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Master's Dissertation
DOI
10.11606/D.60.2018.tde-27062018-105626
Document
Author
Full name
Michelle Ogava Igual
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Carvalho, Ivone (President)
Crotti, Antonio Eduardo Miller
Emery, Flavio da Silva
Kawano, Daniel Fabio
Title in Portuguese
Planejamento, síntese e avaliação biológica de novos inibidores seletivos da hidrolase O-GlcNAcase (OGA)
Keywords in Portuguese
Click chemistry; O-GlcNAcase; O-GlcNAcilação; OGA; Reações CuAAC
Abstract in Portuguese
O-GlcNAcilação, ou modificação por O-GlcNAc, consiste na glicosilação de proteínas envolvidas em processos celulares fundamentais, e a sua desregulação tem sido associada a importantes doenças tais como diabetes tipo 2, doenças neurodegenerativas, cardiovasculares e câncer. A O-GlcNAcilação é regulada por duas enzimas: O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA). Muitos inibidores da OGA descritos apresentam falta de seletividade entre a OGA e as isoenzimas Hex A e B lisossomais, o que pode ocasionar acúmulo de gangliosídeos no interior dos lisossomos e doenças neurodegenerativas. Desta forma, visando a obtenção de inibidores mais seletivos, foram propostos neste estudo a síntese e avaliação biológica de compostos derivados de N-acetilglicosamina, contendo na posição C-2 o anel 1,2,3-triazólico 1,4-di-substituído com diferentes cadeias laterais, na maioria contendo anéis aromáticos na sua extremidade, e a investigação da influência da extensão da cadeia entre os anéis na cavidade da OGA. Foram obtidos os derivados triazólicos 1, 2 (no estudo anterior) e 8a-j (no presente trabalho), em rendimentos bons a moderados, por meio da estratégia de click chemistry, envolvendo a reação de CuAAC entre o intermediário azido glicopiranosídeo 6, e dez diferentes alcinos, dos quais cinco foram previamente sintetizados (9 - 11, 14 e 15), e cinco disponíveis comercialmente. Para a síntese dos precursores acetilênicos 9 - 11, 14 e 15 foram utilizadas diferentes estratégias sintéticas de acordo com cada alcino, como, por exemplo, reações de substituição nucleofílica e aminação redutiva. Os derivados triazólicos foram obtidos como mistura de anômeros ?:? na proporção de 10:1, respectivamente. Devido à seletividade da enzima OGA apenas para substratos ?, os compostos 8a-h foram purificados por CLAE-DAD para separação das misturas. Posteriormente, foi possível realizar a cristalização do azido 6 em etanol, com consequente separação do anômero ? deste intermediário, o que auxiliou na etapa sintética dos derivados 8i e 8j como anômeros puros. Os ensaios de MTT não evidenciaram citotoxicidade para os compostos, avaliados em 1,0 ?M. Os ensaios de western blot e enzimáticos dos derivados 1, 2 e 8a-j demonstraram que apenas os compostos 1, 8i e 8j foram ativos para OGA, com valores de IC50 de 0,49, 0,52 e 0,72 ?M, respectivamente. A partir deste resultado, foi possível sugerir que a extensão ideal da cadeia ligada entre os anéis triazólico e aromático é de dois carbonos (1), podendo acomodar um heteroátomo de nitrogênio (8i) ou oxigênio (8j). Ademais, os ensaios enzimáticos dos compostos 1, 8i e 8j, avaliados para as Hex A e B, apresentaram IC50 de 550, 569 e 571 ?M, respectivamente, sugerindo alta seletividade destes compostos para a OGA em detrimento das isoenzimas lisossomais. Com o trabalho desenvolvido em parceria com a Dra. Nuria E. Campillo (CSIC) foram planejados novos derivados não-carboidratos capazes de realizar interações com importantes resíduos do sítio catalítico da OGA e com potencial atividade biológica.
Title in English
Design, synthesis and biological evaluation of novel selective inhibitors of the hidrolase O-GlcNAcase (OGA)
Keywords in English
Click chemistry; CuAAC reactions; O-GlcNAcase; O-GlcNAcylation; OGA
Abstract in English
O-GlcNAcylation, or O-GlcNAc modification, consists of the glycosylation of proteins involved in fundamental cellular processes, and its deregulation has been linked to important diseases such as type 2 diabetes, neurodegenerative, cardiovascular diseases, and cancer. O-GlcNAcylation is regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Many OGA inhibitors described exhibit lack of selectivity between OGA and the lysosomal isoenzymes Hex A and B, which can result in the accumulation of gangliosides within the lysosomes and neurodegenerative diseases. Therefore, aiming to obtain more selective inhibitors, this study proposed the synthesis and biological evaluation of N-acetylglucosamine derivatives, containing at the C-2 position the 1,4-di-substituted 1,2,3-triazole ring bearing different side chains, most of them containing aromatic ring at their end, and the investigation of the side chain extension influence between the rings into OGA active site pocket. It was obtained the triazole derivatives 1, 2 (in the previous study) and 8a-j (in the current project) in good to moderate yield through the click chemistry strategy, involving the CuAAC reaction between azide glucopyranoside intermediate 6 and ten different alkynes, which five were previously synthetized (9 - 11, 14 and 15), and five commercial available. For the synthesis of the acetylenic precursors 9 - 11, 14 and 15 it was employed different synthetic strategies according to each alkyne, for example nucleophilic substitution reactions and reductive amination. The triazole derivatives were obtained as a mixture of ?:? anomers in the proportion of 10:1, respectively. Due to the OGA selectivity to ? substrates, compounds 8a-h were purified by HPLC-DAD in order to separate the mixtures. Later in this study, it was possible to obtain the crystallization of azide 6 in ethanol, resulting in the separation of the ? anomer from this precursor, which assisted in the synthetic step of the derivatives 8i and 8j as pure anomers. The MTT assays did not show cytotoxicity for the synthesized compounds, evaluated at 1.0 ?M. The western blot and the enzymatic assays, evaluated for compounds 1, 2 and 8a-j, demonstrated that only compounds 1, 8i e 8j were active towards OGA, with IC50 values of 0.49, 0.52 e 0.72 ?M, respectively. From this result, it was possible to suggest that the ideal side chain extension linked between the triazole and aromatic rings is of two-carbon atoms (1), being able to accommodate one heteroatom of nitrogen (8i) or oxygen (8j). Furthermore, the enzymatic assays, evaluated for compounds 1, 8i e 8j against Hex A and B, exihibted IC50 of 550, 569 e 571 ?M, respectively, suggesting a high selectivity of these compounds to OGA rather than the two lysosomal isoenzymes. In the project developed in partnership with Dra. Nuria E. Campillo (CSIC) it was designed new non-carbohydrates compounds capable of interacting with important residues of the OGA catalytic site with potential biological activity.
 
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Release Date
2020-06-27
Publishing Date
2018-08-15
 
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