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UNITED. S.,TATES ATOMIC ENERGY COMMISSION
SYNTHESIS OF CHLORACETIC ACID, IODOACETIC ACID AND GLYCINE,
LABELED WITH C-14 IN THE CARBOXYL GROUP
University of California
This document consists of 4 pages.
Date of Manuscript: May 5, 1947
Date Declassified: June 24, 1947
This document is issued for official use.
Its issuance does not constitute authority
to declassify copies or versions of the
same or similar content and title
and by the same author (s).
Technical Information Division, Oak Ridge Directed Operations
Oak Ridge, Tennessee
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SYNTHESIS OF CHLORACETIC ACID, IODOACE FIC ACID AND GLYCINE,
LABELED WITH C-14 IN rHE CARBOXYL GROUP
By Rosemarie Ostwald
In order to investigate protein metabolism with the help of radio.active carbon. it .vemci d dc-
sirable to prepare some of the more important amino acid.s labeled with C"
Carboxyl-labeled glycine and the necessary intermediate product c:irboxyl-l.ib-led tlhfur.iclic
acid were prepared. (Glycerine labeled with C"' has been prepared ov Olsen, Hi niingway. iand Nipr,'
Lorber and Olsen,2 and Sakami, Evans, and Gurin.3) Carboxvl-labrird todo.ictic acid w.I- prepared
to study its reactions with certain proteins, containing SH-group.
The methods and techniques employed were chosen with the objetl of obt;iinim:i hniest pos.ible
yields. Therefore, any unnecessary .teps in the preparation of the compounds eirn avoided and
techniques suitable for handling small amounts of material were u..sed throughout. -
In order to generate anhydrous acetic acid from sodium acetat a platinum boat, charged
with 0.7049 g carboxyi-labered anhydrous sodium acetate' and 0.7032 R inactive sodium acetate
tryhidrate was placed in a horizontal glass tube and was evacuated to 3 x 10-2 mm Hg and kept at
that pressure for 24 hours at room temperature Under these conditions the sodium acetate had
been shown to be anhydrous in separate experiments. The glass tube %,as connected to a drying
train of concentrated sulphuric acid, Drierite, and phosphorus pentoxide, which was swept with
nitrogen while the salt was heated to its melting point. After the mixture was cooled, the other end
of the tube was connected to a train of three traps, each of which was cooled with an isopropyl-
alcohql-dry ice mixture; the last trap was protected with a calcium chloride tube. Gaseous hydrogen
chloride wab passed slowly through the train, the tube was gradually heated, and the liberated acetic
acid distilled into the traps. When the reaction was complete, the three traps were connected to the
vacuum line and the contents were distilled into a small reaction vessel (see Figure li, which wa.s
cooled with liquid nitrogen. The product consisted of about 85-90% acetic acid, 10-15'". water and
a considerable amount of gaseous hydrogen chloride, as was determined by titration in separate
runs. A low-temperature condenser, cooled with isopropyl alcohol-dry ice and protected with a
S, calcium chloride tube, was attached to the reaction vessel which was then allowed to come to room
temperature; 0.65 g of inactive acetic anhydride was added and the mixture was refluxed for one
half hour to destroy the 10% of water which was present. No attempt was made to determine the
actual yield of acetic acid, a mixture of 0 02 g of iodine, 0.04 g of phosphorus, and 0.88 g phosphorus
pentoxide were added,4 and dry chlorine was passed through the system at reflux temperature for
two and one half hours. The condenser was a normal Liebig type with a low-temperature condenser
*Carboxyl-labeled sodium acetate was prepared by R. Lemmon from methyl-iodide and C" 0O
,by the Grignard reaction.
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sealed onto the top (see Figure 1). The whole apparatus was evacuated to 3 x 10-a mm and all t
material from the condenser and the gas-inlet tube was distilled back into the reaction vessel :
which was cooled in liquid nitrogen. The chloracetic acid was then purified by fractional sublita-
tion onto a cold-finger condenser, filled with powdered dry ice. All parts of the apparatus were
connected by ground-glass joints.
DRY ICE --7.
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The yield of pure product was 1.52 g (67%,.based on 1.129 g anhydrous sodium acetate) mp,
60. Previous runs with inactive material gave yields of 85-90%. The sodium acetate used for the
synthesis had a specific activity of 1.6 x 105 cts'min,'mg of compound (3.3 x 0lcts/min/mg or A ..
1.9 x 10-' microcuries ol barium carbonate). The totlI activity was 11.2 x 10' cts or 650-micro-
curies. The chloracetic acid had a specific activity of 5 x 10' cts/min/mg of compound
SMDDC 1047 3
(1.19 x 10 cts/min,'mg or 7.0 x 10" microcuries of barium'carbonate) and a total activity of
7.6 x 10. cts or 440 microcuries. The activity recovered from mother liquors, washings, etc., was
1.7 x 10 cts or 100 microcuries. The specific activities were determined by combustion of the
ample with an appropriate amount of benzoic acid as carrier. The carbon dioxide was precipitated
as barium carbonate and was counted with a Geiger-Mueller counter with a geometry of 12.9
disintegrations per count.s
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1. Carboxyl-labeled chloracetic acid was prepared by passing a stream of dry chlorine through
a mixture of anhydrous carboxyl-labeled acetic acid, using a mixture of iodine, phosphorus, and
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A mixture of 0.2408 g of carboxyl-labeled and 0.3 g of inactive chloracetic acid was added to a
solution of 1.5 g of sodium iodide in 20 cc of dry acetone. After the mixture was kept at room
temperature for 20 hours, the white precipitate of sodium chloride was filtered, washed with dry
. acetone, anr.d the combined brown solutions were brought to a pH of 6.8 by dropwise addition of 40%
sodium hydroxide, under vigorous stirring. The precipitate which formed was filtered immediately,
washed with dry acetone, ice-cold absolute alcohol and ether.' In trial runs, 85-90% yields of
sodium iodoacetate were obtained. The identity and purity were established by titration of a con-
centrated solution of the salt with 2N hydrochloric acid, using a pH meter with micro electrodes.
The product was 98-99% pure. With active chloracetic acid a yield of 500 mg or 45% was obtained.
The total activity of the chloracetic acid used was 1.2 x 107 cis or 70.4 microcuries. The
sodium iodoacetate had a specific activity of 1.1 x 10' cts,'min, mg of compound (5.6 x 10s cts or
3.3 x 10 microcuries) and a total activity of 5.5 x 10 cts or 32.2 microcuries. The activity re-
covered from the sodium chloride precipitate, mother liquors, etc., was 3.4 x 10' cts or 2 micro-
curies. The Balance was lost, probably due to the volatility of iodine-containing compounds which
were formed by decomposition in the strongly alkaline mother liquors.
The sodium chloride precipitate, which weighed 600 mg instead of 330 mg expected from the re-
action, was burned. It had a specific activity of 5000 cts/min, mg and a total activity of 3 x 10" cts
or 17.6 microcuries. Efforts to recover any of the 280 mg sodium iodoacetate to which this activity
corresponds, were unsuccessful.
A mixture of 3.2 g of powdered ammonium carbonate, 10 cc of concentrated ammonia, and 4 cc
of water, was heated in a small three-neck flask, which was fitted through ground-glass joints to a
pressure-equalized dropping funnel, a Liebig condenser, and a thermometer. Aftar the salt had
dissolved, 1.014 g of carboxyl-labeled chloracetic acid in 3 cc of water was added dropwise through
the dropping funnel,-at such a rate that the temperature of the solution-did not rise above 60. The
mixture was held at 600 for six hours and was then allowed to stand for twelve hours at room tem-
perature. The solution was then concentrated until its temperature reached 112. The distillate
showed only very slight radioactivity. The yellowish solution was cooled to 70, and 15 cc of
absolute methanol was added slowly with agitation. The mixture was cooled in a refrigerator for one
hour. The precipitate was filtered and washed with methanol and ether.7
The yield of pure white crystals, which showed no trace of chloride ion was 0.54 g or 70%
(melting point 225, dec.). Upon concentration, the mother liquor gave 0.08 g of glycine which in-
creased the yield to 0.62 g or 79%.
The chloracetic acid used had a total activity of 5.1 x 107 cts or 299 microcuries. The glycine
had a specific activity of 6.3 x 10' cts/min. mg of compound (1.19 cts/ min mg of barium carbonate
or 3.3 x 10" microcuries) and a total activity of 3.9 x 10' cts or 229 microcuries. From distillates,
mother liquors, etc., 8.0 x 10' cts or 50.4 microcuries were recovered.
phosphorus pentoxide as catalyst. The carboxyl -labeled, anhydrous acetic acid was liberated from
carboxyl-labeled sodium acetate by heating the dry salt in a stream of dry gaseous hydrogen chlo-
ride and distilling the liberated acetic acid.
2. Carboxyl-labeled iodoacetic acid was prepared from carboxyl-labeled chloracetic acid by
allowing a mixture of chloracetic acid and sodium iodide in dry acetone to stand for twenty.hours at
room temperature. The sodium chloride was filtered, and the sodium iodoacetate was precipitated
by adjusting the solution to a pH of 6.8 with 40% sodium hydroxide.
3. Carboxyl-labeled glycine was prepared by heating a mixture of carboxyl-labeled chloracetic
acid, ammonia, and ammonium carbonate in the proportion of 1 : 15 : 5 for six hours to 60 and *
. precipitating the glycine by addition of absolute methanol, after the solution had been concentrated.
The author wishes to thank Professor M. Calvin for his advice in this work.
1. Olsen, Hemingway and Nier, J. Biol. Chem. 148: 611 (1943).
2. Lorber and Olsen. Proc. Soc. Exptl. Biol. Med. 61: 227 (1946).
3. Sakami. Evans, and Gurin, Report IB-40 Isotopes Br., Res. Div., Manhattan District.
4. Yorst Brueckner, Z. angew. Ch. 40: 973 and 41: 228.
5. Dauben, Reid, and Yankwich, paper in preparation; Yankwich, Norris, and Huston, paper in
6. Goldberg, Leon, Science 98: 386 (1943).
7. Cheronis, N. D. and K. H. Spitzmueller, 1. Org. Ch. 6: 349, (1941); Orten, J. M. and R. M.
Hill, Org. Synthesis Coll. 1: 300.
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UNIVERSITY OF FLORIDA
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