Fluorescent visualization of the conformational change of aromatic amide or urea induced by N-methylation

Article abstract of DOI:10.1016/j.tetlet.2008.11.044

The conformational change of amide structure of benzanilide or urea structure of N,N′-diphenylurea induced by methylation of the secondary nitrogen was visualized by introduction of pyrene moieties on the benzene ring. In contrast to the extended trans-fo

Full text of DOI:10.1016/j.tetlet.2008.11.044

Tetrahedron Letters 50 (2009) 488–491
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Tetrahedron Letters
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Fluorescent visualization of the conformational change of aromatic
amide or urea induced by N-methylation
Tomoya Hirano a, Takashi Osaki a, Shinya Fujii a, Daisuke Komatsu a, Isao Azumaya b, Aya Tanatani c,d
,
Hiroyuki Kagechika a,
*
a School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
b Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
c Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
d PRESTO, Japan Science and Technology Agency (JST), Japan
a r t i c l e i n f o
a b s t r a c t
The conformational change of amide structure of benzanilide or urea structure of N,N0-diphenylurea
induced by methylation of the secondary nitrogen was visualized by introduction of pyrene moieties
on the benzene ring. In contrast to the extended trans-form of secondary amide and urea, which showed
monomer fluorescence emission of pyrene, the corresponding N-methylated compounds exist in cis-
form, which exhibited excimer emission.
Article history:
Received 31 October 2008
Accepted 13 November 2008
Available online 18 November 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Aromatic amide
Aromatic urea
Conformation
Fluorescence
The amide linkage is an important building block of functional
molecules, since conformations with restricted rotation can often
be obtained by an appropriate choice of substituents. Previously,
we reported that N-methylation of benzanilide, which has trans-
conformation, causes conformational change, resulting in cis-con-
formational preference of N-methylbenzanilide.1 Similarly, N,N0-
diphenylurea exists in extended (trans, trans) conformation, while
N,N0-dimethyl-N,N0-diphenylurea exists in folded (cis, cis) confor-
mation (Fig. 1).2 The cis-conformational preference is a general ste-
ric feature of various aromatic N-methylated amides and ureas,
and some amide structures can be altered by external stimuli, such
as temperature,3 pH,4 and redox potential.5
Pyrene is a polycyclic aromatic fluorophore, and exhibits mono-
mer fluorescence at shorter wavelength (370–390 nm) and exci-
mer fluorescence at longer wavelength (near 480 nm).7 The
efficiency of each fluorescence depends on concentration, that is,
the ratio of excimer fluorescence increases at higher concentration,
or the distance and orientation of two pyrene units in the mole-
cule. When molecular frameworks, whose structures are changed
by external environments, such as temperature and viscosity, or
binding with analytes such as ions and DNA, are labeled with
Fluorescence measurement has distinct advantages in terms of
sensitivity, selectivity, and response time for sensing certain ana-
lytes. Consequently, many fluorescent sensors have been devel-
oped and applied to detect ions, enzymes, and environmental
changes in various fields of scientific research, including analytical
chemistry, biology, and materials science.6 By means of the intro-
duction of suitable fluorophores on appropriate sites, the structural
changes of certain molecules can be fluorescently visualized. In
this Letter, we show that the conformational change of benzanilide
and N,N0-diphenylurea skeletons induced by N-methylation can be
visualized via a change of fluorescence properties. Therefore, these
structures are potentially available as scaffolds for obtaining fluo-
rescent sensor candidates by suitable derivatization.
* Corresponding author. Tel.: +81 3 5280 8032; fax: +81 3 5280 8127.
E-mail address: kage.omc@tmd.ac.jp (H. Kagechika).
Figure 1. Conformational change by N-methylation of aromatic amide and urea.
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.11.044

T. Hirano et al. / Tetrahedron Letters 50 (2009) 488–491
489
two pyrenes at appropriate positions via appropriate spacer, they
can be used as fluorescent sensors for these analytes.8 Because aro-
matic urea and amide structures are candidates for such molecular
frameworks, we designed benzanilide and diphenylurea deriva-
tives substituted with pyrenes via an ethynylene group or ethylene
group as a spacer structure (Fig. 2).
to the corresponding secondary amides or ureas. 1H NMR spectra
of 2b at low temperature revealed that 2b is in equilibrium be-
tween cis- and trans-conformers in the ratio of 98:2 at 213 K in
CD2Cl2, while 4b did not show such an equilibrium between con-
formers, presumably due to the lower rotational barrier of the urea
bond (data not shown).
1-Bromopyrene (6) was reacted with 4-ethynylaniline (7) by
means of the Sonogashira coupling reaction to yield 8 (Scheme
1).9 Reduction of the ethynylene group of 8 with palladium
hydroxide on carbon catalyst afforded 4-[2-(1-pyrenyl)ethyl]ani-
line (9). Similarly, methyl 4-[2-(1-pyrenyl)ethynyl]benzoate (5a)
was prepared from 6 and methyl 4-ethynylbenzoate (10), and
hydrolyzed to 11 or reduced to 5b, which was also hydrolyzed to
yield 12. The carboxylic acid 11 was converted to the acid chloride,
and then reacted with 8 to afford the secondary amide 1a, which
was methylated using methyl iodide and sodium hydride as a base
to afford the tertiary amide 2a. Ethylene-spacered secondary
amide (1b) and its N-methylated derivative (2b) were similarly
prepared from carboxylic acid 12 and amine 9. The secondary ur-
eas 3a and 3b were synthesized from amines 8 and 9, respectively,
with triphosgene, and were similarly converted to dimethylated
ureas 4a and 4b, respectively.
The conformational differences between secondary amide 1 and
its N-methylated amide 2 or between secondary urea 3 and its
N,N0-dimethylated urea 4 were examined by 1H NMR spectroscopy.
In each pair of secondary and N-methylated compounds, upfield
changes of the protons on the phenyl rings on going from second-
ary to N-methylated amide or urea were observed (Fig. S1). Such
chemical shift differences are similar to those of unsubstituted
benzanilide versus N-methylbenzanilide or of N,N0-diphenylurea
versus N,N0-dimethyl-N,N0-diphenylurea, respectively,10 which
suggested the extended trans-structures of compounds 1 and 3,
and the folded cis-structures of N-methylated compounds 2 and
4 in solution. The signals of the pyrene ring protons of N-methyl-
ated compounds were also shifted to a little higher field, compared
The absorption spectra and fluorescence spectra are shown in
Figure 3. In the case of the compounds with the ethynylene groups
(1–5, a series), the absorption spectra were broad and red-shifted,
compared to those of the corresponding compounds with the eth-
ylene groups (1–5, b series). For example, the monomeric com-
pound 5b showed typical pyrene monomer absorbance spectrum
with sharp absorbance peaks at 300–350 nm, while 5a had an
absorption in the region of 350–450 nm. The absorption pattern
is essentially similar between the compounds in the b series
(Fig. 3c and d). On the other hand, there are small differences in
the absorption wavelength between each set of monomeric com-
pounds 5a, secondary (1a or 3a), and N-methylated compounds
(2a or 4a) (Fig. 3a and b). For example, the maximum absorption
wavelength of 5a (377 and 400 nm) was blue- and red-shifted in
1a (382 and 404 nm) and 2a (370 and 397 nm), respectively.
More drastic differences were observed in their fluorescence
spectra. Compound 5b showed fluorescence spectrum with fine
vibrational structure at 370–390 nm (Fig. 3g), while compound
5a showed broad and red-shifted fluorescence in the region of
400–550 nm (Fig. 3e). These data suggested that the 1-phenyl-2-
pyrenylacetylene moiety would no longer show pyrene-like fluo-
rescence, probably due to their conjugation properties. Interest-
ingly, the amides 1a and 2a did not show any significant
fluorescence (Fig. 3e). In the case of urea derivatives, both 3a and
4a showed fluorescence in the region of 400–550 nm, and the fluo-
rescence intensity and wavelength of N,N0-dimethylated urea 4a
(emission maximum at 481 nm) were smaller and a little red-
shifted than those of 3a (emission maximum at 447 nm) and 5a
(Fig. 3f). Among the compounds of b series, 1b and 3b showed fine
vibrational structure at 370–390 nm like monomeric compound
5b. In contrast, 2b and 4b showed broad fluorescence spectra with
its maximum of 477 nm that could be identified as pyrene excimer
spectra, accompanied with a corresponding decrease of monomer
emission (Fig. 3g and h). The spectra of b series compounds did
not show any significant change by the concentration (1–50 lM)
and temperature (273–353 K). Taken together with 1H NMR spec-
tra, these results suggest that in the extended trans-structures of
1b and 3b, the pyrene moieties exhibit monomer-type fluores-
cence spectra, whereas in the cis-structures of the corresponding
N-methylated derivatives 2b and 4b, they exhibit intramolecular
excimer-type fluorescence spectra. The degree of fluorescent
change of the ureas (3b vs 4b) was greater than that of the amides
(1b vs 2b), probably due to the difference in the distance and ori-
entation of two phenyl rings between N-methylated amide 2b and
N,N0-dimethylated urea 4b.11
Our present results showed that the conformational change
could be visualized by the excimer fluorescence of the two pyrenes
linked to the aromatic amides and ureas through the ethylene
spacers. When the spacer structure is ethynylene group, both the
absorption and fluorescence spectra did not show pyrene-like
monomer or excimer spectra. Previously, similar observation was
reported in the fluorescence spectra of N,N0-dimethyl-N,N0-dipyre-
nylurea, in which two pyrenes were directly connected to nitrogen
atoms of N,N0-dimethylurea with the folded (cis, cis) conforma-
tion.12,13 In these reports, both absorption and fluorescence spectra
of this compound were different from those of the corresponding
monomeric compound, and depended on the temperature and vis-
cosity. However, in this system, the difference in fluorescence be-
tween extended trans-structure and folded cis-structure seems
unclear. For this reason, the changes in the fluorescence spectra
Figure 2. Structures of pyrene (Py)-substituted aromatic amides and ureas.

490
T. Hirano et al. / Tetrahedron Letters 50 (2009) 488–491
Scheme 1. Reagents and conditions: (a) PdCl2(PPh3)2, CuI, triethylamine, 70 °C; (b) Pd(OH)2/C, H2 (1 atm), AcOH (for 8) or CH2Cl2/AcOH(for 5a); (c) 2 M NaOH aq, EtOH; (d)
(1) SOCl2, DMF, (2) 8 or 9, pyridine, DMAP; (e) MeI, NaH, DMF, 60 °C; (f) triphosgene, triethylamine, THF.
of urea structure directly connected with two pyrenes are sup-
posed not to reflect the conformational changes of the molecular
structures, such as our ethynylene-spacered series (1a–4a), and
therefore these systems seem not suitable for scaffolds of fluores-
cent sensor candidates. In contrast, our ethylene-spacered systems
(1b–4b) are expected to be good scaffolds of fluorescent sensor
candidates because the conformational preference of aromatic
amide or urea structure certainly reflects the fluorescence
Figure 3. Absorption (a–d) and fluorescence (e–h) spectra of 1–5 in DMSO at 303 K. Excitation wavelength is 380 nm (for e and f) or 345 nm (for g and h). Concentration of
each compound was 5 M.
l

T. Hirano et al. / Tetrahedron Letters 50 (2009) 488–491
491
properties. In the case of pyrene derivatives connected directly or
References and notes
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Acknowledgments
The work described in this Letter was partially supported by
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Experimental procedure and NMR spectral data. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.tetlet.2008.11.044.