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cr-KEJTO ANALOGUES
AND
BY
OF ARGININE,
LYSINE
ALTON
ORNITHINE,
MEISTER
Institutes
of
Health,
August 6, 1953)
activity
were ob-
578
a-KETO
ANALOGUES
2 The author is indebted to Dr. B. D. Davis for cultures of the E. coli mutants
used in this study.
3 About 80 per cent of the oxidase activity was recovered after lyophilization
of
the contents of the dialysis sac.
4 The microanalyses reported in this paper were performed by Mr. R. J. Koegel
and Dr. W. C. Alford.
A.
MEISTER
579
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oc-KETO ANALOGUES
5 The product became yellow and liquefied when exposed to the air at room temperature for several hours. The instability
of the compound may be due to polyVogel and Davis (20) observed that Al-pyrroline-5-carboxylic
acid apmerization.
parently polymerizes readily, a property which prevented isolation of this compound
in pure form.
6 Stoichiometric
decarboxylation
was observed with freshly prepared solutions,
and with solutions of the products in 0.1 N hydrochloric acid, 0.1 N sodium hydroxide,
and 0.1 M phosphate buffer (pH 7.5), which were allowed to stand at 37 for 5 hours
prior to decarboxylation.
7 Under these conditions,
glutamic-r-semialdehyde
(Al-pyrroline-5-carboxylic
acid) yielded glutamic acid and no carbon dioxide.
15 cc. of dry ether were added yielding 50 mg. of a white crystalline precipitate. This was washed thoroughly with dry ether and placed in vacua
over phosphorus pentoxide and sodium hydroxide.
On analysis the following values were obtained: C 30.6, H 4.4, N 7.2, Br 41.0 per cent. These
data agree with the composition of the hydrobromide of A-pyrroline-2carboxylic acid (calculated for GH~02N*HBr, C 30.9, H 4.1, N 7.2, Br 41.2
per cent). The product became yellow when heated to 118 and decomposed at 136.5 Several properties of the product were investigated: (a)
The addition of a a-fold excessof 1 per cent 2,4-dinitrophenylhydrazine
in
2 N hydrochloric acid to a solution of 50 mg. of the product in 1 cc. of
water resulted in immediate precipitation of a crystalline hydrazone. On
recrystallization from hot water 40 mg. were obtained, with a melting
point of 211-212 (N, calculated for CUH~~O~N~,22.5; found, 22.4 per
cent). The hydrazone did not contain inorganic halogen. The hydrochloride of the 2,4-dinitrophenylhydrazone
of cr-keto-&aminovaleric acid
was prepared as described by Krebs (1) by oxidation of nn-proline with
n-amino acid oxidase. A slight excessof aniline was added to an ethanolic
solution of the hydrazone hydrochloride, resulting in precipitation of the
free hydrazone. After crystallization from water, the melting point was
211-212O. A mixed melting point with the hydrazone prepared from the
product showed no depression. The paper chromatographic behavior of
the two hydrazones was identical. (b) The product gave stoichiometric
quantities of carbon dioxide when treated with ceric sulfate (5) or with
hydrogen peroxide (16).6 After treatment with hydrogen peroxide at pH
values of 2.5 or 7.5, y-aminobutyric acid formation was revealed by paper
chromatography and no glutamic acid was detected. (c) Solutions containing 160 PM of pyridoxamine, 40 PM of the product, and 2 PM of aluminum chloride in 2 cc. of 0.1 M sodium acetate buffer (pH 4.9) were
heated at 100 for 30 minutes. The solutions became markedly yellow
after 5 minutes of heating, suggesting formation of pyridoxal.
Ornithine
formation was revealed by paper chromatography, and assay with Escherichia coli, mutant 160-37, gave a value of 5.6 PM of n-ornithine.
(4
Hydrogenation of 108 j.6M of the product with platinum oxide catalyst (40
A.
MEISTER
581
* Prepared
(20) from r,r-dicarbethoxy-r-acetamidobutyraldehyde
generously
provided by Dr. H. J. Vogel.
9 The RF values
for the o-aminobenzaldehyde
compounds
formed
from Al-pyrroline-5-carboxylic
acid, Ai-pyrroline-2-carboxylic
acid, and A-piperidine-2-carboxyto 0.69, and 0.70 to 0.75,
lit acid, with the pyridine
solvent
(la), were 0.55 to 0.60,0.64
respectively.
The corresponding
values
obtained
with
chromatograms
developed
with 77 per cent ethanol
were 0.55 to 0.61, 0.62 to 0.68, and 0.71 to 0.76.
When 0.1 M
potassium
phosphate
buffer
(pH 7.5) was used as the solvent,
the Rp values
were
0.75 to 0.80,0.83
to 0.87, and 0.79 to 0.83.
In tert-amyl
alcohol
saturated
with phthalate buffer
(21), the Rp value for the Ai-piperidine-2-carboxylic
acid compound
was
0.42 to 0.45, and that for the Ai-pyrroline-2-carboxylic
acid product
was 0.28 to 0.32.
The spot corresponding
to the Ai-pyrroline-5-carboxylic
acid derivative
gradually
faded and ultimately
disappeared
during
chromatography
with this solvent.
582
a-KETO
ANALOGUES
DISCUSSION
A.
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MEISTER
with Al-pyrroline-2-carboxylic
acid, and yielded proline on hydrogenation.
However, the product possessed properties characteristic
of ar-keto acids,
including hydrazone formation, decarboxylation
by hydrogen peroxide, and
conversion to ornithine by transamination.
The corresponding
lysine derivative
exhibited similar chemical behavior, although the elementary
analysis of this compound agreed with that calculated for oc-keto-e-aminocaproic acid, as well as the monohydrate
of A-piperidine-2-carboxylic
acid.
Treatment of both compounds with hydrogen peroxide at pH 7.5 yielded
the corresponding w-amino acids, and at the same pH value both compounds
k
c=o
COOH
/Y
CH2
I
CHz
\N/
CH2
I
C-COOH
CH2NH,
I
(C&h
C=O
CHZ-CH2
I
I
% CHZ
C-COOH
\N//
COOH
reacted with o-aminobenzaldehyde. The findings suggest that these compounds may react both as free cr-keto acids and as cyclic compounds.10
The properties of the ar-keto analogues of lysine and ornithine appear
analogous to those observed with glutamic-y-semialdehyde and other yaminoaldehydes, which have been shown to undergo spontaneous cyclization which is reversible under certain conditions (20, 23, 24).
ar-Keto-Saminovaleric acid has been identified by isolation as the 2,4dinitrophenylhydrazone as a product of n-proline and n-ornithine oxidation by sheep kidney n-amino acid oxidase (l), and as a product of the
oxidation of n-proline by rat kidney n-amino acid oxidase (2) .I2 In certain
systems n-proline is oxidized to glutamic-r-semialdehyde (3, ZS), which is
known to cyclize to Al-pyrroline-5-carboxylic
acid (20). The latter compound may be distinguished from Al-pyrroline-2-carboxylic
acid by assay
with E. COG,mutant 55-25, which responds only to Al-pyrroline-5-carboxy10 It is possible that other tautomeric forms exist such as the enolic form of the
normal chain compounds and the AZ cyclic forms, etc.
11 Oxidation of n-kynurenine
by rattlesnake venom in the presence of catalase
yielded kynurenic acid. In this instance cyclization is apparently not reversible,
owing to the formation of a stable aromatic ring. Oxidation in the absence of catalase resulted in the liberation of 1 mole of carbon dioxide, a finding compatible with
the intermediate
formation of o-aminobenzoylpyruvic
acid.
12n-Ornithine and n-lysine are susceptible substrates for rattlesnake n-amino acid
oxidase (25). We have found that oxidation of L-ornithine in the absence of catalase
was associated with the consumption of 1 mole of oxygen, formation of 1 mole each
of carbon dioxide and ammonia, and formation of r-aminobutyric
acid. The oxidation of L-lysine proceeded in a similar manner, and (in the absence of catalase)
b-aminovaleric
acid was formed.
After oxidation of these diamino acids in the
presence of catalase, the corresponding
o-keto acids were identified as the 2,4dinitrophenylhydrazones.
CHzNHz
I
(CH&
584
a-KETO
ANALOGUES
lie acid. The evidence also indicates that these compounds give different
derivatives when condensed with o-aminobenzaldehyde.
Recent reports describe the presence of levorotatory
pipecolic acid in
certain plant tissues (27, 28). A possible mechanism of formation of this
amino acid from lysine may be considered in which oxidation or transamination of lysine is followed by cyclization of the resulting oc-keto-e-aminocaproic acid to Al-piperidine-2-carboxylic
acid, and optically specific reduction of the latter compound to the natural form of pipecolic acid. It is of
interest in this connection that Neurospora
cras.sa n-amino acid oxidase
oxidizes L-lysine to a product which yields nn-pipecolic acid after catalytic
hydrogenation
(29).
1. The preparation of the cr-keto analogues of arginine (cr-keto-d-guanidinovaleric acid) and nitroarginine
(a-keto-&nitroguanidinovaleric
acid),
the corresponding
2,4-dinitrophenylhydrazones,
and the flavianate of o(keto-&guanidinovaleric
acid is described.
The keto acids were slowly
reduced by lactic dehydrogenase, were not attacked by arginase, and underwent non-enzymatic
transamination
with pyridoxamine
at 100 to yield
arginine and nitroarginine,
respectively.
2. ar-Keto-6-N-carbobenzoxyvaleric
and a-keto+N-carbobenzoxycaproic
acids were prepared by enzymatic oxidation of the appropriate w-N-carbobenzoxy-L-amino
acids; the 2,4-dinitrophenylhydrazones
of these keto acids
are described.
3. The cr-keto analogues of lysine and ornithine were prepared from the
corresponding w-N-carbobenzoxyketo
acids by treatment with hydrobromic
acid in acetic acid solution.
The properties of the products obtained included ability to form 2,4-dinitrophenylhydrazones,
quantitative
decarboxylation
by ceric sulfate or hydrogen peroxide, and non-enzymatic
transamination
with pyridoxamine
to yield the corresponding amino acids.
After decarboxylation
with hydrogen peroxide, 6-aminovaleric
and y-aminobutyric
acids were formed, respectively,
from the cu-keto analogues
of lysine and ornithine.
4. Hydrogenation
of the or-keto analogues of lysine and ornithine, or
their w-N-carbobenzoxy
derivatives,
resulted in the formation of pipecolic
acid and proline, respectively,
a finding compatible with the expected
tendency to cyclization.
BIBLIOGRAPHY
1. Krebs,
H. A., Enzymologia,
2. Blanchard,
M., Green,
D.
421 (1944).
7, 53 (1939).
E., Nocito,
V.,
and
Ratner,
S., J.
Biol.
Chem.,
166,
SUMMARY
il.
26.
27.
28.
29.
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21.
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25.
MEISTER
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