CN104004083B - A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] - Google Patents
A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Download PDFInfo
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Abstract
The present invention relates to medicine synthesis field, disclose a kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].The method of the invention is according to the amino acid sequence of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N end to C end, first synthesize 3 polypeptide fragments of the 1st to the 4th amino acid, the 15th to the 16th amino acid and the 17th to the 31st amino acid, then press C end to N end order 3 polypeptide fragments of coupling and remaining Amino acid synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].The method of the invention can carry out the synthesis of 3 fragments simultaneously, and avoid too much and vector resin coupling and acidolysis, resin carrier consumption is greatly reduced, and shortens synthesis cycle, on the premise of ensureing higher total recovery and purity, simplify the complexity of synthesis technique.
Description
Technical field
The present invention relates to medicine synthesis field, be specifically related to a kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], English entitled Liraglutide, is that the one that Novo Nordisk Co., Ltd of Denmark develops treats II type sugar
The sick medicine of urine.Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is a kind of people's glicentin-1 (GLP-1) analog, and it is as GLP-1 receptor stimulating agent
Can play good reduction blood sugar effect, peptide sequence is as follows:
NH2-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
Gln-Ala-Ala-Lys(Nα-PAL-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-
COOH
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] has an amino acid difference compared with natural GLP-1 molecular structure, and adds 16 carbon palms
Acyl fatty acid side chain, has 97% homology with natural human GLP-1.From unlike natural GLP-1, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is in human body
Pharmacokinetics and pharmacodynamic characteristics are suitable for dosage regimen once a day.After subcutaneous administrations, its action time
The mechanism extending includes: the self association making absorption slow down is combined with albumin, to DPP IV (DPP-IV) and neutrality
Endopeptidase (NEP) has higher enzyme stability, thus has longer plasma half-life.
In the existing synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], Novo Nordisk Co., Ltd is mainly by gene recombination technology, utilizes ferment
Female production Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], but the domestic barms that cannot obtain produces.Patent US6268343B1, US6458924B2 with
And document " Journal of Medicinal Chemistry43,1664-1669,2000 " all discloses and utilizes intermediate GLP-
1 (7-37)-OH and NαThe method that-alkanoyl-Glu (ONSu)-OtBu prepares Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], but these three prior art
In, intermediate GLP-1 (7-37)-OH is required to reverse HPLC-purified, then under liquid-phase condition with Nα-alkanoyl-Glu
(ONSu)-OtBu reaction, and owing to GLP-1 (7-37)-OH N end is unprotected and Side chain protective group all removes, can cause
Produce much impurity, it is difficult to purifying, complex operation, the cycle is long, and waste liquid is many, is unfavorable for environmental protection, and two-step purifying, need to spend big
Amount acetonitrile, with high costs, it is unfavorable for large-scale production etc..
Chinese patent CN102286092A discloses a kind of total synthesis method, uses 2-CTC resin or king's resin, according to profit
Drawing Shandong peptide peptide sequence coupling amino acid one by one, eventually passing reversely purifying and obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it does not needs two-step purifying and miscellaneous
Matter is less, is better than above-mentioned several existing preparation method.But, this method needs coupling amino acid one by one, and synthesis cycle is long, always
Yield is relatively low, only about 15% (seeing CN102286092A embodiment 12-14), it is still necessary to improve further.
In order to solve the problems referred to above, the Chinese patent of Application No. 201210369966.3 discloses a kind of synthesis Li Lalu
The method of peptide, it is according to the amino acid sequence of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N end to C end, first synthesize the 1st to the 4th amino acid, the 5th to the
5 polypeptide fragments of ten amino acid, the 11st to the 16th amino acid, the 17th to the 24th amino acid and the 25th to the 31st amino acid,
Then 5 polypeptide fragment synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]s of coupling, shorten synthesis cycle, and the total recovery after purification improving Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] exceedes
30%, purity is more than 99%.But, the method needs to synthesize 5 fragments with resin carrier respectively, then carries out coupling again, its
In relate to connection repeatedly and cracking resin carrier, easy on operational degree, and coupling and cracking resin carrier
It is required to spend many time, and expend too much resin carrier.Additionally, this patent in impurity content context of detection and is not disclosed
The content of the maximum single contaminant bigger to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] qualitative effects.
At Peptides Synthesis, different synthesis strategies is to the purity of final products, maximum single contaminant content and yield
Have a significant impact, how on the premise of ensureing purity and yield, reduce the complexity of synthesis technique, maximum list as far as possible
One impurity and shortening synthesis cycle, be the research emphasis of technical staff instantly.
Content of the invention
In view of this, it is an object of the invention to provide a kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] so that side of the present invention
Method, on the premise of ensureing its total recovery and purity, reduces the complexity of synthetic method and maximum single contaminant, shortens synthesis
Cycle.
For achieving the above object, the present invention provides following technical scheme:
A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], comprises the following steps:
Step 1, synthesis on amino acid sequence N end shown in SEQ ID NO:1, His side chain and on Glu side chain coupling have
The polypeptide fragment 1 of protection group；
The polypeptide fragment 2 of synthesis protected base of coupling on amino acid sequence N end shown in SEQ ID NO:2, Glu side chain；
Synthesis amino acid sequence C end coupling shown in SEQ ID NO:3 have resin carrier, on Gln side chain, Glu side chain
On upper, Trp side chain, on Arg side chain the protected base of coupling and on Lys side chain coupling have Nα-PAL-γ-Glu(α-OtBu)
Polypeptide fragment 3；
Step 2, the C end coupling by the N end of polypeptide fragment 3 and polypeptide fragment 2, the N end removing polypeptide fragment 2 after coupling is protected
Protect base, obtain polypeptide resin I；
Step 3, according to amino acid sequence C end shown in SEQ ID NO:4 to the order of N end, first by protected for N end coupling base
The N end coupling of leucic C section and polypeptide resin I, then one by one other corresponding protected amino acids are extended successively
Coupling, removes N end protection group, obtains polypeptide resin II after coupling；
Step 4, the N end coupling by the C end of polypeptide fragment 1 and polypeptide resin II, remove the N end of polypeptide fragment 1 after coupling
Protection group, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin；
Step 5, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin acid solution removing C end resin and all protection groups obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, and crude product is pure
Change, it is thus achieved that Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] backbone amino acid has 31, uses fragment approach to carry out synthesis and there is a variety of form, but only closes
The total recovery purity of the higher Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of suitable fragment approach guarantee, can reduce again the complicated journey of synthesis technique simultaneously
Degree.To this end, applicant is according to long-term experimental study and amino acid racemization situation, it is proposed that prepared by the method for the invention
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], on the premise of ensureing its total recovery and purity, reduces the complexity of synthetic method..
It in the method for the invention, is first according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence and is divided into 4 parts, be i.e. divided into 3 fragments
Synthesis and the coupling one by one of other amino acid, with the amino acid sequence numbering of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain N end to C end, such as following formula:
NH2-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-L
eu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys20(Nα-PAL-γ-Glu)-Glu21-Phe22-Ile23-Ala24-
Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-COOH
Amino acid sequence shown in SEQ ID NO:1 is the peptide sequence of numbering 1-4 in above formula, shown in SEQ ID NO:2
Amino acid sequence is the peptide sequence of numbering 15-16 in above formula, and amino acid sequence shown in SEQ ID NO:3 is in above formula volume
The peptide sequence of number 17-31, amino acid sequence shown in SEQ ID NO:4 is the peptide sequence of numbering 5-14 in above formula.
The polypeptide fragment 1 that the present invention synthesizes in step 1 is on the basis of amino acid sequence shown in SEQ ID NO:1,
Coupling protection group is distinguished on its N end, His side chain and on Glu side chain；The polypeptide fragment 2 of synthesis is shown in SEQ ID NO:2
On the basis of amino acid sequence, difference coupling protection group on its N end, Glu side chain；The polypeptide fragment 3 of synthesis is at SEQ ID
On the basis of amino acid sequence shown in NO:3, its C end coupling have resin carrier, on Gln side chain, on Glu side chain, Trp side chain
On upper, Arg side chain the protected base of coupling and on Lys side chain coupling have Nα-PAL-γ-Glu(α-OtBu).And remaining its
His amino acid, then be after polypeptide fragment 3 forms polypeptide resin I with polypeptide fragment 2 coupling, according to ammonia shown in SEQ ID NO:4
Base acid sequence C end to the order of N end, the first N end coupling by the leucic C section of protected for N end coupling base and polypeptide resin I,
Then one by one other corresponding protected amino acids are carried out successively extending coupling, remove N end protection group after coupling, obtain polypeptide tree
Fat II.Then polypeptide resin II and polypeptide fragment 1 carry out the peptide chain synthesis that last coupling completes whole Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] again.
Protection group of the present invention be on the conventional protected amino acid main chain in Amino acid synthesis field and side chain amino,
The blocking group of the group of the interference synthesis such as carboxyl, prevents amino, carboxyl etc. from reacting during preparing target product, raw
Become impurity, for the present invention needs protect side chain amino acid for, its side-chain structure as well known to those skilled in the art and
Knowing the conventional protection group of employing and carrying out the groups such as the amino on protected amino acid side chain, carboxyl, as preferably, the present invention passes through Trt
Protection group protection histidine, the side chain of glutamine；By OtBu protection group protection glutamic acid, the side chain of aspartic acid；Pass through
TBu protection group protection threonine, serine, the side chain of tyrosine；Side chain by Boc protection group protective coloration propylhomoserin；Pass through Pdf
The arginic side chain of protection group protection.Additionally, in the amino acid that the method for the invention relates to, amino acid N end is all preferably logical
Cross Fmoc protection group to protect, and histidine also can be protected by Boc protection group.And the amino acid protected by protection group
It is referred to as protected amino acid.
Preferably, described in step 1 synthesize polypeptide fragment 1 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier of Fmoc protection group
After coupling, de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID
N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have by amino acid sequence C shown in NO:1 end successively one by one
The glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protection group, N end coupling have the alanine (Fmoc-Ala-of Fmoc protection group
OH), N end coupling has Boc protection group or Fmoc protection group and side chain coupling to have the histidine (Fmoc/Boc-of Trt protection group
His (Trt)-OH) carry out extending coupling, lysate cracking after coupling, removing resin carrier obtains polypeptide fragment 1 (Fmoc/Boc-
His(Trt)-Ala-Glu(OtBu)-Gly-OH)。
Preferably, described in step 1 synthesize polypeptide fragment 2 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier of Fmoc protection group
After coupling, de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID
N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have OtBu to protect by the end of amino acid sequence C shown in NO:2
The glutamic acid (Fmoc-Glu (OtBu)-OH) of base carries out extending coupling, lysate cracking after coupling, and removing resin carrier obtains many
Fragments of peptides 2 (Fmoc-Glu (OtBu)-Gly-OH).
Preferably, described in step 1 synthesize polypeptide fragment 3 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier of Fmoc protection group
After coupling, de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID
N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have by amino acid sequence C shown in NO:3 end successively one by one
The arginine (Fmoc-Arg (Pdf)-OH) of Pdf protection group, N end coupling have Fmoc protection group glycine (Fmoc-Gly-OH),
N end coupling has Fmoc protection group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protection group, N end coupling
The valine (Fmoc-Val-OH) of Fmoc protection group, N end coupling is had to have the leucine (Fmoc-Leu-OH) of Fmoc protection group, N
End coupling has Fmoc protection group and side chain coupling to have the tryptophan (Fmoc-Trp (Boc)-OH) of Boc protection group, N end coupling to have
Alanine (Fmoc-Ala-OH) the N end coupling of Fmoc protection group has the isoleucine (Fmoc-Ile-OH) of Fmoc protection group, N end
Coupling has the phenylalanine (Fmoc-Phe-OH) of Fmoc protection group, N end coupling to have Fmoc protection group and side chain coupling to have OtBu
The glutamic acid (Fmoc-Glu (OtBu)-OH) of protection group, N end coupling have Fmoc protection group and side chain coupling to have Nα-PAL-γ-
Lysine (Fmoc-Lys (the N of Glu (α-OtBu)α-PAL-γ-Glu (α-OtBu))-OH), two N end couplings have Fmoc to protect
The alanine (Fmoc-Ala-OH) of base, N end coupling have Fmoc protection group and side chain coupling to have the glutamine of Trt protection group
(Fmoc-Gln (Trt)-OH) carries out extending coupling, removing N section with Fmoc protection group obtain polypeptide fragment 3 (Gln (Trt)-
Ala-Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Tr p(Boc)-Leu-
Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin carrier).
Wherein, described NαIn-PAL-γ-Glu (α-OtBu), OtBu is α carboxyl on glutamic acid (i.e. carboxyl on main chain)
Protection group, be in order to make the carboxyl on glutamate side chain and the amino on lysine side-chain condensation coupling formed paddy acyl group,
Complete the synthesis of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] lysine side-chain eventually.
Preferably, step 3 particularly as follows:
Use activating reagent and condensation reagent, according to amino acid sequence C end shown in SEQ ID NO:4 to the order of N end, first
The C end of leucine (Fmoc-Leu-OH) that N end coupling has Fmoc protection group takes off Fmoc guarantor after the N end coupling of polypeptide resin I
Protect base and obtain H-Leu-polypeptide resin I, then have Fmoc protection group and side chain coupling to have tBu to protect N end coupling one by one successively
Protect the tyrosine (Fmoc-Tyr (tBu)-OH) of base, two N end couplings have Fmoc protection group and side chain coupling to have tBu protection group
Serine (Fmoc-Ser (tBu)-OH), N end coupling have the glycine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have
Fmoc protection group and side chain coupling have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protection group, N end coupling to have Fmoc
Protection group and side chain coupling have the serine (Fmoc-Ser (tBu)-OH) of tBu protection group, N end coupling have Fmoc protection group with
And side chain coupling has the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group, N end coupling to have the phenylalanine of Fmoc protection group
(Fmoc-Phe-OH), N end coupling has Fmoc protection group and side chain coupling to have the threonine (Fmoc-Thr of tBu protection group
(tBu)-OH) carry out extending coupling, remove after coupling N end with Fmoc protection group, obtain polypeptide resin II (Thr (tBu)-
Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-polypeptide resin
Ⅰ)。
In the preferred version of above-mentioned synthesis polypeptide fragment 1-3, first protected amino acid and resin carrier coupling institute shape
The polypeptide resin substitution value becoming is preferably 0.2～1.0mmol/g polypeptide resin, and preferred substitution value is 0.3～0.5mmol/g
Polypeptide resin.
Extension coupling of the present invention refers to that remaining amino acid is according to respectively after first amino acid with resin carrier coupling
Enter with the amino acid generation condensation reaction (condensation reaction of backbone amino and carboxyl) of previous coupling one by one from the order of sequence
Row coupling.During coupling of the present invention, each protected amino acid or polypeptide fragment consumption are preferably 1-6 times of polypeptide resin molal quantity,
More preferably 2.5-3.5 times；The described coupling reaction time is preferably 60～300 minutes, more preferably 100～140 minutes.
In extending coupling, due to each protected base of amino acid N end, it is therefore desirable to first remove N end protection group even again
Connection, this is common knowledge for a person skilled in the art.The present invention preferably uses PIP/DMF (piperidines/N, N-dimethyl formyl
Amine) mixed solution removing N end protection group, containing piperidines in mixed solution is 10～30% (V), and remaining is DMF.Go N end protection group
The consumption of reagent is every gram of polypeptide resin 5～15mL, more preferably every gram polypeptide resin 8～12mL；Go the N end protection group time
It is 10～60 minutes, preferably 15～25 minutes.
It should be noted that polypeptide resin of the present invention refers to any number protected amino acid according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] amino acid
Sequentially be connected the polypeptide resin being formed with resin carrier, also includes polypeptide resin the Ith, the polypeptide in independent claims among these
Resin the IIth, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin, polypeptide fragment 3.
In the preferred version of above-mentioned synthesis polypeptide fragment 1-3, the resin carrier using in the synthesis of described polypeptide fragment is
Trityl-Cl (trityl chloride) resinoid or hydroxy kind resin.It is highly preferred that described Trityl-Cl resinoid is Trityl-
Cl resin, 4-Methyltrityl-Cl (4-methyltrityl chlorine) resin, 4-Methoxytrityl-Cl (4-methoxyl group three
Benzyl chlorine) resin or 2-Cl Trity-Cl (2-chlorine trityl chloride) resin；Described carboxyl resinoid is Wang (king) resin
Or HMP (to hydroxymethyl phenoxy methylated polystyrene) resin.
Wherein, it is further preferred that when resin carrier is Trityl-Cl resinoid, protected amino acid and vector resin
Coupling method be: the carboxyl of protected amino acid and the Cl-substituted alkyl in resin occur esterification to connect under the effect of alkali
Enter protected amino acid；
In the preferred version of above-mentioned synthesis polypeptide fragment 1-3, the further preferred percent by volume of described lysate 25%
The dichloromethane solution of trifluoroethanol.
As preferably, described condensation reagent is preferably N, N-DIC (DIC), N, N-dicyclohexyl carbon two
Imines (DCC), hexafluorophosphoric acid BTA-1-base-epoxide tripyrrole alkyl phosphorus/organic base (PyBOP/ organic base), 2-(7-nitrogen
Miscellaneous-1H-BTA-1-base)-1,1,3,3-tetramethylurea hexafluorophosphoric acid ester/organic base (HATU/ organic base), benzo three
Nitrogen azoles-N, N, N', N'-tetramethylurea hexafluorophosphate/organic base (HBTU/ organic base), O-BTA-N, N, N', N'-
One in tetramethylurea tetrafluoro boric acid ester/organic base (TBTU/ organic base).The mole dosage of described condensation reagent is preferably many
1～6 times of amino total mole number in peptide resin, more preferably 2.5～3.5 times.
It should be noted that described PyBOP/ organic base, HATU/ organic base, HBTU/ organic base, TBTU/ organic base,
The present invention belongs to the condensation reagent of four kinds of Dual system, i.e. PyBOP, HATU, HBTU need in use respectively with organic base group
Using together into a kind of condensation reagent, the mol ratio of wherein said organic base and PyBOP, HATU, HBTU, TBTU is preferred
For for 1.3-3.0:1, more preferably 1.3-1.5:1.
As preferably, the organic base in described condensation reagent is preferably DIPEA (DIPEA), triethylamine
Or N-methylmorpholine (NMM), more preferably DIPEA (TEA).
As preferably, described activating reagent is I-hydroxybenzotriazole (HOBt) or N-hydroxyl-7-azepine BTA
(HOAt).The consumption of described activating reagent is preferably 1.2～6 times of amino total mole number in polypeptide resin, more preferably 2.5～
3.5 again.
As preferably, coupling reagent of the present invention is DIPEA (DIPEA), N, N-diisopropyl carbon two
One of imines/DMAP (DIC/DMAP) two kinds.
Meanwhile, synthetic method of the present invention step 2 and step 4 preferably employs above-mentioned condensation reagent and activating reagent carries out idol
Connection.
In building-up process of the present invention, it is preferred to use DMF solvent dissolves.
Except the above-mentioned synthetic method enumerated, the present invention also can use liquid phase synthesizing method according to fragment synthesis strategy of the present invention
Synthesize.
In the method for the invention step 5, preferably, acidolysis described in step 5 uses and by percent by volume is
The TFA of 80-95%, percent by volume are the EDT of 1-10%, the mixing acid hydrolysis solution acidolysis of balance of water composition.It is highly preferred that use
The mixing acid hydrolysis solution acidolysis being made up of the EDT that the TFA that percent by volume is 90%, percent by volume are 5%, balance of water.Institute
State mixing acid hydrolysis solution consumption and be preferably every gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin needs 4～15mL, more preferably 9～11mL.Described acidolysis
Time is preferably under room temperature condition 1～6 hour, more preferably 3～4 hours.
As preferably, described purify particularly as follows:
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, add water stirring, adjusts pH8.5 to being completely dissolved with ammoniacal liquor, and solution is by 0.45 μm of miillpore filter mistake
Filter, purifies standby；
Using high performance liquid chromatography to be purified, purifying chromatograph packing material is the anti-phase C18 of 10 μm, and flow phase system is
The 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the column flow rate of 77mm*250mm is 90mL/min, uses gradient system
System wash-out, circulation sample introduction purifies, and takes crude product solution and is splined in chromatographic column, starts flowing and elutes mutually, collects main peak and boils off acetonitrile
After, get profit and draw Shandong peptide purification intermediate concentrate；
Take Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and purify intermediate concentrate, filter standby with 0.45 μm of filter membrane；
Using high performance liquid chromatography to carry out changing salt, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is the anti-phase C18 of 10 μm, and the column flow rate of 77mm*250mm is 90mL/min, uses gradient elution, sample prescription in circulation
Method, is splined in chromatographic column, start flowing elutes mutually, gather collection of illustrative plates, observation trap change, collect change salt main peak and with divide
Analysis liquid phase detection purity, merges and changes salt main peak solution, reduced pressure concentration, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, freeze-drying, get profit
Draw Shandong peptide sterling.
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] being synthesized by the method for the invention detects through HPLC, and purity is more than 99%, and total recovery is 30%
Above, 3 fragments can synthesize simultaneously and in the method for the invention, the technical side with existing synthesizing amino acid one by one
Case is compared, and shortens synthesis cycle, improves efficiency.
Compared with the Chinese patent of Application No. 201210369966.3, being reduced to 3 fragments by 5 fragments, resin carries
Body consumption is greatly reduced, and reduces complexity and the cost of technique, it is to avoid too much and vector resin coupling and acidolysis, contracting
Short synthesis cycle.
From above technical scheme, the method for the invention can carry out the synthesis of 3 fragments simultaneously, and avoids too much
And the coupling of vector resin and acidolysis, resin carrier consumption is greatly reduced, and shortens synthesis cycle, is ensureing higher total receipts
On the premise of rate and purity, simplify the complexity of synthesis technique.
Detailed description of the invention
The invention discloses a kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], those skilled in the art can use for reference present disclosure, suitable
Realize when improving technological parameter.Special needs to be pointed out is, all similar replacements and change are for a person skilled in the art
Being apparent from, they are considered as including in the present invention.The method of the present invention is retouched by preferred embodiment
Stating, related personnel substantially can be to compound as herein described and preparation side in without departing from present invention, spirit and scope
Method is modified or suitably change and combination, realizes and applies the technology of the present invention.
In the specific embodiment of the invention, all couplings all can be by commercially available acquisition by the amino acid of protection group, this
Protected amino acid in bright is purchased from Hui Rong bio tech ltd, Chengdu, and resin used is limited purchased from Shangyu pul resin
Company, wherein polypeptide fragment of the present invention 1st, polypeptide fragment 2 also can be by commercially available acquisition, English contracting used in application documents
Write corresponding Chinese implication and be shown in Table 1.
Table 1 english abbreviation lexical or textual analysis
Below in conjunction with embodiment, the present invention is expanded on further.
Embodiment 1: the synthesis of polypeptide fragment 1
Weigh the 2-CTC resin 1Kg that substitution degree is 0.5mol/g, join in solid phase synthetic instrument reactor, wash with DMF
2 times, drained after 30 minutes by DMF swellable resins, take 0.75mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with resin
Reaction column in, add 1.5mol DIPEA, drain after reacting 2 hours, add containing the DMF of 1.5mol absolute methanol molten
Liquid, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-2-CTC resin.
By the Fmoc protection group 20 in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-2-CTC resin
Minute, then wash 6 times with DMF, obtain H-Gly-2-CTC resin.
Taking 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, dissolving with DMF, stirring is lower adds 1.5mol
DIC, continues stirring reaction 1 hour, adds in solid phase synthetic instrument reactor, and room temperature reaction 2h (examine with ninhydrin method by reaction end
Survey is as the criterion, if resin water white transparency, then reaction is completely, and resin develops the color, and represents reaction not exclusively, when need to extend coupling reaction again
Between), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
According to amino acid sequence C end shown in SEQ ID NO:1 to the amino acid sequence of N end, repeat above-mentioned deprotection base
Step with adding corresponding amino acid couplings, is sequentially completed the extension coupling of Fmoc-Ala-OH, Fmoc-His (Trt)-OH.
Reaction is washed 6 times by methyl alcohol after terminating, and resin vacuum is dried overnight, and weighing obtains 1.45Kg and do not remove 2-CTC tree
The polypeptide fragment 1 of fat, adds to 25L glass reactor.
Lytic reagent is poured in flask, room temperature reaction 2h by configuration lysate 25%TFE/ dichloromethane solution 15L.Instead
Should terminate, filter resin, collect filtrate.After filtrate volume rotation is evaporated, adds 1.5L DCM, mixed liquor is dropped to 15L second
In ether, separating out white solid, precipitation being collected by filtration, absolute ether washs, and is vacuum dried, and obtains 0.43Kg polypeptide fragment 1,
Fmoc-His (Trt)-Ala-Glu (OtBu)-Gly-OH, purity 96.3%, yield 92.1%.
Embodiment 2: the synthesis of polypeptide fragment 1
Weigh the 2-CTC resin 1Kg that substitution degree is 0.5mol/g, join in solid phase synthetic instrument reactor, wash with DMF
2 times, drained after 30 minutes by DMF swellable resins, take 0.75mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with resin
Reaction column in, add 1.5mol DIPEA, drain after reacting 2 hours, add containing the DMF of 1.5mol absolute methanol molten
Liquid, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-2-CTC resin.
By the Fmoc protection group in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-2-CTC 20 minutes,
Then wash 6 times with DMF, obtain H-Gly-2-CTC resin.
Taking 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, dissolving with DMF, stirring is lower adds 1.43mol
HBTU, adds 2molDIPEA after continuing stirring reaction 1 hour, joins in solid phase synthetic instrument reactor, room after mixing
(reaction end is as the criterion temperature reaction 2h with ninhydrin method detection, if resin water white transparency, then reaction is completely, and resin develops the color, and represents
Reaction not exclusively, need to extend the coupling reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
According to amino acid sequence C end shown in SEQ ID NO:1 to the amino acid sequence of N end, repeat above-mentioned deprotection base
Step with adding corresponding amino acid couplings, is sequentially completed the extension coupling of Fmoc-Ala-OH, Fmoc-His (Trt)-OH.
Reaction is washed 6 times by methyl alcohol after terminating, and resin vacuum is dried overnight, and weighing obtains 1.43Kg and do not remove 2-CTC tree
The polypeptide fragment 1 of fat, adds to 25L glass reactor.
Lytic reagent is poured in flask, room temperature reaction 2h by configuration lytic reagent 25%TFE/ dichloromethane solution 15L.
Reaction terminates, and filters resin, collects filtrate.After filtrate volume rotation is evaporated, adds 1.5L DCM, mixed liquor is dropped to 15L
In ether, separating out white solid, precipitation being collected by filtration, absolute ether washs, and is vacuum dried, and obtains 0.42Kg polypeptide fragment
1, Fmoc-His (Trt)-Ala-Glu (OtBu)-Gly-OH, purity 94.7%, yield 89.9%.
Embodiment 3: the synthesis of polypeptide fragment 2
Weigh the 2-CTC resin 1Kg that substitution degree is 0.5mol/g, join in solid phase synthetic instrument reactor, wash with DMF
2 times, drained after 30 minutes by DMF swellable resins, take 0.75mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with resin
Reaction column in, add 1.5mol DIPEA, drain after reacting 2 hours, add containing the DMF of 1.5mol absolute methanol molten
Liquid, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-2-CTC resin.
By the Fmoc protection group 20 in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-2-CTC resin
Minute, then wash 6 times with DMF, obtain H-Gly-2-CTC resin.
Taking 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, dissolving with DMF, stirring is lower adds 1.5mol
DIC, continues stirring reaction 1 hour, adds in solid phase synthetic instrument reactor, and room temperature reaction 2h (examine with ninhydrin method by reaction end
Survey is as the criterion, if resin water white transparency, then reaction is completely, and resin develops the color, and represents reaction not exclusively, when need to extend coupling reaction again
Between), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
Reaction is washed 6 times by methyl alcohol after terminating, and resin vacuum is dried overnight, and weighing obtains 1.24Kg and do not remove 2-CTC tree
The polypeptide fragment 2 of fat, adds to 25L glass reactor.
Lytic reagent is poured in flask, room temperature reaction 2h by configuration lytic reagent 25%TFE/ dichloromethane solution 15L.
Reaction terminates, and filters resin, collects filtrate.After filtrate volume rotation is evaporated, adds 1.2L DCM, mixed liquor is dropped to 12L
In ether, separating out white solid, precipitation being collected by filtration, absolute ether washs, and is vacuum dried, and obtains 0.22Kg polypeptide fragment
2, Fmoc-Glu (OtBu)-Gly-OH, purity 97.6%, yield 91.3%.
Embodiment 4: the synthesis of polypeptide fragment 2
Weigh the 2-CTC resin 1Kg that substitution degree is 0.5mol/g, join in solid phase synthetic instrument reactor, wash with DMF
2 times, drained after 30 minutes by DMF swellable resins, take 0.75mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with resin
Reaction column in, add 1.5mol DIPEA, drain after reacting 2 hours, add containing the DMF of 1.5mol absolute methanol molten
Liquid, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-2-CTC resin.
By the Fmoc protection group 20 in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-2-CTC resin
Minute, then wash 6 times with DMF, obtain H-Gly-2-CTC resin.
Taking 1.5mol Fmoc-Glu (OtBu)-OH and 1.5mol HOBt, dissolving with DMF, stirring is lower adds 1.43mol
HBTU, adds 2molDIPEA after continuing stirring reaction 1 hour, joins in solid phase synthetic instrument reactor, room after mixing
(reaction end is as the criterion temperature reaction 2h with ninhydrin method detection, if resin water white transparency, then reaction is completely, and resin develops the color, and represents
Reaction not exclusively, need to extend the coupling reaction time again), obtain Fmoc-Glu (OtBu)-Gly-2-CTC resin.
Reaction is washed 6 times by methyl alcohol after terminating, and resin vacuum is dried overnight, and weighing obtains 1.25Kg and do not remove 2-CTC tree
The polypeptide fragment 2 of fat, adds to 25L glass reactor.
Lytic reagent is poured in flask, room temperature reaction 2h by configuration lytic reagent 25%TFE/ dichloromethane solution 15L.
Reaction terminates, and filters resin, collects filtrate.After filtrate volume rotation is evaporated, adds 1.2L DCM, mixed liquor is dropped to 12L
In ether, separating out white solid, precipitation being collected by filtration, absolute ether washs, and is vacuum dried, and obtains 0.23Kg polypeptide fragment
2, Fmoc-Glu (OtBu)-Gly-OH, purity 97.1%, yield 95.4%.
Embodiment 5: the synthesis of polypeptide fragment 3
Weigh the Wang resin 0.5Kg that substitution degree is 0.4mol/g, join in solid phase synthetic instrument reactor, washed by DMF
Wash 2 times, drained after 30 minutes by DMF swellable resins, take 0.6mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with tree
In the reaction column of fat, add 0.6mol DIC and 0.06mol4-dimethylamino naphthyridine (DMAP), drain after reacting 6 hours, add
Enter the DMF solution containing 1.5mol absolute methanol, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-Wang tree
Fat.
By the Fmoc protection group 20 points in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-Wang resin
Clock, is then washed 6 times by DMF, obtains H-Gly-Wang resin.
Taking 0.6mol Fmoc-Arg (Pdf)-OH and 0.6mol HOBt, dissolving with DMF, stirring is lower adds 0.6mol
DIC, continues stirring reaction 1 hour, adds in solid phase synthetic instrument reactor, and room temperature reaction 3h (examine with ninhydrin method by reaction end
Survey is as the criterion, if resin water white transparency, then reaction is completely, and resin develops the color, and represents reaction not exclusively, when need to extend coupling reaction again
Between), obtain Fmoc-Arg (Pdf)-Gly-Wang resin.
According to amino acid sequence C end shown in SEQ ID NO:3 to the amino acid sequence of N end, repeat above-mentioned deprotection base
The step of amino acid couplings corresponding with addition, more one by one N end coupling is had the glycine (Fmoc-Gly-of Fmoc protection group successively
OH), N end coupling has Fmoc protection group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protection group, N end
Coupling has the valine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have the leucine (Fmoc-Leu-of Fmoc protection group
OH), N end coupling has Fmoc protection group and side chain coupling to have the tryptophan (Fmoc-Trp (Boc)-OH) of Boc protection group, N end
Coupling has alanine (Fmoc-Ala-OH) the N end coupling of Fmoc protection group to have the isoleucine (Fmoc-Ile-of Fmoc protection group
OH), N end coupling has the phenylalanine (Fmoc-Phe-OH) of Fmoc protection group, N end coupling to have Fmoc protection group and side chain even
Be associated with the glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protection group, N end coupling has Fmoc protection group and side chain coupling to have NαLysine (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu)α-PAL-γ-Glu (α-OtBu))-OH), two N end couplings have
The alanine (Fmoc-Ala-OH) of Fmoc protection group, N end coupling have Fmoc protection group and side chain coupling to have Trt protection group
Glutamine (Fmoc-Gln (Trt)-OH) carries out extending coupling, finally with every gram of polypeptide resin 15mL PIP (20%)/DMF
(80%) mixed solution removing N section with Fmoc protection group 20min, then with DMF wash 6 times, obtain polypeptide fragment 3 (Gln
(Trt)-Ala-Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Tr p
(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin)；
Embodiment 6: the synthesis of polypeptide fragment 3
Weigh the Wang resin 0.5Kg that substitution degree is 0.4mol/g, join in solid phase synthetic instrument reactor, washed by DMF
Wash 2 times, drained after 30 minutes by DMF swellable resins, take 0.6mol Fmoc-Gly-OH DMF and dissolve, add above-mentioned equipped with tree
In the reaction column of fat, add 0.6mol DIC and 0.06mol4-dimethylamino naphthyridine (DMAP), drain after reacting 6 hours, add
Enter the DMF solution containing 1.5mol absolute methanol, stirring reaction 1 hour, wash 6 times with DMF, obtain Fmoc-Gly-Wang tree
Fat.
By the Fmoc protection group 20 points in every gram of resin 10mL20% piperidines/DMF solution removing Fmoc-Gly-Wang resin
Clock, is then washed 6 times by DMF, obtains H-Gly-Wang resin.
Taking 0.6mol Fmoc-Arg (Pdf)-OH and 0.6mol HOBt, dissolving with DMF, stirring is lower adds 0.57mol
HBTU, adds 0.9molDIPEA after continuing stirring reaction 1 hour, joins in solid phase synthetic instrument reactor after mixing,
(reaction end is as the criterion room temperature reaction 3h with ninhydrin method detection, if resin water white transparency, then reaction is completely, and resin develops the color, table
Show that reaction not exclusively, need to extend the coupling reaction time again), obtain Fmoc-Arg (Pdf)-Gly-Wang resin.
According to amino acid sequence C end shown in SEQ ID NO:3 to the amino acid sequence of N end, repeat above-mentioned deprotection base
The step of amino acid couplings corresponding with addition, more one by one N end coupling is had the glycine (Fmoc-Gly-of Fmoc protection group successively
OH), N end coupling has Fmoc protection group and side chain coupling to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protection group, N end
Coupling has the valine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have the leucine (Fmoc-Leu-of Fmoc protection group
OH), N end coupling has Fmoc protection group and side chain coupling to have the tryptophan (Fmoc-Trp (Boc)-OH) of Boc protection group, N end
Coupling has alanine (Fmoc-Ala-OH) the N end coupling of Fmoc protection group to have the isoleucine (Fmoc-Ile-of Fmoc protection group
OH), N end coupling has the phenylalanine (Fmoc-Phe-OH) of Fmoc protection group, N end coupling to have Fmoc protection group and side chain even
Be associated with the glutamic acid (Fmoc-Glu (OtBu)-OH) of OtBu protection group, N end coupling has Fmoc protection group and side chain coupling to have NαLysine (Fmoc-Lys (the N of-PAL-γ-Glu (α-OtBu)α-PAL-γ-Glu (α-OtBu))-OH), two N end couplings have
The alanine (Fmoc-Ala-OH) of Fmoc protection group, N end coupling have Fmoc protection group and side chain coupling to have Trt protection group
Glutamine (Fmoc-Gln (Trt)-OH) carries out extending coupling, finally with every gram of polypeptide resin 15mL PIP (20%)/DMF
(80%) mixed solution removing N section with Fmoc protection group 20min, then with DMF wash 6 times, obtain polypeptide fragment 3 (Gln
(Trt)-Ala-Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Tr p
(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin)；
Embodiment 7: the synthesis of polypeptide resin I
Take 0.44mol polypeptide fragment the 2nd, 0.44mol polypeptide fragment 3 and 0.44mol HOAt, dissolve with DMF, add under stirring
Enter 0.44mol DIC, continue stirring reaction 1 hour, join in solid phase synthetic instrument reactor, room temperature reaction 6h (reaction end
Being as the criterion with ninhydrin method detection, if resin water white transparency, then reaction is completely, and resin develops the color, and represents that reaction not exclusively, need to be prolonged again
The long coupling reaction time), then take off Fmoc protection group, time with every gram of resin 10mL PIP (20%)/DMF (80%) mixed solution
For 20min, wash 6 times with DMF, obtain polypeptide resin I, i.e. NH2-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(Ot Bu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg
(Pbf)-Gly-Arg (Pbf)-Gly-Wang resin；
Embodiment 8: the synthesis of polypeptide resin I
Take 0.44mol polypeptide fragment the 2nd, 0.44mol polypeptide fragment 3 and 0.44mol HOAt, dissolve with DMF, add under stirring
Enter 0.42mol HATU, add 0.65molDIPEA after continuing stirring reaction 1 hour, after mixing, join synthesis in solid state
In instrument reactor, room temperature reaction 6h (reaction end is as the criterion with ninhydrin method detection, if resin water white transparency, then reaction is completely,
Resin develops the color, and represents reaction not exclusively, need to extend the coupling reaction time again), then with every gram of resin 10mL PIP (20%)/DMF
(80%) mixed solution takes off Fmoc protection group, and the time is 20min, washs 6 times with DMF, obtains polypeptide resin I, i.e. NH2-Glu
(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(Ot Bu)-Phe-
Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-Wang resin；
Embodiment 9: the synthesis of polypeptide resin II
Taking 0.6mol protected amino acid, 0.6mol polypeptide resin I and 0.6mol HOBt, dissolving with DMF, stirring is lower to be added
0.6mol DIC, continues stirring reaction 1 hour, adds in solid phase synthetic instrument reactor, and (reaction end is with indenes three for room temperature reaction 3h
The detection of ketone method is as the criterion, if resin water white transparency, then reaction is completely, and resin develops the color, and represents that reaction not exclusively, extends coupling reaction
Time).
N end coupling to the order of N end, is first had by protected amino acid according to amino acid sequence C end shown in SEQ ID NO:4
After the C end of the leucine (Fmoc-Leu-OH) of Fmoc protection group and the N end coupling of polypeptide resin I, de-Fmoc protection group obtains H-
Then N end coupling is had Fmoc protection group and side chain coupling to have the junket ammonia of tBu protection group by Leu-polypeptide resin I successively one by one
Acid (Fmoc-Tyr (tBu)-OH), two N end couplings have Fmoc protection group and side chain coupling to have the serine of tBu protection group
(Fmoc-Ser (tBu)-OH), N end coupling have the glycine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have Fmoc to protect
Base and side chain coupling have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protection group, N end coupling have Fmoc protection group with
And side chain coupling has the serine (Fmoc-Ser (tBu)-OH) of tBu protection group, N end coupling to have Fmoc protection group and side chain even
Be associated with the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group, N end coupling has the phenylalanine (Fmoc-of Fmoc protection group
Phe-OH), N end coupling has Fmoc protection group and side chain coupling to have the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group
Carry out extending coupling, after coupling, take off Fmoc protection group with every gram of resin 10mL PIP (20%)/DMF (80%) mixed solution, when
Between be 20min, with DMF washing, obtain polypeptide resin II, it may be assumed that NH2-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp
(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(t Bu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-
Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 10: the synthesis of polypeptide resin II
Taking 0.6mol protected amino acid, 0.6mol polypeptide resin I and 0.6mol HOBt, dissolving with DMF, stirring is lower to be added
0.57mol HBTU, adds 0.9molDIPEA after continuing stirring reaction 1 hour, joins solid phase synthetic instrument after mixing
In reactor, (reaction end is as the criterion room temperature reaction 3h with ninhydrin method detection, if resin water white transparency, then reaction is completely, tree
Fat develops the color, and represents that reaction not exclusively, extends the coupling reaction time).
N end coupling to the order of N end, is first had by protected amino acid according to amino acid sequence C end shown in SEQ ID NO:4
After the C end of the leucine (Fmoc-Leu-OH) of Fmoc protection group and the N end coupling of polypeptide resin I, de-Fmoc protection group obtains H-
Then N end coupling is had Fmoc protection group and side chain coupling to have the junket ammonia of tBu protection group by Leu-polypeptide resin I successively one by one
Acid (Fmoc-Tyr (tBu)-OH), two N end couplings have Fmoc protection group and side chain coupling to have the serine of tBu protection group
(Fmoc-Ser (tBu)-OH), N end coupling have the glycine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have Fmoc to protect
Base and side chain coupling have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protection group, N end coupling have Fmoc protection group with
And side chain coupling has the serine (Fmoc-Ser (tBu)-OH) of tBu protection group, N end coupling to have Fmoc protection group and side chain even
Be associated with the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group, N end coupling has the phenylalanine (Fmoc-of Fmoc protection group
Phe-OH), N end coupling has Fmoc protection group and side chain coupling to have the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group
Carry out extending coupling, after coupling, take off Fmoc protection group with every gram of resin 10mL PIP (20%)/DMF (80%) mixed solution, when
Between be 20min, with DMF washing, obtain polypeptide resin II, it may be assumed that NH2-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp
(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(t Bu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-
Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 11: the synthesis of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin
Take 0.44mol polypeptide fragment the 1st, 0.44mol polypeptide resin II and 0.44mol HOAt, dissolve with DMF, add under stirring
Enter 0.44mol DIC, continue stirring reaction 1 hour, join in solid phase synthetic instrument reactor, room temperature reaction 6h (reaction end
Being as the criterion with ninhydrin method detection, if resin water white transparency, then reaction is completely, and resin develops the color, and represents that reaction not exclusively, need to be prolonged again
The long coupling reaction time), then take off Fmoc protection group, time with every gram of resin 10mL PIP (20%)/DMF (80%) mixed solution
For 20min, washing 6 times with DCM, vacuum decompression is dried, and gets profit and draws Shandong peptide resin 1445g, i.e. NH2-His(Trt)-Ala-Glu
(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Va l-Ser(tBu)-Ser(tBu)-
Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-
Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Ar g (Pbf)-Gly-Wang resin.
Embodiment 12: the synthesis of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin
Take 0.44mol polypeptide fragment the 1st, 0.44mol polypeptide resin II and 0.44mol HOAt, dissolve with DMF, add under stirring
Enter 0.42mol HATU, add 0.65molDIPEA after continuing stirring reaction 1 hour, after mixing, join synthesis in solid state
In instrument reactor, room temperature reaction 6h (reaction end is as the criterion with ninhydrin method detection, if resin water white transparency, then reaction is completely,
Resin develops the color, and represents reaction not exclusively, need to extend the coupling reaction time again), then with every gram of resin 10mL PIP (20%)/DMF
(80%) mixed solution takes off Fmoc protection group, and the time is 20min, washs 6 times with DCM, and vacuum decompression is dried, and obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] tree
Fat 1381g, i.e. NH2-His(Trt)-Ala-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp
(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys
(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-
Gly-Arg (Pbf)-Gly-Wang resin.
Embodiment 13: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product
Example 11 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin, the mixing acid hydrolysis solution adding volume ratio to be TFA water EDT=90 55
(10mL/ gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin of consumption), stirs, and reaction 3 hour is stirred at room temperature, and reactant mixture uses sand core funnel mistake
Filter, collects filtrate, and resin is washed 3 times by a small amount of TFA again, reduced pressure concentration after merging filtrate, adds absolute ether precipitation, then uses nothing
Water ether washes precipitation 3 times, drains to obtain off-white powder, and vacuum decompression is dried to constant weight, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product 863g.
Embodiment 14: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product
Example 12 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin, the mixing acid hydrolysis solution adding volume ratio to be TFA water EDT=90 55
(10mL/ gram of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin of consumption), stirs, and reaction 3 hour is stirred at room temperature, and reactant mixture uses sand core funnel mistake
Filter, collects filtrate, and resin is washed 3 times by a small amount of TFA again, reduced pressure concentration after merging filtrate, adds absolute ether precipitation, then uses nothing
Water ether washes precipitation 3 times, drains to obtain off-white powder, and vacuum decompression is dried to constant weight, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product 797g.
Embodiment 15: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purifying crude
Example 13 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, add water stirring, adjusts pH8.5 to being completely dissolved with ammoniacal liquor, and solution is used
0.45 μm of filtering with microporous membrane, purifies standby；
Using high performance liquid chromatography to be purified, purifying chromatograph packing material is the anti-phase C18 of 10 μm, and flow phase system is
The 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the column flow rate of 77mm*250mm is 90mL/min, uses gradient system
System wash-out, circulation sample introduction purifies, and takes crude product solution and is splined in chromatographic column, starts flowing and elutes mutually, collects main peak and boils off acetonitrile
After, get profit and draw Shandong peptide purification intermediate concentrate；
Take Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and purify intermediate concentrate, filter standby with 0.45 μm of filter membrane；
Using high performance liquid chromatography to carry out changing salt, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is the anti-phase C18 of 10 μm, and the column flow rate of 77mm*250mm is 90mL/min, uses gradient elution, sample prescription in circulation
Method, is splined in chromatographic column, start flowing elutes mutually, gather collection of illustrative plates, observation trap change, collect change salt main peak and with divide
Analysis liquid phase detection purity, merges and changes salt main peak solution, reduced pressure concentration, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, freeze-drying, get profit
Drawing Shandong peptide sterling 270.9g, total recovery is 36.1%, molecular weight: 3751.2 (100%M+H), purity: 99.3%, maximum single
Impurity 0.12%.
Embodiment 16: Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] purifying crude
Example 14 gained Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, add water stirring, adjusts pH8.5 to being completely dissolved with ammoniacal liquor, and solution is used
0.45 μm of filtering with microporous membrane, purifies standby；
Using high performance liquid chromatography to be purified, purifying chromatograph packing material is the anti-phase C18 of 10 μm, and flow phase system is
The 0.1%TFA/ aqueous solution-0.1%TFA/ acetonitrile solution, the column flow rate of 77mm*250mm is 90mL/min, uses gradient system
System wash-out, circulation sample introduction purifies, and takes crude product solution and is splined in chromatographic column, starts flowing and elutes mutually, collects main peak and boils off acetonitrile
After, get profit and draw Shandong peptide purification intermediate concentrate；
Take Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and purify intermediate concentrate, filter standby with 0.45 μm of filter membrane；
Using high performance liquid chromatography to carry out changing salt, flow phase system is 1% acetic acid/water solution-acetonitrile, purifying chromatogram
Filler is the anti-phase C18 of 10 μm, and the column flow rate of 77mm*250mm is 90mL/min, uses gradient elution, sample prescription in circulation
Method, is splined in chromatographic column, start flowing elutes mutually, gather collection of illustrative plates, observation trap change, collect change salt main peak and with divide
Analysis liquid phase detection purity, merges and changes salt main peak solution, reduced pressure concentration, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] aqueous acetic acid, freeze-drying, get profit
Drawing Shandong peptide sterling 237.8g, total recovery is 31.7%, molecular weight: 3751.6 (100%M+H), purity: 99.0%, maximum single
Impurity 0.10%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. the method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterised in that comprise the following steps:
Step 1, synthesis on amino acid sequence N end shown in SEQ ID NO:1, His side chain and on Glu side chain coupling protected
The polypeptide fragment 1 of base；
The polypeptide fragment 2 of synthesis protected base of coupling on amino acid sequence N end shown in SEQ ID NO:2, Glu side chain；
Synthesis amino acid sequence C end coupling shown in SEQ ID NO:3 have resin carrier, on Gln side chain, on Glu side chain,
On Trp side chain, on Arg side chain the protected base of coupling and on Lys side chain coupling have Nα-PAL-γ-Glu (α-OtBu) is many
Fragments of peptides 3；
Step 2, the C end coupling by the N end of polypeptide fragment 3 and polypeptide fragment 2, remove the N end protection of polypeptide fragment 2 after coupling
Base, obtains polypeptide resin I；
Step 3, according to amino acid sequence C end shown in SEQ ID NO:4 to the order of N end, first bright by protected for N end coupling base
Then other corresponding protected amino acids are carried out extending coupling by the C section of propylhomoserin and the N end coupling of polypeptide resin I successively one by one,
Remove N end protection group after coupling, obtain polypeptide resin II；
Step 4, the N end coupling by the C end of polypeptide fragment 1 and polypeptide resin II, remove the N end protection of polypeptide fragment 1 after coupling
Base, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin；
Step 5, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] resin use by percent by volume be EDT that the TFA of 80-95%, percent by volume are 1-10%,
The mixing acid hydrolysis solution acidolysis removing C end resin of balance of water composition and all protection groups obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product, purifying crude,
Obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
2. method according to claim 1, it is characterised in that synthesize polypeptide fragment 1 described in step 1 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier coupling of Fmoc protection group
Rear de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID NO:1 institute
Show that N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have OtBu to protect by amino acid sequence C end successively one by one
Protect the glutamic acid (Fmoc-Glu (OtBu)-OH) of base, N end coupling has the alanine (Fmoc-Ala-OH) of Fmoc protection group, N end
Coupling have Boc protection group or Fmoc protection group and side chain coupling have Trt protection group histidine (Fmoc/Boc-His (Trt)-
OH) carry out extending coupling, lysate cracking after coupling, removing resin carrier obtain polypeptide fragment 1 (Fmoc/Boc-His (Trt)-
Ala-Glu(OtBu)-Gly-OH)。
3. method according to claim 1, it is characterised in that synthesize polypeptide fragment 2 described in step 1 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier coupling of Fmoc protection group
Rear de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID NO:2 institute
Show that N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have the paddy of OtBu protection group by amino acid sequence C end
Propylhomoserin (Fmoc-Glu (OtBu)-OH) carries out extending coupling, lysate cracking after coupling, and removing resin carrier obtains polypeptide fragment
2(Fmoc-Glu(OtBu)-Gly-OH)。
4. method according to claim 1, it is characterised in that synthesize polypeptide fragment 3 described in step 1 particularly as follows:
In the presence of coupling reagent, N end coupling is had glycine (Fmoc-Gly-OH) and the resin carrier coupling of Fmoc protection group
Rear de-Fmoc protection group obtains H-Gly-resin carrier, then uses activating reagent and condensation reagent, according to SEQ ID NO:3 institute
Show that N end coupling, to the order of N end, is had Fmoc protection group and side chain coupling to have Pdf to protect by amino acid sequence C end successively one by one
The arginine (Fmoc-Arg (Pdf)-OH) of base, N end coupling have the glycine (Fmoc-Gly-OH) of Fmoc protection group, N end coupling
Fmoc protection group and side chain coupling is had to have the arginine (Fmoc-Arg (Pdf)-OH) of Pdf protection group, N end coupling to have Fmoc to protect
Protect the valine (Fmoc-Val-OH) of base, N end coupling has the leucine (Fmoc-Leu-OH) of Fmoc protection group, N end coupling to have
Fmoc protection group and side chain coupling have the tryptophan (Fmoc-Trp (Boc)-OH) of Boc protection group, N end coupling to have Fmoc to protect
The alanine (Fmoc-Ala-OH) of base, N end coupling have the isoleucine (Fmoc-Ile-OH) of Fmoc protection group, N end coupling to have
The phenylalanine (Fmoc-Phe-OH) of Fmoc protection group, N end coupling have Fmoc protection group and side chain coupling to have OtBu protection group
Glutamic acid (Fmoc-Glu (OtBu)-OH), N end coupling have Fmoc protection group and side chain coupling to have Nα-PAL-γ-Glu(α-
OtBu) lysine (Fmoc-Lys (Nα-PAL-γ-Glu (α-OtBu))-OH), two N end couplings have the third of Fmoc protection group
Propylhomoserin (Fmoc-Ala-OH), N end coupling have Fmoc protection group and side chain coupling to have the glutamine (Fmoc-of Trt protection group
Gln (Trt)-OH) carry out extend coupling, removing N section with Fmoc protection group obtain polypeptide fragment 3 (Gln (Trt)-Ala-
Ala-Lys(N-ε-(Nα-PAL-γ-Glu(α-OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-
Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin carrier).
5. method according to claim 1, it is characterised in that step 3 particularly as follows:
Using activating reagent and condensation reagent, according to shown in SEQ ID NO:4, amino acid sequence C end is to the order of N end, first by N
End coupling has the C end of leucine (Fmoc-Leu-OH) of Fmoc protection group to take off Fmoc protection after the N end coupling of polypeptide resin I
Base obtains H-Leu-polypeptide resin I, then has Fmoc protection group and side chain coupling to have tBu to protect N end coupling one by one successively
The tyrosine (Fmoc-Tyr (tBu)-OH) of base, two N end couplings have Fmoc protection group and side chain coupling to have tBu protection group
Serine (Fmoc-Ser (tBu)-OH), N end coupling have the glycine (Fmoc-Val-OH) of Fmoc protection group, N end coupling to have
Fmoc protection group and side chain coupling have the aspartic acid (Fmoc-Asp (OtBu)-OH) of OtBu protection group, N end coupling to have Fmoc
Protection group and side chain coupling have the serine (Fmoc-Ser (tBu)-OH) of tBu protection group, N end coupling have Fmoc protection group with
And side chain coupling has the threonine (Fmoc-Thr (tBu)-OH) of tBu protection group, N end coupling to have the phenylalanine of Fmoc protection group
(Fmoc-Phe-OH), N end coupling has Fmoc protection group and side chain coupling to have the threonine (Fmoc-Thr of tBu protection group
(tBu)-OH) carry out extend coupling, after coupling remove N end with Fmoc protection group, obtain polypeptide resin II (Thr (tBu)-
Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Val-Ser (tBu)-Ser (tBu)-Tyr (tBu)-Leu-polypeptide resin
I)。
6. method according to claim 1-4 any one, it is characterised in that described resin carrier is Trityl-Cl class tree
Fat or hydroxy kind resin.
7. method according to claim 2-3 any one, it is characterised in that described lysate is percent by volume 25%
The dichloromethane solution of trifluoroethanol.
8. method according to claim 1-5 any one, it is characterised in that described condensation reagent is N, N-diisopropyl carbon
Diimine, N, N-dicyclohexylcarbodiimide, hexafluorophosphoric acid BTA-1-base-epoxide tripyrrole alkyl phosphorus/N, N-diisopropyl
Base ethamine, 2-(7-azepine-1H-BTA-1-base)-1,1,3,3-tetramethylurea hexafluorophosphoric acid ester/N, N-diisopropyl
Ethamine, BTA-N, N, N', N'-tetramethylurea hexafluorophosphate/N, N-diisopropylethylamine, O-BTA-N,
N, N', N'-tetramethylurea tetrafluoro boric acid ester/N, the one in N-diisopropylethylamine.
9. method according to claim 1-5 any one, it is characterised in that described activating reagent is I-hydroxybenzotriazole
Or N-hydroxyl-7-azepine BTA.
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| CN104650219B (en) * | 2015-02-15 | 2017-11-14 | 兰州大学 | The method that fragment condensation prepares Liraglutide |
| CN106478805B (en) * | 2015-08-28 | 2021-05-04 | 甘李药业股份有限公司 | Preparation method of GLP-1 derivative |
| CN107286234B (en) * | 2016-03-31 | 2021-06-08 | 深圳翰宇药业股份有限公司 | Method for reducing and/or removing default peptide in polypeptide solid phase synthesis |
| WO2018032521A1 (en) | 2016-08-19 | 2018-02-22 | 深圳市健元医药科技有限公司 | Method for synthesizing liraglutide |
| EP3692056A1 (en) | 2017-10-04 | 2020-08-12 | Chemical & Biopharmaceutical Laboratories of Patras S.A. | A process for preparing a glucagon-like peptide |
| CN108107103B (en) * | 2017-12-14 | 2020-10-16 | 华中师范大学 | Mass spectrum probe of glutamate receptor and method for detecting spatial distribution rule of glutamate receptor in brain tissue |
| CN107903317A (en) * | 2017-12-29 | 2018-04-13 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of synthetic method of Liraglutide |
| CN110894227A (en) * | 2018-09-13 | 2020-03-20 | 南京华威医药科技集团有限公司 | Solid-phase synthesis method of liraglutide |
| CN112912390B (en) | 2018-10-09 | 2023-12-15 | 北京费森尤斯卡比医药有限公司 | GLP-1 analogues and methods of preparation |
| EP3897570A1 (en) | 2018-12-19 | 2021-10-27 | KRKA, d.d., Novo mesto | Pharmaceutical composition comprising glp-1 analogue |
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| CN103275208A (en) * | 2013-05-27 | 2013-09-04 | 成都圣诺生物制药有限公司 | Preparation method for liraglutide |
| CN103288951A (en) * | 2013-06-19 | 2013-09-11 | 深圳翰宇药业股份有限公司 | Preparation method of liraglutide |
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