Reaction of Internucleotidic Phosphorothioate Diesters with Fluorescent Reporter groups

Abstract

The substitution of a non-bridging oxygen by sulfur in a DNA intemucleotidic phosphorus residue creates a nucleophilic site which is amenable to labeling by reporter groups such as fluorophores or spin labels. Placement of a single phosphorothioate diester at a selected position allows site-specific attachment of the reporter group. Incorporating a phosphorothioate at each and every intemucleotidic phosphorous linkage allows the incorporation of multiple reporter groups, ideally one for each nucleoside residue.     

Intrinsic barrier for protonation of radical anions

Ab initio calculations on radical anions show that, counterintuitively, protonation on the radicaloid carbon is favored. In the case of radical anions derived from acrylonitrile and acrylaldehyde, protonation on the heteroatom is less favored than protonation on the radicaloid carbon. However, in nitroethylene, the preferred protonation site is on the nitro oxygen in accordance with experimental observation.     

Impact of arsenic/phosphorus substitution on the intrinsic conformational properties of the phosphodiester backbone of DNA investigated using ab initio quantum mechanical calculations

Deoxyribonucleic acid (DNA) is composed of five major elements carbon, hydrogen, nitrogen, oxygen, and phosphorus. The substitution of any of these elements in DNA would be anticipated to have major biological implications. However, recent studies have suggested that the substitution of arsenic into DNA (As-DNA) in bacteria may be possible. To help evaluate this possibility, ab initio quantum mechanical calculations are used to show that arsenodiester and phosphodiester linkages have similar geometric and conformational properties. Based on these results, it is suggested that the As-DNA will have similar conformational properties to phosphorus-based DNA, including the maintenance of base stacking.     

The reaction of atomic oxygen with methanethiol. A theoretical study of the structures and the potential energy surface

Ab initio calculations at the Hartree-Fock, MP2 and MP4 levels were performed to find structures of the equilibrium and transition states and the reaction energies and energies of activation of several competing reaction pathways of O (3P)+CH3SH. A 6-31G* basis set was used in all calculations. The mechanism of hydrogen atom abstraction from the S-H group methanethiol was found to be very competitive with the oxygen atom addition to the sulfur atom.     

A theoretical study of protonated N2O

Ab initio MO calculations predict the preferred site of protonation of N₂O to be at the oxygen atom, and yield a structure of N₂OH⁺ and protonation energies in excellent accord with experiment.     

In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'

Deoxyribozymes that ligate RNA expand the scope of nucleic acid catalysis and allow preparation of site-specifically modified RNAs. Previously, deoxyribozymes that join a 5'-hydroxyl and a 2',3'-cyclic phosphate were identified by in vitro selection from random DNA pools. Here, the alternative strategy of in vitro evolution was used to transform the 8-17 deoxyribozyme that cleaves RNA into a family of DNA enzymes that ligate RNA. The parent 8-17 DNA enzyme cleaves native 3'-5' phosphodiester linkages but not 2'-5' bonds. Surprisingly, the new deoxyribozymes evolved from 8-17 create only 2'-5' linkages. Thus, reversing the direction of the DNA-mediated process from ligation to cleavage also switches the selectivity in forming the new phosphodiester bond. The same change in selectivity was observed upon evolution of the 10-23 RNA-cleaving deoxyribozyme into an RNA ligase. The DNA enzymes previously isolated from random pools also create 2'-5' linkages. Therefore, deoxyribozyme-mediated formation of a non-native 2'-5' phosphodiester linkage from a 5'-hydroxyl and a 2',3'-cyclic phosphate is strongly favored in many different contexts.     

Protonation of phenylboronic acid: Comparison of G3B3 and G2MP2 methods

G3B3 and G2MP2 calculations using Gaussian 03 have been carried out to investigate the protonation preferences for phenylboronic acid. All nine heavy atoms have been protonated in turn. With both methodologies, the two lowest protonation energies are obtained with the proton located either at the ipso carbon atom or at a hydroxyl oxygen atom. Within the G3B3 formalism, the lowest‐energy configuration by 4.3 kcal · mol^?1 is found when the proton is located at the ipso carbon, rather than at the electronegative oxygen atom. In the resulting structure, the phenyl ring has lost a significant amount of aromaticity. By contrast, calculations with G2MP2 show that protonation at the hydroxyl oxygen atom is favored by 7.7 kcal · mol^?1. Calculations using the polarizable continuum model (PCM) solvent method also give preference to protonation at the oxygen atom when water is used as the solvent. The preference for protonation at the ipso carbon found by the more accurate G3B3 method is unexpected and its implications in Suzuki coupling are discussed. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

Design of Conjugated Molecules Presenting Short‐Wavelength Luminescence by Utilizing Heavier Atoms of the Same Element Group

The introduction of heavy atoms into conjugated molecules often induces a redshift in the emission spectra. Conversely, we report here a blueshifting effect in the absorption and emission bands of a conjugated organic dye by employing a heavier atom from the same element group. Boron complexes having oxygen‐ and sulfur‐bridged structures in the ligand moiety were synthesized, and their optical properties were compared. Significant optical bands in the absorption and luminescence spectra of the sulfur‐bridged complex were observed in a shorter wavelength region than those of the oxygen‐bridged complex. Theoretical calculations suggest that replacement of the bridging atom by a heavier one should reduce molecular planarity because of the larger atom size. As a result, the degree of electronic conjugation decreases, and this is followed by a blueshift in the optical bands. Finally, a blue‐emissive crystal is demonstrated.     

Stabilisation of the transition state of phosphodiester bond cleavage within linear single-stranded oligoribonucleotides

The effect of base sequence on the stability of the transition state (TS) of phosphodiester bond cleavage within linear single-stranded oligoribonucleotides has been studied in order to better understand why the reactivity of some phosphodiester bonds is enhanced compared to an unconstrained linkage. Molecular dynamics simulations of 3.0 ns were carried out for 14 oligonucleotides that contain in the place of the scissile phosphodiester bond a phosphorane structure mimicking the TS of the bond cleavage. The hydrolytic stability of the same oligonucleotides had previously been reported. Both the non-bridging oxyanions and the leaving 5[prime or minute]-oxygen of the pentacoordinated phosphorane moiety were observed to form hydrogen bonds with solvent water molecules in a similar way with all the compounds studied. In addition, water mediated hydrogen bonds between the phosphorane non-bridging oxyanions and the bases of the 3[prime or minute]-flanking sequence were detected with some of the compounds, but not with the most labile ones. Hence, it seems that the enhanced cleavage of some internucleosidic linkages does not result from the TS stabilisation by hydrogen bonding. With heterooligomers, the stacking of bases next to the cleavage site was observed to be enhanced on going from the initial state to the TS, whereas within uracil homooligomer, having initially negligible stacking, no change in the magnitude of stacking was seen. Accordingly, while strong stacking in the initial state is known to retard the phosphodiester bond cleavage, it may in the TS accelerate the reaction. Therefore, enhanced stacking on going from the initial state to transition state appears to be a factor that markedly contributes to the hydrolytic stability of phosphodiester bonds within oligonucleotides and may, at least partly, explain accelerated cleavage compared to fully unconstrained bonds, such as those in polyuridylic acid.     

Desulfurization of phosphorothioate oligonucleotides via the sulfur‐by‐oxygen replacement induced by the hydroxyl radical during negative electrospray ionization mass spectrometry

While the occurrence of desulfurization of phosphorothioate oligonucleotides in solution is well established, this study represents the first attempt to investigate the basis of the unexpected desulfurization via the net sulfur‐by‐oxygen (S‐O) replacement during negative electrospray ionization (ESI). The current work, facilitated by quantitative mass deconvolution, demonstrates that considerable desulfurization can take place even under common negative ESI operating conditions. The extent of desulfurization is dependent on the molar phosphorothioate oligonucleotide‐to‐hydroxyl radical ratio, which is consistent with the corona discharge‐induced origin of the hydroxyl radical leading to the S‐O replacement. This hypothesis is supported by the fact that an increase of the high‐performance liquid chromatography (HPLC) flow rate and the on‐column concentration of a phosphorothioate oligonucleotide, as well as a decrease of the electrospray voltage reduce the degree of desulfurization. Comparative LC‐tandem mass spectrometry (MS/MS) sequencing of a phosphorothioate oligonucleotide and its corresponding desulfurization product revealed evidence that the S‐O replacement occurs at multiple phosphorothioate internucleotide linkage sites. In practice, the most convenient and effective strategy for minimizing this P = O artifact is to increase the LC flow rate and the on‐column concentration of phosphorothioate oligonucleotides. Another approach to mitigate possible detrimental effects of the undesired desulfurization is to operate the ESI source at a very low electrospray voltage to diminish the corona discharge; however this will significantly compromise sensitivity when analyzing the low‐level P = O impurities in phosphorothioate oligonucleotides. Copyright © 2012 John Wiley & Sons, Ltd.     

Planar 2,2′-bithiophenes with 3,3′- and 3,3′,4,4′-substituents. A computational study

Ab initio calculations have shown that when carbonyl groups are incorporated into the 3- and 3′-positions of bithiophenes, as part of five-membered rings, the bithiophene system is planar. This is due to electrostatic attraction between the carbonyl oxygen and the sulfur atom in the adjacent ring. In the analogous systems containing a CH₂ group in place of the carbonyl, or containing six-membered rather than five-membered rings, the bithiophenes are twisted. This has implications for preparing planar polythiophenes.     

Protonation and deprotonation of hydroxamic acids. An MO ab initio study

Ab initio molecular orbital calculations with 4-31G//4-31G, 6-31G^*//4-31G and 6-31+G//4-31G basis sets have been used to examine the structure, relative energy, protonation and deprotonation of a series of seven hydroxamic acids in the gas phase. The results show that hydroxamic acids are predominantly in the E-TS form and that the most probable protonation site is the carbonyl oxygen atom, while deprotonation proceeds by loss of NH hydrogen.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.     

Synthesis,spectroscopic, electrochemical and structural characterization of Cu(II) complexes with asymmetric NN′OS coordination spheres

A set of four Cu(II) complexes, [Cu(cdnapen)], [Cu(cdnappd)], [Cu(cdMenappd)] and [Cu(cdMeMeOsalpd)], derived from Schiff base ligands with an asymmetric NN′OS coordination sphere have been synthesized. The molecular and the crystal structures have been determined by X-ray diffractometry. The structural results confirm that the complexes are tetra coordinated. The copper (II) ion coordinates to two nitrogen atoms from the imine moiety of the ligand, a sulfur atom from the methyl dithiocarboxylate moiety and a phenolic oxygen atom. The complexes show an unusual tetrahedral distortion to the square-planar geometry around the metal centre in spite of the pseudomacrocyclic skeleton of the ligand. The complexes were further characterized by cyclic voltammetry and electron paramagnetic resonance spectroscopy. The degree of tetrahedral distortion of the complexes appears to be dependent on the number of carbon atoms of the aliphatic bridge and the nature of the coordinating atoms.     

Separation of oligonucleotide phosphorothioate diastereoisomers by pellicular anion-exchange chromatography

Synthetic oligonucleotides (ONs) are often prepared for development of therapeutic candidates. Among the modifications most often incorporated into therapeutic ONs are phosphorothioate (PT) linkages. The PT linkage introduces an additional chiral center at phosphorus to the chiral centers in D-ribose (and 2-deoxy-D-ribose) of the nucleic acid. Therefore, modified linkages can produce a diastereoisomer pair ([Rp] and [Sp]) at each PT linkage. These isomers are of identical length, sequence, charge and mass, and are not reliably separated by most chromatographic approaches (e.g., reversed phase chromatography) unless the ON is very short. Further these isomers are not distinguishable by single-stage mass spectrometry. During chromatography of a purified anti-NGF (nerve growth factor) aptamer containing 37 bases with 2 PT linkages by monolithic pellicular anion-exchange (pAE) column, we observed four components. The four components were postulated to be: (i) distinct folding conformations; (ii) fully and partially athioated aptamers; or (iii) PT diastereoisomers. Fractionation of the components, followed by de- and re-naturation failed to produce the original forms by refolding, eliminating option (i). Mass spectrometry of the fractionated, desalted samples revealed no significant mass differences, eliminating option (ii). Oxidative conversion of the PT to phosphodiester (PO) linkages in each of the purified components produced a single chromatographic peak, co-eluting with authentic PO aptamer, and having the PO aptamer mass. We conclude that the components resolved by pAE chromatography are diastereoisomers arising from the two PT linkages. Hence, pAE chromatography further enhances characterization of ON therapeutics harboring limited PT linkages and having up to 37 bases.     

Fluorides of ruthenium and chromium: Theoretical studies

The electronic and structural characteristics of CrF₅, CrF₄, RuF₅ and RuF₄ were studied. Ab initio (SCF-CI) calculations were performed with different structures and spin states for each complex. The favored conformation always corresponds to the highest multiplicity: doublet for CrF₅ in D_3h, triplet for CrF₄ in T_d, quadruplet for RuF₅ in C_4v and quintuplet for RuF₄ in D_4h symmetry.     

Lithium cation as radical-polymerization catalyst

Density-functional theory (DFT) and ab initio (QCISD and CBS-RAD) calculations suggest that complexation of "naked" lithium cations to olefins favors the addition of alkyl radicals to the double bond over abstraction of an allyllic hydrogen atom. Thus, "naked" lithium cations in nonpolar solvents can catalyze the radical polymerization of olefins by favoring the chain-lengthening reaction over the competing hydrogen-atom extraction, which is competitive in the absence of metal ions. One putative initiation reaction, addition of triplet dioxygen to the double bond, is thermoneutral and has a very low barrier when the oxygen molecule is complexed to a lithium cation. An alternative process, abstraction of an allyllic hydrogen atom to generate the allyl and hydroperoxy radicals, is also strongly favored by complexation of the oxygen to the lithium cation but is less favorable than addition. These results support Michl's recent interpretation of experimentally observed alkene polymerization in the presence of lithium salts of hydrophobic carborane anions.     

Theoretical and experimental study of the acetohydroxamic acid protonation: the solvent effect

The mechanism of the protonation of acetohydroxamic acid is investigated comparing experimental results and ab initio calculations. Experimentally, the UV spectral curves were recorded at different temperatures, at constant dioxane/water concentration, and at very high concentrations of strong mineral acids. The process is followed by monitoring the changes in the UV curves with increasing acid concentration. The molecular structures and the solvation energies were calculated with the RHF, B3LYP, and MP2 methods. The solvent is treated as a continuum of uniform dielectric constant. The isolated molecule of acetohydroxamic acid exhibits two protonation sites, the carbonyl oxygen and the nitrogen atom. In dioxane/water mixture, the RHF calculations predict the existence of a third cation of low stability, where the proton is bonded to the OH oxygen. With the MP2 ab initio calculations, the free energies of the formation processes in solution of the two most stable cations, CH3COH-NHOH+ (O3H+) and CH3CO-NH2OH+ have been evaluated to be -160.2 kcalmol(-1) and -157.6 kcal mol(-1). The carbonyl site is the most active center in solution and in the gas phase. The carbonyl site is also the most active center in the UV measurements. Experimentally, the ionization constant was found to be pK(O3H+) = -2.21 at 298.15 K, after the elimination of the medium effects using the Cox-Yates equation for hight acidity levels. Experiments and ab initio calculations indicate that K(O3H+) decreases with the temperature.