United Vstates Patent. 10 R 2,971,891 7 -- Patented Feb

United vStates Patent. 10 r 2,971,891 7 -- Patented Feb. 14, 1 2 2,971,891 arabinose cyanohydrin; and dicyanohydrins such as 2,5 PREPARATION OF CYANOCOBALAMINS dihydroxyadiponitrile and 2,3-dihydroxysuccinonitrile. Helmuth Cords, Middlesex County, N.J., and John C. The total amount of copper cyanide or cyanohydrin Megna, Kern County, Dale W. Grant, Santa Clara (above de?ned) which may be incorporated into the County, and Earl M. Butterworth, Kern County, Calif., nutrient medium in the practice of this invention may assignors to Olin Mathieson Chemical Corporation, vary within wide limits which are dependent on the New York, N.Y., a corporation of Virginia toxicity, eifectiveness and solubility of the particular No Drawing. Filed Apr.’ 8, 1959, Ser. No. 804,871 member which is selected for use but it is preferable to 2 Claims. (Cl. 195-114) use at least the stoichiometric amount necessary to con This invention relates to an improved process for the 10 vert all hydroxocobalamin produced to cyanocobalarnin. preparation of cyanocobalamins (e.g. vitamin B12; cyano In general it has been found that the addition of the 5,6-dimethylbenzimidazole cobalamin) and especially to cyano compound is preferably effected so that the total a process for enhancement of the proportion of cyano-v concentration of cyanide ion in the fermentation broth cobalamins in fermentation procedures yielding as prod is within the range of 5 and 210 p.p.m., optimally in ucts a mixture of cyanocobalamins and hydroxocobala periodic incremental amounts of a solution of the com mins (e.g. vitamin B12,; hydroxy-5,6-dibenzimidazole pound. More speci?cally, if the source of cyanide ion cobalamin). used is relatively insoluble and toxic, such as cuprous It is known that physiologically active X—Y‘cobala cyanide, only a small amount preferably should be used mins [using the nomenclature of Bernhauer et al., An so that only a low concentration of cyanide ions (i.e., gew. Chemie, 66, 776 ( 1954)], wherein Y represents 20 5 to 70 p.p.m. and preferably 35 to 50 p.p.m.) will the base present in the nucleotide portion of the cobala be present in the broth, whereas if the compound is min molecule (e.g., 5,6-dimethylbenzimidazole in vita relatively non-toxic, such as glycolonitrile or lactonitrile, min B12) and X represents the hydroxy or cyano radical it is advantageous to adjust the addition so Ithat a rela .can be prepared by culturing a vitamin B12 producing tively high concentration of cyanide ions (Le, 30 ‘to microorganism in a nutrient medium in the presence or 210 p.p.m. and preferably 100 to 180 p.p.m.) will be absence of a precursor (depending on the requirements present in the broth. While under ‘favorable circum of the microorganism and the particular X—-Y-cobala stances there is no advantage in using a combination of min desired) under aerobic or anaerobic conditions (de compounds rather than a single compound, since either pending on the requirements of the microorganism) and will bring about the biosynthesis of about 80 to 90% it is further known that the fermentation will yield a 30 CN-—Y-cobalamins (based on the total amount of X--Y product which contains a ‘mixture of both CN-Y-co cobalamin produced), under certain conditions it has balamins and OH—Y-colabamins. While it is known been found that yields of CN—Y-cobalamins will be that enhanced amounts of CN—Y-cobalamins and less greatly enhanced by the expedient of using a combina er amounts of OH—Y-cobalamins (proportional to the tion of two compounds which will supply cyanide ions total amount of X-Y-cobalamins in the fermentation 35 to the fermentation broth. If more than one source of product) may be obtained by the addition to the fermen cyanide ion is added to the broth (e.g., cuprous cyanide tation medium of a source of cyanide ion (hydrogen _ and glycolonitrile) addition should be adjusted so that cyanide and soluble salts thereof or cyanide complexes) each compound yields the concentration of cyanide ions it was found in actual practice that the addition to the applicable when that compound is the sole source of fermentation medium of these known sources of cyanide 40 cyanide ion (i.e., the addition of a mixture of cuprous ions exerts a toxic effect on vitamin B12 fermentations and glycolonitrile would be adjusted to yield 35 to 50 _ and more importantly, these sources of cyanide ions gave p.p.m. and 100 to 180 p.p.m. of CN-respectively, from relatively low yields (9 to 18%) of CN—Y-cobalamins each of the sources). based on the total amount of X-Y-cobalamins present The manner in which the source of cyanide is added in the fermentation product. 45 to the fermentation broth is somewhat critical, the pre It is an object of this invention to provide an im ferred manner being addition in increments, of equal ' proved fermentation process for obtaining CN—Y~co~ or unequal size, over varying periods during the entire balamins. More speci?cally, it is an object of this in fermentation period. vention to improve the ratio of CN-Y-cobalamins in The source of cyanide ion may be in solid form, or the mixture of cobalamins obtained by culturing a vita 50 dissolved in a preferably aqueous medium. The opti min B12 producing microorganism. mum concentration of the solution will depend on sev The above and other objects are accomplished in ac— eral factors such as the solubility of the solute, the man cordance with this invention by adding to a fermenta ner of addition i.e., whether incremental or single dose tion medium containing a vitamin B12 producing micro addition is employed and the concentration of CN-ions organism at least one member selected from the group 55 which it is desired to maintain. If it is desired to in consisting of copper cyanide and a cyanohydrin having crease the solubility of the cyanide compound, such as the formula cuprous cyanide, in water, preparatory to forming an OH 7 aqueous solution this can readily be accomplished by Z--(R)n—C|)—CN treating the mixture with a base, such as aqueous am 60 monia. l'l The practice of this invention affords a means whereby wherein R is an alkylene linkage having from 1 to 6 a microorganism which produces physiologically active carbon atoms and from 0 to 6 hydroxyl radicals; n is" ' X——~Y'-cobalamins, when cultured under known fermenta a whole number of from 0 to l and Z is a member tion condition in conventional nutrient media, can be selected from the group consisting of hydrogen and 65 induced to yield CN—Y-cobalamins in enhanced quanti ‘ cyanohydroxymethyl. Among the members suitable for ties. More particularly, it has been surprisingly found the practice of this invention there may be named cop that by substituting a cyanide compound of this inven per cyanide (preferably in the cuprous form); mono tion for a usually employed cyanide compound, such as hydric cyanohydrins such as lactonitrile and glycoloni potassium cyanide the ratio of cyanocobalamins to by trile; dihydric cyanohydrins such as 3,3-dimethyl-2,4 70 droxycobalamins in the‘ product mixture can be increased dihydroxybutyroni-trile and 2,3-d hydroxypropionitrile from one wherein the former represents the minor frac (aldo cyanohydrin); polyhydriccyanohydrins such as 1 tion to one wherein it is the major product. a, - ‘3,971,891

- > . 3 4 . ‘microorganisms -.which may be employed in the ‘the vitamins, thiamin, niacin, biotin. folic acid, pyrio gpractice of .this invention include those of the genus doxine, ribo?avin, vand p-aminobenzoic acid. These ions Propionibacteria which are known to produce vitamin and vitamins may be present in the crude materials used B12 in the presence of a vitamin B12 precursor (such as in the nutrient medium. In addition, of course, the "5z;,6-dimethylbenzimidazole) among which are ‘included cyanide compounds of this invention are also added as ,Pr-anabinosum :(A;T.'C.C. 4965), P. thoej'zii ,(A.T.,C.C. hereinbeforedescribed. The fermentation process may A892,), (A.T.C.-C. 4871), »P. zeae (A.T.C.C. 74964), be carried out at temperatures 'from' about 20,“ C. ‘to :P. pentosaceum (A.T.C.C. ‘4875) and P. tpetersonii about 40° ,C.1and may be operated .under aerobic, an 1(A>.T;C.C. 4870); and thosetwhich produce vitamin ‘B12 aerobic or vmicroaerophillic conditions depending on the _,w_itho_ut aprecursor, such as P. freudem'eichii (A.T.C.C. 10 microorganism. :6207). If the microorganismproduces vitamin B12 without the ;The~riutrie'nt media useful in the process of this in presence of a precursor, none 'is added to'the'broth. If --vention include the usual sources of assimilable carbon a precursor (leading torthe formation of Y in CN—-Y .andt-nitrogen. 'As sources of assimilable carbon, there cobalamin), is necessary it may be added to the fermenta .imayt-be used: (1) carbohydratestsuch as glucose, fruc— 15 tion, medium either initially, or continuously or inter 'tose, xylose, galactose, lactose, and maltose; (2) sub mittently during the incubation period. After a su?i ,stances ‘containing carbohydrates such as whey, milk, cient incubation time (about one to ten days) the fermented medium may ,be dried andtincorporatedas .a fcorn‘steep 'liquong'rainmashes, and ~molasses; (3) ‘poly- , supplement into the feed for chickens and pigs, or the *hydric alcohols'such ¥as glycerol and mannitol; (4) fats, cyanocobalamins therein maybe recoveredtin more ,puri r-‘s'uchaslard oil,¥soybean oil, corn oil, butterfat and cotton ?ed form by usual procedures. g‘se‘ed'oihfand (5,) ‘fatty acids such as -acetic,rprropionic, When fermenting with a precursor requiring micro pyruvic, ~stearic,>palmitic, oleic, and linoleic. Sources of organism, the nature of the precursor employed depends available nitrogen include: (1) organic nitrogen com on the physiologically active CN—~Y-cobalamin desired. pounds such as proteinaceous materials e.g. casein, ,llreta, 25 ‘Thus, if vitamin B12 is desired, 5,6-dimethylbenzirnidazo1e ‘soybean meal, ?shmeal, yeast or yeast products, whey or another vitamin B12 precursor, such as 2,3-dimethyl or whey v‘concentrates, amino acids and liver cake; and 5,6-diaminobenzene, 2,3-dinitro-5,6-dimethylbenzene or (2) inorganic compounds such as nitrates or ammonium 2,3-dimethyl-4-amino-5-nitrobenzene, may be used. Thus, compounds. The nutrient media should also include ‘as summarized in the following table, the nature of the metallic cations including _cobalt,>molybdenum, potas 30 resulting cobalamin will depend on the precursor chosen. sium, sodium, magnesium, iron, copper, manganese, the In this table, the symbol Y is that in the formula Y :anions chloride, sulfate, phosphate, and carbonate, and cobalamin.

Product (Ycobalamins) Precursor Name Yis l N CH / \ / \ 0,6-Dunethylbenzrmidazola;- - - - 2,3-Dimethyl-5,6411v- . 5,67D1methylbenzimidazole-i _ CH C[I I aminobenzene; 2,3-Dinitro-5,?-dimethylbenzene; cobalamin. C C—CHa 2,3-DiInethyl-4-amino-5-nitro-benzene. \ / \ % N OH I l N CH / \C/ §CH Benzimidazole; 1,2-Diaminobenzene; 1,2-Dinitro- Benzimidazole-cobalamin____ CH H I benzene; l-Amino-2-nitro benzene. \ C CH / \ % III CH 1 N\ 2%\ 2-N1tro-4-tn?uoromethylaml1ne;_. . .. B-Tn?uoro-I . 5-Tr1?uoromethyl-benzi-_ 1 OH/ oI! C—CF|| methyl-benzimidazole; 4-Tri?uoromethyl- midnzole-cobalamin. C CH phenylene-diamine-l,2. \ / \ % a N OH I Br I | N C / \ / \ _ ,_ C CH 4~Brom0-6-methoxy-benz1m1dazole;methoxyphenylene-ghamine-LZ. B-Bromo-?- 4-Bromo-6-methoxy-benz-imidazole-cobalamin. CH\ /C1] C-OCH:l , \ % N OH I l Qulnazolinea Quinazollne-cobalamin ______r/ N\ _N\/ i _4(3H)-q'uinazo1ine ...... ;--.-._., ...... _. 3.4-Dihydro-4-oxoquinazo- / \ line-cobalamin. -—N l 0 2,971,591

Precursor Product (Y-cobalamlns) Name Yls

2,4-Dichloroquinazoline ______' ______- 2,4-DichloroquinazoIine-co- Cl / l balamin.

C1 NHCOCH: | 1-Acetamido-S-methoxyphenazine ...... -_ 1 - Acetamido - 3 - methoxy phenazine-cobalamln. OCH: @N/\:1? : I

2~Hydroxynh en a i no _ _ 2-Hydroxyphenazine~cobal- /N\ OH amm. N/ l I \ ?e\ 5-Methylbenzimidazole ______-_ S-Methylbenzimidazule-co- OH CH O-CHaI balamin. /C\ /CH ' / N OH I 15 Quinoxaliue ______Quinoxaline-eobalamim.-." // \

\N I N Benzotriazole ______Benzotriazole-cobalamin---

The following examples are presented to further illus 45 tracted with distilled water. The aqueous phase is washed trate the invention. with chloroform and then concentrated to about 0.3 ml. Example I An aliquot of the aqueous phase is taken for potency A medium containing 7.6% beet molasses, 1.5% yeast determination and then 100-200 micrograms of pigment autolysate solids, 2.0% protein hydrolysate,‘1.0% corn are applied to Whatman No. 3 MM paper. T wenty-seven steep liquor, 5 p.p.m. cobalt (cobalt sulfate) and 2.0% 50 cm. of this paper is exposed in 2 N acetic acid buffer CaCO3 is dispensed in 1 liter Erlenmeyer ?asks at the to 280 volts for 16 hours in an electrophoresis migration rate of 500 ml. per ?ask and autoclaved 20 minutes at chamber. Examination of the resulting electrophero 121° C. After cooling, the ?asks are inoculated with an gram shows that the cyanocohalamin represents about active vegetative culture of P. freudenreichii (A.T.C.C. 90% of the total cobalamin pigments. 6207) and incubated at 30° C. on a slow rotary shaker Example 11 for 96 hours. Five ml. aliquots of an aqueous glycoloni trile solution are added to the medium at 0, 19, 24, 43, The example is carried out in the same manner as Ex 48, 66 and 72 hours after inoculation to yield a ?nal total ample I except for a lower level of glycolonitrile addi of 140 p.p.m. CN. [The glycolonitrile solution is pre tion, and a concurrent decrease in cyanide'ion concentra pared by adding.0.312 ml. of a 70% glycolonitrile solu 60 tion to 49 p.p.m. Cyanobalamin biosynthesis relative to tion to 55 mls. sterile water.] The ?asks are adjusted total colabalmin pigments is 73%. Vitamin B12 yield twice daily to pH 7.0-8.0 with sterile sodium hydroxide. is 6.3 mg. per liter. . . . - The progress of the fermentation is followed by periodic Example III tests for residual sugar and contamination by conventional Following the procedure of Example 1, except for plating methods vitamin B12 yield is about 6.4 mg. per 65 an increase in the fermentation time and the use of 5 liter. ’ ml. aliquot portions of cuprous cyanide solution pre At the end of the fermentation, the cells are harvested pared by adding 166 mg. of CuCN to 100 ml. of water by centrifugation. The cell cream is mixed with one and treating with 5% aqua ammonia, in addition to the volume acetone and shaken for 20 minutes at room tem 70% glycolonitrile solution (instead of the latter per se) perature. The acetone extract is separated and evap 70 in amounts necessary to supply 35 p.p.m. CN from orated to approximately 1/10 volume, and washed with cuprous cyanide and 170 p.p.m. CN from glycolonitrile chloroform. The red pigments are extracted into phenol over a fermentation period of seven days, a product re benzene and the rich organic extract is washed with sults, which upon puri?cation and examination as out 0.5% sodium sesquicarbonate and water. One-half vol lined in Example I shows a cobalamin product contain ume of acetone is added and the organic solution is ex 75 ing about 90% vitamin B12 and about 10% vitamin Bm. ‘2,971,891 8 Example IV ,, (A) An experiment ,was conducted exactly as de scribed in Example I except for the addition of 5 ml. ali Following the procedure described in Example '1 ex quots of a solution of acetone cyanohydrin at 0, 24, 31, cept for the addition of 10 ml. aliquots of aesterile-solu 47, 54 and 71 hours after inoculation (in place of the gly tion of cuprous cyanide (CuCN) prepared by dissolving colonitrile solution) _to,.yield acyanide ion concentration 166 milligrams of copper cyanide in 100 ml. water, to of 120 ppm. (the acetone cyanohydrin solution being which was added 5 ml. of aqua ammonia, and sterilizing prepared by adding 0.55 ml. acetone cyanohydrin to 80 the solution for 10 minutes at 121° C. added to the ml. or sterile water). The cells were harvested from the medium at 8, 25, 32, 50, 56 and 75 hours after inocula medium after 75 hours fermentation, and tested by the tion (in place of glycolonitrile) to yield a final total of 10 electrophoresis method, as described in Example I. Cy 60 p.p.m. CN, cyanocobalamin production relative to anocobalarnin content relative to hydroxocobalamin was hydroxocobalamin production is found to be'90zl0. 25:75. Following the procedure of Example I correspondingly (B) An experiment was conducted exactly as outlined high yields of cyano-[5,6-dimethylbenzimidazole]-cobala in A except for the addition of a potassium cyanide solu min, cyano-[benzimidazole]-cobalamin and cyano-[quin tion (prepared by dissolving 625 mg. in 250 ml. of azoline]-cobalamin are readily obtained using as the - .-water and sterilizing the solution for 10 minutes at 121° fermenting microorganism P. petersonii (A.T.C.C. 4870), - C.) at 0, 17,v 24, 41, 48, 65, 71 and 721/2‘ hours after in the presence of, as the precursor, _5,6~,dimethylbenzi inoculation (instead of the acetone cyanohydrin used in midazole, benzimidazole and quinazoline, respectively. experiment A). The cells were harvested after 721/2 Example V 20 hours, extracted and tested by the electrophoresis method .as in experiment A. cyanocobalamin production rela~ 30 liters of a medium containing .(per liter): vglucose: tive to hydroxocobalamin was 25:75. 30 g.; autolyzed yeast: 20 g.; Co(NO3)2.6l-l2O: 0.01 g.; Many other cyanide containing compounds were tested tap water: 1 liter, are placed in a stainless steel fermenta in experiments similar to A and B but these compounds tion unit of 38 liters capacity, heated at 121° for 30 25 resulted in a cyanocobalamin production relative to hy minutes, and cooled to 30°. About 2,000 ml. of a slurry droxocobalamin of less than 15:85. Among the com of CaCO3 (containing 600 g. of CaCOB), sterilized by pounds tested were inorganic compounds such as di autoclaving, is then added together with 1 liter of Pro cyandiamide, calcium cyanamide, zinc cyanide, nickel pionibacterium arabinasum A.T.C.C. 4965 culture grown cyanide, potassium cyanate, cyanamide potassium ferro on the same medium for 72 hours in ?asks kept at 30° ' 30 cyanide and potassium ferricyanide; simple nitriles such C. and shaken on a reciprocating shaker (120-1 inch as propionitrile, allyl nitrile, n- and iso-butyronitrile, strokes per minute). A sterile alcoholic solution of 2 nitro-4-tri?uoromethyl-aniline (sterlized by ?ltration acrylonitrile and acetonitrile; carbonyl-containing-nitriles through a fritted glass ?lter) containing 1 mg.,per ml. such as ethyl cyanoacetate, malononitrile; p-cyano ben~ is then added so that the ?nal concentration of aniline is zoic acid, cyano-acetic acid, a cyanoacetamide and (cy 5 mg. per liter (this supplementation being repeated at 35 anoacetyl) urea; and heterocyclic nitriles such as 2-cy 24-hour intervals) and the culture is allowed to grow in anoimino-4,6-dihydroxy-pyrimidine, Z-cyanopyridine, 3 the medium, under virtually anaerobic conditions while cyanopyridine, 4-cyanopyridine and 2-cyanoimino-6-hy being agitated with a turbine mixer rotating at 87 r.p.m. droxy-4-methylpyrimidine. The compounds were rela Three hundred ml. aliquots of a 70% lactonitrile solution tively ine?ective for the object of this invention. (prepared by adding 18 ml. of lactonitrile to 3.3 liters 40 The invention may be variously otherwise embodied of sterile water) are added at 0, 24, 43 and 66 hours after within the scope of the appended claims. inoculation to yield a ?nal total of 80 ppm. CN. What is claimed is: , After 72 hours incubation at 30°, a sterile solution of l. The process of preparing cyanocobalamins which comprises culturing a vitamin B12 producing micro glucose is added so that the concentration of the glucose 45 after addition is 30 g. per liter. After 5 days of incuba organism of the genus Propionibacteria in a nutrient tion, the pH of the mixture is about 5.3. The fermented medium containing cuprous cyanide as'a source of cya medium is then passed through a Sharples Super Centri nide ions. fuge, and then further puri?ed, concentrated and assayed 2. The process of preparing cyanocobalamins which in accordance with the procedure of Example I. Exami comprises culturing Propionibacrerium freundenreichii in nation of the resulting electropherogram shows a S-tri 50 a nutrient medium containing cuprous cyanide as a source ?uorornethyl-benzimidazolecyano cobalamin production of cyanide ions. relative to the corresponding hydroxo form, of 90:10. References Cited in the ?le of this patent In direct contrast to the results obtained when pro ceeding in accordance with this invention, it has been UNITED STATES PATENTS found that incorporating other sources of cyanide ions 72,842,540 Perlman ______July s, 1958 into the fermentation broth has only slight effect on the 2,872,444 Perlman ______Feb. 3, 1959 yield of cyanocobalamin relative to total cobalamin prod- , FOREIGN PATENTS net. The following experiments were conducted using 731,706 Great Britain ______-_ June 15, 1955 6 sources of cyanide ion outside the scope of this inven— O 505,358 Canada ______Aug. 24, 1954 tion, in fermentation processes which are otherwise iden tical to those outlined in the examples.