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Glycolaldehyde phosphate
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78,
328-333 (1958)
Glycolaldehyde Phosphate’ Clinton E. Ballou From the Department of Biochemistry, The University Berkeley, California
of California,
ReceivedMay 5, 1958 INTRODUCTION
AND DISCUSSION
Glycolaldehyde phosphate is an interesting and important substance. It has often played a role in structural studies on sugar phosphates, since it can be produced when such a compound, having the phosphate group on a terminal position, is treated with the glycol-splitting reagent sodium periodate (1). Indeed, the classical method for the preparation of glycolaldehyde phosphate has been the periodate cleavage of glycerol 1-phosphate (2). Glycolaldehyde phosphate has not been implicated in any important biochemical role, although it does act as a substrate for aldolase by which it is condensed with dihydroxyacetone phosphate to give D-threopentulose 1,5-diphosphate (3) (n-xylulose 1,5-diphosphate). Glycolaldehyde phosphate is not a substrate for glyceraldehydephosphate dehydrogenase, although some preparations have been found to exert a strong inhibition of this enzyme.2 This latter fact would indicate that the product obtained by the periodate cleavage of glycerol l-phosphate is variable in its properties. Rouser and co-workers (4) have published conflicting R, values for glycolaldehyde phosphate, while Loring et al. (1) have found that some preparations of the substance show two components on chromatography. As a possible explanation of these facts, it has been suggested (1) that these preparations may contain either c&tram isomers of the enolic form, or a mixture of monomeric and dimeric products. 1 This work was supported in part by grants from the U. S. Public Health Service (grant ASS4) and University of California Cancer Research Coordinating Committee. 2 E. Racker and I. Krimsky, personal communication. 328
GLYCOLALDEHYDE
PHOSPHATE
329
In order to make pure glycolaldehyde phosphate more readily available for chemical and biochemical studies, we have undertaken its preparation by a procedure analogous to those which have been so useful for the preparation of other sensitive sugar phosphates (5). Glycolaldehyde diethyl acetal (I) was phosphorylated with diphenylphosphorochloridate, and the resulting diphenylphosphoryl glycolaidehyde diethyl acetal (II) was hydrogenated to give glycolaldehyde phosphate diethyl acetal (III) as the crystalline cyclohexylamine salt. Mild acid hydrolysis of III gives free glycolaldehyde phosphate (IV), which was characterized by chromatography, oxidation equivalent, acid hydrolysis rate, and reduction to ethylene glycol phosphate (V). Glycolaldehyde phosphate produced by this procedure does not inhibit glyceraldehydephosphate dehydrogenase, but by treatment with base or by long storage it can be converted to a substance that is strongly inhibitory. The nature of this reaction is being studied. EXPERIMENTAL
Glycolaldehyde Diethyl Acetal (I) This substance has been prepared by the hydrolysis of 2-chloro- and 2-bromoacetaldehyde diethyl acetal (6). However, the material we prepared by this procedure, although it had approximately the reported boiling point, could not be phosphorylated. This procedure was repeated several times with the same result. No explanation was found to account for this failure. I was subsequently obtained by the periodate cleavage of m-glyceraldehyde diethyl acetal (7) and by sodium borohydride reduction of the resulting glyoxal diethyl semiacetal. To a solution of 45 g. sodium periodate in 500 ml. water was added 30 g. nL-glyceraldehyde diethyl acetal. The reaction took place instantaneously and the flask was cooled in ice water to dissipate the heat. After 30 min. any remaining periodate was destroyed by adding more of the acetal. About 500 ml. ethanol was then added to the mixture to precipitate most of the sodium iodate. This was filtered off, and to the filtrate (which was cooled in a bath of ice water) was added a solution of 6 g. sodium borohydride in 50 ml. water. The mixture was left for 1 hr. to allow for reduction of the aldehyde, at which time glacial acetic acid was added to bring the pH down to about 7 and destroy the excess borohydride. The solution was next concentrated in vomo to remove
330
BALLOU
the ethanol, and the aqueous solution remaining was saturated with potassium carbonate. This caused separation of the glycolaldehyde diethyl acetal as an oily layer, which was removed in a separatory funnel. The water layer was extracted twice with loo-ml. portions of ether, and these ether extracts were combined with the oil. The ether solution was dried with anhydrous potassium carbonate, then concentrated to remove the ether, and the residue was distilled at atmospheric pressure to give 19 g. of I with b.p. 174-178”. The reported b.p. is 165-167” 03). Glycolaldehyde Phosphate Diethyl Acetal (III) A solution of 7.7 g. of I in 50 ml. of dry pyridine was cooled to 5’ in an ice bath, and 19.0 g. diphenylphosphorochloridate was added dropwise during a period of 15 min. with continual swirling of the reaction mixture. The reaction was then left at 5” for 1 hr. and at room temperature for 2 hr. It was worked up by adding a few drops of water to decompose the excess phosphorylating reagent and then concentrating at reduced pressure to remove most of the pyridine. The residue, in 100 ml. benzene, was washed with water, cold 1 ill hydrochloric acid, cold 1 M sodium bicarbonate, and water, in that order. The dried benzene solution (sodium sulfate) was concentrated to a sirup that weighed 20 g. The theoretical yield of diphenylphosphoryl glycolaldehyde diethyl acetal (II) is 19.3 g. This product, without further purification, was hydrogenated in 500 ml. of absolute ethanol to remove the phenyl groups. The catalyst was platinum oxide (2.0 g.), and the mixture was shaken with hydrogen at atmospheric pressure. The reaction was complete in 1.5 hr. with a hydrogen uptake of 11.2 1. The solution was decanted from the catalyst, and cyclohexylamine was added immediately to bring the pH to about 9. On concentration at reduced pressure, a sirup was obtained. This was dissolved in 20 ml. water, and about 400 ml. acetone was added. The mixture was warmed to gentle reflux, and a little insoluble material was removed by filtration with suction through a layer of Filtercel on a Buchner funnel. The filtrate, on cooling at 5” overnight, deposited the crystalline cyclohexylamine salt of glycolaldehyde phosphate diethyl acetal (if crystallization fails to occur, more cyclohexylamine should be added to be certain the product is in the diamine form). The material was collected by filtration on a Biichner funnel. washed with a.cet,one on the funnel, and then dried in air. The yield was 20 g. Recrystalliza-
GLYCOLALDEHYDE
PHOSPHATE
331
tion in a similar fashion gave 18.8 g. On heating to 57” in a vacuum, the substance loses water of crystallization and turns to a gum. For analysis, a sample was dried in a vacuum at room temperature. This material turned brown at 195” and melted with decomposition at 200”. Anal. Calcd. for C18H4108N2P.H20 (430): C, 50.2; H, 10.0; N, 6.5; P, 7.2; OR, 20.9; HzO, 4.2. Found: C, 49.5; H, 9.8; N, 6.3; P, 7.1; OEt, 20.2; HzO, 5.4. Conversion of III
to Free Glycolaldehyde Phosphate (IV)
This reaction was carried out as we have described for other acetals (5). A solution of 100 mg. of the amine salt of III in 10 ml. water was swirled with 2 ml. of Dowex 50 (H) for a minute to remove the amine; then the resin was filtered off and the filtrate was kept at 40”. Aliquots of t,he solution were removed and analyzed by the modified WillstiitterSchudel procedure for free aldehyde (8). The acetal was 80 % hydrolyzed after 5 hr. and completely hydrolyzed after 18 hr., when the solution showed 104 % of the calculated reducing power. When this solution was chromatographed on Whatman No. 1 acidwashed paper using a butanol-picric acid solvent (9), it showed only one phosphate-containing component that had an R, (0.40) identical with the glycolaldehyde phosphate prepared from glycerol l-phosphate by periodate cleavage.3 The rate of acid hydrolysis in 1 N sulfuric acid at) loo”, with the formation of inorganic phosphate, was determined and found to have a half time of 16 min. The compound yielded only about 5 % of the theoretical amount of inorganic phosphate when treated with 1 N sodium hydroxide at room temperature for 20 min. The crystalline brucine salt was prepared according to Loring, and was obtained in a theoretical yield. However, the m.p. was lOO-120”, whereas Loring reports 160-163”. The melting point could be raised 20-30” by drying the sample under reduced pressure at GO” for a long period of time. Anal. Calcd. for C2SH3109N2P: N, 5.25; P, 5.81. Found: N, 4.84; P, 5.69. We have prepared crystalline brucine salts of dihydroxyucetone phosphate and u-glyceraldehyde phosphate in a similar fashion, and both a A gift from Dr. H. S. Loring. 4 H. S. Loring, unpublished.
BALLOU
332
analyze very well for the expected products. However, they have only 60-80% of the expected biological activity and decompose still further over a period of days. Thus, a crystalline, “analytically pure” brucine salt of such compounds camrot be relied upon to represent the pure material. Reductim of IV to Ethylene Glycol Phosphate (V) A solution of IV, prepared by hydrolyzing 500 mg. of II in 50 ml. water as described above, was treated with 200 mg. sodium borohydride. After 1 hr., the cations were removed by use of Dowex 50 (H), and cyclohexylamine was added to the acidic solution to bring it to pH 9. The solution was then concentrated to dryness, and the residue was dissolved in absolute ethanol and precipitated as an amorphous solid by the addition of acetone. The solid weighed 400 mg., whereas the calculated yield for the dicyclohexylamine salt is 430 mg. This solid was dissolved in 2.0 ml. water, the solution was filtered, and acetone was added to the filtrate to cause turbidity. Long needlelike crystals were obtained. These were collected and recrystallized a second time in a similar fashion. The yield was 150 mg. of a product with m.p. 165-167” (gas evolution), with sintering from 100” and softening between 160 and 165”. Anal. Calcd. for CJ4HS30bPN2 (360): N, 7.78; P, 8.61. Found: N, 7.64; P, 8.85. The compound, thus, analyzes correctly for dicyclohexylammonium ethylene glycol phosphate. Chromatography of the material in a picric acid solvent showed one phosphate-containing component, RI 0.8, which was indistinguishable from authentic ethylene glycol phosphate. SUMMARY A new preparation of glycolaldehyde phosphate is described. It involves the phosphorylation of glycolaldehyde diethyl acetal. The final product is obtained by a mild hydrolysis of the acetal. Whereas glycolaldehyde phosphate prepared by the periodate oxidation of glycerol lphosphate is apparently variable in its properties and purity, our preparation is free of the same impurities. REFERENCES H. S., LEVY, L. W., Moss, L. K., AND Sot. 78, 3724 (1956).
1. LORINQ,
PLOESER,
J. M.,J. Am. Chem.
GLYCOLALDEHYDE
PHOSPHATE
333
2. FLEURY, I’., COURTOIS, J., AND DESJOBERT, A., Bull. see. chim. France 1948, 694. 3. BYRNE, W. L., AND LARI)Y, H. A., Biochim. et Biophys. .4cta 14, 495 (1954). 4. ROUGER, G., BERRY, J. F., MARINETTI, G., AND STOTZ, E., J. Am. Chem. Sot. 76, 310 (1953); MORRISON, M., ROUSER, G., AND STOTZ, IX., J. Am. Chem. sot. 77, 5156 (1955). 6. BALLOU, C. E., AND FISCHER, H. 0. L., J. Am. Chem. Sot. 77, 3329 (1955); 78, 1659 (1956). 6. BEYERSTEDT, F., AND MCELVAIN, S. M., J. Am. Chem. Sot. 68, 529 (1936). 7. FISCHER, H. 0. L., AND BAER, E., Helv. Chim. Acta 18, 514 (1935). Y. AUERBACH, F., AND BODLHNDER, E., Z. angew. Chem. 36, 602 (1923). 9. HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78,
328-333 (1958)
Glycolaldehyde Phosphate’ Clinton E. Ballou From the Department of Biochemistry, The University Berkeley, California
of California,
ReceivedMay 5, 1958 INTRODUCTION
AND DISCUSSION
Glycolaldehyde phosphate is an interesting and important substance. It has often played a role in structural studies on sugar phosphates, since it can be produced when such a compound, having the phosphate group on a terminal position, is treated with the glycol-splitting reagent sodium periodate (1). Indeed, the classical method for the preparation of glycolaldehyde phosphate has been the periodate cleavage of glycerol 1-phosphate (2). Glycolaldehyde phosphate has not been implicated in any important biochemical role, although it does act as a substrate for aldolase by which it is condensed with dihydroxyacetone phosphate to give D-threopentulose 1,5-diphosphate (3) (n-xylulose 1,5-diphosphate). Glycolaldehyde phosphate is not a substrate for glyceraldehydephosphate dehydrogenase, although some preparations have been found to exert a strong inhibition of this enzyme.2 This latter fact would indicate that the product obtained by the periodate cleavage of glycerol l-phosphate is variable in its properties. Rouser and co-workers (4) have published conflicting R, values for glycolaldehyde phosphate, while Loring et al. (1) have found that some preparations of the substance show two components on chromatography. As a possible explanation of these facts, it has been suggested (1) that these preparations may contain either c&tram isomers of the enolic form, or a mixture of monomeric and dimeric products. 1 This work was supported in part by grants from the U. S. Public Health Service (grant ASS4) and University of California Cancer Research Coordinating Committee. 2 E. Racker and I. Krimsky, personal communication. 328
GLYCOLALDEHYDE
PHOSPHATE
329
In order to make pure glycolaldehyde phosphate more readily available for chemical and biochemical studies, we have undertaken its preparation by a procedure analogous to those which have been so useful for the preparation of other sensitive sugar phosphates (5). Glycolaldehyde diethyl acetal (I) was phosphorylated with diphenylphosphorochloridate, and the resulting diphenylphosphoryl glycolaidehyde diethyl acetal (II) was hydrogenated to give glycolaldehyde phosphate diethyl acetal (III) as the crystalline cyclohexylamine salt. Mild acid hydrolysis of III gives free glycolaldehyde phosphate (IV), which was characterized by chromatography, oxidation equivalent, acid hydrolysis rate, and reduction to ethylene glycol phosphate (V). Glycolaldehyde phosphate produced by this procedure does not inhibit glyceraldehydephosphate dehydrogenase, but by treatment with base or by long storage it can be converted to a substance that is strongly inhibitory. The nature of this reaction is being studied. EXPERIMENTAL
Glycolaldehyde Diethyl Acetal (I) This substance has been prepared by the hydrolysis of 2-chloro- and 2-bromoacetaldehyde diethyl acetal (6). However, the material we prepared by this procedure, although it had approximately the reported boiling point, could not be phosphorylated. This procedure was repeated several times with the same result. No explanation was found to account for this failure. I was subsequently obtained by the periodate cleavage of m-glyceraldehyde diethyl acetal (7) and by sodium borohydride reduction of the resulting glyoxal diethyl semiacetal. To a solution of 45 g. sodium periodate in 500 ml. water was added 30 g. nL-glyceraldehyde diethyl acetal. The reaction took place instantaneously and the flask was cooled in ice water to dissipate the heat. After 30 min. any remaining periodate was destroyed by adding more of the acetal. About 500 ml. ethanol was then added to the mixture to precipitate most of the sodium iodate. This was filtered off, and to the filtrate (which was cooled in a bath of ice water) was added a solution of 6 g. sodium borohydride in 50 ml. water. The mixture was left for 1 hr. to allow for reduction of the aldehyde, at which time glacial acetic acid was added to bring the pH down to about 7 and destroy the excess borohydride. The solution was next concentrated in vomo to remove
330
BALLOU
the ethanol, and the aqueous solution remaining was saturated with potassium carbonate. This caused separation of the glycolaldehyde diethyl acetal as an oily layer, which was removed in a separatory funnel. The water layer was extracted twice with loo-ml. portions of ether, and these ether extracts were combined with the oil. The ether solution was dried with anhydrous potassium carbonate, then concentrated to remove the ether, and the residue was distilled at atmospheric pressure to give 19 g. of I with b.p. 174-178”. The reported b.p. is 165-167” 03). Glycolaldehyde Phosphate Diethyl Acetal (III) A solution of 7.7 g. of I in 50 ml. of dry pyridine was cooled to 5’ in an ice bath, and 19.0 g. diphenylphosphorochloridate was added dropwise during a period of 15 min. with continual swirling of the reaction mixture. The reaction was then left at 5” for 1 hr. and at room temperature for 2 hr. It was worked up by adding a few drops of water to decompose the excess phosphorylating reagent and then concentrating at reduced pressure to remove most of the pyridine. The residue, in 100 ml. benzene, was washed with water, cold 1 ill hydrochloric acid, cold 1 M sodium bicarbonate, and water, in that order. The dried benzene solution (sodium sulfate) was concentrated to a sirup that weighed 20 g. The theoretical yield of diphenylphosphoryl glycolaldehyde diethyl acetal (II) is 19.3 g. This product, without further purification, was hydrogenated in 500 ml. of absolute ethanol to remove the phenyl groups. The catalyst was platinum oxide (2.0 g.), and the mixture was shaken with hydrogen at atmospheric pressure. The reaction was complete in 1.5 hr. with a hydrogen uptake of 11.2 1. The solution was decanted from the catalyst, and cyclohexylamine was added immediately to bring the pH to about 9. On concentration at reduced pressure, a sirup was obtained. This was dissolved in 20 ml. water, and about 400 ml. acetone was added. The mixture was warmed to gentle reflux, and a little insoluble material was removed by filtration with suction through a layer of Filtercel on a Buchner funnel. The filtrate, on cooling at 5” overnight, deposited the crystalline cyclohexylamine salt of glycolaldehyde phosphate diethyl acetal (if crystallization fails to occur, more cyclohexylamine should be added to be certain the product is in the diamine form). The material was collected by filtration on a Biichner funnel. washed with a.cet,one on the funnel, and then dried in air. The yield was 20 g. Recrystalliza-
GLYCOLALDEHYDE
PHOSPHATE
331
tion in a similar fashion gave 18.8 g. On heating to 57” in a vacuum, the substance loses water of crystallization and turns to a gum. For analysis, a sample was dried in a vacuum at room temperature. This material turned brown at 195” and melted with decomposition at 200”. Anal. Calcd. for C18H4108N2P.H20 (430): C, 50.2; H, 10.0; N, 6.5; P, 7.2; OR, 20.9; HzO, 4.2. Found: C, 49.5; H, 9.8; N, 6.3; P, 7.1; OEt, 20.2; HzO, 5.4. Conversion of III
to Free Glycolaldehyde Phosphate (IV)
This reaction was carried out as we have described for other acetals (5). A solution of 100 mg. of the amine salt of III in 10 ml. water was swirled with 2 ml. of Dowex 50 (H) for a minute to remove the amine; then the resin was filtered off and the filtrate was kept at 40”. Aliquots of t,he solution were removed and analyzed by the modified WillstiitterSchudel procedure for free aldehyde (8). The acetal was 80 % hydrolyzed after 5 hr. and completely hydrolyzed after 18 hr., when the solution showed 104 % of the calculated reducing power. When this solution was chromatographed on Whatman No. 1 acidwashed paper using a butanol-picric acid solvent (9), it showed only one phosphate-containing component that had an R, (0.40) identical with the glycolaldehyde phosphate prepared from glycerol l-phosphate by periodate cleavage.3 The rate of acid hydrolysis in 1 N sulfuric acid at) loo”, with the formation of inorganic phosphate, was determined and found to have a half time of 16 min. The compound yielded only about 5 % of the theoretical amount of inorganic phosphate when treated with 1 N sodium hydroxide at room temperature for 20 min. The crystalline brucine salt was prepared according to Loring, and was obtained in a theoretical yield. However, the m.p. was lOO-120”, whereas Loring reports 160-163”. The melting point could be raised 20-30” by drying the sample under reduced pressure at GO” for a long period of time. Anal. Calcd. for C2SH3109N2P: N, 5.25; P, 5.81. Found: N, 4.84; P, 5.69. We have prepared crystalline brucine salts of dihydroxyucetone phosphate and u-glyceraldehyde phosphate in a similar fashion, and both a A gift from Dr. H. S. Loring. 4 H. S. Loring, unpublished.
BALLOU
332
analyze very well for the expected products. However, they have only 60-80% of the expected biological activity and decompose still further over a period of days. Thus, a crystalline, “analytically pure” brucine salt of such compounds camrot be relied upon to represent the pure material. Reductim of IV to Ethylene Glycol Phosphate (V) A solution of IV, prepared by hydrolyzing 500 mg. of II in 50 ml. water as described above, was treated with 200 mg. sodium borohydride. After 1 hr., the cations were removed by use of Dowex 50 (H), and cyclohexylamine was added to the acidic solution to bring it to pH 9. The solution was then concentrated to dryness, and the residue was dissolved in absolute ethanol and precipitated as an amorphous solid by the addition of acetone. The solid weighed 400 mg., whereas the calculated yield for the dicyclohexylamine salt is 430 mg. This solid was dissolved in 2.0 ml. water, the solution was filtered, and acetone was added to the filtrate to cause turbidity. Long needlelike crystals were obtained. These were collected and recrystallized a second time in a similar fashion. The yield was 150 mg. of a product with m.p. 165-167” (gas evolution), with sintering from 100” and softening between 160 and 165”. Anal. Calcd. for CJ4HS30bPN2 (360): N, 7.78; P, 8.61. Found: N, 7.64; P, 8.85. The compound, thus, analyzes correctly for dicyclohexylammonium ethylene glycol phosphate. Chromatography of the material in a picric acid solvent showed one phosphate-containing component, RI 0.8, which was indistinguishable from authentic ethylene glycol phosphate. SUMMARY A new preparation of glycolaldehyde phosphate is described. It involves the phosphorylation of glycolaldehyde diethyl acetal. The final product is obtained by a mild hydrolysis of the acetal. Whereas glycolaldehyde phosphate prepared by the periodate oxidation of glycerol lphosphate is apparently variable in its properties and purity, our preparation is free of the same impurities. REFERENCES H. S., LEVY, L. W., Moss, L. K., AND Sot. 78, 3724 (1956).
1. LORINQ,
PLOESER,
J. M.,J. Am. Chem.
GLYCOLALDEHYDE
PHOSPHATE
333
2. FLEURY, I’., COURTOIS, J., AND DESJOBERT, A., Bull. see. chim. France 1948, 694. 3. BYRNE, W. L., AND LARI)Y, H. A., Biochim. et Biophys. .4cta 14, 495 (1954). 4. ROUGER, G., BERRY, J. F., MARINETTI, G., AND STOTZ, E., J. Am. Chem. Sot. 76, 310 (1953); MORRISON, M., ROUSER, G., AND STOTZ, IX., J. Am. Chem. sot. 77, 5156 (1955). 6. BALLOU, C. E., AND FISCHER, H. 0. L., J. Am. Chem. Sot. 77, 3329 (1955); 78, 1659 (1956). 6. BEYERSTEDT, F., AND MCELVAIN, S. M., J. Am. Chem. Sot. 68, 529 (1936). 7. FISCHER, H. 0. L., AND BAER, E., Helv. Chim. Acta 18, 514 (1935). Y. AUERBACH, F., AND BODLHNDER, E., Z. angew. Chem. 36, 602 (1923). 9. HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949).