Convergent synthesis and optical properties of near-infrared emitting bioluminescent infra-luciferins

Abstract

Infra-luciferin, an alkene linked analogue of luciferin, gives bioluminescence emission >700 nm and has the potential to be used for multiparametric in vivo imaging. We report here a high yielding, scalable and convergent synthesis of infra-luciferin which will allow the synthesis of other conjugated luciferins for investigation in near-infrared bioluminescence imaging. We demonstrated this potential by using the new route to synthesise a diene linked analogue of luciferin, the fluorescent and bioluminescent properties of which were compared to those of d-luciferin and infra-luciferin. We found that extension of conjugation to a diene linker resulted in the specific bioluminescence activity being reduced by 3-4 orders of magnitude compared to d-luciferin. Analogous to its fluorescence emission spectrum, the diene linked analogue exhibited two peaks in its bioluminescence spectrum, the major one being slightly blue-shifted compared to natural d-luciferin, and a minor peak at ca. 800 nm. The fluorescence quantum yield and pH dependence of fluorescence were also determined.

Fig. 1. Firefly d-luciferin and synthetic extended conjugation analogues.

Scheme 1. Previous reported route to infra-luciferin (4).

Fig. 2. Proposed disconnection for the rapid synthesis of conjugated luciferin analogues.

Scheme 2. Synthesis of thiazoline 9.

Scheme 3. Convergent synthesis of infra-luciferin (4) and novel diene luciferin analogue 5.

Fig. 3. (a) Fluorescence emission spectra of d-luciferin (1) (green line), infra-luciferin (4) (red line) and 5 (black line). Dotted lines are normalized spectra, (b) pH dependence of fluorescence spectra of d-luciferin (1) (330 nm excitation), (c) infra-luciferin (4) (352 nm excitation), and (d) 5 (369 nm excitation) measured in 5 nm increments.

Fig. 4. Quantification of light emission of luciferin and analogues with wild-type Photinus pyralis luciferase. 2 mM ATP was added to 0.17 μM pure Flucs mixed with 150 μM substrates and these were assayed in the PIO with and open filter to measure (a) specific activity, and (b) kinetics over 50 min.

Fig. 5. Emission spectra of luciferin and analogues with wild-type Photinus pyralis luciferase. (a) 0.17 μM Fluc was mixed with 150 μM d-luciferin (1) and 2 mM ATP and emission spectra were obtained using the PIO. (b) Infra-luciferin (4) in same conditions as (a). (c) 3 μM WT Fluc was used to acquire spectra with (5).