Design of an Acidic pH-Activated NIR Fluorescent Convertible Rhodamine-Hemicyanine Probe-Peptide Conjugate for Living Cancer Cell Active Targeted Selective Tracking of Lysosomes.

We have synthesized an acidic pH-activatable dual targeting ratiometric fluorescent probe-peptide conjugate using the SPPS protocol on resin. Living carcinoma cell specific active targeting, successive cell penetration, and selective staining of lysosomes are accomplished. Real-time monitoring of lysosomes, 3D, and multicolor cancer cell imaging are attained. The de novo design consists of the integration of multifunctionality into a single molecular scaffold, e.g., RGDS peptide to target cancer cell overexpressed receptor αVß3 integrin, live-cell penetrating  rhodamine-hemicyanine chromophore comprising a lysosome targeting morpholine group, and an acidic pH openable spiro-lactam ring for a visible-to-NIR switchable ratiometric response. Water-soluble probe-peptide conjugate exhibits intramolecular spirolactamization at basic pH through Arg amide N. The visible spirolactam state predominantly exists at physiological and basic pH and can be switched to the highly conjugated NIR open amide state (λem=735 nm) through spiro-lactam ring opening triggered by acidic pH with a huge bathochromic shift (Δλabs=336 nm, ΔλFL=265 nm). pH-sensitive ratiometric switching is achieved. This in situ acidic cancer cell lysosome activatable multifunctional fluorophore-peptide conjugate shows augmented molar absorptivity, enhanced quantum yield, and improved fluorescence lifetime at acidic lysosomal pH; negligible cytotoxicity; and dual targeted ratiometric imaging capability of living cancer cell selective lysosomes with pKa value of 5.1.

Design of Water-Soluble Rotaxane-Capped Superparamagnetic, Ultrasmall Fe3O4 Nanoparticles for Targeted NIR Fluorescence Imaging in Combination with Magnetic Resonance Imaging.

Integrating an NIR fluorescent probe with a magnetic resonance imaging (MRI) agent to harvest complementary imaging information is challenging. Here, we have designed water-soluble, biocompatible, noncytotoxic, bright-NIR-emitting, sugar-functionalized, mechanically interlocked molecules (MIMs)-capped superparamagnetic ultrasmall Fe3O4 NPs for targeted multimodal imaging. Dual-functional stoppers containing an unsymmetrical NIR squaraine dye interlocked within a macrocycle to construct multifunctional MIMs are developed with enhanced NIR fluorescence efficiency and durability. One of the stoppers of the axle is composed of a lipophilic cationic TPP+ functionality to target mitochondria, and the other stopper comprises a dopamine-containing catechol group to anchor at the surface of the synthesized Fe3O4 NPs. Fe3O4 NPs surface-coated with targeted NIR rotaxanes help to deliver ultrasmall magnetic NPs specifically inside the mitochondria. Two carbohydrate moieties are conjugated with the macrocycle of the rotaxane via click chemistry to improve the water solubility of MitoSQRot-(Carb-OH)2-DOPA-Fe3O4 NPs. Water-soluble, rotaxane-capped Fe3O4 NPs are used for live-cell mitochondria-targeted NIR fluorescence confocal imaging, 3D and multicolor imaging in combination with T2-weighted MRI on a 9.4 T MR scanner with a high relaxation rate (r2) of 180.7 mM-1 s-1. Biocompatible, noncytotoxic, ultrabright NIR rotaxane-capped superparamagnetic ultrasmall monodisperse Fe3O4 NPs could be a promising agent for targeted multimodal imaging applications.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Construction of Red Fluorescent Dual Targeting Mechanically Interlocked Molecules for Live Cancer Cell Specific Lysosomal Staining and Multicolor Cellular Imaging.

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 real-time tracking, 3D, and multicolor cellular imaging applications.

Construction of Self-Assembling Lipopeptide-Based Benign Nanovesicles to Prevent Amyloid Fibril Formation and Reduce Cytotoxicity of GxxxGxxxGxxxG Motif.

Alzheimer's disease, a progressive severe neurodegenerative disorder, has been until now incurable, in spite of serious efforts worldwide. We have designed self-assembled myristoyl-KPGPK lipopeptide-based biocompatible nanovesicles, which can inhibit amyloid fibrillation made by the transmembrane GxxxGxxxGxxxG motif of Aß-protein and human myelin protein zero as well as reduce their neurotoxicity. Various spectroscopic and microscopic investigations illuminate that the lipopeptide-based nanovesicles dramatically inhibit random coil-to-ß-sheet transformation of Aß25-37 and human myelin protein zero protein precursor, which is the prerequisite of GxxxGxxxGxxxG motif-mediated fibril formation. Förster resonance energy transfer (FRET) assay using synthesized Cy-3 (FRET donor) and Cy-5 (FRET acceptor)-conjugated Aß25-37 also exhibits that nanovesicles strongly inhibit the fibril formation of Aß25-37. The mouse neuro-2a neuroblastoma cell line is used, which revealed the GxxxGxxxGxxxG-mediated cytotoxicity. However, the neurotoxicity has been diminished by co-incubating the GxxxGxxxGxxxG motif with the nanovesicles.

Selective fluorescence sensing of Cu(II) and Zn(II) using a simple Schiff base ligand: naked eye detection and elucidation of photoinduced electron transfer (PET) mechanism.

A simple Schiff base compound 2-((cyclohexylmethylimino)-methyl)-naphthalen-1-ol (2CMIMN1O) has been synthesized and characterized by (1)H NMR, (13)C NMR and FT-IR spectroscopic techniques. A significantly low emission yield of the compound has been rationalized in anticipation with photo-induced electron transfer (PET) from the imine receptor moiety to the naphthalene fluorophore unit. Consequently, an evaluation of the transition metal ion-induced modification of the fluorophore-receptor communication reveals the promising prospect of the title compound to function as a chemosensor for Cu(2+) and Zn(2+) ions selectively, through remarkable fluorescence enhancement as well as visual changes. While perturbation of the PET process has been argued to be the plausible mechanism behind the fluorescence enhancement, the selectivity for these two metal ions has been interpreted on the grounds of an appreciably strong binding interaction. Particularly notable aspects regarding the chemosensory activity of the compound is its ability to detect the aforesaid transition metal ions down to the level of micromolar concentration (detection limit being 2.74 and 2.27ppm respectively), along with a simple and efficient synthetic procedure.

Selective fluorescence sensing of Cu(II) and Zn(II) using a new Schiff base-derived model compound: naked eye detection and spectral deciphering of the mechanism of sensory action.

A new Schiff base compound 2-((benzylimino)-methyl)-naphthalen-1-ol (2BIMN1O) has been synthesized and characterized by (1)H NMR, (13)C NMR, DEPT, FT-IR and mass spectroscopic techniques. The significantly low fluorescence yield of the compound has been rationalized in connection with photo-induced electron transfer (PET) from the imine receptor moiety to the naphthalene fluorophore unit. Subsequently, an evaluation of the transition metal ion-induced modification of the fluorophore-receptor communication reveals a promising prospect for the title compound to function as a fluorosensor for Cu(2+) and Zn(2+) ions selectively, through remarkable fluorescence enhancement. While perturbation of the PET process in 2BIMN1O has been argued to be the responsible mechanism behind the fluorescence enhancement, the selectivity for these two metal ions has been interpreted on the grounds of an appreciably strong binding interaction. Particularly notable aspects regarding the chemosensory activity of the compound are its ability to detect the aforesaid transition metal ions down to the level of micromolar concentration (detection limit being 0.82 and 0.35 µM respectively), along with a simple and efficient synthetic procedure. Also the spectral modulation of 2BIMN1O in the presence of the transition metal ions paves the way for the construction of a calibration curve in the context of its fluorescence signaling potential.

Photoinduced intramolecular charge transfer phenomena in 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid.

A donor acceptor substituted aromatic system 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) has been synthesized and its spectral properties have been explored on the basis of steady state absorption and fluorescence spectroscopy. Spectral features point largely towards a possible occurrence of photoinduced intramolecular charge transfer process from the donor NMe2 group to the acceptor acid group. Solvent dependency of the large Stokes' shifted emission band and the calculated large excited state dipole moment support the polar character of the charge transfer excited state. Quantum yield calculations and effect of addition of acid and base on the steady state spectra were also performed to further scrutinize the excited state CT character.

Excited state lactim to lactam type tautomerization reaction in 5-(4-fluorophenyl)-2-hydroxypyridine: spectroscopic study and quantum chemical calculation.

The photophysical properties of 5-(4-fluorophenyl)-2-hydroxypyridine (FP2HP) have been studied by steady state and time resolved spectroscopy in combination with quantum chemical calculations. The molecule FP2HP exists as lactim and lactam form in the ground state due to small stability difference but does not undergo lactim to lactam isomerisation due to high barrier energy. Whereas in the excited state the lactim form undergoes tautomerization producing red shifted emission of the lactam tautomer along with the local emission of the lactim form. In polar protic solvents, the barrier for lactim-lactam tautomerization rapidly decreases forming the lactam tautomer only. Temperature has pronounced effect on the excited state tautomerization equilibrium and is clearly reflected in the measured equilibrium constant (K(tau)(0)) and free energy change (ΔG(0)). Structural calculations at Hartree Fock and Density Functional Theory levels, calculated stability of the isomers in different solvents using Polarized Continuum Model and the theoretical potential energy surfaces for the ground and excited states along the proton transfer coordinate are reported for the tautomeric equilibrium of FP2HP.

Photoinduced properties of methyl ester of 2,6-dimethyl-1-amino-4-benzoic acid: evidence of charge transfer emission from a weak primary amino donor.

Photophysical properties of methyl ester of 2,6-dimethyl-1-amino-4-benzoic acid (M26DMB) have been investigated using steady state absorption and emission spectroscopy and time-resolved emission spectroscopy. Interestingly, not only in polar solvents, the molecule M26DMB having a weak primary amino donor group shows broad red-shifted emission band even in non-polar environment which in all probability arises from closely spaced local and charge transfer (CT) states. Clear dual fluorescence in polar protic solvents comprises of less intense local emission band and strong red-shifted CT band. The position of the red-shifted emission band is dependent on both the polarity and the hydrogen-bonding ability of the solvent.

Photoinduced intramolecular charge transfer reaction in (E)-3-(4-Methylamino-phenyl)-acrylic acid methyl ester: A fluorescence study in combination with TDDFT calculation.

A donor-acceptor substituted aromatic system (E)-3-(4-Methylamino-phenyl)-acrylic acid methyl ester (MAPAME) has been synthesized, and its photophysical behavior obtained spectroscopically has been compared with the theoretical results. The observed dual fluorescence from MAPAME has been assigned to emission from locally excited and twisted intramolecular charge transfer states. The donor and acceptor angular dependency on the ground and excited states potential energy surfaces have been calculated both in vacuo and in acetonitrile solvent using time dependent density functional theory (TDDFT) and TDDFT polarized continuum model (TDDFT-PCM), respectively. Calculation predicts that a stabilized twisted excited state is responsible for red shifted charge transfer emission.

Steric-controlled excimer formation in naphthalene analogues of chalcone.

Naphthalene analogues of chalcone (typical vinylarenes) are well known for their intramolecular charge transfer (CT) process. The all four possible isomers NC1, NC2, NC3 and NC4 were observed to give structure-less broad CT bands, whereas, excimer formation at higher concentration was reported only for NC4 [Res. Chem. Intermed. 25 (1999) 903]. However, conventional GMMX calculation data reveals that the naphthalene portion of all the isomers are planar, hence, excimer emission is expected from all of them, i.e., if NC4 can form an excimer, then the rests are also capable of doing it. In this paper we have actually succeeded in resolving the excimer peaks for all these four isomers by optimization of concentration, though the extent of excimer formation was observed to be the maximum for NC4. These differential tendencies of excimer formation can be explained by the change in extent of intermolecular stacking interaction of the naphthalene moieties. Variation occurs here due to steric perturbation arising from the specific orientation of the near resident non-planar aroyl component with respect to the naphthalene moiety.