Jeehiun K. Lee

The acidity of uracil and uracil analogs in the gas phase: four surprisingly acidic sites and biological implications

The gas phase acidities of a series of uracil derivatives (1-methyluracil, 3-methyluracil, 6-methyluracil, 5,6-dimethyluracil, and 1,3-dimethyluracil) have been bracketed to provide an understanding of the intrinsic reactivity of uracil. The experiments indicate that in the gas phase, uracil has four sites more acidic than water. Among the uracil analogs, the N1-H sites have ΔHacid values of 331–333 kcal mol−1; the acidity of the N3 sites fall between 347–352 kcal mol−1. The vinylic C6 in 1-methyluracil and 3-methyluracil brackets to 363 kcal mol−1, and 369 kcal mol−1 in 1,3-dimethyluracil; the C5 of 1,3-dimethyluracil brackets to 384 kcal mol−1. Calculations conducted at B3LYP/6-31+G* are in agreement with the experimental values. The bracketing of several of these sites involved utilization of an FTMS protocol to measure the less acidic site in a molecule that has more than one acidic site, establishing the generality of this method. In molecules with multiple acidic sites, only the two most acidic sites were bracketable, which is attributable to a kinetic effect. The measured acidities are in direct contrast to in solution, where the two most acidic sites of uracil (N1 and N3) are indifferentiable. The vinylic C6 site is also particularly acidic, compared to acrolein and pyridine. The biological implications of these results, particularly with respect to enzymes for which uracil is a substrate, are discussed.

EXPLORATION OF PERICYCLIC REACTION TRANSITION STRUCTURES BY QUANTUM MECHANICAL METHODS : COMPETING CONCERTED AND STEPWISE MECHANISMS

Abstract Density functional theory and multiconfigurational SCF calculations have been applied to a number of pericyclic reactions, including cycloadditions, electrocyclizations and sigmatropic shifts. Emphasis is on the competition between concerted and stepwise mechanisms, comparisons of computed and experimental activation energies and isotope effects, and the performance of MP2, CASSCF and DFT calculations. Various diradical processes, such as the vinylcyclopropane rearrangement and cyclobutane isomerizations, were also studied to test the performance of DFT with diradical processes.

Gas-phase acidity studies of dual hydrogen-bonding organic silanols and organocatalysts.

The fundamental properties of a series of organic monosilanols, silanediols, disiloxanediols, and known hydrogen-bonding organocatalysts have been examined in the gas phase using computational and experimental mass spectrometry methods. The organosilicon diol molecules contain dual hydrogen-bonding groups that were designed as potential hosts and hydrogen-bonding catalysts. Newly measured acidities are reported, and implications regarding solvent effects, catalysis, and molecular recognition are discussed.

The mechanism of RNA 5′ capping with NAD + , NADH and desphospho-CoA

The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.

Proton affinities of phosphines versus N-heterocyclic carbenes.

The gas-phase proton affinities of unusually basic phosphines and N-heterocyclic carbenes are compared and contrasted both computationally and experimentally.

DNA stability in the gas versus solution phases: a systematic study of thirty-one duplexes with varying length, sequence, and charge level.

We report herein a systematic mass spectrometric study of a series of thirty-one non-self-complementary, matched, DNA duplexes ranging in size from 5- to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same Tm yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin “charge level” to compare our results with others’ and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O6-methylguanine causes mutations.

2-Pyridone and derivatives: gas-phase acidity, proton affinity, tautomer preference, and leaving group ability.

The fundamental properties of the parent and substituted 2-pyridones (2-pyridone, 3-chloro-2-pyridone, and 3-formyl-2-pyridone) have been examined in the gas phase using computational and experimental mass spectrometry methods. Newly measured acidities and proton affinities are reported and used to ascertain tautomer preference. These particular substrates (as well as additional 3-substituted pyridones) were chosen in order to examine the correlation between leaving group ability and acidity for moieties that allow resonance delocalization versus those that do not, which is discussed herein.

Gas-phase studies of substrates for the DNA mismatch repair enzyme MutY.

The gas-phase thermochemical properties (tautomeric energies, acidity, and proton affinity) have been measured and calculated for adenine and six adenine analogues that were designed to test features of the catalytic mechanism used by the adenine glycosylase MutY. The gas-phase intrinsic properties are correlated to possible excision mechanisms and MutY excision rates to gain insight into the MutY mechanism. The data support a mechanism involving protonation at N7 and hydrogen bonding to N3 of adenine. We also explored the acid-catalyzed (non-enzymatic) depurination of these substrates, which appears to follow a different mechanism than that employed by MutY, which we elucidate using calculations.

Uracil and Thymine Reactivity in the Gas Phase: The SN2 Reaction and Implications for Electron Delocalization in Leaving Groups

The gas-phase substitution reactions of methyl chloride and 1,3-dimethyluracil (at the N1-CH(3)) are examined computationally and experimentally. It is found that, although hydrochloric acid and 3-methyluracil are similar in acidity, the leaving group abilities of chloride and N1-deprotonated 3-methyluracil are not: chloride is a slightly better leaving group. The reason for this difference is most likely related to the electron delocalization in the N1-deprotonated 3-methyluracil anion, which we explore further herein. The leaving group ability of the N1-deprotonated 3-methyluracil anion relative to the N1-deprotonated 3-methylthymine anion is also examined in the context of an enzymatic reaction that cleaves uracil but not thymine from DNA.

An Isolable, Photoswitchable N-Heterocyclic Carbene: On-Demand Reversible Ammonia Activation.

The first isolable, photoswitchable N-heterocyclic carbene was synthesized and found to undergo reversible electrocyclic isomerization upon successive exposure to UV and visible radiation. The UV-induced ring closure afforded substantial changes to the electronic structure of the dithienylethene-based NHC, as evidenced by changes in the corresponding UV/Vis absorption and (13)C NMR spectra. Likewise, molecular orbital calculations revealed diminished electron density at the carbene nucleus upon photocyclization, consistent with the enhanced electrophilicity displayed by the ring-closed form. The photoswitchable NHC was successfully switched between its ring-opened and ring-closed states with high fidelity over multiple cycles. Furthermore, the ring-closed isomer was found to undergo facile N-H bond activation, allowing for the controlled capture and release of ammonia upon cycling between its isomeric states.