A Ligand That Targets CUG Trinucleotide Repeats

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9‐diaminoalkyl‐substituted 1,10‐phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (T_m) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry showed that DAP bound to r(CUG)₉ but not to r(CAG)₉ and r(CGG)₉. The dual luciferase assay clearly indicated DAP bound to the r(CUG)_n repeat by affecting the translation in vitro.     

Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA

One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small‐molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1 ‐NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1 ‐NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1 ‐NHR was performed from pre‐miR‐29a loop library RNA, and one RNA, to which 1 ‐NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1 ‐NHR, with a predominantly unstacked conformation, would show entropy‐driven binding, isothermal titration calorimetry analysis suggested that the binding of 1 ‐NHR to RNA was enthalpy driven with an apparent K _d of about 100 nm .     

Carbohydrate–Lipid Interactions: Affinities of Methylmannose Polysaccharides for Lipids in Aqueous Solution

The interactions between 3‐O‐methyl‐mannose polysaccharides (MMPs), extracted from Mycobacterium smegmatis (consisting of a mixture of MMP‐10, ‐11, ‐12 and ‐13) or obtained by chemical synthesis (MMP‐5_s, ‐8_s, ‐11_s and ‐14_s), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 25 °C and pH 8.5 were quantified by electrospray ionization mass spectrometry (ESI‐MS). Association constants (K_a) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans‐parinaric acid) were measured by using an indirect ESI‐MS assay, the “proxy protein” method. The K_a values are in the 10⁴–10⁵ M ^?1 range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the measured affinity of the extracted MMPs for trans‐parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly developed competitive binding assay, referred to as the “proxy protein/proxy ligand” ESI‐MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affinities of approximately (5×10⁴) M ^?1 for the mixture of NAs tested. This represents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here represents a novel method for probing carbohydrate–lipid interactions.     

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′‐O‐(D ‐arabinofuranosyl), 2′‐O‐(L ‐arabinofuranosyl), 2′‐O‐(D ‐ribopyranosyl), 2′‐O‐(D ‐erythrofuranosyl), and 2′‐O‐(5‐azido‐5‐deoxy‐D ‐ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔT_m of +0.5 to −1.4° for DNA/RNA and a ΔT_m of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH₂−C(5″) group has a beneficial effect on duplex stability.     

Three New Alkaloids from the Leaves of Uncaria rhynchophylla

Three new alkaloids, 2′‐O‐β‐D ‐glucopyranosyl‐11‐hydroxyvincoside lactam ( 1 ), 22‐O‐demethyl‐22‐O‐β‐D ‐glucopyranosylisocorynoxeine ( 2 ), and (4S)‐corynoxeine N‐oxide ( 3 ) were isolated from the leaves of Uncaria rhynchophylla, together with four known tetracyclic oxindole or indole alkaloids, isocorynoxeine N‐oxide ( 4 ), rhynchophylline N‐oxide ( 5 ), isorhynchophylline N‐oxide ( 6 ), and dihydrocorynantheine ( 7 ), and an indole alkaloid glycoside, strictosidine ( 8 ). The structures of 1 – 3 were elucidated by spectroscopic methods including UV, IR, ESI‐TOF‐MS, 1D‐ and 2D‐NMR, as well as CD experiments. The activity assay showed that 8 (IC₅₀=8.3 μM ) exhibited potent inhibitory activity on lipopolysaccharide(LPS)‐induced nitrogen monoxide (NO) release in N9 microglia cells. However, only weak inhibitory activities were observed for 1 – 7 (IC₅₀>100 μM for 1 – 6 or >30 μM for 7 ).     

Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G‐Quadruplex: a Joint ESI MS and XRD Investigation

The dicarbene gold(I) complex [Au(9‐methylcaffein‐8‐ylidene)₂]BF₄ is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G‐quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X‐ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9‐methylcaffein‐8‐ylidene)₂]⁺ and Tel 23 DNA G‐quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G‐quadruplex recognition and its selectivity are described.     

Enantiomeric and Diastereomeric Self‐Assembled Multivalent Nanostructures: Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA

A family of four self‐assembling lipopeptides containing Ala‐Lys peptides attached to a C₁₆ aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C₁₆‐l ‐Ala‐l ‐Lys/C₁₆‐d ‐Ala‐d ‐Lys) and (C₁₆‐d ‐Ala‐l ‐Lys/C₁₆‐l ‐Ala‐d ‐Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self‐assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d ‐lysine ligands, with a further slight preference for attachment to l ‐alanine, with the CMC being irrelevant.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Cucurbit[7]uril: A High‐Affinity Host for Encapsulation of Amino Saccharides and Supramolecular Stabilization of Their α‐Anomers in Water

Cucurbit[7]uril (CB[7]), an uncharged and water‐soluble macrocyclic host, binds protonated amino saccharides (D ‐glucosamine, D ‐galactosamine, D ‐mannosamine and 6‐amino‐6‐deoxy‐D ‐glucose) with excellent affinity (K_a=10³ to 10⁴ M ^?1). The host–guest complexation was confirmed by NMR spectroscopy, isothermal titration calorimetry (ITC), and MALDI‐TOF mass spectral analyses. NMR analyses revealed that the amino saccharides, except D ‐mannosamine, are bound as α‐anomers within the CB[7] cavity. ITC analyses reveal that CB[7] has excellent affinity for binding amino saccharides in water. The maximum affinity was observed for D ‐galactosamine hydrochloride (K_a=1.6×10⁴ M ^?1). Such a strong affinity for any saccharide in water using a synthetic receptor is unprecedented, as is the supramolecular stabilization of an α‐anomer by the host.     

Synthesis of the Anticodon Hairpin tRNAfMet Containing N‐{[9‐(β‐D‐Ribofuranosyl)‐9H‐purin‐6‐yl]carbamoyl}‐L‐threonine (=N6‐{{[(1S,2R)‐1‐Carboxy‐2‐hydroxypropyl]amino}carbonyl}adenosine,t6A)

As part of our studies on the structure of yeast ^tRNA_f^Met, we investigated the incorporation of N‐{[9‐(β‐D ‐ribofuranosyl)‐9H‐purin‐6‐yl]carbamoyl}‐L ‐threonine (t⁶A) in the loop of a RNA 17‐mer hairpin. The carboxylic function of the L ‐threonine moiety of t⁶A was protected with a 2‐(4‐nitrophenyl)ethyl group, and a (tert‐butyl)dimethylsilyl group was used for the protection of its secondary OH group. The 2′‐OH function of the standard ribonucleotide building blocks was protected with a [(triisopropylsilyl)oxy]methyl group. Removal of the base‐labile protecting groups of the final RNA with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) and then with MeNH₂ was done under carefully controlled conditions to prevent hydrolysis of the carbamate function, leading to loss of the L ‐threonine moiety.     

Interaction of [VIVO(acac)2] with Human Serum Transferrin and Albumin

[VO(acac)₂] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI‐TOF), small‐angle X‐ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)₂] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI‐TOF data clearly prove that at least two VO–acac moieties may bind to apoHTF, most probably forming [V^IVO(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)₂] to HSA is obtained. We conclude that V^IVO–acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form.     

Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high‐resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high‐performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI‐Orbitrap or the MALDI‐TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI‐Orbitrap mass spectrometer enabled differentiation between two possible RNA‐based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In‐source decay (ISD) analysis using a MALDI‐TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4‐dihydroxyacetophenone (2,4‐DHAP) as a matrix. The ESI‐Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on‐line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI‐TOF mass spectrometer, in combination with 2,4‐DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis,crystal structures and DNA/human serum albumin binding of ternary Cu(II) complexes containing amino acids and 6‐(pyrazin‐2‐yl)‐1,3,5‐triazine‐2,4‐diamino

Two water‐soluble 6‐(pyrazin‐2‐yl)‐1,3,5‐triazine‐2,4‐diamino (pzta)‐based Cu(II) complexes, namely [Cu(l ‐Val)(pzta)(H₂O)]ClO₄ ( 1 ) and [Cu(l ‐Thr)(pzta)(H₂O)]ClO₄ ( 2 ) (l ‐Val: l ‐valinate; l ‐Thr: l ‐threoninate), were synthesized and characterized using elemental analyses, molar conductance measurements, spectroscopic methods and single‐crystal X‐ray diffraction. The results indicated that the molecular structures of the complexes are five‐coordinated and show a distorted square‐pyramidal geometry, in which the central copper ions are coordinated to N,N atoms of pzta and N,O atoms of amino acids. The interactions of the complexes with DNA were investigated using electronic absorption, competitive fluorescence titration, circular dichroism and viscosity measurements. These studies confirmed that the complexes bind to DNA through a groove binding mode with certain affinities (K_b = 4.71 × 10³ and 1.98 × 10³ M^?1 for 1 and 2 , respectively). The human serum albumin (HSA) binding properties of the complexes were also evaluated using fluorescence and synchronous fluorescence spectroscopies, indicating that the complexes could quench the intrinsic fluorescence of HSA in a static quenching process. The relevant thermodynamic parameters revealed the involvement of van der Waals forces and hydrogen bonds in the formation of complex–HSA systems. Finally, molecular docking technology was also used to further verify the interactions of the complexes with DNA/HSA.     

A Study of Interaction between Flavonoids and the Parallel Quadruplex Structure [d(TGGGGT)]4 by Electrospray Ionization Mass Spectrometry

In this study, electrospray ionization mass spectrometry (ESI‐MS) was used to investigate interaction of 21 flavonoids (10 aglycones and 11 glycosides) with the parallel quadruplex structure [d(TGGGGT)]₄. Relative binding affinities of flavonoids toward [d(TGGGGT)]₄ were estimated based on the fraction of bound DNA. It was found that [d(TGGGGT)]₄ showed a binding preference to the flavonoid glycosides over flavonoid aglycones. It was deduced that glycosylation played a key role for the [d(TGGGGT)]₄‐binding properties of flavonoid glycosides. Upon collision‐induced dissociation, complexes of flavonoid/[d(TGGGGT)]₄ underwent the loss of flavonoids, suggesting an end‐stacking binding mode. The current work demonstrates that ESI‐MS is a powerful tool in the study of interaction between drugs and nucleic acids.     

Ultra‐performance liquid chromatography coupled with electrospray ionization/quadrupole time‐of‐flight mass spectrometry for the rapid analysis of constituents in the traditional Chinese medical formula Danggui San

In this work, ultra‐performance LC with ESI quadrupole TOF‐MS (UPLC–ESI‐Q‐TOF‐MS) and automated MetaboLynx analysis was used to rapidly separate and identify the chemical constituents of Danggui San, a traditional Chinese medical formula. The analysis was performed on a Waters UPLC BEH C₁₈ column using a gradient elution system. A hyphenated ESI and Q‐TOF analyzer was used for the determination of the accurate mass of the protonated or deprotonated molecule and fragment ions in both positive and negative modes. Based on retention times, accurate mass, and the mass spectrometric fragmentation characteristics, a total of 47 compounds distributed over the chemical groups of phthalides, flavonoids, monoterpene glycosides, sesquiterpenoids, phenolics, and alkaloids, were simultaneously separated within 18 min and identified or tentatively elucidated in Danggui San for the first time. UPLC–ESI‐Q‐TOF‐MS analysis revealed the complexity of the chemical composition of this formula. The method developed is rapid, accurate, reliable, and highly sensitive to characterize the chemical constituents of Danggui San.     

Reduction of N‐Nitrosaminoquinolinediones with LiAlH4 – an Easy Path to New Tricyclic Benzoxadiazocines

3‐Butylaminoquinolinediones ( 1 ) react with NaNO₂ in AcOH to give the corresponding N‐nitrosoderivatives ( 2 ). The analogous reactions of 4‐hydroxy‐3‐butylaminoquinolinediones ( 5 ), prepared by the reduction of 1 with NaBH₄, produce the corresponding nitrosamines ( 4 ). The reduction of both 2 and 4 with Zn under different conditions was non‐productive, but the reduction of both compounds with LiAlH₄ at the oxo and lactame groups yielded impure products, generating new tricyclic benzoxadiazocines ( 9 ) by a reaction with HNCO. All compounds were characterized by IR, ¹H‐, and ¹³C‐NMR (in some cases, ¹⁵N‐NMR also) spectroscopy and EI and/or ESI mass spectrometry. The X‐ray structure of compound 9g was determined.     

Evaluation of alkaloids binding to the parallel quadruplex structure [d(TGGGGT)]4 by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI‐MS) was used to investigate the binding interaction of six alkaloids with parallel intermolecular G‐quadruplex [d(TGGGGT)]₄, and five alkaloids including berberine, jatrorrhizine, palmatine, tetrandrine, and fangchinoline showed complexation with the target DNA. Relative binding affinities were estimated on the basis of mass spectrometric data. The slight differences in chemical structures of berberine, jatrorrhizine, and palmatine had little influence on their binding affinities to [d(TGGGGT)]₄. Tetrandrine and fangchinoline selectively bound to [d(TGGGGT)]₄ versus duplex DNA. Collision‐induced dissociation (CID) experiments showed that the complexes with berberine, jatrorrhizine, and palmatine dissociated via strand separation and ligand retaining in the strand while the complexes with tetrandrine and fangchinoline were dissociated via ligand elimination. A comparison of dissociation patterns in CID experiments of complexes with the alkaloids to those with the traditional G‐quadruplex DNA binders suggested an end‐stacking binding mode for tetrandrine and fangchinoline and an intercalation binding mode for berberine, jatrorrhizine, and palmatine to the target DNA. The current work not only provides deep insight into alkaloid/[d(TGGGGT)]₄ complexes and useful guidelines for design of efficient anticancer agents but also demonstrates the utility of ESI‐MS as a powerful tool for evaluating interaction between ligand and quadruplex DNA. Copyright © 2012 John Wiley & Sons, Ltd.     

Enantiomerically Pure Thrombin Inhibitors for Exploring the Molecular‐Recognition Features of the Oxyanion Hole

A new route via intermediate pseudoenantiomers was developed to synthesize racemic and enantiomerically pure new non‐peptidic inhibitors of thrombin, a key serine protease in the blood‐coagulation cascade. These ligands feature a conformationally rigid tricyclic core and are decorated with substituents to fill the major binding pockets (distal (D), proximal (P), selectivity (S1), and oxyanion hole) at the thrombin active site (Fig. 1). The key step in the preparation of the new inhibitors is the 1,3‐dipolar cycloaddition between an optically active azomethine ylide, prepared in situ from L ‐(4R)‐hydroxyproline and 4‐bromobenzaldehyde, and N‐piperonylmaleimide (Scheme 1). According to this protocol, tricyclic imide (compounds (±)‐ 15 ‐(±)‐ 18 and (+)‐ 21 ) and lactam (compound (+)‐ 2 ) inhibitors with OH or ether substituents at C(7) in the proline‐derived pyrrolidine ring were synthesized to specifically explore the binding features of the oxyanion hole (Schemes 2–4). Biological assays (Table) showed that the polar oxyanion hole in thrombin is not suitable for the accommodation of bulky substituents of low polarity, thereby confirming previous findings. In contrast, tricyclic lactam (+)‐ 2 (K_i=9 nM , K_i(trypsin)/K_i(thrombin)=1055) and tricyclic imide (+)‐ 21 (K_i=36 nM , K_i(trypsin)/K_i(thrombin)=50) with OH‐substituents at the (R)‐configured C(7)‐atom are among the most‐potent and most‐selective thrombin inhibitors in their respective classes, prepared today. While initial modeling predicted H‐bonding between the OH group at C(7) in (+)‐ 2 and (+)‐ 21 with the H₂O molecule bound in the oxyanion hole (Fig. 2), the X‐ray crystal structure of the complex of (+)‐ 21 (Fig. 7, b) revealed a different interaction for this group. The propionate side chain of Glu192 undergoes a conformational change, thereby re‐orienting towards the OH group at C(7) under formation of a very short ionic H‐bond (O? H???^?OOC; d(O???O)=2.4 Å). The energetic contribution of this H‐bond, however, is negligible, due to its location on the surface of the protein and the unfavorable conformation of the H‐bonded propionate side chain.     

Two New Phenolic Compounds from Artemisia iwayomogi

Two new phenolic compounds, (Z)‐5′‐hydroxyjasmone 5′‐O‐{6″‐O‐[(E)‐caffeoyl]‐β‐D ‐glucopyranoside} ( 1 ) and quercetin‐7‐O‐β‐D ‐glucuronide methyl ester ( 2 ), along with ten known phenolic compounds, 3 – 12 , were isolated from the aerial parts of Artemisia iwayomogi. Their structures were elucidated by spectroscopic methods, including 1D‐ and 2D‐NMR, and HR‐ESI‐TOF‐MS techniques. The inhibitory effects of compounds 1 – 12 on the LPS‐stimulated production of IL‐12 p40, IL‐6, and TNF‐α in bone marrow‐derived dendritic cells were evaluated.     

Solution Structure of a RNA Decamer Duplex,Containing 9‐[2‐O‐(β‐D‐ribofuranosyl)‐β‐D‐ribofuranosyl]adenine,a Special Residue in Lower Eukaryotic Initiator tRNAs

The solution structure of the self‐complementary deca‐ribonucleotide 5′‐r(GCGA*AUUCGC)‐3′ containing 9‐[2‐O‐(β‐D ‐ribofuranosyl)‐β‐D ‐ribofuranosyl]adenine (A*), a modified nucleotide that occurs in lower eukaryotic methionine initiator tRNAs (tRNAs_i^Met), was determined by NMR spectroscopy. Unexpectedly, the modification has no effect on the thermal stability of the duplex. However, the extra ribose moiety is in the C(3′)‐endo conformation and takes up a well‐defined position in the minor groove, which is in agreement with its position in tRNAs_i^Met as determined by X‐ray crystallography. Molecular‐dynamics simulations on the RNA duplex in H₂O show that the position of the extra ribofuranose moiety seems to be stabilized by bridged H‐bonds (mediated by two H₂O molecules) to the backbone of the complementary chain.