CN1361791A - Process for deacylation of lipodepsipeptides - Google Patents
Process for deacylation of lipodepsipeptides Download PDFInfo
- Publication number
- CN1361791A CN1361791A CN00810417A CN00810417A CN1361791A CN 1361791 A CN1361791 A CN 1361791A CN 00810417 A CN00810417 A CN 00810417A CN 00810417 A CN00810417 A CN 00810417A CN 1361791 A CN1361791 A CN 1361791A
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- CN
- China
- Prior art keywords
- pseudomucin
- compound
- syringomycin
- natural product
- deacylase
- Prior art date
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- 229950010765 pivalate Drugs 0.000 description 1
- IUGYQRQAERSCNH-UHFFFAOYSA-N pivalic acid Chemical compound CC(C)(C)C(O)=O IUGYQRQAERSCNH-UHFFFAOYSA-N 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229940093916 potassium phosphate Drugs 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical class OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 229930185586 syringostatin Natural products 0.000 description 1
- 108010030788 syringostatin A Proteins 0.000 description 1
- SOKGGVHELUKAFO-UHFFFAOYSA-N syringostatin A Natural products CCCCCCCCCCCC(O)CC(=O)NC1COC(=O)C(C(O)CCl)NC(=O)C(C(O)C(O)=O)NC(=O)C(=CC)NC(=O)C(C(C)O)NC(=O)C(CCCN)NC(=O)C(CCO)NC(=O)C(CCN)NC(=O)C(CCN)NC1=O SOKGGVHELUKAFO-UHFFFAOYSA-N 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N thiocyanic acid Chemical compound SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A process is described for deacylating a lipodepsipeptide to produce the corresponding nucleus. The products produced from this process is also described (e.g., a pseudomycin nucleus represented by structures (I) or (II)).
Description
Invention field
The present invention relates to fat depsipeptides (lipodepsipeptides), particularly pseudomucin (pseudomycin) and syringomycin natural product the N-acyl side-chain deacylation and therefrom the preparation compound.
Background of invention
Pseudomucin and syringomycin are the natural products of separating from the liquid nutrient medium of Pseudomonas syringae (PseudomoHassyringae) (plant Related Bacteria), and verifiedly have an anti-mycotic activity.(promptly referring to Harrison, L. etc., " pseudomucin is from the new peptide of the gang with broad-spectrum antifungal activity of Pseudomonas syringae ", heredity JOURNAL OF MICROBIOLOGY (J.Gen.Microbiology) 137 (12), 2857-65 (1991) and U.S. Patent No. 5576298 and 5837685).Different with previously described anti-mycotic agent from Pseudomonas syringae (for example syringomycin, Pseudomonas syringae toxin and syringostatins), pseudomucin A-C contains the hydroxyl aspartic acid, aspartic acid, Serine, dehydrogenation aminobutyric acid, Methionin and DAB.
Pseudomucin A, A ', B, B ', C, the peptide moiety of C ' have the big ring of terminal carboxyl(group) sealing corresponding to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp (3-OH)-L-Thr (4-Cl) on the OH base of the terminal Ser of N-.These analogue differences are the N-acyl side-chain; be that pseudomucin A is by 3; 4-dihydroxyl tetradecanoyl N-acylations; pseudomucin A ' is by 3; 4-dihydroxyl pentadecanoyl N-acylations, pseudomucin B is by 3-hydroxyl tetradecanoyl N-acylations, and pseudomucin B ' is by 3-hydroxyl lauroyl N-acylations; pseudomucin C is by 3, and 4-dihydroxyl hexadecanoyl N-acylations and pseudomucin C ' are by 3-hydroxyl hexadecanoyl N-acylations.(promptly referring to Ballio, A., etc., " from the new bioactive lipid depsipeptides of Pseudomonas syringae: pseudomucin ", FEBS Letters, 355 (1), 96-100, (1994) and Coiro, V.M., wait the people, " being used to measure by computer simulation the comformation in solution of Pseudomonas syringae MSU 16H phytotoxicity fat depsipeptides pseudomucin A " from the geometry spacing of NMR data and molecular mechanics, european journal of biological chemistry (Eur.J.Biochem.) 257 (2), 449-456 (1998)).
Known pseudomucin and syringomycin have some disadvantageous biological actions.Found the destruction venous endothelial when for example intravenously is used pseudomucin, disorganize, inflammation and to the local toxicity of host tissue.Therefore, need to identify in this compounds useful and do not have a compound of the adverse side effect of current discovery to treatment fungi infestation.
Summary of the invention
The invention provides the method that the N-acyl side-chain deacylation of fat depsipeptides natural product is produced corresponding ring compound.The amino ring compound of the pseudomucin of structural formula I representative below pseudomucinous deacylation produces.
This ring compound can be used as the initiator of the semi-synthetic derivative for preparing corresponding natural product.
This method comprises makes pseudomucin natural product and the deacylase reaction that is selected from ECB deacylase and polymycin acyltransferase produce the corresponding ring compound of structural formula I representative.Unhindered amina can be reset the cyclic peptide ring compound with a free hydroxyl group (also being referred to as pseudomucin hydroxyl ring compound) that produces following structural formula II representative.
Compound I I again can be as the initiator of the new derivative that produces pharmaceutical active.
In another embodiment of the invention, aforesaid method is used to syringomycin compound deacylation is produced the amino ring compound of syringomycin.For example the amino ring compound of syringomycin E has following structural formula II I.
The same with the amino ring compound of pseudomucin, the amino ring compound of syringomycin can be reset the compound IV (also being referred to as syringomycin hydroxyl ring compound) below generating.
Although described Compound I above, II, the particular chiral form of III and IV, other chirality form is all within the scope of the present invention.Each compound is also with its pharmaceutically acceptable salt, and hydrate or solvate exist.
Definition
Term used herein " pseudomucin " refers to have the compound of following structural formula:
Wherein R is a lipophilic portion.This lipophilic portion comprises C
9-C
15Alkyl, C
9-C
15Hydroxyalkyl, C
9-C
15The dihydroxyl alkyl, C
9-C
15Alkenyl, C
9-C
15Hydroxyl alkenyl, or C
9-C
15The dihydroxyl alkenyl.The pseudomucin compd A, A ', B, B ', C, C ' is represented by top formula I, and wherein R is as giving a definition.Pseudomucin A R=3; 4-dihydroxyl tetradecanoyl pseudomucin A ' R=3; 4-dihydroxyl pentadecanoyl pseudomucin B R=3-hydroxyl tetradecanoyl pseudomucin B ' R=3-hydroxyl lauroyl pseudomucin C R=3,4-dihydroxyl hexadecanoyl pseudomucin C ' R=3-hydroxyl hexadecanoyl.
The detailed description of invention
The applicant has found the N-acyl side-chain enzymatic deacylation of wide spectrum fat depsipeptides natural product is produced the method for corresponding ring compound.Astoundingly, the unhindered amina ring compound is reset and is produced the free hydroxyl group derivative, compound shown in for example top structural formula II and the IV.By Compound I or III are exposed to pH 〉=6, Compound I and III can be converted into Compound I I and IV respectively.If the product of expectation is Compound I or III, then people can will reduce from the speed of deacylation pseudomucin or deacylation syringomycin generation rearrangement product by adding sour for example trifluoroacetic acid.But, add acid and may cause the productive rate of amine ring compound to reduce.Under lower pH, enzyme can be settled out from reaction mixture, thereby ends to transform.Therefore, the pH of reaction mixture preferably is not less than about 5.5.People can make it prevent the enzyme precipitation through molecular weight film (being that molecular weight cut-off is 10000-50000) by separate enzyme from reaction.Effluent liquid by film contains the compound that has less than the 10000-5000 molecular weight (Compound I-IV) for example, and may not comprise the enzyme of higher molecular weight.The pH of effluent liquid can be turned down with stable product then.
Different with sour deacylation method (for example trifluoroacetic acid in water-containing solvent under the room temperature), enzymatic means of the present invention can be used for and will have or not have the pseudomucin or the syringomycin analogue deacylation of γ or δ hydroxyl side chain.Therefore, the range expansion of initial natural product gets very big.For example, use the inventive method can be with pseudomucin A, A ', B, B ', C or C ' deacylation.And sour deacylation method only is suitable for pseudomucin A, A ' and C.
Suitable enzyme comprises that ECB deacylase and polymycin acyltransferase are (from (the Wako Pure Chemical Industries of Wako pure chemistry product company limited; Ltd.) can obtain thick product and purified form product; as 161-16081 fatty acyl group transferring enzyme; pure; with 164-16081 fatty acyl group transferring enzyme, thick product).(for example referring to LaVerne, D waits the people can to obtain the ECB deacylase from actinoplanes utahensis (Actinoplanes utahensis); " by actinoplanes utahensis with the ECB deacylation "; microbiotic magazine (J.ofAntibiotics), 42 (3), 382-388 (1989)).Can be by the method purifying actinoplanes utahensis ECB deacylase of describing in the U.S. Patent No. 5573936 incorporated by reference here.People also can use the enzyme of cloning and expressing in paleness streptomycete (Streptomyces lividans).With PenG Ntn hydrolase and phthalyl Ntn hydrolase that the trial of pseudomucin A deacylation is not successful.
It is known for those skilled in the art that use standard deacylated tRNA method is finished enzymatic deacylated tRNA this point.For example, the general method of use polymycin acyltransferase can be referring to Yasuda, and N waits the people; agricultural biochemistry (Agric.Biol.Chem.) 53,3245 (1989) and Kimura, Y.; Deng the people, agricultural biochemistry (Agric.Biol.Chem.) 53,497 (1989).
The deacylation method is generally at about 20 ℃ and about 60 ℃, carries out under the temperature between preferably approximately room temperature (25 ℃) and about 40 ℃.Higher temperature may promote the generation of rearrangement product (Compound I I).PH8.0 and under the temperature between about 50 ℃ and about 60 ℃ enzyme be optimum activity.Although reaction is faster under higher pH and higher temperature, under higher pH, may find more rearrangement product.Therefore, the pH of reaction generally remains between about 5.5 and about 8.0.Reaction times will be along with pH and temperature different and different.But under high temperature and high pH value, with enzyme concn that limits and saturated concentration of substrate, the reaction times reaches 10 minutes.Because pseudomucin A is unstable under higher pH, so the deacylation of pseudomucin A is generally carried out under low pH (between about 5.0 and 6.0) and temperature (about 25 ℃).For example containing 0.05M KPO
4With the deacylation that can carry out pseudomucin A in the damping fluid of 0.8M KCl.The saturated level of substrate is generally between about 0.5 milligram and about 1 milligram of every milliliter of reaction.
As discussed above, pseudomucin is the natural product of separating from Pseudomonas syringae, it has been characterized by the cyclic peptide part that contains the lactone bond sealing and has comprised non-common amino acid 4-chlorine Threonine (ClThr), 3-hydroxyl aspartic acid (HOAsp), 2, the fat depsipeptides (lipodepsinonapeptides) of 3-dehydrogenation-2-aminobutyric acid (Dhb) and 2,4-diamino-butanoic (Dab).Cultivate each bacterial strain of Pseudomonas syringae and produce different pseudomucin analogue (A, A ', B, B ', C and C ') method do general the description hereinafter, and be described in greater detail in the PCT patent application registration number No.PCT/US00/08728 of people such as Hilton application in 14 days April in 2000 incorporated by reference here, denomination of invention is " producing pseudomucin with Pseudomonas syringae ", the PCT patent application registration number No.PCT/US00/08727 of people such as Kulanthaivel application in 14 days April in 2000 incorporated by reference here, denomination of invention is among " pseudomucin natural product " and the U.S. Patent No. 5576298 and No.5837685 incorporated by reference here separately.
The strain separated of producing one or more pseudomucinous Pseudomonas syringaes is well known in the art.The mutant strain MSU 16H of wild type strain MSU174 and this bacterial strain of producing by transposon mutagenesis is described in U.S. Patent No. 5576298 and 5837685; HarriSon, people such as L., " pseudomucin is from new peptide family of the gang with broad-spectrum antifungal activity of Pseudomonas syringae ", hereditary JOURNAL OF MICROBIOLOGY (J.Gen.Microbiology) 137,2857-65 (1991); With people such as Lamb, " transposon mutagenesis of fluorescent pseudomonas and mark: anti-mycotic agent production is essential for the Dutch elm disease of control ", periodical (Proc.Natl.Acad.Sci.USA) 84 of institute of NAS, 6447-6451 (1987).
Be fit to produce one or more pseudomucinous Pseudomonas syringae bacterial strains and can comprise plant (for example barley plants, oranges and tangerines plant and cloves plant) from the source of environment and resemble soil, water, the such source of air and dust is separated to.Preferred bacterial strain separates from plant.The Pseudomonas syringae bacterial strain that is separated to from the environment source can be referred to as wild-type." wild-type " used herein refers to naturally occurring preponderant genotype in the normal population of Pseudomonas syringae (for example Pseudomonas syringae bacterial strain or isolate that find rather than prepared in laboratory in the nature).As most of biologies, (the Pseudomonas syringae bacterial strain is MSU174 for example, MSU 16H, MSU206,25-B1, feature difference 7H9-1) for the pseudomucinous culture of the generation of use.Therefore, can obtain the filial generation (for example recombinant chou, mutant strain and mutation) of these bacterial strains by means commonly known in the art.
The mutant strain of Pseudomonas syringae also is fit to produce one or more pseudomucins." mutant strain " used herein refers to heritable variation of happening suddenly in the bacterial strain phenotype, it can spontaneous generation or induces by known mutagenic compound, for example irradiation (for example uv irradiating or X-ray), chemical mutagen (ethyl methane sulfonate (EMS) for example, the diepoxy octane, N-methyl-N-nitro-N '-nitroso-group guanine (NTG), and nitrous acid), the mutagenesis of some specificity, with transposon-mediated mutagenesis, produce the pseudomucinous mutant strain of production that one or more pseudomucins promptly surpass a kind of pseudomucin of the excessive production of other pseudomucin (for example pseudomucin B) or the amount processing bacterium of the mutagenic compound of one or more pseudomucinous mutant strains of production can produce Pseudomonas syringae under favourable growth conditions by using effective excess that produces.Although the type of the mutagenic compound that use can be different with amount, preferable methods is with the level of NTG serial dilution to 1-100 mcg/ml scope.Preferred mutant strain is excessive production pseudomucin B and those mutant strains of growing in basic defined medium.
Can be at the environment separation thing of Pseudomonas syringae, the bacterial strain of mutant strain and other expectation is selected the habit of anticipant character, the growth medium nutrition source, carbon source, growth conditions, amino acid requires or the like.Preferably, the pseudomucinous bacterial strain of the production of Pseudomonas syringae is chosen in for example growth and/or produce one or more pseudomucins with the level that is higher than about 10 mcg/ml in the N21 substratum of basic defined medium.Preferred strains expressed goes out when comprising three kinds or amino acid and randomly or a kind of lipid still less, produces one or more pseudomucinous characteristics when growing on the substratum of potato product or its combination.
Use method well known in the art can produce the recombinant chou bacterial strain by transforming the Pseudomonas syringae bacterial strain.By using recombinant DNA technology, the Pseudomonas syringae bacterial strain can be transformed to express the range gene product except the microbiotic of these bacterial strains generations.For example, people can modify bacterial strain with a plurality of copies of importing endogenous pseudomucin biosynthesis gene to realize bigger pseudomucin productive rate.
In order to produce one or more pseudomucins from wild-type Pseudomonas syringae or Pseudomonas syringae mutant strain, comprising three kinds of significant quantity or amino acid still less, preferred L-glutamic acid, glycine, culturing micro-organisms under the vibration in the moisture nutritional medium of Histidine or its combination.Perhaps glycine is mixed with one or more potato products and lipid.Under the effective condition of one or more pseudomucins that the growth and the production of Pseudomonas syringae are expected, cultivate.Condition for validity comprise about 22 ℃ to about 27 ℃ temperature and about 36 hours to about 96 hours incubation time.The concentration of oxygen is favourable for producing pseudomucin in the control substratum during cultivating Pseudomonas syringae.Preferably, it is about 5% to 50% saturated that the level of oxygen remains on, and more preferably about 30% is saturated.Bubbling air, purity oxygen or contain the concentration that the gaseous mixture of oxygen can be regulated oxygen in the substratum.
The pH of control substratum also is favourable during the cultivation Pseudomonas syringae.Pseudomucin instability under alkaline pH keep to surpass about 12 hours then obvious degradation takes place if the pH of substratum is higher than about 6.Preferably, the pH of substratum remains between 6 and 4.When batch culture, Pseudomonas syringae can produce one or more pseudomucins.Yet glucose the and randomly batch feeding of acid or alkali (for example hydroxylammonium) or semicontinuous charging are controlled pH and boosted productivity.Glucose and the automatically feed cultured continuously method of hydroxylammonium can further improve pseudomucin productivity by utilizing wherein.
The selection of Pseudomonas syringae can influence one or more pseudomucinous amount and distributions that produce.For example, MSU 16H and 67 H1 bacterial strains advantage separately produce pseudomucin A, but also produce pseudomucin B and C, and general ratio is 4: 2: 1.Compare with the pseudomucin level of MSU 16H bacterial strain production, 67 H1 bacterial strains generally produce high approximately 3-5 pseudomucin level doubly.Compare with 67 H1 bacterial strains with MSU 16H, the 25-B1 bacterial strain produces more pseudomucin B and pseudomucin C still less.Different is that 7H9-1 bacterial strain advantage produces pseudomucin B and produces more substantial pseudomucin B than other bacterial strain.For example, the fertile pseudomucin B of this bacterial strain surpasses pseudomucin A or C 10 times at least.
As discussed above, method described herein also is used for syringomycin compound deacylation.Can be respectively from Pseudomonas syringae cloves mutation (Pseudomonas syringae pv.syringae) bacterial strain B301D, the culture of PS268 and SY12 produces syringomycin E, syringomycin B and syringomycin A.Also can separate syringomycin A from the mutation of Pseudomonas syringae cloves
1And G.According to Zhang, L., and J.Y.Takemoto, " Pseudomonas syringae plant poison syringomycin is to the influence of the plasma membrane function of Rhodotorula pilimanae ", B301D and PS268 bacterial strain are cultivated in being described in of plant pathology (Phytopathol.) 77 (2): 297-303 (1987) in the potato glucose nutrient solution.Adding 100M arbutin (Sigma Chemical Co., A4256; St.Louis, Mo.) and cultivate the SY12 bacterial strain in the syringomycin minimum medium of 0.1% fructose (SRMAF) (19,23).As previous Bidwai, A.P., and J.Y.Takemoto, " schizomycete toxin; syringomycin is induced the phosphorylation of the protein kinase-mediation of Radix Dauci Sativae plasma membrane polypeptide ", periodical (Proc.Natl.Acad.Sci.USA) 84 of institute of NAS, 6755-6759 (1987) is described by high-efficient liquid phase chromatogram purification SR-E, ST-B and SS-A.Contain 35% Sodium desoxycholate (Sigma ChemicalCo., AP528; St.Louis, Mo.) and KETOKONAZOL (Sigma Chemical Co., K-1003; St.Louis, dissolved AmB Mo.) is used as the touchstone thing.Can see relevant three kinds of ring grease depsipeptides syringomycin E here in the U.S. Patent No. 5830855 incorporated by reference, the generation of syringacin B and Syringostatin A and isolating detailed description.
Pseudomucin or syringomycin ring compound or corresponding rearrangement compounds (Compound I I and IV) can be separated and be used with itself or with its pharmaceutically acceptable salt or solvate form thereof.Term " pharmaceutically acceptable salt " refers to from mineral acid and organic acid deutero-and non-toxic acid additive salt.Suitable salt derivative comprises halogenide, thiocyanate-, vitriol, hydrosulfate, sulphite, hydrosulphite, arylsulphonate, alkylsulfonate, phosphonate, monohydric phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate salt, alkanoate, cycloalkyl alkanoate, aryl-alkanoic salt, adipate, alginate, aspartate, benzoate, fumarate, glucoheptose salt, glycerophosphate, lactic acid salt, maleate, nicotinate, oxalate, palmitate, pectinic acid salt, picrate, Pivalate, succinate, tartrate, Citrate trianion, camphorate, camsilate, digluconate, trifluoroacetate or the like.
Term " solvate " refers to comprise for example water of one or more solute molecules (being pseudomucin and syringomycin compound) and one or more pharmaceutical solvents molecule, the aggregate of ethanol or the like.When solvent was water, aggregate was referred to as hydrate.Usually by under heating with ring compound or rearrangement compounds (Compound I I or IV) is dissolved in the suitable solvent and slowly cooling generation amorphous or recrystallisation solvent compound form produce solvate.
Embodiment
Biological sample
From American type culture collection (American Type CultureCollection), Parklawn Drive, Rockville, MD, USA openly obtain Pseudomonas syringae MSU 16H, preserving number No.ATCC 67028.Pseudomonas syringae bacterial strain 25-B1,7H9-1 and 67 H1 are deposited in American type culture collection on March 23rd, 2000, and the preserving number of registration is as follows: 25-B1 preserving number No.PTA-16227H9-1 preserving number No.PTA-1 62367 H1 preserving number No.PTA-1621
The compound abbreviation
Each listed material is represented in abbreviation below using among the embodiment:
The ACN-acetonitrile
The TFA-trifluoroacetic acid
The DMF-dimethyl formamide
Embodiment 1
This embodiment describes for example and uses the deacylation of ECB deacylase to pseudomucin A.
The ECB deacylase (50 microlitre) that in containing aqueous buffer solution 900 microlitres of 0.05M potassiumphosphate and 0.8M Repone K, adds pseudomucin A (50 microgram) and purifying.PH keeps 6.0 to 8.0.Temperature is brought up to 40 ℃ from 25 ℃.By HPLC monitoring reaction (1% acetonitrile/0.2% trifluoroacetic acid (4 minutes) is to 60% acetonitrile/0.2% trifluoroacetic acid (16 minutes) for Waters C18 μ Bondapak 3.9X300 millimeter post, 235nm).Find pseudomucin amine ring compound (Compound I) and the pseudomucin hydroxyl ring compound of resetting (Compound I I).
Compound I all shows identical M+H ion (m/z981.3) with II in electrospray MALDI-MS (ESIMS), corresponding to molecular formula C
37H
61CIN
12O
17(referring to following Table I).
1Can arrange all protons of hydrolysate of H and TOCSY (total correlation spectroscopy) NMR spectrographic detailed analysis, this underwork I and II.β-the proton of the serine residue of I
1H nmr chemical displacement (4.83 and 4.46ppm) with at pseudomucin A, those that find among B and the C are consistent, show that the big ring of peptide is complete.In addition, as desired, the TOCSY spectrum does not prove the part of typical amide proton as Serine spin system.On the other hand, in II, Serine β-proton is shifted to upfield shift (3.78 and 3.74ppm) greatly, points out these protons not have the lactone functionality.This point and in TOCSY spectrum, except the α proton, β-proton is relevant with amide proton, is in 8.04ppm, shows that the macrolide rearrangement is the peptide core shown in the II.
Table I
H
2O+CD
3I and II among the CN
1H NMR data
a
| Amino acid | The position | ????I | ????II |
| ????Ser | ????NH | ?????- | ????8.04 |
| ????α | ????4.30 | ????4.30 | |
| ????β1 | ????4.83 | ????3.78 | |
| ????β2 | ????4.46 | ????3.74 | |
| ????Dab-1 D | ????NH | ????9.19 | ????7.99 |
| ????α | ????4.06 | ????4.19 | |
| ????β1 | ????2.03 | ????2.15 | |
| ????β2 | ????2.01 | ||
| ????γ1 | ????3.03 | ????2.92 | |
| ????γ2 | ????2.96 | ||
| ????Asp | ????NH | ????8.51 | ????8.20 |
| ????α | ????4.61 | ????4.56 | |
| ????β1 | ????2.89 | ????2.84 | |
| ????β2 | ????2.83 | ????2.75 | |
| ????Lys | ????NH | ????7.90 | ????8.11 |
| ????α | ????4.23 | ????4.06 | |
| ????β1 | ????1.79 | ????1.76 | |
| ????β2 | ????1.71 | ????1.68 | |
| ????γ1 | ????1.27 | ????1.30 | |
| ????γ2 | ????1.25 | ||
| ????δ | ????1.54 | ????1.54 | |
| ????ε | ????2.84 | ????2.84 | |
| ????NH 2 | ????7.34 | ????7.34 | |
| ????Dab-2 D | ????NH | ????8.35 | ????8.31 |
| ????α | ????4.29 | ????4.34 | |
| ????β1 | ????2.14 | ????2.09 | |
| ????β2 | ????1.98 | ????1.91 | |
| ????γ | ????2.90 | ????2.92 | |
| ????NH 2 | ????7.53 | ????7.49 | |
| ????Thr | ????NH | ????7.73 | ????7.74 |
| ????α | ????4.24 | ????4.21 | |
| ????β | ????3.98 | ????3.98 | |
| ????γ | ????1.18 | ????1.16 |
Table I (continuing)
| Amino acid | The position | ????I | ????II |
| ????Dhb | ????NH | ????9.65 | ????9.26 |
| ????β | ????6.69 | ????6.62 | |
| ????γ | ????1.69 | ????1.66 | |
| ????OHAsp | ????NH | ????7.82 | ????7.83 |
| ????α | ????4.95 | ????4.99 | |
| ????β | ????4.72 | ????4.75 | |
| ????ClThr | ????NH | ????7.92 | ????7.95 |
| ????α | ????4.90 | ????4.62 | |
| ????β | ????4.27 | ????4.25 | |
| ????γ1 | ????3.48 | ????3.57 | |
| ????γ2 | ????3.42 | ????3.51 |
aChemical shift with respect to solvents signals (1.94ppm) report.
bArranging can interior change.
Use above-mentioned identical general method will have other pseudomucin or the syringomycin compound deacylation of N-acyl group.
Claims (12)
1. with the method for the N-acyl side-chain deacylation of pseudomucin natural product, comprise the step that makes pseudomucin natural product and the deacylase reaction that is selected from ECB deacylase and polymycin acyltransferase produce the pseudomucin ring compound.
2. the process of claim 1 wherein that described pseudomucin ring compound usefulness structural formula I or II represent
Or its pharmaceutically acceptable salt, hydrate or solvate.
3. the process of claim 1 wherein that described pseudomucin natural product is selected from pseudomucin A, A ', B, B ', C and C '.
4. pass through the compound with following structure of the method preparation of claim 1,2 or 3
Perhaps its pharmaceutically acceptable salt, hydrate or solvate.
5. the compound that has following structure
Perhaps its pharmaceutically acceptable salt, hydrate or solvate.
6. by making pseudomucin natural product and the pseudomucin ring compound that is selected from the deacylase prepared in reaction of ECB deacylase and polymycin acyltransferase.
7. the pseudomucin ring compound of claim 6, wherein said pseudomucin natural product is selected from pseudomucin A, A ', B, B ', C and C '.
8. with the method for the N-acyl side-chain deacylation of syringomycin natural product, comprise the step that makes syringomycin natural product and the deacylase reaction that is selected from ECB deacylase and polymycin acyltransferase produce the syringomycin ring compound.
9. the method for claim 7, wherein said syringomycin ring compound is with structural formula II I or IV representative
Or its pharmaceutically acceptable salt, hydrate or solvate.
10. by making syringomycin natural product and the syringomycin ring compound that is selected from the deacylase prepared in reaction of ECB deacylase and polymycin acyltransferase.
11. have the compound of following structure
Perhaps its pharmaceutically acceptable salt, hydrate or solvate.
12. have the compound of following structure
Perhaps its pharmaceutically acceptable salt, hydrate or solvate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14396899P | 1999-07-15 | 1999-07-15 | |
| US60/143968 | 1999-07-15 |
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|---|---|
| CN1361791A true CN1361791A (en) | 2002-07-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN00810417A Pending CN1361791A (en) | 1999-07-15 | 2000-06-08 | Process for deacylation of lipodepsipeptides |
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| Country | Link |
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| EP (1) | EP1198471A1 (en) |
| JP (1) | JP2003505042A (en) |
| CN (1) | CN1361791A (en) |
| AU (1) | AU5310600A (en) |
| BR (1) | BR0012481A (en) |
| CA (1) | CA2378868A1 (en) |
| EA (1) | EA200200162A1 (en) |
| HU (1) | HUP0202315A2 (en) |
| MX (1) | MXPA02000458A (en) |
| NO (1) | NO20020183L (en) |
| WO (1) | WO2001005815A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002066499A1 (en) | 2001-02-19 | 2002-08-29 | Takara Bio Inc. | Cyclic peptide |
| DE10335584B4 (en) * | 2003-07-31 | 2006-06-29 | Philipps-Universität Marburg | Process for the preparation of cyclic molecules |
| US20060004185A1 (en) * | 2004-07-01 | 2006-01-05 | Leese Richard A | Peptide antibiotics and peptide intermediates for their prepartion |
| DE102004060750A1 (en) * | 2004-12-15 | 2006-07-13 | Sanofi-Aventis Deutschland Gmbh | Process for the deacylation of lipopeptides |
| NZ593892A (en) | 2008-12-23 | 2013-11-29 | Biosource Pharm Inc | Antibiotic compositions for the treatment of gram negative infections |
| US8415307B1 (en) | 2010-06-23 | 2013-04-09 | Biosource Pharm, Inc. | Antibiotic compositions for the treatment of gram negative infections |
| IN2015DN01633A (en) * | 2012-09-24 | 2015-07-03 | Dsm Sinochem Pharm Nl Bv |
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| CA2043762C (en) * | 1990-06-07 | 2001-05-29 | Adam J. Kreuzman | Lipopeptide deacylase |
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- 2000-06-08 CN CN00810417A patent/CN1361791A/en active Pending
- 2000-06-08 EA EA200200162A patent/EA200200162A1/en unknown
- 2000-06-08 HU HU0202315A patent/HUP0202315A2/en unknown
- 2000-06-08 WO PCT/US2000/015018 patent/WO2001005815A1/en not_active Application Discontinuation
- 2000-06-08 CA CA002378868A patent/CA2378868A1/en not_active Abandoned
- 2000-06-08 JP JP2001511472A patent/JP2003505042A/en not_active Withdrawn
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| Publication number | Publication date |
|---|---|
| WO2001005815A1 (en) | 2001-01-25 |
| MXPA02000458A (en) | 2002-07-02 |
| EA200200162A1 (en) | 2002-06-27 |
| NO20020183L (en) | 2002-03-13 |
| HUP0202315A2 (en) | 2002-10-28 |
| NO20020183D0 (en) | 2002-01-14 |
| EP1198471A1 (en) | 2002-04-24 |
| CA2378868A1 (en) | 2001-01-25 |
| AU5310600A (en) | 2001-02-05 |
| JP2003505042A (en) | 2003-02-12 |
| BR0012481A (en) | 2002-04-02 |
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