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.

Ainda não é claro o papel dos hormônios tireoideanos (HT) no desenvolvimento do esqueleto fetal. Para responder a questão, induzimos hipotireoidismo materno e fetal em camundongos prenhes através da administração de metimazol e perclorato de sódio. O esqueleto fetal apresentou discretas morfológicas até 16,5 dias de idade embrionária (E). Apenas no final da gestação, em 18,5E, foram observadas a redução significativa da zona hipertrófica, do número de condrócitos hipertróficos, desorganização e diminuição da quantidade dos condrócitos proliferativos, além da redução da expressão do colágeno I, X e osteocalcina. Os TRs, assim como LAT1, LAT2 e MCT8 foram detectados em todas as idades estudadas. A alta expressão gênica da D3, principal inativadora do hormônio tireoideano, em 14,5E e sua redução significativa durante o desenvolvimento, até atingir níveis indetectáveis no período pós-natal indicam que a D3 seja responsável por manter baixos níveis de HT no esqueletono início da gestação, garantindo um desenvolvimento ósseo normal. / Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice and fetuses were made hypothyroid. The skeleton morphology was preserved up to 16.5 embryonic days (E). Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrofic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly at E18.5, and even more after birth. The expression levels of D3 gene during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that its expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe. </p><p>Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages. </p><p>Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99. </p><p>The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls. </p><p>Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation. </p><p>A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.</p> / Dissertation

Made available in DSpace on 2016-03-15T19:39:56Z (GMT). No. of bitstreams: 1 Cyntia Moraes Teixeira.pdf: 299481 bytes, checksum: 9bb0d211bcfc9fdcd4b0b8dbed28d6e4 (MD5) Previous issue date: 2012-08-09 / Universidade Presbiteriana Mackenzie / Obesity has been considered epidemic in the whole world and is a risk factor for the development of hypertension, dyslipidemia, hyperglycemia, type 2 diabetes and hepatic steatosis. The increase of obesity in during pregnancy not only increases the risk of developing cardiovascular diseases but may also be related to abnormalities in the developing CNS of embryos, for example, reduction of potential long-term (LTP) in the hippocampus and neurogenesis . It is possible that the reduced levels of BDNF observed in these embryos, are involved with the injury in the processes of learning and memory observed in these animals. Studies also show that BDNF is reduced in fetuses of mothers with maternal subclinical hypothyroidism. These puppies have worsening neurological development, with deficits in long and short term memory. Here we evaluated whether maternal obesity may alter levels of BDNF and enzyme expression of type III deiodinase (D3) in the brains of pups, with consequent alteration of local levels of T3 to 7 °, 10 ° and 16 ° day-old post -natal. Our results showed that obesity reduced the expression of D3 on the 7th day of postnatal life of the offspring of obese mothers, but not in later days. There were no significant alterations in the levels of BDNF in any of the evaluated days. Our data suggest that it is possible that thyroid hormone is involved in neurophysiological abnormalities observed in offspring of obese rats. / A obesidade é uma epidemia de ordem mundial que é fator de risco para o desenvolvimento de hipertensão arterial, dislipidemia, hiperglicemia, diabetes tipo 2 e esteatose hepática. O aumento de obesidade em gestantes além de aumentar o risco do desenvolvimento de doenças cardiovasculares, também pode estar relacionado com anomalias no desenvolvimento do Sistema Nervoso Central dos embriões como, por exemplo, redução dos potenciais de longa duração (LTP) e da neurogênese no hipocampo. É possível que os níveis reduzidos de BDNF observados nestes embriões, estejam envolvidos com o prejuízo nos processos de aprendizado e memória exibidos por esses animais. Estudos também mostram que o BDNF se encontra reduzidos em fetos de mães com hipotiroidismo subclínico materno. Estes filhotes apresentam piora no desenvolvimento neurológico, demonstrando déficits na memória de longo e de curto prazo. O presente avaliou se a obesidade materna pode alterar os níveis de BDNF e a expressão da enzima desiodase do tipo III (D3) no cérebro dos filhotes, com consequente alteração dos níveis locais de T3 ao 7°, 10° e 16° dias de vida pós-natal. Os nossos resultados mostraram que a obesidade reduziu a expressão da D3 no 7º dia de vida pós-natal dos filhotes de mães obesas, mas não nos dias posteriores, sem alteração significativa nos níveis de BDNF. Não houve alteração significativa nos níveis de BDNF em nenhum dos dias avaliados. Os nossos dados sugerem que é possível que o hormônio tiroideano esteja envolvido nas alterações neurofisiológicas observadas em filhotes de ratas obesas.

Made available in DSpace on 2016-03-15T19:40:17Z (GMT). No. of bitstreams: 1 Felipe Rodrigues Pinto.pdf: 488990 bytes, checksum: 8735cc0097d83b1a36638c054be80716 (MD5) Previous issue date: 2014-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The prevalence and incidence of obesity has increased in the western society and can be partially explained by the consumption of a high fat and sugar diet combined with a sedentary lifestyle. Obesity can lead to a condition known as Metabolic Syndrome, characterized by hyperglycemia, dyslipidemia, hypertension and insulin resistance. Furthermore, studies show that mice born from obese mothers have deficits in learning and memory. Those deficits may be caused by alteration in the production of neurotrophins, essential in the formation of the long term potentiation which is the base of the consolidation of long term memory. Hypothyroidism also affects the development of the Central Nervous System in the offspring and reduces cognitive performance in adults. Serum levels of thyroid hormones are constant and its availability for cells is dependent on the activity of deiodinases which may activate (D2) or inactivate (D3) the T4 into T3 or T3r, respectively. Moreover, neurotrophins that are kwon to be decreased in the brain of obese mothers also are positively regulated by the T3. This study tested the hypothesis that the maternal obesity leads to alteration in the expression of D3 in the offspring hippocampus. Even though the test used in this study has shown no prejudice in memory and learning in the offspring of obese mothers, a lower expression of D3 in the hippocampus was observed. Thus, is possible to conclude that the maternal obesity alters the thyroid hormone signaling in the offspring hippocampus. / A prevalência e a incidência da obesidade têm aumentado na sociedade ocidental e pode ser explicada em parte pelo consumo de uma dieta rica em gordura e carboidratos aliado à vida sedentária. A obesidade pode levar a uma condição conhecida como Síndrome Metabólica, que se caracteriza pela hiperglicemia, dislipidemia, hipertensão e resistência à insulina. Além disso, estudos mostram que camundongos nascidos de mães obesas apresentam déficits de aprendizado e memória. Esses déficits podem ser causados por alterações na produção de neurotrofinas, essenciais para a formação da potenciação de longa duração que é à base da consolidação da memória de longo prazo. O hipotireoidismo também afeta o desenvolvimento do SNC dos filhotes e piora o desempenho cognitivo de indivíduos adultos. Os níveis séricos de hormônio tiroidiano permanecem constantes e a sua disponibilidade para as células depende da atividade das desiodases que ativam, por meio da desiodase 2 (D2) ou inativam, por meio da desiodase 3 (D3) o T4 a T3 ou a T3r, respectivamente. Além disso, as neurotrofinas, que reconhecidamente estão diminuídas no cérebro de filhotes de mães obesas, também são positivamente reguladas pelo T3. O presente estudo testou a hipótese de que a obesidade materna leva à alteração na expressão da D3 em hipocampo dos filhotes. Nossos estudos mostraram que, embora os testes empregados não tenham evidenciado prejuízos na memória e aprendizado dos filhotes de mães obesas, observamos menor expressão da D3 no hipocampo. Assim, é possível concluir que a obesidade materna altera a sinalização do hormônio tireoidiano no hipocampo dos filhotes.

3-Jodthyronamin (3-T1AM) und Thyronamin (T0AM) sind endogene Signalmoleküle, die eine große strukturelle Ähnlichkeit zu Schilddrüsenhormonen aufweisen, allerdings die klassischen Wirkungen des aktiven Schilddrüsenhormons 3,5,3’-Trijodthyronin (T3) antagonisieren. In der vorliegenden Arbeit wurde untersucht, ob Thyronamine (TAMs) Substrate von Dejodasen (Dio1, Dio2, Dio3) sind. Die TAMs wurden mit isozymspezifischen Dio-Präparationen inkubiert. Die Dejodierungsprodukte wurden mittels Hochleistungsflüssigkeitschromatographie und Tandemmassenspektrometrie (LC-MS/MS) analysiert. Mit Präparationen der Dio1 wurden Dejodierungen von 3,3’,5’-Trijodthyronamin, 3’,5’- und 3,3’-Dijodthyronamin am phenolischen Ring sowie Dejodierungen von 3,5,3’-Trijodthyronamin und 3,5-Dijodthyronamin am Tyrosylring beobachtet. Dio2 haltige Präparationen katalysierten ebenfalls Dejodierungen von 3,3’,5’-Trijodthyronamin und 3’,5’-Dijodthyronamin am phenolischen Ring. Mit Dio3 haltigen Präparationen wurden alle TAMs mit jodiertem Tyrosylring dejodiert. In Kompetitionsversuchen inhibierten ausschließlich die TAMs, die als Substrate von Dio Isozymen identifizierten wurden, eine etablierte Dejodierungsreaktion eines bekannten Substrats. Im Gegensatz dazu interferierten TAMs, die in den LC-MS/MS Experimenten als Substrate der Dio Isozyme ausgeschlossen wurden, nicht mit der genannten etablierten Dejodierungsreaktion. Zusammenfassend wurde in der vorliegenden Arbeit gezeigt, dass TAMs Substrate aller drei Dio Isozyme sind und jedes Isozym eine eigene Substratspezifität aufweist. Diese Befunde weisen darauf hin, dass Dio Isozyme an der Biosynthese von TAMs beteiligt sein könnten. Ferner wurden die Biosynthesewege für 3-T1AM und T0AM eingegrenzt. Desweiteren gestatten die Ergebnisse neue Einblicke in die generellen strukturellen Voraussetzungen für Dio Substrate, da TAMs die bisher einzigen endogenen Dio Substrate darstellen, deren Seitenkette am Tyrosylring eine positive Ladung aufweist. / 3-iodothyronamine (3-T1AM) and thyronamine (T0AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T3) actions. The present study investigated whether thyronamines (TAMs) are substrates of three Dio isozymes (Dio1, Dio2 and Dio3). TAMs were incubated with isozyme specific Dio preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3’,5’-triiodothyronamine, 3’,5’- and 3,3’-diiodothyronamine as well as tyrosyl ring deiodinations of 3,5,3’-triiodothyronamine and 3,5-diiodothyronamine were observed with preparations containing Dio1. Preparations of Dio2 also deiodinated 3,3’,5’-triiodothyronamine and 3’,5’-diiodothyronamine at the phenolic rings. All TAMs with tyrosyl ring iodine atoms were deiodinated by Dio3 containing preparations. In functional competition assays, the newly identified TAM substrates inhibited an established iodothyronine deiodination reaction. By contrast, TAMs which had been excluded as Dio substrates in LC-MS/MS experiments, failed to show any effect in the competition assays, thus verifying the former results. In summary, all three Dio isozymes catalyzed TAM deiodination reactions with each isozyme exhibiting a unique substrate specificity. These data support a role for Dio isozymes in TAM biosynthesis and contribute to confining the biosynthetic pathways of 3-T1AM and T0AM. Furthermore, they provide new insights into the structural requirements for Dio substrates in general since TAMs represent the only endogenous Dio substrates described, so far, which possess a positively charged tyrosyl ring side chain.

Thyroid hormones, which are secreted by the thyroid gland, are one of the most important halogenated compounds in the body. Thyroid hormones control almost every processes in the body including growth, body temperature, protein synthesis, carbohydrate and fat metabolism, heart rate, and cardiovascular, renal and brain function. Thyroid gland secretes L-thyroxine or 3,3',5,5'-tetraiodothyronine (T4) as a prohormone. While the biologically active hormone 3,3',5-triiodothyronine (T3) is produced by selective phenolic ring deiodination of T4, selective tyrosyl ring deiodination of T4 produces a biologically less active metabolite 3,3',5'-triiodothyronine (rT3). Tyrosyl and phenolic ring deiodination of T3 and rT3, respectively, also produces a biologically inactive metabolite 3,3'-diiodothyronine (3,3'-T2). Regioselective deiodinations of thyroid hormones are catalysed by three isoforms of a selenoenzyme iodothyronine deiodinase (DIO1, DIO2, DIO3). DIO1 can remove iodine from both the tyrosyl and phenolic rings of thyroid hormones, whereas DIO2 and DIO3 are selective towards phenolic and tyrosyl ring, respectively. Although the Figure 1. (A) Deiodination of thyroid hormones by iodothyronine deiodinases (DIOs) (A) and naphthyl-based selenium and/or sulphur compounds (B). mystery behind the origin of regioselectivity of deiodination by DIOs remains unsolved, formation of halogen bonding between selenium in the active site of DIOs and iodine of thyroid hormones has been widely accepted as the mechanism of deiodination. Halogen bonding, a noncovalent interaction between halogen and an electron donor such as nitrogen, oxygen, sulphur, selenium etc., elongates the C-I bond and impart a carbanionic character on the carbon atom that gets protonated after the removal of iodide. Apart from the deiodination, thyroid hormones also undergo decarboxylation, oxidative deamination, sulphate-conjugation to form iodothyronamines, iodothyroaetic acids and sulphated thyroid hormones, respectively. Figure 2. (A) Proposed mechanism of deiodination of thyroid hormones by deiodinase mimics. (B) Halogenation of uracil- and cytosine-containing nucleosides by hypohalous acid (HOX). Recently, naphthyl-based selenium/sulphur-containing compounds, such as compound 1 (Figure 1B), have been reported to mediate the selective tyrosyl ring deiodination of T4 and T3 to form rT3 and 3,3'-T2, respectively. Interestingly, replacement of the selenol moiety in compound 1 with a thiol decreases the activity, whereas replacement of the thiol moiety with another selenol dramatically increases the deiodination activity. Based on the detailed experimental and theoretical investigations, a mechanism involving the Se···I halogen bonding was proposed (Figure 2A). In addition to the halogen bonding between selenium and iodine atom, chalcogen bonding between two nearby chalcogen atoms was also shown to be important for the deiodination activity. Another important class of halogenated compounds in the body are the halogenated nucleosides. Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes, which can convert halide ions (X¯) into a toxic reactive halogen species hypohalous acid (HOX) in presence of hydrogen peroxide (H2O2). Uracil- and cytosine-containing nucleosides are known to undergo halogenation at the 5-position of the nucleobase to form the halogenated nucleosides (Figure 2B). Interestingly, halogenated nucleosides such as 5-halo-2'-deoxyuridine are known to be incorporated in the DNA of dividing cells essentially substituting for thymidine. Incorporation of halogenated nucleosides into the DNA leads to mutagenesis, carcinogenesis and loss of genome integrity. Thymidylate synthase (TSase), the key enzyme involved in the biosynthesis of 2'-deoxythmidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), can catalyse the dehalogenation of halogenated nucleotides in presence of external thiols. This thesis consists of five chapters. The first chapter provides a general introduction to halogen bonding, thyroid hormones and halogenated nucleosides. This chapter also briefly describes the halogen bond-mediated biochemical and biomimetic deiodinations of thyroid hormones by iodothyronine deiodinases and naphthyl-based organoselenium compounds. Dehalogenation of halogenated nucleotides by thymidylate synthase and thiol-based small molecules has also been discussed in this chapter. The second chapter of this thesis contains the regioselective deiodination of iodothyronamines (TAMs) by deiodinases mimics. TAMs are the endogenous metabolites produced by the decarboxylation of β-alanine side chain of thyroid hormones (THs). 3,3',5-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) undergoes selective tyrosyl ring deiodination by deiodinase mimics to form 3,3'-diiodothyronamine (3,3'-T2AM) and 3-iodothyronamine (3-T1AM), respectively. Interestingly, when the initial rates of deiodinations of T3 and T3AM were compared, deiodination of T3 was found to be several fold faster than that of T3AM under identical reaction conditions. To understand the ability of the iodine atoms to form Figure 3. (A) HPLC chromatogram of deiodination of T3. (B) Proposed mode of interaction of dimeric T3 and monomeric T3AM with organoselenium compounds. halogen bonding, a model selenolate (MeSe¯) was optimized with the T3 and T3AM. Although both T3 and T3AM forms the expected Se···I halogen bonding with MeSe¯, the strength of halogen bonding was found to be less for T3AM than T3. Furthermore, detailed kinetic and spectroscopic studies indicate that T3 and T3AM exist as dimeric and monomeric species in solution. The dimerization of T3 in solution was shown to have remarkable impact on the activation energy and pre-exponential factor of the deiodination reactions. Single crystal X-Ray crystallography and theoretical calculations indicated that in addition to Se···I halogen bonding, I···I halogen bonding may play an important role in the deodination of thyroid hormones by deiodinase mimics. Furthermore, the presence of heteroatoms such as nitrogen, oxygen and sulphur in the close proximity of one of the selenium atoms of deiodinase mimics was shown to have significant effect on the rate of deiodination reactions. The third chapter of the thesis focusses on the conformational polymorphism and conformation-dependent halogen bonding of L-thyroxine. Synthetic version of L-thyroxine (T4) is a life-saver for millions of people who are suffering from hypothyroidism, a thyroidal disorder recognised by low levels of T4 and elevated levels of TSH in blood plasma. Synthetic version of L-thyroxine is available in the Figure 4. Ball and stick model of the single crystal X-Ray structure of the conformational polymorphs of L-thyroxine. Form I and Form II was exclusively crystallized from methanol and acetonitrile, respectively. Water molecules are omitted for clarity. market with various brand names. However, adverse effects have been observed in the patients when they switch their brand of thyroxine. Based on these observations, the American Thyroid Association (ATA), the Endocrine Society (TES), and the American Association of Clinical Endocrinologists (AACE) declared that the different brands of T4 are not bioequivalent, thus leading to differences in the bioavailability of the drug. We have shown that the commercially available thyroxine exists in at least two stable forms (Form I and Form II) with different three-dimensional structures (Figure 4). These two forms exhibit different intermolecular interactions in crystal packing, spectral behaviours, thermal stabilities, optical activity and very interestingly, different solubility in acidic and basic pH. At pH 4, solubility of Form I is about 42% and 45% greater than that of Form II and bulk T4, respectively, whereas at pH 9, the solubility of Form II is about 38% and 42% higher than that of Form I and bulk T4, respectively. As T4 is a narrow therapeutic index drug, these differences in solubility may have remarkable impact on the bioavailability of the drug. In addition to this, we have shown that the ability of the iodine atoms in the C-I bonds to form halogen bond with donor atoms can be altered by changing the relative orientation of tyrosyl and phenolic rings in T4. In the fourth chapter, the three-dimensional structures and conformations of thyroid hormones (THs) and iodothyronamines (TAMs) are discussed. TAMs, the endogenous decarboxylated metabolites of THs, exhibit different binding affinities to the transport proteins and iodothyronine deiodinases (DIOs) compared to the THs. Figure 5. Change in the structure and conformations of thyroid hormones and iodothyronamines with the decarboxylation of amino acid side chain and deiodination of phenolic and tyrosyl ring. Furthermore, the substrate specificities of DIOs have been found to be dependent on the position of iodine atoms on the phenolic and tyrosyl ring of TAMs and THs. Single crystal X-ray structures of TAMs indicate that decarboxylation of amino acid side chain of THs induces significant changes in the structure and conformation. Furthermore, the positional isomers of THs and TAMs exhibit remarkably different conformations, which may have significant effect on the binding of these metabolites to the active site of DIOs. In addition to the structure and conformations, different categories of the intermolecular halogen···halogen (X···X) interactions in the crystal packing of THs and TAMs have also been discussed. Natural bond orbital (NBO) analysis have been done on the halogen-bonded geometries to understand the electronic nature of these interactions. In the fifth chapter, the dehalogenation of halogenated nucleosides and nucleobases by naphthyl-based sulphur/selenium compounds is discussed. Purine and pyrimidine nucleosides are halogenated at various positions of the aromatic ring by different peroxidases such as myeloperoxidase and eosinophil peroxidase present in the white blood cells. Incorporation of the halogenated nucleosides into the DNA of replicating cells leads to DNA-strand breaks, mutagenesis, carcinogenesis and loss of Figure 6. (A) Dehalogenation of halogenated nucleosides. Effect of base-pairing wih adenine and guanine on the deiodination of IU (B) and debromination of BrU (C) by compound 2. genome integrity. We have shown that the naphthalene-based organoselenium compounds such as compound 2 can mediate the dehalogenation of 5-iodo-2'-deoxyuridine (5-IdUd) and 5-bromo-2'-deoxyuridine (5-BrdUd) to produce 2'-deoxyuridine (dUd) (Figure 6A). The deiodination of 5-IdUd was found to be faster than the debromination of 5-BrdUd by compound 2. The mechanism of dehalogenation of halogenated nucleosides by compound 2 was found to be dependent on the nature of halogen. While the deiodination of 5-IdUd by compound 2 follow halogen bond-mediated pathway like thyroid hormones, debromination of 5-BrdUd follow a Michael addition-elimination pathway. Similar results were obtained when 5-iodo-2'-deoxycytidine (5-IdCd) or 5-bromo-2'-deoxycytidine (5-BrdCd) was used as substrate for dehalogenation reaction. Base-pairing of 5-iodouracil (IU) and 5-bromouracil (5-BrU) with adenine and guanine has a significant effect on the rate of dehalogenations of IU and BrU by compound 2 (Figure 6B and 6C).