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Title: Simple and double conjugates of peptides and oligonucleotides
Author: Agramunt Pi, Jordi
Director/Tutor: Grandas Sagarra, Anna
Keywords: Pèptids
Reaccions químiques
Chemical reactions
Issue Date: 8-Nov-2019
Publisher: Universitat de Barcelona
Abstract: [eng] 2,2-Disubstituted cyclopent-4-ene-1,3-diones (CPDs), which have been as non-hydrolysable maleimide analogs, have been found to possess an unexpected reactivity with N-terminal cysteines. Whilst maleimides react in an irreversible manner with all types of thiol nucleophiles, the Michael-type addition between CPDs and N-terminal cysteines, furnish a stable product with a mass 20 Da lower than the expected Michael-type adduct. This newly found reactivity can be applied for different purposes. In first instance, CPD-derivatized peptides can be used to synthesize conjugates by reaction with cystine-derivatized biomolecules such as peptides, peptide nucleic acids (PNAs) or oligonucleotides. In addition, the combination of the CPD-Cys reaction with other “click” reactions has been explored. In the first instance, the CPD-Cys reaction was paired the thia-Michael addition of thiols to maleimides using peptides containing two cysteines (one at the N-terminus). By first performing a CPD-Cys reaction at the N-terminus and then the addition of a maleimide, double conjugation could be easily achieved. In another case, combinations with oxime ligation and copper(I) catalyzed azide-alkyne (CuAAC) cycloadditions have been accomplished with a different degree of success. In the latter case, the CPD-Cys reaction has to be strictly performed before the CuAAC. In a further examination why this was the case, it was found out that CPDs react with azides to furnish two stable compounds one is the product of a 1,3-dipolar cycloaddition and the other results from nitrogen loss. Secondly, the bioconjugation of a splice-switching enhancing molecule known as “Retro-1” to an oligonucleotide with splice-switching activity described as 623 has been explored. In this part of the work, the complete synthesis of described Retro-1 has been accomplished in addition to that of other several analogues containing either a 1,3-diene, a thiol or a phosphoramidite group appending from two positions, nitrogen 4 or carbon 3. For this purpose, two synthetic strategies were followed one making use of valuable intermediates obtained from the Retro-1 synthesis and the other utilizing a properly protected lysine analogue to construct the Retro scaffold and introduce a reactive group at carbon 3. Additionally to all these reactants two oligonucleotides were synthesized: one with the 623 sequence and another with the same nucleobases but different order coined “scrambled”. Both oligonucleotides were appended at the 5’ position with a protected maleimide phosphoramidite (to obtain the corresponding maleimido-oligonucleotide and allow a Diels-Alder or thia-Michael addition to be performed) or the Retro-phosphoramidite. Finally, in this work the possibility of using 7-oxanorbornenes as dienophiles in the inverse electron-demanding Diels-Alder cycloaddition (IEDDA) with 3,6-disbustituted 1,2,4,5-tetrazines has been explored. The oxanorbornene moiety has been appended at the N-terminus and internal positions in peptides, and at the 5’ and 3’ positions of oligonucleotides utilizing solid-phase protocols. For the tetrazine counterpart optimization processes have been followed for the synthesis of N-terminal tetrazine peptides and for derivatization with biologically relevant molecules in reasonably good success. Once oxanorbornene- and tetrazine-containing derivatives were obtained it was observed that the IEDDA reaction took place satisfactorily, allowing for the synthesis of peptide and oligonucleotide conjugates with small molecules. In second term, the possibility of double conjugation involving at least one IEDDA reaction has been explored. The IEDDA has been combined with the Diels-Alder cycloaddition between a maleimide and a 1,3-diene, the thia-Michael between a thiol and a maleimide, the CPD-Cys reaction (utilizing CPDs and N-terminal cysteines as previously commented) and the aromatic nucleophilic substitution between a thiol and a chlorotetrazines. In every combination tested the double conjugate target compound was obtained, usually performing both reactions simultaneously, but sometimes in a “one-pot” fashion, with the sequential addition of reactants due to side reactions. In one case, it was necessary to isolate the intermediate monoconjugate because of difficulties related to undesired interferences between reactants.
[spa] En la primera parte de la tesis se ha trabajado con la reacción de conjugación que tiene lugar entre una ciclopentenodiona 2,2-disustituída (CPD) y una cisteína con el grupo amino libre, que proporciona un aducto bicíclico estable. Se han preparado CPD-poliamidas (péptido, PNA) y CPDs para reacciones de conjugación, así como varios conjugados. Se han obtenido también conjugados de oligonucleótidos tras derivatizar cadenas oligonucleotídicas con residuos de cisteína. La reacción de conjugación CPD-Cys se ha utilizado también satisfactoriamente para preparar dobles conjugados, combinándola bien con la reacción tia-Michael bien con la cicloaddició azida-alquino catalizada por Cu(I). La posibilidad de combinarla con la formación de oximas no ha tenido suficiente éxito. En la segunda parte del trabajo se ha querido averiguar si la unión covalente de una molécula (Retro-1) que facilita la internalización de oligonucleótidos a una cadena oligonucleotídica con potencial terapéutico tiene un efecto superior a la utilización de los dos componentes por separado. Para ello se han preparado varios derivados de Retro-1 y se han unido a diferentes cadenas oligonucleotídicas utilizando diferentes métodos de conjugación. Los ensayos biológicos realizados por el Prof. R. L. Juliano (Universidad de Carolina del Norte en Chapel Hill) han mostrado resultados diferentes en función del tipo de células utilizado, lo cual pone de manifiesto la dificultad de extrapolar resultados in vitro a experimentos más complejos. En la tercera y última parte del trabajo se han explorado las aplicaciones del uso de 7-oxanorbornenos como dienófilos en reacciones de Diels-Alder de demanda electrónica inversa con 1,2,4,5-tetrazinas 3,6-disustituídas. Una vez sintetizados los reactivos necesarios, tanto derivados de oxanorborneno como de tetrazina, se ha comprobado, en primer lugar, que la cicloaddició tiene lugar satisfactoriamente, permitiendo la síntesis de conjugados de péptidos y de oligonucleótidos. En segundo lugar, se han evaluado varias posibilidades para la preparación de dobles conjugados, combinando la reacción de Diels-Alder de demanda electrónica inversa con la reacción de tipo Michael entre una maleimida y un tiol, la reacción CPD-Cys, la reacción de Diels-Alder y la sustitución nucleófila aromática entre un tiol y una clorotetrazina. Todas las combinaciones han permitido obtener el doble conjugado deseado, en unos casos llevando a cabo las dos reacciones de conjugación simultáneamente, en otros a través de un proceso "one-pot" y en otros ha sido necesario aislar el monoconjugado intermedio.
Appears in Collections:Tesis Doctorals - Departament - Química Inorgànica i Orgànica

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