DE1417312B2 - Process for the microbiological production of tetracycline - Google Patents

Description

3 4

Phosphorus pentoxide dried and twice with 120 ecm at 217 to 219 ° C with decomposition. The analysis

Methanol. Made into a paste. 14.5 g of undissolved residue can result in the following values:

be discarded. The methanol solutions are n »r ^ i, n» f f ;; r r w r> χι. ιτγί-

combined and very quickly poured in ^{400 cm 3 of water}

stirs. When standing overnight in the cold, tetra- 5: ⁵ J γ \ - '° ^{3 / o} "' ⁵ ^ ^{/ o N} - ·, ^{7 '37} / · ^U

3340 1229 964 781 1678 1175 949 743 1623 1140 864 719 1597 1061 823 692 1315 1036 796 667 1248 1002

cyclin from this aqueous solution and becomes «nyr. ^: f # / H · <? qq ° / n
filtered off and air dried. 8.9 g will be ^ ' ^{Ό /} ° ^C ' ^ * ⁰ / " ^M >. ⁵ ' ^yy Λ ^N
so obtained in 2 approaches. If this crystalline tetracycline hydrochloride is carried out, this tetracycline is treated in acidic aqueous solution in a finely divided suspension in liquid paraffin oil at a pH of 1.5 with decolorizing charcoal, this also shows many characteristic delts, the solution is filtered and the tetracycline Absorption band in the infrared. Among these, by increasing the pH value to 3.5 by adding in particular the following frequencies, the rate of ammonia is precipitated. 6.9 g tetracycline, expressed in reciprocal centimeters, called:
are obtained thereby, are used for cleaning in
34.5 cm ³ with hydrochloric acid to a pH of 1.5
acidified water, the solution is through
Add ammonia adjusted to a pH value of 3.5 and inoculated with tetracycline crystals. Included
slow precipitation of the crystalline tetracycline base in hydrate form takes place, which is filtered off. * °
6.45 g of the base are thus obtained, which are dried in air. The following describes how the fermentation-drying is carried out. The melting point of the dried solutions used to obtain and purify the tetra-water-free base is around 171.5 to 173.5 ° C. Cyclins work up.

(with decomposition). Examination by ultraviolet- A) Tetracycline is isolated from the fermentation solution, Analysis showed a tetracycline content of 890 μg / mg »5 when this was brought to a pH of calculated on 1000 μg / mg tetracycline hydrochloride 2.0 to 2.5 acidified and filtered. The clear one, Tetraoder 964 μg / mg tetracycline hydrate, while the filtrate containing cyclin is then added to Bacillus by alkali microbiological testing, ζ. B. by adding sodium hydroxide or substilis on agar plates from a pH of 8.0 an ammonia adjusted to a pH of 9.0 to 10.0. Tetracycline content of 892 μg / mg was found. 30 A little sodium carbonate can be added towards the end of the alkali through Paper strip chromatography can be added as an add-on. This reacts with the indicate that the product is free of chlortetracycline, any calcium ions present and calcium oxytetracycline drops and other impurities. carbonate, which is also the precipitated Tetra-These crystalline hydrated tetracycline base contains cyclin adsorbed. That with such an alkaline one 19.2% moisture. The dried product gave a pH of 9.0 to 10.0 which has precipitated crude tetracycline the following analysis result: an effectiveness of at least 200 μg / mg

Calculated for C ₂₂ H ₂₄ O ₈ N ^_2: S '' *? ^{1 And} - ^g - ^{as given below} ^ given below

59 45 ° / C- 5 44 ° / H 6 30 ° / N methods.

■ » λ ^ L *, - T" / TT Τ? / ", For example, the cleaning of the raw tetra-

found: 59.60% C; 5.45% H 6.29% N _{40 cyclins} carried out in such a way that it was in aqueous

When this crystalline tetracycline base is dissolved in nitric acid at a pH of 2.0. to Sodium nitrate is added to the hydrate form in liquid paraffin oil while dissolving it. Suspended tetrafine distribution shows the cyclin obtained is extracted therefrom with n-butanol. the Suspension in the infrared many characteristic ab- butanol solution is absorbed by the aqueous layer. Among these, in particular, the 45 is separated and neutralized, whereby tetracycline base precipitates, following frequencies, expressed in reciprocal After a different working method, the raw per-centimeter, called: . duct slurried directly in n-butanol and z. B.

■: '■■■. after acidification with hydrochloric acid and concentration as pure tetracycline hydrochloride from the butanol

50 solution won. ;

According to a third method, the raw product

■ i. '■■ - .. slurried directly in dilute sulfuric acid and filtered. The filtrate is made up by the addition of alkali

The infrared absorption spectrum of these minerals has been adjusted to a pH of 6.0 and

oil suspension in the characteristic range of the undulating tetracycline precipitated.

numbers between 1350 and 650 cm " ¹ is from the on B) Crystalline tetracycline is obtained from fermentation

S. 7 given curve of FIG. 1 can be seen. In her solutions in the following way. The fermentation solution will

the numbers on the lower abscissa represent the wave z. B. with sulfuric acid to a pH of 2.0 to

length in μ, that of the upper abscissa the wavenumbers 2.5 are set and filtered. The filter residue can

in cm- ¹ and the numbers on the ordinate, the ^{permeable 6o are} discarded. The filtrate is poured onto an alkali

ability in percent. B. from 9.0 to 9.5, set and

2 g of the crystalline tetracycline base in hydrate form with a third of its volume of n-butanol

are extracted in 20 cm ^{3 of} n-butanol, which is about 0.5 cm ³ concentrated. The butanol solution is from the aqueous

Contains trated hydrochloric acid, dissolved. The solution is allowed to phase, d. H. the extracted fermentation solution, which is discarded

30 min at room temperature and then 90 min in 65 can be separated, with sulfuric acid z. B. on

stand the cold. Crystalline tetracycline falls with a pH of 5.0 to 6.0 acidified and through

hydrochloride, which is filtered off. 1.5 g hydro vacuum distillation to about Vso of the original

chloride are obtained in this way. The hydrochloride melts by volume of the fermentation solution evaporated. It falls

3490 1222 963 803 1672 1130 948 786 1607 1065 932 741 1524 1040 861 739 1259 990 838 668

5 6

Tetracycline off. It is filtered off. This crude product according to a preferred embodiment of this rice

has an effectiveness of at least 200 μg / mg. The cleaning method impure tetracycline base or theirs

Butanol is returned to the process. Salts, e.g. B. their hydrochloride in aqueous acid

and used for further extractions. a pH of 1.5 or below and then dissolve

100 g of crude tetracycline obtained in this way, 5 sufficient amounts of bases, e.g. B. ammonia

are added to the resulting solution in 1 1 of water, which is mixed with sulfuric acid or sodium hydroxide solution.

. pH of about 1.5 was acidified, dissolved. Start to raise their pH to about 2.5. the

soluble residue is filtered off and can be discarded. Amount of tetracycline, which is subjected to purification

will be. The aqueous solution will be 10 to 50 g, is by no means to a certain amount

Treated activated carbon and filtered. One can now, io restricted. It is generally convenient to 50 to

if desired, about 10 g ethylenediamine tetraacetic 100 mg tetracycline base or salt per cm ^{3 of} water, the

acid or other calcium complex compounds acidified with hydrochloric acid to a pH value of 2.5

Add forming agents (sequestering agents). has been to use. The pH value is against it

The pH of the aqueous solution obtained becomes a critical factor in the successful passage

then z. B. with ammonia to a pH of about 3.5 15 leadership of this type of cleaning. A pH of 2.5

set. Pure tetra crystallizes on standing- gives the best results; but can also with everyone

cyclinbase, which is filtered off. pH between 2.0 and 3.5 can be worked. the

Additional amounts of less pure tetracycline, amount of chlortetracycline used as an impurity in

which can be present in the circuit to the tetracycline to be cleaned for renewed cleaning,

If possible, 9 mg / cm ^{3 of} acidified water will not be converted from the water received last

NEN filtrate by increasing the pH to about and is expediently lower than 5 mg / cm ³ .

6.0 failed. This concentration of the solution in chlortetracycline

As a complex compound-forming agent (Se- can easily be complied with by reducing the amount questing agent) to eliminate the calcium in acidic water, which is used to slurry, as a consequence of this, to facilitate the expansion, it is increased accordingly in each case. It recommends Precipitation of the pure tetracycline can, for. B. Zi- further, z. B. Mixtures of tetracycline hydrotric acid, Tartaric acid, sodium phytate, gluconic acid, chloride and chlortetracycline hydrochloride in with salt phosphates, such as sodium metaphosphate, and generally acidic water of a pH of about Connections of this kind, as they are e.g. B. in »Journal 2.5 for a long time, e.g. B. 16 h long to slurry, Chemical Education «Vol. 25 (1948), pp. 482 to 488 3 ° to a complete solution of the chlorotetracycline and the references given therein. The are used. Many of these agents are suitable concentrations and the necessary extinguishing wax ionized, polybasic acid compounds can be easily determined. The cleaned, or hydroxyl group-containing organic carbide tetracycline base, which remains undissolved, bonic acids. 35 is separated from the solution by filtration.

C) 1 g of crude, moist tetracycline base with about 20%. B. used a crude product that is expediently converted into the crystalline hydro- base of the differential determination method with the help of chloride by adding the base in the ultraviolet absorption to a content of 830 μg minimum amount of dry n-butanol, about 5 cm ³ , Tetracycline and 171 μg chlorotetracycline per mg with the addition of about 0.3 cm ^{3 of} concentrated salt, i.e. an approximately 83% tetracycline, dissolves acid, the solution, if necessary, with decolorizing charcoal or the decolorizing charcoal based on paper chromatography, filtered and further tests on the filtrate contain about 10% chlortetracycline. 10 g of 5 cm ^{3 of} dry n-butanol are added. On standing, this crude product is left in 200 cm ³ for 16 hours, pure tetracycline hydrochloride crystallizes out, water is suspended, which is filtered off by adding salt. 45 acid is adjusted to a pH of about 2.5.

The first filtration can also be omitted and the undissolved solid, purified tetra-

add the total amount of butanol at once. cyclinbase is filtered and dried. Be obtained

The initially dissolved tetracycline then crystallizes the 8 g tetracycline, which according to the differential

spontaneously as tetracycline hydrochloride. Mood method with the help of ultraviolet absorption

D) Tetracycline is contaminated by 5 ° tion 901 μg tetracycline and 39 μg chlortetracycline, to chlortetracycline by slurrying the free ie 96% tetracycline, and after the paper base freed in methanol. Chlortetracycline base does not match a tetracycline chromatographic method in solution, while the dissolved tetracycline base has a hold of 95 to 97%. 87% of the used Tetracyclines obtained in purified form from the methanol solution are recovered in this way.

will, e.g. B. by evaporation of the solvent 55 If you work in a similar way, but under stick or, by pouring the methanol solution into water, fabric, 92% tetracycline base with 0 to 3% Tetracycline can also be effective against chlortetracycline and an effectiveness of 929 μg / mg contaminating chlorotetracycline are recovered.

by putting it in an aqueous acid solution of an E) tetracycline can also be more effective

pH of about 2.5 slurries. Only the 6th way of contaminating amounts of chlorine tetra-

Chlortetracycline dissolved. Hydrochloric acid has been found to be exempt for this cyclin by placing it in an aqueous solution

Purpose proven most useful. Other acids, alkali solution with a pH value of 7.5 to 9.0

z. B. sulfuric acid or phosphoric acid can also be slurried. Only the chlorotetracycline goes in

if used, provided that the required solution.

such pH value is achieved. One prepares z. B. a 65 According to a further embodiment of this

Slurry of the impure tetracycline base or alkaline cleaning method will be the impure

its salt in sufficient aqueous hydrochloric acid so that base or its salts with acids at a pH of 9.5

a pH of 2.5 is achieved. But you can also be dissolved. By adjusting the pH of this

7 8

Solution by adding an acid, e.g. B. of hydrochloric acid, is heated to 50 to 55 ° C for a few minutes and then

cooled to a pH of 7.5 to 9.0 and expediently to. In the process, abundant crystalline precipitation sets in

a pH of about 8.5 then becomes the purified one. The mixture is left at 0 to 5 ° C. for 15 hours

Tetracycline base precipitated. Ammonia is preferred and the crystals are filtered off. The educated

wisely used for dissolution; however, 5 crystalline yellow tetracycline formate is only possible with

also other water-soluble alkalis, e.g. B. sodium n-propanol and then with anhydrous ether

hydroxide or triethylamine are used, pre-wash and air-dry. Yield: 9.8 g

exposed; that they give the desired pH. corresponding to 89% of theory. Melting point: 176.5

After this procedure z. B. Tetracycline base to 177.0 ° C (sinters at 173 ° C).
or their salts with acids, such as the hydrochloride, io

with water obtained by adding alkali on an analysis

pH adjusted from 8.5 to 9.0 to slurried. Calculated for C ₂₂ H ₂₄ N ₂ O ₈ · HCOOH:

The amount of tetracycline that this alka- .56.3% C 5.35% H

can be subjected to chemical cleaning, not found

kind of limited. In general it recommends 15 56 6 ° / C 5 59 ° / H

himself, 50 to 100 mg of impure tetracycline or his ° '

Use acid salt per cm ^{3 of} water. Important- When drying in a vacuum at 60 ° C, nothing occurs

However, maintaining a pH value of water loss is essential.

7.5 to 9.0. The amount of chlortetracycline that can be freed from contaminating amounts of impurities in the tetracycline to be cleaned, chlorotetracycline and oxytetracycline, as tetracycline, should preferably be 12 mg / cm ³ alkali by adding its aqueous solution at a pH of icy water do not exceed and should appropriately be kept around 8.0 until the inconsistent chlorine is less than 2 mg / cm ³ . Such a concentrate tetracycline or oxytetracycline is decomposed.
tration of the solution to chlorotetracycline can easily. B. solid tetracycline base, which is maintained about 26% by, if necessary, containing the chlorotetracycline, dissolved in a dilute aqueous amount of alkaline water, the sodium hydroxide solution to form a slurry. For this purpose, concentrated is used, each time increased accordingly. It recommends adding caustic soda until the concentration of the solution is further removed, the mixture of tetracycline and chlorine solution of 0.17 to 1.17 normal caustic soda solution enttetracycline in water, the z. B. by adding speaks. The solution is left at room temperature ammonia to a pH value of 8.5 to 9.0 adjusted to 3 ° or at 40 ° C for 6, 20 or 45 minutes. B. 16 h long, then slurry to a pH of 1.5 to 2.0, filtered, men to achieve complete solution of the chlorotetracycline and the tetracycline precipitates from the filtrate by. The pH can be increased to 3.5 or 6.0 with simple preliminary tests. The suitable concentrations obtained in this way and the necessary tetracycline is of a high degree of purity and contains solution duration can be easily determined. The purified 35 less than 2% chlortetracycline.
solid tetracycline base, which in this type of purification is similarly an alkaline solution of remains undissolved, is then filtered off. Appropriately 45 mg of crude tetracycline per cm ³ at a pH of 8.5 is in an oxygen-free atmosphere, e.g. B. in freed from contaminating chlorotetracycline by a nitrogen atmosphere, worked to reduce the risk of any decomposition of this solution for 24 hours at room temperature or it can stand completely and to avoid the purified tetracycline from it. regains.

10 g tetracycline hydrochloride, which, according to the dif- A particularly simple way of working to win

Determined rential ultraviolet absorption method, tion of tetracycline from acidic aqueous solutions

845 ug tetracycline and 17 ^ g chlortetracycline per mg of a pH below 3.0 is that this

Crude product, i.e. about 85% tetracycline, contains, and, 45 acidic liquid in the presence of a water-soluble one

using the paper chromatographic method, salt with a volatile organic solution

true, a content of about 10% chlortetracycline medium, which is not or only with aqueous salt solutions

has, are ^{extracted in 200 cm 3 of} water that is slightly miscible with Ammo, the solvent

niak has been adjusted to pH 9.0, solvent extract from the extracted aqueous

Slurried under nitrogen for 16 hours. The unge- 5 ° separated liquid and the tetracycline from the

The dissolved, purified tetracycline base is then filtered off and the extract is recovered. Solution

trimmed and dried. 8.5 g are obtained with an agent that is particularly suitable for this cleaning process.

Content of 986 μg tetracycline and 20 μg chlorine tetra- ders are well suited. B. lower aliphatic alcohol

cyclin per mg corresponding to a content of 98.1% hole, lower alkyl esters of lower fatty acids, methyl

Tetracycline if after the differential ultraviolet 55 isobutyl ketone and chloroform. n-Butanol has proven to be

absorption method determined, and proven to be particularly useful with a content. As water soluble

from 97 to 98% tetracycline, if paper chromato- salts come the alkali, alkaline earth and ammo

graphically examined. 89% tetracycline are niumhalogenide or -sulfate or the hydrochloride

recovered. and sulfates of lower alkyl amines. Particularly

All of the analytical data given under E) and D) are good results with sodium chloride, which is purposely related based on equivalent μg tetracycline hydro- moderately in amounts of 2 to 20% of the weight of the chloride. The theoretical maximum content of the water-based liquid is added, and with calcium-free solid substances is therefore 1000 μg / mg chloride achieved. The solvent is at least in Tetracycline hydrochloride and 1080 μg / mg Tetra added in an amount that is sufficient an amount of cyclinbase. 65 pure tetracycline equivalent to the original

F) to dissolve 10.0 g (0.0225 mol) of anhydrous tetracycline tetracycline content of the liquid,

are in 100 cm ^{3 of} n-propanol and 1.1 g (0.0244 mol) According to another quite useful embodiment

98% formic acid suspended. The mixture form for recovery and purification is aqueous

Tetracycline solution at a pH between 8.0 and 11.0, which contains calcium ions in an amount of 0.1 to 2.0%, calculated in percent by weight calcium chloride, extracted with less than half its volume of n-butanol and that of butanol extract separated from the extracted solution worked up on tetracycline. The infrared absorption spectrum of this mineral oil suspension in the characteristic range of wave numbers between 1350 and 650 cm " ¹ can be seen from the curve in FIG. 2, in which the numbers given

the abscissa and ordinate have the same meaning as indicated above for FIG. ·

Tetracycline, an optical rotatory power of [α] ²⁷ /) = -245 ⁰ C at a concentration of 1% in methanol. Its ultraviolet spectrum, measured in 0.1 N hydrochloric acid, shows peaks at 267 ΐημ (e: 17400) and at 355 m ^ (e: 13500). :

The optical rotatory power of the hydrochloride is ²⁷ _fl = -253 ⁰ C at a concentration of 0.5% in 0.1 N hydrochloric acid at [a].

For this purpose 2 sheets of drawings

Claims (1)

1 2 According to another embodiment, the entire culture is first acidified and brought to a pH value set below 3.0, then filtered off the mycelium and Process for microbiological production then adjust the filtrate to a pH of 8.0 to 11.0, of tetracycline, characterized thereby, preferably 9.0 to 10.0. The n e t that the strain Streptomyces viridi- falling crude tetracycline is then filtered off, faciens ATCC 11989 in an aqueous, nitrogen- To adjust the pH value each can be sufficient Carbohydrate solution containing nutrients, strong water-soluble alkali can be used. Ammo breeds under submerged aerobic conditions. Niacin and sodium hydroxide are preferred ίο means. The precipitate obtained from tetracycline or of tetracycline and mycelium is for the purpose of further processing by dissolving in aqueous acid of a pH below 3.0 or in aqueous alkali of It is already known that tetracycline can be brought into solution by catalyzing a pH above 11.0. Verlytic Removal of chlorine from chlorotetracycline reversible aqueous acids are z. B. water soluble (J. Am. Chem. Soc. 75, 4621 to 4623 strong acids such as hydrochloric acid, sulfuric acid, phosphorus [1953]). This process satisfies its expense acid, nitric acid, the addition of which increases the pH of the because of not. There was therefore for the technology the fermentation solution below 3.0, preferably to about Need for a process that is more economical and so lowers 2.0 to 2.5. The same acids can more effective is to create. According to the invention, this is also used to make the tetracycline in the form of acidification succeeded in solving it with a microbiological salt and removing it from the undissolved one Process for the production of tetracycline, first separating the mycelium by filtration, followed by malig has been produced by fermentation. First the acidic filtrate to a pH between 8.0 and According to the method of the invention, the strain is adjusted to be 11.0 and thereby the tetracycline is removed from Streptomyces viridifaciens ATCC 11989 in one falls. The following example illustrates the invention. Carbohydrate solution under submerged aerobic conditions. . . bred. example The cultivation takes place at a pH value of about 30. Several batches of tetracycline are in the 5.0 to 6.5, preferably at 5.8 to 6.2. The temperature obtained from fermentation solutions in the following manner, which is at about 24 to 30 ⁰ C. After a period of cultivation of Streptomyces viridifaciens ATCC 1 to 5 days, that is, as soon as the nutrient solution arises considerably. To 600 to 1100 l of such a fermentative bacterial activity, the solution is 285 to 532 g of calcium chloride and separated from the mycelium and tetracycline is added from it to 35 200 to 475 l of moist n-butanol. He won. The extraction can take place in various ways, the pH of the solution, which was 4.9 to 5.5. by adding 4.0 to 10.5 liters of 50% sodium hydroxide solution. The nutrient solution is adjusted to a pH of 8.35 to 8.8, preferably after Ein. After the pH value has been set to 8.5, the mixture is stirred with butanol or stirred, an emulsion of methyl isobutyl ketone being stirred. The solvent 4 ° arises, the layer by filtration over diatomaceous earth, which contains the tetracycline, is separated from the, the butanol phase, which separates the tetra-aqueous layer, contains cyclin by partial distillation and makes up about 200 to 480 1, ablate the solvent is separated to a small volume and evaporated by vacuum distillation with an internal vapor and with a liquid low carbon temperature of the liquid below about 38 ° C and hydrogen, e.g. B. having a boiling range between 45 under pressure of less than 35 mm to a volume 85 and mixed 100 ⁰ C. In doing so, tetracycline falls in from about 2.5 to 7.0 1 evaporated,
The butanol concentrates obtained in this way, which were alkaline in the extraction solution, ie have a pH of about 7.0 to 7.5, will have a pH of 8.5. The hydrochloride is obtained, extracted three times with equal volumes of water, e.g. B. if the nutrient solution had been acidified with sulfuric acid or hydrochloric acid to a pH of 2.0 hydrochloric acid before the extraction with 50. is set. The aqueous extracts are combined. The solid tetracycline obtained in this way can be further purified with hydrochloric acid to a pH of 1.5, adjusted with nit by stirring it with ammonia solution of 1 to 2% activated charcoal to a paste and filing, or by it triert from a solution of the free. The filtrate is evaporated to one fifth of its volume by vacuum distillation on base on a chromatographic adsorbent. The pH of the z. B. can be adsorbed on silica. By washing aqueous concentrate is then with ammonia on the adsorbent, z. B. adjusted with acetone or metha- 6.0. In the process, tetracycline precipitates, the abfilno], the impurities are removed, whereupon the is trated. Often an additional amount of tetracycline can be eluted with acid. Obtain the purified tetracycline salt of the 60 concerned to about a fifth of its volume, which was retained in the eluate by renewed evaporation of the filtrate.
Acid is then obtained by crystallization, precipitation, drying the various parts of the precipitated tetralyophile or the like. cyclin are combined and in 630 ecm of water, which can be adjusted to a pH of 1.5 with hydrochloric acid, the tetravalent produced in the above-mentioned way, cyclin is also precipitated in alkaline form from the fermentation solution. Too solved. After filtration, 63 g of this solution are added to a pH between 65 ethylenediamine-tetraacetic acid. The pH-8.0 and 11.0, preferably 9.0 and 10.0, adjusted value of the mixture is then adjusted to 6.0 with ammonia and the precipitated mixture of mycelium and tetra. This filtered off in an amount of 30 g cyclin. Precipitated tetracycline is filtered off, over in vacuo