Pinkment: a synthetic platform for the development of fluorescent probes for diagnostic and theranostic applications†

Reaction-based fluorescent-probes have proven successful for the visualisation of biological species in various cellular processes. Unfortunately, in order to tailor the design of a fluorescent probe to a specific application (i.e. organelle targeting, material and theranostic applications) often requires extensive synthetic efforts and the synthetic screening of a range of fluorophores to match the required synthetic needs. In this work, we have identified Pinkment-OH as a unique “plug-and-play” synthetic platform that can be used to develop a range of ONOO− responsive fluorescent probes for a variety of applications. These include theranostic-based applications and potential material-based/bioconjugation applications. The as prepared probes displayed an excellent sensitivity and selectivity for ONOO− over other ROS. In vitro studies using HeLa cells and RAW 264.7 macrophages demonstrated their ability to detect exogenously and endogenously produced ONOO−. Evaluation in an LPS-induced inflammation mouse model illustrated the ability to monitor ONOO− production in acute inflammation. Lastly, theranostic-based probes enabled the simultaneous evaluation of indomethacin-based therapeutic effects combined with the visualisation of an inflammation biomarker in RAW 264.7 cells.


Introduction
There is a growing need for new and effective diagnostic tools that can evaluate biomarkers involved in inammatory based diseases. [1][2][3][4][5][6] Inammation is the innate defence mechanism of the body that recognises damaged cells, pathogens and infections. The inammatory response oen results in the generation of reactive oxygen species/reactive nitrogen species (ROS/RNS), which are involved in the functional regulation of M1 and M2 macrophages. 7,8 The M1 pro-inammatory phenotype is induced by lipopolysaccharide (LPS), which triggers the generation of ROS from NADPH using NADPH oxidase (NOX). 9 This production of ROS regulates an array of cellular events including the activation of the nuclear factor kappa-B (NF-kB), the production of cytokines and cell survival whereas, high levels of ROS are associated with programmed cell death, i.e. apoptosis. 7,[10][11][12][13][14] The high sensitivity and high spatial and temporal resolution of uorescent probes allow us to visualise these key cellular events. Our group and others have focused on the uorescence-based detection of ROS/RNS such as ONOO À , H 2 O 2 and HOCl. 1,[15][16][17][18][19][20][21] To achieve the selective detection of a particular ROS requires the careful consideration of both uorophore and reactive motif. In this regard, resorun is a particularly attractive uorophore due to its red shied uorescence and easy to functionalise scaffold. Pioneering work led by Chang et al. developed peroxyresorun-1 (PR1) for H 2 O 2 detection whereby resorun is masked with boronic esters. 22,23 Boronic esters have been identied as a relevant sensing group for both H 2 O 2 and ONOO À detection. However, in an environment with both species present, boronic esters preferentially react with ONOO À due to the inherent faster reactivity of ONOO À in comparison to H 2 O 2 . 24 Previously, we have demonstrated PR1's ability to preferentially detect ONOO À over H 2 O 2 in vitro. 25 Consequently, we decided to investigate functionalized synthetic derivatives of PR1. 26 This led to the development of Pinkment-OH for the design of dual analyte ANDlogic probes, Pinkment-OTBS (ONOO À "AND" uoride) and Pinkment-OAc (H 2 O 2 "AND" esterase) using Pinkment-OH (Fig. 1) as a synthetic starting point. These results revealed the potential of Pinkment-OH to be used as a synthetic platform for the development of ONOO À selective uorescence probes with additional sensing, targeting or drug units. Here, we have serendipitously discovered that the benzyl unit of our ROS Pinkment uorescent probe can be functionalized with a functional unit of choice without compromising ROS selectivity. As a result, Pinkment-OH was successfully shown as a synthetic platform to develop ONOO À selective uorescent probes with additional functional units ( Fig. 1).

Results and discussion
Initially, our focus was on continuing the development of "AND"-based logic-gates for biological application. 1,17,26 This led to the elaboration of probe 1, which was accessed in a simple three step synthesis (Scheme S1 †). Unexpectedly, we discovered that 1 "turned on" in the sole presence of ONOO À (Fig. 1B and S1 †). This led to the development of Pinkment probes 2 and 3 to further conrm this observation. These probes were accessible from the synthetic platform Pinkment-OH, whose 6-step synthesis has been previously reported by our group. 26 Nucleophilic substitution by Pinkment-OH using 1-bromopropane and pentanoyl chloride respectively gave 2 and 3 in moderate yields: 50% and 51% respectively (Scheme S2 and S3 †). Probes 2 and 3 showed good selectivity towards ONOO À over other ROS species ( Fig. S2-S8 †). Surprisingly, the probes demonstrated a high sensitivity towards ONOO À requiring concentrations in the low micromolar range. Both 2 and 3 displayed increased solubility in comparison to 1. We decided to further explore this unexpected result by introducing a terminal nitrile group. Probe 4 was accessible in a facile three-step synthesis (Scheme S4 †) in the same manner as 1. Again, good selectivity and sensitivity for ONOO À was observed ( Fig. S2-S8 †). From these results, we realized that the Pinkment benzyl unit can be functionalized with any unit of choice without compromising the ROS selectivity. Thus, we rationalized that Pinkment-OH offers a unique platform for the design of ONOO À selective uorescence based probes that can be tailored towards a range of applications. [27][28][29] This led to the development of alkyne-based Pinkment probes 5 and 6 that have potential to be used in "click" chemistry. 29 These probes were accessed from Pinkment-OH and prepared in moderate yields: 48% and 47% for 5, and 6 respectively (Scheme S5 and S6 †). Fluorescence studies of 5 and 6 established good sensitivity and selectivity towards ONOO À over other ROS (Fig. S2-S8 †).
We then turned our attention to assessing the imaging capacity of probes 2 and 3 and the potential "click" based probes 5 and 6 in cells and live animals. To demonstrate their suitability as imaging tools, all four probes were evaluated for cellular toxicity in murine RAW 264.7 macrophages using a MTS cell proliferation assay. Probes 2, 3, 5 and 6 were incubated at different concentrations ranging from 5 to 40 mM for 24 h (Fig. S9 †). Probes 2, 5 and 6 were found to be non-toxic. In contrast, probe 3 decreased the cell viability of RAW 264.7 macrophages by 40% at a concentration of 40 mM compared to control conditions. As a result, 3 was not taken forward for further cell studies since high concentrations of the probe are required for in vivo studies.
Probes 2, 5 and 6 were shown to be non-toxic, and were evaluated with exogenous ONOO À , using SIN-1 (500 mM) in RAW 264.7 macrophages ( Fig. 2A and S10 †). Each probe alone demonstrated minimal uorescence in cells, the addition of SIN-1 led to a signicant enhancement in intracellular uorescence at a wavelength corresponding to the dye, resorun, therefore, suggesting the intracellular reaction of the probe with ONOO À and their suitability for use as uorescence-based probes. The SIN-1 generated uorescence signal was then evaluated with the ONOO À scavenger, uric acid. 30 As expected, uric acid attenuated the uorescent increase that was induced by SIN-1 for all probes, thus conrming the ONOO À mediated increase in uorescence intensity. Next, we evaluated the capability of 2, 5 and 6 to detect endogenous ONOO À in LPS primed RAW 264.7 macrophages. All three probes were shown to detect endogenous ONOO À in LPS primed RAW 264.7 macrophages ( Fig. 2A and S11 †), conrming their promise for the imaging of LPS-induced inammatory responses. In addition, HeLa and A549 cell lines treated with or without SIN-1 were used to illustrate the versatility of the Pinkment probes ( Fig. S12 and S13 †).
Encouraged by these cell imaging results, we used a known LPS-induced inammation mouse model 31 for the in vivo detection of ONOO À (Fig. 2B). The injection of LPS (2 mg mL À1 in saline) to the abdominal region of mice followed by the injection of 6 (200 mM) led to its uorescence activation. The quantied uorescence intensity in the probe(+)/LPS(+) group was signicantly larger than that in the probe(+)/LPS(À) group (Fig. 1C), demonstrating the potential of using 6 for the monitoring of ONOO À in situ during acute inammation.
In order to follow our current interest in theranostics, 32 we then turned our attention towards the potential of Pinkment-OH for the design of uorescence-based drug releasing probes. Therefore, we used the drugs chlorambucil and indomethacin to afford two distinct theranostic probes 7 and 8, respectively (Fig. 1A). Chlorambucil is used to treat chronic lymphatic leukemia 33 and indomethacin is used as a non-steroidal anti-inammatory drug (NSAID). 34,35 Both 7 and 8 were easily accessible from Pinkment-OH (Scheme S7 and S8 †).
Mass spectrometry conrmed and validated the simultaneous release of each drug and uorescent resorun dye ( Fig. S14 and S15 †). Therefore, the enhancement in the uorescence intensity over time indicates the release of each drug. As such, time-dependent uorescence experiments with 7 and 8 in the presence of ONOO À were performed to illustrate the time dependence of the drug release. These experiments revealed a maximum uorescence response aer $10 min (Fig. S16 †).
Fluorescence studies were carried out including ROS selectivity, H 2 O 2 titration and ONOO À titration studies (Fig. S17-S21 †) and demonstrated high sensitivity towards these inammation-based biomarkers. Following these initial studies, we evaluated both 7 and 8 in RAW 264.7 macrophages towards exogenous ONOO À detection (Fig. S22 †). The presence of SIN-1 signicantly enhanced the intracellular uorescence of 7 and 8, conrming the applicability of the probes in vitro. Despite 7 displaying signicant promise, the creation of an appropriate model system to differentiate between cancerous and healthy cells would require a signicant amount of development and as such was beyond the scope of this current research. Therefore, only 8 was further evaluated, since its cellular behaviour was easier to monitor. Endogenous ONOO À was also detected by 8 in RAW 264.7 macrophages (Fig. 3A). Indomethacin, a NSAID, is an effective and non-selective This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8567-8571 | 8569 inhibitor of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), of which COX-2 is mainly responsible for the inammatory response. 36 The therapeutic effects on the LPS-induced inammatory responses in RAW 264.7 macrophages were further investigated using 8. RAW 264.7 macrophages were treated with LPS and the expression of the pro-inammatory gene (COX-2) was investigated using qRT-PCR in the presence or absence of 8 (Fig. 3B). 37 The mRNA level of COX-2 decreased in the presence of 8 (50 mM) in comparison to the LPS-induced group. A similar effect to the LPS-induced group was observed with indomethacin alone. This suggests that 8 can monitor ONOO À production in acute inammation, and in addition, reduce the inammatory response by releasing indomethacin.

Conclusions
The ability of the Pinkment scaffold to be functionalised with any unit of choice without compromising the overall ROS selectivity, opens up new possibilities for the design of highly specic ONOO À probes that can be used in a variety of applications. In this work, we have successfully illustrated the applicability of Pinkment-based probes for diagnostic and theranostics applications. Our probes displayed good selectivity and sensitivity towards ONOO À over a range of other ROS. Cellular studies with the Pinkment probes led to the identication of alkyne-functionalised Pinkment probe 6 as a suitable candidate for in vivo studies using an inammatory mouse model. These promising results led us to design potential theranostic probes 7 and 8 with candidate 8 displaying promising properties in vitro. We believe this work demonstrates Pinkment-OH as a useful synthetic platform to enable the rapid development of a ONOO À uorescent probe that can be tailored to the needs of the chemical biologist. In particular, the alkyne Pinkment probes offer the possibility of attaching any desired unit via click chemistry. Therefore, we anticipate that the Pinkment scaffold can be further elaborated for the development of dual analyte, organelle targeting and theranostic probes for a range of diagnostic and theranostic applications.

Conflicts of interest
There are no conicts to declare. Central Universities (222201717003) and the Programme of Introducing Talents of Discipline to Universities (B16017) for nancial support. Characterization facilities were provided through the Material and Chemical Characterization Facility (MC 2 ) at the University of Bath (http://go.bath.ac.uk/mc2). In particular, we would like to acknowledge Shaun Reeksting for helping with the more advanced MS studies of the theranostic probes. All data supporting this study are provided in the ESI † accompanying this paper.