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Thermodynamics and Mechanisms of Protonated Asparaginyl-Glycine Decomposition Georgia C Boles, Ranran Wu, Mary T. Rodgers, and Peter B. Armentrout J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03253 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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The Journal of Physical Chemistry

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Submitted to J. Phys. Chem. B

Thermodynamics and Mechanisms of Protonated Asparaginyl-Glycine Decomposition

Georgia C. Boles,† R. R. Wu,‡ M. T. Rodgers,‡ and P. B. Armentrout†,* †

Department of Chemistry, University of Utah, 315 S. 1400 E. Rm. 2020, Salt Lake City, Utah

84112, United States ‡

Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States

Corresponding Author: P. B. Armentrout, [email protected], (801) 581-7885

Abstract Deamidation at asparagine residues, a spontaneous post-translational modification in proteins, plays a significant role in various biological processes and degenerative diseases. In the current work, we present a full description of the deamidation process as well as other key fragmentations (dehydration, peptide bond cleavage, and loss of 2 NH3) from protonated asparaginyl-glycine, H+(AsnGly), by studying its kinetic energy dependent collision-induced dissociation (CID) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). These results are compared with those for sustained off-resonance irradiation (SORI)-CID of H+(AsnGly) with Ar in a Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS). Computationally, simulating annealing methodology and a series of relaxed potential energy scans at the B3LYP/6-31G(d) level were performed to identify all intermediate and transition state (TS) structures for each key reaction. All species were further optimized at the B3LYP and B3LYP-GD3BJ/6-311+G(d,p) levels of theory. Single point energies of all major reaction species were calculated at the B3LYP, B3P86, MP2(full), and B3LYP-GD3BJ levels of theory and using M06-2X for rate-limiting species. Relative energies of intermediates, TSs, and products allow characterization of the elementary and rate limiting steps in H+(AsnGly)

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decomposition. By combining experimental and computational results, the complete mechanistic nature of H+(AsnGly) deamidation and other fragmentations is explored and compared to the previously studied H+(Asn) complex. The influence of water solvation on key TSs is also explored. On a fundamental level, this analysis will aid in understanding the thermodynamic and kinetic characteristics of the key intramolecular interactions involved in deamidation, dehydration, and other important fragmentations of peptides.

Introduction Spontaneous deamidation at asparaginyl (Asn) residues is a primary route of protein degradation.1 Under biological conditions, the mechanism of deamidation is believed to occur via formation of a succinimide intermediate, which spontaneously undergoes hydrolysis, producing a combination of iso-aspartate (iso-Asp) and aspartate (Asp), typically found in a 3:1 ratio.1 These degradation effects have been shown to have major influences on biologically important factors such as modified protein function2 and an altered potency of pharmaceuticals.3 Furthermore, deamidation processes (specifically regarding the formation of iso-Asp, which puts an extra carbon in the backbone of the protein) have also been linked to the onset and progression of Parkinson’s and Alzheimer’s disease.1,4 Similar degradation is observed in glutamine (Gln) residues, although generally at a significantly slower rate as a result of forming a less stable, six-membered glutarimide (rather than succinimide).1,5 Specifically, Gly-Xxx-GlnYyy-Gly pentapeptides (where Xxx and Yyy correspond to various amino acids) exhibit deamidation rates more than 10 times slower than those of Gly-Xxx-Asn-Yyy-Gly sequences in solution-phase studies.5 Interestingly, the deamidation rates for representative Asn-Yyy sequences occur over a range of 1.2 to > 1000 days.5 The fastest deamidation rates were observed with smaller residues (especially Gly), presumably because they do not introduce a great deal of steric hindrance interfering with succinimide ring formation. However, select side chains exhibit much faster Asn-Yyy deamidation rates than comparable side chains that would seem to provide similar degrees of steric hindrance. Specifically, threonine (Thr) is similar in


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size and composition to valine (Val), although the deamidation rate of AsnVal is more than five times that of AsnThr.5 Other factors, in addition to steric effects, have also been shown to influence deamidation rates. Specifically, Kosky and co-workers found that amide hydrogen exchange rate (i.e., amide proton acidity), hydrophilicity, and polarizability also play roles in controlling deamidation rates.6 Therefore, it would be useful to examine these systems on a fundamental level to determine the specific interactions that play a significant role in hindering or accelerating the deamidation process. This aspect of deamidation is much harder to determine via large scale solution phase studies. In the present study, we utilize threshold collision-induced dissociation (TCID), a technique allowing for the accurate determination of thermochemistry, carried out in a guided ion beam tandem mass spectrometer (GIBMS) to examine a prototypical deamidation process from protonated AsnGly, H+(AsnGly) (m/z = 190). The use of gas-phase methods eliminates dependences on solution conditions present in all solution phase studies of asparagine deamidation, namely temperature, pH,7 viscosity,8 and solvation effects on higher-order protein structural characteristics.9 The major reactions observed in the current study are the deamidation and dehydration processes, loss of NH3 (m/z = 173) and H2O (m/z = 172), respectively. Because quantitative GIBMS studies require tuning the instrument to obtain complete transmission of all product ions, mass resolution often suffers, such that separating the two major peaks at m/z = 172 and 173 could be problematic. To ensure that the relative cross sections for these two species are accurate, the system was also studied using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS), a technique commonly used for dissociating large biological molecules. The FT-ICR MS provides very high mass resolution,10 and although the kinetic energy can be measured as a function of SORI activation time, several problems arise when quantitatively determining the internal energy resulting from SORI collisions, a result of the oscillating nature of the kinetic energy11 and multiple collisions. Thus, SORI-CID data is used here for a qualitative comparison to kinetic energy dependent cross sections obtained via TCID studies. All reaction energetics



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reported are obtained from analysis of TCID thresholds and compared to theoretical results calculated at the B3LYP, B3P86, MP2(full), B3LYP-GD3BJ, and M06-2X levels of theory using a 6-311+G(2d,2p) basis set. Such comparisons allow details of the mechanisms found for these reactions to be explored and validated. After submission of this work, we became aware of a parallel spectroscopic study that characterized the structures of the deamidation products of protonated asparaginyl-alanine, H+(AsnAla).12 Kempkes et al. found that two distinct structures, a succinimide and furanone complex, respectively, were formed by loss of NH3 from infrared multiple photon dissociation (IRMPD). Both pathways are considered in the work below, and we compare several pathways for formation of the succinimide product, including a pathway detailed previously7 that proceeds via a tetrahedral intermediate. The results presented in this work are also compared to similar information previously obtained for protonated asparagine, H+(Asn),13 specifically regarding the energetic characteristics of the deamidation pathway. The combined results from the H+(Asn) and H+(AsnGly) systems provide a foundation for further study of the effect of amino acid sequence, chain length, and steric effects of residue side-chains on the rate of deamidation. A complete characterization of these systems allows evaluation of the mechanistic nature of deamidation processes and will provide valuable, fundamental information regarding the specific intramolecular interactions that play the most significant roles in the deamidation process as well as other peptide fragmentations.

Experimental and Computational Section General Experimental Procedures - GIBMS The kinetic energy dependent cross sections for the CID of H+(AsnGly) with Xe were measured using a guided ion beam tandem mass spectrometer (GIBMS) in the Armentrout lab that has been described in detail elsewhere.14-16 Ions are generated using an electrospray ionization (ESI) source under similar conditions to those described previously.17 In short, the ESI is operated using 5x10-5 M H+AsnGly (purchased from AnaSpec, Fremont, CA, USA) in 50:50


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(by volume) MeOH/H2O solution, acidified with an appropriate amount of acetic acid (purchased from Sigma-Aldrich, St. Louis, MO, USA), and syringe-pumped at a rate of 0.67 µL/min into a 35 gauge stainless steel needle biased at 1800 – 2100 V relative to ground. Ions are directed through a heated capillary at 70 °C into a radio frequency (rf) ion funnel,18 where they are focused into a tight beam. After exiting the ion funnel, the ions enter a rf trapping hexapole ion guide where the ions undergo on the order of 104 thermalizing collisions with the ambient gas. As demonstrated in earlier studies, ions produced in the source region should have a MaxwellBoltzmann distribution of rovibrational states at 300 K.13,17,19 In the present study, production of thermal ions required careful control of source conditions to limit the formation of excited species. For H+(AsnGly), thermalization of the ions was particularly sensitive to changes in the peak-to-peak rf voltage (Vpp) of the ion funnel. A peak-to-peak voltage of 14 V was used as it resulted in the highest threshold energies, whereas a higher voltage resulted in much higher intensities but hot ions (indicated by lower threshold energies). Additional precautions taken included using a small gradient (8 V) on the initial set of focusing lenses, where a larger gradient resulted in hot ions, and a lower temperature on the heated capillary (where more typical temperatures used in the past range from 75 – 80 °C). The precursor H+(AsnGly) ions are extracted from the source and mass selected using a magnetic momentum analyzer, decelerated to a well-defined kinetic energy, and focused into an rf octopole ion guide that traps the ions radially,20,21 which minimizes losses of product and reactant ions. The octopole passes through a collision cell containing xenon22,23 at a sufficiently low pressure (< 0.3 mTorr) such that the opportunity for multiple collisions to occur is minimal. The product and residual reactant ions drift to the end of the octopole guide, where they are extracted and focused into a quadrupole mass filter for mass analysis. For the present studies, the quadrupole mass filter was operated under two resolution conditions. For experiments at high mass resolution, the pole offset in the quadrupole mass analyzer was lowered to achieve unit mass resolution. It was carefully checked that these conditions did not compromise the transmission of ions, although these high resolution conditions did not allow the observation of



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product ions of low intensity. Alternate (higher) pole offset voltages allowed such products to be observed, but no longer permitted the single mass unit separation. Ions are detected with a high voltage dynode and scintillation detector,24 and the signal is processed using standard pulse counting techniques. Ion intensities of reactants and products, measured as a function of collision energy, are converted to absolute cross sections as described previously.14 Briefly, the calculation of the cross section from the ion intensities utilizes a relationship that is directly analogous to the Beer-Lambert Law, specifically, I = I0 exp(-ρσl), where, I is the reactant ion intensity after passing through the collision cell, I0 is the reactant ion intensity entering the collision cell, l is the length of the collision cell (8.3 cm), and ρ is the number density of the neutral reactant and equals P/kBT, where P and T are the pressure and temperature of the gas and kB is Boltzmann’s constant. The uncertainty in these relative cross sections is about ±5% and that for the absolute cross sections is about ±20%. The ion kinetic energy distribution is measured using a retarding potential analysis and found to be Gaussian with a typical full width at half maximum (FWHM) of 0.1 - 0.2 eV (lab). Uncertainties in the absolute energy scale are about ±0.05 eV (lab). Ion kinetic energies in the laboratory (lab) frame are converted to energies in the center-of-mass (CM) frame using ECM = Elab m/(m + M), where M and m are the masses of the ionic and neutral reactants, respectively. All energies in this work are reported in the CM frame unless stated otherwise. General Experimental Procedures – SORI-CID A Bruker 7 T SolariX Hybrid FTMS mass spectrometer (Q-FT-ICR MS) in the Rodgers Laboratory at Wayne State University was used to perform the SORI-CID of H+(AsnGly). The AsnGly dipeptide was synthesized, purified and provided by the Polfer Laboratory of the University of Florida. (A different supplier of the AsnGly sample was used in the GIBMS experiments because they were performed much later than the FT-ICR experiments and we wanted to ensure that the samples had not degraded, presumably by deamidation.) A 0.1 mM solution of H+(AsnGly) was prepared by dissolving an appropriate amount of AsnGly in a 50%:50% MeOH/H2O solution with a total concentration of 1% acetic acid. The solution was


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delivered to the electrospray ionization (ESI) source at a flow rate of 2.0 µL/min. The ions pass through a glass capillary, are orthogonally extracted into a dual ion funnel, decelerated and cooled within the source multipole, and focused and guided through a quadrupole mass filter for mass-selection. The mass-isolated H+(AsnGly) ions are directed by a rf hexapole ion guide through the inhomogeneous fringing field of the superconducting magnet into the ICR cell. Argon gas was pulsed into the cell to induce SORI-CID fragmentation of the H+(AsnGly) ions. The SORI-power was gradually increased from 0.0% to 0.5% at a step size of 0.05%, and from 0.5% to 0.8% at a step size of 0.1%, with an irradiation time of 0.5 s. Thermochemical Analysis Thresholds of the TCID cross sections are modeled using eq 1, = , ⁄

,

∗ ⁄ ∗ 1 − ! "#$%$

∗ &

} − ()"* +( 1

where σ0,j is an energy-independent scaling factor for channel j, n is an adjustable, empirical representation of factors that describe the efficiency of the energy transfer during collision and varies with the complexity of the system being studied,15 E is the relative kinetic energy of the reactants, E0,j is the threshold for dissociation of the ground electronic and rovibrational state of the reactant ion at 0 K for channel j, , is the experimental time for dissociation (~5 x 10-4 s, as

measured by previous time-of-flight studies),15 ( is the energy transferred during the collision, and E* is the internal energy of the energized molecule (EM) after the collision, so that E* = ( +

Ei. The summation is over the rovibrational states of the reactant ions, i, where Ei is the excitation energy of each state and gi is the fractional population of those states ( gi = 1). The Beyer–Swinehart-Stein-Rabinovitch algorithm25-27 is used to evaluate the number and density of the rovibrational states and the relative populations gi are calculated for a Maxwell-Boltzmann distribution at 300 K. The term kj(E*) is the unimolecular rate constant for dissociation of the EM to channel j via its rate-limiting transition state (TS). The rate constants kj(E*) and ktot(E*) are defined by Rice-Ramsperger Kassel-Marcus (RRKM) theory as in eq 2,28,29 ∗ = - ∗ = - + ./ ∗ − , /ℎ2 ∗ 2



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8 where + is the reaction degeneracy of channel j, Nj†(E*- E0,j) is the sum of rovibrational states

for the TS of channel j at an energy E* - E0,j, and 2(E*) is the density of states of the EM at the

available energy, E*. These rate constants allow both kinetic shifts (where the probability of dissociation is given by the term {1 − ! "#$%$

∗ &

} in eq 1) and competition between multiple

channels (which in controlled by the ratio of rate coefficients in eq 1, ∗ ⁄ ∗ ) to be

modeled accurately.30,31 Several effects complicate the data analysis and must be accounted for to produce accurate thermodynamic information. First, the kinetic energy distributions of the reactants result in energy broadening, which is accounted for by explicit convolution of the model over kinetic energy distributions of both reactants, as described elsewhere.14 After this convolution, the threshold model of eq 1 includes all sources of energy available to the reactants. The second effect involves the necessity of single-collision events in order for the data to be accurately modeled using eq 1. To ensure single collision conditions, data for the high mass resolution conditions were collected at three pressures of Xe, here about 0.30, 0.15, and 0.07 mTorr, and the resulting cross sections evaluated for pressure effects and extrapolated to zero pressure when pressure effects outside the range of experimental error are detected.32 The last effect arises from the average lifetime for dissociation, which can result in a kinetic shift of the CID threshold, which increases as the size of the molecule increases. To estimate the kinetic shifts observed, the thresholds are also determined without including RRKM modeling and competition in eq 1. To evaluate the rate coefficients in eqs 1 and 2, the needed sets of rovibrational frequencies for the EM and the rate-limiting TSs are determined from quantum chemical calculations discussed in the following section. Additionally, the entropy of activation at 1000 K for each dissociation channel was calculated as described in detail elsewhere.30 The model cross sections of eq 1 are convoluted with the kinetic energy distributions of the reactants14 and compared to the experimental data. A nonlinear least-squares analysis is used to provide optimized values for σ0,j, n, and E0,j. The uncertainty in E0,j is estimated from the range of threshold values determined from multiple sets of data, variations in the parameter n (±10%


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9 around the optimum value), variations in vibrational frequencies (±10%), changes in , by factors of 2, and the uncertainty in the absolute energy scale (0.02 eV). In the present work, several approaches to the variations in vibrational frequencies were used: vibrational frequencies for the reactant, EM, and TSs are simultaneously scaled; frequencies for only the EM and TSs were scaled (as these control the kinetic shift), and alternatively only scaling the frequencies of the reactant (as this alters the amount of energy available to the system). The uncertainties reported in the following sections include the deviations resulting from all three cases. Computational Details Model structures, vibrational frequencies, and energetics for all reaction species were calculated using the Gaussian 09 suite of programs.33 To ensure that the ground structures (GS) of reactant and product species were correctly identified, a simulated annealing program (modified to complete 20,000 annealing cycles) was utilized to explore conformational space.34 Optimizations of all unique low-energy annealed structures were conducted at the B3LYP/6311+G(d,p) level of theory. Additional reaction intermediates and all TSs were initially found at the B3LYP/6-31G(d) level via relaxed potential energy surface scans and were further optimized at the B3LYP and B3LYP-GD3BJ/6-311+G(d,p) levels of theory. Each TS was verified to contain one imaginary frequency, and it was determined that each intermediate was vibrationally stable. Intrinsic reaction coordinate (IRC) calculations were performed on each of the ratelimiting steps to ensure that the optimized TS led to the proposed intermediates. Rotational constants and vibrational frequencies were calculated from optimized structures and vibrational frequencies were scaled by a factor of 0.989 when used for the determination of internal energy, RRKM calculations, and zero-point vibrational energy (ZPE) corrections. Single point energies of all reaction species were calculated using the 6311+G(2d,2p) basis set at the B3LYP, B3P86, and MP2(full) levels using B3LYP geometries and at the B3LYP-GD3BJ//B3LYP-GD3BJ level. Studies of amino acid systems of similar composition have shown that B3LYP-GD3BJ empirical dispersion corrections better describe the hydrogen bonding in these systems.35 Additionally, conversion from 0 K to 298 K reaction



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enthalpies and free energies is accomplished using the rigid rotor/harmonic oscillator approximation with rotational constants and vibrational frequencies calculated at the B3LYP/6311+G(d,p)

level.

Single

point

energies

were

also

calculated

at

the

M06-2X/6-

311+G(2d,2p)//M06-2X/6-311+G(d,p) for all rate-limiting TSs, including products for phase space limit (PSL) TSs.

Results Cross Sections for Collision-Induced Dissociation – GIBMS Experimentally determined kinetic energy dependent cross sections were obtained for the interaction of Xe and H+(AsnGly). For these studies, multiple resolution conditions for the quadrupole mass filter were used to obtain the cross sections for H+(AsnGly) fragmentation. High resolution conditions were used to allow characterization of cross sections for channels separated by 1 amu, whereas low resolution conditions provide a more global overview of the dissociation pathways available to the system. Under low resolution conditions, many dissociation pathways are of sufficient intensity and observed for the decomposition of H+(AsnGly), whereas only four major products are observed in the energy range suitable for high resolution conditions (where the lower pole offset voltage limits the energy range where ions are efficiently transmitted in the laboratory frame). Figure 1 shows a representative dataset for low resolution conditions taken at 0.2 mTorr of Xe, as not all products were easily observed at lower pressures. As refined further below, the product with the lowest threshold is the combination of m/z 173 and 172, corresponding to deamidation and dehydration channels, respectively, the main products of interest here. At about one electron volt higher, the formation of m/z 156 + 155, 113, and 87 occur competitively and have energy dependences consistent with secondary losses from one of the primary product channels. As shown in Scheme 1, m/z 156 and 155 result from the loss of NH3 and H2O from m/z 173, respectively, whereas m/z 113 results from the loss of NH2COCH3, the Asn side-chain, from the m/z 172 primary product ion. As demonstrated below, both primary and sequential (from m/z


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172) pathways were found for the formation of m/z 87, see Scheme 1 and Supporting Information. Another 1 eV higher in energy, the m/z 131 channel appears and corresponds to loss of the Asn side-chain from the reactant ion in a primary process, Scheme 1. The final primary channel is formation of NH4+, m/z 18, which does not appear until relatively high energies, presumably because the proton affinity of the molecule yielding m/z 173 is much higher than that of ammonia. Over the range of 3.5 - 4 eV, thresholds for products at m/z 99, 110, and 138 appear, and at higher energies, products are found at m/z 44, 60, and 70. Although no detailed mechanistic calculations have been done for these latter six products, theoretical calculations were performed on all proposed products (where the lowest energy conformer found for each species is given in Scheme 1). Scheme 1 plausibly identifies these products as sequential dissociations of the various primary and secondary product ions, primarily from the m/z 173, 156, and 87 ions. These higher order dissociations explain the decrease in the cross sections of the primary ions at high energies. Figure 2 shows a representative dataset taken under high resolution conditions at 0.30 mTorr of Xe. Four key processes were monitored over this energy range, including three primary dissociation channels measured at m/z 173 (loss of NH3), 172 (loss of H2O), and 87 (loss of Gly + CO), and one sequential channel measured at higher energies: m/z 156 (loss of NH3 from m/z 173). Because asparginyl residues are known to form succinimide ring structures upon deamidation, m/z 173 is tentatively identified as protonated 3-amino-N-methyl carboxylic acidsuccinimide, H+(AMCA-Suc), as shown in Scheme 1. On the basis of the spectroscopic work of Kempkes et al.,12 we have also explored additional pathways leading to an alternate m/z 173 product ion, specifically protonated 4-amino, 5-2-λ2-azanylacetic acid 3H-furan-2-one, which exhibits energetics comparable to the succinimide pathway. The significance of these pathways related to the interpretation of the data is detailed in sections below. The second primary channel, m/z 172, results from dehydration of the parent ion and can be characterized as an oxazolone, specifically protonated 2-(3-aminopropanamide)-5-(4H)-oxazolone, H+(APA-Ox). As shown in Figure 2, these two primary dissociation channels have very similar thresholds, although the



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magnitude of the m/z 173 cross section is measurably higher than that of m/z 172 at all energies. It was also verified that the summation of the cross sections for m/z 173 and 172 obtained under high resolution conditions was equivalent to the total cross section observed at low resolution conditions. Such a comparison demonstrates that the transmission of these ions is not compromised at the high resolution conditions. However, as noted above, the high resolution conditions are only achieved by sacrificing ion intensity, such that the full array of product ions observed in Figure 1 can no longer be monitored easily. The high resolution conditions also demonstrated that the m/z 156 + 155 product channel is dominated by the m/z 156 product (loss of 2 NH3), which theory (see below) indicates is probably

protonated

7-hydroxy-6,7-dihydro-2H-4λ4-pyrrolo[2,1-b]oxazole-2,5(3H)-dione,

H+(Py-Ox). Under low resolution conditions, evidence for small amounts of m/z 155 (loss of NH3 + H2O) was observed even though this product could not be fully resolved. Collision-Induced Dissociation – SORI Figure 3 shows the SORI-CID of H+(AsnGly), which resulted in major products at m/z 173, 172, 156, 110, and 87, as well as products at m/z 155, 145, 144, 138, and 127 and additional low mass fragments at m/z 99, 85, 70, 58, and 45 (see Supporting Information Figure S1). As shown in Figure 3, products having the lowest thresholds are those of the competitive m/z 173 and 172 channels, where the relative intensities versus power behavior matches that shown in Figure 2. At slightly higher powers, m/z 156 and 87 are observed, again with behavior similar to that seen in Figures 1 and 2. The next product observed as the energy is increased, m/z 155, was not observed in the TCID studies under high mass resolution conditions because of its smaller intensity, but could be observed when low resolution conditions were used. Slightly higher in energy, m/z 138 is observed followed rapidly by the sequential loss of CO to form m/z 110, again similar to the behavior seen in Figure 1. The highest energy products (m/z 127, 144, and 145) were not observed in the TCID studies under either low or high resolution conditions, but were specifically looked for. Apparently, under higher-energy single collision TCID conditions, m/z 145 dissociates rapidly to m/z 99 (which is observed), whereas the multiple low-energy SORI


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conditions allow both ions to be observed. Likewise the failure to observe m/z 144 and its subsequent dissociation product at m/z 127 (loss of NH3) is probably because dissociation of m/z 172 to 113 (a simple bond cleavage, see Scheme 1) is favored under TCID conditions, whereas the more complicated decarbonylation forming m/z 144 is favored for SORI conditions. A similar preference could explain the failure to observe the m/z 131 product under SORI conditions. The slow heating of multiple low-energy collisions will generally favor the formation of primary ions along the lowest energy pathways, here m/z 172 and 173 product ions, and disfavor a primary ion formed at higher energies (such as m/z 131). Other high energy products will also differ in the TCID and SORI-CID experiments for similar reasons. On this basis, the difference in products observed suggests that the collisional aspects of the CID reactions are clearly important. Here, the pressure of the SORI studies could be sufficiently high that collisional stabilization can occur, under which conditions m/z 144 and 145 (and their sequential losses) could be observed. Conversely, no collisional stabilization can occur in the TCID studies. For example, m/z 145 could readily dissociate to form m/z 99, Scheme 1, as seen in the TCID results, such that the presence of m/z 145 and 127 would not be detected. Although the SORI data cannot be interpreted quantitatively, the comparison with the TCID results confirms several aspects of the qualitative behavior of the observed cross sections. Specifically, the deamidation (m/z 173) and dehydration (m/z 172) channels have similar lowenergy thresholds and magnitudes that favor m/z 173. In addition, m/z 156 and 87 are observed at higher energies, and the relative thresholds and intensities of m/z 110 and 138 are consistent with the TCID results. Theoretical Results for Low-Energy H+(AsnGly) Conformers Several low-energy conformations of H+(AsnGly) were found, Figure 4, with relative energies given in Table 1. An additional 15 conformers were found within 30 kJ/mol of the ground structure (GS) and are listed in Table S1 of the Supporting Information and seven of these are shown in Figure S2. Conformers are named according to their protonation sites including additional hydrogen bonds by using the designation [X,Y,Z] where X = protonated



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atom and Y/Z = site of hydrogen bond in the order of increasing hydrogen bond length. The protonation site is followed by the series of dihedral angles starting from the N-terminal sidechain amide-group nitrogen to the C-terminal carboxylic acid, i.e., ∠NsCCC, ∠CCCC, ∠CCCN2, ∠CCN2C, ∠CN2CC, ∠N2CCO3, ∠CCO3H. Backbone nitrogen and oxygen atoms are numbered by residue along the backbone chain starting from the N-terminus. Side-chain nitrogen and oxygen atoms are designated by a superscript s. Dihedral angles are distinguished as cis (c, for angles between 0°–45°), gauche (g, 45°–135°), or trans (t, 135°–180°). The six lowest energy structures of H+(AsnGly) lie within 9 kJ/mol of the GS at all levels of theory and are all protonated at the amine nitrogen (N1) of the backbone. As shown in Figure 4, each of the N1 protonation sites interacts via hydrogen bonds with the carbonyl oxygen of both the side-chain amide (COs) and backbone amide (CO1), [N1,COs,CO1]. B3LYP and B3P86 methods predict the [N1,COs,CO1]-ttgtttt conformer to be lowest in energy, whereas MP2, B3LYP-GD3BJ, and M06-2X predict [N1,COs,CO1]-gggtgtt to be lowest in energy. These two structures differ in how the carbonyl group of the Gly residue, CO2, hydrogen bonds: CO2•HN2 in ttgtttt and in a head-to-tail arrangement, CO2•HNs in gggtgtt. At the B3LYP-GD3BJ level, geometry optimization of [N1,COs,CO1]-tgctttt collapses to its GS gggtgtt conformation. All six low-energy [N1,COs,CO1] conformers also have a O2•O3H hydrogen bond. The only alternate protonation site that is relatively low in energy (8 – 23 kJ/mol above the ground conformer) puts the proton on the carbonyl group of the Asn residue, which H-bonds to the side-chain carbonyl, [CO1,COs]-tggtttt and tgttgtt, Table 1. Other alternate protonation sites, e.g., [COs], [N2,COs], and [CO2,OH], have energies at least 62 kJ/mol relative to the GS. Select structures depicting the four major alternate proton binding motifs are given in Figure 4, along with their relative energies as listed in Table 1. Additional low-lying H+(AsnGly) conformers up to 25 kJ/mol and alternate protonation sites are shown in Figure S3 of the Supporting Information. The mechanisms presented here are the lowest energy pathways for decomposition of H+(AsnGly) starting from the B3LYP determined GS, [N1,COs,CO1]-ttgtttt, although several alternative mechanisms were explored on the basis of different conformational changes in the


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GS. Energies reported at the MP2(full), B3LYP-GD3BJ, and M06-2X levels in all Tables are determined relative to their predicted GS, [N1,COs,CO1]-gggtgtt. The mechanism for moving between these two species is given in Figure S4 of the Supporting Information. Rate-limiting TSs separating these conformations lie below 25 kJ/mol, well below the energies needed for decomposition. Thus, rearrangement among any of these conformers can occur readily and the small changes in conformation do not strongly affect the energetics of the decomposition reactions. Theoretical Results for H+(AsnGly) Deamidation Under biological conditions, deamidation of asparagine residues is known to proceed through a cyclic succinimide intermediate. We located one mechanism for deamidation of H+(AsnGly) that forms H+(AMCA-Suc)[N1,O2], Figure 5. We also explored an additional pathway that proceeds via a tetrahedral intermediate and leads to a H+(AMCA-Suc)[Oc2] product, a mechanism that parallels theoretical work by Konkular et al. on a related neutral peptide model.7 Further, as demonstrated by Kempkes et al. for H+(AsnAla),12 an alternative deamidation pathway yields H+(AAF), such that a mechanism for formation of this product is also developed. Figure 5 shows additional nomenclature adopted for each of these, and several of the other major product ions. Briefly, cyclic nitrogen and oxygen atoms are designated Ncx and Ocx, respectively, where x indicates the order of appearance. Cyclic carbons are then designated using standard nomenclature: α, β, etc. When terminal ends of the dipeptide (carboxylic acid of the deamidation product and side-chain amide of the dehydration product) remain intact, naming is consistent with the nomenclature described above (O2/O3 and Os/Ns, respectively). The series of elementary steps leading to H+(AMCA-Suc)[N1,O2] is shown in Figure 6 as the potential energy surface (PES) calculated at the MP2(full)/6-311+G(2d,2p)//B3LYP/6311+G(d,p) level of theory. Relative energies of all species mapped along the PES are given in Table 2, which includes their structural designations. For TSs where a proton transfer is occurring, protonation sites are designated as [X-Y] where X = protonated atom of the lower energy species and Y = site of transfer. Additional designations are included when additional



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bonds are being broken {X~Y} or formed {X-Y}. When neutral fragments dissociate and remain bound to the complex, the fragment will be indicated in parentheses along with the site it remains bound to in subscript. In the first elementary step of Figure 6, the H-bond between the oxygen of the side chain (Os) and the N1 protonation site is broken by the rotation of the ∠CCCC dihedral angle to form the first intermediate, INT1N, passing through the gauche TS1N (where “N” designates the deamidation pathway). INT1N is stabilized by the formation of an H-bond between the side-chain carbonyl oxygen and the hydrogen of the peptide bond (COs•HN2). In the second elementary step, rotation of the ∠NsCCC dihedral angle results in the formation of hydrogen bonding between the side-chain amide nitrogen and the backbone amide hydrogen (Ns•HN2) in INT2N. This rotation orients the backbone such that cyclization in subsequent steps is feasible. In the third elementary step, a nearly isoenergetic hydrogen transfer between the N1 protonation site and the backbone amide oxygen (O1) decreases the (Ns•HN2) bond length from 2.3 to 2.1 Å. This promotes hydrogen transfer in the fourth elementary step, TS4N, resulting in the formation of the NH3 leaving group in INT4N. Simultaneously, the distance between the carbon of the side-chain amide and the backbone amide nitrogen decreases from 3.1 to 2.5 Å, initiating cyclization. In the following elementary step, concerted ring closure occurs as the complex passes through the ratelimiting TS5N (133 – 151 kJ/mol relative to the GS). This step is stabilized in INT5N by Hbonding between the free ammonia and the succinimide ring and carboxylic acid side chain, thus forming H+(AMCA-Suc)[Oc1,N1](NH3,CβH•N,O2•HN). In the next step, the proton on the carbonyl transfers

back

to

the

original

protonation

site

to

form

INT6N,

H+(AMCA-

Suc)[N1,Oc1](NH3,CβH•N,O2•HN). Other possible binding sites of the ammonia to H+(AMCA-Suc)[N1]

were not explored. From INT6N, 49 - 68 kJ/mol above the GS, simple dissociation can form either NH3 + H+(AMCA-Suc)[N1,O2] (Figure 5) or AMCA-Suc + NH4+. The former deamidation process lies 31 – 55 kJ/mol lower in energy than TS5N, whereas the latter product channel lies 37 – 49 kJ/mol higher in energy (4 – 17 kJ/mol lower than TS5N at the B3LYP, B3P86, and MP2(full) levels of theory, but 2 – 3 kJ/mol higher for B3LYP-GD3BJ and M06-2X).


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The series of elementary steps of the alternative deamidation process leading to H+(AAF) (designated by “F”) is given in Figure 7 as the PES calculated at the MP2(full)/6311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory. Relative energies, along with structural designations, of all species mapped along the PES are given in Table 3. In the first elementary step, rotation of the trans ∠CCCC dihedral angle of the GS passes through gauche TS1F and

results in formation of INT1F. Rotation of the ∠NCCC dihedral angle in the next step results in the formation of INT2F, where proton transfer from N2 to Ns has occurred, thus forming the NH3

leaving group. Elongation of the C-Ns bond results in the concerted C-Ns bond rupture and C-O1 bond formation leading to the furanone ring. This TS leads to INT3F where the NH3 leaving group remains hydrogen-bound to the amino group of the furanone ring (NH3,NH2•N). Other possible binding positions for the NH3 leaving group were not explored. In the final elementary step, simple dissociation of the complex leads to the formation of NH3 + H+(AAF), where the latter species is protonated at N2 and there is both a gtt and ttt conformation with very similar energies, Table 3. These products lie 129 – 149 kJ/mol above the GS and 1 – 15 kJ/mol above TS3F across all levels of theory except B3LYP, which predicts that the pathway is limited by the tight TS3F by 3 kJ/mol. The product asymptote for the alternative NH4+ + AAF products was found to be 234 – 252 kJ/mol higher in energy than the GS, 91 – 106 kJ/mol above H+(AAF) + NH3. With respect to the succinimide formation process in Figure 6, the H+(AAF) + NH3 products lie 4 – 10 kJ/mol above TS5N at the MP2(full) and B3LYP-GD3BJ levels of theory, whereas B3P86 and M06-2X predict energies ~3 kJ/mol lower. Interestingly, B3LYP predicts a much larger difference in energy, where the energy of H+(AAF) + NH3 is predicted to be ~20 kJ/mol lower in energy than TS5N. Clearly, theory suggests that both H+(AMCA-Suc) and H+(AAF) products are energetically comparable and could be formed experimentally (as confirmed by the spectroscopic observations of Kempkes et al. for the alanine analogue of the present system). This is particularly important as it may affect higher energy sequential channels observed, primarily that of the possible m/z 155 and 156 products, as discussed in more detail in following sections.



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A previous theoretical study has modeled the deamidation of asparagine residues via pathways leading to a cyclic tetrahedral intermediate prior to the deamidation step yielding a succinimide.7 Here, cyclization occurs before proton transfer to the amide NH2 group, whereas the mechanism detailed in Figure 6, reverses the order of these steps with TS5N characterized by concerted motions of C-NH3 bond rupture and cyclization. This previous work, which used a neutral model peptide, where no protonation is involved, concluded that if deamidation is occurring through this tetrahedral intermediate, then the deamidation process can behave as the rate-limiting step, where the energetics of the cyclization (barrier of 190 kJ/mol) and deamidation (barrier of 182 kJ/mol) steps are comparable.7 A much larger difference in barrier energies is observed when the basic form of the peptide is considered, where cyclization (46 kJ/mol) occurs much faster than deamidation, which exhibits a barrier energy of 408 kJ/mol.7 Therefore, we have mapped the PES for the deamidation process from the intermediate most likely to be formed via the tetrahedral cyclization of H+(AsnGly), as shown in Supporting Information Figure S5 (pathway designated by “T”). From the tetrahedral intermediate (where Oc2 is now characterized as a hydroxyl group instead of a carbonyl), proton transfer from Oc2 to Ns occurs as the complex passes through the rate-limiting four-centered TS1T (190 – 234 kJ/mol higher in energy than the GS, 57 – 83 kJ/mol higher than TS5N, and 47 – 105 kJ/mol higher than TS3F). This proton transfer also induces a concerted proton transfer from N1 to Oc2. This TS leads to INT1T, in which a hydroxyl and NH3 leaving group are present. Formation of the NH3 group increases the C-NH3 bond by 0.1 Å, and further elongation of this bond in the following step results in bond rupture via TS2T. INT2T is stabilized by H-bonding between the free ammonia and the carboxylic acid side chain, thus forming H+(AMCA-Suc)[Oc2](NH3,O2•HN). Simple dissociation of this complex leads to the formation of H+(AMCA-Suc)[Oc2] + NH3 (145 – 154 kJ/mol higher in energy than the GS and 49 – 65 kJ/mol above H+(AMCA-Suc)[N1,O2]). Theoretical Results for H+(AsnGly) Dehydration The PES mapping the reaction coordinate for the loss of H2O from H+(AsnGly) is given in Figure 8, with a complete list of energies given in Table 4. In the first elementary step, proton


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transfer from the N1 protonation site to N2 results in the formation of INT1O (where “O” designates the dehydration pathway). Upon rotation of the ∠N2CCO2 dihedral angle in the second elementary step, the CO2•HN2 H-bond is broken and is replaced with the formation of a COs•HN2 H-bond. This H-bond allows proton transfer between the sites, resulting in INT3O. Now proton transfer from COsH to the hydroxyl oxygen of the carboxylic acid group (O3) can occur facilely via the head-to-tail conformation of the TS4O complex in the fourth elementary step. As this proton transfer occurs, cyclization occurs by linking O1 to the carbon of the carboxylic acid, forming an oxazolone ring. After passing over the rate-limiting TS4O, which lies 111 - 130 kJ/mol above the GS, a complex of the protonated oxazolone H+(APA-Ox)[Nc] (m/z 172) Hbound to water at the side-chain carbonyl and nitrogen (Nc) of the oxazolone is formed. Lastly, dissociation of INT4O can lead to H2O + H+(APA-Ox)[Nc,N1] (m/z 172), where the product asymptote lies 1 - 20 kJ/mol below TS4O. The alternative products, H3O+ + APA-Ox, lie 267 – 278 kJ/mol higher in energy. Theoretical Results for the Formation of m/z 87 from H+(AsnGly) The reaction coordinate showing the elementary steps of the reaction yielding m/z 87 is given in Figure 9, with energies given in Table 5 (where “G” designates the loss of glycine). The first elementary step is the same as for dehydration, i.e., TS1O = TS1G and INT1O = INT1G, which contains the glycine leaving group. Upon rotation of the ∠CCCC dihedral angle, the complex passes through TS2G to form INT2G, thereby replacing a N2H•N1 H-bond with N2H•OsC. In the following step, rotation about the ∠CCN2C dihedral angle breaks the H-bond between CO2 and N2H, which increases the distance between the carbon of the peptide bond and N2, facilitating cleavage of that bond in the following rate-limiting step. TS4G depicts the simultaneous cleavage of the bonds on either side of the central carbonyl. These motions result in INT4G, a complex of the incipient products, protonated 3-amino propanamide (H+APA, m/z 87) along with Gly and CO. The CO leaving group remains hydrogen bound to the terminal NH2 group of glycine, which is H-bonded to APA via the amino NH2 protonation site and a CH•N H-bond. Alternative isomers of this complex were not explored. INT4G readily loses CO, which is bound by only 5 –



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10 kJ/mol, to form INT5G, a complex of H+(APA)[N1](Gly). This proton-bound species can dissociate to form the final products H+(APA)[N1] (m/z 87) + Gly + CO, where this product asymptote lies 154 – 204 kJ/mol higher than the H+(AsnGly) GS and 10 – 27 kJ/mol above TS4G at all levels except B3LYP where the products lie 9 kJ/mol below TS4G. Thus, product formation is limited by the loose TS associated with the final products at most levels of theory. It is also possible that this intermediate dissociates to form the alternative products, H+(Gly) + APA + CO, which lies 48 – 56 kJ/mol higher in energy. An alternative sequential pathway (starting from the dehydration product, m/z 172) was also found, although this path has a rate-limiting TS that lies 20 – 44 kJ/mol higher in energy than that shown in Figure 9. This is primarily because the neutral products formed along this pathway, 5-(4H)-Ox + H2O, are less stable than Gly + CO, lying only 1 – 17 kJ/mol lower in energy than the rate-limiting TS. The PES showing the reaction coordinate for this sequential pathway is given in Supporting Information, Figure S6. Theoretical Results for the Formation of m/z 156 from m/z 173 The reaction coordinate showing the elementary steps of the reaction yielding m/z 156 from the succinimide deamidation product is given in Figure 10, with energies given in Table 6 (where “S” represents the sequential deamidation process). From H+(AMCA-Suc)[N1,O2], proton transfer from N1 to Oc1 results in INT1S. In the second elementary step, rotation around the ∠NcCCO2 dihedral angle in TS2S facilitates proton transfer from O3H to the N1 amino group in the following step. Proton transfer occurs via rate-limiting TS3S, which is stabilized by the formation of a fused ring containing two cyclic components, an oxazolone and a pyrrolidine. From INT3S, backside attack of the hydroxyl group at C7 displaces the NH3 leaving group and forms INT4S, a loosely bound complex of m/z 156 and ammonia. Alternative isomers of this complex were not explored. Simple dissociation of INT4S leads to the product of interest, H+(PyOx)[Oc4] and ammonia. Several additional pathways were found that resulted in product ions with lower relative energies than H+(Py-Ox)[Oc4], although each of these pathways has a rate-limiting TS that is


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much higher in energy than the pathway presented here. Interestingly, the energetic cost associated with keeping the succinimide cyclic structure intact throughout the secondary ammonia loss is quite high. Specifically, ammonia loss to form H+(MCA-Suc) occurs by tight TSs along several alternative reaction coordinates, where these TSs have relative energies 85 – 87 kJ/mol higher than TS3S, Table 6. These alternative mechanisms are given in Supporting Information (Figures S7 and S8), along with additional isomers of m/z 156. Also given in Supporting Information is the only pathway found describing the formation of m/z 156 via a loose, PSL TS. From H+(AMCA-Suc), elongation of the Cβ-N1 bond results in its rupture, formation of the NH3 leaving group, and increases the Cγ-Cδ bond length, thus opening the succinimide ring and forming a vinyl ketone side-chain on the nitrogen. The incipient complex is stabilized via oxazolone formation resulting in the formation of m/z 156, an oxo vinyl ketoneoxazolone, H+(OVK-Ox)[Oc3] + 2 NH3, Figure S9. These products are 293 – 326 kJ/mol higher in energy than H+(AsnGly) and 33 – 53 kJ/mol above H+(Py-Ox)[Oc4] + 2 NH3. Sequential deamidation might also occur from the alternative deamidation channel forming H+(AAF) + NH3. Two PESs calculated at the MP2(full)/6-311+G(2d,2p)//B3LYP/6311+G(d,p) are given in Supporting Information, Figures S10 and S11. The lower energy process, limited by a tight TS (362 – 374 kJ/mol relative to the GS), leads to a fused morpholinone-oxazolone bicyclic compound, H+(Mo-Ox), 315 – 345 kJ/mol higher in energy than H+(AsnGly). This process lies 53 – 75 kJ/mol higher in energy than the H+(Py-Ox)[Oc4] + NH3 product asymptote from H+(AMCA-Suc). The second pathway, Figure S11, involves much less molecular rearrangement but is measurably higher in energy, with a rate-limiting TS 375 – 424 kJ/mol relative to the GS. As for secondary ammonia loss from the succinimide products, simple bond cleavage of a C-NH3 bond is much higher in energy than processes that require much more rearrangement and lead to bicyclic compounds via lower energy TSs. Cross Section Modeling Equation 1 was used to analyze the thresholds for the primary competitive decomposition channels of H+(AsnGly), which correspond to deamidation, dehydration, and the loss of (Gly +



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CO) to form m/z 87. Simultaneous modeling of these three primary channels accounts directly for competition among these channels. Using parameters given in Table 7, the data were reproduced over the full energy and magnitude ranges (with Figure 11 showing one example). For initial modeling of the deamidation channel, the cross section for the subsequent dissociation channel was summed with the primary channel, i.e., m/z 173 + m/z 156. For the reactions limited by a tight TS (deamidation via TS5N or TS3F, dehydration via TS4O, and secondary ammonia loss via TS3S), the TS frequencies used for the cross section modeling were taken from the theoretical results discussed above. For m/z 87, different levels of theory differ in whether the rate-limiting TS is loose (PSL) or tight, so both approaches are attempted, as discussed below. When the ratelimiting TS corresponds to the product asymptote of the respective products, the transitional frequencies are treated as rotors, a treatment that corresponds to a phase space limit (PSL), as described previously.25,26 We first modeled the data assuming that deamidation occurs via the pathway shown in Figure 6 and limited by TS5N, Figure 11. Our analysis shows that in order to fit the data accurately over the full energy range, the low-frequency modes (

Thermodynamics and Mechanisms of Protonated ... - ACS Publications

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