CN102702342A - Method for preparing solid phase nesritide crude product - Google Patents
Method for preparing solid phase nesritide crude product Download PDFInfo
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- CN102702342A CN102702342A CN2012102344052A CN201210234405A CN102702342A CN 102702342 A CN102702342 A CN 102702342A CN 2012102344052 A CN2012102344052 A CN 2012102344052A CN 201210234405 A CN201210234405 A CN 201210234405A CN 102702342 A CN102702342 A CN 102702342A
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Abstract
The invention discloses a method for preparing a solid phase nesritide crude product. The method comprises the steps of: sequentially connecting amino acid with a Fmoc protecting group by taking solid phase synthesis resin as a starting material according to a solid phase synthesis method to obtain a protected 32-peptide resin; sequentially removing the Fmoc protecting group; carrying out a peptide-bounding reaction by using any of TBTU (tetramethyluronium tetrafluoroborate)/HOBt (hydroxbenzotriazole), HBTU (o-benzotriazole-n,n,n,n-tetramethyl-uronium-hexafluorophosphate)/HOBt, BOP (benzotriazol1yloxy)tris(dimethylamino)phosphonium hexafluophosphate)/HOBt, TBTU/HOAt (hydroxyazabenzotriazole), HBTU/HOAt, DIC (diisopropylcarbodiimide)/HOBt or BOP/HOAt as a condensing agent to obtain a protected 32-peptide resin; and synchronously removing a side chain protecting group and cutting peptides to obtain the nesritide crude product. The method has the characteristics that the solid phase synthesis resin is bromine epoxy resin; and a DMF (Dimethyl Formamide)/DCM (Dichloromethane) mixed solvent is used as an activated solvent, and amino acids with the Fmoc protecting group and the condensing agent are dissolved in advance, activated outside a reaction system and then added to the reaction system.
Description
Technical field
The present invention relates to the chemosynthesis of medical polypeptide class bulk drug, relate in particular to a kind of method for preparing solid phase of Nesiritide bullion.
Background technology
((nesiritide/Natrecor) is one type of new vasodilator to Nesiritide, and by the cyclic peptide that 32 amino acid are formed, the 10-26 position links to each other through the S-S key.A kind of recombinant human brain natriuretic peptide or Type B natriuretic peptide (BNP) are identical with interior living hormone in the treatment of acute heart failure, dyspneic treatment when being mainly used in acute mistake compensatory hyperemia DHF.Nesiritide has vein, artery and coronary vasodilator diastole to do in order to load before and after alleviating and under the situation of no direct positive inotropic action, increases cardiac output.Quiet notes Nesiritide can produce useful effect of Hemodynamics on Pathogenesis through promoting sodium excretion and inhibition renin-angiotensin-aldosterone system and sympathetic nervous system in the patient of chronic heart failure.Nesiritide and intravenous nitroglycerin comparison can more effectively improve hematodinamics and have less untoward reaction.
The compound method of Nesiritide has methods such as solid phase method, recombination at present; 200910104860 described preparing methods are starting raw material with the HMPB-AM resin like application number; At condensing agent, connect under the effect of peptide reagent; Connect amino acid successively through solid-phase synthesis, obtain the linear Nesiritide HMPB-AM of side chain full guard resin with Fmoc blocking group.CN200410083873.X has reported a kind of method of solid phase preparation Nesiritide.Yet these methods HMPB-AM resin that provides in the prior art is not conventional resin, and difficulty obtains on market.The HMPB-AM resin causes racemization easily when connecting Fmoc-His (Trt)-OH, impurity is more.
Therefore, this area presses for the preparation method that a kind of effective, easy solid phase synthesis Nesiritide bullion is provided.
Summary of the invention
The present invention aims to provide a kind of preparation method of solid phase synthesis Nesiritide bullion.
The invention provides a kind of method for preparing solid phase of Nesiritide bullion, said method comprises step:
With the solid phase synthesis resin is starting raw material; Method according to solid phase synthesis connects the amino acid with Fmoc blocking group successively; Obtaining three dodecapeptide resins of protection, slough the Fmoc-blocking group therebetween successively, (is TBTU and HOBt with TBTU/HOBt;), any a pair of among HBTU/HOBt (being HBTU and HOBt), BOP/HOBt (being BOP and HOBt), TBTU/HOAt (being TBTU and HOAt), HBTU/HOAt (being HBTU and HOAt), DIC/HOBt (being DIC and HOBt) or the BOP/HOAt (being BOP and HOAt) be condensing agent; Connect reactive polypeptide, behind the three dodecapeptide resins that must protect, take off the side chain protected group synchronously and cut peptide; Obtain the Nesiritide bullion, said step has following characteristics:
The one, said solid phase synthesis resin is a bromination king resin; And
The one, adopting the DMF/DCM mixed solvent is the activation solvent, and dissolving in advance has the amino acid and the condensing agent of Fmoc blocking group, and reaction system is carried out activation outward, joins in the reaction system again.
In the method for preparing solid phase of Nesiritide bullion provided by the invention, connect amino acid successively and comprise the steps: with Fmoc blocking group
(a) king's resin, PBr3, Fmoc-His (Trt)-OH and DIPEA are mixed, reaction obtains bromination king resin;
(b) in bromination king resin; Add deprotection agent and handle, resin washs with DMF then, adds to have the amino acid of Fmoc blocking group, the mixture of condensing agent with DMF/DCM mixed solvent dissolved; Add the deprotection agent processing again after connecing reactive polypeptide; Add the amino acid that has the Fmoc blocking group with DMF/DCM mixed solvent dissolved again, so repeat, obtain Fmoc-Ser
1-Pro
2-Lys
3-Met
4-Val
5-Gln
6-Gly
7-Ser
8-Gly
9-Cys
10-Phe
11-Gly
12-Arg
13-Lys
1 4-Met
15-Asp
16-Arg
17-Ile
18-Ser
19-Ser
20-Ser
21-Ser
22-Gly
23-Leu
24-Gly
25-Cys
26-Lys
27-Val
28-Leu
29-Arg
30-Arg
31-His
32-resin;
Said amino acid with Fmoc blocking group is followed successively by:
(1) Fmoc-His (Trt)-OH or Fmoc-His-OH,
(2) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(3) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(4)Fmoc-Leu-OH、
(5)Fmoc-Val-OH、
(6)Fmoc-Lys(Boc)-OH、
(7) Fmoc-Cys (Trt)-OH or Fmoc-Cys (Acm)-OH,
(8)Fmoc-Gly-OH、
(9)Fmoc-Leu-OH、
(10)Fmoc-Gly-OH、
(11) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,
(12) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(13) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(14) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(15)Fmoc-Ile-OH、
(16) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,,
(17)Fmoc-Asp(OtBu)-OH、
(18)Fmoc-Met-OH、
(19)Fmoc-Lys(Boc)-OH、
(20) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(21)Fmoc-Gly-OH、
(22)Fmoc-Phe-OH、
(23) Fmoc-Cys (Trt)-OH or Fmoc-Cys (Acm)-OH,
(24)Fmoc-Gly-OH、
(25) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,
(26)Fmoc-Gly-OH、
(27)Fmoc-Gln(Trt)-OH、
(28)Fmoc-Val-OH、
(29)Fmoc-Met-OH、
(30)Fmoc-Lys(Boc)-OH、
(31)Fmoc-Pro-OH、
(32) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH.
In aforesaid method, when connecting the Fmoc-amino acid of 22 to 18 residues successively, said condensing agent is DIC/HOBt or DIC/Cl-HOBt and the mixing that is selected from following one or more: TBTU/HOBt, HBTU/HOBt and HATU/HOAt.
In aforesaid method, when connecting the Fmoc-amino acid of 22 to 18 residues successively, setting-up point is 0-5 ℃.
In aforesaid method; When connecting the Fmoc-amino acid of 22 to 18 residues successively; 1.0-3.0 the HOBt or the Cl-HOBt that doubly measure; 1.0-3.0 doubly the DIC reagent of amount is dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent, reacts to add the DIC reagent that 1.0-3.0 doubly measures after 30-60 minute.
In another preference, in the TV of the mixing solutions of DMF and DCM, wherein the percentage composition of DMF is 40-60%.
In aforesaid method, when connecting the Fmoc-Arg (Pbf) of 17,13,8 residues-OH successively, with Pentafluorophenol as activating reagent.
In aforesaid method; When connecting the Fmoc amino acid of 12,9,7,5 residues successively; 2.0-5.0 doubly the Fmoc of amount protects amino acid, the DIPEA reagent that the HOBt/Cl-HOBt that 2.0-5.0 doubly measures, 2.0-5.0 doubly measure; Be dissolved in the DMF of 5ml-10ml/g solid phase synthesis resin and the mixing solutions in the DCM solvent, add HBTU/HATU or the TBTU that 2.0-5.0 doubly measures at 0-5 ℃.
In aforesaid method; When connecting the Fmoc amino acid of 10,6,4 to 1 residue successively; 1.0-3.0 doubly the Fmoc of amount protects amino acid, the DIC reagent that the HOBt/Cl-HOBt that 2.0-6.0 doubly measures, 1.0-3.0 doubly measure; Be dissolved in the DMF of 5ml-10ml/g solid phase synthesis resin and the mixing solutions in the DCM solvent, react 30-60 minute continued and add the DIC reagent that 1.0-3.0 doubly measures.
In view of the above, the invention provides a kind of preparation method of effective, easy solid phase synthesis Nesiritide bullion.
Description of drawings
Fig. 1 is that the GC-MS spectrogram and the HPLC of the Nesiritide bullion 1 that obtained of embodiment 1 detects collection of illustrative plates;
Wherein A is the LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Fig. 2 is that the GC-MS spectrogram and the HPLC of the Nesiritide bullion 2 that obtained of embodiment 2 detects collection of illustrative plates;
Wherein A is the LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Fig. 3 is that the GC-MS spectrogram and the HPLC of the Nesiritide bullion 3 that obtained of embodiment 3 detects collection of illustrative plates;
Wherein A is the LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Embodiment
The contriver is through extensive and deep research; If be surprised to find with regard to the different amino acid residue and adopt specific compound method; Reduce the detrimentally affect of single compound method; Adopt classical solid phase synthesis process also can obtain the bullion of Nesiritide easy, effectively, the product purity that obtains behind the purifying high (＞98%).
The present invention utilizes solid phase synthesis technique that the same insoluble macromolecule resin Pam Resin of carboxyl of the carboxylic terminal protection amino acid His (Dnp) of peptide chain is linked to each other; Progressively extend with excessive activated carboxyl component reaction spreading peptide chain as amino group with this amino acid that is combined on the solid phase carrier again, a kind of new peptide compound method is provided to aminoterminal.
Adopt bromination king resin to synthesize first amino acid His particularly, effectively prevent the His racemization, reduce racemization impurity.
Adopt the DMF/DCM mixed solvent, dissolve amino acid and activating reagent in advance, reaction system is carried out activation outward, joins in the reaction system again.Space steric influence in the swelling of effective control long-chain peptide and the reaction process.
Different residues are adopted different compound methods:
Like the 22-18# residue, employing DIC/HOBt is a condensation reagent, and control reaction 0-5C when middle controlling/monitoring is incomplete, then continues to adopt the HBTU/HATU/TBTU activation method to continue reaction.
For 17#, 13#, 8# Arg residue, then adopting Pentafluorophenol is activating reagent.
And for 12#, 9#, 7# and the less amino acid of 5# molecule adopt the way that increases charging capacity, accomplish reaction fast, reduce the side reaction that causes because of time lengthening.
For 10#, 6#, 4#-1# amino acid adopts the amount that increases activating reagent, impels reaction to accomplish fast.
The implication of employed abbreviation or English full name is listed in the table below among the present invention:
As used herein; " solid phase synthesis " or " polypeptide solid phase synthesis (solid phase peptide synthesis) " is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: with the protected amino acid of amino covalently bound (bonding) on solid phase carrier; Going in the presence of the protective material, the protection base of desamidizate is received on the solid phase carrier first amino acid; Then amino be closed (protection) second amino acid whose carboxyl through activation; Second amino acid that carboxyl is activated forms peptide bond with first amino group of amino acids reaction (condensation) that is connected on solid phase carrier again, on solid phase carrier, has just generated a dipeptides that has the protection base like this; Repeat above-mentioned peptide bond and form reaction, peptide chain is grown, from the C end to the N end until reaching needed peptide chain length; The protection base of last deaminize, the ester bond between hydrolysis peptide chain and the solid phase carrier (cutting) obtains synthetic good peptide.
As used herein, before " Nesiritide bullion " was meant purifying, HPLC purity was at the product of 40-60%.
The structural formula of Nesiritide is suc as formula shown in the I:
H-Ser
1-Pro
2-Lys
3-Met
4-Val
5-Gln
6-Gly
7-Ser
8-Gly
9-Cys
10-Phe
11-Gly
12-Arg
13-Lys
14-Met
15-Asp
16-Arg
17-Ile
18-Ser
19-Ser
20-Ser
21-Ser
22-Gly
23-Leu
24-Gly
25-Cy?Is
26-Lys
27-Val
28-Leu
29-Arg
30-Arg
31-His
32
As used herein, " removing protective material " or " deprotection agent " can be exchanged use, and all being meant can be with the chemical reagent that is connected the amino protecting agent removal on the amino acid; Described amino protecting agent can make well known in the art; Such as but not limited to, Fmoc, Boc; Preferably, the protective material that goes of the present invention is to contain the 3-20v/v% piperidines in its TV, the DMF solution of 0.5-10v/v% bicyclic amidine (DBU) and 0.5-10w/v%1-hydroxybenzotriazole (HOBt).
As used herein; " condensing agent ", " acvator ", " activating reagent " or " condensation acvator " can exchange use; All be to instigate the amino acid whose carboxyl condensation of an amino group of amino acids and another to form the chemical reagent of peptide bond; Can make well known in the art, such as but not limited to, DIC, HATU, TBTU, DIPEA.
As used herein, " cutting agent " be meant with the polypeptide of resin-bonded and the chemical reagent of resin isolation, can make well known in the art, such as but not limited to, contain weakly acidic solution, the HCl solution of TFA.
As used herein; " HPLC purity " is meant the Nesiritide product for preparing; Detect through HPLC,, carry out area normalization method and percentage ratio that the peak area suc as formula compound shown in the I that obtains is occupied in all peak area summations according to resulting chromatogram collection of illustrative plates.
In an instance of the present invention, the preparation method of polypeptide solid phase synthesis Nesiritide of the present invention may further comprise the steps:
The first step, 32# His residue synthetic
With PL wang resin (substitution value 0.5-1.0mmol/g) and 3.0-5.0 equivalent PBr3 stirring reaction; React after 2-3 hour, take out reaction solution, use the DMF washing resin;
Add the normal Fmoc-His of 1.0-3.5 (Trt)-OH again, the DIPEA stirring reaction that 1.0-3.5 doubly measures 10-18 hour, responseless bromine group on the resin; Use the methyl alcohol end-blocking; Obtain resin at last, oven dry, UV ultraviolet determination substitution value is 0.20-0.40mmol/g;
According to classical polypeptide solid phase synthesis process; Bonding amino-acid residue successively; Peptide chain is grown to the N end from the C end, but in bonding amino-acid residue process, adopted different strategies for different amino acid residue contriver; For convenience, the method for condensing of the amino-acid residue that uses similar approach is explained as follows:
Amino-acid residue 31#-23#, 16-14#, 11#, 8#'s is synthetic:
Solid in the reaction kettle is handled 2 times with deprotection agent, after DMF/MeOH washing 1-3 time, added Fmoc protection amino acid (as: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH that 1.0-3.0 doubly measures; Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc)-OH; Fmoc-Cys (Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH; Fmoc-Gly-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Met-OH; Fmoc-Lys (Boc)-OH, Fmoc-Ser (tBu)-OH), HOBt that 1.0-3.0 doubly measures or Cl-HOBt; 1.0-3.0 the DIC reagent of doubly measuring, the DMF and the mixing solutions in the DCM solvent that are dissolved in the 5ml-10ml/g resin (protect amino acid, HOBt, DIC to be dissolved among DMF and the DCM mixing solutions of formation Fmoc.As follows).React after 30-60 minute, add the DIC reagent that 1.0-3.0 doubly measures once more, normal temperature is reaction down.Whether middle detection examination reaction is complete.
The DMF blending ratio that accounts for DMF/DCM solution should be 40%-60% here.
Amino-acid residue 22-18#'s is synthetic:
Solid in the reaction kettle is handled 2 times with deprotection agent, after DMF/MeOH washing 1-3 time, added the Fmoc protection amino acid (as: Fmoc-Ser (tBu)-OH that 1.0-3.0 doubly measures; Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-OH; Fmoc-Ile-OH); 1.0-3.0 the HOBt or the Cl-HOBt that doubly measure, the DIC reagent that 1.0-3.0 doubly measures is dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent.The temperature of control condensation solution is 0-5C.Condensation solution is poured in the reaction kettle, reacted after 30-60 minute, add the DIC reagent that 1.0-3.0 doubly measures once more, normal temperature reaction down spends the night.Whether middle detection examination reaction is complete.
Amino-acid residue 17#, 13# 8#, synthetic:
Solid in the reaction kettle is handled 2 times with deprotection agent; After DMF/MeOH washing 1-3 time; Fmoc-Arg (Pbf)-OH that adding 1.0-3.0 doubly measures protects amino acid; 1.0-3.0 the PFP that doubly measures, the DIC reagent that 1.0-3.0 doubly measures is dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent.Normal temperature reaction down spends the night.Whether middle detection examination reaction is complete.
Amino residue 12#, 9#, 7#, 5#'s is synthetic:
Solid in the reaction kettle is handled 2 times with deprotection agent, after DMF/MeOH washing 1-3 time, added the Fmoc protection amino acid (Fmoc-Gly-OH that 2.0-5.0 doubly measures; Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH); 2.0-5.0 the HOBt/Cl-HOBT that doubly measures, the DIPEA reagent that 2.0-5.0 doubly measures is dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent; Controlled temperature adds HBTU/HATU or TBTU that 2.0-5.0 doubly measures under the 0-5C condition.Normal temperature is reaction down.Whether middle detection examination reaction is complete.
Solid in the reaction kettle is handled 2 times with deprotection agent, after DMF/MeOH washing 1-3 time, added the Fmoc protection amino acid (Fmoc-Cys (Trt)-OH that 1.0-3.0 doubly measures; Fmoc-Glu (Trt)-OH, Fmoc-Met-OH, Fmoc-Lys (Boc)-OH; Fmoc-Pro-OH, Fmoc-Ser (tBu)-OH), the HOBt/Cl-HOBT that 2.0-6.0 doubly measures; 1.0-3.0 doubly the DIC reagent of amount is dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent, and mixed solvent is poured in the reaction kettle.React after 30-60 minute, continue to add the DIC reagent that 1.0-3.0 doubly measures.Normal temperature reaction down spends the night.Whether middle detection examination reaction is complete.
What whether fully the detection examination reacted employing among the present invention is ninhydrin.Relevant ninhydrin (Kaiser), ninhydrin test (Ninhydrin test); And monitoring method can be referring to document VIRENDER K.SARIN; Et al. " Quantitative Monitoring of Solid-Phase Peptide Synthesis by the Ninhydrin Reaction " ANALYTICAL BIOCHEMISTRY 117; 147-157 (1981), E.KAISER; Et al. " Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides " SHORT COMMUNICATIONS595-598 (Received October 28,1969) and THORKILD CHRISTENSEN " A Qualitative Test for Monitoring Coupling Completeness in Solid Phase Peptide Synthesis Using Chloranil " Acta Chemica Scandinavica B33 (1979) 763-766.
The above-mentioned characteristic that the present invention mentions, or the characteristic that embodiment mentions can arbitrary combination.All characteristics that this case specification sheets is disclosed can with any composition forms and usefulness, each characteristic that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, the characteristic that is disclosed to be merely the general example of equalization or similar features.
Major advantage of the present invention is:
1, preparing method's step provided by the invention is simple, easy to operate, and cost is controlled.
2, preparation method provided by the invention adopts classical solid phase synthesis process, can make full use of existing installation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The HPLC method that relates among the following embodiment is following:
Stationary phase: 30 ℃ of C18 post column temperatures
Moving phase: A:0.1% trifluoroacetic acid (TFA)/water
B:0.1% trifluoroacetic acid (TFA)/acetonitrile
Gradient elution: 0-50 minute, A from 90% to 65%, and B from 10% to 35%
Flow velocity: 1.0ml/min
Detect wavelength: 215nm
The mass spectrum that relates among the following embodiment (LC-MS) detection method is following:
Moving phase: 80% methanol
Flow velocity: 0.3ml/min
Compression gas flow: Sheath gas flow rate (arb): 50
Aux?gas?flow?rate(arb):10
Scan?ranges(M/Z):150.00-2000.00
Embodiment 1
Synthetic Nesiritide bullion 1
Load Fmoc-His (Trt)-OH
With 10 gram PL Wang resins (substitution value 0.7-0.9mmol/g) and 2.5 equivalent PBr
3Stirring reaction 2 hours; Take out reaction solution then, use the DMF washing resin, again with 3.0 normal Fmoc-His (Trt)-OH; 3.0 normal DIPEA stirring reaction 16 hours; Responseless bromine group is used the methyl alcohol end-blocking on the resin, obtains resin 13 grams at last, and UV ultraviolet determination substitution value is 0.26mmol/g.
Deprotection
With the double deprotection of 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v), use DMF and methanol wash resin then respectively, thoroughly drain back Kaiser test detection method monitoring Fmoc and slough degree.
Amino acid condensation
Condensation 31#-23#, 16-14#, 11#, during 8# amino acid, with the Fmoc protection amino acid that adds 2.0 times of amounts, the HOBt of 2.0 times of amounts, the DIC reagent of 2.0 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 5ml/g resin.React after 30 minutes, add the DIC reagent of 2.0 times of amounts once more.
During condensation 22-18# amino acid, the Fmoc of 2.0 times of amounts protects amino acid, the Cl-HOBt of 2.0 times of amounts, and the DIC reagent of 2.0 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 5ml/g resin.The temperature of control condensation solution is 0-3C.Condensation solution is poured in the reaction kettle, reacted after 30 minutes, add the DIC reagent of 2.0 times of amounts once more, reaction is spent the night.
During 8,13 of condensations and 17 s' Fmoc-Arg (Pbf)-OH; In reactor drum, add 3.0 equivalent Fmoc-Arg (Pbf)-OH/3.0 equivalent Pentafluorophenol (3.0 equivalents are with respect to Fmoc-His (Trt)-PL Wang resin)/DMF solution; Add 2.0 equivalent DIC subsequently, stirring reaction 36-48 hour at least.If amino acid condensation is incomplete, add 0.5-1.0 equivalent Fmoc-AA-OH in reactor drum, continue stirring reaction and spend the night.
Condensation 12#, 9#, 7# is during 5# amino acid; The Fmoc protection amino acid that adds 4.0 times of amounts, the Cl-HOBT of 4.0 times of amounts, the DIPEA reagent of 4.0 times of amounts; Be dissolved among the DMF and DCM (1:1) mixed solvent of 5ml/g resin, controlled temperature adds the HBTU of 2.5 times of amounts under the 0-5C condition.Normal temperature is reaction down.Whether middle detection examination reaction is complete.
Cutting
, stir and rise again as cutting liquid with refrigerative TFA/TIS/EDT/ water/Thioanisole (90%v/3%v/3%v/2%v/2%v) to 25 ℃ ± 5 ℃ reactions 2-3 hour.Concentrate good filtrating and pour sedimentation in the good MTBE (MTBE) of cooling into, cooling was left standstill crystallization 0.5-1.5 hour.Filter or the centrifugal filter cake that obtains, with the thorough washing leaching cake of refrigerative MTBE three times.The bullion polypeptide is transferred in the vacuum drier vacuum-drying at least 12 hours.
The result sees Fig. 1.Detection obtains bullion purity 59%. yields 100.85%.
Load Fmoc-His (Trt)-OH
With 10 gram PL Wang resins (substitution value 0.6-0.8mmol/g) and 4.0 equivalent PBr
3Stirring reaction 2 hours; Take out reaction solution then, use the DMF washing resin, again with 3.0 normal Fmoc-His (Trt)-OH; 3.0 normal DIPEA stirring reaction 15 hours; Responseless bromine is used the methyl alcohol end-blocking on the resin, obtains resin 13 grams at last, and UV ultraviolet determination substitution value is 0.26mmol/g.
Deprotection
With the double deprotection of 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v), use DMF and methanol wash resin respectively, thoroughly drain back Kaiser test detection method monitoring Fmoc and slough degree.
Amino acid condensation
Condensation 31#-23#, 16-14#, 11#, during 8# amino acid, with the Fmoc protection amino acid that adds 1.5 times of amounts, the HOBt of 1.5 times of amounts, the DIC reagent of 1.5 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 5ml/g resin.React after 30 minutes, add the DIC reagent of 1.5 times of amounts once more.
During condensation 22-18# amino acid, the Fmoc of 1.5 times of amounts protects amino acid, the HOBt of 1.5 times of amounts, and the DIC reagent of 1.5 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 6ml/g resin.The temperature of control condensation solution is 0-3C.Condensation solution is poured in the reaction kettle, reacted after 30 minutes, add the DIC reagent of 1.5 times of amounts once more, reaction is spent the night.
During 8,13 of condensations and 17 s' Fmoc-Arg (Pbf)-OH; In reactor drum, add 4.0 equivalent Fmoc-Arg (Pbf)-OH/4.0 equivalent Pentafluorophenol (4.0 equivalents are with respect to Fmoc-His (Trt)-PL Wang resin)/DMF solution; Add 3.0 equivalent DIC subsequently, stirring reaction 36-48 hour at least.
Condensation 12#, 9#, 7# is during 5# amino acid; The Fmoc protection amino acid that adds 4.0 times of amounts, the HOBT of 4.0 times of amounts, the DIPEA reagent of 4.0 times of amounts; Be dissolved among the DMF and DCM (1:1) mixed solvent of 6ml/g resin, controlled temperature adds the HBTU of 3.0 times of amounts under the 0-5C condition.Normal temperature is reaction down.Whether middle detection examination reaction is complete.
Cutting
, stir and rise again as cutting liquid with refrigerative TFA/TIS/EDT/ water/Thioanisole (95%v/1.5%v/1.5%v/1%v/1%v) to 25 ℃ ± 5 ℃ reactions 2-3 hour.Concentrate good filtrating and pour sedimentation in the good MTBE (MTBE) of cooling into, cooling was left standstill crystallization 0.5-1.5 hour.Filter or the centrifugal filter cake that obtains, with the thorough washing leaching cake of refrigerative MTBE three times.The bullion polypeptide is transferred in the vacuum drier vacuum-drying at least 12 hours.
The result sees Fig. 2.Purity 54%, yield 111.20%.
Embodiment 3
Synthetic Nesiritide bullion 3
Load Fmoc-His (Trt)-OH
With 159 gram PL Wang resins (substitution value 0.7-0.9mmol/g) and 4.5 equivalent PBr
3Stirring reaction 2 hours; Take out reaction solution then, use the DMF washing resin, again with 1.0 normal Fmoc-His (Trt)-OH; 1.0 normal DIPEA stirring reaction 18 hours; Responseless bromine is used the methyl alcohol end-blocking on the resin, obtains resin 223 grams at last, and UV ultraviolet determination substitution value is 0.33mmol/g.
Deprotection
With the double deprotection of 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v), use DMF and methanol wash resin then respectively, thoroughly drain back Kaiser test detection method monitoring Fmoc and slough degree.
Amino acid condensation
Condensation 31#-23#, 16-14#, 11#, during 8# amino acid, with the Fmoc protection amino acid that adds 3.0 times of amounts, the Cl-HOBt of 3.0 times of amounts, the DIC reagent of 3.0 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 8ml/g resin.React after 30 minutes, add the DIC reagent of 3.0 times of amounts once more.
During condensation 22-18# amino acid, the Fmoc of 3.0 times of amounts protects amino acid, the Cl-HOBt of 3.0 times of amounts, and the DIC reagent of 3.0 times of amounts is dissolved among the DMF and DCM (1:1) mixed solvent of 8ml/g resin.The temperature of control condensation solution is 0-3C.Condensation solution is poured in the reaction kettle, reacted after 30 minutes, add the DIC reagent of 3.0 times of amounts once more, normal-temperature reaction.
During 8,13 of condensations and 17 s' Fmoc-Arg (Pbf)-OH; In reactor drum, add 5.0 equivalent Fmoc-Arg (Pbf)-OH/5.0 equivalent Pentafluorophenol (5.0 equivalents are with respect to Fmoc-His (Trt)-PL Wang resin)/DMF solution; Add 5.0 equivalent DIC subsequently, stirring reaction 36-48 hour at least.
Condensation 12#, 9#, 7# is during 5# amino acid; The Fmoc protection amino acid that adds 3.0 times of amounts, the Cl-HOBT of 3.0 times of amounts, the DIPEA reagent of 3.0 times of amounts; Be dissolved among the DMF and DCM (1:1) mixed solvent of 5ml/g resin, controlled temperature adds the HBTU of 3.0 times of amounts under the 0-5C condition.Normal temperature is reaction down.
Cutting
, stir and rise again as cutting liquid with refrigerative TFA/TIS/EDT/ water/Thioanisole (80%v/10%v/2.5%v/2.5%v/5%v) to 25 ℃ ± 5 ℃ reactions 2-3 hour.Concentrate good filtrating and pour sedimentation in the good MTBE (MTBE) of cooling into, cooling was left standstill crystallization 0.5-1.5 hour.Filter or the centrifugal filter cake that obtains, with the thorough washing leaching cake of refrigerative MTBE three times.The bullion polypeptide is transferred in the vacuum drier vacuum-drying at least 12 hours.
The result sees Fig. 3.Purity 55%, yield 96.4%.
The above is merely preferred embodiment of the present invention; Be not in order to limit essence technology contents scope of the present invention; Essence technology contents of the present invention is broadly to be defined in the claim scope of application, and if any technological entity or method that other people accomplish are defined identical with the claim scope of application; Also or a kind of change of equivalence, all will be regarded as and be covered by among this claim scope.
Claims (9)
1. the method for preparing solid phase of a Nesiritide bullion, said method comprises step:
With the solid phase synthesis resin is starting raw material; Method according to solid phase synthesis connects the amino acid with Fmoc blocking group successively, obtains three dodecapeptide resins of protection, sloughs the Fmoc-blocking group therebetween successively; With TBTU/HOBt (is TBTU and HOBt;), any a pair of among HBTU/HOBt (being HBTU and HOBt), BOP/HOBt (being BOP and HOBt), TBTU/HOAt (being TBTU and HOAt), HBTU/HOAt (being HBTU and HOAt), DIC/HOBt (being DIC and HOBt) or the BOP/HOAt (being BOP and HOAt) be to connect reactive polypeptide, behind the three dodecapeptide resins that must protect by condensing agent; Take off the side chain protected group synchronously and cut peptide; Obtain the Nesiritide bullion, it is characterized in that said step has following characteristics:
The one, said solid phase synthesis resin is a bromination king resin; And
The one, adopting the DMF/DCM mixed solvent is the activation solvent, and dissolving in advance has the amino acid and the condensing agent of Fmoc blocking group, and reaction system is carried out activation outward, joins in the reaction system again.
2. preparation method as claimed in claim 1 is characterized in that, connects the amino acid with Fmoc blocking group successively and comprises the steps:
(a) with king's resin, PBr
3, Fmoc-His (Trt)-OH and DIPEA mix, reaction obtains bromination king resin;
(b) in bromination king resin; Add deprotection agent and handle, resin washs with DMF then, adds to have the amino acid of Fmoc blocking group, the mixture of condensing agent with DMF/DCM mixed solvent dissolved; Add the deprotection agent processing again after connecing reactive polypeptide; Add the amino acid that has the Fmoc blocking group with DMF/DCM mixed solvent dissolved again, so repeat, obtain Fmoc-Ser
1-Pro
2-Lys
3-Met
4-Val
5-Gln
6-Gly
7-Ser
8-Gly
9-Cys
10-Phe
11-Gly
12-Arg
13-Lys
1 4-Met
15-Asp
16-Arg
17-Ile
18-Ser
19-Ser
20-Ser
21-Ser
22-Gly
23-Leu
24-Gly
25-Cys
26-Lys
27-Val
28-Leu
29-Arg
30-Arg
31-His
32-resin;
Said amino acid with Fmoc blocking group is followed successively by:
(1) Fmoc-His (Trt)-OH or Fmoc-His-OH,
(2) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(3) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(4)Fmoc-Leu-OH、
(5)Fmoc-Val-OH、
(6)Fmoc-Lys(Boc)-OH、
(7) Fmoc-Cys (Trt)-OH or Fmoc-Cys (Acm)-OH,
(8)Fmoc-Gly-OH、
(9)Fmoc-Leu-OH、
(10)Fmoc-Gly-OH、
(11) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,
(12) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(13) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(14) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,,
(15)Fmoc-Ile-OH、
(16) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,,
(17)Fmoc-Asp(OtBu)-OH、
(18)Fmoc-Met-OH、
(19)Fmoc-Lys(Boc)-OH、
(20) Fmoc-Arg (Pbf)-OH or Fmoc-Arg (HCl)-OH,
(21)Fmoc-Gly-OH、
(22)Fmoc-Phe-OH、
(23) Fmoc-Cys (Trt)-OH or Fmoc-Cys (Acm)-OH,
(24)Fmoc-Gly-OH、
(25) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH,
(26)Fmoc-Gly-OH、
(27)Fmoc-Gln(Trt)-OH、
(28)Fmoc-Val-OH、
(29)Fmoc-Met-OH、
(30)Fmoc-Lys(Boc)-OH、
(31)Fmoc-Pro-OH、
(32) Fmoc-Ser (tBu)-OH or Fmoc-Ser-OH.
3. preparation method as claimed in claim 2; It is characterized in that; When connecting the Fmoc-amino acid of 22 to 18 residues successively, said condensing agent is DIC/HOBt or DIC/Cl-HOBt and the mixing that is selected from following one or more: TBTU/HOBt, HBTU/HOBt and HATU/HOAt.
4. preparation method as claimed in claim 3 is characterized in that, when connecting the Fmoc-amino acid of 22 to 18 residues successively, setting-up point is 0-5 ℃.
5. like claim 3 or 4 described preparing methods; It is characterized in that; When connecting the Fmoc-amino acid of 22 to 18 residues successively, the DIC reagent that HOBt that 1.0-3.0 doubly measures or Cl-HOBt, 1.0-3.0 doubly measure; Be dissolved in the DMF of 5ml-10ml/g resin and the mixing solutions in the DCM solvent, react the DIC reagent that adding 1.0-3.0 doubly measures after 30-60 minute.
6. preparation method as claimed in claim 5 is characterized in that, in the TV of the mixing solutions of DMF and DCM, wherein the percentage composition of DMF is 40-60%.
7. preparation method as claimed in claim 2 is characterized in that, when connecting the Fmoc-Arg (Pbf) of 17,13,8 residues-OH successively, with Pentafluorophenol as activating reagent.
8. preparation method as claimed in claim 2; It is characterized in that, when connecting the Fmoc amino acid of 12,9,7,5 residues successively, the Fmoc protection amino acid that 2.0-5.0 doubly measures; 2.0-5.0 the HOBt/Cl-HOBt that doubly measures; 2.0-5.0 doubly the DIPEA reagent of amount is dissolved in the DMF of 5ml-10ml/g solid phase synthesis resin and the mixing solutions in the DCM solvent, adds HBTU/HATU or the TBTU that 2.0-5.0 doubly measures at 0-5 ℃.
9. preparation method as claimed in claim 2; It is characterized in that, when connecting the Fmoc amino acid of 10,6,4 to 1 residue successively, the Fmoc protection amino acid that 1.0-3.0 doubly measures; 2.0-6.0 the HOBt/Cl-HOBt that doubly measures; 1.0-3.0 doubly the DIC reagent of amount is dissolved in the DMF of 5ml-10ml/g solid phase synthesis resin and the mixing solutions in the DCM solvent, reacts 30-60 minute continued and adds the DIC reagent that 1.0-3.0 doubly measures.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463080A (en) * | 2009-01-09 | 2009-06-24 | 深圳市翰宇药业有限公司 | Method for purifying nesiritide |
| CN101519444A (en) * | 2009-01-09 | 2009-09-02 | 深圳市翰宇药业有限公司 | Method for preparing Nesiritide |
| CN102250235A (en) * | 2011-06-23 | 2011-11-23 | 成都圣诺科技发展有限公司 | Preparation method of nesiritide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463080A (en) * | 2009-01-09 | 2009-06-24 | 深圳市翰宇药业有限公司 | Method for purifying nesiritide |
| CN101519444A (en) * | 2009-01-09 | 2009-09-02 | 深圳市翰宇药业有限公司 | Method for preparing Nesiritide |
| CN102250235A (en) * | 2011-06-23 | 2011-11-23 | 成都圣诺科技发展有限公司 | Preparation method of nesiritide |
Non-Patent Citations (3)
| Title |
|---|
| M. MERGLER ET AL: "peptide synthesis by a combination of solid-phase and solution methods III resin derivatives allowing minimum-racemization coupling of N-protected acids", 《TETRAHEDRON LETTERS》, 31 December 1989 (1989-12-31), pages 6741 - 6744 * |
| 张政朴等: "《反应性与功能性高分子材料》", 28 February 2005, article "反应性与功能性高分子材料", pages: 35 * |
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