THE CHEMISTRY OF JAFFE’S REACTION FOR CREATININE.

VI. A COMPOUND OF PICRIC ACID WITH TWO MOLECULES OF CREATININE. ITS COMBINATIONS WITH ACID AND ALKALI.*

BY ISIDOR GREENWALD.

(From the Littauer Pneumonia Research Fund, New York University, New York.)

(Received for publication, January 21, 1930.)

In Paper IV of this series (l), there was described a compound containing 2 molecules of creatinine, 1 of picric acid, 3 of sodium hydroxide, and 3 of water. This was precipitated by running into alcohol the mixture obtained by the reaction of 1 mol of picric acid with 2 or more mols of creatinine and an excess of sodium hydroxide. The creatinine in this compound did not give Jaffe’s reaction until after its solution in dilute acetic acid had been allowed to stand for some time.

In another paper (2) it was shown that when the alkaline re- action mixture contained an excess of picrate over creatinine, precipitation with alcohol yielded a red substance which, when dissolved in water or alkaline picrate solution, gave immediately the full color value of the creatinine content. This compound contained 1 mol of creat,inine, 1 of picric acid, and 2 of sodium hydroxide. It was evidently the disodium compound of the red tautomer, or isomer, of creatinine picrate described in Paper I of this series (3). The corresponding barium salt has since been described by Anslow and Ring (4).

It seemed to be of interest to attempt to prepare the free acid from which the sodium compound that did not give Jaffe’s reaction

*A preliminary report was read before the Division of Organic Chemistry, American Chemical Society, Swampscott, Massachusetts, September, 1928.

Aided by a grant from the American Association for the Advancement of Science.

333

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334 Chemistry of Jaffe’s Reaction. VI

was derived. For this purpose, the alkaline reaction mixture was run into a slight excess of hydrochloric acid. The orange precip- itate obtained was filtered out, washed with ice water, and dried in vacua. As expected, it contained 2 mols of creatinine and 1 of picric acid (besides 1 or 2 mols of water) and did not give Jaffe’s reaction until after it had been dissolved in dilute acetic acid.

It was observed as the alkaline reaction mixture was run into the excess of acid that there was no precipitate until most of the acid had been neutralized. The dicreatinine-picric acid compound was evidently soluble in excess of acid. This indicated the possibility of the existence of a combination of this new compound with acid.

In an attempt to isolate this, the alkaline reaction mixture was run into an excess of acetic acid. No precipitate was obtained. The clear liquid was run into alcohol. An orange precipitate was obtained. This was washed with alcohol and dried in vacua. Analyses showed this to be not a compound with acid, but one with alkali.

It resembled the sodium compound previously described in that it did not give Jaffe’s reaction directly. It differed in that it contained only 24 mols instead of 3 for each mol of picric acid and in that, when treated with concentrated sulfuric acid, it lost only one-ninth, instead of two-ninths of its total nitrogen. This might indicate that the compounds had larger molecules than had been supposed and that they contained 4 mols of creatinine and 2 of picric acid and, in the case of the sodium compounds, 6 or 5 mols of sodium hydroxide.

The solution of this new sodium compound was neutral to litmus. It was quite obvious, therefore, that the dicreatinine- picric acid compound was a strong acid and that a combination with acid could occur, if at all, only at a high concentration of hydrogen ions. Since alcohol seemed to be the most available precipitant, hydrochloric acid could not be used because sodium chloride would also be precipitated. Hydrobromic acid was substituted. The clear acid solution was run into absolute alcohol but there was no precipitation. However, when propyl alcohol was used, an abundant orange precipitate was obtained. This was filtered out, washed with propyl alcohol, and dried in vacua,

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I. Greenwald 335

over HzS04. Analyses showed it to contain 2 molecules of creati- nine, 1 of picric acid, 1 of hydrobromic acid, and 5 or 6 of water. It is true that no preparation gave figures that agreed precisely with this formula but it is quite obvious from the method of preparation that the compound is likely to be contaminated with the free dicreatinine-picric acid compound and with products of decomposition of this and the hydrobromide.

The compound is not very stable, even after drying. Thus a preparation that contained 36.5 per cent picric acid in June, was found to contain only 28 per cent in October. When moist, it is much more unstable. If this compound is suspended in water and heated, practically no picric acid can be precipitated by sub- sequent treatment with nitron nor can creatinine be precip- itated by the addition of picric acid. Only if suspended in sodium or ammonium acetate solution and allowed to stand for several hours does the decomposition into the original constituents proceed at all smoothly.

Both the dicreatinine-picric acid compound and its hydro- bromide differ from the compounds with sodium hydroxide in that no nitrogen is split off when they are dissolved in hot con- centrated sulfuric acid. This would indicate a difference in structure.

A study of the behavior of substituted creatinines with alkaline picrate solutions led to the conclusion (5) that “the production of the red color . . . depends upon the formation of the enolic form of creatinine and upon the retention of the basic properties of the molecule,” On the assumption that picric acid, in picrates, is in the o-quinone form, the formula for yellow creatinine picrate was written

NO&X c=c 0 H NH -CO

/\II I/ HC C-N-0-N=C

xcmc// 0 i \ N(CHs)-CHz

NOz

and the red tautomer, or isomer, was provisionally given the formula

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336 Chemistry of Jaffe’s Reaction. VI

H t. . . . %. NOa/.

. . I 1 “c-c -COH

/ \ i7 HC C-N-0-N=C

\ ’ h

II c-c- ’ ‘N(CH)-CH H 3

..’ / loa //

“The dotted lines indicate that the exact positions of these hydro- gens and the disposition of the remaining valences of the carbon atoms are not known.”

Anslow and King (4) have taken exception to this formula and have proposed one of their own:

But this formulation fails to explain why dimethylol creatinine

N(CHsOH)-CO /

(CHsOH)NC \

NCHr---- CHa

fails to give the reaction, nor why, neither the dinitrophenols nor trinitro-m-cresol gives colors with creatinine (and alkali) at all resembling that given by picric acid.

In their discussion they state, “It is of interest that two sodium salts, one containing 1 molecule of creatinine, 1 of picric acid, 2 of sodium hydroxide, the other, 2 molecules of creatinine, 1 of picric acid and 3 of sodium hydroxide have been prepared by Greenwald and fit in with the above formulation of the red acid. The presence of three nitro-groups in the picric acid would allow of the addition of at least two mols of creatinine in the way formulated above, and the compound formed would require 3 atoms of sodium as found by Greenwald.”

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I. Greenwald 337

Anslow and King seem to have failed to recognize that the second of these compounds, that which contains 2 mols of creati- nine to 1 of picric acid, does not give Jaffe’s reaction until after the compound has been decomposed by dilute acetic acid. If the formulation of Anslow and King were correct, the dicreatinine compound when treated with alkaline picrate solution as in the determination of creatinine might be expected to dissociate into the monocreatinine compound which has the intense red color of Jaffe’s reaction. This is not the case. Moreover, the second creatinine cannot be combined in the same manner as Anslow and King assume the first to be, for 1 or 2 of the nitrogen atoms of the dicreatinine sodium or lead compounds are easily split off by treatment with concentrated acids. This is not the case with the red sodium compound containing equimolecular quantities of creatinine and picric acid. Moreover, since the free dicreatinine- picric acid compound and the hydrobromide do not lose nitrogen when treated with hot concentrated sulfuric acid, the nature of the combination of at least 1 of the nitrogen atoms cannot be the same in these as in the sodium or lead salts.

A number of possible formula? might be suggested but it seems best to await a study of the products of the decomposition of the dicreatinine-picric acid compounds as well as the results of the attempted preparation of similar compounds containing (a) homologues of creatinine and (b) other nitrophenols.

EXPERIMENTAL.

The methods of analysis Gere those previously described (1) except that precipitation of picric acid was attempted only after the substance had been allowed to stand in dilute acetic acid (plus ammonium acetate in the case of the hydrobromide) for from 12 to 16 hours. Bromine was determined, gravimetrically, by precipitation with silver nitrate, either directly or in the filtrate from the picric acid determination, after precipitation of the excess nitron with nitric acid.

I. Compound of 2 Mols of Creatinine and I of Pi& Acid.-To a solution of 4.52 gm. of creatinine in 100 cc. of water, there were added 6.84 gm. of creatinine picrate. 20 cc. of 6 N NaOH were then added. The temperature rose from 18 to 22”. After 15

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338 Chemistry of Jaffe’s Reaction. VI

minutes, the mixture was placed in a freezing mixture and the temperature lowered to 0”. It was then run in a fine stream into 25 cc. of 6 N HCI contained in a beaker immersed in a freezing mixture. The maximum temperature during this process was 5”. The orange precipitate was filtered out and washed with ice water. After drying over H&SO+ in vacua, this weighed 1.4 gm. The results of analyses are given in Table I (Preparation 98 A).

The filtrate from this precipitate became turbid as it was

TABLE I.

Composition of Different Preparations of Compounds of 2 Mols of Creatinine and 1 of Picric Acid.

Preparation No. Total N. Picrio acid. Pi& acid N.

78 81 86 98 A 98 B

104 113 118

Calculated for com- pound of:

2 mols creatinine, 1 picric acid, 1 HaO.

Same, with 2 mols H20.

per cent per cent

25.7 47.0 26.5 ‘48.3 26.1 48.0 26.4 48.2 25.6 47.0 25.4 47.1 25.1* 46.3 25.5 46.7

I I

per cent 8.62 8.86 8.81 8.84 8.62 8.64 8.50 8.57

26.6 48.4 8.84

25.7 46.6 8.56

401s creatinine 381‘ mol picric arid in reac- tion mixture.

* When dissolved directly in hot concentrated sulfuric acid, 24.3 per cent.

diluted with the wash liquid. Addition of 6 cc. of 6 N NaOH pro- duced a clear solution. 6 N HCl was then added until the mixture was distinctly acid to Congo red paper. For this purpose 4 cc. were required. The mixture was filtered and washed with ice water. After drying, this weighed 0.8 gm. It appears as Prep- aration 98 B in Table I.

Determination of Creatinine Content.-37.6 mg. of Preparation 81 were dissolved in a mixture of 10 cc. of HSO, 15 cc. of 1 per cent

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I. Greenwald 339

picric acid, and 5 cc. of 10 per cent NaOH. After 10 minutes, the mixture was diluted to 200 cc. and compared with 0.5 N

KJZrz07 set at 8.0 mm. The readings were 10.4, 10.2, and 10.2 mm., or the equivalent of 3.15 mg. of creatinine or 8.37 per cent.

23.8 mg. of the same preparation were allowed to stand over- night in 10 cc. of 1 per cent acetic acid. The next morning solution was not complete and the mixture was heated to boiling and then cooled. 10 minutes after the addition of 15 cc. of 1 per cent picric acid and 5 cc. of 10 per cent NaOH, the mixture was diluted to 500 cc. and compared with 0.5 N K&rz07 set at 8.0 mm. The readings were 7.4, 7.5, 7.5, and 7.3 mm., or the equiv- alent of 10.96 mg. of creatinine or 46.1 per cent. This is the cal-

TABLE II.

Analyses of Compound of 2 Mols of Creatinine, 1 of Picric Acid, and 6.6 of Sodium Hydroxide.

Preparation No. Total N.

per cent 70 20.3 92 22.2 94 22.1*

Calculated for com- position of 1 mol picric acid, 2 cre- atinine, 2.5 NaOH, 2 HzO. 22.1

-

per cent

38.9 39.6 39.4

40.1

_-

-

Pioric acid N.

per cent

7.12 7.26 7.23

7.37

Na C H ___.~ per cent per cent per cent

10.1 10.3 10.4 29.4t 4.11t

10.07 29.4 4.11

* Whendissolved in hot concentratedsulfuric acid, 19.9 per cent. t For these analyses I am indebted to Dr. W. A. Jacobs.

culated content for a compound of 2 mols of creatinine, 1 of picric acid, and 2 of water.

2. Compound with 2.5 Mols of NoOH.-To a solution of 4.52 gm. of creatinine in 100 cc. of Hz0 there were added 6.84 gm. of creati- nine picrate and 20 cc. of 6 N NaOH. The temperature rose from 15 to 20”. After 10 minutes at 20”, the mixture was cooled to 0” and 10 cc. of glacial acetic acid were added. The mixture was then run in a fine stream into 1200 cc. of absolute alcohol. The orange precipitate was washed with 95 per cent alcohol and dried over HzS04 in vucuo. It weighed 10.3 gm. After analysis, with the results given in Table II for Preparation 92, the remainder of the

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Chemistry of Jaffe’s Reaction. VI

material was dissolved in 100 cc. of water (the solution was neutral to litmus) and run into 1000 cc. of absolute alcohol. After being filtered and washed and dried in VCLCUO, the material was analyzed with the results given in Table II for Preparation 94.

Determination of Creatinine Content.-58.4 mg. of Preparation 94 were dissolved in a mixture of 10 cc. of water, 15 cc. of 1 per cent picric acid, and 5 cc. of 10 per cent NaOH. After 10 minutes, the mixture was diluted to 100 cc. and compared in a calorimeter with a solution containing 5 mg. of creatinine in 250 cc. and set at 20 mm. The readings were 26.4, 26.2, 26.2, and 26.2 mm., or the equivalent of 1.53 mg. of creatinine or 2.62 per cent.

25.1 mg. of the same preparation were dissolved in 10 cc. of water and 1 drop of acetic acid and the mixture was allowed to stand overnight. After the addition of the picric acid and sodium hydroxide the mixture was allowed to stand 10 minutes and was then diluted to 500 cc. The standard contained 5 mg. in 250 cc. and was set at 20 mm. The readings were 21.0, 21.2, 21.2, and 21.0 mm., or the equivalent of 9.48 mg. of creatinine or 37.8 per cent. The calculated amount is 39.6 per cent.

Recovery of Creatinine Picrate.-0.6012 gm. of the same prepara- tion was dissolved in 30 cc. of water. 0.5 cc. of acetic acid was then added and the mixture set aside. The orange color gradually became yellow and in about an hour the solution became turbid. About 3 hours later, 0.5 gm. of picric acid, dissolved in a minimal quantity of hot water, was added. The solution was then placed in the ice chest overnight. The crystals that separated were filtered out and recrystallized. After drying, they weighed 0.3173 gm., melted at 217”, and were found to contain 66.0 per cent picric acid and 32.1 per cent creatinine. Calculated for creatinine picrate, 67.0 per cent picric acid and 33 per cent creati- nine. The calculated yield of creatinine picrate is 0.732 gm. After correction for the solubility in the 100 cc. of mother liquor, 0.600 gm. was to be expected. Creatinine picrate seems to be more soluble in this liquid than in pure water. Calorimetric determinations indicated the presence of 0.117 gm. of creatinine in this solution, giving a total recovery of 0.220 gm. or 36.6 per cent.

3. Compound with Hydrobromic Acid.-To a solution of 4.58 gm. of picric acid in 125 cc. of boiling water, there were added 6.78

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I. Greenwald 341

gm. of creatinine. After cooling to 18”, 20 cc. of 6.51 N NaOH were added. The temperature rose to 23”. After 10 minutes, the mixture was cooled in a freezing mixture to -7” and was then run into 50 cc. of 6.51 N HBr at -10”. During this process, the temperature did not rise above 2” and at its close was 0”. The mixture was then run into 1000 cc. of n-propyl alcohol at -8”, with constant stirring. The mixture was kept at 5” for 30 min- utes and was then filtered and washed with ice-cold propyl alcohol. After drying over HzSO+ .in WZCUO, the yield was 4.1 gm. This constituted Preparation 154. The combined filtrates were kept at 5”. An additional precipitate separated. This was filtered out, washed with propyl alcohol, and dried over HzS04 in vacua.

TABLE III.

Analyses of Compound of B Mols of Creatinine, 1 of Picric Acid, and I of Hydrobromic Acid.

Preparation No.

90 154 156

Calculated for com- position of 2 mols creatinine, 1 picric acid, 1 HBr, 5 HzO.

Same, with 6 HzO.

Total N.

per cent

19.5 19.4* 19.8

20.13 19.57

Picrio acid. Picric acid N. Br

per cent per cent 35.6 6.54 36.5 6.70 40.0 7.34

36.60 6.71 35.55 6.52

per cent

12.2 12.4 12.2

12.77 12.41

* When dissolved in hot concentrated sulfuric acid, 19.7 per cent.

The yield in this fraction was 3.1 gm. (Preparation 156, Table III).

Determination of Creatinine.-24.9 mg. of Preparation 154 were dissolved in a mixture of 10 cc. of HzO, 15 cc. of 1 per cent picric acid, and 5 cc. of 10 per cent NaOH. After standing 10 minutes, the solution was diluted to 50 cc. and compared in a calorimeter with a standard containing 5 mg. of creatinine in a volume of 250 cc. and set at 20 mm. The readings were 21.2, 21.3, 21.2, 21.2, and 21.2 mm,, or the equivalent of 0.94 mg. of creatinine or 3.8 per cent.

34.4 mg. of the same preparation were left in 10 cc. of a dilute sodium acetate solution overnight. The usual reagents were

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342 Chemistry of Jaffe’s Reaction. VI

added and, after 10 minutes, the solution was diluted to 500 cc. The standard contained 5 mg. of creatinine in a volume of 250 cc. and was set at 20 mm. The readings were 15.3, 15.3, 15.3, 15.2, and 15.2 mm., or the equivalent of 13.1 mg. of creatinine or 38.1 per cent. Calculated for the compound with 5 HzO, 36.1 per cent; with 6 HzO, 35.1 per cent.

Recovery of G-eat&&e.-1.064 gm. of Preparation 154 were ground with 1 gm. of NaOAc.3Hz0 in a small mortar. Upon the addition of 10 cc. of water, a clear red solution was obtained. This was transferred to a beaker containing 400 cc. of ice water and allowed to stand at 5” overnight. No crystals having sep- arated, 0.5 gm. of picric acid was dissolved in a little hot water and added. Upon standing, crystals separated. These were filtered out and recrystallized twice. They melted at 215-217” and contained 66.0 per cent picric acid and 33.0 per cent creati- nine. (Calculated for creatinine picrate, 67 per cent picric acid and 33 per cent creatinine.) The total yield was 0.34 gm. The calculated yield, without allowance for solubility of creatinine picrate, was 1.13 gm. The 160 cc. of mother liquor would be expected to contain about 0.21 gm. of creatinine picrate, so there is a possibility that 1 mol of creatinine had suffered an irreversible change. But the product of its transformation must have had about the same action on alkaline picrate solution as creatinine itself for calorimetric analyses indicated the presence of 0.27 gm. of creatinine, making with that contained in the crystals, a total of 0.38 gm. or 35.7 per cent which is close to the calculated value.

SUMMARY.

In the presence of an excess of creatinine and alkali, picric acid may combine with 2 mols of creatinine. The resulting compound has been isolated in the free state and in the form of its combina- tion with (a) 3 mols of sodium hydroxide, (b) 23 mols of sodium hydroxide, and (c) 1 mol of hydrobromic acid. None of these compounds gives Jaffe’s reaction for creatinine until after solution in dilute acetic acid-acetate buffer solutions.

The formulas suggested by Anslow and King for the red isomer of creatinine picrate and for the dicreatinine-picric acid compound are considered to be unsatisfactory, The necessity for assuming

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I. Greenwald 343

quite different arrangements in (a) the red isomer, (b) the alkali salts of the dicreatinine compound, and (c) the free dicreatinine compound and its hydrobromide, is indicated.

BIBLIOGRAPHY.

1. Greenwald, I., J. Biol. Chem., 7’7,539 (1928). 2. Greenwald, I., J. Biol. Chem., 80,103 (1928). 3. Greenwald, I., and Gross, J., J. Biol. Chem., 69,601 (1924). 4. Anslow, W. K., andKing, H., J. Chem. SOL, 1210 (1929). 5. Greenwald, I., J. Am. Chem. Sot., 47,1443 (1925).

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Isidor GreenwaldALKALI

ITS COMBINATIONS WITH ACID ANDTWO MOLECULES OF CREATININE. COMPOUND OF PICRIC ACID WITHREACTION FOR CREATININE: VI. A

THE CHEMISTRY OF JAFFE'S

1930, 86:333-343.J. Biol. Chem. 

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