[CONTRIBUTION FROM THE
DEPARTMENT OF CHEMISTRY, BROOKLYN COLLEGE]
THE FORMATION OF URAMIL FROM DIALURIC ACID DAVID DAVIDSON AND HAROLD SOLOWAY Received September 9, 1988 INTRODUCTION
During their classical researches on the chemistry of uric acid, Wohler and Liebig' observed that when a solution of alloxantine was boiled with ammonium chloride, uramil was rapidly formed as an insoluble precipitate, leaving alloxan in the mother liquors. In reexamining this reaction, Biltz and Damm2 considered the mechanism of the reaction to involve the dissociation of alloxantine (I) into dialuric acid (11) and alloxan (111) followed by a metathesis of the resulting dialuric acid with ammonium chloride to form uramil (IV). Since this left the alloxan moiety of alloxantine unutilized for the production of uramil, these authors suggested the more economical procedure of reducing alloxantine to dialuric acid before treating it with ammonium chloride.
CO-NH NH-CO NH-CO f T H b I I b I I I 0 cC CO e 0 CHOH + CO CO
I
I
NH-GO
HOI'
I
CO-NH
I
I
1
NH-GO I1
p,,
I
1
NH-CO I11
p"2
NH-CO
IV This work fixed the impression that alloxan has but a passive part in the formation of uramil from alloxantine. Recently, however, Davidson and Epstein3 have demonstrated that alloxan plays a r81e in the reverse reac1
WBHLER AND
LIEBIQ,
Ann., 26, 310 (1838).
* BILTZ AND DAYM, Ber., 46, 3662 (1913).
* DAVIDSON AND EPBTEIN, J.
ORQ. CHEM.,1, 305 (1936). 365
366
DAVID DAVIDSON A N b HAROLD SOLOWAY
tion; that is, the hydrolysis of uramil to dialuric acid and ammonia. They proposed the hypothesis that both the formation and the hydrolysis of uramil involve a reversible oxidation-reduction process as well as a metathetical change, the latter involving not dialuric acid but alloxan. The composite reversible reaction may be represented by means of the following reversible steps. Step 1. Metathesis: Alloxan (111) Ammonium Ion S Alloxan-Imine (V) Oxonium Ion
+
+
NH-CO
I I CO CO I I
NH-CO
+ NH:
I 1 e CO C=NH I
NH-CO I11
t
+ HsO+
NH-CO
v
+
Step 2. Oxidation-Reduction: Alloxan-Imine (V) (11) S Uramil (IV) Alloxan (111)
+
NH-CO
I
CO
I
C=NH
hH--bO
v
NH-CO
I
+ CO I
NH-co
I / CO CHNHz I I
I
CHOH
I
NH-CO I1
NH-CO
IV
Dialuric Acid
NH-CO
I + CO I
1 I
CO
NH-CO I11
Thus the products disappearing and appearing in the first step respectively appear and disappear in the second step so that the resultant becomes : Sum of Steps 1 and 2. (Apparent Metathesis): Dialuric Acid (11) Ammonium Ion S Uramil (IV) Oxonium Ion
+
+
NH-CO
I I CO CHOH I I
NH-CO I1
NH-CO
+ NH:
t
S CO
I
i
CHNHz
I
+ H30+
NH-CO IV
EVIDENCE SUPPORTING THE PROPOSED MECHANISM
The mechanism proposed above is supported by certain facts already recorded in the literature. Thus, Biltz and Beck4 found that 7-cyanouramil (VI) could be prepared by heating alloxantine solution (Le., dialuric acid plus alloxan) with cyanamide, but that this product was not BILTZAND BECK,J . prakt. Chem., [2], 118, 151, 162 (1928).
FORMATION OF URAMIL FROM DIALURIC ACID
367
obtainable from the action of cyanamide on dialuric acid alone. In the absence of alloxan, of course, the primary metathesis could not occur. This result appears all the more remarkable in view of the ease with which dialuric acid is autoxidized to alloxan (or alloxantine) .5 NH-CO
NH-CO
NH-CO
I 1 I I CO CHNHCN CO CHNHGHs 0 CHN(CH& I 1 rsH-coI I 1
C:l
NH-CO VI
NH-CO VI1
VI11
Likewise, while Piloty and Finckhs prepared 7-ethyluramil (VII) from alloxantine and ethylammonium acetate, Biltz, Marwitsky, and Heyn' had difficulty in obtaining this uramil derivative from dialuric acid. The salt, et hylammonium dialurate, appeared instead of 7-ethyluramil, unless a large excess of ethylammonium acetate was employed. Furthermore, Piloty and Finckha were unable to produce 7,7-dimethyluramil (VIII) from alloxantine and dimethylammonium acetate under the same conditions which succeeded for the formation of 7-methyluramil from alloxantine and methylammonium acetate. According to the proposed hypothesis the formation of 7,7-dimethyluramil would require the occurrence of a quaternary ion derivative of alloxan-imine as an intermediate (IX). Presumably the stability of such an ion, and hence the conditions for its formation, would be quite different from those for alloxan-imine or its 'I-monoalkyl derivatives. NH-CO
t 0: C=N(CHa)t I
AH-coI
IX In the present study it was attempted to demonstrate that the formation of uramil from dialuric acid and ammonium chloride is catalyzed by alloxan and that the success of the method of Biltz and Damm2depends upon the formation of some alloxan by the autoxidation of dialuric acid. The problem of obtaining pure dialuric acid (free from alloxan or alloxantine) was solved by utilizing isodialuric acids (X), which resists autoxidation. HILL,J . Biol. Chem., 86, 713 (1930); 92, 471 (1931). PILOTYAND FINCKH,Ann., 333, 64 (1904). 7 BILTZ,MARWITSKY, AND HEYN,ibid., 433, 148 (1923). 8 ( a ) BEHREND AND ROOSEN, Ann., 261, 235 (1889); ( b ) DAVIDSON AND BAUDISCH, J . Biol. Chem., 64, 621 (1925); (e) BOGERT AND DAVIDSON, J . Am. Chem. SOC.,66, 1667 (1933). 6
6
368
DAVID DAVIDSON AND HAROLD SOLOWAY
This was rearranged to dialuric acid by alkaliQin the absence of air. The subsequent addition of potassium bisulfate served to neutralize the alkali employed as well as to furnish sufficient acidity to prevent the precipitation of alkali dialurate.
NH-CO
bo bo
‘ A
NH-
HOH
X APPARATUS AND PROCEDURE
The apparatus employed in the present experiments consisted of a test-tube (30 X 150 mm.) the open end of which was fused t o a narrower tube (20 mm. wide) which in turn bore three side-tubes (20 X 80 mm.) arranged one above the other and fixed at successively more acute angles t o the main tube. Radially the upper two sidetubes were 90” apart, the lowermost tube falling in between. This reaction vessel was charged as indicated in the accompanying sketch, some of the solid reagents being weighed out in glass “shoes” (made from 8-mm. tubing) which were then introduced into the reaction vessel by means of a glass hook at the end of a string. Granular ammonium chloride was introduced by means of a delivery tube (10 mm. wide) which had a short right-angle at one end and an enlargement at the other. Potassium bisulfate was formed into rod, which could be slipped into the reaction vessel, by fusing the salt in a test-tube and then drawing the molten salt into a length of 8-mm. glass tubing. After cooling, the glass was broken away leaving the bisulfate stick. The charge consisted of 0.180 g. of isodialuric acid dihydrate (0.001 mole) plus 15 cc. of water, two pellets of sodium hydroxide (approximately 0.20 g. or 0.005 mole), 1.4 g. of potassium bisulfate (0.010 mole), 1.0 g. of ammonium chloride (0.017 mole), and a varying amount of alloxan monohydrate.1° When the charging was complete, the open end of the reaction tube was drawn out in the blast lamp. The tube was then immersed in a water bath (a large museum jar served for this bath) to a point well above the side-tubes. Hot water was poured into the upper part of the bath so that while the lower part of the reaction tube was at about 35” the side-tubes were considerably warmer. This arrangement prevented condensation in the sidetubes during the boiling out which was effected by connecting t o an efficient water pump. Five minutes’ pumping was adequate for the present purpose and avoided excessive evaporation of the water charged into the tube (about 2 cc. was lost). The reaction vessel was then sealed off with a blast lamp and manipulated further. The tube was tilted and tapped so t h a t the alkali “shoe” fell into the test-tube. The apparatus was then carefully shaken for a few minutes until the isodialuric acid and the sodium hydroxide were completely dissolved. The bisulfate was then moved into the test-tube. As this dissolved, a precipitate appeared (potassium dialurate), which could be inhibited or redissolved by occasionally immersing the tube in a boiling water bath. The ammonium chloride and any alloxan present were 9
BEHREND AND KOECH, Ann., 316, 246 (1901). MCELVAIN,J . Am. C h e m SOC.,67, 1303 (1935).
lo
FORMATION OF URAMIL FROM DIALURIC ACID
369
then added b y inverting and reinverting the tube several times. If the mixture was not homogeneous at this point, gentle warming on the water bath for a minute or two was sufficient to produce a clear, colorless solution consisting of acidulated dialuric acid and ammonium chloride, ready for observations on the effect of alloxan on the formation of uramil from dialuric acid. When uramil was produced in an experiment i t was filtered by suction and washed with water and alcohol. Filtrates were tested for unconverted dialuric acid by heating. The crude uramil was dried by continuing the suction while a lighted 60-watt bulb fitted with a reflector rested on the rim of the funnel. Purification of uramil was effected by suspending the crude precipitate in 10-20 cc. of water containing a little sodium sulfite, adding 2 cc. of 2N sodium hydroxide, filtering quickly, and turning the filtrate into 50-100 cc. of boiling water containing 6 cc. of 2N hydrochloric acid. Uramil separated at once from the boiling solution as fine, colorless,
1
EO
silky needles, which were filtered and dried as described above. The identity of the uramil preparations was checked by means of nitrogen determinations (Kjeldahl). DISCUSSION O F RESULTS
The r&Zeof air-In one experiment, two tubes containing no alloxan were prepared as described above. (See table; tubes Nos. 1 and 2.) Air was then admitted to No. 1 by cutting off the capillary tip. Both tubes were then heated on the water bath, the second (sealed) tube being inverted to prevent the collection of liquid in the side-tubes. Uramil began to precipitate in the open tube after eleven minutes of heating, and the process was complete within an hour. The sealed tube, however, remained clear even after five hours’ heating and then standing overnight. That this second tube nevertheless had the potentiality of producing uramil was shown by later admitting air to it, whereupon it produced a precipitate of uramil of about the same quantity and quality as in the first tube.
370
DAVID DAVIDSON AND HAROLD SOLOWAY
Alloxan, a catalyst-In another experiment three tubes were charged as usual, one containing one millimole of alloxan (tube no. 3)’ a second containing one-tenth millimole of alloxan (tube no. 4), and a third, as a control, no alloxan (duplicate of tube no. 1). All three tubes were heated on the water bath. The first assumed a faint wine color (murexide) and evidenced a precipitate of uramil within five minutes, the second remained colorless and produced a precipitate in about fifteen minutes, while the third was unchanged after an hour’s heating and three weeks’ standing TABLE OF RESULTS (Each tube contained one millimole of dialuric acid in water in the presence of potassium bisulfate and ammonium chloride) YIELD (THEORETIALLOXAN TVBE NO. EMPLOYE1 MILLIMOLI
CONDITIONS OF EXPERIMENT
TIME FOR PRECIPITATE TO APPEAR
CAL: 0.143
Crude, g.
2
0.0
2 (cont .) 3 4 5
0.0 1.o 0.1 0.0 I
6
0.0
7
1 .o
8
I
0.1
Opened tube after mixing; heated 1 hour. Heated 5 hours; stood overnight. Opened tube $2; stood 5 days. Heated 25 min. Heated 3 hours. At room temp. for 3 weeks. Opened tube after stand ing overnight; stood 5 days longer. At room temp. for 24 hours. At room temp. for 5 days
9.)
ANALYLIIS (THEORETICAL:
29.4%) lecryst’d N, OUND: (%) B.
11 min.
0.082
0.066
29.2
-
None
None
-
Overnight
0.076
0.057
29.1
3-5 min. 14-17 min. -
0.125 0.110 None
0.094 0.097 None
29.3 29.3
Overnight
0.086
0.057
29.4
15-20 min.
0.122
0. 056a
29.1
Overnight
O.0Mb 0.054
-
29.7
a This shrinkage on recrystallization was probably due to the presence of alloxantine in the crude precipitate. * The reaction was incomplete as the filtrate yielded a precipitate on being heated.
thereafter. Precipitation of uramil was complete in the first tube within an hour, whereas the second tube required three hours for completion. It is significant that the proportion of alloxan determined merely the rate of formation of uramil but not the ultimate yield which was about 75-85 per cent. of crude product and 65-70 per cent. of recrystallized material which analyzed correctly. Temperature e$ects.-Preliminary tests having indicated that uramil could be formed a t room temperature, the two sets of experiments just described were repeated in the cold. The results parallel those obtained
FORMATION OF URAMIL FROM DIALURIC ACID
371
on the water bath, confirming the r61e of air in the formation of uramil from dialuric acid and the catalytic action of alloxan. A NOTE ON THE STRUCTURE OF DIALURIC ACID
Although, in harmony with custom, the structure of dialuric acid has been represented in this paper by means of the carbinol formula (11), it seems worthwhile to point out that the properties of dialuric acid are better represented by the enediol formula (XI). For the sake of brevity these properties are listed below without discussion. 1. Acidity.-Dialuric acid is a fairly strong monobasic acid (pK = 2.83)11 while 5-ethyl-5-hydroxybarbituric acid is weak.12 2. Iron salt.-Dialuric acid forms a deep blue ferric salt. 3. Action of diazomethane.-Diazomethane converts dialuric acid to a tetra:methyl derivative13 (XII). 4. Autoxidation.-Dialuric acid is readily oxidized to alloxan or alloxantine by elementary oxygen.6 5. Reversible oxidation-reduction.-With alloxan, dialuric acid forms a reversible oxidation-reduction system."' l4 According to Conant's view,I5 this suggests a stable enol structure for the reductant, dialuric acid. NH-GO
I I GO COH I I NH-COH XI
CH3N-GO
I
I COCOCHI I II
CHaN-COCHS XI1
NH-GO
1 I
GO
1 II
CNHz
NH-COH XI11
By analogy, the structure of uramil is better represented by the enolic formula (XIII) than by the carbinamine formula (IV). SUMMARY
1. 'The formation of uramil by the action of ammonium chloride on dialuric acid is catalyzed by alloxan. 2. This catalysis is explained by assuming the reaction to occur in two stages; viz., (a) metathesis between alloxan and ammonium ion to form alloxan-imine and oxonium ion; and (b) oxidation-reduction between alloxan-imine and dialuric acid producing uramil and regenerating alloxan. 3. Biltz and Damm's preparation of uramil depends upon the production of alloxan, the catalyst, by the autoxidation of dialuric acid. RICHARDSON AND CANNAN,Biochem. J . , 23, 68 (1929). ASPELUND,J . prakt. Chem., [2],136, 334 (1933). 18 BILTZ AND PAETZOLD, Ann., 433, 77 (1923). " HILL AND MICHAELIS, Science, 78, 485 (1933). 16 CONANT, Chem. Rev., 3, 12 (1926). 11
