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In this work on the design and studies of luciferins related to the blue‐hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O‐acetylated derivatives of imidazo[1,2‐b]pyridazin‐3(5 H)‐one, imidazo[2,1‐f][1,2,4]triazin‐7(1 H)‐one, imidazo[1,2‐a]pyridin‐3‐ol, imidazo[1,2‐a]quinoxalin‐1(5 H)‐one, benzo[f]imidazo[1,2‐a]quinoxalin‐3(11 H)‐one, imidazo[1′,2′:1,6]pyrazino[2,3‐c]quinolin‐3(11 H)‐one, and 5,11‐dihydro‐3 H‐chromeno[4,3‐e]imidazo[1,2‐a]pyrazin‐3‐one is described thanks to extensive use of the Buchwald–Hartwig N‐arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues. [ABSTRACT FROM AUTHOR]

We describe here an extensive structure‐bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep‐sea shrimp Oplophorus gracilirostris. Out of the 135 O‐acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8‐(2,3‐difluorobenzyl)‐2‐((5‐methylfuran‐2‐yl)methyl)‐6‐phenylimidazo[1,2‐a]pyrazin‐3(7H)‐one provided a 1.5‐fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal‐to‐background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate‐dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer‐dependent fraction of a photon). [ABSTRACT FROM AUTHOR]

ABSTRACT Background Methods Results Conclusions Diagnosis of allergies is mostly based on the patient's clinical history and allergen provocation tests. Determination of specific IgE (sIgE) profiles can be performed to support allergy diagnosis. This is commonly done in vivo by the skin prick test or in vitro with automated systems. Several platforms exist to quantify sIgE levels, but all these methods require access to specific instruments, often delaying the test's results. The IgE luciferase‐linked immunosorbent assay (LuLISA) allows bioluminescent quantification of IgE against peanut in microliter samples, but this method awaits extension to other allergens. This study aimed to validate a new method, named LuLIPLEX, for multiplexed bioluminescent detection of sIgE against 20 major molecular allergens.Quantification of sIgE against 12 recombinant or purified food allergens and eight aeroallergens was performed by LuLIPLEX versus standard IgE detection methods (ImmunoCAP, ISAC, ALEX, or NOVEOS). Multiplexed detection of IgE against these 20 allergens was performed within 45 min using 50 μL of serum, plasma, or whole blood samples.A head‐to‐head comparison between LuLIPLEX and standard IgE detection methods showed a high correlation among all allergens tested. sIgE profiles in polysensitized subjects could be determined within 45 min in serum and plasma samples, as well as using a single drop of capillary blood.LuLIPLEX is a rapid and sensitive method to quantify sIgE levels against multiple allergens. Given that the test is very fast and can be performed on small and inexpensive luminometers, the IgE LuLIPLEX could allow point‐of‐care testing of sIgE profiles in allergic subjects. [ABSTRACT FROM AUTHOR]