Novel Bacteriochlorin–Styrylnaphthalimide Conjugate for Simultaneous Photodynamic Therapy and Fluorescence Imaging

Propargyl-152,173-dimethoxy-131-amide of bacteriochlorin e (BChl) and a 4-(4-N,N-dimethylaminostyryl)-N-alkyl-1,8-naphthalimide bearing azide group in the N-alkyl fragment were conjugated by the copper(i)-catalyzed 1,3-dipolar cycloaddition to produce a novel dyad compound BChl-NI for anticancer photodynamic therapy (PDT) combining the modalities of a photosensitizer (PS) and a fluorescence imaging agent. A precise photophysical investigation of the conjugate in solution using steady-state and time-resolved optical spectroscopy revealed that the presence of the naphthalimide (NI) fragment does not decrease the photosensitizing ability of the bacteriochlorin (BChl) core as compared with BChl; however, the fluorescence of naphthalimide is completely quenched due to resonance energy transfer (RET) to BChl. It has been shown that the BChl-NI conjugate penetrates into human lung adenocarcinoma A549 cells, and accumulates in the cytoplasm where it has a mixed granular-diffuse distribution. Both NI and BChl fluorescence in vitro provides registration of bright images showing perfectly intracellular distribution of BChl-NI. The ability of NI to emit light upon excitation in imaging experiments has been found to be due to hampering of RET as a result of photodestruction of the energy acceptor BChl unit. Phototoxicity studies have shown that the BChl-NI conjugate is not toxic for A549 cells at tested concentrations (<8 μM) without light-induced activation. At the same time, the concentration-dependent killing of cells is observed upon the excitation of the bacteriochlorin moiety with red light that occurs due to reactive oxygen species formation. The presented data demonstrate that the BChl-NI conjugate is a promissing dual function agent for cancer diagnostics and therapy.


Synthesis of the compounds
General analytical methods. 1 H and 13 C NMR spectra were recorded on an Avance 400, Avance 500 and Avance 600 spectrometers (Bruker) operating at 400.13, 500.13,600.22 MHz (for 1 H) and 100.61, 125.76, 150.93 MHz (for 13 C) respectively.The chemical shifts were determined with an accuracy of 0.01 ppm relative to residual solvent signals and translated to the internal standard (TMS), coupling constants were measured with an accuracy of 0.1 Hz.The numbering of carbon atoms in the naphthalimide and bacteriochlorin fragments used for the description of the 1 H NMR spectra of the synthesized compounds is shown in Scheme S1.In the case of conjugate BChl-NI, carbon atoms of naphthalimide are marked with a prime.The assignment of 1 H and 13 C signals is based on 2D NMR experiments (HMBC, HSQC, 1 H COSY), which were performed using standard pulse sequences from the Bruker library.2D NMR spectra are presented in section 2.

Scheme S1. Numbering of the carbon atoms in BChl and NI6
Melting points were measured on Melt-temp melting point electrothermal apparatus and were uncorrected.The reaction course and purity of the final products was followed by TLC on silica gel (DC-Alufolien Kieselgel 60 F254, Sigma-Aldrich).Column chromatography was conducted over silica gel (Kieselgel, particle size 40-60 µm, Acros Organics).Preparative TLC was performed on silica gel 60 (Merck) using 20×20 cm plates with a layer thickness of 1 mm.IR spectra were recorded on a Bruker EQUINOX 55 spectrometer and on Magna-IR 750 Nicolet spectrometer with potassium bromide pellets.Electron impact (EI) (70 eV) mass spectra were obtained from Finnigan Polaris Q instrument (ion-trap) in standard conditions.The mass-spectra of BChl and BChl-NI were obtained by MALDI method on time-offlight mass-spectrometer Bruker Ultraflex TOF/TOF using dihydroxybenzene matrix.m/z: 1997.68 (M + ).Elemental analyses were carried out in the Microanalysis Laboratory of the A.N. Nesmeyanov Institute of Organoelement Compounds.
The excess of POCl 3 was removed under reduced pressure, the residue was dissolved in CHCl 3 and then, washed sequentially with 5% aqueous solution of Na 2 CO 3 and distilled water.The organic layer was dried with MgSO 4 and evaporated in vacuum.The residue was chromatographed on SiO 2 using dichloromethanemethanol gradient mixture to give 56 mg (89% yield) of NI5.M.p. 187-189 °С.

Evaluation of Different Parameters for RET process
Energy transfer efficiency value Φ RET1 for the conjugate BChl-NI was calculated using the expression shown in Eq. (S1), where  D,0 is the 4-styrylnaphthalimide donor chromophore excited state lifetime in the absence of RET acceptor ( D,0 = 0.38 ns, the lifetime of NI4), and  RET1energy transfer rate constant.
According to Förster resonance theory, estimation of  RET1 was done following Eq.(S2), where r is the distance between the donor and acceptor chromophores (15.06 Å) which was obtained from the PM6 optimized ground state geometry of BChl-NI (Fig. S23), and  0 is the critical Förster distance.To calculate  0 we used Eq. ( S3), where κ 2 denotes a factor which describes the relative orientations of the donor and acceptor (κ 2 = 2/3 for a random orientation), φ fl is the donor emission quantum yield in the absence of the
acceptor (the quantum yield of compound NI4, φ fl = 0.032), n is the refractive index of the medium (n = 1.344 for acetonitrile), N A is Avogadro constant (N A = 6.02•10 23 mol -1 ) and the integral defines the amount of overlap between the normalized emission spectrum of the donor F D (λ) and the acceptor absorption spectrum ε A (λ).

Fig. S23 .Fig. S24 .Fig. S25 .Fig. S26 .
Fig.S23.Optimized ground state geometry of BChl-NI obtained by MOPAC 2012 using PM6 Hamiltonian.The solvent effect was included in geometry optimizations following the «COnductorlike Screening Model» (COSMO) implemented in MOPAC 2012.A dielectric constant of  = 20 and a refraction index of solvent (n) such that n 2 = 2 were used.