CN103992401B - Method for preparing exenatide - Google Patents
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- CN103992401B CN103992401B CN201410179385.2A CN201410179385A CN103992401B CN 103992401 B CN103992401 B CN 103992401B CN 201410179385 A CN201410179385 A CN 201410179385A CN 103992401 B CN103992401 B CN 103992401B
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Abstract
The invention relates to a method for preparing exenatide, and solves the problems that cost is high, yield is low, impurity (des-Glu＜15＞, Glu＜16＞-exenatide) content is relatively large and large-scale production cannot be realized in the prior art. The concrete steps comprise: A) taking amino resin as initial resin, employing an Fmoc solid-phase synthesis method, according to the peptide sequence of the exenatide main chain to successively couple with amino acids with Fmoc protection at N terminal and with protected side chains, wherein in the amino acid coupling reactions of the peptide sequence 15-17, a solution DMF is added with 2,2,2-trifluoroethyl alcohol accounting for 20% by volume of the solution and 1.5 mol/L of urea; and B) after peptide resin is subjected to cracking, employing a reverse-phase filling material for first purification, and employing anion resin for second purification, desalinating to enable the content of impurities des-Glu＜15＞, Glu＜16＞-exenatide to be reduced to 0.1% or less, and performing freeze drying, so as to obtain exenatide. The method is a low-cost preparation technology suitable for large-scale production of exenatide with high purity, and the technology is capable of effectively controlling the content of des-Glu＜15＞, Glu＜16＞-exenatide and does not influence the yield of exenatide.
Description
Technical field
It is a kind of synthesis with glucagon-like-peptide-1 the present invention relates to a kind of preparation method of polypeptide drug
(GLP-1) preparation method of the treatment specific drug-Exenatide of the type ii diabetes of receptor stimulating agent.
Background technology
Exenatide, English name：Exenatide, is the peptide of a straight chain 39, and structural formula is as follows：
Peptide sequence is：
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-
Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-
Pro-Pro-Pro-Ser-NH2
Exenatide is the plan duodenin developed by Amylin companies, is mainly used in treating type ii diabetes,
In No. 28 approvals of acquisition U.S. FDA of in April, 2005.Exenatide have GLP-1 receptor stimulating agents act on, molecular structure,
The aspects such as bioactivity, action target spot and immunogenicity are similar to GLP-1.The molecular structure of Exenatide is homologous with GLP-1
Up to 53 %, architectural difference shows property：（1）C-terminal has more 9 amino acid（PSSGAPPPS）, it is difficult to be degraded by endopeptidase；
（2）Second amino acid of N-terminal is glycine, will not be by dipeptidyl peptidase IV（DPP-Ⅳ）Decompose.Special molecular structure makes this
, up to 9.57 hours, injection is twice and hypoglycemia occurrence risk is small daily for patient, works as blood glucose rise for long half time of the product in blood plasma
When, this product can raise intracellular cAMP, induced insulin release；When blood sugar is reduced, the effect of its induced insulin release
Can disappear.Additionally, this product can also reduce the secretion of hyperglycemic factor in blood sugar dependence mode, and postpone gastric emptying.It is existing
In having technology US6902744, US6924264, the preparation of Exenatide using synthesis in solid state method.This kind of method is due to Ai Sai
That peptide is containing 39 peptides long of amino acid, have that technical difficulty is big, synthesis cycle is long, production cost is high, process intermediates because
Cause product purity not high for the impurity for bringing can not be purified, be unfavorable for the problems such as mass producing.
Reported in CN102942625A patents and Ser, Thr, Cys are first derived by the pseudo- proline knot of chemical conversion by liquid phase method
Structure, i.e. side chain-hydroxyl or β-sulfydryl form similar proline oxazolidine five-membered ring structures with aldehyde or ketone, then according to Fmoc
Synthesis in solid state, HPLC obtain Exenatide after purification, and purity is 95 %, yield is 36 %.This method is given birth to due to first wanting liquid phase
Into corresponding false dipeptides, therefore there are problems that synthesis cycle is long, production cost is high, be unfavorable for.
The synthesis in solid state of existing Exenatide, because Exenatide synthesis step is more, causes impurity in products species to compare
It is many, including： des-Glu15,Glu16- Exenatide and other impurity etc..Wherein relative substance des-Glu15,Glu16- Ai Saina
The amino acid sequence of peptide is as follows：
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Ala-
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-
Pro-Ser-NH2
The impurity is one of maximum impurity of toxicity, and extremely unstable, and the presence of the impurity has a strong impact on Ai Saina
Peptide content and use safety.Therefore need to find effective method and remove it and reach the % of gold standard rank 0.1
Below.The present inventor's research finds that the means of the impurity prior art are difficult to remove, though some methods can remove part,
But removal effect is undesirable, it is difficult to reach gold standard rank while easily causing Exenatide yield reduction in itself.
The present inventor uses existing synthetic method, prepares Exenatide, finds high cost, and yield is low, and impurity is more, is unsuitable for
Industrial-scale production.Therefore, the present inventor is studied the preparation method of Exenatide, there is provided a kind of low cost,
Purity is high, is adapted to the preparation technology of the Exenatide of large-scale production, and this technique can effectively control des-Glu15,Glu16- Chinese mugwort
Fill in that peptide content and do not influence the yield problem of Exenatide.
The content of the invention
The present invention relates to a kind of method for preparing Exenatide, the high cost of prior art presence is solved, yield is low, miscellaneous
Matter des-Glu15,Glu16- Exenatide content is larger, is not suitable for the problem of large-scale production.It is an object of the invention to provide
A kind of low cost, purity are high, are adapted to the preparation technology of the Exenatide of large-scale production, and this technique can effectively control des-
Glu15,Glu16- Exenatide content does not influence the yield problem of Exenatide again.
Synthetic route of the invention is as shown in Figure 1：A）Obtained by amino resins and Fmoc-Ser (tBu)-OH couplings first
Fmoc-Ser (tBu)-resin, then by Fmoc solid-phase synthesis, is coupled with N-terminal successively according to Exenatide main chain peptide sequence
Fmoc protections and the amino acid of side chain protected, wherein, in the amino acid couplings reaction of peptide sequence 15-17, its body is added in solution D MF
The 2,2,2 tfifluoroethyl alcohol of 20 % of product and the urea of 1.5 mol/L；B）After peptide resin cracking, purifying for the first time is filled out using anti-phase
Material, second purifying uses resin anion (R.A.), and des-Glu can be made after desalination15,Glu16- Exenatide content is reduced to 0.1
Below %, freezes, and obtains Exenatide.Some conventional abbreviations have following meanings in the present invention；
Fmoc ：Fluorenylmethyloxycarbonyl
Fmoc-AA-OH ：The amino acid of fluorenylmethyloxycarbonyl protection
DIC ：N, N '-Diisopropylcarbodiimide
DCC ：N, N '-dicyclohexylcarbodiimide
PyBOP ：Hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus
HATU ：2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HOBt ：1- hydroxy benzenes a pair of horses going side by side triazole
tBu ：The tert-butyl group
Trt ：Trityl
Boc ：Tertbutyloxycarbonyl
Pbf ：2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulfonyls
Tyr ：Tyrosine
Ile ：Isoleucine
Gln ：Glutamine
Asn ：Asparagine
Cys ：Cysteine
Pro ：Proline
Leu ：Leucine
Gly ：Glycine
Arg ：Arginine
Val ：Valine
Trp ：Tryptophan
Ala ：Alanine
Met ：Methionine
Phe ：Phenylalanine
Glu ：Glutamic acid
Lys ：Lysine
Ser ：Serine
Asp ：Aspartic acid
Thr ：Threonine
His ：Histidine
DMF ：N, N '-dimethylformamide
MeOH ：Methyl alcohol
DCM ：Dichloromethane
TFE ：2,2,2- trifluoroethanols
NMP ：1-METHYLPYRROLIDONE
DMSO ：Dimethyl sulfoxide (DMSO)
TFA ：Trifluoracetic acid
EDT ：Dithioglycol
Piperidine ：Hexahydropyridine
DIEA ：N, N '-diisopropylethylamine
For this present invention provides a kind of method for preparing Exenatide, its step is as follows：
Step 1, with amino resins as initial resin, by Fmoc solid-phase synthesis, according to Exenatide main chain peptide sequence according to
Amino acid of the secondary coupling with N-terminal Fmoc protections and side chain protected, wherein, in the amino acid couplings reaction of peptide sequence 15-17, solution
The 2,2,2 tfifluoroethyl alcohol of its volume 20 % and the urea of 1.5 mol/L are added in DMF；
Step 2, after cracking, purifying for the first time uses reverse phase filler, and second purifying uses resin anion (R.A.), can after desalination
So that des-Glu15,Glu16- Exenatide content is reduced to 0.1 below %, freezes, and obtains Exenatide.
Wherein, the solid phase synthesis process described in step 1,1）It is starting to use the amino resins of 0.10 ~ 0.35 mmol/g
Resin；2）It is 1 to use by volume ratio:Fmoc protection groups on the deprotection liquid removing amino resins of 4 piperidines and DMF composition；
3）In the presence of coupling agent system, amino resins and Fmoc-Ser (tBu)-OH couplings obtain Fmoc-Ser (tBu)-resin；
4）Repeat step 2）、3）, carrying out the coupling of amino acid successively according to Exenatide main chain peptide sequence, coupling amino acid order is：
Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys
(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH.Wherein, in the amino acid couplings reaction of peptide sequence 15-17, it is added in solution D MF
2,2, the 2- trifluoroethanols and the urea of 1.5 mol/L of the % of volume 20, due to 2,2,2- trifluoroethanols can destroy beta structure stabilization
Property, mitigate the tendency of peptide interchain aggregation, the solvation degree of peptide chain can be greatly improved；And urea molecule is used to hydrogen bond
Interference effect destroys the beta sheet of peptide chain, can improve the condensation rate of Fmoc-Glu (OtBu)-OH.
Wherein, the upper amino resins is selected from Rink amide AM resins, Rink amide mbha resins, Sieber
Resin；The coupling agent system includes condensing agent and reaction dissolvent, and the condensing agent is selected from DIC/HOBt, PyBOP/HOBt/
DIEA or HATU/HOBt/DIEA；The reaction dissolvent is selected from DMF, DCM, NMP, DMSO or any combination between them.
Wherein, the purification process described in step 2, after thick peptide water dissolves, 1）Purified with C18 posts for the first time, purify bar
Part：Mobile phase is：A phases：0.1 %TFA；B phases：Acetonitrile；Gradient program is:To 60 %B in 15 %B, 60min；Detection wavelength
220 nm；Collect purpose peak cut；2）Use anion-exchange column (UniDEAE-10) second；Mobile phase：A is that buffer solution I is molten
Liquid（10 mmol/L ammonium acetate aqueous solution weak aqua ammonias adjust its pH value for 7.30 ± 0.20):Acetonitrile=90:10；B is buffer solution
II solution（The mmol/L sodium-chloride water solution acetic acid of 10 mmol/L ammonium acetates+100 adjust its pH value for 4.50 ± 0.20):
Acetonitrile=90:10；Gradient program is:To 100 %B in 100 %A, 60min；Detection wavelength is 220 nm；Purpose peak is collected to evaporate
Point；3）Desalination condition：Mobile phase：A phases：The aqueous solution of 20 mmol/L ammonium acetates:Acetonitrile=95:5；B phases：Water:Acetonitrile=95:
5；C phases：0.03 % vinegar aqueous acids:Acetonitrile=95:5；D phases：0.03 % vinegar aqueous acids:Acetonitrile=50:50；Gradient program
For：With mobile phase A Gradient elution 15 minutes, Mobile phase B Gradient elution is converted into 10 minutes, be converted into the ladders such as mobile phase C
Degree wash-out 10 minutes, is converted into mobile phase D Gradient elutions 25 minutes；The nm of Detection wavelength 220；Collect purpose peak cut；Can
So that des-Glu15,Glu16- Exenatide content is reduced to 0.1 below %, and concentrated by rotary evaporation is lyophilized to obtain Exenatide.
The method of the present invention is obtained by screening, and screening process is as follows：
1）The selection of urea concentration：
In amino acid couplings reaction in 15-17 peptide sequences, urea, the selection of urea concentration are added in solution D MF： 1.5
mol/L；2.0 mol/L
2）Add the selection of TFE volumes：
10 %；20 %
3）The selection of resin anion (R.A.) filler：
UniDEAE-10；UniQ-10.
8 kinds of experiment conditions are proposed for this：
Experiment condition 1：
1）8.14 g substitution values are taken for 0.20 mmol/g
Fmoc-Ala-Val-Arg(Pbf)-Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn
(Trt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide
AM resins (1.0 mmol) are added in solid phase reaction post, are washed with DMF 1 time, swelling 30 minutes with DCM.
2）Volume ratio is 1:The deprotection liquid of 4 piperidines and DMF composition is reacted 5 minutes, and DMF is washed 1 time, and volume ratio is 1:
The deprotection liquid of 4 piperidines and DMF composition is reacted 10 minutes, and DMF is washed 6 times.
3）Weigh 1.80 g urea to be added in the mixed solution of 16.5 ml DMF and 3.5 ml TFE, dissolve, be prepared into
The DMF/TFE mixed solutions of the urea of 1.5 mol/L.
4）Weigh 2.13 g Fmoc-Glu (OtBu)-OH（5 mmol）、0.68 g HOBt（5 mmol）Add concentration be
In the DMF/TFE mixed solutions of the urea of 1.5 mol/L, 0.8 ml DIC are added under ice-water bath（5 mmol）After activation, add
In the above-mentioned reaction column equipped with resin, react 2 hours at room temperature.
5）Repeat 2）-4）Twice, similarity condition is coupled two Fmoc-Glu (OtBu)-OH again.
6）Remaining amino acid is coupled successively, and coupling amino acid order is：Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH.Coupling is finished, and is washed with DMF 3 times, and DCM is washed 3 times, and MeOH is washed
Wash 3 times, DCM is washed 3 times, MeOH is washed 3 times, is drained and is obtained 11.82 g Exenatide Rink amide AM resins.
7）Above-mentioned resin is added in the three neck round bottom flask of 250 mL, by TFA:Thioanisole:Methyl phenyl ethers anisole:EDT=
90:5:3:The 2 volume ratio configuration mL of lysate 100, during lysate added into above-mentioned resin, room temperature reaction 2 hours, filtering is used
Resin after a small amount of TFA washings cracking 3 times, merging filtrate, concentration, precipitation 1 is small during the liquid after concentration is added into ice ether
When, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying obtains the thick g of peptide 4.20 of Exenatide.The % of HPLC purity 63.57,
The % of synthesis yield 82.
8）After the thick peptide of above-mentioned Exenatide is weighed with 4 L water dissolves, purified with C18 posts for the first time, purification condition：Mobile phase
For：A phases：0.1 %TFA；B phases：Acetonitrile；Gradient program is:To 60 %B in 15 %B, 60 min；The nm of Detection wavelength 220；Receive
Collection purpose peak cut.
9）Use anion-exchange column (UniDEAE-10) second；Mobile phase：A is buffer solution I solution（10 mmol/L vinegar
Sour aqueous ammonium weak aqua ammonia adjust its pH value for 7.30 ± 0.20):Acetonitrile=90:10；B is the solution of buffer solution II（10
The mmol/L sodium-chloride water solution acetic acid of mmol/L ammonium acetates+100 adjust its pH value for 4.50 ± 0.20):Acetonitrile=90:10；
Gradient program is：To 100 %B in 100 %A, 60 min；Detection wavelength is 220 nm；Collect purpose peak cut.
10）Desalination condition：
Mobile phase：A phases：The aqueous solution of 20 mmol/L ammonium acetates:Acetonitrile=95:5；
B phases：Water:Acetonitrile=95:5；
C phases：0.03 % vinegar aqueous acids:Acetonitrile=95:5；
D phases：0.03 % vinegar aqueous acids:Acetonitrile=50:50；
Gradient program is：With mobile phase A Gradient elution 15 minutes, Mobile phase B Gradient elution is converted into 10 minutes,
It is converted into mobile phase C Gradient elutions 10 minutes, is converted into mobile phase D Gradient elutions 25 minutes；
The nm of Detection wavelength 220；Collect purpose peak cut；Concentrated by rotary evaporation, it is lyophilized to obtain Exenatide acetate fine peptide 1.92
The % of g, HPLC purity 99.89, purifies the % of yield 56, the % of total recovery 46.
Experiment condition 2-8, experimental implementation is as shown in experiment condition 1, and each experiment condition and experimental result are as follows：
Result above shows that the purification effect of experiment condition 7 is optimal.
Compared to the prior art the method for the present invention has obvious advantage, and relevant contrast experiment is as follows：
The beneficial effects of the invention are as follows：The present inventor is studied the preparation method of Exenatide, there is provided a kind of
Low cost, purity are high, are adapted to the preparation technology of the Exenatide of large-scale production, and this technique can effectively control des-Glu15,
Glu16- Exenatide content does not influence the yield of Exenatide again.
Brief description of the drawings
Fig. 1 synthetic routes of the invention；
The HPLC spectrograms of the thick peptide of Fig. 2 Exenatides；
The HPLC spectrograms of Fig. 3 Exenatide fine peptides；
The HPLC spectrograms of Fig. 4 Exenatide control samples；
The Exenatide fine peptide mass spectrogram of Fig. 5 embodiments nine.
Specific embodiment
The present invention is further illustrated by the following examples.
Embodiment one：Substitution value is the synthesis of Fmoc-Ser (tBu)-Rink amide AM resins of 0.20 mmol/g
Weigh the Rink amide AM resins that 5.00 g substitution values are 0.20 mmol/g（1 mmol）, it is added to solid phase
In reaction column, washed with DMF 1 time, with DCM swellable resins 30 minutes after, it is 1 to use volume ratio:4 piperidines and DMF composition
Deprotection liquid is reacted 5 minutes, and DMF is washed 1 time, and it is 1 to use volume ratio:The deprotection liquid of 4 piperidines and DMF composition reacts 10 points
Clock, DMF is washed 6 times, weighs 1.92 g Fmoc-Ser (tBu)-OH（5 mmol）With 0.68 g HOBt（5 mmol）Add body
Product is than being 1:1 DCM and DMF mixed solutions, add 0.8 ml DIC under ice-water bath（5 mmol）Above-mentioned being equipped with is added after activation
In the reaction column of resin, react 2 hours, washed with DMF 3 times, DCM is washed 3 times, MeOH is washed 3 times, DCM is washed 3 times, MeOH
Washing 3 times, 5.15 g Fmoc- Ser (tBu)-Rink amide AM resins are obtained after drying.
Embodiment two：Substitution value is the conjunction of Fmoc-Ser (tBu)-Rink amide MBHA resins of 0.20 mmol/g
Into
Weigh the Rink amide MBHA resins that 5.00 g substitution values are 0.20 mmol/g（1 mmol）, it is added to solid
In phase reaction post, washed with DMF 1 time, with DCM swellable resins 30 minutes after, it is 1 to use volume ratio:4 piperidines and DMF composition
Deprotection liquid react 5 minutes, DMF wash 1 time, it is 1 to use volume ratio:The deprotection liquid reaction 10 of 4 piperidines and DMF composition
Minute, DMF is washed 6 times, weighs 1.92 g Fmoc-Ser (tBu)-OH（5 mmol）With 0.68 g HOBt（5 mmol）Add
Volume ratio is 1:1 DCM and DMF mixed solutions, add 0.8 ml DIC under ice-water bath（5 mmol）Above-mentioned dress is added after activation
Have in the reaction column of resin, react 2 hours, washed with DMF 3 times, DCM is washed 3 times, MeOH is washed 3 times, DCM is washed 3 times,
MeOH is washed 3 times, and 5.14 g Fmoc-Ser (tBu)-Rink amide MBHA resins are obtained after drying.
Embodiment three：Substitution value is the synthesis of Fmoc-Ser (tBu)-Sieber resins of 0.20 mmol/g
Weigh the Rink amide Sieber resins that 5.00 g substitution values are 0.20 mmol/g（1 mmol）, it is added to
In solid phase reaction post, washed with DMF 1 time, with DCM swellable resins 30 minutes after, it is 1 to use volume ratio:4 piperidines and DMF groups
Into deprotection liquid react 5 minutes, DMF wash 1 time, it is 1 to use volume ratio:The deprotection liquid reaction of 4 piperidines and DMF composition
10 minutes, DMF was washed 6 times, weighs 1.92 g Fmoc-Ser (tBu)-OH（5 mmol）With 0.68 g HOBt（5 mmol）Plus
It is 1 to enter volume ratio:1 DCM and DMF mixed solutions, add 0.8 ml DIC under ice-water bath（5 mmol）Added after activation above-mentioned
In reaction column equipped with resin, react 2 hours, washed with DMF 3 times, DCM is washed 3 times, MeOH is washed 3 times, DCM is washed 3 times,
MeOH is washed 3 times, and 5.15 g Fmoc- Ser (tBu)-Sieber resins are obtained after drying.
Example IV：The preparation of Exenatide Rink amide AM resins
Weigh Fmoc-Ser (tBu)-Rink amide AM resins that 5.15 g substitution values are 0.20 mmol/g（1
mmol）, add in solid phase reaction post, washed with DMF 1 time, with DCM swelling Fmoc-Ser (tBu)-Rink amide AM resins
After 30 minutes, it is 1 to use volume ratio:The deprotection liquid of 4 piperidines and DMF composition is reacted 5 minutes, and DMF is washed 1 time, using body
Product is than being 1:The deprotection liquid of 4 piperidines and DMF composition is reacted 10 minutes, and DMF is washed 6 times, weighs 1.69 g Fmoc-Pro-
OH（5 mmol）、0.68 g HOBt（5 mmol）It is 1 to add volume ratio:1 DCM and DMF mixed solutions, add under ice-water bath
0.8 ml DIC（5 mmol）After activation, add in the above-mentioned reaction column equipped with resin, react 2 hours at room temperature, with ninhydrin
Method detection judges reaction end, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction is incomplete,
Need to react again 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection.
The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, according to Exenatide main chain peptide sequence,
Be sequentially completed Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys
(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH。
Wherein, 1）In the amino acid couplings reaction of peptide sequence 15-17, the 2 of its volume 20%, 2,2- trifluoros are added in solution D MF
The urea of ethanol and 1.5 mol/L；2）Solvent is changed to when Fmoc-Leu-OH and Fmoc-Phe-OH is coupled：It is 1 from volume ratio:
4 DMSO and DMF mixed solutions；3）Coupling reagent is changed to when Fmoc-Asp (OtBu)-OH is coupled：PyBOP/HOBt/DIEA；4）
Coupling reagent is changed to when Fmoc-Val-OH is coupled：HATU/HOBt/DIEA.Coupling is finished, and is washed with DMF 3 times, DCM washings 3
Secondary, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 11.84g Exenatide Rink amide AM trees
Fat.
Embodiment five：The preparation of Exenatide Rink amide MBHA resins
Weigh Fmoc-Ser (tBu)-Rink amide MBHA resins that 5.14 g substitution values are 0.20 mmol/g（1
mmol）, add in solid phase reaction post, washed with DMF 1 time, with DCM swelling Fmoc-Ser (tBu)-Rink amide MBHA trees
After fat 30 minutes, it is 1 to use volume ratio:The deprotection liquid of 4 piperidines and DMF composition is reacted 5 minutes, and DMF is washed 1 time, is used
Volume ratio is 1:The deprotection liquid of 4 piperidines and DMF composition is reacted 10 minutes, and DMF is washed 6 times, weighs 1.69 g Fmoc-
Pro-OH（5 mmol）、0.68 g HOBt（5 mmol）It is 1 to add volume ratio:1 DCM and DMF mixed solutions, under ice-water bath
Add 0.8 ml DIC（5 mmol）After activation, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, with
Ninhydrin method detection judges reaction end, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction
Not exclusively, it is necessary to react again 1 hour, this criterion judges reaction suitable for subsequent amino-acid coupling with ninhydrin method detection
Terminal.
The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, according to Exenatide main chain peptide sequence,
It is sequentially completed：Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys
(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH。
Wherein, 1）In the amino acid couplings reaction of peptide sequence 15-17,2, the 2,2- tri- of its volume 20 % are added in solution D MF
The urea of fluoroethanol and 1.5 mol/L；2）Solvent is changed to when Fmoc-Leu-OH and Fmoc-Phe-OH is coupled：It is from volume ratio
1:4 DMSO and DMF mixed solutions；3）Coupling reagent is changed to when Fmoc-Asp (OtBu)-OH is coupled：PyBOP/HOBt/DIEA；
4）Coupling reagent is changed to when Fmoc-Val-OH is coupled：HATU/HOBt/DIEA.Coupling is finished, and is washed with DMF 3 times, DCM washings 3
Secondary, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 11.86g Exenatide Rink amide AM trees
Fat.
Embodiment six：The preparation of Exenatide Sieber resins
Weigh Fmoc-Ser (tBu)-Rink amide MBHA resins that 5.15 g substitution values are 0.20 mmol/g（1
mmol）, add in solid phase reaction post, washed with DMF 1 time, with DCM swelling Fmoc-Ser (tBu)-Rink amide MBHA trees
After fat 30 minutes, it is 1 to use volume ratio:The deprotection liquid of 4 piperidines and DMF composition is reacted 5 minutes, and DMF is washed 1 time, is used
Volume ratio is 1:The deprotection liquid of 4 piperidines and DMF composition is reacted 10 minutes, and DMF is washed 6 times, weighs 1.69 g Fmoc-
Pro-OH（5 mmol）、0.68 g HOBt（5 mmol）It is 1 to add volume ratio:1 DCM and DMF mixed solutions, under ice-water bath
Add 0.8 ml DIC（5 mmol）After activation, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, with
Ninhydrin method detection judges reaction end, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction
Not exclusively, it is necessary to react again 1 hour, this criterion judges reaction suitable for subsequent amino-acid coupling with ninhydrin method detection
Terminal.
The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, according to Exenatide main chain peptide sequence,
It is sequentially completed：Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys
(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH。
Wherein, 1）In the amino acid couplings reaction of peptide sequence 15-17,2, the 2,2- tri- of its volume 20 % are added in solution D MF
The urea of fluoroethanol and 1.5 mol/L；2）Solvent is changed to when Fmoc-Leu-OH and Fmoc-Phe-OH is coupled：It is from volume ratio
1:4 DMSO and DMF mixed solutions；3）Coupling reagent is changed to when Fmoc-Asp (OtBu)-OH is coupled：PyBOP/HOBt/DIEA；
4）Coupling reagent is changed to when Fmoc-Val-OH is coupled：HATU/HOBt/DIEA.Coupling is finished, and is washed with DMF 3 times, DCM washings 3
Secondary, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 11.86 g Exenatide Sieber resins.
Embodiment seven：The prepare with scale of Exenatide Rink amide AM resins
Weigh Fmoc-Ser (tBu)-Rink amide AM resins that 5152.50 g substitution values are 0.20 mmol/g（1
mol）, add in solid phase reaction post, washed with DMF 1 time, with DCM swelling Fmoc-Ser (tBu)-Rink amide AM resins 30
After minute, it is 1 to use volume ratio:The deprotection liquid of 4 piperidines and DMF composition is reacted 5 minutes, and DMF is washed 1 time, using volume
Than being 1:The deprotection liquid of 4 piperidines and DMF composition is reacted 10 minutes, and DMF is washed 6 times, weighs 1690.21 g Fmoc-Pro-
OH（5 mol）、680.40 g HOBt（5 mol）It is 1 to add volume ratio:1 DCM and DMF mixed solutions, add under ice-water bath
800 ml DIC（5mol）After activation, in the above-mentioned reaction column equipped with resin of addition, after reacting 2 hours at room temperature, with ninhydrin
Method detection judges reaction end, if resin water white transparency, then it represents that reaction is complete；Resin develops the color, then it represents that reaction is incomplete,
Need to react again 1 hour, this criterion judges reaction end suitable for subsequent amino-acid coupling with ninhydrin method detection.
The step of repeating above-mentioned removing Fmoc protections and add corresponding amino acid couplings, according to Exenatide main chain peptide sequence,
It is sequentially completed：Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Ala-OH、Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-
Phe-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Ala-OH、Fmoc-Glu(OtBu)-
OH、Fmoc-Glu(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Met-OH、Fmoc-Gln(Trt)-OH、Fmoc-Lys
(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Leu-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-Gly-OH、Boc-His(Trt)–OH。
Wherein, 1）In the amino acid couplings reaction of peptide sequence 15-17,2, the 2,2- tri- of its volume 20 % are added in solution D MF
The urea of fluoroethanol and 1.5 mol/L；2）Solvent is changed to when Fmoc-Leu-OH and Fmoc-Phe-OH is coupled：It is from volume ratio
1:4 DMSO and DMF mixed solutions；3）Coupling reagent is changed to when Fmoc-Asp (OtBu)-OH is coupled：PyBOP/HOBt/DIEA；
4）Coupling reagent is changed to when Fmoc-Val-OH is coupled：HATU/HOBt/DIEA.Coupling is finished, and is washed with DMF 3 times, DCM washings 3
Secondary, MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, is drained and is obtained 11910.05 g Exenatide Rink amide
AM resins.
Embodiment eight：The preparation of the thick peptide of Exenatide
Above-mentioned 100.00 g Exenatides Rink amide AM resins are added in the three neck round bottom flask of 2000 mL,
By TFA:Thioanisole:Methyl phenyl ethers anisole:EDT=90:5:3:The 2 volume ratio configuration mL of lysate 1000, lysate is added above-mentioned
In resin, room temperature reaction 2 hours, filtering, the resin after washing cracking with a small amount of TFA 3 times, merging filtrate, concentration, after concentration
Liquid be added in ice ether and precipitate 1 hour, centrifugation, absolute ether centrifuge washing 6 times, vacuum drying obtains Exenatide
The thick g of peptide 35.21.The % of HPLC purity 63.57, the % of synthesis yield 82.
Embodiment nine：The preparation of Exenatide fine peptide acetate
After the above-mentioned thick peptide of 35.21 g Exenatides is weighed with 35 L water dissolves, purified with C18 posts for the first time, purification condition：
Mobile phase is：A phases：0.1 %TFA；B phases：Acetonitrile；Gradient program is：To 60%B in 15 %B, 60min；Detection wavelength 220
nm；Collect purpose peak cut.Use anion-exchange column (UniDEAE-10) second；Mobile phase：A is buffer solution I solution（10
Mmol/L ammonium acetate aqueous solution weak aqua ammonias adjust its pH value for 7.30 ± 0.20):Acetonitrile=90:10;B is that buffer solution II is molten
Liquid（The mmol/L sodium-chloride water solution acetic acid of 10 mmol/L ammonium acetates+100 adjust its pH value for 4.50 ± 0.20):Acetonitrile=
90:10；Gradient program is：To 100 %B in 100 %A, 60 min；Detection wavelength is 220 nm；Collect purpose peak cut.It is de-
The condition of salt：Mobile phase：A phases：The aqueous solution of 20 mmol/L ammonium acetates:Acetonitrile=95:5；B phases：Water:Acetonitrile=95:5；C phases：
0.03 % vinegar aqueous acids:Acetonitrile=95:5；D phases：0.03 % vinegar aqueous acids:Acetonitrile=50:50；Gradient program is：With
Mobile phase A Gradient elution 15 minutes, is converted into Mobile phase B Gradient elution 10 minutes, is converted into mobile phase C constant gradients and washes
It is de- 10 minutes, it is converted into mobile phase D Gradient elutions 25 minutes；The nm of Detection wavelength 220；Collect purpose peak cut；Revolving is dense
Contracting, it is lyophilized to obtain Exenatide acetate fine peptide 16.10 g, the % of HPLC purity 99.89, purify the % of total recovery 56, total recovery 46
%。des-Glu15, Glu16- Exenatide content is 0.05 %.
Embodiment ten：Impurity des-Glu15,Glu16The assay of-Exenatide
des-Glu15,Glu16The content of-Exenatide=Exenatide reference substance content × des-Glu15,Glu16- Ai Sai
That peptide peak area × Exenatide reference substance concentration × correction factor/（The Exenatide reference substance peak area × thick peptide of Exenatide
Concentration) × 100 %=92.15 % × 0.042 × 1.0 × 1.00/（79.406×1.0）×100 %=0.05 %
Note：Exenatide reference substance is consistent with sample size with the testing conditions of Exenatide fine peptide, all for：25 μl.des-
Glu15,Glu16Correction factor=1.00 of-Exenatide.
Exenatide reference substance concentration：1.0 mg/ml
Exenatide reference substance peak area：79.406 mAU*min （Fig. 3）
Exenatide reference substance content：92.15 %
The thick peptide configuration concentration of Exenatide：1.0 mg/ml
Impurity des-Glu in Exenatide fine peptide15,Glu16- Exenatide peak area：0.042 mAU*min
Claims (1)
1. a kind of method for preparing Exenatide, it is characterised in that the described method comprises the following steps：
Step 1, it is even successively according to Exenatide main chain peptide sequence by Fmoc solid-phase synthesis with amino resins as initial resin
Amino acid of the connection with N-terminal Fmoc protections and side chain protected, wherein, in the amino acid couplings reaction of peptide sequence 15-17, solution D MF
The urea of the middle 2,2,2 tfifluoroethyl alcohol and 1.5mol/L for adding its volume 20%；
Step 2, after peptide resin cracking, 1) purify for the first time using the purifying of C18 posts, purification condition：Mobile phase is：A phases：0.1%
TFA；B phases：Acetonitrile；Gradient program is：To 60%B in 15%B, 60min；Detection wavelength 220nm；Collect purpose peak cut；2)
Second purifying uses resin anion (R.A.), uses UniDEAE-10 anion-exchange columns；Mobile phase：A is 10mmol/L ammonium acetate water
Solution weak aqua ammonia adjusts the buffer solution that its pH value is 7.30 ± 0.20:Acetonitrile=90:10；B be 10mmol/L ammonium acetates+
100mmol/L sodium-chloride water solution acetic acid adjusts the buffer solution that its pH value is 4.50 ± 0.20:Acetonitrile=90:10；Gradient journey
Sequence is：To 100%B in 100%A, 60min；Detection wavelength is 220nm；Collect purpose peak cut；3) desalination condition：Mobile phase：
A phases：The aqueous solution of 20mmol/L ammonium acetates:Acetonitrile=95:5；B phases：Water:Acetonitrile=95:5；C phases：0.03% acetic acid it is water-soluble
Liquid:Acetonitrile=95:5；D phases：0.03% vinegar aqueous acid:Acetonitrile=50:50；Gradient program is：Washed with mobile phase A constant gradient
It is de- 15 minutes, it is converted into Mobile phase B Gradient elution 10 minutes, it is converted into mobile phase C Gradient elutions 10 minutes, it is converted into stream
Dynamic phase D Gradient elutions 25 minutes；Detection wavelength 220nm；Collect purpose peak cut；It is lyophilized, obtain Exenatide.
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| CN102942625A (en) * | 2012-11-30 | 2013-02-27 | 南京工业大学 | Solid-phase synthesis method of exenatide |
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| CN102942625A (en) * | 2012-11-30 | 2013-02-27 | 南京工业大学 | Solid-phase synthesis method of exenatide |
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| 多肽"困难序列"的合成研究进展;姜和 等;《重庆理工大学学报（自然科学）》;20110531;第25卷(第5期);第3.7、3.8节 * |
| 降糖药艾塞那肽和利拉鲁肽的固相合成及艾塞那肽的药效学评价;张许;《中国优秀硕士学位论文全文数据库》;20130715;全文 * |
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