CN102850441B

CN102850441B - A kind of solid phase synthesis process of oxytocin

Info

Publication number: CN102850441B
Application number: CN201210256922.XA
Authority: CN
Inventors: 杨毅跃; 姚程成; 康国伟; 蒙相峰; 鲁尧
Original assignee: WUXI KAILI PHARMACEUTICAL CO Ltd
Current assignee: Yancheng Kaili Pharmaceutical Co ltd
Priority date: 2012-07-23
Filing date: 2012-07-23
Publication date: 2016-05-25
Anticipated expiration: 2032-07-23
Also published as: CN102850441A

Abstract

Does the solid phase synthesis process that the present invention proposes a kind of oxytocin, comprise the following steps: (I) is with AM? Resin and Rink? Amide? does is Linker raw material, preparation Rink? Amide? AM? Resin; The RinkAmide that (II) makes in step (I)? AM? Resin is upper contains Fmoc protecting group amino acid by solid-phase synthesis successively coupling, Deprotection, and cracking obtains linear oxytocin; (III) is oxidized above-mentioned linear oxytocin, purifying, and freeze drying obtains oxytocin. Synthesis technique of the present invention is simple, and cost is low, and yield is high, easily purifying, and environmental pollution is little, is suitable for suitability for industrialized production.

Description

A kind of solid phase synthesis process of oxytocin

Technical field

The present invention relates to oxytocin synthesis technical field, refer to especially a kind of solid phase synthesis process of oxytocin.

Background technology

Oxytocin, claims again oxytocins, English Oxytocin by name, and molecular formula is: C₄₃H₆₆N₁₂O₁₂S₂, pointSon amount is 1007.2, structural formula:

Oxytocin is peptide hormone uterine contractile medicine. There is following function: stimulate uterine smooth muscle,Cause its rhythmicity to be shunk, increase frequency and improve flesh tension force, also can be by acting on around mammary gland alveolusMusculoepithelia cell promotes galactosis, and this product is through liver, kidney metabolism, and through renal excretion, minute quantity is original shape thing.Clinical in induced labor for humans and animals, hasten parturition, postpartum and post-abortion because of uterine atony or shrink restore badUterine hemorrhage, mazischesis and the uterine involution causing is complete and contraction mammary gland alveolus myoepithelium is around thinBorn of the same parents, promote milk ejection, before and after point puerperium, in 24h, use.

The preparation method of current domestic oxytocin mainly contains liquid phase method and solid phase method. Wherein, patent CN200810084940.8 announce a kind of method of liquid phase synthetic oxytocin, prepare respectively 2 intermediatesBoc-Cys-Tyr-Ile-Gln and H-Asn-Cys-Pro-Leu-Gly-NH₂, then adopt fragment condensation reactionAfter 10 ~ 40h, after passing into air oxidation reaction and completing, remove the solvent in reaction system through vacuum drying,Obtain oxytocin. This technique produces more waste liquid, and the reaction time is long, and post processing is loaded down with trivial details. PatentCN200510112356.5 announces a kind of method of solid phase synthetic oxytocin, the method with RinkAmide orRinkMBHA resin is initiation material, taking TBTU/HOBt or HABU/HOBt as condensing agent, progressivelySynthetic; Then cutting, with air or with hydrogen peroxide (10^-3~10^-4Mol/L) solution is oxidized, the methodGeneration waste liquid is more, dimerization and the polymer of intermolecular reaction generation linearity or ring-type easily occurs, this methodShortcoming be that oligomerization between peptide molecule easily causes the serious reduction of cyclic peptide productive rate and purity.

Summary of the invention

The present invention is directed to the problem that prior art exists, a kind of high yield, low cost, environmental friendliness be provided,Be conducive to the oxytocin synthesis technique of large-scale production.

Oxytocin synthesis technique of the present invention is as follows:

A solid phase synthesis process for oxytocin, comprises the following steps:

(I) taking AMResin and RinkAmideLinker as raw material, preparation RinkAmideAMResin;

The RinkAmideAMResin that (II) makes in step (I) above contains by solid-phase synthesis successively couplingFmoc protecting group amino acid, Deprotection, cracking obtains linear oxytocin;

(III) is oxidized above-mentioned linear oxytocin, purifying, and freeze drying obtains pure oxytocin.

As preferred technical scheme, described step (I) is at N, N'-DIC (DIC)+1-Hydroxybenzotriazole (HOBT)+DMAP (DMAP) or N, N'-DIC(DIC) under+I-hydroxybenzotriazole (HOBT) effect, react, wherein AMResin substitution degree is0.4~1.0mmol/g。

As preferred technical scheme, the Fmoc-amino acid that contains in described step (II) is: Fmoc-Gly-OH,Fmoc-Leu-OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln (Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu)-OH and Boc-Cys (Trt)-OH; DescribedThe condensing agent that solid-phase synthesis uses is: I-hydroxybenzotriazole (HOBt)/N, N'-diisopropyl carbon two AsiasAmine (DIC).

As preferred technical scheme, above-mentioned steps (I), (II) or (III) reaction medium are anhydrous N, N-bis-NMF (DMF); De-Fmoc is 20% ~ 25% piperidines/carrene (DCM) or piperidines/N, N-Dimethyl formamide (DMF); De-Side chain protective group, tertbutyloxycarbonyl (Boc) and lytic reagent are:Trifluoroacetic acid (TFA)/tri isopropyl silane (TIS)/phenol (PhOH)/3-mercaptopropionic acid (MPA)/H₂O；Activation of amino acid, coupling, deprotection, oxidative cyclization temperature degree are 0 ~ 30 DEG C.

As preferred technical scheme, the washing agent of use is followed successively by DMF, MeOH, DCM, DMF.

As preferred technical scheme, the oxidant that described step (III) oxidation is used is air or H₂O₂, alkaliNertralizer is NH₃Water, acid neutralizing agent is HAC.

As preferred technical scheme, oxidant is 10 at reactant concentration^-310^-4Mol/L, oxidizing temperature is20~30℃。

Synthesis technique of the present invention is simple, and cost is low, and yield is high, easily purifying, and environmental pollution is little, is suitable forSuitability for industrialized production.

Detailed description of the invention

To the technical scheme in the embodiment of the present invention be specifically described below, obviously, described enforcementExample is only the present invention's part embodiment, instead of whole embodiment. Based on the embodiment in the present invention,Those of ordinary skill in the art are not making the every other embodiment obtaining under creative work prerequisite,All belong to the scope of protection of the invention.

The meaning of the abbreviation representative occurring in these claims and description is following table:

Fmoc	9-fluorenylmethyloxycarbonyl
		Boc	Tertbutyloxycarbonyl
Trt	Trityl
		HOBt	I-hydroxybenzotriazole
tBu	The tert-butyl group
		DMF	DMF
DCM	Carrene
		DIC	N, N'-DIC
TFA	Trifluoroacetic acid
		TIS	Tri isopropyl silane
MeOH	Methyl alcohol
		PhOH	Phenol
MPA	3-mercaptopropionic acid

Embodiment 1

A solid phase synthesis process for oxytocin, comprises the following steps:

Step (1) RinkAmideAMResin preparation

Take AMResin10g (100 orders, 0.49mmol/g), soak 30min with 50mLDCM, makeResin is fully swelling, successively with 50mLDMF 2 times (being no less than 1min) of washing at every turn. Take RinkAmideLinker:7.93g, HOBt:1.98g, DIC:2.24mL, join in 10mL dry DMF, 0 ~ 5DEG C activation 15min. Add solid phase composite tube at 30 DEG C of reaction 2.0 ~ 4.0h, reaction end is examined with ninhydrin methodSurvey is as the criterion, and with 50mL dry DMF washing 2 times, is no less than 1.0min, through inspection after reacting completely at every turnSurvey substitution degree, obtaining substitution degree is 0.48mmol/gRinkAmideAMResin.

Step (2) Boc-Cys (Trt)-Tyr (tBu)-Ile-Gln (Trt)-Asn (Trt)-Cys (Trt)-Por-Leu-Gly-RinkThe preparation of AmideAMResin

De-Fmoc protecting group

Use successively 70mL25% piperidines/DMF 30 DEG C of de-Fmoc protecting groups 2 times, for the first time 5min,15min for the second time, the middle 70mL dry DMF of using is washed once, is no less than 1min.

Washing:

Respectively wash 2,1,1,2 times with 70mL dry DMF, MeOH, DCM, DMF successively, everyThe inferior 1min that is no less than.

Fmoc-Gly-RinkAmideAMResin preparation:

Take Fmoc-Gly-OH4.19g, HOBt1.97g, it is anhydrous that DIC2.24mL joins 10mLDMF, at 0 ~ 5 DEG C of activation 15min. Add solid phase composite tube at 30 DEG C of reaction 1.5 ~ 3.0h, reaction endDetect and be as the criterion with ninhydrin method. With 70mL dry DMF washing 2 times, be no less than 1min successively at every turn,Obtain Fmoc-Gly-RinkAmideAMResin.

Repeat said process successively coupling Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Cys (Trt)-OH,Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Tyr(tBu)-OH、Boc-Cys (Trt)-OH. After coupling, with 70mLDMF washing 3 times, be no less than 1min successively at every turn,70mLMeOH washing 3 times is no less than 5min at every turn, drains to obtain Boc-Cys (Trt)-Tyr (tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Por-Leu-Gly-RinkAmideAMResin。

Step (3) cracking

Boc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Por-Leu-Gly-RinkAmideAMResin joins cracking reaction still, at 0 ~ 10 DEG C, adds while stirring 100mL crackingReagent (TFA/TIS/PhOH/MPA/H₂O=90/4/1.5/1.5/1.5), after reaction 0.5h, temperature rise to 25DEG C stir 2h. Remove by filter resin, concentrated filtrate rear point is joined to 1000mL absolute ether liquid and slowly addEnter to without in water-ice ether, leave standstill 2-5h, high speed centrifugation obtains thick peptide:

Cys-Tyr-Ile-Gln-Asn-Cys-Por-Leu-Gly-OH。

Step (4) oxidative cyclization

Cys-Tyr-Ile-Gln-Asn-Cys-Por-Leu-Gly-OH crude product is dissolved in purified water, is mixed withConcentration is 2g/L solution, and ammoniacal liquor regulates PH to 6.8 ~ 7.2, passes into air stirring, adopts HPLC to follow the tracks of anti-Answer process, after reaction finishes, filter, through the preparation of high pressure liquid phase, freeze drying obtains smart oxytocin, its purity>99.6%, single impurity<0.2%, total recovery reaches 33%.

Claims

1. a solid phase synthesis process for oxytocin, comprises the following steps:

(I) taking 0.49mmol/gAMResin and RinkAmideLinker as raw material, preparation RinkAmideAMResin;

The RinkAmideAMResin that (II) makes in step (I) above contains protecting group amino by solid-phase synthesis successively couplingAcid, Deprotection, cracking obtains linear oxytocin; The protecting group amino acid that contains in described step (II) is: Fmoc-Gly-OH,Fmoc-Leu-OH、Fmoc-Pro-OH、Fmoc-Cys(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH, Fmoc-Tyr (tBu)-OH and Boc-Cys (Trt)-OH; The condensing agent that described solid-phase synthesis uses is:HOBt/DIC；

(III) is oxidized above-mentioned linear oxytocin, purifying, and freeze drying obtains oxytocin; Reaction medium is anhydrous N, N-dimethylFormamide DMF; Piperidines/DMF that the reagent that de-Fmoc uses is 25%; Lytic reagent is TFA/TIS/PhOH/MPA/H2O=90/4/1.5/1.5/1.5; Activation of amino acid temperature is 0～5 DEG C, coupling, 30 DEG C of deprotections and uses air as oxidant.

2. the solid phase synthesis process of a kind of oxytocin according to claim 1, is characterized in that, the washing agent of use successivelyFor DMF, MeOH, DCM, DMF.