CN102702327A - Solid-liquid phase synthesis method for alarelin acetate - Google Patents
Solid-liquid phase synthesis method for alarelin acetate Download PDFInfo
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- CN102702327A CN102702327A CN2012102042616A CN201210204261A CN102702327A CN 102702327 A CN102702327 A CN 102702327A CN 2012102042616 A CN2012102042616 A CN 2012102042616A CN 201210204261 A CN201210204261 A CN 201210204261A CN 102702327 A CN102702327 A CN 102702327A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to a solid-liquid phase synthesis method for alarelin acetate, and mainly solves the technical problems of low yield, high cost, severe reaction conditions and serious pollution existing in the conventional synthesis method. The solid-liquid phase synthesis method mainly comprises the synthesis steps of: (1) coupling each protective amino acid one by one by using a fluorenylmethoxycarbonyl solid phase synthesis method by taking proline-dichloro-trityl-chlorine resin as initial resin and synthesizing to obtain side chain fully protected peptide chain resin; (2) cutting the side chain fully protected peptide chain resin to obtain a fully protected peptide chain segment pGluP-9; (3) performing C-terminal ethylamination on the fully protected peptide chain segment pGluP-9 to obtain a fully protected segment of the alarelin acetate; and (4) cutting the fully protected segment of the alarelin acetate to remove a side chain protective group to obtain alarelin acetate rough product peptide. The solid-liquid phase synthesis method has the advantages of high large-scale production capacity, easy operation, stable process, low production cost and total yield of exceeding 40 percent.
Description
Technical field
The present invention relates to a kind of compound method of my Rayleigh, particularly a kind of solid-liquid phase synthesi of my Rayleigh.
Background technology
My Rayleigh, Chinese are my Rayleigh of acetic acid, have another name called Wy 18481; English name Alarelin Acetate, peptide sequence are pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHC2H5, molecular formula C56H78N16O12; Molecular weight is 1167.30, and structural formula is following:
These article are the nonapeptide analogue of artificial synthetic gonadotropin releasing hormone (GnRH), and the medication initial stage can stimulate hypophysis to discharge interstitialcellstimulating hormone (ICSH) (LH) and follitropin (FSH) causes the of short duration rising of ovary source steroid hormone; Repeated drug taking can suppress hypophysis and discharge this restraining effect of effect that LH and FSH make estradiol level decline in the blood reach the medicine removal ovary and can be used for treating hormone-dependent diseases such as endometriosis.
Summary of the invention
The purpose of this invention is to provide that a kind of high yield, low cost, reaction conditions are gentle, environmental pollution is little, helped realizing the compound method that the solid-liquid of my Rayleigh of industrialization combines.Solve mainly that the yield that existing compound method exists is low, cost is high, severe reaction conditions, heavy-polluted technical problem.
For realizing above-mentioned purpose, the present invention takes following technical scheme:
A kind of my solid-liquid of Rayleigh is combined to method, comprises the steps:
1) be that initial resin adopts Fmoc solid-phase synthesis each protection amino acid of coupling one by one, the synthetic peptide chain resin that obtains the side chain full guard with the H-Pro-2Cl-Trt-Cl resin;
2) the peptide chain resin to the side chain full guard cuts, and obtains the peptide chain fragment pGluP-9 of full guard;
3) the peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine, obtains the full guard fragment of my Rayleigh;
4) Side chain protective group is removed in the full guard fragment cutting of my Rayleigh, obtained my Rayleigh bullion peptide;
According to the present invention, connect each protection amino acid successively, obtain the peptide chain resin of side chain full guard, the method that removes the Fmoc-blocking group therebetween successively comprises the steps:
1) gets the H-Pro-2Cl-Trt-Cl resin and soak, make the abundant swelling of resin, drain with DCM; Make solvent with DMF, add Fmoc-Arg (pbf)-OH, NMM or DIEA successively, use a kind of mixture among HBTU/HOBT, TBTU/HOBT, the HATU/HOAT to be condensing agent; Reacted 0.5-2 hour, ninhydrin method detection reaction terminal point is drained; With DMF washing 3-5 time, drain, obtain Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin;
H-Pro-2Cl-Trt-Cl resin substitution value is: 0.3mmol/g ~ 1.2mmol/g, and the scheme of more optimizing is 0.6mmol/g;
The amount of Fmoc-Arg (pbf)-OH is 1-5 a times of resin molar weight, and the scheme of more optimizing is 2 times of resin molar weight.
2) in the Fmoc-Arg of step (1) (pbf)-Pro-2Cl-Trt-Cl resin, add the reagent of raising one's hat, reacted 10-30 minute, drain; With DMF washing 5-10 time, drain, make solvent with DMF, add amino acid, NMM or DIEA with Fmoc protection; And use a kind of mixture among HBTU/HOBT, TBTU/HOBT, the HATU/HOAT to be condensing agent, and reacting 0.5-2 hour, the detection reaction terminal point is drained; With DMF washing 3-5 time, drain, and then add deprotecting regent, deprotection reaction; Add amino acid again, so repeatedly,, use N until having connected last Pyrrolidonecarboxylic acid with Fmoc blocking group; Dinethylformamide washing 3-5 time is alternately washed 3 times with methyl alcohol and methylene dichloride again, adds the methyl alcohol shrinkage resin at last; Drain, vacuum-drying obtains pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin;
The proportioning of reagent of raising one's hat is PIP/DMF (v/v) solution of 10%-30%, and the scheme of more optimizing is 20% PIP/DMF (v/v) solution; The amino acid whose amount of Fmoc protection is 1-5 a times of resin molar weight, and the scheme of more optimizing is 2 times of resin molar weight; The amount of condensation reagent (a kind of mixture among HBTU/HOBT, TBTU/HOBT, the HATU/HOAT) is 1-5 a times of resin molar weight, and the scheme of more optimizing is that the amount of TBTU/HOBT is 2 times of resin molar weight; The amount of NMM or DIEA is 2-10 a times of resin molar weight, and the scheme of more optimizing is that DIEA is 4 times of resin molar weight; Said amino acid with Fmoc blocking group comprises successively:
Fmoc-Leu-OH，Fmoc-D-Ala-OH，Fmoc-Tyr(tbu)-OH，Fmoc-Ser(tbu)-OH，Fmoc-Trp(boc)-OH，Fmoc-His(Trt)-OH。
According to the present invention, the described peptide of cutting comprises the steps:
In pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin that above-mentioned steps (2) is obtained, add 1%-5%TFA/DCM (v/v) and cut peptide reagent, reacted 1-3 hour, suction filtration filters resin particle; Collect filtrating; Underpressure distillation removes and desolvates, and adds elutriation and goes out white solid, drains; Vacuum-drying, the peptide chain fragment pGluP-9 of acquisition full guard;
The scheme of more optimizing is cut peptide reagent for selecting 2%TFA/DCM (v/v) for use, reacts 2 hours.
According to the present invention, the method steps that described peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine is following:
The peptide chain fragment pGluP-9 of full guard being dissolved in the DMF solution, adding ethylamine hydrochloride, DIEA, is condensing agent with a kind of mixture among PyBOP/HOBT, DIC/HOBT, the HBTU/ HOBT; Stirring reaction 12-24 hour, the consumption of ethylamine hydrochloride was 1-5 a times of peptide chain fragment pGluP-9 mole number, and the consumption of condensing agent is 1-5 a times of peptide chain fragment pGluP-9 mole number; The consumption of DIPEA is 3-15 a times of peptide chain fragment pGluP-9 mole number, after reaction finishes, and the adding citric acid saturated solution; Separate out white solid; Drain, vacuum-drying obtains the full guard fragment of my Rayleigh;
The scheme of more optimizing is: the consumption of ethylamine hydrochloride is 1.5 times of peptide chain fragment pGluP-9 mole number; Condensation reagent is selected PyBOP/HOBT for use; Consumption is 1.5 times of peptide chain fragment pGluP-9 mole number, and the DIEA consumption is 6 times of peptide chain fragment pGluP-9 mole number, 18 hours reaction times.
According to the present invention, the method steps that Side chain protective group is removed in described full guard fragment cutting to my Rayleigh is following:
Remove add in the full guard fragment of my Rayleigh to protect reagent, going to protect the proportioning of reagent is TFA:H
2O:Tis=95:2:3 (v/v), or TFA: thioanisole: phenol: EDT:H
2O=87.5:5:2.5:2.5:2.5 (v/v).Reaction times is 1-3 hour, after reaction finishes, and the deposition that adds diethyl ether, centrifugal collecting precipitation, with ether washing 3-6 time, vacuum-drying obtains my Rayleigh bullion again;
The scheme of more optimizing is to select TFA:H for use
2O:Tis=95:2:3 (v/v) removes to protect reagent, reacts 2 hours.
Some abbreviations commonly used have following implication among the present invention
HBTU:O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate
HATU:O-(7-azo benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluorophosphate
TBTU:O-(benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluoro borate
PyBOP: (benzotriazole-1-oxygen)-tripyrrole alkane subbase phosphorus hexafluorophosphate
DIC: DIC
The HOBt:1-hydroxybenzotriazole
HOAt:1-hydroxyl-7-azo benzotriazole
DIEA: diisopropylethylamine
PIP: piperidines
The NMM:N-methylmorpholine
Fmoc: fluorenylmethyloxycarbonyl
Pbf:2,2,4,6,7-pentamethyl--2H-cumarone-5 alkylsulfonyl
Trt: trityl
Tbu: the tertiary butyl
Boc: tertbutyloxycarbonyl
PGLu: Pyrrolidonecarboxylic acid
His: Histidine
Trp: tryptophane
Ser: Serine
Tyr: tyrosine
The D-Ala:D-L-Ala
Leu: leucine
Arg: l-arginine
Pro: proline(Pro)
Piperide: piperidines
DMF:N, dinethylformamide
DCM: methylene dichloride
TFA: trifluoroacetic acid
EDT: dithioglycol
Tis: tri isopropyl silane
PGluP-9: sequence is the abbreviation of pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-OH nonapeptide.
Embodiment
Below will do further detailed description to the present invention, but the invention is not restricted to this specific examples with reference to instance.
Embodiment 1
1) preparation Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin
(Loading 0.6mmol/g 30mmol), soaked 30 minutes with 800ml DCM, made the abundant swelling of resin to take by weighing H-Pro-2Cl-Trt-Cl resin 50 grams; Drain, add Fmoc-Arg (pbf)-OH (MW:648.8,60mmol) 38.9g, TBTU (MW:321.1; 60mmol) 19.3g, HOBT (MW:135.1,60mmol) 8.1g, DIEA (MW:129.24; 120mmol) 20ml, DMF500ml reacted 1.5 hours, and triketohydrindene hydrate detects the resin water white transparency; Drain,, drain, obtain Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin with DMF washing 3 times.
2) preparation pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin
In the Fmoc-Arg of step (1) (pbf)-Pro-2Cl-Trt-Cl resin, add the 800ml reagent (20% PIP/DMF (v/v) solution) of raising one's hat, reacted 30 minutes, drain; With DMF washing 6 times, drain, make solvent with DMF, add amino acid with Fmoc protection, DIEA, TBTU/HOBT; Reacted 1.5 hours, the detection reaction terminal point is drained, with DMF washing 3-5 time; Drain, and then add deprotecting regent, deprotection reaction adds the amino acid with Fmoc blocking group again; So repeatedly, after having connected last Pyrrolidonecarboxylic acid, DMF washing 3 times is alternately washed 3 times with methyl alcohol and methylene dichloride again; Add the methyl alcohol shrinkage resin at last, drain, vacuum-drying obtains pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin 102g.
The amino acid whose amount that each step, condensation reaction added is respectively: Fmoc-Leu-OH (MW:353.4,60mmol) 21.2g, Fmoc-D-Ala-OH (MW:311.3,60mmol) 18.7g; Fmoc-Tyr (tbu)-OH (MW:459.5,60mmol) 27.6g, Fmoc-Ser (tbu)-OH (MW:383.4,60mmol) 23g; Fmoc-Trp (Boc)-OH (MW:526.6,60mmol) 31.6g, Fmoc-His (Trt)-OH (MW:619.7; 60mmol) 37.2g, pGlu-OH (MW:129.1,60mmol) 7.7g; The amount of the condensing agent that each step condensation reaction is adopted is: TBTU (MW:321.1,60mmol) 19.3g, HOBT (MW:135.1; 60mmol) 8.1g, the amount of the organic bases that each step condensation reaction is added is: DIEA (MW:129.24,120mmol) 20ml.
3) the peptide chain fragment pGluP-9 of preparation full guard
Get pGLu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (the pbf)-Pro-2Cl-Trt-Cl resin 102g in the step (2), place the round-bottomed flask of 2L, add 1000ml and cut peptide reagent; Proportioning is 2%TFA/DCM (v/v), and 25 ℃ of oscillatory reactions are 2 hours in the constant temperature shaking table, and suction filtration filters resin particle; Collect filtrating, underpressure distillation removes and desolvates, and adds elutriation and goes out white solid; Drain, vacuum-drying, the peptide chain fragment pGluP-9 that obtains full guard is total to 51.2g.
4) the peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine
With the peptide chain fragment pGluP-9 of full guard in the step (3) altogether 51.2g be dissolved in the 2L DMF solution, add ethylamine hydrochloride 3.67g, 1.5eq PyBOP 23.4g, 1.5eq HOBT 6.08g, the 6eq DIEA 30ml of 1.5eq, 25 ℃ of stirring reactions 18 hours; After reaction finishes; The adding citric acid saturated solution is separated out white solid, drains; Vacuum-drying, the full guard fragment that obtains my Rayleigh is total to 53.2g.
5) Side chain protective group is removed in my Rayleigh full guard fragment cutting and obtained my Rayleigh bullion
My Rayleigh full guard fragment in the step (4) is total to the round-bottomed flask that 53.2g places 2L, adds 1000ml and remove to protect reagent, proportioning is TFA:H
2O:Tis=95:2:3 (v/v), 25 ℃ of constant-temperature shaking were reacted 2 hours, after reaction finishes, the deposition that adds diethyl ether, centrifugal collecting precipitation, with ether washing 3-6 time, vacuum-drying obtains my Rayleigh bullion 31.2g again.Bullion yield 89.1%, bullion purity 75.8%.
Embodiment 2
1) preparation Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin
(Loading 0.45mmol/g 30mmol), soaked 30 minutes with 800ml DCM, made the abundant swelling of resin to take by weighing H-Pro-2Cl-Trt-Cl resin 67 grams; Drain, add Fmoc-Arg (pbf)-OH (MW:648.8,90mmol) 58.4g, HBTU (MW:379.2; 90mmol) 34.1g, HOBT (MW:135.1,90mmol) 12.2g, NMM (MW:102.1; 180mmol) 20ml, DMF500ml reacted 1 hour, and triketohydrindene hydrate detects the resin water white transparency; Drain,, drain, obtain Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin with DMF washing 3 times.
2) preparation pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin
In the Fmoc-Arg of step (1) (pbf)-Pro-2Cl-Trt-Cl resin, add the 800ml reagent (20% PIP/DMF (v/v) solution) of raising one's hat, reacted 30 minutes, drain; With DMF washing 6 times, drain, make solvent with DMF, add amino acid, NMM, HBTU/HOBT with Fmoc protection; Reacted 1 hour, the detection reaction terminal point is drained, with DMF washing 3-5 time; Drain, and then add deprotecting regent, deprotection reaction adds the amino acid with Fmoc blocking group again; So repeatedly, after having connected last Pyrrolidonecarboxylic acid, DMF washing 3 times is alternately washed 3 times with methyl alcohol and methylene dichloride again; Add the methyl alcohol shrinkage resin at last, drain, vacuum-drying obtains pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin 115g.
The amino acid whose amount that each step, condensation reaction added is respectively: Fmoc-Leu-OH (MW:353.4,90mmol) 31.8g, Fmoc-D-Ala-OH (MW:311.3,90mmol) 28.0g; Fmoc-Tyr (tbu)-OH (MW:459.5,90mmol) 41.4g, Fmoc-Ser (tbu)-OH (MW:383.4,90mmol) 34.5g; Fmoc-Trp (Boc)-OH (MW:526.6,60mmol) 47.4g, Fmoc-His (Trt)-OH (MW:619.7; 90mmol) 55.8g, pGlu-OH (MW:129.1,90mmol) 11.6g; The amount of the condensing agent that each step condensation reaction is adopted is: HBTU (MW:379.2,90mmol) 34.1g, HOBT (MW:135.1; 90mmol) 12.2g, the amount of the organic bases that each step condensation reaction is added is: NMM (MW:102.1,180mmol) 20ml.
3) the peptide chain fragment pGluP-9 of preparation full guard
Get pGLu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (the pbf)-Pro-2Cl-Trt-Cl resin 115g in the step (2), place the round-bottomed flask of 2L, add 1000ml and cut peptide reagent; Proportioning is 2%TFA/DCM (v/v), and 25 ℃ of oscillatory reactions are 2 hours in the constant temperature shaking table, and suction filtration filters resin particle; Collect filtrating, underpressure distillation removes and desolvates, and adds elutriation and goes out white solid; Drain, vacuum-drying, the peptide chain fragment pGluP-9 that obtains full guard is total to 47.2g.
4) the peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine
With the peptide chain fragment pGluP-9 of full guard in the step (3) altogether 49.6g be dissolved in the 2L DMF solution, add ethylamine hydrochloride 3.67g, 1.5eq PyBOP 23.4g, 1.5eq HOBT 6.08g, the 6eq DIEA 30ml of 1.5eq, 25 ℃ of stirring reactions 18 hours; After reaction finishes; The adding citric acid saturated solution is separated out white solid, drains; Vacuum-drying, the full guard fragment that obtains my Rayleigh is total to 48.5g.
5) Side chain protective group is removed in my Rayleigh full guard fragment cutting and obtained my Rayleigh bullion
My Rayleigh full guard fragment in the step (4) is total to the round-bottomed flask that 48g places 2L, adds 1000ml and remove to protect reagent, proportioning is TFA:H
2O:Tis=95:2:3 (v/v), 25 ℃ of constant-temperature shaking were reacted 2 hours, after reaction finishes, the deposition that adds diethyl ether, centrifugal collecting precipitation, with ether washing 3-6 time, vacuum-drying obtains my Rayleigh bullion 28.6g again.Bullion yield 81.7%, bullion purity 65.2%.
Embodiment 3
1) preparation Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin
(Loading 0.72mmol/g 30mmol), soaked 30 minutes with 800ml DCM, made the abundant swelling of resin to take by weighing H-Pro-2Cl-Trt-Cl resin 42 grams; Drain, add Fmoc-Arg (pbf)-OH (MW:648.8,60mmol) 38.9g, HATU (MW:380.23; 60mmol) 22.8g, HOAT (MW:136.1,60mmol) 8.2g, NMM (MW:102.1; 120mmol) 14ml, DMF500ml reacted 0.5 hour, and triketohydrindene hydrate detects the resin water white transparency; Drain,, drain, obtain Fmoc-Arg (pbf)-Pro-2Cl-Trt-Cl resin with DMF washing 3 times.
2) preparation pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin
In the Fmoc-Arg of step (1) (pbf)-Pro-2Cl-Trt-Cl resin, add the 800ml reagent (20% PIP/DMF (v/v) solution) of raising one's hat, reacted 30 minutes, drain; With DMF washing 6 times, drain, make solvent with DMF, add amino acid, NMM, HATU/HOAT with Fmoc protection; Reacted 0.5 hour, the detection reaction terminal point is drained, with DMF washing 3-5 time; Drain, and then add deprotecting regent, deprotection reaction adds the amino acid with Fmoc blocking group again; So repeatedly, after having connected last Pyrrolidonecarboxylic acid, DMF washing 3 times is alternately washed 3 times with methyl alcohol and methylene dichloride again; Add the methyl alcohol shrinkage resin at last, drain, vacuum-drying obtains pGlu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (pbf)-Pro-2Cl-Trt-Cl resin 92g.
The amino acid whose amount that each step, condensation reaction added is respectively: Fmoc-Leu-OH (MW:353.4,60mmol) 21.2g, Fmoc-D-Ala-OH (MW:311.3,60mmol) 18.7g; Fmoc-Tyr (tbu)-OH (MW:459.5,60mmol) 27.6g, Fmoc-Ser (tbu)-OH (MW:383.4,60mmol) 23g; Fmoc-Trp (Boc)-OH (MW:526.6,60mmol) 31.6g, Fmoc-His (Trt)-OH (MW:619.7; 60mmol) 37.2g, pGlu-OH (MW:129.1,60mmol) 7.7g; The amount of the condensing agent that each step condensation reaction is adopted is: HATU (MW:380.23,60mmol) 22.8g, HOAT (MW:136.1; 60mmol) 8.2g, the amount of the organic bases that each step condensation reaction is added is: NMM (MW:102.1,120mmol) 14ml.
3) the peptide chain fragment pGluP-9 of preparation full guard
Get pGLu-His (Trt)-Trp (Boc)-Ser (tbu)-Tyr (tbu)-D-Ala-Leu-Arg (the pbf)-Pro-2Cl-Trt-Cl resin 92g in the step (2), place the round-bottomed flask of 2L, add 1000ml and cut peptide reagent; Proportioning is 2%TFA/DCM (v/v), and 25 ℃ of oscillatory reactions are 2 hours in the constant temperature shaking table, and suction filtration filters resin particle; Collect filtrating, underpressure distillation removes and desolvates, and adds elutriation and goes out white solid; Drain, vacuum-drying, the peptide chain fragment pGluP-9 that obtains full guard is total to 49.3g.
4) the peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine
With the peptide chain fragment pGluP-9 of full guard in the step (3) altogether 49.3g be dissolved in the 2L DMF solution, add ethylamine hydrochloride 7.34g, 3eq DIC 11.4g, 3eq HOBT 12.2 g, the 12eq DIEA 60ml of 3eq, 25 ℃ of stirring reactions 18 hours; After reaction finishes; The adding citric acid saturated solution is separated out white solid, drains; Vacuum-drying, the full guard fragment that obtains my Rayleigh is total to 51.2g.
5) Side chain protective group is removed in my Rayleigh full guard fragment cutting and obtained my Rayleigh bullion
My Rayleigh full guard fragment in the step (4) is total to the round-bottomed flask that 51.2g places 2L, adds 1000ml and remove to protect reagent, proportioning is TFA: thioanisole: phenol: EDT:H
2O=87.5:5:2.5:2.5:2.5 (v/v), 25 ℃ of constant-temperature shaking were reacted 2.5 hours, after reaction finishes, the deposition that adds diethyl ether, centrifugal collecting precipitation, with ether washing 3-6 time, vacuum-drying obtains my Rayleigh bullion 29.8g again.Bullion yield 85.1%, bullion purity 72.3%.
Claims (10)
1. the solid-liquid phase synthesi of my Rayleigh is characterized in that, comprises the steps:
1) be that initial resin adopts fluorenylmethyloxycarbonyl solid-phase synthesis each protection amino acid of coupling one by one, the synthetic peptide chain resin that obtains the side chain full guard with proline(Pro)-2 chloro-trityl-chlorine resin;
2) the peptide chain resin to the side chain full guard cuts, and obtains the peptide chain fragment pGluP-9 of full guard;
3) the peptide chain fragment pGluP-9 to full guard carries out the C terminal ethylamine, obtains the full guard fragment of my Rayleigh;
4) Side chain protective group is removed in the full guard fragment cutting of my Rayleigh, obtained my Rayleigh bullion peptide.
2. method according to claim 1 is characterized in that the step 1) particular content comprises the steps:
1) get proline(Pro)-2 chloro-trityl-chlorine resin and soak with methylene dichloride, make the abundant swelling of resin, drain, with N, dinethylformamide is made solvent; Add fluorenylmethyloxycarbonyl (2,2,4,6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl) l-arginine, N-methylmorpholine or diisopropylethylamine; With O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole, O-(benzotriazole-1-oxygen)-N, N; N, N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole, O-(7-azo benzotriazole-1-oxygen)-N, N, N; A kind of mixture in N-tetramethyl-urea hexafluorophosphate/1-hydroxyl-7-azo benzotriazole is a condensing agent, reacts 0.5-2 hour, and ninhydrin method detection reaction terminal point is drained; Use N, dinethylformamide washing 3-5 time is drained, and obtains fluorenylmethyloxycarbonyl-(2; 2,4,6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl) Arg-Pro-2 chloro-trityl-chlorine resin;
2) in the resin that step (1) obtains, add the reagent of raising one's hat, reacted 10-30 minute, drain, use N; Dinethylformamide washing 5-10 time is drained, and with N, dinethylformamide is a solvent, adds amino acid, N-methylmorpholine or diisopropylethylamine with fluorenylmethyloxycarbonyl protection; And with O-benzotriazole-N, N, N, N-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole, O-(benzotriazole-1-oxygen)-N; N, N, N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole, O-(7-azo benzotriazole-1-oxygen)-N, N; N, a kind of mixture in N-tetramethyl-urea hexafluorophosphate/1-hydroxyl-7-azo benzotriazole is a condensing agent, reacts the detection reaction terminal point 0.5-2 hour; Drain, use N, dinethylformamide washing 3-5 time is drained; And then the adding deprotecting regent, deprotection reaction adds the amino acid with fluorenylmethyloxycarbonyl blocking group, so repeatedly again; Until having connected last Pyrrolidonecarboxylic acid, use N, dinethylformamide washing 3-5 time is alternately washed 3 times with methyl alcohol and methylene dichloride again; Add the methyl alcohol shrinkage resin at last, drain, vacuum-drying obtains Pyrrolidonecarboxylic acid-(trityl) Histidine-(tertbutyloxycarbonyl) tryptophane-(tert.-butoxy) Serine-(tert.-butoxy) tyrosine-D-L-Ala-leucine-(2; 2,4,6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl) Arg-Pro-2 chloro-trityl-chlorine resin; Said amino acid with fluorenylmethyloxycarbonyl blocking group; Comprise successively: the fluorenylmethyloxycarbonyl leucine; Fluorenylmethyloxycarbonyl-D-L-Ala, fluorenylmethyloxycarbonyl tert.-butoxy tyrosine, fluorenylmethyloxycarbonyl tert.-butoxy Serine; Fluorenylmethyloxycarbonyl tertbutyloxycarbonyl tryptophane, fluorenylmethyloxycarbonyl trityl Histidine.
3. method according to claim 2 is characterized in that, the substitution value of proline(Pro)-2 chloro-trityl-chlorine resin is between 0.3mmol/g-1.2mmol/g; Fluorenylmethyloxycarbonyl (2,2,4; 6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl) arginic amount be the resin molar weight 1-5 doubly; The reagent of raising one's hat is piperidines/N of volume ratio 10-30%; The dinethylformamide mixing solutions; The amino acid whose amount of each step protection be the resin molar weight 1-5 doubly; The amount of condensation reagent be the resin molar weight 1-5 doubly, the amount of N-methylmorpholine or diisopropylethylamine be the resin molar weight 2-10 doubly, each step reaction end detection method adopted is the triketohydrindene hydrate detection method.
4. method according to claim 3 is characterized in that, the substitution value of proline(Pro)-2 chloro-trityl-chlorine resin is 0.6mmol/g; Fluorenylmethyloxycarbonyl (2,2,4; 6,7-pentamethyl--2H-cumarone-5-alkylsulfonyl) arginic amount is 2 times of resin molar weight; The reagent of raising one's hat is the piperidines/N of volume ratio 20%, the dinethylformamide mixing solutions, the amino acid whose amount of each step protection be 2 times of resin molar weight; Condensation reagent is O-(benzotriazole-1-oxygen)-N; N, N, N-tetramethyl-urea hexafluoro borate/I-hydroxybenzotriazole; Its consumption is 2 times of resin molar weight, and the amount of diisopropylethylamine is 4 times of resin molar weight.
5. method according to claim 1 is characterized in that, said step 2) comprise the steps:
Peptide reagent is cut in peptide chain resin adding in the side chain full guard, reacts 1-3 hour, and suction filtration filters resin particle, collects filtrating, and underpressure distillation removes and desolvates, and adds elutriation and goes out white solid, drains vacuum-drying, the peptide chain fragment pGluP-9 of acquisition full guard.
6. method according to claim 5 is characterized in that the used peptide reagent of cutting is that volume ratio is the trifluoroacetic acid/dichloromethane mixing solutions of 1%-5%.
7. method according to claim 6 is characterized in that the used peptide reagent of cutting is that volume ratio is 2% trifluoroacetic acid/dichloromethane mixing solutions.
8. method according to claim 1 is characterized in that said step 3) comprises the steps:
The peptide chain fragment pGluP-9 of full guard is dissolved in N, in the dinethylformamide solution, adds ethylamine hydrochloride, diisopropylethylamine; With (benzotriazole-1-oxygen)-tripyrrole alkane subbase phosphorus hexafluorophosphate/I-hydroxybenzotriazole, DIC/I-hydroxybenzotriazole, O-benzotriazole-N, N, N; A kind of mixture is a condensing agent in N-tetramethyl-urea hexafluorophosphate/I-hydroxybenzotriazole, and stirring reaction 12-24 hour, the consumption of ethylamine hydrochloride was 1-5 a times of peptide chain fragment pGluP-9 mole number; The consumption of condensing agent is 1-5 a times of peptide chain fragment pGluP-9 mole number, and the consumption of diisopropylethylamine is 3-15 a times of peptide chain fragment pGluP-9 mole number, after reaction finishes; The adding citric acid saturated solution is separated out white solid, drains; Vacuum-drying obtains the full guard fragment of my Rayleigh.
9. method according to claim 8; It is characterized in that; Condensation reagent is selected (benzotriazole-1-oxygen)-tripyrrole alkane subbase phosphorus hexafluorophosphate/I-hydroxybenzotriazole for use, and stirring reaction 18 hours, the consumption of ethylamine hydrochloride are 1.5 times of peptide chain fragment pGluP-9 mole number; The consumption of condensing agent is 1.5 times of peptide chain fragment pGluP-9 mole number, and the consumption of diisopropylethylamine is 6 times of peptide chain fragment pGluP-9 mole number.
10. method according to claim 1 is characterized in that described step 4) comprises the steps:
Remove add in the full guard fragment of my Rayleigh to protect reagent, go to protect reagent to select the volume ratio trifluoroacetic acid for use: the mix reagent of water: tri isopropyl silane=95:2:3, or volume ratio is a trifluoroacetic acid: the mix reagent of thioanisole: phenol: dithioglycol: water=87.5:5:2.5:2.5:2.5; Reaction times is 1-3 hour, and after reaction finished, deposition added diethyl ether; Centrifugal collecting precipitation; With ether washing 3-6 time, vacuum-drying obtains my Rayleigh bullion again.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106800592A (en) * | 2017-01-20 | 2017-06-06 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279998A (en) * | 2008-05-13 | 2008-10-08 | 吉尔生化（上海）有限公司 | Preparation of C-terminal ethylamine polypeptides and derivates thereof |
| US20090131587A1 (en) * | 2007-11-15 | 2009-05-21 | Mallinckrodt Inc. | Solid Support for Fmoc-Solid Phase Synthesis of Peptides |
| CN101935339A (en) * | 2010-08-17 | 2011-01-05 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method for buserelin |
-
2012
- 2012-06-20 CN CN2012102042616A patent/CN102702327B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090131587A1 (en) * | 2007-11-15 | 2009-05-21 | Mallinckrodt Inc. | Solid Support for Fmoc-Solid Phase Synthesis of Peptides |
| CN101279998A (en) * | 2008-05-13 | 2008-10-08 | 吉尔生化（上海）有限公司 | Preparation of C-terminal ethylamine polypeptides and derivates thereof |
| CN101935339A (en) * | 2010-08-17 | 2011-01-05 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method for buserelin |
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| CN107987129B (en) * | 2017-12-25 | 2020-12-08 | 肽泽（武汉）生物科技有限公司 | Cell-penetrating peptide and preparation method and application thereof |
| CN107987129A (en) * | 2017-12-25 | 2018-05-04 | 肽泽（武汉）生物科技有限公司 | A kind of cell-penetrating peptide and preparation method thereof, application |
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| CN111269291A (en) * | 2020-03-20 | 2020-06-12 | 三峡大学 | Oligopeptide synthesis and application of oligopeptide in medicine for inhibiting citrus saprogenicia citrobacter fingerlike penicillium |
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Application publication date: 20121003 Assignee: Jill peptide biopharmaceutical (Dalian) Co.,Ltd. Assignor: SHANGHAI GL POLYPEPTIDE Co.,Ltd.|GL BIOCHEM (SHANGHAI) Ltd.|BINHAI GL POLYPEPTIDE Co.,Ltd. Contract record no.: X2020980009639 Denomination of invention: Solid liquid phase synthesis of Alarelin Granted publication date: 20131030 License type: Common License Record date: 20201221 |