CN114736289B - Chemical synthesis method of hirudin with tyrosine sulfation modification - Google Patents
Chemical synthesis method of hirudin with tyrosine sulfation modification Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention provides a chemical synthesis method of hirudin with tyrosine sulfation modification. The invention synthesizes a C-terminal hydrazinized fragment 1 and a sulfated modified tyrosine fragment 2, then obtains full-length linear polypeptide by utilizing natural chemical connection reaction and accompanying neopentyl removal reaction, finally removes impurities in the system, and adds the impurities into a refolding reaction system dropwise for oxidation and refolding reaction to generate three pairs of disulfide bonds, thus obtaining the target product. The method introduces the post-translational modification of the sulfated tyrosine to the polypeptide resin through a solid-phase polypeptide synthesis method, so that the sulfated tyrosine is stably existing on the solid-phase resin, side reaction is avoided, and separation and purification are realized; natural chemical connection is combined with automatic removal of neopentyl in 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 in the middle, so that the synthesis yield is greatly improved, and the method has good application prospect.
Description
Technical Field
The invention relates to the technical field of solid phase synthesis preparation of polypeptide sulfation modification, 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, its amino terminal (N-terminal) contains six cysteines, and forms a stable structure with compact sphere by three pairs of disulfide bonds, and its exposed carboxyl terminal (C-terminal) sequence is identical to the amino acid sequence of fibrinogen in animalsThe above-mentioned sequences have homology. Thrombin in an animal body can specifically identify and cut fibrinogen, so that fibrinogen with good water solubility originally forms a water-insoluble fibrin monomer, 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 thrombin", can directly inhibit the activity of thrombin, and are currently known to be the most effective natural thrombin inhibitors. Because hirudin has anticoagulant activity, it is considered to be clinically used as an anticoagulant, and potential value of hirudin as an antithrombotic agent is considered through related evaluation, and a certain clinical study was conducted on recombinant hirudin obtained by a plasmid expression method (Nowak, g., K.,Thromb.Haemostasis,2007,98,116–119.)。
K i The inhibition constant in the enzyme kinetics can be expressed, reflecting the inhibition intensity of the inhibitor on the enzyme, and the smaller this value, the stronger the inhibition intensity. K of natural hirudin against thrombin is reported i The value was about 25fM (Stone, S.R., hofsteinge, J., biochemistry, 1986,25,4622-4628), whereas recombinant hirudin expressed by E.coli or yeast had a K for thrombin i Values of about 300fM (Liu, C.C., schultz, P.G., nat.Biotechnol.,2006,24,1436-40.) indicate that recombinant hirudin obtained by the plasmid expression method has a lower anticoagulant activity than natural hirudin. The difference in activity between natural hirudin and recombinant hirudin is due to the fact that natural hirudin contains a sulfated and modified tyrosine (Tyr), which can negatively charge the C-terminal of hirudin and enhance the interaction between the C-terminal of hirudin and thrombin, thereby enhancing the anticoagulant activity of hirudin. Since the sulfation modification of tyrosine is a post-translational modification (post-translational modification, PTM) found in higher eukaryotes, recombinant hirudin expressed by E.coli or yeast does not undergo this important post-translational modification, and thus the anticoagulant activity of the recombinant hirudin is significantly reduced.
Post-translational modification (PTM) of proteins is a common mechanism for enriching the structural diversity of the genome of an organism. Two common PTMs include the O-glycosylation modification of serine (Ser) or threonine (Thr) (R.J.Payne, C.H.Wong, chem.Commun.,2010,46,21-43) and the sulfation modification of tyrosine (Tyr) mentioned above (C.Seibert, T.P.Sakmar, pept.Sci.,2008,90,459-477). These two modifications are performed again in the golgi apparatus by glycosyltransferases and Tyrosine Protein Sulfotransferases (TPSTs), respectively. It is estimated that of eukaryotic expressed proteins, more than 50% are O-glycosylation modified and more than 1% are tyrosine sulfation modified. These two PTMs are involved in a number of biological processes including molecular recognition, cell differentiation, immunomodulation, and protein folding, which have led to a great deal of interest in the development of various disease-treating drugs for glycosylated and sulfated proteins. Despite the importance of these modifications, it is very challenging to obtain purified O-glycosylation modified proteins and Tyr sulfate modified proteins. Because the PTM process has non-templated properties, which are determined by the relative activity of the transferase, changes in relative activity result in the organism expressing various protein mixtures of the glycoform (modified with O-glycosylation) and the sulfo form (modified with Tyr sulfation), which are generally not separated by chromatographic separation techniques.
To obtain homogeneous glycoproteins (O-glycosylation modified proteins) and sulfated modified proteins, the main viable pathway is currently the method of chemical synthesis. The technology for obtaining a homogenous glycoprotein by a chemical synthesis method has been mature gradually, and the technology for synthesizing a homogenous sulfated modified protein using the technology is also developing gradually.
The different species of hirudins found at present differ somewhat in amino acid sequence, and it is thought that hirudins undergo mutation to develop several variants. Among them, HIRV1, an european pharmaceutical hirudin, was entered into clinical studies as an anticoagulant in unmodified form and was found to exhibit a certain inhibition of human thrombin activity (C.C.Liu, E.Brustad, W.Liu, P.G.Schultz, J.Am.Chem.Soc.,2007,129,10648-10649.). It is believed that if tyrosine sulfation modified hirudin HIRV1 could be obtained and the synthetic yield of sulfation modified hirudin HIRV1 in the chemical synthesis process was improved by optimizing the synthesis process, this would have a significant impact on the improvement of the anticoagulation activity in the clinical studies of hirudin HIRV1 and the reduction of the clinical study costs. Therefore, there is a need to develop a synthetic method which is convenient to synthesize and purify, can be synthesized in a modularized manner and has a considerable yield, so as to efficiently prepare the uniform hirudin HIRV1 with sulfation modification.
Disclosure of Invention
The primary aim of the invention is to provide a chemical synthesis method of hirudin with sulfated modified tyrosine.
The sectional modularized hirudin with sulfated modified tyrosine prepared by the method is separable and finally uniform 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 one-pot reaction of natural chemical connection and protein refolding in sequence.
The aim of the invention is achieved by the following technical scheme:
a chemical synthesis method of hirudin with tyrosine sulfation modification comprises the following steps:
(1) Dividing the hirudin into a fragment 1 and a fragment 2 from any cysteine according to the amino acid sequence of the hirudin (the N end of the fragment 2 is cysteine); using a C-terminal hydrazide (-NHNH) 2 ) The method comprises the steps of condensing Fmoc-protected amino acid from the C end to the N end in sequence according to the sequence of the fragment 1 by adopting Fmoc solid-phase polypeptide synthesis method to obtain terminal resin, namely hydrazide resin, washing and drying to obtain the fragment 1;
(2) Using amides at the C-terminus (-CONH) 2 ) The method comprises the steps of condensing Fmoc-protected amino acid from the C end to the N end in sequence according to the sequence of the fragment 2 by adopting Fmoc solid-phase polypeptide synthesis method to obtain the terminal resin, namely amide resin, washing and drying to obtain the fragment 2; wherein:
The tyrosine near the C end of hirudin is sulfated modified tyrosine, and Fmoc sulfation modified tyrosine with side chain sulfate protecting group of Neopentyl (Neopenyl, nP) is usedFmoc-Tyr(OSO 3 nP) -OH for synthesis;
(3) Taking the linear polypeptide resin of the fragment 1 prepared in the step (1), adding a cutting reagent to enable a polypeptide chain to be detached from the hydrazide resin, and removing residual side chain protecting groups; filtering, spin drying, extracting, centrifuging and freeze-drying to obtain the product with the C-terminal as hydrazide (-NHNH) 2 ) Fragment 1 crude of (2);
(4) Taking the linear polypeptide resin of the fragment 2 prepared in the step (2), adding a cutting reagent to remove the polypeptide chain from the amide resin, and removing the residual side chain protecting group; filtering, spin drying, extracting, centrifuging and freeze-drying to obtain a fragment 2 crude product; further separating, purifying, and lyophilizing to obtain amide (-CONH) at C-terminal 2 ) Fragment 2 of (2);
(5) Taking a fragment 1 crude product with a hydrazide at the C end, which is prepared in the step (3), dissolving, adding acetylacetone, shaking uniformly, adding 4-mercaptophenylacetic acid (MPAA), performing a first reaction, adding tris (2-carboxyethyl) phosphine (TCEP), performing a second reaction, centrifuging, filtering, further separating, purifying and freeze-drying to obtain a fragment 1 with the MPAA at the C end;
(6) Taking the C-terminal prepared in the step (4) as amide (-CONH) 2 ) The fragment 2 of the hirudin linear polypeptide which is prepared in the step (5) and is obtained by obtaining the fragment 1 with the C end being MPAA is respectively dissolved, mixed and subjected to natural chemical ligation (Native chemical ligation, abbreviated as NCL) reaction to obtain a crude solution, abbreviated as solution M, of the hirudin linear polypeptide which is subjected to tyrosine sulfation modification near the C end and removed by neopentyl;
(7) Adding the solution M prepared in the step (6) into a ultrafilter tube, centrifugally filtering and simultaneously replacing a solution system to obtain a crude solution of tyrosine sulfation modified hirudin linear polypeptide which does not contain 4-mercaptophenylacetic acid and tri (2-carboxyethyl) phosphine and is close to the C end, wherein the crude solution is called as solution N for short; 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) 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 tyrosine sulfation modified hirudin near 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 VVYTDCTESGQNLCLCEGSNVCGQGNK 27 CILGS 33 34 DGEKNQCVTGEGTPKPQSHN 53 54 DGDFEEIPEEY 63 LQ is also shown as SEQ ID NO.1, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids and retaining the same biological activity as SEQ ID NO. 1. Wherein the superscript indicates the position number of the amino acid in the sequence.
In the chemical synthesis method, an aspartic acid (Asp) -glycine (Gly) amino acid site contained in the amino acid sequence of hirudin is synthesized by using Fmoc aspartic acid-glycine dipeptide with side chain carboxyl and amino protecting groups of OtBu (tert-butyl ester, R2) and Dmb (2, 4-dimethoxybenzyl, R5) respectively, namely Fmoc-Asp (OtBu) - (Dmb) Gly-OH. For example, when the amino acid sequence of hirudin is shown in SEQ ID NO.1, aspartic acid (Asp) -glycine (Gly) at amino acid positions 33, 34, 53 and 54 is a specific sequence, and the gradual synthesis is easy to cause isomerism, so that a homogenized polypeptide product cannot be obtained during final purification. The related problems can be solved by using the above dipeptide for synthesis. 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 fourth, fifth or sixth cysteine from the N end of hirudin; further preferred is 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, fmoc-Tyr (OSO 3 The structural formula of nP) -OH is shown as follows:
the Fmoc-Tyr (OSO) 3 The amount of nP) -OH is preferably, if cost is concerned, as Fmoc amino resin: fmoc-Tyr (OSO) 3 nP) -oh=1: 1.5 molar ratio calculation; if cost is not a concern, the Fmoc amino resin is preferably used: fmoc-Tyr (OSO) 3 nP) -oh=1: 4 molar ratio calculation; in general, the Fmoc-Tyr (OSO 3 The dosage of nP) -OH is as follows: fmoc-Tyr (OSO) 3 nP) -oh=1: 1.5 to 4.
The Fmoc-Tyr (OSO) 3 The coupling time of nP) -OH and Fmoc amino resin is preferably 6-12 h; more preferably 12h;
the structural formula of Fmoc-Asp (OtBu) - (Dmb) Gly-OH is shown as follows:
The Fmoc-Asp (OtBu) - (Dmb) Gly-OH is preferably used in the form of Fmoc amino resin if cost is a concern: fmoc-Asp (OtBu) - (Dmb) Gly-oh=1: 1.5 molar ratio calculation; if cost is not a concern, the Fmoc amino resin is preferably used: fmoc-Asp (OtBu) - (Dmb) Gly-oh=1: 4 molar ratio calculation; in general, fmoc-Asp (OtBu) - (Dmb) Gly-OH was used in the following resin: fmoc-Asp (OtBu) - (Dmb) Gly-oh=1: 1.5 to 4.
The coupling time of Fmoc-Asp (OtBu) - (Dmb) Gly-OH and Fmoc amino resin is preferably 4-8 h; more preferably 8h.
Fmoc-Tyr removal (OSO) 3 The nP) -OH and Fmoc protected amino acid except Fmoc-Asp (OtBu) - (Dmb) Gly-OH are Fmoc amino acid without side chains, or the protecting groups R1, R2, R3 or R4 of the side chains can be synthesized by using trifluoroacetic acid to remove the Fmoc amino acid; wherein: r1 representsT-butyl (tBu), R2 represents t-butyl (OtBu), R3 represents trityl (Trt), R4 represents t-butoxycarbonyl (Boc), the amounts being preferably as Fmoc amino resin: fmoc protected amino acid = 1: 4.
Fmoc-Tyr removal (OSO) 3 The coupling time of the Fmoc-protected amino acid except nP) -OH and Fmoc-Asp (OtBu) - (Dmb) Gly-OH and the Fmoc amino resin is preferably 2-4 h; more preferably 2h.
The specific operation of condensing Fmoc protected amino acids from the C end to the N end in sequence in the step (1) and the step (2) is as follows: under the action of a coupling system, firstly reacting the 1 st amino acid with hydrazide or amide resin after Fmoc removal to generate amino acid-amino resin, and then coupling other Fmoc protective amino acids one by one to obtain the linear polypeptide resin.
The condensing agent in the coupling system is preferably "HOBT+DIC" or "TBTU+DIEA". In particular, the Fmoc-Tyr (OSO 3 The condensing agent in the coupling system of 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 0.5mmoL/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) according to 95:2.5:2.5 by volume.
The time for the cutting in the step (3) and the step (4) is preferably 2 to 4 hours.
The extractant extracted in step (3) and step (4) is preferably glacial diethyl ether.
The number of extractions described in step (3) and step (4) is preferably two.
The separation and purification in the step (4) are preferably carried out by reverse phase liquid chromatography.
The mobile phase of the reverse liquid chromatography is acetonitrile/water mixed solution containing 0.1% 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, 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, 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 as crude product of fragment 1: acetylacetone=1:2.5 molar ratio.
The shaking-up time in step (5) is preferably 3 minutes.
The amount of 4-mercaptophenylacetic acid described in step (5) is preferably as crude product of fragment 1: 4-mercaptophenylacetic acid=1:15 molar ratio.
The conditions for the first reaction step in step (5) are a temperature of 20 to 30℃and a reaction time of preferably 12 hours.
The amount of tris (2-carboxyethyl) phosphine described in step (5) is preferably as crude in fragment 1: mpaa=1:10 molar ratio.
The conditions for the second reaction step in step (5) are 20 to 30℃and the reaction time is preferably 20 minutes.
The separation and purification described in step (5) is preferably carried out by reverse phase liquid chromatography.
The mobile phase of the reverse liquid chromatography is acetonitrile/water mixed solution containing 0.1% of trifluoroacetic acid.
The solution for dissolving the fragments 1 and 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, 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 amount of fragment 1 described in step (6) is preferably 1.5 to 2 times the equivalent weight of fragment 2.
The natural chemical ligation reaction conditions in step (6) are 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 automatic removal of neopentyl groups as it is carried out in an aqueous system.
The ultrafiltration tube in the step (7) is an ultrafiltration tube with a molecular weight of 3K, and the ultrafiltration has the following functions: firstly, removing all molecules with molecular weight less than 3000 in the system; and secondly, a replacement solution system.
The replacement solution system in the step (7) is preferably 6mol/L guanidine hydrochloride, 0.2mol/L sodium dihydrogen Phosphate (PBS), and the pH value of the solution is 6.0.
The ultrafiltration described in step (7) and the operation of replacing the solution system are 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, pH of the solution being 8.5.
The volume of the refolding reaction system described in step (8) is preferably calculated as 1mL of reaction solution per 0.25 to 0.4mg of linear polypeptide.
The duration of the slow addition in step (8) is preferably 15 to 30 minutes, more preferably 30 minutes.
The reaction conditions in step (8) are temperatures of 20 to 30℃and reaction times of 4 to 12 hours are preferred.
The separation and purification in the step (8) are preferably carried out by reverse phase liquid chromatography.
The mobile phase of the reverse liquid chromatography is acetonitrile/water mixed solution containing 0.1% of trifluoroacetic acid.
Hirudin with tyrosine sulfation modification is obtained by the above synthesis method.
The hirudin with tyrosine sulfation modification is applied to the preparation of anticoagulants and/or medical materials.
The principle of the invention is as follows: adopting Fmoc solid-phase polypeptide synthesis method, modularly dividing the sequence into two peptide segments according to the amino acid sequence of hirudin, wherein the polypeptide segment 1 is amino acid 1-27 of hirudin, using hydrazide resin as a carrier, and condensing Fmoc protective amino acid from C end to N end in sequence to obtain the acyl of hirudin HIRV1 polypeptide segment 1 A hydrazine resin; the polypeptide fragment 2 is amino acid 28-65 of hirudin HIRV1, MBHA resin is used as carrier, fmoc-Tyr (OSO) is used at the site (63 rd tyrosine) to be introduced with modification 3 nP) -OH, fmoc-Asp (OtBu) - (Dmb) Gly-OH is used at the site (33 rd aspartic acid and 34 th glycine, 53 rd aspartic acid and 54 th glycine) needing to avoid isomerization, fmoc-protected amino acid is condensed from the C end to the N end in sequence, and amino resin of hirudin HIRV2 polypeptide fragment 2 is obtained; wherein, the removing 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 under the water solution reaction system of NCL to obtain the crude product solution of the full-length hirudin linear polypeptide which is subjected to 63 rd tyrosine sulfation modification and the nP protecting group is removed; performing ultrafiltration centrifugation, replacing the solution, and removing MPAA and TCEP which affect refolding reaction; the crude solution of the full-length hirudin linear polypeptide after ultrafiltration and solution replacement can be slowly dripped into a large-volume refolding reaction system without separation and purification, so that six free sulfhydryl groups of six cysteine sites (6 th, 14 th, 16 th, 22 th, 28 th and 39 th) of the tyrosine sulfation modified full-length hirudin linear polypeptide are oxidized to form three pairs of disulfide bonds (a pair of 6 th and 14 th, a pair of 16 th and 28 th and a pair of 22 nd and 39 th), and the folded tyrosine sulfation modified full-length hirudin protein is obtained.
Compared with the prior art, the invention has the following advantages and effects:
the method of the invention provides that the sulfated modified tyrosine Fmoc-Tyr (OSO) is introduced into the polypeptide resin 3 The means of synthesis of nP) -OH, and corresponding examples are provided. In the synthetic route, the sulfated and modified tyrosine can be introduced into the target position only by using an Fmoc amino acid solid-phase synthesis method which is mature in technology, and side reactions which are easy to occur in the synthesis process are avoided due to the maturation of the Fmoc amino acid solid-phase polypeptide synthesis method.
In the method of the invention, amino acid Fmoc-Tyr (OSO) with side chain sulfate radical protecting group nP is introduced in polypeptide solid phase synthesis 3 nP) -OH, in the NCL reaction process, nP protecting group is automatically removed in aqueous solution without increasingAnd an additional protecting group is added for removal reaction, 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 the hirudin is obtained, the C-terminal hydrazide is converted into the MPAA through the thioesterification reaction of the pH 3 of a reaction system, and the MPAA is separated, purified and freeze-dried, so that the HIRV1 polypeptide fragment 1 at the tail end of the MPAA exists stably in a freeze-dried form, and the hydrolysis reaction easily occurring when the pH of the MPAA is more than 7 is avoided.
In the earlier stage of the research of the method, after NCL reaction in the step (6), 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, a pure product of the full-length hirudin linear polypeptide with 63 rd tyrosine sulfation modification and nP protecting group removed is obtained, 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, finally, separation and purification are carried out again by a reverse liquid chromatography method with the same mobile phase, and freeze-drying is carried out, so that the 63 rd tyrosine sulfation modified full-length hirudin protein is obtained, and the protein synthesis yield is 4%. The method is innovatively carried out in a one-pot method of NCL reaction and refolding reaction. Polypeptide fragment 1 with MPAA at the C end of hirudin and polypeptide fragment 2 with cysteine at the N end of hirudin with sulfated modified tyrosine are subjected to NCL reaction, micromolecule MPAA and TCEP which are easy to influence refolding reaction are removed by ultrafiltration centrifugation and a solution system replacement method, and then crude solution is directly and slowly dripped into the refolding reaction system for reaction, so that the conversion of folding the hirudin full-length linear polypeptide into hirudin protein is realized, the liquid phase purification step after the NCL reaction is saved, the two separation and purification steps are reduced to one time, the synthesis time and cost are greatly shortened on the premise of not influencing the purity of the product, 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, the problem that the post-translational modification process cannot be carried out by expressing recombinant hirudin by escherichia coli or saccharomycetes is solved, the Fmoc solid-phase polypeptide synthesis is used, the sulfated modified tyrosine is introduced into the polypeptide, and the synthesis yield of the hirudin protein is improved 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 anticoagulation activity equivalent to that of natural hirudin, is favorable for researching physiological and biochemical effects of hirudin on antithrombotic or cardiovascular diseases, and lays a good foundation for the hirudin in clinical research and disease control.
Drawings
FIG. 1 is a diagram showing the characterization of reverse HPLC and ESI-MS in the preparation of pure hirudin HIRV1 polypeptide fragment 2 (F2 nP) with sulfation modification and containing neopentyl-protected tyrosine according to example 1.
FIG. 2 is a diagram showing the characterization of reverse HPLC and ESI-MS of example 1 for preparing purified hirudin HIRV1 polypeptide fragment 1-MPAA (F1).
FIG. 3 shows the preparation of HIRV1 (63 OSO) from example 1 3 ) In the course of (1) the starting material fragment 1 (F1), the neopentyl-protected fragment 2 (F2 nP), the NCL at the beginning of the reaction (NCL-0 h), the NCL at the end of the reaction (NCL-12 h), the refolding at the beginning of the reaction (Fold-0 h), the refolding at the end of the reaction (Fold-12 h) and the final product HIRV1 (63 OSO) 3 ) Characterization of reverse-phase High Performance Liquid Chromatography (HPLC) with ESI-MS of the peak site important material monitored for (FoldP).
FIG. 4 is HIRV1 (63 OSO) prepared in example 1 3 ) Results of experiments for inhibiting thrombin cleavage fibrinogen; wherein, (A) is a control group 1 sample, (B) is a control group 2 sample, and (C) is an experimental group sample.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Fmoc-Tyr (OSO) used in the examples below 3 nP) -OH, of the formula shown below, are prepared by the methods described in the references "Simpson l.s, zhu J.Z, widlanski T.S, et al, chemistry Biology,2009,16 (2): 153-161", "Simpson l.s, widlanski t.s., journal of the American Chemical Society,2006,128 (5): 1605-1610", "Schlienger N, peyrottes S, kasmem T, et al, journal of Medicinal Chemistry,2000,43 (23): 4570-4574").
Fmoc-Asp (OtBu) - (Dmb) Gly-OH used in the examples below was 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, HPLC was performed using Agilent 1260 as the instrument, phenomnex C18 column as the column, and water and acetonitrile (0.1% (v/v) TFA) as the mobile phase.
The sequence of hirudin HIRV1 polypeptide fragment 1 synthesized in the following examples is as follows, the C-terminal end of polypeptide fragment 1 has been hydrazided:
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 sequence of hirudin HIRV1 polypeptide fragment 2 with sulfated modified tyrosine synthesized in the following examples is as follows, the N-terminus of polypeptide fragment 2 is cysteine and the C-terminus is the amide terminus:
NH 2 -Cys 28 (R3)-Ile-Leu-Gly-Ser(R1)- 33 34 Asp(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 54 Asp(R2)-Gly(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: the connection and refolding one-pot synthesis method of hirudin HIRV1 with tyrosine sulfation modification comprises the following steps:
(1)Fmoc-Lys(Boc)-NHNH 2 preparation of the resin: 1g of 2-Chlorotrityl Chloride resin (2-Cl resin for short) is taken and put into a polypeptide synthesis tube, the loading capacity is 0.3-0.5mmol/g, 15mL of DMF is added for swelling twice under the shaking at room temperature for 30min each time, the mixture is drained, 15mL of 5% hydrazine hydrate/DMF is added into the resin for shaking reaction at room temperature for 30min, after washing twice with DMF, 15mL of 5% hydrazine hydrate/DMF is added for the second time, shaking reaction at room temperature for 30min, after washing twice with DMF, the mixture is washed twice with DMFAdding 15mL of 5% hydrazine hydrate/DMF three times, vibrating and reacting for 30min at room temperature, washing twice with DMF, DCM, DMF respectively, draining solvent to obtain resin with a terminal end of hydrazide (referred to as hydrazide resin), adding 15mL of 5% MeOH/DMF to the resin, vibrating and reacting for 10min at room temperature, washing twice with DMF, DCM, DMF respectively, draining solvent, closing resin sites which are not subjected to hydrazidation, weighing Fmoc-Lys (Boc) -OH (1 mmol), TBTU (0.98 mmol) and DIEA (2 mmol), dissolving Fmoc-Lys (Boc) -OH (27 th amino acid) and TBTU with a small amount of DMF, adding DIEA, vibrating and reacting at room temperature to activate carboxyl groups for 3min, adding the activated amino acid to the resin, vibrating and reacting at room temperature for 2h, washing three times with DMF and DCM respectively, drying the resin by nitrogen drying to obtain dry resin, and detecting the resin load of about 0.42mmol/g Fmoc-Lys (Boc) -NHNH by ultraviolet detection 2 And (3) resin.
(2) Hirudin HIRV1 polypeptide fragment 1-NHNH 2 Preparation of the resin: fmoc-Lys (Boc) -NHNH obtained 2 Adding 15mL of DMF (dimethyl formamide) into the resin, swelling twice at room temperature for 15min, draining, adding 10mL of 20% acetic anhydride/DMF into the resin, performing a room temperature shaking reaction for 20min to block amino groups of the resin which are not coupled with amino acids, stopping the next reaction, sequentially washing twice with DMF, DCM, DMF, adding 10mL of 20% piperidine/DMF into the resin, performing a shaking reaction for 5min at room temperature, washing twice with DMF, adding 10mL of 20% piperidine/DMF again, performing a shaking reaction for 5min at room temperature, sequentially washing twice with DMF, DCM, DMF, draining the solvent to obtain Fmoc-protected resin, weighing Fmoc-Asn (Trt) -OH (4×0.42 mmol), TBTU (3.9X0.42 mmol) and DIEA (8×0.42 mmol), dissolving Fmoc amino acids and TBTU with a small amount of DMF, adding DIEA, performing a shaking reaction for 3min at room temperature, then adding the activated amino acids into the resin, performing a shaking reaction for 2h at room temperature, monitoring the reaction with ninhydrin reagent, washing twice with a DMF, DCM, DMF, and performing a shaking reaction for 4 times to obtain a peptide sequence, and coupling the amino acid sequence from the end of 1 to the amino acid sequence of the hirudin (1 time, which is calculated as the end of the amino acid sequence of the peptide, and the peptide is the end of the peptide, and the peptide is subjected to a 4 times to the peptide. After the synthesis was completed, the reaction mixture was washed three times with DMF and DCM, and dried with nitrogen to give 2.7g of a dried resin. Due to the increase in resin mass The resin loading was about 0.15mmol/g at this time.
(3) Preparation of Fmoc-Gln (Trt) -MBHA resin: taking 1g Rink Amide MBHA resin, placing the resin into a polypeptide synthesis tube, carrying capacity of 0.3-0.5mmol/g, adding 15mL of DMF, vibrating and swelling at room temperature for two times, 15min each time, draining, adding 10mL of 20% piperidine/DMF into the resin, vibrating and reacting at room temperature for 5min, washing twice with DMF, adding 10mL of 20% piperidine/DMF again, vibrating and reacting at room temperature for 5min, washing twice with DMF, DCM, DMF in sequence, draining the solvent to obtain Fmoc-protected resin, weighing Fmoc-Gln (Trt) -OH (1 mmol), TBTU (0.98 mmol) and DIEA (2 mmol), dissolving Fmoc-Gln (Trt) -OH and TBTU with a small amount of DMF, adding DIEA, vibrating and reacting at room temperature for 3min, adding activated amino acid into the resin, vibrating and reacting at room temperature for 2h, washing three times with DMF and nitrogen to obtain dry resin, taking the resin, and finally drying the Fmoc-Gln (Trt) -OH resin with resin carrying capacity of about 0.47 mmol/g.
(4) Preparation of hirudin HIRV2 polypeptide fragment 2-MBHA resin with sulfated modified tyrosine: swelling the obtained Fmoc-Gln (Trt) -MBHA resin with 15mL of DMF twice with shaking at room temperature for 15min each time, draining, adding 10mL of 20% acetic anhydride/DMF to the resin, shaking at room temperature for 20min to block amino groups of the resin which are not coupled with amino acids, stopping the next reaction, washing with DMF, DCM, DMF in sequence for two times, adding 10mL of 20% piperidine/DMF to the resin, shaking at room temperature for 5min, washing with DMF twice, adding 10mL of 20% piperidine/DMF again, shaking at room temperature for 5min, washing with DMF, DCM, DMF in sequence for two times, draining the solvent to obtain Fmoc-protected resin, weighing Fmoc-Leu-OH (4×0.47 mmol), TBTU (3.9×0.47 mmol) and DIEA (8×0.47 mmol), dissolving Fmoc amino acid and TBTU with a small amount of DMF, adding DIEA, shaking at room temperature for 3min, adding activated amino acid to the resin, shaking at room temperature for 2h, washing with 12 h of hirudin with shaking for 2h, and repeating the experiment with 1 mol of hirudin (1 time, the amino acid content can be monitored by using the following the shaking on the Ind for 1-4 times of the resin after shaking The peptide fragment 2 sequence is coupled from the C-terminus to the N-terminus. Wherein, the aspartic acid-glycine sequence at the 33 th position and 34 th position, 53 th position and 54 th position is coupled by Fmoc-dipeptide Fmoc-Asp (OtBu) - (Dmb) Gly-OH; fmoc-Tyr (OSO) was used to modify tyrosine at position 63 3 nP) -OH. After the synthesis, the mixture was washed three times with DMF and DCM, and dried with nitrogen to give 3.3g of a dry resin. The loading of the resin at this time was about 0.10mmol/g due to the increased mass of the resin.
(5) Hirudin HIRV1 polypeptide fragment 1-NHNH 2 Preparing a crude product: 200mg of the resin obtained in step (2) was taken and 10mL of a cleavage reagent (TFA: DODT: H) was added 2 O=volume ratio 95:2.5:2.5 Shaking reaction for 2-4 h, filtering to obtain yellow transparent liquid, spin drying by a liquid spin-steaming instrument, adding about 20mL of glacial ethyl ether, extracting twice, centrifuging, collecting precipitate, and freeze-drying the sample to obtain about 30mg of hirudin HIRV1 polypeptide fragment 1-NHNH 2 Crude product.
(6) Preparation of pure 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 added 2 O=volume ratio 95:2.5:2.5 Shaking for 2H, filtering to obtain yellow transparent liquid, spin drying with a liquid spin-steaming instrument, adding about 20mL of glacial ethyl ether, extracting twice, centrifuging, collecting precipitate, and collecting sample with 20% ACN/H 2 O was dissolved by mobile phase as ACN/H with 0.1% TFA 2 Performing reverse liquid chromatography on the O mixed solution to separate and purify, and freeze-drying the purified sample to obtain about 20mg hirudin HIRV2 polypeptide fragment 2 pure product with sulfated modified tyrosine;
(7) Preparation of hirudin HIRV1 polypeptide fragment 1-MPAA pure product: taking hirudin HIRV1 polypeptide fragment 1-NHNH obtained in step (5) 2 Dissolving the crude product with buffer salt solution containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS and pH 3.0, adding 2.5 times of acetylacetone equivalent to the crude polypeptide, shaking for 3 min, adding 15 times of MPAA equivalent to the crude polypeptide, reacting for 12H, adding 10 times of TCEP equivalent to the crude polypeptide, reacting for 20 min, centrifuging, filtering, and filtering to obtain ACN/H containing 0.1% TFA as mobile phase 2 Separating and purifying by reverse liquid chromatography of O mixed solution, and collecting purified sampleThe product was lyophilized to obtain about 10mg of hirudin HIRV1 polypeptide fragment 1-MPAA as a pure product.
(8) Preparation of crude full-length hirudin HIRV1 linear polypeptide with sulfated modified tyrosine: preparing 4mL of buffer salt solution containing 6mol/L guanidine hydrochloride, 0.2mol/L PBS, 0.2mol/L MPAA, 40mmol/L TCEP and pH 6.5, adding 5.24mg of hirudin HIRV2 polypeptide fragment 2 pure product with sulfated modified tyrosine in the step (6), adding 275 mu L of the buffer salt solution for dissolution, taking 5.92mg of hirudin HIRV1 polypeptide fragment 1-MPAA pure product in the step (7) (1.67 times equivalent of the hirudin HIRV2 polypeptide fragment 2 pure product with sulfated modified tyrosine), adding 300 mu L of the buffer salt solution for dissolution, adding 275 mu L of the hirudin HIRV2 pure product solution with sulfated modified tyrosine into 300 mu L of the hirudin HIRV1 polypeptide fragment 1-MPAA pure product, carrying out natural chemical connection (Native chemical ligation, short NCL) for reaction for 12h, and simultaneously carrying out automatic removal of neopentyl (nP) sulfate protecting group with the sulfate protecting group in the buffer salt solution to obtain a full-length hirudin 1 crude product with 63 th sulfation and nP protecting group removed.
(9) Refolding of the crude full-length hirudin HIRV1 linear polypeptide with sulfated modified tyrosine in a one-pot process: taking the 63 rd tyrosine sulfation modified full-length hirudin HIRV1 linear polypeptide crude solution obtained in the step (8) and removed with nP protecting group, adding the crude solution into a ultrafiltration tube with a molecular weight of 3K in volume halving, centrifugally ultrafiltering, carrying out solution replacement by using 6mol/L guanidine hydrochloride, 0.2mol/L PBS and buffer salt solution with a pH of 6.0, removing small molecules influencing refolding reaction such as MPAA and TCEP to obtain about 250 mu L of the 63 rd tyrosine sulfation modified full-length hirudin HIRV1 linear polypeptide crude solution without MPAA and TCEP, slowly dripping the crude solution into about 20mL of a refolding reaction system with 0.2mol/L tris, 4mol/L sodium chloride, 4mmol/L L-cysteine, 2mmol/L L-cystine and a pH of 8.5, carrying out oxidation, refolding reaction of the polypeptide for 12H, centrifuging, filtering, and obtaining the ACN/H with a mobile phase containing 0.1% TFA 2 The O mixed solution is separated and purified by reverse liquid chromatography, and the purified sample is freeze-dried to obtain about 1.81mg (the separation yield is about 21 percent) of 63 rd tyrosine sulfation modificationFull-length hirudin HIRV1 protein, HIRV1 (63 OSO) 3 )。
Characterization of pure hirudin HIRV2 polypeptide fragment 2 with sulfated modified tyrosine obtained in step (6) by reverse high performance liquid chromatography and ESI-MS is shown in figure 1.
Characterization of pure hirudin HIRV1 polypeptide fragment 1-MPAA obtained in step (7) by reverse high performance liquid chromatography and ESI-MS is shown in figure 2.
Step (8) and step (9) preparation of HIRV1 (63 OSO) 3 ) During the course of the above procedure, the conditions of the NCL reaction at the beginning (NCL-0 h), during the NCL reaction (NCL-2 h), at the end of the NCL reaction (NCL-12 h), at the beginning of the refolding reaction (Fold-0 h) and at the end of the refolding reaction (Fold-12 h) were monitored, and the results of the characterization of the reverse HPLC and ESI-MS of the important peak position substances at the above time points are shown in FIG. 3.
For HIRV1 (63 OSO) prepared in step (9) 3 ) The results of the bioactivity assay are shown in fig. 4. As can be seen, HIRV1 (63 OSO) prepared according to the present invention 3 ) Can obviously inhibit the hydrolysis of thrombin to fibrinogen.
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 (5 nM) was combined with HIRV1 (63 OSO) at room temperature (25 ℃) 3 ) (10 nM) was added to solution X (1 mL), dissolved, shaken well, incubated at 37℃for 30min, cooled to room temperature (25 ℃), fibrinogen (5. Mu.M) was added to the sample, and shaken well as the experimental group; observing the turbidity of the test group samples;
Fibrinogen (5. Mu.M) was added to solution X (1 mL) at room temperature (25 ℃), and shaken well as control 1; observing the turbidity of the control group 1 sample;
thrombin (5 nM) was added to solution X (1 mL) at room temperature (25 ℃), dissolved, shaken well, incubated at 37 ℃ for 30min, cooled to room temperature (25 ℃), fibrinogen (5 μm) was added to the sample, and shaken well as control group 2; the turbidity of the control group 2 samples was observed.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> university of North China
<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 (7)
1. A chemical synthesis method of hirudin with tyrosine sulfation modification is characterized in that: the method comprises the following steps:
(1) Dividing cysteine into a fragment 1 and a fragment 2 according to the amino acid sequence of hirudin; using resin with a C end as a hydrazide end, namely a hydrazide resin for short, adopting an Fmoc solid-phase polypeptide synthesis method, condensing Fmoc protective amino acids from the C end to the N end in sequence according to the sequence of the fragment 1, washing, and drying to obtain the fragment 1;
(2) Using resin with a C end as an amide end, namely amide resin for short, adopting Fmoc solid-phase polypeptide synthesis method, condensing Fmoc protective amino acid from the C end to the N end in sequence according to the sequence of the fragment 2, washing, and drying to obtain the fragment 2; wherein:
the sequence of fragment 1 is as follows:
Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys；
the sequence of fragment 2 is as follows:
Cys-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln；
the tyrosine near the C end of hirudin is sulfated modified tyrosine, fmoc sulfation modified tyrosine with neopentyl as side chain sulfate protecting group is called Fmoc-Tyr (OSO) 3 nP) -OH for synthesis;
the amino acid site of aspartic acid-glycine contained in the amino acid sequence of hirudin is synthesized by Fmoc aspartic acid-glycine dipeptide with side chain carboxyl and amino protecting groups of OtBu and Dmb respectively, namely Fmoc-Asp (OtBu) - (Dmb) Gly-OH;
(3) Taking the linear polypeptide resin of the fragment 1 prepared in the step (1), adding a cutting reagent to enable a polypeptide chain to be detached from the hydrazide resin, and removing residual side chain protecting groups; filtering, spin drying, extracting, centrifuging and freeze-drying to obtain a fragment 1 crude product with a hydrazide at the C end;
(4) Taking the linear polypeptide resin of the fragment 2 prepared in the step (2), adding a cutting reagent to remove the polypeptide chain from the amide resin, and removing the residual side chain protecting group; filtering, spin drying, extracting, centrifuging and freeze-drying to obtain a fragment 2 crude product; further separating, purifying and freeze-drying to obtain fragment 2 with amide at the C end;
(5) Dissolving a crude product of the fragment 1 with the C-terminal as the hydrazide, which is prepared in the step (3), adding acetylacetone, shaking uniformly, adding 4-mercaptophenylacetic acid, performing a first reaction, adding tri (2-carboxyethyl) phosphine, performing a second reaction, centrifuging, filtering, further separating and purifying, and lyophilizing to obtain the fragment 1 with the C-terminal as the MPAA;
(6) Taking the fragment 2 with the amide at the C end prepared in the step (4) and the fragment 1 with the MPAA at the C end prepared in the step (5), respectively dissolving, mixing, and carrying out natural chemical connection reaction to obtain a crude hirudin linear polypeptide solution which is subjected to tyrosine sulfation modification near the C end and has the neopentyl removed, wherein the crude hirudin linear polypeptide solution is abbreviated as a solution M;
(7) Adding the solution M prepared in the step (6) into a ultrafilter tube, centrifugally filtering and simultaneously replacing a solution system to obtain a crude solution of tyrosine sulfation modified hirudin linear polypeptide which does not contain 4-mercaptophenylacetic acid and tri (2-carboxyethyl) phosphine and is close to the C end, wherein the crude solution is called as solution N for short; wherein:
the buffer salt solution used in 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) 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 tyrosine sulfation modified hirudin near 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.
2. The method for the chemical synthesis of hirudin with a tyrosine sulfation modification according to claim 1, wherein:
the amino acid sequence of hirudin is shown as SEQ ID NO. 1.
3. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:
in the step (5), the solution for dissolving the crude product of the fragment 1 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;
in the step (6), the solution for dissolving the fragment 1 and the fragment 2 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, 38-42 mmol/L tris (2-carboxyethyl) phosphine, and the pH value of the solution is 6.5+/-0.5;
the volume of the refolding reaction system in step (8) is calculated by dissolving 0.25-0.4 mg of linear polypeptide in 1mL of reaction solution.
4. 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, 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-mercaptophenylacetic acid, 40mmol/L tris (2-carboxyethyl) phosphine, and the pH value of the solution is 6.5;
the amount of acetylacetone used in step (5) is as per the crude product of fragment 1: acetylacetone=1:2.5 molar ratio calculation;
the amount of 4-mercaptophenylacetic acid described in step (5) is as crude product of fragment 1: 4-mercaptophenylacetic acid=1:15 molar ratio;
the amount of tris (2-carboxyethyl) phosphine described in step (5) is as crude in fragment 1: tris (2-carboxyethyl) phosphine=1:10 molar ratio calculation;
the dosage of the fragment 1 in the step (6) is calculated according to the equivalent weight of the fragment 2 which is 1.5-2 times;
the ultrafiltration tube in the step (7) uses an ultrafiltration tube with a molecular weight of 3K;
the buffer salt solution used in the replacement solution system 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 reaction 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.
5. A chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 4, wherein:
the condition of the first reaction in the step (5) is that the temperature is 20-30 ℃ and the reaction time is 12 hours;
the condition of the second reaction in the step (5) is that the temperature is 20-30 ℃ and the reaction time is 20 minutes;
the natural chemical ligation reaction condition in the step (6) is that the temperature is 37+/-0.5 ℃ and the reaction time is 8-12 hours;
the time length of the slow dripping 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.
6. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 2, wherein:
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) 3 The structural formula of nP) -OH is shown as follows:
the Fmoc-Tyr (OSO) 3 The dosage of nP) -OH is as follows: fmoc-Tyr (OSO) 3 nP) -oh=1: 1.5 to 4 molar ratio;
The Fmoc-Tyr (OSO) 3 The coupling time of nP) -OH and Fmoc amino resin is 6-12 h;
the structural formula of Fmoc-Asp (OtBu) - (Dmb) Gly-OH is shown as follows:
the Fmoc-Asp (OtBu) - (Dmb) Gly-OH is used in the following amount of resin: fmoc-Asp (OtBu) - (Dmb) Gly-oh=1: 1.5 to 4 molar ratio;
the coupling time of Fmoc-Asp (OtBu) - (Dmb) Gly-OH and Fmoc amino resin is 4-8 h;
Fmoc-Tyr removal (OSO) 3 The nP) -OH and Fmoc protected amino acid except Fmoc-Asp (OtBu) - (Dmb) Gly-OH are Fmoc amino acid without side chains, or the protecting groups R1, R2, R3 or R4 of the side chains can be synthesized by using trifluoroacetic acid to remove the Fmoc amino acid; wherein: r1 represents tert-butyl, R2 represents tert-butyl, R3 represents trityl, R4 represents tert-butoxycarbonyl, and the amount is as Fmoc amino resin: fmoc protected amino acid = 1:4 molar ratio calculation;
Fmoc-Tyr removal (OSO) 3 Coupling time of Fmoc protective amino acid except nP) -OH and Fmoc-Asp (OtBu) - (Dmb) Gly-OH and Fmoc amino resin is 2-4 h;
the specific operation of condensing Fmoc protected amino acids from the C end to the N end in sequence in the step (1) and the step (2) is as follows: under the action of a coupling system, firstly reacting the 1 st amino acid with Fmoc-removed hydrazide or amide resin to generate amino acid-amino resin, and then coupling other Fmoc-protected amino acids one by one to obtain linear polypeptide resin;
Condensing agents in the coupling system are HOBT and DIC, or TBTU and 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.
7. The chemical synthesis method of hirudin with tyrosine sulfation modification according to claim 1 or 2, characterized in that:
the cleavage reagents in step (3) and step (4) are trifluoroacetic acid, water and 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid according to 95:2.5:2.5 volume ratio of the mixed solution;
the cutting time in the step (3) and the step (4) is 2-4 h;
the extractant extracted in the step (3) and the step (4) is glacial diethyl ether.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057294A (en) * | 1990-05-10 | 1991-12-25 | 法米塔利亚·卡洛·埃巴有限责任公司 | The recombinant chou preparation method of r-hirudin |
| JPH07138298A (en) * | 1993-11-19 | 1995-05-30 | Japan Energy Corp | Production of sulfate derivative of hirudin analog or its salt |
| US5767235A (en) * | 1991-03-05 | 1998-06-16 | Nippon Mining Company Limited | Anticoagulant hirudin variants and methods for their production |
| CN110590911A (en) * | 2019-09-30 | 2019-12-20 | 华南理工大学 | Synthetic method and application of polypeptide containing tyrosine sulfating modification |
| CN113699086A (en) * | 2020-05-22 | 2021-11-26 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacterium for producing sulforecombinant hirudin and preparation method and application thereof |
-
2022
- 2022-03-17 CN CN202210261766.XA patent/CN114736289B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057294A (en) * | 1990-05-10 | 1991-12-25 | 法米塔利亚·卡洛·埃巴有限责任公司 | The recombinant chou preparation method of r-hirudin |
| US5767235A (en) * | 1991-03-05 | 1998-06-16 | Nippon Mining Company Limited | Anticoagulant hirudin variants and methods for their production |
| JPH07138298A (en) * | 1993-11-19 | 1995-05-30 | Japan Energy Corp | Production of sulfate derivative of hirudin analog or its salt |
| CN110590911A (en) * | 2019-09-30 | 2019-12-20 | 华南理工大学 | Synthetic method and application of polypeptide containing tyrosine sulfating modification |
| CN113699086A (en) * | 2020-05-22 | 2021-11-26 | 中国科学院青岛生物能源与过程研究所 | Recombinant bacterium for producing sulforecombinant hirudin and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
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| Convergent solid-phase synthesis of hirudin;SPYROS GOULAS等;《Journal of Peptide Science》;第12卷(第2期);第116-123页 * |
| 水蛙素的分离纯化及其聚乙二醇修饰;李雪芹;《中国优秀硕士论文电子期刊网》;全文 * |
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