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The absorption and UV resonance Raman spectra of 2′,3′,5′-tri- O -acetyladenosine (AcAdo) were investigated in two non-hydrogen-bonding, three proton acceptor, and four proton donor/acceptor solvents. The molar extinction coefficient at the absorption maximum around 260 nm ( e max ) changed up to ∼10% with change of the solvent. The intensities of 257 nm-excited Raman bands varied dramatically (up to ∼1000%) from solvent to solvent. The e max of UV absorption and the intensities of six Raman bands of AcAdo in the nine solvents were plotted against the Kamlet–Taft solvent parameters α , β , and π *, which represent the hydrogen-bond donor acidity, acceptor basicity, and the polarity–polarizability of the solvent, respectively. Both e max and the Raman band intensities increased with α . Similar but weaker correlations were seen for β . π * had the least influence. These observations strongly suggest that hydrogen bonding is the main factor that affects the intensities of UV absorption and resonance Raman scattering. Multiple regression analysis has shown that the correlations with α and β are improved by taking linear combinations of Raman band intensities. We propose a new method to evaluate the environmental α and β values of the adenine ring from the relative intensities of three strong Raman bands at ∼1500, 1480, and 1330 cm −1 . UV resonance Raman intensities of adenine ring vibrations are useful in analyzing the strength of hydrogen bonding at the proton acceptor (N1, N3, and N7) and donor (C6–NH 2 ) sites.

Raman spectroscopy has been combined with site-selective isotopic labeling techniques to obtain structural information on a selected nucleic acid residue in oligonucleotides. In the difference spectrum between the unlabeled and site-selectively labeled oligonucleotides, the Raman signals from the residue at the labeled position show up, while the signals from the residues at unlabeled positions are canceled out. To demonstrate the utility of this new method, we have prepared a self-complementary tetradecamer DNA, d(AGTGCTCGAGCACT)2, containing single C8-deuterated adenine at position 9 (A9) or 12 (A12). UV (257 nm) resonance Raman difference spectra between the unlabeled and labeled oligonucleotides in solution reveal slightly different microenvironments around A9 and A12 as expected for a double-stranded helical structure of the tetradecamer DNA. In the presence of an antitumor antibiotic, actinomycin D, which intercalates into the 5′-GC-3′ sequence of DNA, the Raman signals of A9 on the 5′-side of the intercalation site become significantly weaker, indicating an increased base stacking with adjacent guanine bases. In contrast, the Raman signals of A12 on the 3′-side are not affected by the binding of actinomycin D. This observation provides the first experimental evidence that the intercalation of actinomycin D induces an asymmetric structural alteration of DNA in solution.

Ultraviolet (UV) resonance Raman spectra of an acetyl derivative of adenosine and its C8-deuterated analog were measured in solvents with varied hydrogen bonding properties. The Raman spectrum of the acetyl derivative in H 2 O solution is almost identical to that of adenosine in H 2 O solution, indicating that the acetyl derivative is a good model for the adenine nucleotide in DNA. Most of the Raman bands increase or decrease in wavenumber upon hydrogen bonding at the proton-donor (C6-NH 2 ) and proton-acceptor sites (N1, N3 and N7) of the adenine ring. Among them, the ν 1 , ν 2 , ν 3 , ν 5 and ν 9 bands of the adenine ring and the ν 1 ′, ν 2 ′, ν 3 ′, ν 5 ′, ν 7 ′ and ν 9 ′ bands of the C8-deuterated adenine ring show wavenumber shifts larger than 5 cm −1 . Relative intensities of some Raman bands change as well. The wavenumber and intensity changes can be used as markers of hydrogen bonding at the proton donor and/or acceptor sites of the adenine ring. The Raman spectral difference between the non-deuterated and C8-D labeled adenine rings reflects the hydrogen-bonding state very sensitively and is expected to be useful in studying the hydrogen-bonding state of a particular adenine residue in oligonucleotides by a combination of site-selective C8-D labeling and UV resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra of 2′-deoxy-3′,5′-bis(triisopropylsilyl)guanosine (TPS-dGuo) were recorded in non-hydrogen bonding, proton acceptor, and proton donor/acceptor solvents. Raman spectral changes observed on going from inert to proton acceptor solvents were ascribed to the hydrogen bonding at the proton donor sites of the guanine ring (N1H and C2NH 2 ), and the spectral changes associated with the solvent change from proton acceptor to donor/acceptor were ascribed to the hydrogen bonding at the proton acceptor sites (N3, C6O, and N7). A Raman band appearing at 1624 cm −1 in inert solvents is assigned mainly to the NH 2 scissors mode and its frequency changes to ≈ 1640 cm −1 in acceptor solvents, reflecting the hydrogen bonding at C2NH 2 . Another band at 1581 cm −1 , arising largely from the N1H bend, shows an upshift of ≈ 10 cm −1 upon hydrogen bonding at either N1H or acceptor sites. Hydrogen bonding at the acceptor sites also produces frequency shifts of other Raman bands (at 1710, 1565, 1528, 1481, and 1154 cm −1 in 1,2-dichloroethane solution). Among the Raman bands listed above, the 1710 cm −1 band due to the C6O stretch decreases in frequency, whereas the others increase. The downshift of the C6O stretching frequency is correlated with the strength of hydrogen bonding at C6O. The frequency of the 1481 cm −1 band increases with a decrease of the C6O stretching frequency, indicating that the 1481 cm −1 band is also a marker of hydrogen bonding at C6O. This finding is in sharp contrast to the previously proposed correlation with the hydrogen bonding at N7. The 1565 cm −1 band is assigned to a vibration mainly involving the N1C2N3 linkage, and its frequency increases with increasing strength of the hydrogen bond at N3. Three bands around 1315, 1180, and 1030 cm −1 , which are known to be sensitive to the ribose ring puckering and glycosidic bond orientation, also show small frequency changes upon hydrogen bonding. The Raman marker bands for the hydrogen bonds at C2NH 2 , N1H, C6O, and N3 are expected to be useful in studying the structures of nucleic acids and nucleic acid-protein complexes containing guanine residues.

Ultraviolet resonance Raman spectra (266-nm excitation) were studied for adenine, uracil and thymine derivatives in several solvents at low concentration (0.5−10 mM) where hydrogen bonding between solute molecules is negligible. The Raman spectra patterns depend critically on the proton-accepting/donating properties of the solvents. Raman bands which show large solvent effects are expected to be useful in studying the hydrogen-bonding states of the nucleic acid bases.