CN1361791A - Process for deacylation of lipodepsipeptides - Google Patents

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

lipopeptide deacylation method

Field of Invention

The present invention relates to the deacylation of the N-acyl side chains of lipodepsipeptides, particularly pseudomycin and syringomycin natural products, and compounds prepared therefrom.

Background of the Invention

Pseudomucin and syringomycin are natural products isolated from the liquid culture medium of Pseudomonas syringae (plant-associated bacteria) and have demonstrated antifungal activity. (See Harrison, L. et al., "Pseudomucins, a novel family of peptides from Pseudomonas syringae with broad-spectrum antifungal activity", J. Gen. Microbiology 137(12), 2857-65 (1991) and US Patent Nos. 5,576,298 and 5,837,685). Unlike previously described antifungal agents from P. syringae (such as syringomycin, P. GABA, lysine and diaminobutyric acid.

The peptide portion of pseudomucin A, A', B, B', C, C' corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb- L-Asp(3-OH)-L-Thr(4-Cl), a macrocycle with a terminal carboxyl group closed at the OH group of the N-terminal Ser. These analogues differ in the N-acyl side chains, that is, pseudomucin A is N-acylated with 3,4-dihydroxytetradecanoyl and pseudomucin A' is N-acylated with 3,4-dihydroxypentadecanoyl. Acylation, pseudomucin B is N-acylated with 3-hydroxytetradecanoyl, pseudomucin B' is N-acylated with 3-hydroxydodecanoyl, pseudomucin C is 3,4-dihydroxydecanoyl Hexadecanoyl N-acylation and pseudomucin C' with 3-hydroxyhexadecanoyl N-acylation. (see Ballio, A., et al., "New bioactive lipopeptides from Pseudomonas syringae: pseudomucin", FEBS Letters, 355 (1), 96-100, (1994) and Coiro, V.M. , et al., "Determination of the solution conformation of the phytotoxic lipodepsipeptide pseudomycin A of Pseudomonas syringae MSU 16H by computer simulation using geometric spacing and molecular mechanics from NMR data", Eur.J.Biochem. .) 257(2), 449-456(1998)).

Pseudomucin and syringomycin are known to have some adverse biological effects. For example, disruption of the vein endothelium, tissue destruction, inflammation, and local toxicity to host tissues have been observed when pseudomucin is administered intravenously. Accordingly, there is a need to identify compounds of this class that are useful in the treatment of fungal infections without the currently discovered adverse side effects.

Summary of Invention

The present invention provides methods for deacylation of the N-acyl side chains of lipopeptide natural products to produce the corresponding cyclic compounds. Deacylation of pseudomucin produces the pseudomucin aminocyclic compound represented by structural formula I below. The cyclic compounds can be used as starting materials for the preparation of semi-synthetic derivatives of the corresponding natural products.

化合物II又可以用作产生药学活性的新的衍生物的起始物。The method comprises reacting a pseudomycin natural product with a deacylase selected from the group consisting of ECB deacylase and polystreptomycin acylase to produce the corresponding cyclic compound represented by structural formula I. The free amines can be rearranged to produce a cyclic peptide cyclic compound with one free hydroxyl group (also known as a pseudomucin hydroxyl cyclic compound) represented by the following structural formula II.

Compound II can in turn be used as a starting material for the production of new pharmaceutically active derivatives.

In another embodiment of the present invention, the above method is used to deacylate a syringomycin compound to produce a syringomycin aminocyclic compound. Aminocyclic compounds such as syringomycin E have the following structural formula III.

Like the pseudomucin aminocycle, the syringomycin aminocycle can be rearranged to yield Compound IV below (also known as the syringomycin hydroxycycle). Although specific chiral forms of compounds I, II, III and IV are described above, other chiral forms are within the scope of the invention. Each compound also exists as a pharmaceutically acceptable salt, hydrate or solvate thereof.

Definition

其中R是亲脂部分。该亲脂部分包括C₉-C₁₅烷基，C₉-C₁₅羟基烷基，C₉-C₁₅二羟基烷基，C₉-C₁₅链烯基，C₉-C₁₅羟基链烯基，或C₉-C₁₅二羟基链烯基。假粘蛋白化合物A，A’，B，B’，C，C’由上面式I代表，其中R如下定义。假粘蛋白A  R＝3，4-二羟基十四烷酰基假粘蛋白A’R＝3，4-二羟基十五烷酰基假粘蛋白B  R＝3-羟基十四烷酰基假粘蛋白B’R＝3-羟基十二烷酰基假粘蛋白C  R＝3，4-二羟基十六烷酰基假粘蛋白C’R＝3-羟基十六烷酰基。The term "pseudomucin" as used herein refers to a compound having the following structural formula:

wherein R is a lipophilic moiety. The lipophilic moiety includes C ₉ -C ₁₅ alkyl, C ₉ -C ₁₅ hydroxyalkyl, C ₉ -C ₁₅ dihydroxyalkyl, C ₉ -C ₁₅ alkenyl, C ₉ -C ₁₅ hydroxyalkenyl , or C ₉ -C ₁₅ dihydroxyalkenyl. Pseudomucin compounds A, A', B, B', C, C' are represented by the above formula I, wherein R is as defined below. Pseudomucin A R = 3,4-Dihydroxytetradecanoyl Pseudomucin A'R = 3,4-Dihydroxypentadecanoyl Pseudomucin B R = 3-Hydroxytetradecanoyl Pseudomucin B'R = 3-Hydroxydodecanoylpseudomucin CR = 3,4-dihydroxyhexadecanoylpseudomucin C'R = 3-hydroxyhexadecanoyl.

Detailed description of the invention

Applicants have discovered a method for the enzymatic deacylation of the N-acyl side chains of broad-spectrum lipopeptide natural products to produce the corresponding cyclic compounds. Surprisingly, rearrangement of free amine cyclic compounds yields free hydroxy derivatives, such as those shown in Formulas II and IV above. Compounds I and III can be converted to Compounds II and IV, respectively, by exposing Compounds I or III to pH > 6. If the desired product is compound I or III, one can reduce the rate of formation of rearrangement products from deacylated pseudomycin or deacylated syringomycin by adding an acid such as trifluoroacetic acid. However, the addition of acid may result in lower yields of amine cyclic compounds. At lower pH, the enzyme can precipitate out of the reaction mixture, aborting the conversion. Therefore, the pH of the reaction mixture is preferably not lower than about 5.5. One can prevent enzyme precipitation by separating the enzyme from the reaction by passing it through a molecular weight membrane (ie, a molecular weight cut off of 10000-50000). The effluent through the membrane contains compounds with a molecular weight of less than 10000-5000 (eg compounds I-IV) and may not include higher molecular weight enzymes. The pH of the effluent can then be adjusted lower to stabilize the product.

Unlike acid deacylation methods (such as trifluoroacetic acid in aqueous solvents at room temperature), the enzymatic method of the present invention can be used to convert pseudomycin or syringomucin with or without gamma or delta hydroxyl side chains to deacylation of analogues. Thus, the range of starting natural products is extended very greatly. For example, pseudomucin A, A', B, B', C or C' may be deacylated using the methods of the invention. The acid deacylation method is only applicable to pseudomucin A, A' and C.

Suitable enzymes include ECB deacylase and polystreptomycin acylase (available in crude and purified form from Wako Pure Chemical Industries, Ltd., such as 161-16081 fatty acyltransferase enzyme, pure, and 164-16081 fatty acyltransferase, crude). ECB deacylase can be obtained from Actinoplanes utahensis (see for example LaVerne, D, et al., "Deacylation of Echinocandin B by Actinoplanes utahensis", Journal of Antibiotics (J of Antibiotics), 42(3), 382-388 (1989)). Actinomyces uthaides ECB deacylase can be purified by the method described in US Patent No. 5,573,936, incorporated herein by reference. One can also use enzymes cloned and expressed in Streptomyces lividans. Attempts to deacylate pseudomucin A with PenG amidase and phthaloyl amidase were unsuccessful.

Enzymatic deacylation is accomplished using standard deacylation methods well known to those skilled in the art. For example, general methods for using polystreptomycin acylase can be found in Yasuda, N, et al., Agric. Biol. Chem. 53, 3245 (1989) and Kimura, Y., et al., Agricultural Biochemistry (Agric. Biol. Chem.) 53, 497 (1989).

The deacylation process is generally carried out at a temperature between about 20°C and about 60°C, preferably between about room temperature (25°C) and about 40°C. Higher temperature may promote the formation of the rearrangement product (compound II). The enzyme is optimally active at pH 8.0 and at temperatures between about 50°C and about 60°C. Although the reaction is faster at higher pH and higher temperature, more rearrangement products may be found at higher pH. Thus, the pH of the reaction is generally maintained between about 5.5 and about 8.0. Reaction times will vary with pH and temperature. However, at high temperature and high pH, with defined enzyme concentrations and saturated substrate concentrations, reaction times were up to 10 minutes. Because pseudomucin A is unstable at higher pH, deacylation of pseudomucin A is generally performed at lower pH (between about 5.0 and 6.0) and temperature (about 25°C). For example, deacylation of pseudomucin A can be performed in a buffer containing 0.05M _KPO4 and 0.8M KCl. Saturation levels of substrate are generally between about 0.5 mg and about 1 mg per mL of reaction.

As discussed above, pseudomycin is a natural product isolated from Pseudomonas syringae that has been characterized as containing a lactone-bonded cyclic peptide moiety and includes the unusual amino acid 4-chlorothreonine (ClThr), Lipodepsinonapeptides of 3-hydroxyaspartic acid (HOAsp), 2,3-dehydro-2-aminobutyric acid (Dhb) and 2,4-diaminobutyric acid (Dab). Methods for culturing individual strains of Pseudomonas syringae to produce the different pseudomycin analogs (A, A', B, B', C, and C') are described generally below, and in more detail herein as cited in Referenced PCT patent application registration number No.PCT/US00/08728 filed by Hilton et al. on April 14, 2000. The title of the invention is "production of pseudomycin with Pseudomonas syringae", which was cited here as a reference on April 14, 2000. PCT Patent Application Registration No. PCT/US00/08727 filed by Kulanthaivel et al. on April 14, the title of the invention is "pseudomucin natural product", and among US Patent No.5,576,298 and No.5,837,685 respectively incorporated herein by reference .

Isolated strains of P. syringae that produce one or more pseudomucins are known in the art. The wild-type strain MSU174 and a mutant of this strain, MSU 16H, produced by transposon mutagenesis are described in U.S. Patent Nos. 5,576,298 and 5,837,685; HarriSon, L. et al., "Pseudomycin, from Pseudomonas syringae A novel family of peptides with broad-spectrum antifungal activity", J. Gen. Microbiology 137, 2857-65 (1991); and Lamb et al., "Transposon-attractant Mutation and Marking: Antifungal Production Is Essential for Control of Dutch Elm Disease", Proc. Natl. Acad. Sci. USA 84, 6447-6451 (1987).

Strains of Pseudomonas syringae suitable for the production of one or more pseudomucins can be isolated from environmental sources including plants (eg barley plants, citrus plants and clove plants) as well as sources such as soil, water, air and dust. Preferred strains are isolated from plants. Strains of P. syringae isolated from environmental sources can be referred to as wild type. As used herein, "wild type" refers to the predominant genotype naturally occurring in normal populations of P. syringae (eg, a strain or isolate of P. syringae found in nature rather than produced in a laboratory). Like most organisms, the pseudomycin-producing cultures used (Pseudomonas syringae strains such as MSU174, MSU 16H, MSU206, 25-B1, 7H9-1) were characterized differently. Accordingly, progeny (eg recombinants, mutants and variants) of these strains can be obtained by methods well known in the art.

Mutant strains of P. syringae are also suitable for the production of one or more pseudomucins. "Mutant" as used herein refers to a sudden heritable change in the phenotype of a strain, which may occur spontaneously or be induced by a known mutagen, such as irradiation (e.g., ultraviolet radiation or X-rays), chemical mutagens (e.g. ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N'-nitrosoguanine (NTG), and nitrous acid), site-specific mutagenesis, and transposon-mediated mutagenesis by overproducing one or more pseudomucins over other pseudomucins with efficient production of Amounts of Mutagen Under Conditions That Produce One or More Pseudomucin-Producing Mutants Bacteria can produce pseudomycin-producing mutants of Pseudomonas syringae. Although the type and amount of mutagen to be used can vary, the preferred method is to serially dilute NTG to levels in the range of 1-100 micrograms/ml. Preferred mutants are those that overproduce pseudomucin B and grow in substantially defined media.

Environmental isolates, mutants, and other desired strains of P. syringae can be selected for growth habit, growth medium nutrient source, carbon source, growth conditions, amino acid requirements, etc. for desired traits. Preferably, pseudomycin-producing strains of Pseudomonas syringae are selected for growth in a substantially defined medium, such as N21 medium, and/or for production of one or more pseudomycins at levels greater than about 10 μg/ml. protein. Preferred strains exhibit the property of producing one or more pseudomucins when grown on media comprising three or fewer amino acids and, optionally, either a lipid, a potato product, or a combination thereof.

Recombinant strains can be produced by transforming Pseudomonas syringae strains using methods well known in the art. Through the use of recombinant DNA techniques, Pseudomonas syringae strains can be transformed to express various gene products in addition to the antibiotics produced by these strains. For example, one can modify a strain to introduce multiple copies of an endogenous pseudomucin biosynthesis gene to achieve greater yields of pseudomucin.

For the production of one or more pseudomucins from wild-type Pseudomonas syringae or mutant strains of Pseudomonas syringae, in the presence of an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine or The microorganisms are grown with shaking in the combined aqueous nutrient medium. Alternatively glycine is mixed with one or more potato products and lipids. The cultivation is carried out under conditions effective for the growth and production of the desired pseudomycin or proteins by Pseudomonas syringae. Effective conditions include a temperature of about 22°C to about 27°C and an incubation time of about 36 hours to about 96 hours. Controlling the concentration of oxygen in the medium during the cultivation of Pseudomonas syringae is advantageous for the production of pseudomycin. Preferably, the oxygen level is maintained at about 5% to 50% saturation, more preferably at about 30% saturation. The concentration of oxygen in the medium can be adjusted by feeding air, pure oxygen or a gas mixture containing oxygen.

It is also advantageous to control the pH of the medium during the cultivation of Pseudomonas syringae. Pseudomucin is unstable at alkaline pH, with significant degradation occurring if the pH of the medium is maintained above about 6 for more than about 12 hours. Preferably, the pH of the medium is maintained between 6 and 4. When cultured in batches, Pseudomonas syringae can produce one or more pseudomucins. However fed-batch or semi-continuous feeding of glucose and optionally acid or base (eg hydroxylammonium) to control pH increases productivity. Pseudomucin productivity can be further improved by utilizing a continuous culture method in which glucose and hydroxylammonium are automatically fed.

The choice of P. syringae can affect the amount and distribution of one or more pseudomycins produced. For example, MSU 16H and 67 H1 strains each predominantly produce pseudomycin A, but also produce pseudomycin B and C, generally in a ratio of 4:2:1. The 67 H1 strain generally produced approximately 3-5 times higher levels of pseudomucin than the level of pseudomucin produced by the MSU 16H strain. Compared with the MSU 16H and 67 H1 strains, the 25-B1 strain produced more pseudomycin B and less pseudomycin C. The difference is that the 7H9-1 strain predominantly produces pseudomycin B and produces a larger amount of pseudomycin B than the other strains. For example, the strain is capable of producing at least 10-fold more Pseudomucin B than Pseudomucin A or C.

As discussed above, the methods described here were also used to deacylate syringomycin compounds. Syringomycin E, syringomycin B, and syringomycin A can be produced from cultures of Pseudomonas syringae pv. syringae strains B301D, PS268, and SY12, respectively. Syringomycins A ₁ and G can also be isolated from Pseudomonas syringae var. syringae. According to Zhang, L., and JY Takemoto, "Effect of the Pseudomonas syringae phytotoxin syringomycin on the plasma membrane function of Rhodotorula pilimanae", Phytopathol. (Phytopathol.) 77 (2): 297-303 (1987) The cultivation of B301D and PS268 strains in potato dextrose broth is described. SY12 strain was cultured in syringomycin minimal medium supplemented with 100 M hydroquinone glucoside (Sigma Chemical Co., A4256; St. Louis, Mo.) and 0.1% fructose (SRMAF) (19, 23). As previously Bidwai, AP, and JY Takemoto, "The bacterial phytotoxin, syringomycin, induces protein kinase-mediated phosphorylation of carrot plasma membrane polypeptides", Proc. Natl. Acad. Sci. Purification of SR-E, ST-B and SS-A by high performance liquid chromatography as described in USA) 84, 6755-6759 (1987). Dissolved AmB containing 35% sodium deoxycholate (Sigma Chemical Co., AP528; St. Louis, Mo.) and ketoconazole (Sigma Chemical Co., K-1003; St. Louis, Mo.) was used as an assay standard. A detailed description of the production and isolation of the three cycloaliphatic peptides syringomycin E, syringostatin B and syringostatin A can be found in US Patent No. 5,830,855, incorporated herein by reference.

The pseudomycin or syringomycin cyclic compounds or the corresponding rearranged compounds (compounds II and IV) can be isolated and used as such or in the form of their pharmaceutically acceptable salts or solvates. The term "pharmaceutically acceptable salts" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfonates, phosphonates, monohydrogenphosphates , dihydrogen phosphate, metaphosphate, pyrophosphate, alkanoate, naphthenic alkanoate, aryl alkanoate, adipate, alginate, aspartate, benzene Formate, fumarate, glucoheptate, glycerophosphate, lactate, maleate, niacinate, oxalate, palmitate, pectinate, picrate, new Valerate, succinate, tartrate, citrate, camphorate, camphorsulfonate, digluconate, trifluoroacetate and many more.

The term "solvate" refers to an aggregate comprising one or more solute molecules (ie pseudomucin and syringomycin compounds) and one or more pharmaceutical solvent molecules such as water, ethanol, and the like. When the solvent is water, the aggregates are called hydrates. Solvates are generally produced by dissolving the cyclic or rearranged compound (compound II or IV) in a suitable solvent with heating and slow cooling to produce amorphous or crystalline solvate forms.

Example

Biological samples

Pseudomonas syringae MSU 16H was publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA, Accession No. ATCC 67028. Pseudomonas syringae strains 25-B1, 7H9-1 and 67 H1 were deposited in the American Type Culture Collection on March 23, 2000, and the registered preservation numbers are as follows: 25-B1 preservation number No.PTA-16227H9-1 Accession No.PTA-1 62367 H1 Accession No.PTA-1621

Compound Abbreviation

The following abbreviations are used in the examples to represent each listed material:

ACN-Acetonitrile

TFA-Trifluoroacetic Acid

DMF-Dimethylformamide

Example 1

This example illustrates the deacylation of pseudomycin A using ECB deacylase.

Pseudomucin A (50 µg) and purified ECB deacylase (50 µl) were added to 900 µl of an aqueous buffer solution containing 0.05 M potassium phosphate and 0.8 M potassium chloride. The pH was maintained at 6.0 to 8.0. The temperature was increased from 25°C to 40°C. The reaction was monitored by HPLC (Waters C18 μBondapak 3.9×300 mm column, 235 nm, 1% acetonitrile/0.2% trifluoroacetic acid (4 minutes) to 60% acetonitrile/0.2% trifluoroacetic acid (16 minutes)). A pseudomucin amine cyclic compound (compound I) and a rearranged pseudomucin hydroxy cyclic compound (compound II) were found.

Both compounds I and II exhibited the same M+H ion (m/z 981.3) in electrospray ionization mass spectrometry (ESIMS), corresponding to the molecular formula C ₃₇ H ₆₁ CIN ₁₂ O ₁₇ (see Table I below). Detailed analysis of ¹ H and TOCSY (Total Correlation Spectroscopy) NMR spectroscopy can assign all protons of the hydrolyzate, which supports structures I and II. The ¹ H NMR chemical shifts (4.83 and 4.46 ppm) of the β-proton of the serine residue of I were consistent with those found in pseudomucins A, B and C, indicating that the peptide macrocycle is intact. Furthermore, as expected, the TOCSY spectrum did not demonstrate typical amide protons as part of the serine spin system. In II, on the other hand, the serine β-protons were greatly shifted upfield (3.78 and 3.74 ppm), suggesting that these protons do not have lactone functionality. This and the fact that in the TOCSY spectrum, in addition to the α protons, the β-protons are associated with the amide protons at 8.04 ppm, suggest rearrangement of the macrolide into the peptide core shown in II.

Table I

¹ H NMR ^data of I and II in H ₂ O+CD ₃ CNa amino acid Location I II Ser NH - 8.04 alpha 4.30 4.30 β1 4.83 3.78 beta2 4.46 3.74 Dab- ^1D NH 9.19 7.99 alpha 4.06 4.19 β1 2.03 2.15 beta2 2.01 gamma 1 3.03 2.92 gamma 2 2.96 Asp NH 8.51 8.20 alpha 4.61 4.56 β1 2.89 2.84 beta2 2.83 2.75 Lys NH 7.90 8.11 alpha 4.23 4.06 β1 1.79 1.76 beta2 1.71 1.68 gamma 1 1.27 1.30 gamma 2 1.25 δ 1.54 1.54 ε 2.84 2.84 NH ₂ 7.34 7.34 Dab- ^2D NH 8.35 8.31 alpha 4.29 4.34 β1 2.14 2.09 beta2 1.98 1.91 gamma 2.90 2.92 NH ₂ 7.53 7.49 Thr NH 7.73 7.74 alpha 4.24 4.21 beta 3.98 3.98 gamma 1.18 1.16

Table I (continued) amino acid Location I II Dhb NH 9.65 9.26 beta 6.69 6.62 gamma 1.69 1.66 OHAsp NH 7.82 7.83 alpha 4.95 4.99 beta 4.72 4.75 ClThr NH 7.92 7.95 alpha 4.90 4.62 beta 4.27 4.25 gamma 1 3.48 3.57 gamma 2 3.42 3.51

^a Chemical shifts reported relative to solvent signal (1.94 ppm).

^{The b} arrangement can vary internally.

Other pseudomucin or syringomycin compounds having N-acyl groups can be deacylated using the same general procedure described above.

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.