Sandro Varano
email: sandro.varano@strasbourg.archi.fr

Nicolas Descamps
email: nicolas.descamps@strasbourg.archi.fr

Eric Touvenot
email: eric.touvenot@nancy.archi.fr

Expérimentation d'un EVE pour la co-création spatiale et pédagogique Experimenting an EVE for spatial and educational cocreation

Keywords: EVE, espace d'apprentissage, dispositif pédagogique, réalité virtuelle EVE, learning space, educational device, virtual reality

Dans la continuité du projet de recherche Dune-EOLE portant sur la transformation numérique de l'enseignement supérieur, l'ENSA Strasbourg expérimente l'utilisation d'un Environnement Virtuel Éducatif (EVE) pour la co-création spatiale et pédagogique. L'objectif est de réinterroger les modalités et espaces d'apprentissage, les pratiques pédagogiques à l'aide de dispositifs immersifs. Nous supposons que l'utilisation de la réalité virtuelle constitue une entrée à cette problématique et pourrait favoriser une forme d'autodidaxie, de collaboration entre pairs et un séquençage entre espaces physique et virtuel. L'article explicite notre approche expérimentale, menée avec des étudiants et des enseignantschercheurs, ainsi que les réflexions portant sur le rôle de l'enseignant, les modalités d'apprentissage et les systèmes de représentation.

Introduction

Cet article s'inscrit dans la continuité des travaux menés dans le cadre du projet de recherche Dune-EOLE3 , financé par l'Agence Nationale de la Recherche4 et co-coordonné par les Universités de Strasbourg et de Lorraine5 depuis 2016. L'objectif de la mission EOLE est d'interroger les nouvelles modalités d'apprentissage et d'enseignement avec le numérique et d'accompagner « la transformation numérique des établissements d'enseignement supérieur dans le cadre du programme Investissements d'Avenir »6 .

Cette mission de recherche a été scindée en 3 axes concernant 1) Les espaces d'apprentissage ; 2) Les moteurs de la transformation et 3) Le compte numérique de formation. Dans le cadre de l'axe 1 visant à « consolider et élargir les espaces d'apprentissage, des campus connectés aux campus virtuels »7 , les Ecoles Nationales Supérieures d'Architecture (ENSA) de Strasbourg et Nancy ont été mises à contribution. La participation des ENSA dans la réflexion sur les espaces d'enseignement en lien avec le numérique s'explique entre autre par le fait que les espaces architecturaux, « en tant que cadres physiques de l'apprentissage, sont […] indissociables des processus humains de construction des savoirs et des apprentissages » [START_REF] Mazalto | Introduction: Espaces scolaires et projets éducatifs[END_REF] et conditionnent les pratiques pédagogiques.

A l'aune des premiers résultats du projet Dune-EOLE et des recherches universitaires existantes sur les espaces d'apprentissages mettant en évidence les relations entre espaces architecturaux et pédagogies, l'objectif est de questionner « l'introduction d'artefacts numériques dans une fonction de médiation » (Delépine 2018, 242) et de comprendre comment la culture numérique -plus que les technologies numériques -considérée « en tant que productrice d'artefacts, notamment spatiaux, […] ont une incidence sur les modalités pédagogiques et la nature de l'apprentissage » (Valenzuela-Aguilera 2018, 15).

Ainsi, en considérant les évolutions numériques dans le cadre d'une mutation non seulement technologique mais également culturelle (Ibidem, 13), nous souhaiterions savoir comment l'usage du numérique et plus spécifiquement de la réalité virtuelle, redéfinit les modalités d'apprentissage et d'enseignement dans la co-création d'espaces architecturaux ; la conception d'espaces impliquant une collaboration entre enseignants-chercheurs et étudiants à travers l'utilisation d'outils et instruments médiateurs.

La création d'un Environnement Virtuel Éducatif (EVE) conçu pour la co-création spatiale pourrait nous permettre d'adapter les espaces existants aux nouvelles approches pédagogiques dans une démarche de Design Thinking en considérant l'étudiant comme « designer as a self organizing system » (Jones 1970, 46) : la notion de designer comme auto-organisateur « implique l'usage de la pratique réflexive, lors de laquelle le concepteur va se prendre comme objet de sa propre réflexion afin d'expliciter la façon dont la démarche intellectuelle est organisée permettant de la formaliser et de la rendre compréhensible » [START_REF] Descamps | De l'algorithme à la ville, vers un Minecraft IRL[END_REF].

Dans cet article, nous reprendrons la définition proposée par Loup-Escande, Jamet et al. : « Un EVE peut être défini comme un environnement virtuel -fondé sur un modèle pédagogique spécifique en fonction d'objectifs didactiques -qui fournit aux utilisateurs des expériences qu'ils ne peuvent pas trouver (totalement ou partiellement) dans le monde réel et qui favorise des apprentissages chez les apprenants. » [START_REF] Loup-Escande | Concevoir des environnements virtuels éducatifs avec les utilisateurs finaux : Exemple du projet VirtualiTeach[END_REF] Notre EVE est ainsi considéré comme un artefact numérique et interactif qui permet à l'étudiant/apprenant d'être immergé dans un environnement virtuel pour expérimenter des situations diverses. Il est un instrument qui « remplit des fonctions plus complexes, à la fois techniques et sémiotiques » (Albero 2010a, 15), autour duquel s'organisent l'enseignement et l'apprentissage ; il constitue dès lors une séquence d'apprentissage qui s'inscrit dans un dispositif pédagogique plus large, celui qui « englobe les lieux, les méthodes et l'ensemble fonctionnel des acteurs et des moyens mobilisés en vue d'un objectif » (Albero 2010b, 1).

L'approche instrumentale de Pierre Rabardel, nous montre comment les instruments sont façonnés par la technologie et ses utilisateurs [START_REF] Rabardel | Les hommes et les technologies, approche cognitive des instruments contemporains[END_REF]. Il distingue deux processus mêlés : « instrumentation » et « instrumentalisation » ; le premier met en avant la façon dont l'instrument conditionne les actions de l'utilisateur pour réaliser sa tâche ; le second implique une « contribution » de l'utilisateur pour la « personnalisation », la « différenciation », le « détournement » de l'instrument.

Nous présupposons qu'un EVE pourrait être un instrument original, qui ne viendrait pas se substituer mais compléter les outils de projet et de représentation que sont la maquette, le plan, la coupe, le discours, … La réalité virtuelle pourrait ainsi permettre une nouvelle perception, au moins visuelle et auditive, du projet architectural à l'échelle 1/1. Cela dans le but de mettre en évidence les notions d'échelle, d'ambiance, de luminosité et d'usage avant la présentation finale, voire la construction.

Dans un premier temps, nous passerons en revue des projets développés dans le cadre de la mission de recherche Dune-EOLE mettant en avant l'influence réciproque des espaces d'apprentissage avec l'enseignement.

Nous nous intéresserons ensuite aux EVE développés au sein des ENSA avec « l'idée que la réalité virtuelle en y intégrant l'importance d'une conception pédagogique devient un outil à part entière dans le cadre de l'enseignement » [START_REF] Merchant | Effectiveness of virtual reality-based instruction on students' learning outcomes in K-12 and higher education : A meta-analysis[END_REF] Les enseignements avaient pour objectif de réinterroger les méthodes pédagogiques en considérant une autodidaxie, une volonté de collaboration et de fluidité spatio-temporelle permise par le numérique, nécessitant un changement dans l'approche pédagogique de la part des enseignants (Loup-Escande, Jamet et al. 2015, 1). Ainsi, l'existence de ces enseignements pragmatiques se justifie par notre double posture d'enseignants-chercheurs et d'architectes qui nous fait nous interroger sur « le rôle propre des espaces pédagogiques tant du côté de la forme que de celui de l'usage » [START_REF] Touvenot | Les espaces d'apprentissage, action 1.1. Faire évoluer les espaces physiques[END_REF]) mais également par les approches pédagogiques particulières des études en architecture. En effet, la dimension de Learning by doing, c'est-à-dire un « apprentissage [qui] combine savoir et manipulation objet » (Weber, Rodhain & Fallery 2019, 41) y est déjà prédominante notamment dans la pratique du projet. Dans cette partie nous présenterons succinctement deux projets issus de ces enseignements.

Création d'espaces et de pratiques didactiques en réalité virtuelle

Error 404, (projet d'étudiants 11 2018-2019 à l'ENSAS)

Le projet Error 404 est pertinent dans notre étude car il témoigne d'une réflexion sur les séquences d'apprentissage, sur la complémentarité des espaces physiques et virtuels12 , de la co-création d'une architecture virtuelle impermanente et d'un apprentissage par et pour les étudiants. Le projet a pour objectif de questionner l'importance des cinq sens dans la conception et dans la réception d'une architecture grâce à la création d'un espace virtuel immersif mouvant, augmenté par un espace physique également évolutif. L'intérêt majeur est d'avoir voulu dépasser la domination visuelle très souvent mise en avant dans les systèmes immersifs car considérée comme « le sens dominant de la perception humaine » (Mellet d'Huart 2001, 332) et de renforcer la dimension haptique de l'immersion. Les étudiants ont en effet choisi de retranscrire l'ensemble des sens, en les superposant, dans et par l'espace à l'aide de pistons déformants les parois souples des murs et du plafond. Si la traduction des perceptions sensorielles en modulations spatiales est d'une subjectivité assumée, les étudiants ont mis en avant une réflexion sur la place des sens en architecture ainsi qu'une pratique réflexive dans la manière d'aborder le projet, illustrée par de nombreux diagrammes conceptuels concernant aussi bien l'espace que leur processus de création (Figure 1). Ils ont également interrogé les notions de complémentarité, de singularité et de confrontation entre espaces physiques et virtuels et dans les modes de représentation (réalité virtuelle, maquettes, diagrammes, plans, coupes, croquis et discours) 13 dans une logique de séquençage de l'apprentissage qui commence dans l'espace physique et se poursuit dans l'espace virtuel. 2). En effet, ici, l'espace physique -la box -est indissociable de l'espace virtuel dont il est la représentation. La box fait office de seuil entre ces deux dimensions. Au début de l'expérimentation, l'utilisateur prend place dans un espace particulier mais stable qu'il retrouvera à l'identique une fois équipé des lunettes de réalité virtuelle 15 . Cette copie conforme de l'espace physique permet d'atténuer la sensation d'ailleurs et de distanciation avec sa physicalité. Une fois immergé, l'utilisateur va faire évoluer son environnement à l'aide d'un appareil de monitoring Muse qui capte en temps réel les variations des ondes cérébrales (EEG), de l'activité cardiaque (VRC), de l'activité respiratoire, …16 L'ensemble des données recueillies modifie automatiquement l'espace virtuel en faisant évoluer la luminosité, les couleurs, les formes, … Ainsi, l'usager projette sur l'espace ses sensations/émotions et doit procéder à un travail de méditation pour le faire évoluer, comme l'a fait Luc Schuitten avec son Arbre de lévitation 17 . Avant de quitter l'environnement immersif, l'espace virtuel reprend sa forme initiale afin de permettre une transition de retour fluide. Une variante du projet est en cours, procédant de manière inverse pour comprendre comment l'espace influence l'individu. Ces deux exemples mettent en avant l'un des risques potentiels de la réalité virtuelle. Si la notion de subjectivité n'est pas dérangeante, la transcription des cinq sens ou de l'activité biologique en signaux et en formes -d'autant plus que les informations se superposent -peut conduire à « une surcharge informationnelle », c'est-à-dire « un flot de données qu'un sujet humain peut difficilement transformer en information et en connaissance » (Briffaut 2012, 212). Nous présupposons que cette superposition informationnelle amène certes à des espaces complexes, interactifs et généralement intéressants mais peut également faire perdre la lisibilité et la compréhension de l'espace. Comment en effet répondre à un programme spécifique ou même analyser le résultat à partir de cela?

Expérimentation d'un EVE pour la co-création spatiale et pédagogique au sein d'une ENSA

Objectif

Même si cela semble évoluer, nous retrouvons encore peu l'utilisation des technologies immersives dans le domaine universitaire. Nous avons donc adopté une approche expérimentale pour développer un EVE au sein des ENSA. Cette attitude empirique s'explique également par la nécessité de considérer ces environnements immersifs (et numériques) au cas par cas afin qu'ils répondent 1) à des pratiques pédagogiques spécifiques ; 2) à des attentes et à des pratiques d'apprentissage particulières des étudiants et de leurs champs disciplinaires et 3) à des espaces physiques et des moyens financiers mis à disposition. Sur la base des résultats des travaux étudiants, nous avons trouvé pertinent de développer un EVE -certes plus académique -mais toujours dans l'objectif de développer et d'adapter les pratiques pédagogiques pour un public majoritairement de la « génération Z » qui n'a connu qu'une société fondée sur un outillage et une culture numérique prédominante. L'espace développé a une triple ambition. La première consiste à poser les bases d'un outil immersif de conception des ambiances d'apprentissage qui a pour vocation d'évoluer suite aux retours des étudiants et des enseignants-chercheurs. La deuxième vise à expérimenter les interactions avec des données quantitatives et qualitatives dans un environnement virtuel en lien avec l'espace physique dans une volonté de travail sur les séquences d'apprentissage. Enfin la dernière ambition est plus générale et concerne aussi bien l'apprentissage spécifique aux études d'architecture que les études universitaires dans leur ensemble. Cet EVE est une prolongation expérimentale des réflexions menées lors de la mission de recherche Dune-EOLE sur l'impact du numérique dans l'enseignement et l'apprentissage afin de valoriser un apprentissage plus didactique, autonome, collaboratif et centré sur la notion de Learning by doing.

Méthodologie

Nous nous sommes basé sur la norme ISO 9241-210:2019 « Ergonomie de l'interaction homme-système -Partie 210 : Conception centrée sur l'opérateur humain pour les systèmes interactifs » qui définit cinq critères pour cadrer le développement d'un EVE : « 1) La prise en compte en amont des utilisateurs, de leurs tâches et de leur environnement ; 2) La participation active des utilisateurs, garantissant la fidélité des besoins et des exigences liées à leurs tâches ; 3) La répartition appropriée des fonctions entre les utilisateurs et la technologie ; 4) L'itération des solutions de conception, jusqu'à satisfaction des besoins et des exigences exprimés par les utilisateurs et 5) L'intervention d'une équipe de conception multidisciplinaire, visant une expérience utilisateur optimale »18 .

Les lieux, les enseignements, les ressources et les acteurs du Laboratoire Lumière de l'ENSAS constituent le dispositif pédagogique. Équipé d'appareils de prise de mesures (luxmètres, ...), d'outils informatiques et de réalité virtuelle, de Lumbox (maquettes 1/20) et récemment d'un simulateur d'éclairage naturel (ciel artificiel et soleil mécanique) ; associant chercheurs, enseignants et étudiants, il ancre la recherche dans la pédagogie.

Dans une logique de complémentarité entre les lieux et les outils, il est d'abord demandé aux étudiants d'effectuer des relevés in-situ (dimensionnement, quantification et qualification des ambiances de l'objet d'étude). Les premières analyses se poursuivent à partir de maquettes physiques et modèles informatiques. Elles débouchent ensuite sur des intentions de projet (dispositifs architecturaux, matériaux, orientation, ...) et des hypothèses qui mènent à l'expérimentation. En plus des expérimentations des étudiants, une mission a été proposée (à la société Echoes.Paris) en vue de créer des prototypes d'interfaçage entre la réalité physique d'un lieu existant et un environnement immersif en réalité virtuelle.

Présentation des prototypes d'interfaçage

Synchronisation semi-automatique

La première méthodologie mise en place consiste en un protocole semi automatisé unidirectionnel entre les plateformes Grasshopper et Twinmotion/UnrealEngine. Il est d'abord demandé aux étudiants de retranscrire leurs intentions de projet en paramètres modélisables. A ce stade de développement, le protocole permet de créer des modèles paramétriques afin de générer des variantes dans les propositions de projet ou dans l'étude des ambiances (images de simulations calculées en fausses couleurs) depuis l'interface Grasshopper et de les exporter dynamiquement dans Twinmotion. L'immersion se fait ensuite grâce à un casque de réalité virtuelle.

Ce type de synchronisation ne permet pas à l'étudiant de modifier son projet lorsqu'il est immergé. Cependant, les différentes variantes générées en amont, peuvent être explorées en aval par phases. Tout en étant immergé, l'étudiant peut, d'une part, faire défiler les différentes propositions spatiales via le menu interactif ; d'autre part, superposer les informations quantitatives (images de simulation) aux informations qualitatives (l'espace du projet dans sa morphologie, avec ses matériaux, ...) (Figure 3). Nous espérons pouvoir maximiser le sentiment d'immersion dans l'espace modélisé et permettre aux étudiants d'interagir avec l'environnement virtuel comme ils le feraient naturellement avec une maquette. Toutefois, la différence entre ces deux pratiques (outre les techniques et technologies utilisées) réside dans le fait que dans la manipulation de la maquette, la création de l'espace se fait du dehors lorsque l'EVE permettrait de le créer du dedans : de concevoir, fabriquer ou vérifier l'espace depuis l'espace.

Une autre particularité réside dans le fait que l'environnement permet d'interagir avec des données objectives ou subjectives. Les premières sont mesurables (géométriques ou climatiques), les secondes résultent de la sémantique du lieu d'étude, identifiées in-situ par les étudiants lorsqu'ils expriment des qualificatifs d'ambiance (lumière chaleureuse, intime, ...). L'idée est d'immerger l'étudiant dans un projet paramétrique informé, capable d'évoluer par le biais de commandes interactives (Figure 4). Si l'implémentation des données objectives est opérationnelle, la retranscription des données subjectives en données interactives est encore au stade de réflexion.

Conclusion

« Une des choses que nos petits-enfants trouveront le plus bizarre à notre sujet, c'est que nous faisons la différence entre le numérique réel, entre le virtuel et le réel. Dans le futur, cela serait littéralement impossible. La distinction entre le cyberespace et ce qui n'est pas le cyberespace va devenir inimaginable. » [START_REF] Gibson | William Gibson: The Rolling Stone interview[END_REF] Cette phrase de William Gibson, l'auteur de l'ouvrage culte Neuromancien résume l'un des premiers constats de ces expérimentations. Après une première phase de compréhension de l'environnement immersif, de ces potentialités, de ces limites et de premiers tâtonnements, les étudiants ont vite intégrés d'eux-mêmes qu'aucun des espaces physique et virtuel n'étaient substituables ou antagonistes mais bien complémentaires dans les modalités de conception et d'usage. Cela a confirmé l'intérêt et les intentions des enseignants-chercheurs de travailler par séquences d'apprentissage : la conception commence dans l'espace physique et se poursuit dans l'espace virtuel. Il est également apparu un besoin commun de lier l'expérience virtuelle à une expérience sensible, sensorielle, dans les différentes expérimentations. Aussi, nous nous sommes rendus compte que les étudiants ont considéré la réalité virtuelle comme un espace d'apprentissage et d'expérimentations et non pas comme un outil de visualisation ou de rendus.

La mise en oeuvre d'un EVE au sein de l'ENSAS -bien qu'encore au stade expérimental -a déjà amené des résultats très encourageants et soulevé de nombreuses problématiques. Plus qu'un simple outil de « vérification » ou de rendu, l'utilisation libre de l'EVE par les étudiants a mis en exergue des questions fondamentales dans la conception d'espaces d'apprentissage et dans l'apprentissage de l'architecture : spatialité et temporalité de l'architecture ; liens entre espaces physique et virtuel ; rapport entre corps, émotions et espaces ; influence de l'outil sur le processus de conception ; réflexivité, autodidaxie et adaptation des pratiques ; séquences d'apprentissage ; place des sens dans l'élaboration du projet, … Les résultats des expérimentations montrent également la manière dont l'usage d'environnements immersifs et le séquençage de l'apprentissage entre les espaces physique et virtuel amènent à (re)questionner les modalités d 'interaction, d'enseignement et d'apprentissage (Loup-Escande, Jamet et al. 2015, 1). Il apparaît en effet qu'« une réflexion sur la formation doit notamment appréhender la complexité du processus cognitif de transfert de connaissances lequel nécessite une décontextualisation puis une recontextualisation [START_REF] Fallery | Le storytelling : un outil de gestion des connaissances[END_REF] et met le rôle de l'enseignant au premier plan » (Weber, Rodhain & Fallery 2019, 39). De plus, comme l'ont mis en évidence David Gauckler et Jean-François Cerisier, s'il est vrai que les étudiants de la « génération Z » sont nés avec le numérique, cela ne présuppose en rien leur capacité « à manipuler les objets techniques numériques [qui] induirait une évidence à l'usage des environnements immersifs » (Gauckler 2019, 9). Il convient plutôt de considérer que cette génération (comme la société en général) a été impactée -transformée -par le numérique d'abord et essentiellement d'un point de vue culturel et social plus que d'un point de vue technique. Toutefois, un regard critique sur la culture numérique ne va pas de soi et n'ai que très peu abordé dans l'enseignement numérique alors même qu'il semble incontournable.

Bibliographie

Figure 1 .

 1 Figure 1. Diagramme des scenarii proposés pour le projet Error 404, témoignant de la pratique réflexive dans la manière d'aborder le projet

Figure 2 .

 2 Figure 2. Diagramme conceptuel des liens entre données biologiques, définition d'un état/émotion et spatialité virtuelle dans le projet EMO_Box

Figure 3 .

 3 Figure 3. Variations d'ambiances en réalité virtuelle (Twinmotion synchronisé avec Grasshopper) : exploration des propositions de projet et superposition des informations qualitatives et quantitatives

Figure 4 .

 4 Figure 4. Synchronisation automatique Grasshopper/Rhino/Unity : interaction avec des données objectives ou subjectives en immersion virtuelle

évoluer ce qui nous permettra également d'émettre des pistes de recherches et d'améliorations de la version présentée dans cet article. conception

  d'ambiances d'apprentissage afin de réaménager un hall de l'Université de Strasbourg et une salle de l'IUT de Colmar 9 . Les projets d'étudiants ont été présentés en réalité virtuelle lors de la réunion annuelle du programme Dune-EOLE aux membres du consortium à l'Université de Reims Champagne Ardenne 10 .

	et participe à
	certaine forme d'autodidaxie de la part des étudiants.
	Nous conclurons en analysant les premiers résultats en matière de pédagogie et de
	perspectives de conception spatiale. De par son côté expérimental, ce projet d'espaces
	pédagogiques immersifs continue d'

« Développement d'universités numériques expérimentales » et « Engagement pour ouvrir l'éducation ».

Projet ANR-16-DUNE-0001-EOLE.

Le projet Dune-EOLE est réalisé en partenariat avec l'UHA, l'URCA, UNIT et les ENSA Strasbourg et Nancy

Voir les axes de recherche et les résultats Dune-EOLE sur : http://www.dune-eole.fr/laureatsdune/#page-content

7 http://www.dune-eole.fr/axe-1-les-espaces-dapprentissage/#page-content

Expérimentations de conception de NEA dans le cadre du projet Dune-EOLE 2.1 Des premières réflexions sur le rapport spatialité/pédagogie Parallèlement à la constitution d'un État de l'art des Nouveaux Espaces d'Apprentissage (NEA) (Descamps, 2018), deux enseignants-chercheurs en ENSA, ont mis en place une réflexion prospective grâce à des cours optionnels de Master afin de questionner et d'expérimenter l'aménagement de NEA et leurs pédagogies associées. En 2018 et 2019, dans le cadre de l'atelier « Modélisation/Fabrication » (M1 -ENSAS) les étudiants ont proposé des dispositifs d'apprentissage numérique, mobile et connecté capables de se « greffer » dans un lieu universitaire existant 8 . En 2019, au sein de l'atelier « Laboratoire Lumière : ambiances et immersions », leurs camarades ont réfléchi à la 8 Rendu final des travaux et soutenances publiques des étudiants les 14 janvier 2019 et 20 janvier 2020 à l'ENSAS ; PDF disponible sur le site Dune-EOLE : http://www.dune-eole.fr/actualites/#pagecontent

Rendu final des travaux et soutenance publique des étudiants le 3 juin 2019 à l'ENSAS ; PDF disponible sur le site Dune-EOLE : http://www.dune-eole.fr/actualites/#page-content

Démonstrations du 9 octobre 2019. Voir à ce sujet le poster et le livret de présentation des travaux d'étudiants des ENSAS et ENSAN sur le site Dune-EOLE : http://www.dune-eole.fr/actualites/#pagecontent

11 BALLET Mélanie, BIACHE Gaël & COUTANT

Tom 12 Dans cet article nous parlerons d'espaces physiques et virtuels et non de réels/virtuels considérant comme Gilles Deleuze que « Le virtuel possède une pleine réalité, en tant que virtuelle » (Différence et Répétition, PUF, 2011).

Pour plus d'informations sur ce projet voir : http://www.dune-eole.fr/files/2019/11/Rendu-Master1-ENSAS-14-janvier-2019-1.pdf

14 LEPAUL Romain, MORNET Colas, SARGENTI Justin, SCHNINDLER Martial & SCHWEITZER

Joseph 15 Voir à ce sujet l'épisode 2 de la saison 3 de Black Mirror,

Playtest. 16 https://choosemuse.

com/fr/muse-2/ 17 L'arbre de lévitation -Luc SCHUITEN -http://www.vegetalcity.net/topics/larbre-de-levitation/

Définition Wikipédia « Conception centrée sur l'utilisateur », https://fr.wikipedia.org/wiki/Conception_centr%C3%A9e_sur_l%27utilisateur


Sébastien Jenni
email: sebstien.jenni@u-bourgogne.fr

Flavien Ponsot

Pierre Baroux

Lucile Collard

Takayuki Ikeno

Kenjiro Hanaoka

Valentin Quesneau

Kévin Renault

Anthony Romieu
email: anthony.romieu@u-bourgogne.fr

Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes

Keywords: Diketopyrrolopyrrole, Enzyme detection, Fluorogenic probe, Hypoxia, Penicillin G acylase, Reductase Highlights

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Introduction

Over the past decade, intensive research efforts have been devoted to develop novel highperformance visible fluorescent organic dyes based on the diketopyrrolopyrrole (DPP) framework which was used previously only as basic structural element of pigment for paints, coatings and printing inks industry [1]. The high degree of interest for these photoactive bislactam-based small molecules is related to their numerous valuable features including facile synthetic accessibility, ease of structural modification, outstanding chemical and thermal robustness and attractive spectral properties [2]. However, compared with common classes of organic-based fluorophores (i.e., BODIPY, coumarin, cyanine and xanthene dyes) [3], the main limitation currently associated with the fluorescent DPP dyes is the lack of simple and effective structural alteration strategies to readily tune their emission capability, especially after their specific interaction or reaction with a biological and/or reactive species to be detected.

Modulation of fundamental photophysical processes as the final result of such activity-based sensing approaches, is the key to the design of analyte-responsive fluorescent probes applied to sensing/imaging applications [4]. Nevertheless, there are some interesting achievements focusing on fluorogenic detection of reactive analytes (e.g., Hg(II) cations, H2S and biothiols) both in vitro and in living cells through irreversible and stoichiometric organic reactions.

However, as illustrated by the selected examples shown in Fig. 1 [5], the corresponding DPPbased fluorescent chemodosimeters are not constructed from a common and structurally simple fluorogenic scaffold bearing an optically tunable group (e.g., amino, hydroxyl, ...) as is frequently the case for reaction-based fluorescent probes using coumarins, cyanines or xanthenes as photoactive reporters [6]. In an effort to address this gap, the Hua group has recently proposed an unsymmetrical 3,6-diaryl-DPP core structure bearing a single 4'-pyridyl unit acting as fluorogenic reactive center [7] (Fig. 1). Indeed, depending on the charge state of this N-heterocycle (i.e., pyridinium cation bearing N-alkyl substituent and neutral pyridine molecule), internal charge transfer (ICT) process is operative or completely abolished. Thus, a ratiometric detection strategy of enzymes (esterase activity and g-glutamyltranspeptidase (g-GT)) [7a, 7c] and reactive oxygen species (ROS) such as superoxide radical anion (O2 •-) [7b], through an effective biocompatible domino reaction triggered by the targeted bioanalyte and leading to N-dealkylation of 4'-pyridinium moiety, has been devised and applied to living cells and in vivo context. To the best of our knowledge, these contributions are the only examples of enzyme-responsive DPP probes that, in addition, highlight the potential of these less common activatable fluorophores for biological imaging applications. However, curiously no attention has been paid to optimization of their physicochemical properties (e.g., water solubility) [8] for better performances in aq. media whereas some effective strategies for imparting polarity and possibly bioconjugation ability to DPP derivatives are readily available in the literature [9]. In pursuit of novel structurally simple fluorophores possessing both biocompatibility and fluorogenic reactivity, the chemistry of pyridine-flanked DPP dyes deserves to be revisited.

Furthermore, this will enable to assess the level of versatility of this emerging class of fluorescent platforms, particularly illustrated by the range of different enzymes (both peptidases and reductases) which could be detected. The present Article comes within this topic. Indeed, we report the synthesis of six different enzyme-responsive fluorogenic DPP probes rationally designed from symmetrical 3,6-bis-(4'pyridyl)-DPP pigment 4 or an unsymmetrical analog namely mono-(4'-pyridyl)-DPP pigment 5. They differ in both the type of their bio-metabolizable recognition moiety with the aim of targeting hypoxia-related reductases (azo-and nitroreductase, AzoR and NTR respectively) or penicillin G acylase (PGA) [10], and the nature of their N-lactam substituents (i.e., carboxymethyl, methyl and hexyl) directly impacting the solubility and the photophysical behavior of the probes in aq. environments (Fig. 1). However, the common feature of all probes related to their activation mechanism, is the transient formation of a highly reactive 4aminobenzyl quaternary pyridinium intermediate readily prone to self-immolation [11]. The fluorogenic behavior as well as enzymatic activation and aq. stability of these DPP probes were studied through in vitro fluorescence assays and HPLC-fluorescence/-MS analyses. In the light of results and data produced, we will identify suitable combination of structural elements to achieve conversion of early industrial DPP pigments to high-performance fluorescent probes for tracking enzymatic activities in living systems [10,12].

Experimental Section

See SI for all experimental details related to the preparation of compounds 4, 5, 11, 19-21, photophysical characterizations and HPLC analyses/purifications.

General

Unless otherwise noted below, all commercially available reagents and solvents were used without further purification. TLC were carried out on Merck DC Kieselgel 60 F-254 aluminum sheets. The spots were directly visualized and through illumination with dual wavelength UV lamp (l = 254 and 365 nm). Column chromatography purifications were performed with different types of silica gel for which specifications are given throughout the description of synthetic protocols. MeCN and THF were dried over alumina cartridges immediately prior to use, using a solvent purification system PureSolv PS-MD-5 model from Innovative Technology. DMF (Fisher Chemicals, extra pure, ≥99%, #D/3840/17) was dried by storage over activated 3 Å molecular sieves and kept under Ar atmosphere. Anhydrous NMP over 3 Å molecular sieves was purchased from Acros Organics. DMSO (for spectrophotometry, #D5293) was purchased from TCI Europe. Formic acid (puriss p.a., ACS reagent, reag. Ph.

Eur., ≥98%, #33015), NTR (from Escherichia coli, #N9284, 0.1 U/μg, lyophilized enzyme + buffer resuspended in ultrapure water), NADH and LAP (microsomal, from porcine kidney, #L5006, type IV-S, ammonium sulfate suspension, 10-40 U per mg protein) were obtained from Merck-Millipore (brand Sigma-Aldrich) and stored at -20 °C for NTR/NADH and LAP. PGA (from Escherichia coli, EZ50150, 841 U/mL) was provided by Iris Biotech GmbH. The HPLC-gradient grade MeCN was obtained from Carlo Erba, Fisher Scientific or VWR. Aq. buffers and aq. mobile-phases for HPLC were prepared using ultrapure water produced by an ELGA PURELAB Ultra system (purified to 18.2 MW.cm).

Instruments and methods

Centrifugation operations were performed with a Thermo Scientific Espresso Personal Microcentrifuge instrument. Centrifugation steps required for isolation of DPP pigments 4 and 5 were performed with an Hettich Universal 320 instrument (with the following parameters: 15 min, 3000 rpm). 1 H-, 13 C-and 19 F-NMR spectra were recorded on Bruker spectrometers: Avance Neo 500 MHz equipped with a 5 mm BBOF iProbe and Avance III HD 600 MHz equipped with a 5 mm BBOF N2 cryoprobe. NMR spectroscopy chemical shifts are quoted in parts per million (ppm) relative to TMS (for 1 H, and 13 C) and CFCl3 (for 19 F). For 1 H and 13 C spectra, calibration was made by using residual signals of partially deuterated solvent summarized by Fulmer et al. [13]. J values are expressed in Hz. 13 C substitutions of some compounds (16 and 18) were determined with JMOD experiments, differentiating signals of methyl and methine carbons pointing "up" (+) from methylene and quaternary carbons pointing "down" (-) [14]. IR spectra were recorded with a Bruker Alpha FT-IR spectrometer equipped with an universal ATR sampling accessory. The bond vibration frequencies are expressed in reciprocal centimeters (cm -1 ). HPLC-MS analyses were performed on a Thermo-Dionex Ultimate 3000 instrument (pump + autosampler at 20 °C + column oven at 25 °C) equipped with a DAD (Thermo-Dionex DAD 3000-RS) and a MSQ Plus single quadrupole mass spectrometer. HPLC-fluorescence analyses were performed with the same instrument but connected to Thermo Scientific Dionex UltiMate 3000 fluorescence detector FLD-3400 RS dual-PMT. UHPLC analyses (implemented during in vitro assays with rat liver microsomes) were performed on a Waters Acquity UPLC (H class)/QDa quadrupole MS analyzer (Waters).

Purifications by semi-preparative HPLC were performed on a Thermo-Dionex Ultimate 3000 instrument (semi-preparative pump HPG-3200BX) equipped with a RS Variable Detector (VWD-3400RS, four distinct wavelengths within the range 190-800 nm). Lyophilization operations were performed with a Christ Alpha 2-4 LD plus. TFA mass content of samples purified by semi-preparative was determined by ion chromatography according to a method developed by the PACMSUB staff. Such analyses were performed with an ion chromatograph Thermo Scientific Dionex ICS 5000 equipped with a conductivity detector CD (Thermo Scientific Dionex) and a conductivity suppressor ASRS-ultra II 4 mm (Thermo Scientific Dionex). Low-resolution mass spectra (LRMS) were recorded either on a Thermo Scientific MSQ Plus single quadrupole equipped with an electrospray (ESI) source (LC-MS coupling mode), or on a Bruker Amazon SL instrument equipped with an ESI source (direct introduction mode). symmetrical 3, 6-bis-(4'-pyridyl)-DPP dye 13 and related NTR-sensitive probe 6

Synthesis of

Di-tert-butyl 3,6-bis-(4'-pyridyl)-DPP N,N'-diacetate 12

A suspension of 3,6-Bis-(4'-pyridyl)-DPP pigment 4 (0.255 g, 0.88 mmol, 1.0 equiv.) and tBuOK (247 mg, 2.2 mmol, 2.5 equiv.) in dry NMP (20 mL) was stirred at 75 °C under Ar atmosphere for 15 min (color changes from dark pink to night blue). Thereafter, tert-butyl bromoacetate (386 μL, 2.61 mmol, 3 equiv.) was added and the resulting reaction mixture was stirred at 75°C for 90 min (color changes from blue to brown). The reaction was checked for completion by TLC (DCM-EtOAc 8:2, v/v + 1% TEA) and RP-HPLC (system A). After cooling to RT, toluene (60 mL) was added and the resulting organic layer was washed with deionized water (2 × 100 mL) to remove NMP, dried over anhydrous Na2SO4 and finally evaporated to dryness. The resulting residue was purified by column chromatography over silica gel (VWR,technical grade,#84814.360,eluent: step gradient of EtOAc in DCM (+1% TEA) from 0% to 7.5%). Fractions containing the desired product, were combined, washed with aq. 1.0 M KHSO4 (100 mL) and deionized water (2 × 100 mL) to remove TEA, dried over anhydrous Na2SO4 and evaporated under reduced pressure. Compound 12 was obtained as an orange-brown solid (160 mg, 0.31 mmol, yield 35%). 1 H NMR (600 MHz, CDCl3): δ = 8.78 (d, 3 JH-H = 4.2 Hz, 4H), 7.61 (d, 3 JH-H = 5.4 Hz, 4H), 4.39 (s, 4H), 1.36 (s, 18H); 13

3,6-Bis-(4'-pyridyl)-DPP N,N'-diacetic acid 13

Di-tert-butyl ester 12 (17.0 mg, 32.8 μmol, 1.0 equiv.) was dissolved in a TFA-DCM (1:1, v/v) mixture (2 mL) and the mixture was stirred at RT for 3 h. The reaction was checked for completion by RP-HPLC (system B) and the reaction mixture was evaporated to dryness. The resulting residue was dissolved in a (1:1, v/v) mixture of 0.1% aq. TFA and MeCN (4 mL) and purified by semi-preparative RP-HPLC (system C, 1 injection, tR = 15.8-17.4 min of collected fractions containing the desired compound). The product-containing fractions were lyophilized to give 13 as TFA salt (6.4 mg, 11.3 μmol, yield 34% based = 10.26 (bs,1H,CO2H),8.84 (m,2H),8.66 (d,J = 5.4 Hz,2H),7.83 (d,J = 5.4 Hz,2H),7.72 (d,J = 5.4 Hz,2H),4.12 (s,2H),3.75 (s,2H)

Open-chain lactam form of probe 14

Di-tert-butyl ester 12 (16.8 mg, 32.4 μmol, 1.0 equiv.) was dissolved in dry MeCN (3 mL), 4-nitrobenzyl bromide (54 mg, 250 μmol, 7.7 equiv.) was added and the resulting reaction mixture was stirred under reflux and Ar atmosphere for 24 h. The progress of the reaction was checked by RP-HPLC (system B). Thereafter, the reaction mixture was evaporated to dryness.

The resulting residue was dissolved in a TFA-DCM (1:1, v/v) mixture (2 mL) and stirred at RT for 3 h. The reaction was checked for completion by RP-HPLC (system A) and the mixture was evaporated to dryness. The resulting residue was dissolved in a (1:1, v/v) mixture of 0.1% aq.

TFA and MeCN (4 mL) and purified by semi-preparative RP-HPLC (system C, 1 injection, tR = 24.5-28.5 min of collected fractions containing the desired compound). The productcontaining fractions were lyophilized to give 14 as TFA salt (5.2 mg, 5.6 μmol, yield 17% based on mass percentage of TFA = 23% determined by ionic chromatography, 1.9 TFA). 1 H NMR (500 MHz, DMSO-d6): δ = 10.18 (t, 3 JH-H = 5.7 Hz, 1H), 9.15 (d, 3 JH-H = 6.8 Hz, 2H), 8.82 (d, 3 JH-H = 7.1 Hz, 2H), 8.33 (m, 4H), 8.07 (d, 3 JH-H = 6.8 Hz, 2H), 7.88 (d, 3 JH-H = 7.1 Hz, 2H), 7.82 (d, 3 JH-H = 8.8 Hz, 2H), 7.76 (d, 3 JH-H = 8.8 Hz, 2H), 6.02 (s, 2H), 5.83 (s, 2H), 4.20 (s, 2H), 3.82 (d, 3 JH-H = 5.7 Hz, 2H); 13 C NMR (151 MHz, DMSO-d6): δ = 178.7, 171.3, 170.6, 166.0, 164.7, 159.2, 148.0, 147.9, 147.7, 144.6, 142.8, 141.6, 141.3, 134.9, 134.8, 130.2 (2C), 129.9 (2C), 129.6, 127.0, 126.7, 125.7, 125.4, 124.4 (2C), 124.3 (2C), 124.2, 123.8, 123.3, 121.2, 101.8, 62.0, 61.7, 60.7, 42.5, 41.1, 36.5, 16.9

Di-tert-butyl mono-(4'-pyridyl)-DPP N,N'-diacetate 16

A suspension of mono-(4'-pyridyl)-DPP pigment 5 (250 mg, 0.68 mmol, 1.0 equiv.) and tBuOK (190 mg, 1.70 mmol, 2.5 equiv.) in dry NMP (30 mL) was stirred at 75 °C under N2 atmosphere for 15 min. tert-Butyl bromoacetate (300 μL, 2.04 mmol, 3.0 equiv) was added and the mixture was stirred at 75 °C under N2 for 90 min. Thereafter, the mixture was cooled down to RT and deionized water was added. The product was extracted with toluene three times. The combined organic layers were dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure. The product was purified by column chromatography over silica gel (VWR technical grade, 40-63 μm, #84814.360) using a step gradient of EtOAc in DCM as eluent (from 0% to 10% + 1% TEA). Compound 16 was obtained as an orange solid (86.0 mg, 144 μmol, yield 22%). 1 H NMR (500 MHz, CDCl3): δ = 8.73 (brs, 2H), 7.76 (brs, 2H), 7.61 (m, 4H), 4.38 (s, 2H), 4.32 (s, 2H), 1.34 (s, 9H), 1.32 (s, 9H); 13 C NMR (126 MHz, CDCl3): δ = 167.3, 167.2, 162.0, 161.7, 150.8, 149.2, 144.8, 135.3, 132.6 (2C), 130.3 (2C), 126.8, 126.4, 122.0, 111.5, 109.9, 83.3, 83.2, 44.3, 44.1, 28.0 (6C); HPLC (system A): tR = 5.6 min (purity 85% at 260 nm and 95% at 470 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 261, 299 and 462 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 596.2 and 598.2 [M + H] + , calcd for C29H31BrN3O6 + 596.1 and 598.1.

Mono-(4'-pyridyl)-DPP N,N'-diacetic acid 17

To a solution of di-tert-butyl ester 16 (16.2 mg, 27 μmol, 1.0 equiv) in DCM (2 mL), TFA ( 2mL) was added. The mixture was stirred at RT for 4 h. Thereafter, the reaction mixture was evaporated to dryness. The resulting residue was dissolved in a (2:1, v/v) mixture of 0.1% aq.

TFA and MeCN (3 mL) and purified by semi-preparative RP-HPLC (system E, 1 injection, tR = 35.0-42.5 min of collected fractions containing the desired compound). The productcontaining fractions were lyophilized to give TFA salt of 17 as an orange amorphous powder (7.8 mg, 14 μmol, yield 50% based on mass percentage of TFA = 15% determined by ionic chromatography, 0.7 TFA). 1 H NMR (500 MHz, DMSO-d6): δ = 13.24 (brs, 2H), 8.83 (d, 3 JH- 1 Hz,2H),7.84 (d,2H),7.75 (m,4H),4.53 (s,2H),4.49 (s,2H);13 C NMR (151 MHz, , 161.7, 161.3, 151.0, 149.1, 145.2, 134.9, 132.7, 131.0, 126.6, 126.1, 122.4, 110.6, 109.1, 56.5, 43.6, 43.5; 19

H = 6.

N-(4-Nitrobenzyl)pyridinium mono-(4'-pyridyl)-DPP N,N'-diacetic acid 7

To a solution of di-tert-butyl ester 16 (34 mg, 57 μmol, 1.0 equiv.) in dry MeCN (5 mL), 4nitrobenzyl bromide (99 mg, 456 μmol, 8.0 equiv.) was added. The mixture was refluxed under N2 atmosphere overnight. Thereafter, the reaction mixture was evaporated to dryness. The resulting residue was dissolved in DCM (2 mL) and TFA (2 mL) was added dropwise. The mixture was stirred at RT for 3 h, then evaporated to dryness. The resulting residue was dissolved in a (2:3, v/v) mixture of 0.1% aq. TFA and MeCN (4 mL) and purified twice by semi-preparative RP-HPLC (system D, 1 injection, tR = 46.0-50.0 min of collected fractions containing the desired compound). The product-containing fractions were lyophilized to give TFA salt of 7 as a purple solid (4.9 mg, 6.7 μmol, yield 12% based on mass percentage of TFA = 17% determined by ionic chromatography, 1.1 TFA). 1 H NMR (500 MHz, DMSO-d6): δ 13.34 (brs, 2H), 9.36 (d, 3 JH-H = 6.5 Hz, 2H), 8.47 (d, 3 JH-H = 6.5 Hz, 2H), 8.33 (d, 3

Synthesis of unsymmetrical mono-(4'-pyridyl)-DPP dye 18 and related AzoR-and NTR-

sensitive probes 8 and 9

Mono-(4'-pyridyl)-DPP N,N'-dimethyl 18

A suspension of mono-(4'-pyridyl)-DPP pigment 5 (300 mg, 0.81 mmol, 1.0 equiv.) and tBuOK (229 mg, 2.04 mmol, 2.5 equiv.) in dry NMP (30 mL) was stirred at RT under nitrogen for 15 min. MeI (151 µL, 2.44 mmol, 3.0 equiv.) was added and the mixture was stirred at RT under N2 for 1 h. Deionized H2O was added and the product was extracted with toluene three times.

The combined organic layers were dried over anhydrous Na2SO4, filtered and the solvent was evaporated under reduced pressure. Compound 18 was obtained as a dark-orange solid (141 mg, 0.35 mmol, yield 44%). IR (ATR): ν = 3046, 2850,1650,1587,1539,1494,1468,1425,1397,1363,1283,1218,1098,1071,1045,1006,824,757,740,729,701,687,647,582,554,535,519,496,469,434,423,412,402; 1 H NMR (500 MHz, CDCl3): δ = 8.90 (d, 3 JH-H = 5.5 Hz, 2H), 8.11 (d, 3 JH-H = 5.5 Hz, 2H), 7.84 (d, 3 JH-H = 8.6 Hz, 2H), 7.75 (d, 3 JH-H = 8.6 Hz, 2H), 3.44 (s, 3H), 3.39 (s, 3H); 13 C NMR (126 MHz, CDCl3): δ = 162.3, 162.0, 150.2 (2C), 149.8, 144.7, 135.5, 132.4 (2C), 130.8 (2C), 126.9, 126.3, 122.5 (2C), 111.4, 109.4, 29.6, 29.5; HPLC (system A): tR = 4.7 min (purity 99% at 260 nm and 100% at 460 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 264 and 485 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 396.1 and 398.1 [M + H] + , calcd for C19H15BrN3O2 + 396.0 and 398.0.

N-(4-[2-[4-(dimethylamino)phenyl]diazenyl]-benzyl)pyridinium mono-(4'-pyridyl)-DPP N,N'-dimethyl 8

To a solution of 18 (22.0 mg, 56 µmol, 1.0 equiv.) in dry DMF (5 mL), benzyl bromide 19 (53.0 mg, 167 µmol, 3.0 equiv.) was added. The resulting mixture was stirred at 75 °C under N2 for 12 h. Thereafter, this mixture was evaporated under reduced pressure and the resulting residue was purified by column chromatography over silica gel (VWR technical grade, 40-63 μm, #84814.360, eluent: a step gradient of MeOH in DCM as eluent from 5% to 15%). The isolated product was dissolved in a (1:3, v/v) mixture of 0.1% aq. TFA and MeCN and (4 mL) and subjected to a further purification by semi-preparative RP-HPLC (system F, 1 injection, tR = 55.0-63.0 min of collected fractions containing the desired compound). The product-containing fractions were lyophilized to give TFA salt of 8 as a purple solid (7.9 mg, 9.5 µmol, yield 17% based on mass percentage of TFA = 24% determined by ionic chromatography, 1.8 TFA). IR (ATR): ν 2923, 1674,1629,1599,1519,1488,1429,1365,1200,1136,1073,1043,1005,943,830,798,756,720,687,639,600,583,553,511,496,469,434,413; 1 H NMR (500 MHz, CDCl3): δ = 9.20 (d, 3 JH-H = 6.4 Hz, 2H), 8.57 (d, 3 JH-H = 6.4 Hz, 2H), 7.89 (m, 4H), 7.81 (d, 3 JH-H = 8.7 Hz, 2H), 7.70 (d, 3 JH-H = 8.7 Hz, 2H), 7.62 (d, 3 JH-H = 8.2 Hz, 2H), 6.75 (d, 3 JH-H = 9.2 Hz, 2H), 6.02 (s, 2H), 3.48 (s, 3H), 3.36 (s, 3H), 3.11 (s, 6H); 13 C NMR (151 MHz, CD3CN): δ 162. 1, 161.1, 153.9, 153.3, 153.3, 144.7, 143.1, 143.0, 139.3, 133.4, 132.2, 131.3, 130.4, = 126.8, 126.5, 126.1, 125.1, 122.7, 115.5, 111.6, 109.7, 63.9, 39.5, 29.2, 29.1; 19 F NMR ( 565MHz, CD3CN) δ = -75.9 (3F, CF3-trifluoroacetate), -76.5 (0.05F, CF3-free TFA); HPLC (system A): tR = 4.9 min (purity 97% at 260 and 98% at 460 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 280 and 452 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 633.4 and 635.4 [M] + , calcd for C34H30BrN6O2 + 633.2 and 635.2.

N-(4-Nitrobenzyl)pyridinium mono-(4'-pyridyl)-DPP N,N'-dimethyl 9

To a solution of 18 (20.0 mg, 51 µmol, 1.0 equiv.) in dry MeCN (3 mL) was added 4-nitrobenzyl bromide (32.7 mg, 150 µmol, 2.9 equiv.). The resulting reaction mixture was stirred at 75 °C under N2 overnight and the disappearance of the starting material was checked by RP-HPLC (system A). Thereafter, the mixture was evaporated under reduced pressure, re-dissolved in a (1:3, v/v) mixture of 0.1 % aq. TFA and MeCN (4 mL) and purified by semi-preparative RP-HPLC (system F, 1 injection, tR = 43.0-50.0 min of collected fractions containing the desired compound). The product-containing fractions were lyophilized to give TFA salt of 9 as a purple solid (8.3 mg, 12 µmol, yield 24% based on mass percentage of TFA = 22% determined by ionic chromatography, 1.3 TFA). IR (ATR): ν = 3046, 2923, 1670, 1630, 1564, 1520, 1487, 1430, 1402, 1374, 1347, 1289, 1197, 1165, 1125, 1072, 1043, 1005, 830, 798, 759, 736, 718, 705, 686, 619, 521, 489, 441, 411; 9, 161.8, 154.2, 145.8, 144.0, 140.3, 139.9, 133.0, 132.1, 131.1, 127.6, 127.4, 126.8, 125.1, 63.7, 29.9, 29.9; 19

Synthesis of PGA-sensitive probe 10 based on unsymmetrical mono-(4'-pyridyl)-DPP 18

To a solution of 18 (20.0 mg, 51 µmol, 1.0 equiv.) in dry MeCN (5 mL) was added benzyl bromide 20 (46.0 mg, 150 µmol, 2.9 equiv.). The resulting reaction mixture was stirred at 75 °C under N2 for 12 h. The disappearance of the starting material was checked by RP-HPLC (system A) and the mixture was evaporated under reduced presssure. The residue was dissolved in a (1:3, v/v) mixture of 0.1 % aq. TFA and MeCN (4 mL) and purified by semi-preparative RP-HPLC (system F, 1 injection, tR = 47.5-55.5 min of collected fractions containing the desired compound). The product-containing fractions were lyophilized to give TFA salt of 10 as a purple solid (9.7 mg, 12.4 µmol, yield 25 % based on mass percentage of TFA = 20% determined by ionic chromatography, 1.4 TFA). IR (ATR): ν = 1672, 1631, 1602, 1537, 1515, 1488, 1416, 1373, 1199, 1131, 1073, 1044, 1006, 833, 798, 755, 719, 625, 517, 433, 8, 163.0, 162.1, 154.1, 145.5, 143.8, 141.6, 140.3, 136.6, 133.1, 132.3, 131.2, 130.2, 129.4, 128.4, 127.8, 127.7, 127.4, 127.1, 121.0, 110.6, 64.8, 44.5, 30.1, 30.

2.7

In vitro activation of DPP-based probes 7-11 by PGA or reductases -experimental details

Stock solutions of fluorogenic probes, enzymes and co-factor (NADH)

Stock solutions of DPP-based fluorogenic probes (1.0 mg/mL) were prepared in DMSO (spectrophotometry grade). Solution NTR was prepared by dissolving the commercial lyophilized powder in ultrapure water (0.1 U/µL). Stock solution of NADH (140 mM) was prepared from commercial powder and ultrapure water. Commercial PGA solution (841 U/mL) was directly used without dilution. Solution of LAP was pepared by dissolving 14.2 μL of the commercial suspension in 985.8 μL of ultrapure water (0.001 U/µL). Please note: stock solutions of parent DPP fluorophores were also prepared in DMSO (1.0 mg/mL) but prolonged sonication is required to complete solubilize N,N'-dimethyl DPP dye in this polar aprotic solvent.

Fluorescence-based assays

All assays were performed at 37 °C (using a temperature control system combined with water circulation and conducted with magnetic stirring). For all probes 7-11 (final concentration in 3.5 mL fluorescence quartz cell: 1.0 μM, volume: 3.0 mL of PB, pH 7.6), the fluorescence emission of the released mono-(4'-pyridyl)-DPP dye was monitored at l(Ex./Em.) = 440/555 nm = 5 nm, over time with measurements recorded every 5 s. For assays conducted with NTR/NADH, 1 µL of co-factor solution (final concentration in quartz cell: 47 µM) was added after 5 min of incubation of the probe in PB, and NTR (1 µL, 0.1 U) after 5 min of incubation with NADH. For assays conducted with amidase/peptidase, 1.2 μL of PGA solution (1 U) or 2 µL of LAP solution (0.002 U) was added after 5 min of incubation of the probe in PB. Blank experiments to assess the stability of the probes in PB, were achieved in the same way but without adding the corresponding enzyme. Further blank experiments were performed with PGA-sensitive probe 10, either under Ar atmosphere (bubbling of Ar for 15 min prior to incubation in PB, use of a quartz cell with screw top, sealing cap, Starna Scientific, #3-Q-10-GL14-C, to keep such Ar atmosphere), or in the presence of NaN3 (final concentration: 14.3 mM), or in acetate buffer (0.1 M, pH 5.6).

RP-HPLC-fluorescence and RP-HPLC-MS (full scan and SIM modes) analyses

Enzymatic reaction mixtures from fluorescence-based in vitro assays were directly analyzed by RP-HPLC-fluorescence after an incubation time of 60-150 min (injected volume: 20 μL, system H). Thereafter, each enzymatic reaction mixture (ca. 3 mL) was freeze-dried; the resulting white amorphous powder was dissolved in a (1:1, v/v) mixture of ultrapure H2O and MeCN (total volume = 400-1000 µL depending on the kinetics). The solution was vortexed followed by centrifugation (9 000 rpm, 1 min). 20 μL of supernatant was injected into the HPLC-MS apparatus (system I). Please note: injection of PB and H2O-MeCN (1:1, v/v) were also achieved immediately prior to this latter analysis, especially to confirm the lack of residual contaminants within the C18 column or ESI probe at the corresponding m/z value selected for the SIM detection mode and then avoid misinterpretations. For RP-HPLC (fluorescence or MS detection) analyses of mono-(4'-pyridyl)-DPP samples, 1.0 µM solution in PB was prepared and 20 μL was injected into the HPLC-fluorescence/MS apparatus (systems H and I).

In vitro assays with rat liver microsomes

All animal experiments were conducted in accordance with institutional guidelines. Rats (Wistar, ♂ 6-7 weeks) were purchased from CLEA Japan company. Rats were fasted overnight and sacrificed by exsanguination from the abdominal aorta. The liver containing 0.15 M KCl (pH 7.4) was homogenized in 3 volumes of the same buffer. Microsomes were prepared according to the method of Omura and Sato [15]. Microsomes contained 51.0 mg protein/mL and 0.82 nmol P450/mg protein. They were diluted with 0.1 M potassium phosphate buffer at pH 7.4 for assay and the final concentration was 170 µg/mL. Azo-based fluorogenic probes 8

and MAR (1.0 μM) were incubated in the presence of rat liver microsomes (vide supra) under hypoxic conditions. Fluorescence measurements were performed in PB (0.1 M, pH 7.4) containing DMSO (0.1%) as a co-solvent and the enzymatic reaction mixtures were also subjected to UHPLC analyses (see Fig. S88-S94 form more details). NADPH (50 μM) was added after 5 min. The hypoxic conditions were prepared by bubbling Ar gas into the reaction solution for 30 min.

Results and discussion

In order to simplify the synthesis of fluorogenic pyridyl-based DPP dyes, and contrary to the structure intensively studied by the Hua group [7], we have chosen to focus first on the preparation of a symmetrical derivative based on the easily-accessible 3,6-bis(4'-pyridyl)-DPP dye, equipped with two identical enzyme-recognition moieties and hence operating as a dualreactable probe. Due to the prominent role of reductases (i.e., AzoR, NTR and quinonereductases such as DT-diaphorase) in devising optical detection/imaging methods of tumor hypoxia (i.e., oxygen-deprivation state) [START_REF] Elmes | For comprehenisve reviews[END_REF] and bacterial infections [17], we selected the wellknown NTR-4-nitrobenzyl pair for rapid access to DPP-based fluorogenic enzyme substrates and to facilitate in vitro validations of their fluorogenicity. To improve aq. solubility of the overall structure while keeping a neutral state at physiological pH, the introduction of carboxymethyl arms through N-lactam alkylation has been also considered.

Synthesis and aqueous stability of NTR-sensitive probe 6 based on symmetrical 3,6-bis-(4'pyridyl)-DPP

As displayed in Scheme 1, the planned synthetic route towards the water-soluble NTRsensitive probe 6 was based on the sequential N-alkylation of lactam and pyridyl moieties of 3,6-bis-(4'-pyridyl)-DPP 4 with tert-butyl bromoacetate and 4-nitrobenzyl bromide respectively. As we had anticipated, the main difficulty associated with this synthesis was the selective N-substitution of lactam units with the protected carboxymethyl arm. On the basis of the valuable work of Vakuliuk et al. [18] identifying tBuOK as the most suited base to promote this challenging N-alkylation and by conducting a rapid screening of reaction conditions (solvent, heating temperature and duration), we have managed to optimize this step and the corresponding N,N'-dialkyl-DPP 12 was obtained in an acceptable 35% yield after purification by column chromatography over silica gel. Thereafter, quaternization of pyridyl moieties with 4-nitrobenzyl bromide was readily achieved by treatment of 12 with a large excess of this alkylating agent (7.7 equiv.) in refluxing MeCN for 24 h. It is important to specify that the addition of an inorganic base such as K2CO3, in an attempt to accelerate this reaction, has led to rapid and complete degradation of the bis-lactam-fused structure. Fully N-alkylated crude product was directly subjected to anhydrous acidic conditions TFA-DCM (1:1, v/v) for the deprotection of tBu esters, and to obtain the targeted water-soluble fluorogenic probe 6.

Unfortunately, all our attempts to isolate this highly polar compound by semi-preparative RP-HPLC (different aq. mobile phases with pH in the range 2.0-6.0) failed owing to its instantaneous conversion into the mono-lactam ring-opening product 14. Its structure was confirmed by detailed measurements including ESI mass spectrometry and NMR analyses (see supporting information for the corresponding spectra, Figs. S51-S56). To the best of our knowledge, this propensity to undergo lactam hydrolysis under acidic or neutral aq. conditions, has never been reported for DPP dyes. One possible explanation may be the strong electronwithdrawing effect of both N-(4-nitrobenzyl) pyridinium moieties that dramatically enhance electrophilicity of lactam carbonyl and hence its facile reaction with water molecules. This hypothesis is strengthened by the fact that the parent DPP fluorophore 13 bearing free pyridyl nitrogen positions, synthesized according the same overall strategy (Scheme 1), was found to be a more stable and isolatable compound. However, we noted that the acidic conditions used for its RP-HPLC purification favored its partial conversion (during the freeze-drying process) into the corresponding non-fluorescent ring-opened derivative 13-RO.

This unexpected result allows us to unambiguously state that 3,6-bis-(4'-pyridyl)-DPP 4 is not a suitable photoactive platform to construct reaction-based fluorescent probes for use in aq. environments, whose modulation of output optical signal is based on a N-pyridyl alkylationdealkylation process. Perhaps as a result, the Hua group preferred to use an unsymmetrical analog bearing a single fluorogenic 4-pyridyl group for designing esterase-and g-GT-activated ratiometric probes 1 and 3 [7a, 7c]. Thus, we have been focused on this type of DPP pigments for our next investigations.

Synthesis of AzoR-and NTR-sensitive probes based on unsymmetrical mono-(4'-pyridyl)-DPP

The synthetic route towards the unsymmetrical analog of NTR-sensitive probe discussed above, is very similar to that depicted in Scheme 1. The key step was the preparation of mono-(4'-pyridyl)-DPP pigment 5 through a two-step procedure based on literature protocols (Scheme 2 and supporting information). Contrary to the DPP structure selected by the Hua group [7a, 7c], we have chosen to work with a pigment bearing the 4-bromophenyl moiety as the second (hetero)aryl substituent. Indeed, the availability of a reactive bromine atom will enable a further functionalization of the probe, through post-synthetic Pd-catalyzed derivatization, if structural elements (and properties associated with) are specifically required for a particular biosensing/bioimaging application. Briefly, 3-ethyloxycarbonyl-2-pyrrolin-5-one 15 carrying 4-bromophenyl substituent was reacted with 4-cyanopyridine in the presence of sodium tertamylate (t-AmylONa, in situ generation through reaction of tert-amyl alcohol with metallic sodium) to give DPP pigment 5 in a satisfying 73% yield. This compound was undergone the N-alkylation/pyridine quaternization/tBu ester deprotection sequence to give the NTR-sensitive probe. Purification was achieved by semi-preparative RP-HPLC under acidic conditions (i.e., linear gradient of MeCN in aq. TFA 0.1%, pH 2.0) that enabled isolation of 7 (TFA salt) in a pure form (>95%) required for photophysical studies and enzymatic bioassays. In order to have the reference for fluorescence assays and HPLC analyses (see subsections 3.4 and 3.5), the parent DPP fluorophore 17 was prepared using the same reaction sequence whilst omitting quaternization of pyridyl moiety with 4-nitrobenzyl bromide.

Synthesis of PGA-sensitive probes based on unsymmetrical mono-(4'-pyridyl)-DPP

Due to its many positive attributes including commercial availability at a reasonable cost, stability especially under an immobilized form and structurally simple substrate (phenylacetyl moiety, PhAc), penicillin G acylase (PGA) is widely used as biocatalyst in the synthesis of blactam antibiotics [19], for kinetic resolution of racemates and as reagent for the removal of PhAc-based protecting groups. PGA is often regarded as a valuable model protease to perform in vitro validations of self-immolative molecular systems used as diagnostic probes, molecular amplifiers or drug delivery systems [20]. Furthermore, several fluorescent probes were recently developed to image endogenous bacterial PGA and thus help microbial diagnostics both in health area and food industry [21]. Thus, there is a real interest for the developement of novel fluorogenic PGA substrates with superior performances.

As illustrated by numerous examples documented in the literature, the most universal and frequently used PGA-sensitive triggering unit is para-(phenylacetamido)benzyl moiety which is readily introduced through a carbamate or an ether linkage depending on the nature and reactivity of optically tunable group to be masked [20], [22]. In the present case, the availability Finally, to facilitate comparison with results obtained by the Hua group [7a, 7c], we have also prepared the analog 11 for which the fluorogenic DPP platform is N,N'-substituted by hexyl moieties, even if we were highly sceptical about the relevance of this choice done for the construction of their esterase-and g-GT-activated ratiometric probes 4 and 6, intended to be used in aq. media. As was the case for reductase-sensitive probes, the structure of each fluorogenic PGA substrate was confirmed by detailed spectroscopic measurements including NMR and ESI-MS analyses (see supporting information for the corresponding spectra, Figs.

S25-S39 and S80-S85).

Photophysical behavior of enzyme-sensitive DPP-based probes and parent fluorophores

The spectral properties of the novel pyridine-flanked DPP fluorophores and the related enzyme-sensitive DPP-based probes were evaluated and collected parameters are summarized in Table 1 (see Figs. 2 and3 for the absorption/fluorescence spectra of 8, 10, 13, 17 and 18, and Figs. S7, S24, S40-S41, S86-S87 for 7, 9, 11 and 21). In contrast to photophysical characterizations performed by the Hua group on DPP derivatives 1-3, we have deliberately chosen phosphate buffer (PB, 0.1 M, pH 7.6) alone and not mixed with 20% DMSO as solvent for these measurements. Indeed, this allowed to mimic the real operating conditions for these Alp [23], the three fluorophores 13, 17 and 18 are characterized by a broad absorption within the blue-violet spectral range, centered at 467 nm, 471 nm and 439 nm respectively. The hypsochromic shift of the absorption wavelength of N,N'-dimethyl derivative 18 and the dramatic change in the shape of its absorption band (loss of symmetry and appearance of a shoulder) are attributed to the formation of non-emissive H-type aggregates. This hypothesis was supported by recording the excitation spectrum, which does not match the absorption spectrum (Fig. 2C). However, these aggregates are assumed to be disrupted for concentrations in the range 10 -7 -10 -8 M (against 10 -5 -10 -6 M for absorption measurements), typically used for recording emission/excitation spectra and determination of the relative fluorescence quantum yield. Thus, whatever N-lactam substituents (i.e., carboxymethyl or methyl) of mono-(4'pyridyl)-DPP scaffold, an intense fluorescence emission centered in the range 553-556 nm, was observed when the three pyridine-flanked DPP dyes (Ex. at 465 nm). Like N,N'-dialkyl mono-(4'-pyridyl)-DPP dyes being spectrally characterized in organic media [23], emission bands of 13, 17 and 18 in PB are not structured and large Stokes' shift values are obtained. Very satisfactory values of fluorescence quantum yield in pure aq. medium have been determined. This is a valuable feature that enables to compensate the relatively low molar extinction coefficients of DPP fluorophores, in the perspective of practical applications in biological contexts. Obviously, this set of results also confirms that the best way to readily convert 3,6diaryl-DPP pigments into fluorophores both soluble and strongly emissive in water, should be based on N-alkylation of their lactam groups with hydrophilic pendant arms. Indeed, this strategy leads to DPP molecules with a bolaamphiphilic character (i.e., hydrophilic substituents located on both sides of the hydrophobic bis-lactam fused core) [24] not prone to micelle formation unlike amphiphilic dyes that are often non-emissive species. Unsurprisingly, the spectral behavior of N,N'-dihexyl derivative 21 in aq. buffer is dramatically different and unsatisfactory: weak fluorescence emission and persistence of aggregates even at low concentrations that prevents determination of relative fluorescence quantum yield in PB (Fig. S86). This latter result explains why the Hua group had been chosen a PBS-DMSO (8:2, v/v) mixture for the spectral measurements and in vitro validations of esterase-and g-GT-sensitive probes 1 and 3 [7a, 7c]. The same medium was used for spectral characterization of 21 (Table 1, entry 10 and Fig. 87) but it is necessary to specify that addition of 20% DMSO in phosphate buffer changes greatly the pH value (8.5 vs. 7.6). a stock solutions (1.0 mg/mL) of fluorophores and probes were prepared in spectroscopic grade DMSO. b only 0-0 band of the S0®S1 transition is reported. c for experimental conditions used for such determination, see supporting information. d non-linear relationship between area of Em. curve and Abs value at Ex. wavelength due to aq. instability or persistence of aggregates in diluted aq. solutions, has prevented determination of relative fluorescence quantum yield.

e non-fluorescent.

f spectral measurements were achieved in PB-DMSO (8:2, v/v, pH 8.5) due to poor solubility in pure phosphate buffer. g sample of 13 used for these photophysical characterizations contains less than 5% of non-fluorescent species 13-RO.

h formation of H-type aggregates that is disrupted for concentrations in the range 10 -7 -10 -8 M used for the recording of Em./Ex. spectra. Ex. maximum is centered at 473 nm and Stokes' shift was calculated using this latter value.

When the same characterization methodology was applied to N-alkylpyridinium-based illustrating these spectral features). However, we did not manage to accurately determine this latter parameter due to the marked instability of 7 and 9-11 highlighted in PB and at low concentration (see section 3.5). The major consequence of the lack of a zero background signal for these fluorogenic probes, is that the detection of enzyme activity will be achieved through a ratiometric output signal and not an intensometric "OFF-ON" response [4b]. This will not be the case with the probe 8 that is functionalized with an azobenzene moiety knowing to act as an effective dark quencher through photochemical isomerization of its - N=N-bridge [16b, 25].

The lack of emission for 8 was borne out by fluorescence spectrum shown in Fig 3A. (5-nitro-2-furyl)methyl group), have already reported in the literature [29]. A similar negative result was also obtained when AzoR-sensitive DPP probe 8 was incubated with rat liver microsomes and NADH under simulated hypoxic conditions. Indeed, no increase in green-yellow fluorescence intensity at 560 nm (Ex. at 465 nm) was observed, thus suggesting the inertness of azobenzene moiety towards these bioreductive conditions (Figs. S88 and S89).

This was further supported by incubation of 8 with a large excess of sodium dithionite (Na2S2O4), a bio-compatible and effective reducing agent for diazo bridges highlighted by the Wagner group for applications in chemical biology [30], that did not produce the expected fluorescent "OFF-ON" response (Figs. S95 and S96). In addition to this lack of enzymatic activation, we noted a strange spectral behavior of these N-alkylpyridinium based probes when they are incubated in PB alone at 37 °C. A slow but gradual decrease of their NIR-I emission was observed, especially for 9. As illustrated in Fig. 4C, fluorescence signal was significantly reduced by a factor of 17 within 115 min of incubation. Following this blank kinetics, the recording of absorption spectrum revealed the formation of a novel non-emissive species characterized by a blue-shifted maximum wavelength (-35 nm compared to starting probe 9, Fig. 4D). Additional experiments have been done to gain insight into the mechanism of formation of this unknown product that may complicate the practical implementation of fluorogenic probes based on a pyridine-flanked DPP platform (vide infra). Curiously, this poor aq. stability observed in the absence of targeted enzyme, was not mentioned by the Hua group whereas their esterase-and g-GT-activated ratiometric probes 1 and 3 share the same structural features than our probes [7a, 7c]. That point deserves further checking. All these disappointing results led us to unambiguously state that fluorogenic DPP dyes bearing a pyridyl moiety as optically tunable group are not really suitable candidates for designing bioreductive fluorescent imaging agents intended for hypoxia detection or bacterial monitoring.

Fortunately, the sensing performances of probe 10 toward hydrolase PGA have been much more encouraging. Indeed, when this phenylacetamide derivative was incubated with the commercial amidase, a moderately rapid and gradual increase of fluorescence emission at 555 nm (Ex. at 440 nm) was observed (Fig. 5A) and the plateau was reached within 2 h of incubation. Interestingly, a direct comparison of the fluorescence emission spectra of PGAsensitive DPP probe 10 recorded before and after reaction with enzyme (Ex. at 440 nm, Fig. 5B), highlighted a spectral blue-shift of 120 nm allowing a proper ratiometric detection of PGA. S115 and S116). Furthermore, the dramatic change in the absorption profile of probe 10, already noted during in vitro bioassays of NTR-sensitive probe 9, (vide surpa), allowed us to conclude that the DPP chromophore is missing and probably degraded through hydrolytic ring-opening.

In the light of these observations, we propose an hypothetical mechanism for the formation of furo [2,3-c]pyridinium derivatives 22 and 23 (Fig. 9), even if an additional, comprehensive study involving the independent synthesis and spectroscopic characterization of these unusual heterocyclic compounds, will be required. Once again, we are truly surprised that the Hua group did not discuss the hydrolysis problem of esterase-and g-GT-sensitive probes 1 and 3 that is expected to occur in the same manner in PBS-DMSO (8:2, v/v) mixture whose pH is greater than 7.5 (vide supra) [7a, 7c].

A further control experiment aimed at proving the specificity of the enzymatic cleavage of PGA-sensitive probe 10, was achieved. Incubation with an aminopeptidase (leucine aminopeptidase (LAP) from porcine kidney [START_REF] Chen | For a review on fluorescent probes for detection and bioimaging of LAP[END_REF]) did not produce an increase of green-yellow fluorescence at 555 nm (Fig. S97), confirming that this latter protease is not able to cleave the phenylacetamide moiety. Furthermore, a direct comparison of the fluorescence emission spectra of 10 recorded before and after reaction with LAP (Ex. at 440 nm, Fig. S98) provided evidence once again of poor aq. stability of this N-alkylpyridinium-based probe. Finally, to assess the possible influence of N-lactam substituents on hydrolytic stability of probes based on N-alkylpyridinium-based DPP scaffold, more hydrophobic analog 21 bearing hexyl moieties was studied under the same conditions. As expected, intuitively, the spectral behavior and enzymatic reactivity of 11 were negatively impacted by the propensity of this compound to form aggregates in PB. The alternative use of PB-DMSO (8:2, v/v) as incubation buffer, did not produce better and valuable results in terms of ratiometric detection of PGA (Figs. S117 and S118). This confirmed the relevance of using polar or at least small size Nlactam substituents for fluorogenic DPP dyes used in the construction of reaction-based fluorescent probes working in biological media.

Conclusion

In an attempt to develop novel bioreductive fluorescent imaging agents with the aim of detecting tumor hypoxia, we assessed under-utilized photoactive platforms based on a DPP scaffold functionalized with one or two 4-pyridyl units acting as fluorogenic reactive center(s).

We have synthesized four DPP-based fluorescent probes functionalized with either 4nitrobenzyl or 4- [2-[4-(dimethylamino)phenyl]diazenyl]-benzyl moiety as NTR-or AzoRresponsive group respectively. In vitro study of all probes has highlighted on one hand the aq. unstability of the dual-reactable NTR substrate 6 derived from a symmetrical 3,6-bis-(4'pyridyl)-DPP dye and on the other hand the inability of both reductases (AzoR and NTR) to activate fluorogenic compounds based on a symmetrical mono-(4'-pyridyl)-DPP dye and designed by the protection-deprotection strategy (i.e., masking-unmasking of pyridine nitrogen) [6]. Thus, we can conclude that pyridine-flanked DPP fluorophores are not well suited to construct "smart" activity-based probes for reductase-triggered hypoxia imaging. Conversely, and as already shown by the Hua group [7a, 7c], an effective fluorogenic ratiometric detection of hydrolases (i.e., esterase and g-GT previously studied by the Hua group and PGA in the present work) was readily achievable through an enzyme-triggered self-immolative Ndealkylation process of 4'-pyridinium moiety. In this context, we are convinced that structurally and easily functionalizable fluorogenic platforms such as mono-(4'-pyridyl)-DPP dyes may be a valuable alternative to more conventional aniline-or phenol-based fluorophores typically used

Fig. 1 .

 1 Fig. 1. Selected examples of DPP-based fluorescent chemodosimeters (top) [5, 7]; structures of pyridine-flanked DPP pigments and related enzyme-responsive fluorogenic probes studied in the present work (bottom) (Cys = Lcysteine, Ac = acetyl, g-GT = g-glutamyltranspeptidase, Py = pyridyl, p-Br-Ph = p-or 4-bromophenyl, p-DMA-N2 = 2-[4-(dimethylamino)phenyl]diazenyl), PhAc = phenylacetyl, for compounds 1-3, the nature of counter-ion Y -was not specified by the authors, for 6-11, Y -= CF3CO2 -).

"

  OFF-ON" probe responsive to biothiols (phenol deprotection through S N Ar & SO 2 release) Ratiometric probe responsive to Hg(II) cations (thioacetal deprotection mediated by Hg(II)) ON" probe responsive to H 2 S & biothiols (NO 2 reduction or Michael addition) = NH, P = L-glutamyl moiety (γ-GT) Ratiometric probes responsive to hydrolases or ROS (aniline or phenol deprotection followed by 1,6-elimination) = Py + , R = CH 2 CO 2 H, P-X = NO 2 7, Ar = p-Br-Ph, R = CH 2 CO 2 H, P-X = NO 2 8, Ar = p-Br-Ph, R = Me, P-X = p-DMA-N 2 9, Ar = p-Br-Ph, R = Me, P-X = NO 2 10, Ar = p-Br-Ph, R = Me, P-X = PhAc-NH 11, Ar = p-Br-Ph, R = C 6 H 13 , P-X = PhAc-NH Py +

  C NMR (150 MHz, CDCl3): δ = 167.1 (2C), 161.6 (2C), 151.0 (4C), 146.7 (2C), 135.0 (C), 122.1 (4C), 111.4 (2C), 83.5 (C), 44.2 (2C), 28.1 (6C); HPLC (system A): tR = 4.7 min (purity 99% at 500 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 230, 260 and 477 nm LRMS (ESI+, recorded during RP-HPLC analysis): m/z = 519.2 [M + H] + , calcd for C28H31N4O6 + 519.2.

  ; HPLC (system B): tR = 1.0 min; UV-vis (recorded during RP-HPLC analysis): lmax = 260 and 456 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 425.3 [M + H] + , calcd for C20H17N4O7 + 425.1; LRMS (ESI-, recorded during RP-HPLC analysis): m/z 423.2 [M + H] -, calcd for C20H15N4O7 -423.1.

  F NMR (565 MHz, DMSO-d6): d = -73.9 (3F, CF3-trifluoroacetate), -75.2 (0.19F, CF3-free TFA); HPLC (system A): tR = 3.6 min (purity 94% at 460 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 260, 307 and 475 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 484.1 and 486.1 [M + H] + , calcd for C21H15BrN3O6 + 484.0 and 486.0.

  JH-H = 8.1 Hz, 2H), 7.88 (m, 2H), 7.82 (m, 4H), 6.04 (s, 2H), 4.59 (s, 2H), 4.53 (s, 2H); 19 F NMR (565 MHz, DMSO-d6): d = -73.5 (3F, CF3-TFA); full spectroscopic characterization of this probe through 13 C NMR measurements was not completed immediately, and an unexpected degradation occurring at the solid state has prevented us to record 13 C NMR spectrum. HPLC (system A): tR = 3.8 min (purity 98% at 260 nm and 100% at 520 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 271, 330 and 518 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 619.2 and 621.2 [M] + , calcd for C28H20BrN4O8 + 619.1 and 619.2.

  1 H NMR (500 MHz, CD3CN): δ = 8.84 (d, J = 7.1 Hz, 2H), 8.54 (d, J = 7.1 Hz, 2H), 8.30 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 5.85 (s, 2H), 3.37 (s, 3H), 3.30 (s, 3H); 13 C NMR (126 MHz, CD3CN): δ = 162.

  F NMR (565 MHz, CD3CN): δ = -75.5 (3F, CF3trifluoroacetate; HPLC (system A): tR = 3.4 min (purity 99% at 260 nm, 98 % at 460 nm and 100% at 520 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 264 and 539 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 531.2 and 533.3 [M] + , calcd for C26H20BrN4O4 + 531.1 and 533.1.

  0; 19 F NMR (565 MHz, CD3CN): δ = -76.0 (3F, CF3-trifluoroacetate); HPLC (system A): tR = 3.5 min (purity 97% at 260 nm and 100% at 460 nm); UV-vis (recorded during RP-HPLC analysis): lmax = 256 and 534 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z 619.3 and 621.3 [M] + , calcd for C34H28BrN4O3 + 619.1 and 621.1.

Scheme 1 .

 1 Scheme 1. Synthesis of water-soluble 3,6-bis-(4'-pyridyl)-DPP dye 13 and open-chain lactam form 14 of targeted NTR-sensitive fluorogenic probe 6 (Ar = argon, NMP = N-methyl-2-pyrrolidone, RT = room temperature).

Scheme 2 .

 2 Scheme 2. Synthesis of water-soluble mono-(4'-pyridyl)-DPP dye 17 and related NTR-sensitive fluorogenic probe 7 (t-AmylOH = tert-amyl alcohol or 2-methylbutano-2-ol, NMP = N-methyl-2-pyrrolidone, O/N = overnight, RT = room temperature).

Scheme 3 .

 3 Scheme 3. Synthesis of N,N'-dimethyl-DPP dye 18 and related AzoR-and NTR-sensitive fluorogenic probes 8 and 9 (NMP = N-methyl-2-pyrrolidone, O/N = overnight).

  , NMP, N2, RT, 15 min 2. MeI, N2, RT, 60 min, 44% R'X, MeCN, N2, reflux, O/N yield after RP-HPLC purification 24% alkylating agent para-(phenylacetamido)benzyl bromide 20 (see supporting information for its optimized synthesis) has enabled us to use again the synthetic route initially devised for the two other probes based on N,N'-dimethyl DPP 18, and hence to easily obtain the targeted compound 10 (Scheme 4).

Scheme 4 .

 4 Scheme 4. Synthesis of PGA-sensitive fluorogenic probes 10 and 11 and N,N'-dihexyl-DPP dye 21 (NMP = Nmethyl-2-pyrrolidone, O/N = overnight).

  , NMP, N2, RT °C, 15 min 2. MeI, N2, RT, 60 min, 44% 20, MeCN, N2, reflux, O/N yield after RP-HPLC purification 25 to draw any relevant conclusions concerning their resistance to aggregation and their stability at physiological pH. As expected for conventional 3,6-diaryl-DPP dyes, and in line with the spectral parameters of several N,N'-dialkyl mono-(4'-pyridyl)-DPP dyes already determined in pure organic solvents (e.g., DMF and MeCN) by Erbas and

Fig. 2 .

 2 Fig. 2. Normalized absorption (blue), excitation (Em 625 nm for 13 or 600 nm for 17 and 18, green) and emission (Ex. 465 nm, red) spectra of pyridine-flanked DPP fluorophores 13 (A), 17 (B) and 18 (C) in PB at 25 °C. Please note: sample of 13 used for these photophysical characterizations contains less than 5% of non-fluorescent species 13-RO.

probes 7- 11 ,

 11 some common features have been noted and confirm that a strong ICT process operates within these DPP molecules: (1) broad structureless absorption and emission profiles with full-width at half maximum (FWHM) Dl1/2max in the range 100-115 nm and 125-130 nm respectively, (2) huge Stokes' shift values, and (3) NIR-I emission with a very low fluorescence quantum yield in aq. media (see Fig. 3B for the absorption/fluorescence spectra of 10

Fig. 3 .

 3 Fig. 3. Normalized absorption (blue), excitation (Em. 700 nm only for 10, green) and emission (Ex. 465 nm, red) spectra of enzyme-sensitive fluorogenic probes 8 (A, target enzyme: AzoR) and 10 (B, targeted enzyme: PGA) in PB at 25 °C. Please note: probe 8 is not fluorescent and only the peak assigned to Raman scattering (553 nm) was observed.

Fig. 4 .

 4 Fig. 4. Fluorescence emission time course (Ex./Em. 440/555 nm, slit 5 nm) of NTR-sensitive fluorogenic probes 7 (A) and 9 (B), concentration: 1.0 µM, in the presence of NTR (0.1 U) and NADH (final concentration: 47 µM) in PB at 37 °C. (C) Fluorescence emission time course (Ex./Em. 440/675 nm, slit 5 nm) of probe 9, concentration: 1.0 µM, in PB alone at 37 °C. (D) UV-vis absorption spectra of probe 9 before and after incubation in PB (12 h at 37 °C, concentration: 15 µM).

Fig. 5 .

 5 Fig. 5. (A) Fluorescence emission time course (Ex./Em. 440/555 nm, slit 5 nm) of PGA-sensitive fluorogenic probe 10, concentration: 1.0 µM, in the presence of PGA (1 U) in PB at 37 °C. (B) Fluorescence emission spectra (Ex. at 440 nm, slit 5 nm) of fluorogenic probe 10 before and after incubation with PGA (1 U, 150 min of incubation) in PB at 37 °C.

Fig. 6 .

 6 Fig. 6. RP-HPLC elution profiles (fluorescence detection Ex./Em. 475/550 nm) of PGA-sensitive fluorogenic probe 10 (A), enzymatic reaction mixture of probe 10 with PGA, 157 min of incubation at 37 °C (B), authentic sample of fluorophore 18 (C) and co-injection of enzymatic reaction mixture and 18 (D). Please note: minor peak at 6.8 min is present in the blank sample run.

Fig. 7 .

 7 Fig. 7. RP-HPLC elution profiles of PGA-sensitive fluorogenic probe 10 before and after incubation with PGA. (A) UV detection at 260 nm of probe 10 in PB alone. (B) UV detection at 260 nm of probe 10 after enzymatic reaction. (C) ESI-MS detection, positive and SIM mode at m/z = 396.0 ± 0.5 (detection of fluorophore 18). (D) ESI-MS detection, positive and SIM mode at m/z = 619.1 ± 0.5 (detection of probe 10).

Fig. 8 .

 8 Fig. 8. RP-HPLC elution profiles of PGA-sensitive fluorogenic probe 10 after incubation in PB (0.1 M, pH 7.6) alone. (A) UV detection at 260 nm and ESI+ mass spectrum of peak at tR = 3.7 min (inset). (B) ESI-MS detection, positive and SIM mode at m/z = 673.2 ± 0.5; RP-HPLC elution profile of fluorogenic probe 10 after incubation with PGA, ESI-MS detection, positive and SIM mode at m/z = 673.2 ± 0.5 (inset). Please note: complete description of isotopic pattern of [M] + of degradation product is omitted for clarity, m/z =671.3 (90), 672.3 (30), 673.3 (100) and 674.2 (35).

Fig. 9 .

 9 Fig.9. Proposed degradation pathway of PGA-sensitive fluorogenic probe 10 observed in aq. buffers and in the lack of enzyme. a For clarity in the drawing of steps namely "fragmentation of tertiary alcohol" and "intramolecular pericyclic reaction", final protonation step of postulated carbanion intermediate is omitted. Please note:a similar degradation pathway may be proposed for NTR-sensitive fluorogenic probe 9.

Table 1 .

 1 Photophysical properties of pyridine-flanked DPP fluorophores and related enzyme-sensitive fluorogenic probes studied in this work, determined in PB (0.1 M, pH 7.6) at 25 °C.

	Fluorophore or probe a	l Abs (nm) b	l Em. (nm)	e (M -1 cm -1 )	Stokes' shift (cm -1 )	FF c
	7	516	669	14 050	4 432	-d
	8	460	-e	20 500	-e	-e
	9	529	675	16 250	4 089	-d
	10	526	679	16 250	4 284	-d
	11 f	531	613	9 300	2 519	-d

Acknowledgements

This work is part of the project "Pharmacoimagerie et Agents Theranostiques", supported by the Université de Bourgogne and Conseil Régional de Bourgogne through the Plan d'Actions Régional pour l'Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. Financial support from the French "Investissements d'Avenir" program, project ISITE BFC (contract ANR-15-IDEX-0003), especially for the post-doc fellowship of Dr. Sébastien Jenni, Agence Nationale de la Recherche (ANR, AAPG 2018, DetectOP_BChE, ANR-18-CE39-0014 and LuminoManufac-Oligo, ANR-18-CE07-0045), especially for the post-doc fellowships of Drs. Valentin Quesneau and Kévin Renault respectively is also greatly acknowledged. Flavien Ponsot gratefully acknowledges the French

in the construction of fluorogenic probes for disease-relevant hydrolases. However, the issue of slow but effective degradation of N-alkylpyridinium-based DPP dyes in aq. media, unveiled by the present study, needs to be addressed before considering further applications in biological media. That is why we encourage (bio)chemists working in the field of enzyme-responsive fluorescent chemodosimeters to routinely implement an analytical methodology involving a series of RP-HPLC-fluorescence/MS analyses and along the lines of that used in this Article, especially for deciphering precisely their activation mechanism and possible degradation pathway(s), and thus improving their performances.

CRediT authorship contribution statement

Sébastien Jenni: Conceptualization, Investigation, Formal analysis, Writing -original draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2020.119179.

References and note

A preprint was previously posted on ChemRxiv, see:


Sébastien Benzekry

Mathieu Grangeon

Mélanie Karlsen

Pauline Fleury

Maria Alexa

Isabella Bicalho-Frazeto

Solène Chaleat

Pascale Tomasini

Dominique Barbolosi

Fabrice Barlesi

Machine learning for prediction of immunotherapy efficacy in nonsmall cell lung cancer from simple clinical and biological data

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

 However, 4 out of 5 patients don't respond to single agent ICI.  Strong predictive markers for ICIs efficiency are still lacking.

 Study the predictive power of blood counts before the start of immunotherapy  Assess the utility of classical machine learning algorithms to establish a predictive model of response

Machine Learning

• Statistical analysis :

-univariable and multivariable logistic regression

• Feature selection :
-Feature importance from random forest classification algorithm (no tuning, 1,000 trees).

-Sort by mean decrease accuracy -incremental logistic regression -Optimal set of features = maximal before observing a decrease in 10-fold crossvalidated accuracy

• ML :

-Models :

-logistic regression -random forest -single layer neural network -naïve Bayes -k-nearest neighbors -support vector models -tuned and trained on the train set (3-fold cross-validation, N=200) -predictions assessed on the test set (N=100) -decision tree for interpretability Data • 298 2nd line or more stage IV NSCLC patients • Pre-treatment blood count + clinical variables • 12 variables :

-hemoglobin -platelets -leukocytes -neutrophils -lymphocytes -eosinophils -monocytes -basophils -NLR (neutrophils to lymphocytes ratio) -PLR (platelets to lymphocytes ratio) -derived NLR (absolute neutrophils count / (white blood countabsolute neutrophils count)) -BMI -patient status (dichotomized into < 2 or ≥ 2)

• Disease control (DCR) : patients with complete or partial response, or stable disease.

Blood counts prior to ICIs (elevation of hemoglobin, decrease of NLR, leukocytes or neutrophils) and clinical status (good PS) were significantly associated with better DCR in multivariable analysis. The practical application of these associations using machine learning algorithms was able to accurately predict individual response to treatment. This could be improved further by increasing the number of variables in the model and should be further validated in an independent cohort.

RESULTS

Logistic regression analysis for disease control

Variable selection

Summary of machine learning algorithms predictive performances

Machine learning algorithms predictive performances 1 COMPutational pharmacology and clinical Oncology Team, Inria Sophia Antipolis -Méditerranée, Cancer Research Center of Marseille, Inserm, CNRS, Aix Marseille University, Marseille, France.

Sébastien Benzekry 1 , 2 BACKGROUND

 12 * , Mathieu Grangeon 2, * , Mélanie Karlsen 1 , Pauline Fleury 1 , Maria Alexa 1 , Isabella Bicalho-Frazeto 1 , Solène Chaleat 2 , Pascale Tomasini 2,3 , Dominique Barbolosi 1 , Fabrice Barlesi 3,4 , Laurent Greillier 1,Machine learning for prediction of immunotherapy efficacy in nonsmall cell lung cancer from simple clinical and biological data OBJECTIVES CONCLUSION AND PERSPECTIVES MATERIAL AND METHODS  Immune checkpoint inhibitors (ICIs) are now a therapeutic standard in stage IV non-small cell lung cancer (NSCLC).

4

  Gustave Roussy Cancer Campus, International Center of Thoracic Cancers, Université Paris-Saclay, Villejuif, France. . 3 Aix Marseille University, CNRS, INSERM, CRCM, Marseille, France 2 Multidisciplinary Oncology and Therapeutic Innovations Department, Assistance Publique -Hôpitaux de Marseille, Aix Marseille University, Marseille, France. * = these authors contributed equally .