Coumarin-based fluorescent ‘AND’ logic gate probes for the detection of homocysteine and a chosen biological analyte

With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO− reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO− (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

Homocysteine (HCys) is a non-proteinogenic amino acid, formed from the de-methylation of methionine, 1 which is then converted into cysteine (Cys) via a vitamin B 6 cofactor. Typical physiological concentrations of HCys range between 5-15 mmol L À1 . 2 However, elevated levels of HCys (>15 mmol L À1 ), which is known as hyperhomocysteinemia (hHCys), 3 have been associated with pregnancy disorders, Alzheimer's disease, cardiovascular disease and neurodegenerative diseases (NDs). 4-6 It is believed that the main cause of HCys induced toxicity is through the non-enzymatic modication of proteins. This is achieved through irreversible covalent attachment of the predominant metabolite of HCys, homocysteine thiolactone (HTL), to lysine residues; a phenomenon known as 'protein N-homocysteinylation' that results in the loss of a proteins structural integrity leading to loss of enzymatic function and aggregation. 7 A number of uorescent sensors have been developed for the detection of HCys to help improve our understanding of its role in biological systems. [8][9][10][11] However, these uorescent probes have focused on the detection of a single biomarker (HCys), however, processes associated with HCys induced toxicity oen involve more than one biochemical species. For example, it has been reported that peroxynitrite (ONOO À ) and nitric oxide (NOc) play a signicant role in HCys-mediated apoptosis in trigeminal sensory neurons 12 and HCys has been reported to induce cardiomyocytes cell death through the generation of ONOO À . 13 The production of ONOO À is believed to be the result of an increased production of superoxide (O 2 c À ) by HCys activating the enzyme NADPH oxidase. [14][15][16] This increased production of O 2 c À leads to a reduction in the bioavailability of NOc by increasing the formation of ONOO À (NOc + O 2 c À / ONOO À ). 17 The reported ONOO À concentrations in vivo are believed to be approximately 50 mM but, higher concentrations of 500 mM have been found in macrophages. 18,19 Furthermore, hypoxia has been reported to facilitate HCys production in vitamin-decient diets 20 where hypoxia leads to an upregulation of nitroreductase (NTR)a reductive enzyme upregulated in cells under hypoxic stress. 21,22 Therefore, the development of tools that enable an understanding of the relationship of HCys with these biologically important species would be highly desirable.
To achieve this, a number of uorescent probes have been developed that are capable of detecting two or more analytes. 23 These include AND logic gate based-uorescence probes, which require both analytes to work in tandem to produce a measurable optical output. [24][25][26][27][28] In our group, we have developed several 'AND' reaction-based probes for the detection of ROS/RNS and a second analyte. [29][30][31][32] These 'AND' logic scaffolds have been used to detect two analytes within the same biological system. 24,33 Owing to the pathological role of HCys, we set out to develop the rst example of a uorescent probe for the detection of HCys and biological related analyte. Aiming towards that target, we became interested in a previously reported coumarin-based uorescent probe developed by Hong et al. CAH, with a salicylaldehyde ( Fig. 1). 34 Salicylaldehyde is a known reactive unit towards HCys/Cys, therefore we believed CAH could be used as a scaffold for the development of 'AND'-based systems for the detection of HCys/Cys and a second analyte. 34 In the presence of HCys, CAH exhibited a 'turn-on' uorescence response which is attributed to the nucleophilic nature of the nitrogen and sulfur atoms resulting in thiazine ring formation (Scheme S1, Fig. S1 and S2 †). [34][35][36] We believed that CAH was a useful core unit that can be used to introduce the chosen reactive chemical trigger on the phenol for the detection of the corresponding analyte with HCys/Cys. Owing to the relationship between HCys and ONOO À /NTR, we set out on the development of a HCys AND ONOO À probe and a HCys AND NTR probe.
Therefore, we set out to prepare JEG-CAB and JEG-CAN, which are able to detect HCys/Cys and peroxynitrite (ONOO À ) or nitroreductase (NTR), respectively (Scheme 1). For JEG-CAB, a benzyl boronate ester was introduced as a ONOO À reactive unit. 37 For JEG-CAN, a p-nitrobenzyl group was installed as it is known to be an effective substrate for NTR. [38][39][40] To afford CAH, compound 2 was synthesized by reuxing umbelliferone and acetic anhydride at 140 C. Compound 2 was then dissolved in triuoroacetic acid at 0 C followed by the addition of hexamethylenetetramine (HMTA). The mixture was heated to reux overnight and the solvent was then removed. The intermediate was then hydrolyzed in H 2 O for 30 min at 60 C. Upon isolating CAH, it was then alkylated using 4-bromomethylphenylboronic acid pinacol ester and K 2 CO 3 in DMF at r.t. to afford JEG-CAB in 51% yield. JEG-CAN was produced by alkylating CAH using 4-nitrobenzyl bromide and K 2 CO 3 in DMF at r.t. to give 49% yield (Scheme 1).
We then evaluated the ability of JEG-CAB to detect ONOO À 'AND' HCys in PBS buffer solution (10 mM, pH 7.40). The maximum absorption of JEG-CAB at 336 nm shied to 323 nm with the addition of HCys and then slightly shied to 328 nm following the addition of ONOO À (Fig. S3 †). As shown in Fig. 2, JEG-CAB was initially non-uorescent, but the addition of HCys (1 mM) led to a small increase in uorescence intensity, the subsequent additions of ONOO À (0-24 mM) led to a signicant increase in uorescence intensity (>17-fold, see Fig. S5 †). These results demonstrated the requirement for both ONOO À 'AND' HCys to obtain a signicant turn ''on'' uorescence response.
The addition of HCys and ONOO À were then performed in reverse where JEG-CAB exhibited a negligible increase in uorescence intensity upon addition of ONOO À (16 mM). However, in a similar manner to that shown in Fig. 2, a large increase in uorescence intensity was produced aer the subsequent addition of HCys (0-5.5 mM) (Fig. 3 and S6 †). LC-MS experiments were carried out to ascertain the reaction mechanism and the results conrmed the sequential formation of the thiazine ring in the presence of HCys followed by boronate ester cleavage in the presence of ONOO À or vice versa (Scheme S2 and Fig. S19-S21 †).
As expected, probe JEG-CAB was shown to have excellent selectivity with ONOO À against other ROS in the presence of HCys (1 mM) ( Fig. S9 and S10 †). Furthermore, JEG-CAB  exhibited a high degree of selectivity towards a series of amino acids where only HCys and Cys led to a uorescence response in the presence of ONOO À . This is due to the formation of stable ve or six-membered thiazine rings ( Fig. S7 and S8 †). 34 We then evaluated the changes in the uorescence of JEG-CAN with both HCys and NTR in PBS buffer solution (10 mM, pH 7.40, containing 1% DMSO). As shown in Fig. 4, addition of HCys led to a small increase in uorescence intensity. However, subsequent addition of NTR (4 mg mL À1 ) led to a large time dependant increase in uorescence intensity. To ensure both analytes were required, NTR and NADPH was kept constant (4 mg mL À1 and 400 mM respectively) resulting in a 3.4 fold uorescence increase (Fig. 5). We attribute the large initial increase to background uorescence of NADPH. 41 NTR then facilitates reduction of the nitro group of JEG-CAN releasing the core probe CAH via a fragmentation cascade (Scheme S3 †). 38,42 Subsequent addition of HCys (2.0 mM) led to a 2 fold increase in uorescence intensity. Again, LC-MS experiments conrmed the proposed reaction mechanism (Fig. S22 †).
Kinetic studies for JEG-CAN with both NTR and HCys were carried out (Fig. S11-S18 †) where it is clear that JEG-CAN exhibits a dose dependant uorescence increase in response of both HCys and NTR.
Unfortunately, the probes failed to give good data in cells, which could be due to their short excitation wavelengths or the extremely low intracellular HCys concentrations (5-15 mM). We are now pursuing the development 'AND' logic uorescence probes with longer excitation and emission wavelengths suitable for in vitro and in vivo applications.
In summary, we have developed two coumarin-based 'AND' logic uorescence probes (JEG-CAB and JEG-CAN) for the detection of HCys/Cys and ONOO À or NTR, respectively. CAH is a useful platform that enables easy modication for the development of 'AND'-based uorescent probes for the detection of HCys/Cys and a second analyte. Both JEG-CAB and JEG-CAN were shown to be 'AND'-based uorescent probes.

Conflicts of interest
No conicts of interest.
Acknowledgements LW wishes to thank China Scholarship Council and the University of Bath for supporting his PhD work in the UK. JEG   and ACS wish to thank the EPSRC for funding their PhD studentships. TDJ wishes to thank the Royal Society for a Wolfson Research Merit Award. X.-P. He thanks the National Natural Science Foundation of China (21722801 and 21572058), the Programme of Introducing Talents of Discipline to Universities (B16017) and the Shanghai Rising-Star Program (16QA1401400). LKK wishes to acknowledge the Irish Research Council for a Government of Ireland Postdoctoral Research Fellowship (GOIPD/2017/1091) and Science Foundation Ireland are acknowledged for the funding of Advion Mass Spec facilities through the Opportunistic Infrastructure Fund (16/RI/3399). RBPE would like to thank Science Foundation Ireland (SFI) and the European Regional Development Fund (12/RC/ 2275_P2). NMR characterisation facilities were provided through the Material and Chemical Characterisation Facility (MC 2 ) at the University of Bath (http://go.bath.ac.uk/mc2). The EPSRC UK National Mass Spectrometry Facility at Swansea University is thanked for analyses.