Synthesis, NMR kinetics and dynamic structure of a 17-mer heptaloop RNA hairpin carrying a 3-N-methyluridine nucleotide residue in the loop region.

A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. 1H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (∼5-6%) ACSL conformations. Exchange rate constant (kex) for interconversion between P and S states is 112 sec-1 (<Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (kPS) and backward (kSP) rate constants are 49.166 sec-1 and 62.792 sec-1, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by 1H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs.Communicated by Ramaswamy H. Sarma.

Water self-diffusion in glassy and liquid maltose measured by Raman microscopy and NMR.

Raman microscopy and isotope labeling have been used for the first time to measure water self-diffusion in carbohydrate glasses. Together with pulsed-gradient stimulated-echo NMR, this method yielded the self-diffusion coefficients of water in amorphous maltose over 8 orders of magnitude, from the liquid to the glassy state. There are consistencies and major differences between our data and those obtained by evaporative drying. Water diffusion is remarkably fast in maltose glasses, decouples from maltose diffusion, and is not strongly affected by the glass transition.

NMR and amber analysis of the neamine pharmacophore for the design of novel aminoglycoside antibiotics.

The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D(2)O at 25°C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ~80% and ~20%, respectively, were detected at pD 7.4 with kinetic constants k(on(P→S))=1.9771s(-1) and k(off(S→P))=1.1319s(-1). A tertiary, T-state, neamine species populated at ~3% was also detected by NMR at pD 7.4. The pKa values determined by NMR titration experiments are pKa1 6.44±0.13 for N3 of ring-II, pKa2 7.23±0.09 for N2' of ring-I, pKa3 7.77±0.19 for N1 of ring-II, and pKa4 8.08±0.15 for N6' of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the (4)C(1) chair conformation between pD 3.3 and pD=7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the (6)rH(1) half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.

Glycine exists mainly as monomers, not dimers, in supersaturated aqueous solutions: implications for understanding its crystallization and polymorphism.

Glycine, the simplest amino acid, is described as existing as hydrogen-bonded cyclic dimers in supersaturated aqueous solutions and, as a result, crystallizing in a centrosymmetric polymorph (polymorph alpha) for which the dimer can be viewed as the building unit, in favor of other polymorphs of polar structures. In exhibiting this relation between polymorphic selectivity and self-association in solution, glycine is thought to illustrate a general principle. We measured the freezing-point depression of glycine-water up to 30% supersaturation and found that glycine exists mainly as monomers, not dimers, and that the dimer stability constant K D is smaller than 0.1 kg of H 2O/mol if the observed osmotic abnormality is attributed to dimerization. We also revisited a report cited as evidence for glycine dimerization: the slowdown of glycine diffusion with solution age. Pulsed gradient spin-echo NMR spectroscopy was used in place of the previous method of Gouy interferometry to avoid perturbations to sloution structure caused by the interdiffusion between two solutions of different concentrations. No aging effect was observed on glycine diffusion up to 24% supersaturation after five days. The solute size calculated from diffusivity, viscosity, and the Stokes-Einstein relation showed no increase with concentration or solution age. We conclude that glycine exists in supersaturated aqueous solutions mainly as monomers, not dimers, and remains so upon aging. This result does not invalidate the theories of how pH and additives affect glycine's polymorphic preference, because they begin with the assumption that alpha glycine is the preferred polymorph under normal conditions, but requires a new explanation for that assumption itself.

Photophysical, photochemical, and tumor-selectivity properties of bromine derivatives of rhodamine-123.

The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemotherapy and photochemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a common tumor cell phenotype. The potential of this strategy is highlighted by the fact that the toxic effects associated with a number of cationic dyes known to localize in energized cell mitochondria are much more pronounced in tumor cells than in normal cells. Here we evaluate the phototoxic properties of four bromine derivatives of rhodamine-123 toward human uterine sarcoma (MES-SA) and green monkey kidney (CV-1) cells and compare the degrees of tumor cell selectivity associated with these dyes with those associated with two model mitochondrial triarylmethanes (crystal violet and ethyl violet). Selective phototoxicity toward tumor cells was found to be highly dependent upon the lipophilic/hydrophilic character of the cationic photosensitizer. Our experimental data have indicated that the probability of success of mitochondrial targeting in (photo)chemotherapy of neoplastic diseases is higher when the octan-1-ol/water partition coefficient of the drug candidate falls within approximately two orders of magnitude from that of the prototypical mitochondria-specific dye rhodamine-123.

Self-association of cromolyn sodium in aqueous solution characterized by nuclear magnetic resonance spectroscopy.

The major objective of this study was to investigate and characterize the solution properties of cromolyn sodium (in D(2)O or D(2)O/H(2)O phosphate buffer at pH 7.5) using nuclear magnetic resonance (NMR) spectroscopy. The self-association of cromolyn molecules was examined primarily via one-dimensional (1)H and (13)C, and two-dimensional homonuclear NOESY NMR. Significant spectral shifts were observed for a majority of cromolyn (1)H and (13)C resonances, and are attributed to inter-molecular ring-stacking association accompanied by intra-molecular conformational changes. The critical self-association concentration was determined to be 10 mg/mL at pH 7.5 and 25 degrees C by measuring the chemical shift of a specific cromolyn (1)H resonance. The observed magnitude and sign changes of NOESY correlations indicate the formation of cromolyn aggregates with restricted molecular mobility. Mesomorphic liquid crystal formation is suggested by uniformly pronounced line broadening in concentrated cromolyn solutions; the transition concentration was approximately 60 mg/mL at 25 degrees C, which is consistent with literature findings based on other techniques. A stronger tendency toward association was observed at lower temperature but aggregation appeared to be independent of pH. Lastly, it was concluded that self-association of cromolyn is promoted by the presence of monovalent cations as a result of reduced electrostatic repulsive forces.

A disilapentalene and a stable diradical from the reaction of a dilithiosilole with a dichlorocyclopropene.

The reaction of 1,1-dilithio-2,3,4,5-tetraphenylsilole (1) with 1,1-dichloro-2,3-diphenylcyclopropene (2) leads to the novel 1,4-disila-1,4-dihydropentalene (4), as well as an exceptionally stable diradical for which the structure 3 is suggested. The diradical is unreactive toward water, methanol, and chloroform; upon heating it transforms into 4. Structure 3 for the paramagnetic species is proposed on the basis of EPR data and theoretical calculations. The trans-trans isomer of diradical 3 was calculated to be more stable than its cis-cis isomer. The strong and stable EPR signal in the reaction mixture is probably due to the trans-trans isomer of diradical 3 in the triplet state. A reaction scheme describing the formation of 3 and 4 is presented.

Structure and chemistry of 1-silafluorenyl dianion, its derivatives, and an organosilicon diradical dianion.

1-Silafluorene dianion was synthesized by potassium reduction of 1,1-dichloro-1-silafluorene in refluxing THF. The X-ray structure of 1,1-dipotassio-1-silafluorene (3b) shows C-C bond length equalization in the five-membered silole ring and C-C bond length alternation in the six-membered benzene rings, indicating aromatic delocalization of electrons in the silole ring. The downfield (29)Si chemical shift at 29.0 ppm and theoretical calculations also support electron delocalization in the silole ring of 3b. Dianion salt 3b underwent nucleophilic reactions with Me(3)SiCl and EtBr to form the corresponding disubstituted products. Benzaldehyde underwent reductive coupling in the presence of 3b. Slow oxidation of 3b yielded 1,1'-dipotassio-1,1'-bis(silafluorene) (16). The X-ray structure and (29)Si chemical shift (-38.0 ppm) of 16 indicate localized negative charges at the silicon atoms and no aromatic character. Heating a DME/hexane solution of 3b in the presence of 18-crown-6 led to a novel diradical dianion salt.