B American Society for Mass Spectrometry, 2014

J. Am. Soc. Mass Spectrom. (2014) DOI: 10.1007/s13361-014-0832-0

RESEARCH ARTICLE

Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen Aleš Marek, František Tureček Department of Chemistry, University of Washington, Bagley Hall, Seattle, WA 98195-1700, USA

Abstract. Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gasphase peptide cations resulted in facile N2 elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N2 elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N2 from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N2 loss is proposed as a thermometer dissociation for peptide ion dissociations. Key words: Diazirine-labeled peptides, Collision-induced dissociation, Nitrogen elimination, Ion structures, Dissociation mechanisms Received: 10 December 2013/Revised: 26 December 2013/Accepted: 9 January 2014

Introduction

T

he diazirine ring has been used as a versatile and chemically inert photo-sensitive group for covalent footprinting of proteins [1–4]. The footprinting relies on photodissociation of the diazirine ring [5, 6] that has been shown for aliphatic diazirines to produce singlet carbenes as reactive intermediates that undergo facile insertion into C–H bonds (Scheme 1) [7, 8]. Similar carbene-based reactions are thought to occur in diazirine-labeled proteins. The diazirine label can be introduced in several ways, e.g., as a trifluoromethylphenyl substituent [9], or in the form of L2-amino-4,4-azi-pentanoic (photoleucine, L*) or L-2-amino5,5-azihexanoic (photomethionine, M*) residues that are built in the peptide or protein sequence and replace the pertinent natural amino acids [10]. In addition to photodis-

Electronic supplementary material The online version of this article (doi:10.1007/s13361-014-0832-0) contains supplementary material, which is available to authorized users. Correspondence to: František Tureček; e-mail: [email protected]

sociation, thermal isomerization and dissociation of aliphatic diazirines have also been extensively studied [11–13]. Previous studies found that upon heating in the gas phase or solution some diazirines underwent ring opening to isomeric diazoalkanes (Scheme 1) followed by elimination of nitrogen [13]. The activation energies for the diazirine ring opening were found to be in the 113–126 kJ mol–1 range [8, 14]. While the chemistry of aliphatic diazirines is well understood, much less is known about the behavior of diazirine-containing complex polar molecules, such as photo-labeled peptides and proteins [15]. With respect to the use of mass spectrometry for the detection and characterization of photo-labeled proteins [15], studies of diazirine chemistry in gas-phase ions are warranted. Recently, we reported unusual dissociations of diazirinelabeled peptide ions following electron transfer [16]. The diazirine ring in photoleucine-containing peptide ions was calculated to have a substantial adiabatic electron affinity (1.3–1.5 eV) that allowed an electron to be attached to the ring and resulted in its cleavage in a fraction of chargereduced peptide ions. Here, we report the results of a

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Scheme 1. Diazirine ring cleavage and carbene C–H bond insertion

combined experimental and computational study of dissociations of diazirine-labeled peptide ions upon collisional activation and propose a mechanism for the nitrogen elimination and its dependence on the peptide ion structure.

Experimental Materials Photoleucine containing peptides GL*GGK, GL*GGK-NH2, GL*GGKK, GL*GLK, and GL*LGK, where L* stands for the L-2-amino-4,4-azi-pentanoic acid residue (Pierce Biotechnology, Rockford, IL, USA), were synthesized on Wang resin (Bachem Americas, Torrance, CA, USA and Chem-Impex Intl., Wood Dale, IL, USA) using the Fmoc methodology according to literature procedures [17]. F-moc protected photo leucine (L-2-amino-4,4-azi-pentanoic acid) was prepared according to the literature [18]. The diazirine-labeled pentacosapeptide, GL*GGKKYTVSINGGKKITVSIGLLG, was synthesized by Dr. Mathias Schaefer (Institute of Organic Chemistry, University of Cologne, Germany) and obtained by courtesy of Dr. Andrea Sinz (Martin Luther University, Halle-Wittenberg, Germany). The peptides were characterized by electrospray tandem mass spectrometry (ESI-CID-MS/MS) and displayed correct accurate masses and amino acid sequences. The other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents were of reagent grade purity, the deuterated solvents (Cambridge Isotope Laboratories) contained 999.5 % D.

Methods Mass spectra were measured on a Thermo Fisher Scientific (San Jose, CA, USA) LTQ XL linear ion trap instrument. Ions were produced by electrospraying 5–10 μM peptide solutions in 50:50:1 methanol-water-acetic acid. Collision activation was achieved by resonant excitation of massselected ions. The excitation time and normalized collision energy were varied as described in the Results section. Accurate mass measurements were carried out on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher

Scientific). The resolving power was set to 60,000 or 100,000. H/D exchange in peptides used for gas-phase studies was accomplished by dissolving the peptides in 1:1 D2O:CH3OD (99.5 %D) that contained 0.5 % CD3COOD to achieve 7–10 μM concentrations and allowing the solutions to stand in tightly capped vials overnight. The peptide solutions were infused with a syringe pump into an open electrospray source consisting of a pulled fused silica capillary as the ESI needle that was positioned 2 mm from the orifice of the source transfer capillary. Simultaneously with electrospraying the D2O/CH3OD peptide solutions, a stream of nitrogen that was saturated with D2O vapor at ambient temperature was blown over the orifice and tip of the ESI needle from a 15 mm o.d. glass tubing that was brought within 2 mm of the ESI tip.

Calculations Structures and energies for the GL*GGK ions, reaction intermediates, and transition states were obtained in several steps. In the first step, exhaustive conformational search was run for (GLGGK-NH2 +H)+ ions protonated on the Lys side chain and (GLGGK+2H)2+ dications protonated at the Nterminal and Lys amino groups to determine the lowest freeenergy conformers in each group. Briefly, this involved extensive molecular dynamics-replica exchange trajectory, semi-empirical, density functional theory, and second-order Møller-Plesset perturbational energy calculations, as described in detail elsewhere [19]. In the next step, three lowest free-energy (GLGGK-NH2 +H)+ conformers and two lowest free-energy (GLGGK+2H)2+ conformers were modified using PCModel 9.2 (Serena Software, Riverside, CA, USA) to replace a Leu methyl group and a hydrogen atom with a diazirine ring, and the new structures, (GL*GGKNH2 + H)+ and (GL*GGK +2H)2+ containing the photo leucine residue (L*) were fully reoptimized with B3LYP [20] and M06-2X/6-31+G(d,p) [21] calculations. The DFT calculations were performed using the Gaussian 09 suite of programs [22]. Transition states for ion dissociations and isomerizations were obtained by mapping with B3LYP and M06-2X/6-31+G(d,p) the relevant parts of the potential energy surfaces starting from local energy minima identified by these methods. The stationary points were further characterized by harmonic frequency analysis to have the appropriate number of imaginary frequencies (0 for local minima, 1 for first-order saddle points). Intrinsic reaction coordinate calculations [23] were run to connect the transition state geometries with those of the reactants and products. Improved relative energies were obtained by single-point B3LYP and MP2 (frozen core) calculations with the 6-311++G(2d,p) basis set, using the B3LYP/6-31+ G(d,p) optimized geometries. The B3LYP and MP2 (frozen core) [24] single-point energies were averaged (B3-MP2) to partially cancel known errors inherent to both approximations [25]. Another set of single-point energies were obtained by M06-2X/6-311++G(2d,p) calculations using

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

the M06-2X/6-31+G(d,p) optimized geometries. The relative free energies of the L*-containing peptide ion conformers showed the same ranking as their Leu-analogs, indicating that the L* residue did not affect the major Hbonding interactions that determine the conformer stability. The B3LYP-optimized structures of the lowest-energy ion conformers are shown in Figure S1 (Supplementary Data). Complete Cartesian coordinates for all structures are available from the corresponding author upon request. Rice-Ramsperger-Kassel-Marcus (RRKM) calculations of unimolecular rate constants were performed using the program of Zhu and Hase [26] that was modified by expanding the limit for the number of internal degrees of freedom to 1000 and recompiled to run under Windows XP or Windows 7 [27]. Vibrational state densities were obtained by a direct count of quantum states in 2 kJ mol–1 steps for internal energies up to 400 kJ mol–1 above the threshold. The rotational states were treated adiabatically and the microscopic rate constants [k(E,J,K)] were Boltzmannaveraged over the thermal distribution of rotational J and K states at 298 and 473 K.

Results and Discussion Gas-Phase Dissociations CID under the slow-heating regime provides a means of vibrational excitation and allows for multi-step analysis of the dissociation products. The gas-phase ion dissociations showed a conspicuous dependence on the peptide ion structure. CID of both (GL*GG+H)+ and its amide analogue mainly resulted in eliminations of water, ammonia, and standard backbone dissociations forming b and y sequence ions. A minor fragment ion attributable to elimination of N2 (m/z 287.1350, loss of 28.0058 Da, ~1 % of summed fragment ion intensities) was also observed on CID of (GL*GG+H)+ that remained low at higher ion excitation energies where the m/z 315 precursor ion was almost completely depleted (Figure 1a). In contrast, CID of ions containing the C-terminal lysine residue resulted in a very facile loss of N2 (Figure 1b). The excitation energy dependence of this dissociation is shown for the m/z 415 fragment ion, [G(L*-N2)GGK+H]+, in Figure 1 (inset). The loss of N2 showed an onset at a low normalized collision energy, NCE=10, where it represented 960 % of the summed fragment ion intensities. The yield of the [G(L*N2)GGK+H]+ ion reached 34 % at NCE = 14 and remained constant at higher NCE. The molar fraction of [G(L*N2)GGK+H]+ relative to summed fragment ion intensities did not depend on the excitation time (Figure S2, Supplementary Data), indicating that the loss of N2 was competitively fast (tG5 ms), and the majority of the product ions were not metastable to undergo further dissociation upon extending the observation time. The major competing dissociation of (GL*GGK+H)+ was a backbone cleavage yielding the y3 fragment ion at m/z 261 (Figure 1b). Further

analysis of the mass-selected [G(L*-N2)GGK+H]+ ion by CID-MS3 showed backbone cleavages at all peptide bonds, resulting in a complete series of sequence b and y ions (Figure 2). This strongly indicated that the loss of N2 from the L* side chain was not associated with a covalent bond formation to another amino acid residue. Doubly charged (GL*GGK+2H)2+ and its amide analogue also showed prominent loss of N2 upon CID giving rise to abundant m/z 2082+ and m/z 207.62+ fragment ions, respectively (Figure S3, Supplementary Data). The intensity of the m/z 2082+ and m/z 207.62+ fragment ions relative to backbone fragment ions was 22 % for both (GL*GGK+ 2H)2+ and its amide analogue. Analogous results were obtained for CID of singly and doubly charged ions from several other Lys containing peptides, e.g., GL*GGK amide, GL*GLK, GL*LGK, GL*GGKK, and GL*GGKK amide, all of which underwent facile elimination of N 2 (Figure S4a, b, c, f, g, h, Supplementary Data). We also generated singly charged GL*GG, GL*GGK, and GL*GGKK amide ions as the respective c4-c6 fragments by electron transfer dissociation of a +4 ion from a larger 25-mer, GL*GGKKYTVSINGGKKITVSIGLLG, and analyzed them by CID-MS3 experiments (Figure S4d and e, Supplementary Data). Again, only the Lys containing ions underwent facile loss of N2, whereas that of GL*GGamide did not, in complete agreement with the results of the above-described experiments. While elimination of N2 is a well-known thermolytic decomposition of simple organic diazirines [11, 12], the lysine effect on the CID was unprecedented and indicated an interaction between the Lys residue and the diazirine ring that was further investigated by experiment and theory.

Gas-Phase Deuterium Labeling Remote group interactions in gas-phase ions can be probed by isotope labeling. We generated singly and doubly protonated ions from GL*GGK, GL*GLK, and GL*LGK in which all labile protons were exchanged for deuterium. The formation of D10-singly charged and D11 doubly charged ions required special forcing conditions under which it was possible to achieve ≥93 % deuterium content in the ions stored in the ion trap and investigated by CID [16]. This high level of isotope enrichment was needed to avoid isobaric overlaps of the fully deuterated species with natural 13 C and 15 N isotopologues of partially labeled ions. The CID-MS3 spectra of D-labeled ions from G(L*-N2)GGK and G(L*-N2)GLK showed the expected mass shifts for sequence fragment ions according to the number of exchangeable hydrogen atoms present in the species. However, exceptions were observed for the complementary b2 and y3 ions (Figure 3a, b, c). The b2 ion contains 3 exchangeable deuterons, yet the CID mass spectrum showed the D4 ion (m/z 159) as a major species accompanied by the D3 ion at m/z 158 (Figure 3a). Likewise, the complementary y3 ions, having eight exchangeable deuterons, showed mainly the D7

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Figure 1. (a) CID mass spectrum of (GL*GG+H)+ at m/z 315. (b) High-resolution CID mass spectrum of (GL*GGK+H)+ at m/z 443.2361. Inset shows a plot of ion relative intensities as a function of normalized collision energy (NCE): full circles (GL*GGK+H)+ ion at m/z 443.2; triangles: (GL*GGK – N2 +H)+ ion at m/z 415.2 relative to the summed intensities of dissociation products; empty circles: GL*GGK – N2 +H)+ ion at m/z 415.2 relative to the summed intensities of all ions

ions (m/z 268 or 324), along with the D8 species (Figure 3b, c). This indicated that upon N2 elimination one exchangeable deuteron in the peptide ion was incorporated in a nonexchangeable position in a fraction of G(L*-N2)GGK and G(L*-N2)GLK intermediates. Although the CID-MS3 spectra did not allow us to distinguish whether the deuterium atom was in the G or L*-N2 residues, the nature of the N2 elimination points to the L* side chain as the likely site of deuterium incorporation.

Effects of Protecting Groups We further investigated the lysine effect on the N2 elimination by modifying the ammonium groups, which are protonated in the singly and doubly charged lysinecontaining ions. GL*GGK in which the Lys ε-amine was blocked by an allyloxycarbamate group, GL*GGK(alloc), formed only singly charged ions by electrospray. CID of

protonated GL*GGK(alloc) (m/z 526, Figure S5a, Supplementary Data) resulted in a substantially diminished elimination of N2 compared with the CID of the unblocked singly charged (GL*GGK+H)+ ion. The allyloxycarbamate group in GL*GGK(alloc) is substantially less basic than the lysine ε-amine group and slightly less basic than the Gly amino group, as inferred from the respective topical proton affinities for alkylcarbamates (882 kJ mol–1) [28], lysine (992 kJ mol–1), and glycine (890 kJ mol–1) [29]. Hence, protonation in GL*GGK(alloc) is likely to take place in positions other than the Lys carbamate group. In contrast, Nacetylation of the N-terminal amino group in N-AcGL*GGK did not result in blocking the N2 elimination upon CID (Figure S5b, Supplementary Data). Another means of blocking the Lys side-chain ammonium group is through noncovalent complexation with 18crown-6-ether (CE) [30–32]. Peptide–CE complexes in a 1:2 stoichiometric ratio formed readily as doubly charged ions

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Figure 2. CID-MS3 mass spectrum of [G(L*-N2)GGK+H]+ ion at m/z 415.3

upon electrospray of peptide-crown solutions. The ion dissociations are illustrated with the CID spectra of the GL*GLK+2CE complex (Figure S6a, Supplementary Data). The CID spectrum of the doubly charged complex at m/z 514 showed major dissociations by loss of crown ligands (m/ z 382), combined with loss of N2 (m/z 368), water (m/z 471), and backbone cleavage (m/z 317, y3). The doubly charged (GL*GLK+CE) complex at m/z 382 underwent abundant loss of N2 upon further collisional activation (Figure S6b, Supplementary Data), which competed with the loss of the crown ligand. In summary, covalent modifications of the Lys side chain had a substantially larger effect on quenching the loss of N2 from the diazirine ring than did the noncovalent modifications by crown ether attachment. This may be due to a collisioninduced migration of the crown-ether ligand from the Lys ammonium to the N-terminal one, thus freeing the Lys ammonium for attack at the Leu* residue. N-acetylation of the N-terminal amino group did not impede the loss of N2.

Effects of Charge and Amino Acid Residues The above-described results indicated that the elimination of N2 competed with backbone dissociations of GL*GGX cations. CID of dications showed a larger proportion of backbone cleavages compared with monocations when related to the loss of N2. The effect on CID of the nature of the charge-carrying site and the ion polarity was investigated for CID of (GL*GGR+2H)2+ (Figure S7) and (GL*GGK – H)− (Figure S8, Supplementary Data) ions. CID of (GL*GGR+2H)2+ showed competitive loss of N2 (17 %–22 %) and backbone dissociations forming the y3 and b2 fragment ions (40 % combined). The CID spectrum of the negative ion showed a dominant loss of N2 (65 %) and

Figure 3. (a) The b2 fragment ion mass region in the CIDMS3 mass spectra of (from top) [G(L*-N2)GLK-d9 +2D]2+, [G(L*-N2)LGK-d9 +2D]2+, [G(L*-N2)GGK-d9 +2D]2+, and [G(L*N2)GGK+H]+. (b) The y3 fragment ion mass region in the CIDMS3 mass spectra of [G(L*-N2)GLK-d9 + D]+. (c) The y3 fragment ion mass region in the CID-MS3 mass spectra of [G(L*-N2)GGK-d9 +D]+

only weak backbone fragment intensities. These results indicated that the loss of N2 from the Leu* residue can occur even in the absence of the lysine residue, provided the diazirine ring cleavage is kinetically competitive with backbone dissociations.

Gas-Phase Ion Structures and Dissociation Mechanisms The lysine effect, as well as the incorporation of exchangeable hydrogen atoms in the Leu* side chain upon loss of N2, indicated that the diazirine ring and the Lys ammonium group can interact upon collisional activation of the photolabeled peptide ions or intermediates produced by their isomerization. To gain insight into the nature of these interactions as well as the energetics of diazirine ring cleavage, we carried out a detailed study to map several dissociation pathways of (GL*GGK-amide+H)+ on the potential energy surface (PES) of the ground (singlet) electronic state, as summarized in Figure 4. The structures

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Figure 4. Potential energy diagram for isomerizations and dissociations of (GL*GGK-NH2 +H)+. Roman characters: energies from B3-MP2/6-311++G(2d,p) calculations; italics: energies from M06-2X/6-311++G(2d,p) calculations. All energies are referenced to 1 and include zero-point corrections

of the species whose relative energies are shown in Figure 4, appear in Schemes 2, 3 and 4 and in Supplementary Data. The calculations were carried out at three levels of theory, including two density functionals and a second-order Moller-Plesset treatment of correlation energy. The energies are summarized in Table 1, the data discussed in text are from combined B3LYP and MP2 single-point energies including zero-point vibrational energy corrections and referring to 0 K unless stated otherwise. The diazirine→diazo rearrangement was calculated to be exothermic, ΔH0,rxn = 10–50 kJ mol–1, depending on the reactant (1a-c) and product (2a-c) conformation (Figure S1, Supplementary Data). The diazirine ring opening in the most stable conformer 1 required 124 kJ mol–1 in the transition state (TS1). The N2 loss energetics critically depended on the structure of the product ion. Direct loss of N2 from 1 forming a singlet carbene product (3) was 56 kJ mol–1 endothermic (Scheme 2). Investigation of the PES for N2 loss from 1 indicated that simultaneous stretching of the C– N bonds in the diazirine ring led to high energy structures that upon releasing one constrained C–N coordinate, collapsed to 2. Loss of N2 from 2 then can proceed through TS2 (ETS2 = 127 kJ mol–1 relative to 2) to form carbene 3. The structures of TS1 and TS2 indicated very little interaction between the diazirine and diazo groups on the one hand and the lysine ammonium on the other in the course of the diazirine ring opening. This lack of interaction was understandable because the diazirine ring’s basicity was too low to provide a favorable donor site for internal

solvation of the lysine ammonium group. By comparison, the proton affinity of the diazirine ring (PA = 742 kJ mol–1 at 0 K from combined B3LYP and MP2 calculations of 3,3dimethyldiazirine) was substantially lower than that of the GLGGK peptide, which had PA=1094 kJ mol–1 at 0 K for protonation at the lysine NH2 group. Likewise, the 298 K gas-phase basicity of the diazirine ring in 3,3dimethyldiazirine was calculated by combined B3LYP and MP2/6-311++G(2d,p) as 717 kJ mol–1, which was substantially lower than the GB of amino acid residues in peptides [33]. Our calculated TS energies for the loss of N2 (113–127 kJ mol–1 at different levels of theory) were very close to those measured for simple aliphatic diazirines [8, 14] and indicated that the carbene formation was not much affected by the polar groups in the peptide ion. At the same time, the TS1 and TS2 energies were comparable to activation energies for proton-driven backbone dissociations in peptide ions [34, 35], indicating that these two reaction types can proceed in competition as observed for CID of the dications and anions. Furthermore, the lack of interaction between the diazirine ring and the lysine ammonium group in TS1 and TS2 indicated that a singlet carbene forming loss of N2 from the GL*GG ion should be analogous to that from GL*GGK. However, this was in stark contrast to the CID spectra of the (GL*GG+H)+ ion, which showed that the loss of N2 competed rather poorly with loss of water and backbone dissociations. The relative abundance of the N2 loss ion from (GL*GG+H)+, relative to the summed fragment ion intensi-

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Scheme 2. Diazirine ring opening in 1 and loss of N2 forming carbene intermediate 3

ties, stayed nearly constant at 1.1 %–1.3 % over a range of excitation energies that resulted in 14 %–99 % dissociation of the precursor ion. In contrast, the relative abundance of the N2 loss ion from the GLGGK and other lysine-

containing ions was much higher, reaching 60 % and representing a major dissociation pathway. The enhancement of N2 loss by protonated lysine points to mechanisms that may not involve carbene products. This

Scheme 3. Lysine proton transfer to the diazo group in 2 forming diazonium intermediates 4a, b

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Scheme 4. Structures of presumed isomeric fragment ions formed by lysine ammonium catalyzed elimination of N2 from diazonium intermediate 4a

prompted us to investigate a mechanism involving the lysine ammonium group in a Brønsted-acid-assisted loss of N2 from the diazoalkane intermediate 2 (Scheme 3). Upon Table 1. Relative Energies of Peptide Ions Ion/reaction

1 2 1→TS1 1→3+N2 1→12 2→TS2 2→TS3 2→4 2→TS4 2→11 2→13 2→5+N2 2→6+N2 2→7+N2 2→8+N2 2→9+N2 2→10+N2 7→TS5

Relative Energya,b B3LYPc

MP2c

B3-MP2d

M06-2Xe

0 –50 117 30 –158 129 117 102 105 –149 –160 –150 −116 –86 –63 –142 122

0 –32 132 81 –147 125 89 77 89 –144 –169 –156 −113 –92 –96 –172 136

0 –41 124 56 –153 127 103 89 97 –146 –165 −153 −115 –89 –80 –157 129

0 –10 98 81 –124 129 93 62 77 47 –145 –173 –154 −107 –97 –89 –170 -

In kJmol–1 Including zero-point vibrational energy corrections c From single-point energy calculations on the B3LYP/6-31+G(d,p) optimized geometries using the 6-311++G(2d,p) basis set d From averaged B3LYP and MP2 single point energies e From single-point energy calculations on the M06-2X/6-31+G(d,p) optimized geometries using the 6-311++G(2d,p) basis set a

b

collisional activation, ion 2 is presumed to undergo a series of conformational transformations that bring the lysine ammonium group into the vicinity of the diazo group. The activation energies involved in the conformational changes could not be studied for this complex pentapeptide ion system, but previous investigations of di- and tri-peptide ions had indicated conformational TS energies in the range of 8–40 kJ mol–1 [36, 37]. The lysine ammonium then can transfer a proton to the C = N = N group, forming a diazonium ion (4), whereby this exchangeable proton is placed in a nonexchangeable γ-position in the modified L* side chain. The pertinent transition state for the proton transfer (TS3a, ETS3 = 103 kJ mol–1 relative to 2) has a lower energy than those for TS1 and TS2 on the ion potential energy surface (Figure 4, Table 1). Similar results were obtained from an independent search on the M06-2X potential energy surface (TS3b and 4b, Scheme 3). The diazonium ion 4 is a relatively high point on the respective potential energy surface (49 kJ mol–1 relative to 1) and can undergo a variety of dissociations resulting in expulsion of N2 and forming terminal olefin (5), cis or trans internal olefin (6), or cyclic (7–10) products as summarized in Scheme 4. The dissociation pathways for loss of N2 from 4 differed when examined by B3LYP and M06-2X. In the M06-2X pathway, stretching the C–N2 bond in 4 proceeded over a low barrier (TS4b, ETS4b = 15 kJ mol–1) to form a complex of the incipient carbocation with N2 (11). The dissociation of 4 was slightly exothermic (ΔHrxn = –15 kJ mol–1, Scheme

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

S1, Supplementary Data). In the B3LYP pathway, stretching the C–N2 bond in 4 also proceeded over a very low barrier (TS4a, ETS4a = 7 kJ mol–1) but was accompanied by a spontaneous nucleophilic attack of the L* amide carbonyl at the developing carbocation center. This interaction dramatically lowered the potential energy of the system and led to the formation of a complex (12) in which the L* side chain was cyclized in an oxolan ring (–152 kJ mol–1 relative to 1). Complex 12 is not expected to survive, as it can readily eliminate N2 forming the cyclized product ion 7. The loss of N2 from diazonium ion 4 is extremely exothermic when forming the L*-cyclized product 7, ΔHrxn = –204 and –179 kJ mol–1 by B3-MP2 and M06-2X, respectively. The reaction can further proceed by ring opening in 7 and prototropic isomerization to terminal (5) or internal olefins (6) in the L* side chain, which is driven by the considerable exothermicity of N2 loss. We addressed the 7→6 isomerization (ΔHrxn =–50 kJ mol–1) and located a transition state (Scheme S2, TS5, ETS5 = 129 kJ mol–1 relative to 7). The isomerization involves cleavage of the L* side-chain oxolan Cγ–O bond with concomitant abstraction of a β-methylene proton by the lysine amino group. The 7→TS5→6 path was confirmed by intrinsic reaction coordinate analysis [23]. Since the formation of 7+N2 from 1 is 156 kJ mol–1 exothermic and the N2 molecule has a very low heat capacity, the internal excitation in 7 may be sufficient to drive an exothermic isomerization to 6. This assumption was further investigated by RRKM calculations of unimolecular rate constants (vide infra). Several alternative pathways for loss of N2 were investigated as sketched in Scheme 4. One of those involved interactions with the lysine NH2 group, which can abstract a proton from the L* side chain methyl group, forming a complex (13) that can lose N2 to form a conformer of the terminal olefin 5 (Scheme S3, Supplementary Data). The TS for this last step was not established and is indicated by a broken line in the Figure 4 PES diagram. An interesting feature of this pathway is that although the attack by O-8 drives the C-16–N2 dissociation, the energy gradient along the O-8–C-16 coordinate stays negative, and there is no Walden inversion at C-16 in the intermediate structures shown in Scheme S3. Another highly exothermic reaction can commence with a nucleophilic attack at the diazonium carbon (C-16) by the neutral Lys amino group, forming a macrocyclic ammonium ion (10, Scheme 4). We were unable to find a transition state for this ring closure, also depicted by a broken line in Figure 4, because all attempted approaches to the diazonium group for a nucleophilic attack by the lysine NH2 were sterically hindered and resulted in a reverse migration of the Cγ-proton that was previously transferred in TS3. Note that a lysine-involving ring closure was incompatible with the dissociations of the (GL*GGK-NH2 +H – N2)+ ions that showed backbone cleavages between the (L*-N2)-Gly, GlyGly, and Gly-Lys residues, which would be severely hampered in an L*∩Lys cyclized structure. Hence, this

highly exothermic N2 loss was excluded by the experimental data and its absence was explained by an unfavorable transition state for cyclization. A nucleophilic attack at the diazonium ion involving the Gly carbonyl (8) or the N-terminal amino group (9) were also calculated to be highly exothermic and may involve weakly bound complexes with N2. However, reactions leading to 8 and 9 would result in cyclizations that would hamper backbone cleavage between the G-(L*-N2) and residues, which is observed as a minor pathway in the CID spectrum, forming the y4 fragment ion. Hence, at least a fraction of the (GL*GGK-NH2 + H – N2)+ ions did not include structures 8 and 9. A tentative distinction of structures 5-10 can be made by considering the formation of the abundant b2 fragment ion upon CID (Scheme S4). According to the generally accepted peptide fragmentation mechanism, this dissociation proceeds with protonation of the L* amide nitrogen followed by a heterolytic amide bond cleavage that is assisted by oxazolone or diketopiperazine ring formation in the b ion [38–42]. Inspection of structures 5-10 indicates that oxazolone-assisted b2 ion formation can proceed most readily in the olefinic ions 5 and 6. Cyclizations in the other ions would inevitably result in strained transition states, e.g., a strained 4-aza-5,8-dioxabicyclo[3.3.0] structure from 7 or a bridged structure with an sp2 bridgehead carbon from 9.

RRKM Dissociation Kinetics The energy data were used to calculate unimolecular rate constants for diazirine ring opening in 1 (k1) and reverse closure in 2 (k–1), reversible proton transfer in 2→4 (k2) and 4→2 (k–2), competing dissociation of 2 by N2 loss to form carbene 3 (k3), and loss of N2 from 4 (k4) forming 7 via complex 12. The rate constants based on the B3-MP2 potential energy surface are shown in Figure 5. The M06-2X data are given in Figure S9, Supplementary Data. The kinetic data indicated that the diazirine→diazo rearrangement was subject to a substantial kinetic shift when occurring on the time scale of the CID experiment (30 ms). Furthermore, the rate constant for the reverse ring closure in the diazo derivative 2 (k–1) was substantially smaller than those for the other reactions of 2 (k2, k3), indicating that the diazirine ring opening was practically irreversible. The ring opening in (GL*GGK-amide+H)+ (1) was the rate determining step, requiring 278 kJ mol–1 internal energy to proceed with 50 % conversion on the 30 ms time scale of the CID experiments. This indicated a kinetic shift [43] of ΔEshift =278 – 124=154 kJ mol–1. The M06-2X energies and pertinent rate constants indicated a smaller kinetic shift (95 kJ mol–1, Figure S9, Supplementary Data) and 50 % conversion in ions having 194 kJ mol–1 internal energy. The RRKM rate constants further provided supporting evidence for Lys-ammonium assisted loss of N2. The data in Figure 5 showed that 1 undergoing ring opening on the 30 ms time scale formed 2 that can undergo fast rearrangement by Lys

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

Figure 5. Top panel: RRKM rate constants from combined B3LYP and MP2 calculations for reactions: k1: 1→2; k-1: 2→1; k2: 2→4; k-2: 4→2; k3: 2→3+N2; k4: 4→12. The dashed line indicates rate constants needed for 50 % conversion at 30 ms. Bottom panel: breakdown diagram showing molar fractions of (GL*GGK-NH2 +H)+ (1, black trace) and (GL*GGKNH2 − N2 + H)+ (dark red trace) at 30 ms reaction time calculated using the B3-MP2 potential energy surface

proton migration forming 4 (k2) Proton migration in 2 competes with loss of N2 forming carbene 3. RRKM calculations on both B3-MP2 and M06-2X potential energy surfaces pointed to direct loss of N2 from 2 (k3) as being more than two orders of magnitude slower than the protoncatalyzed isomerization at all excitation energies. The diazonium intermediate 4 is at a somewhat higher potential energy than 1, making the rearrangement reversible (k–2). However, the reverse proton migration (k–2) was 60- to 100fold slower than the loss of N2 from 4 (k4) and did not affect the dissociation kinetics. Figure 5 (bottom panel) shows that depletion of 1 by N2 loss chiefly occurred in the 250– 300 kJ mol–1 interval of internal energies. The rate constants that were based on M06-2X energies (Figure S9, Supplementary Data) indicated crossovers of the k1 and k2 curves at ca. 160 k mol–1, indicating that at higher internal energies the proton migration became the rate determining step. The rate constants for the direct N2 loss from 2 (k3) were very similar to those for reverse ring

closure (k–1). However, the loss of N2 was dominated by the fast dissociation of 4 (k4) that was somewhat affected by competition with the reverse proton migration (k–2). Overall, both calculations greatly prefer N2 elimination through the 2→4→12 pathway which is several orders of magnitude faster than the formation of carbene 3. These results are fully compatible with the facile loss of N2 in Lys-containing peptide ions. The products of N2 elimination from GL*GGK peptide ions presumably can undergo further isomerization, e.g., 7→5 or 7→6. The RRKM rate constants for the 7→6 isomerization (Figure S10, Supplementary Data) indicated that the reaction was affected by a kinetic shift of 227 kJ mol–1, achieving 50 % conversion at internal energies of 356 kJ mol–1. However, 7 is formed from 1 that must have acquired 9280 kJ mol–1 internal energy to proceed through TS1 on the experimental time scale (Figure 4). This excitation is combined with the 155 kJ mol–1 reaction exothermicity to give 435 kJ mol–1, which is only slightly diminished by the kinetic and internal energy of the departing N2 molecule. One can conclude that a substantial fraction of 7 has sufficient internal energies to undergo spontaneous isomerization to 6. The lack of interaction between the charged groups and the diazirine ring in TS1 and TS2 indicated that the noncatalyzed loss of N2 can proceed with similar energetics and kinetics in various diazirine-labeled peptide ions. This could be utilized to gauge the energetics of the competing backbone cleavages. For example, energy-resolved CID of the (GL*GGR+2H)2+ ion (Figure S7, Supplementary Data) indicated that the backbone dissociation forming the y3 ion had a lower activation energy (G100 kJ mol–1) and entropy than the loss of N2. The entropy effect can be inferred from the dissociation mechanisms because the loss of N2 involves only bond cleavages in loose transition states TS1 and TS2, whereas the formation of the y3 ion requires a proton transfer with a tight transition state. CID of the (GL*GGK−H)− ion (Figure S8, Supplementary Data) indicated that backbone cleavages in the negative ion had substantially higher activation energies (9100 kJ mol–1) and associated kinetic shifts than those for the non-catalyzed N2 loss through transition states analogous to TS1 and TS2.

Conclusions Diazirine containing peptides undergo elimination of N2 upon collisional activation of gas-phase ions. Ion activation under slow heating conditions induces diazirine ring opening to form diazoalkane intermediates. When a free lysine ammonium group is present, it acts as a Brønsted acid in promoting a rearrangement of diazoalkane intermediates to diazonium cations that can eliminate N 2 via several energetically favorable pathways. In the absence of a proton donor, N2 elimination proceeds via the classic carbene mechanism with TS energies of 100–120 kJ mol–1. The competitive dissociations of diazirine-labeled peptide ions

A. Marek and F. Tureček: CID of Diazirine-Labeled Peptides

can potentially be used to gauge the energetics of competing backbone dissociations. Conversely, the intramolecular interaction with lysine of diazirine-labeled amino acid residues can potentially be used as a probe of the gas-phase ion conformation. Studies to this end are in progress in this laboratory.

Acknowledgments Support of this research by the Chemistry Division of the National Science Foundation (grant CHE-1055132) is gratefully acknowledged. Thanks are due to Dr. Jan Urban for advice with peptide synthesis, Dr. Priska von Haller of the University of Washington Proteome Resource and Dr. Rob Moritz of the Seattle Institute for Systems Biology for providing access to the mass spectrometers, and Dr. Mathias Schaefer and Dr. Andrea Sinz for the tetracosapeptide sample and fruitful discussions.

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