CN103396475B - A kind of method of pure solid-phase synthetic peptide class microbiotic Colistin - Google Patents
A kind of method of pure solid-phase synthetic peptide class microbiotic Colistin Download PDFInfo
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- CN103396475B CN103396475B CN201310339190.5A CN201310339190A CN103396475B CN 103396475 B CN103396475 B CN 103396475B CN 201310339190 A CN201310339190 A CN 201310339190A CN 103396475 B CN103396475 B CN 103396475B
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Abstract
The invention belongs to technical field of medicine synthesis, disclose a kind of method of pure solid-phase synthetic peptide class microbiotic Colistin, this process simplify synthesis technique and the step of Colistin, shorten generated time, decrease the usage quantity of solvent simultaneously.
Description
Technical field
The present invention relates to technical field of medicine synthesis, be specifically related to the pure solid phase synthesis process of polypeptide antibiotics Colistin.
Background technology
Colistin, molecular formula is C
52h
98n
16o
13; Relative molecular mass is 1155.45; CAS registration number is 1264-72-8; Its structure is shown below:
C8H15O-Dab-Thr-Dab-Dab-(Dab-Leu-Leu-Dab-Dab-Thr)。
The polymyxin microbiotic that Colistin produces for bacterial classification specific in Bacillus polymyxa var.colistinus, it is anti-gram negative bacillus microbiotic, tool germicidal action, stronger anti-microbial effect is had to most of gram negative bacillus, more remarkable to the effect of Pseudomonas aeruginosa, also there is good effect to intestinal bacteria, Salmonellas, dysentery bacterium, hemophilus influenza, bordetella pertussis, gas bacillus and pneumobacillus etc.Be mainly used in treating the various infection that gram positive bacterial infection, particularly Pseudomonas aeruginosa and intestinal bacteria cause clinically, as respiratory system infection, peritonitis, bile duct infection, urinary tract infections, burn infection, cornea infect and septicemia etc.Therefore, Colistin has very high pharmaceutical use and wide market outlook.
The synthetic method of current Colistin is mainly the solid liquid phase combining method (T.Kline, D.Holub, J.Therrien, T.Leung and D.Ryckman, J.Peptide Res., 2001,57,175-187) of people's employings such as T.Kline.The method first adopts solid phase method to carry out the coupling of linear peptides, then carries out into ring in the liquid phase.There is complex steps in the method, the shortcoming such as needs to carry out repeatedly purifies and separates and solvent usage quantity is large.
Summary of the invention
The present invention is directed to the above-mentioned defect existed in prior art, provide a kind of method of pure solid-phase synthetic peptide class microbiotic Colistin, this process simplify synthesis technique and the step of Colistin, shorten generated time, decrease the usage quantity of solvent simultaneously.
For this reason, one aspect of the present invention provides a kind of method of pure solid phase synthesis Colistin, and it comprises the steps:
1) Fmoc-Thr-OAll and resins synthesis, obtains Fmoc-Thr (resin)-OAll;
2) Fmoc-Thr (resin)-OAll adopts the mode of coupling one by one to obtain Colistin-linear peptides resin;
3) Colistin-linear peptides resin completes annulation on resin, forms cyclic peptide, i.e. Colistin-peptide resin;
4) Colistin-peptide resin is through scission reaction, obtains the thick peptide of Colistin;
5) the thick peptide of Colistin purified, turn salt and freeze-drying after obtain Colistin sterling.
On the other hand, present invention also offers the method for another pure solid phase synthesis Colistin, comprise the steps:
1) Fmoc-Thr-OAll and resins synthesis, obtains Fmoc-Thr (resin)-OAll;
2) Dab, Dab, Leu, Leu and Dab of Fmoc-Thr (the resin)-OAll mode coupling Fmoc blocking group successively that adopts coupling one by one;
3) on resin, complete annulation, form cyclic peptide;
4) coupling is continued successively after forming cyclic peptide, until obtain Colistin-peptide resin;
5) Colistin-peptide resin is through scission reaction, obtains the thick peptide of Colistin;
6) the thick peptide of Colistin purified, turn salt and freeze-drying after obtain Colistin sterling.
In the method for above-mentioned two kinds of pure solid phase synthesis Colistin, the solid support resin described in step 1) is preferably DHP HM resin.The substitution degree of Fmoc-Thr (the resin)-OAll obtained in step 1) is preferably 0.2 ~ 0.5mmol/g.
In addition; in a preferred embodiment; step 2) described in coupling one by one mode in; when being coupled to the 6th; the synthesis material of the 6th Dab is the side chain protected group of Fmoc-Dab (mtt)-OH or Fmoc-Dab (Alloc)-OH, the Dab on all the other positions is Boc.The reagent of deprotection group All and Alloc is preferably Pd (PPh
3)
4and phenyl silane, the reagent of deprotection group mtt is preferably the DCM solution containing 5%TFA.
In the method for above-mentioned two kinds of pure solid phase synthesis Colistin, the coupling system completing annulation on resin described in step 3) is preferably HATU, HOAt and DIPEA or PyBOP, HOBt and DIPEA.The lytic reagent that scission reaction adopts is preferably TFA:TIS:H
2o=90 ~ 98:0 ~ 5:2 ~ 5(V:V).
In addition, in a preferred embodiment, the purification process of Colistin is preferably RPLC method of purification, and chromatographic column is anti-phase C18 post.
Seen from the above description, compared with prior art, the method that present invention employs pure solid phase has synthesized polypeptide antibiotics Colistin, this process simplify synthesis technique and the step of Colistin, shortens generated time, decrease the usage quantity of solvent simultaneously.
Embodiment
Below by embodiment, the present invention is described in further detail, is intended to non-limiting the present invention for illustration of the present invention.It should be pointed out that to those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and fall into too within protection scope of the present invention.
The implication of abbreviation used in the present invention is listed in the following table.
Embodiment 1: substitution degree is the preparation of Fmoc-Thr (the Resin)-OAll of 0.2mmol/g
Take the DHP HM resin 100g(100mmol that substitution degree is 1.0mmol/g); add in solid state reaction post; 2 times are washed with DCE; with DCE swellable resins after 60 minutes; get 22.7gFmoc-Thr-OAll(60mmol) and 7.5g PPTS(30mmol) be dissolved in 200ml DCM solution; add in resin, under nitrogen protection, 80 DEG C of reaction 5h.Cold filtration, adopts DMF, DCM, Hexane to wash three times respectively respectively, and dry, detection substitution degree is 0.202mmol/g.
Embodiment 2: substitution degree is the preparation of Fmoc-Thr (the Resin)-OAll of 0.5mmol/g
Take the DHP HM resin 100g(100mmol that substitution degree is 1.0mmol/g); add in solid state reaction post; 2 times are washed with DCE; with DCE swellable resins after 60 minutes; get 56.8gFmoc-Thr-OAll(150mmol) and 18.8g PPTS(75mmol) be dissolved in 200ml DCM solution; add in resin, under nitrogen protection, 80 DEG C of reaction 5h.Cold filtration, adopts DMF, DCM, Hexane to wash three times respectively respectively, and dry, detection substitution degree is 0.510mmol/g.
Embodiment 3: substitution degree is the preparation of Fmoc-Thr (the Resin)-OAll of 0.3mmol/g
Take the DHP HM resin 100g(100mmol that substitution degree is 1.0mmol/g); add in solid state reaction post; 2 times are washed with DCE; with DCE swellable resins after 60 minutes; get 34.1gFmoc-Thr-OAll(90mmol) and 11.3g PPTS(45mmol) be dissolved in 200ml DCM solution; add in resin, under nitrogen protection, 80 DEG C of reaction 5h.Cold filtration, adopts DMF, DCM, Hexane to wash three times respectively respectively, and dry, detection substitution degree is 0.308mmol/g.
The preparation of embodiment 4:Colistin-linear peptides resin
In preparation embodiment 3, substitution degree is Fmoc-Thr (DHP the HMResin)-OAll resin 324.7g(100mmol of 0.308mmol/g); join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; with DBLK(20% piperidines/DMF) remove Fmoc protection; then wash 4 times with DMF, DCM washes 2 times.Get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6g HOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete the coupling of Fmoc-Dab (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Dab (Alloc)-OH, Fmoc-Dab (Boc)-OH, Fmoc-Dab (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Dab (Boc)-OH, 6-Methyloctanoic Acid successively.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, weighs and obtain Colistin-linear peptides resin 471.6g.
The preparation of embodiment 5:Colistin-linear peptides resin
In preparation embodiment 3, substitution degree is Fmoc-Thr (DHP HM the Resin)-OAll resin 324.7g(100mmol of 0.308mmol/g); join in solid state reaction post; 2 times are washed with DMF; with DMF swellable resins after 30 minutes; with DBLK(20% piperidines/DMF) remove Fmoc protection; then wash 4 times with DMF, DCM washes 2 times.Get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6g HOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete the coupling of Fmoc-Dab (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Dab (mtt)-OH, Fmoc-Dab (Boc)-OH, Fmoc-Dab (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Dab (Boc)-OH, 6-Methyloctanoic Acid successively.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, weighs and obtain Colistin-linear peptides resin 478.6g.
The preparation of embodiment 6:Colistin-peptide resin
471.6g Colistin-linear peptides resin C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (Alloc)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHPHM Resin)-OAll in Example 4, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, add 1000mL methylene dichloride, add 147.8mL phenyl silane (1.2mol) again, react 3 minutes, then add 28.9g Pd (PPh
3)
4(25mmol), room temperature reaction 45 minutes, take out reaction solution, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Namely C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OH is obtained.Take 38.0g HATU(100mmol), 16.3g HOAt(120mmol) be dissolved in DMF and the DMSO mixing solutions that volume ratio is 9:1, add 34.8ml DIPEA(200mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 5 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, namely obtains Colistin-peptide resin 470.3g.
The preparation of embodiment 7:Colistin-peptide resin
471.6g Colistin-linear peptides resin C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (Alloc)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHPHM Resin)-OAll in Example 4, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, add 1000mL methylene dichloride, add 147.8mL phenyl silane (1.2mol) again, react 3 minutes, then add 28.9g Pd (PPh
3)
4(25mmol), room temperature reaction 45 minutes, takes out reaction solution, and DMF washs 6 times, detects color of resin with ninhydrin method, and resin has color, represents that Fmoc removes.Namely C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OH is obtained.Take 52.0gPyBOP(100mmol), 16.2g HOBt(120mmol) be dissolved in DMF and the DMSO mixing solutions that volume ratio is 9:1, add 34.8ml DIPEA(200mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 5 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, namely obtains Colistin-peptide resin 469.4g.
The preparation of embodiment 8:Colistin-peptide resin
487.6g Colistin-linear peptides resin C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (mtt)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHPHM Resin)-OAll in Example 5, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, add 1000mL methylene dichloride, add 147.8mL phenyl silane (1.2mol) again, react 3 minutes, then add 28.9g Pd (PPh
3)
4(25mmol), room temperature reaction 45 minutes, take out reaction solution, DMF washs 6 times, namely obtains C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (mtt)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HMResin)-OH.Adding volume ratio is DCM solution 1000ml containing 5%TFA, and room temperature reaction 2h, takes out reaction solution, and DMF washs 6 times, detects color of resin with ninhydrin method, and resin has color, represents that Fmoc removes.Namely C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OH is obtained.Take 38.0gHATU(100mmol), 16.3g HOAt(120mmol) be dissolved in DMF and the DMSO mixing solutions that volume ratio is 9:1, add 34.8ml DIPEA(200mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 5 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, namely obtains Colistin-peptide resin 485.9g.
The preparation of embodiment 9:Colistin-peptide resin
487.6g Colistin-linear peptides resin C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (mtt)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHPHM Resin)-OAll in Example 5, join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, add 1000mL methylene dichloride, add 147.8mL phenyl silane (1.2mol) again, react 3 minutes, then add 28.9g Pd (PPh
3)
4(25mmol), room temperature reaction 45 minutes, take out reaction solution, DMF washs 6 times, namely obtains C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab (mtt)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HMResin)-OH.Adding volume ratio is DCM solution 1000ml containing 5%TFA, and room temperature reaction 2h, takes out reaction solution, and DMF washs 6 times, detects color of resin with ninhydrin method, and resin has color, represents that Fmoc removes.Namely C8H15O-Dab (Boc)-Thr (tBu)-Dab (Boc)-Dab (Boc)-Dab-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OH is obtained.Take 52.0gPyBOP(100mmol), 16.2g HOBt(120mmol) be dissolved in DMF and the DMSO mixing solutions that volume ratio is 9:1, add 34.8ml DIPEA(200mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 5 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, namely obtains Colistin-peptide resin 482.9g.
The preparation of embodiment 10:Colistin-peptide resin
In preparation embodiment 3, substitution degree is Fmoc-Thr (DHP HM the Resin)-OAll resin 324.7g(100mmol of 0.308mmol/g), join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6g HOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete Fmoc-Dab (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Dab (Alloc)-OH successively, obtain peptide sequence Fmoc-Dab (Alloc)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OAll.As embodiment 7 or 8, adopt phenyl silane and Pd (PPh
3)
4remove Alloc and All group, HATU, HOAt and DIPEA or PyBOP, HOBt and DIPEA system carry out cyclisation.With DBLK(20% piperidines/DMF) remove Fmoc protection, then wash 4 times with DMF, DCM washes 2 times.Get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6g HOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete the coupling of Fmoc-Dab (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Dab (Boc)-OH, 6-Methyloctanoic Acid successively.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, weighs and obtain Colistin-peptide resin 479.2g.
The preparation of embodiment 11:Colistin-peptide resin
In preparation embodiment 3, substitution degree is Fmoc-Thr (DHP HM the Resin)-OAll resin 324.7g(100mmol of 0.308mmol/g), join in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins after 30 minutes, get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6g HOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete Fmoc-Dab (Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Dab (mtt)-OH successively, obtain peptide sequence Fmoc-Dab (mtt)-Leu-Leu-Dab (Boc)-Dab (Boc)-Thr (DHP HM Resin)-OAll.As embodiment 9 or 10, adopt phenyl silane and Pd (PPh
3)
4remove All group, with the DCM solution removal mtt containing 5%TFA, then adopt HATU, HOAt and DIPEA or PyBOP, HOBt and DIPEA system to carry out cyclisation.With DBLK(20% piperidines/DMF) remove Fmoc protection, then wash 4 times with DMF, DCM washes 2 times.Get 132.2g Fmoc-Dab (Boc)-OH(300mmol), 48.6gHOBt(360mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 56.3ml DIC(360mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Remove Fmoc protection with DBLK, then wash 6 times with DMF, detect color of resin with ninhydrin method, resin has color, represents that Fmoc removes.According to Colistin main chain peptide sequence, complete the coupling of Fmoc-Dab (Boc)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Dab (Boc)-OH, 6-Methyloctanoic Acid successively.Reaction terminates rear methyl alcohol and shrinks, and resin vacuum dried overnight, weighs and obtain Colistin-peptide resin 483.2g.
The preparation of the thick peptide of embodiment 12:Colistin
In Example 7, the peptide resin of preparation is placed in cracking reactor, adds lytic reagent (TFA:TIS: water=90:5:5 (V/V)), stirring at room temperature 2h with the ratio of 10ml/g resin.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 114.9g of Colistin, and thick peptide weight yield is 99.4%, HPLC purity is 57.2%.
The preparation of the thick peptide of embodiment 13:Colistin
In Example 8, the peptide resin of preparation is placed in cracking reactor, adds lytic reagent (TFA: water=98:2 (V/V)), stirring at room temperature 2h with the ratio of 10ml/g resin.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 109.8g of Colistin, and thick peptide weight yield is 95.0%, HPLC purity is 53.6%.
The preparation of the thick peptide of embodiment 14:Colistin
In Example 9, the peptide resin of preparation is placed in cracking reactor, adds lytic reagent (TFA:TIS: water=95:2:3 (V/V)), stirring at room temperature 2h with the ratio of 10ml/g resin.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide 116.8g of Colistin, and thick peptide weight yield is 101.1%, HPLC purity is 56.4%.
The preparation of the thick peptide of embodiment 15:Colistin
In Example 10 to 12, the peptide resin of preparation is placed in cracking reactor, adds lytic reagent (TFA:TIS: water=94:3:3 (V/V)), stirring at room temperature 2h with the ratio of 10ml/g resin.Reactant sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, wash 3 times with anhydrous diethyl ether, vacuum-drying obtains white powder solid, i.e. the thick peptide of Colistin, and thick peptide weight yield is greater than 95%, HPLC purity and is greater than 55%.
The purifying of the thick peptide of embodiment 16:Colistin, turn salt and freeze-drying
To take in embodiment 13 to 16 after any 100.0g Colistin thick peptide 10L water dissolution, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.2%TFA/ acetonitrile mobile phase purifying, collect object peak cut, obtain purity and be greater than 98.5% smart peptide.Smart peptide solution is adopted Waters2545RP-HPLC system, and chromatographic column is the anti-phase C18 post of 50 × 250mm, 0.05% (NH
4)
2sO
4solution (pH2.5)/acetonitrile mobile phase turns salt, collects object peak cut, and rotary evaporation concentrates, and freeze-drying obtains Colistin vitriol essence peptide and is greater than 22g, and it is 98.5% that calculating total recovery is greater than 20.0%, RP-HPLC purity.
Claims (9)
1. a method of solid phase synthesis Colistin, comprises the steps:
1) Fmoc-Thr-OAll and resins synthesis, obtains Fmoc-Thr (resin)-OAll;
2) Fmoc-Thr (resin)-OAll adopts the mode of coupling one by one to obtain Colistin-linear peptides resin;
3) Colistin-linear peptides resin completes annulation on resin, forms cyclic peptide, i.e. Colistin-peptide resin;
4) Colistin-peptide resin is through scission reaction, obtains the thick peptide of Colistin;
5) the thick peptide of Colistin purified, turn salt and freeze-drying after obtain Colistin sterling.
2. a method of solid phase synthesis Colistin, comprises the steps:
1) Fmoc-Thr-OAll and resins synthesis, obtains Fmoc-Thr (resin)-OAll;
2) Dab, Dab, Leu, Leu and Dab of Fmoc-Thr (the resin)-OAll mode coupling Fmoc blocking group successively that adopts coupling one by one;
3) on resin, complete annulation, form cyclic peptide;
4) coupling is continued successively after forming cyclic peptide, until obtain Colistin-peptide resin;
5) Colistin-peptide resin is through scission reaction, obtains the thick peptide of Colistin;
6) the thick peptide of Colistin purified, turn salt and freeze-drying after obtain Colistin sterling.
3. method according to claim 1 and 2, the solid support resin wherein described in step 1) is DHP HM resin.
4. method according to claim 1 and 2, the substitution degree of Fmoc-Thr (the resin)-OAll of wherein step 1) acquisition is 0.2 ~ 0.5mmol/g.
5. method according to claim 1 and 2; wherein in step 2) described in coupling one by one mode in; when being coupled to the 6th; the synthesis material of the 6th Dab is the side chain protected group of Fmoc-Dab (mtt)-OH or Fmoc-Dab (Alloc)-OH, the Dab on all the other positions is Boc.
6. method according to claim 5, wherein the reagent of deprotection group All and Alloc is Pd (PPh
3)
4and phenyl silane, the reagent of deprotection group mtt is the DCM solution containing 5%TFA.
7. method according to claim 1 and 2, the coupling system completing annulation on resin wherein described in step 3) is HATU, HOAt and DIPEA or PyBOP, HOBt and DIPEA.
8. method according to claim 1 and 2, the lytic reagent that wherein said scission reaction adopts is TFA:TIS:H
2o=90 ~ 98:0 ~ 5:2 ~ 5(V:V).
9. method according to claim 1 and 2, wherein said purification process is RPLC method of purification, and chromatographic column is anti-phase C18 post.
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| CN101010336A (en) * | 2004-07-01 | 2007-08-01 | 生物资源药物股份有限公司 | Peptide antibiotics and their preparation methods |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
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| CN101010336A (en) * | 2004-07-01 | 2007-08-01 | 生物资源药物股份有限公司 | Peptide antibiotics and their preparation methods |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
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| Title |
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| Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides;T. Kline et al.;《J. Peptide Res.》;20011231;第57卷;图3，第183页右栏第2-4段，第185页左栏第1段 * |
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