Endocrine disrupting compounds are ubiquitous in aquatic environments. Exposure to these compounds may cause deiodination of thyroid hormones in fish and also affect certain behaviors. Behavioral change may be a useful indicator of deiodination on an organismal level, and may indicate effects to populations, especially if these behaviors affect reproduction. We exposed Pimephales promelas (Fathead Minnow) to effluent from a recently upgraded wastewater treatment facility. The effluent contained a matrix of low-level (< 1.0 µg/L) concentrations of endocrine disrupting compounds pre- and post-upgrade. We examined the behavior of adult Fathead Minnow exposed to treated wastewater for 90 days and the behavior of their offspring at 12-20 days old. There was no significant difference in 11 of the 12 behaviors we documented for adult fish in control and treatment tanks (p > 0.05). However, treatment fish were significantly more aggressive towards a decoy fish compared to control fish (p < 0.05). There was no significant difference found in the predator avoidance behaviors examined in the F1 generation. Although our results did not find any significant differences between control and treatment fish in regard to most behaviors we would typical associate with thyroid deiodination, such behaviors may have manifested following a longer exposure period or by having a larger sample size. Other future considerations should include looking at different behaviors (e.g. social behaviors) and further characterization of the finished effluent.

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.

Thyroid hormones (THs; T4 and T3), secreted from thyroid gland, play an important role in human growth and development. T3 (3,5,3′-triiodothyronine) is the active hormone and the conversion of T4 (3,3′,5,5′-tetraiodothyronine) to T3 in cells is mediated by iodothyronine deiodinases enzymes (DIOs). DIOs are selenocysteine containing enzymes and are classified into three types (DIO1, DIO2 and DIO3). DIO1 catalyzes the outer-ring deiodination (ORD; T3 formation) and inner-ring deiodination (IRD; rT3 formation) reactions, involving in the activation (T4 to T3 conversion) and inactivation (T4 to rT3 conversion), respectively. DIO2 and DIO3 catalyse the ORD and IRD reactions, respectively. This homeostasis is regulated tightly and any deviation would lead to diseases like hyperthyroidism or hypothyroidism. Recently it is of interest to many research groups to develop iodothyronine deiodinase mimics and we have developed naphthalene-based peri-substituted thioselenol pair at 1,8-positions (1.25), which remove iodine selectively from inner-ring of T4. When selenium atom is substituted in place of sulfur (selenol-selenol pair; 1.26), the deiodination activity was ca. 90 times faster than with 1.25. This thesis deals with various aspects of the effect of substituents on the naphthalene-1,8-diselenol and solvent effect on the thyroid hormone deiodination by naphthalene-based iodothyronine deiodinase mimics. Figure 1. (A) Deiodination reactions by DIOs. (B) Chemical structure of 1.25 and 1.26. The thesis consists of five chapters. The first chapter provides a general overview about sialoproteins, thyroid hormone biosynthesis, thyroid hormone metabolism, halogen bonding, iodothyronine deiodinase mimics and proposed mechanisms for the deidoination of thyroid hormones. This chapter also introduces peri-naphthalene-1,8-diselenol (1.26), which is the key compound in this thesis and discusses about proposed mechanism for the deiodination of thyroxine involving co-operative halogen bonding and chalcogen bonding mechanism. Figure 2. (A) TH action. (B) Proposed mechanism for the deiodination of T4 by 1.26 involving cooperative halogen bonding and chalcogen bonding. Chapter 2 discusses about the synthesis, characterization and deiodination activity of a series of naphthalene-based peri-substituted-1,8-diselenols (Figure 3). These diselenols regioselectivity remove iodine from inner ring of thyroxine and other thyroid hormones, (T3 and 3, 5-T2). Substitution with different groups on the naphthalene ring did not change the regioselectivity of deiodination, indicating that the deiodination activity does not depend on the nature of substituents. Secondary or tertiary amine side chain group attached at the 2nd position of the naphthalene ring showed better activity. It is due to the secondary interaction, which facilitates the iodine removal. It was further confirmed with the substitutions at the 4th position of the ring to discriminate the possibility of electronic effect. The higher deiodination rate owing to the t-butyl group at second position of the ring also suggests that the steric effect may also play a role in the deiodination reaction (Figure 4). It is proposed that peri substituted naphthalene-1,8-diselenols remove iodine from thyroid hormones through halogen bonding-chalcogen bonding mechanism (Figure 2). The investigation of Se···Se bond distance from the crystal structures and through DFT calculation and NMR experiment showed that the stronger chalcogen bond could be the reason for the increase in the reactivity observed with substituted peri-naphthalene-1,8-diselenols. Figure 3. peri-substituted naphthalene-1,8-diselenols used for the study. Figure 4. Relative deiodinase activity of substituted-peri-naphthalene-1,8-diselenols with T4. In Chapter 3, we have discussed about the effect of chalcogen atom substitution in a series of deiodinase mimics on the deiodination of thyroid hormones. Moving from thiol-selenol pair (1.25) to selenol-selenol pair (1.26) in naphthalene based peri-substituted mimics, an increase in the activity was observed. In this chapter, we have shown that substituting with tellurium, as tellurium-thiol pair (3.3) and ditellurol (3.4) increases the reactivity of deiodination to several times and also regioselectivity of deiodination is changed from IRD in the case of 1.26 to both IRD and ORD for 3.3 and 3.4. The presence of two tellurol moieties (3.4) or a thiol-tellurol pair (3.3) can mediate sequential deiodination of T4, to produce all the possible thyroid hormone derivatives under physiologically relevant conditions (Figure 5). This study provided the first experimental evidence that the regioselectivity of the thyroid hormone deiodination is controlled by the nucleophilicity and the strength of halogen bond between the iodine and chalcogen atoms. Figure 5. (A) HPLC chromatograms of deiodination reaction of T4 with 3.3 and 3.4. (B) Chemical structure of 3.3 and 3.4. (C) Sequential deiodination reaction of T4 by 3.3 and 3.4. Chapter 4 describes the effect of alkyl conjugation at 4′-OH position of THs on the deiodination by iodothyronine mimics. In addition to the deiodination, iodothyronines undergo conjugation with sulfate and glucuronic acid group at 4′-hydroxyl position. Conjugation alters the physico-chemical properties of iodothyronines. For example, it is known that sulfate conjugation increases the rate of deiodination to a large extend. We have conjugated alkyl group at 4′-hydroxyl position of iodothyronines and investigated the deiodination reactions with reported peri-substituted naphthalene-1,8-diselenols. We observed that similar to sulfated thyroid hormones O-methylthyroxine also undergoes both phenolic and tyrosyl ring deiodination reactions and overall the rate of deiodination is increased at least by 5 times as compared with T4 under identical conditions. The phenolic iodine removal is favored by conjugation as compared to the tyrosyl ring iodine, which is similar to the observation made for T4S. Interestingly, when the acetamide group is conjugated at 4′-OH position, the regioselectivity of deiodination is changed exclusively to 5′-iodine. DFT calculations show that the positive potential on the iodine increase upon conjugation, which leads to stronger halogen bonding interaction with selenol, might be the reason for the change in the regioselectivity of deiodination. Figure 6. (A) HPLC chromatogram of deiodination reaction of T4(Me) with 1.26. (B) Initial rate comparison of T4 and T4(Me).(C) HPLC chromatogram of deiodination reaction of T4(AA) with 1.26 showing the formation of T3(AA) (ORD product). (D) Electron potential map of T4, T4(Me) and T4(AA) showing the increase in electro positive potential on 5′-iodine upon conjugation. Chapter 5 deals with the solvent effect on the deiodination reactions of THs by iodothyronine deiodinase mimics. As discussed in the earlier chapters, the deiodination reaction of thyroxine by naphthalene based-1,8-diselenols under physiological conditions produce, rT3 (IRD) as the only observable products. Surprisingly, when the deiodination reaction was performed in DMF or DMSO in the presence of 1.26, the regioselectivity of reaction was changed and the formation of both T3 (ORD) and rT3 was observed. In DMF or in DMSO, the deiodination reactivity of 1.26 was found to be 1000 fold higher than the reaction performed in phosphate buffer at pH 7.4. Figure 7. (A) HPLC chromatogram for the deiodination reaction of T4 in DMF by 1.26 showing both IRD and ORD. (B) A comparison of initial rate for the deiodination reactions of T4, T3 and 3,5-T2 in DMF and in DMSO by 1.26. (C) HPLC chromatograms for the deiodination reaction of T4 in DMF by 1.26 in the presence of TEMPO, showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. (D) HPLC chromatograms for the deiodination reaction of T4 in DMSO by 1.26 in the presence of TEMPO showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. 3,5-DIT was not denominated under physiological conditions, however, in DMF and in DMSO, 3,5-DIT was deiodinated by 2.4 to produce 3-MIT. We also observed that the control reactions in DMF or DMSO also showed a little deiodination activity. The very high reactivity observed in the presence of DMF or DMSO implied that the mechanism of denomination in these solvents may be different. It has been reported that DMSO or DMF radicals can be formed with small amounts of a base. Reaction mixture consisting of NaBH4 (for generating selenol from diselenide) and NaOH (T4 solution) may facilitate the radical formation. We also performed the reaction in the presence of TEMPO (free radical scavenger) and observed the inhibition of deiodination reaction. However, it is not clear whether the radical pathway could be one of the possible mechanisms of deiodination in these solvents by compounds 1.26 and 2.4. Further studies are required to propose a radical mechanism in different solvents such as DMF and DMSO.