Principio de las 3 R´S

All the TG analogs studied in this work were synthesized by Solid Phase Peptide Synthesis (SPPS). SPPS was developed in the middle 60s by Merrifield [136-139] and it brought a significant improvement with respect to the synthesis in solution, leading to the win of the Noble Prize in 1985 [140]. In the SPPS, the C-terminal amino-acid of the desired peptide is covalently linked to an insoluble resin, from which it is cleaved only when the synthesis is completed. The solid support allows to use reagents in large excess to drive the reactions to completion and to wash the resin effectively, obtaining the final peptide in a relatively pure form. The SPPS can also be conducted as an automated synthesis, further reducing the synthetic time and solvent consumption. The general process to synthesize peptides on a resin is schematized in Figure 3.3. It starts by loading the resin with the C-terminal amino-acid. The residue, as well as all the other residues that will be added in the subsequent passages, are protected at the alpha amino group with a protecting group, to avoid polymerization. Moreover, also the reactive side chains are kept protected with an orthogonal protecting group until the synthesis is complete. The next step is the removal of the protecting group at the N-terminal; then, the next amino-acid, protected at the N-alpha, is coupled to the N-terminal amino-acid on the resin. After the coupling, it is possible to remove the protection at the new N-terminal residue and repeat the deprotection-coupling cycle until the sequence is complete. All the protecting groups at the side chains are removed in one

Figure 3.3 Schematic representation of the SPPS process.

linker O C O H C R' H N Fmoc Deprotection linker O C O H C R' NH2 Coupling O C O H C R'' H N Fmoc linker O C O H C R' NH O C O H C R'' H N Fmoc Deprotection Cleavage Peptide

41 Figure 3.4 2-chlorotrytil resin linked to Leucinol.

Figure 3.5 Mechanism of the coupling reaction. After deprotonation by means of a base, the deprotonated, protected, residue is activated by reacting with HBTU that induces a favorable orientation of the growing peptide for the coupling with the desired residues thanks to a hydrogen bond with a nitrogen on the HBTU ring. The HATU ring has an additional nitrogen that enables one additional coupling geometry.

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step before the peptide is cleaved from the resin. For the synthesis of the TG analogs, a protocol was developed and optimized [2,141] in the research group of Prof. C. Toniolo and Prof. F. Formaggio, University of Padova. The used resin is a styrene copolymer containing 1-2% of divynilbenzene, acting as a cross-linker. The C-terminal is separated from the resin by 2-chlorotrytil [142-144], that is optimal for the synthesis of TOAC-labeled peptides since it is removable in mild acidic conditions (see below). The Lol residue is already linked to the resin (Figure 3.4). The coupling procedure involves the activation of the N _{protected amino-acid via the uronium salt HBTU ([2-} (1H-1,2,3-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate]) for the standard residues, while for the less reactive C-tetrasubstituted residues (Aib, TOAC, Api) it is required a strong activant [145,146], i.e. HATU ([2-(1H-7-aza-1,2,3- benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate]). Figure 3.5 schematizes the reaction mechanism for both activation pathways [147-149], that enhance the electrophilic propensity of the carbonyl group; an activated ester is formed, that reacts quickly with the N-terminal of the peptide. Both HBTU and HATU lead to the formation of the ester that perform its catalytic effect promoting an optimal geometry between the peptide on the resin and the coupling amino-acid. As shown in the figure, HATU has a higher activating capability since it has two nitrogens that can form hydrogen bonds with the N-terminus of the peptide, favoring the attack of the new residue. After the coupling, the Fmoc (9-fluorenylmethyloxycarbonyl) group that protects the N-terminal part of the peptide is removed with a basic treatment with a secondary amine that attacks the acidic proton of the fluorenyl ring. The chosen base is piperidine, since it has the double purpose: it removes the Fmoc group from the peptide sequence via a -elimination and it scavenges the byproduct dibenzofulvene that would form a covalent bond with the N-terminal part of the peptide if not readily removed from the solution. The -elimination reaction is schematized in Figure 3.6. At the end of the coupling steps, the completed peptide is cleaved from the resin in mild acidic conditions using HFIP (1,1,1,3,3,3,-hexafluoropropan-2-ol), that are appropriate to preserve the radical function of the paramagnetic amino acid TOAC, present in most of the peptide sequences. The mechanism of reaction involved in the resin cleavage is shown in Figure 3.7. However, some amino-acids had reactive side-chains that were

Figure 3.6 Mechanism of the elimination reaction.

O HN Peptide H O H N O HN Peptide O H2 N H2N-Peptide

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protected during the coupling reactions. In particular, Lys and Api side-chains were protected with Boc (t-Butyloxycarbonyl) group and Arg was protected with Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl), see Figure 3.8. All these protecting groups are acid labile, so it was chosen to remove them as a single step with the cleavage of the peptide from the resin. This required stronger acidic conditions (TFA or HCl) that cause the partial TOAC disproportion to an oxoammonium cation and hydroxylamine [150] (see figure 3.9). The regeneration of the TOAC radical moiety was achieved with a basic treatment with ammonia and followed with reverse phase HPLC. All the peptides were, subsequently to the cleavage, purified by reverse phase liquid chromatography and characterized by ESI-MS and, for peptides without the TOAC residue, 1_{H-NMR. All the peptides were purified until a purity >98% was}

obtained.

Figure 3.7 Mechanism of the reaction of cleavage of the peptide from the resin.

Resin O C H3 C H₃ Cl NH PEPTIDE n-Oct O H F₃C CF₃ O- F₃C CF₃ H+ + Leu NH PEPTIDE n-Oct _OH Resin OH+ C H₃ C H3 Cl NH PEPTIDE n-Oct

Figure 3.8 The two protecting groups Boc (left) e Pbf (right). O O S O O O

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3.3 Biological assays