Jake C Spiteri

Alex D Johnson

Sergey A Denisov

Gediminas Jonusauskas
email: gediminas.jonusauskas@u-bordeaux.fr

Nathan D Mcclenaghan
email: nathan.mcclenaghan@u-bordeaux.fr

David C Magri
email: david.magri@um.edu.mt

A fluorescent AND logic gate based on a ferrocene-naphthalimide-piperazine format responsive to acidity and oxidizability

Keywords:

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Many chemical and biological phenomena are dependent on pH and redox potential including corrosion [START_REF] Pourbaix | Atlas of electrochemical equilibria in aqueous solutions[END_REF], geochemistry [START_REF] Brookins | Eh-pH diagrams for geochemistry[END_REF], clinical chemistry [START_REF] Burtis | Tietz fundamentals of clinic chemistry[END_REF] and anticancer treatments [START_REF] Gellerman | [END_REF]. In recent years there has been a tremendous effort focused exclusively on pH sensing [START_REF] De Silva | Fluorescent PET (Photoinduced Electron Transfer) sensors as potent analytical tools[END_REF], notably for intracellular fluorescence imaging applications [START_REF] Yue | A review: the trend of progress about pH probes in cell application in recent years[END_REF]. In contrast, the field of luminescent redox indicators has not received as much attention and offers much opportunity for future development [START_REF] Magri | Photoinduced electron transfer as a design concept for luminescent redox indicators[END_REF]. Indeed, de spite there being numerous molecular probes for specifically mon itoring the pH or the redox environment in living cells, there are no commercially available probes that simultaneously probe pH and pE in living cells [START_REF] Johnson | The molecular probes handbook: a guide to fluorescent probes and labeling technologies[END_REF]. Molecular probes that are able to detect and measure high concentrations of both protons and redox active oxidants could be valuable diagnostic imaging tools for diseases such as cancer [START_REF] Li | Mitochondrial redox imaging for cancer diagnostics and therapeutic studies[END_REF].

We are interested in engineering molecular logic gates that detect acidic and oxidizing conditions by emitting an optical signal. We deemed this class of smart molecule 'Pourbaix sensors' in honour of Marcel Pourbaix, the father of corrosion science [START_REF] Pourbaix | Atlas of electrochemical equilibria in aqueous solutions[END_REF]. Molecular logic gates specifically combining acid base and redox equilibria are still rather rare [START_REF] Magri | Fluorescent photoinduced electron transfer (PET) logic gates for acidity (pH) and redox potential (pE)[END_REF][START_REF] Magri | Pourbaix sensors': fluorescent molecular logic gates for pE and pH[END_REF]. Our first proof of concept prototypes were based on the hydrophobic fluorophore anthracene [ 12 14]. We subsequently designed 'Pourbaix sensors' with the non symmetrical naphthalimide fluorophore [START_REF] Spiteri | A naphthalimide-based 'Pourbaix sensor': a redox and pH driven AND logic gate with photoinduced electron transfer and internal charge transfer mechanisms[END_REF][START_REF] Johnson | Water-soluble naphthalimide-based 'Pourbaix sensors': pH and redox-activated fluorescent AND logic gates based on photoinduced electron transfer[END_REF]. Naphthalimides are routinely used in molecular probe design due to their facile synthetic pathways, compatible water solubility and emission at longer wavelengths, among other favourable properties [START_REF] Banerjee | Recent advances in the development of 1,8-naphthalimide based DNA targeting binders, anticancer and fluorescent cellular imaging agents[END_REF][START_REF] Panchenko | FRET versus PET: ratiometric chemosensors assembled from naphthalimide dyes and crown ethers[END_REF]. More specifically, chemosensors and molecular logic gates combining a naphthalimide fluorophore with a piperazine receptor represent a successful design strategy for many applications [ 19 23]. We and others who are interested in redox responsive optical molecules have employed the ferrocene naphthalimide piperazine platform to demonstrate proof of concepts of three input INHIBIT [START_REF] Li | A redox-activated fluorescence switch based on a ferrocene-fluorophore-boronic ester conjugate[END_REF] and three input INHIBIT AND OR [START_REF] Magri | Proof of principle of a three-input AND-INHIBIT-OR combinatorial logic gate array[END_REF] combinatorial logic gate arrays. Other research groups have demonstrated pH/redox multi stimuli ma terials [START_REF] Al-Kutubi | Electrofluorochromic systems: molecules and materials exhibiting redox-switchable fluorescence[END_REF], nanogels [START_REF] Chen | Photo, pH and redox multi-responsive nanogels for drug delivery and fluorescence cell imaging[END_REF], polymers [START_REF] Yan | Multiple stimuli-responsive fluorescence behavior of novel polyamic acid bearing oligoaniline, triphenylamine, and fluorene groups[END_REF], film materials [START_REF] Meng | pH controllable photocurrent switching and molecular half-subtracter calculations based on a monolayer composite film of a dinuclear Ru-II complex and graphene oxide[END_REF] and degradable micelles [START_REF] Chen | Redox and pHresponsive degradable micelles for dually activated intracellular anticancer drug release[END_REF].

Herein we report the synthesis and characterisation of a naphtha limide based logic gate 1 (Scheme 1) with an electron donor spacer fluorophore spacer receptor format [START_REF] Spiteri | A naphthalimide-based 'Pourbaix sensor': a redox and pH driven AND logic gate with photoinduced electron transfer and internal charge transfer mechanisms[END_REF]. 'Pourbaix sensor' 1 consists of ferrocene (electron donor) connected to 1,8 naphthalimide (fluor ophore) by a methylene spacer, which is connected to piperazine (proton receptor/electron donor). The molecule is demonstrated as an AND logic gate responsive to protons and Fe 3+ as the oxidant in aqu eous methanol. Previously reported methylpiperazine prototype 2 and piperazine fluorescent pH indicator 3 are studied as model compounds (Scheme 1).

Experimental

Materials

N Ferrocenyl 4 bromo 1,8 naphthalimide was synthesized as reported (Scheme S1) [START_REF] Spiteri | A naphthalimide-based 'Pourbaix sensor': a redox and pH driven AND logic gate with photoinduced electron transfer and internal charge transfer mechanisms[END_REF]. Piperazine (99%, Alfa Aesar) was used as received. Tetrabutylammonium hydroxide (25 wt % in H 2 O), iron(III) sulfate pen tahydrate (97%) and methanesulfonic acid (≥99.5%) were obtained from Sigma Aldrich and used as received. Solvents were HPLC grade.

Instrumentation

Melting points were recorded with a Stuart SMP40 automatic melting point apparatus. 1 H and 13 C NMR spectra were recorded on a Bruker Avance III HD NMR spectrometer at 500.13 MHz and 125.76 MHz, respectively. Chemical shifts are reported in ppm down field from TMS at δ = 0.00 ppm and δ = 77.00 ppm for 1 H and 13 C NMR. Infra red spectra were recorded using a Shimadzu IR Affinity 1 spectrophotometer calibrated against 1601 cm -1 polystyrene absorp tion peak as KBr disks. HRMS was performed by ES ToF technique and performed by Medac Ltd (UK). UV visible absorption spectra were obtained on a Jasco V 650 spectrophotometer. Fluorescence spectra were obtained on a Jasco FP 8300 spectrofluorimeter. Experiments were performed at room temperature 20 °C in 10 mm quartz cuvettes. The time resolved luminescence set up was previously described [START_REF] Johnson | Water-soluble naphthalimide-based 'Pourbaix sensors': pH and redox-activated fluorescent AND logic gates based on photoinduced electron transfer[END_REF]. pH measurements were recorded with a Hanna Instruments pH 210 microprocessor meter with a HI 1131 electrode calibrated at pH 4.00 and pH 7.00.

Synthesis

Reactions were carried out in round bottom flasks (50 mL, 100 mL or 250 mL) partially immersed in an oil bath and heated using on a IKA C MAG HS 7 hotplate fitted with an IKA ETS D5 temperature probe. Silica gel 60 (70 230 mesh, Fluka Analytical) and silica coated alumi nium foil (silica gel matrix with fluorescent indicator 254 nm, Fluka Analytical) were used for column and thin layer chromatography, re spectively.

N Ferrocenylmethyl 4 piperazine 1,8 naphthalimide 1

N Ferrocenyl 4 bromo 1,8 naphthalimide (0.199 g, 0.420 mmol) and piperazine (0.897 g, 10.4 mmol) were dissolved in 25 mL of DMF and refluxed for 2 h. The reaction was diluted with 50 mL water and extracted with CH 2 Cl 2 (3 × 30 mL), dried over anhydrous MgSO 4 , fil tered and the solvent removed in vacuo. The product was purified by column chromatography on silica gel eluted with 1:1 (v/v) CH 2 Cl 2 / MeOH and obtained as a yellow solid in 31% yield. R f = 0.50 (20:1 (v/ v) CH 2 Cl 2 /MeOH); m.p. = 132 135 °C; 1 H NMR (CDCl 3 , 500 MHz, ppm): δ 8.56 (dd, 1H, J = 7.3 Hz, 0.9, Ar H), 8.50 (d, 1H, J = 8.1 Hz, Ar H), 8.38 (dd, 1H, J = 8.5, 0.9 Hz, Ar H), 7.66 (dd, 1H, J = 7.9, 0.9 Hz, Ar H), 7. [START_REF] Liu | Naphthalimide-based fluorescent photoinduced electron transfer sensors for saccharides[END_REF]

Spectroscopic measurements

Solutions of 1 3 were prepared in 1:1 (v/v) methanol/water with an absorbance of 0.1. The excitation and emission slits were set at 2.5 nm and 5.0 nm, respectively, with a scan rate of 200 nm min -1 and emis sion range of 400 650 nm. The acid dissociation constants (pβ H+ ) were determined from fluorescence intensity pH plots and fitted to a line arized Henderson Hasselbalch equation adapted for spectroscopic measurements eq. ( 1) [START_REF] Magri | The anthracen-9-ylmethyloxy unit: an underperforming motif within the fluorescent PET (photoinduced electron transfer) sensing framework[END_REF]:

⎜ ⎟ ⎛ ⎝ - - ⎞ ⎠ = - + I I I I β log pH p H F F max min (1)
Fe 3+ titrations were performed in acidified 1:1 (v/v) methanol/ water to prevent the formation of insoluble iron hydroxides. Known aliquots of Fe 3+ were added by micropipette. A modified Henderson Hasselbalch equation was employed for determining the apparent Fe 3+ binding constant (pβ Fe 3+ ) according to eq. ( 2):

⎜ ⎟ ⎛ ⎝ - - ⎞ ⎠ = - - + + I I I I β log log[Fe ] p Fe F F 3 max min 3 (2)
The molar extinction coefficients (ε) were determined according to the Beer Lambert Law eq. ( 3) where A is the absorbance, c is the con centration and l is the cuvette path length. The relative fluorescence quantum yields (Φ f ) were determined by eq. ( 4) from area under the fluorescence spectra (I), the absorbance at the excited wavelength (A) and the solvent refractive index (η). Reference values are denoted by the subscript 'ref'. Anthracene dissolved in ethanol (refractive index 1.36) with a Φ ref of 0.27 was used as the standard [START_REF] Dawson | Fluorescence yields of aromatic compounds[END_REF].

= A ε l c ⎜ ⎟ ⎜ ⎟ = ⎛ ⎝ ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ Φ Φ I I A A η η f r e f ref ref 2 ref 2 (4)

Results and discussion

Synthesis and characterisation

2 Ferrocenylmethyl 6 (piperazin 1 yl) 1H benzo[de]isoquinoline 1,3 (2H) dione 1 was synthesized from N ferrocenyl 4 bromo 1,8 naphthalimide reacted with piperazine in 31% yield after purification (Scheme S1). We fully characterised 1 by 1 H and 13 C NMR, FTIR and HRMS (Figs. S1 S4). Compound 1 was previously reported from the synthesis of 6 (piperazin 1 yl)benzo[de]isochromene 1,3 dione with ferrocenylmethylamine in 16% yield [START_REF] Li | A redox-activated fluorescence switch based on a ferrocene-fluorophore-boronic ester conjugate[END_REF]. In this previous work, 1 was used as a synthetic intermediate and not satisfactorily characterised. We note that 13 C NMR resonances at ca. 39.1 ppm and 130.2 ppm were not reported, and it is unclear whether the reported 1 H NMR resonances are for CDCl 3 or DMSO d 6 . From the FTIR spectrum one can clearly identify the diagnostic broad secondary N H stretching vibration at 3416 cm -1 and the intense carbonyl stretches at 1690 cm -1 and 1659 cm -1 . Scheme 1. The 1,8-naphthalimide-based fluorescent logic gates 1-3.

Photophysics of 1 and 2

The UV visible absorption spectra of 1 and 2 in 1:1 (v/v) methanol/ water display broad bands between 300 and 550 nm (Fig. S5). The peak maxima in acidic and basic solutions are situated at 386 nm and 405 nm, respectively, with an isosbestic point at 400 nm. A complete titration set of spectra are shown between pH 5 and pH 10 (Figs. S5a ande). The ε values of 8000 L mol -1 cm -1 and 10000 L mol -1 cm -1 in acid and base, respectively, correspond to π→π* electronic transitions within the naphthalimide fluorophore. The pH induced 20 nm hypso chromic shift in acidic solution is characteristic of an ICT mechanism associated with a less stable Franck Condon excited state. This is at least partly attributed to the piperazine group, which directly interferes with the naphthalimide π conjugation due to a 'pre twisted' ICT in the ground state [START_REF] Zheng | Structural effects on the pH-dependent fluorescence of naphthalenic derivatives and consequences for sensing/switching[END_REF]. Similar photophysical characteristics were observed with 2 (Fig. S6a) [START_REF] Spiteri | A naphthalimide-based 'Pourbaix sensor': a redox and pH driven AND logic gate with photoinduced electron transfer and internal charge transfer mechanisms[END_REF] as summarised in Table 1.

The absorbance spectra of 1 under the four input conditions for AND logic are shown in Fig. 1. The λ max is observed to decrease with in creasing proton concentration. Upon addition of 50 μM Fe 3+ a slight increase is observed in the absorbance. A higher concentration of Fe 3+ does result in a greater fluorescence emission; however, this increases the possibility of an inner filter effect.

Fluorescence emission spectra of 1 and 2 were obtained on excita tion at 400 nm providing broad emission spectra with a peak maximum at 530 nm (Fig. S5). Emission intensity profiles revealed sigmoidal shaped curves ranging three pH units (Figs. S5c andg). The calculated binding constants from the analysis of eq (1) by fluorimetric titration provided pβ H + values of 8.1 and 7.7 for 1 and 2, respectively (Figs. S5c andg). pH titrations in the presence of 50 μM Fe 3+ gave pβ H + values of 8.0 and 7.2. (Fig. S6). Fluorimetric Fe 3+ titrations at constant pH 4 gave pβ Fe + values of 4.7 and 4.6 for 1 and 2, respectively (Fig. S7).

The AND logic test of 1 in vials is qualitatively shown in Fig. 2. The truth table including Φ f are given in Table 2. The 'vial test' of 1 displays a fluorescence output to the naked eye on irradiation with 365 nm light. The four possible conditions are (A) low H + and no oxidant, (B) high H + and no oxidant, (C) low H + and high oxidant and (D) high H + and high oxidant. A green fluorescence is only observed by the naked eye in the presence of 10 -4 M H + and 50 μM Fe 3+ (Fig. 2, vial D). In the other three cases, no fluorescence is observed by the naked eye, the maximum emission quantum yield being Φ f ≤ 0.01. Hence, compound 1 functions as a two input AND logic gate for acidity and oxidizability.

The fluorescence spectra of 1 associated with the four vials are overlaid in Fig. 3. The fluorescence output is significantly greater when both inputs are simultaneously present, whereas a low output is ob served when either one or both inputs are absent. The fluorescence enhancement for 1 is a 6 fold switching (I max (50 μM Fe 3+ at pH 4) /I max (pH 4) ) whereas with 2 we observed up to a 13 fold enhancement [START_REF] Spiteri | A naphthalimide-based 'Pourbaix sensor': a redox and pH driven AND logic gate with photoinduced electron transfer and internal charge transfer mechanisms[END_REF]. The switching between the 'off' and 'on' states is slightly better for 2 than 1. Surprisingly, this difference is not due to the different proton receptors because the Φ f of 1 and 2 in the presence of 50 μM Fe 3+ are rather ref. [START_REF] Dawson | Fluorescence yields of aromatic compounds[END_REF].

h Published results ref. [START_REF] Johnson | Water-soluble naphthalimide-based 'Pourbaix sensors': pH and redox-activated fluorescent AND logic gates based on photoinduced electron transfer[END_REF].

similar. It would be expected that the ICT efficiencies from the piper azine and methylpiperazine to the naphthalimide fluorophore would be nearly the same.

Model piperazine compound 3

Comparison to the naphthalimide piperazine model 3 provides in sight into the structural effect on the pH dependent fluorescence switching (See Table 2) [START_REF] Zheng | Structural effects on the pH-dependent fluorescence of naphthalenic derivatives and consequences for sensing/switching[END_REF]. A peak maximum is observed at 407 nm (6900 L mol -1 cm -1 ) in basic solution and 384 nm (8700 L mol -1 cm -1 ) in acidic solution with a clear isosbestic point at 401 nm. These values are in good agreement with those of 1 confirming that the ferrocene moiety is electronically decoupled from the naph thalimide fluorophore by the methylene spacer. The Φ f of 3 is 0.20 when monoprotonated at pH 4 and 0.002 when unprotonated at pH 10, yielding a remarkable 138 fold fluorescence enhancement factor, which is due to the enhanced twisted piperazine ground state [START_REF] Zheng | Structural effects on the pH-dependent fluorescence of naphthalenic derivatives and consequences for sensing/switching[END_REF]. In contrast, the maximum Φ f of 1 is a more modest 0.060 even on protonation and oxidation, while in the absence of both input conditions the minimum Φ f is 0.004 at pH 10. We observed a similar trend on comparing pyr azoline based fluorescent pH probes with pyrazoline ferrocene INH logic gates [START_REF] Scerri | Redox-enabled, pH-disabled pyrazoline-ferrocene INHIBIT logic gates[END_REF]. The cause of this limitation of fluorescent enhance ment is not fully understood. While protonation of the piperazine ni trogen atom results in a covalent bond, which implicates the electron lone pairs, oxidation of ferrocene by Fe 3+ generates a radical cation intermediate. A recent comprehensive DFT study by us suggests there are localised deactivation pathways associated with ferrocene that may render a lower fluorescent quantum yield [START_REF] Spiteri | Molecular engineering of logic gate types by module rearrangement in 'Pourbaix Sensors': the effect of excited-state electric fields[END_REF].

Time resolved fluorescence spectroscopy

A time resolved fluorescence study was undertaken to measure the fluorescence lifetimes of 1 at the four input conditions [START_REF] Johnson | Water-soluble naphthalimide-based 'Pourbaix sensors': pH and redox-activated fluorescent AND logic gates based on photoinduced electron transfer[END_REF]. The fluorescence decay curve and 3D plot for the most fluorescent logic state are shown in Fig. 4. A complete set of fluorescence decay curves (a d) and 3D plots (e h) are available in the supporting information (Fig. S8). The experimental results highlight the efficient charge transfer processes between the piperazine and naphthalimide, and the ferrocene and naphthalimide fluorophore. For both 1 and 2, the fluorescence lifetime decay profiles are bi exponential in the absence of one or both inputs (Figs. S8a c). However, on addition of H + and Fe 3+ a mono exponential decay profile is observed (Fig. 4, Fig. S8d). In the logic states 'A C', with no inputs or only one input, the long lifetime component (∼90%) results from the intrinsic properties of the naph thalimide fluorophore. The fluorescence lifetimes of 1 and 2 (in the absence of at least one input) span 4.4 5.5 ns and 6.6 7.4 ns, respec tively. The shorter lifetime component (∼10%) corresponds to the suppressed fluorescence emission when one or both quenching me chanisms are operational: ICT from the proton receptor and/or PET from the ferrocene unit to the excited state fluorophore with a time constant less than 1 ns. The addition of acid and oxidant deactivates the PET and ICT pathways and yields a single fluorescent lifetime compo nent (> 99%). The fluorescence lifetimes are 7.3 ns for 2, whilst 1 has a slightly shorter lifetime of 5.8 ns in 1:1 (v/v) methanol/water. It is plausible that the discrepancy originates from the positive inductive effect of the methyl group in 2, which better stabilises the protonated amine resulting in a longer lifetime in the excited state. The 3D surface plots (Figs. S8e h) provide similar information as the lifetime decay curve, but with the additional dimension of the emission wavelength. From these plots it is easy to appreciate how the fluorescence is re markably enhanced in the presence of both inputs H + and Fe 3+ com pared to the other three input conditions.

Femtosecond transient absorption spectroscopy

Further insight into the photophysics of 1 was gained from femto second transient absorption studies. The transient map of 1 is shown in Fig. 5. After excitation (< 1 ps) 1 experiences a significant charge density shift within the naphthalimide fluorophore due to the inherent ICT character. Ground state bleaching is observed at 420 450 nm (red) and stimulated emission at 500 650 nm (blue). We previously noted that the transient signal recovery rate of 2 was faster compared to a methylpiperazine model (∼50 ps) due to the faster excited state elec tron transfer from ferrocene (∼5 ps) [START_REF] Johnson | Water-soluble naphthalimide-based 'Pourbaix sensors': pH and redox-activated fluorescent AND logic gates based on photoinduced electron transfer[END_REF]. In the case of 1, the transient signal recovery rate is remarkable fast at < 2 ps. In the absence of either input, the excited state absorption has bi exponential decays of 270 fs and 1.25 ps with almost equal pre exponential factors of 0.46 and 0.42 and stimulated emission 140 fs (23%) and 500 fs (62%). On addition of protons the contributions from the excited states are shorter at 120 ps and 1 ps with pre exponential factors of 0.47 and 0.42, respectively. In the presence of acid and oxidant the stimulated emission is mono ex ponential with an emission lifetime of 160 fs.

Conclusions

The successful operation of fluorescent logic gate 1 is another convincing illustration of the cross fertilization of fluorescent pH in dicators and fluorescent redox indicators [START_REF] Magri | Pourbaix sensors': fluorescent molecular logic gates for pE and pH[END_REF]. Specifically, 1 in corporates a ferrocene naphthalimide piperazine format, which con sists of an electron donor, fluorophore and proton receptor/electron donor with PET and ICT mechanisms. Molecule 1 responds to an oxi dant and protons in accordance to two input AND logic by emitting a fluorescent signal in mixed aqueous methanol. Future engineering of systems responsive to pH and redox potential should continue to pro vide stimulating examples of information processing at the molecular level [START_REF] De Silva | Molecular logic-based computation[END_REF][START_REF] Erbas-Cakmak | Molecular logic gates: the past, present and future[END_REF] and practical application in various realms including cell biology [START_REF] Johnson | The molecular probes handbook: a guide to fluorescent probes and labeling technologies[END_REF] and corrosion detection [START_REF] Pourbaix | Atlas of electrochemical equilibria in aqueous solutions[END_REF].

Fig. 1 .

 1 Fig. 1. UV-visible absorption spectra of 8.2 μM 1 in 1:1 (v/v) MeOH/H 2 O at four different input conditions of H + and Fe 3+ .

Fig. 2 . 2 a

 22 Fig. 2. Irradiation of 10 -5 M 1 with UV light (365 nm) under AND logic conditions in 1:1 (v/v) MeOH/H 2 O: (A) 10 -10 M H + , (B) 10 -4 M H + (C) 10 -10 M H + and 50 μM Fe 3+ and (D) 10 -4 M H + and 50 μM Fe 3+ .

Fig. 3 .

 3 Fig. 3. Fluorescence spectra of 8.2 μM 1 in 1:1 (v/v) MeOH/H 2 O at four different input conditions of H + and Fe 3+ (λ ex = 400 nm).

Fig. 4 .

 4 Fig. 4. Fluorescence decay curve and 3D surface plot of 1 in 1:1 (v/v) MeOH/H 2 O in presence of 10 -4 M H + and 50 μM Fe 3+ (λ ex = 400 nm).

Fig. 5 .

 5 Fig. 5. Transient absorption map of 1 in acetonitrile in presence of 10 -4 M H + and 50 μM Fe 3+ .

  (d, 1H, J = 8.1 Hz, Ar H), 5.11 (s, 2H, eCH 2 spacer), 4.50 (t, 2H, J = 1.8 Hz, Cp), 4.20 (s, 5H, Cp), 4.06 (t, 2H, J = 1.8 Hz, Cp), 3.22 (m, 4H, eN(CH 2 CH 2 ) 2 NH), 3.20 (m, 4H, eN (CH 2 CH 2 ) 2 NH); 13 C NMR (CDCl 3 , 126 MHz, ppm): δ 164.2, 163.8, 156.2, 132.6, 131.2, 130.2, 129.9, 126.2, 125.6, 123.4, 116.9, 115.0, 83.4, 70.4, 68.6, 68.0, 54.2, 46.1, 39.1; IR (KBr, cm -1 ): 3416 (N H), 3092(=C H), 2951( C H), 1690( C=O), 1659( C=O), 1587, 1516, 1418, 1383, 1371, 1331, 1240, 1175, 1134, 1105, 1022, 827, 785; HRMS (ES ToF): Calculated C 27 H 25 N 3 O 2 Fe [M] + 479.1296, found 479.1288.

Table 1

 1 Comparison of the photophysical data of 1-3 in 1:1 (v/v) MeOH/H 2 O. a d Abs(pH 10 + 50 μM Fe 3+ ) /nm (log ε) 408 (4.08) 401 (4.03) λ Abs(pH 4 + 50 μM Fe 3+ ) /nm (log ε) pH adjusted with NaOH or (C 4 H 9 ) 4 NOH and CH 3 SO 3 H solutions. c Estimated errors for pβ H +, pβ Fe 3+ and log ε are ± 0.2, ± 0.2 and ± 0.3, respectively. Duplicate measurements.

			1	2	3
	λ Abs(pH 10) /nm (log ε)	405 (3.95)	408 (3.91)	407 (3.84)
	λ Abs(pH 4) /nm (log ε)	386 (4.01)	385 (3.89)	384 (3.94)
	λ 385 (4.16)	381 (4.05)	-
	λ iso /nm	400	400	401
	λ f(pH 4 + 50 μM Fe 3+ ) /nm	530	528	528
	Δλ (pH 4 + 50 μM Fe 3+ ) /nm	130	128	127
	pβ H + * e		8.1	7.7	8.1
	pβ H + * f		8.0	7.2	-
	pβ Fe 3+ g		4.7	4.6	-
	Φ f (acid)	h	0.060	0.086	0.20
	FE		6	13	138
	τ/ns i		5.8	7.3	-

a Sensors concentration ∼5-10 μM. b d Molar absorptivity ε in L mol -1 cm -1 . e Determined by log((I max I)/(I I min )) = log[H + ] + log β H + in absence of Fe 3+ . f In presence of 50 μM Fe 3+ . g Determined by log((I max I)/(I I min )) = log[Fe 3+ ] + log β Fe 3+.

h At 10 -4 M H + and 50 μM Fe 3+ in case of 1 and 2.

i Fluorescence lifetimes in presence of 10 -4 M H + and 50 μM Fe 3+ .

Acknowledgements

This research was supported by the University of Malta, the Embassy of France to Malta, CNRS and the MCST. Prof. Robert M. Borg is thanked for assistance with the acquisition of the NMR spectra.