Novel highly emissive non-proteinogenic amino acids: Synthesis of 1,3,4-thiadiazolyl asparagines and evaluation as fluorimetric chemosensors for biologically relevant transition metal cations

Article abstract of DOI:10.1007/s00726-010-0730-0

Highly emissive heterocyclic asparagine derivatives bearing a 1,3,4-thiadiazolyl unit at the side chain, functionalised with electron donor or acceptor groups, were synthesised and evaluated as amino acid-based fluorimetric chemosensors for metal cations,

Full text of DOI:10.1007/s00726-010-0730-0

Amino Acids (2011) 40:1065–1075
DOI 10.1007/s00726-010-0730-0
ORIGINAL ARTICLE
Novel highly emissive non-proteinogenic amino acids: synthesis
of 1,3,4-thiadiazolyl asparagines and evaluation as fluorimetric
chemosensors for biologically relevant transition metal cations
•
´
Catia I. C. Esteves M. Manuela M. Raposo
Susana P. G. Costa
•
Received: 15 June 2010 / Accepted: 21 August 2010 / Published online: 11 September 2010
Ó Springer-Verlag 2010
Abstract Highly emissive heterocyclic asparagine
derivatives bearing a 1,3,4-thiadiazolyl unit at the side
chain, functionalised with electron donor or acceptor
groups, were synthesised and evaluated as amino acid-
based fluorimetric chemosensors for metal cations, such as
Cu2?, Zn2?, Co2? and Ni2?. The results suggest that there
is a strong interaction through the donor heteroatoms at the
side chain of the various asparagine derivatives, with high
sensitivity towards Cu2? in a ligand–metal complex with
1:2 stoichiometry. Association constants and detection
limits for Cu2? were calculated. The photophysical and
metal ion sensing properties of these asparagine derivatives
confirm their potential as fluorimetric chemosensors and
suggest that they can be suitable for incorporation into
chemosensory peptidic frameworks.
organic and inorganic molecules involved in biological
pathways being a current thrust in molecular recognition
´
ˆ
´
(Fan et al. 2009; Martınez-Manez and Sancenon 2006;
Zhao et al. 2004; Lin et al. 2004; Togrul et al. 2005; Wright
and Anslyn 2004; Fedorova et al. 2006; Voyer et al.
¨
2001; Mandl and Konig 2005; Zheng et al. 2003; Heinrichs
et al. 2006).
Copper is third in abundance (after iron and zinc) among
the essential heavy metals in the human body and plays a
fundamental role in the biochemistry of the human nervous
system, but the accumulation of excess copper ions or their
misregulation can cause neurological disorders, such as
Parkinson’s and Alzheimer’s diseases (Wang et al. 2010).
Zinc is recognised as one of the most important cations in
catalytic centers and structural cofactors of many zinc-
containing enzymes and DNA-binding proteins. It also
functions as signalling agent mediating processes, such as
gene expression, neurotransmission and apoptosis. Several
enzymes depend on nickel for activity (i.e. carbon mon-
oxide dehydrogenase, acetyl-coenzyme A synthase and
methyl-coenzyme M reductase), but in excess nickel’s
toxic nature can cause respiratory system diseases, such as
lung and nasal cavity cancer, acute pneumonitis and
asthma (Gupta et al. 2007). Like copper, zinc and nickel
are also known to play a part in central nervous system
disorders, such as amyotrophic lateral sclerosis and epi-
leptic seizures (Xu et al. 2010). As for cobalt, it is a
component of vitamin B12 and in large doses can cause
gastrointestinal disorders and respiratory irritation, pul-
monary edema and pneumonia (Kumar and Shim 2009).
Amino acids, and hence peptides, are known for their
ability to complex metal ions through the nitrogen, oxygen
and sulphur donor atoms at the main and side chain
(Shimazaki et al. 2009). Thus, the design of peptides that
coordinate metals, by incorporation of modified amino
Keywords Non-proteinogenic amino acids ꢀ Asparagine ꢀ
Thiadiazole ꢀ Fluorescence ꢀ Chemosensors ꢀ Transition
metals
Introduction
Molecular recognition is the basis for most biological
functions and, given the high degree of control over bio-
logical systems in nature, in recent years the research on
molecular recognition has evolved to mimic natural
mechanisms of organisation as much as possible (Schneider
and Strongin 2009), with the construction of fluorescent
synthetic molecules capable of recognising and binding
C. I. C. Esteves ꢀ M. M. M. Raposo ꢀ S. P. G. Costa (&)
´
Centro de Quımica, Universidade do Minho,
Campus de Gualtar, 4710-057 Braga, Portugal
e-mail: spc@quimica.uminho.pt
123

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C. I. C. Esteves et al.
acids, has potential for applications as varied as the study
of protein–protein interactions mediated by metals and
protein binding to nanoparticles and metal surfaces, and the
development of selective chemical sensors for metals for
use in vivo and in vitro (Mathews et al. 2008; Joshi et al.
2009). The insertion of coordination centres into amino
acids or peptide enables the construction of supramolecules
which may play an innovative role in molecular recogni-
tion. Apart from the importance of metal complexation by
amino acids in a protein, the application of amino acids in
the detection of metals both in solution (Zheng et al. 2003)
and in solid phase through incorporation in polymeric
materials (Gooding et al. 2001; Yang et al. 2001) has been
encouraged. This requires modified amino acids bearing a
metal ion chelating site for stable complex formation for
incorporation into a peptide sequence. Fluorescent ligands
are mostly heteroaromatic ring systems often substituted by
potentially chelating groups which act as both the recog-
nition and signalling site, can be used in the synthesis of
peptide-based chemosensors, as reported recently with
ligands that are capable to chelate various metal ions and
whose complexes possess diversified photophysical prop-
A cyclopentapeptide was found to display ionophoric
properties towards lead cations (Abo-Ghalia and Amr
2004). The indole group of tryptophan has been applied in
the area of anion sensors (Pfeffer et al. 2007).
Our current research interests include the synthesis and
characterisation of unnatural amino acids (Costa et al.
2003, 2008a, b; Esteves et al. 2009), and innovative het-
erocyclic colorimetric/fluorimetric chemosensors for
anions and cations containing (oligo)thiophene, benzox-
azole, imidazole and amino acid moieties (Costa et al.
2007; Batista et al. 2007, 2008, 2009). Given our previous
results with the above-mentioned nitrogen, oxygen and
sulphur heterocycles, we envisaged the use of a heterocycle
that has never been considered for chemosensing applica-
tions in combination with an amino acid, namely 1,3,4-
thiadiazole. This heterocycle contains N and S heteroatoms
and, to the best of our knowledge, has only been used in
nanoparticle design for the fluorimetric sensing of Ag? and
Hg2? (Qu et al. 2008; Li and Yan 2009).
Considering this, we now report the synthesis and
evaluation of novel heterocycle-based unnatural amino
acids as fluorimetric chemosensors for the recognition of
metallic cations (Cu2?, Zn2?, Co2? and Ni2?) of analyti-
cal, biological, environmental and medicinal relevance,
through the combination of a 1,3,4-thiadiazole as coordi-
nating/reporting unit with an amino acid core to obtain new
chemosensors. The studied metallic cations can be con-
sidered as borderline acids by Pearson’s Hard Soft Acid
Base (HSAB) theory. The novel 1,3,4-thiadiazolyl aspara-
gines possess in its structure a donor atom set consisting of
both soft (S) and hard (N and O) bases and should ensure
complexation to a variety of metal ions. Using an hetero-
cyclic p-conjugated bridge, it is intended to improve the
intramolecular electronic delocalisation, for the enhance-
ment of the photophysical properties of the new sensors
and the optimisation of the recognition process of target
analytes, through higher fluorescence and thus higher
sensitivity, which represent a challenging goal in biomi-
metic and supramolecular chemistry.
¨
erties (Voyer et al. 2001; Mandl and Konig 2005; Zheng
et al. 2003; Heinrichs et al. 2006).
Unnatural amino acids with intrinsic functions have
been increasingly studied, to be incorporated into peptides
(Ishida et al. 2004; Hodgson and Sanderson 2004). A
strategy for the development of such compounds involves
the incorporation of multidentate complexing ligands in
amino acid residues in the form of heterocyclic moieties.
Amino acids bearing 2,20-bipyridine and 1,10-phenanthro-
line have been used in the synthesis of metalopeptides that
bind Zn2? (Cheng et al. 1996; Torrado and Imperiali 1996).
Histidine participates actively in the coordination of Zn2?
in various proteins and imidazole isosters were proposed to
mimic the side chain of the natural residue, containing a
complexing group attached to the b-carbon via a 1,2,3-
triazole (Nadler et al. 2009), histidine in the N-terminal
position of a peptide binds metals through its terminal
nitrogen and the imidazole nitrogen, a process analogous to
histamine coordination (Kozlowski et al. 1999). Hydroxy-
quinolines are also known to complex with metals and thus
the synthesis of a (dihydroxyquinolinyl)-alanine from
L-tyrosine was proposed (Heinrich and Steglich 2003). The
complexes formed between metal ions and amino acids can
be considered as models for biochemical interactions, such
as substrate–enzyme and other metal-mediated interactions
with metal ions, with many Cu2? complexes playing a
decisive role in biological systems and, as such, hetero-
cyclic nitrogen ligands like benzimidazole and creatinine
have been used in the synthesis of ternary complexes of
Cu2? with dipeptides, such as Gly-Gly, Gly-Ala, Gly-Val,
Experimental
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. Chromatography on silica
gel was carried out on Merck Kieselgel (230–240 mesh).
IR spectra were determined on a BOMEM MB 104 spec-
trophotometer. NMR spectra were obtained on a Varian
´
Gly-Tyr, Gly-Trp-Gly and Ala (Garcıa-Raso et al. 2003).
123

Novel highly emissive non-proteinogenic amino acids
1067
Unity Plus Spectrometer at an operating frequency of
1
H60); 13C NMR (75.4 MHz, CDCl3): d = 28.19 (C(CH3)3),
38.58 (b-CH2), 50.50 (a-C), 67.47 (CH2), 80.10 (C(CH3)3),
127.28 (C20 and C60), 128.19 (C200 and C600), 128.30 (C400),
128.45 (C300 and C500), 129.25 (C30 and C50), 129.96 (C10),
130.95 (C40), 135.15 (C100), 155.64 (C=O urethane), 159.73
(C2), 163.37 (C5) 168.94 (C=O amide), 171.00 (C=O
ester); IR (KBr 1%, cm-1): m = 3,359, 3,161, 3,032, 2,977,
2,931, 1,754, 1,746, 1,701, 1,694, 1,562, 1,524, 1,501,
1,454, 1,444, 1,407, 1,382, 1,369, 1,348, 1,324, 1,309,
1,281, 1,249, 1,225, 1,200, 1,165, 1,109, 1,093, 983, 909,
873, 784, 755, 729, 696, 666; UV/vis (acetonitrile, nm):
kmax (log e) = 290 (4.25); MS: m/z (EI) 482 (M?, 100);
HMRS: m/z (EI) calc. for C24H26N4O5S 482.16258, found
482.16252.
300 MHz for H 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 Me4Si = 0 ppm as ref-
erence and J values are given in Hz. Assignments were
made by comparing chemical shifts, peak multiplicities and
J values and were supported by spin decoupling-double
resonance and bidimensional heteronuclear HMBC and
HMQC correlation techniques. Low- and high-resolution
mass spectrometry analyses were performed at the
‘‘CACTI––Unidad de Espectrometria de Masas’’, at Uni-
versity of Vigo, Spain. Fluorescence spectra were collected
using
a FluoroMax-4 spectrofluorometer. UV–visible
absorption spectra (200–800 nm) were obtained using a
Shimadzu UV/2501PC spectrophotometer. The linearity of
the absorption versus concentration was checked within the
used concentration. All commercially available reagents
were used as received.
N-t-Butyloxycarbonyl (5-(40-methoxyphenyl)-
1,3,4-thiadiazol-2-yl) L-asparagine benzyl ester (3b)
Starting from amine 2b (0.129 g, 6.22 9 10-4 mol) and
Boc-Asp-OBzl 1 (0.201 g, 6.22 9 10-4 mol), compound
3b was obtained as a white solid (0.159 g, 50%); mp
131.9–133.0°C; 1H NMR (400 MHz, CDCl3): d = 1.39 (s,
9H, C(CH3)3), 3.23–3.50 (m, 2H, b-CH2), 3.90 (s, 3H,
OCH3), 4.86–4.87 (m, 1H, a-H), 5.14–5.27 (m, 2H, CH2),
5.97 (d, J 8.8 Hz, 1H, NH Boc), 7.01 (d, J 8.8, 2H, H30 and
H50), 7.24–7.30 (m, 5H, 59 Ph-H), 7.90 (d, J 6.8 and
2.0 Hz, 1H, H20 and H60); 13C NMR (100.6 MHz, CDCl3):
d = 28.21 (C(CH3)3), 38.62 (b-CH2), 50.53 (a-C), 55.51
(OCH3), 67.57 (CH2), 80.11 (C(CH3)3), 114.69 (C30 and
C50), 122.37 (C10), 128.21 (C200 and C600), 128.31 (C400),
128.47 (C300 and C500) 128.88 (C20 and C60), 135.12 (C100),
155.64 (C=O urethane), 159.14 (C2), 161.94 (C40), 163.23
(C5), 168.84 (C=O amide), 170.96 (C=O ester); IR (KBr
1%, cm-1): m = 3,372, 2,972, 2,928, 2,855, 1,748, 1,699,
1,609, 1,581, 1,563, 1,521, 1,499, 1,456, 1,417, 1,387,
1,368, 1,345, 1,311, 1,256, 1,222, 1,174, 1,113, 1,028, 985,
902, 834, 732, 697, 666; UV/vis (acetonitrile, nm): kmax
(log e) = 291 (4.23); MS: m/z (EI) 512 (M?, 100); HMRS:
m/z (EI) calc. for C25H28N4O6S 512.17314, found
512.17320.
General procedure for the synthesis
of 1,3,4-thiadiazolyl asparagines 3a–e
N-t-Butyloxycarbonyl L-aspartic acid benzyl ester 1 (DCC,
1 equiv.) was dissolved in dry DMF (3 mL mmol-1), fol-
lowed by HOBt (1 equiv.), and after stirring for 10 min,
N,N0-dicyclohexylcarbodiimide (DCC, 1 equiv.) was added.
The reaction mixture was placed in an ice bath, stirred for
30 min and the corresponding 2-amino-1,3,4-thiadiazole 2
was added (1 equiv.). The mixture 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
separation of by-product N,N0-dicyclohexylurea as a pre-
cipitate, which was separated by filtration. This procedure
was repeated two times. The filtrate was evaporated in a
rotary evaporator. The resulting solid was purified by col-
umn chromatography, using mixtures of ethyl acetate and
light petroleum 40–60 of increasing polarity.
N-t-Butyloxycarbonyl (5-(40-fluorophenyl)-
1,3,4-thiadiazol-2-yl) L-asparagine benzyl ester (3c)
N-t-Butyloxycarbonyl (5-phenyl-1,3,4-thiadiazol-2-yl)
L-asparagine benzyl ester (3a)
Starting from amine 2c (0.121 g, 6.22 9 10-4 mol) and
Boc-Asp-OBzl 1 (0.201 g, 6.22 9 10-4 mol), compound
3c was obtained as a white solid (0.204 g, 66%); mp
193.3–194.0°C; 1H NMR (400 MHz, CDCl3): d = 1.38
(s, 9H, C(CH3)3), 3.26–3.49 (m, 2H, b-CH2), 4.87–4.88
(m, 1H, a-H), 5.14–5.26 (m, 2H, CH2), 5.96 (d, J 8.8 Hz,
1H, NH Boc), 7.17–7.21 (m, 2H, H30 and H50), 7.25–7.30
(m, 5H, 59 Ph-H), 7.94–7.97 (m, 2H, H20 and H60), 13.40
(br s, 1H, NH amide); 13C NMR (100.6 MHz, CDCl3):
Starting from amine 2a (0.110 g, 6.18 9 10-4 mol) and
Boc-Asp-OBzl 1 (0.200 g, 6.18 9 10-4 mol), compound
3a was obtained as a white solid (0.158 g, 53%); mp
168.7–169.7°C; 1H NMR (300 MHz, CDCl3): d = 1.39
(s, 9H, C(CH3)3), 3.25–3.53 (m, 2H, b-CH2), 4.87–4.90
(m, 1H, a-H), 5.13–5.28 (m, 2H, CH2), 5.98 (d, J 9.3 Hz,
1H, NH Boc), 7.23–7.29 (m, 5H, 59 Ph-H), 7.50–7.52
(m, 3H, H30, H40 and H50), 7.95–7.98 (m, 2H, H20 and
123

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C. I. C. Esteves et al.
d = 28.19 (C(CH3)3), 38.57 (b-CH2), 50.52 (a-C), 67.47
(CH2), 80.11 (C(CH3)3), 116.44 (d, J 22.1 Hz, C30 and
C50), 126.32 (d, J 3.0 Hz, C10), 128.15 (C200 and C600),
128.31 (C400), 128.46 (C300 and C500), 129.26 (d, J 9.1 Hz,
C20 and C60), 135.16 (C100), 155.61 (C=O urethane), 159.67
(C2), 162.22 (C5), 164.24 (d, J 252.5 Hz, C40), 168.95
(C=O amide), 170.99 (C=O ester); IR (KBr 1%, cm-1):
m = 3,359, 2,979, 2,930, 2,852, 1,749, 1,699, 1,609, 1,596,
1,566,1,518, 1,455, 1,407, 1,368, 1,349, 1,310, 1,232,
1,161, 1,052, 973, 839; UV/vis (acetonitrile, nm): kmax
(log e) = 289 (4.33); MS: m/z (EI) 500 (M?, 100); HMRS:
m/z (EI) calc. for C24H25N4O5SF 500.15315, found
500.15309.
and C50), 128.24 (C200 and C600), 128.41 (C400), 128.51 (C300
and C500), 135.05 (C100), 137.54 (C40), 149.43 (C20 and
C60), 155.55 (C=O urethane), 159.37 (C2), 160.51 (C5),
160.81 (C=O amide), 170.81 (C=O ester); IR (KBr 1%,
cm-1): m = 3,366, 2,985, 2,920, 2,859, 1,744, 1,686, 1,626,
1,607 1,580, 1,504, 1,449, 1,434, 1,390, 1,367, 1,353,
1,305, 1,281, 1,253, 1,220, 1,163, 1,110, 1,063, 1,020, 997,
965, 913, 856, 824, 786, 747, 701; UV/vis (acetonitrile,
nm): kmax (log e) = 288 (4.15); MS: m/z (EI) 483 (M?,
100); HMRS: m/z (EI) calc. for C23H25N5O5S 483.15785,
found 483.15772.
Synthesis of N-t-butyloxycarbonyl (5-(40-fluorophenyl)-
1,3,4-thiadiazol-2-yl) L-asparagine (4)
N-t-Butyloxycarbonyl (5-(40-nitrophenyl)-
1,3,4-thiadiazol-2-yl) L-asparagine benzyl ester (3d)
Asparagine derivative 3c (0.120 g, 2.40 9 10-4 mol) was
dissolved in 1,4-dioxane (3 mL equiv-1) in an ice bath,
followed by the addition of 6 M aqueous NaOH
(1.5 equiv.). After stirring at r.t. for 3 h, the pH was
adjusted to 2–3 by adding 1 M aq. KHSO4 and the solution
extracted with ethyl acetate (3 9 10 mL). The organic
extract was dried with anhydrous MgSO4, filtered and the
solvent removed in a rotary evaporator. The residue was
triturated with diethyl ether and the resulting solid was
submitted to silica gel chromatography using a mixture of
dichloromethane/methanol (5:1), to afford 4 as a white
solid (0.083 g, 85%); mp 212.3–214.0°C; 1H NMR
(400 MHz, DMSO-d6): d = 1.35 (s, 9H, C(CH3)3),
2.66–2.75 (m, 1H, b-CH2), 2.88–2.95 (m, 1H, b-CH2), 4.19
(d, J 6.0 Hz, 1H, a-H), 6.72 (br s, 1H, NH Boc), 7.35
(t, J 9.2 Hz, 2H, H30 and H50), 7.98 (t, J 7.2 Hz, 2H, H20
and H60) ppm; 13C NMR (100.6 MHz, DMSO-d6):
d = 28.18 (C(CH3)3), 35.87 (b-CH2), 50.60 (a-C), 78.13
(C(CH3)3), 116.44 (d, J 22.1 Hz, C30 and C50), 127.00
(d, J 3.0 Hz, C10), 129.20 (d, J 9.1 Hz, C20 and C60),
154.97 (C=O urethane), 158.71 (C2), 160.50 (C5), 163.29
(d, J 249.5 Hz, C40), 169.52 (C=O amide), 172.95 (C=O
acid); IR (KBr 1%, cm-1): m = 3,097, 2,981, 2,930, 2,850,
1,744, 1,700, 1,610, 1,546, 1,509, 1,463, 1,403, 1,367,
1,329, 1,295, 1,234, 1,170, 1,062, 1,052, 996, 954, 915,
852, 823, 782, 728, 706 cm-1; UV/vis (acetonitrile, nm):
kmax (log e) = 290 (4.20); MS: m/z (EI) 410 (M?, 100);
HMRS: m/z (EI) calc. for C17H19N4O5SF 410.10617, found
410.10643.
Starting from amine 2d (0.137 g, 6.19 9 10-4 mol) and
Boc-Asp-OBzl 1 (0.200 g, 6.19 9 10-4 mol), compound
3d was obtained as a white solid (0.097 g, 30%); mp
171.3–173.5°C; 1H NMR (300 MHz, CDCl3): d = 1.40
(s, 9H, C(CH3)3), 3.31–3.50 (m, 2H, b-CH2), 4.89 (br s,
1H, a-H), 5.16–5.27 (m, 2H, CH2), 5.83 (d, J 6.9 Hz, 1H,
NH Boc), 7.26–7.30 (m, 5H, 59 Ph-H), 8.15 (d, J 8.7 Hz,
2H, H20 and H60), 8.37 (d, J 9.0 Hz, 2H, H30 and H50) ppm;
13C NMR (75.4 MHz, CDCl3): d = 28.21 (C(CH3)3), 38.76
(b-CH2), 50.45 (a-C), 67.66 (CH2), 80.40 (C(CH3)3), 124.58
(C30 and C50), 128.01 (C200 and C600), 128.25 (C400), 128.43
(C300 and C500), 128.52 (C20 and C60), 135.03 (C100), 135.49
(C10), 149.06 (C40), 155.52 (C=O urethane), 159.52 (C2),
160.73 (C5), 169.02 (C=O amide), 170.78 (C=O ester); IR
(KBr 1%, cm-1): m = 3,348, 3,160, 2,977, 2,933, 1,738,
1,700, 1,600, 1,557, 1,524, 1,500, 1,455, 1,442, 1,406,
1,368, 1,348, 1,310, 1,250, 1,215, 1,164, 1,106, 1,052,
1,027, 1,012, 992, 970, 913, 853, 820, 782, 753, 736, 692;
UV/vis (acetonitrile, nm): kmax (log e) = 324 (4.34); MS:
m/z (EI) 527 (M?, 100); HMRS: m/z (EI) calc. for
C24H25N5O7S 527.14765, found 527.14754.
N-t-Butyloxycarbonyl (5-(40-pyridyl)-1,3,4-thiadiazol-2-yl)
L-asparagine benzyl ester (3e)
Starting from amine 2e (0.109 g, 6.13 9 10-4 mol) and
Boc-Asp-OBzl 1 (0.198 g, 6.13 9 10-4 mol), compound
3e was obtained as a white solid (0.056 g, 19%);
mp = 194.3–195.5°C; 1H NMR (300 MHz, CDCl3):
d = 1.40 (s, 9H, C(CH3)3), 3.31–3.48 (m, 2H, b-CH2),
4.83–4.90 (m, 1H, a-H), 5.16–5.27 (s, 2H, CH2), 5.82
(d, J 8.7 Hz, 1H, NH Boc), 7.25–7.30 (m, 5H, 59 Ph-H),
7.89 (d, J 5.4 Hz, 2H, H30 and H50), 8.81 (d, J 5.4 Hz, 2H,
H20 and H60), 13.12 (br s, 1H, NH amide); 13C NMR
(75.4 MHz, CDCl3): d = 28.21 (C(CH3)3), 38.70 (b-CH2),
50.43 (a-C), 67.63 (CH2), 80.37 (C(CH3)3), 121.05 (C30
Synthesis of (5-(40-fluorophenyl)-1,3,4-thiadiazol-2-yl)
L-asparagine (5)
Asparagine derivative 4 (0.070 g, 1.71 9 10-4 mol) was
stirred in a trifluoroacetic acid/dichloromethane solution
(1:1, 1 mL) at r.t. for 2 h. The solvent was evaporated, the
residue dissolved in pH 8 aqueous buffer solution and
extracted with ethyl acetate (39 10 mL). After drying with
123

Novel highly emissive non-proteinogenic amino acids
1069
anhydrous magnesium sulphate and evaporation of the varying the molar fraction of the cation, while maintaining
solvent, 5 was isolated as a white solid (0.041 g, 77%); constant the total asparagine derivative and metal cation
mp 239.8–242.1°C; 1H NMR (300 MHz, DMSO-d6): concentration. The association constants were obtained
d = 2.66–2.71 (dd, J 16.4 and 6.0 Hz, 1H, b-CH2), from Benesi–Hildebrand plots in the form of straight lines
3.06–3.12 (dd, J 16.4 and 6.0 Hz, 1H b-CH2), 3.68 with good correlation coefficients using a equation reported
(t, J 6.4 Hz, 1H, a-H), 7.38 (t, J 8.7 Hz, 2H, H30 and H50), elsewhere (Azab et al. 2010).
7.99 (t, J 7.8 Hz, 2H, H20 and H60) ppm; 13C NMR
(75.4 MHz, DMSO-d6): d = 37.45 (b-CH2), 49.61 (a-C),
116.20 (d, J 22.2 Hz, C30 and C50), 126.10 (d, J 3.0 Hz, Results and discussion
C10), 129.62 (d, J 9.5 Hz, C20 and C60), 160.10 (C2),
162.40 (C5), 164.32 (d, J 251.7 Hz, C40), 169.10 (C=O Synthesis
amide), 169.93 (C=O acid); IR (KBr 1%, cm-1):
m = 3,071, 2,923, 2,850, 1,675, 1,611, 1,563, 1,495, 1,462, The new asparagine derivatives 3a–e with 1,3,4-thiadiazole
1,435, 1,390, 1,355, 1,303, 1,260, 1,260, 1,169, 1,060, 971, at its side chain were synthesised by a standard coupling
857, 827, 815, 733, 710; UV/vis (acetonitrile, nm): kmax procedure involving DCC and HOBt, between the side
(log e) = 289 (4.19); MS: m/z (EI) 310 (M?, 100); HMRS: chain carboxylic acid group of N-t-butyloxycarbonyl
m/z (EI) calc. for C12H11N4O3SF 310.05373, found
310.05401.
L-aspartic acid benzyl ester 1 and 2-amino-1,3,4-thiadiaz-
oles 2a–e, bearing a phenyl ring with substituents of dif-
ferent electronic character (electron donor or acceptor such
as methoxy, fluor and nitro) or a electron deficient pyridyl
ring (Scheme 1; Table 1). From the results in Table 1, it
can be seen that the yield for thiadiazolyl asparagine
derivatives 3a–e was influenced by the electronic nature of
Spectrophotometric titrations and chemosensing
studies of 1,3,4-thiadiazolyl asparagine derivatives
3a–e, 4 and 5
Solutions of asparagine derivatives 3a–e, 4 and 5 (ca. the substituent at position 5 of the thiadiazole, being low for
1.0 9 10-5 to 1.0 9 10-6 M) and of the metallic cations derivatives 3d–e with electron acceptor nitro and pyridyl
under study (ca. 1.0 9 10-1 to 1.0 9 10-3 M) were pre- groups. In addition, in order to assess the influence of the
pared in UV-grade acetonitrile (in the form of hexahidrated presence of additional heteroatoms at the N- and C-termini
tetrafluorborate salts for Cu2?, Co2? and Ni2? and per- blocking groups (in the form of Boc and Bzl ester), the
chlorate salt for Zn2?). Titration of the compounds with the selective deprotection at the carboxylic and amino group of
several metallic cations was performed by the sequential fluoro asparagine derivative 3c was undertaken by standard
addition of equivalents of metal cation to the asparagine
deprotection procedures, yielding novel derivatives 4 (with
derivative solution, in a 10-mm path length quartz cuvette a free carboxylic acid terminal) and 5 (with free amino and
and emission spectra were measured by excitation at the carboxylic acid terminals) in good yields. All the hetero-
wavelength of maximum absorption for each compound, cyclic asparagine derivatives were fully characterised by
indicated in Table 1.
the usual spectroscopic techniques.
The binding stoichiometry of the asparagine derivatives
The absorption and emission spectra of asparagine
with the metal cations was determined using Job’s plots, by derivatives 3a–e, 4 and 5 were measured in degassed
acetonitrile solution (10-6–10-5 M) (Table 1). The aspar-
agine derivatives showed similar absorption and emission
wavelengths with the exception of 3d, which displayed a
Table 1 Yields, UV–visible absorption and fluorescence data for
bathochromic shift for the maximum wavelength of
1,3,4-thiadiazolyl asparagine derivatives 3–5 in acetonitrile
absorption and emission when compared to 3a, according
Compound
R
Yield UV/vis
(%)
Fluorescence
to the electronic character of the nitro substituent. The
nature of the ring at position 5 of the thiadiazole did not
influence the overall photophysical properties of deriva-
tives 3a and 3e, bearing a phenyl and a pyridyl ring,
respectively. The relative fluorescence quantum yields (UF)
were determined using a 10-6 M solution of 9,10-diphen-
ylanthracene in ethanol as standard (UF = 0.95) (Morris
et al. 1976). For the UF determination, the fluorescence
standard was excited at the wavelengths of maximum
absorption found for each of the compounds to be tested
and in all fluorimetric measurements, the absorbance of the
kmax log e kem Stokes’ shift UF
(cm-1
)
3a
3b
3c
3d
3e
4
H
53
290 4.25 364 7,010
291 4.23 363 6,816
289 4.33 361 6,901
324 4.34 390 5,223
288 4.15 362 7,098
290 4.20 385 8,509
289 4.19 369 7,502
0.27
0.43
0.71
0.01
0.34
0.23
0.21
OCH3 50
66
NO2 30
F
–
F
F
19
85
77
5
123

1070
C. I. C. Esteves et al.
N
Scheme 1 Synthesis of
1,3,4-thiadiazolyl asparagine
derivatives 3–5. Reagents and
conditions: (i) 1,4-dioxane, aq.
NaOH 6 M, rt; (ii)
O
C
N
R
N
N
S
N
H
H2N
R
a
b
c
d
R = H
R = OCH3
R = F
R2
S
trifluoroacetic acid/
dichloromethane (1:1), r.t.
R1-HN
C
O
R = NO2
(S)-(+)
2a-d
3
4
5
R1 = Boc, R2 = OBzl
CO2H
i)
R
R
1 = Boc, R2 = OH
1 = H, R2 = OH
ii)
DCC/HOBt
DMF, 0°C to rt
Boc-HN
CO2Bzl
(S)-(+)
N
O
C
N
1
N
N
N
H2N
N
S
N
H
S
3e
2e
Boc-HN
CO2Bzl
(S)-(+)
solution did not exceed 0.1. The 1,3,4-thiadiazolyl aspar-
agine derivatives exhibited good-to-excellent fluorescence
quantum yields in acetonitrile, with the exception of the
nitro derivative 3d (0.01) which is a known strong fluo-
rescence quencher. The highest UF value was obtained for
the fluoro derivative 3c (0.71), which is in agreement with
previous results reported by us for benzothiazolyl aspara-
gine derivatives bearing different substituents including
fluorine (Esteves et al. 2009). N-protected fluoro aspara-
gine 4 and fully deprotected fluoro asparagine derivative 5
displayed similar UF (ca. 0.2), which was lower than that of
the parent asparagine derivative 3c, a fact that could be
attributed to the possibility for formation of intra- and
intermolecular H-bonds between the carboxylic acid proton
and the heteroatoms at the side chain. All compounds
showed large Stokes’ shifts (from 6,800 to 8,500 cm-1),
except for the nitro derivative 3d which was ca.
5,200 cm-1. Stokes’ shifts directly relate to energy dif-
ferences between the ground and excited states and this
large value is an interesting feature for biological fluores-
cent probes that allows an improved separation of the light
inherent to the matrix and the light dispersed by the sample
(Holler et al. 2002).
transition metals, such as Cu2?, Zn2?, Co2? and Ni2?, the
interaction of asparagine derivatives 3a–e with these cat-
ions was evaluated through UV–vis and fluorescence
spectroscopies in spectrophotometric and spectrofluori-
metric titrations in acetonitrile. In the spectrophotometric
titrations, no changes were seen in the bands corresponding
to the maximum wavelength of absorption of asparagine
derivatives 3a–e after addition of up to 200 equiv. of each
metal cation. In the presence of Cu2?, two new absorption
bands appeared at shorter wavelengths (212 and 235 nm)
and increased with the addition of up to 10 equiv. of Cu2?
.
Increase of the metal concentration to 15 equiv. leads to the
decrease of the band at 212 nm, accompanied by a hipso-
chromic shift and the disappearance of the band at 235 nm,
resulting in a new band centered at 206 nm. This band
increased with further addition of Cu2?. These observations
may indicate the formation of a new species, a asparagine–
metal complex, absorbing at shorter wavelengths. In the
presence of Ni2?, a similar behaviour was observed for a
new band visible at 266 nm, which increased upon addition
of up to 10 equiv. of metal and decreased at higher metal
concentration (up to 30 equiv.). In the presence of Zn2? and
Co2?, the formation of asparagine–metal complexes was
not detected by UV–vis spectroscopy.
Spectrophotometric and spectrofluorimetric titrations
In the spectrofluorimetric titrations with Cu2?, a strong
decrease of the fluorescence intensity (a chelation-
enhanced quenching, CHEQ effect) was observed for all
the asparagine derivatives, with an almost complete fluo-
rescence quenching. In Fig. 1a the spectrofluorimetric
titration of asparagine derivative 3c with Cu2? is shown,
where the drastic effect of ion complexation is evident in
the band centred at the wavelength of maximum emission
at 361 nm. This figure is representative of the Cu2? titra-
tions of asparagine derivatives 3a–e, the only difference
between them being the number of metal equivalents
necessary to quench at least 90% of the initial fluorescence
intensity (before complexation) of the heterocyclic amino
of 3a–e, 4 and 5 with metallic cations
The modification of asparagine through the introduction of
a UV-active and highly fluorescent heterocycle at its side
chain is expected to provide additional binding sites for a
variety of metal ions through the heterocycle donor atoms,
as well as improved photophysical properties for the
chemosensing studies. With heterocyclic asparagine deriv-
atives 3a–e, it was intended to assess the influence in the
chemosensing ability of metallic cations of the type of the
substituent at the 1,3,4-thiadiazolyl system. Considering
the biological, environmental and analytical relevance of
123

Novel highly emissive non-proteinogenic amino acids
1071
acid (20 equiv. for 3a, 30 equiv. for 3b, 40 equiv. for 3c,
25 equiv. for 3d and 70 equiv. for 3e) (see Fig. 2 for
spectrofluorimetric titrations of 3a, b, d, e with Cu2?).
In order to assess the influence on the chemosensing
ability of the presence of additional heteroatoms from
the urethane and ester protecting groups at the N- and
C-terminals, spectrofluorimetric titrations with Cu2? of the
fluoro asparagine derivatives 4, having a free carboxylic
acid group, and 5, having free amino and carboxylic acid
groups, were also carried out in acetonitrile (Fig. 2). It was
found that both derivatives required similar amounts of
Cu2? (15 equiv.) for a 90% quench of the initial fluores-
cence, revealing that the free amino terminal was not
essential for coordination (comparing 4 and 5) and that free
carboxylic acid group had a positive effect on the coordi-
nation ability (when comparing 3c, 40 equiv. and 4), as less
metal equivalents were necessary for the same decrease in
fluorescence. Considering these findings, it can be sug-
gested that the coordination process should occur through
the heteroatoms at the side chain of the amino acid, aided
by the carbonyl oxygen at the C-terminal of the amino acid
(either in ester or carboxylic acid form).
metal addition, without complete quenching of fluores-
cence. In Fig. 1b–d, the spectrofluorimetric titrations of
asparagine derivative 3c with Zn2?, Co2? and Ni2? are
shown, and, as already stated for Fig. 1a, these are also
representative of the titrations of asparagine derivatives
3a–e with Zn2?, Co2? and Ni2?. For these metallic cations,
the complete quenching of fluorescence was not achieved
even after addition of up to 200 equiv. of metal, with a
plateau being reached depending on the metal and the
asparagine derivative: for Zn2?, there was a maximum
decrease of 70% in fluorescence of asparagine derivatives
3a–d with the addition of 35–60 equiv. of cation, while for
asparagine derivative 3e a decrease of less than 30% was
achieved with 30 equiv. of cation; as for Co2?, a fluores-
cence quenching of 70% was visible with the addition of
20–40 equiv. to asparagine derivatives 3a–c, e and a 90%
quenching was achieved upon the addition of 35 equiv. to
3d; and finally, asparagine derivatives 3a–e responded
weakly to titration with Ni2? with a 25–35% quenching
after 20–30 equiv. of metal were added.
For each 1,3,4-thiadiazolyl asparagine derivative, the
variation in fluorescence intensity was plotted against the
concentration of the metal cation, which gave a linear
correlation (until it reached a plateau). Stern–Volmer plots
With regard to the other cations Zn2?, Co2? and Ni2?, a
less pronounced CHEQ effect was also observed after
1
1
1,00
1,00
B
A
0,8
0,8
0,6
0,6
0,80
0,60
0,40
0,20
0,00
0,80
0,4
0,2
0
0,4
0,2
0,60
0,40
0,20
0,00
0
0
20
40
60
0
10
20
30
40
50
310
350
390
430
305
345
385
425
465
Wavelength (nm)
Wavelength (nm)
1
0,8
0,6
0,4
0,2
0
1
0,8
0,6
0,4
0,2
1,00
0,80
0,60
0,40
0,20
0,00
1,00
0,80
0,60
0,40
0,20
0,00
C
D
0
0
50 100 150 200
0
10
20
30
40
305
345
385
425
465
305
345
385
425
465
Wavelength (nm)
Wavelenght (nm)
Fig. 1 Fluorimetric titrations of asparagine derivative 3c with Cu2? (a), Zn2? (b), Co2? (c) and Ni2? (d) in acetonitrile (kexc: 3c = 289 nm).
Inset Normalised emission at 361 nm as a function of added metal equivalents
123

1072
C. I. C. Esteves et al.
1
0,8
0,6
0,4
0,2
0
1
0,8
0,6
0,4
0,2
0
1,00
0,80
0,60
0,40
0,20
0,00
1,00
0,80
0,60
0,40
0,20
0,00
B
A
0
5
10
15
20
25
0
10
20
30
310
350
390
430
470
310
350
390
430
470
Wavelength (nm)
Wavelength (nm)
1
0,8
0,6
0,4
0,2
0
1
0,8
0,6
0,4
0,2
1,00
0,80
0,60
0,40
0,20
0,00
1,00
0,80
0,60
0,40
0,20
0,00
C
D
0
20
40
0
0
20
40
60
80 100
310
350
390
430
470
350
400
450
500
Wavelength (nm)
Wavelength (nm)
1
1
0,8
0,6
0,4
0,2
0
1,00
0,80
0,60
0,40
0,20
0,00
1,00
0,80
0,60
0,40
0,20
0,00
0,8
0,6
0,4
0,2
0
F
E
0
5
10
15
20
0
5
10
15
20
310
350
390
430
470
310
350
390
430
470
Wavelength (nm)
Wavelength (nm)
Fig. 2 Fluorimetric titrations of asparagine derivatives 3a (a), 3b (b),
3d (c), 3e (d), 4 (e) and 5 (f) with Cu2? in acetonitrile (kexc: 3a =
290 nm, 3b = 291 nm, 3d = 324 nm, 3e = 288 nm, 4 = 290 nm,
5 = 289 nm). Inset Normalised emission at the wavelength of
maximum emission as a function of added metal equivalents
for the titration of Cu2? with asparagine derivatives 3a-e
indicated that the linear dependence of the fluorescence
quenching and the metal concentration is of dynamic nature
(Fig. 3). The spectrofluorimetric titration results indicated
that all the heterocyclic asparagine derivatives were sensi-
tive to Cu2?, whereas the sensing of Zn2?, Co2? and Ni2?
was non-selective with lower sensitivity, especially for nitro
asparagine derivative 3d. From these plots, and with regard
to the sensing of Cu2?, it appeared that the presence of an
extra nitrogen atom at the pyridyl ring at position 5 of the
thiadiazole (asparagine derivative 3e) had no additional
positive effect on metal complexation, when compared with
asparagine derivative 3a (bearing a phenyl ring). A similar
conclusion could be drawn for asparagine derivatives 3a–d,
with comparable results, which can also indicate that the
type of substituent (electron donor or acceptor) present at
the phenyl ring did not influence significantly the coordi-
nation process. Nevertheless, considering the photophysical
properties in acetonitrile of asparagine derivatives 3a–d
(presented in Table 1), the fluoro asparagine derivative
123

Novel highly emissive non-proteinogenic amino acids
1073
1,8
1,6
1,4
1,2
1
0
4
8
12
16
[Cu2+] ( x 10-5 M)
Fig. 4 Relative fluorimetric response (I/I0) of asparagine derivatives
3a–e in the presence of 100 equiv. of Cu2?, Zn2?, Co2? and Ni2? as a
function of metal concentration in acetonitrile
Fig. 3 Stern–Volmer plots for the titration of Cu2? with asparagine
derivatives 3 (a open diamonds, b open squares, c open triangles,
d times symbol, e open circles in acetonitrile
Table 2 Association constants (Ka) and detection limits (DL) for the
interaction of asparagine derivatives 3–5 with Cu2? in acetonitrile
3c would be the more interesting candidate as chemosensor
due to the higher fluorescence quantum yield, which is
important for maximisation of response to analyte in the
analysis of very dilute samples.
Cpd.
Ka (mol-1 L)
DL (mol L-1
)
3a
3b
3c
3d
3e
4
3.3 9 103
2.4 9 103
1.4 9 103
5.4 9 103
6.5 9 103
1.4 9 105
4.5 9 104
2.0 9 10-6
8.0 9 10-6
1.2 9 10-6
4.4 9 10-5
8.2 9 10-6
9.8 9 10-7
1.7 9 10-7
The comparative fluorimetric response of asparagine
derivatives 3a–e to Cu2?, Zn2?, Co2? and Ni2? is sum-
marised in Fig. 4, considering the variation of the fluo-
rescence intensity (I/I0) upon the addition of 100 equiv. of
each metal (the shorter the bar, the higher the change in
fluorescence and higher sensitivity).
5
The binding stoichiometry of asparagine derivatives
3a–e, 4 and 5 with Cu2? were determined from Job’s plots
and the binding affinity was calculated from a Benesi–
Hildebrand plot using an equation reported elsewhere
(Azab et al. 2010). The results suggest the formation of a
ligand–metal complex with 1:2 stoichiometry and the
association constants (Ka) were calculated (Table 2). In
addition, the detection limit (DL) was calculated taking
into account the fluorimetric titrations in the presence of
metal cations and the standard deviation of a set of ten
fluorescence measurements of a blank asparagine deriva-
tives solution according to a previously reported expression
(Miller 1993). The results presented in Table 2 indicate
that there was a strong interaction between 1,3,4-thiadiaz-
olyl asparagine derivatives 3–5 and Cu2?, with low DLs
which constitute an important feature considering their
potential application as chemosensors for transition metals
with biological, environmental and analytical relevance.
With the aim of further elucidating the metal binding
mode and to complement the findings of the spectrofluo-
oxygens at the amide and ester carbonyl groups. Upon the
addition of 10 equiv. of Cu2?, a change was visible for the
signals corresponding to the protons of the phenyl group
attached to the thiadiazole and the a-CH and b-CH2 and a
very slight shift of the benzylic CH2 group; after the
addition of 60 equiv. of Cu2?, along with a more pro-
nounced shift of the previous signals, the displacement of
the benzylic phenyl group signal and in the presence of
160 equiv. of Cu2? was also seen, the signals of the pre-
viously mentioned protons were further displaced. The
chemical shifts of the Boc and the urethane NH were not
affected by the presence of the metal cation, thus indicating
that this part of the amino acid derivative did not partici-
pate in the coordination process (Fig. 5). These observa-
tions are in agreement with the data collected from the
spectrofluorimetric titrations of asparagine derivatives 3c, 4
and 5 with Cu2? in acetonitrile.
1
rimetric titrations, H NMR titrations of fluoro asparagine
Conclusions
derivative 3c (as representative of asparagines 3a–e) with
Cu2? were carried out in CDCl3 at 25°C. It was found that
after addition of increasing amounts of metal cation, there
was a displacement to lower chemical shift of several
signals suggesting that the metallic cation is coordinated by
the thiadiazole heteroatoms at the side chain and the
A family of novel 1,3,4-thiadiazolyl asparagine derivatives
3a–e, 4 and 5 was synthesised and evaluated as fluorescent
chemosensors based on an amino acid core for a series of
transition metal cations, namely Cu2?, Zn2?, Co2? and
Ni2?. From the spectrofluorimetric titrations in acetonitrile,
123

1074
C. I. C. Esteves et al.
Fig. 5 1H NMR titration of
asparagine derivative 3c with
Cu2? in CDCl3: a in the absence
of Cu2?, and upon addition of
b 10 equiv., c 60 equiv., and
d 160 equiv. of Cu2?
contract REDE/1517/RMN/2005 with funds from POCI 2010
(FEDER) and FCT.
it was found that asparagine derivatives 3–5 were suitable
chemosensors for Cu2?, showing higher sensitivity for this
cation when compared to Zn2?, Co2? and Ni2?, as a pro-
nounced fluorescence quenching was achieved with the
addition of a low number of metal equivalents. The results
suggested that there was a strong interaction with Cu2?
through the donor heteroatoms at the side chain of the
various asparagine derivatives, aided by the carbonyl
oxygen at the amino acid C-terminal (both in ester and
carboxylic acid forms). Considering the electronic nature
of nitrogen, one could foresee a similar behaviour for
C-terminal amide derivatives. Considering the photophys-
ical properties and the chemosensing ability, the novel
highly emissive 1,3,4-thiadiazolyl asparagine derivatives
3a–e, 4 and 5 can be considered very promising candidates
as amino acid-based fluorescent probes for chemosensing
applications within a peptidic framework (where the
N- and C-terminals will be blocked with amide links).
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