Synthesis and Evaluation of 1,8‐Disubstituted‐Cyclam/Naphthalimide Conjugates as Probes for Metal Ions

Abstract Fluorescent molecular probes for metal ions have a raft of potential applications in chemistry and biomedicine. We report the synthesis and photophysical characterisation of 1,8‐disubstituted‐cyclam/naphthalimide conjugates and their zinc complexes. An efficient synthesis of 1,8‐bis‐(2‐azidoethyl)cyclam has been developed and used to prepare 1,8‐disubstituted triazolyl‐cyclam systems, in which the pendant group is connected to triazole C4. UV/Vis and fluorescence emission spectra, zinc binding experiments, fluorescence quantum yield and lifetime measurements and pH titrations of the resultant bis‐naphthalimide ligand elucidate a complex pattern of photophysical behaviour. Important differences arise from the inclusion of two fluorophores in the one probe and from the variation of triazole substitution pattern (dye at C4 vs. N1). Introducing a second fluorophore greatly extends fluorescence lifetimes, whereas the altered substitution pattern at the cyclam amines exerts a major influence on fluorescence output and metal binding. Crystal structures of two key zinc complexes evidence variations in triazole coordination that mirror the solution‐phase behaviour of these systems.


General materials and instrumentation
Acetonitrile, methanol and THF were collected fresh from a PureSolv MD 7 solvent purification system having been passed through anhydrous alumina columns. All commercially available reagents and solvents were purchased from Sigma Aldrich, Alfa Aesar, Merck or Ajax Finechem and used without purification.
Flash column chromatography was performed on Davisil Grace Davison 40-63 μm (230-400 mesh) silica gel. Automated flash column chromatography was performed on a Biotage Isolera Spektra One using Biotage SNAP KP-Sil cartridges at their default flow rates. Preparative reversed-phase HPLC was carried out on a Waters 600 controller with a Waters 600 pump and a 2998 photodiode array detector. A Waters SunFire TM C18 OBD TM preparative column (5 μm, 19 × 150 mm) was used at a flow rate of 7 mL/min; mobile phases of 0.1% TFA in Milli-Q water and 0.1% TFA in acetonitrile in different ratios were used.
Melting points were recorded on a Stanford Research Systems Optimelt automated melting system and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Bruker Avance DPX 400 or 500 spectrometer.
Chemical shifts are reported in ppm relative to tetramethylsilane or residual solvent resonance as internal

1,8-Bis(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraazacyclotetradecane 14
The protected bis-phenyl 17 (243 mg, 0.327 mmol) was dissolved in dioxane (2 mL) and a solution of HCl in dioxane (4.0 M, 1.00 mL, 4.00 mmol) was added. The reaction mixture was stirred at rt for 16 h. The volatiles were removed in vacuo and the residue was triturated with EtOAc (2 × 3 mL    Single crystals were attached with Exxon Paratone N to a nylon loop and quenched in a cold nitrogen gas stream from an Oxford Cryosystems Cryostream. A SuperNova Dual equipped with an Atlas detector and employing mirror monochromated Cu (Kα) radiation from a micro-source was used for the X-ray data collections. Data were collected using  scans at 150(1) Kelvin. Data processing was undertaken with CrysAlisPro [4] and subsequent computations were carried out with WinGX [5] and ShelXle. [6] A multi-scan absorption correction was applied [4] to the data. In both cases the asymmetric unit contains a complex molecule and two perchlorate counterions.
[Zn (13)](ClO 4 ) 2 : Data were collected from a colourless needle like crystal using  scans to 151º 2, with cell constants obtained from a least squares refinement against 6146 reflections located between 10 and 145º 2. The structure was solved in the space group P2 1 /n(#14) by direct methods with SIR97 [7] and extended and refined with SHELXL-2013. [8][9] The non-hydrogen atoms in the asymmetric unit were modelled with anisotropic displacement parameters and a riding atom model with group displacement parameters was used for the hydrogen atoms. The amine hydrogen sites were located in final difference maps. The counterions are linked to the complex molecule by hydrogen bonding to the amine hydrogens. An ORTEP [10][11] depiction of the molecule with 50% displacement ellipsoids is provided in Figure S4.
[Zn(14)](ClO 4 ) 2 : Data were collected from a colourless blade like crystal using  scans to 152º 2, with cell constants obtained from a least squares refinement against 19032 reflections located between 8 and 152º 2.
The structure was solved in the space group P1(#2) by direct methods with SHELXT [12] and extended and refined with SHELXL-2014/7. [8][9] The non-hydrogen atoms in the asymmetric unit were modelled with anisotropic displacement parameters. In general a riding atom model with anisotropic displacement parameters was used for the hydrogen atoms. The amine hydrogen sites were located in final difference maps and modelled with isotropic displacement parameter. One of the counterions is linked to the complex molecule by weak hydrogen bonding to the amine hydrogens. An ORTEP [10][11] depiction of the molecule with 50% displacement ellipsoids is provided in Figure S5.

Further Discussion of Crystal Structures
Currently the Cambridge Structural Database (CSD) [13] has nine zinc cyclam complexes that are five coordinate with all of the coordinated atoms being nitrogen. Of these, three are essentially trigonal bipyramidal. The average axial distances of these three are 2.16 (3)