0031] Furthermore, in step (13), the slow dripping time is 15 to 30 minutes.

[0032] Furthermore, in step (13), the refolding reaction time is 16 to 20 hours.

[0033] The present invention also provides an N-TIMP2 N-terminal domain of TIMP2 protein, which is prepared by the above-mentioned chemical synthesis method of the N-terminal domain of TIMP2 protein.

[0034] This invention also provides the application of the N-terminal domain N-TIMP2 of the above-mentioned TIMP2 protein in studying the specific mechanism of action of N-TIMP2 and MMP14.

[0035] The principle of this invention is as follows: Using the Fmoc solid-phase peptide synthesis method, the amino acid sequence of the N-terminal domain of the TIMP2 protein was modularly divided into four peptide fragments. Peptide fragment 1 consists of amino acids 1-31 of the N-terminal domain of the TIMP2 protein. Using an acylhydrazine resin as a carrier, Fmoc peptides with acetaminomethyl thiol side chains were synthesized at the cysteine ​​residues (positions 1, 3, and 13). Cys(Acm) To prevent the removal of the sulfhydryl group of cysteine ​​during subsequent desulfurization, Fmoc-protected amino acids are sequentially condensed from the C-terminus to the N-terminus to obtain hydrazide resin for peptide fragment 1 of the N-terminal domain of TIMP2 protein; Peptide fragment 2 consists of amino acids 32-71 of the N-terminal domain of TIMP2 protein, and Fmoc-protected amino acids are sequentially condensed from the C-terminus to the N-terminus using hydrazide resin as a carrier to obtain hydrazide resin for peptide fragment 2 of the N-terminal domain of TIMP2 protein; Peptide fragment 3 consists of amino acids 72-100 of the N-terminal domain of TIMP2 protein, and Fmoc-protected amino acids are sequentially condensed from the C-terminus to the N-terminus using hydrazide resin as a carrier to obtain hydrazide resin for peptide fragment 3 of the N-terminal domain of TIMP2 protein; Peptide fragment 4 consists of amino acids 101-127 of the N-terminal domain of TIMP2 protein, and Fmoc with an acetaminomethyl side chain is used at the cysteine ​​position 126 using MBHA resin as a carrier. Cys(Acm) OH is added to ensure that subsequent natural chemical linkages proceed with the highest efficiency. Fmoc-protected amino acids are condensed sequentially from the C-terminus to the N-terminus to obtain the amino resin of the N-terminal domain polypeptide fragment 4 of the TIMP2 protein. After the first natural chemical linkage and desulfurization, the second natural chemical linkage and deacetylation, and the third natural chemical linkage and deacetylation, a crude linear polypeptide solution with completely deprotected N-terminal domain side chains of the TIMP2 protein was obtained. This solution was purified by size exclusion chromatography, ultrafiltration centrifugation, and solution replacement to remove MPAA and TCEP, which would affect the refolding reaction. Then, it was slowly added dropwise to a large-volume refolding reaction system, causing the six free sulfhydryl groups at the six cysteine ​​sites (positions 1, 3, 13, 72, 101, and 126) of the linear polypeptide to oxidize and form three pairs of disulfide bonds (positions 1 and 72 form one pair, position 3 and 101 form one pair, and position 13 and 126 form one pair), resulting in the folded N-terminal domain N-TIMP2 of the TIMP2 protein.

[0036] The present invention has the following advantages and effects compared with the prior art: This invention is the first to obtain the N-terminal domain N-TIMP2 of the TIMP2 protein through total chemical synthesis and verify that its disulfide bonds are correctly paired.

[0037] N-TIMP2 retains the inhibitory activity of TIMP2 against MMP14. The method provided by this invention can precisely introduce non-natural amino acids or site-specific modifications and mutations at any site in the sequence during the synthesis process, providing an effective tool for studying the specific mechanism of action of N-TIMP2 and MMP14, and further laying the foundation for the development of MMP14 inhibitors.

[0038] In the method provided by this invention, after obtaining the crude product of fragment 1 with C-terminal hydrazide, the C-terminal hydrazide is converted into MPAA through a thioester conversion reaction at pH 3 of the reaction system. The MPAA is then separated, purified, and lyophilized, so that fragment 1 with C-terminal MPAA exists stably in lyophilized form. This avoids the hydrolysis reaction that easily occurs in MPAA at pH > 7. Furthermore, no additional thiol reagent is required during the connection process between fragment 1 with C-terminal MPAA and fragment 2. One-pot connection-desulfurization can be carried out in a solution containing 2.5 mmol / L imidazole, thereby improving the yield. Attached Figure Description

[0039] Figure 1 Segment 2 in Example 1 Reverse high performance liquid chromatography and ESI of pure NHNH2 Characterization of MS.

[0040] Figure 2 Segment 4 in Example 1 Reverse high performance liquid chromatography and ESI of pure CONH2 Characterization of MS.

[0041] Figure 3 Reverse high performance liquid chromatography and ESI of the pure 1-MPAA fragment in Example 1 Characterization of MS.

[0042] Figure 4 Reverse high performance liquid chromatography and ESI of the pure 3-MPAA fragment in Example 1 Characterization of MS.

[0043] Figure 5 For example, fragment 1-MPAA and fragment 2 in Example 1 Natural chemical bonding of NH2 and one-pot desulfurization ( Figure 5 -A represents the reversed-phase high-performance liquid chromatography (RP-HPLC) of the naturally chemically linked and desulfurized pure product. Figure 5 -B represents the ESI of the bonding product before desulfurization. Characterization of MS Figure 5 -C represents the ESI of the desulfurized product after connection. Characterization of MS).

[0044] Figure 6 For example, fragments 3-MPAA and 4 in Example 1 The natural chemical linkage of CONH2 and one-pot deacetylation of aminomethyl groups ( Figure 6 -A is the reverse-phase high-performance liquid chromatography (RP-HPLC) of the purified product after natural chemical linkage and deacetylation. Figure 6 -B represents the ESI of the ligation product before deacetamidomethylation. Characterization of MS Figure 6-C represents the ESI of the deacetamidomethylated linker product. Characterization of MS).

[0045] Figure 7 The natural chemical linking and deacetylation of fragment 6 (C-terminus of hydrazide) and fragment 8 (C-terminus of amide) in Example 1. Figure 7 -A represents reversed-phase high-performance liquid chromatography (HPLC) of natural chemical linkages and refolding after deacetylation of aminomethyl groups. Figure 7 -B represents the ESI of the ligation product before refolding. Characterization of MS Figure 7 -C represents the ESI after refolding of the ligation product. Characterization of MS).

[0046] Figure 8 This is a graph showing the results of liquid chromatography-mass spectrometry (LC-MS) analysis after N-TIMP2 digestion in Example 1.

[0047] Figure 9 This is a schematic diagram of the chemical synthesis method for the N-terminal domain of the TIMP2 protein. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Fmoc used in the following examples Cys (Acm) OH, its structural formula is shown below:

[0051] The structural formula of Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH used in the following embodiments is shown below:

[0052] Fmoc used in the following examples Asp(OtBu) (Dmb)Gly The structural formula of OH is shown below:

[0053] The structural formula of Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH used in the following embodiments is shown below:

[0054] The remaining Fmoc-protected amino acids (Fmoc-protected amino acids without side chains, or Fmoc-protected amino acids whose protecting groups R1, R2, R3, R4, or R5 on the side chain can be removed with trifluoroacetic acid) are Fmoc-protected amino acids. Ala OH, Fmoc Cys(Trt) OH, Fmoc Asp(OtBu) OH, Fmoc Glu(OtBu) OH, Fmoc Phe OH, Fmoc Gly OH, Fmoc His(Trt) OH, Fmoc Ile OH, Fmoc Lys(Boc) OH, Fmoc Leu OH, Fmoc Asn(Trt) OH, Fmoc Pro OH, Fmoc Gln(Trt) OH, Fmoc Arg(Pbf) OH, Fmoc Ser(tBu) OH, Fmoc Thr(tBu) OH, Fmoc Val OH, Fmoc Trp(Boc) OH, Fmoc Tyr(tBu) OH, Fmoc Nle OH.

[0055] In the following examples, Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH, Fmoc Asp(OtBu) (Dmb)Gly OH, Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH and Fmoc Cys(Acm) The OH condensing agent used was HOBT+DIC, with a condensation time of 4 hours; the remaining Fmoc-protected amino acid condensing agents used were TBTU+DIEA, with a condensation time of 2 hours.

[0056] Fmoc in the following examples Cys(Acm) OH, Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH, Fmoc Asp(OtBu) (Dmb)Gly The dosage of OH and Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH is twice the molar amount calculated based on the loading amount of each g of Fmoc amino resin; the dosage of the remaining Fmoc protected amino acids is four times the molar amount calculated based on the loading amount of each g of Fmoc amino resin.

[0057] In the following examples, the HPLC instrument used was an Agilent 1260, the chromatographic column was a Phenomenex C4 column, and the mobile phase was water and acetonitrile (containing 0.1% (v / v) TFA).

[0058] The sequence of the N-TIMP2 polypeptide fragment 1 with a C-terminus of hydrazide synthesized in the following examples is as follows: NH2-Cys(Acm)-Ser(R1)-Cys(Acm)-Ser(R1)-Pro-Val-His(R3)-Pro-Gln(R3)-Gln(R3)-Ala-Phe-Cys(Acm)-Asn(R3)-Ala-A sp(R2)-Val-Val-Ile-Arg(R5)-Ala-Lys(R4)-Ala-Val-Ser(R1)-Glu(R2)-Lys(R4)-Glu(R2)-Val-Asp(R2)-Ser(R1)-NHNH2 The sequence of the N-TIMP2 polypeptide fragment 2 synthesized in the following examples, with a C-terminus of hydrazide, is as follows (the N-terminus is cysteine, which is converted to alanine after desulfurization and removal of the sulfhydryl group): NH2-Cys(R3)-Asn(R3)-Asp(R2)-Ile-Tyr(R1)-Gly-Asn(R3)-Pro-Ile-Lys(R4)-Arg(R5)-Ile-Gln(R3)-Tyr(R1)-Glu(R2)-Ile-Lys(R4)-Gln(R3)-Ile- Lys(R4)-Nle-Phe-Lys(R4)-Gly-Pro-Glu(R2)-Lys(R4)-Asp(R2)-Ile-Glu(R2)-Phe-Ile-Tyr(R1)-Thr(R1)-Ala-Pro-Ser(R1)-Ser(R1)-Ala-Val-NHNH2 The sequence of the N-TIMP2 polypeptide fragment 3 with a C-terminus of hydrazide synthesized in the following examples is as follows: NH2-Cys(R3)-Gly-(Val-Ser)-Leu-Asp(R2)-Val-Gly-Gly-Lys(R4)-Lys(R4)-Glu(R2)-Tyr(R1)-Leu -Ile-Ala-Gly-Lys(R4)-Ala-Glu(R2)-Gly-(Asp-Gly)-Lys(R4)-Nle-His(R3)-(Ile-Thr)-Leu-NHNH2 The sequence of the N-TIMP2 polypeptide fragment 4 synthesized in the following examples, which has an amide terminus at the C-terminus, is as follows: NH2-Cys(R3)-Asp(R2)-Phe-Ile-Val-Pro-Trp(R4)-Asp(R2)-Thr(R1)-Lys(R4)-Ser(R1)-Thr(R1)-Thr(R1)-Gln(R3) -Lys(R4)-Lys(R4)-Ser(R1)-Leu-Asn(R3)-His(R3)-Arg(R5)-Tyr(R1)-Gln(R3)-Nle-Gly-Cys(Acm)-Glu(R2)-CONH2 Figure 9 A schematic flowchart of the chemical synthesis method for the N-terminal domain of the TIMP2 protein provided by the present invention is shown.

[0059] The reagent names and abbreviations used in the following examples are as follows: Fmoc-NHNH2: 9-fluorenylmethylhydrazine carbamate; DMF: N, N Dimethylformamide; DCM: Dichloromethane; MeOH: Methanol; HOBT: 1 Hydroxybenzotriazole; DIC: N, N Diisopropylcarbodiimide; TBTU: Benzotriazole tetramethyltetrafluoroboric acid; DIEA: N, N Diisopropylethylamine; NMM: N Methylmorpholine; TFA: Trifluoroacetic acid; DODT: 1, 4, 7, 10 Tetraazacyclododecane 1, 4, 7, 10 Tetraacetic acid; MeCN: Acetonitrile; TCEP: Three (2) Carboxyethyl phosphine (TCEP); MPAA: 4 Mercaptophenylacetic acid; PBS: Sodium dihydrogen phosphate; TCEP: Three (2) (Carboxyethyl)phosphine; acac: Acetylacetone; VA-044: Azobisisobutyrazoline hydrochloride; t-BuSH: tert-butylthiol; Example 1: Chemical synthesis method of the N-terminal domain of TIMP2 protein: (1) Fmoc Preparation of NHNH2 resin: Take 1g of NHNH2 resin. Chlorotrityl Chloride resin (abbreviated as 2) Fmoc-NHNH2 (dissolved in dry DMF) was added to a peptide synthesis tube with a loading of 0.5 mmol / g. 15 mL of DMF was added, and the mixture was shaken twice at room temperature for 30 min each time. After draining, 0.5 mmol of two molar equivalents of Fmoc-NHNH2 dissolved in dry DMF was added to the resin. The mixture was shaken at room temperature for 90 min. After draining, the resin was washed three times each with dry DMF and DCM, and the solvent was drained. The resin was then dried under nitrogen to obtain dry resin. The resin loading was determined by UV spectroscopy, yielding a final Fmoc loading of 0.35 mmol / g. NHNH2 resin. After re-swelling the resin with DMF, 15 mL of 5% (v / v) MeOH / DMF was added to its polypeptide synthesis tube. The reaction was shaken at room temperature for 10 min. The resin was washed three times each with DMF and DCM, the solvent was drained, the un-hydrazide-treated resin sites were blocked, and the resin was dried with nitrogen to obtain dry resin.

[0060] (2) Peptide fragment 1 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of NHNH2 resin: Take the Fmoc obtained according to (1) NHNH2 resin (loading 0.35 mmol / g) was swollen twice with 15 mL of DMF at room temperature by shaking for 15 min each time. The solution was then drained. Next, 10 mL of 20% (v / v) acetic anhydride / DMF was added to the resin, and the mixture was shaken at room temperature for 20 min to block the amino groups of the uncoupled amino acids, preventing further reaction. The resin was then washed twice each with DMF, DCM, and DMF, and the solvent was drained. Then, 10 mL of 20% (v / v) piperidine / DMF was added to the resin, and the mixture was shaken at room temperature for 8 min. After washing twice with DMF, another 10 mL of 20% (v / v) piperidine / DMF was added, and the mixture was shaken at room temperature for 6 min. This process was repeated twice with DMF, DCM, and DMF, and the solvent was drained to obtain the resin deprotected from Fmoc. Fmoc was weighed... Ser(tBu) OH (4 × 0.35 mmol), TBTU (3.9 × 0.35 mmol), and DIEA (8 × 0.35 mmol) were used to dissolve Fmoc amino acids and TBTU in a small amount of DMF. DIEA (condensing agent TBTU + DIEA) was added, and the reaction was carried out at room temperature with shaking for 3 min to activate the carboxyl group. Then, the activated amino acids were added to the resin and the reaction was carried out at room temperature with shaking for 2 h. After the reaction was completed, the solution was monitored with ninhydrin reagent. The solution was washed twice each with DMF, DCM, and DMF. The above experimental procedures were repeated to condense Fmoc protected amino acids (the amount of amino acids was calculated as 4 molar equivalents per g of resin loading, and the shaking reaction was carried out for 2 h; for cysteine, Fmoc cysteine ​​with acetaminomethyl side chain protecting group was used). Cys(Acm) The synthesis was performed using OH, with the amount calculated as 4 molar equivalents of the resin loading per g. The reaction was carried out with shaking for 4 h. The condensing agent used was HOBT+DIC. Coupling was performed from the C-terminus to the N-terminus of the TIMP2 protein N-terminal domain polypeptide fragment 1. After synthesis, the sample was washed three times each with DMF and DCM, and dried under nitrogen to obtain 2.8 g of dried resin.

[0061] (3) Peptide fragment 2 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of NHNH2 resin: Take the Fmoc obtained according to (1) NHNH2 resin (loading 0.35 mmol / g) was swollen twice with 15 mL of DMF at room temperature by shaking for 15 min each time. The solution was then drained. Next, 10 mL of 20% (v / v) acetic anhydride / DMF was added to the resin, and the mixture was shaken at room temperature for 20 min to block the amino groups of the uncoupled amino acids, preventing further reaction. The resin was then washed twice each with DMF, DCM, and DMF, and the solvent was drained. Then, 10 mL of 20% (v / v) piperidine / DMF was added to the resin, and the mixture was shaken at room temperature for 8 min. After washing twice with DMF, another 10 mL of 20% (v / v) piperidine / DMF was added, and the mixture was shaken at room temperature for 6 min. This process was repeated twice with DMF, DCM, and DMF, and the solvent was drained to obtain the resin deprotected from Fmoc. Fmoc was weighed... Val OH (4 × 0.35 mmol), TBTU (3.9 × 0.35 mmol), and DIEA (8 × 0.35 mmol) were used to dissolve Fmoc amino acids and TBTU in a small amount of DMF. DIEA (condensing agent TBTU + DIEA) was added, and the reaction was carried out at room temperature with shaking for 3 min to activate the carboxyl groups. Then, the activated amino acids were added to the resin and the reaction was carried out at room temperature with shaking for 2 h. After the reaction was monitored with ninhydrin reagent, the resin was washed twice each with DMF, DCM, and DMF. The above experimental procedures were repeated to condense Fmoc protected amino acids (the amount of amino acids was calculated as 4 molar equivalents per g of resin loading, and the shaking reaction was carried out for 2 h). The coupling was performed from the C-terminus to the N-terminus of the TIMP2 protein N-terminal domain polypeptide fragment 2 sequence. After synthesis, the resin was washed three times each with DMF and DCM and dried under nitrogen to obtain 2.4 g of dry resin.

[0062] (4) Peptide fragment 3 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of NHNH2 resin: Take the Fmoc obtained according to (1) NHNH2 resin (loading 0.35 mmol / g) was swollen twice with 15 mL of DMF at room temperature by shaking for 15 min each time. The solution was then drained. Next, 10 mL of 20% (v / v) acetic anhydride / DMF was added to the resin, and the mixture was shaken at room temperature for 20 min to block the amino groups of the uncoupled amino acids, preventing further reaction. The resin was then washed twice each with DMF, DCM, and DMF, and the solvent was drained. Then, 10 mL of 20% (v / v) piperidine / DMF was added to the resin, and the mixture was shaken at room temperature for 8 min. After washing twice with DMF, another 10 mL of 20% (v / v) piperidine / DMF was added, and the mixture was shaken at room temperature for 6 min. This process was repeated twice with DMF, DCM, and DMF, and the solvent was drained to obtain the resin deprotected from Fmoc. Fmoc was weighed... Leu OH (4 × 0.35 mmol), TBTU (3.9 × 0.35 mmol), and DIEA (8 × 0.35 mmol) were used to dissolve Fmoc amino acids and TBTU in a small amount of DMF. DIEA (condensing agent TBTU + DIEA) was added, and the reaction was carried out at room temperature with shaking for 3 min to activate the carboxyl groups. The activated amino acids were then added to the resin, and the reaction was carried out at room temperature with shaking for 2 h. The reaction was monitored with ninhydrin reagent until completion, and the mixture was washed twice each with DMF, DCM, and DMF. The above experimental procedures were repeated for the condensation of Fmoc-protected amino acids (the amount of amino acids was calculated as 4 molar equivalents per g of resin loading, and the shaking reaction was carried out for 2 h; isoleucine was included). Threonine amino acid site, aspartic acid glycine amino acid sites and valine The serine amino acid site uses Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH, Fmoc Asp(OtBu) (Dmb)Gly OH and Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH amino acids were synthesized, with the amount calculated as twice the molar equivalent of the resin loading per g. The reaction was shaken for 4 h, and HOBT+DIC was used as the condensing agent. Coupling was performed from the C-terminus to the N-terminus of the TIMP2 protein N-terminal domain polypeptide fragment 3 sequence. After synthesis, the mixture was washed three times each with DMF and DCM, and dried under nitrogen to obtain 2.6 g of dried resin.

[0063] (5) Fmoc Glu(OtBu) Preparation of MBHA resin: 1 g of Rink Amide MBHA resin was placed in a peptide synthesis tube with a loading of 0.5 mmol / g. 15 mL of DMF was added, and the resin was shaken twice at room temperature for 15 min each time. After draining, 10 mL of 20% (v / v) piperidine / DMF was added to the resin, and the mixture was shaken at room temperature for 8 min. After washing twice with DMF, another 10 mL of 20% (v / v) piperidine / DMF was added, and the mixture was shaken at room temperature for 6 min. The resin was then washed twice each with DMF, DCM, and DMF, and the solvent was drained to obtain resin with Fmoc protection removed. 0.5 mmol of 2 molar equivalents of Fmoc was weighed. Glu(OtBu) OH (1 mmol), TBTU (0.98 mmol), and DIEA (2 mmol) were added to Fmoc. Glu(OtBu) OH and TBTU were dissolved in a small amount of DMF, and DIEA was added. The reaction was carried out at room temperature with shaking for 3 min to activate the carboxyl groups. Then, the activated amino acids were added to the resin and the reaction was carried out at room temperature with shaking for 2 h. The resin was washed three times each with DMF and DCM, and dried with nitrogen to obtain dry resin. The resin loading was measured by UV, and the final result was Fmoc with a loading of approximately 0.42 mmol / g. Glu(OtBu) MBHA resin.

[0064] (6) Peptide fragment 4 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of CONH2 resin: Take the Fmoc obtained according to operation (5) Glu(OtBu) MBHA resin (loading capacity 0.42 mmol / g) was swollen twice with 15 mL of DMF at room temperature by shaking for 15 min each time. The solution was then drained. Next, 10 mL of 20% (v / v) acetic anhydride / DMF was added to the resin, and the mixture was shaken at room temperature for 20 min to block the amino groups of the uncoupled amino acids, preventing further reaction. The resin was then washed twice each with DMF, DCM, and DMF, and the solvent was drained. Then, 10 mL of 20% (v / v) piperidine / DMF was added to the resin, and the mixture was shaken at room temperature for 8 min. After washing twice with DMF, another 10 mL of 20% (v / v) piperidine / DMF was added, and the mixture was shaken at room temperature for 6 min. This process was repeated twice with DMF, DCM, and DMF, and the solvent was drained to obtain the resin deprotected from Fmoc. Fmoc was weighed... Cys(Acm) OH (4 × 0.42 mmol), TBTU (3.9 × 0.42 mmol), and DIEA (8 × 0.42 mmol) were used to dissolve Fmoc amino acids and TBTU in a small amount of DMF. DIEA (condensing agent TBTU + DIEA) was added, and the reaction was carried out at room temperature with shaking for 3 min to activate the carboxyl group. Then, the activated amino acid was added to the resin, and the reaction was carried out at room temperature with shaking for 2 h. After the reaction was completed, the solution was monitored with ninhydrin reagent. The solution was washed twice each with DMF, DCM, and DMF. The above experimental procedures were repeated to condense Fmoc-protected amino acids (the amount of amino acid was calculated as 4 molar equivalents per g of resin loading, and the shaking reaction was carried out for 2 h; for cysteine ​​at non-linking sites, Fmoc cysteine ​​with acetaminomethyl side chain protecting group was used). Cys(Acm) OH was used for synthesis, with the amount calculated as 4 molar equivalents of the resin loading per gram. The reaction was shaken for 4 hours. HOBT+DIC was used as the condensing agent. Coupling was performed from the C-terminus to the N-terminus of the TIMP2 protein N-terminal domain polypeptide fragment 1 sequence. Cysteine ​​residues at non-linking sites were protected with Fmoc cysteine ​​residues with acetaminomethyl side chain thiol protecting groups. Cys(Acm) The synthesis was carried out using OH. After the synthesis was completed, the resin was washed three times each with DMF and DCM, and then dried with nitrogen to obtain 3.2 g of dried resin.

[0065] (7) Fragment 1 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of crude NHNH2: Take 500 mg of the resin obtained in step (2), add 15 mL of cleavage reagent (TFA:DODT:H2O = volume ratio 95:2.5:2.5), shake and react for 2 h, filter to obtain a yellow transparent liquid, evaporate the liquid to dryness using a rotary evaporator, add about 20 mL of ice-cold diethyl ether to extract twice, centrifuge and collect the precipitate, freeze-dry the sample to obtain about 240 mg of fragment 1 with C-terminus hydrazide (fragment 1) NHNH2) crude product.

[0066] (8) Fragment 2 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of pure NHNH2: Take 500 mg of the resin obtained in step (3), add 15 mL of cleavage reagent (TFA:DODT:H2O = volume ratio 95:2.5:2.5), shake for 2 h, filter to obtain a yellow transparent liquid, evaporate the liquid to dryness using a rotary evaporator, add about 20 mL of ice-cold ether for extraction twice, centrifuge and collect the precipitate, freeze dry to obtain crude fragment 2, dissolve the crude fragment 2 in a solution of 6 mol / L guanidine hydrochloride, 0.2 mol / L sodium dihydrogen phosphate, pH 5, separate and purify by reversed liquid chromatography with a mobile phase of ACN / H2O mixture containing 0.1% (v / v) TFA, freeze dry the purified sample to obtain about 15 mg of fragment 2 with C-terminus hydrazide (fragment 2) NHNH2) pure product; (9) Fragment 3 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of crude NHNH2: Take 500 mg of the resin obtained in step (4), add 15 mL of cleavage reagent (TFA:DODT:H2O = volume ratio 95:2.5:2.5), shake and react for 2 h, filter to obtain a purple transparent liquid, evaporate the liquid to dryness using a rotary evaporator, add about 20 mL of ice-cold diethyl ether for extraction twice, centrifuge and collect the precipitate, freeze-dry the sample to obtain about 200 mg of fragment 3 with C-terminus hydrazide (fragment 3) NHNH2) crude product.

[0067] (10) Fragment 4 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of crude CONH2: Take 500 mg of the resin obtained in step (5), add 15 mL of cleavage reagent (TFA:DODT:H2O = volume ratio 95:2.5:2.5), shake and react for 2 h, filter to obtain an orange transparent liquid, evaporate the liquid to dryness using a rotary evaporator, add about 20 mL of ice-cold ether for extraction twice, centrifuge and collect the precipitate, dissolve the sample in a solution of 6 mol / L guanidine hydrochloride, 0.2 mol / L sodium dihydrogen phosphate, pH 5, and separate and purify by reversed liquid chromatography with a mobile phase of ACN / H2O mixture containing 0.1% (v / v) TFA, freeze-dry the purified sample to obtain about 25 mg of fragment 4 with an amide at the C-terminus (fragment 4). (CONH2) Pure product.

[0068] (11) Fragment 1 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of pure MPAA (thioester conversion): Take the crude fragment 1 with C-terminus hydrazide obtained in step (7), dissolve it in a buffer solution containing 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, pH 3.0, add 2.5 times the molar equivalent of the crude fragment 1 with C-terminus hydrazide (the molar number of the crude fragment 1 is calculated based on the pure product: M=3545) of acetylacetone, shake for 3 minutes to mix, then add 10 times the molar equivalent of the crude fragment 1 with C-terminus hydrazide of MPAA, react at room temperature for 12 h, then add 10 times the molar equivalent of the crude fragment 1 with C-terminus hydrazide of TCEP, react at room temperature for 30 minutes, centrifuge, filter, and separate and purify by reversed liquid chromatography with a mobile phase of ACN / H2O mixture containing 0.1% (v / v) TFA, freeze-dry the purified sample to obtain about 10 mg of C-terminus MPAA fragment 1 (fragment 1) MPAA (pure).

[0069] (12) Fragment 3 of the N-terminal domain (N-TIMP2) of the TIMP2 protein Preparation of pure MPAA (thioester conversion): Take the crude fragment 3 with C-terminus hydrazide obtained in step (9) (the relative molecular mass of the crude fragment 3 with C-terminus hydrazide is 3056.7), dissolve it in a buffer solution containing 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, and pH 3.0, add 2.5 times the molar equivalent of the crude fragment 3 with C-terminus hydrazide, shake for 3 minutes to mix, then add 10 times the molar equivalent of the crude fragment 3 with C-terminus hydrazide, react at room temperature for 12 h, then add 10 times the molar equivalent of the crude fragment 3 with C-terminus hydrazide, react at room temperature for 30 minutes, centrifuge, filter, and separate and purify by reversed liquid chromatography with a mobile phase of ACN / H2O mixture containing 0.1% (v / v) TFA. Freeze-dry the purified sample to obtain approximately 8 mg of fragment 3 with C-terminus hydrazide (fragment 3). MPAA (pure).

[0070] (13) Take 10.10 mg of fragment 2 with C-terminus hydrazide prepared in step (8) (the relative molecular mass of fragment 2 with C-terminus hydrazide is 4645.4) and 12.00 mg of fragment 1 with C-terminus MPAA prepared in step (11) (the relative molecular mass of fragment 1 with C-terminus MPAA is 3681.7), dissolve them in 2.174 mL of a solution of 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, 2.5 mol / L imidazole, 10 mmol / L TCEP, pH 6.5, mix them, and carry out a natural chemical ligation reaction at 37°C for 4 hours. Then add 124.63 mg of TCEP (in hydrochloride form, molecular weight 286.65) (200 mmol / L) (100 molar equivalents of fragment 2 with C-terminus hydrazide), 108.7 μl (5% volume of solution) of t-BuSH, and 28.12 mg of VA-044 (in hydrochloride form, molecular weight 323.27) (40 molar equivalents of fragment 2 with C-terminus hydrazide) was subjected to desulfurization reaction at 37°C for 24 hours. After centrifugation and filtration, the product was separated and purified by reversed-phase liquid chromatography with ACN / H2O mixture containing 0.1% (v / v) TFA as the mobile phase. The product was then lyophilized to obtain pure fragment 6 with C-terminus hydrazide.

[0071] (14) Take 14.05 mg of fragment 4 with a C-terminus of amide (relative molecular mass of C-terminus of amide 4) prepared in step (10) and 12.70 mg of fragment 3 with a C-terminus of MPAA (relative molecular mass of C-terminus of MPAA 3) prepared in step (12), dissolve them in 2.65 mL of a solution of 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, 10 mmol / L TCEP, pH 6.5, mix them, and carry out a natural chemical ligation reaction at 37°C for 4 hours. Then add 23.5 mg of palladium chloride (30 molar equivalents of C-terminus of amide fragment 4) to the solution, carry out the first step reaction at 37°C for 1 hour, and then add 204.4 mg of palladium chloride. mg of dithiothreitol (300 molar equivalents of fragment 4 with an amide at the C-terminus) was used for the second step reaction at 37°C for 30 minutes. After centrifugation and filtration, the fragment was separated and purified by reversed-phase liquid chromatography with a mobile phase of ACN / H2O containing 0.1% (v / v) TFA. Cysteine ​​was deprotected by acetaminomethyl to obtain fragment 8 with an amide at the C-terminus.

[0072] (15) Take 10.20 mg of fragment 6 with C-terminus hydrazide (the relative molecular mass of fragment 6 with C-terminus hydrazide is 8127.3), dissolve it in 0.63 mL of 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, pH 3.0 solution, add 2.5 times the molar equivalent of fragment 6 with C-terminus hydrazide, shake well, and then add 200 times the molar equivalent of fragment 6 with C-terminus hydrazide, MPAA. After reacting at room temperature for 10 h, reversed-phase high-performance liquid chromatography coupled with liquid chromatography-mass spectrometry confirms that the hydrazide terminal is converted to a thioester; add 23.10 mg of dissolved fragment 8 with C-terminus amide (the relative molecular mass of fragment 8 with C-terminus amide is 6136.5) (3 times the molar equivalent of fragment 6 with C-terminus hydrazide) to this solution, and add TCEP to a concentration of 10. mmol / L, mixed, pH adjusted to 6.5, and subjected to natural chemical ligation at 37°C for 4 hours to obtain a crude solution of the linear peptide of the N-terminal domain of TIMP2 protein, referred to as solution M; (16) Add solution M to a 3K ultrafiltration tube, centrifuge and filter while replacing the solution system, repeat 10 times, and use a buffer solution of 6 mol / L guanidine hydrochloride, 0.25 mol / L PBS, and pH 6.5 to replace the solution to obtain a crude N-TIMP2 peptide solution without MPAA and TCEP; add 10 mg of palladium chloride (45 molar equivalents of fragment 6 with C-terminus hydrazide) to the replaced solution for the first step reaction, and then add 173.95 mg of dithiothreitol (450 molar equivalents of fragment 6 with C-terminus hydrazide) for the second step reaction. Centrifuge, filter, and separate and purify by size exclusion chromatography. The mobile phase used in the size exclusion chromatography separation and purification is: 6 mol / L guanidine hydrochloride, 0.2 mol / L sodium dihydrogen phosphate, 2 mol / L tri(2) phosphate. Carboxyethylphosphine, with a solution pH of 5.0 ± 0.05, yields a solution of the full-length linear peptide of the TIMP2 protein with the N-terminal domain deprotected by acetaminomethylation, referred to as solution N.

[0073] (17) Refolding of the N-terminal domain of TIMP2 protein N-TIMP2 full-length linear polypeptide: Take solution N and add it to ultrafiltration tubes with a molecular weight cutoff of 3K. Centrifuge and filter while replacing the solution system. Repeat 10 times. Use 6 mol / L guanidine hydrochloride, 0.2 mol / L PBS, and pH 5.0 buffer solution for solution replacement to remove small molecules such as MPAA and TCEP that affect the refolding reaction, and obtain about 500 μL of N-TIMP2 full-length linear polypeptide solution without MPAA and TCEP (measure the absorbance A of this crude solution at OD280 with a UV spectrophotometer, according to Lambert-Beer law A=εcl, where ε=12950, ​​l=1 cm—the inner diameter of a standard quartz cuvette, measured A=7.362, c=n / v, n=m / M, M (relative molecular mass of the N-TIMP2 full-length linear peptide solution without MPAA and TCEP)=14099, c=0.568mol / L, m / v=8.02 mg / ml, m(500 μL)=4.1mg), and slowly added dropwise to approximately 20 mL of a refolding reaction system containing 50 mol / L Tris, 0.45 mol / L guanidine hydrochloride, 0.78 mmol / L reduced glutathione, and 0.44 mmol / L oxidized glutathione, with a pH of 8.5. The slow dropwise addition took 20 minutes. After stirring at room temperature for 2 h, the mixture was placed in a 4℃ refrigerator for 18 h of peptide oxidation and refolding reaction. After centrifugation and filtration, the mobile phase contained 0. The 0.1% (v / v) TFA was separated and purified by reversed-phase liquid chromatography with an ACN / H2O mixture. The purified sample was lyophilized to obtain approximately 0.80 mg (separation yield approximately 4.5%) of the N-terminal domain N-TIMP2 of the folded TIMP2 protein.

[0074] (18) Verification of the correct folding of the N-terminal domain N-TIMP2 of TIMP2 protein: Take 0.5 mg of pure N-TIMP2 after folding, dissolve it in 250 μL of 25 mmol / L NH4HCO3, add 0.13 mg of trypsin (0.5 mg / mL), react at 37℃ for 2 h, then add 0.08 mg of thermophilic protease to the system, react at 70℃ for 10 h, centrifuge and filter the reaction system, take a sample for liquid chromatography-mass spectrometry (LC-MS) analysis, and look for the corresponding disulfide bond fragments; from the LC-MS results, the molecular weights of the Cys1-Cys72 plus Cys3-Cys101 fragment and the Cys13-Cys126 fragment were successfully found, indicating that the N-TIMP2 obtained by this chemical synthesis formed the correct disulfide bond pairing and the folding was correct.

[0075] The N-TIMP2 peptide fragment 2 obtained in step (8) Reverse high performance liquid chromatography and ESI of pure NHNH2 The characterization of MS and the results are as follows Figure 1 As shown.

[0076] The N-TIMP2 polypeptide fragment 4 obtained in step (10) Reverse high performance liquid chromatography and ESI of pure CONH2 The characterization of MS, the results are as follows Figure 2 As shown.

[0077] The N-TIMP2 polypeptide fragment 1-MPAA obtained in step (11) was subjected to reversed-phase high-performance liquid chromatography and ESI. The characterization of MS and the results are as follows Figure 3 As shown.

[0078] The N-TIMP2 polypeptide fragment 3-MPAA obtained in step (12) was subjected to reversed-phase high-performance liquid chromatography and ESI. The characterization of MS and the results are as follows Figure 4 As shown.

[0079] In step (13), fragment 1-MPAA and fragment 2 The natural chemical bonding of NH2 and one-pot desulfurization, such as Figure 5 As shown, Figure 5 -A represents the reversed-phase high-performance liquid chromatography (RP-HPLC) of the naturally chemically linked and desulfurized pure product. Figure 5 -B represents the ESI of the bonding product before desulfurization. Characterization of MS Figure 5 -C represents the ESI of the desulfurized product after connection. Characterization of MS. Combined with Figure 5 A and Figure 5 B indicates that the natural chemical linkage reaction was completed after 4 hours, the desulfurization reaction was completed after 24 hours, and the pure fragment 6 was obtained after semi-preparative purification.

[0080] In step (14), fragment 3-MPAA and fragment 4 The natural chemical linkage of CONH2 and one-pot deacetylation of aminomethyl groups, such as Figure 6 As shown, Figure 6 -A is the reverse-phase high-performance liquid chromatography (RP-HPLC) of the purified product after natural chemical linkage and deacetylation. Figure 6 -B represents the ESI of the ligation product before deacetamidomethylation. Characterization of MS Figure 6 -C represents the ESI of the deacetamidomethylated linker product. Characterization of MS. Combined with Figure 6 A and Figure 6 B indicates that the natural chemical linkage reaction was completed after 4 hours, the deprotection reaction of the Acm protecting group was successfully completed, and fragment 8 was obtained after semi-preparative purification.

[0081] In step (15), the natural chemical linking of fragment 6, which has a C-terminus of hydrazide, and fragment 8, which has a C-terminus of amide, and the deacetylation and refolding, are as follows: Figure 7 As shown, Figure 7 -A represents reversed-phase high-performance liquid chromatography (HPLC) of natural chemical linkages and refolding after deacetylation of aminomethyl groups. Figure 7 -B represents the ESI of the ligation product before refolding. Characterization of MS Figure 7 -C represents the ESI after refolding of the ligation product. Characterization of MS. Combined with Figure 7 A and Figure 7 B indicates that the natural chemical linkage reaction was completed after 4 hours, the deprotection reaction of the Acm protecting group was successfully completed, and after refolding and subsequent purification, fragment 11 was obtained as pure product.

[0082] In step (18), after enzyme digestion, liquid chromatography-mass spectrometry (LC-MS) is used to search for disulfide bond fragments, such as... Figure 8 As shown, the molecular weights of the Cys1-Cys72 plus Cys3-Cys101 fragments and the Cys13-Cys126 fragment were successfully found in the enzyme digestion mixture, indicating that the N-TIMP2 obtained by this chemical synthesis formed the correct disulfide bond pairing and the folding was correct.

[0083] Example 2 The steps are the same as in Example 1, except that: Step (1) Fmoc In the preparation of NHNH2 resin, a loading of 0.3 mmol / g of 2 Chlorotrityl Chloride resin; Step (5) Fmoc Glu(OtBu) In the preparation of MBHA resin, Rink Amide MBHA resin with a loading of 0.3 mmol / g was used.

[0084] Example 3 The steps are the same as in Example 1, except that: step (2) polypeptide fragment 1 Preparation of NHNH2 resin and (6) polypeptide fragment 4 In the preparation of CONH2 resin, Fmoc Cys(Acm) The amount of OH is calculated as twice the molar amount of the loading per g of Fmoc amino resin; Step (4) Peptide fragment 3 In the preparation of NHNH2 resin, Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH and Fmoc... Asp(OtBu) (Dmb)Gly The amount of OH and Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH is calculated as 4 times the molar amount of each g of Fmoc amino resin. Example 4 The steps are the same as in Example 1, except that: in step (13), the amount of t-BuSH added is 10% of the solution volume; the natural chemical linkage reaction is carried out for 6 hours and the desulfurization reaction is carried out for 20 hours; in step (14), the natural chemical linkage reaction is carried out for 6 hours; in step (15), fragment 6 reacts with MAPP for 6 hours and the natural chemical linkage reaction is carried out for 6 hours.

[0085] Example 5 The steps are the same as in Example 1, except that in step (17), two 500 μL solutions of N-TIMP2 full-length linear peptide without MPAA and TCEP are slowly added dropwise to an 8 mL refolding reaction system for 15 and 30 minutes respectively, and the refolding reaction is carried out for 16 and 20 hours respectively.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A chemical synthesis method for the N-terminal domain of the TIMP2 protein, characterized in that, Includes the following steps: (1) The amino acid sequence of the N-terminal domain N-TIMP2 of the TIMP2 protein was divided into fragment 1, fragment 2, fragment 3 and fragment 4; (2) Using the Fmoc solid-phase peptide synthesis method, fragments 1, 2, and 3 with a C-terminus of hydrazide and fragment 4 with a C-terminus of amide were synthesized. Thioester conversion yielded fragment 4 with a C-terminus of amide. Fragments 1 and 3 of mercaptophenylacetic acid (MPAA); (3) The fragments 1 and 3 with C-terminus MPAA, fragment 2 with C-terminus hydrazide and fragment 4 with C-terminus amide were ligated by natural chemical linking, desulfurized and desulfurized, and acetaminomethyl was removed to obtain a full-length linear polypeptide and remove impurities. (4) The full-length linear polypeptide with impurities removed is added dropwise to the refolding reaction system to carry out oxidation and refolding reactions to obtain the target product.

2. The chemical synthesis method of the N-terminal domain of the TIMP2 protein according to claim 1, characterized in that, In step (1), the amino acid sequence of N-TIMP2 is CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSA (32)NDIYGNPIKRIQYEIKQIKM (52)FKGPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKM (96)HITLCDFIVPWDTLSTTQKKSLNHRYQM (124)GCE, as shown in SEQ ID No.

1.

3. The chemical synthesis method for the N-terminal domain of the TIMP2 protein according to claim 1, characterized in that, In step (1), fragments 1, 2, 3 and 4 are divided according to the amino acid sequence of the N-terminal domain of the TIMP2 protein, namely amino acids 1-31, 32-71, 72-100 and 101-127.

4. The chemical synthesis method for the N-terminal domain of the TIMP2 protein according to claim 3, characterized in that, In the synthesis of fragment 2, alanine at position 32 is replaced by cysteine, and alanine is formed after desulfurization and removal of the sulfhydryl group; methionine at position 52 in fragment 2, position 96 in fragment 3, and position 124 in fragment 4 are replaced by ortholeucine (Nle) during the synthesis process.

5. The chemical synthesis method for the N-terminal domain of the TIMP2 protein according to claim 1, characterized in that, In step (2), the isoleucine contained in the amino acid sequence of the synthesized C-terminal hydrazide fragments 1, 2, and 3 and the C-terminal amide fragment 4, N-TIMP2. The threonine amino acid site is used to form Fmoc isoleucine with the Psi(Me,Me)Pro structure using the threonine amino group and the side chain carboxyl group. The threonine dipeptide Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH was synthesized. The amino acid sequence of N-TIMP2 contains aspartic acid. Fmoc aspartic acid with side chain carboxyl and amino protecting groups of OtBu and Dmb, respectively, at the glycine amino acid site. Glycine dipeptide Fmoc Asp(OtBu) (Dmb)Gly Synthesized using OH; The amino acid sequence of N-TIMP2 contains valine. Fmoc valine, formed by using the serine amino group and the side chain carboxyl group to form the Psi(Me,Me)Pro structure at the serine amino group site. The serine dipeptide Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH was synthesized. The cysteine ​​residues in fragment 1 and the non-linked cysteine ​​residues in fragment 4 are treated with Fmoc cysteine, whose side chain thiol protecting group is acetaminomethyl. Cys(Acm) Synthesized using OH.

6. The chemical synthesis method for the N-terminal domain of the TIMP2 protein according to claim 5, characterized in that, The structural formula of Fmoc-Ile-Thr(Psi(Me,Me)Pro)-OH is shown below: ； The aforementioned Fmoc Asp(OtBu) (Dmb)Gly The structural formula of OH is shown below: ； The structural formula of Fmoc-Val-Ser(Psi(Me,Me)Pro)-OH is shown below: ； The aforementioned Fmoc Cys(Acm) The structural formula of OH is shown below: 。 7. The chemical synthesis method for the N-terminal domain of the TIMP2 protein according to claim 1, characterized in that, In step (3), the natural chemical linking and desulfurization process, in which fragment 1 with C-terminus of MPAA and fragment 2 with C-terminus of hydrazide are linked naturally, is carried out in a solution containing imidazole in a one-pot linking-desulfurization process, without the need to add additional thiol reagents.

8. The chemical synthesis method for the N-terminal domain of TIMP2 protein according to claim 1, characterized in that, In step (4), during the refolding reaction, the six free thiol groups at the six cysteine ​​sites of the linear polypeptide of the N-terminal domain of the TIMP2 protein are oxidized to form three pairs of disulfide bonds: the first and 72nd positions form one pair, the third and 101st positions form one pair, and the 13th and 126th positions form one pair.

9. An N-terminal domain N-TIMP2 of a TIMP2 protein, characterized in that, It is prepared by the chemical synthesis method of the N-terminal domain of the TIMP2 protein as described in any one of claims 1 to 8.

10. The application of the N-terminal domain N-TIMP2 of the TIMP2 protein as described in claim 9 in studying the specific mechanism of action of N-TIMP2 and MMP14.

Images