阅读说明：本技术 比伐卢定的合成方法 (Synthetic method of bivalirudin ) 是由 伊万·迪·博纳文图拉 何润泽 于 2018-06-19 设计创作，主要内容包括：活性药物成分(API)比伐卢定(Bivalirudin)是20个氨基酸的肽,其含有一个碱基和5个氨基酸残基。化学式为C_(98)H_(138)N_(24)O_(33),且分子量为2180.3g/mol。比伐卢定也被称为Angiomax。它是被指定用作抗凝剂的直接凝血酶抑制剂。本发明提供合成比伐卢定的方法,特别是提供使用Fmoc-Leu-Wang树脂作为载体的通过固相肽合成的比伐卢定合成方法。该方法具有收率高、副产物少和分离纯化简单的优点,且与现有技术相比,该方法节省时间且适用于中试和工业化生产。(The Active Pharmaceutical Ingredient (API) Bivalirudin (Bivalirudin) is a 20 amino acid peptide containing one base and 5 amino acid residues. Has a chemical formula of C 98 H 138 N 24 O 33 And a molecular weight of 2180.3 g/mol. Bivalirudin is also known as Angiomax. It is a direct thrombin inhibitor designated for use as an anticoagulant. The invention provides a method for synthesizing bivalirudin, in particular to a bivalirudin synthesis method which uses Fmoc-Leu-Wang resin as a carrier and is synthesized by solid phase peptide. The method has the advantages of high yield, less by-products and simple separation and purification, and compared with the prior art, the method saves time and is suitable for pilot plant test and industrial production.)

1. A method of synthesis characterized by producing bivalirudin using solid phase peptide synthesis, wherein said method uses Fmoc-Leu-Wang resin, Fmoc-Leu-2CT resin, Rink amide protide (ll) resin and ChemMatrix Wang resin as a carrier for said solid phase synthesis of said polypeptide.

2. The method according to claim 1, wherein the polypeptide linker used in the polypeptide solid phase synthesis method is selected from the group consisting of 2-oxime cyanoethyl acetate, N-diisopropylcarbodiimide, N-diisopropylethylamine, Dimethylformamide (DMF), Dichloromethane (DCM) and formic acid.

3. The method of claim 1, wherein the Fmoc-Leu-Wang resin, Fmoc-Leu-2CT resin, Rink amide protide (ll) resin, and ChemMatrix Wang resin are used as carriers for the solid phase synthesis reaction of the polypeptide.

4. The method of claim 1, wherein the polypeptide solid phase peptide synthesis method is performed by Fmoc deprotection method.

5. The method of claim 4, wherein piperidine/N, N-dimethylformamide/formic acid/piperazine is added to the Fmoc deprotection process and the fluorenylmethoxycarbonyl protection reaction is performed under heat or microwave.

6. The method of claim 1, wherein the polypeptide is produced by a solid phase synthesis reaction and then washed 2-3 times with N, N-dimethylformamide after completion of each reaction.

7. The method according to claim 6, wherein the condensation reaction of amino acids in the solid phase synthesis reaction of the polypeptide on the resin is performed at 50 to 120 ℃ at 2-10 fold equivalent compared to the amount of the resin.

8. The method according to claim 6, wherein after completion of the amino acid condensation reaction in the solid phase synthesis reaction of the polypeptide, before completion of a cleavage reaction, the resin is purged with nitrogen.

9. The process according to claim 8, wherein the trifluoroacetic acid added in the cleavage reaction has a purity of > 95%, a reaction temperature of 25 ℃ and a cleavage reaction time of 1-3 hours.

10. The method of claim 1, wherein the resin is subjected to a swelling treatment with DMF prior to the solid phase synthesis reaction of the polypeptide.

Technical Field

The present invention relates to the field of biomedicine, and in particular to a method for synthesizing bivalirudin.

Conventional synthetic methods for bivalirudin are time consuming and complex processes, and cannot be implemented on a large scale, and thus, those skilled in the art have been devoted to developing new synthetic methods for bivalirudin.

Background

Solid Phase Peptide Synthesis (SPPS) is a rapid method for polypeptide synthesis. However, conventional SPPS for synthesizing large amounts of long peptides such as bivalirudin is both time consuming and generates a large waste of solvent. As a widely used active pharmaceutical ingredient, bivalirudin is a direct thrombin inhibitor designated for use as an anticoagulant. It is of great significance to develop an efficient and economical SPPS process for the preparation of large quantities of bivalirudin of high purity.

Disclosure of Invention

The invention aims to provide a preparation method of bivalirudin.

According to a first aspect of the present invention, there is provided a method for producing bivalirudin, which is prepared by using Solid Phase Peptide Synthesis (SPPS), wherein the method performs solid phase synthesis of a polypeptide using Fmoc-Leu-Wang resin or Fmoc-Leu-2CT resin, Rink amide protide (ll) resin, and ChemMatrix Wang resin as a carrier.

Further, the coupling agent used in the polypeptide solid phase synthesis method is selected from the group consisting of ethyl 2-oxime cyanoacetate, N-diisopropylcarbodiimide, N' -dicyclohexylcarbodiimide, N-diisopropylethylamine.

Furthermore, in the Fmoc deprotection method, piperidine/N, N-dimethylformamide/formic acid (preferably 1/4 volumes of piperidine/N, N-dimethylformamide, 5% -20% piperidine volume formic acid)/piperazine was added, and the heating conditions were preferably carried out by means of a water bath, oil bath or microwave heating reaction for 2 to 5 minutes.

In addition, after the reaction is completed, each of the reactants in the solid phase synthesis reaction of the polypeptide is washed 2 to 3 times with N, N-dimethylformamide.

Further, the condensation reaction of amino acids in the solid phase synthesis reaction of polypeptides on a resin is carried out at a temperature of 50 to 120 ℃; the reaction time is preferably 10 to 30 minutes depending on the temperature.

In addition, the amino acid condensation reaction of the polypeptide in the solid phase synthesis reaction is performed, and the resin is purged with nitrogen before the cleavage reaction is performed.

In addition, the purity of trifluoroacetic acid added in the reaction is 95%, the temperature is 10-50 ℃, and the cracking reaction time is 1-3 hours.

In addition, in the solid phase synthesis reaction of the polypeptide, the equivalent of the amino acid used is twice the molar amount of the resin.

In addition, crude bivalirudin was purified by Pre-HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 minutes.

Further, the purified bivalirudin solution was lyophilized at-50 to-70 ℃ for 18-48 hours by a lyophilizer.

Drawings

Fig. 1 is a schematic diagram of the molecular structure of bivalirudin.

Figure 2 is an HPLC analysis of prepared bivalirudin.

Fig. 3 is a mass spectrum of prepared bivalirudin.

Detailed Description

Bivalirudin shown in figure 1 is a synthetic peptide of 20 amino acids. The invention will now be described with reference to specific embodiments. It must be understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, the experimental materials and reagents used in the following examples are available from commercial sources.

TABLE 1 list of amino acid abbreviations

[ Table 1]

Name (R)	Three letter symbol	Single letter symbols
			Arginine	Arg	R
Asparagine	Asn	N
			Aspartic acid	acid	D
Phenylalanine	Phe	F
			Glutamic acid	acid	E
Glycine	Gly	G
			Isoleucine	Ile	I
Leucine	Leu	L
			Phenylalanine	Phe	F
Proline	Pro	P
			Tyrosine	Tyr	Y

TABLE 2 list of chemical reagent abbreviations

[ Table 2]

TABLE 3 List of intermediates and Fmoc protected amino acids

[ Table 3]

The invention provides a bivalirudin synthesis method using Fmoc-Leu-wang resin through solid phase peptide synthesis, which comprises the following steps:

step 1, fluorenylmethoxycarbonyl-leucine-Wang resin is available directly (Sigma Aldrich), which can reduce the synthesis of the first step and accelerate the synthesis efficiency;

step 2: swelling the resin in DMF under nitrogen for 5 to 15 minutes;

and step 3: the fluorenylmethoxycarbonyl chloride protecting group was removed under the following conditions: formic acid (5% -20% piperidine volume)/piperazine was held in DMF for 2 min at 50 ℃ to 120 ℃.

And 4, step 4: preparation of fluorenylmethoxycarbonyl-tyrosine (tert-butyl) -leucine-Wang resin: deprotecting the fluorenylmethoxycarbonyl-leucine-Wang resin obtained in the step 1, washing with DMF, and subjecting Fmoc-L-Tyr- (tert-butyl) -OH to condensation reaction under the condition of a polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-tyrosine (tert-butyl) -leucine-Wang resin;

and 5: preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin: deprotecting and washing the Fmoc-dipeptide obtained in step 2 and then reacting it with Fmoc-L-glutamic acid (tert-butoxy) -OH under the condition of a peptide coupling agent to give fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin;

step 6: preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin. Deprotecting the obtained fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin, washing and adding Fmoc-L-glutamic acid (tert-butoxy) -OH, and then reacting under the condition of a peptide coupling agent to obtain fluorenylmethoxycarbonyl-glutamic acid (tert-butyl (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -pyridine (tert-butyl) -leucine-Wang resin;

and 7: preparation of fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin. The fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin was obtained by deprotecting glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin, washing, and then reacting with fluorenylmethoxycarbonyl-proline-OH under the condition of a polypeptide coupling agent. Glutamic acid- (tert-butoxy) -glutamic acid (tert-butoxy) tyrosine- (tert-butyl) -leucine-Wang resin;

and 8: preparation of fluorenylmethoxycarbonyl-isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid- (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-isoleucine-OH under the conditions of a peptide coupling agent to give fluorenylmethoxycarbonyl-isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

and step 9: preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin. Deprotecting the fluorenylmethoxycarbonyl-isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine- (tert-butyl) -leucine-Wang resin obtained in step 6, washing, and adding and reacting Fmoc-L-glu (otbu) -OH in the presence of a peptide coupling agent to obtain fluorenylmethoxycarbonylglutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butyl) -tyrosine-leucine-Wang resin;

step 10: preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid- (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Adding Fmoc-L-glu (otbutoxy) -glutamic acid (t-butoxy) -isoleucine-proline-glutamic acid (t-butoxy) -glutamic acid- (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin obtained in step 7 and subjecting it to condensation reaction under the condition of a polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-glutamic acid (t-butoxy) -isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin;

step 11: preparation of fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting the peptide fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin obtained in step 8, washing, and reacting it with fluorenylmethoxycarbonyl-phenylalanine-OH under the condition of a peptide coupling agent to give fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) tyrosine (tert-butyl) -leucine-Wang resin;

step 12: preparation of fluorenylmethoxycarbonylaspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting, washing and treating the peptide obtained in step 9 with fluorenylmethoxycarbonylaspartic acid (tert-butoxy) -OH to give fluorenylmethoxycarbonylaspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 13: preparation of fluorenylmethoxycarbonyl-glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting, washing and treating the peptide obtained in step 10 with fluorenylmethoxycarbonyl-glycine-OH under conditions of a polypeptide coupling agent to give fluorenylmethoxycarbonyl-glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 14: preparation of fluorenylmethoxycarbonyl-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting, washing and treating the peptide obtained in step 11 with fluorenylmethoxycarbonyl-asparagine (trityl) -OH under the conditions of a peptide coupling reagent to give fluorenylmethoxycarbonyl-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 15: preparation of fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting the peptide obtained in step 12, washing and subjecting to condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under the condition of a polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (t-butoxy) -phenylalanine-glutamic acid (t-butoxy) -isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin;

step 16: preparation of fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (t-butoxy) -phenylalanine-glutamic acid (t-butoxy) -isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-glycine-OH under peptide coupling reagent conditions to give fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (t-butoxy) -phenylalanine-glutamic acid (t-butoxy) -isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

and step 17: preparation of fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and subjecting to condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under the condition of polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) Tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 18: preparation of fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotection of fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under the condition of polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine- Proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 19: preparation of fluorenylmethoxycarbonyl-proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-proline-OH under conditions of a polypeptide coupling agent to obtain fluorenylmethoxycarbonyl-proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -proline-OH Yl) isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin;

step 20: preparation of fluorenylmethoxycarbonyl-arg (pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-Arg (Pbf) -OH under the condition of peptide coupling agent to obtain fluorenylmethoxycarbonyl-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butyl) Oxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 21: fluorenylmethoxycarbonyl-proline-arg (pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (t-butoxy) -phenylalanine-glutamic acid (t-butoxy) isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin was prepared. Deprotecting fluorenylmethoxycarbonyl-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-proline-OH under peptide coupling agent conditions to obtain fluorenylmethoxycarbonyl-proline-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -benzene-n Alanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 22: preparation of fluorenylmethoxycarbonyl-D-Phe-proline-arg (pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. Deprotecting fluorenylmethoxycarbonyl-proline-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin, washing and adding fluorenylmethoxycarbonyl-D-phenylalanine-OH under the conditions of a peptide coupling agent to obtain fluorenylmethoxycarbonyl-D-Phe-proline-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine -aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin;

step 23: fluorenylmethoxycarbonyl-D-Phe-proline-Arg (Pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin was washed and deprotected again to give NH₂-D-Phe-proline-arg (pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (t-butoxy) -phenylalanine-glutamic acid (t-butoxy) isoleucine-proline-glutamic acid (t-butoxy) -tyrosine (t-butyl) -leucine-Wang resin;

step 24: reacting NH₂-D-Phe-proline-arg (pbf) -proline-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin was washed three times and the cleavage of the peptide from the solid support was performed under the following conditions: TFA/TIS/H₂O94: 2.5 for three hours.

Step 25: the cleavage solution was precipitated with ether and centrifuged at 3500-. The precipitate was washed and centrifuged another 3 times with ether to give the final precipitate and dried under vacuum.

Step 26: crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min.

Step 27: and (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.

Advantages of the invention

Conventional processes for synthesizing bivalirudin SPPS typically produce large amounts of byproducts that are difficult to separate and large amounts of waste that are costly to dispose of. The process is faster than the conventional SPPS process used, produces less waste, and can yield bivalirudin in high yield.

Example 1

Peptide synthesis was performed by using the stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang resin (40g, substitution capacity 0.67 meq/g). Fmoc deprotection was performed by using formic acid (5% -20% piperidine volume)/piperazine in DMF. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids was carried out at 50 ℃ to 120 ℃ for 2 to 3 minutes and monitored by the Kaiser test. TFA cleavage of peptides with TFA/TIS/H₂O94: 5: 1 for 3 hours, followed by precipitation and washing with diethyl ether 2 times. The yield of crude peptide was 85%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. And (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.

Example 2

Peptide synthesis was performed by using the stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-2CT resin (40g, substitution capacity 0.8 meq/g). The resin was transferred to the reaction vessel of a peptide synthesizer and a 2 molar excess of the protected amino acid was used with formic acid (5% -20% piperidine body)Product)/piperazine in DMF. Fmoc deprotection reaction was performed at 50 deg.C-120 deg.C for 2-3 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. TFA cleavage of peptides with TFA/TIS/H₂O94: 5: 1 for 3 hours, followed by precipitation and 3-fold washing with diethyl ether. The yield of crude peptide was 86.44%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. And (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.

Example 3

Peptide synthesis was performed by using a stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Feu-2CT resin (40g, substitution capacity 1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. Fmoc deprotection was performed by using formic acid (5% -20% piperidine volume)/piperazine in DMF. The Fmoc deprotection reaction was performed at 50 ℃ to 120 ℃ for 1 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids was carried out at 50 ℃ to 120 ℃ for 2 minutes and repeated twice. TFA cleavage of peptides with TFA/thioanisole/phenol/H₂O/TES 89: 2.5: 5: 1 for 3 hours, followed by precipitation and 4-time washing with diethyl ether. The yield of crude peptide was 88.12%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. Passing the purified bivalirudin solution through a lyophilizer at-50 toLyophilizing at-70 deg.C for 18-48 hr to obtain bivalirudin with purity higher than 98%. Purity was analyzed by HPLC as shown in fig. 2. The mass analysis was corrected as shown in fig. 3.

Example 4

Peptide synthesis was performed by using stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Chemcatarix resin (40g, substitution capacity 0.67 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. Fmoc deprotection was performed by using formic acid (5% -20% piperidine volume)/piperazine in DMF. The Fmoc deprotection reaction was performed at 50 deg.C-120 deg.C for 1 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids is carried out at 50 ℃ to 120 ℃ for 2 minutes. TFA cleavage of peptides with TFA/thioanisole/phenol/H₂O/TES 89: 2.5: 5: 1 for 3 hours, followed by precipitation and 4-time washing with diethyl ether. The yield of crude peptide was 84.15%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. And (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.

Example 5

Peptide synthesis was performed by using the stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Feu-Wang resin (40g, substitution capacity 0.67 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. Fmoc deprotection was performed with formic acid 5% -20% piperidine volume)/piperazine in DMF. The Fmoc deprotection reaction was performed at 50 deg.C-120 deg.C for 1 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-DmBGly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids was carried out at 50 ℃ to 120 ℃ for 2 minutes and was repeated twice after the 10 th amino acid. The synthesis time was 4 hours and 20 minutes. TFA cleavage of peptides with TFA/thioanisole/phenol/H₂O/TES 89: 2.5: 5: 1 for 3 hours, followed by precipitation and 4-time washing with diethyl ether. The yield of crude peptide was 81.86%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. And (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.

Example 6

Peptide synthesis was performed by using a stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from the Protide resin of CEM resin (40g, substitution capacity 0.18 meq/g). The resin was transferred to the reaction vessel of a peptide synthesizer (Cem Liberty Blue) and the first amino acid was coupled to the resin with KI and DIPEA in DMF for 10 min at 50 ℃ -120 ℃. Fmoc deprotection was performed with formic acid 5% -20% piperidine volume)/piperazine in DMF. Fmoc deprotection was performed at 50 deg.C-120 deg.C for 1 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids is carried out at 50 ℃ to 120 ℃ for 2 minutes. TFA cleavage of peptides with TFA/thioanisole/phenol/H₂O/TES 89: 2.5: 5: 1 for 3 hours, followed by precipitation and 4-time washing with diethyl ether. The yield of crude peptide was 82.16%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. Lyophilizing the purified bivalirudin solution at-50 deg.C to-70 deg.C for 18-48 hr to obtain bivalirudin with purity higher than 98%And (4) determining.

Example 7

Peptide synthesis was performed by using stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang or 2CT resin (40g, substitution capacity 0.67/1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. Fmoc deprotection was performed with formic acid 5% -20% piperidine volume)/piperazine in DMF. The Fmoc deprotection reaction was performed at 50 deg.C-120 deg.C for 1 min and repeated twice. The other amino acids following in the sequence are linked in the following order: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH. The coupling and uncoupling of the amino acids is carried out at 50 ℃ to 120 ℃ for 2 minutes. TFA cleavage of peptides with TFA/TIS/H₂O94: 5: 1 for 3 hours, followed by precipitation and washing with diethyl ether 4 times. The yield of crude peptide was 87.56%. Crude bivalirudin was purified by preparative HPLC with a water/acetonitrile gradient from 100% water to 100% acetonitrile over 20 min. And (3) carrying out freeze-drying on the purified bivalirudin solution for 18-48 hours at the temperature of-50 to-70 ℃ by a freeze-dryer to obtain the prepared bivalirudin with the purity higher than 98%.