Heptamethine cyanines (Cy7) are fluorophores essential for modern bioimaging techniques and chemistry. Here, we systematically evaluated the photochemical and photophysical properties of a library of Cy7 derivatives containing diverse substituents in different positions of the heptamethine chain. A single substitution allows modulation of their absorption maxima in the range of 693–805 nm and photophysical properties, such as quantum yields of singlet-oxygen formation, decomposition, and fluorescence or affinity to singlet oxygen, within 2–3 orders of magnitude. The same substituent in different positions of the chain often exhibits distinctly contradictory effects, demonstrating that both the type and position of the substituent are pivotal for the design of Cy7-based applications. The combination of experimental results with quantum-chemical calculations provides insights into the structure–property relationship, the elucidation of which will accelerate the development of cyanines with properties tailored for specific applications, such as fluorescent probes and sensors, photouncaging, photodynamic therapy, or singlet-oxygen detection.

Cyanine dyes play an indispensable and central role in modern fluorescence-based biological techniques.Despite their importance and widespread use, the current synthesis methods of heptamethine chain modification are restricted to coupling reactions and nucleophilic substitution at the meso position in the chain. Herein, we report the direct transformation of Zincke salts to cyanine dyes under mild conditions, accompanied by the incorporation of a substituted pyridine residue into the heptamethine scaffold. This work represents the first general approach that allows the introduction of diverse substituents and different substitution patterns at the C3′−C5′ positions of the chain. High yields, functional tolerance, versatility toward the condensation partners, and scalability make this method a powerful tool for accessing a new generation of cyanine derivatives.

Carbon monoxide (CO) is an endogenouss ignaling molecule that controls an umber of physiological processes. To circumventt he inherentt oxicity of CO, light-activated CO-releasing molecules (photoCORMs)h ave emerged as an alternative for its administration. However,t heir wider application requires photoactivation using biologically benign visible and near-infrared (NIR) light.I nt his work, a strategy to access such photoCORMs by fusing two CO-releasing flavonol moieties with aN IR-absorbing cyanine dye is presented. These hybrids liberate two molecules of CO in high chemical yields upon activation with NIR light up to 820 nm and exhibit excellent uncaging cross-sections, which surpasst he state-of-the-art by two orders of magnitude.F urthermore, the biocompatibility and applicabilityo ft he system in vitro and in vivo are demonstrated, andamechanism of CO release is proposed. It is hoped that this strategy will stimulate the discovery of new classes of photoCORMs and accelerate the translation of CO-based phototherapy into practice.

Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.

Carbon monoxide release from biocompatible heptamethine cyanine–flavonol hybrids is activated by NIR light. Excellent release yields and uncaging cross sections, enhanced water solubilities, and a host–guest approach using cucurbit[7]uril are shown.