CN106518975B - A kind of preparation method of pitressin - Google Patents
A kind of preparation method of pitressin Download PDFInfo
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- CN106518975B CN106518975B CN201710001529.9A CN201710001529A CN106518975B CN 106518975 B CN106518975 B CN 106518975B CN 201710001529 A CN201710001529 A CN 201710001529A CN 106518975 B CN106518975 B CN 106518975B
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- pitressin
- reverse phase
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- 108010004977 Vasopressins Proteins 0.000 title claims abstract description 57
- 102000002852 Vasopressins Human genes 0.000 title claims abstract description 57
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 title claims abstract description 57
- 229940072644 pitressin Drugs 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000012071 phase Substances 0.000 claims abstract description 64
- 230000002441 reversible effect Effects 0.000 claims abstract description 45
- 239000012043 crude product Substances 0.000 claims abstract description 37
- 239000002243 precursor Substances 0.000 claims abstract description 36
- 238000010612 desalination reaction Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000007791 liquid phase Substances 0.000 claims abstract description 11
- 239000000945 filler Substances 0.000 claims abstract description 10
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 7
- 229920001577 copolymer Polymers 0.000 claims abstract description 6
- 125000003396 thiol group Chemical group [H]S* 0.000 claims abstract description 5
- JSAIENUMNDAGTD-UHFFFAOYSA-N benzene ethene styrene Chemical group C1=CC=CC=C1.C=C.C=C.C=CC1=CC=CC=C1 JSAIENUMNDAGTD-UHFFFAOYSA-N 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 238000005336 cracking Methods 0.000 claims description 5
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 230000036961 partial effect Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N divinylbenzene Substances C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 11
- 229920001184 polypeptide Polymers 0.000 abstract description 10
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 10
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 238000010924 continuous production Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 239000000126 substance Chemical group 0.000 description 3
- 230000009102 absorption Effects 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 239000013058 crude material Substances 0.000 description 2
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 229960003726 vasopressin Drugs 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical group C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000002686 anti-diuretic effect Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010064 diabetes insipidus Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 108010061557 pitressin tannate Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation methods of pitressin.The preparation method includes the following steps: that pitressin precursor crude product solution is successively carried out reverse phase cyclisation, reverse phase purifying, reverse phase desalination using efficient liquid phase RP chromatography；The filler of efficient liquid phase RP chromatography is styrene diethylene benzene copoly mer；The pitressin precursor crude product is the pitressin precursor crude product containing two free sulfhydryl groups.Preparation method of the invention has used reverse phase absorption cyclisation, purifying, desalination one-step method that polypeptide sterling is made, and optimizes production technology, is suitble to industrialization continuous production.
Description
Technical field
The present invention relates to field of biological pharmacy.More particularly it relates to a kind of preparation method of pitressin.
Background technique
The synthesis polypeptide that pitressin is made of nine amino acid residues, chemical structure are
The theoretical molecular weight 1084.24 of pitressin.Tannic acid pressurization
Element is antidiuretic hormone medicine, and distal renal tubular and concetrated pipe can be promoted to the reabsorption of moisture and have antidiuretic activity, system
Agent is clinically used for treatment diabetes insipidus.
Current country's pitressin protected pitressin resin using synthesis in solid state before this mostly, so dry that pressurize using cracking
Plain precursor crude product (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2Trifluoroacetate), high dilution cyclisation,
Purifying turns salt, finally obtains pitressin tannate.High dilution cyclisation is bulky since sample concentration is dilute, for
Later-period purification is very unfavorable.A kind of method for also lacking effective polypeptide bulk pharmaceutical chemicals of the preparation containing disulfide bond now, therefore still compel
The preparation method of the new polypeptide containing disulfide bond will be developed by being essential.
Summary of the invention
Technical problem to be solved by the present invention lies in order to overcome, the preparation process of pitressin in the prior art is complicated, receives
The defect that rate is low, sample concentration is dilute, bulky, and provide a kind of preparation method of pitressin.Pitressin of the invention
Preparation method using containing a pair of of free sulfhydryl groups (- SH) polypeptide crude product --- pitressin precursor crude product is starting material, by efficient
The cyclisation of liquid phase reverse-phase chromatography, purifying and salt-removal steps, the method for preparing the pitressin of high-purity.
The present invention is to solve above-mentioned technical problem by the following technical programs:
The present invention provides a kind of preparation methods of pitressin comprising following step: using efficient liquid phase reverse-phase chromatography
Pitressin precursor crude product solution is successively carried out reverse phase cyclisation, reverse phase purifying, reverse phase desalination by method；Efficient liquid phase reverse phase color
The filler of spectrometry is styrene-divinylbenzene (PS-DVB) copolymer；
The pitressin precursor crude product is the pitressin precursor crude product containing two free sulfhydryl groups.
Wherein, the pitressin precursor crude product preferably uses solid-phase synthesis by the dry pressurization obtained of cracking
Plain precursor crude product, HPLC purity are 60~90%；The structural formula of the pitressin precursor crude product is Cys-Tyr-Phe-Gln-
Asn-Cys-Pro-Arg-Gly-NH2Trifluoroacetate.
Wherein, the pitressin precursor crude product solution is preferably pitressin precursor crude product and is dissolved in concentration of volume percent
For the 5g/L solution of 5% acetonitrile solution formation.
In the present invention, the reverse phase cyclisation, reverse phase purifying, reverse phase desalination are complete in the reverse phase elution process of a step
At.
Wherein, reverse phase cyclisation, reverse phase purifying, the condition of reverse phase desalination are preferably as follows: mobile phase A 1 is pure water, A2
For the H of percent by volume 0.01~0.05% (preferably 0.02~0.03%)2O2The NaOH aqueous solution that pH is 7.5~9.0,
Mobile phase B is acetonitrile, and mobile phase C1 is the pitressin precursor crude product solution, and mobile phase C2 is that the NaOH of 0.1mol/L is molten
Liquid, flow velocity are 180~220mL/min (preferably 200mL/min), Detection wavelength 220nm；
Online loading, elution are carried out according to the condition of following table, percentage is percent by volume；
| Elution step | Elution time | Eluent |
| 1 | 0~10min | 100%C1 |
| 2 | 10.1~25min | 75%A1+5%B+20%C2 |
| 3 | 25.1~40min | 95%A1+5%B |
| 4 | 40.1~45min | 95%A2+5%B |
| 5 | 45.1~60min | 95%A1+5%B |
| 6 | 60~75min | 95%A1+5%B → 80%A1+20%B |
| 7 | 75~90min | 80%A1+20%B → 73%A1+27%B |
| 8 | 90~105min | 50%A1+50%B |
| 9 | 105~115min | 95%A1+5%B |
It collects the eluent that retention time is 75~90min and obtains pitressin solution.
Wherein, the filling condition of the efficient liquid phase RP chromatography is preferably as follows: filler is Agilent PLRP-S benzene
Ethylene-divinylbenzene (PS-DVB) copolymer, aperture 10nm, 10 μm of partial size.
Pitressin is a kind of peptide material, unstable under alkaline condition, degradable, especially under strong alkali environment, this hair
The de- concentration of bright integrated survey alkali cleaning and time, to guarantee to reduce the destruction and loss of sample in desalination processes.
Innovative point of the present invention is will to be cyclized, purify and one step reverse phase of desalination obtains polypeptide sterling, and pitressin precursor contains
Two free sulfhydryl groups simultaneously contain basic amino acid, and traditional handicraft cyclisation and purifying substep carry out, and height of specimen when cyclization
Dilution, it is bulky, the difficulty of purifying is increased, for cyclisation and preparation process rapidly and efficiently, designs polymer filler
The more recent application of PLRP-S.Novelty of the present invention has used reverse phase absorption method to be cyclized, purify and desalination, disposable solution cyclisation,
The problem of purifying and desalination.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) reduced form polypeptide crude product is first adsorbed onto stationary phase using the method being cyclized online, is utilized polypeptide by the present invention
With the hydrophobic binding of reverse phase filler, first Alkaline Elution combines by force on a column acid group, the again free hydroxyl of neutral elution, and
H is contained using pH meta-alkali2O2Mobile phase rinse, promote two sulfydryls at disulfide bond, obtain target polypeptides crude product, sample is retained in
In chromatographic column.
(2) present invention avoids eluting using online cyclisation, the sample of cyclisation, and progress gradient is washed after can directly converting mobile phase
De- purifying, obtains final sterling, is suitble to continuous production.
(3) present invention has innovatively used reverse phase absorption cyclisation, purifying, desalination one-step method that polypeptide sterling, optimization is made
Production technology is suitble to industrialization continuous production.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Embodiment 1 (HPLC method detects pitressin precursor crude product and after purification product solution purity)
Instrument: 2695/2489 high performance liquid chromatograph of Waters
Splitter: Agilent XDB-C18 4.6 × 250mm, 5 μm
Mobile phase: A is that percent by volume is 0.1%TFA aqueous solution, and B is the second that percent by volume is 0.1%TFA-50%
Nitrile aqueous solution (TFA is trifluoroacetic acid)
Flow velocity is 1.0mL/min, Detection wavelength 214nm, and room temperature detection, gradient see the table below, and percentage is volume
Percentage.
| Elution step | Elution time | Eluent |
| 1 | 0~5min | 95%A+5%B |
| 2 | 5~25min | 95%A+5%B → 50%A+50%B |
| 3 | 25~30min | 100%B |
| 4 | 30~35min | 95%A+5%B |
Embodiment 2 (75mm internal diameter L&L4003 prepares column filling)
With Load&Lock dynamic axial compression and static locking technology, filler is styrene-divinylbenzene copolymer
(reverse phase filler Agilent PLRP-S), aperture 10nm, fill column density 0.33g/mL, are filled to column bed pressure by 10 μm of partial size
650psi, using Varian chromatography loading system, 370g dry powder filler pours into internal diameter 75mm L& after the stirring homogenate of 2L methanol
L4003 prepares column, compression ratio 3:1, carrier gas N2, adjust nebulizer gas pressure and oil pressure meter pressure made to be 2000psi, dynamic axial
It is compressed to column bed height 26cm, prepares column as used in reverse phase cyclisation, reverse phase purifying and reverse phase desalination scheme.
Reverse phase cyclisation, reverse phase purifying and the reverse phase desalination of 3 pitressin precursor crude material of embodiment
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: 2 self-chambering of embodiment prepares 10 μm of 10nm of column Load&Lock4003 75 × 260mm, PLRP-S
Pitressin precursor crude product is using solid-phase synthesis by the dry pitressin precursor crude product obtained of cracking, structural formula
For Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2Trifluoroacetate.
Pitressin precursor crude product solution is the acetonitrile water that above-mentioned pitressin precursor crude product is dissolved in that concentration of volume percent is 5%
The 5g/L solution that solution is formed.
Mobile phase A 1 is purified water, and A2 is the H of percent by volume 0.032O2The NaOH aqueous solution that pH is 7.5, Mobile phase B are
Acetonitrile, mobile phase C1 are the pitressin precursor crude product solution, and measuring its HPLC purity as described in Example 1 is
85.12%, retention time 12.4min, mobile phase C2 are the NaOH solution of 0.1M.
The reverse phase cyclisation of the present embodiment, reverse phase purify and reverse phase desalination condition is as follows: flow velocity 200mL/min, 220nm inspection
It surveys, purifying gradient see the table below shown, and percentage is percent by volume.
| Elution step | Elution time | Eluent |
| 1 | 0~10min | 100%C1 |
| 2 | 10.1~25min | 75%A1+5%B+20%C2 |
| 3 | 25.1~40min | 95%A1+5%B |
| 4 | 40.1-45min | 95%A2+5%B |
| 5 | 45.1-60min | 95%A1+5%B |
| 6 | 60~75min | 95%A1+5%B → 80%A1+20%B |
| 7 | 75~90min | 80%A1+20%B → 73%A1+27%B |
| 8 | 90~105min | 50%A1+50%B |
| 9 | 105~115min | 95%A1+5%B |
It collects the eluent that retention time is 75~90min and obtains pitressin solution.Measure it as described in Example 1
HPLC purity is 99.18%, retention time 9.35min.
Reverse phase cyclisation, reverse phase purifying and the reverse phase desalination of 4 pitressin precursor crude material of embodiment
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: 2 self-chambering of embodiment prepares 10 μm of 10nm of column Load&Lock4003 75 × 260mm, PLRP-S
Pitressin precursor crude product is using solid-phase synthesis by the dry pitressin precursor crude product obtained of cracking, structural formula
For Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2Trifluoroacetate.
Pitressin precursor crude product solution is the acetonitrile water that above-mentioned pitressin precursor crude product is dissolved in that concentration of volume percent is 5%
The 5g/L solution that solution is formed.
Mobile phase A 1 is purified water, and A2 is the H of percent by volume 0.022O2The NaOH aqueous solution that pH is 9.0, Mobile phase B are
Acetonitrile, mobile phase C1 are the pitressin precursor crude product solution, and measuring its HPLC purity as described in Example 1 is
85.12%, retention time 12.4min, mobile phase C2 are the NaOH solution of 0.1M.
The reverse phase cyclisation of the present embodiment, reverse phase purify and reverse phase desalination condition is as follows: flow velocity 200mL/min, 220nm inspection
It surveys, purifying gradient see the table below shown, and percentage is percent by volume.
| Elution step | Elution time | Eluent |
| 1 | 0~10min | 100%C1 |
| 2 | 10.1~25min | 75%A1+5%B+20%C2 |
| 3 | 25.1~40min | 95%A1+5%B |
| 4 | 40.1-45min | 95%A2+5%B |
| 5 | 45.1-60min | 95%A1+5%B |
| 6 | 60~75min | 95%A1+5%B → 80%A1+20%B |
| 7 | 75~90min | 80%A1+20%B → 73%A1+27%B |
| 8 | 90~105min | 50%A1+50%B |
| 9 | 105~115min | 95%A1+5%B |
It collects the eluent that retention time is 75~90min and obtains pitressin solution.Measure it as described in Example 1
HPLC purity is 99.42%, retention time 9.38min.
Embodiment 5 (Mass Spectrometer Method of pitressin)
It is obtained using waters micromass ZQ substance level four bars electrospray ionization mass spectrum (ESI-MS) measurement embodiment 3,4
Pitressin molecular mass peak [M+1]+Measured value 1085.39, leading ion fragment peak [M+2]2+Measured value 543.20, all accords with
Close theoretical value 1084.24.
Claims (8)
1. a kind of preparation method of pitressin comprising following step: using efficient liquid phase RP chromatography by pitressin precursor
Crude product solution successively carries out reverse phase cyclisation, reverse phase purifying, reverse phase desalination；The filler of efficient liquid phase RP chromatography is benzene
Ethylene-divinyl benzene copolymer；
The pitressin precursor crude product is the pitressin precursor crude product containing two free sulfhydryl groups；
The reverse phase is cyclized, reverse phase purifies, the condition of reverse phase desalination is as follows: mobile phase A 1 is pure water, and A2 is percent by volume
0.01~0.05% H2O2NaOH aqueous solution, pH be 7.5~9.0, Mobile phase B is acetonitrile, and mobile phase C1 is institute
The pitressin precursor crude product solution stated, mobile phase C2 are the NaOH solution of 0.1mol/L, and flow velocity is 180~220mL/min, detection
Wavelength is 220nm；
Online loading, elution are carried out according to the condition of following table, percentage is percent by volume；
It collects the eluent that retention time is 75~90min and obtains pitressin solution.
2. preparation method as described in claim 1, which is characterized in that the pitressin precursor crude product is using synthesis in solid state
For method by the dry pitressin precursor crude product obtained of cracking, HPLC purity is 60~90%.
3. preparation method as described in claim 1, which is characterized in that the structural formula of the pitressin precursor crude product is Cys-
Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2Trifluoroacetate.
4. preparation method as described in claim 1, which is characterized in that the pitressin precursor crude product solution is before pitressin
Body crude product is dissolved in the 5g/L solution that the acetonitrile solution that concentration of volume percent is 5% is formed.
5. preparation method as described in claim 1, which is characterized in that the reverse phase is cyclized, reverse phase purifies, reverse phase desalination is equal
It is to be completed in the reverse phase elution process of a step.
6. preparation method as described in claim 1, which is characterized in that the A2 is percent by volume 0.02~0.03%
H2O2NaOH aqueous solution, pH be 7.5~9.0.
7. preparation method as described in claim 1, which is characterized in that the flow velocity is 200mL/min.
8. preparation method as described in claim 1, which is characterized in that the filling condition of the efficient liquid phase RP chromatography
It is as follows: filler be Agilent PLRP-S styrene diethylene benzene copoly mer, aperture 10nm, 10 μm of partial size.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710001529.9A CN106518975B (en) | 2017-01-03 | 2017-01-03 | A kind of preparation method of pitressin |
Applications Claiming Priority (1)
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| CN103254295A (en) * | 2013-05-31 | 2013-08-21 | 青岛国大生物制药股份有限公司 | Preparation method of terlipressin |
| CN103374054A (en) * | 2012-04-28 | 2013-10-30 | 上海第一生化药业有限公司 | One-step method based solid-phase polypeptide synthesis method |
| CN103980351A (en) * | 2014-05-27 | 2014-08-13 | 上海第一生化药业有限公司 | Method for preparing vasopressin and vasopressin tannate |
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| CN103374054A (en) * | 2012-04-28 | 2013-10-30 | 上海第一生化药业有限公司 | One-step method based solid-phase polypeptide synthesis method |
| CN103254295A (en) * | 2013-05-31 | 2013-08-21 | 青岛国大生物制药股份有限公司 | Preparation method of terlipressin |
| CN103980351A (en) * | 2014-05-27 | 2014-08-13 | 上海第一生化药业有限公司 | Method for preparing vasopressin and vasopressin tannate |
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