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Reaction product of acetoacetyl coenzyme A and hydroxylamine
514
SlIOkT
COMMUNICATIO&S
wavelength, the method is considerably less sensitive (Fig. 2)) and increasing the mannose concentrations results in turbid solutions. The responses of the sugars enumerated are not significantly different from that of the manual procedure (a), even though the heating times differ somewhat between the two methods. As in the manual method, the protein does not interfere. No difference in response to mannose is seen in the presence or absence of /3-lactoglobulin and recoveries of mannose added to ovalbumin are quantitative. The analysis for mannose in ovalbumin by the automated methods gives consistently lower results than that obtained in the manual procedure (3). The procedure is highly reproducible, as is indicated by Fig. 2, in which the standard curve is drawn through two sets of points obtained on two independent occasions. The method has proven especially useful in the analysis of column fractions for glycopeptides. In such instances the water tube between samples can be omitted and satisfactory plots of elution patt.erns can be obtained at the full speed of 40 samples per hour. REFERENCES 1. LUSTIG, B., AND LANCER, A., Bioehem. 2. WINZLER, R. J., Methods of Biochem. 3. NUENKE, R. H., AND CUNNINGHAM,
2. 242, 320 (1931). Anal. 2, 279 (1955). L. W., J. Biol. Chem.
J. w.
236,
2452
(1961).
JUDD CLOUSE
J. FORD J. VAN EYS” L. W. CUNNINGHAM Department Vanderbilt
Nashville, Received
of Biochemistry University Tennessee September 7, 1962
3 Investigator,
Reaction
Howard
Hughes Medical
Product and
Institute.
of Acetoacetyl
Coenzyme
A
Hydroxylaminel
Acyl derivatives of coenzyme A (CoA) , which are intermediates in the metabolism of fatty acids, have been conveniently identified as the products of their reaction with hydroxylamine. In this reaction, while the esters of saturated, @unsaturated, and P-hydroxy acids are well known to yield the corresponding hydroxamic acids, the product with acetoacetyl
SHORT
515
COMMUNICATIONS
coenzyme A has not been unequivocally identified. This product is referred to as acetoacetyl hydroxamate by Green (1) and Hele et al. (2), and as methyl isoxazolone by Stadtman (3). Mahler, without specifying the reaction product, stated that P-ketoacyl derivatives of CoA did not form hydroxamates (4). A survey of lit.erature did not reveal any experimental evidence establishing the identity of this compound. While consideration of organic reaction mechanisms would indicate isoxazolones to be the most likely product, it remained to be conclusively shown that hydroxylamine reacts exclusively with the keto group of the /3-ketoacyl CoA forming the corresponding oxime, which then undergoes cyclization. The work reported here offers evidence in support of the suggested formation of methyl isoxazolone in the reaction between acetoncetyl thiol esters and hydroxylamine. Methyl isoxazolone was prepared according to the procedure described by Hantzsch (5) with minor modifications. Distilled ethyl acetoacetate (3.4 mmoles) was added t,o 3 ml of an aqueous solution containing 4.4 mmoles each of NH,OHaHCl and NaOH. The reaction mixture was kept in a water bath at 37°C for 18 hr and was then carefully acidified with concentrated HCI. The yellow, crystalline, needle-like compound formed was purified by repeat,ed crystallization from hot wat,er. The melt.ing point of the pure product was found to be 169-171°C (reported, 169-170°C). Acetoacetylthioglycolic acid, a model compound of acetoacetyl CoA, was prepared as follows: 2.3 gm of thioglycolic acid, neutralized to pH 7.5 with 7 N NaOH, was cooled in crushed ice and 2.2 gm of distilled, colorless, diketene was added to it dropwise with constant stirring. The reaction was allowed to proceed for 30 min in the cold with stirring until the top layer of diketene disappeared. More diketene was added, if necessary, until all the thioglycolic acid had reacted as indicated by the sodium nitroprusside test. NH,OH*HCI solution (2.3 gm), adjusted to pH 10.5, was then added to the acetoacetylthioglycolic acid formed and the mixture was acidified as described earlier. A yellow crystalline substance separated which, on recrystallization, gave a product with melting point 168-17O”C, suggesting that the compound was methyl isoxazolone. Determination of the ult,raviolet absorption spectrum revealed this compound to have the same absorption peak (288-290 rnp1 as methyl isoxazolone, showing the two compounds to be identical. In order to check its chromatographic behavior, Clb-labeled methyl isoxazolone was prepared from ethyl acetoacetate-3-CP. Identity of this ’ Supported research grant.
by the A-3504.
National
Institute
P of
Health.
IT, S, Public
Health
Service
516
SHORT COMMUNICATIONS
compound was established from its melting point, 16S--170°C, and its absorption spectrum, which was identical to that of the authentic methyl isoxazolone. When the labeled methyl isoxazolone was chromatographed on filter paper using a solvent system consisting of methylene chloride, butanol, acetic acid, and water2 and the chromatogram scanned for radioactivity with an actigraph, a sharp peak having an Rf of 0.50 rt 0.03 was obtained. A similar radioactive peak (22, 0.50 + 0.03) had been found invariably in the chromatograms of the extracts of hydroxamates formed in the mammary slices of lactating rabbits and goats incubated with Cl”labeled acetate, P-hydroxybutyrate, and butyrate. This peak did not correspond to any of the known hydroxamates. The attempts made for the identification of this peak had, in fact, resulted in the present investigation. The region corresponding to this peak was eluted from several chromatograms with ethanol, pooled, and concentrated. This was cochromatographed with authentic CY4-labeled methyl isoxazolone using two different solvent systems, butanol saturated with water, and benzene:formic acid:water (1: 1: 1). Only one radioactive peak (Rf 0.58 and 0.0, respectively) was obtained with each of the solvent mixture. This shows that acetoacetyl CoA of the tissue had formed methyl isoxazolone under the same conditions in which the nonketo thiol esters formed hydroxamic acids. REFERENCES 1. GREEN,
D.
E., Science 115, 664 (1952). 2. HELE, P,, POPJAK, G., AND LAURYSSENS, M., Rio&em. J. 65, 348 (1957). 3. STADTMAN, E. R., in “Methods in Enzvmologv” (8. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 932. Academic Press, New York, 1955. 4. MAHLER, H. R., Federation Proc. 12, 694 (1953). 5. HANTZSCH, A., Ber. 24, 495 (1891).
REMEDIOS M. SOMAKUMAR Chemistry Georgetown Washington, Received
AVENA~
Department University D. C. August 28, 1962
‘Unpublished method for the separation of short chain 3 This work is taken from a thesis submitted to Georgetown fulfillment of the degree of Doctor of Philosophy.
acyl hydroxamates. University in partial
SlIOkT
COMMUNICATIO&S
wavelength, the method is considerably less sensitive (Fig. 2)) and increasing the mannose concentrations results in turbid solutions. The responses of the sugars enumerated are not significantly different from that of the manual procedure (a), even though the heating times differ somewhat between the two methods. As in the manual method, the protein does not interfere. No difference in response to mannose is seen in the presence or absence of /3-lactoglobulin and recoveries of mannose added to ovalbumin are quantitative. The analysis for mannose in ovalbumin by the automated methods gives consistently lower results than that obtained in the manual procedure (3). The procedure is highly reproducible, as is indicated by Fig. 2, in which the standard curve is drawn through two sets of points obtained on two independent occasions. The method has proven especially useful in the analysis of column fractions for glycopeptides. In such instances the water tube between samples can be omitted and satisfactory plots of elution patt.erns can be obtained at the full speed of 40 samples per hour. REFERENCES 1. LUSTIG, B., AND LANCER, A., Bioehem. 2. WINZLER, R. J., Methods of Biochem. 3. NUENKE, R. H., AND CUNNINGHAM,
2. 242, 320 (1931). Anal. 2, 279 (1955). L. W., J. Biol. Chem.
J. w.
236,
2452
(1961).
JUDD CLOUSE
J. FORD J. VAN EYS” L. W. CUNNINGHAM Department Vanderbilt
Nashville, Received
of Biochemistry University Tennessee September 7, 1962
3 Investigator,
Reaction
Howard
Hughes Medical
Product and
Institute.
of Acetoacetyl
Coenzyme
A
Hydroxylaminel
Acyl derivatives of coenzyme A (CoA) , which are intermediates in the metabolism of fatty acids, have been conveniently identified as the products of their reaction with hydroxylamine. In this reaction, while the esters of saturated, @unsaturated, and P-hydroxy acids are well known to yield the corresponding hydroxamic acids, the product with acetoacetyl
SHORT
515
COMMUNICATIONS
coenzyme A has not been unequivocally identified. This product is referred to as acetoacetyl hydroxamate by Green (1) and Hele et al. (2), and as methyl isoxazolone by Stadtman (3). Mahler, without specifying the reaction product, stated that P-ketoacyl derivatives of CoA did not form hydroxamates (4). A survey of lit.erature did not reveal any experimental evidence establishing the identity of this compound. While consideration of organic reaction mechanisms would indicate isoxazolones to be the most likely product, it remained to be conclusively shown that hydroxylamine reacts exclusively with the keto group of the /3-ketoacyl CoA forming the corresponding oxime, which then undergoes cyclization. The work reported here offers evidence in support of the suggested formation of methyl isoxazolone in the reaction between acetoncetyl thiol esters and hydroxylamine. Methyl isoxazolone was prepared according to the procedure described by Hantzsch (5) with minor modifications. Distilled ethyl acetoacetate (3.4 mmoles) was added t,o 3 ml of an aqueous solution containing 4.4 mmoles each of NH,OHaHCl and NaOH. The reaction mixture was kept in a water bath at 37°C for 18 hr and was then carefully acidified with concentrated HCI. The yellow, crystalline, needle-like compound formed was purified by repeat,ed crystallization from hot wat,er. The melt.ing point of the pure product was found to be 169-171°C (reported, 169-170°C). Acetoacetylthioglycolic acid, a model compound of acetoacetyl CoA, was prepared as follows: 2.3 gm of thioglycolic acid, neutralized to pH 7.5 with 7 N NaOH, was cooled in crushed ice and 2.2 gm of distilled, colorless, diketene was added to it dropwise with constant stirring. The reaction was allowed to proceed for 30 min in the cold with stirring until the top layer of diketene disappeared. More diketene was added, if necessary, until all the thioglycolic acid had reacted as indicated by the sodium nitroprusside test. NH,OH*HCI solution (2.3 gm), adjusted to pH 10.5, was then added to the acetoacetylthioglycolic acid formed and the mixture was acidified as described earlier. A yellow crystalline substance separated which, on recrystallization, gave a product with melting point 168-17O”C, suggesting that the compound was methyl isoxazolone. Determination of the ult,raviolet absorption spectrum revealed this compound to have the same absorption peak (288-290 rnp1 as methyl isoxazolone, showing the two compounds to be identical. In order to check its chromatographic behavior, Clb-labeled methyl isoxazolone was prepared from ethyl acetoacetate-3-CP. Identity of this ’ Supported research grant.
by the A-3504.
National
Institute
P of
Health.
IT, S, Public
Health
Service
516
SHORT COMMUNICATIONS
compound was established from its melting point, 16S--170°C, and its absorption spectrum, which was identical to that of the authentic methyl isoxazolone. When the labeled methyl isoxazolone was chromatographed on filter paper using a solvent system consisting of methylene chloride, butanol, acetic acid, and water2 and the chromatogram scanned for radioactivity with an actigraph, a sharp peak having an Rf of 0.50 rt 0.03 was obtained. A similar radioactive peak (22, 0.50 + 0.03) had been found invariably in the chromatograms of the extracts of hydroxamates formed in the mammary slices of lactating rabbits and goats incubated with Cl”labeled acetate, P-hydroxybutyrate, and butyrate. This peak did not correspond to any of the known hydroxamates. The attempts made for the identification of this peak had, in fact, resulted in the present investigation. The region corresponding to this peak was eluted from several chromatograms with ethanol, pooled, and concentrated. This was cochromatographed with authentic CY4-labeled methyl isoxazolone using two different solvent systems, butanol saturated with water, and benzene:formic acid:water (1: 1: 1). Only one radioactive peak (Rf 0.58 and 0.0, respectively) was obtained with each of the solvent mixture. This shows that acetoacetyl CoA of the tissue had formed methyl isoxazolone under the same conditions in which the nonketo thiol esters formed hydroxamic acids. REFERENCES 1. GREEN,
D.
E., Science 115, 664 (1952). 2. HELE, P,, POPJAK, G., AND LAURYSSENS, M., Rio&em. J. 65, 348 (1957). 3. STADTMAN, E. R., in “Methods in Enzvmologv” (8. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 932. Academic Press, New York, 1955. 4. MAHLER, H. R., Federation Proc. 12, 694 (1953). 5. HANTZSCH, A., Ber. 24, 495 (1891).
REMEDIOS M. SOMAKUMAR Chemistry Georgetown Washington, Received
AVENA~
Department University D. C. August 28, 1962
‘Unpublished method for the separation of short chain 3 This work is taken from a thesis submitted to Georgetown fulfillment of the degree of Doctor of Philosophy.
acyl hydroxamates. University in partial