The development of a novel AND logic based fluorescence probe for the detection of peroxynitrite and GSH

We have developed a novel AND logic based fluorescence probe for the simultaneous detection of ONOO– and GSH (GSH-PF3).

Peroxynitrite (ONOO À ), is a highly reactive nitrogen species that is formed via the diffusion controlled reaction between superoxide anion (O 2 c À ) and nitric oxide (NOc). 1,2 ONOO À acts as a signalling molecule in vivo for a number of pathways. 1,3 However, ONOO À is more commonly known for its deleterious properties, causing irreversible damage to a range of biological targets such as lipids, proteins and DNA. 4 Therefore, ONOO À has been implicated as a key pathogenic factor for a number of diseases, which include inammation, cancer, ischemiareperfusion and neurodegenerative diseases. [5][6][7] Glutathione (GSH) is a natural tripeptide (g-L-glutamyl-L-cysteinyl-glycine), that exists in the thiol reduced form (GSH) and disulphideoxidised (GSSG) form. GSH is the predominant form, existing at millimolar concentrations in most cells. 8 However, GSH can be directly oxidised by ONOO À therefore acting as a cellular defence by serving as an ONOO À scavenger. Elevated levels of GSH are common in cells under oxidative stress and the susceptibility of a cell towards ONOO À largely depends on the concentration of intracellular GSH. 1,9,10 Therefore, with this work we set out to develop a uorescencebased probe capable of monitoring the close relationship between ONOO À and GSH. Traditionally, most uorescence probes require a single analyte to produce a uorescence response. [11][12][13][14][15][16][17][18][19] However, in recent years a number of uorescence based probes for dual or multi-analyte detection have been developed. [20][21][22][23][24][25][26][27] These types of uorescence based probes have been used for the construction of molecular logic gates or for medical diagnostics. 28 AND logic based uorescence probes require both analytes to be simultaneously present or work in tandem in order to elicit a uorescence response. This method has a number of advantages including: being faster than serial measurements for different analytes within the same biological sample and can provide a method for monitoring bimolecular events, which may contribute to a specic disease. 20 Currently, only a few reversible uorescence based probes for the detection of ONOO À and GSH have been developed to monitor the relationship between these analytes. 29,30 These include a selenium based uorescence probe, which is oxidised by ONOO À (turn "on") and reduced by GSH (turn "off"). The uorescence of the CyPSe probe is initially quenched by a photoinduced electron transfer (PET) process. The presence of ONOO À results in the oxidation of selenium to CyPSe]O causing the uorescence emission to be "turned on". Then in the presence of biological thiols such as cysteine and GSH, the CyPSe]O probe is reduced back to its non-uorescent selenide form. A similar reversible NIR Tellurium-based uorescence probe was later developed for monitoring the redox cycles between ONOO À and GSH. This probe was successfully applied for the visualisation of the redox cycles of ONOO À and GSH in live cells and animals. 29 The probes developed by Han et al. are turn "on" with ONOO À and "off" with GSH. As far as we are aware, there are currently no AND logic based uorescence probes for GSH "and" ONOO À .
With this research, we aimed to develop a GSH "and" ONOO À logic based uorescence probe (Scheme 1). We identi-ed a suitable mono-boronate uorescein based uorescence probe, PF3 that had been previously developed by Chang et. al., shown in Scheme 2. As previously reported the reactivity of boronate based uorescence probes with ONOO À , 12,14,15 are signicantly greater than hypochlorite (ClO À ) and H 2 O 2 . 31 Therefore, we anticipated that the attachment of a GSH reactive motif to PF3 would produce a selective GSH-ONOO À AND logic based uorescence probe, GSH-PF3 (Scheme 2). GSH-PF3 was readily synthesised in three steps. Fluorescein was triated using N-phenyl bis(triuoromethanesulfonamide) to afford uorescein mono-triate in good yield. Suzuki-Miyaura conditions were then carried out to provide uorescein monoboronate, PF3. The 2,4-dinitrobenzenesulfonyl unit was then attached to PF3 using 2,4-dinitrobenzenesulfonyl chloride, CH 2 Cl 2 and NEt 3 at 0 C. Using these conditions GSH-PF3 was prepared in a reasonable yield of 52%.
With GSH-PF3 in hand, uorescence experiments for the detection of GSH and ONOO À were performed. As shown in Fig. 1, GSH-PF3 was initially non-uorescent and with the addition of ONOO À (10 mM), a small uorescence increase was observed. However, incremental additions of GSH resulted in a much larger increase in uorescence intensity (>30-fold see ESI - Fig. S1 †), clearly demonstrating the need for the addition of both GSH and ONOO À in order to achieve a full 'turn-on' response.
In order to demonstrate that GSH-PF3 required both GSH and ONOO À for a complete 'turn-on' response, the uorescence experiments were performed the other way around. Therefore, an excess of GSH (200 mM) was added to GSH-PF3, and the probe was incubated for 10 min. Remarkably, this only led to a small increase in uorescence intensity and the subsequent additions of ONOO À resulted in a large uorescence increase ( Fig. 2 and ESI -Fig S2 †). These results conrm that the probe requires both GSH and ONOO À for a full uorescence 'turn-on' response.
The selectivity of GSH-PF3 was then evaluated against a series of amino acids (L-cysteine, L-methionine, L-tryptophan, L-serine, L-lysine, L-leucine, L-glutamic acid, L-valine, L-arginine, L-histidine and L-aspartic acid)see ESI Fig. S3. † Unsurprisingly, the probe responded to the other sulydryl containing amino acid cysteine. However, the biological concentrations of cysteine are low in cells. 32 More importantly, GSH-PF3 displayed an excellent selectivity against other amino acids including serine, methionine and lysine. The probe demonstrated excellent selectivity for ONOO À over many other ROS including H 2 O 2 (see ESI - Fig. S4 †). The excellent selectivity for both GSH and ONOO À allowed us to evaluate GSH-PF3 in cell imaging experiments.
The macrophage cell line -RAW264.7 (ATCC® TIB-71™; obtained from ATCC [American Type Culture Collection])was used for cell imaging experiments. The cells were incubated with GSH-PF3, followed by either treatment of SIN-1 (an ONOO À donor) 33 to produce intracellular ONOO À or by addition of Scheme 1 Design concept for AND logic based fluorogenic probe. A fluorescence dye is masked by two functional groups, which respond to two different analytes. The fluorogenic probe requires both analytes to be present or to work in tandem in order to produce a response.
Scheme 2 Structure of the GSH-PF3 probe and proposed sensing mechanism for the simultaneous detection of ONOO À and GSH. exogenous GSH. As shown below in Fig. 3, addition of GSH separately resulted in no uorescence response, whereas ONOO À led to a small uorescence response in cells. This observation is believed to be due to the presence of a low levels of endogenous biological thiols in the cells, resulting in the activation of the probe's uorescence. As predicted, treatment of cells with both GSH and SIN-1 led to a clear uorescence increase in RAW264.7 cells with GSH-PF3, in clear agreement with the analytical data obtained on the uorimeter.
We then turned our attention to evaluate the ability of GSH-PF3 for monitoring the co-existence of metabolically produced ONOO À by lipopolysaccharide (LPS) simulation 34 and GSH through a drug treatment. 35 It has been reported that LPS can elicit ONOO À in macrophage, which is a signature of inammation, whereas caffeic acid (CA) is commonly used to treat inammation through the augmentation of intracellular GSH. Consequently, treatment with LPS, followed by the addition of increasing CA (0-100 mM) led to a gradual enhancement in the uorescence intensity of GSH-PF3 (Fig. 4). This result is due to the co-existence of ONOO À produced by LPS stimulation as well as GSH elicited by CA. Interestingly, a further increase of the CA concentration (>100 mM) led to the suppression of probe's uorescence, which is believed to be due to the production of an excess of GSH, resulting in quenching of the ONOO À (Fig. 4). To corroborate the presence of intracellular GSH in macrophage, a commercial GSH probe was used. The result suggested that both the exogenous addition of GSH and stimulation of metabolic GSH by treatment of CA/LPS activated the commercial probe's uorescence (Fig. S5 †). Furthermore, a cell proliferation assay indicated that GSH-PF3 is not toxic for RAW264.7 cells (Fig. S6 †).   These results clearly demonstrate the potential of AND logic based uorescence imaging probes for optimizing drug dosage in the treatment of inammation and various other diseases. In addition this system could be used to help monitor the treatment of Alzheimer's disease (AD), since it is known that high levels of ONOO À correlate with oxidative stress and the progression of AD, while, high levels of GSH have been implicated in AD therapy. [36][37][38][39] Therefore, we believe that our GSH-PF3 probe, which can detect GSH "and" ONOO À could potentially be used to monitor cellular resistance towards ROS and map therapeutic improvements in AD victims.
In summary, GSH-PF3 is an easy-to-prepare uorescence based probe providing a platform for the development of other novel AND logic based uorescence imaging probes for use in medical diagnostics. We are currently exploring the use of 6amino/carboxyuorescein, which we believe could provide the opportunity to attach additional uorophores in order to develop ratiometric uorescence sensors 40 or for the attachment of targeting or therapeutic units. [41][42][43]

Conflicts of interest
No conicts of interest.