CN1724566A - Antibiotic, antiendotoxin allosteric peptide molecule and synthetic method thereof - Google Patents

Abstract

The present invention relates to can be antibiotic, antiendotoxin allosteric peptide molecule and synthetic method thereof.Be primarily characterized in that: a kind of antibiotic, antiendotoxin allosteric peptide molecule is characterized in that: have following general structure, R ¹-BNEP-X (R ³) n.Can be applied to kill bacteria, the treatment endotoxemia is used for the phytopathogen control.

Description

Antibiotic, antiendotoxin allosteric peptide molecule and synthetic method thereof

Described technical field

The present invention relates to be applied to kill bacteria, the treatment endotoxemia is used for a kind of antibiotic, antiendotoxin allosteric peptide molecule that phytopathogen is prevented and treated.

Background technology

Gram-negative (G ^-) bacillus Sepsis and shock be to cause one of clinical patient main causes of death, particularly with serious intracellular toxin (Lipopolysaccharide, LPS; Endotoxin, ET) G of mass formed by blood stasis ^-The infection of bacillus, case fatality rate reaches 50-80%.And at the treatment of endotoxemia, clinically owing to lack specific medicine, still there are not effective measure at present, only limit to anti symptom treatment and antibacterials, thereby cause the mortality ratio of infectious diseases high, therefore studying its pathogeny and seeking therapy measure is the focus that clinical medicine is paid close attention to always.

In recent years, along with to the going deep into of G-coli infections research, be present in that endotoxic effect comes into one's own day by day on the G-bacillus adventitia.LPS is considered to the Sepsis shock patient cause of death--property inflammatory response syndrome (Systemic inflammatoryresponse syndrome out of control, SIRS) and compensatory anti-inflammatory syndrome (Compensatory anti-inflammatory responsesyndrome, promotor CARS).Its main biological action of intracellular toxin that enters in the blood comprises the following aspects: (1. with can induce, discharge multiple inflammatory mediator such as TNF, IL-1 etc. after scavenger cell contacts, directly or indirectly bring into play part and systemic effect by these media.2. while intracellular toxin molecule also can combine with multiple tissue, parenchyma and directly cause cell manipulation.3. activate blood coagulation, fibrinolytic, complement system, bring out DIC.4. act on alpha-receptor, impel suprarenal gland catecholamine secretion, cause microcirculation disturbance, plasma extravasation, until shock taking place and other is as heating, oligoleukocythemia, Shwartzman reaction etc.In the inflammatory reaction network of complexity, regulation measure at various inflammatory factors such as TNF, IL-1 also constantly proposes respectively, but the result is disappointing, and it is inseparable that its major cause is that various inflammatory factor interacts, and only can not deal with problems at certain or several cytokines.Therefore catch the principal aspect of a contradiction---LPS, it is significant to seek the breakthrough point of dealing with problems.

Existing discovering: intracellular toxin is present in G ^-On the cytolemma of bacillus, its main chemical compositions is a lipopolysaccharides.Its basic comprising is O-specific side chains, core polysaccharide, lipoid A three parts.Wherein lipoid A is the active centre of toxin.Main biological action induces, discharges multiple inflammatory mediator such as TNF, IL-1, IL-6 etc. for stimulating monokaryon-phagocytic cell system, by these medium activated inflammation cascade reactions and direct or indirect performance part and systemic effect.

Over surplus in the of nearly ten year, in the world at G ^-Endotoxemia that coli infections is followed and the study on prevention of SIRS, mainly be how anti-, the monoclonal antibody at antiendotoxin core glycolipid (Lipid A), various anti-cytokine antibodies and receptor antagonist, as anti-TNF antibodies, anti-IL-8 I (IL-1) antibody, anti-IL-6 antibodies and NO antagonist etc.In animal experiment study; above-mentioned preparation all shows different provide protections to the visceral organ injury due to the Sepsis; but in clinical experimental study, all cut and do not obtain drugs approved by FDA, generally believe that bonding force is weak, to select improper be the major cause that causes developing failure for poor specificity, medication time because of uncertain therapeutic efficacy.

Bactericidal properties/power/permeability increasing protein (Bactericidal/permeability increasing protrin, BPI) be positively charged positive protein matter in a kind of people of being present in and the Mammals PMN azurophilic granule, the about 55kDa of molecular weight is made up of 456 amino acid, has the G of killing ^-Bacterium and in and the ability of LPS, be to promise to be most at present to be applied to clinical preparation, the concern of gone abroad numerous scholars and pharmacy corporation.Confirmed that at present BPI is to many G ^-Bacterium comprises performance germicidal actions such as Colibacter, Pseudomonas aeruginosa, salmonella, Shigella, Rhodopseudomonas, Klebsiella, proteus, serratia, eisseria; BPI also can with G ^-LPS specific combination on the bacterial membrane, the activity of the free LPS of inhibition.Terminal 1-199 the amino-acid residue fragment (BPI of BPI N- ₂₃) have all biological activity of complete BPI, its allosteric body BPI ₂₁Remove and have BPI ₂₃Biological activity outside, part G+ coccus such as drug-resistant staphylococcus aureus, suis are had fungicidal activity, and structure is more stable.Recombinant BPI by U.S.'s development _23/21Entered clinical study, preliminary clinical study shows that BPI can obviously reduce bacterial count in the blood, improves cardiac index, art pO2, blood pressure, reduction plasma endotoxin level, the animal survival rate that improves.External recombinant BPI ₂₃(rBPI ₂₃, see 1992, Infect.Immun.60:4754-4761) and mutant (rBPI ₂₁) development research entered the III clinical trial phase, PRELIMINARY RESULTS proves, rBPI ₂₃And rBPI ₂₁To serious G ^-Infectation of bacteria has significant curative effect.Because the treatment of infectation of bacteria and LPS mass formed by blood stasis needs in the medicine of absolute quantity and intracellular toxin and killing bacteria, so rBPI ₂₃And rBPI ₂₁The clinical application amount is bigger, and present production technique still adopts the method for cell fermentation, causes cost high, and market is difficult to promote.So, screen and find novel high reactivity, high specificity sterilization and in and the medicine of LPS become the focus that the various countries scientist pays close attention to.

Chinese patent application 94191894 and 94194835 relates to the biologically active peptides and the application thereof of the functional zone of bactericidal properties permeability-increasing protein, the aminoacid sequence behaviour sterilization/aminoacid sequence of enhancing permeability protein (BPI) functional zone that has or the peptide of its subsequence are wherein disclosed, and the varient of this sequence or its subsequence, they have the biologic activity of at least a BPI, as heparin in conjunction with the neutralization of, heparin, LBS in conjunction with, LPS neutralization or fungicidal activity, and provide the peptide that is used for various therepic use and the medicinal compositions of this peptide.Simultaneously, Chinese patent application 01104591 discloses varient and the sequence thereof by computer simulation, the New BP Neural I simulating peptide that filters out, and they all have LPS neutralization or fungicidal activity.Although more, still there is not a kind of medicine that can be used for the treatment of endotoxemia safely and effectively clinically to endotoxic research.Therefore, thus reaching the purpose that reduce to infect mortality ratio in focusing on and on the intracellular toxin has very important significance.

In research fields such as cytobiology and clinical pharmacies, many hydrophilic proteins, polypeptide and oligonucleotide are because of its uniqueness, biologic activity is counted as the ideal tools of research viable cell life behavior efficiently.For many years, biologists are devoted to explore the effective ways with in these tool molecule importing viable cell.Recent study is found: have many peptides to pass cytoplasmic membrane and (to see 1991, Proc Natl Acad Sci USA, 88 (5): 1864-1868) without any need for the assistance of membrane protein receptor; Other has a kind of peptide portability wetting ability macromole to pass hemato encephalic barrier, make " the wetting ability macromole is difficult to pass through hemato encephalic barrier " this difficult problem that is perplexing scientists for a long time had important breakthrough (see Science, 1999,285:1569-1572).These are important to be found to be in the future hydrophilic protein, oligopeptides and oligonucleotide and to be applied to research fields such as pharmacy, gene therapy, cytobiology and to have opened up a new road.Now, this class has peptide that the cell biological film penetrates function and is called as and wears film peptide (cell-penetrating peptides).They are class peptide molecules, and length is severally not wait to tens amino acid, just constantly is subjected to people's attention at present.

Technology contents

By the present known transcriptional activators-Tat (containing 86-102 amino-acid residue) that derives from virus of AIDS with known have sterilization with in activity of endotoxin BPI simulating peptide (Chinese patent application 01104591) be connected, and in conjunction with various chemically modifieds, design and filter out novel have stronger have sterilization and in and the allosteric peptide of activity of endotoxin.Its design philosophy is: the BPI simulating peptide is to increase its biologic activity by the avidity that changes peptide molecule and LPS; Comparatively speaking, to increase active result limited for the transformation on existing BPI molecule.The Tat49-57 fragment is for realizing that intracellular transport is the most effective minimal segment; Up to now, the small peptide that has kind more than ten to derive from Tat demonstrates the function that can pass the different cytoplasm film.Its mechanism mainly is its amphoteric character, wearing the film peptide can combine with phosphatide that has negative charge or glycolipid, and wear in the film peptide molecule tryptophan residue can bimolecular lamellar lipid membrane induce reverse micelle formation (1996, J Biol Chem 271 (30): 18188-18193).These mechanism promptings are worn the film peptide and may be had and LPS bonded great ability, and the main active ingredient of LPS is a lipid A after all.Therefore, we imagine by wearing being connected of film peptide and BPI simulating peptide, increase it and come allosteric BPI simulating peptide to the affinity ability of LPS and in conjunction with other chemical modification methods, with the design and filter out have stronger sterilization and in and the active peptide molecule of LPS.

Based on this, we choose 66 of Chinese granted patent 01104591.4 design have antibiotic/antiendotoxin and in and the active BPI simulating peptide of LPS BNEP9901315:KTKGG WLIQL FHKK as parent molecule (BNEP), to its carbon teminal (C-terminal, the C-end), nitrogen end (N-end, N-terminal) carry out chemically modified, and the combination of employing polypeptide solid-state reaction method, liquid-phase synthesis process and these two kinds of methods, synthetic a series of polypeptide derivatives with following general formula;

R ¹-BNEP-X(R ³)n

R wherein ¹Can be H or R ²CO;

R ²Can be C ₁-C ₁₇Alkyl or benzyl or aromatic base or fragrant heterocycle; Can contain following replacement on these groups

Base: NH ₂, CO ₂H, CO ₂R ⁴, CONHR ⁵, CONR ⁶R ⁷

The peptide preface of BNEP is: KTKGGWLIQLFHKK;

X can be O or NH or N;

R ³Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁴Can be H, or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁵Can be H, or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁶, R ⁷Can be C ₁-C ₆Alkyl or heterocycle or cyclic hydrocarbon;

N can be 1 or 2.

Choose to have and wear the short as far as possible of film effect (cell penetrating effect) and wear film peptide (CMT) RKKRRQRRR or YGRKKRRQRRR as another reactive site (CMT), these two kinds of reactive sites of BNEP and CMT directly link or through other chemical structure molecular tie, the common parent of forming, and its C-end, N-end carried out chemically modified, obtain having the series derivates of following general formula:

R ¹-BNEP-CMT-X(R ³)n

R wherein ¹Can be H or R ²CO etc.;

R ²Can be C ₁-C ₁₇Alkyl or benzyl or aromatic base or fragrant heterocycle; Can contain following replacement on these groups

Base: NH ₂, CO ₂H, CO ²R ⁴, CONHR ⁵, CONR ⁶R ⁷

The peptide preface of BNEP is: KTKGGWLIQLFHKK;

The peptide preface of CMT is: RKKRRQRRR or YQRKKRRQRRR

X can be O or NH or N;

R ³Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁴Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁵Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁶R ⁷Can be C ₁-C ₆Alkyl or heterocycle or cyclic hydrocarbon;

N can be 1 or 2;

R ¹-CMT-BNEP-X(R ³)n

R wherein ¹Can be H or R ²CO etc.;

R ²Can be C ₁-C ₁₇Alkyl or benzyl or aromatic base or fragrant heterocycle; Can contain following replacement on these groups

Base: NH ₂, CO ₂H, CO ²R ⁴, CONHR ⁵, CONR ⁶R ⁷

The peptide preface of CMT is: RKKRRQRRR or YGRKKRRQRRR

The peptide preface of BNEP is: KTKGGWLIQLFHKK;

X can be O or NH or N;

R ³Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁴Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁵Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁶, R ⁷Can be C ₁-C ₆Alkyl or heterocycle or cyclic hydrocarbon;

N can be 1 or 2.

R ¹-BNEP-Z-CMT-X(R ³)n

R wherein ¹Can be H or R ²CO etc.;

R ²Can be C ₁-C ₁₇Alkyl or benzyl or aromatic base or fragrant heterocycle; Can contain following replacement on these groups

Base: NH ₂, CO ₂H, CO ₂R ⁴, CONHR ⁵, CONR ⁶R ⁷

The peptide preface of BNEP is: KTKGGWLIQLFHKK;

The peptide preface of CMT is: RKKRRQRRR or YGRKKRRQRRR

Z can be HN-(CH ₂) m-CO, HN-(CHR ⁸) m-CO, m=1-8, R ⁸Be alkyl, benzyl, virtue

Perfume base, fragrant heterocycle; Polypeptide;

X can be O or NH or N;

R ³Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁴Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁵Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁶, R ⁷Can be C ₁-C ₆Alkyl or heterocycle or cyclic hydrocarbon;

N can be 1 or 2.

R ¹-CMT-Z-BNEP-X(R ³)n

R wherein ¹Can be H or R ²CO etc.;

R ²Can be C ₁-C ₁₇Alkyl, benzyl, aromatic base, fragrant heterocycle; Can contain following replacement on these groups

Base: NH ₂, CO ₂H, CO ₂R ⁴, CONHR ⁵, CONR ⁶R ⁷

The peptide preface of BNEP is: KTKGGWLIQLFHKK;

The peptide preface of CMT is: RKKRRQRRR or YGRKKRRQRRR

Z can be HN-(CH ₂) m-CO, HN-(CHR ⁸) m-CO, m=1-8, R ⁸Be alkyl, benzyl, fragrance

Base, fragrant heterocycle; Polypeptide;

X can be O or NH or N;

R ³Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁴Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁵Can be H or C ₁-C ₆Alkyl or benzyl or aromatic base or fragrant heterocycle;

R ⁶, R ⁷Can be C ₁-C ₆Alkyl or heterocycle or cyclic hydrocarbon;

N can be 1 or 2.

In case of necessity their cyclisation are obtained cyclic peptide.The characteristics of these allosteric peptides are: R ¹-BNEP-XRn ³The series polypeptide derivative has the fundamental characteristics of BNEP, and the new feature of modified peptides is arranged again, expectation obtain sterilization and in and activity of endotoxin enhanced novel drugs; R ¹-BNEP-CMT-X (R ³) n, R ¹-CMT-BNEP-X (R ³) n, R ¹-BNEP-Z-CMT-X (R ³) n, R ¹-CMT-Z-BNEP-X (R ³) n, both kept BNEP sterilization and in and activity of endotoxin, the mode of again can be effectively that polypeptide, protein molecule and dna fragmentation etc. are receptor-mediated by not having, not having a power consumption imports multiple mammalian cell and is positioned kytoplasm and nuclear ability (wearing the characteristic of film peptide CMT), thereby brings into play the biological effect of BNEP as early as possible.

Above-mentioned antibiotic, pharmaceutically acceptable various mineral acids, organic acid salt that the antiendotoxin allosteric peptide molecule can show as polypeptide are such as trifluoroacetate, hydrochloride, hydrobromate, acetate, phosphoric acid salt, tartrate, Citrate trianion, sulfonate, amino acid salts etc.

In above-mentioned antibiotic, the chain that the antiendotoxin allosteric peptide molecule can show as polypeptide or the cyclic peptide of end position.

In above-mentioned antibiotic, the chain that the antiendotoxin allosteric peptide molecule can show as each peptide species or pharmaceutically acceptable various mineral acids, the organic acid salt of end position cyclic peptide, such as trifluoroacetate, hydrochloride, hydrobromate, acetate, phosphoric acid salt, tartrate, Citrate trianion, sulfonate, amino acid salts etc.

Method for making antibiotic, the antiendotoxin allosteric peptide molecule of the present invention is as follows:

1. the parent peptide resin synthesizes logical method

Taking by weighing an amount of Fmoc-AA-Wang resin or other polypeptide synthesizing amino acid resin pours in the synthetic post; add solvent (DCM; DMF; or DCM/DMF) swelling; remove the Fmoc blocking group of amino-acid resin with the DMF solution of 10%-50% hexahydropyridine; wash successively with DCM, MeOH, DMF, DCM, DMF, eliminate hexahydropyridine.

According to of the present invention antibiotic/aminoacid sequence of antiendotoxin allosteric peptide takes by weighing an amount of Fmoc-amino acid successively in suitable container, add an amount of DMF dissolving, add then coupling agent (as TBTU/HOBt, HBTU/HOBt, HATU/HOAt, DIC/HOBt, DCC/HOBt, DCC/HOSu, TBTU/HOSu, PyBOP/HOBt, BOP/HOBt, etc) and the DMF solution of proper N methylmorpholine or diisopropylethylamine.After coupling is finished, remove reaction solution, wash peptide resin successively with DCM, MeOH, DMF, DCM, DMF; The DMF solution that adds the 20%-50% hexahydropyridine removes the Fmoc blocking group of amino-acid resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.According to polypeptide solid state chemistry synthetic method according to of the present invention antibiotic/aminoacid sequence of antiendotoxin allosteric peptide parent is from the follow-up coupling of C end beginning carrying out successively, so circulation finish whole antibiotic/peptide chain of antiendotoxin allosteric peptide, obtain the parent peptide resin; Thorough washing, MeOH shrink, and drain, from synthetic post, take out synthetic antibiotic/antiendotoxin peptide derivant resin, put freeze-drying in the Freeze Drying Equipment, standby.

2. derivative preparation

According to the difference of modification group, adopt different synthetic methods, prepare the antibiotic/antiendotoxic mimic polypeptide derivative of full guard; the thick peptide that obtains gets smart peptide through high performance liquid chromatography (HPLC) purifying; smart peptide salify, mass spectrum are identified and HPLC checks purity, carry out biological activity and identify.

2.1 nitrogen end acylations

Acylting agent is a lot, and modal agent combination has: (RCO) ₂O/pyridine; RCO ₂H/activatingreagent (activator).Common activator has: DCC/HOBt, DCC/HOSu, TBTU/HOBt, HBTU/HOBt, HATU/HOAt, DIC/HOBt, TBTU/HOSu, PyBOP/HOBt, BOP/HOBt, DIC/HOSu, PyBOP/HOSu.

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post; add solvent (DCM; DMF; orDCM-DMF) swelling; drain; the DMF solution that adds the 20%-50% hexahydropyridine removes the Fmoc blocking group of peptide resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.The DMF solution that adds acylting agent stirs or logical nitrogen or vibration temperature control.After coupling is finished, remove reaction solution, wash successively with DCM, MeOH, DMF, DCM, DMF, MeOH shrinks, and drains, from synthetic post, take out synthetic antibiotic/antiendotoxin peptide derivant resin, put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal; Add anhydrous diethyl ether, stir evenly, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

2.2 substituted peptide amide

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post, and the adding solvent (DCM, DMF, DCM-DMF, THF, THF/DMF) swelling is drained.The DMF solution that adds aminated reagent stirs or logical nitrogen temperature control.After amidation was finished, suction filtration was removed reaction solution, with DMF, MeOH, THF, Et ₂O washs successively, and filtrate merges, and revolves to steam to doing or put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal; Add anhydrous diethyl ether, stir evenly, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

2.3 peptide ester

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post, and the adding solvent (DCM, DMF, DCM-DMF, THF, THF-DMF) swelling is drained.The DMF solution that adds esterifying reagent stirs or logical nitrogen temperature control.After esterification was finished, suction filtration was removed reaction solution, with DMF, MeOH, THF, Et ₂O washs successively, and filtrate merges, and revolves to steam to doing or put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

The bioactivity research of the antibiotic/antiendotoxin allosteric peptide of the present invention's design

The present invention also by measure of the present invention antibiotic/antiendotoxin allosteric peptide is to the body outer disinfecting activity of groups of people source germ, external anti-endotoxin activity (to the provide protection of LPS attack cells), external inhibition activity to several agriculture pathogenic bacterium; be used for treatment of G-infectation of bacteria, people's endotoxemia and phytopathogen control so that study it, so that explore the endotoxic novel drugs that can sterilization can neutralize again.

In vitro tests shows, of the present invention antibiotic/antiendotoxin allosteric peptide all has in various degree fungicidal activity external to bacteriums such as the golden ATCC of Portugal 25923, the golden 318MRS of Portugal, golden Portugal 321, golden Portugal 453, golden Portugal 464, golden Portugal 465, table Portugal 370, table Portugal 372, large intestine ATCC 25922, large intestine 249, large intestine 339, large intestine 355, large intestine 405, large intestine 467, green pus resistance, 328 green pus 406, green pus 433, green pus 451; External, of the present invention antibiotic/antiendotoxin allosteric peptide all has neutralizing effect in various degree to LPS external, different derivatives there are differences the neutralizing effect of LPS, the secretion of person monocytic cell's tumour TNF-α of endotaxin induction is had in various degree restraining effect, can be used for the treatment of G-infectation of bacteria and people's endotoxemia.Different derivatives are to the restraining effect difference of rice leaf spot bacteria, bacterial stripe bacterium.Of the present invention antibiotic/antiendotoxin allosteric peptide can be aided with medicinal diluent, adjuvant and carrier and make medicinal compositions, as tablet, pulvis, pill, solution or suspension agent, by sucking, inject or other mode administrations, be used for the treatment of the control of G-infectation of bacteria and endotoxemia and phytopathogen; Of the present invention antibiotic/antiendotoxin allosteric peptide also can be compound with other drug, be used for the treatment of G-infectation of bacteria and endotoxemia and phytopathogen the control.

Of the present invention antibiotic/the antiendotoxin peptide derivant is to prepare the application in kill bacteria medicine and treatment endotoxemia and the phytopathogen control medicine.

Of the present invention antibiotic/antiendotoxin allosteric peptide is used for the treatment of G-infectation of bacteria and people's endotoxemia and phytopathogen control as thinner, adjuvant and carrier.

At least two identical or different of the present invention antibiotic/antiendotoxin allosteric peptides are connected to a peptide, are used for the treatment of the control of G-infectation of bacteria and endotoxemia and phytopathogen.

In addition, at least two identical or different of the present invention antibiotic/antiendotoxin allosteric peptides are connected to a peptide, can be used as thinner, adjuvant and carrier and are used for the treatment of G-infectation of bacteria and phytopathogen control.

Description of drawings:

Fig. 1-1, part BNEP allosteric peptide of the present invention are to the restraining effect figure of Bacillus subtilus.

Fig. 1-2, part BNEP allosteric peptide of the present invention are to the restraining effect figure of bacterial stripe.

Fig. 1-3, part BNEP allosteric peptide of the present invention are to the restraining effect figure of rice leaf spot bacteria.

Fig. 2, BNEP1 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Fig. 3, BNEP3 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Fig. 4, BNEPCMT-04-2 polypeptide of the present invention are to the provide protection figure of LPS attack cells.

Fig. 5, BNEP6 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Fig. 6, BNEPCMT-03-2a polypeptide of the present invention are to the provide protection figure of LPS attack cells.

Fig. 7, BNEPCMT-04-2a polypeptide of the present invention are to the provide protection figure of LPS attack cells.

Fig. 8, BNEP9 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Fig. 9, BNEP10 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 10, BNEP11 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 11, BNEP24 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 12, BNEPCMT-03 polypeptide of the present invention are to the provide protection figure of LPS attack cells.

Figure 13, BNEP26 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 14, BNEP27 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 15, BNEP31 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 16, BNEP50 of the present invention ^#Polypeptide is to the provide protection figure of LPS attack cells.

Figure 17, polymyxin of the present invention are to the provide protection figure of LPS attack cells.

Embodiment

Be specific embodiments of the invention below, described preferred embodiment is to be used to describe the present invention rather than restriction the present invention.

Embodiment 1

Present embodiment relates to the synthetic of Ac-BNEP-Ahx-CMT-030625, in order to illustrate of the present invention antibiotic/the synthetic and purification process of antiendotoxin allosteric peptide.

1.Fmoc-BNEP-Ahx-CMT-030625 peptide resin is synthetic

Take by weighing 2.420g (0.5mmol) Fmoc-Arg (Pbf)-Wang Resin-030620 and pour in the synthetic post, and adding 35mL DCM-DMF (1: 2, v/v) swelling.Extract solvent behind the 30min out, remove the Fmoc blocking group of amino-acid resin, wash successively with DCM, MeOH, DMF, DCM, DMF behind the 15min, eliminate hexahydropyridine with the DMF solution of 15mL 30% hexahydropyridine.Take by weighing 1.301g Fmoc-Arg (Pbf)-OH in the 100mL Erlenmeyer flask, add 10mL DMF dissolving, add the DMF solution of 0.780gTBTU, 0.328g HOBt and 35mL 25% diisopropylethylamine in batches.Triketohydrindene hydrate detection reaction process.After coupling is finished, remove reaction solution, wash peptide resin successively with DCM, MeOH, DMF, DCM, DMF; The DMF solution that adds the 20%-50% hexahydropyridine removes the Fmoc blocking group of amino-acid resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.According to similar method, drop into amino acid, 4mmol TBTU and the 4mmolHOBt of the follow-up Fmoc protection of 3mmol, continue coupling.After reaction is finished, remove Fmoc, washing, according to of the present invention antibiotic/aminoacid sequence of antiendotoxin allosteric peptide parent is from the follow-up coupling of C end beginning carrying out successively, so circulation finish whole antibiotic/peptide chain of antiendotoxin allosteric peptide, obtain the parent peptide resin; Thorough washing, MeOH shrink, and drain, from synthetic post, take out synthetic antibiotic/antiendotoxin peptide derivant resin, put freeze-drying in the Freeze Drying Equipment, peptide resin 3.5g.

Take out the 1g peptide resin, add 30mL DCM, swelling is drained behind the 30min, and the DMF solution that adds 20mL 30% hexahydropyridine removes the Fmoc blocking group of peptide resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.Conventional cracking [cracking agent (Reagent R): TFA/Thioanisole/EDT/anisole 94: 3: 2: 1, v/v, 15mL; Room temperature, 3 hours], anhydrous diethyl ether repeatedly sedimentation gets thick peptide BNEP-Ahx-CMT-CP-030625-S-W 403mg, thick peptide purity 87.53%, MALDI-TOF-MS3119.3; The proof parent synthesizes successfully.

2.Ac-BNEP-Ahx-CMT-030625 synthetic

2.5g the Fmoc-BNEP-Ahx-CMT-030625 peptide resin, the DMF solution that adds 35mL 30% piperidines removes the Fmoc blocking group of peptide resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.After sloughing the Fmoc protecting group, take out 1.691g (0.35mmol) BNEP-Ahx-CMT-030625 resin, add 20mL DMF, splash into and contain 0.945mL (40eq.) Ac ₂The 10mL DMF solution of O, 1.6mL (80eq.) Pyridine carries out acetylize to the N-end, stirs temperature control.Triketohydrindene hydrate detects, and the 3h resin is transparent, shows that acetylize finishes.Remove reaction solution, wash successively with DCM, MeOH, DMF, DCM, DMF, MeOH shrinks, and drains, and takes out the Ac-BNEP-Ahx-CMT-030625 peptide resin from synthetic post, drains, weigh 2.503g acetylated peptide resin.

3.Ac-BNEP-Ahx-CMT-030625 peptide resin cracking

2.503g the Ac-BNEP-Ahx-CMT-030625 peptide resin adds 35mL cracking agent R, stirs, and room temperature reaction 3.5 hours, the sedimentation of 400mL anhydrous diethyl ether, centrifugal; Add the 150mL anhydrous diethyl ether, stir evenly, centrifugal, repeat 4 times.Add 50% aqueous acetic acid 55mL, lyophilize gets Ac-BNEP-Ahx-CMT-CP-0306251023mg, yield 92.5%, and HPLC purity 80.15%, MALDI-TOF-MS 3161.4.The proof derivative synthesizes successfully, obtains having the product of following peptide preface: it modifies base and the peptide preface is: CH ₃CO-KTKGGWLIQLFHKK-NH (CH ₂) ₅CO-RKKRRQRRR-OH

4.Ac-BNEP-Ahx-CMT-030625 thick peptide purification

The thick peptide of Ac-BNEP-Ahx-CMT-030625 adds a small amount of pyrogen-free pure water thick peptide is fully dissolved, and obtains the sample solution of 0.5mg/mL concentration, filters (the organic filter membrane of 0.45 μ m) through the pin type filter, collects filtrate, by following chromatographic condition purifying.

Instrument: Dionex P680

The anti-phase C18 201SP54 of analytical column: Vydac (4.6mm * 150mm)

Moving phase: A:H ₂O+0.1%TFA B:ACN

Flow velocity: 5mL/min

Detect wavelength: 214nm

Gradient: 30%ACN → 38%ACN 20min

The HPLC purifying gets smart peptide Ac-BNEP-Ahx-CMT-030625354.7mg, yield 45.0%.

Embodiment 2

Present embodiment relate to of the present invention antibiotic/acetylize of antiendotoxin allosteric peptide is synthetic.

Method is led in acetylize: after peptide resin is sloughed the Fmoc protecting group with 30% left and right sides piperidines, with thorough washing such as DMF, DCM, MeOH.Add an amount of DMF swelling resin, slowly drip diacetyl oxide (Ac ₂O)-and the DCM solution of pyridine, logical nitrogen, room temperature reaction.The triketohydrindene hydrate monitoring, suction filtration, conventional washing, vacuum-drying after reaction is finished.The method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.Quality test (purity, content), mass spectrum is surveyed molecular weight.

1.Ac-CMT-BNEP(15)

According to the logical method of acetylize, the experiment condition of Ac-CMT-BNEP-040321 and the results are shown in following table.Cracking all is to add TES 2mL in Reagent R, and the cracking time is 170min.

Table 2 preparative conditions and results of Ac-CMT-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-BNEP-Pr	0.33	2.0g	2.0g	1	Reaction 3h
Diacetyl oxide	13.2	1.25ml	1.3ml	40
					Pyridine	26.4	2.12ml	2.3ml	80
Thick peptide: 927mg; Smart peptide: 160.4; Yield 17.3%

2.Ac-CMT-Ahx-BNEP(22)

According to the logical method of acetylize, the experiment condition of Ac-CMT-Ahx-BNEP-040321 and the results are shown in following table.Cracking all is to add TES 2mL in Reagent R, and the cracking time is 160min.

Table 3 preparative conditions and results of Ac-CMT-Ahx-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-Ahx-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 22h
Diacetyl oxide	13.2	1.25ml	1.3ml	40
					Pyridine	26.4	2.12ml	2.2ml	80
Thick peptide: 973mg; Smart peptide: 204.1mg; Yield 21.0%

1.841g the conventional cracking of Ac-CMT-Ahx-BNEP-030812-pr obtains thick peptide 691mg, HPLC purity 54.47%, and MALDI-TOF-MS 3161.3; Get smart peptide 66.6mg, purity 98.0% behind the purifying salify; 1.165gAc-CMT-Ahx-BNEP-030705-pr conventional cracking obtains thick peptide 597mg, HPLC purity 60.37%, and MALDI-TOF-MS 3161.2; Get smart peptide 93.3mg, purity 98.0% behind the purifying salify;

It modifies base and the peptide preface is: CH ₃CO-RKKRRQRRR-NH (CH ₂) ₅CO-KTKGGWLIQLFHKK-OH

3.Ac-BNEP-CMT(29)

According to the logical method of acetylize, the experiment condition of Ac-BNEP-CMT-040108 and the results are shown in following table.

Table 4 preparative conditions and results of Ac-BNEP-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 16h
Diacetyl oxide
		8	0.756ml	0.8ml	40
Pyridine						16	1.280ml	1.3ml	80
	Thick peptide: 453mg; Smart peptide: 137.0mg; Yield 30.2%

Embodiment 3

Present embodiment relate to of the present invention antibiotic/the Succinic Acid list acidylate of antiendotoxin allosteric peptide is synthetic.

Succinic Acid list acidylate is led to method: after peptide resin is sloughed the Fmoc protecting group with 30% left and right sides piperidines, with thorough washing such as DMF, DCM, MeOH.With an amount of DMF swelling resin, slowly drip Succinic anhydried (Suc ₂O) or the DCM solution of Succinic anhydried-pyridine, logical nitrogen, room temperature reaction.The triketohydrindene hydrate monitoring, suction filtration, conventional washing, vacuum-drying after reaction is finished.The method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.Quality test (purity, content), mass spectrum is surveyed molecular weight.

1.Suc-CMT-BNEP(16)

According to the logical method of Succinic Acid list acidylate, the experiment condition of Suc-CMT-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 2.2mL in Reagent R, and the cracking time is 230min.

Table 5 preparative conditions and results of Suc-CMT-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 11h
Succinic anhydried	13.2	1.32	1.34	40
					Pyridine	52.8	4.24ml	4.4ml	160
Thick peptide: 1108mg; Smart peptide: 182.3mg; Yield 16.5%

It modifies base and the peptide preface is HOOC-CH ₂CH ₂CO-RKKRRQRRR-KTKGGWLIQLFHKK-OH

2.Suc-BNEP-CMT

According to the logical method of Succinic Acid list acidylate, the experiment condition of Suc-BNEP-CMT-040108 and the results are shown in following table.

Table 6 preparative conditions-d results of Suc-BNEP-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 31h
Succinic anhydried	2	0.2g	0.2g	40
					Pyridine	8	0.64ml	0.4ml	160
Thick peptide: 506mg; Smart peptide: 145.8mg; Yield 28.8%

3.Suc-BNEP-Ahx-CMT(37)

According to the logical method of Succinic Acid list acidylate, the experiment condition of Suc-BNEP-Ahx-CMT-040108 and the results are shown in following table.

Table 7 preparative conditions and results of Suc-BNEP-Ahx-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Ahx-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 7h
Succinic anhydried	2	0.2g	0.2g	40
					Pyridine	4	0.32ml	0.30ml	80
Thick peptide: 506mg; Smart peptide: 253.6mg; Yield 50.1%

4.Suc-CMT-Ahx-BNEP(51)

According to the logical method of Succinic Acid list acidylate, the experiment condition of Suc-CMT-Ahx-BNEP-040321 and the results are shown in following table.Cracking is to add TES 1.2mL in Reagent R, and the cracking time shortens to 72min.

Table 8 preparative conditions and results of Suc-CMT-Ahx-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-Ahx-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 94h
Succinic anhydried	13.2	1.32g	1.32g	40
					Pyridine	26.4	2.12ml	2.0ml	80
Thick peptide: 1151mg; Smart peptide: 240.2mg; Yield 20.9%

It modifies base and the peptide preface is:

HOOC-CH ₂CH ₂CO-RKKRRQRRR-NH(CH ₂) ₅CO-KTKGGWLIQLFHKK-OH

Embodiment 4

Present embodiment relate to of the present invention antibiotic/palmitoylation, the stearyl of antiendotoxin allosteric peptide be combined to.

Stearyl, the logical method of palmitoylation: after peptide resin is sloughed the Fmoc protecting group with 15% left and right sides piperidines, with thorough washing such as DMF, DCM, MeOH.With an amount of DMF swelling resin, add the DMF solution of stearic acid (stearic acid) or palmitinic acid (palmiticacid), HBTU or TBTU or PyBOP, HOBt, logical nitrogen splashes into TEA-DMF solution, room temperature reaction.The triketohydrindene hydrate monitoring, suction filtration, conventional washing, vacuum-drying after reaction is finished.The method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.The molecular weight conclusive evidence is surveyed in quality test (purity, content), mass spectrum.

1.C ₁₅H ₃₁CO-BNEP(5)

According to logical method, C ₁₅H ₃₁The experiment condition of CO-BNEP-031020 and the results are shown in following table.

Table 9 preparative conditions and results of C ₁₅H ₃₁CO-BNEP-031020(TBTU method)

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.1	0.575g	0.575g	1	Reaction 6h
C 15H 31CO 2H	1	0.256g	0.257g	10
					TBTU	2.4	0.772g	0.774g	24
HOBt	1.2	0.162g	0.163g	12
					TEA	0.52ml/1mmol acid	0.3ml	0.3ml
Thick peptide: 111mg; Smart peptide: 40.6mg; Yield 36.6%

Table 10 preparative conditions and results ofC ₁₅H ₃₁CO-BNEP-031020(PyBOP method)

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.1	0.575g	0.574g	1	Reaction 50h
C 15H 31CO 2H	1	0.256g	0.257g	10
					PyBOP	0.6	0.318g	0.311g	6
HOBt	1.2	0.162g	0.164g	12
					DIEA	0.6ml/1mmol acid	0.3ml	0.6ml
Thick peptide: 137mg; Smart peptide: 65.7mg yield 47.9%

It modifies base and the peptide preface is CH ₃(CH ₂) ₁₄CO-KTKGGWLIQLFHKK-OH

2.C ₁₇H ₃₅CO-BNEP(6)

According to logical method, C ₁₇H ₃₅The experiment condition of CO-BNEP-040206 and the results are shown in following table.

Table 11 preparative conditions and results of C ₁₇H ₃₅CO-BNEP-040206(HBTU method)

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.1	0.575g	0.575g	1	Reaction 31h
C 17H 35CO 2H	1	0.284g	0.285g	10
					HBTU	0.6	0.193g	0.193g	6
HOBt	1.2	0.162g	0.163g	12
					TEA	0.52ml/1mmol acid	0.6ml	0.6ml
Thick peptide: 128mg; Smart peptide: 23.1mg yield 18.1%

Table 12 preparative conditions and results of C ₁₇H ₃₅CO-BNEP-040206(PyBOP method)

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.1	0.575g	0.574g	1	Reaction 44h
C 17H 35CO 2H	1	0.284g	0.287g	10
					PyBOP	1.2	0.625g	0.622g	12
HOBt	2.4	0.324g	0.328g	24
					TEA	1.2ml/1mmol acid	1.2ml	1.2ml
Thick peptide: 137mg; Smart peptide: 31.5mg; Yield 24.1%

3.C ₁₇H ₃₅CO-CMT-BNEP(17)

According to the logical method of stearic acidylate, C ₁₇H ₃₅The experiment condition of CO-CMT-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 1.1mL in Reagent R, and the cracking time is 180min.

Tabl 13 preparative conditions and results of C ₁₇H ₃₅CO-CMT-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-BNEP-Pr	0.32	2.0g	2.0g	1	Make solvent with DMF
C 17H 35CO 2H	3.2	0.904g	0.908g	10
					TBTU	7.04	2.261g	2.260g	22
HOBt	7.04	0.951g	0.955g	22
					TEA	1.2ml/1mmol acid	6.8ml	6.8ml
Thick peptide: 955mg; Smart peptide: 38.2mg; Yield 4.0%

It modifies base and the peptide preface is: CH ₃(CH ₂) ₁₆CO-RKKRRQRRR-KTKGGWLIQLFHKK-OH

4.C ₁₇H ₃₅CO-CMT-Ahx-BNEP(23)

According to the logical method of stearic acidylate, C ₁₇H ₃₅The experiment condition of CO-CMT-Ahx-BNEP-040321 and the results are shown in following table.Cracking is to add TES 0.7mL in Reagent R, and the cracking time is 60min.

Table 14 preparative conditions and results of C ₁₇H ₃₅CO-CMT-Ahx-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-Ahx-BNEP-Pr	0.16	1.0g	1.0g	0.5	Use CHCl 3Make solvent
C 17H 35CO 2H	6.4	1.817g	1.818g	20
					TBTU	7.04	2.261g	2.265g	22
HOBt	7.04	0.951g	0.952g	22
					TEA	0.9ml/1mmol acid	5.1ml	5.1ml
Thick peptide: 1057mg; Smart peptide: 104.7mg; Yield 9.9%

1.60g get C after the stearic acidylate of F-CMT-Ahx-BNEP-030812-pr ₁₇H ₃₅CO-CMT-Ahx-BNEP-030812-pr 1.64g, conventional cracking obtains thick peptide 681mg, HPLC purity 89.58%, MALDI-TOF-MS 3385.6; Get smart peptide 30.6mg, purity 92.3% behind the purifying salify.

5.C ₁₅H ₃₁CO-BNEP-CMT (31) is according to the logical method of palmitoylation, C ₁₅H ₃₁The experiment condition of CO-BNEP-CMT-040321 and the results are shown in following table.

Table 15 preparative conditions and results of C ₁₅H ₃₁CO-BNEP-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 38h;
C 15H 31CO 2H	4	1.025g	1.027g	20
					TBTU	4.4	1.413g	1.415g	22
HOBt	2.2	0.297g	0.298g	11
					TEA	0.6ml/1mmol acid	2ml	2ml
Thick peptide: 400mg; Smart peptide: 129.6mg; Yield 32.4%

It modifies base and the peptide preface is: CH ₃(CH ₂) ₁₄CO-KTKGGWLIQLFHKK-RKKRRQRRR-OH

6.C ₁₅H ₃₁CO-BNEP-Ahx-CMT(38)

Getting peptide resin BNEP-Ahx-CMT-030728-pr 2.3g (0.25mmol) modifies with palmitinic acid the N end.According to palmitinic acid: resin=10: 1, TBTU/HOBt: the mol ratio of palmitinic acid=0.6: 1 feeds intake, 32 ℃ of reaction 3h, triketohydrindene hydrate detects, resin by light yellow become colorless transparent.Take out 1.02g from the 2.3g peptide resin, add 22mL Reagent R, conventional cracking.Lyophilize gets C ₁₅H ₃₁CO-BNEP-Ahx-CMT-CP-030728413mg, purity 53.97%MALDI-TOF-MS 3357.7; Salify behind the HPLC purifying gets smart peptide 64.5mg, purity 99.7%.

C ₁₅H ₃₁CO-BNEP-Ahx-CMT-040108 batch preparation condition and the results are shown in following table.

Table 16 C ₁₅H ₃₁CO-BNEP-Ahx-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Ahx-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 16h;
C 15H 31CO 2H	8	2.050g	2.054g	40
					TBTU	4.4	1.413g	1.415g	22
HOBt	4.4	0.594g	0.595g	22
					TEA	0.9ml/mmol acid	3ml	3ml
Thick peptide: 400mg; Smart peptide: 174.3mg; Yield 43.6%

7.C ₁₇H ₃₅CO-BNEP-Ahx-CMT(39)

Operate same C ₁₅H ₃₁CO-BNEP-Ahx-CMT (38).2.3g the BNEP-Ahx-CMT-030728 peptide resin, 32 ℃ of reaction 18h, triketohydrindene hydrate monitoring, resin by light yellow become colorless transparent.1.93g peptide resin adds 25mLReagent R, conventional cracking.Lyophilize gets C ₁₇H ₃₅CO-BNEP-Ahx-CMT-CP-030728360mg, purity 69.96%, MALDI-TOF-MS 3386.6; Salify behind the HPLC purifying gets smart peptide 90.5mg, purity 96.1%.

Embodiment 5

Present embodiment relate to of the present invention antibiotic/the amino hexanoylization of 6-(N-fluorenes formyl) of antiendotoxin allosteric peptide.

The amino hexanoylization of 6-(N-fluorenes formyl) is led to method: after peptide resin is sloughed the Fmoc protecting group with 30% left and right sides piperidines, with thorough washing such as DMF, DCM, MeOH.With an amount of DMF swelling resin, add the DMF solution of 6-(N-fluorenes formyl) hexosamine (Fmoc-Ahx-OH), TBTU or PyBOP, HOBt, logical nitrogen splashes into TEA-DMF solution, room temperature reaction.The triketohydrindene hydrate monitoring, suction filtration, conventional washing, vacuum-drying after reaction is finished.The method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.The molecular weight conclusive evidence is surveyed in quality test (purity, content), mass spectrum.

1.Fmoc-Ahx-BNEP(7)

According to logical method, the experiment condition of Fmoc-Ahx-BNEP-040212 and the results are shown in following table.

Table 17 preparative conditions and results of F-Ahx-BNEP-040212

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.2	1.2g	1.2g	1	Reaction 14h resin detects transparent
F-Ahx-OH	0.8	0.282g	0.283g	4
					TBTU	0.44	0.141g	0.143g	2.2
HOBt	0.88	0.118g	0.119g	4.4
					TEA	0.9ml/1mmol acid	0.72ml	0.9ml
Thick peptide: 408mg; Smart peptide: 72.4mg; Yield 17.7%

It modifies base and the peptide preface is Fmoc-NH (CH ₂) ₅CO-KTKGGWLIQLFHKK-OH

2.Fmoc-Ahx-CMT-BNEP(19)

According to the logical method of the amino hexanoylization of 6-(N-fluorenes formyl), the experiment condition of F-Ahx-CMT-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 1.2mL in Reagent R, and the cracking time is 100min.

Table 18 preparative conditions and results of F-Ahx-CMT-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 18h resin detects transparent
F-Ahx-OH	1.28	0.452g	0.451g	4
					TBTU	1.54	0.493g	0.493g	4.8
HOBt	0.77	0.104g	0.110g	2.4
					TEA	0.9ml/1mmol acid	1.15ml	1.2ml
Thick peptide: 800mg; Smart peptide: 116.5mg; Yield 14.6%

3.Fmoc-Ahx-CMT-Ahx-BNEP(24)

According to the logical method of the amino hexanoylization of 6-(N-fluorenes formyl), the experiment condition of F-CMT-Ahx-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 1.2mL in Reagent R, and the cracking time is 100min.

Table 19 preparative conditions and results of F-Ahx-CMT-Ahx-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-Ahx-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 35h resin detects transparent
F-Ahx-OH	0.64	0.226g	0.228g	2
					TBTU	1.54	0.493g	0.495g	4.8
HOBt	.0.77	0.104g	0.104g	2.4
					TEA	0.3ml/1mmol acid	0.38ml	0.4ml
Thick peptide: 776mg; Smart peptide: 149.3mg; Yield 19.2%

4.Fmo-Ahx-BNEP-CMT(33)

According to the logical method of the amino hexanoylization of 6-(N-fluorenes formyl), the experiment condition of F-Ahx-BNEP-CMT-040108 and the results are shown in following table.

Table 20 preparative conditions and results of F-Ahx-BNEP-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 6h; Resin detects transparent
F-Ahx-OH	1.2	0.424g	0.425g	6
					TBTU	0.65	0.209g	0.210g	3.3
HOBt	1.3	0.176g	0.179g	6.6
					TEA	1.2ml/1mmol acid	1.2ml	1.2ml
Thick peptide: 413mg; Smart peptide: 212.6mg; Yield 51.5%

It modifies base and the peptide preface is Fmoc-NH (CH ₂) ₅CO-KTKGGWLIQLFHKK-RKKRRQRRR-OH

5.Fmoc-Ahx-BNEP-Ahx-CMT(40)

According to the logical method of the amino hexanoylization of 6-(N-fluorenes formyl), the experiment condition of F-Ahx-BNEP-Ahx-CMT-040108 and the results are shown in following table.

Table 21 preparative conditions and results of F-Ahx-BNEP-Ahx-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP- Ahx-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 16h resin detects transparent
F-Ahx-OH	0.6	0.212g	0.214g	3
					TBTU	0.65	0.209g	0.210g	3.3
HOBt	1.3	0.176g	0.176g	6.6
					TEA	0.9ml/mmol acid	0.9ml	0.9ml
Thick peptide: 412mg; Smart peptide: 240.6mg; Yield 58.4%

Embodiment 6

Present embodiment relate to of the present invention antibiotic/the amino hexanoylization of the 6-of antiendotoxin allosteric peptide.

The logical method of the amino hexanoylization of 6-: according to the logical method of the amino hexanoylization of 6-(N-fluorenes formyl), preparation Fmoc-Ahx-peptideresin, 25% left and right sides piperidines is sloughed the Fmoc protecting group, and thorough washing such as DMF, DCM, MeOH obtain Ahx-peptide resin.After the vacuum-drying, the method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.The molecular weight conclusive evidence is surveyed in quality test (purity, content), mass spectrum.

1.Ahx-BNEP

According to logical method, the experiment condition of Ahx-BNEP-031020 and the results are shown in following table.

Table 22 preparative conditions and results of Ahx-BNEP-031020

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-Pr	0.2	1.0g	1.0g	1	Reaction 17h resin detects transparent DBLK5min
F-Ahx-OH	1.6	0.564g	0.566g	8
					TBTU	0.88	0.282g	0.287g	4.4
HOBt	0.44	0.056g	0.060g	2.2
					TEA	0.9ml/1mmol acid	0.72ml	0.72ml
Thick peptide: 316mg; Smart peptide: 245.8mg; Yield 77.8%

2.Ahx-CMT-BNEP(18)

According to the logical method of the amino hexanoylization of 6-, the experiment condition of Ahx-CMT-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 2.2mL in Reagent R, and the cracking time is 100min.

Table 23 preparative conditions and results of Ahx-CMT-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-BNEP-Pr	0.32	2.0g	2.0g	1	Reaction 2h resin detects transparent DBLK5min
F-Ahx-OH	1.28	0.452g	0.450g	4
					TBTU	3.08	0.980g	0.990g	9.6
HOBt	1.54	0.208g	0.210g	4.8
					TEA	1.2ml/1mmol acid	1.4ml	1.6ml
Thick peptide: 446mg; Smart peptide: 76.0; Yield 17.0%

3.Ahx-CMT-Ahx-BNEP(25)

According to the logical method of the amino hexanoylization of 6-, the experiment condition of Ahx-CMT-Ahx-BNEP-040321 and the results are shown in following table.The peptide resin cracking is to add TES 1.2mL in Reagent R, and the cracking time is 100min.

Table 24 preparative conditions and results of Ahx-CMT-Ahx-BNEP-040321

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						CMT-Ahx-BNEP- Pr	0.32	2.0g	2.0g	1	Reaction 36h resin detects transparent; DBLK 10min
F-Ahx-OH	0.64	0.226g	0.225g	2
					TBTU	1.54	0.493g	0.490g	4.8
HOBt	3.08	0.416g	0.420g	9.6
					TEA	0.3ml/1mmol acid	0.38ml	0.4ml
Thick peptide: 488mg; Smart peptide: 150.8mg; Yield 30.9%

It modifies base and the peptide preface is

H ₂N(CH ₂) ₅CO-RKKRRQRRR-HN(CH ₂) ₅CO-KTKGGWLIQLFHKK-OH

4.Ahx-BNEP-CMT(32)

According to the logical method of the amino hexanoylization of 6-, the experiment condition of Ahx-BNEP-CMT-040108 and the results are shown in following table.

Table 25 preparative conditions and results ofAhx-BNEP-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 4h; Resin detects transparent; DBLK 16min
F-Ahx-OH	1.2	0.424g	0.425g	6
					TBTU	1.9	0.620g	0.626g	9.9
HOBt	1.3	0.176g	0.179g	6.6
					TEA	1.2ml/mmol acid	1.2ml	1.2ml
Thick peptide: 470mg; Smart peptide: 234.0mg; Yield 49.8%

5.Ahx-BNEP-Ahx-CMT(41)

According to the logical method of the amino hexanoylization of 6-, the experiment condition of Ahx-BNEP-Ahx-CMT-040108 and the results are shown in following table.

Table 26 preparative conditions and results of Ahx-BNEP-Ahx-CMT-040108

Raw material	Mole number/mmol	Theoretical charging capacity	Actual charging capacity	Mol ratio	Remarks
						BNEP- Ahx-CMT-Pr	0.2	1.0g	1.0g	1	Reaction 6h resin detects transparent DBLK 10min
F-Ahx-OH	1.2	0.424g	0.423g	6
					TBTU	1.3	0.417g	0.418g	6.6
HOBt	1.93	0.260g	0.266g	9.9
					TEA	0.9ml/mmol acid	0.9ml	0.9ml
Thick peptide: 411mg; Smart peptide: 292.1; Yield 71.1%

Embodiment 7

Present embodiment relate to of the present invention antibiotic/ethamineization of antiendotoxin allosteric peptide.

Ethamine is separated logical method: with a certain amount of peptide resin of DMF swelling, add capacity ethylamine solution (70%EtNH ₂.H ₂O), shake for some time, room temperature is placed.TLC monitoring reaction process.Suction filtration, the DMF washing, filtrate merges.Temperature control revolves and steams near doing, and the suitable solvent dissolving is revolved and steamed to doing; Perhaps concentrate, add anhydrous diethyl ether, supernatant liquor is poured out in sedimentation back refrigeration fully, and residual solid is with dissolution with solvents, and drying is filtered in sedimentation once more, refrigeration.The ethamine that so obtains is separated the full guard peptide, and the method for selecting cracking gets thick peptide; HPLC method purifying, freeze-drying gets pure product.The molecular weight conclusive evidence is surveyed in quality test (purity, content), mass spectrum.

1.Ac-Ahx-BNEP-NHEt(10)

1.900g (0.80mmol) Ac-Ahx-BNEP-WR-040102 adds 30mLDMF, shakes up the back and drips 19mL 70%EtNH ₂.H ₂O shakes for some time, and room temperature is placed.TLC monitoring reaction process.Behind 22 ℃, 28h, suction filtration, DMF washing resin 4 times, filtrate merges.50 ℃ revolve and steam near doing, and the DCM dissolving is revolved and steamed to doing, and vacuum-drying gets ethamine and separates full guard peptide 1.538g, yield 66.7%.Add the classical lytic reagent of 20mL (Reagent R:TFA/thioanisole/anisole/EDT, 90: 5: 3: 2, v/v), stirring at room reaction 3h, sedimentation drain thick peptide 813mg; HPLC method purifying, freeze-drying gets pure product 137.4mg, purity 87.84%.

It modifies base and the peptide preface is CH ₃CO-NH (CH ₂) ₅CO-KTKGGWLIQLFHKK-NHCH ₂CH ₃

2.BNEP-NHEt(11)

1.496g (0.632mmol) Fmoc-BNEP-pr-031112-S-W adds 12mL DMF, shakes up the back and drips 16mL 70%EtNH ₂.H ₂O shakes for some time, and room temperature is placed.Behind 20 ℃, 69h, suction filtration, DMF washing resin 2 times, filtrate merges.50 ℃ revolve and steam near doing, and the MeOH dissolving is revolved and steamed to doing, and vacuum-drying must go Fmoc ethamine to separate full guard peptide 1.208g, yield 70.4%.Add 20mL lytic reagent Reagent R and react 2.5h in stirring at room, sedimentation drain thick peptide 491mg; HPLC method purifying, freeze-drying gets pure product 102.5mg, purity 92.80%.

3.BNEP-CMT-NHEt(28)

1.421g (0.449mmol) Fmoc-BNEP-CMT-Pr-040108-S-W adds 18mL DMF, shakes up the back and drips 18mL 70%EtNH ₂.H ₂O shakes for some time, and room temperature is placed.TLC monitoring reaction process.Behind 28 ℃ of 50h, suction filtration, DMF washing resin 2 times, filtrate merges.50 ℃ revolve and steam near doing, and the MeOH dissolving is revolved and steamed to doing, and vacuum-drying must go the ethamine of Fmoc to separate full guard peptide 1.30g, yield 46.7%.Add the classical lytic reagent (Reagent R) of 26mL, stirring at room reaction 3h, sedimentation drain thick peptide 1400mg (102.8%); HPLC method purifying, freeze-drying gets pure product 172.7mg.

4.BNEP-Ahx-CMT-NHEt(42)

1.476g (0.466mmol) Fmoc-BNEP-Ahx-CMT-WR-040102 adds 14mL DMF, shakes up the back and drips 15mL 70%EtNH ₂.H ₂O shakes for some time, and room temperature is placed.TLC monitoring reaction process.Behind 22 ℃ of 61h, suction filtration, DMF washing resin 4 times, filtrate merges.50 ℃ revolve and steam near doing, and the DCM dissolving is revolved and steamed to doing, and vacuum-drying must go Fmoc ethamine to separate full guard peptide 1.40g, yield 47.5%.Add the classical lytic reagent (Reagent R) of 25mL, stirring at room reaction 3h, the sedimentation freeze-drying gets thick peptide 1500mg (102.2%); HPLC method purifying, freeze-drying gets pure product 154.6mg, total recovery 10.5%.

It modifies base and the peptide preface is KTKGGWLIQLFHKK-NH (CH ₂) ₅CO-RKKRRQRRR-NHCH ₂CH ₃

Embodiment 8

Present embodiment relates to part of the present invention antibiotic/antiendotoxin allosteric peptide is to the inhibition test of groups of people source germ.

1. experimental strain

The experiment bacterial strain uses therefor is in January, 2004～2004 year Third Military Medical University southwest hospital clinical separation in February pathogenic bacterium, is separated and evaluation by southwestern hospital laboratory.Wherein streptococcus aureus 7 strains, Pseudomonas aeruginosa 7 strains, Portugal coccus 2 strains of epidermis Portugal, escherichia coli 7 strains, Klebsiella Pneumoniae 4 strains amount to 27 strains.Do the Quality Control bacterium with standard bacterium escherichia coli ATCC25922, streptococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853.

2. substratum and reagent

Mueller-Hinton (MH) substratum (liquid, solid), Nat'l Pharmaceutical ﹠ Biological Products Control Institute's product (lot number 010925).

Phosphate buffered saline buffer (PBS): dipotassium hydrogen phosphate 8.17g, potassium primary phosphate 0.87g adds distilled water 1000ml, and transferring pH with 0.1N NaOH is 6.0.

3. instrument

Multiple spot inoculation instrument (Japanese storehouse light Co., Ltd. produce)

Electronic balance (Sartorius, Germany)

(Beckman 21, USA) for pH meter

Adjustable pipette (eppendorf, USA)

4. experimental technique

4.1 test liquid preparation

Precision takes by weighing 1-10 number polypeptide sample to be measured in the 2ml volumetric flask, uses pH6.0 phosphate buffered saline buffer (PBS) dissolving respectively, and being made into drug content is the 3840mg/L storing solution.

4.2 test organisms liquid preparation

Before the experiment, get an amount of lawn from the semisolid medium (or inclined-plane) of preserving bacterial classification, be seeded on the MH substratum, cultivate 16～18h in 37 ℃, the single colony inoculation that picking is fresh is in the MH broth culture again, increase bacterium in 37 ℃ and cultivate 16～18h, take out, it is that 0.5 Maxwell is than turbid standard (bacteria containing amount 10 that every pipe bacterium liquid dilutes respectively with the MH broth culture ^7-8CFU/ml), keeping the final inoculum size of bacterium liquid is every 10 ^4-5CFU.

4.3 minimum inhibitory concentration (MIC) is measured

Adopting the agar doubling dilution, is that the storing solution of 3840mg/L becomes series concentration with the pH6.0PBS doubling dilution with medicament contg, gets the plate that 1ml puts into diameter 90mm respectively, the MH nutrient agar that adds the 14ml fusing, mixing promptly gets drug content and is respectively 256,128,64,32,16,8,4,2,1,0.5,0.25,0.125mg/L serial agar plate, treat that agar solidifies after, with multiple spot inoculation instrument on pastille agar plate and blank agar plate surface dibbling bacterium, hatch the 24h observations for 37 ℃, do not see that the interior minimum drug level of plate of bacterial growth is minimum inhibitory concentration (MIC).

Experimental result sees the following form.

Table 27 BNEP derivatives are to the inhibition test (MIC:mg/L) of groups of people source germ

	1	2	3	4	7	8	9	11	12	15
											The gold ATCC of Portugal 25923	32	128	32	32	64	16	64	16	64	32
The gold 318MRS of Portugal	64	256	64	128	128	64	128	32	＞256	64
											Gold Portugal 273	＞256	256	＞256	＞256	＞256	＞256	＞256	＞256	＞256	＞256
Gold Portugal 321	64	256	64	128	128	64	64	64	128	64
											Gold Portugal 453	64	64	16	16	16	4	4	64	16	8
Gold Portugal 464	8	128	16	8	8	4	4	8	8	8
											Gold Portugal 465	8	128	16	8	8	4	4	8	8	8
Table Portugal 370	4	64	16	8	8	2	4	4	4	4
											Table Portugal 372	8	64	64	32	16	4	8	16	4	8
Large intestine ATCC 25922	128	128	64	128	＞256	64	256	128	128	64
											Large intestine 249	128	128	128	128	256	64	128	64	128	64
Large intestine 339	128	256	128	128	256	128	128	128	128	64
											Large intestine 355	64	128	64	64	256	64	64	64	64	64
Large intestine 404	＞256	256	＞256	＞256	＞256	＞256	＞256	＞256	＞256	256
											Large intestine 405	64	128	64	64	256	64	64	64	64	64
Large intestine 467	64	128	64	128	256	64	64	128	64	64
											Green pus ATCC 27853	＞256	256	128	256	256	256	256	128	＞256	128
Green pus resistance 328	64	256	64	128	128	128	256	64	＞256	128
											Green pus 406	＞256	256	128	256	256	128	＞256	128	＞256	256

Green pus 419	＞256	256	128	256	256	128	256	128	＞256	128
											Green pus 433	＞256	256	64	256	256	128	256	128	＞256	128
Green pus 451	64	128	64	32	32	64	64	64	64	32
											Green pus 460	＞256	256	128	256	256	128	256	128	＞256	128
Lung gram 353	＞256	256	256	128	256	256	256	128	＞256	128
											Lung gram 452	256	256	128	128	256	128	256	128	128	256
Lung gram 461	＞256	256	256	256	＞256	＞256	256	＞256	128	256
											Lung gram 468	＞256	256	128	256	＞256	＞256	256	256	128	128

Embodiment 9

Present embodiment relates to part of the present invention antibiotic/antiendotoxin allosteric peptide is to the inhibition activity of several agriculture pathogenic bacterium.

1 materials and methods

1.1 test materials

1.1.1 part allosteric peptide

1.1.2 experimental strain

Subtilis MIG1.22 (Bacillus subtilis)

Bacterial stripe bacterium (Xanthomonas campestris)

Rice leaf spot bacteria (Xanthomonas oryzae)

Above bacterial classification is all provided by Institute of Plant Protection, academy of agricultural sciences, Guangdong Province.

1.1.3 substratum

Nutrient agar: smart agar powder (import packing) 15 restrains, peptone 3 grams, and glucose 3 grams, dipotassium hydrogen phosphate 4 grams, deionized water 1000ml transfers pH=7.0, sterilizes 30 minutes for 121 ℃.

Miller V-8 calcium carbonate culture-medium: V-8 liquid 200ml, lime carbonate 2 grams, agar 20 grams, deionized water 800ml filters with wire cloth earlier, through twice of which floor filtered through gauze and absorbent cotton filters again.Filter into the stillness of night through No. 1 filter paper of Whatman on the Buchner funnel at last.With containing the 95ml nutrient solution in every bottle of the 250ml triangular flask, 121 ℃ of sterilizations 15 minutes.pH＝6.4-6.7。As prepare solid medium, and add agar 20 grams in every liter of liquid, use for cultivating phytophthora.

PDA substratum: potato 200 grams, glucose 15 grams, agar 20 grams, deionized water 1000ml.Nature pH sterilized 30 minutes for 121 ℃.

The Ben Shi of association potato semisynthetic medium: potato 300 grams, nitrocalcite 0.5 gram, Sodium phosphate dibasic 2 grams, peptone 5 grams, sucrose 15 grams, agar 18 grams, deionized water 1000ml.Nature pH sterilized 30 minutes for 121 ℃.

1.2 laboratory apparatus and equipment

303-DB type water isolation type incubator, Nantong scientific instrument factory

XDD-84 vertical electric High pressure steam sterilizer, ShaoXing,ZheJiang medical apparatus and instruments factory

The BS200 electronic analytical balance, Germany this company of match multidimensional

1.3 experimental technique

1.3.1 polypeptide solution is prepared: by aseptic technique 1-10 number polypeptide to be measured is diluted to 50mg/ml with aqua sterilisa.

1.3.2 paper disk method: punch on quantitative paper with punch tool, obtain the circular filter paper sheet of diameter 5mm, it is standby to be loaded in the culture dish 121 ℃ of sterilization 30min.Get 2.5 μ l with liquid-transfering gun at every turn and soak filter paper, filter paper is left standstill dry then, be tiled in the culture dish of good substratum.Note exchanging for the rifle head before detecting next liquid to be measured.

1.3.3 the restraining effect to Bacillus subtilus is measured

1.3.3.1 the preparation of Bacillus subtilus spore suspension: the Bacillus subtilus of storage is equipped with in the eggplant bottle of nutrient agar medium with the method for scoring access, cultivated 72 hours for 37 ℃, with sterilized water 100ml Bacillus subtilus and spore thereof are washed in the 250ml tripod bottle of sterilization, make suspension.With water-bath with suspension in 70 ℃ of insulation 30min, kill nourishing body, keep spore.

1.3.3.2 measure antibacterial circle diameter with paper disk method, each sample repeats for 3 times.With sterilized water in contrast.

1.3.4 bacterial stripe bacterium, bacterial leaf spot pathogenic bacteria restraining effect are measured

Substratum is poured in the culture dish of diameter 9cm, stand-by after the condensation.Wash cultured strains tested on the test tube slant with sterilized water 3ml, get 0.2ml and drop on the substratum, use the spreading rod of the bacterium of going out to smoothen, wait to do the back and measure the inhibition zone radius, in contrast with sterilized water with paper disk method.

Bacterial stripe bacterium and rice leaf spot bacteria restraining effect are detected with the Ben Shi of association potato semisynthetic medium.

2 experimental results

By a large amount of tests, obtained some PRELIMINARY RESULTS, the excellent development prospect that shows as that has, part is not as people's will.In general, the acetate of BNEP derivative, the result is comparatively clear and definite, and its trifluoroacetate waits further checking.

2.1 restraining effect to Bacillus subtilus

Experimental result shows the restraining effect difference (see Table 1-1, Fig. 1-1) of different derivatives to Bacillus subtilus.

The different derivatives of Table 1-1 are to the restraining effect effect of Bacillus subtilus growth

Numbering

	1	2	3	4	6	7	8	9	10	11	12	13	14	CK +	CK
																Concentration (mg/ml) inhibition zone radius (mm)	50 1	50 1	50 1	50 1.2	50 1.3	50 1.3	50 2	50 1.3	50 0	50 1.5	50 0	50 0	50 2	50 4	50 0

2.2 restraining effect to the bacterial stripe bacterium

Experimental result shows that different derivatives (see Table 1-2, Fig. 1-2) to the restraining effect difference of bacterial stripe bacterium.

The different derivatives of Table 1-2 are to the restraining effect effect of bacillary streak growth

Numbering

	1	2	3	4	6	7	8	9	10	CK +	CK -
												Concentration (mg/ml) inhibition zone radius (mm)	50 2	50 3	50 0.5	50 2	50 2	50 1.7	50 1.6	50 0	50 0	50 3	50 0

2.3 restraining effect to rice leaf spot bacteria

Experimental result shows the restraining effect difference (see Table 1-3, Fig. 1-3) of different derivatives to rice leaf spot bacteria.

The different derivatives of Table 1-3 are to the restraining effect effect of rice leaf spot bacteria growth

Numbering

	1	2	3	4	6	7	8	9	10	CK +	CK -
												Concentration (mg/ml) inhibition zone radius (mm)	50 0	50 3	50 3.5	50 4	50 5	50 2.5	50 1.5	50 5.5	50 3	50 5.5	50 0

3 BNEP and derivative thereof are measured the MIC/MBC of bacterial leaf streak of rice

3.1 the preparation of medicine and bacterium liquid

A. the preparation of medicine: with sterilized water, PBS damping fluid 1-14 number, 13c-27 number polypeptide sample dissolution to be measured being mixed with content respectively at aseptic is 2mg/ml mother liquor (abbreviation soup).Preparating liquid carries out two times of concentration dilutions that successively decrease (coubling dilution), and 10 doubling dilution concentration of each sample work (2.0,1.0,0.5,0.25,0.125,0.063,0.031,0.016,0.0078,0.0039mg/ml), get storage liquid, stand-by.

B. the preparation of bacterium liquid: will for the examination bacterium in association's Ben Shi liquid nutrient medium in 28 ℃ with the 220r/min shaking culture to logarithmic growth OD600 in mid-term be about 0.5.Be diluted to proper concn (10 with 2 * nutrient solution ⁵CFU/ml).

Bacterial count: preliminary experiment will prepare bacterium liquid and carry out 10 times of serial dilutions (10 with physiological saline ^-1, 10 ^-2, 10 ^-3), get 0.1ml bacterium liquid to the plate nutrient agar, gently bacterium liquid is spread out with spreading rod, cultivated number bacterium colony number 24 hours.

3.2MIC measure: carry out with micro-dilution method.Add successively with micro sample adding appliance in 8 * 12 hole U-shapeds, 96 orifice plates of sterilization aseptic 10 doubling dilution concentration storage liquid (2mg/ml-3.9 μ g/ml) every kind of medicine, every hole 75 μ l, inoculate 75 μ l dilution bacterium liquid then, the 11st hole is bacterium control wells (only adding liquid substratum and bacterium liquid), the 12nd hole is polypeptide soup control wells (only adding the polypeptide soup), hatched observations under black background 24～48 hours for 28 ℃.As the soup hole that adds indicator still is clarification, and then this hole medicine has anti-microbial effect.With the minimum concentration hole of visual inspection, be defined as MIC (mg/ml) with delegation's asepsis growth.Experimental result sees Table 1.

3.3MBC: observe each experimental port culture of not seeing bacterial growth more than the medicine minimum inhibitory concentration, draw 100 μ l respectively, culture transferring was hatched 24～48 hours for 28 ℃ to the plate nutrient agar, and direct viewing has or not bacterium colony to form.MBC is defined as the lowest concentration of drug that no bacterium colony forms.Experimental result sees Table 1.

Table 1 each peptide is to the MIC of bacterial leaf streak of rice, MBC

peptides

	1	2	3	4	6	7	8	9	10	11	12	13	14
														MIC(mg/ml) MBC(mg/ml)	0.125 0.5	0.125 0.5	1.0 2.0	1.0 2.0	1.0 2.0	0.125 0.25	0.5 2.0	1.0 2.0	1.0 2.0	0.5 2.0	2.0 2.0	1.0 2.0	1.0 2.0

peptides	13c	17	18	19a	20	21	22	23	24	25	26	27
													MIC(mg/ml) MBC(mg/ml)	0.25 0.5	1.0 2.0	0.5 1.0	0.25 1.0	1.0 2.0	/ /	1.0 2.0	0.5 ＞2.0	0.5 1.0	0.5 1.0	0.25 0.5	0.5 1.0

Embodiment 10

Present embodiment relates to part of the present invention antibiotic/antiendotoxin allosteric peptide is to the provide protection of LPS attack cells.

As everyone knows, when cell was subjected to intracellular toxin LPS attack, the reaction that can initiatively stress be inflamed produced a series of inflammatory factors such as TNF-a, interleukin-IL-1, IL-2, IL-6, IL-8 etc.The inspection of TNF-a can be investigated the provide protection of BNEP derivative polypeptide to the LPS attack cells.

1. test one

1.1 material and key instrument

1.1.1 synthetic peptide: sample presentation is numbered 1,3,4, CMT-04-2,6, CMT-03-2a, CMT-04-2a, 9,10,11, CMT-03,17,18,19a, 20b, 21,22,23,24,25,26,27 BNEP derivative polypeptide;

1.1.2 other reagent

Cell strain: the THP-1/CD14 cell strain is provided by immunology teaching and research room of No.1 Military Medical Univ.;

LPS 7261:Sigma company product

TNF-a detection kit: eBioscience company product;

PXB (polymyxin B sulfate, PB): available from Shenzhen brilliant U.S. company;

IMDM nutrient solution: Invitrogen company product

1.1.3 key instrument

Constant temperature incubator: Thermo Forma Model 3111

96 porocyte culture plate: Falcon

Enzyme-linked immunoassay instrument (ELISA A450 pH-value determination pH instrument): BIO-RAD Model 550

Inverted microscope: COIC XDS-1 (optical instrument factory, Chongqing)

Electronic balance: Sartorius BP61

Liquid-transfering gun: GILSON

1.2 experimental technique

1.2.1 test cell line: cultivate the THP-1/CD14 cell, dilute and be 1X10 ⁵/ hole bed board, 37 ℃, 5%CO ₂Hatch 24h; Nutrient solution is removed in suction, with the BNEP part derivative polypeptide of above-mentioned numbering with the dilution of IMDM nutrient solution be 50,10,2,0.4ug/ml (annotate: No. 17 later synthetic peptide dilutions are 100,20,4,0.8ug/ml), add 100ul/ hole in the culture hole, hatch 30min; LPS 7261 dilutions are 200ng/ml, add 100ul/ hole in the culture hole, set control group; 37 ℃, 5%CO ₂After hatching 6h, draw supernatant, centrifugal, supernatant is detected TNF-a according to the test kit explanation.

1.2.2TNF-a detect: carry out according to the test kit specification sheets.

1.2.3 experimental result

Synthetic peptide inhibition test result is reported as follows: be followed successively by 1,3,4, CMT-04-2,6, CMT-03-2a, CMT-04-2a, 9,10,11, CMT-03,17,18,19a, 20b, 21,22,23,24,25,26,27.In the table capable 1,2,3,4 row of A are No. 1 peptide concentration corresponding to 50,10,2, the experimental result of 0.4ug/ml; Capable 9,10,11,12 row of D are No. 17 peptide concentrations corresponding to 100,20,4, the experimental result of 0.8ug/ml; Capable 5,6,7,8 row of H are PB concentration corresponding to 50,10,2, the experimental result of 0.4ug/ml; H-9,10 is no peptide control wells.

Table 2 TNF-a detected result (OD ₄₅₀)

2. test two

2.1 material and key instrument

2.1.1 synthetic peptide: sample presentation numbering 1,3,4, CMT-03-2a, CMT-04-2a, 16,28-50 BNEP part derivative polypeptide;

2.1.2 other reagent

XTT:MOLECULAR Probes company product;

2.2 experimental technique

2.2.1 test cell line: cultivate the THP-1/CD14 cell, dilute and be 1X10 ⁴/ hole bed board, 37 ℃, 5%CO ₂Hatch 24h; Nutrient solution is removed in suction, will in and peptide be 50,25,12.5,6.25 μ g/ml with IMDM nutrient solution dilution, add 100ul/ hole in the culture hole, hatch 30min; LPS 7261 dilutions are 200ng/ml, add 100 μ l/ holes in the culture hole, set control group; 37 ℃, 5%CO ₂After hatching 6h, draw supernatant, centrifugal, supernatant is detected TNF-a according to the test kit specification sheets.

2.2.2XTT detect: with IMDM dilution XTT concentration is 1mg/ml, and filtration sterilization adds IMDM substratum 80 μ l and XTT 20 μ l in the cell cultures hole of above-mentioned removal supernatant, hatch 6h, directly detects the 450nm absorbance.

2.2.3TNF-a detect: carry out according to the test kit specification sheets.

2.3 experimental result

2.3.1 inhibition test result

Synthetic peptide inhibition test result is reported as follows: listedly in the table be followed successively by 1,3,4, CMT-03-2a, CMT-04-2a, 16,28-50 number synthetic peptide, each sample standard deviation is done 4 gradient doubling dilutions.Be that 1,2,3,4 capable row of A are to be numbered 1 BNEP derivative polypeptide, its concentration is corresponding to 25,12.5,6.25,3.125 μ g/ml; 5,6,7,8 row that A is capable are to be numbered 3 BNEP derivative polypeptide, and its concentration is corresponding to 25,12.5,6.25,3.125 μ g/ml; 9,10,11,12 row that B is capable are to be numbered 16 BNEP derivative polypeptide, and its concentration is corresponding to 25,12.5,6.25,3.125 μ g/ml; All the other and the like.The capable 1-6 of K hole is irrelevant peptide control group; Capable 1,2,3,4 holes of L are the PXB contrast, and concentration is followed successively by 100,50,25,5 μ g/ml, and L is no peptide control wells capable 5, No. 6, and L is irrelevant peptide control wells capable 7,8,9, No. 10, and L is the blank hole capable 11, No. 12.

Table 3 TNF-a detect

D	(31) 1.577	1.271	1.415	1.029	(32) 0	0.038	0.237	0.692	(33) 0.055	0.674	1.155	1.077
													E	(34) 0.028	0.131	0.601	1.018	(35) 0	0.066	0.374	0.83	(36) 0	0.178	0.672	0.621
F	(37) 0.054	0.107	0.516	1.063	(38) 0	0.094	0.278	0.967	(39) 0.682	0.690	0.878	0.865
													G	(40) 0.706	1.458	1.615	1.061	(41) 0	0.059	0.461	0.882	(42) 0	0.134	0.607	0.698
H	(43) 0.050	0.362	0.616	1.237	(44) 0.014	0.019	0.050	0.605	(45) 0.015	0.097	0.591	0.698
													1	(46) 0.266	1.045	1.232	0.399	(47) 0.013	0.086	0.168	0.569	(48) 0.117	0.240	0.328	0.504
J	(49) 0.039	0.177	0.551	0.305	(50) 0.292	0.334	0.545	0.911
													K	(CTL) 0.969	0.905	0.989	0.464	0.298	0.273
L	(PB) 0.066	0.262	0.412	0.539	(POS) 1.184	1.055	1.206	1.091	0.791	0.713	0.048	0

2.3.2XTT method detects cytotoxicity

The XTT method detects the toxicity of each peptide to the THP-1/CD14 cell, after detecting, TNF-a carries out, so the numbering of following table, pairing peptide, same with last epiphase.

Table 4 XTT methods detect cytoactive

I	0.148	0.196	0.298	0.441	0.134	0.231	0.411	0.553	(48) 0.508	0.520	0.525	0.527
													J	0.176	0.255	0.431	0.501	(50) 0.507	0.489	0.543	0.560
K	0.545	0.538	0.534	0.537	0.521	0.608
													L	0.117	0.156	0.180	0.391	0.584	0.387	0.265	0.209	0.359	0.322	0	0

Investigate in conjunction with above-mentioned two experimental results, be numbered 1,3,4,5 small peptides of CMT-03-2a, CMT-04-2a are to THP-1/CD14 cell free of toxic effects, and can suppress the effect of LPS7261, meet the conclusion (of pressure testing) in early stage.Be numbered three small peptides of 39,48,50 and can obviously suppress the effect (seeing accompanying drawing) that LPS stimulates THP-1/CD14 emiocytosis TNF-a.

Claims (9)

1. antibiotic, antiendotoxin allosteric peptide molecule is characterized in that: have following general structure,

R ¹-BNEP-X(R ³)n。

2. antibiotic, antiendotoxin allosteric peptide molecule according to claim 1 is characterized in that: have following general structure,

R ¹-BNEP-CMT-X(R ³)n。

3. antibiotic, antiendotoxin allosteric peptide molecule according to claim 1 is characterized in that: have following general structure,

R ¹-CMT-BNEP-X(R ³)n

4. antibiotic, antiendotoxin allosteric peptide molecule according to claim 1 is characterized in that: have following general structure,

R ¹-BNEP-Z-CMT-X(R ³)n

5. antibiotic, antiendotoxin allosteric peptide molecule according to claim 1 is characterized in that: have following general structure,

R ¹-CMT-Z-BNEP-X(R ³)n

6. according to arbitrary antibiotic, the antiendotoxin allosteric peptide molecule described in the claim 1 to 5, it is characterized in that: the pharmaceutically acceptable various mineral acids, the organic acid salt that show as polypeptide.

7. according to arbitrary antibiotic, the antiendotoxin allosteric peptide molecule described in the claim 1 to 5, it is characterized in that: show as in the chain of polypeptide or an end position cyclic peptide.

8, arbitrary antibiotic, antiendotoxin allosteric peptide molecule according to claim 7 is characterized in that: show as in the chain of each peptide species or pharmaceutically acceptable various mineral acids, the organic acid salt of end position cyclic peptide.

9. the synthetic method of arbitrary antibiotic, the antiendotoxin allosteric peptide molecule described in the claim 1 to 5 is characterized in that:

(1) the synthetic logical method of parent peptide resin

Taking by weighing an amount of Fmoc-AA-Wang resin or other polypeptide synthesizing amino acid resin pours in the synthetic post; add solvent (DCM; DMF; or DCM-DMF) swelling; remove the Fmoc blocking group of amino-acid resin with the DMF solution of 10%-50% hexahydropyridine; wash successively with DCM, MeOH, DMF, DCM, DMF, eliminate hexahydropyridine.According to of the present invention antibiotic/aminoacid sequence of antiendotoxin allosteric peptide takes by weighing an amount of Fmoc-amino acid successively in suitable container, add an amount of DMF dissolving, add then coupling agent (as TBTU/HOBt, HBTU/HOBt, HATU/HOAt, DIC/HOBt, DCC/HOBt, DCC/HOSu, TBTU/HOSu, PyBOP/HOBt, BOP/HOBt, etc) and the DMF solution of proper N methylmorpholine or diisopropylethylamine.After coupling is finished, remove reaction solution, wash peptide resin successively with DCM, MeOH, DMF, DCM, DMF; The DMF solution that adds the 20%-50% hexahydropyridine removes the Fmoc blocking group of amino-acid resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.According to polypeptide solid state chemistry synthetic method according to of the present invention antibiotic/aminoacid sequence of antiendotoxin allosteric peptide parent is from the follow-up coupling of C end beginning carrying out successively, so circulation finish whole antibiotic/peptide chain of antiendotoxin allosteric peptide, obtain the parent peptide resin; Thorough washing, MeOH shrink, and drain, from synthetic post, take out synthetic antibiotic/antiendotoxin peptide derivant resin, put freeze-drying in the Freeze Drying Equipment, standby.

(2) derivative preparation

According to the difference of modification group, adopt different synthetic methods, prepare the antibiotic/antiendotoxic mimic polypeptide derivative of full guard; the thick peptide that obtains gets smart peptide through high performance liquid chromatography (HPLC) purifying; smart peptide salify, mass spectrum are identified and HPLC checks purity, carry out biological activity and identify.

(2.1) nitrogen end acylations

Acylting agent is a lot, and modal agent combination has: (RCO) ₂O/pyridine, RCO ₂H/activatingreagent (activator).Common activator has: DCC/HOBt, DCC/HOSu, TBTU/HOBt, HBTU/HOBt, HATU/HOAt, DIC/HOBt, TBTU/HOSu, PyBOP/HOBt, BOP/HOBt, DIC/HOSu, PyBOP/HOSu.

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post; add solvent (DCM; DMF; orDCM-DMF) swelling; drain; the DMF solution that adds the 20%-50% hexahydropyridine removes the Fmoc blocking group of peptide resin, washs successively with DCM, MeOH, DMF, DCM, DMF, eliminates hexahydropyridine.The DMF solution that adds acylting agent stirs or logical nitrogen or vibration temperature control.After coupling is finished, remove reaction solution, wash successively with DCM, MeOH, DMF, DCM, DMF, MeOH shrinks, and drains, from synthetic post, take out synthetic antibiotic/antiendotoxin peptide derivant resin, put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal; Add anhydrous diethyl ether, stir evenly, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

(2.2) substituted peptide amide

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post, and the adding solvent (DCM, DMF, DCM-DMF, THF, THF-DMF) swelling is drained.The DMF solution that adds aminated reagent stirs or logical nitrogen temperature control.After amidation was finished, suction filtration was removed reaction solution, with DMF, MeOH, THF, Et ₂O washs successively, and filtrate merges, and revolves to steam to doing or put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal; Add anhydrous diethyl ether, stir evenly, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

(2.3) peptide ester

Antibiotic/antiendotoxin allosteric peptide parent peptide resin is poured in the synthetic post, and the adding solvent (DCM, DMF, DCM-DMF, THF, THF-DMF) swelling is drained.The DMF solution that adds esterifying reagent stirs or logical nitrogen temperature control.After esterification was finished, suction filtration was removed reaction solution, with DMF, MeOH, THF, Et ₂O washs successively, and filtrate merges, and revolves to steam to doing or put freeze-drying in the Freeze Drying Equipment.Add an amount of lysate, stir, reacted 2-5 hour, the anhydrous diethyl ether sedimentation, centrifugal, repeat 6 times.Add water or 50% aqueous acetic acid, freeze-drying.The HPLC purifying, salify or freeze-drying, mass spectrograph is surveyed molecular weight, and HPLC checks purity and ion content.

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