PROCESS FOR PRODUCING TOCOPHEROLS

Abstract: A novel process for producing tocopherols and their precursors is described.

Description: Process for producing tocopherols The present invention relates to a process for produci tocopherols and related compounds. More particularly, the invention relates to a process for producing the compounds of the formula wherein R 1 is methyl and 2 and R3 are hydrogen, 1 2 3 1 R and R are methyl and R is hydrogen, R and R 3 are methyl and R2 is hydrogen or hydrogen or methyl and the corresponding 3 ' ,7' ,11'-trienols, which process comprises treating the phytylbenzohydroquino derivatives of the formula wherein R 1. R2 and R3 are as above, or the corresponding 2',6' ,10' ,14'-tetraen-yl hydroquinones with (a culture of) spheroplasts of procaryotic blue-green algae (Cyanophyceae) , or enzyme fractions obtained therefrom; the term (a culture of) is used to indicate that not only a culture, but also planctonic populations of the algae can be used. The formula I encompasses the - preferred - (R.R.R)-α-tocopherol and the corresponding β.γ. and δ-tocopherols. As is known, the following applies: R 1 =R 2 =R 3 =H tOCOl α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol In the foreground of the interest is R,R,R-α-tocopherol. The remaining compounds of the formula I as well as the trienols may primarily be considered to be precursors of R.R.R-α-tocopherol; see in this respect also US-PS 3819657 and US-PS 2640058 relating to the methylation of such precursors. It has been known that in eucaryotic organisms such as in higher plants or green algae and in procaryotic blue-green algae the biosynthesis of tocopherols involves the condensation of homogentisates and phytyl pyrophosphate to form phytylbenzoquinol derivatives, which, by cyclisa- tion and subsequent methylations; yield tocopherols. E.g., in Biochem. J. (1970) 117. 593-600 it is reported that in the higher plants Lactuca sativa and Rumex sanguineus, in the green algae Euglena gracilis and Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans homogentisate are biosynthetic precursors of plastoquinone, α-toco- pherolquinone and the tocopherols. Moreover in Biochem. J. (1987) 242. 367-373 it is reported that the unicellular blue-green alga Scenedesmus obliquus contains α-tocopherol and different phytylbenzoquinone derivatives. Methylation o the aromatic ring at the precursor level as well at the tocopherol level has been shown to be mediated by SAM (S-adenosylmethionine) , see Phytochemistry (1980) 19, 215-218. However there is no evidence relating to the bio¬ synthetic conversion of 5-phytyl-l.4-hydroquinone-precursor and 5-geranylgeranyl-l,4-benzohydroquinones into γ- and δ-tocotrienols respectively into tocopherols. The process provided by the present invention now renders possible the production of tocopherols and toco- trienols from the corresponding benzoquinol derivatives by microorganisms. An unexpected advantage of the present process consists in the fact that only the E derivatives II react, i.e. the isomers of the derivatives II do not react with the spheroplasts under consideration. In other words it is not necessary to use the starting materials II as 100% E isomers; mixtures of E derivatives II with their Z isomers can be used a starting materials. Since the compounds II are more polar than the compounds I, the "unreacted" II can easily be separated, e.g. by way of chromatography. The microorganisms used in the present invention are those belonging to the class of the procaryotic blue-green algae (Cyanophyceae) . It is assumed that the cyclisation of the benzoquinol precursors to the tocopherols is catalysed by an up to now unknown enzyme which we call "tocopherol cyclase" due to its cyclising function. The toco- pherol-cyclase activity may be determined by measuring the conversion of labelled or unlabelled benzoquinol deriva¬ tives respectively to labelled and unlabelled tocopherols respectively and tocotrienols by standard methods such as TLC-radioactivity-scanning. HPLC-radioactivity-monitoring and HP C-fluorescence detection respectively. The strains preferably used in the present invention ar Anabaena variabilis (SAG 1403-4b) and Chlorogleopsis (SAG 1411-la). According to the classification in "Algen; Einfiihrung in die Phykologie", 1984. ed. Christian van den Hoek. Georg Thieme Verlag Stuttgart, New York, Anabaena variabilis and Chlorgleopsis are procaryonts of the class o Cyanobacteriae. The morphological and physiological characteristics of these strains are described in the mentioned manual. Any strain belonging to the above mentioned class, isolated from natural sources or publicly available from the usual collections, e.g. Sammlung von Algenkulturen, Pflanzenphysiolog. Institut der Universitat Gδttingen (SAG), may be employed for the present invention. The preferred strains are those containing tocophe- rol(s). Such content of tocopherol can easily be assayed by extracting a probe of the algae with, e.g. hexan and carry then out a suitable gaschromatographic determination. The process of this invention may be carried out by cultivating the microorganism in an appropriate medium in a manner known per se and using the modified form of the microorganism, namely spheroplasts of this organism, solubilised spheroplasts or enzyme fractions, in particular purified enzyme fractions. It has been found that using spheroplasts in their unmodified form led to substantial transport problems and thus lack of enzymatic activity. Appropriate culture media are those conventional media containing carbon sources, nitrogen sources and those inorganic salts indispensable for the growth of the micro¬ organism. Carbon dioxide may serve as the carbon source but other substances, e.g. carbonates, may also be used. As nitrogen sources, there may be used inorganic salts such as nitrates or ammonium salts, etc. As the inorganic nutrients, for example, potassium phosphates, magnesium sulfate. ferrous and ferric chlorides, calcium chloride and the like may be employed. There exist different methods for cultivating the above mentioned microorganisms. For example there may be used submerged cultures, such as shake flask cultures or fermen- tor cultures. Batch cultures or continuous cultures may be used. Preferably the cultivation should be carried out under aerobic conditions. The cultivation should preferably be conducted in the presence of light at a pH of about 5.0 to about 9.0, preferably from about 6.5 to 8.0. A preferred temperature range for carrying out the cultivation is from about 20°C to 37°C. preferably from 25°C to 33°C. While the time for cultivation varies depending upon the particular microorganism and the medium to be used, about 4 to 10 days of cultivation usually yields the best results. The process provided by the present invention is. as mentioned above, carried out by employing spheroplasts, solubilised spheroplasts (i.e. solubilized in aqueous media or enzyme fractions of the cultivated microorganisms. As it is known the term "spheroplast" refers to a spherical or near spherical structure of the cell as formed by the disruption or partial removal of the cell wall of a cell suspended in an isotonic (or hypertonic) medium. The spheroplasts of the algae are obtained in the conventional manner, i.e. by the treatment of the intact cells with lysozymes [this term referring to a group of hydrolases which represent cell lysing enzymes]. This is conveniently done at a pH of about 6 to 8, in a temperatur range of about 20 to 40°C, and for about 1 to 10 hours. Th usual lysozyme preparations may be used, e.g. "Lysozym" as manufactured by Merck, Darmstadt. The appropriate dosage i about 15000-25000 units/g of the lysozyme preparation. The preparation of the spheroplasts is conveniently effected in the presence of a hypertonic buffer, avoiding this way disruption of the cells. The hypertonic buffer is by definition a buffer, havin a higher osmotic pressure than that of the cells. This higher pressure can be achieved through the addition of. e.g. sucrose, see L.R. Semenova et al. Microbiologiya 51. 259 (1982). The reaction of the spheroplasts with the chemical substrate. In the case where spheroplasts are utilised, the benzo quinol derivatives II may be converted to the tocopherols i a hypertonic buffer under anaerobic conditions at a pH of about 6 to 8. Generally, cells from about 5 to 9 day cultu¬ res are preferred for obtaining the most effective cells fo the formation of spheroplasts and thus for the conversion o the benzoquinol derivatives to tocopherols and tocotrienols. In this conversion no additional nutrients are necessar On the other hand, when solubilised spheroplasts - this term referring to aqueous systems - or enzyme preparations are used, the conversion of the benzoquinol derivatives II to the tocopherols and tocotrienols may be carried out in commonly used non-hypertonic buffers under anaerobic conditions at a pH about 6 to 8. In this case, no nutrients need to be present. In the case enzyme fractions are utilized, said enzyme fractions are conveniently obtained from solubilized sphero- plasts by chromatography, e.g. ion exchange chromatography, liquid chromatography, gel filtration, gel electrophoresis, affinity chromatography, etc. One works conveniently in buffer solutions such as outlined above. The active fractions are determined by assay of the cyclase activity as outlined above. The necessary purification can be effected by detergents, preferably non-ionic detergents. In each case a temperature range of ca. 25°-35°C is feasible. For the present reaction the substrates are preferably applied in a solution of a hydrophilic solvent, such as in acetone, methanol, ethanol, etc. or, in a solution contai¬ ning such hydrophilic solvent. Furthermore, the presence of a non-ionic detergent is preferred in order to maintain the activity of the enzyme. Examples are: dodecyl-D-maltoside, Triton 100, Tween 80. etc. The substrate may also be applied as a preformed complex, the advantages being: 1.) no hydrophilic co-solvent is needed [and thus the enzyme activity being easily maintained this way] 2.) the oxidation level of the substrate is easily maintai¬ ned at the hydroquinone state in the presence of a reducing agent, e.g. an ascorbate. etc. 3.) the assay is homogenous, i.e. no formation of slurries does occur. Suitable ' complexing agents are β-cyclodexrin. 2,6-di-O-methyl-β-cyclodext in. etc. The substrates I are preferably prevented from oxidatio by the addition of reducing agent, such as NaBH.. Na-S-O., ascorbates. etc. The final concentration of the substrate for the conver sion should be in a range of ca. 0.04 and 1 g/1 with a reaction time depending on the microorganism. A reaction temperature of about 15 to 37°C. preferably of about 25 to 35°C and a reaction time of about 2 to 20 hours usually brings the most preferable results. A reasonable range of (solubilized) spheroplasts: com¬ pounds I would be ca. 10-20 g (centrifuged spheroplasts): The reaction mixture may be extracted and the products may be isolated from the extract by standard methods, such as chromatography and distillation, to give enantiomericall pure (R.R.R)-tocopherols and tocotrienols. The following examples illustrate the present inventio In these Examples. Substrate 1 is 2,3-dimethyl- -5-[(E.7 ■ .11•R)-3 ' .7 » 11'15'-tetramethyl -2'-hexa- decenyl]-l,4-benzohydroquinone. Examples A. Composition of a suitable medium for the growth of the microorganisms Medium 1 0.150 g 0.600 g 0.010 g 0.500 g 0.165 g 0.004 g Solution A, 1 ml dist. water to 1000 ml Solution A ς : (trace elements) H 3 B0 3 2.860 g MnCl 2 • 4H 2 0 1.810 g ZnS0 4 • 7H O 0.222 g MoO 0.015 g dist. water to 1000 ml B. Used microorganisms/substrate and suitable media Species/ Optimal substrate Medium colletion number cone, [mg/1] , , Anabaena variabilis 2-20 1 SAG B 1403-4b Chlorogleopsis 2-20 1 SAG B 1411-la C. Composition of suitable buffer solutions: in all cases based on phosphates. hypertonic buffer (pH 7) (for the production of spheroplasts) fbuffer 11 Sucrose 171.150 g K 2 H P0 4 3.483 g KH PO 1.361 g MgSO • 7H O 0.990 g EDTA • 2Na 0.065 g dist. water to 1000 ml Hypertonic buffer (pH 7) (preparation of compounds I ex non-solubilized spheroplasts) ("buffer 21 Substrate 1 0.100 g Sucrose 171.150 g Ascorbate 44.033 g K 2 HP0 4 3.483 g KH 2 P0 4 1.361 g MgSO • 7H 0 0.990 g EDTA • 2Na 0.065 g 2,6-di-O-methyl-β-cyclodextrin 6.000 g dist. water to 1000 ml Buffer (pH 7) (preparation of solubilized spheroplasts) Tbuffer 31 K 2 HP0 4 11.612 g KH 2 P0 4 4.536 g Glycerol 200.000 g (see C.C.Conta et al. , Bioche J.124. (1971),6 Dodecyl-D-maltoside 6.639 g l,4-Dithio-D,L-threitol 0.308 g dist. water to 1000 ml Buffer (PH 7) (diluent for the preparation of compounds I solubilized spheroplasts) [buffer 41 (see P.Rosevear et al.. Biochemistry JL9.. (1980), 4105 seq. K 2 HP0 4 11.612 g H 2 P0 4 4.536 g dist. water to 1000 ml Buffer (PH 7) (for gel filtration) Tbuffer 51 2 HP0 4 11.612 g Glycerol 200.000 g Dodecyl-D-maltoside 0.511 g 1.4-Dithio-D.L-threitol 0.077 g dist. water to 1000 ml Example 1 An agar slant culture of Anabaena variabilis SAG 1403- was inoculated in 100 ml medium 1 in a 200 ml-flask and cultivated at 31°C for 6 days while bubbling medium 1 with mixture of air/C0_ (0.5% C under permanent illumina- tion with 6 bulbs (Philips TLD 15W/33). After sedimentatio for 10 minutes and decantation of the medium the residual culture was inoculated in 1.8 1 of medium 1 in a 2 1-flask Cultivation was carried out under the above mentioned cond tions for 6 days. After sedimentation for 10 minutes and decantation of the medium the residual culture was inocula ted in 8 1 of medium 1 in a 10 1-flask. When cultivated under the above mentioned conditions fo 7 days, collecting cells by centrifugation at 1000 g yielde a sediment in the form of an pellet at a net weight of 3-3,5 g/1. 20 ml of buffer 1 (degassed) were transferred under argon to a 50 ml-flask containing 0,2 mg of the hydroquinon substrate 1, containing traces of the respective benzo- quinone. Addition of 120 mg 2,6-di-O-methyl-β-cyclodextrin and stirring intensively at room temperature yielded a homo genous yellowish solution. Addition of 0.88 g ascorbic acid and gentle stirring for 3 hours yielded a homogenous colour less solution (buffer 2). The complete conversion of the benzoquinone in the substrate 1 to the hydroquinone could b measured by extracting 1 ml of buffer 2 with 1.5 ml of a mixture of hexane/methanol 2:1 and subjecting 0.02 ml of th overstanding hexane layer to HPLC-analysis. Blue-green algae of 60 g net weight obtained by the above mentioned method were washed twice with 200 ml of buffer 1 and resuspended in a 500 ml-flask in 200 ml of buffer 1. The conversion of the cells to spheroplasts was carried out by adding 100 mg Lysozym (15000 E/mg, Merck) an slowly rotating the flask at 35°C for 2 hours. The conver- sion to the spheroplasts was monitored by phase contrast microscopy every 20 minutes. The spheroplasts were collecte by centrifugation at 4°C and 1000 g for 5 minutes. After washing twice with 200 ml buffer 1 (degassed) at 4°C under argon atmosphere the spheroplasts were maintained at 4°C under argon until use. 20 g of spheroplasts were suspended under argon in a 100 ml-flask, containing 50 ml buffer 1 (degassed), at 4°C and closed with a "pyrogallol" stopper (a wad of cotton-woo soaked with pyrogallol). After shaking the spheroplasts for 5 seconds with a vortex shaker, the enzyme reaction was started by the addition of 2 ml of buffer 2, containing 0.2 mg substrate 1, and additionally vortexing for 5 seconds. The flasks was incubated at 35°C with a rotary shaker for 15 hours at 120 rpm. The amount of (2R,4'R,8'R)-γ-tocopherol accumulated was 0.186 mg (93%). Example 2 20 g of spheroplasts (Example 1) were suspended under argon in a 100 ml-flask, containing 10 ml buffer 3, at 4°C and closed with a "pyrogallol" stopper. After slowly stirring for 1 hour at 4°C solubilised spheroplasts were obtained and stored at 4°C under argon until use. 40 ml of buffer 4 were added to 20 ml of solubilised spheroplasts under argon at 4°C. After vortexing the sus¬ pension for 30 seconds the enzyme reaction was started by the addition of 2 ml of buffer 2. containing 0.2 mg of the substrate 1 and additionally vortexing the suspension for 5 seconds. The flask was incubated at 35°C with a rotary shaker for 15 hours at 120 rpm. The amount of the correspon ding tocopherol accumulated was 0.136 mg (68%). Example 3 20 g of spheroplasts (Example 1) were suspended under argon in a 100 ml-flask, containing 50 ml buffer 1 (degas¬ sed), at 4°C and closed with a pyrogallol stopper. After shaking the spheroplasts for 5 seconds with a vortex shaker, the enzyme reaction was started by the addition of 1.0 ml EtOH, containing 0.2 mg of the substrate 1 and 1 mg NaBH 4 , and additionally vortexing for 5 seconds. The flask was incubated at 35°C with a rotary shaker for 15 hours at 120 rpm. The amount of tocopherol accumulated was 0.070 mg (35%). Example 4 20 g of spheroplasts (Example 1) were suspended under argon in a 100 ml-flask, containing 50 ml buffer 1 (degas- sed), at 4°C and closed with a pyrogallol stopper. After shaking the spheroplasts for 5 seconds with a vortex shaker the enzyme reaction was started by the addition of 1.0 ml EtOH. containing 0.2 mg of the substrate 1. 50 mg 2.6-di-O-methyl-β-cyclodextrin and 1 mg NaBH., and additionally vortexing for 5 seconds. The flask was incuba¬ ted at 35°C with a rotary shaker for 15 hours at 120 rpm. The amount of tocopherol accumulated was 0.120 mg (60%). Example 5 For gel filtration (liquid exclusion chromatography (SEC)), a column (2.6 x 50 cm) was packed with Fractogel TS HW65F (a polyethylene glycol dimethyl acrylate available from Merck) and equilibrated with 3 column volumes of buffer 5 at a constant flow of 2 ml/min./4°C. 20 ml of solubilized spheroplasts were centrifuged at 100.000 g/4°C for 5 hours. The supernatant was separated from the sediment and subjec¬ ted to gel filtration at 4°C and a constant flow of 1 ml/min. The eluate was collected in fractions of 10 ml and assayed for cyclase activity according to by comparing with the similar experiment carried out in puffer 5. The cyclase was found to elute between 290 and 380 ml and highest acti¬ vity was detected around 330 ml. Analogous results were obtained with the remaining derivatives of formula II, namely with 3-methyl- -5-[(E.7 , R,ll , R)-3' ,7 ' 11' 15 '-tetramethyl -2'-hexadecenyl]- -1,4-benzohydroquinone. leading to (2R,4*R.8 'R)-δ-toco- pherol. 2,3,6-trimethyl-5-[ (E.7 *R.11'R)-3 ' .7 '11'15'- -tetramethyl -2 '-hexadecenyl]-!,4-benzohydroquinone, leading to (2R.4 'R,8 'R)-α-tocopherol, 3-methyl-5-[ (all E)-3',7',ll',15 '-tetramethyl-2 ' , 6 ' , 10' ,14 '-hexadecatetraenyl 1,4-beπzohydroquinone, leading to (2R)-δ-tocotrienol, 2,3-diraethyl-5-[(alI E)-3* ,7' .11' .15'-tetramethyl- -2' ,6' ,10' ,1 '-hexadecatetraenyl]-!,4-benzohydroquinone, leading to (2R)-γ-tocotrienol. 2,3,6-trimethyl-5-[(all E)-3 ' .7' .11' .15'-tetramethyl-2' .6' .10' ,14'-hexadecatetrae 1,4-benzohydroquinone. leading to (2R)-α-tocotrienol. 2-[E.7'R.ll'R)-3' ,7' ,11' .15'-tetramethyl-2'-hexadecenyl] 1.4-benzohydroquinone, leading to (2R)-tocol.

Claims: Claims 1. A process for producing the compounds of the formul wherein R 1 is methyl and R2 and R3 are hydrogen. R 1 and R2 are methyl and R3 is hydrogen, R1 and R 3 are methyl and R2 is hydrogen or = hydrogen or methyl and the corresponding 3 ' ,7' ,ll'-trienols, which process comprises treating the phytylbenzohydroquinon derivatives of the formula wherein R 1, R2 and R3 are as above, or the corresponding 2 ',6 ',10' ,14 '-tetraen-yl hydroquinones with (a culture of) spheroplasts of procaryotic blue-green algae (Cyanophyceae), or enzyme fractions obtained therefro 2. A process according to claim 1, wherein the blue- -green algae used are Anabaena variabilis (SAG 1403-4b) or Chlorogloeopsis (SAG 1411-1). 3. A process according to claim 1 or 2, wherein solubi lized spheroplasts of procaryotiσ blue-green algae are used. 4. A process according to claim 3. wherein the concen¬ tration of the formula II substrate is 0,04 to 1 g/1. 5. A process according to any one of claims l to 4, wherein the substrate is applied as a preformed complex. 6. A process according to any one of claims 1 to 5. wherein the substrate contains a reducing agent, preferably an ascorbate. Na-S-O. or NaBH.. 7. A process according to any one of claims 1 to 4, wherein a non-ionic detergent is present. 8. A process according to any one of claims 1 to 4, wherein R 1 is methyl and R2 and R3 are hydrogen, R1 and R 2 are methyl and R3 is hydrogen, R1 and R3 are methyl and R 2 is hydrogen or hydrogen or methyl. 9. A process according to any one of claims 1 to 7, wherein the starting material is the compound of formula II with and R3 is hydrogen or the correspon¬ ding tetraen-yl hydroquinone. 10. A process according to any one of claims 1 to 7, wherein the starting material II has R2 = R3 methyl or is the corresponding tetraen-yl hydroquinone.

Inventors: STOCKER ACHIM, WOGGON WOLF-DIETRICH, STOCKER, ACHIM, WOGGON, WOLF-DIETRICH
Applicants: HOFFMANN LA ROCHE, F. HOFFMANN-LA ROCHE AG
Application year: 1989
Main classification: C12P17
IPC codes: C07D311, C12P17, C12R1