CN105384809B - A kind of method that segment method solid-liquid combination prepares Teriparatide - Google Patents
A kind of method that segment method solid-liquid combination prepares Teriparatide Download PDFInfo
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- CN105384809B CN105384809B CN201511024053.8A CN201511024053A CN105384809B CN 105384809 B CN105384809 B CN 105384809B CN 201511024053 A CN201511024053 A CN 201511024053A CN 105384809 B CN105384809 B CN 105384809B
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- fmoc
- trt
- leu
- osu
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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 Peptides Synthesis, it is related to a kind of method that solid-liquid combination prepares Teriparatide, the present invention uses liquid phase mode composite part dipeptides and tripeptide fragment, it is fed intake again with synthesized dipeptides and tripeptide fragment, reduce 13 step solid phase reactions, coupling efficiency greatly improved, not only avoids the generation for 34 end racemization impurity for being difficult to purify removing, and the generation of multiple site deletion peptides is also avoided, final goal peptide purity reaches 75% or more.Compared with prior art, synthetic route of the present invention is simple, and solid phase coupling step number is few, has focused on solving the too long coupling difficulty of peptide sequence, has been easy to produce the problem of peptide disappearance, and segment synthetic technology is mature, Material Cost reduces, and is able to carry out industrial mass production.
Description
Technical field
The present invention relates to Peptides Synthesis, in particular to a kind of side that Teriparatide is prepared with segment method solid-liquid combination
Method.
Background technique
Teriparatide, English name Teriparatide are made of 34 natural amino acids, are endogenous parathyroid glands
The biologically active N- terminal region 1-34 amino acid fragment of hormone, peptide sequence are as follows: H-Ser-Val-Ser-Glu-Ile-
Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-
Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH。
Teriparatide is researched and developed by Li Lai company, and in December, 2002 lists in the U.S. for the first time, is that the first obtains U.S.'s food
The bone shaping agent kind new medicine of Drug Administration's approval is mainly used for treating primary osteoporosis, women post menopausal sclerotin
Osteoporosis after loose and male gonad deterioration.Conventional medicine for treating osteoporosis typically just slows down or blocks bone at present
Mass flow is lost, and can increase osteoblast number as the Teriparatide of parathyroid hormone derivative, enhance its activity and
TNF-a Induced Apoptosis in Osteoblasts is prevented, Teriparatide by human endogenous property substance exploitation from similar to numerous polypeptide drugs, poison pair
It acts on small, and seldom causes accumulation of poisoning, it is very high that completely new mechanism of action and significant curative effect determine that Teriparatide has
Medical value and vast market prospect.
Currently, the preparation method of Teriparatide, is widely reported both at home and abroad.Li Lai company, Yuan Yan producer is using gene table
The mode reached obtains Teriparatide, and in addition to this, still there are many patents in terms of gene expression, such as: US6590081, however,
Gene expression method has the defects of technical threshold is high, and work is complicated, serious three wastes.
Another relatively conventional Teriparatide preparation method is solid-state chemical reaction method method, such as patent 201210213044.3
Five peptide resin segments of Teriparatide are first made, then by segment coupling in a manner of by each peptide resin segment in solid phase gradually
It is coupled Teriparatide resins synthesis Teriparatide peptide resin, finally cracking obtains Teriparatide crude product, this with segment coupling
Mode synthesizes, and need to segment peptide be purified and is lyophilized, complicated for operation, higher cost；Patent 201510005427.5 takes biography
The solid phase synthesis process of system gradually synthesizes Teriparatide from peptide chain C-terminal to N-terminal, although easy to operate, after can not solving long peptide
The difficult problem of phase coupling, and final goal peptide purity is not high, purification difficult；Patent 201410262511.0 peptide chain 16~
17 are fed intake by the way of pseudo proline dipeptides, avoid the generation of special process impurity, but not can avoid because peptide chain is too long
Generated a variety of peptide disappearance；Patent 201510295556.2 is linked Teriparatide peptide chain with resin by HMP Linker,
Subsequent site is by the way of being gradually coupled, the problem of it is various to equally exist target too impurity, purification difficult；Patent
201310403743.9 are synthesized by the way of being gradually coupled, by the α carboxyl of the free hydroxyl group of 17 Ser and 16 Asn with ester
Key is connected, and peptide chain obtains Teriparatide by a step ester amine conversion reaction in the solution after synthesizing, although can be by changing
Become target peptide steric configuration to reduce the coupling difficulty in subsequent site, but still there is a problem of more than solid phase coupling step number, simultaneously
Final step ester amine is converted there is also more serious yield problem, and ultimate cost is caused to increase.
The polypeptide that Teriparatide is made of 34 amino acid, for the angle of synthesis in solid state, peptide chain is longer, with peptide
The extension coupling reaction of chain can be more difficult, is easy to produce more process impurity and is unfavorable for later-period purification.Presently, there are spy
Vertical pa peptide solid phase synthesis process is broadly divided into two classes, and one is coupling amino acid one by one, secondly to be synthesized by way of solid phase
Full guard segment, then target peptide is synthesized in such a way that segment is coupled, first kind synthetic method can not solve because peptide chain is longer
Caused by the lower problem of target peptide purity, the second class synthetic method then increases object because of the issues of purification of full guard segment
Expect cost.
Summary of the invention
To solve disadvantages described above present in Teriparatide synthesis, the present invention provides a kind of preparations of segment method solid-liquid combination
The new method of Teriparatide.Specific technical solution is as follows:
A kind of method that segment method solid-liquid combination prepares Teriparatide, includes the following steps:
(a) dipeptides needed for being synthesized under liquid-phase condition and tripeptide fragment: Fmoc-Asn (Trt)-Phe-OH, 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) using Wang Resin or CTC resin as solid phase carrier, in the presence of coupling agent, 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 (Ot Bu)-Ile-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Ser (tBu)-Val-OH, obtains the Teriparatide peptide resin of side chain protection, and 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 lyophilized by cracking, purifying and obtains Teriparatide fine peptide.
Wherein in step (a), Fmoc-Asn (Trt)-Phe-OH's the preparation method comprises the following steps: first by Fmoc-Asn (Trt)-OH
Fmoc-Asn (Trt)-OSu is generated under the conditions of DCC with HOSu, then by the Fmoc-Asn of generation (Trt)-OSu and H-Phe-
OH generates Fmoc-Asn (Trt)-Phe-OH under the conditions of alkali A；
Fmoc-Asp (OtBu)-Val-OH's the preparation method comprises the following steps: first by Fmoc-Asp (OtBu)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Asp (OtBu)-OSu, then by the Fmoc-Asp of generation (OtBu)-OSu and H-Val-OH in alkali A item
Fmoc-Asp (OtBu)-Val-OH is generated under part；
Fmoc-Lys (Boc)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-Lys (Boc)-OH and HOSu in DCC item
Fmoc-Lys (Boc)-OSu is generated under part, then by the Fmoc-Lys of generation (Boc)-OSu and H-Leu-OH under the conditions of alkali A
Generate Fmoc-Lys (Boc)-Leu-OH；
Fmoc-Trp (Boc)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-Trp (Boc)-OH and HOSu in DCC item
Fmoc-Trp (Boc)-OSu is generated under part, then by the Fmoc-Trp of generation (Boc)-OSu and H-Leu-OH under the conditions of alkali A
Generate Fmoc-Trp (Boc)-Leu-OH；
Fmoc-Arg (pbf)-Val-OH's the preparation method comprises the following steps: first by Fmoc-Arg (pbf)-OH and HOSu in DCC item
Fmoc-Arg (pbf)-OSu is generated under part, then by the Fmoc-Arg of generation (pbf)-OSu and H-Val-OH under the conditions of alkali A
Generate Fmoc-Arg (pbf)-Val-OH；
Fmoc-Ser (tBu)-Met-OH's the preparation method comprises the following steps: first by Fmoc-Ser (tBu)-OH and HOSu in DCC item
Fmoc-Ser (tBu)-OSu is generated under part, then by the Fmoc-Ser of generation (tBu)-OSu and H-Met-OH under the conditions of alkali A
Generate Fmoc-Ser (tBu)-Met-OH；
Fmoc-His (Trt)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-His (Trt)-OH and HOSu in DCC item
Fmoc-His (Trt)-OSu is generated under part, then by the Fmoc-His of generation (Trt)-OSu and H-Leu-OH under the conditions of alkali A
Generate Fmoc-His (Trt)-Leu-OH；
Fmoc-Asn (Trt)-Leu-Gly-OH's the preparation method comprises the following steps: Fmoc-Asn (Trt)-OH and HOSu exists first
Fmoc-Asn (Trt)-OSu is generated under the conditions of DCC, then by the Fmoc-Asn of generation (Trt)-OSu and H-Leu-OH in alkali A item
Fmoc-Asn (Trt)-Leu-OH, Fmoc-Asn (Trt)-Leu-OH and HOSu are generated under part generates Fmoc- under the conditions of DCC
Asn (Trt)-Leu-OSu finally generates the Fmoc-Asn of generation (Trt)-Leu-OSu and H-Gly-OH under the conditions of alkali A
Fmoc-Asn(Trt)-Leu-Gly-OH；
Fmoc-Gln (Trt)-Leu-Met-OH's the preparation method comprises the following steps: Fmoc-Gln (Trt)-OH and HOSu exists first
Fmoc-Gln (Trt)-OSu is generated under the conditions of DCC, then by the Fmoc-Gln of generation (Trt)-OSu and H-Leu-OH in alkali A item
Fmoc-Gln (Trt)-Leu-OH, Fmoc-Gln (Trt)-Leu-OH and HOSu are generated under part generates Fmoc- under the conditions of DCC
Gln (Trt)-Leu-OSu finally generates the Fmoc-Gln of generation (Trt)-Leu-OSu and H-Met-OH under the conditions of alkali A
Fmoc-Gln(Trt)-Leu-Met-OH；
Fmoc-Glu (OtBu)-Ile-OH's the preparation method comprises the following steps: first by Fmoc-Glu (OtBu)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Glu (OtBu)-OSu, then by the Fmoc-Glu of generation (OtBu)-OSu and H-Ile-OH in alkali A item
Fmoc-Glu (OtBu)-Ile-OH is generated under part；
Fmoc-Ser (tBu)-Val-OH's the preparation method comprises the following steps: first by Fmoc-Ser (tBu)-OH and HOSu in DCC item
Fmoc-Ser (tBu)-OSu is generated under part, then by the Fmoc-Ser of generation (tBu)-OSu and H-Val-OH under the conditions of alkali A
Generate Fmoc-Ser (tBu)-Val-OH.
Preferably, dipeptide fragment preparation method is in step (a), by taking Fmoc-Asn (Trt)-Phe-OH as an example: by H-
Phe-OH and alkali A are dissolved in water according to the ratio of molar ratio 1:1~2, and the organic solvent B hydrotropy of 5~20% volumes is added, complete
After fully dissolved, the organic solvent B solution of Fmoc-Asn (Trt)-OSu is added drop-wise in the solution of H-Phe-OH under ice bath stirring,
Wherein the mole of H-Phe-OH is 1.5 times of Fmoc-Asn (Trt)-OSu.TLC monitors reaction end, to subtract after reaction
Organic solvent B is distilled off in pressure, then reacting liquid pH value is adjusted to 2~3 with 10% citric acid/water, and ethyl acetate extracts, crystallization
Obtain Fmoc-Asn (Trt)-Phe-OH.
Fmoc-Asn (Trt)-OSu's the preparation method comprises the following steps: Fmoc-Asn (Trt)-OH and HOSu is pressed in above-mentioned steps (a)
It is dissolved in organic solvent B according to the amount of substance ratio of 1:1.0-1.2, it is under ice-water bath that the DCC/ of 1.0-1.2 times of amount of substance is organic molten
Agent B (with the meter of Fmoc-Asn (Trt)-OH) solution is added drop-wise in Fmoc-Asn (Trt)-OH/HOSu solution, is dripped in 1h
Finish, removes ice bath, 25 DEG C are continued to be stirred to react 2h.It filtering after the reaction was completed, filtrate concentration, decompression boils off 80% organic solvent,
Petroleum ether is added in remaining grease, and until upper solution no longer bleaches, white solid is precipitated, filters, dry, obtains Fmoc-
Asn(Trt)-OSu。
Alkali A is sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethylamine, N- ethyl two in above step
Isopropylamine, N, one of N- diisopropylethylamine.
Organic solvent B be tetrahydrofuran, dioxane, N,N-dimethylformamide, acetone, n-methyl-2-pyrrolidone,
One or more of acetonitrile.
Preferably, tripeptide fragment preparation method described in step (a) is to be with Fmoc-Asn (Trt)-Leu-Gly-OH
Example, H-Gly-OH and alkali A are dissolved in water according to the ratio of molar ratio 1:1~2, and the organic solvent B of 5~20% volumes is added
Hydrotropy；After being completely dissolved, the organic solvent B solution of Fmoc-Asn (Trt)-Leu-OSu is added dropwise to H-Gly- under ice bath stirring
In OH solution, wherein the mole of H-Gly-OH is 1.5 times of Fmoc-Asn (Trt)-Leu-Osu mole.TLC monitoring is anti-
Terminal is answered, reacting liquid pH value is adjusted to 2 after reacting and terminating vacuum distillation removing organic solvent B, then with 10% citric acid/water
~3, ethyl acetate extraction, crystallization obtains Fmoc-Asn (Trt)-Leu-Gly-OH.
Fmoc-Asn (Trt)-Leu-OSu's the preparation method comprises the following steps: by Fmoc-Asn (Trt)-Leu-OH and HOSu according to 1:
The amount of substance ratio of 1.0-1.2 is dissolved in organic solvent B, by the DCC/ organic solvent B of 1.0-1.2 times of amount of substance under ice-water bath
(with the meter of Fmoc-Asn (Trt)-OH) solution is added drop-wise in Fmoc-Asn (Trt)-Leu-OH/HOSu solution, is added dropwise in 1h
It finishes, removes ice bath, 25 DEG C are continued to be stirred to react 2h.Filter after the reaction was completed, filtrate concentration, decompression boil off 60% it is organic molten
Agent, petroleum ether is added in remaining grease, and until upper solution no longer bleaches, white solid is precipitated, filters, dry, obtains
Fmoc-Asn(Trt)-Leu-OSu。
Fmoc-Asn (Trt)-Leu-OH's the preparation method comprises the following steps: by H-Leu-OH and alkali A according to the ratio of molar ratio 1:1~2
Example is dissolved in water, and the organic solvent B hydrotropy of 5~20% volumes is added.After being completely dissolved, by Fmoc-Asn under ice bath stirring
(Trt) the organic solvent B solution of-OSu is added dropwise into H-Leu-OH solution, wherein the mole of H-Leu-OH is Fmoc-
1.5 times of Asn (Trt)-OSu mole.TLC monitors reaction end, to the end of reacting, after reduction vaporization removes organic solvent B,
Reacting liquid pH value is adjusted to 2~3 with 10% citric acid/water again, ethyl acetate extraction, crystallization obtains Fmoc-Asn (Trt)-Leu-
OH。
Fmoc-Asn (Trt)-OSu's the preparation method comprises the following steps: by Fmoc-Asn (Trt)-OH and HOSu according to 1:1.0-1.2's
Amount of substance ratio is dissolved in organic solvent B, by the DCC/ organic solvent B of 1.0-1.2 times of amount of substance (with Fmoc-Asn under ice-water bath
(Trt) meter of-OH) solution is added drop-wise in Fmoc-Asn (Trt)-OH/HOSu solution, is added dropwise in 1h, remove ice bath, 25 DEG C
Continue to be stirred to react 2h.It filters after the reaction was completed, filtrate concentration, decompression boils off 80% organic solvent, and remaining grease is added
White solid is precipitated until upper solution no longer bleaches in petroleum ether, filters, dry, obtains Fmoc-Asn (Trt)-OSu.
Alkali A is sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethylamine, N- ethyl two in above step
Isopropylamine, N, one of N- diisopropylethylamine；
Organic solvent B be tetrahydrofuran, dioxane, N,N-dimethylformamide, acetone, n-methyl-2-pyrrolidone,
One or more of acetonitrile.
Wherein in step (b), fed intake coupling 33~34 with Fmoc-Asn (Trt)-Phe-OH, with Fmoc-Asp
(OtBu)-Val-OH feeds intake coupling 30~31, is fed intake coupling 27~28 with Fmoc-Lys (Boc)-Leu-OH, with Fmoc-
Trp (Boc)-Leu-OH feeds intake coupling 23~24, is fed intake coupling 20~21 with Fmoc-Arg (pbf)-Val-OH, with
Fmoc-Ser (tBu)-Met-OH feeds intake coupling 17~18, is fed intake coupling 14~15 with Fmoc-His (Trt)-Leu-OH,
It is fed intake coupling 10~12 with Fmoc-Asn (Trt)-Leu-Gly-OH, is fed intake coupling with Fmoc-Gln (Trt)-Leu-Met-OH
It 6~8, is fed intake coupling 4~5 with Fmoc-Glu (OtBu)-Ile-OH, is fed intake coupling 1 with Fmoc-Ser (tBu)-Val-OH
~2.
Preferably, Wang Resin described in step (b) or CTC Resin substitution degree are 0.3~0.5mmol/g, tree
Substitution degree is 0.25~0.45mmol/g, preferably 0.35mmol/g after rouge transformation；
In step (b), when single amino acids or dipeptide fragment and tripeptide fragment are fed intake, molar feed ratio is 3~4 times
It measures (synthesize the substance meter of scale), the peptide reaction time is 2~3h, and endpoint is with Kaiser reagent detected artifacts
It is quasi-；Coupling agent used by coupling reaction be DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA,
One or more of HATU/HOAT/DIPEA.
Preferably, lytic reagent is that the TFA solution that volume ratio is 1~5% scavenger is added in step (c), wherein removing
Agent is one or more of methyl phenyl ethers anisole, thioanisole, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane.
Compared with the existing technology, the beneficial effects of the present invention are:
1, it is fed intake using above-mentioned dipeptides and tripeptide fragment, makes 33,30,27,23,20,17,14,11
Position, 10,7,6,4, the solid phase coupling efficiency in 1 these site has reached absolutely, improves target peptide purity,
Thick peptide purity reaches 75%, while avoiding the formation of these site deletion peptides, the number of process impurity is reduced, after improving
The efficiency of phase purifying, ultimate yield reach 40%.
2, solid phase is coupled step number and foreshortens to 21 steps by 34 steps, substantially reduces the dosage of solvent in synthesis in solid state, reduces object
Expect cost, reduces three waste discharge.
3, Phe, Val, Leu, Gly, Met are chosen, these amino acid without functional side chain group of Ile carry out dipeptides or three
The synthesis of peptide fragment can substantially reduce the cost of segment liquid phase synthesis, can apply in conjunction with mature HOSu/DCC coupling method
In industrial mass production.
Abbreviation meaning used in specification and claims is as follows:
Fmoc 9-fluorenylmethyloxycarbonyl
CTC resin 2- chlorine trityl chloride resin
Wang Resin Wang Shuzhi
TBu tert-butyl
Trt trityl
DCM methylene chloride
DMF N,N-dimethylformamide
DMAP 4-dimethylaminopyridine
DIEA N, N- diisopropylethylamine
DIC N, N- diisopropylcarbodiimide
HBTU benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate
HATU 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
TBTU O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid
HOBT I-hydroxybenzotriazole
HOAT 1- hydroxyl -7- azo benzotriazole
TFA trifluoroacetic acid
HOSu n-hydroxysuccinimide
EA ethyl acetate
THF tetrahydrofuran
PE petroleum ether
Specific embodiment
With specific embodiment, the present invention is described in detail below, but does not limit this patent；Change according to the present invention former
The feed ratio or reaction dissolvent of material or and condensing agent etc., be within the scope of the invention.
The preparation of embodiment 1:Fmoc-Asn (Trt)-OSu
It accurately weighs Fmoc-Asn (Trt)-OH 1193.4g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Asn (Trt)-OSu 1305.6g, yield
94.2%.
The preparation of embodiment 2:Fmoc-Asn (Trt)-Phe-OH
It accurately weighs H-Phe-OH 446.1g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Asn (Trt)-OSu (1248.7g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and revolving pumps ethyl acetate, and remaining grease adds
Enter 2000ml methanol, solution clear is placed in -20 DEG C of refrigerators and stands overnight, and a large amount of white solids are precipitated, and filters, dry
Obtain Fmoc-Asn (Trt)-Phe-OH 1041.2g, yield 77.3%.
The preparation of embodiment 3:Fmoc-Asp (OtBu)-OSu
It accurately weighs Fmoc-Asp (OtBu)-OH 822.2g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Asp (OtBu)-OSu 961.7g, yield
95.3%.
The preparation of embodiment 4:Fmoc-Asp (OtBu)-Val-OH
It accurately weighs H-Val-OH 315.9g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Asp (OtBu)-OSu (908.2g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, and remaining aqueous solution filters, ice water
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, concentrated by rotary evaporation to 4000ml, and petroleum ether is added
4000ml, solution clear, crystallization under the conditions of being placed in 4-8 DEG C obtain Fmoc-Asp (OtBu)-Val-OH 801.4g, yield
87.2%.
The preparation of embodiment 5:Fmoc-Lys (Boc)-OSu
It accurately weighs Fmoc-Lys (Boc)-OH 937.2g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Lys (Boc)-OSu 1036.8g, yield
92.3%.
The preparation of embodiment 6:Fmoc-Lys (Boc)-Leu-OH
It accurately weighs H-Leu-OH 355.6g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Lys (Boc)-OSu (1010.3g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and revolving pumps ethyl acetate, and 2000ml is newly added
Tetrahydrofuran redissolves, and 4000ml petroleum ether crystallization is added, obtains Fmoc-Lys (Boc)-Leu-OH 871.2g, yield 83.2%.
The preparation of embodiment 7:Fmoc-Trp (Boc)-OSu
It accurately weighs Fmoc-Trp (Boc)-OH 1053.2g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Trp (Boc)-OSu 1135.3g, yield
91.6%.
The preparation of embodiment 8:Fmoc-Trp (Boc)-Leu-OH
It accurately weighs H-Leu-OH 354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Trp (Boc)-OSu (1115.5g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and concentrated by rotary evaporation removes ethyl acetate, is added
2500ml methanol redissolves, and solution clarification is placed in -20 DEG C of crystallizations, obtains Fmoc-Trp (Boc)-Leu-OH 905.1g, yield
78.6%.
The preparation of embodiment 9:Fmoc-Arg (pbf)-OSu
It accurately weighs Fmoc-Arg (pbf)-OH 1297.4g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Arg (pbf)-OSu 1181.3g, yield
92.6%.
The preparation of embodiment 10:Fmoc-Arg (pbf)-Val-OH
It accurately weighs H-Val-OH 316.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Arg (pbf)-OSu (1342.5g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 10000ml ethyl acetate of the lower addition 10% of bath extracts in three times, it is associated with
Machine phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and revolving removes ethyl acetate, remaining grease
3000ml methanol is added to redissolve, is heated to 40 DEG C of solution clears, is placed in -20 DEG C of crystallizations, obtain Fmoc-Arg (pbf)-Val-
OH 1086.4g, yield 80.7%.
The preparation of embodiment 11:Fmoc-Ser (tBu)-OSu
It accurately weighs Fmoc-Ser (tBu)-OH 766.9g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Ser (tBu)-OSu 894.7g, yield
93.1%.
The preparation of embodiment 12:Fmoc-Ser (tBu)-Met-OH
It accurately weighs H-Met-OH 402.8g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Ser (tBu)-OSu (864.9g, 1.8mol) under ice bath, stir
Reaction is mixed, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice-water bath after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of lower addition 10% extracts in three times, merges organic phase,
It is washed in three times with 3000ml saturated salt solution, anhydrous sodium sulfate is dry, concentrated by rotary evaporation to 3000ml, and petroleum ether 4000ml is added
Crystallization obtains Fmoc-Ser (tBu)-Met-OH 765.15g, yield 82.6%.
The preparation of embodiment 13:Fmoc-His (Trt)-OSu
It accurately weighs Fmoc-His (Trt)-OH 1239.4g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-His (Trt)-OSu 1316.2g, yield
91.8%.
The preparation of embodiment 14:Fmoc-His (Trt)-Leu-OH
It accurately weighs H-Leu-OH 354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-His (Trt)-OSu (1290.2g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and revolving removes ethyl acetate, remaining solid and oil
Shape object is added 3000ml methanol and redissolves, and is heated to 40 DEG C of solution clears, is placed in -20 degrees Celsius of crystallizations, obtains Fmoc-His
(Trt)-Leu-OH 765.15g, yield 82.6%.
The preparation of embodiment 15:Fmoc-Asn (Trt)-OSu
It accurately weighs Fmoc-Asn (Trt)-OH 1193.4g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Asn (Trt)-OSu 1279.3g, yield
92.2%.
The preparation of embodiment 16:Fmoc-Asn (Trt)-Leu-OH
It accurately weighs H-Leu-OH354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Asn (Trt)-OSu (1248.7g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, and revolving removes ethyl acetate, and remaining grease adds
Enter the redissolution of 2000ml methanol, solution clear is placed in crystallization in -20 DEG C of refrigerators, obtains Fmoc-Asn (Trt)-Leu-OH
1100.2g, yield 86.1%.
The preparation of embodiment 17:Fmoc-Asn (Trt)-Leu-OSu
It accurately weighs Fmoc-Asn (Trt)-Leu-OH 1063.9g (1.5mol) and HOSu 172.5g (1.5mol) is dissolved in
In 4000ml tetrahydrofuran, ice-water bath stirring.DCC 309.6g (1.5mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran,
It is slowly dropped in above-mentioned solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation is extremely
3000~3500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 spends ice
Case stands 1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.Be placed in-
In 20 degree of refrigerators, a large amount of white solids are precipitated after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Asn (Trt)-Leu-
OSu1079.6g, yield 89.2%.
The preparation of embodiment 18:Fmoc-Asn (Trt)-Leu-Gly-OH
It accurately weighs H-Gly-OH 135.1g (1.8mol) and sodium carbonate 228.9g (2.16mol) is dissolved in 2000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution of Fmoc-Asn (Trt)-Leu-OSu (968.28g, 1.2mol) under ice bath
3000ml is stirred to react, and TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, and remaining aqueous solution is taken out
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of addition 10% extracts in three times under ice-water bath after filter,
Merge organic phase, washed in three times with 3000ml saturated salt solution, anhydrous sodium sulfate is dry, and revolving removes ethyl acetate, is added
3500ml methanol is heated to 40 DEG C of solution clears, is placed in -20 DEG C of crystallizations, obtains Fmoc-Asn (Trt)-Leu-Gly-OH
736.2g yield 80.0%.
The preparation of embodiment 19:Fmoc-Gln (Trt)-OSu
It accurately weighs Fmoc-Gln (Trt)-OH 1221.4g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Gln (Trt)-OSu 1320.5g, yield
93.3%.
The preparation of embodiment 20:Fmoc-Gln (Trt)-Leu-OH
It accurately weighs H-Leu-OH 354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Gln (Trt)-OSu (1273.8g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, with 3000ml
Saturated salt solution washs in three times, and anhydrous sodium sulfate is dry, merges organic phase, and revolving removes ethyl acetate, and remaining grease adds
Enter the redissolution of 2000ml methanol, is heated to 40 DEG C of solution clears, is placed in crystallization in -20 DEG C of refrigerators, obtain Fmoc-Gln (Trt) -
Leu-OH 1150.5g, yield 88.3%.
The preparation of embodiment 21:Fmoc-Gln (Trt)-Leu-OSu
It accurately weighs Fmoc-Gln (Trt)-Leu-OH 1085.8g (1.5mol) and HOSu 172.5g (1.5mol) is dissolved in
In 4000ml tetrahydrofuran, ice-water bath stirring.DCC 309.6g (1.5mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran,
It is slowly dropped in above-mentioned solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation is extremely
3000~3500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 spends ice
Case stands 1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.Be placed in-
In 20 degree of refrigerators, a large amount of white solids are precipitated after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Gln (Trt)-Leu-
OSu1054.3g, yield 85.6%.
The preparation of embodiment 22:Fmoc-Gln (Trt)-Leu-Met-OH
It accurately weighs H-Met-OH 268.6g (1.8mol) and sodium carbonate 228.9g (2.16mol) is dissolved in 2000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution of Fmoc-Gln (Trt)-Leu-OSu (985.1g, 1.2mol) under ice bath
3000ml is stirred to react, and TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, and remaining aqueous solution is taken out
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of addition 10% extracts in three times under ice-water bath after filter,
Merging organic phase, is washed in three times with 3000ml saturated salt solution, anhydrous sodium sulfate is dry, and concentrated by rotary evaporation removes ethyl acetate,
Methanol 3500ml is added to redissolve, is heated to 40 DEG C of solution clears, is placed in -20 DEG C of crystallizations, obtain Fmoc-Gln (Trt)-Leu-
Met-OH 859.8g, yield 83.8%.
The preparation of embodiment 23:Fmoc-Glu (OtBu)-OSu
It accurately weighs Fmoc-Glu (OtBu)-OH 850.9g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Glu (OtBu)-OSu 977.2g, yield
93.5%.
The preparation of embodiment 24:Fmoc-Glu (OtBu)-Ile-OH
It accurately weighs H-Ile-OH 354.2g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Glu (OtBu)-OSu (940.6g, 1.8mol) under ice bath,
It is stirred to react, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice water after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of the lower addition 10% of bath extracts in three times, merges organic
Phase is washed in three times with 3000ml saturated salt solution, and anhydrous sodium sulfate is dry, concentrated by rotary evaporation to 4000ml, and petroleum ether is added
4000ml crystallization obtains Fmoc-Glu (OtBu)-Ile-OH 841.6g, yield 86.8%.
The preparation of embodiment 25:Fmoc-Ser (tBu)-OSu
It accurately weighs Fmoc-Ser (tBu)-OH 766.9g (2mol) and HOSu 230g (2mol) is dissolved in 4000ml tetrahydro
In furans, ice-water bath stirring.DCC 412.8g (2mol) is accurately weighed, is dissolved in 2400ml tetrahydrofuran, is slowly dropped to
It states in solution, 25 degree of the reaction was continued 2h after ice bath stirring 1h.It filters after the reaction was completed, filtrate concentrated by rotary evaporation to 2000~
2500ml.It is filtered after the completion of concentration, 8000ml petroleum ether is added in filtrate, and a large amount of white solids are precipitated, and solution -20 is spent refrigerator and stood
1h.It is filtered after standing, 6000ml petroleum ether, solution clarification is added with the dissolution of 2000ml ethyl acetate in filter cake.It is placed in -20 degree ice
A large amount of white solids are precipitated in case, after 2h, filter, filtration cakes torrefaction, weighing obtains Fmoc-Ser (tBu)-OSu 862.8g, yield
89.7%.
The preparation of embodiment 26:Fmoc-Ser (tBu)-Val-OH
It accurately weighs H-Val-OH 315.9g (2.7mol) and sodium carbonate 343.4g (3.24mol) is dissolved in 4000mL water,
(2~8 DEG C) are slowly added to the tetrahydrofuran solution 3000ml of Fmoc-Ser (tBu)-OSu (864.9g, 1.8mol) under ice bath, stir
Reaction is mixed, TLC monitors reaction end.After fully reacting, vacuum rotary steam removes tetrahydrofuran, ice-water bath after remaining aqueous solution filters
Aqueous citric acid solution tune solution ph to 2~3, the 8000ml ethyl acetate of lower addition 10% extracts in three times, merges organic phase,
It is washed in three times with 3000ml saturated salt solution, anhydrous sodium sulfate is dry, concentrated by rotary evaporation to 4000ml, and petroleum ether 4000ml is added
Crystallization obtains Fmoc-Ser (tBu)-Val-OH 743.5g, yield 85.6%.
The synthesis of embodiment 27:Fmoc-Asn (Trt)-Phe-Wang Resin
It weighs 30.0g (Sub=0.47mmol/g) Wang Resin and is placed in solid phase reactor, twice with DMF washing resin,
200ml/ times, rejoin DCM 200ml swellable resins 30min.While resin swelling, Fmoc-Asn (Trt)-Phe- is weighed
OH 31.43g (42.3mmol, 3eq), HOBt 6.28g (46.5mmol, 3.3eq), with 150ml volume ratio for 1:1 DMF and
The mixed solution of DCM dissolves, ice bath 10min, and DIC 7.25ml (46.5mmol, 3eq) is added and activates 5min.Resin swelling and ammonia
After the completion of base acid activation, feed intake reaction, nitrogen gas stirring, and DMAP 0.51g (4.23mmol, 0.3eq) is added after 10min, continues anti-
Answer 5h.After 5h, reaction solution is taken out, DMF is washed resin 4 times, and 200ml/ is each；DCM is washed resin 2 times, 200ml/ times.Envelope is added
Hold reagent 180mL (50ml acetic anhydride and 42.5ml pyridinium dissolution are in 87.5mL DMF) by remaining unreacted acetylating hydroxyl groups
2h.Reaction solution is filtered after the reaction was completed, and respectively with DMF, DCM, methanol washing resin 3 times 200ml/ times, must after vacuum drying
Fmoc-Asn (Trt)-P he-Wang Resin 38.6g, substitution degree is 0.30mmol/g after transformation.
The synthesis of embodiment 28:Fmoc-Asn (Trt)-Phe-CTC Resin
It weighs CTC resin 30.0g (sub=0.40mmol/g) to be placed in synthesis column, is washed twice, added with 200mL DMF
Enter 200mL DCM swelling 30min；After pumping DCM, it is added dissolved with 8.93g (12mmol) Fmoc-Asn (Trt)-Phe-OH's
DCM/DMF (3/1, volume ratio) solution 100ml, N2DIPEA 4.0ml (24mmol) is added after stirring 5min, continues to rouse N2Stirring
React 60min.Reaction solution is taken out after the reaction was completed, and DCM/CH is added3OH/DIPEA (volume ratio 17:2:1) mixed solution sealing end 3
Secondary, each 200ml reacts 10min；Then it is washed respectively 3 times with DMF, DCM, methanol, is dried in vacuo to obtain Fmoc-Asn (Trt)-
Phe-CTC Resin 39.2g, substitution degree is 0.26mmol/g after surveying transformation.
Embodiment 29: the preparation of Teriparatide peptide resin
Accurately weigh Fmoc-Asn (the Trt)-Phe-Wang Resin 33.33g (synthesis that substitution degree is 0.30mmol/g
Scale 10mmol) it is placed in solid phase reactor, DMF is washed resin 2 times, 150ml/ times, 150ml DCM is added and is swollen 30min；Tree
It is taken off Fmoc 2 times after liposoluble is swollen with 20% piperidines/DMF, 150ml/ times, reacts 5min and 10min respectively；It has been deprotected
With DMF washing 4 times after, DCM is washed 2 times, 150ml/ times.Take sample with the detection of Kaiser reagent, resin is in navy blue.
Fmoc-His (Trt)-OH 18.57g (30mmol, 3eq), HOBt 4.05g (30mmol, 3eq) are weighed, with
120ml volume ratio is the DMF of 1:1, the dissolution of DCM solution, ice-water bath 10min, addition DIC 4.68ml (30mmol, 3eq) activation
5min.It feeds intake reaction after the completion of activation, nitrogen gas stirring 2h, reaction end is subject to Kaiser reagent testing result, and detection is reacted
After the completion, it filters and removes reaction solution, with DMF washing 4 times, DCM is washed 2 times, 200ml/ times.Fmoc is taken off with 20% piperidines/DMF
2 times, 150ml/ times, 5min and 10min is reacted respectively；With DMF washing 4 times after the completion of deprotection, DCM is washed 2 times, 150ml/
It is secondary.Take sample with the detection of Kaiser reagent, resin is in navy blue, and 27 His couplings are completed.
Amino acid and dipeptide fragment and tripeptides are sequentially connected according to Teriparatide peptide sequence according to the coupling method of 32 His
Segment: 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.After peptide resin synthesizes, successively with DMF washing 4 times, DCM is washed 2 times, and methanol washs 3 times, 200ml/ times, 25 degree of resin
It is dried in vacuo 8h, weigh to obtain 110.3g.
Embodiment 30: the preparation of Teriparatide peptide resin
Accurately weigh Fmoc-Asn (Trt)-Phe-CTC Resin 38.5g (the synthesis rule that substitution degree is 0.26mmol/g
Mould 10mmol) it is placed in synthesis column, DMF is washed resin 2 times, 150ml/ times, 150ml DCM is added and is swollen 30min；Resin swelling
After Fmoc 2 times taken off with 20% piperidines/DMF, 150ml/ time, reaction 5min and 10min respectively；After the completion of deprotection with
DMF is washed 4 times, and DCM is washed 2 times, 150ml/ times.Take sample with the detection of Kaiser reagent, resin is in navy blue.
Fmoc-His (Trt)-OH 18.57g (30mmol, 3eq), HOBt 4.05g (30mmol, 3eq) are weighed, with
120ml volume ratio is the DMF of 1:1, the dissolution of DCM solution, ice-water bath 10min, addition DIC 4.68ml (30mmol, 3eq) activation
5min.It feeds intake reaction after the completion of activation, nitrogen gas stirring 2h, reaction end is subject to Kaiser reagent testing result, and detection is reacted
After the completion, it filters and removes reaction solution, with DMF washing 4 times, DCM is washed 2 times, 200ml/ times.Fmoc is taken off with 20% piperidines/DMF
2 times, 150ml/ times, 5min and 10min is reacted respectively；With DMF washing 4 times after the completion of deprotection, DCM is washed 2 times, 150ml/
It is secondary.Take sample with the detection of Kaiser reagent, resin is in navy blue, and 27 His couplings are completed.
Amino acid and dipeptide fragment and tripeptides are sequentially connected according to Teriparatide peptide sequence according to the coupling method of 32 His
Segment: 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.After peptide resin synthesizes, successively with DMF washing 4 times, DCM is washed 2 times, and methanol washs 3 times, 200ml/ times, 25 degree of resin
It is dried in vacuo 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 pre-cooling.Match
Set lytic reagent 1100ml, volume ratio TFA:TIS: water=94:1:5 is placed in -20 refrigerators and 30min is pre-chilled.According to 1g tree
Lytic reagent is added in peptide resin by the ratio of rouge 10ml, and ice-water bath stirs 2h, 25 degree of stirring 2h, is filtered after stopping reaction,
Resin is washed twice with TFA, and 100ml/ times, merging filtrate is slowly poured onto the ether of 13L pre-cooling, and stirring is placed in -20 ice
1h is stood in case.Ether sedimentation liquid is centrifuged, and with ether washing six times, centrifugation obtained solid is dry, obtains 44.7g, yield
108.2%.Take thick peptide sample analysis, purity 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 pre-cooling.Configuration
Lytic reagent 1000ml, volume ratio TFA:TIS: water=94:1:5 is placed in -20 refrigerators and 30min is pre-chilled.According to 1g resin
Lytic reagent is added in peptide resin by the ratio of 10ml, and ice-water bath stirs 2h, 25 degree of stirring 2h, is filtered after stopping reaction, is set
Rouge is washed twice with TFA, and 100ml/ times, merging filtrate is slowly poured onto the ether of 12L pre-cooling, and stirring is placed in -20 refrigerators
Middle standing 1h.Ether sedimentation liquid is centrifuged, and with ether washing six times, centrifugation obtained solid is dry, obtains 37.7g, yield
91.5%.Take thick peptide sample analysis, purity 75.5%.
Embodiment 33: the purifying of the thick peptide of Teriparatide
The embodiment thick peptide 10.0g of 31 gained is weighed, is dissolved with the acetonitrile/water of 500ml 5%, ultrasonic dissolution assisting, 0.45um filter
Film filtering.
Internal diameter is the C of 100mm18Column is prepared, mobile phase A is 0.1%TFA/ water, and Mobile phase B is 0.1%TFA/ acetonitrile, on
Sample amount is 10g/ needle, flow velocity 300ml/min, Detection wavelength 210nm.Gradient elution recycles sample introduction behind before peak and peak, obtains middle control
The fine peptide solution that purity assay is 98.5% or more；Teriparatide fine peptide is lyophilized to obtain after turning salt with acetic acid/acetonitrile/aqueous systems
3.1g, 99.5% or more purity, total recovery 43.7%.
Embodiment 34: the purifying of the thick peptide of Teriparatide
The embodiment thick peptide 10.0g of 32 gained is weighed, is dissolved with the acetonitrile/water of 500ml 5%, ultrasonic dissolution assisting, 0.45um filter
Film filtering.Internal diameter is the C of 100mm18Column is prepared, mobile phase A is 0.1%TFA/ water, and Mobile phase B is 0.1%TFA/ acetonitrile, loading
Amount is 10g/ needle, flow velocity 300ml/min, Detection wavelength 210nm.Gradient elution recycles sample introduction behind before peak and peak, obtains middle control point
Analyse the fine peptide solution that purity is 98.5% or more；Teriparatide fine peptide 2.9g is lyophilized to obtain after turning salt with acetic acid/acetonitrile/aqueous systems,
99.5% or more purity, total recovery 36.6%.
Claims (6)
1. a kind of method that segment method solid-liquid combination prepares Teriparatide, which comprises the steps of:
(a) dipeptides needed for being synthesized under liquid-phase condition and tripeptide fragment: Fmoc-Asn (Trt)-Phe-OH, 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) using Wang Resin or CTC resin as solid phase carrier, in the presence of coupling agent, 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, obtains the Teriparatide peptide resin of side chain protection, and 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 lyophilized by cracking, purifying and obtains Teriparatide fine peptide.
2. a kind of method for preparing Teriparatide with segment method solid-liquid combination according to claim 1, which is characterized in that step
Suddenly in (a),
The Fmoc-Asn (Trt)-Phe-OH's the preparation method comprises the following steps: first by Fmoc-Asn (Trt)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Asn (Trt)-OSu, then by the Fmoc-Asn of generation (Trt)-OSu and H-Phe-OH in alkali A condition
Lower generation Fmoc-Asn (Trt)-Phe-OH；
The Fmoc-Asp (OtBu)-Val-OH's the preparation method comprises the following steps: Fmoc-Asp (OtBu)-OH and HOSu exists first
Fmoc-Asp (OtBu)-OSu is generated under the conditions of DCC, then by the Fmoc-Asp of generation (OtBu)-OSu and H-Val-OH in alkali A
Under the conditions of generate Fmoc-Asp (OtBu)-Val-OH；
The Fmoc-Lys (Boc)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-Lys (Boc)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Lys (Boc)-OSu, then by the Fmoc-Lys of generation (Boc)-OSu and H-Leu-OH in alkali A condition
Lower generation Fmoc-Lys (Boc)-Leu-OH；
The Fmoc-Trp (Boc)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-Trp (Boc)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Trp (Boc)-OSu, then by the Fmoc-Trp of generation (Boc)-OSu and H-Leu-OH in alkali A condition
Lower generation Fmoc-Trp (Boc)-Leu-OH；
The Fmoc-Arg (pbf)-Val-OH's the preparation method comprises the following steps: first by Fmoc-Arg (pbf)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Arg (pbf)-OSu, then by the Fmoc-Arg of generation (pbf)-OSu and H-Val-OH in alkali A condition
Lower generation Fmoc-Arg (pbf)-Val-OH；
The Fmoc-Ser (tBu)-Met-OH's the preparation method comprises the following steps: first by Fmoc-Ser (tBu)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Ser (tBu)-OSu, then by the Fmoc-Ser of generation (tBu)-OSu and H-Met-OH in alkali A condition
Lower generation Fmoc-Ser (tBu)-Met-OH；
The Fmoc-His (Trt)-Leu-OH's the preparation method comprises the following steps: first by Fmoc-His (Trt)-OH and HOSu in DCC
Under the conditions of generate Fmoc-His (Trt)-OSu, then by the Fmoc-His of generation (Trt)-OSu and H-Leu-OH in alkali A condition
Lower generation Fmoc-His (Trt)-Leu-OH；
The Fmoc-Asn (Trt)-Leu-Gly-OH's the preparation method comprises the following steps: Fmoc-Asn (Trt)-OH and HOSu exists first
Fmoc-Asn (Trt)-OSu is generated under the conditions of DCC, then by the Fmoc-Asn of generation (Trt)-OSu and H-Leu-OH in alkali A item
Fmoc-Asn (Trt)-Leu-OH, Fmoc-Asn (Trt)-Leu-OH and HOSu are generated under part generates Fmoc- under the conditions of DCC
Asn (Trt)-Leu-OSu finally generates the Fmoc-Asn of generation (Trt)-Leu-OSu and H-Gly-OH under the conditions of alkali A
Fmoc-Asn(Trt)-Leu-Gly-OH；
The Fmoc-Gln (Trt)-Leu-Met-OH's the preparation method comprises the following steps: Fmoc-Gln (Trt)-OH and HOSu exists first
Fmoc-Gln (Trt)-OSu is generated under the conditions of DCC, then by the Fmoc-Gln of generation (Trt)-OSu and H-Leu-OH in alkali A item
Fmoc-Gln (Trt)-Leu-OH, Fmoc-Gln (Trt)-Leu-OH and HOSu are generated under part generates Fmoc- under the conditions of DCC
Gln (Trt)-Leu-OSu finally generates the Fmoc-Gln of generation (Trt)-Leu-OSu and H-Met-OH under the conditions of alkali A
Fmoc-Gln(Trt)-Leu-Met-OH；
The Fmoc-Glu (OtBu)-Ile-OH's the preparation method comprises the following steps: Fmoc-Glu (OtBu)-OH and HOSu exists first
Fmoc-Glu (OtBu)-OSu is generated under the conditions of DCC, then by the Fmoc-Glu of generation (OtBu)-OSu and H-Ile-OH in alkali A
Under the conditions of generate Fmoc-Glu (OtBu)-Ile-OH；
The Fmoc-Ser (tBu)-Val-OH's the preparation method comprises the following steps: first by Fmoc-Ser (tBu)-OH and HOSu in DCC
Under the conditions of generate Fmoc-Ser (tBu)-OSu, then by the Fmoc-Ser of generation (tBu)-OSu and H-Val-OH in alkali A condition
Lower generation Fmoc-Ser (tBu)-Val-OH；
The alkali A is sodium carbonate, sodium bicarbonate, saleratus, potassium carbonate, triethylamine, diethylamine, N- ethyl diisopropyl
Amine, N, one of N- diisopropylethylamine.
3. a kind of method for preparing Teriparatide with segment method solid-liquid combination according to claim 1, which is characterized in that step
Suddenly it in (b), is fed intake coupling 33~34 with Fmoc-Asn (Trt)-Phe-OH, is fed intake idol with Fmoc-Asp (OtBu)-Val-OH
30~31, connection, is fed intake coupling 27~28 with Fmoc-Lys (Boc)-Leu-OH, is fed intake with Fmoc-Trp (Boc)-Leu-OH
Coupling 23~24, is fed intake coupling 20~21 with Fmoc-Arg (pbf)-Val-OH, with Fmoc-Ser (tBu)-Met-OH throwing
Material coupling 17~18, is fed intake coupling 14~15 with Fmoc-His (Trt)-Leu-OH, with Fmoc-Asn (Trt)-Leu-
Gly-OH feeds intake coupling 10~12, is fed intake coupling 6~8 with Fmoc-Gln (Trt)-Leu-Met-OH, with Fmoc-Glu
(OtBu)-Ile-OH feeds intake coupling 4~5, is fed intake coupling 1~2 with Fmoc-Ser (tBu)-Val-OH.
4. a kind of method for preparing Teriparatide with segment method solid-liquid combination according to claim 1, which is characterized in that step
Suddenly in (b), the initial substitution degree of Wang Resin or CTC Resin is 0.3~0.5mmol/g.
5. a kind of method for preparing Teriparatide with segment method solid-liquid combination according to claim 1, which is characterized in that step
Suddenly in (b), the coupling agent is HOBt/DIC, HOAt/DIC, HOBt/HBTU/DIEA, HOAt/HATU/DIEA, PyBOP/
One or more of DIEA, TBTU/DIEA.
6. a kind of method for preparing Teriparatide with segment method solid-liquid combination according to claim 1, which is characterized in that step
Suddenly in (c), the cleavage step is the TFA solution that volume ratio 1-5% scavenger is added, and the scavenger is methyl phenyl ethers anisole, benzene
One or more of methyl sulfide, dithioglycol, mercaptoethanol, phenol, water, tri isopropyl silane.
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| WO2018032521A1 (en) * | 2016-08-19 | 2018-02-22 | 深圳市健元医药科技有限公司 | Method for synthesizing liraglutide |
| CN106749614A (en) * | 2017-01-05 | 2017-05-31 | 济南康和医药科技有限公司 | A kind of fragment method solid-liquid combination is prepared for the method for degree Shandong peptide |
| CN109096388A (en) * | 2017-07-24 | 2018-12-28 | 江苏金斯瑞生物科技有限公司 | A kind of preparation method of Teriparatide |
| CN108047329A (en) * | 2018-02-01 | 2018-05-18 | 润辉生物技术（威海）有限公司 | A kind of preparation method of A Bapa peptides |
| CN110642936B (en) * | 2018-06-26 | 2023-01-17 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
| CN111057139B (en) * | 2018-10-17 | 2023-12-22 | 南京华威医药科技集团有限公司 | Novel process for preparing teriparatide |
| 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 |
| CN113135988B (en) * | 2021-04-08 | 2022-04-01 | 润辉生物技术（威海）有限公司 | Preparation method of thymosin beta 4 |
| 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 |
| CN113501871B (en) * | 2021-07-21 | 2022-10-28 | 汉肽生物医药集团有限公司 | Method for preparing darunavagon by combining solid phase with liquid phase |
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