Research Article
Received: 30 November 2010
Revised: 21 January 2011
Accepted: 24 January 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2011, 25, 1119–1129
(wileyonlinelibrary.com) DOI: 10.1002/rcm.4965
A study of fragmentation of protonated amides of some acylated
amino acids by tandem mass spectrometry: observation of an
unusual nitrilium ion
†
‡
*
Erach R. Talaty , Sarah M. Young , Ryan P. Dain and Michael J. Van Stipdonk
Department of Chemistry, Wichita State University, Wichita, KS 67260–0051, USA
A tandem mass spectrometric study of a series of secondary amides of acetylglycine and hippuric acid utilizing
electrospray ionization (ESI) was conducted. Among the fragment⊕ions observed w⊕as an unusual one, which we have
determined to be a nitrilium ion having the structure CH3‐C≡ N‐Ph or Ph‐C≡ N‐Ph by loss of the full mass of
glycine as a neutral fragment. A mechanism that we propose involves an initial protonation of the oxygen atom at
the N‐terminus, followed by cyclization to a five‐membered imidazolium ring, and its subsequent collapse to the
nitrilium ion. This mechanism is supported by extensive isotopic labels and considerable variation of substituents.
A similar study of the amides of acyl β‐alanine and acyl γ‐aminobutyric acid revealed that the former furnishes the
same nitrilium ion, but not the latter. Thus, a six‐membered intermediate is also possible and capable of losing the
full mass of β‐alanine as a neutral fragment. When the size of the ring is forced to be seven‐membered, this pathway
is blocked. When this study was expanded to include a variety of N‐acylproline amides, the nitrilium ion was
observed in 100% abundance only when the acyl group was acetyl. Thus a proline effect (involvement of a strained
bicyclic [3.3.0] structure) is being observed. Copyright © 2011 John Wiley & Sons, Ltd.
As part of a broader mass spectrometric study aimed at
delineating the competitive modes of fragmentation of various
amino acids, simple peptides or their derivatives in the
presence of a proton or metal ion,[1–7] we investigated several
secondary amides of actylglycine and hippuric acid. When the
protonated forms of these amides were subjected to tandem
mass spectrometry (MS/MS) utilizing electrospray ionization
(ESI), they usually furnished b or y ions as a primary mode of
fragmentation.[8–10] However, in some cases, another ion
emerged that either replaced these ions or accompanied them
as a prominent ion. For example, the N‐phenylamide of
acetylglycine (I) afforded such an ion having an m/z value of
118 (corresponding to loss of 75 u, the mass of glycine).
Tandem mass spectrometry of this ion yielded an ion of mass
77 u along with smaller fragments. Since 77 u very likely
corresponds to a phenyl cation, we wondered what combina-
tion of other atoms would constitute the mass difference
between 118 and 77, namely, 41 u. One possibility is C2H3N,
while another is C2HO. Based on these assumptions, an ion
having an m/z value of 118 could have the following structures:
A related experiment involved fragmentation of the
N‐methyl amide of hippuric acid (II) (the result of exchanging
methyl and phenyl groups in I), which did not furnish an ion
having 118 u. In order to determine the structure of this
unusual ion, the mechanism leading to it and the factors
favoring it, we have undertaken a detailed study of these and
related amides using 2H‐, 13C‐ and 15N‐isotopic labels that
constitutes the present article.
EXPERIMENTAL
Synthesis of N‐arylamides of amino acids
The appropriate acylglycine (most are commercially available)
was allowed to undergo reaction with the arylamine in the
presence of polymer‐bound carbodiimide in methylene
chloride for at least 24 h. Filtration of the polymer and
evaporation of the solvent furnished the amide which was
used for the mass spectrometry studies.
If the acylglycine was not commercially available, it
was prepared by stirring excess of the acid anhydride
(trifluoroacetic anhydride) with glycine in chloroform and a
drop or two of water until the solution was homogeneous,
followed by workup. The corresponding analogs of alanine,
β‐alanine or γ‐aminobutyric acid were prepared in a similar
manner.
Other N‐arylamides of glycine (Table 2) were prepared by
reaction of the acid chloride with glycine in the presence of
triethylamine, followed by condensation with the arylamine.
The aminocarbonylglycines were obtained by reaction of an
isocyanatewithglycine, whereasthen‐butyloxycarbonylglycine
was prepared by reaction of n‐butylchloroformate with
Ph
H
Ph
H
N=C=CH2 ,Ph-N
C-CH3,
C=C=NH2 or
Ph-C
C-OH
* Correspondence to: E. R. Talaty, Department of Chemistry,
Wichita State University, Wichita, KS 67260–0051, USA.
E-mail: erach.talaty@wichita.edu
†
Present address: Department of Chemistry and Biochem-
istry, Northern Arizona University, PO Box 5698, S. Beaver
Street, Bldg 20, Room 125, Flagstaff, AZ 86011–5698, USA.
‡
Present address: Department of Chemistry, University
of Utah, 315 S. 1400 E. Rm. 2020, Salt Lake City, Utah
84112–0850.
Rapid Commun. Mass Spectrom. 2011, 25, 1119–1129
Copyright © 2011 John Wiley & Sons, Ltd.