CN106554407A - A kind of synthetic method of ularitide - Google Patents
A kind of synthetic method of ularitide Download PDFInfo
- Publication number
- CN106554407A CN106554407A CN201510644646.8A CN201510644646A CN106554407A CN 106554407 A CN106554407 A CN 106554407A CN 201510644646 A CN201510644646 A CN 201510644646A CN 106554407 A CN106554407 A CN 106554407A
- Authority
- CN
- China
- Prior art keywords
- ularitide
- peptide
- synthetic method
- methylimidazole
- ionic liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to pharmaceutical technology field, discloses a kind of synthetic method of ularitide.Synthetic method of the present invention is added to ularitide linear peptides thick peptide or fine peptide in ionic liquid of the fusing point below 20 DEG C, and 0.5-3h is aoxidized under alkaline environment, and acquisition forms the ularitide crude product of disulfide bond.The present invention instead of the liquid phase oxidation medium of organic solvent and water in conventional oxidation link with ionic liquid at room temperature, so that the thick peptide purity of the ularitide of solid phase synthesis and fine peptide total recovery are significantly improved, the simultaneous oxidation response time can be greatly decreased, it is easy to operate, it is to avoid the generation of organic liquid waste.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to a kind of synthetic method of ularitide.
Background technology
Ularitide (Ularitide) is a kind of natriuretic peptide separated from human urine, its analysis knot
Structure is similar with atrial natriuretic peptide, and it is that its sequence N- end has more four amino acid residues that difference is only.Phase
The research of pass shows that ularitide has various effects such as expansion of blood vessels, expansion bronchus and diuresis, faces
There is certain therapeutical effect to heart failure, renal failure, pulmonary hypertension and bronchial asthma on bed.
The structure of ularitide is the polypeptide containing a pair of intramolecular disulfide bonds of 32 aminoacid composition, one
As by chemosynthesis be obtained, sequential structure is：
H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-As
p-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH(S-S)。
At present, for the synthetic method of ularitide, mainly there are two kinds.One kind is that line is completed in solid phase
The coupling of property peptide peptide resin, then forms corresponding disulfide bond using iodine phase oxidative；Another kind of method is
First solid phase synthesis linear peptide precursors, then in the low-down organic solvent/aqueous solution of concentration, liquid phase oxidation
Form corresponding disulfide bond.
From existing method as can be seen that the synthesis of ularitide is broadly divided into amino acid couplings and oxidation reaction
Two stages.The linear peptide resin for obtaining is added solid elemental iodine, oxidation to form disulfide bond, obtain by the former
The peptide resin of cyclization to after oxidation, obtains crude product after cracking.But aoxidized on peptide resin using iodine,
Iodine is difficult to complete eluting in whole process, contains micro in being easily caused final sterling i.e. crude drug
Iodine, causes the inclined yellow of crude drug.Additionally, aoxidizing to peptide using strong oxidizer, peptide sequence is easily destroyed
The secondary structure of itself, most affects the biological activity and drug effect of peptide itself at last.
And the latter will obtain the thick peptide of linear peptides through HPLC purification, linear peptides fine peptide is obtained.Linear peptides
Fine peptide methanol or acetonitrile dissolving, add water to be diluted to 10-3The concentration of mg/ml, in air and alkaline bar
72h is aoxidized under part, the cyclic peptide crude product after being aoxidized.Cyclic peptide crude product after purification, obtains essence through HPLC
Peptide.It is that the space secondary structure of final product can obtain complete reservation relative to the former advantage,
The biological activity of final fine peptide is much better for the former.But, aoxidized using the program,
As ularitide dissolving difficulty, and final oxidation need to carry out under very dilute concentration, operation is very
Difficulty, simultaneous reactions time are long, are unfavorable for large-scale production.Additionally, the method can be produced and substantial amounts of be had
The mixed waste liquor of machine solvent and water, is also unfavorable for the requirement of Green Chemistry in environmental protection.
The content of the invention
In view of this, it is an object of the invention to provide a kind of synthetic method of ularitide so that described
Synthetic method can shorten ularitide linear peptides oxidation time, while avoiding producing organic liquid waste.
Further object is that providing a kind of synthetic method of ularitide so that the synthesis
Method can improve the total recovery of the thick peptide purity of ularitide and fine peptide.
For achieving the above object, the present invention provides following technical scheme：
Ularitide linear peptides thick peptide or fine peptide are added to fusing point and are existed by a kind of synthetic method of ularitide
In less than 20 DEG C of ionic liquid, 0.5-3h is aoxidized in the basic conditions, acquisition forms the Wu Lali of disulfide bond
Peptide crude product.
For oxidation time in existing synthetic method is long, operating difficultiess, produces organic liquid waste etc. and lack
Fall into, ionic liquid of the present invention using fusing point below 20 DEG C substitutes existing organic solvent and water is liquid phase
The medium of oxidation, the system extraordinary dissolubility for ularitide has can be in higher concentration
In the range of aoxidized, it is not necessary to being diluted under low-down concentration is carried out, easy to operate, while can be with
Greatly shorten oxidation time, it is to avoid produce organic liquid waste, and the biological activity of increase ularitide,
Large-scale production can be realized.
Wherein, the alkalescence condition can refer to the alkalescence condition in existing liquid-phase oxidation, for example, adjust
PH is in 7.0-7.5 or more high scope, and terminates oxidation reaction and adjust pH to acidity, oxidization time
0.5h, 1h, 2h or 3h can be selected in certain specific embodiments of the invention.
Preferably, the cation of the ionic liquid is 1- ethyl-3-methylimidazoles cation or 1- butyl
- 3- methyl imidazolium cations.
Used as preferred or further preferred scheme, the ionic liquid is 1- ethyl-3-methylimidazole acetate
[C2mim] [OAc], 1- ethyl-3-methylimidazole trifluoroacetates [C2mim] [TFA], 1- ethyl -3- methyl
Limidazolium hexafluorophosphate [C2mim] [PF6], 1- butyl -3- Methylimidazole. acetate [C4mim] [OAc], 1-
Butyl -3- Methylimidazole. trifluoroacetates [C4mim] [TFA] or 1- butyl -3- Methylimidazole. hexafluorophosphates
[C4mim][PF6]。
Preferably, the concentration of the thick peptide of ularitide linear peptides or fine peptide is 20-200mg lines during the oxidation
Property peptide/mL ionic liquids.In certain specific embodiments of the invention, the thick peptide of ularitide linear peptides
Or the concentration of fine peptide can select 20mg/mL, 50mg/mL, 100mg/mL or 200mg/mL.
The thick peptide of ularitide linear peptides or fine peptide in synthetic method of the present invention can be according to this area routine
Synthetic method is prepared, and used as preferred scheme, the present invention is prepared by solid phase synthesis process,
Include among these synthesizing by synthetic method one by one according to ularitide aminoacid sequence or closed by fragment method
Into.
In building-up process one by one, can be by first aminoacid Tyr of ularitide C-terminal be carried with resin
Body such as wang resins etc. are coupled, then according to aminoacid sequence carries out follow-up coupling；Synthesize in fragment method
In, ularitide peptide sequence can be divided into into several fragments according to actual needs simultaneously synthesizing, finally be coupled each
Section.
In building-up process, those skilled in the art would generally be synthesized using protected amino acid.It is described
The aa (aa refers to certain concrete aminoacid) of protected amino acid or protection is referred to protection group protected amino acid
On main chain and side chain, amino, carboxyl etc. are easy to the aminoacid that disturbing reaction group occurs.For in synthesis
Need protect side chain aminoacid for, its side-chain structure as well known to those skilled in the art and know adopt
The groups such as amino that conventional protection group is come on protected amino acid side chain, carboxyl.
Building-up process is illustrated by taking synthetic method one by one as an example：
Step 1, by protection Tyr and Wang resins be coupled under coupling system；
Step 2, according to ularitide C-terminal to N-terminal peptide sequence, be coupled one by one to be left using coupling system
Remaining protected amino acid：
Fmoc-Arg (Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Asn (Trt)-OH, Fmoc-Cys (Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH,
Fmoc-Gly-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asp (OtBu)-OH,
Fmoc-Met-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,
Fmoc-Phe-OH, Fmoc-Cys (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Arg (Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Thr (tBu)-OH；
Fmoc protection groups are taken off after the completion of coupling and obtains the thick peptide of ularitide linear peptides, the acquisition of fine peptide carries out pure
Change.
The coupling system of above-mentioned solid phase synthesis remaining amino acid includes DIC+A or B+A+C, wherein A
Be HBTU, HATU, TBTU or PyBOP for HOBt or HOAt, B, C be DIPEA or
TMP。
The cracking is using volume ratio TFA：TIS：EDT：PhOH：H2O=85-95:2-5:0-5:0-2:1-5
Lytic reagent cracking 1.5-3.5h；Volume ratio TFA is adopted more preferably：TIS：EDT：PhOH：
H2O=90:3:2:1:4 lytic reagent cracking 2.5h.
In the coupling process of the Try and subsequent amino-acid of protection, as N-terminal has Fmoc etc. to protect
Shield base protection, therefore each Deprotection is needed, this is conventional steps in solid phase synthesis, can adopt and contain 20%
The DMF solution of hexahydropyridine, it is also possible to removed using other suitable reagents.
Additionally, present invention additionally comprises the step of ularitide crude product is carried out purification, the purification is further
Preferably HPLC purification.
More specifically HPLC purification steps are as follows：
The ularitide crude product after oxidation is taken, using Waters 2454RP-HPLC systems, wavelength 220nm,
Chromatographic column be the anti-phase C18 posts of 100 × 500mm, mobile phase：A phases：0.3%TFA/ acetonitrile solutions (v/v)；
B phases：Acetonitrile, gradient：B%：38%～68%, flow velocity：6 ml/mins, collect purpose peak fraction,
Rotary evaporation is concentrated, and lyophilizing obtains ularitide fine peptide.
The oxidation reaction of ularitide linear peptides is carried out according to the method for the invention, reaction can be obtained
The purity of the thick peptide of ularitide improve to 62.47-77.52%, and the thick peptide of existing liquid-phase oxidation
Purity is only 48.52%, can reach 58.3-74.7% in the ularitide total recovery through the present invention after purification,
And existing method total recovery after purification is because 36.3%, both compare has extremely significant difference.
Based on the beneficial effect brought using ionic liquid in ularitide linear peptides oxidation reaction, this
Bright offer ionic liquid aoxidizes the application to be formed in ularitide in ularitide linear peptides.
Wherein, preferably, the ionic liquid is ionic liquid of the fusing point below 20 DEG C.More preferably
Ground, the cation of the ionic liquid is 1- ethyl-3-methylimidazoles cation or 1- butyl -3- Methylimidazole .s
Cation.
Used as preferred or further preferred scheme, the ionic liquid is 1- ethyl-3-methylimidazole acetate
[C2mim] [OAc], 1- ethyl-3-methylimidazole trifluoroacetates [C2mim] [TFA], 1- ethyl -3- methyl
Limidazolium hexafluorophosphate [C2mim] [PF6], 1- butyl -3- Methylimidazole. acetate [C4mim] [OAc], 1-
Butyl -3- Methylimidazole. trifluoroacetates [C4mim] [TFA] or 1- butyl -3- Methylimidazole. hexafluorophosphates
[C4mim][PF6]。
Ionic liquid carrier of the present invention can be according to the field after it specify that cation and anion
General direct synthesis technique or two-step method are synthesized, and also can be obtained by commercially available purchase.
From above technical scheme, the present invention is instead of in conventional oxidation link with ionic liquid at room temperature
The liquid phase oxidation medium of organic solvent and water so that the thick peptide purity of ularitide of solid phase synthesis and fine peptide
Total recovery is significantly improved, and the simultaneous oxidation response time can be greatly decreased, easy to operate, it is to avoid organic
The generation of waste liquid.
Specific embodiment
The invention discloses a kind of synthetic method of ularitide, those skilled in the art can use for reference herein
Content, is suitably modified technological parameter realization.Specifically, all similar replacements and change
Apparent to those skilled in the art, they are considered as being included in the present invention.The present invention
Synthetic method be described by preferred embodiment, related personnel substantially can be without departing from this
Compound as herein described and preparation method are modified or are suitably become in bright content, spirit and scope
More with combine, realize and apply the technology of the present invention.
Some abbreviations and Key Term in the present invention are often defined as follows during table, protected amino acid etc. synthesize
Gill biochemical corp, Tianjin Nankai are purchased from and into company with reagent and material.
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| Boc | Tertbutyloxycarbonyl |
| tBu | The tert-butyl group |
| Trt | Trityl |
| Pbf | 2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls |
| DMF | N,N-dimethylformamide |
| DCM | Dichloromethane |
| DBLK | 20% hexahydropyridine/DMF solution |
| DIC | N, N- DIC |
| DIPEA | N, N- diisopropylethylamine |
| PyBOP | Hexafluorophosphoric acid benzotriazole -1- bases-epoxide tripyrrole alkyl |
| TBTU | O- BTA-N, N, N', N'- tetramethylurea Tetrafluoroboric acid |
| HBTU | O- BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid |
| HOBT | I-hydroxybenzotriazole |
| HOAT | 1- -7 azos of hydroxyl-benzotriazole |
| TFA | Trifluoroacetic acid |
| EDT | 1,2- dithioglycols |
| PhOH | Phenol |
| TIS | Tri isopropyl silane |
In a specific embodiment, if not otherwise specified, the present invention is being contrasted with existing method for oxidation
During test, the thick peptide of ularitide linear peptides for being used or fine peptide are derived from a same solid phase synthesis,
To guarantee the comparability of contrast test.
With reference to embodiment, the present invention is expanded on further.
Embodiment 1：The preparation of the thick peptide of ularitide linear peptides
By way of solid phase synthesis one by one, synthesize ularitide 1mol, obtain thick peptide 3510g, thick peptide
Yield 100.1%, purity 78.43%.
Embodiment 2：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 5.0L [C2mim] [OAc] and (aoxidizing dense
Spend for 20mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 2.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust solution ph for acidity,
Oxidation is completed, and ularitide crude product purity is 62.47%.
Embodiment 3：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 5.0L [C2mim] [TFA] and (aoxidizing dense
Spend for 20mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 2.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 76.81%.
Embodiment 4：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 2.0L [C2mim] [TFA] and (aoxidizing dense
Spend for 50mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 75.48%.
Embodiment 5：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0L [C2mim] [TFA] and (aoxidizing dense
Spend for 100mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 76.01%.
Embodiment 6：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 0.5L [C2mim] [TFA] and (aoxidizing dense
Spend for 200mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 0.5h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 73.05%.
Embodiment 7：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0L [C2mim] [PF6] in (and oxidation it is dense
Spend for 100mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 77.11%.
Embodiment 8：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0L [C4mim] [OAc] and (aoxidizing dense
Spend for 100mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 70.49%.
Embodiment 9：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0L [C4mim] [TFA] and (aoxidizing dense
Spend for 100mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 76.39%.
Embodiment 10：The thick peptide oxidation reaction of ularitide linear peptides
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0L [C4mim] [PF6] in (and oxidation it is dense
Spend for 100mg/mL), it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature,
Oxidation reaction 1.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation
Complete.The crude product purity for obtaining ularitide is 77.52%.
Embodiment 11：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 2, using Waters 2454RP-HPLC systems, ripple
Long 220nm, chromatographic column be the anti-phase C18 posts of 100 × 500mm, mobile phase：A phases：0.3%TFA/
Acetonitrile solution (v/v)；B phases：Acetonitrile, gradient：B%：38%～68%, flow velocity：6 ml/mins,
Purpose peak fraction is collected, rotary evaporation concentration, lyophilizing obtain ularitide fine peptide 58.32g, HPLC purity
99.34%, total recovery 58.3%.
Embodiment 12：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 3, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 72.58g, HPLC purity 99.15%, total recovery 72.6%.
Embodiment 13：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 4, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 71.94g, HPLC purity 99.27%, total recovery 72.0%.
Embodiment 14：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 5, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 73.08g, HPLC purity 99.85%, total recovery 73.1%.
Embodiment 15：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 6, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 67.44g, HPLC purity 99.01%, total recovery 67.5%.
Embodiment 16：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 7, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 74.13g, HPLC purity 99.30%, total recovery 74.2%.
Embodiment 17：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 8, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 65.59g, HPLC purity 99.20%, total recovery 65.6%.
Embodiment 18：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 9, using the purification process of embodiment 11, lyophilizing is obtained
Ularitide fine peptide 73.25g, HPLC purity 99.47%, total recovery 73.3%.
Embodiment 19：The preparation of ularitide fine peptide
Thick peptide solution after aoxidizing in Example 10, using the purification process of embodiment 11, lyophilizing is obtained
To ularitide fine peptide 74.67g, HPLC purity 99.36%, total recovery 74.7%.
Embodiment 20：The oxidation reaction of existing organic solvent/aqueous solution liquid phase ularitide
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0LDMF, adds the dilution of 4L water
To 5L, it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature, oxidation reaction
24.0h, plus TFA aqueous solutions that 0.5L concentration is 0.1% adjust pH value for acidity, oxidation is completed.Nothing
Method obtains target product, the dimer to form intermolecular disulfide bond for obtaining.
Embodiment 21：The oxidation reaction of existing organic solvent/aqueous solution liquid phase ularitide
The thick peptide 100g of linear peptides that Example 1 is obtained, is dissolved in 1.0LDMF, adds 99L water dilute
Release to 100L, it is 7.0-7.5 alkaline environments to adjust pH value of solution with ammonia, gentle agitation under room temperature, oxidation
Reaction 48.0h (HPLC monitors reaction process), plus the TFA aqueous solutions tune that 0.5L concentration is 0.1%
Section pH value is acidity, and oxidation is completed.The crude product purity for obtaining ularitide is 48.52%.
Embodiment 22：The preparation of fine peptide after the oxidation of existing organic solvent/aqueous solution liquid phase ularitide
Thick peptide solution after aoxidizing in Example 21, obtains 5L reactant liquors, using embodiment after concentration
11 same preparation methoies, lyophilizing obtain ularitide fine peptide 36.58g, HPLC purity 99.31%, always
Yield 36.6%.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, some improvement and profit can also be made
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (12)
1. a kind of synthetic method of ularitide, it is characterised in that include：
Ularitide linear peptides thick peptide or fine peptide are added in ionic liquid of the fusing point below 20 DEG C,
0.5-3.0h is aoxidized under alkalescence condition, acquisition forms the ularitide crude product of disulfide bond.
2. synthetic method according to claim 1, it is characterised in that the cation of the ionic liquid
For 1- ethyl-3-methylimidazoles cation or 1- butyl -3- methyl imidazolium cations.
3. synthetic method according to claim 1 or claim 2, it is characterised in that the ionic liquid is 1-
Ethyl-3-methylimidazole acetate [C2mim] [OAc], 1- ethyl-3-methylimidazole trifluoroacetates
[C2mim] [TFA], 1- ethyl-3-methylimidazole hexafluorophosphate [C2mim] [PF6], 1- butyl -3- methyl
Imidazoles acetate [C4mim] [OAc], 1- butyl -3- Methylimidazole. trifluoroacetates [C4mim] [TFA] or 1-
Butyl -3- Methylimidazole. hexafluorophosphate [C4mim] [PF6]。
4. synthetic method according to claim 1, it is characterised in that ularitide line during the oxidation
Property the thick peptide of peptide or fine peptide concentration be 20-200mg linear peptides/mL ionic liquids.
5. synthetic method according to claim 1, it is characterised in that the ularitide linear peptides are thick
Peptide or fine peptide are prepared by process for solid phase synthesis.
6. synthetic method according to claim 1, it is characterised in that also include ularitide crude product
Carry out purification.
7. synthetic method according to claim 6, it is characterised in that the purification is HPLC purification.
8. synthetic method according to claim 7, it is characterised in that the HPLC purification steps are：
The ularitide crude product after oxidation is taken, using Waters 2454RP-HPLC systems, wavelength 220nm,
Chromatographic column be the anti-phase C18 posts of 100 × 500mm, mobile phase：A phases：0.3%TFA/ acetonitrile solutions (v/v)；
B phases：Acetonitrile, gradient：B%：38%～68%, flow velocity：6 ml/mins, collect purpose peak fraction,
Rotary evaporation is concentrated, and lyophilizing obtains ularitide fine peptide.
9. ionic liquid aoxidizes the application to be formed in ularitide in ularitide linear peptides.
10. apply according to claim 9, it is characterised in that the ionic liquid is fusing point 20
Ionic liquid below DEG C.
11. apply according to claim 10, it is characterised in that the cation of the ionic liquid is
1- ethyl-3-methylimidazoles cation or 1- butyl -3- methyl imidazolium cations.
12. apply according to claim 10 or 11, it is characterised in that the ionic liquid is 1-
Ethyl-3-methylimidazole acetate [C2mim] [OAc], 1- ethyl-3-methylimidazole trifluoroacetates
[C2mim] [TFA], 1- ethyl-3-methylimidazole hexafluorophosphate [C2mim] [PF6], 1- butyl -3- methyl
Imidazoles acetate [C4mim] [OAc], 1- butyl -3- Methylimidazole. trifluoroacetates [C4mim] [TFA] or 1-
Butyl -3- Methylimidazole. hexafluorophosphate [C4mim] [PF6]。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510644646.8A CN106554407B (en) | 2015-09-30 | 2015-09-30 | Synthetic method of ularitide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510644646.8A CN106554407B (en) | 2015-09-30 | 2015-09-30 | Synthetic method of ularitide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106554407A true CN106554407A (en) | 2017-04-05 |
| CN106554407B CN106554407B (en) | 2020-02-04 |
Family
ID=58417771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510644646.8A Expired - Fee Related CN106554407B (en) | 2015-09-30 | 2015-09-30 | Synthetic method of ularitide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106554407B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018205402A1 (en) * | 2017-05-10 | 2018-11-15 | 深圳翰宇药业股份有限公司 | Synthetic method for ularitide |
| CN110845599A (en) * | 2018-08-21 | 2020-02-28 | 鲁南制药集团股份有限公司 | Preparation and purification method of polypeptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103145827A (en) * | 2013-03-04 | 2013-06-12 | 吉尔生化（上海）有限公司 | Solid-phase synthesis method of ularitide |
| CN104371018A (en) * | 2014-01-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Ularitide preparation method |
-
2015
- 2015-09-30 CN CN201510644646.8A patent/CN106554407B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103145827A (en) * | 2013-03-04 | 2013-06-12 | 吉尔生化（上海）有限公司 | Solid-phase synthesis method of ularitide |
| CN104371018A (en) * | 2014-01-22 | 2015-02-25 | 江苏汉邦科技有限公司 | Ularitide preparation method |
Non-Patent Citations (3)
| Title |
|---|
| ALESIA A. TIETZE: "Ionic Liquid Applications in Peptide Chemistry:", 《MOLECULES》 * |
| MIRIAM BÖHM: "Ionic liquids as reaction media for oxidative folding and native chemical", 《JOURNAL OF MOLECULAR LIQUIDS》 * |
| PASCAL HEIMER: "Application of Room-Temperature Aprotic and Protic Ionic", 《CHEMBIOCHEM》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018205402A1 (en) * | 2017-05-10 | 2018-11-15 | 深圳翰宇药业股份有限公司 | Synthetic method for ularitide |
| CN108864275A (en) * | 2017-05-10 | 2018-11-23 | 深圳翰宇药业股份有限公司 | A kind of synthetic method of Ularitide |
| CN108864275B (en) * | 2017-05-10 | 2019-12-03 | 深圳翰宇药业股份有限公司 | A kind of synthetic method of Ularitide |
| CN110845599A (en) * | 2018-08-21 | 2020-02-28 | 鲁南制药集团股份有限公司 | Preparation and purification method of polypeptide |
| CN110845599B (en) * | 2018-08-21 | 2022-10-14 | 鲁南制药集团股份有限公司 | Preparation and purification method of polypeptide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106554407B (en) | 2020-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11518794B2 (en) | Synthesis method for liraglutide with low racemate impurity | |
| CN102875665B (en) | Method for synthesizing liraglutide | |
| US20080287650A1 (en) | High purity peptides | |
| EP3398957B1 (en) | Method for synthesizing etelcalcetide | |
| CN104017064B (en) | A kind of method preparing teriparatide | |
| CN105017387B (en) | A method of preparing Linaclotide | |
| CN105384809B (en) | A kind of method that segment method solid-liquid combination prepares Teriparatide | |
| CN104031127B (en) | A kind of solid-liquid combination prepares the method for bivalirudin | |
| DE602004001727T2 (en) | PROCESS FOR PREPARING CYCLIC PEPTIDES | |
| CN111732651B (en) | Method for preparing Somalutide through continuous flow solid phase reaction | |
| CN106892968A (en) | A kind of synthetic method of Linaclotide | |
| CN112386707B (en) | Tumor targeting polypeptide drug conjugate and preparation method thereof | |
| US20170260247A1 (en) | Method For Synthesizing Degarelix | |
| CN111087462B (en) | Solid-phase synthesis method of abamectin | |
| CN107216374A (en) | A kind of synthetic method of ziconotide | |
| CN111732650A (en) | Continuous flow solid phase reaction preparation of Somaloutide | |
| CN106554391B (en) | Method for synthesizing marine biological peptide Xen2174 | |
| CN106478805B (en) | Preparation method of GLP-1 derivative | |
| CN106554407A (en) | A kind of synthetic method of ularitide | |
| US9150615B2 (en) | Process for the preparation of leuprolide and its pharmaceutically acceptable salts | |
| Akaji et al. | Regioselective double disulfide formation using silylchloride-sulfoxide system | |
| CN108070030B (en) | Preparation method of loxenapeptide and analogue thereof | |
| CN111057129B (en) | Preparation method and kit for synthesizing polypeptide containing two pairs of disulfide bonds, and preparation method of pramipexole | |
| CN106554406B (en) | Synthetic method of ularitide | |
| CN103992401B (en) | Method for preparing exenatide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200204 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |