ABSTRACT
The reaction between maleimides and resin-linked diene-polyamides allows the latter to be used in the preparation of conjugates. Conjugation takes place by reacting the insoluble, hydrophobic diene component either with water-soluble dienophiles or with dienophiles requiring mixtures of water and organic solvents. Experimental conditions can be adjusted to furnish the target conjugate in good yield with no need of adding large excesses of soluble reagent. In case protected maleimides are used, maleimide deprotection and Diels-Alder cycloaddition can be simultaneously carried out to render conjugates with different linking positions. On-resin conjugation is followed by an acidic treatment that removes the polyamide protecting groups with no harm to the cycloadduct, in contrast with the unreacted diene that is indeed degraded under these conditions. Cycloadducts incorporating suitable functional groups can undergo subsequent additional conjugation reactions in solution to furnish double conjugates.
Subject(s)
Oligonucleotides/chemistry , Polyenes/chemistry , Water/chemistry , Biological Phenomena , Cycloaddition Reaction , Maleimides/chemistry , Molecular StructureABSTRACT
The internal ribosome entry site (IRES) element located at the 5'untranslated genomic region of various RNA viruses mediates cap-independent initiation of translation. Picornavirus IRES activity is highly dependent on both its structural organization and its interaction with host factors. Small molecules able to interfere with RNA function are valuable candidates for antiviral agents. Here we show that a small molecule based on benzimidazole (IRAB) inhibited foot-and-mouth disease virus (FMDV) IRES-dependent protein synthesis in cells transfected with infectious RNA leading to a decrease of the virus titer, which was higher than that induced by a structurally related benzimidazole derivative. Interestingly, IRAB preferentially inhibited IRES-dependent translation in cell free systems in a dose-dependent manner. RNA structural analysis by SHAPE demonstrated an increased local flexibility of the IRES structure upon incubation with IRAB, which affected 3 stem-loops (SL) of domain 3. Fluorescence binding assays conducted with individual aminopurine-labeled oligoribonucleotides indicated that the SL3A binds IRAB (EC50 18 µM). Taken together, the results derived from SHAPE reactivity and fluorescence binding assays suggested that the target site of IRAB within the FMDV IRES might be a folded RNA structure that involves the entire apical region of domain 3. Our data suggest that the conformational changes induced by this compound on a specific region of the IRES structure which is essential for its activity is, at least in part, responsible for the reduced IRES efficiency observed in cell free lysates and, particularly, in RNA-transfected cells.
Subject(s)
Foot-and-Mouth Disease Virus/genetics , Internal Ribosome Entry Sites/genetics , Protein Biosynthesis , RNA, Viral/genetics , Base Sequence , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Cell-Free System , Fluorescence , Foot-and-Mouth Disease Virus/drug effects , Foot-and-Mouth Disease Virus/growth & development , Genome, Viral , Hydroxyl Radical/metabolism , Ligands , Molecular Sequence Data , Nucleic Acid Conformation , Protein Biosynthesis/drug effects , RNA, Viral/chemistry , SolventsABSTRACT
Friedreich's ataxia is a severe neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein that stimulates iron-sulfur (Fe-S) cluster biogenesis. In mammals, the primary steps of Fe-S cluster assembly are performed by the NFS1-ISD11-ISCU complex via the formation of a persulfide intermediate on NFS1. Here we show that frataxin modulates the reactivity of NFS1 persulfide with thiols. We use maleimide-peptide compounds along with mass spectrometry to probe cysteine-persulfide in NFS1 and ISCU. Our data reveal that in the presence of ISCU, frataxin enhances the rate of two similar reactions on NFS1 persulfide: sulfur transfer to ISCU leading to the accumulation of a persulfide on the cysteine C104 of ISCU, and sulfur transfer to small thiols such as DTT, L-cysteine and GSH leading to persulfuration of these thiols and ultimately sulfide release. These data raise important questions on the physiological mechanism of Fe-S cluster assembly and point to a unique function of frataxin as an enhancer of sulfur transfer within the NFS1-ISD11-ISCU complex.
Subject(s)
Carbon-Sulfur Lyases/metabolism , Iron-Binding Proteins/metabolism , Sulfhydryl Compounds/metabolism , Carbon-Sulfur Lyases/chemistry , Chromatography, Gel , Chromatography, High Pressure Liquid , Cysteine/chemistry , Cysteine/metabolism , Glutathione/chemistry , Glutathione/metabolism , Humans , Iron-Binding Proteins/chemistry , Mass Spectrometry , Software , Sulfhydryl Compounds/chemistry , Sulfides/chemistry , Sulfides/metabolism , FrataxinABSTRACT
Cyclic peptides and peptoids were prepared using the thiol-ene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.
Subject(s)
Alkynes/chemistry , Maleimides/chemistry , Oligonucleotides/chemistry , Peptides, Cyclic/chemistry , Peptoids/chemistry , Sulfhydryl Compounds/chemistry , Catalysis , Click Chemistry , Copper/chemistry , Cycloaddition Reaction , Molecular StructureABSTRACT
Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles.
Subject(s)
Cysteine/chemistry , Peptides, Cyclic/chemical synthesis , Peptides/chemical synthesis , Cyclization , Maleimides/chemistry , Molecular Structure , Peptides/chemistry , Peptides, Cyclic/chemistryABSTRACT
Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently.
Subject(s)
Maleimides/chemistry , Peptide Nucleic Acids/chemistry , Peptides/chemistry , Peptoids/chemistry , Cyclization , Maleimides/chemical synthesis , Nylons/chemical synthesis , Nylons/chemistry , Peptide Nucleic Acids/chemical synthesis , Peptides/chemical synthesis , Peptoids/chemical synthesisABSTRACT
In addition to X-ray, NMR, and FRET, electron paramagnetic resonance (EPR) can be applied to elucidate the structure of different macromolecular systems and determine local surroundings of paramagnetic centers in DNA and RNA. This technique permits structural characterization as well as dynamic structural changes of macromolecular systems. To do so, free radicals with good stability must be introduced. Here, the site-directed spin labeling of DNA and RNA based on the Sonogashira cross-coupling reaction is described. First, the appropriate building blocks, either 5-iodo-substituted pyrimidine deoxy- or ribo-nucleoside phosphoramidites are prepared and incorporated by solid-phase synthesis. Following this, the protected oligonucleotides are "on column" reacted with the acetylenic nitroxide spin labels and subsequently purified. Applications of this technique for duplexes, hairpins, and riboswitches in vitro and in cell are described.
