Transition of the coordination modes in sodiated uridine radicals revealed by infrared multiphoton dissociation spectroscopy and theoretical calculations

Abstract

The stable generation and structural characterization of sodium cationized nucleic acid radicals at the molecular level have always been a difficult problem to solve. Herein, we produced the radical cation of [Urd + Na − H]˙⁺ through ultraviolet photodissociation (UVPD) of the precursor ion of [I − Urd + Na]⁺ in the gas phase and further studied its infrared multiphoton dissociation (IRMPD) spectrum in the region of 2750–3850 cm⁻¹. The comparison between the IRMPD spectra of the precursor and radical cations shows their common features at both 3445 and 3705 cm⁻¹ peaks, as well as the difference in the 3628 cm⁻¹ peak that exists only in the case of the latter. By combining with theoretical calculations, it is indicated that the bidentate coordination structure M–B(O2,O2′)-1 and the tridentate coordination structure R–T(O2,O′,O5′)–(C5H–C1′)-1 are dominantly populated for the precursor and the radical cations, respectively. After the homo-cleavage of the C–I bond using a UV laser, a multi-step hydrogen transfer process started from the C1′ position, followed by a rotation of the intramolecular C–N bond, resulting in the formation of the most stable isomer, characterized by its radical position at C1′ and its tridentate coordination mode. This result indicates that the generation of free radicals of metal cationized nucleic acids by UVPD may result in the hydrogen transfer from the sugar ring, as well as the accompanied change of its coordination mode of the attached metal ions.

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1 Introduction

Free radicals play an important role in cellular redox reactions, particularly in research related to DNA damage.^1–3 Base radicals are the main reaction intermediates produced when nucleic acids are exposed to high-energy radiation, as well as the main reaction intermediates produced when hydroxyl radicals react with nucleic acids.^4–6 As one of the reactive species, uridine radicals can react with DNA, RNA, and other biomolecules, thereby affecting cell metabolism, repair, and death. Considering the versatile role of uridine radicals in metabolic diseases, their relative radical species may play unexpected roles in various biological processes.⁷ Therefore, it is particularly important to study the structures and kinetics of these free radical species.

To study such transient species formed during the reaction process, various methods for generating nucleoside free radicals have been developed in the past few years.^8–15 Compared to the liquid phase, the gas phase provides a unique surrounding in which molecular ions are not affected by solvent and counterion effects.^16,17 Typically, mass spectrometry-based experimental methods, including collision-induced dissociation (CID), ion mobility, and UV–vis/IR photodissociation action spectroscopy, can be combined with the design of suitable precursor ions, such as the charge-tagging method, to provide powerful means for the characterization of special radicals.^18–25 By comparing experimental results with theoretical calculations, their structures and kinetic information of radical ions in the gas phase can be deeply investigated at the molecular level.

On the other hand, the coordination between nucleic acids and the ubiquitous sodium ion is an unavoidable issue. It has been reported that sodium cations can affect the replication and cleavage of DNA and RNA.^26–31 The effects of sodium cationization on nucleosides have been studied in the gas phase in the past few years. The method of IRMPD spectroscopy has been applied in the structural study of such complexes.^20,32 For instance, Zhu et al. compared the obtained IRMPD and theoretical spectra of [Urd + Na]⁺ and [dUrd + Na]⁺ in detail. For [Urd + Na]⁺, the most stable isomer is characterized by its tridentate coordination mode, while the most populated ion in the ESI process has a bidentate coordination mode. The reason is that the former isomer is more stabilized by hydration in aqueous solution and the corresponding microsolvation environment and is kinetically trapped in the desolvation process due to the energy barrier.³³

However, studies on sodium cationized nucleic acid radicals, as well as the effects of the formation of radicals on the coordination modes, have been rarely reported. One of the problems may be the difficulty in stably generating and systematically studying them at the molecular level. We have previously reported the experimental way to generate metalized nucleoside radical cations in the gas phase with the aid of the iodine-labelled precursor and the method of UVPD to cleave the C–I bond. Herein, by combining a Fourier transform ion cyclotron resonance (FT ICR) mass spectrometer with tunable infrared and ultraviolet lasers,^19,34 the radical cations of [Urd + Na − H]˙⁺ were generated and studied by IRMPD spectroscopy. Further comparisons between the experimental and calculational spectra for both the radical cations and the precursor ions reveal not only their structures in detail, but also the transition of the coordination modes of the sodium cations accompanied by the formation of the radical cations.

2 Results and discussion

2.1 Generation of the radical ion of [Urd + Na − H]˙⁺

The molecule of 5-iodine uridine is selected as the precursor in these experiments. After its generation by electrospray ionization (ESI), the complex sodiated ion was selected by the method of stored waveform inverse Fourier transform (SWIFT)³⁵ in the cell of the FT ICR mass spectrometer for subsequent experiments (Fig. 1a). The two fragment ions generated by the process of collision-induced dissociation (CID) are the same as those generated by IRMPD (Fig. 1b and c), which are characterized by their m/z's at 260.96 and 155.06. The two fragment ions are formed by the two separate pieces attached with the sodium ions after the cleavage of the glycosidic bond of the precursor ion.

Fig. 1 Mass spectra of the parent ion [I − Urd + Na]⁺ (m/z 392.96): (a) after its isolation, followed tandem experiments with (b) CID, (c) IRMPD (@3446 cm⁻¹, 10 s), (d) UVPD (@218 nm, 5 s), (e) UVPD (@245 nm, 15 s), and (f) UVPD (@282 nm, 5 s); along with those of [Urd + Na − H]˙⁺ that was generated by the UVPD at 282 nm: (g) after its isolation, followed tandem experiments with (h) CID, (i) IRMPD (@3446 cm⁻¹, 15 s), and (j) UVPD (@ 280 nm, 5 s).

However, UV irradiation shows quite different results, which also depend on the wavelength of the UV laser applied. With the UV laser at 218 nm, only the fragment ion with m/z 238.09 was observed. When a 245 nm UV laser was applied, both fragments at m/z 238.09 and 266.06 were observed. The ion at m/z 266.06 can be clearly identified as the product ion formed by the homolytic cleavage of the C–I bond, which is the radical cation with the form of [Urd + Na − H]˙⁺. Interestingly, while the 282 nm UV laser was used, only the radical ion at m/z 266.06 was observed. The fundamental aspects underlying this phenomenon of the wavelength-dependent ultraviolet photodissociation are important. But a clear understanding about it is also challenging, since the dissociation channels are relative to both thermodynamics and dynamic processes, and possible intersystem crossing may make the situation more complicated.³⁶

MS³ tandem mass spectra of the radical ion [Urd + Na − H]˙⁺ generated by the 282 nm UVPD were also obtained after its selection (Fig. 1g). Both CID and IRMPD result in the fragment ion at m/z 238.09, which is formed by the loss of the unit of CO from the radical cation (Fig. 1h and i), while the 5 s UV irradiation at 280 nm results in no observed fragment ion (Fig. 1j). For the m/z 238 fragment ion observed in Fig. 1h and i, MS⁴ tandem mass spectrometry shows the only peak at m/z 198.06 (Fig. S1 in ESI†), which was formed by the loss of the unit of C₂H₂N.

To test if the product radical ions of [Urd + Na − H]˙⁺ have different structures that are relative to the wavelength applied in the previous UVPD step for its generation, MS³ tandem mass spectrometry experiments based on IRMPD and CID were also performed for the radical cations generated by UVPD with different wavelengths, including 250, 260, and 290 nm. The resultant mass spectra are all very similar to those shown in Fig. 1h and j, indicating their structural difference is indistinguishable, if there is any.

To determine the wavelength-dependent yield of the radical ions, the yield spectrum of [Urd + Na − H]˙⁺ as a function of applied UV wavelength was recorded, as well as the UVPD spectrum of the precursor ion. As shown in Fig. 2a/b, the absorption range of the parent ion [I − Urd + Na]⁺ is wide between 218 and 314 nm, with a maximum value at 278 nm. Meanwhile, the radical ions of [Urd + Na − H]˙⁺ can only be generated within the range of 234–314 nm, while the range of the ion m/z 238.08 is 218–278 nm. The changes in dissociation channels result in the generation of fragments at m/z 238 or 266 at low or high wavelengths, respectively.

Fig. 2 (a) The UVPD spectrum of the precursor ion of [I − Urd + Na]⁺; (b) the yield spectrum of the radical ion of [Urd + Na − H]˙⁺. Both spectra were obtained in the range of 210–350 nm.

2.2 IRMPD spectra of the precursor and the radical cations

It must be emphasized that the accurate analysis of the structures of these ions is difficult. Both ions involve tautomers, since structural tautomers between amino and imine groups or between ketone and enol groups are very common. In addition, the free radical sites generated by UV laser irradiation may migrate and eventually settle in different positions: C5 and C6 on the pyrimidine ring or C1′, C2′, C3′, C4′, C5′, O2′, O3′, and O5′ on the sugar ring. Scheme 1 demonstrates the various isomers mentioned above. To make it worse, the coordination mode of alkali metal ions, intramolecular hydrogen bonds, and the conformation of sugar rings must be systematically considered too.

Scheme 1 Isomers of [I − Urd + Na]⁺ and [Urd + Na − H]˙⁺.

To explore their structures, IRMPD spectroscopy experiments were performed for both ions. In such a study, the intensity of the spectra was calculated based on I_IR = −ln(I_p/∑(I_fi + I_p)), where I_p and I_fi are the intensities of the parent ion and the i-th product ion, respectively, in the corresponding experiment. As shown in Fig. 3, the IRMPD spectrum of the precursor ion [I − Urd + Na]⁺ is characterized by sharp peaks at 3445 and 3705 cm⁻¹, which is consistent with that of [Urd + Na]⁺ previously reported by Zhu et al.³³ For the radical ion [Urd + Na − H]˙⁺ generated with the UVPD of [I − Urd + Na]⁺ at 282 nm, its spectrum shows some difference. The two peaks centered at 3438 and 3710 cm⁻¹, which are very close to those observed in the case of the precursor ion, still dominate the spectrum, but they have noticeably widened. Besides, two weak peaks centered at 2972 and 3628 cm⁻¹ can be clearly identified, the latter of which also has a peak width similar to that of the peak at 3710 cm⁻¹. To investigate whether the radicals generated at different wavelengths of ultraviolet laser irradiation have different structures, infrared photodissociation experiments on the mass-selected radical ions generated by UVPD at 250 and 290 nm were conducted separately at ten wavenumbers that are selected according to the spectral characteristics observed in Fig. 3b. The results show that the infrared dissociation mass spectra of these free radical ions generated at different wavelengths have very similar characteristics, indicating that they do not have significant structural differences. In other words, the previously observed wavelength-dependent generation of radical isomers has not been observed here.³²

Fig. 3 A comparison between the experimental IRMPD spectra of the precursor ion of [I − Urd + Na]⁺ and the radical cation of [Urd + Na − H]˙⁺.

2.3 The structure of [I − Urd + Na]⁺

Considering that the only difference between [I − Urd + Na]⁺ and [Urd + Na]⁺ is the substitution of the I atom, and the IRMPD spectra of both species are so similar, it is believed that the structures of the two species should be very similar. Based on the previously reported structure of [Urd + Na]⁺,³³ the corresponding structural research on [I − Urd + Na]⁺ was also performed here. The most stable isomers belonging to different structural types (Scheme 1) were obtained at the level of B3LYP/6-311+G(d,p). The top three isomers are shown in Fig. 4. The sodium ions can be doubly or triply coordinated inside the complexes, in which the coordination mode can affect the stability greatly. For the convenience of comparison, here we directly reflect the coordination mode of the sodium ions of the isomers in their corresponding name. For example, the most stable isomer of [I − Urd + Na]⁺ is found to be M–T(O2,O′,O5′)-1, in which the letter of T indicates its triply coordinated mode and the content in the parentheses represents the corresponding atoms coordinated with the Na ion.

Fig. 4 Structures of the top isomers of the precursor ions. Their relative energies and Gibbs free energies are shown in the parentheses in the unit of kcal mol⁻¹.

The top two isomers of [I − Urd + Na]⁺ have structures very similar to those of [Urd + Na]⁺ previously reported by Zhu et al.³³ The most stable isomer, M–T(O2,O′,O5′)-1, has a triply coordinated sodium ion and a syn-orientation of the nucleobase, which has an energy 0.3 kcal mol⁻¹ lower than the second stable isomer, M–B(O2,O2′)-1, which is characterized by the double coordination and the anti-orientation of the nucleobase. Tautomers with tridentate or bidentate binding typically have much higher energies (Fig. S2†). The found best tautomer, M–T(O2,O′,O5′)–O4H-1, for example, is found to be 8.8 kcal mol⁻¹ higher in energy (Fig. 4).

Fig. 5 compares the experimental IRMPD with the theoretically calculated IR spectra of the three isomers shown in Fig. 4. For the most stable isomer, M–T(O2,O′,O5′)-1, the predicted peak at 3573 cm⁻¹, due to the O2′–H stretch mode, is absent in the experimental spectrum. Similar to that, the predicted peak at 3563 cm⁻¹ of M–T(O2,O′,O5′)–O4H-1 is also absent. It is found that the spectrum of the second stable isomer M–B(O2,O2′)-1 is characterized by the two peaks at 3455 cm⁻¹ and 3678 cm⁻¹, corresponding to the stretching vibrations of N3–H/O2′–H and O5′–H, respectively, which are in good agreement with the experimental IRMPD spectrum. The energies and IR spectra of other structures including different tautomers show that their contributions to the observed ions are negligible (Fig. S3†). Thus, the M–B(O2,O2′)-1 is believed to be dominantly populated in the experiments reported here.

Fig. 5 A comparison among the experimental IRMPD spectrum and the calculated IR spectra of the three isomers of [I − Urd + Na]⁺. Their relative energies and Gibbs free energies are shown in the parentheses in the unit of kcal mol⁻¹.

This result is consistent with the previous work on sodiated uridine,³³ in which the calculated spectrum of the bidentate structure B(O2,O2′) matches the experimental spectrum much better than the tridentate structure T1(O2,O4′,O5′), which has an energy 1.5 kcal mol⁻¹ lower than that of the former isomer. Their further calculations explained the reasons behind the result.³³ The bidentate structure B1(O2,O2′) is more stabilized by hydration in aqueous solution than its tridentate counterpart and is kinetically trapped during the ESI process, resulting in it as the dominant conformer of [Urd + Na]⁺ in the gas phase. The explanation can also be applied to the experimental results reported here. As shown in Fig. S4,† the results show that the binding of two water molecules can further stabilize the isomer M–B(O2,O2′)-1, since M–B(O2,O2′)-1–2W is found to be 1.9 kcal mol⁻¹ more stable than M–T(O2,O′,O5′)-1–2W.

2.4 Structure of the radical cation of [Urd + Na − H]˙⁺

Considering that the formation of the radical ion is caused by the homo-cleavage of the C–I bond under ultraviolet laser irradiation, the structure of [Urd + Na − H]˙⁺ is constructed based on the two stable isomers of the precursor ions (M–B(O2,O2′)-1, M–T(O2,O′,O5′)-1) in the first step. After that, different structures relative to the migration of radicals through the transfer of H atoms from different positions are constructed and calculated, along with their corresponding structures with different conformations or hydrogen bonds. Besides, tautomerism based on different initial structures is also considered here (Scheme 1).

Based on these considerations, the relevant isomers were constructed, optimized, and screened. The top isomers belonging to different structural types are shown in Fig. S5–9.† For simplicity, these structures are named according to their structural styles defined in the following way. First, these structures are divided into bidentate or tridentate cases according to their coordination modes and named with the letters of B or T, along with the coordinated atoms shown in the parentheses. Second, they are classified into two categories based on the presence or absence of the radical transfer. For those isomers with the transfer, the positions of the transferred hydrogen atom before and after the transfer are shown in the following parentheses. If there is no radical transfer, this part will be omitted. Third, the tautomerization, if there is one, is identified in the following parentheses. In the last step, the isomers are labelled with the corresponding numbers according to their energy order with the same structural types.

We can first focus on the structures without radical transfers. For such kinds of isomers, the top three are shown in Fig. 6. For ease of comparison, the relative energy of the first structure, R–T(O2,O′,O5′)-1, is set to 0 kcal mol⁻¹. This structure can be thought of as the direct product ion after the homo-cleavage of the C–I bond of the most stable structure of the precursor ion (M–T(O2,O′,O5′)-1). And another isomer with a similar structure, R–T(O2,O′,O5′)-2, has an energy 0.5 kcal mol⁻¹ higher. Similarly, the structure R–B(O2,O2′)-1 can be treated as the direct product ion from the precursor ion of M–B(O2,O2′)-1, which has a relative energy of 0.4 kcal mol⁻¹.

Fig. 6 The top three structures with the lowest energy without hydrogen migration and the top six structures with the corresponding hydrogen migrations. The calculation was performed at the level of B3LYP/6-311+G(d,p). Their relative energies and Gibbs free energies are shown in the parentheses in kcal mol⁻¹.

Calculation reveals that most structures formed after radical transfers have lower energies than that of R–T(O2,O′,O5′)-1. Since the radical transfer is achieved through the transfer of H atoms here, the corresponding isomers are classified and labeled using the original site of the hydrogen atom that undergoes transfer, as described above (Scheme 1). Taking R–T(O2,O′,O5′)–(C5H–C1′)-1 as an example, the first half T (O2,O′,O5′) means its coordination style, and the second half means the transfer of the H atom from the sugar ring C1′ to the pyrimidine ring C5 positions.

Fig. 6 also shows the top six structures that experienced hydrogen transfers. It can be observed that among these six structures, the 2nd and 4th are characterized by bidentate coordination, while the rest are characterized by tridentate coordination. The energy of the most stable structure R–T(O2,O′,O5′)–(C5H–C1)-1 is 30.7 kcal mol⁻¹ lower than that of R–T(O2,O′,O5′)-1. For the second stable structure, it also has an H transfer from the C1′ position, but with a bidentate coordination of B(O2,O2′). It has an energy 0.9 kcal mol⁻¹ higher than that of the most stable structure. For structures of R–T(O2,O′,O5′)–(C5H–C4′)-1, R–T(O2,O′,O5′)–(C5H–C2′)-1, and R–T(O2,O′,O5′)–(C5H–C3′)-1, the difference exists only in the origin of the H atom transferred. And they have energies of 2.4, 3.9, and 5.2 kcal mol⁻¹ higher than that of the top isomer. The structure of R–B(O2,O2′)–(C5H–C5′)-1, which is characterized by the bidentate coordination and the C5′ H transfer, has an energy 4.4 kcal mol⁻¹ higher than that of R–T(O2,O′,O5′)–(C5H–C1)-1. The results mean that both coordination styles and origins of the H atoms transferred can greatly affect the total energies of the radicals.

Fig. 7 compares the experimental IRMPD spectrum with the theoretical infrared spectra of various structures of the radical cations shown in Fig. 6 (the corresponding spectra of other structures, including different tautomers, are listed in Fig. S10–16†). Among all the structures, the calculated IR spectrum of the most stable isomer, R–T(O2,O′,O5′)–(C5H–C1′)-1, matches best with the experimental result. The predicted peak at 3443 cm⁻¹ due to the stretching vibration of the pyrimidine ring N3–H matches the experimental peak at 3438 cm⁻¹. The peaks at 3695 cm⁻¹ and 3703 cm⁻¹ due to the stretching modes of O5′–H and O3′–H match the experimental peaks at 3696 cm⁻¹ and 3712 cm⁻¹, respectively. Importantly, the peak due to the O2′–H stretching vibration matches the experimental peak at 3628 cm⁻¹. For other isomers involving hydrogen transfer processes, although their predicted spectra include peaks at ∼3440 and ∼3703 cm⁻¹, their O2′–H stretching vibration modes are located at different places, which do not agree with the experimental result. The isomers without hydrogen transfer, however, are much higher in energy compared to the most stable isomer. Thus, it can be believed that the main contribution of the experimentally observed radical cations is coming from the isomer R–T(O2,O′,O5′)–(C5H–C1′)-1.

Fig. 7 A comparison among the experimental IRMPD spectrum and the calculated IR spectra of the corresponding isomers of the radical ion of [Urd + Na − H]˙⁺. Their relative energies and Gibbs free energies are shown in parentheses in kcal mol⁻¹. The structures of these isomers can be found in Fig. 6. All calculations were performed at the level of B3LYP/6-311+G(d,p).

2.5 The isomerization process and the transition of the coordination mode

Considering the structural continuity between the structures of M–T(O2,O′,O5′)-1 and R–T(O2,O′,O5′)-1, and that between the structures of M–B(O2,O2′)-1 and R–B(O2,O2′)-1, the change in the coordination modes from the precursor ion of [I − Urd + Na]⁺ with the structure of M–B(O2,O2′)-1 to the product radical ion of [Urd + Na − H]˙⁺ with the structure of R–T(O2,O′,O5′)-1 is quite confusing. Undoubtedly, understanding the process of hydrogen transfer that occurs after the formation of free radicals, as well as the accompanying energy barriers and changes in their coordination modes, is crucial.

To understand the processes of hydrogen migration and coordination transition, the potential energy distribution of possible isomerization pathways from R–B(O2,O2′)-1 to R–T(O2,O′O5′)–(C5H–C1′)-1 is calculated. The results are shown in Fig. 8. In this experiment, the generation of the free radical was achieved through C–I bond homo-cleavage induced by ultraviolet laser irradiation, indicating that the isomer R–B(O2,O2′)-1 is a direct product in the first step. This product ion can be isomerized to form the isomer of R–B(O2,O2′)–(C5H–C6)-1 through a transition state of TS1 that has an energy barrier of 53.1 kcal mol⁻¹. In the process, the H atom migrates along the edge of the pyrimidine ring, resulting in the structure of R–B(O2,O2′)–(C5H–C6)-1 with the radical position transferred to the position of C6. The subsequent step is characterized by the hydrogen transfer from the ribose ring (C1′), forming a much more stable isomer, R–B(O2,O2′)–(C5H–C1′)-1, through the transition state TS2 with a barrier of 32.7 kcal mol⁻¹.

Fig. 8 Potential energy profile associated with the isomerization pathways relative to [Urd + Na − H]˙⁺. Relative energies and Gibbs free energies are shown in the parentheses in kcal mol⁻¹. The subgraph in the upper right corner shows the potential energy change from R–B(O2,O2′)–(C5H–C1′)-1 to R–T(O2,O′,O5′)–(C5H–C1′)-1, which is achieved through the rotation of the intramolecular C–N bond. All calculations were performed at the level of B3LYP/6-311+G(d,p).

In the last step, through the rotation of the single C–N bond within the molecule, the structure R–B(O2,O2′)–(C5H–C1′)-1 isomerizes to the most stable isomer R–T(O2,O′,O5′)–(C5H–C1′)-1 and completes the transition from bidentate to tridentate coordination modes. Although a similar channel may exist in the case of the precursor ion, the energy barrier of the process blocks the rotation in the ESI source, resulting in the survivor as the isomer of M–T(O2,O′,O5′)-1, instead of the most stable isomer. To understand the energy barrier of this step, a relaxed potential energy scan connecting the two isomers was performed here with 5° dihedral angle increments. As shown in the upper right corner of Fig. 8, the calculated energy barrier for the internal rotation is 23.3 kcal mol⁻¹. For the precursor ion of [I − Urd + Na]⁺, the corresponding energy barrier is much higher, mainly due to the existence of the iodine atom (Fig. S17†), which explains why the precursor ion maintained the structure with the bidentate coordination mode.

2.6 Some discussion

There are still some issues that require more consideration. For the first point, it should be considered whether the cleavage of the ribose ring is possible for the radical cation of [Urd + Na − H]˙⁺. In fact, the radical site at C1′ in the structure R–T(O2,O′,O5′)–(C5H–C1′)-1 may promote the cleavage of the adjacent bonds. Previous calculations by Zima et al.³⁷ have shown that the ring-opening isomerization may at last result in the loss of the unit of C₂H₃O₂ in the case of 2′-O-acetyladenosine radical cations. Here, we also investigated possible ring-opening structures. The top two structures with opening rings are shown in Fig. S18.† It can be found that their energies are 8–9 kcal mol⁻¹ higher than that of R–T(O2,O′,O5′)–(C5H–C1′)-1, and the IR spectra (Fig. S19†) are not consistent with the experimental results. In terms of energy barriers, as the example shown in Fig. S20,† the energy barrier for the critical step of the sugar ring bond break (from R–T(O2,O′,O2′)–(C5H–O2′)-1 to R–T(O2,O′,O2′)–(C5H–O2′)-open-2) is 158.9 kcal mol⁻¹, which is much higher than photon energy. Thus, the proportion of such ring-opening structures should be insignificant.

For the second point, our previous results show that the radical cations of cytidine generated by the UVPD at different wavelengths may have different structures, due to whether the isomerization energy barrier can be overpassed after the homo-cleavage of the C–I bond.³² In that case, the energy of the UV photon at 245 nm allows the isomerization to occur after the homolytic cleavage, resulting in the formation of the most isomer of R–C5 from the initial isomer of R–O2, while that of the UV photon at 280 nm cannot make the isomerization happen. Here, the isomer-selective generation via wavelength-regulated photodissociation has not been observed. That is, the structure of [Urd + Na − H]˙⁺ generated by UVPD does not depend on the UV wavelength in the region of 250–290 nm. This difference can be reasonably explained by comparing the energy barriers of two isomerization processes. The energy barrier in the isomerization process shown in Fig. 8 is 53.1 kcal mol⁻¹, about 14% lower than that of [Cyt]˙⁺ (61.5 kcal mol⁻¹). Further calculations indicate that even for the UVPD at 290 nm, the energy provided by the single photon is enough for the isomerization after the break of the C–I bond.

3 Conclusions

In this paper, the precursor ion of sodiated 5-I-uridine was used to generate sodium-ionized uridine radical cations in the gas phase. It has been found that the radical cation of [Urd + Na − H]˙⁺ can be generated through UVPD of [I − Urd + Na]⁺ in the region of 234–314 nm. Both the precursor and radical cations were mass-selected for further IRMPD spectroscopy study in the region of 2750–3850 cm⁻¹. Although their spectra show common features of the peaks at the nearby 3445 and 3705 cm⁻¹, the characterized peak at 3628 cm⁻¹ of [Urd + Na − H]˙⁺ reflects their difference. Calculation results indicated that the bidentate coordination structure M–B(O2,O2′), instead of the most stable tridentate coordination structure M–T(O2,O′,O5′), should be dominantly populated in the experiments for the precursor ion. It can be explained by the fact that the bidentate coordination structure is more favored in energy by hydration in aqueous solution than the tridentate isomers and the structural retention caused by the isomerization energy barrier that cannot be crossed by the corresponding ESI processes. The results are consistent with the previously reported IRMPD and theoretical study of the ion of [Urd + Na]⁺.³³ For the radical cation of [Urd + Na − H]˙⁺, calculation results showed that the isomer R–T(O2,O′,O5′)–(C5H–C1′)-1 is mainly populated. The isomer is characterized by its tridentate coordination, the hydrogen transfer from the C1′ to C5 after the homo-cleavage of the C–I bond. The isomerization process with the transition of the coordination mode is also revealed. Following the homo-cleavage of the C–I bond, a multi-step hydrogen transfer process results in the fact that the H atom at the C1′ position was finally transferred to the C5 position, forming a stable free radical isomer with the bidentate coordination mode, R–B(O2,O2′)–(C5H–C1′)-1. And the bidentate structure can isomerize to the most stable tridentate structure by a rotation of the C–N bond. The energy barrier of the whole process is 53 kcal mol⁻¹, which makes the isomerization possible for UVPD in the range of 250–290 nm. This result indicates that during the generation of free radicals of nucleic acids, changes in the coordination mode of the attached alkali metal ions accompanied by hydrogen transfer may be common and should be given more attention.

Author contributions

Kairui Yang: investigation, calculation, validation, and writing – original draft. Min Kou: investigation, experiments and calculation. Zicheng Zhao: investigation and calculation. Jinyang Li: writing – original draft. Xianglei Kong: funding acquisition, project administration, methodology, conceptualization, writing – review & editing, and supervision.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (no. 22174076 and 21627801).

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, additional calculation results and molecular coordinates. See DOI: https://doi.org/10.1039/d4dt03561e

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