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The properties and interrelationship of oxaluric and parabanic acids
The Properties
and Interrelationship Parabanic Acids’
of Oxaluric and
James C. Andrews and Irl T. Sell of Biocht?mistry and Nutrition, School of Medicine, of North Carolina, Chapel Hill, North Carolina
From the Department
University
Received November 19, 1954
The prevalence of renal calculi composed wholly or in part of calcium oxalate has impelled consideration of a metabolic source of oxalic acid, in addition to that derived purely from dietary sources. Reference to the possible role of parabanic and oxaluric acids as intermediates in the endogenous production of oxalic acid has been made by Rodillon (1) and by Cerecedo (2-4) in whose scheme, dietary pyrimidines could serve as a source, being finally metabolized to parabanic acid (I) which can be assumed to hydrolyze to oxaluric acid (II) and this latter to oxalic acid and urea: II
I CO-NH \ CO-NH
/
co
--+
COOH 1 CO-NH-CO-NH2
--+
COOH I COOH
+
NH2 I co NH2
The literature, thus far, affords very little information as to the properties of parabanic and oxaluric acids and their salts and as to the conditions required for the above hydrolytic steps. Some information concerning the equilibrium bet,ween I and II is recorded below. PARABANIC
ACID
Parabanic acid may be conveniently prepared by oxidation of uric acid with concentrated nitric acid aa described by Aache (5), although other methods have 1 This investigation was supported by a research grant (PHS A-248) from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, Public Health Service. 405
406
J. C. ANDREWS AND I. T. SELL
been reported. The method of Asche was used with no modifications, and yields of thrice-recrystallized parabanic acid of 36-32% were obtained. Recrystallization from hot-water solution was used, water about 1.5 times the weight of the parabanic acid being employed. The acid crystallized in well-formed monoclinic crystals which melted at 245°C. (corr.). This compares well with the value of 2457°C. given by Asche. Some decomposition W&B evident at the melting point. The sample showed 24.55% nitrogen by Kjeldahl (theory, 24.560/,). Parabanic acid is soluble in water at 25%. to the extent of 8.34 g./lOO ml. solution. It is less soluble in 95$J,ethyl alcohol (4.6 g./lOO ml.) and practically insoluble in ether. It forms salts with both strong and weak bases, apparently behaving as a monobasic acid by passing into the enol form. O=C-NH
O=C-N \ c=o
\
e
C(0I-I)
/
/
O=&NH’
O=&NiI
These salts are unstable in aqueous solution and hydrolyze readily to give the salts of oxaluric acid. The latter are much less water-soluble at room temperature and are thus easily isolated as indicated below. One report (6) in the literature as to the ionization constant of parabanic acid gives a value of 7.5 X 10-r. We have investigated and confirmed this value and also investigated the possibility of a second ionizable hydrogen at high pH values. OXALURIC
ACID
Oxaluric acid is difficult to prepare satisfactorily. However partial success was attained by use of a procedure which might have been expected possibly to produce parabanic acid: condensation of diethyl oxalate with urea. Molar proportions of the two, with no added water, were allowed to react for 3 days at 110°C. A mixture of two products resulted: Product I, after five recrystallizations from hot water, was white and crystalline. It melted at 186-7°C. and contained 21.22% nitrogen. Neither value changed on further recrystallization. Oxaluric acid has been reported (7) as melting at 187°C. although higher values, up to 210°C. (8) have also been reported. Its theoretical nitrogen percentage is 21.21%. The yield of this product was low: about 23%. Product II was an orange-yellow powder, insoluble in water, 950/oalcohol, ether, acetone, ammonium hydroxide (cont.), sodium hydroxide (2 N), hydrochloric acid (2 N), and glacial acetic acid. It is, however, soluble in cold, concentrated sulfuric acid. This substance appeared to have the properties of oxalyl diureide :
It melted at 27O”C., whereas 274°C. has been reported (9). Its nitrogen content averaged 32.15$& (Theory for oxalyl diureide is 32.2970.) The method of Biltz and Schrauder (8) was used for the preparation of the potassium salt of oxaluric acid. In this method uric acid is oxidized in alkaline solution by potassium permanganate. However, a more satisfactory procedure for the
OXALURIC
AND
PARABANIC
407
ACIDS
preparation of either the potassium or the ammonium salts of oxaluric acid was by the action of the respective bases upon parabanic acid, prepared as described above. A sample of parabanic acid was dissolved in the minimal amount of warm water, and the concentrated base was added dropwise until a pH of about 10 was reached. On cooling the resulting solution, either salt crystallized out in thin needles. One recrystallization from water gave products of constant composition. The ammonium oxalurate averaged 28.45yo N (theory = 28.18%) and melted with decomposition at 220°C. The potassium oxalurate averaged 17.19% N (theory = 16.46%) and melted with decomposition at 225-30°C. Biltz and Schrauder (8) reported melting points with decomposition, of 242“C. for the ammonium salt and 245°C. for the potassium salt. The free oxaluric acid was most easily prepared by acidification with concentrated hydrochloric acid of a water solution of either of the above salts until a pH of about 2 was reached. During acidification the free acid precipitated out. The mixture was cooled and the resulting free acid was washed and recrystallized from hot water. An over-all yield of 64-70’%, based on the original parabanic acid was obtained. The final product contained an average of 21.74yc N (theory = 21.21%). Oxaluric acid melts with decomposition at about 250°C. although it darkens and chars from 204°C. on. As is the case with other compounds which decompose on points have very little significance. Oxaluric acid is only heating, “melting” slightly soluble in water at room temperature.
Tl’? method of preparation
Titration Parabanic
acid fresh, titration
of oxalurate salts, described above, indi-
TABLE I Data on Parabanic and Oxaluric Acids
direct
Parabanic
!
acid fresh. titration
back
PK
-1.016 -0.660 -0.530 -0.385 -0.140
-0.091 +0.093 f0.212 +0.343 +0.500 to.658 +0.917 Average
5.10 5.40 5.60 5.75 5.94 6.00 6.18 6.30 6.44 6.60 6.75 7.00 --
6.12 6.06 6.13 6.13 6.08 6.09 6.09 6.09 6.10 6.10 6.09 6.08 6.10
$0.473 +0.385 +0.225 +0.154 +0.043 -0.096 -0.213 -0.376 -0.570
--
2.85 2.70 2.50 2.30 2.20 2.10 2.00 1.95 1.90
2.38 2.32 2.17 2.15 2.16 2.20 2.21 2.33 2.47
-/-
+0.954 +0.477 +0.176 0 -0.176 -0.368 -0.602 -0.954
PH
PK
2.55 2.20 2.00 1.90 1.84 1.80 1.74 1.70
1.60 1.72 1.82 1.90 2.02 2.17 2.34 2.65
408
J.
C. ANDREWS
AND
I.
T.
SELL
cates the easewith which the ring of parabanic acid can be split, particularly under slightly alkaline conditions. The behavior of this latter compound when titration curves of the free acid are compared with those of the salt demonstrates the ease of this hydrolysis. Even when the free acid is kept in solution, at room temperature, slow hydrolysis occurs, as shown by the change in form of the titration curve as it approaches that of oxaluric acid. Table I shows the titration data obtained from freshly prepared parabanic acid, titrated with sodium hydroxide and from the same solution when titrated back with hydrochloric acid. Whereas the parabanic acid, on direct titration, behaves in a satisfactory manner giving a pK value which averages 6.10, it is evident that the salt decom-
2. Z?20-
-.
10 -
-6
- 16
-22
FIQ. 1. Titration curves of parabanic acid. Curve. 1. Direct titration of 0.10 M solution of parabanic acid, freshly prepared, with standard 0.10 M base. Curve 2. Direct titration of 0.10 M solution of parabanic acid which had been allowed to stand at room temperature for 46 days, with standard 0.10 M base. Partial conversion to oxaluric acid is evident. Curve. 3. Immediate back titration of the solution in curve 2 with standard 0.10 M acid.
OXALURIC
AND
PARABANIC
ACIDS
409
poses readily. The back titration produces drifting values corresponding to those obtained from oxaluric acid. For comparison, there are also included in Table I a few of the points obtained by titration of oxaluric acid. These show drifting pK values of about 2.0. The unsatisfactory nature of data obtained from transition acids such as this makes this pK value of 2 only an approximation. The value of 6.10 for undecomposed parabanic acid, however, corresponds well with that of 6.12 reported by Wood (6). That slow hydrolysis of free parabanic acid proceeds at room temperature is shown by the curves obtained with a 0.10 M solution which had been kept for 46 days before titration. Figure 1 shows the effect of this
FIG.
Curve prepared. Curve at room Curve
2. Ultraviolet absorption curves of psrabanic and oxaluric acids. 1. 5.10 X 10-’ M parabanic acid in phosphate buffer at pH 7.45, freshly 2. Same solution as in curve 1 but readings taken after standing 2 weeks temperature. 3. 5.10 X lO+ M oxalurie acid under same conditions as in curve 1.
410
J. C. ANDREWS
AND I. T. SELL
hydrolysis on the form of the curves. Curve 1 corresponds to the data in Table I for direct titration of a fresh solution of parabanic acid. Curve 2 shows the result of letting the solution stand for several weeks before titration, and curve 3 the increased hydrolysis resulting from a few minutes in the form of the salt. Examination of the absorption curves of these compounds in the ultraviolet region leads to the same conclusions. Figure 2 shows the optical density measured over a range of 220-320 rnp of a solution of freshly prepared parabanic acid, of a solution about 2 weeks old, and of freshly prepared oxaluric acid. These solutions were all 5.0 X 1W4 M and were buffered at from pH 7.00 to 7.45. The shift from the curve of fresh parabanic acid with its maximum at 270 rnp to that of oxaluric acid is obvious. Further hydrolysis of oxaluric acid to oxalic acid and urea proceeds to completion at room temperature and at pH 7.4 in the course of about 3 months as indicated by there being no measurable absorption over any part of the ultraviolet range. Neither oxalic acid nor urea shows any such absorption. ACKNOWLEDGMENT The J.Logan
authors desire to express their appreciation for help and advice from Dr. Irvin
of this department. SUMMARY
Conditions for the hydrolysis of parabanic to oxaluric acid and to urea and oxalic acid have been studied by means of both titration and ultraviolet absorption data. These studies show that at room temperature free parabanic acid slowly changes to oxaluric acid whereas the salts of the former change to the latter in a few minutes. The splitting of oxaluric acid to oxalic acid and urea is a slower process. Parabanic acid behaves as a monobasic acid with a pK value of 6.10. Oxaluric acid also shows only one inflection of its titration curve at an approximate pH of 2.0. In this it somewhat resembles oxalic acid (pK1 = 1.4) but lacks any second constant corresponding to the Kt value of oxalic acid at 4.3. REFERENCES 1. RODILLON, G., J. Nutrition 3, 43 (1933). 2. CEBECEDO, L. R., J. Biol. Chem. 88, 695 (1930). 3. CERECEDO, L. R., J. Biol. Chem. 93, 269 (1931).
OXALURIC
AND
PARABANIC
ACIDS
411
4. CERECEDO, L. R., J. Biol. Cbm. 93, 283 (1931).
5. ASCHE, A., Ann. 416, 226 (1918). 6. WOOD, J. K., J. C&m. Sot. 89, 1831 (1906). 7. HODOMAN, C. D., “Handbook of Chemistry and Physics,” p. 980. Chemical Rubber Publishing Co., Cleveland, 1949. 8. BILTZ, H., AND SCFIRAUDER, R., J. prakt. Chem. [2] 106, 108 (1923). 9. BILTZ, H., AND TOPP, E., Ber. 46, 1404 (1913).
and Interrelationship Parabanic Acids’
of Oxaluric and
James C. Andrews and Irl T. Sell of Biocht?mistry and Nutrition, School of Medicine, of North Carolina, Chapel Hill, North Carolina
From the Department
University
Received November 19, 1954
The prevalence of renal calculi composed wholly or in part of calcium oxalate has impelled consideration of a metabolic source of oxalic acid, in addition to that derived purely from dietary sources. Reference to the possible role of parabanic and oxaluric acids as intermediates in the endogenous production of oxalic acid has been made by Rodillon (1) and by Cerecedo (2-4) in whose scheme, dietary pyrimidines could serve as a source, being finally metabolized to parabanic acid (I) which can be assumed to hydrolyze to oxaluric acid (II) and this latter to oxalic acid and urea: II
I CO-NH \ CO-NH
/
co
--+
COOH 1 CO-NH-CO-NH2
--+
COOH I COOH
+
NH2 I co NH2
The literature, thus far, affords very little information as to the properties of parabanic and oxaluric acids and their salts and as to the conditions required for the above hydrolytic steps. Some information concerning the equilibrium bet,ween I and II is recorded below. PARABANIC
ACID
Parabanic acid may be conveniently prepared by oxidation of uric acid with concentrated nitric acid aa described by Aache (5), although other methods have 1 This investigation was supported by a research grant (PHS A-248) from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, Public Health Service. 405
406
J. C. ANDREWS AND I. T. SELL
been reported. The method of Asche was used with no modifications, and yields of thrice-recrystallized parabanic acid of 36-32% were obtained. Recrystallization from hot-water solution was used, water about 1.5 times the weight of the parabanic acid being employed. The acid crystallized in well-formed monoclinic crystals which melted at 245°C. (corr.). This compares well with the value of 2457°C. given by Asche. Some decomposition W&B evident at the melting point. The sample showed 24.55% nitrogen by Kjeldahl (theory, 24.560/,). Parabanic acid is soluble in water at 25%. to the extent of 8.34 g./lOO ml. solution. It is less soluble in 95$J,ethyl alcohol (4.6 g./lOO ml.) and practically insoluble in ether. It forms salts with both strong and weak bases, apparently behaving as a monobasic acid by passing into the enol form. O=C-NH
O=C-N \ c=o
\
e
C(0I-I)
/
/
O=&NH’
O=&NiI
These salts are unstable in aqueous solution and hydrolyze readily to give the salts of oxaluric acid. The latter are much less water-soluble at room temperature and are thus easily isolated as indicated below. One report (6) in the literature as to the ionization constant of parabanic acid gives a value of 7.5 X 10-r. We have investigated and confirmed this value and also investigated the possibility of a second ionizable hydrogen at high pH values. OXALURIC
ACID
Oxaluric acid is difficult to prepare satisfactorily. However partial success was attained by use of a procedure which might have been expected possibly to produce parabanic acid: condensation of diethyl oxalate with urea. Molar proportions of the two, with no added water, were allowed to react for 3 days at 110°C. A mixture of two products resulted: Product I, after five recrystallizations from hot water, was white and crystalline. It melted at 186-7°C. and contained 21.22% nitrogen. Neither value changed on further recrystallization. Oxaluric acid has been reported (7) as melting at 187°C. although higher values, up to 210°C. (8) have also been reported. Its theoretical nitrogen percentage is 21.21%. The yield of this product was low: about 23%. Product II was an orange-yellow powder, insoluble in water, 950/oalcohol, ether, acetone, ammonium hydroxide (cont.), sodium hydroxide (2 N), hydrochloric acid (2 N), and glacial acetic acid. It is, however, soluble in cold, concentrated sulfuric acid. This substance appeared to have the properties of oxalyl diureide :
It melted at 27O”C., whereas 274°C. has been reported (9). Its nitrogen content averaged 32.15$& (Theory for oxalyl diureide is 32.2970.) The method of Biltz and Schrauder (8) was used for the preparation of the potassium salt of oxaluric acid. In this method uric acid is oxidized in alkaline solution by potassium permanganate. However, a more satisfactory procedure for the
OXALURIC
AND
PARABANIC
407
ACIDS
preparation of either the potassium or the ammonium salts of oxaluric acid was by the action of the respective bases upon parabanic acid, prepared as described above. A sample of parabanic acid was dissolved in the minimal amount of warm water, and the concentrated base was added dropwise until a pH of about 10 was reached. On cooling the resulting solution, either salt crystallized out in thin needles. One recrystallization from water gave products of constant composition. The ammonium oxalurate averaged 28.45yo N (theory = 28.18%) and melted with decomposition at 220°C. The potassium oxalurate averaged 17.19% N (theory = 16.46%) and melted with decomposition at 225-30°C. Biltz and Schrauder (8) reported melting points with decomposition, of 242“C. for the ammonium salt and 245°C. for the potassium salt. The free oxaluric acid was most easily prepared by acidification with concentrated hydrochloric acid of a water solution of either of the above salts until a pH of about 2 was reached. During acidification the free acid precipitated out. The mixture was cooled and the resulting free acid was washed and recrystallized from hot water. An over-all yield of 64-70’%, based on the original parabanic acid was obtained. The final product contained an average of 21.74yc N (theory = 21.21%). Oxaluric acid melts with decomposition at about 250°C. although it darkens and chars from 204°C. on. As is the case with other compounds which decompose on points have very little significance. Oxaluric acid is only heating, “melting” slightly soluble in water at room temperature.
Tl’? method of preparation
Titration Parabanic
acid fresh, titration
of oxalurate salts, described above, indi-
TABLE I Data on Parabanic and Oxaluric Acids
direct
Parabanic
!
acid fresh. titration
back
PK
-1.016 -0.660 -0.530 -0.385 -0.140
-0.091 +0.093 f0.212 +0.343 +0.500 to.658 +0.917 Average
5.10 5.40 5.60 5.75 5.94 6.00 6.18 6.30 6.44 6.60 6.75 7.00 --
6.12 6.06 6.13 6.13 6.08 6.09 6.09 6.09 6.10 6.10 6.09 6.08 6.10
$0.473 +0.385 +0.225 +0.154 +0.043 -0.096 -0.213 -0.376 -0.570
--
2.85 2.70 2.50 2.30 2.20 2.10 2.00 1.95 1.90
2.38 2.32 2.17 2.15 2.16 2.20 2.21 2.33 2.47
-/-
+0.954 +0.477 +0.176 0 -0.176 -0.368 -0.602 -0.954
PH
PK
2.55 2.20 2.00 1.90 1.84 1.80 1.74 1.70
1.60 1.72 1.82 1.90 2.02 2.17 2.34 2.65
408
J.
C. ANDREWS
AND
I.
T.
SELL
cates the easewith which the ring of parabanic acid can be split, particularly under slightly alkaline conditions. The behavior of this latter compound when titration curves of the free acid are compared with those of the salt demonstrates the ease of this hydrolysis. Even when the free acid is kept in solution, at room temperature, slow hydrolysis occurs, as shown by the change in form of the titration curve as it approaches that of oxaluric acid. Table I shows the titration data obtained from freshly prepared parabanic acid, titrated with sodium hydroxide and from the same solution when titrated back with hydrochloric acid. Whereas the parabanic acid, on direct titration, behaves in a satisfactory manner giving a pK value which averages 6.10, it is evident that the salt decom-
2. Z?20-
-.
10 -
-6
- 16
-22
FIQ. 1. Titration curves of parabanic acid. Curve. 1. Direct titration of 0.10 M solution of parabanic acid, freshly prepared, with standard 0.10 M base. Curve 2. Direct titration of 0.10 M solution of parabanic acid which had been allowed to stand at room temperature for 46 days, with standard 0.10 M base. Partial conversion to oxaluric acid is evident. Curve. 3. Immediate back titration of the solution in curve 2 with standard 0.10 M acid.
OXALURIC
AND
PARABANIC
ACIDS
409
poses readily. The back titration produces drifting values corresponding to those obtained from oxaluric acid. For comparison, there are also included in Table I a few of the points obtained by titration of oxaluric acid. These show drifting pK values of about 2.0. The unsatisfactory nature of data obtained from transition acids such as this makes this pK value of 2 only an approximation. The value of 6.10 for undecomposed parabanic acid, however, corresponds well with that of 6.12 reported by Wood (6). That slow hydrolysis of free parabanic acid proceeds at room temperature is shown by the curves obtained with a 0.10 M solution which had been kept for 46 days before titration. Figure 1 shows the effect of this
FIG.
Curve prepared. Curve at room Curve
2. Ultraviolet absorption curves of psrabanic and oxaluric acids. 1. 5.10 X 10-’ M parabanic acid in phosphate buffer at pH 7.45, freshly 2. Same solution as in curve 1 but readings taken after standing 2 weeks temperature. 3. 5.10 X lO+ M oxalurie acid under same conditions as in curve 1.
410
J. C. ANDREWS
AND I. T. SELL
hydrolysis on the form of the curves. Curve 1 corresponds to the data in Table I for direct titration of a fresh solution of parabanic acid. Curve 2 shows the result of letting the solution stand for several weeks before titration, and curve 3 the increased hydrolysis resulting from a few minutes in the form of the salt. Examination of the absorption curves of these compounds in the ultraviolet region leads to the same conclusions. Figure 2 shows the optical density measured over a range of 220-320 rnp of a solution of freshly prepared parabanic acid, of a solution about 2 weeks old, and of freshly prepared oxaluric acid. These solutions were all 5.0 X 1W4 M and were buffered at from pH 7.00 to 7.45. The shift from the curve of fresh parabanic acid with its maximum at 270 rnp to that of oxaluric acid is obvious. Further hydrolysis of oxaluric acid to oxalic acid and urea proceeds to completion at room temperature and at pH 7.4 in the course of about 3 months as indicated by there being no measurable absorption over any part of the ultraviolet range. Neither oxalic acid nor urea shows any such absorption. ACKNOWLEDGMENT The J.Logan
authors desire to express their appreciation for help and advice from Dr. Irvin
of this department. SUMMARY
Conditions for the hydrolysis of parabanic to oxaluric acid and to urea and oxalic acid have been studied by means of both titration and ultraviolet absorption data. These studies show that at room temperature free parabanic acid slowly changes to oxaluric acid whereas the salts of the former change to the latter in a few minutes. The splitting of oxaluric acid to oxalic acid and urea is a slower process. Parabanic acid behaves as a monobasic acid with a pK value of 6.10. Oxaluric acid also shows only one inflection of its titration curve at an approximate pH of 2.0. In this it somewhat resembles oxalic acid (pK1 = 1.4) but lacks any second constant corresponding to the Kt value of oxalic acid at 4.3. REFERENCES 1. RODILLON, G., J. Nutrition 3, 43 (1933). 2. CEBECEDO, L. R., J. Biol. Chem. 88, 695 (1930). 3. CERECEDO, L. R., J. Biol. Chem. 93, 269 (1931).
OXALURIC
AND
PARABANIC
ACIDS
411
4. CERECEDO, L. R., J. Biol. Cbm. 93, 283 (1931).
5. ASCHE, A., Ann. 416, 226 (1918). 6. WOOD, J. K., J. C&m. Sot. 89, 1831 (1906). 7. HODOMAN, C. D., “Handbook of Chemistry and Physics,” p. 980. Chemical Rubber Publishing Co., Cleveland, 1949. 8. BILTZ, H., AND SCFIRAUDER, R., J. prakt. Chem. [2] 106, 108 (1923). 9. BILTZ, H., AND TOPP, E., Ber. 46, 1404 (1913).