Intriguing Heteroleptic ZnII bis(dipyrrinato) Emitters in the Far-Red Region With Large Pseudo-Stokes Shift for Bioimaging

Novel heteroleptic ZnII bis(dipyrrinato) complexes were prepared as intriguing emitters. With our tailor-made design, we achieved far-red emissive complexes with a photoluminescence quantum yield up to 45% in dimethylsulfoxide and 70% in toluene. This means that heteroleptic ZnII bis(dipyrrinato) complexes retain very intense emission also in polar solvents, in contrast to their homoleptic counterparts, which we prepared for comparing the photophysical properties. It is evident from the absorption and excitation spectra that heteroleptic complexes present the characteristic features of both ligands: the plain dipyrrin (Lp) and the π-extended dipyrrin (Lπ). On the contrary, the emission comes exclusively from the π-extended dipyrrin Lπ, suggesting an interligand nonradiative transition that causes a large pseudo-Stokes shift (up to 4,600 cm−1). The large pseudo-Stokes shifts and the emissive spectral region of these novel heteroleptic ZnII bis(dipyrrinato) complexes are of great interest for bioimaging applications. Thus, their high biocompatibiliy with four different cell lines make them appealing as new fluorophores for cell imaging.

1 H-NMR and 13 C-NMR data were recorded at room temperature in CDCl3 by a Bruker 400 spectrometer. Mass spectral determination were performed with a MALDI-ToF System employing a reflection tuning mode and using 6-aza-2-thiothymine (ATT) as matrix. The microwave-assisted reaction was conducted in a Biotage ® Initiator+ Microwave System with Robot Sixty.

Photophysics
The absorption spectra were recorded with a PerkinElmer Lambda 750 double-beam Uv/Vis-NIR spectrometer. Fluorescence was measured with a Jobin-Yvon Fluoromax 4 fluorimeter with a step width of 1 mm. All measurements were performed in quartz cuvettes with septum from Hellma and, where not explicated, conducted at 20 °C through the use of an Advanced Ac200 Immersion Circulators Thermostats-Thermo Fisher Scientific. Solvents for spectroscopy were supplied by Merck (Uvasol). Photoluminescence quantum yields were determined utilizing as standard Cresyl Violet in spectroscopic methanol (Φ in MeOH, 22 °C = 0.54). Lifetime measurements were performed by time-correlated single-photon counting method (TCSPC) with a DeltaTime kit for DeltaDiode source on Fluoromax systems, including DeltaHuB and DeltaDiode controller. The light-sources were NanoLED sources (455nm, 570 nm and 625 nm).

Cytotoxicity Assay
The cytotoxicity was assessed by using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) according to producer´s instructions. It is based on the intracellular reduction of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) to a blue formazan product. Briefly, 1 x 10 4 cells of respective cells type (HeLa, NHDF, NIH3T3 and MCF7) were seeded in each well of a 96-well plate and incubated with DMEM, supplemented with 10% of FCS and 1% of Penicillin/Streptomycin (37 °C, 5% of CO2 atmosphere) for 24 h. Then, the cell culture medium was removed and fresh DMEM containing new compounds was added to the cells which were incubated for 72 h. In order to have an accurate spectrum of the results, the colorimetric assay was performed on HeLa cells incubated with ligands 4a-d, 5a, 5e at different concentration (1, 2.5, 5, 7.5 µM). Subsequently, viability levels of HeLa and NHDF cells were evaluated by incubating with complexes 1a-e at different concentration (1, 2.5, 5, 7.5, 10 and 20 µM) for 72 h. Considering that the concentration used for the microscopy is 20 µM and expecting a comparable trend, NIH3T3 and MCF7 were treated with complexes 1a-e only at 20 µM. One negative control was realized by incubating cells in DMEM + DMSO 20 µM. After adding 5 µl of Triton-X100 (20% in ultra-pure and sterile water ddH2O) to the positive control, 15 µl of MTT-solution was added in each well and the cells were incubated for additional 3h before adding 100 µL of a stop-solution mix. The absorption was measured at 570 nm by using a Spectramax ID3 (HeLa and NHDF) or a BioTek Synergy LX (NIH3T3 and MCF7) multi-well reader after an incubation period of 24 h, to allow the solubilization of the formazan product. Each experimental well and controls were prepared in triplicates.

Cell Culture, confocal laser microscopy and co-staining experiments
Confocal live-cell fluorescence microscopy was performed by seeding 1.5 x 10 4 cells on each chamber of an µ-slide 8-well (ibidi®) (for HeLa and NHDF) and on 35mm-glass bottom dishes (ibidi®) (for NIH3T3 and MCF7) followed by an incubation period of 24 h in DMEM supplemented with 10% of FCS and 1% of Penicillin/Streptomycin (37°C, 5% of CO2 atmosphere). Subsequently, medium was removed and fresh DMEM, containing 1a-e (20 µM, o.4% v/v), was added to the cell culture which was incubated for additional 24 h. Afterwards, MitoTracker™ Green (125 nM) or LysoTracker™ Green (50 nM) was added and, after 30 min, cells were washed three times with DPBS and fresh medium with Hoechst 33342 (2 µg/ml) was included.
Due to the possibility of exciting the fluorophores at different excitation wavelength, in order to prove the feasibility of different imaging conditions, BioTracker™ 405 Blue Mitochondria Dye (125 nM) and LysoTracker®Blue DND-22 (50 nM) were also employed. Co-staining experiments were performed following the same procedure as above mentioned, with the exception of the Hoechst 33342 which, in this case, was not added. The microscopy was performed by using, for HeLa and NHDF cell type, a confocal Leica Stellaris microscope with a HC PL Apo CS2 63x oil objective (N.A.= 1.4) and, for NIH3T3 and MCF7 cell type, a confocal Zeiss LSM800 microscope with a C-Apo 40x water immersion objective (N.A.=1.2). Acquisition parameters (e.g. excitation/emission wavelengths) were adjusted according to the specific investigation as indicated in the figure captions. The Pearson correlation coefficient (PCC) was calculated by using the JACoP plugin for ImageJ program. (Bolte and Cordelières, 2006)

Cross-linking fixative procedure for fixed-cells confocal laser microscopy
For the fixation experiments, DMEM was removed after cell culture (containing complex 1d) and replaced with paraformaldehyde (4% in PBS) for 15min. Cells were then washed three times with PBS+ 0.1% of Triton X-100 for 5min. Subsequently, cells were incubated with a solution of phalloidin coupled to AlexaFluor488 (Invitrogen, dilution 1:200) and DAPI (4,6diamidino-2-phenylindole) (Roth, dilution 1:1000) in PBS+ 1% of BSA for one hour, to stain the actin cytoskeleton and the nuclei, respectively. Finally, cells were washed three times with PBS, kept in buffer and imaged.
2 Synthetic procedures

Synthesis of plain dipyrrin 4a-d
General procedure: The synthesis of plain dipyrrins, a condensation of 2,4-dimethyl-1Hpyrrole and the appropriate aromatic aldehyde, was performed following the procedure described in literature (I.V. Sazanovich) with some improvements.

Synthesis of 2d:
To a solution of 4d (34.93mg, 0.1090 mmol, 2equiv.) in dichloromethane (10 mL), zinc acetate (10.0 mg, 0.054 mmol, 1 equiv.) pre-solved in methanol (5ml) was added and stirring overnight to give 4d as a dark-green powder (25.4 mg, 0.036 mmol, 66% yield). Figure S1: UV/Vis absorption (dotted plot), excitation (dashed plot, λem = 710 nm) and emission (solid plot, λexc = 570 nm) spectra of heteroleptic complexes 1a-e in DMSO. Absorption and excitation values have been normalized to the ( 1 LC) π→π* transition localized on the plain dipyrrins (ca.490 nm). Emission values have been normalized to the maximum of the excitation spectra, which is attributed to the π→π* transition of the π-expanded dipyrrin. Figure S2: Fluorescence lifetimes of heteroleptic complexes 1a-e in DMSO exciting with NanoLED source at three different excitation wavelengths (λexc 455, 570, 625 nm). Baselines appear different depending on the excitation laser wavelength, due to different signal to noise ratio. In fact, an enhanced noise is caused by scattering and other sources. Nevertheless, single exponential decay curves are independent of the excitation wavelength and are reported in Table 1 of the manuscript.
Compound Compound 4 1 H-NMR and 13 C-NMR spectra of novel complexes.