Infrared, Raman and UV resonance Raman spectra of adenosine and its 1,3‐15N2, 2–13C, and 8–13C isotopic analogues were measured in neutral aqueous solution (Raman and UV Raman) and in the crystalline state (infrared and Raman). The observed isotopic wavenumber shifts are useful in distinguishing adenine ring vibrations from ribose vibrations. In‐plane modes of the adenine ring are selectively enhanced in UV resonance Raman spectra, which facilitates the assignment of the in‐plane vibrations. In addition to the in‐plane modes, a ribose vibration coupled with adenine in‐plane vibrations was identified in the UV resonance Raman spectra. The fundamental wavenumbers for 22 in‐plane normal modes of the 9‐substituted adenine ring of adenosine in the 1700–250 cm−1 region are proposed. Although the fundamental wavenumbers of adenosine correspond well with those of adenine above 1350 cm−l and below 800 cm−1 the vibrations in the 1350–800 cm−1, region are appreciably affected by the presence of the N‐9—C‐1′ glycosidic bond and the couplings between ribose and adenine ring vibrational motions. The adenosine fundamental wavenumbers and their isotopic shifts reported here may be useful in analysing vibrational spectra of adenine nucleosides and nucleotides and in improving the force field of the 9‐substituted adenine ring.
UV resonance Raman spectra of guanosine and its seven isotope-substituted analogs (2-13 C, 2-15 N, 6-18 O, 7-15 N, 8-13 C, 9-15 N and 1 -13 C) were measured with 257 nm excitation in H 2 O and D 2 O solutions. In-plane vibrations of the guanine ring were selectively enhanced in the UV resonance Raman spectra, and most Raman bands showed significant wavenumber shifts upon isotopic substitution. The observed isotope shifts were used to assign the Raman bands to vibrations of the peripheral sites (N1-H, C2-NH 2 and C6 O), the pyrimidine ring and/or the imidazole ring. Previous assignments for some Raman bands were shown to be inconsistent with the isotopic data and they were revised. Relationships between the vibrational modes and the sensitivities to hydrogen bonding or conformation are discussed for known Raman marker bands. Each hydrogen bond marker arises from a vibration that involves, at least partly, the proton donor or acceptor atom. All the marker bands of glycosidic bond orientation and ribose ring puckering actually involve atomic displacements around the N9-C1 moiety connecting the guanine ring to ribose, permitting vibrational coupling between them. The isotopic wavenumber shifts reported here may be useful in improving the force field for the 9-substituted guanine ring and in interpreting the vibrational spectra of guanine nucleoside and nucleotides.
Isotope-edited Raman spectroscopy, a combination of site-selective isotopic labeling and Raman difference spectroscopy, is a useful method for studying the structure and interaction of individual nucleic acid residues in oligonucleotides. To obtain basic data for applying isotope-edited Raman spectroscopy to guanine residues, we studied the vibrational modes of UV resonance Raman bands of the C8-deuterated guanine ring by examining the wavenumber shifts upon seven isotopic substitutions (2-13 C, 2-15 N, 6-18 O, 7-15 N, 8-13 C, 9-15 N and 1 -13 C). The hydrogen bond sensitivities of the Raman bands were also investigated by comparing the Raman spectra recorded in several solvents of different hydrogen bonding properties. Some of the Raman bands were found to be markers of hydrogen bonding at specific donor or acceptor sites on the guanine ring. The Raman bands, which shift on C8-deuteration, remain in the difference spectrum between the unlabeled and C8-deuterated guanine rings. Among them, a negative peak around 1525 cm −1 and a strong positive/negative peak pair around 1485/1465 cm −1 serve as markers of hydrogen bonding at N7 and C6 O, respectively. Another weak positive/negative peak pair around 1025/1040 cm −1 is sensitive to hydrogen bonding at the proton donor sites (N1 -H and N2 -H 2 ). The applicability of the hydrogen bond markers has been tested by using a 22-mer oligonucleotide duplex containing eight guanine residues and its analog in which a single guanine residue is C8-deuterated. The difference spectrum shows that the hydrogen bonding state of the guanine residue at the labeled position is consistent with the Watson-Crick base pair structure of DNA. Isotope-edited Raman spectroscopy is a useful tool for studying the hydrogen bonding state of selected guanine residues in oligonucleotides.
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