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[195c] Synthesis of O-acetylhomoserine
[195c]
SYNTHESIS OF O-ACETYLHOMOSERINE
423
[195c] Synthesis of O-Acetylhomoserine By SADAMU NAGAI and MARTIN FLAVIN
Preparation Principle. H o m o s e r i n e is acetylated with acetic a n h y d r i d e in a strongly acidic m e d i u m that prevents N-acetylation. Homoserine lactone is f o r m e d as a side product, which is readily separated. T h e general m e t h o d was described some years ago 1"2 and applied to homoserine, 3"4 but the isolation and properties of the product were not m e n t i o n e d by the original authors. T h e p r o c e d u r e below is modified from the one previously described by us. 5 Reagents
Glacial acetic acid, 99.9% Acetic anhydride, 99.9% Perchloric acid, 60-62% Homoserine, DL or L Pentylamine Procedure. To 2 ml of glacial acetic acid in a glass-stoppered tube was a d d e d 0.17 ml of perchloric acid (1.5 millimoles of perchloric acid and 5.6 millimoles of water). With slight cooling (the mixture solidifies at 14°), 0.81 ml (8.5 millimoles) of acetic a n h y d r i d e was added. Hydroxylamine assay showed that 2.1 millimoles o f a n h y d r i d e r e m a i n e d after the exothermic reaction with the water which had been a d d e d with the acetic and perchloric acids. A solution of 143 mg (1.2 millimoles) of DL-homoserine in 1 ml of glacial acetic acid was prepared and a d d e d dropwise to the a n h y d r i d e solution while agitating the latter. T h e addition was m a d e over a period of 5 minutes, at r o o m temperature. H y d r o x y l a m i n e assay showed that the calculated a m o u n t (1.2 millimoles) of a n h y d r i d e was c o n s u m e d within a few minutes of a d d i n g the homoserine. T h e stoppered mixture was kept at r o o m t e m p e r a t u r e for 90 minutes to ensure complete reaction, and the remaining a n h y d r i d e
1W. Sakamiand G. Toennies,J. Biol. Chem. 144, 203 (1942). 2.]. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," Vol. II, p. 1043, John Wileyand Sons, New York, 1961. 3y. Matsuo, M. Rothstein, and D. M. Greenberg,J. Biol. Chem. 221,679 (1956). 4N. Grobbelaar and F. C. Steward, Nature 182, 1358 (1958). 5S. Nagai and M. Flavin,J. Biol. Chem. 242, 3884 (1967).
424
SULFUR AMINO ACIDS
[ 195c]
was then decomposed by adding 0.022 ml (1.2 millimoles) of water. A positive hydroxylamine reaction at this stage indicates the presence of homoserine lactone. Relatively little of the latter is formed when the reaction is carried out by adding homoserine to the anhydride solution as described above. To neutralize the perchloric acid, 0.20 ml (1.8 millimoles) of pentylamine was added. After the addition of 30 ml of ether, the solution was kept at 0 ° overnight and then chilled to --15 ° after adding an additional 15 ml of ether. The granular precipitate was recovered on a sintered-glass filter, washed with ether, and dried in a desiccator. Paper chromatography in pyridine-water, 8:2, showed the presence of only a trace of homoserine lactone, and no free homoserine. This crude product (160 mg, 83% yield) was dissolved in 1.5 ml of water, and the solution was filtered to remove any insoluble material. Ten milliliters of ethanol was added, and the mixture was left overnight at 0 ° to aid crystallization; homoserine lactone perchlorate remains in solution under these conditions. O-Acetyl-DL-homoserine (116 mg, 60% yield) was obtained as thin hexagonal plates, m.p. 182-184 ° (with decomposition; uncorrected). O-Acetyl-L-homoserine has been prepared by the same procedure; 5 its specific rotation, measured at 24 ° on a 1% solution in water, was +14.9 ° at 436 m/.~, and +28.2 ° at 350 m~. 5 The solid state infrared spectra of the L and DL compounds have been published. 5 The compound appears to be stable indefinitely if stored in the cold. Aqueous solutions have been kept frozen at pH 5 for several months with minimal decomposition. Brief alkaline treatment (pH 12, 100 ° for 1 minute) results in complete conversion to N-acetylhomoserine. This property is useful for identification or assay, since the product lacks a free amino group. Other procedures for determining O-acetylhomoserine are the same as those described for O-succinylhomoserine (this volume [195b]).
SYNTHESIS OF O-ACETYLHOMOSERINE
423
[195c] Synthesis of O-Acetylhomoserine By SADAMU NAGAI and MARTIN FLAVIN
Preparation Principle. H o m o s e r i n e is acetylated with acetic a n h y d r i d e in a strongly acidic m e d i u m that prevents N-acetylation. Homoserine lactone is f o r m e d as a side product, which is readily separated. T h e general m e t h o d was described some years ago 1"2 and applied to homoserine, 3"4 but the isolation and properties of the product were not m e n t i o n e d by the original authors. T h e p r o c e d u r e below is modified from the one previously described by us. 5 Reagents
Glacial acetic acid, 99.9% Acetic anhydride, 99.9% Perchloric acid, 60-62% Homoserine, DL or L Pentylamine Procedure. To 2 ml of glacial acetic acid in a glass-stoppered tube was a d d e d 0.17 ml of perchloric acid (1.5 millimoles of perchloric acid and 5.6 millimoles of water). With slight cooling (the mixture solidifies at 14°), 0.81 ml (8.5 millimoles) of acetic a n h y d r i d e was added. Hydroxylamine assay showed that 2.1 millimoles o f a n h y d r i d e r e m a i n e d after the exothermic reaction with the water which had been a d d e d with the acetic and perchloric acids. A solution of 143 mg (1.2 millimoles) of DL-homoserine in 1 ml of glacial acetic acid was prepared and a d d e d dropwise to the a n h y d r i d e solution while agitating the latter. T h e addition was m a d e over a period of 5 minutes, at r o o m temperature. H y d r o x y l a m i n e assay showed that the calculated a m o u n t (1.2 millimoles) of a n h y d r i d e was c o n s u m e d within a few minutes of a d d i n g the homoserine. T h e stoppered mixture was kept at r o o m t e m p e r a t u r e for 90 minutes to ensure complete reaction, and the remaining a n h y d r i d e
1W. Sakamiand G. Toennies,J. Biol. Chem. 144, 203 (1942). 2.]. p. Greenstein and M. Winitz, "Chemistry of the Amino Acids," Vol. II, p. 1043, John Wileyand Sons, New York, 1961. 3y. Matsuo, M. Rothstein, and D. M. Greenberg,J. Biol. Chem. 221,679 (1956). 4N. Grobbelaar and F. C. Steward, Nature 182, 1358 (1958). 5S. Nagai and M. Flavin,J. Biol. Chem. 242, 3884 (1967).
424
SULFUR AMINO ACIDS
[ 195c]
was then decomposed by adding 0.022 ml (1.2 millimoles) of water. A positive hydroxylamine reaction at this stage indicates the presence of homoserine lactone. Relatively little of the latter is formed when the reaction is carried out by adding homoserine to the anhydride solution as described above. To neutralize the perchloric acid, 0.20 ml (1.8 millimoles) of pentylamine was added. After the addition of 30 ml of ether, the solution was kept at 0 ° overnight and then chilled to --15 ° after adding an additional 15 ml of ether. The granular precipitate was recovered on a sintered-glass filter, washed with ether, and dried in a desiccator. Paper chromatography in pyridine-water, 8:2, showed the presence of only a trace of homoserine lactone, and no free homoserine. This crude product (160 mg, 83% yield) was dissolved in 1.5 ml of water, and the solution was filtered to remove any insoluble material. Ten milliliters of ethanol was added, and the mixture was left overnight at 0 ° to aid crystallization; homoserine lactone perchlorate remains in solution under these conditions. O-Acetyl-DL-homoserine (116 mg, 60% yield) was obtained as thin hexagonal plates, m.p. 182-184 ° (with decomposition; uncorrected). O-Acetyl-L-homoserine has been prepared by the same procedure; 5 its specific rotation, measured at 24 ° on a 1% solution in water, was +14.9 ° at 436 m/.~, and +28.2 ° at 350 m~. 5 The solid state infrared spectra of the L and DL compounds have been published. 5 The compound appears to be stable indefinitely if stored in the cold. Aqueous solutions have been kept frozen at pH 5 for several months with minimal decomposition. Brief alkaline treatment (pH 12, 100 ° for 1 minute) results in complete conversion to N-acetylhomoserine. This property is useful for identification or assay, since the product lacks a free amino group. Other procedures for determining O-acetylhomoserine are the same as those described for O-succinylhomoserine (this volume [195b]).