CN102167731B - Method for preparing omiganan through solid phase peptide synthesis - Google Patents
Method for preparing omiganan through solid phase peptide synthesis Download PDFInfo
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Abstract
The invention discloses a method for preparing omiganan through solid phase peptide synthesis. The method comprises the following steps of: connecting RinkAmideMBHA resin serving as an initial material with amino acids with Fmoc-protecting groups in turn according to a solid phase synthesis method to obtain the protective dodecapeptide resin, wherein the last amino acid uses Boc-Ile-OH, and removing the Fmoc-protecting groups in turn; synchronously performing side chain protecting group removal and peptide cutting to obtain a crude product; and performing separation and purification by using a preparative high-performance liquid chromatography (a C18 column), and performing freeze drying to obtain the refined omiganan. By the method, the process is stable, raw materials and auxiliary materials are wide in sources, the production period is short, the yield is high, the quality is stable, the production cost is low, the peptide connecting yield is high, highly toxic reagents such as hydrogen fluoride are avoided, and three-waste pollution is light. The peptide connecting yield in each step is over 98 percent, the yield after peptide cutting is 95.8 percent, the purification yield is over 35 percent, and the total yield is 36.7 percent. The method is suitable for industrial implementation and has good industrial prospect.
Description
Technical field
The present invention relates to the preparation method of a kind of Omiganan (Omiganan), be specifically related to the preparation method of omiganan through solid phase peptide synthesis.
Background technology
Chinese name: Omiganan
English name: Omiganan
CAS number: 204248-78-2
Molecular formula: C
90H
127N
27O
12
Molecular weight: 1779.0
Aminoacid sequence: Ile-Leu-Arg-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Lys-NH
2
Omiganan is 12 peptides of a synthetic.The clinical three phases tests latter stage that has entered at present that U.S. FDA declares.Its former Cadence Pharma of company that grinds externally announces in September, 2010, and the clinical three phase test-results of Omiganan are considerable, and this product will be in the recent period by FDA approval listing, the infection that is used for the treatment of and prevents the catheter intubate to cause.
Omiganan is in existing 4 related application of the U.S., and the patent holder is the former person of grinding Cadence Pharma.Main contents involve adaptation purposes and the reagent combination of this medicine, wherein US 6835536 special protections the targeting of said preparation prescription about urinary system infection treatment.But do not find the related application of this peptide sequence and chemosynthesis preparation.
In the Intellectual Property in China patent database, not yet inquire patent or the patent application of disclosed relevant Omiganan.
Summary of the invention
The objective of the invention is to disclose a kind of preparation method of omiganan through solid phase peptide synthesis.The present invention proposes a more suitable route, use the synthetic Omiganan of solid phase method, and yield is higher.
Technical scheme of the present invention may further comprise the steps:
(1) take Rink Amide mbha resin as starting raw material, method according to solid phase synthesis connects the amino acid with Fmoc-blocking group successively, last amino acid uses Boc-Ile-OH, obtains the dodecapeptide resin of protection, sloughs successively the Fmoc-blocking group therebetween;
(2) cut peptide, take off synchronously the side chain protected group, obtain crude product;
(3) crude product adds the acid treatment of 15ml salt through preparation HPLC (C18 post) separation and purification, and solvent is removed in decompression, after the freeze-drying, makes the Omiganan elaboration.
Omiganan has 4 basic aminoacidss, 1 Lys wherein, 3 Arg.Other has 4 Trp, therefore synthesizes to polypeptide and brings certain difficulty.The present invention adopts the technique of unique combination to prepare Omiganan, has obtained beyond thought effect.The present invention is take Rink Amide mbha resin as starting raw material, adopts Fmoc-Lys (Boc)-OH with first amino acid of resin condensation, and with unreacted amino on the aceticanhydride sealing resin.Then, carry out condensation reaction with Fmoc-Arg (Pbf)-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Trp-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Boc-Ile-OH one by one, obtain Boc-Ile-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin; With the special peptide reagent (HCl/i-PrOH/DCM/Na that cuts
2S=1/3.5/4/1.5) polypeptide is cut from resin; With special moving phase (0.2MNH
4SO
4/ acetonitrile=7.5/2.5) carries out separation and purification; Obtain purity greater than 98% Omiganan.Technical process is as follows:
Rink Amide MBHA-resin
↓ 1. PIP; 2. HBTU/HOBt, Fmoc-Lys (Boc)-OH; 3. aceticanhydride
Fmoc-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Arg(Pbf)-OH
Fmoc-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Arg(Pbf)-OH
Fmoc-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Trp-OH
Fmoc-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Pro-OH
Fmoc-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Trp-OH
Fmoc-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Trp-OH
Fmoc-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Pro-OH
Fmoc-Pro Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Trp-OH
Fmoc-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Arg(Pbf)-OH
Fmoc-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Fmoc-Leu-OH
Fmoc-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓①PIP；②HBTU/HOBt，Boc-Ile-OH
Boc-Ile-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
↓ 1. HCl/i-PrOH/DCM/Na
2S; 2. NH
4SO
4Solution/acetonitrile
Ile-Leu-Arg-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-Lys-NH
2
By above-mentioned disclosed technical scheme as seen; method of the present invention; adopting Rink Amide mbha resin is starting raw material, connects successively the amino acid with Fmoc-blocking group according to the method for solid phase synthesis, obtains the dodecapeptide resin of protection; slough successively the Fmoc-blocking group therebetween; take off synchronously the side chain protected group and cut peptide, obtain crude product, crude product is through preparation HPLC (C18 post) separation and purification; after the freeze-drying, make the Omiganan elaboration.Method of the present invention, process stabilizing, the raw and auxiliary material convenient sources, with short production cycle, yield is high, steady quality, production cost is low, connects the peptide yield high, avoids using the poisonous reagents such as hydrogen fluoride, and three-waste pollution is few.Per step connects the peptide yield all more than 98%.Cut that yield is 95.8% behind the peptide, purification yield is up to more than 35%, and total recovery is 36.7%.This shows, method of the present invention is convenient to industrializing implementation, has larger industrialization prospect.
Description of drawings
Fig. 1 is process flow sheet of the present invention.
Embodiment
The raw material that adopts in embodiment and the aforementioned process is listed as follows:
Embodiment 1
Among the embodiment:
Connecing peptide reagent is: NMM: DMF=1: 10, and volume ratio;
The reagent of raising one's hat is: PIP: DMF=1: 3.5, and volume ratio;
1, preparation Fmoc-Lys (Boc)-resin
1) (the 0.8mmol/g resin 72mmol), soaked 1 hour with 1000mlDMF, made the abundant swelling of resin, drained to get the 90g resin.
2) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and respectively wash three times with DMF, methyl alcohol and DMF respectively, drain.
3) get 78g Fmoc-Lys (Boc)-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
4) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
5) add again aceticanhydride 100ml, DMF800ml, 25 ℃ were reacted 1 hour.
6) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
2, preparation Fmoc-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and respectively wash three times with DMF, methyl alcohol and DMF respectively, drain.
2) get 105g Fmoc-Arg (Pbf)-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
3, preparation Fmoc-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 105g Fmoc-Arg (Pbf)-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
4, preparation Fmoc-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 69g Fmoc-Trp-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
5, preparation Fmoc-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 54g Fmoc-Pro-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
6, preparation Fmoc-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 69g Fmoc-Trp-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
7, preparation Fmoc-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 69g Fmoc-Trp-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
8, preparation Fmoc-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 54g Fmoc-Pro-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
9, preparation Fmoc-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 69g Fmoc-Trp-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
10, preparation Fmoc-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 105g Fmoc-Arg (Pbf)-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
11, preparation Fmoc-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 57g Fmoc-Leu-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
12, preparation Boc-Ile-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin
1) add the 1000ml reagent of raising one's hat, 25 ℃ of reactions 15 minutes are drained, and with DMF, methyl alcohol and DMF washing three times, drain respectively.
2) get 38g Boc-Ile-OH, 63g HBTU, 22g HOBT, connect the peptide reagent dissolving with 750ml, add reaction vessel, 25 ℃ were reacted 1 hour.
3) drain, wash three times with DMF, methyl alcohol and DMF respectively, drain.
13, cut peptide:
To protect the dodecapeptide resin transfer to the 2000ml reactor, adding prepares and is chilled in advance approximately 5 ℃ reagent (HCl/i-PrOH/DCM/Na in advance
2S=100g/350ml/400ml/150g), 25 ℃ were stirred 2 hours.Decompression desolventizes to small volume, adds the 1000ml cold diethyl ether, and precipitation is filtered collecting precipitation, wash fully with anhydrous diethyl ether after, vacuum-drying.Get Omiganan crude product 122.8g (69mmol), yield 95.8%.
14, separation and purification:
The Omiganan crude product is dissolved in 5% acetic acid, filters, filtrate is through the C18 column purification, moving phase: 0.2M NH
4SO
4: acetonitrile (7.5: 2.5, volume ratio); Flow velocity is 700ml/min; The detection wavelength is: 280nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, add the 15ml hcl acidifying after the sample peak merges, after the removal of solvent under reduced pressure, freeze-drying, approximately get 51.8g white block finished product (MW:1961.45,26.4mmol), total recovery 36.7% (in the 72mmol of resin).
Claims (6)
1. the preparation method of an omiganan through solid phase peptide synthesis is characterized in that, may further comprise the steps:
At first, take Rink Amide mbha resin as starting raw material, connect successively the amino acid with Fmoc-blocking group according to the method for solid phase synthesis, last amino acid uses Boc-Ile-OH, obtain the dodecapeptide resin of protection, slough successively the Fmoc-blocking group therebetween;
Secondly, cut peptide and remove simultaneously the side chain protected groups such as Boc and Pbf, with the Boc-Ile-Leu-Arg (Pbf) that obtains-Trp-Pro
-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin adding is chilled in advance 0 ~ 10 ℃ cut in the peptide reagent and reacts, and obtains crude product, and cutting peptide reagent is HCl/i-PrOH/DCM/Na
2S=0.5-2/3-4/3-5/1-2, described ratio refers to HCl, Na
2The quality of S and i-PrOH, DCM and volume ratio, quality are take g as unit, and volume is take ml as unit;
At last, crude product is dissolved in the Omiganan crude product in the acetic acid through preparation HPLC C18 column separating purification, filters, and filtrate is through the HPLC purifying, and moving phase is 0.1 ~ 1M ammonium sulfate: acetonitrile=9 ~ 6: 1 ~ 4; Flow velocity is 100-1500ml/min; The detection wavelength is: 250-300nm; Follow the tracks of the needed effluent liquid of collection with liquid chromatograph, add the 15ml hcl acidifying after the sample peak merges, decompression desolventizes, freeze-drying, the acquisition finished product;
Take Rink Amide mbha resin as starting raw material, connect successively the amino acid with Fmoc-blocking group, obtain protection dodecapeptide resin, the method for sloughing successively the Fmoc-blocking group therebetween comprises the steps:
(1) preparation Fmoc-Lys (Boc)-resin:
Rink Amide mbha resin is soaked with DMF, make resin swelling, drain, add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, then add the mixture be dissolved in the Fmoc-Lys (Boc) that connects peptide reagent-OH, HBTU and HOBt, reaction is drained, and uses respectively DMF and methanol wash, drain, add aceticanhydride and DMF, reaction obtains Fmoc-Lys (Boc)-resin again;
The said peptide reagent that connects is: NMM: DMF=1: 5 ~ 15, and volume ratio, lower same;
The said reagent of raising one's hat is: PIP: DMF=1: 2 ~ 5, and volume ratio, lower same;
In the mixture, solvent is 5 ~ 20ml/g to the volumetric concentration of weight resin, and is lower same;
The mole number of HBTU is 2 ~ 6 times of resin, and is lower same;
The mole number of HOBt is 2 ~ 6 times of resin, and is lower same;
The mole number of Fmoc-Lys (Boc)-OH is 2 ~ 6 times of resin;
The reagent of raising one's hat is 5 ~ 20ml/g to the ratio of the add-on of resin, lower with;
(2) preparation Fmoc-Arg (Pbf)-Lys (Boc)-resin:
In the Fmoc-Lys of step (1) (Boc)-resin, add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, then add the mixture be dissolved in the Fmoc-Arg (Pbf) that connects peptide reagent-OH, HBTU, HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Arg (Pbf)-OH is 2 ~ 6 times of resin;
(3) preparation Fmoc-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (2), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add the mixture with the Fmoc-Arg (Pbf) that connects the peptide reagent dissolving-OH, HBTU and HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Arg (Pbf)-OH is 2 ~ 6 times of resin;
(4) preparation Fmoc-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (3), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Trp-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Trp-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(5) preparation Fmoc-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (4), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Pro-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Pro-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(6) preparation Fmoc-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (5), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Trp-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Trp-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(7) preparation Fmoc-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (6), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Trp-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Trp-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(8) preparation Fmoc-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (7), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Trp-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Pro-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(9) preparation Fmoc-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (8), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Trp-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Trp-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(10) preparation Fmoc-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (9), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add the mixture with the Fmoc-Arg (Pbf) that connects the peptide reagent dissolving-OH, HBTU and HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Arg (Pbf)-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(11) preparation Fmoc-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (10), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Fmoc-Leu-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Fmoc-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Fmoc-Leu-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same;
(12) preparation Boc-Ile-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin:
In the resin of step (11), add the reagent of raising one's hat, reaction is drained, use respectively DMF and methanol wash, drain, add with the Boc-Ile-OH, the HBTU that connect the peptide reagent dissolving and the mixture of HOBT, reaction, drain, use respectively DMF and methanol wash, drain, obtain Boc-Ile-Leu-Arg (Pbf)-Trp-Pro-Trp-Trp-Pro-Trp-Arg (Pbf)-Arg (Pbf)-Lys (Boc)-resin;
In the mixture, the mole number of Boc-Ile-OH is 2 ~ 6 times of resin;
All the other operations and processing condition are the same.
2. method according to claim 1 is characterized in that the described peptide reagent of cutting: HCl/i-PrOH/DCM/Na
2S=1/3.5/4/1.5, described ratio refers to HCl, Na
2The quality of S and i-PrOH, DCM and volume ratio, quality are take g as unit, and volume is take ml as unit.
3. method according to claim 1 is characterized in that, the weight concentration of acetic acid is 0.5 ~ 10%, and the weight concentration of Omiganan crude product is 1 ~ 50% in the acetic acid.
4. each described method is characterized in that according to claim 1 ~ 3, and the temperature of reaction of raising one's hat is 10 ~ 60 ℃, and the reaction times is 5 ~ 30 minutes.
5. each described method is characterized in that according to claim 1 ~ 3, and connecing the reactive polypeptide temperature is 10 ~ 60 ℃, and the reaction times is 0.5 ~ 1.5 hour.
6. each described method is characterized in that according to claim 1 ~ 3, and cutting the peptide temperature is 10 ~ 60 ℃, and the time is 1 ~ 6 hour.
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| US6180604B1 (en) * | 1996-08-21 | 2001-01-30 | Micrologix Biotech Inc. | Compositions and methods for treating infections using analogues of indolicidin |
| CN101522704A (en) * | 2006-10-05 | 2009-09-02 | 隆萨股份公司 | Method for peptide synthesis |
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| US6180604B1 (en) * | 1996-08-21 | 2001-01-30 | Micrologix Biotech Inc. | Compositions and methods for treating infections using analogues of indolicidin |
| CN101522704A (en) * | 2006-10-05 | 2009-09-02 | 隆萨股份公司 | Method for peptide synthesis |
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| 杨世林等.天然药物化学.《天然药物化学》.2010,427-429. * |
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