J. Am. Chem. Soc. 1997, 119, 11315-11316
11315
Scheme 1. Blasi’s Intermolecular Mechanism of Thyroxine
Formation
Model Reactions of Thyroxine Biosynthesis.
Identification of the Key Intermediates in
Thyroxine Formation from 3,5-Diiodo-L-tyrosine
and 4-Hydroxy-3,5-diiodophenylpyruvic Acid
Vibha B. Oza, Grzegorz M. Salamonczyk, Zhi-wei Guo, and
Charles J. Sih*
School of Pharmacy, UniVersity of Wisconsin
425 North Charter Street, Madison, Wisconsin 53706-1515
ReceiVed June 10, 1997
Although it was suggested 50 years ago that thyroxine (T4)
is formed in the thyroid from its precursor 3,5-diiodo-L-tyrosine
(DIT),1 the mechanism of this conversion is still not completely
understood. Two possible mechanisms have been proposed for
the formation of T4 in the thyroid gland, and these are generally
referred to as intramolecular and intermolecular coupling.2
Intramolecular coupling involves oxidation of DIT to a free
radical and interaction of two DIT radicals to form T4 through
a quinol ether intermediate.3 Earlier workers envisaged the
coupling reaction to involve the conversion of free DIT to free
T4.4 However, later studies indicated that peptide-linked DIT
is more likely the precursor of T4 and that coupling of two
molecules of DIT occurs within the thyroglobulin (TGB)
molecule3 to yield T4 and dehydroalanine as the lost three carbon
unit.5
intramolecular or the intermolecular coupling mechanism.13 We
decided to reexamine the chemical identity of the putative
hydroperoxide intermediate 1 because it was difficult for us to
rationalize on mechanistic grounds the unusually facile reactivity
of this intermediate with DIT to form T4. In this paper, we
report the experimental data used in establishing the structures
of the T4 precursors as the epoxides 8a and 9.
When DIHPPA was subjected to the oxygenation conditions
(0.2 M borate buffer, pH 7.5) as described by Cahnmann and
co-workers,7b a complex mixture of products was formed. The
reverse-phase HPLC profile of the reaction mixture showed the
presence of six peaks.14 Owing to the instability of the isolated
compounds, they were kept at -78 °C and analyzed im-
mediately. It was found that both peaks 3 and 5 decomposed
completely to 4-hydroxy-3,5-diiodobenzaldehyde (DIHBA) and
3,5-diiodo-1,4-benzoquinone (DIBQ) within 24 h at 24 °C,
whereas the other three peaks decomposed less readily only to
DIHBA. Further, only peaks 3 and 5 reacted readily with DIT
to form T4.
The major product, peak 5, was found to react readily with
DIT to give T4 in over 85% yield after 3 h of stirring at 24 °C.
It was characterized as the quinol epoxide 8a on the basis of its
NMR spectral data and its reduction by sodium borohydride to
furnish 4-hydroxy-3,5-diiodomandelic acid15 (7a). Its identity
was confirmed by the reaction of 7b with an excess of sodium
bismuthate16 in an ethyl acetate/acetic acid/water solution to give
8b, whose spectral properties and HPLC retention time were
identical to those from a sample of 8b obtained by reaction of
8a with diazomethane.
Intermolecular coupling is based on the discovery that DIT
couples very readily with its keto analog 4-hydroxy-3,5-
diiodophenylpyruvic acid (DIHPPA) to form T4.6 This reaction
has been studied extensively by Cahnmann and co-workers7 who
showed that under suitable oxidizing conditions DIHPPA
couples readily with DIT residues in TGB to form T4.8 They
isolated an active intermediate in this reaction and proposed
that its structure is the hydroperoxide of DIHPPA, 1.7b
A
possible scheme for T4 formation in the thyroid by inter-
molecular coupling was presented by Blasi et al.,9 who
postulated that DIHPPA may be formed from DIT in a reaction
catalyzed by tyrosine transaminase (Scheme 1).10 A tautomerase
present in the thyroid and in other tissues then converts the
DIHPPA to the enol form.11 The latter is presumably oxidized
by H2O2 and thyroid peroxidase9 (TPO) to the putative hydro-
peroxide 1, which was shown by Cahnmann and co-workers to
be the intermediate that couples spontaneously with DIT.12
Hence, there are cogent reasons to believe that the coupling
reaction between DIHPPA and DIT may represent a biosynthetic
model for T4 formation in the thyroid.
However, at the present time it is not definitely known
whether T4 formation in vivo involves predominantly the
The compound in peak 3 (proposed to be 9) reacted with
DIT even faster than 8a to yield T4 cleanly and quantitatively
at 0 °C in less than 45 min. The other product of the reaction,
mesoxalic acid semialdehyde, was trapped with phenylhydrazine
to furnish an adduct whose physical properties coincided with
those of the bis-diphenylhydrazone. Its retention time and mass
spectral data were identical to an authentic sample prepared by
the procedure of Fenton and Ryffel.17 The presence of a keto
(1) Harington, C. R.; Barger, G. Biochem. J. 1927, 21, 169-183.
(2) Degroot, L. J.; Niepomniszcze, H. Prog. Endocrinol. Met. 1977, 665-
718.
(3) Taurog, A.; Dorris, M.; Doerge, D. R. Arch. Biochem., Biophys. 1994,
315, 82-89. See also: Johnson, T.; Tewkesbury, L. Proc. Natl. Acad. Sci.
U.S.A. 1942, 23, 73-77.
(4) Harington, C. R.; Randall, S. S. Biochem. J. 1929, 23, 373-383.
(5) (a) Gavaret, J. M.; Nunez, J.; Cahnmann, H. J. J. Biol. Chem. 1980,
255, 5281-5285. (b) Gavaret, J. M.; Cahnmann, H. J.; Nunez, J. J. Biol.
Chem. 1979, 254, 11218-11222.
(6) (a) Hillmann, G.; Keil, B.; Tashimi, P. Z. Naturforsch. 1961, 16,
28-32. (b) Meltzer, R. I.; Stanaback, R. J. J. Org. Chem. 1961, 26, 1977-
1979.
(13) Taurog, A. In Endocrinology; DeGroot, L. J., Cahill, G. F., Jr.,
Martini, L., Nelson, D. H., Odell, W. D., Potts, J. T., Jr., Steinberger, E.,
Winegrad, A. I., Eds.; Grune and Stratton: New York, 1979; Vol. 1, pp
331-342.
(14) The oxygenated DIHPPA mixture was separated on a reverse-phase
C18 column (19 × 300 mm) using a gradient elution system consisting of
80% of solvent A (0.1% in water) to 76% of solvent B (0.1% TFA in 90%
aqueous acetonitrile) in 25 min. A flow rate of 8 mL/min was used.
Retention times: peak 1, 11.8 min; peak 2, 15.3 min; peak 3, 16.3 min;
peak 4, 17.3 min; peak 5, 20.8 min; peak 6, 24.3 min.
(15) Matsuura, T.; Cahnmann, H. J. J. Am. Chem. Soc. 1959, 81, 871-
878.
(7) (a) Nishinaga, A.; Cahnmann, H. J.; Kon, H.; Matsuura, T.
Biochemistry 1968, 7, 388-397. (b) Cahnmann, H. J.; Funakoshi, K.
Biochemistry 1970, 9, 90-98.
(8) Toi, K.; Salvatore, G.; Cahnmann, H. J. Biochim. Biophys. Acta 1965,
97, 523-531.
(9) Blasi, F.; Fragomele, F.; Covelli, I. Endocrinology 1969, 85, 542-
551.
(10) Igo, R. P.; Mahoney, C. P.; Limbeck, G. A. Biochim. Biophys. Acta
1968, 151, 88-98.
(11) Blasi, F.; Fragomele, F.; Covelli, I. J. Biol. Chem. 1969, 244, 4864-
4870.
(16) Adler, E.; Holmberg, K.; Ryrfors, L. O. Acta Chem. Scand. 1974,
B28, 883-887.
(17) Fenton, H. J. H.; Ryffel, J. H. J. Chem. Soc. 1902, 81, 426-434.
(12) Ogawara, H.; Cahnmann, H. J. Biochim. Biophys. Acta 1972, 257,
328-338.
S0002-7863(97)01903-3 CCC: $14.00 © 1997 American Chemical Society