CN102702342B - A kind of method for preparing solid phase of Nesiritide crude product - Google Patents
A kind of method for preparing solid phase of Nesiritide crude product Download PDFInfo
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- CN102702342B CN102702342B CN201210234405.2A CN201210234405A CN102702342B CN 102702342 B CN102702342 B CN 102702342B CN 201210234405 A CN201210234405 A CN 201210234405A CN 102702342 B CN102702342 B CN 102702342B
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Abstract
The invention discloses a kind of method for preparing solid phase of Nesiritide crude product.Described method comprises step: with solid phase synthesis resin for starting raw material, the amino acid with Fmoc blocking group is connected successively according to the method for solid phase synthesis, obtain three dodecapeptide resins of protection, slough Fmoc-blocking group successively therebetween, with TBTU/HOBt, HBTU/HOBt, BOP/HOBt, TBTU/HOAt, HBTU/HOAt, any one in DIC/HOBt or BOP/HOAt is condensing agent, carry out connecing reactive polypeptide, after the three dodecapeptide resins that must protect, synchronously carry out de-side chain protected group and cut peptide, obtain Nesiritide crude product, described step has following feature: one be described solid phase synthesis resin is bromination king resin, and one is adopt DMF/DCM mixed solvent to be activating solvent, dissolves amino acid and the condensing agent with Fmoc blocking group in advance, activates, then join in reaction system outside reaction system.
Description
Technical field
The present invention relates to the chemosynthesis of medical polypeptide class bulk drug, particularly relate to a kind of method for preparing solid phase of Nesiritide crude product.
Background technology
((nesiritide/Natrecor) is the vasodilator that a class is new to Nesiritide, the cyclic peptide be made up of 32 amino acid, and 10-26 position is connected by S-S key.In the treatment of acute heart failure, a kind of rhBNP or B-typeNatriuretic Peptide (BNP), identical with interior raw hormone, dyspneic treatment when being mainly used in acute mistake compensatory hyperemia DHF.Nesiritide has vein, artery and coronary vasodilatation to do in order to alleviate front and back load and when increasing cardiac output without when direct positive inotropic action.In the patient of chronic heart failure, quiet note Nesiritide produces useful effect of Hemodynamics on Pathogenesis by promoting sodium excretion and suppression renin-angiotensin-aldosterone system and sympathetic nervous system.Nesiritide compares with intravenous nitroglycerin and more effectively can improve Hemodynamics and have less untoward reaction.
The synthetic method of current Nesiritide has the method such as solid phase method, gene recombination; preparation method as described in application number 200910104860 with HMPB-AM resin for starting raw material; condensing agent, connect peptide reagent effect under; connected the amino acid with Fmoc blocking group by solid-phase synthesis successively, obtain side chain full guard linear Nesiritide HMPB-AM resin.CN200410083873.X reports a kind of method that solid phase prepares Nesiritide.But these the method HMPB-AM resins provided in prior art are not usual resins, more difficultly commercially to obtain.HMPB-AM resin easily causes racemization when connecting Fmoc-His (Trt)-OH, and impurity is more.
Therefore, this area is in the urgent need to providing a kind of preparation method of effective, easy solid phase synthesis Nesiritide crude product.
Summary of the invention
The present invention aims to provide a kind of preparation method of solid phase synthesis Nesiritide crude product.
The invention provides a kind of method for preparing solid phase of Nesiritide crude product, described method comprises step:
With solid phase synthesis resin for starting raw material, the amino acid with Fmoc blocking group is connected successively according to the method for solid phase synthesis, obtain three dodecapeptide resins of protection, slough Fmoc-blocking group successively therebetween, with TBTU/HOBt (i.e. TBTU and HOBt, ), HBTU/HOBt (i.e. HBTU and HOBt), BOP/HOBt (i.e. BOP and HOBt), TBTU/HOAt (i.e. TBTU and HOAt), HBTU/HOAt (i.e. HBTU and HOAt), any in DIC/HOBt (i.e. DIC and HOBt) or BOP/HOAt (i.e. BOP and HOAt) is for a pair condensing agent, carry out connecing reactive polypeptide, after the three dodecapeptide resins that must protect, synchronously carry out de-side chain protected group and cut peptide, obtain Nesiritide crude product, described step has following feature:
One be described solid phase synthesis resin is bromination king resin; And
One is adopt DMF/DCM mixed solvent to be activating solvent, dissolves amino acid and the condensing agent with Fmoc blocking group in advance, activates, then join in reaction system outside reaction system.
In the method for preparing solid phase of Nesiritide crude product provided by the invention, connect the amino acid with Fmoc blocking group successively and comprise the steps:
A () is by king's resin, PBr
3, Fmoc-His (Trt)-OH and DIPEA mixing, be obtained by reacting bromination king resin;
B () is in bromination king resin; add deprotection agent to process; then resin DMF washs; add with DMF/DCM mixed solvent dissolve there is the amino acid of Fmoc blocking group, the mixture of condensing agent; deprotection agent process is added again after connecing reactive polypeptide; add the amino acid with Fmoc blocking group dissolved with DMF/DCM mixed solvent 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;
The 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 the above-mentioned methods, when connecting the Fmoc-amino acid of 22 to 18 residues successively, described condensing agent is DIC/HOBt or DIC/Cl-HOBt and is selected from one or more following mixing: TBTU/HOBt, HBTU/HOBt and HATU/HOAt.
In the above-mentioned methods, when connecting the Fmoc-amino acid of 22 to 18 residues successively, setting-up point is 0-5 DEG C.
In the above-mentioned methods, when connecting the Fmoc-amino acid of 22 to 18 residues successively, 1.0-3.0 HOBt or Cl-HOBt of times amount, the DIC reagent of 1.0-3.0 times amount, be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin, react the DIC reagent adding 1.0-3.0 times amount after 30-60 minute.
In another preference, with the entire volume of the mixing solutions of DMF and DCM, wherein the percentage composition of DMF is 40-60%.
In the above-mentioned methods, when connecting Fmoc-Arg (the Pbf)-OH of 17,13 residues successively, using Pentafluorophenol as activating reagent.
In the above-mentioned methods; when connecting the Fmoc amino acid of 12,9,7,5 residues successively; the Fmoc protected amino acid of 2.0-5.0 times amount; 2.0-5.0 the HOBt/Cl-HOBt of times amount; the DIPEA reagent of 2.0-5.0 times amount; be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g solid phase synthesis resin, add HBTU/HATU or TBTU of 2.0-5.0 times amount at 0-5 DEG C.
In the above-mentioned methods; when connecting the Fmoc amino acid of 10,6,4 to 1 residues successively; the Fmoc protected amino acid of 1.0-3.0 times amount; 2.0-6.0 the HOBt/Cl-HOBt of times amount; the DIC reagent of 1.0-3.0 times amount; be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g solid phase synthesis resin, react and continue the DIC reagent adding 1.0-3.0 times amount after 30-60 minute.
Accordingly, the invention provides a kind of preparation method of effective, easy solid phase synthesis Nesiritide crude product.
Accompanying drawing explanation
Fig. 1 is GC-MS spectrogram and the HPLC detection collection of illustrative plates of the Nesiritide crude product 1 that embodiment 1 obtains;
Wherein A is LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Fig. 2 is GC-MS spectrogram and the HPLC detection collection of illustrative plates of the Nesiritide crude product 2 that embodiment 2 obtains;
Wherein A is LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Fig. 3 is GC-MS spectrogram and the HPLC detection collection of illustrative plates of the Nesiritide crude product 3 that embodiment 3 obtains;
Wherein A is LC-MS spectrogram, and B is that HPLC detects collection of illustrative plates.
Embodiment
Contriver is through extensive and deep research, if be surprised to find just different amino-acid residue to adopt specific synthetic method, reduce the detrimentally affect of single synthetic method, adopt classical solid phase synthesis process also easy, effectively can obtain the crude product of Nesiritide, the product purity obtained after purifying high (> 98%).
Adopt bromination king's resins synthesis first amino acid His particularly, effectively prevent His racemization, reduce racemization impurity.
Adopt DMF/DCM mixed solvent, Dissolved Amino Acids and activating reagent, activate outside reaction system in advance, then join in reaction system.Steric interference in the swelling and reaction process of effective control long-chain peptide.
Different synthetic method is adopted to different residue:
As 22-18# residue, employing DIC/HOBt is condensation reagent, controls reaction 0-5C, when middle controlling/monitoring is incomplete, then continues to adopt HBTU/HATU/TBTU activation method to continue reaction.
For 17#, 13#Arg residue, then Pentafluorophenol is adopted to be activating reagent.
And for the less amino acid of 12#, 9#, 7# and 5# molecule, adopt the way increasing charging capacity, complete reaction fast, reduce the side reaction caused because of time lengthening.
For 10#, 6#, 4#-1# amino acid, adopt the amount increasing activating reagent, impel reaction to complete fast.
The implication of the abbreviation used in the present invention or English full name is listed in the table below:
As used herein, " solid phase synthesis " or " Solid-phase synthesis peptides (solid phase peptide synthesis) " is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: by a protected amino acid of amino covalently bound (bonding) on solid phase carrier; Under going protective material to exist, the protecting group of desamidizate, makes first amino acid receive on solid phase carrier; Then amino is closed second amino acid whose carboxyl of (protection) by activation, by second amino acid activating, amino acid whose amino reacts (condensation) and forms peptide bond carboxyl with first that is connected on solid phase carrier again, just generates a dipeptides with protecting group like this on solid phase carrier; Repeat above-mentioned peptide bond forming reactions, make peptide chain from C end to the growth of N end, until reach required peptide chain length; The protecting group of last deaminize, the ester bond (cutting) between hydrolysis peptide chain and solid phase carrier, obtains synthetic peptide.
As used herein, before " Nesiritide crude product " refers to purifying, HPLC purity is at the product of 40-60%.
The structural formula of Nesiritide is such as formula shown in 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 exchange use, all refers to the chemical reagent amino protecting agent be connected on amino acid can removed, 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, with its entire volume, is containing 3-20v/v% piperidines, 0.5-10v/v% bicyclic amidine (DBU), and the DMF solution of 0.5-10w/v%1-hydroxybenzotriazole (HOBt).
As used herein, " condensing agent ", " activator ", " activating reagent " or " condensation activator " can exchange use, it is all the chemical reagent of instigating an amino acid whose amino and another amino acid whose carboxyl condensation to form peptide bond, can make well known in the art, such as but not limited to, DIC, HATU, TBTU, DIPEA.
As used herein, " cutting agent " refers to the chemical reagent of polypeptide by same resin-bonded and resin isolation, can make well known in the art, such as but not limited to, the weakly acidic solution containing TFA, HCl solution.
As used herein, " HPLC purity " refers to the Nesiritide product that will prepare, detect through HPLC, according to obtained chromatogram collection of illustrative plates, carry out area normalization method and the percentage ratio occupied in all peak area summations such as formula the peak area of compound shown in I that obtains.
In an example of the present invention, the preparation method of Solid-phase synthesis peptides Nesiritide of the present invention comprises the following steps:
The first step, the synthesis of 32#His residue
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 DMF washing resin;
Add the DIPEA stirring reaction 10-18 hour of Fmoc-His (Trt)-OH, the 1.0-3.5 times amount of 1.0-3.5 equivalent again, responseless bromine group on resin, use methyl alcohol end-blocking, finally obtain resin, dry, UV ultraviolet determination substitution value is 0.20-0.40mmol/g;
According to classical Solid-phase synthesis peptides method, bonding amino-acid residue successively, make peptide chain from C end to the growth of N end, but in bonding amino-acid residue process, different strategies be have employed for different amino-acid residue contrivers, for convenience, the method for condensing of the amino-acid residue using similar approach is expressed as follows:
Amino-acid residue 31#-23#, the synthesis of 16-14#, 11#, 8#:
By the deprotection agent process 2 times of the solid in reactor, after DMF/MeOH washing 1-3 time, add Fmoc protected amino acid (as: Fmoc-Arg (the Pbf)-OH of 1.0-3.0 times amount, Fmoc-Arg (Pbf)-OH, 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), 1.0-3.0 HOBt or Cl-HOBt of times amount, the DIC reagent of 1.0-3.0 times amount, be dissolved in mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin (by Fmoc protected amino acid, HOBt, DIC is dissolved in DMF and DCM, the mixing solutions formed.As follows).React after 30-60 minute, again add the DIC reagent of 1.0-3.0 times amount, react under normal temperature.Whether middle control test reaction is complete.
The blending ratio that DMF accounts for DMF/DCM solution herein should be 40%-60%.
The synthesis of amino-acid residue 22-18#:
By the deprotection agent process 2 times of the solid in reactor; after DMF/MeOH washing 1-3 time; add Fmoc protected amino acid (as: Fmoc-Ser (the tBu)-OH of 1.0-3.0 times amount; Fmoc-Ser (tBu)-OH; Fmoc-Ser (tBu)-OH; Fmoc-Ser (tBu)-OH; Fmoc-Ile-OH); 1.0-3.0 HOBt or Cl-HOBt of times amount; the DIC reagent of 1.0-3.0 times amount, is dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin.The temperature controlling condensation solution is 0-5C.Pour in reactor by condensation solution, react after 30-60 minute, again add the DIC reagent of 1.0-3.0 times amount, under normal temperature, reaction is spent the night.Whether middle control test reaction is complete.
Amino-acid residue 17#, 13#, synthesis:
By the deprotection agent process 2 times of the solid in reactor; after DMF/MeOH washing 1-3 time; add Fmoc-Arg (the Pbf)-OH protected amino acid of 1.0-3.0 times amount; 1.0-3.0 the PFP of times amount; the DIC reagent of 1.0-3.0 times amount, is dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin.Under normal temperature, reaction is spent the night.Whether middle control test reaction is complete.
The synthesis of amino residue 12#, 9#, 7#, 5#:
By the deprotection agent process 2 times of the solid in reactor; after DMF/MeOH washing 1-3 time; add Fmoc protected amino acid (Fmoc-Gly-OH, Fmoc-Gly-OH, the Fmoc-Gly-OH of 2.0-5.0 times amount; Fmoc-Val-OH); the DIPEA reagent of the HOBt/Cl-HOBT of 2.0-5.0 times amount, 2.0-5.0 times amount, is dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin; control temperature, under 0-5C condition, adds HBTU/HATU or TBTU of 2.0-5.0 times amount.React under normal temperature.Whether middle control test reaction is complete.
The synthesis of amino residue 10#, 6#, 4#-1#:
By the deprotection agent process 2 times of the solid in reactor; after DMF/MeOH washing 1-3 time; add Fmoc protected amino acid (Fmoc-Cys (the Trt)-OH of 1.0-3.0 times amount; Fmoc-Glu (Trt)-OH; Fmoc-Met-OH; Fmoc-Lys (Boc)-OH; Fmoc-Pro-OH; Fmoc-Ser (tBu)-OH); 2.0-6.0 the HOBt/Cl-HOBT of times amount; the DIC reagent of 1.0-3.0 times amount, is dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin, is poured in reactor by mixed solvent.React after 30-60 minute, continue the DIC reagent adding 1.0-3.0 times amount.Under normal temperature, reaction is spent the night.Whether middle control test reaction is complete.
What control in the present invention whether test reaction adopt completely is ninhydrin.Relevant ninhydrin (Kaiser), ninhydrin test (Ninhydrin test), and monitoring method can see document VIRENDER K.SARIN, et al. " Quantitative Monitoring of Solid-Phase PeptideSynthesis by the Ninhydrin Reaction " ANALYTICAL BIOCHEMISTRY 117, 147-157 (1981), E.KAISER, et al. " Color Test for Detection of Free Terminal AminoGroups in the Solid-Phase Synthesis of Peptides " SHORT COMMUNICATIONS595-598 (Received October 28, 1969), with THORKILD CHRISTENSEN " A QualitativeTest for Monitoring Coupling Completeness in Solid Phase Peptide Synthesis UsingChloranil " Acta Chemica Scandinavica B 33 (1979) 763-766.
The above-mentioned feature that the present invention mentions, or the feature that embodiment is mentioned can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and use, each feature disclosed in specification sheets, anyly can provide identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
Major advantage of the present invention is:
1, preparation method's step provided by the invention is simple, and easy to operate, 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, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or number by weight.
Unit in percent weight in volume in the present invention is well-known to those skilled in the art, such as, refer to the weight of solute in the solution of 100 milliliters.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
The HPLC method related in following embodiment is as follows:
Stationary phase: C18 post column temperature 30 DEG C
Moving phase: A:0.1% trifluoroacetic acid (TFA)/water
B:0.1% trifluoroacetic acid (TFA)/acetonitrile
Gradient elution: 0-50 minute, A are from 90% to 65%, B from 10% to 35%
Flow velocity: 1.0ml/min
Determined wavelength: 215nm
Mass spectrum (LC-MS) detection method related in following embodiment is as follows:
Moving phase: 80% methanol/water
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
Synthesis Nesiritide crude product 1
load Fmoc-His (Trt)-OH
With 10 grams of PL Wang resins (substitution value 0.7-0.9mmol/g) and 2.5 equivalent PBr
3stirring reaction 2 hours; Then take out reaction solution, use DMF washing resin, then with Fmoc-His (the Trt)-OH of 3.0 equivalents, the DIPEA stirring reaction of 3.0 equivalents 16 hours, on resin, responseless bromine group methyl alcohol end-blocking, finally obtains resin 13 grams, and UV ultraviolet determination substitution value is 0.26mmol/g.
deprotection
With 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v) double deprotection, then use DMF and methanol wash resin respectively, thoroughly drain rear Kaiser test detection method monitoring Fmoc and slough degree.
amino acid condensation
Condensation 31#-23#, during 16-14#, 11#, 8# amino acid, will add the Fmoc protected amino acid of 2.0 times amount, the HOBt of 2.0 times amount, the DIC reagent of 2.0 times amount, be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin.React after 30 minutes, again add the DIC reagent of 2.0 times amount.
During condensation 22-18# amino acid, the Fmoc protected amino acid of 2.0 times amount, the Cl-HOBt of 2.0 times amount, the DIC reagent of 2.0 times amount, is dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin.The temperature controlling condensation solution is 0-3C.Pour in reactor by condensation solution, react after 30 minutes, again add the DIC reagent of 2.0 times amount, reaction is spent the night.
During Fmoc-Arg (the Pbf)-OH of condensation 13 and 17,3.0 equivalent Fmoc-Arg (Pbf)-OH/3.0 equivalent Pentafluorophenols (3.0 equivalents are relative to Fmoc-His (Trt)-PL Wang resin)/DMF solution is added in reactor, add 2.0 equivalent DIC subsequently, stirring reaction is 36-48 hour at least.If amino acid condensation is incomplete, add 0.5-1.0 equivalent Fmoc-AA-OH in reactor, continue stirring reaction and spend the night.
Condensation 12#, during 9#, 7#, 5# amino acid; add the Fmoc protected amino acid of 4.0 times amount, the Cl-HOBT of 4.0 times amount, the DIPEA reagent of 4.0 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin, control temperature, under 0-5C condition, adds the HBTU of 2.5 times amount.React under normal temperature.Whether middle control test reaction is complete.
Condensation 10#, during 6#, 4#-1# amino acid; add the Fmoc protected amino acid of 2.0 times amount, the Cl-HOBT of 5.0 times amount, the DIC reagent of 2.0 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin, mixed solvent is poured in reactor.React after 30 minutes, continue the DIC reagent adding 2.0 times amount.Under normal temperature, reaction is spent the night.
cutting
With the TFA/TIS/EDT/ water/Thioanisole (90%v/3%v/3%v/2%v/2%v) cooled as cutting liquid, stir and rise again to 25 DEG C ± 5 DEG C reaction 2-3 hour.Sedimentation in the methyl tertiary butyl ether (MTBE) that filtrate the pouring into concentrated has cooled, cooling leaves standstill crystallization 0.5-1.5 hour.Filter or centrifugally obtain filter cake, with the thorough washing leaching cake of MTBE three times of cooling.Crude product polypeptide is transferred in vacuum drier, vacuum-drying at least 12 hours.
The results are shown in Figure 1.Detection obtains crude product purity 59%. yield 100.85%.
Embodiment 2
Synthesis Nesiritide crude product 2
load Fmoc-His (Trt)-OH
With 10 grams of PL Wang resins (substitution value 0.6-0.8mmol/g) and 4.0 equivalent PBr
3stirring reaction 2 hours; Then take out reaction solution, use DMF washing resin, then with Fmoc-His (the Trt)-OH of 3.0 equivalents, the DIPEA stirring reaction of 3.0 equivalents 15 hours, on resin, responseless bromine methyl alcohol end-blocking, finally obtains resin 13 grams, and UV ultraviolet determination substitution value is 0.26mmol/g.
deprotection
With 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v) double deprotection, use DMF and methanol wash resin respectively, thoroughly drain rear Kaiser test detection method monitoring Fmoc and slough degree.
amino acid condensation
Condensation 31#-23#, during 16-14#, 11#, 8# amino acid, will add the Fmoc protected amino acid of 1.5 times amount, the HOBt of 1.5 times amount, the DIC reagent of 1.5 times amount, be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin.React after 30 minutes, again add the DIC reagent of 1.5 times amount.
During condensation 22-18# amino acid, the Fmoc protected amino acid of 1.5 times amount, the HOBt of 1.5 times amount, the DIC reagent of 1.5 times amount, is dissolved in DMF and DCM (1:1) mixed solvent of 6ml/g resin.The temperature controlling condensation solution is 0-3C.Pour in reactor by condensation solution, react after 30 minutes, again add the DIC reagent of 1.5 times amount, reaction is spent the night.
During Fmoc-Arg (the Pbf)-OH of condensation 13 and 17,4.0 equivalent Fmoc-Arg (Pbf)-OH/4.0 equivalent Pentafluorophenols (4.0 equivalents are relative to Fmoc-His (Trt)-PL Wang resin)/DMF solution is added in reactor, add 3.0 equivalent DIC subsequently, stirring reaction is 36-48 hour at least.
Condensation 12#, during 9#, 7#, 5# amino acid; add the Fmoc protected amino acid of 4.0 times amount, the HOBT of 4.0 times amount, the DIPEA reagent of 4.0 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 6ml/g resin, control temperature, under 0-5C condition, adds the HBTU of 3.0 times amount.React under normal temperature.Whether middle control test reaction is complete.
Condensation 10#, during 6#, 4#-1# amino acid; add the Fmoc protected amino acid of 1.5 times amount, the Cl-HOBT of 4.0 times amount, the DIC reagent of 1.5 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin, mixed solvent is poured in reactor.React after 30 minutes, continue the DIC reagent adding 1.5 times amount.Under normal temperature, reaction is spent the night.
cutting
With the TFA/TIS/EDT/ water/Thioanisole (95%v/1.5%v/1.5%v/1%v/1%v) cooled as cutting liquid, stir and rise again to 25 DEG C ± 5 DEG C reaction 2-3 hour.Sedimentation in the methyl tertiary butyl ether (MTBE) that filtrate the pouring into concentrated has cooled, cooling leaves standstill crystallization 0.5-1.5 hour.Filter or centrifugally obtain filter cake, with the thorough washing leaching cake of MTBE three times of cooling.Crude product polypeptide is transferred in vacuum drier, vacuum-drying at least 12 hours.
The results are shown in Figure 2.Purity 54%, yield 111.20%.
Embodiment 3
Synthesis Nesiritide crude product 3
load Fmoc-His (Trt)-OH
With 159 grams of PL Wang resins (substitution value 0.7-0.9mmol/g) and 4.5 equivalent PBr
3stirring reaction 2 hours; Then take out reaction solution, use DMF washing resin, then with Fmoc-His (the Trt)-OH of 1.0 equivalents, the DIPEA stirring reaction of 1.0 equivalents 18 hours, on resin, responseless bromine methyl alcohol end-blocking, finally obtains resin 223 grams, and UV ultraviolet determination substitution value is 0.33mmol/g.
deprotection
With 10% piperidines/5%DBU/5%HOBt/DMF (v/v/w/v) double deprotection, then use DMF and methanol wash resin respectively, thoroughly drain rear Kaiser test detection method monitoring Fmoc and slough degree.
amino acid condensation
Condensation 31#-23#, during 16-14#, 11#, 8# amino acid, will add the Fmoc protected amino acid of 3.0 times amount, the Cl-HOBt of 3.0 times amount, the DIC reagent of 3.0 times amount, be dissolved in DMF and DCM (1:1) mixed solvent of 8ml/g resin.React after 30 minutes, again add the DIC reagent of 3.0 times amount.
During condensation 22-18# amino acid, the Fmoc protected amino acid of 3.0 times amount, the Cl-HOBt of 3.0 times amount, the DIC reagent of 3.0 times amount, is dissolved in DMF and DCM (1:1) mixed solvent of 8ml/g resin.The temperature controlling condensation solution is 0-3C.Condensation solution is poured in reactor, reacts after 30 minutes, again add the DIC reagent of 3.0 times amount, normal-temperature reaction.
During Fmoc-Arg (the Pbf)-OH of condensation 13 and 17,5.0 equivalent Fmoc-Arg (Pbf)-OH/5.0 equivalent Pentafluorophenols (5.0 equivalents are relative to Fmoc-His (Trt)-PL Wang resin)/DMF solution is added in reactor, add 5.0 equivalent DIC subsequently, stirring reaction is 36-48 hour at least.
Condensation 12#, during 9#, 7#, 5# amino acid; add the Fmoc protected amino acid of 3.0 times amount, the Cl-HOBT of 3.0 times amount, the DIPEA reagent of 3.0 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin, control temperature, under 0-5C condition, adds the HBTU of 3.0 times amount.React under normal temperature.
Condensation 10#, during 6#, 4#-1# amino acid; add the Fmoc protected amino acid of 3.0 times amount, the Cl-HOBT of 5.0 times amount, the DIC reagent of 3.0 times amount; be dissolved in DMF and DCM (1:1) mixed solvent of 5ml/g resin, mixed solvent is poured in reactor.React after 30 minutes, continue the DIC reagent adding 3.0 times amount.React under normal temperature.
cutting
With the TFA/TIS/EDT/ water/Thioanisole (80%v/10%v/2.5%v/2.5%v/5%v) cooled as cutting liquid, stir and rise again to 25 DEG C ± 5 DEG C reaction 2-3 hour.Sedimentation in the methyl tertiary butyl ether (MTBE) that filtrate the pouring into concentrated has cooled, cooling leaves standstill crystallization 0.5-1.5 hour.Filter or centrifugally obtain filter cake, with the thorough washing leaching cake of MTBE three times of cooling.Crude product polypeptide is transferred in vacuum drier, vacuum-drying at least 12 hours.
The results are shown in Figure 3.Purity 55%, yield 96.4%.
The foregoing is only preferred embodiment of the present invention, and be not used to limit substantial technological context of the present invention, substantial technological content of the present invention is broadly defined in the right of application, any technology entities that other people complete or method, if with application right define identical, also or a kind of change of equivalence, be all covered by being regarded as among this right.
Claims (4)
1. a method for preparing solid phase for Nesiritide crude product, described method comprises step:
With solid phase synthesis resin for starting raw material, the amino acid with Fmoc blocking group is connected successively according to the method for solid phase synthesis, obtain three dodecapeptide resins of protection, slough Fmoc-blocking group successively therebetween, with TBTU and HOBt, HBTU and HOBt, BOP and HOBt, TBTU and HOAt, HBTU and HOAt, any in DIC and HOBt or BOP and HOAt is for a pair condensing agent, carry out connecing reactive polypeptide, after the three dodecapeptide resins that must protect, synchronously carry out de-side chain protected group and cut peptide, obtain Nesiritide crude product, it is characterized in that, described step has following feature:
One be described solid phase synthesis resin is bromination king resin; And
One is adopt DMF and DCM mixed solvent to be activating solvent, dissolves amino acid and the condensing agent with Fmoc blocking group in advance, activates, then join in reaction system outside reaction system;
Connect the amino acid with Fmoc blocking group successively to comprise the steps:
A () is by king's resin, PBr
3, Fmoc-His (Trt)-OH and DIPEA mixing, be obtained by reacting bromination king resin;
B () is in bromination king resin; add deprotection agent to process; then resin DMF washs; add with DMF and DCM mixed solvent dissolve there is the amino acid of Fmoc blocking group, the mixture of condensing agent; deprotection agent process is added again after connecing reactive polypeptide; add the amino acid with Fmoc blocking group dissolved with DMF and DCM mixed solvent 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
14-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;
The 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;
When connecting the Fmoc-amino acid of 22 to 18 residues successively, described condensing agent is DIC and HOBt or DIC and Cl-HOBt;
When connecting Fmoc-Arg (the Pbf)-OH of 17,13 residues successively, using Pentafluorophenol as activating reagent;
When connecting the Fmoc amino acid of 12,9,7,5 residues successively, add the Fmoc protected amino acid of 2.0-5.0 times amount, 2.0-5.0 HOBt or Cl-HOBt of times amount, the DIPEA reagent of 2.0-5.0 times amount, be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g solid phase synthesis resin, control temperature, under 0-5 DEG C of condition, adds HBTU or HATU or the TBTU of 2.0-5.0 times amount, reacts under normal temperature;
When connecting the Fmoc amino acid of 10,6,4 to 1 residues successively; add the Fmoc protected amino acid of 1.0-3.0 times amount; 2.0-6.0 HOBt or Cl-HOBt of times amount; the DIC reagent of 1.0-3.0 times amount; be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g solid phase synthesis resin; react and continue the DIC reagent adding 1.0-3.0 times amount after 30-60 minute, react under normal temperature.
2. preparation method as claimed in claim 1, is characterized in that, when connecting the Fmoc-amino acid of 22 to 18 residues successively, setting-up point is 0-5 DEG C.
3. preparation method as claimed in claim 1 or 2, it is characterized in that, when connecting the Fmoc-amino acid of 22 to 18 residues successively, 1.0-3.0 HOBt or Cl-HOBt of times amount, the DIC reagent of 1.0-3.0 times amount, be dissolved in the mixing solutions in DMF and the DCM solvent of 5ml-10ml/g resin, react the DIC reagent adding 1.0-3.0 times amount after 30-60 minute.
4. preparation method as claimed in claim 3, it is characterized in that, with the entire volume of the mixing solutions of DMF and DCM, wherein the percentage composition of DMF is 40-60%.
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