Mechanistic Examination of CÎ²âCÎ³ Bond Cleavages of Tryptophan

Article pubs.acs.org/JPCA

Mechanistic Examination of Cβ−Cγ Bond Cleavages of Tryptophan Residues during Dissociations of Molecular Peptide Radical Cations Tao Song,† Ching-Yung Ma,†,‡ and Ivan K. Chu*,† †

Department of Chemistry and ‡School of Biological Sciences, The University of Hong Kong, Hong Kong, China

Chi-Kit Siu* Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China

Julia Laskin Chemical and Materials Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, United States S Supporting Information *

ABSTRACT: In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [GnW]•+ (n = 2−4) and [GXW]•+ (X = C, S, L, F, Y, Q) species. The Cβ−Cγ bond cleavage of a C-terminal decarboxylated tryptophan residue ([M − CO2]•+) can generate [M − CO2 − 116]+, [M − CO2 − 117]•+, and [1H-indole]•+ (m/z 117) species as possible product ions. Competition between the formation of [M − CO2 − 116]+ and [1H-indole]•+ systems implies the existence of a proton-bound dimer formed between the indole ring and peptide backbone. Formation of such a proton-bound dimer is facile via a protonation of the tryptophan γ-carbon atom as suggested by density functional theory (DFT) calculations. DFT calculations also suggested the initially formed ion 2, the decarboxylated species that is active against Cβ−Cγ bond cleavage, can efficiently isomerize to form a more stable π-radical isomer (ion 9) as supported by Rice−Ramsperger−Kassel−Marcus (RRKM) modeling. The Cβ−Cγ bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WGnR]•+ (n = 1−3) radical cations consistently resulted in predominant formation of [M − 116]+ product ions. It appears that the basic arginine residue tightly sequesters the proton and allows the charge-remote Cβ−Cγ bond cleavage to prevail over the chargedirected one. DFT calculations predicted that the barrier for the former is 6.2 kcal mol−1 lower than that of the latter. Furthermore, the pathway involving a salt-bridge intermediate also was accessible during such a bond cleavage event.

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INTRODUCTION

dissociations along the backbone also becoming more complicated. In contrast, side-chain losses typically are observed in the CID of radical peptides, providing complementary information for peptide sequencing. Hydrogen-rich odd-electron peptide radical cations [M + nH]•(n−1)+, generated in electron-capture dissociation (ECD)13,14 and electron-transfer dissociation (ETD) experiments,15−17 undergo N−Cα bond cleavages along the backbone, resulting in the formation of characteristic c and z fragments.17−22 Consecutive dissociations of oddelectron z• ions result in a variety of side-chain losses, which

The use of mass spectrometry for protein identification relies on the gas-phase dissociation of peptide ions.1−3 A fundamental understanding of these processes is crucial for the successful peptide sequencing. Under the conditions of low-energy collision-induced dissociation (CID), protonated even-electron peptides fragment at different amide bonds along the backbone, producing b or y ions that facilitate the sequencing of peptides.4,5 The fragmentation of protonated peptides typically follows a charge-directed pathway, described within the framework of the mobile proton model.6−10 Although losses of H2O and NH3 are common in the CID of protonated peptides, the amino acid side chains usually remain intact. Under high-energy CID conditions, cleavages of the bonds between the β- and γ-carbon atoms of the side chains can provide additional structural information, 11,12 but with © 2012 American Chemical Society

Special Issue: Peter B. Armentrout Festschrift Received: April 13, 2012 Revised: June 13, 2012 Published: June 14, 2012 1059

dx.doi.org/10.1021/jp303562e | J. Phys. Chem. A 2013, 117, 1059−1068

The Journal of Physical Chemistry A

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Mass Spectrometry. All mass spectrometry experiments were conducted using a quadrupole ion trap mass spectrometer (Finnigan LCQ, ThermoFinnigan, San Jose, CA, USA). The molecular peptide radical cations M•+ were generated through one-electron oxidative dissociations of transition metal/peptide complexes.19,22,29−31 Their abundances were optimized using [CuII(terpy)M]•2+ complexes (for M = GGGW, GGGW− OMe, GGW−OMe, and GXW; X = G, C, S, L, F, Y, Q) or [CoIII(salen)M]+ complexes (for M = WGGR and WGR). The fragmentation chemistries of these radical peptide cations were independent of the nature of the metal complex.47,57 Samples typically comprised 600 μM Cu(II)(terpy) or Co(III)(salen) complex and 50 μM oligopeptide in a 1:1 H2O/MeOH solution. A syringe pump (Cole Parmer, Vernon Hills, IL, USA) was used for direct infusion of the electrospray samples (flow rate, 30 μL h−1). CID spectra were acquired using He as the collision gas. The injection time and excitation time for CID in the ion trap were 200 and 50 ms, respectively. The excitation amplitude was optimized for each experiment. Computational Methods. Electronic energies were calculated in the framework of DFT using the unrestricted (U) hybrid functional formulated with a mixture of Hartree− Fock exchange energy and Becke’s three-parameter 1988 gradient-corrected exchange energy, as well as Lee−Yang− Parr (LYP) correlation energy.58 Atomic orbitals were described by a Gaussian-type split valence shell 6-31++G(d,p) basis set, including polarization and diffuse functions for all atoms.59,60 Low-lying structures of the molecular ions were obtained through Monte Carlo conformational searches with a semiempirical method (PM3) using Spartan software,61 followed by geometry optimizations at UB3LYP/6-31G(d) and UB3LYP/6-31++G(d,p) levels. Additional DFT geometry optimizations for other plausible low-lying structures, which likely were missed in the conformational searches, also were performed. Harmonic vibrational frequencies of all optimized structures were calculated to confirm the structures were at local minima (all real frequencies) or were transition states (one imaginary frequency). The local minima associated with each transition structure were verified using the intrinsic reaction coordinate (IRC) method. Relative enthalpies at 0 K (ΔH0°) were calculated from the electronic energies and zeropoint vibrational energies (ZPVE) obtained within the harmonic approximation. Atomic charges and spin densities were evaluated using natural population analyses (NPA). All DFT calculations were performed using the Gaussian 03 software package.62 The microcanonical rate constant ki(E) of each unimolecular reaction i was calculated using the Rice−Ramsperger−Kassel− Marcus (RRKM) equation.63,64 The value of ki(E), given by eq 1, is a function of the internal energy E of the reactant relative to that of the structure at the global minimum on the potential energy surface (PES)

can be used as unique signatures of the individual amino acids in the peptide sequence. For example, losses of 30, 33, 61, 87, and 129 Da are characteristic of serine, cysteine, methionine, arginine, and tryptophan residues, respectively.16,23−25 In addition, leucine and isoleucine can be distinguished by their diagnostic losses of 43 and 29 Da, respectively.16,23,26,27 Eleven of the 20 common amino acids have characteristic side-chain losses, providing additional structural information that can improve the confidence of peptide identification.28 Classical molecular peptide radical cations (M•+), which can be generated through oxidative dissociation of transition metal/ peptide complexes19,22,29−32 or dissociation of peptides containing labile groups,20,33−40 also exhibit side-chain losses under conditions of low-energy CID. The chemistry of M•+ species is unique relative to that of their even-electron protonated counterparts, [M + nH]n+. Extensive studies using different peptide models containing aliphatic, aromatic, and/or basic residues have demonstrated that the fragmentation behavior of M•+ radical cations is determined by competition between the radical-driven and charge-directed processes.21,28,41−53 In M•+ species featuring only aliphatic amino acid residues, proton transfer (PT) is facile. As a result, chargedirected fragmentations prevail.53 In contrast, the presence of a highly basic arginine residue allows the proton to be sequestered firmly by its side chain, thereby hindering charge mobility and resulting in facilitated, radical-driven fragmentations.49 The radical migration can occur prior to fragmentation,37,49,52 inducing a diverse range of fragmentation channels including side-chain, C α −C, and N−C α bond cleavages.26,28,43,50−52 Loss of an indolyl radical (116 Da) is a characteristic that has been employed in several ECD/ETD studies to identify the presence of a tryptophan residue in the sequence.16,25,27 CID of tryptophan-containing M •+ species, [GGGGW] •+ and [WGGGR]•+, generated from the dissociation of transition metal/peptide complexes also can result in the loss of the 1indolyl radical. This originates from Cβ−Cγ bond cleavage of the tryptophan residue, induced by a radical located at its αcarbon atom, as suggested by density functional theory (DFT) calculations on a small model.49 In this study, we further examined the detailed mechanisms of such Cβ−Cγ bond cleavages using the tripeptide radical cations [GGW]•+ and [WGR]•+ as models that strongly resemble their longersequence analogues in terms of fragmentation behavior.

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EXPERIMENTAL SECTION Materials. Fmoc-protected amino acids and the Wang resin were purchased from Advanced ChemTech (Louisville, KY, USA). All other chemicals were supplied from Sigma-Aldrich (St Louis, MO, USA) or Bachem (King of Prussia, PA, USA). Oligopeptides and the Cu(II)(terpy)(NO3)2 (terpy =2,2′:6′,2′terpyridine) and [Co(III)(salen)]Cl [salen = N,N′-ethylenebis(salicylideneaminato)] complexes were synthesized according to procedures described in the literature.54−56 Methylation of Peptides. A solution of approximately 2 M HCl in MeOH was prepared through the dropwise addition of acetyl chloride (800 μL) into anhydrous MeOH (5 mL), which was then stirred for 5 min at room temperature. This solution (1 mL) was added to the peptide (10 mg). Then, the mixture was stirred for 3 h at room temperature. The resulting solution was dried using a SC250DDA Speedvac Plus (Thermo Electron Corporation, Waltham, MA, USA). The methylated peptide was directly used in each experiment without purification.

ki(E) =

σWi ‡(Ei − E0i) hρi (Ei)

(1)

where Ei = E − ΔH0i is the available vibrational energy; ΔH0i is the ith reactant enthalpy of formation at 0 K; ρi(Ei) is the density of vibrational states of the reactant; Wi‡(Ei − E0i) is the sum of the vibrational states of the transition state; E0i is the corresponding critical energy for the reaction; h is Planck’s constant; and σ is the reaction path degeneracy, which was equal to 1 in all of the pathways considered in this study. The 1060



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vibrational states were calculated using the Beyer−Swinehart direct count algorithm.65

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RESULTS AND DISCUSSION CID of [GnW]•+ (n = 2−4). A recent study found that CID of the [GGGGW]•+ radical cation results in the loss of CO2 to produce a C-terminal α-radical. Subsequent activation of [M − CO2]•+ ion results in Cβ−Cγ bond cleavage of the tryptophan residue, with concomitant loss of a neutral 1-indolyl radical having a mass of 116 Da.49 These consecutive neutral losses also are observed in the CID of the shorter peptide radical cation [GGGW]•+, forming [M − CO2]•+ (m/z 331) and [M − CO2 − 116]+ (m/z 215) species (Figure 1a). The ion at m/z 117 can be assigned as [1H-indole]•+ (the protonated form of the neutral 1-indolyl radical), and the fragment at m/z 214 corresponds to [M − CO2 − 117]•+ (with the loss of 117 Da being that of a neutral 1H-indole molecule), both resulting from the Cβ−Cγ bond cleavage of the tryptophan residue. These assignments are confirmed by comparison with the CID spectrum of [GAGW]•+ (Figure S1, Supporting Information), where both the [M − CO2 − 116]+ and [M − CO2 − 117]•+ species are shifted by 14 Da toward higher mass-to-charge ratio, while the [1H-indole]•+ radical cation exhibits no mass shift. The observed mass shift results from the mass difference between the glycine and alanine residues. In the CID of [GGGW-OMe]•+ (Figure 1b), we did not observe CO2 loss, subsequent 116 or 117 Da loss, or the formation of [1Hindole]•+, indicating that the generation of the tryptophyl αradical through decarboxylation is essential for Cβ−Cγ bond cleavage of the tryptophan residue. This cleavage also is observed in even shorter tryptophan-containing tripeptide radical cations, such as the well-studied [GGW]•+ system42,43,48 (Figure 1c), to produce the currently assigned [1H-indole]•+ at m/z 117, which was inconspicuous because of its relatively low abundance (10%) and the absence of its counterpart, [M − CO2 − 116]+ at m/z 158. Similar to the [GGGW-OMe]•+ radical cation, the ion at m/z 117 was absent in the CID spectrum of [GGW-OMe]•+ (Figure 1d), supporting our hypothesis that CO2 loss from the precursor ion is a key step to [1H-indole]•+ formation. Table 1 summarizes the relative abundances of the [M − CO2 − 116]+, [M − CO2 − 117]•+, and [1H-indole]•+ species in the CID spectra of [GnW]•+ (n = 2−4) radical cations. The ratio of the abundances of the [M − CO2 − 116]+ and [1Hindole]•+ species was highest when n was 4 and lowest when n was 2. Considering that the proton affinity (PA) of a peptide grows upon increasing the number of amino acid residues,66 it seems the formation of the [M − CO2 − 116]+ or [1Hindole]•+ ion results from the competition between the 1indolyl radical and the remaining peptide backbone for the proton during dissociation of the proton-bound dimer, a process that is common in gas-phase fragmentations.43,48,67−73 CID of [GXW]•+ (X = G, C, S, L, F, Y, Q). By examining the CID spectra from a series of tripeptides radical cations [GXW]•+ (X = G, C, S, L, F, Y, Q) with increasing PA of the middle residue,66,74 we confirmed the proton competition mechanism in the Cβ−Cγ bond cleavage of the tryptophan residue. Table 2 lists the PAs for the residues X and the relative abundances of [M − CO2 − 116]+, normalized to the sum of [M − CO2 − 116]+ and [1H-indole]•+ species abundances. Because none of the CID spectra of the [GXW]•+ systems in Table 2 featured a signal for [M − CO2 − 117]•+, as observed in the CID spectrum of [GGGW]•+ (Figure 1a), the ratios

Figure 1. CID spectra of (a) [GGGW]•+, (b) [GGGW-OMe]•+, (c) [GGW]•+, and (d) [GGW-OMe]•+.

presented in Table 2 simply reflect the competition for the proton between the 1-indolyl radical and the remaining peptide backbone. In general, the abundance ratio for [M − CO2 − 116]+ escalated upon increasing the PA of residue X, ultimately to 98% for the CID of the [GQW]•+ radical cation. These results suggest that the important factors that mediate product ion formation are (1) the competition of the fragments formed from the Cβ−Cγ bond cleavage of tryptophan residue for the ionizing proton and (2) the PA of the backbone. 1061



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elucidate the mechanism of the Cβ−Cγ bond cleavage of the tryptophan residue theoretically, we used DFT calculations. We selected [GGW]•+ as the model system because (1) it is the smallest tripeptide radical cation that undergoes Cβ−Cγ bond cleavage of tryptophan residue and (2) its lowest-energy structure (ion 1 in Figure 2), and the mechanisms of its primary fragmentation channels already have been extensively studied.48 For example, it has been demonstrated that the loss of CO2 from [GGW]•+ (ion 1) to form the C-terminal α-carboncentered radical [M − CO2]•+ (ion 2), the key step for subsequent formation of the [1H-indole]•+ radical cation, proceeds through a transition structure TS(1→2) with a relative energy of 29.0 kcal mol−1 (Figure 2).48 [M − CO2]•+ (Ion 2) → [1H-Indole]•+. Figure 2 displays the PES for the formation of the [1H-indole]•+ radical cation. Starting from ion 2, rotation about the C-terminal N−Cα bond, with a barrier of 5.0 kcal mol−1 relative to ion 2, results in ion 3, where the proton on the C-terminal amide oxygen atom is positioned close to the γ-carbon atom of the tryptophan residue. A subsequent 1,6-PT from the amide oxygen atom to the γ-carbon atom of the tryptophan residue results in ion 4, with a barrier of 6.0 kcal mol−1 through the transition structure TS(3→4). Such protonation at the γ-carbon atom substantially weakens the Cβ−Cγ bond of the tryptophan residue,49 elongating from 1.511 Å in ion 3 to 1.661 Å in ion 4. Ion 4 is a shallow minimum and can undergo Cβ−Cγ bond cleavage with a barrier of only 1.3 kcal mol−1, resulting in ion 5, a proton-bound dimer formed between the [1H-indole]•+ radical cation and the rest of the peptide backbone. The barriers against the formation of the [1H-indole]•+ and [M − CO2 − 116]+ species from ion 5 are 22.4 and 27.8 kcal mol−1, respectively, indicating that the PA of the neutral backbone is lower than that of the 1-indolyl radical. This protoncompetition mechanism is in good agreement with the

Table 1. Relative Abundances of Product Ions Originating from Cβ−Cγ Bond Cleavages of Tryptophan Residues in the CID Spectra of [GnW]•+ (n = 2−4) Cβ−Cγ bond cleavage [M − CO2 − 116]+

[M − CO2 − 117]•+

[1Hindole]•+

41% 61% 0%

0% 20% 0%

0% 35% 10%

[GGGGW]•+ [GGGW]•+ [GGW]•+

Table 2. PAs (kcal mol−1) for Residues X in GXW and Relative Abundances of [M − CO2 − 116]+ Normalized to the Sum of the Abundances of the Product Ions Associated with the Cβ−Cγ Bond Cleavage of Tryptophan Residue in the CID of [GXW]•+ Radical Cations (X = G, C, S, L, F, Y, Q) [GXW]•+

a

X X X X X X X

= = = = = = =

G C Sc L F Y Qc

PA of X

[M − CO2 − 116]+ (normalized)

211.9 215.6 218.1 218.5 221.2 222.0 232.6

0% 0% 0% 30% 49% 50% 98%

b

[M − CO2 − 117]•+ species were not observed in the CID spectra of [GXW]•+. bProton affinity (PA) values are taken from ref 73. cTypical spectra, [GSW]•+ and [GQW]•+, are available in Figure S2, Supporting Information, wherein [GSW]•+ shows abundant [1Hindole]•+ (64%) formation and [GQW]•+ shows abundant [M − CO2 − 116]+ (99%) formation. a

Mechanisms, Energetics, and Kinetics of the Formation of [1H-Indole]•+ in the CID of [GGW]•+. To further

Figure 2. PES for the formation of [GGW − CO2]•+ from [GGW]•+ and subsequent fragmentations forming [1H-indole]•+. Numbers on the PES are enthalpies at 0 K (all energies in kcal mol−1; bond lengths in Å). 1062



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Scheme 1. Pathways Considered in This Study for (a) the Formation of [1H-Indole]•+ with the Cβ−Cγ Bond Weakened by a Proton (Figure 2); (b) the Formation of [1H-Indole]•+ with the Cβ−Cγ Bond Weakened by a Hydrogen Atom (Figure S3, Supporting Information); and (c) the Formation of Stable [GGW − CO2]•+ (Ion 9) from [GGW − CO2]•+ (Ion 2) and Its Dissociation Giving [z1 − H − CO2]•+ (Figure S4, Supporting Information)a

Relative enthalpies ΔH0° were evaluated (in kcal mol−1) at the UB3LYP/6-31++G(d,p) level. Underlined numbers and numbers on the arrow are relative enthalpies and critical barriers, respectively. For simplification, only mechanisms for key bond-breaking steps and RRKM rate constants for rate-determining steps are presented. a

scale of this experiment. This finding is inconsistent with the low barrier for the formation of [1H-indole]•+ from [M − CO2]•+ as displayed in Figure 2. Furthermore, it has been demonstrated that the [1H-indole]•+ radical cation is not produced in CID of the [M − CO2]•+ radical cation; the major fragmentation pathway is cleavage of the N−Cα bond of the tryptophan residue, resulting in the formation of the [z1 − H − CO2]•+ (m/z 143) radical cation.42 Therefore, a pathway leading to the facile formation of a stable [M − CO2]•+ radical cation should exist. We propose the structure of this stable [M − CO2]•+ species is that of ion 9, which can be formed from ion 2 via a 1,4-H transfer from the C-terminal amide oxygen atom to the α-carbon atom of the tryptophan residue [pathway c in Scheme 1; PES is displayed in Figure S4, Supporting Information]. The barrier against this isomerization, 26.6 kcal mol−1, is comparable with the reaction barrier for [1H-indole]•+ formation via pathway a (25.3 kcal mol−1). The charge and unpaired electron of ion 9 are stabilized on the indole ring of the tryptophan residue, making ion 9 lower in enthalpy than ion 2 by 23.3 kcal mol−1. A 1,5-PT from the β-carbon atom of the tryptophan residue to the C-terminal amide oxygen atom, with a barrier of 24.2 kcal mol−1 (relative to ion 9), produces a benzylic radical structure (ion 10) that can undergo N−Cα bond cleavage to form the [z1 − H − CO2]•+ radical cation.

experimental observations presented in Table 2, which shows the 1-indolyl radical is produced with high abundance from [GXW]•+ with higher PA of X residue. The PES shown in Figure 2 corresponds to pathway (a) in Scheme 1. We also explored alternative pathways for formation of the [1H-indole]•+ radical cation from ion 2. For example, pathway b in Scheme 1 involves a hydrogen-atom transfer (HAT) from the C-terminal amide nitrogen atom to the γcarbon atom of the tryptophan residue (PES as shown in Figure S3, Supporting Information). The barrier against this HAT from the conformer ion 6 via TS(6→7) is 20.3 kcal mol−1 (or 22.8 kcal mol−1, relative to ion 1). It is higher than all of the barriers for the PT mechanism to form the proton-bound complex (ion 5) via pathway a. Following pathway b, formation of the [M − CO2 − 116]+ species is predicted with an overall endothermicity of 28.0 kcal mol−1, 7.8 kcal mol−1 lower than that for the formation of [1H-indole]•+ radical cation (Figure S3, Supporting Information). These results suggest that pathway b is energetically hindered and not likely to occur in CID of the [GGW]•+ radical cation. [M − CO2]•+ (Ion 2) → Stable [M − CO2]•+ (Ion 9). Notably, the abundance of [M − CO2]•+ (76%) is much higher than that of [1H-indole]•+ (10%) in the CID spectrum of the [GGW]•+ radical cation (Figure 1c), indicating that the majority of the [M − CO2]•+ radical cations did not dissociate on the time 1063



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isomerization from ion 2 to the stable [M − CO2]•+ radical cation (ion 9). To further verify ion 9’s stability, Figure 3b compares the rate constants for the formation of ion 9 (k2→9) with the rate constants for its subsequent fragmentation (k9→2 and k9→10). When the internal energy is greater than 30.0 kcal mol−1, the values of k2→9 are two to five orders of magnitude larger than those of k9→2 and k9→10, indicating that, once ion 9 is formed, it does not isomerize back to ion 2 or readily dissociate to form [z1 − H − CO2]•+ (P3) during the CID of [GGW]•+. Furthermore, because the value of k9→10 is much larger than that of k9→2, upon further activation the isolated ion 9 will form only the [z1 − H − CO2]•+ radical cation (P3) rather than interconvert to ion 2 and generate [1H-indole]•+ via pathway a. This finding is in good agreement with the CID spectrum of [M − CO2]•+, which did not feature a signal for [1Hindole]•+.42 CID of [WGnR]•+ (n = 1−3). In addition to the loss of 116 Da that results from the Cβ−Cγ bond cleavage of the Cterminal decarboxylated tryptophan residue forming [M − CO2 − 116]+, we also observed related cleavage from the intact peptide radical cations M•+ generating [M − 116]+ species, particularly from peptides containing an N-terminal tryptophan residue and a basic residue (e.g., histidine, lysine, or arginine).49 For instance, CID of [WGGGR]•+ predominately generates the [M − 116]+ product ion, which also was observed in the CID spectrum of the shorter peptide analogues [WGGR]•+ and [WGR]•+ with relative abundances of 100% and 62%, respectively (Table 3). Figure 4 presents CID spectra of

Both the mechanism and energetics of this process are similar to those for the formation of [z1 − H]•+ from [GGW]•+.48 RRKM Modeling of the Dissociation Rate Constants for [M − CO2]•+. The barrier against the dissociation of ion 2 to give [1H-indole]•+ via pathway a is comparable with the barrier against the isomerization of ion 2 to give the stable [M − CO2]•+ radical cation via pathway c. However, their yields are dramatically different (Figure 1c). This behavior may be attributed to the dissociation kinetics of these pathways, which we have been examined using RRKM modeling. Figure 3 shows the calculated critical rate constants for these two pathways.

Table 3. Relative Abundance of Product Ions Originating from Cβ−Cγ Bond Cleavages of Tryptophan Residues in the CID Spectra of [WGnR]•+ (n = 1−3) Radical Cations Cβ−Cγ bond cleavage [WGGGR]•+ [WGGR]•+a [WGR]•+a a

[M − 116]+ (100%) [M − 116]+ (100%) [M − 116]+ (62%)

CID spectra are presented in Figure S5, Supporting Information.

[WGGR]•+ and [WGR]•+. The major competing fragmentations for the [M − 116]+ species are cleavages of tryptophan’s Cα−Cβ bond and the amide bonds, forming [M − 129]•+ and y ions, respectively. In our preceding discussion, we stated DFT calculations of the [GGW]•+ model system revealed that protonation of the tryptophan residue’s γ-carbon atom was the critical step for its Cβ−Cγ bond cleavage. However, this situation may be different in the presence of a basic residue, which could hinder proton migration. In addition, the basic residue has the ability to facilitate migration of the radical to various sites,49 especially to the captodatively stabilized N-terminal α-carbon atom, which can induce the Cβ−Cγ bond cleavage of tryptophan49 in the CID of [WGnR]•+ (n = 1−3). This feature is evident by the absence of a signal for the [M − 116]+ species in the CID spectrum of [Wα‑CH3 GGGR] •+ (Figure S5, Supporting Information). Migrating the radical to the N-terminal α-carbon atom can follow numerous pathways. In general, it is quite facile in arginine-containing peptide radical cations.37,49 The detailed mechanism for the radical migration is beyond this article’s scope and our focus is on the Cβ−Cγ bond cleavages of tryptophan residues from M•+ radical cations containing an Nterminal α-carbon radical using [W•GR]+ as a model system.

Figure 3. RRKM modeling for the key competitive isomerizations and fragmentations of (a) [GGW − CO2]•+ (ion 2) and (b) stable [GGW − CO2]•+ (ion 9), as well as RRKM rate constants, log ki (s−1), plotted with respect to internal energies E (kcal mol−1).

Scheme 1 displays the rate-determining steps for the transformations of ion 2 via pathways a and c. The rate constants, labeled k5→P1 and k2→9, of the corresponding critical elementary reactions have barriers of 22.4 and 12.9 kcal mol−1, respectively. In Figure 3a, it is evident that the rate constants for the isomerization from ion 2 to ion 9 (k2→9) are one to two orders of magnitude larger than those for the formation of [1Hindole]•+ (k5→P1), in agreement with the CID spectrum of [GGW]•+, where the abundance of the [M − CO2]•+ radical cation is greater than that of [1H-indole]•+. The difference between the rate constants k2→9 and k5→P1 decreases at higher internal energy, indicating that the activation entropy for the formation of [1H-indole]•+ is larger than that for the 1064



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Figure 5b presents a detailed PES of an alternative mechanism for the formation of [M − 116]+ from [W•GR]+ that involves a salt-bridge (SB) intermediate, which has demonstrated an important role in the cleavages of the peptide backbones in arginine-containing peptide radical cations.49 Starting from ion 15, where the arginine side chain forms a hydrogen bond (bond length, 1.782 Å) with the carboxylic group, the highly basic arginine residue can assist a PT from the carboxylic group to the γ-carbon atom of the tryptophan residue through the transition structure TS(15→16) with a relative enthalpy of 34.4 kcal mol−1. The formed ion 16 is a SB intermediate that contains a γ-protonated indole ring and a strong hydrogen bond (bond length, 1.530 Å) between the cationic protonated guanidine group and the anionic carboxylate group. The γ-protonation weakens the Cβ−Cγ bond of the tryptophan residue, and homolytic cleavage leads to the formation of the complex ion 17, which features a strong hydrogen bond (bond length, 1.609 Å) between the indole ring and peptide backbone. Dissociation of ion 17 and subsequent PT from the indole nitrogen atom to the carboxylic oxygen atom results in the formation of the [M − 116]+ cation and 116 Da neutral radical. The overall barrier against this pathway (34.4 kcal mol−1) is only 2.2 kcal mol−1 lower than that of the charge-remote pathway (36.6 kcal mol−1) as presented in Figure 5a, suggesting that both are accessible for the formation of [M − 116]+ species during the dissociation of the [WGR]•+ radical cation. We also investigated two other possible fragmentation pathways, albeit with much higher reaction barriers. Figure S6a, Supporting Information, displays a charge-remote pathway with a 1,4-HAT from the N-terminal amino group to the γcarbon atom followed by Cβ−Cγ bond cleavage. Figure S6b, Supporting Information, presents a charge-directed pathway with a PT from the guanidine group of the arginine side chain to the γ-carbon atom followed by Cβ−Cγ bond cleavage. Both are energetically hindered pathways with barriers of 41.8 and 42.8 kcal mol−1, respectively.

Figure 4. CID spectra of (a) [WGGR]•+ and (b) [WGR]•+.

Mechanisms and Energetics of [W•GR]+ → [WGR − 116]+. Figure 5a displays the PES for the formation of [M − 116]+ from [W•GR]+ following a charge-remote pathway. Ion 12 is the lowest-energy structure for [W•GR]+. The radical and proton are located at the N-terminal α-carbon atom and on the guanidine group of the arginine residue, respectively. Two hydrogen bonds are formed between the guanidine group and the O1C unit (the italicized subscript refers to the residue numbered from the N- to the C-terminus) with bond lengths of 1.866 and 1.923 Å, which greatly strengthen the electron withdrawing ability of the [−C(O)NH]1 unit and therefore reinforce the captodative stabilization of the α-carbon-centered radical.30,53 Starting from ion 12, a 1,5-HAT from the first amide group to the γ-carbon atom of the tryptophan residue, against a barrier of 36.6 kcal mol−1, forms a shallow local minimum, ion 13, which is 33.1 kcal mol−1 higher in enthalpy than ion 12. Ion 13 undergoes facile Cβ−Cγ bond cleavage with a barrier of only 2.0 kcal mol−1, resulting in ion 14, a protonbound complex formed between 1H-indole and the backbone. Dissociation of ion 14 and subsequent HAT from the indole nitrogen atom to the first amide nitrogen atom result in the product [M − 116]+ and the 1-indolyl radical (116 Da neutral loss) with an overall endothermicity of 32.4 kcal mol−1. The reaction barrier against this pathway is 36.6 kcal mol−1, comparable with the barriers (32−34 kcal mol−1) against the backbone cleavages in the similar [GRW] •+ system.49 Dissociation of ion 14 without HAT results in the [M − 117]•+ radical cation and 1H-indole (117 Da neutral loss) in a process that is endothermic by 39.9 kcal mol−1 (7.5 kcal mol−1 greater than the 116 Da loss). This result is consistent with the CID spectrum of [WGR]•+, wherein we observed no signal for the [M − 117]•+ radical cation.

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CONCLUSIONS Following losses of CO2 from the precursors, dissociations of [GnW]•+ (n = 2−4) and [GXW]•+ (X = C, S, L, F, Y, Q) radical cations feature facile Cβ−Cγ bond cleavages of the tryptophan residues, resulting in the formation of [M − CO2 − 116]+, [M − CO2 − 117]•+, and [1H-indole]•+ product ions. The formation of the [M − CO2 − 116]+ and [1H-indole]•+ fragments is favored from the CID of peptide radical cations possessing high and low PAs, respectively. This finding indicates that the two fragments resulting from Cβ−Cγ bond cleavage of the tryptophan residue compete for the proton. This finding is supported by DFT calculations of [GGW − CO2]•+ as the model system. According to the calculations, a proton’s migration to the γ-carbon atom of the tryptophan residue in [GGW − CO2]•+ (ion 2) can lead to cleavage of the Cβ−Cγ bond and formation of a proton-bound complex (ion 5), which is followed by competition for the proton between the indole ring and the backbone. Ion 2 also can efficiently isomerize to a more stable isomer (ion 9) with both the radical and charge located on the indole ring. RRKM modeling suggested that ion 9 cannot isomerize back to ion 2. Instead, it dissociates predominantly through N−Cα bond cleavage, which is in good agreement with the experimental results. We also observed Cβ−Cγ bond cleavage of tryptophan residues in the dissociations of [WGnR]•+ (n = 1−3) species, 1065



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Figure 5. PES for the formation of [M − 116]+ from [W•GR]+ through (a) a charge-remote pathway and (b) a pathway involving a salt-bridge intermediate. Numbers on the PES are enthalpies at 0 K (all energies in kcal mol−1; bond lengths in Å).

resulting in the predominant formation of [M − 116]+ product ions. In these arginine-containing systems, the proton is tightly sequestered by the highly basic guanidine group, likely facilitating the radical’s migration to the N-terminal α-carbon atom.49 DFT calculations for the [W•GR]+ model system indicate that both charge-remote and SB pathways are accessible for the formation of [M − 116]+ species with barriers of 36.6 and 34.4 kcal mol−1, respectively, that are comparable with those calculated for backbone cleavages of peptide radical cations having similar sequences (32−34 kcal mol−1).

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material 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] (I.K.C.); [email protected] (C.K.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Hong Kong Research Grants Council (RGC), Hong Kong Special Administrative Region, China (project No. HKU7016/10P and No. HKU7016/11P). T.S. thanks the Hong Kong RGC for supporting his studentship. C.-K.S. gratefully acknowledges the RGC, Hong Kong Special Administrative Region (HKSAR), for financial support (project No. CityU 103110). J.L. acknowledges support from the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences &

ASSOCIATED CONTENT

S Supporting Information *

CID spectra of [GAGW]•+, [GSW]•+, [GQW]•+, [WGGGR]•+, and [Wα‑CH3GGGR]•+; PES for pathways b and c in Scheme 1; two alternative pathways for the formation of [M − 116]+ from [W•GR]+; and a list of total energies and Cartesian coordinates predicted for the stationary points in Figures 2 and 4. This 1066



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