WO2019241903A1 - Synthetic method of bivalirundin - Google Patents

Synthetic method of bivalirundin Download PDF

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Publication number
WO2019241903A1
WO2019241903A1 PCT/CN2018/091752 CN2018091752W WO2019241903A1 WO 2019241903 A1 WO2019241903 A1 WO 2019241903A1 CN 2018091752 W CN2018091752 W CN 2018091752W WO 2019241903 A1 WO2019241903 A1 WO 2019241903A1
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Prior art keywords
tert
fmoc
butoxy
glutamic acid
glycine
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PCT/CN2018/091752
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French (fr)
Inventor
Ivan Di BONAVENTURA
Runze HE
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Shanghai Space Peptides Pharmaceutical Co., Ltd.
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Application filed by Shanghai Space Peptides Pharmaceutical Co., Ltd. filed Critical Shanghai Space Peptides Pharmaceutical Co., Ltd.
Priority to EP18923472.7A priority Critical patent/EP3810627A4/en
Priority to CN201880094571.2A priority patent/CN112424216A/en
Priority to PCT/CN2018/091752 priority patent/WO2019241903A1/en
Publication of WO2019241903A1 publication Critical patent/WO2019241903A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin

Definitions

  • the present invention pertains to the field of biomedicine, and in particular the present invention relates to a synthetic method of bivalirudin.
  • the conventional bivalirudin synthesis method is time-consuming and complicated in process and is not able to be achieved in large scale, and therefore technical personnel in the art are committed to developing a new bivalirudin synthesis method.
  • SPPS Solid Phase Peptide Synthesis
  • a method for producing bivalirudin which is prepared by using a solid phase peptide synthesis (SPPS), wherein the method uses Fmoc -Leu-Wang resin or Fmoc-Leu-2CT resin, Rink amide ProTide (LL) resin and ChemMatrix wang resin as carrier for the solid phase synthesis of the polypeptide.
  • SPPS solid phase peptide synthesis
  • the coupling reagents used in the polypeptide solid phase synthesis method are selected from the group consisting of ethyl 2-oxime cyanoacetate, N, N-diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide Amine, N,N-Diisopropylethylamine .
  • piperidine / N, N-dimethylformamide/ Formic acid preferably 1/4 volume of piperidine / N, N-dimethylformamide, Formic acid 5%-20% of piperidine volume
  • piperazine is added, and the heating conditions are preferably carried out by means of a water bath, an oil bath or a microwave heating reaction for 2 to 5 minutes.
  • condensation reaction of the amino acid in the solid phase synthesis reaction of the polypeptide on the resin is carried out at a temperature of 50 to 120 °C; preferably, the reaction time is 10 to 30 minutes depending on the temperature.
  • amino acid condensation reaction of the polypeptide in the solid-phase synthesis reaction is carried out and the resin is blown off with nitrogen before the cleavage reaction is carried out.
  • the purity of the trifluoroacetic acid added in the reaction was 95%, the temperature was 10-50 °C, and the cleavage reaction time was 1-3 hours.
  • the equivalents of the amino acid used is twice the molar amount of the resin.
  • the crude bivalirudin is purified by Pre-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours.
  • Figure 1 is a schematic of bivalirudin molecular structure.
  • Figure 2 is HPLC analysis of prepared Bivalirudin.
  • Figure 3 is Mass spectrum of prepared Bivalirudin.
  • Bivalirudin show in Figure 1 is 20 amino acids synthetic peptide.
  • the invention will now be described with reference to specific embodiments. It has to be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise stated, the percentages and parts are by weight. The experimental materials and reagents used in the examples below are available from commercially available sources unless otherwise specified.
  • the present invention provides a synthetic method of bivalirudin using Fmoc-Leu-wang resin by solid phase peptide synthesis comprising the following steps of:
  • fluorenylmethoxycarbonyl-leucine-Wang resin can be purchased directly (Sigma Aldrich), it can reduce the first step of the synthesis and speed up the synthesis efficiency;
  • Step 2 The resin is swollen for 5 to 15 minutes under nitrogen in DMF;
  • Step 3 fluorenylmethoxycarbonyl chloride protecting group is removed under the following conditions: Formic acid 5%-20% of piperidine volume)/ piperazine in DMF for 2 minutes at 50°C - 120°C.
  • Step 4 Preparation of fluorenylmethoxycarbonyl-tyrosine (t-butyl) -leucine-Wang resin:
  • the fluorenylmethoxycarbonyl-leucine-Wang resin obtained in step 1 is deprotected, washed with DMF and Fmoc-L-Tyr-(t-butyl) -OH is subjected to a condensation reaction under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-tyrosine (t-butyl) -leucine-Wang resin;
  • Step 5 Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin:
  • the Fmoc-dipeptide obtained in step 2 is deprotected and washed, and then reacted with Fmoc-L-Glutamic acid (tert-butoxy) -OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin;
  • Step 6 Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (t-butoxy) -tyrosine-(t-butyl) -leucine- Wang resin.
  • Step 7 Preparation of fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin.
  • Glutamic acid-(tert-butoxy)-glutamic acid (tert-butoxy) tyrosine-(t-butyl) -leucine-Wang resin is deprotected, washed and then reacted with fluorenylmethoxycarbonyl-proline-OH under the conditions of the polypeptide coupling reagent to obtain fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin.
  • Glutamic acid-(tert-butoxy)-glutamic acid (tert-butoxy) tyrosine-(t-butyl) -leucine-Wang resin is deprotected, washed and then reacted with fluorenylmethoxycarbonyl-proline-OH under the conditions of the polypeptide coupling reagent to obtain fluorenyl
  • Step 8 Preparation of fluorenylmethoxycarbonyl-isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl) -leucine-Wang resin.
  • fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid-(tert-butoxy)-tyrosine (t-butyl) -leucine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-isoleucine-OH is added under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-isoleucine-proline- glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine (t-butyl)-leucine-Wang resin;
  • Step 9 Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine-(t-butyl) -leucine-Wang resin.
  • fluorenylmethoxycarbonyl- isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine-(t-butyl) -leucine-Wang resin obtained in step 6 is deprotected, washed and Fmoc-L-Glu(Otbu)-OH is added and reacted in the presence of a peptide coupling reagent conditions to give fluorenylmethoxycarbonyl glutamic acid (tert-butoxy) -isoleucine-proline- glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine (t-butyl)-leucine-Wang resin;
  • Step 10 Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy)-glutamic acid-(tert-butoxy)-tyrosine (t-butyl) -leucine-Wang resin.
  • Fmoc-L-Glu(Otbu) is added and subjected to a condensation reaction under the conditions of a polypeptide coupling reagent to giving fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy ) - isoleucine - proline - glutamic acid (tert - butoxy) - glutamic acid (tert - butoxy) - tyrosine (tert - butyl) - leucine -Wang resin;
  • Step 11 Preparation of fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin.
  • the peptide obtained in step 8 fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy ) - glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl)-leucine-Wang resin was deprotected, washed and reacted with fluorenylmethoxycarbonyl-phenylalanine-OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) tyrosine (tert-butyl) - leucine - Wang resin;
  • Step 12 Preparation of fluorenylmethoxycarbonyl aspartic acid(tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl) -leucine-Wang resin.
  • the peptide obtained in step 9 is deprotected, washed and treated with fluorenylmethoxycarbonyl-aspartic acid (tert-butoxy) -OH to give fluorenylmethoxycarbonyl aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • the peptide obtained in step 10 is deprotected , washed and treated with fluorenylmethoxycarbonyl-glycine- OH under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • the peptide obtained in step 11 is deprotected , washed and treated with fluorenylmethoxycarbonyl-asparagine (trityl) -OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • the peptide obtained in step 12 is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
  • Step 16 Preparation of fluorenylmethoxycarbonyl-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Step 17 Preparation of fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Fluorenylmethoxycarbonyl-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OHunder the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-gluta
  • Step 18 Preparation of fluorenylmethoxycarbonyl-glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy)
  • Step 19 Preparation of fluorenylmethoxycarbonyl - proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Step 20 Preparation of fluorenylmethoxycarbonyl -Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Step 21 Preparation of fluorenylmethoxycarbonyl -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Step 22 Preparation of fluorenylmethoxycarbonyl-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin.
  • Step 23 Fluorenylmethoxycarbonyl-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is washed and deprotected again to give NH 2 -D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy
  • Step 24 NH 2 -D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is washed three times and cleavage of the peptide from the solid support is performed under the following conditions: TFA/TIS/H 2 O 94:2.5:2.5 for three hours.
  • Step 25 The cleavage solution is precipitated with diethyl ether and centrifuged at 3500-5000rpm to give a white precipitate. The precipitate is washed and centrifuged with diethyl ether for another three times to give a final precipitate and dry under vacuum.
  • Step 26 The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • Step 27 The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the conventional method of SPPS for the synthesis of bivalirudin produce commonly large amounts of byproducts difficult to separate, large amount of waste that is expensive to dispose.
  • the present process is faster than the normal SPPS process used, produce less amount of waste and it allows to achieve bivalirudin in high yield.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang resin (40g, substitution 0.67 meq/g).
  • Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF.
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2-3 minutes and was monitored by Kaiser test. TFA cleavage of the peptide was done with TFA/TIS/H 2 O 94:5:1 for 3 hours followed by precipitation and 2 washings with diethyl ether. Yield of crude peptide is 85%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-2CT resin (40g, substitution 0.8 meq/g).
  • the resin was transferred to the reaction vessel of the peptide synthesizer and the synthesis was carried out using 2 molar excess protected amino acids with formic acid 5%-20% of piperidine volume)/ piperazine in DMF.
  • the Fmoc deprotection reaction was for 2-3 minute at 50°C -120°C, repeated twice.
  • TFA cleavage of the peptide was done with TFA/TIS/H 2 O 94:5:1 for 3 hours followed by precipitation and 3 washings with diethyl ether. Yield of crude peptide is 86.44%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-2CT resin (40g, substitution 1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C - 120°C, repeated twice.
  • Fmoc SPPS solid phase peptide synthesis
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes and was repeated twice.
  • TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H 2 O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 88.12%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the purity was analyzed by HPLC as figure 2.
  • the Mass analysis was corrected as Figure 3.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Chemmatrix resin (40g, substitution 0.67 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice.
  • Fmoc SPPS solid phase peptide synthesis
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes.
  • TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H 2 O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 84.15%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang resin (40g, substitution 0.67 meq/g).
  • the resin was transferred to the reaction vessel of the peptide synthesizer.
  • the Fmoc deprotection was carried out with P formic acid 5%-20% of piperidine volume)/ piperazine in DMF.
  • the Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice.
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes and was repeated twice after the 10th amino acid. Synthesis time was 4hours and 20 minutes. TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H 2 O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 81.86%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Protide resin from CEM resin (40g, substitution 0.18 meq/g).
  • the resin was transferred to the reaction vessel of the peptide synthesizer (Cem Liberty Blue) and the first amino acid was coupled to the resin with use of KI and DIPEA in DMF for 10 min at 50°C -120°C.
  • the Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF.
  • the Fmoc deprotection reaction was carried out for 1 minute at 50°C -120°C, repeated twice.
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes.
  • TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H 2 O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 82.16%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
  • the synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang or 2CT resin (40g, substitution 0.67/1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice.
  • Fmoc SPPS solid phase peptide synthesis
  • the coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes.
  • TFA cleavage of the peptide was done with TFA/TIS/H 2 O 94:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 87.56%.
  • the crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
  • the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.

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Abstract

The Active Pharmaceutical Ingredient (API), Bivalirudin, is a 20 amino acid peptide containing one basic and 5 amino acid residues. With a chemical formula of C98H138N24O33 and a molecular weight of 2180.3 g/mol. Bivalirudin is also known as Angiomax. It is a direct thrombin inhibitor indicated for use as an anticoagulant. It provides a method for synthesizing bivalirudin and in particular to the synthetic method of bivalirudin by solid phase peptide synthesis using Fmoc-Leu-Wang resin as a carrier. The method has the advantages of high yield, less by-product and simple separation and purification, and it's time gaining than the prior art, and is suitable for pilot and industrial production.

Description

A SYNTHETIC METHOD OF BIVALIRUDIN
Field of the Invention
The present invention pertains to the field of biomedicine, and in particular the present invention relates to a synthetic method of bivalirudin.
The conventional bivalirudin synthesis method is time-consuming and complicated in process and is not able to be achieved in large scale, and therefore technical personnel in the art are committed to developing a new bivalirudin synthesis method.
Description of the Prior art
Solid Phase Peptide Synthesis (SPPS) is fast method for polypeptides synthesis. However, conventional SPPS to synthesis large quantity of long peptides like Bivalirudin is time consuming and generates large amount of solvent waste. As a widely used active pharmaceutical ingredient, Bivalirudin is a direct thrombin inhibitor indicated for use as an anticoagulant. It is significant to develop an efficient and economic SPPS method for preparation of large quantity and high purity Bivalirudin.
Summary of the Invention
It is an object of the present invention 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 a solid phase peptide synthesis (SPPS), wherein the method uses Fmoc -Leu-Wang resin or Fmoc-Leu-2CT resin, Rink amide ProTide (LL) resin and ChemMatrix wang resin as carrier for the solid phase synthesis of the polypeptide.
Further, the coupling reagents used in the polypeptide solid phase synthesis method are selected from the group consisting of ethyl 2-oxime cyanoacetate, N, N-diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide Amine, N,N-Diisopropylethylamine .
Further, in the Fmoc deprotection method, piperidine / N, N-dimethylformamide/ Formic acid (preferably 1/4 volume of piperidine / N, N-dimethylformamide, Formic acid 5%-20% of piperidine volume)/ piperazine is added, and the heating conditions are preferably carried out by means of a water bath, an oil bath or a microwave heating reaction for 2 to 5 minutes.
Further, each of the reactions in the solid phase synthesis reaction of the polypeptide was washed with N, N-dimethylformamide for 2-3 times after completion of the reaction.
Further, the condensation reaction of the amino acid in the solid phase synthesis reaction of the polypeptide on the resin is carried out at a temperature of 50 to 120 °C; preferably, the reaction time is 10 to 30 minutes depending on the temperature.
Further, the amino acid condensation reaction of the polypeptide in the solid-phase synthesis reaction is carried out and the resin is blown off with nitrogen before the cleavage reaction is carried out.
Further, the purity of the trifluoroacetic acid added in the reaction was 95%, the temperature was 10-50 °C, and the cleavage reaction time was 1-3 hours.
Further, in the solid phase synthesis reaction of the polypeptide, the equivalents of the amino acid used is twice the molar amount of the resin.
Further, the crude bivalirudin is purified by Pre-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
Further, the purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours.
Brief Description of the Drawings
Figure 1 is a schematic of bivalirudin molecular structure.
Figure 2 is HPLC analysis of prepared Bivalirudin.
Figure 3 is Mass spectrum of prepared Bivalirudin.
Detailed Description of the Preferred Embodiments
Bivalirudin show in Figure 1 is 20 amino acids synthetic peptide. The invention will now be described with reference to specific embodiments. It has to be understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise stated, the percentages and parts are by weight. The experimental materials and reagents used in the examples below are available from commercially available sources unless otherwise specified.
Table 1 List of amino acid abbreviations
Name Three letter symbol Single letter symbol
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
Fmoc fluorenylmethoxycarbonyl chloride
OtBu tert-butil este
DIC N, N-diisopropylcarbodiimide
DMF N, N-dimethylformamide
EDT ethanedithiol
Oxyma 2-oxime cyanoacetate
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Trt triphenylmethyl
Fmoc-Leu-Wang resin fluorenylmethoxycarbonyl-leucine-Wang resin
Fmoc-Leu-2CT resin fluorenylmethoxycarbonyl-leucine-2CT resin
Rink amide ProTide (LL) resin fluorenylmethoxycarbonyl-leucine- Rink amide ProTide (LL) T resin
ChemMatrix wang resin fluorenylmethoxycarbonyl-leucine- ChemMatrix wang resin
Table 3 the list of intermediates and Fmoc protected amino acdis
Fmoc-Tyr (tBu) -Leu-Wang resin
Fmoc-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang resin
Fmoc-Leu-OH
Fmoc-Tyr(tBu)-OH
Fmoc-Glu(OtBu)-OH
Fmoc-Pro-OH
Fmoc-Ile-OH
Fmoc-Phe-OH
Fmoc-Asp(OtBu)-OH
Fmoc-Gly-OH
Fmoc-Asn(Trt)-OH
Fmoc-Arg(Pbf)-OH
Fmoc-D-Phe-OH
The present invention provides a synthetic method of bivalirudin using Fmoc-Leu-wang resin by solid phase peptide synthesis comprising the following steps of:
Step 1, fluorenylmethoxycarbonyl-leucine-Wang resin can be purchased directly (Sigma Aldrich), it can reduce the first step of the synthesis and speed up the synthesis efficiency;
Step 2: The resin is swollen for 5 to 15 minutes under nitrogen in DMF;
Step 3: fluorenylmethoxycarbonyl chloride protecting group is removed under the following conditions: Formic acid 5%-20% of piperidine volume)/ piperazine in DMF for 2 minutes at 50°C - 120°C.
Step 4: Preparation of fluorenylmethoxycarbonyl-tyrosine (t-butyl) -leucine-Wang resin: The fluorenylmethoxycarbonyl-leucine-Wang resin obtained in step 1 is deprotected, washed with DMF and Fmoc-L-Tyr-(t-butyl) -OH is subjected to a condensation reaction under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-tyrosine (t-butyl) -leucine-Wang resin;
Step 5: Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin: The Fmoc-dipeptide obtained in step 2 is deprotected and washed, and then reacted with Fmoc-L-Glutamic acid (tert-butoxy) -OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin;
Step 6: Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (t-butoxy) -tyrosine-(t-butyl) -leucine- Wang resin. The resulting fluorenylmethoxycarbonyl-glutamic acid (t-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin is deprotected, washed and Fmoc-L-Glutamic acid (tert-butoxy) -OH is added and then reacted under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-glutamic acid (tert-butyl (Tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -pyridine (t-butyl) ) - leucine - Wang resin;
Step 7: Preparation of fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin. Glutamic acid-(tert-butoxy)-glutamic acid (tert-butoxy) tyrosine-(t-butyl) -leucine-Wang resin is deprotected, washed and then reacted with fluorenylmethoxycarbonyl-proline-OH under the conditions of the polypeptide coupling reagent to obtain fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine-(t-butyl) -leucine-Wang resin. Glutamic acid-(tert-butoxy)-glutamic acid (tert-butoxy) tyrosine-(t-butyl) -leucine-Wang resin;
Step 8: Preparation of fluorenylmethoxycarbonyl-isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl) -leucine-Wang resin. The fluorenylmethoxycarbonyl-proline-glutamic acid (tert-butoxy) -glutamic acid-(tert-butoxy)-tyrosine (t-butyl) -leucine-Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-isoleucine-OH is added under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-isoleucine-proline- glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine (t-butyl)-leucine-Wang resin;
Step 9: Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine-(t-butyl) -leucine-Wang resin. To fluorenylmethoxycarbonyl- isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine-(t-butyl) -leucine-Wang resin obtained in step 6 is deprotected, washed and Fmoc-L-Glu(Otbu)-OH is added and reacted in the presence of a peptide coupling reagent conditions to give fluorenylmethoxycarbonyl glutamic acid (tert-butoxy) -isoleucine-proline- glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy)-tyrosine (t-butyl)-leucine-Wang resin;
Step 10: Preparation of fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy)-glutamic acid-(tert-butoxy)-tyrosine (t-butyl) -leucine-Wang resin. To fluorenylmethoxycarbonyl- glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy)-glutamic acid-(tert-butoxy)-tyrosine (t-butyl) -leucine-Wang resin obtained in step 7 Fmoc-L-Glu(Otbu) is added and subjected to a condensation reaction under the conditions of a polypeptide coupling reagent to giving fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy ) - isoleucine - proline - glutamic acid (tert - butoxy) - glutamic acid (tert - butoxy) - tyrosine (tert - butyl) - leucine -Wang resin;
Step 11: Preparation of fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (tert-butyl) -leucine-Wang resin. The peptide obtained in step 8 fluorenylmethoxycarbonyl-glutamic acid (tert-butoxy ) - glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl)-leucine-Wang resin was deprotected, washed and reacted with fluorenylmethoxycarbonyl-phenylalanine-OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) tyrosine (tert-butyl) - leucine - Wang resin;
Step 12: Preparation of fluorenylmethoxycarbonyl aspartic acid(tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -tyrosine (t-butyl) -leucine-Wang resin. The peptide obtained in step 9 is deprotected, washed and treated with fluorenylmethoxycarbonyl-aspartic acid (tert-butoxy) -OH to give fluorenylmethoxycarbonyl aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) -isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. The peptide obtained in step 10 is deprotected , washed and treated with fluorenylmethoxycarbonyl-glycine- OH under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. The peptide obtained in step 11 is deprotected , washed and treated with fluorenylmethoxycarbonyl-asparagine (trityl) -OH under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -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) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. The peptide obtained in step 12 is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 16: Preparation of fluorenylmethoxycarbonyl-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-glycine-OH was added under the conditions of the peptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 17: Preparation of fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OHunder the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 18: Preparation of fluorenylmethoxycarbonyl-glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. fluorenylmethoxycarbonyl-glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and subjected to a condensation reaction with fluorenylmethoxycarbonyl-glycine-OH under the conditions of a polypeptide coupling reagent to give fluorenylmethoxycarbonyl-glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 19: Preparation of fluorenylmethoxycarbonyl - proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl-glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-proline-OH is added in a polypeptide coupling reagent conditions, to give fluorenylmethoxycarbonyl - proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 20: Preparation of fluorenylmethoxycarbonyl -Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl - proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and fluorenylmethoxycarbonyl -Arg(Pbf)-OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl -Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 21: Preparation of fluorenylmethoxycarbonyl -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl -Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-proline-OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 22: Preparation of fluorenylmethoxycarbonyl-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin. Fluorenylmethoxycarbonyl -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is deprotected, washed and fluorenylmethoxycarbonyl-D Phenylalanine-OH is added in a peptide coupling reagent conditions, to give fluorenylmethoxycarbonyl-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 23: Fluorenylmethoxycarbonyl-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is washed and deprotected again to give NH2-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin;
Step 24: NH2-D-Phe -proline-Arg(Pbf)- proline - glycine- glycine-glycine-glycine-asparagine (trityl) -glycine-aspartic acid (tert-butoxy) -phenylalanine-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) isoleucine-proline-glutamic acid (tert-butoxy) -glutamic acid (tert-butoxy) - tyrosine (tert-butyl) - leucine -Wang resin is washed three times and cleavage of the peptide from the solid support is performed under the following conditions: TFA/TIS/H2O 94:2.5:2.5 for three hours.
Step 25: The cleavage solution is precipitated with diethyl ether and centrifuged at 3500-5000rpm to give a white precipitate. The precipitate is washed and centrifuged with diethyl ether for another three times to give a final precipitate and dry under vacuum.
Step 26: The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes.
Step 27: The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
ADVANTAGES OF THE PRESENT INNOVATION
The conventional method of SPPS for the synthesis of bivalirudin produce commonly large amounts of byproducts difficult to separate, large amount of waste that is expensive to dispose. The present process is faster than the normal SPPS process used, produce less amount of waste and it allows to achieve bivalirudin in high yield.
Example 1
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang resin (40g, substitution 0.67 meq/g). The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2-3 minutes and was monitored by Kaiser test. TFA cleavage of the peptide was done with TFA/TIS/H2O 94:5:1 for 3 hours followed by precipitation and 2 washings with diethyl ether. Yield of crude peptide is 85%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
Example 2
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-2CT resin (40g, substitution 0.8 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer and the synthesis was carried out using 2 molar excess protected amino acids with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 2-3 minute at 50°C -120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. TFA cleavage of the peptide was done with TFA/TIS/H2O 94:5:1 for 3 hours followed by precipitation and 3 washings with diethyl ether. Yield of crude peptide is 86.44%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
Example 3
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-2CT resin (40g, substitution 1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C - 120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes and was repeated twice. TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H2O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 88.12%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity. The purity was analyzed by HPLC as figure 2. The Mass analysis was corrected as Figure 3.
Example 4
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Chemmatrix resin (40g, substitution 0.67 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes. TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H2O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 84.15%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
Example 5
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang resin (40g, substitution 0.67 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with P formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-DmBGly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes and was repeated twice after the 10th amino acid. Synthesis time was 4hours and 20 minutes. TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H2O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 81.86%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
Example 6
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Protide resin from CEM resin (40g, substitution 0.18 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer (Cem Liberty Blue) and the first amino acid was coupled to the resin with use of KI and DIPEA in DMF for 10 min at 50°C -120°C. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was carried out for 1 minute at 50°C -120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes. TFA cleavage of the peptide was done with TFA/Thioanisole/phenol/H2O/TES 89:2.5:2.5:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 82.16%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.
Example 7
The synthesis of the peptide was carried out by employing stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Fmoc-Leu-Wang or 2CT resin (40g, substitution 0.67/1.1 meq/g). The resin was transferred to the reaction vessel of the peptide synthesizer. The Fmoc deprotection was carried out with formic acid 5%-20% of piperidine volume)/ piperazine in DMF. The Fmoc deprotection reaction was for 1 minute at 50°C -120°C, repeated twice. Later other amino acids in the sequence are attached in the following order, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc- Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly- OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe- OH. The coupling and decoupling of amino acids was carried out at 50°C -120°C for 2 minutes. TFA cleavage of the peptide was done with TFA/TIS/H2O 94:5:1 for 3 hours followed by precipitation and 4 washings with diethyl ether. Yield of crude peptide is 87.56%. The crude bivalirudin is purified by Preparative-HPLC with water/ acetonitrile gradient from 100% water to 100% acetonitrile in 20 minutes. The purified bivalirudin solution is lyophilized by lyophilizer at -50 to -70°C for 18-48 hours to give prepared bivalirudin more than 98% purity.

Claims (10)

  1. A method of synthesis, characterized by the use of solid phase peptide synthesis produces bivalirudin, wherein the method uses Fmoc-Leu- Wang resin, Fmoc-Leu-2CT resin, Rink amide ProTide (LL) resin and ChemMatrix wang resin as carrier for the solid phase synthesis of the polypeptide.
  2. The method according to claim 1, wherein the polypeptide linking agent used in the polypeptide solid phase synthesis method is selected from the group consisting of ethyl 2-oxime cyanoacetate, N, N-diisopropylcarbodiimide, N,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 a carrier for the solid phase synthesis reaction of the polypeptide.
  4. The method according to claim 1, wherein the polypeptide solid phase peptide synthesis method is carried out by the Fmoc deprotection method.
  5. The process according to claim 4, wherein the piperidine / N, N-dimethylformamide/ Formic acid/piperazine is added to the Fmoc deprotection method, and the defluorenylmethoxycarbonyl protecting reaction is carried out under heating or microwave.
  6. The method of claim 1, wherein the polypeptide is produced by solid phase synthesis reaction and is then washed with N, N-dimethylformamide for 2-3 times after completion of each reaction.
  7. The method according to claim 6, wherein the condensation reaction of the amino acid in the solid phase synthesis reaction of the polypeptide on the resin is carried out at 50 to 120°C with 2-10 times equivalents compared to the resin amount.
  8. The method according to claim 6, wherein the resin is blown off with nitrogen gas before the completion of the cleavage reaction after completion of the amino acid condensation reaction in the solid phase synthesis reaction of the polypeptide.
  9. The method according to claim 8, wherein the purity of the trifluoroacetic acid added in the cleavage reaction is >95%, the reaction temperature is 25°C, and the cleavage reaction time is 1-3 hours.
  10. The method according to claim 1, wherein the resin is subjected to a swelling treatment before the solid phase synthesis reaction of the polypeptide with DMF.
PCT/CN2018/091752 2018-06-19 2018-06-19 Synthetic method of bivalirundin WO2019241903A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033249A (en) * 2006-03-10 2007-09-12 周逸明 Preparation method of synthesizing bivalirudin from solid phase polypeptide
CN102532274A (en) * 2012-02-13 2012-07-04 成都圣诺生物制药有限公司 Method for preparing bivalirudin
CN103242431A (en) * 2013-05-20 2013-08-14 齐鲁制药有限公司 Preparation method of bivalirudin
CN103319570A (en) * 2013-05-30 2013-09-25 深圳翰宇药业股份有限公司 Preparation method of bivalirudin
CN103374054A (en) * 2012-04-28 2013-10-30 上海第一生化药业有限公司 One-step method based solid-phase polypeptide synthesis method

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007033383A2 (en) * 2005-09-14 2007-03-22 Novetide, Ltd. Process for production of bivalirudin
US20090062511A1 (en) * 2007-09-05 2009-03-05 Raghavendracharyulu Venkata Palle Process for the preparation of bivalirudin and its pharmaceutical compositions
US20110160431A1 (en) * 2009-04-06 2011-06-30 Novetide, Ltd. Production of peptides containing poly-gly sequences using fmoc chemistry
CN101555274B (en) * 2009-05-15 2013-08-21 海南双成药业股份有限公司 Preparation method of polypeptide solid-phase synthesis bivalirudin crude product
CN102286076B (en) * 2011-06-23 2014-03-12 成都圣诺科技发展有限公司 Preparation method for bivalirudin
US10125163B2 (en) * 2015-10-23 2018-11-13 Cem Corporation Solid phase peptide synthesis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033249A (en) * 2006-03-10 2007-09-12 周逸明 Preparation method of synthesizing bivalirudin from solid phase polypeptide
CN102532274A (en) * 2012-02-13 2012-07-04 成都圣诺生物制药有限公司 Method for preparing bivalirudin
CN103374054A (en) * 2012-04-28 2013-10-30 上海第一生化药业有限公司 One-step method based solid-phase polypeptide synthesis method
CN103242431A (en) * 2013-05-20 2013-08-14 齐鲁制药有限公司 Preparation method of bivalirudin
CN103319570A (en) * 2013-05-30 2013-09-25 深圳翰宇药业股份有限公司 Preparation method of bivalirudin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3810627A4 *

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