Participation of C-H Protons in the Dissociation of a Proton Deficient Dipeptide

Article abstract of DOI:10.1007/s13361-017-1662-7

The dissociation of anionic dipeptides Phe*Gly and GlyPhe*, where Phe* refers to sulfonated phenyl alanine, has been investigated by using ion trap mass spectrometry. The dipeptides undergo collision-induced dissociation (CID) to give the same products, indicating that they rearrange to a common structure before dissociation. The rearrangement does not occur with the dipeptide methyl esters. The structures of the b₂ ions were investigated to determine the effect that having a remote, anionic site has on product formation. Comparison with the CID spectra for authentic structures shows that the b₂ ion obtained from GlyPhe* has predominantly a diketopiperazine structure. The CID spectra for the Phe*Gly b₂ ion and the authentic oxazolone are similar, but differences in intensity suggest a two-component mixture. Isotopic labeling studies are consistent with the formation of two products, with one resulting from loss of a non-mobile proton on the Gly α-carbon. The results are attributed to the formation of an oxazole and oxazolone enol product. Electronic structure calculations predict that the enol structure of the Phe*Gly b₂ ion is lower in energy than the keto version due to intramolecular hydrogen bonding with the sulfonate group. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s13361-017-1662-7

B American Society for Mass Spectrometry, 2017
J. Am. Soc. Mass Spectrom. (2017) 28:1313Y1323
DOI: 10.1007/s13361-017-1662-7
FOCUS: BIO-ION CHEMISTRY: INTERACTIONS OF BIOLOGICAL IONS
WITH IONS, MOLECULES, SURFACES, ELECTRONS, AND LIGHT: RESEARCH ARTICLE
Participation of C-H Protons in the Dissociation of a Proton
Deficient Dipeptide
Damodar Koirala, Sabyasachy Mistry, Paul G. Wenthold
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47906, USA
NH2
Abstract. The dissociation of anionic dipeptides Phe*Gly and GlyPhe*, where Phe*
refers to sulfonated phenyl alanine, has been investigated by using ion trap mass
spectrometry. The dipeptides undergo collision-induced dissociation (CID) to give the
same products, indicating that they rearrange to a common structure before dissoci-
ation. The rearrangement does not occur with the dipeptide methyl esters. The
structures of the b2 ions were investigated to determine the effect that having a
remote, anionic site has on product formation. Comparison with the CID spectra for
authentic structures shows that the b2 ion obtained from GlyPhe* has predominantly
a diketopiperazine structure. The CID spectra for the Phe*Gly b2 ion and the authentic
oxazolone are similar, but differences in intensity suggest a two-component mixture.
N
O
D
-HOD
O
H
N
O
H
_
SO3
O
HO
D D
NH2
-H
_
NH2
2O
N
D
D
O
_
SO3
O
SO3
Isotopic labeling studies are consistent with the formation of two products, with one resulting from loss of a non-
mobile proton on the Gly α-carbon. The results are attributed to the formation of an oxazole and oxazolone enol
product. Electronic structure calculations predict that the enol structure of the Phe*Gly b2 ion is lower in energy
than the keto version due to intramolecular hydrogen bonding with the sulfonate group.
Keywords: Dipeptide dissociation, b2 Ions, Charge remote fragmentation, Canonical structures,
Diketopiperazine, Oxazolone
Received: 17 November 2016/Revised: 13 March 2017/Accepted: 14 March 2017/Published Online: 20 April 2017
libraries of mass spectra can be used to identify known pro-
Introduction
teins, amino acid sequences in unknown proteins can be deter-
mined by de novo analysis of ionic fragments. In particular,
fragments observed in peptide dissociation, especially b- and y-
type sequence ions resulting from fragmentation of amide
bonds, can be used to determine the sequence from the C-
and N-terminus [2–8].
he significance of amino acids and peptides to the world
around us, and to life itself, cannot be overstated, and,
T
therefore, it is not surprising that their structures, properties,
and reactivity are exceptionally well-characterized, largely due
to the capabilities of mass spectrometry and electrospray ion-
ization [1]. The ability for rapid, accurate analysis of proteins
and peptides by mass spectrometry has had a major impact on
the field of proteomics, and allows for the study of the struc-
tures and functions of large proteins in complex biological
system.
Products obtained upon dissociation of protonated peptides
have generally been explained in terms of the Bmobile proton
theory^ [9–12]. The model assumes that protons, initially
localized on the most basic sites (N-terminus and the side
chains of basic amino acid residues) of protonated peptides,
can be transferred to the less basic of sites upon activation,
including the various peptide linkages, thereby producing a
heterogeneous population of protonated peptides [9, 10, 12].
The mobile proton model provides a quantitative framework
such that if the peptide sequence and number of protons are
known, one can predict the general appearance of the fragmen-
tation spectrum [12]. Although the mobile proton model is not
intended to model the full peptide fragmentation spectrum
quantitatively, it successfully accounts for common dissocia-
tion, including aforementioned b- and y-type ions [13–15].
One of the most important features of using mass spectrom-
etry to identify proteins and peptides in proteomics is the ability
to use tandem mass spectrometry (MSn) to differentiate isobar-
ic ions and for sequence determination. Therefore, whereas
Electronic supplementary material The online version of this article (doi:10.
1007/s13361-017-1662-7) contains supplementary material, which is available
to authorized users.
Correspondence to: Paul Wenthold; e-mail: pgw@purdue.edu

1314
D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
Because peptide b ions are common, stable, species in
The nature of the charge on peptides also plays an im-
portant role in the dissociation mechanism. Mass spec-
trometry studies of peptide and peptide’s dissociation
usually involve the use of protonated [13] or metallated
[36, 52] peptides, often multiply charged depending on
the number of basic sites. Therefore, the excess positive
charge allows for facile intramolecular proton transfer that
does not require, inter alia, formation of salt-bridges or
other forms of charge separation. In a recent study, we
reported the formation and dissociation of sulfonated phe-
peptide fragmentation [16], a better understanding of
how they are formed can improve peptide sequencing
algorithms and the current models for peptide fragmen-
tation [9]. Consequently, several theoretical and experi-
mental studies have been carried out in recent years to
better understand the structures of b ions and the mech-
anism of their formation [16–35], In this work, we use
dipeptides with backbone charges to investigate the for-
mation of b2 ions for canonical structures.
–
Many structures have been proposed for b2 ions [12],
Initially, an acylium ion was thought to be the major b2
ion structure, but it was later discovered to be thermody-
namically less stable than its isomeric cyclic structures [32].
Of the structures proposed for b2 ions, the most common
are protonated diketopiperazines and protonated oxazolones
[28, 35–49]. Six-membered cyclic diketopiperazine is
formed when the N-terminal amino group acts as a nucle-
ophile and attacks at the carbonyl carbon of second amino
acid residue. In contrast, a five-membered cyclic oxazolone
is formed when the N-terminal carbonyl oxygen acts as a
nucleophile and attacks at the carbonyl carbon of second
amino acid residue. Which ion forms is mainly determined
by the structure of the peptide (Equation 1). A cis confor-
mation of the first amide bond favors diketopiperazine
because it facilitates the nucleophilic attack by amino group
of N-terminal amino acid (Equation 1a), whereas a trans
conformation of the first amide bond favors oxazolone
(Equation 1b) because it facilitates the nucleophilic attack
by carbonyl oxygen of N-terminal amino acid [13, 45–48,
50, 51]. Although diketopiperazine structures are generally
lower in energy [47], the oxazolone structures result for
trans amides because the barrier for the formation is lower
than the barrier for cis-trans isomerization.
nylalanine, PheSO3 , an anionic amino acid where the
charge is isolated from the α-amino acid moiety by a
phenyl spacer [53]. The ion dissociates only by loss of
NH3 to make a product assigned to be an α-lactone
(Equation 2) [54]. The proposed mechanism involves
proton transfer within the canonical amino acid to form
a zwitterionic intermediate, which dissociates by intramo-
lecular substitution to make the lactone (Equation 2), and
therefore nominally illustrates the potential of mobile
electron within anionic systems as well. However, an
important feature of all these reactions is that although
energetically unfavorable proton transfer can occur in
these reactions upon activation, it is still generally limited
to protons on heteroatom positions, such as amines, amide
nitrogen, or carboxyl groups.
In this work, we have used mass spectrometry to examine
the dissociation of dipeptides that include PheSO3ˉ,
namely GlyPhe*OH and Phe*GlyOH, where Phe* refers
to sulfonated Phe, PheSO3ˉ. Unlike protonated and
metallated ions, these dipeptides are anionic and hence
proton deficient. However, because the sulfonate charge is
more stable than a carboxylate, and the charge is localized
on the side chain, the dipeptides are predicted to have
canonical structures [53]. In this respect, they are similar
to protonated dipeptides containing His [47, 49, 50, 55,
56] or Arg [27, 29, 35, 57], which contain basic side
chains that sequester positive charge. We find that the
sulfonated dipeptides dissociate similar to what has been
reported for ArgGly and other dipeptides, with the forma-
tion of characteristic sequence ions, such as b2 ions.
However, unlike what is typically found for protonated
dipeptides, we find that b2 ion formation in the sulfonated

D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
1315
dipeptides involves not only loss of exchangeable protons
on nitrogen and oxygen but also loss of protons from
carbon, which are generally not considered within the
mobile proton model.
of second residue amino acid methyl ester (R2-OMe) was
added. After stirring for an additional 40 min, the excess
DCM was evaporated in vacuum. Formation of the dipep-
tide product (BocR1-R2OMe) was confirmed by mass spec-
trometry. The Boc group was removed by dissolving the
product in excess trifluoroacetic acid (TFA) for 20 min,
followed by evaporation of the TFA in vacuum, resulting
in formation of the methyl ester. The ester, R1-R2OMe, can
be hydrolyzed to R1-R2OH by treating their aqueous solu-
tion with concentrated sulfuric acid. The synthesis of
GlyPhe*OMe is shown in Scheme S1 in the Supplementary
Material.
Experimental
Mass spectrometry experiments were carried out using a com-
mercial LCQ-DECA (Thermo Electron Corporation, San Jose,
CA, USA) quadrupole ion trap mass spectrometer, equipped
with electrospray ionization (ESI) source. Samples were dis-
solved in a methanol:water mixture (1:1) and introduced into
the source at a flow rate of 10 μL/min. Electrospray and ion
focusing conditions were varied so to maximize the signal of the
ion of interest. Dissociation of ions was carried out by using MSn
experiments with mass-selected ions in the cell, with the helium
buffer serving as the collision target. Reactant ions for CID were
isolated at qz = 0.250 and with a mass-width sufficient to avoid
off-resonance excitation of the mass-selected ions. The energy of
collision-induced dissociation (CID) in the cell is reflected in the
Bnormalized collision energy,^ which ranges from 0 to 100%.
Synthesis of Diketopiperazine
Diketopiperazine (I) was synthesized using the approach
described previously [58]. Briefly, the solution containing
50 mg of Boc-GlyPhe*OMe and 10 mL formic acid
(98%) was stirred at room temperature for 2 h (Equa-
tion 4). Excess formic acid was removed in vacuo and
the crude product was dissolved in 15 mL of sec-butyl
alcohol and 5 mL of toluene. The solution was boiled
for 3 h and the solvent level maintained by addition of
fresh butanol. After concentrating the solution to 5–10
mL and cooling to 0 °C, the product was obtained by
filtration.
Synthesis of Amino Acid Esters
A 50 mg sample of the amino acid was dissolved in 10 mL of
alcohol and cooled to 0 °C. Esterification was initiation by
adding 3 mL of SOCl2, dropwise, into the cold amino acid
solution. After the solution warmed to room temperature, it was
refluxed overnight to get desired amino acid ester (Equation 3).
Synthesis of GlyPhe*OH and Phe*GlyOH
Dipeptides
A sample of 0.09 mmol of Boc-Amino acid (Boc-R1) was
dissolved in 15 mL of dichloromethane (DCM), and 0.09 mmol
of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBop) was added. The solution was
cooled to 0 °C and 0.14 mmol of di-isopropylethylamine base
(DIEA) was added dropwise. After stirring for 20 min, the
solution was brought to room temperature, and 0.09 mmol

1316
D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
Synthesis of Oxazolones (GlyPhe* and Phe*Gly
Oxazolones)
dissolved in 10 mL of dichloromethane. The solution
was stirred at room temperature for 3 h. Excess solvent
was removed in vacuo and the resulting crude product
was dissolved in 3 mL TFA to produce GlyPhe*-
oxazolone (II). Similar procedure was adopted for syn-
thesis of Phe*Gly-oxazolone (III).
The syntheses of the oxazolones were carried out using
procedures described previously [59]. Briefly, the solu-
tion containing 10 mmol of Boc-GlyPhe*OH and
15 mmol of 1, 3,-dicyclohexylcarbodiimide (DCC) were
Computational Methods
[27]. As described in the introduction, protonated ArgGly/
GlyArg is structurally similar to Phe*GlyOH/GlyPhe*OH be-
cause they both contain canonical dipeptides with the charges
Geometries and electronic energies were calculated at the
B3LYP/6-31+G* level of theory. Low-energy conformations
were initially identified by using the Monte Carlo multiple
minimum search method, with the Merck molecular force field
(MMFF) as implemented in MacroModel (ver. 9.6) [60], and
unique conformations were used as starting points in B3LYP
optimizations. Single point energies for selected optimized
structures were carried out at the MP2/6-311+G** level of
theory in order to obtain a better description of van der Waals
interactions and charge polarization. Energies are not corrected
for zero-point energies or thermal energy contributions.
B3LYP and MP2 calculations were carried out using QCHEM
ver. 4.0 [61].
Results and Discussion
Analysis of GlyPhe*OH and Phe*GlyOH
The CID (MS2) mass spectra of GlyPhe*OH and Phe*GlyOH
(m/z 301) are shown in Figure 1. Both isomers give the same
products with the same intensities. Primary product ions in-
clude m/z 284 (M – NH3), m/z 283 (M – H2O), m/z 257 (M –
CO2), and m/z 244 (M – C2ONH3, net loss of glycine – H2).
Additional products can be attributed to secondary fragmenta-
tion of the primary products. In contrast, the CID spectra of the
positive ions derived from the two dipeptides are markedly
distinct (see Supplementary Material). The fact that both
GlyPhe*OH and Phe*GlyOH give the same spectra upon
negative ion CID indicates that they rearrange to a common
structure before dissociation. Rearrangement of dipeptides
upon low-energy CID has been reported previously by
Farrugia and O’Hair for protonated ArgGly and GlyArg
Figure 1. CID mass spectra for (a) GlyPhe*OH and (b)
Phe*GlyOH

D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
1317
on the side chains. Therefore, the rearrangement of
GlyPhe*OH and Phe*GlyOH dipeptides is likely similar to
what was proposed previous by Farrugia and O’Hair for
protonated ArgGly and GlyArg dipeptides [27], involving
proton transfer within the dipeptide to form zwitterionic struc-
tures, which can rearrange to cyclic structure and eventually
rearrange to form the common acid anhydride intermediate
(Figure 2). Consequently, any of the structures in Figure 2 can
be the common intermediate leading to the dissociation prod-
uct(s), with the dissociation determined by the overall lowest
energy pathway for the product [62]. Further CID of the
product ions in Figure 1a and b are also similar, confirming
that GlyPhe*OH and Phe*GlyOH give the same products
upon CID.
CID spectra of GlyPhe*OMe and Phe*GlyOMe, shown
in Figure 3, are significantly different in both the identi-
ties of the products and the intensities. Although both
ions dissociate to form products m/z 283 (M – MeOH),
m/z 266 (M – MeOH-NH3), and m/z 170, the relative
intensities are very different. In particular, whereas the
b2 ion (M-CH3OH @ m/z 283) is one of the dominate
peaks upon CID of Phe*GlyOMe, it is very weak upon
CID of GlyPhe*OMe. In addition, peaks at m/z 298, 240,
226, 198, 185, and 171 are observed solely on CID of
Phe*GlyOMe spectra, whereas m/z 266, 241, and 197 are
observed solely on CID of GlyPhe*OMe spectra.
The M – CH3OH ions observed upon CID of GlyPhe*OMe
and Phe*GlyOMe are the b2 ions. The MS3 spectra of b2 ions
obtained from GlyPhe*OMe and Phe*GlyOMe, shown in Fig-
ure 4a and b, are also different, consistent with different iso-
mers giving different products, without rearranging to a com-
mon structure. The CID spectra of the b2 ions obtained from
Phe*GlyOH and GlyPhe*OH agree with that from that obtain-
ed from Phe*GlyOMe.
Analysis of GlyPhe*OMe and Phe*GlyOMe
Support for the mechanism shown in Figure 2 comes from
the experiments involving methyl esters. Farrugia and
O’Hair [27] have shown previously that methylation of
the C-terminus carboxylic acid inhibits the rearrangement
reaction of protonated ArgGly and GlyArg, presumably
by blocking the initial proton transfer step. Similarly, we
have found that esterification of GlyPhe*OH and
Phe*GlyOH inhibits their rearrangement as well. The
Comparison to Authentic Diketopiperazine
and Oxazolones
In order to determine the structures of the b2 ions, we
have compared their CID spectra with those obtained for
the most likely possibilities, including the diketopiperazine
O
O
R
H
O
H
O
HN
R
N
H
NH2
NH2
O
O
Gly-Phe*OH
Phe*-GlyOH
Proton
Transfer
Proton
Transfer
O
O
O
R
O
R
N
H
HN
O
O
NH2
H
H2N
H
O
O
O
H
O
R
O
R
HN
N
H
NH2
NH2
H
O
O
O
NH2
R
O
NH2
Figure 2. Proposed mechanism for formation of common
structure from Phe*GlyOH and GlyPhe*OH
Figure 3. (a) CID of GlyPhe*OMe, and (b) CID of Phe*GlyOMe

1318
D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
Figure 4. CID of b2 ions (a) MS3 spectra of GlyPhe*OMe, and
(b) MS3 spectra of Phe*GlyOMe)
Figure 5. CID spectra of authentic structures of m/z 283 ions
derived from the dipeptide (a) diketopiperazine (I), (b) GlyPhe*
oxazolone II, (c) Phe*Gly oxazolone III
(I) and the oxazalones (II and III), shown in Figure 5.
There is fair agreement between the CID spectrum of the
b2 ions obtained from GlyPhe*OMe (Figure 4a) and for
that for the diketopiperazine I (Figure 5a). Fragments in
common include m/z 255 and m/z 170. However, the
relative intensities do not match, and there are peaks in
the CID spectrum of b2 ion (such as m/z 266) that are not
found for CID of the diketopiperazine. Therefore, al-
though the CID spectrum of the b2 ion obtained from
GlyPhe*OMe is partially consistent with that for I, the
presence of other CID products likely indicates an isobaric
mixture.
Material). This result is evidence that the carbonyl carbon
of glycine is at the carbonyl position in the oxazolone ring
in the b2 product, as shown in Equation 5.
The CID spectrum of the b2 ion obtained from
Phe*GlyOMe (Figure 4b) partially agrees with that for
III, the authentic oxazalone prepared from Phe*GlyOH
(Figure 5c). All the products in the CID spectrum of III
are present and there are no additional peaks in the spec-
trum of the b2 ion. These results suggest that the b2 ion
obtained from Phe*GlyOMe is the expected oxazolone.
Additional support for this assignment comes from isotopic
labeling studies. Carbonyl loss in CID of the b2 ion from
13C labeled Phe*GlyOMe, where the label is at the car-
bonyl carbon of the glycine residue, occurs solely by loss
of the carbon label (13CO and 13CO2) without any loss of
unlabeled carbonyl fragments (see Supplementary
The fact that Phe*GlyOMe, which has no acidic proton, forms
same b2 ion as that of Phe*GlyOH and GlyPhe*OH indicates
that the formation of b2 ion from Phe*GlyOMe does not
involve rearrangement such as that shown in Figure 2, suggest-
ing that for the dipeptides, GlyPhe*OH rearranges to
Phe*GlyOH before dissociation. The rearrangement from
GlyPhe*OH to Phe*GlyOH is calculated to be exothermic by
approximately 7 kcal/mol (see Supplementary Material). More

D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
1319
importantly, that the rearrangement of GlyPhe*OH occurs
before dissociation indicates that the barrier for the rearrange-
ment is lower than the dissociation energy, and the barrier for
dissociation of Phe*GlyOH is lower than that for dissociation
of GlyPhe*OH [62].
Although the spectra of the Phe*GlyOMe b2 ion and
oxazolone III are superficially similar in that they form the
same products, the relative intensities of the products do not
match. In particular, the m/z 239 peak in the spectrum of III
(Figure 5c) is much more intense than that in the spectrum of
the b2 ion (Figure 4b). It is possible that the difference in CID
intensities for the two ions may result from differences in the
manner in which they were generated. Because the b2 ion is
formed by CID whereas the authentic oxazolone comes direct-
ly from electrospray, the ions in the trap may initially have
different amounts of internal energy, which can lead to differ-
ences in product intensities.
However, further analysis of the CID spectra suggests some
patterns. For example, the ratios of m/z 239/m/z 170 intensities
in the two spectra are very similar (5.4 versus 5.9). Similarly,
the ratio of the m/z 198 and m/z 266 products are also similar
(1.7 versus 2.0). These results are consistent with the spectrum
for an isobaric mixture of two components – one that dissoci-
ates to form m/z 239 and 170 (and possibly 266 and 198), and
the other dissociates to form m/z 266 and 198 (and likely 255) –
in different proportions, depending on the source of the ion.
The formation of an oxazalone structure for the b2 ion from
Phe*Gly is not surprising. As described in the introduction,
oxazolones are expected for most b2, especially those formed
from peptides with trans amide bonds. Indeed, the lowest
energy conformations of Phe*GlyOH and Phe*GlyOMe are
calculated to have trans amide bond structures, approximately
15 kcal/mol lower in energy than the cis geometry (see
Supplementary Material). In contrast, formation of the
diketopiperazine from GlyPhe*OMe is more unexpected. Both
GlyPhe*OH and GlyPhe*OMe also have trans amide struc-
tures, with the cis structures, again, more than 10 kcal/mol less
stable than trans. Perkins et al. [49] have observed
diketopiperazine formation in the formation of HisAla b2 ions,
and initially suggested that having the charge on the backbone
may have an effect. Subsequent studies of HisAla [47] and
ArgGly [35] have also found diketopiperazine products, con-
sistent with the proposal by Perkins et al. [49]. However, the b2
ion for Phe*GlyOMe (or Phe*GlyOH) does not have
diketopiperazine structure, despite having the backbone
charge. For the trans dipeptides, Phe*GlyOH is calculated to
be ~7 kcal/mol lower in energy than GlyPhe*OH, which ac-
counts for the rearrangement of GlyPhe*OH before
dissociation.
d3-Phe*GlyOMe, Phe*Gly(2,2-d2) OMe, and d5-
Phe*GlyOMe (Figure 6). H/D exchange of the three labile
protons of Phe*GlyOMe was carried out in 50:50 D2O and
MeOD solution. Upon CID, d3-Phe*GlyOMe loses MeOD and
MeOH in a ratio of about 1:2 (Figure 6a). Loss of MeOD is
consistent with what is expected formation of oxazolone III,
with loss of methoxy from the C-terminus and loss of a mobile
proton from one of the nitrogen positions. However, loss of
CH3OH is surprising, especially as the major product, because
it requires loss of hydrogen from a carbon position, which
would not normally be considered a Bmobile^ proton lost
during dissociation. Scrambling involving hydrogen on α-
carbon has been observed previously for dissociation of b3
[63] and a3 [64] fragment ions, but has not been observed in
the simple formation of bn ions.
Although the spectrum in Figure 6a shows that protons on
carbon are involved in the dissociation, it does not specify
which carbons are involved, whether they are from an α-
carbon on Gly or Phe*, or from the aliphatic or aromatic region
of the side chain. Additional deuterium labeling experiments,
however, provide further insight. The spectrum for
Phe*GlyOMe, where the Gly is labeled with deuterium in the
α-positon (2, 2-d2), is shown in Figure 6b. Again, the ion
Deuterium Labeling
Deuterium labeling provides additional insight into the struc-
ture(s) of the product(s) of the b2 ions. In order to determine
which proton is involved in methanol loss in the formation of
the b2 ion, we examined three deuterated labeled compounds
Figure 6. CID spectra showing b2 ion formation from deuter-
ated dipeptide esters (a) d3-Phe*-GlyOMe, (b) Phe*-Gly(2,2-
d2)OMe, (c) d5-Phe*-GlyOMe

1320
D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
dissociates by loss of MeOH and MeOD, but in this case, the
intensities are reversed, with loss of MeOD being the major b2
product. In this experiment, loss of CH3OH is consistent with
formation of the oxazolone. Loss of CH3OD as the major
pathway for b2 ion formation confirms that carbon-based hy-
drogens are involved and, in particular, it is the proton on the α-
position of glycine. However, this result still does not rule out
participation of other protons, such as those at the α-position of
Phe* or those on the side chain. To test for the possibility, we
also deuterated the nitrogen positions. The CID spectrum of the
d5-Phe*GlyOMe, with deuterium at the α-position of Gly and
at all the exchangeable positions, is shown in Figure 6c. In this
case, the b2 ion is formed almost exclusively (>98%) by loss of
CH3OD. Therefore, the b2 ions are being formed either by loss
of an exchangeable proton (from nitrogen) or from a proton α-
position of the Gly, with the latter process being more promi-
nent. The fact that b2 ions are formed by multiple processes is
consistent with the conclusion in the previous section that the
b2 ions are a mixture of multiple structures.
The deuterium labeling indicates that the two products
formed involve loss of proton from nitrogen or from the Gly.
A mechanism that accounts for the observed products is shown
in Scheme 1. Nucleophilic attack of the Phe* carbonyl oxygen
at the Gly carbonyl carbon accompanied by proton transfer
leads to the hemiacetal-like structure, IV. Loss of alcohol with
proton loss from the OH in IV (proton Ha in Scheme 1) leads to
the standard oxazolone, III. In this pathway, the proton that is
lost is a mobile proton, originally on the amide nitrogen.
Alternatively, loss of alcohol from IV with the proton coming
from the ring (proton Hb) would lead instead to a hydroxy-
oxazole, IIIb, the enol structure of the oxazolone,.
Computational studies support the assignment of the enol
structure, IIIb, for the b2 ion obtained from Phe*GlyOMe.
Unlike phenols, hydroxyoxazoles are typically not more stable
than the corresponding oxazolones. At the MP2/6-31+G*//
B3LYP/6-31+G* level of theory, the simple oxazolone is
computed to be 15.4 kcal/mol lower in energy than the
hydroxyoxazole (Equation 6). However, interaction between
the hydroxy group and the sulfonate charge in the Phe*Gly b2
ion is predicted to stabilize the enol structure. The relative
energies of possible isomeric b2 ion structures obtained from
GlyPhe* and Phe*Gly are shown in Table 1. The most stable
product is predicted to be the diketopiperazine, I, and the II and
III oxazolone b2 ions are calculated to be 14 and 20 kcal/mol
higher in energy, respectively. The relative energies of the
diketopiperizine and oxazolones are consistent with what has
been found in previous studies [47, 49, 65].
Scheme 1.
Scheme 1. In contrast, the hydroxyoxazole obtained from
GlyPhe*, IIb, is significantly higher in energy. The stability
of the enol structure for the Phe*Gly b2 can be attributed to a
favorable hydrogen bond interaction between the OH and the
sulfonate group, as shown in Figure 7, and accounts for the
preference for losing proton from carbon, as opposed to from
nitrogen. The oxazolone structure II obtained from GlyPhe*
has a hydrogen bond between the N-H and sulfonate, which
accounts for its stability compared to III. The other structures
are not capable of having interactions with the sulfonate. The
optimized geometries of all the possible b2 structures are shown
in Figure 7. An oxazalone-enol structure in peptide fragmenta-
tion has been proposed previously by Paizs and co-workers
[64], but the proposed enol structure is inconsistent with the
Table 1. Calculated Relative Energies of b2 Ionsa
Ion
MP2/6-31+G*
Diketopiperizine I
0
GlyPhe* Oxazolone II
GlyPhe* Oxazolone enol IIb
Phe*Gly Oxazolone III
Phe*Gly Oxazolone enol IIIb
14
26
20
12
At the MP2/6-31+G* level of theory, the lowest energy struc-
ture, aside from the diketopiperizine, is predicted to be the
hydroxyoxazole obtained from Phe*Gly, IIIb, as shown in
a
Relative electronic energies (not corrected) in kcal/mol at B3LYP/6-31+G*
optimized geometries.

D. Koirala et al: Loss of C-H Protons in Dipeptide Dissociation
1321
Figure 7. Lowest energy structures of possible b2 ion structures
measured infrared multiphoton dissociation spectrum for the
Acknowledgements
ion [63]. This is not surprising because the enol structure is
nominally expected to be ~15 kcal/mol higher in energy than
the keto form. However, in this work the enol is stabilized
through intramolecular hydrogen bonding and is, therefore,
predicted to be a low-energy isomer. Stabilization of the b2
ion by intramolecular hydrogen bonding has been observed
previously for HisPro [65], but in that system, the hydrogen
bonding preferentially stabilized the diketopiperazine structure,
by almost 20 kcal/mol.
This work was supported by the National Science Foundation
(CHE11-11777 and CHE15-65755). Calculations were carried
out using the resources of the Center for Computational Studies
of Open-Shell and Electronically Excited Species
(iopenshell.usc.edu), supported by the National Science Foun-
dation through the CRIF:CRF program. The authors thank
Professors Ben Bythell, Vicki Wysocki, and JC Poutsma for
their valuable insights about b2 ions and other discussions. The
authors thank Scott McLuckey for his inspirational work and
leadership, and are delighted that they actually have a bio-ion
paper that they can contribute to this issue in his honor.
Conclusions
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