Characterization of Nα-Fmoc-protected ureidopeptides by electrospray ionization tandem mass spectrometry (ESI-MS/MS): Differentiation of positional isomers

Article abstract of DOI:10.1002/jms.1862

Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R ₁)-φ(NH-CO-NH)-CH(R₂)-OY and FmocNH-CH(R ₂)-φ(NH-CO-NH)-CH(R₁)-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R₁ = H, alkyl; R₂

Full text of DOI:10.1002/jms.1862

Research Article
Received: 11 August 2010
Accepted: 18 October 2010
Published online in Wiley Online Library: 6 November 2010
(wileyonlinelibrary.com) DOI 10.1002/jms.1862
Characterization of Nα-Fmoc-protected
ureidopeptides by electrospray ionization
tandem mass spectrometry (ESI-MS/MS):
differentiation of positional isomers
M. Ramesh,a B. Raju,a R. Srinivas,a∗ V. V. Sureshbabu,b∗ N. Narendrab
and B. Vasanthab
Four pairs of positional isomers of ureidopeptides, FmocNH-CH(R1)-ϕ(NH-CO-NH)-CH(R2)-OY and FmocNH-CH(R2)-ϕ(NH-CO-
NH)-CH(R1)-OY (Fmoc = [(9-fluorenyl methyl)oxy]carbonyl; R1 = H, alkyl; R2 = alkyl, H and Y = CH3/H), have been characterized
and differentiated by both positive and negative ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS).
The major fragmentation noticed in MS/MS of all these compounds is due to –N–CH(R)–N–bond cleavage to form the
characteristic N- and C-terminus fragment ions. The protonated ureidopeptide acids derived from glycine at the N-terminus
form protonated (9H-fluoren-9-yl)methyl carbamate ion at m/z 240 which is absent for the corresponding esters. Another
interesting fragmentation noticed in ureidope+ptides derived from glycine at the N-terminus is an unusual loss of 61 units from
an intermediate fragment ion FmocNH = CH2 (m/z 252). A mechanism involving an ion-neutral complex and a direct loss of
NH3 and CO2 is proposed for this process. Whereas ureidopeptides derived from alanine, leucine and phenylalanine at the
N-terminus eliminate CO2 followed by corresponding imine to form (9H-fluoren-9-yl)methyl cation (C14H11+) from FmocNH =
CHR+. Inaddition, characteristicimmoniumionsarealsoobserved. Thedeprotonatedureidopeptideacidsdissociatedifferently
from the protonated ureidopeptides. The [M − H]− ions of ureidopeptide acids undergo a McLafferty-type rearrangement
followed by the loss of CO2 to form an abundant [M − H − Fmoc + H]− which is absent for protonated ureidopeptides. Thus,
the present study provides information on mass spectral characterization of ureidopeptides and distinguishes the positional
c
isomers. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: electrospray ionization; tandem mass spectrometry; ureidopeptides; structure elucidation; positional isomers
Introduction
Tandem mass spectrometry (MS/MS) of protonated and de-
protonated organic and biological analytes including peptides[5]
in electrospray ionization (ESI) and MALDI[6–8] has been a well-
established technique for structure elucidation and differentiation
of isomers.[9–16] The applications of ESI[17] MS to various types of
analytes are well documented in the literature. A literature survey
revealed that there are very few reports on mass spectral studies of
pseudopeptides containing urea bonds.[18,19] This has prompted
us to undertake a detailed mass spectrometric study on a series
Peptides are the ubiquitous biomolecules that have myriad roles
in the life’s process. Each one of their roles is due to a unique three-
dimensionalstructure.[1] However,theextensiveusageofpeptides
as drug candidates needs to outdo some of the unfavorable
features such as low oral bioavailability, lesser permeability, poor
metabolic stability in vivo, etc. Some of these limitations are being
overcome through the modification of one or more peptide bonds
into unnatural functionalities. This has led to the emergence of
pseudopeptides,[1–3] a class of novel entities which are being
employed as potential alternatives to peptide-based drugs as
well as in de novo design of molecules for structural studies.
In this regard, a large research attention is being bestowed
upon backbone engineering and design of novel amino acid
derivatives for the construction of peptidomimetics. Nα-Fmoc
amino acid-derived isocyanates are important intermediates that
were first synthesized, isolated and fully characterized by one
of us and are employed for the synthesis of several classes of
peptidomimetics.[4] In particular, ureidopeptides and peptidyl
ureas are accessed by these synthons which are recognized as
most successful variety of peptidomimetics due to the interesting
metabolic and hydrogen bonding properties possessed by the
urea moiety.
∗
Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, Indian
Institute of Chemical Technology, Hyderabad 500 607, Andhra Pradesh, India.
E-mail: srini@iict.res.in, sragampeta@yahoo.co.in
V. V. Sureshbabu, Peptide Research Laboratory, Department of Studies in
Chemistry, Central College Campus, Bangalore University, Dr. B. R. Ambedkar
Veedhi, Bangalore 560 001, Karnataka, India. E-mail: hariccb@gmail.com
a
NationalCentreforMassSpectrometry,IndianInstituteofChemicalTechnology,
Hyderabad 500 607, Andhra Pradesh, India
b
Peptide Research Laboratory, Department of Studies in Chemistry, Central
College Campus, Bangalore University, Dr. B. R. Ambedkar Veedhi, Bangalore
560 001, Karnataka, India
c
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M. Ramesh et al.
R2
R1
R1
O
R1
R2
1. NMM, IBC-Cl
2. aq. NaN3
H2N
COOMe
FmocHN
FmocHN
COOH
N
NCO
FmocHN
N
H
COOMe
H
3.
Scheme 1. Synthesis of Nα-Fmoc-protected ureidopeptides.
of Fmoc-protected ureidopeptides and also the differentiation of
four pairs of the positional isomeric compounds using ESI-MS/MS.
R2
R1
O
O
OY
N
H
N
H
O
N
H
Experimental
O
Mass spectrometry
1. R1 = CH3
2. R1 = CH3
R2 = CH2C6H5
R2 = CH2C6H5
R2 = CH3
Y = CH3
Y = H
ESI mass spectra of ureidopeptides 1–20 (Scheme 1) were
recorded using a quadrupole ion-trap mass spectrometer (Thermo
Finnigan, San Jose, CA, USA) equipped with an electrospray
source. The data acquisition was under the control of Xcalibur
software (Thermo Finnigan). The typical source conditions were:
spray voltage, 5 kV; capillary voltage, 15–20 V; heated capillary
temperature, 200 ◦C; tube lens offset voltage, 20 V; sheath gas (N2)
pressure, 20 psi; and helium was used as damping gas. For the
ion-trap analyzer, the automatic gain control setting was 2 × 107
counts for a full-scan mass spectrum and 2 × 107 counts for a full
product ion mass spectrum with a maximum ion injection time of
200 ms. In the full-scan MS2 mode, the precursor ion of interest
was first isolated by applying an appropriate wave form across the
end-cap electrodes of the ion-trap to resonantly eject all trapped
ions, except those ions of m/z ratio of interest. The isolated ions
were then subjected to a supplementary ac signal to resonantly
excite them and thus cause collision-induced dissociation (CID).
The collision energies used were 15–37 eV. The excitation time
used was 30 ms. All the spectra were recorded under identical
experimental conditions for isomers, and average of 20–25 scans.
All the samples were infused into the ESI source at a flow rate of
5 µl/min by using an instrument’s syringe pump.
3. R1 = CH2C6H5
4. R1 = CH2C6H5
5. R1 = H
Y = CH3
Y = H
R2 = CH3
R2 = CH2CH(CH3)2
R2 = CH2CH(CH3)2
Y = CH3
Y = H
6. R1 = H
7. R1 = CH2CH(CH3)2 R2 = H
8. R1 = CH2CH(CH3)2 R2 = H
Y = CH3
Y = H
9. R1 = H
R2 = CH2C6H5
Y = CH3
Y = H
10. R1 = H
R2 = CH2C6H5
R2 = H
11. R1 = CH2C6H5
12. R1 = CH2C6H5
13. R1 = H
Y = CH3
Y = H
R2 = H
R2 = CH3
R2 = H
Y = CH3
Y = CH3
Y = CH3
Y = H
14. R1 = CH3
15. R1 = H
R2 = H
16. R1 = H
R2 = H
Accurate mass measurements were obtained using
a
quadrupole time-of-flight (QTOF) mass spectrometer (QSTAR XL,
Applied Biosystems/MDS Sciex, Foster City, CA, USA), equipped
with an ESI source. The data acquisition was under the control of
Analyst QS software (Foster City, CA). The typical source conditions
were: capillary voltage, 5.00 kV; declustering potential, 60 V; focus-
ing potential, 260 V; declustering potential 2, 10 V; resolution 8000
(full-width half-maximum). Ultra high-pure nitrogen was used as
the curtain gas and collision gas, whereas zero air was used as
the nebulizer. The [M + H]+ ions were selected as precursors
by the quadrupole and allowed to collide with nitrogen gas in
the collision cell. The product ions were then detected by a TOF
analyzer. The samples were infused into the ESI source at a flow
rate of 10 µl/min using an in-built syringe pump.
17. R1 = H
R2 = CH(CH3)2
R2 = CH(CH3)2
R2 = C6H5
R2 = C6H5
Y = CH3
Y = H
18. R1 = H
19. R1 = H
Y = CH3
Y = H
20. R1 = H
Scheme 2. Structures of the Nα-Fmoc-protected ureidopeptides (1–20).
General procedure for the preparation of isocyanates derived
from Fmoc-α-amino acids
To a solution of Nα-Fmoc-amino acid (1 mmol) in dry THF (5 ml)
at −20 ◦C, N-methylmorpholine (NMM, 0.12 ml, 1.1 mmol) and
isobutyl chloroformate (0.14 ml, 1.1 mmol) were added and the
mixture was stirred at the same temperature for 20 min. It
was treated with aqueous NaN3 (98 mg, 1.5 mmol in 0.5 ml of
water) and the stirring was continued for another 30 min. After
completion of the reaction (TLC analysis), the organic layer was
evaporated and the residue was dissolved in dichloromethane
(CH2Cl2, 20 ml). It was washed successively thrice with 10 ml
portions of 5% HCl, 5% aqueous NaHCO3, water and dried
over anhydrous Na2SO4. Evaporation of the solvent in vacuo
affords corresponding acid azide. It was dissolved in toluene
(10 ml) and heated at 65 ◦C under nitrogen atmosphere. After
Materials
Solvents used in the present study were purchased from Merck
(Mumbai, India) and used without further purification. Stock
(1 mM) solutions of ureidopeptides were diluted with high-
performance liquid chromatography-grade methanol to achieve
a final concentration of 10 µM each.
Synthesis of the ureidopeptides
The synthesis and spectroscopic characterization of the com-
pounds studied in this work have been reported by one of us.[20]
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Characterization of Nα-Fmoc-protected ureidopeptides
Figure 1. ESI-MS/MS spectra of [M + H]+ ions (m/z 488) of (a) 1 and (b) 3 at 18 eV.
H
CH3
O
O
OY
N
O
N
N
H
H
H
O
O
m/z 488,Y = CH3
m/z 474,Y = H
H
H
O
OY
H3C
OY
N
N
H
H2N
N
O
H
m/z 249
m/z 235
O
m/z 223
m/z 209
- CH3CN, CO
- HNCO
- CH3CH = NH
H
H
H
O
OY
OY
OY
N
H2N
m/z 180
H2N
O
O
O
m/z 180
m/z 166
m/z 206
m/z 192
m/z 166
Scheme 3. Proposed fragmentation mechanism for protonated ureidopeptides 1and 2.
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M. Ramesh et al.
O
H
O
N
C
H
m/z 342
H
CH3
O
O
OY
O
N
N
H
N
H
H
O
m/z 488, Y = CH3
m/z 474, Y = H
H
CH3
O
H
OY
CH3
O
N
H2N
OY
H
O
N
N
H
m/z 147
m/z 133
O
m/z 249
m/z 235
- HNCO
CH3
- PhCH2CN, CO
H
H
CH3
OY
H2N
OY
CH2-CH=NH2
m/z 120
H2N
m/z 104
m/z 90
O
m/z 104
O
m/z 90
Scheme 4. Proposed fragmentation mechanism for protonated ureidopeptides 3 and 4.
the completion of the reaction, the solvent was removed under
reducedpressuretoobtaintheisocyanate,whichwasrecrystallized
using dichloromethane/n-hexane.
It can be seen from Fig. 1 that the MS2 CID spec-
tra (m/z 488) of isomers Fmoc-Ala-ϕ(NH-CO-NH)-Phe-OCH3
(1) and Fmoc-Phe-ϕ(NH-CO-NH)-Ala-OCH3 (3) (Fmoc = [(9-
fluorenylmethyl)oxy]carbonyl)[20] are distinctly different from one
another. Both the isomers show an abundant ion at m/z 249
corresponding to the loss of (9H-fluoren-9-yl) methyl carba-
mate (Scheme 3). This appears to be a diagnostic loss for the
presence of Fmoc group at the N-terminus as can be seen
from the fragmentation of other Fmoc-protected ureidopep-
tides. Isomer 1 shows additional peaks at m/z 223 and 180
which are absent for 3. The former corresponds to protonated
(S)-methyl 3-phenyl-2-ureidopropanoate which on loss of HNCO
forms a low abundant ion at m/z 180 (Scheme 3). Whereas iso-
mer 3 shows a low abundance ion at m/z 342 formed by the
loss of (S)-methyl 2-ureidopropanoate (Scheme 4). The comple-
mentary fragment ion corresponding to protonated (S)-methyl
2-ureidopropanoate appears at m/z 147. It can be noted that
the major fragmentation in these compounds does not involve
the cleavage of –NH–CO–NH–bonds and it mainly involves
–N–CH(R)–N–bonds.
General procedure for the preparation of ureidopeptides
To a stirred suspension of amino acid methyl ester hydrochloride
salt(1 mmol)inCH2Cl2 (5 ml),NMM(2 mmol)wasaddedslowlyand
the mixture was stirred at 25 ◦C for few minutes. This was added to
the solution of isocyanate (1 mmol) derived from Fmoc-α-amino
acid, and the stirring was continued till the completion of the
reaction. The precipitated solid was filtered and recrystallized
using DMSO–water to obtain the uriedopeptide as crystalline
white solid (Scheme 1).
Results and Discussion
ThepositionalisomericpairsoftheNα-Fmoc-protectedureidopep-
tides studied in this work are shown in Scheme 2. The positive ion
ESI mass spectra of all these Nα-Fmoc-protected ureidopeptides
(1–20) show abundant [M + H]+, [M + Na]+ and [2M + Na]+ ions.
To study the mass spectrometric behavior of these ureidopeptides
and to investigate the possibility of distinguishing these posi-
tional isomers, we have examined the MS/MS CID spectra of the
protonated positional isomers (1–20).
To probe further the fragmentation of the ureidopeptides, the
MS3 CIDofthecommonionofm/z 249from1and3wasexamined.
It can be seen from Fig. 2 that the MS3 of 1 is also significantly
different from that of 3. Isomer 1 forms low abundance ion at m/z
206andanabundantionatm/z 180bythelossofCH3CH= NHand
a combination of CH3CN and CO, respectively (Scheme 3). These
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Characterization of Nα-Fmoc-protected ureidopeptides
Figure 2. ESI-MS3 spectra of m/z 249 of (a) 1 and (b) 3 at 22 eV.
ions are absent for 3. Instead, the latter shows an abundant ion at
m/z 120 corresponding to the immonium ion of phenylalanine[21]
(Scheme 4), a characteristic ion for the ureidopeptides derived
from phenylalanine at the N-terminus. A low abundance ion is
observed at m/z 104 corresponding to the loss of combination
of PhCH2CN and CO. Thus, the positional isomers 1 and 3 can
be clearly distinguished from one another by their characteristic
fragmentation in both MS2 and MS3 CID spectra.
Similarly, the positional isomers of corresponding acids 2 and
4 can be clearly distinguished from one another, except that
the masses of the C-terminal ions are decreased by 14 Da. It
is noteworthy that the intensity of the m/z 342 ion (Scheme 4)
becomes highly abundant for acid 4 than the corresponding
ester 3.
An another pair of positional isomers, Fmoc-Gly-ϕ(NH-CO-NH)-
Leu-OCH3 (5) and Fmoc-Leu-ϕ(NH-CO-NH)-Gly-OCH3 (7), yielded
product ion spectra that are distinctly different from one another
(Fig. 3). Similar to 1 and 3, the CID mass spectra of [M + H]+ ions
(m/z 440) of 5 and 7 display an ion at m/z 201 corresponding to the
loss of (9H-fluoren-9-yl) methyl carbamate. The major difference
observed between 5 and 7 is that the former shows m/z 408
(loss of methanol), m/z 397 (loss of HNCO), m/z 269 (protonated
(9H-fluoren-9-yl) methyl aminomethyl carbamate), m/z 252 (loss
of (S)-methyl 4-methyl-2-ureidopentanoate), m/z 189 (protonated
(S)-methyl 4-methyl-2-ureidopentanoate), m/z 158 (loss of HNCO
from m/z 201) and m/z 146 (loss of HNCO from m/z 189), whereas
these ions are totally absent for 7 (Scheme 5). Instead, the latter
shows only m/z 308 (loss of methyl 2-ureidoacetate) and its
complementary low abundance ion at m/z 133 corresponding to
protonated methyl 2-ureidoacetate (Scheme 6). It can be noted
that the ion at m/z 201 is relatively more abundant for 7 than for 5.
Thus, the positional isomers 5 and 7 can be readily distinguished
from one another by means of their characteristic product ions.
Furthermore,theMS3 CIDofm/z201of5and7gives(Supporting
Information)lowabundanceionatm/z 158(lossofHNCO).Besides,
the former shows an abundant ion at m/z 172 by the loss of
CH2 = NH (Scheme 5) and the latter gives abundant ions at m/z
90 (loss of (CH3)2CHCH2CN and CO) and m/z 86 corresponding
to the immonium ion of leucine[21] (Scheme 6). Thus, the MS3
spectra of m/z 201 ion of 5 and 7 are also useful to distinguish
between the ureidopeptides derived from glycine and leucine at
the N-terminus.
It is interesting to note that the MS3 CID of m/z 252 (protonated
(9H-fluoren-9-yl) methyl methylenecarbamate) ion from 5 derived
from glycine at the N-terminus forms an intense product ion of
m/z 191 that corresponds to an unusual loss of 61 units (Fig. 4).
The mechanism of formation of this ion may presumably involve
an ion–neutral complex (INC)[22,23] of fluorenylethaniminium ion
[24]
and CO2
that eliminate CO2 and NH3 simultaneously to form
m/z 191 (C15H11+) (Scheme 7). The formation of the INC seems
to be triggered by a region-specific 1,2-hydride abstraction[25]
by the imine nitrogen followed by migration of methylfluorenyl
to the methyne carbon involving loss of CO2 (Scheme 7). The
elemental composition of m/z 191 (C15H11+) ion has been
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M. Ramesh et al.
Figure 3. ESI-MS/MS spectra of [M + H]+ ions (m/z 440) of (a) 5 and (b) 7 at 23 eV.
confirmed by accurate mass measurements performed on a
QTOF mass spectrometer (calculated m/z 191.0860, observed
m/z 191.0862 and error 0.6518 ppm). This fragmentation process
has been observed for all the ureidopeptides (9, 10, 13, 15–20)
derived from glycine at the N-terminus as will be discussed later.
The concomitant loss of CO2 and NH3 (61 units) or consecutive
elimination of NH3 and CO2 for the [Ag–Phe]+ systems[26,27]
through zwitter ionic structure was reported earlier.[28–30] The loss
of 61 units is absent for 7 derived from leucine at the N-terminus,
instead, the MS3 spectrum of its m/z 308 shows m/z 264 (loss
of CO2) and m/z 179 corresponding to (9H-fluoren-9-yl)-methyl
cation (C14H11+) (Scheme 7). The elemental composition of m/z
179 (C14H11+) ion also has been confirmed by accurate mass
measurements (calculated m/z 179.0861, observed m/z 179.0863
and error 0.6518 ppm).
(loss of methanol), m/z 431 (loss of HNCO), m/z 269 (protonated
(9H-fluoren-9-yl)methyl aminomethyl carbamate), m/z 252 (loss
of (S)-methyl 3-phenyl-2-ureidopropanoate), m/z 223 (protonated
(S)-methyl 3-phenyl-2-ureidopropanoate), m/z 192 (loss of HNCO
from m/z 235) and m/z 180 (loss of HNCO from m/z 223) which are
absent for 11. Instead, the latter shows a peak at m/z 342 (loss of
methyl2-ureidoacetate),acharacteristicionfortheureidopeptides
derived from phenylalanine at the N-terminus. It can be noted that
the ion at m/z 235 is relatively more abundant for 11 compared
with 9 (Table 1). Thus, the positional isomers 9 and 11 can be
readily distinguished from one another by means of different
product ions.
The MS3 spectrum of m/z 235 of 9 shows (Supporting
Information) m/z 218 (loss of NH3), m/z 206 (loss of imine), m/z
203 (loss of methanol), m/z 190 (loss of CO from m/z 218) and
abundant ion at m/z 192 (loss of HNCO) which are totally absent
for 11. Instead, the latter shows an abundant ion at m/z 120
(immonium ion) and low abundance ions at m/z 116 (loss of
benzyl imine) and m/z 90 (protonated glycine ester). Thus, the MS3
spectrum of m/z 235 ion of 11 is also distinctly different from that
of 9.
Similarly, the positional isomers of corresponding acids 6 and
8 can be clearly distinguished from one another, except that the
masses of the C-terminal ions are decreased by 14 Da. In addition,
the m/z 240 ion corresponding to protonated (9H-fluoren-9-yl)
methyl carbamate (Scheme 5) is significant for acid 6 and absent
for corresponding ester 5.
The CID mass spectra of [M + H]+ ions (m/z 474) of the third
pair of isomers Fmoc-Gly-ϕ(NH-CO-NH)-Phe-OCH3 (9) and Fmoc-
Phe-ϕ(NH-CO-NH)-Gly-OCH3 (11)yieldproductionspectrathatare
distinctly different from one another (Table 1 and also Supporting
Information). Similartothepreviousisomers, both9and11display
an ion at m/z 235 corresponding to the loss of (9H-fluoren-9-yl)
methyl carbamate. Besides, 9 shows fragment ions at m/z 442
As explained above for 5, the MS3 spectrum of m/z 252 of 9
derived from glycine at the N-terminus shows a peak at m/z 191
formed by the direct loss of 61 units. Whereas the MS3 spectrum of
m/z 342of 11derivedfromphenylalanineattheN-terminusshows
(SupportingInformation)m/z298correspondingtothelossofCO2,
m/z 179 ((9H-fluoren-9-yl) methyl cation (C14H11+)) (Scheme 7),
low abundance ions at m/z 164 (loss of 9-methylene-9H-fluorene
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Characterization of Nα-Fmoc-protected ureidopeptides
H
O
O
H
O
H
OY
NH2
O
N
O
N
NH2
H
m/z 240
m/z 172
m/z 158
O
m/z 269
only 6
- CH2 = NH
H
H
O
O
O
OY
OY
O
CH2
N
N
O
N
H
N
H
N
H
H
O
m/z 201
m/z 187
m/z 440, Y = CH3
m/z 426,Y = H
- HNCO
H
H
O
O
H
OY
OY
CH2
N
N
H2N
CH2
O
N
H
O
m/z 158
m/z 144
O
m/z 189
m/z 252
m/z 175
- CO2 & NH3
- HNCO
H
OY
H2N
m/z 146
O
m/z 191
m/z 132
Scheme 5. Proposed fragmentation mechanism for protonated ureidopeptides 5 and 6.
(Fme)) and m/z 120 (loss of Fmoc).[31] The formation of the m/z 120
ioncanberationalizedintermsofaMcLafferty-typerearrangement
involving a γ -hydrogen migration from fluorenyl moiety to the
carbonyl oxygen in the Fmoc-N-moiety followed by the loss of
9-methylene-9H-fluorene (Fme) (178 Da) and subsequent loss of
CO2.
Similarly, the positional isomers of corresponding acids 10
and 12 can be clearly distinguished from one another, except
that the masses of the C-terminal ions are decreased by 14
Da. In addition, the m/z 240 ion corresponding to protonated
(9H-fluoren-9-yl) methyl carbamate is significant for acid 10 and
absent for corresponding ester 9.
derived from glycine at the N-terminus undergo simultaneous loss
ofCO2 andNH3 (61 units)toform C15H11+ ions. Instead, onlylossof
CO2 from m/z 266 leads to m/z 222 in case of 14 (Scheme 7). It also
shows (Supporting Information) a peak at m/z 179 corresponding
to (9H-fluoren-9-yl) methyl cation (C14H11+).
The direct loss of 61 units from m/z 252 ion of ureidopeptides
derived from glycine at the N-terminus has been supported by the
study of some more ureidopeptides (15–20) derived from glycine
at the N-terminus by varying C-terminus amino acid. All these
+
compounds showed the direct loss of 61 units giving C15H11 in
the MS3 CID of m/z 252 ions. Other MS2 fragmentations from these
compounds are given in Table 1.
The CID of [M + H]+ ions (m/z 398) of isomers Fmoc-Gly-ϕ(NH-
CO-NH)-Ala-OCH3 (13)andFmoc-Ala-ϕ(NH-CO-NH)-Gly-OCH3 (14)
yields m/z 355 (loss of HNCO), m/z 159 (loss of (9H-fluoren-9-yl)
methyl carbamate) and m/z 116 (loss of HNCO from m/z 159)
(Table 1 and also Supporting Information). Besides, 13 shows m/z
380(lossofwater),m/z 366(lossofmethanol), m/z 269(protonated
(9H-fluoren-9-yl)methyl aminomethyl carbamate), m/z 252 (loss of
(S)-methyl 2-ureidopropanoate), m/z 147 (protonated (S)-methyl
2-ureidopropanoate) and m/z 191 (loss of 61 units from m/z 252)
which are totally absent for 14. Instead, the latter shows m/z 266
(loss of methyl 2-ureidoacetate) and m/z 133 (protonated methyl
2-ureidoacetate). It is noteworthy that the intensity of the m/z 159
ion becomes highly abundant for 14 than 13 (Table 1 and also
Supporting Information). Thus, this pair of isomers can be clearly
distinguished from one another by means of different product
ions.
Negative ion CID of isomeric ureidopeptide acids
As peptide acids are known to form negative ions more readily
than the esters, we examined ureidopeptide acids under negative
ion ESI conditions. The negative ion ESI mass spectra of the
ureidopeptide acids show [M − H]− ion as the base peak. In
contrast to the positive ions that do not eliminate Fmoc, negative
ion ESI CID of [M − H]− ion (m/z 472) of isomeric acids 2 and 4
yields (Table 2 and also Supporting Information) a low abundance
ion at m/z 294 (loss of 9-methylene-9H-fluorene (Fme)) and an
abundant ion at m/z 250 ([M − H − Fmoc + H]−) by a loss of
Fmoc.[31] The formation of the latter ion can be rationalized in
terms of a McLafferty-type rearrangement involving a γ -hydrogen
migration from fluorenyl moiety to the carbonyl oxygen in the
FmocN-moiety followed by the loss of 9-methylene-9H-fluorene
(Fme) (178 Da) and subsequent loss of CO2 (Scheme 8).
As observed in case of 5 and 9, the MS3 CID of m/z 252 from
13 also gives m/z 191 (C15H11+) ions by the direct loss of 61
units. Thus, it can be generalized that protonated ureidopeptides
The MS3 spectra of isomers 2 and 4 (m/z 250) show distinctly
different fragmentation from one another (Table 2 and also
c
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M. Ramesh et al.
H
O
OY
N
H
H2N
O
m/z 133
H
O
O
OY
N
N
N
O
H
H
H
O
m/z 440, Y = CH3
m/z 426, Y = H
H
O
O
OY
H
N
N
H
N
C
O
O
m/z 201
m/z 187
- (CH3)2 CHCH2CN, CO
H
- HNCO
m/z 308
H
OY
H
- CO2
H2N
OY
N
O
H
m/z 90
NH2
m/z 86
O
N
m/z 158
m/z 144
m/z 76
C
H
m/z 264
- (CH3)2CHCH2CH=NH
CH2
m/z 179
Scheme 6. Proposed fragmentation mechanism for protonated ureidopeptides 7 and 8.
Table 1. Partial CID of [M + H]+ of ureidopeptides 9, 11, 13–20: m/z with relative abundance (%) in parenthesis
Loss of
H2O
Loss of
CH3OH
Loss of
HNCO
Compound
[M + H]+
Other ions
9
474
474
398
398
384
370
426
412
460
446
–
442 (5)
431 (5)
–
269 (1), 252 (10), 235 (12), 223 (100), 192 (18), 180 (20)
342 (38), 235 (100)
11
13
14
15
16
17
18
19
20
–
–
380 (2)
–
366 (8)
355 (12)
355 (4)
341 (12)
327 (5)
383 (12)
369 (8)
417 (12)
403 (10)
269 (2), 252 (100), 191 (6), 159 (2), 147 (20), 116 (3)
266 (42), 159 (100), 133 (66), 116 (4)
–
366 (2)
352 (2)
408 (1)
394 (10)
442 (2)
428 (7)
352 (3)
252 (100), 191 (2), 133 (2)
–
394 (20)
–
252 (100), 240 (12), 191 (2), 179 (5)
269 (3), 252 (86), 191 (10), 187 (14), 175 (100), 144 (12), 132 (12)
269 (2), 252 (100), 240 (6), 191 (16), 179 (6), 161 (12), 118 (4)
428 (7)
–
269 (2), 252 (40), 221 (7), 209 (100), 191 (6), 178 (60), 166 (44), 149 (10)
281 (60), 269 (2), 252 (100), 240 (38), 208 (30), 195 (50), 191 (25), 179 (44), 164 (24),
152 (75), 135 (12)
Supporting Information). The CID spectra of both the isomers
display an ion at m/z 232 (loss of H2O). Besides, 2 shows an
abundant ion at m/z 164 (loss of isocyanate) and low abundance
ions at m/z 207 (loss of CH3CH NH) and m/z 190 (loss of NH3
from m/z 207) which are absent for 4. Instead, the latter shows m/z
131 (loss of PhCH2CH NH), m/z 113 (loss of H2O from m/z 131)
and an abundant ion at m/z 88 (loss of isocyanate) (Scheme 8).
Similarly, other two pairs of isomeric acids, 6/8 and 10/12 can
be clearly distinguished from each other from their characteristic
fragmentation in MS3 (Scheme 8 and Table 2). The compounds 16
and 18 also show similar fragmentation (Scheme 8) and the data
are presented in Table 2.
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Characterization of Nα-Fmoc-protected ureidopeptides
Figure 4. ESI-MS3 spectra of m/z 252 of (a) 5 and m/z 308 of (b) 7 at 20 eV.
O
H
H
NH2
NH2
O
N
C
O
O
O
O
H
H
C
C
m/z 252
m/z 252
m/z 252
(INC)
- NH3, - CO2
O
CH = CH
H
O
N
C
X
m/z 191
H
R
m/z 191
m/z 266, R = CH3
m/z 308, R = CH2CH(CH3)2
m/z 342, R = CH2Ph
- CO2
H
CH2
N
C
- RCH = NH
H
R
m/z 179
m/z 222, R = CH3
m/z 264, R = CH2CH(CH3)2
m/z 298, R = CH2Ph
Scheme 7. Proposed fragmentation mechanism for loss of 61 units via ion–neutral complex.
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M. Ramesh et al.
Table 2. Partial CID of [M − H]− of ureidopeptide acids 2, 4, 6, 8, 10, 12, 16, 18 and 20: m/z with relative abundance (%) in parenthesis
Compound
[M − H]−
[M − H − Fme]−
[M − H − Fmoc + H]−
Other ions
2
4
472
472
424
424
458
458
368
410
444
294 (3)
294 (3)
246 (8)
246 (8)
280 (3)
280 (3)
190 (30)
232 (3)
266 (3)
250 (100)
250 (100)
202 (100)
202 (100)
236 (100)
236 (100)
146 (100)
188 (100)
222 (100)
250→ 232 (5), 207 (8), 190 (5), 164 (100)
250→ 232 (10), 131 (50), 113 (10), 88 (100)
202→ 184 (100), 173 (15), 156 (10), 155 (6), 130 (98)
202→ 184 (5), 117 (60), 100 (10), 99 (6), 74 (100)
236→ 218 (22), 207 (3), 190 (4), 189 (2), 164 (100)
236→ 218 (12), 117 (100), 100 (14), 99 (4), 74 (72)
146→ 128 (60), 117 (40), 100 (20), 99 (3), 74 (100)
188→ 170 (100), 159 (12), 142 (8), 141 (10), 116 (90)
222→ 178 (100)→ 149 (100)
6
8
10
12
16
18
20
H
O
H
O
R1
O
R2
O
O
O
O
N
H
N
N
H
N
N
N
O
H
H
H
H
m/z 444, (M-H)-
O
O
(M-H)-
McLafferty- type
McLafferty- type
O
R1
O
R2
H
O
H
O
O
O
O
O
N
N
N
H
N
N
N
H
H
H
H
H
m/z 266
- CO2
O
O
O
- CO2
R
1
O
R2
O
O
H2N
N
H
N
H
N
N
H2N
O
H
H
m/z 202
O
R1
O
R2
- CO2
OH
O
H2N
N
N
H
O
H2N
N
N
H
H
m/z 178
R1-CH = NH
R2
R1
O
H2N
OH
N
R2
- CH2 = NH
O
O
OH
HN
HN
N
H
H2N
N
H
O
m/z 149
O
Scheme 9. Proposed fragmentation mechanism for deprotonated urei-
dopeptide 20.
Scheme 8. Proposed fragmentation mechanism for deprotonated urei-
dopeptides 2, 4, 6, 8, 10, 12, 16 and 18.
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Characterization of Nα-Fmoc-protected ureidopeptides
In contrast to the isomers discussed above, compound 20 that
has a phenyl glycine at the C-terminus fragments differently. After
the loss of Fmoc, the [M − H − Fmoc + H]− (m/z 222) eliminates an
additional CO2 from the C-terminus to form m/z 178 (Scheme 9).
This loses imine to form m/z 149 as evidenced from the MS4
spectrum of m/z 178 (Table 2 and also Supporting Information).
Formation of these fragments may be attributed to the generation
of a highly stable phenyl carbanion as shown in Scheme 9.
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Conclusions
Positive and negative ion ESI-MS/MS has been shown to be very
useful for the structural characterization and differentiation of
fourpairsofNα-Fmoc-protectedureidopeptidepositionalisomers.
The major fragmentation noticed in all these compounds is due
to –N–CH(R)–N–bond cleavage. The MS2 of [M + H]+ ions
of all the studied ureidopeptide acids derived from glycine at
the N-terminus yield an ion at m/z 240 which is absent for
corresponding esters. Another interesting fragmentation noticed
in these compounds is the loss of 61 units from an intermediate
fragment ion FmocNH = CH2+ (m/z 252). A mechanism involving
an INC and a direct loss of NH3 and CO2 is proposed for this
process. Whereas ureidopeptides derived from alanine, leucine
and phenylalanine at the N-terminus eliminate CO2 followed
by corresponding imine to form (9H-fluoren-9-yl)methyl cation
(C14H11+) from FmocNH CHR+. In contrast to the positive ions,
the negative ions of Nα-Fmoc-protected ureidopeptide acids
undergo a McLafferty-type rearrangement ion involving loss of
Fme and Fmoc. It is worth to note that the losses of HNCO ions
are totally absent in negative ion spectra. It can be concluded that
the ESI-MS/MS study of both the protonated and deprotonated
ureidopeptides provides the fragmentation behavior of this new
class of compounds and also useful for differentiating these
isomeric ureidopeptides.
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K. R. Reddy, P. R. Krishna. Mass spectral study of Boc-carbo-β3-
peptides: differentiation of two pairs of positional and
diastereomeric isomers. J. Mass Spectrom. 2004, 39, 1068.
[10] R. Srikanth, P. N. Reddy, R. Srinivas, G. V. M. Sharma, K. R. Reddy,
P. R. Krishna. Mass spectral study of alkali-cationized Boc-carbo-β3-
peptides by electrospray tandem mass spectrometry. Rapid
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P. Nagendar, P. R. Krishna. Differentiation of Boc-α,β- and β,
α-peptides and a pair of diastereomeric β,α-dipeptides by positive
and negative ion electrospray tandem mass spectrometry (ESI-
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P. Nagendar. Electrospray tandem mass spectrometry of alkali-
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positional isomers. Rapid Commun. Mass Spectrom. 2006, 20, 3351.
[13] P. N. Reddy, V. Ramesh, R. Srinivas, G. V. M. Sharma, P. Nagendar,
V. Subash. Differentiation of some positional and diastereomeric
isomers of Boc-carbo-β3 dipeptides containing galactose, xylose
and mannose sugars by electrospray ionization tandem mass
spectrometry (ESI-MS/MS). Int. J. Mass Spectrom. 2006, 248, 115.
[14] P. N. Reddy, R. Srinivas, M. R. Kumar, G. V. M. Sharma, V. B. Jadhav.
Positive and negative ion electrospray ionization tandem mass
spectrometry of Boc-protected peptides containing repeats of
L-Ala-γ 4Caa/γ 4Caa-L-Ala: differentiation of some positional
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Acknowledgements
The authors thank Dr J.S. Yadav, Director, IICT, Hyderabad, for
facilities and Dr M. Vairamani for cooperation. M.R. is thankful to
CSIR, New Delhi, for the award of Senior Research Fellowship, B.R.
is thankful to DST, New Delhi, for the award of Junior Research
FellowshipandN.N.andB.V.thankCSIR,NewDelhi,forthefinancial
assistance.
Supporting information
Supporting information may be found in the online version of this
article.
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