The methoxymethyl cation cleaves peptide bonds in the gas phase

Article abstract of DOI:10.1039/a703335d

Methoxymethyl cations and simple N-acyl amino acids and dipeptides react in the gas phase to form [M + MeOCH₂]⁺ ions which fragment via a number of pathways including amide bond cleavage.

Full text of DOI:10.1039/a703335d

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The methoxymethyl cation cleaves peptide bonds in the gas phase†
Michael A. Freitas,a Richard A. J. O’Hair,*a Suresh Duab and John H. Bowieb
a School of Chemistry, The University of Melbourne, Parkville, Victoria 3052, Australia
b Department of Chemistry, The University of Adelaide, Adelaide, S.A. 5005, Australia
+
Methoxymethyl cations and simple N-acyl amino acids and
dipeptides react in the gas phase to form [M + MeOCH2]+
ions which fragment via a number of pathways including
amide bond cleavage.
MeOCH2 , collisional activated mass-analysed ion kinetic
energy (CA-MIKE) spectra were carried out on each of the [M
+ MeOCH2]+ ions using argon as the collision gas (at a pressure
of 2 3 1026 Torr).
The first model systems we chose to study were the N-acyl
derivatives of glycine and alanine (Table 1).‡ The [M +
MeOCH2]+ ions of these systems both fragment via loss of
CH2CO and MeCO2CH3. These losses are best rationalized as
occuring via attack at the amide nitrogen atom to give a nitrogen
adduct which then undergoes amide bond cleavage as shown in
eqn. (4) [c.f. eqn. (3)] and eqn. (5) [c.f. eqn. (1)]. Further
A major goal in modern mass spectrometry is the development
of techniques to structurally characterize molecules of biolog-
ical significance. As part of our ongoing research into the gas
phase electrophilic modification of biomolecules and model
compounds,1,2 we became interested in designing reagents to
sequence biomolecules via specific gas phase ion–molecule
reactions. Thus for peptides, the ultimate aim is to develop site
specific ion–molecule reactions to cleave the peptide bonds in
an analogous fashion to solution phase chemistry such as the
Edman technique.3–5 Although at first glance such an effort may
seem ambitious, we were encouraged during a recent flowing
afterglow study on the rates of ion–molecule reactions of the
methoxymethyl cation. In particular, we rediscovered a reac-
tion6 which leads to products arising from amide bond cleavage
[eqns. (1)–(3)].
Me
O
C
O
–CH2CO
–AcOMe
[MeOCH2NHCH2CO2H + H]+
(4)
(5)
+
N
CH2
Me
+
CH2 NHCH2CO2H
H
CH2CO2H
adduct from
N attack
products due to loss of MeOCH2OH, [MeOCH2OH + CO] and
HCO2Me (this loss might arise from hydride ion transfer
followed by a proton transfer8) are also observed which may
arise from initial attack at the C terminus to give an oxygen
adduct which then undergoes the fragmentations shown in eqns.
(6)–(9). Thus the methoxymethyl cation not only cleaves amide
+
CH2 NR2
+
R1CO2Me
(1)
2
O
OMe
CH2 NR2
+
R1
+ MeOCH2
+
+
(2)
R1
NR2
OR
2
R1, R2 = H and Me
[MeOCH2NR22 + H]+ + [R1CO–H+] (3)
–MeOCH2OH
[AcNHCH2CO]+
[AcNH=CH2(MeOCH2OH)]+ (7)
Me
(6)
O
C
O
–CO
+
CH2
Here we present preliminary results designed to address the
question: can the methoxymethyl cation be used to cleave
peptide bonds in the gas phase? Experiments were performed on
a VG ZAB-2HF reverse sector tandem mass spectrometer.7
Methoxymethyl cations, which were generated in the chemical
ionization source using MeOCH2CH2OMe as the CI gas, were
allowed to react with peptides and model systems introduced
via a direct insertion probe. Typical source conditions were:
source temperature = 250 °C; electron energy = 70 eV;
emission current = 200 mA; source pressure = 7 3 1024 Torr,
measured on the source ion gauge. In each case, three major
alkylated product ions [M + MeOCH2]+, [M + Me]+ and [M +
CH]+ were formed, consistent with previous studies.2b,6 In order
to gain some insights into the regioselectivity of attack by
AcNHCH2
O
H
–MeOCH2OH, –CO
[AcNH=CH2]+
[AcNHMe + H]+
(8)
(9)
adduct from
O attack
–HCO2Me
bonds, but also cleaves the C–OH bond of the carboxylic acid
group.
We next turned our attention to the [M + MeOCH2]+ ions of
the free dipeptides glycylglycine, glycylalanine and alanylgly-
cine, which all only fragment to form [M + CH]+ ions as the
major product with [M + Me]+ ion formation as a minor reaction
channel (Table 1). The lack of attack by the methoxymethyl
Table 1 CID MS/MS Spectra of [M + MeOCH2]+ ions of peptides and model systems
Neutral species (m/z of [M + MeOCH2]+)
Daughter ions (intensity)a
MeC(O)NHCH2CO2H (162)
132(54), 130(100), 120(2.5), 100(2.5), 88(5), 74(5), 72(17), 45(1)
133(15), 131(100), 121(2), 101(1.5), 89(3.5), 75(2), 73(5), 45(1)
134(21), 130(100), 122(3), 102(2), 90(5), 76(5), 74(9), 45(1)
146(24), 144(100), 134(1.5), 114(16), 102(11), 86(4), 45(*)
147(2), 145(100)
161(2), 159(100)
161(3), 159(100)
201(4), 189(6), 187(100), 176(*), 157(*), 145(2), 120(1), 88(1)
MeC(O)15NHCH2CO2H (163)
MeC(O)NHCD2CO2H (164)
MeC(O)NHCHMeCO2H (176)
H2NCH2C(O)NHCH2CO2H (177)
H2NCH2C(O)NHCHMeCO2H (191)
H2NCHMeC(O)NHCH2CO2H (191)
MeC(O)NHCH2C(O)NHCH2CO2H (219)
a (*) Designates an ion with an intensity of less than 1.
Chem. Commun., 1997
1409

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[Y1 + MeOCH2]+
and
[Y1 + CH]+
cation onto the peptide bond with concomitant fragmentation is
consistent with the known gas phase reactivity of glycine2a as
well as the basicity of the various functional groups within
dipeptides. For example, ab initio calculations have shown the
following basicity order for glycylglycine: N-terminal amino
group > oxygen of amide group > nitrogen of amide group >
carboxyl carbonyl oxygen).9
[Y2 + CH]+
1
2
3
O
C
O
C
Ac
N
H
CH
R2
N
H
CH
R1
OH
In order to make the peptide bonds in these dipeptides more
+
susceptible to attack by the MeOCH2 ion, we have examined
B3
the effects of moderating the gas phase reactivity of the
N-terminal amino group via N-acetylation. Thus the [M +
MeOCH2]+ ions of N-acetyl derivatives‡ of glycylglycine,
glycylalanine and alanylglycine not only decompose to form [M
+ CH]+ and [M + Me]+ ions, but more importantly, several other
product ions resulting from amide bond cleavage were observed
(Table 1 and Fig. 1). In particular, the CA-MIKE spectra of the
[M + MeOCH2]+ ions of these N-acetyl derivatives indicate that
attack by the methoxymethyl cation onto the heteroatom
adjacent to a CNO group followed by bond cleavage are general.
Thus attack at all three sites (labelled as 1–3 in Fig. 2) occurs,
thereby providing complete sequence information. Conse-
quently distinction between N-acetylglycylalanine and
N-acetylalanylglycine is readily made (Fig. 1). Interestingly,
cleavage of the peptide bonds in these instances gives rise to
fragments ions (Fig. 2) where the charge is retained at the C
terminus (i.e. modified Y ions such as [Yn + CH]+ and [Y1 +
CH3OCH2]+ are formed), while the only fragment ion in which
the charge is retained at the N terminus is that due to cleavage
of the C terminus OH bond to give a B3 ion [for sequence ion
nomenclature, see ref. 4(a)]. Thus not only does the methox-
ymethyl cation cleave the peptide bonds, but it cleaves them in
a specific fashion, thereby simplifying the mass spectra.
Fig. 2
Further work is required to determine the general utility of
these reactions to sequence small peptides in the gas phase via
cleavage of their peptide bonds (the inherent limitations of
volatility of the peptide could potentially be overcome using
laser desorption CI10). In particular the effects of residues with
potentially reactive side chains (e.g. lysine, aspartic acid etc.)
need to be investigated. Future studies will also examine the
+
modes of reactivity of other reagent ions such as ClCH2 .
R. A. J. O. and J. H. B. both thank the ARC for financial
support. M. F. thanks the School of Chemistry at the University
of Melbourne for a Research Fellowship.
Footnotes
* E-mail: r.ohair@mail.chemistry.unimelb.edu.au
† Part 8 of the Series ‘Gas Phase Ion Chemistry of Biomolecules’.
‡ N-Acetyl derivatives were prepared via a modified literature procedure
(D. P. Knapp, Methods Enzymol., 1990, 193, 314): The a-amino acid or
dipeptide (0.002 mol) was dissolved in 2 ml of 2 m NaOH and chilled in an
ice bath. Upon complete dropwise addition of acetic anhydride (2 ml), the
mixture was stirred for a further 10 min followed by careful acidification to
pH
< 4 with H2SO4. The solution was allowed to warm to room
temperature and extracted with EtOAc (3 3 25 ml). The combined EtOAc
layers were dried with anhydrous Na2SO4 and the solvent was removed in
vacuo. The resultant N-acetyl derivatives were purified via recrystallization
from MeOH. All other compounds were of reagent grade obtained
commercially and were used without further purification. H215NCH2CO2H
(99 atom% 15N) and H2NCD2CO2H (98 atom% D) were both obtained from
Cambridge Isotope Laboratories.
233
(a)
201
× 5
+
[Y + CH]
1
102
References
1 M. A. Freitas, R. A. J. O’Hair, J. A. R. Schmidt, S. E. Tichy,
B. E. Plashko and T. D. Williams, J. Mass Spectrom., 1996, 31,
1086.
2 For gas phase reactions of MeOCH2+ with glycine and cysteine, see (a)
R. A. J. O’Hair, M. A. Freitas, S. Gronert, J. A. R. Schmidt and
T. D. Williams, J. Org. Chem., 1995, 60, 1990; (b) M. A. Freitas,
R. A. J. O’Hair and T. D. Williams, submitted to J. Org. Chem.
3 Sequencing of Proteins and Peptides, in Laboratory Techniques in
Biochemistry and Molecular Biology, 2nd edn., ed. G. Allen, Elsevier,
Amsterdam, 1989, ch. 6.
4 Mass spectrometry has been used to sequence small peptides. For some
leading references, see (a) K. Biemann and I. A. Papayannopoulos, Acc.
Chem. Res., 1994, 27, 370; (b) R. J. Waugh and J. H. Bowie, Rapid
Commun. Mass Spectrom., 1994, 8, 169.
5 The only other report of a peptide bond cleavage reaction involved
reacting the [M 2 H]2 ion of some peptide models [MeC(O)NHCH2-
C(O)X; X = OEt and NHMe] with a neutral electrophile: X. Cheng and
J. J. Grabowski, Proceedings of the 39th ASMS Conference on Mass
Spectrometry and Allied Topics, Nashville, Tennesee, May 19–24,
1991, p. 477.
+
[Y + CH]
2
+
B
171
[Y + MeOCH ]
3
1
2
159
134
190
233
201
(b)
× 5
+
[Y + MeOCH ]
1
2
120
6 The methoxymethyl cation cleaves the acyl bond in acids, esters and
amides: M. C. Caserio and J. K. Kim, J. Org. Chem., 1982, 47, 2940.
7 K. L. Busch, G. L. Glish and S. A. McLuckey, Mass Spectrometry/Mass
Spectrometry. Techniques and Applications of Tandem Mass Spectrom-
etry, VCH, New York, 1988.
8 H. E. Audier, F. Dahhani, A. Millet and D. Kuck, Chem. Commun.,
1997, 429.
9 K. Zhang, D. M. Zimmerman, A. Chung-Phillips and C. J. Cassady,
J. Am. Chem. Soc., 1993, 115, 10 812.
+
[Y + CH]
2
159
190
+
[Y + CH]
1
B
3
171
88
10 See for example: J. P. Speir and I. J. Amster, J. Am. Soc. Mass
Spectrom., 1995, 6, 1069.
m / z
CA-MIKE spectra of the [M
N-acetylglycylalanine and (b) N-acetylalanylglycine
Fig.
1
+
MeOCH2]+ ions of (a)
Received in Cambridge, UK, 14th May 1997; 7/03335D
1410
Chem. Commun., 1997