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Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.