Is Dissociation of Peptide Radical Cations an Ergodic Process?

This study presents a first detailed investigation of the energetics and dynamics of dissociation of peptide radical cations using a model system, in which the initial position of the radical site is well-defined. We demonstrate that fragmentation is dominated by bond cleavages that are remote from the initial position of the radical site and that all the dissociation channels are adequately described by the RRKM theory. Our findings suggest that fragmentation of peptide radical cations does not circumvent the ergodic assumption

Energetics and Dynamics of Electron Transfer and Proton Transfer in Dissociation of Metal^III(salen)−Peptide Complexes in the Gas Phase

Time- and collision energy-resolved surface-induced dissociation (SID) of ternary complexes of CoIII(salen)+, FeIII(salen)+, and MnIII(salen)+ with several angiotensin peptide analogues was studied using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Time-resolved fragmentation efficiency curves (TFECs) were modeled using an RRKM-based approach developed in our laboratory. The approach utilizes a very flexible analytical expression for the internal energy deposition function that is capable of reproducing both single-collision and multiple-collision activation in the gas phase and excitation by collisions with a surface. The energetics and dynamics of competing dissociation pathways obtained from the modeling provides important insight on the competition between proton transfer, electron transfer, loss of neutral peptide ligand, and other processes that determine gas-phase fragmentation of these model systems. Similar fragmentation behavior was obtained for various CoIII(salen)−peptide systems of different angiotensin analogues. In contrast, dissociation pathways and relative stabilities of the complexes changed dramatically when cobalt was replaced with trivalent iron or manganese. We demonstrate that the electron-transfer efficiency is correlated with redox properties of the metalIII(salen) complexes (Co > Fe > Mn), while differences in the types of fragments formed from the complexes reflect differences in the modes of binding between the metal−salen complex and the peptide ligand. RRKM modeling of time- and collision-energy-resolved SID data suggests that the competition between proton transfer and electron transfer during dissociation of CoIII(salen)−peptide complexes is mainly determined by differences in entropy effects while the energetics of these two pathways are very similar

The Effect of the Secondary Structure on Dissociation of Peptide Radical Cations: Fragmentation of Angiotensin III and Its Analogues

Fragmentation of protonated RVYIHPF and RVYIHPF−OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of CoIII(salen)−peptide complexes. Both the energetics and the mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable toward fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation, while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments

Effect of the Basic Residue on the Energetics, Dynamics, and Mechanisms of Gas-Phase Fragmentation of Protonated Peptides

The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice−Ramsperger−Kassel−Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues

Mechanistic Examination of C_β–C_γ Bond Cleavages of Tryptophan Residues during Dissociations of Molecular Peptide Radical Cations

In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [G_<i>n</i>W]^•+ (<i>n</i> = 2–4) and [GXW]^•+ (X = C, S, L, F, Y, Q) species. The C_β–C_γ bond cleavage of a C-terminal decarboxylated tryptophan residue ([M – CO₂]^•+) can generate [M – CO₂ – 116]⁺, [M – CO₂ – 117]^•+, and [1<i>H</i>-indole]^•+ (<i>m</i>/<i>z</i> 117) species as possible product ions. Competition between the formation of [M – CO₂ – 116]⁺ and [1<i>H</i>-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 <b>2</b>, the decarboxylated species that is active against C_β–C_γ bond cleavage, can efficiently isomerize to form a more stable π-radical isomer (ion <b>9</b>) as supported by Rice–Ramsperger–Kassel–Marcus (RRKM) modeling. The C_β–C_γ bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WG_<i>n</i>R]^•+ (<i>n</i> = 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 C_β–C_γ bond cleavage to prevail over the charge-directed 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

α,ω-Diaminoalkanes as Models for Bases that Dicoordinate the Proton: An Evaluation of the Kinetic Method for Estimating Their Proton Affinities

The effectiveness of the kinetic method for estimating the proton affinities of bases that di-coordinate the proton is evaluated using α,ω-diaminoalkanes as model bases. The proton affinities of these diamines have previously been examined using the equilibrium method and critically evaluated. Calculations using density functional theory at the B3LYP/6-31++G(d,p) level confirm that protonated α,ω-diaminoalkanes have cyclic structures with the proton covalently bound to one of the amino nitrogen atoms and hydrogen-bonded to the other. Furthermore, this cyclic structure persists in the protonated heterodimer ion between an α,ω-diaminoalkane and ammonia (the model reference base); binding of the two bases takes place via a second hydrogen bond between the RNH3+ and ammonia. Measuring the proton affinities under several collision energies and extrapolating to zero collision energy yields proton affinities that are smaller than the reference values by −2.8 kcal/mol, on average. Application of the Fenselau correction gives proton affinities that differ from the reference values by ±1.0 kcal/mol. These results indicate that the kinetic method is effective for estimating the proton affinities of molecules that tend to have more than one potential protonation site. Application of this method is particularly suited to biological molecules, such as peptides, where application of the equilibrium method is impossible due to low sample volatility

Copper-Mediated Peptide Radical Ions in the Gas Phase

Molecular radical cations, M•+, of amino acids and oligopeptides are produced by collision-induced dissociation of mixed complex ions, [CuII(dien)M]•2+, that contain CuII, an amine, typically diethylenetriamine (dien), and the oligopeptide, M. With dien as the amine ligand, abundant M•+ formation is observed only for the amino acids tryptophan and tyrosine, and oligopeptides that contain either the tryptophanyl or tyrosyl residue. Dissociation of the M•+ ion is rich and differs considerably from that of protonated amino acids and peptides. Facile fragmentation occurs around the α-carbon of the tryptophanyl residue. Cleavage of the N−Cα bond and proton transfer from the exocyclic methylene group in the side chain to the N-terminal residue results in formation of the [zn − H]•+ ion and elimination of the N-terminal fragment as ammonia or an amide, depending on the position of the tryptophanyl residue. Cleavage of the Cα−C bond of an oligopeptide containing a C-terminal tryptophan residue and proton transfer from the carboxylic group to the N-terminal fragment (a carbonyl oxygen atom) results in formation of the [an + H]•+ ion and elimination of carbon dioxide. Both types of fragmentation have no analogous reactions in protonated peptides. For the M•+ of tryptophanylglycylglycine, WGG, elimination of the tryptophanyl side chain results in GGG•+. This radical cation fragments by eliminating its C-terminal glycine to give the [b2 − H]•+ ion, which is an oxazolone and shares much of the structure and reactivity of the b2+ ion from protonated triglycine. Density functional theory shows the mechanism of forming the [b2 − H]•+ ion is similar to that of the b2+ ion, although the free-energy barrier at 29.4 kcal/mol is lower. The [b2 − H]•+ ion eliminates CO readily to give the [a2 − H]•+ ion, which is an iminium radical ion

Formation, Isomerization, and Dissociation of α-Carbon-Centered and π-Centered Glycylglycyltryptophan Radical Cations

Gas phase fragmentations of two isomeric radical cationic tripeptides of glycylglycyltryptophan[G•GW]+ and [GGW]•+with well-defined initial radical sites at the α-carbon atom and the 3-methylindole ring, respectively, have been studied using collision-induced dissociation (CID), density functional theory (DFT), and Rice−Ramsperger−Kassel−Marcus (RRKM) theory. Substantially different low-energy CID spectra were obtained for these two isomeric GGW structures, suggesting that they did not interconvert on the time scale of these experiments. DFT and RRKM calculations were used to investigate the influence of the kinetics, stabilities, and locations of the radicals on the competition between the isomerization and dissociation channels. The calculated isomerization barrier between the GGW radical cations (>35.4 kcal/mol) was slightly higher than the barrier for competitive dissociation of these species (<30.5 kcal/mol); the corresponding microcanonical rate constants for isomerization obtained from RRKM calculations were all considerably lower than the dissociation rates at all internal energies. Thus, interconversion between the GGW isomers examined in this study cannot compete with their fragmentations

Proton Migration and Tautomerism in Protonated Triglycine

Proton migration in protonated glycylglycylglycine (GGG) has been investigated by using density functional theory at the B3LYP/6-31++G(d,p) level of theory. On the protonated GGG energy hypersurface 19 critical points have been characterized, 11 as minima and 8 as first-order saddle points. Transition state structures for interconversion between eight of these minima are reported, starting from a structure in which there is protonation at the amino nitrogen of the N-terminal glycyl residue following the migration of the proton until there is fragmentation into protonated 2-aminomethyl-5-oxazolone (the b2 ion) and glycine. Individual free energy barriers are small, ranging from 4.3 to 18.1 kcal mol-1. The most favorable site of protonation on GGG is the carbonyl oxygen of the N-terminal residue. This isomer is stabilized by a hydrogen bond of the type O−H···N with the N-terminal nitrogen atom, resulting in a compact five-membered ring. Another oxygen-protonated isomer with hydrogen bonding of the type O−H···O, resulting in a seven-membered ring, is only 0.1 kcal mol-1 higher in free energy. Protonation on the N-terminal nitrogen atom produces an isomer that is about 1 kcal mol-1 higher in free energy than isomers resulting from protonation on the carbonyl oxygen of the N-terminal residue. The calculated energy barrier to generate the b2 ion from protonated GGG is 32.5 kcal mol-1 via TS(6→7). The calculated basicity and proton affinity of GGG from our results are 216.3 and 223.8 kcal mol-1, respectively. These values are 3−4 kcal mol-1 lower than those from previous calculations and are in excellent agreement with recently revised experimental values

Arginine-Facilitated Isomerization: Radical-Induced Dissociation of Aliphatic Radical Cationic Glycylarginyl(iso)leucine Tripeptides

The gas phase fragmentations of aliphatic radical cationic glycylglycyl­(iso)­leucine tripeptides ([G•G­(L/I)]+), with well-defined initial locations of the radical centers at their N-terminal α-carbon atoms, are significantly different from those of their basic glycylarginyl­(iso)­leucine ([G•R­(L/I)]+) counterparts; the former lead predominantly to [b2 – H]•+ fragment ions, whereas the latter result in the formation of characteristic product ions via the losses of •CH­(CH3)2 from [G•RL]+ and •CH2CH3 from [G•RI]+ through Cβ–Cγ side-chain cleavages of the (iso)­leucine residues, making these two peptides distinguishable. The α-carbon-centered radical at the leucine residue is the key intermediate that triggers the subsequent Cβ–Cγ bond cleavage, as supported by the absence of •CH­(CH3)2 loss from the collision-induced dissociation of [G•RLα‑Me]+, a radical cation for which the α-hydrogen atom of the leucine residue had been substituted by a methyl group. Density functional theory calculations at the B3LYP 6-31++G­(d,p) level of theory supported the notion that the highly basic arginine residue could not only increase the energy barriers against charge-induced dissociation pathways but also decrease the energy barriers against hydrogen atom transfers in the GR­(L/I) radical cations by ∼10 kcal mol–1, thereby allowing the intermediate precursors containing α- and γ-carbon-centered radicals at the (iso)­leucine residues to be formed more readily prior to promoting subsequent Cβ–Cγ and Cα–Cβ bond cleavages. The hydrogen atom transfer barriers for the α- and γ-carbon-centered GR­(L/I) radical cations (roughly in the range 29–34 kcal mol–1) are comparable with those of the competitive side-chain cleavage processes. The transition structures for the elimination of •CH­(CH3)2 and •CH2CH3 from the (iso)­leucine side chains possess similar structures, but slightly different dissociation barriers of 31.9 and 34.0 kcal mol–1, respectively; the energy barriers for the elimination of the alkenes CH2CH­(CH3)2 and CH3CHCHCH3 through Cα–Cβ bond cleavages of γ-carbon-centered radicals at the (iso)­leucine side chains are 29.1 and 32.8 kcal mol–1, respectively