Thyroxine is the main secretory hormone of thyroid gland and it is produced in thyroglobulin by thyroid peroxidase/hydrogen peroxide/iodide system. After biosynthesis and secretion of thyroxine, it undergoes multiple metabolic reactions. The most important metabolic pathway is the stepwise deiodination from the inner ring or outer ring. Removal of one of the outer ring or phenolic ring iodines of biologically less active T4, leads to the formation of 3,5,3'-triiodothyronine or T3, a compound which is biologically more active. On the other hand, removal of one of the inner ring or tyrosyl ring iodines gives 3,3',5'-triiodothyronine (3,3',5'-T3 or rT3) which is a biologically inactive thyroid hormone. Three enzymes involved in this activation and inactivation pathway of thyroid hormones are known as iodothyronine deiodinases (IDs), which are dimeric integral-membrane selenoproteins. Depending upon the sequence and substrate specificity, three iodothyronine deiodinase enzymes have been identified, iodothyronine deiodinase-1
(ID-1), iodothyronine deiodinase-2 (ID-2) and iodothyronine deiodinase-3 (ID-3). ID-1 can catalyze both inner ring and outer ring deiodination of thyroid hormones whereas, ID-2 is selective to the outer ring deiodination. The type-1 and -2 deiodinases (ID-1 and ID-2) produces the biologically active hormone 3,5,3′-triiodothyronine (T3). These two enzymes also convert 3,3′,5′-triiodothyronine (reverse T3 or rT3) to 3,3′-diiodothyronine (3,3′-T2) by outer-ring deiodination (Scheme 1). The type-3 deiodinase (ID-3) catalyzes the convertion of T4 to rT3 by an inner-ring deiodination pathway.
Apart from deiodination, there are several alternate pathways of thyroid hormone metabolism, which include sulfate conjugation and glucoronidation of the phenolic hydroxyl group of iodothyronines, the oxidative deamination and decarboxylation of the alanine side chain to form thyroacetic acid and thyronamines, respectively. Glucoronidation and sulfate conjugation changes the physico-chemical properties of iodothyronines dramatically.
This thesis consists of five chapters. The first chapter provides a general introduction of biosynthesis of thyroid hormones and followed by deiodination by three iodothyronine deiodinase enzyme. This chapter also provides an overview of thyroid hormone transport and different transport proteins and their mode of binding with thyroid hormones. Apart from this, this chapter also provides a brief overview on other thyroid hormone metabolites.
In the second chapter of the thesis, initial attempts in the development of different iodothyronine deiodinase mimics have been discussed. Goto et al have shown that the sterically hindered selenol 1 converts the thyroxine derivative 3 (N¬butyrylthyroxine methyl ester) to the corresponding triiodo derivative 4 by an outer-ring deiodination (Scheme 2). Although the reaction was carried out in organic solvent and a relatively higher temperature (50 °C) and longer reaction time (7 days) were required for about 65% deiodination, this study also provides an experimental evidence for the formation of selenenyl iodide (2) in the deiodination of a thyroxine derivative by an organoselenol. However, only one iodine was removed from the outer ring of 3, no inner ring deiodination was detected (Scheme 2).
Interestingly, when compound 5 was treated with selenol 1 under similar conditions, no deiodination was observed (Scheme 3). This leads to assumption that presence of free phenolic hydroxyl group is important for the deiodinase activity. Based on this experimental observation, they proposed a mechanism which involves an enol¬keto tautomerism of the phenolic hydroxyl group. In the case of thyroxine, the outer-ring can undergo enol-keto tautomerism, whereas due to lack of free hydroxyl group, the inner ring cannot undergo similar kind of tautomerism. The enol-keto tautomerism probably makes the outer ring iodines more reactive than the inner ring iodines of thyroxine.
We have developed tthe first chemmical modell for the inneer ring deioddination of TT4 and T3 by type 33 deiodinase . We have shown that naphthyl-baseed selenol 6 bearing a thhiol group in the cloose proximitty to the sellenium act aas an excelleent model foor ID-3 by selectively deiodinatting T4 andd T3 to prodduce rT3 annd 3,3'-T2, rrespectively,, under physiological relevant conditions. When 2 equuivalent of ccompound 66 was emplooyed in the assay, an almost quuantitative cconversion oof T4 to rT3 was observeed within 300 hours and there was no indicaation of the fformation off T3 or 3,3'-TT2.
When the selenol group was repplaced with a thiol group in compouund 7, the ddeiodinase activity wwas decreassed. On thee other handd, when thee thiol groupp was replaaced with selenol mmoiety in commpound 8, thhe deiodinasse activity drramatically iincreased wiithout any change iin the selecttivity. Comppounds 10 and 11 havving N-methhylamino grooup were found too be more aactive than the correspponding unssubstituted ccompounds 7 and 8, respectively. However, introduction of a secondary amine adjacent to the selenol moiety into the compound 9 significantly reduces the deiodinase activity.
In the third chapter synthesis, deiodinase activity and mechanism of deiodination of a series of peri-substituted naphthalene derivatives is discussed. Iodobenzene was used as halogen bond donor for the DFT calculations. From the orbital analysis it is observed that there is perfect orbital symmetry match between the HOMO of compound 8 (selenolate form) and LUMO of iodobenzene. When the selenolate form of 1-selenonaphthol interacts with iodobenzene, a halogen bonded adduct is formed. The negative charge on the selenium center decreases as it donates electron pair to the σ* orbital of C–I bond in iodobenzene and as a consequence the positive charge on the iodine center decreases (Figure 1). Addition of iodobenzene to 1-selenonaphthol led to a significant downfield shift in 77Se NMR spectrum of 1-selenonaphthol and with an increase in the concentration of iodobenzene, more downfield shift in the signal was observed.
Figure 1. The charges obtained from Natural Bond Orbital (NBO) analysis for the selenolate form of (a) 1-selenonaphthol (b) iodobenzene, (c) halogen-bonded adduct
On the basis of experimental end theoretical data, a mechanism for the deiodination of T4 by compound 8 is proposed. According to the mechanism, the initial interaction of one of the selenol moieties with an iodine leads to the formation of halogen bond. The transfer of electron density from selenium to the σ* orbital of the C−I bond generates a σ-hole or partial positive charge on the selenium atom, which facilitates an interaction between the halogen bonded selenium atom and the free selenol (selenolate) moiety (intermediate 12). The selenium−selenium interaction (chalcogen bond) strengthens the halogen bond, leading to a heterolytic cleavage of the C−I bond. The protonation of the resulting carbanion leads to the formation of rT3. On the other hand, the formation of an Se−Se bond produces the diselenide 13 with elimination of iodide as HI. The reductive cleavage of the Se−Se bond in compound 13 regenerates the diselenol 8 (Figure 2).
In the fourth chapter deiodination of sulfated thyroid hormones is discussed. Sulfate conjugation is an important step in in the irreversible inactivation of thyroid hormones. Sulfate conjugation of the phenolic hydroxyl group stimulates the inner ring deiodination of T4 and T3 but it blocks the outer ring deiodination of T4 by ID-1. The thyroxine sulfate (T4S) undergoes faster deiodination as compared to the parent thyroid hormone T4. Only ID-1 catalyzes the deiodination of sulfated thyroid hormones. In contrast, ID-2 and ID-3 do not accept T4S and/or T3S as substrate. We have shown that iodothyronine sulfates can be readily deiodinated by synthetic deiodinase model compound 8 and its derivatives. In contrast to the inner ring-selective deiodination of T4, the synthetic compounds loses the selectivity and mediate both inner and outer-ring deiodination of T4S and outer ring deiodination of rT3S. From this study, we have also proposed that the enol-keto tautomerism is probably not required for the outer ring deiodination and the strength of halogen bonding controls the regioselective deiodination by model compounds.
In the fifth chapter, the mechanism of inhibition of iodothyronine deiodinases by PTU and IAA is discussed with the help of model compounds. In the model study, it has been observed that compound 8 does not form a stable Se-I intermediate (14), which is essential for the formation of Se-S covalent bond with PTU. As a consequence, the deiodination of T4 by compound 8 is not inhibited by PTU. This study supports the proposal that ID-3 does not follow a ping-pong bi-substrate pathway for deiodination and may not form a stable E-Se-I intermediate, which is responsible for the insensitivity of ID-3 towards PTU.
The biphenyl based diselenol 15 reacts with IAA and iodoacetamide to form the corresponding carboxymethylated product 17. On the other hand, compound 8 does not undergo the expected carboxymethylation by IAA and iodoacetamide, but they readily deiodinate both IAA and iodoacetamide. Based on this model study, a possible model is proposed for the insensitivity of ID-3 towards IAA.
Iopanoic acid (18) is a well known radiocontrast agent and is used as adjunctive therapy with PTU and CBZ for the treatment of thyrotoxicosis.[9] We show in this chapter that iopanoic acid undergoes monodeiodination by compound 8 under physiological relevant conditions. The deiodinated products (19 and 20) from iopanoic acid are characterized by NMR spectroscopy and single crystal X-ray crystallography. It is observed that after monodeiodination, the strength of halogen bonding decreases and therefore, the monodeiodinated products do not undergo further deiodination.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.iisc.ernet.in:2005/3304 |
| Date | January 2013 |
| Creators | Manna, Debasish |
| Contributors | Mugesh, G |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G25668 |
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