CN104650219A - Method for preparing liraglutide by convergent synthesis - Google Patents
Method for preparing liraglutide by convergent synthesis Download PDFInfo
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- CN104650219A CN104650219A CN201510079168.0A CN201510079168A CN104650219A CN 104650219 A CN104650219 A CN 104650219A CN 201510079168 A CN201510079168 A CN 201510079168A CN 104650219 A CN104650219 A CN 104650219A
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Abstract
The invention discloses a method for preparing liraglutide by convergent synthesis. The method comprises the steps of performing solid phase synthesis to obtain four side chain protected peptide fragment sequences, gradually coupling the peptide fragments in a solution system to obtain an all-protected liraglutide straight chain polypeptide, removing the side chain protection of the 20th Lys, performing modification to form all-protected liraglutide, then cracking to remove protecting groups to obtain a crude liraglutide peptide, purifying and exchanging salt to obtain liraglutide; wherein in the four peptide fragment sequences, the first peptide fragment sequence is 1st to 8th amino acids in the liraglutide sequence, the second peptide fragment sequence is 9th to 16th amino acids in the liraglutide sequence, the third peptide fragment sequence is 17th to 26th amino acids in the liraglutide sequence, and the fourth peptide fragment sequence is 27th to 31st amino acids in the liraglutide sequence. By adopting the method, the yield is improved, and the synthesis cost is greatly reduced; and the method is favorable for large-scale and industrialized production.
Description
Technical field
The present invention relates to pharmacy field, specifically, relate to the method that fragment condensation prepares 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 name liraglutide, it is a kind of glucagon-like-peptide-1 (GLP-1) analogue, sequence is: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Le u-Glu-Gly-Gln-Ala-Ala-Lys (N-ε-(N-ɑ-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, molecular formula: C
172h
265n
43o
51, as a kind of subcutaneous injection formulation, good reduction blood sugar effect can be played, glycemic control can be improved by the empty stomach of reduction diabetes B patient and postprandial blood sugar, can weight in patients be reduced simultaneously.
Synthetic method such as patent CN102286092, patent CN103145828, patent CN103288951, patent CN103304660 and the patent CN103980358 of current Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] utilize Fmoc strategy solid phase method to be connected synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] successively.Coupling synthesis cycle is long one by one for the method amino acid, and progressively during coupling, resin shrinkage is serious, reaction is incomplete, produce defect peptide, the substitution value restriction that solid phase carrier is selected, and total recovery is lower, and impurity is more simultaneously, purification difficult.
Patent CN102875665, patent CN104045705, patent CN104045706, patent CN103275208, patent CN103304659, patent CN103864918 and patent CN104004083 adopt the method synthesis of solid phase fragment condensation, each fragment that solid phase fragment condensation drops into is that 1.5-3.5 is doubly excessive, serious waste peptide fragment, causes synthesis cost very high; The resin substitution value restriction of solid phase fragment condensation simultaneously, throughput reduces, and waste solvent, produces a large amount of waste liquid.
Other patent discloses gene recombination and produce Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], but genetic expression has the shortcomings such as workload is large, serious three wastes, technical difficulty are large, production cost is high.
So those skilled in the art still expect to obtain the method with better quality product with high product yield, low synthesis cost, especially reduce costs, reduce the novel method of waste liquid generation, it is very necessary and important for producing for extensive, industrialization.
Summary of the invention
Technical problem to be solved by this invention be low for existing method synthesis yield, production cost is high, produce that waste liquid is many, purifying products is difficult, can not obtain the shortcoming of highly purified Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] by low-cost high-efficiency, provides a kind of fragment condensation to prepare the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Fragment condensation prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the peptide fragment sequences of solid phase synthesis 4 side chain protected, by each peptide fragment in solution system progressively coupling obtain full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide, slough the side chain protected of the 20th Lys, and complete modification, form full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], then cracking deprotection base obtains the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and purifying changes salt and obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
Wherein, 4 described peptide fragment sequences are:
First peptide fragment sequences is the 1-8 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
Second peptide fragment sequences is the 9-16 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
Tripeptide fragment sequence is the 17-26 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
4th peptide fragment sequences is the 27-31 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence.
Above-mentioned fragment condensation prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], preferably includes following steps:
(1) the first ~ four peptide fragment sequences of difference solid phase synthesis side chain protected, and from cracking resin;
(2) by the 4th peptide fragment sequences carboxyl terminal modification protection of side chain protected, and its amino protecting group is sloughed;
(3) the 4th peptide fragment sequences of the side chain protected of deaminize protecting group and the tripeptide fragment coupling sequence of side chain protected are obtained the pentapeptide fragment sequence of side chain protected, and slough its amino protecting group;
(4) the second peptide fragment sequences coupling of the pentapeptide fragment sequence of the side chain protected of deaminize protecting group and side chain protected is obtained the 6th peptide fragment sequences of side chain protected, and slough its amino protecting group;
(5) the 6th peptide fragment sequences of the side chain protected of deaminize protecting group and the first peptide fragment sequences coupling of side chain protected are obtained the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide of full guard;
(6) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide of full guard is sloughed the side chain protected of the 20th Lys, and complete modification and obtain full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
(7) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] cracking deprotection base of full guard is obtained the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
(8) the purified salt that changes of the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In step (1), first to fourth peptide fragment sequences of side chain protected is coupled at successively on solid phase carrier by amino acid respectively and obtains; Wherein, described solid phase carrier is acid sensitive resin, is preferably the chloro-trityl chloride resin of 2-.
In first to fourth peptide fragment sequences solid phase synthesis of side chain protected,
The amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration; Preferred volume percentage composition is the DMF solution of 20% piperidines.
The coupling agent used is the combination of DIC and the HOBt combination of 1:1 in molar ratio or HBTU and HOBt and the DIEA combination of 1:1:2 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio; The combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio; Treat that the amino acid of coupling and the mol ratio of HOBt are 1:1.
The cracking agent used is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7 for volumn concentration is the DCM solution of the TFA of 0.5 ~ 1% or volumn concentration, and preferred volume percentage composition is the DCM solution of the TFA of 0.5 ~ 1%.
In step (1), concrete solid phase synthesis process is the routine techniques means of those skilled in the art.
In step (2), the DCM solution of the 2Cl-Trt Cl that the carboxyl terminal protection reagent used is 0.5M or CHCl
3with the mixture of TFE and TBTA according to volume ratio 7:2:1, be preferably CHCl
3with the mixture of TFE and TBTA according to volume ratio 7:2:1.
In step (2), (3), (4), the amino deprotecting regent used for volumn concentration be the DMF solution of the piperidines of 16%, or volumn concentration is the DMF solution of the DBU of 1%.Preferred volume percentage composition is the DMF solution of 16% piperidines.
In step (3), (4), (5), the coupling agent used is the combination of HBTU and HOBt and the DIEA combination of 1:1:2 in molar ratio or HBTU and HOAt and the DIEA combination of 1:1:2 in molar ratio or DIC and the HOBt combination of 1:1 in molar ratio or EDC and the HOBt combination of 1:1 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio.The combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio.Carboxyl terminal and the N-terminal mol ratio for the treatment of coupling are 0.95 ~ 1.05:1.Treat that the carboxyl terminal of coupling and the mol ratio of HOBt are 1:1.The solvent of linked reaction is any one or a few the combination in DMF, DCM, NMP, THF, TFE and DMSO, preferred DMF.
In step (6), the reagent sloughing the epsilon-amino protecting group ivDde of Lys is hydrazine hydrate and the DMF mixture according to volume ratio 1:15.
In step (6), the method that Lys side chain is modified is the Fmoc-Glu-OtBu condensation first adding epsilon-amino end 4 times of molar weights; Then slough Fmoc group, the DMF solution of agents useful for same to be volumn concentration the be piperidines of 16%, or volumn concentration is the DMF solution of the DBU of 1%, is preferably the DMF solution that volumn concentration is the piperidines of 16%; Last again with palmitinic acid condensation.
In step (7), the lysate of full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] cracking is TFA and H
2o is the mixing solutions of 95:5 or TFA and EDT and TIS and PhOH and H by volume
2o is the mixing solutions of 80:5:5:5:5 or TFA and EDT and TIS and H by volume
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume, preferred TFA and EDT and TIS and H
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.
In step (8), purifying is that RPLC purifying changes salt; Namely chromatographic column is C18 post; Moving phase is volume percent 0.25% vinegar aqueous acid and acetonitrile.
The present invention uses acid sensitive resin to carry out the solid phase synthesis of each peptide fragment, and resin substitution value is high, and amino acid/11 .5-2 doubly throwing amount, Material Cost is low.The peptide fragment purity of synthesis is high, need not carry out HPLC purifying, can carry out liquid phase reaction, decrease the step of aftertreatment, decreases waste liquid and produces.Multiple fragment can be synthesized simultaneously, saves generated time, shortens synthesis cycle.Fragment condensation adopts liquid-phase system, the throwing amount of carboxyl-terminal fragment is only n terminal fragment 0.95-1.05 doubly, can not cause the waste of fragment, greatly reduce cost, and unreacted fragment is all removed by extraction by suitable reaction system, aftertreatment is simple, quick.And the problem of the resin substitution value restriction that liquid-phase fragment condensation exists without solid phase fragment condensation, throughput increases, and decreases waste liquid and produces.In final liquid chromatography purification step, impurity is not lack one or several amino acid whose defect peptide, but the fragment of non-condensation, the problem of purification difficult can not be caused.So feature of the present invention be high-throughput, low cost, waste liquid is few, efficiency is high, purifying is easy, be applicable to very much extensive, industrialization and produce.
The aminoacid sequence of target peptide (Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]) involved in the present invention and each peptide fragment of intermediate is in Table l.The implication of the material abbreviation used in the present invention is in table 2.
The corresponding encoding amino acid sequence of table 1 Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
Table 2 material abbreviation used in the present invention implication
| English abbreviation | Full name |
| Fmoc- | 9-fluorenylmethyloxycarbonyl |
| 2-CTC Resin | The chloro-trityl chloride resin of 2- |
| RP-HPLC | RPLC |
| DMF | DMF |
| NMP | N-Methyl pyrrolidone |
| DMSO | Dimethyl sulfoxide (DMSO) |
| DCM | Methylene dichloride |
| THF | Tetrahydrofuran (THF) |
| DBU | 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene |
| DIEA | DIPEA |
| HOBt | 1-hydroxy benzo triazole |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| PyBOP | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl |
| HATU | 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea hexafluorophosphate |
| HBTU | Benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate |
| DIC | N, N-DIC |
| EDC | 1-ethyl-(3-dimethylaminopropyl) carbodiimide |
| TFA | Trifluoroacetic acid |
| TFE | Trifluoroethanol |
| EDT | 1，2-ethandithiol |
| TIS | Tri isopropyl silane |
| Boc- | Tertbutyloxycarbonyl |
| -Pbf | 2,2,4,6,7-pentamethyl-cumarone-5-alkylsulfonyl |
| -tBu | The tertiary butyl |
| -Trt | Trityl |
| -ivDde | 1-(4,4-dimethyl-2,6-dioxocyclohexyl methylene radical)-3-methyl butyl |
| TBTA | The tertiary butyl 2,2,2-trichoroacetic acid(TCA) imines ester |
| MTBE | Methyl tertiary butyl ether |
Beneficial effect: hinge structure of the present invention has the following advantages:
1, the present invention utilizes the acid sensitive resin of high loads amount for starting raw material, first adopt the high purity peptide fragment of the selected structure of Solid phase peptide synthesis technology synthesis of standard, adopt liquid phase coupling technology to make peptide fragment condensation again, thus obtain the target peptide of high purity (﹥ 99%), high yield (﹥ 39%).
The technique of continuous solid phase of 2, comparing synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], each fragment of the present invention can use the solid phase carrier of high capacity value, the resin substitution value restriction not having condensation amino acid number one by one too much to cause, and throughput increases, and discharging of waste liquid reduces; Fragment condensation, the synthesis of each peptide fragment can be carried out simultaneously, substantially reduces generated time.
The technique of solid phase of 3, comparing fragment condensation synthesis Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the present invention utilizes liquid-phase fragment condensation, and fragment mol ratio is 0.95-1.05 times amount, doubly excessive far below the 1.5-3.5 of solid phase fragment condensation synthesis fragment, saves Material Cost; And unreacted fragment is all removed by extraction by suitable reaction system, aftertreatment is simple, quick.
4, adopt less than 10 or 10 amino acid whose side chain protected peptide fragment sequences purity of super acid responsive type resins synthesis very high, need not purify with chromatographic technique, only need to carry out precipitating, grinding and can use; The coupling of fragment liquid phase, its impurity is mainly the fragment of non-coupling, instead of lack one or several amino acid whose defect peptide, and the fragment of non-coupling can be removed by the extraction of suitable solvent system, much easier in final high-efficient liquid phase chromatogram purification, thus number of times is prepared in minimizing, reduces the preparation cost of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
The present invention has high-throughput, low cost, waste liquid is few, efficiency is high, purifying is easy feature, is conducive to realizing mass-producing, industrialization is produced.
Accompanying drawing explanation
Fig. 1 is Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] mass spectrum prepared by the present invention.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1:
1. resin-made is standby
The chloro-trityl resin of 1.1 preparation Fmoc-Gly-2-: chloro-for 2-trityl chloride resin (10g, substitution value 0.84mmol/g resin, 1eq) is added Peptide systhesis device, with 100mL DCM washing resin.Drain solvent, add the 50mL DCM solution of Fmoc-Gly-OH (1.3eq) and DIEA (2.5eq).This mixture of argon shield mechanical stirring 1 hour.Add chromatogram methyl alcohol 20mL (2ml/g resin) to carry out closing for 30 minutes to the active part on resin.Drain solvent, wash with 3 × 80mL DMF, 3 × 80mL DCM, 3 × 80mL MeOH, vacuum-drying, to constant weight, obtains the chloro-trityl resin of 11.44g Fmoc-Gly-2-.Utilize ultraviolet spectrophotometry to measure Fmoc amount in piperidines deprotection liquid, the capacity value of resin is 0.55mmol/g.
The chloro-trityl resin of 1.2 preparation Fmoc-Leu-2-: chloro-for 2-trityl chloride resin (5g, substitution value 0.84mmol/g resin, 1eq) is added Peptide systhesis device, with 60mL DCM washing resin.Drain solvent, add the 30mL DCM solution of Fmoc-Leu-OH (1.2eq) and DIEA (2.5eq).This mixture of argon shield mechanical stirring 1 hour.Add chromatogram methyl alcohol 10mL (2ml/g resin) to carry out closing for 30 minutes to the active part on resin.Drain solvent, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains the chloro-trityl resin of 5.72g Fmoc-Leu-2-.Utilize ultraviolet spectrophotometry to measure Fmoc amount in piperidines deprotection liquid, the capacity value of resin is 0.45mmol/g.
The chloro-trityl resin of 1.3 preparation Fmoc-Ser (tBu)-2-: chloro-for 2-trityl chloride resin (5g, substitution value 0.84mmol/g resin, 1eq) is added Peptide systhesis device, with 60mL DCM washing resin.Drain solvent, add the 30mL DCM solution of Fmoc-Ser (tBu)-OH (1.3eq) and DIEA (2.5eq).This mixture of argon shield mechanical stirring 1 hour.Add chromatogram methyl alcohol 10mL (2ml/g resin) to carry out closing for 30 minutes to the active part on resin.Drain solvent, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains the chloro-trityl resin of 5.87g Fmoc-Ser (tBu)-2-.Utilize ultraviolet spectrophotometry to measure Fmoc amount in piperidines deprotection liquid, the capacity value of resin is 0.5mmol/g.
2. solid phase fragment preparation
The preparation of 2.1 peptide fragment Boc-AA (1-8)-OH:
The chloro-trityl resin of 5g Fmoc-Ser (tBu)-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mL DMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-Thr (tBu)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mL DMF washing resin.
Successively with Fmoc-protection amino acid Phe, Thr (tBu), Gly, Glu (OtBu), each 2eq of Ala and Boc-His (Trt)-OH, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mLDMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 8.29g resin-bonded peptide.
With 150mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 20mL volume, and then with 10mL DMF reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 3.36g purity 97%Boc-AA (1-8)-OH, productive rate 95%.
The preparation of 2.2 peptide fragment Fmoc-AA (9-16)-OH:
The chloro-trityl resin of 5g Fmoc-Gly-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mLDMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-Glu (OtBu)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mL DMF washing resin.
Successively with Fmoc-protection amino acid Leu, Tyr (tBu), Ser (tBu), Ser (tBu), Val and Asp (OtBu) each 2eq, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mLDMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 8.48g resin-bonded peptide.
With 150mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 20mL volume, and then with 10mL DMSO reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 3.59g purity 96%Fmoc-AA (9-16)-OH, productive rate 95%.
The preparation of 2.3 peptide fragment Fmoc-AA (17-26)-OH:
The chloro-trityl resin of 5g Fmoc-Leu-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mLDMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-Trp (Boc)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mL DMF washing resin.
Successively with amino acid Ala, Ile, Phe, Glu (OtBu), Lys (ivDde), Ala, Ala and Gln (Trt) each 2eq of Fmoc-protection, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 9.17g resin-bonded peptide.
With 150mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 20mL volume, and then with 10mL DMSO reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 4.28g purity 95%Fmoc-AA (17-26)-OH, productive rate 95%.
The preparation of 2.4 peptide fragment Fmoc-AA (27-31)-OH:
The chloro-trityl resin of 5g Fmoc-Gly-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mLDMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-Arg (Pbf)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mL DMF washing resin.
Successively with amino acid Gly, Arg (Pbf) and each 2eq of Val of Fmoc-protection, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 8.31g resin-bonded peptide.
With 150mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 20mL volume, and then with 10mL DMF reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 3.39g purity 98%Fmoc-AA (27-31)-OH, productive rate 97%.
3. liquid-phase fragment condensation course
3.1 preparation H-AA (27-31)-OtBu
Add 1.27g Fmoc-AA (27-31)-OH (1mmol) in round-bottomed flask, add CHCl
3: TFE:TBTA=7:2:1 solution 25mL, heat to 35 DEG C of magnetic agitation 1 hour, add the cold MTBE precipitated product of 100mL, stir 1 hour, collected by vacuum filtration solid, wash with 2 × 100mL MTBE, dry, obtain Fmoc-AA (27-31)-OtBu.Then add 20mL DMF to dissolve, drip piperidines to ultimate density 16%, react 2 hours, add frozen water precipitated product, frozen water washs 2 times, adds the cold MTBE of 100mL and stirs the fulvene product that removal in 2 hours removes Fmoc, filtering-depositing, drying, obtains 1.083g H-AA (27-31)-OtBu, yield 98%.
3.2 preparation H-AA (17-31)-OtBu
1.85g Fmoc-AA (17-26)-OH (0.92mmol), 1.07g H-AA (27-31)-OtBu (0.97mmol) and 124mg HOBt (0.92mmol) is added in round-bottomed flask.By described dissolution of solid in 30mL DMF, add 305 μ L DIEA (1.84mmol), then under argon shield, be cooled to 0 DEG C.349mg HBTU (0.92mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to room temperature, then stir 4 hours.Add 200mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 200mL water washing, at room temperature grinds described solid 3 hours, collected by vacuum filtration with 200mL acetonitrile, dry acquisition Fmoc-AA (17-31)-OtBu.Add 30mLDMF again to dissolve, drip piperidines to ultimate density 16%, react 2 hours, add frozen water precipitated product, frozen water washs 2 times, adds the cold MTBE of 200mL and stirs the fulvene product that removal in 2 hours removes Fmoc, filtering-depositing, drying, obtains 2.56g H-AA (17-31)-OtBu, yield 97%.
3.3 preparation H-AA (9-31)-OtBu
1.27g Fmoc-AA (9-16)-OH (0.92mmol), 2.53g H-AA (17-31)-OtBu (0.88mmol) and 124mg HOBt (0.92mmol) is added in round-bottomed flask.By described dissolution of solid in 35mL DMF, add 305 μ L DIEA (1.84mmol), then under argon shield, be cooled to 0 DEG C.349mg HBTU (0.92mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to room temperature, then stir 6 hours.Add 200mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 200mL water washing, 2 × 200mL MTBE washs, and at room temperature grinds 3 hours, collected by vacuum filtration with 200mL acetonitrile, dry acquisition Fmoc-AA (9-31)-OtBu.Add 40mL DMF again to dissolve, drip piperidines to ultimate density 16%, react 4 hours, add frozen water precipitated product, frozen water washs 2 times, adds the cold MTBE of 200mL and stirs the fulvene product that removal in 2 hours removes Fmoc, filtering-depositing, drying, obtains 3.33g H-AA (9-31)-OtBu, yield 97%.
3.4 preparation Boc-AA (1-31)-OtBu
1.23g Boc-AA (1-8)-OH (0.87mmol), 3.32g H-AA (9-31)-OtBu (0.83mmol) and 117mg HOBt (0.87mmol) is added in round-bottomed flask.By described dissolution of solid in 50mL DMF, add 288 μ L DIEA (1.74mmol), then under argon shield, be cooled to 0 DEG C.330mg HBTU (0.87mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to room temperature, then stir 8 hours.Add 300mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 300mL water washing, 2 × 300mL MTBE washs, dry acquisition 4.47g crude product Boc-AA (1-31)-OtBu.At room temperature grind described solid 3 hours, collected by vacuum filtration with 300mL acetonitrile, dry acquisition 4.3g Boc-AA (1-31)-OtBu, yield 96%.
3.5 synthesis full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]s
4.27g Boc-AA (1-31)-OtBu (0.79mmol) is added in round-bottomed flask, add 50mL hydrazine hydrate: the mixing solutions of DMF=1:15, react 3 hours, add frozen water precipitated product, frozen water washs 2 times, and 300mL MTBE washs 2 times, filtering-depositing, drying, obtains the product that 4.06g sloughs ivDde.Add 1.28g Fmoc-Glu-OtBu (3mmol), 405mg HOBt (3mmol) is dissolved in 50mL DMF, then add 992 μ L DIEA (6mmol), then under argon shield, be cooled to 0 DEG C.1.14g HBTU (3mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to room temperature, then stir 8 hours.Add 300mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 300mL water washing, 2 × 300mL MTBE washs, and at room temperature grinds described solid 3 hours, collected by vacuum filtration with 300mL acetonitrile, dry.Then add 50mL DMF to dissolve, drip piperidines to ultimate density 16%, react 2 hours, add frozen water precipitated product, frozen water washs 2 times, adds the cold MTBE of 300mL and stirs the fulvene product that removal in 2 hours removes Fmoc, filtering-depositing, dry, obtains 4.05g product.Add 770mg palmitinic acid (3mmol) again, 50mL DMF, stir 8 hours.Add 300mLMTBE precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 300mLMTBE, 2 × 300mL water washing, dry acquisition 4.34g full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], yield 95%.
4. the cracking of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and purifying
4.1 prepare the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] by removing side chain protected
Add trifluoroacetic acid/water/tri isopropyl silane/1，2-ethandithiol (92.5:2.5:2.5:2.5) solution 60mL in round-bottomed flask, and be cooled to 0 DEG C.2g full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is added in this cooling solution.Stir described slurry until described dissolution of solid (about 5 minutes) at 0 DEG C, then rise to room temperature, stir 3 hours.Spin concentration, adds 0 DEG C of ether 200mL and precipitates described peptide by this solution.Centrifugal, precipitation 2 × 200mL washed with diethylether, then by dissolution of solid in containing in the 1:1 water/acetonitrile 50mL of 1% acetic acid, lyophilize obtains the thick peptide of 1.26g Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], productive rate 98%.
The thick peptide of 4.2 HPLC purifying Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
The thick peptide of 50mg Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] produces Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sterling 24.4mg through preparation HPLC purifying, purity 99%, productive rate 49%.
HPLC purification condition: chromatographic column: Waters C18 250 × 19,5u, 130A; Flow velocity: 8mL/min; Detect: UV, 220nm; Moving phase: A. acetonitrile; B.0.25% acetic acid/water; Method: 20%-30%A, 10min; 30-60%A, 40min.
Claims (10)
1. fragment condensation prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, the peptide fragment sequences of solid phase synthesis 4 side chain protected, by each peptide fragment in solution system progressively coupling obtain full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide, slough the side chain protected of the 20th Lys, and complete modification, form full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], then cracking deprotection base obtains the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and purifying changes salt and obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
Wherein, 4 described peptide fragment sequences are:
First peptide fragment sequences is the 1-8 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
Second peptide fragment sequences is the 9-16 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
Tripeptide fragment sequence is the 17-26 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence,
4th peptide fragment sequences is the 27-31 amino acids in Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sequence.
2. fragment condensation according to claim 1 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, it comprises the following steps:
(1) the first ~ four peptide fragment sequences of difference solid phase synthesis side chain protected, and from cracking resin;
(2) by the 4th peptide fragment sequences carboxyl terminal modification protection of side chain protected, and its amino protecting group is sloughed;
(3) the 4th peptide fragment sequences of the side chain protected of deaminize protecting group and the tripeptide fragment coupling sequence of side chain protected are obtained the pentapeptide fragment sequence of side chain protected, and slough its amino protecting group;
(4) the second peptide fragment sequences coupling of the pentapeptide fragment sequence of the side chain protected of deaminize protecting group and side chain protected is obtained the 6th peptide fragment sequences of side chain protected, and slough its amino protecting group;
(5) the 6th peptide fragment sequences of the side chain protected of deaminize protecting group and the first peptide fragment sequences coupling of side chain protected are obtained the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide of full guard;
(6) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] straight-chain polypeptide of full guard is sloughed the side chain protected of the 20th Lys, and complete modification and obtain full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
(7) the Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] cracking deprotection base of full guard is obtained the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
(8) the purified salt that changes of the thick peptide of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] obtains Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
3. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (1), first to fourth peptide fragment sequences of side chain protected is coupled at successively on solid phase carrier by amino acid respectively and obtains; Wherein, described solid phase carrier is acid sensitive resin;
In first to fourth peptide fragment sequences solid phase synthesis of side chain protected, the amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration; The combination of the combination of the combination that the coupling agent used is DIC and HOBt or HBTU and HOBt and DIEA or PyBOP and HOBt and DIEA; The cracking agent used for volumn concentration be the DCM solution of the TFA of 0.5 ~ 1%, volumn concentration is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7.
4. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (2), and the DCM solution of the 2Cl-Trt Cl that the carboxyl terminal protection reagent used is 0.5M or CHCl
3with the mixture of TFE and TBTA according to volume ratio 7:2:1.
5. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; it is characterized in that; in step (2), (3), (4); the amino deprotecting regent used for volumn concentration be the DMF solution of the piperidines of 16%, or volumn concentration is the DMF solution of the DBU of 1%.
6. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (3), (4), (5), the combination that the coupling agent used is HBTU and HOBt and the combination of DIEA or the combination of the combination of HBTU and HOAt and DIEA or the combination of DIC and HOBt or EDC and HOBt or PyBOP and HOBt and DIEA; The solvent of linked reaction is any one or a few the combination in DMF, DCM, NMP, THF, TFE and DMSO.
7. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (6), the reagent sloughing Lys side chain protected ivDde is hydrazine hydrate and the DMF mixture according to volume ratio 1:15.
8. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (6), the method that Lys side chain is modified is for first to add Fmoc-Glu-OtBu condensation; Then slough Fmoc group, the DMF solution of agents useful for same to be volumn concentration the be piperidines of 16%, or volumn concentration is the DMF solution of the DBU of 1%; Last again with palmitinic acid condensation.
9. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (7), the lysate of full guard Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] cracking is TFA and H
2o is the mixing solutions of 95:5 or TFA and EDT and TIS and PhOH and H by volume
2o is the mixing solutions of 80:5:5:5:5 or TFA and EDT and TIS and H by volume
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.
10. fragment condensation according to claim 2 prepares the method for Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], it is characterized in that, in step (8), purifying is that RPLC purifying changes salt; Moving phase is aqueous acetic acid and acetonitrile solution.
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| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN111116731A (en) * | 2020-01-09 | 2020-05-08 | 山东大学 | Method for preparing Somaloutide by liquid phase method based on soluble hydrophobic labeled carrier |
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