Guided Ion Beam and Computational Studies of the Decomposition of

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Guided Ion Beam and Computational Studies of the Decomposition of a Model Thiourea Protein Cross-Linker Ran Wang, Bo Yang, Ranran Wu, Mary T. Rodgers, Mathias Schäfer, and Peter B. Armentrout J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp512997z • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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The Journal of Physical Chemistry

1

Guided Ion Beam and Computational Studies of the Decomposition of a Model Thiourea Protein Cross-Linker Ran Wang,a Bo Yang,b R. R. Wu,b M. T. Rodgers,b M. Schäfer,c and P. B. Armentrouta,* a

Department of Chemistry, University of Utah, Salt Lake City, UT, USA

b

Department of Chemistry, Wayne State University, Detroit, MI, USA

c

Department of Chemistry, Cologne University, Cologne, Germany

Abstract The dissociation of protonated d3-methyl thiourea-4-butyric acid methyl amide (1), a model of thiourea-based protein cross-linking compounds, is examined both experimentally and computationally.

Using a guided ion beam tandem mass spectrometer (GIBMS), the threshold

collision-induced dissociation (TCID) of [1+H]+ with Xe is examined as a function of collision energy.

Analysis of the kinetic energy-dependent CID cross sections provides the 0 K barriers

for four primary and four secondary dissociation pathways, after accounting for competition between channels, sequential dissociations, unimolecular decay rates, internal energy of reactant ions, and multiple ion-neutral collisions.

Computations are used to explore the pathways for the

various processes and elucidation of their rate-limiting transition states. These results indicate that dissociation is initiated by migration of the excess proton from sulfur to one of three nitrogen atoms in 1, similar to the “mobile proton” model of peptide fragmentation.

The

computational energies for the rate-limiting transition states are generally in good agreement with

the

experimentally

derived

threshold

energies,

MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) results being particularly favorable.

with This

good comparison validates the mechanisms explored theoretically and allows identification of the structures of the various product ions and neutrals.

ACS Paragon Plus Environment


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2 Introduction The significance of understanding the tertiary and quaternary structures of proteins cannot be overstated as they directly affect the physiological function of protein complexes. However, the three-dimensional (3D) structures of many proteins remain unclear. Chemical cross-linking coupled with mass spectrometry (MS) has developed as a technique suitable for mapping 3D structures of proteins and elucidating protein-protein interactions.1-6 Chemical cross-linking (XL) is a procedure that covalently conjugates two or more biomolecules. Structurally defined links are formed via a chemical cross-linker of a defined length that targets specific functional groups of the biomolecules. One advantage for using cross-linking to investigate protein 3D structure and protein-protein interactions is that the perturbation induced by chemical cross-linking modification appears to be small, because there is evidence that cross-linked enzyme crystals retain functional stability.7-9 However, this strategy relies on the selective identification of chemically modified amino acids within peptides generated by proteolytic digestion of a respective protein or protein complex. The resultant enzymatically created peptide mixtures are very complex because only a relatively small percentage of cross-linked products is present. Tandem mass spectrometry (MS) supported by tailor-made algorithms has proved to be a feasible method to specifically identify the chemically marked peptides.10-12 For effective analysis of cross-linked peptides by soft ionization tandem MS, novel collision-induced dissociation (CID) labile XL-reagents that decompose more easily than other covalent bonds present in peptides were developed.

These reagents exhibit characteristic and predictable fragmentation behaviour

upon collisional activation, thereby allowing selective detection and sequence analysis by MS3.13-22 For that special purpose, cross-linking reagents with a central urea and thio-urea moiety that fragment preferentially at low-energy during CID have been designed and synthesized by Schäfer and co-workers.16-18

These reagents produce characteristic fragment ions and highly

indicative constant neutral losses (CNLs) associated with a labile covalent bond located within the linker region. Such CNLs can facilitate the identification of cross-linking through the use of selected and multiple reaction monitoring techniques.


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3 The development of new as well as the optimization of existing CID-labile XL-reagents can be facilitated by a fundamental understanding of the energetics and entropic demands of the individual fragmentation pathways, including charge-driven simple bond cleavages and complex rearrangements. To provide a molecule exhibiting an appropriate CNL, the preferred fragmentation should be both energetically and entropically favored. In order to characterize the fundamental CID pathways of these large thiourea-based cross-linking compounds, model compounds comprising smaller modified symmetric and asymmetric thiourea-compounds have been studied by Falvo et al. using ion trap mass spectrometry.23 In the present work, the CID of the protonated form of one of these compounds, d3-methyl thiourea-4-butyric acid methyl amide (1), Scheme I, is investigated more quantitatively using a guided ion beam mass spectrometer (GIBMS). Compared to conventional instruments, a GIBMS has the advantage of providing absolute kinetic-energy resolved CID cross sections that can be interpreted to obtain accurate thermodynamic information.24 The present work also provides a more complete quantum chemical exploration of the reaction mechanisms and compares these theoretical results directly with the experimental thermochemistry. It is hoped that a more precise determination of the thermochemistry by threshold CID studies of the cleavage of a particular covalent bond in a cross-linker molecule coupled with identification of the cleavage mechanisms by comparison with reliable computational analysis will be of value in future explorations of such cross-linkers.

Experimental and Computational Methods Instrumentation The collision-induced dissociation of the protonated form of compound 1, [1+H]+, is measured using the Wayne State University GIBMS that has been described in detail previously.25 The protonated thiourea-compound ions are formed using an electrospray ionization (ESI) source26 operated using 50:50 by volume H2O/MeOH solutions with ~10-4 M of 1, protonated by 1% acetic acid. The solutions are syringe-pumped through a needle biased at 1300 – 2100 V relative to ground. Ions are transmitted into vacuum through a capillary heated to



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4 107 °C, then collected by a radio frequency (rf) ion funnel.27 Ions are directed into an rf hexapole ion guide where they are trapped radially and thermalized by a high number of collisions (~104). The internal energies of ions are believed to be well described by a 300 K Maxwell-Boltzmann distribution, as characterized previously.26,28-32 The ions are then focused, accelerated, and mass analyzed in a magnetic momentum analyzer. After mass analysis, ions are decelerated to a well-defined kinetic energy distribution and focused into an rf octopole guide33 that passes through a static gas collision cell. Here collision-induced dissociation is carried out with xenon at sufficiently low pressure (below 0.20 mTorr) to minimize multiple collisions. The octopole provides effective radial trapping of the ions with small axial perturbations, making it favorable for thermodynamic studies.26,28-32 After the collision cell, all ions drift to the end of the octopole, where they are directed into a quadrupole mass filter for mass separation, and then detected using a high voltage (28 kV) dynode coupled with a scintillator and photomultiplier (Daly detector)34 along with standard pulse counting methods. Reactant and product ion intensities are measured and converted to absolute cross sections as described previously.35 Uncertainties in absolute cross sections are about ± 20%, and that for relative cross sections are approximately ± 5%. The absolute zero and distribution of the kinetic energy of the reactant ion are measured using the octopole as a retarding energy analyzer.35 The ion distribution of kinetic energies is approximately Gaussian with 0.4 - 0.5 eV (lab) full width at half maximum (FWHM). Laboratory (lab) energies of ions are converted to collision energies in the center-of-mass (CM) frame energies using the equation, E(CM) = E(lab) × m/(m + M), where m is the mass of Xe and M is the ion mass. eV (Lab) and 0.02 eV (CM).

The absolute energy scale has an uncertainty of 0.05

All energies cited below are in the center-of-mass frame.

Computational Methods To find the global energy minimum and all low-energy geometries of [1+H]+, a large number of possible conformations were screened using a simulated annealing methodology with the AMBER program and the AMBER force field based on molecular mechanics.36 All possible


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5 structures identified in this way were subsequently optimized using NWChem37 at the HF/3-21G level.38-39 Unique structures for each system were optimized using Gaussian 0940 at the B3LYP/6-31G(d,p) level of theory41-42 with the “loose” keyword (maximum step size 0.01 au and an RMS force of 0.0017 au) to facilitate convergence. A series of relaxed potential energy surface (PES) scans at the B3LYP/6-31+G(d) or B3LYP/6-311+G(d,p) level were performed to locate transition state and intermediate structures occurring along the potential energy surfaces for fragmentation. Unique structures of all stable conformations, intermediates, transition states, and products obtained from these procedures were then chosen for higher-level geometry optimization and frequency calculations using the B3LYP/6-311+G(d,p) level of theory.43 Each transition state was found to contain one imaginary frequency and each reactant, intermediate, and product was vibrationally stable. Vibrational frequencies were scaled by 0.9944 in determining zero-point vibrational energy (ZPE) corrections. Single-point energies were determined at the B3LYP, B3P86, and MP2(full) levels using the 6-311+G(2d,2p) basis set and the B3LYP/6-311+G(d,p) geometries.

Thermochemical Analysis Experimental cross sections are modeled and analyzed using procedures described elsewhere.45-48 Briefly, the threshold regions of the CID reaction cross sections are modeled using eq 1,  nσ 0, j  E

σ j ( E ) = 

E   ∑ g i ∫ [ k j ( E *) / k tot ( E *) ][1 − e − ktot ( E *)τ ]( E − ε ) n −1 d (ε )  i E0 , j − Ei

(1)

where n is an adjustable parameter that describes the energy deposition during collision,49 σ0,j is an energy-independent scaling factor for product channel j, E is the collision energy, E0,j is the reaction threshold for channel j, ε is the energy deposited in the ion during the collision, and τ is the average experimental time available for dissociation (the ion time-of-flight, ~100 µs).

The

summation is over the rovibrational states of the reactant ion having energies Ei with the populations gi (Σgi = 1).

This equation accounts for the lifetime for dissociation of the



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6 energized molecule (EM), which can lead to a delayed onset for the reaction threshold, a kinetic shift, which becomes more noticeable as the size of the EM increases.

In addition, the ratio of

rate coefficients in eq 1 naturally includes competition among parallel reactions with a full statistical treatment.50 Previous studies have verified the efficacy of this approach in modeling reactions that compete through loose as well as loose versus tight transition states.49,51-55 The rate coefficients are calculated using Rice-Ramsperger-Kassel-Marcus (RRKM) theory,56-58

k tot ( E *) =

∑k j

j

( E *) =

∑d

j

N †j ( E * − E 0 , j ) / h ρ ( E *)

j

(2)

where kj(E*) is the rate coefficient for a single dissociation channel j, dj is the reaction degeneracy calculated from the ratio of rotational symmetry numbers of the reactants and † products of channel j, N j ( E * −E0, j ) is the sum of the ro-vibrational states of the transition state

(TS) at an energy ( E * −E0, j ) above the threshold for channel j, and ρ (E*) is the density of ro-vibrational states for the EM at the energy available, E* = ε + Ei.

Vibrational frequencies

and rotational constants are taken from the quantum chemical calculations detailed below. The Beyer-Swinehart-Stein-Rabinovitch algorithm59-61 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.

For reactions limited by loose TSs, most frequencies are those of the

products with the transitional frequencies treated as rotors, an approach that corresponds to a phase space limit (PSL), as described in detail elsewhere.47,50

The two-dimensional (2D)

external rotations are treated adiabatically but with centrifugal effects included,61 and calculated using a statistical distribution with an explicit summation over all the possible values of the rotational quantum number.

For reactions limited by tight TSs, molecular parameters are taken

directly from theoretical results for the rate-limiting TS structures. The decomposition of [1+H]+ involves several sequential dissociation pathways, which require additional assumptions to model accurately, as described previously.62

Here we use a

statistical model to estimate the energy remaining in the primary product ion undergoing further


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7 dissociation, which has a probability, PD2 = 1 – exp[-k2tot(E2*)τ2], where k2tot, E2*, and τ2 are the total rate coefficient for the secondary dissociation (calculated again using RRKM theory), the energy available to the secondary EM, and the time available for the secondary dissociation, respectively.

Thus, the total cross section for channel j is partitioned into that for the

non-dissociating product, σ1(E) = σj(E)(1 - PD2), and that for the sequential dissociation product ion, 2, σ2(E) = σj(E)PD2.

The combination of sequential and competitive modeling allows

accurate reproduction of all experimental reaction cross sections observed here, as detailed further below. Two effects that would otherwise obscure the interpretation of the data must also be accounted for during data analysis.

The first effect involves energy broadening resulting from

the thermal motion of the neutral collision gas and the kinetic energy distribution of the reactant ion, which are accounted for by explicitly convoluting the model of eq 1 over both kinetic energy distributions.35

The second effect considers that our models only represent products formed as

the result of a single collision event, which we account for by evaluating the cross sections for pressure effects and extrapolating to zero pressure of Xe (rigorously single collision conditions).63 The model cross sections of eq 1 are compared to the data after convolution with the kinetic energy distributions of the reactants. optimized values for σ0,j, n, and E0,j.

A nonlinear least-squares analysis is used to provide The uncertainty associated with E0,j is estimated from the

range of threshold values determined from different data sets, different values of n, 10% variations in the vibrational frequencies, changes in τ by factors of 2, and the uncertainty of the absolute energy scale, 0.05 eV (lab).

For loose TSs, we assume that the measured threshold E0

values for dissociation are from ground state reactant to ground state ion and neutral products. Given the relatively long experimental time frame (~1×10−4 s), dissociating complexes should be able to rearrange to their ground state product conformations upon dissociation.



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8 Results Cross Sections for Collision-Induced Dissociation Kinetic-energy dependent experimental cross sections were obtained for the interaction of [1+H]+ with Xe. Figure 1 shows a representative data set taken at a xenon pressure of 0.2 mTorr. Eight ionic products are observed and can be identified with reactions (3) – (10), where fragment ions are referred to using the nomenclature outlined in Scheme 1: [1+H]+ + Xe → C6H8D3N2OS+ (m/z 162, γ) + CH3NH2 + Xe

(3)

→ C6H11N2OS+ (m/z 159, φ) + CD3NH2 + Xe

(4)

→ C5H13N2O+ (m/z 117, χ) + CD3NCS + Xe

(5)

→ C5H10NO+ (m/z 100, ψ) + CD3NHCSNH2 + Xe

(6a)

→ C5H10NO+ (m/z 100, ψ) + HNCS + CD3NH2 + Xe

(6b)

→ C5H10NO+ (m/z 100, ψ) + NH3 + CD3NCS + Xe

(6c)

→ C4H8NO+(m/z 86, λ) + CD3NCS + CH3NH2 + Xe

(7)

→ CD3HNCS+ (m/z 77, β) + C4H7NO + CH3NH2+ Xe

(8)

→ CH3HNCO+ (m/z 58, ω) + SCNC3H7 + CD3NH2 + Xe

(9a)

→ CH3HNCO+ (m/z 58, ω) + C3H7NH2 + CD3NCS + Xe

(9b)

→ CH3HNCO+ (m/z 58, ω) + C3H6 + CD3NHCSNH2 + Xe

(9c)

→ CD3NH3+ (m/z 35, α) + C6H10N2OS + Xe

(10)

The two lowest energy processes arise from similar thresholds and correspond to losses of methyl amine from either end of the molecule, indicating why 1 was specifically deuterated at one end.

Despite the comparable energetics of these two nearly equivalent decompositions, the

dominant process is the loss of CD3NH2 to form C6H11N2OS+ (m/z 159, φ), reaction 4, with loss of CH3NH2 forming C6H8D3N2OS+ (m/z 162, γ), reaction 3, having a similar magnitude at low energies but over an order of magnitude smaller at higher energies.

The threshold for the loss

of CD3NCS, forming the χ ion (m/z 117) in reaction 5, is slightly higher in energy. The next process observed forms the α ion (m/z 35) in reaction 10, a process that must compete directly with reaction 4 forming the φ ion (m/z 159), as these channels differ only in which fragment


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9 carries the excess proton (Scheme 1). (In that regard, it is then significant that CH3NH3+ was not observed, the analogous pathway that would complement reaction 3.) Formation of the ψ ion (m/z 100) has a similar apparent threshold. As discussed further below, three pathways are capable of yielding ψ: the primary reaction 6a and the sequential decompositions of reactions 6b (fragmentation of φ) and 6c (fragmentation of χ).

At elevated energies, it is clear that ψ is

primarily produced by reaction 6b because no other process can account for the decline in the cross section of φ (m/z 159).

The λ (m/z 86) product ion is an internal ion that can only be

formed by sequential dissociation processes from either the γ (m/z 162) or χ (m/z 117) product ions.

Consistent with this hypothesis, both cross sections decline at high energies while the λ

cross section rises. Finally, two minor products, β (m/z 77) and ω (m/z 58), are observed at high energy, with reactions 8 and 9 proposed as their decomposition pathways.

Reaction 8 must be a

sequential dissociation of the γ primary product ion, whereas reactions 9a – 9c correspond to the sequential decompositions of the φ, χ, and ψ primary ions. As mentioned above, the decomposition reactions of [1+H]+ have been investigated previously by Falvo et al. using an ESI-tandem quadrupole ion trap (QIT) mass spectrometer.23 Using He as the collision gas under multiple collision conditions, Falvo et al. observed φ, χ, ψ, γ, and λ fragments with relative abundances similar to the present results at about 2 eV. 35 (α) ion observed here at this energy is below the mass range of the QIT after CID.

The m/z In the

energy resolved decomposition work of Falvo et al., no quantitative breakdown results are reported but the φ and γ ions exhibit nearly identical voltage onsets (as also observed here), with those for χ and ψ being slightly higher and similar to one another, in contrast to the differentiation obvious in Figure 1. These differences are probably the result of the more ill-defined internal energy distribution of the precursor ions and the effects of multiple collisions in the QIT work.64

Ground Structure of [1+H]+ To locate the ground structure (GS) of [1+H]+, simulated annealing methodology described



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10 above was employed with all electronegative atoms, O, S, N1, N2, and N3 on 1 being checked as potential proton acceptor sites. Single point energies including ZPE corrections were compared at three levels of theory, MP2(full), B3P86, and B3LYP using the 6-311+G(2d,2p) basis set. Protonation on S (where the protonation site is indicated in square brackets) is found to be energetically favored with low-lying structures stabilized by an intramolecular N2H•O hydrogen bond.

Twenty such conformers were located and range in energy from 0 – 39 kJ/mol, Table S1.

These stable conformers differ in the orientations of the two end groups and which way the proton on sulfur points, as illustrated by several examples shown in Figure 2.

The orientations

of the end groups can be designated by specifying the approximate values for the seven dihedral angles along the backbone starting at the urea group, CN1CN2CCCCN3C, where c = cis indicates angles less than 50°, g = gauche for angles between 50° and 130°, and t = trans for angles greater than 130°.

The urea group (CN1CN2CC) can be tct, ctt, ttt, or cct, and the

methyl amine group (CCCN3C) can be tt or tc, where the latter orientation will be indicated by use of the prime symbol (′).

The favorable N2H•O hydrogen bond locks the central portion of

the molecule (N2CCCC) into a gg conformation.

The orientation of the sulfur proton can be

similarly indicated by the N1CSH dihedral angle as either St or Sc.

In four cases, [St+/–]cttggtx

and [St+/–]cctggtx conformers where x = t and c, separate minima corresponding to positive and negative values of this N1CSH dihedral angle were located, although these differ in energy by only 2 – 3 kJ/mol, Table S1.

For the [St]cttggtx conformers, the energy of the transition state

between the two minima lay below the energy of the [St+]cttggtx conformers, indicating that only [St–]cttggtc exists as a true minimum.

For the [St]cctggtx conformers, the TS remains but lies

only 0.5 – 2 kJ/mol above the higher energy [St+] conformers. The relative energies in Table S1 show that the thiourea and sulfur proton orientations are coupled such that the two lowest energy structures are [St]tctggtt and [Sc]cttggtt.

The former is

the ground structure by 0.8 – 1.0 kJ/mol for the density functional approaches, whereas MP2(full) finds the latter structure to be lower in energy by 1.3 kJ/mol.

In the tct orientation of the urea

group, the Sc conformer lies about 10 kJ/mol higher in energy than St, whereas this difference is


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11 about 6 kJ/mol for cct, 0 kJ/mol for ttt, and -2 kJ/mol for ctt.

Changing from tct to ctt, ttt, and

cct costs 2 – 3, ~6, and ~19 kJ/mol, respectively, when the sulfur proton is St, but negative 9 – 11, negative 4, and 15 kJ/mol, respectively when Sc.

Many of these differences can be attributed to

a SH+•O hydrogen bond that can form in the tct and cct conformers with St, whereas the ctt and ttt conformers cannot have such an interaction such that the orientation of the sulfur proton no longer matters.

At the other end of the molecule, changing the methyl amine group orientation

from tt to tc increases the energy by a uniform 10 – 12 kJ/mol. Although not explored exhaustively, several transformations among these various conformers were located and all require less energy than the decomposition reactions below. Here, transition states are indicated by TS and enclosing the orientation being changed in parentheses, by a dash when the protonation site changes, or by {C~N} to designate cleavage of a C−N bond.

For example, rotation of the sulfur proton in the [St] tctggtt (GS[S]) conformer

from trans to cis costs 10 – 11 kJ/mol.

This passes over TS[S(tc)] tctggtt (TS1φ) lying 20 – 22

kJ/mol above the lower energy [St] conformer.

Rotation about the N1CN2C dihedral angle

through TS[Sc] t(tc)tggtt (TS2φ[S]), which lies 82 – 88 kJ/mol above GS[S], forms [Sc]tttggtt (INT2φt[S]).

Rotation of the CN1CN2 dihedral angle through TS[Sc] (tc)ttggtt (TS2φex[S]),

which lies 90 – 92 kJ/mol above GS[S], forms the low-energy [Sc]cttggtt (INT2φc[S]) structure. Rotation of the CCN3C dihedral angle requires more energy, for example, TS[St] tctggt(tc) lies 114 – 116 kJ/mol above GS[S].

It seems likely that similar energies are required for

comparable dihedral rotations of other conformations and that few if any will require more than 120 kJ/mol.

In addition, proton transfer transition states connecting the conformers protonated

on different basic atoms (S, O, N1, N2, and N3) were found, and most are discussed below. The theoretical results show that relatively large energies are needed to move the proton from S to these energetically less favored sites.

Mechanistic Overview Overall, the mechanisms for the CID reactions observed here are outlined in Scheme 2.


In


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12 all cases, isomers protonated on the three nitrogen atoms constitute the reactive intermediates formed initially by collisional activation and ready to dissociate in a charge-driven fashion leading to the formation of the observed fragment ions. In this regard, the theoretical work here agrees qualitatively with the mobile proton model used to describe peptide dissociations,65-67 even though 1 is not a peptide.

If the proton is located on N1, INT3φ[N1], C−N1 bond

cleavage coupled with proton transfer from N2 to the oxygen atom leads to the thiocyanate φ (m/z 159) product ion along with concomitant loss of CD3NH2.

At higher energies, the same

C−N1 bond cleavage but coupled instead with proton transfer from N2 to N1 yields CD3NH3+ (α) and [φ-H+] neutral.

From φ, backside attack of the oxygen at the alpha carbon leads to loss of

HNCS and the cyclic ψ (m/z 100) product ion.

When the proton is located on N2, INT2χ[N2],

backside attack of the oxygen at the alpha carbon again leads to ψ but with the CD3NHCSNH2 neutral, the lowest energy pathway for this product ion.

Alternatively, proton transfer from N1

to N2 in INT2χ[N2] leads to loss of CD3NCS and the χ (m/z 117) product ion.

From χ,

nucleophilic attack of the oxygen at the alpha carbon again forms ψ along with ammonia, whereas backside attack of N2 at the carbonyl forms the internal λ (m/z 86) product ion with loss of CH3NH2.

Locating the proton at N3, INT2γ[N3], allows backside attack of either S or N2 at

the carbonyl to form two different forms of the γ (m/z 162) product ion, a seven- (γ7) or five-membered (γ5) ring, along with CH3NH2.

The latter can decompose by simple C−N2 bond

cleavage to form the β product ion along with C4H7NO (deprotonated λ), or proton transfer between these products from N1 to the oxygen forms λ + CD3NCS.

These various pathways

are explored more thoroughly via direct computation in the following sections, which build on preliminary theoretical explorations described by Falvo et al.23 for formation of the φ and γ product ions.

Formation of φ (m/z 159) and α (m/z 35) Overall, the mechanism for φ and α formation requires the transfer of the mobile proton from S to N1 adjacent to the deuterated methyl group.

This step can be initiated from any of


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13 the eight [Sc] conformers of [1+H]+ listed in Table S1, with the lowest energy pathway found shown in Figure 3.

The relative single point energies of all species along this surface are

provided in Table 1 and Table S2 lists such energies for the alternative pathways. The lowest energy pathways start from GS[S] and rotate the proton on the sulfur away from the oxygen (not shown in Figure 3), which requires only 20 – 23 kJ/mol. Then, the ion rotates about the C−N2 bond over TS2φ, thereby forming INT2φt[S].

A nearly equivalent intermediate

can be obtained by rotating about the N1−C bond over TS2φ(tc)[S] yielding INT2φc[S], the overall ground structure at the MP2(full) level, which differs from INT2φt[S] only in the orientation of the CD3 group (cis versus trans, respectively, as indicated by the subscripts).

The

next elementary step transfers the proton from S to N1 to access both TS3φ+ (137 – 144 kJ/mol) and TS3φ– (138 – 145 kJ/mol) from both INT2φt[S] and INT2φc[S].

These TSs differ only in

which side of the ion the CD3 moiety resides (leading to opposite signs for the gauche CN1CN2 dihedral angle and indicated here by the subscripts); thus they are nearly isoenergetic, Table 1, differing in energy by only ~ 1 kJ/mol because of the weak interaction with the CCC backbone. For the B3LYP calculations, this step is rate-limiting for φ product formation, as the C−N1 bond is significantly weakened by protonation on the thiocarbamoyl nitrogen.

Depending on the

direction in which the CD3 group rotates as the proton is transferred, these steps form the nearly isoenergetic INT3φ+[N1] or INT3φ–[N1], which again differ only in which side of the molecular plane the CD3 group resides.

These two intermediates can rearrange to one another via

TS3φ(+–)[N1], which lies only 6 – 8 kJ/mol higher in energy.

From either INT3φ+[N1] or

INT3φ–[N1], the C−N1 bond breaks and simultaneously the proton on N2 transfers to the amide oxygen by passing over TS4φ (although this TS lies below INT4φ[O] once ZPE corrections are made). This forms INT4φ[O], a dimer of the final products, CD3NH2 and a protonated thiocyanate chain structure (φ), where the latter product is stabilized by a OH+•N2 hydrogen bond.

(Here, the φ product has a backbone configuration of [Oc]tggtt with the proton on the

oxygen and the CD3NH2 product is bound to φ via a N•+HO hydrogen bond. structures of the INT4φ complex were not explored.)

Alternative

In the final step, this dimer dissociates to



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Page 14 of 52

14 form the products, at an energetic cost of 11 – 21 kJ/mol.

The loose TS associated with product

separation is the rate-limiting step according to B3P86 and MP2(full) theory, Table 1. As noted above, the thiourea quasimolecular ion can orient the methyl groups at both ends in two directions.

For the case where the N3CH3 terminus has a tc orientation (instead of the

lower energy tt structure), we find that the φ′[Oc]tggtc + CD3NH2 product asymptote lies 10 – 12 kJ/mol higher in energy than the φ[Oc]tggtt + CD3NH2 products, Table 1.

The transition states

for this process, TS3φ′+[S-N1]g+ttggtc and TS3φ′–[S-N1]g–ttggtc, also lie 10 – 12 kJ/mol above their [S-N1]g+/–ttggtt counterparts, Table S2.

Again the B3LYP theories find that TS3φ′ is rate

limiting by 16 – 20 kJ/mol, whereas B3P86 and MP2(full) find the product asymptotes are rate limiting by 2 – 10 kJ/mol.

Other variants of the products include gccgt and gccgc conformers

that lie 12 – 15 and 23 – 26 kJ/mol above tggtc, respectively.

A seven-membered ring structure

for m/z 159 was also located but lies 48 – 69 kJ/mol above φ[Oc]tggtt. Although variants of all species along the pathway to form φ were not explored (specifically the TS4φ and INT4φ equivalents), energetics for the INT2φ, TS3φ, and INT3φ equivalents can be found in the Supporting Information Table S2.

As noted above, any of the other four [Sc]

species in Table S1, tctggtx and cctggtx (where x = t or c), are also positioned to transfer the proton from S to N1.

This pathway was followed in the case of INT1φt[Sc]tctggtt and

INT1φc[Sc]cctggtt and found to have transition states, TS3φ+c and TS3φ–c, lying 23 – 28 kJ/mol above TS3φ+, Tables 1 and S2.

The former path includes the INT3φ–c[N1], TS4φ–c, and

INT4φ–c[O] species, which are also included in Table S2, and lead to the same products, φ + CD3NH2.

Equivalent pathways having the tc orientation for the N3CH3 terminus were not

explored but can be expected to lie another 10 – 12 kJ/mol higher in energy and lead to φ′ + CD3NH2 products.

Although these six alternative pathways are higher in energy than those in

Table 1, the competition among the various product channels is probably affected by the fact that there are eight parallel TS3φ transition states (within 40 kJ/mol) leading to two different final products within 10 kJ/mol, φ and φ′ + CD3NH2. The four INT3φ+/−[N1] and INT3φ′+/−[N1] intermediates can also dissociate by letting the


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15 excess proton be carried off by the CD3NH2 fragment forming α.

(This channel is probably

inhibited for the INT3φ+/–c[N1] and INT3φ′+/–c[N1] intermediates because the cis orientation of the N1CN2C dihedral angle puts the CD3NH2 moiety on the opposite side of the NCS group relative to the proton.

Thus, the CD3NH2 group would have to migrate to the other side to

obtain the proton before dissociating to α + [φ-H+] products.) This process occurs over TS4α, in which the hydrogen on N2 transfers to N1 in the incipient CD3NH3+ (α) product.

This step

requires 22 – 31 kJ/mol more energy than TS4φ, because the former requires that there be an N2H+•N1 hydrogen bond in order to allow transfer to the CD3NH2 product, whereas TS4φ maintains a stronger O•H+•N2 hydrogen bond.

A variant of this TS is TS4α’ in which the

N3CH3 terminus has a tc orientation (~9 kJ/mol higher). Also the CD3 group can lie on the other side of the plane of the complex (such that the CN1CN2 dihedral is g+ or g–), but these variants lie within 0.2 kJ/mol of one another.

From any of these TSs, a quite stable complex

INT4α[N1], only 16 – 43 kJ/mol higher than the GS[S] reactant, is formed.

This structure is a

dimer of CD3NH3+ and the [φ-H+]cgggt neutral and is stabilized by N1H+•O and N1H•S hydrogen bonds.

(Alternative structures of this hydrogen bound species were not explored.)

The last step of α formation is the dissociation of the INT4α[N1] complex to yield the final CD3NH3+ + [φ-H+] products, which lie 152 – 179 kJ/mol above GS[S] reactants, 10 – 42 kJ/mol above TS3φ+, and 20 – 32 kJ/mol above the φ + CD3NH2 product asymptote.

The latter values

are in qualitative agreement with the experimental results, which indicates that these product channels differ by roughly 40 kJ/mol on the basis of the apparent thresholds, Figure 1.

As for

formation of the φ product channel, we also examined parallel pathways corresponding to different orientations of the termini in [φ-H+].

Eight variations having the N3CH3 terminus in a

tt orientation were located and lie within 18 kJ/mol of the most stable of these, [φ-H+]gg+gtt, Table S2.

Another seven variants have the tc orientation leading to [φ′-H+] with the tggtc

conformer lying 9 – 14 kJ/mol higher in energy than the [φ-H+]gg+gtt and the other [φ′-H+] variants within 20 kJ/mol. The φ and φ′ product ions can undergo further dissociation by losing isothiocyanate, which



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Page 16 of 52

16 yields the five-membered cyclized m/z 100 product (ψ), which has a ctt ground structure for the CCCCN3C dihedral angles.

The rate-limiting TS1ψ for this process involves an SN2 reaction

(Figure 3) in which the oxygen displaces the isothiocyanate, forming a loosely bound complex of the two products, INT1ψ[N3], that requires only 17 – 34 kJ/mol to dissociate.

Calculations

indicate that TS1ψ lies 131 – 156 kJ/mol above φ, 256 – 300 kJ/mol compared to GS reactants, and 112 – 156 kJ/mol higher than the rate-limiting TS for φ formation.

The latter energy is

larger than the difference in apparent thresholds between the m/z 159 (φ) and m/z 100 (ψ) product ions, indicating that this sequential decomposition cannot explain the formation of ψ at its threshold, Figure 1.

However, this process must occur at higher energies because the cross

section for m/z 100 is the only high energy product with sufficient intensity to explain why the cross section for φ declines significantly above about 3 eV. the amino methyl group has a cis orientation.

A parallel pathway also exists when

Here TS1ψ′ lies 8 – 9 kJ/mol above TS1ψ and

the final product ψ′[N3]ctc lies only 4 – 5 kJ/mol above ψ[N3]ctt. The mechanism for decomposition of m/z 159 (φ) to yield the m/z 58 (ω) product ion in reaction 9a was not explored thoroughly because no experimental thermochemistry can be obtained reliably for such a high energy product. However, one possibility is nucleophilic attack at the thioisocyanate carbon by the carbon alpha to the carbonyl moiety, enabling the release of the CH3HNCO+ (ω) product ion and forming a cyclic pyrrolidine-2-thione (c-C3H6NHC=S) neutral. kJ/mol higher.

An open form of this product, 1-thiocyanatopropane, lies 12 – 35

The lower energy asymptote lies 315 – 347 kJ/mol above GS[S], consistent with

its high energy apparent threshold, Figure 1.

Formation of γ (m/z 162) Scheme 2 shows that the m/z 162 (γ) product ion can either be a seven (γ7) or five (γ5) membered ring, depending on whether S or N2 undergoes nucleophilic attack at the carbonyl carbon. 2.

Theory finds that the seven-membered ring is lower in energy by 46 – 68 kJ/mol, Table

Other possible structures for the m/z 162 product include another seven-membered ring, γ7alt


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17 = c-(CD3)NC(=S)NHCH2CH2CH2C(=O), which can be protonated on S, O, N1, or N2.

The

most stable of these is S protonated (two orientations) and lies 12 – 20 kJ/mol higher than γ7, with the O protonated another 4 – 15 kJ/mol higher and the N protonated forms much higher (> 50 kJ/mol), Table S3. Because it is not the ground structure of the γ (m/z 162) product ion, pathways for forming the γ7alt species were not explored. Loss of CH3NH2 requires transfer of the mobile proton to N3, which in turn is facilitated by a [St] orientation and a cis N1CN2C dihedral angle (i.e., tct or cct).

As for the φ + CD3NH2

product channel, formation of the γ + CH3NH2 product channel can occur via four parallel pathways associated with different orientations (c or t) of the two terminal methyl groups. Overall, the lowest energy potential energy surface is provided in Figure 4 and Table 2, with Table S3 in the Supporting Information providing energies of all four pathways.

Along all four

pathways, the first elementary step involves transfer of the excess proton from S to N3 forming INT1γ[N3], which is unstable relative to TS1γ when ZPEs are included.

Then, the methyl

isothiocyanate group rotates away from the carbonyl over TS2γ, which positions the thio group towards the backside of the carbonyl.

For TS1γ, INT1γ, and TS2γ, the lowest energy pathway

has both terminal methyl groups in trans orientations, whereas the highest energy pathway has both in cis orientations and lies 14 – 19 kJ/mol higher in energy.

The trans-trans, trans-cis,

cis-trans, and cis-cis orientations are indicated by TS1γt, TS1γt′, TS1γc, and TS1γc′, respectively, with Figure 4 showing the cis-trans configuration. This path is shown because the relative energy ordering changes at INT2γ[N3], where having the CD3N1 group in a cis position is more favorable by about 6 kJ/mol.

From the four INT2γ[N3] intermediates, the sulfur undergoes

nucleophilic attack at the carbonyl carbon via TS3γ7, thereby forming the seven-membered ring form of the m/z 162 (γ7) product ion.

The pathway shown produces the γ7c ([N1]ctggg)

structure, where like [1+H+], the letters designate the dihedral angles starting at CN1CN2.

This

is the lowest energy conformer of this product ion, with the γ7t ([N1]ttggg) conformer lying ~8 kJ/mol higher in energy. two pathways.

Of the four parallel pathways, each product conformer is formed along

Additional pathways leading to a γ7g ([N1]tgggg) structure, which lies 45 – 47



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Page 18 of 52

18 kJ/mol higher than γ7c, were also located but here the TSs lie 49 – 57 kJ/mol above the lowest TS3γ7c′, Table S3.

Structures of the product ion in which the proton migrates to O or S were

also located but lie over 100 kJ/mol above γ7c, Table S3.

The formation of the final products is

the highest energy point along these pathways, lying 153 – 164 kJ/mol above GS reactants. We also calculated the energy necessary for proton transfer between these two products, yielding CH3NH3+ + deprotonated γ7.

Deprotonation of both γ7c and γ7t at both N1 and N2 were

considered, with the most stable variant being [γ7c-HN2+] and the other three conformers lying within 24 – 26 kJ/mol. This product asymptote lies 76 – 84 kJ/mol above γ7c + CH3NH2, Table 2, making it unlikely to compete very effectively, consistent with the failure to observe the m/z 32 ion. An alternative pathway for forming γ is also shown in Figure 4.

Starting from INT2γ[N3],

nucleophilic attack of N2 at the carbonyl carbon leads to an appreciably higher energy transition state, TS3γ5t.

This TS lies 46 – 69 kJ/mol above TS3γ7c, TS3γ5c lies another 15 – 22 kJ/mol

higher in energy, and TS3γ5t′ and TS3γ5c′ lie within 4 kJ/mol of TS3γ5t and TS3γ5c, respectively. These TS3γ5 lead to the five-membered ring form of the m/z 162 (γ5) product ion, which has two conformers, γ5t ([N2]tttcc) and γ5c ([N2]cttcc), where the latter lies ~11 kJ/mol higher in energy. Formation of the five-membered ring is limited by the energy of the final products, which lie 46 – 68 kJ/mol higher than γ7c.

The seven and five membered ring forms of γ can also

interconvert over TSγ(7-5), with the trans variant shown in Figure 4, which lies 17 – 23 kJ/mol above γ5t.

In this TS, the sulfur rotates away from the carbonyl, extending the C−S bond from

2.00 Å to 2.74 Å, while the C−N2 distance drops from 3.10 to 2.11 Å, transitioning to 1.64 Å in γ5t. The importance of the five-membered ring form of γ5 is that it can easily dissociate further by the loss of the terminal CD3NCS group to form the λ (m/z 86) product, c-C4H8NO protonated on the oxygen, Scheme 2.

This process is limited by TSγλ, which lies 14 – 23 kJ/mol above γ5t

(70 – 86 kJ/mol above γ7c) and involves simple cleavage of the C−N2 bond and formation of a N1H+•O hydrogen bond, Figure 4.

(The cis variant TSγλ′ lies 36 – 41 kJ/mol higher in energy


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19 because the cis orientation no longer allows the stabilizing N1H+•O interaction, Table S3.) Notably, TSγλ is nearly isoenergetic with TSγt(7-5) (within 4 kJ/mol at all levels of theory), such that both the seven and five membered ring forms of γ can reach the λ product with similar energetics.

In the course of TSγλ, the proton is transferred to oxygen, which yields INTλ, a

loosely bound complex of the incipient λ + CD3NCS products bound via OH+•N1 and N2H•S hydrogen bonds, lying 49 – 67 kJ/mol below the λ + CD3NCS + CH3NH2 products.

(A variant

of this intermediate, only 0.3 – 5 kJ/mol higher in energy, rotates the CD3NCS group perpendicular to the plane of λ, such that only the OH+•N1 hydrogen bond remains.) The cyclic λ product ion has two conformers in which the proton on oxygen is either trans or cis relative to the nitrogen, with the former lying lower in energy by ~9 kJ/mol.

If the proton remains instead

on the side-chain, then CD3HNCS+ (m/z 77, β) is formed along with the c-C4H7NO neutral. This channel lies 89 – 96 kJ/mol higher in energy than formation of λ (40 – 75 kJ/mol above TSγλ), consistent with the inhibited formation of the β product ion, Figure 1.

Formation of χ (m/z 117) Another primary decomposition is the formation of χ by loss of CD3NCS, which requires transfer of the proton to N2, Scheme 2.

As in the pathways for forming the φ and γ product ions,

it is possible to transfer the proton directly from S to the appropriate nitrogen.

The lowest

energy surface for this pathway is shown in Figure S1 with relative energies of all parallel pathways in Table S4.

This process can be initiated from any of the eight [St] conformers of

[1+H]+ (pairs of [St+/–] conformers behave the same) and each can lead to two of eight distinct transition states, TS2χ. The most stable of these, TS2χt–[S-N2]tg–gggtt, leads from INT1χtt[St]tttggtt to INT2χt–[N2]tg–gggtt and lies 145 – 153 kJ/mol above GS[S].

Of the eight

TS2χ, half differ in the CN1CN2 angles (t or c), half in the CCN3C angles (t or c), and half in which side of the overall molecular plane the thiourea group lies (g+ or g–).

The g+ and g–

conformers can both be reached from INT1χ having either t or c orientations of the N1CN2C dihedral angles.

The various TS2χ lie up to 37 kJ/mol higher in energy compared to the lowest



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Page 20 of 52

20 energy one, as much as 177 – 185 kJ/mol above GS[S], Table S4.

TS2χ lead to eight different

INT2χ[N2] intermediates, which again vary with the orientations of the two terminal methyl groups (t or c) and thiourea group.

The most stable of these depends on the level of theory,

either INT2χt+[N2]tg+tggtt or INT2χt–[N2]tg–gggtt, which lie 35 – 50 kJ/mol above GS[S], and the highest is another 39 – 44 kJ/mol higher in energy. Although the proton can be transferred directly from S to N2, this is not the lowest energy pathway because it is a four-centered transition state.

A lower energy pathway involves transfer

from S to N3 then back to N2, as illustrated in Figure 5.

Relative energies of the lowest energy

pathway are given in Table 3 with all alternate species included in Table S5 of the Supporting Information.

This pathway can start at any of the eight [N3] intermediates formed along the

pathway to the γ product ion, INT1γ[N3] or INT2γ[N3].

Eight different TS2γχ[N3-N2] were

identified with the most stable (TS2γχt–) at 105 – 131 kJ/mol above GS[S] and the remaining within 25 kJ/mol. Thus, the lowest energy pathway here is 20 – 40 kJ/mol below the most stable TS2χ[S-N2] and 22 – 59 kJ/mol below the γ7c + CH3NH2 product asymptote.

The prospect that

the γ and χ products share the common INT2γ intermediates has implications for the competition between these two channels. TS2γχ[N3-N2] lead to the same eight INT2χ intermediates formed by TS2χ[S-N2]. In INT2χ, the excess proton on N2 weakens the N2−C bond, which is then broken as the system passes over TS3χ, for which four conformers were located, Table S4. These can be traced back to the four conformers of INT2χt having a trans CN1CN2 dihedral angle.

In the TS3χt

conformers, the N1H hydrogen is pointing at the oxygen such that cleavage of the N2−C bond also leads to transfer of a proton from N1 to O. below the lowest TS2χ.

The lowest energy TS3χt lies 3 – 31 kJ/mol

The four TS3χt lead to four conformers of INT3χ[O], in which the

incipient products are bound by an OH+•N1 hydrogen bond.

The most stable of these

intermediates, INT3χ+[O], is 91 – 114 kJ/mol above GS[S] with the other three lying within 13 kJ/mol.

In the four INT2χc conformers (with cis CN1CN2 dihedrals), the N1H points away

from the rest of the molecule, such that cleavage of the N2−C bond is no longer stabilized by the


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21 hydrogen bond and the excess proton on N1 of the CD3NHCS+ moiety is not easily transferred back to the [χ-H+] neutral. From INT3χ[O], a much more stable complex of the products, INT4χ[N2], is formed by transfer of the proton on the oxygen to the terminal N2 nitrogen yielding a N2H+•O hydrogen bond along with a N2H•S hydrogen bond between the incipient products. The final χ product ion has a [N2]ggtt conformation that retains the N2H+•O hydrogen bond.

An alternative

conformation (χ′) reorients the terminal methyl group, [N2]ggtc, and lies ~10 kJ/mol higher in energy.

Variations in the pathways from the INT3χ[O] intermediates to products were not

explored as they cannot limit the reactivity observed. The χ and χ′ + CD3NCS product asymptotes have energies that lie well below any of the preceding transition states, such that this reaction must be limited by a tight TS. the level of theory.

Overall, the rate-limiting TS for this process depends on

At the B3LYP level, TS2γχt–[N3-N2] lies 7 – 9 kJ/mol above TS3χt–[N1-O]

and 126 – 131 kJ/mol above GS[S], whereas B3P86 increases the TS3χt–[N1-O] energy such that it is 142 kJ/mol above GS[S], 17 kJ/mol above TS2γχt–[N3-N2], and only 3 kJ/mol above TS2χt–[S-N2].

MP2(full) results put TS2γχt–[N3-N2] particularly low, 105 kJ/mol, such that

TS3χt–[N1-O] is rate limiting at 122 kJ/mol. It is also conceivable that these two products undergo a proton transfer, yielding CD3NHCS+ (m/z 77, β) + NH2C3H6COHNCH3 [χ-H+].

Eight conformers of the neutral species were located

with the most stable having a ggct backbone orientation and a N2•HN3 hydrogen bond. other conformers lie within 29 kJ/mol of this species, Table S5.

The

Overall, this asymptote lies 261

– 290 kJ/mol above GS[S] and 204 – 219 kJ/mol above the χ + CD3NCS asymptote.

This large

difference is consistent with this pathway being an inefficient means of producing the β ion. The χ and χ′ product ions can undergo further dissociation by losing ammonia.

This

occurs by rotation of the terminal NH3+ group away from the oxygen, which allows nucleophilic attack of the oxygen at the CN bond in TS2ψ, Figure 5.

This leads to the five-membered cyclic

m/z 100 (ψ and ψ′) products, with the latter 4 – 5 kJ/mol higher in energy.

As seen in Table 3,

the calculations indicate that TS2ψ is rate-limiting, lying 126 – 150 kJ/mol above the χ product,



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Page 22 of 52

22 176 – 226 kJ/mol above GS reactants, and 47 – 103 kJ/mol above the rate-limiting TS, TS2γχ or TS3χ. (TS2ψ′ lies only 8 kJ/mol higher in energy, Table S6.) The lower end of the latter energies are comparable to the difference between the apparent thresholds for formation of m/z 117 (χ) and m/z 100 (ψ), Figure 1, indicating that this process is conceivably responsible for the onset of the ψ product ion or could contribute at higher energies. The χ product ion can also rearrange to form λ (m/z 86) + CH3NH2, which can occur by nucleophilic attack of N2 at the carbonyl carbon, Scheme 1.

However, exploration of the

potential energy surface for this transformation (detailed in Table S7) finds that transfer of a proton from N2 to N3 over TS2λ is relatively high in energy, 261 – 267 kJ/mol above GS reactants (186 – 214 kJ/mol above χ), with an alternative TS2λ′ less than 2 kJ/mol higher in energy.

TS2λ lies 18 – 31 kJ/mol higher in energy than the pathway evolving from γ5 over

TSγλ.

Furthermore, the calculations indicate that the proton affinity of CH3NH2 is higher than

that of c-C4H7NO [λ-H+] by 5 – 14 kJ/mol, such that CH3NH3+ should be the favored product, yet this species (m/z 32) is not observed. 86) is not important experimentally.

We conclude that this pathway for formation of λ (m/z

Details of the species along this pathway can be found in

Table S7 of the Supporting Information. As for the φ precursor, the mechanism for decomposition of χ to yield the ω product in reaction 9b was not explored computationally.

It seems likely that from χ, transfer of the

proton from N2 to the gamma carbon will cleave the C−C bond α to the carbonyl moiety to yield the CH3HNCO+ (ω) product ion along with the C3H7NH2 neutral product.

Primary Formation of ψ (m/z 100) As discussed above, the m/z 100 ion (ψ) can be produced by the sequential decompositions of m/z 159 (φ) and m/z 117 (χ); however, the lowest energy pathway for its formation on the basis of the present quantum chemical calculations is a primary decomposition, as shown in Figure 5 and Table 3. The first two elementary steps are the same as those for χ formation. From INT2χ, rather than break the SC−N2 bond (TS3χ), the alternate N2-butyric acid amide


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23 bond is broken.

This is facilitated by rotation of the deuteriated methyl thiourea moiety such

that the oxygen can undergo nucleophilic attack to cleave the C−N2 bond yielding deuteriated methyl thiourea (CD3NHCSNH2) + ψ.

This requires passing over TS3ψ or TS3ψ′, the

rate-limiting step for this primary decomposition. TS3ψ lies 162 – 185 kJ above GS[S] with TS3ψ′ another 7 – 8 kJ/mol higher, Table S6.

Notably TS3ψ lies 33 – 80 kJ/mol above TS2γχ ,

the rate-limiting step in producing its INT2γ precursor, and 29 – 63 kJ/mol above TS3χ with which it competes, and even 9 – 21 kJ/mol above (7 kJ/mol below at the B3P86 level) formation of α.

Thus, the primary formation of m/z 100 may not compete effectively with the other

primary product ions. As for the φ and χ precursors, the mechanism for decomposition of ψ to yield the ω product in reaction 9c was not explored computationally.

This reaction would appear to necessitate

cleavage across the ring, yielding the CH3HNCO+ (ω) product ion along with a C3H6 neutral product, which could either be cyclopropane or propene if a 1,2-hydrogen shift occurred.

Analysis of CID Cross Sections for [1+H]+ On the basis of the PESs calculated above coupled with the experimental data, Scheme 2 represents the pathways for decomposition of [1+H]+ that can potentially be modeled.

Five

primary decomposition pathways for [1+H]+ are observed and involve proton migration from sulfur to N1, N2, and N3 sites.

The m/z 159 (φ) and m/z 35 (α) channels are the primary

products of proton migration to N1, whereas proton transfer to N2 leads to the formation of products m/z 117 (χ) and 100 (ψ). product m/z 162 (γ).

If the proton migrates to N3, [1+H]+ dissociates to primary

Both γ and χ can further dissociate to form the λ (m/z 86) product, with

the former pathway lying lower in energy.

Furthermore, the latter pathway should also lead to

CH3NH3+, which is not observed. The φ and χ ions can dissociate to m/z 100 (ψ) at higher energy, but primary formation of the m/z 100 channel may also contribute at threshold.

The data of

Figure 1 clearly indicate that dissociation of φ to ψ must be a major sequential dissociation channel as no other product has a cross section large enough to account for the decline in the



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24 cross section for φ.

To model these complicated and interrelated reaction processes, the cross

sections for the major sequential decomposition channels (ψ and λ) were summed with their principal precursor, i.e., m/z 159 (φ) + 100 (ψ) and m/z 162 (γ) + 86 (λ).

In both cases, the cross

section sum changes smoothly with energy, consistent with such sequential dissociation Then cross sections for the four primary channels, α, χ, φ + ψ, and γ + λ, were

pathways.

modeled competitively using eq 1.

Once the modeling parameters for these primary pathways

were determined, sequential dissociation channels (which can only be modeled one at a time using the current version of our data analysis program CRUNCH in a more time consuming process) were considered.

Details of the various permutations can be found in Table S8 of the

Supporting Information, but the modeling parameters of successful models were fairly similar. Thus, Table 4 lists the transition states used to analyze each channel along with average values of the modeling parameters from all successful fits with uncertainties expanded to include the variations observed. The observation that formation of φ is the favored channel by over an order of magnitude suggests that this channel is limited by a loose PSL transition state (TS), consistent with theory at the MP2(full) and B3P86 levels, but not B3LYP, which indicates that TS3φ lies higher in energy. In contrast, formation of γ and α cannot be modeled accurately (either in shape or magnitude) when loose PSL TSs are used, even though all theory agree that these products lie above all preceding transition states.

Therefore, these channels were modeled using switching TSs in

which the rate is determined by a lower energy tight TS coupled with the higher energy loose PSL TS.

In both cases, the tight TS was chosen as the last TS before product formation, i.e.,

TS3γ and TS4α, respectively, as this TS would be expected to couple with the PSL TS most closely.

According to theory, earlier tight TSs, TS2γ and TS3φ, respectively, might also be

limiting as TS2γ and TS3γ have similar energies (Table S3) and TS3φ is a little higher than TS4α (Table S2).

In both cases, modeling of the data with these earlier tight TSs leads to larger

kinetic shifts and poorer agreement between experimental and theoretical energies. No matter what tight TS is used, the switching TSs allow the shape and magnitude of the data for both


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25 channels to be reproduced well, as shown in Figure 6a for modeling with TS3γ and TS4α.

In

both of these cases, the energy of the PSL TSs could be as high as 1.40 eV for γ and 1.65 eV for α without affecting how accurately the models reproduce the data.

At higher PSL TS energies,

the threshold region of each cross section is no longer reproduced no matter what energy the associated tight TS has.

For the γ channel, no scaling of any molecular parameters was needed,

but in order to reproduce the magnitude of the α cross section, vibrational frequencies below 900 cm-1 needed to be scaled by 0.915. This addresses the well-known problem associated with using a harmonic approximation for hindered rotations and other low-frequency motions. For the remaining primary channel, χ, theory agrees that this pathway is limited by a tight TS, TS3χ (with TS3χt– being the most stable), although B3LYP indicates that TS2γχ is slightly higher in energy.

In order to reproduce the magnitude of this cross section using TS3χt–, vibrational

frequencies below 900 cm-1 needed to be scaled by 0.86 (with a much more severe scaling factor needed for analyses using TS2γχ instead).

For both the χ and α pathways, the need for

frequency scaling indicates that these tight rate-limiting TSs are somewhat looser than suggested by theory.

This may partially reflect the range of entropic factors associated with the parallel

pathways for TS4α and TS3χ, but none of the variants were sufficiently loose to reproduce the data without scaling of the frequencies. pathways along each product pathway.

It may also reflect the fact that there are multiple Overall, using this approach, Figure 6a shows that the

competition between all four primary channels can be reproduced nicely over extended ranges of energy and magnitude.

(The models do not quite reproduce the cross sections for φ and γ at the

lowest energies, and indeed no set of modeling parameters do.

This is believed to be the result

of contributions from a small amount, no more than a couple of %, of an excited conformer of [1+H]+, which was more evident in some data sets.) We also tried including the formation of the product ψ, m/z 100, as a primary dissociation pathway passing over the rate-limiting TS3ψ, Figure 5.

Because this channel is higher in energy than the other four primary channels and

involves a tight TS, no reasonable set of modeling parameters would reproduce this channel. (Any set of parameters that did approach the correct magnitude and energy dependence led to an



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Page 26 of 52

26 entropy of activation that exceeded those for the PSL TSs, which is inconsistent with the assumption of the tight TS.) Once the primary dissociation channels were reproduced, the sequential dissociation pathways could be modeled.

In the case of product λ, theory finds that the product asymptote

and TS3γλ are similar in energy, hence our analyses examined both as rate limiting.

They gave

similar threshold energies and both models required some scaling of the cross sections. Comparison of Figures 6a and 6b shows that the sum of γ and λ as well as the individual cross sections are modeled nicely over the energy range considered.

At higher energies, γ can also

form β in a parallel dissociation pathway, although the cross section for this product was not acquired in all data sets. Nevertheless, a rough analysis provided an approximate threshold of 3.1 ± 0.2 eV.

As noted above, a major contributor to the ψ product must come from subsequent

dissociation of φ, the major product ion.

Indeed, modeling the sequential dissociation of φ over

TS1ψ to yield ψ provides a cross section having a magnitude in good agreement with experiment, but with a threshold that is too high.

Therefore, contributions from sequential dissociation of χ

over TS2ψ to yield ψ were also considered.

Here, the modeling was designed to reproduce

only the threshold region of the ψ cross section.

In an iterative fashion, both contributions were

considered, although the final parameters obtained must be considered somewhat speculative because multiple adjustable parameters are used to reproduce the ψ cross section, although here no scaling was required for TS2ψ and that for TS1ψ was only 0.75. Nevertheless, as shown in Figure 6b, the results are quite satisfactory and allow the φ, χ, and ψ cross sections to be reproduced nicely. Ultimately, the success of such modeling can also be evaluated by comparison of the threshold energies obtained with the theoretically computed energies for the rate-limiting transition states of each channel. results.

This is shown in Table 5 and visually in Figure 7 for the MP2

(Comparisons with B3LYP and B3P86 results are shown in Figure S2 of the Supporting

Information.) In general, theory and experiment agree well with the various theoretical values often spanning the experimental threshold.

Bigger differences occur for the two transition


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27 states leading to ψ, but here the modeling is somewhat speculative.

Even so, theory brackets

the threshold obtained for TS2ψ, which is the more reliable of the two numbers.

Other notable

differences are that the upper limit of 135 kJ/mol obtained for PSL (γ7c) is well below the theoretical values, with discrepancies > 18 – 29 kJ/mol, whereas the upper limit for PSL (α) agrees fairly well with theory.

The discrepancy could be because we have not found the true

ground structure of the m/z 162 product ion, although many variations were explored, as discussed above.

Overall, the agreement between experiment and theory is reasonably

illustrated by the mean absolute deviations (MADs), 6 – 18 kJ/mol, Table 5, if the upper limit and more speculative TSψ values are excluded.

If all values are included in this comparison,

the MADs increase to 14 (B3LYP), 22 (B3P86), and 16 (MP2).

In either case, considering that

the average experimental uncertainty is 13 kJ/mol (two standard deviations), the agreement is reasonable, especially for the MP2 and B3LYP values.

Conclusions The kinetic energy dependence of the collision-induced dissociation of protonated d3-methyl thiourea-4-butyric acid methyl amide (1), [1+H]+, is examined using guided ion beam mass spectrometry.

The threshold energies for four primary and four secondary product channels are

determined by taking into account the effects of competition, kinetic shifts, internal energy of reactants, and multiple collisions. The mechanism for the decomposition of [1+H]+ is elucidated by computational studies that identify the rate-limiting steps for each process.

Reasonable

agreement between the computational energies and the thresholds determined from experiment demonstrate that migration of the proton to N1 leads to the dominant reaction, formation of m/z 159 (φ) + CD3NH2, which is limited by the product asymptote, a loose TS.

This product goes

on to dissociate to m/z 100 (ψ) + HNCS, making this the dominant channel at high energies.

In

contrast, migration of the proton to N3 and subsequent rearrangement at the other end of the molecule forms m/z 162 (γ) + CH3NH2, which is restricted by two tight TSs, TS2γ and TS3γ, even though the product asymptote lies 25 – 52 kJ/mol higher in energy according to theory,



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Page 28 of 52

28 Figure 4.

This product ion undergoes further dissociation to yield m/z 86 (λ) + CD3NCS as well

as m/z 77 (β, CD3NHCS+) + c-C4H7NO, with the former being lower in energy.

The threshold

for formation of the third primary product ion m/z 117 (χ) + CD3NH2 lies somewhat above those for φ and γ even though this product asymptote is the lowest energy asymptote among all channels observed. Computations show that this is because this channel involves migration of the proton to N2 and requires a tight TS, either TS2γχ or TS3χ, that lies well above the products. χ undergoes further dissociation to also form ψ, in a process that is lower in energy than that evolving from φ.

In direct competition with φ + CD3NH2 is CD3NH3+ + [φ-H+], which is the

highest energy primary process observed and is inhibited a tight transition state, TS4α, also lying below the product asymptote by 17 – 27 kJ/mol. Using this theoretical input, the experimental cross sections can be reproduced with good fidelity over an extended energy range of 3 eV and magnitude range of 1000. α channels.

This requires the use of switching transition states for both the γ and

The mechanisms elucidated for decomposition of [1+H]+ are similar to those for

peptide fragmentations, including the “mobile proton” concept. The results of this extensive theoretical and experimental study of the unique CID-lability found for thiourea-containing XL-reagents lay a reliable foundation for the mechanistic understanding of these competing and consecutive fragmentation pathways, all of which are scrutinized here.

The good agreement between experiment and theoretical energies validates

the mechanisms explored theoretically and identifies the structures of the various product ions and neutrals, with γ, ψ, and λ being cyclic and φ, χ, β, and α being acyclic.

Acknowledgements This work is supported by the National Science Foundation, Grants No. CHE-1409420 (MTR) and CHE-1359769 (PBA). A grant of computer time from the Center for High Performance Computing at the University of Utah is gratefully acknowledged.


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29 Supporting Information Available Figure showing the reaction coordinate surface for decomposition of [1+H]+ to form χ by proton transfer from S to N2. Figures showing the comparison of experimental threshold energies with energies for rate limiting transition states computed at the B3LYP and B3P86 levels of theory. Tables of relative energies of S protonated conformers of [1+H]+. Tables of intermediates, transition states, and products for [1+H]+ decomposition by proton transfer to N1 to form m/z 159 (φ), by proton transfer from S to N3 to form m/z 162 (γ), by proton transfer from S to N2, by proton transfer from N3 to N2 to form m/z 117 (χ), to form m/z 100 (ψ) by decomposition of m/z 117 (χ) and direct decomposition of [1+H]+, and for decomposition of m/z 117 (χ) by proton transfer from N2 to N3 to form m/z 86 (λ). Table of fitting parameters of eq 1, threshold energies at 0 K, and entropies of activation at 1000 K for the decomposition of [1+H]+. This information is available free of charge via the Internet at http://pubs.acs.org.

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36 Table 1. Relative energies (kJ/mol) of intermediates, transition states, and products for [1+H]+ decomposition by proton transfer to N1 to form m/z 159 (φ) and m/z 35 (α) and sequential dissociation to m/z 100 (ψ).a Species

conformation

B3LYPb

B3LYPc

B3P86c

MP2(full)c

GS[S]

[St]tctggtt

0.0

0.0

0.0

1.3

TS1φ

[S(tc)]tctggtt

20.5

21.0

21.9

22.5

INT1φt[S]

[Sc]tctggtt

10.8

10.1

10.2

12.4

TS2φ

[Sc]t(tc)tggtt

82.2

81.6

82.9

89.4

INT2φt[S]

[Sc]tttggtt

6.2

6.1

5.7

9.1

TS2φ(tc)

[Sc](tc)ttggtt

89.7

90.7

91.2

92.3

INT2φc[S]

[Sc]cttggtt

1.0

0.8

1.0

0.0

TS3φ+

[S-N1]g+ttggtt

141.9

144.1

137.1

140.2

TS3φ–

[S-N1]g–ttggtt

142.6

145.0

138.0

141.6

INT3φ+[N1]

[N1]g+ttggtt

65.1

68.1

68.8

56.1

TS3φ(+–)[N1]

[N1]g(+–)ttggtt

72.6

75.1

75.1

62.7

INT3φ–[N1]

[N1]g–ttggtt

65.4

68.5

69.3

56.1

TS4φ

[N1-O]tttggtt{C~N1}

110.5

112.9

129.6

113.2

INT4φ[O]

(φ[Oc]tggtt)(CD3NH2)

113.6

113.8

135.4

123.0

φ + CD3NH2

[Oc]tggtt

126.1

124.9

147.0

143.9

φ′ + CD3NH2

[Oc]tggtc

136.5

135.4

157.0

155.4

TS4α

(φ)(CD3NH2)[N2-N1]

132.8

137.2

160.5

137.3

INT4α[N1]

(φ−Η+)(CD3NH3+)

16.9

20.5

42.7

20.2

CD3NH3+ (α) + [φ-H+]

gg+gtt

152.1

154.8

179.2

163.7

CD3NH3+ (α) + [φ′-H+]

tggtc

161.5

164.8

189.4

177.5


Page 37 of 52

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37 Table 1. continued Species

conformation

B3LYPb

B3LYPc

B3P86c

MP2(full)c

TS1ψ + CD3NH2

{N2~C~O}gtctt

257.1

256.3

289.1

299.7

TS1ψ′ + CD3NH2

{N2~C~O}ttctc

265.7

264.8

297.4

307.7

INT1ψ[N3] + CD3NH2

(ψ[N3]ctt)(HNCS)

150.1

146.5

177.1

178.2

INT1ψ′[N3] + CD3NH2

(ψ[N3]ctc)(HNCS)

155.5

151.5

181.8

184.7

ψ + HNCS + CD3NH2

[N3]ctt

167.9

163.4

194.4

212.5

ψ′ + HNCS + CD3NH2

[N3]ctc

172.9

168.0

198.9

217.7

ω + cC3H6NHC=S + CD3NH2

315.6

318.9

346.6

347.2

ω + SCNC3H7 + CD3NH2

329.9

331.2

381.3

368.4

a

All energies are corrected for ZPE with geometries and frequencies calculated at the B3LYP/P

level. Bold indicates the rate-limiting TS for a particular pathway. c

6-311+G(2d,2p).


b

6-311+G(d,p).


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 52

38 Table 2. Relative energies (kJ/mol) of intermediates, transition states, and products for [1+H]+ decomposition by proton transfer to N3 to form m/z 162 (γ) and sequential dissociation to m/z 86 (λ).a Species

conformation

B3LYPb

B3LYPc

B3P86c

MP2(full)c

INT0γc[S]

[St]cctggtt

18.4

19.3

19.7

20.4

TS1γc

[S-N3]cctggtt

94.5

97.1

89.2

76.6

INT1γc[N3]

[N3]ccgggtt

100.6

103.3

97.4

83.4

TS2γc

[N3]c(tc)ggg(gt)t

130.8

135.2

131.9

114.4

INT2γc[N3]

[N3]ctggggt

81.7

83.7

79.9

66.4

TS3γ7c

{S~C~N3}

123.7

127.5

126.3

111.6

INT3γ7c[N1]

(γ7c[N1]ctggg)(CH3NH2)

119.2

120.8

124.4

109.6

γ7c + CH3NH2

[N2]ctggg

154.6

153.0

162.2

163.8

γ7t + CH3NH2

[N2]ttggg

162.1

160.9

170.1

172.1

[γ7c–HN2+] + CH3NH3+

ctggg

237.0

237.2

243.7

240.0

TS3γ5t

{N2~C~N3}

190.9

196.3

193.7

157.4

INT3γ5t[N2]

(γ5[N2]tttcc)(CH3NH2)

188.7

193.4

193.9

155.1

γ5t + CH3NH2

[N2]tttcc

219.4

220.5

227.9

210.2

γ5c + CH3NH2

[N2]cttcc

230.6

232.0

239.3

221.1

TSγt(7-5) + CH3NH2

{OC~N2}tggcc

236.4

238.7

249.6

233.2

TSγλ + CH3NH2

{SC~N2}tttcc

233.2

234.3

248.3

233.5

INTλ + CH3NH2

λ(CD3NCS)OH•N1,S•HN2

130.0

126.7

150.9

152.9

λ + CD3NCS + CH3NH2

[Ot]

179.4

176.2

203.5

219.5

λ + CD3NCS + CH3NH2

[Oc]

188.5

185.2

212.7

228.9

β + C4H7NO + CH3NH2

[N]

275.0

274.8

305.2

308.6

a


level. Bold indicates the rate-limiting TS for a particular pathway. c

6-311+G(2d,2p).


b

6-311+G(d,p).

Page 39 of 52

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39 Table 3. Relative energies (kJ/mol) of intermediates, transition state, and products for [1+H]+ decomposition by proton transfer to N2 to form m/z 100 (ψ) and m/z 117 (χ) and sequential dissociation to m/z 100 (ψ).a

a

Species

conformation

B3LYPb

B3LYPc

B3P86c

MP2(full)c

INT2γt[N3]

[N3]ttggggt

87.3

89.8

86.4

72.2

TS2γχt–

[N3-N2]tg–tgggt

126.2

131.0

125.0

105.2

INT2χt–[N2]

[N2]tg–gggtt

45.6

49.9

47.8

34.8

TS3χt–

[N1-O]ggtt{C~N2}

119.3

122.4

142.2

122.2

INT3χ+[O]

(χ[Oc]ggg+t)CD3NCS

91.5

92.3

113.7

94.5

TS4χ

(χ[O-N2])CD3NCS

107.9

109.4

131.3

111.3

INT4χ[N2]

(χ[N2]ggtt)CD3NCS

5.3

7.7

27.5

20.6

χ + CD3NCS

[N2]ggtt

49.3

51.3

71.9

76.0

χ′ + CD3NCS

[N2]ggtc

59.3

61.5

81.6

86.6

β + [χ-H+]

ggct

261.2

263.6

290.4

280.5

TS2ψ + CD3NCS

{N2~C~O}ctt

176.5

177.8

209.4

225.6

INT2ψ[N] + CD3NCS

[N]ctt

114.2

112.6

142.6

147.0

ψ + NH3 + CD3NCS

[N]ctt

139.9

135.8

166.6

177.2

TS3ψ

{N2~C~O}ctt

162.1

164.1

171.7

185.0

INT3ψ[N3]

[N]ctt

21.3

21.0

24.4

41.4

ψ + CD3NHCSNH2

[N]ctt

100.4

99.8

107.1

134.0


level. Bold indicates the rate-limiting TS.

b

6-311+G(d,p). c 6-311+G(2d,2p).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 52

40 Table 4. Fitting parameters of equation 1, threshold energies at 0 K, and entropies of activation at 1000 K for the decomposition of [1+H]+.a Product

E0

b

Transition state

n

ߪ଴

(eV) m/z 162 (γ)

TS3γc′

2.1 (0.2)

(J/K mol)

1.06 (0.11)

-28 (18)

PSL (γ7c)

Guided Ion Beam and Computational Studies of the Decomposition of

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