Abstract:The barrier to rotation in the N-acetyl methyl ester of the thyroxine was found to be 8.6 kcal mol-1. Previous experiments determining the barrier to rotation in triiodothyropropionic acid in HCl-ethanol were shown to be in error.
“…Knowledge of this three-dimensional shape provides a starting point for modeling the interactions of the hormones with their target proteins, but the representation shown in Figure a is somewhat simplified, because it is known that there are significant dynamic motions associated with the outer ring. Studies on soluble analogues, , and later on T4 and T3 themselves, have shown that rotation about the diphenyl ether linkage with a barrier of approximately 36 kJmol -1 produces an interchange of the environment of H2‘ with that of H6‘. For hormones which are symmetrically iodinated in the outer ring, the interchange occurs between isoenergetic (and structurally equivalent) forms, but for monoiodinated derivatives such as T3, the rotation about the diphenyl ether linkage interconverts “proximal” and “distal” forms of the hormone, as illustrated in Figure .…”
1H and 13C NMR spin-lattice relaxation times and 13C{1H} nuclear Overhauser enhancement factors have been measured for the thyroid hormones thyroxine, 3,5,3'-triiodothyronine, and 3,5-diiodothyronine, with the aim of determining the internal molecular dynamics in these molecules. Spin-lattice relaxation times of protons on the two aromatic rings of these hormones show remarkable differences, with values for the hydroxyl-bearing ring being a factor of 4-12 times larger than those for the alanyl-bearing ring. This difference is not mirrored in the 13C relaxation times, which are identical within experimental error for the two rings. The 13C data show that the mobility of the two rings is similar, and therefore the difference in proton spin-lattice relaxation times arises because the protons of the alanyl-bearing ring are efficiently relaxed by interactions with neighboring protons on the side chain. Quantitative analysis of the 13C relaxation data shows that there must be a significant degree of internal flexibility in the thyroid hormone molecules. The NMR data suggest that in methanol the molecules tumble with an overall correlation time of approximately 0.35 ns, but that rapid internal motion (in the form of jumps between two stable conformations) occurs on a 30-fold faster time scale. When combined with previous variable temperature NMR studies that show interconversion between proximal and distal forms of the outer ring on the microsecond time scale, the results provide a complete description of the conformations and both fast and slow internal motions in the thyroid hormones. The findings suggest that modeling studies of thyroid hormone interactions with receptor proteins should take into account the possibility that these internal motions are present. In effect, the thyroid hormones may likely populate a larger range of conformations in the bound state than might be inferred from just the lowest energy forms seen in the crystal and solution states.
“…Knowledge of this three-dimensional shape provides a starting point for modeling the interactions of the hormones with their target proteins, but the representation shown in Figure a is somewhat simplified, because it is known that there are significant dynamic motions associated with the outer ring. Studies on soluble analogues, , and later on T4 and T3 themselves, have shown that rotation about the diphenyl ether linkage with a barrier of approximately 36 kJmol -1 produces an interchange of the environment of H2‘ with that of H6‘. For hormones which are symmetrically iodinated in the outer ring, the interchange occurs between isoenergetic (and structurally equivalent) forms, but for monoiodinated derivatives such as T3, the rotation about the diphenyl ether linkage interconverts “proximal” and “distal” forms of the hormone, as illustrated in Figure .…”
1H and 13C NMR spin-lattice relaxation times and 13C{1H} nuclear Overhauser enhancement factors have been measured for the thyroid hormones thyroxine, 3,5,3'-triiodothyronine, and 3,5-diiodothyronine, with the aim of determining the internal molecular dynamics in these molecules. Spin-lattice relaxation times of protons on the two aromatic rings of these hormones show remarkable differences, with values for the hydroxyl-bearing ring being a factor of 4-12 times larger than those for the alanyl-bearing ring. This difference is not mirrored in the 13C relaxation times, which are identical within experimental error for the two rings. The 13C data show that the mobility of the two rings is similar, and therefore the difference in proton spin-lattice relaxation times arises because the protons of the alanyl-bearing ring are efficiently relaxed by interactions with neighboring protons on the side chain. Quantitative analysis of the 13C relaxation data shows that there must be a significant degree of internal flexibility in the thyroid hormone molecules. The NMR data suggest that in methanol the molecules tumble with an overall correlation time of approximately 0.35 ns, but that rapid internal motion (in the form of jumps between two stable conformations) occurs on a 30-fold faster time scale. When combined with previous variable temperature NMR studies that show interconversion between proximal and distal forms of the outer ring on the microsecond time scale, the results provide a complete description of the conformations and both fast and slow internal motions in the thyroid hormones. The findings suggest that modeling studies of thyroid hormone interactions with receptor proteins should take into account the possibility that these internal motions are present. In effect, the thyroid hormones may likely populate a larger range of conformations in the bound state than might be inferred from just the lowest energy forms seen in the crystal and solution states.
“…The outer ring proton which is proximal to the inner ring is upfield of the distal proton, due to a significant ring current shift. For T4, T3, and several analogues , the barrier to the conformational exchange of H2‘ and H6‘ ranges from 33 to 38 kJ mol -1 . 2 Interconversion of the distal and proximal protons of T4.…”
1H NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol-1. This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is > or = 37 kJ mol-1, which is significantly larger than has previously been reported for Csp2-Csp3 bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.
'H NMR spectra at 300 and 400 MHz have been recorded for the thyroid hormones thyroxine (T4) and triiodothyronine (T3) over a range of temperatures from 175 to 298 K in MeOH-d, . Above 200 K the aromatic region of the NMR spectrum of T4 consists of two peaks, one for the H-2',H-6' protons and one for H-2,H-6. As the temperature is lowered each of these resonances first broadens and then splits into two peaks of equal intensity at low temperature. This is consistent with dowed exchange between two equally populated conformers. Similar behaviour is noted for T3, although in this case the spectrum is complicated by the presence of spin-spin splitting. Consideration of coalescence temperatures and a line-shape analysis yielded activation barriers in the range 35-38 kJ mol-'. It is concluded that the observed spectral changes are due to dynamic interconversion between proximal and distal forms of the hormones, and that the interconversion takes place by cooperative rotations around the bonds between each phenyl ring and the linking oxygen atom.
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