Patent GB-744540-A (GB)
Title: Amino acid compounds and methods for producing the same
Inventor: EHRLICH JOHN
IPC: C07D251/30
Filed: 1953-03-20
Abstract: The invention comprises O-diazoacetyl-l- and -dl-serine and their metal salts, and the preparation thereof by (a) reacting a mono-acid addition salt of O-glycyl-l- or -dl-serine with a diazotizing agent, followed, if desired, by reaction of the product with a hydroxide, carbonate, bicarbonate, oxide, alkoxide or amide of an alkali or alkaline-earth metal, or (b) in the case of O-diazoacetyl-l-serine, inoculating a suitable sterile aqueous nutrient medium, having a pH of 5.0-8.5 (preferably 6.5-7.0) and containing carbon and nitrogen sources and inorganic salts, with Streptomyces fragilis, incubating the mixture under aerobic conditions at about 20-35 DEG  C. (preferably 23-29 DEG  C), removing solid material present in the culture mixture, and isolating the product from the aqueous liquid. Process (a) is advantageously effected below about 30 DEG  C. in an aqueous reaction medium of pH 3-6 (preferably 4.0-5.5), suitable diazotizing agents being nitrous acid (which may be formed in situ from an inorganic nitrite and a mineral acid), alkyl nitrites, and nitrosyl compounds.  The starting materials are obtainable by the processes of Specifications 744,584, 744,585, 744,586, 744,587 and 744,588. The product of (b) may be isolated by concentrating the culture liquid to a small volume, adding 3-10 volumes of a water-miscible organic solvent (e.g. acetone) and separating insoluble impurities, or evaporating the culture liquid to dryness, and extracting the residue with a water-miscible organic solvent containing less than 50 per cent of water, and in either case passing the resulting solution through an adsorption column containing alumina of pH 5-8 or solid sodium aluminium silicate, discarding the percolate, eluting with water or an aqueous water-miscible organic solvent mixture containing more than 50 per cent of water, removing the solvents from the eluate, dissolving the residue in a small amount of water, passing the solution through an adsorption column containing activated carbon, discarding the percolate, eluting with water containing 1-25 per cent of a water-miscible organic solvent (e.g. acetone), concentrating the eluate to a small volume and precipitating the O-diazoacetyl-l-serine by the addition of a water-miscible organic solvent (e.g. ethanol).  The products are useful for therapeutic purposes.  In examples: (1) and (2) O-glycyl-l- and -dl-serine monohydrochlorides are treated with sodium nitrite in aqueous solution in the cold; (3) O-glycyl-dl-serine monohydrochloride is treated with silver nitrite in aqueous solution at room temperature; (4) the sodium nitrite in (2) is replaced by nitrous acid; (5) O-glycyl-l-serine monohydrochloride is treated with amyl nitrite in aqueous ethanol in the cold; (6) O-glycyl-dl- or -l-serine monosulphate (produced in situ from the disulphate and caustic soda) is treated as in (1); (7) the amyl nitrite in (5) is replaced by butyl nitrite; (8) nitrogen trioxide fumes are bubbled through an aqueous solution of O-glycyl-l-serine monohydrochloride; the products of the foregoing examples may be treated with sodium bicarbonate, potassium hydroxide or calcium hydroxide to produce the corresponding metal salts; (9) an aqueous medium containing glucose, soybean expeller oil meal, acid hydrolysed casein, debittered yeast and sodium chloride is adjusted to pH 7.5 with caustic soda, a trace of calcium carbonate is added and the medium is sterilized (after which the pH is 6.85) and fermented with S. fragilis at 27 DEG  C., the resulting culture being used to ferment a further medium containing the same ingredients plus ammonium nitrate; (10) a similar medium, with the addition of lard as antifoam, is subjected to a single stage fermentation with S. fragilis; (11) in the process of (9) the first medium contains a mixture of lard and mineral oil as antifoam, whilst the ammonium nitrate is omitted from the second; (12) an aqueous medium containing glucose, saline extracted hog stomach, ammonium chloride, sodium hydroxide (to pH 7.5) and calcium carbonate is sterilized (after which the pH is 6.8-7.2) and fermented with S. fragilis; (13) an aqueous medium containing glucose, soybean expeller oil meal, saline extracted hog stomach, sodium chloride, ammonium chloride and calcium carbonate is fermented as in (11); the products of (9)-(11) and (13) are worked up by the first of the methods outlined above.