Collision-induced dissociation of singly and doubly charged Cu^II–cytidine complexes in the gas phase: an experimental and computational study

Abstract

The complexes of doubly charged cations, [CuL_n]²⁺, [CuL(MeOH)_n]²⁺ and single charged cations, [Cu(L–H)L_n]⁺, [L+H]⁺, [L₂+H]⁺, and [B+H]⁺, etc. (L = cytidine, B = cytosine) were observed via electrospray of water and methanol solutions of Cu(NO₃)₂ and cytidine. The peak distribution for [CuL_n]²⁺ shows that the [CuL₂]²⁺ and [CuL₄]²⁺ are the highest peaks, then followed by [CuL₃]²⁺, [CuL₅]²⁺, etc.Collision-induced dissociation (CID) of [Cu(L–H)L_n]⁺(n = 0,1), [CuL_n]²⁺(n = 2–6) and [CuL(MeOH)_n]²⁺(n = 1, 2) was investigated using the ESI-MS/MS instrument, and the structure and thermal energy for the most of these complexes were also calculated with ab initio and DFT methods through Gaussian 09 program. For [Cu(L–H)L_0-1]⁺, the primary dissociation only involved a ribose group, including ribose group lost, ribose side chain and ribose ring broken. The calculation result shows that the deprotonated site for [Cu(L–H)]⁺ is the N-atom in the amino group of the cytidine base. The inter-ligand proton transfer was observed to dominate the CID of all doubly charged Cu^II–cytidine complexes, [CuL_n]²⁺ (n = 2–6), and other channels, including charge reduction dissociation, lost neutral ligands were also observed. The protonated cytidine dimer, [L₂+H]⁺, produced in the CID of [CuL_n]²⁺ (n = 4–6), can further break into [L+ H]⁺ and neutral L, and the structure is proposed to be from the interaction of two cytidinesviahydrogen bonds. The protonated trimeric cytidine was also observed in the dissociation of [CuL₆]²⁺. Three charge reduction dissociation channels were observed in the CID of [CuL(MeOH)]²⁺. Unlike the Cu^II–guanosine complex, the radical cation [CuL_n]⁺ was not observed in the CID process of the [CuL_n]²⁺, and the reasons are attributed to the higher ionization energy and proton affinity, which makes the inter-ligand proton transfer more favorable than the electron transfer from ligand to Cu²⁺.

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1. Introduction

There is considerable interest in the interaction of metal cations with nucleosides in view of speculation that metal cations play a crucial role in gene expression (replication, transcription and translation),^1–5 in particular the multiple charged cations, because many metals of chemical and biochemical importance commonly occur in their higher oxidation states.^6–8 In the liquid-phase, the stability and coordination chemistry of metal cations, especially the multiply charged cations, to nucleosides and nucleotides have been studied quite extensively using NMR, X-ray and other technologies.^9–18 But in the gas-phase, there are challenges to generate the multiply charged ion–solvent complex for easy charge transfer from the solvent to metal ions. In the 1990s, Kebarle et al. have demonstrated that electrospray could be used with considerable success to generate a range of solvated doubly and triply charged transition metal cations.^19–22 Since then, electrospray ionization (ESI) mass spectrometry (MS) has become the most frequently used technique for the study of the gas-phase complex of monovalent and divalent metal cations with a variety of protic and aprotic ligands, including water,^23–27alcohol,^28–30acetone and other ketones,^23,28,31acetonile,^29,32pyridine,^19,29dimethysulfoxide,^20,22,28 and some DNA bases,^33–38etc. With the development of mass spectrometry, the computation and/or MS has proved to be very useful to study the interaction of metal cations with ligands. Very recently, Marino et al.³⁹ have studied the theoretical interaction of rubidium and cesium mono-, strontium and barium bi-cations with DNA and RNA bases; Niu et al.⁴⁰ studied the interaction of sulfur- and aminopyridine-containing chelating resins with Hg(II) and Pb(II) through calculation; Dunbar et al.⁴¹ observed the encapsulation of metal cations by the PhePhe ligand through ESI-MS and DFT calculations.

The biologically important d⁹ open shell Cu²⁺ cation has rich redox chemistry and is closely associated with DNA bases.^42–45 The activity is connected with a relatively high electron affinity and the Cu^II oxidation state could easily reduce to Cu^I. From this point, it is quite necessary to understand the interaction of Cu²⁺ with the nucleoside in order to study the influence of the Cu²⁺ in the DNA damage process. Recently , the interactions of Cu²⁺ with guanosine were investigated, and the radical cations of monomeric and dimeric molecular guansoine were generated by the dissociation of the doubly charged Cu^II–guanosine complexes.⁴⁶ Now the study of gas-phase copper complexes using MS has grown into an active and promising research area.^47–52

Cytidine (L) is one of the main nucleosides which occur in RNA or DNA, where it participates in stacking interactions and hydrogen bonding to preserve and transfer the genetic information.^4,6 Most of the investigations about the interaction of metal caitons with cytidine were done in the liquid-phase.^53–55 Recently, a report about the interaction of cytidine with metal cations measured by ESI-MS showed that two types of cytidine clustering depending on coexisting ions were observed. One was the electrostatic interaction, and the other was the coordination interaction.^34,56 Almost at the same time, the guanosine–cytidine dinucleoside that self-assembles into a trimetric supramolecule was presented.⁵⁷

The exposure of DNA to Cu²⁺ can have different consequences such as single and double strand cleavage, base modification, and formation of abasic sites. So the nature of the interactions of Cu²⁺ with nucleoside bases should be understood in order to unravel the mechanism for such consequences. ^58,59 In this study, we report the gas phase interaction of the Cu²⁺ with cytidine by ESI mass spectrometry and calculation. Singly and doubly charged complexes of Cu^II–cytidine were formed, and varied fragmentation chemistries, including inter-ligand proton transfer, ligand elimination, ligand dissociation charge reduction, etc. were observed for the CID of Cu^II–cytidine complexes. The structure, thermal energy and entropy for most of these Cu^II–cytidine complexes and their fragmentation products were calculated with ab initio and DFT theory through Gaussian 09 program.⁶⁰

2. Methods

2.1 Experimental method

Experiments were performed using API 2000 (AB Sciex) mass spectrometer equipped with an ESI source. The operating conditions were as follows: ESI positive ion mode; ion spray voltage, 5500 V; ring-electrode potential, 300 V; varied potential values between the orifice and the skimmer; curtain gas flow rate, 10 arbitrary instrument units of N₂; nebulizer gas flow rate, 20 arbitrary instrument units dry air; and sample flow rate, 5 μL min⁻¹.

MS/MS was carried out in the product ion monitoring modes with N₂ as the collision gas with a flow rate about 9 arbitrary units. The first quadrupole (Q1) was used to select the precursor ions, and the collision induced dissociation performed in the second quadrupole (Q2), the product ions were mass analyzed via the third quadrupole (Q3). The collision energy (E_lab) was typically 10–50 ev.

Cu²⁺ was generated from its nitrate hydrate (Aldrich, p.a 99.99%) without further purification; cytidine was purchased from SIGMA p.a. 98%. HPLC degree methanol and Millipore (18.2 mΩ) water were used to prepare the solvent mixture. Cytidine and Cu²⁺ metal salt were dissolved in a 1 : 3 water–methanol mixture.

2.2 Theoretical method

Calculations were carried out using both the ab initio method and the B3LYP (Becke, three-parameter, Lee–Yang–Parr)/density functional theory (DFT) method. These methods were implemented in the Gaussian 09 program package.⁶⁰ The UNIX version with standard parameters of this package was installed on the computing cluster in Shanghai University. DFT methods provide an adequate compromise between the desired chemical accuracy and heavy demands on computer time and power. Moreover, DFT methods and in particular B3LYP have been used satisfactorily in many studies of drug design, as well as in other nucleosides and nucleic bases, and they predict vibrational frequencies better than Hartree–Fock (HF) and MP2 methods.^61,62 It was well-established that weak interactions were poorly described by DFT methods. However, the hydration of uracil and their derivatives had been reasonably well modeled by DFT. Standard density functional theory calculations were performed using the Gaussian09 suite of programs. Geometries were optimized using Becke's hybrid B3LYP functional and the 6-311+G(d) basis set on C, H, N and O. The copper electrons were handled by the Hay–Wadt effective core potential (ECP) with the LANL2DZ basis set.⁶³ Spin-unrestricted calculations were used for all open-shell systems. The calculated frequencies were used to calculate zero-point energies and thermal enthalpy corrections. Improved energies were obtained by single point calculations with B3LYP using the larger 6-311+G(d) basis set on all atoms or in combination with ECP/LANL2DZ on Cu. Atomic spin and charge densities were calculated using natural population analysis.

3. Results and discussion

Complexes of the type [CuL_n]²⁺, [CuL(MeOH)_n]²⁺ and [Cu(L–H)]⁺, etc. were readily observed by electrospray of a solution containing Cu²⁺ and cytidine (L) molecules. The value n depends on the concentration ratio of Cu²⁺ to cytidine and working in conditions where the declustering potential (DP) is high or low. Keeping the concentration ratio unchanged, if the DP is high, above 50 volts, the maximum value n will reduce, and dissociation charge reduction and other dissociation channels will take place in the source, and lead to diversified MS products. For these reasons, in this experiment we usually work at low DP so as to get the reasonably high abundance of Cu(II)-cytidine complexes. High DP is just used for further CID of the second and higher order products from primary complex dissociation.

3.1 Mass spectrometry of ESI-MS of Cu(NO₃)₂ and cytidine mixed solution

The ESI mass spectra of cytidine in the presence of Cu²⁺ at different concentrations in 10 eV declustering potential are presented in Fig. 1. Fig. 1(a) shows the spectrum with Cu²⁺(0.2 mM) and cytidine (0.4 mM), and Fig. 1(b) with Cu²⁺ (0.4 mM) and cytidine (0.8 mM). It is quite clear there is a big difference of the peak distribution. Fig. 1(a) shows the peaks at m/z 169.1, 185.1, corresponding to [CuL(MeOH)_1-2]²⁺; 112.1, 244.4 and 487.5, assigned as [B+H]⁺ (B = cytosine), [L+H]⁺ and [L₂+H]⁺; 368.0, 611.5 and 854.2, corresponding to [Cu(NO₃)L_1-3]⁺; and 305.3, 548.5, 791.3 and 275.0 assigned as [Cu(L–H)L_0-2]⁺ and [CuL₂]²⁺, respectively. There are also some other peaks, like 65.0, 126.1, 133.1 and 416.5 corresponding to [(MeOH)₂+H]⁺, [Cu(CH₂O)(MeOH)]⁺, [R]⁺ and [Cu(L–H)B]⁺. Except for the peaks, 112.1([B+H]⁺), 243.9([L+H]⁺), 486.9([L₂+H]⁺), 168.9 ([CuL(MeOH)]²⁺), 185.1([CuL(MeOH)₂]²⁺) and 274.8([CuL₂]²⁺) in Fig. 1(a), Fig. 1(b) also shows a series of Cu²⁺–cytidine peaks, 396.3, 518.0, 639.5, 761.0, 882.7, 1004.7 and 1126.4, corresponding to [CuL_3-9]²⁺, respectively. The chemical composition of these ions are assigned and confirmed from isotopic patterns, especially for those that contain Cu. The two isotopes ⁶³Cu and ⁶⁵Cu have an abundance ratio 69 : 31. The observed [B+H]⁺ ion in Fig. 1 would be formed through the dissociation of an N–glycoside bond of [L+H]⁺ at the interface of the instrument according to reaction (1).

[L+H]⁺ → [B+H]⁺ + [R–H] (1)

Fig. 1 ESI-MS spectra of a water–methanol solution of (a) Cu²⁺ (0.2 M) and cytidine (0.4 mM) and (b) Cu²⁺ (0.4 M) and cytidine (0.8 mM).

It is quite clear to see from Fig. 1 that the concentration will affect the distribution of the complexes of Cu^II–cytidine, and a higher concentration would cause more doubly charged cations to be produced. The reason could be relative with the complex stability constants in solution, and the higher concentration could be helpful to the predominant formation of the Cu^II–cytidine complexes. Moriwaki³³ had investigated the complexes of Cd^II–guanosine by ESI-MS; he observed the molar ratio of Cd²⁺ to guanosine also had a dramatic effect on the ion peak distribution. Kearns et al. observed that the molar ratio and pH value affect the formation of the complexes in liquid in the magnetic resonance studies of Cu²⁺ interaction with nucleosides and nucleotides.⁶⁴

From the ion peak distribution of [CuL_n]²⁺ in Fig. 1(b), we can see that the peaks of n = 2 and 4 are the highest which means they both have a very stable coordination structure. This stable coordination structure would be relative with the characteristics of both cytidine and Cu²⁺. The N(3) and O(7) of cytidine are the preferred sites for metal to bind, and have been proved in many experiments.^7,43,45 Binding to either N(3) or O(7) depends on the softness/hardness of the metal ions and possibly steric aspects. Sometimes the cytidine can bind metal simultaneously through N(3) and O(7) in chelating (or semichelating) fashion or bridge fashion.⁷ Generally, the Cu^II has a coordination number of 4, and prefers the square-planar or octahedral geometry while coordinating with some ligands.^65–68 According to these facts and the calculation results, for the structure of [CuL₂]²⁺, Cu^II coordinates with N(3) and O(7) simultaneously to form a planar structure with cytidine base; for [CuL₄]²⁺, Cu^II only coordinates with N(3) to form a planar structure with cytidine base; for [CuL₃]²⁺, Cu^II binds with 3 N(3) atoms in the same plane. For other complex cations, [CuL_n]²⁺, we suggest that the extra ligands will loosely bind together through electrostatic strength based on structures of [CuL₄]²⁺. The IR spectrophotometry results indicated that the metal cations bind N(3) and O(7) simultaneously with the cytidine in the solid chelates for the complexes [ML₂]ⁿ⁺ (M = U(VI), Th(IV), Ce(III) and La(III)).⁶⁹

3.2 CID of Cu²⁺-cytidine complex cations [Cu(L–H)]⁺ and [Cu(L–H)L]⁺

The cations of [Cu(L–H)]⁺ and [Cu(L–H)L]⁺ were observed in the ESI spectra of low concentration solution and 10 eV DP (Fig. 1(a)) .

The CID spectrum of [Cu(L–H)]⁺ at a collision energy of 14 eV is shown in Fig. 2. It shows that there is only one dissociation pathway for losing CH₂O (m/z 275); and followed by elimination of H₂O (m/z 257) or breaking the ribose ring to lose C₂H₃O₂ (m/z 216) or CuC₂H₂O₂ (m/z 154); then the higher order dissociation will continue and form other small daughter ions like m/z = 229, 173 and 174, etc. These dissociation processes were very similar to the chemical ionization mass spectrometry of nucleosides results.^70–74 For the structure of [Cu(L–H)]⁺ (Table 1), the calculation results show that the coordination sites for copper(II) with cytidine are N3 and O(7), and the deprotonation site is an amino group on the base and not on the hydroxy group of the sugar moiety which is also consistent with previous results.^34,45

Fig. 2 CID spectra of [Cu(L–H)]⁺ at E_lab = 14 eV.

Table 1 B3LYP/LANDL2DZ6-311+G(d) calculations of potential structures for the compounds of Cu^II–cytidine complexes

Compound	Structure
[Cu(L–H)]^+
[CuL(L–H)]^+
[CuL2]^2+
[CuL3]^2+
[CuL4]^2+
[CuL(MeOH)]^2+

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CID of the [CuL(L–H)]⁺ complex shows quite complicated fragmentation pathways, and there is no L or (L–H) loss channel . Fig. 3 is the CID spectrum of [CuL(L–H)]⁺ at a collision energy of 15 eV. Three main fragmentation channels were observed and identified as R–H (R = ribose) loss (m/z 416), reaction (2a); breaking the ribose ring to lose R–CH₂O (m/z 445), reaction (2b) and to lose R–C₂H₄O (m/z 459), reaction (2c). Besides these, a minor channel of CH₂O lost (m/z 518) was also observed, reaction (2d).

[CuL(L–H)]⁺ → [Cu(L–H)(B+H)]⁺ + [R–H] (2a)

→ [CuL(B+COH)]⁺ + [R–CH₂O] (2b)

→ [CuL(B+CH₂COH)]⁺ + [R–C₂H₄O] (2c)

→ [CuL(L–H) –CH₂O]⁺ + [CH₂O] (2d)

Fig. 3 CID spectra of [CuL(L–H)]⁺ at E_lab = 18 eV.

The calculation results show that the structure of [CuL(L–H)]⁺ (Table 1) is the one where the Cu^II coordinates with N(3) and O(7) simultaneously forming a planar structure with the cytidine base. This structure has high stability and the fragmentation only involves the ribose being broken.

3.3 CID of Cu²⁺-cytidine complex cations [CuL_2–6]²⁺

The results of CID [CuL_2-6]²⁺ complexes show that their fragmentation channels are quite different.

Fig. 4 shows the MS/MS spectra of [CuL₂]²⁺ at a collision energy of 20 eV. Three main primary fragmentation channels were identified as, inter-ligand proton transfer accompanied by dissociation to give [L+H]⁺ and [Cu(L–H)]⁺, reaction (3a); [R–H] is lost to produce [CuLB]²⁺, reaction (3b); and dissociation charge reduction to break the N–glycoside bond of one ligand to give [CuL(B–H)]⁺ and [R]⁺, reaction (3c). Besides these, there is also a minor dissociation channel of dissociation charge reduction (DCR) to break the N–glycoside bond of one ligand to give [CuL(R–2H)]⁺ and [B+H]⁺, reaction (3d).

[CuL₂]²⁺ → [Cu(L–H)]⁺ + [L+H]⁺ (3a)

→ [CuLB]²⁺ + [R–H] (3b)

→ [CuL(B–H)]⁺ + [R]⁺ (3c)

→ [CuL(R–2H)]⁺ + [B+H]⁺ (3d)

Fig. 4 CID spectra of [CuL₂]²⁺ at E_lab = 20 eV.

The calculation results show that the structure of [CuL₂]²⁺ (Table 1) is where Cu^II coordinates with N(3) and O(7) simultaneously to form a planar structure. The fragmentations mainly involve the ribose being broken. During the fragmentation process, the inter-ligand electron transfer dissociation channel wasn't observed, even though Cu has the highest IE₂ (20.3 ev)⁷⁵ among all the transition metals, and the electron transfer from a neutral cytidine ligand (IP = 8.6 eV)⁷⁰ to a double charge Cu²⁺ is energetically favorable. Cytidine is a protic solvent ligand with a quite high proton affinity (PA = 234.8 kcal mol⁻¹)⁷¹ and may be the possible reason for inter-ligand proton transfer rather than electron transfer to occur.

Fig. 5 is the CID spectrum of [CuL₃]²⁺ at collision energy of 16 eV. The CID process is quite different from [CuL₂]²⁺, the inter-ligand proton transfer dissociation channel was mainly observed (reaction 4).

[CuL₃]²⁺ → [CuL(L–H)]⁺ + [L+H]⁺ (4)

Fig. 5 CID spectra of [CuL₃]²⁺ at E_lab = 16 eV.

Generally the complex stabilization could be achieved by multiple ligations which facilitates delocalization of the Cu²⁺ charges onto the cytidine ligands. However, an additional ligand also increases the possibility of other competing dissociation pathways, such as proton transfer, neutral ligand loss, etc. In this experiment, one more ligand adding from [CuL₂]²⁺ to form [CuL₃]²⁺ makes the inter-ligand proton transfer dissociation dominate. The reason is probably relative to the structure of [CuL₃]²⁺. As we mentioned before, the molecule [CuL₂]²⁺ is very stable for its high peak intensity in the ESI spectrum and possible stable four coordinated molecular structure. The addition of one more cytidine ligand changes the planar coordination structure (Table 1), and the Cu^II only binds with 3 N(3) atoms. This coordination structure is not very stable, and the very low energy collision makes it go back to the planar structure again to form [CuL(L–H)]⁺ and [L+H]⁺ rather than [CuL₂]²⁺ and [L]. The reason is that the inter-ligand proton transfer is more favorable than the electron transfer, and the formation of [CuL(L–H)]⁺ and [L+H]⁺ is easier than the formation of [CuL₂]²⁺ and [L].

The CID spectrum of [CuL₄]²⁺ (m/z 518.0) at a collision energy of 24 eV is shown in Fig. 6. Only two primary dissociation channels could be identified, the inter-ligand proton transfer process forming [CuL(L–H)]⁺ and [L₂+H]⁺ (reaction 5a) and [CuL₂(L–H)]⁺ and [L+H]⁺ (reaction 5b). The structure for [CuL₄]²⁺ (Table 1) is a Cu^II coordinating with four N(3) in the same plane, and the two CID channels are reasonable and the fragmentation products are quite stable. The [L+H]⁺ ions can also come from the dissociation of [L₂+H]⁺ (reaction (6)) which was identified by the CID of [L₂+H]⁺.

[CuL₄]²⁺→ [CuL(L–H)]⁺ + [L₂ + H]⁺ (5a)

→ [CuL₂(L–H)]⁺ + [L + H]⁺ (5b)

[L₂ + H]⁺ → [L+H]⁺ + [L] (6)

Fig. 6 CID spectra of [CuL₄]²⁺ at E_lab = 24 eV.

About the structure of [L₂ +H]⁺, we assume that the two ligands bind together by hydrogen bonds. The structure of cytidine makes it possible to bind other ligands through hydrogen bonding. The hydrogen bonding guanine–cytidine or guanosine–cytidine pairs have been studied by several groups.^{57,72–74,76–78} Sindona et al.⁷⁰ used the formation of proton-bound heterodimer between nucleosidesetc. to evaluate the proton affinities. A calculated lowest energy minimized structure of N–1-methylcytosine proton dimmer was given. The structure shows the occurrence of three hydrogen bonds between the O(7), N(3) and N(8). The proton-bound cytidine dimer observed in our experiment has a similar structure (see the supporting information†).

Fig. 7 shows the CID spectrum of [CuL₅]²⁺ at a collision energy of 20 eV. The CID channels are different from other double charge cations, like [CuL_2-4]²⁺. One more ligand adding from [CuL₄]²⁺ to [CuL₅]²⁺ makes the neutral ligand loss channel dominate, reaction (7a). Besides this, a small dissociation channel to produce a protonated cytidine dimer, reaction (7b), was also observed.

[CuL₅]²⁺ → [CuL₄]²⁺ + [L] (7a)

→ [CuL₂(L–H)]⁺ + [L₂+H]⁺ (7b)

Fig. 7 CID spectra of [CuL₅]²⁺ at E_lab = 20 eV.

Fig. 8 is the CID spectrum of [CuL₆]²⁺ at a laboratory energy of 16 eV. Three dissociation channels were identified. They are neutral ligand loss (reaction (8a)), inter-ligand proton transfer to produce protonated cytidine dimer [L₂+H]⁺ and [CuL₃(L–H)]²⁺ (reaction (8b)) and protonated cytidine trimer [L₃+H]⁺ and [CuL₂(L–H)]²⁺ (reaction (8c)).

[CuL₆]²⁺ → [CuL₅]²⁺ + [L] (8a)

→ [CuL₃(L–H)]⁺ + [L₂ +H]⁺ (8b)

→ [CuL₂(L–H)]⁺ + [L₃ +H]⁺ (8c)

Fig. 8 CID spectra of [CuL₆]²⁺ at E_lab = 10 eV.

From the CID of the Cu²⁺–cytidine complex cations [CuL_2-6]²⁺, we could see that the Cu^II mainly shows the coordination number 4 and has a tendency to coordinate with cytidine at N(3) or N(3) and O(7) to form a planar structure, and good examples are [CuL₂]²⁺, [CuL(L–H)]⁺, [CuL₄]²⁺ and [CuL₃(L–H)]⁺; sometimes the Cu^II also shows the coordination number 3, and the typical example is [CuL₃]²⁺, but this structure is not very stable and it is easy to lose a protonated L. For the structure of [CuL_n]²⁺ (n > 4), we believe that the extra ligands will connect together with electrostatics and hydrogen bonds.

3.4 CID of Cu²⁺–cytidine-methane complex cations [CuL(MeOH)_1-2]²⁺

The methanol molecule was also observed to be involved in the complex formation. [CuL(MeOH)_1-2]²⁺ was observed in the ESI-MS spectrum of mixture of Cu²⁺ and cytidine (Fig. 1(a) and 1(b)). The CID spectra of [CuL(MeOH)]²⁺ (m/z 169.1) at a collision energy of 20 eV is shown in Fig. 9. Four main primary CID channels are observed: neutral methanol loss to from [CuL]²⁺ (m/z 153.1), reaction (9a), dissociation charge reduction to form [CuB(MeOH)]⁺ (m/z 205.1) and [R]⁺ (m/z 133.1), reaction (9b), dissociation charge reduction to produce [Cu(CH₂OH)(MeOH)]⁺ (m/z 125.1) and [R–CH₂OH]⁺ (m/z 213.1), reaction (9c), and dissociation charge reduction forming [Cu(MeOH)(BCOH)]⁺ (m/z 234.1), [C₃H₅O₂]⁺ (m/z 73) and neutral radical CH₂OH, reaction (9d). For [CuL(MeOH)₂]²⁺, only MeOH loss is observed in its CID spectrum.

[CuLMeOH)]²⁺ → [CuL]²⁺ + [MeOH] (9a)

→ [CuB(MeOH)]⁺ + [R]⁺ (9b)

→ [Cu(CH₂OH)(MeOH)]⁺ + [L–CH₂OH]⁺ (9c)

→ [Cu(MeOH)(BCOH)]⁺ + [C₃H₅O₂]⁺ + [CH₂OH] (9d)

Fig. 9 CID spectra of [CuL(CH₃OH)]²⁺ at E_lab = 10 eV.

The structure for [CuL(MeOH)]²⁺ shows that Cu^II has a coordination number of 3, N(3), O(7) of the L ligand and O of the MeOH. The fragmentation of [CuL(MeOH)]²⁺ mainly involves an extra MeOH loss or braking of the ribose. Reaction (9c) shows that the ligand base can also be lost. The structure of [CuL(MeOH)₂]²⁺ shows that the Cu^II has a coordination number of 4, 2 with O of the MeOH molecules and 2 with N(3), O(7) of the ligand.

3.5 Comparison of the formation and CID of Cu²⁺–cytidine with Cu²⁺–guanosine

The formation of Cu^II–guanosine and its CID chemistry have been previously investigated.⁴⁶ A comparison of results reported here of Cu²⁺–cytidine complexes indicates that these two molecules exhibit both similarities and differences in their formation and CID chemistry. Generally speaking, they both can form double charge complexes, [CuL_n]²⁺ and [CuL(MeOH)_m]²⁺, by the electronspray of the methanol and water solution of Cu²⁺ with ligands, and multiple competing dissociation channels occurred for [CuL₂]²⁺, but the dissociation channels for [CuL_n]²⁺ (n > 2) are quite simply dominated by one channel. The big difference of the CID chemistry for these two complexes is that the inter-electron transfer is the dominant channel for Cu²⁺–guanosine complexes, [CuL_n]²⁺(n > 2), while the inter-ligand proton transfer dominates the CID process of Cu²⁺–cytidine complexes, [CuL_n]²⁺(n > 2). For these big and complicated systems, it is too hard to provide the potential energy diagrams with energy barriers that bare no relationship to the final energies for the products through calculation, but the reasons could be attributed to their differences in ionization energy (IE) and proton affinity (PA). In other words, the balance between the attractive force of the hydrogen bonding and the repulsive electrostatic force determines the dissociation types.⁷⁹ The IE for cytidine (IE = 8.6 eV) is 0.6 eV bigger than that of guanosine (IE = 8.0 eV), and the PAs for them are comparable PA (cytidine) = 234.8 kcal mol⁻¹, PA (guanosine) = 236.8 kcal mol⁻¹.⁷⁷ The lower IE for guanosine makes the electron transfer preferable for Cu^II–guanosine complexes, [CuL_n]²⁺ (n > 2), while the higher IE for cytidine makes the inter-proton transfer preferred for Cu^II–cytidine complexes, [CuL_n]²⁺ (n > 2). The second difference is that, after coordination with Cu²⁺, guanosine still has enough sites to hydrogen-bond with other guanosine ligands, and there is no so-called magic number in Cu^II–guanosine complexes, [CuL_n]²⁺. For cytidine, after coordination with Cu^II, there are no extra sites for hydrogen-bonding, and the extra ligands only bind together through weak electrostatics, so the magic number 2 and 4 exist for their stable complex configurations.

4. Conclusion

The singly charged cations, [L+H]⁺, [L₂+H]⁺, and [B+H]⁺, and doubly charged cations, [CuL_n]²⁺, [CuL(MeOH)_n]²⁺, [Cu(L–H)L_n]⁺, (L = cytidine, B = cytosine) were formed by electronsprary of water and methanol solution of Cu(NO₃)₂ and cytidine. The collision-induced dissociations (CIDs) of [Cu(L–H)L_0-1]⁺, [CuL_2-6]²⁺ and [CuL(MeOH)_1-2]²⁺ were investigated in low energy using the ESI-MS/MS instrument; and the structure, thermal energy and entropy for the most of these complexes were calculated with ab initio and DFT methods through Gaussian 09 program. The broken parts for the [Cu(L–H)L_0-1]⁺ only on the ribose group of the ligand during the CID process, including ribose group loss ribose side chain and ribose ring braking. The inter-ligand proton transfer was observed in the CID of double charge Cu^II–cytidine complexes, [CuL_n]²⁺ (n = 2–6), and other channels, including charge reduction dissociation and neutral ligand loss were also observed. The peak distribution for [CuL_2-6]²⁺ shows that the Cu²⁺ likes to coordinate with N(3) or N(3) and O(7) to form a planar structure, which makes the [CuL₂]²⁺ and [CuL₄]²⁺ produce the highest peaks. Neutral MeOH loss and three kinds of charge reduction dissociation channels were presented in the CID of [CuL(MeOH)]²⁺. In the experiment, the Cu²⁺ shows the coordination number 3 or 4 in the complexes of Cu^II–cytidine and Cu^II–cytidine–MeOH. The N(3) and O(7) are mainly coordination sites for cytidine. The dimeric cytidine was produced in the CID of [CuL_n]²⁺ (n = 4–6), and could be attributed to be the interactions of two cytidinesviahydrogen bonds. The inter-ligand electron transfer from ligand to Cu²⁺ to produce a ligand radical cation was not observed in the CID of the doubly charged Cu^II–cytidine complexes, [CuL_n]²⁺ (n = 2–6), and the reasons can be attributed to the ligand's high ionization energy and proton affinity, which makes the inter-ligand proton transfer more favorable than electron transfer.

Acknowledgements

The authors gratefully acknowledge the financial support from General Foundation of Tianjin Science Committee for Applied Basic Research (No. 08JCYBJC00500) and the National Instrumentation Program (2011YQ170067).

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Footnote

† Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c2ra01293f/

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