Patent GB-906212-A (GB)
Title: The antibiotic valacidin and its salts and methods for producing same
Inventor: BROMER WILLIAM WALLIS
Filed: 1960-08-04
Abstract: <PICT:0906212/IV (b)/1> A new antibiotic designated Valacidin is produced by cultivating a Valacidin-producing strain of Streptomcyes Lavendulae especially S. Lavendulae NRRL 2765 in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts, preferably under submerged aerobic conditions. Specified sources of carbon are glucose, sucrose, starch, glycerine, molasses, sorbitol, dextrine, brown sugar, mannitol and corn steep solids. Specified sources of nitrogen are linseed meal, tankage, fish meal, corn meal, cotton seed meal, oatmeal, ground wheat, soy bean meal, beef extract, peptones (meat or soy) asparagine, agar casein and amino acid mixtures.  Specified inorganic salts are those providing sodium, potassium, ammonium, calcium, magnesium sulphate, chloride, phosphate or nitrate ions.  Trace elements may be added to the culture medium.  Cultivation at 25-32 DEG C. for 2-5 days at an initial pH of 5.5-8.0 is preferred.  The antibiotic valacidin is isolated from the clarified broth by (a) extraction with a water-immiscible polar organic solvent of which typical examples are ethyl or amyl acetate, chloroform, butyl alcohol, methyl isobutyl ketones, or di-ethylether or (b) adsorption on adsorbants or ion exchange resins e.g. carbon or "Permutit DR" (Registered Trade Mark). The solvent extracts are purified by (i) concentrating in vacuo, extracting the concentrate with a weak aqueous solution of a base e.g. sodium hydroxide and extracting the aqueous extract after acidification with a water-immiscible polar organic solvent, reextracting with aqueous base and acidifying to pH 3 to precipitate valacidin, (ii) precipitation by addition of a non-solvent e.g. petroleum ether or (iii) adsorption chromatography using silicic acid, alumina, "Floril" (Registered Trade Mark) or ion exchange resins as adsorbant and eluting with a water-immiscible polar organic solvent.  Salts of valacidin e.g. the sodium, potassium, calcium, magnesium, tributylamine or diethanolamine salts are formed with organic and inorganic bases. Valacidin and its cationic salts are active against gram positive and gram negative bacteria including certain plant pathogens e.g. X. phaseoli and Alternaria solani. Valacidin is a red brown solid organic acid soluble in aqueous alkaline solutions and polar solvents; slightly soluble in lower alcohols; insoluble in non-polar solvents and aqueous acid solutions; melts with decomposition at about 261 DEG C.; stable in aqueous solution at pH 1.0-9.0; molecular weight 470-510; contains 60.01% carbon, 4.66% hydrogen, 10.80% nitrogen and 24.53% oxygen; empirical formula C26H24O8N4; Pka  in dimethyl formamide; water solution (2 : 1) of 7.0; I.R. absorption maxima in mineral oil mull at 2.90, 2.98, 3.07, 5.74, 5.95, 6.09, 6.18, 6.27, 6.39, 6.46, 6.65, 7.13, 7.46, 7.80, 8.11, 9.10, 9.24, 9.33, 9.63, 9.98, 10.89, 11.61, 12.32, 13.4, 13.9 and 14.2 microns; U.V. absorption maxima at 375mm , 294mm  and 246mm  in methanol with 16,800; 27,100; and 40,400 as the respective extinction coefficients; and Rf values of 0.40 in butanol and water, 0.75 in butanol and water containing 2% p-toluene sulphonic acid monohydrate and 0.00 in methyl-isobutyl ketone and water containing 2% piperidine. Chemical tests show the presence of phenolic, methoxy and carboxy groups and valacidin gives positive Fehlings and permanganate tests for reducing groups, a positive Molisch test and a negative ninhydrin test.  Valacidin is used as a preservative (see Group VI).ALSO:Compositions for preserving organic materials by external application and more particularly fluids used for preserving cadavers and animal specimens contain the antibiotic valacidin or a cationic salt thereof (see Group IV (b)).  In an example, a formaldehyde embalming solution contains 1.0 mg. of valacidin or a cationic salt thereof.  In the form of a dust or spray the compositions containing the antibiotic are applied as plant fungicides.