CN105384809A - Method for preparing teriparatide by fragment method and solid-liquid combination - Google Patents
Method for preparing teriparatide by fragment method and solid-liquid combination Download PDFInfo
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- CN105384809A CN105384809A CN201511024053.8A CN201511024053A CN105384809A CN 105384809 A CN105384809 A CN 105384809A CN 201511024053 A CN201511024053 A CN 201511024053A CN 105384809 A CN105384809 A CN 105384809A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
Abstract
The invention belongs to the field of polypeptide synthesis, relating to a method for preparing teriparatide by solid-liquid combination. The method adopts a liquid phase mode to synthesize a part of dipeptide and tripeptide fragments, feeding is performed by the synthesized dipeptide and tripetide fragments, 13 steps of solid phase coupled reaction are reduced, the coupling efficiency is greatly improved, not only is the generation of racemization impurities difficult to be purified and removed at the 34th site tail end avoided, but also the generation of missing peptides at multiple sites is avoided, and the purity of the final target peptide reaches 75 percent or more. Compared with the prior art, the method provided by the invention is simple in synthetic route, less in solid phase coupling steps, mainly solves the problems that a peptide sequence is too long and coupling is difficult, and that the missing peptides are easily generated, the fragment synthesis technology is well developed, the materiel cost is lowered, and the industrialized mass production can be carried out.
Description
Technical field
The present invention relates to Peptides Synthesis, particularly a kind of method preparing teriparatide with fragment method solid-liquid combination.
Background technology
Teriparatide, English name is Teriparatide, be made up of 34 natural amino acids, be that endogenous Rat parathyroid hormone 1-34 has bioactive N-stub area 1-34 amino acid fragment, its peptide sequence is: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-Hi s-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Le u-Gln-Asp-Val-His-Asn-Phe-OH.
Teriparatide is researched and developed by Li Lai company, in December, 2002 goes on the market in the U.S. first, be that the first obtains the bone shaping agent kind new medicine of U.S. food Drug Administration approval, be mainly used in treating primary osteoporosis, osteoporosis after women's postmenopausal osteoporosis and male gonad deterioration.The medicine for treating osteoporosis of current routine generally just slows down or blocks bone-loss, and osteoblast number can be increased as the teriparatide of parathyroid hormone derivative, strengthen its active and prevention TNF-a Induced Apoptosis in Osteoblasts, similar to numerous polypeptide drug, developed and next teriparatide by human endogenous's property material, toxic side effect is little, and seldom causes accumulate poisoning, and brand-new mechanism of action and significant curative effect determine teriparatide and have very high pharmaceutical use and wide market outlook.
At present, the preparation method of teriparatide, widely reports both at home and abroad.Li Lai company of Yuan Yan producer adopts the mode of genetic expression to obtain teriparatide, and in addition, still have the patent of several genes expression aspect, such as: US6590081, but gene expression method has technical threshold high, and work is complicated, the defects such as serious three wastes.
Another kind of comparatively common teriparatide preparation method is solid-state chemical reaction method method, as patent 201210213044.3 first makes five peptide resin fragments of teriparatide, then in the mode of fragment coupling by each peptide resin fragment progressively coupling teriparatide resins synthesis teriparatide peptide resin in solid phase, last cracking obtains teriparatide crude product, this mode with fragment coupling is synthesized, need carry out purifying and freeze-drying to fragment peptide, complicated operation, cost is higher; Patent 201510005427.5 takes traditional solid phase synthesis process, is held and progressively synthesizes teriparatide to N end, although easy and simple to handle, cannot solve the problem of long peptide later stage coupling difficulty, and ultimate aim peptide purity is not high, purification difficult by peptide chain C; Patent 201410262511.0 feeds intake in the mode of 16 ~ 17 employing pseudo proline dipeptides of peptide chain, avoids the generation of special process impurity, but cannot avoid because of long the produced multiple disappearance peptide of peptide chain; Teriparatide peptide chain is linked with resin by HMPLinker by patent 201510295556.2, and follow-up site adopts the mode of progressively coupling, and too impurity is various to there is target equally, the problem of purification difficult; Patent 201310403743.9 adopts the mode of progressively coupling to synthesize, the free hydroxyl group of 17 Ser is connected with ester bond with the α carboxyl of 16 Asn, teriparatide is obtained by a step ester amine conversion reaction in the solution after peptide chain end of synthesis, although the coupling difficulty in follow-up site can be reduced by changing target peptide sterie configuration, but still there is the many problems of solid phase coupling step number, also there is comparatively serious yield problem in the conversion of the ester of final step simultaneously amine, causes final cost to increase.
The polypeptide that teriparatide is made up of 34 amino acid, from the angle of solid phase synthesis, peptide chain is longer, along with the extension linked reaction of peptide chain can be comparatively difficult, easily produce more process contaminants and is unfavorable for later-period purification.The teriparatide solid phase synthesis process of current existence is mainly divided into two classes; one is coupling amino acid one by one; it two is synthesize full guard fragment by the mode of solid phase; target peptide is synthesized again by the mode of fragment coupling; first kind synthetic method cannot solve the lower problem of the target peptide purity that causes because peptide chain is longer, and Equations of The Second Kind synthetic method then increases Material Cost because of the issues of purification of full guard fragment.
Summary of the invention
For solving the above defect existed in teriparatide synthesis, the invention provides the novel method that a kind of fragment method solid-liquid combination prepares teriparatide.Concrete technical scheme is as follows:
Fragment method solid-liquid combination prepares a method for teriparatide, comprises the steps:
The required dipeptides of synthesis and tripeptide fragment: Fmoc-Asn (Trt)-Phe-OH under (a) liquid-phase condition, Fmoc-Asp (OtBu)-Val-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-Val-OH;
(b) with WangResin or CTC resin for solid phase carrier, under coupling agent exists, successively with Fmoc-Asn (Trt)-Phe-OH, Fmoc-His (Trt)-OH, Fmoc-Asp (OtBu)-Val-OH, Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Glu (Boc)-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-Asn (Trt)-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-Val-OH, obtain the teriparatide peptide resin of side chain protected, its structure is as follows:
Fmoc-Ser(tBu)-Val-Ser(tBu)-Glu(OtBu)-Ile-Gln(Trt)-Leu-Met-His(Trt)-Asn(Trt)-Leu-Gly-Lys(Boc)-His(Trt)-Leu-Asn(Trt)-Ser(tBu)-Met-Glu(OtBu)-Arg(pbf)-Val-Glu(OtBu)-Trp(Boc)-Leu-Arg(pbf)-Lys(Boc)-Lys(Boc)-Leu-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-Asn(Trt)-Phe-Resin；
C () teriparatide peptide resin is through cracking, purifying, freeze-drying obtains teriparatide essence peptide.
Wherein in step (a), the preparation method of Fmoc-Asn (Trt)-Phe-OH is: first Fmoc-Asn (Trt)-OH and HOSu is generated Fmoc-Asn (Trt)-OSu under DCC condition, then Fmoc-Asn (the Trt)-OSu and H-Phe-OH generated is generated Fmoc-Asn (Trt)-Phe-OH under alkali A condition;
The preparation method of Fmoc-Asp (OtBu)-Val-OH is: first Fmoc-Asp (OtBu)-OH and HOSu is generated Fmoc-Asp (OtBu)-OSu under DCC condition, then Fmoc-Asp (the OtBu)-OSu and H-Val-OH generated is generated Fmoc-Asp (OtBu)-Val-OH under alkali A condition;
The preparation method of Fmoc-Lys (Boc)-Leu-OH is: first Fmoc-Lys (Boc)-OH and HOSu is generated Fmoc-Lys (Boc)-OSu under DCC condition, then Fmoc-Lys (the Boc)-OSu and H-Leu-OH generated is generated Fmoc-Lys (Boc)-Leu-OH under alkali A condition;
The preparation method of Fmoc-Trp (Boc)-Leu-OH is: first Fmoc-Trp (Boc)-OH and HOSu is generated Fmoc-Trp (Boc)-OSu under DCC condition, then Fmoc-Trp (the Boc)-OSu and H-Leu-OH generated is generated Fmoc-Trp (Boc)-Leu-OH under alkali A condition;
The preparation method of Fmoc-Arg (pbf)-Val-OH is: first Fmoc-Arg (pbf)-OH and HOSu is generated Fmoc-Arg (pbf)-OSu under DCC condition, then Fmoc-Arg (the pbf)-OSu and H-Val-OH generated is generated Fmoc-Arg (pbf)-Val-OH under alkali A condition;
The preparation method of Fmoc-Ser (tBu)-Met-OH is: first Fmoc-Ser (tBu)-OH and HOSu is generated Fmoc-Ser (tBu)-OSu under DCC condition, then Fmoc-Ser (the tBu)-OSu and H-Met-OH generated is generated Fmoc-Ser (tBu)-Met-OH under alkali A condition;
The preparation method of Fmoc-His (Trt)-Leu-OH is: first Fmoc-His (Trt)-OH and HOSu is generated Fmoc-His (Trt)-OSu under DCC condition, then Fmoc-His (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-His (Trt)-Leu-OH under alkali A condition;
The preparation method of Fmoc-Asn (Trt)-Leu-Gly-OH is: first Fmoc-Asn (Trt)-OH and HOSu is generated Fmoc-Asn (Trt)-OSu under DCC condition, then Fmoc-Asn (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-Asn (Trt)-Leu-OH under alkali A condition, Fmoc-Asn (Trt)-Leu-OH and HOSu generates Fmoc-Asn (Trt)-Leu-OSu under DCC condition, finally Fmoc-Asn (the Trt)-Leu-OSu and H-Gly-OH generated is generated Fmoc-Asn (Trt)-Leu-Gly-OH under alkali A condition,
The preparation method of Fmoc-Gln (Trt)-Leu-Met-OH is: first Fmoc-Gln (Trt)-OH and HOSu is generated Fmoc-Gln (Trt)-OSu under DCC condition, then Fmoc-Gln (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-Gln (Trt)-Leu-OH under alkali A condition, Fmoc-Gln (Trt)-Leu-OH and HOSu generates Fmoc-Gln (Trt)-Leu-OSu under DCC condition, finally Fmoc-Gln (the Trt)-Leu-OSu and H-Met-OH generated is generated Fmoc-Gln (Trt)-Leu-Met-OH under alkali A condition,
The preparation method of Fmoc-Glu (OtBu)-Ile-OH is: first Fmoc-Glu (OtBu)-OH and HOSu is generated Fmoc-Glu (OtBu)-OSu under DCC condition, then Fmoc-Glu (the OtBu)-OSu and H-Ile-OH generated is generated Fmoc-Glu (OtBu)-Ile-OH under alkali A condition;
The preparation method of Fmoc-Ser (tBu)-Val-OH is: first Fmoc-Ser (tBu)-OH and HOSu is generated Fmoc-Ser (tBu)-OSu under DCC condition, then Fmoc-Ser (the tBu)-OSu and H-Val-OH generated is generated Fmoc-Ser (tBu)-Val-OH under alkali A condition.
Preferably, in step (a), dipeptide fragment preparation method is, for Fmoc-Asn (Trt)-Phe-OH: H-Phe-OH and alkali A is dissolved in water according to the ratio of mol ratio 1:1 ~ 2, add the organic solvent B hydrotropy of 5 ~ 20% volumes, after dissolving completely, the organic solvent B solution of Fmoc-Asn (Trt)-OSu is added drop-wise in the solution of H-Phe-OH under stirring by ice bath, and wherein the molar weight of H-Phe-OH is 1.5 times of Fmoc-Asn (Trt)-OSu.TLC monitors reaction end, and question response terminates rear underpressure distillation removing organic solvent B, then with the citric acid/water of 10%, reacting liquid pH value is adjusted to 2 ~ 3, extraction into ethyl acetate, and crystallization obtains Fmoc-Asn (Trt)-Phe-OH.
In above-mentioned steps (a), the preparation method of Fmoc-Asn (Trt)-OSu is: be dissolved in organic solvent B by Fmoc-Asn (Trt)-OH and HOSu according to the amount ratio of 1:1.0-1.2, under ice-water bath, DCC/ organic solvent B (gauge with Fmoc-Asn (Trt)-OH) solution of 1.0-1.2 times of amount is added drop-wise in Fmoc-Asn (Trt)-OH/HOSu solution, dropwise in 1h, remove ice bath, 25 DEG C are continued stirring reaction 2h.Reacted rear suction filtration, filtrate concentrates, the organic solvent of pressure reducing and steaming 80%, and residue oily matter adds sherwood oil, till upper solution no longer bleaches, separates out white solid, suction filtration, dry, obtains Fmoc-Asn (Trt)-OSu.
In above step, alkali A is the one in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA.
Organic solvent B is one or more in tetrahydrofuran (THF), dioxane, DMF, acetone, METHYLPYRROLIDONE, acetonitrile.
Preferably, tripeptide fragment preparation method described in step (a) is, for Fmoc-Asn (Trt)-Leu-Gly-OH, H-Gly-OH and alkali A is dissolved in water according to the ratio of mol ratio 1:1 ~ 2, adds the organic solvent B hydrotropy of 5 ~ 20% volumes; After dissolving completely, the organic solvent B solution of Fmoc-Asn (Trt)-Leu-OSu is added dropwise in H-Gly-OH solution under stirring by ice bath, and wherein, the molar weight of H-Gly-OH is 1.5 times of Fmoc-Asn (Trt)-Leu-Osu molar weight.TLC monitors reaction end, and after question response terminates underpressure distillation removing organic solvent B, then with the citric acid/water of 10%, reacting liquid pH value is adjusted to 2 ~ 3, extraction into ethyl acetate, crystallization obtains Fmoc-Asn (Trt)-Leu-Gly-OH.
The preparation method of Fmoc-Asn (Trt)-Leu-OSu is: be dissolved in organic solvent B by Fmoc-Asn (Trt)-Leu-OH and HOSu according to the amount ratio of 1:1.0-1.2, under ice-water bath, DCC/ organic solvent B (gauge with Fmoc-Asn (Trt)-OH) solution of 1.0-1.2 times of amount is added drop-wise in Fmoc-Asn (Trt)-Leu-OH/HOSu solution, dropwise in 1h, remove ice bath, 25 DEG C are continued stirring reaction 2h.Reacted rear suction filtration, filtrate concentrates, the organic solvent of pressure reducing and steaming 60%, and residue oily matter adds sherwood oil, till upper solution no longer bleaches, separates out white solid, suction filtration, dry, obtains Fmoc-Asn (Trt)-Leu-OSu.
The preparation method of Fmoc-Asn (Trt)-Leu-OH is: be dissolved in water by H-Leu-OH and alkali A according to the ratio of mol ratio 1:1 ~ 2, adds the organic solvent B hydrotropy of 5 ~ 20% volumes.After dissolving completely, the organic solvent B solution of Fmoc-Asn (Trt)-OSu is added dropwise to in H-Leu-OH solution under stirring by ice bath, and wherein, the molar weight of H-Leu-OH is 1.5 times of Fmoc-Asn (Trt)-OSu molar weight.TLC monitors reaction end, and question response terminates, and after reduction vaporization removes organic solvent B, then with the citric acid/water of 10%, reacting liquid pH value is adjusted to 2 ~ 3, extraction into ethyl acetate, and crystallization obtains Fmoc-Asn (Trt)-Leu-OH.
The preparation method of Fmoc-Asn (Trt)-OSu is: be dissolved in organic solvent B by Fmoc-Asn (Trt)-OH and HOSu according to the amount ratio of 1:1.0-1.2, under ice-water bath, DCC/ organic solvent B (gauge with Fmoc-Asn (Trt)-OH) solution of 1.0-1.2 times of amount is added drop-wise in Fmoc-Asn (Trt)-OH/HOSu solution, dropwise in 1h, remove ice bath, 25 DEG C are continued stirring reaction 2h.Reacted rear suction filtration, filtrate concentrates, the organic solvent of pressure reducing and steaming 80%, and residue oily matter adds sherwood oil, till upper solution no longer bleaches, separates out white solid, suction filtration, dry, obtains Fmoc-Asn (Trt)-OSu.
In above step, alkali A is the one in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA;
Organic solvent B is one or more in tetrahydrofuran (THF), dioxane, DMF, acetone, METHYLPYRROLIDONE, acetonitrile.
Wherein in step (b), to feed intake coupling 33 ~ 34 with Fmoc-Asn (Trt)-Phe-OH, to feed intake coupling 30 ~ 31 with Fmoc-Asp (OtBu)-Val-OH, to feed intake coupling 27 ~ 28 with Fmoc-Lys (Boc)-Leu-OH, to feed intake coupling 23 ~ 24 with Fmoc-Trp (Boc)-Leu-OH, to feed intake coupling 20 ~ 21 with Fmoc-Arg (pbf)-Val-OH, to feed intake coupling 17 ~ 18 with Fmoc-Ser (tBu)-Met-OH, to feed intake coupling 14 ~ 15 with Fmoc-His (Trt)-Leu-OH, to feed intake coupling 10 ~ 12 with Fmoc-Asn (Trt)-Leu-Gly-OH, to feed intake coupling 6 ~ 8 with Fmoc-Gln (Trt)-Leu-Met-OH, to feed intake coupling 4 ~ 5 with Fmoc-Glu (OtBu)-Ile-OH, to feed intake coupling 1 ~ 2 with Fmoc-Ser (tBu)-Val-OH.
Preferably, WangResin or the CTCResin substitution degree described in step (b) is 0.3 ~ 0.5mmol/g, and after resin transformation, substitution degree is 0.25 ~ 0.45mmol/g, preferred 0.35mmol/g;
In step (b), when single amino acids or dipeptide fragment and tripeptide fragment feed intake, molar feed ratio is 3 ~ 4 times amount (to synthesize the material gauge of scale), and connecing the reactive polypeptide time is 2 ~ 3h, and endpoint is as the criterion with Kaiser reagent detected artifacts; The coupling agent that linked reaction adopts is one or more in DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA.
Preferably, in step (c), lytic reagent is add the TFA solution that volume ratio is 1 ~ 5% scavenging agent, and wherein scavenging agent is one or more in methyl-phenoxide, thioanisole, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane.
Relative to prior art, the invention has the beneficial effects as follows:
1, use above-mentioned dipeptides and tripeptide fragment to feed intake, make 33,30,27,23,20,17,14,11,10,7,6,4, the solid phase coupling efficiency in 1 these site reaches absolutely, improve target peptide purity, thick peptide purity reaches 75%, avoids the formation of these site deletion peptides simultaneously, reduce the number of process contaminants, improve the efficiency of later-period purification, ultimate yield reaches 40%.
2, solid phase coupling step number foreshortens to 21 steps by 34 steps, greatly reduces the consumption of solvent in solid phase synthesis, reduces Material Cost, decrease three waste discharge.
3, Phe is chosen, Val, Leu, these amino acid not containing functional side chain group of Gly, Met, Ile carry out the synthesis of dipeptides or tripeptide fragment, in conjunction with ripe HOSu/DCC coupling method, greatly can reduce the cost of fragment liquid phase synthesis, can industrial mass production be applied to.
The abbreviation implication used in specification sheets and claims is as follows:
Fmoc9-fluorenylmethyloxycarbonyl
CTC resin 2-chlorine trityl chloride resin
WangResin king's resin
The tBu tertiary butyl
Trt trityl
DCM methylene dichloride
DMFN, dinethylformamide
DMAP4-Dimethylamino pyridine
DIEAN, N-diisopropylethylamine
DICN, N-DIC
HBTU benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate
HATU2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester
TBTUO-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid
HOBT1-hydroxybenzotriazole
HOAT1-hydroxyl-7-azo benzotriazole
TFA trifluoroacetic acid
HOSuN-N-Hydroxysuccinimide
EA ethyl acetate
THF tetrahydrofuran (THF)
PE sherwood oil
Embodiment
The present invention is described in detail to use specific embodiment below, but do not limit this patent; According to the feed ratio of feed change of the present invention, or reaction solvent or and condensing agent etc., all in protection scope of the present invention.
The preparation of embodiment 1:Fmoc-Asn (Trt)-OSu
Accurately take Fmoc-Asn (Trt)-OH1193.4g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Asn (Trt)-OSu1305.6g, yield 94.2%.
The preparation of embodiment 2:Fmoc-Asn (Trt)-Phe-OH
Accurately take H-Phe-OH446.1g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Asn (Trt)-OSu (1248.7g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolves steaming and pumps ethyl acetate, and residue oily matter adds 2000ml methyl alcohol, solution clear, be placed in-20 DEG C of refrigerator hold over night, separate out a large amount of white solid, suction filtration, dry Fmoc-Asn (Trt)-Phe-OH1041.2g, yield 77.3%.
The preparation of embodiment 3:Fmoc-Asp (OtBu)-OSu
Accurately take Fmoc-Asp (OtBu)-OH822.2g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Asp (OtBu)-OSu961.7g, yield 95.3%.
The preparation of embodiment 4:Fmoc-Asp (OtBu)-Val-OH
Accurately take H-Val-OH315.9g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Asp (OtBu)-OSu (908.2g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), remaining aqueous solution suction filtration, the aqueous citric acid solution adding 10% under ice-water bath adjusts solution ph to 2 ~ 3, and 8000ml ethyl acetate divides three extractions, merges organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation, to 4000ml, adds sherwood oil 4000ml, solution clear, be placed in crystallization under 4-8 DEG C of condition, obtain Fmoc-Asp (OtBu)-Val-OH801.4g, yield 87.2%.
The preparation of embodiment 5:Fmoc-Lys (Boc)-OSu
Accurately take Fmoc-Lys (Boc)-OH937.2g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Lys (Boc)-OSu1036.8g, yield 92.3%.
The preparation of embodiment 6:Fmoc-Lys (Boc)-Leu-OH
Accurately take H-Leu-OH355.6g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Lys (Boc)-OSu (1010.3g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3, and 8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolves steaming and pumps ethyl acetate, newly add 2000ml tetrahydrofuran (THF) to redissolve, add 4000ml sherwood oil crystallization, obtain Fmoc-Lys (Boc)-Leu-OH871.2g, yield 83.2%.
The preparation of embodiment 7:Fmoc-Trp (Boc)-OSu
Accurately take Fmoc-Trp (Boc)-OH1053.2g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Trp (Boc)-OSu1135.3g, yield 91.6%.
The preparation of embodiment 8:Fmoc-Trp (Boc)-Leu-OH
Accurately take H-Leu-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Trp (Boc)-OSu (1115.5g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation removing ethyl acetate, add 2500ml methyl alcohol to redissolve, solution is clarified, and is placed in-20 DEG C of crystallizatioies, obtain Fmoc-Trp (Boc)-Leu-OH905.1g, yield 78.6%.
The preparation of embodiment 9:Fmoc-Arg (pbf)-OSu
Accurately take Fmoc-Arg (pbf)-OH1297.4g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Arg (pbf)-OSu1181.3g, yield 92.6%.
The preparation of embodiment 10:Fmoc-Arg (pbf)-Val-OH
Accurately take H-Val-OH316.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Arg (pbf)-OSu (1342.5g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,10000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolves and steams removing ethyl acetate, and residue oily matter adds 3000ml methyl alcohol and redissolves, be heated to 40 DEG C of solution clears, be placed in-20 DEG C of crystallizatioies, obtain Fmoc-Arg (pbf)-Val-OH1086.4g, yield 80.7%.
The preparation of embodiment 11:Fmoc-Ser (tBu)-OSu
Accurately take Fmoc-Ser (tBu)-OH766.9g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Ser (tBu)-OSu894.7g, yield 93.1%.
The preparation of embodiment 12:Fmoc-Ser (tBu)-Met-OH
Accurately take H-Met-OH402.8g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Ser (tBu)-OSu (864.9g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3, and 8000ml ethyl acetate divides three extractions, merges organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation, to 3000ml, adds sherwood oil 4000ml crystallization, obtain Fmoc-Ser (tBu)-Met-OH765.15g, yield 82.6%.
The preparation of embodiment 13:Fmoc-His (Trt)-OSu
Accurately take Fmoc-His (Trt)-OH1239.4g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-His (Trt)-OSu1316.2g, yield 91.8%.
The preparation of embodiment 14:Fmoc-His (Trt)-Leu-OH
Accurately take H-Leu-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-His (Trt)-OSu (1290.2g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolve and steam removing ethyl acetate, remaining solid and oily matter add 3000ml methyl alcohol and redissolve, and are heated to 40 DEG C of solution clears, are placed in-20 degrees Celsius of crystallizatioies, obtain Fmoc-His (Trt)-Leu-OH765.15g, yield 82.6%.
The preparation of embodiment 15:Fmoc-Asn (Trt)-OSu
Accurately take Fmoc-Asn (Trt)-OH1193.4g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Asn (Trt)-OSu1279.3g, yield 92.2%.
The preparation of embodiment 16:Fmoc-Asn (Trt)-Leu-OH
Accurately take H-Leu-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Asn (Trt)-OSu (1248.7g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolve and steam removing ethyl acetate, residue oily matter adds 2000ml methyl alcohol and redissolves, and solution clear, is placed in-20 DEG C of refrigerator crystallizatioies, obtain Fmoc-Asn (Trt)-Leu-OH1100.2g, yield 86.1%.
The preparation of embodiment 17:Fmoc-Asn (Trt)-Leu-OSu
Accurately take Fmoc-Asn (Trt)-Leu-OH1063.9g (1.5mol) and HOSu172.5g (1.5mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC309.6g (1.5mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 3000 ~ 3500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Asn (Trt)-Leu-OSu1079.6g, yield 89.2%.
The preparation of embodiment 18:Fmoc-Asn (Trt)-Leu-Gly-OH
Accurately take H-Gly-OH135.1g (1.8mol) and sodium carbonate 228.9g (2.16mol) is dissolved in 2000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Asn (Trt)-Leu-OSu (968.28g, tetrahydrofuran solution 3000ml 1.2mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, revolve and steam removing ethyl acetate, add 3500ml methyl alcohol, be heated to 40 DEG C of solution clears, be placed in-20 DEG C of crystallizatioies, obtain Fmoc-Asn (Trt)-Leu-Gly-OH736.2g, yield 80.0%.
The preparation of embodiment 19:Fmoc-Gln (Trt)-OSu
Accurately take Fmoc-Gln (Trt)-OH1221.4g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Gln (Trt)-OSu1320.5g, yield 93.3%.
The preparation of embodiment 20:Fmoc-Gln (Trt)-Leu-OH
Accurately take H-Leu-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Gln (Trt)-OSu (1273.8g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, merge organic phase, revolve and steam removing ethyl acetate, residue oily matter adds 2000ml methyl alcohol and redissolves, and is heated to 40 DEG C of solution clears, is placed in-20 DEG C of refrigerator crystallizatioies, obtain Fmoc-Gln (Trt)-Leu-OH1150.5g, yield 88.3%.
The preparation of embodiment 21:Fmoc-Gln (Trt)-Leu-OSu
Accurately take Fmoc-Gln (Trt)-Leu-OH1085.8g (1.5mol) and HOSu172.5g (1.5mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC309.6g (1.5mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 3000 ~ 3500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Gln (Trt)-Leu-OSu1054.3g, yield 85.6%.
The preparation of embodiment 22:Fmoc-Gln (Trt)-Leu-Met-OH
Accurately take H-Met-OH268.6g (1.8mol) and sodium carbonate 228.9g (2.16mol) is dissolved in 2000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Gln (Trt)-Leu-OSu (985.1g, tetrahydrofuran solution 3000ml 1.2mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3,8000ml ethyl acetate divides three extractions, merge organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation removing ethyl acetate, add methyl alcohol 3500ml to redissolve, be heated to 40 DEG C of solution clears, be placed in-20 DEG C of crystallizatioies, obtain Fmoc-Gln (Trt)-Leu-Met-OH859.8g, yield 83.8%.
The preparation of embodiment 23:Fmoc-Glu (OtBu)-OSu
Accurately take Fmoc-Glu (OtBu)-OH850.9g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Glu (OtBu)-OSu977.2g, yield 93.5%.
The preparation of embodiment 24:Fmoc-Glu (OtBu)-Ile-OH
Accurately take H-Ile-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Glu (OtBu)-OSu (940.6g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3, and 8000ml ethyl acetate divides three extractions, merges organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation, to 4000ml, adds sherwood oil 4000ml crystallization, obtain Fmoc-Glu (OtBu)-Ile-OH841.6g, yield 86.8%.
The preparation of embodiment 25:Fmoc-Ser (tBu)-OSu
Accurately take Fmoc-Ser (tBu)-OH766.9g (2mol) and HOSu230g (2mol) is dissolved in 4000ml tetrahydrofuran (THF), ice-water bath stirs.Accurately take DCC412.8g (2mol), be dissolved in 2400ml tetrahydrofuran (THF), be slowly added drop-wise in above-mentioned solution, after ice bath stirs 1h, 25 degree are continued reaction 2h.React rear suction filtration, filtrate concentrated by rotary evaporation to 2000 ~ 2500ml.Concentrated rear suction filtration, filtrate adds 8000ml sherwood oil, separates out a large amount of white solid, and solution-20 is spent refrigerator and left standstill 1h.Suction filtration after leaving standstill, filter cake, with 2000ml acetic acid ethyl dissolution, adds 6000ml sherwood oil, and solution is clarified.Be placed in-20 degree refrigerators, separate out a large amount of white solid after 2h, suction filtration, filtration cakes torrefaction, weigh, obtain Fmoc-Ser (tBu)-OSu862.8g, yield 89.7%.
The preparation of embodiment 26:Fmoc-Ser (tBu)-Val-OH
Accurately take H-Val-OH315.9g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water, under ice bath, (2 ~ 8 DEG C) slowly add Fmoc-Ser (tBu)-OSu (864.9g, tetrahydrofuran solution 3000ml 1.8mol), stirring reaction, TLC monitors reaction end.After reacting completely, vacuum rotary steam removing tetrahydrofuran (THF), the aqueous citric acid solution adding 10% after remaining aqueous solution suction filtration under ice-water bath adjusts solution ph to 2 ~ 3, and 8000ml ethyl acetate divides three extractions, merges organic phase, with three washings of 3000ml saturated common salt moisture, anhydrous sodium sulfate drying, concentrated by rotary evaporation, to 4000ml, adds sherwood oil 4000ml crystallization, obtain Fmoc-Ser (tBu)-Val-OH743.5g, yield 85.6%.
The synthesis of embodiment 27:Fmoc-Asn (Trt)-Phe-WangResin
Take 30.0g (Sub=0.47mmol/g) WangResin and be placed in solid phase reactor, with DMF washing resin twice, 200ml/ time, rejoin DCM200ml swellable resins 30min.While resin swelling, take Fmoc-Asn (Trt)-Phe-OH31.43g (42.3mmol, 3eq), HOBt6.28g (46.5mmol, 3.3eq), the mixing solutions of DMF and DCM being 1:1 with 150ml volume ratio dissolves, ice bath 10min, add DIC7.25ml (46.5mmol, 3eq) and activate 5min.After resin swelling and activation of amino acids complete, feed intake reaction, and nitrogen gas stirring adds DMAP0.51g (4.23mmol, 0.3eq) after 10min, continues reaction 5h.After 5h, take out reaction solution, DMF washing resin 4 times, 200ml/ is each; DCM washing resin 2 times, 200ml/ time.Add capping reagent 180mL (50ml diacetyl oxide and 42.5ml pyridinium dissolution are in 87.5mLDMF) and unreacted acetylating hydroxyl groups 2h will be remained.Reacted rear suction filtration reaction solution, respectively with DMF, DCM, methanol wash resin 3 times, 200ml/ time, obtain Fmoc-Asn (Trt)-Phe-WangResin38.6g after vacuum-drying, after transformation, substitution degree is 0.30mmol/g.
The synthesis of embodiment 28:Fmoc-Asn (Trt)-Phe-CTCResin
Take CTC resin 30.0g (sub=0.40mmol/g) and be placed in synthesis post, wash twice with 200mLDMF, add the swelling 30min of 200mLDCM; After pumping DCM, add the DCM/DMF (3/1, volume ratio) solution 100ml, the N that are dissolved with 8.93g (12mmol) Fmoc-Asn (Trt)-Phe-OH
2add DIPEA4.0ml (24mmol) after stirring 5min, continue drum N
2stirring reaction 60min.Take out reaction solution after having reacted, add DCM/CH
3oH/DIPEA (volume ratio 17:2:1) mixing solutions end-blocking 3 times, each 200ml, reaction 10min; Then wash 3 times respectively with DMF, DCM, methyl alcohol, vacuum-drying obtains Fmoc-Asn (Trt)-Phe-CTCResin39.2g, and surveying the rear substitution degree of transformation is 0.26mmol/g.
Embodiment 29: the preparation of teriparatide peptide resin
Accurately take Fmoc-Asn (the Trt)-Phe-WangResin33.33g (synthesis scale 10mmol) that substitution degree is 0.30mmol/g and be placed in solid phase reactor, DMF washing resin 2 times, 150ml/ time, add the swelling 30min of 150mlDCM; Piperidines/DMF with 20% after resin swelling takes off Fmoc2 time, 150ml/ time, reacts 5min and 10min respectively; Wash 4 times with DMF after deprotection completes, DCM washs 2 times, 150ml/ time.Get sample to detect with Kaiser reagent, resin is mazarine.
Take Fmoc-His (Trt)-OH18.57g (30mmol, 3eq), HOBt4.05g (30mmol, 3eq), with the DMF that 120ml volume ratio is 1:1, DCM solubilize, ice-water bath 10min, add DIC4.68ml (30mmol, 3eq) and activate 5min.Feed intake after having activated reaction, nitrogen gas stirring 2h, and reaction end is as the criterion with Kaiser reagent detected result, and after detection reaction completes, suction filtration removing reaction solution, wash 4 times with DMF, DCM washs 2 times, 200ml/ time.Take off Fmoc2 time with the piperidines/DMF of 20%, 150ml/ time, react 5min and 10min respectively; Wash 4 times with DMF after deprotection completes, DCM washs 2 times, 150ml/ time.Get sample to detect with Kaiser reagent, resin is mazarine, and 27 His couplings complete.
According to the coupling method of 32 His, according to teriparatide peptide sequence, connect amino acid and dipeptide fragment and tripeptide fragment: Fmoc-Asp (OtBu)-Val-OH successively, Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Glu (Boc)-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-Asn (Trt)-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-Val-OH.After peptide resin end of synthesis, wash 4 times successively with DMF, DCM washs 2 times, methanol wash 3 times, 200ml/ time, and resin 25 degree of vacuum-drying 8h, weigh to obtain 110.3g.
Embodiment 30: the preparation of teriparatide peptide resin
Accurately take Fmoc-Asn (the Trt)-Phe-CTCResin38.5g (synthesis scale 10mmol) that substitution degree is 0.26mmol/g and be placed in synthesis post, DMF washing resin 2 times, 150ml/ time, adds the swelling 30min of 150mlDCM; Piperidines/DMF with 20% after resin swelling takes off Fmoc2 time, 150ml/ time, reacts 5min and 10min respectively; Wash 4 times with DMF after deprotection completes, DCM washs 2 times, 150ml/ time.Get sample to detect with Kaiser reagent, resin is mazarine.
Take Fmoc-His (Trt)-OH18.57g (30mmol, 3eq), HOBt4.05g (30mmol, 3eq), with the DMF that 120ml volume ratio is 1:1, DCM solubilize, ice-water bath 10min, add DIC4.68ml (30mmol, 3eq) and activate 5min.Feed intake after having activated reaction, nitrogen gas stirring 2h, and reaction end is as the criterion with Kaiser reagent detected result, and after detection reaction completes, suction filtration removing reaction solution, wash 4 times with DMF, DCM washs 2 times, 200ml/ time.Take off Fmoc2 time with the piperidines/DMF of 20%, 150ml/ time, react 5min and 10min respectively; Wash 4 times with DMF after deprotection completes, DCM washs 2 times, 150ml/ time.Get sample to detect with Kaiser reagent, resin is mazarine, and 27 His couplings complete.
According to the coupling method of 32 His, according to teriparatide peptide sequence, connect amino acid and dipeptide fragment and tripeptide fragment: Fmoc-Asp (OtBu)-Val-OH successively, Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Glu (Boc)-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-Asn (Trt)-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-Val-OH.After peptide resin end of synthesis, wash 4 times successively with DMF, DCM washs 2 times, methanol wash 3 times, 200ml/ time, and resin 25 degree of vacuum-drying 8h, weigh to obtain 96.6g.
Embodiment 31: the preparation of the thick peptide of teriparatide
Teriparatide peptide resin 110g prepared by embodiment 29 is placed in 2000ml round-bottomed flask, ice bath precooling.Configuration lytic reagent 1100ml, its volume ratio is TFA:TIS: water=94:1:5, is placed in-20 refrigerator precooling 30min.Join in peptide resin according to the ratio of 1g resin 10ml by lytic reagent, ice-water bath stirs 2h, and 25 degree are stirred 2h, suction filtration after stopped reaction, resin washes twice with TFA, 100ml/ time, merging filtrate, slowly be poured onto in the ether of 13L precooling, stir, be placed in-20 refrigerators and leave standstill 1h.By centrifugal for ether sedimentation liquid, and with washed with diethylether six times, centrifugal gained solid drying, obtains 44.7g, yield 108.2%.Get thick peptide sample analysis, purity is 76.3%.
Embodiment 32: the preparation of the thick peptide of teriparatide
Teriparatide peptide resin 96g prepared by embodiment 30 is placed in 1000ml round-bottomed flask, ice bath precooling.Configuration lytic reagent 1000ml, its volume ratio is TFA:TIS: water=94:1:5, is placed in-20 refrigerator precooling 30min.Join in peptide resin according to the ratio of 1g resin 10ml by lytic reagent, ice-water bath stirs 2h, and 25 degree are stirred 2h, suction filtration after stopped reaction, resin washes twice with TFA, 100ml/ time, merging filtrate, slowly be poured onto in the ether of 12L precooling, stir, be placed in-20 refrigerators and leave standstill 1h.By centrifugal for ether sedimentation liquid, and with washed with diethylether six times, centrifugal gained solid drying, obtains 37.7g, yield 91.5%.Get thick peptide sample analysis, purity is 75.5%.
Embodiment 33: the purifying of the thick peptide of teriparatide
Take the thick peptide 10.0g of embodiment 31 gained, dissolve with the acetonitrile/water of 500ml5%, ultrasonic dissolution assisting, 0.45um membrane filtration.
Internal diameter is the C of 100mm
18preparative column, mobile phase A is 0.1%TFA/ water, and Mobile phase B is 0.1%TFA/ acetonitrile, and applied sample amount is 10g/ pin, flow velocity 300ml/min, determined wavelength 210nm.Gradient elution, with peak Posterior circle sample introduction before peak, obtains the smart peptide solution that middle control analysis purity is more than 98.5%; After turning salt with acetic acid/acetonitrile/aqueous systems, freeze-drying obtains teriparatide essence peptide 3.1g, purity more than 99.5%, total recovery 43.7%.
Embodiment 34: the purifying of the thick peptide of teriparatide
Take the thick peptide 10.0g of embodiment 32 gained, dissolve with the acetonitrile/water of 500ml5%, ultrasonic dissolution assisting, 0.45um membrane filtration.Internal diameter is the C of 100mm
18preparative column, mobile phase A is 0.1%TFA/ water, and Mobile phase B is 0.1%TFA/ acetonitrile, and applied sample amount is 10g/ pin, flow velocity 300ml/min, determined wavelength 210nm.Gradient elution, with peak Posterior circle sample introduction before peak, obtains the smart peptide solution that middle control analysis purity is more than 98.5%; After turning salt with acetic acid/acetonitrile/aqueous systems, freeze-drying obtains teriparatide essence peptide 2.9g, purity more than 99.5%, total recovery 36.6%.
Claims (8)
1. fragment method solid-liquid combination prepares a method for teriparatide, it is characterized in that, comprises the steps:
The required dipeptides of synthesis and tripeptide fragment: Fmoc-Asn (Trt)-Phe-OH under (a) liquid-phase condition, Fmoc-Asp (OtBu)-Val-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-Val-OH;
(b) with WangResin or CTC resin for solid phase carrier, under coupling agent exists, successively with Fmoc-Asn (Trt)-Phe-OH, Fmoc-His (Trt)-OH, Fmoc-Asp (OtBu)-Val-OH, Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-Trp (Boc)-Leu-OH, Fmoc-Glu (Boc)-OH, Fmoc-Arg (pbf)-Val-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ser (tBu)-Met-OH, Fmoc-Asn (Trt)-OH, Fmoc-His (Trt)-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asn (Trt)-Leu-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-Gln (Trt)-Leu-Met-OH, Fmoc-Glu (OtBu)-Ile-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ser (tBu)-Val-OH, obtain the teriparatide peptide resin of side chain protected, its structure is as follows:
Fmoc-Ser(tBu)-Val-Ser(tBu)-Glu(OtBu)-Ile-Gln(Trt)-Leu-Met-His(Trt)-Asn(Trt)-Leu-Gly-Lys(Boc)-His(Trt)-Leu-Asn(Trt)-Ser(tBu)-Met-Glu(OtBu)-Arg(pbf)-Val-Glu(OtBu)-Trp(Boc)-Leu-Arg(pbf)-Lys(Boc)-Lys(Boc)-Leu-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-Asn(Trt)-Phe-Resin；
C () teriparatide peptide resin is through cracking, purifying, freeze-drying obtains teriparatide essence peptide.
2. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 1, is characterized in that, in step (a),
The preparation method of described Fmoc-Asn (Trt)-Phe-OH is: first Fmoc-Asn (Trt)-OH and HOSu is generated Fmoc-Asn (Trt)-OSu under DCC condition, then Fmoc-Asn (the Trt)-OSu and H-Phe-OH generated is generated Fmoc-Asn (Trt)-Phe-OH under alkali A condition;
The preparation method of described Fmoc-Asp (OtBu)-Val-OH is: first Fmoc-Asp (OtBu)-OH and HOSu is generated Fmoc-Asp (OtBu)-OSu under DCC condition, then Fmoc-Asp (the OtBu)-OSu and H-Val-OH generated is generated Fmoc-Asp (OtBu)-Val-OH under alkali A condition;
The preparation method of described Fmoc-Lys (Boc)-Leu-OH is: first Fmoc-Lys (Boc)-OH and HOSu is generated Fmoc-Lys (Boc)-OSu under DCC condition, then Fmoc-Lys (the Boc)-OSu and H-Leu-OH generated is generated Fmoc-Lys (Boc)-Leu-OH under alkali A condition;
The preparation method of described Fmoc-Trp (Boc)-Leu-OH is: first Fmoc-Trp (Boc)-OH and HOSu is generated Fmoc-Trp (Boc)-OSu under DCC condition, then Fmoc-Trp (the Boc)-OSu and H-Leu-OH generated is generated Fmoc-Trp (Boc)-Leu-OH under alkali A condition;
The preparation method of described Fmoc-Arg (pbf)-Val-OH is: first Fmoc-Arg (pbf)-OH and HOSu is generated Fmoc-Arg (pbf)-OSu under DCC condition, then Fmoc-Arg (the pbf)-OSu and H-Val-OH generated is generated Fmoc-Arg (pbf)-Val-OH under alkali A condition;
The preparation method of described Fmoc-Ser (tBu)-Met-OH is: first Fmoc-Ser (tBu)-OH and HOSu is generated Fmoc-Ser (tBu)-OSu under DCC condition, then Fmoc-Ser (the tBu)-OSu and H-Met-OH generated is generated Fmoc-Ser (tBu)-Met-OH under alkali A condition;
The preparation method of described Fmoc-His (Trt)-Leu-OH is: first Fmoc-His (Trt)-OH and HOSu is generated Fmoc-His (Trt)-OSu under DCC condition, then Fmoc-His (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-His (Trt)-Leu-OH under alkali A condition;
The preparation method of described Fmoc-Asn (Trt)-Leu-Gly-OH is: first Fmoc-Asn (Trt)-OH and HOSu is generated Fmoc-Asn (Trt)-OSu under DCC condition, then Fmoc-Asn (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-Asn (Trt)-Leu-OH under alkali A condition, Fmoc-Asn (Trt)-Leu-OH and HOSu generates Fmoc-Asn (Trt)-Leu-OSu under DCC condition, finally Fmoc-Asn (the Trt)-Leu-OSu and H-Gly-OH generated is generated Fmoc-Asn (Trt)-Leu-Gly-OH under alkali A condition,
The preparation method of described Fmoc-Gln (Trt)-Leu-Met-OH is: first Fmoc-Gln (Trt)-OH and HOSu is generated Fmoc-Gln (Trt)-OSu under DCC condition, then Fmoc-Gln (the Trt)-OSu and H-Leu-OH generated is generated Fmoc-Gln (Trt)-Leu-OH under alkali A condition, Fmoc-Gln (Trt)-Leu-OH and HOSu generates Fmoc-Gln (Trt)-Leu-OSu under DCC condition, finally Fmoc-Gln (the Trt)-Leu-OSu and H-Met-OH generated is generated Fmoc-Gln (Trt)-Leu-Met-OH under alkali A condition,
The preparation method of described Fmoc-Glu (OtBu)-Ile-OH is: first Fmoc-Glu (OtBu)-OH and HOSu is generated Fmoc-Glu (OtBu)-OSu under DCC condition, then Fmoc-Glu (the OtBu)-OSu and H-Ile-OH generated is generated Fmoc-Glu (OtBu)-Ile-OH under alkali A condition;
The preparation method of described Fmoc-Ser (tBu)-Val-OH is: first Fmoc-Ser (tBu)-OH and HOSu is generated Fmoc-Ser (tBu)-OSu under DCC condition, then Fmoc-Ser (the tBu)-OSu and H-Val-OH generated is generated Fmoc-Ser (tBu)-Val-OH under alkali A condition.
3. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 2, it is characterized in that, in step (a), described alkali A is the one in sodium carbonate, sodium bicarbonate, saleratus, salt of wormwood, triethylamine, diethylamine, N-ethyl diisopropyl amine, DIPEA.
4. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 1, it is characterized in that, in step (b), to feed intake coupling 33 ~ 34 with Fmoc-Asn (Trt)-Phe-OH, to feed intake coupling 30 ~ 31 with Fmoc-Asp (OtBu)-Val-OH, to feed intake coupling 27 ~ 28 with Fmoc-Lys (Boc)-Leu-OH, to feed intake coupling 23 ~ 24 with Fmoc-Trp (Boc)-Leu-OH, to feed intake coupling 20 ~ 21 with Fmoc-Arg (pbf)-Val-OH, to feed intake coupling 17 ~ 18 with Fmoc-Ser (tBu)-Met-OH, to feed intake coupling 14 ~ 15 with Fmoc-His (Trt)-Leu-OH, to feed intake coupling 10 ~ 12 with Fmoc-Asn (Trt)-Leu-Gly-OH, to feed intake coupling 6 ~ 8 with Fmoc-Gln (Trt)-Leu-Met-OH, to feed intake coupling 4 ~ 5 with Fmoc-Glu (OtBu)-Ile-OH, to feed intake coupling 1 ~ 2 with Fmoc-Ser (tBu)-Val-OH.
5. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 1, it is characterized in that, in step (b), the initial substitution degree of described WangResin or CTCResin is 0.3 ~ 0.5mmol/g, and after transformation, resin substitution degree is 0.25 ~ 0.45mmol/g.
6. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 5, is characterized in that, after transformation, the preferred substitution degree of resin is 0.35mmol/g.
7. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 1, it is characterized in that, in step (b), described coupling agent is HOBt/DIC, HOAt/DIC, HOBt/HBTU/DIEA, HOAt/HATU/DIEA, one or more in PyBOP/DIEA, TBTU/DIEA.
8. a kind of method preparing teriparatide with fragment method solid-liquid combination according to claim 1, it is characterized in that, in step (c), described lytic reagent is the TFA solution adding volume ratio 1-5% scavenging agent, and described scavenging agent is one or more in methyl-phenoxide, thioanisole, dithioglycol, mercaptoethanol, phenol, water, tri isopropyl silane.
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| CN111057139A (en) * | 2018-10-17 | 2020-04-24 | 南京华威医药科技集团有限公司 | Novel process for preparing teriparatide |
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| CN111087462B (en) * | 2018-10-24 | 2024-03-26 | 南京华威医药科技集团有限公司 | Solid-phase synthesis method of abamectin |
| CN112940104A (en) * | 2019-12-10 | 2021-06-11 | 深圳翰宇药业股份有限公司 | Teriparatide impurity F |
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| CN113121673B (en) * | 2021-04-08 | 2022-03-29 | 润辉生物技术（威海）有限公司 | Method for preparing elcatonin by solid-liquid combination method |
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