Synthesis and photophysics of benzazole based triazoles with amino acid-derived pendant units. Multiparametric optical sensors for BSA and CT-DNA in solution

Article abstract of DOI:10.1016/j.molliq.2020.113092

Herein we report the synthesis of a series of amino acid-derived triazoles by an organocatalytic cycloaddition reaction between azides and carbonyl compounds, catalyzed by a simple amine. These compounds present absorption maxima located in the UV-B ascribed to fully spin and symmetry allowed electronic transitions and a main fluorescence emission in the UV-A (~380 nm) with a relatively large Stokes shift (5700 cm^?1). No significant solvatochromism was observed in both ground and excited states. Unexpectedly, the benzoxazole derivatives presented much higher fluorescence quantum yield values (40–80%) of compared to the sulfur analogues (3–6%). In addition, the DNA binding assays indicated that these compounds presented strong interaction with CT-DNA, which could be attributed to π-stacking and intermolecular hydrogen-bonding. The interaction of the benzazoles with bovine serum albumin (BSA) was also investigated, where a suppression mechanism was observed. In each case, docking was performed to better understand the observed interactions.

Full text of DOI:10.1016/j.molliq.2020.113092

Journal Pre-proof
Synthesis and photophysics of benzazole based triazoles with
amino acid-derived pendant units. Multiparametric optical sensors
for BSA and CT-DNA in solution
Natalí P. Debia, Juan J.P. Rodríguez, Carolina H. da Silveira,
Otavio A. Chaves, Bernardo A. Iglesias, Fabiano S. Rodembusch,
Diogo S. Lüdtke
PII:
S0167-7322(20)31454-9
DOI:
https://doi.org/10.1016/j.molliq.2020.113092
MOLLIQ 113092
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
9 March 2020
2 April 2020
5 April 2020
Please cite this article as: N.P. Debia, J.J.P. Rodríguez, C.H. da Silveira, et al., Synthesis
and photophysics of benzazole based triazoles with amino acid-derived pendant units.
Multiparametric optical sensors for BSA and CT-DNA in solution, Journal of Molecular
Liquids (2018), https://doi.org/10.1016/j.molliq.2020.113092
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Synthesis and Photophysics of Benzazole based Triazoles with Amino Acid-derived
Pendant Units. Multiparametric optical sensors for BSA and CT-DNA in solution
[a]
[b]
[c]
Natalí P. Debia,[a] Juan J. P. Rodríguez, Carolina H. da Silveira, Otavio A. Chaves,
Bernardo A. Iglesias, [b] Fabiano S. Rodembusch,*, [a] and Diogo S. Lüdtke*,[a]
[a]
Instituto de Química, UFRGS – Universidade Federal do Rio Grande do Sul, Av. Bento
Gonçalves 9500, 91501-970, Porto Alegre, RS, Brazil.
E-mail: dsludtke@iq.ufrgs.br, rodembusch@iq.ufrgs.br
[b]
Federal de Santa Maria, Av. Roraima 1000, 97105-900, Santa Maria, RS, Brazil.
[b] SENAI Innovation Institute for Green Chemistry, Rua Morais e Silva N° 53, Maracanã,
LBMP – Laboratório de Bioinorgânica e Materiais Porfirínicos, UFSM – Universidade
20271030, Rio de Janeiro, RJ, Brazil.
Abstract
Herein we report the synthesis of a series of amino acid-derived triazoles
by
an
organocatalytic cycloaddition reaction between azides and carbonyl compounds, catalyzed by
a simple amine. These compounds present absorption maxima located in the UV-B ascribed
to fully spin and symmetry allowed electronic transitions and a main fluorescence emission in
the UV-A (~380 nm) with a relatively large Stokes shift (5700 cm-1). No significant
solvatochromism was observed in both ground and excited states. Unexpectedly, the
benzoxazole derivatives presented much higher fluorescence quantum yield values (40-80%)
of compared to the sulfur analogues (3-6%). In addition, the DNA binding assays indicated
that these compounds presented strong interaction with CT-DNA, which could be attributed to
π-stacking and intermolecular hydrogen-bonding. The interaction of the benzazoles with
bovine serum albumin (BSA) was also investigated, where a suppression mechanism was
observed. In each case, docking was performed to better understand the observed
interactions.
Keywords: amino acid-derived triazole • fluorescent sensor • protein detection • DNA
intercalation • docking studies
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1. Introduction
The development of fluorescent sensors has been a subject of great interest in the field of
chemistry, biology and materials science.[1] The design of a sensor is composed of two parts,
the recognition and signaling units, which should act in synergy. Upon exposition to an
analyte, the resulting interaction should result in changes in the photophysical profile which
can be measured and then result in useful analytical information.[2] The recognition moiety is
responsible to perform specific interactions with the analyte, causing a change in the system,
while the signaling moiety is a fluorophoric unit which will send an optical signal in response to
this perturbation in the system.[3] Taking the optical sensing into account, fast, sensitive and
selective, as well as cost‐effective analyses of biomolecules are important in clinical
diagnostics and treatments. Compounds presenting particular photophysical properties, such
as planar conjugated structures, are potential candidates for use as fluorescent probes to
study biological mechanisms in protein and DNA.[4-9] For instance, electronic spectroscopies
have been applied to study the interaction of small molecules to serum albumin proteins.[10]
In this regard, due to the bovine serum albumin (BSA) role in the transport and disposition of
endogenous and exogenous compounds in blood,[11] BSA is widely used as a model of drug-
albumin interaction. This latter can contribute to the understanding of the absorption,
transportation, metabolism, distribution and efficacy of drugs in the body.[12] Additionally, its
low cost, ready availability and the homology with human serum albumins (HSA)[13, 14] also
contribute for the large number of studies in the literature. On the other hand, DNA is certainly
one of the most important of all biomolecules and its interaction study with different
compounds is worth of interest.[15-18] As discussed by Zhai et al.,[15] the ability of a single
stranded DNA (ssDNA) molecule to ‘seek out’, or hybridize to, it’s complementary strand in a
sample is the foundation of DNA-based detection systems. This event, so-called hybridization,
is then translated into a signal. A variety of transduction techniques can be used to observe
this process, including the optical one.[19, 20]
In this way, benzazoles are very interesting organic molecules as fluorophore to build probes
due to their planarity and -delocalized system. The benzazole family is constituted of a
benzene-fused 5-membered heterocycle incorporating one nitrogen and a second heteroatom
(N, O, S or Se).[21] Among these, 2-aryl-benzazoles are widely used because of their
versatility and can be obtained through several methods.[7, 22, 23] Benzazoles derivatives are
studied as sensors both for pH and for ion sensing,[24] imaging of amyloid aggregates,[25]
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detection of H2S,[26] among others. In addition, the benzazole nucleus is found in many drug
structures due to widespread biological activity such as antibacterial, antifungal, anticancer,
neuroprotective, muscle relaxant, cholinesterase inhibitory, etc.[9, 27] One very useful
synthetic tool to connect the fluorophore with the recognition moiety is through a 1,2,3-triazol
linker. This nucleus is a 5-membered heterocycle containing three nitrogen-atoms in a vicinal
position. Several methods have been developed since Michael first performed its synthesis by
the reaction between phenylazide and acetylene dicarboxylic ester under thermal
conditions.[28] However, the widespread utilization of the 1,2,3-triazole only came after the
breakthrough discovery that the use of a copper(I) catalyst enhances the reaction rate and
regioselectivity.[29, 30] Since this discovery, the Cu-catalyzed azide-alkyne cycloaddition has
become one of the most useful reaction to connect a number of different structural elements
and has been employed as a linking unit of functional materials,[31, 32] in drugs discovery,[33,
34] protein sensors,[35] among others. A number of other transition metals such as Ru(II),[36]
Ag(I)[37] and Ir(I)[38] have also been reported to be competent to catalyze the reaction.
Despite its undeniable usefulness, there is still some drawbacks in the synthesis of 1,2,3-
triazoles that should be addressed. Notably, the search for metal-free methodologies,
especially for the synthesis fully substituted 1,2,3-triazoles, has become a topic of research
and has led to the development of organocatalytic reactions. In this context, a large range of
carbonyl compounds have been employed as starting materials, such as β-diketones,[39] β-
keto esters,[20, 40] β-keto sulfones,[41] β-keto amides,[42] α-keto nitriles,[20, 43] aldehydes
combined with nitroalkanes.[44]
In this work, we report the synthesis and photophysical characterization of a novel series of
amino acid-derived β-keto esters and benzazole moieties connected by a triazole-4-
carboxylate linker by organocatalytic enamine-azide [3+2] cycloaddition (Scheme 1). For
optical sensor purposes, these compounds were also used to study the interaction with CT-
DNA and BSA in solution.
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Scheme 1. This work.
2. Experimental Section
2.1. General Information
Air- and moisture-sensitive reactions were conducted in flame- or oven-dried glassware
equipped with tightly fitted rubber septa and under a positive pressure of dry argon. Reagents
and solvents were handled by using standard syringe techniques. Temperatures above room
temperature were maintained by use of a mineral oil bath heated on a hot plate. Column
chromatography was performed using silica gel (230-400 mesh) following the methods
described by Still.[45] TLC was performed using supported silica gel GF254, 0.25 mm
thickness. For visualization, TLC plates were either placed under ultraviolet light or treated
with acid vanillin followed by heating. NMR spectra were recorded either with a Varian 400
MHz or a Bruker 400 MHz instrument in CDCl3 solutions. Chemical shifts are reported in ppm,
referenced to the solvent peak of residual CHCl3 or TMS as reference. The data are reported
as follows: chemical shift (δ), multiplicity, coupling constant (J) in Hz, and integrated intensity.
13C NMR spectra were recorded at 100 MHz in CDCl3 solutions. Chemical shifts are reported
in ppm, referenced to the solvent peak CDCl3. Abbreviations to denote the multiplicity of a
particular signal are s (singlet), d (doublet), dd (double doublet), m (multiplet), and bs (broad
singlet). ESI-QTOF-MS measurements were performed in the positive ion mode (m/z 50−2000
range). IR spectra were obtained on an FTIR-ATR instrument (Bruker). Melting points were
obtained on a Buchi M-565. Optical rotations were measured using a JASCO P-200 digital
polarimeter using 10 mm cells and are reported as [α]2D0, concentration (g/100 mL), and
solvent. HPLC analysis were performed in a high-performance liquid chromatography
Shimadzu, LC - 20AT, using chiral column Daicel Chiracel IB, hexano/iso-propanol as eluent
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and detector UV-vis with wavelength 254 nm. Spectroscopic grade solvents were used in the
photophysical study. The UV-Vis absorption spectra in solution was acquired on a Shimadzu
UV-2450 spectrophotometer (data interval, 1.0 nm), and the steady-state fluorescence spectra
were measured on a Shimadzu spectrofluorometer model RF-5301PC (Slit 3.0 nm;
emission/excitation). Quinine sulfate (Riedel) in 0.5 M H2SO4 (QY = 0.55) was used as the
quantum yield standard.[46] For the analysis with BSA and DNA, UV-Vis absorption spectra
were recorded using Shimadzu UV2600 spectrophotometer (data interval, 2.0 nm). Steady-
state emission fluorescence spectra of samples were measured with a Varian Cary 50
fluorescence spectrophotometer (Slit 5.0 nm; emission/excitation) and were corrected
according to the manufacturer’s instructions.
2.2. Procedure for the synthesis of β-keto esters[40]
In a two-necked flask under argon atmosphere were added the amino alcohol (1 equiv, 1.70
mmol), DMAP (0.25 equiv, 0.42 mmol) and dry toluene (6 mL). Then, tert-butyl acetoacetate
(1.2 equiv, 2.04 mmol) was slowly added and the system was heated under reflux for 24 h.
After the reaction time, the mixture was washed with distilled water (2 x 10 mL), sat. NH4Cl (1
x 10 mL) and sat. NaCl (1 x 10 mL). The organic phase was dried with anhydrous MgSO4,
filtered and the solvent was removed in a rotary evaporator. The crude was purified by column
chromatography with a mixture of EtOAc:hexanes (30:70) as eluent. The compounds 1-4 were
described in previous work.[40]
(R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropyl 3-oxobutanoate (5). White solid in 86
% yield (493.0 mg, 1.46 mmol). Mp: 65 – 67°C. [α]2D0 = 14.37 (c 1.06, CH2Cl2). IR (cm-1): 3386,
3354, 3019, 2978, 2937, 1717, 1685, 1520, 1295, 1152, 699. Keto-enol equilibrium detected.
1H NMR (400 MHz, CDCl3) δ 11.95 (s, 0.1H), 7.32 – 7.26 (m, 2H), 7.24 – 7.17 (m, 3H), 5.04
(s, 0.1H), 4.78 (d, J = 7.6 Hz, 1H), 4.18 (dd, J = 10.5, 3.1 Hz, 1H), 4.08 – 4.02 (m, 2H), 3.50 (s,
2H), 2.87 (dd, J = 13.6, 6.1 Hz, 1H), 2.78 (dd, J = 13.5, 7.6 Hz, 1H), 2.28 (s, 3H), 1.40 (s, 9H).
13C NMR (100 MHz, CDCl3) δ 200.3, 175.8, 172.07, 166.7, 155.0, 137.0, 129.0, 128.3, 126.4,
89.2, 79.2, 65.4, 50.4, 49.6, 37.6, 29.9, 28.1, 21.0. HRMS-ESI calcd for [C18H25NO5+Na]+:
358.1631, found 358.1628.
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(R)-3-(benzylthio)-2-((tert-butoxycarbonyl)-amino)propyl 3-oxobutanoate (6). White solid
in 77 % yield (590.0 mg, 1.55 mmol). Mp: 76 – 79°C. [α]2D0 = 2.28 (c 0.98, CH2Cl2). IR (cm-1):
3377, 3024, 2978, 2923, 1739, 1680, 1519, 1171, 1152, 699. Keto-enol equilibrium detected.
1H NMR (400 MHz, CDCl3) δ 11.92 (s, 0.1H), 7.37 – 7.21 (m, 5H), 4.97 (s, 0.1H), 4.92 (d, J =
7.9 Hz, 1H), 4.29 – 4.23 (m, 2H), 4.07 – 3.99 (m, 1H), 3.74 (s, 2H), 3.46 (s, 2H), 2.58 (dd, J =
13
12.8, 5.2 Hz, 1H), 2.53 (dd, J = 12.8, 6.0 Hz, 1H), 2.25 (s, 3H), 1.45 (s, 9H). C NMR (100
MHz, CDCl3) δ 200.4, 176.2, 172.1, 166.7, 155.2, 137.8, 128.9, 128.5, 127.1, 89.2, 79.7, 65.3,
64.0, 49.8, 48.7, 36.3, 32.6, 30.1, 28.3, 21.2. HRMS-ESI calcd for [C19H27NO5S+Na]+:
404.1508, found 404.1508.
(S)-2-((tert-butoxycarbonyl)amino)-2-phenylethyl 3-oxobutanoate (7). Light yellow solid in
77 % yield (496.0 mg, 1.54 mmol). Mp: 62 – 65°C. [α]2D0 = 28.20 (c 0.98, CH2Cl2). IR (cm-1):
1
3350, 2973, 2918, 2850, 1694, 1528, 1244, 1149, 699. Keto-enol equilibrium detected. H
NMR (400 MHz, CDCl3) δ 11.90 (s, 1H), 7.40 – 7.33 (m, 2H), 7.32 – 7.25 (m, 3H), 5.31 (d, J =
8.2 Hz, 1H), 5.06 – 4.97 (m, 1H), 4.45 – 4.39 (m, 1H), 4.34 (dd, J = 10.7, 6.2 Hz, 1H), 3.44 (s,
2H), 2.19 (s, 3H), 1.43 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 200.3, 172.0, 166.8, 155.1,
138.4, 128.5, 127.6, 126.4, 89.2, 79.5, 67.0, 65.8, 53.2, 49.6, 29.8, 28.1, 21.0. HRMS-ESI
calcd for [C17H23NO5+Na]+: 344.1474, found 344.1484.
2.3. Procedure for the synthesis of 1,2,3-triazoles[40]
In a reaction flask under argon atmosphere were added 2-(4-azidophenyl)benzazole (1.5
equiv, 0.75 mmol), the amino acid-derived -keto ester (1 equiv, 0.50 mmol), dry DMSO (2,0
mL) and finally the catalyst Et2NH (0.1 equiv, 0.05 mmol). The reaction mixture was stirred at
80 °C for 24 h. After this time, DMSO was removed in vacuum and the crude was purified by
column chromatography with a mixture of EtOAc:hexanes (40:60) as eluent.
(S)-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)methyl
1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (8a). Burnt yellow solid in 96 % yield (252.1 mg, 0.48
mmol). Mp: 183 – 187°C. [α]2D0 = -41.35 (c 1.06, CH2Cl2). IR (cm-1): 3055, 2971, 2870, 1696,
1385, 1171, 1095, 965, 842, 763. 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.2 Hz, 2H), 8.13 –
8.07 (m, 1H), 7.96 – 7.90 (m, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.56 – 7.49 (m, 1H), 7.46 – 7.39
(m, 1H), 4.58 – 4.33 (m, 2H), 4.31 – 4.13 (m, 1H), 3.49 – 3.35 (m, 2H), 2.65 (s, 3H), 2.11 –
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13
1.97 (m, 3H), 1.93 – 1.83 (m, 1H), 1.57 – 1.41 (m, 9H). C NMR (100 MHz, CDCl3) δ 165.5,
161.2, 154.4, 154.2, 153.8, 138.8, 138.7, 136.8, 136.7, 136.5, 135.0, 134.9, 128.5, 126.5,
125.6, 125.5, 123.3, 121.6, 79.6, 79.2, 64.8, 55.4, 46.6, 46.3, 28.3, 23.6, 22.7, 10.0. HRMS-
ESI calcd for [C27H29N5O4S+Na]+: 542.1838, found 542.1833.
(S)-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)methyl1-(4-(benzo[d]oxazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (8b). Beige solid in 80 % yield (122.2 mg, 0.24
mmol). Mp: 206 – 210°C. [α]2D0 = -37.72 (c 1.08, CH2Cl2). IR (cm-1): 3063, 2974, 2874, 1722,
1696, 1389, 1174, 1099, 977, 851, 754. 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 8.4 Hz, 2H),
7.85 – 7.78 (m, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.64 – 7.60 (m, 1H), 7.45 – 7.36 (m, 2H), 4.60 –
4.34 (m, 2H), 4.32 – 4.14 (m, 1H), 3.49 – 3.32 (m, 2H), 2.68 (s, 3H), 2.15 – 1.97 (m, 3H), 1.93
13
– 1.84 (m, 1H), 1.55 – 1.42 (m, 9H). C NMR (100 MHz, CDCl3) δ 161.22, 161.15, 154.5,
154.3, 150.8, 141.8, 138.9, 138.8, 137.4, 136.8, 136.7, 128.7, 128.6, 125.7, 125.5, 124.8,
120.2, 110.7, 79.7, 79.2, 64.9, 55.5, 46.7, 46.4, 28.4, 23.7, 22.8, 10.1. HRMS-ESI calcd for
[C27H29N5O5+Na]+: 526.2067, found 526.2073.
(S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyl
1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (9). Dark yellow solid in 83 % yield (215.8 mg, 0.41
mmol). Mp: 193 – 196°C. [α]2D0 = -32.73 (c 1.11, CH2Cl2). IR (cm-1): 3375, 3059, 2971, 1709,
1507, 1158, 1112, 969, 842, 758. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.6 Hz, 2H), 8.14 –
8.10 (m, 1H), 7.97 – 7.93 (m, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.56 – 7.51 (m, 1H), 7.47 – 7.42
(m, 1H), 4.86 (d, J = 9.5 Hz, 1H), 4.49 (dd, J = 11.2, 6.0 Hz, 1H), 4.40 (dd, J = 11.2, 4.0 Hz,
1H), 3.88 – 3.80 (m, 1H), 2.66 (s, 3H), 2.01 – 1.91 (m, 1H), 1.42 (s, 9H), 1.05 – 0.99 (m, 6H).
13C NMR (100 MHz, CDCl3) δ 165.6, 161.4, 155.7, 154.0, 138.9, 137.0, 136.7, 135.18, 135.15,
128.7, 126.7, 125.8, 125.7, 123.6, 121.7, 79.2, 65.4, 54.7, 29.7, 28.3, 19.5, 18.4, 10.2. HRMS-
ESI calcd for [C27H31N5O4S+Na]+: 544.1995, found 544.1995.
(S)-2-((tert-butoxycarbonyl)amino)propyl 1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-methyl-1H-
1,2,3-triazole-4-carboxylate (10). Burnt yellow solid in 93 % yield (229.5 mg, 0.46 mmol). Mp:
192 – 196°C. [α]2D0 = -18.42 (c 0.99, CH2Cl2). IR (cm-1): 3379, 3059, 2975, 2925, 1704, 1519,
1166, 1099, 1066, 969, 842, 758. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.6 Hz, 2H), 8.13 –
8.09 (m, 1H), 7.96 – 7.92 (m, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.56 – 7.51 (m, 1H), 7.47 – 7.41
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(m, 1H), 4.98 – 4.87 (m, 1H), 4.44 – 4.32 (m, 2H), 4.21 – 4.08 (m, 1H), 2.66 (s, 3H), 1.43 (s,
9H), 1.29 (d, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 165.6, 161.4, 155.2, 154.0, 139.0,
137.0, 136.6, 135.2, 135.1, 128.6, 126.6, 125.8, 125.6, 123.5, 121.7, 79.4, 67.7, 45.5, 28.3,
17.7, 10.1. HRMS-ESI calcd for [C25H27N5O4S+Na]+: 516.1682, found 516.1688.
(S)-2-((tert-butoxycarbonyl)amino)-2-phenylethyl
1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (11). Burnt yellow solid in 84 % yield (233.4 mg, 0.42
mmol). Mp: 193 – 198°C. [α]2D0 = 19.40 (c 1.12, CH2Cl2). IR (cm-1): 3382, 3061, 2974, 1704,
1
1162, 1112, 965, 841, 759, 699. H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.5 Hz, 2H), 8.14 –
8.09 (m, 1H), 7.96 – 7.92 (m, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.56 – 7.51 (m, 1H), 7.46 – 7.26
(m, 6H), 5.52 – 5.42 (m, 1H), 5.24 – 5.11 (m, 1H), 4.68 – 4.56 (m, 2H), 2.57 (s, 3H), 1.40 (s,
9H). 13C NMR (100 MHz, CDCl3) δ 165.6, 161.3, 155.2, 154.0, 139.0, 138.8, 137.0, 136.5,
135.17, 135.15, 128.7, 128.6, 127.8, 126.6, 126.5, 125.8, 125.6, 123.5, 121.7, 79.8, 67.1,
53.8, 28.2, 10.1. HRMS-ESI calcd for [C30H29N5O4S+Na]+: 578.1838, found 578.1839.
(S)-2-((tert-butoxycarbonyl)amino)-3-phenyl-propyl 1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (12). Pale yellow solid in 96 % yield (230.5 mg, 0.40
mmol). Mp: 173 – 178°C. [α]2D0 = -8.89 (c 0.96, CH2Cl2). IR (cm-1): 3387, 3059, 2979, 1696,
1
1511, 1221, 1166, 1120, 969, 847, 750, 733, 695. H NMR (400 MHz, CDCl3) δ 8.30 (d, J =
8.4 Hz, 2H), 8.13 – 8.09 (m, 1H), 7.96 – 7.92 (m, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.56 – 7.50 (m,
1H), 7.46 – 7.40 (m, 1H), 7.34 – 7.20 (m, 5H), 5.14 (d, J = 8.1 Hz, 1H), 4.39 – 4.34 (m, 2H),
4.33 – 4.24 (m, 1H), 3.05 – 2.91 (m, 2H), 2.65 (s, 3H), 1.40 (s, 9H). 13C NMR (100 MHz,
CDCl3) δ 165.6, 161.3, 155.2, 154.0, 139.1, 137.2, 136.9, 136.5, 135.1, 135.1, 129.2, 128.6,
128.5, 126.6, 126.5, 125.7, 125.6, 123.5, 121.7, 79.4, 65.5, 50.7, 37.8, 28.2, 10.1. HRMS-ESI
calcd for [C31H31N5O4S+Na]+: 592.1995, found 592.1989.
(R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropyl 1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (13a). Dark yellow solid in 99 % yield (283.7 mg,
0.49 mmol). Mp: 173– 178°C. [α]2D0 = 8.89 (c 0.96, CH2Cl2). IR (cm-1): 3382, 2978, 2923, 1708,
1
1519, 1208, 1167, 1117, 969, 845, 763, 731, 694. H NMR (400 MHz, CDCl3) δ 8.30 (d, J =
8.6 Hz, 2H), 8.13 – 8.09 (m, 1H), 7.95 – 7.92 (m, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.55 – 7.50 (m,
1H), 7.46 – 7.41 (m, 1H), 7.32 – 7.20 (m, 5H), 5.04 (d, J = 8.1 Hz, 1H), 4.38 – 4.34 (m, 2H),
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4.33 – 4.25 (m, 1H), 3.03 – 2.93 (m, 2H), 2.65 (s, 3H), 1.40 (s, 9H). 13C NMR (100 MHz,
CDCl3) δ 165.6, 161.3, 155.2, 154.0, 139.0, 137.2, 136.9, 136.5, 135.12, 135.09, 129.2, 128.6,
128.5, 126.6, 126.5, 125.7, 125.6, 123.5, 121.7, 79.4, 65.5, 50.7, 37.9, 28.2, 10.1. HRMS-ESI
calcd for [C31H31N5O4S+Na]+: 592.1995, found 592.1998.
(R)-2-((tert-butoxycarbonyl)amino)-3-phenylpropyl 1-(4-(benzo[d]oxazol-2-yl)phenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxylate (13b). Dark yellow solid in 80 % yield (84.2 mg, 0.15
mmol). Mp: 198 – 202 °C. [α]2D0 = 3.96 (c 1.17, CH2Cl2). IR (cm-1): 3380, 2980, 2925, 1701,
1
1515, 1209, 1172, 1126, 1056, 853, 746, 692. H NMR (400 MHz, CDCl3) δ 8.52 – 8.44 (m,
2H), 7.84 – 7.78 (m, 1H), 7.70 – 7.60 (m, 3H), 7.45 – 7.37 (m, 2H), 7.33 – 7.19 (m, 5H), 5.11 –
5.01 (m, 1H), 4.40 – 4.35 (m, 2H), 4.34 – 4.24 (m, 1H), 3.05 – 2.92 (m, 2H), 2.67 (s, 3H), 1.40
(s, 9H). 13C NMR (100 MHz, CDCl3) δ 161.3, 161.2, 155.2, 150.8, 141.8, 139.0, 137.4, 137.2,
136.6, 129.2, 128.8, 128.7, 128.5, 126.5, 125.8, 125.5, 124.9, 120.3, 110.7, 79.4, 65.5, 50.7,
37.9, 28.2, 10.2. HRMS-ESI calcd for [C31H31N5O5+Na]+: 576.2223, found 576.2216.
(R)-3-(benzylthio)-2-((tert-butoxycarbonyl)amino)propyl
1-(4-(benzo[d]-thiazol-2-
yl)phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylate (14). Burnt yellow solid in 60% yield
(184.7 mg, 0.30 mmol). Mp: 171 – 175°C. [α]2D0 = -8.01 (c 0.97, CH2Cl2). IR (cm-1): 3386,
1
3061, 2978, 2923, 1707, 1520, 1208, 1167, 1117, 965, 841, 759, 699. H NMR (400 MHz,
CDCl3) δ 8.31 (d, J = 8.6 Hz, 2H), 8.14 – 8.10 (m, 1H), 7.97 – 7.93 (m, 1H), 7.61 (d, J = 8.6
Hz, 2H), 7.57 – 7.52 (m, 1H), 7.47 – 7.42 (m, 1H), 7.36 – 7.21 (m, 5H), 5.14 (d, J = 8.0 Hz,
1H), 4.59 (dd, J = 11.2, 4.2 Hz, 1H), 4.42 (dd, J = 11.2, 4.3 Hz, 1H), 4.25 – 4.17 (m, 1H), 3.78
13
(s, 2H), 2.74 – 2.68 (m, 2H), 2.63 (s, 3H), 1.45 (s, 9H). C NMR (100 MHz, CDCl3) δ 165.6,
161.2, 155.3, 154.0, 139.2, 137.9, 137.0, 136.5, 135.22, 135.20, 129.0, 128.7, 128.5, 127.1,
126.7, 125.8, 125.7, 123.6, 121.8, 79.7, 65.2, 48.9, 36.4, 32.7, 28.3, 10.1. HRMS-ESI calcd for
[C32H33N5O4S2+Na]+: 638.1872, found 638.1869.
2.4 Procedure for the N-Boc deprotection[40]
In a reaction flask under air atmosphere were added the 1,2,3-triazolic compound with the N-
Boc group (1 equiv, 0.30 mmol), dichloromethane (1.0 mL) and, then, TFA (13 equiv, 0.70
mmol) was added slowly. The reaction mixture was stirred at room temperature for 2 h. Then,
the volatile substances were evaporated and the residue was dissolved in dichloromethane,
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and the solvent was evaporated (this process was repeated three times). The result oil was
dissolved in dichloromethane (1.0 mL) and K2CO3 (3.6 equiv, 1.08 mmol) was added. The
mixture was stirred at room temperature for 2 h. After this time, the solids were separated by
filtration and the filtrate was evaporated. The crude was purified by column chromatography
using a mixture of AcOEt:Et3N:hexanes (79:1:20).
(S)-pyrrolidin-2-ylmethyl 1-(4-(benzo[d]thiazol-2-yl)phenyl)-5-methyl-1H-1,2,3-triazole-4-
carboxylate (15). Beige solid in 81 % yield (67.9 mg, 0.16 mmol). Mp: 152 – 157°C. [α]2D0 = -
42.82 (c 0.97, CH2Cl2). IR (cm-1): 3408, 2953, 2920, 1725, 1591, 1444, 1053, 960, 847, 758.
1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.5 Hz, 2H), 8.12 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 7.9
Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.57 – 7.51 (m, 1H), 7.47 – 7.42 (m, 1H), 5.03 – 4.94 (m,
1H), 4.55 – 4.44 (m, 1H), 4.40 – 4.32 (m, 1H), 4.15 – 4.02 (m, 1H), 3.83 – 3.72 (m, 2H), 2.64
(s, 3H), 2.20 – 2.10 (m, 1H), 2.04 – 1.97 (m, 1H), 1.95 – 1.87 (m, 1H), 1.77 – 1.66 (m, 1H). 13C
NMR (100 MHz, CDCl3) δ 165.8, 163.2, 154.0, 140.3, 138.3, 137.3, 135.2, 135.0, 128.6,
126.6, 125.8, 125.7, 123.5, 121.7, 67.3, 62.2, 50.4, 28.0, 25.0, 10.5. HRMS-ESI calcd for
[C22H21N5O2S+Na]+: 442.1314, found 442.1324.
(S)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(1-hydroxy-3-methylbutan-2-yl)-5-methyl-1H-
1,2,3-triazole-4-carboxamide (16). Pale yellow solid in 79 % yield (66.8 mg, 0.16 mmol). Mp:
160 – 164°C. [α]2D0 = -35.36 (c 0.94, CH2Cl2). IR (cm-1): 3480, 3391, 3055, 2958, 2925, 2869,
1650, 1511, 1482, 969, 838, 758. 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.6 Hz, 2H), 8.11 –
8.07 (m, 1H), 7.92 – 7.87 (m, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.54 – 7.48 (m, 2H), 7.43 – 7.38
(m, 1H), 4.04 – 3.96 (m, 1H), 3.87 (dd, J = 11.3, 3.6 Hz, 1H), 3.81 (dd, J = 11.3, 6.3 Hz, 1H),
3.61 (bs, 1H), 2.67 (s, 3H), 2.13 – 2.00 (m, 1H), 1.06 (d, J = 4.8 Hz, 3H), 1.04 (d, J = 4.8 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 165.6, 161.8, 153.9, 138.6, 137.1, 136.7, 135.0, 134.8,
128.5, 126.5, 125.6, 125.4, 123.4, 121.6, 63.5, 56.8, 29.1, 19.5, 18.7, 9.8. HRMS-ESI calcd for
[C22H23N5O4S+Na]+: 444.1470, found 444.1461.
(S)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(1-hydroxypropan-2-yl)-5-methyl-1H-1,2,3-
triazole-4-carboxamide (17). Beige solid in 76 % yield (59.9 mg, 0.15 mmol). Mp: 196 –
200°C. [α]2D0 = -26.52 (c 0.94, CH2Cl2). IR (cm-1): 3475, 3387, 3063, 2971, 2916, 2870, 1642,
1
1507, 1490, 998, 964, 838, 754. H NMR (400 MHz, CDCl3) 8.27 (d, J = 8.7 Hz, 2H), 8.11 –
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8.07 (m, 1H), 7.93 – 7.89 (m, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.54 – 7.49 (m, 1H), 7.46 (d, J =
7.8 Hz, 1H), 7.44 – 7.39 (m, 1H), 4.37 – 4.26 (m, 1H), 3.81 (dd, J = 11.0, 3.7 Hz, 1H), 3.70
(dd, J = 11.0, 6.2 Hz, 1H), 3.63 (bs, 1H), 2.67 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 165.7, 161.5, 153.9, 138.5, 137.1, 136.8, 135.1, 134.9, 128.6, 126.6, 125.7,
125.5, 123.5, 121.6, 66.8, 47.6, 17.1, 9.8. HRMS-ESI calcd for [C20H19N5O2S+Na]+: 416.1157,
found 416.1159.
(S)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(1-hydroxy-3-phenylpropan-2-yl)-5-methyl-1H-
1,2,3-triazole-4-carboxamide (18). Beige solid in 76 % yield (71.8 mg, 0.15 mmol). Mp: 214 –
219°C. [α]2D0 = -49.42 (c 1.00, CH2Cl2). IR (cm-1): 3459, 3412, 3025, 2925, 1654, 1506, 1490,
1
1053, 960, 838, 758, 670. H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.6 Hz, 2H), 8.14 – 8.09
(m, 1H), 7.96 – 7.92 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.56 – 7.51 (m, 2H), 7.47 – 7.41 (m,
1H), 7.35 – 7.28 (m, 4H), 7.26 – 7.21 (m, 1H), 4.46 – 4.36 (m, 1H), 3.83 (dd, J = 11.2, 3.7 Hz,
1H), 3.72 (dd, J = 11.2, 5.4 Hz, 1H), 3.05 (dd, J = 11.9, 5.4 Hz, 1H), 3.00 (dd, J = 11.9, 5.3 Hz,
1H), 2.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.8, 161.6, 154.0, 138.6, 137.6, 137.2,
136.8, 135.2, 135.1, 129.3, 128.7, 128.6, 126.7, 126.6, 125.8, 125.6, 123.6, 121.8, 64.3, 52.9,
37.3, 9.9. HRMS-ESI calcd for [C26H23N5O2S+Na]+: 492.1470, found 492.1478.
(R)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(1-hydroxy-3-phenylpropan-2-yl)-5-methyl-1H-
1,2,3-triazole-4-carboxamide (19). Beige solid in 84 % yield (79.3 mg, 0.17 mmol). Mp: 214 –
219°C. [α]2D0 = 49.42 (c 0.97, CH2Cl2). IR (cm-1): 3459, 3413, 3029, 2923, 1653, 1506, 1483,
1
1052, 960, 841, 754, 704. H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.6 Hz, 2H), 8.13 – 8.10
(m, 1H), 7.96 – 7.92 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.56 – 7.52 (m, 2H), 7.47 – 7.42 (m,
1H), 7.35 – 7.29 (m, 4H), 7.27 – 7.21 (m, 1H), 4.46 – 4.37 (m, 1H), 3.83 (dd, J = 11.2, 3.6 Hz,
1H), 3.72 (dd, J = 11.2, 5.4 Hz, 1H), 3.05 (dd, J = 11.9, 5.4 Hz, 1H), 3.00 (dd, J = 11.9, 5.3 Hz,
1H), 2.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.76, 161.62, 154.04, 138.54, 137.57,
137.17, 136.81, 135.20, 135.05, 129.27, 128.67, 128.65, 126.69, 126.65, 125.79, 125.57,
123.58, 121.75, 64.30, 52.93, 37.27, 9.87. HRMS-ESI calcd for [C26H23N5O2S+Na]+: 492.1470,
found 492.1466.
(R)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(1-(benzylthio)-3-hydroxypropan-2-yl)-5-methyl-
1H-1,2,3-triazole-4-carboxamide (20). Pale yellow solid in 88 % yield (77.1 mg, 0.15 mmol).
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Mp: 155 – 160°C. [α]2D0 = -28.09 (c 0.96, CH2Cl2). IR (cm-1): 3460, 3409, 3024, 2960, 2928,
2873, 1643, 1515, 1483, 969, 837, 759. 1H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 8.7 Hz, 2H),
8.14 – 8.11 (m, 1H), 7.98 – 7.94 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.7 Hz, 2H),
7.57 – 7.52 (m, 1H), 7.48 – 7.43 (m, 1H), 7.38 – 7.29 (m, 4H), 7.26 – 7.22 (m, 1H), 4.34 – 4.24
(m, 1H), 3.91 – 3.82 (m, 2H), 3.81 (s, 2H), 2.79 (dd, J = 11.2, 4.2 Hz, 1H), 2.75 (dd, J = 11.2,
13
4.4 Hz, 1H), 2.70 (s, 3H), 2.68 – 2.62 (m, 1H). C NMR (100 MHz, CDCl3) δ 165.8, 161.5,
154.0, 138.5, 137.9, 137.2, 136.9, 135.2, 135.1, 129.0, 128.7, 128.57, 127.1, 126.7, 125.8,
125.6, 123.6, 121.8, 64.0, 50.6, 36.6, 32.3, 9.9. HRMS-ESI calcd for [C27H25N5O2S2+Na]+:
538.1348, found 538.1334.
(S)-1-(4-(benzo[d]thiazol-2-yl)phenyl)-N-(2-hydroxy-1-phenylethyl)-5-methyl-1H-1,2,3-
triazole-4-carboxamide (21). Pale yellow solid in 73 % yield (33.5 mg, 0.07 mmol). Mp: 187 –
191°C. [α]2D0 = 6.76 (c 1.10, CH2Cl2). IR (cm-1): 3488, 3414, 3056, 3032, 2933, 2877, 1653,
1506, 1487, 960, 841, 759, 694. 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.5 Hz, 2H), 8.13 –
8.09 (m, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.95 – 7.92 (m, 1H), 7.60 (d, J = 8.5 Hz, 2H), 7.56 –
7.51 (m, 1H), 7.46 – 7.41 (m, 3H), 7.40 – 7.36 (m, 2H), 7.33 – 7.28 (m, 1H), 5.34 – 5.27 (m,
1H), 4.02 (d, J = 5.4 Hz, 2H), 3.05 (bs, 1H), 2.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.7,
161.5, 154.0, 138.9, 138.5, 137.1, 137.0, 135.2, 135.0, 128.9, 128.7, 127.9, 126.8, 126.7,
125.8, 125.6, 123.6, 121.7, 66.5, 55.8, 9.9. HRMS-ESI calcd for [C25H21N5O2S+Na]+:
478.1314, found 478.1313.
2.5. Absorption and emission analysis with CT-DNA
For DNA binding assays, amino-acid compounds interactions with calf-thymus DNA (CT-DNA)
were performed by UV-vis absorption measurements at room temperature in Tris-HCl buffer at
pH 7.4, using DMSO stock solution of derivatives (10-4 to 10-5 M range) at 250 to 500 nm
range. The DNA pair base concentrations of low molecular weight DNA from calf thymus (CT-
DNA) was determined by absorption spectroscopy, using the molar extinction coefficients
13,200 M−1·cm−1 (per base pair) at abs = 260 nm, respectively. Derivative solutions in DMSO
(2%)/Tris-HCl buffer mixture were titrated with increasing concentrations of CT-DNA (ranging
from 0 to 100 μM). The intrinsic binding constants (Kb) of derivatives were calculated
according to the decay of the absorption bands of compounds using the following Equation (1)
through a plot of [DNA]/(εa − εf ) versus [DNA]:
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[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/Kb(εb − εf )
(1)
where [DNA] is the concentration of DNA in the base pairs, εa is the extinction coefficient
(Aobs/[compound]), εb and εf are the extinction coefficients of free and fully bound forms,
respectively. In plots of [DNA]/(εa − εf) versus [DNA], Kb is given by the ratio of the slope to the
interception.
For EB-DNA competition assays, steady-state emission fluorescence analysis was recorded
and amino-acid derivatives were dissolved in DMSO and competitive studies were performed
through the gradual addition of the DMSO stock solution of the derivatives to the quartz
cuvette (1.0 cm path length) containing ethidium bromide (EB, 2.0 x 10-7 M) and DNA (2.0 x
10-5 M) in a Tris-HCl pH 7.4 buffer solution. Derivative concentrations ranged from 0 to 100
μM. All compounds were excited at λexc = 510 nm and emission spectra were recorded at the
range of 550-800 nm, 5 min after each addition of the complex solution in order to allow
incubation to occur. The fluorescence quenching Stern-Volmer (KSV) and quenching rate
Stern-Volmer (kq) constants of compounds were calculated according to the decay of the
emission bands of EB-DNA using the following Equation (2) through a plot of F0/F versus
[DNA],
F0/F = 1+ kq0 [Q] = 1 + KSV[Q]
(2)
where F0 and F are the fluorescence intensities in the absence and presence of a quencher,
respectively. KSV, kq, 0 and [Q] denote Stern-Volmer quenching constant, quenching rate
constant, lifetime of EB-DNA adducts (0 =23 ns)[47, 48] and the concentration of quencher,
respectively. According to Equation (2), the Stern-Volmer values (KSV) were calculated from
the slope and kq is equal KSV/0.
In order to quantify the displacement, the concentration of the amino-acid derivatives at which
ethidium bromide dye (EB) fluorescence decreases by 50% (in this case, assumed to be 50%
displacement of EB) is calculated.[49] The values of apparent DNA-binding constant (Kapp)
were calculated using the Equation (3):
KEB[47] = Kapp[compound] (3)
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where KEB (4.94 × 105 M−1) is the DNA-binding constant of ethidium bromide, [47] is the
concentration of EB (1.50 x 10-6 M), and [compound] is the concentration of the derivative
used to obtain 50% reduction in fluorescence emission intensity of EB dye.
2.6. Steady-state emission BSA-binding assays
For emission BSA experiments, amino-acid compounds interactions with bovine serum
albumin (BSA) were performed by steady-state emission fluorescence measurements at room
temperature in Tris-HCl buffer at pH 7.4, using DMSO stock solution of derivatives (10-5 M
range) at 300 to 550 nm region. To analyze the data from the quenching experiments, we
used the Stern–Volmer Equation (4):
F0/F = 1+ kq0 [Q] = 1 + KSV[Q]
(4)
where F0 and F are the fluorescence intensities in the absence and presence of a quencher,
respectively. KSV, kq, 0 and [Q] denote Stern-Volmer constant, quenching rate constant, the
original lifetime of BSA (1.00 x 10-8 s)[50] and the concentration of quencher, respectively.
According to Equation (4), the Stern–Volmer constants (KSV) were calculated from the slope
and kq is equal KSV/0. It is generally accepted that the maximum Stern–Volmer constant
allowed for the dynamic quenching of biomolecules is 100 M-1. To conclude therefore that the
quenching is not a dynamic but static process occurring in the compound–BSA adduct, Stern-
Volmer-Scatchard Equation (5) predicted the Kapp (apparent binding constant) and n (number
of binding site):
log (F0 – F)/F = log Kapp + n log [Q]
(5)
where F0 and F represent fluorescence intensities in the absence and presence of chalcogen
derivatives, respectively, and [Q] the concentration of the quencher. According to Equation (5),
the binding constants (Kb) were calculated from the interception and the number of binding
sites (n) by the slope, respectively.
2.7. Molecular docking calculation
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The crystallographic structures of DNA and BSA were obtained from the Protein Data Bank
(PDB code 1BNA and 4F5S, respectively).[51, 52] The structure of the amino acid derivatives
were built and energy-minimized by Density Functional Theory (DFT), method Becke-3-Lee
Yang Parr (B3LYP) with standard 6-31G* basis set, available in the Spartan'14 program
(Wavefunction Inc.). Molecular docking was performed with GOLD 5.6 program (CCDC).[53]
Hydrogen atoms were added to the macromolecules according to the data inferred by the
program on the ionization and tautomeric states. For DNA structure it was defined a spherical
cavity of 5.0 Å radius around the double helix of DNA. While, for BSA a 10 Å radius spherical
cavity around Trp-134, and Trp-212 residues (subdomain IB and IIA, respectively) were
defined and molecular docking calculations for each compound were carried out. The scoring
function used was ‘ChemPLP’ for both macromolecules, which is the default function of the
GOLD 5.6 program. The score of each pose identified was calculated as the negative of the
sum of a series of energy terms involved in the macromolecule-compound interaction process,
so that the more positive the score, better is the interaction. The figures of the docking poses
for the largest docking score value were generated with the PyMOL Delano Scientific LLC
program.[54]
3 Results and Discussion
3.1 Synthesis
At the outset, the synthesis of the required amino acid-derived β-keto esters 1-7 was
performed. The amino acid derivatives were obtained through a reaction sequence involving:
(a) esterification of the acid group with thionyl chloride in MeOH, (b) Boc protection of the free
amino group, (c) ester reduction with LiAlH4, and (d) transesterification of the corresponding
amino alcohol with t-butyl acetoacetate. In this way, the desired products were synthesized in
good overall yields (Scheme 2).
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Scheme 2. Started β-keto esters 1-7 synthesized. Reagents and conditions: (a) SOCl2 (2 equiv.), MeOH, 0°C -
RT 12 h; (b) Et3N (2.18 equiv.), Boc2O (1.1 equiv.), CH2Cl2, 0°C - RT 12 h; (c) LiAlH4 (3 equiv.), THF, 0°C - RT 12
h, argon atmosphere; (d) t-butyl acetoacetate (1.2 equiv.), DMAP (0.25 equiv.), toluene, reflux 24 h, argon
atmosphere.
With the required β-keto esters in hands, we have proceeded to the synthesis of the 1,2,3-
triazole unit using the methodology developed in our previous work,[40] employing 10 mol% of
diethylamine as the catalyst and DMSO as solvent. Under these conditions the reaction of the
β-keto esters with 2-(4‘-azidophenyl)-benzothiazole and 2-(4‘-azidophenyl)-benzoxazole
produced a series of amino acid-derived 1,2,3-triazoles 8a-14 in very good yields (80 - 95%
yield), as depicted in Scheme 3a. The reaction tolerated the presence of side chains with
different frameworks, such as a pyrrolidine ring (8a-b, derived from L-proline), branched alkyl
(9, from L-valine), methyl (10, from L-alanine), phenyl (11, from L-phenylglycine), benzyl (12,
from L-phenylalanine and 13a-b, from D-phenylalanine), and a sulfur-functionalized alkyl side-
chain (14, from L-cysteine).
In order to increase the scope of the fluorophores for the photophysical studies, we have also
performed the deprotection of the Boc group, by the reaction of N-Boc-protected derivatives
with trifluoroacetic acid (Scheme 3). Deprotection of proline derivative 8a proceeded
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uneventfully, yielding compound 15 in a very good yield, after neutralization of the resulting
salt with K2CO3 (Scheme 3b). On the other hand, to compounds bearing an acyclic side-chain,
the deprotection was accompanied by an ester-to-amide rearrangement upon exposure to
trifluoroacetic acid. The reaction proceeded through an O-N acyl intramolecular migration
(Scheme 3c).[55, 56] All rearranged products 16-21 have been obtained in very good yields
and with complete retention of the stereochemistry of the stereogenic center, as evidence by
chiral HPLC (see supporting information).
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Scheme 3. a. Synthesis of the 1,2,3-triazole unit scope, b. Deprotection of the Boc group and c. Ester-to-amide
rearrangement.
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3.2 Photophysics
The photophysical investigation in solution of the all synthesized amino acid derivatives was
carried out using different organic solvents with different dielectric constant values (1,4-
dioxane, dichloromethane, methanol and acetonitrile). The relevant data from this
characterization can be found in Tables 1 and 2. Figure 1 and 2 show the UV-Vis spectra of
two sets of compounds, which were chosen as protected and unprotected model derivatives
for this discussion. All original data of the additional compounds are presented as supporting
information (Figures S42-S43) and presented similar behaviour.
Figures 1 and 2 indicate that the amino acid derivatives present absorption maxima located
around 300 nm, related to the benzazolic unity, with any tendency on the solvent polarity,
which can be related to an almost absent charge transfer character in the ground state. In
addition, similar shape and position were found between the protected (Figure 1) and
unprotected (Figure 2) derivatives.
Figure 1. UV-Vis absorption spectra in solution of different organic solvents [ca. 10-6 M] of the protected amino
acid derivatives (a) 8a, (b) 8b, (c) 13a and (d) 13b.
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Figure 2. UV-Vis absorption spectra in solution of different organic solvents [ca. 10-6 M] of the unprotected amino
acid derivatives (a) 17, (b) 20 and (c) 21.
From the relationship between absorption intensity and fluorescence lifetime of molecules,
published by Strickler and Berg, the UV-Vis spectra was also used to obtain the respective
0
theoretical rate constant for emission (ke ), as well as the respective oscillator strengths (fe)
from Equations (1) and (2), respectively.[57] In these equations, the area under the absorption
̅
curve from a plot of the molar absorptivity coefficient  (M-1·cm-1) against wavenumber 푣 (cm-1)
0
can be related to the respective theoretical rate constant for emission (ke ) and the oscillator
0
strength (fe). In addition, the pure radiative lifetime 0 can also be defined as 1/ke .[58]
푘푒0 ≈ 2.88푥10−9푣02
̅
∫ 휀푑푣
̅
(1)
푓 ≈ 4.3푥10−9 ∫ 휀푑푣
̅
(2)
푒
In this regard, taking the molar absorptivity coefficient (), as well as the calculated radiative
0
rate constants (ke ) values into account (Table 1) fully spin and symmetry allowed electronic
transitions were observed for these compounds, ascribed to * transitions. In addition, the
same magnitude of the radiative lifetime indicates that after the radiation absorption the amino
acid derivatives populate the same excited state. Calculated values for 0 range from 1.76 to
3.00 ns for 1,4-Dioxane, 1.97 to 2.83 ns in Dichloromethane, 1.72 to 2.81 ns in methanol and
from 1.38 to 2.78 ns in Acetonitrile.
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Table 1 Photophysical data of the protected amino acid derivatives 8a-14, where  is the molar extinction
coefficient (x104 M-1·cm-1), abs and em are the absorption and emission maxima (nm), respectively, fe is the
0
calculated oscillator strength, ke is the calculated radiative rate constant (108 s-1), 0 is the calculated pure
radiative lifetime (ns), ST is the Stokes shift (nm/cm-1) and FL is the fluorescence quantum yield (%).

풌풆ퟎ
5.48
4.79
3.98
5.09
4.46
3.64
4.47
7.26
5.69
3.54
4.89
5.23
5.11
4.28
4.07
4.18
5.34
4.01
5.51
5.61
5.60
4.55
5.80
4.03
3.33
3.61
4.12
4.12

FL
4.6
4.6
4.4
4.1
74
Compound
Solvent
1,4-Dioxane
CH2Cl2
풇풆
0.76
0.67
0.55
0.70
0.62
0.51
0.61
0.98
0.79
0.49
0.67
0.72
0.71
0.60
0.56
0.58
0.75
0.56
0.75
0.77
0.79
0.64
0.80
0.56
0.47
0.51
0.57
0.57
abs
305
307
304
304
304
306
301
301
305
306
304
304
305
307
305
304
307
307
302
303
307
307
303
304
307
308
305
304
 em
370
376
378
380
360
361
361
359
374
377
380
379
371
373
376
375
370
374
376
376
372
377
379
381
371
374
379
377
ST
65/5760 2.28
69/5978 2.24
74/6440 1.99
76/6579 2.16
57/5117 2.43
55/4979 2.53
60/5522 2.53
58/5367 4.04
69/6049 2.35
71/6155 2.01
76/6579 2.12
75/6510 2.23
66/5833 2.36
66/5764 2.20
71/6191 1.92
71/6228 2.00
63/5546 2.39
67/5835 2.07
74/6517 2.45
73/6408 2.32
65/5692 2.32
70/6048 2.16
76/6618 2.36
77/6648 1.91
64/5619 2.03
67/5730 1.91
74/6402 1.94
73/6370 1.92
1.82
2.09
2.51
1.97
2.24
2.75
2.24
1.38
1.76
2.83
2.04
1.91
1.96
2.34
2.46
2.39
1.87
2.49
1.81
1.78
1.79
2.20
1.72
2.48
3.00
2.77
2.43
2.43
8a
MeOH
MeCN
1,4-Dioxane
CH2Cl2
73
8b
MeOH
79
MeCN
41
1,4-Dioxane
CH2Cl2
4.3
4.9
4.1
3.8
3.6
3.8
3.7
3.3
4.3
4.5
3.3
3.5
4.5
4.4
3.2
4.2
5.1
4.9
3.9
3.8
9
MeOH
MeCN
1,4-Dioxane
CH2Cl2
10
11
12
13a
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
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1,4- Dioxane
CH2Cl2
304
360
362
363
363
371
377
376
380
56/5117 2.40
0.56
0.57
0.60
0.49
0.50
0.72
0.69
0.66
4.03
4.10
4.41
3.59
3.54
5.09
4.94
4.84
2.48
2.44
2.27
2.78
2.83
1.97
2.02
2.07
70
304
303
303
308
308
306
303
58/5270 2.50
60/5455 2.42
60/5455 2.20
63/5513 1.92
69/5942 2.33
70/6084 2.22
77/6688 2.05
61
13b
14
MeOH
72
MeCN
79
1,4-Dioxane
CH2Cl2
5.4
4.3
3.9
3.8
MeOH
MeCN
Table 2 Photophysical data of the unprotected amino acid derivatives 15-21, where  is the molar extinction
coefficient (x104 M-1·cm-1), abs and em are the absorption and emission maxima (nm), respectively, fe is the
0
calculated oscillator strength, ke is the calculated radiative rate constant (108 s-1), 0 is the calculated pure
radiative lifetime (ns), ST is the Stokes shift (nm) and FL is the fluorescence quantum yield (%).
풌풆ퟎ
4.34
3.60
3.56
4.14
3.75
4.10
4.18
4.34
4.25
3.98
5.18
4.43
3.87
3.65
4.50
5.07
5.27
4.09
3.57
4.44
Compound
Solvent
abs
304
305
303
304
304
307
304
302
304
306
304
303
304
307
304
303
307
307
306
302
em
ST

풇풆

FL
4.4
4.4
4.0
3.4
4.5
3.9
3.6
3.5
3.8
3.9
3.1
3.1
4.3
4.2
3.6
3.1
4.6
4.6
4.4
4.1
1,4-Dioxane
CH2Cl2
368
371
375
373
368
372
375
374
368
374
376
374
370
371
375
374
368
375
374
375
64/5721 2.06
66/5833 1.90
72/6337 1.87
69/6085 2.03
64/5721 1.98
65/5692 2.15
71/6228 2.02
72/6375 2.08
64/5721 2.13
68/5942 2.18
72/6299 2.35
71/6265 2.19
66/5868 2.02
64/5619 2.03
71/6228 2.19
71/6265 2.30
62/5399 2.58
68/5907 2.11
68/5942 1.96
73/6446 2.06
0.60
0.50
0.49
0.57
0.52
0.58
0.58
0.59
0.59
0.56
0.71
0.61
0.53
0.51
0.62
0.70
0.74
0.58
0.50
0.60
2.30
2.78
2.81
2.41
2.67
2.44
2.39
2.30
2.35
2.51
1.93
2.26
2.58
2.74
2.22
1.97
1.90
2.44
2.80
2.25
15
MeOH
MeCN
1,4-Dioxane
CH2Cl2
16
17
18
19
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
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1,4-Dioxane
CH2Cl2
306
368
372
375
374
369
370
376
373
62/5506 2.59
0.65
0.67
0.77
0.75
0.76
0.58
0.68
0.54
4.62
4.81
5.55
5.39
5.42
4.13
4.95
3.90
2.17
2.08
1.80
1.86
1.85
2.42
2.02
2.56
4.2
4.1
3.4
3.4
3.8
4.1
3.5
3.5
306
305
305
307
308
304
305
66/5798 2.48
70/6120 2.55
69/6049 2.53
62/5473 2.60
62/5441 2.32
70/6299 2.36
68/5977 2.05
20
21
MeOH
MeCN
1,4-Dioxane
CH2Cl2
MeOH
MeCN
The first set of fluorescence emission curves were obtained by exciting the compounds at the
absorption maxima (Table 1). Both protected (Figure 3) and unprotected (Figure 4)
compounds showed a main fluorescence emission in the UV-A region (368-375 nm). It could
be possible to calculate Stokes shift values in the range of 55 to 76 nm, indicating that these
compounds present a significant energy loss in the excited state. Due to their electronic
characteristics, any tendency on the emission maxima position related to the solvent polarity
was observed, which can be related to an almost absent charge transfer character in the
excited state. All original data of the additional compounds are presented as supporting
information (Figures S44-S45) and presented similar behaviour.
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Figure 3. Steady-state fluorescence emission spectra (Exc./Em. slits 3.0nm/3.0nm) in solution of different
organic solvents [ca. 10-6 M] of the protected amino acid derivatives (a) 8a, (b) 8b, (c) 13a and (d) 13b.
Figure 4. Steady-state fluorescence emission spectra (Exc./Em. slits 3.0nm/3.0nm) in solution of different
organic solvents [ca. 10-6 M] of the unprotected amino acid derivatives (a) 17, (b) 20 and (c) 21.
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It is worth mentioning that the calculated values for the fluorescence quantum yields of the
protected benzothiazole based amino acid derivatives were found around 4%, while for the
benzoxazole analogues, the values ranged from 40 to 80%. Such huge difference cannot be
related to the amino acid units, or even solvent effects since both set of compounds
(benzothiazole and benzoxazole) present the same chemical groups, as well as electronic
structures, except for the heteroatom present in the benzazole moiety. In this sense, it is
believed that for this class of compounds, the sulfur atom plays a quite decisive role on the
excited state non-radiative deactivation. In addition, it was also observed that the protected
derivatives such as 13a, with very low fluorescence quantum yields may appear to be in
violation of Kasha’s rule of excitation wavelength independence of the emission spectrum
(Figure 5), as already observed in similar compounds.[59] On the other hand, as expected and
to corroborate with this proposal, the oxygenated derivatives 13b, for instance, which
presented very high fluorescence quantum yields show any shift on the emission maxima
location by changing the excitation wavelength (Figure 5). In general, the benzothiazole
derivatives presented the same violation of Kasha’s rule as presented in the supporting
information (Figures S46-S54). In this case, it is believed that in these compounds the sulfur
atom is playing a key role on the deactivation of the excited state, does not allowing a rapid
internal conversion and a vibrational relaxation warrant that the corresponding emission
comes from the first excited state in spite of which electronic state of equal multiplicity was
excited. [60] The excitation spectra (data not shown, see supporting information (Figures S55-
S56) corroborates with the assumption that the benzothiazole derivatives probably violated the
Kasha's rule, where quite different spectra were found when observed at different emission
maxima.
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Figure 5. Steady-state fluorescence emission spectra (Exc./Em. slits 3.0nm/3.0nm) in solution [ca. 10-6 M] at
different excitation wavelengths of the amino acid derivatives 13a (top) and 13b (bottom) in dichloromethane (a
and c) and acetonitrile (b and d).
3.3. DNA-binding assay
The interaction of the synthesized amino acid derivatives with calf-thymus DNA (CT-DNA) was
performed by UV-Vis spectroscopy in DMSO (2%)/Tris-HCl buffer solution (pH 7.4), because
all compounds are poorly soluble in pure aqueous media. This investigation was performed
taking the compounds 8a, with Boc protective group and 15 presenting the N-H moiety into
account, as representative chemical structures of the compounds synthesized in this study
(Figure 6). However, it could be observed that all synthesized compounds (8a-14 and 15-21)
presented interaction with CT-DNA by changes on the UV-Vis absorption spectra (see
Supporting Information Figures S57-S71). The relevant data for all compounds are
summarized in Table 3 and all experiments are due in duplicate.
In general, upon increase of the CT-DNA concentration (0-100 μM), both amino acid
derivatives show hyperchromicity in the UV region between 250-350 nm. Moreover, slightly
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bathochromic shift was also observed in all cases probably related to the intercalative grooves
of the DNA and amino acid derivatives. These changes could also be accounted by the
interaction of the aromatic structure of the molecule, probably via hydrogen-bonding and/or
hydrophobic forces (π-stacking interactions) with the CT-DNA bases and aromatic structure
moiety of derivatives, as previously reported by Rakesh and co-workers.[61] The intrinsic
binding constants (Kb) of the compounds were calculated as summarized in Table 3. In the
present study, the amino acid derivatives showed strong binding forces to CT-DNA, with
values between 105 and 106 M-1, following the increasing order of Kb values: 8a-14, with Boc
protective group < 15-21, without Boc, indicating the role of this protective group on the
interaction with CT-DNA.
Figure 6. UV-Vis titration absorption spectra of amino acid derivatives 8a (a) and 15 (b), in a DMSO(2%)/Tris-HCl
buffer (pH 7.4) solution. The concentration of CT-DNA ranged from 0 to 100 μM. Insert graph shows the plot of
[DNA]/(εa – εf) versus [DNA] with R=0.99755 (8a) and 0.99683 (15).
Table 3. Hyperchromicity (H%), bathochromic shift (), Intrinsic binding constant (Kb), Stern-Volmer quenching
constant (KSV), quenching rate constant (kq), apparent binding constant (Kapp) and the number of binding sites (n)
values for the interactions of amino-acid compounds with calf thymus DNA (CT-DNA).
Absorption-DNA
Emission EB-DNA
Kapp
(M-1)g
Compound
H
(%)a

(nm)b
6.0
Kb
(M-1)c
1.72(±0.16)x105
9.30(±0.69)x105
Q
(%)d
KSV
(M-1)e
5.26(±0.20)x102
6.34(±0.30)x102
kq
(M-1s-1)f
5.26(±0.26)x1010
6.34(±0.26)x1010
8a
8b
84.10
87.62
6.72
6.37
1.64(±0.13)x104
9.18(±0.22)x104
0.0
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9.75(±0.69)x105
5.65
6.18
11.97
5.14
5.49
14.23
9.73
6.24
3.15
5.76
6.13
5.34
4.29
5.94
6.05(±0.30)x102
6.05(±0.26)x1010
4.49(±0.15)x1010
6.62(±0.15)x1010
5.32(±0.30)x1010
5.60(±0.03)x1010
1.24(±0.03)x1011
1.05(±0.03)x1011
6.46(±0.20)x1010
3.29(±0.20)x1010
3.79(±0.40)x1010
4.44(±0.70)x1010
3.37(±0.40)x1010
1.70(±0.40)x1010
5.93(±0.40)x1010
9.11(±0.25)x104
1.60(±0.08)x104
8.13(±0.60)x104
7.42(±0.16)x104
3.33(±0.10)x104
4.46(±0.20)x104
4.75(±0.12)x104
2.06(±0.12)x104
1.01(±0.18)x104
2.22(±0.04)x104
1.41(±0.35)x104
6.13(±0.20)x104
3.71(±0.20)x104
1.55(±0.09)x104
9
10
11
12
13a
13b
14
15
16
17
18
19
20
21
87.04
82.46
85.06
81.69
83.31
83.30
84.85
83.13
82.77
80.65
82.16
85.28
83.70
83.21
6.0
5.0
4.0
5.0
5.0
6.0
4.0
4.0
7.0
5.0
4.0
5.0
6.0
6.0
1.51(±0.10)x105
8.35(±0.80)x105
7.40(±0.15)x105
3.44(±0.15)x105
4.57(±0.20)x105
4.79(±0.20)x105
2.34(±0.30)x106
1.10(±0.25)x106
2.10(±0.30)x106
1.61(±0.30)x106
6.29(±0.50)x105
3.68(±0.60)x106
1.67(±0.60)x106
4.49(±0.40)x102
6.62(±0.10)x102
5.32(±0.10)x102
5.60(±0.10)x102
1.24(±0.10)x103
1.05(±0.15)x103
6.46(±0.10)x102
3.29(±0.10)x102
3.79(±0.30)x102
4.44(±0.40)x102
3.37(±0.40)x102
1.70(±0.20)x102
5.93(±0.20)x102
aH (%) = (Absinitial - Absfinal)/(Absinitial) × 100 between 250-270 nm region; b(nm) = final - initial; cIntrinsic binding
d
e
constant by UV-vis CT-DNA analysis; Q (%) = (Eminitial - Emfinal)/(Eminitial) × 100; Stern-Volmer quenching EB-
f
DNA constant (KSV) by steady-state fluorescence emission spectra; Bimolecular Stern-Volmer quenching rate
EB-DNA constant (kq) by steady-state fluorescence emission spectra (EB-DNA – 0 = 23.0x10-9 s); gDNA-binding
apparent constant (Kapp);
In order to provide additional information about the binding affinity of these new derivatives
with CT-DNA, competitive-binding assays using fluorescence quenching was also applied.
Changes observed in the fluorescence spectra of ethidium bromide dye (EB) following binding
to DNA are often used for interaction studies between DNA and other compounds such
heterocyclic organic molecules.[62-64] In this sense, steady-state emission spectra were
monitored upon increasing the concentration of the amino acid derivatives 8a and 15 to a fixed
concentration of CT-DNA, pre-treated with EB dye, as presented in Figure 7. It is worth
mentioning that the additional compounds presented similar behavior (see Supporting
Information Figures S72-S88).
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Figure 7. Steady-state fluorescence emission spectra under excitation at 510 nm of EB bound to CT-DNA in the
presence of 8a (a) and 15 (b), in a DMSO(2%)/Tris-HCl buffer solution. The arrow indicates the changes in
fluorescence intensities at increasing concentrations of samples. Insert graph shows the plot of F0/F versus
[DNA] R=0.99106 (8a) and 0.99108 (15).
The EB bounded to CT-DNA in absence of the amino acid derivatives (quencher) shows an
intense fluorescence emission located around 650 nm. After addition of the amino acid
derivatives, the EB emission decreases as presented in Figure 7. This behavior shows the
quenching effect, related to the displacement of EB from the EB-DNA adduct due to the
competition of the amino acid derivatives with the intercalator EB over binding to the DNA
structure. In addition, the calculated low Stern-Volmer quenching constants (KSV), presented in
the inset of Figure 7, corroborate with the competition mode of EB-binding. Moreover, the kq
quenching rate constant values indicate a static interaction mechanism between the amino
acid derivatives and the DNA structure (Table 3),[65] which takes place via ground state
association, following to the moderate DNA-binding apparent constants (Kapp) (Table 3).
29