Synthesis and photophysical characterization of new fluorescent bis-amino acids bearing a heterocyclic bridge containing benzoxazole and thiophene

Article abstract of DOI:10.1016/j.tet.2008.07.096

A series of new bis-alanine derivatives bearing (oligo)thiophene and benzoxazole units as the heteroaromatic bridge were synthesized in moderate to good yields. The photophysical characterization of these bis-amino acids was performed by UV-vis absorption and fluorescence emission studies and revealed that the compounds displayed exceptionally high fluorescence quantum yields, making them good candidates for application as fluorescent probes when incorporated into peptides, as well as peptide conformation-restricting and cross-linking elements.

Full text of DOI:10.1016/j.tet.2008.07.096

Tetrahedron 64 (2008) 9733–9737
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and photophysical characterization of new fluorescent bis-amino
acids bearing a heterocyclic bridge containing benzoxazole and thiophene
*
Susana P.G. Costa , Rosa M.F. Batista, M. Manuela M. Raposo
Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2008
Received in revised form 21 July 2008
Accepted 22 July 2008
Available online 26 July 2008
A series of new bis-alanine derivatives bearing (oligo)thiophene and benzoxazole units as the hetero-
aromatic bridge were synthesized in moderate to good yields. The photophysical characterization of
these bis-amino acids was performed by UV–vis absorption and fluorescence emission studies and
revealed that the compounds displayed exceptionally high fluorescence quantum yields, making them
good candidates for application as fluorescent probes when incorporated into peptides, as well as
peptide conformation-restricting and cross-linking elements.
Keywords:
Bis-amino acids
Ó 2008 Elsevier Ltd. All rights reserved.
(
Oligo)thiophene
Benzoxazole
Fluorescence
Fluorescent probes
Bis-alanines
1. Introduction
synthetic origin, which are effective building blocks for peptide
design considering the following two points: (i) use of rigid un-
Bis-amino acids have been the subject of significant research in
recent years because they can be used in the synthesis of analogues
of biologically active peptides and also in biomimetic chemistry as
natural amino acids to control the conformation and function of the
resulting peptides and (ii) incorporation of functional unnatural
amino acids into peptides resulting in the appearance of the
1
5
cross-linking elements for the control of peptide secondary struc-
inherent functions.
ture, leading to stabilized
a
-helical,
b
-turn, and
g
-turn conforma-
Benzoxazoles display excellent optical properties (broad spec-
tral windows, high molar absorptivity coefficients and fluorescence
quantum yields) and they have been described as fluorescent
probes and sensing materials, namely as fluorescent and/or color-
imetric sensors for metals, anionic species and in pharmaceutical
2
tions. Bis-amino acids can be regarded as a replacement for
cysteine residues in the cross-linking between peptide chains, and
other residues like lysine, tyrosine and aspartic, and glutamic acids,
which can also undergo cross-linking in the presence of appropri-
ate reagents. Cross-linking is a key feature in (bio)polymer and
6
analysis. Thiophene and its derivatives also possess important
3
biomaterials sciences and food chemistry.
electroluminescent properties with wide application in polymer
science, which has prompted their application as energy transfer
The insertion of noncoded amino acids into the backbone of
both natural and synthetic polymers is a very appealing area of
research since it can lead to the development of macromolecules
possessing biomimetic characteristics, with unique structural and
biological properties. By synthetic manipulation at the side chain of
coded amino acids, new functions and functional relationships can
be generated as well as altered physicochemical properties, such as
luminescence, conducting ability, and metal ion and other analyte
7
and light-harvesting systems, for optical and electronic devices, as
8
sensors and as fluorescent markers.
The work on bis-amino acids reported so far deals with the
synthesis of aliphatic- and aromatic-bridged bis-amino acids and,
to the best of our knowledge, this is the first report on the synthesis
of heteroaromatic-bridged bis-amino acids, apart from those con-
9
cerning histidinoalanine. Therefore, there is a practical interest on
4
recognition ability, among other properties.
The molecular design of functional peptides can thus be ach-
ieved by judicious choice of noncoded amino acids of natural and
the synthesis of new and more complex systems and in order to
contribute to and expand the body of work published in this area in
the last years, we decided to design new bis-amino acid-based
systems consisting on functionalized alanines containing thio-
phene and benzoxazole as the heterocyclic bridge, as a result of the
previously mentioned applications for these heterocycles. These
bis-alanine derivatives represent both a conformation-restricting
*
Corresponding author. Tel.: þ351 253 604054; fax: þ351 253 604382.
E-mail address: spc@quimica.uminho.pt (S.P.G. Costa).
0
040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.07.096

9734
S.P.G. Costa et al. / Tetrahedron 64 (2008) 9733–9737
and a functional amino acid, in view of their fluorescence. The
resulting bis-(oligo)thienylbenzoxazole-alanine derivatives, be-
cause of the presence of amino and carboxyl groups, could be in-
corporated into peptide chains and as such used as an energy donor
in conformational studies of peptides by means of fluorescence or
be used as fluorescence markers. Following our previous research
with 5-formylthiophene boronic acid in 75% yield. Precursor 1 was
obtained from commercial 5-formyl-2,2 -bithiophene by bromi-
nation with NBS in DMF in 98% yield (Scheme 1).
0
NBS
DMF
10
CHO
Br
CHO
S
S
S
S
S
on the synthesis and characterization of unnatural amino acids,
1
11
benz-X-azole derivatives with interesting optical properties and
heterocyclic colorimetric/fluorimetric chemosensors containing
Pd(PPh )
aq. Na2CO3
DME, 80 °C
3
4
12
(
oligo)thiophene, benzoxazole, and amino acid moieties, we now
OHC
B(OH)2
report the synthesis of a new highly fluorescent family of alanine
derived bis-amino acids connected through an (oligo)thie-
nylbenzoxazole bridge, also having in mind the future studies of
these compounds as fluorimetric peptide-based chemosensors.
Amino acids and peptides are known to bind a variety of metal ions
as they contain a large number of potential donor atoms through
the peptide backbone and amino acid side chains and the insertion
of an extended heterocyclic system between the two ends of the
bis-amino acid will provide an UV-active and highly fluorescent
chromophore, as well as additional binding sites through the donor
OHC
CHO
S
S
S
2c
Scheme 1. Synthesis of precursor diformyl-terthiophene 2c.
The synthesis of compound 2c has been reported earlier by
13
Giannetas et al. through Vilsmeier formylation of terthiophene.
This reaction gave a mixture of three products, which were isolated
by column chromatography: the starting material, the mono-for-
myl derivative (65%) and the diformyl-terthiophene 2c in 20% yield.
In comparison to the method described earlier, compound 2c
was obtained by us in higher yield from low cost commercially
available reagents and using simple work-up procedures, allowing
the good yielding preparation and ease of isolation of this de-
rivative. Attempt to synthesize compound 2c using the same
method described by Giannetas gave, in our hands, a complex
mixture of compounds, which was not possible to separate neither
by recrystallization nor by chromatography.
atoms in the conjugated
oligo)thienylbenzoxazolyl-alanines we also intended to evaluate
the effect of the length of the -conjugated (oligo)thienyl system at
p-bridge. With the study of these new bis-
(
p
position 2 of the benzoxazole moiety in the photophysical prop-
erties of the resulting compounds.
2
2
. Results and discussion
.1. Synthesis
2.1.2. Synthesis of bis-[2-(oligothienyl)benzoxazol-5-yl]-alanine
derivatives 4a–d
The new bis-(oligo)thienylbenzoxazolyl-alanines 4a–d with
Starting from commercially available 3-nitro-
3-amino- -tyrosine methyl ester 3 was obtained by using simple
synthetic procedures.
L-tyrosine, N-Boc-
thiophene, bithiophene, terthiophene, and phenylthiophene units
acting as linking -conjugated bridges between the two benzox-
L
12b
p
Condensation of compound 3 with difor-
azolyl-alanine systems were synthesized in moderate to good
yields, by a multistep synthesis. Different (oligo)thiophenes were
used in order to study the influence of the structure modification
mylated (oligo)thiophenes 2a–d, namely thiophene, bithiophene,
terthiophene, and phenylthiophene, resulted in the corresponding
imines, which by subsequent oxidation and cyclization with lead
tetraacetate in DMSO afforded the bis-[2-(oligothienyl)benzoxazol-
5-yl]-alanine derivatives 4a–d in moderate to good yields (Scheme
2, Table 1). These compounds were fully characterized by the usual
spectroscopic techniques.
(
i.e., the increase of the p-conjugated bridge) on the overall optical
properties of compounds 4a–d and also in order to compare with
our recently reported systems containing only one benzoxazolyl-
alanine system.1
0c,12b
The IR spectra of bis-amino acids 4a–d showed the character-
istic bands due to stretching vibrations of the carbonyl of the C- and
N-terminal protecting groups: the methyl ester from 1744 to
2
.1.1. Synthesis of diformyl-terthiophene 2c
The diformyl-terthiophene 2c was synthesized through a Suzuki
0
0
ꢀ1
ꢀ1
cross-coupling reaction of 5 -bromo-5-formyl-2,2 -bithiophene 1
1749 cm and the N-butyloxycarbonyl from 1693 to 1715 cm . A
NH2
OH
3
Boc-HN
CO Me
2
2
a n = 1
b n = 2
c n = 3
OHC
2 d
OHC
CHO
S
S
n
1
2
. EtOH, reflux
. LTA, DMSO, r.t.
CHO
n
N
S
N
N
S
O
O
O
N
O
4
a-c
4
d
NH-Boc
Boc-HN
CO Me
MeO
2
C
NH-Boc Boc-HN
CO
2
Me
MeO C
2
2
Scheme 2. Synthesis of N- and C-terminus protected bis-(oligo)thienylbenzoxazolyl-alanine derivatives 4a–d.

S.P.G. Costa et al. / Tetrahedron 64 (2008) 9733–9737
9735
Table 1
Yields, UV–vis absorption and emission data for bis-(oligo)thienylbenzoxazolyl-alanines 4a–d in absolute ethanol and comparison with data for (oligo)thienylbenzoxazolyl-
alanines 5a1 and 5b, c
2b
10c
Cpd. -Bridge Yield (%) UV–vis
p
Fluorescence
em (nm) Stokes’ shift (cm
Cpd. UV–vis
max (nm)
315
Fluorescence
ꢀ1
em (nm) Stokes’ shift (cm )
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
lmax (nm)
3
(mol L cm
)
l
)
F
F
l
3
(mol L cm
)
l
F
F
0.89 5a1
2b
21,300
33,884
37,153
d
394
445
490
d
6365
4925
4592
d
0.80
0.46
0.10
d
4
4
4
4
a
b
c
Tiophene
Bithiophene
Terthiophene 60
Arylthiophene 76
75
54
371
388
416
363
47,210
46,641
46,454
51,800
430
469
514
440
3698
4451
4583
4821
10c
0.59 5b
365
400
d
0.14 5c1
0c
d
0.77
d
band related to the oxazole ring (N]C stretching) appeared at
F
the F determination, the fluorescence standard was excited at
ꢀ
1
1
633–1668 cm
.
the wavelengths of maximum absorption found for each one of the
compounds to be tested and in all fluorimetric measurements the
absorbance of the solution did not exceed 0.1. The highest values
were obtained for bis-alanines 4a, 4b and 4d, with a thiophene,
bithiophene and a phenylthiophene at position 2 of the benzox-
azole, respectively, which were found to be strongly emissive
1
H NMR spectra showed signals that were attributed to the
benzoxazole, in the form of doublets at about 7.48 and 7.53 ppm
for H7 and H4, respectively, and double doublets for H6 around
7.15 ppm. The confirmation of the occurrence of the oxidative
d
d
13
cyclization was also supported by C NMR spectra, where signals of
the oxazole C2 carbon were visible at about 159 ppm.
d
(0.59<
F
F <0.89) while compound 4c, with a terthiophene bridge,
displayed much lower quantum yield (
F
F
¼0.14).
At this stage, a comparison could be made between the photo-
2
.2. Photophysical study
physical data of the synthesized bis-alanines 4a–c and the corre-
sponding (oligo)thienylbenzoxazolyl-alanines 5a–c, recently
The absorption and emission spectra of bis-(oligo)thie-
nylbenzoxazolyl-alanines 4a–d were measured in absolute ethanol
10c,12b
reported by us,
bearing only one benzoxazolyl-alanine system
(Fig. 2, Table 1). For all compounds, it was found that the in-
ꢀ6 ꢀ5
(
10 to 10 M solution) (Table 1). The nature of the thiophenic
troduction of a second benzoxazolyl-alanine system resulted in
a bathochromic shift of both the absorption and the emission
wavelength maxima, an increase in the molar absorptivity value
and, more importantly, in an enhancement of the fluorescence
quantum yield. Also, the new bis-alanines 4a–c had smaller Stokes’
shifts than the mono-alanine series 5a–c, a fact that can potentially
be rationalized by the more polar nature of the mono-alanine
compounds when compared to the inherently less polar nature of
the bis-alanine compounds.
bridge at position 2 of the benzoxazole had a clear influence on the
absorption and emission bands of compounds 4a–d. Comparing
the absorption data for compounds 4b and 4d, it can be seen that
the replacement of the second thiophene ring by a phenyl ring
leads to a small hypsochromic shift, related to the bathochromic
effect of sulfur and an increased
p-overlap between the thiophene
units in compound 4b. Also, compounds 4a–d possess very large
ꢀ
1
ꢀ1
molar absorptivity coefficients (higher than 46,000 mol L cm ).
The wavelength of maximum absorption for compounds 4a–c
was shifted to longer wavelengths as the number of thiophene
units increased, as expected from the increase in conjugation. The
same trend was observed in the emission spectra of these com-
pounds as the position of the wavelength of maximum emission
was red-shifted, ca. 40 nm for each added thiophene (Fig. 1). The
synthesized compounds showed large Stokes’ shift (the lowest
2
N
S
n
3a
O
4
5
7
a
5a n = 1
b n = 2
c n = 3
7
ꢀ
1
ꢀ1
6
being 3698 cm for 4a and the highest 4821 cm for 4d). As
Stokes’ shifts directly relate to energy differences, an increasing
trend could be observed along the series 4a–d. A large Stokes’ shift
is an interesting characteristic for a fluorescent probe that allows an
improved separation of the light inherent to the matrix and the
Boc-HN
CO2Me
Figure 2. Structure of (oligo)thienylbenzoxazolyl-alanine derivatives 5a–c.12b,10c
14
light dispersed by the sample.
The relative fluorescence quantum yields were determined
using a 10 M solution of 9,10-diphenylanthracene in ethanol as
Our results revealed a decrease in the fluorescence quantum
yield as the number of thiophene rings at the p-conjugated bridge
increased. Nevertheless, the fluorescence quantum yield of non-
substituted (oligo)thiophenes is expected to increase as the oligo-
thiophene chain length increases, due to a further extension of the
ꢀ
6
15
standard (
F
F
¼0.95) and bis-(oligo)thienylbenzoxazolyl-alanines
4
a–d exhibited good to excellent fluorescence quantum yields. For
conjugated p-system. On the other hand, the heavy atom induced
1
.2
spin-orbit coupling by the sulfur atoms can give raise to a very
efficient intersystem crossing mechanism, thus lowering the
4
b
4c
1
16
emission. Moreover, azomethine nitrogens contribute to the
heavy atom effect concomitant with the increased degree of con-
4
a
0
0
0
0
.8
.6
.4
.2
0
16c
jugation. Also, the different chains (thiophene, bithiophene, and
terthiophene) should exhibit different degrees of torsion between
the thiophene units, which lead to variations in the effective con-
jugation length, affecting the planarity of the whole heteroaromatic
4
b
4c
4a
16d
system. In our case, we believe that a combination of the above
mentioned effects could be responsible for the trend observed in
our results.
3
00
350
400
450
Wavelength (nm)
500
550
600
Keeping in mind, further applications of these amino acids as
emissive probes for energy transfer or FRET (Fluorescence Reso-
nance Energy Transfer) studies when incorporated in peptides
Figure 1. Normalized UV–vis absorption and emission spectra of compounds 4a–c in
absolute ethanol at T¼298 K (4a, exc¼371 nm; 4b, exc¼388 nm; 4c, exc¼416 nm)
absorption, full line; emission, grey).
l
l
l
(

9736
S.P.G. Costa et al. / Tetrahedron 64 (2008) 9733–9737
00
0
0 00
chains, in Figure 1 can also be seen the good superposition of some
4.2.2. Synthesis of 5,5 -diformyl-2,2 :5 2 -terthiophene 2c through
absorption and emission spectra (for example, compounds 4c and
Suzuki cross-coupling
0
0
4
a), which opens up a very wide range of potential interesting
5 -Bromo-5-formyl-2,2 -bithiophene 1 (0.60 g, 2.1 mmol) was
applications to be explored.
coupled to 5-formylthiophene boronic acid (0.42 g, 2.7 mmol), in
a mixture of DME (35 mL) and aqueous 2 M Na
2
CO
3
(2 mL) and
ꢁ
Pd(PPh
3
)
4
(6 mol %) at 80 C under a argon atmosphere during 20 h.
3
. Conclusions
After cooling the mixture was filtered. Ethyl acetate and a saturated
solution of NaCl were added and the phases were separated. The
organic phase was washed with water (3ꢂ75 mL) and an aqueous
solution of NaOH (10%). The organic phase obtained was dried
In summary, we have achieved for the first time the synthesis of
new fluorescent heteroaromatic bis-amino acid derivatives 4a–
d containing (oligo)thiophene and benzoxazole moieties combined
with an alanine residue by simple procedures in moderate to good
yields and their photophysical properties were evaluated. Due to
their interesting photophysical properties, namely their strongly
emissive character, these heterocyclic alanine derivatives could find
application as useful building blocks for the cross-linking of peptide
chains with the extra value of inserting a UV-active and fluorescent
chromophore, adding functionality to the resulting peptide. These
heteroaromatic bis-amino acids could also be used as fluorescent
markers and probes for FRET studies in peptides.
4
(MgSO ), filtered, and solvent removal gave the crude product,
which was recrystallized affording the pure diformyl derivative 2 as
ꢁ
13
ꢁ
an orange solid (0.38 g, 60%). Mp¼203.6–205.2 C (lit. 224 C). IR
(Nujol):
n
¼1664 (C]O), 1505, 1319, 1219, 1164, 1047, 798, 759,
ꢀ
1 1
0
00
6
68 cm . H NMR (CDCl
3
):
d
¼7.28–7.38 (m, 4H, H-3, H-3 , H-3 ,
0
0
00
and H-4 ), 7.70 (d, 2H, J¼4.2 Hz, H-4 and H-4 ), 9.89 (s, 2H,
2ꢂCHO) ppm.
4.3. General procedure for the synthesis of bis-(oligo)-
thienylbenzoxazolyl-alanines 4a–d
4
4
. Experimental
N-t-Butyloxycarbonyl-3-amino-
2 equiv) was stirred with the corresponding diformylated (oligo)-
thiophene 2 (1 equiv) and heated in ethanol overnight at reflux
5 mL/equiv). The solvent was evaporated and the crude imine used
L
-tyrosine methyl ester
3
(
.1. Synthesis general
(
All melting points were measured on a Gallenkamp melting
in the next step without further purification. The corresponding
crude imine and lead tetraacetate (3 equiv) were stirred at room
temperature in DMSO (5 mL/equiv) for 3 days. The mixture was
poured over water and extracted with ethyl acetate (3ꢂ10 mL).
After drying with anhydrous magnesium sulfate and evaporation of
the solvent under reduced pressure, the crude compound was
submitted to column chromatography with silica gel by elution
with dichloromethane and dichloromethane/methanol, 100:1. The
fractions containing the pure compound were combined and
evaporated under reduced pressure.
point apparatus and are uncorrected. TLC analyses were carried out
on 0.25 mm thick precoated silica plates (Merck Fertigplatten
Kieselgel 60F254) and spots were visualized under UV light. Chro-
matography on silica gel was carried out on Merck Kieselgel (230–
2
40 mesh). IR spectra were determined on a BOMEM MB 104
spectrophotometer using KBr discs or Nujol. UV–vis absorption
spectra (200–800 nm) were obtained using a Shimadzu UV/2501PC
spectrophotometer. H NMR spectra were recorded on a Varian 300
spectrometer in CDCl
given in parts per million using
values are given in hertz. C NMR spectra were run in the same
instrument at 75.4 MHz using the solvent peak as internal refer-
ence. Assignments were made by comparison of chemical shifts,
peak multiplicities, and J values, and were supported by spin
decoupling-double resonance and bidimensional heteronuclear
HMBC and HMQC correlation techniques. Mass spectra were
obtained on a Finnigan LXQ MS spectrometer (ESI technique,
positive mode). Elemental analyses were carried out on a Leco
CHNS 932 instrument. Fluorescence spectra were collected using
1
ꢁ
3
at 300 MHz at 25 C. All chemical shifts are
d
H
Me
4
Si¼0 ppm as reference and J
13
0
4
.3.1. bis-[N-t-Butyloxycarbonyl [2-(thien-2 -yl)benzoxazol-
5-yl]-L-alanine methyl ester] (4a)
The product was isolated as a yellow solid (0.19 g, 75%).
ꢁ
Mp¼106.2–108.0 C. IR:
n
¼3355, 2968, 2940, 1749, 1693, 1640,
1
583, 1524, 1476, 1446, 1392, 1365, 1255, 1166, 1056, 1016, 813, 794,
ꢀ1
1
7
14 cm . H NMR (CDCl
m, 4H, 2ꢂ CH ), 3.75 (s, 6H, 2ꢂCH
.06 (d, J¼7.8 Hz, 2H, 2ꢂNH), 7.16 (dd, J¼1.8 and 8.4 Hz, 2H, 2ꢂH-6),
.50 (d, J¼8.4 Hz, 2H, 2ꢂH-7), 7.53 (d, J¼1.8 Hz, 2H, 2ꢂH-4), 7.93
3
):
d
¼1.43 (s, 18H, 2ꢂC(CH
3
)
3
), 3.16–3.31
(
b
2
3
), 4.62–4.67 (m, 2H, 2ꢂ
aH),
5
7
(
a Spex Fluorolog 1680 spectrometer. All reagents were used as
received. 5-Formyl-2,2 -bithiophene, 3-nitro-
diformylthiophene 2a are commercially available (Aldrich). The
synthesis of 5,5 -diformyl-2,2 -bithiophene 2b
0
13
dd, J¼1.2 and 3.9 Hz, 2H, 2ꢂH-3 ) ppm. C NMR (CDCl
3
):
d
¼28.20
0
L-tyrosine, and 2,5-
(
C(CH
3
)
3
), 38.28 (
b
CH
2
), 52.31 (CH
3
), 54.53 (
a
C), 79.95 (C(CH
3
)
3
),
0
0
1
10.25 (C7), 120.30 (C4), 126.18 (C6), 130.02 (C2 ), 130.17 (C3 ),
0
0
11g
and 2-formyl-5-
132.83 (C5), 142.27 (C3a), 149.61 (C7a), 155.14 (C]O Boc), 159.50
0
11c
(
4 -formylphenyl)-thiophene 2d was described elsewhere.
(
(
C
C2), 171.97 (C]O ester) ppm. UV–vis (ethanol, nm):
l
max
(
3
)¼371
þ
47,210). MS: m/z (ESI, %): 721 ([MþH] , 31), 665 (100), 609 (32).
4
.2. Procedure for the synthesis of diformyl-terthiophene 2c
36
40
H N
4
O10S: calcd C 59.99, H 5.59, N 7.77; found C 60.04, H 5.67,
N 7.61.
0
0
4
.2.1. Synthesis of 5 -bromo-5-formyl-2,2 -bithiophene 1
0
0
To
a
stirred solution of 5-formyl-2,2 -bithiophene (0.8 g,
4.3.2. bis-[N-t-Butyloxycarbonyl[2-(bithien-5 -yl)benzoxazol-
4
4
.1 mmol) in DMF (1 mL), in the dark, was added NBS (0.73 g,
.1 mmol) at ꢀ20 C during 4 h. After this time the reaction mixture
5-yl]-L-alanine methyl ester] (4b)
ꢁ
The product was isolated as an orange solid (0.09 g, 54%).
ꢁ
was poured on ice and stirred till room temperature and a light
yellow solid precipitated, which was isolated by filtration. Re-
crystallization from dichloromethane–light petroleum gave the
Mp¼111.7–113.9 C. IR (KBr):
n
¼3415, 2965, 2932, 1745, 1714, 1633,
1579, 1473, 1394, 1369, 1260, 1164, 1053, 1019, 936, 863, 801,
ꢀ
1 1
713 cm . H NMR (CDCl
CH
3
):
d
¼1.43 (s, 18H, 2ꢂC(CH
3
)
3
), 3.18–3.28
-H),
pure compound as a light yellow solid (1.09 g, 98%). Mp¼137–
(m, 4H, 2ꢂ
b
2
), 3.75 (s, 6H, 2ꢂCH
3
), 4.60–4.65 (m, 2H, 2ꢂ
a
ꢁ
139 C. IR (KBr):
n
¼1651 (C]O), 1510, 1455, 1278, 1230, 1193, 1050,
5.03 (d, J¼8.1 Hz, 2H, 2ꢂNH), 7.15 (dd, J¼1.2 and 8.4 Hz, 2H, 2ꢂH-6),
ꢀ
1 1
0
9
1
75, 879, 800, 784, 751, 666, 644 cm . H NMR (CDCl
3
):
H, J¼3.9 Hz, H-3 ), 7.12 (d, 1H, J¼3.9 Hz, H-4 ), 7.19 (d, 1H, J¼3.9 Hz,
BrOS
calcd C 39.57, H 1.84, S 23.48; found C 39.65, H 1.75, S 23.42.
d
¼7.05 (d,
7.36 (d, J¼4.2 Hz, 2H, 2ꢂH-4 ), 7.48 (d, J¼8.4 Hz, 2H, 2ꢂH-7), 7.51 (d,
0
0
0
13
J¼1.2 Hz, 2H, 2ꢂH-4), 7.85 (d, J¼4.2 Hz, 2H, 2ꢂH-3 ) ppm. C NMR
H-3), 7.67 (d, 1H, J¼3.9 Hz, H-4), 9.88 (s, 1H, CHO) ppm. C
9
H
5
2
:
(CDCl
3
):
d
¼28.22 (C(CH
3
)
3
), 38.25 (
b
CH
2
), 52.32 (CH
3
), 54.60 (
a
C),
0
80.04 (C(CH
3
)
3
), 110.22 (C7), 120.23 (C4), 126.45 (C4 ), 126.28 (C6),

S.P.G. Costa et al. / Tetrahedron 64 (2008) 9733–9737
9737
0
0
0
1
27.38 (C2 ), 130.91 (C3 ), 132.85 (C5), 143.84 (C5 ), 142.37 (C3a),
References and notes
1
49.65 (C7a), 155.09 (C]O Boc), 159.01 (C2), 172.10 (C]O
1
. (a) Garrard, E. A.; Borman, E. C.; Cook, B. N.; Pike, E. J.; Alberg, D. G. Org. Lett.
000, 23, 3639–3642; (b) Fu, X.; Jiang, S.; Li, C.; Xin, J.; Yanga, Y.; Jia, R. Bioorg.
Med. Chem. Lett. 2007, 17, 465–470.
2. (a) For examples, see: Travins, J. M.; Etzkorn, F. A. J. Org. Chem. 1997, 62, 8387–
ester) ppm. UV–vis (ethanol, nm):
l
max
(3
)¼388 (46,641). MS: m/z
2
þ
(
C
ESI, %): 803 ([MþH] , 34), 747 (30), 681 (78), 509 (30), 451 (100).
H
40 42
N
4
O
S
10 2
: calcd C 59.84, H 5.27, N 6.98; found C 59.78, H 5.27,
8
5
393; (b) Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122,
891–5892; (c) McNamara, L. M. A.; Andrews, M. J. I.; Mitzel, F.; Siligardi, G.;
N 7.11.
Tabor, A. B. J. Org. Chem. 2001, 66, 4585–4594; (d) Levins, C. G.; Schafmeister, C.
E. J. Org. Chem. 2005, 70, 9002–9008.
. (a) Boschin, G.; Scaglioni, L.; Arnoldi, A. J. Agric. Food Chem. 1999, 47, 939–944
0
4
5
.3.3. bis-[N-t-Butyloxycarbonyl[2-(terthien-5 -yl)benzoxazol-
-yl]-L-alanine methyl ester] (4c)
3
and references therein; (b) Ma, L.; Gao, C.; Mao, Z.; Zhou, J.; Shen, J. Biomaterials
The compound was isolated as an orange solid (0.07 g, 60%).
2004, 25, 2997–3004; (c) Morino, K.; Oobo, M.; Yasgima, E. Macromolecules
ꢁ
Mp¼99.1–101.3 C. IR (KBr):
n
¼3428, 2976, 2929, 1744, 1714, 1662,
2005, 38, 3461–3468; (d) Gyenes, T.; Torma, V.; Gyarmaty, B.; Zr ı´ nyi, M. Acta
Biomater. 2008, 4, 733–744.
1
8
3
577, 1516, 1496, 1439, 1392, 1366, 1256, 1218, 1165, 1060, 1018, 913,
ꢀ
1 1
4. (a) For selected examples, see: Cheng, R. P.; Fisher, S. L.; Imperiali, B. J. Am.
Chem. Soc. 1996, 118, 11349–11356; (b) Budisa, N.; Alefelder, S.; Bae, J. H.; Golbik,
R.; Minks, C.; Huber, R.; Moroder, L. Protein Sci. 2001, 10, 1281–1292; (c) Li, B. S.;
Cheuk, K. K. L.; Ling, L.; Chen, J.; Xiao, X.; Bai, C.; Tang, B. Z. Macromolecules
2003, 36, 77–85.
63, 792 cm . H NMR (CDCl
CH
3
):
d
¼1.44 (s, 18H, 2ꢂC(CH
3 3
) ), 3.15–
.24 (m, 4H, 2ꢂ
b
2
), 3.75 (s, 6H, 2ꢂCH
3
), 4.60–4.66 (m, 2H, 2ꢂ
a-
H), 5.04 (d, J¼8.1 Hz, 2H, 2ꢂNH), 7.14 (dd, J¼1.8 and 8.1 Hz, 2H,
0
2
7
ꢂH-6), 7.34 (d, J¼3.9 Hz, 2H, 2ꢂH-4 ), 7.48 (d, J¼8.1 Hz, 2H, 2ꢂH-
5
6
. Ishida, H.; Kyakuno, M.; Oishi, S. Biopolymers (Pep. Sci.) 2004, 76, 69–82.
. (a) Taki, M.; Wolford, J. L.; OHalloran, T. V. J. Am. Chem. Soc. 2003, 126, 712–713;
(b) Shamsipur, M.; Poursaberi, T.; Karami, A. R.; Hosseini, M.; Momeni, A.;
Alizadeh, N.; Yousefi, M.; Ganjali, M. R. Anal. Chim. Acta 2004, 501, 55–60; (c)
Milewska, M.; Skwierawska, A.; Guzow, K.; Szmigiel, D.; Wiczk, W. Inorg. Chem.
Commun. 2005, 8, 947–950; (d) Zang, X.-B.; Peng, J.; He, C.-L.; Shen, G.-L.; Yu,
R.-Q. Anal. Chim. Acta 2006, 567, 189–195.
0
), 7.51 (d, J¼1.8 Hz, 2H, 2ꢂH-4), 7.71 (d, J¼3.9 Hz, 2H, 2ꢂH-3 ), 7.86
´
0
0
13
(
(
(
(
(
s, 2H, 2ꢂH-3 ) ppm. C NMR (CDCl
3
):
d
¼28.23 (C(CH
3 3
) ), 38.17
b
CH
2
), 52.24 (CH
3
), 54.60 (
a
C), 80.03 (C(CH
3
)
3
), 110.17 (C7), 120.16
0
00
0
C4), 124.30 (C4 ), 125.74 (C3 ), 126.30 (C6), 127.26 (C2 ), 130.81
C3 ), 132.78 (C5), 137.83 (C2 ), 141.93 (C5 ), 142.28 (C3a), 149.67
C7a), 155.11 (C]O Boc), 158.91 (C2), 172.12 (C]O ester) ppm. UV–
0
00
0
7
. (a) Roncali, J. Chem. Rev. 1997, 97, 173–206; (b) Zrig, S.; R e´ my, P.; Andrioletti, B.;
Rose, E.; Asselberghs, I.; Clays, K. J. Org. Chem. 2008, 73, 1563–1566.
. (a) Yu, J.; Shirota, Y. Chem. Lett. 2002, 31, 984–985; (b) Lin, S.-C.; Sun, S.-S. Chem.
Mater. 2002, 14, 1884–1890; (c) Destri, S.; Pasini, M.; Botta, C.; Porzio, W.;
Bertini, F.; Marchio, L. J. Mater. Chem. 2002, 12, 924–933; (d) B e´ ra-Ab e´ rem, M.;
Ho, H.-A.; Leclerc, M. Tetrahedron 2004, 60, 11169–11173; (e) Barbarella, G.;
Melucci, M.; Sotgiu, G. Adv. Mater. 2005, 17, 1581–1593.
vis (ethanol, nm):
l
max
(
3
)¼416 (46,454). MS: m/z (ESI, %): 885
8
þ
(
6
[MþH] , 22), 509 (100). C44
44 4 10 3
H N O S
: calcd C 61.97, H 5.20, N
.57; found C 61.88, H 5.27, N 6.72.
0
0
0
4
.3.4. bis-[N-t-Butyloxycarbonyl[2-(phen-4 -yl-thien-2 -yl)-
9. Finley, J. W.; Friedman, M. Adv. Exp. Med. Biol. 1977, 86B, 123–130; (b) Fujimoto,
D.; Hirama, M.; Iwashita, T. Biochem. Biophys. Res. Commun. 1982, 104, 1102–
benzoxazol-5-yl]-
L
-alanine methyl ester] (4d)
1
106; (c) Henle, T.; Walter, A. W.; Klostermeyer, H. Z. Lebensm. -Unters. Forsch
993, 197, 114–117; (d) Tohdo, K.; Hamada, Y.; Shioiri, T. Synlett 1994, 247–249;
e) Boschin, G.; D’Agostina, A.; Arnoldi, A. Food Chem. 2002, 78, 325–331.
10. (a) Costa, S. P. G.; Maia, H. L. S.; Pereira-Lima, S. M. M. A. Org. Biomol. Chem.
003, 1, 1475–1479; (b) Jiang, W.-Q.; Costa, S. P. G.; Maia, H. L. S. Org. Biomol.
The compound was isolated as a yellow solid (0.14 g, 76%).
1
(
ꢁ
Mp¼96.00–98.5 C. IR (KBr):
n
¼3435, 2977, 2930, 1746, 1715, 1668,
ꢀ
1 1
1
615, 1575, 1505, 1479, 1367, 1257, 1168, 1056, 1018, 802 cm . H
NMR (CDCl ): ), 3.20–3.25 (m, 4H,
¼1.44 (s, 18H, 2ꢂC(CH
CH -H), 5.05
), 3.75 (s, 6H, 2ꢂCH
2
3
d
3 3
)
Chem. 2003, 1, 3804–3810; (c) Costa, S. P. G.; Oliveira, E.; Lodeiro, C.; Raposo, M.
M. M. Tetrahedron Lett. 2008, 49, 5258–5261.
2
(
(
ꢂb
2
3
), 4.62–4.66 (m, 2H, 2ꢂ
a
d, J¼8.4 Hz, 2H, 2ꢂNH), 7.15 (dd, J¼1.2 and 8.1 Hz, 2H, 2ꢂH-6), 7.48
d, J¼8.1 Hz, 2H, 2ꢂH-7), 7.52 (d, J¼1.2 Hz, 2H, 2ꢂH-4), 7.56 (d,
11. (a) Batista, R. M. F.; Costa, S. P. G.; Raposo, M. M. M. Tetrahedron Lett. 2004, 45,
2825–2828; (b) Costa, S. P. G.; Batista, R. M. F.; Sousa, A. M. R. C.; Raposo, M. M. M.
Mater. Sci. Forum 2006, 514–516, 147–151; (c) Costa, S. P. G.; Batista, R. M. F.;
Cardoso, P.; Belsey, M.; Raposo, M. M. M. Eur. J. Org. Chem. 2006, 17, 3938–3946;
(d) Batista, R. M. F.; Costa, S. P. G.; Malheiro, E. L.; Belsey, M.; Raposo, M. M. M.
Tetrahedron 2007, 63, 4258–4265; (e) Batista, R. M. F.; Costa, S. P. G.; Belsley, M.;
Raposo, M. M. M. Tetrahedron 2007, 63, 9842–9849; (f) Pina, J.; Seixas de Melo, J.;
Burrows, H. D.; Batista, R. M. F.; Costa, S. P. G.; Raposo, M. M. M. J. Phys. Chem. A
2007, 111, 8574–8578; (g) Batista, R. M. F.; Costa, S. P. G.; Belsley, M.; Lodeiro, C.;
Raposo, M. M. M. Tetrahedron 2008, 64, 9230–9238.
0
0
00
00
J¼3.9 Hz, 1H, H-4 ), 7.81–7.87 (m, 3H, H-3 , H-2 , and H-6 ), 8.31 (d,
0
0
00
13
J¼8.1 Hz, 2H, H-3 and H-5 ) ppm. C NMR (CDCl
3
):
d
¼28.30
(
C(CH
3
)
3
), 38.31 (bCH
2
), 52.35 (CH
3
), 54.57 (
a
C), 80.00 (C(CH
3
)
3
0
),
0
1
1
10.25 (C7), 120.28 (C4), 126.38 (C4 ), 126.30 (C6), 127.41 (C2 ),
30.48 (C2 and C6 ), 130.93 (C3 ), 132.84 (C5), 136.41 (C3 and
C5 ), 138.49 (C1 ), 144.00 (C5 ), 142.44 (C3a), 149.80 (C7a), 151.78
C4 ), 155.11 (C]O Boc), 159.10 (C2), 172.03 (C]O ester) ppm. UV–
0
0
00
0
00
0
0
00
0
00
12. (a) Batista, R. M. F.; Oliveira, E.; Costa, S. P. G.; Lodeiro, C.; Raposo, M. M. M.
Org. Lett. 2007, 9, 3201–3204; (b) Costa, S. P. G.; Oliveira, E.; Lodeiro, C.; Raposo,
M. M. M. Sensors 2007, 7, 2096–2114.
13. Anestopoulos, D.; Fakis, M.; Polyzos, I.; Tsigaridas, G.; Persephonis, P.; Gian-
netas, V. J. Phys. Chem. B 2005, 109, 9476–9481.
(
vis (ethanol, nm):
l
max
(3
)¼363 (51,800). MS: m/z (ESI, %): 797
10S: calcd C 63.30, H 5.57, N
þ
(
7
[MþH] , 43), 740 (100). C42
H
44
N
4
O
.03; found C 63.23, H 5.43, N 7.12.
1
4. Holler, M. G.; Campo, L. F.; Brandelli, A.; Stefani, V. J. Photochem. Photobiol., A:
Chem. 2002, 149, 217–225.
5. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969–974.
6. (a) Becker, R. S.; Seixas de Melo, J.; Maçanita, A. L.; Elisei, F. J. Phys. Chem. 1996,
00, 18683–18695; (b) Seixas de Melo, J.; Silva, L. M.; Arnaut, L. G.; Becker, R. S.
1
1
Acknowledgements
1
J. Chem. Phys. 1999, 111, 5427–5433; (c) Dufresne, S.; Bourgeaux, M.; Skene, W.
G. J. Mater. Chem. 2007, 17, 1166–1177; (d) Seixas de Melo, J.; Burrows, H. D.;
Svensson, M.; Andreson, M. R.; Monkman, A. P. J. Chem. Phys. 2003, 118,
1550–1556.
Thanks are due to the Fundaç ˜a o para a Ci eˆ ncia e Tecnologia
Portugal) for financial support through project PTDC/QUI/66250/
006 and a Ph.D. grant to R.M.F.B. (SFRH/BD/36396/2007).
(
2