BY Biological · The results of a series of qualitative tests are shown in Table I; the...

OXIDATION OF AMINO ACIDS BY SILVER OXIDE*

BY R. M. HERBSTt AND H. T. CLARKE

(From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, January 25, 1934)

In the course of the preparation of dimethylglycine from its hydrochloride, it was observed (1) that addition of excess of silver oxide to a cold aqueous solution of the latter caused the evolution of carbon dioxide, accompanied by the odors of dimethylamine and formaldehyde. Repetition of this experiment at the boiling temperature showed that the following reaction took place with essen- tially quantitative yields.

(CH&NCH&02H + 3Ag,O = (CH&NH + 202 + Hz0 + 6Ag

As this reaction, if general, might offer certain analogies to the biochemical oxidation of amino acids, its study was extended to other compounds.

The formation of metallic silver on heating an aqueous solution of the silver salt of glycine was observed by Kraut and Hartmann (2) and confirmed by Heintz (3), but no attempt appears to have been made to study the reaction nor to ascertain whether it occurs with other amino acids. Strecker (4) found that alanine yields carbon dioxide, ammonia, and acetaldehyde on boiling with a suspension of lead peroxide. Silver oxide was shown by Nef (5) to cause the oxidation of glycolic acid, but under the conditions adopted the reaction halted with the production of oxalic, formic, and carbonic acids.

The results of a series of qualitative tests are shown in Table I; the quantitative experiments are described in the latter part of this report. Glycine and sarcosine are oxidized in boiling aqueous

* This work was aided by a grant from The Chemical Foundation, Inc., to the Department of Biological Chemistry.

t W. J. Gies Fellow in Biological Chemistry, 1931-32. 769

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770 Oxidation of Amino Acids

solution by silver oxide in much the same way as dimethylglycine, the former considerably more slowly.

NH&H&02H + 3AgaO = NHs + 2CO2 + Hz0 + 6Ag CHsNHCHzC02H + 3Ag20 = CHsNHz + 2COz + H20 + 6Ag

TABLE I

Behavior of Various Amino Acids and Derivatives with Silver Oxide

Amino acid I

Products recognized

Very rapidly oxidized

Sarcosine CO,, CHsNH2 Dimethylglycine “ (CH&NH N-Methylalanine “ CHsNH,, CH,CHO, CHsCOOH N-Dimethylalanine “ (CHdsNH, CH&HO, CH,-

COOH a-Dimethylaminoisobutyric acid CO,, (CHa)s NH, acetone

Rapidly oxidized

Glycine Alanine

c+Aminoisobutyric acid Leucine

a-Aminophenylacetic acid Phenylalanine

Glutamic acid Proline N-Phenylglycine N-Phenylalenine N-Phenylaminoisobutyric acid a-Phenyl-cr-aminobutyric acid Acetylglycine Hippuric acid a-Benzoylaminophenylacetic acid N-Benzenesulfonylglycine a-Ureidopropionic acid

a-Phenylureidopropionic acid 5-Methylhydantoin

3-Phenyl-5-methylhydantoin Creatine

CO,, NH3, urea (trace) “ “ CHaCHO, CH,COOH, urea (trace)

CO,, NHs, acetone ‘I “ isovaleraldehyde and acid

COZ, NHs, CsHCHO, CaHsCOOH “ “ PhCHO, PhCOOH, PhCH&HO, PhCH,COOH

CO,, NHJ, succinic acid “ volatile base with pyrrole odor “ PhNH,, PhN:NPh “ ‘I ‘I CHsCOOH “ “ I‘ acetone “ NHJ, PhCOEt “ “ PhCONHl “ PhCHO “ PhSOtNHt “ NH, urea, CH&HO, CHa- COOH

PhNH, CO,, NH*, urea, CHsCOOH, CHs-

CONHCONHZ PhNH,


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R. M. Herbst and H. T. Clarke 771

TABLE I-Concluded

Amino acid Products recognized

Slowly oxidized

Glycylalanine Alanylglycine Alanylalanine p-Toluenesulfonylalanine a-Benzenesulfonaminoisobutyric

acid Benzenesulfonylleucine a-Benzenesulfonaminophenylacetic

acid Benzenesulfonylphenylalanine

Con, NH3, CHaCOOH

Isovaleric acid (odor) PhCHO (odor)

PhCH&HO (odor)

Not oxidized

Betaine &Aminopropionic acid Benzoylalanine d-Phthaliminopropionic acid a-Benzoylaminoisobutyric acid Benzoylphenylalanine 5,5-Dimethylhydantoin 3-Phenyl-5,5-dimethylhydantoin

Betaine, on the other hand, shows no tendency to reduce silver oxide.

An unexpected difference between glycine and its methyl derivatives is the oxidation of a notable proportion (over 10 per cent) of the primary amino group to nitrogen, with the formation of a correspondingly increased amount of metallic silver.

2NH&H&02H + 9Ag,O = Nz + 4CO2 + 5HzO + 18Ag

Small but appreciable quantities of urea are also produced. N- Phenylglycine, in contrast to sarcosine, shows a similar behavior, yielding azobenzene as well as the anticipated aniline. This fact has, however, no particular significance, since azobenzene is also formed by the action of silver oxide upon aniline itself.

Analogous oxidations occur with homologues of glycine which contain the amino group in the CY position.

R.CH(NHz)CO,H + 2Ag,O = R.COOH + NHs + COz + 4Ag


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That the reaction takes place in at least two stages is shown by the formation of aldehydes

R.CH(NH,)COzH + Ag,O = R.CHO + NH3 + CO2 + 2Ag

which undergo further oxidation to carboxylic acids, to an extent depending upon experimental conditions and the volatility and solubility of the aldehyde. As with glycine, some oxidation of the amino group to nitrogen takes place with alanine and those of its homologues which contain the grouping -CH(NH,)COOH; this, however, is not the case with N-methylated alanine nor with CP aminoisobutyric acid (in which no hydrogen atom is present in the LX position). In this last instance the oxidation proceeds only through the first stage, yielding acetone.

(CH&C(NH2)COOH + AgzO = (CH&&O + NH, + CO, + 2Ag

That the amino group must be situated in the cy position for oxidation to occur is indicated by the total absence of reaction in the case of &aminopropionic acid.

Oxidation is suppressed by the presence of either mineral acid or alkali. It thus seems reasonable to suppose that the primary reaction in the case of glycine is a function of the amphion +NHa .- CH2+COO-. Both of the hydrogen atoms of the methylene group, and two of those attached to the nitrogen atom, may be replaced by alkyl groups without adverse influence on the reducing power towards silver oxide. Methylation of the amino group, in fact, leads to increased reactivity, possibly owing to a greater tendency for ionic dissociation in the basic group. Replacement of all three of the hydrogen atoms on the nitrogen, as in betaine, completely inhibits the reaction, which may be expressed in general terms by the scheme

&N+H RzN+ I -e+

R’&COO- [I 1 R’,CCOO- -e+z++CO2

2

RsN+ 11 + OH- ---f &NH + R’,CO

R’Z

The oxidation seems to be associated with the primary loss of a single hydrogen atom from the nitrogen, rather than with a dehy-


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drogenation in the sense of Wieland’s theory (Wieland and Bergel (6) ), which cannot explain the oxidation of cY-aminoisobutyric acid. The failure of this theory adequately to account for the ready oxidation of a-dialkylamino acids by oxygen in presence of charcoal has recently been pointed out by Bergel and Bolz (7). The action of silver oxide bears, likewise, only a partial analogy to the oxidation of amino acids (8) in the animal body, for ingested cu-aminoisobutyric acid is excret.ed unchanged, in substantial en- tirety, by the dog, without the production of acetone or increased urea in detectable amounts.

Even more striking is the readiness with which a-dimethylaminoisobutyric acid,l in which all the hydrogen atoms of glycine (except that transferred from the carboxyl to the amino group on ionization) are replaced by methyl groups, is oxidized by silver oxide. This compound differed from all others examined in that the nitrogen was liberated (in the form of dimethylamine) more rapidly than the carbon dioxide. To test the possibility that the primary stage of the oxidation consists in hydrolytic deamination, t’he behavior of Lu-hydroxyisobutyric acid under similar conditions was investigated. Carbon dioxide was quantitatively evolved, but at so slow a rate as to preclude the possibility of the hydroxy acid forming an intermediate in the oxidation of either a-aminoisobutyric acid or its dimethyl derivative.

Acylation of the nitrogen, except in the case of glycine and cy- aminophenylacetic acid, inhibits the oxidizability by silver oxide. Hippuric acid and the benzenesulfonylglycine readily reduce silver oxide without detachment of the acyl groups. Combination of the carboxyl into a peptide linkage prevents oxidation until this linkage is ruptured by hydrolysis. The presence of a dissociated carboxyl group at the a-carbon atom thus appears to be a necessary factor.

With respect to the hydantoins, our findings parallel those of Baudisch and Davidson (10) in that the substitution of both hydrogen atoms at position (5) (as in 5; 5-dimethylhydantoin) prevents oxidation. Of interest is the formation of a trace of acetylurea from 5-methylhydantoin.

1 After the completion of the work here reported, Bergel and Bolz (9) have demonstrated the oxidizability of a-dimethylaminoisobutyric acid by oxygen in presence of charcoal.


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EXPERIMENTAL

General Procedure-The first oxidation experiments were carried out by boiling under a reflux a suspension of AgnO in an aqueous solution of the amino acid in a slow current of hydrogen; NH, and CO2 were absorbed from the gases which escaped from the top of the condenser. In all experiments for which this apparatus was employed, a pronounced lag was encountered in the rate of evolution of NH, as compared wit.h that of COz. This effect was found to be due to the retention of NH3 when a high COz tension was maintained, and was intensified by passing COz instead of Hz through the apparatus. Since these early experiments were con- cerned only with the oxidation of glycine, from which 2 moles of CO* are formed for each mole of NHI, the lag in the rate of NH3 evolution was not surprising.

In all the experiments described below the apparatus was so modified as to minimize as far as possible the lag in NH3 evolution caused by the simultaneous presence of COz. A 300 cc. Kjeldahl flask was fitted with a 3-hole rubber stopper through which passed an inlet tube for hydrogen, a dropping funnel, and an exit tube lead- ing directly to a declining condenser. The lower end of the condenser was connected to a series of receivers for the absorption of volatile reaction products.

The operation of the apparatus was varied slightly, depending on whether or not the compound to be oxidized was readily soluble in water. In case of amino acids or derivatives easily soluble in cold water, the following general procedure was employed.

Silver oxide, prepared by precipitation from a solution of a weighed amount of silver nitrate with the theoretical amount of 2 N sodium hydroxide, followed by thorough washing with water, was suspended in about 100 cc. of water in the reaction flask and heated to boiling. Throughout the experiment a slow stream of hydrogen was passed through the apparatus. To start the reaction a solut,ion of the amino acid in 25 cc. of water, followed by 25 cc. of wash water, was added to the boiling silver oxide suspension through the dropping funnel, whereupon the absorption train WR.S immediately attached to the condenser.

The oxidation of sparingly soluble compounds was carried out, by placing them in the reaction flask, adding silver oxide and cold water, and heating to boiling, care being necessary to avoid ex-


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cessive frothing at the outset. The reaction was timed from the first application of heat.

Throughout the experiment the reaction mixture was boiled gently, so as to provide a slow, continuous distillation of water into the first receiver. Water lost by distillation was replaced through the dropping funnel, the volume of liquid being held practically constant throughout the experiment. The absorption train was changed at intervals. The first receiver contained standard acid for the absorption and estimation of volatile base, which, when volatile acids were products of the oxidation, was re- distilled from alkaline solution before titration. The second and third receivers contained Ba(OH)* solution for absorption and estimation of COz. When the absorption train was detached from the apparatus, the first (acid) receiver was always boiled for a few moments in order to drive all the CO2 into the Ba(OH)2, before the several receivers were disconnected. The BaC03 was filtered off and washed, dissolved in an excess of standard hydrochloric acid, and titrated.

In several experiments Mg(OH)2 was added to the reaction mixture. The liberation of CO2 was somewhat restrained, and the rate of NH8 evolution was generally more rapid.

When the evolution of NH3 and COz ceased, heating was dis- continued, the reaction mixture was filtered, and the residue washed with water. The filtrate and washings were combined for analysis.

The residue was washed with hot dilute acetic acid and hot dilute ammonia solution until free of excess silver oxide and silver salts. The residual metallic silver was then dissolved in nitric acid, and estimated by titration with standard thiocyanate solution.

In the filtrate, the total nitrogen was estimated by the Kjeldahl process, and ammonia nitrogen by distillation with Mg(OH)z or NaOH. The acetic acid washings of the silver-silver oxide residues were, as a rule, also subjected t,o Kjeldahl determinations.

Identifications of other products were made as follows, all melting points being controlled by admixture with authentic samples.

Methylamine-Insolubility of hydrochloride in chloroform, and conversion of base into 2,4dinitromethylaniline, m.p. 175’ (un- correct.ed).

Dimethylamine-Solubility of hydrochloride in chloroform, and conversion of base to picrate, m.p. 158-159” (uncorrected).


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Oxidation of Amino Acids

Aniline-By carbylamine test; occasionally by conversion to acetanilide, m.p. 116-116.5’.

Azobenzene-Isolated by extraction of acidified distillate with chloroform, m.p. 67-68”.

Benzamide-M.p. 127.5’ (uncorrected). Benzenesulfonamide-M.p. 152.5-153’ (uncorrected). Urea-Isolated and estimated as compound with xanthydrol,

or estimated by urease.2

Fra. 1. Glycine (10 mM) and AgO (66 milli-equivalents). Curve 1 represents CO,; Curve 2, NH,; Curve 3, NH, in presence of Mg(OH)*; Curve 4, NH, in presence of NaOH (50 mM); Curve 5, CO* in presence of HZSO, (66 milli-equivalents); Curve 6, COz in presence of CH&OOH (66 mM).

FIQ. 2. Sarcosine (10 mM) and Ag,O (66 milli-equivalents). Curve 1 represents COZ; Curve 2, CHZNHS; Curve 3, CHaNH2 in presence of Mg(OH)a.

Fra. 3. Dimethylglycine (10 mM) and AgzO (66 milli-equivalents). Curve 1 represents CO,; Curve 2, (CH&NH; Curve 3, (CH&NH in presence of Mg(OH),; Curve 4, (CH,),NH in presence of NaOH (50 mM).

FIQ. 4. Alanine (10 mM) and AgzO (50 milli-equivalents). Curve 1 represents CO,; Curve 2, NHI; Curve 3, NH8 in presence of Mg(OH),. Alanylalanine (5 mM) and Ag,O (50 milli-equivalents), Curve 4 represents CO,; Curve 5, NH,.

FIQ. 5. N-Methylalanine (10 mM) and AgzO (50 milli-equivalents). Curve 1 represents CO*; Curve 2, CHaNH2; Curve 3, CHaNH2 in presence of Mg(OH),.

FIG. 6. N-Dimethylalanine (10 mM) and AgzO (50 milli-equivalents). Curve 1 represents CO*; Curve 2, (CH&NH; Curve 3, (CH&NH in presence of Mg(OH)2.

FIG. 7. Leucine (10 mM) and Ag,O (70 milli-equivalents). Curve 1 represents COZ; Curve 2, NH,; Curve 3, NH8 in presence of Mg(OH)z.

FIQ. 8. a-Aminoisobutyric acid (10 mM) and AgtO (30 milli-equivalents). Curve 1 represents COz; Curve 2, NHa; Curve 3, NHain presence of Mg(OH)2.

FIQ. 9. a-Dimethylaminoisobutyric acid (10 mM) and AgnO (30 milli- equivalents). Curve 1 represents CO*; Curve 2, (CH&NH. a-Hydroxy- isobutyric acid (10 mM) and Ag,O (60 milli-equivalents). Curve 3 represents CO,.

FIG. 10. a-Aminophenylacetic acid (10 mM) and Ag,O (60 milli-equivalents). Curve 1 represents COz; Curve 2, NHa.

FIQ. 11. Phenylalanine (5 mM) and Ag,O (60 milli-equivalents). Curve 1 represents CO,; Curve 2, NH,. Phenylacetic acid (5 mM) and Ag,O (36 milli-equivalents). Curve 3 represents CO,.

FIQ. 12. cz-Amino-a-phenyl-n-butyric acid (10 mm) and Ag,O (49 milli- equivalents). Curve 1 represents CO*; Curve 2, NHs.

a The authors are indebted to Dr. Samuel Graff and Miss Rhoda Howard for these determinations.


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15.

le FIG.1

I

0 lo HOURS HOURS

20 20

1 15,

E L z ” h IO n

z

I 5

0 5 10 HOURS

0 5 10 HOURS

a E

0 HOURS 1

10

CI z FIG.7

b -5

s E 3

z

0 10 HOURS

tiOURS 0 15

HOURS

Fros. 1 TO 12 777


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Acetaldehyde-As derivative of dimethyldihydroresorcinol, m.p. 143-144” (uncorrected).

Acetone-As 2,4-dinitrophenylhydrazone, m.p. 126-128’ (uncorrected).

Isovaleraldehyde-As derivative of dimethyldihydroresorcinol, m.p. 154155” (uncorrected).

Benzaldehyde-As p-nitrophenylhydrazone, m.p. 204-205’ (uncorrected).

Acetic Acid-Separated by distillation from dilute phosphoric acid, isolated as silver acetate (Ag found, 64.2 to 64.5; calculated, 64.6).

Benz&c Acid-M.p. 123’ (uncorrected). In all of the quantitative experiments, the results of which are

outlined below, complete accounts of the nitrogen and silver distribution were kept; records of these are withheld for economy of space.

Glycine (Fig. l)-In Fig. 1 are shown typical curves for the rate of evolution of NH, and CO, during the oxidation of glycine. Curve 3 was obtained in the presence of Mg(OH)2, and shows a slight increase in the rate of NH, evolution. Curves 4, 5, and 6 show the effect of sodium hydroxide, sulfuric acid, and acetic acid respectively on the rate of oxidation of glycine by silver oxide.

On oxidation with silver oxide under the conditions described above, the main products obtained from glycine are CO, and NHS. The yield of silver varied from 5.7 to 6.4 atoms per mole of gly- tine. There was generally a loss of 10 to 15 per cent of the nitrogen, presumably in the form of NP.

The only other product obtained as a result of oxidation was a small amount of urea. A suspension of silver oxide in an aqueous solution of glycine was boiled for 2 hours under a reflux. A current of hydrogen through the apparatus washed the gaseous reaction products into suitable receivers attached to the upper end of the condenser. The reaction mixture was cooled to room temperature as rapidly as possible at the end of 2 hours.3 In two such experiments, the yields of CO, were 1.1 and 1.2 moles, of NH, 0.82

3 At this point, in several other experiments, aniline hydrochloride was added. In no case was evidence of the formation of phenylurea obtained. although urea itself was always found. Cyanate cannot, therefore, have been present in significant amounts.


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and 0.82 moles, and of metallic silver 6.0 and 6.2 atoms, per mole of glycine. The cold reaction mixture was filtered, and an aliquot of the filtrate was treated with H&3 to remove silver, and then con- centrated to a small volume for the estimation of urea, in which form 5 to 8 per cent of the nitrogen was recovered.

Sarcosine (Fig. @-The same general formulation as written for glycine can be applied to the oxidation of sarcosine, except that

I !

r” z $ 10

0”

f

5

0’

I5

e”

zm E

5,

0

1 lo 2 FIG. 15

‘r- 5

0 lo HOURS

Fra. 13. a-Ureidopropionic acid (10 mM) and AgtO (60 milli-equivalents). Curve 1 represents CO*; Curve 2, NHS. 5-Methylhydantoin (10 mM) and Ag,O (60 milli-equivalents). Curve 3 represents CO*; Curve 4, NHs. Urea (10 mM) and AgnO (10 milli-equivalents). Curve 5 represents CO*; Curve 6, NHs.

FIQ. 14. Hippuric acid (10 mM) and Ag,O (76 milli+equivalents). Curve 1 represents COz. Benzenesulfonylglycine (10 mrr) and Ag,O (SO milli- equivalents). Curve 2 represents CO,. N-Phenylglycine (10 mv) and AgzO (106 milli-equivalents). Curve 3 represents COz.

FIQ. 15. N-Phenylalanine (10 mM) and Ag,O (80 milli-equivalents). Curve 1 represents CO,. a-Phenylaminoisobutyric acid (10 mM) and AgzO (40 milli-equivalents). Curve 2 represents CO*.

the nitrogen was recovered almost quantitatively in the form of methylamine; 5.9 to 6.2 atomic proportions of metallic silver were produced.

Dimethylglycine (Fig. $)-This was employed as the hydrochloride, a correspondingly increased amount of silver oxide being taken. No attempt was made to remove silver chloride prior to the reaction, owing to the readiness with which dimethylglycine is oxidized


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by silver oxide, even in the cold. The yield of silver was 6.5 to 6.6 atomic proportions. No appreciable loss of nitrogen occurred, all of it reappearing as dimethylamine. As with glycine, the reaction is almost completely inhibited by sodium hydroxide.

N-Phenylglycine (Fig. 14)-Owing to the character of the volatile oxidation products (aniline and azobenzene) obtained from this amino acid, the condenser was replaced by a flask cooled with running water. The delivery tube passed directly into the cooled receiver, containing dilute acid for the absorption of basic reaction products, which were collected continuously throughout the reaction. The receiver for carbon dioxide was changed at intervals.

After extracting the azobenzene (0.13 equivalent of nitrogen) from the distillate, the aniline (0.28 equivalent) was estimated by the Kjeldahl process. The filtrate from the reaction mixture and the acetic acid washings of the silver-silver oxide residue were both colored reddish purple, and together contained 0.59 equivalent of nitrogen. The value for silver (6.9 atoms) was high, owing to difficulty in washing the residue.

A mixture of silver oxide and aniline in water was distilled in the above apparatus. Azobenzene appeared in the first few drops of distillate, together with some aniline; intensely colored oxidation products were also formed.

Hippuric Acid (Fig. 14)-No nitrogen was liberated, either as NH3 or NS; all was recovered as benzamide in the filtrate from the reaction mixture. Only about one-half the theoretical amount (1.0 to 1.1 moles) of COzwas liberated, and 4.5 to 4.7 atomic proportions of silver were formed. These low values require explana- tion; possible intermediates such as glyoxylic acid and oxalic acid were sought in vain.

Benzenesulfonylglycine (Fig. id)-In analogy with the case of hippuric acid, benzenesulfonamide was formed during the oxidation. No nitrogen was liberated as N, or NH3, all being recovered in the filtrate and the acetic acid washings of the residue. The amounts of carbon dioxide (1.86 to 1.88 moles) and of silver formed (6.0 to 6.1 atoms) agreed closely with the theoretical values.

Alanine (Fig. Q-Besides ammonia and carbon dioxide, acetaldehyde and acetic acid (0.6 to 0.8 mole) were isolated as reaction products. Varying amounts of nitrogen (0.16 to 0.26 equivalent) disappeared during the oxidation, presumably as Nt; only traces


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of urea were found. The yield of silver was 4.0 atoms in two experiments carried out in water alone; in the presence of magnesia, 4.7 atoms were obtained.

N-Methyl&nine (Fig. 5)-The reaction was more rapid than with alanine. Some of the base was held back during the reaction, probably by the resulting acetic acid, since it was easily released on distillation with magnesia. No loss of nitrogen occurred; all reappeared as methylamine. Acetaldehyde was present only in small quantity, the major portion having been oxidized to acetic acid (0.88 mole) ; 4.6 to 4.9 atoms of silver were formed.

N-Dimethylulanine (Fig. @-The oxidation was slightly more rapid than that with methylalanine, and followed the same course, yielding carbon dioxide (1.2 moles), dimethylamine (1.1 to 1.2 moles), acetic acid (0.85 to 1.0 mole), a trace of acetaldehyde, and silver (5.0 atoms in two experiments).

N-Phenylalanine (Fig. I@-The thermal instability of the silver salt of this compound has been noted by Tiemann and Stephen (11).

The oxidation was carried out as described for N-phenylglycine, and gave analogous results, aniline (0.21 mole), aaobenzene (0.17 equivalent of N), CO2 (1.2 moles), and acetic acid (0.69 mole) being the chief products. Owing to difficulty in washing, the yield of silver (6.7 atoms) was unduly high. A deficiency of about 6 per cent of the nitrogen may be due to the same cause.

a-Ureidopropionic Acid (Fig. IS)-This compound, in contrast to the acylated alanine derivatives, was readily oxidized by a boiling suspension of silver oxide, with formation of 1.7 to 1.9 moles of carbon dioxide and 1.35 to 1.68 moles of ammonia. Urea was formed only in traces. Judging by the rate of evolution of COZ and NH3, urea does not enter as an important intermediate step, since both were liberated in this reaction much more rapidly than they are from urea under the same conditions (Fig. 13, Curves 5 and 6). Acetaldehyde was formed in considerable amounts, together with acetic acid (0.5 to 0.6 mole). The yield of silver was 4.5 to 4.7 atoms.

6-Methylhydantoin (Fig. lS)-As in the case of a-ureidopropionic acid, a nearly quantitative yield (1.8 moles) of carbon dioxide was obtained. Ammonia (0.68 mole) was formed, but much of the nitrogen (1.1 equivalents) was retained in the reaction mixture.


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Urea was present only in traces (0.05 equivalent of N). No trace of acetaldehyde could be found; 0.92 mole of acetic acid was recovered. The yield of silver was 5.7 atoms. From the filtrate there was isolated a minute amount of a crystalline solid, m.p. 213- 214’ (uncorrected). This appeared to consist of acetylurea.

Analysis Calculated for C H 0 N a 6 z 2. C35.29, H5.88, N27.4 Found. “ 35.66, “ 5.87, “ 27.6 (Kjeldahl), 24.8 (Dumas)

Alunylalanine (Fig. 4)-Alanylalanine was oxidized only very slowly by silver oxide; after 14 hours of boiling, 0.25 mole of ammonia, 0.48 mole of carbon dioxide, 0.18 mole of acetic acid, and 2.1 atoms of silver were formed. There was no loss of nitrogen; unchanged alanylalanine was recovered.

Leucine (Fig. 7)-Rapid oxidation occurred, with production of CO*, NHI, and isovaleraldehyde. The large loss of nitrogen (0.23 to 0.40 equivalent), particularly in presence of magnesia (0.45 equivalent), is noteworthy. Isovaleric acid was not isolated, but its presence was evident from the odor. Further oxidation of isovaleric acid by silver oxide does not take place to any great extent, as was proved by direct experiment, and could be inferred from the proximity of the yield of CO2 (1.1 moles in two experiments) to the theoretical value. The amount of silver formed in each case (4.8 to 5.4 atoms) was only slightly more than that re- quired to bring about the primary reaction and to oxidize the missing NHa to Nt.

a-Aminoisobutyric Acid (Fig. 8)-Aminoisobutyric acid was oxidized at about the same rate as alanine, with formation of COZ, NH3, and acetone. Of interest are the loss of nitrogen (0.16 equivalent) which occurred only in the presence of magnesia, and the fact that, although NH, was evolved more slowly than CO2 during the early stages of the reaction, it is subsequently released more rapidly. The yields of silver were 2.1 atoms in the absence of magnesia, 2.9 in its presence.

An experiment in which ar-hydroxyisobutyric acid was oxidized with silver oxide (Fig. 9) showed definitely, by the much slower rate of COa evolution, that this compound cannot have been formed as an intermediate stage in the oxidation of the corresponding amino acid.


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wDimethylaminoisobutyric Acid (Fig. 9)-The oxidation of this compound (1) was slower in the cold than that of dimethylglycine, but was slightly more rapid in hot solution. The relative rates of evolution of base and CO* during the oxidation of dimethylaminoisobutyric acid differ from the others in that dimethylamine is evolved far more rapidly than the COZ. The formation of acetone was established only qualitatively; according to Linnemann (12) acetone is appreciably oxidizable by silver oxide, a fact which may explain the rather high yield (2.1 to 2.3 atoms) of silver. There was no loss of nitrogen.

a-N-Phenylaminoisobutyric Acid (Fig. 15)-Although the heat instability of the silver salt (13) of N-phenylaminoisobutyric acid has long been known, nothing concerning the oxidation of this acid appears in the literature.

The reaction was carried out in the apparatus employed for the oxidation of N-phenylglycine. Aniline and azobenzene, formed in yields of 0.64 and 0.04 equivalents respectively, were both present in the first drops of distillate, which also contained acetone. The filtrate from the reaction mixture and the acetic acid washings of the silver-silver oxide residue were colored intensely reddish purple, and contained 0.27 equivalent of nitrogen. It was im- possible to wash the residue free of all organic material; this may account for a loss of about 5 per cent of the nitrogen. The yield of silver was 3.1 atoms.

a-Aminophenylacetic Acid (Fig. I@-Rapid oxidation occurred, with formation of CO,, NHI, benzaldehyde, and benzoic acid. The yield of silver was 4.5 to 5.0 atoms. Most striking is the loss of 40 to 50 per cent of the nitrogen during the reaction. An experiment devised to demonstrate the formation of Nz during the oxidation of aminophenylacetic acid is described below.

Phenylalanine (Fig. Zl)-Oxidation was slightly more rapid than with alanine; approximately 2 moles of CO1 were formed for every mole of NHI, indicating almost complete oxidation of the side chain. The distillate contained benzaldehyde, contaminated by a trace of phenylacetaldehyde (discernible by its odor) ; benzoic acid, containing a trace of phenylacetic acid (detected by its odor), was formed in larger proportion. The yield of silver was 10.6 atoms; a loss of about 10 per cent of the nitrogen occurred.

An experiment with phenylacetic acid (Fig. 11) indicated fairly


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ready oxidizability by silver oxide, with formation of benzoic acid and COz. As the reaction was carried out under a reflux, no benzaldehyde was isolated.

cY-Amino-a-Phenyl-+Butyric Acid (Fig. 18)-Oxidation took place with formation of CO!+ NHI, and propiophenone, identified as its semicarbazone, m.p. 177-178” (uncorrected). No loss of nitrogen occurred; the yield of silver was 2.7 atoms.

FIG.16

HOURS FIG. 16. N2 formed from a-aminophenylacetic acid (10 mM) and Ag,O

(60 milli-equivalents).

Formation of Nitrogen during Oxidation of a-Aminophenylacetic Acid-In various experiments on the oxidation of a-aminophenylacetic acid with AgZO, as much as 40 to 50 per cent of the nitrogen disappeared. This amino acid, therefore, seemed particularly suitable for an experiment designed to test the formation of molec- ular nitrogen during the course of oxidation.


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ln this experiment the reaction was carried out under a reflux, in a slow current of carbon dioxide. The gaseous reaction products were washed with dilute sulfuric acid, collected over 40 per cent potassium hydroxide, and measured periodically, corrections being made for the values observed in blank experiments. Fig. 16 shows the amounts of nitrogen liberated from a solution of 1.51 gm. (10 mM) of cr-aminophenylacetic acid in 150 cc. of water on boiling with the freshly precipitated silver oxide from 10.2 gm. (60 mM> of silver nitrate. At the end of the reaction, 7.52 milli- equivalents of nitrogen were present in the residual solution, 0.04 in the acid wash liquor. The total volume of nitrogen gas evolved was 11.2 cc. (0’ at 760 mm.) or 1.00 milli-equivalent, bringing the total recovery up to 85.6 per cent of the theoretical amount.

Metabolism of a-Aminoisobutyric Acid-A dog weighing 22 kilos was placed on a suitable diet supplying a constant daily nitrogen intake of 13.3 gm., water being allowed ad libitum. The urine, collected in 3 day periods, was analyzed for total nitrogen, urea, and ammonia. After 15 days, three daily portions of 20 gm. of cr-aminoisobutyric acid were added to the diet. After this period, the standard ration was resumed for 6 days.

No significant change in weight occurred throughout the experiment. During the first 2 days on which the amino acid was fed, the dog was extremely thirsty and drank abnormally large quantities of water, with a corresponding increase in urine volume. This effect was not noted on the 3rd day.

From Fig. 17 it will be seen that the urea nitrogen did not rise during the period of amino acid feeding, nor were there significant changes in ammonia nitrogen. Attempts to estimate amino nitrogen were frustrated by the observation that cY-aminoisobutyric acid differs from the naturally occurring amino acids (13) in its response to the Folin procedure (14) ; the color develops more slowly and reaches a final intensity of only about one-third of the usual value.

It is clear that the aminoisobutyric acid was not metabolized by the dog. This conclusion is supported by the isolation of the unchanged amino acid in a yield of 75 per cent from the urine collected during the experimental period: To 1 liter of urine, representing 16.7 gm. of ingested aminoisobutyric acid, lead acetate (30 gm.) was added until no further precipitation


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OCCU~d. The filtrate and washings were treated with 13 gm. of sodium bicarbonate, the precipitate removed, 50 cc. of 25 per cent sodium hydroxide were added, and the solution evaporated in a copper vessel over a free flame. When the volume had been re- duced to 100 cc., 60 gm. of crystallized barium hydroxide were added, and the mixture heated in an oven at 115’ for 21 hours. The residue was taken up in water, the barium carbonate was filtered off, and the filtrate boiled in the presence of 1 gm. of zinc filings until its temperature reached 105”. It was then replaced

q = Undetermined N •~&a q =NtlrN Nitrogen Intake = Sum of Shaded and Unshaded Portions

Nitrog!en Output IR Urine = Sum of Shaded Portions FIQ. 17. Nitrogen distribution in urine on feeding a-aminoisobutyric

acid.

in the oven for 20 hours. The residue was dissolved, filtered, and freed of heavy metals by hydrogen sulfide. The filtrate was acidified to litmus with hydrochloric acid, filtered, shaken with ether to remove fatty acids, cleared with norit, acidified to Congo red with hydrochloric acid, and evaporated to dryness. The residue was extracted with absolute alcohol, and the soluble fraction recrys- tallized from glacial acetic acid. Mother liquors were worked up systematically. There were thus obtained 16.9 gm. of pure, crystalline cz-aminoisobutyric acid hydrochloride (m.p. and mixed m.p.


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253’), corresponding to 12.5 gm. (75 per cent of the theoretieal amount) of the free amino acid. The phenylureide had a melting point and a mixed melting point of 174”. No attempt was made to estimate the amino acid present in the non-crystalline residue, which amounted to less than 1 gm.

In similar experiments with 1 liter quantities of urine from the preexperimental period, with and without added a-aminoisobutyric acid, the alcohol-soluble residues were analyzed without at- tempting to secure crystalline fractions. The results, recorded in Table II, indicate the recovery of about 77 per cent of the calculated amount of amino nitrogen under the conditions adopted. In the determinations of amino acid nitrogen by Folin’s method, a-aminoisobutyric acid was employed as standard.

TABLE II

Nitrogen Distribution in Alcohol-Soluble Fractions of Urine after Successive Treatment with Alkali and Hydrochloric Acid

--~~ ma. w7. Tl. ml.

1 liter urine (preexperimental). . . . 155 1.3 93 65 Same + 1.0 gm. a-aminoisobutyric

acid................................ 347 7.7 198 172

SUMMARY

a-Amino acids are oxidized in hot aqueous solution by silver oxide, with formation of ammonia, carbon dioxide, and aldehydes; the latter may undergo further oxidation to the corresponding acids. Replacement of both amino hydrogen atoms by methyl groups facilitates rather than inhibits the oxidizability, but in betaine this faculty is entirely lost. The presence of hydrogen at the a-carbon atom is not essential to oxidizability. Acylation of the amino group tends to prevent oxidation, but this effect is by no means universal.

BIBLIOGRAPHY

1. Clarke, H. T., Gillespie, H. IL, and Weisshaus, S. Z., J. Am. Chem. Sot., 66, 4571 (1933).

2. Kraut, K., and Hartmann, F., Ann. Chem. u. Pharm., 133, 101 (1865). 3. Heintz, W., Ann. Chem. u. Pharm., 146, 214 (1868).


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4. Strecker, A., Ann. Chem. u. Pharm., 76, 37 (1350). 5. Nef, J. W., Ann. Chem., 367, 287 (1907). 6. Wieland, H., and Bergel, F., Ann. Chem., 439, 196 (1924). 7. Bergel, F., and Bolz, K., 2. physiol. Chem., 215, 25 (1933). 8. Krebs, H. A., 2. physiol. Chem., 217, 191 (1933). 9. Bergel, F., and Bolz, K., 2. phusiol. Chem., 220, 20 (1933).

10. Baudiach, O., and Davidson, D., J. Biol. Chem., 76, 247 (1927). 11. Tiemann, F., and Stephen, R., Ber. them. Ges., 16, 2036 (1882). 12. Linnemann, E., Ann. Chem. u. Pharm., 139, 125 (1366). 13. Danielson, I. S., J. Biol. Chem., 101, 505 (1933). 14. Folin, O., J. BioZ. Chem., 61, 393 (1922).


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R. M. Herbst and H. T. ClarkeSILVER OXIDE

OXIDATION OF AMINO ACIDS BY

1934, 104:769-788.J. Biol. Chem.

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