Javier Alguacil

Organic Chemistry

PhD Organic Chemistry
11.62

Publications

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    ABSTRACT: Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.
    Full-text · Article · Jul 2014 · Nucleic Acids Research
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    ABSTRACT: An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 μM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(β) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.
    Full-text · Article · Nov 2010 · The FASEB Journal
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    ABSTRACT: An alternative and straightforward method to prepare ami- noglycoside–dinucleotide and –diPNA conjugates is re- ported, which is based on copper-catalyzed Huisgen azide- alkyne cycloaddition (“click chemistry” ligation) assisted by microwave irradiation. This method permitted conjugations to be performed in aqueous solution, in very short times and with readily prepared precursors.
    Full-text · Article · Jun 2010 · European Journal of Organic Chemistry
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    Sonia Pérez-Rentero · Javier Alguacil · Jordi Robles
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    ABSTRACT: Morpholinoamidines were devised as new cationic units in oligonucleotides, by combining morpholino-nucleosides (to simplify the nomenclature, we will use the term morpholino-nucleosides to refer to nucleoside analogues in which the ribose ring was transformed into a morpholine) with internucleoside guanidines. Here, a methodology was developed to synthesize oligonucleotides containing morpholinoamidines formed by morpholino-uridine and 5'-amino-5'-deoxythymidine. Morpholinoamidine was produced by solid-phase reaction of Alloc-morpholinocarbothioamide with 5'-aminonucleoside resin and Mukaiyama's reagent activation. Two 14-mer oligonucleotides containing a single morpholinoamidine were synthesized and their affinity properties were investigated by forming DNA double and triple helices. Duplexes were slightly stabilized by a 3' unit, but were less stable if internally positioned. Notably, triplexes were significantly stabilized at pH 7.0.
    Full-text · Article · Feb 2009 · Tetrahedron

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