Isomer-Selective Generation of Cytidine Radicals via Wavelength-Regulated Photodissociation

Open AccessCCS ChemistryRESEARCH ARTICLES27 Jul 2024Isomer-Selective Generation of Cytidine Radicals via Wavelength-Regulated Photodissociation Luyang Jiao, Mengying Du, Shiyin Xu, Min Kou and Xianglei Kong Luyang Jiao The State Key Laboratory and Institute of Elemento-Organic Chemistry, Frontiers Science Center for New Organic Matter, Collage of Chemistry, Nankai University, Tianjin 300071 Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 , Mengying Du The State Key Laboratory and Institute of Elemento-Organic Chemistry, Frontiers Science Center for New Organic Matter, Collage of Chemistry, Nankai University, Tianjin 300071 Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 , Shiyin Xu The State Key Laboratory and Institute of Elemento-Organic Chemistry, Frontiers Science Center for New Organic Matter, Collage of Chemistry, Nankai University, Tianjin 300071 Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 , Min Kou The State Key Laboratory and Institute of Elemento-Organic Chemistry, Frontiers Science Center for New Organic Matter, Collage of Chemistry, Nankai University, Tianjin 300071 Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 and Xianglei Kong *Corresponding author: E-mail Address: [email protected] The State Key Laboratory and Institute of Elemento-Organic Chemistry, Frontiers Science Center for New Organic Matter, Collage of Chemistry, Nankai University, Tianjin 300071 Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 Cite this: CCS Chemistry. 2024;0:1–13https://doi.org/10.31635/ccschem.024.202404201 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail It is well-known that nucleoside-derived radicals play crucial roles in cellular activity. However, due to the instability of the radicals and their widespread isomerization, experimental research on their generation and structural characterization has been a challenge for a long time. Herein, we provide a unique approach to selectively generate the isomers of nucleoside radicals in the gas phase. Through the process of ultraviolet photodissociation (UVPD) at 285 and 245 nm, we were able to selectively produce O2 and C5 isomers of cytidine radicals from a single precursor ion of protonated 5-I-cytidine. These radical isomers were structurally characterized by a combination of infrared multiphoton dissociation (IRMPD) spectroscopy of the mass-selected ions and systematic computations. The structural characteristics of cytidine radical isomers such as the anti/syn glycosidic link and the North/South sugar ring conformation were disclosed. The key to selectivity lay in whether the excess energy of the precursor ions after absorbing one ultraviolet photon and undergoing the homo-cleavage of the C–I bond could overcome the corresponding energy barrier of the isomerization. This method provides an effective way for the selective generation of radical isomers with rich flexibility by wavelength-regulated UVPD and reasonable introductions of C–I bonds in the precursors. Download figure Download PowerPoint Introduction The exposure of RNA and DNA to high-energy radiation can result in a variety of physical and chemical changes, including strand breakage, mutation, and nucleic acid damage. It is well known that nucleobase- or nucleoside-derived radicals are crucial transient intermediates in these processes.1–3 However, the precise structural identification of these species is still challenging. While the characterization of the nucleobase radicals in solution and the solid state has been studied by electron paramagnetic resonance (EPR) spectroscopy or femtosecond time-resolved spectroscopy,4–10 the information deduced from gas-phase studies is also of particular importance. Gas-phase studies are valuable and unique for obtaining information on the nucleobase radical's intrinsic chemical properties by eliminating the influence of solvents, counterions, and surfaces, allowing for a direct comparison between experimental data and quantum chemical calculations.11,12 In such experiments, mass spectrometry provides a powerful method to perform structure and kinetic studies of key species relative to nucleic acids. Mass-selected ions can be isolated and further studied by methods of action spectroscopy, including infrared multiphoton dissociation (IRMPD), ultraviolet photodissociation (UVPD), and flexible combinations of IRMPD and UVPD.13–16 Compared to the numerous studies of the related nonradical species of nucleic acids, including nucleosides,17–20 nucleotides,21–23 nucleobase dimers,24 and metal-bound species,25–28 research on their relative radicals is much more difficult. One of the main tasks is figuring out how to establish an effective way to generate these unstable species. If too many cumbersome steps are needed to generate the goal radicals from some precursor ions in the mass spectrometer, the space for scrutinizing these radicals based on multistage tandem mass spectrometry will be very limited. Meanwhile, the structural identification of radicals is difficult due to the existing multi-isomers and possible isomerization accompanied by generation processes. These two points together make the creation of radical isomers particularly challenging. To face the challenge, several mass spectrometry-based methods have been developed in the past twenty years. Tureček and coworkers12,29–32 have conducted long-term research in this field. In one of their works, different precursor cations were designed and prepared in the mass spectrometer for two special isomeric uracil radicals, and then accelerated and discharged by a glancing collision with an electron donor molecule. Two neutral radical isomers were formed, which were further studied by the method of neutralization–reionization mass spectrometry.12 Similarly, they also reported the targeted generation of two key hydrogen-atom adducts of methylcytosine.12 Recently, to study neutral radicals in the gas phase more conveniently, they developed a new method of tagging radicals with a fixed-charged group.33–35 Using this novel method, charge-tagged radicals, including those of cytidine,33 adenosine,34 and guanosine,35 have been systematically studied using a combination of UV–vis photodissociation spectroscopy and theoretical calculations. Nucleobase cation radicals were also generated in other ways. O'Hair et al.36,37 have revealed that tandem mass spectrometry of ternary transition metal complexes generated by electrospray ionization (ESI) could be applied as an effective means of forming gas phase cation radicals of the constituents of nucleic acids. These cation radicals can be further studied by tandem mass spectrometry or photodissociation spectroscopy. For example, the radical cation of cytosine was generated by dissociative oxidation from a ternary Cu(II) complex in the gas phase.38 The radical was further characterized experimentally by IRMPD spectroscopy, UV–Vis photodissociation spectroscopy, and ion–molecule reactions. Even with these efforts, gas-phase radical research remains quite difficult, particularly when it comes to developing reliable techniques for precisely producing special radical isomers. Here, we combine a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry with both tunable infrared and ultraviolet lasers to achieve an efficient method for the generation, selection, manipulation, and spectroscopic study of cation radicals. We complement the structure elucidation of the generated cytidine cation radicals using IRMPD spectroscopy combined with density functional theory (DFT) calculations. Three-dimensional structural features of cytidine radicals are determined, including the conformation of the sugar ring in North or South style, and the glycosidic bond in anti or syn connection.39 The structures of radicals generated by UV lasers at different wavelengths, are conformed to structural isomers with the help of their IRMPD spectra and calculations. Experimental Methods Experiments were performed on a Varian 7.0 T FT-ICR mass spectrometer (IonSpec, Varian Inc., Lake Forest, CA, USA) combined with a double-beam laser system. As shown in Figure 1a, the instrumental setup was similar to the one described previously, except that a parabolic mirror was applied here to replace the two focusing lenses for IR and UV laser beams to eliminate chromatic aberration.14,15 Briefly, two commercially available tunable lasers, the M Squared OPO (Firefly-IR, M Squared, Glasgow, UK) and the EKSPLA UV OPO laser (NT-342C, EKSPLA, Vilnius, Lithuania), were applied without modification. The FT-ICR mass spectrometer was modified to introduce laser beams into the cell. The irradiation times of the two lasers were separately controlled by two mechanical shutters controlled by a multichannel controller (SSH-C4RA, Sigma-Koki, Tokyo, Japan), which was triggered by a pulse generator (DG535, Stanford, CA, USA). The ions were generated by the Z-spray ESI source. The process of obtaining the IRMPD spectra of radical cations is shown in Figure 1b. Briefly, the precursor ion of protonated 5-I-cytidine was generated by ESI and injected into the ICR cell, followed by isolation using the method of stored waveform inverse Fourier transform (SWIFT).40 Then the radical cation was generated through the cleavage of the C–I bond via the irradiation of a UV laser. The wavelength of the UV laser could be adjusted, and the yield spectrum for the radical cations was recorded. After its generation, the radical cation was further selected and characterized by collision-induced dissociation (CID)/IRMPD/UVPD. By tuning the output wavelengths of the IR OPO laser, the IRMPD spectrum of the target radical cation was recorded. For the IRMPD spectra reported here, the intensities were calculated according to IIR = −ln (Ip/(∑Ifi + Ip)), where Ip and Ifi are the intensities of the parent ion and the ith product ion in the corresponding experiments. More experimental and calculational details can be found in the Supporting Information. Figure 1 | (a) The experimental setup for the cytidine radicals generation via wavelength-regulated photodissociation; (b) steps involved. Download figure Download PowerPoint Results and Discussion The generation of the radical ion [Cyt]•+ and tandem mass spectrometry study To generate the radical cation of cytidine, 5-I-cytidine was selected as the precursor. The protonated ion of [I-Cyt+H]+ was readily generated through ESI and isolated in the cell of the FT-ICR mass spectrometer with the SWIFT method ( Supporting Information Figure S1a). The irradiation of an IR laser on the precursor ion only produced the fragment ion at m/z 237.95, corresponding to the cleavage of the glycosidic bond and the subsequent loss of the unit C5H8O4 (a typical IRMPD mass spectrum obtained at 3446 cm−1 is shown in Figure 2a), while the irradiation by a UV laser showed different results (Figures 2b,c). The 285 nm UVPD mass spectrum of [I-Cyt+H]+ was characterized by a strong peak at m/z 243.09, which corresponded to the cytidine radical cation formed by a direct loss of the I atom. The fragment ion at m/z 237.95, due to the loss of C5H8O4, was also observed, along with the fragment ion at m/z 111.04, formed by the elimination of a unit of C5H8O4 from the radical cation at m/z 243.09. For the 245 nm UVPD mass spectrum of [I-Cyt+H]+, a new fragment ion at m/z 112.05 was observed, formed by the loss of C5H7O4 from the radical cation. Figure 2 | Photodissociation mass spectra of [I-Cyt+H]+ at m/z 369.99 generated by (a) an IR irradiation at 3446 cm−1 for 8s, (b) a UV irradiation at 245 nm for 4s, and (c) a UV irradiation at 285 nm for 4s. Photodissociation mass spectra of [Cyt]•+ at m/z 243.09 generated by (d) UV–UV sequential irradiations at 285 nm for 4s twice, (e) UV-IR sequential irradiations at 285 nm for 4s and IR irradiation at 3446 cm−1 for 8s, and (f) UV-IR sequential irradiations at 245 nm for 4s and IR irradiation at 3574 cm−1 for 5s. During the two laser irradiations in (d–f), a clear isolation of the cation radicals was applied in all cases. The sites of cleavage from each MS/MS process are also shown in the subfigs, in which the fragment ions at m/z 112.05 and 111.04 were formed by the cleavages at the same position with and without hydrogen migration. It should be mentioned that the structure of the parent ion in each MS/MS process may be more complicated than what is displayed here due to the existence of their tautomers, which was discussed in this article. Download figure Download PowerPoint The cytidine radical cation was further isolated with the SWIFT method ( Supporting Information Figure S1c) and characterized by UVPD/IRMPD-MS3 spectra. To differentiate the radical ions of [Cyt]•+ generated by different UV wavelengths (which might have different structures), the experimentally applied UV wavelengths are expressed in the following parentheses (in nm) when these radical ions are mentioned herein. For example, the ions of [Cyt]•+ generated at 285 and 245 nm are expressed as [Cyt]•+ (285) and [Cyt]•+ (245), respectively. As shown in Figure 2d, in the UV–UV experiment, the 285 nm UV irradiation on the isolated radical ion of [Cyt]•+ (285) resulted in the formation of the single fragment ion at m/z 111.04. However, while the IR laser was applied, the observed spectrum was characterized by the dominant peak at m/z 112.05 (Figure 2e). Interestingly, in the IRMPD mass spectra of the radical ion of [Cyt]•+ (245), only the fragment ion at m/z 112.05 was observed (one example is shown in Figure 2f). If the last step activation was performed using the CID method, similar results were observed ( Supporting Information Figure S1b), strongly suggesting these two radical ions had different structures. In fact, radical ions of [Cyt]•+ could be generated by the UVPD of the precursor ion of [I-Cyt + H]+ under a wild range of wavelengths. To identify the dependence of the yield of the radical ion on the wavelength, the yield spectrum of [Cyt]•+ depending on the UV wavelength was recorded, along with the UV dissociation spectrum of the precursor ion. As shown in Figure 3a,b, the precursor ion of [I-Cyt+H]+ exerted a wide absorption range between 210 and 348 nm, and the maximum was located at 290 nm. Meanwhile, the radical ion of [Cyt]•+ could only be generated in the range of 230–348 nm. The change in the dissociation channel caused the precursor ion to no longer produce the radical ion after absorbing ultraviolet photons in the range of 210–230 nm (one example is shown in Supporting Information Figure S1d). Figure 3 | (a) The peak intensities of [Cyt]•+ generated from UVPD of the precursor ion of [I-Cyt+H]+ as a function of UV wavelength in the range of 210–350 nm. (b) Experimental UVPD spectra of the precursor ion of [I-Cyt+H]+. Download figure Download PowerPoint IRMPD spectra of the precursor and radical ions As pointed out in previous studies by different research groups, the identification of the structural minima of these species is difficult.19,20 First, there are several possible protonation sites for the precursor ion [I-Cyt+H]+. Among all possible positions, the three most important sites are the ones on the pyrimidine ring: O2, N3, and N4, shown in Scheme 1. Second, analogous tautomers should also be taken into account since structural tautomerisms between amino and imine groups, or ketone and enol groups, are common and might be significant in gene mutation and biological evolution. These isomers are also listed in Scheme 1. Third, the conformation effects should be considered. The ribose moieties can exist in two puckered conformations, named North and South, in RNA and DNA molecules. It has been found that the North or South conformation plays an important role in modulating RNA cleavage via internal transesterification.41 Thus, the effects of the conformation of the sugar ring should be taken seriously, and the nucleobase orientation (anti or syn) is a structural element that needs to be considered. Besides, intramolecular H-bonding, which affects the stability of the whole ion, cannot be ignored. Scheme 1 | Different isomers and tautomers of [I-Cyt+H]+ are presented in which labels of the atoms are shown in the first example. Download figure Download PowerPoint In order to address their structural complexity, IRMPD spectroscopy was applied to characterize the protonated ion of [I-Cyt+H]+; its IRMPD spectrum exhibited a sharp peak centered at 3440 cm−1 and three accompanied peaks in the region of 3500–3700 cm−1 with relatively low intensities. In the range of 2900–3200 cm−1, it exhibited two weak peaks centered at 2970 and 3090 cm−1 (Figure 4a). The IRMPD action spectroscopy of protonated 5-I-cytidine was consistent with that of the protonated cytidine reported previously by Rodgers and coworkers.21 It was also similar to that of the singly hydrated protonated cytosine reported by Philippe et al.,16 except the latter has more complex peaks in the 3500–3700 cm−1.42 A comparison of the IRMPD spectra of [I-Cyt+H]+ and [Cyt]•+ (285) showed that they were quite close but could still be clearly distinguished from each other. As shown in Figure 4a,b, it seems that both peaks at 3440 and 3556 cm−1 in the spectrum of [I-Cyt+H]+ are blueshifted by 25 cm−1 for [Cyt]•+ (285), while the other peaks have no obvious change in position. For the radical ion [Cyt]•+ (245), its IRMPD spectrum is much different; the peak at 3614 cm−1 disappeared, while the 3440 cm−1 peak became broader than those of the former two (Figure 4c). Figure 4 | Experimental IRMPD spectra of (a) [I-Cyt+H]+ and (b, c) [Cyt]•+ generated by 4s UV irradiation from the precursor ion [I-Cyt+H]+ at 285 and 245 nm. The insets show magnified views of the weak peaks in the region of 2900–3200 cm−1. Download figure Download PowerPoint The IRMPD spectra of [Cyt]•+ generated under different UV wavelengths were also recorded in the range of 2800–3800 cm−1 by scanning the wavelength of the IR laser in the IRMPD-MS3 experiments. As shown in Figure 2, the radicals generated at 245 and 285 nm might have different structures. For the convenience of comparison, the IRMPD spectroscopy experiments were performed for the two species of [Cyt]•+ (285) and [Cyt]•+ (245), generated by the UV irradiation of the [I-Cyt+H]+ at 285 and 245 nm, respectively. The two UV wavelengths were also very close to the two peaks in the UV yield spectrum of the radical cations (Figure 3a), indicating their possible different origins. To better understand the results, the IRMPD spectrum of the precursor ion [I-Cyt+H]+ was recorded in the same region. Structure of the precursor ion Based on a systematic search of its structures, the most stable isomers of [I-Cyt+H]+ belonging to different structural conformation types were obtained at the level of B3LYP/6-311+G(d,p). The structures of these isomers are shown in Supporting Information Figures S2–S4 and their energies are listed in Supporting Information Table S1. The four most important isomers next to be discussed are also shown in Figure 5. Among all of these structures, the isomer M-N3a which has a protonation position at N3, has the lowest energy. This structure is characterized by a North-conformation of the sugar ring and an anti-orientation of the glycosidic bond. Its South-counterpart (M-N3b) has a slightly higher energy (0.1 kcal/mol at 0 K) and almost the same Gibbs free energy at 298 K, and its syn-counterpart (M-N3h) has electronic and free Gibbs energies 4.6 and 4.12 kcal/mol higher, respectively. For these isomers, it can be found that the energy difference between the South- and North-conformation pairs is low, while that between anti- and syn-conformation pairs is much higher. Supporting Information Figure S5 shows the structural differences between these two conformations. The anticonformations are typical of the dominant type. For isomers with the protonation site at O2, the most stable isomer, M-O2a, characterized by its South-anti-conformation, has very close electronic and free energies than those of M-N3a. For M-O2a, its North-counterpart M-O2b and anti-counterpart M-O2e, have energies 0.7 and 1.3 kcal/mol higher, respectively. For isomers with the protonation site at N4, their energies are much higher. The most stable one, M-N4a, with a South-anti-conformation, has an energy 27.7 kcal/mol higher than M-N3a. For those tautomers of MT1-3, their energies are at least 24.9 kcal/mol higher than that of M-N3a, thus their contributions can be neglected. These calculation results are consistent with previous protonated cytidine findings reported by other groups.18,20 For example, in Wu et al. calculation at the level of B3LYP/6-311+G(2d,2p), the reported structure N3A with the protonation site at N3 was the most stable conformer of the protonated cytidine, with free energy (at 298 K) of 1.9 kJ/mol, lower than that of the second most stable conformer O2A with the protonation site at O2. However, the latter conformer turned out to be the most stable one, with a free energy of 4.6 kJ/mol, lower than that of N3A at the level of MP2(full)/6-311+G(2d,2p). Figure 5 | Experimental and theoretical IR spectra of [I-Cyt+H]+: (a) experimental spectrum, (b–e) theoretical spectra of some low-energy structures with protonation sites at N3 and O2, which were calculated on the level of B3LYP/6-311+G(d,p). Their corresponding structures are also shown in the blanks above their spectra. Download figure Download PowerPoint Figure 5 shows the theoretical IR spectra of the selected lowest-energy structures of [I-Cyt + H]+, along with the experimental IRMPD spectrum. As discussed above, experimental peaks were found at 2970, 3090, 3440, 3556, 3622, and 3714 cm−1 (Figure 5a). The peak at 3714 cm−1, which originated from the stretching vibration of free –OH groups, was found in all predicted spectra, as shown in Figure 5b–e. For the sharp peak observed at 3440 cm−1, the spectrum of isomer M-O2a gave the best prediction at 3444 cm−1, contributed by the symmetrical stretching vibration of –NH2. The corresponding antisymmetric stretching vibration of NH2 had a band at 3565 cm−1, which was also in good agreement with the experimental peak at 3556 cm−1. Two close peaks at 3624 and 3637 cm−1, due to the vibration of OH on the pyrimidine ring and the weakly H-bonded OH on the ring of sugar, exhibited reasonable agreement with the measured broad peak centered at 3626 cm−1. On the other hand, the H-bonding of C(6)H···O (as shown in Figure 5b,c), contributed to an enhanced vibrotational frequency of C(6)-H, which blueshifted to 3073 cm−1, along with an experimental weak peak at 3090 cm−1. Compared with M-O2a and M-O2b, they were both antioriented but with different sugar puckers, with quite similar spectra. Considering that M-O2a was more stable than M-O2b by 0.7 kcal/mol, it was believed that the former made a more significant contribution. For the isomers of M-N3a and M-N3b, their existence could be ruled out, as their contributions were not as important as that of M-O2a because the predicted double peaks in the region of 3390–3460 cm−1 due to the symmetrical stretching mode of NH2 and the stretching mode of NH on the pyrimidine ring were poorly consistent with the experimental spectrum. To further check this, single-point energy calculations on the CCSD(T)/6-31+G(d)/def2qzvp were also performed for the two most stable isomers of M-O2a and M-N3a. The results showed that M-O2a was more stable than M-N3a by 0.78 kcal/mol. Comparisons between the measured IRMPD spectrum and the predicted spectra of other high-energy isomers with different protonation sites or tautomers suggested that they could be neglected in the experiments ( Supporting Information Figure S6). Briefly, the results show that the isomer M-O2a should be the main isomer of the molecular ions observed in this experiment. Considering that the formation of the radical ion was induced by the homo-cleavage of the C–I bond due to the irradiation of the UV laser, the radical ion of [Cyt]•+ might have a structure similar to that of M-O2a. On the other hand, the mobility of the H atom also made other structures possible. For convenience of description, the radical isomers were classified and named according to the position of the H atom. As shown in Scheme 2, these isomers were R-O2 (without migration), R-N3, R-N4, and R-C5, and their relative tautomers, RT1, RT2, and RT3, were also considered. Scheme 2 | Different isomers and tautomers of the cation radical of [Cyt]•+ are presented. Download figure Download PowerPoint Structure of the radical isomers With a similar theoretical structure search strategy to that applied for the molecular ion, the isomers shown in Scheme 2 were also studied with consideration of the conformation (anti/syn, North/South) and the intramolecular H-bonding effects. The structures of the top low-energy isomers with different conformational types are shown in Supporting Information Figures S7–S9, and their energies obtained at the level of B3LYP/6-311+G(d,p) are summarized in Supporting Information Table S2. Among them, the most stable isomer was R-C5a, which has a (south, anti)-conformation. Its syn-counterpart, R-C5g, was less stable in energy and Gibbs energy by 4.6 and 5.3 kcal/mol, while its North-counterpart, R-C5c, was less stable by 1.7 and 2.7 kcal/mol ( Supporting Information Figure S7). A similar (south, anti)-structure, R-C5b, which has activation energy and Gibbs free energy of 0.47 and 1.3 kcal/mol higher than those of R-C5a, was also found. For the R-O2 isomers, the most stable one, with a (south, anti)-conformation, has much higher energies. The activation energy and Gibbs free energy are found to be 13.5 and 14.5 kcal/mol higher than those of R-C5a, respectively. The North- and syn-counterparts (R-O2c, R-O2e) of R-O2a are found to be 0.7 and 1.2 kcal/mol less stable in energy, respectively. For the structures of R-N4 and RT1-3, their energies were much higher than that of R-C5a (>40 kcal/mol) ( Supporting Information Figure S10). A comparison of the experimental (Figure 6a,b) and calculated (Figure 6c–h) IR spectra showed that the IR action spectrum was sensitive to the structures. The predicted IR spectra of the most stable isomers in different types, including R-C5a/b and R-O2a/b, are shown in Figure 6, while those of other structures and tautomeric conformations are overlaid in Supporting Information Figure S11. All isomers listed in Figure 6 show the stretching mode of the free OH group at 3714 cm−1, making the observed peak less diagnostic. For the experimentally observed spectrum of the radical ion [Cyt]•+ (285), the predicted spectrum of R-O2a, instead of those of the most stable isomers of R-C5a and R-C5b, matched the experimental spectrum best. The predicted symmetrical and antisymmetric stretching vibrations of NH2 centered at 3460 and 3582 cm−1 are in good agreement with the experimental results at 3466 and 3580 cm−1, respectively. Compared with the IR spectrum of M-O2a, the two peaks corresponding to symmetrical and antisymmetric stretching vibrations of NH2 in R-O2a have been blueshifted by 16 and 17 cm−1, respectively. On the other hand, the close peaks at 3638 and 3625 cm−1, due to the vibration of OH on the pyrimidine ring and the weakly H-bonded OH on the sugar ring, were in reasonable agreement with the measured broad peak centered at 3620 cm−1. The weak peak observed at 3070 cm−1 was attributed to the contribution from the R-O2a isomer, representing the C(6)-H vibration enhanced by the H-bond of C(6)-H···O. The isomer R-O2b, with a higher energy than R-O2a, showed a different spectrum, and thus, was neglected. For the North- and syn-counterparts of R-O2a, we found that the predicted spectrum of the North-counterpart R-O2c was very similar to that of R-O2a, while that of the syn-counterpart R-O2e was quite different ( Supporting Information Figure S8).