Patent GB-1020967-A (GB)
Title: Improvements in or relating to the biochemical production of ribosides or ribotides
IPC: C12P19/30
Filed: 1964-10-15
Abstract: 5 - amino - 4 - imidazole carboxamide riboside and 5 amino-4-imidazolecarboxamide riboside 51 phosphoric acid are prepared by cultivating under submerged aerobic conditions, in an aqueous nutrient fermentation medium a nutritionally deficient species Arthrobacter albidus ATCC 15243 or a mutant strain thereof, said medium containing a source of available nitrogen, a source of carbohydrate and a growth factor for said specifies, and maintaining the pH of the culture medium within the range of from about 5.5 to 8.5.  The micro-organism may be maintained on nutrient agar slants or on other suitable media, particularly a slant medium containing 30 g/l. of Bacto-peptone, 3 g/l. of Bacto-meat extract, 5 g/l. of Bacto-yeast extract, 5 g/l. of glucose, 20 g/l. of agar and tap water, whose make up pH is 6.9.  The inoculated slants are incubated overnight at 28 DEG  C. and stored under refrigeration, and the cell growth may then be transferred from such slants to suitable nutrient media for growth of the organism in flasks.  The fermentation process is generally effected under submerged aerobic conditions at a temperature from about 25 DEG  to 34 DEG  C. for a period of from 30 to 96 hours after the broth has been incubated with the organism. The initial concentration of the growth factor in the fermentation should be within the range of about 50 to 200 mg/l., suitable growth factors being biotin and (a) hypoxanthine or (b) mixtures comprising from about 70% adenine to about 30% guanine or xanthine or from about 30% adenine to about 70% guanine or xanthine.  The 5-amino-4-imidazole-carboxadride riboside and ribotide may be recovered from the fermentation medium by precipitating all amino compounds with mercuric acetate at pH 7 to 7.5, decomposing the mercuric salts with hydrogen sulphide and extracting the aqueous solution of amino compounds with alkaline peroxide-free ether. The mercuric salt precipitation and decomposition is repeated, the remaining aqueous solution evaporated to near dryness in vacuo, the residue is extracted with ethanol, taken up in water and treated with ethanolic picric acid to give the picrate.  The free riboside is isolated by ether extraction of an aqueous sulphuric acid suspension of the picrate followed by removal of the sulphate with barium hydroxide and barium carbonate, and evaporation of the filtrate to near dryness.  The products may also be recovered by adsorbing all amino compounds on a solid absorbent, filtering and eluting the filler cake with a mixture of ethanol, concentrated ammonium hydroxide and water.  The eluate is concentrated in vacuo to an oil, water is added and the pH is adjusted to 10-11 by ammonium hydroxide. The solution is passed through a "Dowex" (Registered Trade Mark) 1 formate column, the riboside is washed through with water and obtained as an oil by freeze-drying. The oil is taken up in hydrochloric acid, adsorbed on Dowex-50, ammonium form, and eluted with ammonium hydroxide. The eluate is taken to dryness and the residue recrystallized from water.  The ribotide is obtained by eluting the Dowex 1 column with ammonium formate at pH 4.18.  Also produced in this process are small amounts of 5-amino-4-imidazole-N-succino - carboxamide riboside and 5 - amino-4-imidazolecarboxylic acid riboside. Instead of the Arthrobacter albidus, mutants produced from the said strain by irradiation with X-rays or ultra-violet, or by treatment with nitrogen mustards or single-cell culture techniques may be used.