Iclaprim mesylate displaying a hydrogen-bonded mol-ecular tape.

The title compound, 2,6-di-amino-5-[(2-cyclo-propyl-7,8-dimeth-oxy-2H-1-benzo-pyran-5-yl)meth-yl]pyrimidin-1-ium methane-sulfonate, C19H23N4O3 +·CH3O3S-, is a salt made up from a protonated iclaprim mol-ecule and a mesylate anion. The pyrimidine and chromene units of the iclaprim mol-ecule form an orthogonal arrangement [inter-planar angle of 89.67 (6)°], and the 3-nitro-gen position of the pyrimidine ring is protonated. Four distinct N-H⋯O inter-actions and an additional N-H⋯N hydrogen bond connect iclaprim and mesylate mol-ecules to one another, resulting in an infinite hydrogen-bonded mol-ecular tape structure. The central section of the tape is formed by a sequence of fused hydrogen-bonded rings involving four distinct ring types.

Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes.

The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Šof the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage.

The synthesis of 15N(7)-Hoogsteen face-labeled adenosine phosphoramidite for solid-phase RNA synthesis.

ABSTRACT: We have developed an efficient route for the synthesis of 15N(7)-labeled adenosine as phosphoramidite building block for site- and atom-specific incorporation into RNA by automated solid-phase synthesis. Such labeled RNA is required for the evaluation of selected non-canonical base pair interactions in folded RNA using NMR spectroscopic methods.

Molecular insights into protein synthesis with proline residues.

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.

Binding of Macrolide Antibiotics Leads to Ribosomal Selection against Specific Substrates Based on Their Charge and Size.

Macrolide antibiotic binding to the ribosome inhibits catalysis of peptide bond formation between specific donor and acceptor substrates. Why particular reactions are problematic for the macrolide-bound ribosome remains unclear. Using comprehensive mutational analysis and biochemical experiments with synthetic substrate analogs, we find that the positive charge of these specific residues and the length of their side chains underlie inefficient peptide bond formation in the macrolide-bound ribosome. Even in the absence of antibiotic, peptide bond formation between these particular donors and acceptors is rather inefficient, suggesting that macrolides magnify a problem present for intrinsically difficult substrates. Our findings emphasize the existence of functional interactions between the nascent protein and the catalytic site of the ribosomal peptidyl transferase center.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 ŠX-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

The "Speedy" Synthesis of Atom-Specific (15)N Imino/Amido-Labeled RNA.

Although numerous reports on the synthesis of atom-specific (15)N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of (15)N(1) adenosine and (15)N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve (15)N(3) uridine and (15)N(3) cytidine amidites in order to tap full potential of (1)H/(15)N/(15)N-COSY experiments for directly monitoring individual Watson-Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.

Synthesis of aminoacylated N(6),N(6)-dimethyladenosine solid support for efficient access to hydrolysis-resistant 3'-charged tRNA mimics.

RNA-amino acid and RNA-peptide conjugates that mimic charged tRNA 3'-ends are valuable substrates for structural and functional investigations of ribosomal complexes. To obtain such conjugates, most synthetic approaches that are found in the literature make use of puromycin. This well available aminonucleoside antibiotic contains a dimethylamino group at the nucleobase and a methylated tyrosine that is connected via an amide linkage to the ribose moiety. To increase structural diversity, we present the synthesis of a N(6),N(6)-dimethylated 3'-azido-3'-deoxyadenosine precursor that can be coupled to any amino acid. Further derivatization results in the solid support that is eligible for the preparation of stable 3'-aminoacyl- or 3'-peptidyl-tRNA termini with an amide instead of the natural ester linkage. The present work expands our previously established route that delivered a broad range of peptidyl-tRNA mimics to the corresponding counterparts with N(6),N(6)-dimethylation pattern of the terminal adenosine (A76). This aspect is of significance to modulate the binding preferences of the mimics for ribosomal A- versus P-site.