THE BIURET REACTION. There was published recently' in this

THE BIURET REACTION.

II. THE BIURET REACTION OF DI-ACID AMIDES.

BY MARY M. RISING, JOSEPH S. HICKS, AND GEORGINE A. MOERKE.*

(From the Kent Chemical Laboratory of the University of Chicago, Chicago.)

(Received for publication, July 12, 1930.)

There was published recently’ in this Journal a paper describing a study of the biuret reaction of acid imides. The work discussed therein constitutes an early part of an extensive investigation of the chemistry of the biuret reaction, undertaken for the purpose of ascertaining (1) the chemical nature and molecular structure of the colored products formed in the reaction of acid imides, di-acid amides, amino acid amides, and polypeptides with the biuret reagents cupric ion and alkali, and (2) the atoms or groups of atoms in molecules which show the biuret reaction which are essential for the occurrence of the reaction. It is hoped that the results of these investigations will uncover clues relating to the molecular structure of the proteins, which so characteristically form colored products with the biuret reagents.

The present paper discusses the biuret reaction of di-acid amides. The behavior of malonamide and certain of its N-alkyl and N-aryl derivatives, and of oxamide and some alkylated oxamides, with cupric ion and alkali is described, the isolation of the sodium copper salts of biuret, malonamide, monoethyloxamide, and symmetrical diethyloxamide is report.ed, and structures for these salts are proposed, based upon analytical data and the behavior of the salts. In addition, the theory of the biuret reaction, the development of

* The experimental work on the alkali copper salts of biuret, oxamide, and malonamide herein described was done by J. S. Hicks; that on the analogous salts of substituted oxamides by G. A. Moerke. Both investigations were carried out in partial fulfilment of the requirements for the degree of Doctor of Philosophy at the University of Chicago, 1927.

1 Rising, M. M., and Johnson, C. A., J. Biol. Chem., 80,709 (1928).

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2 Biuret Reaction. II

which was begun by SchiffJ2 and continued in our earlier paper, is augmented. Suggestions are made to explain certain phenomena observed in connection with the reactions of the amides studied, such as the well defined difference in the behavior of di-acid amides and acid imides towards the biuret reagents, and the failure of N- polysubstituted di-acid amides to show the biuret reaction. Sug- gestions are made further as to the atoms in di-acid amide molecules which take part in the biuret reaction, and with regard to conditions which are favorable for the occurrence of the reaction.

The logical point of departure for a study of the biuret reaction of di-acid amides is the biuret reaction of biuret, NH2. CO *NH .- CO .NHz, itself a di-acid amide. This beginning was made by Schiff in his isolation of potassium copper biuret, the formula of which he proved to be K&u (biuret)z.4 HzO. Similarly, the sodium copper salt of malonamide, NH,. CO . CH2. CO .NH2, which is closely related to biuret, of monoethyloxamide, C2HSNH.C0.- CO .NHz, and of symmetrical diethyloxamide, C!zHbNH . CO. CO .- NHCZHS, all isolated by us, have been found to have the formula MezCu (di-acid amide)z .zHzO. An inspection of the structures of the amides from which the salts are derived makes it apparent that di-acid amides of a variety of structural types react with the biuret reagents to produce salts containing a ratio of 2 atoms of alkali metal to 1 atom of copper to 2 molecules of amide. Schiff himself distinguished three types of di-acid amides showing the biuret reaction: (1) those with the group NH,.C.N.C.NH,, (2)

I II I II

those with the group NH2. C .C *C .NHz, and (3) those with the II I II

group NH:!. C. C .NH2. Biuret (Group l), malonamide (Group II II

2), and oxamide (Group 3) all form alkali copper salts of the formula Me&u (di-acid amide)z, as we have now found, and it is undoubtedly significant from the point of view of the atoms in a di- acid amide molecule which are essential for the occurrence of the biuret reaction that di-acid amides with these marked structural variations form products of identical formula with alkali and cu-

2 Schiff, H., Ber. &em. Ges., 29, 298 (1896); Ann. Chem., 299,236 (1898); 319,287 (1901); 362,73 (1907).


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Rising, Hicks, and Moerke 3

pric ion. It is quite probable that the matter of atoms required for the occurrence of the reaction may be reduced to much simpler terms than was done by Schiff in his classification: it is altogether likely that the atoms requisite for a biuret reaction in the case of a di-acid amide are common to all three of Schiff’s groups, and that if the atoms in t.hese groups not concerned in the reaction are excluded, the three groups become identical. The Schiff classification has its chief value as an aid in predicting which di-acid amides will, and which will not, show the biuret reaction. It pre- dicts, for example, the succinamide, NH2. CO. CH2. CH2. CO. NH2, and hydrazodicarbonamide, NH2. CO *NH *NH. CO. NH2, will show no biuret reaction, since they are structurally unlike the three types mentioned; and it is indeed the case that these two di- acid amides show no biuret reaction under any conditions so far used for the test. While the Schiff classification takes into ac- count the inhibition of the occurrence of the biuret reaction when the two acid amide groups in the molecule are separated by more than a single nitrogen atom or carbon atom (as in succinamide and hydrazodicarbonamide), an inhibition which we shall pres- ently attempt to explain, it fails to recognize two other important aspects of the biuret reaction of di-acid amides: (1) Certain N- alkylated di-acid amides show the reaction, notably monoethyloxamide, with the group RNH . C *C .NHz, and symmetrical di-

ll II

ethyloxamide with the group RNH . C. C .NHR. These two II II

groups, in common with Schiff’s three, contain the atoms essential for the occurrence of the reaction, which are probably the same for all di-acid amides which react with the biuret reagents. (2) The multiple substitution of hydrogen atoms attached to nitrogen in the di-acid amide molecule by alkyl radicals may entirely prevent the occurrence of the biuret reaction, as will be shown in the present work. The Schiff classification includes only d&acid amides; he does not extend his theory to acid imides with the characteristic group -C.NH.C-; many imides form colored products with

II II 0 0

the biuret reagents, having the formula Me&u (imide)a.~Hz0.1~3

3 Tschugaeff, L., Ber. &em. Ges., 38,2899 (1905); 39, 3190 (1906); 40, 1973. (1907). Ley, H., and Werner, F., Ber. hem. Ges., 38, 2199 (1905); 40, 705 (1907).


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Nor does the Schiff theory include amino acid amides with the characteristic group -C -C-NH,. Products of the biuret reac-

NH 6 tion of amino acid amides’are being studied by one of the present authors (Rising) in collaboration with Peter S. Yang.

In order to profit by the experience of Schiff, we began our work with the isolation of the product of the biuret reaction of biuret itself, sodium copper biuret. The salt was obtained by treatment of biuret with 4 mols of sodium hydroxide and 1 mol of cupric acetate in water, and subsequent precipitation of it from the reaction

TABLE I.

Analytical Data for Sodium Copper Biuret.

Element.

Na cu C H N 0

Percentage composition.

Sample I. Sample II.

13.10 13.32 18.33 18.32 13.73 13.80 2.80 2.85

24.01 24.27

Calculated percentage composition for

NazCkGHmOsNs (mol. wt. 347.718).

13.23 18.28 13.81 2.90

24.17 27.61

100.00

mixture by means of the addition of 20 volumes of alcohol. The reaction product was a rose-pink amorphous substance, quickly decomposed by water and all acids. The analytical data for two samples of the product of a single preparation of sodium copper biuret are to be found in Table I. Details regarding the procedure used to obtain this salt and others described are to be found in the experimental part of this paper.

The percentage composition of the salt agrees very well with that calculated for a molecule of empirical formula NazCuC4H1,,- 06Ns, and this formula corresponds closely to Me&u (di-acid amide)z.zHzO (biuret is GHaOzN3, and Na2CuC&,06N6 is Na&u- (C2H,02N&.2H,O). At present no satisfactory data for the molecular weight of this salt and the others discussed in the present paper are available; it is therefore assumed tentatively that the


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empirical formulas of the salts are also the molecular ones. With this assumption in mind a structure for the salt is proposed.

O-

/ -O\ C-NH HN=C

k. ‘... H-O-H .,.. /”

x. : “\ ,,..,’

./ ., : ‘.. ++ ,/’

Na2+ HN cu NH i : . . : i ,.,. ./ j “...

,,/ fH- C-J&

A-2 . . . . . . .

riN=C

\ / O- -0

Sodium copper biuret.

This structure indicates that sodium hydroxide reacts with an acid tautomer of biuret to form a salt4 Salt formation increases the basicity of the biuret molecule which, acting as an amine, forms a complex with cupric ion. In this complex there is only a minute ionization of cupric ion, due perhaps to the restraining effect of the union of auxiliary valences of copper with those of nitrogen and oxygen.

It will be noted that the complex ion in sodium copper biuret carries an excess of negative charge. Proof of the correctness of this assignment of charge has been found in the results of electrolysis of solutions of sodium copper biuret containing an excess of alkali to prevent hydrolysis. When a current is passed through

4 The biuret molecule may exist in a number of tautomeric forms.

H,N~C~N~C~NH,~HN:C~N~C~NH,~HN:C~N~C:NH

II I II I I II I I I OH0 OHH 0 OHH OH I 1‘

H,N~&~:C~NH, (1)

II I 0 OH

(3)

(2)

The formation of acid tautomers is assumed for other compounds of this type which show the biuret reaction.


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the solution, the pink ingredient, which contains the copper, accumulates around the anode, and the solution surrounding the cathode becomes colorless. Only after a relatively long time is metallic copper deposited upon the cathode showing the minimal ionization of the complex.

It may be recalled that the products of the reaction of acid i.mides with the biuret reagents have already been assigned the structures of salts comparable in constitution with the combina- tions of amino acids with salts; e.g., K+(-OOC~CH~~N+H~)C1-.l For example, the product of the biuret reaction of the imide diethylbarbituric acid, 90. NH. CO -NH. CO. C (GH&, is a sub-

stance of molecular formula NazCu(imide)a.2 HzO, and may have, according to Rising and Johnson, the structure

Naz+ ([O-a C : N - CO - NH . CO . C(C2Hs)& Cu++ (HaO)z)

[O-e C : N - CO . NH . CO . ~(C,H,),lz I

in which the complex ion is assigned no excess of negative or positive charge. The results of electrolysis of solutions of this salt indicate, however, that the complex ion containing copper is negative: since it moves to the anode. In view of this fact it would be more accurate to assign to sodium copper diethylbarbitate the structure

Naz+ [(O- . C : N - CO . NH 9 CO . y(CzHs)z)a Cuff (HzO)t] I.

This structure indicates that sodium hydroxide neutralizes 2 molecules of diethylbarbituric acid; the imide then behaves as an amine, forming a complex with cupric ion in which there are 4 molecules of the imide. Here, as in sodium copper biuret, six auxiliary valences of copper may function, two holding the oxygen atoms of 2 molecules of water, and each of the other four holding a nitrogen atom of the imide (or amide).

The pertinent question may well be raised at this point as to why 4 molecules of an acid imide react with the biuret reagents to form MezCu (irnide)~.~H~O: while di-acid amides5 form with the

6 Biuret is an acid imide, with the characteristic group -CO*NH*CO-, as well as a di-acid amide. In its reaction with alkali and cupric ion it behaves as a di-acid amide, since the product is a substance of formula MezCu (di-acid amide)2 rather than of formula MezCu(imide)d.


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same reagents products of formula MezCu (di-acid amide)z.xHzO Some suggestions are offered in an attempt to formulate an answer to this question: an acid imide molecule contains 1 acid hydrogen atom, namely that in the group - CO *NH. CO - , capable of migra- tion to a nearby oxygen atom under the influence of alkali, and of subsequent replacement by a metal ion. It may be reasoned that under the conditions used for the biuret reaction 2 molecules of imide, thus neutralized, and with amine properties thus enhanced could enter into a complex with cupric ion, forming the product

rH,C-C-O- -0-C-CHI

Na: \ //

N _______.______..... CUT’ ._______.____.__ N

/ \ H,C-C=O O-C-CHz

L 1,

This product cannot, however, represent an actual molecule be- cause it is not electrically neutral, since it carries an excess of two positive charges. Two further auxiliary valences of the copper may then function, resulting in the formation of an electrically neutral molecule, which contains 4 imide ions, of structure

N&Z

rH,C-C-O- rH,C-C-O- -0-C-CH, -0-C-CH, \ \ / /

/” /” .**. ..\ .**. ..\ ,... “\ ,... “\ &C-C=0 &C-C=0 ‘. . . . . . ‘. . . . . . /“” /“”

‘..* r” ‘..* r” O=C-CH2 O=C-CHs

‘.. ++ .,.,* ‘.. ++ .,.,* cu cu

. ..’ . ..’ t. t.

H&-C=0 H&-C=0 \ \ .,..,,. ..f ‘.*’ .,..,,, .i/ \ \

...,. ...,.

YN YN

. . . . . . . . . . . ;~-s . . . . . . . . . . . ;F-L

\ \ H&-C-O- H&-C-O- -0-C-CH:! -0-C-CH:!

It is apparent that the complex now carries an excess of negative charge, as the results of electrolytic study indicate. In this molecule the second pair of imide ions fulfil the function of “internal neutrali!zation,” constitut,ing the negative radical of the salt, much as SOP is the negative radical of [Cu(NH&++] SOT, and


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as Cl- is the negative radical of K+(-OOC . CHzN+H3)C1- In the latter instances SOT and Cl- are not placed within the complex since they are ionizable and not bound to any atom within the complex by auxiliary valences. In the imide complex, however, the negative radical is placed within the complex since it is non- ionizable due to its secondary union with copper. In summary, it requires 4 imide molecules to form an alkali copper imide salt since each molecule is a monobasic acid and contains 1 amine nitrogen atom.

If this summary represents the facts, and 4 hydrogen atoms and 4 nitrogen atoms are concerned in, and required for, formation of the product of a biuret reaction, it is readily seen that 2 molecules

TABLE II.

Analytical Data for Sodium Copper Malonamide.

Na cu C H N 0


Sample I.

13.45 13.56 18.41 18.43 20.70 20.74

3.36 3.43 15.91 16.17

Sample II.

Mculated percentage composition for

NarCuCsH~nOsNa (mol. wt. 345.728).

13.30 18.39 20.83

3.50 16.21 27.77

100.00

of a di-acid amide fulfil these requirements, since a typical di-acid amide has in its molecule two acid groups and 2 amine nitrogen atoms. The structure proposed for sodium copper biuret shows a molecule in which four auxiliary valences of copper find satis- faction in 2 molecules of the amide and two others in 2 molecules of water; an acid group in each molecule of biuret has been neutralized by 1 molecule of alkali, and the second acid group in each of the 2 biuret molecules is internally neutralized by the complex; these two acid groups thus fill the role of negative radical.

The biuret reaction of malonamide was next studied. In a sense, malonamide is a more typical di-acid amide than is biuret since its molecule contains no acid imide group. Sodium copper malonamide was obtained by treatment of 1 mol of malonamide with 4 mols of sodium hydroxide and 1 mol of cupric acetate in


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Rising, Hicks, and Moerke

water, and subsequent precipitation of the salt from the reaction mixture by means of alcohol and ether. The salt is a rose-pink amorphous powder which is decomposed by water and all acids. The analytical data for sodium copper malonamide are to be found in Table II.

The percentage composition of the salt agrees well with that calculated for a molecule of empirical formula Na2CuCSH1206N4, and this formula corresponds closely to MezCu (di-acid amide)2 .z- Hz0 (malonamide is CaH602Nz, and NazCuCsHlzOGN~ is NazCu- (GH~O~N,), .2&O) .6 A structure for the salt is proposed.

r O-

/ C=NH

-O\ IIN=C

1

i

Sodium copper malonamide.

The close structural analogy of biuret and malonamide points to the probability that the alkali copper derivatives of the two di- acid amides are essentially the same in structure.

The effect of N-alkylation of di-acid amides upon the course of their biuret reaction was studied in the case of N-substituted OX-

amides and malonamides. Oxamide itself shows the biuret reaction very readily and the solid product of the reaction was isolated

6 Certain products of the biuret reaction of di-acid amides and acid imides (e.g., sodium copper monoethyloxamide and potassium copper suc- cinimide) are readily dehydrated in vucuo over sulfuric acid or phosphorus pentoxide at room temperature. It has seemed to us logical to assign to the water in such molecules a position outside the complex ion. Attempts to show the presence of water in sodium copper malonamide (and similarly in sodium copper biuret) by dehydration have failed. If there are 2 molecules of water in these salts they are probably within the complex.


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by us but not in pure form, as the rose-colored salt was invariably contaminated with oxamide, the insolubility of which prevents its complete removal from the salt. The analytical results for sodium copper oxamide so far available do not furnish any reliable infor- mation about the formula of the salt.

Monoethyloxamide, CzH5NH. CO. CO. NH2, was prepared and converted into its sodium copper derivative by treatment of 2 mols of monoethyloxamide with 2 mols of sodium hydroxide and 1 mol of cupric acetate in alcoholic solution containing approximately 15 per cent of water. The salt was also prepared in water. It is a rose-pink crystalline substance, separating as needles from alcohol, and plates from water. The analytical data for the products of two preparations of the salt are to be found in Table III.

The percentage composition of the salt agrees well with that calculated for a molecule of empirical formula Na#ZuCeHzoOsN~, and this formula corresponds closely t,o Me&u (di-acid amide)n- * zH?O (monoethyloxamide is CJSsOzN~, and NazCuCsHzoOsNd i,s NazCu(C4H602N&.4Hz0). A structure for the salt is proposed.

Na,

O- -0 / \

C-NH HN=C ‘... .’

*... \ ,...**

5.. f \ ,..’

‘... ++ . ..*’ cu

f 5.. *...** . . ‘...

/” i. \

C=N&HJ

‘S.

(C*HJN=C \ /

O- -0

.4HzO

Sodium copper monoethyloxamide (rose, hydrated).

This structure finds support in (1) percentage composition data, (2) results of electrolysis of the salt, and (3) dehydration data.

Electrolysis.-The solution of the salt used for electrolysis con- tained an excess of alkali to prevent hydrolysis. When a current was allowed to pass through this pink solution between suitable electrodes, the solution became colorless around the cathode within a short time and the line of demarcation between colored and color-


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less regions of the solution moved gradually to the anode. In the pink solution about the anode there was formed slowly by the gradual decomposition of the complex a precipitate of copper oxide. It is to be concluded from these observations that copper is in a negative complex.

TABLE III.

Analytical Data for Sodium Copper Monoethyloxamide.

I Percentage. composition. Calculated percentage

Element. composition for NazCuCsHtoOsN~

Preparation I. Preparation II. (mol. wt. 409.73).

Na 11.19 11.36 11.23 cu 15.62 15.43 15.52 C 23.53 23.96 23.43 H 5.01 4.96 4.92 N 13.21 13.06 13.67

31.23 100.00

TABLE IV.

Analytical Data for Sodium Copper Diethyloxamide (Symmetrical).

Element.

Na cu C H N 0

T

-


Sample I.

11.68 11.70 16.18 15.99 36.92 36.83 5.34 5.28

14.15 14.16

Sample II.

-

- t Xculated percentage

composition for NaSJKhH~oO~N~ (mol. wt. 393.724).

11.68 16.14 36.83 5.12

14.22 16.01

100.00

Dehydration.-When the pink salt is allowed to stand in a desiccator over phosphorus pentoxide for a period of 13 days at room temperature it loses 17.04 per cent of its weight, an amount corresponding to 4 molecules of water. The loss in weight is accom- panied by a change in color from pink to lavender, and this fact suggests that at least part of the water should be placed within the complex as water of structure. However, all 4 molecules are re- movable at low temperatures, resembling in this regard water of crystallization. We prefer to reserve judgment as to the struc-


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12 Riuret Reaction. II

tural position occupied by the water until further evidence is available. The structure of the lavender salt may be

O- O- / / -"\ -"\

C=NH C=NH HN=C HN=C ‘.., ‘.., ‘%.* ‘%.* *..: *..: “.. “.. ./ ./

/ / . . . . . . . . . . . . . . . . ++ ++ ,/’ ,/’

cu cu ‘... ‘...

/ ‘... . ..- ,/’

. . , . /

‘... ,i’ ,,/ % %.

“..

C=N&Hs) C=N&Hs)

\ (&-&TZC (CsH$N-C

\ \ / / O- O- -0 -0

Sodium copper monoethyloxamide (lavender, dehydrated).

Besides the alkali copper derivatives of monoethyloxamide just described, sodium nickel monoethyloxamide was prepared and analyzed. The analytical data for this salt are to be found in the experimental part of this paper. It is a part of our plan to inves- tigate alkali nickel salts of di-acid amides and imides.

Symmetrical diethyloxamide, NH. CzHs * CO. CO .NH. CzH5, was prepared and found to show the biuret reaction, although Schiff states that. this di-acid amide does not. react with the biuret reagents.7 A study of the conditions favorable for the occurrence of the reaction showed t.hat diethyloxamide (symmetrical) forms no colored product. with alkali and cupric ion in dilute aqueous solution. When, however, precautions are taken to prevent hydrolysis by the use of a nearly non-aqueous reaction medium containing an excess of alkali and of diethyloxamide, the biuret reaction occurs, and a soIid product of deep blue color is readily obtained. Sodium copper diethyloxamide (symmetrical) was sub- jected to analysis, and the results are summarized in Table IV.

The percentage composition found agrees quite closely with that calculated for a molecule of empirical formula NaLJuClzHzo- 04N4 (diethyloxamide is CsH1Z02N~ and Na2CuG2H2,04N4 is NaL!u(CsH1202N&). Diethyloxamide (symmetrical) thus behaves as a typical d&acid amide in that it forms with alkali and cupric

’ Schiff, H., Ann. Chem., 299,257 (1898).


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ion a product of formula Me&u(di-acid amide)a. The structure of the salt may be

Na;t

r

/O- -0

\ C=N(CzHs) (&H&N=C

2.. . ..* \ .-..

*.f ‘.. /*,.

‘s. +.+ ..* 8” cu

/ \ ‘... ,...” 5..

/ 5,.

C=N&Ha)

‘...

(GH;;N=C \ /

O- -0

Sodium copper diethyloxamide (symmetrical).

Electrolysis showed that the copper is held in a negative complex. The abrupt change in color from the rose of the sodium copper

derivatives of oxamide and monoethyloxamide to the deep blue of sodium copper diethyloxamide (symmetrical) may perhaps be ac- counted for on the basis of a difference in water content. The pink biuret reaction products are considered to be hydrates. Dehy- dration of pink sodium copper monoethyloxamide produces a lavender salt. Blue sodium copper diethyloxamide (symmetrical) is not a hydrate. Possibly blue and lavender are the normal colors for anhydrous salts in the series.

Unsymmetrical diethyloxamide, (C&H&N. CO. CO. NH2, and triethyloxamide, (C2H&N . CO. CO .NH(C2Hs), show no biuret reaction under any conditions so far used for the test. In view of these facts it seemed likely to us that tetraethyloxamide, (C&H&- N.CO-CO-N(C H ) 2 5 2 would also be inactive towards the biuret reagents, and no attempt was made to devise a method for its synthesis.

The effect of N-substitution of alkyl groups in the malonamide series was next studied.8 N-monoethyhnalonamide, GH, .HN .-

8 It has been shown by Schiff (Ann. Chem., 299,254 (1898)) that C-sub-

7 stituted di-acid amides of the type NH2 * CO . C. CO . NH2 and NH% a-

l R


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CO. CH2. CO. NHZ, prepared by a method devised for the purpose, was found to react with cupric ion in alkali to form a bluish purple product. So far the colored compound has not been isolated from the reaction medium in which it was formed, and there remains work to be done in this direction. N-monophenylmalonamide, CsH5.HN.C0.CH2.C0.NH2, said by Schiff not to show the biuret reaction, was found to form a greenish blue product with cupric ion in alkali. The isolation of the pure compound has not yet been accomplished. In this connection it is of interest to recall that copper glycine, (NH2 . CH, +CO&Zu, is blue, while copper phenylglycine, (C&H5 .HN. CH2 1 C02)&!u, is green. N,N- diethylmalonamide, CzHs .HN. CH,. CO. NH. C2H5, was prepared and found to show no biuret reaction under the conditions used.

It is desired to state by way of summary certain hypotheses concerning the biuret reaction of di-acid amides, formulated by us on the basis of the results of the work just discussed. Biuret, oxamide, and malonamide readily show the reaction; mono- and symmetrical dialkylated oxamides show the reaction but unsymmetrical dialkylated and trialkylated oxamides do not; N-mono- alkylated malonamides show the reaction, while further alkylation of the malonamide molecule prevents it. These findings are significant in that they serve to clarify the essential nature of the biuret reaction and aid in predicting with some degree of accuracy the behavior of di-acid amides toward the biuret reagents.

It has been pointed out previously] that a molecule which is sensitive to the biuret reaction is both an acid and a base and that the reaction of such a molecule with alkali and cupric ion involves salt, and complex ion, formation. 4 ionizable hydrogen atoms apparently take part in the reaction. A typical acid imide mole-

CO * C * CO * NH2 readily show the biuret reaction. Biltz, H., and I

R Jeltsch, A., Ber. them. Ges., 66, 1923 (1923), have shown that di-acid

R I

amides of the type NH2 . CO . N . CO . NH2 readily show the reaction. It is therefore apparent that substitution outside the amide groups does not affect the course of the reaction.


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cule contains 1 such hydrogen atom, hence 4 imide molecules are to be found in the biuret reaction product of an acid imide, while a di-acid amide with 2 ionizable hydrogen atoms forms a product containing only 2 amide molecules. The bare essentials in the matter of atoms for the occurrence of the biuret reaction of a di- acid amide seem to us to be (1) 2 acid hydrogen atoms in each molecule for salt formation, and (2) 1 or more amine nitrogen atoms in each m’olecule for complex ion formation.

It would undoubtedly be apart from the truth, however, to conclude that the occurrence of these essential atoms in a di-acid amide molecule suffices to insure its reaction with cupric ion and alkali. Certain other conditions favoring the occurrence of the reaction must be met, and it is believed that a number of factors aside from the “essential atoms” mentioned influence the behavior of di-acid amides toward the biuret reagents. One of these may be termed the ionizing power of the amide as an acid. If the deli- cat,e balance between acidity and basicity in the amide molecule is disturbed, and, for example, the amide becomes so weaklybasic (and therefore strongly acid) that complex ion formation is prevented, no biuret reaction can occur. On the other hand, if any change of condition makes the amide molecule so weakly acid (and conse- quently strongly basic) that salt formation is prevent,ed, again no biuret reaction can occur. N-alkylation undoubtedly has a marked effect upon the acidity of the amide molecule. Since an alkylated amino group is more strongly basic than an unsubstituted one, an alkylated amide should be more strongly basic (and less acid) than its unsubstituted analogue. Mono- and symmetrical dialkylations of oxamide do not prevent the occurrence of a biuret reaction, and it is logical to conclude that such introduction of alkyl groups does not reduce the acidity of the molecule to the extent that salt formation cannot occur; 2 of the remaining hydrogen atoms in each molecule are evidently ionizable. Unsym- metrical diethyloxamide shows no biuret reaction despite the fact that its molecule contains 2 hydrogen atoms, and it would appear that substitution of this variety disturbs the balance between acidity and basicity to such an extent that the hydrogen atoms of the unsubstituted amino group are non-ionizable; the amide has no longer sufficient acid properties for salt formation, a fundamen- tal preliminary to the occurrence of a biuret reaction. Triethyl-


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oxamide is even more strongly basic and its 1 hydrogen atom may be wholly non-ionizable, while tetraethyloxamide has no acid properties whatever.

Thus in the oxamide series we have correlated the progressive decrease in acid strength attendant upon alkylation with decreased tendency to show the biuret reaction.

It has been of interest to us to note that the occurrence of the biuret reaction is prevented earlier in the biuret and malonamide, than in the oxamide, series. It is reported by Biltz and Jeltsch8 than monomethylbiuret, CH3.NH .CO.NH.CO.NH2, shows the reaction but that symmetrical dimethylbiuret, CHs.NH *CO *NH.- CO *NH .CH3, does not. We have found that N-monoethylmalonamide shows the reaction, while symmetrical N-diethylmalonamide does not. The inhibition of the reaction by the multipli- cation of alkyl groups attached to nitrogen may again be the result of the decrease in acid strength of the amide att.endant upon alkylation. The cause of the earlier inhibition may be the separation of the two amide groups, in biuret by a nitrogen atom, and in malonamide by a carbon atom. Just as oxalic acid, with its carboxyl groups adjacent, is a stronger acid than malonic acid with its carboxyl groups separated by a carbon atom (oxalic acid, K1=9X10-2, Kz=4X10-5; malonic acid, K1=1.6X10-3, Kz= 2X10+) so oxamide is probably a stronger acid than is malonamide or biuret, and for the same reason. The influence of this “separation factor” upon di-acid amides may be such that the increase in the number of carbon or nitrogen atoms separating the amide groups weakens the strength of the amides as acids so that salt formation is ultimately hindered. The failure of succinamide and of hydrazodicarbonamide to show the reaction may be ex- plained on this basis ‘(succinic acid, K1 = 7 X 10P5, Kz = 2X lo+, is even weaker than malonic acid). Hence there are two factors which may affect the salt-forming power of di-acid amides and therefore their behavior with the biuret reagents, alkylation, and separation of the amide groups, and the net result of these influ- ences may be no biuret reaction at all.

The effect of N-alkylation upon the acidity of the amide molecule has been stressed. The effect of substitution upon basicity has also been observed in the case of phenylbiuret, CsH5 -NH. CO .


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NH.C0.NH2, which does not show the biuret reaction.g The phenyl group is strongly acid in its effect and its substitution in the amino group may reduce the basicity of the group to such an extent that complex ion formation is prevented (aniline forms complexes less readily than does ammonia).

The conclusions outlined here are tentative, but are believed to be useful working tools for our further research upon the chemistry of the biuret reaction.

EXPERIMENTAL.

1. Sodium Copper Biuret, NcL&uC~H~~O~N~.

The preparation of sodium copper biuret was carried out as follows: biuret’O (4.0 gm.) was dissolved in a 10 per cent aqueous solution of sodium hydroxide (5.3 gm.) and powdered copper acetate (CU(C&H,O&*HZO) (6.6 gm.)” was added at room temperature. The solution became deep violet-red in color. The con- tents of the reaction vessel were thoroughly stirred, and finally the supernatant solution was decanted from some unused copper acetate. To this solution there were added, with constant shaking, 20 volumes (600 cc.) of anhydrous alcohol containing a few cc. of sodium hydroxide solution, used to prevent hydrolysis of sodium copper biuret. Upon addition of the alcohol, sodium copper bi- m-et was precicitated as a bright pink, finely divided substance. The salt was brought on a filter, washed with absolute alcohol, and dried in a vacuum desiccator over sulfuric acid and solid sodium hydroxide. From 4 gm. of biuret, 5.6 gm. of the sodium copper salt were obtained. It is particularly important that the alcohol and sodium hydroxide used in the preparation of all the biuret reaction products should be entirely free from carbon dioxide, and

9 Gatewood, E., J. Am. Chem. SOC., 46,146 (1923). I0 Biuret was prepared by the method of Thiele (Thiele, J., Ann. Chem.,

303, 95 (foot-note) (1898)) modified by Newburn, L., Master’s thesis, Uni- versity of Chicago (1923).

11 The proportion of reagents used is 1 mol of biuret to 1 mol of copper acetate to 4 mols of alkali. The excess of alkali serves to prevent hydrolysis of the salt. The product of the reaction contains 2 molecules of biuret to every cupric ion, but it was found necessary to use an excess of copper salt in the preparation of all of our biuret reaction products, since products of varying copper content were obtained otherwise.


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that all reagents used and products obtained be protected from moisture and carbon dioxide at all times.

Sodium copper biuret is a pink amorphous substance which is soluble in water, being extensively hydrolyzed therein. It is decomposed by carbon dioxide and all acids. When a solution of the salt was electrolyzed, the pink color deepened around the anode, while the solution around the cathode became colorless. It was concluded that the complex ion containing copper is negative.

A complete set of analytical data obtained for the salt (already referred to in the introduction to this paper) follows. The copper content of t,his salt and of the others about to be described was determined by electrolysis. Sodium was determined as sodium sulfate in solutions of the salts from which copper had been removed.

0.2269, 0.1927 gm. substance: 0.0918, 0.0793 gm. NatSOd. NazCuC4HIoOsNa. Calculated. Na 13.23.

Found. “ 13.10, 13.32. 0.2269, 0.1927 gm. substance: 0.0416, 0.0353 gm. Cu.

Na#uC+HlOO~Na. Calculated. Cu 18.28. Found. “ 18.33, 18.32.

0.0719, 0.0848 gm. substance: 0.0362, 0.0429 gm. CO2 and 0.0180, 0.0216 gm. HsO.

Na&uCJLoOaNs. Calculated. C 13.81, H 2.90. Found. “ 13.73, 13.80, “, 2.80, 2.85.

0.0976,0.1112 gm. substance: HCl (factor 0.0913) 18.33 cc., 21.11 cc. Na&uCdH1006Ne. Calculated. N 24.17.

Found. “ 24.01, 24.27.

The analytical results agree well with the empirical formula NazCuCaH1oOsNs. The structure which we have proposed for the salt is to be found in the introduction to this paper.

2. Sodium Copper Malonamide, NazCuC6HlzOsN~.

To obtain sodium copper malonamide, malonamidel* (3 gm.) was dissolved in a 10 per cent aqueous solution of sodium hydroxide (4.7 gm.), and this solution was treated with powdered copper acetate (5.9 gm.) at room temperature. After thorough mixing of these ingredients the reaction solution, which was deep purple,

12 Malonamide was prepared by the method of Freund, M., Ber. them. Ges., 17, 133 (1884).


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was decanted from the unused copper acetate and treated with 5 volumes of anhydrous alcohol, then very rapidly with 3 volumes of carbon dioxide-free anhydrous ether. Bright pink sodium copper malonamide was thereupon precipitated; the salt was collected on a filter, washed with absolute alcohol, and dried in vacua over sulfuric acid and solid sodium hydroxide. From 3 gm. of malonamide 3.8 gm. of the salt were obtained.

Sodium copper malonamide is a pink amorphous substance which is soluble in water, in which it is appreciably hydrolyzed. In dilute solution the salt is decomposed and copper hydroxide is precipitated. This decomposition does not occur in a concentrated solution of the salt. Electrolysis of a 7 per cent solution of the salt led to the accumulation of the colored ingredient around the anode, while the solution around the cathode became colorless. The salt is decomposed by carbon dioxide and all acids. Attempts were made to prove that sodium copper malonamide is a hydrate. It was found, however, that the salt does not lose weight nor show a color change when placed in vacua over boiling bromobenzene (156”). Above this temperature the compound turns brown and decomposes. Complete analytical data for the salt follows.

0.0869,0.1232 gm. substance: 0.0361,0.0516 gm. NazS04. NazCuCsHIzOaN1. Calculated. Na 13.30.

Found. “ 13.45, 13.56. 0.0869,0.1232 gm. substance: 0.0160,0.0227 gm. Cu.

Na&uCeHlzOsNa. Calculated. Cu 18.39. Found. “ 18.41, 18.43.

0.992,0.0944 gm. substance: 0.0753,0.0718 gm. COZ and 0.0298,0.0289 gm. HzO.

Na&uGHlzOsN1. Calculated. C 20.83, H 3.50. Found. ” 20.70, 20.74, ” 3.36, 3.43.

0.3242,0.1113 gm. substance: HCI (factor 0.0913) 40.35 cc., 14.08 cc. Na&uCsHlzOsNc Calculated. N 16.21.

Found. “ 15.91, 16.17.

The analytical results agree well with the empirical formula NazCuCsHlzOcN4. The structure proposed for the salt is to be found in the introduction to this paper.

3. N-Monoethylmalonamide, C2Hs. HN . CO. CH2. CO. NH2, and Its Behavior with the Biuret Reagents.

N-Monoethylmalonamide was obtained from cyanoacetic ethyl ester in a series of reactions:


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CzH60.CO.CHz.CN + CzHsOH + HCl + CZH~O.CO.CH.C(OC~H~): NH&l

C&H60.CO.CHz~C(OCzHs) : NH&l ‘z” C~HSO.CO.CHZ.CO.NHS + CJSCl

CzHbO.CO.CHz.CO.NHz + NHa.CzHa + CzHsHN . CO. CH2. CO. NH, + C&OH

A. Malonamic Ethylimido Ester Hydrochloridet C2H50. CO. CH2 .- C(OCzHJ : NHL’L-The imido ester hydrochloride was prepared according to the method of Pinner,13 a yield of 88.3 per cent being obtained.

B. Malonamic Ethyl Ester, CzH50. CO. CHz. CO. NHz.-The ester was obtainedI in quantitative yield when t.he imido ester hydrochloride was warmed at a temperature of 106-108” till evo- lution of ethyl chloride ceased. The residue was extracted several times with anhydrous ether under a reflux; traces of ammonium chloride were separated from the ether solution by filtration, and the ether was removed by evaporation. The residue was pure amide ester of melting point 50”. The use of ether for extraction of the ester is much to be preferred to that of acetone advised by Pinner.

C. N-Ethylmalonamide, CZHSHN. CO. CH2 . CO. NH2.----Malo- namic ethyl ester (14.5 gm.) in 10 cc. of 95 per cent alcohol was placed in an ice bath and ethylamine (10 gm.) was added; the containing flask was then stoppered and allowed to stand overnight. A solid mass of the ethyl amide formed, which was separated from the reaction solution and extracted, under a reflux, with 350 cc. of a mixture of equal amounts of dry benzene and toluene. The crystals of ethyl amide which formed upon cooling of the bensene- toluene mixture were separated by filtration and the mother liq- uor was used for several extractions of the crude mass of amide. The yield was 11.6 gm., or 77 per cent of the calculated yield. The ethyl amide melted at 122’. The substance was identified by analysis.

1X Pinner, A., Ber. them. Ges., 28,479 (1895). I4 It is probable that dehydration of the blue solution would result in the

separation of sodium copper N-monoethylmalonamide in solid form. A method of dehydration has not yet been found. The reaction does not occur readily in other solvents.


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0.1913, 0.1597 gm. substance: 0.3211, 0.2693 gm. CO* and 0.1374, 0.1159 gm. HzO.

CaHloOzN~. Calculated. C 46.13, H 7.75. Found. “ 45.77, 45.99, ” 8.03, 8.12.

0.1512, 0.1014 gm. substance: 29.5 cc. N~(23”, 747.0 mm.), 19.8 cc. Nz(24”, 741 mm.) (over 50 per cent KOH).

CdLoNzO~. Calculated. N 21.52. Found. “ 21.62, 21.33.

N-Monoethylmalonamide reacts with cupric ion and alkali, When 0.1 gm. of the amide is moistened with 1 drop of a saturated aqueous solution of copper acetate and 1 or 2 drops of 35 per cent aqueous sodium hydroxide a deep purplish blue color develops. Attempts to isolate this blue product have been so far unsuccessful and further study is needed. l4 Electrolysis of the blue solution led to a deepening of color around the anode, the solution around the cathode becoming colorless.

4. Sodium Copper Monoethyloxamide, NaJ7uC~Hz~OsN~.

To obtain sodium copper monoethyloxamide, monoethyloxa- mide15 (3 gm.) was &ssolved in 250 cc. of 95 per cent alcohol and this solution was warmed to 50”. 25 cc. of a warm aqueous solution containing 2.4 gm. of copper acetate were then added, where- upon a blue-green precipitate appeared. A 35 per cent solution of sodium hydroxide (6 cc.) was dropped slowly into the reaction mixture from a burette while the mixture was constantly shaken. The blue-green precipitate was gradually replaced by a rose-pink one and precipitation was complete after 14 hours. The precipitate was collected, washed with alcohol, and dried in vacua over calcium chloride and soda-lime. The yield was 5 gm. The salt is a pink crystalline substance which is decomposed by water and all acids. Electrolysis of a solution of the salt containing an excess of alkali to prevent hydrolysis resulted in intensification of the pink color around the anode while the solution around the cathode became colorless. A precipitate of copper oxide formed under the anode as the colored ion containing copper decomposed, and copper was slowly deposited on the cathode. Analytical data

15 Monoethyloxamide was prepared by the method of Wallach, O., Ann. Chem., 184, 65 (1877).


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for the salt follow, analyses being carried out on the products of two different preparations of the salt.

0.6083,0.7207gm. substance: 0.2102,0.2529 gm. Na&Od. Na&uCsHioOsNd. Calculated. Na 11.23.

Found. “ 11.19, 11.36. 0.6083,0.7207 gm. substance: 0.0950,0.1112 gm. Cu.

Na&uCsHzoOsNc Calculated. Cu 15.52. Found. “ 15.62, 15.43.

TABLE V.

Dehydration of Sodium Copper Monoethyloxamide.

Weight of samples. Conditions used.

gm.

0.3504 P~06, room tempera-

0.3410

0.1082

0.1003

ture, in vacua. PzO6, over boiling al-

cohol, in vacua. Atmospheric pres-

sure, 135”. ‘I I‘

TiIlll?. Loss in weight. Color change.

hrs. gm. per cent

313 0.0597 17.04

168

t

t

0.0602 17.65

0.0191 17.65

0.0179 17.84

Pink to lavender.

“ “ ‘I

“ “ “

“ ‘I “

0.2283, 0.2552 gm. substance: 0.1970, 0.2242 gm. CO2 and 0.1022, 0.1131 gm. HzO.

Na&uCsHnoOaNd. Calculated. C 23.43, H 4.92. Found. “ 23.53, 23.96, “ 5.01, 4.96.

0.1743, 0.1322 gm. substance: 20.45 cc. N) (21’, 751.5 mm.) 15.25 cc. N2(20”, 752.4 mm.) (over 50 per cent KOH).

NazCuCsHzoOsNa. Calculated. N 13.67. Found. “ 13.21, 13.06.

The analytical results agree well with the empirical formula Na#JuCsHzoOsN~. The discussion of a possible structure for the salt G to be found in the introduction to this paper.

Dehydration Data.16--When the salt was obtained as just described, it was brought to constant weight in vacua over calcium chloride and soda-lime to remove traces of water and alcohol.

It was then dehydrated (1) in vacua over P,Os and soda-lime at room temperature, (2) in vacua over PzOS and soda-lime over boil-

I6 The dehydration data were obtained by DeKay and are stated in full in DeKay, H. G., Master’s dissertation, University of Chicago (1930).


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ing alcohol, (3) at atmospheric pressure at 135”. The results are summarized in Table V.

It was impossible to remove the last traces of water at room temperature. A further loss in weight occurred when the salt was warmed, ranging from 0.3 to 0.7 per cent. If a molecule of molecular weight 409.73 were to lose 4 molecules of water the loss in weight would be 17.68 per cent. Accordingly we have concluded that the salt contains 4 molecules of water.

5. Sodium Nickel Monoethyloxamide, NasNi2C12H37NS014.

To obtain this salt a solution of monoethyloxamide (2 gm.) in 75 cc. of 95 per cent alcohol was warmed to 35-40” and 15 he. of an aqueous solution containing 2 gm. of nickel acetate (Ni(C&H,O& .- 4 HzO) were added. To this mixture 7.2 cc. of a 10 per cent aqueous solution of sodium hydroxide were added drop by drop from a burette with constant shaking of the reaction mixture. A yellow precipitate formed, and precipitation was complete at the end of an hour. It was observed that an appreciable amount of the amide remained unused. The salt was collected on a filter, washed with absolute alcohol and dried in vacua over calcium chloride. A yield of 3 gm. was obtained. The compound is yellow, crystalline, soluble in water, but quickly hydrolyzed and decomposed by it. Upon warming, the salt is dehydrated, a loss in weight corresponding to 6 molecules of water occurring at 125’. At 140” the loss in weight corresponds to 7 molecules of water. At the latter temperature a color change from yellow to orange was observed.

The analytical data for the salt follows. Sodium was determined as sodium sulfate in the filtrate from precipitation of nickel as nickel dimethylglyoxime. The nickel was determined as nickel dimethylglyoxime.

0.2749,0.2705 gm. substance: 0.0799,0.0781 gm. NanSO+ Na3Ni2C12H3TN6014. Calculated. Na 10.21.

Found. “ 9.41. 9.35. 0.2749, 0.2705 gm. substance: 0.2365, 0.2349 gm. Ni.

NatNiLLHs~NaOln. Calculated. Ni 17.37. Found. “ 17.48, 17.65.


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Biuret Reaction. II

0.2108, 0.2167 gm. substance: 0.1651, 0.1714 gm. COz and 0.1032, 0.1104 gm. H20.

Na3NizC12H3~N6014. Calculated. C 21.32, H 5.52. Found. I‘ 21.36, 21.57, “ 5.59, 5.71.

0.1253, 0.1071 gm. substance: 13.7 cc. N2(22”, 751.0 mm.), 11.9 cc. N1 (24.5”, 748.5 mm.) (over 50 per cent KOH).

Na3Ni&H37N6014. Calculated. N 12.43. Found. “ 12.26, 12.26.

The analytical results agree well with the empirical formula Nas3NiG2H37NS014. Evidence upon which to base a structure for the salt is not available; its molecule is obviously more complex than are those of the sodium copper salts studied. The salt is a hydrate, as already stated. The nickel is bivalent, since hydrolysis and electrolysis of the salt produce nickelous hydroxide. In the electrolysis the yellow ingredient of the solution accumulates a- round the anode and nickel hydroxide is precipitated, hence the complex ion contains nickel and is negative.

6. Sodium Copper (Symmetrical) Diethyloxamide, Na&WLHzoN~O~.

The salt was prepared as follows: A solution of copper acetate (2 gm.) in 200 cc. of 95 per cent alcohol was warmed to 60” and filtered into a solution of 4 gm. of symmetrical diethyloxamide17 in 100 cc. of 95 per cent alcohol, also at 60’. The mixture was warmed almost to boiling, 8 cc. of 35 per cent aqueous sodium hydroxide solution being meanwhile dropped slowly into it. The dark blue reaction mixture was filtered from any residue without washing of the residue, since treatment of the filtrate with alcohol or ether decomposed the sodium copper salt in it. A heavy mass of dark blue crystals formed in the filtrate, and these were collected and dried in a vacuum desiccator. The yield of salt was 3.25 gm.

Sodium copper (symmetrical) diethyloxamide is a dark blue crystalline substance which is extremely hygroscopic. It is readily hydrolyzed. Electrolysis of an aqueous solution made stable by the addition of a large excess of alkali gave the usual results: the copper was found in the blue complex at the anode, while the solution around the cathode became colorless.

Analyses of the salt were carried out as usual except that after

I7 Diethyloxamide (symmetrical) was prepared according to the method of Wallach, O., Ann. Chem., 184,33 (1877); 214,268 (1882).


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the determination of copper by electrolysis the organic matter was destroyed by treatment with hot concentrated H&S04 and 30 per cent H,Oz before sodium was determined as sulfate.

0.6048,0.6217 gm. substance: 0.2185,0.2263 gm. Na2S04. Na~Cu’&Hzx,N~O~. Calculated. Na 11.68.

Found. ‘I 11.70, 11.78. 0.6048, 0.2401 gm. substance: 0.0959, 0.0384 gm. Cu.

Na&uC12H20N404. Calculated. Cu 16.14. Found. “ 15.87, 15.99.

0.2216, 0.2493 gm. substance: 0.2993, 0.3346 gm. CO2 and 0.1075, 0.1176 gm. HzO.

NazCuCIzHtoN404. Calculated. C 36.83, H 5.12. Found. I‘ 36.83, 36.60, “ 5.43, 5.28.

0.1133, 0.1132 gm. substance: 14.00 cc. Nz (19”, 758.5 mm.) and 14.15 cc. NZ (22’, 753.5 mm.) (over 50 per cent KOH).

Na&uC12H3aNd04. Calculated. N 14.22. Found. “ 14.16, 14.04.

The hydrogen content is slightly high for a molecule of empirical formula Na2CuC12Hz,N404, due possibly to the extremely hygroscopic nature of the compound. Otherwise the analytical results agree well with this empirical formula.

SUMMARY.

1. Further work upon the chemistry of the biuret reaction is reported.

2. The present paper discusses the biuret reaction of the typical di-acid amides biuret, malonamide, and oxamide. The sodium copper salts of the first two of these were isolated and analyzed, empirical formulas for them deduced, and structures proposed.

3. This paper discusses also the biuret reaction of the substituted di-acid amidesN-monoethylmalonamide, monoethyloxamide, and symmetrical diethyloxamide. Their sodium copper salts were isolated and analyzed, empirical formulas deduced, and structures proposed.

4. The theory of the biuret reaction is augmented with regard to the atoms concerned in the biuret reaction of di-acid amides.

5. An explanation is offered for the inhibition of the biuret reaction of di-acid amides by multiple substitution of alkyl groups in their molecules, and by separation of the amide groups by carbon or nitrogen atoms.


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A. MoerkeMary M. Rising, Joseph S. Hicks and Georgine

AMIDESBIURET REACTION OF DI-ACID THE BIURET REACTION: II. THE

1930, 89:1-25.J. Biol. Chem.

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