Photochemical Generation of Allenylidenes from Cyclopropanated Phenanthrenes: An Experimental and Computational Study.

To address the scarcity of generally applicable photochemical routes to allenylidenes in solution, phenanthrene-based sources have been investigated. Specifically, the syntheses of 1-vinylidene-1a,9b-dihydro-1H-cyclopropa[l]phenanthrene, 1-(2-phenylvinylidene)-1a,9b-dihydro-1H-cyclopropa[l]phenanthrene, and 1-(2-methylvinylidene)-1a,9b-dihydro-1H-cyclopropa[l]phenanthrene, photochemical precursors to propadienylidene, 3-phenylpropadienylidene, and 3-methylpropadienylidene have been carried out. Photolysis of these new precursors in olefin traps and benzene afforded the expected cyclopropane adducts of the corresponding allenylidenes. Quantum chemical calculations show that the ground state of all three carbenes is a singlet with a singlet-triplet gap of ∼29, 30, and 33 kcal/mol for propadienylidene, 3-phenylpropadienylidene, and 3-methylpropadienylidene, respectively.

Side-chain modification of collagen-targeting peptide prevents dye aggregation for improved molecular imaging of arthritic joints.

Near-infrared (NIR) dye-peptide conjugates are widely used for tissue-targeted molecular fluorescence imaging of pathophysiologic conditions. However, the significant contribution of both dye and peptide to the net mass of these bioconjugates implies that small changes in either component could alter their photophysical and biological properties. Here, we synthesized and conjugated a type I collagen targeted peptide, RRANAALKAGELYKCILY, to either a hydrophobic (LS1000) or hydrophilic (LS1006) NIR fluorescent dye. Spectroscopic analysis revealed rapid self-assembly of both LS1000 and LS1006 in aqueous media to form stable dimeric/H aggregates, regardless of the free dye's solubility in water. We discovered that replacing the cysteine residue in LS1000 and LS1006 with acetamidomethyl cysteine to afford LS1001 and LS1107, respectively, disrupted the peptide's self-assembly and activated the previously quenched dye's fluorescence in aqueous conditions. These results highlight the dominant role of the octadecapeptide, but not the dye molecules, in controlling the photophysical properties of these conjugates by likely sequestering or extruding the hydrophobic or hydrophilic dyes, respectively. Application of the compounds for imaging collagen-rich tissue in an animal model of inflammatory arthritis showed enhanced uptake of all four conjugates, which retained high collagen-binding affinity, in inflamed joints. Moreover, LS1001 and LS1107 improved the arthritic joint-to-background contrast, suggesting that reduced aggregation enhanced the clearance of these compounds from non-target tissues. Our results highlight a peptide-driven strategy to alter the aggregation states of molecular probes in aqueous solutions, irrespective of the water-solubilizing properties of the dye molecules. The interplay between the monomeric and aggregated forms of the conjugates using simple thiol-modifiers lends the peptide-driven approach to diverse applications, including the effective imaging of inflammatory arthritis joints.

Unifying Mechanism for Thiol-Induced Photoswitching and Photostability of Cyanine Dyes.

Cyanines (Cy3, Cy5, Cy3B) are the most utilized dyes for single-molecule fluorescence and localization-based super-resolution imaging. These modalities exploit cyanines' versatile photochemical behavior with thiols. A mechanism reconciling seemingly divergent results and enabling control over cyanine photoreactivity is however missing. Utilizing single-molecule fluorescence on Cy5 and Cy5B, transient-absorption spectroscopy, and DFT modeling on a range of cyanine dyes, herein we show that photoinduced electron transfer (PeT) from a thiolate to Cy in their triplet excited state and then triplet-to-singlet intersystem crossing in the nascent geminate radical pair are crucial steps. Next, a bifurcation occurs, yielding either back electron transfer and regeneration of ground state Cy, required for photostabilization, or Cy-thiol adduct formation, necessary for super-resolution microscopy. Cy regeneration via photoinduced thiol elimination is favored by adduct absorption spectra broadening. Elimination is also shown to occur through an acid-catalyzed reaction. Overall, our work provides a roadmap for designing fluorophores, photoswitching agents, and triplet excited state quenchers for single-molecule and super-resolution imaging.

Cyanine Conformational Restraint in the Far-Red Range.

Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

Fluorous Phase-Directed Peptide Assembly Affords Nano-Peptisomes Capable of Ultrasound-Triggered Cellular Delivery.

Here, we report the design, synthesis and efficacy of a new class of ultrasound (US)-sensitive self-assembled peptide-based nanoparticle. Peptisomes are prepared via templated assembly of a de novo designed peptide at the interface of fluorinated nanodroplets. Utilizing peptide assembly allows for facile particle synthesis, direct incorporation of bioactive sequences displayed from the particle corona, and the ability to easily encapsulate biologics during particle preparation using a mild solvent exchange procedure. Further, nano-peptisome size can be precisely controlled by simply modulating the starting peptide and fluorinated solvent concentrations during synthesis. Biomolecular cargo encapsulated within the particle core can be directly delivered to the cytoplasm of cells upon US-mediated rupture of the carrier. Thus, nano-peptisomes represent a novel class of US-activated carriers that can shuttle cell-impermeable biomacromolecules into cells with spatial and temporal precision.

Effect of charge localization on the in vivo optical imaging properties of near-infrared cyanine dye/monoclonal antibody conjugates.

Near-infrared (NIR) fluorophores show superior in vivo imaging properties than visible-light fluorophores because of the increased light penetration in tissue and lower autofluorescence of these wavelengths. We have recently reported that new NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. In this study, we synthesized two NIR cyanine dyes with the same core structure and charge but different indolenine substituents: FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, and FNIR-G-765 bearing a combination of two sulfonates and two guanidines, resulting in zwitterionic charge with distinct cationic moieties. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-Z-759 and FNIR-G-765 with panitumumab (pan) at antibody-to-dye ratios of 1 : 2 or 1 : 5. One-to-five conjugation of pan-to-FNIR-G-765 was not successful due to aggregate formation during the conjugation reaction. Conjugates of both dyes to pan (2 : 1) demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower accumulation in the mouse liver, resulting in higher tumor-to-liver ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-G-765 conjugates, especially in the abdominal region. Moreover, from a chemistry point of view, mAb conjugation with FNIR-Z-759 has an advantage over FNIR-G-765, because it does not form aggregates at high dye-to-mAb ratio. These results suggest that zwitterionic cyanine dyes are a superior class of fluorophores for conjugating with mAbs for fluorescence imaging applications due to improving target-to-background contrast in vivo. However, zwitterionic cyanine dyes should be designed carefully, as small changes to the structure can alter in vivo pharmacokinetics of mAb-dye conjugates.

Role of Fluorophore Charge on the In Vivo Optical Imaging Properties of Near-Infrared Cyanine Dye/Monoclonal Antibody Conjugates.

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior light penetration in tissue and lower autofluorescence. We recently demonstrated that a new class of NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. We synthesized two NIR cyanine dyes with the same core structure but different indolenine substituents: FNIR-774 bearing four sulfonate groups and FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, resulting in an anionic (-3) or monocationic (+1) charge, respectively. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-774 and FNIR-Z-759 with panitumumab (pan) at antibody-to-dye ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower background in mice, resulting in higher tumor-to-background ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-774 conjugates, especially in the abdominal region, regardless of the dye-mAb ratio. These results suggest that zwitterionic cyanine dyes are a promising class of fluorophores for improving in vivo optical imaging with antibody-NIR dye conjugates.