CN114736289A

CN114736289A - Chemical synthesis method of hirudin with tyrosine sulfation modification

Info

Publication number: CN114736289A
Application number: CN202210261766.XA
Authority: CN
Inventors: 何春茂; 杨烨
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2022-07-12
Anticipated expiration: 2042-03-17
Also published as: CN114736289B

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

Chemical synthesis method of hirudin with tyrosine sulfation modification

Technical Field

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

Background

Hirudin (Hirudin) is a protein found in the saliva of leeches. The amino acid sequence of hirudin contains about 65 amino acid residues, and its amino terminal (N-terminal) contains six cysteines, and forms a spherical compact stable structure by three pairs of disulfide bonds, and its exposed carboxyl terminal (C-terminal) sequence has homology with fibrinogen in animal body in amino acid sequence. The thrombin in the animal body can specifically recognize and cut fibrinogen, so that the fibrinogen with better water solubility originally forms a fibrin monomer which is insoluble in water, and the thrombin plays roles of blocking blood vessels and preventing excessive bleeding through the action mechanism. The hirudin and thrombin have stronger interaction than the fibrinogen and the thrombin, can directly inhibit the activity of the thrombin, and are the most effective natural thrombin inhibitors known at present. Since hirudin has anticoagulant activity, it is considered to be clinically used as an anticoagulant, and through relevant evaluation, hirudin is considered to have potential value as an antithrombotic agent, and some clinical studies have been conducted on it by obtaining recombinant hirudin by a plasmid expression method (Nowak, G.,

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

K_ithe inhibition constant in enzyme kinetics can be expressed and reflects the inhibition intensity of the inhibitor on the enzyme, and the smaller the value, the stronger the inhibition intensity is. K of natural hirudin to thrombin is reported_iThe value is about 25fM (Stone, S.R., Hofsteenge, J., biochemistry, 1986,25, 4622-one 4628), whereas the K of thrombin for recombinant hirudins expressed by E.coli or yeast_iThe value was approximately 300fM (Liu, C.C., Schultz, P.G., nat. Biotechnol.,2006,24,1436-40.), which indicates expression by plasmidThe obtained recombinant hirudin has anticoagulant activity lower than that of natural hirudin. The difference between the activity of natural hirudin and recombinant hirudin is that the natural hirudin contains a sulfated and modified tyrosine (Tyr), which can make the C-terminal of hirudin negatively charged, and enhance the interaction between the C-terminal of hirudin and thrombin, thereby improving the anticoagulant activity of hirudin. Since the sulfation modification of tyrosine is a post-translational modification (PTM) which is found in higher eukaryotes, recombinant hirudins expressed by e.coli or yeast do not undergo this important post-translational modification, thus resulting in a significant reduction of the anticoagulant activity of the recombinant hirudin.

Post-translational modification (PTM) of proteins is a common mechanism that enriches the structural diversity of an organism's genome. Two common PTMs include O-glycosylation modification of serine (Ser) or threonine (Thr) (r.j. payne, c.h.wong, chem.commun.,2010,46, 21-43) and sulfation modification of tyrosine (Tyr) mentioned above (c.seibert, t.p.sakmar, pept.sci.,2008,90, 459-. These two modifications are performed at golgi weight by glycosyltransferases and Tyrosine Protein Sulfotransferases (TPSTs), respectively. It is estimated that more than 50% of proteins expressed in eukaryotes are O-glycosylation modified and more than 1% are tyrosine sulfation modified. These two PTMs are involved in many biological processes including molecular recognition, cell differentiation, immunomodulation and protein folding, which have led to a great interest in the development of various disease therapeutics for glycosylated and sulfated proteins. Despite the importance of these modifications, it is extremely challenging to obtain purified O-glycosylation modified proteins and Tyr sulfation modified proteins. Because the PTM process has a non-templated nature, it is determined by the relative activities of the transferases, and changes in relative activities result in the organisms expressing a mixture of proteins of various types, both glycoform (with O-glycosylation modifications) and sulfoform (with Tyr sulfation modifications), which are generally not separable by chromatographic separation techniques.

The main currently available route for obtaining homogenous glycoproteins (O-glycosylation modified proteins) and sulphated modified proteins is the chemical synthesis. The technology of obtaining a homogenous glycoprotein by chemical synthesis methods is well established, and the technology of synthesizing a homogenous sulfated and modified protein by applying the technology is also being developed.

The different hirudins found to date differ somewhat in amino acid sequence and are thought to undergo mutation to develop several variants. Among them, hirudin HIRV1, a european medicine, was entered into clinical studies as an anticoagulant in an unmodified form and was found to exhibit a certain inhibitory effect on human thrombin activity (c.c. liu, e.brusmad, w.liu, p.g. schultz, j.am.chem.soc.,2007,129, 10648-10649.). It is considered that if tyrosine sulfated and modified hirudin HIRV1 can be obtained, and the synthetic process is optimized, the synthetic yield of sulfated and modified hirudin HIRV1 in the chemical synthetic process is improved, and the improvement of the anticoagulation activity in the clinical research of hirudin HIRV1 and the reduction of the cost of the clinical research are greatly influenced. Therefore, there is a need to develop a synthetic method with convenient synthesis and purification, modular synthesis and considerable yield, so as to efficiently prepare the homogenized hirudin HIRV1 with sulfation modification.

Disclosure of Invention

The invention aims at providing a chemical synthesis method of hirudin with sulfated modified tyrosine.

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

The method is optimized, and the aim of improving the synthesis yield can be achieved by carrying out sequential one-pot reaction on natural chemical ligation and protein refolding.

The purpose of the invention is realized by the following technical scheme:

a chemical synthesis method of hirudin with tyrosine sulfation modification comprises the following steps:

(1) according to the amino acid sequence of hirudin, any cysteine is divided into a segment 1 and a segment 2 (the N end of the segment 2 is cysteine); using C-terminal as acylHydrazine (-NHNH)₂) Resin at the tail end, called hydrazide resin for short, adopting an Fmoc solid-phase polypeptide synthesis method, sequentially condensing Fmoc protected amino acids from the C end to the N end according to the sequence of the fragment 1, washing, and drying to obtain the fragment 1;

(2) using C-terminal amide (-CONH)₂) End resin, called amide resin for short, adopting Fmoc solid phase polypeptide synthesis method, condensing Fmoc protected amino acids from C end to N end in sequence according to the sequence of the fragment 2, washing, drying to obtain the fragment 2; wherein:

the tyrosine close to the C end of the hirudin is sulfated and modified tyrosine, Fmoc sulfated and modified tyrosine with side chain sulfate protecting group of Neopentyl (Neopentyl, nP) is used, and is called Fmoc-Tyr (OSO) for short₃Synthesizing nP) -OH;

(3) taking the linear polypeptide resin of the segment 1 prepared in the step (1), adding a cutting reagent to remove polypeptide chains from hydrazide resin, and removing residual side chain protecting groups; filtering, spin-drying, extracting, centrifuging, and lyophilizing to obtain C-terminal hydrazide (-NHNH)₂) Crude fragment 1 of (a);

(4) taking the linear polypeptide resin of the segment 2 prepared in the step (2), adding a cutting reagent to remove polypeptide chains from the amide resin, and removing the residual side chain protecting groups; filtering, spin-drying, extracting, centrifuging, and lyophilizing to obtain crude fragment 2; further separating, purifying and freeze-drying to obtain C terminal amide (-CONH)₂) Fragment 2 of (1);

(5) taking the crude product of the fragment 1 with the C end as hydrazide prepared in the step (3), dissolving, adding acetylacetone, shaking up, adding 4-mercaptophenylacetic acid (MPAA), reacting in the first step, adding tris (2-carboxyethyl) phosphine (TCEP), reacting in the second step, centrifuging, filtering, further separating and purifying, and freeze-drying to obtain the fragment 1 with the C end as MPAA;

(6) taking the C terminal prepared in the step (4) as amide (-CONH)₂) Respectively dissolving and mixing the fragment 2 and the fragment 1 with the C end being MPAA prepared in the step (5), carrying out Natural Chemical Ligation (NCL) reaction to obtain a crude hirudin linear polypeptide solution with the tyrosine sulfation modification near the C end and the neopentyl removed,solution M for short;

(7) adding the solution M prepared in the step (6) into an ultrafiltration tube, performing centrifugal filtration while replacing a solution system to obtain a crude product solution of the hirudin linear polypeptide which does not contain 4-mercaptophenylacetic acid and tris (2-carboxyethyl) phosphine and is close to the C terminal and is modified by tyrosine sulfation, and the crude product solution is called solution N for short; wherein:

the substitution solution system is 5.5-6.5 mol/L guanidine hydrochloride and 0.15-0.25 mol/L sodium dihydrogen Phosphate (PBS), and the pH value of the solution is 6.0 +/-0.5;

(8) slowly dripping the solution N prepared in the step (7) into a refolding reaction system, reacting, centrifuging, filtering, and further separating and purifying to obtain the hirudin with tyrosine sulfation modification close to the C end; wherein:

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

In the chemical synthesis method, the amino acid sequence of hirudin is VVYTDCTESGQNLCLCEGSNVCGQGNK²⁷CILGS ³³ ³⁴DGEKNQCVTGEGTPKPQSHN ⁵³ ⁵⁴DGDFEEIPEEY⁶³LQ is also shown as SEQ ID NO.1, or is shown as an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and retaining the same biological activity in SEQ ID NO. 1. Wherein the superscript indicates the position number of the amino acid in the sequence.

In the chemical synthesis method, the amino acid site of aspartic acid (Asp) -glycine (Gly) contained in the amino acid sequence of the hirudin is synthesized by using Fmoc aspartic acid-glycine dipeptides of which the side chain carboxyl and amino protecting groups are OtBu (tert-butyl ester, R2) and Dmb (2, 4-dimethoxybenzyl, R5), which are called Fmoc-Asp (OtBu) - (Dmb) Gly-OH for short. For example, when the amino acid sequence of hirudin is shown in SEQ ID NO.1, aspartic acid (Asp) -glycine (Gly) at the amino acid positions 33, 34, 53 and 54 are specific sequences, and the gradual synthesis is easy to generate isomerization phenomena, so that a uniform polypeptide product cannot be obtained during final purification. The synthesis using the above-mentioned dipeptides can solve the related problems. Wherein, 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-terminal of hirudin; further preferably, the fifth cysteine from the N-terminus.

In the chemical synthesis method, the hydrazide resin is preferably obtained by treating 2-Chlorotrityl Chloride resin (2-Cl resin for short) 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 Fmoc-Tyr (OSO)₃The formula of nP) -OH is shown below:

the Fmoc-Tyr (OSO) is₃The amount of nP) -OH is preferably used, in view of cost, as Fmoc amino resin: Fmoc-Tyr (OSO)₃nP) -OH ═ 1: 1.5 molar ratio calculation; if cost is not a concern, it is preferable to use Fmoc amino resin: Fmoc-Tyr (OSO)₃nP) -OH ═ 1: 4, calculating the molar ratio; in general, said Fmoc-Tyr (OSO)₃The amount of nP) -OH is calculated according to the resin: Fmoc-Tyr (OSO)₃nP) -OH ═ 1: 1.5-4.

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

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

the amount of Fmoc-Asp (OtBu) - (Dmb) Gly-OH is preferably determined by the following steps of Fmoc amino resin: fmoc-asp (otbu) - (Dmb) Gly-OH ═ 1: 1.5 molar ratio calculation; if cost is not a concern, it is preferable to use Fmoc amino resin: fmoc-asp (otbu) - (Dmb) Gly-OH ═ 1: 4; in general, the amount of Fmoc-Asp (OtBu) - (Dmb) Gly-OH used was calculated as resin: fmoc-asp (otbu) - (Dmb) Gly-OH ═ 1: 1.5-4.

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

Except Fmoc-Tyr (OSO)₃The Fmoc protected amino acids except nP) -OH and Fmoc-Asp (OtBu) - (Dmb) Gly-OH are Fmoc amino acids without side chains, or protecting groups of the side chains R1, R2, R3 or R4 can be synthesized by removing the Fmoc amino acids by trifluoroacetic acid; wherein: r1 represents tert-butyl (tBu), R2 represents tert-butyl ester (OtBu), R3 represents trityl (Trt), R4 represents tert-butoxycarbonyl (Boc), preferably in an amount as follows in Fmoc amino resin: fmoc protected amino acid ═ 1: 4 in terms of molar ratio.

Except Fmoc-Tyr (OSO)₃The coupling time of the amino acid with Fmoc protection except for nP) -OH and Fmoc-Asp (OtBu)) - (Dmb) Gly-OH and Fmoc amino resin is preferably 2-4 h; more preferably 2 h.

The specific operation of sequentially condensing Fmoc protected amino acids from the C end to the N end in the steps (1) and (2) is as follows: under the action of a coupling system, the 1 st amino acid reacts with hydrazide or amide resin after Fmoc removal to generate amino acid-amino resin, and other Fmoc protected amino acids are coupled one by one to obtain linear polypeptide resin.

The condensing agent in the coupling system is preferably "HOBT + DIC" or "TBTU + DIEA". In particular, said Fmoc-Tyr (OSO)₃The coupling system of nP) -OH, Fmoc-Asp (OtBu) - (Dmb) Gly-OH and the condensing agent 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 amount of the resin is preferably 0.3-0.5 mmoL/g.

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

The cutting time in the steps (3) and (4) is preferably 2-4 h.

The extractant for extraction in the steps (3) and (4) is preferably ethyl glacial ether.

The number of extractions described in step (3) and step (4) is preferably two.

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

The mobile phase of the reverse phase liquid chromatography is acetonitrile/water mixed solution containing 0.1 percent of trifluoroacetic acid.

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

The amount of acetylacetone used in step (5) is preferably determined in accordance with the ratio of crude fragment 1: acetylacetone was calculated at a molar ratio of 1: 2.5.

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

The amount of 4-mercaptophenylacetic acid used in step (5) is preferably such that the ratio of crude fragment 1: 4-mercaptophenylacetic acid was calculated at a molar ratio of 1: 15.

The first-step reaction in the step (5) is carried out under the condition that the temperature is 20-30 ℃ (room temperature), and the reaction time is preferably 12 hours.

The amount of tris (2-carboxyethyl) phosphine used in step (5) is preferably as follows: MPAA is calculated at a molar ratio of 1: 10.

The second-step reaction in the step (5) is carried out under the condition that the temperature is 20-30 ℃ (room temperature), and the reaction time is preferably 20 minutes.

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

The solution for dissolving the fragment 1 and the fragment 2 in the 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/L4-mercaptophenylacetic acid and 38-42 mmol/L tris (2-carboxyethyl) phosphine, and the pH value of the solution is 6.5 +/-0.5; further preferably 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen Phosphate (PBS), 0.2 mol/L4-mercaptophenylacetic acid, 40mmol/L tris (2-carboxyethyl) phosphine, and the pH of the solution is 6.5.

The dosage of the fragment 1 in the step (6) is preferably calculated according to 1.5-2 times of the equivalent of the fragment 2.

The condition of the natural chemical ligation reaction in the step (6) is that the temperature is 37 +/-0.5 ℃, and the reaction time is preferably 8-12 hours.

The natural chemical ligation reaction described in step (6) will be accompanied by the automatic removal of neopentyl as it is carried out in an aqueous system.

The ultrafiltration tube in the step (7) is an ultrafiltration tube with the molecular weight of 3K, and the ultrafiltration has the following functions: firstly, removing all molecules with the molecular weight less than 3000 in the system; the second is a replacement solution system.

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

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

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

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

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

The reaction condition in the step (8) is 20-30 ℃ (room temperature), and the reaction time is preferably 4-12 hours.

The separation and purification in the step (8) is preferably carried out by reverse phase liquid chromatography.

Hirudin with tyrosine sulfating modification is obtained by the above synthesis method.

The hirudin with tyrosine sulfation modification is applied to the preparation of anticoagulant drugs and/or medical materials.

The principle of the invention is as follows: adopting an Fmoc solid-phase polypeptide synthesis method, modularly dividing a sequence into two peptide segments according to an amino acid sequence of hirudin, wherein a polypeptide segment 1 is the 1 st-27 th amino acid of the hirudin, and condensing Fmoc protective amino acids from the C end to the N end by taking hydrazide resin as a carrier to obtain the hydrazide resin of the hirudin HIRV1 polypeptide segment 1; polypeptide fragment 2 is 28-65 th amino acid of hirudin HIRV1, MBHA resin is used as carrier, Fmoc-Tyr (OSO) is used at the site (63 rd tyrosine) to be modified₃nP) -OH, using Fmoc-Asp (OtBu) - (Dmb) Gly-OH at sites (33 th aspartic acid and 34 th glycine, 53 th aspartic acid and 54 th glycine) needing to avoid isomerization, and sequentially condensing Fmoc protected amino acids from the C end to the N end to obtain an amino resin of the hirudin HIRV2 polypeptide fragment 2; wherein, the removal mode of the nP protecting group is different from the side chain protecting groups of other amino acids; the nP protecting group is automatically removed in an aqueous solution reaction system of NCL to obtain a crude product solution of the full-length hirudin linear polypeptide, wherein the 63 rd tyrosine is modified by sulfation and the nP protecting group is removed; performing ultrafiltration centrifugation to replace the solution and remove MPAA and TCEP which can influence refolding reaction; the crude solution of the full-length hirudin linear polypeptide after ultrafiltration and replacement can be slowly dripped into a large-volume refolding reaction system without separation and purification, so that six free sulfydryl groups of six cysteine sites (6 th site, 14 th site, 16 th site, 22 th site, 28 th site and 39 th site) of the full-length hirudin linear polypeptide modified by tyrosine sulfation are oxidized to form three pairs of disulfide bonds (6 th site and 14 th site are a pair, 16 th site and 28 th site are a pair, and 22 th site and 39 th site are a pair), and the folded full-length hirudin protein modified by tyrosine sulfation is obtained.

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

the method of the invention provides a method for introducing sulfated modified tyrosine Fmoc-Tyr (OSO) on polypeptide resin₃Means for synthesizing nP) -OH, and corresponding examples are provided. In the synthetic route, only the mature Fmoc amino acid solid-phase synthesis method is needed to introduce the sulfated and modified tyrosine at the target position, and the side reaction easily occurring in the synthetic process is avoided due to the mature Fmoc amino acid solid-phase polypeptide synthesis method.

In the method of the invention, amino acid Fmoc-Tyr (OSO) with side chain sulfate protecting group of nP is introduced in the solid phase synthesis of polypeptide₃And the nP) -OH, the nP protecting group is automatically removed in the aqueous solution in the NCL reaction process, and no additional protecting group removing reaction is needed, so that the synthesis time is shortened, and the synthesis efficiency is improved.

In the method, after the crude product of the polypeptide fragment 1 of the C-terminal hydrazide of hirudin is obtained, the C-terminal hydrazide is converted into MPAA through thioesterification reaction of the pH 3 of a reaction system, and the MPAA is separated, purified and lyophilized, so that the HIRV1 polypeptide fragment 1 at the tail end of the MPAA stably exists in a lyophilized form, and hydrolysis reaction easily occurring when the pH of the MPAA is more than 7 is avoided.

In the earlier stage of research of the method, after the NCL reaction in the step (6) is carried out, the crude product solution obtained in the step (6) is subjected to first separation and purification by reverse liquid chromatography with a mobile phase of acetonitrile/water mixed solution containing 0.1% trifluoroacetic acid, freeze-drying is carried out to obtain a pure product of the full-length hirudin linear polypeptide with the sulfated and modified tyrosine at the 63 th position and the removed nP protecting group, then the pure product freeze-dried powder is dissolved in a refolding reaction system for oxidation and refolding, the components and the concentration of the refolding reaction system are the same as those of the system, and finally the pure product is separated and purified by the reverse liquid chromatography method with the same mobile phase again and freeze-dried to obtain the sulfated and modified full-length hirudin protein at the 63 th position, and the protein synthesis yield is 4%. The method of the invention innovatively adopts a one-pot method of NCL reaction and refolding reaction. The polypeptide fragment 1 of hirudin with MPAA at the C end and the polypeptide fragment 2 of hirudin with sulfated modified tyrosine and cysteine at the N end are subjected to NCL reaction, and by a method of ultrafiltration centrifugation and solution system replacement, after small molecules MPAA and TCEP which are easy to influence refolding reaction are removed, a crude product solution is directly and slowly dripped into the refolding reaction system for reaction, so that the conversion of the hirudin full-length linear polypeptide folded into hirudin protein is realized, the liquid phase purification steps after the NCL reaction are saved, two times of separation and purification are reduced to one time, the synthesis time and cost are greatly shortened on the premise of not influencing the product purity, the synthesis yield is improved, and the protein synthesis yield is finally improved to 21%.

The method for preparing the hirudin protein with the sulfated modified tyrosine solves the problem that the recombinant hirudin expressed by escherichia coli or yeast cannot be subjected to a post-translational modification process, adopts Fmoc solid-phase polypeptide synthesis, introduces the sulfated modified tyrosine into the polypeptide, and improves the synthesis yield of the hirudin protein by a sequential one-pot method of NCL reaction and refolding reaction.

The hirudin protein with sulfated modified tyrosine obtained by the method is expected to show the anticoagulant activity equivalent to that of natural hirudin, is beneficial to researching the physiological and biochemical effects of the hirudin on thrombus resistance or cardiovascular diseases, and lays a good foundation for the hirudin in the aspects of clinical research and disease control.

Drawings

FIG. 1 is a representation of the reverse phase HPLC and ESI-MS of example 1 in the preparation of pure hirudin HIRV1 polypeptide fragment 2(F2nP) with sulfated modification and containing a neopentyl protected tyrosine.

FIG. 2 is a representation of the reverse phase HPLC and ESI-MS of the pure hirudin HIRV1 polypeptide fragment 1-MPAA (F1) prepared in example 1.

FIG. 3 shows example 1 in the preparation of HIRV1(63 OSO)₃) In the process of (1), fragment 1(F1), fragment 2(F2nP) protected with a neopentyl group, the time of initiation of the NCL reaction (NCL-0h), the time of completion of the NCL reaction (NCL-12h), the time of initiation of the refolding reaction (Fold-0h), the time of completion of the refolding reaction (Fold-12h), and the reaction product HIRV1(63 OSO)₃) (FoldP) characterization of reverse phase high performance liquid chromatography with ESI-MS of the material at the important peak positions.

FIG. 4 shows HIRV1(63 OSO) prepared in example 1₃) A graph showing the results of an experiment for inhibiting the activity of thrombin to cleave fibrinogen; wherein, (A) is a sample of a control group 1, (B) is a sample of a control group 2, and (C) is a sample of an experimental group.

Detailed Description

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

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Fmoc-Tyr (OSO) used in the following examples₃nP) -OH, the structural formula of which is shown below, is prepared by the method described in references "Simpson L.S, Zhu J.Z, Widlanski T.S, et al, Chemistry Biology,2009,16(2): 153-" Simpson L.S, Widlanski T.S., Journal of the American Chemical Society,2006,128(5):1605- "Schlienger N, Peyrotes S, Kassem T, et al, Journal of Medicinal Chemistry,2000,43(23): 4570-" and 4574 ":

Fmoc-Asp (OtBu) - (Dmb) Gly-OH used in the following examples has the following structural formula:

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.

The following examples were HPLC using an Agilent 1260, a Phenomenex C18 column as column and water and acetonitrile as mobile phase (0.1% (v/v) TFA).

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

NH₂-Val¹-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)-Lys²⁷(R4)-NHNH₂

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

NH₂-Cys²⁸(R3)-Ile-Leu-Gly-Ser(R1)- ³³ ³⁴Asp(R2)-Gly(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)- ⁵³ ⁵⁴Asp(R2)-Gly(R5)-Asp(R2)-Phe-Glu(R2)-Glu(R2)-Ile-Pro-Glu(R2)-Glu(R2)-Tys⁶³(R6)-Leu-Gln(R3)-CONH₂

the names and abbreviations of the reagents used in the following examples:

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: tris (2-carboxyethyl) phosphine (TCEP);

MPAA: 4-mercaptophenylacetic acid;

PBS: sodium dihydrogen phosphate;

example 1: the method for synthesizing hirudin HIRV1 with tyrosine sulfation modification by a connection and refolding one-pot method comprises the following steps:

(1)Fmoc-Lys(Boc)-NHNH₂preparation of resin: taking 1g of 2-Chlorotrityl Chloride resin (abbreviated as 2-Cl resin) in a polypeptide synthesis tube, adding 15mL of DMF (dimethyl formamide) with the loading amount of 0.3-0.5mmol/g, oscillating and swelling twice at room temperature for 30min each time, draining, adding 15mL of 5% hydrazine hydrate/DMF into the resin, oscillating and reacting for 30min at room temperature, washing twice with DMF, adding 15mL of 5% hydrazine hydrate/DMF for the second time, oscillating and reacting for 30min at room temperature, washing twice with DMF, adding 15mL of 5% hydrazine hydrate/DMF for the third time, oscillating and reacting for 30min at room temperature, washing twice with DMF, DCM and DMF in sequence, draining solvent to obtain resin (abbreviated as hydrazide resin) with the C end, adding 15mL of 5% MeOH/DMF into the resin, oscillating and reacting for 10min at room temperature, washing twice with DMF, DCM and DMF in sequence, draining solvent, blocking non-hydrazide resin sites, weighing Fmoc-Lys (Boc) -OH (1mmol), TBTU (0.98mmol) and DIEA (2mmol), dissolving Fmoc-Lys (Boc) -OH (27 th amino acid) and TBTU with a small amount of DMF, adding DIEA, performing oscillation reaction at room temperature to activate carboxyl for 3min, adding the activated amino acid into the resin, performing oscillation reaction at room temperature for 2h, washing with DMF and DCM for three times respectively, drying with nitrogen to obtain dried resin, taking the resin, and detecting the resin load by ultraviolet to obtain Fmoc-Lys (Boc) -NHNH with the load of about 0.42mmol/g₂And (3) resin.

(2) Hirudin HIRV1 polypeptide fragment 1-NHNH₂Preparation of resin: taking the obtained Fmoc-Lys (Boc) -NHNH₂Adding 15mL of DMF into the resin, shaking and swelling twice at room temperature for 15min each time, draining, adding 10mL of 20% acetic anhydride/DMF into the resin, shaking and reacting for 20min at room temperature to seal the amino group of the resin not coupled with the amino acid, stopping the next reaction, sequentially washing twice with DMF, DCM and DMF, adding 10mL of 20% piperidine/DMF into the resin, shaking and reacting for 5min at room temperature, washing twice with DMF, adding 10mL of 20% piperidine/DMF again, shaking and reacting for 5min at room temperature, sequentially washing with DMF, DCM and DMFWashing with DMF twice respectively, draining the solvent to obtain the resin without Fmoc protection, weighing Fmoc-Asn (Trt) -OH (4 multiplied by 0.42mmol), TBTU (3.9 multiplied by 0.42mmol) and DIEA (8 multiplied by 0.42mmol), dissolving Fmoc amino acid and TBTU with a small amount of DMF, adding DIEA, oscillating at room temperature to activate carboxyl for 3min, adding the activated amino acid into the resin, oscillating at room temperature for 2h, monitoring the reaction with ninhydrin reagent, washing with DMF, DCM and DMF twice respectively, repeating the above experimental operations (the amino acid content is calculated by 4 times of molar equivalent of the resin, the oscillating reaction is 2h), and coupling from C end to N end according to hirudin HIRV1 polypeptide fragment 1 sequence. After the synthesis, the resin was washed with DMF and DCM for three times, and dried with nitrogen to obtain 2.7g of dried resin. The resin loading was about 0.15mmol/g at this time due to the increased mass of the resin.

(3) Preparation of Fmoc-Gln (Trt) -MBHA resin: taking 1g Rink Amide MBHA resin into a polypeptide synthesis tube, adding 15mL of DMF, oscillating and swelling twice at room temperature for 15min each time, draining, adding 10mL of 20% piperidine/DMF into the resin, oscillating and reacting for 5min at room temperature, washing twice with DMF, adding 10mL of 20% piperidine/DMF, oscillating and reacting for 5min at room temperature, washing twice with DMF, DCM and DMF in sequence, draining the solvent to obtain the resin with the Fmoc protection removed, weighing Fmoc-Gln (Trt) -OH (1mmol), TBTU (0.98mmol) and DIEA (2mmol), dissolving Fmoc-Gln (Trt) -OH and TBTU with a small amount of DMF, adding DIEA, oscillating and reacting for 3min at room temperature, adding the activated amino acid into the resin, oscillating and reacting for 2h at room temperature, washing three times with DMF and DCM respectively, drying the resin by nitrogen to obtain dry resin, taking the resin, and detecting the resin load by ultraviolet to finally obtain the Fmoc-Gln (Trt) -MBHA resin with the load of about 0.47 mmol/g.

(4) Preparation of hirudin HIRV2 polypeptide fragment 2-MBHA resin with sulfated modified tyrosine: adding 15mL of DMF into the obtained Fmoc-Gln (Trt) -MBHA resin, shaking and swelling for 15min at room temperature twice, draining, adding 10mL of 20% acetic anhydride/DMF into the resin, shaking and reacting for 20min at room temperature to block the amino group of the resin without the amino acid coupled, stopping the next reaction, washing with DMF, DCM and DMF twice in sequence, adding 10mL of DMF into the resin, shaking and reacting for 20min at room temperature to block the amino group of the resin without the amino acid coupled, and washing with DMF, DCM and DMF twice in sequence, adding 10mL of DMF into the resin, and swelling with shaking and swelling with DMF for 15min at room temperature to obtain the Fmoc-Gln (Trt) -MBHA resin, and swelling with DMF to obtain the amino acid coupled resin with the amino acid coupled resin, and reacting for the next reaction of the resin with DMF, washing with DCM and DMF in sequence, and washing with DMF twice, and adding 10mL of DMF to the resinOscillating and reacting for 5min at room temperature by using 20 percent of piperidine/DMF in mL, washing twice by using DMF, adding 10mL of 20% piperidine/DMF again, carrying out shake reaction for 5min at room temperature, washing with DMF, DCM and DMF twice in sequence, draining the solvent to obtain the resin without Fmoc protection, weighing Fmoc-Leu-OH (4X 0.47mmol), TBTU (3.9X 0.47mmol) and DIEA (8X 0.47mmol), dissolving Fmoc amino acid and TBTU with a small amount of DMF, adding DIEA, carrying out shake reaction at room temperature to activate carboxyl for 3min, adding the activated amino acid into the resin, shaking for 2h at room temperature, monitoring reaction with ninhydrin reagent, washing twice with DMF, DCM, and DMF respectively, repeating the above experiment (amino acid content is calculated as 4 times molar equivalent of resin, shaking for 2h), and coupling from C-terminus to N-terminus according to sequence 2 of hirudin HIRV1 polypeptide fragment. Wherein, the aspartic acid-glycine sequences at 33 th site and 34 th site, and 53 th site and 54 th site need to be coupled by using Fmoc-dipeptide Fmoc-Asp (OtBu) - (Dmb) Gly-OH; the 63-site sulfated modified tyrosine needs Fmoc-Tyr (OSO)₃And (n) are coupled to OH. After the synthesis, the resin was washed with DMF and DCM for three times, and dried with nitrogen to obtain 3.3g of dry resin. The loading of the resin at this point was about 0.10mmol/g due to the increased mass of the resin.

(5) Hirudin HIRV1 polypeptide fragment 1-NHNH₂Preparation of a crude product: 200mg of the resin obtained in step (2) was taken, and 10mL of a cleavage reagent (TFA: DODT: H) was added₂O-volume ratio 95: 2.5: 2.5) shaking for 2-4 h, filtering to obtain a yellow transparent liquid, drying the liquid by a rotary evaporator, adding about 20mL of diethyl ether for extraction twice, centrifuging, collecting precipitate, and freeze-drying the sample to obtain about 30mg of hirudin HIRV1 polypeptide fragment 1-NHNH₂And (5) crude product.

(6) Preparation of purified hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine: 200mg of the resin obtained in step (4) was taken, and 10mL of a cleavage reagent (TFA: DODT: H) was added₂O-volume ratio 95: 2.5: 2.5), shaking for 2H, filtering to obtain a yellow transparent liquid, spin-drying the liquid by a rotary evaporator, adding about 20mL of diethyl ether for extraction twice, centrifuging, collecting precipitate, and taking a sample with 20% ACN/H₂Dissolving O, passing through mobile phase of ACN/H containing 0.1% TFA₂Separating and purifying by reversed liquid chromatography of O mixed solutionPurifying, and lyophilizing the purified sample to obtain about 20mg of hirudin HIRV2 polypeptide fragment 2 with sulfated and modified tyrosine;

(7) preparation of pure hirudin HIRV1 polypeptide fragment 1-MPAA: taking the hirudin HIRV1 polypeptide fragment 1-NHNH obtained in the step (5)₂Dissolving the crude product with buffer salt solution containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS and pH 3.0, adding acetylacetone with 2.5 times of polypeptide crude product equivalent, shaking for 3 minutes, adding MPAA with 15 times of polypeptide crude product equivalent, reacting for 12 hours, adding TCEP with 10 times of polypeptide crude product equivalent, reacting for 20 minutes, centrifuging, filtering, and passing through mobile phase ACN/H containing 0.1% TFA₂And separating and purifying the O mixed solution by reverse liquid chromatography, and freeze-drying the purified sample to obtain the hirudin HIRV1 polypeptide fragment 1-MPAA pure product of about 10 mg.

(8) Preparation of crude full-length hirudin HIRV1 linear polypeptide with sulfated modified tyrosine: preparing 4mL buffer salt solution containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS, 0.2mol/L MPAA, 40mmol/L TCEP and pH 6.5, taking 5.24mg of hirudin HIRV2 polypeptide fragment 2 with sulfated and modified tyrosine in the step (6), adding 275 mu L of the above buffer salt solution for dissolution, taking 5.92mg of hirudin HIRV1 polypeptide fragment 1-MPAA pure product (which is 1.67 times of the polypeptide fragment 2 with sulfated and modified tyrosine), adding 300 mu L of the above buffer salt solution for dissolution, adding 275 mu L of the polypeptide fragment 2 pure solution with sulfated and modified tyrosine into 300 mu L of the polypeptide fragment 1-MPAA pure product of hirudin HIRV1 for mixing, carrying out Natural Chemical Ligation (NCL) reaction for 12h, and simultaneously removing a neopentyl P protecting group in the buffer salt solution, a crude solution of the full-length hirudin HIRV1 linear polypeptide is obtained, which is modified by sulfation of tyrosine at position 63 and has been freed of the nP protecting group.

(9) One-pot refolding of the full-length hirudin HIRV1 linear polypeptide crude product with sulfated modified tyrosine: taking the crude solution of the full-length hirudin HIRV1 linear polypeptide which is obtained in the step (8) and is sulfated and modified by tyrosine at the 63 th position and removed with the nP protecting group, dividing into two parts according to the volume, adding the two parts into an ultrafiltration tube with the molecular weight of 3K, performing centrifugal ultrafiltration, and using 6mol/L guanidine hydrochloridePerforming solution replacement with 0.2mol/L PBS and pH 6.0 buffer salt solution to remove small molecules affecting refolding reaction such as MPAA and TCEP to obtain about 250 μ L crude solution of full-length hirudin HIRV1 linear polypeptide without sulfated modification of tyrosine at position 63 of MPAA and TCEP, slowly adding dropwise into about 20mL refolding reaction system containing 0.2mol/L trihydroxymethyl aminomethane, 4mol/L sodium chloride, 4mmol/L L-cysteine, 2mmol/L L-cystine and pH 8.5, performing polypeptide oxidation and refolding reaction for 12H, centrifuging, filtering, and performing mobile phase of ACN/H containing 0.1% TFA₂Separating and purifying by reverse phase liquid chromatography, and lyophilizing the purified sample to obtain about 1.81mg (21% isolated yield) of the 63 rd tyrosine sulfated-modified full-length hirudin HIRV1 protein, HIRV1(63 OSO)₃)。

And (3) reverse phase high performance liquid chromatography and ESI-MS characterization of the pure hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine obtained in the step (6) is shown in figure 1.

And (3) carrying out reverse phase high performance liquid chromatography and ESI-MS characterization on the purified hirudin HIRV1 polypeptide fragment 1-MPAA obtained in the step (7), and obtaining a result shown in figure 2.

Step (8) and step (9) in the preparation of HIRV1(63 OSO)₃) In the process (2), the conditions of the initial phase of the NCL reaction (NCL-0h), the in-process phase of the NCL reaction (NCL-2h), the end phase of the NCL reaction (NCL-12h), the start phase of the refolding reaction (Fold-0h) and the end phase of the refolding reaction (Fold-12h) were monitored, and the results of the reverse phase high performance liquid chromatography and ESI-MS of the substance having the important peak position at the above time points are shown in FIG. 3.

To HIRV1(63 OSO) prepared in step (9)₃) The results of the bioactivity analysis are shown in FIG. 4. It can be seen that HIRV1(63 OSO) prepared according to the invention₃) Can obviously inhibit the hydrolysis of the fibrinogen by the thrombin.

The analytical procedure was as follows:

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

2) thrombin (5nM) was mixed with HIRV1(63 OSO) at room temperature (25 ℃ C.)₃) (10nM) was added to solution X (1mL), dissolved and shakenHomogenizing, incubating at 37 deg.C for 30min, cooling to room temperature (25 deg.C), adding fibrinogen (5 μ M) into the sample, and shaking to obtain experimental group; observing the turbidity of the experimental group samples;

fibrinogen (5. mu.M) was added to solution X (1mL) at room temperature (25 ℃ C.) and shaken well as control 1; observing the turbidity of the control group 1 sample;

adding thrombin (5nM) to solution X (1mL) at room temperature (25 ℃), dissolving, shaking, incubating at 37 ℃ for 30min, cooling to room temperature (25 ℃), adding fibrinogen (5. mu.M) to the sample, shaking, and shaking to obtain control group 2; the turbidity of the control 2 sample was observed.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Sequence listing

<110> south China university of science and technology

<120> chemical synthesis method of hirudin with tyrosine sulfation modification

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 65

<212> PRT

<213> Artificial Sequence (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

1. A chemical synthesis method of hirudin with tyrosine sulfation modification is characterized in that: the method comprises the following steps:

(1) the amino acid sequence of hirudin is divided into fragment 1 and fragment 2 from any cysteine; sequentially condensing Fmoc protected amino acids from the C end to the N end according to the sequence of the fragment 1 by using a resin with the C end as the terminal of hydrazide, namely hydrazide resin for short, adopting an Fmoc solid-phase polypeptide synthesis method, washing and drying to obtain the fragment 1;

(2) using resin with the C end as the amide end, abbreviated as amide resin, condensing Fmoc protected amino acids from the C end to the N end in sequence according to the sequence of the fragment 2 by adopting an Fmoc solid-phase polypeptide synthesis method, washing and drying to obtain a fragment 2; wherein:

the tyrosine close to the C end of the hirudin is sulfated and modified tyrosine, and Fmoc sulfated and modified tyrosine with side chain sulfate protecting group of neopentyl is used, and is called Fmoc-Tyr (OSO) for short₃Synthesizing nP) -OH;

(3) taking the linear polypeptide resin of the segment 1 prepared in the step (1), adding a cutting reagent to remove polypeptide chains from hydrazide resin, and removing the remaining side chain protecting groups; filtering, spin-drying, extracting, centrifuging and freeze-drying to obtain a fragment 1 crude product with the C end being hydrazide;

(4) taking the linear polypeptide resin of the segment 2 prepared in the step (2), adding a cutting reagent to remove polypeptide chains from the amide resin, and removing the residual side chain protecting groups; filtering, spin-drying, extracting, centrifuging, and lyophilizing to obtain fragment 2 crude product; further separating, purifying and freeze-drying to obtain a fragment 2 of which the C end is amide;

(5) taking the crude product of the fragment 1 with the C end as hydrazide prepared in the step (3), dissolving, adding acetylacetone, shaking up, adding 4-mercaptophenylacetic acid, reacting in the first step, adding tris (2-carboxyethyl) phosphine, reacting in the second step, centrifuging, filtering, further separating and purifying, and freeze-drying to obtain the fragment 1 with the C end as MPAA;

(6) respectively dissolving and mixing the fragment 2 with the C terminal being amide prepared in the step (4) and the fragment 1 with the C terminal being MPAA prepared in the step (5), and performing natural chemical ligation reaction to obtain a crude hirudin linear polypeptide solution which is close to the C terminal and is modified by sulfation of tyrosine and has removed neopentyl, and is called solution M for short;

the displacement solution system is 5.5-6.5 mol/L guanidine hydrochloride and 0.15-0.25 mol/L sodium dihydrogen phosphate, and the pH value of the solution is 6.0 +/-0.5;

2. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 1, which is characterized in that:

the amino acid sequence of the hirudin is shown as SEQ ID NO.1, or is shown as the amino acid sequence obtained by substituting, deleting or adding one or more amino acids in SEQ ID NO.1 and keeping the same biological activity.

3. The chemical synthesis process of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:

the amino acid site of aspartic acid-glycine contained in the amino acid sequence of the hirudin is synthesized by Fmoc aspartic acid-glycine dipeptides of which the side chain carboxyl and amino protecting groups are OtBu and Dmb respectively, which are called Fmoc-Asp (OtBu) - (Dmb) Gly-OH for short.

4. The chemical synthesis process of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:

the solution for dissolving the crude product of the fragment 1 in the step (5) is 5.5-6.5 mol/L guanidine hydrochloride and 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 the fragment 1 and the fragment 2 in the 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/L4-mercaptophenylacetic acid and 38-42 mmol/L tris (2-carboxyethyl) phosphine, and the pH value of the solution is 6.5 +/-0.5;

and (3) calculating the volume of the refolding reaction system in the step (8) according to the fact that each 0.25-0.4 mg of linear polypeptide is dissolved in 1mL of reaction solution.

5. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:

the solution for dissolving the crude product of the fragment 1 in the step (5) is 6mol/L guanidine hydrochloride and 0.2mol/L sodium dihydrogen phosphate, and the pH value of the solution is 3.0;

the solution for dissolving the fragment 1 and the fragment 2 in the step (6) is 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen phosphate, 0.2 mol/L4-mercaptophenyl acetic acid and 40mmol/L tris (2-carboxyethyl) phosphine, and the pH value of the solution is 6.5;

the dosage of the acetylacetone in the step (5) is calculated according to the crude product of the fragment 1: acetylacetone at a molar ratio of 1: 2.5;

the dosage of the 4-mercaptophenylacetic acid in the step (5) is calculated according to the crude product of the fragment 1: 4-mercaptophenylacetic acid at a molar ratio of 1: 15;

the dosage of the tri (2-carboxyethyl) phosphine in the step (5) is as follows according to the crude product of the fragment 1: MPAA 1:10 molar ratio;

calculating the dosage of the fragment 1 in the step (6) according to 1.5-2 times of the equivalent of the fragment 2;

the ultrafiltration tube in the step (7) is an ultrafiltration tube with the molecular weight of 3K;

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

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

6. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 5, which is characterized in that:

the first step of reaction in the step (5) is carried out under the conditions that the temperature is 20-30 ℃ and the reaction time is 12 hours;

the second-step reaction in the step (5) is carried out under the conditions that the temperature is 20-30 ℃ and the reaction time is 20 minutes;

the condition of the natural chemical ligation reaction in the step (6) is that the temperature is 37 +/-0.5 ℃, and the reaction time is 8-12 hours;

repeating the operation of ultrafiltering and replacing the solution system in the step (7) for five times;

the slow dripping time in the step (8) is 15-30 minutes;

the reaction condition in the step (8) is that the temperature is 20-30 ℃, and the reaction time is 4-12 hours.

7. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 2, characterized in that:

the hydrazide resin is obtained by treating 2-Chlorotrityl Chloride resin with 5% hydrazine hydrate/DMF for 30 min;

the Amide resin is Fmoc-Rink Amide-MBHA resin;

the Fmoc-Tyr (OSO) is₃The structural formula of nP) -OH is shown as followsThe following steps:

the Fmoc-Tyr (OSO) is₃The amount of nP) -OH is calculated according to the resin: Fmoc-Tyr (OSO)₃nP) -OH ═ 1: 1.5-4 molar ratio calculation;

the Fmoc-Tyr (OSO) is₃The coupling time of the nP) -OH and the Fmoc amino resin is 6-12 h;

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

the dosage of Fmoc-Asp (OtBu) - (Dmb) Gly-OH is as follows: fmoc-asp (otbu) - (Dmb) Gly-OH ═ 1: 1.5-4 molar ratio calculation;

the coupling time of the Fmoc-Asp (OtBu) - (Dmb) Gly-OH and the Fmoc amino resin is 4-8 h;

except Fmoc-Tyr (OSO)₃The nP) -OH and Fmoc protected amino acids except Fmoc-Asp (OtBu) - (Dmb) Gly-OH are Fmoc amino acids without side chains, or protecting groups of the side chains, such as R1, R2, R3 or R4, can be synthesized by removing the Fmoc amino acids by trifluoroacetic acid; wherein: r1 represents tert-butyl, R2 represents tert-butyl ester, R3 represents trityl, R4 represents tert-butoxycarbonyl, used in an amount of Fmoc amino resin: fmoc protected amino acid ═ 1: 4;

except Fmoc-Tyr (OSO)₃Coupling the amino acid with Fmoc protection except for nP-OH and Fmoc-Asp (OtBu) - (Dmb) Gly-OH with Fmoc amino resin for 2-4 h;

the specific operation of sequentially condensing Fmoc protected amino acids from the C end to the N end in the steps (1) and (2) is as follows: under the action of a coupling system, firstly, reacting the 1 st amino acid with hydrazide or amide resin without Fmoc to generate amino acid-amino resin, and then coupling other Fmoc-protected amino acids one by one to obtain linear polypeptide resin;

the condensing agent in the 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 capacity of the resin is 0.3-0.5 mmoL/g.

8. The chemical synthesis process of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:

the reagents for cleavage in step (3) and step (4) are trifluoroacetic acid, water and 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid at a molar ratio of 95: 2.5: 2.5 volume ratio of the obtained solution;

the cutting time in the step (3) and the step (4) is 2-4 h;

the extractant extracted in the steps (3) and (4) is ethyl glacial ether.

9. A hirudin with a tyrosine sulfation modification is characterized in that: obtained by the synthesis method described in any one of claims 1 to 8.

10. Use of hirudin according to claim 9 with tyrosine sulfation modification for the preparation of anticoagulant and/or medical materials.