CN104418948A - Method for preparing polypeptide medicament - Google Patents
Method for preparing polypeptide medicament Download PDFInfo
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- CN104418948A CN104418948A CN201310410636.9A CN201310410636A CN104418948A CN 104418948 A CN104418948 A CN 104418948A CN 201310410636 A CN201310410636 A CN 201310410636A CN 104418948 A CN104418948 A CN 104418948A
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- diapep277
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Abstract
The invention relates to a method for preparing a polypeptide medicament DiaPep277. The method comprises: coupling Fmoc-Asp(OtBu)-OH with resin P to obtain Fmoc-Asp(OtBu)-P; performing solid phase synthesis on the Fmoc-Asp(OtBu)-P in a one-by-one coupling manner to obtain DiaPep277-P; and cracking the DiaPep277-P to obtain a DiaPep277 crude peptide.
Description
Technical field
The present invention relates to a kind of method preparing polypeptide drugs, in particular to the preparation method of DiaPep277.
Background technology
DiaPep277 is that a section of being derived from HUMAN HEAT SHOCK PROTEINS 60 (HSP60) sequence contains 24 amino acid whose peptide sections.The structure of DiaPep277 is as follows:
H-Val-Leu-Gly-Gly-Gly-Val-Ala-Leu-Leu-Arg-Val-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr-Pro-Ala-Asn-Glu-Asp-OH。
Research proves, DiaPep277 can immunity moderation system, makes islet cells from destroying and protecting insulin secretion function.Therefore, DiaPep277 is that the treatment of autoimmune diabetes patient brings substantial breakthrough, and can as the preventive usage of this onset diabetes high risk population.This medicine can significantly improve the life quality of type i diabetes patient, delays or stop the generation of various complication, to improve the predicted life of patient.
Summary of the invention
For containing 24 amino acid whose small peptides, because solid phase method exists easy and simple to handle, solvent usage quantity is little, is easy to the advantages such as automatization, therefore often adopts conventional protected amino acid to carry out the coupling of solid phase order and synthesizes.But after the peptide chain of DiaPep277 by analysis, the present inventor finds, this peptide chain exists some special situations, a large amount of hydrophobic groups (particularly N end) and the existence of-Gly-Gly-Gly-structure can make synthesis there is larger difficulty.If adopt the conventional Fmoc-Gly-OH raw material easily impurity etc. of generation ± Gly when synthesizing-Gly-Gly-Gly-structure.
The object of the invention is to adopt special amino acid starting material to solve above-mentioned difficulties, improve purity and the yield of thick peptide.
The invention provides a kind of method preparing polypeptide drugs DiaPep277, comprise following steps:
1) using a kind of resin P as carrier, by Fmoc-Asp (OtBu)-OH and resin coupling, Fmoc-Asp (OtBu)-P is obtained;
2) with Fmoc-Asp (OtBu)-P for raw material, adopt the mode of coupling one by one and other amino acid starting materials to carry out solid phase synthesis, obtain DiaPep277-P;
3) by DiaPep277-P cracking, the thick peptide of DiaPep277 is obtained;
Wherein Fmoc is 9-fluorenylmethyloxycarbonyl, is connected or is connected with the nitrogen-atoms on the connected carbon atom of carboxyl with amino,
Wherein step 2) described in coupling one by one mode in Asp-Ser structure adopt synthesis material be Fmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH, it is a pseudo proline dipeptides, and make the protected one-tenth five-ring of Serine, structure is as follows:
Wherein the synthesis material of-Gly-Gly-Gly-structure employing is one or both in Fmoc-(Hmb) Gly-OH, Fmoc-(Dmb) Gly-OH, Fmoc-Gly-(Dmb) Gly-OH or Fmoc-Gly-(Hmb) Gly-OH.
Preferably, the inventive method also comprises the step of purifying DiaPep277.
Embodiment
Abbreviation used in the present invention and implication thereof are described as follows:
Herein, " substitution degree " refers to the quantity of the resin-carried material of unit vol, and unit is " mmol/g ".
Herein, as illustrated without contrary, then described reaction is carried out at normal temperatures and pressures.
Wide in range, preferred, preferred and most preferred definition in the present invention and scope can combine mutually.
In the present invention, " peptide " refers to the coupled product obtained similarly by the Fmoc solid phase synthesis process in Peptide systhesis.
Described vector resin is not particularly limited, if its have can carry out with the carboxyl in material the avtive spot that reacts and not with other radical reactions in described material.Described vector resin is preferably 2-CTC resin or king's resin two class, and resin substitution degree is 0.2-1.0mmol/g, is preferably 0.3-0.8mmol/g, is more preferably 0.4-0.6mmol/g.
The structure of the present invention king used resene is as follows:
The structure of 2-CTC resene used is as follows:
Wherein
represent the rest part of resin structure and this part does not participate in reaction.
Above-mentioned two kinds of resins are all available commercially (Tianjin Nankai Hecheng S&T Co., Ltd.) or prepare by the method that document is known.
In a specific embodiment, the consumption of Fmoc-amino acid-OH can be selected according to the substitution degree before the consumption of resin, reaction and required reacted substitution degree, to be generally on resin can with the 1-5 equivalent of the mole number of the functional group of amino acid couplings, preferred 2-4 equivalent, more preferably 2.5-3.5 equivalent wherein resin can be the product of weight resin and substitution degree with functional group's mole number of amino acid couplings.
Described solvent is, such as, but not limited to, DMF, DCM, DMSO, NMP, THF, ethyl acetate, methyl alcohol, ether etc. and any mixture thereof, and preferred DMF, DCM, or the mixed solution of DMF and DCM (such as, the two volume ratio is 1:1).
Coupling is prepared peptide resin and Fmoc solid phase synthesis process known to the skilled in Peptides Synthesis can be adopted to realize, such as can see document Fmoc Solid Phase Peptide Synthesis:A Practical Approach, W.C.Chan, Peter D.White work, March2,2000 (ISBN-10:0199637245), Britain Oxford University Press.
In coupling, the washing of resin and the swelling any reagent realizing this object that adopts are carried out, preferred DMF.
Described being coupled in solid state reaction post is carried out.Solid state reaction post is not particularly limited, can be any solid state reaction post that can realize this object.
Preferably, by step 1)-2) in carboxyl in material activate, activation is preferably carried out in ice-water bath.Activator is preferably DIPEA or DIC.
Wherein after amino acid Fmoc-Asp (OtBu)-OH is coupled on vector resin, also comprise the step adding unreacted group excessive in a kind of confining liquid removing resin wherein, to avoid it to treat that conjugate qualitative response forms by product with other in next step linked reaction, and remove the step of the confining liquid added.When described vector resin is 2-CTC resin, the confining liquid added is preferably methyl alcohol; When described vector resin is king's resin, the confining liquid added is preferably the diacetyl oxide and pyridine that mol ratio is 1:1.
Coupling is preferably carried out under the existence of coupling agent, and described coupling agent is preferably DIC, A or it combines, or be DIPEA, A, B or its combination, wherein the A one that to be HOBt or HOAt, B be in PyBOP, PyAOP, HATU, HBTU and TBTU; Such as, step 1) in coupling agent be HOBt/DIC.
Step 2) described in Fmoc-Asp (OtBu)-Ser (ψ
me, Mepro) coupling system of-OH is the one in HATU/HOAt/DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
Step 2) described in coupling one by one mode in the synthesis material that adopts of-Gly-Gly-Gly-structure be one or both in Fmoc-(Hmb) Gly-OH, Fmoc-(Dmb) Gly-OH, Fmoc-Gly-(Dmb) Gly-OH or Fmoc-Gly-(Hmb) Gly-OH; Preferably, described raw material for being selected from the one of Fmoc-(Hmb) Gly-OH and Fmoc-(Dmb) Gly-OH, and is selected from the one of Fmoc-Gly-(Dmb) Gly-OH and Fmoc-Gly-(Hmb) Gly-OH.
Step 2) described in the coupling system of Gly synthesis material be one in DIC/HOAt, HATU/BOAt/DIPEA or PyBOP/HOBt/DIPEA.
In addition, the time that often kind of material carries out linked reaction is generally 1-6 hour, preferred 2-5 hour; Pressure is preferably normal pressure, also can carry out under the pressure (such as 0.01-1.5 normal atmosphere) suitably improving or reduce; Temperature is preferably room temperature (referring to 20 ± 5 DEG C), also (such as 0-50 DEG C) can carry out at the temperature suitably improving or reduce.
In coupling process, detection agent used can be any reagent that can judge described reaction end, preferred triketohydrindene hydrate.
The reagent removing Fmoc can be any reagent that can realize this object, preferred DBLK (i.e. 20% piperidines/DMF).
The deprotection reaction time is generally 5-30 minute, preferred 10-20 minute; Pressure is preferably normal pressure, also can carry out under the pressure (such as 0.01-1.5 normal atmosphere) suitably improving or reduce; Temperature is preferably room temperature (referring to 20 ± 5 DEG C), also (such as 0-50 DEG C) can carry out at the temperature suitably improving or reduce.
Described cracking is preferably carried out at room temperature and atmospheric pressure, also can carry out under the temperature and pressure suitably improving or reduce.
Step 3) in the mixed solvent of lytic reagent TFA, tri isopropyl silane (TIS) and water, wherein the proportioning of mixed solvent is: TFA volume ratio is 85-90%, the volume ratio of tri isopropyl silane is 1-5%, and the volume ratio of water is 1-5%, and three's sum is 100%.
In a specific embodiment, step 3) described in lytic reagent be TFA:TIS:H
2o=95:5:5 (V:V).
Preferably, the method preparing DiaPep277 compound also comprises the step of purifying DiaPep277.Purifying can adopt the routines such as Fast protecting to carry out for the method for purifying DiaPep277.
In the present invention, preferably, purifying is undertaken by recrystallization method, namely by using dissolution with solvents DiaPep277, then separating out the crystal of DiaPep277 and realizing.
In a specific embodiment, the step of purifying DiaPep277, by dissolving DiaPep277 with good solvent, then adds poor solvent and separates out the crystal of DiaPep277 and realize.Herein " good solvent " refer at room temperature and atmospheric pressure 90 % by weight are greater than to the solubleness of DiaPep277, are preferably greater than 95 % by weight, solvent more preferably greater than 99 % by weight; " poor solvent " refers to and at room temperature and atmospheric pressure the solubleness of DiaPep277 is less than to 10 % by weight, is preferably less than 5%, is more preferably less than the solvent of 1%.
Recrystallization preferably carries out at room temperature and atmospheric pressure, also can carry out under the pressure suitably improving or reduce.Recrystallization can use routine to realize for this object all devices.
Preferably, purifying obtains DiaPep277 by RP-HPLC (such as, chromatographic column is anti-phase C18 post) purifying, freeze-drying, and purity is greater than 98%.
Describe the present invention in detail referring to embodiment, should be understood that following embodiment is intended to illustrate, the present invention is not construed as limiting.The compound used in following examples or reagent are buied by commercial sources, or are prepared by ordinary method well known by persons skilled in the art; The laboratory apparatus used is buied by commercial sources.
Embodiment
Embodiment one: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.4mmol/g
Take the Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g, add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 98.7g Fmoc-Asp (OtBu)-OH, 38.9g HOBt, 36.3g DIC, 2.93g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 189.8g pyridine is added and resin 6h closed by 245.04g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-Wang resin, detection substitution degree is 0.402mmol/g.
Embodiment two: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.6mmol/g
Take the Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g, add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 138.2g Fmoc-Asp (OtBu)-OH, 54.5g HOBt, 50.8g DIC, 4.1g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 189.8g pyridine is added and resin 6h closed by 245.04g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-Wang resin, detection substitution degree is 0.603mmol/g.
Embodiment three: substitution degree is the preparation of Fmoc-Asp (the OtBu)-Wang Resin of 0.5mmol/g
Take the Wang resin 120g (120mmol) that substitution degree is 1.0mmol/g, add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 118.4g Fmoc-Asp (OtBu)-OH, 46.7g HOBt, 43.6g DIC, 3.5g DMAP be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add in solid state reaction post, room temperature reaction 2h.Reaction terminates rear DMF and washs 4 times, and DCM washes 2 times.Then 189.8g pyridine is added and resin 6h closed by 245.04g diacetyl oxide mixed solution.Wash 4 times with DMF, after DCM washs 2 times, methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-Wang resin, detection substitution degree is 0.505mmol/g.
Embodiment four: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.4mmol/g
Take the 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 29.6g (72mmol) Fmoc-Asp (OtBu)-OH DMF to dissolve, after adding 13.9g (108mmol) DIPEA activation under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 9.3g (72mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 182mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-CTC resin, detection substitution degree is 0.407mmol/g.
Embodiment five: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.6mmol/g
Take the 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 44.4g (108mmol) Fmoc-Asp (OtBu)-OH DMF to dissolve, after adding 20.9g (162mmol) DIPEA activation under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 13.9g (108mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 182mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-CTC resin, detection substitution degree is 0.601mmol/g.
Embodiment six: substitution degree is the preparation of Fmoc-Asp (the OtBu)-CTC Resin of 0.5mmol/g
Take the 2-CTC resin 150g (150mmol) that substitution degree is 1.0mmol/g, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 37.0g (90mmol) Fmoc-Asp (OtBu)-OH DMF to dissolve, after adding 17.4g (135mmol) DIPEA activation under ice-water bath, add and be above-mentionedly equipped with in the reaction column of resin, after reaction 5min, again add 11.6g (90mmol) DIPEA, room temperature reaction 60min.In reaction solution, add 182mL anhydrous methanol close 30min.Wash 3 times with DMF, DCM washes 3 times, and methyl alcohol collects concentrated draining, and obtain Fmoc-Asp (OtBu)-CTC resin, detection substitution degree is 0.508mmol/g.
The preparation of embodiment seven: DiaPep277-Wang resin
Take Fmoc-Asp (OtBu)-Wang resin 198.0g (100mmol) that substitution degree in embodiment three is 0.505mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH (300mmol; Biochemical purchased from gill), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 130.1g Fmoc-(Hmb) Gly-OH (300mmol), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-(Hmb) Gly-OH, Fmoc-(Hmb) Gly-OH successively.Take 106.1gFmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 451.6g.
The preparation of embodiment eight: DiaPep277-Wang resin
Take Fmoc-Asp (OtBu)-Wang resin 198.0g (100mmol) that substitution degree in embodiment three is 0.505mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH (300mmol), 156.1g PyBOP (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 134.3gFmoc-(Dmb) Gly-OH (300mmol), 156.1g PyBOP (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-(Dmb) Gly-OH, Fmoc-(Dmb) Gly-OH successively.Take 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 455.6g.
The preparation of embodiment nine: DiaPep277-Wang resin
Take Fmoc-Asp (OtBu)-Wang resin 198.0g (100mmol) that substitution degree in embodiment three is 0.505mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH (300mmol), 96.3g TBTU (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 134.3gFmoc-(Dmb) Gly-OH (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-(Dmb) Gly-OH, Fmoc-(Dmb) Gly-OH successively.Take 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 458.1g.
The preparation of embodiment ten: DiaPep277-Wang resin
Take Fmoc-Asp (otBu)-Wang resin 198.0g (100mmol) that substitution degree in embodiment three is 0.505mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH (300mmol), 96.3g TBTU (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 134.3gFmoc-(Dmb) Gly-OH (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 151.4g Fmoc-Gly-(Dmb) Gly-OH (purchased from Merck), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 106.1gFmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 448.3g.
The preparation of embodiment 11: DiaPep277-CTC resin
Take Fmoc-Asp (OtBu)-CTC resin 196.9g (100mmol) that substitution degree in embodiment six is 0.508mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
ue, Mepro)-OH (300mmol), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 130.1g Fmoc-(Hmb) Gly-OH (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 147.2g Fmoc-Gly-(Hmb) Gly-OH (purchased from Merck), 156.1g PyBOP (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 104.3mlDIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 449.3g.
The preparation of embodiment 12: DiaPep277-CTC resin
Take Fmoc-Asp (OtBu)-CTC resin 196.9g (100mmol) that substitution degree in embodiment six is 0.508mmol/g; join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method; resin has color, represents that Fmoc removes.Get 127.7g Fmoc-Glu (OtBu)-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1: 1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Asn (Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH successively.Get 166.7gFmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH (300mmol), 156.1g PyBOP (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Val-OH successively.Take 147.2g Fmoc-Gly-(Hmb) Gly-OH (300mmol), 114.1g HATU (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 134.3gFmoc-(Dmb) Gly-OH, 114.1g HATU (300mmol), 49.0g HOAt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 104.3ml DIPEA (600mmol) activates 3min adds in solid state reaction post, detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Take 106.1g Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, adding under ice-water bath after 56.3ml DIC (360mmol) activates 3min adds in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings, according to DiaPep277 main chain peptide sequence, complete the coupling of Fmoc-Val-OH.Reaction terminates rear methyl alcohol and collects concentrated, and resin vacuum dried overnight, weighs and obtain DiaPep277-Wang resin 450.7g.
The preparation of the thick peptide of embodiment 13: DiaPep277
In Example seven to ten two, the peptide resin of preparation is placed in scission reaction wherein, adds lytic reagent (TFA:TIS: water=90:5:5 (V/V)), stirring at room temperature 2h with the ratio of 10ml/g resin.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide of DiaPep277, and weight yield is 98.6% ~ 103.9%.HPLC purity is greater than 55%.Single impurity is less than 5%.
The preparation of embodiment 14: DiaPep277 essence peptide
To take in embodiment 13 after any 80.0g DiaPep277 thick peptide 4000ml water dissolution, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.2%TFA/ acetonitrile mobile phase purifying, collect object peak cut, obtain purity and be greater than 98.5% smart peptide.Rotary evaporation concentrates, and freeze-drying obtains DiaPep277 essence peptide 23.5g, and RP-HPLC purity is greater than 98.5%.
Claims (10)
1. prepare a method of polypeptide drugs DiaPep277, comprise following steps:
1) using a kind of resin P as carrier, by Fmoc-Asp (OtBu)-OH and resin coupling, Fmoc-Asp (OtBu)-P is obtained;
2) with Fmoc-Asp (OtBu)-P for raw material, adopt the mode of coupling one by one and other amino acid starting materials to carry out solid phase synthesis, obtain DiaPep277-P;
3) by DiaPep277-P cracking, the thick peptide of DiaPep277 is obtained;
Wherein Fmoc is 9-fluorenylmethyloxycarbonyl, is connected or is connected with the nitrogen-atoms on the connected carbon atom of carboxyl with amino,
Wherein step 2) described in coupling one by one mode in Asp-Ser structure adopt synthesis material be Fmoc-Asp (OtBu)-Ser (ψ
me, Mepro)-OH, it is a pseudo proline dipeptides, and make the protected one-tenth five-ring of Serine, structure is as follows:
Wherein the synthesis material of-Gly-Gly-Gly-structure employing is one or both in Fmoc-(Hmb) Gly-OH, Fmoc-(Dmb) Gly-OH, Fmoc-Gly-(Dmb) Gly-OH or Fmoc-Gly-(Hmb) Gly-OH.
2. the method for claim 1, also comprises the step of purifying DiaPep277, and purifying carries out preferably by recrystallization method or HPLC (such as reversed-phase HPLC).
3. the process of claim 1 wherein step 1)-3) carry out all at room temperature and atmospheric pressure; Step 1)-2) linked reaction carry out in solid state reaction post.
4. the process of claim 1 wherein that described resin P is 2-CTC resin or king's resin; The substitution degree of wherein said resin P is 0.2-1.0mmol/g, is preferably 0.3-0.8mmol/g, is more preferably 0.4-0.6mmol/g.
5. the method for claim 1, wherein step 2) described in coupling one by one mode in the synthesis material that adopts of-Gly-Gly-Gly-structure for being selected from the one of Fmoc-(Hmb) Gly-OH and Fmoc-(Dmb) Gly-OH, and be selected from the one of Fmoc-Gly-(Dmb) Gly-OH and Fmoc-Gly-(Hmb) Gly-OH.
6. the process of claim 1 wherein step 1) in coupling agent be HOBt/DIC;
Step 2) described in Fmoc-Asp (OtBu)-Ser (ψ
me, Mepro) coupling system of-OH is the one in HATU/HOAt/DIPEA, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA;
Step 2) described in the coupling system of Gly synthesis material be one in DIC/HOAt, HATU/BOAt/DIPEA or PyBOP/HOBt/DIPEA.
7. the process of claim 1 wherein and the washing of resin and swelling employing DMF are carried out in coupling;
Preferably, by step 1)-2) in carboxyl in material activate, activation is preferably carried out in ice-water bath; Preferred active agent is DIPEA or DIC.
8. the process of claim 1 wherein step 1)-2) in remove Fmoc reagent be DBLK; The deprotection reaction time is 5-30 minute, preferred 10-20 minute.
9. the method for claim 1, wherein step 3) in the mixed solvent of lytic reagent TFA, tri isopropyl silane and water, wherein the proportioning of mixed solvent is: TFA volume ratio is 85-90%, the volume ratio of tri isopropyl silane is 1-5%, the volume ratio of water is 1-5%, and three's sum is 100%;
Preferably, described lytic reagent is TFA:TIS:H
2o=95:5:5 (V:V).
10. the process of claim 1 wherein after amino acid Fmoc-Asp (OtBu)-OH is coupled on vector resin, also comprise the step adding unreacted group excessive in a kind of confining liquid removing resin wherein; When described vector resin is 2-CTC resin, the confining liquid added is preferably methyl alcohol; When described vector resin is king's resin, the confining liquid added is preferably the diacetyl oxide and pyridine that mol ratio is 1:1.
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| CN106916086A (en) * | 2015-12-28 | 2017-07-04 | 深圳翰宇药业股份有限公司 | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal |
| 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 |
| CN112111001A (en) * | 2019-06-19 | 2020-12-22 | 翰宇药业（武汉）有限公司 | Method for synthesizing thymosin T alpha-1 |
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| WO2010117725A2 (en) * | 2009-04-06 | 2010-10-14 | Novetide, Ltd. | Production of peptides containing poly-gly sequences using fmoc chemistry |
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| CN106916086A (en) * | 2015-12-28 | 2017-07-04 | 深圳翰宇药业股份有限公司 | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal |
| 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 |
| CN112111001A (en) * | 2019-06-19 | 2020-12-22 | 翰宇药业（武汉）有限公司 | Method for synthesizing thymosin T alpha-1 |
| CN112111001B (en) * | 2019-06-19 | 2021-10-29 | 翰宇药业（武汉）有限公司 | Method for synthesizing thymosin T alpha-1 |
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