Biological hydrogen peroxide detection with aryl boronate and benzil BODIPY-based fluorescent probes

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Highlights

  • First study comparing the optical and sensing properties between the two reactive architectures against hydrogen peroxide.

  • Two new probes, peroxy BODIPY-1 (PB1) and nitrobenzoyl-BODIPY (NbzB) were developed.

  • Sensing properties such as biocompatibility, cell permeability, selectivity and photostability were detailed.

  • Sensing experiments in bovine oocytes were carried out as the first step towards detecting H2O2 associated with an increased incidence of DNA damage in developing embryos.

Abstract

The detection of hydrogen peroxide (H2O2) using fluorescent probes is critical to the study of oxidative stress in biological environments. Two important sensing architectures for detecting H2O2, aryl boronates and benzils, are compared here using novel boron-dipyrromethene (BODIPY) fluorescent probes. The aryl boronate PeroxyBODIPY-1 (PB1) and benzil-based nitrobenzoylBODIPY (NbzB) were synthesised from a common BODIPY intermediate in order to compare sensitivity and selectivity to H2O2. The aryl boronate PB1 gives the highest change in fluorescence on reaction with H2O2 while the benzil NbzB exhibits exclusive selectivity for H2O2 over other reactive oxygen species (ROS). Both proved to be cell-permeable, with PB1 being able to detect H2O2 in denuded bovine oocytes. The strengths of these aryl boronate and benzil probes can now be exploited concurrently to elucidate biological mechanisms of H2O2 production and oxidative stress.

Introduction

Hydrogen peroxide (H2O2) is an important reactive oxygen species (ROS) that acts as both a cellular signaller [1] and a causative of oxidative stress [2]. Such cellular stress can have serious consequences for cell function, e.g. it results in defective sperm function [3] and compromised embryonic development in reproductive biology [4]. The detection of H2O2 is thus of real importance in understanding both the mechanisms of oxidative stress and a range of cellular signalling processes.

More specifically, fluorescent probes are commonly used for in situ detection of H2O2 and real-time monitoring of associated processes within an oocyte or embryo [[5], [6]]. These fluorophores function by giving a measurable change in fluorescence on reaction with H2O2. While many fluorescent probes for H2O2 (e.g. 2′,7′-dichlorohydrofluorescein diacetate) [7] suffer from a lack of specificity for H2O2 over other biologically relevant ROS, fluorescent probes bearing the aryl boronates [[7], [8], [9]] and benzyl reactive groups are known to exhibit selectivity for H2O2 (see Fig. 1A and B for reaction mechanisms) [[10], [11]] and are important as they provide an opportunity to elucidate the identity of specific oxidants in biological production of ROS [12]. Unlike many fluorescent probes (e.g. 2′,7′-dichlorohydrofluoresceindiacetate), [7] aryl boronates [[7], [8], [9]] andbenzil-based systems exhibit good selectivity for H2O2 over other biologically relevant ROS (see Fig. 1A and B for reaction mechanisms). [[10], [11]] These probes thus provide an opportunity to help identify specific oxidants involved in the biological production of ROS [12].

An experimental comparison of the ability of aryl boronate and benzil fluorescent probes to detect H2O2 is required, as comparative strengths and weaknesses of each probe is critical to understand the role of H2O2 in biological processes. A peroxide based probe should have the following criteria to be useful in biological applications. In general, the probe should 1) be biocompatible i.e. excellent aqueous solubility and function at physiological pH; 2) be cell permeable without the need for further chemical modifications; 3) have high selectivity for H2O2 over other biologically relevant species; and 4) be photostable under typical confocal microscopy experiment conditions i.e. minimal photobleaching when exposed to laser light from the confocal microscope.

Existing fluorescein-based aryl boronate and benzil probes [[10], [13]], are known to have relatively poor photostability at higher concentrations of H2O2 where fluorescein was found to photobleach rapidly [14]. The overall measured change in fluorescence from the fluorescein-based probe may not then reflect the true concentration of H2O2, and thus would not be ideal for a comparative study. In contrast, the boron-dipyrromethene (BODIPY) fluorescent core structure is known to have improved photostability compared to fluorescein and properties such as biocompatibility and spectroscopic properties can be readily tuned with minor structural modifications [15].

Two new probes, peroxyBODIPY-1 (PB1) and nitrobenzoyl-BODIPY (NbzB) (Fig. 1C) are reported here to allow a direct comparison of aryl boronate and benzil-based detection of H2O2. The H2O2-sensitive boronate (Fig. 1A) and para-nitrophenyl diketone (Fig. 1B) groups were attached at analogous positions from the BODIPY core for both of PB1 and NbzB, respectively. This study investigates the sensitivity and selectivity of PB1 and NbzB to H2O2 and applies both to use in bovine oocytes. The utility of the aryl boronate and benzil probes is here directly compared in vitro, to give a clear insight into the class of probe that should be chosen for detecting H2O2 in biological environments.

Section snippets

Materials and general procedures

All reagents were purchased from Sigma-Aldrich unless otherwise stated. Tetrahydrofuran (THF) was purchased from Scharlau and dried using an Innovative Technology Pure-Solv solvent purification system. Davsil silica gel (40–63 μm) was used for column chromatography. 1H and 13C NMR spectra were recorded in CDCl3 (Cambridge Isotope Laboratories, Cambridge, MA) at 26 °C on an Agilent Technologies 500 MHz NMR with DD2 console, then analysed using Bruker TopSpin 3.2. FTIR spectra were recorded using

Synthesis

PB1 and NbzB were synthesized from common intermediate (3), which was readily prepared on reaction of 2,4-dimethylpyrrole 1 with 4-iodobenzoic acid 2, see Scheme 1. Reaction of 3 with bis(pinacolato)diboron under Suzuki conditions gave PB1 in a 48 % yield. Alternately, reaction of intermediate 3 with 4-ethynyl-1-nitrobenzene 4, under Sonigashira conditions, gave 5 as an immediate precursor to NbzB. The alkyne functionality on compound 5 was oxidised with DMSO and PdCl2 to give NbzB in 54% yield

Conclusions

PB1 and NbzB were synthesised from a common intermediate BODIPY. These probes were compared to determine the respective sensitivity and selectivity of the aryl boronate and benzil groups to H2O2. PB1 showed higher sensitivity to H2O2 than did NbzB, with a greater fluorescent response. PB1 also showed a good fluorescent response to H2O2 in bovine oocytes. Despite less sensitivity to H2O2, NbzB was more selective for H2O2 over other ROS than PB1. Hence it is advantageous to utilise benzils to

Acknowledgements

This research was supported in part by Cook Medical Pty Ltd, Australian Research Council linkage grant LP110200736, and the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP)CE140100003. This work was performed in part at the OptoFab node of the Australian National Fabrication Facility (ANFF) utilizing Commonwealth and SA State Government funding. This research was also undertaken in part on the NCI National Facility in Canberra, Australia, which is supported by the Australian

Malcolm Purdey is an Adjunct Fellow at the University of Adelaide and Associate Investigator in the ARC Centre of Excellence in Nanoscale BioPhotonics (CNBP). Malcolm obtained his PhD from the University of Adelaide in 2015 and subsequently worked in the Institute for Photonics and Advanced Sensing (IPAS) with Professor Andrew Abell. His research focuses on the application of fluorescent sensors for detecting reactive oxygen species in biological systems.

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  • Cited by (0)

    Malcolm Purdey is an Adjunct Fellow at the University of Adelaide and Associate Investigator in the ARC Centre of Excellence in Nanoscale BioPhotonics (CNBP). Malcolm obtained his PhD from the University of Adelaide in 2015 and subsequently worked in the Institute for Photonics and Advanced Sensing (IPAS) with Professor Andrew Abell. His research focuses on the application of fluorescent sensors for detecting reactive oxygen species in biological systems.

    Hanna McLennan graduated from the University of Adelaide with B.Health.Sc. (Hons) in 2015 and is currently a PhD candidate within CNBP and Robinson Research Institute (RRI) under the supervision of Associate Professor Jeremy Thompson and Drs. Melanie Sutton-McDowall and Sabrina Heng. Her research has a trans-disciplinary focus using tools developed by chemists and physicists within the CNBP to investigate and detect the signals produced by the oocyte during in vitro fertilisation.

    Melanie Sutton-McDowall is an Adjunct Fellow in the Adelaide Medical School and previously was a Senior Research Associate at CNBP and the Robinson Research Institute. Dr Sutton-McDowall obtained her PhD in Health Sciences, The University of Adelaide, in 2005 and the focus of her research was how the metabolic activity of oocytes and embryos are influenced by the surrounding environment, both in vivo and in vitro.

    Daniel Drumm received his Ph.D. from The University of Melbourne (2013), and is now a Research Fellow with CNBP in Physics at RMIT University. He has modelled a range of systems from semiconductor defects for quantum computation and communications, through transition metal oxides, organometallic crystals, ternary chalcogenide glasses, polymer-dye-sensitised solar cells, nuclear physics, and now fluorescent-dye-based sensors. He has also worked on a cloud-based computation system, and is currently focussing on quantum correlation imaging, transition-metal dichalcogenides, search algorithms for accelerating microscopy, and collaborating on applying modelling techniques to other systems.

    Xiaozhou Zhang graduated from the University of Adelaide with B.Sc (Hon) and Ph.D. under the supervision of Professor Andrew Abell. She is currently undertaking post-doctoral research at CNBP and her research interests include developing biocompatible sensors and sensing strategies for detecting biologically-relevant substrates.

    Patrick Capon graduated from the University of Adelaide with BSc (Advanced) and MPhil. in 2016 and is currently a PhD candidate within the CNBP under the supervision of Professor Andrew Abell and Dr. Malcolm Purdey. His research focuses on developing multi-component biological sensors consisting of nanodiamond and organic fluorescent probes.

    Sabrina Heng is a Senior Research Associate at CNBP. Sabrina obtained her PhD from Boston College (MA, USA) in 2009. Shortly after, she joined the Institute for Photonics and Advanced Sensing (IPAS) and School of Chemistry and Physics to work with Professors Andrew Abell and Tanya Monro. In 2010, she was awarded the inaugural ARC Super Science Fellowship to develop light-driven sensors for metal ions.

    Jeremy Thompson is an Associate Professor at the University of Adelaide, the Head of Early Development Group and chief investigator with the CNBP. His overall research interest is the impact of the micro-environment surrounding oocytes and embryos, especially nutritional factors, within both the in vivo (follicular/oviduct/uterine) and in vitro environment. This encompasses hypoxia, hypoxia inducible factors and their role in reproduction

    Andrew Abell is a Professor of Chemistry, Adelaide node leader CNBP and is an Australian Fulbright ambassador. He completed his PhD in Chemistry at the University of Adelaide in 1985 and then a two-year post-doctoral fellowship at the University of Cambridge. He held a professorship at the University of Canterbury, before returning to Adelaide in 2007.

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