Roger R. Nani

A Near-IR Uncaging Strategy Based on Cyanine Photochemistry

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.

Enantioselective Total Synthesis of (+)-Salvileucalin B

An enantioselective total synthesis of the diterpenoid natural product (+)-salvileucalin B is reported. Key findings include a copper-catalyzed arene cyclopropanation reaction to provide the unusual norcaradiene core and a reversible retro-Claisen rearrangement of a highly functionalized norcaradiene intermediate.

α-Diazo-β-ketonitriles: Uniquely Reactive Substrates for Arene and Alkene Cyclopropanation

An investigation of the intramolecular cyclopropanation reactions of α-diazo-β-ketonitriles is reported. These studies reveal that α-diazo-β-ketonitriles exhibit unique reactivity in their ability to undergo arene cyclopropanation reactions; other similar acceptor-acceptor-substituted diazo substrates instead produce mixtures of C-H insertion and dimerization products. α-Diazo-β-ketonitriles also undergo highly efficient intramolecular cyclopropanation of tri- and tetrasubstituted alkenes. In addition, the α-cyano-α-ketocyclopropane products are demonstrated to serve as substrates for SN2, SN2, and aldehyde cycloaddition reactions.

Rapid assembly of the salvileucalin B norcaradiene core.

Preparation of the polycyclic core of the cytotoxic natural product salvileucalin B is described. The key feature of this synthetic strategy is a copper-catalyzed intramolecular arene cyclopropanation to provide the central norcaradiene. These studies lay the foundation for continued investigations toward an enantioselective total synthesis of 1.

Near-IR Light-Mediated Cleavage of Antibody–Drug Conjugates Using Cyanine Photocages

Despite significant progress in the clinical application of antibody drug conjugates (ADCs), novel cleavage strategies that provide improved selectivity are still needed. Herein is reported the first approach that uses near-IR light to cleave a small molecule from a biomacromolecule, and its application to the problem of ADC linkage. The preparation of cyanine antibody conjugates, drug cleavage mediated by 690u2005nm light, and initial in vitro and in vivo evaluation is described. These studies provide the critical chemical underpinning from which to develop this near-IR light cleavable linker strategy.

Electrophile-integrating Smiles rearrangement provides previously inaccessible C4'-O-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′-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.

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.

In Vivo Activation of Duocarmycin–Antibody Conjugates by Near-Infrared Light

Near-IR photocaging groups based on the heptamethine cyanine scaffold present the opportunity to visualize and then treat diseased tissue with potent bioactive molecules. Here we describe fundamental chemical studies that enable biological validation of this approach. Guided by rational design, including computational analysis, we characterize the impact of structural alterations on the cyanine uncaging reaction. A modest change to the ethylenediamine linker (N,N′-dimethyl to N,N′-diethyl) leads to a bathochromic shift in the absorbance maxima, while decreasing background hydrolysis. Building on these structure–function relationship studies, we prepare antibody conjugates that uncage a derivative of duocarmycin, a potent cytotoxic natural product. The optimal conjugate, CyEt-Pan-Duo, undergoes small molecule release with 780 nm light, exhibits activity in the picomolar range, and demonstrates excellent light-to-dark selectivity. Mouse xenograft studies illustrate that the construct can be imaged in vivo prior to uncaging with an external laser source. Significant reduction in tumor burden is observed following a single dose of conjugate and near-IR light. These studies define key chemical principles that enable the identification of cyanine-based photocages with enhanced properties for in vivo drug delivery.

Effect of Metalation on Porphyrin-Based Bifunctional Agents in Tumor Imaging and Photodynamic Therapy

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.

Impact of C4′-O-Alkyl Linker on in Vivo Pharmacokinetics of Near-Infrared Cyanine/Monoclonal Antibody Conjugates

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.