Synthesis of Original Cyclic Dinucleotide Analogues Using the Sulfo-click Reaction.

The stimulator of interferon genes (STING) protein plays a crucial role in the activation of the innate immune response. Activation of STING is initiated by cyclic dinucleotides (CDNs) which prompted the community to synthesize structural analogues to enhance their biological properties. We present here the synthesis and biological evaluation of four novel CDN analogues composed of an N-acylsulfonamide linkage. These CDNs were obtained in high overall yields via the sulfo-click reaction as a key step.

N-Acylsulfonamide: a valuable moiety to design new sulfa drug analogues.

Sulfonamides are the oldest class of antibiotics, discovered more than 80 years ago. They are still used today despite the appearance of drug resistance phenomena that limit their prescription. Since the discovery and use of the first sulfa drugs, many analogues have been synthesized in order to obtain new active molecules able to circumvent bacterial resistance. Structurally similar to sulfonamide, the N-acylsulfonamide group arouses interest in the field of medicinal chemistry due to specific physico-chemical properties. We report here the synthesis and antibacterial/antibiofilm activities of 18 sulfa drug analogues with an N-acylsulfonamide moiety. These derivatives were obtained efficiently by sulfo-click reactions between readily available thioacid and sulfonyl azide synthons.

Facile access to 4'-(N-acylsulfonamide) modified nucleosides and evaluation of their inhibitory activity against SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

N-Acylsulfonamides possess an additional carbonyl function compared to their sulfonamide analogues. Due to their unique physico-chemical properties, interest in molecules containing the N-acylsulfonamide moiety and especially nucleoside derivatives is growing in the field of medicinal chemistry. The recent renewal of interest in antiviral drugs derived from nucleosides containing a sulfonamide function has led us to evaluate the therapeutic potential of N-acylsulfonamide analogues. While these compounds are usually obtained by a difficult acylation of sulfonamides, we report here the easy and efficient synthesis of 20 4'-(N-acylsulfonamide) adenosine derivatives via the sulfo-click reaction. The target compounds were obtained from thioacid and sulfonyl azide synthons in excellent yields and were evaluated as potential inhibitors of the SARS-CoV-2 RNA cap N7-guanine-methyltransferase nsp14.

Expansion of the Strigolactone Profluorescent Probes Repertory: The Right Probe for the Right Application.

Strigolactones (SLs) are intriguing phytohormones that not only regulate plant development and architecture but also interact with other organisms in the rhizosphere as root parasitic plants (Striga, Orobanche, and Phelipanche) and arbuscular mycorrhizal fungi. Starting with a pioneering work in 2003 for the isolation and identification of the SL receptor in parasitic weeds, fluorescence labeling of analogs has proven a major strategy to gain knowledge in SL perception and signaling. Here, we present novel chemical tools for understanding the SL perception based on the enzymatic properties of SL receptors. We designed different profluorescent SL Guillaume Clavé (GC) probes and performed structure-activity relationship studies on pea, Arabidopsis thaliana, and Physcomitrium (formerly Physcomitrella) patens. The binding of the GC probes to PsD14/RMS3, AtD14, and OsD14 proteins was tested. We demonstrated that coumarin-based profluorescent probes were highly bioactive and well-adapted to dissect the enzymatic properties of SL receptors in pea and a resorufin profluorescent probe in moss, contrary to the commercially available fluorescein profluorescent probe, Yoshimulactone Green (YLG). These probes offer novel opportunities for the studies of SL in various plants.

A Phelipanche ramosa KAI2 protein perceives strigolactones and isothiocyanates enzymatically.

Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.

Modified internucleoside linkages for nuclease-resistant oligonucleotides.

In the past few years, several drugs derived from nucleic acids have been approved for commercialization and many more are in clinical trials. The sensitivity of these molecules to nuclease digestion in vivo implies the need to exploit resistant non-natural nucleotides. Among all the possible modifications, the one concerning the internucleoside linkage is of particular interest. Indeed minor changes to the natural phosphodiester may result in major modifications of the physico-chemical properties of nucleic acids. As this linkage is a key element of nucleic acids' chemical structures, its alteration can strongly modulate the plasma stability, binding properties, solubility, cell penetration and ultimately biological activity of nucleic acids. Over the past few decades, many research groups have provided knowledge about non-natural internucleoside linkage properties and participated in building biologically active nucleic acid derivatives. The recent renewing interest in nucleic acids as drugs, demonstrated by the emergence of new antisense, siRNA, aptamer and cyclic dinucleotide molecules, justifies the review of all these studies in order to provide new perspectives in this field. Thus, in this review we aim at providing the reader insights into modified internucleoside linkages that have been described over the years whose impact on annealing properties and resistance to nucleases have been evaluated in order to assess their potential for biological applications. The syntheses of modified nucleotides as well as the protocols developed for their incorporation within oligonucleotides are described. Given the intended biological applications, the modifications described in the literature that have not been tested for their resistance to nucleases are not reported.

Synthesis of Profluorescent Strigolactone Probes for Biochemical Studies.

In this chapter, we will describe a method we set up to synthesize two profluorescent strigolactone (SL) mimic probes (GC240 and GC242) and the optimized protocols developed to study the enzymatic properties of various strigolactone receptors. The Arabidopsis AtD14 SL receptor is used here as a model for this purpose.

The Sulfo-Click Reaction and Dual Labeling of Nucleosides.

This article contains detailed synthetic procedures for the implementation of the sulfo-click reaction to nucleoside derivatives. First, 3'-O-TBDMS-protected nucleosides are converted to their corresponding 4'-thioacid derivatives in three steps. Then, various conjugates are synthetized via a biocompatible and chemoselective coupling procedure using sulfonyl azide partners. Finally, to illustrate the potential of the sulfo-click reaction, a nucleoside bearing two orthogonal azido groups is synthesized and engaged in one-pot dual labeling through a sulfo-click/copper-catalyzed azide-alkyne cycloaddition (CuAAC) cascade. The high efficiency of the sulfo-click reaction as applied to nucleosides opens up new possibilities in the context of bioconjugation. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the synthesis of 4'-thioacid-nucleoside derivatives Basic Protocol 2: Implementation of the sulfo-click reaction Basic Protocol 3: Synthesis of 3'-azido-4'-(carboxamido)ethane-sulfonyl azide-3'-deoxythymidine Basic Protocol 4: Detailed synthetic procedure for one-pot double-click conjugations.

An Entry of the Chemoselective Sulfo-Click Reaction into the Sphere of Nucleic Acids.

We report here the first example of a sulfo-click conjugation reaction to be applied to modified nucleosides. The reaction, which proceeds rapidly (k ∼ 2.0 × 10-1 M-1 s-1 at 25 °C) under aqueous biocompatible conditions in the ribo- and deoxyribonucleoside series, affords the corresponding conjugated products in excellent yields. Furthermore, we demonstrate the orthogonality of the reaction with the copper-catalyzed azide-alkyne click reaction (CuAAC) by performing a one-pot dual labeling of a nucleoside carrying two orthogonal azido groups.

Validated Method for Strigolactone Quantification by Ultra High-Performance Liquid Chromatography - Electrospray Ionisation Tandem Mass Spectrometry Using Novel Deuterium Labelled Standards.

INTRODUCTION: Strigolactones (SLs) are important plant hormones. They are difficult to analyse because they occur in very small concentrations especially in comparison with other plant hormones and other substances can interfere with their detection. OBJECTIVE: To develop a procedure for the extraction, purification and quantification of SLs from plant roots. METHODOLOGY: Samples were prepared by extraction of plant root tissues with ethyl acetate. Then the extracts were further purified with silica column chromatography. The natural SLs in the final extracts were quantified using novel deuterium labelled SLs. The results of the methodology were compared with those of the procedure of Yoneyama and coworkers. RESULTS: This procedure required about 1-g root samples to detect and quantify simultaneously the SLs (orobanchyl acetate and fabacyl acetate) concentration with high reliability. CONCLUSION: A method was developed for determining endogenous fabacyl acetate and orobanchyl acetate in plant tissue based on novel deuterium labelled standards. A method of orobanchol quantification using a synthetic SL GR24 as internal standard was proposed. Copyright © 2017 John Wiley & Sons, Ltd.

An histidine covalent receptor and butenolide complex mediates strigolactone perception.

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/ß-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Biochemical characterization of a Caspase-3 far-red fluorescent probe for non-invasive optical imaging of neuronal apoptosis.

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures.

The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

New strigolactone analogs as plant hormones with low activities in the rhizosphere.

Strigolactones (SLs) are known not only as plant hormones, but also as rhizosphere signals for establishing symbiotic and parasitic interactions. The design of new specific SL analogs is a challenging goal in understanding the basic plant biology and is also useful to control plant architectures without favoring the development of parasitic plants. Two different molecules (23 (3'-methyl-GR24), 31 (thia-3'-methyl-debranone-like molecule)) already described, and a new one (AR36), for which the synthesis is presented, are biologically compared with the well-known GR24 and the recently identified CISA-1. These different structures emphasize the wide range of parts attached to the D-ring for the bioactivity as a plant hormone. These new compounds possess a common dimethylbutenolide motif but their structure varies in the ABC part of the molecules: 23 has the same ABC part as GR24, while 31 and AR36 carry, respectively, an aromatic ring and an acyclic carbon chain. Detailed information is given for the bioactivity of such derivatives in strigolactone synthesis or in perception mutant plants (pea rms1 and rms4, Arabidopsis max2 and, max4) for different hormonal functions along with their action in the rhizosphere on arbuscular mycorrhizal hyphal growth and parasitic weed germination.

The first metal-free water-soluble cryptophane-111.

Cryptophane-111 is one of the best candidates for (129)Xe MRI-based applications. Herein, we report the first metal-free and water-soluble cryptophane-111 core which involves an efficient and unusual post-synthetic sulfonation procedure.

N-Fmoc-α-sulfo-ß-alanine: a versatile building block for the water solubilisation of chromophores and fluorophores by solid-phase strategy.

An easy and efficient solid-phase synthesis strategy to obtain rapidly water-soluble chromophores/fluorophores in highly pure form has been developed. This first successful use of N-Fmoc-α-sulfo-ß-alanine as a SPPS building block opens the way to the future development of promising direct "on-resin" peptide labelling and water-solubilising methods.

A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates.

We describe for the first time, the synthesis and some bioconjugation applications of an original heterotrifunctional cross-linking reagent (also named tripod) bearing three different bioorthogonal functional groups which are fully compatible amongst themselves. Contrary to the first generation tripod recently reported by us (Org. Biomol. Chem., 2008, 6, 3065), the use of an azido group instead of the nucleophile-sensitive active carbamate moiety enables us to reach the targeted chemical orthogonality without the use of temporary aminooxy- and thiol protecting groups. Thus, the preparation of sophisticated bioconjugates through the sequential derivatisation of the tripod by means of copper-mediated 1,3-dipolar cycloaddition, oxime ligation and aqueous compatible mild thiol-alkylation reactions, is significantly simpler and more convenient. The chemoselective bioconjugation protocols were optimised through the preparation of FRET cassettes based on cyanine and/or xanthene fluorescent dye pairs and subsequent anchoring to fragile biomolecules. The applicability of this universal cross-linking reagent was also illustrated by the preparation of biochips suitable for aflatoxin B1 detection through the SPIT-FRI method.

A highly sensitive competitive enzyme immunoassay of broad specificity quantifying microcystins and nodularins in water samples.

Microcystins (MCs) form a group of cyclic heptapeptides produced by common cyanobacteria (blue green algae) and cause both acute and chronic toxicity. For immunization purposes, an amino derivative of MC-LR was prepared before coupling to BSA. Among the different monoclonal antibodies produced, mAb MC159 was selected due to its broad specificity to develop a sensitive enzyme immunoassay (EIA). This method measures MC-LR, MC-YR, MC-LA, nodularins in a similar way and exhibits an important recognition (cross reactivity up to 69%) for Adda analogues. Using MC-LR as standard, the present EIA proved to be very sensitive with a limit of detection close to 10 fmol/ml, largely below the provisional guideline level for drinking water proposed by the WHO (1 pmol/ml for MC-LR). This assay showed a high accuracy (CV% < 12) and a high recovery rate for MC-LR in spiked surface water (up to 96.5%). Moreover due to its broad spectrum of recognition, this method allows a real quantification of the sum of MCs in water bloom and cyanobacteria culture samples. Indeed, in parallel analysis of these samples using HPLC, EIA shows a good relationship between both measurements while LC-MS/MS demonstrates the presence of different variants of MCs whose heterogeneity did not impair EIA measurement.

Thyroid hormone improves postischaemic recovery of function while limiting apoptosis: a new therapeutic approach to support hemodynamics in the setting of ischaemia-reperfusion?

Although it has long been recognized that thyroid hormone is an effective positive inotrope, its efficacy in supporting hemodynamics in the acute setting of ischaemia and reperfusion (R) without worsening reperfusion injury remains largely unknown. Thus, we investigated the effects of triiodothyronine (T3) on reperfusion injury in a Langendorff-perfused rat heart model of 30 min zero-flow ischaemia and 60 min of (R) with or without T3 (40 microg/l) at R, T3-R60, n = 11 and CNT-R60, n = 10, respectively. Furthermore, phosphorylated levels of intracellular kinases were measured at 5, 15 and 60 min of R. T3 markedly improved postischaemic recovery of left ventricular developed pressure (LVDP%); 56.0% (SEM, 4.4) in T3-R60 versus 38.8% (3.1) in CNT-R60, P < 0.05. Furthermore, LDH release was significantly lower in T3-R60. Apoptosis detection by fluorescent probe optical imaging showed increased fluorescent signal in CNT-R60 hearts, while the signal was hardly detectable in T3-R60 hearts. Similarly, caspase-3 activity was found to be 78.2 (8.2) in CNT-R60 vs 40.5 (7.1) in T3-R60 hearts, P < 0.05. This response was associated with significantly lower levels of phospho-p38 MAPK at any time point of R. No significant changes in phospho- ERK1/2 and JNK levels were observed between groups. Phospho-Akt levels were significantly lower in T3 treated group at 5 min and no change in phospho-Akt levels were observed at 15 and 60 min between groups. In conclusion, T3 administration at reperfusion can improve postischaemic recovery of function while limiting apoptosis. This may constitute a paradigm of a positive inotropic agent with anti-apoptotic action suitable for supporting hemodynamics in the clinical setting of ischaemia-reperfusion.

A novel heterotrifunctional peptide-based cross-linking reagent for facile access to bioconjugates. Applications to peptide fluorescent labelling and immobilisation.

A convenient, versatile and straightforward synthesis of a novel heterotrifunctional peptide-based linker molecule is described. This generic bio-labelling reagent contains an amine-reactive N-hydroxysuccinimidyl carbamate moiety, an aldehyde/ketone-reactive aminooxy group and a thiol group with a propensity to form urea, oxime and thioether linkages respectively. The full chemical orthogonality between the free aminooxy and thiol functionalities was demonstrated through the preparation of a fluorescent reagent suitable for the selective staining of a carboxaldehyde-modified surface by means of oxime ligation. The absence of reactivity of these two functions toward the nucleophile-sensitive active carbamate was obtained by using temporary aminooxy- and thiol-protecting groups removable under mild conditions. The utility of the linker molecule to cross-link three different molecular partners has been illustrated by the preparation of fluorescent tripod-functionalised surfaces which may be useful in developing new peptide microarrays and related immunosensors.