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.

•The photobleaching of heptamethine cyanines arises from a photooxidative cleavage reaction.•Slowing cyanine photooxidation generates photochemically stable fluorophores.•Cyanine photooxidation has been used to create near-IR photocages, which can enable targeted drug delivery.Light provides a uniquely powerful stimulus to help visualize and/or perturb biological systems. The use of tissue penetrant near-IR wavelengths enables in vivo applications, however the design of molecules that function in this range remains a substantial challenge. Heptamethine cyanine fluorophores are already important tools for near-IR optical imaging. These molecules are susceptible to photobleaching through a photooxidative cleavage reaction. This review details efforts to define the mechanism of this reaction and two emerging fields closely tied to this process. In the first, efforts that slow photooxidation enable the creation of photobleaching resistant fluorophores. In the second, cyanine photooxidation has recently been employed as the cornerstone of a near-IR uncaging strategy. This review seeks to highlight the utility of mechanistic organic chemistry insights to help tailor cyanine scaffolds for new, and previously intractable, biological applications.

Herein we report the syntheses and comparative photophysical, electrochemical, in vitro, and in vivo biological efficacy of 3-(1′-hexyloxy)ethyl-3-devinylpyropheophorbide-cyanine dye (HPPH-CD) and the corresponding indium (In), gallium (Ga), and palladium (Pd) conjugates. The insertion of a heavy metal in the HPPH moiety makes a significant difference in FRET (Förster resonance energy transfer) and electrochemical properties, which correlates with singlet oxygen production [a key cytotoxic agent for photodynamic therapy (PDT)] and long-term in vivo PDT efficacy. Among the metalated analogs, the In(III) HPPH-CD showed the best cancer imaging and PDT efficacy. Interestingly, in contrast to free base HPPH-CD, which requires a significantly higher therapeutic dose (2.5 μmol/kg) than imaging dose (0.3 μmol/kg), the corresponding In(III) HPPH-CD showed excellent imaging and therapeutic potential at a remarkably low dose (0.3 μmol/kg) in BALB/c mice bearing Colon26 tumors. A comparative study of metalated and corresponding nonmetalated conjugates further confirmed that STAT-3 dimerization can be used as a biomarker for determining the level of photoreaction and tumor response.

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.

An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5–10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (−)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (−)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6–10 exhibited significant antimalarial activity against Plasmodium falciparum.

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 μM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein–protein binding events.

Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the characteristic polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen (1O2), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for the design of improved compounds for future applications.

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′-O-alkyl heptamethine cyanines. The key reaction involves N- to O- 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.

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior tissue penetration and lower autofluorescence. We recently accessed a new class of readily synthesized NIR cyanines containing a novel C4′-O-alkyl linker, which provides both high chemical stability and excellent optical properties. In this study, we provide the first in vivo analysis of this new class of compounds, represented by the tetrasulfonate FNIR-774 (Frederick NIR 774). Monoclonal antibody (mAb) conjugates of FNIR-774 were compared to conjugates of the commercially available dye (IRDye800CW (IR800)), one of the most widely used NIR fluorophores for clinical translation. Both dyes were conjugated to panitumumab (pan) or cetuximab (cet) with ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. In contrast, in vivo imaging in mice showed different pharmacokinetics between pan-FNIR-774 (1:5) and pan-IR800 (1:5), or cet-FNIR-774 (1:5) and cet-IR800 (1:5). Particularly at the higher labeling density, mAb-FNIR-774 conjugates showed superior specific accumulation in tumors compared with mAb-IR800 conjugates. Thus, FNIR-774 conjugates showed superior in vivo pharmacokinetics compared with IR800 conjugates, independent of the mAb. These results suggest that FNIR-774 is a promising fluorescent probe for NIR optical imaging.

Cyanines are indispensable fluorophores that form the chemical basis of many fluorescence-based applications. A feature that distinguishes cyanines from other common fluorophores is an exposed polyene linker that is both crucial to absorption and emission and subject to covalent reactions that dramatically alter these optical properties. Over the past decade, reactions involving the cyanine polyene have been used as foundational elements for a range of biomedical techniques. These include the optical sensing of biological analytes, super-resolution imaging, and near-IR light-initiated uncaging. This review surveys the chemical reactivity of the cyanine polyene and the biomedical methods enabled by these reactions. The overarching goal is to highlight the multifaceted nature of cyanine chemistry and biology, as well as to point out the key role of reactivity-based insights in this promising area.

The development of photocaging groups activated by near-IR light would enable new approaches for basic research and allow for spatial and temporal control of drug delivery. Here we report a near-IR light-initiated uncaging reaction sequence based on readily synthesized C4′-dialkylamine-substituted heptamethine cyanines. Phenol-containing small molecules are uncaged through sequential release of the C4′-amine and intramolecular cyclization. The release sequence is initiated by a previously unexploited photochemical reaction of the cyanine fluorophore scaffold. The uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreERT/LoxP-reporter cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of small molecules using cytocompatible, tissue penetrant near-IR light.

Cyanines are indispensable fluorophores that form the chemical basis of many fluorescence-based applications. A feature that distinguishes cyanines from other common fluorophores is an exposed polyene linker that is both crucial to absorption and emission and subject to covalent reactions that dramatically alter these optical properties. Over the past decade, reactions involving the cyanine polyene have been used as foundational elements for a range of biomedical techniques. These include the optical sensing of biological analytes, super-resolution imaging, and near-IR light-initiated uncaging. This review surveys the chemical reactivity of the cyanine polyene and the biomedical methods enabled by these reactions. The overarching goal is to highlight the multifaceted nature of cyanine chemistry and biology, as well as to point out the key role of reactivity-based insights in this promising area.

Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the characteristic polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen (1O2), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for the design of improved compounds for future applications.