THE OXIDATION OF AMIDO-ACIDS WITH THE PRODUb

TION OF SUBSTANCES OF BIOLOGICAL IMPORTANCE.

BY H. D. DAKIN.
(From the Laboratory of Dr. C. A. Herter.)
(Receivedfor publication, November 20, 1905.)

The mechanism of the various changes which the products of

proteid digestion undergo during their degradation in the animal
organism is almost wholly unknown, From the standpoint of
pure organic chemistry, the transformation of an amido-acid into
substances belonging to the carbohydrate group is not at all
easy to understand, but yet it cannot be doubted that such
changes take place in the organism.
That enzymes are present in the liver capable of removing
ammonia from the amido-acids is clear from the work of Lang
and others. It is probable too that carbon dioxide is readily
removed from the carboxyl group of amido-acids by means of
enzyme activity. Thus the author has observed the conversion
of ornithin into tetramethylene diamine as a result of digestion
with liver tissue, and a similar type of reaction is constantly
met with in biochemical changes brought about by living
organisms.

H,N.CH,.CH,.CH,.CH(NH,).C -3 co, +
\
OH
H,N.CH,.CH,.CH,.CH,.NH,.

Taking, therefore

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172 Oxidation of Amido-Acids

representing a typical amido-acid derived from the hydrolysis

of proteid, one might expect that in some of the chemical trans-
formations it may undergo, ammonia and carbon dioxide will
be removed while a carbon-rich alkyl group is left. The
mechanism of the transformation of this group, whether into
carbohydrate or even to simple carbon dioxide and water, is
completely unknown.
It seemed of interest to know more of the chemical decompo-
sition which the simple amido-acids undergo, and in particular
to try to study the products formed by changes whose character
approximates as closely as possible to biochemical reactions.
The reaction which has first been studied is the oxidation of the
amido-acids by means of hydrogen peroxide and a trace of a
catalyst such as ferrous sulphate. This method of oxidation is
due to Fenton and has led to most beautiful results in the chem-
istry of the carbohydrate group. A feature of the reaction is
that the changes are brought about at ordinary temperatures.
Some three years ago I observed that cu-amido-isovaleric acid
and leucin (cu-amido-isobutylacetic acid) on treatment with this
reagent are readily decomposed with formation of volatile pro-
ducts. The further study of the reaction has been rendered
possible by the kindness of Professor C. A. Herter, to whom I
am indebted for the opportunity of making the present com-
munication.
The results at present are incomplete, but some particulars
may be given of the oxidation of glycocoll, alanin, and leucin.
As is so often the case, the first member of a series offers some
differences from its homologues. The three amido-acids men-
tioned may be represented by the formula,
NH,
/
R. C.H
\ //O

“\OH
where R is either a hydrogen atom in glycocoll or a methyl
or isobutyl group in alanin or leucin. On oxidation, they
all are readily resolved at the ordinary temperature into car-
bon dioxide and ammonia and an aldehyde. The liberation
 H. D. Dakin ‘73
of ammonia and carbon dioxide is completely analogous to their
formation in the case of the enzyme reactions previously men-
tioned, while the extremely reactive aldehydes are obviously
substances which could be quickly transformed into other pro-
ducts, as is believed to be the case with formaldehyde in the
photosynthetic production of carbohydrate in plants.
In the case of glycocoll, formic aldehyde is produced, and the
conversion of this substance into reducing sugars (cy-acrose and
formose) has already been accomplished in the laboratory by
Rutlerow and by Fischer, so that it is possible to effect the con-
version by purely chemical means and at low temperatures, of
glycocoll-a fragment of the proteid molecule-into carbon diox-
ide, ammonia (or urea), and a carbohydrate. In this connection
it is of interest to note that Embden and Salomon have shown
that the giving of glycocoll largely increases the output of sugar
in diabetic dogs deprived of their pancreas. In the case of the
oxidation of glycocoll, formic acid and glyoxylic acid are formed
at the same time, and the large production of the latter sub-
stance accounts for the small quantity of formaldehyde pro-
duced, compared with the yield of aldehydes from alanin and
leucin. The formation of glyoxylic acid in itself is not without
interest as this substance is known to be commonly present in
unripe fruit, and apparently is converted into sugar during the
process of ripening. Their mode of origin is obvious from the
following scheme:

“\\ //O
NH,+ C-C
H/ ‘OH

No
NH, + CO, + H.C
\
H

/p
NH,+CO,+H.C
\
OH
In similar fashion alanin yields acetaldehyde and acetic acid
 ‘74 Oxidation of Amido-Acids
(but no pyruvic acid, CH,.CO.COOH, the product correspond-
ing to glyoxylic acid), while leucin yields isovaleric aldehyde
and isovaleric acid.

//O
CH,.C +CO, +NH,
\H

//O
CH,.C +CO,+NH,
\
OH

CH,
H
\ CH.CH,.
Ho
CH, +NJ&+CO,
\ //O / c\
H
CHCH,. A- CH,
/ I c\ CH,
CH* NH2 OH \ //O
CH.CH,. + NH, +CO,
/ c\
CH, OH

The experimental details of a typical oxidation are as follows:

1.0 gram of alanin was added to the calculated amount of hydro-
gen peroxide solution, which had been standardized by potassium
permanganate and also carefully neutralized with sodium car-
bonate. A few milligrams of ferrous sulphate were added and
the whole allowed to stand at the ordinary temperature of the
air. After a couple of minutes the solution became slightly
warm and a slow evolution of carbon dioxide commenced, while
the smell of acetaldehyde steadily increased. After standing
overnight the mixture was slowly distilled and the first portion
of the distillate caught in a freezing mixture. The distillate
smelt strongly of acetaldehyde, and gave all the typical reac-
tions, including the reduction of Tollen’s silver solution, the
production of aldehyde resin with caustic soda, the iodoform
reaction, and also the reaction with sodium nitroprusside in the
presence of secondary amines which is characteristic of acetalde-
hyde as opposed to formaldehyde, ketones, and most other alde-
 H. D. Dakin I75
hydes (cf. Beilstein’s Handbuch ErgCenzungsband, i, p. 471).
Finally, with phenyl hydrazine a good yield of the /3-hydrazone
(m. p., 66OC.), ethylidenephenylhydrazine,C,H,.NH.N:CH.CH,,
was obtained.
The residue, after distilling off the aldehyde, was acidified
with dilute sulphuric acid and redistilled. An acid distillate was
obtained, while at the same time a small additional quantity of
acetaldehyde was liberated, doubtless set free from combina-
tions it had entered into with some of the amido compounds
present.
The distillate was examined for pyruvic acid with a negative
result, but contained about 0.06 gram of acetic acid. It was
identified by the ferric chloride reaction and by conversion into
the barium salt.
On analysis the salt gave 53,7 per cent. barium.
Calculated for (CH,COO),Ba, Ba=33.8 per cent.
In a similar way the oxidation of glycocoll led to the isola-
tion of formic acid which was converted into lead formate.
Analysis gave 69.5 per cent. metallic lead.
Calculated for (HCOO)BPb, Pb=69.7 per cent.

The acid gave also the characteristic reactions with mercuric

and silver salts. A large quantity of glyoxylic acid was formed
which was identified by means of the phenylhydrazone and by
the tryptophan reaction of Hopkins. The quantity of formal-
dehyde was small, but it was readily detected by the Tollen’s
aldehyde silver reagent, and by the Lebbin method for the esti-
mation of formaldehyde, and further by the color reactions with
concentrated sulphuric acid and resorcin and pyrogallol.
On making alkaline the residue from the distillation of the
volatile acids and redistilling, a large quantity of ammonia was
liberated and was identified by conversion into the platinum
salt. Hydroxylamine was completely absent.
On analysis the salt gave 43.9 per cent. Pt.
Calculated for Pt (NHI),CI,, Pt=43,g per cent.
It is of interest to note that the oxidation of glycocoll is pe-
culiar in that small quantities of nitrogen-containing substances
 176 Oxidation of Amido-Acids

are produced which are soluble in ether and are probably oximido-
acetic acid and formaldoxime. The quantity is, however, very
small and their isolation offers peculiar difficulties which need
not be entered upon here. Their formation, however, would
explain the production of hydrocyanic acid from glycocoll by
oxidation with nitric acid, observed by Plimmer, since they
break up readily with production of hydrocyanic acid.

Ho
H0N:CH.C + HCN+CO,+H,O
\
OH
The direct oxidation of glycocoll by peroxide, however, does
not yield hydrocyanic acid. An analogous formation of oximes
has been observed by Wollfenstein and Haase in the oxidation
of piperidin.
Leucin is very readily attacked by the peroxide reagent, and
a very vigorous evolution of carbon dioxide and a strong smell
of isovaleric aldehyde are immediately obvious. On distillation,
isovaleric aldehyde was readily separated and a similar hydra-
zone to that given by pure isovaleric aldehyde was readily
obtained. A small quantity of a volatile substance giving
the iodoform reaction was also present and is still under
investigation.
On acidifying and re-distilling, isovaleric acid was obtained
and identified by conversion into the zinc salt.
The further investigation of the oxidation of proteid decom-
position products, including the oxyamido- and diamido-acids is
in progress.
BIBLIOGRAPHY.
FISHER, d. deulsch.
Ber. chew Gesellsch., xxii, p. 359, 1890.
LOW, ibid.,xxii, p. 4~5, 1890.
BUTLEROW, L&big’s Ann., cxx, p. 296, 1866.
PLIMMER, Journ. of Physiol., xxxi, p. 65, 1905.
R. WOLLPENSTEIN, Ber. d. deutsch. them. Gesellsch., xxx%, p. 3228,
1904.