Nitrous oxide as a 1,3-dipole: a theoretical study of its cycloaddition mechanism.

The 1,3-dipolar cycloadditions of nitrous oxide and substituted alkynes have been studied at the B3LYP/6-31G(d,p) level. The reaction is controlled by LUMO (dipole)--HOMO (dipolarofile) and involves aromatic transition structures. The shape of the potential energy surface and the regioselectivity are not affected by the polarity of the solvents, except in the case of N2O + HC triple bond CSiH3. Different reactivity criteria including FMO coefficients product C, local softness differences Delta, magnetic susceptibility anisotropy chi(anis), and nucleus-independent chemical shifts NICS were used to predict the regioselectivity in all studied cases; the C, Delta criteria turn out to give the best results among them. The aromaticity of the transition structure is not a factor in determining the regiochemistry of the cycloaddtition reactions.

Ab initio molecular orbital evaluation of the hydrogen bond energy of base pairs formed between substituted 1-methylcytosine derivatives and 9-methylguanine.

The substitution effect on hydrogen bond energy of the Watson-Crick type base pair between 9-methylguanine (G) and chemically modified 1-methylcytosine (CX) derivatives was evaluated by ab initio molecular orbital theory. A remarkable trend was observed in the substitution effect of the hydrogen bond stability: Cytosine derivatives possessing an electron-donating group form stable base pairs with guanine. However, both the hydrogen bond distance and the charge distribution were not good indexes for the hydrogen bond status in CX-G base pairing.

Theoretical and experimental study of the vibrational spectrum of N-acetyl-L-alanine.

The energies, vibrational frequencies and IR intensities of cis- and trans-N-acetyl-L-alanine (NAAL) are computed using the density functional theory (B3LYP) combined with the 6-311G(d, p) basis set. The trans conformer is characterized by an intramolecular NH ... O hydrogen bond leading to the formation of a five-membered ring and is by 23 kJ mol(-1) more stable than the cis conformer. The difference between the vibrational frequencies and IR intensities computed for the two conformers is discussed. The IR spectra at different temperatures and the Raman spectra of solid NAAL and its deuterated counterpart are investigated and discussed. The frequencies of the v(OH) vibration and the isotopic ratio suggest the formation of short OH ... O hydrogen bonds in the solid state. The NH group seems also to be involved in a weak hydrogen bond.

Evaluation of the hydrogen bond energy of base pairs formed between substituted 9-methyladenine derivatives and 1-methyluracil by use of molecular orbital theory.

Systematic substituent effects on the stability of the hydrogen bonding between substituted 9-methyladenine derivatives (Ax) and 1-methyluracil (U) were studied by ab initio molecular orbital theory. Predicted substituent effects on the hydrogen bond energies of Ax-U base pairs were in good agreement with those observed for experimental binding constants. Ab initio calculation is effective for evaluation of the stability of the hydrogen-bonding pairs of chemically modified nucleic acid base analogues. In contrast to the substitution effect of uracil on hydrogen bond energies of A-Ux base pairs, it is difficult to systematically interpret the substitution effect of adenine derivatives for Ax-U base pairs.

The relationship between metal-metal distance of two metal ions chelated complex and RNA cleavage activity.

We prepared a series of ligands possessing two binding sites for metal coordination: in each ligand molecule, two binding sites with the same functionality (2,2'-dipicolylamino group) were placed at the of various methyl arenes. Thus, the distances between the metal binding sites were different from ligand to ligand. We examined the rate of the hydrolysis of RNA dimer catalyzed by La3+ ion binuclear complexes of the ligands. The catalytic activity of the binuclear complexes increased as the distance between the metal binding sites was decreased.

Efficiency enhancement of long-range energy transfer by sequential multistep FRET using fluorescence labeled DNA.

The efficiency of long-range (ca. 80 A) fluorescence energy transfer was enhanced about 1.5 times by a third chromophore located midway between two chromophores. A third chromophore should act like a relay station in sequential multistep energy transfer.

Theoretical analyses on the role of metal cations in RNA cleavage processes.

Previously, we found that locations of metal cations in biomolecules, such as proteins and nucleotides, were in accord with predictions based on electrostatic-potential calculations of the nearest polar functional groups. Electrostatic potential of a cyclic oxyphosphorane dianion 2, which is a model compound for an intermediate or a transition state for the hammerhead ribozyme reaction, indicates that Mg2+ coordinations at the regions between axial (bridging) and equatorial (phosphoryl) oxygens are most favorable. If we consider the fact that negative charge is concentrated on two of the equatorial phosphoryl oxygens in the transition state 2, the axial-equatorial coordination is an unexpected result. Nevertheless, experimental results also support such an axial-equatorial coordination. Therefore, hammerhead ribozyme is a metalloenzyme in which at least one Mg2+ ion acts as a base and possibly a second Mg2+ ion acts as a Lewis acid. The double Mg2+ ion catalysis may be a common mechanism among various types of ribozyme and some protein enzymes as well.

Theoretical analyses on the role of Mg2+ ions in ribozyme reactions.

To elucidate the role of the Mg2+ ion in ribozyme reactions, we carried out ab initio molecular orbital investigations on dianionic trimethoxyphosphorane A and its Mg2+ complex (overall a neutral molecule) as a model system for the reaction center of Tetrahymena-type ribozyme. Although dianionic oxyphosphorane A concentrates its negative charges on the equatorial phosphoryl oxygens, the coordination of the Mg2+ ion between these two oxygens is unlikely. Geometry optimizations of the complex and the electrostatic potential of A both suggest that Mg2+ coordination preferably occurs in the region between the axial oxygen and the equatorial phosphoryl oxygen. The considerations of electrostatic potential rationalize the geometries of carboxylate-metal and phosphate-metal interactions extracted from the Cambridge Structural Database as well. Consequently, the Mg2+ ion at the active site of Tetrahymena-type ribozyme most likely lies in the regions between the axial and equatorial oxygens. The axial-equatorial coordinations of Mg2+ ions conceivably increase the electronegativities of the axial oxygens and facilitate cleavage of the phosphodiester bond located at the junction of the intron and the exon. It is thus likely that the Mg2+ ions play the key role in the phosphodiester cleavage reactions mediated by ribozymes.

RNA hydrolysis via an oxyphosphorane intermediate.

From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5') bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.

Pentacoordinate oxyphosphorane intermediate always exists in aqueous solution.

Gas-phase ab initio calculations indicate that dianionic pentacoordinate oxyphosphoranes do not have a kinetically meaningful intermediate. The simplest oxyphosphorane PO5H3(2-) has the least tendency to have a pentacoordinate intermediate. However, it does have a pentacoordinate intermediate when it is solvated with six water molecules. These results support the hypothesis that the phosphoryl transfer reactions take place via pentacoordinate intermediate not only in acidic but also in basic media.

Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base-catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.

Preferential chelation of cationic ligands to axial-equatorial oxygens over equatorial-equatorial dianionic oxygens: implication to the mechanism of action of ribozymes.

Effects of chelation of H2O, H+, and Mg2+ to two kinds of potential pentacoordinate intermediates of ribozyme reactions were investigated by ab initio molecular orbital calculations. Unexpectedly, in all cases examined, axial-equatorial chelations were found to be more stabilizing than equatorial-equatorial chelations. These results support the hypothesis that Mg2+ ion is bound to the equatorial phosphoryl oxygen and the axial leaving/attacking oxygen in the transition state of ribozyme reactions.

Ab initio investigations on the intermediates of RNA cleaving processes.

Ab initio molecular orbital calculations have been carried out on acyclic oxyphosphoranes in order to elucidate the origin of previously calculated energy differences of cyclic counterparts of RNA cleaving reactions. It appears that the orientation of the equatorial methoxyl group has pronounced effects in energy on the transition states than the metastable intermediates.

In vivo RNA transcript-releasing plasmid possessing a universal pseudo-terminator by means of artificial ribozymes.

RNA transcript-releasing plasmid has been constructed by means of artificial hammerhead ribozymes. In this specific construct of pGENE8459v3 the ribozyme targeted for SFL1 gene (a yeast suppressor gene for flocculation) was fused between two other ribozymes called 5'-processing and 3'-processing ribozymes. Since the "Ribozyme for SFL1" portion (cassette) can be replaced by other RNA sequences, it is now possible to produce any RNAs with defined 5'- and 3'- ends.

Computational studies on pterins and speculations on the mechanism of action of dihydrofolate reductase.

The significance of the enol form of the pterin ring in enzymatic reduction of dihydrofolate by DHFR is discussed on the basis of the results of ab initio calculations carried out on the keto/enol tautomers of 6-methyl-7, 8-dihydropterin as the model compound for the natural substrate, dihydrofolate.