CN110423251A - A kind of preparation method of Suo Malu peptide side chain - Google Patents
A kind of preparation method of Suo Malu peptide side chain Download PDFInfo
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- CN110423251A CN110423251A CN201910751757.7A CN201910751757A CN110423251A CN 110423251 A CN110423251 A CN 110423251A CN 201910751757 A CN201910751757 A CN 201910751757A CN 110423251 A CN110423251 A CN 110423251A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/50—Organo-phosphines
- C07F9/53—Organo-phosphine oxides; Organo-phosphine thioxides
- C07F9/5325—Aromatic phosphine oxides or thioxides (P-C aromatic linkage)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a kind of preparation methods of Suo Malu peptide side chain, include the following steps: 1 analog of blocking group compound 1 and compound of (A) synthesis；(B) synthesis of compound 3；(C) synthesis of compound 4；(D) synthesis of compound 6；(E) synthesis of compound 8；(F) synthesis of compound 9；(G) synthesis of compound 11；(H) synthesis of compound 12；(I) the inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, up to Suo Malu peptide fine work after exclusion chromatography purification and desalination.The polypeptide products purity of present invention auxiliary group containing special elements is higher to be prepared without multiple high pressure liquid phase, is significantly simplified production process while being reduced production cost.
Description
Technical field
The present invention relates to pharmaceutical chemistry synthesis technical fields, and in particular to a kind of preparation method of Suo Malu peptide side chain.
Background technique
Chinese name: Suo Malu peptide, English name: Sermaglutide, molecular formula: C187H291N45O59, molecular weight:
4113.23。
Structural formula:
Suo Malu peptide is the GLP-1 analog of Novo Nordisk Co., Ltd's research and development, and Suo Malu peptide is designed with unique structure, one
Blood concentration is highly stable after all one injections, has very good effect of lowering blood sugar, while having both the curative effect of weight-reducing.
Nearly 10 years glucose-lowering treatments progress, the appearance of GLP-1 receptor stimulating agent (GLP-1RAs) are modern most promising sugar
Urinate one of medicine.GLP-1RAs stimulates the insulin secretion of beta Cell of islet by dependence on the glucose and inhibits alpha Cell of islet
Glucagon is secreted to reduce blood glucose, while satiety can be promoted (to directly affect brain GLP1 receptor and delay stomach row
It is empty), and then lose weight.
In existing patent document, the preparation method of Suo Malu peptide is mostly solid-liquid synthetic method, and synthetic route is also similar,
And heteropolymer supporter is expensive, occupies most costs of rapidoprint, generates a large amount of polymer support
Waste, it is difficult to expand scale, be unfavorable for industrialized production.
In consideration of it, this patent develops a kind of highly-solid selectively Liquid phase peptides synthesis technology, according to comprising certain " specific
The reaction product of functional group " in specific solvent with specific solubility property (it is solvable in certain solvents, in certain solvents
It is insoluble), by, come washing reaction crude product, directly obtaining the purifying chemistry of clean product with specific solvent or solvent combination.
Lack a kind of preparation side of Suo Malu peptide side chain that high-purity and high yield are prepared using liquid phase synthesis techniques at present
Method.
Summary of the invention
The present invention solves a kind of side of Suo Malu peptide side chain that high-purity and high yield are prepared using liquid phase synthesis techniques
Method.
To realize the above-mentioned technical purpose, The technical solution adopted by the invention is as follows: a kind of Suo Malu peptide side chain of the invention
The blocking group compound 1 and 1 analog of compound of synthesis are respectively as shown in formula (I) and formula (II):
Formula (I)
Formula (II).
A kind of system of 1 analog of blocking group compound 1 and compound of Suo Malu peptide side chain synthesis of the present invention
Preparation Method includes the following steps: to be dissolved in diphenylphosphine oxygroup Bian alcohol (0.26mol) in the dry tetrahydrofuran solution of 400mL,
Be cooled to -5 DEG C, sodium hydrogen (0.39mol) be added portionwise, after being stirred 50 minutes under ice bath, be added dropwise to chlorobenzene methanol (50.7g,
Dry tetrahydrofuran solution (100mL) 0.26mol) keeps interior temperature less than 5 degree；After drop finishes, it is stirred overnight at room temperature, in
It is about 8 that saturated ammonium chloride liquid to aqueous pH values are carefully instilled under ice bath, adds ethyl acetate liquid separation extraction three times, combined ethyl acetate
Layer, twice with saturated salt solution backwash, dry crude product compound 1 or compound 1 analog.
A kind of preparation method of Suo Malu peptide side chain of the invention, includes the following steps:
(A) 1 analog of blocking group compound 1 and compound synthesized；
(B) synthesis of compound 3；
(C) synthesis of compound 4；
(D) synthesis of compound 6；
(E) synthesis of compound 8；
(F) synthesis of compound 9；
(G) synthesis of compound 11；
(H) synthesis of compound 12；
(I) purifying of crude product Suo Malu peptide side chain；
The inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, up to Suo Malu after exclusion chromatography purification and desalination
Peptide fine work；
Wherein, compound 1 is (4- ((4- benzylalcohol) methoxyl group) phenyl) diphenylphosphine oxide, compound 2 is [2- [2-
((fluorenes methoxy carbonyl) amino) ethyoxyl] ethyoxyl] acetic acid；Compound 3 is (4- ((4- ((([2- [2- (amino) ethyoxyl] ethoxy
Base] second rouge) methoxyl group) phenyl) methoxyl group) phenyl diphenylphosphine oxide, compound 4 be (4- ((4- ((((2- (2- ((2-
(2- (amino) ethyoxyl) ethyoxyl) acetyl group) ethyoxyl] ethyoxyl] second rouge) methoxyl group) phenyl) methoxyl group) phenyl) hexichol
Base phosphorous oxides, compound 5 are N- fluorenylmethyloxycarbonyl--1 tert-butyl ester of Pidolidone, compound 6 is [4- [[4- [[[2- [2-
[[2- [2- (- 1 tert-butyl ester of glutamine) ethyoxyl] ethyoxyl] acetyl group] ethyoxyl] ethyoxyl] second rouge] methoxyl group] phenyl]
Methoxyl group] phenyl] diphenylphosphine oxide, compound 7 be octadecane diacid tert-butyl monoesters, compound 8 is [4- [[4-
[[17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- ethyl ester) ethyoxyl] ethylamino- formoxyl] methoxyl group] second
Oxygroup] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester] methoxyl group] phenyl] methoxyl group] phenyl] diphenyl
Phosphorus oxidation, compound 9 are 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- acetic acid) ethyoxyl] ethylamino- first
Acyl group] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester, compound 10 be N- hydroxyl
Succinimide, compound 11 are 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidins -
1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formyl
Base] the Heptadecanoic acide tert-butyl ester, compound 12 be 17- [(S) -1- carboxyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrroles
Alkane -1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino
Formoxyl] Heptadecanoic acide.
Further, in step (E), with compound 1, [2- [2- (Fmoc- amino) ethyoxyl] ethyoxyl]-acetic acid
(2), N- fluorenylmethyloxycarbonyl--1 tert-butyl ester of Pidolidone (5) and octadecane diacid tert-butyl monoesters (7) are raw material, 1- hydroxyl -
7- azo benzotriazole (HOAt)/N, N- diisopropylcarbodiimide (DIC)/N, N- diisopropylethylamine (DIEA), diformazan ammonia
Yl pyridines (DMAP) are condensation reagent, synthesize compound 8；
In step (F), blocking group is removed under trifluoracetic acid cracking, obtains compound 9；
In step (G), compound 9 is condensed to obtain 17- [the tertiary fourth of (S) -1- with n-hydroxysuccinimide (HOSU, 10) again
Oxygen carbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- first
Acyl group] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester (11), in step (H),
17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base oxygroup carbonyls are obtained after deprotection
Ylmethoxy) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] heptadecane
Tert-butyl acrylate (12).
Further, in step (A), the analog of compound 1 or compound 1 the preparation method is as follows:
Diphenylphosphine oxygroup Bian alcohol (0.26mol) is dissolved in the dry tetrahydrofuran solution of 400mL, is cooled to -5 DEG C,
Sodium hydrogen (0.39mol) is added portionwise, after being stirred 50 minutes under ice bath, is added dropwise to the dry of chlorobenzene methanol (50.7 g, 0.26mol)
Dry tetrahydrofuran solution (100mL) keeps interior temperature less than 5 degree.After drop finishes, it is stirred overnight, is carefully dripped under ice bath at room temperature
Entering saturated ammonium chloride liquid to aqueous pH values is about 8, adds ethyl acetate liquid separation extraction three times, and combined ethyl acetate layer is eaten with saturation
Brine twice, dry product compound 1 or compound 1 analog.
Further, in step (B), the synthesis of compound 3
Accurately weigh in 100mg step A synthesized blocking group compound 1, compound 2 (1.2-2.0eq) and
CH2Cl2(10mL) is stirred in 20mL screw cap vial at 0 DEG C；It is added DIEA (1.5-3.5eq), is stirred to react 10 minutes,
The DMAP of 0.2-0.6eq is added at this time, at room temperature, condensation reaction mixture is stirred into 2h；Condensation reaction is A+D+E condensation body
System,
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice；It will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected；By the way that crude mixture is dissolved in most
In the ethyl acetate of small equivalent, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid,
98% or more HPLC purity.
Further, in step (C), the synthesis of compound 4
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF of 752mg compound 2:
In DCM (volume ratio 4:1,10ml) solution, after mixing 15min, it is eventually adding 420mg DIEA, is mixed；The mixture is added to
Above-mentioned gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, takes the CH of 20% piperidines2Cl2Solution；Product is in CH2Cl2Layer, collection have
Machine mutually carries out being concentrated in vacuo to obtain solid, 98% or more HPLC purity.
Further, in step (D), the synthesis of compound 6
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF of 860mg compound 5:
In DCM (volume ratio 4:1,10ml) solution, after hybrid reaction 15min, it is eventually adding 420mg DIEA, is mixed；By the mixture
Add to above-mentioned gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；Take 20% piperidines
CH2Cl2Solution；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid,
98% or more HPLC purity.
Further, in step (E), the synthesis of compound 8
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds 312mg compound 7
In DMF:DCM (volume ratio 4:1,10ml) solution, after mixing 15min, the DIEA of 420mg is added；The mixture is added to
State gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and ninhydrin detects reaction process；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer, takes the CH of 20% piperidines2Cl2Solution；Collection has
Machine mutually carries out being concentrated in vacuo to obtain solid, 98% or more HPLC purity.
Further, in step (F), the synthesis of compound 9
Compound 8 is added in methanol (150ml), appropriate trifluoracetic acid is then added and cracks 30 minutes, filtering reaction is mixed
It closes object and filtrate is evacuated to by drying by diatomite, which is dissolved in the mixture of 10% acetic acid (aqueous solution) and ethyl alcohol by crude oil
It is middle to use chloroform, chloroform is then washed twice with water, evacuates water layer, obtains compound 9, is yellow oil；
In step (G), the synthesis of compound 11
It accurately weighs 150mg compound 10 to be added in reaction vessel, adds 10ml DCC, finally add to above-mentioned gained again
In the THF solution (20ml) of compound 9, after reaction 1h is stirred at room temperature；Filter out the white solid DCU that reaction generates, filtrate decompression
Concentration removes solvent；DCM (25ml) is added in concentrate, is washed with water (25ml × 2), anhydrous magnesium sulfate dries, filters, filter
Liquid is concentrated under reduced pressure, and obtains yellow oil 11；
In step (H), the synthesis of compound 12
Compound 11 is slowly added to 5m1 lysate (TFA:TIS:H2O=95:3:2), reaction 2h is stirred at room temperature；Decompression
Concentration removing TFA, the ether for adding 20ml to be pre-chilled in advance, and after -20 DEG C of precipitating lh, centrifugation, obtained solid 20ml cold ether
It washs again 2 times, after reduced pressure at room temperature 4h, obtains 502mg white solid 12, crude product Suo Malu peptide side chain is made.
In step (I), the purifying of crude product Suo Malu peptide side chain
Purifying using reversed high performance liquid chromatography to the crude product Suo Malu peptide side chain of synthesis, detection use C18 points
Column is analysed, carries out efficient liquid phase chromatographic analysis, wherein mobile phase: A phase: water, 0.05%TFA；B phase: acetonitrile, 0.05%TFA are washed
De- gradient: (0-5min:A 75%；5-15min:A 75%~60%；15-25min:A 60%~55%；25-35min:A
55%-10%)；Elution speed is 1ml/min, and elution time takes 35min, Detection wavelength 215nm, 35 DEG C of column temperature；Collect target
The solution at peak, concentrated, freeze-drying, is made the sterling of Suo Malu peptide side chain.
Further, in step (B), the protected amino acid is that the group protected for alpha-amido is closed in solid-phase peptide
Usually there are Boc (tertiary butyloxycarbonyl), Moz (to methoxyl group benzyloxy carbonyl) and Fmoc (9- fluorenes methylene oxygen carbonyl) in；The protection side
Formula is Fmoc (9- fluorenes methylene oxygen carbonyl)；Condensing agent system in condensation reaction is C or A+D+E or A+D or A+B+C, and wherein A is
HOBT or HOAT, B HATU, HBTU or TBTU any of which, C are DIEA or TMP, D DIC, E DAMP.
The utility model has the advantages that highly-solid selectively Liquid phase peptides synthesis technology of the present invention, assists base containing special elements
The polypeptide products purity of group is higher to be prepared without multiple high pressure liquid phase, is significantly simplified production process while being reduced and is produced into
This.
Compared with prior art, the present invention has the advantage that
(1) these external auxiliary functional groups of the present invention can not only control the dissolubility of product, moreover it is possible to control the change of reaction
Learn the stereoselectivity of selectivity and product.And after purified product, the prothetic group of introducing can be recycled with reclaiming.This
The technology of invention can be widely applied to preparation Suo Malu peptide, Liraglutide, etc. polypeptides.
(2) purification process of the present invention is simple, and purification process is taken into account among reaction process, realize reaction process with it is pure
The combination of change method.The type auxiliary group is easy to crack, and can be recycled with reclaiming, high-efficiency environment friendly；The present invention is adopted
Highly-solid selectively Liquid phase peptides synthesis technology, substantially reduces resin and solvent usage amount, and it is extensive to be conducive to industrialization
Production.
(3) highly-solid selectively Liquid phase peptides synthesis technology of the present invention avoids a large amount of three in synthesis in solid state
Useless generation.
Specific embodiment
The present invention is further illustrated by the following examples.It should be understood that these embodiments are explainations of the invention
And citing, and the range that the invention is not limited in any way.
Embodiment 1
The blocking group compound 1 and 1 analog of compound of a kind of Suo Malu peptide side chain synthesis of the invention are respectively such as formula
(I) and shown in formula (II):
Formula (I)
N=1,2,3,4,5,6,7,8
Formula (II).
A kind of system of 1 analog of blocking group compound 1 and compound of Suo Malu peptide side chain synthesis of the present invention
Preparation Method includes the following steps: to be dissolved in diphenylphosphine oxygroup Bian alcohol (0.26mol) in the dry tetrahydrofuran solution of 400mL,
Be cooled to -5 DEG C, sodium hydrogen (0.39mol) be added portionwise, after being stirred 50 minutes under ice bath, be added dropwise to chlorobenzene methanol (50.7g,
Dry tetrahydrofuran solution (100mL) 0.26mol) keeps interior temperature less than 5 degree；After drop finishes, it is stirred overnight at room temperature, in
It is about 8 that saturated ammonium chloride liquid to aqueous pH values are carefully instilled under ice bath, adds ethyl acetate liquid separation extraction three times, combined ethyl acetate
Layer, twice with saturated salt solution backwash, dry crude product compound 1 or compound 1 analog.
A kind of preparation method of Suo Malu peptide side chain of the invention, includes the following steps:
(A) 1 analog of blocking group compound 1 and compound synthesized；
(B) synthesis of compound 3；
(C) synthesis of compound 4；
(D) synthesis of compound 6；
(E) synthesis of compound 8；
(F) synthesis of compound 9；
(G) synthesis of compound 11；
(H) synthesis of compound 12；
(I) purifying of crude product Suo Malu peptide side chain；
The inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, up to Suo Malu after exclusion chromatography purification and desalination
Peptide fine work；
Wherein, compound 1 is (4- ((4- benzylalcohol) methoxyl group) phenyl) diphenylphosphine oxide, compound 2 is [2- [2-
((fluorenes methoxy carbonyl) amino) ethyoxyl] ethyoxyl] acetic acid；Compound 3 is (4- ((4- ((([2- [2- (amino) ethyoxyl] ethoxy
Base] second rouge) methoxyl group) phenyl) methoxyl group) phenyl diphenylphosphine oxide, compound 4 be (4- ((4- ((((2- (2- ((2-
(2- (amino) ethyoxyl) ethyoxyl) acetyl group) ethyoxyl] ethyoxyl] second rouge) methoxyl group) phenyl) methoxyl group) phenyl) hexichol
Base phosphorous oxides, compound 5 are N- fluorenylmethyloxycarbonyl--1 tert-butyl ester of Pidolidone, compound 6 is [4- [[4- [[[2- [2-
[[2- [2- (- 1 tert-butyl ester of glutamine) ethyoxyl] ethyoxyl] acetyl group] ethyoxyl] ethyoxyl] second rouge] methoxyl group] phenyl]
Methoxyl group] phenyl] diphenylphosphine oxide, compound 7 be octadecane diacid tert-butyl monoesters, compound 8 is [4- [[4-
[[17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- ethyl ester) ethyoxyl] ethylamino- formoxyl] methoxyl group] second
Oxygroup] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester] methoxyl group] phenyl] methoxyl group] phenyl] diphenyl
Phosphorus oxidation, compound 9 are 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- acetic acid) ethyoxyl] ethylamino- first
Acyl group] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester, compound 10 be N- hydroxyl
Succinimide, compound 11 are 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidins -
1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formyl
Base] the Heptadecanoic acide tert-butyl ester, compound 12 be 17- [(S) -1- carboxyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrroles
Alkane -1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino
Formoxyl] Heptadecanoic acide.
In step (B), the synthesis of compound 3
Accurately weigh in 100mg step A synthesized blocking group compound 1, compound 2 (1.2-2.0eq) and
CH2Cl2(10mL) is stirred in 20mL screw cap vial at 0 DEG C；It is added DIEA (1.5-3.5eq), is stirred to react 10 minutes,
The DMAP of 0.2-0.6eq is added at this time, at room temperature, condensation reaction mixture is stirred into 2h；Condensation reaction is A+D+E condensation body
System,
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice；It will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected；By the way that crude mixture is dissolved in most
In the ethyl acetate of small equivalent, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid,
98% or more HPLC purity.
The protected amino acid is that the group protected for alpha-amido usually has Boc (tertiary fourth oxygen in Solid phase peptide synthesis
Carbonyl), Moz (to methoxyl group benzyloxy carbonyl) and Fmoc (9- fluorenes methylene oxygen carbonyl)；The protected mode is Fmoc (9- fluorenes methylene oxygen
Carbonyl)；Condensing agent system in condensation reaction is C or A+D+E or A+D or A+B+C, and wherein A is HOBT or HOAT, B HATU,
HBTU or TBTU any of which, C are DIEA or TMP, D DIC, E DAMP.
In step (C), the synthesis of compound 4
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF of 752mg compound 2:
In DCM (volume ratio 4:1,10ml) solution, after mixing 15min, it is eventually adding 420mg DIEA, is mixed；The mixture is added to
Above-mentioned gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, takes the CH of 20% piperidines2Cl2Solution；Product is in CH2Cl2Layer, collection have
Machine mutually carries out being concentrated in vacuo to obtain solid, 98% or more HPLC purity.
In step (D), the synthesis of compound 6
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF of 860mg compound 5:
In DCM (volume ratio 4:1,10ml) solution, after hybrid reaction 15min, it is eventually adding 420mg DIEA, is mixed；By the mixture
Add to above-mentioned gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；Take 20% piperidines
CH2Cl2Solution；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid,
98% or more HPLC purity.
In step (E), with compound 1, [2- [2- (Fmoc- amino) ethyoxyl] ethyoxyl]-acetic acid (2), N- fluorenes first
- 1 tert-butyl ester of oxygen carbonyl-Pidolidone (5) and octadecane diacid tert-butyl monoesters (7) are raw material, 1- hydroxyl -7- azo benzo
Triazole (HOAt)/N, N- diisopropylcarbodiimide (DIC)/N, N- diisopropylethylamine (DIEA), dimethylamino naphthyridine
(DMAP) it is condensation reagent, synthesizes compound 8；
In step (F), blocking group is removed under trifluoracetic acid cracking, obtains compound 9；
In step (G), compound 9 is condensed to obtain 17- [the tertiary fourth of (S) -1- with n-hydroxysuccinimide (HOSU, 10) again
Oxygen carbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- first
Acyl group] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester (11), in step (H),
17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base oxygroup carbonyls are obtained after deprotection
Ylmethoxy) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] heptadecane
Tert-butyl acrylate (12).
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that: the preparation method of a kind of Suo Malu peptide side chain of the invention, including such as
Lower step:
In step (E), the synthesis of compound 8
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds 312mg compound 7
In DMF:DCM (volume ratio 4:1,10ml) solution, after mixing 15min, the DIEA of 420mg is added；The mixture is added to
State gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and ninhydrin detects reaction process；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice, it will
Combined organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture to be dissolved in most
In a small amount of ethyl acetate, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml, 10min is to remove Fmoc group for processing,
Then it is washed with ammonium chloride to remove excessive piperidines, product is in CH2Cl2Layer, takes the CH of 20% piperidines2Cl2Solution；Collection has
Machine mutually carries out being concentrated in vacuo to obtain solid, 98% or more HPLC purity.
In step (F), the synthesis of compound 9
Compound 8 is added in methanol (150ml), appropriate trifluoracetic acid is then added and cracks 30 minutes, filtering reaction is mixed
It closes object and filtrate is evacuated to by drying by diatomite, which is dissolved in the mixture of 10% acetic acid (aqueous solution) and ethyl alcohol by crude oil
It is middle to use chloroform, chloroform is then washed twice with water, evacuates water layer, obtains compound 9, is yellow oil；
In step (G), the synthesis of compound 11
It accurately weighs 150mg compound 10 to be added in reaction vessel, adds 10ml DCC, finally add to above-mentioned gained again
In the THF solution (20ml) of compound 9, after reaction 1h is stirred at room temperature；Filter out the white solid DCU that reaction generates, filtrate decompression
Concentration removes solvent；DCM (25ml) is added in concentrate, is washed with water (25ml × 2), anhydrous magnesium sulfate dries, filters, filter
Liquid is concentrated under reduced pressure, and obtains yellow oil 11；
In step (H), the synthesis of compound 12
Compound 11 is slowly added to 5m1 lysate (TFA:TIS:H2O=95:3:2), reaction 2h is stirred at room temperature；Decompression
Concentration removing TFA, the ether for adding 20ml to be pre-chilled in advance, and after -20 DEG C of precipitating l h, centrifugation, obtained solid 20ml cold ether
It washs again 2 times, after reduced pressure at room temperature 4h, obtains 502mg white solid 12, crude product Suo Malu peptide side chain is made.
In step (I), the purifying of crude product Suo Malu peptide side chain
Purifying using reversed high performance liquid chromatography to the crude product Suo Malu peptide side chain of synthesis, detection use C18 points
Column is analysed, carries out efficient liquid phase chromatographic analysis, wherein mobile phase: A phase: water, 0.05%TFA；B phase: acetonitrile, 0.05%TFA are washed
De- gradient: (0-5min:A 75%；5-15min:A 75%~60%；15-25min:A 60%~55%；25-35min:A
55%-10%)；Elution speed is 1ml/min, and elution time takes 35min, Detection wavelength 215nm, 35 DEG C of column temperature；Collect target
The solution at peak, concentrated, freeze-drying, is made the sterling of Suo Malu peptide side chain.
Test example 1
The purifying of crude product Suo Malu peptide side chain
Purifying using reversed high performance liquid chromatography to the crude product Suo Malu peptide side chain of synthesis, detection use C18 points
Column is analysed, carries out efficient liquid phase chromatographic analysis, wherein mobile phase: A phase: water, 0.05%TFA；B phase: acetonitrile, 0.05%TFA are washed
De- gradient: (0-5min:A 75%；5-15min:A 75%~60%；15-25min:A 60%~55%；25-35min:A
55%-10%)；Elution speed is 1ml/min, and elution time takes 35min, Detection wavelength 215nm, 35 DEG C of column temperature.Collect target
The solution at peak, concentrated, freeze-drying, obtains the sterling of Suo Malu peptide side chain, it is pure to obtain Suo Malu peptide side chain through peak area normalization method
98% or more degree.
Used reagent is as follows in a kind of new technology of synthesis Suo Malutai side chain provided in the present embodiment:
Sodium borohydride (NaBH4)；Diisopropylethylamine (DIEA)；Diisopropylcarbodiimide (DIC)；Dimethylamino naphthyridine
(DMAP)；1- hydroxyl -7- azo benzotriazole (HOAt)；Piperidines (PIP)；Palladium/carbon (pd/C)；Tri isopropyl silane
(TIS)；Methanol (MeOH)；It is hydrated Ang triketone (IUPAC)；Trifluoroacetic acid (TFA)；Methylene chloride (DCM)；N, N- dimethyl formyl
Amine (DMF)；Acetonitrile (ACN)；Dehydrated alcohol；Ether；Acetic acid；
Experiment reagent involved in this embodiment is formulated as follows:
Deprotection agent: piperidines: DCM=1:4 (volume ratio)
Cracked solution: TFA:TIS:H2O=95:3:2 (volume ratio)
The preparation of detection reagent Kaiser:
Solution a:6g ninhydrin is dissolved in 100ml dehydrated alcohol, slightly hot or stirring dissolution.
Solution b:80g melting phenol is dissolved in 20ml dehydrated alcohol.
Solution c: pyridine collects distillate in addition Ang triketone, is then refluxed for.
Kaiser method of inspection: take three drops, 6% ninhydrin ethanol solution and three 80% phenol of drop anhydrous when detection respectively
Ethanol solution, three drops are steamed pyridine again and are mixed well with resin particle to be detected, 105 DEG C of heating 5min, observation color of resin variation,
If becoming blue or purple, illustrate that free amino acid exists, deprotection is complete；If yellow light tone or colourless solution, then table
Show that condensation is complete.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (10)
1. a kind of 1 analog of blocking group compound 1 and compound of Suo Malu peptide side chain synthesis, it is characterised in that: described
The blocking group compound 1 and 1 analog of compound of Suo Malu peptide side chain synthesis are respectively as shown in formula (I) and formula (II):
2. a kind of system of 1 analog of blocking group compound 1 and compound of Suo Malu peptide side chain synthesis described in claim 1
Preparation Method, it is characterised in that include the following steps: for diphenylphosphine oxygroup Bian alcohol (0.26mol) to be dissolved in the dry tetrahydro of 400mL
In tetrahydrofuran solution, -5 DEG C are cooled to, sodium hydrogen (0.39mol) is added portionwise, after being stirred 50 minutes under ice bath, is added dropwise to chlorobenzene first
The dry tetrahydrofuran solution (100mL) of alcohol (50.7g, 0.26mol) keeps interior temperature less than 5 degree；After drop finishes, stir at room temperature
It mixes overnight, carefully instilling saturated ammonium chloride liquid to aqueous pH values under ice bath is about 8, adds ethyl acetate liquid separation extraction three times, closes
And ethyl acetate layer, twice with saturated salt solution backwash, dry product compound 1 or compound 1 analog.
3. a kind of preparation method of Suo Malu peptide side chain, it is characterised in that include the following steps:
(A) 1 analog of blocking group compound 1 and compound synthesized；
(B) synthesis of compound 3；
(C) synthesis of compound 4；
(D) synthesis of compound 6；
(E) synthesis of compound 8；
(F) synthesis of compound 9；
(G) synthesis of compound 11；
(H) synthesis of compound 12；
(I) purifying of crude product Suo Malu peptide side chain；
The inverted high performance liquid chromatography purifying of Suo Malu peptide crude product, up to Suo Malu peptide essence after exclusion chromatography purification and desalination
Product；
Wherein, compound 1 is (4- ((4- benzylalcohol) methoxyl group) phenyl) diphenylphosphine oxide, compound 2 is [2- [2- ((fluorenes
Methoxy carbonyl) amino) ethyoxyl] ethyoxyl] acetic acid；Compound 3 is (4- ((4- ((([2- [2- (amino) ethyoxyl] ethyoxyl]
Second rouge) methoxyl group) phenyl) methoxyl group) phenyl diphenylphosphine oxide, compound 4 be (4- ((4- ((((2- (2- ((2- (2-
(amino) ethyoxyl) ethyoxyl) acetyl group) ethyoxyl] ethyoxyl] second rouge) methoxyl group) phenyl) methoxyl group) phenyl) diphenyl
Phosphorous oxides, compound 5 are N- fluorenylmethyloxycarbonyl--1 tert-butyl ester of Pidolidone, compound 6 is [4- [[4- [[[2- [2- [[2-
[2- (- 1 tert-butyl ester of glutamine) ethyoxyl] ethyoxyl] acetyl group] ethyoxyl] ethyoxyl] second rouge] methoxyl group] phenyl] methoxy
Base] phenyl] diphenylphosphine oxide, compound 7 be octadecane diacid tert-butyl monoesters, compound 8 is [4- [[4- [[17-
[(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- ethyl ester) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl]
Ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester] methoxyl group] phenyl] methoxyl group] phenyl] diphenyl phosphorus oxygen
Change, compound 9 is 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (1- acetic acid) ethyoxyl] ethylamino- formyl
Base] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester, compound 10 be N- hydroxyl amber
Amber acid imide, compound 11 are 17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1-
Base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl]
The Heptadecanoic acide tert-butyl ester, compound 12 are 17- [(S) -1- carboxyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1-
Base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl]
Heptadecanoic acide.
4. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (E), with chemical combination
Object 1, [2- [2- (Fmoc- amino) ethyoxyl] ethyoxyl]-acetic acid (2), N- fluorenylmethyloxycarbonyl--1 tert-butyl ester of Pidolidone (5)
It is raw material, 1- hydroxyl -7- azo benzotriazole (HOAt)/N, N- diisopropyl carbon two with octadecane diacid tert-butyl monoesters (7)
Imines (DIC)/n,N-diisopropylethylamine (DIEA), dimethylamino naphthyridine (DMAP) are condensation reagent, synthesize compound 8；
In step (F), blocking group is removed under trifluoracetic acid cracking, obtains compound 9；
In step (G), compound 9 is condensed to obtain 17- [(S) -1- tertiary butyloxycarbonyl with n-hydroxysuccinimide (HOSU, 10) again
Base -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base Epoxide carbonyl methoxyl group) ethyoxyl] ethylamino- formoxyl]
Methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester (11), in step (H), deprotection
17- [(S) -1- tertbutyloxycarbonyl -3- [2- [2- [2- [2- [2- (2,5- dioxo pyrrolidin -1- base Epoxide carbonyl methoxies are obtained afterwards
Base) ethyoxyl] ethylamino- formoxyl] methoxyl group] ethyoxyl] ethylamino- formoxyl] Propylamino formoxyl] the Heptadecanoic acide tert-butyl ester
(12)。
5. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (B), compound
3 synthesis
Accurately weigh blocking group compound 1 synthesized in 100mg step A, compound 2 (1.2-2.0eq) and CH2Cl2
(10mL) is stirred in 20mL screw cap vial at 0 DEG C；Be added DIEA (1.5-3.5eq), be stirred to react 10 minutes, at this time plus
Enter the DMAP of 0.2-0.6eq, at room temperature, condensation reaction mixture is stirred into 2h；Condensation reaction is that A+D+E is condensed system,
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs twice；It will merge
Organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected；Worked as by the way that crude mixture is dissolved in minimum
In the ethyl acetate of amount, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml handles 10min to remove Fmoc group, then uses
Ammonium chloride is washed to remove excessive piperidines, and product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid, and HPLC is pure
98% or more degree.
6. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (C), compound
4 synthesis
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF:DCM of 752mg compound 2
In (volume ratio 4:1,10ml) solution, after mixing 15min, it is eventually adding 420mg DIEA, is mixed；The mixture is added to above-mentioned
Gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs, will merge twice
Organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture is dissolved in minimum
Ethyl acetate in, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml handles 10min to remove Fmoc group, then uses
Ammonium chloride is washed to remove excessive piperidines, takes the CH of 20% piperidines2Cl2Solution；Product is in CH2Cl2Layer, collect organic phase into
Row is concentrated in vacuo to obtain solid, 98% or more HPLC purity.
7. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (D), compound
6 synthesis
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF:DCM of 860mg compound 5
In (volume ratio 4:1,10ml) solution, after hybrid reaction 15min, it is eventually adding 420mg DIEA, is mixed；The mixture is added to
Above-mentioned gained CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and reaction process is detected with ninhydrin；Take 20% piperidines
CH2Cl2Solution；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs, will merge twice
Organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture is dissolved in minimum
Ethyl acetate in, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml handles 10min to remove Fmoc group, then uses
Ammonium chloride is washed to remove excessive piperidines, and product is in CH2Cl2Layer collects organic phase and carries out being concentrated in vacuo to obtain solid, and HPLC is pure
98% or more degree.
8. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (E), compound
8 synthesis
265mg HOAt is accurately weighed in 25ml beaker, 300ul DIC is added, adds the DMF:DCM of 312mg compound 7
In (volume ratio 4:1,10ml) solution, after mixing 15min, the DIEA of 420mg is added；The mixture is added into above-mentioned gained
CH2Cl2In layer (after dry), mixed at room temperature reacts 1h, and ninhydrin detects reaction process；
By reaction mixture saturation NH4Cl (aq) is washed twice, then with the Na of saturation2CO3(aq) it washs, will merge twice
Organic layer MgSO4It dries, filters and evacuates, the amino acid slightly protected, by the way that crude mixture is dissolved in minimum
Ethyl acetate in, then with petroleum ether precipitation and filter gained white precipitate；
The CH of 15-25% piperidines is added into white precipitate2Cl2Solution 10ml handles 10min to remove Fmoc group, then uses
Ammonium chloride is washed to remove excessive piperidines, and product is in CH2Cl2Layer, takes the CH of 20% piperidines2Cl2Solution；Collect organic phase into
Row is concentrated in vacuo to obtain solid, 98% or more HPLC purity.
9. the preparation method of Suo Malu peptide side chain according to claim 3, it is characterised in that: in step (F), compound
9 synthesis
Compound 8 is added in methanol (150ml), appropriate trifluoracetic acid is then added and cracks 30 minutes, filters reaction mixture
Filtrate is evacuated to drying by diatomite, which is dissolved in 10% acetic acid (aqueous solution) and the mixture of ethyl alcohol by crude oil uses
Then chloroform is washed twice with water in chloroform, evacuate water layer, obtain compound 9, is yellow oil；
In step (G), the synthesis of compound 11
It accurately weighs 150mg compound 10 to be added in reaction vessel, adds 10ml DCC, finally add to above-mentioned gained chemical combination again
In the THF solution (20ml) of object 9, after reaction 1h is stirred at room temperature；Filter out the white solid DCU that reaction generates, filtrate decompression concentration
Remove solvent；DCM (25ml) is added in concentrate, is washed with water (25ml × 2), anhydrous magnesium sulfate dries, filters, and filtrate subtracts
Pressure concentration, obtains yellow oil 11；
In step (H), the synthesis of compound 12
Compound 11 is slowly added to 5m1 lysate (TFA:TIS:H2O=95:3:2), reaction 2h is stirred at room temperature；Reduced pressure removes
TFA is removed, the ether for adding 20ml to be pre-chilled in advance, and after -20 DEG C of precipitating lh, centrifugation, obtained solid washs 2 with 20ml cold ether again
It is secondary, after reduced pressure at room temperature 4h, 502mg white solid 12 is obtained, crude product Suo Malu peptide side chain is made；In step (I), crude product
The purifying of Suo Malu peptide side chain
Purifying using reversed high performance liquid chromatography to the crude product Suo Malu peptide side chain of synthesis, detection use C18 analytical column,
Efficient liquid phase chromatographic analysis is carried out, wherein mobile phase: A phase: water, 0.05%TFA；B phase: acetonitrile, 0.05%TFA, gradient:
(0-5min:A 75%；5-15min:A 75%~60%；15-25min:A 60%~55%；25-35min:A 55%-
10%)；Elution speed is 1ml/min, and elution time takes 35min, Detection wavelength 215nm, 35 DEG C of column temperature；Collect the molten of target peak
Liquid, concentrated, freeze-drying, is made the sterling of Suo Malu peptide side chain.
10. the preparation method of Suo Malu peptide side chain according to claim 9, it is characterised in that: described in step (B)
Protected amino acid be that the group protected for alpha-amido usually has Boc tertiary butyloxycarbonyl, Moz to methoxyl group in Solid phase peptide synthesis
Benzyloxy carbonyl and Fmoc9- fluorenes methylene oxygen carbonyl；The protected mode is Fmoc9- fluorenes methylene oxygen carbonyl；Condensing agent in condensation reaction
System is C or A+D+E or A+D or A+B+C, and wherein A is HOBT or HOAT, B HATU, HBTU or TBTU any of which, C
For DIEA or TMP, D DIC, E DAMP.
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Cited By (2)
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| CN114685646A (en) * | 2020-12-31 | 2022-07-01 | 厦门赛诺邦格生物科技股份有限公司 | Preparation method and application of polypeptide side chain analogue |
| WO2024032081A1 (en) * | 2022-08-09 | 2024-02-15 | 扬州奥锐特药业有限公司 | Preparation method for semaglutide, and intermediate |
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| CN108697770A (en) * | 2015-12-21 | 2018-10-23 | 得克萨斯技术大学联合体 | System and method for solution phase gap peptide synthesis |
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2019
- 2019-08-15 CN CN201910751757.7A patent/CN110423251A/en active Pending
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| CN108697770A (en) * | 2015-12-21 | 2018-10-23 | 得克萨斯技术大学联合体 | System and method for solution phase gap peptide synthesis |
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|---|---|---|---|---|
| CN114685646A (en) * | 2020-12-31 | 2022-07-01 | 厦门赛诺邦格生物科技股份有限公司 | Preparation method and application of polypeptide side chain analogue |
| CN114685646B (en) * | 2020-12-31 | 2023-04-07 | 厦门赛诺邦格生物科技股份有限公司 | Preparation method and application of polypeptide side chain analogue |
| WO2024032081A1 (en) * | 2022-08-09 | 2024-02-15 | 扬州奥锐特药业有限公司 | Preparation method for semaglutide, and intermediate |
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