Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/106623
Title: Novel methodologies for the conjugation and cyclisation of polyamides
Author: Brun Cubero, Omar
Director: Grandas Segarra, Anna
Keywords: Oligonucleòtids
Síntesi orgànica
Síntesi de pèptids
Biopolímers
Oligonucleotides
Organic synthesis
Peptide synthesis
Biopolymers
Issue Date: 27-Jul-2016
Publisher: Universitat de Barcelona
Abstract: [eng] An alternative allowing diene-derivatised polyamides to be used in Diels-Alder conjugation reactions has been explored and developed. In this methodology, conjugation takes place between a fully protected, resin-linked diene-derivatised polyamide and a soluble maleimide- containing compound. The acid-labile diene is thus not exposed to acids, and the acidic deprotection treatment that follows does not affect the cycloadduct. In contrast with previously described alternatives, this methodology has no limitations in terms of sequence and does not require special protecting groups. (E)-4,6-Heptadienoyl-polyamide-resins are used due to the good yields observed in the Diels- Alder reaction and the stability of the generated compounds. Polyamides are assembled using the standard, commercially available building blocks on water-swelling solid matrixes, and no special building blocks need to be synthesised. Both water and organic solvent/water mixtures can be used for the on-resin cycloaddition, and simultaneous maleimide deprotection and on- resin Diels-Alder cycloaddition is feasible, allowing conjugates with different linking sites to be prepared. The on-resin Diels-Alder reaction is also compatible with the wide-spread maleimide- thiol reaction, and combination of both transformations is feasible, making it possible to synthesise double conjugates. However, diene-derivatised oligonucleotides do not withstand the reaction conditions required for the solid-phase Diels-Alder conjugation, meaning that peptide- oligonucleotide conjugates cannot be prepared using this methodology. In a different project, 2,2-disubstituted cyclopent-4-ene-1,3-diones (CPDs), which were chosen as non-hydrolysable maleimide analogs, have been found to possess an unexpected reactivity. While maleimides react in an irreversible manner with all types of thiols, the Michael-type reaction between CPDs and cysteines placed at internal or C-terminal positions, which do not possess a free amine, is reversible. In contrast, cysteines with a free amine (at the N-terminus of peptides) react with CPDs to end up furnishing a stable product with a mass 20 Da lower (M- 20 Da) than the Michael-type adduct (weighing M Da). Formation of the M-20 Da adduct plausibly takes place through the following steps: First, conjugate addition of the N-terminal cysteine thiol to the CPD double bond yields a Michael-type adduct. This product immediately undergoes intramolecular imine formation, by reaction between one of the CPD keto groups and the N-terminal amine, giving an intermediate with two fused rings and a mass 18 Da lower (M- 18 Da) than the initially formed Michael-type adduct. Subsequent oxidation of this M-18 Da adduct provides the final, stable M-20 Da adduct, which absorbs around 330 nm and whose structure has been confirmed by NMR. This newly found reactivity can be applied for different purposes. Firstly, CPD-derivatised peptides can be used to synthesise conjugates by reaction with cysteine-derivatised PNAs. Secondly, the different reactivity of CPDs towards 1,2-aminothiols and other thiols has been exploited to selectively tag a peptide containing an N-terminal cysteine in the presence of peptides with cysteines in other positions, and to double-derivatise a peptide that contains an N- terminal and an internal cysteine. Use of CPDs with this last type of peptides also allows for their simultaneous cyclisation and derivatisation. Formation of the CPD-M-20 Da adduct is followed by Michael-type addition of the internal thiol to this adduct, and finally by oxidation to yield a conjugated system absorbing around 370 nm. Cyclisation has been assessed by NMR experiments. The cyclisation reaction is accelerated by the presence of oxidants, namely O2 or TEMPO, and the addition of LiCl also has a beneficial effect, in particular with difficult to cyclise sequences. In this respect, it has been found that aromatic residues proximal to the N-terminal cysteine hinder the cyclisation. The N-terminal cysteine of CPD-cyclised peptides undergoes epimerisation at the α carbon when exposed to bases, as confirmed by synthesis of the cyclic analogs from peptides with a D- cysteine at the N-terminus. Curiously, the latter are less prone to epimerise than the L-cysteine counterparts, for which reason use of the non-proteinogenic residue is recommended.
[spa] En esta tesis se ha llevado a cabo el desarrollo de nuevas metodologías para la conjugación y ciclación de péptidos y otras poliamidas. En un primer proyecto, se buscó y encontró una alternativa que permitiera la conjugación de poliamidas derivatizadas con un 1,3-dieno generadas por síntesis en fase sólida, evitando la descomposición del dieno durante la desprotección final. Para ello se puso a punto una metodología que permitió conjugar, a través de una reacción de Diels-Alder, dichas poliamidas mientras se aún se encontraban protegidas y unidas a resina. Como dienófilos se usaron derivados de maleimida, debido a su fácil obtención, buena reactividad y su disponibilidad comercial. La reacción de Diels-Alder sobre resina se efectuó tanto en agua como en mezclas agua/disolvente orgánico, y es compatible con el uso de maleimidas protegidas y con la reacción de tipo Michael entre un tiol y una maleimida. En un proyecto distinto, se usaron ciclopent-4-en-1,3-dionas 2,2-disustituidas (CPDs) como análogos no hidrolizables de maleimida. Pronto se descubrió que su reactividad es muy distinta a la de las maleimidas, y que, mientras que su reacción con cisteínas internas o C-terminales es reversible, cuando reaccionan con cisteínas N-terminales se genera un producto estable. Éste tiene una masa 20 unidades menor (M-20 Da) que el producto de tipo Michael inicialmente esperado (M Da) un máximo de absorción alrededor de 330 nm. Esta distinta reactividad se ha usado tanto para la formación de conjugados como para la diferenciación de péptidos que poseen cisteína en la posición N-terminal frente a péptidos que la contienen en otras posiciones. Además, las CPDs también se pueden usar para conseguir la ciclación y derivatización simultánea de péptidos que contienen dos cisteínas, una de las cuales se encuentra en la posición N-terminal. En este caso se obtienen derivados cíclicos de masa M-22 Da con un máximo de absorción alrededor de 370 nm. Se recomienda el uso de D-cisteína en la posición N-terminal debido a su mayor estabilidad frente a la epimerización.
URI: http://hdl.handle.net/2445/106623
Appears in Collections:Tesis Doctorals - Departament - Química Inorgànica i Orgànica

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