BE499383A - - Google Patents

Description

       

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  PROCESS FOR PREPARING VITAMIN B 120
This invention relates to the production of vitamin B12 and more especially to the recovery of vitamin B12 in greater yields from raw materials containing substances closely related to vitamin B12 but having markedly less physiological activity (i.e. (say pernicious anti-anemia activity and animal protein factor activity) than vitamin B12. More particularly, the invention relates to novel methods by which such vitamin B12-like substances are converted into vitamin B12.



   According to the invention, the vitamin B12-like material is subjected to a reaction with a substance providing cyanogen ions to convert said material into vitamin B12.



   The literature has disclosed various methods for obtaining vitamin B12 from liver and fermentation broths. For example, it has been shown by Rickes et al. in Science 108, 634-35 (Dec. 3, 1948) that vitamin B12 can be obtained from fermentation broths produced by Streptomyces griseus.

   While fermentation broths provide a convenient source for the commercial production of vitamin B12, such broths have also been found to contain fermentation products, which are referred to herein as vitamin B12-like substances, and which are closely related to vitamin B12 but exhibit only a fraction of the pernicious anti-anemia activity and animal protein factor activity exhibited by vitamin B12 itself. In the production of vitamin B12 of uniform purity, the removal of these vitamin B12-like substances has presented a difficult problem.

   Due to the close relationship between these substances and vitamin B12, the separation of pure crystallized vitamin B12 has only been possible by the use of complicated and expensive procedures.



   We have now discovered that it is possible to transform these heretofore undesirable vitamin B12-like substances into vitamin B12 almost

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 that quantitatively, thereby realizing a significant simplification of the recovery process of pure vitamin B12 and at the same time an increase
 EMI2.1
 considerable yield of vitamin B12 obtained from fermentation broths.



   Considered in some of its broadest aspects, the new process according to the present invention involves the reaction of substances such as vitamin B12 with a substance providing cyanogen ions, which results in the transformation of said substances into vitamin B12. Thus, for example, in treating broths, fermentation broths, or suitable concentrates thereof, containing both vitamin 12 and a vitamin B12-like substance, with a source of cyanogen ions, it is found that the 'greatly increased yields of pure vitamin B12 are obtained. In addition, as previously reported, the isolation of vitamin B12 is greatly facilitated.
 EMI2.2
 



  As a further element of the invention, it has been found that this transformation can be carried out under various conditions using different sources of cyanogen ion. In general, the reaction is carried out by effecting intimate contact between substances such as vitamin B12 and cyanogenic ions, thereby effecting the transformation of said substances into vitamin B12.
 EMI2.3
 



  For example solutions of substances such as vitamin B32 can be treated with compounds providing cyanogen ions (especially metal cyanides, ammonium cyanide or hydrocyanic acid). Suitable solvent media include water, mixtures of water and organic solvent, or organic solvents containing cyanogen ions and in which the vitamin B12-like substances are soluble. For practical purposes, it has been found that it is best to carry out the reaction in an aqueous medium.



   It has also been found that the reaction of particular vitamin B12-like substances which have been prepared with a substance providing cyanogen ions as described above results in each case in a transformation of the vitamin B12-like substance into vitamin B12, which can be recovered in a very pure form.

   Particular substances which have been found to be convertible into vitamin B12 by reaction with a substance providing cyanogen ions include substances which have been identified as vitamin B12a, vitamin B12b, vitamin B12c and vitamin B12d-
 EMI2.4
 Vitamin B12a, which in its pure state exists as red orthorhombic crystals, can be prepared from vitamin B12 by reacting vitamin B12 with hydrogen in the presence of a hydrogenation catalyst, such as platinum oxide, palladium oxide or Raney nickel.



  Vitamin B12a shows a characteristic distribution coefficient of about
 EMI2.5
 ron 1.48 (water conc./solva-nt conc.) in an organic solvent-water system in which the organic solvent is a 2: 5 mixture of o.resol and carbon tetrachloride.



   Vitamin B 12b can be prepared from. crude vitamin B12 concentrates obtained by a fermentation broth of Streptomyces griseus, by countercurrent distribution between a 2: 5 mixture of o.resol and te-
 EMI2.6
 carbon trachloride and water, system in which the "vitamin Blub shows a characteristic distribution coefficient of about 2,., (c. water / c, solvent), while vitamin B12 shows a distribution coefficient of almost zero.



   Vitamin B12c can be prepared by reacting vitamin B12 with an excess of ionizable sulphide such as alkali metal sulphides, ammonium sulphide and hydrogen sulphide, preferably in aqueous solution or in aqueous alcoholic solution. The reaction takes place at room temperature and is completed in a short time, and vitamin B12c can be recovered by evaporating the reaction mixture to dryness, dissolving the residue in water and crystallizing in a solution. water-acetone mixture.

   The crystals can then be purified by countercurrent distribution between a 2: 5 mixture of o.resol and carbon tetrachloride and water, system.
 EMI2.7
 in which vitamin B 12c shows a characteristic distribution coefficient of about 0.10, (tc water / tsp soil is).

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   Vitamin B12d can be prepared by reacting vitamin B12 with sulfur dioxide in aqueous, alcoholic or aqueous-alcoholic solution preferably using a considerable excess of sulfur dioxide and heating the reaction mixture slightly to ensure a maximum conversion of vitamin B12 into vitamin B12d.

   Vitamin B12d can be recovered by evaporating the reaction mixture to dryness, dissolving the residue in water and crystallizing from a water-acetone mixture. The crystals can then be purified by countercurrent distribution between a 2: 5 mixture of o.resol and carbon tetrachloride and water, a system in which vitamin B12d exhibits a characteristic distribution coefficient of d 'about 1.63, (tsp water / tsp solvent).



   Although the study of the reaction shows that a cyanogen ion mole is required. per mole of vitamin B12-like substance, it is generally preferred to use an excess cyanogen to ensure complete conversion. By treating an aqueous solution of substances such as B12 with an excess of metallic cyanide, such as sodium cyanide, or ammonium cyanide formed in alkaline solution, an intermediate complex of purple color is formed. This purple complex turns out to be the same substance that forms when you. add sodium cyanide to an aqueous solution of vitamin B12. By acidifying a solution containing this purple complex, the solution regains the characteristic red color of vitamin B12.

   However, in the absence of an excess of metal cyanide or an alkaline pH, this acidification process is not necessary, since the purple complex does not form. When hydrocyanic acid is used as the source of cyanogen ions, the purple complex does not form, even if a large excess is used.



   The vitamin B12 obtained by this reaction is in all respects identical to the vitamin B12 obtained directly from fermentation broths. This identity was established by comparison of ultraviolet, visible and infra-red adsorption spectra, optical rotations, elementary analyzes, crystalline structure, melting or decomposition points, phases of. solubility, polarographic behavior, and distribution coefficients, as well as by microbiological and clinical activities.



   By applying the method of the present invention, vitamin B12-like substances at different stages of recovery from fermentation broths can be reacted with a substance providing cyanogen ions. So; for example, the fermentation broth may be treated with a small amount of a suitable cyanide, such as potassium or sodium cyanide, or alternatively, concentrates of vitamin B12-like substances alone. or in combination with vitamin B12, can be treated with a substance providing cyanogen ions.



   When a fermentation broth containing both vitamin B12 and B12-like substances is processed according to the present invention, a certain amount of substance providing an excess of cyanogen ions is added to the broth to effect the conversion of the cyanogen ions. substances like B12 into B12.



  Vitamin B12 is then extracted from this broth by adsorption using a suitable absorbent such as fuller's earth, charcoal, etc. This adsorbate can be used as a supplement to B12 to strengthen animal feeds, or as a starting material for the isolation of pure vitamin B12.



   It is preferable in the application of the process of the present invention, however, to employ a concentrate of the vitamin B12-like substances since this reduces the volume of material to be handled and the difficulties and dangers resulting from the preparation are minimized. presence of unreacted cyanide.



  It is also preferable to process a concentrate containing both vitamin B12 and vitamin B12-like substances because, as noted above, one of the practical advantages of the new process is the elimination of the difficult and complicated processes required for the separation of these materials.



   The solution or solid concentrate containing the substances like

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   @iramine B12 is treated with a substance providing an excess of cyanogen ions so as to ensure intimate contact of the reagents and complete reaction. Preferably, the mixture is stirred vigorously and then allowed to stand for a period of time. light breeze i.e. approx. 15 to 11.5 minutes. Any substance providing excess cyanogenicity may be used, such as, for example, liquid or gaseous hydrocyanic acid, a metal or ammonium salt of hydrocyanic acid, or mixtures. of a metallic cyanide and of an acid providing hydrocyanic acid in the nascent state.

   It suffices for the metal cyanide used, in a neutral or alkaline medium, to be capable of supplying cyanogen ions under the reaction conditions used. Alkaline and alkaline earth cyanides meet this condition. For example, it has been found that the cyanides of sodium, potassium, barium and calcium and strontium can be used effectively.



   When a solid concentrate is treated with liquid or gaseous hydrocyanic acid, the ionization of the hydrocyanic acid is sufficient to allow the reaction to proceed in the absence of a solvent, although one can however use a solvent advantageously. The reaction between substances such as vitamin B12 and salts of hydrocyanic acid is preferably carried out in the presence of a solvent which will promote the ionization of the salt, such as, for example, water, alcohols of one to three carbons. , and water-organic solvent mixtures in which the vitamin B12-like substances and the substances providing the cyanogen ions are soluble. In a large scale operation, the most convenient and economical is to conduct the reaction in an aqueous alkaline solution.



   By specifying that an excess of cyanogen ions is used, it is meant by this that an amount greater than that below which the yield of pure vitamin B12 begins to fall is required. It is best to determine this quantity experimentally for each raw starting material, as it will be found that the composition of the different broths and concentrates varies considerably. The necessary amount of cyanogen ions can also be determined approximately by measuring the optical density of a sample of the material to be treated, using light with a wavelength of 5500, one characteristic peaks of adsorption of pure vitamin B12.

   The value obtained, which represents the color due to any vitamin B12 present, plus that due to substances such as vitamin B12, is calculated as the potential for vitamin B12. For each mgr. Of the potential vitamin B12 thus calculated, preferably about 0.5-2 mg of cyanogen ions is used, which turns out to be a considerable excess.



   It will be appreciated that there are usually many unidentified impurities present in vitamin B12-like substances. Since these impurities too can react with the cyanogen ion, it is necessary to provide a sufficient excess of ions to meet these needs and at the same time to ensure complete conversion of the B12-like substances.



   When the reaction is carried out in aqueous solution using a metal cyanide with alkaline pH, the formation of the purple complex mentioned above is a convenient visible indication that excess cyanide has been added. The purple complex which forms is transformed into vitamin B12 by acidification of the reaction mixture to a pH of approx. 5 or less.



  Common acids such as hydrochloric, sulfuric and acetic acid can be used for acidification.



   It should be understood that the amount of cyanogen ions to be used to convert vitamin B12-like substances into vitamin B12 depends in principle on the type of product desired. As the addition of cyanogen ions in an amount less than equivalent on a molecular basis to the amount of vitamin B12-like substances present, produces a partial and proportional transformation of vitamin B12-like substances into vitamin B12; It is evident that amounts of cyanogen ions as low as 10% of the amount required to react with all of the vitamin B12-like substances present will produce a beneficial increase in the vitamin B12 content of the resulting product.

   To achieve a complete transformation of substances

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 B12 genus in vitamin B12, however, it is important to use at least an amount of dyanogen on a molecular basis equivalent to the amount of vitamin B12-like substances present. As previously pointed out, the advantages of the present invention applied to the commercial production of vitamin B12 are twofold, comprising a marked increase in the recovery of vitamin B12 from concentrates containing vitamin B12 in combination with substances such as this. vitamin B12, and also the elimination of difficult stages that would otherwise be essential to achieve the separation of vitamin B12 from the associated vitamin B12-like substances.

   The last of these advantages can obviously only be fully obtained if the amount of cyanogen ions used in the reaction is sufficient to react with all the vitamin B12-like substances present. In true practice, the exact amount of vitamin B12-like substances present in a mixture can only be determined by complicated and time-consuming processes, so the best way to conduct the reaction on a production scale is to use a determined excess of cyanogen ions to convert all vitamin B12-like substances into vitamin B12.



   After completion of the reaction, the unconverted cyanide is removed by evaporation under acidic pH. Evaporation can be carried out at a temperature which can range up to 50-60, and preferably under reduced pressure. Evaporation is carried out until all the excess hydrocyanic acid is substantially removed. When a solid concentrate is reacted with liquid or gaseous hydrocyanic acid, the unconsumed cyanide can be removed by evaporation in vacuo or at atmospheric pressure.



  When a solution is reacted with cyanide, on the other hand, it is sometimes desirable to use a stream of nitrogen or air to accelerate the removal of the unconsumed cyanide.



   After cyanide treatment, acidification (if necessary) and removal of the nor cyanide consumed as hydrocyanic acid, the reaction mixture is then treated to remove pure vitamin B12. Different methods are known for treating mixtures containing vitamin B12 and obtaining pure vitamin B12 and the present invention is not limited to the use of a particular method of recovery.

   One such process which can be practically employed to recover vitamin B12 involves saturating a solution of vitamin B12 with an inorganic salt, (especially ammonium sulfate, sodium chloride, sodium sulfate or sodium sulfate. alumina) and extracting the saturated solution with benzyl alcohol, then drying the extract in benzyl alcohol, for example by heating in vacuum to 75-80 C, then ether is added to anhydrous benzyl alcohol solution to precipitate crude vitamin B12.

   The precipitate is then dissolved in water saturated with benzyl alcohol and containing approx. 2-3% glacial acetic acid. The solution and an approximately equivalent volume of water saturated with benzyl alcohol are placed in suitable containers and subjected to successive extractions with approximately equivalent portions of benzyl alcohol saturated with water.



   These extractions can be carried out continuously or by successive extractions of the two solutions initially mentioned with six to eight portions of benzyliqp.e alcohol saturated with water. The combined benzyl alcohol extracts are then dried and treated with ether to precipitate the purified vitamin B12. This precipitate can be dissolved in water and recrystallized to obtain the vitamin B12 of approx. 95% purity, which is preferred for clinical uses. A further purification can also be carried out by recrystallization from water.



   In case the precipitate obtained after extraction with benzyl alcohol is not sufficiently purified to give a vitamin B12 of 95% purity by crystallization, the precipitate can be subjected to further purification by dissolving in methanol, the adsorption of vitamin B12 from the solution onto an activated alumina column and by growth and leaching of the column with methanol. The rich leach liquor treated with ether gives a new precipitate of purified vitamin B12. The further purification can also be carried out by repeating the procedure.

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   extraction process by benzyl alcohol.

   It should be noted, at. In this connection, the aqueous residues of the extraction with benzyl alcohol, and the fractions of the leaching liquor which reveal a low content of vitamin B12 in the chromatographic purification process, can be again treated with the cyanogen ion as exhibited here and recycled to produce additional amounts of vitamin B12.



   To determine the amount of vitamin B12 present in various intermediate concentrates and to determine the amount of vitamin B12 present before and after treatment with. cyanide, it is necessary to have a means of dosing. It has been found advantageous to employ a modification of the countercurrent extraction with water-benzyl alcohol as the dosing medium. This process is based on a distribution coefficient determined for vitamin B12 of 1.2, for the water-benzyl alcohol system.



   To carry out the assay method, the material having an unknown vitamin B12 content is subjected to an eight-plate countercurrent distribution between equal portions of water and benzyl alcohol. Vitamin B12-like substances and other interfering substances have considerably higher distribution coefficients and are found in the first three plateaus of the distribution. In addition, in some cases, substances with lower distribution coefficients are found which are found in the last three plateaus of the distribution. The distribution curve for pure vitamin B12 shows a peak for the fourth plateau.

   The optical density of the contents of the fourth (or fifth) tray is measured at 5500 and the value obtained represents the color due to vitamin B12. By comparison with the optical density of pure vitamin B12 (E1cm1% = 63), we can calculate the vitamin B12 content of the plateau. The fourth tray contains 29.1% (24.2% in the fifth tray) of the total amount of vitamin B12 present in the material under examination. In this way, we can calculate the total amount of vitamin B12 present. This method of determination generally gives an absolute value of the quantity of vitamin B12 when the material examined contains a high content of vitamin B12, ie greater than 75%, depending on the nature of the impurities.

   For decreasing purity, the method is less reliable for determining absolute values, due to additional interfering impurities; the method then indicates the maximum amount of vitamin B12 present. Raw materials poor in vitamin B12 and containing interference staining may be subjected to countercurrent distribution followed by the fourth (or fifth) plateau LLD test, instead of a color measurement, and the maximum amount of vitamin B12 is then calculated on the basis of 11,000,000 LLD units per mg. vitamin B12. A method for carrying out the above assay method is given below.



   To each of the eight 15 ml tubes of a centrifuge is added 5 ml of water, saturated with benzyl alcohol. In the first tube, the solid matter to be examined is added in an estimated quantity to contain approx. 1-10 mg of -vitamin B12 5 ml of benzyl alcohol saturated with water are then added to the first tube, the tube is shaken, and the phases are separated by centrifugation. The lower phase (benzyl alcohol) is transferred to the second tube, where the operation is repeated., This process is repeated in each successive tube until the benzyl alcohol phase is in equilibrium with the water. of the eighth tube. A second portion of 5 ml of benzyl alcohol saturated with water is then passed through each tube successively, in the same manner; until equilibrium with the water of the seventh tube.

   This process is continued with six more portions of benzyl alcohol, after which the eight tubes contain two phases in equilibrium.



  To the contents of the fourth (or fifth) tube, 10 ml of chloroform are added, to pass the vitamin B12 into the water layer. The optical density of the water layer is then determined at 5500 A in a 1 cm cell. The total amount of vitamin B112 present is then calculated from this value as described above.



   The following examples show different methods for treating vitamin B12-like materials with cyanogen ion to convert them to vitamin B12, while pointing to the increased yields of vitamin B12 obtained.

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 by subjecting mixtures containing both vitamin B12 and vitamin B12-like substances to reaction with the ion. cyanogen.

   It should however be understood that these examples are given by way of example and are not limiting.
Vitamin B12 itself is found to contain a characteristic nitrile group, and the following partial structural formula is proposed for vitamin B12:
CN- +++
Co
X-
000 in which five groups are coordinated with the cobalt atom and represented by (X--), two of these groups being of negative character indicated by - and the other three of neutral character represented by ooo; the sixth group coordinated with cobalt being the CN nitrile group.

   The atoms comprising the five groups represented by (XOOO--) and coordinated with cobalt are also probably linked to each other in a way, however, hitherto unknown.,
Vitamin B12a can be thought of as the hydroxy- analogue of vitamin B12 in which a hydroxyl group replaced the ni- trile group in the vitamin B12 molecule @ Apparently vitamin B12a exists in aqueous solutions as a mixture in equilibrium of the hydroxy- isomer and of the ionic hydrated isomer as shown by the following reaction:
 EMI7.1
   Other analogs of vitamin B12 can be represented in a similar fashion, for example the chlorine (or chloride) analog of vitamin B12.
 EMI7.2
 



   Vitamin B12 analogues show ionic character to varying degrees. Thus, the chloride analog has a pronounced ionic character. In this case, the isomer represented by the formula on the right can be assumed to predominate. Vitamin B12 itself, however, shows little if not no ionic character. It behaves like a neutral molecule according to the usual criteria.



   It should of course be understood that these theoretical explanations of a possible structure of vitamins B12 and B12a are based on our present knowledge of these products and do not exclude the possibility that subsequent experimental data establishes that the supposed structures are in fact. incorrect. Consequently, one does not wish to be bound by these theoretical considerations, however plausible they may appear in the light of current knowledge. These explanations are given primarily to allow a better understanding of the invention.



   Vitamin B12a, which in its pure state is in the form of orthahombic crystals can be prepared from vitamin B12 by reacting vitamin B12 with hydrogen in the presence of a hydrogenation catalyst such as platinum oxide, palladium oxide or Rsney nickel. Vitamin B12a can also be obtained from vitamin B12 and vitamin B12a concentrates derived from a streptomycin fermentation broth by extracting an aqueous solution of such concentrates with benzyl alkrool,

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 to remove substantially all of the vitamin B12, the addition of acetone to the resulting aqueous solution, and the recovery of the vitamin B12a which precipitates.



   From vitamin B12 or vitamin B12a, various other analogs of vitamin B12 can be prepared by reaction with compounds providing the particular anion desired. For example, when a solution of vitamin B12 acidified with hydrochloric acid is hydrogenated, the chlorinated analog of vitamin B12 is obtained; or if a large excess of hydrochloric acid is used, a dichlorinated analogue of vitamin B12 is formed. Likewise, by using different acids instead of hydrochloric acid, other analogues of vitamin B12 are obtained which have the characteristic anion of the acid employed.



  Vitamin B1a can also be converted to a chlorinated analogue of vitamin B12 by contact with reagents containing hydrochloric acid.



   Starting from vitamin B12a, other analogues of vitamin B12 can be obtained by reacting vitamin B12a with an acid or salt having the desired anion. Analogs so prepared include the nitrite, thiocyanate, cyanate, iodite and sulfate analogues of vitamin B12.
The sulfate analog of vitamin B12 can thus be prepared by dissolving vitamin B12 in water or aqueous solutions of alcohols; and adding sulfur dioxide or sulfurous acid to the resulting solution. During the reaction, there is a color change from red to brown, but the red color reappears on exposure to air.

   The sulfate analog can be recovered from such a reaction by concentrating the reaction mixture to dryness, the residue is dissolved in a small amount of water, acetone is added to the aqueous solution and allowed to crystallize. the sulfate analog.



   When vitamin B12 is reacted with an ionizable sulphide such as an alkali sulphide, ammonium sulphide, or hydrogen sulphide, a reaction takes place giving a vitamin B12 analogue which is this product is also referred to as vitamin B12d. This product can also be obtained by evaporating the reaction mixture to dryness, dissolving the residue in water and crystallizing from a water-acetone mixture.



   It has now been found in accordance with the present invention that analogs of vitamin B12 having a characteristic anion other than CN- can be converted into vitamin B12 by reaction with a compound providing the cyanogen ion. In the resulting reaction, the characteristic nitrile group of vitamin B12 is substituted for the characteristic anion of the starting analog, and in each case, the same product is obtained having all the physical properties and chemical characteristics of vitamin B12.



   It should be noted in this connection that the discovery of the convertibility of analogues of vitamin B12 to vitamin B12 by reaction with a substance providing the cyanogen ion was made prior to, and in fact allowed, important data from which the hypotheses derive. presented here regarding the chemical nature and apparent structure of vitamin B12 and its analogues.



   In order to carry out the method of the present invention, it is preferred to treat a solution of the particular analog of vitamin B12 with a compound providing the cyanogen ion such as, for example, a metal cyanide, ammonium cyanide or hydrocyanic acid. A suitable solvent medium comprises the aqueous medium, mixtures of water and organic solvent, or organic solvents containing cyanogen ions in which the particular analog of vitamin B12. is soluble For practical purposes, it is most convenient to carry out the reaction in an aqueous medium.



   It is preferred to use an excess of cyanogen ions to ensure complete reaction. By treating an aqueous solution of a vitamin B12 analogue with an excess of metallic cyanide such as sodium or potassium cyanide, or with an excess of ammonium cyanide, ie. under conditions which are necessarily alkaline, an intermediate complex of purple color is formed.This purple complex turns out to be the same substance as that which is

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 forms when sodium cyanide is added to an aqueous solution of vitamin P12. By acidification of a solution containing this purple complex with a common acid such as hydrochloric, sulfuric or acetic acid, the solution regains the red coloration which is characteristic of vitamin B12.

   However, in the absence of an excess of metal cyanide or an alkaline pH, this acidification process is not necessary since the purple complex does not form. When hydrocyanic acid is used as the source of cyanogen ions, the purple complex does not form even if a considerable excess is used.



   After completion of the reaction, the unconsumed cyanide can be removed by partial evaporation at acidic pH. Evaporation can be carried out at a temperature of up to 50-60 C and preferably under reduced pressure. Evaporation is continued until practically total elimination of the excess cyanide in the form of hydrocyanic acid. To thereby remove excess cyanide from the solution, the operation can be speeded up by passing a stream of nitrogen or air through the solution during evaporation.



   It is also possible to convert analogues of vitamin B12 into vitamin B12 by reaction of a-. anhydrous analogue of vitamin B12 with liquid or gaseous hydrocyanic acid which, even in the absence of water, ionizes sufficiently to sustain the reaction, and after completion of the reaction, the unconsumed cyanide is removed by evaporation under vacuum or at atmospheric pressure
After treatment with cyanide, acidification (if necessary) and removal of unconsumed cyanide, the reaction mixture is then treated to obtain pure vitamin B12.

   Various processes are known for treating mixtures containing vitamin B12 and obtaining pure vitamin B12, and the present invention is not limited to the use of a particular recovery process. As the reaction mixtures resulting from the treatment of a - analogues of vitamin B12 with a substance supplying the cyanogen ion mainly contain vitamin B12 and relatively few organic impurities, vitamin B12 can easily be isolated by equilibration of the reaction mixture (in aqueous solution) with a o.cresol-carbon tetrachloride solution (2: 5 by volume and saturated with water).

   Equilibration causes virtually all of the vitamin B12 to pass into the cresol-carbon tetrachloride layer, and the organic product. of the reaction and organic impurities remain in the water layer. The cresol-carbon tetrachloride solution can, if desired, be purified by extraction with saturated water of the cresol-carbon tetrachloride mixture. The solution in cresol-carbon tetrachloride of vitamin B12 is then diluted with 10 to 12 volumes of carbon tetrachloride and extracted with water, the excess of carbon tetrachloride passing the vitamin B12 into the water layer. Vitamin B12 can then be recovered from the water phase by evaporation to dryness and crystallization from an aqueous acetone solution.



   It is also possible to recover vitamin B12 from reaction mixtures formed in the cyanide treatment by the direct concentration of these reaction mixtures to dryness, dilution with approx. ten volumes of acetone and crystallization of vitamin B12 from the aqueous solution of acetone. The combined process first comprising equilibration with carbon o-cresol-tetrachloride offers the advantage, however, of separating unreacted vitamin B12 analogs from vitamin B12.



   Vitamin B12 obtained by the above process is in all respects identical to vitamin B12 obtained directly from fermentation brothso This identity was established by comparison of the ultraviolet, visible and infrared adsorption spectra. red, optical rotation, elemental analysis, crystal structure, solubility phases and polarographic behavior, and also by microbiological and clinical activities.

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  }?: {P! S] 5 la Ef:.: - 5Je: linear concentration of vitamin B12 concentrate. .



   About 2200 gals. of fermentation broth obtained by the preparation of a filtrate of S.griseus and assaying 4630 units per ml of LLD activity, were acidified to a pH of 2.5 with phosphoric acid, subjected to filtration. preliminary clarifying solution on diatomaceous earth, neutralized at a
 EMI10.2
 µl of 7-8 with sodium hydroxide, then filtered again through diatomaceous earth. The filtrate was then treated with 88 lbs of activated charcoal to adsorb the active factors. After removal by filtration,
 EMI10.3
 the charcoal was stirred with 45 gals. from n. but year 01 for 15 minutes.



  35 gals were added to the mixture. of water and 25 lbs. of filter aid, and the mixture was stirred for 45 minutes.



   The solid was separated by filtration on a basket centrifuge and then washed several times on the centrifuge with a total of approx. 40 gals. water
 EMI10.4
 previously saturated with butanolo. The filtrate and the washing waters were combined and the butanol and water layers separated. The water layer which contained virtually all of the LLD active material was filtered to remove fine carbon particles.



   At 85 gals. of the filtered water layer obtained, 13 gals were added. of benzyl alcohol and 425 lbs. of ammonium sulfate. The mixture was stirred for 15 minutes then left to stand for 1 hour. The water layer was
 EMI10.5
 separated and re-extracted with 8.5 gals. benzyl alcohol. The benzyl alcohol extracts were combined and dried over anhydrous sodium sulfate.



  The volume of the solids was approximately 28 gals. (the increase in volume was due to the presence of butanol).
 EMI10.6
 



  The solution in benzyl alcohol was then chromatographed on 20 kg of activated alumina. When all of the solution had passed through the column, the column was washed with a 1: 2 mixture of methanol and acetone until the effluent became clear as water. The column was then developed with methanol with all the effluent showing the red coloration being collected as the rich portion. 52 liters of rich portion were obtained.



   The rich effluent was concentrated in vacuum below 35 ° C to approx. 2 liters and precipitated by adding one volume of acetone and 4 volumes of ether.



   The precipitate was extracted in portions with methanol until a white residue was obtained. The optical density of the solution in methanol
 EMI10.7
 measured at 5500 i indicated a maximum of 416 mg of vitamin Big and vitamin B12-like substances. An aliquot of the methanol solution was treated with ether to produce the precipitate and the precipitate was countercurrently distributed to eight tubes in the benzyl alcohol-water system. The maximum amount of vitamin B12 present determined by the countercurrent assay method was 187 mg.



  Conversion of vitamin B12-like substances by cyanide.



   The remainder of the solution was divided into two parts and one half was treated with ether to produce the precipitation. The precipitate was dissolved in approx. 100 ml of water. 8 ml were added with stirring. 1% aqueous potassium cyanide solution and the solution was allowed to stand for approx. 15 minutes. The solution was then treated with hydrochloric acid.
 EMI10.8
 up to a fold of approx. 4 '70 grams of ammonium sulfate were added and the mixture was extracted with 20 ml, 10 ml and 10 ml portions of benzyl alcohol. The benzyl alcohol extracts were dried by heating at 75-80 ° C in the wrinkle. the resulting solution then being filtered and treated with ether to produce the precipitation.



   The precipitate was dissolved in 20 ml of water previously saturated with benzyl alcohol and to which 0.5 ml of glacial acetic acid had been added. The solution was placed in a 40 ml centrifuge tube. In a second 40 ml centrifuge tube, 20 ml of water saturated with benzyl alcohol were added. Seven servings of 20 ml. benzyl alcohol saturated with water were

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 EMI11.1
 then passed through both. countercurrent tubes, each portion of benzyl cool being used to extract the first tube: Ne 1 then the tube
 EMI11.2
 N 2. The benzyl alcohol solutions were then combined, dried by heating in vacuo and treated with ether to produce the precipitation.



  (Note that this countercurrent extraction process is in fact equivalent to the countercurrent distribution for the separation of vitamin B12 from
 EMI11.3
 any residue of substances such as vitamin 1312o It is not necessary to keep the last tubes of the countercurrent distribution separate, since at this stage they all contain essentially pure vitamin B12.



   The obtained precipitate was dissolved in 1.1 ml. of water and left to crystallize. The crystals were removed by centrifugation and recrystallized by dissolving in 10 ml of water, adding acetone (approx. 120 ml) until cloudy appearance and leaving the solution to stand. The weight of cris-
 EMI11.4
 rate obtained was 127 mg. (dried at 100 ° C in vacuum) and showed 95% purity in the countercurrent dispensing test. The identity of these crystals with genuine vitamin B12 was proven by comparison of physical and chemical properties. The results are shown in the table below:
Comparison of the product obtained by the cyanide process with the
 EMI11.5
 Vitamin Bi 3. genuine.
 EMI11.6
 



  Yroauit of vitamin B proceeds to cvanure .. 12
 EMI11.7
 
<tb> Coefficient <SEP> of <SEP> distribution
<tb>
<tb> water./alcohol <SEP> benzylic <SEP> 1,2 <SEP> 1,2
<tb>
<tb> Adsorption <SEP> spectrum
<tb>
 
 EMI11.8
 , \ Max. (1) 2780, 3615, 5500 2780, 3610 5500 Infra-red absorption The two materials correspond in detail.



  Refractive indices:
 EMI11.9
 1.618 "O 002 1.616 + 0.002 r 1î65o ¯ + 0.002 1.652: 0.002 1.668 + 0.002 1.664: 0.002 Optical rotation
 EMI11.10
 
<tb> 23
<tb>
 
 EMI11.11
 [0 (] 6563 1 -61 - 9 -59 = 9 Solubility comparison Determination of absolute solubility in aqueous solution as well as mixed solubility in saturated solution of genuine vitamin B12 shows that the two materials are identical.



   The aqueous solution remaining after the modified countercurrent dispensing described above was reprocessed with potassium cyanide in solution.
 EMI11.12
 In aqueous t3, the solution was again acidified and then extracted with a 2: 1 solution of carbon tetrachloride-cresol and the extracts were treated with ether to produce the precipitation. The precipitate was dissolved in a small quantity of methanol and again treated with ether to produce the precipitation. The precipitate was then dissolved in 0.13 ml. of water and left to crystallize. The crystals were recrystallized from a mixture.
 EMI11.13
 ge eeii-acetone. The weight of crystals obtained was f7 mg.

   (dry at 100 ° C. in vacuum) The crystals revealed a purity of 73% pure vitamin B12 by the countercurrent dispensing assay.



   Thus, the equivalent of 153.3 mg was obtained. of pure vitamin B12 from 1100 gays. of fermentation broth using the new process.
 EMI11.14
 



  The second half of the original methanolic solution provides a yield of emr. 50 mg, of vitamin B12 by a treatment essentially analogous to the preceding one, except that the cyanide was removed, which shows that the

 <Desc / Clms Page number 12>

 Cyanide treatment gives a threefold increase in the amount of isolated vitamin B12.



   When repeating the previous process using am-
 EMI12.1
 minium., barium, and calcium instead of potassium cyanide, the results show in each case an approximately three-fold increase in the amount of vitamin B12 recovered due to the cyanide treatment.



  EXAMPLE 2.



   An intermediate concentrate obtained by propagating S.griseus containing vitamin B12 and vitamin B12-like substances was purified by countercurrent distribution between water and benzyl alcohol, using two tubes of water and passing Through each of these - successively a total of seven portions of benzyl alcohol, as described in Example 1. The concentrate had not previously been treated with cyanide. The product purified in the extracts in benzyl alcohol was then subjected to the process to obtain pure vitamin B12.

   The water layers, which contained the vitamin B12-like substances, a small amount of vitamin B12 and unknown impurities were combined and treated with ether, and the amorphous precipitate formed was separated and dried. Working according to the previous methods, this precipitate was normally reworked with other fractions to achieve a small additional production of vitamin B12 after many purification steps and there was still a considerable loss of the active substances present.



   A portion of the amorphous precipitate was treated for comparison and several other portions were treated with cyanide as follows: (a) A portion of the precipitate was dissolved in water and analyzed
 EMI12.2
 spectrographically. Adsorption peaks were observed at 3610 B. and 5200.

   The solution was then subjected to a countercurrent distribution between water and benzyl alcohol as described in the assay method, Optical density measurements on the fifth tube of the distribution to con-
 EMI12.3
 Tre-current reported that a maximum of 51% of the total vitamin B12 and vitamin B12-like substances present was in fact vitamin B12, accounting for 0.112 mg of vitamin per mg. of the original solid, (b) 10.5 mg of the amorphous precipitate was dissolved in 3 ml of water and 0.2 ml of liquid hydrocyanic acid was added. The solution was allowed to stand for a short time, then heated to 50-60 C, to remove excess.
 EMI12.4
 -hydric acid.

   Spectroscopic analyzes of the resulting sol-utiorl showed adsorption peaks at 3610, and 5200, and 5500%. indicating a shift towards the vitamin B12 spectrum. The solution was subjected to a countercurrent distribution between water and benzyl alcohol. The
 EMI12.5
 measurement of the optical density of the solution in the fifth tube JJ1Ontre. that 88.3 L of the total vitamin B12 and vitamin B12-like substances present were vitamin B, representing 0.220 mg of vitamin B12 per mg of starting material, an increase of 96%.



   (c) 9.6 mg. of the amorphous precipitate were dissolved in 2 ml. of methanol and 0.2 ml. anhydrous hydrocyanic acid was added. We let it rest
 EMI12.6
 the solution in an ice bath for 15 minutes then heated at 50-600C until dry. The residue was dissolved in water and spectrographic analyzes showed adsorption peaks at 3610%, 5200L and 5500L, indicating a shift towards the vitamin B12 spectrum.



   The aqueous solution was subjected to countercurrent distribution between water and benzyl alcohol. Measurement of the optical density of the material in the fifth tube showed that 61% of the total vitamin B12 and vitamin B12-like substances present were vitamin B12; representing 0.219mg
 EMI12.7
 of vitamin B12 per mg of starting material, an increase of 95.55.



   Three additional portions of the amorphous precipitate were treated with hydrocyanic acid dissolved in ethanol, benzyl alcohol and cresol, respectively, instead of solution in methanol. In all cases, an increase in the vitamin B12 content was obtained, the increases being of the same order of magnitude as those obtained with the use of methanol.

 <Desc / Clms Page number 13>

 



   (d) 10.8 mg of the amorphous precipitate were thoroughly mixed with 2.2 ml of anhydrous hydrocyanic acid and left to stand until evaporation of the hydrocyanic acid. The residue was dissolved in water and spectrographic analyzes of the resulting solution showed adsorption peaks at 3610 1, 5200 A and 5500 A, indicating a shift towards the vitamin B12 spectrum. The aqueous solution was subjected to countercurrent distribution between water and benzyl alcohol. Measurement of the optical density of the fifth tube showed that 81% of the total vitamin B12 and vitamin B12-like substances was vitamin B12, representing 0.198 mg. of vitamin B12 per mg. of starting material, an increase of 77%.



  EXAMPLE 3.



   3000 gals. of fermentation broth from several cu.-. Vies obtained by the propagation of a S. griseus filtrate were treated by adsorption with activated charcoal, washing with butanol-water, extraction, with benzyl alcohol, chromatography, and precipitation as described in the example. - ple 1. The precipitated solids were extracted with methanol until a white residue was obtained. The optical density of the obtained methanolic solution, when measured at 5500 and compared to the value obtained for pure vitamin B12, indicated that 540 mg of vitamin B12 and vitamin B12-like substances were present.

   Acetone and ether were added to the methanolic solution to produce the precipitation, until a liquor without pink coloration was obtained.



   The precipitate was dissolved in 300 ml of water and adjusted to approx. pH 8 using sodium hydroxide solution. To this solution was added 2.7 mg. of sodium cyanide and the solution was allowed to stand, with little stirring, for 45 minutes. (The solution was purple in color indicating that a determined excess of cyanide had been added. The solution was then acidified to a pH of approx. 3 with hydrochloric acid, and nitrogen was passed through the solution. to remove hydrocyanic acid. 210 gm of ammonium sulfide was added to the solution, and the extraction was carried out with 50 ml., 25 ml., and 10 ml of benzyl alcohol portions.

   The extracts were dried by heating at 75-80 ° C, in vacuum, and the dry extracts were filtered through a bed of sintered glass. Ether was added to the filtrate to produce the precipitation.



   The precipitate was dissolved in 100 ml of water to which 2 ml of glacial acetic acid had been added, and 100 ml. with water saturated with benzyl alcohol were placed in a second tube. A modified countercurrent distribution purification process was carried out by passing seven 100 ml portions. benzyl alcohol saturated with water through each tube successively, as described in Example 1, and the benzyl alcohol extracts were combined after their removal from the second tube. The combined extracts were dried by heating at 75-80 ° C in vacuum. Ether was added to the solids to produce the precipitation.



   The precipitate was dissolved in methanol and chromatographed on activated alumina, developing the column with methanol. The rich effluent was treated with ether to produce the precipitation. The tail portions of the chromatography, containing a 5500 l stain which when calculated as vitamin B12 was the equivalent of approx. 90 mg. of vitamin B12, should be kept for reprocessing. The precipitate was dissolved in 1.9 ml of water and allowed to crystallize. The red crystals were dissolved in 20 ml of water, and the solution was filtered. An additional 20 ml of water was used to wash the apparatus and added to the solution. 520 ml of acetone were added and crystallization occurred.

   The crystals were removed by centrifugation, washed with acetone and dried at 56 ° C in vacuum. 324.7 mg were obtained. of red crystals. A sample of the crystals lost 5% weight upon drying at 100 ° C. The dried sample revealed 94% pure vitamin B12 by the countercurrent distribution method, indicating a recovery of 290 mg. pure vitamin B12,
It was found that the same results were obtained substantially when the cyanide treatment was carried out in an aqueous solution of methanol.

 <Desc / Clms Page number 14>

 and ethanol.



  EXAMPLE 4.



   Treatment at. Cyanide for conversion of vitamin B12-like substances was applied to fermentation broth, and conversion evaluation was made using the countercurrent assay method.



  Three liters of fermentation broth obtained by preparing a S.griseus filtrate were treated with 2.1 gm of sodium cyanide dissolved in a small amount of water. The solution was stirred for two hours and brought to pH 4 using concentrated hydrochloric acid. Vacuum was carried out so that air was bubbled through the solution overnight. 2150 gm of ammonium sulfate and 30 ml of benzyl alcohol were added to the solution. The mixture was stirred and allowed to stand and the benzyl alcohol layer removed.



   The aqueous layer was then re-extracted with one 20 ml portion and three 10 ml of benzyl alcohol. The depleted water layer not containing LLD activity was removed. The combined extracts of benzyl alcohol were diluted with 2 volumes of chloro and extracted with three 5 ml portions of water. 10 ml of the aqueous solution containing approx. 300,000 LLD units per ml. were subjected to an eight-plate countercurrent distribution with benzyl alcohol using 10 ml phases. The results of the countercurrent distribution are given in the table below.



   An identical treatment was applied to 3 liters of the same broth except that no sodium cyanide was added. The original aqueous solution obtained by extraction with benzyl alcohol was again subjected to countercurrent distribution of benzyl alcohol. Further results are given in the table.



   Effect of sodium cyanide on the conversion of LLD activity in broth to vitamin B12.



   (Results of the dosing by countercurrent distribution in the benzyl alcohol-water system.



   Percentage of distribution.



  Tube N Untreated broth Pure vitamin B12 treated broth.



   (see figures p.31 English text).



   The above table shows a marked shift in the behavior of LLD activity in countercurrent distribution to the typical behavior of vitamin B12 after treatment of the broth with sodium cyanide. From the above data, it is not possible to calculate exactly the amounts of vitamin B12 contained in the treated and untreated broths. However, it will be seen that the distribution of treated broth is different from that of untreated broth and very similar to the distribution of pure vitamin B12. Furthermore, it is evident that the activity of the processed broth in the fourth tube (tube showing maximum vitamin B12 content) is almost double that of the untreated broth.



  EXAMPLE 5.



   Fermentation broth was obtained by propagating a S. griseus filtrate. 100 liters of the broth were acidified to a pH of 2.5 by the use of hydrochloric acid. The broth was then treated with 220 mg of sodium cyanide, and the bath was stirred for 10 minutes. 100 grams of fuller's earth and 100 gm of diatomaceous earth were added, the resulting slurry was stirred for 3C minutes, and the adsorbate was filtered off and dried at 50 ° C. The adsorbate showed an activity of 843,000 units per. gm, when tested on L. Lactis Dorner by the cup assay and was found to promote the growth of chicks.



    @
Almost all of the active material in the broth is adsorbed by treatment with Fuller's earth under the conditions described. Since the vitamin B12 content of the broth is markedly increased by the cyanide treatment, as shown in Example 4, the vitamin content of the adsorbate

 <Desc / Clms Page number 15>

   thus prepared from treated broth is increased proportionally.
 EMI15.1
 



  LyBIePLE 60 4 mg of vitamin B12 guai showed maxima in the adsorption spectrum at 2750 Ay 3520 Aj, and 5300 A and 42 mg of potassium cyanide were dissolved in 1 ml deal1o The red solution immediately changed to a solution colored purple by the addition of potassium cyanide. The adsorption spectrum of this solution showed maxima at eniv. 2780 k, 3700 1, 5400, Â and 5800 A, which is identical its spectrum given by a solution of vitamin B12 treated with potassium cyanide. After approx. one minute, the
 EMI15.2
 Purple colored solution was brought to pH 4-5 with 3-1. drops of dilute hydrochloric acid, after which the color of the solution turned red.
 EMI15.3
 This solution was equilibrated with 1 ml of a solution of cresol-carbon tetrachloride (2-5 by volume) saturated with water.

   After equilibration; all the coloring except a trace was found in the cresol-carbon tetrachloride. The cresol-carbon tetrachloride solution was then extracted nine times with 1 ml portions of water saturated with carbon o-cresol-tetrachloride which removed a very small amount of colored material. The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed four times with 1 ml portions of ether to remove traces of solvent. The resulting aqueous solution of vitamin B12 was evaporated to dryness in vacuum.
 EMI15.4
 at 25-300C.

   The residue was dissolved in approx.0.25 ml of water and the solution was then diluted with approx. 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution. The solution was allowed to stand for approx. twenty hours and the crystals were removed by centrifugation, washed 3-4 times with portions of l
 EMI15.5
 ml of acetone and dried; yield P 2, lt. mg. The adsorption spectra of these vitamin B12 crystals showed maxima at 2780 A (Eli cm = 112), 3610 A (Etl% :: 193) and 5500 A (E 1 cm = 60). The material was essentially Vl eI1lJ.ne B 12 püea PL 70 IDCAMPLE 7 4 mg of vitamin B12b and 2 mg of potassium cyanide were dissolved in 1 ml of water.

   After approx. 1 minute the purple colored solution was brought to pH 4-5 with 3-4 drops of dilute hydrochloric acid. This solution
 EMI15.6
 was equilibrated with 1 ml of a solution of oocresol-carbon tetrachloride (2-5 by volume) saturated with water. After equilibration, all the coloration except a trace was found in the cresol-carbon tetrachloride layer. This cresol-carbon tetrachloride solution was then extracted nine times with
 EMI15.7
 one-ml portions of water saturated with oocresol-tetrachloI'u, re carbon which removed a very small amount of colored matter.

   The cresol-tetrac: carbon dioxide solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed four times. times with 1 ml portions of ether. The aqueous solution of vitamin B12 was evaporated to sic-
 EMI15.8
 cited in vacuum at? 5-30 C The residue was dissolved in approx. Oe25 ml of water and then diluted with approx. 10 volumes of acetone. After less than half an hour thin red needles of vitamin B12 began to form in the solution.

   The solution was allowed to stand for about 20 hours and the crystals were then removed by centrifugation, washed 3-4 times with 1 ml portions of acetone and dried; yield: 3.3 mg. The adsorption spectrum of
 EMI15.9
 Vitamin B1 2 crystals showed maxima at 2780 A (E! 7 = 115)? 3610% (cm = 188), and 550C (fcm 5) The product was essentially pure 'V'1 IT1112e B.



  Example. 80. 3.4 mga of vitamin B12c and 38 mg of potassium cyanide were dissolved in 1 ml of water o After approx. one minute, the color solution for--
 EMI15.10
 Pre was brought to pH 4-5 with 3-4 drops of dilute hydrochloric acid. This solution was equilibrated with 1 ml of a solution of Olesol-t.etl'ach10.!

 <Desc / Clms Page number 16>

 of carbon (2: 5 by volume) saturated with water. After equilibration, all the color except a trace was found in the cresol-carbon tetrachloride layer. This cresol-carbon tetrachloride solution was then extracted new.
 EMI16.1
 times with 1 ml portions of water saturated with carbon o.cresol-tetrachloxide which removed a very small amount of colored matter.

   This cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed four times with 1 ml portions of ether. The resulting aqueous solution of vitamin B12 was evaporated to dryness in vacuum at 25-30 C. The residue was dissolved in approx. 0.25 ml of water and diluted with approx. 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution. The solution was left to stand for approx. twenty hours, and the crystals were removed by centrifugation, washed 3-4 times with 1 ml portions of acetone and dried: yield: 2.3 mg.

   Spectra
 EMI16.2
 Adsorption of vitamin -B12 crystals showed maxima at 2780 A (E1% = 115), 3610 A (E-L% = 198) and 5500 (E1% = 61). The material was essentially pure vitamin B12.



  EXAMPLE 9.



   2 mg of vitamin B12d and 18 mg of potassium cyanide were dissolved in 1 ml of water. After approx. one minute the purple colored solution was brought to pH 4-5 with 3-4 drops of dilute hydrochloric acid. The solution
 EMI16.3
 was equilibrated with 1 ml of a solution of o.cresol-carbon tetrachloride (2-5 by volume) saturated with water. After equilibration, all the coloration except a trace was found in the cresol-carbon tetrachloride layer. The cresol-carbon tetrachloride solution was then extracted nine times with 1 ml portions of water saturated with o-cresol-carbon tetrachloride which removed a very small amount of colored material.

   The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed four times with portions.
 EMI16.4
 of 1 ml of ether. The aqueous vitamin B12 solution was evaporated to dryness in vacuum at 25-30 C. The residue was dissolved in approx. 0.25 ml of water and then diluted with approx. 10 volumes of acetone. After less than half an hour thin red needles of vitamin B12 began to form in the solution.

   The solution was left to stand for approx. twenty hours and then the crystals were removed by centrifugation, washed 3-4 times with 1 ml portions of acetone and dried; yield: 1.1 mg. Adsorption spectra
 EMI16.5
 Vitamin Bl2 crystals showed maxima at 2790 A (El If. '= 110), 3610A (E 1 cm = 196) and 5500 A (Eµ11, = 60). The product was essentially pure vitamin B12.



    EXAMPLE 10.



   An aqueous solution of hydrocyanic acid was prepared by distilling a solution of 50 mg of sodium cyanide in 20 ml of sulfuric acid.
 EMI16.6
 The distillate was added to a solution of 4.2 mg of Vitanin-B! 2a (originally the material showed adsorption maxima at 2750 A, 3540 A in 3 ml of water. After a few minutes at room temperature the reaction mixture was concentrated to dryness The residue after dissolving in 0.5 ml of water and treating with 5 ml of acetone left 3.4 mg of red needle crystals typical of vitamin B12 which showed adsorption maxima at
 EMI16.7
 2780 1, 3610 À and 5500 Î.



   When we reacted vitamins B12b ,. B12c, B12d with hydrocyanic acid as described in Example 10, in each case a red crystalline product was obtained which revealed the adsorption spectra characteristic of vitamin B12, i.e. maximums at 2780 A, 3610 and 5500.



  EXAMPLE 11.



    4 mg of the monochlorinated analogue of vitamin B12 showing maxima in the adsorption spectrum at approx. 2750 A, 3520 and 5300 A, and 42 mg of

 <Desc / Clms Page number 17>

 Potassium cyanide were dissolved in 1 ml of water. The red solution immediately changed to a purple colored solution by the addition of the po- cyanide.
 EMI17.1
 tassiblll. The absorption spectrum of this solution showed maxima at approx.



  2780 A, 5400 A and 5 $ GO A, which is identical to the spectrum given by a solution of vitamin B12 treated with potassium cyanide. After approx. a half
 EMI17.2
 Then, the purple-colored solution was brought to pH L-5 by 3-li drops of dilute hydrochloric acid after which the color of the solution turned red.



  The solution was equilibrated with 1 ml of carbon o-, creso1-tetrachloride solution (2-5 by volume) saturated with water. After equilibration, all the coloration except a trace was found in the cresol-carbon tetrachloride layer. This cresol-carbon tetrachloride solution was then extracted.
 EMI17.3
 Te nine times with 1 ml portions of water saturated with carbon qyresol tetrachio- ride which removed a very small amount of colored matter.



  The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed four times with 1 ml portions of ether to remove traces of solvent.



  The resulting aqueous solution of vitamin B12 was evaporated to dryness in the
 EMI17.4
 vacuum at 25-300C. The residue was dissolved in 0.25 ml of water and the solution was then diluted with approx. 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution., The solution was allowed to stand for approx. 20 hours and the crystals were removed by centrifugation, washed 3-4 times with portions.
 EMI17.5
 1 ml of acetone and dried; yield: 2.4 mg. The adsorption spectrum of these crystals showed maxima at 2780 A (E'10 = 112)., 3610. (El / ocm = 193) ic? 1 cm 1 cm and 5500 A (E "= 60). This material, essentially pure vitamin B, showed an activity of 10.5 x 10 U./mg for the growth of L. lactiso EXAMPLE 12.



   3.7 mg of vitamin B12a were dissolved in approx. 1 ml of water. To this solution was added approx. 1 ml of an ammonium cyanide solution (prepared by reacting a solution of potassium cyanide with sulfuric acid and collecting the hydrocyanic acid in a dilute solution of hydroxide ammonium). After standing this solution for 12 hours, it was evaporated to dryness in the video. The residue was dissolved in approx. 0.5 ml of water and then diluted with approx. 12 ml of acetone. After the solution had stood for a little while, thin red needles of vitamin B12 began to form. After 18 hours the crystals were separated by centrifugation, washed with acetone and dried. Yield 3.3 mg. The adsorption spectrum of the crystals was that of vitamin B12.



    EXAMPLE 13.



   3.7 mg of vitamin B12b were dissolved in 2 ml of water. To this solution was added 0.5 ml of potassium cyanide solution (13.3 mg potassium cyanide / ml.). The solution turned purple in color and was brought to pH 5-6 with dilute hydrochloric acid. This solution was extracted twice with 1.5 ml portions of a solution of phenol-carbon tetrachloride (1-7 by volume) saturated with water, to remove vitamin B12. . This phenol-carbon tetrachloride solution was extracted 4 times with 1 ml portions of water which had previously been equilibrated with the phenol-carbon tetrachloride solution. The phenol-carbon tetrachloride solution was diluted with about 12 ml of carbon tetrachloride.

   This solution was then extracted 3 times with 1 ml portions of water, thereby causing the extraction of vitamin B12 into the water. The aqueous extract of vitamin B12 was washed with ether to remove the extracted phenol and carbon tetrachloride. The aqueous solution of vitamin B12 was evaporated to dryness in the vacuum. The residue was dissolved in approx. 0.5 ml of water and diluted with approx. 12 ml of acetone. After the solution had stood for a little while, thin red needles of vitamin B12 began to crystallize. Vitamin B12 crystals were separated by centrifugation, washed with acetone and dried.

 <Desc / Clms Page number 18>

 



  Yield 2.3 mg.
 EMI18.1
 



  EXELTLE 1L, ..



  0.3 mg of vitamin B12b, (the substance described by Lichtman et al., In Proc. Soc. 0 Exptl. Biol. & Bleds 72, 64,3-4.5, 1949 and considered to be i- dentic to the hydroxy analogue - vitamin B12 and given here as a vitamin
 EMI18.2
 Bl2a) and 10 mg of potassium cyanide were dissolved in 1 ml of water, and the reaction mixture was acidified, equilibrated with 2-5 o-cresol-carbon tetrachloride, and treated as described in Example 1 to give 0 , 2 mg of crystalline vitamin B12. The adsorption spectra of these crystals showed
 EMI18.3
 principal maxima at 2780 1 (Eµ g "118), 3615-3620 1 (Ello == 192), and 5500 A (Ell% cm == 6.3).



  EXAMPLE 15.



     4 mg of (mono,) chloride analogue of vitamin B12 showing maximum
 EMI18.4
 ai in the adsorption spectrum at 2750 1, 3520 Î and 5200-5300 1, and L2 mg of potassium cyanide were dissolved in 1 ml of water. After approx. one minute the purple colored solution was brought to pH 4-5 with 3-4 drops of dilute hydrochloric acid. This solution was equilibrated with 1 ml of an o-cresol-carbon tetrachloride solution (2-5 by volume) saturated with water. After equilibration, all color except a trace was found in the cresol-carbon tetrachloride layer. The cresol-carbon tetrachloride solution was then extracted 9 times with 1 ml portions of water saturated with o-cresol-carbon tetrachloride which removed a very small amount of colored material.



  The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with three 1 ml portions of water. The aqueous extracts were combined and washed 4 times with 1 ml portions of ether. The aqueous vitamin B12 solution was evaporated to dryness in a vacuum at 25-30 C. The residue was dissolved in approx. 0.25 ml of water and then diluted with approx. 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution. The solution was left to stand for approx. 20 hours and the crystals were then removed by centrifugation, washed 3-4 times with
 EMI18.5
 1 ml portions of acetone and dried; yield 3.3 mg.

   Vitamin B12 crystal adsorption speaters showed maxima at 2780 1 (E1'cm --1% 1% 1 cm 115), 3610 A (E "= 188) and 5500 A (E 1 cm 59) The product was essentially pure vitamin B12.



  EXAMPLE 16.
 EMI18.6
 



  3.1 mg of vitamin B. and 38 mg of potassium cyanide were dissolved in 1 ml of water. After approx. one minute the solution color purple
 EMI18.7
 was brought to pH 4-5 with 3-1 drops of dilute hydrochloric acid. This solution was equilibrated with 1 ml of a solution of o-cresol-carbon tetrachloride (2-5 by volume) saturated with water. After equilibration, all of the color except a trace was found in the cresol-carbon tetrachloride layer. This cresol-carbon tetrachloride solution was then extracted 9
 EMI18.8
 times with 1 ml portions of water saturated with carbon o-cresoir-tetrachloride which removed a very small amount of colored matter.

   The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with 1 ml portions of water. The aqueous extracts were combined and washed four times with portions of water. 1 ml of ether The resulting aqueous solution of vitamin B12 was evaporated to dryness in vacuo at 25-30 C. The residue was dissolved in about 0.25 ml of water and diluted with about 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution. The solution was allowed to stand for about 20 hours, and the crystals were removed by centrifugation, washed 3-4 times with 1 ml portions of acetone and dried; yield 2.3 mg.

   The adsorption spectra of
 EMI18.9
 Vitamin B12 crystals showed maxima at 2780 A (Ei70 = 115), 3610 A (3) "cl == 198) and 5500 A (31 'cl :: 61). This material was essentially

 <Desc / Clms Page number 19>

   the. pure vitamin B12.
 EMI19.1
 EXErdt: 1E 17.



   2 mg of vitamin B12 analog sulfate and 18 mg. of potassium cyanide were dissolved in 1 ml of water. ¯After approx. one minute the purple colored solution was brought to pH 4-5 with 3-4 drops of dilute hydrochloric acid This solution was equilibrated with one ml of o-cresol-carbon tetachloride solution (2-5 by volume) saturated with water. After equilibration all of the color except a trace was in the cresol-carbon tetrachloride layer. This cresol-carbon tetrachloride solution was then extracted nine times with 1 ml portions of water saturated with o-cresol-carbon tetrachloride which removed a very small amount of colored material.



  The cresol-carbon tetrachloride solution was then diluted with 10-12 volumes of carbon tetrachloride and the product was extracted by stirring with 3 1 ml portions of water. The aqueous extracts were combined and washed 4 times with 1 ml portions of ethero The aqueous vitamin B12 solution was removed.
 EMI19.2
 vaporized to dryness in vacuum at 25 = 30 Oa The residue was dissolved in approx.



  0.25 ml of water and then diluted with approx. 10 volumes of acetone. After less than half an hour, thin red needles of vitamin B12 began to form in the solution. The solution was left to stand for approx. 20 hours and then the crystals were removed by centrifugation, washed 3-4 times with 1 ml portions of acetone and dried; yield 1.1 mg. Spectra
 EMI19.3
 Big vitamin crystal adsorption showed peaks at 2780 -. (El% 110) 3610 A (E '0 = 196) and 5500 fla (3)% z.0). The product was essentially 1 cm. element of pure vitamin B12.



    EXAMPLE 18.



   An aqueous solution of hydrocyanic acid was prepared by distilling a solution of 50 mg of sodium cyanide in 20 ml of sulfuric acid.
 EMI19.4
 risk at 5%. The distillate was added to a solution in 3 ml of water of 4.2 mg of a vitamin B12 analog prepared as follows: 400.8 mg of vitamin B12 was dissolved in glacial acetic acid. Reduced platinum oxide was added as a catalyst, and the mixture was stirred.
 EMI19.5
 under light hydrogen pressure for 20 hours at approx. 250C. The catalyst was removed by centrifugation and the solution was evaporated to dryness in vacuum. The residue was dissolved in 30 ml of water and acetone was added to a volume of -300 ml.



   A crystalline precipitate formed on standing and was removed and dried at approx. 25-35 C in vacuum; yield 295 mg. An aqueous solution of the product at its natural pH will show the main adsorption maxima at approx.
 EMI19.6
 



  2750 A, 35ZO, Î and 5300 A. This product, originally considered to be "ritamine B12a, turns out on the basis of present knowledge to be the acetate analog of vitamin B12.



   After a few minutes at room temperature, the mixture will react
 EMI19.7
 tional was concentrated to dryness. The residue after dissolution in 0.5 ml of water and treatment with 5 ml of acetone deposited 3.4 mg of characteristic red needle crystals of vitamin B12 which showed adsorption maxima at 2780
 EMI19.8
 .fil., 3610 to and 5500 A.



    EXAMPLE 19.



   5 mg of the vitamin B12 analog monochloride was dissolved in about 1 ml of water. To this solution; we added approx. 10 mg of yellowish green cuprous cyanide. This solution was left to stand for 12 hours at approx. 25-30 C, then heated to approx. 75 C for half an hour. The adsorption spectrum of this solution showed maxima at 2780 A, 3610 1 and 5400-5500, which is characteristic of a vitamin B12 solution. After separation of a small amount of insolubles by centrifugation, the solution was evaporated to dryness in vacuo. The residue was dissolved in approx. 0.5 ml of water and then diluted with approx. 12 ml of acetone.

   After the solution had stood for a short time, dense red crystals of vitamin B12 began to form.
 EMI19.9
 After 18 hours, the crystals were separated by centrifugation, washed with

 <Desc / Clms Page number 20>

 acetone and dry. Yield 3.1 mg of vitamin B12.



  EXAMPLE 20.



   2.9 mg of the thiocyanate analogue of vitamin B12 was dissolved in 1 ml of water. To this solution was added 0.5 ml of potassium cyanide solution (13.3 mg potassium cyanide / ml).



   By the same process as that described in Example 3, 1.8 mg of thin red needles of vitamin B12 were obtained.



  EXAMPLE 21.



   A mixture of 2.1 mg of the vitamin B12 analogue monochloride and 2.0 mg of the vitamin B12 analogue thiocyanate was dissolved in approx. l ml of water. To this solution was added 0.5 ml of potassium cyanide (13.3 mg of potassium cyanide / ml). By the same process as that described in Example 3, 2.9 mg of crystalline vitamin B12 was obtained.



   The above examples of the conversion of various analogs of vitamin B12 to vitamin B12 by reaction with a substance providing the cyanogen ion appear, when taken together, to indicate that vitamin B12 itself contains an ion (ON) Replaceable which is more firmly attached than any other ion. This ion also seems to easily replace any other ion that characterizes the B12 analogues.

   Now; vitamin B12 is considered to be the preferred compound for therapeutic use, and therefore the method set forth herein for the conversion of vitamin B12 analogs to vitamin B12 is of particular importance as a means for providing vitamin B12 of uniform composition. .



   Even if some particular analogue of vitamin B12, i.e. having a particular anion substituted for the CN group, was to be found to be superior to vitamin B12 in certain clinical applications, the discovered method would still retain real value and importance since the preparation of a particular vitamin B12 analogue can be carried out more effectively (and while removing unwanted analogs) by pre-preparing pure vitamin B12 according to the method set forth herein and then converting pure vitamin B12 to the particular analog desired.



   Various variations and modifications in the foregoing processes will be apparent to those skilled in this field, and to the extent that such changes and modifications fall within the scope of the appended claims, it should be understood that they form an integral part of it. invention.



  CLAIMS.



   1. Process for the preparation of vitamin B12, characterized in that it comprises reacting vitamin B12-like material with a substance providing cyanogen ions to thereby convert said substance into vitamin B12.


    

Claims (1)

2. The method of claim 1, wherein the vitamin B12-like material is a mixture of vitamin B12 and vitamin B12-like materials obtained by propagating a vitamin B12-producing organism in a medium of. suitable culture. 3.Process according to claim 2 wherein the starting mixture is the fermentation broth produced in the propagation of this microorganism. 4. A process according to claims 2 and 3 wherein the starting mixture is a concentrated solution of vitamin B12 and vitamin B12-like materials. 5. Process according to claims 2 and 3, in which the starting mixture is a solid concentrate of vitamin B12 and vitamin B12-like materials: 6. Process according to claims 1 to 5, in which an excess of the substance providing cyanogen ions is used. <Desc / Clms Page number 21> 7. The process of claims 1 to 5 in which an excess of the substance providing the cyanogen ions is used and, after completion of the reaction, the reaction mixture is brought to an acidic pH and the unconsumed cyanide is removed in the form of. gaseous hydrocyanic acid. 8. Process according to claims 1 to 7, in which an acid solution of the reaction mixture is used to extract the vitamin B12 therefrom. 9. A method according to claims 1 to 8 wherein the substance. what providing the cyanogen ions is an ionizable salt of hydrocyanic acid. 10. A process according to claims 1 to 9 wherein the substance providing the cyanogen ions is an alkali metal cyanide. 11. The method of claims 1 to 10 wherein the substance providing the cyanogen ions is sodium cyanide. 12. The method of claims 1 to 9 wherein the substance providing the cyanogen ions is ammonium cyanide. 130 The method of claims 1 to 8 wherein the substance providing the cyanogen ions is hydrocyanic acid. 14. The method of claims 1 to 8 and 13 wherein the substance providing the cyanogen ions is hydrocyanic acid formed in the nascent state by reaction of a metal cyanide and an acid. 15.Process according to claims 1 to 14 wherein the reaction is carried out in the presence of a solvent which will promote the ionization of the substance providing the cyanogen ions, said solvent being water, alcohols to 1. 3 carbon, or mixtures of water and organic solvent, in which the vitamin B12-like materials and the cyanogen ion-providing substance are soluble. 16. The method of claims 1 to 15 wherein the reaction is carried out in an aqueous medium. 17. Process according to claims 1 to 15, in which the reaction is carried out in an alcoholic medium. 18. Process according to claims 1 to 15, in which the reaction is carried out in an aqueous-alcoholic medium. 190 The process of claims 1 to 13 wherein the reaction is carried out under anhydrous conditions using a solid concentrate comprising vitamin B12-like materials and anhydrous hydrocyanic acid. 20. The method of claims 1 to 19 wherein the vitamin B12-like material is vitamin B12a. 21. The method of claims 1 to 19 wherein the vitamin B12-like material is vitamin B12b. 22. The method of claims 1 to 19 wherein the vitamin B12-like material is vitamin B12c. 23. The method of claims 1 to 19 wherein the vitamin B12-like material is vitamin B12d. 24. The method of claim 1, wherein the vitamin B12-like material is reacted with an amount of a cyanogen ion-providing substance which is substantially equivalent on a molecular basis to the amount of vitamin B12-like material. used to suit said vitamin B12 material. The method of claim 24, wherein the vitamin B12-like material is provided in the form of a mixture comprising vitamin B12 and vitamin B12-like materials obtained by propagating a vitamin B12-producing microorganism in a medium. of suitable culture. 26. The method of claim 24, wherein the reaction with a substantially equivalent amount of the ion-providing substance <Desc / Clms Page number 22> cyanogen is ensured by replacing an excess of said substance, and the cyanide not consumed is then eliminated by carrying out an acid reaction mixture and by removing the gaseous hydrocyanic acid. 27. The method of claims 1 to 26, wherein the vitamin B12 in the reaction mixture is adsorbed onto a solid adsorbent. 28. The method of claims 1 to 27, wherein the vitamin B12-like material is a vitamin B12 analogue. 29. The method of claims 1 to 28, wherein the vitamin B12 analog has a characteristic anion other than CN-. 30. "A process according to claims 1 to 29, wherein the reaction is carried out in aqueous medium under alkaline conditions using an excess of substance providing cyanogen ion thereby forming an intermediate cyanide complex of vitamin B12 before a purple color. , and the reaction mixture is then acidified to pH 12 of 4-5 to convert said purple complex to vitamin B12. 31. A process according to claims 1 to 30 comprising acidifying the reaction mixture colored purple to pH 4-5, removing excess cyanide and recovering vitamin B12 from the solution. - sultante by concentration of the solution. dryness and crystallization of the vitamin B12 thus obtained in about 1-10 water-acetone. 32. The method of claims 1 to 30, wherein a. analog of vitamin B12, having a characteristic anion other than ON is reacted with an excess of alkali metal cyanide in aqueous solution, the reaction mixture colored purple is acidified to a pH of about 4-5, removed. the excess cyanide and the vitamin B12 is recovered from the resulting solution by equilibration with about 2-5 o-cresol-carbon tetrachloride, dilution of the organic solvent layer with about 10-12 volumes of carbon tetrachloride , extracting the resulting solution with water, and crystallization of vitamin B12 by adding about 10 volumes of acetone to the extract in water. 33. The method of claims 1 to 19, wherein the vitamin B12-like material is the chlorinated analog of vitamin B12. 34. The method of claims 1 to 19, wherein the vitamin B12-like material is the sulfate analog of vitamin B12. 35. Process substantially as described.