We describeh ere an extensive structure-bioluminescence relationship studyo fachemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, am utated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 Oacetylated precursors that were prepared by using our recently reporteds ynthesis and following their hydrolysis to give solutions of the corresponding luciferins,n otable bioluminescence improvements were achieved in comparison with furimazine, whichi sc urrently amongst the best substrates of nanoKAZ/NanoLuc.F or instance, the ratherm ore lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provid-ed a1 .5-fold improvement of the total light outputo ver a 2h period, ac lose to threefold increase of the initial signal intensity and as ignal-to-background ratio five timesg reater than furimazine. The kinetic parameters for the enzymatic reactionw ere obtainedf or as electiono fl uciferina nalogues and provided unexpected insights into the luciferase activity. Most prominently,a long with ag eneral substrate-dependent and irreversible inactivation of this enzyme,i nt he case of the optimized luciferin mentioned above,t he consumption of 2664 molecules was foundt ob er equired for the detection of as ingleR elative Light Unit (RLU;aluminometer-dependentfraction of ap hoton).Scheme1.Mechanism for coelenterazine (1)b ioluminescence.[a] Dr.Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.

An original three component synthetic access to coelenterazine and analogues can lead to grams of marine luciferins which are extensively used in bioluminescence-based assays.

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renillatype bioluminescence through crystallographic, spectroscopic, and computational experiments.Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue lightemitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.

The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase‒luciferin pairs for ultrasensitive optical bioassays.

Palladium-catalyzed carbothiolation of terminal alkynes with azolyl sulfides affords various 2-(azolyl)alkenyl sulfides with perfect regio-and stereoselectivities. The present addition reaction proceeded through a direct cleavage of carbon-sulfur bonds in azolyl sulfides. The resulting adducts that are useful intermediates in organic synthesis are further transformed to multi-substituted olefins containing azolyl moieties.Carbothiolation of alkynes has been regarded as the most ideal approach to the highly substituted alkenyl sulfides in organic synthesis, which can generate carboncarbon and carbon-sulfur bonds simultaneously. 1 Regioand stereoselective addition of various carbon-sulfur bonds to alkynes has been achieved by using transition metal catalysts; thioesterification, 2 cyanothiolation, 3 allylthiolation, 4 alkenylthiolation, 5 acylthiolation, 6 iminothiolation, 7 alkynylthiolation, 8 and alkylthiolation. 9,10 Although only decarbonylative addition reaction of thioesters is known, 11 the atom-economical arylthiolation across alkynes has yet to be disclosed to date because carbon-sulfur bonds in aryl sulfides tend to cause a reversible oxidative addition. 12 While it was previously found that aryl sulfides underwent cross-coupling with organometallic reagents 13 probably because of the high reactivity of the once formed oxidative adducts for subsequent transmetalation, arylthiolation of alkynes is unprecedented.Recently, Weller and Willis have reported rhodiumcatalyzed addition of aryl sulfides bearing unique activating groups to terminal alkynes as a specific case. 14 On the other hand, addition reaction of heteroaryl sulfides to alkynes, which can construct the ubiquitous skeletons in pharmaceuticals and agrochemicals, 15 is significantly limited despite its utility. Although platinum-catalyzed furylthiolation, 11b,16 thienylthiolation, 17 and pyridylthiolation 18 of terminal alkynes with thioesters or with heteroaryl halides and arenethiolate salts are only known, those reactions produce toxic carbon monoxide or undesired by-products. We have recently disclosed the regioand stereocontrolled chlorothiolation of alkynes with transition metal catalysts through the chlorine-sulfur bond cleavage of sulfenyl chlorides. 19 During the course of our research on selective addition of organosulfur compounds to alkynes, we investigated carbothiolation with a direct activation of heteroaryl sulfides. Herein, we report that a palladium complex ligated with Nheterocyclic carbene (NHC) catalyzed regio-and stereoselective addition of azolyl sulfides to terminal alkynes.

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We have explored here the scope of the age-old diethyl malonate-based accesses to α-amino esters involving Knoevenagel condensations of diethyl malonate on aldehydes, reductions of the resulting alkylidenemalonates, the preparation of the corresponding α-hydroxyimino esters and their final reduction. This synthetic pathway turned out to be general although some unexpected limitations were encountered. The synthetic modifications of some of the intermediates – using Suzuki–Miyaura coupling or cycloadditions – before undertaking the oximation step – provided accesses to further α-amino esters. Moreover, other pathways to α-hydroxyimino esters were explored including an attempt to improve the cycloadditions between ethyl β-bromo-α-hydroxyiminocarboxylate and various alkylfuranes.

We report here on the use of ethyl nitroacetate as a glycine template to produce α-amino esters. This started with a study of its condensation with various arylacetals to give ethyl 3-aryl-2-nitroacrylates followed by a reduction (NaBH4 and then zinc/HCl) into α-amino esters. The scope of this method was explored as well as an alternative with arylacylals instead. We also focused on various [2 + 3] cycloadditions, one leading to a spiroacetal, which led to the undesired ethyl 5-(benzamidomethyl)isoxazole-3-carboxylate. The addition of ethyl nitroacetate on a 5-methylene-4,5-dihydrooxazole using cerium(IV) ammonium nitrate was also explored and the synthesis of other oxazole-bearing α-amino esters was achieved using gold(I) chemistry.