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Identification of methyl-p-coumarate as a metabolic product of Lentinus lepideus
LETTERS
TO
THE
263
EDITORS
REFERENCES 1. STEELMAN, S. L., KELLY, T. L., SEGALOFF, A., AND WEBER, G. F., Endocrinology 69, 256 (1956). 2. RIGAS, D. A.? PAULSON, C. A., AND HELLER, C. G., Endocrinology 62,738 (1958). 3. APOSTOLAKIS, M., AND VOIGT, K., Acta Endocrinol. 28, 54 (1958). 4. KOENIG, V. L., AND KING, E., Arch. Biochem. 26, 219 (1950). 5. RABEN, M. S., Science 126, 883 (1957). 6. STEELMAN, S. L., AND POHLEY, F. M., Endocrinology 63, 604 (1953). Departments of Biochemistry, Baylor University College of Medicine, and M. D. Anderson Hospital and Tumor Houston, Texas, and The Alton Ochsner Medical Foundation, New Orleans, Louisiana Received August 11, 1968
Identification
of Methyl-p-Coumarate Lentinus
SANFORD L. STEELMAN ALBERT SEGALOFF M. GAYLE MAYS
Institute,
as a Metabolic
Lepidet&
Product
of
2, 3
When the wood-destroying fungus Lentinus Zepideus is grown on a glucose-peptone medium in surface culture, a gradually increasing amount of a crystalline deposit is observed during the course of the growth of the mycelium. The crystals have been identified as methyl p-methoxycinnamate (1, 2). If the mycelia and crystals are allowed to incubate for several months, these crystals eventually disappear. It has now been observed that, if the culture flasks in which the crystals have accumulated are continuously shaken, the amount of the deposit diminishes rapidly, and, after a few days, has almost completely disappeared. When, after 1 or 2 days of shaking, the media in the flasks were extracted with ether, the presence of a small amount of a hitherto undetected phenolic compound was determined by paper chromatography. This phenolic compound was isolated, purified and identified as methyl p-coumarate; m. p., 137-139’C.; ultraviolet absorption: maximum, 310 rnp, minimum, 246 rnp (in water). However, this compound could not be detected in a surface culture medium, neither after continued shaking, nor if the media were shaken immediately after inoculation. It was further observed that if methyl p-coumarate were added to a mycelial extract, this compound was subject to a very rapid oxidation. The clear mycelial extract was prepared from the mycelium of a a-day shake culture. This 1 This study was supported by grants of the National Science Foundation, the U. S. Public Health Service, and the U. S. Atomic Energy Commission. z Communication No. 351. 3 The authors wish to thank Dr. Wm. J. Robbins of the New York Botanical Garden for the culture of Lent&us Zepideus used in this study. H. Shimazono wishes to thank Dr. Walter J. Schubert of this Department for discussions.
264
LETTERS
TO
THE
EDITORS
extract was added to 3 ml. of 5 X lo-* M methyl p-coumarate in a citrate-phosphate buffer (pH 5.0), and the optical density was determined spectrophotometrically. After 2 hr., the peak of methyl p-coumarate at 310 rnp had almost disappeared, and the color of the reaction medium became darker. The phenolic compound could be detected under certain specific conditions by paper chromatography. It may be visualized that the phenolase enzyme which oxidizes methyl p-coumarate under these circumstances requires a lag phase during the early stages of the shaking period, resulting in the persistence of small amounts of this compound. It has recently been reported (3) that methyl p-coumarate is nitrated to methyl 3-nitro-4-hydroxycinnamate by nitrite, which was thought to be formed from nitrate by a nitrate reductase in certain plant tissues. However, in the case of Lentinus lepideus, this compound is subject to the activity of a phenolase enzyme. Methyl p-coumarate may then be regarded as an intermediate in the metabolism of methyl p-methoxycinnamate by Lent&us Zepideus. and also possibly in the biosynthesis of that compound. It might therefore be considered that, in the first step of the metabolism of methyl p-methoxycinnamate, this compound may be demethylated to methyl p-coumarate, and t.hen, this latter compound may be further oxidized by a phenolase enzyme, possibly tgrosinase. Such methylation and demethylation of phenolic compounds might indeed have a bearing on the mechanism of the biogenesis of lignin, and also on the metabolism of that substance by wood-destroying fungi. REFEREXES 1. NORD, F. F., AND VITUCCI, J. C., flrch. B&hem. 14, 243 (1947); 2. SHIMAZONO, H., SCHUBERT, W. J., AND NERD, F. F., J. Am. 1992 (1958). 3. ZIOUDROU, C., MEYER, W. I,., AND FRUTON, J. S., ,I. Am. Chem. (1957); ZIOUDROU, C., AND FRUTON, J. S., J. Am. Chem. Sot. Department of Organic Chemistry and Enzymology, Pordham University, New York, New York Received September dB, 1958
16, 465 (1947). Chem. Sot. 80,
79,
Sot. 79, 4114 5951 (1957).
H. SHIMAXONO F. F. Nom
TO
THE
263
EDITORS
REFERENCES 1. STEELMAN, S. L., KELLY, T. L., SEGALOFF, A., AND WEBER, G. F., Endocrinology 69, 256 (1956). 2. RIGAS, D. A.? PAULSON, C. A., AND HELLER, C. G., Endocrinology 62,738 (1958). 3. APOSTOLAKIS, M., AND VOIGT, K., Acta Endocrinol. 28, 54 (1958). 4. KOENIG, V. L., AND KING, E., Arch. Biochem. 26, 219 (1950). 5. RABEN, M. S., Science 126, 883 (1957). 6. STEELMAN, S. L., AND POHLEY, F. M., Endocrinology 63, 604 (1953). Departments of Biochemistry, Baylor University College of Medicine, and M. D. Anderson Hospital and Tumor Houston, Texas, and The Alton Ochsner Medical Foundation, New Orleans, Louisiana Received August 11, 1968
Identification
of Methyl-p-Coumarate Lentinus
SANFORD L. STEELMAN ALBERT SEGALOFF M. GAYLE MAYS
Institute,
as a Metabolic
Lepidet&
Product
of
2, 3
When the wood-destroying fungus Lentinus Zepideus is grown on a glucose-peptone medium in surface culture, a gradually increasing amount of a crystalline deposit is observed during the course of the growth of the mycelium. The crystals have been identified as methyl p-methoxycinnamate (1, 2). If the mycelia and crystals are allowed to incubate for several months, these crystals eventually disappear. It has now been observed that, if the culture flasks in which the crystals have accumulated are continuously shaken, the amount of the deposit diminishes rapidly, and, after a few days, has almost completely disappeared. When, after 1 or 2 days of shaking, the media in the flasks were extracted with ether, the presence of a small amount of a hitherto undetected phenolic compound was determined by paper chromatography. This phenolic compound was isolated, purified and identified as methyl p-coumarate; m. p., 137-139’C.; ultraviolet absorption: maximum, 310 rnp, minimum, 246 rnp (in water). However, this compound could not be detected in a surface culture medium, neither after continued shaking, nor if the media were shaken immediately after inoculation. It was further observed that if methyl p-coumarate were added to a mycelial extract, this compound was subject to a very rapid oxidation. The clear mycelial extract was prepared from the mycelium of a a-day shake culture. This 1 This study was supported by grants of the National Science Foundation, the U. S. Public Health Service, and the U. S. Atomic Energy Commission. z Communication No. 351. 3 The authors wish to thank Dr. Wm. J. Robbins of the New York Botanical Garden for the culture of Lent&us Zepideus used in this study. H. Shimazono wishes to thank Dr. Walter J. Schubert of this Department for discussions.
264
LETTERS
TO
THE
EDITORS
extract was added to 3 ml. of 5 X lo-* M methyl p-coumarate in a citrate-phosphate buffer (pH 5.0), and the optical density was determined spectrophotometrically. After 2 hr., the peak of methyl p-coumarate at 310 rnp had almost disappeared, and the color of the reaction medium became darker. The phenolic compound could be detected under certain specific conditions by paper chromatography. It may be visualized that the phenolase enzyme which oxidizes methyl p-coumarate under these circumstances requires a lag phase during the early stages of the shaking period, resulting in the persistence of small amounts of this compound. It has recently been reported (3) that methyl p-coumarate is nitrated to methyl 3-nitro-4-hydroxycinnamate by nitrite, which was thought to be formed from nitrate by a nitrate reductase in certain plant tissues. However, in the case of Lentinus lepideus, this compound is subject to the activity of a phenolase enzyme. Methyl p-coumarate may then be regarded as an intermediate in the metabolism of methyl p-methoxycinnamate by Lent&us Zepideus. and also possibly in the biosynthesis of that compound. It might therefore be considered that, in the first step of the metabolism of methyl p-methoxycinnamate, this compound may be demethylated to methyl p-coumarate, and t.hen, this latter compound may be further oxidized by a phenolase enzyme, possibly tgrosinase. Such methylation and demethylation of phenolic compounds might indeed have a bearing on the mechanism of the biogenesis of lignin, and also on the metabolism of that substance by wood-destroying fungi. REFEREXES 1. NORD, F. F., AND VITUCCI, J. C., flrch. B&hem. 14, 243 (1947); 2. SHIMAZONO, H., SCHUBERT, W. J., AND NERD, F. F., J. Am. 1992 (1958). 3. ZIOUDROU, C., MEYER, W. I,., AND FRUTON, J. S., ,I. Am. Chem. (1957); ZIOUDROU, C., AND FRUTON, J. S., J. Am. Chem. Sot. Department of Organic Chemistry and Enzymology, Pordham University, New York, New York Received September dB, 1958
16, 465 (1947). Chem. Sot. 80,
79,
Sot. 79, 4114 5951 (1957).
H. SHIMAXONO F. F. Nom