Here, we have designed and synthesized acidic pH-activatable visible to NIR switchable ratiometric pH-sensitive fluorescent dye. The design consists of a cell-permeable organic probe containing a lysosome targeting morpholine functionality and an acidic pH-activatable oxazolidine moiety. The visible closed oxazolidine form (λ abs 418 nm) can be switched to the highly conjugated NIR Cy-7 form (λ abs 780 nm) through ring opening of the oxazolidine moiety at acidic pH. This switching of the ratiometric fluorescent probe is highly reversible and can be controlled by pH. NMR, UV/vis, and fluorescence spectroscopies allowed monitoring of pH switching behavior of the probe. This bioresponsive in situ acidic organelle activatable fluorophore showed reversible pH-switchable ratiometric optical properties, high photostability, huge bathochromic emission shift of 320 nm from basic to acidic pH, off-to-on narrow NIR absorption and emission bands with enhanced molar extinction coefficient at lysosomal pH, good quantum yield, low cytotoxicity, and targeted imaging ability of live cell lysosomes with ideal pK a . The report demonstrated ratiometric imaging with improved specificity of the acidic lysosome while minimizing signals at the NIR region from nontargeted neutral or basic organelles in human carcinoma HeLa and A549 as well as rat healthy H9c2(2-1) live cells, which is monitored by confocal laser scanning microscopy.

Herein, we report water-soluble mitochondria-selective molecules that consist of a target-specific moiety conjugated with a near-infrared (NIR) imaging agent through variable spacer length. The presented NIR fluorescent cyanine-5 (Cy-5) chromophore exhibits excellent photostability, narrow NIR absorption and emission bands, high molar extinction coefficient, high fluorescence quantum yield, and long fluorescence lifetime. The biological compatibility and negligible cytotoxicity further make the dye an attractive choice for biological applications. Confocal fluorescence microscopic studies in the fixed human lung carcinoma cell line (A549) stained with the targeting NIR Cy-5 dyes (Cy-5a and Cy-5b) at 700 nM concentration show their cellular uptake and localization, which is compared with the nontargeting Cy-5c. Mitochondrial target specificity is demonstrated by colocalization experiments using the mitochondrion-tracking probe, MitoTracker Red and lysosome-tracking probe, LysoTracker Green. Multicolor imaging of cellular organelles in A549 cells is achieved in combination with suitable target-specific dyes with distinct excitation and emission, such as green emitting FM 1–43FX to selectively image the plasma membrane, blue-fluorescent DAPI to stain the nucleus, and the synthesized NIR Cy-5 to image the mitochondria. Higher accumulation of the dye inside the cancer cell mitochondria compared to the noncancerous cell is also demonstrated.

Bromide (CTAB), and Zinc phthalocyanine were obtained from TCI Chemicals. NMR solvents were purchased from Cambridge Isotope Laboratories, Inc. Epithelioid cervix carcinoma HeLa and lung adenocarcinoma A549 cell lines and normal human embryonic kidney (HEK293) cell line were acquired from The National Centre for Cell Science, India. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco's modified eagle medium (DMEM), Trypsin EDTA mixture, Fetal Bovine Serum (FBS), and Antibiotic Antimycotic Solution were obtained from Himedia. MitoTracker Green was procured from Thermo Fisher Scientific. METHODS Microwave Synthesizer: Unsymmetrical MSQ and MSQRot were constructed using microwave synthesizer (CEM corporation, USA make, model discover bio). NMR Spectroscopy: 1D (1 H, 13 C, 31 P) and 2D NMR (1 H-1 H DQF COSY) were measured on Bruker DPX500 MHz, Bruker DPX400 MHz, and Bruker DPX300 MHz spectrometers at 25C in appropriate deuterated solvents. HRMS (ESI): High-resolution electrospray ionisation mass spectrometry (HRMS-ESI) was recorded using a Q-Tofmicro TM (Waters Corporation) mass spectrometer. Single Crystal X-Ray Diffraction: Single crystals suitable for X-ray diffraction was obtained by vapor diffusion method. A solution of SQRot in CHCl 3 was placed in a 2 mL vial and put inside a 20 mL vial filled with EtOAc. The larger vial was capped and allow to slow diffusion of EtOAc into CHCl 3 solution of the compound. Diffraction data of SQRot was measured with MoK α radiation (a graded multilayer mirror monochromator, λ = 0.71073 Å) at 273 K on a micro focus Single Crystal X-ray Diffraction instrument (Make: Bruker, Model : D8 Quest). A PHOTON-100 CMOS detector is used. Structures were solved by direct methods using the SHELXT 2014/5 program. S1,S2 Refinements were carried out with a full-matrix least squares method against F 2 using SHELXL-2018/3 S1,S2 incorporated in Olex2 S3 crystallographic collective package. The non-hydrogen atoms were refined with anisotropic thermal parameters. Formula sum = C97.24 H84 N6 O12, formula weight = 1528.58, crystal system = triclinic, space group = P-1, a = 11.1202(11) Å, b = 11.6727(12) Å, c = 16.8575(17) Å,  = 94.197(3),  = 100.651(3),  = 111.900(3), V = 1970.9(3) Å 3 , Z = 1, D calcd = 1.288 g cm 3 , μ = 0.085 mm 1. The final R value was R 1 = 0.0688 and wR 2 = 0.1875 for reflections with I > 2ζ(I).

Herein, conjugation of the amyloid-β (Aβ) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aβ16–20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson’s correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer −220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

We have designed and synthesized red fluorescent mechanically interlocked molecules with dual targeting functionality for live cancer cell specific active targeting followed by selective internalization and imaging of malignant lysosomes along with realtime tracking, 3D, and multicolor cellular imaging applications.