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CN103980358B - A kind of method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] - Google Patents

A kind of method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] Download PDF

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CN103980358B
CN103980358B CN201410001671.XA CN201410001671A CN103980358B CN 103980358 B CN103980358 B CN 103980358B CN 201410001671 A CN201410001671 A CN 201410001671A CN 103980358 B CN103980358 B CN 103980358B
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fmoc
palmitoyl
resin
otbu
glu
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CN103980358A (en
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路杨
杨东晖
方晨
周亮
刘少华
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Hangzhou Anuo Biological Medicine Technology Co Ltd</en>
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    • C07—ORGANIC CHEMISTRY
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    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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Abstract

The present invention relates to a kind of technology synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], solve the synthesis cycle length that prior art exists, the problem that cost is high, yield is low, impurity is many.The present invention concretely comprises the following steps: A) by liquid phase synthesis fragment Fmoc Lys (Glu (Nα‑Palmitoyl)‑OtBu)‑OH；B) in the presence of activator systems, resin solid phase carrier and Fmoc Gly OH coupling Fmoc Gly resin is obtained；C) by solid-phase synthesis, having N end Fmoc protection and the aminoacid of side chain protected according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence coupling successively, wherein lysine tripeptide fragment uses Fmoc Lys (Glu (Nα‑Palmitoyl)‑OtBu)‑OH；D) cracking, purification, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] after lyophilizing.The invention provides the synthesis technique that a kind of synthesis cycle is short, low cost, yield are higher, be suitable for the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of large-scale production.

Description

A kind of method preparing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Technical field
The present invention relates to the preparation method of a kind of polypeptide drug, be the preparation method of the treatment specific drug-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of the long-acting type ii diabetes with glucagon-like-peptide-1 (GLP-1) receptor stimulating agent of a kind of synthesis.
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], illustrious name is: Liraglutide, and structural formula is as follows:
Peptide sequence is:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] be first developed by Novo Nordisk Co., Ltd of Denmark be also currently the only one long-actingGLP-1 Analog, there is GLP-1 receptor stimulating agent effect, similar to GLP-1 at aspects such as molecular structure, biological activity, action target spot and immunogenicities.The molecular structure of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] reaches 97% with the homology of GLP-1 (7-37), and architectural difference shows Lys34Substituted by Arg, Lys26Via glutamate-induced generation palmitoylation; fatty acid side chain can make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] reversibly be combined with albumin in blood; make the extended durations of action of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; and strengthen the opposing to DPP-4 enzymatic degradation; fatty acid side chain can also make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] molecule be unified into heptamer in injection site selfing, thus delays it from subcutaneous attraction so that it is is action time close to 24 hours; every day, injection once and can be injected at any time, and hypoglycemia occurrence risk is little.Additionally, this product can also reduce the secretion of glucagon in blood glucose dependency mode, and postpone gastric emptying.
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of Novo Nordisk is prepared by biological methods such as genetic engineerings, and technical difficulty is big, and production cost is high, is unfavorable for the large-scale production of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].US6268343B1 and US6458924B2 reports the solid-liquid synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and intermediate GLP-1 (7-37)-OH is required to reverse HPLC-purified, then under liquid-phase condition with Nα-Palmitoyl-Glu (OSu)-OtBu reacts, and the method needs twice purification, and synthesis cycle is long, and waste liquid is many, cost intensive, the shortcoming being unfavorable for large-scale production.
WO2013037266A1 discloses the preparation method of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; concretely comprise the following steps: by Fmoc solid-phase synthesis; according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence coupling successively, there is N end Fmoc protection and the aminoacid of side chain protected; wherein lysine uses Fmoc-Lys (Alloc)-OH; slough Alloc; by solid-phase synthesis coupling Palmitoyl-Glu-Offiu on lysine side chain amino groups, after cracking, obtain product.The method, owing to using tetrakis triphenylphosphine palladium to slough Alloc, not only makes high expensive, is unfavorable for large-scale production, metal residual also can be made to cause content of beary metal to exceed standard, cause product quality and content the highest.
In sum, during the solid phase synthesis of existing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], owing to synthesis cycle is long, cost is high, and yield is low, and impurity is many, is not suitable for industrialized production.
The present inventor uses existing synthetic method, prepares Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], finds that synthesis step is more, and synthesis cycle is long, and purity and yield are the highest, are unsuitable for industrial-scale production.To this end, the synthetic method of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is studied by the present inventor, thus obtain technical scheme.
Summary of the invention
It is an object of the invention to provide the solid phase synthesis process of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].The technical issues that need to address of the present invention are: synthesis cycle is long, and cost is high, and yield is low, and impurity is many, is not suitable for industrialized production.
The synthetic route of the present invention is as shown in Figure 1: first pass through liquid phase process synthetic lysine tripeptide fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH; secondly in the presence of activator systems; Fmoc-Gly-resin is obtained by resin solid phase carrier and Fmoc-Gly-OH coupling; then solid-phase synthesis is passed through; having N end Fmoc protection and the aminoacid of side chain protected according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence coupling successively, wherein lysine tripeptide fragment uses Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH, finally crack, purification, lyophilizing, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In the present invention, some conventional abbreviations have following meanings；
Fmoc: fluorenylmethyloxycarbonyl
The aminoacid of Fmoc-AA: fluorenylmethyloxycarbonyl protection
DIC: N, N '-Diisopropylcarbodiimide
DCC: N, N '-dicyclohexylcarbodiimide
PyBOP: hexafluorophosphoric acid benzotriazole-1-base-epoxide tripyrrole alkyl phosphorus
HATU: 2-(7-azo BTA)-N, N, N', N'-tetramethylurea hexafluorophosphoric acid ester
HOBt: 1-hydroxy benzenes a pair of horses going side by side triazole
HOSu: N-hydroxy-succinamide
TBu: the tert-butyl group
Trt: trityl
Boc: tertbutyloxycarbonyl
Palmitoyl: palmityl
Pbf: 2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulfonyl
Tyr: tyrosine
Ile: isoleucine
Gln: glutamine
Asn: agedoite
Cys: cysteine
Pro: proline
Leu: leucine
Gly: glycine
Arg: arginine
Val: valine
Trp: tryptophan
Ala: alanine
Phe: phenylalanine
Glu: glutamic acid
Lys: lysine
Ser: serine
Asp: aspartic acid
Thr: threonine
His: histidine
DMF: N, N '-dimethylformamide
MeOH: methanol
DCM: dichloromethane
NMP: N-Methyl pyrrolidone
DMSO: dimethyl sulfoxide
TFA: trifluoracetic acid
EDT: dithioglycol
Piperidine: hexahydropyridine
DMAP:4-dimethylamino naphthyridine
DIEA: N, N '-diisopropylethylamine
TMP: 2,4,6-trimethylpyridine.
There is provided the synthetic method of a kind of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] for this present invention, its step is as follows:
Step 1, by liquid phase process synthetic lysine tripeptide fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH；
Step 2, in the presence of activator systems, is obtained Fmoc-Gly-resin by resin solid phase carrier and Fmoc-Gly-OH coupling；
Step 3, by solid-phase synthesis, has N end Fmoc protection and the aminoacid of side chain protected according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence coupling successively, and wherein lysine tripeptide fragment uses Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH；
Step 4, cracking, purification, lyophilizing, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Wherein, the solid phase synthesis process described in step 1, described fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu) liquid phase synthesis of-OH is: Palmiticacid, HOSu, DCC coupling obtain Palmitoyl-OSu and activate fat, then obtain dipeptide fragment Palmitoyl-Glu-OtBu with H-Glu-OtBu reaction；Palmitoyl-Glu-OtBu, HOSu, DCC coupling obtains Palmitoyl-Glu (OSu)-OtBu and activates fat, then obtains lysine tripeptide fragment Fmoc-Lys-(Glu (N with Fmoc-Lys-OH reactionα-Palmitoyl)-OtBu)-OH。
Wherein, the solid phase synthesis process described in step 2, described resin solid supports uses 2-CTC resin, and described activator systems is selected from DIEA, TMP or NMM, and described Fmoc-Gly-resin is the Fmoc-Gly-CTC resin of 0.10 ~ 0.35mmol/g substitution value.
Wherein, the solid phase synthesis process described in step 2, described resin solid supports uses king's resin, and described activator systems is made up of DIC, HOBt and DMAP, and described Fmoc-Gly-king's resin is Fmoc-Gly-king's resin of 0.10 ~ 0.35mmol/g substitution value.
Wherein, the solid phase synthesis process described in step 3,
1) use the deprotection loss of thick fluid being made up of the piperidines that volume ratio is 1:4 and DMF except the Fmoc protection group on Fmoc-Gly-resin, obtain H-Gly-resin；
2) in the presence of coupling agent system, the arginine coupling of H-Gly-resin and Fmoc protection and side chain protected obtains Fmoc-Arg (Pbf)-Gly-resin；
3) repeat step 1), 2), carry out amino acid whose coupling successively according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence, wherein lysine uses Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH, coupling amino acid order is:
Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH 、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc- Lys-(Glu(Nα-Palmitoyl)-OtBu)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala -OH、Boc-His(Trt)-OH；
Described coupling agent system includes condensing agent and reaction dissolvent, and described condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA；Described reaction dissolvent is selected from DMF, DCM, NMP, DMSO or the combination in any between them.
The method of the present invention obtains through screening, and screening process is as follows:
1) selection of mol ratio: H-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin: Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu) mol ratio of-OH:HATU:HOBt:DIEA is: 1:3:3:3:3 and 1:5:5:5:5；
2) selection of reaction temperature:
25oC and 35oC
3) selection in response time:
Response time is: 2 hours and 3 hours.
8 kinds of experiment conditions are proposed for this:
Experiment condition 1: take 3.43g (1.0mmol) H-Glu (OtBu)-Phe-Ile-Ala-Trp (Boc)-Leu-Val-Arg (Pbf)-Gly-Arg (Pbf)-Gly-resin, 2.16g (3.0 mmol) Fmoc-Lys-(Glu(Nα-Palmitoyl)-OtBu)-OH, 0.41g (3.0 mmol) HOBt and 1.14g (3.0 mmol) HATU adds stirring and dissolving in 20ml DMF, is cooled to 0oC, adds 0.5ml (3.0 mmol) DIEA in above-mentioned solution, 25onullC reacts 2 hours，The remaining aminoacid of coupling the most successively，Coupling amino acid order is: Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala -OH、Boc-His(Trt)-OH，Cracking，Purification，Lyophilizing，Obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine peptide；
Experiment condition 2-8, experimental implementation is as shown in experiment condition 1, and different experiment conditions and experimental result thereof are as follows:
Experiment condition Mol ratio Temperature Time Total recovery Purity
Experiment condition 1 1:3:3:3 25℃ 2 hours 23% 99.10%
Experiment condition 2 1:5:5:5 25℃ 2 hours 27% 99.11%
Experiment condition 3 1:3:3:3 35℃ 2 hours 27% 99.23%
Experiment condition 4 1:5:5:5 35℃ 2 hours 31% 99.75%
Experiment condition 5 1:3:3:3 25℃ 3 hours 28% 99.54%
Experiment condition 6 1:5:5:5 25℃ 3 hours 29% 99.64%
Experiment condition 7 1:3:3:3 35℃ 3 hours 28% 99.13%
Experiment condition 8 1:5:5:5 35℃ 3 hours 26% 99.26%
Result above shows, the purification effect of experiment condition 4 is optimum.
Compared to the prior art the method for the present invention has obvious advantage, and relevant contrast experiment is as follows:
Patent Total recovery Purity
The technology of the present invention 31% 99.75%
WO2013037266A1 15.87% 99.17%
US6458924B2 22% NA
The invention has the beneficial effects as follows: select fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH direct solid phase synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the synthesis cycle solving prior art existence is long, and cost is high, and purity is low, and impurity is many, the problem not being suitable for industrialized production；Synthesis cycle is short, low cost, yield are higher to the invention provides one, is suitable for the synthesis technique of the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of large-scale production.
Accompanying drawing explanation
The synthetic route of Fig. 1 present invention；
The HPLC spectrogram of Fig. 2 lysine tripeptide fragment；
The HPLC spectrogram of the thick peptide of Fig. 3 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]；
The HPLC spectrogram of Fig. 4 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine peptide；
Fig. 5 embodiment 12 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine peptide mass spectrogram.
Detailed description of the invention
Further illustrate the present invention by the following examples.
The synthesis of embodiment one: Palmitoyl-OSu activation fat
Weigh 256.42g Palmiticacid (1.0mol), 138.10g HOSu(1.2mol) add in 2000ml THF, 247.56g DCC(1.2mol is added under ice-water bath), react 1 hour, be warmed up to room temperature reaction 3 hours, reacting liquid filtering, mother solution is spin-dried for, and adds DCM and dissolves, filters, saturated sodium bicarbonate washes 3 times, pure water 2 times, back extraction 2 times, merge organic facies, natrium carbonicum calcinatum is dried, it is spin-dried for, 0-5 DEG C of ice ethyl alcohol recrystallization 3 times, filters, solid oil pump draws the dry 314.62g Palmitoyl-OSu that obtains to activate fat, yield 89%.
The synthesis of embodiment two: Palmitoyl-Glu-OtBu
Weigh 101.62g H-Glu-OtBu(0.5mol) and 79.50g Na2CO3(0.75mol) join in the mixed solution of 500ml water and 500ml THF and dissolve, weigh 176.75g Palmitoyl-OSu(0.5mol) join 500ml THF, drip in above-mentioned mixed solution after dissolving, react overnight under room temperature, with 10% dilute hydrochloric acid regulation PH to 7, rotation is evaporated off THF, afterwards regulation PH to 3.Obtain a large amount of white precipitate, filter.The 0-5 DEG C of ice ethyl alcohol recrystallization of white precipitate that will obtain.Solid oil pump draws the dry 192.11g Palmitoyl-Glu-OtBu that obtains, yield 87%.
The synthesis of embodiment three: Palmitoyl-Glu (OSu)-OtBu
Weigh 88.33g Palmitoyl-Glu-OtBu(0.2mol), 27.62g HOSu(0.24mol) add in 1000ml THF, 49.51g DCC(0.24mol is added) under ice-water bath, react 1 hour, it is warmed up to room temperature reaction 3 hours, reacting liquid filtering, mother solution is spin-dried for, add DCM to dissolve, filter, saturated sodium bicarbonate washes 3 times, pure water 2 times, back extraction 2 times, merge organic facies, natrium carbonicum calcinatum is dried, it is spin-dried for, 0-5 DEG C of ice ethyl alcohol recrystallization 3 times, filter, solid oil pump draws dry 94.81g Palmitoyl-Glu (the OSu)-OtBu that obtains to activate fat, yield 88%.
Embodiment four: Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu) synthesis of-OH
Weigh 36.74g Fmoc-Lys-OH(0.1mol) and 15.90g Na2CO3(0.15mol) join in the mixed solution of 100ml water and 100ml THF and dissolve, weigh 53.87g Palmitoyl-Glu (OSu)-OtBu(0.1mol) join 100ml THF, drip in above-mentioned mixed solution after dissolving, react under room temperature overnight, with 10% dilute hydrochloric acid regulation PH to 7, rotation is evaporated off THF, afterwards regulation PH to 3.Obtain a large amount of white precipitate, filter.The 0-5 DEG C of ice ethyl alcohol recrystallization of white precipitate that will obtain.Solid oil pump draws and dry obtains 67.24g Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH, HPLC purity is 97.40%, yield 85%.
Embodiment five: substitution value is the synthesis of the Fmoc-Gly-CTC resin of 0.10mmol/g
Weigh the 2-CTC resin 20g that substitution value is 0.4mmol/g, join in solid state reaction post, join in solid state reaction post, wash 1 time with DMF, after DMF swellable resins 30 minutes, take 13.37g Fmoc-Gly-OH DMF and dissolve, after adding 7.5ml DIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 100ml absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washs 3 times, closes 30 minutes with absolute methanol, and methanol shrinks and is dried, and obtains 22.34g Fmoc-Gly-CTC resin, and detection substitution degree is 0.10mmol/g.
Embodiment six: substitution value is the synthesis of the Fmoc-Gly-CTC resin of 0.25mmol/g
Weigh the 2-CTC resin 10g that substitution value is 0.95mmol/g, join in solid state reaction post, join in solid state reaction post, wash 1 time with DMF, after DMF swellable resins 30 minutes, take 14.11g Fmoc-Gly-OH DMF and dissolve, after adding 8.0ml DIEA activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, after reacting 2 hours, add 100ml absolute methanol and close 1 hour.Washing 3 times with DMF, DCM washs 3 times, closes 30 minutes with absolute methanol, and methanol shrinks and is dried, and obtains Fmoc-Gly-CTC resin, and detection substitution degree is 0.25mmol/g.
Embodiment seven: substitution value is the synthesis of Fmoc-Gly-king's resin of 0.10mmol/g
Weigh the king resin 20g that substitution value is 0.45mmol/g, join in solid state reaction post, join in solid state reaction post, wash 1 time with DMF, after DMF swellable resins 30 minutes, take 13.37g Fmoc-Gly-OH, 6.01g HOBt DMF dissolves, after adding 6.0ml DIC activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, 2.75g DMAP is added after 5 minutes, after reacting 2 hours, wash 3 times with DMF, DCM washs 3 times, block overnight with 100ml acetic anhydride/pyridine, methanol shrinks and is dried, obtain Fmoc-Gly-king's resin, detection substitution degree is 0.10mmol/g.
Embodiment eight: substitution value is the synthesis of Fmoc-Gly-king's resin of 0.25mmol/g
Weigh the king resin 20g that substitution value is 0.75mmol/g, join in solid state reaction post, join in solid state reaction post, wash 1 time with DMF, after DMF swellable resins 30 minutes, take 22.28g Fmoc-Gly-OH, 10.13g HOBt DMF dissolves, after adding 8.0ml DIC activation under ice-water bath, add above-mentioned equipped with in the reaction column of resin, 4.5g DMAP is added after 5 minutes, after reacting 2 hours, wash 3 times with DMF, DCM washs 3 times, block overnight with 100ml acetic anhydride/pyridine, methanol shrinks and is dried, obtain 22.54g Fmoc-Gly-king's resin, detection substitution degree is 0.25mmol/g.
Embodiment nine: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin
nullWeigh the Fmoc-Gly-CTC resin that 4.46g (1mmol) substitution value is 0.10mmol/g，Add in solid state reaction post，Wash 1 time with DMF，After DMF swelling Fmoc-Gly-CTC resin 30 minutes，Fmoc protection is sloughed with the mixed solution that DMF: pyridine volume ratio is 4:1，Then wash 6 times with DMF，Weigh 3.24g Fmoc-Arg (Pbf)-OH(5mmol)、0.68g HOBt(5mmol) add DCM and the DMF mixed solution that volume ratio is 1:1，Under ice-water bath add 0.8ml DIC(10mmol) activation after，Add above-mentioned equipped with in the reaction column of resin，After reacting 2 hours under room temperature，Reaction end is judged with ninhydrin method detection，If resin water white transparency，Then represent reaction completely；Resin develops the color, then it represents that reaction not exclusively, needs to react 1 hour again, and this criterion judges reaction end with ninhydrin method detection be applicable to subsequent amino-acid coupling.Repeat above-mentioned removing Fmoc protection and add the step of corresponding amino acid couplings; according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence, it is sequentially completed Fmoc-Gly-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys-(Glu (Nαnull-Palmitoyl)-OtBu)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala -OH、The coupling of Boc-His (Trt)-OH.Wherein during Fmoc-Leu-OH and Fmoc-Phe-OH coupling, solvent is changed to: DMSO and the DMF mixed solution selecting volume ratio to be 1:4；During Fmoc-Asp (OtBu)-OH coupling, coupling reagent is changed to: PyBOP/HOBt/DIEA；During Boc-His (Trt)-OH coupling, coupling reagent is changed to: HATU/HOBt/DIEA, and coupling is complete, is washed 3 times by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin DMF, DCM washs 3 times, and MeOH washs 3 times, and DCM washs 3 times, MeOH washs 3 times, drains and obtains 9.67g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin.
Embodiment ten: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] king's resin
nullWeighing 4.57g(1mmol) substitution value is Fmoc-Gly-king's resin of 0.10mmol/g，Add in solid state reaction post，Wash 1 time with DMF，After DMF swelling Fmoc-Gly-king resin 30 minutes，Fmoc protection is sloughed with the mixed solution that DMF: pyridine volume ratio is 4:1，Then wash 6 times with DMF，Weigh 3.24g Fmoc-Arg (Pbf)-OH(5mmol)、0.68g HOBt(5mmol) add DCM and the DMF mixed solution that volume ratio is 1:1，Under ice-water bath add 0.8ml DIC(10mmol) activation after，Add above-mentioned equipped with in the reaction column of resin，After reacting 2 hours under room temperature，Reaction end is judged with ninhydrin method detection，If resin water white transparency，Then represent reaction completely；Resin develops the color, then it represents that reaction not exclusively, needs to react 1 hour again, and this criterion judges reaction end with ninhydrin method detection be applicable to subsequent amino-acid coupling.Repeat above-mentioned removing Fmoc protection and add the step of corresponding amino acid couplings; according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence, it is sequentially completed Fmoc-Gly-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys-(Glu (Nαnull-Palmitoyl)-OtBu)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala -OH、The coupling of Boc-His (Trt)-OH.
Wherein during Fmoc-Leu-OH and Fmoc-Phe-OH coupling, solvent is changed to: DMSO and the DMF mixed solution selecting volume ratio to be 1:4；During Fmoc-Asp (OtBu)-OH coupling, coupling reagent is changed to: PyBOP/HOBt/DIEA；During Boc-His (Trt)-OH coupling, coupling reagent is changed to: HATU/HOBt/DIEA, and coupling is complete, is washed 3 times by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin DMF, DCM washs 3 times, and MeOH washs 3 times, and DCM washs 3 times, MeOH washs 3 times, drains and obtains 9.78g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] king's resin.
Embodiment 11: prepared by the scale of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] king's resin
nullWeighing 4570g(1mol) substitution value is Fmoc-Gly-king's resin of 0.10mmol/g，Add in solid state reaction post，Wash 1 time with DMF，After DMF swelling Fmoc-Gly-king resin 30 minutes，Fmoc protection is sloughed with the mixed solution that DMF: pyridine volume ratio is 4:1，Then wash 6 times with DMF，Weigh 3240g Fmoc-Arg (Pbf)-OH(5mol)、682g HOBt(5mol) add DCM and the DMF mixed solution that volume ratio is 1:1，Under ice-water bath add 800ml DIC(5mol) activation after，Add above-mentioned equipped with in the reaction column of resin，After reacting 2 hours under room temperature，Reaction end is judged with ninhydrin method detection，If resin water white transparency，Then represent reaction completely；Resin develops the color, then it represents that reaction not exclusively, needs to react 1 hour again, and this criterion judges reaction end with ninhydrin method detection be applicable to subsequent amino-acid coupling.Repeat above-mentioned removing Fmoc protection and add the step of corresponding amino acid couplings; according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence, it is sequentially completed Fmoc-Gly-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Lys-(Glu (Nαnull-Palmitoyl)-OtBu)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(tBu)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ala -OH、The coupling of Boc-His (Trt)-OH.Wherein during Fmoc-Leu-OH and Fmoc-Phe-OH coupling, solvent is changed to: DMSO and the DMF mixed solution selecting volume ratio to be 1:4；During Fmoc-Asp (OtBu)-OH coupling, coupling reagent is changed to: PyBOP/HOBt/DIEA；During Boc-His (Trt)-OH coupling, coupling reagent is changed to: HATU/HOBt/DIEA, and coupling is complete, is washed 3 times by Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin DMF, DCM washs 3 times, and MeOH washs 3 times, and DCM washs 3 times, MeOH washs 3 times, drains and obtains 9795g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] king's resin.
Embodiment 12: the preparation of the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Weigh Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] CTC resin or Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] king's resin of 100g full guard, join in the three neck round bottom flask of 1000mL, by TFA: thioanisole: methyl phenyl ethers anisole: the volume ratio configuration lysate 10L of EDT=90:5:3:2, lysate is added in above-mentioned resin, room temperature reaction 2 hours, filter, resin after cracking with a small amount of TFA washing 3 times, merging filtrate, concentrate, liquid after concentrating joins in ice ether and precipitates 1 hour, centrifugal, absolute ether centrifuge washing 6 times, vacuum drying, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] thick peptide 34.13g, HPLC purity 83.03%, thick peptide yield 78%.
Embodiment 13: the preparation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] fine peptide acetate
Weigh the thick peptide of 3413g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] 50% acetonitrile+50% water mixed solution 30L dissolve after, by 2 purification of C18 or C8 post, turn salt, lyophilization after obtain target product.Purification condition for the first time: flowing is mutually: A phase: 0.1%TFA；B phase: acetonitrile, detects wavelength 220nm, collects purpose peak fraction.Purification condition for the second time: flowing is mutually: A phase: 0.3% acetic acid；B phase: acetonitrile.Detection wavelength 220nm, collects purpose peak fraction.Turn salt condition: flow phase: A phase: 20mM ammonium acetate-aqueous solution；B phase: acetonitrile；Detection wavelength 220nm.Collecting purpose peak fraction, concentrated by rotary evaporation, lyophilizing obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] acetate fine peptide 11.24g, HPLC purity 99.75%, purification total recovery 40%, total recovery 31%.
Above content is to combine concrete repairing to select embodiment further description made for the present invention, it is impossible to assert the present invention be embodied as be confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, it is also possible to make some simple deduction or replace, insured's scope of the present invention should be all considered as belonging to.

Claims (6)

1. a synthetic method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], described method step is as follows:
Step 1, by liquid phase process synthetic lysine tripeptide fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH；
Step 2, in the presence of activator systems, is obtained Fmoc-Gly-resin by resin solid phase carrier and Fmoc-Gly-OH by solid phase synthesis process coupling；
Step 3, by solid-phase synthesis, has N end Fmoc protection and the aminoacid of side chain protected according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence coupling successively, and wherein lysine tripeptide fragment uses Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH；
Step 4, cracking, purification, lyophilizing, obtain Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Method the most according to claim 1, is characterized in that:
Wherein, the liquid-phase synthesis process described in step 1, described fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu) liquid phase synthesis of-OH is: Palmiticacid, HOSu and DCC coupling obtain Palmitoyl-OSu, and then Palmitoyl-OSu and H-Glu-OtBu reaction obtains dipeptide fragment Palmitoyl-Glu-OtBu；Palmitoyl-Glu-OtBu, HOSu and DCC coupling obtains Palmitoyl-Glu (OSu)-OtBu, and then Palmitoyl-Glu (OSu)-OtBu and Fmoc-Lys-OH reaction obtains lysine tripeptide fragment Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH。
Method the most according to claim 1, is characterized in that:
Wherein, the solid phase synthesis process described in step 2, described resin solid supports uses 2-CTC resin, and described activator systems is selected from DIEA, TMP or NMM, and described Fmoc-Gly-resin is the Fmoc-Gly-CTC resin of 0.10 ~ 0.35mmol/g substitution value.
Method the most according to claim 1, is characterized in that:
Wherein, the solid phase synthesis process described in step 2, described resin solid supports uses king's resin, and described activator systems is made up of DIC, HOBt and DMAP, and described Fmoc-Gly-resin is Fmoc-Gly-king's resin of 0.10 ~ 0.35mmol/g substitution value.
Method the most according to claim 1, is characterized in that:
Wherein, the solid phase synthesis process described in step 3,1) use the deprotection loss of thick fluid being made up of the piperidines that volume ratio is 1:4 and DMF except the Fmoc protection group on Fmoc-Gly-resin, obtain H-Gly-resin；
2) in the presence of coupling agent system, the arginine coupling of H-Gly-resin and Fmoc protection and side chain protected obtains Fmoc-Arg (Pbf)-Gly-resin；
3) repeat step 1), 2), carry out amino acid whose coupling successively according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] main chain peptide sequence, wherein lysine tripeptide fragment uses Fmoc-Lys-(Glu (Nα-Palmitoyl)-OtBu)-OH。
Method the most according to claim 5, is characterized in that:
Described coupling agent system includes condensing agent and reaction dissolvent, and described condensing agent is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA；Described reaction dissolvent is selected from DMF, DCM, NMP, DMSO or the combination in any between them.
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