CN104447979B - A kind of method for preparing Nesiritide - Google Patents
A kind of method for preparing Nesiritide Download PDFInfo
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Abstract
The present invention relates to a kind of method for preparing Nesiritide, the technology specifically comprises the steps of：A）Tripeptide fragment H Arg (Boc) are synthesized by liquid phase method2‑Arg(Boc)2‑His(Trt)‑OtBu；B）Using solid-phase synthesis, using 2 CTC resins as initial resin, the amino acid with N-terminal Fmoc protections and side chain protected is coupled successively according to the peptide sequence of Nesiritide main chain 1 29；C）Under the conditions of organic base, using the solid ring of iodine；D）Cracking, obtains the fragments of peptides of Full-protective Nesiritide main chain 1 29；E）Tripeptide fragment H Arg (Boc)2‑Arg(Boc)2His (Trt) OtBu and the coupling of the fragments of peptides of Full-protective Nesiritide main chain 1 29；F）Cracking, purifying, it is lyophilized after obtain Nesiritide.The invention provides a kind of purity height, cost are low, it is adapted to the Nesiritide preparation technology of large-scale production, this technique not only can effectively control racemization peptide impurity D His32Nesiritide, disappearance peptide impurity Des Arg31Nesiritide or Des Arg30Nesiritide, also optimizes the yield that solid ring condition improves Nesiritide.
Description
Technical field
The invention belongs to polypeptide drugs preparation method technical field, more particularly to a kind of method for preparing Nesiritide.
Background technology
Nesiritide, illustrious name is：Nesiritide, structural formula is as follows：
H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-
Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg- Arg-His-OH (disulfide keys：
10-26)
Nesiritide was separated by Suhoh in 1988 from pig brain tissue, the cyclic peptide being made up of 32 amino acid,
10-26 positions are connected by S -- S, identical with interior raw hormone, are mainly used in acute decompensation congestive heart failure
Dyspneic treatment when exhausting, its effect is a kind of polypeptide drugs for having very much a market prospects preferably and Small side effects.
On the preparation method of Nesiritide, CN200410083873.X reports the manufacture method of atrial natriuretic peptide, due to adopting
It is combined to Boc France, it is necessary to cracked using toxicity very big HF, therefore, is not suitable for large-scale production.Chinese patent
CN200910104860.9, CN200910104861.3 report a kind of solid phase and prepare and purify method, on the one hand using HMPB-
AM resins are vector resin, when HMPB-AM resins and the first protected amino acid are esterified, have 50% Fmoc-His (Trt)-
Racemization will occur for OH, so there is racemization isomers in the product；On the other hand due to using low concentration Nesiritide line
Property peptide pendular ring, cause to produce a large amount of waste liquids, pollution is big, and production efficiency is not high, is not suitable for large-scale production.Wang Mei and Chen Wuling
At " the Fmoc synthesis in solid state of human brain profit sodium skin "（《Modern biomedical is in progress》, 2008,8（2）：280-282.）In report and adopt
With the Nesiritide-CTC- resins of the solid ring side chain full guard of iodine, although the solid ring of iodine avoids pendular ring from producing a large amount of waste liquids, but iodine oxygen
The by-product hydrogen iodide of change can cause peptide to be scaled off from CTC- resins, influence total recovery.In addition, containing one in this product structure
Arg-Arg fragments, when solid phase is coupled, because arginic side chain protecting group is the hydrophobic group of macromolecular, therefore lead
Cause arginine coupling incomplete, generation disappearance peptide impurity Des-Arg31- Nesiritide（Or Des-Arg30- Nesiritide）, due to
Racemization isomers D-His32- Nesiritide and disappearance peptide Des-Arg31- Nesiritide（Or Des-Arg30- Nesiritide）With how
The polarity of Seeley peptide is close, is difficult to remove in purge process, so this product conventional solid synthesis total recovery is very low, it is difficult to real
Existing large-scale production.
The content of the invention
The present invention is to solve above-mentioned technical problem so as to provide a kind of method for preparing Nesiritide, and on the one hand this method makes
Use fragment coupling method, it is to avoid disappearance peptide impurity Des-Arg31- Nesiritide（Or Des-Arg30- Nesiritide）With racemization peptide
Impurity D-His32The generation of-Nesiritide, on the other hand optimizes solid ring condition and improves production yield, realize Nesiritide scale
Big production.
The synthetic route of the present invention is as shown in Figure 1：Tripeptide fragment H-Arg (Boc) is synthesized by liquid phase method first2-Arg
(Boc)2-His(Trt)-OtBu；Then solid-phase synthesis is used, using 2-CTC- resins as initial resin, according to Nesiritide master
Chain 1-29 peptide sequence is coupled the amino acid with N-terminal Fmoc protections and side chain protected successively；C）Under the conditions of organic base, using iodine
Gu ring；D）Cracking, obtains Full-protective Nesiritide main chain 1-29 fragments of peptides；E）Tripeptide fragment H-Arg (Boc)2-Arg
(Boc)2- His (Trt)-OtBu and the coupling of Full-protective Nesiritide main chain 1-29 fragments of peptides；F）Cracking, purifying, it is lyophilized after
To Nesiritide.
The conventional abbreviation of some in the present invention has following meanings；
Fmoc ：Fluorenylmethyloxycarbonyl
Fmoc-AA ：The amino acid of fluorenylmethyloxycarbonyl protection
DIC ：N, N '-Diisopropylcarbodiimide
PyBOP ：Hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus
HATU ：2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HOBt ：1- hydroxy benzenes a pair of horses going side by side triazole
tBu ：The tert-butyl group
Trt ：Trityl
Boc ：Tertbutyloxycarbonyl
Pbf ：2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls
His ：Histidine
Arg ：Arginine
Leu ：Leucine
Val ：Valine
Lys ：Lysine
Cys ：Cysteine
Gly ：Glycine
Ser ：Serine
Ile ：Isoleucine
Asp ：Aspartic acid
Met ：Methionine
Phe ：Phenylalanine
Gln ：Glutamine
Pro ：Proline
DMF ：N, N '-dimethylformamide
MeOH ：Methanol
DCM ：Dichloromethane
TFE ：Trifluoroethanol
NMP ：1-METHYLPYRROLIDONE
DMSO ：Dimethyl sulfoxide (DMSO)
TFA ：Trifluoracetic acid
PhSMe ：Thioanisole
EDT ：1,2- dithioglycols
Piperidine ：Hexahydropyridine
DMAP ：DMAP
DIEA ：N, N '-diisopropylethylamine
TMP ：2,4,6- trimethylpyridines
A kind of method for preparing Nesiritide is provided for this present invention, its step is as follows：
Step 1, tripeptide fragment H-Arg (Boc) is synthesized by liquid phase method2-Arg(Boc)2-His(Trt)-OtBu；
Step 2, using solid-phase synthesis, using 2-CTC- resins as initial resin, according to Nesiritide main chain 1-29 peptide
Sequence is coupled the amino acid with N-terminal Fmoc protections and side chain protected successively；
Step 3, under the conditions of organic base, using the solid ring of iodine；
Step 4, crack, obtain Full-protective Nesiritide main chain 1-29 fragments of peptides；
Step 5, tripeptide fragment H-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu and Full-protective Nesiritide master
Chain 1-29 fragments of peptides is coupled；
Step 6, crack, purify, it is lyophilized after obtain Nesiritide.
Wherein, the liquid phase method synthetic method described in step 1, the fragment H-Arg (Boc)2-Arg(Boc)2-His(Trt)-
OtBu liquid phase method synthesis step is：Fmoc-Arg(Boc)2- OH, HOSu and DCC couplings obtain Fmoc-Arg (Boc)2- OSu,
Then Fmoc-Arg (Boc)2- OSu and H-Arg (Boc)2- OH reactions obtain dipeptide fragment Fmoc-Arg (Boc)2-Arg
(Boc)2-OH；Fmoc-Arg(Boc)2-Arg(Boc)2- OH, HOSu and DCC couplings obtain Fmoc-Arg (Boc)2-Arg
(Boc)2- OSu, then Fmoc-Arg (Boc)2-Arg(Boc)2- OSu and H-His (Trt)-OtBu reactions obtain tripeptide fragment
Fmoc-Arg(Boc)2-Arg(Boc)2- His (Trt)-OtBu, goes after Fmoc protection groups to obtain tripeptide fragment H-Arg (Boc)2-
Arg(Boc)2-His(Trt)-OtBu。
Wherein, the solid phase synthesis process described in step 2 comprises the following steps：（1）2-CTC resins and Fmoc-Leu-OH exist
The Fmoc-Leu-CTC resins of 0.10 ~ 0.90 mmol/g substitution values are obtained under conditions of DIEA, TMP or NMM；（2）Using by body
Product is than being 1:Fmoc protection groups on the deprotection liquid removing Fmoc-Leu-CTC resins of 4 piperidines and DMF composition, obtain H-
Leu-CTC resins；（3）In the presence of coupling agent system, the valine coupling of H-Leu-CTC resins and Fmoc protections is obtained
Fmoc-Val-Leu-CTC resins；（4）Repeat step（2）、（3）, ammonia is carried out successively according to main chain 1-27 peptides sequence before Nesiritide
The coupling of base acid.Wherein, step（3）The coupling agent system includes condensing agent and reaction dissolvent, and the condensing agent is selected from
DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA；The reaction dissolvent be selected from DMF, DCM, NMP, DMSO or he
Between any combination.
Wherein, the solid ring of iodine is used under the conditions of organic base described in step 3, the organic base is selected from N ' N- diisopropyl second
Amine, triethylamine, one kind of 2,4,6- trimethylpyridines or N-methylmorpholine.
Wherein, the cracking described in step 4, uses volume ratio for the DCM of 20-25% trifluoroethanol lysate, with resin
Full-protective Nesiritide main chain 1-29 fragments of peptides is obtained after reaction cracking.
Wherein, the tripeptide fragment H-Arg (Boc) described in step 52-Arg(Boc)2- His (Trt)-OtBu and Full-protective
Nesiritide main chain 1-29 fragments of peptides is coupled, and coupling condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/
DIEA one kind, preferably HATU/HOBt/DIEA；When condensing agent selects HATU/HOBt/DIEA, Full-protective Nesiritide master
Chain 1-29 fragments of peptides：Tripeptide fragment:HATU:HOBt:DIEA mol ratio is preferably：1:1.1:1.1:1.1:1.1~1:1.5:
1.5:1.5:1.5, i.e., the molal quantity of this 4 kinds of materials of described tripeptide fragment and condensing agent HATU/HOBt/DIEA is equal, and they are each
It is 1.1/1 ~ 1.5/1 from the molar ratio relative to the Full-protective Nesiritide main chain 1-29 fragments of peptides, reaction temperature is
25 ~ 35 DEG C, the reaction time is 2 ~ 3 hours；It is further preferred that each of which is relative to the Full-protective Nesiritide main chain 1-29 peptides
The molar ratio of fragment is 1.5/1, and reaction temperature is 35 DEG C, and the reaction time is 2 hours.
The method of the present invention is obtained by screening, and screening process is as follows：
（1）The selection of mol ratio：Full-protective Nesiritide main chain 1-29 fragments of peptides:Tripeptide fragment H-Arg (Boc)2-
Arg(Boc)2-His(Trt)-OtBu:HATU:HOBt:DIEA mol ratio is：1:1.1:1.1:1.1:1.1 and 1:1.5:
1.5:1.5:1.5；
（2）The selection of reaction temperature：
25oC and 35oC；
（3）The selection in reaction time：
2 hours and 3 hours.
8 kinds of experiment conditions are proposed for this：
Experiment condition 1：Take 221.16 Full-protective Nesiritide main chain 1-29 cyclic peptide fragments (50mmol), 53.28g tripeptides
Fragment H-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu (55mmol), 7.43g HOBt (55mmol) and 20.92g
HATU (55mmol) adds stirring and dissolving in 1000ml DMF, is cooled to 0oC, 7.11g DIEA (55mmol) is added above-mentioned
In solution, 25oC reacts 2 hours, then cracks, and purifies, and freezes, obtains Nesiritide fine peptide.
Experiment condition 2-8, experimental implementation is as shown in experiment condition 1, and different experiment conditions and its experimental result is for example following
Table 1 shown in：
Table 1
| Experiment condition | Mol ratio | Temperature | Time | Total recovery | Purity |
| Experiment condition 1 | 1:1.1:1.1:1.1 | 25℃ | 2 hours | 34% | 99.11% |
| Experiment condition 2 | 1:1.5:1.5:1.5 | 25℃ | 2 hours | 48% | 99.12% |
| Experiment condition 3 | 1:1.1:1.1:1.1 | 35℃ | 2 hours | 38% | 99.24% |
| Experiment condition 4 | 1:1.5:1.5:1.5 | 35℃ | 2 hours | 50% | 99.75% |
| Experiment condition 5 | 1:1.1:1.1:1.1 | 25℃ | 3 hours | 38% | 99.53% |
| Experiment condition 6 | 1:1.5:1.5:1.5 | 25℃ | 3 hours | 49% | 99.63% |
| Experiment condition 7 | 1:1.1:1.1:1.1 | 35℃ | 3 hours | 38% | 99.12% |
| Experiment condition 8 | 1:1.5:1.5:1.5 | 35℃ | 3 hours | 46% | 99.27% |
Result above shows that the purification effect of experiment condition 4 is relatively excellent.
Compared to the prior art the method for the present invention has obvious advantage, and relevant contrast experiment is as shown in table 2 below：
The contrast and experiment of table 2
| Patent | Des-Arg31- Nesiritide | D-His32- Nesiritide | Total recovery | Purity |
| The technology of the present invention | Do not detect | Do not detect | 50.0% | 99.75% |
| CN201310094866.9 | 0.3% | 0.3% | 30.0% | 99.00% |
| CN201310096382.8 | 0.3% | 0.4% | 24.5% | 99.00% |
| CN201110171152.4 | 0.2% | 0.2% | 18.9% | 99.50% |
The beneficial effects of the invention are as follows：Use fragment coupling method, it is to avoid disappearance peptide impurity Des-Arg31- Nesiritide
（Or Des-Arg30- Nesiritide）With racemization peptide impurity D-His32The generation of-Nesiritide, while the solid ring condition of optimization is improved
Yield is produced, the big production of Nesiritide scale is realized.
Brief description of the drawings
Fig. 1：The synthetic route of Nesiritide of the present invention；
Fig. 2：The HPLC spectrograms of Nesiritide fine peptide；
Fig. 3：Nesiritide fine peptide mass spectrogram.
Embodiment
The present invention is further illustrated by the following examples.
Specifically, on each commercially available amino acid and amino acid fragment that are related in example below, and each commercially available tree
Fat, its manufacturer and marque are as follows：
Fmoc protection groups amino acid starting material, 2-CTC resins and Wang resin are conventional commercial reagent（Producer：Gill is given birth to
Change（Shanghai）Co., Ltd；Chemistry is pure）；Tripeptide fragment is this patent description synthesis.
Organic solvent and other raw material sources are commercially available product（Producer：Chemical Reagent Co., Ltd., Sinopharm Group；Chemistry
It is pure）.
In addition, " concentrated by rotary evaporation " and " lyophilized " mentioned in example below and determine HPLC and mass spectrographic condition and
Device therefor model and manufacturer are described as follows：
Concentrated by rotary evaporation equipment：Rotary Evaporators R-200/205（Switzerland Buchi (cloth is strange) company）；
Concentrated by rotary evaporation condition：At 30 DEG C, vacuum（-0.1Mpa）Under the conditions of concentrated by rotary evaporation, volume is total before revolving after concentration
Below volume 75%.
Freeze-drier：Freeze dryer FD-3（Beijing Bo Yikang laboratory apparatus Co., Ltd）；
Lyophilisation condition：Lyophilized plate is put into freezer compartment of refrigerator (- 20 DEG C), the h of pre-freeze 6.Freeze dryer is opened, system is opened
Cold, more than the min of precooling 30 sets freeze-drying curve as follows：
First paragraph：16 h are run at -27 DEG C；Second segment：4 h are run at -5 DEG C；3rd section：2 h are run at 5 DEG C；4th
Section：16 h are run at 30 DEG C.
HPLC：Dionex high performance liquid chromatographs；Use octadecylsilane chemically bonded silica（5 μm, 250 × 4.6mm）To fill out
Fill agent；Using 0.1%TFA solution as mobile phase A, using acetonitrile as Mobile phase B, gradient elution is carried out；Flow velocity is 1.0ml per minute；Inspection
Survey wavelength is 220nm；30 DEG C of column temperature.The μ l of need testing solution 20 are taken, liquid chromatograph is injected, chromatogram is recorded.
Mass spectrum：MALDI-TOF-MS MALDI-TOF-MSs；INSTRUMENT MODEL is AUTO
FLEX SPEED TOF-TOF。
Embodiment one：Fmoc-Arg(Boc)2The synthesis of-OSu Acibenzolars
Weigh 596.67g Fmoc-Arg (Boc)2-OH（1.0mol）, 138.10g HOSu（1.2mol）Add 3000ml
In THF, 247.56g DCC are added under ice-water bath（1.2mol）, react 1 hour, be warming up to room temperature reaction 3 hours, reaction solution mistake
Filter, mother liquor is spin-dried for, plus DCM dissolvings, and filtering, saturated sodium bicarbonate is washed 3 times, and pure water 2 times is stripped 2 times, merges organic phase, anhydrous
Sodium carbonate is dried, and is spin-dried for, ice ethyl alcohol recrystallization 3 times, is filtered, and the drawing of solid oil pump is dry to obtain 617.45g Fmoc-Arg (Boc)2-
OSu Acibenzolars, yield 89%.
Embodiment two：Fmoc-Arg(Boc)2-Arg(Boc)2- OH synthesis
Weigh 187.33g H-Arg (Boc)2-OH（0.5mol）With 79.50g Na2CO3（0.75mol）It is added to 500ml
Dissolved in water and 500ml THF mixed solution, weigh 346.88g Fmoc-Arg (Boc)2-OSu（0.5mol）It is added to
It is added dropwise after 500ml THF, dissolving in above-mentioned mixed solution, reaction at room temperature is stayed overnight, and adjusts PH to 7 with 10% watery hydrochloric acid, revolving is removed
THF is removed, PH to 3 is adjusted afterwards.A large amount of white precipitates are obtained, are filtered.By obtained white precipitate ice ethyl alcohol recrystallization.Solid
Oil pump drawing is dry to obtain 414.70g Fmoc-Arg (Boc)2-Arg(Boc)2- OH, yield 87%.
Embodiment three：Fmoc-Arg(Boc)2-Arg(Boc)2The synthesis of-OSu Acibenzolars
Weigh 381.34g Fmoc-Arg (Boc)2-Arg(Boc)2-OH（0.4mol）, 55.24g HOSu（0.48mol）Plus
Enter in 800ml THF, 99.02g DCC are added under ice-water bath（0.48mol）, react 1 hour, be warming up to room temperature reaction 3 hours,
Reacting liquid filtering, mother liquor is spin-dried for, plus DCM dissolvings, and filtering, saturated sodium bicarbonate is washed 3 times, and pure water 2 times is stripped 2 times, is merged organic
Phase, natrium carbonicum calcinatum is dried, and is spin-dried for, ice ethyl alcohol recrystallization 3 times, is filtered, and the drawing of solid oil pump is dry to obtain 373.95g Fmoc-Arg
(Boc)2-Arg(Boc)2- OSu Acibenzolars, yield 89%.
Example IV：Fmoc-Arg(Boc)2-Arg(Boc)2- His (Trt)-OtBu synthesis
Weigh 51.10g H-His (Trt)-OtBu（0.2mol）With 31.80g Na2CO3（0.3 mol）It is added to 500ml
Dissolved in water and 500ml THF mixed solution, weigh 210.09g Fmoc-Arg (Boc)2-Arg(Boc)2-OSu（0.2
mol）It is added to after 500ml THF, dissolving and is added dropwise in above-mentioned mixed solution, reaction at room temperature is stayed overnight, and PH is adjusted with 10% watery hydrochloric acid
To 7, revolving removes THF, and PH to 3 is adjusted afterwards.A large amount of white precipitates are obtained, are filtered.By obtained white precipitate ice ethanol
Recrystallization.The drawing of solid oil pump is dry to obtain 207.21g Fmoc-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu, yield 87%.
Embodiment five：H-Arg(Boc)2-Arg(Boc)2- His (Trt)-OtBu synthesis
Take the 207.21g Fmoc-Arg (Boc) of above-described embodiment four2-Arg(Boc)2- His (Trt)-OtBu is added to
In 500 ml hexahydropyridines and 500 ml DCM, add after ether sedimentation, filtering, ice ethyl alcohol recrystallization 3 times, filtering, solid oil
Pump drawing is dry to obtain 146.67g H-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu, yield 87%.
Embodiment six：Substitution value is the synthesis of 0.10mmol/g Fmoc-Leu-CTC resins
The 2-CTC resin 50.00g that substitution value is 0.40mmol/g are weighed, is added in solid phase reaction post, is added to solid phase
In reaction column, washed with DMF 1 time, after DMF swellable resins 30 minutes, take 35.34g Fmoc-Leu-OH (100mmol) to use
DMF dissolves, and adds after 17ml DIEA (100mmol) activation, is added in the above-mentioned reaction column equipped with resin under ice-water bath, reaction 2
After hour, add 500ml absolute methanols and close 1 hour.Washed with DMF 3 times, DCM is washed 3 times, 30 points are closed with absolute methanol
Clock, methanol shrinks drying, obtains Fmoc-Leu-CTC resins, and detection substitution degree is 0.10mmol/g.
Embodiment seven：Substitution value is the synthesis of 0.90mmol/g Fmoc-Leu-CTC resins
The 2-CTC resin 133.33g that substitution value is 1.50mmol/g are weighed, are added in solid phase reaction post, are added to solid
In phase reaction post, washed with DMF 1 time, after DMF swellable resins 30 minutes, take 353.41g Fmoc-Leu-OH (1000mmol)
Dissolved with DMF, add after 165ml DIEA (1000mmol) activation, added in the above-mentioned reaction column equipped with resin under ice-water bath,
After reaction 2 hours, add 2000ml absolute methanols and close 1 hour.Washed with DMF 3 times, DCM is washed 3 times, is sealed with absolute methanol
Close 30 minutes, methanol shrinks drying, obtain Fmoc-Leu-CTC resins, detection substitution degree is 0.90mmol/g.
Embodiment eight：Substitution value is the synthesis of 0.50mmol/g Fmoc-Leu-CTC resins
The 2-CTC resin 200.00g that substitution value is 1.00mmol/g are weighed, are added in solid phase reaction post, are added to solid
In phase reaction post, washed with DMF 1 time, after DMF swellable resins 30 minutes, take 353.41g Fmoc-Leu-OH
(1000mmol) is dissolved with DMF, added under ice-water bath after 165ml DIEA (1000mmol) activation, is added above-mentioned equipped with resin
In reaction column, after reacting 2 hours, add 2000ml absolute methanols and close 1 hour.Washed with DMF 3 times, DCM is washed 3 times, with nothing
Water methanol is closed 30 minutes, and methanol shrinks drying, obtains Fmoc-Leu-CTC resins, and detection substitution degree is 0.50mmol/g.
Embodiment nine：The synthesis of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
The Fmoc-Leu-CTC resins (100mmol) that 200.00g substitution values are 0.50mmol/g are weighed, solid phase reaction is added
In post, washed with DMF 1 time, after being swelled Fmoc-Leu-CTC resins 30 minutes with DMF, use DMF:Pyridine volume ratio is 4:1 it is mixed
Close solution and slough Fmoc protections, then washed with DMF 6 times, weigh 101.82g Fmoc-Val-OH（300mmol）、40.52g
HOBt（300mmol）It is 1 to add volume ratio:46ml DIC are added under 1 DCM and DMF mixed solutions, ice-water bath（300mmol）
After activation, add in the above-mentioned reaction column equipped with resin, after reacting 2 hours at room temperature, judge reaction eventually with ninhydrin method detection
Point, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that react incomplete, it is necessary to react again 1 hour,
This criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection.Above-mentioned removing Fmoc is repeated to protect
The step of protecting and add corresponding amino acid couplings, according to Nesiritide 1-27 main chain peptide sequences, be sequentially completed Fmoc-Lys (Boc)-
OH、Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ile-OH、Fmoc-Arg(Pbf)-
OH、Fmoc-Asp(OtBu)-OH、Fmoc-Met-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-
OH、Fmoc-Phe-OH、Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、
Fmoc-Gln (Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc)-OH, Fmoc-Pro-OH and Boc-
Ser (tBu)-OH coupling.Solvent is changed to when wherein Fmoc-Gln (Trt)-OH is coupled：It is 1 from volume ratio:4 DMSO and
DMF mixed solutions；Coupling reagent is changed to when Fmoc-Asp (OtBu)-OH is coupled：PyBOP/HOBt/DIEA；Fmoc-Pro-OH is even
Coupling reagent is changed to during connection：HATU/HOBt/DIEA couplings are finished, and Linaclotide CTC resins are washed 3 times with DMF, DCM washings
3 times, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 645.20g Full-protective Nesiritide main chains 1-
29 peptide-CTC resins.
Embodiment ten：The synthesis of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
The Fmoc-Leu-CTC resins (100mmol) that 200.00g substitution values are 0.50mmol/g are weighed, solid phase reaction is added
In post, washed with DMF 1 time, after being swelled Fmoc-Leu-CTC resins 30 minutes with DMF, use DMF:Pyridine volume ratio is 4:1 it is mixed
Close solution and slough Fmoc protections, then washed with DMF 6 times, weigh 101.82g Fmoc-Val-OH（300mmol）、40.52g
HOBt（300mmol）It is 1 to add volume ratio:46ml DIC are added under 1 DCM and DMF mixed solutions, ice-water bath（300mmol）
After activation, add in the above-mentioned reaction column equipped with resin, after reacting 2 hours at room temperature, judge reaction eventually with ninhydrin method detection
Point, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that react incomplete, it is necessary to react again 1 hour,
This criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection.Above-mentioned removing Fmoc is repeated to protect
The step of protecting and add corresponding amino acid couplings, according to Nesiritide 1-27 main chain peptide sequences, be sequentially completed Fmoc-Lys (Boc)-
OH、Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-Ser
(tBu)-Ser(tBu)-Ser(tBu)-OH、Fmoc-Ile-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Asp(OtBu)-OH、
Fmoc-Met-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-
Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Gln(Trt)-OH、Fmoc-
Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc)-OH, Fmoc-Pro-OH and Boc-Ser (tBu)-OH coupling.Wherein
Solvent is changed to when Fmoc-Gln (Trt)-OH is coupled：It is 1 from volume ratio:4 DMSO and DMF mixed solutions；Fmoc-Asp
(OtBu) coupling reagent is changed to when-OH is coupled：PyBOP/HOBt/DIEA；Coupling reagent is changed to when Fmoc-Pro-OH is coupled：
HATU/HOBt/DIEA couplings are finished, and Linaclotide CTC resins are washed 3 times with DMF, and DCM is washed 3 times, and MeOH is washed 3 times,
DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 657.20g Full-protective Nesiritide main chain 1-29 peptide-CTC resins.
Embodiment 11：The solid ring of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
657.20g Full-protective Nesiritide main chain 1-29 peptide-CTC resins are weighed to add in solid phase reaction post, it is molten with DCM
Swollen Full-protective Nesiritide main chain 1-29 peptide-CTC resins are washed 1 time after 30 minutes with DMF, add 129.24g N ' N- bis-
Wopropyl ethyl amine（1000mmol）With 253.81g iodine（1000mmol）DMF solution, react 3 hours, 6 times, DCM are washed with DMF
Washing 3 times, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 608.80g Full-protective Nesiritide masters
Chain 1-29 cyclic peptide-CTC resins.
Embodiment 12：The solid ring of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
Weighing 657.20g Full-protectives, how type Seeley peptide backbone 1-29 peptide-CTC resins are added in solid phase reaction post, use DCM
Full-protective Nesiritide main chain 1-29 peptide-CTC resins are swelled after 30 minutes, are washed with DMF 1 time, 101.19g triethylamines are added
（1000mmol）With 253.81g iodine（1000mmol）DMF solution, react 3 hours, wash with DMF 6 times, DCM washing 3 times,
MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 601.25g Full-protective Nesiritide main chain 1-29 rings
Peptide-CTC resins.
Embodiment 13：The solid ring of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
657.20g Full-protective Nesiritide main chain 1-29 peptide-CTC resins are weighed to add in solid phase reaction post, it is molten with DCM
Swollen Full-protective Nesiritide main chain 1-29 peptide-CTC resins are washed 1 time after 30 minutes with DMF, add 121.18g 2,4,6-
Trimethylpyridine（1000mmol）With 253.81g iodine（1000mmol）DMF solution, react 3 hours, 6 times, DCM are washed with DMF
Washing 3 times, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 593.86g Full-protective Nesiritide masters
Chain 1-29 cyclic peptide-CTC resins.
Embodiment 14：The solid ring of Full-protective Nesiritide main chain 1-29 peptide-CTC resins
657.20g Full-protective Nesiritide main chain 1-29 peptide-CTC resins are weighed to add in solid phase reaction post, it is molten with DCM
Swollen Full-protective Nesiritide main chain 1-29 peptide-CTC resins are washed 1 time after 30 minutes with DMF, add 101.15g N- methyl
Morpholine（1000mmol）With 253.81g iodine（1000mmol）DMF solution, react 3 hours, wash with DMF 6 times, DCM wash 3
Secondary, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 592.78g Full-protective Nesiritide main chains 1-29
Cyclic peptide-CTC resins.
Embodiment 15：The cracking of Full-protective Nesiritide main chain 1-29 cyclic peptide-CTC resins
Weigh the 304.40g Full-protective Nesiritide main chain 1-29 cyclic peptide-CTC resins in embodiment 11（50mmol）
It is added in 5000mL three neck round bottom flask, is by volume 1:4 TFE and DCM configuration lysate 3000ml, will be cracked
Liquid is added in above-mentioned resin, is reacted at room temperature 2 hours, filtering, the resin 3 times after cracking wash with a small amount of TFA, merging filtrate is dense
Contracting, the liquid after concentration is added in ice ether and precipitated 1 hour, centrifugation, absolute ether centrifuge washing 6 times, and vacuum drying is obtained
To 220.01g Full-protective Nesiritide main chain 1-29 cyclic peptide.
Embodiment 16：The cracking of Full-protective Nesiritide main chain 1-29 cyclic peptide-CTC resins
Weigh the 304.40g Full-protective Nesiritide main chain 1-29 cyclic peptide-CTC resins in embodiment 11（50mmol）
It is added in 5000mL three neck round bottom flask, is by volume 1:3 TFE and DCM configuration lysate 3000ml, will be cracked
Liquid is added in above-mentioned resin, is reacted at room temperature 2 hours, filtering, the resin 3 times after cracking wash with a small amount of TFA, merging filtrate is dense
Contracting, the liquid after concentration is added in ice ether and precipitated 1 hour, centrifugation, absolute ether centrifuge washing 6 times, and vacuum drying is obtained
To 221.16g Full-protective Nesiritide main chain 1-29 cyclic peptide.
Embodiment 17：The preparation of the thick peptide of Full-protective Nesiritide
Weigh the 221.16g Full-protective Nesiritide main chain 1-29 cyclic peptide fragments (50mmol) of embodiment 16,72.66g
Tripeptide fragment H-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu (75mmol), 10.13g HOBt (75mmol) and
28.52g HATU (75mmol) add stirring and dissolving in 1000ml DMF, are cooled to 0oC, by 9.69g DIEA (75mmol)
Add in above-mentioned solution, 35oC reacts 2 hours, drains, ether sedimentation, obtains the thick peptide of pulpous state Full-protective Nesiritide.
Embodiment 18：The cracking of the thick peptide of Full-protective Nesiritide
The thick peptide of gained pulpous state Full-protective Nesiritide of embodiment 17 is weighed to be added in 5000mL three neck round bottom flask,
By TFA:TIS:PhOH:PhSMe:H2O:Me2S:NH4I=70:8:6:4:4:4:The mL of 4 proportional arrangement lysate 3000, will be cracked
Liquid is added in above-mentioned resin, is reacted at room temperature 2 hours, filtering, the resin 3 times after cracking wash with a small amount of TFA, merging filtrate is dense
Contracting, the liquid after concentration is added in ice ether and precipitated 1 hour, centrifugation, absolute ether centrifuge washing 6 times, and vacuum drying is obtained
To the thick peptide of white solid Nesiritide.
Embodiment 19：The preparation of Nesiritide fine peptide acetate
Weigh the thick thick peptide of peptide Nesiritide of the gained white solid Nesiritide of embodiment 18 with 18000 mL water dissolve after,
By the purifying of C18 or C8 posts 2 times, turn salt, freeze-drying after obtain target product.First time purification condition：Mobile phase is：A phases：
0.1%TFA；B phases：Acetonitrile, Detection wavelength 220nm collects purpose peak cut.Second of purification condition：Mobile phase is：A phases：
0.3% acetic acid；B phases：Acetonitrile.Detection wavelength 220nm, collects purpose peak cut.Turn salt condition：Mobile phase：A phases：20mM acetic acid
Ammonium-the aqueous solution；B phases：Acetonitrile；Detection wavelength 220nm.Purpose peak cut is collected, concentrated by rotary evaporation is lyophilized to obtain Nesiritide acetic acid
Salt fine peptide 86.6g, its HPLC spectrogram are as shown in Fig. 2 HPLC purity 99.75%, total recovery 50%.Its mass spectrum is as shown in figure 3, [M
]+：3462.7218, the theoretical accurate molecular weight of Nesiritide is：3462.7411, sample mass spectral results and theoretical molecular phase
Symbol.
Above content is to combine specific repair to select embodiment further description made for the present invention, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (1)
1. a kind of method for preparing Nesiritide, it is characterised in that comprise the following steps：
Step 1, tripeptide fragment H-Arg (Boc) is synthesized by liquid phase method2-Arg(Boc)2- His (Trt)-OtBu, the fragment H-
Arg(Boc)2-Arg(Boc)2- His (Trt)-OtBu liquid phase method synthesis step is：Fmoc-Arg(Boc)2- OH, HOSu and
DCC couplings obtain Fmoc-Arg (Boc)2- OSu, then Fmoc-Arg (Boc)2- OSu and H-Arg (Boc)2- OH reactions obtain two
Fragments of peptides Fmoc-Arg (Boc)2-Arg(Boc)2-OH；Fmoc-Arg(Boc)2-Arg(Boc)2- OH, HOSu and DCC are coupled
To Fmoc-Arg (Boc)2-Arg(Boc)2- OSu, then Fmoc-Arg (Boc)2-Arg(Boc)2- OSu and H-His (Trt)-
OtBu reactions obtain tripeptide fragment Fmoc-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu, is obtained after going Fmoc protection groups
Tripeptide fragment H-Arg (Boc)2-Arg(Boc)2-His(Trt)-OtBu；
Step 2, using solid-phase synthesis, using 2-CTC- resins as initial resin, according to Nesiritide main chain 1-29 peptide sequence according to
Amino acid of the secondary coupling with N-terminal Fmoc protections and side chain protected, comprises the following steps：(1) 2-CTC resins and Fmoc-Leu-
OH obtains the Fmoc-Leu-CTC resins of 0.10~0.90mmol/g substitution values under conditions of DIEA, TMP or NMM；(2) use
It is 1 by volume ratio:Fmoc protection groups on the deprotection liquid removing Fmoc-Leu-CTC resins of 4 piperidines and DMF composition, are obtained
H-Leu-CTC resins；(3) in the presence of coupling agent system, the valine coupling of H-Leu-CTC resins and Fmoc protections is obtained
Fmoc-Val-Leu-CTC resins；(4) repeat step (2), (3), ammonia is carried out according to main chain 1-27 peptides sequence before Nesiritide successively
The coupling of base acid；
Step 3, under the conditions of organic base, using the solid ring of iodine, the organic base is selected from N ' N- diisopropylethylamine, triethylamine, 2,
One kind of 4,6- trimethylpyridines or N-methylmorpholine；
Step 4, crack, use volume ratio for the DCM of 20-25% trifluoroethanol lysate, with being obtained after resin reaction cracking
To Full-protective Nesiritide main chain 1-29 fragments of peptides；
Step 5, tripeptide fragment H-Arg (Boc)2-Arg(Boc)2- His (Trt)-OtBu and Full-protective Nesiritide main chain 1-
29 fragments of peptides are coupled, and condensing agent is HATU/HOBt/DIEA, Full-protective Nesiritide main chain 1-29 fragments of peptides：Tripeptide fragment:
HATU:HOBt:DIEA mol ratio is：1:1.5:1.5:1.5:1.5, reaction temperature is 35 DEG C, and the reaction time is 2 hours；
Step 6, crack, purify, it is lyophilized after obtain Nesiritide.
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| CN102875650A (en) * | 2012-09-26 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Preparation method of barusiban |
| CN103204922A (en) * | 2013-03-22 | 2013-07-17 | 深圳翰宇药业股份有限公司 | Method for preparing nesiritide |
| CN103275207A (en) * | 2013-03-22 | 2013-09-04 | 深圳翰宇药业股份有限公司 | Nesiritide preparation method |
| CN103992383A (en) * | 2014-06-27 | 2014-08-20 | 杭州诺泰制药技术有限公司 | Method for preparing icatibant |
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| CN102875650A (en) * | 2012-09-26 | 2013-01-16 | 深圳翰宇药业股份有限公司 | Preparation method of barusiban |
| CN103204922A (en) * | 2013-03-22 | 2013-07-17 | 深圳翰宇药业股份有限公司 | Method for preparing nesiritide |
| CN103275207A (en) * | 2013-03-22 | 2013-09-04 | 深圳翰宇药业股份有限公司 | Nesiritide preparation method |
| CN103992383A (en) * | 2014-06-27 | 2014-08-20 | 杭州诺泰制药技术有限公司 | Method for preparing icatibant |
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