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O2 Protonation Controls Threshold Behavior for N‑Glycosidic Bond Cleavage of Protonated Cytosine Nucleosides R. R. Wu and M. T. Rodgers* Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States S Supporting Information *

ABSTRACT: IRMPD action spectroscopy studies of protonated 2′-deoxycytidine and cytidine, [dCyd+H]+ and [Cyd+H]+, have established that both N3 and O2 protonated conformers coexist in the gas phase. Threshold collisioninduced dissociation (CID) of [dCyd+H]+ and [Cyd+H]+ is investigated here using guided ion beam tandem mass spectrometry techniques to elucidate the mechanisms and energetics for N-glycosidic bond cleavage. N-Glycosidic bond cleavage is observed as the major dissociation pathways resulting in competitive elimination of either protonated or neutral cytosine for both protonated cytosine nucleosides. Electronic structure calculations are performed to map the potential energy surfaces (PESs) for both N-glycosidic bond cleavage pathways observed. The molecular parameters derived from theoretical calculations are employed for thermochemical analysis of the energy-dependent CID data to determine the minimum energies required to cleave the N-glycosidic bond along each pathway. B3LYP and MP2(full) computed activation energies for N-glycosidic bond cleavage associated with elimination of protonated and neutral cytosine, respectively, are compared to measured values to evaluate the efficacy of these theoretical methods in describing the dissociation mechanisms and PESs for N-glycosidic bond cleavage. The 2′-hydroxyl of [Cyd+H]+ is found to enhance the stability of the N-glycosidic bond vs that of [dCyd+H]+. O2 protonation is found to control the threshold energies for N-glycosidic bond cleavage as loss of neutral cytosine from the O2 protonated conformers is found to require ∼25 kJ/mol less energy than the N3 protonated analogues, and the activation energies and reaction enthalpies computed using B3LYP exhibit excellent agreement with the measured thresholds for the O2 protonated conformers.

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nucleosides.17,18,20−23 In particular, we have examined the structures and relative stabilities of [dCyd+H]+ and [Cyd+H]+ in the gas phase using IRMPD action spectroscopy.18 Both N3 and O2 protonated conformers were found to coexist in the experimental population in roughly equal abundance, consistent with that reported by Filippi et al.17 in a parallel IRMPD study. In contrast, Ung et al.19 interpreted the measured IRMPD spectra of [dCyd+H]+ and partially deuterated [dCyd+H]+ in the hydrogen-stretching region as arising solely from N3 protonated conformers, and suggested that the O2 protonated conformers are largely absent. Our IRMPD action spectroscopy study18 indicated that the most stable N3 and O2 protonated conformers are highly parallel with the only significant difference being the position of the excess proton (see Figure 1). Both exhibit a preference for the anti nucleobase orientation and puckering of the sugar in a C2′-endo configuration. A weak noncanonical C6H···O5′ hydrogen bond provides modest stabilization to both the N3 and O2

INTRODUCTION 2′-Deoxycytidine and cytidine, as naturally occurring DNA and RNA nucleosides, are of great interest due to the fact that cytosine is the most basic nucleobase in aqueous environments.1 As a result, cytosine participates in noncanonical basepairing interactions under acidic conditions.2−7 For example, the protonated C+•C base pair and C+•G•C base triplet play important roles in stabilizing DNA tetrameric structures2,3,7−10 and triple helices, respectively.4−6 Protonation usually occurs at the N3 position11,12 in these C+•C and C+•G•C base-pairing interactions. In contrast, O2 protonation of cytosine occurs for the A•C+ base pairs observed in oligonucleotide duplexes.13 Because the N3 and O2 atoms of cytosine exhibit similar proton affinities,14 and protonation plays an important role in facilitating the formation of noncanonical base pairing interactions that greatly influence the biochemical properties of nucleic acids, studies of the protonated cytosine nucleosides, protonated 2′-deoxycytidine and cytidine, [dCyd+H]+ and [Cyd+H]+, have been performed in the gas phase to probe their intrinsic properties.14−19 IRMPD action spectroscopy has been firmly established as a highly potent approach for examining the gas-phase conformations of protonated DNA and RNA © 2016 American Chemical Society

Received: April 30, 2016 Revised: May 3, 2016 Published: May 9, 2016 4803

DOI: 10.1021/acs.jpcb.6b04388 J. Phys. Chem. B 2016, 120, 4803−4811

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The Journal of Physical Chemistry B

barrier associated with these pathways.32 However, the intrinsic dissociation mechanisms for N-glycosidic bond cleavage of cytosine nucleosides upon protonation remain unclear. In this work, we examine N-glycosidic cleavage of [dCyd+H]+ and [Cyd+H]+ in the gas phase using guided ion beam tandem mass spectrometry techniques in combination with theoretical electronic structure calculations. As our previous IRMPD action spectroscopy study18 has established that both N3 and O2 protonated conformers are formed by electrospray ionization and coexist in the experiments, we examine the N-glycosidic bond dissociation mechanisms for both N3 and O2 protonated conformers using energy-dependent collision-induced dissociation (CID) studies assisted by theoretically predicted potential energy surfaces (PESs) for the glycosidic bond cleavage pathways observed upon CID. These synergistic approaches allow the influence of the site of protonation on the intrinsic dissociation mechanisms and energetics for N-glycosidic bond cleavage of the protonated cytosine nucleosides to be elucidated. The influence of the 2′-hydroxyl substituent of protonated cytidine on the stability of the N-glycosidic bond is revealed via comparison of results for protonated 2′deoxycytidine and cytidine. Comparison to previous TCID studies of N-glycosidic bond cleavage of the protonated guanine nucleosides, [dGuo+H]+ and [Guo+H]+,33 allows the effects of the nucleobase identity on the N-glycosidic bond dissociation mechanisms and stabilities to be elucidated.

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EXPERIMENTAL AND THEORETICAL SECTION General Procedures. Tandem mass spectrometry approaches are employed to measure absolute cross sections for collision-induced dissociation (CID) of the protonated forms of the cytosine nucleosides with Xe34−36 as a function of collision energy using a custom-built guided ion beam tandem mass spectrometer (GIBMS). This GIBMS instrument37 and the data acquisition and handling procedures typically employed for such CID studies have previously been described in detail.38 However, this instrument has since undergone modifications to include a custom-built electrospray ionization (ESI) source, rf ion funnel, and hexapole ion guide collision cell interface to enable a wider variety of biologically relevant ions such as those examined here to be investigated.39−42 Details regarding the data acquisition and handling procedures are described in the Supporting Information. Theoretical Calculations. N3 and O2 protonated conformers of the cytosine nucleosides have been shown to coexist in the gas phase, and thus, both are examined as stable reactants for the CID experiments performed here.18 The Gaussian 09 suite of programs43 are used to completely characterize the PESs for the two N-glycosidic bond cleavage pathways observed upon CID including determination of the stable structures and relative energies44−46 of all intermediates and the CID products as well as the transitions states (TSs)47,48 that connect these species. Details regarding the theoretical calculations as well as tables summarizing the molecular parameters (vibrational frequencies and rotational constants, Tables S1−S4) of the reactants, TSs, and CID products needed for thermochemical analysis of the CID cross sections are provided in the Supporting Information. Thermochemical Analysis. The threshold regions of the CID cross sections measured for the products arising from glycosidic bond cleavage of the protonated forms of the cytosine nucleosides are modeled using procedures developed elsewhere9,10,35,36,38,49−60 that have been found to be robust for

Figure 1. Chemical structures of 2′-deoxycytidine (dCyd) and cytidine (Cyd). N3 and O2 protonated ground-state structures of [dCyd+H]+ and [Cyd+H]+. Structures optimized at the B3LYP/6-311+G(d,p) level of theory with relative energies (in kJ/mol) at the B3LYP/6311+G(2d,2p) (in blue) and MP2(full)/6-311+G(2d,2p) (in red) levels of theory are also shown.

protonated conformers. In contrast, theory predicts that the neutral forms of dCyd and Cyd in the gas phase18 are stabilized by somewhat stronger canonical hydrogen-bonding interactions than the protonated species, thus altering their conformations. Specifically, neutral dCyd exhibits a preference for the syn orientation of cytosine, which is stabilized by an O5′H···O2 hydrogen bond. In neutral Cyd, the presence of the 2′-hydroxyl substituent enables formation of hydrogen-bonding interactions that bridge between the 2′- and 3′-hydroxyls of the sugar and the carbonyl moiety of the anti oriented cytosine nucleobase. Nucleic acids carry important biological and genetic information. The integrity of the information that is contained within the primary sequence of nucleic acids is maintained by the N-glycosidic bonds by which the nucleobases are bound to the phosphate−sugar backbone. Therefore, under normal physiological conditions, the N-glycosidic bonds of nucleic acids are extremely stable. However, the nucleobases are vulnerable to damage and/or modification, and excision of unnatural nucleobases, and replacement by their canonical counterparts, only becomes feasible when the N-glycosidic bond is cleaved.24 Therefore, N-glycosidic bond cleavage is involved in nucleobase salvage25−27 and base excision repair pathways,28,29 which require various enzymes to catalyze these reactions.24,30,31 Acidic conditions have been found to facilitate N-glycosidic bond cleavage by reducing the high activation 4804

DOI: 10.1021/acs.jpcb.6b04388 J. Phys. Chem. B 2016, 120, 4803−4811

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The Journal of Physical Chemistry B

For both protonated cytosine nucleosides, the primary and lowest energy dissociation pathways involve endothermic loss of the protonated nucleobase, [Cyt+H]+, or loss of the neutral nucleobase, Cyt. The corresponding CID reactions are represented by eqs 1 and 2, respectively.

thermochemical analysis of CID cross sections in general, and specifically for N-glycosidic bond cleavage of the protonated forms of the guanine nucleosides.33 The 0 K activation energies (AEs) and heats of reaction (ΔHrxns) determined from the threshold analyses are converted to 298 K enthalpies and free energies. Details regarding the thermochemical analysis procedures employed here are discussed in the Supporting Information.

[Nuo+H]+ + Xe → [Cyt+H]+ + [Nuo−Cyt] + Xe (1)

[Nuo+H]+ + Xe → [Nuo−Cyt+H]+ + Cyt + Xe

(2)

These competitive CID pathways both involve N-glycosidic bond cleavage with the excess proton retained by either the nucleobase or the sugar moieties, respectively. Comparisons of the apparent thresholds for these primary CID pathways suggest that the 2′-hydroxyl substituent strengthens the N-glycosidic bond of [Cyd+H]+ vs that of [dCyd+H]+ as the apparent thresholds shift to higher energies. Competition between these two primary CID channels is slightly greater for [dCyd+H]+ than [Cyd+H]+, similar to that observed for [dGuo+H]+ and [Guo+H]+.33 Sequential fragmentation of the initially formed protonated sugar moieties formed by reaction 2, [Nuo-Cyt+H]+, is prevalent, but still minor, at elevated collision energies. The mass-to-charge (m/z) ratios for each sequential ionic product observed and the chemical compositions proposed for these products (shown in Figure 2) are summarized in Table S5 of the Supporting Information. As these sequential reactions are not of interest in the current study, they will not be discussed further, except to note that we have added the CID cross sections of these sequential fragments into the [Nuo−Cyt+H]+ product cross sections (eq 2) to ensure accurate threshold analysis. Theoretical Results. In our previous IRMPD study of the protonated cytosine nucleosides, B3LYP was found to favor N3 protonation, whereas MP2 prefers O2, and both N3 and O2 protonated conformers were found to contribute to the experimental population.18 The B3LYP barrier to tautomerization of gas-phase N3 and O2 protonated conformers was computed to be 153.9 kJ/mol (153.4 kJ/mol MP2), significantly in excess of the internal energy available to these species (∼40 kJ/mol) such that interconversion is only possible when facilitated by solvent. Thus, here we probe the mechanisms and energetics for N-glycosidic bond cleavage arising from both N3 and O2 protonated stable conformations. The relative energies of all relevant structures along the PESs for N-glycosidic bond cleavage are listed in Tables S6 and S7 for the O2 and N3 protonated systems, respectively. Theoretical estimates for the AEs for the CID pathway described by eq 1 are provided by the difference in energy between the reactant protonated nucleosides and their corresponding rate-determining TSs, whereas theoretical estimates for the reaction enthalpies (ΔHrxns) for the CID pathway described by eq 2 are given by the difference in energy between the reactant protonated nucleosides and their corresponding CID products. N-Glycosidic Bond Cleavage of the Protonated Cytosine Nucleosides. The calculated PESs for elimination of [Cyt+H]+ and Cyt from O2 protonated [dCyd+H]+ and [Cyd+H]+ are shown in Figures 3 and 4, respectively. The predicted mechanism for N-glycosidic bond cleavage resulting in elimination of [Cyt+H]+ (eq 1) involves two major steps: (1) elongation of the N-glycosidic bond accompanied by conformational changes that eventually lead to the formation of

Figure 2. Cross sections for collision-induced dissociation of [dCyd+H]+ and [Cyd+H]+ with Xe as a function of kinetic energy in the center-of-mass frame (lower x-axis) and laboratory frame (upper x-axis), parts a and b, respectively. The two primary CID pathways leading to elimination of protonated and neutral cytosine via N-glycosidic bond cleavage are shown as solid blue and red circles, respectively. Ionic products from sequential dissociation of [dCyd-Cyt+H]+ and [Cyd-Cyt+H]+ are shown as small open red symbols; see Table S5 of the Supporting Information for details. Data are shown for a Xe pressure of 0.2 mTorr.

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RESULTS Cross Sections for Collision-Induced Dissociation. Experimental cross sections were acquired for the interaction of Xe with the protonated cytosine nucleosides, where [Nuo+H]+ = [dCyd+H]+ or [Cyd+H]+. Figure 2 shows the measured CID cross sections as a function of collision energy. 4805

DOI: 10.1021/acs.jpcb.6b04388 J. Phys. Chem. B 2016, 120, 4803−4811

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Figure 3. Calculated potential energy surfaces for glycosidic bond cleavage of O2 protonated [dCyd+H]+ leading to elimination of [Cyt+H]+ and Cyt, parts a and b, respectively. Structures are optimized at the B3LYP/6-311+G(d,p) level of theory with relative energies (in kJ/mol) calculated at the B3LYP/6-311+G(2d,2p) (in blue) and MP2(full)/6-311+G(2d,2p) (in red) levels of theory.

Figure 4. Calculated potential energy surfaces for glycosidic bond cleavage of O2 protonated [Cyd+H]+ leading to elimination of [Cyt+H]+ and Cyt, parts a and b, respectively. Structures are optimized at the B3LYP/6-311+G(d,p) level of theory with relative energies (in kJ/mol) calculated at the B3LYP/6-311+G(2d,2p) (in blue) and MP2(full)/6-311+G(2d,2p) (in red) levels of theory.

a “proton-bound dimer” intermediate between the resulting oxacarbenium-ion like sugar and nucleobase moieties and (2) proton transfer between the sugar and nucleobase moieties (from C2′ to N1). In contrast, loss of the neutral nucleobase (eq 2) simply involves elongation of the N-glycosidic bond. The increase in the barriers to activated dissociation predicted for [Cyd+H]+ (Figure 4) vs those predicted for [dCyd+H]+ (Figure 3) foreshows an increase in N-glycosidic bond stability due to the presence of the 2′-hydroxyl substituent. Indeed, both B3LYP and MP2 predict that ∼25 kJ/mol more energy is necessary to cleave the N-glycosidic bond of [Cyd+H]+ than [dCyd+H]+ along either dissociation pathway. The PESs shown in Figures 3 and 4 are examined in greater detail in the Supporting Information. The calculated PESs for eliminating [Cyt+H]+ or Cyt from N3 protonated [dCyd+H]+ and [Cyd+H]+ are shown in Figures S1 and S2, respectively. The dissociation mechanisms predicted for N-glycosidic bond cleavage of N3 protonated [dCyd+H]+ and [Cyd+H]+ are remarkably similar to those for the O2 protonated nucleosides. However, the noncanonical tautomer of neutral cytosine eliminated from the N3 protonated nucleosides is less stable than the noncanonical tautomer eliminated from the O2 protonated nucleosides. As a result, the dissociation asymptotes for elimination of neutral cytosine from N3 protonated [dCyd+H]+ and [Cyd+H]+ lie >20 kJ/mol above those for the O2 protonated nucleosides. Analysis of the Cross Section Thresholds for N-Glycosidic Bond Cleavage. Competitive analyses of the

CID product cross sections for N-glycosidic bond cleavage of [dCyd+H]+ and [Cyd+H]+ (as described by eqs 1 and 2) using the empirical threshold law of eq S1 (see the Supporting Information) were performed to extract threshold energies for these processes. The data were competitively analyzed using tight TS (TTS) and switching (SW) TS models for both the O2 and N3 protonated forms of the cytosine nucleosides. The results of these analyses are listed in Tables S8 and S9, respectively. Elimination of [Cyt+H]+ (eq 1) is analyzed using a TTS model, TS1 or TS2, whereas elimination of Cyt (eq 2) is analyzed using a SW TS model. For the O2 protonated forms of the cytosine nucleosides, the data are modeled using TS1 and a SW TS and TS2 and a SW TS, as TS1 and TS2 are predicted by both theoretical models to be very similar in energy. Very good agreement between the AEs and ΔHrxns extracted from the threshold analyses and those theoretically predicted is found. Specifically, TS2 and a SW TS model for [dCyd+H]+ and TS1 and a SW TS model for [Cyd+H]+ yield the best agreement with the theoretical values. The results of these analyses are shown in Figure 5. For the N3 protonated forms of [dCyd+H]+ and [Cyd+H]+, only TS2 and a SW TS are used to analyze the thresholds, as both theoretical models predict that TS2 is higher in energy than TS1. In particular, the ΔHrxns extracted from the threshold analyses exhibit poor agreement with the theoretical values for the N3 protonated forms because the dissociation asymptotes for elimination of neutral Cyt from [dCyd+H]+ and [Cyd+H]+ lie >20 kJ/mol above those for the O2 protonated analogues. The measured threshold energies 4806

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DISCUSSION

Validation of the Proposed Mechanisms for NGlycosidic Bond Cleavage and the Theoretical Approaches Examined. The measured and theoretically predicted AEs and ΔHrxns for N-glycosidic bond cleavage of the O2 and N3 protonated forms of the cytosine nucleosides are compared in Figure 6. For both primary CID pathways, [Cyd+H]+ requires more energy to cleave the glycosidic bond than [dCyd+H]+. The TCID measured AEs for elimination of [Cyt+H]+ and the ΔHrxns for loss of Cyt clearly indicate that competition between these two CID pathways is slightly greater for [dCyd+H]+ than for [Cyd+H]+. For the O2 protonated cytosine nucleosides, the mean absolute deviations (MADs) between theory and experiment for the AEs (eq 1) are 1.4 ± 1.1 kJ/mol (B3LYP) and 36.9 ± 0.6 kJ/mol (MP2), whereas those for the ΔHrxns (eq 2) are 3.1 ± 1.8 kJ/mol (B3LYP) and 21.1 ± 3.5 kJ/mol (MP2). In contrast, for the N3 protonated species, the MADs between theory and experiment for the AEs are 4.0 ± 4.3 kJ/mol (B3LYP) and 37.6 ± 2.1 kJ/mol (MP2), whereas those for the ΔHrxns are 15.7 ± 10.3 kJ/mol (B3LYP) and 41.9 ± 8.8 kJ/mol (MP2). The B3LYP MADs clearly show that the mechanisms elucidated for N-glycosidic bond cleavage are consistent with the experimental results. Both levels of theory exhibit better agreement with experiment for the O2 protonated species, and that much better agreement between theory and experiment is found for B3LYP than MP2. In particular, the AEs predicted by B3LYP for elimination of [Cyt+H] + from the N3 protonated conformers agree reasonably well with the measured thresholds, as these values differ only slightly from those of the O2 analogues. However, the ΔHrxns predicted for elimination of Cyt from the N3 protonated conformers exhibit poor agreement with the measured values. The high energy of the noncanonical tautomer of neutral Cyt eliminated from the N3 protonated nucleosides is too energetic to affect the threshold behavior in the presence of O2 protonated species. As can be seen in Figure 6, MP2 systematically overestimates the energetics but predicts behavior parallel to B3LYP. The MP2 BSSE corrections appear to overcorrect the ΔHrxns. Effects of N3 vs O2 Protonation on N-Glycosidic Bond Cleavage. The agreement between experiment and theory determined here indicates that O2 protonation controls the energetics for N-glycosidic bond cleavage of the protonated cytosine nucleosides at or near threshold energies in the gas phase. The measured threshold energies for elimination of neutral Cyt, reaction 2, are >55 kJ/mol larger than those for

Figure 5. Zero-pressure-extrapolated CID product cross sections of [dCyd+H]+ and [Cyd+H]+ in the threshold region, parts a and b, respectively. The solid lines show the best fits to the data using eq S1 convoluted over the ion and neutral kinetic energy distributions. The dotted lines show the model cross sections in the absence of experimental kinetic energy broadening for reactants with an internal energy corresponding to 0 K.

and the theoretically calculated AEs and ΔHrxns are compared for the O2 and N3 protonated species in Tables 1 and 2.

Table 1. Activation Energies and Reaction Enthalpies for N-Glycosidic Bond Cleavage of O2 Protonated [dCyd+H]+ and [Cyd+H]+ at 0 K in kJ/mola TCIDb CID products

AE

[Cyt+H]+ from [dCyd+H]+ [dCyd-Cyt+H]+ [Cyt+H]+ from [Cyd+H]+ [Cyd-Cyt+H]+ AEUf MADg

85.9 (5.8)

B3LYPc ΔHrxn

AE

145.7 (7.7) 111.9 (2.9) 4.4 (2.1)

175.6 (5.8) 6.8 (1.3)

MP2d ΔHrxne

AE

ΔHrxne

83.8 79.1 111.3 90.7

80.7 147.5 74.5 171.2

123.2 117.9 148.4 124.0

99.4 169.3 97.2 194.2

1.4 (1.1)

3.1 (1.8)

36.9 (0.6)

21.1 (3.5)

Present results, uncertainties are listed in parentheses. bTCID AEs and ΔHrxns obtained from competitive threshold analysis. cCalculated at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory including ZPE corrections. dCalculated at the MP2(full)/6-311+G(2d,2p)// B3LYP/6-311+G(d,p) level of theory including ZPE corrections. eIncluding ZPE and BSSE corrections. fAverage experimental uncertainty (AEU). g The mean absolute deviation (MAD) between calculated and experimentally obtained AEs and ΔHrxns. a

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Table 2. Activation Energies and Reaction Enthalpies for N-Glycosidic Bond Cleavage of N3 Protonated [dCyd+H]+ and [Cyd+H]+ at 0 K in kJ/mola TCIDb CID products +

AE +

[Cyt+H] from [dCyd+H] [dCyd-Cyt+H]+ [Cyt+H]+ from [Cyd+H]+ [Cyd-Cyt+H]+ AEUg MADh

B3LYPc ΔHrxn

AE

86.8 (3.9) 150.5 (4.6) 98.4 (2.9) 3.4 (0.7)

186.2 (4.7) 4.7 (0.1)

MP2d ΔHrxn

e

AE

ΔHrxne

85.9 73.7 91.4 77.6

85.2 173.4 90.6 194.6

125.8 117.2 134.5 116.1

101.6 198.6 110.2 221.9

4.0 (4.3)

15.7 (10.3)

37.6 (2.1)

41.9 (8.8)

Present results, uncertainties are listed in parentheses. bTCID AEs and ΔHrxns obtained from competitive threshold analysis. cCalculated at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory including ZPE corrections. dCalculated at the MP2(full)/6-311+G(2d,2p)// B3LYP/6-311+G(d,p) level of theory including ZPE corrections. eIncluding ZPE and BSSE corrections. fAverage experimental uncertainty (AEU). g The mean absolute deviation (MAD) between calculated and experimentally obtained AEs and ΔHrxns. a

N-glycosidic bond cleavage of N3 protonated conformers in aqueous solutions. Therefore, O2 protonation may be even more important in controlling the energetics for N-glycosidic bond cleavage in the condensed phase. The Effect of the 2′-Hydroxyl Substituent. The stable conformers of the protonated cytosine nucleosides shown in Figure 1 are remarkably similar and suggest that the 2′-hydroxyl substituent does not markedly alter the structures of [dCyd+H]+ vs [Cyd+H]+ except that it enables formation of a hydrogen bond between the sugar hydroxyls. The PESs predicted for N-glycosidic bond cleavage of [dCyd+H]+ and [Cyd+H]+ illustrated in Figures 3 and 4 (O2 protonated), and Figures S1 and S2 (N3 protonated), respectively, are also remarkably similar, and thus, the 2′-hydroxyl substituent also does not significantly influence the dissociation mechanisms. However, both the calculated and measured energetics indicate that the 2′-hydroxyl increases the AE and ΔHrxn for N-glycosidic bond cleavage of [Cyd+H]+ vs [dCyd+H]+, and thus the 2′-hydroxyl substituent enhances the stability of the N-glycosidic bond of [Cyd+H]+ over [dCyd+H]+. The 2′-hydroxyl substituent also leads to a slight decrease in the competition between the two primary dissociation pathways observed for [Cyd+H]+ vs [dCyd+H]+. Influence of Nucleobase Identity on N-Glycosidic Bond Stability. The gas-phase proton affinities (PAs) of cytosine and guanine are similar (ΔPA < 10 kJ/mol).61 As a result, the cytosine and guanine nucleobases might be expected to exhibit similar leaving group propensities and thus AEs in N-glycosidic bond cleavage processes. Indeed, the protonated cytosine nucleosides are found to require a similar amount of energy to activate the N-glycosidic bond as compared to the analogous protonated guanine nucleosides.33 In particular, the AEs measured here for [dCyd+H]+ and [Cyd+H]+ are only 7.7 and 2.9 kJ/mol less than those previously determined for [dGuo+H]+ and [Guo+H]+, respectively.33 The measured AEs suggest that protonated cytosine is a slightly better leaving group than protonated guanine. The differences between the measured AEs and ΔHrxns of these protonated nucleosides, i.e., the extent of competition between the two N-glycosidic bond cleavage reactions, increases in the order [dCyd+H] + < [Cyd+H]+ < [dGuo+H]+ < [Guo+H]+, consistent with the relative PAs of the sugar and nucleobase moieties, where sugar < cytosine < guanine.61 The anti nucleobase orientation of the protonated cytosine and guanine nucleosides, which although constrained (and stabilized) by a weak noncanonical hydrogen bond, leads to relatively facile elimination of the protonated nucleobase, and thus modest N-glycosidic bond stabilities.

Figure 6. Theoretical versus experimental 0 K activation energies (AEs) and reaction enthalpies (ΔHrxns) for N-glycosidic bond cleavage of [dCyd+H]+ and [Cyd+H]+. All values are taken from Tables 1 and 2. The black diagonal line indicates perfect agreement between theory and experiment, whereas the red dashed line is offset from perfect agreement by the MAD between MP2 and experiment for the AEs.

elimination of protonated Cyt, reaction 1, for both protonated cytosine nucleosides. Competition between reactions 1 and 2 is insignificant until a few eV above the onset for N-glycosidic bond cleavage. Clearly, reaction 2 is much less important than reaction 1, and would be expected to be even less important in the condensed phase. The excellent and fairly good MADs between B3LYP and experiment achieved for the AEs for reaction 1 for the O2 and N3 protonated species, respectively, are consistent with and further enhance the conclusion determined from our IRMPD spectroscopy study that both types of tautomeric conformations are populated by ESI.18 The relative energies along the PESs for reaction 1 for both O2 and N3 protonated conformers of the protonated cytosine nucleosides in an aqueous polarizable continuum model (PCM) were also calculated; the results are summarized in Tables S10 and S11, respectively. Comparisons of the calculated AEs in aqueous solution listed in Tables S10 and S11 to those in vacuo listed in Tables S6 and S7, respectively, suggest that solvation does not markedly affect the AEs for the O2 protonated conformers. In contrast, solvation increases the AEs associated with N3 protonated [dCyd+H]+ and [Cyd+H]+ by ∼15 kJ/mol, indicating that solvation may impede 4808

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CONCLUSIONS Collision-induced dissociation of [dCyd+H]+ and [Cyd+H]+ leads to cleavage of the N-glycosidic bond, and results in elimination of protonated and neutral cytosine in competition. The mechanisms proposed for the N-glycosidic bond cleavage pathways observed for [dCyd+H]+ and [Cyd+H]+, and consistent with the energy dependent CID data, are highly parallel and involve two major steps. In the first step, the protonated cytosine nucleosides undergo N-glycosidic bond elongation. Glycosidic bond elongation is accompanied by changes in the orientation of the nucleobase that align the N1 atom of cytosine with the H2′ atom of the sugar moiety. In the second step, proton transfer from the sugar to cytosine facilitates smooth dissociation of the protonated nucleoside via elimination of [Cyt+H]+. Both [dCyd+H]+ and [Cyd+H]+ also undergo N-glycosidic bond elongation along another pathway that facilitates elimination of neutral Cyt. The threshold regions of the CID cross sections for these activated dissociation pathways are analyzed to extract activation energies for elimination of [Cyt+H]+ and reaction enthalpies for elimination of Cyt. Although the 2′-hydroxyl substituent does not significantly alter the structure or mechanism for N-glycosidic bond cleavage of [Cyd+H]+ vs that of [dCyd+H]+, it does stabilize the glycosidic bond by ∼25 kJ/mol. Comparisons between experiment and theory indicate that B3LYP does a better job of predicting the PESs than MP2, and that the O2 protonated nucleosides control the reactivity at or near threshold energies. Much poorer agreement between theory and experiment is found for the N3 protonated analogues, particularly for the loss of the neutral nucleobase pathway. Results based on the use of an aqueous PCM model suggest that solvent actually impedes N-glycosidic bond cleavage of the N3 protonated species, which further confirms the significant role that O2 protonation plays in the structures and N-glycosidic bond cleavage processes of the cytosine nucleosides in both the gas and solution phases.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by a grant from the Chemical Measurement & Imaging Program within the Division of Chemistry of the National Science Foundation, CHE-1409420. R.R.W. gratefully acknowledges financial support from Wayne State University via Thomas C. Rumble Graduate and Summer Dissertation Fellowships. The authors also thank WSU C&IT for access to and support of the computational resources used to perform this work.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b04388. Complete citation for ref 43; details regarding the experimental and theoretical methods, data handling, and analysis procedures; tables of molecular parameters including vibrational frequencies (scaled by a factor of 0.99), average internal energies (at 298 K), and rotational constants as well as the relative energies of all species accessed along the PESs for N-glycosidic bond cleavage of the O2 and N3 protonated forms of the cytosine nucleosides; proposed chemical compositions of the CID fragments derived from sequential dissociation of the primary CID products of reaction 2, [dCyd-Cyt+H]+ and [Cyd-Cyt+H]+ at elevated energies; fitting parameters for threshold analyses using eq S1 of the CID product cross sections associated with N-glycosidic bond cleavage of [dCyd+H]+ and [Cyd+H]+; relative energies along the PESs calculated for elimination of protonated cytosine from the O2 and N3 protonated forms of the cytosine nucleosides in a polarizable continuum consistent with an aqueous environment; 0 and 298 K enthalpies and free energies for N-glycosidic bond cleavage of the O2 protonated forms of the cytosine nucleosides (PDF) 4809

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