US3579426A - Microbiological demethylation of 7-halo-7-deoxylincomycins - Google Patents

Microbiological demethylation of 7-halo-7-deoxylincomycins Download PDF

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US3579426A
US3579426A US769411A US3579426DA US3579426A US 3579426 A US3579426 A US 3579426A US 769411 A US769411 A US 769411A US 3579426D A US3579426D A US 3579426DA US 3579426 A US3579426 A US 3579426A
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nrrl
streptomyces
chloro
deoxy
growth
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US769411A
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Alexander D Argoudelis
John H Coats
Donald J Mason
Oldrich K Sebek
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Pharmacia and Upjohn Co
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Upjohn Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/14—Acyclic radicals, not substituted by cyclic structures attached to a sulfur, selenium or tellurium atom of a saccharide radical
    • C07H15/16—Lincomycin; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces
    • Y10S435/906—Streptomyces venezuelae

Definitions

  • This invention provides an effective process for the production of 7-halo-7-deoxy 1 demethyllincomycms, which were heretofore available only by a long and com plicated chemical methods of synthesis.
  • alkyl of from 1 to 8 carbon atoms, inclusive are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl and isomeric forms thereof.
  • the wavy line (I) appearing at the 4'- position of the pyrrolidine ring is used to indicate that the group R can .be in position cis (below the plane of the ring or trans (above the plane of the ring), with respect to the carbonyl group and the wavy line appearing at the 7-position is used to indicate that both halo epimers are to be included, i.e,, the (S) configuration wherein the halo group is located to the right as shown in the formulae or the (R) configuration wherein the halo group is located to the left.
  • the compounds of Formulas I and H exist either in the protonated or non-protonated forms.
  • the free bases can be converted to stable acid-addition salts in accordance with methods known in the art, for example, by neutralizing the free base with the appropriate acid to below about pH 7.0, and advantageously to about pH 2 to pH 6.
  • Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicyclic, 5-phenylsalicyclic, 3-methylglutaric, orthosulfobenzoic, cyclopentanepropionic, 1,2-cyclohexanedicarboxylic, 4 cyclohexanecarboxylic, otadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reineckes dimethyldithiocarbarnic, cyclohexylsulfamic, hexadecylsulfamic, octadecyl
  • the 1'-demethyl compounds produced by the process of the present invention, represented by Formula II, above, and the acid addition salts thereof are known active antibacterial agents having improved gram negative activity over the corresponding compounds of Formula I.
  • 7(S)-chl0r0-7-de0xy-1'-demethyllincomycin and related compounds are active antimalarial agents.
  • the microbiological process of this invention comprises subjecting a 7-halo-7-deoxylincomycin (I) to the activity of one of the following microorganisms:
  • Streptomyces punipalus sp. nov. NRRL 3529; Streptomyces venezuelae, NRRL 3528; Streptomyces venezuelae, NRRL 3527; Aspergillus ornatus, NRRL 2291; Scopulariopsis brevicaulis, ATCC 7903;
  • T richoderma viride NRRL 1762; Streptomyces spectabilis, NRRL 2494; and Streptomyces speclabilis, NRRL 2792 Streptomyces punipalus sp. nov. NRRL 3529.
  • Streptomyces punipalus sp. nov., NRRL 3529 is an actinomycete of the genus Streptomyces which was isolated from a soil sample. The culture was differentiated from known Stretptomyces species. This soil isolate has a distinctive pink color pattern not seen in any validly described, publicly available Streptomyces, sporophores that are readily distinguished from those of other pink sporulating, melanin-positive streptomycetes, and an ability to grow and sporulate well at temperatures of 18-37 C. The distinctive properties of the culture require it to be considered a new species of Streptomyces designated S treplomyces punipalus sp. nov., NRRL 3529.
  • the culture has been named to conform with the International Code of Nomenclature of Bacteria (Intern. J. Bacteriol. 16: 459-490, 1966). This organism has been deposited, and is available without restriction as NRRL 3529, from the Northern Utilization Research and Development Branch, US. Department of Agriculture, Peoria, 111.
  • Inorganic-salts starch (TSP-4) Glycerol-asparagine (I SP-5) 5cb(g ⁇ (no name) 2gc bamboo, chamois 2ec(g) biscuit, ecru, oatmeal, sand"... 3cb(g) sand 2ec(g) biscuit, ecru, oatmeal, sand 2ge(g) covert tan, griege S 3ba(g) pearl, shell tint 2gc(g) bamboo, chamois.
  • TSP-4 Glycerol-asparagine
  • Tyrosine Gray-pink Tan Tan pigment tyrosine solubilized.
  • Yeast extract-malt extract do Tan Pale tan pigment.
  • the bioconversion can be effected by a growing or resting culture of the microorganism or by sporm, Washed cells or enzymes of the microorganism.
  • Culture of the microorganism for the purpose and practice of this invention is in or on a medium favorable to its development.
  • Sources of nitrogen and carbon should be present in the culture medium and an adequate sterile air supply should be maintained during the conversion, for example, by the conventional techniques of exposing a large surface of the medium or by passing air through a. submerged culture.
  • Nitrogen in assimilable form can be provided by sources normally employed in such processes, such as corn steep liquor, cotton seed meal, soybean metal, yeast extracts, peptone, soluble or insoluble vegetable or animal protein, lactalbumin, casein, whey, distillers solubles, amino acids, nitrates and ammonium compounds, such as ammonium tartrate, nitrate, sulfate and the like.
  • sources normally employed in such processes such as corn steep liquor, cotton seed meal, soybean metal, yeast extracts, peptone, soluble or insoluble vegetable or animal protein, lactalbumin, casein, whey, distillers solubles, amino acids, nitrates and ammonium compounds, such as ammonium tartrate, nitrate, sulfate and the like.
  • Available carbon can also be provided by sources normally used in bioconversions such as carbohydrates, e.g., glucose, fructose, sucrose, lactose, maltose, dextrines, starches; meat extracts, peptones, amino acids, proteins, fatty acids, glycerol, Whey and the like. These materials may be used either in a purified state of as concentrates such as whey concentrate, corn steep liquor, grain mashes, cotton seed meal, and the like, or as mixtures of the above. Many of the above sources of carbon can also serve as a source of nitrogen.
  • sources normally used in bioconversions such as carbohydrates, e.g., glucose, fructose, sucrose, lactose, maltose, dextrines, starches; meat extracts, peptones, amino acids, proteins, fatty acids, glycerol, Whey and the like. These materials may be used either in a purified state of as concentrates such as whey concentrate, corn steep liquor, grain
  • the medium can desirably have a pH before inoculation of between about pH 4 to about 8 though a higher or lower pH can be used.
  • a temperature between about 25 to 32 C. is preferred for growth of the microorganism but higher or lower temperatures within a relatively wide range are suitable.
  • the substrate compound (I) can be added to the culture during the growth period of the microorganism as a single feed or by intermittent addition during the conversion period, or it can be added to the medium before or after sterilization or inoculation making appropriate adjustments for effects of pH and/or temperature upon the stability of the substrate used.
  • the preferred, but not limiting, range of concentration of the substrate in the culture medium is about 50500 mg. per liter. In the practice of this invention, it is convenient to add the substrate to the medium in the form of a water soluble acid addition salt.
  • the temperature during the fermentation can be the same as that found suitable for growth of the microorganism. It need be maintained only within such range as supports life, active growth or the enzyme activity of the microorganism. A range of to 35 C. is preferred. A pH of about 6 to 8 is generally preferred for growth of the microorganism during the bioconversion. Aeration can be effected by surface culture or preferably by use of submerged fermentation conditions with air sparging,
  • the time required for demethylation by the enzymatic system of the microorganism employed can vary considerably. The range of about 2 to hours is practical but not limiting; 24-72 hours is generally satisfactory.
  • the progress of the bioconversion and its completion are conveniently determined by paper-strip chromatography, or thin-film chromatography [Heftman, Chromatography (1961), Reinhold Publishing Co., New York, N.Y.].
  • demethylation of the selected substrate can be effected by subjecting it to the activity of enzymes prepared from the microorganism, to the action of spores of the microorganism, and to the action of isolated cells of the microorganism.
  • Isolated enzyme preparations can be prepared in accordance with the general procedure disclosed by Zuidweg et al., Biochim, Biopy. Acta, 58, 131-133 (1962).
  • the bioconversion can be effected with spores in accordance with the general process disclosed in US. Pats. 3,031,379 and 3,031,382.
  • the separation of washed cells from the fermentation medium is well known in the art, see for example, US. Pat. 2,831,789.
  • demethylation means the enzymatic action of a growing or resting culture of the microorganism which effects removal of a methyl group from the molecule of the substrate under fermentation conditions.
  • the resulting product (H) is recovered from the fermentation beer by conventional methods.
  • the whole beer can be extracted with a Water-immiscible organic solvent such as methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichlorethylene, ether, amyl acetate, benzene, and the like, or the beer and mycelium can be separated by conventional methods such as centrifugation or filtration, and then separately extracted with suitable solvents.
  • the mycelium can be extracted with either watermiscible or water-immiscible solvents or in cases where little or no product is contained in the mycelium, it can be merely washed with water and the wash water added to the beer filtrate.
  • the beer, free of mycelium, can then be extracted with water-immiscible solvents such as those listed above.
  • the extracts are combined, dried over a drying agent such as anhydrous sodium sulfate, and the solvent removed by conventional methods such as evaporation or distillation at atmospheric or reduced pressure.
  • the product can be adsorbed from the beer on an adsorbent non-ionic resin or on carbon and the product eluted with a polar organic solvent such as methanol, ethanol, acetone, ethyl acetate, methyl ethyl ketone, aqueous mixtures thereof, and the like.
  • a polar organic solvent such as methanol, ethanol, acetone, ethyl acetate, methyl ethyl ketone, aqueous mixtures thereof, and the like.
  • the 1'-demethyl products (II) obtained by either the extraction or elution procedures can be isolated and further purified by conventional methods, e.g., chromatography and/or crystallization, and the like.
  • the products (II) can be obtained as free bases or as acid addition salts in accordance with procedures hereintofore disclosed.
  • EXAMPLE 1 7 (S) -chlr0-7-deoxy-l '-de methyllincomycin
  • the resulting seed is used to inoculate a medium consisting of 20 g. per liter of black strap molasses, 30 g. per liter of dextrin, 15 g. per liter of fish meal and 15 g. per liter of Pharmamedia.
  • the medium is adjusted to pH 7.2 and 120 shake flasks (500 ml.) are prepared each containing 100 ml. of the medium.
  • the shake flasks are sterilized (pH'after sterilization is 6.2) and are inoculated with 5 ml. of the vegetative seed (prepared above) per 100 ml. of medium.
  • the culture is then allowed to grow for 48 hours on a rotary shaker (250 rpm. stroke 2.5 cm.) at about 28 C.
  • a rotary shaker 250 rpm. stroke 2.5 cm.
  • 5 mg. of 7(8)- chloro-7-deoxylincomycin hydrochloride is added to each shake flask and the fermentation is continued for an additional period of about 24 hours.
  • the contents of the flasks are then pooled (about 10 liters), diatomaceous earth is added and the beer and mycelium are separated by filtration.
  • the filter cake is washed with l l. of water and the wash water is added to the clear beer.
  • the clear beer thus obtained is passed over a column containing Amberlite XAD-2 resin (Rohm and Haas Co., Philadelphia, Penn. 19105); the column is prepared by wet packing with 350 g. of the resin and allowing the resin to setle by gravity.
  • the bed-volume of the column is 500 ml.
  • the clear beer is added at the top of the column and passed through the resin at a flow rate of about 4% of the bed volume.
  • the column is then eluted with 3.5 liters of methylethyl ketone: water (95:5 v./v.). Fractions of ml. each are collected and aliquots spotted on agar trays seeded with Sarcina lutea. Those fractions which inhibit the growth of S.
  • lurea are combined and the upper and lower phases are separated.
  • the upper phase is concentrated to dryness and labeled preparation A
  • the lower aqueous phase is adjusted to pH 10 with 20 g. of sodium carbonate per 100 ml. of the lower phase.
  • the alkaline solution thus obtained is then extracted with methylene chloride and the methylene chloride solution is evaporated to dryness and labeled preparation B.
  • Thin layer chromatography [silica gel G, methylethyl ketone: acetone: water (l40:40:22 v./v.)] shows the presence of 7(S) chloro 7 deoxylincomycin and 7(8) chloro- 7-deoXy-l'-demethyllincomycin in both preparations A and B.
  • Preparations A and B (6 g.) are combined, dissolved in about 10 ml. of chloroformzmethanol (6:1 v./v), mixed with 24 g. of silica gel (Merck-Darmstadt No. 7734), and evaporated to dryness. The dry material thus obtained is then added at the top of a wet packed (chloroform-methanol 6:1 v./v.) column containing 600 g. of the same silica gel. Additional silica gel is added at the top of the load bed and the column is eluted with about 12.8 I. of chloroform-methanol (6:1 v./v.). Fractions of 20 ml.
  • the samples are then checked for bioconversion using ascending paper chromatography. Aliquots of 2 to 5 ,ul. of the filtered beer from each of the bioconversions is applied on filter paper (Whatman No. 3 HR, 18 x 13 mm.). The bioconversion products are separated from the starting material by ascending chromatography using a mixture of ethyl acetate; acetone; water (8:5 :1 v./v.) as solvent system.
  • EXAMPLE 4 7 (S) -chloro-7-de oxy-l -d emethyllincomycin
  • R is methyl or ethyl
  • R is alkyl of from 2 to 8 carbon atoms, inclusive
  • X is chlorine or bromine
  • R, R and X have the meanings given above, or an acid addition salt thereof, to the demethylating activity of Streptomyces punipalus, NRRL 3529; Streptomyces venezuelae, NRRL 3528; Streptomyces venezuelae, NRRL 3527; Streptomyces spectabilis, NRRL 2494; Streptomyces spectabilis, NRRL 2792; Aspergillus ornatus, NRRL 2291; Scapulariopsis brevicaulis, ATCC 7903; or Trichoderma viride, NRRL 1762.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US769411A 1968-10-21 1968-10-21 Microbiological demethylation of 7-halo-7-deoxylincomycins Expired - Lifetime US3579426A (en)

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US (1) US3579426A (enrdf_load_stackoverflow)
CS (1) CS162693B2 (enrdf_load_stackoverflow)
DE (1) DE1952518A1 (enrdf_load_stackoverflow)
FR (1) FR2021156A1 (enrdf_load_stackoverflow)
GB (1) GB1237824A (enrdf_load_stackoverflow)
NL (1) NL156381B (enrdf_load_stackoverflow)
PL (1) PL80274B1 (enrdf_load_stackoverflow)

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FR2021156A1 (enrdf_load_stackoverflow) 1970-07-17
GB1237824A (en) 1971-06-30
NL156381B (nl) 1978-04-17
NL6915872A (enrdf_load_stackoverflow) 1970-04-23
CS162693B2 (enrdf_load_stackoverflow) 1975-07-15
PL80274B1 (enrdf_load_stackoverflow) 1975-08-30
DE1952518A1 (de) 1970-04-23

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