A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging

Abstract

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λ(max)=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

Keywords: imaging agents; luciferase; luminescence; multiparametric imaging; structure-activity relationships.

Figure 1

a) Bioluminescence of luciferin (1) catalyzed by luciferase. b) Structures of red-shifted bioluminescent amino-luciferin analogues. c) New luciferin analogue iLH₂ 6 that exhibits near-infrared bioluminescence with mutant Fluc. ATP=adenosine triphosphate.

Scheme 1

Synthesis of infra-luciferin 6. a) BnBr (1.2 equiv), K₂CO₃ (2.8 equiv), acetone, room temperature, 16 h, 85 %; b) nBuLi (1.93 m in hexanes, 1.1 equiv), THF, −78 °C, 15 min then DMF (4.1 equiv), 1 h, 96 %; c) (Carbethoxymethylene)triphenylphosphorane (3 equiv), PhMe, reflux, 3 h, 92 %; d) NaOH (1 m), iPrOH, 16 h, quantitative yield; e) Et₃N (2.4 equiv), DMF, amino acid (aa; 1.2 equiv), 0 °C then BOP (1.2 equiv) in CH₂Cl₂, 2 h (R=Me, 80 %, R=Et, 82 %); f) Ph₃PO (1.3 equiv), Tf₂O (2.7 equiv), CH₂Cl₂, 0 °C, 30 min added to benzothiazole in CH₂Cl₂, 0 °C, 10 min, (R=Me, 65 %, R=Et, 74 %); g) pentamethylbenzene (4.4 equiv), BCl₃ (1 m in CH₂Cl₂, 3 equiv), CH₂Cl₂, −78 °C, (R=Me, 79 %, R=Et, 72 %); h) PLE, buffer, 37 °C, in situ. Bn=benzyl; Tf₂O=trifluoromethanesulfonic anhydride; Trt=triphenylmethyl; BOP=(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; PLE=pig liver esterase.

Figure 2

Bioluminescence spectra of native 1 and 6 with x5 and x5 S284T Fluc mutants.

Figure 3

In vivo imaging with 6 in mouse cancer models expressing firefly luciferase (Fluc). Left column of images: no substrate; middle column: mice imaged with LH₂; right column: mice imaged with iLH₂ Me ester. Inset graphs show the relative in vivo light yields from mice with different substrates and imaged for equivalent times. In vivo spectra are displayed in Figure S4 in the Supporting Information.

Figure 4

Histogram showing the increased penetration of iLH₂ 6 emission through blood compared to LH₂ 1.