TH438A - Preparation methods for antibiotic A/16686 Factor A2 and the co-formed antibiotic A/16686 Factor A1 and A3. - Google Patents

Abstract

This invention concerns a chlorine-containing antibiotic, named A/16686 Factor A2 in its pure form. The invention also includes a method for producing A/16686 Factor A2 by culturing Actinoflennes strains, and the combined production of A/16686 Factor A1 and A3. A/16686 Factor A1, A2, and A3, like their corresponding salts, are formed by combination with physiologically acceptable acids. They are non-toxic, effective antimicrobial agents, particularly exhibiting activity against gram-positive bacteria.

Claims (6)

1. A procedure for the production of pure antibiotic A/16686 Factor A2 and its salts by combination with physiologically acceptable, non-toxic acids, based on the following separation criteria: a) It is a white amorphous powder that melts and decomposes at approximately 210-220°C. b) It is highly soluble in water, dimethylformamide, aqueous methanol, and 0.1 N HCl; slightly soluble in absolute ethanol and normal-butanol; and precipitates from aqueous solution by adding saturated NaHCO3 solution. c) Elemental analysis shows approximate values of carbon 54.57%, hydrogen 6.19%, nitrogen 10.88%, and chlorine 1.37%. d) Infrared absorption spectrum in Nujol. The highest absorption spectra were shown as follows: 3290, 2930 and 2860 (Nujol), 1765, 1635, 1510, 1455 and 1375 (Nujol), 1240, 1175, 1130, 1060, 1015, 975, 840 and 815 cm⁻¹. e) Ultraviolet absorption spectrum showed the highest absorption (1) in neutral methanol 234 nM (##chemical formula) 268 nM (##chemical formula) (2) in methanol with 0.1 N HCl 233 nM (##chemical formula) 271 nM (##chemical formula) (3) in methanol with 0.1 N NaOH 251 nM (##chemical formula) 300. Nanometers (curved like a shoulder) F) Specific rotation (##chemical formula) G) The following are Rf values performed with paper chromatography on Watman paper No. 1, using B. subtilis ATCC 6633 as the organism used to investigate the illusion system (volume/volume/volume). Rf values: 1) Normal-butanol saturated with Sorensen buffer, pH 6.0 0.00 2) Normal-butanol saturated with water containing 2% para-toluene sulfonic acid 0.00 3) Normal-butanol The following are the Rf values obtained using a silica gel thin-layer chromatography system: 1) Normal-butanol:methanol:water 4:1:2 0.48 2) Ethyl acetate saturated with water 0.00 3) Normal-butanol:acetic acid:water 2:1:1 0.44 (H) Shown below: Illusion System (Volume/Volume/Volume) Rf Value 1) 2.5% HCOONH4:CH3CN 65:35 in water 0.36 2) Normal-butanol:acetic acid:water 4:2:5 0.80 3) Normal-butanol:acetic acid:water 4:2:5 0.52 4) Normal-propanol:normal-butanol:IN NH4 OH2:3:4 (top phase) 0.12 5) Normal-butanol:acetic acid:water 4:1:5 0.15 1,2- on silenized silica gel 60 F254 3,4,5 on silica gel 60 F254 (standards included: caffeine Rf 0.60, corticone Rf) 0.35, Ketchmethachone 0.30) I) Amino acid analysis after hydrolysis with 6N hydrochloric acid at 110°C for 6 hours, showing at least the following amino acids: alanine, leucine, glycine, aspartic acid, phenylalanine, ornithine, para-hydroxyphenylglycine, and hydroxyl, chloro-substituted phenylglycine. J) Carbohydrate analysis from the acid hydrolysate obtained after 2 hours in 2N H2SO4 at 100°C, showing the presence of demannose. K) The following is the characterization reaction: ninhydrin (3% ethanol solution) + molich + biuret + millon minus 1%FeCl3-1%K3Fe(CN)6 in water, green KMnO4 (acid) plus concentrated H2SO4 (negative L), retention time (tR) 124.43 cm⁻¹ (4' 99/100") when analyzed by reverse phase PLC using a -Bondapat C18 column (3.9 mm diameter x 300 mm) and a mixture of HCOONH4 0.025 M/CH3CN 60/40 (volume/volume) as the mobile phase at a flow rate of 2 ml/min. (Standards included: anthrazine tR 1067.26 cm⁻¹ (35'18/100"), alpha-nitronaphthalene tR 335.64 cm⁻¹ (11'14/100"), polyluene 245.47 cm⁻¹ (8'64/100") and M) in hydrochloride form, melting point approximately 250°C (decomposes). The production process involves culturing the microbial strain Actinoplanes sp. ATCC 33076 under fermentation in oxygenated medium with supplemental nutrients in water containing carbon and nitrogen sources and absorbable inorganic salts until the substance reaches a level exhibiting antibiotic activity. The antibiotic-active compounds, consisting of antibiotic A/16686 factor A2 in complex form with two other minority factors named antibiotic A/16686 factor A1 and A3, were isolated from the fermentation medium. Antibiotic A/16686 factor A2 was separated from the antibiotic complex, and the individual purified antibiotic compounds were then isolated. 2. The process as defined in claim 1 involves extracting the antibiotic-acting compounds from the final stage of fermentation by extracting the fungal hyphae using selected organic solvents: small-molecule alkanols and acetone. 3. The process as per claim 2, in which the solvent used for extraction is methanol. 4. The procedure specified in Requisition 1, which separates antibiotic A/16686 factor A2 by column chromatography, uses an "Amberlite rR xAD-2" column and a mixture of water and acetonitrile or a mixture of aqueous ammonium formate and acetonitrile as an elute. 5. The procedures specified in Claim 1 include the use of specific characteristics to separate all three factors A1, A2, and A3. 5a) The criteria for separating antibiotic A/16686 factor A1, which exists as a free base and a salt formed by combination with a physiologically acceptable and non-toxic acid, is: a) It is a white amorphous powder. a) Melts and decomposes at approximately 210-220 °C. b) Highly soluble in water, dimethylformamide, and aqueous methanol; soluble in methanol and ethanol; insoluble in ethyl ether, petroleum ether, and benzene. c) Elemental analysis shows approximate values of carbon 50.31%, hydrogen 6.00%, nitrogen 9.92%, chlorine (total) 0.61%, chlorine ions 0.9%, and residue 6.07%. d) Infrared absorption spectrum in Noojol shows the following maximum absorption values: 3290, 2930, and 2860 (Noojol); 1765, 1630, 1510, 1455, and 1375 (Noojol); 1260, 1235, 1175, 1150. 1130,1060,1015,975,840 and 810 cm⁻¹ e) Ultraviolet absorption spectrum shows maximum absorption (1) in neutral methanol 233 nM (E1% 1cm=202) 266 nM (E1% 1cm=106) (2) in methanol with 0.1 N HCl 232 nM (E1% 1cm=219) 270 nM (E1% 1cm=110) (3) in methanol with 0.1 N NaOH 251 nM (E1% 1cm=264) ~290 nM (curved to shoulder) f) Specific rotation (chemical formula) (C=0.51,H2O) g) The following are Rf values. Performed with paper chromatography on Watman paper No. 1 using B. subtilis ATCC as the verification system. Illusion system (volume/volume/volume): 1) Normal-butanol saturated with Sorenzen buffer pH 6.0 0.00 2) Normal-butanol saturated with water containing 2% para-toluene sulfonyl acid 0.00 3) Normal-butanol saturated with water containing 2% ammonium hydroxide 0.00 4) Sorenzen, pH 6.0, saturated with normal-butanol 0.05 5) Normal-butanol; methanol:water 4:1:2 0.48 6) Ethyl acetate saturated with water 0.00 7) Normal-butanol; Acetic acid:water 2:1:1 0.44 H) The following are the Rf values made in a silica gel thin-layer chromatography system, shown below. Illusion systems (volume/volume/volume): 1) 2.5% HCOONH4 :CH3CN65:35 in water 0.40 2) Normal-butanol; Acetic acid:water 4:2:5 0.80 3) Normal-butanol; 1) Acetic acid: water 4:2:5 0.52 4) Normal-propanol: normal-butanol 1N NH4OH 2:3:4 (upper phase) 0.12 5) Normal-butanol: acetic acid: water 4:1:5 0.15 1,2 Performed on silenized silica gel 60 F254 plates. 3,4,5 Performed on silica gel 60 F254 plates (standards added: caffeine Rf 0.60, cortisone Rf 0.35; I) Amino acid analysis after hydrolysis with 6N hydrochloric acid at 110 °C for 6 hours, showing the presence of at least the following amino acids: alanine, leucine, glycine, aspartic acid, phenylalanine, orniline, para-hydroxyphenylglycine, and hydroxychloro-substitutite phenylglycine. J) Carbohydrate analysis from... Acid hydrolysate obtained after 2 hours in 2N H2-SO4 at 100 °C, indicating the presence of D-mannose. k) Retensitone (tR) 93.57 cm⁻¹ (3'84"100) when analyzed by reverse phase PLC using a "u-BondaPac RC18" column (3.9 mm diameter x 300 mm) and a mixture of HCOONH4 0.025M/CH3CN 60/40 (volume/volume) as the phase, moved at a flow rate of 2 ml/min. (Standards included: Anthrazine tR 1067.26 cm⁻¹ (35'18/100) (chemical symbol - Nitronaphthalene tR 335.64 cm⁻¹ (11'14'/100), Toluene tR 245.47 cm⁻¹) (8'64/100)) 5b) Criteria for separating antibiotics A/16686 Factor A3, which exists as a free base and a salt formed by combination with an acceptable acid, is used in physiological applications and is non-toxic. The substance is: a) a white amorphous powder that melts and decomposes at approximately 220 °C; b) highly soluble in water, dimethyl formamide, and aqueous methanol; soluble in methanol and ethanol; but insoluble in ethyl ether, petroleum ether, and benzene; c) elemental analysis. The approximate values show carbon 48.41%, hydrogen 5.84%, nitrogen 9.09%, chlorine (total) 1.68%, chlorine ions 1.16%, residue 9.1%. D) Infrared absorption spectrum in nujol shows the highest absorption as follows: 3290, 2930 and 2860 (nujol), 1765, 1635, 1510, 1455 and 1375 (nujol), 1260, 1240, 1175, 1100, 1060, 1020, 975, 840, and 850 cm⁻¹. E) Ultraviolet absorption spectrum shows the highest absorption (1) in neutral methanol at 234 nm (E 1% 1 cm⁻¹ = 147). 267 nm (E 1% 1 cm = 74) (2) in methanol with 0.1 N HCl 232 nm (E1% 1 cm = 132) 270 nm (E1% 1 cm = 76) (3) in methanol with 0.1 N NaOH 270 nm (E1% 1 cm = 202) ~ 295 nm (curved to shoulder) f) Specific rotation (chemical formula) (C=0.48, HCl N/100) g) The following are the Rf values made with paper chromatography on Wotman paper No. 1 using B. subtilis ATCC 6633 as the organism used to investigate the illusion system (Volume/Volume/Volume) 1) Normal - butanol saturated with Sorensen buffer pH 6.0 0.00 2) Normal-butanol saturated with water containing 2% para-toluene sulfonyl acid 0.00 3) Normal-butanol saturated with water containing 2% ammonium hydroxide 0.00 4) Sorrenzaine, pH 6.0, saturated with normal-butanol 0.05 5) Normal-butanol; methanol:water 4:1:2 0.48 6) Ethyl acetate saturated with water 0.00 7) Normal-butanol; Acetic acid:water 2:1:1 0.44 H) The following are the Rf values made in a silica gel thin-layer chromatography system, shown below. Illusion systems (volume/volume/volume): 1) 2.5% HCOONH4 :CH3CN65:35 in water 0.40 2) Normal-butanol; Acetic acid:water 4:2:5 0.80 3) Normal-butanol; 1) Acetic acid: water 4:2:5 0.52 4) Normal-propanol: normal-butanol 1N NH4OH 2:3:4 (upper phase) 0.12 5) Normal-butanol: acetic acid: water 4:1:5 0.15 1,2 Performed on silenized silica gel 60 F254 plates. 3,4,5 Performed on silica gel 60 F254 plates (standards added: caffeine Rf 0.60, cortisone Rf 0.35; I) Amino acid analysis after hydrolysis with 6N hydrochloric acid at 110 °C for 6 hours, showing the presence of at least the following amino acids: alanine, leucine, glycine, aspartic acid, phenylalanine, ornithine, para-hydroxyphenylglycine, and hydroxylchlorosubstituted phenylglycine. J) Carbohydrate analysis from... Acid hydrolysate obtained after 2 hours in 2N H2-SO4 at 100 °C, indicating the presence of D-mannose. k) Retensitone (tR) 93.57 cm⁻¹ (3'84"100) when analyzed by reverse phase PLC using a "u-BondaPac RC18" column (3.9 mm diameter x 300 mm) and a mixture of HCOONH4 0.025M/CH3CN 60/40 (volume/volume) as the phase, moved at a flow rate of 2 ml/min. (Standards included: Anthrazine tR 1067.26 cm⁻¹ (35'18/100) (chemical symbol - Nitronaphthalene tR 335.64 cm⁻¹ (11'14'/100), Toluene tR 245.47 cm⁻¹) (8'64/100) 6. A successful procedure under claim 1 or 5 for the separation of antibiotics A/16686 Factor A1, A2, and A3 by reverse-phase PLC using a u-boncapac R C18" column and a mixture of aqueous ammonium formiate and acetonitrile in variable ratios as the mobile phase (Clause 6, page 7, figure 7).