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[238] Biosynthesis of vitamin K2
[238]
BIOSYNTHESIS OF VITAMIN Ks
547
~Ci/mg. Ultraviolet maximum absorption at 248 nm ~ (~1% z c m = 414) (in hexane). Phylloquinone-l',2'-3H~) 6 This compound is prepared from 2-methyl1,4-naphthohydroquinone 1-benzoate and isophytol-l,2-3H~ in analogy to phylloquinone-Y,2'-14C2. Specific activity: 311 ~Ci/mg. Menaquinone-4 (Ring Methylo3H) (MK-~). 45 In a three-necked pearshaped flask, equipped with a mechanical stirrer, a reflux condenser and a gas inlet tube, is placed the solution of 700 mg (4 millimoles) of 2-methylaH-1,4-naphthohydroquinone (specific activity: 400 ~Ci/mg) in 1.7 ml of absolute dioxane; 42 mg of anhydrous zinc chloride and 90 ~l of BFa etherate are added, and the mixture is heated at 50° with stirring under argon. The solution of 1.16 g (4 millimoles) of geranyllinalool in 1.2 ml of dioxane is then added dropwise with stirring, and the reaction mixture is stirred for 20 minutes longer at 50 ° . The solution is cooled, and petroleum ether (b.p. 60°-90 °) and 75% aqueous methanol are added. The petroleum ether phase is extracted five times with aqueous methanol, and the extracts are reextracted with petroleum ether. The combined petroleum ether extracts are dried over anhydrous sodium sulfate and evaporated in vacuo. The residue is dissolved in 10 ml of absolute ether, 600 mg of silver oxide is added, and the mixture is shaken at room temperature for 30 minutes. After filtration and evaporation, the crude MK-4 (1.61 g) is chromatographed on silica gel (Merck). With petroleum ether-ether (9:1) 400 mg of still rather impure MK-4 is eluted. This fraction is dissolved in 1 ml of low-boiling petroleum ether, the solution is cooled to - 1 0 °, seeded with all-trans-MK-4, and kept at - 4 0 ° for 1 hour. The precipitated crystals are collected, washed with cold ( - 4 0 °) petroleum ether, and recrystallized once more in the same way. The yield of menaquinone-4 (ring methyl-3H) is 100 mg, showing a specific activity of 160 ~Ci/mg. ~6j. Wtirsch, unpublished.
[ 2 3 8 ] B i o s y n t h e s i s of V i t a m i n K 2
By ECKHARD LEISTNER and MEINHART H. ZENK Until recently virtually nothing was known about the route of vitamin K biosynthesis, and our present knowledge is restricted to incorporation studies with labeled precursors. Therefore, we are mainly concerned here with degradation and incorporation procedures. In the biosynthesis of vitamin K one has to distinguish between three separate biosynthetic
548
VITAMIN X GROUP
[238]
units, the aromatic nucleus, the isoprenoid side chain, and the nuclear methyl group. Degradative Methods The Aromatic Nucleus
In order to conduct degradation experiments successfully, one has to be sure of an ultrapure sample, which in the case of vitamin K is particularly difficult to obtain, since contaminations with fatty acid esters, for instance, frequently occur. 1,2 In the hands of the authors, isolation and purification of vitamin K~ by the method of Bishop et al. a has proved particularly useful. Subsequent purification by thin-layer chromatography4 in different solvent systems yields a pure product. ~ Chemical Degradation of Vitamin K2 to Phthalic Acid (Fig. 1) Principle. Oxidation of vitamin K2 (I) with KMn04 results in the formation of phthalic acid (II). No intermediates can be isolated (see [239]). There is no necessity to prepare a derivative of vitamin K for this step. Procedure. e Vitamin K in hexane is diluted with unlabeled material (e.g., K2(20); K1) so as give a total of 10-15 micromoles. The solvent is taken to dryness in a stream of N2, and the residue is taken up in i ml of redistilled acetone. Thirty milligrams of powdered KMn04 is added, and the mixture is refluxed for 1.5 hours. The solvent is evaporated with a stream of air, and 3 ml of 2 N H2S04 is added to the residue. By slow addition of solid NariS03 the mixture is decolorized and the phthalic acid is extracted with 4 X 5 ml of diethyl ether. The ether phase is dried over anhydrous Na2SO4, filtered, and taken to dryness. The residue is taken up in methanol and applied to chromatography paper and developed at least twice ascending in isopropanol-ammonia-water (8:1 : 1). The Rf value of phthalic acid after multiple development is 0.2-0.3; the substance is located in short UV light and eluted with methanol. The concentration is determined spectrophotometrically; 1 micromole of phthalic acid per milliliter gives under these conditions an extinction of 0.609 at 285 nm. Yield: 75-80%. This
1 j. M. CAmpbelland R. Bentley, Biochemistry 7, 3323 (1968). 2 R. Azerad, R. Bleiler-Hill, F. Catala, O. Samuel, and E. Lederer, Biochem. Biophys. Res. Commun. 27, 253 (1967). 3 D. H. L. Bishop, K. P. Pandya, and H. K. King, Biochem. J. 83, 606 (1962). 4 G. R. Whistance, D. R. Threlfall, and T. W. Goodwin, Biochem. J. 105, 145 (1967). s See this volume [236]. e E. Leistner, J. H. Schmitt, and M. H. Zenk, Biochem. Biophys. Res. Commun. 28, 845 (1967).
[238]
BIOSYNTHZSIS OF VITAMIN KS
549
jcoo.
~
~H s
[CH,--CH--~C--CH:].H
v
"COOH
o (1)
(II)
8.5 7.6
6.7
~
8~0I 1.4
/COOH
CO~
~COOH
10.9 •
OH
NH~
NO: (vn)
NO s
(v)
(vx)
CBrsNO ~
CBr3NO 2
(c,.~
(c,.,)
3 CBrNO~
Cc,.,;c,.,;c~.~0)
Fro. 1. Degradation of the benzenoid ring of vitamin Ks via nitrosalicylic acids. procedure m a y be scaled down to the oxidation of 1-2 micromoles of vitamin Ks with a yield of 50%.
Decarboxylation of Phthalic Acid Principle. Phthalic acid m a y be decarboxylated b y either of two m e t h ods. I t can be decarboxylated b y heating with quinoline 7 or it m a y be subjected to a Schmidt degradation. 8 I n b o t h cases a mixture of b o t h carboxyl groups representing C1 and C4 of v i t a m i n Ks is obtained. Procedure: Thermal Decarboxylalion. Phthalic acid in methanol is diluted with unlabeled carrier material to a total of 50 micromoles and t a k e n to d r y n e s s in a 50-ml ground-glass flask. The residue is dissolved in 5 ml of redistilled quinoline. To the flask, 50 mg of copper-chromite catalyst is added. The flask is connected to a Stutz-Burris apparatus, 9 7 E. Leistner and M. H. Zenk, Z. Naturforsch. 22b, 865 (1967). s E. Leete, J. Am. Chem. Soc. 81, 3948 (1959). 9 R. E. Stutz and R. H. Burris, Plant Physiol. 20, 226 (1951).
550
VITAMIN K GROUP
[238]
flushed with N~, and the reaction vessel is kept at 255o-260 ° in an oil bath for 50 minutes. The C02 liberated is flushed with a slow stream of N~ into the receiver vessel and trapped in 5 ml of a 5.6% Ba(OH)2 solution containing 10% BaCls. The resulting BaCO3 is centrifuged off, washed with distilled water, and applied to a preweighed small sintered porcelain disk. The carbonate is washed with 80% ethanol and dried for 1 hour at 100% The yield of COs is 70%. After the weight of the BaCO~ is determined, COs is liberated by addition of 8 ml of HCI04 (10%) in the Stutz-Burris apparatus, and the COs is swept with a Ns stream into a mixture of 8.8 m] of methanol and 1.2 ml of ethanolamine in the receiver vessel. Recovery of COs is 86%. To 8 ml of this mixture, 10 ml of toluene scintillator are added, and the sample is counted in a liquid scintillation spectrometer. Schmidt-Degradation and Isolation of Anthranilic Acid (Fig. 1). Phthalic acid (II) (100 micromoles) is dissolved in 0.2 ml of 100% HsS04 in a groundglass flask cooled in an ice-salt bath. The mixture is stirred with a glass rod, and 30 mg of NaNa is added until most of the acid is dissolved. The tip of the stirring rod is broken off and left in the reaction flask, which is connected with the Stutz-Burris apparatus. The apparatus is flushed with Ns, and 5 ml of the saturated barium hydroxide solution are added to the receiving flask. The flask containing the phthalic acid is heated in an oil bath to 100° and left at this temperature for 30 minutes. During the reaction, the COs liberated is swept with a stream of nitrogen into the receiver. The yield of BaCO8 is 100-130% (based on one carboxyl group). The reaction flask is chilled in an ice bath, and small pieces of ice are added to the flask, followed by 15 ml of H20; solid NH4HCO8 is added until a pH-value of 7 is reached. The solution is brought back to pH 5 with tartaric acid and extracted continuously with ether. The ether is evaporated, and the residue is applied to chromatography paper; the chromatogram is developed in isopropanol-ammonia-water (8:1:1). Anthrani]ic acid (III) is eluted with methanol (80%). One micromole of anthranilic acid per milliliter shows under these conditions an extinction of 4.02 at 334 nm. The yield is 70%. The acid is radiochemically pure.
Degradation of the Benzenoid Ring of Vitamin K (Fig. 1) Principle. The isolated anthranilic acid (III) containing the benzenoid ring of vitamin K is converted to salicylic acid (IV), which is nitrated separately in positions 3 (V), and 5 (VI), and 1, 3, and 5 (VII). Each carbon atom adjacent to the nitro group is isolated as bromopicrin, whose specific activity is determined. Since anthranilic acid is derived from the symmetric phthalic acid molecule, the isolated carbon atoms constitute mixtures of C5+8, C8+7, and C9+i0 of vitamin K.
[238]
BIOSYNTHESIS OF VITAMIN K2
551
Procedure: Conversion of Anlhranilic Acid to Salicylic Acid. ~° To a solution of 1.85 mfllimoles of ~4C-labeled anthranilic acid in 3 ml of cold conc. H~S04, 0.35 ml of cold 40% aqueous NaNO2 H solution is added dropwise over a period of 10 minutes. After 15 minutes the mixture is diluted dropwise with 6 ml 4 N H~SO4, and this solution is transferred to 6 ml of 4 N H2S04 at 75 °. The reaction mixture is kept at 75° over a period of 2 hours and stirred frequently. The crystals of salicylic acid which begin to separate after 1 hour are collected by filtration. The product can be recrystallized from a mixture of ethyl acetate + petroleum ether (30°-50°). Yield: 68%; m.p. 156°. Degradation of Salicylic Acid 12
Cs+s OF VITAMIN K. One millimole of salicylic acid is dissolved in 3 ml of chloroform. To this mixture the theoretical amount of bromine (0.052 ml) is added. After standing for 12 hours with frequent shaking, the chloroform is evaporated by a stream of air, and the residue is crystallized from water. The yield of 5-bromosalicylic acid is 90%; m.p. 162°. The acid is dissolved in 1.26 ml of glacial acetic acid, and a mixture of 0.72 ml of fuming nitric acid (d = 1.52) in 0.72 ml of glacial acetic acid at 0 ° is carefully added. This mixture is kept for 2-3 hours at room temperature, then 12.5 ml of H20 is added and crystallization is completed at 0 ° for 2-3 hours. The product is sublimed in vacuo, yielding two fractions; the fraction subliming at lower temperature is discarded. The desired 5-bromo-3-nitrosalicylic acid (V) sublimes at 150°-160 °. Yield: 44%, m.p. 175°. BROMOPICmN CLEAVAGE. Approximately 200 micromoles of the nitro compound is dissolved in 6 ml of water. Ca(OH)2 (30 rag) is added, and the mixture is chilled to 0 °. Then 21.5 ml of a well mixed paste consisting of 7.5 g of Ca(OH)2, 30 ml of H20, and 2.5 ml of bromine is added, and the flask is swirled for about 1 minute at 40 °. The content of the flask is then immediately steam distilled. Three milliliters of the bromopicrin-H~O mixture is collected. The bromopicrin is separated by centrifugation, and the drop of bromopicrin at the bottom of the centrifuge tube is washed twice by stirring with distilled water and centrifuging. The water layer is removed by a Pasteur pipette. The product is dried by suspending into the centrifuge tube a small test tube containing P~O~ and then allowing the closed tube to stand overnight. It is advisable to check the purity of bromopicrin by infrared spectroscopy, la Sharp infrared maxima at 1145 em -1 ~o p. R. Srinivasan, Biochemistry 4, 2860 (1965). 1, There is an error in the original procedure regarding the concentration of NaNO~. ~ F. Weygand and H. Wendt, Z. Naturforsch. 14b, 421 (1959). ~3 A. J. Birch, C. J. Moye, R. W. Rickards, and Z. Vanek, J. Chem. Sac. p. 3586 (1962).
552
VITAMIN K GROUP
[238]
(CHBr3); 1310 cm -1, 838 and 800 cm -1 (CBr3NO2); and 670 cm -1 (CBr 4) can be used for detection of impurities. Combustion of bromopicrin: Direct scintillation counting of bromopicrin gives erratic results. It is advisable therefore to combust the CBr3NO~ to CO2 with the Van Slyke-Folch method 1~for determination of its specific activity, or to prepare a derivative, e.g., methylamine. 13 C6-F7 OF VITAMIN K. Salicylic acid (550 micromoles) is dissolved in 1.75 ml of chloroform, and 0.15 ml of concentrated nitric acid (d = 1.4) is carefully added while the mixture is being cooled to 15°-18 °. After 1 hour the chloroform solution is extracted with 2 ml of 2 N NaOH. The aqueous phase is separated and acidified with HC1, and the pale yellow precipitate is extracted into ether. 5-Nitrosalicylic acid (VI) is recrystallized from a small amount of water after the ether is evaporated and the acid sublimed i n vacuo at 95 °. The yield is 93%; m.p. 228 °. Bromopicrin cleavage yields C6+7 of vitamin K as bromopicrin. Combustion of bromopicrin is conducted as above. C5+8,6+7,9-F10 OF VITAMIN K. To an ice-cold solution of 650 micromoles of salicylic acid in 0.49 ml of concentrated H2SO4, an ice-cold mixture of 0.31 ml of concentrated HNO~ (d = 1.4) is added. After 2 hours an additional amount of 0.31 ml of concentrated HNO~ is added, and the mixture is heated for 3 hours in a boiling water bath. After chilling to 0 °, 4.2 ml of ice-water is added, and the precipitated picric acid (VII) is collected by filtration. This acid is recrystallized from 4 ml of H20. The yield is 55%; m.p. 123 °. Bromopicrin cleavage yields C5+s+6+7+9+10 of vitamin K. The specific activity of C9+19 is obtained as the difference between this value and those separately determined for C6+8 and C6+7 (see above). Degradation of V i t a m i n K to 1,~-Diacetoxy-~-methylnaphthalene-3-acetic A c i d Principle. After reductive acetylation, vitamin K may be cleaved either ozono]ytically~5,1e or by chromic acid oxidation ~7 to 1,4-diacetoxy2-methylnaphthalene-3-acetaldehyde (Fig. 2, II) or to the corresponding naphthalene-3-acetic acid. This latter method is described here. The naphthalene acetic acid may subsequently be cleaved by treatment with alkaline H202 to give phthalic and malonic acid. ~8 Procedure. ~7 Radiochemically pure vitamin K~ is diluted with inactive
14D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 15S. B. Binkley, R. W. McKee, S. A. Thayer, and E. A. Doisy, J. Biol. Chem. 133, 721 (1940). 16I). R. Threlfall, W. T. Griffiths, and T. W. Goodwin, Biochem. J. 103, 831 (1967). 1~C. Martius and W. Leuzinger, Biochem. Z. 340, 304 (1964). 18I. M. Campbell, C. J. Coscia, M. Kelsey, and R. Bentley, Biochem. Biophys. Res. Commun. 28, 25 (1967).
BIOSYNTHESIS OF VITAMIN K2
[238]
553
11 o (1)
O--LCH s CHs CHO
O--~--CH~
+
o .o (in)
+
/CHs O=C\cHs
(IV)
(H) FIG. 2. Ozonolytlc cleavage of 1,4-diacetoxyvitamin K2. commercially available carrier vitamin K1 (phylloquinone) and reductively acetylated. 19 Four hundred micromoles (or an appropriate a m o u n t ) o f dihydro vitamin K diacetate is dissolved in 4 ml of glacial acetic acid; 750 mg of fused KHSO4 is added, followed by 76 mg of Cr03 in 1 ml of 80% aqueous acetic acid. The mixture is stirred for 1 hour at 50°. The acetic acid is removed by distillation under reduced pressure, and the residue is dissolved in water and extracted with ethyl ether. The naphthalene acetic acid is extracted from the ether phase with 2% NaHCO3 solution and precipitated by acidification of the bicarbonate phase. The acid is again extracted with ether, the ether phase is washed with water and evaporated, and the product is dissolved in methanol. The solution is decolorized, if necessary, with a little charcoal, and naphthalene acetic acid is crystallized at - 5 ° from 50% aqueous methanol. Recrystallization from ether-light petroleum and from benzene gives a pure product. The yield is 23-50%; m.p. 209 °. This acid can be separated from 1,4-diacetoxynaphthalene-3-acetic acid, which would originate from demethyl vitamin K during this degradation, by thin-layer chromatography in the solvent system benzene-propanol-glacial acetic acid (100: 20:1).17 1,4-Diacetoxy-2-methylnaphthalene-3-acetic acid is cleaved by treat19For reductive acetylation of vitamin K see Vol. VI, p. 303.
554
VITAMIN K GROUP
[238]
ment with alkaline I-I20220 to give phthalic acid and malonic acid, the latter representing C-3 of the naphthalene ring and carbon atoms 3' and 3" of the side chain, is Malonic acid is decarboxylated in quantitative yield in triethylphosphate solution ~' at 150°. The evolved COs is converted to BaC03 as above and represents a mixture of carbons 3 and 3 ~. The Side Chain
Isolation of Levulinic Aldehyde (Fig. 2) Principle. During ozonolysis of the diacetate of dihydro vitamin K2 (I) and subsequent reduction of the ozonide, the isoprenoid side chain is degraded to levulinie aldehyde (III), which is isolated and assayed as its bisdinitrophenylhydrazone. Procedure. The sample of labeled vitamin K2 which is diluted with carrier quinone is reductively acetylated 19after determination of its specific activity. The ozonolysis, decomposition of the ozonide, and isolation of the dinitrophenylhydrazone are conducted exactly as described for the degradation of tocopherols and prenylated benzoquinones? ,15,16 The yield of the hydrazone is in the range of 90%; m.p. 232 °. A satisfactory yield of the hydrazone can be obtained by this procedure starting with only 0.5 mg of the quinone. 16 Because of the quenching effect of the dinitrophenylhydrazone during direct scintillation counting, it is advantageous to combust the hydrazone by the Sch6ninger method. 22 Isolation of Acetone Principle. During the above-mentioned ozonolytic cleavage of the diacetate of vitamin K2 (I), the tail end of the side chain is liberated as acetone (IV) in a 1:1 molar ratio. Acetone is isolated as its 2,4-dinitrophenylhydrazone and assayed after conversion to iodoform. Procedure. 15 Approximately 90 micromoles of the labeled diacetate of dihydro vitamin K2 is dissolved in 4 ml of ethyl acetate in a two-necked 25-m] ground-glass flask. The flask is chilled in a mixture of dry-ice and isopropanol, and ozone is bubbled through the" solution until a persistent blue color appears ( ~ 15 minutes). Excess ozone is then removed by bubbling 02 through the solution. The reaction mixture is diluted with 20 ml of diethyl ether, and 2 drops of 2 N aqueous acetic acid are added. Subsequently 400 mg of zinc dust is added in small quantities with shaking over a period of 1 hour. After the mixture has stood for 1 hour, the zinc is filtered off at - 5 °, and the ether is distilled into 10 ml of 10% NaHSO3 at 0 °. After 20 R. Bentley, J. Biol. Chem. 238, 1889 (1963). ,1 L. W. Clark, J. Phys. Chem. 60, 1150 (1956). F. Kalberer and J. Rutsehmann, Hdv. Chim. Acta 44~ 1956 (1961).
[238]
BIOSYNTHESIS OF VITAMIN K2
555
all the ether is distilled, an additional portion of 10 ml of ether is added to the distillation flask, and the distillation is continued. This procedure is repeated a third time. Ether and NaHSO3 solution are separated in a separatory funnel, and the ether phase is washed twice, each time with 12.5 ml of 10% NariS03. The combined extracts are treated with 3 g of KOH dissolved in 10 ml of H~O, and the solution is distilled, about 25 ml being collected in the ice-cold distillate. The distillate is added to 20 ml of 6 N H2S04 containing 50 mg of 2,4-dinitrophenylhydrazine. The solution is allowed to freeze in a deep-freezer, then thawed; the yellow precipitate is filtered off and washed with dilute H2S04 and water. Yield: 70%; m.p. 124° . Acetone 2,4-dinitrophenylhydrazone may be subjected to thin-layer chromatography on silica gel G plates in the solvent system benzenepetrol ether, 30°-50 ° (3:1). The Rr value is 0.3. To determine the specific activity of the derivative, a weighed portion of the hydrazone is combusted by the SchSninger method, 22 or the derivative is converted to iodoform in the following manner. ~3 Forty milligrams of acetone 2,4-dinitrophenylhydrazone is dissolved in 2 N H2S04, and about 15 ml of this solution is slowly distilled into 7.5 ml of 1 N NaOH. To the distillate an excess of a solution of KI:I~ is added. The precipitated iodoform is collected by centrifugation and washed with distilled water. The Ring-Methyl Group The ring-methyl group of vitamin Ks can be isolated24.~: 1,4-diacetoxy2-methyl-naphthalene-3-acetic acid is submitted to Kuhn-Roth oxidation after removal of the protective acetyl groups. Schmidt degradation~ of the acetic acid, which represents carbon atoms 2 and 2' of vitamin Ks, allows the assay of the individual carbon atoms. Isotope Incorporation Studies Choice of Organisms and Feeding Procedures Vitamin K2 is widely distributed among gram-positive and gramnegative bacteria? The average content of this naphthoquinone in bacteria is of the order of 1 micromole per gram dry weight. Six different species of bacteria (Bacillus ~negaterium, Bacillus sublilis, Escherichia coli, Micrococcus lysodeikticus, Proteus vulgaris, and Sarcina lutea) with a known high s~H. Simon and H. G. Floss, "Bestimmungder Isotopverteihm~in markierten Verbindungen." Springer, Berlin, 1967. s4R. Ku_hnand H. Roth, Chem. Ber. 66, 1274 (1933). D. J. Robins, I. M. Campbell, and R. Bentley, Biochem. Biophys. Res. Commun. 39, 1081 (1970).
556
VITAMIN K GROUP
[238]
content of vitamin K2 have been tested for their ability to incorporate exogenously supplied shikimic acid-14C into vitamin K. 6 Of these, only E. coli and B. megaterium were found to incorporate shikimate to a notable extent; besides these two species, Mycobacterium phlei is known to convert this precursor into vitamin K2.18 In all other strains, penetration difficulties complicate the issue, since only little 14C activity is found in the cell mass from labeled shikimate. For precursor feeding experiments, the organisms are grown in I liter of synthetic medium containing: K2HP04 (7 g), KH2P04 (2 g), Nas-citrate.5H20 (0.6 g), MgSO4.7H20 (0.1 g), (NH4)2SO4 (1 g), glycerol (5 m]). L-Phenylalanine (16.5 rag), L-tyrosine (18.3 mg), and L-tryptophan (20.4 mg) are included in the medium to avoid metabolic drainage of shikimate into protein amino acids. The medium is autoclaved in 4-liter penicillin flasks (Schott and Gen., Mainz, Germany; No. 20551). The labeled precursor solution, sterilized by using a Millipore 0.22-~ filter, is added to the medium, and the flask is inoculated with a subculture of the organism grown in the same basal medium. The bacteria are grown at 36 ° under vigorous aeration on a platform shaker with 60 strokes per minute. The organisms are harvested in the stationary phase by centrifugation, and vitamin K2 is isolated and purified. An extremely useful organism for investigation of the later steps in vitamin K2 biosynthesis is an anaerobic naphthoquinone dependent strain of Fusiformis nigrescens (Bacterioides ~elaninogenicus). 17,2e This organism is grown in a medium containing per liter:6: trypticase (Baltimore Biological Laboratory) (27 g), Difco yeast extract (3 g), NaC1 (2 g), KH2P04 (2.5 g), K2COs (2.5 g), and hemin (5 mg). Precursors of vitamin K are added in a concentration of 0.2-2 ~g per milliliter of medium. The medium is sterilized by filtration or autoclaved. Growth proceeds at 37 ° for 2-10 days in an oxygen-free atmosphere. Synthesis of Specifically Labeled Precursors 1,~-Naphthoquinone-1,.~J4C. 2~ Commercially available a-naphthol-1-14C is diluted with carrier material to 100 micromoles, dissolved in 1 ml of methanol in a ground-glass test tube, and cooled to 0 °. To this is added a solution of 200 micromoles of Fremy salt (nitrosodisu]fonate) in 4.5 m] of H20 containing 166 micromoles of KH2P04. After 30 minutes at 0 °, the precipitated naphthoquinone is extracted into ether. The ethereal solution is" concentrated in a nitrogen stream and subjected to thin-layer chromatography in the solvent system benzene-petroleum ether, 300-50 ° (3:1). The naphthoquinone zone (RI 0.9) is eluted with dichloromethane, 2eR. J. Gibbons and L. P. Engle, Science 146, 1307 (1964). 27H. J. Teuber and N. GStz, Chem. Ber. 87, 1236 (1954).
[238]
BIOSYNTHESIS OF VITAMIN Ks
557
the solvent evaporated, and the residue is sublimed in vacuo. Yield: 40-50%. The product is radiochemicaUy pure. 1,4-Naphthoquinone-2,3,9,10-1~C. 2s,~9 Commercially available p-benzoquinone-2,3,5,6-14C is diluted with freshly sublimed carrier material to 100 micromoles in a heavy-walled 5-ml ground-glass flask. Butadiene (0.4 ml) in glacial acetic acid (0.8 ml) is added, and the mixture is left for 44 hours at room temperature. Then 0.25 ml of a dichromate-sulfuric acid mixture (4 g of Na2Cr~07 and 0.2 ml of concentrated H~SO4 in 2.5 ml of H~O) is mixed with 0.1 ml of glacial acetic acid; the mixture is added to the flask and kept at 65 ° for 30 minutes. Thereafter an additional 0.1 ml of the same mixture is added, and the content of the flask is kept for another 50 minutes at the same temperature. To the mixture 3 ml of ice water is then added; after 10 minutes at 0 ° the precipitate is filtered, washed, and dried over P205. Yield: 50%; m.p. 121°. Further purification is achieved as above; final yield: 24%. The preparation of 1,4-naphthoquinone [5,8(?)-aH] has also been reported. ~7 Scheme of Biosynthesis of Vitamin Ks Based on Isotope Incorporation Studies (Fig. 3) It is established that the ring carbon atoms of shikimie acid (I) give rise to the benzenoid ring of vitamin K. 6as,~° Furthermore, shikimate is utilized during this conversion as an intact C7 unit. 6as It is not yet clear, however, at what stage the aromatization of the ring occurs. Chorismic acid, a plausible intermediate, 3°,31seems not t~ be a precursor of 5-hydroxy1,4-naphthoquinone in Juglans regia L.,29 and neither protocatechualdehyde nor protocatechuic acid seem to be intermediates, e,~s Carbon atoms 2, 3, and 4 of the naphthalene nucleus are very probably derived from carbons 2, 3, and 4 of a-ketoglutarate, 25,3~which is incorporated by way of o-succinylbenzoie acid (II) 31since a ~4C-labeled sample of this compound was efficiently converted to bacterial menaquinones as well as quinones from higher plants. 31 Ring closure of o-succinylbenzoic acid (II) would give rise to 1,4-dihydroxy-2-naphthoic acid (III). The position of a-naphthol (IV) which has been implicated in naphthoquinone biosynthesise,83,8~ 28j. Baddiley, G. Ehrensvard, E. Klein, L. Reio, and E. Saluste, J. Biol. Chem. 183, 777 (1950). 2, E. Leistner and M. H. Zenk, Z. Naturforsch. 25b, 259 (1968). 80G. B. Cox and F. Gibson, Biochem. J. 1O0, 1 (1966). 31p. Dansette and R. Azerad, Biochem. Biophys. Res. Commun. 40, 1090 (1970). 8~I. M. Campbell, Tetrahedron Letters p. 4777 (1969). 3aW. Sandermarm,NaturwissenschaSten 53, 513 (1966). R. K. Hammond and D. C. White, J. Bacteriol. 100, 573 (1969).
558
VITAMIN K GROUP
o oo HO~
[238]
oo:
OH
~ OH (I1
0
OH
(II)
(nI)
OH
?
~ C02
~
l
////
OH
: (VIII)
(VI) / 0
OH
(Iv) (vn) FIG. 3. Hypothetical schemeof the biosynthesisof vitamin K~. is not clear. It cannot be excluded that naphthol is unspecifically converted to naphthohydroquinone (V) or naphthoquinone, which was shown to be transformed to both menadione (2-methy]naphthoquinone) and vitamin K~(45) in F~sifornds nigrescens. 17 Naphthoquinone and 2-methylnaphthoquinone [possibly as their hydroquinones (V), (VI)] are precursors of vitamin K in this organism. 85 Martins and Leuzinger were the first to demonstrate that the nuclear methyl group of vitamin K and menadione originates from the methyl group of L-methionine. This report has been confirmed in a most elegant way by Jackman, O'Brien, Cox and Gibson, ~6 using (Me-~H) methionine and a methionine auxotrophic strain of E. coli. Examination of the isolated vitamin K2(40) ( ~ 5 mg) by nuclear magnetic resonance and mass spectrometry revealed that only one methyl group is incorporated into the vitamin and that it is located at the 2 position ]vI. Lev, J. Gen. Microbiol. 20, 697 (1959). 86L. M. Jackman, J. G. O'Brien, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 141, 1 (1967).
[239]
BIOSYNTHESIS OF PHYLLOQUINONE
559
of the naphthoquinone. 2-Methylnaphthoquinone or the hydroquinone (VI) serves as the acceptor for an isoprenoid side chain to give the complete vitamin K2 molecule (VIII). 17,37 There is some evidence that demethyl vitamin K (VII) is an intermediate in vitamin Ks biosynthesis,~,88 which would place the ring methylation as the last step into the biosynthetic sequence leading to vitamin K. However, it is not yet established whether demethyl vitamin K (VII) or menadione (VI) or even both, but in different organisms, are the immediate precursors of vitamin Ks (VIII). A hypothetical scheme for the biosynthesis of vitamin Ks in bacteria based on incorporation studies is shown in Fig. 3. 8TM. Billeter, W. Bolliger, and C. Martius, Biochem. Z. $40, 290 (1964). 88O. Samuel and R. Azerad, FEBS Letters 2, 336 (1969).
[ 2 3 9 ] B i o s y n t h e s i s of P h y l l o q u i n o n e By D. R. THRELFALLand G. R. WHISTANCE Phylloquinone (vitamin K1) is a normal constituent of the photosynthetic tissues of all higher plants. It has also been reported to be present in green, brown, red, and blue-green algae. The methods described in this article are those which we have used to study the biosynthesis of phylloquinone in maize shoots, bean shoots, and ivy leaves.
Cultivation (or Source) of Biological Material and Its Exposure to Radioactive Substrates The routine experimental systems used in our investigations are greening-excised-etiolated maize or French bean shoots. These systems were chosen because in them a marked and rapid synthesis of phylloquinone takes place. Thus on exposure of etiolated maize shoots to light for 24 hours, the level of phylloquinone increases from 90 ~g/100 shoots to 140 ~g/100 shoots. ~ The details of the cultivation (or sources) of etiolated maize shoots (Zea mays), etiolated French bean shoots (Phaseolus vulgaris), and ivy leaves (Hedera helix) and their subsequent exposure to radioactive compounds are identical to those described in the article on the biosynthesis of tocopherols and biogenetically related compounds.2 1W. T. Griffiths,D. R. Threlfall, and T. W. Goodwin,Biochem. J. 103, 589 (1967). 2D. R. Threlfall and G. R. Whistance, this volume [231].
BIOSYNTHESIS OF VITAMIN Ks
547
~Ci/mg. Ultraviolet maximum absorption at 248 nm ~ (~1% z c m = 414) (in hexane). Phylloquinone-l',2'-3H~) 6 This compound is prepared from 2-methyl1,4-naphthohydroquinone 1-benzoate and isophytol-l,2-3H~ in analogy to phylloquinone-Y,2'-14C2. Specific activity: 311 ~Ci/mg. Menaquinone-4 (Ring Methylo3H) (MK-~). 45 In a three-necked pearshaped flask, equipped with a mechanical stirrer, a reflux condenser and a gas inlet tube, is placed the solution of 700 mg (4 millimoles) of 2-methylaH-1,4-naphthohydroquinone (specific activity: 400 ~Ci/mg) in 1.7 ml of absolute dioxane; 42 mg of anhydrous zinc chloride and 90 ~l of BFa etherate are added, and the mixture is heated at 50° with stirring under argon. The solution of 1.16 g (4 millimoles) of geranyllinalool in 1.2 ml of dioxane is then added dropwise with stirring, and the reaction mixture is stirred for 20 minutes longer at 50 ° . The solution is cooled, and petroleum ether (b.p. 60°-90 °) and 75% aqueous methanol are added. The petroleum ether phase is extracted five times with aqueous methanol, and the extracts are reextracted with petroleum ether. The combined petroleum ether extracts are dried over anhydrous sodium sulfate and evaporated in vacuo. The residue is dissolved in 10 ml of absolute ether, 600 mg of silver oxide is added, and the mixture is shaken at room temperature for 30 minutes. After filtration and evaporation, the crude MK-4 (1.61 g) is chromatographed on silica gel (Merck). With petroleum ether-ether (9:1) 400 mg of still rather impure MK-4 is eluted. This fraction is dissolved in 1 ml of low-boiling petroleum ether, the solution is cooled to - 1 0 °, seeded with all-trans-MK-4, and kept at - 4 0 ° for 1 hour. The precipitated crystals are collected, washed with cold ( - 4 0 °) petroleum ether, and recrystallized once more in the same way. The yield of menaquinone-4 (ring methyl-3H) is 100 mg, showing a specific activity of 160 ~Ci/mg. ~6j. Wtirsch, unpublished.
[ 2 3 8 ] B i o s y n t h e s i s of V i t a m i n K 2
By ECKHARD LEISTNER and MEINHART H. ZENK Until recently virtually nothing was known about the route of vitamin K biosynthesis, and our present knowledge is restricted to incorporation studies with labeled precursors. Therefore, we are mainly concerned here with degradation and incorporation procedures. In the biosynthesis of vitamin K one has to distinguish between three separate biosynthetic
548
VITAMIN X GROUP
[238]
units, the aromatic nucleus, the isoprenoid side chain, and the nuclear methyl group. Degradative Methods The Aromatic Nucleus
In order to conduct degradation experiments successfully, one has to be sure of an ultrapure sample, which in the case of vitamin K is particularly difficult to obtain, since contaminations with fatty acid esters, for instance, frequently occur. 1,2 In the hands of the authors, isolation and purification of vitamin K~ by the method of Bishop et al. a has proved particularly useful. Subsequent purification by thin-layer chromatography4 in different solvent systems yields a pure product. ~ Chemical Degradation of Vitamin K2 to Phthalic Acid (Fig. 1) Principle. Oxidation of vitamin K2 (I) with KMn04 results in the formation of phthalic acid (II). No intermediates can be isolated (see [239]). There is no necessity to prepare a derivative of vitamin K for this step. Procedure. e Vitamin K in hexane is diluted with unlabeled material (e.g., K2(20); K1) so as give a total of 10-15 micromoles. The solvent is taken to dryness in a stream of N2, and the residue is taken up in i ml of redistilled acetone. Thirty milligrams of powdered KMn04 is added, and the mixture is refluxed for 1.5 hours. The solvent is evaporated with a stream of air, and 3 ml of 2 N H2S04 is added to the residue. By slow addition of solid NariS03 the mixture is decolorized and the phthalic acid is extracted with 4 X 5 ml of diethyl ether. The ether phase is dried over anhydrous Na2SO4, filtered, and taken to dryness. The residue is taken up in methanol and applied to chromatography paper and developed at least twice ascending in isopropanol-ammonia-water (8:1 : 1). The Rf value of phthalic acid after multiple development is 0.2-0.3; the substance is located in short UV light and eluted with methanol. The concentration is determined spectrophotometrically; 1 micromole of phthalic acid per milliliter gives under these conditions an extinction of 0.609 at 285 nm. Yield: 75-80%. This
1 j. M. CAmpbelland R. Bentley, Biochemistry 7, 3323 (1968). 2 R. Azerad, R. Bleiler-Hill, F. Catala, O. Samuel, and E. Lederer, Biochem. Biophys. Res. Commun. 27, 253 (1967). 3 D. H. L. Bishop, K. P. Pandya, and H. K. King, Biochem. J. 83, 606 (1962). 4 G. R. Whistance, D. R. Threlfall, and T. W. Goodwin, Biochem. J. 105, 145 (1967). s See this volume [236]. e E. Leistner, J. H. Schmitt, and M. H. Zenk, Biochem. Biophys. Res. Commun. 28, 845 (1967).
[238]
BIOSYNTHZSIS OF VITAMIN KS
549
jcoo.
~
~H s
[CH,--CH--~C--CH:].H
v
"COOH
o (1)
(II)
8.5 7.6
6.7
~
8~0I 1.4
/COOH
CO~
~COOH
10.9 •
OH
NH~
NO: (vn)
NO s
(v)
(vx)
CBrsNO ~
CBr3NO 2
(c,.~
(c,.,)
3 CBrNO~
Cc,.,;c,.,;c~.~0)
Fro. 1. Degradation of the benzenoid ring of vitamin Ks via nitrosalicylic acids. procedure m a y be scaled down to the oxidation of 1-2 micromoles of vitamin Ks with a yield of 50%.
Decarboxylation of Phthalic Acid Principle. Phthalic acid m a y be decarboxylated b y either of two m e t h ods. I t can be decarboxylated b y heating with quinoline 7 or it m a y be subjected to a Schmidt degradation. 8 I n b o t h cases a mixture of b o t h carboxyl groups representing C1 and C4 of v i t a m i n Ks is obtained. Procedure: Thermal Decarboxylalion. Phthalic acid in methanol is diluted with unlabeled carrier material to a total of 50 micromoles and t a k e n to d r y n e s s in a 50-ml ground-glass flask. The residue is dissolved in 5 ml of redistilled quinoline. To the flask, 50 mg of copper-chromite catalyst is added. The flask is connected to a Stutz-Burris apparatus, 9 7 E. Leistner and M. H. Zenk, Z. Naturforsch. 22b, 865 (1967). s E. Leete, J. Am. Chem. Soc. 81, 3948 (1959). 9 R. E. Stutz and R. H. Burris, Plant Physiol. 20, 226 (1951).
550
VITAMIN K GROUP
[238]
flushed with N~, and the reaction vessel is kept at 255o-260 ° in an oil bath for 50 minutes. The C02 liberated is flushed with a slow stream of N~ into the receiver vessel and trapped in 5 ml of a 5.6% Ba(OH)2 solution containing 10% BaCls. The resulting BaCO3 is centrifuged off, washed with distilled water, and applied to a preweighed small sintered porcelain disk. The carbonate is washed with 80% ethanol and dried for 1 hour at 100% The yield of COs is 70%. After the weight of the BaCO~ is determined, COs is liberated by addition of 8 ml of HCI04 (10%) in the Stutz-Burris apparatus, and the COs is swept with a Ns stream into a mixture of 8.8 m] of methanol and 1.2 ml of ethanolamine in the receiver vessel. Recovery of COs is 86%. To 8 ml of this mixture, 10 ml of toluene scintillator are added, and the sample is counted in a liquid scintillation spectrometer. Schmidt-Degradation and Isolation of Anthranilic Acid (Fig. 1). Phthalic acid (II) (100 micromoles) is dissolved in 0.2 ml of 100% HsS04 in a groundglass flask cooled in an ice-salt bath. The mixture is stirred with a glass rod, and 30 mg of NaNa is added until most of the acid is dissolved. The tip of the stirring rod is broken off and left in the reaction flask, which is connected with the Stutz-Burris apparatus. The apparatus is flushed with Ns, and 5 ml of the saturated barium hydroxide solution are added to the receiving flask. The flask containing the phthalic acid is heated in an oil bath to 100° and left at this temperature for 30 minutes. During the reaction, the COs liberated is swept with a stream of nitrogen into the receiver. The yield of BaCO8 is 100-130% (based on one carboxyl group). The reaction flask is chilled in an ice bath, and small pieces of ice are added to the flask, followed by 15 ml of H20; solid NH4HCO8 is added until a pH-value of 7 is reached. The solution is brought back to pH 5 with tartaric acid and extracted continuously with ether. The ether is evaporated, and the residue is applied to chromatography paper; the chromatogram is developed in isopropanol-ammonia-water (8:1:1). Anthrani]ic acid (III) is eluted with methanol (80%). One micromole of anthranilic acid per milliliter shows under these conditions an extinction of 4.02 at 334 nm. The yield is 70%. The acid is radiochemically pure.
Degradation of the Benzenoid Ring of Vitamin K (Fig. 1) Principle. The isolated anthranilic acid (III) containing the benzenoid ring of vitamin K is converted to salicylic acid (IV), which is nitrated separately in positions 3 (V), and 5 (VI), and 1, 3, and 5 (VII). Each carbon atom adjacent to the nitro group is isolated as bromopicrin, whose specific activity is determined. Since anthranilic acid is derived from the symmetric phthalic acid molecule, the isolated carbon atoms constitute mixtures of C5+8, C8+7, and C9+i0 of vitamin K.
[238]
BIOSYNTHESIS OF VITAMIN K2
551
Procedure: Conversion of Anlhranilic Acid to Salicylic Acid. ~° To a solution of 1.85 mfllimoles of ~4C-labeled anthranilic acid in 3 ml of cold conc. H~S04, 0.35 ml of cold 40% aqueous NaNO2 H solution is added dropwise over a period of 10 minutes. After 15 minutes the mixture is diluted dropwise with 6 ml 4 N H~SO4, and this solution is transferred to 6 ml of 4 N H2S04 at 75 °. The reaction mixture is kept at 75° over a period of 2 hours and stirred frequently. The crystals of salicylic acid which begin to separate after 1 hour are collected by filtration. The product can be recrystallized from a mixture of ethyl acetate + petroleum ether (30°-50°). Yield: 68%; m.p. 156°. Degradation of Salicylic Acid 12
Cs+s OF VITAMIN K. One millimole of salicylic acid is dissolved in 3 ml of chloroform. To this mixture the theoretical amount of bromine (0.052 ml) is added. After standing for 12 hours with frequent shaking, the chloroform is evaporated by a stream of air, and the residue is crystallized from water. The yield of 5-bromosalicylic acid is 90%; m.p. 162°. The acid is dissolved in 1.26 ml of glacial acetic acid, and a mixture of 0.72 ml of fuming nitric acid (d = 1.52) in 0.72 ml of glacial acetic acid at 0 ° is carefully added. This mixture is kept for 2-3 hours at room temperature, then 12.5 ml of H20 is added and crystallization is completed at 0 ° for 2-3 hours. The product is sublimed in vacuo, yielding two fractions; the fraction subliming at lower temperature is discarded. The desired 5-bromo-3-nitrosalicylic acid (V) sublimes at 150°-160 °. Yield: 44%, m.p. 175°. BROMOPICmN CLEAVAGE. Approximately 200 micromoles of the nitro compound is dissolved in 6 ml of water. Ca(OH)2 (30 rag) is added, and the mixture is chilled to 0 °. Then 21.5 ml of a well mixed paste consisting of 7.5 g of Ca(OH)2, 30 ml of H20, and 2.5 ml of bromine is added, and the flask is swirled for about 1 minute at 40 °. The content of the flask is then immediately steam distilled. Three milliliters of the bromopicrin-H~O mixture is collected. The bromopicrin is separated by centrifugation, and the drop of bromopicrin at the bottom of the centrifuge tube is washed twice by stirring with distilled water and centrifuging. The water layer is removed by a Pasteur pipette. The product is dried by suspending into the centrifuge tube a small test tube containing P~O~ and then allowing the closed tube to stand overnight. It is advisable to check the purity of bromopicrin by infrared spectroscopy, la Sharp infrared maxima at 1145 em -1 ~o p. R. Srinivasan, Biochemistry 4, 2860 (1965). 1, There is an error in the original procedure regarding the concentration of NaNO~. ~ F. Weygand and H. Wendt, Z. Naturforsch. 14b, 421 (1959). ~3 A. J. Birch, C. J. Moye, R. W. Rickards, and Z. Vanek, J. Chem. Sac. p. 3586 (1962).
552
VITAMIN K GROUP
[238]
(CHBr3); 1310 cm -1, 838 and 800 cm -1 (CBr3NO2); and 670 cm -1 (CBr 4) can be used for detection of impurities. Combustion of bromopicrin: Direct scintillation counting of bromopicrin gives erratic results. It is advisable therefore to combust the CBr3NO~ to CO2 with the Van Slyke-Folch method 1~for determination of its specific activity, or to prepare a derivative, e.g., methylamine. 13 C6-F7 OF VITAMIN K. Salicylic acid (550 micromoles) is dissolved in 1.75 ml of chloroform, and 0.15 ml of concentrated nitric acid (d = 1.4) is carefully added while the mixture is being cooled to 15°-18 °. After 1 hour the chloroform solution is extracted with 2 ml of 2 N NaOH. The aqueous phase is separated and acidified with HC1, and the pale yellow precipitate is extracted into ether. 5-Nitrosalicylic acid (VI) is recrystallized from a small amount of water after the ether is evaporated and the acid sublimed i n vacuo at 95 °. The yield is 93%; m.p. 228 °. Bromopicrin cleavage yields C6+7 of vitamin K as bromopicrin. Combustion of bromopicrin is conducted as above. C5+8,6+7,9-F10 OF VITAMIN K. To an ice-cold solution of 650 micromoles of salicylic acid in 0.49 ml of concentrated H2SO4, an ice-cold mixture of 0.31 ml of concentrated HNO~ (d = 1.4) is added. After 2 hours an additional amount of 0.31 ml of concentrated HNO~ is added, and the mixture is heated for 3 hours in a boiling water bath. After chilling to 0 °, 4.2 ml of ice-water is added, and the precipitated picric acid (VII) is collected by filtration. This acid is recrystallized from 4 ml of H20. The yield is 55%; m.p. 123 °. Bromopicrin cleavage yields C5+s+6+7+9+10 of vitamin K. The specific activity of C9+19 is obtained as the difference between this value and those separately determined for C6+8 and C6+7 (see above). Degradation of V i t a m i n K to 1,~-Diacetoxy-~-methylnaphthalene-3-acetic A c i d Principle. After reductive acetylation, vitamin K may be cleaved either ozono]ytically~5,1e or by chromic acid oxidation ~7 to 1,4-diacetoxy2-methylnaphthalene-3-acetaldehyde (Fig. 2, II) or to the corresponding naphthalene-3-acetic acid. This latter method is described here. The naphthalene acetic acid may subsequently be cleaved by treatment with alkaline H202 to give phthalic and malonic acid. ~8 Procedure. ~7 Radiochemically pure vitamin K~ is diluted with inactive
14D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 15S. B. Binkley, R. W. McKee, S. A. Thayer, and E. A. Doisy, J. Biol. Chem. 133, 721 (1940). 16I). R. Threlfall, W. T. Griffiths, and T. W. Goodwin, Biochem. J. 103, 831 (1967). 1~C. Martius and W. Leuzinger, Biochem. Z. 340, 304 (1964). 18I. M. Campbell, C. J. Coscia, M. Kelsey, and R. Bentley, Biochem. Biophys. Res. Commun. 28, 25 (1967).
BIOSYNTHESIS OF VITAMIN K2
[238]
553
11 o (1)
O--LCH s CHs CHO
O--~--CH~
+
o .o (in)
+
/CHs O=C\cHs
(IV)
(H) FIG. 2. Ozonolytlc cleavage of 1,4-diacetoxyvitamin K2. commercially available carrier vitamin K1 (phylloquinone) and reductively acetylated. 19 Four hundred micromoles (or an appropriate a m o u n t ) o f dihydro vitamin K diacetate is dissolved in 4 ml of glacial acetic acid; 750 mg of fused KHSO4 is added, followed by 76 mg of Cr03 in 1 ml of 80% aqueous acetic acid. The mixture is stirred for 1 hour at 50°. The acetic acid is removed by distillation under reduced pressure, and the residue is dissolved in water and extracted with ethyl ether. The naphthalene acetic acid is extracted from the ether phase with 2% NaHCO3 solution and precipitated by acidification of the bicarbonate phase. The acid is again extracted with ether, the ether phase is washed with water and evaporated, and the product is dissolved in methanol. The solution is decolorized, if necessary, with a little charcoal, and naphthalene acetic acid is crystallized at - 5 ° from 50% aqueous methanol. Recrystallization from ether-light petroleum and from benzene gives a pure product. The yield is 23-50%; m.p. 209 °. This acid can be separated from 1,4-diacetoxynaphthalene-3-acetic acid, which would originate from demethyl vitamin K during this degradation, by thin-layer chromatography in the solvent system benzene-propanol-glacial acetic acid (100: 20:1).17 1,4-Diacetoxy-2-methylnaphthalene-3-acetic acid is cleaved by treat19For reductive acetylation of vitamin K see Vol. VI, p. 303.
554
VITAMIN K GROUP
[238]
ment with alkaline I-I20220 to give phthalic acid and malonic acid, the latter representing C-3 of the naphthalene ring and carbon atoms 3' and 3" of the side chain, is Malonic acid is decarboxylated in quantitative yield in triethylphosphate solution ~' at 150°. The evolved COs is converted to BaC03 as above and represents a mixture of carbons 3 and 3 ~. The Side Chain
Isolation of Levulinic Aldehyde (Fig. 2) Principle. During ozonolysis of the diacetate of dihydro vitamin K2 (I) and subsequent reduction of the ozonide, the isoprenoid side chain is degraded to levulinie aldehyde (III), which is isolated and assayed as its bisdinitrophenylhydrazone. Procedure. The sample of labeled vitamin K2 which is diluted with carrier quinone is reductively acetylated 19after determination of its specific activity. The ozonolysis, decomposition of the ozonide, and isolation of the dinitrophenylhydrazone are conducted exactly as described for the degradation of tocopherols and prenylated benzoquinones? ,15,16 The yield of the hydrazone is in the range of 90%; m.p. 232 °. A satisfactory yield of the hydrazone can be obtained by this procedure starting with only 0.5 mg of the quinone. 16 Because of the quenching effect of the dinitrophenylhydrazone during direct scintillation counting, it is advantageous to combust the hydrazone by the Sch6ninger method. 22 Isolation of Acetone Principle. During the above-mentioned ozonolytic cleavage of the diacetate of vitamin K2 (I), the tail end of the side chain is liberated as acetone (IV) in a 1:1 molar ratio. Acetone is isolated as its 2,4-dinitrophenylhydrazone and assayed after conversion to iodoform. Procedure. 15 Approximately 90 micromoles of the labeled diacetate of dihydro vitamin K2 is dissolved in 4 ml of ethyl acetate in a two-necked 25-m] ground-glass flask. The flask is chilled in a mixture of dry-ice and isopropanol, and ozone is bubbled through the" solution until a persistent blue color appears ( ~ 15 minutes). Excess ozone is then removed by bubbling 02 through the solution. The reaction mixture is diluted with 20 ml of diethyl ether, and 2 drops of 2 N aqueous acetic acid are added. Subsequently 400 mg of zinc dust is added in small quantities with shaking over a period of 1 hour. After the mixture has stood for 1 hour, the zinc is filtered off at - 5 °, and the ether is distilled into 10 ml of 10% NaHSO3 at 0 °. After 20 R. Bentley, J. Biol. Chem. 238, 1889 (1963). ,1 L. W. Clark, J. Phys. Chem. 60, 1150 (1956). F. Kalberer and J. Rutsehmann, Hdv. Chim. Acta 44~ 1956 (1961).
[238]
BIOSYNTHESIS OF VITAMIN K2
555
all the ether is distilled, an additional portion of 10 ml of ether is added to the distillation flask, and the distillation is continued. This procedure is repeated a third time. Ether and NaHSO3 solution are separated in a separatory funnel, and the ether phase is washed twice, each time with 12.5 ml of 10% NariS03. The combined extracts are treated with 3 g of KOH dissolved in 10 ml of H~O, and the solution is distilled, about 25 ml being collected in the ice-cold distillate. The distillate is added to 20 ml of 6 N H2S04 containing 50 mg of 2,4-dinitrophenylhydrazine. The solution is allowed to freeze in a deep-freezer, then thawed; the yellow precipitate is filtered off and washed with dilute H2S04 and water. Yield: 70%; m.p. 124° . Acetone 2,4-dinitrophenylhydrazone may be subjected to thin-layer chromatography on silica gel G plates in the solvent system benzenepetrol ether, 30°-50 ° (3:1). The Rr value is 0.3. To determine the specific activity of the derivative, a weighed portion of the hydrazone is combusted by the SchSninger method, 22 or the derivative is converted to iodoform in the following manner. ~3 Forty milligrams of acetone 2,4-dinitrophenylhydrazone is dissolved in 2 N H2S04, and about 15 ml of this solution is slowly distilled into 7.5 ml of 1 N NaOH. To the distillate an excess of a solution of KI:I~ is added. The precipitated iodoform is collected by centrifugation and washed with distilled water. The Ring-Methyl Group The ring-methyl group of vitamin Ks can be isolated24.~: 1,4-diacetoxy2-methyl-naphthalene-3-acetic acid is submitted to Kuhn-Roth oxidation after removal of the protective acetyl groups. Schmidt degradation~ of the acetic acid, which represents carbon atoms 2 and 2' of vitamin Ks, allows the assay of the individual carbon atoms. Isotope Incorporation Studies Choice of Organisms and Feeding Procedures Vitamin K2 is widely distributed among gram-positive and gramnegative bacteria? The average content of this naphthoquinone in bacteria is of the order of 1 micromole per gram dry weight. Six different species of bacteria (Bacillus ~negaterium, Bacillus sublilis, Escherichia coli, Micrococcus lysodeikticus, Proteus vulgaris, and Sarcina lutea) with a known high s~H. Simon and H. G. Floss, "Bestimmungder Isotopverteihm~in markierten Verbindungen." Springer, Berlin, 1967. s4R. Ku_hnand H. Roth, Chem. Ber. 66, 1274 (1933). D. J. Robins, I. M. Campbell, and R. Bentley, Biochem. Biophys. Res. Commun. 39, 1081 (1970).
556
VITAMIN K GROUP
[238]
content of vitamin K2 have been tested for their ability to incorporate exogenously supplied shikimic acid-14C into vitamin K. 6 Of these, only E. coli and B. megaterium were found to incorporate shikimate to a notable extent; besides these two species, Mycobacterium phlei is known to convert this precursor into vitamin K2.18 In all other strains, penetration difficulties complicate the issue, since only little 14C activity is found in the cell mass from labeled shikimate. For precursor feeding experiments, the organisms are grown in I liter of synthetic medium containing: K2HP04 (7 g), KH2P04 (2 g), Nas-citrate.5H20 (0.6 g), MgSO4.7H20 (0.1 g), (NH4)2SO4 (1 g), glycerol (5 m]). L-Phenylalanine (16.5 rag), L-tyrosine (18.3 mg), and L-tryptophan (20.4 mg) are included in the medium to avoid metabolic drainage of shikimate into protein amino acids. The medium is autoclaved in 4-liter penicillin flasks (Schott and Gen., Mainz, Germany; No. 20551). The labeled precursor solution, sterilized by using a Millipore 0.22-~ filter, is added to the medium, and the flask is inoculated with a subculture of the organism grown in the same basal medium. The bacteria are grown at 36 ° under vigorous aeration on a platform shaker with 60 strokes per minute. The organisms are harvested in the stationary phase by centrifugation, and vitamin K2 is isolated and purified. An extremely useful organism for investigation of the later steps in vitamin K2 biosynthesis is an anaerobic naphthoquinone dependent strain of Fusiformis nigrescens (Bacterioides ~elaninogenicus). 17,2e This organism is grown in a medium containing per liter:6: trypticase (Baltimore Biological Laboratory) (27 g), Difco yeast extract (3 g), NaC1 (2 g), KH2P04 (2.5 g), K2COs (2.5 g), and hemin (5 mg). Precursors of vitamin K are added in a concentration of 0.2-2 ~g per milliliter of medium. The medium is sterilized by filtration or autoclaved. Growth proceeds at 37 ° for 2-10 days in an oxygen-free atmosphere. Synthesis of Specifically Labeled Precursors 1,~-Naphthoquinone-1,.~J4C. 2~ Commercially available a-naphthol-1-14C is diluted with carrier material to 100 micromoles, dissolved in 1 ml of methanol in a ground-glass test tube, and cooled to 0 °. To this is added a solution of 200 micromoles of Fremy salt (nitrosodisu]fonate) in 4.5 m] of H20 containing 166 micromoles of KH2P04. After 30 minutes at 0 °, the precipitated naphthoquinone is extracted into ether. The ethereal solution is" concentrated in a nitrogen stream and subjected to thin-layer chromatography in the solvent system benzene-petroleum ether, 300-50 ° (3:1). The naphthoquinone zone (RI 0.9) is eluted with dichloromethane, 2eR. J. Gibbons and L. P. Engle, Science 146, 1307 (1964). 27H. J. Teuber and N. GStz, Chem. Ber. 87, 1236 (1954).
[238]
BIOSYNTHESIS OF VITAMIN Ks
557
the solvent evaporated, and the residue is sublimed in vacuo. Yield: 40-50%. The product is radiochemicaUy pure. 1,4-Naphthoquinone-2,3,9,10-1~C. 2s,~9 Commercially available p-benzoquinone-2,3,5,6-14C is diluted with freshly sublimed carrier material to 100 micromoles in a heavy-walled 5-ml ground-glass flask. Butadiene (0.4 ml) in glacial acetic acid (0.8 ml) is added, and the mixture is left for 44 hours at room temperature. Then 0.25 ml of a dichromate-sulfuric acid mixture (4 g of Na2Cr~07 and 0.2 ml of concentrated H~SO4 in 2.5 ml of H~O) is mixed with 0.1 ml of glacial acetic acid; the mixture is added to the flask and kept at 65 ° for 30 minutes. Thereafter an additional 0.1 ml of the same mixture is added, and the content of the flask is kept for another 50 minutes at the same temperature. To the mixture 3 ml of ice water is then added; after 10 minutes at 0 ° the precipitate is filtered, washed, and dried over P205. Yield: 50%; m.p. 121°. Further purification is achieved as above; final yield: 24%. The preparation of 1,4-naphthoquinone [5,8(?)-aH] has also been reported. ~7 Scheme of Biosynthesis of Vitamin Ks Based on Isotope Incorporation Studies (Fig. 3) It is established that the ring carbon atoms of shikimie acid (I) give rise to the benzenoid ring of vitamin K. 6as,~° Furthermore, shikimate is utilized during this conversion as an intact C7 unit. 6as It is not yet clear, however, at what stage the aromatization of the ring occurs. Chorismic acid, a plausible intermediate, 3°,31seems not t~ be a precursor of 5-hydroxy1,4-naphthoquinone in Juglans regia L.,29 and neither protocatechualdehyde nor protocatechuic acid seem to be intermediates, e,~s Carbon atoms 2, 3, and 4 of the naphthalene nucleus are very probably derived from carbons 2, 3, and 4 of a-ketoglutarate, 25,3~which is incorporated by way of o-succinylbenzoie acid (II) 31since a ~4C-labeled sample of this compound was efficiently converted to bacterial menaquinones as well as quinones from higher plants. 31 Ring closure of o-succinylbenzoic acid (II) would give rise to 1,4-dihydroxy-2-naphthoic acid (III). The position of a-naphthol (IV) which has been implicated in naphthoquinone biosynthesise,83,8~ 28j. Baddiley, G. Ehrensvard, E. Klein, L. Reio, and E. Saluste, J. Biol. Chem. 183, 777 (1950). 2, E. Leistner and M. H. Zenk, Z. Naturforsch. 25b, 259 (1968). 80G. B. Cox and F. Gibson, Biochem. J. 1O0, 1 (1966). 31p. Dansette and R. Azerad, Biochem. Biophys. Res. Commun. 40, 1090 (1970). 8~I. M. Campbell, Tetrahedron Letters p. 4777 (1969). 3aW. Sandermarm,NaturwissenschaSten 53, 513 (1966). R. K. Hammond and D. C. White, J. Bacteriol. 100, 573 (1969).
558
VITAMIN K GROUP
o oo HO~
[238]
oo:
OH
~ OH (I1
0
OH
(II)
(nI)
OH
?
~ C02
~
l
////
OH
: (VIII)
(VI) / 0
OH
(Iv) (vn) FIG. 3. Hypothetical schemeof the biosynthesisof vitamin K~. is not clear. It cannot be excluded that naphthol is unspecifically converted to naphthohydroquinone (V) or naphthoquinone, which was shown to be transformed to both menadione (2-methy]naphthoquinone) and vitamin K~(45) in F~sifornds nigrescens. 17 Naphthoquinone and 2-methylnaphthoquinone [possibly as their hydroquinones (V), (VI)] are precursors of vitamin K in this organism. 85 Martins and Leuzinger were the first to demonstrate that the nuclear methyl group of vitamin K and menadione originates from the methyl group of L-methionine. This report has been confirmed in a most elegant way by Jackman, O'Brien, Cox and Gibson, ~6 using (Me-~H) methionine and a methionine auxotrophic strain of E. coli. Examination of the isolated vitamin K2(40) ( ~ 5 mg) by nuclear magnetic resonance and mass spectrometry revealed that only one methyl group is incorporated into the vitamin and that it is located at the 2 position ]vI. Lev, J. Gen. Microbiol. 20, 697 (1959). 86L. M. Jackman, J. G. O'Brien, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 141, 1 (1967).
[239]
BIOSYNTHESIS OF PHYLLOQUINONE
559
of the naphthoquinone. 2-Methylnaphthoquinone or the hydroquinone (VI) serves as the acceptor for an isoprenoid side chain to give the complete vitamin K2 molecule (VIII). 17,37 There is some evidence that demethyl vitamin K (VII) is an intermediate in vitamin Ks biosynthesis,~,88 which would place the ring methylation as the last step into the biosynthetic sequence leading to vitamin K. However, it is not yet established whether demethyl vitamin K (VII) or menadione (VI) or even both, but in different organisms, are the immediate precursors of vitamin Ks (VIII). A hypothetical scheme for the biosynthesis of vitamin Ks in bacteria based on incorporation studies is shown in Fig. 3. 8TM. Billeter, W. Bolliger, and C. Martius, Biochem. Z. $40, 290 (1964). 88O. Samuel and R. Azerad, FEBS Letters 2, 336 (1969).
[ 2 3 9 ] B i o s y n t h e s i s of P h y l l o q u i n o n e By D. R. THRELFALLand G. R. WHISTANCE Phylloquinone (vitamin K1) is a normal constituent of the photosynthetic tissues of all higher plants. It has also been reported to be present in green, brown, red, and blue-green algae. The methods described in this article are those which we have used to study the biosynthesis of phylloquinone in maize shoots, bean shoots, and ivy leaves.
Cultivation (or Source) of Biological Material and Its Exposure to Radioactive Substrates The routine experimental systems used in our investigations are greening-excised-etiolated maize or French bean shoots. These systems were chosen because in them a marked and rapid synthesis of phylloquinone takes place. Thus on exposure of etiolated maize shoots to light for 24 hours, the level of phylloquinone increases from 90 ~g/100 shoots to 140 ~g/100 shoots. ~ The details of the cultivation (or sources) of etiolated maize shoots (Zea mays), etiolated French bean shoots (Phaseolus vulgaris), and ivy leaves (Hedera helix) and their subsequent exposure to radioactive compounds are identical to those described in the article on the biosynthesis of tocopherols and biogenetically related compounds.2 1W. T. Griffiths,D. R. Threlfall, and T. W. Goodwin,Biochem. J. 103, 589 (1967). 2D. R. Threlfall and G. R. Whistance, this volume [231].