WO1994023056A1 - Process for isolating a83543 and its components - Google Patents

Abstract

Process for isolating A83543 components from fermentation broth in which they are produced which comprises: a) adding an approximately equal volume of a water miscible, polar organic solvent to the fermentation broth, including the biomass thereof; b) separating the liquid phase of the resulting mixture from the biomass; c) adjusting the pH of the separated liquid phase to between 7 and 13; d) applying the separated liquid phase directly to a column of nonfunctional, macroreticular polymer; e) eluting the A83543 components from the column with an aqueous solution of water miscible, polar organic solvent; and f) collecting the fractions containing A83543 components.

Description

PROCESS FOR ISOLATING A83543 AND ITS COMPONENTS

This invention provides an improved process for isolating A83543 and its separate components from the fermentation broth in which it is produced. Fermentation product A83543, a family of related components produced by strains of Saccharopolyspora spinosa, was recently discovered and was shown to exhi bit excellent insecticidal activity. A83543 and each of the components are useful for the control of mites and insects, particularly Lepidoptera and Diptera species. The general structure of the A83543 components consists of a 5,6,5-tricyclic ring system fused to a 12-membered macrocyclic lactone, a neutral sugar and an amino sugar. By the term "A83543 component" means a compound of Formula 1 :

wherein R¹ is hydrogen or a group of formula

(a) (b)

and R², R³, R⁴, R⁵, R⁶ and R⁷ are independently hydrogen or methyl; or an acid addition salt thereof when R¹ is other than hydrogen. In one embodiment, the present invention is directed to a subgroup of Formula 1 compounds, Formula 1A compounds The Formula 1 A compounds are the Formula 1 compounds wherein R5, R⁶ and R⁷ are not concurrently hydrogen

The family of natural components of A83543 includes a genus of compounds, Formula 2 Compounds (which are Formula 1 compounds wherein R⁶ ιs -OCH3), taught in EPO Application No. 0375316 and having the following general formula:

wherein R8 is H or a group selected from

and R9, Rio, RH, R -2 and R¹³ are hydrogen or methyl; or an acid addition salt thereof when R¹ is other than hydrogen.

A83543 compounds having been shown to comprise individual components within the definition of Formula 2 include A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H and A83543J (see European Patent Publication No. 0 375 316); individual components A83543L, A83543M and A83543N ( taught in United States Patent Application Number 07/790,287, filed November 8, 1991 ), and individual components A83543Q, A83543R, A83543S and A83543T ( taught in United States Patent Application of Turner, Broughton, Huber and Mynderse, entitled "New A83543 Compounds and Processes for Production Thereof" (taught in United States Patent Application Serial Number 07/973,121 , filed on November 6, 1992). The following table identifies by structure these individual A83543 components of Formula 2, wherein R8, R9, RIO, RI I , Ri2 _and 13 are for each component as follows:

The present invention also includes a process for isolating a new genus of A83543 compounds, termed Formula 3 compounds (which are Formula 1 compounds wherein 6 is - OH), having the following general formula:

wherein R¹⁴ is hydrogen or a group of formula

(a) (b)

and R¹5, 16_# 17, R18₍ and R¹⁹ are independently hydrogen or methyl; or an acid addition salt thereof when R¹ is other than hydrogen. In one embodiment, the present invention is directed to a subgroup of Formula 3 compounds, Formula 3A compounds, (taught in United States Patent Application Serial

Number 08/030,522, filed on March 12, 1993) . The Formula 3A compounds are the Formula 1 compounds wherein R¹8 and R¹⁹ are not concurrently hydrogen.

A83543 compounds shown to comprise individual components within the definition of Formula 3 include A83543K, A835430, A83543P, A83543U, A83543V and

A83543W. The following table identifies by structure these individual A83543 components of

Formula 3 wherein R¹⁴, R¹5, R16, R¹⁷, R 8, and 19 for each component are as follows:

A83543 is produced by culturing an A83543-producing strain of Saccharopolyspora spinosa under submerged aerobic conditions, by methods disclosed in European Patent Application No. 0375316. The term "A83543-producing strain of Saccharopolyspora spinosa" means a strain of Saccharopolyspora spinosa capable of producing recoverable amounts of at least one A83543 component. The following A83543-producing Saccharopolyspora spinosa cultures, A83543.1 , A83543.3, A83543.4, A83543.5, A83543.6, A83543.7, A83543.8 and A83543.9 have been deposited and made a part of the stock culture collection of the Midwest Area Regional Research Center, Agricultural Research Service, United States Department of Agriculture, from which they are available to the public under the following accession numbers:

The characteristics of the species Saccharopolyspora spinosa, specifically A83543.1 and mutants thereof, are described in detail in the above identified EPO Application No. 0375316. Each of the strains A83543.3, A83543.4, A83543.5, A83543.6, and A83543.7 was derived from A83543.1 by chemically-induced mutagenesis with N-methyl-N'-nitro-N- nitrosoguanidine. Strains A83543.8 and A83543.9 were derived from A83453.4 by chemically- induced mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Except for differences in the production of the A83543 components, these isolates appear the same as the parent culture.

As is the case with other organisms, the characteristics of the A83543-producing strains are subject to variation. Thus, mutants of these strains may be obtained by physical and chemical methods known in the art. For example, other strains may be obtained by treatment with chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine. Natural and induced mutants derived directly or indirectly from A83543.1 and the other deposited cultures which retain the characteristic of producing recoverable amounts of at least one A83543 component are applicable in the present invention. The Formula 1 compounds are generally produced by culturing an A83543- producing strain of S. spinosa sp. nov. under submerged aerobic conditions in a suitable culture medium, until a recoverable amount of the Formula 1 compound is produced.

The culture medium used to grow these Saccharopolyspora spinosa cultures can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, certain culture media are preferred. For example, preferred carbon sources in large-scale fermentation are glucose and methyl oleate, although ribose, xylose, fructose, galactose, mannose, mannitol, maltose, soluble starch, potato dextrin, oils such as soybean oil and the like can also be used. Preferred nitrogen sources are cottonseed flour, peptonized milk and corn steep liquor, although fish meal, digested soybean meal, yeast extract, enzyme- hydrolyzed casein, beef extract, and the like can also be used. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate and like ions. Essential trace elements necessary forthe growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other substituents of the medium in amounts sufficient to meet the growth requirements of the organism.

Usually, if foaming is a problem, small amounts (i.e., 0.2 ml/L) of an antifoam agent such as polypropylene giycol may be added to large-scale fermentation media. In the case of the A83543-producing cultures, however, conventional defoamers inhibit A83543 production. Foaming is controlled by including soybean oil or PLURONIC L-101 (BASF, Parsippany, New Jersey, USA) in the medium (1-3 percent). Additional oil is added if foaming develops. The A83543 components are produced by the A83543-producing organisms when grown at temperatures between 24° and 33°C. Optimum temperatures for production appear to be 28-30°C.

As is customary in submerged aerobic culture processes, sterile air is blown into the vessel from the bottom while the medium is stirred with conventional turbine impellors. In general, the aeration rate and agitation rate should be sufficient to maintain the level of dissolved oxygen at or above 60 percent of air saturation, preferably above 65 percent, with an internal vessel pressure of 0.34 atmospheres.

One aspect of the present invention is the production of a compound of Formula 3 produced by culturing an A83543A-producing strain of S. spinosa in a suitable culture medium containing sinefungin, selected from the group consisting of NRRL 18395, NRRL 18537, NRRL 18538, and NRRL 18539 or an A83543A-producing mutant thereof. An "A83543A- producing mutant" is a strain derived from any one of the A83543A-producing strains of S. spinosa, NRRL 18395, NRRL 18537, NRRL 18538, NRRL 18539, which is capable of producing recoverable amounts of A83543A and which is capable, when cultured in a suitable culture medium containing sinefungin, to produce concomitant amounts of A83543K and A835430. Another aspect of the present invention is the production of a compound of Formula 3 by culturing an A83543H-produci ng strain of S. spinosa, such as NRRL 18823 or an A83543H-producing mutant thereof, in a suitable culture medium containing sinefungin. An "A83543H-producing mutant" is a strain derived from any one of the A83543H-producing strains of S. spinosa, NRRL 18823, which is capable of producing recoverable amounts of A83543H and which is capable, when cultured in a suitable culture medium containing sinefungin, to produce concomitant amounts of A83543U and A83543V. A still further aspect of this invention is the production of a compound of Formula 3 by culturing an A83543J-producing strain of S. spinosa, such as NRRL 18719 or an A83543J- producing mutant thereof, in a suitable culture medium containing sinefungin. An "A83543J- producing mutant" is a strain derived from any one of the A83543J-producing strains of S. spinosa, NRRL 18719 or NRRL 18720, which is capable of producing recoverable amounts of A83543J and which is capable, when cultured in a suitable culture medium containing sinefungin, to produce concomitant amounts of A83543P and A83543W.

Typically, sinefungin is added to the production medium after 48-72 hours or for large scale production, the addition of sinefungin is postponed until the culture begins to grow as indicated by the uptake of oxygen. Preferably, sinefungin is added to the fermentation medium 48 hours to 72 hours after inoculation. Sinefungin may be added as a solid or as a solution. For convenience, when sinefungin is added to a large scale fermentation, addition as an alcoholic solution is preferred. Such a solution is prepared by dissolving sinefungin in a sufficient volume of methyl alcohol, then sterilizing the solution by filtration through a 0.45μ filter.

Alternatively, the Formula 3 compounds are produced by culturing S. spinosa strain NRRL 18743 (which produces components A83543K, A835430 and A83543Y), or an A83543K-producing mutant thereof, in a suitable culture medium without the addition of sinefungin. An "A83543K-producing mutant" is a strain derived from S. spinosa NRRL 18743 which is capable of producing recoverable amounts of A83543K.

The Formula 1 compounds can be recovered using various isolation and purification procedures which are understood in the art. Production of the A83543 components can be followed during the fermentation by testing extracts of the broth. A preferred method for following the production is analysis of the broth extracts by high performance liquid chromatography (HPLC). Suitable systems for analysis are described in Examples 1 , 4, 7 and Comparative Example 1 .

The A83543 components produced during fermentation of the A83543-producing organism occurs in both the mycelial mass (the biomass) and the broth. A83543 components are lipophilic. Separation of the I ipophi lie A83543 components from the broth has proved to be a significant problem, because it requires a simultaneous separation of the components from the substantial quantity of oil used to control foaming during the fermentation.

Prior to this invention, A83543 components were isolated by the method described in European Patent Application number 90/916854.4 of Patrick J. Baker on "A83543 Recovery Process". That reference teaches a process for recovering fermentation product A83543 which comprises extracting the A83543 from the whole broth into a water miscible solvent such as acetone, filtering to remove the biomass, raising the pH of the filtrate to 10-1 1 and extracting the A83543 with an immiscible solvent such as ethyl acetate, back extracting with an acidic solution and separating the A83543 by precipitating it with a base. Although the process described in that patent application is a significant improvement over prior processes, there remained a need to improve the efficiency of the recovery operation

This invention provides a process for isolating A83543 components of Formula 1 from fermentation broth in which they are produced which comprises a) adding an approximately equal volume of a water miscible, polar organic solvent to the fermentation broth, including the biomass thereof, b) separating the liquid phase of the resulting mixture from the biomass, c) adjustingthe pH of the separated liquid phase to between 9 and 13, d) applying the separated liquid phase directly to a column of nonfunctional, macroreticular polymer; e) eluting the A83543 components from the column with an aqueous solution of water miscible, polar organic solvent, and f) collecting the fractions containing A83543 components

The invention, in a specific embodiment, is a process for isolating A83543 components of Formula 1 , excluding compounds of Formula 2 Also contemplated by the invention is a process for isolating from a fermentation broth A83543 components of Formula 1A, Formula 2,Formula 3 and/or Formula 3A

As used herein the term "water miscible, polar organic solvent" encompasses mixtures of one or more water miscible, polar organic solvents, with or without water In step a), the whole fermentation broth may be used Preferred "water miscible, polar organic solvents" are acetone and acetonitπle The function of this step is to extract the A83543 components from the biomass Other water miscible, polar organic solvents in which the components are readily soluble may also be used

Step b) is preferably carried out by filtering the mixture produced in step a) through a ceramic -^'liter The filtrate produced by this step contains the oil that was added to the broth during fermentation to control foaming

The pH adjustment called for in step c) assures that the A83543 components will be in the base form The base form is adsorbed more strongly on the nonfunctional, macroreticular polymer A preferred pH range is from 9 to 13 The value is not critical, provided the A83543 components are in the base form Possible instability of the A83543 components becomes of increasing concern when the pH exceeds 13 The pH adjustment is conveniently made using sodium hydroxide, but the base used is not critical

The preferred nonfunctional, macroreticular polymer for use in step d) is Diaion HP-20ss (Mitsubishi Chemical Industries Co , Ltd , Japan) Following step d) and prior to step e), the loaded column may optionally be washed with an aqueous solution of water miscible, polar organic solvent to displace the broth from the column A preferred organic solvent is methanol/acetonitπle/water (1 1 2) The organic component of the solvent should not be so small as to cause a potential for precipitation of material in the column, nor so large as to cause A83543 components to be eluted at this stage. The solvent is preferably 30 percent to 70 percent aqueous, and more preferably 50 percent aqueous. By removing the oily impurities in this step the purity of the A83543 components is significantly improved. The water miscible, polar organic solvent used in step e) is preferably a 95:5 mixture of methanol/acetonitrile (1 : 1) containing 0.1 percent ammonium acetate to control pH (pH 8.1). Other polar organic solvents, such as acetone can also be used. Ammonium acetate is the preferred buffer, because it is volatile and easily removed from the A83543 components. The fractions containing the A83543 components are combined, concentrated, and optionally lyophilized.

The advantages of this method over the previously used method are:

1 ) recovery of purified A83543 components can be achieved as part of this process, whereas the previous method provided no component separation;

2) improved recovery of purified A83543 components by reducing the number of isolation steps; and

3) elimination of costly and time consuming distillation steps.

The invention is illustrated in further detail by the following detailed procedures, preparations, and examples. The examples are for purposes of illustration only, and are not to be construed as limiting the scope of the present invention. All parts and percentages are by weight unless otherwise specifically noted. Example 1 : A83543 Assay Method

The following analytical high performance liquid chromatography (HPLC) method is useful for monitoring a fermentation for the production of A83543 components: A sample of the whole broth is diluted with three volumes of acetonitrile to extract the components from the myceiia. The resulting solution is then filtered through a 0.45 micron PTFE filter to remove particulate matter prior to injection into the HPLC assay system. A solution of purified A83543A at a concentration of 100 mg/ml in methanol is used as an external standard for the assay and peak areas of all A83543 components are related back to this calibration standard to determine concentrations of individual components. HPLC System:

Column Support: 8 x 100-mm column, silica gel 4μ spherical Cιs (Nova C18,

Waters)

Mobile Phase: CH₃CN/MeOH/H₂0 (45/45/10) containing 0.05 percent ammonium acetate Flow Rate: 4 ml/min

Detection: U at 250 nm

Retention Times: A83543A 3.6 - 3.7 min

Retention Times: A83543D 4.4 - 4.5 min

Example 2: Preparation of A83543A and A83543D with Culture A83543.6 A. Shake-flask Fermentation

The culture s, spinosa NRRL 18719, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, was used to inoculate a vegetative medium having the following composition:

Vegetative Medium In redient Amount

* Baltimore Biological Laboratories, Cockeysville, Maryland, USA.

Slants or plates can be prepared by adding 2.5 percent agar to the vegetative medium. The inoculated slant is incubated at 30°C for between 10 to 14 days. The mature slant culture is scraped with a sterile tool to loosen the spores and to remove and macerate the mycelial mat. About one-fourth of the loosened spores and culture growth thus obtained is used to inoculate 50 ml of a first-stage vegetative medium. Alternatively, the first-stage medium may be inoculated from a liquid nitrogen ampoule.

When culture is maintained in liquid nitrogen, ampoules are prepared by homogenizing a vegetative culture (48-72 hours incubation, 30°C), diluting 1 : 1 (volume: volume) with a sterile suspending agent, and dispensing into sterile tubes (1.5 ml/tube). The suspending agent contains lactose (100 g), glycerol (200 ml), and deionized water (q.s. to 1-L). A liquid nitrogen ampoule is used to inoculate 100 ml of vegetative medium in 500-ml Erienmeyer flasks (or 50 ml of medium in 250-ml flasks). The cultures are incubated at 30°C for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 260 rpm.

The incubated culture (10 percent v/v inoculum) is used to inoculate 50 ml or 100 ml, dependent on the size of the Erienmeyer flask, of a production medium having the following composition:

Production Medium

pH adjusted to pH 7.0 with 1 NaOH, sterilized 40 min. at 120°C.

* Peptonized Milk Nutrient, Sheffield Products, Norwich, New York 13815, USA. ** Proflo, Traders Protein, Memphis, Tennessee 38108, USA. *** The amount of methyl oleate was 30 ml.

The inoculated production medium is incubated in 250-ml or 500-ml Erienmeyer flasks at 30°C for 7 to 10 days on a shaker orbiting in a two-inch circle at 260 rpm. B. Stirred Reactor Fermentation In order to provide a larger volume of inoculum, 10 ml of incubated first stage medium, prepared as described in Example 2, Section A, is used to inoculate 400 ml of a second- stage vegetative medium having the same composition as that of the first-stage medium. This second-stage vegetative medium is incubated in a 2-L wide-mouth Erienmeyer flask for 48 hours at 30°C on a shaker orbiting in a two-inch circle at 260 rpm. Incubated second-stage vegetative medium (2-L) thus prepared is used to inoculate 80 to 115 liters of sterile production medium, prepared as described in Example 2, Section A.

The inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for 7 to 10 days at a temperature of 30°C. The air-flow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at or above 60 percent to 80 percent of air saturation.

Example 3: Isolation of A83543A and A83543D from Fermentation Broth

Fermentation broth (185 liters) prepared using A83543.6 as described in Example 2, was added to an equal volume of acetone. The resulting mixture was filtered using a ceramic filter to remove the biomass. The pH of the filtrate was adjusted to pH13 and then adsorbed onto a 10 liter column of HP-20ss resin (Mitsubishi Chemical Industries Co., Ltd., Japan) and then the A83543A and A83543D components were eluted using a gradient mixture of organic solvent comprising methanol and acetonitrile (1 :1) containing 0.1 percent ammonium acetate. The gradient profile was from 0 percent organic solvent to 95 percent organic solvent. Fractions (4 liters) were collected, and based on analytical HPLC, fractions 19 to 26 were combined and concentrated to dryness and then redissolved in 1 liter of methanol for further purification.

Example 4: Assay Method for A83543Q, A83543R. A83543S and A83543T The following analytical high performance liquid chromatography (HPLC) method is useful for monitoring a fermentation for the production of A83543Q, A83543R, A83543S, A83543T and other A83543 components:

A sample of the whole broth is diluted with three volumes of acetonitrile to extract the components from the myceiia. The resulting solution is then filtered through a 0.45 micron polytetrafluoroethylene (PTFE) filter to remove particulate matter prior to injection into the HPLC assay system. A solution of purified A83543 A at a concentration of 1 mg/ml in methanol is used as an external standard for the assay and peak areas of all A83543 components are related back to this calibration standard to determine concentrations of individual components. HPLC System:

Column Support: 4.6 x 100-mm column, ODS-AQ, 5μ spherical particles, 12θA pore (YMC, Inc., Morris Plains, New Jersey, USA).

Mobile Phase: CH₃CN/MeOH/H₂0 (37.5/37.5/25) containing 0.05 percent ammonium acetate Flow Rate: 2 ml/min

Detection: U at 250 nm Retention Times: A83543A 14.97 min A83543Q 1 1.82 min A83543R 4.52 min A83543S 6.50 min

A83543T 5.97 min A83543H 8.50 min Example 5: Preparation of A83543Q. A83543R, A83543S and A83543T with Strain NRRL 18823

(A83543.9)

A. Shake-flask Fermentation

The culture s, spinosa NRRL 18823, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, was used to inoculate a vegetative medium having the following composition:

Vegetative Medium Ingredient Amount(q)

Trypticase Broth* 30 Yeast extract 3

MgS0₄-7H₂0 2

Glucose 5

Deionized water q.s. 1 L

Autoclave 30 min at 120°C. * Baltimore Biological Laboratories, Cockeysville, Maryland, USA.

The first-stage medium may be inoculated from a liquid nitrogen ampoule. Such ampoules are prepared by homogenizing a vegetative culture (48-72 hours incubation, 30°C) diluting 1 : 1 (volume:volume) with a sterile suspending agent, and dispensing into sterile tubes (1.5 ml/tube). The suspending agent contains lactose (100 g), glycerol (200) ml, and deionized water (q.s. to 1 L).

A liquid nitrogen ampoule is used to inoculate 100 ml of vegetative medium in 250-ml wide-mouthed Erienmeyer flasks. The cultures are incubated at30°Cto 32°C for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250 rpm.

The incubated culture (5 percent v/v inoculum) is used to inoculate 50 ml Erienmeyer fiask, of a production medium having the following composition:

Production Medium ingredient Amount (q)

Glucose 80

Peptonized milk* 20 Cottonseed flour** 30

Corn steep liquor 10

CaC0₃ (tech. grade) 5

Methyl oleate 30***

Tap water q.s. to 1 L pH adjusted to pH 7.0 with 1 NaOH, sterilized 40 min. at 120°C.

* Peptonized Milk Nutrient, Sheffield Products, Norwich, New York, USA. ** Proflo, Traders Protein, Memphis, Tennessee, USA. ***The amount of methyl oleate was 30 ml. The inoculated production medium is incubated in 250-ml Erienmeyer flasks at 30°C for 7 days on a shaker orbiting in a two-inch circle at 250 rpm. B. Stirred Reactor Fermentation

In orderto provide a larger volume of inoculum, 10 ml of incubated first stage medium, prepared as described in Example 5, Section A, is used to inoculate 400 ml of a second- stage vegetative medium having the same composition as that of the first-stage medium. This second-stage vegetative medium is incubated in a 2 L wide-mouth Erienmeyer flask for 48 hours at 30°C on a shaker orbiting in a two-inch circle at 260 rpm. Incubated second-stage vegetative medium (2 L) thus prepared is used to inoculate 80 to 1 15 liters of sterile production medium, prepared as described in Example 5, Section A.

The inoculated production medium is allowed to ferment in a 165 L stirred bioreactor for 7 days to 10 days at a temperature of 30°C. The air-flow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at or above 60 percent to 80 percent of air saturation. Example 6: Isolation of A83543Q, A83543R, A83543S and A83543T from Strain NRRL 18823 (A83543.9)

Fermentation broth (100 L; harvest titer A83543H, 303 μg/ml, A83543Q, 50 μg/ml), prepared as described in Example 5, Section B, was refrigerated two days prior to processing. Acetone (100 L) was added to the whole broth after adjusting the pH to 3.0 with 5N HCI. The resulting mixture was filtered through a ceramic filterto give filtrate (170 L) which was held over the weekend under refrigeration. The broth/acetone filtrate was adjusted to pH 13 and refiltered through the ceramic filter prior to loading onto a steel column (10L) containing HP-20ss resin (Mitsubishi Chemical Industries, Ltd., Japan) at a flow rate of 1 L/minute. The column was eluted at a flow rate of 1 L/minute with a gradient mixed from solvent "A" (0.1 percent NH₄OAc, adjusted to pH 8.1 with NH₄OH) and solvent "B" (CH3CN - CH3OH 1 : 1), collecting 4 L fractions. The pumping system was programmed to generate a gradient from 0 to 50 percent B in one minute, followed by a gradient from 50 to 100 percent B in 90 minutes, followed by isocratic delivery of 100 percent B for an additional 15 minutes. HPLC analysis (described in Example 4) indicated that fraction 17 (4 L), contained predominantly component R with additional more polar materials and a small amount of components T and H; fractions 18-22 contained predominantly component H with lesser amounts of components R and Q and small amounts of component S and more polar materials; fractions 23-24 contained components H and Q. HPLC analysis of the pools suggested the following total quantities; component H, 23.0 g; component Q, 3.4 g; component R, 2.0 g; component S, 0.2 g; component T, 0.2 g. Example 7: Assay Method for A83543K, A83543Q, A83543P, A83543U, A83543V, A83543W, A83543Y

The following analytical high performance liquid chromatography (HPLC) method is useful for monitoring a fermentation for the production of A83543K, A835430, A83543P, A83543U, A83543V, A83543W, A83543Y and other A83543 components:

A sample of the whole broth is diluted with three volumes of acetonitrile to extract the factors from the myceiia. The resulting solution isthen filtered through a 0.45 micron polytetrafluorine (PTFE) filterto remove particulate matter prior to injection into the HPLC assay system. A solution of purified A83543A at a concentration of 100 mg/ml in methanol is used as an external standard for the assay and peak areas of all A83543 components are related back to this calibration standard to determine concentrations of individual components. HPLC System:

Column Support: YMC-PACK 4.6 x 100-rmm ID column, 5μ spherical, 12θA (YMC Inc., Morris Plains, New Jersey, USA).

Mobile Phase: CH₃CN/MeOH/H₂0 (3:3:2) containing 0.05 percent ammonium acetate

Flow Rate: 2 ml/min Detection: U at 250 nm Retention Times: A83543A 15.52 min

A83543K 8.10 min A835430 1 1.40 min A83543P 6.40 min A83543U 5.22 min A83543V 7.05 min

A83543W 8.47 min A83543Y 6.12 min Example 8: Preparation of A83543K and A83543Q with Culture NRRL 18538 (A83543.4) A. Shake-flask Fermentation The culture S. spinosa NRRL 18538, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, was used to inoculate a vegetative medium having the following composition:

pH 6.2, adjust to pH 6.5 with NaOH

* NZ Amine A, Sheffield Products, Norwich, New York, USA. Slants or plates can be prepared by adding 2.5 percent agar to the vegetative medium. The inoculated slant is incubated at 30°C for between 10 to 14 days. The mature slant culture is scraped with a sterile tool to loosen the spores and to remove and macerate the mycelial mat. About one-fourth of the loosened spores and culture growth thus obtained is used to inoculate 50 ml of a first-stage vegetative medium. Alternatively, the first-stage medium may be inoculated from a liquid nitrogen ampoule.

When culture is maintained in liquid nitrogen, ampoules are prepared using equal volumes of vegetative culture (-48-72 hours incubation, 30°C) and suspending medium. The suspending medium contains lactose (100 g), glycerol (200 ml), and deionized water (q.s. to 1-L). A liquid nitrogen ampoule is used to inoculate 50 ml of vegetative medium in

250-ml Erienmeyer flasks. The cultures are incubated at 30°C for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250 rpm.

The incubated culture (5 percent v/v inoculum) is used to inoculate 30 ml of a production medium in a 250-ml wide-mouth Erienmeyer flask. The medium composition is as follows:

Production Medium Inqredient Amount (g)

Glucose 80

Peptonized milk* 20 Cottonseed flour** 30

Corn steep liquor 10

CaC0₃ (tech. grade) 5

Methyl oleate 30***

Tap water q.s. to 1-L * Peptonized Milk Nutrient, Sheffield Products, Norwich, New York, USA. ** Proflo, Traders Protein, Memphis, Tennessee, USA. ***The amount of methyl oleate was 30 ml. The inoculated production medium is incubated in 250-ml wide-mouth Erienmeyer flasks at 30°C for 7 days on a shaker orbiting in a two-inch circle at 250 rpm. Sinefungin is added at a final concentration of 100 μg/ml, at 72 hours after inoculation. B. Stirred Reactor Fermentation In order to provide a larger volume of inoculum, 10 ml of incubated first stage medium, prepared as described in Example 8, Section A, is used to inoculate 400 ml of a second- stage vegetative medium having the same composition as that of the first-stage medium. This second-stage vegetative medium is incubated in a 2-L wide-mouth Erienmeyer flask for 48 hours at 30°C on a shaker orbiting in a two-inch circle at 250 rpm. Incubated second-stage vegetative medium (2-L) thus prepared is used to inoculate 1 15 liters of sterile production medium, prepared as described in Example 8, Section A. Sinefungin, as a filtered methanolic solution, is added at 66 hours to a final concentration of 100 μg/ml.

The inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for 7 days at a temperature of 30°C. The air-flow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at 80 percent of air saturation.

Example 9: Isolation of A83543K and A83543Q from NRRL 18538 (A83543.4) Fermented in the Presence of Sinefungin Fermentation broth (210-L stirred fermenter) was prepared substantially as described in Example 8, Section B. Acetone was added to the whole broth and the pH was adjusted to 8.0. The resulting mixture was filtered through a ceramic filter to give filtrate (370- L). The broth/acetone filtrate was loaded onto a steel column (10-L, 10 cm x 122 cm) containing HP-20ss resin (Mitsubishi Chemical Industries, Ltd., Japan) at a flow rate of 1-L/minute, collecting the effluent in a single pool. The column was el uted at a flow rate of 1-L/minute with a gradient mixed from solvent "A" (0.1 percent NH₄OAc) and solvent "B" (CH3OH - CH3CN; 1 : 1). The pumping system was programmed to deliver 50 percent B for 2 minutes, followed by a linear gradient from 50 - 80 percent B (45 minutes), followed by a linear gradient from 80 - 90 percent B (33 minutes), collecting 20 x 4 L fractions. Fractions 13 - 17, containing components K and O were pooled. The column effluent (see above) was adjusted to pH 9.5 with 5N NaOH and reapplied to the HP-20ss column. The pumping system was programmed to deliver 50 percent B for 1 minute, a linear gradient from 50 - 75 percent B (30 minutes), a linear gradient from 75 - 85 percent B (45 minutes), a linear gradient from 85 - 88 percent B (15.4 minutes), and a linear gradient from 88 - 100 percent B (20 minutes), at a flow rate of 1- L minute, collecting 22 x 4 Lfractions. Fractions 7 - 17 were pooled and combined with the pool (fractions 13 - 17 from the first HP-20ss chromatography (see above). The combined pools were concentrated to 4-L, then further concentrated to dryness, redissolved in CH3OH (100 ml), then precipitated into CH₃CN (3-L). The resulting precipitate was removed by filtration, washed with CH_jC , and discarded; the filtrate was concentrated to dryness. The resulting residue was redissolved in dichloromethane (50 ml) and applied to a column (6 cm x 24 cm) of silica gel (EM grade 62, 60 - 200 mesh) equilibrated in acetonitrile. The column was eluted with CH₃CN (4-L), then CH₃CN - CH₃OH (9: 1 ; 10-L), taking 10 x 250 ml fractions, followed by 7 x 1 L fractions. Fractions 6 - 15, containing components K and O, were concentrated to dryness. The resulting residue was dissolved in CH3OH (100 ml) and applied (in 20 runs) to a preparative reverse phase HPLC column (Rainin Dynamax-6θA 8 μm C18, 41.4 mm ID x 25 cm with 41.4 mm x 5 cm guard module) equilibrated in H₂0 - CH3OH - CH3CN; (50: 175: 175, containing 0.1 percent NH₄OAc). The column was eluted at a flow rate of 40 ml/minute. Progress of the separation was monitored with a variable wavelength UV detector tuned to 250 nm. UV absorbing peaks (from the 20 chromatographic runs) were collected in 7 pools. The two largest peaks corresponded to components K and O. Pool 3 (6-L), contained component K (98 percent pure). Pool 4 (8-L), containing components O and K, was concentrated to 200 ml and rechromatographed (in 4 runs) under the same conditions, collecting the two peaks as two pools. Pool 1 (3-L) contained component K (98 percent pure). Pool 2 (5-L), contained component O (95 percent) and component K (5 percent). Pool 2 was concentrated to 100 ml and desalted by chromatography on the same HPLC column (in 3 runs), eluting with a 60 minute linear gradient from H₂0 - CH3OH - CH3CN (30:35:35) to H₂0 - CH3OH - CH3CN (10:45:45). The UV absorbing eluate was collected in 10 x 3 minute fractions. Fractions containing >98 percent pure component O were pooled, concentrated to dryness, and lyophilized from t-BuOH to give component O (2.5 g; >98 percent pure). Component K containing pools from the first preparative HPLC separation (pool 3, 6-L) and the repurifi cation of component O (pool 1 , 3-L) were combined, concentrated to 200 ml, and desalted in the same manner as component O. Fractions containing >98 percent pure component K were pooled, concentrated to dryness, and lyophilized from t-BuOH to give component K (1 1.1 g; >99 percent pure). Example 10: Preparation of A83543K, A83543Q and A83543Y with Culture NRRL 18743

(A83543.8) A. Shake-flask Fermentation The culture s, spinosa NRRL 18743, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, was used to inoculate a vegetative medium having the following composition: Vegetative Medium Inqredient Amount(q)

Trypticase soy broth* 30

Yeast extract 3 MgS0₄-7H 0 2

Glucose 5

Maltose 4

Deionized water q.s. 1-L autoclave 30 min at 120°C * Baltimore Biological Laboratories, Cockeysville, Maryland, USA.

Slants or plates may be prepared by adding 2.5 percent agar to the vegetative medium. The inoculated slant is incubated at 30°C for between 10 to 14 days. The mature slant culture is scraped with a sterile tool to loosen the spores and remove and macerate the mycelial mat. About one-fourth of the loosened spores and culture growth thus obtained is used to inoculate 50 ml of a first-stage vegetative medium. Alternatively, the first-stage medium may be inoculated from a liquid nitrogen ampoule.

Liquid-nitrogen-stock inoculum is prepared by homogenizing a vegetative culture, diluting 1 : 1 (volume:volume) with a sterile suspending agent of glycerol :lactose:water (2: 1 :7), and dispensing into sterile tubes (1.5 ml/tube). The diluted inoculum isthen stored over liquid nitrogen in appropriate storage containers and used as a working stock inoculum for the cultivation of shake-flask cultures and fermenter seed inoculum.

A liquid nitrogen ampoule is quick thawed and 0.5 ml is used to inoculate 50 ml of vegetative medium in 250-ml wide-mouth Erienmeyer flasks. The cultures are incubated at 32°C for 48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 250 rpm. The incubated culture (5 percent v/v inoculum) is used to inoculate 25 ml of a production medium having the following composition:

Production Medium Ingredients Amount(q)

Peptonized milk* 20 Cottonseed flour** 30

Corn steep liquor 10

CaC0₃ (tech. grade) 5

Methyl oleate 30

Tap water q.s. to 1-L * Peptonized Milk Nutrient, Sheffield Products, Norwich, New York, USA. **Proflo, Traders Protein, Memphis Tennessee, USA.

The inoculated production medium is incubated in 250-ml wide-mouth Erienmeyer flasks at 30°C for 7 days on a shaker orbiting in a two-inch circle at 250 rpm. B Stirred Reactor Fermentation

In orderto provide a larger volume of inoculum, 10 ml of incubated first stage medium, prepared as described in Example 10, Section A, is used to inoculate 400 ml of a second-stage vegetative medium having the same composition as that of the first-stage medium This second-stage vegetative medium is incjbated in a 2-L wide-mouth Erienmeyer flask for 48 hours at 32°C on a shaker orbiting in a two-inch circle at 250 rpm

Incubated second-stage vegetative medium (2-L) thus prepared is used to inoculate 1 15 liters of sterile production medium, prepared as described in Example 10, Section A The inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for 7 days at a temperature of 30°C The air- flow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at or above 80 percent of air saturation Example 1 1 Isolation of A83543K. A83543Q, and A83543Y from Strain NRRL 18743 (A83543 8) Fermentation broth (260-L) was prepared as substantially described in Example

10, Section B Acetone (260-L) was added to the whole broth after adjusting the pH to 3 0 with 5N HCI The resulting mixture was filtered through a ceramic filterto give filtrate (480-L) which was held over the weekend under refrigeration The broth/acetone filtrate was adjusted to pH 12 with 25 percent NaOH and refiltered twice through the ceramic filter prior to loading onto a steel column (10-L, 10 cm x 122 cm) containing HP-20ss resin (Mitsubishi Chemical Industries, Ltd , Japan) at a flow rate of 0.5-L/mιnute. The column was washed with CH3CN - CH3OH - 0 1 percent aq NH₄OAc (adjusted to pH 8 1 with NH₄OH) (25 25.50, 20-L) New components K, O and Y were eluted with CH3CN - CH₃OH - 0 1 percent aq NH₄OAc (adjusted to pH 8 1 with NH₄OH) (95 95: 10, 30-L) at a flow rate of 1-L/rπιnute The eluate (30-L) was concentrated, redissolved in CH3OH, reconcentrated to dryness, redissolved in CH3OH (100 ml), then precipitated into CH3CN (2-L) The resulting precipitate was removed by filtration, washed with CH3CN, and discarded; the combined filtrate and wash (3-L) was concentrated to dryness The resulting residue was redissolved in dichloromethane (50 ml) and applied to a column (7 5 cm x 50 cm) of silica gel (EM grade 62, 60 - 200 mesh) equilibrated in acetonitrile The column was eluted with CH3CN (10-L), then CH3CN - CH₃OH (9 1 , 20-L), followed by CH3CN - CH3OH (8:2; 10-L), collecting 1-L fractions Fractions 1 1 - 30 were pooled and concentrated to dryness The resulting residue was dissolved in CH3OH (50 ml) and applied (in 10 runs) to a preparative reverse phase HPLC column (Rainin Dynamax-60A 8 μm C18, 41 4 mm ID x 25 cm with 41 4 mm x 5 cm guard module) equilibrated in H₂0 - CH3OH - CH3CN, (50 175 175, containing 0 1 percent NH₄OAC) The column was eluted at a flow rate of 40 ml/mιnute wιth a 60 minute linear gradient from H₂0 - CH₃OH - CH₃CN, (50: 175 175, containing 0 1 percent NH₄OAc) to H₂0 - CH3OH - CH3CN; (10:45 45, containing 0 1 percent NH₄OAc) Progress of the separation was monitored with a variable wavelength UV detector tuned to 250 nm The first three peaks collected (10 runs pooled) corresponded to the el ution of minor component Y (pool 1 , 1-L), component K (pool 2, 8-L) and component O (pool 3, 4-L). Pool 2 was concentrated to a small volume, then desalted by rechromatographing on the same column, eluting without buffer. The effluent corresponding to the UV absorption peak was concentrated to dryness, dissolved in t-BuOH, and lyophilized to give pure component K (7.3 g). Pool 3 was desalted and lyophilized in like manner to give pure component 0 (1.4 g). Pool 1 was desalted by similar chromatography (Rainin Dynamax-6θA 8 μm C18 column, 21.4 mm ID x 25 cm with 21.4 mm x 5 cm guard module) and lyophilized in like manner to give pure component Y (46 mg). Example 12: Isolation of A83543P and A83543W from NRRL 18719 (A83543.6) Fermented in the Presence of Sinefungin

Fermentation broth (190-L stirred fermenter), prepared substantially as described in Example 10, Section B (with the exception that strain A83543.6 was used), was refrigerated two days prior to processing. Acetone (190-L) was added to the whole broth after adjusting the pH to 3.0 with 5N HCI. The resulting mixture was filtered through a ceramic filterto give filtrate (335-L) which was held over the weekend under refrigeration. The broth/acetone filtrate was adjusted to pH 10 with 5N NaOH and refiltered through the ceramic filter prior to loading onto a steel column (10-L; 10 cm x 122 cm) containing HP-20ss resin (Mitsubishi Chemical Industries, Ltd., Japan) at a flow rate of 1-L/minute. The column was washed with CH₃CN - CH₃OH - 0.1 percent aq. NH₄OAc (adjusted to pH 8.1 with NH OH) (25:25:50; 20-L), then eluted with CH₃CN - CH₃OH - 0.1 percent aq. NH OAc (adjusted pH 8.1 with NH4OH) (95:95: 10; 40-L), collecting 2-L fractions. Fractions 3 - 9 were concentrated to dryness, redissolved in CH3OH (100 ml), reconcentrated, then precipitated into CH3CN (1-L). The resulting precipitate was removed by filtration and discarded; the filtrate was concentrated to dryness. The resulting residue was redissolved in dichloromethane (25 ml) and applied to a column (7.5 cm x 50 cm) of silica gel (EM grade 62, 60 - 200 mesh) equilibrated in acetonitrile. The column was eluted with CH3CN (4-L), then CH3CN - CH3OH (9: 1 ; 5-L), followed by CH3OH (1-L), collecting 1-L fractions. Pool 1 (fractions 3 - 4) contained A83543 components J and L; pool 3 (fractions 7 - 10), components M and N. Pool 2 (fractions 5 - 6), containing new components P and W, was concentrated to dryness. The resulting residue was dissolved in CH3OH (10 ml) and applied to a preparative reverse phase HPLC column (Rainin Dynamax-6θA 8 μm C18, 41.4 mm ID x 25 cm with 41.4 mm x 5 cm guard module) equilibrated in H₂0 - CH₃OH - CH3CN; (30:35:35, containing 0.1 percent NH₄OAc). The column was eluted at a flow rate of 40 ml/minute with a gradient mixed from solvent "A" H2O - CH3OH - CH3CN (30:35:35, containing 0.1 percent NH_ΔOAc) and solvent "B" H₂0 - CH3OH - CH3CN; (10:45:45, containing 0.1 percent NH₄OAC). The pumping system was programmed to generate a linear gradient from 25 to 75 percent B in 60 minutes. Progress of the separation was monitored with a variable wavelength UV detector tuned to 250 nm. The major peak was collected in 6 x 3 minute fractions. Fractions 1 - 2, containing new component P, were concentrated to 40 ml, then desalted on the same HPLC column equilibrated in H₂0 - CH3OH - CH₃CN (30:35:35) by eluting with a 60 minute linear gradient from H₂0 - CH3OH - CH3CN (30:35:35) to H₂0 - CH₃OH - CH₃CN (10:45:45). The UV absorbing peak (minus the first 2 minutes eluted) was collected and concentrated to dryness. The resulting residue was dissolved in t-BuOH (10 ml) and lyophilized to give pure component P (479 mg). Pooled fractions 3 - 4 from above, containing a mixture of component P and W, were concentrated to 20 ml and applied to a preparative reverse phase HPLC column (Rainin Dynamax-6θA δ μm C18, 21.4 mm ID x 25 cm with 21.4 mm x 5 cm guard module), equilibrated in H₂0 - CH3OH - CH3CN (30:35:35) containing 0.1 percent NH₄OAc, and eluted at a flow rate of 10 ml/minute with a gradient mixed from solvent "A" H2O - CH3OH - CH3CN; (30:35:35, containing 0.1 percent NH₄OAc) and solvent "B" H₂0 - CH3OH - CH3CN; (10:45:45, containing 0.1 percent NH₄OAC). The pumping system was programmed to generate a linear gradient from 25 to 75 percent B in 60 minutes. Two major UV absorbing peaks (component P, followed by component W) were collected. The componentW containing pool was concentrated to a small volume, then desalted on the same HPLC column equilibrated in H₂0 - CH3OH - CH3CN (30:35:35). Component W was eluted with a 60 minute linear gradient from H₂0 - CH3OH - CH3CN (30:35:35) to H₂0 - CH3OH - CH3CN (10:45:45) at a flow rate of 10 ml/ minute, collecting UV absorbing peak into 10 x 3 minute fractions. Pooled fractions 2 - 7 were concentrated to residue, dissolved in t-BuOH, and lyophilized to give pure component W (82 mg). The component P-containing UV absorbing peak from above was desalted in like manner to give additional pure component P (132 mg). Comparative Example 1 : A. Assay Method

The following analytical high performance liquid chromatography (HPLC) method is useful for monitoring a fermentation for the production of A83543 components: A sample of the whole broth is diluted with three volumes of acetonitrile to extract the factorcomponents from the myceiia. The resulting solution is then filtered through a 0.45 micron PTFE filter to remove particulate matter prior to injection into the HPLC assay system. A solution of purified A83543A at a concentration of 100 mg/ml in methanol is used as an external standard for the assay and peak areas of all A83543 components are related back to this calibration standard to determine concentrations of individual factorcomponents. HPLC System:

Column Support: 4.6 x 100-mm column, ODS-AQ, 5μ spherical particles, 12θA pore (YMC, Inc., Morris Plains, New Jersey, USA)

Mobile Phase: CH3CN/MeOH/H20 (40/40/20) containing 0.05 percent ammonium acetate. Flow Rate: 3 mUm\n Detection: UV at 250 nm Retention Times: A83543A 9.1 min

A83543J 5.7 min

A83543L 7.3 min

A83543M 2.6 min

A83543N 3.3 min

B. Preparation of A83543J, A83543L, A83543M, and A83543N with Culture A83543.6

1. Shake-flask Fermentation

The culture Saccharopolyspora spinosa NRRL 18719, either as a lyophilized pellet or as a suspension maintained in liquid nitrogen, was used to inoculate a vegetative medium having the following composition:

Vegetative Medium Inqredient Amount(g) Trypticase broth* 30

Yeast extract 3

MgSO₄.7H₂0 2

Glucose 5

Maltose 4

Deionized water q.s. 1 L

Autoclave 30 min at 120°C

* Baltimore Biological Laboratories, Cockeysville, Maryland, U.S.A.

Slants or plates can be prepared by adding 2.5 percent agar to the vegetative medium. The inoculated slant is incubated at 30°C for about 10 to about 14 days. The mature slant culture is scraped with a sterile tool to loosen the spores and to remove and macerate the mycelial mat. About one-fourth of the loosened spores and culture growth thus obtained is used to inoculate 50 ml of a first-stage vegetative medium. Alternatively, the first-stage medium may be inoculated from a liquid nitrogen ampoule. When culture is maintained in liquid nitrogen, ampoules are prepared by homogenizing a vegetative culture (48-72 hours incubation, 30°C), diluting 1 : 1 (volume:volume) with a sterile suspending agent, and dispensing into sterile tubes (1.5 ml/tube). The suspending agent contains lactose (100 g), glycerol (200 ml), and deionized water (q.s. to 1 L).

A liquid nitrogen ampoule is used to inoculate 100 ml of vegetative medium in 500-ml

Erienmeyer flasks (or 50 ml of medium in 250-ml flasks). The cultures are incubated at 30°C for

48 hours on a shaker orbiting in a two-inch (5.08 cm) circle at 260 rpm.

The incubated culture (10 percent v/v inoculum) is used to inoculate 50 ml or 100 ml, dependent on the size of the Erienmeyer flask, of a production medium having the following composition:

Production Medium

Inqredient

Glucose

Peptonized milk*

Cottonseed flour**

Corn steep liquor CaCU3 (tech. grade)

Methyl oleate

Tap water

pH adjusted to pH 7.0 with 1 N NaOH, sterilized 40 min. at 120°C

* Peptonized Milk Nutrient, Sheffield Products, Norwich, NY 13815

** Proflo, Traders Protein, Memphis TN 38108

*** The amount of methyl oleate was 30 ml

The inoculated production medium is incubated in 250-ml or 500-ml Erienmeyer flasks at 30°C for 7 to 10 days on a shaker orbiting in a two-inch circle at 260 rpm.

2. Stirred Reactor Fermentation

In order to provide a larger volume of inoculum, 10 ml of incubated first stage medium, prepared as described in Section A, is used to inoculate 400 ml of a second-stage vegetative medium having the same composition as that of the first-stage medium. This second-stage vegetative medium is incubated in a 2-L wide-mouth Erienmeyer flask for about 48 hours at 30°C on a shaker orbiting in a two-inch circle at 260 rpm. Incubated second-stage vegetative medium (2 L) thus prepared is used to inoculate 80 to 1 15 liters of sterile production medium, prepared as described in Section A.

The inoculated production medium is allowed to ferment in a 165-L stirred bioreactor for 7 days to 10 days at a temperature of 30°C. The air-flow and agitator speed in the stirred vessel are computer controlled to maintain a dissolved oxygen level at or above 60 percent to about 80 percent of air saturation.

C. Isolation of A83543J, A83543L, A83543M, and A83543N

Fermentation broth (105 L), prepared as described in Preparation 1 , was adjusted to pH 10 (initially pH 6.8) by adding 5N NaOH. The resulting mixture was filtered through a ceramic filter. The filtrate was discarded, a mixture of acetone and water (1 : 1 , 50 L) was added to the mycelial solids, and the resulting mixture was filtered. A second mixture of acetone and water (1 : 1, 50 L) was added to the mycelial solids, and the pH of the resulting mixture was adjusted to pH 3.0 with 25 percent sulfuric acid. The resulting mixture was filtered, and a third mixture of acetone and water (1 : 1 50 L) was added to the mycelial solid. The resulting mixture was filtered and the acidic filtrates were combined.

The combined filtrates were extracted with heptane (10 L). The phases were separated and the aqueous phase added to a second portion of heptane (10 L). The pH of the resulting mixture was adjusted to pH 10 with 5N NaOH. The resulting emulsion was diluted with 50 L of water. The phases were separated and the aqueous phase extracted with a third portion of heptane (10 L). The phases were separated and the second and third heptane extracts were combined and concentrated to a volume of about 4 liters. Upon standing, the concentrate separated into 3 phases: aqueous, emulsion, and organic. The organic phase was lyophilized to give 15.29 g of crude product.

The crude product was dissolved in methanol (500 mL), filtered, and concentrated to dryness in vacuo. The residue was dissolved in a second portion of methanol (20 ml) and applied to a column of LH-20 SEPHADEX (Pharmacia LKB Biotechnology, Inc., Piscataway, New Jersey, 7.5 cm x 46cm), eluting with methanol and collecting 25 ml fractions. Using the HPLC system described above, the fractions were analyzed to determine which fractions contained compounds of interest. Fractions 18-50 were combined and concentrated to dryness.

The residue was dissolved in a mixture of ethanol, acetonitrile, and water (5:5: 1 ) and chromatographed in 1 ml portions on a preparative reverse-phase HPLC column (Rainin DYNAMAX-60A, C18, 41.4 mm x 300 mm, 8 mm particles, 6θA pore, Woburn, MA). The column was eluted with a mixture of methanol, acetonitrile and water (87.5:87.5:25) with ammonium acetate added to a final concentration of 0 1 percent (pH 7 6) The fractions were analyzed using an HPLC system, similar to that as described above, combining like fractions and concentrating to give three semi-pure concentrates A, B, and C

Semi-pure concentrate C was rechromatographed on the system described in the preceding paragraph, loading 200 mL on each of 10 runs The fractions from each of the runs were combined and concentrated to give preparations C1 and C2. Preparation C2 was chromatographed a third time; however, water was used in place of the 0 1 percent ammonium acetate (desalting step) Fractions containing A83543L in at least 99 5 percent HPLC purity were combined and concentrated The residue was crystallized from ethanol/water (1.1) to give 2 4 g of A83543L

Preparation C1 and semi-pure concentrate B were combined and desalted as described in the preceding paragraph (12 x 200 mL runs); however, the desired compound was eluted with a mixture of methanol, acetonitrile, and water (1 1 1 1 3) The fractions containing A83543J in at least 99 5 percent HPLC purity were combined and concentrated The residue was dissolved in hot t-butanol and lyophilized to gιve 4 3 g of A83543J

Semi-pure concentrate A was chromatographed as described above, except the desired compounds were eluted with a mixture of methanol, acetonitrile, and water (37 5 37 5 25), with ammonium acetate added to final concentration of 0.1 percent The fractions from each of the runs (4) were combined and concentrated to give preparations A1, A2, and A3

Preparation A1 was chromatographed using the column described above; however, the column was eluted with a mixture of methanol, acetonitrile, and water (2 2: 1) Fractions containing A83543M in at least 99 5 percent HPLC purity were combined and concentrated The residue was dissolved in t-butanol and lyophilized to give 136 mg of A83543M

Preparation A2 was chromatographed and processed as described in the preceding paragraph

Claims

Claims

1. A method for isolating macrolide compounds from a fermentation broth in which they are produced which comprises: a) providing a fermentation broth which comprises a culture medium capable of meeting the growth requirements of an organism forming a biomass and producing at least one factor (an A83543 component) having the following formula:

wherein R¹ is hydrogen or a group of formula

(a) (b)

and R², R³, R⁴, R5, R6 and R⁷ are independently hydrogen or methyl; or an acid addition salt thereof when R¹ is other than hydrogen; b) adding an approximately equal volume of a water miscible, polar organic solvent to the fermentation broth, including the biomass thereof, under conditions and for a time sufficient to extract the A83543 component from the biomass; c) separating the liquid phase of the resulting mixture from the biomass; d) adjusting the pH of the separated liquid phase to between 7 and 13; e) applying the separated liquid phase directly to a column of nonfunctional, macroreticular polymer; f) eluting the A83543 components from the column with a water miscible, polar organic solvent; and g) collecting the fractions containing the A83543 components.

2. The method of Claim 1 , wherein between steps d and e) the loaded column is washed with a 30 percent to 70 percent aqueous solution of water miscible, polar organic solvent to displace the broth from the medium.

3. The method of Claim 2 wherein said aqueous solution used to displace the broth from the column isa mixture of methanol, acetonitrile, and water wherein the components are present in a ratio of 1 : 1 :2, respectively.

4. The method of Claim 1 wherein the water miscible, polar organic solvent used in step d) is a mixture of up to 95 percent methanol and acetonitrile wherein the components are present in a ratio of 1 : 1 and the balance of an aqueous buffer containing 0.1 percent ammonium acetate.

5. The method of Claim 1 , wherein the organism is selected from the group consisting of A83543.1 (NRRL No. 18395); A83543.3 (NRRL No. 18537); A83543.4 (NRRL No. 18538); A83543.5 (NRRL No. 18539); A83543.6 (NRRL No. 18719); A83543.7 (NRRL No. 18720), A83543.8 (NRRL No. 18743), A83543.9 (NRRL No. 18823) or mutants thereof.

6. The method of Claim 1 , wherein the A83543 component is selected from the group consisting of A83543A, A83543B, A83543C, A83543D, A83543E, A83543F, A83543G, A83543H, A83543J, A83543K, A83543L, A83543M, A83543N, A835430, A83543P, A83543Q, A83543R, A83543S, A83543T, A83453U, A84543V and A84543W.

7. The method of Claim 1 , wherein the culture medium contains a carbon source selected from the group consisting of glucose, methyl oleate, ribose, xylose, fructose, galactose, mannose, mannitol, maltose, soluble starch, potato dextrin and oils.

8. The method of Claim 1 , wherein the culture medium contains a nitrogen source selected from the group consisting of cottonseed flour, peptonized milk, corn steep liquor, fish meal, digested soybean meal, yeast extract, enzyme-hydrolyzed casein and beef extract.

9. The method of Claim 1 , wherein the culture medium contains a nutrient inorganic salt selected from the group consisting of zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate and nitrate.

10. The method of Claim 1 , wherein the fermentation medium contains antifoaming agents.

1 1. The method of Claim 1 , wherein the liquid phase separating is separated from the resulting mixture by passing the mixture through a ceramic filter.

12. The method of Claim 1 , wherein the pH is adjusted between 7 and 10.

13. The method of Claim 1, wherein the pH is adjusted using sodium hydroxide.

14. The method of Claim 1, wherein the nonfunctional, macroreticular polymer is HP-20ss resin.

15. The method of Claim 1, wherein the isolated fractions are concentrated.

16. The method of Claim 1, wherein the concentrated fractions are lyophilized.

17. The method of Claim 1 wherein the eluate is 95 percent ofthe organic solvent containing methanol and acetonitrile wherein the components are present in a ratio of 1 : 1 and 5 percent buffer containing 0.1 percent ammonium acetate.

18. The method of Claim 1, wherein the factor is an A83543 compound of Formula 2:

wherein R8 is H or a group selected from

and R9, R¹θ, RI I , Ri2 _and R¹³ are hydrogen or methyl, or an acid addition salt thereof when R1 is other than hydrogen.

19. The method of Claim 1 , wherein the factor is an A83543 compound of Formula 3:

10 wherein R¹⁴ is hydrogen or a group of formula

(a) (b)

-ϋ and R^{1 5}, R¹⁶, R^{1 7}, R¹⁸, and R¹⁹ are independently hydrogen or methyl, or an acid addition salt thereof when R¹ is other than hydrogen.

30

35