The opioid system is involved in the modulation of multiple biological functions, above all pain behavior. It consists of four different families of opioid receptors known as MOP, KOP, DOP and NOP. All of these are Gi/G0 protein coupled receptors expressed throughout the spinal axis and pain related pathways. The activation of these receptors induces cell hyperpolarization and ion channels opening, regulating the release of second messengers and promoting signaling cascades which converge to cell desensitization, analgesia. The most potent analgesic effect is known to be exerted by the activation of MOP receptor, unfortunately it is associated to important side effects which represent the major limitation for their clinical use, such as constipation, cough, itch, respiratory depression, tolerance and addiction. While the co-activation of different receptor subtypes is emerging as a safer therapeutic strategy to promote analgesia. The simultaneous activation of MOP and NOP receptors has been suggested as an interesting pharmacological approach to treat pain with potentially reduced side effects. Dual MOP/NOP ligands have been obtained through subtle changes of the N-terminal part of Nociceptin/Orphanin FQ (N/OFQ). The most important substitution which confers such interesting behavior is the replacement of the phenylalanine residue at the N-terminal position (Phe1) of N/OFQ with the non-canonical amino acid 2,6-dimethyl tyrosine (Dmt). Of note, some Dmt-modified opioid peptides exhibited increased biological activity and extended half-life with unexpected affinity and selectivity patterns. This thesis has been focused on the development of different synthetic strategies for the synthesis of Dmt-like analogues. In particular, we were able to functionalize the 4-position of the phenyl ring of the target residues after an ortho-ortho C(sp2)-H alkylation reaction. Some of these analogues were successfully employed as building blocks in solid phase peptide synthesis (SPPS) for the synthesis of N/OFQ(1-13)-NH2 derivatives modified at the message domain. Moreover, Pd-catalyzed C(sp2)-H activation was successfully applied for the synthesis of the non-natural amino acid mono methyl tyrosine (Mmt) in which 3-nitro-L-tyrosine was employed as starting material. Another Pd-catalyzed synthesis here described is focused on a beta C(sp3)-H activation of a properly functionalized alanine precursor which led to the obtainment of 3,5-Dmt, as well as bis-arylated analogues of Tyr and 3,5-Dmt. This thesis work includes a strategy for assembling bifunctional (NOP/MOP) heterotetrabranched ligands. Peptide clustering is emerging as an innovative tool to enhance the pharmacokinetic profile of therapeutic peptides, particularly by increasing their half-life. Specifically, we synthesized the heteromultimeric peptide conjugate H-PWT1-N/OFQ-[Dmt1]dermorphin (Scheme 4) to target NOP and MOP opioid receptors, observing similar and high agonist potency compared to the parent peptides. The synthetic approach is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity could be combined to generate dual acting multimeric compounds. Finally, with the aim to identify novel mixed NOP/opioid receptor peptide agonists, Dmt was employed as Fmoc-precursor in SPPS to build a series of linear peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2. The best results in terms of NOP versus MOP opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/MOP receptor peptide agonist so far described in literature.

Il sistema oppioide è coinvolto in molteplici funzioni biologiche, tra queste la principale è la modulazione del dolore. Il sistema oppioide si compone di quattro diverse famiglie di recettori: MOP, KOP, DOP e NOP. Tutti i recettori oppioidi appartengono alla famiglia delle proteine G accoppiate a recettore, Gi/G0. I ligandi endogeni dei recettori sono i peptidi oppioidi endogeni, che legatisi ai recettori, inducono iperpolarizzazione cellulare e apertura di canali ionici, e regolando il rilascio di secondi messaggeri, promuovono cascate di segnalazione volte allo spegnimento del firing cellulare. L’attivazione dei recettori MOP comporta l’espressione del maggiore effetto analgesico, associato però all’insorgenza di una serie di effetti collaterali, tra i quali costipazione, tosse, depressione respiratoria, dipendenza e tolleranza, che ne limitano l’uso in ambito clinico. Sebbene l’attivazione del solo recettore MOP sia accompagnata dal manifestarsi dei suddetti effetti collaterali, la co-attivazione di diversi recettori tende a ridimensionare l’insorgenza degli effetti indesiderati. In particolare, l’attivazione dei recettori MOP/NOP sembra rappresentare al meglio tale profilo terapeutico. Ligandi bivalenti, a target MOP/NOP sono stati ottenuti modificando il peptide endogeno Nocicettina (N/OFQ), effettuando delle sostituzioni nella porzione N-terminale. La modifica più significativa, è rappresentata dalla sostituzione del residuo Phe1 con il derivato amminoacidico non-naturale 2,6-dimetil tirosina (Dmt). Alcuni peptidi aventi il residuo Dmt incorporato in struttura, mostrano maggiore attività biologica, tempo di emivita, e particolari caratteristiche di affinità e selettività recettoriali rispetto ai peptidi di riferimento. Questo lavoro di tesi, si propone di sviluppare diverse strategie di sintesi per ottenere derivati amminoacidici Dmt-like. In particolare, tramite reazioni di C(sp2)-H attivazione Pd-catalizzate, è stato possibile ottenere lo scaffold Dmt-like desiderato, funzionalizzando successivamente la posizione 4 dell’anello aromatico. Alcuni di questi analoghi sono stati inseriti con successo nel message domain di N/OFQ(1-13)-NH2, tramite sintesi peptidica in fase solida (SPPS). Una simile strategia sintetica ha permesso di ottenere il derivato noto come monometil-tirosina (Mmt), partendo dalla 3-nitro-L-tirosina, tramite controllo regio-selettivo della reazione di C(sp2)-H attivazione Pd-catalizzata. Inoltre, per reazioni di C(sp3)-H attivazione Pd-catalizzate, è stato possibile arilare L-Ala in posizione beta, ottenendo 3,5-Dmt (isomero costituzionale di Dmt) e i derivati di bis-arilazione di Tyr e di 3,5-Dmt. In questo lavoro di tesi è riportato anche l’ottenimento di un ligando bivalente MOP/NOP eterotetramerizzato. La sintesi di peptidi tetramerizzati si propone come strategia innovativa per migliorare il profilo farmacocinetico dei peptidi ad uso terapeutico, aumentandone il tempo di emivita. Viene qui riportata la sintesi del derivato eterotetramerizzato H-PWT1- N/OFQ-[Dmt1]dermorfina selettivo per i recettori NOP e MOP, in grado di espletare un simile profilo agonista, rispetto ai peptidi di riferimento. Questa strategia sintetica si è dimostrata versatile e potenzialmente applicabile a qualsiasi sequenza peptidica, volta all’ottenimento di profili farmacologici eterotetramerizzati bivalenti. Con l’obiettivo di identificare nuovi profili peptidici con attività agonista, a target misto MOP/NOP, è stata utilizzato il residuo non-naturale Dmt, Fmoc-protetto, per sintetizzare una serie di peptidi di sequenza generica [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2. Il miglior risultato, in termini di potenza, espressa per entrambi i recettori, si è dimostrato essere il derivato caratterizzato dalla sostituzione con Dmt in ambedue i residui Tyr1 e Thr5, [Dmt1,5]N/OFQ(1-13)-NH2, ad oggi agonista, a target MOP/NOP, con maggiore attività biologica, in letteratura.

Exploring the potential of Pd-catalyzed C-H activation reaction for the synthesis of non-natural amino acid 2,6-dimethyl tyrosine-like (Dmt-like) analogues

ILLUMINATI, Davide
2022

Abstract

The opioid system is involved in the modulation of multiple biological functions, above all pain behavior. It consists of four different families of opioid receptors known as MOP, KOP, DOP and NOP. All of these are Gi/G0 protein coupled receptors expressed throughout the spinal axis and pain related pathways. The activation of these receptors induces cell hyperpolarization and ion channels opening, regulating the release of second messengers and promoting signaling cascades which converge to cell desensitization, analgesia. The most potent analgesic effect is known to be exerted by the activation of MOP receptor, unfortunately it is associated to important side effects which represent the major limitation for their clinical use, such as constipation, cough, itch, respiratory depression, tolerance and addiction. While the co-activation of different receptor subtypes is emerging as a safer therapeutic strategy to promote analgesia. The simultaneous activation of MOP and NOP receptors has been suggested as an interesting pharmacological approach to treat pain with potentially reduced side effects. Dual MOP/NOP ligands have been obtained through subtle changes of the N-terminal part of Nociceptin/Orphanin FQ (N/OFQ). The most important substitution which confers such interesting behavior is the replacement of the phenylalanine residue at the N-terminal position (Phe1) of N/OFQ with the non-canonical amino acid 2,6-dimethyl tyrosine (Dmt). Of note, some Dmt-modified opioid peptides exhibited increased biological activity and extended half-life with unexpected affinity and selectivity patterns. This thesis has been focused on the development of different synthetic strategies for the synthesis of Dmt-like analogues. In particular, we were able to functionalize the 4-position of the phenyl ring of the target residues after an ortho-ortho C(sp2)-H alkylation reaction. Some of these analogues were successfully employed as building blocks in solid phase peptide synthesis (SPPS) for the synthesis of N/OFQ(1-13)-NH2 derivatives modified at the message domain. Moreover, Pd-catalyzed C(sp2)-H activation was successfully applied for the synthesis of the non-natural amino acid mono methyl tyrosine (Mmt) in which 3-nitro-L-tyrosine was employed as starting material. Another Pd-catalyzed synthesis here described is focused on a beta C(sp3)-H activation of a properly functionalized alanine precursor which led to the obtainment of 3,5-Dmt, as well as bis-arylated analogues of Tyr and 3,5-Dmt. This thesis work includes a strategy for assembling bifunctional (NOP/MOP) heterotetrabranched ligands. Peptide clustering is emerging as an innovative tool to enhance the pharmacokinetic profile of therapeutic peptides, particularly by increasing their half-life. Specifically, we synthesized the heteromultimeric peptide conjugate H-PWT1-N/OFQ-[Dmt1]dermorphin (Scheme 4) to target NOP and MOP opioid receptors, observing similar and high agonist potency compared to the parent peptides. The synthetic approach is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity could be combined to generate dual acting multimeric compounds. Finally, with the aim to identify novel mixed NOP/opioid receptor peptide agonists, Dmt was employed as Fmoc-precursor in SPPS to build a series of linear peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2. The best results in terms of NOP versus MOP opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/MOP receptor peptide agonist so far described in literature.
TRAPELLA, Claudio
GUERRINI, Remo
CAVAZZINI, Alberto
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2478825
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