The mechanism of Troeger's base formation probed by electrospray ionization mass spectrometry

Article abstract of DOI:10.1021/jo062556w

(Chemical Equation Presented) Using direct infusion electrospray ionization mass and tandem mass spectrometric experiments [ESI-MS(/MS)], we have performed on-line monitoring of some reactions used to form Troeger's bases. Key intermediates, either as cationic species or as protonated forms of neutral species, have been intercepted and characterized. The role of urotropine as the methylene source in these reactions has also been accessed. Reaction pathways shown by ESI-MS(/MS) have been probed by gas-phase ion/molecule reactions, and an expanded mechanism for Troeger's base formation based on the mass spectrometric data has been elaborated.

Full text of DOI:10.1021/jo062556w

The Mechanism of Tro1ger’s Base Formation Probed by
Electrospray Ionization Mass Spectrometry
Carlos A. M. Abella,† Mario Benassi,‡ Leonardo S. Santos,§ Marcos N. Eberlin,*,‡ and
Fernando Coelho*,†
Laboratory of Synthesis of Natural Products and Drugs and the ThoMSon Mass Spectrometry Laboratory,
Institute of Chemistry, State UniVersity of Campinas, UNICAMP, 13084-971 Campinas, SP, Brazil, and
Instituto de Qu´ımica de Recursos Naturales, UniVersidad de Talca, P.O. Box 747, Talca, Chile
coelho@iqm.unicamp.br; eberlin@iqm.unicamp.br
ReceiVed December 13, 2006
Using direct infusion electrospray ionization mass and tandem mass spectrometric experiments [ESI-
MS(/MS)], we have performed on-line monitoring of some reactions used to form Tro¨ger’s bases. Key
intermediates, either as cationic species or as protonated forms of neutral species, have been intercepted
and characterized. The role of urotropine as the methylene source in these reactions has also been accessed.
Reaction pathways shown by ESI-MS(/MS) have been probed by gas-phase ion/molecule reactions, and
an expanded mechanism for Tro¨ger’s base formation based on the mass spectrometric data has been
elaborated.
Introduction
methanodibenzo[b,f][1,5]diazocine shown in Scheme 1, are
formed under acid catalysis by the condensation of anilines
(p-toluidine for instance) with formaldehyde. Tro¨ger’s bases
were the first amines to be optically resolved,3 and their
dissymmetry results from the impossibility of nitrogen inversion.
Although known for more than a century, it was only in the
1980s that interest in Tro¨ger’s bases increased greatly since they
started to be used as building blocks of molecular receptors.4
As evidenced by the pioneering work of Wilcox et al.,5 these
bases provide relatively rigid chiral frameworks for the con-
struction of chelating and biomimetic systems. The essential
characteristic of a small molecule receptor is concavity,
In 1887, Julius Tro¨ger1 described the formation of a class of
fascinating molecules2 known today as Tro¨ger’s bases. These
relatively simply but geometrically rich V-shaped bicyclic
molecules, such as the chiral 2,8-dimethyl-6H,12H-(5,11)-
* Corresponding author (F.C.) Phone: (+55) 19-3521-3085; (M.N.E.)
Phone: (+55) 19-3521-3073; (for both) Fax: (+55) 19-3521-3023.
† Laboratory of Synthesis of Natural Products and Drugs, UNICAMP.
‡ ThoMSon Mass Spectrometry Laboratory, UNICAMP.
§ Universidad de Talca.
(1) (a) Tro¨ger, J. J. Prakt. Chem. 1887, 36, 225. (b) For a recent review,
see: Val´ık, M.; Strongin, R. M.; Kra´l, V. Supramol. Chem. 2005, 17, 347.
(2) Fascinating Molecules in Organic Chemistry; Vo¨gtle, F., Ed.; John
Wiley: New York, 1992; pp 237-249.
(3) Prelog, V.; Wieland, P. HelV. Chim. Acta 1944, 27, 1127.
10.1021/jo062556w CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/02/2007
4048
J. Org. Chem. 2007, 72, 4048-4054

Mechanism of Tro¨ger’s Base Formation
SCHEME 1. Tro1ger’s Base from p-Toluidine and
Formaldehyde
SCHEME 2. Original Mechanism Proposed by Wagner for
the Formation of Tro1ger’s Bases
FIGURE 1. Intermediates proposed by Farrar for the formation of
Tro¨ger’s bases and its byproduct 8.
SCHEME 3. Intermediate Proposed by Cooper and
Partridge to Explain the Formation of Tro1ger’s Base
and the majority of synthetic receptors have used macrocyclic
rings to enforce the formation of concave surfaces. For Tro¨ger’s
bases, however, their rigid chiral grooves are maintained by
conformational constraints intrinsic to their unique architecture
and are produced without recourse to macrocyclic structural
elements. Tro¨ger’s bases have been used therefore mainly as
synthetic receptors but also as chiral solvating agents,6 as
fluorescent compounds,7 and for biological8 and catalytic
activity.9 Recently, Tro¨ger’s base interactions with DNA have
been explored, as exemplified by the work of Yashima et al.
who used a Tro¨ger base derived from chiral bis(1,10-phenan-
throline) for DNA recognition.10 Demeunynck et al. also used
both the unique geometry and chirality of Tro¨ger’s bases with
the DNA binding properties of acridines to form a new family
of C2 chiral DNA binding molecules.11 The binding of the
proflavine-based Tro¨ger base has also been shown to be both
enantio- and sequence-specific.12 Asymmetric Tro¨ger bases
containing the two well-characterized DNA binding chro-
mophores, proflavine and phenanthroline, have also been
formed.13 The proflavine moiety was shown to intercalate
between DNA base pairs, and the phenanthroline ring occupied
the DNA groove. Tro¨ger’s bases have been used also as
molecular tweezers by extending their aromatic surfaces via
fusion of the methanodiazocine core with other Tro¨ger’s bases.
Examples of such molecular tweezers are bis-Tro¨ger’s bases14
and tris-Tro¨ger’s base analogues.15
The first attempts to establish the structures of Tro¨ger’s bases
by Lo¨b,16 Goecke,17 Lepetit,18 and Eisner and Wagner19 were
incorrect, but in 1935, Spielman20 succeeded in determining their
correct and unique V-shaped structures. The angle formed by
the least-square planes containing the two aryl rings changes
depending on the ring substituents and varies from 88 to 113°.21
The mechanism of Tro¨ger’s base formation was extensively
studied by Wagner in the 1930s,22 and Scheme 2 depicts his
first proposal.
In the classical reaction conditions (Scheme 2), the first step
involves the acid-catalyzed reaction of p-toluidine 1 with
formaldehyde, which acts as the source of methylene to give
imine 2 as the first key intermediate. Acid-catalyzed condensa-
tion of [2 + H]+ with 1 followed by two concomitant methylene
additions accompanied by cyclizations gives Tro¨ger’s base 6
(4) (a) Adrian, J. C., Jr.; Wilcox, C. S. J. Am. Chem. Soc. 1989, 111,
8055. (b) Crossley, M. J.; Hambley, T. W.; Mackay, L. G.; Try, A. C.;
Walton, R. J. Chem. Soc., Chem. Commun. 1995, 10, 1077. (c) Crossley,
M. J.; Mackay L. G.; Try, A. C. J. Chem. Soc., Chem. Commun. 1995, 18,
1925. (d) Goswami, S.; Ghosh, K. Tetrahedron Lett. 1997, 38, 4503. (e)
Manjula, A.; Nagarajan, M. Tetrahedron 1997, 53, 11859. (f) Hansson, A.
P.; Norrby, P.; Wa¨rnmark, K. Tetrahedron Lett. 1998, 39, 4565. (g)
Goswami, S.; Ghosh, K.; Dasgupta, S. J. Org. Chem. 2000, 65, 1907-
1914.
(5) (a) Wilcox, C. S. Tetrahedron Lett. 1985, 26, 5749. (b) Wilcox, C.
S.; Cowart, M. D. Tetrahedron Lett. 1986, 27, 5563. (c) Wilcox, C. S.;
Adrian, J. C., Jr.; Webb, T. H.; Zawacki, F. J. J. Am. Chem. Soc. 1992,
114, 10189. (d) Webb, T. H.; Wilcox, C. S. Chem. Soc. ReV. 1993, 383.
(6) Wilen, S. H.; Qi, J. Z.; Williard, P. G. J. Org. Chem. 1991, 56, 485.
(7) (a) Zhao, L.; Xu, J. J.; Zhang, G.; Bu, X.; Shionoya, M. Opt. Lett.
1999, 24, 1793. (b) Abella, C. A. M.; Rodembusch, F. S.; Stefani, V.
Tetrahedron Lett. 2004, 45, 5601. (c) Deprez, N. R.; McNitt, K. A.; Petersen,
M. E.; Brown, R. G.; Lewis, D. E. Tetrahedron Lett. 2005, 46, 2149.
(8) Johnson, R. A.; Gorman, R. R.; Wnuk, R. J.; Crittenden, N. J.; Aiken,
J. W. J. Med. Chem. 1993, 36, 3202.
(13) Baldeyrou, B.; Tardy, C.; Bailly, C.; Colson, P.; Houssier, C.;
Charmantray, F.; Demeunynck, M. Eur. J. Med. Chem. 2002, 37, 315.
(14) Pardo, C.; Sesmilo, E.; Gutie´rrez-Puebla, E.; Monge, A.; Elguero,
J.; Fruchier, A. J. Org. Chem. 2001, 66, 1607.
(15) (a) Mas, T.; Pardo, C.; Elguero, J. MendeleeV Commun. 2004, 6,
235. (b) Dolensky´, B.; Val´ık, M.; Sy´kora, D.; Kra´l, V. Org. Lett. 2005, 7,
67. (c) Hansson, A.; Wixe, T.; Bergquist, K-E.; Wa¨rnmark, K. Org. Lett.
2005, 7, 2019-2022.
(16) Lo¨b, W. Z. Elektrochem. 1898, 4, 428.
(17) Goecke, E. Z. Elektrochem. 1903, 9, 470.
(18) (a) Lepetit, R.; Maimeri, C. Atti. Accad. Naz. Lincei. 1917, 26, 558.
(b) Lepetit, R.; Maffei, G.; Maimeri, C. Gazz. Chim. Ital. 1927, 57, 862.
(19) Eisner, A.; Wagner, E. C. J. Am. Chem. Soc. 1934, 56, 1938.
(20) Spielman, M. A. J. Am. Chem. Soc. 1935, 57, 583.
(21) (a) Larson, S. B.; Wilcox, C. S. Acta Crystallogr., Sect. C 1986,
42, 224. (b) Pardo, C.; Alkorta, I.; Elguero, J. Tetrahedron: Asymmetry
2006, 17, 191-198.
(9) Goldberg, Y.; Alper, H. Tetrahedron Lett. 1995, 36, 369.
(10) Yashima, E.; Akashi, M.; Miyauchi, N. Chem. Lett. 1991, 6, 1017.
(11) Tatiboue¨t, A.; Demeunynck, M.; Andraud, C.; Collet, A.; Shomme,
J. Chem. Commun. 1999, 2, 161.
(12) Laine, W.; Demeunynck, M.; Lhome, J. Biochem. Biophys. Res.
Commun. 2000, 273, 681.
(22) (a) Wagner, E. C. J. Am. Chem. Soc. 1935, 57, 1296. (b) Miller, T.
R.; Wagner, E. C. J. Am. Chem. Soc. 1941, 63, 832.
J. Org. Chem, Vol. 72, No. 11, 2007 4049

Abella et al.
FIGURE 2. ESI-MS for the reaction solution of 1 and formaldehyde in neat TFA after 3 min of reaction.
SCHEME 4. Proposed Mechanism for the Formation of the
Ion of m/z 150
via intermediates 3-5. Intermediate 5 has been, however,
questioned, for instance, by Farrar,23 who proposed instead the
participation of intermediate 7 (Figure 1). Farrar also isolated a
byproduct identified as diazajulolidine 8, which led him to
propose 9 as an additional intermediate that leads to 8.
Cooper and Partridge24 demonstrated that Tro¨ger’s bases
could also be obtained via intermediate 14 (Scheme 3), which
they were able to synthesize. Tetrahydrophenhomazine 14 was
shown to react readily with formaldehyde to yield Tro¨ger’s
base 6.
In general, it is now accepted that the mechanism for Tro¨ger’s
base formation involves a series of in situ Friedel-Crafts
reactions of 2, but the detailed course of the reaction has not
yet been fully established. In trying to elucidate major mecha-
nistic aspects, different methylene sources as well as different
anilines have been used to form Tro¨ger’s bases.
SCHEME 5. Proposed Mechanism for the Formation of
[5 - H]+ of m/z 267
In this study, we used direct infusion electrospray ionization
mass and tandem mass spectrometric experiments (ESI-MS/MS)
to monitor Tro¨ger’s base formation from different anilines (p-
toluidine and 4-aminoveratrol),25 using both formaldehyde and
urotropine as the methylene source. ESI-MS/MS has been
selected since it is rapidly becoming an important technique
for mechanistic studies of chemical reactions in solution.26-28
Since ESI is known to be highly efficient in transferring ions
to the gas phase in a gentle way that most often causes little or
no dissociation, providing therefore rapid snapshots of the ionic
population in solution, we expected that ESI-MS could ef-
ficiently “fish” major intermediates, as well as reagents and
products (Scheme 1), directly from solution to the gas phase,
either in their cationic or protonated forms (expected to be
abundant in the acid medium), so they could be properly
characterized via both ESI-MS and ESI-MS/MS experiments.29
Results and Discussion
Formaldehyde: First in our mechanistic investigation, we
performed a classical reaction for Tro¨ger’s base formation, that
is, that of p-toluidine in neat TFA as solvent with formaldehyde
(23) Farrar, W. V. J. Appl. Chem. 1964, 14, 389.
(24) Cooper, F. C.; Partridge, M. W. J. Chem. Soc. 1955, Part 3, 991.
(25) The data acquired with veratrol are similar to those obtained with
formaldehyde, hence such results are not discussed.
(26) (a) Griep-Raming, J.; Meyer, S.; Bruhn, T.; Metzger, J. O. Angew.
Chem., Int. Ed. 2002, 41, 2738. (b) Meyer, S.; Koch, R.; Metzger, J. O.
Angew. Chem., Int. Ed. 2003, 42, 4700. (c) Santos, L. S.; Metzger, J. O.
Angew. Chem., Int. Ed. 2006, 45, 977. (d) Santos, L. S.; Knaack, L.;
Metzger, J. O. Int. J. Mass Spectrom. 2005, 246, 84.
(27) (a) Santos, L. S.; Pavam, C. H.; Almeida, W. P.; Coelho, F.; Eberlin,
M. N. Angew. Chem. 2004, 116, 4430; Angew. Chem., Int. Ed. 2004, 43,
4330. (b) Domingos, J. B.; Longhinotti, E.; Brandao, T. A. S.; Bunton, C.
A.; Santos, L. S.; Eberlin, M. N.; Nome, F. J. Org. Chem. 2004, 69, 6024.
(c) Meurer, E. C.; Santos, L. S.; Pilli, R. A.; Eberlin, M. N. Org. Lett.
2003, 5, 1391. (d) Sabino, A. A.; Machado, A. H. L.; Correia, C. R. D.;
Eberlin, M. N. Angew. Chem. 2004, 116, 2568; Angew. Chem., Int. Ed.
2004, 43, 2514.
(28) (a) Hilderling, C.; Adlhart, C.; Chen, P. Angew. Chem. 1998, 110,
2831; Angew. Chem., Int. Ed. 1998, 37, 2685. (b) Chen, P. Angew. Chem.
2003, 115, 2938; Angew. Chem., Int. Ed. 2003, 42, 2832. (c) Raminelli,
C.; Prechtl, M. H. G.; Santos, L. S.; Eberlin, M. N.; Comasseto, J. V.
Organometallics 2004, 23, 3990. (d) Domingos, J. B.; Longhinotti, E.;
Brandao, T. A. S.; Santos, L. S.; Eberlin, M. N.; Bunton, C. A.; Nome, F.
J. Org. Chem. 2004, 69, 7898.
(29) (a) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B.
Anal. Chem. 1985, 57, 675. (b) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong,
S. F.; Whitehouse, C. M. Science 1989, 246, 64. (c) de la Mora, J. F.; Van
Berckel, G. J.; Enke, C. G.; Cole, R. B.; Martinez-Sanchez, M.; Fenn, J. B.
J. Mass Spectrom. 2000, 35, 939.
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Mechanism of Tro¨ger’s Base Formation
SCHEME 6. Proposed Routes to the Dissociation of
[4 - H]+
of m/z 237. ESI-MS(/MS) monitoring has been able therefore,
in the reaction of p-toluidine and formaldehyde in neat TFA,
to intercept and characterize in cationic forms two key inter-
mediates, 2 and 4 (Scheme 2). The ion of m/z 237 is presented
as resulting from a hydride loss from 4. We believe that 4 is
oxidized in the ESI source,30 and that the driving force for this
oxidation is the formation of the stable [4 - H]+ displaying a
highly resonance-stabilized positive charge.
The ions of m/z 134, 150, and 267, which appeared at first
to have no direct relation to the formation of Tro¨ger’s bases,
were also isolated and dissociated (Figure 3A-C). The ion of
+
m/z 134 dissociates mainly to C7H7 of m/z 91 as well as by
methyl and HCN loss and, from this dissociation behavior, we
propose the ion to be the protonated form of N-methylated 2.
The mechanism for N-methylation of 2 is, however, unclear to
us at the moment.
The ion of m/z 150 seems to result from the incorporation of
a molecule of formaldehyde into 2 (Scheme 4). Its ESI-MS/
MS (Figure 3B) corroborates this possibility as the proposed
bicyclic ion dissociates mainly by the loss of formaldehyde.
The ion of m/z 267 (Figure 3C) is attributed to the interception
of another key intermediate for Tro¨ger’s base formation (Scheme
2), that is, [5 - H]+ (Scheme 5). The ion dissociates mainly by
formaldehyde loss to afford likely [4 - H]+ of m/z 237.
Interestingly, a compound structurally related to [5 - H]+ has
been proposed by Wagner22 (Scheme 2) as an advanced
intermediate for Tro¨ger’s bases. Note that, in addition to the
ESI-MS/MS characterization of the intercepted species, high-
accuracy m/z measurements also corroborate the proposed
structures (see Supporting Information).
FIGURE 3. ESI-MS/MS for the ions of m/z 134, 150, and 267.
Urotropine: To gain further insights of the mechanism of
Tro¨ger’s base formation, we performed the reaction of p-
toluidine with urotropine (15) as an alternative methylene source
(instead of formaldehyde) again in neat TFA at room temper-
ature. A clear reaction solution was formed, which was
monitored by on-line ESI-MS initiated approximately 20 s after
mixing. From 30 s up to 1 min of reaction, ESI-MS detects
only the two starting reactants in their protonated forms, that
is, [1 + H]+ of m/z 108 and [15 + H]+ of m/z 141, affording
ESI-MS similar to that shown in Figure 4a. After 2 min, the
same imine intermediate 2 (as for Figure 2) in its protonated
form [2 + H]+ of m/z 120 shows up clearly (Figure 3B). When
as the methylene source. At room temperature, a clear reaction
solution was formed and directly injected to the ESI source
operating in the positive ion mode. Approximately 3 min after
mixing, ESI-MS (Figure 2) detects four cationic species
attributed to key participants of the reaction: the protonated
reactant [1 + H]+ (Scheme 2) of m/z 108, [2 + H]+ of m/z
120, [4a - H]+ of m/z 237 (the species preceding 4), and the
final product, that is the protonated Tro¨ger base [6 + H]+ of
m/z 251. These key species were characterized by collision-
induced dissociation (CID) via ESI(+)-MS/MS experiments (see
discussion below for the reaction with urotropine).
After a few more minutes (5 min), ESI-MS shows that most
of 2 had been consumed and [6 + H]+ of m/z 251 was now
detected as by far the major ion, with some residual [4 - H]+
(30) Cerchiaro, G.; Saboya, P. L.; Ferreira, A. M. C.; Tomazela, D. M.;
Eberlin, M. N. Transition Met. Chem. 2004, 29, 495.
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Abella et al.
FIGURE 4. ESI(+)-MS acquired for the reaction solution in neat TFA during the on-line monitoring of the reaction of p-toluidine and urotropine
(15) after (a) 20 s; (b) 2 min; (c) 3 min; (d) 3.5 min; (e) 15 min; and (f) 35 min of reaction.
SCHEME 7. Proposed Routes for the Dissociation of the
SCHEME 8. Proposed Mechanism for the Action of
Urotropine as a Methylene Source in Tro1ger’s Base
Formation
Protonated Tro1ger’s Base [6 + H]+
hydrogens from [4 - H]+ to form the aromatic pyrimidine
cationic derivative [4 - 3H]+. Finally, after 3.5 min of reaction
(Figure 4d), the Tro¨ger’s base final product showed up as [6 +
H]+ of m/z 251. From this moment on, as exemplified in Figure
4e,f by the ESI-MS acquired at 15 and 35 min of reaction, the
abundance of [6 + H]+ increased significantly, whereas the [2
+ H]+/[4 - 3H]+ abundance ratio decreased proportionally.
Interestingly, a byproduct attributed to 8, which has been isolated
previously by Farrar and Johnson,8,23 was also detected by ESI-
MS as [8 - H]+ of m/z 368 after 15 min of reaction and
characterized by CID (Figures 4e and S1e). The [8 - H]+
dissociates mainly by loss of neutral tolylimine (CH3-C6H4-
NdCH2) to form the ion of m/z 249. It also dissociates to the
iminium ion of m/z 120 (Figure S1e). We do not observe any
dissociation of [8 -H]+ to form an ion of m/z 251. The reduced
form of ion [8 - H]+ has already been detected and isolated
from the reaction medium by Johnson8 and Farrar.23 Once
formed, this compound is not converted to the Tro¨ger base.8
Furthermore, in regard to the ESI-MS/MS characterization
of the intercepted species, intermediate [4a - H]+ of m/z 237
(Figure S1c) contravenes the even-electron rule26 to lose a CH3
radical, thus forming the fragment ion of m/z 222. Scheme 6
this ion of m/z 120 was selected (by quadrupole filtering) and
dissociated (Figure S1a, see Supporting Information), it was
found to dissociate mainly by the loss of CH2dNH to form a
+
C7H7 ion of m/z 91. This ion displayed therefore the same
CID behavior as that of [2 + H]+ of m/z 120 intercepted in the
reaction using formaldehyde as the methylene source. After 3
min of reaction, an additional cationic species of m/z 235 was
detected (Figure 4c). This new intermediate was characterized
by ESI-MS/MS (Figure S1b). Dissociation proceeds mainly by
+
loss of neutral methylbenzopyrimidine to give a C7H7 ion of
m/z 91 and by CH3 radical loss to produce the distonic cation
of m/z 220. The ion of m/z 235 is rationalized to be formed in
the reaction medium via the abstraction by urotropine of benzylic
4052 J. Org. Chem., Vol. 72, No. 11, 2007

Mechanism of Tro¨ger’s Base Formation
SCHEME 9. Mechanism for the Formation of Tro1ger’s
Base as Probed by ESI(+)-MS(/MS) and Ion/Molecule
Reactions
FIGURE 5. ESI-MS screening for the reaction solution formed by
mixing TFA solutions of p-toluidine (1) with urotropine (15) on-line
using a microreactor coupled just before the ESI source and reaction
time of ca. 2.0 s and not 0.7 s.
intermediate 2 was indeed detected (in its protonated form of
m/z 120) in reactions of p-toluidine 1 with both formaldehyde
(Figure 2) and urotropine (15, Figure 4), but no intermediate
that could be attributed to any stage of urotropine hydrolysis
was detected. In trying to intercept early reaction intermediates
that could reveal exactly how urotropine acts as a methylene
source, TFA solutions of 1 and urotropine (15) were mixed in
a microreactor placed very close the ESI source; hence, in this
way, the reaction solution was electrosprayed just ca. 2.0 s after
mixing. Under these conditions, the ESI-MS of Figure 5 was
acquired. Interestingly, a new cationic intermediate of m/z 248
was observed, that is, [1 + urotropine + H]+. ESI-MS/MS of
this ion shows dissociation to occur mainly by the loss of neutral
tolylamine to give protonated urotropine of m/z 141 (Figure S1f).
This rather transient species that escaped detection without the
use of the microreactor provides therefore a clue for the action
of urotropine as a methylene source in Tro¨ger’s base forma-
tion: without the need of hydrolysis to formaldehyde, it transfer
methylene directly to 1 via acid-catalyzed nucleophilic attack
(Scheme 8).
To gain further evidence for the methylene transfer step, gas-
phase ion/molecule reactions of protonated urotropine (15) with
two volatile amines were performed. We used a hybrid linear
ion-trap equipment (2000 QTrap Applied Biosystems) in which
N2 gas was replaced with reactive neutrals by needle valve
adaptors that allowed the introduction of reactive gases into the
collision cell (Figure 6). Interestingly, 15 of m/z 141 was indeed
found to react with ethylamine and aniline to form directly the
respective iminium ions of m/z 56 and 106 (Figure 6). Figure
6a shows that the imine C2H5-NHdCH2+ (as for 2) of m/z 56
is formed in high yield in the ion/molecule reactions. Herein,
the adduct of m/z 186, the first transient intermediate leading
to methylene transfer, is obtained from nucleophilic addition
of the amine to protonated urotropine. Furthermore, in reactions
with aniline (Figure 6b), the corresponding imine of m/z 106
(as for 2), that is, Ph-NHdCH2+, is formed as an abundant
ion, and the transient adduct of m/z 234 is detected as a low
abundance ion. When aminoveratrol was used as the aniline
source and following the same procedure as described above,
the reaction showed the same behavior observed for p-
toluidine.25
FIGURE 6. ESI-MS/MS for gas-phase ion/molecule reactions of
protonated urotropine of m/z 141 with (a) ethylamine and (b) aniline.
rationalizes this loss, which forms a relatively stable distonic
radical cation,31,32 but the main dissociation pathway for [4a -
H]+ is by retro-addition to form the species [2 + H]+ of m/z
120 (Figure S1c).
The protonated Tro¨ger’s base, that is, [6a + H]+ of m/z 251,
also dissociates in a structurally diagnostic fashion (Figure
S1d): as for [4a + H]+, it loses a CH3 radical to form the
ion of m/z 236 (Scheme 7), by the loss of a neutral imine
CH3-C6H4-NdCH2 to afford an ion of m/z 132, as well to
the iminium ion 2a of m/z 120.
In Tro¨ger’s base formation, urotropine has been proposed to
act as an efficient methylene source due to its hydrolysis to
formaldehyde in the acidic TFA medium.8 The same imine
On the basis of the mechanistic data collected from on-line
ESI-MS(/MS) monitoring and the gas-phase ion/molecule
reactions just described, an experimentally probed mechanism
for the formation of Tro¨ger’s bases using either formaldehyde
or, more particularly, urotropine as the source of methylene is
presented (Scheme 9). This mechanism proposes the participa-
(31) Tomazela, D. M.; Sabino, A. A.; Sparrapan, R.; Gozzo, F. C.;
Eberlin, M. N. J. Am. Soc. Mass Spectrom. 2006, 17, 1014.
(32) For an example concerning similar dissociation patterns, see: Santos,
L. S.; Padilha, M. C.; Neto, F. R. D.; Pereira, A. D.; Menegatti, R.; Fraga,
C. A. M.; Barreiro, E. B.; Eberlin, M. N. J. Mass Spectrom. 2005, 40, 815.
J. Org. Chem, Vol. 72, No. 11, 2007 4053

Abella et al.
were applied to transfer dry solvents and reagents. The preparation
of samples and the setup of the microreactor were carried out using
two syringe pumps.25 Electrospray ionization mass spectra (ESI-
MS) and accurate mass measurements were carried out with a
QTOF-I instrument. TFA and other reagents were used as received
without further purification. Ion/molecule reactions were performed
in a 2000 QTRAP system replacing N2 gas with reactive neutrals
using needle valve adaptors that allowed the introduction of reactive
gases into the collision cell through the residual vacuum of the
equipment, which was measured by a vacuum gauge and varied
from around 9 × 10-6 to 5 × 10-5 torr depending on the neutral
introduced into the equipment.
tion of all of the intermediates intercepted by ESI-MS and
properly characterized by ESI-MS/MS.
Conclusions
Key intermediates for Tro¨ger’s base formation in reactions
of amines and aldehydes have been transferred and measured
directly from the reaction solution to the gas-phase environment
of a mass spectrometer by ESI-MS followed by characterization
via ESI-MS/MS. The participation of urotropine as a direct
source of methylene in Tro¨ger’s base formation has also been
demonstrated by gas-phase ion/molecule reactions of its pro-
tonated molecule with aniline and ethylamine. The urotropine
action has also been shown in solution, via the ESI-MS
interception and ESI-MS/MS characterization of a key reaction
intermediate leading to methylene transfer. A concise and
experimentally probed mechanism for the formation of Tro¨ger’s
base has been therefore elaborated.
Acknowledgment. We thank FAPESP and CNPq for grants.
L.S.S. thanks the Universidad de Talca (Grants Committee) for
financial support.
Supporting Information Available: Figure S1, ESI(+)-MS/
MS of major intermediates intercepted in reactions of p-toluidine
(1) and urotropine (15) in TFA. Spectra (a), (c), and (d) were nearly
the same when acquired for the reaction using formaldehyde as
the methylene source. This material is available free of charge via
the Internet at http://pubs.acs.org.
Experimental Section
General Procedures. All reactions were performed under an
inert atmosphere of dry nitrogen and were followed by ESI-MS.
Glassware was flame dried before use. Standard syringe techniques
JO062556W
4054 J. Org. Chem., Vol. 72, No. 11, 2007