Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy

The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable twocarbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygeninitiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool

Electrophile-Integrating Smiles Rearrangement Provides Previously Inaccessible C4′‑<i>O</i>‑Alkyl Heptamethine Cyanine Fluorophores

New synthetic methods to rapidly access useful fluorophores are needed to advance modern molecular imaging techniques. A new variant of the classical Smiles rearrangement is reported that enables the efficient synthesis of previously inaccessible C4′-<i>O-</i>alkyl heptamethine cyanines. The key reaction involves <i>N</i>- to <i>O</i>- transposition with selective electrophile incorporation on nitrogen. A representative fluorophore exhibits excellent resistance to thiol nucleophiles, undergoes productive bioconjugation, and can be used in near-IR fluorescence imaging applications

A plant lipocalin promotes retinal-mediated oscillatory lateral root initiation

In Arabidopsis, de novo organogenesis of lateral roots is patterned by an oscillatory mechanism called the root clock, which is dependent on unidentified metabolites. To determine whether retinoids regulate the root clock, we used a chemical reporter for retinaldehyde (retinal)–binding proteins. We found that retinal binding precedes the root clock and predicts sites of lateral root organogenesis. Application of retinal increased root clock oscillations and promoted lateral root formation. Expression of an Arabidopsis protein with homology to vertebrate retinoid-binding proteins, TEMPERATURE INDUCED LIPOCALIN (TIL), oscillates in the region of retinal binding to the reporter, confers retinal-binding activity in a heterologous system, and, when mutated, decreases retinal sensitivity. These results demonstrate a role for retinal and its binding partner in lateral root organogenesis

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging

Multi-component bioluminescence imaging requires an expanded collection of luciferase-luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging