Methionine, a-methylmethionine and S-methylcysteine radical … · Methionine, a-methylmethionine...

Methionine, a-methylmethionine and S-methylcysteine radical cations:

generations and dissociations in the gas phasew

Junfang Zhao,a C. M. Dominic Ng,b Ivan K. Chu,b K. W. Michael Siua

and Alan C. Hopkinson*a

Received 19th March 2009, Accepted 21st May 2009

First published as an Advance Article on the web 29th June 2009

DOI: 10.1039/b905615g

Methionine, a-methylmethionine and S-methylcysteine radical cations have been formed by

oxidative dissociations of [CuII(M)(CH3CN)2]�2+ complexes. The radical cations M�+ were

trapped, and CID spectra (MS3) of these ions are presented. Fragmentations of the methionine

and S-methylcysteine radical cations, initiated by migration of the a-carbon hydrogen atom

to the sulfur, trigger the losses of water and thiomethanol from methionine and thiomethanol

from S-methylcysteine. Deuterium labeling experiments show that considerable H–D scrambling

and rearrangements involving N–H and S–H hydrogens occur in the methionine radical cation

prior to fragmentation. An additional channel for S-methylcysteine is the loss of ammonia

following b-hydrogen migration. Methylation at the a-carbon of methionine results in a radical

cation that fragments differently. Two neutral losses from a-methylmethionine, NH3 and methyl

vinyl sulfide, CH2QCH–S–CH3, are initiated by g-hydrogen migration; a third channel is the loss

of�COOH. DFT computations at the B3LYP/6-311++G(d,p) level have been used to test

aspects of the proposed fragmentation mechanisms of the radical cations.

Introduction

Protein-based radicals play critical roles in some of the most

elaborate biosyntheses in nature, including photosynthesis and

substrate oxidation.1 Radical centers are located on aromatic-

and sulfur-containing amino acid residues, as well as on the

glycine residue.1a Generation of many of these charged or

neutral radicals involves oxidation of an amino acid residue by

a neighboring metal cofactor. Until recently radical cations of

peptides have proved to be difficult to generate in the gas

phase. The low vapor pressures of peptides made the

traditional method for producing organic radical cations,

electron ionization, not viable. Recently, copper(II)-containing

ternary complexes [CuII(L)(M)]�2+, where L is typically an

amine and M is a peptide or amino acid, have been employed

as a source for peptide and amino acid radical cations in

the gas phase.2 Collision-induced dissociation (CID) of the

complex leads to dissociative electron transfer, resulting in the

formation of M�+. Radical cation formation occurs most

efficiently when the peptide contains an amino acid residue

that has a low ionization energy. One shortcoming of this

method is the existence of competitive channels, mainly proton

transfer in which a proton migrates either from the auxiliary

ligand to the peptide to create [M + H]+ ions, or vice versa

giving [Cu(M � H)]�+ and [L + H]+. By judicious choice of

auxiliary ligands that are devoid of acidic hydrogens:

terpyridines; macrocyclics 1,4,7-triazacyclononane, 1,4,7,10-

tetraoxacyclododecane and 2,5,8,11-tetraoxadecane;3 or large

bidentate ligands,4 this competing proton-transfer reaction is

largely suppressed.

Peptide radical cations M�+ have much richer and more

varied chemistries than those of protonated peptides,

[M + H]+. Under low-energy CID conditions, the dissociations

of protonated peptides are primarily charge-driven with the

mobile proton inducing cleavage principally at the peptide

bonds, giving b- or y-ions. By contrast, the fragmentation of

radical peptide ions can either be charge- or radical-driven,

and this can to some extent be controlled by, for example,

sequestering the proton on the side chain of a basic amino acid

to suppress charge-induced reactions. Laskin et al.5a showed that

the gas-phase fragmentation of odd-electron M�+, [M+H]�2+,

and [M� 2H]�� ions, whereMwas angiotensin III (HVYIHPF),

share many similarities, thus suggesting strongly that the

fragmentation pathways are radical-driven. Furthermore,

radical-driven bond cleavages often occur at locations distant

from the a-radical center in the initially formed radical cation,

thereby indicating that mobility of the radical is an important

factor.5b In a very recent study on isomeric radical ions of the

simplest tripeptides, [G�GG]+, [GG�G]+ and [GGG�]+, it

was found that the barriers against interconversion among

these triglycine isomers are Z 44.7 kcal mol�1,6 significantly

higher than those against tautomerism of protonated

triglycine isomers (o17 kcal mol�1).7 Charge-driven dissociations

of both protonated triglycine and triglycine radical cations

lead to cleavage of the peptide bonds, producing the b2 and

[b2 � H]�+ ions, respectively.

aDepartment of Chemistry and Centre for Research in MassSpectrometry, York University, 4700 Keele Street, Toronto, CanadaM3J 1P3. E-mail: [email protected]; Fax: +1 416-736-5936;Tel: +1 416-736-2100 ext. 77839

bDepartment of Chemistry, University of Hong Kong, Pokfulam Road,Hong Kong

w Electronic supplementary information: Reaction profiles for the lossof NH3 from (S-methyl-Cys)�+ and (a-methyl-Met)�+. Tables of totalenergies and Cartesian coordinates of the optimized structures andtransition states shown in Fig. 2, 8–10 and Fig. S1 and S2. See DOI:10.1039/b905615g

This journal is �c the Owner Societies 2009 Phys. Chem. Chem. Phys., 2009, 11, 7629–7639 | 7629

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

Amino acid radical cations are much more fragile than

protonated amino acids, and only a few have been reported.

Radical cations of aromatic amino acids, tryptophan, tyrosine

and histidine, have been observed in the CID spectra of

[CuII(M)2]�2+ and [CuII(L)(M)]�2+.8 The basic amino acid

radical cations Arg�+ and Lys�+ were present in the CID

spectra of complexes [CuII(L)(M)]�2+, where the auxiliary ligand

was 1,3,5-triazacyclononane (tacn) or 2,20:60,200-terpyridine

(terpy);8b Arg�+ was also formed in the oxidative dissociation

of [FeIII(salen)(Arg)]+, where salen is the N,N0-ethylene-

bis(salicyldeneaminato) dianion.9 Basic amino acids facilitate

the formation of a-radicals that have captodative structures inwhich the charge and spin are formally separated; delocalization

of the charge onto the carboxyl group adjacent to the radical

center through hydrogen bonding enriches the electron-

withdrawing properties, and is highly stabilizing. Very recently,

infrared multiple photon dissociation (IRMPD) spectroscopy

established unambiguously that the observable His�+ has the

captodative structure.10

The auxiliary ligand, L, appears to play a significant role in

determining the structure of the amino acid radical cation that

is formed in oxidative dissociation. Barlow et al.9 found that

the Arg�+ ion formed in high abundance in the fragmentation

of [FeIII(salen)(Arg)]+ dissociated solely by the loss of

dehydroalanine (H2CQC(NH2)(COOH)) to give an ion at

m/z 87, whereas dissociation of [CuII(terpy)(Arg)]�2+ resulted

in Arg�+ (in low abundance), [Arg–CO2]�+ and the ion at

m/z 87. The difference in fragmentations was attributed to

different types of binding of arginine to the metal. It was

proposed that, in [FeIII(salen)(Arg)]+, arginine in the canonical

form is bound in a monodentate fashion to the iron through a

nitrogen of the guanidine side chain; dissociation of this

complex then gives a radical cation in which both the charge

and spin are localized on the guanidine group. By contrast, in

[CuII(terpy)(Arg)]�2+, it was proposed that arginine binds in

the zwitterionic form to copper, and oxidative dissociation

results in a distonic ion in which the radical resides on the

carboxy group and the charge on the protonated guanidine

group. Carboxy radicals are known to easily lose CO2,11 hence

the observation of [Arg–CO2]�+ in high abundance. Ke et al.8b

similarly observed Arg�+ (low abundance) and [Arg–CO2]�+

(high abundance) from complexes [CuII(L)(Arg)]�2+, where L was

yielded no Arg�+, only [Arg–CO2]�+ (high abundance) and

[Arg–CO2–H2CQCH(NH2)]�+ (low abundance). Acetone binds

much more weakly than the other auxiliary ligands, and it was

proposed that arginine binds only in the zwitterionic form in

[CuII(acetone)2(Arg)]�2+ and that the carboxy radical produced in

the dissociation is too fragile to be detected. The auxiliary

ligand also has a large effect in the CID of [CuII(L)n(His)]�2+

complexes.8b The use of terpy as auxiliary ligand led primarily to a

captodative a-radical ion that was stable on the MS timescale and

could be isolated and fragmented at a subsequent MS stage; by

contrast, employing acetone as the auxiliary ligand resulted in

essentially only the decomposition products of the carboxy radical.

Methionine and cysteine, the two sulfur-containing amino

acids, are of considerable interest in biochemistry and are

involved in a number of human diseases. Examples are oxidation

on sulfur in methionine to give a radical cation that has been

implicated notably in glycosylation of proteins and subsequent

disease development such as glaucoma;12 also the oxidation of

methionine has been directly linked to amyloid fibril

formation in the brain. This process is suspected to be the

first in a cascade of many chemical reactions leading to

symptoms of Alzheimer’s disease.13 The methionine radical

cation is, therefore, a reactive intermediate of great importance,

whose structure and reactivity need to be understood. The

cation itself and several model systems have been investigated

indirectly by several different physical methods in solution,

and in glassy matrices or single crystals by electron paramagnetic

resonance spectroscopy.14a–c Gas-phase photoemission studies

on methionine have been reported by O. Plekan et al.,14d,e and

the fragmentation reactions of methionine radical cation have

been investigated by direct electron ionization (EI).14f-h The

major fragment ion in the EI spectrum at m/z 131 was

attributed to the loss of water and the fragment at m/z 116

due to a subsequent loss of a methyl radical. A third product

ion at m/z 101 was attributed to the loss of H3CSH from the

methionine radical cation. Recently, we investigated theoretically

the structures and fragmentation pathways of the cysteine

radical cation.15 Nevertheless, attempts at producing Cys�+

via CID of [CuII(Cys)(L)]�2+ were not successful as copper-

catalyzed oxidation of cysteine to cystine in the solution

precluded the observation of [CuII(Cys)(L)]�2+. Very recently,

O’Hair and co-workers16 observed that S-distonic Cys�+ is

formed in the CID of protonated S-nitrosocysteine.

Here we report generations of the radical cations of

methionione, a-methylmethionine and S-methylcysteine, in

the CID of [CuII(M)(L)2]�2+, where L = acetonitrile.

The binding modes of the amino acids to copper and the

fragmentation behaviors of the radical cations have been

investigated theoretically using density functional

theory (DFT).

Experimental section

1. Chemicals

All amino acids, their derivatives, and methionine-2-d1(minimum 98 atom% deuterium) where the a-hydrogenwas replaced by deuterium were available from Sigma-Aldrich

(St. Louis, MO). Copper(II)-containing ternary complexes

were prepared in 1 mL 1 : 1 water–acetonitrile solutions, by

mixing copper(II) perchlorate hexahydrate with the amino acid

or its derivatives to a final concentration of 200 mM[CuII(M)(CH3CN)2]

�2+.

2. Mass spectrometry

The mass spectrometers used were prototype versions of the

API 2000 linear ion trap instrument and the API 3000 triple-

quadrupole mass spectrometer (both MDS SCIEX). The

collision gas used for both instruments was nitrogen. Samples

were introduced by means of pneumatically assisted electro-

spray at flow rates of 50–60 mL h�1. For the ion trap mass

spectrometer, MS2 and MS3 spectra were recorded typically

with a fill time of 50 to 100 ms and a scan speed of 1000 Th s�1.

We adopt the convention of labeling the mass-selected pre-

cursor ion in the spectra with an asterisk (*). Throughout

this work, unspecified Cu isotopes are always 63Cu; the

7630 | Phys. Chem. Chem. Phys., 2009, 11, 7629–7639 This journal is �c the Owner Societies 2009

dissociation chemistries of the 65Cu-containing complexes

were used for verification.

3. Computational methods

All calculations were performed using the Gaussian03

quantum chemical program.17 The total energies of Cu(II)

complexes and radical cations were calculated by the

unrestricted open-shell formalism within the framework of

Becke’s three-parameter DFT hybrid functional, B3LYP,

which is based on a mixture of Hartree–Fock exchange and

the Becke and Lee–Yang–Parr exchange–correlation functional.18

Standard Pople Gaussian-type basis sets, 6-31++G(d,p)

and 6-311++G(d,p), were employed. Local minima and

transition structures were optimized and characterized by

harmonic frequency analyses. Zero-point vibration energies

were evaluated directly using normal-mode frequencies

without anharmonic scaling. The local minima associated with

all transition states were identified using the intrinsic reaction

coordinates method.19 Atomic charges and spin densities were

evaluated using natural population analysis.20

Results and discussion

1. Dissociations of the [CuII(M)(CH3CN)2]

�2+ ions

(a) Methionine. TheCID spectrum of [CuII(Met)(CH3CN)2]�2+

(m/z 147) in Fig. 1a is dominated by singly charged ions arising

from dissociative electron transfer: Met�+ (m/z 149), its

fragmentation products (m/z 131, 116 and 101, vide infra),

and the complementary ion in the complex dissociation,

[CuI(CH3CN)2]+ (m/z 145). Two dipositive fragments corres-

ponding to the loss of one or two acetonitriles,

[CuII(Met)(CH3CN)]�2+ (m/z 126.5) and [CuII(Met)]�2+

(m/z 106), are also observed. Ion [CuII(Met)(CH3CN)]�2+ also

dissociated efficiently to give Met�+ and its complementary

ion [CuI(CH3CN)]+ at m/z 104. It is noteworthy that

there was no dissociative proton-transfer reaction or the loss

of CH3SH from the complex, as these were the dominant

dissociation channels when the auxiliary ligand was dien or

terpy.8a,9 Confirmation of the fragmentation products has

been established via the CID of [CuII(Met-2-d1)(CH3CN)2]�2+

shown in Fig. 1b.

Copper(II) has a d9 electronic configuration and forms four

strong metal–ligand dative bonds in a square-planar arrange-

ment at the equatorial positions; however, five-coordinate

complexes of Cu(II) are known although the fifth interaction

is typically weaker.21,22 Sulfur atoms from thiolate, thioether,

disulfide or inorganic sulfur have been shown to act as a donor

ligand in a variety of copper complexes.23 DFT performed at the

UB3LYP/6-31++G(d,p) level of theory showed the preferred

binding mode for methionine in [CuII(Met)(CH3CN)2]�2+ to

be canonical (Fig. 2), where the Cu is di-coordinated by the

amino nitrogen and the sulfur atom; there is also a weaker

third interaction between Cu and the carbonyl oxygen with a

relatively long bond distance (2.302 A). Binding of the two

acetonitriles to Cu completes the penta-coordination geometry.

Elimination of one acetonitrile from [CuII(Met)(CH3CN)2]�2+

(CS) is endothermic by 33.0 kcal mol�1, while that of two

acetonitriles is endothermic by 93.9 kcal mol�1. By contrast,

the dissociative electron-transfer reaction to yield Met�+ is

exothermic by 33.9 kcal mol�1, albeit there is an activation

barrier due to charge separation.8c

(b) S-Methylcysteine. The dissociation of [65CuII(S-methyl-

Cys)(CH3CN)2]�2+ (m/z 141) (Fig. 3) displays a pattern

similar to that of [CuII(Met)(CH3CN)2]�2+: dissociative

electron transfer to give (S-methyl-Cys)�+ (m/z 135), and

losses of CH3CN to give [65CuII(S-methyl-Cys)(CH3CN)]�2+

(m/z 120.5) and [65CuII(S-methyl-Cys)]�2+ (m/z 100) in low

Fig. 1 CID spectra of (a) [63CuII(Met)(CH3CN)2]�2+ (Elab = 10 eV)

produced in MS2 using enhanced product ion scan mode on API 2000,

ions [63CuII(Met)(CH3CN)2]�2+ and [65CuI(CH3CN)2]

+ are isobaric

at m/z 147w; ions [63CuII(Met)]�2+ and [65CuI(CH3CN)]+ are isobaric

at m/z 106ww; (b) [63CuII(Met-2-d1)(CH3CN)2]�2+ (Elab = 10 eV): the

precursor ion is labeled with an asterisk (*).

Fig. 2 Optimized low-energy structures of [CuII(Met)(CH3CN)2]�2+

(bond lengths in A) at the UB3LYP/6-31++G(d,p) level of theory.

CS is charge-solvated structure and SB is salt-bridged structure.


abundances. Ions at m/z 117.7 and 87.1 were attributed to the

losses of NH3 and CH3SH from (S-methyl-Cys)�+.

(c) a-Methylmethionine. The [CuII(a-methyl-Met)-

(CH3CN)2]�2+ ion (m/z 154) readily loses one acetonitrile,

and the second one with more difficulty, giving ions at

m/z 133.6 and 113.0 (Fig. 4). Dissociative electron transfer

to give the a-methylmethionine radical cation (m/z 163), as

well as its dissociation products (vide infra) and

[CuI(CH3CN)2]+ (m/z 145) is the dominant fragmentation

pathway.

2. Dissociations of the radical cations

(a) Methionine. The radical cation, Met�+, was mass-

selected and subjected to a third stage of CID (Fig. 5a). The

most abundant fragment ion was at m/z 131, the [b1 � H]�+

ion, produced via the loss of water. Subsequent dissociation of

this ion involved the loss of �CH3 from the sulfur atom to give

the cation at m/z 116, a reaction previously observed for

CH3SCH2�.24 There was also a minor dissociation channel

from Met�+: the loss of thiomethanol to give the radical

cation at m/z 101. Examination of the CID spectrum of

(Met-2-d1)�+ in which the a-hydrogen of methionine was

replaced by deuterium (Fig. 5b) revealed the loss of H2O or

HOD to give ions at m/z 132 and 131 in high abundance; CID

of the ions at m/z 132 resulted in the losses of �CH2D and�CH3 (product ions at m/z 116 and 117 in Fig. 5c–1); the ion at

m/z 131, in lower abundance in Fig. 5b, lost only �CH3

(product ion at m/z 116, Fig. 5c–2), as this precursor should

not contain any deuterium. The minor pathway in the CID of

(Met-2-d1)�+ produced ions at m/z 102 and 101, corresponding

to the loss of CH3SH and CH3SD or CH2DSH (Fig. 5b).

These results show that the deuterium from the a-carbon is

efficiently scrambled onto the carboxyl and methyl groups and

presumably onto the NH2 group.

These experimental observations can be rationalized in

terms of Scheme 1. The initially formed Met�+-1 (Met-2-d1)

Fig. 3 CID spectrum of [65CuII(S-methyl-Cys)(CH3CN)2]�2+

(Elab = 10 eV) produced in MS2 on API 3000: the precursor ion is

labeled with an asterisk (*).

Fig. 4 CID spectrum of [63CuII(a-methyl-Met)(CH3CN)2]�2+

(Elab = 15 eV) produced in MS2 using enhanced product ion scan

mode on API 2000: the precursor ion is labeled with an asterisk (*).

Fig. 5 CID spectra of the radical cations: (a) methionine, (b) methionine-

2-d1 (c) 1. product ion atm/z 132 from (methionine-2-d1)�+; 2. product

ion at m/z 131 from (methionine-2-d1)�+, produced in MS3 using

linear ion trap on API 2000. * signifies the precursor ion.


radical cation probably has a canonical structure in which

both the positive charge and the radical are formally localized

on the sulfur atom. Collisional activation can induce a

1,4-deuterium atom shift from the a-carbon to the sulfur to

give a distonic ion Met�+-2a which dissociates via several

channels. One pathway, initiated by D+ transfer from the

sulfur to the hydroxyl group followed by nucleophilic attack

by the sulfur atom on the carbonyl carbon, results in the loss

of HDO to give the product ion at m/z 131; this in turn can

lose the methyl radical to give the ion at m/z 116. A more

minor pathway, initiated by nucleophilic attack by the

carbonyl oxygen on the g-carbon, results in displacement of

CH3SD to give the product ion at m/z 101.

The deuteron on the sulfur atom in Met�+-2a can transfer

to the amino group by 1,5-D shift to yield a distonicMet�+-5a

in which the charge is formally localized on the NH2D group

and the spin on the a-carbon. This process is virtually barrier-

less andMet�+-5a is of similar energy to Met�+-2a (vide infra,

section 3). IonMet�+-2a can be reformed fromMet�+-5a, and

in the process an H+ from the amino group can be transferred

back to the sulfur atom, resulting in H–D scrambling of

N–H and S–D prior to the elimination of water.

Migration of a proton from the S-CH3 group of Met�+-1,

made acidic by the adjacent positively charged sulfur, via a

1,6-proton shift gives Met�+-6. This is followed by a 1,5-D

shift from the a-carbon to S-�CH2, giving Met�+-5b that

contains the SCH2D group. This can subsequently result in

the elimination of H2O (product ion m/z 132) followed by�CH2D loss (product ion m/z 116), or the elimination of

CH2DSH (product ion m/z 101). DFT investigations on the

energetics and details of these reactions are discussed below

in section 3.

Scheme 1


(b) S-Methylcysteine. The loss of CH3SH is the dominant

pathway for fragmentation of (S-methyl-Cys)�+ (Fig. 6), giving

the radical cation of dehydroalanine, H2N�+C(QCH2)COOH,

at m/z 87. Assuming that the S-methylcysteine radical cation

has the charge and spin located on the sulfur, then this

pathway requires a 1,3-hydrogen shift from the a-carbon to

sulfur followed by homolytic cleavage of the Cb–S bond. By

contrast, a very recent deuterium labeling and H–D-exchange

study of S-distonic cysteine radical cation16 showed that the

a-hydrogen was not involved in the H2S loss; it should be

noted, however, that this channel in cysteine was only a very

minor one. A second abundant product ion at m/z 118 was

formed from (S-methyl-Cys)�+ via the elimination of NH3.

This can be achieved by migration of a proton from the b-CH2

group, made acidic by the adjacent positively charged sulfur,

and possibly via two 1,4-proton shifts, that involves the

carbonyl oxygen as a catalyst. The destination of this proton

migration is the nitrogen, leading to heterolytic cleavage of the

N–Ca bond. This type of elimination was not observed in the

CID of Met�+ or of the S-distonic cysteine radical cation.16

(c) a-Methylmethionine. The CID spectrum of (a-methyl-

Met)�+ shown in Fig. 7 is very different from that of Met�+.

The major pathway is the loss of ammonia (not observed in

the fragmentation of Met�+) to give an ion at m/z 146. In

addition, there are two equally abundant fragments, an ion at

m/z 118, formed by elimination of �COOH to give the a1 or

iminium ion, and another ion at m/z 89, assigned as Ala�+,

generated by the loss of the side chain as the neutral methyl

vinyl sulfide. A minor product at m/z 128 corresponds to the

loss of water from the ion at m/z 146. Each of these three

major fragmentation pathways is the result of cleavage of a

different bond at the crowded a-carbon.Both major products in the fragmentation of Met�+ are

formed via formation of the a-radical. In the fragmentation of

(a-methyl-Met)�+ there is no evidence for migration of the

a-�CH3; consequently, the fragmentation pathways are

completely different from those of Met�+. Steric crowding at

the a-carbon and the acidity of protons adjacent to the sulfur

carrying the positive charge appear to be the determining

factors. Loss of �COOH is a common reaction in the CID of

peptide/amino acid radical cations and can be achieved

by transferring the charge and unpaired electron onto the

carboxyl group from the sulfur. Loss of �COOH then reduces

the strain around the a-carbon and produces an iminium ion,

the a1 ion of a-methylmethionine. The other pathways involve

migration of a proton either from the g-carbon to the amino

group or to the carbonyl oxygen, or from the S-methyl group

to the same two receiving groups (Scheme 2). N-protonated

ions with the radical on the side chain eliminate NH3,

probably by nucleophilic attack by either the sulfur or the

carbonyl oxygen. Loss of methyl vinyl sulfide or a cyclic

thioether leads to the formation of the captodative a-radicalcation of alanine, H2NC�(CH3)C(OH)2

+. DFT investigations

are discussed below in section 3.

3. Computational investigations

(a) Methionine. DFT calculations at the UB3LYP/

6-311++G(d,p) level of theory were employed in a systematic

examination of the structures and fragmentation mechanisms.

Fig. 8a–c show the potential energy surfaces (PESs) for the

reactions shown in Scheme 1. The most stable canonical

structure, Met�+-1, formed by dissociative electron transfer

from [CuII(Met)(CH3CN)2]�2+(CS), has the majority of its

charge and spin on the sulfur atom. The fairly long and flexible

side chain permits the sulfur radical to make an intramolecular,

two-center-three electron hemibond to the N atom, thereby

forming a five-membered ring. The calculated S–N distance is

2.574 A; extensive spin delocalization is evident with 64% of

the spin on the sulfur and 33% of the spin on the nitrogen

atom. Migration of the a-hydrogen to the sulfur via TS-1

produces Met�+-2a, a distonic radical cation with the radical

formally located on the a-carbon and charge on the sulfur.

Met�+-2a is higher in enthalpy (DH10) than Met�+-1 by

10.0 kcal mol�1, and the barrier (DHz0) against the 1,4-H

atom transfer via TS-1 is 13.1 kcal mol�1. One possible

ensuing step, migration of a proton from the sulfur to the

OH group, followed by the loss of a H2O molecule via TS-2 to

give the [b1 � H]�+ ion, has a barrier of 20.2 kcal mol�1. The

overall dissociation reaction from Met�+-1 to [b1 � H]�+ and

water is endothermic by 8.4 kcal mol�1. The loss of �CH3 from

[b1 � H]�+ to give the iminium ion at m/z 116 is 30.5 kcal mol�1

above [b1 � H]�+ (Fig. 8a).

Rotation about the Ca–C bond in Met�+-2a plus proton

transfer from the sulfur to the carbonyl oxygen gives the ion at

Fig. 6 CID spectrum of S-methylcysteine radical cation produced in

MS3 using linear ion trap on API 2000. * signifies the precursor ion.

Fig. 7 CID spectrum of a-methylmethionine radical cation produced

in MS3 using linear ion trap on API 2000. * signifies the precursor ion.


the global minimum, Met�+-3, a captodative structure in

which the radical is stabilized by the adjacent electron-donating

NH2 group and electron-withdrawing C(OH)2+ group. The

barrier against formation of Met�+-3 is 27.0 kcal mol�1,

relative to Met�+-1. Transfer of the proton back to the

S atom, followed by nucleophilic attack by the carbonyl

oxygen on the g-carbon and with concomitant elimination of

CH3SH has an activation enthalpy of 30.9 kcal mol�1. Sam-

pling and mass-isolating ions for CID in our mass spectro-

meters involve a large number of collisions and conditions

under which isomerism with low-energy barriers can readily

take place. These conditions favor formation of the global

minimum structure, especially one that is surrounded by

relatively high reaction barriers. Met�+-3 meets this requirement.

Upon collisional activation, Met�+-3 can isomerize to give

Met�+-2a, which can then dissociate to give [b1 � H]�+ and

water; the barrier against this reaction is 32.0 kcal mol�1

(relative to Met�+-3). Alternatively, collisionally activated

Met�+-3 can also dissociate to give the ion at m/z 101 and

thiomethanol. This latter reaction has a higher barrier of

35.9 kcal mol�1. These results are consistent with the experiment

on the a-deuterium labeled ion where eliminations of HDO

(product ion m/z 131) followed by the loss of �CH3 (product

ion m/z 116) and CH3SD (product ion m/z 101) (Fig. 5b and

5c–2) were observed.

The mechanism of the scrambling reaction proposed in

Scheme 1 is shown in Fig. 8b. For Met�+-2a, rotation about

the Cg–S bond leads to rotamer Met�+-4a and the activation

barrier against this interconversion tautomeric reaction is

14.9 kcal mol�1, only 1.8 kcal mol�1 higher than that for the

1,4 a-hydrogen migration (vide supra). The proton (or deuteron

for Met-2-d1) transfer then occurs from the sulfur to the

amino group, giving the distonic structure Met�+-5a which

is 1.6 kcal mol�1 lower in enthalpy than Met�+-2a. This

process is barrierless on the enthalpy surface. Note that

Met�+-2a can be reformed from Met�+-5a using a different

proton on the amino group, thereby leading to scrambling.

The barriers against the proton-switching reactions are

relatively low (6.5 kcal mol�1 relative to Met�+-5a) compared

with those leading to dissociations in Fig. 8a; hence, there is a

large amount of scrambling.

Fig. 8c shows additional scrambling reactions, which

involve a methyl hydrogen on the sulfur. Starting with

Met�+-1, a 1,6-H+ shift from the CH3 group leads to the

distonic Met�+-6, via a barrier of 15.6 kcal mol�1. In the

second step, the a-deuterium atom undergoes a 1,5-H shift to

the carbon radical center S-�CH2 via TS-8 to give Met�+-5b,

whose subsequent isomerization to Met�+-2c is facile. Ion

Met�+-2c then eliminates H2O followed by the losses of

CH2D� and CH2DSH in the CID of (Met-2-d1)

�+ (Fig. 5b

and 5c–1).

From the fragmentation mechanisms discussed above, there

are significant rearrangements prior to dissociation of Met�+.

Canonical Met�+-1 formed in the CID of [CuII(Met-2-d1)-

(CH3CN)2]�2+ isomerizes to Met�+-2a–c, ions that have the

deuterium in three different locations. It is of note that the

Scheme 2


abundance of the product ion at m/z 132 (via the loss of H2O)

is higher than that at m/z 131 (via the loss of HDO), in

accordance with the interpretation of extensive scrambling

(Fig. 5b). In addition, the higher barrier against the loss of

CH3SH is consistent with the experimental observation that

the loss of H2O is more prevalent than that of CH3SH

(Fig. 5a).

(b) S-Methylcysteine. As shown in Fig. 6, (S-methyl-Cys)�+

fragments prominently to give the dehydroalanine radical

Fig. 8 Reaction profiles for dissociation of the methionine radical cation at the UB3LYP/6-311++G(d,p) level of theory (energies are DH10 in

kcal mol�1).


cation, NH2�+C(QCH2)COOH (m/z 87 Th), by the loss of

CH3SH. The calculated reaction profile is shown in Fig. 9a.

Starting with the lowest-energy canonical structure,

(S-methyl-Cys)�+-1, migration of the a-hydrogen to the sulfur

creates a distonic ion (S-methyl-Cys)�+-2, in which the charge

is formally located on the sulfur and the radical center on Ca;

this structure has a long Cb–S distance (2.534 A) and is

essentially H2N�+C(QCH2)COOH solvated by CH3SH. The

barrier against the 1,3-hydrogen shift is 19.1 kcal mol�1,

slightly higher than that of the 1,4-hydrogen shift in

Met�+(13.1 kcal mol�1, Fig. 8a). Dissociation of the

(S-methyl-Cys)�+-2 complex to give H2N�+C(QCH2)COOH

and CH3SH is endothermic by 11.3 kcal mol�1. Collisionally

activated (S-methyl-Cys)�+ will give the products in one step,

as the barrier (19.1 kcal mol�1) is at a higher energy than the

products.

The energy profile leading to the loss of NH3 from

(S-methyl-Cys)�+-1 is given in Fig. 9b. The proton source is

the b-CH2 group, made acidic by the adjacent positively

charged sulfur. The migration takes place via two 1,4-proton

shifts, involving the carbonyl oxygen as a catalyst, and with

the nitrogen being the destination; heterolytic cleavage of the

N–Ca bond then ensues, giving CH3–S�+–CHQCH–COOH

at m/z 118 and NH3. The critical barrier on this pathway is at

22.7 kcal mol�1. In comparison, the critical barrier against a

direct 1,3-H+ shift from the b-carbon to the amino nitrogen is

27.4 kcal mol�1 (see ESIw, Fig. S1). We also investigated

the reaction channel for migration of a proton from the

methyl group to the NH2 group. A higher energy barrier,

32.1 kcal mol�1, was found, making this pathway energetically

uncompetitive (see ESIw, Fig. S1).

(c) a-Methylmethionine. The reaction profile for a-methyl-

methionine is shown in Fig. 10a. Starting from the lowest-

energy structure, canonical (a-methyl-Met)�+-1, migration of

a proton from the g-carbon to the NH2 group via TS-13

produces distonic (a-methyl-Met)�+-2, in which the charge

resides formally on the nitrogen and the radical on the

g-carbon. The barrier against this 1,4-proton shift is

15.4 kcal mol�1. Extension of the N–Ca bond results in

elimination of NH3 with a barrier of 29.1 kcal mol�1 (relative

to (a-methyl-Met)�+-1). The barrier against a subsequent loss

of water via 1,4-proton migration from the b-carbon to the

OH group is much higher at 52.9 kcal mol�1, consistent

with the observation of low abundance for the resulting ion

at m/z 128 (Fig. 7). We also investigated the viability of

Fig. 9 Reaction profile for dissociation of the S-methylcysteine radical cation at the UB3LYP/6-311++G(d,p) level of theory: (a) the loss of

CH3SH and (b) the loss of NH3 (energies are DH10 in kcal mol�1).


transferring an S-methyl proton to the amino group and losing

NH3 (see ESIw, Fig. S2). The barrier against abstracting a

proton from the methyl group to give (a-methyl-Met)�+-5 is

low at 15.6 kcal mol�1, i.e., comparable to that against the

1,4-proton shift described above. However, the barrier

against the elimination of NH3 from (a-methyl-Met)�+-5 is

42.6 kcal mol�1, making this pathway uncompetitive.

(a-methyl-Met)�+-1 can also fragment by losing �COOH to

give an iminium ion at m/z 118. The barrier against that

reaction is 26.1 kcal mol�1, slightly lower than that for NH3

loss; this is contrary to the observed abundance for the two

losses, but it is well within the accuracy in the DFT calculations.

There are two possible reaction pathways that can lead to

the product ion Ala�+ atm/z 89 (Fig. 10b); both involve losing

the a-methylmethionine side chain as CH2QCH–S–CH3.

Starting from (a-methyl-Met)�+-2, cleavage of the Ca–Cb

bond yields a distonic Ala�+ in which the charge resides on

the NH3 group and the radical on the a-carbon. The critical

energy is the endothermicity of this dissociation reaction, at

34.8 kcal mol�1; this is 5.7 kcal mol�1 higher than the barrier

against the elimination of NH3 (see Fig. 10a). An alternative

and more competitive pathway is initiated by migration of a

proton on the g-carbon of (a-methyl-Met)�+-1 to the carbonyl

oxygen to create isomer (a-methyl-Met)�+-3, in which

the charge is on the C(OH)2 group and the radical on the

g-carbon. The barrier against this 1,5-proton shift is the

endothermicity at 26.9 kcal mol�1. Subsequent loss of

CH2QCH–S–CH3 has a barrier of 28.4 kcal mol�1 and

gives the lowest-energy isomer of Ala�+, which is stabilized

captodatively. Parenthetically, it is also of note that the

isomerization from distonic-Ala�+ to captodative-Ala�+ has

a barrier of only 5.2 kcal mol�1 (details not shown).

We have also examined the reaction profile for migration of

a proton from the S-methyl group to the carbonyl oxygen,

followed by the loss of the side chain as trimethylene sulfide.

The barrier against this 1,7-proton shift is 32.8 kcal mol�1,

4.4 kcal mol�1 higher than that against the aforementioned

1,5-proton shift that eliminates the side chain as

Fig. 10 Reaction profiles for dissociation of the a-methylmethionine radical cation at the UB3LYP/6-311++G(d,p) level of theory: (a) the loss

of NH3 and the �COOH and (b) the losses of CH2QCH–S–CH3 (energies are DH10 in kcal mol�1).


CH2QCH–S–CH3, which strongly suggests that this new

pathway is uncompetitive.

Conclusions

The collision-induced dissociations of [Cu(M)(CH3CN)2]�2+

complexes (M = methionine, a-methylmethionine, and

S-methylcysteine) produce abundant M�+ radical cations.

The low-energy CID spectra of the radical cations show

interesting rearrangement and fragmentation reactions. DFT

shows that the methionine radical cation isomerizes to an

a-centered radical before undergoing water loss. By contrast,

in the S-methyl-cysteine radical cation, the a-hydrogenmigrates to the sulfur, leading to the facile loss of CH3SH.

The fragmentation of (a-methyl-Met)�+ is entirely different

from that of Met�+, resulting in the loss of NH3, H2O,�COOH, and the side chain, the last of which gives the alanine

radical cation. DFT shows that abstracting a proton from the

g-carbon by either the amino group followed by NH3 loss, or

by the carbonyl group followed by CH2QCH–S–CH3 loss,

as well as the loss of �COOH are energetically favorable

processes.

Acknowledgements

This research was made possible by funding from the Natural

Sciences and Engineering Research Council (NSERC) of

Canada, and by the facilities of the Shared Hierarchical

Academic Research Computing Network (SHARCNET:

http://www.sharcnet.ca).

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