Unnatural benz-X-azolyl asparagine derivatives as novel fluorescent amino acids: synthesis and photophysical characterization

Article abstract of DOI:10.1016/j.tet.2009.08.086

A family of new asparagine derivatives bearing benzothiazole and benzimidazole units, functionalised with electron donor or acceptor groups, were synthesized in good to excellent yields. The photophysical characterization of these new heterocyclic amino a

Full text of DOI:10.1016/j.tet.2009.08.086

Tetrahedron 65 (2009) 9373–9377
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Tetrahedron
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Unnatural benz-X-azolyl asparagine derivatives as novel fluorescent amino acids:
synthesis and photophysical characterization
*
´
Catia I.C. Esteves, Ana M.F. Silva, M. Manuela M. Raposo, Susana P.G. Costa
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 4 August 2009
Received in revised form 26 August 2009
Accepted 29 August 2009
Available online 10 September 2009
A family of new asparagine derivatives bearing benzothiazole and benzimidazole units, functionalised
with electron donor or acceptor groups, were synthesized in good to excellent yields. The photophysical
characterization of these new heterocyclic amino acids was performed by UV–visible absorption and
fluorescence emission studies and revealed that the compounds displayed remarkably high fluorescence
quantum yields and Stokes’ shifts, making them good candidates for application as fluorescent probes by
incorporation into peptidic frameworks.
Keywords:
Asparagine
Ó 2009 Elsevier Ltd. All rights reserved.
Unnatural amino acids
Benzothiazole
Benzimidazole
Fluorescent probes
Solvatochromism
1. Introduction
moieties, benzothiazole and benzimidazole derivatives may be
considered suitable choices as these compounds are known to
Asparagine is the amide of aspartic acid and has a high pro-
pensity to form hydrogen bond interactions with the peptide
backbone through the amide group. Asparagine residues are often
display interesting optical properties (broad spectral windows,
high molar absorptivity coefficients and fluorescence quantum
yields) and have been described as fluorescent and/or colourimetric
chemosensors for anions and metal cations, biomarkers of pheo-
melanins and nonlinear optical materials.5 Also, benzothiazol-2-yl
amides exhibit a wide range of biological properties such as anti-
bacterial, antitumoral, antifungal and antitubercular activities.6
Bearing these facts in mind, it becomes clear that there is
a practical interest on the expansion of the body of work published
in recent years in the area of unnatural functional amino acids, and
so we decided to design new heterocyclic amino acids consisting of
functionalized asparagine containing benzothiazole and benz-
imidazole, as a result of the previously mentioned properties of
these heterocycles. The resulting benz-X-azolyl asparagine de-
rivatives, because of the presence of amino and carboxyl groups,
could be incorporated into peptide chains and as such used as
energy donor/acceptor in conformational studies of peptides by
means of fluorescence or be used as fluorescence markers. Fol-
lowing our previous research on the synthesis and characterization
of unnatural amino acids,7 imidazole and benz-X-azole derivatives
with interesting optical properties8 and heterocyclic colorimetric/
fluorimetric chemosensors containing (oligo)thiophene, benzox-
azole and amino acid moieties,9 we now report the synthesis of
a new family of highly fluorescent asparagine based heterocyclic
amino acids bearing benzothiazole and benzimidazole, also having
found near the beginning and the end of
a-helices, and in turn
motifs in -sheets. Its role can be thought as ‘capping’ the hydrogen
b
bond interactions, which would otherwise be satisfied by the
polypeptide backbone. This feature opens the way to the de-
velopment of functional unnatural amino acids based on aspara-
gine possessing additional properties, such as fluorescence, that
could be used for the construction of chromophore-labelled pep-
tides and proteins. By using fluorescence spectroscopy based
techniques, such fluorescent peptides could find application in
molecular flexibility studies involving protein folding, substrate
binding activity of proteins, and antigenicity or enzymatic activity.1,2
Therefore, 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
recognition ability,3 with the incorporation of these functional
unnatural amino acids into peptides resulting in the appearance of
the inherent functions.4 With regard to the fluorescent heterocyclic
* Corresponding author. Tel.: þ351 253 604054; fax: þ351 253 604382.
E-mail address: spc@quimica.uminho.pt (S.P.G. Costa).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.08.086

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C.I.C. Esteves et al. / Tetrahedron 65 (2009) 9373–9377
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 side
chains. The insertion of an heterocyclic system at the side chain will
provide an UV-active and highly fluorescent chromophore, as well
as additional binding sites through the heterocycle donor atoms.
With the study of these new benz-X-azolyl asparagines we also
intended to evaluate the effect of the substituent present at the
benzothiazole as well as the position of attachment of the hetero-
cycle in the photophysical properties of the resulting compounds.
From the results in Table 1, it can be seen that the yield of
coupling for benzothiazolyl asparagines 3a–h was influenced by
the nature of the substituent at position 6 of the benzothiazole, as
electron donor groups gave rise to compounds 3b–d in higher
yields (89–91%) and electron acceptor groups originated com-
pounds 3e–h in lower yields (58–68%). The position of attachment
of the benzothiazole appears to have no influence in the yield as
asparagines 3a and 3i were prepared in practically identical yields.
The same observation could be made considering the nature of the
heterocycle, since benzothiazolyl asparagine 3a and benzimida-
zolyl asparagine 3j were also obtained in similar yields.
TheIRspectraofasparagines3a–jshowedthecharacteristicbands
due to stretching vibrations of the carbonyl of the C- and N-terminal
protecting groups: the benzyl ester from 1742 to 1764 cmꢀ1 and the
N-tert-butyloxycarbonyl urethane bond from 1721 to 1747 cmꢀ1. The
bands relatedtothenewlyformedamidebondappearedfrom1671to
1701 cmꢀ1 (for the carbonyl) and at about 3300 cmꢀ1 (for the NH).
1H NMR spectra showed the expected signals for the benz-X-
azolyl unitincompounds 3b–i, intheformofa doubledoubletforH5,
and doublets for H4 and H7, with the chemical shifts of the protons of
homocyclic ring of the benz-X-azole moiety being in agreement with
the electronic character of the substituent (see Experimental for
details). The signals for the protons of the amino acid residue were
2. Results and discussion
2.1. Synthesis of benz-X-azolyl asparagines 3a–j
The new asparagines 3a–j with benzothiazole and benzimid-
azole at its side chain were synthesized in good to excellent yields
by a simple coupling procedure involving DCC and HOBt, between
the side chain carboxylic acid group of N-tert-butyloxy aspartic acid
benzyl ester 1 and aminobenzothiazoles 2a–i and 2-amino-
benzimidazole 2j, bearing substituents of different electronic
character (electron donor: alkyl and alkoxyl; electron acceptor: F,
SCN, SO2Me and SCN) and in different position of attachment of the
heterocycle (position 2 or 6) (Scheme 1, Table 1). These new com-
pounds were fully characterised by the usual spectroscopic
techniques.
also visible, namely the a-H (from 4.60 to 4.82 ppm) and the b-CH2
(from 3.02 to 3.40 ppm), for asparagines 3a–h, j bearing the het-
erocycleattachedatposition2. Withregardtoasparagine3i, withthe
heterocycle attached to position 6, a shift to lower d was seen for the
N
a
b
c
d
e
f
X = S, R = H
R
X = S, R = CH3
H2N
X = S, R = OCH3
X = S, R = OCH2CH3
X = S, R = F
X = S, R = NO2
X = S, R = SCN
X = S, R = SO2CH3
X = NH, R = H
N
X
O
C
R
X
2a-h,j
N
H
g
h
j
3a-h,j
CO2H
Boc-HN
CO2Bzl
DCC/HOBt
DMF, 0 ºC to rt
N
Boc-HN
CO2Bzl
O
C
1
N
S
S
N
H
H2N
Boc-HN
CO2Bzl
3i
2i
Scheme 1. Synthesis of benz-X-azolyl asparagine derivatives 3a–j.
b-CH2, which appeared as a multiplet between 2.97 and 3.14 ppm.
Table 1
The confirmation of the occurrence of the amide coupling was also
Yields, UV–visible absorption and fluorescence data for benz-X-azolyl asparagines
3a–j in absolute ethanol
supported by 13C NMR spectra, where signals of the benz-X-azole C2
carbon were visible between
benzothiazole) and at 143.09 ppm (for the benzimidazole) and the
amide group carbonyl from 168.57 to 169.84 ppm.
d 153.81 and 162.28 ppm (for the
Cpd.
X
R
Yield (%) UV–Vis
Fluorescence
d
lmax log
3
lem Stokes’ shift (cmꢀ1
)
FF
d
3a
3b
3c
3d
3e
3f
3g
3h
3i
S
S
S
S
S
S
S
S
H
CH3
OCH3
OCH2CH3 91
F
NO2
SCN
SO2CH3
–
90
89
89
330 3.22 465
326 3.28 461
347 3.22 467
345 3.34 467
346 3.52 466
325 3.53 454
335 3.28 467
331 3.13 464
332 3.05 462
8798
8983
7405
7572
7443
8743
8437
8660
8475
0.56
0.79
0.72
0.38
0.85
0.28
0.39
0.60
0.53
0.03
2.2. Photophysical study of benz-X-azolyl asparagines 3a–j
65
58
66
68
86
94
The absorption and emission spectra of asparagines 3a–j were
measured in absolute ethanol (10ꢀ6 to 10ꢀ5 M solution) (Table 1).
The nature of the heterocycle had a clear influence on the absorp-
tion and emission bands of compounds 3a–j: benzothiazolyl de-
rivatives 3a–i displayed absorption and emission maxima at longer
S
NH
3j
H
291 3.85 425 10,835

C.I.C. Esteves et al. / Tetrahedron 65 (2009) 9373–9377
9375
wavelengths (ca. 25 nm) when compared to the benzimidazolyl
counterpart 3j, a fact related to the electronic effect of the hetero-
cycle. Also, electron donating groups such as the alkoxyls or fluorine
at position 6 of the benzothiazole (compounds 3c–e) shifted bath-
ochromically the wavelength of maximum of absorption, when
compared to the unsubstituted benzothiazolyl asparagine 3a.
Compounds 3a and 3i, which only differ in the position of attach-
ment of the benzothiazole unit to the side chain of the amino acid,
showed similar absorption and emission data.
The existence of dual fluorescence is suggested by the fluores-
cence spectra of asparagine 3e in ethanol and DMSO, depicted in
Figure 1. This type of emission has been reported for 2-(40-amino-
phenyl)12a,b and 2-[40-(N,N-dimethylamino)phenyl]benzazole deri-
vatives,12c which was proposed to arise from the expected lowest
energy
p/p transition, resulting in the normal Stokes’-shifted
*
band, and also from a twisted intramolecular charge transfer (TICT)
state, with the appearance of a large Stokes’-shifted band.
The synthesized compounds showed large Stokes’ shifts (the
lowest being 7405 cmꢀ1 for 3c and the highest 10,835 cmꢀ1 for 3j).
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 light dispersed by the sample.10
3. Conclusions
In summary, we have achieved for the first time the synthesis of
new fluorescent benz-X-azolyl asparagine derivatives 3a–j con-
taining benzothiazole and benzimidazole units at its side chain by
a simple coupling procedure in good to excellent yields and their
photophysical properties were evaluated. Due to their interesting
photophysical properties, namely their strongly emissive character,
with large fluorescence quantum yields and Stokes’ shifts, and sol-
vatochromic behaviour, these heterocyclic asparagine derivatives
could find application as useful building blocks for peptide-based
structures with the extra value of inserting a UV-active and fluo-
rescent chromophore, adding functionality to the resulting peptide.
These heteroaromatic asparagines could also be used as fluorescent
markers and probes for conformational studies in peptides.
The relative fluorescence quantumyields were determined using
a 10ꢀ6 M solution of 9,10-diphenylanthracene in ethanol as standard
(
FF¼0.95)11 and benzothiazolyl asparagines 3a–i exhibited good to
excellent fluorescence quantum yields between 0.28 (nitro de-
rivative 3f) and 0.85 (fluoro derivative 3e), while the value obtained
for the benzimidazolyl asparagine 3j was much lower (FF¼0.03).
Considering the results obtained so far, the photophysical prop-
erties of benzothiazol-2-yl asparagine 3a, 6-fluorobenzothiazol-2-yl
asparagine 3e (with the highest fluorescence quantum yield in eth-
anol) and benzothiazol-6-yl asparagine 3i were evaluated in other
solvents of different character. The solvents tested were ethyl acetate
and DMSO, as examples of polaraprotic solvents, in order to compare
with ethanol, a polar protic solvent. The collected data revealed
substantial differences in the wavelengths of maximum absorption
and emission and the fluorescence quantum yield (Table 2). For the
considered asparagines 3a, 3e and 3i, it can be seen that there is
a considerable hipsochromic shift in the absorption and emission
maxima in ethyl acetate and DMSO (Fig.1). Also in these solvents, the
fluorescence quantum yields suffered a strong decrease and are very
low, with the exception of asparagine 3e in ethyl acetate, which
displays a moderate quantum yield (FF¼0.26).
4. Experimental
4.1. Synthesis general
All melting points were measured on a Stuart SMP3 melting
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 visualised under UV light. Chro-
matography on silica gel was carried out on Merck Kieselgel (230–
240 mesh). IR spectra were determined on a BOMEM MB 104
spectrophotometer using KBr discs. UV–visible absorption spectra
(200–800 nm) were obtained using a Shimadzu UV/2501PC spec-
trophotometer. NMR spectra were obtained on a Varian Unity Plus
Spectrometer at an operating frequency of 300 MHz for 1H NMR
and 75.4 MHz for 13C NMR or a Bruker Avance III 400 at an operating
frequency of 400 MHz for 1H NMR and 100.6 MHz for 13C NMR
using the solvent peak as internal reference at 25 ꢁC. All chemical
shifts are given in ppm using dH Me4Si¼0 ppm as reference and
J values are given in Hertz. Assignments were made by comparison
of chemical shifts, peak multiplicities and J values and were sup-
ported by spin decoupling-double resonance and bidimensional
heteronuclear HMBC and HMQC correlation techniques. Mass
spectra were obtained with a Finnigan LXQ system. Fluorescence
spectra were collected using a FluoroMax-4 spectrofluorometer. All
reagents were commercially available and used as-received.
Table 2
UV–visible absorption and fluorescence data for benzothiazolyl asparagines 3a, 3e
and 3i in EtOH, AcOEt and DMSO
Cpd.
Solvent
UV–Vis
Fluorescence
lmax
log
3
lem
Stokes’ shift (cmꢀ1
)
FF
3a
EtOH
AcOEt
DMSO
330
268
278
3.22
4.09
4.21
465
308
376
8798
4846
9375
0.56
0.04
0.01
3e
3i
EtOH
AcOEt
DMSO
346
268
278
3.52
4.02
4.17
466
304
377
7443
4419
9446
0.85
0.26
0.01
EtOH
AcOEt
DMSO
332
278
270
3.05
4.05
4.13
462
324
366
8475
5107
9715
0.53
0.02
0.02
4.2. General procedure for the synthesis of heterocyclic
asparagine derivatives 3a–j
N-tert-Butyloxycarbonyl aspartic acid benzyl ester 1 (1 equiv)
was dissolved in dry DMF (3 mL/mmol), followed by HOBt
(1 equiv), and after stirring for 10 min, N,N0-dicyclohexyl-
carbodiimide (1 equiv) was added. The reaction mixture was placed
in an ice bath, stirred for 30 min and the corresponding amino-
benzothiazole or benzimidazole 2 was added (1 equiv). The mix-
ture was stirred at low temperature for 2 h, and then for 24 h at
room temperature. After filtering, the solvent was removed under
reduced pressure in a rotary evaporator. The residue was dissolved
in acetone and placed in the cold overnight to induce the pre-
cipitation of the formed by-product (N,N0-dicyclohexylurea), which
was separated by filtration. This procedure was repeated two times.
Figure 1. Normalised UV–visible absorption and emission spectra of asparagine 3e in
absolute ethanol (E) and DMSO (D) at T¼298 K (lexc¼346 nm in E and 278 nm in D)
(absorption, full line; emission, dotted line).

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C.I.C. Esteves et al. / Tetrahedron 65 (2009) 9373–9377
The filtrate was evaporated in a rotary evaporator. The resulting
solid was purified by recrystallization from methanol and diethyl
ether or column chromatography, using mixtures of ethyl acetate
and light petroleum of increasing polarity.
(0.577 g, 1.15 mmol, 91%). Mp¼117.0–118.9 ꢁC. IR (KBr, 1%):
¼3369,
n
3208, 3068, 2979, 2932, 1764, 1747, 1727, 1695, 1607, 1572, 1556,
1520, 1461, 1417, 1392, 1368, 1344, 1262, 1222, 1158, 1062, 982, 944,
912, 892, 853, 820, 805, 780, 752, 732, 700, 665, 515 cmꢀ1. 1H NMR
(400 MHz, CDCl3):
3.02–3.28 (m, 2H,
4.0 Hz, 1H, -H), 5.16–5.24 (m, 2H, CH2), 5.89 (d, J 8.4 Hz, 1H, NH
d
¼1.42–1.48 (m, 12H, C(CH3)3 and OCH2CH3),
4.2.1. N-tert-Butyloxycarbonyl (benzothiazol-2-yl) asparagine benzyl
b-CH2), 4.08 (q, J 6.8 Hz, 2H, OCH2CH3), 4.75 (t, J
ester (3a). The product was isolated as
a
colourless solid
a
(0.512 g, 1.12 mmol, 90%). Mp¼124.4–124.6 ꢁC. IR (KBr, 1%):
n¼3377,
Boc), 7.01 (dd, J 8.8 and 2.4 Hz, 1H, H5), 7.25–7.30 (m, 6H, 5ꢂPh-H
2970, 2931,1758,1729,1698,1627,1600,1552,1519,1444,1422,1391,
1368, 1347, 1299, 1272, 1245, 1212, 1198, 1170, 1158, 1066, 1016, 983,
909, 873, 784, 755, 729, 696, 666 cmꢀ1. 1H NMR (300 MHz, CDCl3):
and H7), 7.62 (d, J 8.8 Hz,1H, H4), 8.55 (br s,1H, NH amide) ppm. 13C
NMR (100.6 MHz, CDCl3):
d
¼14.79 (OCH2CH3), 28.22 (C(CH3)3),
38.28 ( -CH2), 50.10 ( -C), 64.15 (OCH2CH3), 67.57 (CH2), 80.22
b
a
d
¼1.44 (s, 9H, C(CH3)3), 3.18–3.40 (m, 2H,
b
-CH2), 4.80 (t, J 3.9 Hz,1H,
(C(CH3)3), 105.09 (C7), 116.03 (C5), 120.77 (C4), 128.16 (C30 and C50),
128.27 (C40), 128.46 (C20 and C60), 132.45 (C7a), 135.12 (C10), 140.44
(C3a), 155.57 (C]O urethane), 156.42 (C6), 157.08 (C2), 169.03
(C]O amide), 170.94 (C]O ester) ppm. UV–Vis (ethanol, nm): lmax
a
-H), 5.18–5.23 (m, 2H, CH2), 5.64 (d, J 8.4 Hz,1H, NH Boc), 7.22–7.38
(m, 6H, 5ꢂPh-H and NH amide), 7.49 (dt, J 7.2 and 1.2 Hz, 1H, H6),
7.60 (dt, J 7.2 and 1.2 Hz,1H, H5), 7.84–7.89 (m, 2H, H4 and H7) ppm.
13C NMR (75.4 MHz, CDCl3):
d
¼28.22 (C(CH3)3), 38.44 (
b
-CH2), 50.07
(log
3
)¼345 (3.34). MS m/z (ESI, %): 522 (Mþþ23, 100), 500
(
a
-C), 67.64 (CH2), 80.31 (C(CH3)3), 120.04 (C7), 121.59 (C4), 124.51
([MþH]þ, 8). C25H29N3O6S: calcd C 60.10, H 5.85, N 8.41, S 6.42;
(C6), 126.85 (C5), 128.21 (C40), 128.31 (C30 and C50), 128.48 (C20 and
C60), 130.91 (C7a), 135.05 (C10), 145.81 (C3a), 155.53 (C]O ure-
thane), 159.23 (C2), 169.31 (C]O amide), 170.86 (C]O ester) ppm.
found C 60.20, H 5.98, N 8.21, S 6.19.
4.2.5. N-tert-Butyloxycarbonyl (6-fluorobenzothiazol-2-yl) asparagine
benzyl ester (3e). The product was isolated as a colourless solid
(0.282 g, 0.60 mmol, 65%). Mp¼152.2–154.0 ꢁC. IR (KBr, 1%):
UV–Vis (ethanol, nm): lmax (log
3)¼330 (3.22). MS m/z (ESI, %): 478
(Mþþ23, 100), 456 ([MþH]þ, 20). C23H25N3O5S: calcd C 60.64, H
5.53, N 9.22, S 7.04; found C 60.80, H 5.73, N 9.25, S 7.15.
n
¼3370, 3264, 3217, 3069, 2981, 2932, 1755, 1741, 1696, 1609, 1557,
1520, 1461, 1425, 1417, 1391, 1368, 1344, 1318, 1287, 1251, 1222, 1197,
1167, 1153, 1054, 1029, 1020, 990, 979, 916, 851, 826, 808, 780 cmꢀ1
1H NMR (300 MHz, CDCl3):
¼1.41 (s, 9H, C(CH3)3), 3.08–3.31 (m,
2H, -CH2), 4.82 (t, J 3.6 Hz,1H, -H), 5.17–5.26 (m, 2H, CH2), 6.09 (d,
4.2.2. N-tert-Butyloxycarbonyl (6-methylbenzothiazol-2-yl) aspara-
gine benzyl ester (3b). The product was isolated as a colourless solid
(0.523 g, 1.11 mmol, 89%). Mp¼154.9–158.0 ꢁC. IR (KBr, 1%):
.
d
b
a
n
¼3369, 3210, 3066, 2982, 2931, 1755, 1737, 1698, 1607, 1553, 1521,
J 7.5 Hz,1H, NH Boc), 7.08 (dt, J 9.0 and 2.4 Hz,1H, H5), 7.24–7.33 (m,
5H, 5ꢂPh-H), 7.46 (dd, J 7.8 and 2.1 Hz, 1H, H7), 7.65–7.69 (m, 1H,
H4), 9.50 (br s, 1H, NH amide) ppm. 13C NMR (75.4 MHz, CDCl3):
1467, 1455, 1422, 1389, 1368, 1343, 1317, 1273, 1223, 1208, 1155,
1060, 1029, 990, 900, 866, 814, 779, 736, 695, 665 cmꢀ1 1H NMR
(400 MHz, CDCl3):
¼1.42 (s, 9H, C(CH3)3), 2.47 (s, 3H, CH3), 3.03–
3.28 (m, 2H, -CH2), 4.75 (t, J 4.0 Hz, 1H, -H), 5.17–5.25 (m, 2H,
.
d
d
¼28.17 (C(CH3)3), 38.21 (
b-CH2), 50.14 (a-C), 67.64 (CH2), 80.30
b
a
(C(CH3)3), 107.66 (d, J 26.8 Hz, C7),114.74 (d, J 24.5 Hz, C5), 121.44 (d,
J 8.90 Hz, C4), 128.12 (C30 and C50),128.30 (C40), 128.45 (C20 and C60),
132.59 (d, J 10.3 Hz, C7a), 135.00 (C10), 143.80 (C3a), 155.64 (C]O
urethane), 158.27 (C2), 159.56 (d, J 243.8 Hz, C6), 169.32 (C]O
amide), 171.09 (C]O ester) ppm. UV–Vis (ethanol, nm): lmax
CH2), 5.91 (d, J 8.4 Hz, 1H, NH Boc), 7.23 (dd, J 8.0 and 1.4 Hz, H5),
7.25–7.32 (m, 5H, 5ꢂPh-H), 7.60 (d, J 1.4 Hz, H7), 7.63 (d, J 8.0 Hz,
H4), 9.58 (br s, 1H, NH amide) ppm. 13C NMR (100.6 MHz, CDCl3):
d¼21.39 (CH3), 28.21 (C(CH3)3), 38.28 (
b-CH2), 50.05 (a-C), 67.55
(CH2), 80.19 (C(CH3)3), 119.86 (C4), 121.26 (C7), 128.09 (C5), 128.13
(C30 and C50), 128.26 (C40), 128.45 (C20 and C60), 131.51 (C7a), 134.35
(C6), 135.13 (C10), 144.74 (C3a), 155.58 (C]O urethane), 158.37 (C2),
169.21 (C]O amide), 170.95 (C]O ester) ppm. UV–Vis (ethanol,
(log
3
)¼346 (3.52). MS m/z (ESI, %): 496 (Mþþ23,100), 474 ([MþH]þ,
15). C23H24N3O5SF: calcd C 58.34, H 5.11, N 8.87; found C 58.33, H
5.27, N 8.86.
nm): lmax (log
3
)¼326 (3.28). MS m/z (ESI, %): 492 (Mþþ23, 100),
4.2.6. N-tert-Butyloxycarbonyl (6-nitrobenzothiazol-2-yl) asparagine
benzyl ester (3f). The product was isolated as a colourless solid
(0.368 g, 0.74 mmol, 58%). Mp¼114.7–120.5 ꢁC. IR (KBr, 1%):
470 ([MþH]þ, 13). C24H27N3O5S: calcd C 61.39, H 5.80, N 8.95, S
6.83; found C 61.09, H 5.87, N 8.85, S 6.89.
n
¼3377, 2970, 2931, 1758, 1729, 1698, 1627, 1600, 1552, 1519, 1444,
4.2.3. N-tert-Butyloxycarbonyl (6-methoxybenzothiazol-2-yl) aspar-
agine benzyl ester (3c). The product was isolated as a colourless
solid (0.439 g, 0.90 mmol, 89%). Mp¼130.2–134.0 ꢁC. IR (KBr, 1%):
1422, 1552, 1519, 1455, 1444, 1391, 1368, 1347, 1299, 1272, 1245,
1212, 1198, 1158, 1170, 1066, 1016, 983, 909, 873, 784, 755, 729, 696,
666 cmꢀ1. 1H NMR (400 MHz, CDCl3):
d¼1.43 (s, 9H, C(CH3)3), 3.10–
n
¼3367, 2979, 2932, 1754, 1730, 1695, 1606, 1570, 1556, 1521, 1471,
3.27 (m, 2H, b-CH2), 4.78 (br s,1H, a-H), 5.19–5.31 (m, 2H, CH2), 5.68
1439, 1368, 1345, 1264, 1222, 1157, 1061, 1029, 902, 853, 829, 810,
779, 753, 733, 700, 666, 514 cmꢀ1 1H NMR (400 MHz, CDCl3):
-CH2), 3.87 (s, 3H,
-H), 5.17–5.24 (m, 2H, CH2), 5.90 (d, J
(br s, 1H, NH Boc), 7.27–7.33 (m, 6H, 5ꢂPh-H and NH amide), 7.85
(d, J 8.7 Hz, 1H, H4), 8.35 (dd, J 9.0 and 2.4 Hz, 1H, H5), 8.76 (d, J
.
d
¼1.42 (s, 9H, C(CH3)3), 3.03–3.28 (m, 2H,
b
2.4 Hz, 1H, H7) ppm. 13C NMR (100.6 MHz, CDCl3):
(C(CH3)3), 38.74 (b-CH2), 50.31 (a-C), 67.97 (CH2), 80.76 (C(CH3)3),
d
¼28.26
OCH3), 4.76 (t, J 4.0 Hz, 1H,
a
8.4 Hz, 1H, NH Boc), 7.01 (dd, J 8.8 and 2.4 Hz, 1H, H5), 7.25–7.30 (m,
6H, 5ꢂPh-H and H7), 7.63 (d, J 8.8 Hz, 1H, H4), 9.05 (br s, 1H, NH
118.09 (C7), 120.84 (C4), 122.17 (C5), 128.29 (C40), 128.51 (C30 and
C50), 128.56 (C20 and C60), 132.08 (C7a), 134.85 (C10), 144.07 (C3a),
151.97 (C6), 155.74 (C]O urethane), 162.38 (C2), 169.53 (C]O
amide), 170.89 (C]O ester) ppm. UV–Vis (ethanol, nm): lmax
amide) ppm. 13C NMR (100.6 MHz, CDCl3):
d¼28.22 (C(CH3)3), 38.27
(b-CH2), 50.10 (a-C), 55.81 (OCH3), 67.57 (CH2), 80.23 (C(CH3)3),
104.37 (C7), 115.60 (C5), 120.81 (C4), 128.16 (C40), 128.28 (C30 and
C50), 128.47 (C20 and C60), 132.50 (C7a), 135.12 (C10), 140.58 (C3a),
155.58 (C]O urethane), 157.06 (C6), 157.15 (C2), 169.04 (C]O
amide), 170.96 (C]O ester) ppm. UV–Vis (ethanol, nm): lmax
(log
3
)¼325 (3.53). MS m/z (ESI, %): 523 (Mþþ23, 100).
C23H24N4O7S: calcd C 55.19, H 4.83, N 11.19, S 6.41; found C 55.49, H
5.00, N 10.88, S 6.46.
(log
3
)¼347 (3.22). MS m/z (ESI, %): 508 (Mþþ23, 100), 486
4.2.7. N-tert-Butyloxycarbonyl (6-thiocyanobenzothiazol-2-yl)aspara-
gine benzyl ester (3g). The product was isolated as a colourless
solid (0.424 g, 0.83 mmol, 66%). Mp¼169.0–172.0 ꢁC. IR (KBr, 1%):
([MþH]þ, 7). C24H27N3O6S: calcd C 59.37, H 5.60, N 8.65, S 6.60;
found C 59.42, H 5.76, N 8.55, S 6.73.
n
¼3326, 3230, 3094, 2973, 2940, 1742, 1730, 1701, 1594, 1524, 1447,
4.2.4. N-tert-Butyloxycarbonyl (6-ethoxybenzothiazol-2-yl) aspara-
gine benzyl ester (3d). The product was isolated as a colourless solid
1409, 1393, 1367, 1295, 1278, 1236, 1193, 1154, 1075, 1007, 990, 973,
925, 900, 869, 849, 757, 724 cmꢀ1. 1H NMR (300 MHz, CDCl3):
¼1.44
d

C.I.C. Esteves et al. / Tetrahedron 65 (2009) 9373–9377
9377
(s, 9H, C(CH3)3), 3.13–3.31 (m, 2H,
b
-CH2), 4.76 (br s, 1H,
a
-H), 5.18–
7.37–7.39 (m, 1H, H4 or H7), 7.49 (d, J 8.0 Hz, 1H, NH Boc), 7.61–7.63
5.27 (m, 2H, CH2), 5.69 (br s, 1H, NH Boc), 7.27–7.33 (m, 6H, 5ꢂPh-H
and NH amide), 7.63 (dd, J 7.8 and 2.1 Hz,1H, H5), 7.78 (d, J 8.1 Hz,1H,
H4), 7.98 (d, J 2.1 Hz, 1H, H7) ppm. 13C NMR (75.4 MHz, CDCl3):
(m, 1H, H4 or H7), 12.98 (br s, 1H, NH benzimidazole) ppm. 13C NMR
(100.6 MHz, DMSO-d6):
d
¼28.09 (C(CH3)3), 37.63 (
b-CH2), 49.76
(a-C), 66.37 (CH2), 78.62 (C(CH3)3), 113.43 (C5 and C6), 124.39 (C4
d
¼28.23 (C(CH3)3), 38.55 (
b
-CH2), 50.19 (
a-C), 67.83 (CH2), 80.60
and C7), 127.74 (C30 and C50), 127.98 (C40), 128.28 (C20 and C60),
129.59 (C3a and C7a), 135.65 (C10), 143.09 (C2), 155.25 (C]O
urethane), 169.84 (C]O amide), 171.03 (C]O ester) ppm. UV–Vis
(C(CH3)3), 110.73 (SCN), 118.96 (C6), 122.03 (C4), 124.30 (C7), 128.23
(C40), 128.45 (C30 and C50), 128.53 (C20 and C60), 129.22 (C5), 133.26
(C7a), 134.91 (C10), 148.10 (C3a), 155.66 (C]O urethane), 160.15 (C2),
169.38 (C]O amide), 170.90 (C]O ester) ppm. UV–Vis (ethanol,
(ethanol, nm): lmax (log
3
)¼291 (3.85). MS m/z (ESI, %): 461 (Mþþ23,
26), 439 ([MþH]þ, 100). C23H26N4O5: calcd C 63.00, H 5.98, N 12.78;
nm): lmax (log
3
)¼335 (3.28). MS m/z (ESI, %): 535 (Mþþ23, 100).
found C 63.01, H 6.20, H 12.58.
C24H24N4O5S2: calcd C 56.23, H 4.72, N 10.93, S 12.51; found C 56.46,
H 4.87, N 10.66; S 12.87.
Acknowledgements
4.2.8. N-tert-Butyloxycarbonyl (6-sulfomethylbenzothiazol-2-yl) asp-
aragine benzyl ester (3h). The product was isolated as a colourless
solid (0.451 g, 0.85 mmol, 68%). Mp¼190.6–192.5 ꢁC. IR (KBr, 1%):
˜
ˆ
Thanks are due to the Fundaçao para a Ciencia e Tecnologia
(Portugal) for financial support through project PTDC/QUI/66250/
2006 and research grants to C.I.C.E. and A.M.F.S. The NMR spec-
trometer Bruker Avance II 400 is part of the National NMR Network
and was acquired with funds from FCT and FEDER.
n
¼3362, 3278, 3241, 3064, 3024, 2985, 2930, 1748, 1722, 1698, 1596,
1527, 1447, 1388, 1372, 1283, 1147, 1100, 1055, 1027, 980, 957, 910,
883, 865, 825, 785, 747, 732, 696, 665, 512, 503 cmꢀ1 1H NMR
(300 MHz, CDCl3):
¼1.44 (s, 9H, C(CH3)3), 3.14 (s, 3H, CH3), 3.16–
3.34 (m, 2H, -CH2), 4.77 (br s, 1H, -H), 5.18–5.27 (m, 2H, CH2), 5.70
.
d
References and notes
b
a
1. (a) Watanabe, T.; Miyata, Y.; Abe, R.; Muranaka, N.; Hohsaka, T. ChemBioChem
2008, 9, 1235–1242; (b) Kajihara, D.; Abe, R.; Iijima, I.; Komiyama, C.; Sissido,
M.; Hohsaka, T. Nat. Methods 2006, 3, 923–929.
2. (a) Sahoo, H.; Hennig, A.; Nau, W. M. Int. J. Photoenergy 2006, article no. 89638;
(b) Hennig, A.; Florea, M.; Roth, D.; Enderle, T.; Nau, W. M. Anal. Biochem. 2007,
360, 255–265; (c) Taki, M.; Hohsaka, T.; Murakami, H.; Taira, K.; Sisido, M. FEBS
Lett. 2001, 507, 35–38.
3. (a) 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.
4. (a) Ishida, H.; Kyakuno, M.; Oishi, S. Biopolymers (Pep. Sci.) 2004, 76, 69–82; (b)
Hodgson, D. R. W.; Sanderson, J. M. Chem. Soc. Rev. 2004, 33, 422–430.
5. (a) Zeng, D. X.; Chen, Y. J. Photochem. Photobiol., A 2007, 186, 121–124; (b) Shi,
H.-B.; Ji, S.-J.; Bian, B. Dyes Pigm. 2007, 73, 394–396; (c) Moon, K. S.; Singh, N.;
Lee, G. W.; Jang, D. O. Tetrahedron 2007, 63, 9106–9111; (d) Greco, G.; Panzella,
G. L.; Napolitano, A.; d’Ischia, M. Tetrahedron Lett. 2009, 50 3095–3097; (e)
Chen, L.; Cui, Y.; Qian, G.; Wang, M. Dyes Pigm. 2007, 73, 338–343; (f)
Rodembusch, F. S.; Buckup, T.; Segala, M.; Tavares, L.; Correia, R. R. B.; Stefani, V.
Chem. Phys. 2004, 305, 115–121.
6. Yoshida, M.; Hayakawa, I.; Hayashi, N.; Agatsuma, T.; Oda, Y.; Tanzawa, F.;
Iwasaki, S.; Koyama, K.; Furukawa, H.; Kurakatad, S.; Suganob, Y. Bioorg. Med.
Chem. Lett. 2005, 15, 3328–3332.
7. (a) Costa, S. P. G.; Maia, H. L. S.; Pereira-Lima, S. M. M. A. Org. Biomol. Chem.
2003, 1, 1475–1479; (b) Jiang, W.-Q.; Costa, S. P. G.; Maia, H. L. S. Org. Biomol.
Chem. 2003, 1, 3804–3810; (c) Costa, S. P. G.; Oliveira, E.; Lodeiro, C.; Raposo, M.
M. M. Tetrahedron Lett. 2008, 49, 5258–5261; (d) Costa, S. P. G.; Batista, R. M. F.;
Raposo, M. M. M. Tetrahedron 2008, 64, 9733–9737.
8. (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; (h) Batista,
R. M. F.; Costa, S. P. G.; Belsley, M.; Raposo, M. M. M. Dyes Pigm. 2009, 80,
329–336.
(br s,1H, NH Boc), 7.25–7.34 (m, 6H, 5ꢂPh-H and NH amide), 7.90 (d,
J 8.1 Hz, 1H, H4), 7.97 (dd, J 8.1 and 1.5 Hz, H5), 8.45 (d, J 1.8 Hz, 1H,
H7) ppm. 13C NMR (75.4 MHz, CDCl3):
d
¼28.24 (C(CH3)3), 38.79 (
b-
CH2), 44.88 (CH3), 50.21 (a-C), 67.89 (CH2), 80.73 (C(CH3)3), 121.03
(C4), 121.98 (C7), 125.70 (C5), 128.32 (C40), 128.50 (C20 and C60),
128.57 (C30 and C50), 131.77 (C7a), 134.88 (C10), 136.27 (C6), 149.88
(C3a), 155.61 (C]O urethane), 161.82 (C2), 169.33 (C]O amide),
170.64 (C]O ester) ppm. UV–Vis (ethanol, nm): lmax (log
3
)¼331
(3.13). MS m/z (ESI, %): 534 ([MþH]þ, 14). C24H27N3O7S2: calcd C
54.02, H 5.10, N 7.87, S 12.02; found C 54.22, H 5.18, N 7.76, S 12.12.
4.2.9. N-tert-Butyloxycarbonyl (benzothiazol-6-yl) asparagine benzyl
ester (3i). The product was isolated as
a colourless solid
(0.492 g, 1.08 mmol, 86%). Mp¼150.1–152.0 ꢁC. IR (KBr, 1%):
n
¼3433, 3332, 3055, 2973, 2927, 1745, 1721, 1671, 1609, 1578, 1534,
1456,1490,1474,1456,1401,1382,1366,1339,1286,1247,1197,1160,
1056, 1028, 979, 948, 911, 873, 858, 832, 822, 793, 781, 770,
756, 694, 665, 600 cmꢀ1
.
1H NMR (400 MHz, CDCl3):
d
¼1.42
(s, 9H, C(CH3)3), 2.97–3.14 (m, 2H, b-CH2), 4.66 (br s, 1H, a-H), 5.18–
5.23 (m, 2H, CH2), 5.90 (d, J 5.6 Hz, 1H, NH Boc), 7.27–7.31 (m, 6H,
5ꢂPh-H and NH amide), 7.97 (d, J 8.8 Hz, 1H, H4), 8.49–8.53 (m, 2H,
H5 and H7), 8.97 (s, 1H, H2) ppm. 13C NMR (100.6 MHz, CDCl3):
d
¼28.24 (C(CH3)3), 39.20 (
b-CH2), 50.55 (a-C), 67.51 (CH2), 80.33
(C(CH3)3), 112.55 (C5 or C7), 119.18 (C5 or C7), 123.01 (C4), 128.13
(C40), 128.33 (C30 and C50), 128.49 (C20and C60), 134.39 (C7a), 135.16
(C10), 135.74 (C6), 148.83 (C3a), 153.81 (C2), 155.82 (C]O urethane),
168.57 (C]O amide), 171.34 (C]O ester) ppm. UV–Vis (ethanol,
nm): lmax (log
3
)¼332 (3.05). MS m/z (ESI, %): 478 (Mþþ23, 100).
C23H25N3O5S: calcd C 60.64, H 5.53, N 9.22, S 7.04; found C 60.60, H
5.53, N 9.11, S 7.37.
9. (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; (c) Batista, R. M. F.; Oliveira, E.; Nun˜ez, C.;
Costa, S. P. G.; Lodeiro, C.; Raposo, M. M. M. J. Phys. Org. Chem. 2009, 22,
362–366.
10. Holler, M. G.; Campo, L. F.; Brandelli, A.; Stefani, V. J. Photochem. Photobiol., A:
Chem. 2002, 149, 217–225.
11. Morris, J. V.; Mahaney, M. A.; Huber, J. R. J. Phys. Chem. 1976, 80, 969–974.
12. (a) Dey, J. K.; Dogra, S. K. Chem. Phys. 1990, 143, 97–107; (b) Dey, J. K.; Dogra,
S. K. Bull. Chem. Soc. Jpn. 1991, 64, 3142–3152; (c) Dey, J. K.; Dogra, S. K.
J. Phys. Chem. 1994, 98, 3638–3644 and references therein.
4.2.10. N-tert-Butyloxycarbonyl (benzimidazol-2-yl) asparagine benzyl
ester (3j). The product was isolated as
a colourless solid
(0.519 g, 1.18 mmol, 94%). Mp¼143.0–143.3 ꢁC. IR (KBr, 1%):
¼3363,
n
2978, 2929, 2851,1755,1742,1697,1642, 1594,1584,1523,1433,1391,
1368, 1344, 1309, 1274, 1223, 1167, 1058, 1029, 895, 865, 779,
760, 740, 696, 622 cmꢀ1
.
1H NMR (400 MHz, DMSO-d6):
d
¼1.36
-CH2), 4.60 (t, J 4.4 Hz, 1H, -H),
5.12 (s, 2H, CH2), 7.23–7.34 (m, 8H, 5ꢂPh-H, NH amide, H5 and H6),
(s, 9H, C(CH3)3), 2.94–3.18 (m, 2H,
b
a