CN114736289A - Chemical synthesis method of hirudin with tyrosine sulfation modification - Google Patents

Abstract

The invention provides a chemical synthesis method of hirudin with tyrosine sulfation modification. The method comprises the steps of firstly synthesizing a C-terminal hydrazide fragment 1 and a fragment 2 with sulfated and modified tyrosine, then obtaining full-length linear polypeptide by utilizing a natural chemical ligation reaction and a concomitant neopentyl removal reaction, finally removing impurities in a system, dropwise adding the full-length linear polypeptide into a refolding reaction system, and carrying out oxidation and refolding reactions to generate three pairs of disulfide bonds to obtain a target product. The invention introduces the post-translational modification of sulfated tyrosine onto the polypeptide resin through a solid-phase polypeptide synthesis method, so that the sulfated tyrosine is stably present on the solid-phase resin, the side reaction is avoided, and the separation and purification are realized; the natural chemical connection is combined with the automatic removal of the neopentyl in the aqueous solution, so that the reaction time is shortened; the natural chemical connection and refolding reaction of the linear polypeptide are combined into one-pot reaction without separation and purification, so that the synthetic yield is greatly improved, and the method has a good application prospect.

Description

A kind of chemical synthesis method of hirudin with tyrosine sulfate modification

technical field

The invention relates to the technical field of solid-phase synthesis and preparation of polypeptide sulfated modification, in particular to a chemical synthesis method of hirudin with tyrosine sulfated modification.

Background technique

K.,Thromb.Haemostasis,2007,98,116–119.)。Hirudin is a protein found in leech saliva. The amino acid sequence of hirudin contains about 65 amino acid residues, its amino terminus (N terminus) contains six cysteines, which form a spherical compact stable structure through three pairs of disulfide bonds, while its exposed carboxyl terminus ( C-terminal) sequence has homology in amino acid sequence with fibrinogen in animals. Thrombin in animals will specifically recognize and cut fibrinogen, so that fibrinogen with better water solubility can form water-insoluble fibrin monomer, which can block blood vessels and prevent excessive bleeding through the above mechanism. effect. "Hirudin and thrombin" have a stronger interaction than "fibrinogen and thrombin", and can directly inhibit the activity of thrombin, which is the most effective natural thrombin inhibitor currently known. Because hirudin has anticoagulant activity, it is considered to be used as an anticoagulant clinically. Through relevant evaluation, it is believed that hirudin has potential value as an antithrombotic drug, and recombinant hirudin is obtained by plasmid expression method. certain clinical studies (Nowak, G.,

K., Thromb. Haemostasis, 2007, 98, 116–119.).

K _i can represent the inhibition constant in enzyme kinetics, which reflects the inhibitory strength of the inhibitor to the enzyme. The smaller the value is, the stronger the inhibition strength is. The K _i value of natural hirudin for thrombin is reported to be about 25 fM (Stone, SR, Hofsteenge, J., Biochemistry., 1986, 25, 4622-4628), while recombinant hirudin expressed by E. coli or yeast The Ki value for thrombin is about 300 fM (Liu, CC, Schultz, PG, Nat. Biotechnol., 2006, 24, _1436-40 .), which indicates that the anticoagulant activity of recombinant hirudin obtained by plasmid expression method is higher than Naturally low in hirudin. The difference in activity between natural hirudin and recombinant hirudin is due to the fact that natural hirudin contains a sulfated modified tyrosine (Tyr), which can negatively charge the C-terminus of hirudin and enhance the interaction between the C-terminus of hirudin and thrombin. interaction, thereby enhancing the anticoagulant activity of hirudin. Since tyrosine sulfation is a post-translational modification (PTM) found exclusively in higher eukaryotes, recombinant hirudin expressed by E. coli or yeast does not undergo this important post-translational modification, thus significantly reducing the anticoagulant activity of recombinant hirudin.

Post-translational modification (PTM) of proteins is a common mechanism for enriching the structural diversity of an organism's genome. Two common PTMs include O-glycosylation of serine (Ser) or threonine (Thr) (R.J.Payne, C.H. Wong, Chem. Commun., 2010, 46, 21–43) and the aforementioned tyrosine Sulfation modification of amino acid (Tyr) (C. Seibert, T.P. Sakmar, Pept. Sci., 2008, 90, 459-477). These two modifications are performed at the Golgi weight by glycosyltransferases and protein tyrosine sulfotransferases (TPSTs), respectively. It is estimated that over 50% of proteins expressed in eukaryotes are O-glycosylated and over 1% are tyrosine sulfated. These two PTMs are involved in many biological processes, including molecular recognition, cell differentiation, immune regulation, and protein folding, etc., which have aroused great interest in the development of glycosylated and sulfated proteins to treat various diseases. Despite the importance of these modifications, obtaining purified O-glycosylated and Tyr-sulfated proteins is extremely challenging. Because of the non-templated nature of the PTM process, it is determined by the relative activities of transferases, which lead to the expression of glycoforms (modified by O-glycosylation) and sulfoforms (modified by Tyr sulfation) according to the relative activities of the relative activities. ) of various protein mixtures that are generally not separable by chromatographic separation techniques.

In order to obtain homogenized glycoproteins (O-glycosylation-modified proteins) and sulfated-modified proteins, the main feasible route is chemical synthesis. The technology of obtaining homogenized glycoproteins by chemical synthesis has gradually matured, and the technology of using this technology to synthesize homogenized sulfated-modified proteins is also gradually developing.

The different kinds of hirudin found so far have some differences in amino acid sequence, and it is believed that several variants of hirudin have been developed through mutation. Among them, a European medicinal hirudin, HIRV1, entered clinical research as an anticoagulant in its unmodified form, and was found to exhibit a certain inhibitory effect on human thrombin activity (C.C.Liu, E.Brustad, W. Liu, P. G. Schultz, J. Am. Chem. Soc., 2007, 129, 10648-10649.). We believe that if the tyrosine sulfated hirudin HIRV1 can be obtained, and by optimizing the synthesis process, the synthetic yield of sulfated hirudin HIRV1 in the chemical synthesis process can be improved, and the anticoagulation of hirudin HIRV1 in clinical research will be improved The increase in activity and the reduction in the cost of clinical research have a significant impact. Therefore, it is necessary to develop a synthetic method with convenient synthesis and purification, modular synthesis, and considerable yield to efficiently prepare the homogeneous sulfated-modified hirudin HIRV1.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to provide a chemical synthesis method of hirudin with sulfated modified tyrosine.

The segmented modular hirudin with sulfated modified tyrosine prepared by the method of the present invention is separable and finally homogeneous after natural chemical linkage and protein refolding.

The method of the present invention is optimized, and can achieve the purpose of improving the synthetic yield by performing a sequential one-pot reaction of natural chemical linkage and protein refolding.

The object of the present invention is achieved through the following technical solutions:

A chemical synthesis method of hirudin with tyrosine sulfate modification, comprising the steps:

(1) According to the amino acid sequence of hirudin, any cysteine is divided into fragment 1 and fragment 2 (the N-terminus of fragment 2 is cysteine); using a resin whose C-terminus is a hydrazide (-NHNH ₂ ) end, Referred to as hydrazide resin, the Fmoc solid-phase peptide synthesis method is adopted. According to the sequence of fragment 1, Fmoc-protected amino acids are sequentially condensed from the C-terminus to the N-terminus, washed and dried to obtain fragment 1;

(2) Use resin whose C-terminus is amide (-CONH ₂ ) terminus, referred to as amide resin, adopt Fmoc solid-phase peptide synthesis method, according to the sequence of fragment 2, condense Fmoc-protected amino acids in turn from C-terminus to N-terminus, wash, and dry , obtain Fragment 2; where:

The tyrosine near the C-terminus of the hirudin is a sulfated modified tyrosine, and the Fmoc sulfated modified tyrosine whose side chain sulfate protecting group is a neopentyl group (Neopentyl, nP) is used for short, referred to as Fmoc-Tyr ( OSO ₃ nP)-OH for synthesis;

(3) Take the linear polypeptide resin of fragment 1 prepared in step (1), add a cleavage reagent, remove the polypeptide chain from the hydrazide resin, and remove the remaining side chain protecting groups; filter, spin dry, extract, Centrifuge and freeze-dry to obtain a crude product of fragment 1 whose C-terminal is hydrazide (-NHNH ₂ );

(4) Take the linear polypeptide resin of fragment 2 prepared in step (2), add a cleavage reagent, remove the polypeptide chain from the amide resin, and remove the remaining side chain protecting groups; filter, spin dry, extract, centrifuge , lyophilized to obtain the crude fragment 2; further separation and purification, lyophilized to obtain the fragment 2 whose C-terminal is amide (-CONH ₂ );

(5) Take the crude product of fragment 1 with hydrazide at the C-terminus prepared in step (3), dissolve it, add acetylacetone, shake well, then add 4-mercaptophenylacetic acid (MPAA), and in the first step, add tri( 2-Carboxyethyl) phosphine (TCEP), the second step reaction, centrifugation, filtration, further separation and purification, lyophilization, to obtain fragment 1 whose C-terminal is MPAA;

(6) Take the fragment 2 with an amide (-CONH ₂ ) prepared in the step (4) and the fragment 1 with an MPAA in the C-terminal prepared in the step (5), dissolve, mix, and perform natural chemical connection ( Native chemical ligation, referred to as NCL) reaction, to obtain a crude solution of hirudin linear polypeptide with tyrosine sulfate modification near the C-terminus and the neopentyl group has been removed, referred to as solution M;

(7) Take the solution M prepared in step (6), add an ultrafiltration tube, and replace the solution system by centrifugal filtration to obtain a phenolic acid near the C-terminus that does not contain 4-mercaptophenylacetic acid and tris(2-carboxyethyl)phosphine The crude product solution of amino acid sulfated modified hirudin linear polypeptide, referred to as solution N; wherein:

The replacement solution system is 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate (PBS), and the pH value of the solution is 6.0±0.5;

(8) taking the solution N prepared in step (7), slowly adding it dropwise to the refolding reaction system, reacting, centrifuging, filtering, and further separating and purifying to obtain the hirudin modified by tyrosine sulfate modification near the C-terminus; wherein:

The refolding reaction system is 0.15～0.25mol/L Tris, 3.5～4.5mol/L sodium chloride, 3.5～4.5mmol/L L-cysteine, 1.5～2.5mmol/L L-cystine , the pH of the solution is 8.5 ± 0.5.

In the described chemical synthesis method, the amino acid sequence of the hirudin is as VVYTDCTESGQNLCLCEGSNVCGQGNK ²⁷ CILGS D ³³ G ³⁴ EKNQCVTGEGTPKPQSHN D ⁵³ G ⁵⁴ DFEEIPEEY ⁶³ LQ, also as shown in SEQ ID NO.1, or as shown in SEQ ID NO.1 The amino acid sequence obtained by substitution, deletion or addition of one or several amino acids while retaining the same biological activity. Wherein, the superscript indicates the position number of the amino acid in the sequence.

In the described chemical synthesis method, the aspartic acid (Asp)-glycine (Gly) amino acid site contained in the amino acid sequence of the described hirudin uses the protective group of the side chain carboxyl group and the amino group to be OtBu (tert-butyl) respectively. The Fmoc aspartic acid-glycine dipeptide of ester, R2) and Dmb (2,4-dimethoxybenzyl, R5), referred to as Fmoc-Asp(OtBu)-(Dmb)Gly-OH for short, was synthesized. For example, when the amino acid sequence of hirudin is shown in SEQ ID NO. 1, the aspartic acid (Asp)-glycine (Gly ) is a specific sequence, and the step-by-step synthesis is prone to isomerism, resulting in the inability to obtain a homogeneous polypeptide product in the final purification. Synthesis using the above dipeptides can solve the related problems. Among them, the OtBu protecting group and the Dmb protecting group can be removed by trifluoroacetic acid.

In the chemical synthesis method, the cysteine is preferably the fourth, fifth or sixth cysteine from the N-terminus of hirudin; more preferably the fifth cysteine from the N-terminus.

In the chemical synthesis method, the hydrazide resin is preferably a hydrazide resin obtained after 2-Chlorotrityl Chloride resin (referred to as 2-Cl resin) is treated with 5% (v/v) hydrazine hydrate/DMF for 30 min.

In the chemical synthesis method, the amide resin is preferably Fmoc-Rink Amide-MBHA resin.

In the chemical synthesis method, the structural formula of the Fmoc-Tyr(OSO ₃ nP)-OH is as follows:

The dosage of the Fmoc-Tyr(OSO ₃ nP)-OH, if considering the cost, is preferably calculated according to the molar ratio of Fmoc amino resin: Fmoc-Tyr(OSO ₃ nP)-OH=1:1.5; if the cost is not considered, Preferably, it is calculated according to the molar ratio of Fmoc amino resin: Fmoc-Tyr(OSO ₃ nP)-OH=1:4 _; Tyr(OSO ₃ nP)-OH=1:1.5～4 molar ratio calculation.

The coupling time of the Fmoc-Tyr(OSO ₃ nP)-OH and the Fmoc amino resin is preferably 6-12h; more preferably 12h;

The structural formula of the described Fmoc-Asp(OtBu)-(Dmb)Gly-OH is as follows:

The consumption of the described Fmoc-Asp(OtBu)-(Dmb)Gly-OH, if considering the cost, is preferably calculated according to the molar ratio of Fmoc amino resin: Fmoc-Asp(OtBu)-(Dmb)Gly-OH=1:1.5 If the cost is not considered, it is preferable to calculate according to the molar ratio of Fmoc amino resin: Fmoc-Asp(OtBu)-(Dmb)Gly-OH=1:4; in general, the Fmoc-Asp(OtBu)-( The amount of Dmb)Gly-OH is calculated according to the molar ratio of resin:Fmoc-Asp(OtBu)-(Dmb)Gly-OH=1:1.5-4.

The coupling time between the Fmoc-Asp(OtBu)-(Dmb)Gly-OH and the Fmoc amino resin is preferably 4-8h; more preferably 8h.

Fmoc-protected amino acids other than Fmoc-Tyr(OSO ₃ nP)-OH and Fmoc-Asp(OtBu)-(Dmb)Gly-OH are Fmoc amino acids without side chains, or side chain protecting groups R1, R2, R3 Or R4 can be synthesized with Fmoc amino acid that can be removed with trifluoroacetic acid; wherein: R1 represents tert-butyl (tBu), R2 represents tert-butyl ester (OtBu), R3 represents trityl (Trt), and R4 represents tert-butyl The amount of oxycarbonyl (Boc) is preferably calculated according to the molar ratio of Fmoc amino resin: Fmoc protected amino acid=1:4.

The coupling time of Fmoc-protected amino acids other than Fmoc-Tyr(OSO ₃ nP)-OH and Fmoc-Asp(OtBu)-(Dmb)Gly-OH and Fmoc amino resin is preferably 2-4h; more preferably 2h.

The specific operation of condensing Fmoc-protected amino acids from the C-terminus to the N-terminus in step (1) and step (2) is as follows: under the action of the coupling system, the first amino acid and the hydrazide or hydrazide after Fmoc are removed first. The amide resin is reacted to generate amino acid-amino resin, and then other Fmoc protected amino acids are coupled one by one to obtain a linear polypeptide resin.

The condensing agent in the coupling system is preferably "HOBT+DIC" or "TBTU+DIEA". In particular, the condensing agent in the coupling system of Fmoc-Tyr(OSO ₃ nP)-OH, Fmoc-Asp(OtBu)-(Dmb)Gly-OH, is preferably "HOB T+DIC".

The Fmoc deprotection reagent in the coupling system is preferably 20% piperidine/DMF.

The deprotection reaction time in the coupling system is preferably 5-10 min.

The loading of the resin is preferably 0.3 to 0.5 mmoL/g.

The reagents for cleavage described in step (3) and step (4) are preferably trifluoroacetic acid (TFA), water and 1,4,7,10-tetraazacyclododecane-1,4,7,10 - Tetraacetic acid (DODT) mixed in a volume ratio of 95:2.5:2.5 to the resulting solution.

The cutting time described in step (3) and step (4) is preferably 2-4h.

The extraction agent of the extraction described in step (3) and step (4) is preferably glacial ether.

The number of times of extraction described in step (3) and step (4) is preferably twice.

The separation and purification described in step (4) is preferably realized by reverse phase liquid chromatography.

The mobile phase of the reverse-phase liquid chromatography is an acetonitrile/water mixture containing 0.1% trifluoroacetic acid.

The solution of dissolving the crude product of fragment 1 described in step (5) is preferably 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate (PBS), and the pH value of the solution is 3.0±0.5; more preferably It is 6 mol/L guanidine hydrochloride, 0.2 mol/L sodium dihydrogen phosphate (PBS), and the pH value of the solution is 3.0.

The consumption of acetylacetone described in step (5) is preferably calculated according to the molar ratio of fragment 1 crude product: acetylacetone=1:2.5.

The shaking time described in step (5) is preferably 3 minutes.

The amount of 4-mercaptophenylacetic acid described in step (5) is preferably calculated according to the molar ratio of fragment 1 crude product: 4-mercaptophenylacetic acid=1:15.

The condition of the first step reaction described in step (5) is a temperature of 20-30° C. (room temperature), and the reaction time is preferably 12 hours.

The amount of tris(2-carboxyethyl)phosphine described in step (5) is preferably calculated according to the molar ratio of fragment 1 crude product: MPAA=1:10.

The condition of the second step reaction described in step (5) is that the temperature is 20-30° C. (room temperature), and the reaction time is preferably 20 minutes.

The separation and purification described in step (5) is preferably realized by reverse phase liquid chromatography.

The mobile phase of the reverse-phase liquid chromatography is an acetonitrile/water mixture containing 0.1% trifluoroacetic acid.

The solution for dissolving fragment 1 and fragment 2 in step (6) is 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate (PBS), 0.15-0.25 mol/L 4-mercaptobenzene Ethyl acetic acid, 38～42mmol/L tris(2-carboxyethyl)phosphine, the pH value of the solution is 6.5±0.5; more preferably 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen phosphate (PBS), 0.2mol /L 4-mercaptophenylacetic acid, 40mmol/L tris(2-carboxyethyl)phosphine, the pH value of the solution is 6.5.

The amount of fragment 1 described in step (6) is preferably calculated as 1.5 to 2 times the equivalent of fragment 2.

The conditions of the natural chemical ligation reaction described in the step (6) are the temperature of 37±0.5°C, and the reaction time is preferably 8 to 12 hours.

Since the natural chemical linking reaction described in step (6) is carried out in an aqueous system, it will be accompanied by the automatic removal of the neopentyl group at the same time.

The ultrafiltration tube described in the step (7) uses an ultrafiltration tube with a molecular weight of 3K, and the functions of ultrafiltration are: first, to remove all molecules with a molecular weight of less than 3000 in the system; second, to replace the solution system.

The replacement solution system described in step (7) is preferably 6 mol/L guanidine hydrochloride, 0.2 mol/L sodium dihydrogen phosphate (PBS), and the pH value of the solution is 6.0.

The operation of ultrafiltration and replacement of the solution system described in step (7) is preferably repeated five times.

The refolding system described in step (8) is preferably 0.2mol/L Tris, 4mol/L sodium chloride, 4mmol/L L-cysteine, 2mmol/L L-cystine, and the pH of the solution is 8.5.

The volume of the refolding reaction system described in step (8) is preferably calculated by dissolving 0.25-0.4 mg of linear polypeptide in 1 mL of reaction solution.

The duration of the slow dropwise addition described in step (8) is preferably 15 to 30 minutes, more preferably 30 minutes.

The conditions of the reaction described in step (8) are the temperature of 20-30° C. (room temperature), and the reaction time is preferably 4-12 hours.

The separation and purification described in step (8) is preferably achieved by reverse phase liquid chromatography.

The mobile phase of the reverse-phase liquid chromatography is an acetonitrile/water mixture containing 0.1% trifluoroacetic acid.

A hirudin with tyrosine sulfate modification is obtained by the above synthesis method.

The application of the above-mentioned hirudin with tyrosine sulfate modification in the preparation of anticoagulant drugs and/or medical materials.

The principle of the present invention is as follows: using the Fmoc solid-phase polypeptide synthesis method, according to the amino acid sequence of hirudin, the sequence is modularly divided into two peptide segments. The resin is used as a carrier, and Fmoc-protected amino acids are condensed in turn from the C-terminal to the N-terminal to obtain the hydrazide resin of hirudin HIRV1 polypeptide fragment 1; Use Fmoc-Tyr(OSO 3 nP)-OH at the site to be modified (tyrosine 63), and use Fmoc-Tyr(OSO ₃ nP)-OH at the site where isomerization needs to be avoided (aspartic acid 33 and glycine 34, glycine 34 The 53rd aspartic acid and the 54th glycine) use Fmoc-Asp(OtBu)-(Dmb)Gly-OH to condense Fmoc-protected amino acids from the C-terminus to the N-terminus to obtain the amino resin of hirudin HIRV2 polypeptide fragment 2; Among them, the removal method of the nP protective group is different from the side chain protective group of the other amino acids; the nP protective group will be automatically removed in the NCL aqueous reaction system to obtain the 63rd tyrosine sulfate modification and the nP protective group The crude solution of the removed full-length hirudin linear polypeptide; perform ultrafiltration and centrifugation to replace the solution to remove MPAA and TCEP that will affect the refolding reaction; the crude solution of the full-length hirudin linear polypeptide after ultrafiltration and replacement of the solution does not require After separation and purification, it can be slowly added dropwise to a large-volume refolding reaction system to make the six cysteine sites (6th, 14th, The six free sulfhydryl groups at the 16th, 22nd, 28th, and 39th positions are oxidized to form three pairs of disulfide bonds (the 6th and 14th positions are a pair, and the 16th and 28th positions are a pair , the 22nd position and the 39th position are a pair), and the folded tyrosine sulfated modified full-length hirudin protein was obtained.

Compared with the prior art, the present invention has the following advantages and effects:

The method of the present invention proposes a synthetic method for introducing sulfated modified tyrosine Fmoc-Tyr(OSO ₃ nP)-OH on a polypeptide resin, and provides corresponding examples. In this synthetic route, the sulfated-modified tyrosine can be introduced at the target position only by the solid-phase synthesis method of Fmoc amino acid with mature technology. Due to the maturity of the solid-phase peptide synthesis method of Fmoc amino acid, it avoids the easy occurrence of the synthesis process. side reactions.

In the method of the present invention, the amino acid Fmoc-Tyr(OSO ₃ nP)-OH whose side chain sulfate protecting group is nP is introduced into the solid-phase synthesis of the polypeptide. During the NCL reaction, the nP protecting group is automatically removed in the aqueous solution without the need for Adding an extra protecting group removal reaction shortens the synthesis time and improves the synthesis efficiency.

In the method of the present invention, after obtaining the crude product of the polypeptide fragment 1 of the C-terminal hydrazide of hirudin, the C-terminal hydrazide is converted into MPAA through a thioesterification reaction at pH 3 of the reaction system, and is separated, purified and freeze-dried, so that the MPAA terminal The HIRV1 polypeptide fragment 1 of MPAA exists stably in a lyophilized form, avoiding the hydrolysis reaction that MPAA is prone to occur at pH>7.

In the early stage of the research of the method of the present invention, after the NCL reaction in step (6) is carried out, the crude product solution obtained in step (6) will be subjected to reverse liquid chromatography in which the mobile phase is an acetonitrile/water mixture containing 0.1% trifluoroacetic acid. Carry out the first separation and purification, freeze-drying to obtain the pure product of the full-length hirudin linear polypeptide with the 63rd tyrosine sulfate modification and the nP protecting group removed, and then the pure product freeze-dried powder is dissolved in the refolding reaction Oxidation and refolding are carried out in the system, and the components and concentrations of the refolding reaction system are the same as those of the above system. Finally, separation and purification are carried out by the reversed-phase liquid chromatography method of the same mobile phase as above, and freeze-dried to obtain the 63rd tyrosine. Sulfated modified full-length hirudin protein with a protein synthesis yield of 4%. The innovation of the method of the present invention is a one-pot method of NCL reaction and refolding reaction. The C-terminus of hirudin is MPAA polypeptide fragment 1 and the N-terminus of hirudin with sulfated modified tyrosine is cysteine polypeptide fragment 2, carry out NCL reaction, through ultrafiltration centrifugation, replace the solution system. Method: After removing the small molecules MPAA and TCEP that are easy to affect the refolding reaction, the crude product solution is directly and slowly added dropwise to the refolding reaction system, and the reaction is carried out to realize the conversion of the full-length linear polypeptide of hirudin into hirudin protein. The liquid-phase purification step after the NCL reaction reduces the two separations and purifications to one. Without affecting the product purity, the synthesis time and cost are greatly shortened, the synthesis yield is improved, and the protein synthesis yield is finally improved. to 21%.

The hirudin protein with sulfated modified tyrosine is prepared by the method of the invention, which solves the problem that the post-translational modification process cannot be carried out by expressing recombinant hirudin in Escherichia coli or yeast. Modified tyrosine was introduced into the polypeptide, and the synthetic yield of hirudin protein was improved by the sequential one-pot method of NCL reaction and refolding reaction.

The hirudin protein with sulfated modified tyrosine obtained by the method of the present invention is expected to exhibit an anticoagulant activity equivalent to that of natural hirudin, which is helpful for studying the physiological and biochemical effects of hirudin on antithrombotic or cardiovascular diseases, and also provides Hirudin has laid a good foundation for clinical research and disease prevention and treatment.

Description of drawings

FIG. 1 is the characterization diagram of reverse-phase high performance liquid chromatography and ESI-MS when the pure product of hirudin HIRV1 polypeptide fragment 2 (F2nP) with sulfate modification and containing neopentyl-protected tyrosine is prepared in Example 1. FIG.

FIG. 2 is the characterization diagram of reverse-phase high performance liquid chromatography and ESI-MS of the pure product of hirudin HIRV1 polypeptide fragment 1-MPAA (F1) prepared in Example 1. FIG.

Figure 3 shows the reaction of reaction raw material fragment 1 (F1), fragment 2 with neopentyl protection (F2nP), initial NCL reaction (NCL-0h), NCL reaction in the process of preparing HIRV1 (63OSO ₃ ) in Example 1 Reverse high-efficiency liquid for monitoring the status of the final product of the reaction (NCL-12h), the start of the refolding reaction (Fold-0h), the end of the refolding reaction (Fold-12h), and the final product of the reaction (63OSO ₃ ) (FoldP). Phase chromatogram and ESI-MS characterization of important peak position substances.

Figure 4 is a graph showing the results of the experiment of inhibiting thrombin cleavage of fibrinogen by HIRV1 (63OSO ₃ ) prepared in Example 1; wherein (A) is the sample of control group 1, (B) is the sample of control group 2, (C) sample for the experimental group.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The structural formula of Fmoc-Tyr(OSO ₃ nP)-OH used in the following examples is shown in the reference "Simpson LS, Zhu JZ, Widlanski TS, et al., Chemistry Biology, 2009, 16(2): 153-161.", "Simpson LS, Widlanski TS, Journal of the American Chemical Society, 2006, 128(5): 1605-1610.", "Schlienger N, Peyrottes S, Kassem T, et al., Journal of Medicinal Chemistry , 2000, 43 (23): 4570-4574. "The method described in the preparation can be obtained:

The structural formula of Fmoc-Asp(OtBu)-(Dmb)Gly-OH used in the following examples is as follows:

The remaining Fmoc protected amino acids are Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH , Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Val-OH.

In the following examples, the HPLC instrument is an Agilent 1260, the chromatographic column is a Phenomenex C18 column, and the mobile phase is water and acetonitrile (containing 0.1% (v/v) TFA).

The sequence of the hirudin HIRV1 polypeptide fragment 1 synthesized in the following examples is as follows, and the C-terminus of the polypeptide fragment 1 has been hydrazide:

NH ₂ -Val ¹ -Val-Y(R1)-Thr(R1)-Asp(R2)-Cys(R3)-Thr(R1)-Glu(R2)-Ser(R1)-Gly-Gln(R3)- Asn(R3)-Leu-Cys(R3)-Leu-Cys(R3)-Glu(R2)-Gly-Ser(R1)-Asn(R3)-Val-Cys(R3)-Gly-Gln(R3)- Gly-Asn(R3)-Lys ²⁷ (R4)-NHNH ₂

The hirudin HIRV1 polypeptide fragment 2 sequence with sulfated modified tyrosine synthesized in the following examples is as follows, the N-terminus of the polypeptide fragment 2 is a cysteine, and the C-terminus is an amide end:

NH ₂ -Cys ²⁸ (R3)-Ile-Leu-Gly-Ser(R1) -Asp ³³ (R2)-Gly ³⁴ (R5) -Glu(R2)-Lys(R4)-Asn(R3)-Gln(R3 )-Cys(R3)-Val-Thr(R1)-Gly-Glu(R2)-Gly-Thr(R1)-Pro-Lys(R4)-Pro-Gln(R3)-Ser(R1)-His(R3 )-Asn(R3) -Asp ⁵³ (R2)-Gly ⁵⁴ (R5) -Asp(R2)-Phe-Glu(R2)-Glu(R2)-Ile-Pro-Glu(R2)-Glu(R2)- Tys ⁶³ (R6)-Leu-Gln(R3)-CONH ₂

Reagent names and abbreviations used in the following examples:

DMF: N,N-dimethylformamide;

DCM: dichloromethane;

MeOH: methanol;

HOBT: 1-hydroxybenzotriazole;

DIC: N,N-diisopropylcarbodiimide;

TBTU: benzotriazole tetramethyl tetrafluoroboric 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: tris(2-carboxyethyl)phosphine (TCEP);

MPAA: 4-mercaptophenylacetic acid;

PBS: sodium dihydrogen phosphate;

Example 1: One-pot synthesis method of ligation and refolding of hirudin HIRV1 with tyrosine sulfate modification:

(1) Preparation of Fmoc-Lys(Boc)-NHNH ₂ resin: Take 1 g of 2-Chlorotrityl Chloride resin (referred to as 2-Cl resin) in a peptide synthesis tube with a loading of 0.3-0.5 mmol/g, add 15 mL of DMF at room temperature Swell by shaking twice, 30min each time, drain, add 15mL 5% hydrazine hydrate/DMF to the resin, shake the reaction for 30min at room temperature, wash twice with DMF, add 15mL 5% hydrazine hydrate/DMF for the second time, The reaction was shaken for 30min at room temperature, washed twice with DMF, added 15mL of 5% hydrazine hydrate/DMF for the third time, shaken at room temperature for 30min, washed twice with DMF, DCM, and DMF in turn, drained the solvent to obtain C Resin with a hydrazide end (referred to as hydrazide resin), add 15 mL of 5% MeOH/DMF to the resin, shake for 10 min at room temperature, wash twice with DMF, DCM, and DMF in turn, drain the solvent, and block the non-acyl For the hydrazine resin site, weigh Fmoc-Lys(Boc)-OH (1 mmol), TBTU (0.98 mmol) and DIEA (2 mmol), and mix Fmoc-Lys(Boc)-OH (amino acid at position 27) with TBTU with A small amount of DMF was dissolved, DIEA was added, the carboxyl groups were activated by shaking at room temperature for 3 minutes, then the activated amino acid was added to the resin, and the activated amino acid was shaken at room temperature for 2 hours, washed three times with DMF and DCM, and dried with nitrogen to obtain a dry resin. The resin loading was detected by UV, and finally the Fmoc-Lys(Boc)-NHNH ₂ resin with a loading of about 0.42 mmol/g was obtained.

(2) Preparation of Hirudin HIRV1 Polypeptide Fragment 1-NHNH ₂ Resin: The obtained Fmoc-Lys(Boc)-NHNH ₂ resin was swollen twice by adding 15 mL of DMF at room temperature for 15 min each time, drained, and then added to the resin 10mL of 20% acetic anhydride/DMF was shaken at room temperature for 20min to block the amino group of the resin that was not coupled to the amino acid to prevent the next reaction. Wash twice with DMF, DCM and DMF in turn, add 10mL of 20% piperidine/DMF to the resin. DMF was shaken at room temperature for 5 minutes, washed twice with DMF, added 10 mL of 20% piperidine/DMF again, shaken at room temperature for 5 minutes, washed twice with DMF, DCM, and DMF in turn, and drained the solvent to obtain a Remove the resin protected by Fmoc, weigh Fmoc-Asn(Trt)-OH (4×0.42mmol), TBTU (3.9×0.42mmol) and DIEA (8×0.42mmol), dissolve Fmoc amino acid and TBTU with a small amount of DMF, add DIEA, the carboxyl group was activated by shaking reaction at room temperature for 3 min, then the activated amino acid was added to the resin, and the reaction was shaken at room temperature for 2 h. The reaction could be monitored with ninhydrin reagent, washed twice with DMF, DCM, and DMF, and the above experimental operation was repeated. (The amino acid content is calculated by 4 times the molar equivalent of the resin, and the shaking reaction is 2h), and the coupling is carried out from the C-terminal to the N-terminal according to the hirudin HIRV1 polypeptide fragment 1 sequence. After the synthesis, the mixture was washed three times with DMF and DCM, and dried with nitrogen to obtain 2.7 g of dry resin. Due to the increase of resin mass, the loading of resin was about 0.15 mmol/g at this time.

(3) Preparation of Fmoc-Gln(Trt)-MBHA resin: Take 1 g of Rink Amide MBHA resin in a peptide synthesis tube with a loading of 0.3-0.5 mmol/g, add 15 mL of DMF to swollen twice at room temperature with shaking for 15 min each time, Drain, add 10 mL of 20% piperidine/DMF to the resin, shake for 5 minutes at room temperature, wash twice with DMF, add 10 mL of 20% piperidine/DMF again, shake at room temperature for 5 minutes, and use DMF, DCM, DMF was washed twice each, and the solvent was drained to obtain a resin with the amino group removed from Fmoc protection. )-OH and TBTU were dissolved with a small amount of DMF, DIEA was added, the carboxyl group was activated by shaking reaction at room temperature for 3 min, then the activated amino acid was added to the resin, and the reaction was shaken at room temperature for 2 h, washed three times with DMF and DCM, and dried with nitrogen to obtain The resin was dried, and the resin loading was detected by ultraviolet light, and finally the Fmoc-Gln(Trt)-MBHA resin with a loading of about 0.47 mmol/g was obtained.

(4) Preparation of hirudin HIRV2 polypeptide fragment 2-MBHA resin with sulfated modified tyrosine: The obtained Fmoc-Gln(Trt)-MBHA resin was swollen by shaking twice at room temperature with 15 mL of DMF for 15 min each time, and drained. , then add 10 mL of 20% acetic anhydride/DMF to the resin, shake the reaction at room temperature for 20 min to block the amino group of the resin that is not coupled to the amino acid, prevent its next reaction, wash twice with DMF, DCM, and DMF in turn, add to the resin 10 mL of 20% piperidine/DMF was shaken for 5 min at room temperature, washed twice with DMF, 10 mL of 20% piperidine/DMF was added again, shaken and reacted at room temperature for 5 min, washed twice with DMF, DCM, and DMF in turn, Drain the solvent to obtain the resin deprotected by Fmoc, weigh Fmoc-Leu-OH (4×0.47mmol), TBTU (3.9×0.47mmol) and DIEA (8×0.47mmol), mix Fmoc amino acid with TBTU with a small amount of DMF Dissolve, add DIEA, activate the carboxyl group for 3min at room temperature, then add the activated amino acid to the resin, shake the reaction at room temperature for 2h, monitor the reaction with ninhydrin reagent, wash twice with DMF, DCM, DMF each, repeat The above experimental operations (the amino acid content is calculated by 4 times the molar equivalent of the resin, and the shaking reaction is 2h), are coupled according to the hirudin HIRV1 polypeptide fragment 2 sequence from the C-terminus to the N-terminus. Among them, the 33rd and 34th, 53rd and 54th aspartic acid-glycine sequences need to be coupled with Fmoc-dipeptide Fmoc-Asp(OtBu)-(Dmb)Gly-OH; the 63rd Sulfation-modified tyrosine requires coupling with Fmoc-Tyr(OSO ₃ nP)-OH. After the synthesis, the mixture was washed three times with DMF and DCM, and dried with nitrogen to obtain 3.3 g of dry resin. Due to the increase of resin mass, the loading of resin was about 0.10 mmol/g at this time.

(5) Preparation of hirudin HIRV1 polypeptide fragment 1-NHNH ₂ crude product: take 200 mg of the resin obtained in step (2), add 10 mL of cleavage reagent (TFA:DODT:H ₂ O=volume ratio 95:2.5:2.5), shake the reaction For 2-4 hours, filter to obtain a yellow transparent liquid. After the liquid is spin-dried on a rotary evaporator, add about 20 mL of ice ether for extraction twice, collect the precipitate after centrifugation, and freeze the sample to obtain about 30 mg of hirudin HIRV1 polypeptide fragment 1-NHNH ₂ crude product .

(6) Preparation of pure hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine: take 200 mg of the resin obtained in step (4), add 10 mL of cleavage reagent (TFA:DODT: _H2O =volume ratio 95:2.5 : 2.5), shake for _2h , and filter to obtain a yellow transparent liquid. After the liquid was spin-dried with a rotary evaporator, about 20 mL of ice ether was added to extract twice. After centrifugation, the precipitate was collected. The phase is a reverse-phase liquid chromatography of ACN/H ₂ O mixture containing 0.1% TFA for separation and purification, and the purified sample is lyophilized to obtain about 20 mg of hirudin HIRV2 polypeptide fragment with sulfated modified tyrosine. 2 pure Taste;

(7) Preparation of pure product of hirudin HIRV1 polypeptide fragment 1-MPAA: take the crude product of hirudin HIRV1 polypeptide fragment 1-NHNH ₂ obtained in step (5), use a solution containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS, pH 3.0 2.5 times the crude polypeptide equivalent of acetylacetone was added, shaken for 3 minutes and shaken, then added 15 times the crude polypeptide equivalent of MPAA, reacted for 12 h, then added 10 times the crude polypeptide equivalent of TCEP, reacted for 20 minutes, and centrifuged , filtered, separated and purified by reverse-phase liquid chromatography in which the mobile phase was ACN/H ₂ O mixture containing 0.1% TFA, and the purified sample was lyophilized to obtain about 10 mg of hirudin HIRV1 polypeptide fragment 1-MPAA pure Taste.

(8) Preparation of crude full-length hirudin HIRV1 linear polypeptide with sulfated modified tyrosine: prepare 4 mL containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS, 0.2mol/L MPAA, 40mmol/L TCEP, pH 6.5 Take 5.24 mg of pure hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine in step (6), add 275 μL of the above buffered saline solution to dissolve, take step (7) hirudin HIRV1 polypeptide fragment 1-MPAA 5.92 mg of pure product (1.67 times equivalent of hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine), add 300 μL of the above buffered saline solution to dissolve, and 275 μL of hirudin HIRV2 polypeptide with sulfated modified tyrosine Fragment 2 pure product solution was added to 300 μL of hirudin HIRV1 polypeptide fragment 1-MPAA pure product and mixed, and the natural chemical ligation (NCL for short) reaction was carried out for 12h. The automatic removal of the base (nP) resulted in a crude solution of the full-length hirudin HIRV1 linear polypeptide with the 63rd tyrosine sulfated modified and the nP protective group removed.

(9) One-pot refolding of crude full-length hirudin HIRV1 linear polypeptide with sulfated tyrosine: take the 63rd tyrosine obtained in step (8) that is sulfated and the nP protecting group has been removed. The crude solution of the full-length hirudin HIRV1 linear polypeptide was divided into two equal parts by volume and added to an ultrafiltration tube with a molecular weight of 3K, centrifugal ultrafiltration, using 6mol/L guanidine hydrochloride, 0.2mol/L PBS, pH 6.0 buffered saline solution Perform solution replacement to remove small molecules such as MPAA and TCEP that affect the refolding reaction, and obtain about 250 μL of crude solution of full-length hirudin HIRV1 linear polypeptide without MPAA and TCEP tyrosine sulfate modification at position 63, and slowly add it dropwise. In the refolding reaction system of about 20mL of 0.2mol/L tris(hydroxymethyl)aminomethane, 4mol/L sodium chloride, 4mmol/L L-cysteine, 2mmol/L L-cystine, pH 8.5, The oxidation and refolding reaction of the polypeptide was carried out for 12 h, centrifuged, filtered, and separated and purified by reverse liquid chromatography in which the mobile phase was ACN/H ₂ O mixture containing 0.1% TFA, and the purified sample was lyophilized to obtain approximately 1.81 mg (isolated yield of about 21%) of the 63-position tyrosine sulfated modified full-length hirudin HIRV1 protein, abbreviated as HIRV1 (63OSO ₃ ).

Reverse high performance liquid chromatography and ESI-MS characterization of the pure product of the hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine obtained in step (6), the results are shown in FIG. 1 .

The characterization of the pure product of hirudin HIRV1 polypeptide fragment 1-MPAA obtained in step (7) by reverse-phase high performance liquid chromatography and ESI-MS, the results are shown in FIG. 2 .

In the process of preparing HIRV1 (63OSO ₃ ) in steps (8) and (9), at the beginning of the NCL reaction (NCL-0h), during the NCL reaction (NCL-2h), and at the end of the NCL reaction (NCL-12h) , at the beginning of the refolding reaction (Fold-0h) and at the end of the refolding reaction (Fold-12h) to monitor, the reverse high performance liquid chromatography at the above time points and the ESI-MS characterization of important peak position substances, The results are shown in Figure 3.

The biological activity analysis of the HIRV1 (63OSO ₃ ) prepared in step (9) is carried out, and the results are shown in FIG. 4 . It can be seen from the above that the HIRV1 (63OSO ₃ ) prepared by the present invention can obviously inhibit the hydrolysis of fibrinogen by thrombin.

The analysis process is as follows:

1) Preparation solution X: 50 mM Tris, 0.1 M NaCl, 0.1% (w/v) PEG, pH 7.8;

2) At room temperature (25°C), add thrombin (5nM) and HIRV1 (63OSO ₃ ) (10nM) to solution X (1mL), dissolve, shake evenly, incubate at 37°C for 30min, and cool to room temperature (25°C). ℃), add fibrinogen (5 μM) to the sample, shake it evenly, and set it as the experimental group; observe the turbidity of the sample in the experimental group;

At room temperature (25°C), fibrinogen (5 μM) was added to solution X (1 mL), shaken evenly, and used as control group 1; the turbidity of the sample in control group 1 was observed;

At room temperature (25°C), add thrombin (5nM) to solution X (1mL), dissolve, shake evenly, incubate at 37°C for 30min, cool to room temperature (25°C), add fibrinogen (5μM) In this sample, it was shaken evenly and used as control group 2; the turbidity of the sample in control group 2 was observed.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China University of Technology

<120> A kind of chemical synthesis method of hirudin with tyrosine sulfate modification

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 65

<212> PRT

<213> Artificial Sequence

<220>

<223> Hirudin HIRV1 amino acid sequence

<400> 1

Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys

1 5 10 15

Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys Ile Leu Gly Ser

20 25 30

Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro

35 40 45

Gln Ser His Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu

50 55 60

Gln

65

Claims (10)

1. a chemical synthesis method with the hirudin of tyrosine sulfate modification, is characterized in that: comprise the steps: (1) According to the amino acid sequence of hirudin, it is divided into fragment 1 and fragment 2 from any cysteine; the resin whose C-terminus is a hydrazide end, referred to as hydrazide resin, adopts the Fmoc solid-phase peptide synthesis method. sequence, from C-terminal to N-terminal, condensed Fmoc-protected amino acids in sequence, washed, and dried to obtain fragment 1; (2) Using a resin with an amide end at the C-terminus, amide resin for short, using the Fmoc solid-phase peptide synthesis method, according to the sequence of fragment 2, sequentially condensing Fmoc-protected amino acids from the C-terminus to the N-terminus, washing, and drying to obtain fragment 2; in: The tyrosine near the C-terminus of the hirudin is a sulfated modified tyrosine, and Fmoc sulfated modified tyrosine with a side chain sulfate protecting group of a neopentyl group is used, abbreviated as Fmoc-Tyr(OSO ₃ nP)- OH for synthesis; (3) Take the linear polypeptide resin of fragment 1 prepared in step (1), add a cleavage reagent, remove the polypeptide chain from the hydrazide resin, and remove the remaining side chain protecting groups; filter, spin dry, extract, Centrifuge and freeze-dry to obtain the crude product of fragment 1 whose C-terminal is hydrazide; (4) Take the linear polypeptide resin of fragment 2 prepared in step (2), add a cleavage reagent, remove the polypeptide chain from the amide resin, and remove the remaining side chain protecting groups; filter, spin dry, extract, centrifuge , freeze-dried to obtain fragment 2 crude product; further separation and purification, freeze-dried to obtain fragment 2 whose C-terminal is an amide; (5) Take the crude product of fragment 1 with hydrazide at the C-terminus prepared in step (3), dissolve, add acetylacetone, shake well, then add 4-mercaptophenylacetic acid, and in the first step, add tris(2-carboxylate) ethyl) phosphine, the second step reaction, centrifugation, filtration, further separation and purification, lyophilization to obtain fragment 1 whose C-terminal is MPAA; (6) Take the fragment 2 prepared in step (4) with an amide at the C-terminus and the fragment 1 with an MPAA at the C-terminal prepared in step (5), dissolve, mix, and carry out a natural chemical ligation reaction to obtain a fragment close to the C-terminus. The crude solution of the hirudin linear polypeptide modified by tyrosine sulfate modification and the neopentyl group has been removed, referred to as solution M; (7) Take the solution M prepared in step (6), add an ultrafiltration tube, and replace the solution system by centrifugal filtration to obtain phenolic acid near the C-terminus that does not contain 4-mercaptophenylacetic acid and tris(2-carboxyethyl)phosphine The crude product solution of amino acid sulfated modified hirudin linear polypeptide, referred to as solution N; wherein: The replacement solution system is 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate, and the pH value of the solution is 6.0±0.5; (8) taking the solution N prepared in step (7), slowly adding it dropwise to the refolding reaction system, reacting, centrifuging, filtering, and further separating and purifying to obtain the hirudin modified by tyrosine sulfate modification near the C-terminus; wherein: The refolding reaction system is 0.15-0.25mol/L Tris, 3.5-4.5mol/L sodium chloride, 3.5-4.5mmol/L L-cysteine, 1.5-2.5mmol/L L-cystine , the pH of the solution is 8.5 ± 0.5. 2. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 1, is characterized in that: The amino acid sequence of the hirudin is shown in SEQ ID NO. 1, or the amino acid sequence obtained by replacing, deleting or adding one or several amino acids in SEQ ID NO. 1 and retaining the same biological activity. 3. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 1 and 2, is characterized in that: The aspartic acid-glycine amino acid site that contains in the aminoacid sequence of described hirudin, uses the protecting group of side chain carboxyl and amino to be respectively the Fmoc aspartic acid-glycine dipeptide of OtBu and Dmb, is called for short Fmoc-Asp ( OtBu)-(Dmb)Gly-OH was synthesized. 4. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 1 and 2, is characterized in that: The solution of dissolving the crude product of fragment 1 described in step (5) is 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate, and the pH value of the solution is 3.0±0.5; The solution for dissolving fragment 1 and fragment 2 described in step (6) is 5.5-6.5 mol/L guanidine hydrochloride, 0.15-0.25 mol/L sodium dihydrogen phosphate, 0.15-0.25 mol/L 4-mercaptophenylacetic acid, 38～42mmol/L tris(2-carboxyethyl)phosphine, the pH value of the solution is 6.5±0.5; The volume of the refolding reaction system described in step (8) is calculated by dissolving 0.25-0.4 mg of linear polypeptide in 1 mL of reaction solution. 5. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 1 and 2, is characterized in that: The solution of the dissolving fragment 1 crude product described in step (5) is 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen phosphate, and the pH value of the solution is 3.0; The solution of dissolving fragment 1 and fragment 2 described in step (6) is 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen phosphate, 0.2mol/L 4-mercaptophenylacetic acid, 40mmol/L tris(2- carboxyethyl) phosphine, the pH of the solution is 6.5; The consumption of the acetylacetone described in the step (5) is calculated by the crude product of fragment 1: the mol ratio of acetylacetone=1:2.5; The consumption of 4-mercaptophenylacetic acid described in step (5) is calculated according to the molar ratio of fragment 1 crude product: 4-mercaptophenylacetic acid=1:15; The consumption of tris (2-carboxyethyl) phosphine described in step (5) is calculated according to the molar ratio of fragment 1 crude product: MPAA=1:10; The amount of fragment 1 described in step (6) is calculated as 1.5 to 2 times the equivalent of fragment 2; What the ultrafiltration tube described in step (7) uses is the ultrafiltration tube of molecular weight 3K; The replacement solution system described in the step (7) is 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen phosphate, and the pH value of the solution is 6.0; The refolding system described in step (8) is 0.2mol/L Tris, 4mol/L sodium chloride, 4mmol/L L-cysteine, 2mmol/L L-cystine, and the pH value of the solution is 8.5 . 6. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 5, is characterized in that: The condition of the first step reaction described in the step (5) is that the temperature is 20～30 ℃, and the reaction time is 12 hours; The condition of the second step reaction described in the step (5) is that the temperature is 20～30 ℃, and the reaction time is 20 minutes; The conditions of the natural chemical ligation reaction described in the step (6) are that the temperature is 37±0.5°C, and the reaction time is 8 to 12 hours; The ultrafiltration described in the step (7) and the operation of replacing the solution system are repeated five times; The duration of the slow dripping described in the step (8) is 15 to 30 minutes; The conditions of the reaction described in step (8) are that the temperature is 20-30° C., and the reaction time is 4-12 hours. 7. the chemical synthesis method with the hirudin of tyrosine sulfate modification according to claim 2, is characterized in that: The hydrazide resin is the hydrazide resin obtained after 2-Chlorotrityl Chloride resin is treated with 5% hydrazine hydrate/DMF for 30 min; Described amide resin is Fmoc-Rink Amide-MBHA resin; The structural formula of the Fmoc-Tyr(OSO ₃ nP)-OH is as follows:

The dosage of the Fmoc-Tyr(OSO ₃ nP)-OH is calculated according to the molar ratio of resin: Fmoc-Tyr(OSO ₃ nP)-OH=1:1.5-4; The coupling time between the Fmoc-Tyr(OSO ₃ nP)-OH and the Fmoc amino resin is 6-12 hours; The structural formula of the described Fmoc-Asp(OtBu)-(Dmb)Gly-OH is as follows:

The consumption of described Fmoc-Asp(OtBu)-(Dmb)Gly-OH is calculated according to the molar ratio of resin: Fmoc-Asp(OtBu)-(Dmb)Gly-OH=1:1.5～4; The coupling time of the Fmoc-Asp(OtBu)-(Dmb)Gly-OH and the Fmoc amino resin is 4-8h; Fmoc-protected amino acids other than Fmoc-Tyr(OSO ₃ nP)-OH and Fmoc-Asp(OtBu)-(Dmb)Gly-OH are Fmoc amino acids without side chains, or side chain protecting groups R1, R2, R3 Or R4 can be synthesized with Fmoc amino acid that can be removed with trifluoroacetic acid; wherein: R1 represents tert-butyl, R2 represents tert-butyl ester, R3 represents trityl, R4 represents tert-butoxycarbonyl, and the consumption is according to Fmoc amino resin: Fmoc protected amino acid = 1:4 molar ratio calculation; The coupling time of Fmoc-protected amino acids other than Fmoc-Tyr(OSO ₃ nP)-OH and Fmoc-Asp(OtBu)-(Dmb)Gly-OH and Fmoc amino resin is 2～4h; The specific operation of condensing Fmoc-protected amino acids from the C-terminus to the N-terminus in step (1) and step (2) is as follows: under the action of the coupling system, the first amino acid and the hydrazide or amide of Fmoc are firstly removed. The resin is reacted to generate amino acid-amino resin, and then other Fmoc protected amino acids are coupled one by one to obtain a linear polypeptide resin; The condensing agent in the described coupling system is "HOBT+DIC" or "TBTU+DIEA"; The Fmoc deprotection reagent in the coupling system is 20% piperidine/DMF; The deprotection reaction time in the coupling system is 5-10 min; The loading of the resin is 0.3-0.5 mmoL/g. 8. the chemical synthesis method of the hirudin with tyrosine sulfate modification according to claim 1 and 2, is characterized in that: The reagents for cleavage described in step (3) and step (4) are trifluoroacetic acid, water and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid according to The solution obtained by mixing the volume ratio of 95:2.5:2.5; The time of cutting described in step (3) and step (4) is 2～4h; The extractant of the extraction described in step (3) and step (4) is glacial ether. 9 . A hirudin with tyrosine sulfate modification, characterized in that: it is obtained by the synthetic method described in any one of claims 1 to 8 . 10. The application of the tyrosine sulfated hirudin described in claim 9 in the preparation of anticoagulant drugs and/or medical materials.

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