CN103319570A - Preparation method of bivalirudin - Google Patents
Preparation method of bivalirudin Download PDFInfo
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- CN103319570A CN103319570A CN2013102103296A CN201310210329A CN103319570A CN 103319570 A CN103319570 A CN 103319570A CN 2013102103296 A CN2013102103296 A CN 2013102103296A CN 201310210329 A CN201310210329 A CN 201310210329A CN 103319570 A CN103319570 A CN 103319570A
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Abstract
The invention discloses a preparation method of bivalirudin. The preparation method adopts a sequential coupling preparation technology. Compared with feeding ratios of other corresponding amino acids, a Gly feeding ratio of the preparation method is obviously improved and is 4-8 times.
Description
Technical field
The present invention relates to a kind of peptide preparation method, relate in particular a kind of preparation method of Bivalirudin.
Background technology
Bivalirudin (Bivalirudin) is a kind of clinical thrombin inhibitors that is applied to, early stage clinical study shows Bivalirudin anticoagulant therapy definite effect, and the incidence of bleeding episode is lower, with traditional anticoagulant heparin treatment compare use safer.Bivalirudin can the directly effect of Trombin inhibiting by the negatively charged ion output site that is incorporated into catalyst site and circulation and zymoplasm clot.
Bivalirudin (Bivalirudin has another name called Angiomax) is the synthetic anticoagulant, and its anti-freezing composition is the peptide of 20 amino-acid residues of hirudin derivative C end.Structure sequence is D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Gl u-Ile-Pro-Glu-Glu-Tyr-Leu-OH.Bivalirudin belongs to the direct inhibitor of strong zymoplasm, is the wider medicine of in recent years clinical application.
About the preparation method of Bivalirudin, have a large amount of reports both at home and abroad.Mainly contain successively coupling method of solid phase.Owing to containing one-Gly-Gly-Gly-Gly fragment in the Bivalirudin structure, therefore in solid phase successively coupling-Gly process, the Gly self-characteristic is so that very easily at the impurity of main peak front and back generation Bivalirudin-Gly and Bivalirudin+Gly, these two impurity are difficult to remove when later separation.Chinese patent 201110170669 and Chinese patent 201110144317 have all adopted Fmoc-Gly-Gly-Gly-Gly-OH to solve the problem of above-mentioned Gly impurity as reaction raw materials.But above-mentioned patented method needs self-control, and complex steps is unfavorable for suitability for industrialized production.
Summary of the invention
In order to solve above shortcoming, the present invention is intended to find suitable coupling condition, can effectively control the generation of Gly impurity on the basis of order coupling, is beneficial to suitability for industrialized production.During solid phase synthesis, the amino acid coupling generally adopt 2h even longer reaction times with guarantee coupling fully.The present invention finds unexpectedly, synthetic-during the Gly-Gly-Gly-Gly peptide chain, by improving amino acid (Fmoc-Gly-OH) ingredient proportion, and Reaction time shorten, can effectively solve BIV synthetic in the problem of Gly impurity.
A first aspect of the present invention provides a kind of preparation method of Bivalirudin, may further comprise the steps:
1) selects suitable solid phase carrier;
2) make the Fmoc-Leu-solid phase carrier;
3) remove and add corresponding amino acid with 1-3 feed ratio doubly after the Fmoc protection and carry out coupling, thereby finish successively the coupling of 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH;
4) remove the rear feed ratio adding Fmoc-Gly-OH with 4-8 times of Fmoc protection and carry out coupling, repeat coupling 4 times;
5) remove and add corresponding amino acid with 1-3 feed ratio doubly after the Fmoc protection and carry out coupling, thereby the inferior coupling of finishing Fmoc-Pro-OH, Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH obtains the Bivalirudin peptide resin;
6) with lytic reagent Bivalirudin cracking from the peptide resin is got off, obtain thick peptide;
7) described thick peptide turns salt through purifying, finally obtains refining Bivalirudin.
In some embodiments, the solid phase carrier step 1) is the Wang resin.
In other embodiments, the coupling system of Fmoc-Gly-OH is selected from a kind of among DIC/HOBt, HATU/HOAt/DIPEA, the PyBOP/HOBt/DIPEA step 4).In a preferred embodiment, the linked reaction time of Fmoc-Gly-OH is 10min-30min step 4).
In other embodiment, step 6) lytic reagent described in is the mixture of TFA, a TIS H2O.In a preferred embodiment, TFA: TIS: H2O=92: 4: 4 (V: V).
In some embodiments again, step 7) purifying described in is the RPLC purifying.In a preferred embodiment, the chromatographic column of described RPLC is anti-phase C18 post.In another preferred embodiment, the salt that turns step 7) is to change into the trifluoroacetic acid salt form.
Embodiment
Abbreviation and implication
| Abbreviation | Implication |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| Boc | Tertbutyloxycarbonyl |
| tBu | The tertiary butyl |
| OtBu | Tert.-butoxy |
| Pbf | 2,2,4,6,7-pentamethyl-benzo furans-5-alkylsulfonyl |
| Trt | Trityl |
| DCM | Methylene dichloride |
| DBLK | 20% hexahydropyridine/DMF solution |
| DIPEA | DIPEA |
| TIS | Tri isopropyl silane |
| DMF | DMF |
| DMAP | DMAP |
| HPLC | High performance liquid chromatography |
| TFA | Trifluoroacetic acid |
Terminological interpretation
Substitution degree: the amount that contains active group on the unit weight resin.In this article, the unit of substitution degree is mmol/g.Take embodiment four as example, weight resin is 239.5g, and its substitution degree is 0.501, so the amount of active group is 120mmol.
Feed ratio: the ratio of reaction raw materials and active group.
Peptide synthesizer: Peptide synthesizer is hereinafter described built pharmaceutical Co. Ltd of nation available from Hainan, and model is JBP-20.
Ninhydrin method: ninhydrin method hereinafter described is solid phase conventional sense method, can be referring to " Fmoc Solid phase peptide synthesis " Oxford university press.
Describe the present invention in detail below by following examples.
Embodiment one: substitution degree is the preparation of the Fmoc-Leu-Wang resin of 0.4mmol/g
Take by weighing substitution degree and be the Wang resin of 1.0mmol/g (available from Compositech Inc. of Nankai, article No. HCW01-1-2) 120g (active group 120mmol), add in the Peptide synthesizer, with DMF washing 2 times, with DMF swelling resin after 30 minutes, getting 84.8gFmoc-Leu-OH, 38.9g HOBt, 36.3g DIC, 2.93g DMAP, to be dissolved in the 250ml volume ratio be 1: 1 DCM and DMF mixing solutions, adds in the Peptide synthesizer room temperature reaction 2h.With DMF washing 4 times, DCM washed 2 times after reaction finished.Then add 189.8g pyridine and 245.04g acid anhydrides mixed solution sealing resin 6h.With DMF washing 4 times, after DCM washs 2 times, drain with the methyl alcohol contraction, obtain the Fmoc-Leu-Wang resin, the detection substitution degree is 0.406mmol/g.
Embodiment two: substitution degree is the preparation of the Fmoc-Leu-Wang resin of 0.6mmol/g
Take by weighing substitution degree and be the Wang resin of 1.0mmol/g (available from Compositech Inc. of Nankai, article No. HCW01-1-2) 120g (active group 120mmol), add in the Peptide synthesizer, with DMF washing 2 times, with DMF swelling resin after 30 minutes, getting 118.7gFmoc-Leu-OH, 54.5g HOBt, 50.8g DIC, 4.1g DMAP, to be dissolved in the 250ml volume ratio be 1: 1 DCM and DMF mixing solutions, adds in the Peptide synthesizer room temperature reaction 2h.With DMF washing 4 times, DCM washed 2 times after reaction finished.Then add 189.8g pyridine and 245.04g diacetyl oxide mixed solution sealing resin 6h.With DMF washing 4 times, after DCM washs 2 times, drain with the methyl alcohol contraction, obtain the Fmoc-Leu-Wang resin, the detection substitution degree is 0.617mmol/g.
Embodiment three: substitution degree is the preparation of the Fmoc-Leu-Wang resin of 0.5mmol/g
The Wang resin that takes by weighing substitution degree and be 1.0mmol/g is (synthetic available from Nankai, article No. HCW01-1-2) 120g (active group 120mmol), add in the Peptide synthesizer, with DMF washing 2 times, with DMF swelling resin after 30 minutes, getting 101.7g Fmoc-Leu-OH, 46.7gHOBt, 43.6g DIC, 3.5g DMAP, to be dissolved in the 250ml volume ratio be 1: 1 DCM and DMF mixing solutions, adds in the Peptide synthesizer room temperature reaction 2h.With DMF washing 4 times, DCM washed 2 times after reaction finished.Then add 189.8g pyridine and 245.04g diacetyl oxide mixed solution sealing resin 6h.With DMF washing 4 times, after DCM washs 2 times, drain with the methyl alcohol contraction, obtain the Fmoc-Leu-Wang resin, the detection substitution degree is 0.501mmol/g.
Embodiment four: the preparation of Bivalirudin peptide resin ()
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 500ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and (add 700ml DBLK; then with DMF washing 4 times; DCM washes 2 times) (measure with identical mmol with adding amino acid; be 240mmol) step of coupling; according to the order of fragment, finish successively the coupling of 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 142.7g Fmoc-Gly-OH (480mmol), 77.8g HOBt (576mmol), 90.0g DIC (576mmol) is dissolved in 700ml DMF solution, adds in the Peptide synthesizer room temperature reaction 30min.With DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and the step that adds amino acid (with identical mmol amount, i.e. 480mmol) coupling, according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add amino acid (with identical mmol amount; be 240mmol) step of coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment five: the preparation of Bivalirudin peptide resin (two)
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; according to the order of fragment, finish successively the coupling of 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 178.4g Fmoc-Gly-OH (600mmol), 97.3g HOBt (720mmol), 112.6g DIC (720mmol) is dissolved in 700ml DMF solution, adds in the Peptide synthesizer room temperature reaction 20min.With DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and the step that adds amino acid (with identical mmol amount, i.e. 600mmol) coupling, according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add (with identical mmol amount; be 240mmol) step of amino acid coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment six: the preparation of Bivalirudin peptide resin (three)
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add amino acid (with identical mmol amount; be 240mmol) step of coupling; according to the order of fragment, finish successively the coupling of 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 214.1g Fmoc-Gly-OH (720mmol), 116.7g HOBt (864mmol), 135.1g DIC (864mmol) is dissolved in 700ml DMF solution, adds in the Peptide synthesizer room temperature reaction 10min.With DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and the step that adds amino acid coupling (with identical mmol amount, i.e. 720mmol), according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add amino acid (with identical mmol amount; be 240mmol) step of coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment seven: the preparation of Bivalirudin peptide resin (four)
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add amino acid (with identical mmol amount; be 240mmol) step of coupling; according to the order of fragment, finish successively 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 285.4g Fmoc-Gly-OH (960mmol), 438.0g HATU (960mmol), 156.8g HOAt (1152mmol) is dissolved in 700ml DMF solution, be added dropwise to 333.8mlDIPEA (1920mmol), reaction solution is added in the Peptide synthesizer room temperature reaction 10min.With DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and the step that adds amino acid (with identical mmol amount, i.e. 960mmol) coupling, according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment eight: the preparation of Bivalirudin peptide resin (five)
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; according to the order of fragment, finish successively 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 249.7g Fmoc-Gly-OH (840mmol), 437.1g PyBOP (840mmol), 136.2g HOBt (1008mmol) is dissolved in 700ml DMF solution, be added dropwise to 292.1mlDIPEA (1680mmol), reaction solution is added in the Peptide synthesizer room temperature reaction 20min.With DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling, according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment nine: the preparation of Bivalirudin peptide resin (six)
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; according to the order of fragment, finish successively 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH.
Again with 214.1g Fmoc-Gly-OH (720mmol), 374.7g PyBOP (720mmol), 116.7g HOBt (864mmol) is dissolved in 700ml DMF solution, be added dropwise to 250.4ml DIPEA (1440mmol), reaction solution is added in the Peptide synthesizer room temperature reaction 15min.With DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling, according to the order of fragment, finish successively the coupling of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH.
At last with 81.0g Fmoc-Pro-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 700ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the step that the above-mentioned Fmoc of removing protected and added corresponding amino acid coupling; order according to fragment; finish successively the coupling of Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.
Embodiment ten: the preparation of the thick peptide of Bivalirudin
The peptide resin that embodiment four to embodiment nine is obtained places round-bottomed flask, with the amount of 10ml/g resin add lytic reagent (TFA: TIS: water=92: 4: 4 (V/V)), stirring at room 2h.Reactant filters with sand core funnel, collects filtrate, and resin is again with a small amount of TFA washing 3 times, concentrating under reduced pressure behind the merging filtrate.The freezing anhydrous diethyl ether of amount adding with the 100ml/g peptide resin precipitates, and with anhydrous diethyl ether washing 3 times, vacuum-drying obtains the white powder solid, i.e. the thick peptide of Bivalirudin.The HPLC purity of thick peptide is 77.8%-83.6%, and-Gly impurity " 0.2% ,+Gly impurity " 0.2% (seeing the following form).
Comparative Examples one: the preparation of prior art Bivalirudin resin and thick peptide
Below adopt the order Solid-phase Polypeptide synthetic method of putting down in writing among the 200910051311.X to carry out the preparation of Bivalirudin resin and thick peptide.
Take by weighing the Fmoc-Leu-wang resin 239.5g (active group 120mmol) of embodiment three preparations, add in the Peptide synthesizer, with DMF washing 2 times; with DMF swelling resin after 30 minutes; remove the Fmoc protection with 700ml DBLK, then with DMF washing 4 times, DCM washes 2 times.Then with 110.3g Fmoc-Tyr (tBu)-OH (240mmol), 38.9g HOBt (288mmol), 45.0g it is 1: 1 DCM and DMF mixing solutions that DIC (288mmol) is dissolved in the 450ml volume ratio, adds in the Peptide synthesizer, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if resin water white transparency, then react completely, the resin colour developing, the expression reaction is not exclusively, need again linked reaction 1h), use DMF solution washing resin 6 times.Repeat the above-mentioned Fmoc of removing protection and add amino acid (with identical mmol amount; be 240mmol) step of coupling; order according to fragment; finish successively 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-Asp (OtBu)-OH; Fmoc-Gly-OH; Fmoc-Asn (Trt)-OH; Fmoc-Gly-OH; Fmoc-Arg (pbf)-OH; Fmoc-Pro-OH; the coupling of Fmoc-D-Phe-OH; after finishing, reaction shrinks with 1100ml methyl alcohol; resin vacuum-drying is spent the night, and obtains the Bivalirudin resin.Then by the peptide resin that makes according to preparing the thick peptide of Bivalirudin with embodiment ten described identical methods, then detect by HPLC.
The HPLC detected result is as follows:
| The HPLC detected result | Purity | -Gly impurity | + Gly impurity |
| Embodiment four | 80.76％ | 0.12％ | 0.07％ |
| Embodiment five | 77.80％ | 0.19％ | 0.09％ |
| Embodiment six | 80.63％ | 0.16％ | 0.06％ |
| Embodiment seven | 82.21％ | 0.15％ | 0.05％ |
| Embodiment eight | 83.60％ | 0.13％ | 0.07％ |
| Embodiment nine | 82.63％ | 0.16％ | 0.06％ |
| Comparative Examples one | 74.39％ | 2.33％ | 5.66％ |
Can find out from the above results, in the thick peptide of Bivalirudin that makes by the inventive method-Gly and+the Gly foreign matter content obviously reduces than art methods.
The preparation of embodiment 12 Bivalirudins essence peptide trifluoroacetate
Take by weighing 200g according to the thick peptide of the embodiment nine described Bivalirudins that make with the mixed solvent 5000mL of 25% acetonitrile+25%DMSO+50% water dissolving after, adopt the Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 * 250mm, column temperature is 50 ℃, conventional 0.1%TFA/ acetonitrile moving phase purifying is collected purpose peak cut, obtains purity greater than 98.5% smart peptide.Smart peptide solution is adopted the Waters2545RP-HPLC system, chromatographic column is the anti-phase C8 post of 50 * 250mm, 0.1% trifluoracetic acid/acetonitrile moving phase turns salt, collect purpose peak cut, revolve the inspissation contracting, freeze-drying obtains Bivalirudin trifluoroacetate essence peptide 79.8g, HPLC purity 99.53%, purification yield 81.4%, total recovery 35.9%.And adopting method same as described above refining according to the thick peptide of the Comparative Examples one described Bivalirudin that makes, HPLC purity obviously reduces, and yield is below 10%.
Claims (9)
1. the preparation method of a Bivalirudin may further comprise the steps:
1) selects suitable solid phase carrier;
2) make the Fmoc-Leu-solid phase carrier;
3) remove and add corresponding amino acid with 1-3 feed ratio doubly after the Fmoc protection and carry out coupling, thereby finish successively the coupling of 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-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt)-OH;
4) remove the rear feed ratio adding Fmoc-Gly-OH with 4-8 times of Fmoc protection and carry out coupling, repeat coupling 4 times;
5) remove and add corresponding amino acid with 1-3 feed ratio doubly after the Fmoc protection and carry out coupling, thereby finish successively the coupling of Fmoc-Pro-OH, Fmoc-Arg (pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH, obtain the Bivalirudin resin;
6) with lytic reagent Bivalirudin cracking from the Bivalirudin resin is got off, obtain thick peptide;
7) described thick peptide turns salt through purifying, finally obtains refining Bivalirudin.
2. the process of claim 1 wherein step 1) described in solid phase carrier be the Wang resin.
3. claim 1 or 2 method, wherein step 4) in the coupling system of Fmoc-Gly-OH be selected from a kind of among DIC/HOBt, HATU/HOAt/DIPEA, the PyBOP/HOBt/DIPEA.
4. each method of claim 1-3, wherein step 4) in linked reaction time of Fmoc-Gly-OH be 10min-30min.
5. each method of claim 1-3, wherein step 6) described in lytic reagent be the mixture of TFA, TIS and H2O.
6. the method for claim 5, wherein TFA: TIS: H2O=92: 4: 4 (V: V).
7. each method of claim 1-3, wherein step 7) described in purifying be the RPLC purifying.
8. the method for claim 7, the chromatographic column of wherein said RPLC is anti-phase C18 post.
9. each method of claim 1-3, wherein step 7) described in the salt that turns be to change into the trifluoroacetic acid salt form.
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| CN105218644A (en) * | 2015-11-09 | 2016-01-06 | 叶仲林 | A kind of preparation method of Bivalirudin |
| CN108383905A (en) * | 2016-12-30 | 2018-08-10 | 江苏金斯瑞生物科技有限公司 | A kind of preparation method of bivalirudin |
| WO2019241903A1 (en) * | 2018-06-19 | 2019-12-26 | Shanghai Space Peptides Pharmaceutical Co., Ltd. | Synthetic method of bivalirundin |
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| CN105218644A (en) * | 2015-11-09 | 2016-01-06 | 叶仲林 | A kind of preparation method of Bivalirudin |
| CN108383905A (en) * | 2016-12-30 | 2018-08-10 | 江苏金斯瑞生物科技有限公司 | A kind of preparation method of bivalirudin |
| WO2019241903A1 (en) * | 2018-06-19 | 2019-12-26 | Shanghai Space Peptides Pharmaceutical Co., Ltd. | Synthetic method of bivalirundin |
| CN112424216A (en) * | 2018-06-19 | 2021-02-26 | 上海胜泽泰医药科技有限公司 | Synthetic method of bivalirudin |
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