Search

Traditional tandem mass spectrometry, including collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), is valuable for nucleic acid characterization, particularly for chemically modified oligonucleotides, which cannot be effectively characterized by enzymatic techniques. However, one disadvantage is secondary fragmentation, which complicates spectral interpretation and reduces sensitivity. In this dissertation, alternative gas-phase fragmentation strategies involving radical ion chemistry; electron capture dissociation (ECD) and electron detachment dissociation (EDD), are explored for nucleic acid characterization. EDD was implemented for the first time on a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer and shown to yield complementary fragmentation pathways compared to CAD and IRMPD, and higher sensitivity than ECD due to the negative ion mode operation. Optimum fragmentation efficiency was obtained at 16-22 eV. Higher oligodeoxynucleotide charge states provided improved EDD data, presumably due to more extended gas-phase structures. Oligoribonucleotide ECD fragmentation patterns were found to be nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. EDD provided complete sequence coverage of hexamer RNAs without nucleobase dependence, suggesting that EDD proceeds following direct electron detachment from the phosphate backbone. FT-IC double resonance (DR) was applied for the first time to characterize IRMPD and EDD fragmentation pathways of oligodeoxynucleotide anions. Results from these experiments suggest that IRMPD proceeds via a similar mechanism as proposed for CAD. Charge reduced radical precursor ions were found to constitute intermediates in EDD of thymidine-containing oligodeoxynucleotides. Furthermore, (a/z minus thymine) ions (a and z type ions correspond to backbone C-O bond cleavage) mainly originate from secondary fragmentation of a/z radical ions for the DNA dT6. ECD and EDD were extended to the characterization of three types of chemically modified oligonucleotides. 2’-methoxy and methylphosphonate modified oligonucleotides underwent limited fragmentation in ECD whereas complete sequence coverage was obtained in most cases from ECD of DNAs containing abasic sites (i. e., a hydrogen has replaced a nucleobase). EDD appeared to be more powerful than both ECD and IRMPD as it provided full sequence coverage and spectra are straightforward to interpret. Overall, EDD shows great promise for analysis of antisense compounds and modified nucleic acids.

The highly functionalized [5.9.5] tricyclic diterpenoids (±)-jatrophatrione and (±)-citlalitrione have been synthesized. The route begins with the tandem anionic oxy-Cope rearrangement/methylation/transannular ene cyclization to set in place a tetracyclic structural framework capable of ready chemical modification. Introduction of a conjugated enone double bond is followed by hydroxyl-directed 1,4-reduction to set the stage for the implementation of a Grob fragmentation. Stereocontrolled intramolecular hydrosilylation allows for subsequent introduction of a cyclic carbonate. Other key transformations include intramolecular hydrosilylation for efficient functionalization of the C12-position, Treibs oxidation for installation of the enone functionality on the southern edge, and syn-elimination with thiocarbonyldiimidazole to generate the dienone functionality. The length of the linear sequence from the point of convergence is 20 steps. Starting from an advanced intermediate, (-)-sclerophytin A and its three diastereomers have been synthesized. The total synthesis unequivocally confirmed the detailed structure of the natural product to be consistent with the recently revised assignment. The absolute stereochemistry of (-)-sclerophytin A was also confirmed by our total synthesis. Attempts have been made toward an enantioselective total synthesis of fomannosin. It was demonstrated that zirconocene-mediated ring-contraction delivered cyclobutanol with the desired stereochemistry, ring-closing metathesis has been shown to be a powerful reaction for synthesis of the strained cyclopentene, and samarium(II) iodide-mediated dehydroxylation of hydroxy ketones was found to be a convenient way for functionalization of the sterically demanding cyclopentene. A full scale fomannosin framework has been assembled. It has been demonstrated that the positioning of a 4-vinyl substituted cyclopentenone ring results in adoption of several different reaction pathways of the ini tial photochemically generated diradicals. Based on the specific environment of the 4-methoxy-4-vinyl-2-cyclopentenones, the diradicals undergo rearrangement, proton abstraction, or dimerization.