CN103304660B - A kind of synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] - Google Patents
A kind of synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Download PDFInfo
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Abstract
The invention discloses solid phase and the whole chemical synthesis process of solution hybridization of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], described method includes step: the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of N end protection and the synthesis of cetyl derivatives chemical, deprotection removes end protection, obtains target polypeptides.Obtained the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of half protection by polypeptide solid-state reaction method, this precursor purification carries out lower step chemosynthesis after reaching pharmaceutical grade purity.
Description
Technical field
The present invention relates to the chemosynthesis of medical many peptides crude drug, particularly relate to the solid phase of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
With solution hybridization synthetic method.
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is a kind of people's glicentin-1 (GLP-1) analog, is used for treating diabetes.
Molecular formula: C172H265N43O51, molecular weight: 3751.20, No. CAS: 204656-20-2.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is the medicine of approval in 2010.United States Patent (USP) US Patent No.6268343,
A kind of DNA of 6458924and 7235627 invention restructuring technique synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor, i.e. sequence
1-31, then obtain target polypeptides by chemical synthesis link Pal-Glu (OSu)-OtBu material.DNA weight
Group technique needs to use complicated equipment, and may introduce virus in the product, is unfavorable for the medicinal of product
Purpose.Chinese patent 200610110898.3and 200510107588 reports direct solid phase synthesis profit and draws
The method of Shandong peptide, but, due to the hydrophobicity of cetyl on peptide chain, the impurity that this sampling technology produces is
It is difficult to removing.
Therefore, this area is in the urgent need to providing a kind of feasible Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] synthetic method to produce high-purity product
Product, to meet medical usage.
Summary of the invention
It is desirable to provide a kind of new Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis process.
In a first aspect of the present invention, it is provided that the synthetic method of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], described method includes step:
(1) with Fmoc-Gly-solid phase synthesis resin as initiation material, repeat according to solid phase synthesis process
Deprotection, use condensing agent carry out connecing the step of reactive polypeptide, are sequentially connected with the protection amino with protection group
Acid or protection polypeptide, cut peptide and obtain the N-terminal Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor with protection group；
(2) by N-terminal with the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of protection group and cetyl-N-hydroxy-succinamide
Ester, hexadecanoic acid Acibenzolar or hexadecanoic acid anhydride carry out chemosynthesis in the liquid phase, then remove N-terminal protection
Group, obtains target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In another preference, described target polypeptides is sub-by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor and cetyl-N-hydroxysuccinimidyl acyl
Amine ester, hexadecanoic acid Acibenzolar or hexadecanoic acid anhydride and condensation reagent in-situ activation and obtain, the choosing of described condensation reagent
From DCC, DIC, HBTU or TBTU.
In another preference, described N-terminal with protection group selected from 2-Cl-benzyloxycarbonyl group (i.e. claim Cl-Z protect
Protect group), benzyloxycarbonyl group (i.e. claim Z blocking group) or fluorenylmethyloxycarbonyl (i.e. claiming Fmoc blocking group)；
Use palladium charcoal or metallic catalyst hydrogenation removing Cl-Z blocking group and Z blocking group；
Use aqueous slkali hydrolysis removing Fmoc blocking group.
In another preference, described aqueous slkali is selected from piperidines or DBU.
In another preference, 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu), N-terminal amino
Acid use Cl-Z-His (Trt)-OH, Z-His (Trt)-OH, Alloc-His (Trt)-OH or
Fmoc-His (Trt)-OH activated ester method is condensed, and obtains 31 peptide resins of protection, synchronizes to use remove-insurance
Protect agent agent carry out de-Side chain protective group and cut peptide, it is thus achieved that N-terminal Cl-Z, Z, Alloc or Fmoc protect
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor.
In another preference, 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu), N-terminal amino
Acid uses the condensation of Fmoc-His (Trt)-OH activated ester method to obtain, and after N-terminal Fmoc removing, uses
Cl-Z-OSu, Z-OSu or Alloc-OSu carry out end capping reaction, thus obtain 31 peptide resins of protection,
Synchronization carries out de-Side chain protective group and cuts peptide, it is thus achieved that the Li Lalu that N-terminal is protected with Cl-Z, Z or Alloc
Peptide precursor；More preferably, 20 amino acids are Fmoc-Lys (Alloc)-OH, Alloc protection group tetraphenyl
After the hydrogenation removing of phosphorus palladium, reacting with Boc-γ-Glu-OtBu, 20 amino acids are Fmoc-Lys (Boc-γ
-Glu-OtBu)；Most preferably, 20 amino acids are Fmoc-Lys (Dde)-OH, Dde protection group 2% water
After closing hydrazine/DMF removing, reacting with Boc-γ-Glu-OtBu, 20 amino acids are Fmoc-Lys (Boc-γ
-Glu-OtBu)。
In another preference, 20 amino acids are Fmoc-Lys (Boc)-OH, and N-terminal aminoacid uses
Cl-Z-His (Trt)-OH, Z-His (Trt)-OH, Alloc-His (Trt)-OH or Fmoc-His (Trt)-OH activate
Ester method is condensed and obtains, thus obtains 31 peptide resins of protection, synchronizes carry out de-Side chain protective group and cut
Peptide, it is thus achieved that Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side chain that N-terminal is protected with Cl-Z, Z, Alloc or Fmoc
Without γ-Glu group).
In another preference, 20 amino acids are Fmoc-Lys (Boc)-OH, and N-terminal aminoacid uses
Fmoc-His (Trt)-OH activated ester method is condensed, and after N-terminal Fmoc removing, uses Cl-Z-OSu, Z-OSu
Or Alloc-OSu carries out end capping reaction, thus obtain 31 peptide resins of protection, synchronize to carry out de-side chain
Protection group and cut peptide, it is thus achieved that Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (the Lys20 side that N-terminal is protected with Cl-Z, Z or Alloc
Chain does not contains γ-Glu group).
In another preference, the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that N-terminal-fluorenylmethyloxycarbonyl (Fmoc blocking group) is protected
Precursor (Lys 20 side chain does not contains γ-Glu group) and Nα-Oxohexadecyl-Glu (OSu)-OBzl (is
Pal-Glu (OSu)-OBzl) liquid phase carries out acylation reaction obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of protection；Fmoc protection group is used
Alkali removes, hydrogenation removing benzyl (Bzl)；
Aqueous slkali is selected from piperidines or DBU aqueous slkali；
Hydrogenation uses palladium carbon catalyst catalysis.
In another preference, before the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that N-terminal-2-benzyloxycarbonylchloride base (Cl-Z blocking group) is protected
Body (Lys 20 side chain does not contains γ-Glu group) and Pal-Glu (OSu)-OBzl carry out acylation reaction and obtain protection
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；Hydrogenation removing Cl-Z protection and benzyl；
Hydrogenation uses palladium carbon catalyst catalysis.
In another preference, the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (Lys that N-terminal-benzyloxycarbonyl group (Z blocking group) is protected
20 side chains do not contain γ-Glu group) and Pal-Glu (OSu)-OBzl liquid phase in carry out acylation reaction obtain protect
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；Hydrogenation removing Z protection and benzyl again；
Hydrogenation uses the catalysis of palladium charcoal catalytic reagent.
In another preference, by N-terminal-benzyloxycarbonyl group (Z blocking group) protection or N-terminal-2-benzyl chloride oxygen
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor that carbonyl (Cl-Z blocking group) is protected (20 amino acids side chains do not contain γ-Glu group) and
Pal-Glu (OSu)-OtBu liquid phase carries out acylation reaction and obtains the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of protection；Hydrogenation removing Z
Protection or Cl-Z protection, and hydrolysis removes tBu group in acid condition；
Hydrogenation uses palladium carbon catalyst catalysis；
Acid condition can be HCl or TFA acid solution.
In another preference, before the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of N-terminal-allyloxycarbonyl protection (Alloc blocking group)
Body (Lys 20 side chain does not contains γ-Glu group) and Pal-Glu (OSu)-OAllyl liquid phase carry out acylation reaction obtain
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] to protection；Hydrogenation removing Alloc protection and OAllyl group；
Hydrogenation uses the catalysis of tetraphenylphosphonium palladium chtalyst reagent.
In another preference, before the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] that N-terminal-allyloxycarbonyl (Alloc blocking group) is protected
Body (Lys 20 side chain does not contains γ-Glu group) and Pal-Glu (OSu)-OBzl liquid phase carry out acylation reaction obtain
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of protection；Hydrogenation removing Alloc protection and benzyl；
Hydrogenation uses the catalysis of tetraphenylphosphonium palladium chtalyst reagent.
In another preference, the Li Lalu that N-terminal-allyloxycarbonyl (Alloc blocking group) is protected
Peptide precursor (Lys 20 side chain does not contains γ-Glu group) and Pal-Glu (OSu)-OtBu liquid phase carry out acylation reaction
Obtain the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of protection；Hydrogenation removing Alloc protection and hydrolyzed under acidic conditions remove tBu group；
Hydrogenation uses palladium carbon catalyst catalysis；
Acid condition is selected from HCl or TFA acid solution.
In another preference, before the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of N-terminal-tertbutyloxycarbonyl protection (Boc blocking group)
Body (Lys 20 side chain does not contains γ-Glu group) and Pal-Glu (OSu)-OtBu carry out acylation reaction and obtain protection
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；Hydrolyzed under acidic conditions removes Boc and tBu group；
Hydrogenation uses palladium carbon catalyst catalysis；
Acid condition is selected from HCl or TFA acid solution.
In another preference, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor is exchanged by cation or anion exchange method purifies, ultrafiltration
Or preparative hplc method of purification changes and obtains after salt, and lyophilizing.
In another preference, Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor can be purified to more than 70.0%HPLC purity；It is preferred that
Can be purified to more than 80.0%HPLC purity；More preferably, can be purified to more than 90.0%HPLC purity；
More preferably, can be purified to more than 95.0%HPLC purity；Most preferably, can be purified to more than 98.0%HPLC
Purity.
In another preference, target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] can be purified by preparative high performance liquid chromatography, uses
C18, C8, C4 or other polystyrene resin isochromatic spectrum fillers, the flowing phase of pH2-7, such as the second of trifluoroacetic acid
Nitrile and the acetonitrile of aqueous systems, triethylamine and phosphoric acid and the acetonitrile of aqueous systems, sodium ascorbyl phosphate or potassium phosphate and aqueous systems,
Or the acetonitrile of Spirit of Mindererus. and aqueous systems；Methanol or other alcohol may replace acetonitrile；Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] can purify to be more than
95.0%HPLC purity；It is preferred that purify to more than 96.0%HPLC purity；More preferably, can purify to greatly
In 97.0%HPLC purity；More preferably, can purify to more than 98.0%HPLC purity；Most preferably, can purify
To more than 99.0%HPLC purity.
In another preference, the substitution value of described solid phase synthesis resin is 0.1-0.8mmol/g.
In another preference, setting-up point is room temperature, and optimum condition is 10-30 DEG C.
In another preference, setting-up point is high temperature, and optimum condition is 30-80 DEG C.
In another preference, described solid phase synthesis resin is selected from hydroxy resin, 2-Cl-Trt resin or chloromethyl
Resin；More preferably, described hydroxy resin is from king's resin or PL king's resin.
In another preference, described protected amino acid is respectively as follows:
30#:Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Mtr)-OH, Fmoc-Arg (Pmc)-OH；
29#:Fmoc-Gly-OH；
28#:Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Mtr)-OH, Fmoc-Arg (Pmc)-OH；
27#:Fmoc-Val-OH；
26#:Fmoc-Leu-OH；
25#:Fmoc-Trp (Boc)-OH, Fmoc-Trp-OH；
24#:Fmoc-Ala-OH；
23#:Fmoc-Ile-OH；
22#:#Fmoc-Phe-OH；
21#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
20#:Fmoc-Lys(Dde)-OH、Fmoc-Lys(Boc-Glu-OtBu)-OH、Fmoc-Lys(Boc)-OH、
Fmoc-Lys(Alloc)-OH；
19#:Fmoc-Ala-OH；
18#:Fmoc-Ala-OH；
17#:Fmoc-Gln (Trt)-OH, Fmoc-Gln (Xan)-OH, Fmoc-Gln-OH；
16#:Fmoc-Gly-OH；
15#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
14#:Fmoc-Leu-OH；
13#:Fmoc-Tyr (tBu)-OH；
12#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Ser-OH,
Fmoc-Ser(OBzl)-OH；
11#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Ser-OH,
Fmoc-Ser(OBzl)-OH；
10#:Fmoc-Val-OH；
9#:Fmoc-Asp (OtBu)-OH, Fmoc-Asp (oall)-OH, Fmoc-Asp (OBzl)-OH；
8#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH；
7#:Fmoc-Thr (tBu)-OH, Fmoc-Thr (oBzl)-OH；
6#:Fmoc-Phe-OH；
5#:Fmoc-Thr (tBu)-OH, Fmoc-Thr (oBzl)-OH；
4#:Fmoc-Gly-OH；
3#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
2#:Fmoc-Ala-OH；
1#:Boc-His (Trt)-OH, Z-His (Trt)-OH, Boc-His (Boc)-OH, Cl-Z-His (Trt)-OH,
Alloc-His(Trt)-OH,Fmoc-His(Trt)-OH,Boc-His-OH、Z-His-OH、Boc-His-OH,
Cl-Z-His-OH,Alloc-His-OH,and Fmoc-His-OH。
Accordingly, the invention provides a kind of feasible Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] synthetic method to produce high-purity product, with
Meet medical usage.
Detailed description of the invention
Inventor through extensively in-depth study, differences based on 20 bit substituents, use full solid phase or
Solid liquid phase synthetic method, it is thus achieved that highly purified Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product (content 50-70%).
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] solid phase synthesis process that the present invention provides is with Fmoc-Gly-solid phase synthesis resin for initial former
Material, through following step:
Deprotection steps: protected amino acid or polypeptide-solid phase synthesis resin and DCM are mixed in the reactor
Close, swelling, add piperidines/DMF solution deprotection；
Feed intake condensation step: by with the protected amino acid of protection group, HOBt and DIC, at DMF/DCM
Mixed solution in dissolve after pour in above-mentioned reactor, judge to be condensed the most complete with 1,2,3-indantrione monohydrate detection；
DMF/DCM (30%-70%)
Repeat deprotection, condensation step, make peptide chain extend to N end from C end, until 1# Amino acid synthesis
Complete, obtain the direct-connected peptide of band protection group.
In above-mentioned deprotection steps, need the most swelling；
Feeding intake in condensation step above-mentioned, the mixed solution of described DMF/DCM is
DMF/DCM (30%-70%).
In one embodiment of the invention, feed intake in condensation step above-mentioned, the protected amino acid of 20
Optional Fmoc-Lys (Dde)-OH, end selects Fmoc-His (Trt)-OH；Alternative cutting removes
Protection group Dde of 20, then Boc-Glu (Osu)-OBzl reaction, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor, final with
Pal-OSu reacts and obtains crude product.
In another embodiment of the invention, feed intake in condensation step above-mentioned, the protected amino acid of 20
Optional Fmoc-Lys (Dde)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Alloc)-OH all cut
Except protection group obtains the peptide without protection group, end selects Z-His (Trt)-OH to be directly synthesized, preliminary purification
After synthesize in solid phase or liquid phase with Pal-Glu (OSu)-OBzl again, hydrogenation reaction deprotection base obtains slightly
Product.
In another embodiment of the invention, feed intake in condensation step above-mentioned, the protected amino acid of 20
Optional Fmoc-Lys (Boc-Glu-OtBu)-OH, complete resection protection group obtains the peptide without protection group,
Reacting with Pal-OSu after preliminary purification, hydrogenation reaction deprotection base obtains crude product again.
Abbreviation or the implication of English full name used in the present invention are listed in the table below:
As used herein, " solid phase synthesis " or " Solid-phase synthesis peptides (solid phase peptide synthesis) "
It is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: protected by an amino
The aminoacid covalently bound (bonding) protected is on solid phase carrier；In the presence of deprotection agent, take off amino
Protection group, makes first aminoacid receive on solid phase carrier；Then amino is closed second of (protection)
Amino acid whose carboxyl by activation, second aminoacid that carboxyl is activated again be connected on the of solid phase carrier
One amino acid whose amino reaction (being condensed) forms peptide bond, has so been generated as a band on solid phase carrier
The dipeptides of protected base；Repeating above-mentioned peptide bond and form reaction, making peptide chain grow to N end from C end, until reaching
To required peptide chain length；Finally slough the protection group of amino, the ester between hydrolysis peptide chain and solid phase carrier
Key (cuts), obtains synthetic peptide.
As used herein, " deprotection agent " or " deprotection agent " can exchange use, all referring to can be by
The chemical reagent that the amino protecting agent being connected on aminoacid is removed, described amino protecting agent can make ability
Known to territory, such as but not limited to, Fmoc, Boc；Described deprotection agent can make well known in the art,
Such as but not limited to, in terms of its cumulative volume, it is the DMF solution containing 15-25v/v% piperidines.
As used herein, " condensing agent ", " activator ", " activating reagent " or " condensation activator "
Use can be exchanged, form peptide all referring to making an amino acid whose amino and the condensation of another amino acid whose carboxyl
The chemical reagent of key, is preferably used DIC/HOBt in the present invention.
As used herein, " cutting agent " refers to the chemical reagent separated by the polypeptide of same resin-bonded with resin,
Can make well known in the art, such as but not limited to, the weakly acidic solution containing TFA, HCl solution, excellent
The choosing phenol solution containing TFA, TIS and EDT.
As used herein, " HPLC purity " refers to the polypeptide products that will prepare, and examines through HPLC
Survey, according to obtained chromatograph collection of illustrative plates, carry out area normalization method and the face, peak of target polypeptides compound that obtains
Amass the percent occupied in all peak area summations.
As used herein, " Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product " refers to the HPLC purity Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] at 40%-70%
Product.
The structural formula of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is as shown in formula I:
H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-L
eu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys20[N-(1-oxohexadecyl)-L-γ-glutam Ⅰ
yl]-Glu21-Phe22-Ile23-Ala24-Trp25–Leu26-Val27-Arg28–Gly29–Arg30-Gly31-O
H
Relevant ninhydrin (Kaiser), ninhydrin test (Ninhydrin test), and monitoring
Method may refer to document VIRENDER K.SARIN, et al. " Quantitative Monitoring of
Solid-Phase Peptide Synthesis by the Ninhydrin Reaction”ANALYTICAL
BIOCHEMISTRY 117,147-157 (1981), E.KAISER, et al. " Color Test for Detection
of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides”SHORT
COMMUNICATIONS 595-598 (Received October 28,1969) and THORKILD
CHRISTENSEN“A Qualitative Test for Monitoring Coupling Completeness in Solid
Phase Peptide Synthesis Using Chloranil”Acta Chemica Scandinavica B 33(1979)
763-766。
The features described above that the present invention mentions, or the feature that embodiment is mentioned can be in any combination.This case description institute
The all features disclosed can be with any composition forms use, and each feature disclosed in description can be any
The alternative characteristics that can provide identical, impartial or similar purpose replaces.Therefore except having special instruction, disclosed spy
Levy the only impartial or general example of similar features.
Main advantages of the present invention are:
1, the synthesis polypeptide method that the present invention provides, takes N end to protect temporarily, improves Pal condensing site
Selectivity, the crude product purity obtained is higher.
2, the present invention can change the Pal purification difficult that hydrophobicity height causes on peptide chain, finally improves sterling
Purity.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise institute
Some percent, ratio, ratio or number are by weight.
The unit in percent weight in volume in the present invention is well-known to those skilled in the art, e.g.
Refer to the weight of solute in the solution of 100 milliliters.
Unless otherwise defined, all specialties used in literary composition are ripe with one skilled in the art institute with scientific words
The same meaning known.Additionally, any method similar or impartial to described content and material all can be applicable to
In the inventive method.Preferable implementation described in literary composition only presents a demonstration with material and is used.
It is as follows that the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] HPLC related in following embodiment analyzes method:
Fixing phase: Kromasil C18,5um, 4.6*150mm column temperature 30 DEG C
Flowing phase: A phosphoric acid triethylamine solution (pH=6.5), B acetonitrile
Flow velocity: 1.0ml/min
Gradient: 32-52%B, 30 minutes
Detection wavelength: 215nm
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude product HPLC purification process related in following embodiment is as follows:
Fixing phase: Kromasil C18,10um, column temperature 30 DEG C
Flowing phase: A phosphoric acid triethylamine solution (pH=7.0), B acetonitrile
Flow velocity: 20ml/min
Gradient: 26-48%B, 100 minutes
Detection wavelength: 215nm
Embodiment 1
Weigh 19.23g (5mmol) Fmoc-Gly-Wang Resin to put in reaction column, add 20% piperidines/DMF
(V/V) solution deprotection twice.Washing, takes the inspection of a small amount of resin indenes, is positive after taking out.Weigh
9.73gFmoc-Arg (Pbf)-OH and 2.43gHOBt, adds about 50mLDMF and dissolves, add 2.8mLDIC
Activation, is poured into room temperature reaction 2h in reaction column, 1,2,3-indantrione monohydrate detection detection.Washing.Repeat above-mentioned deprotection,
Washing and condensation step, until N-terminal Fmoc-His (Trt)-OH condensation is complete, 20 protected amino acids are
Fmoc-Lys (Boc)-OH, removing N-terminal Fmoc protection group also adds DMF washing.Weigh 4.25g
Z (2-Cl)-OSu also reacts with joining in above-mentioned resin after appropriate DMF dissolving, and 1,2,3-indantrione monohydrate detects, and is dried to obtain
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] intermediate peptide resin 41.2g, peptide resin yield is about 91%.Above-mentioned resin joins cracking formula examination
Cracking in agent, filtrate joins and separates out thick peptide in absolute ether, obtains 15.7g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] crude intermediate, yield
It is 88.4%.By crude product through high-pressure liquid phase (HPLC) lyophilizing after purification, obtain 6.47g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (Lys
20 side chains do not contain γ-Glu group).HPLC detection purity is 98.8%, ESI-MS (+): [M+3H]+=1185.2,
Purification yield 41.2%.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (Lys 20 side chain does not contains γ-Glu group) is dissolved in about 100mLDMF and ice-water bath,
Add 1.1g Pal-Glu (OSu)-OBzl to react overnight, monitor reaction process with HPLC.Reaction adds after terminating
Vinegar acid for adjusting pH, to 5, adds 0.8gPd/C and makees catalyst and be passed through hydrogen, removing Z (2-Cl) and Obzl protection
Base, monitors reaction process by HPLC equally.After end to be hydrogenated, filter Pd/C catalyst, filtrate is added
Enter in frost absolute ether, have solid to separate out, centrifugal, washing, it is dried to obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] final crude product 6.42g
(yield 95.6%).By thick for 6.42g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide HPLC method purification, obtain 4.3g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product.
HPLC detection purity is 99.0%, ESI-MS (+): [M+3H]+=1251.6, total recovery 23.0%.
Embodiment 2
Weigh 27.0g (10mmol) Fmoc-Gly-WangResin to put in reaction column, add appropriate DCM swelling,
Sucking filtration.Add 20% piperidines/DMF (V/V) solution deprotection twice.Add DMF, MeOH, DCM
Wash successively, take the inspection of a small amount of resin indenes after taking out, be positive.Weigh 12.98gFmoc-Arg (Pbf)-OH,
7.21gHBTU and 2.70gHOBt, adds about 80mLDMF and dissolves, and after ice-water bath 2min, adds
After 3.5mLDIPEA activates about 10min, being poured into room temperature reaction 2h in reaction column, 1,2,3-indantrione monohydrate detects.Reaction
After end, take out reactant liquor, add DMF and wash 2 times, repeat above-mentioned deprotection, washing and condensation step,
Until N-terminal His condensation is complete.20# and 1# protected amino acid be respectively Fmoc-Lys (Dde)-OH and
Z-His(Trt)-OH。
Configure 2% hydrazine hydrate/DMF solution 100mL and join reaction in intermediate peptide resin, remove Lys20Side
Chain Dde protection group, 1,2,3-indantrione monohydrate detects.After end to be removed, washing resin.HPLC side after resin cleavage
Method purification, obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (Lys 20 side chain does not contains γ-Glu group)
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor (Lys 20 side chain does not contains γ-Glu group) is dissolved in about 100mLDMF and ice-water bath,
Add 2.5g Pal-Glu (OSu)-OBzl to react overnight, monitor reaction process with HPLC.Reaction adds after terminating
Vinegar acid for adjusting pH, to 5, adds 1.7gPd/C and makees catalyst and be passed through hydrogen, remove Z and OBzl protection group,
Reaction process is monitored equally by HPLC.After end to be hydrogenated, filter Pd/C catalyst, filtrate is joined
In frost absolute ether, solid is had to separate out, centrifugal, washing, it is dried to obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] final crude product 14.0g and (receives
Rate 96%).By thick for 14g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide HPLC method purification, obtain 9.0g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] finished product.HPLC
Detection purity is 98.8%, ESI-MS (+): [M+3H]+=1251.6, total recovery 24.10%.
Embodiment 3
Weigh 47.6g (20mmol) Fmoc-Gly-WangResin to put in reaction column, add 20% piperidines/DMF
(V/V) solution deprotection twice.Add DMF, MeOH, DCM to wash successively, take a small amount of after taking out
Resin indenes is examined, and is positive.Weigh 32.4gFmoc-Arg (Pbf)-OH, 26.0gPyBOP and 6.75gHOBt, add
Enter about 160mLDMF to dissolve, after ice-water bath 2min, after addition 8.7mLDIPEA activates about 10min, instead
Answering 2h, 1,2,3-indantrione monohydrate detects.After reaction terminates, DMF washs.Repeat above-mentioned deprotection, washing and condensation step,
Until N-terminal Fmoc-His (Trt)-OH condensation is complete, dried gained intermediate peptide resin is heavily 145.0g (peptide
Resin yield 93%).Wherein Lys20Use Fmoc-Lys (Boc-γ-Glu-OtBu)-OH as condensation monomer.
Intermediate peptide resin cutting purification, obtains 31.4g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor, and HPLC detection purity is 92.8%,
ESI-MS(+):[M+3H]+=1246.0, purification yield 47.6%.
The 31.4g precursor of gained is dissolved in about 200mLDMF and ice-water bath, adds 3.25g (9.2mmol)
Pal-OSu reacts overnight, monitors reaction process with HPLC.Reaction terminates to add about 40mL in backward reactant liquor
Piperidines continues stirring 1～2 hour, to remove N-terminal Fmoc group, monitors reaction process with HPLC.Again
Filtrate is joined in frost absolute ether, have solid to separate out, centrifugal, washing, it is dried to obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] final
Crude product 28.4g (yield 90%).28.4 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] thick peptide HPLC purification is obtained 15.3g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Product.HPLC detection purity is 99.1%, ESI-MS (+): [M+3H]+=1251.6, total recovery 20.4%.
The foregoing is only presently preferred embodiments of the present invention, be not limited to the substantial technological of the present invention
Context, the substantial technological content of the present invention is broadly to be defined in the right of application, appoints
What other people technology entities that completes or method, if with the right of application defined in phase completely
With, also or the change of a kind of equivalence, all it is covered by being considered among this right.
Claims (9)
1. a synthetic method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], described method includes step:
(1) with Fmoc-Gly-solid phase synthesis resin as initiation material, repeat according to solid phase synthesis process
Deprotection, use condensing agent carry out connecing the step of reactive polypeptide, are sequentially connected with the protection amino with protection group
Acid or protection polypeptide, cut peptide and obtain the N-terminal Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor with protection group；Described N-terminal with
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of protection group is exchanged by cation or anion exchange method purifies, ultrafiltration or preparative hplc
Method of purification changes salt, and lyophilizing；
(2) by N-terminal with the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor of protection group and cetyl-N-hydroxy-succinamide ester,
Hexadecanoic acid Acibenzolar or hexadecanoic acid anhydride carry out chemosynthesis in the liquid phase, then remove N-terminal blocking group,
Obtain target polypeptides Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；
20 amino acids of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor are Fmoc-Lys (Boc-γ-Glu-OtBu), N-terminal amino
Acid use Cl-Z-His (Trt)-OH, Z-His (Trt)-OH, Alloc-His (Trt)-OH or
Fmoc-His (Trt)-OH activated ester method is condensed, and obtains 31 peptide resins of protection, synchronizes to use remove-insurance
Protect agent carry out de-Side chain protective group and cut peptide, it is thus achieved that N-terminal Cl-Z, Z, Alloc or Fmoc protect
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor；
Described condensation reagent is selected from DCC, DIC, HBTU or TBTU；
Described N-terminal with protection group selected from for Cl-Z blocking group 2-Cl-benzyloxycarbonyl group, for Z protect
The benzyloxycarbonyl group of group or be the fluorenylmethyloxycarbonyl of Fmoc blocking group；
Use palladium charcoal or metallic catalyst hydrogenation removing Cl-Z blocking group and Z blocking group；
Use in terms of its cumulative volume, the DMF solution hydrolysis removing Fmoc protection containing 15-25v/v% piperidines
Group.
2. synthetic method as claimed in claim 1, it is characterised in that described target polypeptides is by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor
Former with condensation reagent with cetyl-N-hydroxy-succinamide ester, hexadecanoic acid Acibenzolar or hexadecanoic acid anhydride
Position activates and obtains.
3. synthetic method as claimed in claim 1, it is characterised in that 20 bit aminos of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] precursor
After acid is the hydrogenation removing of Fmoc-Lys (Alloc)-OH, Alloc protection group tetraphenylphosphonium palladium, with Boc-γ
-Glu-OtBu reacts, and 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu).
4. synthetic method as claimed in claim 1, it is characterised in that 20 amino acids are
Fmoc-Lys (Dde)-OH, Dde protection group is with after 2% hydrazine hydrate/DMF removing, with Boc-γ-Glu-OtBu
Reaction, 20 amino acids are Fmoc-Lys (Boc-γ-Glu-OtBu).
5. synthetic method as claimed in claim 1, it is characterised in that the substitution value of described solid phase synthesis resin
For 0.1-0.8mmol/g.
6. synthetic method as claimed in claim 1, it is characterised in that setting-up point is 10-80 DEG C.
7. synthetic method as claimed in claim 1, it is characterised in that described solid phase synthesis resin is selected from hydroxyl
Resin, 2-Cl-Trt resin or chloromethyl resin.
8. synthetic method as claimed in claim 7, it is characterised in that described hydroxy resin is from king's resin or PL
King's resin.
9. synthetic method as claimed in claim 1, it is characterised in that described protected amino acid is respectively as follows:
30#:Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Mtr)-OH, Fmoc-Arg (Pmc)-OH；
29#:Fmoc-Gly-OH；
28#:Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Mtr)-OH, Fmoc-Arg (Pmc)-OH；
27#:Fmoc-Val-OH；
26#:Fmoc-Leu-OH；
25#:Fmoc-Trp (Boc)-OH, Fmoc-Trp-OH；
24#:Fmoc-Ala-OH；
23#:Fmoc-Ile-OH；
22#:#Fmoc-Phe-OH；
21#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
20#:Fmoc-Lys(Boc-γ-Glu-OtBu)-OH；
19#:Fmoc-Ala-OH；
18#:Fmoc-Ala-OH；
17#:Fmoc-Gln (Trt)-OH, Fmoc-Gln (Xan)-OH, Fmoc-Gln-OH；
16#:Fmoc-Gly-OH；
15#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
14#:Fmoc-Leu-OH；
13#:Fmoc-Tyr (tBu)-OH；
12#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Ser-OH, Fmoc-Ser (OBzl)-OH；
11#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Ser-OH,
Fmoc-Ser(OBzl)-OH；
10#:Fmoc-Val-OH；
9#:Fmoc-Asp (OtBu)-OH, Fmoc-Asp (oall)-OH, Fmoc-Asp (OBzl)-OH；
8#:Fmoc-Ser (tBu)-OH, Fmoc-Ser (Trt)-OH；
7#:Fmoc-Thr (tBu)-OH, Fmoc-Thr (oBzl)-OH；
6#:Fmoc-Phe-OH；
5#:Fmoc-Thr (tBu)-OH, Fmoc-Thr (oBzl)-OH；
4#:Fmoc-Gly-OH；
3#:Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OAll)-OH, Fmoc-Glu (OBzl)-OH；
2#:Fmoc-Ala-OH；
1#:Z-His (Trt)-OH, Cl-Z-His (Trt)-OH, Alloc-His (Trt)-OH, Fmoc-His (Trt)-OH.
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| UA116217C2 (en) | 2012-10-09 | 2018-02-26 | Санофі | Exendin-4 derivatives as dual glp1/glucagon agonists |
| CN104902920A (en) | 2012-12-21 | 2015-09-09 | 赛诺菲 | Exendin-4 derivatives as dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists |
| EP3080149A1 (en) | 2013-12-13 | 2016-10-19 | Sanofi | Dual glp-1/glucagon receptor agonists |
| WO2015086729A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Dual glp-1/gip receptor agonists |
| TW201609795A (en) | 2013-12-13 | 2016-03-16 | 賽諾菲公司 | EXENDIN-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| WO2015086730A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Non-acylated exendin-4 peptide analogues |
| CN103980358B (en) * | 2014-01-03 | 2016-08-31 | 杭州阿诺生物医药科技股份有限公司 | A kind of method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] |
| CN104926927A (en) * | 2014-03-20 | 2015-09-23 | 深圳翰宇药业股份有限公司 | Synthesis method of desmopressin |
| TW201625668A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| TW201625670A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Dual GLP-1/glucagon receptor agonists derived from EXENDIN-4 |
| TW201625669A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4 |
| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
| AR105319A1 (en) | 2015-06-05 | 2017-09-27 | Sanofi Sa | PROPHARMS THAT INCLUDE A DUAL AGONIST GLU-1 / GLUCAGON CONJUGATE HIALURONIC ACID CONNECTOR |
| CN105111303B (en) * | 2015-06-23 | 2019-07-26 | 济南康和医药科技有限公司 | A kind of method that solid-liquid combination prepares Liraglutide |
| AR105284A1 (en) | 2015-07-10 | 2017-09-20 | Sanofi Sa | DERIVATIVES OF EXENDINA-4 AS SPECIFIC DUAL PEPTIDE AGONISTS OF GLP-1 / GLUCAGÓN RECEPTORS |
| CN108676086B (en) * | 2018-07-24 | 2020-06-30 | 江苏诺泰澳赛诺生物制药股份有限公司 | Synthesis method of liraglutide |
| EP3897570A1 (en) | 2018-12-19 | 2021-10-27 | KRKA, d.d., Novo mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2021007701A1 (en) * | 2019-07-12 | 2021-01-21 | Shanghai Space Peptides Pharmaceutical Co., Ltd. | A method for preparing liraglutide via a solid phase peptide synthesis |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN113135990A (en) * | 2020-01-20 | 2021-07-20 | 宁波鲲鹏生物科技有限公司 | Liraglutide derivatives and preparation method thereof |
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