DESCRIPTIONS OF ANTIBIOTICS 



321 



l)i<)c-iii) and Na novobiocin are soUil)ilit\' in water 

 and biological activity against Diplococcus, Pro- 

 teus, Mycobacterium., and B. subtilis. By mutual 

 agreement, the three groups who independently 

 isolated this antibiotic under the names strepto- 

 nivicin, cathomycin, and cardelmycin, have 

 changed the generic name to novobiocin. 



Method of extraction: I. Filtered broth evap- 

 orated to dryness. Residue dissolved in water and 

 adjusted to pH 2.0. Precipitate which forms is 

 dried, then triturated witli acetone and filtered. 

 Acetone solution evaporated to dryness in vacuo 

 and residue triturated with methanol. Methanol 

 filtered and evaporated in rucuo. Methanol- 

 insoluble residue triturated witli jjetroleum ether, 

 then dissolved in dilute NaOH. Addition of HCl 

 gives a precipitate. Dried precipitate extracted 

 with ether, and ether evaporated off. Crystallized 

 from aqueous acetone or ethanol (2). II. Whole 

 broth adjusted to pH 8.0 and filtered. Filtrate 

 extracted with amjd acetate at pH 6.0. Back- 

 extracted into water at pH 10.0. Re-extracted into 

 amyl acetate at pH 6.0. Crystallized from con- 

 centrated amyl acetate solution on atldition of 

 acetone and petroleum ether. Recrystallized from 

 acetone, acetone-water, and acetone-hexane (30). 

 III. Broth-filtrate extracted with methyl iso- 

 l)utyl ketone at pH 2.0. Extract washed with Na- 

 HCO:i , and extracted with ammonium hydroxide. 

 Re-extracted from aqueous solution into ether at 

 pH 2,0. IV. A super saturated solution of a crvule 

 salt of novoljiocin in methanol or ethanol is ob- 

 tained by concentration of the solvent solution, 

 and a novobiocin salt-adduct with the solvent 

 precipitates on cooling or seeding. Drying in the 

 adduct in air gives purified sodium (or potassium) 

 novol)iocin. 



Chemical and physical properties: Acidic sul)- 

 stance. Pale yellow to white crystals existing in 

 two polymorphic modifications: I. m.p. 152-156°C 

 (decomposition) (most common); and II. m.p. 

 170-172°C (decomposition) (2), 172-174°C (22), or 

 174-178°C (decomposition) (3). Soluble in water 

 at pH >7.5; insoluble at lower pH. Acidic form 

 soluble in acetone, ethyl and amyl acetates, 

 methaiuil, ethanol, and pyridine. Ultraviolet ab- 

 sorption spectrum maxima at 334 ni/z {a = 40.7) in 

 0.01 A' ethanolic (70 per cent H0SO4 (2); at 326 mju 

 (.E'l'cm 356 to 367) and a shoulder at 290 ni/z in 0.1 .V 

 HCl and methanol (90 per cent); at 238 m^ (^'I'c'm 

 371 to 374) and 304 m^ (^IL 334) with shoulders at 

 250 and 290 m/i in 1 per cent phosphate buffer pK 

 6.0; at 248 m/. ia = 36.2) and 308 m^ (o = 32.8) in 

 0.01 A' ethanolic (70 per cent) phosphate buffer 

 (pH 7.5); at 311 m^ (a = 53.2) in 0.01 A' ethanolic 



(70 per cent) KOH; or at 307 m/x (Elfn, 574 to 580) 

 with a shoulder at 230 m/x in 0.1 A' NaOH (2-4). 

 Infrared spectrum given in references 12 and 22, 

 and differs for the two forms, [alf = —27° (c = 1 

 per cent in NaOH) and [a]f = -44° (c = 1 per cent 

 in pyridine), both forms having the same optical 

 activity (3). Mean refractive index, 1.595 ± 0.003 

 (4). pK,/ = 4.3 and 9.1 in water (12). Behavior in 

 various s^ystems on paper chromatography given 

 in reference 11. Crystallographic data given in 

 reference 12. Relatively stable; loses 23 per cent 

 of its activity in saline solution after 2 weeks at 

 26°C (5). Does not form salts with ammonia and 

 ordinary amines (12). CnHaeNoOu: C = 60.62%; 

 H = 5.91%; N = 4.54% (14). Structural formula 

 of novobiocin (15, 16) given in Chapter 6. Hydro- 

 genation yields "dihydronovobiocin," m.p. 163- 

 165°C, with potentiometric titration and optical 

 activity values similar to novobiocin. Ultraviolet 

 absorption spectrvun maximum at 328 m^ (in 0.01 

 A' H-iSOi in 70 per cent ethanol) (3). Acid degrada- 

 tion under various conditions yields: (a) Cyclo- 

 novobiocic acid: pale yellow crystals; m.p. 288- 

 290°C (decomposition); pK^' = 6.3; optically in- 

 active; C.-jH.iNOe (3, 25, 33). (b) Novobiocic acid: 

 m.p. 217-232°C; the aglycon of novobiocin (33). 

 (c) The methyl-glycoside of 3-O-carbamylnoviose: 

 m.p. 191-192°C; CioHisNOe (15, 25, 32); (d) The 

 ethylglycoside of the same sugar: CnHaiNOe ; m.p. 

 173-1 75°C (25). Originally noviose was termed 

 novobiose, but this term was dropped, inasmuch 

 as this implied a disaccharide (25). In dilute alkali, 

 novolnocin isomerizes to isonovobiocin in an 

 equilibrium reaction, as shown in Chapter 6. 

 Since isonovobiocin is biologically inactive, the 

 position of the carbamate group appears to govern 

 the antibiotic activity of novobiocin (26). An 

 novobiocin: Glistening, snow-white platelets. 

 Melts slowly above 220°C (decomposition) (12) or 

 m.p. 210-215°C (decomposition) (25). Very soluble 

 in water. Ultraviolet absorption spectrum like 

 that of novobiocin. Infrared spectrum given in 

 reference 12. Ca novobiocin: Nearly white, micro- 

 cr3'stalline powder. Lo.ses birefringence at >240°C. 

 but does not melt up to 325 °C (25). Less soluble in 

 water than the sodium salt (28). Sodium methanol 

 adduct: (See IV under "Methods of Extraction"). 

 Needles; m.p. 225° ± 5°C (decomposition) with 

 loss of birefringence at 10°C before melting. Less 

 soluble in methanol than sodium novobiocin. 

 Sodium ethanol adduct: Strongly birefringent ; 

 m.p. 217-222°C (decomposition). 



Bioluijical activity: Active on gram-positive 

 bacteria and certain gram-negative bacteria such 

 as Hemophilus and Pasteurella. Active at 25 to 50 



