CN109456401B - A kind of synthetic method of Suo Malu peptide - Google Patents
A kind of synthetic method of Suo Malu peptide Download PDFInfo
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- CN109456401B CN109456401B CN201811466181.1A CN201811466181A CN109456401B CN 109456401 B CN109456401 B CN 109456401B CN 201811466181 A CN201811466181 A CN 201811466181A CN 109456401 B CN109456401 B CN 109456401B
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Abstract
The invention discloses a kind of synthetic methods of Suo Malu peptide, comprising the following steps: (1) synthesizes the peptide fragment sequences of 6 side chain protections；(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.Using the purity is high of this method synthesis Suo Malu peptide, high income, and synthesis cost is low.
Description
Technical field
The present invention relates to pharmaceutical fields, and in particular to a kind of synthetic method of Suo Malu peptide.
Background technique
Suo Malu peptide, English name Semaglutide is a kind of glucagon-like-peptide-1 (GLP-1) analog, and sequence is
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-Lys (AEEA-AEEA-r-Glu-Otc)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH,
Molecular formula: C187H291N45O59 need only be injected weekly 1 time as a kind of long-acting GLP-1 receptor agonists, can be used to assist
Diet control and movement can reduce patient's weight to improve the glycemic control of type 2 diabetic patient.
The synthetic method of Suo Malu peptide such as patent at present: CN106928343A, CN101133082A, CN106478806A are adopted
Synthesis Suo Malu peptide is gradually coupled with Fmoc strategy solid phase.It is long that the method amino acid is gradually coupled synthesis cycle, reaction not exclusively, Gu
Phase carrier is limited by substitution value, and total recovery is lower, while impurity is more, purification difficult.Patent: CN106749613A uses solid phase
The method of fragment condensation synthesizes, and solid phase segment synthesis step is long, is not easy to amplify, and wastes solvent, generates a large amount of waste liquids.
Summary of the invention
Suo Malu peptide synthesis yield is low, technical problem of high production cost and purifying products hardly possible in order to solve, and the present invention is public
The synthetic method for opening a kind of Suo Malu peptide, using the purity is high of this method synthesis Suo Malu peptide, high income, and synthesis cost is low.
The present invention is achieved through the following technical solutions:
A kind of synthetic method of Suo Malu peptide, comprising the following steps:
(1) peptide fragment sequences of 6 side chain protections are synthesized；
(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.
In the present invention, the peptide fragment sequences of 6 side chain protections of solid phase and liquid phase synthesis are first passed through in advance, by each peptide fragment solid
Gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in phase, and first synthetic peptide fragment is purified, and can greatly reduce final
The impurity that the racemization of Suo Malu peptide product, oxidation, hydrolysis generate greatly reduces impurity research model in the synthesis of bulk pharmaceutical chemicals
It encloses, has saved time and cost, and the short peptide stretch of liquid phase and synthesis in solid state can rapidly purify by conventional method, purity can
Reach 99% or more, it is not necessary to by preparation HPLC purifying, save solvent, reduce later-period purification cost, due to multiple segments
Can be simultaneously synthesizing, and it is easy amplification production, therefore synthesis in solid state reduces synthesis in solid state it is not necessary that amino acid to be coupled one by one
Step improves combined coefficient, reduces the generation of waste liquid, and in final liquid chromatography purification step, impurity is not the absence of
The peptide disappearance of one or several amino acid, but the Partial Fragment not being condensed, not will cause the difficulty in purifying, and efficiency is high and low
Cost, waste liquid be few, easy purification, is suitble to large-scale production.
Wherein, in the step (1), the peptide fragment sequences of 6 side chain protections are specific as follows:
First peptide fragment sequences are the 7-10 amino acids in Suo Malu peptide sequence,
Second peptide fragment sequences are the 11-15 amino acids in Suo Malu peptide sequence,
Third peptide fragment sequences are the 16-22 amino acids in Suo Malu peptide sequence,
4th peptide fragment sequences are the 23-28 amino acids in Suo Malu peptide sequence,
5th peptide fragment sequences are the 29-32 amino acids in Suo Malu peptide sequence,
Hexapeptide fragment sequence is the 33-37 amino acids in Suo Malu peptide sequence.
In the present invention, in the peptide fragment sequences of 6 side chain protections, Asp, Phe, Gly are placed on to the carbon teminal of peptide fragment sequences
First position, improves the condensation efficiency of amino acid, effectively avoids Trp, Ser, Val, Ile, Arg as carbon teminal first
Set to influencing condensation efficiency, and effectively prevent the beta sheet in synthesis in solid state, effectively improve Suo Malu peptide purity and
Yield.
In the present invention, Suo Malu peptide can be effectively reduced impurity, be improved yield, reduced using 6 peptide fragment sequences synthesis
The generation of waste liquid improves combined coefficient, if the number of the peptide fragment sequences divided is excessive, it is low to will lead to solid phase synthesis efficiency
Under, and the more peptide disappearance impurity that will cause of segment increases, and is unfavorable for purifying, seriously affects combined coefficient, excessive segment
Internal quality control for bulk pharmaceutical chemicals intermediate is also a greatly challenge, considerably increases purifying cost, generates a large amount of waste liquids,
Therefore inventor obtains synthesizing Suo Malu peptide, yield, purity using above-mentioned 6 peptide fragment sequences in a large amount of repetition tests
Most preferably, and simultaneously have the advantages that waste liquid that is at low cost, high-efficient, generating is few, and can using above-mentioned 6 peptide fragment sequences
The beta sheet problem encountered in Peptide systhesis is effectively avoided, the yield and purity of Suo Malu peptide are improved.
For Suo Malu peptide, in medicinal application and research, purity usually requires to reach 99.5% or more ability
Meet demand, Suo Malu peptide prepared by the present invention not only high income, and its product purity can reach 96% or more, effectively improve
The quality of drug, and be conducive to the effective progress of Suo Malu peptide research.
In the step (2), the synthesis technology of all risk insurance guard wire horse Shandong peptide straight-chain polypeptide is as follows:
(21) the hexapeptide fragment sequence c-terminus of side chain protection and resin are coupled, and slough its amino protecting group；
(22) the hexapeptide fragment sequence and side chain protection of the side chain protection of amino protecting group will be sloughed obtained by step (21)
The 5th peptide fragment sequences be coupled to obtain the heptapeptide fragment sequence of side chain protection, and slough its amino protecting group；
(23) the heptapeptide fragment sequence of the side chain protection of amino protecting group and the 4th peptide fragment of side chain protection will be sloughed
Coupling sequence obtains the octapeptide fragment sequence of side chain protection, and sloughs its amino protecting group；
(24) the octapeptide fragment sequence of the side chain protection of amino protecting group and the third peptide fragment of side chain protection will be sloughed
Coupling sequence obtains the nonapeptide fragment sequence of side chain protection, and sloughs its amino protecting group；
(25) the nonapeptide fragment sequence of the side chain protection of amino protecting group and the second peptide fragment of side chain protection will be sloughed
Coupling sequence obtains the tenth peptide fragment sequences of side chain protection, and sloughs its amino protecting group；
(26) the tenth peptide fragment sequences of the side chain protection of amino protecting group and the first peptide fragment of side chain protection will be sloughed
Coupling sequence obtains the Suo Malu peptide straight-chain polypeptide of full guard.
Further, in step (2), obtained all risk insurance guard wire horse Shandong peptide straight-chain polypeptide is prepared into Suo Malu peptide.Specifically
Method are as follows:
1) all risk insurance guard wire horse Shandong peptide straight-chain polypeptide is sloughed into the side chain protection of the 26th Lys, and completes modification and obtains all risk insurance
The Suo Malu peptide of shield；
2) the Suo Malu peptide cracking deprotection base of full guard is obtained into the thick peptide of Suo Malu peptide；
3) the purified salt that turns of the thick peptide of Suo Malu peptide obtains Suo Malu peptide.
Further, the first peptide fragment sequences synthesize in liquid-phase system, remaining peptide fragment sequences closes in solid system
At coupling agent used in the synthesis of the peptide fragment sequences of 6 side chain protections is EDCI, HBTU, TBTU, HATU, PyBop, DIC
In any one combination with HOBT or Cl-HOBT by a certain percentage, used organic base is DIEA or TEA；It uses molten
Agent is any one or the combination of several of them in DMF, DCM, NMP and DMSO, and hydrolyzing reagent used is inorganic base, such as LiOH,
KOH, NaOH, used solvent are one or more of THF, MeOH, EtOH and H2The combination of O.
The combination of coupling agent and organic base is preferably the combination of HATU and HOBt and DIEA, to coupling amino acid and HATU and
The molar ratio of DIEA is 1:2:3.Used solvent is preferably DMF.
In the step (21), the resin used is acid sensitive resin, the chloro- trityl chloride resin of preferably 2-, step
(21), (22), (23), (24), in (25), sloughing reagent used by amino protecting group is the piperazine that volumn concentration is 20%
The piperidines that the DMF solution for the DBU that the DMF solution or volumn concentration of pyridine are 1%, preferably volumn concentration are 20%
DMF solution, used coupling agent are any one and HOBT or Cl- in EDCI, HBTU, TBTU, HATU, PyBop, DIC
The combination of HOBT by a certain percentage, used organic base are DIEA or TEA, and preferably HBTU and HOBt and DIEA is in molar ratio
The combination of 1:1:2, used solvent are any one or the combination of several of them in DMF, DCM, NMP and DMSO, preferably DMF.
Further, in step 1), the Side chain protective group of the 26th Lys is any one of Alloc, Mtt, Mmt, Dde,
Preferably Alloc, when protecting group is Alloc, the removing of protecting group uses the combination of phenylsilane, triphenylphosphine palladium and DCM；It is excellent
Choosing, the ratio of phenylsilane, triphenylphosphine palladium and Lys amino acid derivativges is 5:0.2:1, solvent DCM, when protecting group is
When Dde, the removing of protecting group uses the combination of hydrazine hydrate and DMF；When protecting group is Mmt or Mtt, the removing of protecting group is used
The combination of TFA and DCM.
In step 1), after all risk insurance guard wire horse Shandong peptide straight-chain polypeptide sloughs the side chain protection of the 26th Lys, the method for modification
Are as follows: HO-AEEA-AEEA-r-Glu (OtBu)-Otc condensation is added, then sloughs Fmoc group, agents useful for same contains for volume basis
The DMF solution or volume basis for the piperidines that the DMF solution for the piperidines that amount is 20%, preferably volumn concentration are 20%
The DMF solution for the DBU that content is 1%.
Further, in step 2), lysate kind used by the Suo Malu peptide cracking deprotection base technique of full guard
Class is the mixed solution of any combination of TFA, PhSMe, PhOH, EDT and TiS, arbitrary proportion, preferably TFA, PhSMe,
PhOH、EDT、TiS、H2O by volume 80: 5: 5: 2.5: 2.5 mixed solution.
In step 3), the purifying of the thick peptide of Suo Malu peptide turns salt technique specifically: salt is changed in reversed-phase high performance liquid chromatography purifying, flows
Dynamic is mutually the ammonium acetate aqueous solution and acetonitrile solution of percent by volume 0.25%.Chromatographic column is C18 chromatographic column.
For the present invention in final liquid chromatography purification step, impurity is not the absence of the missing of one or several amino acid
Peptide, but the Partial Fragment not being condensed, it is only necessary to which 1-2 preparation purifying, product purity reaches 99.5% or more, and common side
Method synthesis needs 4-5 preparation to purify, and substantially increases purifying yield, the Suo Malu peptide crude product purity energy synthesized by this method
Reach 85% or more, and traditional synthetic method crude product purity can only achieve 60%, it is total by the Suo Malu peptide of preparation after purification
Yield can reach 65% or more, greatly reduce the production cost of Suo Malu peptide.
Suo Malu peptide structure is write a Chinese character in simplified form and is accordingly numbered as follows:
The amino acid sequence of each peptide fragment peptide fragment sequences of target peptide according to the present invention and intermediate is shown in Table 1.
Material abbreviation used in the present invention is shown in Table 2.
The corresponding encoding amino acid sequence of 1: Suo Malu peptide of table
Each segment of Suo Malu peptide structure is write a Chinese character in simplified form as follows:
Table 2: material abbreviation meaning used in the present invention:
Compared with prior art, the present invention having the following advantages and benefits:
1. a kind of synthetic method of Suo Malu peptide of the present invention, first passes through the peptide of 6 side chain protections of solid phase and liquid phase synthesis in advance
Fragment sequence, by each peptide fragment, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase, first synthetic peptide fragment into
Row purifying can greatly reduce the impurity of final Suo Malu peptide product racemization, oxidation, hydrolysis generation, work as in the synthesis of bulk pharmaceutical chemicals
In greatly reduce impurity research range, saved time and cost, and the short peptide stretch of liquid phase and synthesis in solid state can be by normal
Rule method rapidly purifies, and purity can reach 99% or more, it is not necessary to by preparation HPLC purifying, solvent has been saved, after reducing
Phase purify cost, since multiple segments can be simultaneously synthesizing, and be easy amplification production, therefore synthesis in solid state be not necessarily to by amino acid by
A coupling, the step of reducing synthesis in solid state, improve combined coefficient, reduce the generation of waste liquid, and in final liquid chromatogram
In purification step, the Partial Fragment that impurity is not the absence of the peptide disappearance of one or several amino acid, but is not condensed not will cause
Difficulty in purifying, it is high-efficient, inexpensive, waste liquid is few, easy purification, it is suitble to large-scale production；
2, a kind of synthetic method of Suo Malu peptide of the present invention, in the peptide fragment sequences of 6 side chain protections, by Asp, Phe,
Gly is placed on the carbon teminal first position of peptide fragment sequences, improves the condensation efficiency of amino acid, effectively avoid Trp, Ser,
Val, Ile, Arg influence condensation efficiency as carbon teminal first position, and effectively prevent the beta sheet in synthesis in solid state,
Improve the purity and yield of Suo Malu peptide；
3, a kind of synthetic method of Suo Malu peptide of the present invention, passes through pre-synthesis first peptide fragment sequences to hexapeptide segment
Sequence, then gradually coupling obtains Suo Malu peptide in solid phase, and in final liquid chromatography purification step, impurity is not the absence of
The peptide disappearance of one or several amino acid, but the Partial Fragment not being condensed, it is only necessary to which 1-2 preparation purifying, product purity reach
To 99.5% or more, and usual way synthesis needs 4-5 preparation to purify, and substantially increases purifying yield, passes through this method
The Suo Malu peptide crude product purity of synthesis can reach 75% or more, and traditional synthetic method can only achieve 60%；It is pure by preparing
Suo Malu peptide total recovery after change can reach 60% or more, greatly reduce the production cost of Suo Malu peptide.
Detailed description of the invention
Attached drawing described herein is used to provide to further understand the embodiment of the present invention, constitutes one of the application
Point, do not constitute the restriction to the embodiment of the present invention.In the accompanying drawings:
Fig. 1 is process flow chart of the invention；
Fig. 2 is Suo Malu peptide high-efficient liquid phase chromatogram obtained by the embodiment of the present invention 1；
Fig. 3 is Suo Malu peptide high-efficient liquid phase chromatogram obtained by the embodiment of the present invention 2；
Fig. 4 is Suo Malu peptide high-efficient liquid phase chromatogram obtained by the embodiment of the present invention 3；
Fig. 5 is Suo Malu peptide high-efficient liquid phase chromatogram obtained by the embodiment of the present invention 4.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment and attached drawing, to this
Invention is described in further detail, and exemplary embodiment of the invention and its explanation for explaining only the invention, are not made
For limitation of the invention.
Embodiment 1
As shown in Figure 1, a kind of synthetic method of Suo Malu peptide of the present invention, comprising the following steps:
1, the first peptide fragment sequences~hexapeptide fragment sequence synthesis
1.1 liquid phases prepare Boc-His (Trt)-Aib-OH:
By Boc-His (Trt)-OH (5.01g, 10.07mmol), the HOBT (2.72g, 20.14mmol) of side chain protection,
DIEA (3.90g, 30.21mmol), 2- aminoisobutyric acid methyl ester hydrochloride (1.55g, 10.07mmol) are dissolved in 100mlDMF
In, it is added HATU (6.54g, 17.20mmol), stirs 4 hours at room temperature, reaction solution, which is poured into ice water, white solid analysis
Out, white solid is obtained by filtration, then three times with saturated salt solution mashing washing, finally washed once and be put into very with clear water mashing again
Empty drying box is dried to obtain 5.98g, and filtering and concentrating to dry obtained product is dissolved in THF (50ml), is added water (30ml), then
It is added Lithium hydroxide monohydrate (0.85g, 20.26mmol), stirs 2 hours at room temperature, concentration removes THF, the salt for being 1N with concentration
Acid for adjusting pH is precipitated solid, white is obtained by filtration, solid is washed with water three times, and vacuum drying obtains Boc-His (Trt)-to 6~7
Aib-OH (5.58g, purity 99.3%), yield 95.1%；
1.2 as described in 1.1, with same method be successively coupled remaining amino acid in the first peptide fragment sequences obtain Boc (for
Why not it is Fmoc)-His (Trt)-Aib-Glu (OtBu)-Gly-OH；
1.3 successively obtain the second peptide fragment sequences~hexapeptide sequence fragment by synthesis in solid state, these segments do not pass through
Reversed preparation purifying obtains purity greater than 99% product by excessively quick silicagel column, and yield is greater than 90%:
Second peptide sequence segment: Fmoc-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH
Third peptide sequence segment: Fmoc-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-Glu (OtBu)-Gly-
OH
Tetrapeptide array segment: Fmoc-Gln (Trt)-Ala-Ala-Lys (Alloc)-Glu (OtBu)-Phe-OH
Pentapeptide sequence fragment: Fmoc-Ile-Ala-Trp (Boc)-Leu-OH
Hexapeptide sequence fragment: Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH.
2, synthesis in solid state
The 2.1 preparation chloro- trityl resins of Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-:
Polypeptides reactive pipe is added in the chloro- trityl chloride resin of 2- (10g, substitution value 0.28mmol/g resin), uses 100mL
DCM swellable resins 30 minutes.Drain solvent be added Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH (5.08g,
It 4.00mmol) is advertised 1 hour with DCM (80mL) solution of DIEA (1.09g, 8.43mmol), nitrogen, is added and analyzes pure methanol
20mL, DIEA (1.09g, 8.43mmol) carry out resin sealing end, solvent are drained, with 3 × 100mL DMF, 3 × 100mL DCM, 3
× 100mLMeOH washing, is directly used in and reacts in next step；
2.2 amino protecting groups are sloughed:
The chloro- trityl tree of Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2- is added into polypeptides reactive pipe
Rouge is added the DMF swelling of 100ml, drains, distinguished 10,20 minutes with piperidines/DMF 2 × 50mL of (volume ratio 1/4) solution and handled
Resin removes Fmoc, and resin 4 times described in the DMF Xian Di with 100mL, (dibenzofulvene and its piperidines add removal Fmoc by-product
Close object) and remaining piperidines, ninhydrin test measurement；
The synthesis of 2.3 heptapeptide fragment sequences:
The 5th peptide fragment sequences Fmoc-Ile-Ala-Trp (Boc)-Leu-OH is activated, to react in its carboxyl terminal, so
Afterwards by the 5th peptide fragment sequences Fmoc-Ile-Ala-Trp (Boc)-Leu-OH (4.62g, 5.61mmol), HOBt (0.76g,
It 5.62mmol) is dissolved in the DMF of 100mL at room temperature with DIEA (1.45g, 11.22mmol), the solution under nitrogen protection
It is cooled to 0 DEG C, HBTU (2.13g, 5.62mmol) then is added, the amino acid fragment solution of activation is added to by stirring and dissolving
In step (2.2) in the chloro- trityl resin of H-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-, nitrogen advertises reactant
1 hour, condensation performance is monitored with qualitative ninhydrin test, after the completion of determining the condensation reaction, then dry adsorbent, is used
5 × 100mLDMF washs resin, obtains heptapeptide fragment sequence, and slough amino protecting group according to the method for step (2.2).
2.4 octapeptide fragment sequences~full guard Suo Malu peptide straight-chain polypeptide:
On the basis of heptapeptide fragment sequence, the 4th peptide fragment, third peptide fragment, the second peptide fragment, the are successively utilized
Each 2eq of one peptide fragment repeats the operating process of step 2.3, successively obtains octapeptide fragment sequence, nonapeptide fragment sequence, the
After ten peptide fragment sequences, the final Suo Malu peptide straight-chain polypeptide for synthesizing full guard, after the last one coupling reaction, with 3 ×
100mLDMF, 3 × 100mLDCM, 3 × 100mL MeOH washing, vacuum drying obtain the rope horse of the full guard of 38.46g purification
Shandong peptide straight-chain polypeptide.
3. the preparation of Suo Malu peptide
3.1 the Suo Malu peptide straight-chain polypeptide of 38.46g full guard is added into polypeptides reactive pipe, then by phenylsilane
(0.31g, 2.86mmol) and tetra-triphenylphosphine palladium (0.65g, 0.56mmol) pour into polypeptides reactive pipe, nitrogen after being dissolved in 500mLDCM
Tympanites blows reaction one hour, is washed, is drained with 3 × 100mL DMF, 3 × 100mL DCM；
3.2 by side chain HO-AEEA-AEEA-r-Glu (OtBu)-Oct (OtBu) (4.74g, 5.60mmol), Cl-HOBt
(0.95g, 5.60mmol) and DIEA (1.45g, 11.22mmol) are dissolved in 500mLDMF at room temperature, handle under nitrogen protection
The solution is cooled to 0 DEG C, and HBTU (2.13g, 5.62mmol) then is added, and stirring and dissolving is molten by the Suo Malu peptide side chain of activation
Liquid is added in the resin drained, and nitrogen advertises reactant 6 hours, monitors condensation performance with qualitative ninhydrin test,
After the completion of determining the condensation reaction, then dry adsorbent, washs resin with the DMF of 5 × 100mL, drains；
10,20 minutes processing resins are distinguished with 2 × 50mL20% piperidines/DMF (volume ratio 1/4) solution, remove Fmoc, are used
Resin 4 times described in 100mLDMF Xian Di, removal Fmoc by-product (dibenzofulvene and its piperidine adduct) and remaining piperidines, indenes
Triketone test measurement, obtains the Suo Malu peptide of full guard；
3.3 by TFA, PhSMe, PhOH, EDT, TiS and H2O by volume 80: 5: 5: 2.5: 2.5 prepare mixed solution
The Suo Malu peptide nitrogen protection for the full guard that 150mL obtains step (3.2) carries out cracking 2 hours under room temperature (28 DEG C),
And washed once with TFA (100ml), merge lysate, being concentrated into volume is about 30mL, MTBE precipitated product is added, at room temperature
It stirs the slurry 10 minutes, places into 4 DEG C of refrigerator overnights.The solid is collected by centrifugation, is washed with the methyl tertiary butyl ether(MTBE) of about 300mL
It washs three times, is dried in vacuo the product, obtain the thick peptide of Suo Malu peptide that 12.8g purity is 81.3%.
3.4Prep-HPLC purifies the thick peptide of Suo Malu peptide
It takes the thick peptide of 2g Suo Malu peptide to purify through preparative Prep-HPLC and generates Suo Malu peptide sterling 1.22g, Prep-HPLC
Purification condition: chromatographic column: C18250 × 19 Waters, 5u, 130A；Flow velocity: 8mL/min；Detection: UV, 220nm；Mobile phase:
A. acetonitrile；B.0.25% acetic acid/water；Method: 20%-30%A, 10min；30-60%A, 40min, as shown in Fig. 2, obtained
The purity of Suo Malu peptide sterling is 99.77%, yield 67.80%.
Embodiment 2
A kind of synthetic method of Suo Malu peptide, comprising the following steps:
(1) peptide fragment sequences of 6 side chain protections are synthesized；
(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.
Wherein, in the step (1), the peptide fragment sequences of 6 side chain protections are specific as follows:
First peptide fragment sequences are the 7-10 amino acids in Suo Malu peptide sequence,
Second peptide fragment sequences are the 11-15 amino acids in Suo Malu peptide sequence,
Third peptide fragment sequences are the 16-22 amino acids in Suo Malu peptide sequence,
4th peptide fragment sequences are the 23-28 amino acids in Suo Malu peptide sequence,
5th peptide fragment sequences are the 29-32 amino acids in Suo Malu peptide sequence,
Hexapeptide fragment sequence is the 33-37 amino acids in Suo Malu peptide sequence.
1, the first peptide fragment sequences~hexapeptide fragment sequence synthesis
1.1 preparation Boc-His (Trt)-Aib-OH:
By Boc-His (Trt)-OH (5.00g, 10.07mmol), the HOBT (2.70g, 20.14mmol) of side chain protection,
DIEA (3.91g, 30.21mmol), 2- aminoisobutyric acid methyl ester hydrochloride (1.55g, 10.07mmol) are dissolved in 100mlDMF
In, it is added HATU (6.54g, 17.20mmol), stirs 4 hours at room temperature, reaction solution, which is poured into ice water, white solid analysis
Out, white solid is obtained by filtration, then three times with saturated salt solution mashing washing, finally washed once and be put into very with clear water mashing again
Empty drying box is dried to obtain 5.98g, and filtering and concentrating to dry obtained product is dissolved in THF (50ml), is added water (30ml), then
It is added Lithium hydroxide monohydrate (0.85g, 20.26mmol), stirs 2 hours at room temperature, concentration removes THF, the salt for being 1N with concentration
Acid for adjusting pH is precipitated solid, white is obtained by filtration, solid is washed with water three times, and vacuum drying obtains Boc-His (Trt)-to 6~7
Aib-OH (5.57g, purity 99.4%), yield 95.1%；
1.2 as described in 1.1, are successively coupled remaining amino acid in the first peptide fragment sequences with same method and obtain Fmoc-
His(Trt)-Aib-Glu(OtBu)-Gly–OH；
1.3 successively obtain the second peptide fragment sequences~hexapeptide sequence fragment by synthesis in solid state, these segments do not pass through
Reversed preparation purifying obtains purity greater than 99% product by excessively quick silicagel column, and yield is greater than 85%:
Second peptide sequence segment: Fmoc-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH
Third peptide sequence segment: Fmoc-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-Glu (OtBu)-Gly-
OH
Tetrapeptide array segment: Fmoc-Gln (Trt)-Ala-Ala-Lys (Alloc)-Glu (OtBu)-Phe-OH
Pentapeptide sequence fragment: Fmoc-Ile-Ala-Trp (Boc)-Leu-OH
Hexapeptide sequence fragment: Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH
2, synthesis in solid state
The 2.1 preparation chloro- trityl resins of Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-:
Polypeptides reactive pipe is added in the chloro- trityl chloride resin of 2- (10g, substitution value 0.28mmol/g resin), uses 100mL
DCM swellable resins 30 minutes.Drain solvent be added Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH (5.08g,
4.00mmol) and DCM (80mL) solution of DIEA (1.10g, 8.43mmol).Nitrogen is advertised 1 hour, is added and is analyzed pure methanol
20mL, DIEA (1.07g, 8.38mmol) carry out resin sealing end, solvent are drained, with 3 × 100mL DMF, 3 × 100mL DCM, 3
× 100mLMeOH washing, is directly used in and reacts in next step；
2.2 amino protecting groups are sloughed:
The chloro- trityl resin of Fmoc-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2- into polypeptides reactive pipe,
100mlDMF swelling is added, drains, distinguishes the tree of processing in 10,20 minutes with piperidines/DMF 2 × 50mL of (volume ratio 1/4) solution
Rouge removes Fmoc, resin 5 times described in 100mLDMF Xian Di, removal Fmoc by-product (dibenzofulvene and its piperidines adduction
Object) and remaining piperidines, ninhydrin test measurement；
The synthesis of 2.3 heptapeptide fragment sequences:
The 5th peptide fragment sequences Fmoc-Ile-Ala-Trp (Boc)-Leu-OH is activated, to react in its carboxyl terminal, so
Afterwards by the 5th peptide fragment sequences Fmoc-Ile-Ala-Trp (Boc)-Leu-OH (4.61g, 5.60mmol), HOBt (0.76g,
It 5.62mmol) is dissolved in the DMF of 100mL at room temperature with DIEA (1.46g, 11.22mmol), the solution under nitrogen protection
It is cooled to 0 DEG C, HBTU (2.13g, 5.62mmol) then is added, the amino acid fragment solution of activation is added to by stirring and dissolving
In step (2.2) in the chloro- trityl resin of H-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-, nitrogen advertises reactant
1 hour, condensation performance is monitored with qualitative ninhydrin test, after the completion of determining the condensation reaction, then dry adsorbent, is used
The DMF of 5 × 100mL washs resin, obtains heptapeptide fragment sequence, and slough amino protecting group according to the method for step (2.2)；
2.4 octapeptide fragment sequences~full guard Suo Malu peptide straight-chain polypeptide:
On the basis of heptapeptide fragment sequence, the 4th peptide fragment, third peptide fragment, the second peptide fragment, the are successively utilized
Each 2eq of one peptide fragment, repeat step (2.3) operating process, successively obtain octapeptide fragment sequence, nonapeptide fragment sequence,
After tenth peptide fragment sequences, the final Suo Malu peptide straight-chain polypeptide for synthesizing full guard, after the last one coupling reaction, with 3 ×
100mLDMF, 3 × 100mLDCM, 3 × 100mL MeOH washing, vacuum drying obtain the rope horse of the full guard of 37.87g purification
Shandong peptide straight-chain polypeptide.
3. the preparation of Suo Malu peptide
3.1 the Suo Malu peptide straight-chain polypeptide of 37.87g full guard is added into polypeptides reactive pipe, then by phenylsilane
(0.31g, 2.86mmol) and tetra-triphenylphosphine palladium (0.65g, 0.56mmol) pour into polypeptides reactive pipe, nitrogen after being dissolved in 100mLDCM
Tympanites blows reaction one hour, is washed, is drained with 3 × 100mL DMF, 3 × 100mL DCM；
3.2 by side chain HO-AEEA-AEEA-Glu (OtBu)-Otc (4.74g, 5.60mmol), Cl-HOBt (0.95g,
5.60mmol) it is dissolved in 500mLDMF at room temperature with DIEA (1.45g, 11.22mmol), it is under nitrogen protection that the solution is cold
But to 0 DEG C, HBTU (2.13g, 5.62mmol) then is added, the Suo Malu peptide side chain solution of activation is added to by stirring and dissolving
In the resin drained, nitrogen advertises reactant 1 hour, condensation performance is monitored with qualitative ninhydrin test, described in judgement
After the completion of condensation reaction, then dry adsorbent, washs resin with 3 × 100mL DMF, drains；
10,20 minutes processing resins are distinguished with piperidines/DMF (volume ratio 1/4) solution, Fmoc are removed, with Xian 100mLDMF
Wash the resin 4 times, removal Fmoc by-product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin test are surveyed
It is fixed, obtain the Suo Malu peptide of full guard；
3.3 by TFA, PhSMe, PhOH, EDT, TIS and H2The mixed solution of O 80: 5: 5: 2.5: 2.5 preparations by volume
The Suo Malu peptide nitrogen protection for the full guard that 150mL obtains step (3.2) at room temperature (28 DEG C) to carry out cracking 2 small
When, and washed once with TFA (100ml), merge lysate, being concentrated into volume is about 30mL, and MTBE precipitated product, room temperature is added
The lower stirring slurry 10 minutes, places into 4 DEG C of refrigerator overnights.The solid is collected by centrifugation, with the methyl tertiary butyl ether(MTBE) of about 300mL
Washing three times, is dried in vacuo the product, obtains the thick peptide of Suo Malu peptide that 13.81g purity is 75.22%.
3.4Prep-HPLC purifies the thick peptide of Suo Malu peptide
It takes the thick peptide of 2.01g Suo Malu peptide to purify through preparative Prep-HPLC and generates Suo Malu peptide sterling 1.02g, Prep-
HPLC purification condition: chromatographic column: C18250 × 19 Waters, 5u, 130A；Flow velocity: 8mL/min；Detection: UV, 220nm；Flowing
Phase: A. acetonitrile；B.0.25% acetic acid/water；Method: 20%-30%A, 10min；30-60%A, 40min, as shown in Fig. 2, being made
Suo Malu peptide sterling purity be 99.66%, yield 61.2%.
The following are comparative examples:
Embodiment 3
A kind of synthetic method of Suo Malu peptide, comprising the following steps:
(1) peptide fragment sequences of 6 side chain protections are synthesized；
(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.
Wherein, in the step (1), the peptide fragment sequences of 6 side chain protections are specific as follows:
First peptide fragment sequences are the 7-11 amino acids in Suo Malu peptide sequence,
Second peptide fragment sequences are the 12-17 amino acids in Suo Malu peptide sequence,
Third peptide fragment sequences are the 18-21 amino acids in Suo Malu peptide sequence,
4th peptide fragment sequences are the 22-26 amino acids in Suo Malu peptide sequence,
5th peptide fragment sequences are the 27-31 amino acids in Suo Malu peptide sequence,
Hexapeptide fragment sequence is the 32-37 amino acids in Suo Malu peptide sequence.
Specifically includes the following steps:
1, the first peptide fragment sequences~hexapeptide fragment sequence synthesis
1.1 preparation Boc-His (Trt)-Aib-OH:
By Boc-His (Trt)-OH (5.00g, 10.07mmol), the HOBT (2.70g, 20.14mmol) of side chain protection,
DIEA (3.91g, 30.21mmol), 2- aminoisobutyric acid methyl ester hydrochloride (1.55g, 10.07mmol) are dissolved in 100mlDMF
In, it is added HATU (6.54g, 17.20mmol), stirs 4 hours at room temperature, reaction solution, which is poured into ice water, white solid analysis
Out, white solid is obtained by filtration, then three times with saturated salt solution mashing washing, finally washed once and be put into very with clear water mashing again
Empty drying box is dried to obtain 5.98g, and filtering and concentrating to dry obtained product is dissolved in THF (50ml), is added water (30ml), then
It is added Lithium hydroxide monohydrate (0.85g, 20.26mmol), stirs 2 hours at room temperature, concentration removes THF, the salt for being 1N with concentration
Acid for adjusting pH is precipitated solid, white is obtained by filtration, solid is washed with water three times, and vacuum drying obtains Boc-His (Trt)-to 6~7
Aib-OH (5.57g, purity 99.4%), yield 95.1%；
1.2 as described in 1.1, are successively coupled remaining amino acid in the first peptide fragment sequences with same method and obtain Boc-
His(Trt)-Aib-Glu(OtBu)-Gly–Thr(tBu)-OH；
1.3 successively obtain the second peptide fragment sequences~hexapeptide sequence fragment by synthesis in solid state, these segments do not pass through
Reversed preparation purifying obtains intermediate purity greater than 99% by excessively quick silicagel column, and intermediate yield is greater than 85%:
Second peptide sequence segment: Fmoc-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-OH
Third peptide sequence segment: Fmoc-Ser (tBu)-Tyr (tBu)-Leu-Glu (OtBu)-OH
Tetrapeptide array segment: Fmoc-Gly-Gln (Trt)-Ala-Ala-Lys (Alloc)-OH
Pentapeptide sequence fragment: Fmoc-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-OH
Hexapeptide sequence fragment: Fmoc-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH.
2, synthesis in solid state
The 2.1 preparation chloro- trityl resins of Fmoc-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2-:
Polypeptides reactive pipe is added in the chloro- trityl chloride resin of 2- (10.00g, substitution value 0.28mmol/g resin), is used
100mL DCM swellable resins 30 minutes.It drains solvent and Fmoc-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-OH is added
The 80mLDCM solution of (4.32g, 0.40mmol) and DIEA (1.09g, 8.43mmol).Nitrogen is advertised 1 hour, and it is pure that analysis is added
Methanol 20mL, DIEA (1.09g, 8.43mmol) carry out resin sealing end, solvent are drained, with 3 × 100mL DMF, 3 × 100mL
DCM washing, is directly used in and reacts in next step.
2.2 amino protecting groups are sloughed:
Step (2.1) Fmoc-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-2- is added into polypeptides reactive pipe
Chloro- trityl resin is added 100mlDMF swelling, drains, distinguished 10,20 minutes with 2 × 50mL, 20% piperidines/DMF solution
Resin is handled, Fmoc, resin 4 times described in 100mLDMF Xian Di, removal Fmoc by-product (dibenzofulvene and its piperidines are removed
Adduct) and remaining piperidines, ninhydrin test measurement；
The synthesis of 2.3 heptapeptide fragment sequences:
The 5th peptide fragment sequences Fmoc-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-OH is activated, at its carboxyl end
End reaction, then by the 5th peptide fragment sequences Fmoc-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-OH (5.84g,
5.60mmol), HOBt (0.76g, 5.62mmol) and DIEA (1.45g, 11.22g) are dissolved in 100mLDMF at room temperature, nitrogen
The solution is cooled to 0 DEG C under gas shielded, HBTU (2.12g, 5.59mmol) then is added, stirring and dissolving, by the amino of activation
Acid fragment solution is added in the resin drained, and nitrogen advertises reactant 1 hour, is monitored condensation with qualitative ninhydrin test and is completed
Situation, after the completion of determining the condensation reaction, then dry adsorbent, washs 5 resins with DMF (100ml), obtains heptapeptide piece
Duan Xulie, and amino protecting group is sloughed according to the method for step 2.2；
2.4 octapeptide fragment sequences~full guard Suo Malu peptide straight-chain polypeptide:
On the basis of heptapeptide fragment sequence, the 4th peptide fragment, third peptide fragment, the second peptide fragment, the are successively utilized
Each 2eq of one peptide fragment repeats the operating process of step 2.3, successively obtains octapeptide fragment sequence, nonapeptide fragment sequence, the
After ten peptide fragment sequences, the final Suo Malu peptide straight-chain polypeptide for synthesizing full guard, after the last one coupling reaction, with 3 ×
100mLDMF, 3 × 100mLDCM, 3 × 100mL MeOH washing, vacuum drying obtain the rope horse of the full guard of 37.55g purification
Shandong peptide straight-chain polypeptide.
3. the preparation of Suo Malu peptide
3.1 the Suo Malu peptide straight-chain polypeptide of 37.55g full guard is added into polypeptides reactive pipe, then by phenylsilane
(0.31g, 2.86mmol) and tetra-triphenylphosphine palladium (0.65g, 0.56mmol) pour into polypeptides reactive pipe, nitrogen after being dissolved in 100mLDCM
Tympanites blows reaction one hour, is washed, is drained with 3 × 100mL DMF, 3 × 100mL DCM；
3.2 by side chain HO-AEEA-AEEA-r-Glu (OtBu)-Otc (4.74g, 5.60mmol), Cl-HOBt (0.95g,
5.60mmol) it is dissolved in 500mLDMF at room temperature with DIEA (1.45g, 11.22mmol), it is under nitrogen protection that the solution is cold
But to 0 DEG C, HBTU (2.13g, 5.62mmol) then is added, the Suo Malu peptide side chain solution of activation is added to by stirring and dissolving
In the resin drained, nitrogen advertises reactant 1 hour, condensation performance is monitored with qualitative ninhydrin test, described in judgement
After the completion of condensation reaction, then dry adsorbent, washs resin with 3 × 100mL DMF, drains；
10,20 minutes processing resins are distinguished with 2 × 50mL20% piperidines/DMF solution, Fmoc are removed, with Xian 100mLDMF
Wash the resin 4 times, removal Fmoc by-product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin test are surveyed
It is fixed, obtain the Suo Malu peptide of full guard；
3.3 by TFA, PhSMe, PhOH, EDT, TiS and H2O by volume 80: 5: 5: 2.5: 2.5 prepare mixed solution
150mL carries out cracking 2 times to the Suo Malu peptide of the obtained full guard of step 2.5.2,30 minutes every time, merges lysate, concentration
It is about 30mL to volume, MTBE precipitated product is added, stirs the slurry at room temperature 30 minutes, vacuum filter collects the solid,
It is washed three times with about 300mLMTBE, is dried in vacuo the product, obtain the thick peptide of Suo Malu peptide that 13.52g purity is 58.12%.
3.4Prep-HPLC purifies the thick peptide of Suo Malu peptide
It takes the thick peptide of 2.02g Suo Malu peptide to purify through preparative Prep-HPLC and generates Suo Malu peptide sterling 0.91g, Prep-
HPLC purification condition: chromatographic column: C18250 × 19 Waters, 5u, 130A；Flow velocity: 8mL/min；Detection: UV, 220nm；Flowing
Phase: A. acetonitrile；B.0.25% acetic acid/water；Method: 20%-30%A, 10min；30-60%A, 40min, as shown in Fig. 2, being made
Suo Malu peptide sterling purity be 98.15%, yield 46.40%.
Embodiment 4
A kind of synthetic method of Suo Malu peptide, comprising the following steps:
(1) peptide fragment sequences of 8 side chain protections of liquid phase synthesis；
(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.
Wherein, in the step (1), the peptide fragment sequences of 8 side chain protections are specific as follows:
Wherein, in the step (1), the peptide fragment sequences of 8 side chain protections are specific as follows:
First peptide fragment sequences are the 7-10 amino acids in Suo Malu peptide sequence,
Second peptide fragment sequences are the 11-15 amino acids in Suo Malu peptide sequence,
Third peptide fragment sequences are the 16-19 amino acids in Suo Malu peptide sequence,
4th peptide fragment sequences are the 20-23 amino acids in Suo Malu peptide sequence,
5th peptide fragment sequences are the 24-27 amino acids in Suo Malu peptide sequence,
Hexapeptide fragment sequence is the 28-31 amino acids in Suo Malu peptide sequence,
Heptapeptide fragment sequence is the 32-34 amino acids in Suo Malu peptide sequence,
Octapeptide fragment sequence is the 35-37 amino acids in Suo Malu peptide sequence.
Specifically includes the following steps:
1, the first peptide fragment sequences~octapeptide fragment sequence synthesis
1.1 preparation Fmoc-His (Trt)-Aib-OH:
By Boc-His (Trt)-OH (5.00g, 10.07mmol), the HOBT (2.70g, 20.14mmol) of side chain protection,
DIEA (3.91g, 30.21mmol), 2- aminoisobutyric acid methyl ester hydrochloride (1.55g, 10.07mmol) are dissolved in 100mlDMF
In, it is added HATU (6.54g, 17.20mmol), stirs 4 hours at room temperature, reaction solution, which is poured into ice water, white solid analysis
Out, white solid is obtained by filtration, then three times with saturated salt solution mashing washing, finally washed once and be put into very with clear water mashing again
Empty drying box is dried to obtain 5.98g, and filtering and concentrating to dry obtained product is dissolved in THF (50ml), is added water (30ml), then
It is added Lithium hydroxide monohydrate (0.85g, 20.26mmol), stirs 2 hours at room temperature, concentration removes THF, the salt for being 1N with concentration
Acid adjusts pH to 6~7, and solid is precipitated, and solid is washed with water three times, and vacuum drying obtains Fmoc-His (Trt)-Aib-OH
5.4g, yield 95.1%；
1.2 as described in 1.1, are successively coupled remaining amino acid in the first peptide fragment sequences with same method and obtain Fmoc-
His(Trt)-Aib-Glu(OtBu)-Gly–OH；
1.3 such as 1.1 and 1.2 the methods successively synthesize the second peptide fragment sequences~octapeptide sequence fragment:
Second peptide sequence segment: Fmoc-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-OH
Third peptide sequence segment: Fmoc-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-OH
Tetrapeptide array segment: Fmoc-Leu-Glu (OtBu)-Gly-Gln (Trt)-OH
Pentapeptide sequence fragment: Fmoc-Ala-Ala-Lys (Alloc)-Glu (OtBu)-OH
Hexapeptide sequence fragment: Fmoc-Phe-Ile-Ala-Trp (Boc)-OH
Heptapeptide fragment sequence: Fmoc-Leu-Val-Arg (Pbf)-OH
Octapeptide sequence fragment: Fmoc-Gly-Arg (Pbf)-Gly-OH.
2, synthesis in solid state
The 2.1 preparation chloro- trityl resins of Fmoc-Gly-Arg (Pbf)-Gly-2-:
Polypeptides reactive pipe is added in the chloro- trityl chloride resin of 2- (10g, substitution value 0.28mmol/g resin), uses 100mL
DCM swellable resins 30 minutes.It drains solvent and Fmoc-Gly-Arg (Pbf)-Gly-OH (3.05g, 4.00mmol) and DIEA is added
The 80mLDCM solution of (1.09g, 8.43mmol).Nitrogen is advertised 1 hour, is added and is analyzed pure methanol 20mL, DIEA (1.09g,
Resin sealing end 8.43mmol) is carried out, solvent is drained, is washed with 3 × 100mL DMF, 3 × 100mL DCM, 3 × 100mLMeOH,
It drains to be directly used in and react in next step；
2.2 amino protecting groups are sloughed:
The chloro- trityl resin of Fmoc-Gly-Arg (Pbf)-Gly-2- is added into polypeptides reactive pipe, is added
100mlDMF swelling, is drained, and distinguishes 10 minutes and 20 minutes processing resins with piperidines/DMF 2 × 50mL of (volume ratio 1/4) solution,
Remove Fmoc, resin 5 times described in 100mLDMF Xian Di, removal Fmoc by-product (dibenzofulvene and its piperidine adduct) and
Remaining piperidines, ninhydrin test measurement；
The synthesis of 2.3 nonapeptide fragment sequences:
Heptapeptide fragment sequence Fmoc-Leu-Val-Arg (Pbf)-OH is activated, to be reacted in its carboxyl terminal, then will
Heptapeptide fragment sequence Fmoc-Leu-Val-Arg (Pbf)-OH (4.82g, 5.60mmol), HOBt (0.76g, 5.62mmol) and
DIEA (1.45g, 11.22mmol) is dissolved at room temperature in the DMF of 100mL, and the solution is cooled to 0 DEG C under nitrogen protection,
Then HBTU (2.12g, 5.59mmol) is added, the amino acid fragment solution of activation is added to the resin drained by stirring and dissolving
In, nitrogen advertises reactant 1 hour, monitors condensation performance with qualitative ninhydrin test, is determining that the condensation reaction is complete
Cheng Hou, then dry adsorbent, washs resin with 5 × 100mLDMF, obtains nonapeptide fragment sequence, and according to the side of step (2.2)
Method sloughs amino protecting group；
2.4 ten peptide fragment sequences~full guard Suo Malu peptide straight-chain polypeptide:
On the basis of nonapeptide fragment sequence, hexapeptide segment, the 5th peptide fragment, the 4th peptide fragment, the are successively utilized
Tripeptide fragment, the second peptide fragment, each 2eq of the first peptide fragment repeat the operating process of step (2.3), successively obtain decapeptide piece
After Duan Xulie, the 11st peptide fragment sequences, dodecapeptide fragment sequence, tridecanoic peptide fragment sequence, tetradecapeptide fragment sequence,
The Suo Malu peptide straight-chain polypeptide of final synthesis full guard, after the last one coupling reaction, with 3 × 100mLDMF, 3 ×
100mLDCM, 3 × 100mL MeOH washing, vacuum drying obtain the Suo Malu peptide straight-chain polypeptide of the full guard of 39.23g purification.
3. the preparation of Suo Malu peptide
3.1 the Suo Malu peptide straight-chain polypeptide of 39.23g full guard is added into polypeptides reactive pipe, then by 1.21g phenylsilane
It is dissolved in after 500mLDCM with 3.23g tetra-triphenylphosphine palladium and pours into polypeptides reactive pipe, nitrogen advertises reaction one hour, with 3 × 100mL
DMF, 3 × 100mL DCM washing, are drained；
3.2 by side chain HO-AEEA-AEEA-r-Glu (OtBu)-Otc (4.74g, 5.60mmol), Cl-HOBt (0.95g,
5.60mmol) it is dissolved in 500mLDMF at room temperature with DIEA (1.45g, 11.22mmol), it is under nitrogen protection that the solution is cold
But to 0 DEG C, HBTU (2.13g, 5.62mmol) then is added, the Suo Malu peptide side chain solution of activation is added to by stirring and dissolving
In the resin drained, nitrogen advertises reactant 1 hour, condensation performance is monitored with qualitative ninhydrin test, described in judgement
After the completion of condensation reaction, then dry adsorbent, washs resin with 3 × 100mL DMF, drains；
10,20 minutes processing resins are distinguished with 2 × 50mL20% piperidines/DMF solution, Fmoc are removed, with Xian 100mLDMF
Wash the resin 4 times, removal Fmoc by-product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin test are surveyed
It is fixed, obtain the Suo Malu peptide of full guard；
3.3 by TFA, PhSMe, PhOH, EDT, TIS and H2O by volume 80: 5: 5: 2.5: 2.5 prepare mixed solution
The Suo Malu peptide nitrogen protection for the full guard that 150mL obtains step (3.2) at room temperature (28 DEG C) to carry out cracking 2 small
When, and washed once with TFA (100ml), merge lysate, being concentrated into volume is about 30mL, and MTBE precipitated product, room temperature is added
The lower stirring slurry 10 minutes, places into 4 DEG C of refrigerator overnights.The solid is collected by centrifugation, with the methyl tertiary butyl ether(MTBE) of about 300mL
Washing three times, is dried in vacuo the product, obtains the thick peptide of Suo Malu peptide that 14.81g purity is 53.51%.
3.4Prep-HPLC purifies the thick peptide of Suo Malu peptide
It takes the thick peptide of 2.03g Suo Malu peptide to purify through preparative Prep-HPLC and generates Suo Malu peptide sterling 0.61g, Prep-
HPLC purification condition: chromatographic column: C18250 × 19 Waters, 5u, 130A；Flow velocity: 8mL/min；Detection: UV, 220nm；Flowing
Phase: A. acetonitrile；B.0.25% acetic acid/water；Method: 20%-30%A, 10min；30-60%A, 40min, as shown in Fig. 2, being made
Suo Malu peptide sterling purity be 97.19%, yield 39.22%.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (7)
1. a kind of synthetic method of Suo Malu peptide, which comprises the following steps:
(1) peptide fragment sequences of 6 side chain protections are synthesized, the peptide fragment sequences of 6 side chain protections are specific as follows:
First peptide fragment sequences are the 7-10 amino acids in Suo Malu peptide sequence,
Second peptide fragment sequences are the 11-15 amino acids in Suo Malu peptide sequence,
Third peptide fragment sequences are the 16-22 amino acids in Suo Malu peptide sequence,
4th peptide fragment sequences are the 23-28 amino acids in Suo Malu peptide sequence,
5th peptide fragment sequences are the 29-32 amino acids in Suo Malu peptide sequence,
Hexapeptide fragment sequence is the 33-37 amino acids in Suo Malu peptide sequence；
(2) by each peptide fragment sequences, gradually coupling obtains all risk insurance guard wire horse Shandong peptide straight-chain polypeptide in solid phase.
2. a kind of synthetic method of Suo Malu peptide according to claim 1, which is characterized in that in the step (2), all risk insurance
The synthesis technology of guard wire horse Shandong peptide straight-chain polypeptide is as follows:
(21) the hexapeptide fragment sequence c-terminus of side chain protection and resin are coupled, and slough its amino protecting group；
(22) will slough obtained by step (21) side chain protection of amino protecting group hexapeptide fragment sequence and side chain protection the
Five peptide fragment sequences are coupled to obtain the heptapeptide fragment sequence of side chain protection, and slough its amino protecting group；
(23) the heptapeptide fragment sequence of the side chain protection of amino protecting group and the 4th peptide fragment sequences of side chain protection will be sloughed
Coupling obtains the octapeptide fragment sequence of side chain protection, and sloughs its amino protecting group；
(24) the octapeptide fragment sequence of the side chain protection of amino protecting group and the third peptide fragment sequences of side chain protection will be sloughed
Coupling obtains the nonapeptide fragment sequence of side chain protection, and sloughs its amino protecting group；
(25) the nonapeptide fragment sequence of the side chain protection of amino protecting group and the second peptide fragment sequences of side chain protection will be sloughed
Coupling obtains the tenth peptide fragment sequences of side chain protection, and sloughs its amino protecting group；
(26) the tenth peptide fragment sequences of the side chain protection of amino protecting group and the first peptide fragment sequences of side chain protection will be sloughed
Coupling obtains the Suo Malu peptide straight-chain polypeptide of full guard.
3. a kind of synthetic method of Suo Malu peptide according to claim 1, which is characterized in that in step (2), by what is obtained
All risk insurance guard wire horse Shandong peptide straight-chain polypeptide is prepared into Suo Malu peptide.
4. a kind of synthetic method of Suo Malu peptide according to claim 3, which is characterized in that all risk insurance guard wire horse Shandong peptide straight chain
The method that polypeptide is prepared into Suo Malu peptide are as follows:
1) all risk insurance guard wire horse Shandong peptide straight-chain polypeptide is sloughed into the side chain protection of the 26th Lys, and completes modification and obtains full guard
Suo Malu peptide；
2) the Suo Malu peptide cracking deprotection base of full guard is obtained into the thick peptide of Suo Malu peptide；
3) the purified salt that turns of the thick peptide of Suo Malu peptide obtains Suo Malu peptide.
5. a kind of synthetic method of Suo Malu peptide according to claim 4, which is characterized in that in step 1), the 26th Lys
Side chain protective group be any one of Alloc, Mtt, Mmt, Dde, when protecting group is Alloc, the removing of protecting group is used
The combination of phenylsilane, triphenylphosphine palladium and DCM；When protecting group is Dde, the removing of protecting group uses the group of hydrazine hydrate and DMF
It closes；When protecting group is Mmt or Mtt, the removing of protecting group uses the combination of TFA and DCM.
6. a kind of synthetic method of Suo Malu peptide according to claim 4, which is characterized in that in step 1), all risk insurance guard wire
After horse Shandong peptide straight-chain polypeptide sloughs the side chain protection of the 26th Lys, the method for modification are as follows: HO-AEEA-AEEA-r-Glu is added
(OtBu)-Otc is condensed, and then sloughs Fmoc group, and agents useful for same is the DMF solution for the piperidines that volumn concentration is 20%,
Or volumn concentration be 1% DBU DMF solution.
7. a kind of synthetic method of Suo Malu peptide according to claim 4, which is characterized in that in step 2), full guard
Suo Malu peptide cracks any group that lysate type used by deprotection base technique is TFA, PhSMe, PhOH, EDT and TiS
It closes, the mixed solution of arbitrary proportion, in step 3), the purifying of the thick peptide of Suo Malu peptide turns salt technique specifically: RP-HPLC
Chromatogram purification changes salt, and mobile phase is ammonium acetate aqueous solution and acetonitrile solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811466181.1A CN109456401B (en) | 2018-12-03 | 2018-12-03 | A kind of synthetic method of Suo Malu peptide |
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| CN109627317B (en) * | 2019-02-01 | 2020-11-27 | 兰州大学 | Method for preparing Somalutide by fragment condensation |
| CN111748019A (en) * | 2019-03-29 | 2020-10-09 | 深圳翰宇药业股份有限公司 | Synthetic method of polypeptide derivative compound |
| CN111944037B (en) * | 2019-04-30 | 2023-06-13 | 深圳市健元医药科技有限公司 | Synthetic method of somalupeptide |
| CN110078816B (en) * | 2019-06-04 | 2023-04-25 | 扬子江药业集团四川海蓉药业有限公司 | Preparation method of somalupeptide |
| CN110372785B (en) * | 2019-07-25 | 2020-10-23 | 成都诺和晟泰生物科技有限公司 | Synthesis method of Somalutide |
| CN110922470A (en) * | 2019-12-26 | 2020-03-27 | 杭州肽佳生物科技有限公司 | Preparation method of somaglutide |
| CN111116731A (en) * | 2020-01-09 | 2020-05-08 | 山东大学 | Method for preparing Somaloutide by liquid phase method based on soluble hydrophobic labeled carrier |
| WO2021143073A1 (en) * | 2020-01-19 | 2021-07-22 | 深圳市健元医药科技有限公司 | Preparation method for semaglutide |
| CN113667006B (en) * | 2020-05-14 | 2024-04-19 | 鲁南制药集团股份有限公司 | Preparation method of cable Ma Lutai dipeptide side chain |
| AR122579A1 (en) * | 2020-06-12 | 2022-09-21 | Lilly Co Eli | PROCESS TO PREPARE A GLP-1/GLUCAGON DUAL AGONIST |
| CN111732650B (en) * | 2020-08-04 | 2020-12-08 | 苏州金顶生物有限公司 | Continuous flow solid phase reaction preparation of Somaloutide |
| CN113444164B (en) * | 2021-06-29 | 2022-07-19 | 台州吉诺生物科技有限公司 | Solid-phase synthesis method of Somalutide |
| WO2023012709A1 (en) | 2021-08-05 | 2023-02-09 | Usv Private Limited | An improved process for fmoc synthesis of semaglutide |
| CN114230653B (en) * | 2022-01-25 | 2023-02-10 | 杭州禾泰健宇生物科技有限公司 | Preparation method of chlorotoxin |
| CN114660214B (en) * | 2022-02-18 | 2024-04-05 | 兰州积石药业有限公司 | Liquid chromatography detection method of semaglutin and application thereof |
| WO2024077149A2 (en) * | 2022-10-05 | 2024-04-11 | Eli Lilly And Company | Peptides for incretin synthesis |
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| GR20140100479A (en) * | 2014-09-23 | 2016-05-05 | Novetide, Ltd., | Synthesis of liraglutide |
| CN106749613B (en) * | 2016-12-02 | 2020-08-18 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthetic method of somaglutide |
| CN108203462A (en) * | 2016-12-20 | 2018-06-26 | 上海脉凯生物科技有限公司 | A kind of method for preparing Suo Malu peptides |
| CN108059666B (en) * | 2018-02-10 | 2018-09-25 | 润辉生物技术（威海）有限公司 | A kind of method that solid-liquid combination prepares Suo Malu peptides |
| CN108359006B (en) * | 2018-05-25 | 2021-11-26 | 扬子江药业集团四川海蓉药业有限公司 | Preparation method of somaglutide |
| CN108676087B (en) * | 2018-07-24 | 2020-06-30 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthetic method of somaglutide |
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