Endo Conformation Research Articles

Ultraviolet Photodissociation Spectroscopy of the Cold K⁺·Calix[4]arene Complex in the Gas Phase.

The cooling of ionic species in the gas phase greatly simplifies the UV spectrum, which is of special importance when studying the electronic and geometric structures of large systems, such as biorelated molecules and host-guest complexes. Many efforts have been devoted to achieving ion cooling with a cold, quadrupole Paul ion trap (QIT), but one problem was the insufficient cooling of ions (up to ∼30 K) in the QIT. In this study, we construct a mass spectrometer for the ultraviolet photodissociation (UVPD) spectroscopy of gas-phase cold ions. The instrument consists of an electrospray ion source, a QIT cooled with a He cryostat, and a time-of-flight mass spectrometer. With great care given to the cooling condition, we can achieve ∼10 K for the vibrational temperature of ions in the QIT, which is estimated from UVPD spectra of the benzo-18-crown-6 (B18C6) complex with a potassium ion, K(+)·B18C6. Using this setup, we measure a UVPD spectrum of cold calix[4]arene (C4A) complex with potassium ion, K(+)·C4A. The spectrum shows a very weak band and a strong one at 36018 and 36156 cm(-1), respectively, accompanied by many sharp vibronic bands in the 36000-36600 cm(-1) region. In the geometry optimization of the K(+)·C4A complex, we obtain three stable isomers: one endo and two exo forms. On the basis of the total energy and UV spectral patterns predicted by density functional theory calculations, we attribute the structure of the K(+)·C4A complex to the endo isomer (C2 symmetry), in which the K(+) ion is located inside the cup of C4A. The vibronic bands of K(+)·C4A at 36 018 and 36 156 cm(-1) are assigned to the S1(A)-S0(A) and S2(B)-S0(A) transitions of the endo isomer, respectively.

The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products

In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H–>CH3O–>CH3OC(O)CH2–>HOC(O)CH2NHCH2–≈ CH3NHCH2–, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2′-endo sugar ring conformation, as opposed to the preferred C3′-endo conformation of the parent R5S2Us.

Synthesis of the Tricyclic Ring Structure of Daphnanes via Intramolecular [4 + 3] Cycloaddition/SmI2-Pinacol Coupling.

A synthetic approach toward the tricyclic 5,7,6-membered ring structure of daphnane-family natural products is described. An intramolecular [4 + 3] cycloaddition reaction of furan with an oxypentadienyl cation constructed the oxa-bridged bicyclic structure in a stereoselective fashion. Structural analysis revealed that the desired exo isomer was predominantly acquired through epimerization. Finally, formation of the five-membered ring was achieved through SmI2-mediated pinacol coupling.

The role of RNA conformation in RNA-protein recognition

Interactions between protein and RNA play a key role in many biological processes in the gene expression pathway. Those interactions are mediated through a variety of RNA-binding protein domains, among them the highly abundant RNA recognition motif (RRM). Here we studied protein-RNA complexes from different RNA binding domain families solved by NMR and x-ray crystallography. Characterizing the structural properties of the RNA at the binding interfaces revealed an unexpected number of nucleotides with unusual RNA conformations, specifically found in RNA-RRM complexes. Moreover, we observed that the RNA nucleotides that are directly involved in interactions with the RRM domains, via hydrogen bonds and hydrophobic contacts, are significantly enriched with unique RNA conformations. Further examination of the sequences binding the RRM domain showed a preference for G nucleotides in syn conformation to precede or to follow U nucleotides in the anti-conformation, and U nucleotides in C2' endo conformation to precede U and G nucleotides possessing the more common C3' endo conformation. These findings imply a possible mode of RNA recognition by the RRM domains which enables the recognition of a wide variety of different RNA sequences and shapes. Overall, this study suggests an additional way by which the RRM domain recognizes its RNA target, involving a conformational readout.

Investigation and conformational analysis of fluorinated nucleoside antibiotics targeting siderophore biosynthesis.

Antibiotic resistance represents one of the greatest threats to public health. The adenylation inhibitor 5'-O-[N-(salicyl)sulfamoyl]adenosine (SAL-AMS) is the archetype for a new class of nucleoside antibiotics that target iron acquisition in pathogenic microorganisms and is especially effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. Strategic incorporation of fluorine at the 2' and 3' positions of the nucleoside was performed by direct fluorination to enhance activity and improve drug disposition properties. The resulting SAL-AMS analogues were comprehensively assessed for biochemical potency, whole-cell antitubercular activity, and in vivo pharmacokinetic parameters. Conformational analysis suggested a strong preference of fluorinated sugar rings for either a 2'-endo, 3'-exo (South), or a 3'-endo,2'-exo (North) conformation. The structure-activity relationships revealed a strong conformational bias for the C3'-endo conformation to maintain potent biochemical and whole-cell activity, whereas improved pharmacokinetic properties were associated with the C2'-endo conformation.

Stereochemistry of spiro-acetalized [60]fullerenes: how the exo and endo stereoisomers influence organic solar cell performance.

Exploiting bis-addition products of fullerenes is a rational way to improve the efficiency of bulk heterojunction-type organic photovoltaic cells (OPV); however, this design inherently produces regio- and stereoisomers that may impair the ultimate performance and fabrication reproducibility. Here, we report unprecedented exo and endo stereoisomers of the spiro-acetalized [60]fullerene monoadduct with methyl- or phenyl-substituted 1,3-dioxane (SAF6). Although there is no chiral carbon in either the reagent or the fullerene, equatorial (eq) rather than axial (ax) isomers are selectively produced at an exo-eq:endo-eq ratio of approximately 1:1 and can be easily separated using silica gel column chromatography. Nuclear Overhauser effect measurements identified the conformations of the straight exo isomer and bent endo isomer. We discuss the origin of stereoselectivity, the anomeric effect, intermolecular ordering in the film state, and the performance of poly(3-hexylthiophene):substituted SAF6 OPV devices. Despite their identical optical and electrochemical properties, their solubilities and space-charge limited current mobilities are largely influenced by the stereoisomers, which leads to variation in the OPV efficiency. This study emphasizes the importance of fullerene stereochemistry for understanding the relationship between stereochemical structures and device output.

Molecular cleft or tweezer compounds derived from trioxabicyclo[3.3.1]nonadiene diisocyanate and diacid dichloride.

The structures of two derivatives of the bisdioxine diisocyanate 1, the bisurea 4 and the biscarbamate 5, are established by X-ray crystallography and DFT calculations. These compounds possess endo,endo structures, in the case of the bisurea 4 with two nearly parallel pendant chains. The X-ray structures are reproduced very well by DFT calculations. Similar endo,endo conformations are calculated for the bisamide crown ether derivatives 7, where two proximate and nearly parallel crown ether units endow the molecules with a claw-like molecular cleft or tweezer structure as evidenced by an enhanced ability to extract some alkali, alkaline earth and rare earth metal ions.

Acyl‐Functionalized Molybdenum Compounds [(η 3 ‐C 3 H 5 )(η 5 ‐Cp′)Mo(CO) 2 ]: An Experimental Study Including the X‐ray Structure of a Rare endo Conformer

A series of new acyl-functionalized molybdenum(II) complexes [(η3-C3H5)(η5-Cp′)Mo(CO)2] (Cp′ = substituted Cp) have been synthesized. X-ray diffraction analysis of the CF3CO derivative revealed a rare endo conformer. In addition, it was found that the electronic properties of the acyl substituents have a strong effect on the outcome of protonation reactions.

Isolation and characterization of conformational isomers ofN,N′-bis(3,5-dichlorosalicylidene)-2,2′-ethylenedianiline: crystal structure, photoluminescence, and density functional theory calculation

We have isolated two isomeric solids 1 and 2 of N,N′-bis(3,5-dichlorosalicylidene)-2,2′-ethylenedianiline and characterized by IR, UV/Vis, X-ray powder diffraction, thermogravimetric analysis/differential thermal analysis, and X-ray crystallography. Although the solids are same formulas, each shows different colors and crystal structures. Orange solid (1) shows endo conformation while yellow solid (2) exhibits exo form depending on packing modes. UV/Vis spectra of 1 and 2 appear very similar patterns in the solid state; however, the bands of 1 are slightly red-shifted compared with those of 2. 1 displays a strong fluorescent emission band at ~582 nm while 2 shows an intense fluorescent signal at ~563 nm. The charge density populations of 1 and 2 have been studied by computational simulations using density functional theory at pbe1pbe/6-311G** level. The calculated highest occupied molecular orbital and lowest unoccupied molecular orbital energies of 1 and 2 confirm that charge transfer occurs within the organic molecules. The energy difference of HOMO-LUMO in 1 is smaller slightly than that of 2 about 0.05 eV (~17 nm). Copyright © 2014 John Wiley & Sons, Ltd.

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bc(en)-T and iso-tricyclo-T nucleosides.

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.

Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics.

Modified nucleotides are prevalent in tRNA. Experimental studies reveal that these covalent modifications play an important role in tuning tRNA function. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA dynamics. The X-ray crystal structures of tRNA(Asp), tRNA(Phe), and tRNA(iMet), both with and without modifications, were used as initial structures for 333 ns explicit solvent MD simulations with AMBER. For each tRNA molecule, three independent trajectory calculations were performed, giving an aggregate of 6 μs of total MD across six molecules. The global root-mean-square deviations (RMSD) of atomic positions show that modifications only introduce significant rigidity to the global structure of tRNA(Phe). Interestingly, RMSDs of the anticodon stem-loop (ASL) suggest that modified tRNA has a more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that the rigidity of the anticodon stem-loop is finely tuned by modifications, where rigidity in the anticodon arm is essential for tRNA translocation in the ribosome, and flexibility of the anticodon is important for codon recognition. Sugar pucker and water residence time of pseudouridines in modified tRNAs and corresponding uridines in unmodified tRNAs were assessed, and the results reinforce that pseudouridine favors the 3′-endo conformation and has a higher tendency to interact with water. Principal component analysis (PCA) was used to examine correlated motions in tRNA. Additionally, covariance overlaps of PCAs were compared for trajectories of the same molecule and between trajectories of modified and unmodified tRNAs. The comparison suggests that modifications alter the correlated motions. For the anticodon bases, the extent of stacking was compared between modified and unmodified molecules, and only unmodified tRNA(Asp) has significantly higher percentage of stacking time. Overall, the simulations reveal that the effect of covalent modification on tRNA dynamics is not simple, with modifications increasing flexibility in some regions of the structure and increasing rigidity in other regions.

Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids.

The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2′-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2′-endo conformation of the 2′-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858–2865) that estimated the extent of destabilization caused by a single C2′-endo nucleotide within a native RNA duplex to amount to 6 kcal mol−1 because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2′-endo conformation of 2′-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2′-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2′-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson–Crick base pair, adopted the same C3′-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2′-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2′-SCF3 nucleoside modification and correlate them to base pairing properties.

5,8-Dimeth-oxy-3,9-dimethyl-3a,4,9,9a-tetra-hydro-4,9-ep-oxy-naphtho-[2,3-d]isoxazole.

The title compound, C15H17NO4, is the exo isomer with a syn arrangement of the O atom in the isoxazole ring to the methyl group of the bicyclic alkene. The dihedral angle between the isoxazole ring and the benzene ring is 7.42 (9)°. In the crystal, weak C—H⋯O hydrogen bonds link mol­ecules, forming a three-dimensional network. The isoxazole O atom is an acceptor for both weak hydrogen bonds.

Facile access to cyclooctanoid ring systems via microwave-assisted tandem 6-exo dig cyclization–rearrangement sequence

Appropriately substituted 5-alkyn-1-ol systems bearing a nitrile moiety at the triple bond serve as versatile precursors to a variety of cyclooctenone derivatives via a ‘one-pot’ base-catalyzed oxyanionic 6-exo dig cyclization/Claisen rearrangement sequence under microwave irradiation. It was found that the initially formed cyclic intermediate consists of a mixture of endo and exocyclic isomers, which appear to be in equilibrium under the reaction conditions. However, the only observed products from these reactions are α-cyano-substituted cyclooctenones, derived from the exocyclic dihydrofuran intermediates.

The importance of fitting in: conformational preference of selenium 2′ modifications in nucleosides and helical structures

Selenomethionine incorporation has proven useful in X-ray crystallography of proteins to obtain phase information. In nucleic acids, the introduction of selenium to different positions is beneficial for solving the phase problem as well, but its addition to the 2′ position also significantly enhances the crystal formation. The selenium modification in a single nucleotide shows a preference towards 2′-endo sugar puckering, which is in conflict with existing crystal structures where the duplex incorporated 2′-selenium-modified nucleotide is exclusively found in a 3′-endo conformation. Our work provides a rationale why 2′-selenium modifications facilitate crystallization despite this contradictory behavior.

Steady‐State and Pseudo‐Steady‐State Photocrystallographic Studies on Linkage Isomers of [Ni(Et4dien)(η2‐O,ON)(η1‐NO2)]: Identification of a New Linkage Isomer

At temperatures below 150 K, the photoactivated metastable endo-nitrito linkage isomer [Ni(Et4 dien)(η(2)-O,ON)(η(1)-ONO)] (Et4 dien=N,N,N',N'-tetraethyldiethylenetriamine) can be generated with 100 % conversion from the ground state nitro-(η(1)-NO2) isomer on irradiation with 500 nm light, in the single crystal by steady-state photocrystallographic techniques. Kinetic studies show the system is no longer metastable above 150 K, decaying back to the ground state nitro-(η(1)-NO2) arrangement over several hours at 150 K. Variable-temperature kinetic measurements in the range of 150-160 K show that the rate of endo-nitrito decay is highly dependent on temperature, and an activation energy of Eact =+48.6(4) kJ mol(-1) is calculated for the decay process. Pseudo-steady-state experiments, where the crystal is continually pumped by the light source for the duration of the X-ray experiment, show the production of a previously unobserved, exo-nitrito-(η(1)-ONO) linkage isomer only at temperatures close to the metastable limit (ca. 140-190 K). This exo isomer is considered to be a transient excited-state species, as it is only observed in data collected by pseudo-steady-state methods.

The free energy landscape of pseudorotation in 3'-5' and 2'-5' linked nucleic acids.

The five-membered furanose ring is a central component of the chemical structure of biological nucleic acids. The conformations of the furanose ring can be analytically described using the concept of pseudorotation, and for RNA and DNA they are dominated by the C2′-endo and C3′-endo conformers. While the free energy difference between these two conformers can be inferred from NMR measurements, a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive. Here, we describe a new free energy calculation method for molecular dynamics (MD) simulations using the two pseudorotation parameters directly as the collective variables. To validate our approach, we calculated the free energy surface of ribose pseudorotation in guanosine and 2′-deoxyguanosine. The calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers, but also the main transition path between the stable conformations. Applying this method to a standard A-form RNA duplex uncovered the expected minimum at the C3′-endo state. However, at a 2′–5′ linkage, the minimum shifts to the C2′-endo conformation. The free energy of the C3′-endo conformation is 3 kcal/mol higher due to a weaker hydrogen bond and a reduced base stacking interaction. Unrestrained MD simulations suggest that the conversion from C3′-endo to C2′-endo and vice versa is on the nanosecond and microsecond time scale, respectively. These calculations suggest that 2′–5′ linkages may enable folded RNAs to sample a wider spectrum of their pseudorotation conformations.

Optimization of an AMBER force field for the artificial nucleic acid, LNA, and benchmarking with NMR of L(CAAU).

Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced.

Precision polymers: a kinetic approach for functional poly(norbornenes)

Control over monomer sequence in the ring-opening metathesis polymerization of functional norbornenes is explored based on the difference in reactivity of endo and exo isomers.