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Preparation of solid vitamin B12r by anaerobic photolysis of methylcobalamin
COMMUNICATIONS TABLE
ti7.i
I
ACTION OF PULLULANASE AND @-AMYLAMEOS Digest conditions*
Amylose
potato Potato Wheat Wheat Wheat
1 2 1 1 p-limit dextrin 2 ___----
1 1 1 1 1
Potato Potato Wheat Wheat Wheat
1 2 p-limit 1 1 p-limit 2
2 2 2 2 2
dextrin dextrin
AMYLOSE
hi”
210 440 130 160 110
-
R.,
C. T.,
Control
IBI”
!I9 !)7 85 59 88
98 88 80 80
100 100 !)7 97 9s
a Prepared as in Ref. 1. 5 1 = successive action of pullulanase and P-amylase; 2 = concurrent c See text; &amylolysis limits expressed as $‘& conversion into maltose. i. GEDDES,
SAMPLES”
Test
action
[?I"
[81"
230 670 145 185 135
96 86 i8 3 76
_-
!)6 2 i8 2 76
of these enzymes.
illumination accelerates the enzymic conversion of 1,2-propanediol to propionaldehyde catalyzed by a dioldehydrat.ase requiring cobamide coen9. BANKS, W., AND GREENWOOD, C. T., zyme (3), and react,ivates an inactive propyl-BiXMakromol. Chem. 67, 49 (1963). bound enzyme in a methyl transfer from X:10. KJ$LBERG, O., AND MANNERS, D. J., Hiochem. methyl-Ha-folate to homocysteine (4, 5). These J. (London) 86, 258 (1963). facts indicate t.he participation of a prot,ein11. BENDER, H., AND WALLENFALS, K., Biochem. homolytic bound Blz, formed by an anaerobic 2. 334, 79 (1961). cleavage of the cobalt-carbon bond of adenosyl-HI2 12. ABDULLAH, XZ., CATLEY, B. J., LEE, E. Y. C., or propyl-B13 in these systems, even if t.he prot,einR.OBYT, J., WALLENFALS, K., AND WHELAN, bound B 12r may be further reduced to the SI ate of B1t9 before react ion with the substrate. W. J., Cereal Ch,em. 43, 111 (1966). Since Blz,, as well as B12%, are unstable com13 BANKS, W., GREENWOOD,C. T., AND JONES, I. G., J. Chem. Sot. 150 (1960). pounds easily oxidized by air, it has been difficult to isolate and investigate them in their pure stat c. W. BANKS C. T. GREENL.B.OOD Although Diehl and Murie (6) obt,ained solid Department oj Chemistry B12r by evaporating its aqueous solution prepared The University from cyallocobalamin by catalytic hydrogensEdinburgh 9, Scolland tion, it was contaminated with potassium sulfat,e. Received June 18, 1966 This communication deals with the preparation of pure solid B1zr using methyl-Blz as a starting material. It has been known I hat photolysis of methyl-Blz can hardly occur under strict anaerobic condit.ions (7-Q). In the course of o,lr Preparation of Solid Vitamin B1zr by Anaerobic study on allaerobic photolysis ot alkyl-RI,, we Photolysis of Methylcobalamin have observed that the presence of alcohols, markedly accelerates the especially 2-propanol, Recent works from several laboratories strongly homolytic fission of the cobalt -carbon bond of suggest an important role of a reduced cobamide methyl-B19 yielding RI?, and a methyl radical, in the biological systems including vitamin BIZ. and concluded that the stirnulat,ion is due 10 t,hc For example, I&,, two electron reduced cobamide stabilization of the met,hyl radical by abstraotirig containing Co+, is supposed to be an active intera-hydrogen of I he alcohol (10). mediate in the enzymic formation of a cobamide Based on this fact, vitamin BIT, was readily coenzyme, adenosyl-&s (l), and to be a prosobtained ill pure solid state as follows: Met,hyl-B12 t.hetic group of N6-methyl-Hq-folate-homocysteine (200 mg) prepared by t,he ordinary method (11) methyltransferase (2). It has been shown that in 2-propanol (100 ml) was deoxygenated by GREENI~OOD,
AND
MAC-
KENZIE, S., Carbohydrate Res. 1, 71 (1965). 8. GREENWOOD, C. T., Die St&Ice 12, 169 (1960).
COMMUNICATIONS
I
i50
400
I
I
I
450
500
550
WAVE LENGTH Fra. vitamin
I
60(
1
( rnP]
1. Visible absorption Blsl (in ethanol).
I
washed once with acetone in open state, and dried; a dark brown amorphous powder was obtained. Solid vitamin -Blzr thus obtained was fairly stable to oxygen in dry state. When dissolved in oxygen-free ethanol, even after 6 days of storage on silica gel under a reduced pressure of 10 mm Hg, it gave an absorption spectrum characteristic of vitamin B12r (Fig. 1). It was sensitive to oxygen in the presence of moisture, and on standing in the atmosphere turned slowly to dark-red yielding aquocobalamin. The stability of the solid vitamin Blzr in the dry state makes us suspect whether it is in a dimerized form having no unpaired electron as is known in the case of pentacyanocobaltate (II) ion (12). Its ESR spectrum, however, shows that the substance is paramagnetic (Fig. 2), which excludes the possibility of dimerization. The spectrum
spectrum
t
2400
#
,
2600
of solid
I
I
I
2600
H
,
,
3000
I
3200
,
,
,
3400
,
3600
,
,
3eoc
(GAUSS)
FIG. 2. ESR spectrum bubbling oxygen-free nitrogen for 30 minutes, and then irradiated for 30 minutes in nitrogen atmosphere with a 300 W tungsten filament lamp at a distance of 10 cm, under cooling with ice water. Methyl-B*2 was completely converted to Blzr by this procedure. The solution was concentrated to about 15 ml under reduced pressure in nitrogen atmosphere, then acetone (200 ml) previously deoxygenated by 20 minutes of nitrogen-bubbling was added to the concentrated solution. On standing overnight, most of the Blzr was precipitated. The precipitate was collected by centrifugation,
of solid vitamin
Blr.
appears identical with that of vitamin Blzr water obtained by Hogenkamp et al. (13).
in
ACKNOWLEDGMENT The authors are indebted to Professor S. Okamura, Department of Polymer Chemist.ry, Kyoto University, for the facilities that were made available in his laboratory, and to Mr. K. Takeda for his technical help in ESR studies. REFERENCES 1. VITOLS, E., WALKER,G. A., ANDHUENNEKENS, F. M., J. Riol. Chem. 241, 1455 (1966).
COMMUNICATIONS 2. BKOT, N., AND WEISSBACH, H., J. Biol. Chem. 241, 2024 (1966). 3. YAMANE, T., SHIMIZU, S., AND FUKUI, S., Biochim. Biophys. Acta 110, 616 (1965). -1. WEISSBACH, H., REDFIELD, B. G., AND DICKERI\~AN, II., Biochem. Biophys. Res. Commun. 17, 17 (1964). 5. WEISSBACH, H., REDFIELD, B. G., AND DICKERMAN, H., J. Biol. Chem. 239, 146 (1964). 13. DIEHL, H.. AND MURIE, R., Iozoa Stale Coil. J. Sci. 26, 555 (1952). 7. I)~LPHIX, D., JOHNSON, A. W., AND RODRIGO, R., J. Chem. Sot. 3186 (1964). 8. JOHKSON, A. W., OLDFIELD, D., RODRIGO, R., AND SHAW, N., J. Chem. Sot. 4080 (1964). 9. PBATT, J. M., J. Chem. Sot. 5154 (1964). 10. YAMADA, R., SHIMIZU, S., AND FTJKUI, S., Biochim. Biophys. Acta 124, 195 (1966). 11. SMITH, E. L., MERVYN, I,., JOHBSON, A. W., AND S~aw, N., L%‘&we 194, 1175 (1962). 12. ADAMSOS, A. W., J. Am. Chem. Sot. 73, 5710 (1951). 13. HOGENKAYP, II. P. C., BARKER, H. A., AND MASON, H. S., Arch. Biochem. Biophys. 110, 353 (1963). RYO-HEI YAMADA SHOICHI SHIMIZU SABUR~ F~KUI Laboratory of Industrial Biochemistry Department of Industrial Chemistry Faculty of Engineering h7yoto University h-yolo, Japan Received August 4, 1966
y-Methylaminobutyraldehyde, intermediate
in Nicotine
a New Biosynthesis
It has been well established that the pyrrolidine ritlg of nicotine is derived from ornithine (1, 2), putrescine (3), and derivatives of glutamic acid (4), and it is generally accepted that nornicotine is an intermediate precursor of nicotine (5, 6). However, our previous paper (7) demonstrated that while nicotine always occurs in the pure Z-form in tobacco plants, nornicotine isolated from tobacco roots is predominantly d-. We also reported that radioactive nicotine and nornicotine isolated from sterile root cultures of A’icotiana rustica fed dl-ornit,hine-2-14C differed in the dist,ribution of radioactivity between C-2 and C-5 in the pyrrolidine ring, and that only small amounts of 15N were incorporated into nicotine by administration of 16N’-labeled nornicotine (8). These findings led to the inference
(iiT
that, the biosynthesis of nicotine occurs through a route(s) that does not involve nornicotine. The present paper shows that the biosynthesis of nicotine involves y-methylaminobutyraldehyde (IV-methylpyrroline) as an intermediat,e in 1obacco plants. dl-Ornithine-2-l% dissolved in a small amoutI(, of water was administered either to sterile root cultures of i+‘. rustica var. Brasilia or detached roots of hydroponically grown AT. labacum 1,. (Bright. Yellow). After incubafion for l-6 hollrs :II. 30” as described in a previotls paper 18). a sr~111hle fraction was obtained by repeated extract ion of the roots with 70y0 ethanol containing 0.2% acetic: acid. The metabolites nerr analyzed 011 :L Hrckman-Ppinco amino acid analyzer alld :i Packard Tri-Carb Flow Monitor System. III I he basic fraction (t,he eluate with 0.38 N, pH 4.26 citrate buffer at 30-5O”j the radioactivity was observed in r-aminobutyric acid, ornithine, an unknown substance (termed Y-2), and arginine. Y-2 wv:ts eluted between tryptophan and argitlitle and gave a yellow color with ninhydrin. Furthermore, Y-2 was also rapidly labeled on feeding either put,rcseine-1,4-l”C or mcthionine-14C11:~ t 0 1he escisrd root,s rltlder the SUE conditions. On the ot.ller hand, Ileither nicotinir aritl-l;-3H liar carboxy IrC, the pyridine ring of which serves as a precursor of the pyridille ring of nicotine (0). gave radioact,ive Y-2. Y-2 does not absorb in t,he Illtraviolrt Upon administ,rat,irm of isolated, radioact ivc Y-2, as nillch as 330/, of the t,racer was found ii, Ilicrltine after 6 hours. No Y-2 was defected ill f,hc, aerial parts of fol)acco plants. These observations seem to itldicatc 1hal Y-2 is a precursor of the pyrrolidine ring and 1htx methyl grr)llp of nicotine. Also, Y-2 sho111d possess a butane skeleton derived from ornithiue or !“Itrescine and a methyl grollp derived from mcthionille, tnlt neither a pyriditle ring or a carboxyl group from rlicot,inic acid. The elut iotl positions of Y-2 and auf hentic -,-methylamit~ohu1 ylaldchyde on the amino acid analyzer were ident ical. After addit ion of the authenl ic compound to ratiicjactive Y-2, the 2,4-dinitropllenylhydrazone irn.Il. 168.%170”) was obt.ained by the IISII~~ method. activily persisted 1hrough Constant specific several recrystallizations of the derivative. l’ht: identity of the compound was further confirmed by paper chromatography. An ethanolic extract of the detached root, of hydroponically grow!l N. labacum fed prltreaciue-1,4-l% was applied t t) a column (1.4 X 16 cm) of I)owcx-50 X 4 III’ form) and, after t,horoltghly washing with wtttcr, the column was elated wit,h 1 N IICl and the: effluent was collected in 50 fractions of 5 ml c:rch. Fractions 27-38 were combined and concent rat orl in ZIQCUO.Co-chromatography isolvctl t , t>lltall~~l:
ti7.i
I
ACTION OF PULLULANASE AND @-AMYLAMEOS Digest conditions*
Amylose
potato Potato Wheat Wheat Wheat
1 2 1 1 p-limit dextrin 2 ___----
1 1 1 1 1
Potato Potato Wheat Wheat Wheat
1 2 p-limit 1 1 p-limit 2
2 2 2 2 2
dextrin dextrin
AMYLOSE
hi”
210 440 130 160 110
-
R.,
C. T.,
Control
IBI”
!I9 !)7 85 59 88
98 88 80 80
100 100 !)7 97 9s
a Prepared as in Ref. 1. 5 1 = successive action of pullulanase and P-amylase; 2 = concurrent c See text; &amylolysis limits expressed as $‘& conversion into maltose. i. GEDDES,
SAMPLES”
Test
action
[?I"
[81"
230 670 145 185 135
96 86 i8 3 76
_-
!)6 2 i8 2 76
of these enzymes.
illumination accelerates the enzymic conversion of 1,2-propanediol to propionaldehyde catalyzed by a dioldehydrat.ase requiring cobamide coen9. BANKS, W., AND GREENWOOD, C. T., zyme (3), and react,ivates an inactive propyl-BiXMakromol. Chem. 67, 49 (1963). bound enzyme in a methyl transfer from X:10. KJ$LBERG, O., AND MANNERS, D. J., Hiochem. methyl-Ha-folate to homocysteine (4, 5). These J. (London) 86, 258 (1963). facts indicate t.he participation of a prot,ein11. BENDER, H., AND WALLENFALS, K., Biochem. homolytic bound Blz, formed by an anaerobic 2. 334, 79 (1961). cleavage of the cobalt-carbon bond of adenosyl-HI2 12. ABDULLAH, XZ., CATLEY, B. J., LEE, E. Y. C., or propyl-B13 in these systems, even if t.he prot,einR.OBYT, J., WALLENFALS, K., AND WHELAN, bound B 12r may be further reduced to the SI ate of B1t9 before react ion with the substrate. W. J., Cereal Ch,em. 43, 111 (1966). Since Blz,, as well as B12%, are unstable com13 BANKS, W., GREENWOOD,C. T., AND JONES, I. G., J. Chem. Sot. 150 (1960). pounds easily oxidized by air, it has been difficult to isolate and investigate them in their pure stat c. W. BANKS C. T. GREENL.B.OOD Although Diehl and Murie (6) obt,ained solid Department oj Chemistry B12r by evaporating its aqueous solution prepared The University from cyallocobalamin by catalytic hydrogensEdinburgh 9, Scolland tion, it was contaminated with potassium sulfat,e. Received June 18, 1966 This communication deals with the preparation of pure solid B1zr using methyl-Blz as a starting material. It has been known I hat photolysis of methyl-Blz can hardly occur under strict anaerobic condit.ions (7-Q). In the course of o,lr Preparation of Solid Vitamin B1zr by Anaerobic study on allaerobic photolysis ot alkyl-RI,, we Photolysis of Methylcobalamin have observed that the presence of alcohols, markedly accelerates the especially 2-propanol, Recent works from several laboratories strongly homolytic fission of the cobalt -carbon bond of suggest an important role of a reduced cobamide methyl-B19 yielding RI?, and a methyl radical, in the biological systems including vitamin BIZ. and concluded that the stirnulat,ion is due 10 t,hc For example, I&,, two electron reduced cobamide stabilization of the met,hyl radical by abstraotirig containing Co+, is supposed to be an active intera-hydrogen of I he alcohol (10). mediate in the enzymic formation of a cobamide Based on this fact, vitamin BIT, was readily coenzyme, adenosyl-&s (l), and to be a prosobtained ill pure solid state as follows: Met,hyl-B12 t.hetic group of N6-methyl-Hq-folate-homocysteine (200 mg) prepared by t,he ordinary method (11) methyltransferase (2). It has been shown that in 2-propanol (100 ml) was deoxygenated by GREENI~OOD,
AND
MAC-
KENZIE, S., Carbohydrate Res. 1, 71 (1965). 8. GREENWOOD, C. T., Die St&Ice 12, 169 (1960).
COMMUNICATIONS
I
i50
400
I
I
I
450
500
550
WAVE LENGTH Fra. vitamin
I
60(
1
( rnP]
1. Visible absorption Blsl (in ethanol).
I
washed once with acetone in open state, and dried; a dark brown amorphous powder was obtained. Solid vitamin -Blzr thus obtained was fairly stable to oxygen in dry state. When dissolved in oxygen-free ethanol, even after 6 days of storage on silica gel under a reduced pressure of 10 mm Hg, it gave an absorption spectrum characteristic of vitamin B12r (Fig. 1). It was sensitive to oxygen in the presence of moisture, and on standing in the atmosphere turned slowly to dark-red yielding aquocobalamin. The stability of the solid vitamin Blzr in the dry state makes us suspect whether it is in a dimerized form having no unpaired electron as is known in the case of pentacyanocobaltate (II) ion (12). Its ESR spectrum, however, shows that the substance is paramagnetic (Fig. 2), which excludes the possibility of dimerization. The spectrum
spectrum
t
2400
#
,
2600
of solid
I
I
I
2600
H
,
,
3000
I
3200
,
,
,
3400
,
3600
,
,
3eoc
(GAUSS)
FIG. 2. ESR spectrum bubbling oxygen-free nitrogen for 30 minutes, and then irradiated for 30 minutes in nitrogen atmosphere with a 300 W tungsten filament lamp at a distance of 10 cm, under cooling with ice water. Methyl-B*2 was completely converted to Blzr by this procedure. The solution was concentrated to about 15 ml under reduced pressure in nitrogen atmosphere, then acetone (200 ml) previously deoxygenated by 20 minutes of nitrogen-bubbling was added to the concentrated solution. On standing overnight, most of the Blzr was precipitated. The precipitate was collected by centrifugation,
of solid vitamin
Blr.
appears identical with that of vitamin Blzr water obtained by Hogenkamp et al. (13).
in
ACKNOWLEDGMENT The authors are indebted to Professor S. Okamura, Department of Polymer Chemist.ry, Kyoto University, for the facilities that were made available in his laboratory, and to Mr. K. Takeda for his technical help in ESR studies. REFERENCES 1. VITOLS, E., WALKER,G. A., ANDHUENNEKENS, F. M., J. Riol. Chem. 241, 1455 (1966).
COMMUNICATIONS 2. BKOT, N., AND WEISSBACH, H., J. Biol. Chem. 241, 2024 (1966). 3. YAMANE, T., SHIMIZU, S., AND FUKUI, S., Biochim. Biophys. Acta 110, 616 (1965). -1. WEISSBACH, H., REDFIELD, B. G., AND DICKERI\~AN, II., Biochem. Biophys. Res. Commun. 17, 17 (1964). 5. WEISSBACH, H., REDFIELD, B. G., AND DICKERMAN, H., J. Biol. Chem. 239, 146 (1964). 13. DIEHL, H.. AND MURIE, R., Iozoa Stale Coil. J. Sci. 26, 555 (1952). 7. I)~LPHIX, D., JOHNSON, A. W., AND RODRIGO, R., J. Chem. Sot. 3186 (1964). 8. JOHKSON, A. W., OLDFIELD, D., RODRIGO, R., AND SHAW, N., J. Chem. Sot. 4080 (1964). 9. PBATT, J. M., J. Chem. Sot. 5154 (1964). 10. YAMADA, R., SHIMIZU, S., AND FTJKUI, S., Biochim. Biophys. Acta 124, 195 (1966). 11. SMITH, E. L., MERVYN, I,., JOHBSON, A. W., AND S~aw, N., L%‘&we 194, 1175 (1962). 12. ADAMSOS, A. W., J. Am. Chem. Sot. 73, 5710 (1951). 13. HOGENKAYP, II. P. C., BARKER, H. A., AND MASON, H. S., Arch. Biochem. Biophys. 110, 353 (1963). RYO-HEI YAMADA SHOICHI SHIMIZU SABUR~ F~KUI Laboratory of Industrial Biochemistry Department of Industrial Chemistry Faculty of Engineering h7yoto University h-yolo, Japan Received August 4, 1966
y-Methylaminobutyraldehyde, intermediate
in Nicotine
a New Biosynthesis
It has been well established that the pyrrolidine ritlg of nicotine is derived from ornithine (1, 2), putrescine (3), and derivatives of glutamic acid (4), and it is generally accepted that nornicotine is an intermediate precursor of nicotine (5, 6). However, our previous paper (7) demonstrated that while nicotine always occurs in the pure Z-form in tobacco plants, nornicotine isolated from tobacco roots is predominantly d-. We also reported that radioactive nicotine and nornicotine isolated from sterile root cultures of A’icotiana rustica fed dl-ornit,hine-2-14C differed in the dist,ribution of radioactivity between C-2 and C-5 in the pyrrolidine ring, and that only small amounts of 15N were incorporated into nicotine by administration of 16N’-labeled nornicotine (8). These findings led to the inference
(iiT
that, the biosynthesis of nicotine occurs through a route(s) that does not involve nornicotine. The present paper shows that the biosynthesis of nicotine involves y-methylaminobutyraldehyde (IV-methylpyrroline) as an intermediat,e in 1obacco plants. dl-Ornithine-2-l% dissolved in a small amoutI(, of water was administered either to sterile root cultures of i+‘. rustica var. Brasilia or detached roots of hydroponically grown AT. labacum 1,. (Bright. Yellow). After incubafion for l-6 hollrs :II. 30” as described in a previotls paper 18). a sr~111hle fraction was obtained by repeated extract ion of the roots with 70y0 ethanol containing 0.2% acetic: acid. The metabolites nerr analyzed 011 :L Hrckman-Ppinco amino acid analyzer alld :i Packard Tri-Carb Flow Monitor System. III I he basic fraction (t,he eluate with 0.38 N, pH 4.26 citrate buffer at 30-5O”j the radioactivity was observed in r-aminobutyric acid, ornithine, an unknown substance (termed Y-2), and arginine. Y-2 wv:ts eluted between tryptophan and argitlitle and gave a yellow color with ninhydrin. Furthermore, Y-2 was also rapidly labeled on feeding either put,rcseine-1,4-l”C or mcthionine-14C11:~ t 0 1he escisrd root,s rltlder the SUE conditions. On the ot.ller hand, Ileither nicotinir aritl-l;-3H liar carboxy IrC, the pyridine ring of which serves as a precursor of the pyridille ring of nicotine (0). gave radioact,ive Y-2. Y-2 does not absorb in t,he Illtraviolrt Upon administ,rat,irm of isolated, radioact ivc Y-2, as nillch as 330/, of the t,racer was found ii, Ilicrltine after 6 hours. No Y-2 was defected ill f,hc, aerial parts of fol)acco plants. These observations seem to itldicatc 1hal Y-2 is a precursor of the pyrrolidine ring and 1htx methyl grr)llp of nicotine. Also, Y-2 sho111d possess a butane skeleton derived from ornithiue or !“Itrescine and a methyl grollp derived from mcthionille, tnlt neither a pyriditle ring or a carboxyl group from rlicot,inic acid. The elut iotl positions of Y-2 and auf hentic -,-methylamit~ohu1 ylaldchyde on the amino acid analyzer were ident ical. After addit ion of the authenl ic compound to ratiicjactive Y-2, the 2,4-dinitropllenylhydrazone irn.Il. 168.%170”) was obt.ained by the IISII~~ method. activily persisted 1hrough Constant specific several recrystallizations of the derivative. l’ht: identity of the compound was further confirmed by paper chromatography. An ethanolic extract of the detached root, of hydroponically grow!l N. labacum fed prltreaciue-1,4-l% was applied t t) a column (1.4 X 16 cm) of I)owcx-50 X 4 III’ form) and, after t,horoltghly washing with wtttcr, the column was elated wit,h 1 N IICl and the: effluent was collected in 50 fractions of 5 ml c:rch. Fractions 27-38 were combined and concent rat orl in ZIQCUO.Co-chromatography isolvctl t , t>lltall~~l: