To Jump or Not To Jump? CÎ± Hydrogen Atom Transfer in Post

Article pubs.acs.org/JPCA

To Jump or Not To Jump? Cα Hydrogen Atom Transfer in Post-cleavage Radical-Cation Complexes Benjamin J. Bythell* Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, United States S Supporting Information *

ABSTRACT: Conventionally, electron capture or transfer to a polyprotonated peptide ion produces an initial radical-cation intermediate which dissociates “directly” to generate complementary cn′ and zm• sequence ions (or ions and neutrals). Alternatively, or in addition, the initial radical-cation intermediate can undergo H• migration to produce cn• (or cn − H•) and zm′ (or zm• + H•) species prior to complex separation (“nondirect”). This reaction significantly complicates spectral interpretation, creates ambiguity in peak assignment, impairs effective algorithmic processing (reduction of the spectrum to solely 12C m/z values), and reduces sequence ion signal-to-noise. Experimental evidence indicates that the products of hydrogen atom transfer reactions are substantially less prevalent for higher charge state precursors. This effect is generally rationalized on the basis of decreased complex lifetime. Here, we present a theoretical study of these reactions in post N−Cα bond cleavage radical-cation complexes as a function of size and precursor charge state. This approach provides a computational estimate of the barriers associated with these processes for highly charged peptides with little charge solvation. The data indicate that the H• migration is an exothermic process and that the barrier governing this reaction rises steeply with precursor ion charge state. There is also some evidence for immediate product separation following N−Cα bond cleavage at higher charge state.

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INTRODUCTION Electron capture and electron transfer dissociation1,2 (ECD and ETD; together EXD) are increasingly utilized methods of fragmentation of protein, peptide, and other gas-phase ions. Electron capture/transfer by a multiply charged precursor ion, [M + xH]x+, generates a charge-reduced, radical-cation complex in which bond cleavage occurs, prior to “direct” dissociation2−5 (Scheme 1a). Unlike the ubiquitous, complementary,4 collisionactivated-dissociation (CAD) methods, which proceed by way of closed-shell chemistries6 and cleave protonated amide bonds, −C(O)−NH2+− (Figure 1), the open-shell radical cations generated by electron capture/transfer fragment primarily, but not exclusively, by cleavage of the N−Cα bond,2 i.e., those between the amide nitrogen and adjacent Cα atom. Conventionally, this produces a series of N-terminal, cn′, and C-terminal, zm• sequence ions (where the number of residues is N = n + m) that are used to decipher the primary sequence of the analyte ion;3 the difference in mass-to-charge of consecutive sequence ions (for example, m/z(c5′) − m/z(c4′)) is equal to a single amino acid residue (or modified residue), Figure 1. Regrettably, this simple, but elegant explanation represents an idealized situation, rather than the commonly encountered one. It turns out that particularly for peptides with lower precursor charge state, the situation can be and often is a lot more complicated.5,7−13 Two factors that contribute to the complexity of ECD/ETD spectra are hydrogen atom transfers7−10,12,13 and side-chain loss reactions.7,11,14−20 In the present manuscript, we concentrate on the former. Hydrogen atom transfer between sequence ions while electron capture/transfer dissociation7−10,12,13 occurs significantly complicates spectral interpretation, creates ambiguity in peak assignment, © 2012 American Chemical Society

impairs effective algorithmic processing (reduction of the spectrum to solely 12C m/z values), and reduces sequence ion signal-to-noise. These reactions are generally believed to occur in the ground state radical-cation intermediate generated via electron capture/transfer.7,9,10,12 After electron capture/transfer, N−Cα bond cleavage occurs promptly, whereas complex dissociation can take substantially longer.21,22 The complementary cn′ and zm• species that constitute the initial radicalcation intermediate undergo intracomplex H• migration to produce cn• (or cn′ − H•) and zm′ (or zm• + H•) species prior to complex separation (Scheme 1b). In turn, this results in additional peaks in the mass spectrum at either +1.0078 m/z for the zm′ (or zm• + H•) ion or −1.0078 m/z for the cn• (or cn′ − H•) ion (Figure 2). Fourier transform-ion cyclotron resonance mass spectrometers23,24 are capable of distinguishing the zm′ peak from the 13C12Cc−1 zm• peak. Unfortunately, even this is not always possible due to peak coalescence25−27 and a balance has to be struck between resolving power and experiment signal-tonoise (increased resolving power requires more time but decreases the signal magnitude per scan). Consequently, the nominal zm• ion isotopic distribution will often be a superposition of the zm• and zm′ distributions if H• transfer reactions have occurred. Unless the H• transfer reactions go to completion in every case (i.e., only cn• and zm′ ions are detected for a given N−Cα bond cleavage), the signal magnitude is Special Issue: Peter B. Armentrout Festschrift Received: May 30, 2012 Revised: July 16, 2012 Published: July 18, 2012 1189

dx.doi.org/10.1021/jp305277v | J. Phys. Chem. A 2013, 117, 1189−1196

The Journal of Physical Chemistry A

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Scheme 1. Schematic Representation of the Generally Accepted Model7,9,10,12 for H• Transfer after Electron Capturea

a (a) No H• transfer (“direct” pathway) gives cn′ and zm• sequence ions. (b) Intracomplex H transfer prior to dissociation (“indirect” pathway) yields cn• and zm′ sequence ions.

Hydrogen atom transfer in the radical-cation intermediate generated via electron capture/transfer has been examined by experiment.7−10,12,28,29 O’Connor et al.9 used selected deuterium labeling of glycine residue Cα hydrogens in a series of synthetic peptides to elucidate the effect further. Based on cn′ ion peak distributions, their results provided a conservative estimate of H• transfer degree, with Cα deuterium scrambling percentages of ∼20%; i.e., the experimentally undeterminable H• transfer degree may be substantially greater. This work also illustrated a sequence/local structural dependence on the degree of Cα deuterium scrambling,9 which lead to the obvious conclusion that the gas-phase structure(s) present after the initial N−Cα bond cleavage influence the favorability of subsequent H• transfer processes.21,30−32 Resonant ejection22 of the m/z that corresponds to the charge reduced radicalcation complex intermediate has also been shown to decrease the relative proportion of H• transfer products in some cases. This is logical as increased complex lifetime means greater time for a H• transfer reaction to occur prior to dissociation is available.

Figure 1. Summary of the nomenclature of peptide sequence and fragmentation.

diluted into two peaks (and their isotopomers) rather than one as illustrated in Figure 2.

Figure 2. Representative mass spectral segments showing z4 (left) and c6 (right) peak distributions generated from electron capture dissociation of [SDREYPLLIR + 3H]3+. 1190



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provide a useful starting point for subsequent studies on larger, more peptide sequencing-relevant systems.

Various approaches to reduce the lifetime of the charge-reduced radical-cation complex of the multiply protonated peptide/ protein have been employed.12,13,33−35 These approaches involve an increase in the internal energy (by collision or with photons) of either the precursor protonated peptide, or charge-reduced radical-cation complex itself to disrupt intramolecular/intracomplex bonds and facilitate prompt separation, broadly termed activated ion (AI)-ECD/ETD. Contextually, ECD performed at low temperatures shows fewer product ions than that performed at room temperature.36 Additionally, variation of incident electron energy has been shown to affect H• transfer reactions prevalence too,12 although sometimes with concomitant greater propensity for side-chain losses.7,12 Although certainly helpful means to limit the degree of reaction, regrettably none of these techniques offers complete removal of H• transfer products. Statistical analyses of large data sets of ECD data10,28,37 generated from doubly charged protonated peptide precursor ions (electron energy ∼1.5 eV, 50 ms irradiation period) have also been undertaken. Savitski et al.10 showed an “occurrence frequency of 47% compared to the zm• ion”. On the basis of analysis of variance arguments, the authors claim that the identity of the N-terminal residue of the zm• ion (i.e., the initial site of the radical after N−Cα bond cleavage) was clearly the most important factor in H• transfer propensity, followed by the identity of the C-terminal residue of the initially formed cn′ ion (i.e., the residue most likely to initially be close to the newly formed Cα radical). Furthermore, clear differences on the degree of these processes as a function of the identity of the N-terminal residue of the zm• ion, reflect drastically different reactivities to H• transfer of the Cα radicals present in the charge-reduced radical-cation complex. For example, when glycine, aspartic acid, serine, and threonine are the N-terminal residue of the initially formed zm• ion, H• transfer to form the corresponding zm′ is substantially more prevalent than for other residues. A recent study of peptides fragmentation by ETD29 provided evidence that aspartic acid, glutamic acid, and glutamine residues also enhance H-atom transfer after N−Cα bond cleavage in their vicinity too. In agreement with a recent, comparatively small systematic study8 and earlier individual analyte findings,38 work within our group indicates that higher precursor, [M + xH]x+, charge states (x ≥ 3) generally result in decreased/no detectable hydrogen migration between complementary product ions and thus fewer cn• and zm′ ions. This raised several questions as to why this effect is observed: (1) Are the cn• and zm′ product ions potentially generated from higher charge state precursors still30−32 energetically favorable relative to the detected cn′ and zm• ions? In other words, is ΔHreaction(cn′ + zm• ⇒ cn • + zm′) negative (exothermic). (2) Does the barrier to H• transfer becomes unfeasibly large as precursor charge state increases? (3) Does the lifetime of the radical-cation complex reduce sufficiently to prevent the H• transfer reaction? i.e., immediate product separation? (4) How does the recombination energy which increases with charge state contribute? (5) Is some combination of these possibilities the explanation? Here we present a computational investigation of these factors using model systems. The comparatively simple peptides investigated were chosen specifically to provide an estimate of the relevant energetics in systems with limited charge solvation ability, i.e., model systems deliberatively designed to show the maximal effect of charge repulsion in peptide systems. This should

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THEORETICAL METHODS A Very Simple Model. To test these basic reactions on multiple systems and provide an initial comparative picture for ECD only (i.e., ultralow pressure/zero collision), within a reasonable period of time, thereby informing subsequent more detailed work, some key simplifying assumptions were made. (1) In agreement with the generally accepted model,7,9,10,12 all H• transfers have been modeled from ground electronic state species generated after N−Cα bond cleavage. (2) Highly charged, multiply protonated polyglycines represent a simple model for the extreme case of H• transfer in radical cations with very limited charge shielding and systematically increased charge state. If there is no charge state dependency here, it is doubtful that one will be observed for larger, more typical “sequencing-relevant” peptides. Note that the glycine residue situated as the N-terminal residue of the initially formed zm• ion was previously shown to have the greatest effect10 in promotion of H• transfer to form the corresponding zm′ in a recent statistical study of over 15 000 validated doubly protonated peptide ECD spectra (i.e., from spectra where amino acid sequence was assigned with high confidence). (3) We utilize standard, constrained coordinate scans (one interatom distance tethered), followed by transition structure and intrinsic reaction coordinate calculations to provide an estimate of H• transfer barriers. The actual barriers will be less than or equal to those calculated in this study; i.e., we present the most structurally “straightforward” pathways to H• abstraction, an upper bound in terms of energy necessary to complete the reactions. (4) Potential proton transfer reactions within the radical-cation complex were not explicitly investigated. Only those reactions that either are necessary to enable N−Cα cleavage to occur or occur as part of an intrinsic reaction coordinate (IRC) pathway in a concerted manner with either N−Cα cleavage or H• transfer reaction are considered possible. Calculation Details. Standard ab initio and density functional theory calculations were performed with the Gaussian 0339 suite of programs. Due to the small system size, charge state, and considerable prior experience, major molecular dynamics searches were not performed to generate the protonated peptide minima. However, multiple families of structures where investigated in each case. Structures were optimized with the B3LYP/6-31+G(d,p)40−42 functional. Local energy minima were confirmed with frequency calculations. Single point energies were calculated from the B3LYP/6-31+G(d,p) minima at the B3LYP/6-311++G(2d,p) and MP2/6-311++G(2 d,p)43 levels of theory. These values were then averaged to cancel out known errors in the methods.44 The spin-unrestricted formalism (UB3LYP and UMP2) was used for all calculations of radical systems with higher spin states in UMP2 calculations were annihilated by standard projection procedures.45,46 Transition structures for the cn−zm reactions were optimized from the energetically feasible radical-cation minima, i.e., only those generated from the lowest energy protonated peptide minima, or by simple rotation of these radical-cation structures. Intrinsic reaction coordinate (IRC) calculations were run to identify the minimum energy path that connects the reactant and the product geometries, to ensure that the structures are indeed the correct transition structures. For each radical-cation system, the product dimer (if formed) from the lowest energy cn−zm transition structure was used as the start point for any possible H• transfer reaction (Figure 3). 1191



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were performed; one scan for each Cα hydrogen on the C-terminal residue of the cn′ ion. Constrained optimizations as a function of this bond length were performed at the B3LYP/631+G(d,p) for smaller systems and B3LYP/6-31G(d) for larger ones. All maxima along this artificial path were investigated to ensure they could be overcome facilely (simple rotations) or optimized to a H• transfer transition structure. Transition structure optimizations at the B3LYP/6-31+G(d,p) level of theory were performed (B3LYP/6-31G(d) and B3LYP/631+G(d,p) for the larger systems). IRC calculations were again run to ensure that the TS connected directly to the original product dimer from the cn−zm transition structure IRC calculation (or to a similar structure interconvertible via simple rotation(s), i.e., not energetically significant to achieve). Multiple conformers of the potential product species (cations, radical cations, radical neutrals, neutrals) were calculated. The iminol form (terminated −C(OH)NH) of the cn′ and cn• ions was used for ΔHreaction comparison, as this is the form produced in the simple N−Cα cleavage reactions studied here. Note that use of the potential, protonated amide form (terminated −C(O)−NH2) leads to exactly the same conclusions (ΔHreaction values). Most calculations were performed at the Florida State University shared High-Performance Computing facility. Additional, MP2 single-point calculations that required substantially greater scratch space were performed on the cluster at the German Cancer Research Center (DKFZ), Heidelberg, Germany.

Figure 3. Generic energy level diagrams for electron capture followed by N−Cα bond cleavage. From the resulting radical-cation complex, either “direct” dissociation of to cn′ and zm• sequence ions occurs or, alternatively, a H• transfer reaction occurs (hypothetical barrier heights denoted A−C); then the complex dissociates (“indirect”) to cn• and zm′ sequence ions. Note that the relative energy of post-reaction complexes and product species vary with composition and charge state so are not all endothermic.

Standard constrained optimizations (opt=modred, action code S) were used to tether the zm• Cα radical carbon atom to the adjacent cn′, Cα hydrogen (Figure 4). The structure was

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RESULTS AND DISCUSSION 1. Minima. Many structures are summarized in the present work. Briefly, all multiply charged precursor systems were Nterminally protonated as well as being protonated on one or more oxygen atoms. The radical systems generated from the most favorable closed-shell precursors provided similar Hbonding schemes. In some cases with concomitant H-transfer to adjacent solvating oxygen systems (some singly charged radical cations depending on where electron capture occurs). Detailed structural comparison is not the focus of the paper as these polyglycine systems analyzed were deliberately chosen for the extreme situation of minimal charge solvation; i.e., the higher charge states are “atypical” H-bonded peptide systems for normal ECD. For those interested in the more specific details, all of the lowest energy structures, transition structures, and electronic energies are provided in the Supporting Information. Further questions/requests for additional information are welcome. 2. Product Ion Energies. One of the postulated explanations for the documented decrease in detection of product ion peaks from H• transfer reactions (cn• and zm′ peaks) as a function of charge state is an incremental decrease in the relative energy of these products with respect to the “direct” cn′ and zm• products. Early work30−32 has shown that captodative effects of the NH and CO groups adjacent to the newly formed radical site stabilize the cn• ion/neutral H• transfer product and thereby increase its relative favorability. In contrast, this stabilization is not available to the “direct” zm• ion/neutral product. Consequently, ΔHreaction for this process is negative (exothermic). Our calculations as a function of precursor charge state indicate that this tendency is a precursor charge state invariant (Tables S1−S3, Supporting Information). The mean ΔĤ reaction(cn′ + zm• ⇒ cn • + zm′) = −31 kJ mol−1;

Figure 4. Constrained optimization scan performed for the post N−Cα cleavage radical-cation complex, generated from [GG + 2H]•+. Blue circles indicate optimized structures with fixed zm• Cα radical carbon atom to the adjacent cn′, Cα hydrogen distance. The red dots connect the structures optimized at each distance; the higher the optimization number, the shorter the distance. Note: the initial energy rise is due to the structure not being at the fully (relaxed) optimized post N−Cα bond cleavage minimum anymore (∼−11 kJ mol−1) as a fixed (shorter) interatom distance has been artificially introduced. The energy increases until the bonding switches from basically a planar interaction between the inminol nitrogen and the adjacent H atom of the C-terminal fragment, to an angled one to the imine CN π electrons, i.e., a nonenergetically demanding rotation. The H-transfer barrier requires ∼67 kJ mol−1 (Table 1).

then optimized using the standard optimization algorithm with this additional constraint. Once each optimization is complete, the distance is reduced and the process repeated. Two scans 1192



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i.e., this is not the reason for decreased detection of cn• and zm′ peaks from higher charge state precursors. This trend does not hold for the c1• forms with the corresponding zm′ species where the ΔHreaction values are slightly endothermic (ΔĤ reaction = 11 kJ mol−1, standard deviation, σ = 4). Removal of these from the comparison gives ΔĤ (no c1•)reaction = −48 kJ mol−1 (σ = 16). This is due to a significant difference in favorability of the c1• and c1′ (glycine) forms. The relative proton affinities, PA, of the neutral c1• radical and c1′ species offer a means of illustration here: PA(neutral c1•) ∼801 kJ mol−1 and PA(neutral c1′) ∼ 870 kJ mol−1. This large difference in favorability (PA(cn′) − PA(cn•) = 69 kJ mol−1) more than cancels out the energetic gain from captodative stabilization. In contrast, for larger species, the proton affinity of the radical and closed shell cn species are much more similar: PA(cn′) − PA(cn•) = 17, 6, and 11 kJ mol−1 for n = 2−4, so the captodative stabilization wins out and thus H• transfer is an exothermic process. 3. Hydrogen Atom Transfer Barriers. For the present model systems our calculations indicate that the barrier to hydrogen atom transfer is usually greater than the previous cn−zm• N−Cα bond cleavage barrier; i.e., barriers B or C in Figure 3 are generally pertinent. The specific barrier heights vary as a function of conformation, cn−zm• reaction and charge state. As more energy is necessary to enable H• transfer and thus formation of the cn• and zm′ product species, this is a potential explanation for the decrease in detection of product ion peaks from H• transfer reactions. Is there a charge state dependence? Table 1 summarizes both the preceding cn−zm• N−Cα bond cleavage barriers and the most energetically favorable H• transfer barrier obtained. Though there is fairly substantial variation in individual cn−zm• N−Cα bond cleavage barriers, which reflect the specific transition structure conformation in each case, the mean enthalpies (Ĥ ) and standard deviations (σ) make the energies as a function of charge state essentially indistinguishable. These values are similar to those obtained for a range of peptide radical-cation systems studies by Tureček and coauthors11,16,21,30,47−50 (summarized in 47) and so are consistent with the “proper aminoketyl radical”, −CαC•(OH)NH−, intermediates of N−Cα bond dissociations induced by electron attachment to protonated peptides,47 i.e., pyramidized aminoketyl groups, with high spin density on the central carbon atom, the principle site of the radical. In contrast, these measures for the H• transfer reactions show a clear trend; more energy is necessary to enable these reactions for like species generated with greater charge state. A concomitant increase in the energy of the product complex formed following N−Cα bond cleavage is also observed; i.e., the average singly charged complex is more energetically favorable than the precursor charge reduced radical cation (exothermic), whereas the average doubly charged form is indistinguishable (equithermic) and the triply charged congener is less favorable (endothermic). The charged radical cations H• transfer barriers are similar to those calculated previously,16,30−32 but those of the more highly charged systems are greater still. Furthermore, the most conspicuous difference for the most highly charged radical cations is the lack of successful H• transfer pathways. From our calculations, this situation appears in two ways (Table1). (1) Some of the cn−zm• N−Cα bond cleavage reactions do not generate a post N−Cα cleavage radical-cation complex but dissociate immediately to the direct cn′ + zm• product ions on one side of the IRC calculation (labeled a in Table 1). (2) Alternatively, the N−Cα bond cleavage transition structure IRC

Table 1. Summary of N−Cα Cleavage Barriers and H• Transfer Barriers for Complex, Generated from [GN + xH]•(x−1)+ Precursor Ionsa precursor, charge state, [M + xH]x+

no. of residues (N)

cn−zm• pathway

2 2 2 2 2 2 2 2 2 2

2 3 3 4 4 4 5 5 5 5

c1−z1• c1−z2• c2−z1• c1−z3• c2−z2• c3−z1• c1−z4• c2−z3• c3−z2• c4−z1•

3 3 3 3 3 3 3 3 3 3

2 3 3 4 4 4 5 5 5 5

c1−z1• c1−z2• c2−z1• c1−z3• c2−z2• c3−z1• c1−z4• c2−z3• c3−z2• c4−z1•

4 4 4 4 4 4 4 4 4

3 3 4 4 4 5 5 5 5

c1−z2• c2−z1• c1−z3• c2−z2• c3−z1• c1−z4• c2−z3• c3−z2• c4−z1•

cn−zm• TS, ΔH298(ΔG298), kJ mol−1

H• transfer TS, ΔH298(ΔG298), kJ mol−1

32 (37) 30 (25) 80 (77) 6 (7) 40 (41) 81 (81) 81 (81) 43 (39) 43 (35) 70 (67) ΔĤ = 47, σ = 24 27 (28) 55 (56) 12 (11) 21 (23) 40 (40) 117 (117) 46 (42) 52 (50) 29 (23) 61 (58) ΔĤ = 46, σ = 29 26 (28) 62 (60) 175 (167) 33 (27) 14 (14) 89 (88) 26 (23) 56 (57) 25 (21) ΔĤ = 57, σ = 48

67 (69) 47 (42) 105 (100) 58 (63) 61 (57) 103 (102) 77 (73) 123 (125) 71 (59) 93 (84) ΔĤ = 80, σ = 24 a 138 (135) 151 (147) 104 (102) 109 (112) 123 (122) 88 (82) b 94 (93) 94 (87) ΔĤ = 113, σ = 22 a a b 178 (175) a b 164 (165) a 160 (151) ΔĤ = 167, σ = 9

a

Barriers are averages of single point calculations at the B3LYP/6311++G(2d,p) and MP2/6-311++G(2d,p) levels from B3LYP/631+G(d,p) optimized structures. For a, the IRC leads to direct dissociation following cn′−zm• TS. For b, the H• transfer TS is not a physically bound state; i.e., both ends of the IRC dissociate. No post N−Cα bond cleavage complex is formed from this TS.

calculation does locate a minimum post N−Cα cleavage radicalcation complex, but no H• transfer pathways could be connected to it; i.e., H• transfer TSs can be calculated but are not bound complexes at either end of the reaction pathway. Consequently, both ends of the IRC calculation lead to dissociation, so no post N−Cα bond cleavage complex or product complex is formed from the TS (labeled b in Table 1). This is logical as the energy necessary to generate these transition structures for H• transfer in the highest charge state cases is at least 160 kJ mol−1, which is sufficient to break amide bonds even in some tryptic systems.51−57 Ergo, separation of a noncovalently bound58 ion-radical complex of similar size will be trivial, as the energy to do this is comfortably available, provided a necessary conformation is accessed. 4. Radical-Cation Complex Lifetime. Discussion section 3 is obviously also relevant to the question of complex lifetime. This provides some support for the conceptual appealing 1193



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rate of [cn• + zm′](x−1)+• complex separation; i.e., unfortunately, the RRKM only provides information about the H-transfer rate constant. Nevertheless, the point remains that if the time scale for these H-transfer processes is so long that the reaction cannot occur for a particular charge state, then the subsequent question of product separation is moot. In general terms, the RRKM calculations show (Figure S1, Supporting Information) what you would expect on the basis of experiment8,38 and the present theoretical data. H• transfer in singly charged radical cations, [M + 2H]+•, occurs on a time scale similar to that of N−Cα bond cleavage and has a very steep slope; on this basis one would expect a significant amount of cn• + zm′ product peaks (assuming the [cn• + zm′]+• complex then separates within the experimental time scale). In contrast, the higher charge state radical cations, [M + xH](x−1)+•, must be substantially more energized on an equivalent time scale to enable H• transfer to occur. Moreover, the slope of these reactions is shallower. Thus fewer triply charged post N−Cα bond cleavage radical-cation complexes would be expected to undergo H• transfer and thus have the opportunity for the new [cn• + zm′](x−1)+• complex to exist, then separate, enabling its products to be detected, than the doubly or singly charged congeners. 5. H• Transfer Transition Structures: Extrapolation to Larger “Simple” Systems. The present work (deliberately) represents an extreme situation where charge density is unreasonably high (particularly for precursor charge ≥3). To generate the H• transfer transition structure geometries, it is necessary to force the two fragments that comprise the radicalcation complex more closely together in a specific configuration. For the highly charged species this requires bringing these charges closer together and interrupting the more energetically favorable charge solvation that was present in the initially formed post N−Cα cleavage complex. For the simple systems examined here, the structures themselves are not greatly different between charge states as there are not too many conformations possible. Consequently, the rise in energy is quite steep. Thus charge repulsion in compositionally similar, but larger, systems would be expected to have a reduced, but still significant, effect on H• abstraction barriers due to the increased opportunities for charge solvation (shields charge− charge interactions), less pronounced distortion of those charge solvation patterns, and the larger average charge−charge distance. Consequently the energy necessary to perform such reactions is reduced. Conversely, charge−dipole repulsive forces are an order of magnitude weaker. Ergo, the situation for singly charged radical cations is likely to be roughly constant irrespective of system size. This is in agreement with similar barriers obtained elsewhere for singly charged radical-cation systems.16,30−32

fission-like picture of radical-polycation fragmentation where the charged spheres shoot apart as soon as a barrier (N−Cα bond cleavage) is overcome. It should be explicitly understood, however, that the present method is totally reliant on gradientbased optimizations and is thus not an ideal way to answer the question of how long the complex remains bound as the dynamics of the process are not addressed directly. Regrettably techniques to model this process at believable levels of theory are only just becoming available59 and at present are prohibitively computationally expensive for even dipeptide systems. Resonance-ejection experiments22 indicate that a significant percentage of H• transfer reactions occur on a time scale of greater than 1 ms. Furthermore, activation of the precursor multiply protonated peptide prior to ECD/ETD supports9,12,13,33−35 the contention that reduction or elimination of intermolecular forces facilitates prompt separation to “direct” products. The H• transfer barriers calculated here show that additional energy is necessary to achieve these reactions even in the singly charged radical-cation complex. Thus it is reasonable to expect that some time might be necessary for a sufficient proportion of the complexes to generate the necessary conformations to enable H• transfer to occur. Naturally, if the complex has already dissociated, this cannot happen. The increased charge density coupled with higher barriers to H• transfer support these earlier arguments. Additionally, the recombination energy that results from attachment of a free electron to a gas-phase ion has to be considered. This highly exothermic process2,21,30,48,49 (4−7 eV for doubly charged protonated peptide precursors48,49) becomes increasingly so as a function of precursor ion charge state.21,30 Illustrating this effect with the [G4 + xH]x+ precursor protonated peptides, recombination energies of 6.8, 10.2, and 14 eV for the doubly, triply, and quadruply charged ions were calculated. As noted by Tureček and Syrstad,30 for similarly sized model systems “full internal conversion of recombination energy would cause a substantial increase of temperature ... and will result in accelerated dissociation.” Even if this were not the case though, the very weak N−Cα aminoketyl bond of a radical cation can easily fragment on the millisecond time scale of an ECD experiment.30 How about the H• transfer reactions? To approximate the time scale60 of the H• transfer reactions, Rice−Ramsperger−Kassel−Markus (RRKM) calculations were performed using the energetics, vibrational frequencies, and rotational constants derived from the modeling. The calculated unimolecular rate constants for the c2−z2• pathway of [G4 + xH](x−1)+• were examined. It should be explicitly understood that this is done for illustration, using the assumption that the global minimum and the corresponding TS (N−Cα bond cleavage or H• transfer) are the only important structures and thus all others can be ignored. This obviously assumes61 coupling between the various vibrational degrees of freedom is sufficiently strong for the excitation energy to be randomized rapidly among the active degrees of freedom on the time scale of reaction despite the fact that the degree of coupling might be open to debate for post N−Cα bond cleavage radical-cation complexes. The Beyer−Swinehart direct count algorithm62 is used for rotational−vibrational treatment of both the reactant and the transition structure. Furthermore, the branching ratio between the “direct” cn′ + zm• products and the H• transfer and cn• + zm′ products for a given N−Cα bond cleavage reaction is a function of the ratio of the rate of direct [cn′ + zm•](x−1)+• complex separation to the rate of H• transfer, multiplied by the

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CONCLUSIONS The present study provides a computational estimate of the product energies and barriers associated with H• transfer processes occurring in post N−Cα bond cleavage radical-cation complexes for a series of peptides with little charge solvation. The findings are in general agreement with the prior educated speculation7−10,12,28 that concerned these reactions. In addition, the present data provide evidence for the following: (1) The products of H• abstraction in post N−Cα bond cleavage radical-cation complexes are energetically favorable irrespective of precursor ion charge state. (2) The barriers to H• abstraction increase substantially as a function of precursor ion charge state. 1194



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Although this does not rule out arguments based on time scale, it does indicate than the barrier height to H• transfer should be considered too. (3) The conceptually appealing charge-fission/ “immediate” product separation model follows N−Cα cleavage. (4) Even for these deliberately small and unreasonably charge dense model systems several feasible pathways to H• transfer were sometimes located. (5) Preliminary RRKM calculations support the trend for H• transfer barrier increase with substantially slower rates at increased charge state. Extension of the present work to focus on larger, more sequencingrelevant systems will be the topic of subsequent investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Tables of enthalpy values. Plot of rate constants as a function of energy. XYZ coordinates of the many minima and transition structures summarized in this manuscript. This information is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Jeremiah D. Tipton, Yuan Mao, Christopher L. Hendrickson, and Alan G. Marshall are thanked for providing data that, in part, motivated the current analysis. Béla Paizs generously provided computer time to enable the MP2 single-point calculations to be performed. František Tureček is thanked for useful discussions and pointing out some relevant literature. This work was supported by NSF Division of Materials Research through DMR-06-54118, and the State of Florida. Most calculations were performed at the Florida State University shared High-Performance Computing facility.

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NOTE ADDED AFTER ASAP PUBLICATION This Article was published ASAP on August 3, 2012, with errors in Table 1. The corrected version was posted on September 14, 2012.

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