A Novel Rearrangement of Fluorescent Human Thymidylate Synthase Inhibitor Analogues in ESI Tandem Mass Spectrometry

Article abstract of DOI:10.1016/j.jasms.2009.11.004

Cu(I) catalyzed alkyne-azide cycloaddition reaction was employed to synthesize a series of anthracene-based human thymidylate synthase (hTS) inhibitor analogues. The triazolo-anthracene derivatives were characterized by ESI-MS/MS and a novel rearrangement

Full text of DOI:10.1016/j.jasms.2009.11.004

A Novel Rearrangement of Fluorescent Human
Thymidylate Synthase Inhibitor Analogues in
ESI Tandem Mass Spectrometry
Yi Chen,a Céline Le Droumaguet,a Kai Li,b William E. Cotham,a
Norman Lee,c Mike Walla,a and Qian Wanga
a Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Caroline, USA
b State Key Laboratory of Fine Chemicals, Dalian University of Technology (DUT), Dalian, People’s Republic
of China
c FAS Center for Systems Biology Mass Spectrometry and Proteomics Laboratory, Harvard University,
Cambridge, Massachusetts, USA
Cu(I) catalyzed alkyne-azide cycloaddition reaction was employed to synthesize a series of
anthracene-based human thymidylate synthase (hTS) inhibitor analogues. The triazolo-
anthracene derivatives were characterized by ESI-MS/MS and a novel rearrangement reaction
in ESI-MS/MS was observed. The mechanism is proposed whereby the protonated triazolo-
anthracene derivative forms a carbocation, and then the carbocation electrophilically attacks an
anthracene moiety resulting in formation of a rearrangement ion. Moreover, the carbocation
prefers to attack the ␥ position rather than the ␣ or ␤ position of the anthracene moiety by an
electrophilic substitution mechanism. (J Am Soc Mass Spectrom 2010, 21, 403–410) © 2010
American Society for Mass Spectrometry
uman thymidylate synthase (hTS) has been
targeted in cancer therapy for many years [1].
The catalytic mechanism of this enzyme is very
trusion, methoxy group migration, carbocation rear-
rangement, etc. have been published [14–20]. Herein,
we report the characterization of triazolo-anthracene
derivatives as hTS inhibitor analogues, and particularly,
a rearrangement reaction of triazolo-anthracene deriv-
atives discovered in ESI-MS/MS. The systematic inves-
tigation of these triazolo-anthracene derivatives and
their corresponding fragmentations are not only neces-
sary for further developing potential fluorescent hTS
inhibitors as fluorescent probes but also for enriching
methodological research in mass spectrometry.
H
complicated and some of its biochemical properties,
such as subunit cooperativity and conformational switch-
ing, remain to be elucidated [2]. Recent findings suggest
that the intracellular localization of hTS has certain
correlations with cancer malignancy [3]. Therefore, de-
veloping fluorescently labeled inhibitors as probes,
which would bind to hTS to monitor its intracellular
expression and localization as a function of cell cycle, is
significant for elucidating the role of hTS and its in-
volvement in tumorigenesis. Our efforts focus on using
Cu(I) catalyzed alkyne-azide cycloaddition (CuAAC)
reaction [4–10] to synthesize a library of fluorescent hTS
probes, which can also behave as potential inhibitors.
Mass spectrometry (MS) is an indispensable tool for
analysis of small molecules, peptides, and proteins. In
particular, collision induced dissociation (CID) has
unique advantages in elucidating the structural features
of compounds and studying the mechanism of rear-
rangement reactions in mass spectrometry by providing
detailed fragmentation data [11–13]. Some investiga-
tions on rearrangement reactions occurring in electros-
pray ionization tandem mass spectrometry (ESI-MS/
MS), such as carbonyl oxygen migration, amino group
migration, P-N to P-O rearrangement, formamide ex-
Experimental
Chemical Materials
All triazolo-anthracene derivatives were synthesized in
our laboratory. The starting materials for synthesis
were purchased from VWR (Suwanee, GA, USA) or
1
Sigma (St. Louis, MO, USA). H NMR and 13C NMR
spectra were recorded on Varian 300 NMR spectrome-
ter. Azidomethylanthracenes, Z1, Z2, and Z3, and
3-ethynylquinoline A2 have previously been described
in the literature [5, 21, 22]. Compounds A1, A5, A6, and
A9 have been prepared by reaction between propargyl
bromide and the corresponding amines or alcohols
under basic conditions. Compounds A3, A4, A8, A10,
and A7 were obtained from nucleophilic substitution
between the corresponding brominated compounds and
3-phenylpropargylamine and tert-butyl 4-aminobenzoate,
respectively.
Address reprint requests to Dr. Q. Wang, Department of Chemistry and
Biochemistry, 631 Sumter St., Columbia, SC 29208, USA. E-mail: wang@
mail.chem.sc.edu
Published online December 3, 2009
© 2010 American Society for Mass Spectrometry. Published by Elsevier Inc.
1044-0305/10/$32.00
doi:10.1016/j.jasms.2009.11.004
Received September 9, 2009
Revised November 6, 2009
Accepted November 11, 2009

404
CHEN ET AL.
J Am Soc Mass Spectrom 2010, 21, 403–410
Scheme 1. Synthesis of Compound 3 via cycloaddition (CuAAC) reaction of Z1 and A3.
was used as lock mass for parent ion. (3) For accurate
mass analysis of rearrangement ion in MS/MS mode,
the parent ion was used for the lock mass. The average
mass accuracy of the measurements for rearrangement
ion (388.1176) was achieved by averaging six experimental
data (388.1194; 388.1182; 388.1152; 388.1161; 388.1197, and
388.1171).
General Protocol of CuAAC Reaction for the
Synthesis of Triazolo-Anthracene Derivatives
To a solution of 1-(azidomethyl)anthracene Z1 (60 mg,
257 ␮mol) N-(naphtho[2,1-d]thiazol-2-ylmethyl)-N-(prop-
2-ynyl)aniline A3 (84 mg, 257 ␮mol) in a 3:1 mixture
(vol/vol) of DMF/H2O (24 mL) were added with
CuSO4 (10 mg, 39 ␮mol), sodium ascorbate (15 mg, 77
␮mol), and tris(thiazolyl)benzylamine (7 mg, 13 ␮mol)
(shown in Scheme 1). The mixture was stirred at RT for
15 h. After extraction with EtOAc, the organic layers
were washed with water, dried over MgSO4, and the
solvents were evaporated. The crude product was then
purified by column chromatography (CH2Cl2/EtOAc
gradient from 90:10 to 80:20) to yield 102 mg (71%) of
Calculations
All energies (E) for methane, methyl carbocation, Com-
pound 3, and Compound 4 were evaluated at the
B3LYP/6-31G* level using Spartan’06 software (Irvine,
CA, USA). The stabilization energies (SE) for Com-
pounds 3 and 4 are calculated using an isodesmic
equation as follows [23].
1
Compound 3: Compound 3 was identified by H NMR
and ESI-MS/MS and high-resolution ESI-MS/MS as
1
follows: H NMR (300.11 MHz, CDCl3): ␦ 8.34 (s, 1 H),
SE ϭ Ealkane ϩ Ecarbocation Ϫ Ecarbocation1
8.29 (s, 1 H), 7.91–7.69 (m, 7 H), 7.47–7.36 (m, 4 H),
7.31–7.19 (m, 3 H), 7.07–7.02 (m, 2 H), 6.77 (d, J ϭ 7.8
Hz, 2 H), 6.66 (t, J ϭ 7.3 Hz, 1 H), 5.97 (s, 2 H), 4.80
(s, 2 H), 4.71 (s, 2 H); 13C NMR (75.47 MHz, CDCl3):
␦ ϭ 171.5, 151.7, 147.8, 145.5, 132.3, 132.2, 132.9, 131.8,
131.1, 130.6, 130.0, 129.5, 129.2, 129.1, 128.8, 128.3, 128.0,
127.7, 127.4, 127.3, 127.1, 126.3, 126.1, 125.3, 124.6, 122.4,
121.9, 119.1, 114.3, 54.4, 53.0, 48.1; the observed mass of
Ϫ Emethane (Kcal/mol)
(Eq. 1)
The Mulliken charge on atoms and condensed Fukui
index values of intermediate 3-IIb from Compound 3
were obtained at the DFT B3LYP level of theory with a
6-31G* basis set with Spartan’06 software. All the cal-
culations were based on equilibrium geometry calcula-
tion by searching for a stationary point. In addition, we
tested the final geometry by calculating the vibration
frequencies and verifying all frequencies that are posi-
tive. It indicates that all the carbocations are at minima-
energy geometries.
protonated Compound
3 by high-resolution ESI-
MS/MS is mass of 562.2065 (calculated mass 562.2060).
All triazolo-anthracenes (1-12) were synthesized by the
same method using appropriate Compound (Z1-Z3)
and Compound (A1-A10) (Table 1).
Mass Spectrometry
Results and Discussion
The MS/MS mass spectra were acquired in the positive
ion mode by a Micromass QTOF (Waters, Milford, MA,
USA) equipped with an electrospray source. The source
temperature was held constant at 80 °C. The desolva-
tion temperature was set at 300 °C. The capillary volt-
age was 4 kV. Full scan spectra were collected from m/z
100 to 1000. The collision energy in the collision cell was
set at 23–25 V for MS/MS. MassLynx 4.0 (Waters,
Milford, MA, USA) was used for data acquisition and
analysis. The high-resolution MS/MS data were also
acquired by Micromass QTOF instrument with resolu-
tion of 7500 FWHM. The calibration for high-resolution
MS/MS was achieved as follows: (1) Initial calibration
was achieved with a solution of sodium trifluoroacetate
(TFA) (50–100 mM), which gives ions across a wide
mass range. (2) The TFA cluster ion at mass of 566.8890
Fragmentation Pathways of Compound 3
A series of fluorescent hTS inhibitor analogues (triazolo-
anthracene derivatives) were synthesized by copper
(I)-catalyzed alkyne-azide cycloaddition (Scheme 1 and
Table 1) and the structures were characterized by ESI-
MS/MS (Table 3). The MS/MS mass spectrum of Com-
pound 3 (Figure 1a) shows four expected peaks at m/z
191, 198, 244, 562, and one unexpected peak at m/z 388.
According to the molecular formula C36H27N5S, the
peak at m/z 562 corresponds to the protonated molecule.
The base peak at m/z 191 is produced by the cleavage of
the C–N bond between the anthracene moiety and the
triazole moiety. The ␲-system on anthracene moiety
enhances the thermodynamic stability of the carboca-
tion resulting in the base peak in the MS/MS spectrum.

J Am Soc Mass Spectrom 2010, 21, 403–410
NOVEL REARRANGEMENT MECHANISM IN ESI MS/MS
405
Table 1. Structures of starting azides, alkynes and corresponding products
Entry
1
Dye azide
Alkyne
Fluorescent hTS inhibitor
2
3
4
5
6
7
8
9
10
11
12

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CHEN ET AL.
J Am Soc Mass Spectrom 2010, 21, 403–410
Table 2. Accurate masses of protonated Compound 3 and its rearrangement ion at m/z 388
Ion
Elemental composition Accurate mass (calculated) Observed mass Relative error (ppm)
Protonated Compound 3
Rearrangement ion at m/z 388
C36H28N5Sϩ
C27H18NSϩ
562.2060
388.1160
562.2065
388.1176
0.9
4.1
Apart from the base peak and the peak of protonated
molecule, a couple of peaks at m/z 244 and 198 that
resulted from the heterolytic cleavage of C–N bonds on
both sides of the protonated tertiary amine can be
clearly observed. The ion at m/z 244 is the result of the
neutral loss of N2 from the triazole moiety and the
cleavage of the C–N bond [24]. The peak at m/z 198 is
attributed to the cleavage of another C–N bond on the
tertiary amine group, which plays a significant role in
the subsequent rearrangement reaction. The MS/MS
fragmentation pathways of Compound 3 as an example
of triazolo-anthracene derivatives in positive mode ESI-
MS/MS are summarized in Scheme 2.
accept the proton, leading to migration of the proton to
the nitrogen atom. The two electrons move into the
anthracene moiety to reform the ␲ bond and recover
the aromatic system, thus neutralizing the positive
charge on the carbon to form (3-IV). Finally, the cleav-
age of the C–N bond between the anthracene moiety
and the triazole moiety results in the final rearrange-
ment ion at m/z 388 (3-V).
Elemental Composition Analysis
To further examine the elemental composition of the
rearrangement ion at m/z 388, Compound 3 and its
rearrangement ion were analyzed by high-resolution
ESI-MS/MS. Table 2 and Figure 2 show their corre-
sponding elemental compositions, calculated and ob-
served masses, and relative errors. Since the relative
errors are less than 5ppm, these results suggest that the
elemental compositions of both protonated Compound
3 and its rearrangement ion, which are proposed in the
rearrangement mechanism, are consistent with the ex-
perimental values.
Rearrangement Mechanism of Compound 3
The peak at m/z 388 observed in Figure 1a, which is
unlikely to arise from any moiety, could not be ex-
plained by a simple fragmentation pathway. Moreover,
similar fragment ions were not observed in the other
triazolo-anthracene derivatives, such as Compounds 1
and 2 (Table 1 and Table 3). A possible explanation for
this peak at m/z 388 is that an intramolecular rearrange-
ment reaction occurs in ESI-MS/MS. To rationalize this
observation, a novel rearrangement mechanism is pro-
posed here that the rearrangement reaction may occur
along the pathways as shown in Scheme 3. The rear-
rangement proceeds with the protonation of the tertiary
amine (3-I) and breaking of the N–C bond resulting in
the formation of a carbocation (3-IIa) at m/z 198, which
corresponds to the peak at m/z 198 (Figure 1a). The
carbocation may further form a weakly associated
␲-carbocation complex (3-IIa–3-IIb) with the anthracene
moiety. The electrophilic attack of this carbocation
(3-IIa) on the anthracene moiety (3-IIb) would lead to
the transition-state (3-III). The nitrogen atom on the
triazole moiety donates its lone pair of electrons to
Analysis of Analogous Compounds by MS/MS
Based on the proposed rearrangement mechanism, two
key factors are crucial in the rearrangement process. One
is the formation of a stable carbocation and the other
is the position attacked by the carbocation on the
anthracene moiety. Compounds 7, 8, and 4 (Table 1),
which are believed to form similar carbocations, were
synthesized and fragmented under the same MS/MS
conditions. The rearrangement ions were also observed in
the MS/MS spectra (Figure 1b, c, and d). These results
show that all these compounds are likely to undergo
similar rearrangement reactions via a carbocation forma-
tion, electrophilic attack, and migration of the carbocation.
Table 3. Major fragment ions in ESI-MS/MS
Fragment ions m/z (relative intensity %)
Rearrangement
No.
Compound
[MϩH]ϩ m/z
ion m/z
a
b
c
d
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
407 (21)
387 (8)
562 (8)
507 (22)
422 (2)
431 (0)
505 (4)
605 (9)
407 (11)
421 (13)
562 (7)
—
—
388 (8)
333
—
—
331 (1)
331 (2)
—
—
388 (1)
244 (25)
—
191 (100)
191 (92)
136 (92)
—
198 (5)
—
151 (8)
160 (100)
141 (4)
141 (4)
359 (11)
244 (3)
244 (4)
244 (28)
244 (24)
244 (1)
—
244 (3)
244 (5)
—
191 (100)
191 (100)
191 (100)
191 (140)
191 (100)
191 (100)
191 (100)
191 (100)
191 (100)
403 (17)
10
11
10
11
—

J Am Soc Mass Spectrom 2010, 21, 403–410
NOVEL REARRANGEMENT MECHANISM IN ESI MS/MS
407
Figure 1. MS/MS spectra of Compounds 3 (a), 7 (b), 8 (c), and 4 (d).
However, the abundance of the rearrangement ion for
these various compounds varies considerably in MS/MS.
Among these compounds, Compound 3 gives the most
abundance from the rearrangement ion, while the abun-
dance of the rearrangement ion from Compound 4 is very
low. This difference may be attributed to the stability of
the carbocation. Among Compounds 3, 4, 7, and 8, Com-
pound 3 likely has the most stable resonance structure
because the positive charge can be delocalized and dis-
tributed on the sulfur atom by resonance to afford a much
more stable sulfonium ion-like resonance structure. In
addition, the non-bonding electrons of the sulfur may also
make a contribution to stabilize the positive charge due to
the spatial proximity. As a comparison, the carbocation of
Compound 4 lacks either of these stabilization contribu-
tions. Therefore, the rearrangement reaction of Com-
pound 4 is less favorable, resulting in very low abundance
produced by the rearrangement ion derived from Com-
pound 4 in the MS/MS spectrum.
The stabilization energies of carbocations for Com-
pound 3 (90.6807 kcal/mol) and Compound 4 (74.8317
Scheme 2. Fragmentation pathways of Compound 3.

408
CHEN ET AL.
J Am Soc Mass Spectrom 2010, 21, 403–410
Scheme 3. Proposed rearrangement mechanism of Compound 3.
kcal/mol) were also calculated using an isodesmic
equation (Eq. 1) on the B3LYP/6-31G* optimized geom-
etries using Spartan’06. The greater the value of stabi-
lization energy, the more stable the carbocation will be
[23]. The calculated stabilization energies also are consis-
tent with the results observed in MS/MS, suggesting
higher stabilization energy for Compound 3 and lower
stabilization energy for Compound 4. In contrast, to fur-
ther support the crucial role of the carbocation in the
rearrangement reaction, Compounds 9 and 10, which
have the same structure as Compound 3 except the
substitutes (propyl group for Compound 9 and butyl
group for Compound 10) linked to tertiary amine, were
synthesized and analyzed by ES-MS/MS. It is impossible
for them to form 3-IIa-like stable carbocations following
the pathway shown in Scheme 3. As expected, no rear-
rangement ions were detected in the MS/MS for Com-
pounds 9 and 10. The results from these experiments and
calculations confirm that the rearrangement reaction is
likely a result of generating a carbocation. Moreover, there
was a greater likelihood to observe a rearrangement ion
with a more stable carbocation.
With respect to the substitution position, there are
some possible positions on the anthracene moiety such
Figure 2. High resolution MS/MS spectrum of Compound 3 [M ϩ H]ϩ.

J Am Soc Mass Spectrom 2010, 21, 403–410
NOVEL REARRANGEMENT MECHANISM IN ESI MS/MS
409
Figure 3. MS/MS spectrum of Compound 11.
as ␣, ␤, and ␥ for an electrophilic attack by the carbo-
cation. However, the ␥ positions of anthracene are in
general more reactive towards an electrophilic attack.
When Compound 11 from azide Z2 and alkyne A3 with
an azide group at the ␥ position was synthesized and
analyzed by ESI-MS/MS, a rearrangement ion was
detected with a very low abundance since only one ␥
position is available (Figure 3). Additional experiments
were carried out with Compound 12, which has both ␥
positions occupied. As predicted, no rearrangement ion
was detected in MS/MS. It implies that the carbocation
can only attack the ␥ positions of the anthracene moiety.
To confirm this, the 3-IIb in the rearrangement mecha-
nism (Scheme 3) was modeled at the DFT B3LYP level
of theory with a 6-31G* basis set (Figure 4). Mulliken
charge population analysis shows that C6 has the negative
charge (Ϫ0.322). C9 has a smaller negative value (Ϫ0.300)
than C6 but a much larger negative value than the other
carbons. The greater the negative charge on the atom,
the more likely an electrophilic attack will occur on that
atom [25]. Beside Mulliken charge population analysis,
the condensed Fukui index values were obtained from
condensed Fukui function equation (Eq. 2) [26]. The
calculated results show that the C6 at the ␥ position of
the anthracene moiety has the largest condensed Fukui
index value (0.037). C9 at the opposite ␥ position of the
anthracene moiety has a smaller value (0.025) than C6
but greater than other carbons at the ␣ or ␤ positions
(Table 4). This means that the C6 atom should be the
most reactive site towards an electrophilic attack of
carbocation. Fukui index and Mulliken charge values
for 3-IIb suggest that C6 could be most susceptible and
C9 could have less chance than C6 but more opportu-
nity than other carbons at ␣ or ␤ positions to electro-
philic reaction. The calculated results are consistent
with the MS/MS data, thereby supporting the proposed
theory that the carbocation attacks the ␥ position of the
anthracene moiety.
fc ϭ qc(N) Ϫ qc(N Ϫ 1)
(Eq. 2)
Conclusions
The triazolo-anthracene derivatives as fluorescent in-
hibitor analogues were investigated by ESI-MS/MS,
and the fragmentation pathways have been character-
ized. These pathways involve the cleavage on C–N
bonds on both sides of the protonated tertiary amine
and the cleavage of the C–N bond between the anthra-
cene and triazole moieties leading to base peak. Besides
direct cleavages, a rearrangement ion at m/z 388 in
ESI-MS/MS was observed, and a possible rearrange-
ment mechanism is proposed that involves a carboca-
tion migration from the amine group to the ␥ position
Table 4. Condensed Fukui index and Mulliken charge of
intermediate 3-IIb
Condensed
Fukui index
Mulliken charge
C0
C1
C2
C5
C6
C9
C11
C12
C13
0.009
0.01
–0.139
–0.136
–0.187
–0.183
–0.322
–0.300
–0.194
–0.136
–0.181
0.002
0.009
0.037
0.025
0.019
0.00
Figure 4. Optimized geometry of 3-IIb derived from Compound
3 and Mulliken charge on hydrogen atom (positive value) and
Mulliken charge on carbon (negative value) at DFT B3LYP/6-31G*
(Spartan’06).
0.01

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Acknowledgments
The authors acknowledge partial support for this work by the
Nanocenter of University of South Carolina, South Carolina
EPSCoR/CRP program, the Alfred P. Sloan Scholarship, and the
Camille Dreyfus Teacher Scholar Award. The hTS samples were
kindly supplied by Dr. Lukasz Lebioda.
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