THE JOURNAL OF BIOLOGICAL CHEWI~TRY

Vol. 241, No. 2, Issue of January 25, 1966

Printed in U.S.A.

The Chemical Degradation of Folic Acid

PHOTOLYSIS OF 2,4,7-TRIHYDROXYPTERIDINE*

(Received for publication, June 7, 1965)

ELLIOTT SHAW,~ CHARLES M. BAUGH,~ AND C. L. KRUMDIECK~

From the Department oJ‘ Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112

SUMMARY liability of several methods derived from Wieland and showed

that even under the most favorable conditions a 13 ‘% contamina-
2,4,7-Trihydroxypteridine is readily accessible from folic
tion by carbon atoms 4a and 8a was present in the oxalic acid.
acid and other naturally occurring pteridines by way of 2-
amino-4.hydroxypteridine-6-carboxylic acid, 2-amino-4-hy- During investigation in this laboratory of a variety of other

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approaches of potential value for the ring opening of pteridines,
droxypteridine, and isoxanthopterin (Z-amino-4,7-dihydroxy-
it was observed that 2,4,7-trihydroxypteridine was sensitive to
pteridine). After irradiation with ultraviolet light and brief
ultraviolet light and, following irradiation, liberated glyoxylic
acid hydrolysis, 2,4,7-trihydroxypteridine gives rise to gly-
acid on acid hydrolysis. Since 2,4,7-trihydroxypteridine ap-
oxylic acid coming from carbon atoms 6 and 7 of the pteridine
peared readily accessible from folic acid, as well as from other
ring. A detailed method is described suitable for use on a
micro scale in the study of the biosynthetic origin of these naturally occurring pteridines, this reaction promised to be of
value if it could be established that the glyoxylic acid obtained
carbon atoms in pteridines.
represented only carbon atoms 6 and 7 of the pteridine ring.
This was shown by degradation under specified conditions of ma-
terial labeled in different positions. Application of the procedure
then to folic acid was shown, in fact, to be convenient by way of
2-amino-4-hydroxypteridine-6-carboxylic acid, 2-aminol-hy-
The study of the biosynthesis of pteridines is rendered difficult droxypteridine, isoxanthopterin (2-amino-4, ‘?-dihydroxypteri-
by the minute amounts of these materials generally found in dine), and 2,4,7-trihydroxypteridine (Scheme I).
natural sources. An effective way of separating out the carbon
MATERIALS AND METHODS
atoms of the ring, particularly carbon atoms 6 and 7, on a small
scale and in a high state of purity is essential for isotope experi- nL-Tartaric acid-l ,4-14C was purchased from Nichem, Inc.;
ments designed to determine their origin in folic acid as described ethyl cyanoacetate-2-14C, from Nuclear Chicago. Glyoxylic acid
in the accompanying paper (1). was a product of Nutritional Biochemicals. Xanthine oxidase
The chemical methods used in the past for degradation in was purchased from Worthington.
similar metabolic work have been adopted from the earlier struc- Isoxanthopterin-&14C and Isoxanthopterin-7-14C-The iso-
tural studies of Wieland and Tarter. In general, the very stable xanthopterin synthesis of Albert and Wood (6) was employed.
pteridine ring was labiliaed by an initial chlorination and, follow- For labeling in the 4a position, 2,4,5-triamino-B-hydroxypyrimi-
ing hydrolysis, calcium oxalate was obtained presumably from dine-5-l% was prepared from ethyl cyanoacetate-2-14C by the
carbon atoms 6 and 7 (2). Thus, in isotopic experiments on the method of Bennett (7) followed by condensation with ethyl gly-
origin of the atoms in the butterfly pigment, leucopterin, Wey- oxylate. For the synthesis of isoxanthopterin-7-14C, ethyl
gand et al. (3,4) presented results with these and related methods, glyoxylate labeled in the carboxyl group was prepared from tar-
but a detailed account of the procedure has not appeared. In taric acid-l ,4-14C which was esterified by refluxing overnight in
analogous work on a number of other insect pteridine pigments, ethanol saturated with dry hydrogen chloride. After removal of
Brenner-Holzach and Leuthardt (5) critically reviewed the re- the solvent, the residual, syrupy dimethyl tartrate was subjected
to giycol cleavage with sodium bismuthate (8) to obtain ethyl
* These investigations were supported by American Cancer glyoxylate-l-14C. This was condensed with unlabeled 2,4,5-tri-
Society Grant T-67F. amino-6-hydroxypyrimidine as in the usual isoxanthopterin syn-
t Present address, Brookhaven National Laboratory, Depart- thesis (6).
ment of Biology, Upton, Long Island, New York 11973.
f Present address, Division of Dermatology, Department of .Z,C ,7-Trihydroxypteridine-For the deamination of isoxan-
Mldicine, Washington University School of i?&dicine, St. Louis, thopterin, prolonged refluxing in 6 N HCl (20 ml per mg) was
Missouri. found to be a convenient method as observed with other amino-
$ Present address, Universidad Peruana de Ciencias Medicas y pteridines (9) ;1 a decrease in absorbance at 350 rnp occurs as the
Biolopicas, Facultad de Medicina, “Cayetano Heredia,” Lima,
Peru.- Rockefeller Foundation Fellow (1960 to 1962) and a United 1 The use of nitrous acid for this purpose appears to be contra-
States Public Health Service International Fellow (1962 to 1963) indicated because of side reactions encountered with 7-hydroxy-
during part of this work. pteridines (10, 11).

379
380 Pteridine Degradation Vol. 241, No. 2

Go directly for photolysis. When solid product was desired, the

OH OH
pooled fractions were freed of formic acid by repeated evapora-
tion to dryness in a vacuum with added water. The yield was
95%.
Photolysis of d ,d, 7-Trihydroxypteridine and Isolation of Gly-
oxylic Acid d,4-Dinitrophenylhydrazone-The purified 2,4,7-tri-
@J xanthine
oxidase hydroxypteridine (2 to 4 mg) as formic acid eluate from the above
1 procedure (5 to 10 ml) or as the solid dissolved first in a small
OH OH volume of dilute sodium hydroxide added, in either case, to 1 y0
GNHCI v/v sulfuric acid (50 ml) was irradiated in a quartz Erlenmeyer
7 flask held about 1 inch above an ultraviolet source. (A Minera-
0 lite ultraviolet lamp with an output at 247 mp, such as is com-
2,4,7tri-OH- isoxanthopterin monly employed for inspecting chromatograms, was used for this
Dteridine The disappearance of the characteristic spectrum of
purpose.)
2,4,7?rihydroxypteridine in aliquots of the mixture removed
Carbons 6+7
COOH i from time to time may be used to follow the progress of the reac-
SCHEME 1 tion (Fig. 2).
When the irradiation was carried out at an alkaline pH or with
a longer wave length (366 mp), less satisfactory results were ob-

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tained. Acidity was desirable in any case to avoid a contamina-
tion of the glyoxylic acid representing carbon atoms 6 and 7 by
glyoxylic acid arising from a transamination with glycine (13)
aE 0.60
formed from the central carbon atoms of the ring system.
: lsoxanlhopterin When the photolysis was essentially complete (45 to 60 min),
e?
2 0.40 the irradiated solution was transferred to a small round bottom
flask, provided with a condenser, and refluxed. The duration of
0.20
this acid hydrolysis is critical. The optimal time of heating was
,7-trihydropteridine
found to be 15 min to obtain a good yield of glyoxylic acid.
220 260 300 340 360 Longer heating (1 hour) introduced contamination from the
bridge carbons. To the cooled hydrolysis mixture was added a
0.1 y0 solution of recrystallized (14) 2,4-dinitrophenylhydrazine
FIG. 1. Deamination of isoxanthopterin by acid hydrolysis in 1 N HCI (15 ml). After 30 min at room temperature, the solu-
with formation of 2,4,7-trihydroxypteridine. Isoxanthopterin
was refluxed in 6 N HCl at 1 mg/lO ml and diluted 1:lO for ultra- tion was shaken with four 20.ml portions of a benzene-ether
violet measurement. O--O, at start; n---n, after 50 hours. mixture (2:1, v/v) which extracted unreacted 2,4 dinitrophenyl
hydrazine along with the hydrazone. The combined organic
1.0 n layers were now extracted with two IO-ml portions of 0.05 M

yL
- zero time phosphate buffer, pH 7.0, which quantitatively removed the
-- IO min.
hydrazone leaving the unreacted hydrazine in the organic phase.
--- 20 min.
z --- 45 min. The buffer containing the hydrazone was applied to a column
0’
*
0.5
/- \ (2 x 15 cm) of DEAE-cellulose, and the column was washed with
zu) :\ /’ \ about 200 ml of water and then developed with 0.05 M phosphate
‘S\ /-’ \ / \
t / buffer, pH 7.0. The glyoxylic acid hydrazone was eluted from
‘\$ \/ ,*-‘\ \
‘;\,~~~“-‘, ,’ the column in two distinct yellow peaks, apparently the cis and
‘. -__-----
- --* ‘\\
__.___ ------.
-0-- . . \\ tram isomers.2 The first peak constituted 80% of the total
0 _- ‘:J%,e
hydrazone and had a maximum at 366 mp at pH 7.0, in phos-
220 250 300 350 phate buffer, while the second smaller peak had a maximum at
X ImpI 368 rnp under the same conditions. No difference in specific
radioactivity was found between these two peaks. Quantitation
FIG. 2. Drop in ultraviolet absorption of 2,4,7-dihydroxypteri-
dine during irradiation. The pteridine in 1% HBO, was irradi- was carried out spectrally at 368 rnp in 0.05 M phosphate buffer,
ated at 247 rnp and aliquots were removed at the intervals shown, pH 7.0. The molar extinction coefficient determined on a puri-
diluted 1:lO in HtO, and the ultraviolet spectra were taken.
2 When authentic glyoxylic acid was taken through this hydra-
reaction proceeds (Fig. 1) and the spectrum of aliquots removed zone-forming procedure and chromatography, two yellow peaks
for examination approaches that of 2,4,7-trihydroxypteridine were obtained with the same characteristics as the material ob-
tained from the trihydroxypteridine. In view of the characteris-
(12). After 28 hours, the solvent was completely removed and tic behavior of a-ketoacid dinitrophenylhydrazones to undergo
the residue in 0.1 N NaOH (20 ml) was applied to a column of chromatographic separation into two isomeric derivatives which
Dowex 1-formate (1.8 x 15 cm) which was washed with water are in dynamic equilibrium, it has become customary to consider
until the eluate was free of ultraviolet-absorbing materials. A these as cis and trans isomers (15). In the case of the 2,4-dinitro-
phenylhydrazone of glyoxylic acid, two crystalline forms are
linear gradient formed from water and 2 N formic acid (700 ml known (16), but in our work it was not considered profitable to
each) eluted spectrally pure 2,4,7-trihydroxypteridine at a nor- relate the column peaks to the known cryst,alline forms (cf. also
mality between 0.9 and 1.2. The eluate was sometimes used Footnote 3).
Issue of January 25,1966 E. Shaw, C. M. Baugh, and C. L. Krumdieck 381

fied hydrazone sample (mp 193-195”) was 21.3 x lo3 in this TABLE I
buffer.3 (This reference sample was probably a mixture of Radioactivity of glyoxylic acid obtained by degradation of 2,4,7-
isomers (16, 18).) The yield of hydrazone was 30 to 35% based trihydroxypteridine labeled in aa- or in r-position
on the trihydroxypteridine.
In isotopic experiments in which it was desired to measure I Specific activity I
radioactivity of samples plated as thin films, the presence of salts
was avoided by treatment of the solution of 2,4-dinitrophenylhy-
drazone of glyoxylic acid in phosphate buffer with 6 N hydro- cpm/pno1e %
chloric acid (2 ml) followed by extraction of the hydrazone into 4a-i4C,2,4,7-Trihydroxy-
an organic phase of benzene-ether (2: 1, 10 ml). The organic pteridine................. 10,000 62 0.6
layer, 1 ml, was plated, and the remainder was extracted with
fresh phosphate buffer (45 ml) for spectroscopic quantitation of 7-i%, 2,4,7-Trihydroxy-
the amount plated. Complete transfer was assured by total pteridine................. 3,060 3,200 105
evaporation under reduced pressure of the organic phase in the
* As its 2,4-dinitrophenylhydrazone.
presence of the buffer.
Conversion of Folk Acid to Isoxanthopterin-The oxidation of
?-.
folic acid and related compounds to 2-amino-4-hydroxypteridine-
6-carboxylic acid is a familiar procedure. In the decarboxylation
of the acid to 2-amino4hydroxypteridine, we have used ultra-

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violet light (19). Since the product is also somewhat sensitive
to destruction by irradiation, during the later stages of the de-
carboxylation the 2.amino-4-hydroxypteridine may be removed FIG. 3
by passage of the reaction mixture at pH 6 over a column (2 X 3
cm) of washed DEAE-cellulose which retains the acid, while the
view of the fact that the chemistry of this process has not been
product may be washed through. The pteridine 6-carboxylic worked out, the nature of the product or products of irradiation
acid may then be recovered by stripping the column with 1 N
of 2,4,7-trihydroxypteridine is unknown. However, it was
NHdOH and subjected to a successive irradiation. In this way, shown possible (Table I) to obtain glyoxylic acid representing al-
with three irradiations, the yield could be raised to 85%, which
most exclusively carbon atoms 6 and 7, as hoped, by a limited
was of interest when the supply of the pteridine carboxylic acid
acid hydrolysis after irradiation. This procedure is a clear im-
was very limited. provement over the methods available previously (5) which pro-
Isoxanthopterin was formed from 2-amino-4-hydroxypteridine
vided oxalic acid considerably contaminated with bridgehead
through the use of xanthine oxidase (20). For this purpose the carbon (Fig. 3). It has the additional advantage of being ap-
pteridine was brought into solution in 0.05 M phosphate buffer,
plicable to smaller samples and has been carried out successfully
pH 7.5, at a concentration of about 4 mg/lOO ml and treated at
on as little as 1 mg of 2,4,7-trihydroxypteridine. Use of ion
37” with xanthine oxidase (2 units of enzyme per 10 pmoles of
exchange chromatography at successive steps greatly reduces
pteridine were used). The progress of the reaction was followed
the danger of carrying along radio-impurities. Finally, if de-
at 340 rnp (6). Purification of the isoxanthopterin was found to
sired the radioactivity of carbon atoms 6 and 7 can be determined
be essential before proceeding with its deamination apparently separately through use of the thermal decarboxylation of 2,4-
owing to side reactions which take place with protein present.
dinitrophenylhydrazone of glyoxylic acid (21).
The pH was brought to 1 with hydrochloric acid, and the mixture
The degradation is applicable not only to folic acid but pre-
was applied to a column (2.0 x 19 cm) of Dowex 50-H+ which sumably also to other 6-substituted pteridines found in nature
was washed with water then eluted with a linear gradient formed
since conversion to 2-amino-4-hydroxypteridine-6-carboxylic
from water and 2 N hydrochloric acid (1 liter each). Spectrally acid is a common feature of their chemistry.
pure isoxanthopterin was eluted at a normality of HCl between
0.4 and 0.6 in a yield of 74%. REFERENCES
1. KRUMDIECK, C. L., SH.~W, E., AND BAUGH, C. M., J. Biol.
RESULTS AND DISCUSSION Chem., 241, 383 (1966).
The procedure given represents the best conditions found for a 2. WIELAND, H., AND TARTER, A.. Ann. Chem., Liebias.“, 543, 287
(1940). ’
reliable degradation of the pteridine ring in folic acid, and it was 3. WEYGAND, F., SCHLIEP, H. J., SIMON, H., AND DAHMS, G.,
arrived at after considerable variation in which the highest yield Anaew. Chem.. 71, 522 (1959).
at each step was sought consonant with the goal of obtaining 4. WEY~AND, F., SIMON, fi., D.SHMS, G., WALDSCHMIDT, M.,
glyoxylic acid only from carbon atoms 6 and 7 of the ring. In SCHLIEP, H. J., .~ND W.~CKER, H., Angew. Chem., 73, 402
(1961).
3 The hydrazone spectral peaks are broad; therefore, the differ- 5. BRENNER-HOLZ.~CH, I., AND LELJTHARDT, F., Helv. Chim.
ence in absorbance due to one isomer being measured 2 rnp away Acta, 44, 1480 (1961).
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as found by inspection of its spectrum. While the extinction 521 (1953).
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used here is reasonably accurate. 1620 (1952).
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