Synthesis study of 2'-O-(2-methoxyethyl)-purine derivatives

Alkylation of adenosine and 2-aminoadenosine was studied in dimethylsulfoxide with application of 1-methanesulfonyloxy-2-methoxyethane as an alkylating agent and t-BuOK, KOH and NaH as bases under mild heating. Using new reaction conditions, the improved synthesis of 2'-O-MOE-purine derivatives is described.     

Kilo-scale synthesis process for 2'-O-(2-methoxyethyl)-pyrimidine derivatives

We describe an improved process to produce 2'-O-(2-methoxyethyl)-pyrimidines. Starting with commercially available O-2,2'-anhydro-5-methyluridine and tris-(2-methoxyethyl)borate, we modified the ring-opening reaction conditions and changed to a continuous extraction purification method to give 2'-O-(2-methaxyethyl)-5-methyluridine. The dimethoxytritylation 5'/3' ratios and yield were improved by the use of 2,6-lutidine as the base. Conditions to convert to the 5'-methylcytidine analog and its isolation by crystallization were optimized. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities.     

Crystal structure and improved antisense properties of 2'-O-(2-methoxyethyl)-RNA.

2'-O-(2-Methoxyethyl)-RNA (MOE-RNA) is a nucleic acid analog with promising features for antisense applications. Compared with phosphorothioate DNA (PS-DNA), the MOE modification offers improved nuclease resistance, enhanced RNA affinity, improved cellular uptake and intestinal absorption, reduced toxicity and immune stimulation. The crystal structure of a fully modified MOE-RNA dodecamer duplex (CGCGAAUUCGCG) was determined at 1.7 A resolution. In the majority of the MOE substituents, the torsion angle around the ethylene alkyl chain assumes a gauche conformation. The conformational preorganization of the MOE groups is consistent with the improved RNA affinity and the extensive hydration of the substituents could play a role in the improved cellular uptake of MOE-RNA. A specific hydration pattern that bridges substituent and phosphate oxygen atoms in the minor groove of MOE-RNA may explain its high nuclease resistance.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

Structural properties of a highly polyunsaturated lipid bilayer from molecular dynamics simulations.

The structure of a fully hydrated mixed (saturated/polyunsaturated) chain lipid bilayer in the biologically relevant liquid crystalline phase has been examined by performing a molecular dynamics study. The model membrane, a 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC, 18:0/22:6 PC) lipid bilayer, was investigated at constant (room) temperature and (ambient) pressure, and the results obtained in the nanosecond time scale reproduced quite well the available experimental data. Polyunsaturated fatty acids are found in high concentrations in neuronal and retinal tissues and are essential for the development of human brain function. The docosahexaenoic fatty acid, in particular, is fundamental for the proper function of the visual receptor rhodopsin. The lipid bilayer order has been investigated through the orientational order parameters. The water-lipid interface has been explored thoroughly in terms of its dimensions and the organization of the different components. Several types of interactions occurring in the system have been analyzed, specifically, the water-hydrocarbon chain, lipid-lipid and lipid-water interactions. The distribution of dihedral angles along the chains and the molecular conformations of the polyunsaturated chain of the lipids have also been studied. Special attention has been focused on the microscopic (molecular) origin of the effects of polyunsaturations on the different physical properties of membranes.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

Diastereomeric process control in the synthesis of 2'-O-(2-methoxyethyl) oligoribonucleotide phosphorothioates as antisense drugs

Coupling of 2'-O-methoxyethylsubstituted nucleoside phosphoramidites to 5'-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

Neuron-specific delivery of nucleic acids mediated by Tet1-modified poly(ethylenimine)

BACKGROUND: The development of minimally invasive, non-viral gene delivery vehicles for the central nervous system (CNS) is an important technology goal in the advancement of molecular therapies for neurological diseases. One approach is to deliver materials peripherally that are recognized and retrogradely transported by motor neurons toward the CNS. Tet1 is a peptide identified by Boulis and coworkers to possess the binding characteristics of tetanus toxin, which interacts specifically with motor neurons and undergoes fast, retrograde delivery to cell soma. In this work, Tet1-poly(ethylenimine) (Tet1-PEI) was synthesized and evaluated as a neurontargeted delivery vehicle. METHODS: Tet1-PEI and NT-PEI (neurotensin-PEI) were synthesized and complexed with plasmid DNA to form polyplexes. Polyplexes were assessed for binding and uptake in differentiated neuron-like PC-12 cells by flow cytometry and confocal microscopy. In order to determine gene delivery efficiency, polyplexes were exposed to PC-12 cells at various stages of differentiation. Targeted binding of polyplexes with primary neurons was studied using dorsal root ganglion cells. RESULTS: Tet1-PEI and NT-PEI polyplexes bound specifically to differentiated PC-12 cells. The specificity of the interaction was confirmed by delivery to non-neuronal cells and by competition studies with free ligands. Tet1-PEI polyplexes preferentially transfected PC-12 cells undergoing NGF-induced differentiation. Finally, neuron-specific binding of Tet1-PEI polyplexes was confirmed in primary neurons. CONCLUSIONS: These studies demonstrate the potential of Tet1-PEI as a neuron-targeted material for non-invasive CNS delivery. Tet1-PEI binds specifically and is internalized by neuron-like PC-12 cells and primary dorsal root ganglion. Future work will include evaluation of siRNA delivery with these vectors.     

Data mining of molecular dynamics trajectories of nucleic acids

Analysis, storage, and transfer of molecular dynamic trajectories are becoming the bottleneck of computer simulations. In this paper we discuss different approaches for data mining and data processing of huge trajectory files generated from molecular dynamic simulations of nucleic acids.     

Structural analysis of human lysozyme using molecular dynamics simulations

In this study, various molecular dynamics simulations were conducted to investigate the effects of ethanol and temperature on the conformational changes of human lysozyme, which may lead insights into amyloidosis. The analyses of some important structural characteristics, such as backbone root-mean-square deviation, secondary structural stability, radius of gyration, accessible surface area, and hydrophobic contact of the hydrophobic core all show that ethanol tends to destabilize human lysozyme at high temperatures. It can be attributed to that higher temperatures result in the destruction of the native structure of this protein, leading to the exposure of the interior hydrophobic core. At this stage, ethanol plays a role to destroy this region by forming hydrophobic interactions between protein and solvent due to its lower polarity comparing to water. Such newly formed intermolecular interactions accelerate the unfolding of this protein, starting from the core between the alpha- and beta-domains. Our results are in good agreement with the previous hypothesis suggesting that the distortion of the hydrophobic core at the alpha- and beta-interface putatively results in the formation of the initial "seed" for amyloid fibril. Although the present results cannot directly be linked to fibril formation, they still provide valuable insights into amyloidosis of human lysozyme.     

Synthesis and characteristics of modified oligodeoxyribonucleotides containing 2'-O-(2,3-dihydroxypropyl)uridine and 2'-O-(2-exoethyl)uridine

A new uridine derivative, 2'-O-(2,3-dihydroxypropyl)uridine, and its 3'-phosphoramidite were obtained for direct introduction into oligonucleotides during automated chemical synthesis. Oligonucleotides 10 to 15 nt long harboring 2'-O-(2,3-dihydroxypropyl)uridine residues were synthesized; periodate oxidation of these oligomers gave oligonucleotides containing 2'-O-(2-oxoethyl)uridine residues. The presence of a reactive aldehyde group in 2' position of the carbohydrate moiety was confirmed by the interaction with p-nitrophenylhydrazine and methionine methyl ester. The thermostability of DNA duplexes containing modified units is practically indistinguishable from that of the natural analogues.     

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.     

Structural determinants of MscL gating studied by molecular dynamics simulations

The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock. The channel can be gated by tension in the membrane bilayer. The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating. Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer. Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics. The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water. Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel. The gating process was investigated through simulations of the bare protein under conditions of constant surface tension. Under a range of conditions, the transmembrane helices flattened as the pore widened. Implications for the gating mechanism in light of these and experimental results are discussed.     

Structural and mechanistic insights into the oxy form of tyrosinase from molecular dynamics simulations

The first, long time scale (16-ns) ligand field molecular dynamics (LFMD) simulations of the oxy form of tyrosinase are reported. The calculations use our existing type 3 copper force field for the peroxido-bridged [Cu₂O₂]²⁺ unit which is here translated from MMFF into the AMBER format together with a new charge scheme. The protein secondary and tertiary structures are not significantly altered by removing the ‘caddie’ protein, ORF378, which must be bound to tyrosinase before crystals will grow. A comprehensive principal component analysis of the Cartesian coordinates from the final 8 ns shows that the protein backbone is relatively rigid. However, the significant butterfly fold of the [Cu₂O₂]²⁺ moiety observed in the X-ray structure, presumably due to the caddie protein tyrosine at the active site, is absent in the simulations. LFMD gives a clear and persistent distinction between equatorial and axial Cu–N distances, with the latter about 0.2 Å longer and remaining syn to each other. However, the two coordination spheres display important differences. LFMD simulations of the symmetric model complex [μ-η²:μ²-O₂{Cu(Meim)₃}₂]²⁺ (Meim is 5-methyl-1H-imidazole) provide a mechanism for syn–anti interchange of axial ligands which suggests, in combination with the old experimental X-ray data, the new LFMD simulations and traditional coordination chemistry arguments, that His₅₄ on Cu_A is ‘insipiently axial’ and that a combination of a butterfly distortion of the [Cu₂O₂]²⁺ group and a rotation of the Cu_A(His)₃ moiety converts the vacant, initially axial, binding site on Cu_A into a much more favourable equatorial site.     

Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs

With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml^–1) with PicoGreen and RiboGreen dyes. In addition, with a few simple modifications, autoradiographic film images can be scanned and quantified with the Typhoon series of scanners.     

Inhibition of telomerase by 2'-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells

2′-O-(2-methoxyethyl) (2′-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2′-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC₅₀ values of 5–10 nM at 37°C. IC₅₀ values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.     

Molecular dynamics simulations of nucleic acid-protein complexes

Molecular dynamics simulation studies of protein-nucleic acid complexes are more complicated than studies of either component alone-the force field has to be properly balanced, the systems tend to become very large, and a careful treatment of solvent and of electrostatic interactions is necessary. Recent investigations into several protein-DNA and protein-RNA systems have shown the feasibility of the simulation approach, yielding results of biological interest not readily accessible to experimental methods.