CN105273077B - A method of preparing pramlintide - Google Patents
A method of preparing pramlintide Download PDFInfo
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- CN105273077B CN105273077B CN201410343498.1A CN201410343498A CN105273077B CN 105273077 B CN105273077 B CN 105273077B CN 201410343498 A CN201410343498 A CN 201410343498A CN 105273077 B CN105273077 B CN 105273077B
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- China
- Prior art keywords
- fmoc
- pro
- pramlintide
- tbu
- resin
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- 108010029667 pramlintide Proteins 0.000 title claims abstract description 85
- 229960003611 pramlintide Drugs 0.000 title claims abstract description 85
- NRKVKVQDUCJPIZ-MKAGXXMWSA-N pramlintide acetate Chemical compound C([C@@H](C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 NRKVKVQDUCJPIZ-MKAGXXMWSA-N 0.000 title claims abstract description 85
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- VRAQFWSWKRNOGU-VXKWHMMOSA-N (2s)-1-[(2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carbonyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)OCC2C3=CC=CC=C3C3=CC=CC=C32)CCC1 VRAQFWSWKRNOGU-VXKWHMMOSA-N 0.000 claims abstract description 44
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Abstract
The present invention relates to a kind of methods for preparing pramlintide.Specific steps of the invention are as follows: A) pass through liquid phase synthesis dipeptide fragment Fmoc-Pro-Pro-OH；B solid-phase synthesis) is used, using amino resins as initial resin, the amino acid with N-terminal Fmoc protection and side chain protection is successively coupled according to pramlintide main chain peptide sequence, wherein 28,29 amino acid couplings are coupled using segment Fmoc-Pro-Pro-OH；C) make oxidant solid phase cyclization using iodine, then peptide resin cracking, recrystallize in acid condition after available pramlintide, polypeptide impurities des-Pro after purifying, desalination, freeze-drying29Pramlintide content is less than 0.1%.The present invention provides a kind of purity is highs, at low cost, are suitble to the preparation process of the pramlintide of large-scale production, this technique both can be effectively controlled impurity des-Pro29The content of pramlintide does not influence the yield of pramlintide again.
Description
Technical field
The present invention relates to a kind of preparation methods of polypeptide drug, and in particular to a kind of synthetic method of pramlintide.
Background technique
Pramlintide, illustrious name are as follows: Pramlintide is the cyclic peptide containing 37 peptides, and chemical molecular formula is as follows:
KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH2(disulfide bond: 2-7).
On March 16th, 2005, U.S. Food and Drug Administration (FDA) have approved on Amylin Products pramlintide
City, pramlintide are the analogs of artificial synthesized polypeptide, alanine that structure feature is 25,28 and 29 serine
It is replaced with proline, pramlintide effect is similar with amylin, controls postprandial blood sugar and slowing down postprandial gastric emptying speed, makees
With for about 3 hours, but do not change the absorption of carbohydrate and other nutriments.Meanwhile inhibiting postprandial pancreas high
The secretion of blood glucose element and cause satiety can also play the role of control blood glucose.It is used for 1 type and 2 type maturity-onset diabetes patients control
Blood glucose processed and until the present after insulin second be approved the drug for treating type 1 diabetes.
WO199310146A1 and US5424394 reports a kind of method for preparing pramlintide using solid-phase synthesis,
On amino insoluble resin, according to be successively coupled according to pramlintide main chain peptide sequence with N-terminal Boc protection and side chain protection ammonia
Base acid, 2 for then making peptide chain and 7 two cysteines occur molecule inner ring condensation, obtain disulfide group bridged bond, then use hydrogen
Fluoric acid cracks to obtain pramlintide, cumbersome since entire technique uses Boc synthesis in solid state, uncomfortable using hypertoxic hydrofluoric acid
Close industrialized production.
CN200910079134.6, CN200980137895.0, CN201010594565.9 and CN201210171394.8
The reasonable amino acid residue segment of several compositions for reporting selection design composition pramlintide, then by convergence synthesis to be total to
Several segments reasonably are coupled by the form of valence link, and pramlintide is prepared.It is obvious that including the general of 37 amino acid
Blue woods peptide has many possible synthesis segments and coupling order, and detailed research report is not done in current patented technology,
The segment wherein used, relatively arbitrarily, entire design is less reasonable for coupling order, and synthesis separation is difficult, complicated for operation, yield compared with
It is low, and higher cost, improper industrialization.
CN201010145391.8 reports a kind of method for preparing pramlintide using Fmoc solid-phase synthesis, in amino
On insoluble resin, according to be successively coupled according to pramlintide main chain peptide sequence with N-terminal Fmoc protection and side chain protection amino
Acid then makes 2 of peptide chain and 7 two cysteines issue raw molecule inner ring condensation in iodine, obtains disulfide group bridged bond, then split
Solution obtains pramlintide, and entire technological operation is relatively easy, but since the amino acid of 28 and 29 two couplings is all dried meat ammonia
Acid is all parahelium, therefore leads to proline coupling not exclusively, generates peptide disappearance impurity: des-Pro29Pramlintide.It is related miscellaneous
Matter des-Pro29The amino acid sequence difference of pramlintide is as follows:
KCNTATCATQRLANFLVHSSNNFGPILPTNVGSNTY-NH2(disulfide bond: 2-7).
This impurity is one of the impurity being more toxic, and separate with main peak it is very difficult, the impurity there are serious shadows
Ring pramlintide content and using safe.Therefore it needs to find effective method to remove it and reach gold standard grade
Other 0.1% or less.The present inventor is the study found that the means of the impurity prior art are difficult to remove, though some methods can be gone
Except part, but removal effect is undesirable, it is difficult to which reaching gold standard rank while be easy to causeing pramlintide itself yield reduces.
In conclusion since synthesis yield is low, impurity is more, especially all during the synthesis in solid state of existing pramlintide
Impurity des-Pro cannot be controlled very well29Pramlintide is not suitable for industrialized production.Therefore, a kind of high income, reaction are researched and developed
The simple controllable, synthetic method new suitable for the pramlintide of industrialized production has great importance.
Summary of the invention
The existing synthetic method of the present inventor, prepares pramlintide, it is found that technical problem of the existing technology is: closing
Low at yield, impurity is more, especially cannot all control impurity des-Pro very well29It is raw to be unsuitable for industrially scalable for pramlintide
It produces.For this purpose, the present inventor studies the synthetic method of pramlintide, to obtain technical solution of the present invention.
The object of the present invention is to provide a kind of methods for preparing pramlintide.Synthetic route of the invention is led to as shown in Figure 1:
Cross liquid phase synthesis dipeptide fragment Fmoc-Pro-Pro-OH；Using solid-phase synthesis, using amino resins as initial resin, according to general
Blue woods peptide backbone peptide sequence is successively coupled the amino acid with N-terminal Fmoc protection and side chain protection, wherein 28,29 amino acid couplings are adopted
It is coupled with segment Fmoc-Pro-Pro-OH；Make oxidant solid phase cyclization using iodine, then peptide resin cracks, in acid condition
Available pramlintide after purifying, desalination, freeze-drying after recrystallization.
Some common abbreviations have following meanings in the present invention；
Fmoc: fluorenylmethyloxycarbonyl
Fmoc-AA: the amino acid of fluorenylmethyloxycarbonyl protection
DIC: N, N '-Diisopropylcarbodiimide
EDCI.HCl: 1- [3- (dimethylamino) propyl] -3- ethyl-carbodiimide hydrochloride
PyBOP: hexafluorophosphoric acid benzotriazole -1- base-oxygroup tripyrrole alkyl phosphorus
HATU: 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HOBt: 1- hydroxy benzenes a pair of horses going side by side triazole
Tyr: tyrosine
Ile: isoleucine
Gln: glutamine
Asn: asparagine
Cys: cysteine
Pro: proline
Leu: leucine
Gly: glycine
Arg: arginine
Val: valine
Ala: alanine
Phe: phenylalanine
Lys: lysine
Ser: serine
Thr: threonine
His: histidine
Asn: asparagine
DMF: N, N '-dimethylformamide
MeOH: methanol
DCM: methylene chloride
NMP: N-Methyl pyrrolidone
DMSO: dimethyl sulfoxide
TFA: trifluoracetic acid
EDT: dithioglycol
Piperidine: hexahydropyridine
The present invention provides a kind of synthetic method of pramlintide thus, and its step are as follows:
Step 1, pass through liquid phase synthesis dipeptide fragment Fmoc-Pro-Pro-OH；
Step 2, successively even according to pramlintide main chain peptide sequence using amino resins as initial resin using solid-phase synthesis
Join the amino acid with N-terminal Fmoc protection and side chain protection, wherein 28,29 amino acid couplings use segment Fmoc-Pro-Pro-
OH coupling；
Step 3, make oxidant solid phase cyclization using iodine, then peptide resin is cracked, then recrystallized in acid condition, pure
Available pramlintide after change, desalination, freeze-drying.
Wherein, 1) synthetic method of dipeptide fragment I: Fmoc-Pro-Pro-OH described in step 1 includes the following steps:
Fmoc-Pro-OH, HOSu and DCC are coupled to obtain Fmoc-Pro-OSu, and then Fmoc-Pro-OSu and H-Pro-OH reacts to obtain
Dipeptide fragment Fmoc-Pro-Pro-OH.
Wherein, solid phase synthesis process described in step 2, the described method comprises the following steps: 1) using 0.10 ~
The Rink Amide MBHA Resin amino resins of 0.40mmol/g is initial resin；2) it uses by volume ratio as the piperazine of 1:4
Fmoc protecting group on the deprotection liquid removing amino resins of pyridine and DMF composition；3) in the presence of coupling agent system, Rink
Amide MBHA Resin amino resins and Fmoc-Tyr (tBu)-OH are coupled to obtain Fmoc-Tyr (tBu)-Rink Amide
MBHA Resin amino resins；4) step 2,3) is repeated, successively be coupled according to pramlintide main chain peptide sequence has N-terminal Fmoc guarantor
The amino acid of shield and side chain protection, coupling amino acid sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-
Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-
Pro-Pro-OH、Fmoc-Leu-OH、Fmoc-Ile-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-
Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-
OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-
OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys
(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Cys(Trt)-OH,Boc-Lys(Boc)-OH;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 be selected from DMF, DCM, NMP, DMSO or he
Between combination.
Preferably, in step 2, in dipeptides Fmoc-Pro-Pro-OH coupling process, H-Thr (tBu)-Asn (Trt)-
Val-Gly-Ser (tBu)-Asn (Tt)-Thr (tBu)-Tyr (tBu)-Rink Amide MBHA Resin amino resins,
The molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt are preferred are as follows: 1:3:3:3 ~ 1:5:5:5, reaction temperature are 25 ~ 35 DEG C, instead
It is 2 ~ 3 hours between seasonable；It is further preferred that H-Thr (tBu)-Asn (Trt)-Val-Gly-Ser (tBu)-Asn (Tt)-Thr (tBu)-
Tyr (tBu)-Rink Amide MBHA Resin amino resins, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt are preferred
Are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Method of the invention is obtained by screening, and screening process is as follows:
1) selection of molar ratio: H-Thr (tBu)-Asn (Trt)-Val-Gly-Ser (tBu)-Asn (Tt)-Thr (tBu)-
Tyr (tBu)-Rink Amide MBHA Resin amino resins, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt are preferred
Are as follows: 1:3:3:3 ~ 1:5:5:5；
2) selection of reaction temperature:
25 DEG C and 35 DEG C；
3) selection in reaction time:
2 hours and 3 hours.
8 kinds of experiment conditions are proposed thus:
Experiment condition 1: 53.22g H-Thr (tBu)-Asn (Trt)-Val-Gly-Ser (tBu)-Asn (Tt)-Thr is taken
(tBu)-Tyr (tBu)-Rink Amide MBHA Resin amino resins (10.0mmol) is added in reaction column, will
13.03g Fmoc-Pro-Pro-OH (30.0 mmol) and 4.05g HOBt (30.0 mmol) is added in 20ml DMF and stirs
Dissolution, is cooled to 0 DEG C, and 3.79g DIC (30.0 mmol) is added in above-mentioned solution, reacts 2 hours at 25 DEG C, then successively
It is coupled remaining amino acid, coupling amino acid sequence are as follows: Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-
Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-
Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Asn
(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Thr
(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Thr
(tBu)-OH,Fmoc-Asn(Trt)-OH,Fmoc-Cys(Trt)-OH,Boc-Lys(Boc)-OH；Coupling finishes to obtain full guard
Peptide resin takes 6.35g iodine (5.0mmol) to be dissolved in the DMF of 50mL, reacts 3 hours at 25 DEG C；Cyclisation finishes, using pressing body
Product is than EDT, PhSMe, H for 2.5: 5: 5: 87.52The configuration lysate 1150ml of O and TFA, is added above-mentioned resin for lysate
In, it reacts at room temperature 2 hours, the liquid after concentration is added in ice ether and precipitates 1 hour by concentration, is centrifuged, anhydrous ether centrifugation
Washing 6 times, vacuum drying, obtains the thick peptide of 30.82g pramlintide, and 3L phosphate-buffered is added in the thick peptide of 30.82g pramlintide
In aqueous solution (pH=2.0) (solution A: phosphatase 11 66ml is taken, 10000ml is added water to, is shaken up.Second liquid: disodium hydrogen phosphate is taken
716.30g adds water to make to be dissolved into 10000ml.Take above-mentioned solution A 2175ml to mix with second liquid 825ml, shake up to get.), stirring 5
Hour, there is white solid precipitation, filter, drain to obtain white solid under reduced pressure, above-mentioned white solid is dissolved in water, uses
PH value is adjusted to 5.5-6.5 by acetic acid, obtains target product after purifying by C18 or C8 column 2 times, turn salt, freeze-drying.Purify item
Part: mobile phase are as follows: A phase: 0.1%TFA；B phase: acetonitrile, Detection wavelength 220nm collect purpose peak fraction.Turn salt condition: flowing
Phase: A phase: 20mM ammonium acetate-aqueous solution；B phase: acetonitrile；Detection wavelength 220nm.Collect purpose peak fraction, concentrated by rotary evaporation, freeze-drying
It obtains freeze-drying and obtains 99.62 % of pramlintide fine peptide 17.88g, HPLC purity, total recovery 44%.
Experiment condition 2-8, for experimental implementation as shown in experiment condition 1, different experiment conditions and its experimental result are for example following
Table 1 shown in:
1 contrast and experiment of table
| Experiment condition | Molar ratio | Temperature | Time | des-Pro29Pramlintide content | Total recovery | Purity |
| Experiment condition 1 | 1:1:3 | 25℃ | 2 hours | 0.06% | 44% | 99.62 % |
| Experiment condition 2 | 1:1:3 | 25℃ | 3 hours | 0.06% | 44% | 99.61% |
| Experiment condition 3 | 1:1:3 | 35℃ | 2 hours | 0.07% | 43% | 99.60% |
| Experiment condition 4 | 1:1:3 | 35℃ | 3 hours | 0.07% | 43% | 99.60% |
| Experiment condition 5 | 1:1:5 | 25℃ | 2 hours | 0.06% | 44% | 99.59 % |
| Experiment condition 6 | 1:1:5 | 25℃ | 3 hours | 0.06% | 44% | 99.62 % |
| Experiment condition 7 | 1:1:5 | 35℃ | 2 hours | 0.07% | 43% | 99.60 % |
| Experiment condition 8 | 1:1:5 | 35℃ | 3 hours | 0.07% | 43% | 99.62 % |
The above result shows that the result difference of experiment condition 1-8 is little, but 1 synthesis cost of experiment condition it is relatively low,
Reaction time is shorter, efficiency highest, so the synthesis result of experiment condition 1 is optimal.
Compared to the prior art method of the invention has apparent advantage, related comparative experiments is as shown in table 2 below:
2 contrast and experiment of table
| Patent | des-Pro29Pramlintide content | Total recovery | Purity |
| The technology of the present invention | 0.06% | 44% | 99.62% |
| CN200910079134.6 | 1.29% | 21% | 97.30% |
| CN201010594565.9 | 1.25% | 18% | 98.50% |
The beneficial effects of the present invention are: be coupled to obtain pramlintide linear peptides using dipeptide fragment Fmoc-Pro-Pro-OH,
Then make oxidant solid phase cyclization using iodine, precipitated under the conditions of phosphorylation after cracking, improve purification efficiency, improve product
The cost of efficient liquid phase preparation purifying, available pramlintide after final purification, desalination, freeze-drying are reduced while quality.
The present invention provides a kind of purity is highs, at low cost, are suitble to the preparation process of the pramlintide of large-scale production, this technique can have
Effect control impurity des-Pro29The content of pramlintide does not influence the yield of pramlintide again.
Detailed description of the invention
The synthetic route of Fig. 1 pramlintide of the present invention；
The HPLC spectrogram of Fig. 2 dipeptide fragment Fmoc-Pro-Pro-OH；
The mass spectrogram of Fig. 3 dipeptide fragment Fmoc-Pro-Pro-OH；
The HPLC spectrogram of the thick peptide of Fig. 4 pramlintide；
The HPLC spectrogram of Fig. 5 pramlintide fine peptide；
Fig. 6 pramlintide fine peptide mass spectrogram.
Specific embodiment
The present invention is further illustrated by the following examples.
Specifically, each commercially available amino acid and amino acid fragment and each commercially available tree involved in following example
Rouge, manufacturer and marque are as follows:
Fmoc protecting group amino acid starting material and Wang Shuzhi are that (producer: gill biochemistry (Shanghai) has for conventional commercial reagent
Limit company；Chemistry is pure)；Dipeptide fragment Fmoc-Pro-Pro-OH is this patent description synthesis.
Organic solvent and other raw material sources are commercially available product (producer: Sinopharm Chemical Reagent Co., Ltd.；Chemistry
It is pure).
In addition, " concentrated by rotary evaporation " and " freeze-drying " mentioned in following example and measurement HPLC and mass spectrographic condition and
Device therefor model and manufacturer are described as follows:
Concentrated by rotary evaporation equipment: Rotary Evaporators R-200/205(Switzerland Buchi (cloth is odd) company)；
Concentrated by rotary evaporation condition: at 30 DEG C, concentrated by rotary evaporation under the conditions of vacuum (- 0.1Mpa), volume is total before revolving after concentration
Below volume 75%.
HPLC:Dionex high performance liquid chromatograph；It is to fill out with (5 μm, 250 × 4.6mm) of octadecylsilane chemically bonded silica
Fill agent；Using 0.1%TFA solution as mobile phase A, using acetonitrile as Mobile phase B, gradient elution is carried out；Flow velocity is 1.0mL per minute；Inspection
Survey wavelength is 220nm；30 DEG C of column temperature.20 μ l of test solution is taken, liquid chromatograph is injected, records chromatogram.
Mass spectrum: MALDI-TOF-MS Matrix-Assisted Laser Desorption Ionization Time of Flight；Instrument model is AUTO
FLEX SPEED TOF-TOF。
The synthesis of one: Fmoc-Pro-OSu Acibenzolar of embodiment
Weigh 337.37g Fmoc-Pro-OH(1.0mol), 138.10g HOSu(1.2mol) it is added in 2000ml THF,
247.59g DCC(1.2mol is added under ice-water bath), it reacts 1 hour, is warming up to room temperature reaction 3 hours, reaction solution filtering, mother liquor
It is spin-dried for, DCM is added to dissolve, filter, saturated sodium bicarbonate is washed 3 times, pure water 2 times, is stripped 2 times, and organic phase is merged, and natrium carbonicum calcinatum is dry
It is dry, it is spin-dried for, ethyl acetate and petroleum ether recrystallize 3 times, filtering, and solid oil pump draws the dry 386.66g Fmoc-Pro-OSu that obtains to live
Change ester, yield 89%.
Embodiment two: the synthesis of dipeptide fragment Fmoc-Pro-Pro-OH
Weigh 57.57g H-Pro-OH(0.5mol), 217.23gFmoc-Pro-OSu(0.5mol) and 79.50g Na2CO3
(0.75mol), which is added in the mixed solution of 500ml water and 500ml THF, to be dissolved, and reaction is stayed overnight at room temperature, with 10% dilute hydrochloric acid
PH to 7 is adjusted, revolving removes THF, adjusts PH to 3 later.A large amount of white precipitates are obtained, are filtered.Obtained white precipitate is used
Ethyl acetate and petroleum ether recrystallization, obtain dipeptide fragment Fmoc-Pro-Pro-OH 236.55g, HPLC spectrogram such as Fig. 2 institute
Show, HPLC purity 92.827%, yield 89%(yield calculates: 0.5 mol is 265.79g, 236.55/265.79=89%).Its matter
Spectrum is as shown in figure 3, [M+Na]+: 458.205, [M+K]+: 473.255, the theory of compound dipeptide fragment Fmoc-Pro-Pro-OH
Accurate molecular weight are as follows: 434.18, sample mass spectral results are consistent with theoretical molecular weight, and structure is correct.
Embodiment three: the synthesis of pramlintide full guard linear peptides-Rink Amide AM Resin resin
The Rink Amide AM Resin resin 100g that degree of substitution is 0.10mmol/g is weighed, solid phase reaction column is added to
In, it is washed 1 time with DMF, after DCM swellable resins 30 minutes, takes 13.79g Fmoc-Tyr (tBu)-OH (30mmol), 4.05g
HOBt (30mmol) is dissolved with DMF, after 3.88g DIC (30mmol) activation is added under ice-water bath, is added above-mentioned equipped with resin
In reaction column, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that
Fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs to react 1 hour again, this judgment criteria is suitable for subsequent amino-acid
Reaction end is judged with ninhydrin method detection in coupling.It repeats above-mentioned removing Fmoc protection and the step of corresponding amino acid couplings is added
Suddenly, the amino acid with N-terminal Fmoc protection and side chain protection is successively coupled according to pramlintide main chain peptide sequence, coupling amino acid is suitable
Sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-
OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Pro-Pro-OH、Fmoc-Leu-OH、Fmoc-Ile-OH、
Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-
Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln
(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-
Ala-OH,Fmoc-Thr(tBu)-OH,Fmoc-Asn(Trt)-OH,Fmoc-Cys(Trt)-OH,Boc-Lys(Boc)-OH;It is even
Connection finishes, and is washed 6 times with DMF, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain 170.06 Pulans g
Woods peptide full guard linear peptides-Rink Amide AM Resin resin.
Solvent is changed to when wherein Fmoc-Arg (Pbf)-OH is coupled: selecting DMSO and the DMF mixing that volume ratio is 1:4 molten
Liquid；Coupling reagent is changed to when Fmoc-His (Trt)-OH is coupled: PyBOP/HOBt/DIEA；Coupling examination when Fmoc-Ile-OH is coupled
Agent is changed to: HATU/HOBt/DIEA；When Fmoc-Pro-Pro-OH is coupled, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt
It is preferred that are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Example IV: the synthesis of pramlintide full guard linear peptides-Rink Amide AM Resin resin
The Rink Amide AM Resin resin 25g that degree of substitution is 0.40mmol/g is weighed, solid phase reaction column is added to
In, it is washed 1 time with DMF, after DCM swellable resins 30 minutes, takes 13.79g Fmoc-Tyr (tBu)-OH (30mmol), 4.05g
HOBt (30mmol) is dissolved with DMF, after 3.88g DIC (30mmol) activation is added under ice-water bath, is added above-mentioned equipped with resin
In reaction column, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent, then it represents that
Fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs to react 1 hour again, this judgment criteria is suitable for subsequent amino-acid
Reaction end is judged with ninhydrin method detection in coupling.It repeats above-mentioned removing Fmoc protection and the step of corresponding amino acid couplings is added
Suddenly, the amino acid with N-terminal Fmoc protection and side chain protection is successively coupled according to pramlintide main chain peptide sequence, coupling amino acid is suitable
Sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-
OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Pro-Pro-OH、Fmoc-Leu-OH、Fmoc-Ile-OH、
Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-
Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-
Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Gln
(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-
Ala-OH,Fmoc-Thr(tBu)-OH,Fmoc-Asn(Trt)-OH,Fmoc-Cys(Trt)-OH,Boc-Lys(Boc)-OH;It is even
Connection finishes, and is washed 6 times with DMF, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain the Pulan 95.06g woods
Peptide full guard linear peptides-Rink Amide AM Resin resin.
Solvent is changed to when wherein Fmoc-Arg (Pbf)-OH is coupled: selecting DMSO and the DMF mixing that volume ratio is 1:4 molten
Liquid；Coupling reagent is changed to when Fmoc-His (Trt)-OH is coupled: PyBOP/HOBt/DIEA；Coupling examination when Fmoc-Ile-OH is coupled
Agent is changed to: HATU/HOBt/DIEA；When Fmoc-Pro-Pro-OH is coupled, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt
It is preferred that are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Embodiment five: the synthesis of pramlintide full guard linear peptides-Rink Amide MBHA Resin resin
The Rink Amide MBHA Resin resin 100g that degree of substitution is 0.10mmol/g is weighed, solid phase reaction is added to
In column, washed 1 time with DMF, after DCM swellable resins 30 minutes, take 14.06g Fmoc- Tyr (tBu)-OH (30mmol),
4.05g HOBt (30mmol) is dissolved with DMF, and after 3.88g DIC (30mmol) activation is added under ice-water bath, above-mentioned be equipped with is added
In the reaction column of resin, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent,
Then indicate fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs to react 1 hour again, this judgment criteria is suitable for subsequent
Reaction end is judged with ninhydrin method detection in amino acid couplings.It repeats above-mentioned removing Fmoc protection and corresponding amino acid idol is added
The step of connection, is successively coupled the amino acid with N-terminal Fmoc protection and side chain protection according to pramlintide main chain peptide sequence, is coupled ammonia
Base acid sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH,
Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Pro-Pro-OH、Fmoc-Leu-OH、
Fmoc-Ile-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn
(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-
Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-
OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Boc-Lys
(Boc)-OH;Coupling finishes, and is washed 6 times with DMF, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain
171.05g pramlintide full guard linear peptides-Rink Amide AM Resin resin.
Solvent is changed to when wherein Fmoc-Arg (Pbf)-OH is coupled: selecting DMSO and the DMF mixing that volume ratio is 1:4 molten
Liquid；Coupling reagent is changed to when Fmoc-His (Trt)-OH is coupled: PyBOP/HOBt/DIEA；Coupling examination when Fmoc-Ile-OH is coupled
Agent is changed to: HATU/HOBt/DIEA；When Fmoc-Pro-Pro-OH is coupled, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt
It is preferred that are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Embodiment six: the synthesis of pramlintide full guard linear peptides-Rink Amide MBHA Resin resin
The Rink Amide MBHA Resin resin 25g that degree of substitution is 0.40mmol/g is weighed, solid phase reaction column is added to
In, washed 1 time with DMF, after DCM swellable resins 30 minutes, take 14.06g Fmoc- Tyr (tBu)-OH (30mmol),
4.05g HOBt (30mmol) is dissolved with DMF, and after 3.88g DIC (30mmol) activation is added under ice-water bath, above-mentioned be equipped with is added
In the reaction column of resin, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent,
Then indicate fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs to react 1 hour again, this judgment criteria is suitable for subsequent
Reaction end is judged with ninhydrin method detection in amino acid couplings.It repeats above-mentioned removing Fmoc protection and corresponding amino acid idol is added
The step of connection, is successively coupled the amino acid with N-terminal Fmoc protection and side chain protection according to pramlintide main chain peptide sequence, is coupled ammonia
Base acid sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH,
Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Pro-Pro-OH、Fmoc-Leu-OH、
Fmoc-Ile-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn
(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-
Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-
OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Boc-Lys
(Boc)-OH;Coupling finishes, and is washed 6 times with DMF, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain
96.05g pramlintide full guard linear peptides-Rink Amide AM Resin resin.
Solvent is changed to when wherein Fmoc-Arg (Pbf)-OH is coupled: selecting DMSO and the DMF mixing that volume ratio is 1:4 molten
Liquid；Coupling reagent is changed to when Fmoc-His (Trt)-OH is coupled: PyBOP/HOBt/DIEA；Coupling examination when Fmoc-Ile-OH is coupled
Agent is changed to: HATU/HOBt/DIEA；When Fmoc-Pro-Pro-OH is coupled, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt
It is preferred that are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Embodiment seven: the synthesis of pramlintide full guard linear peptides-Rink Amide MBHA Resin resin
The Rink Amide MBHA Resin resin 50g that degree of substitution is 0.20mmol/g is weighed, solid phase reaction column is added to
In, washed 1 time with DMF, after DCM swellable resins 30 minutes, take 14.06g Fmoc- Tyr (tBu)-OH (30mmol),
4.05g HOBt (30mmol) is dissolved with DMF, and after 3.88g DIC (30mmol) activation is added under ice-water bath, above-mentioned be equipped with is added
In the reaction column of resin, after reacting 2 hours at room temperature, reaction end is judged with ninhydrin method detection, if resin is colorless and transparent,
Then indicate fully reacting；Resin colour developing, then it represents that reaction not exclusively, needs to react 1 hour again, this judgment criteria is suitable for subsequent
Reaction end is judged with ninhydrin method detection in amino acid couplings.It repeats above-mentioned removing Fmoc protection and corresponding amino acid idol is added
The step of connection, is successively coupled the amino acid with N-terminal Fmoc protection and side chain protection according to pramlintide main chain peptide sequence, is coupled ammonia
Base acid sequence are as follows: Fmoc-Thr (tBu)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Gly-OH,
Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Pro-Pro-OH、Fmoc-Leu-OH、
Fmoc-Ile-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn
(Trt)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-His(Trt)-OH、Fmoc-Val-OH、Fmoc-
Leu-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Gln(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Ala-OH、Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-
OH、Fmoc-Ala-OH、Fmoc-Thr(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Cys(Trt)-OH、Boc-Lys
(Boc)-OH;Coupling finishes, and is washed 6 times with DMF, and MeOH is washed 3 times, and DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain
120.01 g pramlintide full guard linear peptides-Rink Amide AM Resin resins, Pulan woods full guard peptide linear peptides tree
Rouge yield: 99% { the linear peptide resin yield=actual weight of Pulan woods full guard peptide/[weight resin+(pramlintide full guard line
Property peptide molecular weight-Fmoc molecular weight)] × 0.01mol=120.35/ [50+(7325.77-222) × 0.01].
Solvent is changed to when wherein Fmoc-Arg (Pbf)-OH is coupled: selecting DMSO and the DMF mixing that volume ratio is 1:4 molten
Liquid；Coupling reagent is changed to when Fmoc-His (Trt)-OH is coupled: PyBOP/HOBt/DIEA；Coupling examination when Fmoc-Ile-OH is coupled
Agent is changed to: HATU/HOBt/DIEA；When Fmoc-Pro-Pro-OH is coupled, the molar ratio of Fmoc-Pro-Pro-OH, DIC and HOBt
It is preferred that are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
Embodiment eight: the synthesis of pramlintide full guard cyclic peptide-Rink Amide MBHA Resin resin
The pramlintide full guard cyclic peptide-Rink Amide MBHA Resin resin for weighing 6 120.01 g of embodiment adds
Enter into solid phase reaction column, washed 1 time with DMF, after DCM swellable resins 30 minutes, 6.35g iodine (50mmol) is taken to use 600mL
DMF dissolution, is added in the above-mentioned reaction column equipped with resin, after reacting 3 hours at room temperature, is then washed 6 times with DMF, MeOH is washed
It washs 3 times, DCM is washed 3 times, and MeOH is washed 3 times, drains to obtain 115.00g pramlintide full guard cyclic peptide-Rink Amide
MBHA Resin resin, pramlintide full guard cyclic peptide resin: 99% { pramlintide full guard cyclic peptide resin yield=reality weight
Amount/[weight resin+(pramlintide full guard cyclic peptide molecular weight-Fmoc molecular weight)] × 0.01mol=115.00/ [50+
(6841.77-222) × 0.01] }.
Embodiment nine: the preparation of the thick peptide of pramlintide
The thick peptide resin of pramlintide for weighing 115.00g full guard, is added in the three neck round bottom flask of 3000mL, adopts
With by volume for 2.5: 5: 5: 87.5 EDT, PhSMe, H2The configuration lysate 1150ml of O and TFA, lysate is added
It states in resin, reacts at room temperature 2 hours, the liquid after concentration is added in ice ether and precipitates 1 hour by concentration, is centrifuged, anhydrous second
Ether centrifuge washing 6 times, vacuum drying obtains the thick peptide of 30.82g pramlintide, HPLC spectrogram is as shown in figure 4, HPLC purity
84.13%, thick 78% [the thick peptide yield=actual weight of pramlintide/(pramlintide molecular weight × 0.01mol)=30.82/ of peptide yield
(3949.39 × 0.01)].
Embodiment ten: the preparation of pramlintide fine peptide acetate
(first in 3L phosphate-buffered aqueous solution (pH=2.0) is added in the thick peptide of 30.82g pramlintide in embodiment eight
Liquid: phosphatase 11 66ml is taken, 10000ml is added water to, is shaken up.Second liquid: taking disodium hydrogen phosphate 716.30g, and water is added to make to be dissolved into
10000ml.Take above-mentioned solution A 2175ml to mix with second liquid 825ml, shake up to get.), it stirs 5 hours, there is white solid precipitation,
It filters, above-mentioned white solid is dissolved in water, pH value is adjusted to 5.5- with acetic acid by the white solid drained under reduced pressure
6.5, target product is obtained after purifying by C18 or C8 column 2 times, turn salt, freeze-drying.Purification condition: mobile phase are as follows: A phase:
0.1%TFA；B phase: acetonitrile, Detection wavelength 220nm collect purpose peak fraction.Turn salt condition: mobile phase: A phase: 20mM acetic acid
Ammonium-aqueous solution；B phase: acetonitrile；Detection wavelength 220nm.Purpose peak fraction, concentrated by rotary evaporation are collected, freeze-drying obtains pramlintide fine peptide
17.88g, HPLC spectrogram are as shown in figure 5, HPLC purity 99.62%(area normalization method), des-Pro29Pramlintide contains
It is fixed to measure: 0.06%, purify total recovery 58%(pramlintide fine peptide/thick peptide=17.88/30.82 of pramlintide), total recovery 44%
(total recovery=synthesis yield × purifying total recovery=99% × 99% × 78% × 58%).Its mass spectrum is as shown in figure 4, [M]+:
3949.7266, the theoretical accurate molecular weight of pramlintide are as follows: 3949.93, sample mass spectral results are consistent with theoretical molecular weight.
Embodiment 11: impurity des-Pro29The assay of pramlintide
Impurity des-Pro29The content assaying method of pramlintide: high performance liquid chromatography； des-Pro29Pulan woods
The assay of peptide: 0.06%(area normalization method).
The above content is combine specifically repair select embodiment further detailed description of the invention, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (12)
1. a kind of method for preparing pramlintide, the described method comprises the following steps:
Step 1, pass through liquid phase synthesis dipeptide fragment Fmoc-Pro-Pro-OH；
Step 2, using solid-phase synthesis, using amino resins as initial resin, tool is successively coupled according to pramlintide main chain peptide sequence
There is the amino acid of N-terminal Fmoc protection and side chain protection, wherein 28,29 amino acid couplings are even using segment Fmoc-Pro-Pro-OH
Connection；
Step 3, make oxidant solid phase cyclization using iodine, then peptide resin is cracked, is then recrystallized in acid condition, purifying,
Pramlintide is obtained after desalination, freeze-drying.
2. according to the method described in claim 1, it is characterized in that:
Wherein, the synthetic method of dipeptide fragment Fmoc-Pro-Pro-OH described in step 1 includes the following steps: Fmoc-Pro-OH
It is coupled under the action of DCC with HOSu and obtains Fmoc-Pro-OSu, then Fmoc-Pro-OSu and H-Pro-OH reacts to obtain two
Peptide fragment Fmoc-Pro-Pro-OH.
3. according to the method described in claim 1, it is characterized in that:
Wherein, solid phase synthesis process described in step 2, the described method comprises the following steps: 1) using the ammonia of 0.1-0.4mmol/g
Base resin is initial resin；2) it uses and is removed on amino resins by volume ratio for the deprotection liquid that the piperidines of 1:4 and DMF are formed
Fmoc protecting group；3) in the presence of coupling agent system, amino resins and Fmoc-Tyr (tBu)-OH are coupled to obtain Fmoc-Tyr
(tBu)-amino resins；4) repeat step 2), 3), successively be coupled according to pramlintide main chain peptide sequence have N-terminal Fmoc protect and
The amino acid of side chain protection, wherein 28,29 amino acid couplings are coupled using segment Fmoc-Pro-Pro-OH.
4. according to the method described in claim 1, it is characterized in that:
Wherein, step 3 is used as oxidant solid phase cyclization using iodine, and the molar ratio of iodine and peptide resin is (10-1): 1, reaction temperature
It is 15 DEG C -35 DEG C, the reaction time is 2-5 hours.
5. according to the method described in claim 1, it is characterized in that:
Wherein, step 3 recrystallizes in acid condition, and acid condition is selected from: the phosphate buffer solution of pH=2.0, pH=2.5
Phosphate buffer solution, the phosphate buffer solution of pH=5.0, the phosphate buffer solution of pH=5.8, the vinegar of pH=3.6
Acid-sodium-acetate buffer, the Acetic acid-sodium acetate buffer of pH=3.7, pH=3.8 Acetic acid-sodium acetate buffer, pH=4.5 vinegar
Acid-sodium-acetate buffer one kind.
6. according to the method described in claim 3, it is characterized in that:
In the dipeptides Fmoc-Pro-Pro-OH coupling process, H-Thr (tBu)-Asn (Trt)-Val-Gly-Ser (tBu)-
The molar ratio of Asn (Trt)-Thr (tBu)-Tyr (tBu)-amino resins, Fmoc-Pro-Pro-OH, DIC and HOBt are as follows: 1:3:
3:3-1:5:5:5, reaction temperature are 25-35 DEG C, and the reaction time is 2-3 hours.
7. according to the method described in claim 3, the amino resins is selected from Rink Amide AM Resin, Rink Amide
MBHA Resin resin；Wherein the substitution degree of the amino resins is 0.10-0.40mmol/g.
8. according to the method described in claim 7, the preferred Rink Amide MBHA Resin resin of the amino resins；Institute
The substitution degree for stating amino resins is preferably 0.15-0.35mmol/g.
9. according to the method described in claim 3, it is characterized in that: the coupling agent system includes condensing agent and reaction dissolvent, institute
It states condensing agent and is selected from DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA；The reaction dissolvent be selected from DMF, DCM,
NMP, DMSO or the combination between them.
10. according to the method described in claim 3, it is characterized in that: in the dipeptides Fmoc-Pro-Pro-OH coupling process, H-
Thr (tBu)-Asn (Trt)-Val-Gly-Ser (tBu)-Asn (Trt)-Thr (tBu)-Tyr (tBu)-amino resins, Fmoc-
The molar ratio of Pro-Pro-OH, DIC and HOBt are as follows: 1:3:3:3, reaction temperature are 25 DEG C, and the reaction time is 2 hours.
11. according to the method described in claim 1, it is characterized in that: wherein, step 3 is using iodine as oxidant solid phase cyclization, iodine
Molar ratio with peptide resin is 5:1；Reaction temperature is 25 DEG C；Reaction time is 3 hours.
12. according to the method described in claim 1, it is characterized in that: wherein, step 3 recrystallizes in acid condition, pH=2.0
Acid condition be phosphate buffer solution.
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| CN102977204A (en) * | 2012-11-14 | 2013-03-20 | 吉林省敖腾生物科技有限责任公司 | Method for synthesizing glucagon-like peptide (GLP)-1 analogue in solid-phase mode |
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| CN102977204A (en) * | 2012-11-14 | 2013-03-20 | 吉林省敖腾生物科技有限责任公司 | Method for synthesizing glucagon-like peptide (GLP)-1 analogue in solid-phase mode |
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