Fragmentation pattern of amides by EI and HRESI: Study of protonation sites using DFT-3LYP data

Article abstract of DOI:10.1039/c7ra00408g

Amides are important natural products which occur in a few plant families. Piplartine and piperine, major amides in Piper tuberculatum and P. nigrum, respectively, have shown a typical N-CO cleavage when analyzed by EI-MS or HRESI-MS. In this study several synthetic analogs of piplartine and piperine were subjected to both types of mass spectrometric analysis in order to identify structural features influencing fragmentation. Most of the amides showed an intense signal of the protonated molecule [M + H]⁺ when subjected to both HRESI-MS and EI-MS conditions, with a common outcome being the cleavage of the amide bond (N-CO). This results in the loss of the neutral amine or lactam and the formation of aryl acylium cations. The mechanism of N-CO bond cleavage persists in α,β-unsaturated amides because of the stability caused by extended conjugation. Computational methods determined that the protonation of the piperamides and their derivatives takes place preferentially at the amide nitrogen supporting the dominant the N-CO bond cleavage.

Full text of DOI:10.1039/c7ra00408g

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Fragmentation pattern of amides by EI and HRESI:
study of protonation sites using DFT-3LYP data†
Cite this: RSC Adv., 2018, 8, 21407
H. H. Fokoue, a J. V. Marques,b M. V. Correia,c L. F. Yamaguchi,d X. Qu,e J. Aires-de-
g
d
Sousa,e M. T. Scotti,f N. P. Lopes
and M. J. Kato
*
Amides are important natural products which occur in a few plant families. Piplartine and piperine, major
amides in Piper tuberculatum and P. nigrum, respectively, have shown a typical N–CO cleavage when
analyzed by EI-MS or HRESI-MS. In this study several synthetic analogs of piplartine and piperine were
subjected to both types of mass spectrometric analysis in order to identify structural features influencing
fragmentation. Most of the amides showed an intense signal of the protonated molecule [M + H]+ when
subjected to both HRESI-MS and EI-MS conditions, with a common outcome being the cleavage of the
amide bond (N–CO). This results in the loss of the neutral amine or lactam and the formation of aryl
acylium cations. The mechanism of N–CO bond cleavage persists in a,b-unsaturated amides because of
the stability caused by extended conjugation. Computational methods determined that the protonation
of the piperamides and their derivatives takes place preferentially at the amide nitrogen supporting the
dominant the N–CO bond cleavage.
Received 20th March 2018
Accepted 4th June 2018
DOI: 10.1039/c7ra00408g
rsc.li/rsc-advances
Furthermore, to fully investigate their bioactivity many analogs of
naturally occurring amides have been synthesized.10–12
1. Introduction
Our research group has focused on the phytochemistry of the
genus Piper because of its wide occurrence in the tropics, ease of
propagation, ecological importance and most importantly due
to the previously described bioactivity of both crude extracts
and isolated compounds from this genus. Thus, the develop-
ment of fast and reliable tools for rapid dereplication and
identication of major compounds in crude extracts became an
important issue.
The application of mass spectrometry based on electron impact
has already been applied to determine a series of amides in P.
amalago.13 Two well-known amides, piperine and piplartine, have
shown the cleavage of the N–CO bond, a specic fragmentation
Amides are an important class of natural products found in a few
plant families such as Asteraceae, Piperaceae, Rutaceae and Sol-
anaceae, among others.1–4 The two most well-known amides are
piperine and capsaicin, the pungent principles of black pepper
(Piper nigrum, Piperaceae) and chili pepper (Capsicum varieties,
Solanaceae), respectively.5 Amides play important roles in plant
ecology, being involved in many aspects of plant–insect interac-
tions.6 Additionally, they exert many important biological activi-
ties. For instance, piplartine, piperine, piperlonguminine,
fagaramide, corcovadine and other piperamides have been shown
to have cytotoxic, antimicrobial and anti-inammatory proper-
ties.7–9 Several amides from different sources have been isolated
through bioactivity-guided fractionation of crude extracts.
a
´
˜
´
ˆ
Laboratorio de Avaliaçao e Sıntese de Substancias Bioativas (LASSBio®), Instituto de
ˆ
´
ˆ
´
Ciencias Biomedicas, Centro das Ciencias da Saude, Universidade Federal do Rio de
Janeiro, CP 68024, 21944-971, Rio de Janeiro, Brazil
bGinkgo Bioworks, Boston, MA, 02210, USA
c
´
´
Instituto de Quımica, Universidade de Brasılia, CP 04478, 70704-970, Brasilia-DF,
Brazil
d
´
˜
Instituto de Quımica, Universidade de Sao Paulo, Av. Prof. Lineu Prestes, 748, 05508-
˜
000 Sao Paulo-SP, Brazil. E-mail: massuojorge@gmail.com
eLAQV and REQUIMTE, Departamento de Quımica, Faculdade de Ciencias
e
´
ˆ
Tecnologia, Universidade Nova de Lisboa, Caparica 2829-516, Portugal
f
´
˜
´
Pos-Graduaçao em Produtos Naturais e Sinteticos Bioativos, Universidade Federal da
´
˜
Paraıba, Joao Pessoa, 58051-900, Brazil
g
´
´
ˆ
ˆ
˜
Departamento de Fısica e Quımica, Faculdade de Ciencias Farmaceuticas de Ribeirao
˜
˜
Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, 14040-903, Brazil
† Electronic supplementary information (ESI) available: EI and HRESI spectra of
all compounds. See DOI: 10.1039/c7ra00408g
Fig. 1 EI-MS and HRESI-MS of piplartine (1a).
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a roto-evaporator. The crude extract was subjected to a silica
column chromatography eluted with hexanes/ethyl acetate
mixtures of increasing polarity. Fractions containing piperine
(6a) were pooled and recrystallized in methanol yielding
a yellowish crystal. Piperine was identied by comparison of
NMR and MS analysis with reported data.19,20
2.2 Synthesis of piplartine and piperine derivatives
Compounds 9 and 10 were obtained by catalytic hydrogenation
(4 atm of hydrogen, Pd–C) of piplartine (1a) and piperine (6a),
respectively, for 12 hours.21
To a solution of 1 equivalent of carboxylic acids in dry THF (10
mL) [i.e. 4-bromocinnamic acid; 5-(4-bromophenyl)-(2E,4E)-2,4-
pentadienoic acid; (E)-cinnamic acid; 5-(4-hydroxyphenyl)-(2E,4E)-
2,4-pentadienoic acid; 3,4-(methylenedioxy)-cinnamic acid or
piperinic acid; (E)-3,4,5-trimethoxycinnamic acid; (E)-3,4-dime-
thoxycinnamic acid], kept under nitrogen atmosphere and over ice
bath, oxalyl chloride (5 equiv.) was added dropwise and stirred for
6 hours. Aer reaching room temperature, the excess of oxalyl
chloride was removed under reduced pressure to leave the corre-
sponding acyl chlorides. Compounds 1c, 1d–1f, 2a–2d, 3a–3c, 4a,
Fig. 2 EI-MS and HRESI-MS of piperine (6a).
pattern observed by EI-MS and HRESI-MS (Fig. 1 and 2).14–16 This
peculiar cleavage, observed in the a,b-unsaturated piperamides is
unusual in aliphatic amides, in which McLafferty rearrangement is
preferable wherever g-hydrogen to the carbonyl is available.
Additionally, the a-cleavage of secondary and tertiary aliphatic
amides and the formation of acylium cations can also be used to
support the characterization of amides. The conjugation between
the amide carbonyl and the b double bond reinforced by the
presence of an aryl group at the g or 5 positions from the carbonyl
could account for preferable N–CO fragmentation.15,16 Herein,
several analogs synthesized were subjected to mass spectrometry
studies to determine whether the N–CO a-cleavage could be
generalized and used as a criteria for the rapid identication of
piperamides. Computational studies (proton affinity and bond
energies) were carried out to verify the N–CO a-cleavage. Both EI
and HRESI-MS experiments were then carried out to compare the
corresponding fragmentation patterns and, together a calculation
of energetic proles at protonation sites were examined to support
the characterization of amides.
Table 1 Structures of natural amides and derivativesa
Amides
n
Ra
Rb
Rc
R
1a
1b
1c
1d
1e
1f
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Br
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
RA
RF
RE
RG
RD
RC
RE
RG
RD
RC
RF
RD
RG
RE
RD
RE
RE
RG
RD
RF
RC
RG
RG
RE
RC
RG
RC
RB
RC
2a
2b
2c
2d
2e
3a
3b
3c
4a
4b
5a
5b
5c
5d
6a
6b
7a
7b
7c
8a
8b
9
2. Experimental
2.1 Isolation of piplartine and piperine
Roots of Piper tuberculatum were harvested on the Campus of
˜
˜
the University of Sao Paulo (USP), Sao Paulo, Brazil. The
H
H
Br
Br
botanical classication was performed by Dr Elsie Franklin
˜
ˆ
Guimaraes (Instituto de Pesquisas Jardim Botanico do Rio de
Janeiro). A voucher specimen (Kato-0169) was deposited at the
Herbarium of the same institute. For the isolation of piplartine,
100 g of ground dry roots of P. tuberculatum were extracted with
a mixture of chloroform/methanol 2 : 1 (1 L) for 72 hours. The
solvent mixture was dried under reduced pressure using a roto-
evaporator to yield a white solid which, aer recrystallization in
MeOH,17 yielded pure piplartine (1a). The identity of piplartine
was conrmed by comparison of NMR and MS data with those
reported.18,19
OCH2O
OCH2O
H
H
H
H
H
H
H
H
OCH2O
OCH2O
H
H
H
OMe
OMe
OMe
Br
Br
OMe
2
2
2
H
H
Seeds of P. nigrum (black-pepper) were purchased in a local
˜
market in Sao Paulo. Piperine (6a) was puried from 250 g of
1 (D ¼ 0)
OMe
OCH2O
OMe
H
dried P. nigrum fruits. The fruits were ground to a ne powder
and extracted twice with 2 L of chloroform/methanol (2 : 1) v/v.
The extract was ltered and concentrated under vacuum using
10
2 (D ¼ 0)
a
For R see Fig. 6.
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Table 2 Molecular ions [M]+c (relative abundance%) and significant fragmentary ions observed in EI-MS spectra of amidesa
Amide
[M]+c
RCO+
Fragmentary ions
1a
1b
1c
1d
1e
1f
317 (90)
305 (100)
349 (37)
307 (85)
307 (50)
305 (60)
319 (20)
277 (43)
277 (31)
275 (55)
275 (51)
295 (21)
295 (10)
337 (5)
221 (100)
221 (55)
221 (100)
221 (95)
221 (100)
221 (65)
191 (100)
191 (100)
191 (100)
191 (100)
191 (100)
209 (72)
209 (74)
209 (100)
175 (100)
175 (100)
131 (100)
131 (100)
131 (100)
131 (100)
201 (87)
201 (57)
187 (100)
187 (100)
187 (100)
235 (37)
235 (25)
223 (15)
205 (5)
274 (32), 193 (20), 190 (32)
205 (40), 190 (22)
222 (63), 190 (15)
236 (42), 222 (100), 191 (26), 190 (27), 181 (55), 179 (27)
222 (60), 190 (26)
222 (100), 194 (25), 191 (35), 190 (25), 84 (69)
276 (12), 262 (10), 163 (11), 151 (15)
206 (35), 192 (40), 151 (45)
192 (23), 163 (14)
192 (35), 163 (20), 161 (20), 84 (42)
163 (12)
211 (70), 183 (13), 181 (11), 126 (27), 102 (100), 86 (52), 56 (34)
211 (75), 102 (100)
211 (91), 102 (100), 44 (83)
176 (32), 145 (81), 117 (37), 89 (47)
145 (42), 89 (26)
216 (14), 103 (33)
188 (10), 146 (20), 103 (45), 77 (25)
103 (60), 86 (26), 77 (28)
187 (14), 103 (57), 77 (32)
2a
2b
2c
2d
2e
3a
3b
3c
4a
4b
5a
5b
5c
5d
6a
6b
7a
7b
7c
8a
8b
9
261 (57)
303 (18)
259 (7)
217 (7)
217 (18)
215 (26)
285 (63)
287 (55)
273 (87)
315 (26)
271 (67)
321 (18)
319 (25)
321 (35)
289 (35)
202 (25), 173 (42), 143 (35), 115 (100), 84 (35)
173 (99), 115 (100)
188 (35), 155 (20), 159 (62), 144 (60), 121 (42), 115 (60)
144 (27), 128 (27), 115 (27), 44 (35)
159 (30), 144 (40), 115 (42), 84 (35)
237 (35), 156 (25), 128 (100), 96 (45)
237 (25), 156 (27), 138 (27), 129 (28), 128 (100), 84 (74)
222 (100), 194 (40), 181 (25), 179 (55), 44 (75)
204 (25), 140 (31), 135 (25), 127 (100), 112 (52), 84 (35)
10
a
RCO+ ¼ [[M] ꢀ NRdRe]+.
4b, 5a–5c, 6b, 7a–7c, 8a and 8b were synthesized by addition of
triethylamine (3 equiv.) and the appropriate amines (i.e. n-dibu-
tylamine, morpholine, n-pentylamine or piperidine) to a methy-
lene chloride solution of the various acyl chlorides (1.0 equivalent).
The reaction mixtures were stirred overnight at room temperature,
quenched with saturated aqueous ammonium chloride solution
and then extracted with methylene chloride 3 times. Combined
organic phases were washed with brine and dried over anhydrous
magnesium sulphate. Aer ltration and concentration, the resi-
dues were puried by ash chromatography over silica-gel using
the hexanes-ethyl acetate (typically 10–30%) as eluent yielding the
desired amides.15
2.3 Mass spectrometry instrumentation (EI-MS and HRESI-MS)
GCMS analysis was performed using a GCMS-QP2010 Ultra gas
chromatograph (Shimadzu) with an AOC-20is series injector/
autosampler (Shimadzu, Kyoto, Japan) operating in the EI mode
at 70 eV with a Rxi-5ms fused silica capillary column (30 m ꢂ
0.25 mm ID ꢂ 0.25 mm df). The front inlet temperature was 280 ꢁC.
The helium gas ow rate through the column was 0.51 mL minꢀ1
.
The column temperature was held isothermally at 60 ꢁC for 1 min
and then ramped up from 60 to 320 ꢁC by 35 ꢁC minꢀ1 and held
isothermally for 6 min. The transfer line and ion-source temper-
atures were 240 and 260 ꢁC, respectively. Experiments were
recorded on scan mode over the mass range 35–500 m/z.
Compounds 1b, 2e and 5d were synthesized by the slow addi-
tion of n-butyl lithium (1.3 equivalent), at ꢀ78 ꢁC under nitrogen
atmosphere, to a solution of 2-pyrrolidinone (1.2 equiv.) in dry
THF. Aer 15 min, solutions of the corresponding acyl chlorides
(1.0 equiv.) were added. The reaction mixtures were stirred for 1
hour at room temperature, quenched with saturated aqueous
ammonium chloride solution and then extracted with ethyl acetate
(3 times). Combined organic phases were washed with brine and
dried over anhydrous magnesium sulphate. Aer ltration and
concentration, the residues were puried by ash chromatography
over silica-gel using the hexanes-ethyl acetate as eluent yielding the
desired imides.
For HRESI-MS, the samples were analyzed by liquid chroma-
tography and mass spectrometry Bruker MicrOTOF-QII Bruker.
The High Performance Liquid Chromatograph (Shimadzu, Kyoto,
Japan) was composed by two analytical pumps (model LC-20AD),
an automatic injector (SIL-20AHT), a UV/Vis detector (SPD-20A),
and a column oven (CTO-20A) controlled by a CBM-20A module.
The mobile phase contained acetonitrile (+0.1% of formic acid)
v/v and water (+0.1% of formic acid) v/v and the gradient was 40%
acetonitrile at 0 min (for up to 2 min), and then it was linearly
raised to 100% acetonitrile (from 2 to 30 min) and kept at a plateau
˚
for 5 min. The column was a reverse phase Luna 5m PFP(2) 100 A,
150 ꢂ 2 mm (Phenomenex). The monitored wavelength was
ꢁ
254 nm, and the column oven was set at 40 C and the mobile
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Table 3 Molecular formula, compound exact mass, HRESI [M + H]+ and acylium ions (RCO+) observed in MS/MS spectra of amidesa
Paper
Amides
Molecular formula
[M + H]+ calculated
[M + H]+ observed
Error ppm
RCO+
Molecular formula
+
1a
1b
1c
1d
1e
1f
C17H19NO5
C16H19NO5
C20H31NO4
C17H25NO4
C16H21NO5
C17H23NO4
C19H29NO3
C16H23NO3
C15H19NO4
C16H21NO3
C15H17NO4
C13H14BrNO2
C14H18BrNO
C17H24BrNO
C14H15NO4
C18H25NO3
C17H25NO
318.1336
306.1336
350.2326
308.1856
308.1492
306.1700
320.2220
278.1751
278.1387
276.1594
276.1230
296.0281
296.0645
338.1114
262.1074
304.1907
260.2008
218.1539
218.1175
216.1019
286.1438
288.1594
274.1802
316.2260
272.1645
322.0801
320.0645
322.1649
290.1751
318.1337
306.1345
350.2334
308.1859
308.1496
306.1704
320.2226
278.1758
278.1389
276.1602
276.1235
296.0282
296.0647
338.1116
262.1072
304.1902
260.2010
218.1538
218.1180
216.1015
286.1441
288.1596
274.1800
316.2258
272.1646
322.0801
320.0649
322.1649
290.1752
0.31
2.94
2.28
0.97
1.30
1.31
1.87
2.52
0.72
2.90
1.81
0.34
0.68
0.59
ꢀ0.76
ꢀ1.64
0.77
ꢀ0.46
2.29
ꢀ1.85
1.05
0.69
ꢀ0.73
ꢀ0.63
0.37
0.00
1.25
0.00
0.34
221.0803
221.0859
221.0817
221.0830
221.0822
221.0815
191.0703
191.0702
191.0706
191.0700
191.0702
208.9588
208.9585
208.9533
175.0384
175.0380
131.0430
131.0487
131.0482
131.0480
201.0556
201.0535
187.0744
187.0743
187.0744
234.9745
234.9749
223.0969
205.0845
C12H13O4
C12H13O4
C12H13O4
C12H13O4
C12H13O4
C12H13O4
C11H11O3
C11H11O3
+
+
+
+
+
+
2a
2b
2c
2d
2e
3a
3b
3c
4a
4b
5a
5b
5c
5d
6a
6b
7a
7b
7c
8a
8b
9
+
+
C11H11O3
C11H11O3
C11H11O3
+
+
C9H6BrO+
C9H6BrO+
C9H6BrO+
+
C10H7O3
+
C10H7O3
C9H7O+
C9H7O+
C9H7O+
C9H7O+
C14H19NO
C13H15NO2
C13H13NO2
C17H19NO3
C17H21NO3
C17H23NO3
C20H29NO2
C17H21NO2
C16H20BrNO
C16H18BrNO
C17H23NO5
C17H23NO3
+
C12H10O3
C12H10O3
C12H12O2
C12H12O2
+
+
+
+
C12H12O2
C11H9BrO+
C11H9BrO+
+
C12H15O4
C12H14O3
+
10
RCO+ ¼ [[M + H] ꢀ NRdRe].
a
phase ow was 200 mL minꢀ1 directly infused into the mass
spectrophotometer.
Proton affinity (PA) was dened as the negative of the
enthalpy variation (DrHꢁ) for the reaction M + H+ / MH+:
The mass spectrophotometer worked in positive mode with
N2 as nebulizer and drying gas at 4 Bar and 8 L minꢀ1, respec-
tively. The drying temperature was set to 200 C; the collision
PA ¼ ꢀDrH298, DrH298 ¼ Eel(MH+) ꢀ Eel(M).
ꢁ
energy and the quadruple energy were set to 12 and 6 eV,
respectively. RF1 and RF2 funnels were programmed to 400 Vpp
and the monitored mass range was 100–1000 kDa.
For H+, no calculations are required, and the only other non-
zero energy term is the difference in translational energy, which
was equal to 3/2 RT z 3.7 kJ molꢀ1
.
24–26
Additionally, bond energies were calculated using the
Spartan 16 (Wavefunction Inc, Irvine, CA, USA) for windows.
The energies of the compounds were calculated with DFT
calculations at the level B3LYP/6-311+G** by using the single
point method.27 Previously a conformational search with the
molecular mechanics method MMFF (Merck Molecular Force
Field) was performed and the geometry of the lowest energy
conformer of each compound was optimized by using the PM6
or AM1 semi-empirical method.28,29 Bond energies between
amide nitrogen and carbonyl were compared for each
compound with protons bound at the amide nitrogen and at the
carbonyl oxygen.
2.4 NMR analysis
1H and 13C NMR spectra were acquired using a Varian Gemini
200 spectrometer operating at 200 and 50 MHz, and a Bruker
(DRX 300) spectrometer operating at 300 MHz and 75 MHz, both
available at the Analytical Center of the Institute of Chemistry of
˜
the University of Sao Paulo.
2.5 Computational method
Density functional theory (DFT) calculations were performed
using Spartan 10 for Windows (Wavefunction Inc, Irvine, CA,
USA).22,23 Each studied amide had its neutral, protonated [M +
H]+ and acyl cation forms examined at the B3LYP/6-311G* level
and the lowest energy conformers were selected for the calcu-
3. Results and discussion
lations. The global minimum on the potential energy surface The 29 amides analyzed in our study were divided in to 10 types
was used for the determination of each geometry.
primarily, according to the aromatic ring substitution pattern,
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Fig. 3 General structures of amides.
Fig. 6 Amine (RC, RD, RE, RG) and lactame (RA, RB, RF) moieties.
Table 4 Proton affinity (kJ molꢀ1) of amide (PA)a
Amides
PA(a)
PA(b)
DP
1c
1d
1f
6a
10
933.66
973.89
984.40
996.12
980.89
903.37
946.86
976.82
976.24
945.19
30.29
27.03
7.58
19.88
35.70
Fig. 7 McLafferty rearrangement for amide 10 under EI conditions.
a
a and b: bonding sites for hydrogens (Fig. 3).
characteristics prevents the McLafferty rearrangement and lead
frequently to the N–CO a-cleavage due to conjugation of the a,b-
unsaturated carbonyl group of the amide (Fig. 5). For all amides
analyzed under 70 eV, the relatively intense molecular ions [M]c+
were observed (Table 2) and aryl acylium daughter ions were
observed for all amides.
side chain size and number of double bonds varying from one
double bond (1–5), two double bonds (6–8) or saturated chain
(9–10) (Table 1 and Fig. 6). Piplartine (1a) and piperine (6a), the
model compounds for conjugated amides, were isolated from
Piper species while their derivatives were synthesized as
described. Non-optimized yields of this synthesis ranged from
50 to 90% and all compounds were characterized by NMR and
MS analysis (See Experimental).
Mass spectrometric analysis of amides by EI-MS quite oen
provides important information for their characterization. In
general, aliphatic primary amides produce an intense frag-
mentary ion peak at m/z 44 (CONH2) resulting from the cleavage
of the R–CONH2 bond.30,31 Aliphatic secondary and tertiary
amides having hydrogens at the g-carbon of the acyl moiety or
N-methyl groups show intense fragmentary ions resulting from
McLafferty rearrangement.30 For aromatic amides a resonance-
stabilized benzoyl cation is formed, and this may undergo
further cleavage of CO loss leading to a phenyl cation.31
Piperamides investigated in the present work have aryl
groups at the position 3 (1–5) or 5 (6–10) and, are in most of the
cases a,b-unsaturated carbonyl amides. These peculiar
The relative intensities of the molecular ions for most of the
amides were higher than 10% and quite oen ranging from 50–
100%. The only exceptions were observed to be 3c (5%) and 5a
(7%) and 5b (7%), which were associated to the N,N-dibutyl and
N-pentyl amides, respectively. The initial fragmentation yielded
acylium ions which were prominent and very informative on the
structures of carboxylic acid moieties in all cases. For instance,
the acylium ion observed at m/z 221 for piplartine (1a), associ-
ated to the 3,4,5-trimethoxycinnamoyl ion, was also observed
for 1b–1f (Table 2 and Fig. 5). Any changes in the substitution
pattern of all synthetic amides were accordingly observed in the
corresponding acylium ion. Thus, m/z at 191 (3,4-dimethox-
ycinnamoyl) was observed for 2a–2e; m/z at 209 (p-bromo-
cinnamoyl) for 3a–3c (Table 2 and Fig. 5). In the cases of amides
of types 3 and 8, the typical isotope M + 2 for brominated
compounds can be tracked and the acylium ion produces
further the peak base at m/z 102 (100%) resulting from the loss
of Br and CO (Table 2 and Fig. 5). In fact, the loss of CO from the
acylium ions can be observed in most of the cases with variable
intensity, excepting for 4a and 4b.
Fig. 4 General structures of imides.
Fig. 8 Main fragmentation pathway for amides (A) and imides (B) in
Fig. 5 Main fragmentation pattern of the amides in EI-MS.
HRESI-MS.
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Table 5 Proton affinity (kJ molꢀ1) of imides (PA)a
Table 7 Bond energy (kJ molꢀ1) of imides and proton bonded at a,
b and c and of the N–C bonded at position 1 (E1, Ea1, Eb1, Ec1) calculated
by Spartan 16 softwarea
Amide
PA(a)
PA(b)
PA(c)
1a
1b
2e
5d
9
945.75
951.73
981.27
951.08
941.94
898.82
887.15
885.84
891.88
889.96
927.77
899.90
899.73 E1
917.21 Ea1
946.81 Eb1
Ec1
position
1a
1b
2e
5d
9
764.92
148.35
115.22
139.98
774.95
168.29
97.53
678.95
86.81
11.76
39.36
731.28
108.23
38.21
770.14
99.55
84.98
124.70
67.22
112.60
a
a, b and c: bonding sites for hydrogens (Fig. 4).
a
a, b and c: bonding sites for hydrogens (Fig. 4).
The amides of type 9 and 10 had the lowest relative abun-
dance of the acylium ions RCO+ possibly because of the lack of
conjugation to the carbonyl group. In this case, the tropylium
cations were observed at m/z 181 and 135, respectively. The
tertiary amide 10, with hydrogen at the g-carbon and not
conjugated, displayed the base peak at m/z 127, resulting from
the McLafferty rearrangement with elimination of safrole as
a neutral molecule (Fig. 7).
The 10 different types of amides were further investigated
under HRESI-MS conditions to determine similarities in their
behavior. First, all 10 amide-types analyzed by HRESI-MS dis-
played intense ions of protonated molecule [M + H]+ (Table 3).
Despite of the relative lower basicity of the amide nitrogen, the
positive mode is still preferable over the negative one yielding
higher intensity of ions (data not shown). As observed for the
fragmentation pattern of amides using EI-MS and from previous
data,32 the aryl acylium ions resulting from the N–CO a-cleavage
with the loss of neutral amines, were observed for all amides
(Table 3) (Fig. 8). Accordingly, the formation of aryl acylium ion at
m/z 221 from piplartine (1a) and at m/z 201 from piperine (6a)
were observed as reported.14,16 Indeed, the acylium ions observed
in the EIMS were consistently observed in the ESIMS spectra for
all cases (1–10) and such fragmentation is the most important for
characterizing the carboxylic acid moieties. The amide 10 showed
an aryl acylium ion at m/z 205 in its HRESI spectrum but also
displayed ions resulting from the McLafferty rearrangement
similar to what was observed with EI (Fig. 7, Tables 2 and 3).
In order to determine the preferable protonation sites for each
amides in HRESI-MS, and to further elucidate the fragmentation
mechanisms, the proton affinity of ve amides was calculated for
two main binding sites: carbonyl oxygen and amide nitrogen (a
and b in Fig. 3, respectively). Five amides (1c, 1d, 1f, 6a and 10)
were used as a template because their representative structures
and because it was assumed that their differing substituents in
the aryl group would not affect signicantly the overall results. In
general, compounds with proton a bonded to the nitrogen have
relatively lower energy, with DP ranging from 7.6 to 35.7 kJ molꢀ1
(Table 4), supporting the formation of a protonated molecular
ion with the proton preferentially bonded to the nitrogen. Thus,
the elimination of the amine molecules would facilitate the
formation of acylium cations.
The hydrogen bonding energy to amide nitrogen and
carbonyl were calculated with Spartan soware. The application
of DFT at the B3LYP level, generated a set of bond energies that
can be compared. The calculated bond energy values calculated
by DFT-B3LYP of all the amides show that the Eb (N–C bond
energy with proton attached to amide nitrogen) energies were
signicantly lower than the Ea (N–C bond energy with proton
attached to the carboxyl oxygen) (Table 6) energies or not
protonated amides (E). Therefore, the fragmentation of the N–C
bond is preferable in order to generate the acylium ions when
the proton is attached to amide nitrogen as it would be mech-
anistically expected.
Similar proles of energies were observed for imide deriva-
tives (1a, 1b, 2e, 5d and 9) (Fig. 4 and Table 5). The PA (Proton
Affinity) values were lower with the hydrogens bonded to the
nitrogen than to the carbonyl oxygen at position a or c (Fig. 4
and Table 5).
We also calculated the bond energies at position 1 of N–C
various of imides derivatives. The values of the bond energies
calculated by DFT-B3LYP for all of the imides show that the Eb1
(N–C bond energy with proton attached to amide nitrogen)
energies were notably lower than Ea1 and Ec1 (Table 7). Speci-
cally, in the case of compound 9, which lacks conjugation
between the carbonyl and the phenyl group, the bond energy
difference is the smallest (Table 7). Thus, the fragmentation of
the N–C is similarly preferred when the proton is attached to
imide nitrogen.
Table 6 Bond energy (kJ molꢀ1) of amides with proton bonded at
position a and b of the N–C bond (E, Ea, Eb) calculated with Spartan 16
softwarea
Amides
E
Ea
Eb
1c
1d
1e
1f
1695.86
948.43
890.08
912.47
846.79
902.95
1528.89
801.33
1490.65
1522.02
891.07
833.14
803.07
838.06
838.07
1482.96
801.67
869.14
822.19
930.22
211.93
222.97
210.00
228.24
125.30
159.94
152.62
121.38
133.37
151.48
157.18
147.35
120.94
125.59
124.79
132.95
129.69
143.18
147.12
208.93
167.08
179.04
180.59
198.91
108.94
109.53
121.72
99.34
101.17
100.01
94.95
116.79
104.79
96.50
3a
3b
3c
4a
4b
5a
5b
5c
6a
6b
7a
7b
7c
8a
8b
10
94.25
105.32
102.17
91.71
108.35
148.45
a
a and b: bonding sites for hydrogens (Fig. 3).
21412 | RSC Adv., 2018, 8, 21407–21413
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M. W. Lima and L. V. Costa-Lotufo, J. Appl. Toxicol., 2008,
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4. Conclusions
9 R. M. Gutierrez, A. M. Gonzalez and C. Hoyo-Vadillo, Mini-
Rev. Med. Chem., 2011, 13, 163–193.
In this work we compared the fragmentation pattern of two
natural piperamides (piplartine and piperine) and several
synthetic derivatives that were analysed under HRESI-MS and
EI-MS conditions. Piplartine and piperine (a,b-unsaturated
amides) have shown a N–CO a-cleavage, a characteristic frag-
mentation observed as a common pattern in all natural and
synthetic amides. Fragmentation mechanisms were proposed
based on computational analysis using DFT-B3LYP to calculate
proton affinities and bond energies. The computational
methods supported the fragmentation mechanism proposed in
the HRESI-MS experiment involving the protonation of piper-
amides and derivatives preferentially in the amide nitrogen.
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Conflicts of interest
There are no conicts to declare.
16 E. H. Schaab, A. E. M. Crotti, Y. Iamamoto, M. J. Kato,
L. V. C. Lotufo and N. P. Lopes, Biol. Pharm. Bull., 2010, 33,
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Acknowledgements
The authors are grateful for nancial support from the CNPq,
´
˜
17 D. P. Bezerra, F. O. Castro, A. P. N. N. Alves, C. Pessoa,
M. O. Moraes, E. R. Silveira, M. A. S. Lima, F. J. M. Elmiro
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CAPES, FAPESP (Proc. 2005/51850-9 and 2014/50316-7) and Pro-
Reitoria de Pesquisas USP. Financial support from Fundaçao
ˆ
para a Ciencia e a Tecnologia (FCT/MEC) Portugal, under
Project PEst-OE/QUI/UI0612/2013, and by the Associated Labo-
ratory for Sustainable Chemistry – Clean Processes and Tech-
nologies – LAQV which is nanced by national funds from FCT/
MEC (UID/QUI/50006/2013) and conanced by the ERDF under
the PT2020 Partnership Agreement (POCI-01-0145-FEDER-
007265) is acknowledged.
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