Michael Smietana

DNA vs. Mirror‐Image DNA: A Universal Approach to Tune the Absolute Configuration in DNA‐Based Asymmetric Catalysis

Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

Straightforward synthesis of triazoloacyclonucleotide phosphonates as potential HCV inhibitors

Preparation of several triazoloacyclic nucleoside phosphonates is described. The key step of the synthesis involves a copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition between azidoalkylphosphonates and propargylated nucleobases. The antiviral properties of these new analogues have been evaluated and revealed interesting potencies.

A new access to spiro-isozazolines derivatives

Abstract A series of spiro-isoxazolines 1a-e was prepared in an one-step procedure by treating the corresponding tricarbonyl 3a-e derivatives with hydroxylamine hydrochloride.

An improved synthesis of iodohydrins from alkenes

Abstract A series of iodohydrins was prepared in excellent yields in a one-step procedure by treating the corresponding alkenes at −20°C with NIS in a mixture of H2O and DME.

Dynamic and Programmable DNA‐Templated Boronic Ester Formation

The Darwinian evolution of biomolecules is described as a continuous process of mutation, selection, and amplification. According to a widely accepted hypothesis, the spontaneous prebiotic generation of nucleic acid oligomers evolved through a variety of possible combinations of information building blocks. Over the past few decades, several research groups have investigated various chemical systems able to promote nonenzymatic oligonucleotide ligation. Among the hypotheses investigated, it has been postulated that the early-life selection process might have taken advantage of reversible backbone linkages, which provided a means to repair themselves or transform in response to their environment. Indeed, reversible chemical connections in biomolecular species enable the development of adaptive dynamic systems capable of responding to external factors, such as temperature, the pH value, or molecular-recognition events. So far, diverse dynamic nucleic acid based architectures with specific constitutions or activities have been reported, but only a few reversible covalent reactions have been used to produce dynamic informational nucleic acid based oligomers. Boronic ester formation is a reliable reaction for the generation of reversible covalent DNA linkages under enzyme-free conditions. Because of their well-known mild Lewis acidity, boronic acids have proved to be both sugarand pHresponsive, and undergo a reversible transformation into cyclic esters in the presence of cis diols. This ability has led to unique applications, such as self-healing materials, therapeutic agents, and sugar sensing. Moreover, the discovery of the thermal stabilization of ribose by borate minerals and the demonstration that borate can be used as a phosphate mimic in enzymatic catalysis shed new light on the potential prebiotic relevance of boron. In this context, our research group recently described the synthesis of the complete set of 2’-deoxyborononucleotide analogues of natural nucleotide monophosphates and the reversible formation of the corresponding dinucleotides in the presence of uridine. Following this study, we envisioned a dynamic and reversible DNAtemplated ligation that would occur through the reaction of two oligodeoxynucleotides (ODNs), one with a boronic acid at its 5’ end, the other with a ribonucleotide at its 3’ end. The resulting joined duplex would differ from natural DNA in the replacement of a phosphodiester with a boronate internucleoside linkage. Herein, we report a new dynamic and programmable ligation of DNA sequences (Figure 1). Our approach requires the synthesis of a oligodeoxynucleotide with a boronic acid at its 5’ end. This strand was prepared from dT [10] by using standard phosphoramidite chemistry (see the Supporting Information). Our experimental design involves a 14 mer template (ODN3), the suitably modified 5’-boronic acid sequence (ODN1), and ODN2, a DNA sequence bearing a ribonucleoside residue at the 3’ end (Figure 1). The sequences exhibit purposefully major differences in affinity for the complementary strand ODN3 (Table 1, entries 3 and 4; Tm = 14.9 8C for ODN3/ODN1 and Tm = 48.5 8C for ODN3/ODN2). In thermal-denaturation studies, we then examined the ability of the template to bring the two functions into close proximity. Control experiments were carried out with (dT)7 (ODN4), an analogue of ODN1 without the boronic acid modification. As expected, all curves featured a double sigmoidal transition corresponding to ODN3/ODN1 and ODN3/ODN2 half-duplexes. Our main goal was to explore whether the ligation between ODN1 and ODN2 occurred as a result of a selective recognition event. Since the boronic acid functionality is carried by the less stable half-duplex, we hypothesized that the formation of a novel boronate-linked full duplex would mainly influence the lower transition. Precisely this effect was observed, in support of our model hypothesis. The stability of the resulting duplex was subsequently evaluated in the presence of various stimuli. It is well-known that the equilibrium of formation of boronic esters is dependent on the pH value. Indeed, when the pH value is increased, a thermodynamically stable hydroxyboronate complex is formed, with a major release of angle tension as a result of the rehybridization of boron from sp to sp. Thus, we envisioned that it should be possible to control the assembly of the boronate linkage by modulating the interaction between ODN1 and ODN2 through variations in the [*] A. R. Martin, Dr. D. Luvino, Dr. M. Smietana, Dr. J.-J. Vasseur Institut des Biomol cules Max Mousseron (IBMM) UMR 5247 CNRS–Universit s Montpellier 1 et 2 Place Bataillon, 34095 Montpellier (France) Fax: (+ 33)4-6714-2029 E-mail: msmietana@univ-montp2.fr vasseur@univ-montp2.fr

Borononucleotides: synthesis, and formation of a new reversible boronate internucleosidic linkage

The synthesis of a borononucleotide analogue of thymidine and its association towards the formation of new borono-linked dimers is described.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of Polysulfated-Derived Oligosaccharides Using Pyrenemethylguanidine

A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers—such as polysulfated oligosaccharides—are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.

The increase in intra-macrophage thiols induced by new pro-GSH molecules directs the Th1 skewing in ovalbumin immunized mice

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-γ production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2.

Molecules altering the intracellular thiol content modulate NF-kB and STAT-1/IRF-1 signalling pathways and IL-12 p40 and IL-27 p28 production in murine macrophages.

Background The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. Methodology/Principal Findings By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. Conclusions and Significance an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies.

Synthesis of new N-isobutyryl-L-cysteine/MEA conjugates: evaluation of their free radical-scavenging activities and anti-HIV properties in human macrophages.

Four novel N-isobutyryl-L-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test, and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro. The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.