Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes
Introduction
Over the past decade, intensive research efforts have been devoted to develop novel high-performance 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 bis-lactam-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 DPP-based 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 γ-glutamyltranspeptidase (γ-GT)) [7]a), [7]c) 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 4-aminobenzyl 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].
Section snippets
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.
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 dual-reactable probe. Due to the prominent role of reductases (i.e., AzoR, NTR and quinone-reductases such as DT-diaphorase) in
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 4-nitrobenzyl or 4-[2-[4-(dimethylamino)phenyl]diazenyl]-benzyl moiety as NTR- or AzoR-responsive group respectively. In vitro study of all
CRediT authorship contribution statement
Sébastien Jenni: Conceptualization, Investigation, Formal analysis, Writing - original draft. Flavien Ponsot: Conceptualization, Investigation, Formal analysis, Writing - original draft. Pierre Baroux: Investigation. Lucile Collard: Investigation. Takayuki Ikeno: Investigation. Kenjiro Hanaoka: Formal analysis, Funding acquisition. Valentin Quesneau: Investigation. Kévin Renault: Investigation. Anthony Romieu: Conceptualization, Investigation, Formal analysis, Writing - original draft, Writing
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.
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
References (32)
- et al.
Tetrahedron Lett.
(1974)et al.Adv. Optical Mater.
(2015) - et al.
Nat. Chem.
(2012)et al.Adv. Drug Deliv. Rev.
(2017)et al.Acc. Chem. Res.
(2019)et al.Angew. Chem., Int. Ed.
(2020) - et al.
Chem. Soc. Rev.
(2015) - et al.
Mater. Interfaces
(2018)et al.Anal. Chem.
(2019)et al.J. Mater. Chem. C
(2020) - et al.
Angew. Chem., Int. Ed.
(2015)et al.Chem. Soc. Rev.
(2018) - et al.
J. Chem. Soc., Chem. Commun.
(1981) - et al.
J. Biol. Chem.
(1964) Chem. Commun.
(2016)et al.Chem. - Asian. J.
(2017)et al.Chinese Chem. Lett.
(2018)et al.Chem.-Biol. Interact.
(2019)et al.Coord. Chem. Rev.
(2020)- et al.
Chem. Soc. Rev.
(2012) - et al.
Adv. Mater.
(2017)et al.Dyes Pigm.
(2019)et al.J. Mater. Chem. C
(2019)et al.Dyes Pigm.
(2020)
Prog. Mol. Biol. Transl. Sci.
Chem. Rev.
Sens. Actuators, B
Dyes Pigm.
Sens. Actuators, B
New J. Chem.
Chem. Rev.
Adv. Funct. Mater.
Bioconjugate Chem.
Dyes Pigm.
New J. Chem.
J. Polym. Sci., Part A Polym. Chem.
Angew. Chem. Int. Ed.
Acc. Chem. Res.
Biol.
Chem. Rev.
Chem. Soc. Rev.
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These authors contributed equally to this work.