Core-Modified Coelenterazine Luciferin Analogues: Synthesis and Chemiluminescence Properties.

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.

Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues.

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).

Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties.

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Unnatural α-amino ethyl esters from diethyl malonate or ethyl ß-bromo-α-hydroxyiminocarboxylate.

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 ß-bromo-α-hydroxyiminocarboxylate and various alkylfuranes.

Synthesis of unnatural α-amino esters using ethyl nitroacetate and condensation or cycloaddition reactions.

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.

Synthetic Routes to Coelenterazine and Other Imidazo[1,2-a]pyrazin-3-one Luciferins: Essential Tools for Bioluminescence-Based Investigations.

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2-a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence ("glow" systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2-a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.

Maximizing binary interactome mapping with a minimal number of assays.

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.

Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH).

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure-activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

On the reliability of NMR relaxation data analyses: a Markov Chain Monte Carlo approach.

The analysis of NMR relaxation data is revisited along the lines of a Bayesian approach. Using a Markov Chain Monte Carlo strategy of data fitting, we investigate conditions under which relaxation data can be effectively interpreted in terms of internal dynamics. The limitations to the extraction of kinetic parameters that characterize internal dynamics are analyzed, and we show that extracting characteristic time scales shorter than a few tens of ps is very unlikely. However, using MCMC methods, reliable estimates of the marginal probability distributions and estimators (average, standard deviations, etc.) can still be obtained for subsets of the model parameters. Thus, unlike more conventional strategies of data analysis, the method avoids a model selection process. In addition, it indicates what information may be extracted from the data, but also what cannot.