CN101230089A - Solid-phase synthesis of glycyl histidyl lycine - Google Patents

Abstract

The invention relates to an amino acid synthetic method, in particular to a solid phase synthetic method of glycyl-histidyl-lysine. The invention mainly solves the technical problem that two basic amino acids are included in the molecule to result in the artificial synthesis to be difficult. During synthesis, Fmoc-Lys(BOC)-OH and Wang Resin are mixed according to the proportion, HOBT, DIC and DMAP are added into to be used as activating agent, DMF is taken as solvent and reacts in the oscillating way, and Fmoc-lys(Boc)-Wang Resin is obtained after the reaction product is filtered and washed; a Fmoc base group is removed from DMF solution added with hexahydropyridine, to obtain H-lys(Boc)Wang Resin; and then Fmoc-His(trt)-OH is added to obtain Fmoc-His(trt)-lys(Boc)Wang Resin through the acylating-aminating reaction; Fmoc is removed, and then glycin is added, and Fmoc-Gly-His(trt)-lys(Boc)Wang Resin is obtained through the acylating-aminating reaction; Fmoc is removed, and then TFA is added, after oscillation, the filter is performed, filtrate is taken to be washed and frozen out to obtain the end product.

Description

The solid phase synthesis process of glycyl histidyl-Methionin

Technical field: the present invention relates to a seed amino acid synthetic method, particularly a kind of solid phase synthesis process of glycyl histidyl-Methionin.

Background technology: glycyl histidyl-Methionin (GHK) is at first to separate a kind of tripeptides obtain by people such as Pickart from human plasma in 1973.GHK can increase mouse normal liver cell survival rate and impel the growth of liver cancer cell, and stimulate the synthetic of these cell DNAs and RNA, therefore be called as pHGF.Current research shows that also GHK and GHK-Gu2+ (complex compound that GHK and bivalent cupric ion form) can impel neurocyte, the growth division and the differentiation of immunity-associated cell and messangial cell.Owing in the GHK molecule 2 basic aminoacidss are arranged, bring a lot of difficulties to synthetic.Traditional liquid-phase synthesis process synthesis cycle is long, and technology is loaded down with trivial details, pollutes greatly, has not more and more met present production requirement.

Summary of the invention: the solid phase synthesis process that the purpose of this invention is to provide a kind of glycyl histidyl-Methionin.Mainly solve and contain 2 basic aminoacidss in its molecule and cause synthetic difficult technologies problem.Technical scheme of the present invention is: a kind of solid phase synthesis process of glycyl histidyl-Methionin may further comprise the steps:

1, Fmoc-Lys (BOC)-OH and Wang Resin (king's resin) are mixed in proportion, add HOBT, DIC and DMAP as activator, make solvent 45 ℃～50 ℃ following oscillatory reactions with DMF, obtain Fmoc-lys (Boc)-Wang Resin behind the reaction product filtration washing;

2, with Fmoc-lys (Boc) the Wang Resin that obtains in the step 1, add in the DMF solution of hexahydropyridine, the Fmoc group is removed, obtain H-lys (Boc) Wang Resin; Add Fmoc-His (trt)-OH then, reaction obtains Fmoc-His (trt)-lys (Boc) Wang Resin through amidated;

3, the Fmoc with Fmoc-His (trt)-lys (Boc) Wang Resin of obtaining in the step 2 removes, and adds Fmoc-Gly-OH, obtains Fmoc-Gly-His (trt)-lys (Boc) Wang Resin by the amidated reaction;

4, get product in the step 3, remove Fmoc, add TFA, the vibration after-filtration is got filtrate washing freeze-drying and can be got H-Gly-His-Lys-OH.

Methionin is excessive 1.5～2 times in the step 1, and the oscillatory reaction time is 3～5 hours; The concentration of the DMF solution of hexahydropyridine is 10～30% in the step 2, and step 4 duration of oscillation is 1～3 hour.

Some abbreviations commonly used have following implication among the present invention:

Fmoc: fluorenylmethyloxycarbonyl

Wang Resin: king's resin

Boc: tertbutyloxycarbonyl

Fmoc-Lys (BOC)-OH: Methionin

The HOBt:1-hydroxybenzotriazole

DIC:N, N '-di-isopropyl carbimide

The DMAP:4-Dimethylamino pyridine

DMF:N, N '-dimethyl formamide

Trt: trityl

Lys: Methionin

His: Histidine

Gly: glycine

TFA: trifluoroacetic acid

Fmoc-lys (Boc)-Wang Resin: fluorenylmethyloxycarbonyl Methionin (tertbutyloxycarbonyl) king resin

H-lys (Boc) Wang Resin: Methionin (tertbutyloxycarbonyl) king resin

Fmoc-His (trt)-lys (Boc) Wang Resin: fluorenylmethyloxycarbonyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin

Fmoc-His (trt)-OH: fluorenylmethyloxycarbonyl (trityl) Histidine

H-His (trt)-lys (Boc) Wang Resin: histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin

Fmoc-Gly-His (trt)-lys (Boc) Wang Resin: fluorenylmethyloxycarbonyl glycyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin

Fmoc-Gly-OH: fluorenylmethyloxycarbonyl glycine

H-Gly-His (trt)-lys (Boc) Wang Resin: glycyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin

H-Gly-His-Lys-OH: glycyl histidyl-Methionin

The invention has the beneficial effects as follows: speed of response of the present invention is fast, pollute little, the productive rate height.Because what adopt is the amino acid of Fmoc system, safer than traditional Boc system, convenient, what adopt when taking off Fmoc is that the DMF solution of 20% hexahydropyridine is more safer than 50% trifluoroacetic acid (TFA), pollutes littler.Polypeptide is to add water to quantitative back to wash with ethyl acetate after resin cuts down.The bad ammonia ester that contains among the GHK is hydrophilic amino acid, is not fine with traditional method of separating out that adds diethyl ether, and the curing of polypeptide in ether is not very desirable, and causes waste easily.The treatment process of present method has been avoided this point, so productive rate is higher.

Embodiment:

Embodiment 1:

1) drop into Fmoc-Lys (Boc)-OH and Wang Resin in the eggplant-shape bottle in proportion; excessive 1.5 times of Methionin; DMF makees solvent; with Fmoc-Lys (Boc)-OH dissolving; add DIC again; HOBT; DMAP is as activator; under 45 ℃～50 ℃ temperature; put into vibrator oscillatory reaction 4h, take out the back and filter with sand core funnel. use DMF; methyl alcohol; methylene dichloride washs resulting resin successively, weighs after drying; calculate the loading value (being the amount of Fmoc-Lys (Boc)-OH contained in every gram resin) of resin, with Fmoc-Lys (the Boc)-OH functional group acetylize that does not have on the Wang Resin to connect.Concrete operations are: above-mentioned resin is dropped in the eggplant-shape bottle; adding the acetylizad liquor capacity proportioning of functional group is: aceticanhydride: methylene dichloride: the solution of pyridine=1: 2: 2; under 45 ℃～50 ℃ temperature, in vibrator, react 2h; taking out the back filters with sand core funnel; use DMF, methylene dichloride, 5% aqueous acetic acid then; methyl alcohol; methylene dichloride washs 2～3 times successively; dry; weigh; the resin that takes a morsel is tested look with the triketohydrindene hydrate developer, if colourless then explanation reacts completely, obtains Fmoc-Lys (Boc) Wang Resin.

2) Fmoc-Lys (Boc) the Wang Resin that obtains in the step 1) is put into connect the peptide bottle, hexahydropyridine-DMF the solution of adding 20%, feed nitrogen reaction 30min, drain reaction solution, with DMF, methyl alcohol, methylene dichloride washing resin successively 2～3 times, the resin that takes a morsel is tested look with the triketohydrindene hydrate developer, and color is dark blue or bluish voilet, product 1 then is described) in Fmoc remove, obtain H-Lys (Boc) Wang Resin.

3) Fmoc-His (trt)-OH is added step 2 is housed) the connecing in the peptide bottle of product, dissolve with DMF, add HBTU (O-Benzotriazole-N, N, N ', N '-tetramethyl-uronium-hexafluorophosphate), the N-methylmorpholine, logical nitrogen reaction 40min, drain reaction solution, with DMF, methyl alcohol, methylene dichloride washing resin successively 2～3 times, the resin that takes a morsel is tested look with the triketohydrindene hydrate developer, color is that colourless then explanation reacts completely, and obtains product Fmoc-His (trt)-Lys (Boc) Wang Resin.

4) with step 2) described, with 20% hexahydropyridine-DMF solution removal Fmoc, to test look be dark blue or hyacinthine then illustrates and reacts completely, and obtains H-His (trt)-Lys (Boc) Wang Resin with resultant product in the step 3).

5) connecing in the peptide bottle of step 4) product is equipped with in the Fmac-Gly-OH adding, dissolve with DMF, add the HBTU.N-methylmorpholine, logical nitrogen reaction 40min, drain reaction solution, with DMF, methyl alcohol, methylene dichloride washing resin successively 2～3 times, the resin that takes a morsel is tested look, color is that colourless then explanation reacts completely, and obtains product Fmoc-Gly-His (trt)-Lys (Boc) Wang Resin.

6) be dark blue with adding 20% hexahydropyridine-DMF solution removal Fmoc in the products therefrom in the step 5), testing look or hyacinthine then illustrates and reacts completely, and obtains H-Gly-His (trt)-Lys (Boc) Wang Resin.

7) products therefrom in the step 6) is put into eggplant-shape bottle, add trifluoroacetic acid (TFA), put into vibrator and react, take out behind the 2h and filter with sand core funnel.In filtrate, add water to quantitatively, filtrate is added in the separating funnel,, at last the filtrate freeze-drying is got the white powder solid, be the finished product with the ethyl acetate washing.

Embodiment 2, and Methionin is excessive 1.7 times in the step 1, and the oscillatory reaction time is 3 hours; The concentration of the DMF solution of hexahydropyridine is 10% in the step 2,4,6, and step 7 duration of oscillation is 1 hour.All the other are identical with embodiment 1.

Embodiment 3, and Methionin is excessive 2 times in the step 1, and the oscillatory reaction time is 5 hours; The concentration of the DMF solution of hexahydropyridine is 30% in the step 2,4,6, and step 7 duration of oscillation is 3 hours.All the other are identical with embodiment 1.

Claims (7)

1. the solid phase synthesis process of a glycyl histidyl-Methionin is characterized in that may further comprise the steps:

A, Methionin and king's resin are mixed in proportion, add I-hydroxybenzotriazole, N, N '-di-isopropyl carbimide and 4-Dimethylamino pyridine are as activator, with N, N '-dimethyl formamide is made solvent 45 ℃～50 ℃ following oscillatory reactions, obtains fluorenylmethyloxycarbonyl Methionin (tertbutyloxycarbonyl) king resin behind the reaction product filtration washing;

B, with fluorenylmethyloxycarbonyl Methionin (tertbutyloxycarbonyl) the king resin that obtains in the step 1, add the N of hexahydropyridine, in N '-dimethyl formamide solution, the fluorenylmethyloxycarbonyl group is removed, obtain Methionin (tertbutyloxycarbonyl) king resin; Add fluorenylmethyloxycarbonyl (trityl) Histidine then, reaction obtains fluorenylmethyloxycarbonyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin through amidated;

C, the fluorenylmethyloxycarbonyl of fluorenylmethyloxycarbonyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) the king resin that obtains in the step 2 is removed, add the fluorenylmethyloxycarbonyl glycine, obtain fluorenylmethyloxycarbonyl glycyl histidyl-(trityl) Methionin (tertbutyloxycarbonyl) king resin by the amidated reaction;

D, get the product in the step 3, remove fluorenylmethyloxycarbonyl, add trifluoroacetic acid, the vibration after-filtration is got filtrate washing freeze-drying and can be got glycyl histidyl-Methionin.

2. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1, Methionin is excessive 1.5～2 times when it is characterized in that among the step a Methionin and king's mixed with resin.

3. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1 is characterized in that the oscillatory reaction time is 3～5 hours among the step a.

4. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1, the concentration that it is characterized in that the DMF solution of hexahydropyridine among the step b is 10～30%.

5. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1 is characterized in that duration of oscillation is 1～3 hour in the steps d.

6. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1 is characterized in that washing is to use N among the step a, and N '-dimethyl formamide, methylene dichloride, 5% aqueous acetic acid, methyl alcohol wash 2～3 times successively.

7. the solid phase synthesis process of glycyl histidyl-Methionin according to claim 1 is characterized in that the used washings of washing is an ethyl acetate in the steps d.