Scheme 1.
Luciferin chemistry mechanism.
Scheme 2.
Luciferin breakdown pathway in solution.
Fig 1.
Chemical modification to improve luciferin stability in solution.
(A) Hypothesis: 5,5-dialkylluciferins are thermally stable due to their inability to break down into dehydroluciferins. (B) Stability studies confirm that 5,5-dialkylluciferins are thermally stable. 5,5-dimethylluciferin (III-a) was compared to unmodified LH2 in an accelerated thermostability study where both compounds (7 mM) were incubated in Bright-Glo™ assay buffer at 60°C for 150 hours.
Scheme 3.
Synthesis of 5,5-Dialkylluciferins.
General procedure: Hydrochloric salts of β,β-disubstituted cysteine analogs (II, 0.15 mmol, 1.5 equiv) dissolved in H2O (1 mL) under N2 were neutralized with 1 N NaOH (aq, 0.30 mmol, 300 μL, 3.0 equiv). To a solution of 2-cyano-6-hydroxy-benzothiazole (I, 0.1 mmol, 1.0 equiv) in DMF (2 mL) at RT under N2, was added the neutralized solution of β,β-disubstituted cysteine analogs. The solution was then stirred under N2 for 30 min. LC-MS indicated complete consumption of benzothiazole starting material (I). Luciferins (III) were obtained via preparative HPLC (mobile phase A: 10 mM NH4OAc aqueous solution; mobile phase B: CH3CN; gradient condition: 5% B to 95% B over 30 minutes).
Fig 2.
Biochemical evaluation of 5,5-dialkylluciferins.
(A) Maximum luminescence of Ultra-Glo™ luciferase with different 5,5-dialkylluciferin substrates. A dotted line indicating background luminescence from a no compound control lacking substrate in Bright-GloTM buffer provides a baseline comparison to show the reduction in luminescence from reactions containing 5,5-dialkylluciferins. (B) Inhibition of the Ultra-Glo™:LH2 luminescence reaction with 5,5-dialkylluciferins. Reactions containing Ultra-Glo™ luciferase, 1 mM ATP, and 100 μM LH2 were spiked with 1 mM of indicated analog. A normalization factor that converted the raw luminescence value of Ultra-Glo™:LH2 reaction to a value of 100 was applied to all samples in the experiment to show the significant reduction in luminescence of the system when 5,5-dialkyl luciferins were spiked in. Experiments were performed in duplicate and plotted as the average with error bars representing one standard deviation from the mean.
Fig 3.
HPLC-based luciferin stability profiles.
(A) HPLC-based quantification of luciferin decomposition at 325 nm. 1 mM luciferins (LH2, III-a or III-b) were reconstituted in Bright-Glo™ buffer and incubated at 37 oC. Aliquots were taken at day 60, percentage of decomposition were quantified based on absorbance at 325 nm. Blue bar represents percentage of luciferins, and red bar represents percentage of decomposition. At day 60, 25% of LH2 were degraded into dehydroluciferin, while III-b showed no signs of decomposition. Less than 5% of decomposition were observed for III-b. (B) Identification of decomposition products of III-b. III-b-s1 and III-b-s2 were identified as key decomposition products, the details of enrichment and identification were illustrated in S2 File.
Fig 4.
Structure-guided mutagenesis of Ultra-Glo™ identifies a key residue in 5,5-dialkylluciferin utilization.
(A) Homology model of Ultra-Glo™ luciferase with 5,5-dimethylluciferin. The expanded view highlights residues in proximity to the active site that were screened by site-saturation mutagenesis. (B) Mutation of H244 to tryptophan had significant improvements in brightness with 5,5-dialkylluciferins and is positioned in close proximity to the dialkyl group of the substrate.
Fig 5.
Directed evolution improves the activity of Ultra-Glo™ luciferase with 5,5-dialkylluciferins.
Progression of brightness improvements during Ultra-Glo™ luciferase evolution as measured through multiple rounds of high-throughput rational and randomized screening with substrates (A) III-a and (B) III-b. The activity of the III-b:M6 combination closely matched that of LH2:Ultra-Glo™, demonstrating that 5,5-dialkylluciferins can be utilized as highly efficient substrates of bioluminescence reactions.
Table 1.
Kinetic and thermostability parameters of evolved Ultra-Glo™ mutants with III-a and III-b.
Fig 6.
Real-time stability of ATP detection reagents using 5,5-dimethylluciferin.
Percent of remaining activity of formulations over 150 days at 37°C in a liquid format comparing the performance of individual components and the complete systems (A) Ultra-Glo™:LH2 and M7:III-a combinations, (B) LH2 or III-a substrates alone, and (C) Ultra-Glo™ or M7 luciferase enzymes alone. System instability for Ultra-Glo™: LH2 is driven primarily by the decomposition of LH2 into dehydroluciferin, while the 5,5-dimethyl modification in the M7:III-a system stabilizes the substrate component. Six replicates were performed for each sample and are plotted as the average at each timepoint with error bars indicated one standard deviation of the mean.