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CN-122011162-A - Chemical synthesis method of N-terminal domain of TIMP2 protein

CN122011162ACN 122011162 ACN122011162 ACN 122011162ACN-122011162-A

Abstract

The invention provides a chemical synthesis method of a N-terminal domain of TIMP2 protein. The invention divides the amino acid sequence of the N-end structural domain of TIMP2 protein into 4 fragments, synthesizes 4 fragments by using a solid-phase polypeptide synthesis method, obtains full-length linear polypeptide by natural chemical connection, desulfurization, sulfhydrylation and acetamidomethyl removal, finally removes impurities in the system and drops the system into a refolding reaction system for oxidation and refolding reaction to obtain a target product. The N-TIMP2 of the N-terminal domain of the TIMP2 reserves the inhibitory activity of the TIMP2 on MMP14, and the N-TIMP2 obtained by chemical total synthesis can introduce unnatural amino acid or carry out specific modification or mutation on any site in the sequence in the synthesis process, thereby providing an effective tool for researching the specific action mechanism of the N-TIMP2 and the MMP14 and further laying a foundation for the research and development of an MMP14 inhibitor.

Inventors

  • HE CHUNMAO
  • Sun yuanmei
  • ZHANG YUQI

Assignees

  • 华南理工大学

Dates

Publication Date
20260512
Application Date
20260225

Claims (10)

  1. 1. A chemical synthesis method of a N-terminal domain of a TIMP2 protein, which is characterized by comprising the following steps: (1) Dividing the amino acid sequence of the N-TIMP2 of the N-terminal domain of the TIMP2 protein into a fragment 1, a fragment 2, a fragment 3 and a fragment 4; (2) Fmoc solid-phase polypeptide synthesis method is adopted to synthesize fragments 1, 2 and 3 with hydrazide at the C end and fragment 4 with amide at the C end, and thioester conversion is carried out to obtain the product with the C end of 4 Fragments 1,3 of mercaptophenylacetic acid MPAA; (3) The fragment 1 with the MPAA at the C end, the fragment 3 with the hydrazide at the C end and the fragment 4 with the amide at the C end are subjected to natural chemical connection, desulfurization, sulfydryl removal and acetamidomethyl removal to obtain full-length linear polypeptide, and impurities are removed; (4) And (3) dripping the full-length linear polypeptide with the impurities removed into a refolding reaction system, and carrying out oxidation and refolding reaction to obtain a target product.
  2. 2. The method of claim 1, wherein in step (1), the amino acid sequence of said N-TIMP2 is CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSA（32）NDIYGNPIKRIQYEIKQIKM（52）FKGPEKDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKM（96）HITLCDFIVPWDTLSTTQKKSLNHRYQM（124）GCE, as shown in SEQ ID No. 1.
  3. 3. The method for chemically synthesizing an N-terminal domain of TIMP2 protein according to claim 1, wherein in the step (1), the fragments 1, 2, 3 and 4 are divided according to amino acids 1 to 31, 32 to 71, 72 to 100 and 101 to 127 of the amino acid sequence of the N-terminal domain of TIMP2 protein.
  4. 4. A chemical synthesis method of a TIMP2 protein N-terminal domain according to claim 3, wherein the alanine at position 32 in the fragment 2 is replaced by cysteine in the synthesis process, and is desulfurated and desulphurized to form alanine, and the methionine at position 52 in the fragment 2, 96 in the fragment 3 and 124 in the fragment 4 is replaced by norleucine Nle in the synthesis process.
  5. 5. The method according to claim 1, wherein in the step (2), isoleucine contained in the amino acid sequence of N-TIMP2 is synthesized from fragments 1, 2,3 of hydrazide and 4 of amide at C-terminus Threonine amino acid site, fmoc isoleucine of Psi (Me, me) Pro structure using threonine amino group and side chain carboxyl group The threonine dipeptide Fmoc-Ile-Thr (Psi (Me, me) Pro) -OH was synthesized; Aspartic acid contained in the amino acid sequence of N-TIMP2 Glycine amino acid site, fmoc aspartic acid using protecting groups of side chain carboxyl and amino groups of OtBu and Dmb respectively Glycine dipeptide Fmoc Asp(OtBu) (Dmb)Gly Synthesizing OH; valine contained in the amino acid sequence of N-TIMP2 Serine amino acid site, fmoc valine of Psi (Me, me) Pro structure was formed using serine amino group and side chain carboxyl group The serine dipeptide Fmoc-Val-Ser (Psi (Me, me) Pro) -OH was synthesized; Cysteine in fragment 1 and cysteine at the non-attachment site in fragment 4, fmoc cysteine Fmoc with side chain thiol protecting group being acetamidomethyl Cys(Acm) OH is synthesized.
  6. 6. The method for chemically synthesizing an N-terminal domain of TIMP2 protein according to claim 5, wherein the Fmoc-Ile-Thr (Psi (Me, me) Pro) -OH has the following structural formula: ; The 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 as follows: ; The Fmoc Cys(Acm) The structural formula of OH is shown below: 。
  7. 7. The method according to claim 1, wherein in the step (3), the natural chemical ligation and desulfurization/removal of thiol groups are performed by one-pot ligation/desulfurization in an imidazole-containing solution without adding any additional thiol reagent during the natural chemical ligation of the segment 1 having the MPAA at the C-terminus and the hydrazide segment 2 at the C-terminus.
  8. 8. The method according to claim 1, wherein in the step (4), the six free sulfhydryl groups of 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, namely, the 1 st and 72 nd positions are in a pair, the 3 rd and 101 rd positions are in a pair, and the 13 th and 126 th positions are in a pair.
  9. 9. A TIMP2 protein N-TIMP 2N-terminal domain prepared by a chemical synthesis method of a TIMP2 protein N-terminal domain as defined in any one of claims 1 to 8.
  10. 10. Use of the N-TIMP2 domain of the N-terminal of the TIMP2 protein of claim 9 for studying the specific mechanism of action of N-TIMP2 and MMP 14.

Description

Chemical synthesis method of N-terminal domain of TIMP2 protein Technical Field The invention relates to the technical field of solid phase synthesis preparation of polypeptide/protein, in particular to a chemical synthesis method of a N-terminal domain of TIMP2 protein. Background Since Merrifield in 1963 proposed solid-phase polypeptide synthesis (SPPS), which was the result of Nobel's chemical prize in 1984, SPPS has become the gold standard （Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154 (1963)）. for polypeptide preparation, which has been basically designed by covalently immobilizing the C-terminus of a target polypeptide to an insoluble solid-phase carrier (e.g., a resin microsphere), coupling protected amino acids one by one in a synthesis tube in a "deprotection-coupling" cycle until sequence coupling is completed, and then removing the peptide chain from the resin and removing the side chain protecting groups altogether to obtain the target product. Compared with liquid phase synthesis, SPPS has the advantages that ① can use excessive amino acid and activator in each step to promote the reaction to be almost complete, the step yield and the overall efficiency are improved, ② products are always fixed on resin, excessive reagents and byproducts can be removed through simple filtration and washing after the reaction, the operation is convenient, ③ has high programmability, and is convenient for accurately introducing unnatural amino acid and implementing site-specific modification (including simulated posttranslational modification) and the like （Merrifield, R. B. Automated Synthesis of Peptides. Science. 150, 178–185 (1965);Kaiser, E., Colescott, R. L., Bossinger, C. D.&Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem. 34, 595–598 (1970).;Carpino, L. A.&Han, G. Y. 9-Fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404–3409 (1972).）. As the peptide chain extends, long peptides on the resin carrier tend to aggregate and form secondary structures, reducing coupling and deprotection efficiencies, thus limiting the effective length of conventional SPPS to typically about 50 amino acids. In order to break the bottleneck, kent proposed "natural chemical Ligation" (NATIVE CHEMICAL Ligation, NCL) in 1994, which is to realize chemical selective Ligation of polypeptide fragment with C-terminal thioester and N-terminal cysteine in water phase, so as to modularly splice multiple fragments, synthesize protein （Dawson, P. E., Muir, T. W., Clark-Lewis, I.&Kent, S. B. H. Synthesis of proteins by Native Chemical Ligation. Science. 266, 776–779 (1994)）.NCL with a length of over 100 amino acids and development of its expansion method, remarkably expand the scale and type of chemical protein synthesis, and provide a high-efficiency and practical route for obtaining protein containing unnatural amino acids, site-specific modifications and isotope labels （Bode, J. W., Fox, R. M.&Baucom, K. D. Chemoselective amide ligations 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-Terminal hydrazides 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, N. J. et al. Rapid additive-free Selenocystine–Selenoester peptide ligation. J. Am. Chem. Soc. 137, 14011–14014 (2015).;Liao, P.&He, C. Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis. Chem. Sci. 15, 7965–7974 (2024).）. Matrix metalloproteinase Tissue Inhibitors (TIMPs) are a family of four endogenous proteins (TIMP 1-4) that inhibit their proteolytic activity primarily by forming a 1:1 complex with Matrix Metalloproteinases (MMPs) that are involved in extracellular matrix (ECM) remodeling, vital （The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell 41, 374–403 (2023)）. in tumor cell migration and invasion, wherein TIMP2 maintains microenvironment homeostasis by limiting the surrounding proteolysis of ECM and cell surface proteins. More interestingly, TIMP2 has dual functions of 'inhibition' and 'bridging', namely the N-terminal domain of the TIMP2 binds to MMP14 (MT 1-MMP), the C-terminal domain binds to precursor MMP2 (proMMP 2) to form an MMP14-TIMP2-proMMP2 ternary complex on the cell surface, and then the adjacent second MMP14 molecule is used as activating enzyme to cleave proMMP2, so that the development of a broad-spectrum MMP inhibitor in early stage of release activity MMP2（Hernandez-Barrantes, S. et al. Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to t