Red‐Shifted Bioluminescence Using Substrate‐Fluorophore Conjugates

NanoLuc (NLuc) is one of the brightest luciferases and has been used in numerous reporter assays and sensor designs. However, its blue light emission (460 nm) is suboptimal for deep tissue imaging or some diagnostic application, and red shifting its emission is desirable. Here, we report the design and synthesis of novel furimazine and coelenterazine analogs conjugated with fluorophores to achieve red‐shifted bioluminescence with NLuc and closely related H‐Luc via a bioluminescence resonance energy transfer (BRET). Structural insights into NLuc‐substrate interactions informed the functionalization of substrates. We report the synthesis of novel substrate‐fluorophore conjugates. We show that red‐shifted emission was achieved with high BRET ratios. The conjugates are less bright than the parent substrates, but our study highlights the importance of linker length and flexibility for optimizing bioluminescence emission reaching wavelength beyond 600 nm.