Palladium-Catalyzed Functionalization of Shortwave Infrared Heptamethine Fluorophores Expands Their In Vivo Utility

Abstract

Fluorescence imaging in the near-infrared (NIR, 700-1000 nm) and shortwave infrared (SWIR, 1000-2000 nm) regions is advantageous for studying mammals. This work applies palladium-catalyzed coupling methods to functionalize flavylium and chromenylium SWIR polymethine fluorophores, which are challenging substrates due to their small HOMO-LUMO gaps. These chemistries include Suzuki-Miyaura and Sonogashira couplings as well as an unprecedented coupling of alcohol substrates to ultimately achieve a panel of C-C_Ar, C-C_sp, and C-O-alkyl functionalized SWIR fluorescent heptamethine dyes. The photophysical properties of the resulting fluorophores are analyzed against Hammett parameters to produce predictive metrics for absorption maxima. These metrics are strategically applied in the design of laser-matched, SWIR-emissive, chromenylium heptamethine dyes. Added functionalities advance the utility of SWIR fluorophores by increasing brightness in micelle formulations, modulating lipophilicity for alternative delivery vehicles, and enabling bioconjugation to targeting moieties. Ultimately, three functionalized fluorophores are employed in concert to achieve multicolor excitation-multiplexed imaging in murine cancer models.

Keywords: bioconjugation; cross-coupling; multiplexed imaging; near-infrared; polymethine dyes; shortwave infrared.

1

Heptamethine fluorophores functionalized at the C4′ position improve biological utility. A) General structure of heptamethine dyes and absorption maxima of dyes in (B). B) Structures of indolinium, chromenylium, and flavylium heterocycles. C) Palladium-catalyzed functionalization of SWIR heptamethine dyes. D) Application of C4′ position coupling chemistries for in vivo multiplexed imaging.

1. Substrate Scope of Palladium-Catalyzed Coupling Chemistries

2

Palladium dependency of the C–O-alkyl transformation and condition screens. A) Conditions used to yield the oxidative addition complex (5) and subsequent alcohol coupling. B) LCMS data of 5 overlaid with the crude reaction mixture. C) Absorbance spectra (taken in DCM) showing the change in λ_max used to monitor product formation. D) Base screen. Conditions: iii. Base, Pd­(PPh₃)₄, CH₃(CH₂)₄OH, 1,4-dioxane:H₂O, 120 °C (μ-wave), 20 min.

2. Common Bioconjugation Approaches Applied to Functionalized Flav7 Derivatives

3

Investigation of λ_max,abs changes and Hammett analysis of functionalized fluorophores. A) Normalized absorption and emission spectra of functionalized Flav7 derivatives 8, 11, 12 taken in DCM. B) Change in λ_max,abs of C4′ position modified fluorophores in reference to Flav7. C) Structure and photophysical characterization of fluorophores. D) Hammett analysis relating λ_max,abs and σ^– of functionalized Flav7 derivatives presented in (C).

4

Application of the C4′ position coupling methods with different heterocyclic scaffolds affords excitation-matched, functionalized fluorophores. A) Structure and photophysical properties of parent fluorophore scaffold and the final functionalized probes 17–19. B) Normalized absorption and emission spectra of 17, 19 taken in DCM, and 18 taken in MeOH. C) Table of photophysical properties of free (in DCM or MeOH) and formulated fluorophores (Micelles in PBS; dye dissolved in 10% n-BuOH in VitE).

5

In vivo imaging of 17 and 18 injected via micelle delivery vehicles. A) Scheme of micelle formulation. B,C). Absorbance spectra of 17@micelles or 18@micelles in PBS. D,E) Brightness comparison of capillaries with either 17@micelles or 18@micelles in PBS. F) Imaging parameters and timeline of injection (not to scale). G,H) SWIR fluorescence images of 17@micelles (40 μM, 200 μL, 1060 nm excitation, 160 mW cm^–2) at t = 0 h, 1 ms ET and t = 1 d, 1 ms ET. I,J) 18@micelles (39 μM, 200 μL, 890 nm excitation, 135 mW cm^–2) at t = 0 min, 5 ms ET and t = 24 h, 3 ms ET. All SWIR fluorescence images were recorded via an InGaAs camera equipped with a 1100 nm LP filter. Scale bar = 10 mm.

6

Excitation-multiplexed imaging in xenograft mice. A) Imaging parameters and timeline of injection (not to scale) of 17@micelles (yellow, 140 μM, 200 μL, 1060 nm excitation, 160 mW cm^–2, 6 ms ET), 18@micelles (cyan, 110 μM, 200 μL, 890 nm excitation, 135 mW cm^–2, 2 ms ET), 19@VitE (magenta, 30 μM, 300 μL, 974 nm excitation, 160 mW cm^–2, 20 ms ET). B,C) Merged images of three-color excitation-multiplexed imaging of the (B) dorsal, (C) ventral, (D) lateral, right, and (E) lateral, left sides of the animal. All SWIR fluorescence images were recorded via an InGaAs camera equipped with a 1300 nm LP filter. Scale bars = 10 mm. See Figures S15 and S16 for single channel images and replicate images.