A mild high yielding synthesis of oxazole-4-carboxylate derivatives

Article abstract of DOI:10.1016/j.tet.2010.09.014

Several 2,5-disubstituted oxazole-4-carboxylates were prepared in high yields from the methyl esters of N-acyl-β-halodehydroaminobutyric acid derivatives by treatment with a 2% solution of DBU in acetonitrile. The scope of this reaction was investigated a

Full text of DOI:10.1016/j.tet.2010.09.014

Tetrahedron 66 (2010) 8672e8680
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A mild high yielding synthesis of oxazole-4-carboxylate derivatives
,
b
a
a
a
Paula M.T. Ferreira a , Elisabete M.S. Castanheira , Luís S. Monteiro , Goreti Pereira , Helena Vilaça
*
a Chemistry Centre (CQ-UM), University of Minho, Gualtar, 4710-057 Braga, Portugal
b Centre of Physics (CFUM), University of Minho, 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 27 July 2010
Received in revised form 1 September 2010
Accepted 3 September 2010
Available online 15 September 2010
Several 2,5-disubstituted oxazole-4-carboxylates were prepared in high yields from the methyl esters of
N-acyl- -halodehydroaminobutyric acid derivatives by treatment with a 2% solution of DBU in aceto-
nitrile. The scope of this reaction was investigated and it was found that dehydrodipeptides having
-bromodehydroaminobutyric acid residue gave the corresponding oxazoles in good yields. The pho-
b
a
b
tophysical properties of some of the oxazoles prepared were studied in four solvents of different polarity.
All compounds have reasonable high fluorescence quantum yields and a moderate solvent sensitivity,
which makes them good candidates to be used as fluorescent probes. One of the fluorescent oxazoles
prepared was inserted after cleavage of the methyl ester into two model peptides using a conventional
solution phase strategy. The photophysical properties of the labelled peptides were studied in ethanol
and water and compared with those of the oxazole. The results obtained showed that the oxazole
maintains a good fluorescence level and the same solvent sensitivity when linked to a peptide chain.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
N-Acyl-threonine derivatives
N-Acyl-b-halodehydroaminobutyric acids
Oxazole-4-carboxylates
Fluorescent probes
Labelled peptides
1. Introduction
promoted by iodine/DBU.11 Continuing this work and taking advan-
tage of our experience in the synthesis and reactivity of
odehydroamino acids12 we aimed at preparing functionalized
oxazoles from -halodehydroaminobutyric acid derivatives. We also
aimed at studying their photophysical properties in order to test their
possible use as fluorescent probes.
b-hal-
Oxazoles are considered as an important class of heterocyclic
compounds since they are structural subunits of various biologically
active natural products and are valuable synthetic precursors and
pharmaceuticals.1 Oxazoles are associated with anti-bacterial, anti-
fungal, anti-inflammatory and anti-tumoral activities and can be
used as peptide mimetics or enzyme inhibitors.2 Oxazole derivatives
have also been used as efficient luminophores for liquid and plastic
scintillators and as fluorescent probes for biological systems.3
Several methods have been developed for the syntheses of
this type of compounds, such as the Hantzsch reaction,4 the Rob-
b
2. Results and discussion
SeveralN-acyl-b-bromodehydroaminobutyric acids were prepared
from the corresponding N-acyl-threonine derivatives using a sequen-
tial dehydration followed by a halogenation reaction (Scheme 1).11,12c
insoneGabriel synthesis where b
-ketoamides are cyclodehydrated,5
the dehydration of oxazolines, condensations of substituted amides
with phenacyl bromide in ethanol,2e the Ugi reaction with ammonia
as the amine component,6 the coupling of an acyl chloride with an
isocyanide under basic conditions7 or the gold catalyzed cyclization
ofpropargylic amides.8 Amino acids have alsobeenused assubstrates
for the synthesis of oxazole derivatives. Thus, Wipf et al. prepared
oxazoles by treating serine derivatives with diethylaminosulphur
trifluouride (DAST) followed by bromotrichloromethane and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU).9 Disubstituted oxazoles were
obtained from N-acylamino acids by cyclodehydration of the in-
H
N
H
N
H
N
R
CO2CH3
OH
R
CO2CH3
R
CO2CH3
X
1. NXS
2. NEt3
1.Boc2O/DMAP
2. TMG
O
O
O
X=Br, 3a-d
X=I, 4a
2a-d
1a-d
O
H3CO
R =
(Bz), a;
[Bz(4-OMe)], b;
Scheme 1.
(2-Fur), d.
(1-Naph), c;
termediate
a
-acylamino aldehydes.10 In our research group we have
-hydroxyamino acids by a sequential
prepared oxazoles from N-acyl
b
Considering our previously developed method for the synthesis
of oxazoles and the mechanism proposed the Z-isomer of the
methyl ester of N-benzoyl- -bromodehydroaminobutyric acid Z-3a
was treated with several bases in different amounts [Et3N, sodium
dehydration reaction followed by an intramolecular cyclization
b
* Corresponding author. E-mail address: pmf@quimica.uminho.pt (P.M.T. Ferreira).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.014

P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
8673
methoxide, sodium tert-butoxide, N,N,N0,N0-tetramethylguanidine
R
R
H
N
Boc
Boc
CO2CH3
(TMG) and DBU] and the reactions followed by HPLC. It was found
that the best result was obtained with a 2% solution of DBU in
acetonitrile (89% yield of oxazole 5a). Thus, compounds Z-3bed
were reacted with DBU to give the corresponding 2,5-disubstituted
oxazole-4-carboxylates (5bed) in high yields (84e91%) (Scheme 2,
Table 1). This reaction was also tested using as substrates de
DBU
N
N
H
N
H
CO2CH3
O
O
Br
6a-c
7a-c
R = H, a; CH2C6H5, b; CH(CH3)2, c.
Scheme 3.
E-isomers of
b-bromodehydroaminobutyric acid derivatives
(Table 1, entries 2 and 6). The oxazole-4-carboxylates were obtained
in similar yields when compared with the Z-isomers. These results
show that it is possible to use the mixtures of stereoisomers as
reagents, which is an advantage since the halogenation reaction
Table 2
Yields obtained in the synthesis of oxazole-4-carboxylates from
drodipeptide derivatives
b
-bromodehy-
Yield/%
Entry
Reagent
Product
affords the corresponding
of the E- and Z-isomers. The scope of this reaction was also enlarged
to -iododehydroaminobutyric acid derivatives since compound
Z-4a afforded oxazole 5a in 96% yield (Table 1, entry 3).
b-halodehydroamino acids as mixtures
Boc
N
N
b
H
1
Z-6a
CO2CH3
85
O
7a
CO2CH3
H
N
R
CO2CH3
X
DBU 2%
ACN
N
O
O
2
Z-6b
78
Boc
N
R
N
5a-d
H
CO2CH3
X=Br, 3a-d
X=I, 4a
O
7b
Scheme 2.
3
4
E-6b
E-6c
7b
82
89
Table 1
Boc
N
N
Yields obtained in the synthesis of oxazole-4-carboxylates from N-acyl-b-hal-
odehydroaminobutyric acid derivates
H
CO2CH3
O
7c
Entry
1
Reagent
Product
Yield/%
89
1
All H NMR spectra of oxazole-4-carboxylates show a downfield
shift of OCH3 and 5-CH3 protons when compared with the corre-
sponding protons in the reagent. The 13C NMR spectra of these com-
poundsshowthatC2(159.3e162.2 ppm)resonatesatthelowestfields
compared with C4 and C5. This is a common feature reported by other
authors for other oxazole derivatives.13 The exception is compound
5d, which shows a C2 chemical shift (152.3 ppm) smaller than C5
(155.8 ppm), which is probably due to the effect of the electron-rich
furan ring. C4 resonates at higher fields (between 120.1 and
128.6 ppm) when compared with C2 and C5. The high chemical shifts
observed for C5 (between 155.4 and156.3 ppm) when compared with
the C5 chemical shift of the unsubstituted oxazole ring (138.1 ppm)13
can be attributed to the carboxylate function linked to C4.
O
N
Z-3a
CO2CH3
5a
2
3
E-3a
Z-4a
5a
5a
86
96
H3CO
O
4
Z-3b
91
N
The mechanism proposed for the intramolecular cyclization of
CO2CH3
5b
the
the attack of the carbonyl oxygen on the
dehydroaminobutyric acid with loss of the halogen anion.
When -bromodehydrophenylalanine and -dibromodehy-
droalanines were used as substrates no reaction occurred. This may
be partially due to a lower electron density at the -carbon atom of
-halodehydroaminobutyric acid derivatives, which can be related
with the 13C NMR chemical shift (Table 3). In fact, the chemical
b
-halodehydroaminobutyric acids promoted by DBU involves
b-carbon atom of the
O
b
b,b
5
6
Z-3c
E-3c
91
88
N
b
CO2CH3
5c
b
5c
shifts of the b-carbon atom of the b-bromodehydroaminobutyric
Table 3
13C NMR chemical shifts in CDCl3 of the
b
-carbon atom and reduction peak poten-
O
O
tials of
b
-bromodehydroamino acid derivatives.12c
7
Z-3d
84
N
Compound
d b-C atom/ppm
ꢀEp (V vs SCE)12c
CO2CH3
5d
Z-3a
Z-3b
Z-3c
Z-3d
123.56
127.51
126.46
126.43
114.32
112.81
84.74
1.78
1.88
d
In order to test the possibility of using dipeptides with a
dehydroaminobutyric acid residue as substrates in this reaction,
several dipeptides having a -bromodehydroaminobutyric acid
were reacted with DBU. The corresponding oxazole derivatives were
obtained in good yields (Scheme 3, Table 2).
b-halo-
1.94
1.41
1.44
1.58
1.57
Bz(OMe)-Z-
D
Phe(
-Br)-OMe
Ala( -Br)-OMe
-Br)-OMe
b-Br)-OMe
Fur-Z-
Bz(OMe)-
Fur- Ala(
DPhe(b
b
D
b,b
D
b
,
b
85.48

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P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
acids are considerably higher than those of the corresponding
-bromodehydrophenylalanines and -dibromodehydroalanines.
treatment with a solution of NaOH in methanol to give the car-
boxylic acid 8 in 97% yield. This compound was then coupled with
b
b
,b
By comparing the reduction peak potentials of these compounds
obtained previously by us in a study concerning the electro-
two model peptides (H-Gly-L-Ala-OMe and H-Gly-L-Ala-L-Phe-OEt)
using a conventional solution phase strategy. The peptides labelled
with the oxazole moiety were obtained in 30% and 81% yield
(Scheme 4).
chemical behaviour of
conclude that -bromodehydroaminobutyric acids have lower re-
b
-halodehydroamino acids12c it is possible to
b
duction potentials when compared with the corresponding dehy-
drophenylalanines and dehydroalanines (Table 3).
The reactions of compounds E-3a, Z-3b, Z-3d and E-6c were
followed by reverse phase HPLC and plots of the peak areas versus
time were obtained for each compound (Fig. 1).
Scheme 4.
The absorption and fluorescence properties of compounds 5aed
and 8 were studied in solvents of different polarity (cyclohexane,
ethyl acetate, acetonitrile and ethanol). For 5a and 8, spectroscopic
measurements in water were also performed, to obtain a better
comparison with the behaviour of the labelled peptides (9 and 10).
The normalized absorption and fluorescence spectra of compounds
5aed and 8 are presented in Figs. 3 and 4. The maximum absorp-
tion (labs) and emission (lem) wavelengths, molar absorption co-
efficients (3) and fluorescence quantum yields (FF) of the oxazole
Fig. 1. Plot of Area versus time for compounds E-3a, Z-3b, Z-3d and E-6c.
derivatives are presented in Table 4.
A linear correlation was obtained for all compounds by plotting
the ln(Area) versus time (Fig. 2), which indicates a first order kinetics.
The rate constants determined for compounds E-3a, Z-3b, Z-3d and
E-6c were 3.88ꢁ10ꢀ2 minꢀ1, 9.34ꢁ10ꢀ2 minꢀ1, 10.2ꢁ10ꢀ2 minꢀ1 and
3.67ꢁ10ꢀ2 minꢀ1, respectively. The increase in the rate constant of
compound Z-3b when compared with compound E-3a is due to the
presenceofanelectron-donatinggrouplinkedtothephenylmoiety. A
similar effect was observed for compound Z-3d, which has an elec-
tron-rich furan ring instead of the phenyl moiety of compound E-3a.
Fig. 2. Plot of ln(Area) versus time for compounds E-3a, Z-3b, Z-3d and E-6c, showing
the linear dependencies.
In order to study the photophysical properties of the oxazoles
prepared when incorporated into peptides, two model peptides
linked to the 5-methyl-2-phenyloxazole moiety were prepared.
Therefore, the methyl ester of compound 5a was cleaved by the
Fig. 3. Normalized absorption (in the lowest energy band) and fluorescence spectra
(
fluorescence) of compounds 5a and 8, in several solvents.
lexc¼270 nm) of solutions (2ꢁ10ꢀ5 mol dmꢀ3 for absorption and 2ꢁ10ꢀ6 mol dmꢀ3 for

P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
8675
Fig. 4. Normalized absorption (in the lowest energy band) and fluorescence spectra of
solutions (2ꢁ10ꢀ5 mol dmꢀ3 for absorption and 2ꢁ10ꢀ6 mol dmꢀ3 for fluorescence) of
compounds 5b and 5d (lexc¼270 nm) and 5c (lexc¼310 nm), in several solvents.
These compounds present high molar absorption coefficients at
the lowest energy maximum (
3
ꢂ1.2ꢁ104 molꢀ1 dm3 cmꢀ1) in all
solvents studied (Table 4), pointing to a
p/p transition, as ob-
*
served for isoxazoles.16 A methyl carboxylate group or carboxylic
acid (in case of compound 6) is also present in all compounds and it
is known that many carbonyl compounds present a low-lying
n/
p
*
state. The
p
/
p
*
and n/
p electronic transitions can be
*
nearby in energy, resulting in state-mixing.17 The high values of the
molar absorption coefficient can be explained by a predominance of
p/p character in these compounds that also justifies the high
*
fluorescence quantum yield values (Table 4). The influence of sol-
vent in the absorption spectral shape is generally negligible.
The emission spectra of the studied compounds (5aed and 8)
show a defined vibrational structure in cyclohexane (non polar
solvent) and a progressive loss of this structure with increasing
solvent polarity (Figs. 3 and 4). Compound 5c, which presents the
more extended aromatic system, exhibits a more detailed vibra-
tional structure in the fluorescence spectra (Fig. 4). The loss of vi-
brational structure in emission and the red shift observed in polar
solvents points to an intramolecular charge transfer (ICT) character
of the excited state,18 although not very pronounced.
The effect of the solvent in the spectral shape is more pro-
nounced for compound 8, where the emission is almost completely
non-structured and significantly red-shifted in ethanol and water
(Fig. 3). It was described that oxazoles and hydroxyl containing
compounds form complexes, leading to a loss of vibrational struc-
ture in their spectra.19 For these oxazoles, complex formation with
protic solvents is not anticipated from their photophysical behav-
iour (Figs. 3 and 4), as the loss of vibrational structure is progressive
with increasing solvent polarity. The spectral changes in protic
solvents are significant for 8, but not for 5a. The presence of

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P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
a carboxylic acid in 8 may lead to the increase of the ICTcharacter of
excited state and also enhances the hydrogen bonding capability of
this oxazole.
From ab initio molecular quantum chemistry calculations,
obtained with Gaussian 09 software20 and use of a 3-21þG(d) basis
set,21 the optimized geometries of compounds 5aed and 8 were
obtained (Fig. 5), showing that all compounds are roughly planar.
Fig. 7. Representation of HOMO and LUMO molecular orbitals of compounds 5b
(above), 5c (middle) and 5d (below).
The same does not happen for compound 5c, where the elec-
tronic density is mainly located in the aromatic rings in both HOMO
and LUMO, with a very small participation of the carboxylate group
in the HOMO/LUMO transition, pointing to a low ICT character of
the excited state. This may justify the well defined vibrational
structure in emission spectra, even in polar solvents, also present in
the absorption lowest energy band (although not so well defined).
The spectra of 5c are significantly shifted to lower energies relative
to the other oxazoles (Fig. 4), showing that the extension of the
conjugation with a naphthyl group makes an important contribu-
tion to both absorption and fluorescence properties.
Fig. 5. Optimized geometries of compounds 5a, 5b, 5c, 5d and 8 obtained by Gaussian
09 software (grey: C atoms; white: H atoms; red: O atoms; blue: N atoms).
Figs. 6 and 7 display the representation of HOMO and LUMO
molecular orbitals for the same compounds. In both compounds 5a
and 8 (Fig. 6), the HOMOeLUMO transition causes a significant
increase in the electronic density of the carbonyl group of the
methyl carboxylate substituent, more significant for compound 8,
where this increase is extended to the whole carboxylate group.
This can justify the differences observed in the photophysical be-
haviour of 8 and 5a, although complex formation between 8 and
protic solvents cannot be completely discarded.
All compounds have reasonable high fluorescence quantum
yields (
FF>30%, except for 8 in cyclohexanedTable 4). A clear trend
between the FF values and the polarity of solvent is not detected.
The generally high fluorescence quantum yields of these oxazole
derivatives and the moderate solvent sensitivity of their fluores-
cence emission make them good candidates to be used as fluores-
cence probes.
The absorption and emission spectra of peptides 9 and 10 were
obtained in ethanol and water (Figs. 8 and 9). It can be observed
a red shift in water relative to emission in ethanol of similar mag-
nitude to that observed for the free 5a (Tables 4 and 5).
Fig. 6. Representation of HOMO and LUMO molecular orbitals of compounds 5a
(above) and 8 (below).
Compounds 5b and 5d have roughly similar absorption and
emission spectra (Fig. 4), while the fluorescence quantum yields are
generally higher for 5d (Table 4). The representation of HOMO and
LUMO molecular orbitals for compounds 5bed is exhibited in Fig. 7.
For both compounds 5b and 5d, it can be observed that upon
HOMO/LUMO transition there is a similar increase in the elec-
tronic density of the carbonyl group, with a decrease in the oppo-
site side of the molecule.
Fig. 8. Normalized fluorescence spectra (lexc¼270 nm) of 2ꢁ10ꢀ6 mol dmꢀ3 solutions
of peptide
9
in ethanol and in water (pH¼7). Inset: absorption spectra of
2ꢁ10ꢀ5 mol dmꢀ3 solutions of peptide 9 in ethanol and in water (pH¼7).

P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
8677
obtained in high yields after treating the corresponding
b
-halo-
dehydroaminobutyric acid derivative with a 2% solution of DBU in
acetonitrile. Amino acids linked to an oxazole moiety were obtained
from dipeptides having a b-bromodehydroaminobutyric acid resi-
due. The strategy used to prepare peptides labelled with an oxazole
moiety involved the cleavage of the methyl ester from one of the
oxazoles prepared, followed by coupling to two C-protected
peptides.
The photophysical properties of the oxazole derivatives were
studied in solvents of different polarity and showed that all com-
pounds have reasonably high fluorescence quantum yields and
moderate solvent sensitivity, which indicate their possible use as
fluorescent probes. From the results obtained with the peptides
labelled with an oxazole, it is possible to conclude that the oxazole
moiety maintains a good fluorescence level and the same solvent
sensitivity when linked to a peptide chain.
In summary, we have prepared in high yields oxazole-
4-carboxylates from b-halodehydroaminobutyric acid derivatives
Fig. 9. Normalized absorption and fluorescence spectra (lexc¼260 nm and 290 nm) of
solutions (2ꢁ10ꢀ5 mol dmꢀ3 for absorption and 2ꢁ10ꢀ6 mol dmꢀ3 for fluorescence) of
peptide 10 in ethanol and in water (pH¼7).
using a simple and efficient methodology. Furthermore, the pho-
tophysical studies carried out with these compounds indicate their
possible use as fluorescent probes.
Table 5
Maximum absorption (labs) and emission (lem) wavelengths, molar absorption coefficients (
3
) and fluorescence quantum yields (FF) for labelled peptides 9 and 10
a
Solvent
Ethanol
labs/nm [
e
/104 molꢀ1 dm3 cmꢀ1
]
lem/nm
9
FF
9
10
10 (lexc¼260 nm)
10 (lexc¼290 nm)
9
10
270 (0.95)
209 (2.16)
270 (1.70)
270 (1.70)
325
303 sh, 317, 326
325
0.35
0.29
Water
269 (1.05)
329
329
329
0.29
0.19
a
Relative to naphthalene in cyclohexane (FF¼0.23 at 25 ꢃC).14 Error about 10%. For peptide 10, lexc¼290 nm was used, to prevent excitation of phenylalanine.
The absorption spectra of peptide 10 (Fig. 9) resemble those of
5a, as phenylalanine has a very low molar absorption coefficient
(195 molꢀ1 dm3 cmꢀ1 at 257.5 nm in an aqueous media),22 due to
4. Experimental section
4.1. General methods
a symmetry-forbidden
p/p
*
transition,23 which also justifies its
low fluorescence quantum yield (FF¼0.04 in water24). When ex-
cited at 290 nm, emission of peptide 10 is similar to one of the
peptide 9 (Figs. 8 and 9). With excitation at 260 nm (Fig. 9), there is
a possibility of non-radiative energy transfer (FRET) from the
phenylalanine moiety to the oxazole. Peptide 10 fluorescence in
water is dominated by the oxazole moiety, despite a shoulder
appearing near 300 nm, which was expected due to the different FF
values of both fluorophores. However, noticeable differences in
spectral shape are observed between emission in water and etha-
nol, with excitation at 260 nm, the latter exhibits two main peaks
and a shoulder (near 300 nm), together with an enlargement in the
higher energy region. These spectral features indicate differences in
energy transfer efficiency from phenylalanine to oxazole in both
solvents. In water, the less structured spectrum, which is clearly
shifted to the oxazole emission region (with a maximum emission
wavelength similar to that observed for 9), may indicate a confor-
mation where the two fluorophores are closer, with a more efficient
energy transfer.
Melting points (ꢃC) were determined in a Gallenkamp apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded on
a Varian Unity Plus at 300 and 75.4 MHz, respectively, or on
a Bruker Avance IIþ at 400 and 100.6 MHz, respectively. 1He1H
spinespin decoupling, DEPT
q
45ꢃ, HMQC and HMBC were used to
attribute some signals. NOE difference experiments were also
performed. Chemical shifts are given in parts per million and
coupling constants in hertz. MS and HRMS data were recorded by
the mass spectrometry service of the University of Vigo, Spain;
elemental analysis was performed on a LECO CHNS 932 elemental
analyser.
The reactions were monitored by thin layer chromatography
(TLC). Column chromatography was performed on Machereye
Nagel silica gel 230e400 mesh. Petroleum ether refers to the
boiling range 40e60 ꢃC. When solvent gradient was used, the in-
crease of polarity was made from neat petroleum ether to mixtures
of diethyl ether/petroleum ether, increasing 10% of diethyl ether
each time until the isolation of the product.
The fluorescence quantum yields of the labelled peptides 9 and
10 (the latter with excitation at 290 nm) are reasonably high (a
reduction of 25% and 38% in ethanol and 15% and 44% in water,
respectively, is observed relative to oxazole 5a). The results show
that the oxazole moiety maintains good fluorescence level and the
same solvent sensitivity when linked to a peptide chain.
HPLC analysis were performed using a Lichrospher 100 RP18
(5 mm) column in an HPLC system composed by a Jasco PU-980
pump, a Jasco UV-975 UV/vis detector and a Shimadzu C-R6A
Chromatopac register.
4.2. Kinetic studies
3. Conclusions
Samples from the reaction mixture were taken at regular in-
tervals and analysed by HPLC. The eluent was acetonitrile/water
(4/1) at a flow rate of 1.2 mL minꢀ1. The chromatograms were
traced by detecting UV absorption at 260 nm (retention time: E-3a
2.17 min, Z-3b 2.23 min, Z-3d 2.63 min, E-6c 2.35 min).
A mild and efficient method for the synthesis of substituted
oxazole-4-carboxylate derivatives from N-acyl-
aminobutyric acid derivatives was developed. The oxazoles were
b-halodehydro-

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P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
4.3. Spectroscopic measurements
OCH3), 7.51e7.63 (4H, m, ArH), 7.76 (1H, d, J¼5.4 Hz, ArH), 7.91 (1H,
d, J¼6.0 Hz, ArH), 8.00 (1H, d, J¼6.0 Hz, ArH), 8.40 (1H, br d, NH) ppm
dC (75.4 MHz, CDCl3): 26.73 (CH3), 52.58 (OCH3),124.63 (CH),125.20
(CH), 125.34 (CH), 125.59 (C), 125.85 (CH), 126.69 (CH), 127.62 (CH),
128.45 (CH),130.21 (C),131.67 (CH),132.53 (C),133.75 (C),134.37 (C),
163.93 (C]O), 167.22 (C]O) ppm. Found C, 55.26; H, 4.16; N, 4.17;
C16H14NO3Br (348.19) requires C, 55.19; H, 4.05; N, 4.02.
All the solutions were prepared using spectroscopic grade sol-
vents. Absorption spectra were recorded in a Shimadzu UV-3101PC
UVevis-NIR spectrophotometer. Fluorescence measurements were
performed using a Fluorolog 3 spectrofluorimeter, equipped with
double monochromators in both excitation and emission and
a temperature controlled cuvette holder. Fluorescence spectra were
corrected for the instrumental response of the system.
4.8.2. Compound Z-3c. Mp 156.0e157.0 ꢃC (from ethyl acetate/pe-
troleum ether). dH (300 MHz, CDCl3): 2.67 (3H, s, bCH3), 3.92 (3H, s,
OCH3), 7.48e7.60 (4H, m, ArH), 7.79 (1H, d, J¼6.6 Hz, ArH), 7.90
(1H, d, J¼7.2 Hz, ArH), 7.99 (1H, d, J¼8.4 Hz, ArH), 8.44 (1H, br d,
NH) ppm. dC (75.4 MHz, CDCl3): 24.77 (CH3), 52.80 (OCH3), 124.62
(CH), 125.28 (CH), 125.54 (CH), 125.84 (C), 126.46 (CH), 126.65 (CH),
127.53 (CH), 128.36 (CH), 130.22 (C), 131.18 (CH), 131.73 (C), 132.05
(C), 133.68 (C), 162.88 (C]O), 167.21 (C]O) ppm.
4.4. Fluorescence quantum yield
For fluorescence quantum yield determination, the solutions
were previously bubbled for 30 min with ultrapure nitrogen. The
fluorescence quantum yields (Fs) were determined using the
standard method (Eq. 1).25
ArFsn2
4.9. Synthesis of
b
-bromodehydrodipeptides
Fs
¼
s Fr
(1)
AsFrn2r
Synthesis of Boc-Gly-Z-
DAbu(
b
-Br)-OMe (Z-6a),12b Boc-
L-
Phe-Z-
DAbu(
b
-Br)-OMe (Z-6b)26 and Boc-
L-Phe-E-DAbu(b-Br)-
where A is the absorbance at the excitation wavelength, F the in-
tegrated emission area and n the refraction index of the solvents
used. Subscripts refer to the reference (r) or sample (s) compound.
OMe (E-6b):26 the synthesis of these compounds was described in
the references given above.
4.5. Synthesis of the methyl esters of N-acyl-threonines
4.10. Synthesis of oxazole-4-carboxylates (5)
Synthesis of compounds Bz-Thr-OMe (1a),11 Bz(OMe)-Thr-OMe
(1b),11 Naph-Thr-OMe (1c)11 and Fur-Thr-OMe (1d):11 the synthesis
of these compounds was described in the reference given above.
4.10.1. General procedure for the synthesis of oxazole-4-carboxylate
derivatives. To a solution of b-halodehydroamino acid methyl ester
in acetonitrile (10ꢀ2 mol dmꢀ3) 2% of DBU was added with stirring
at room temperature. The reactions were followed by HPLC until all
the starting material was consumed (approximately 30 min). The
reaction mixture was evaporated and partitioned between 60 cm3
of ethyl acetate and 20 cm3 of KHSO4 (1 mol dmꢀ3) and washed
with KHSO4 (1 mol dmꢀ3), and brine (two times, 20 cm3 each). After
drying over MgSO4 the extract was taken to dryness at reduced
pressure to afford the respective substituted oxazole-4-carboxylate.
4.6. Synthesis of the methyl esters of N-acyldehydroamino
acids
Synthesis of compounds Bz-D
Abu-OMe (2a),11 Bz(4-OMe)-
D
Abu-OMe (2b),11 Naph- Abu-OMe (2c)11 and Fur-
D DAbu-OMe
(2d):11 the synthesis of these compounds was described in the
reference given above.
4.10.2. Synthesis of methyl 5-methyl-2-phenyloxazole-4-carboxylate
4.7. Synthesis of the methyl esters of N-acyl
(5a). The general procedure described above using Bz-Z-
DAbu
b-halodehydroamino acids
(b-Br)-OMe Z-3a (15 mg, 0.05 mmol) as substrate was followed to
give oxazole 5a (9.6 mg, 89%) as a white solid. Compound 5a was
Synthesis of compounds Bz-Z-DAbu(b
-Br)-OMe (Z-3a),12c Bz-
described elsewhere.11
E-
D
Abu(
b
-Br)-OMe (E-3a),12c Bz(4-OMe)-Z-
D
Abu(
b
Abu(
-Br)-OMe (Z-
-I)-OMe
The general procedure described above using Bz-E-DAbu(b-Br)-
3b),12c Fur-Z-
DAbu(
b
-Br)-OMe (Z-3d)12c and Bz-Z-
D
b
OMe E-3a (15 mg, 0.05 mmol) as substrate was followed to give
oxazole 5a (9.3 mg, 86%) as a white solid. Compound 5a was de-
scribed elsewhere.11
(Z-4a):11 the synthesis of these compounds was described in the
references given above.
The general procedure described above using Bz-Z-DAbu(b-I)-
4.8. Synthesis of methyl-3-bromo-2-(1-naphthamido)but-
2-enoate [Naph-DAbu(b-Br)-OMe] (3c)
OMe Z-4a (24.8 mg, 0.072 mmol) as substrate was followed to give
oxazole 5a (15.0 mg, 96%) as a white solid. Compound 5a was de-
scribed elsewhere.11
To a solution of methyl 2-(1-naphthamido)but-2-enoate (2c)
(2 mmol) in dichloromethane (0.1 mol dmꢀ3) 1.5 equiv of N-bro-
mosuccinimide was added with vigorous stirring. After reacting for
16 h, triethylamine (1.5 equiv) was added and stirring continued for
an additional hour. The solvent was then evaporated at reduced
pressure and the residue partitioned between 100 cm3 of
dichloromethane and 50 cm3 of KHSO4 (1 mol dmꢀ3). The organic
phase was washed with KHSO4 (1 mol dmꢀ3), NaHCO3
(1 mol dmꢀ3) and brine (3ꢁ30 cm3 each). After drying over MgSO4,
the extract was taken to dryness at reduced pressure to give
a 2/3 E/Z mixture of 3c (0.675 g, 97%). The diastereomers were
separated by column chromatography using diethyl ether/n-hex-
ane 1/2 as eluent to give:
4.10.3. Synthesis of methyl 2-(4-methoxyphenyl)-5-methyloxazole-4-
carboxylate (5b). The general procedure described above using Bz
(4-OMe)-Z-DAbu(b-Br)-OMe Z-3b (16.4 mg, 0.05 mmol) as sub-
strate was followed to give oxazole 5b (11.3 mg, 91%) as a white
solid. Compound 5b was described elsewhere.11
4.10.4. Synthesis of methyl 5-methyl-2-(naphthalen-1-yl)oxazole-
4-carboxylate (5c). The general procedure described above using
Naph-Z-DAbu(b-Br)-OMe Z-3c (17.4 mg, 0.05 mmol) as substrate
was followed to give oxazole 5c (12.6 mg, 91%) as a white solid.
Compound 5c was described elsewhere.11
The general procedure described above using Naph-E-
DAbu
(
b-Br)-OMe E-3c (18.5 mg, 0.053 mmol) as substrate was followed
4.8.1. Compound E-3c. Mp 154.0e155.0 ꢃC (from diethyl ether/pe-
to give oxazole 5c (12.5 mg, 88%) as a white solid. Compound 5c
troleum ether). dH (300 MHz, CDCl3): 2.55 (3H, s,
b
CH3), 3.91 (3H, s,
was described elsewhere.11

P.M.T. Ferreira et al. / Tetrahedron 66 (2010) 8672e8680
8679
4.10.5. Synthesis of methyl 2-(furan-2-yl)-5-methyloxazole-4-car-
boxylate (5d). The general procedure described above using Fur-
Z-DAbu(b-Br)-OMe Z-3d (10.4 mg, 0.05 mmol) as substrate was
0.46 mmol) and NEt3 (0.06 mL, 0.46 mmol) were added in an ice
bath. After 48 h at room temperature the mixture was filtered and
the solvent was evaporated at reduced pressure. The residue was
dissolved in ethyl acetate (150 mL) and washed with KHSO4
1 mol dmꢀ3, NaHCO3 1 mol dmꢀ3 and brine (3ꢁ50 mL each). The
organic layer was dried over MgSO4 and the solvent evaporated at
reduced pressure to give peptide 9 as an oil (47.6 mg, 31%). dH
(400 MHz, CDCl3): 1.43 (3H, d, J¼7.2 Hz, CH3), 2.71 (3H, s, CH3), 3.75
(3H, s, OCH3), 4.13e4.17 (2H, m, CH2), 4.58e4.66 (1H, m, CH), 6.91
(1H, d, J¼7.2 Hz, NH), 7.45e7.47 (3H, m, ArH), 7.71 (1H, t, J¼5.4 Hz,
NH), 7.98e8.01 (2H, m, ArH) ppm. dC (100.6 MHz, CDCl3): 11.1
(CH3), 11.7 (CH3), 42.7 (CH2), 48.1 (CH), 52.5 (OCH3), 126.3 (C), 126.6
(C), 128.8 (C), 129.7 (C), 130.7 (C), 153.4 (C), 158.8 (C), 162.6 (C]O),
168.5 (C]O), 173.2 (C]O) ppm. HRMS (EI): found 346.13961;
C17H20N3O5 requires 346.13975.
followed to give oxazole 5d (8.6 mg, 84%) as a white solid. Com-
pound 5d was described elsewhere.11
4.10.6. Synthesis of methyl 2-[(tert-butoxycarbonylamino)methyl]-
5-methyloxazole-4-carboxylate (7a). The general procedure de-
scribed above using Boc-Gly-Z-DAbu(b-Br)-OMe Z-6a (35.1 mg,
0.1 mmol) as substrate was followed to give oxazole 7a (23.0 mg,
85%) as an oil. dH (400 MHz, CDCl3): 1.45 (9H, s, CH3 Boc), 2.61 (3H,
s, CH3), 3.91 (3H, s, CH3 OMe), 4.43 (2H, d, J¼5.6 Hz, CH2 Gly), 5.18
(1H, br s, NH) ppm. dC (100.6 MHz, CDCl3): 11.94 (CH3), 28.28 [C
(CH3)3], 37.80 (CH2 Gly), 51.96 (OCH3), 80.27 [C(CH3)3], 127.38 (C),
155.43 (C]O),156.83 (C),159.25 (C),162.51 (C]O) ppm. HRMS (EI):
found 293.11077; C12H18N2NaO5 requires 293.11079.
4.11.3. Synthesis of peptide 10. To a solution of compound 8 (95 mg,
4.10.7. Synthesis of methyl 2-[1-(tert-butoxycarbonylamino)-2-
0.47 mmol) in dry acetonitrile (5 mL), HOBt (63 mg, 0.47 mmol),
phenylethyl]-5-methyloxazole-4-carboxylate (7b). The general pro-
DCC (96 mg, 0.47 mmol), TFA, H-Gly-L-Ala-L-Phe-OEt (203 mg,
cedure described above using Boc-
L
-Phe-Z-
D
Abu(
b
-Br)-OMe Z-6b
0.47 mmol) and NEt3 (0.13 mL, 0.94 mmol) were added in an ice
bath. After 48 h the solid was filtered and the solvent evaporated at
reduce pressure. The residue was dissolved in ethyl acetate (15 mL)
and washed with KHSO4 1 mol dmꢀ3, NaHCO3 1 mol dmꢀ3 and brine
(3ꢁ5 mL each). The organic layer was dried over MgSO4 and the
solvent evaporated at reduced pressure. Column chromatography
(44.0 mg, 0.10 mmol) as substrate was followed to give oxazole 7b
(28.1 mg, 78%) as a white solid. Mp 98.0e99.0 ꢃC (from ethyl acetate/
petroleum ether). dH (400 MHz, CDCl3): 1.38 (9H, s, CH3 Boc), 2.55
(3H, s, CH3), 3.14e3.24 (2H, m,
bCH2 Phe), 3.90 (3H, s, CH3 OMe),
5.12e5.20 (2H, m, NHþ CH Phe), 7.04e7.06 (2H, m, ArH), 7.22e7.27
a
(3H, m, ArH) ppm. dC (100.6 MHz, CDCl3): 11.86 (CH3), 28.18 [C
(CH3)3], 40.33 (CH2 Phe), 49.89 (CH Phe), 51.93 (OCH3), 80.01 [C
(CH3)3],126.92 (CH),127.28 (C),128.46 (CH), 129.23 (CH),135.78 (C),
154.81 (C]O),156.34 (C),161.71 (C), 162.55 (C]O) ppm. HRMS (EI):
found 383.15782; C19H24N2NaO5 requires 383.15774.
afforded peptide 10 as a white solid (192 mg, 81%) mp
149.0e150.0 ꢃC. dH (400 MHz, CDCl3): 1.22 (3H, t, J¼7.2 Hz,
OCH2CH3), 1.36 (3H, d, J¼7.2 Hz,
bCH3), 2.70 (3H, s, CH3), 3.04e3.18
CH2), 4.06 (2H, t, J¼5.6 Hz, CH2), 4.15 (2H, q, J¼7.2 Hz,
(2H, m,
b
OCH2CH3), 4.50e4.57 (1H, m, CH), 4.80 (1H, q, J¼6.8 Hz, CH), 6.80
(1H, d, J¼8.0 Hz, NH), 6.96 (1H, d, J¼7.2 Hz, NH), 7.12e7.14 (2H, m,
ArH), 7.19e7.22 (1H, m, ArH), 7.25e7.28 (2H, m, ArH), 7.44e7.48
(3H, m, ArH), 7.67 (1H, t, J¼5.6 Hz, NH), 7.99e8.01 (2H, m, ArH) ppm.
dC (100.6 MHz, CDCl3): 11.8 (CH3), 14.0 (OCH2CH3), 17.8 (CH3), 37.7
(CH2), 42.6 (CH2), 48.8 (CH Ala), 53.3 (CH), 61.5 (OCH2CH3), 126.3
(C), 126.6 (C), 127.0 (C), 128.4 (C), 128.8 (C), 129.3 (C), 129.7 (C),
130.6 (C), 135.86 (C), 153.4 (C), 158.7 (C), 162.5 (C]O), 168.7 (C]O),
171.2 (C]O), 171.6 (C]O) ppm. Found C, 63.62; H, 6.02; N, 10.97;
C27H30N4O6 (506.55) requires C, 64.02; H, 5.98; N, 11.06.
The general procedure described above using Boc-Phe-E-
DAbu
(b-Br)-OMe E-6b (44.0 mg, 0.10 mmol) as substrate gave oxazole 7b
(29.6 mg, 82%).
4.10.8. Synthesis of methyl 2-[1-(tert-butoxycarbonylamino)-
2-methylpropyl]-5-methyloxazole-4-carboxylate (7c). The general
procedure described above using Boc-L-Val-E-DAbu(b-Br)-OMe
E-6c (40.0 mg, 0.10 mmol) as substrate was followed to give oxazole
7c (28.0 mg, 89%) as an oil. dH (400 MHz, CDCl3): 0.91e0.94 (6H,
m,
g
CH3), 1.43 (9H, s, CH3 Boc), 2.14e2.19 (1H, m,
b
CH Val), 2.61 (3H,
CH Val), 5.27
s, CH3), 3.90 (3H, s, CH3 OMe), 4.71e4.75 (1H, m,
a
Acknowledgements
(1H, J¼8.8 Hz, NH) ppm. dC (100.6 MHz, CDCl3): 11.99 (CH3), 17.92
(CH3 Val), 18.76 (CH3 Val), 28.27 [C(CH3)3], 32.86 (CH Val),
51.96 (OCH3), 54.08 (CH Val), 79.89 [C(CH3)3], 127.25 (C), 155.37
(C]O), 156.26 (C), 162.15 (C), 162.29 (C]O) ppm. HRMS (EI): found
335.15742; C15H24N2NaO5 requires 335.15774.
Foundation for the Science and Technology (FCT)dPortugal
and FEDER (Fundo Europeu de Desenvolvimento Regional) for fi-
nancial support to Centro de Química (CQ-UM) and Centro de
Física (CFUM) of University of Minho and through research project
PTDC/QUI/81238/2006 co-financed by FCT and by program FEDER/
COMPETE (FCOMP-01-0124-FEDER-007467). The NMR spectrom-
eter Bruker Avance II 400 is part of the National NMR Network and
were purchased in the framework of the National Programme for
Scientific Re-equipment, contract REDE/1517/RMN/2005, with
funds from POCI 2010 (FEDER) and Fundação para a Ciência e
a Tecnologia (FCT). G.P. acknowledges FCT for a Ph.D. grant SFRH/
BD/38766/2007.
4.11. Synthesis of model peptides labelled with an oxazole
moiety
4.11.1. Synthesis of 5-methyl-2-phenyloxazole-4-carboxylic acid
8. To a solution of oxazole 5a (105 mg, 0.48 mmol) in methanol
(1 mL) 2.3 equiv of NaOH (1 mol dmꢀ3, 1.1 mL) was added and the
reaction was followed by TLC. When all the reactant was consumed
the mixture was acidified to pH 2e3 with KHSO4 1 mol dmꢀ3. The
solid formed was filtered and dried. Compound 8 was obtained as
a white solid (95.0 mg, 97%) mp decomposes above 170 ꢃC dH
(400 MHz, CDCl3): 2.64 (3H, s, CH3), 7.53e7.55 (3H, m, ArH),
7.94e7.97 (2H, m, ArH), 12.93 (1H, br s, CO2H) ppm. dC
(100.6 MHz, CDCl3): 11.92 (CH3), 125.91 (CH), 126.28 (C), 128.77 (C),
129.17 (CH), 130.85 (CH), 156.00 (C), 158.35 (C), 163.03 (C]O) ppm.
HRMS (EI): found 226.04739; C11H9NNaO3 requires 226.04746.
Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.09.014.
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