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T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes forT cells and anti-cancer immunotherapies.

[eng] In the present thesis we have investigated the development of new methodologies for the selective and straightforward chemical modification of Trp amino acid either alone or in peptides. In the first chapter, we have optimized a method for the chemoselective C-arylation of Trp amino acid in the C-2 position of the free indole ring, based on a C-H activation protocol catalyzed by palladium. To further demonstrate the versatility of the protocol, indoles from tryptamines, indole- carboxylic acids, Trp amino acid and Trp-containing peptides (e.g. Brevianamide F) were selectively arylated. In particular, the commercially available Fmoc-Trp-OH was arylated with a range of different aryl iodides with both electron-withdrawing and electron-donating substituents. It is noticeable that the location of the iodine atom can be programmed, giving rise to ortho, meta or para aryl derivatives. The direct incorporation of Fmoc-Trp(C2-Ar)-OH in solid-pahse peptide synthesis (SPPS), enables the straightforward preparation of Trp-arylated peptide sequences. As a proof of concept, we arylated an antimicrobial peptide derived from the fragment 107–115 of the C-terminus of the human lysozyme in order to enhance its biological activity. In the second chapter, we disclose the preparation of a fluorogenic BODIPY-Trp amino acid via the developed methodology for the straightforward synthesis of peptide-based imaging probes. Thus, this novel amino acid has been applied directly in SPPS to afford a BODIPY-antifungal PAF26 derivative for cell imaging fungal infections and a BODIPY-cyclic cLac mimic for cell imaging apoptosis. In the third chapter, we have applied the methodology disclosed in chapter 1 for the construction of biaryl peptidic topologies through Pd-catalyzed C-H activation reactions between Trp and I-Phe/I-Tyr residues. Thus, we have prepared stapled peptides, both in solution or in solid-phase, directly from linear precursors and we have determined the factors that control the cyclization/cyclodimerization outcome on these systems. Moreover, this C-H activation process enables the synthesis of macrocyclic conjugates and also bicyclic peptides generated from an internal stapling of a linear sequence and subsequent head-to-tail cyclization or from a double intramolecular C-H arylation within a linear peptide containing a commercially available diiodinated Tyr and two Trp units. These topologies could open the door to the access of simplified bismacrocyclic structural analogues of biologically relevant compounds in a straightforward manner from simple linear peptidic sequences prepared through SPPS out of commercially available amino acids. In the fourth chapter, we have developed a methodology based on cross-dehydrogenative processes for the selective formation of new C-C or C-N bonds in Trp-containing diketopiperazines with potential therapeutic interest. Specifically, we have got access to two different topologies depending on the oxidant source applied. [, [spa] En esta tesis se ha investigado el desarrollo de nuevas metodologías para la modificación post- sintética del aminoácido Trp. En el primer capítulo, se ha optimizado un método de arilación en C-2 del grupo indol, basado en un proceso de activación C-H catalizado por paladio. En especial, se ha arilado de manera directa el aminoácido comercial Fmoc-Trp-OH con diferentes iodoarilos sustituidos, obteniendo así, nuevos derivados arilados que se pueden aplicar directamente en protocolos en fase sólida. En el segundo capítulo, se ha sintetizado un triptófano fluorogénico basado en el grupo BODIPY vía el proceso de activación C-H desarrollado en el capítulo 1. Así, este aminoácido se ha aplicado directamente en fase sólida para la síntesis de dos péptidos fluorogénicos con interés terapéutico: * Se ha sintetizado una serie de derivados fluorogénicos del péptido antifúngico PAF26 con la finalidad de visualizar infecciones fúngicas de diversos patógenos. * Se ha sintetizado el péptido cíclico fluorescente cLac-BODIPY para la detección de apoptosis, basado en la incorporación del grupo BODIPY a una secuencia peptídica simplificada de lactaderina. En el tercer capítulo, se ha aplicado este protocolo de activación C-H para el acceso a estructuras tipo grapa (staple), directamente a partir de secuencias lineales precursoras que contengan Trp y Phe/Tyr en un proceso de arilación C-H intramolecular. De este modo, se han sintetizado péptidos grapados de secuencias con interés biológico. Esta metodología también permite obtener un amplio rango de diferentes topologías. Así, se han obtenido conjugados macrocíclicos, macrocíclos diméricos, biciclopéptidos que incluyen una ciclación tipo grapa y otra ciclación N-terminal/C-terminal y otros biciclopéptidos formados por dos ciclaciones tipo grapa en una sola etapa sintética. En el cuarto capítulo, se ha desarrollado una metodología basada en reacciones de acoblamiento deshidrogenativo para la formación selectiva de enlaces C-C o C-N en dicetopiperacinas que contengan Trp. Concretamente, se ha accedido a dos tipos de topologías a partir de la correspondiente dicetopiperacina en un solo paso, de acuerdo a la naturaleza del oxidante utilizado.

Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C-H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds.

6 figures., Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time., A. S. acknowledges a PhD fellowship from the Aragon Government. M. A. A. acknowledges funds from the Ministry of Economy and Competitiveness, Spain (IJC2019- 039192-I). This research was supported by CIBER (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU, FEDER (Group B29_20R), Ministry of Science, Innovation and Universities and Agencia Estatal de Investigación, Spain (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). T. K. acknowledges funding from an MRC Career Development Award (MR/S006982/1) and an MRC Centre Grant (MR/N022556/1). E. W. R. acknowledges a Cancer Research UK grant (A_BICR_1920_Roberts). M. V. acknowledges funds from an ERC Consolidator Grant (DYNAFLUORS, 771443) and an ERC PoC Grant (IBDIMAGE, 957535). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (859908).