Synthesis of Amino Acid-Derived 1,2,3-Triazoles: Development of a Nontrivial Fluorescent Sensor in Solution for the Enantioselective Sensing of a Carbohydrate and Bovine Serum Albumin Interaction

Article abstract of DOI:10.1021/acs.joc.7b02852

A series of amino acid-derived 1,2,3-triazoles presenting the amino acid and the aromatic moieties connected by a triazole-4-carboxylate spacer is discussed in this work. These compounds were achieved in good yields by organocatalytic enamine-azide [3 + 2] cycloadditions. One of the molecules obtained, bearing a 7-chloroquinoline moiety, was photoactive in the UV-violet region and was successfully employed as a probe for substrate-specific enantiomeric sensing using d-(-)-arabinose and l-(+)-arabinose. The potential application as a fluorescent probe to detect protein in phosphate buffer solution was also explored using as model bovine serum albumin (BSA). The studied compounds presented both suppression and association behavior in the presence of BSA. In addition, theoretical calculations were performed at levels ωB97XD/cc-pVDZ and PBE1PBE/6-311+G(d,p) together with the polarizable continuum model to understand the interaction of the molecules with the enantiomers.

Full text of DOI:10.1021/acs.joc.7b02852

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Article
Synthesis of amino acid-derived 1,2,3-triazoles: development
of a non-trivial fluorescent sensor in solution for the
enantioselective sensing of a carbohydrate and BSA interaction
Natalí P Debia, Maiara Torchelsen Saraiva, Bruna Simões Martins, Roiney Beal, Paulo
F. B. Goncalves, Fabiano Severo Rodembusch, Diego Alves, and Diogo S. Lüdtke
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02852 • Publication Date (Web): 09 Jan 2018
Downloaded from http://pubs.acs.org on January 9, 2018
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Synthesis of amino acidꢀderived 1,2,3ꢀtriazoles: development of a nonꢀtrivial
fluorescent sensor in solution for the enantioselective sensing of a carbohydrate
and BSA interaction
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a
b
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c
Natalí P. Debia, Maiara T. Saraiva, Bruna S. Martins, Roiney Beal, Paulo F. B.
,c
,d
,b
,a
Gonçalves,* Fabiano S. Rodembusch,* Diego Alves,* and Diogo S. Lüdtke*
a
Instituto de Química, Universidade Federal do Rio Grande do Sul, UFRGS, Av. Bento
Gonçalves 9500, 91501-970, Porto Alegre, RS, Brazil. Phone: +55 51 3308 9637.
E-mail: dsludtke@iq.ufrgs.br
b
LASOL - CCQFA, Universidade Federal de Pelotas - UFPEL, Pelotas, RS, Brazil. E-mail:
diego.alves@ufpel.edu.br
c
Grupo de Química Teórica e Computacional, Universidade Federal do Rio Grande do Sul -
UFRGS, Porto Alegre, RS, Brazil. E-mail: paulo@iq.ufrgs.br
d
Grupo de Pesquisa em Fotoquímica Orgânica Aplicada, Universidade Federal do Rio Grande
do Sul - UFRGS, Porto Alegre, RS, Brazil. E-mail: rodembusch@iq.ufrgs.br
Graphical Abstract:
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Abstract. A series of amino acidꢀderived 1,2,3ꢀtriazoles presenting the amino
acid and the aromatic moieties connected by a triazoleꢀ4ꢀcarboxylate spacer is
discussed in this work. These compounds were achieved in good yields by
organocatalytic enamine−azide [3+2] cycloadditions. One of the molecules obtained,
bearing a 7ꢀchloroquinoline moiety was photoactive in the UVꢀviolet region and was
successfully employed as a probe for substrateꢀspecific enantiomeric sensing using Dꢀ(ꢀ
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)ꢀarabinose and Lꢀ(+)ꢀarabinose. The potential application as fluorescent probe to detect
protein in phosphate buffer solution (PBS) was also explored using as model bovine
serum albumin (BSA). The studied compounds presented both suppression and
association behavior in presence of BSA. In addition, theoretical calculations were
performed at levels ωB97XD/ccꢀpVDZ and PBE1PBE/6ꢀ311+G(d,p) together with PCM
(Polarizable Continuum Model) to understand the interaction of the molecules with the
enantiomers.
Keywords: metalꢀfree triazole synthesis, fluorescent sensor, enantioselective
discrimination, protein detection
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Introduction
Heterocyclic compounds represent one of the most general structural units found
1,2
in several natural and synthetic bioactive compounds. In this line, 1,2,3ꢀtriazoles are
an interesting class of nitrogenꢀbased heterocycles extensively studied and used in the
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discovery of drug candidates and in new materials. Several methodologies have been
4
reported for the preparation of 1,2,3ꢀtriazoles, especially via thermal or metalꢀcatalyzed
5
(
Cu, Ru, Ag and Ir) 1,3ꢀdipolar cycloaddition reactions of azides with alkynes. However,
considering the restricted applications of metalꢀbased methodologies in biologically
6ꢀ12
studies in view of their ecoꢀadverse effects,
recent studies have been directed
towards the development of metalꢀfree methodologies for the synthesis of fully
13,14
substituted 1,2,3ꢀtriazoles.
Organocatalytic approaches employing carbonyl
compounds have been reported to promote the synthesis of highly functionalized 1,2,3ꢀ
1
5ꢀ17
triazoles in high yields and mild reaction conditions.
A range of carbonyl compounds,
such as βꢀketo esters, βꢀketo amides, βꢀketo sulfones, αꢀcyano ketones, among others,
18ꢀ29
have been employed as starting material in these protocols.
In these
organocatalyzed [3+2] cycloadditions reactions, the generated enamines or enolates
might act as the dipolarophile partner in the 1,3ꢀdipolar cycloadditions with organic
azides. There remains, however, a need for an inꢀdepth study on the synthesis of more
highly functionalized and complex 1,2,3ꢀtriazoles from various combinations of
substrates.
On the other hand, the development of organic optical sensors for analytical
purposes, such as optical assays and chemical sensing probes, has been highlighted as
30
a subject of general interest. These particular optical sensors must present a
fluorophoric unit, which is able to interact with matter or energy, causing a change that
can be correlated to the identity and/or quantity of the analyte of interest. Finally, this
31
response can be transmitted as an optical signal that can be measured. In fluorimetric
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titrations, changes on the intensity or even on the maxima location can be associated to
specific interaction between the analyte and the fluorophore. Thus, the research and
development of enantioselective fluorescent sensors for the enantiodiscrimination of
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chiral compounds has been increasing fast. These optical sensors present potential
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application in high sensitivity assays to determine the enantioselectivity of a realꢀtime
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system. The enantioselective fluorescent sensors are designed to incorporate one or
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more interaction and/or response mechanisms with a chiral target. In this context,
macrocycles and supramolecular oligomers/polymers which operate through
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supramolecular interactions, have been reported. Another frequent structural motif
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encountered in enantioselective fluorescent sensors is the binaphthol system. Several
BINOLꢀderived compounds have been reported, and in some cases, the dendrimeric
shape is used for signal amplification or even a derivative containing terpyridine (BINOLꢀ
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terpyridine) or bisꢀBINOL connected by a monoamine can be found.
In this work we report the results of our studies towards the synthesis of amino
acidꢀderived 1,2,3ꢀtriazoles, through a metalꢀfree, organocatalyzed enamideꢀazide [3+2]ꢀ
cycloaddition. In addition, the ground state of these compounds was investigated by UVꢀ
Vis absorption spectroscopy (Scheme 1). Noteworthy, one of the synthesized
compounds bearing a 7ꢀchloroquinolineꢀderived fluorophoric moiety, was successfully
used as fluorescent probe to detect bovine serum albumin (BSA) in an aqueous solution.
Additionally, these compounds were also used as probes for enantiomeric recognition of
the carbohydrate arabinose. Theoretical calculations were performed to propose an
interaction mechanism to better described the observed excited state deactivation
channel present in this compound, and to explain the observed enantiomeric
discrimination.
.
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Scheme 1. This work
Results and Discussion
Synthesis
The investigation started by the synthesis of the required amino acidꢀderived βꢀ
keto esters 1ꢀ4. The NꢀBoc amino acid derivatives were reduced to the corresponding
amino alcohols, followed by transesterification with tꢀbutyl acetoacetate, in the presence
of DMAP (Scheme 2), giving the desired products in good overall yields.
Scheme 2. Synthesis of βꢀketo esters 1ꢀ4
With these compounds in hands, we started the optimization studies for the
enamineꢀazide [3+2] cycloaddition reaction using Lꢀprolineꢀderived βꢀketo ester 1 and 4ꢀ
methoxyphenyl azide (5) as the starting materials. The first attempt for the 1,2,3ꢀtriazole
2
synthesis was performing the reaction using Et NH as the catalyst, in DMSO as the
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solvent at room temperature (Table 1, entry 1). Unfortunately, under these conditions no
o
product was formed (entry 1). Heating up the reaction to 80 C greatly improved the
outcome and the desired triazole product 6 was isolated in 80% yield (entry 2). Then, it
was evaluated the performance of different secondary amines as catalysts for this
reaction (entries 3ꢀ7), however none of them resulted in improved yields for product 6. In
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the absence of an amine, no reaction was observed.
Table 1. Screening of reaction conditions for the reaction of βꢀketo ester 1 and 4ꢀ
a
methoxyphenyl azide (5).
b
Entry
Catalyst (10 mol%)
Et NH
Temp (°C)
Yield (%)
NR
80
1
2
3
4
5
6
7
8
2
25
80
80
80
80
80
80
80
Et NH
2
Lꢀproline
pyrrolidine
piperidine
dibenzylamine
morpholine
ꢀ
32
42
69
33
36
NR
a
All reactions performed using 1.0 equiv of βꢀketo ester 1, 2.5 equiv of 4ꢀ
b
methoxyphenyl azide (5), under argon atmosphere. Isolated yield. NR = no reaction.
Having established the best reaction conditions, the scope was expanded to the
other βꢀketo esters prepared and different aryl azides (Scheme 3). Initially, βꢀketo ester
1 was used in combination with different aryl azides displaying substituents with different
electronic properties. The corresponding triazole products 6ꢀ11 were isolated with yields
ranging from 25% to 89%. It is worth mentioning that the reactions with 4ꢀnitrophenyl
azide and 4ꢀazidoꢀ7ꢀchloroquinoline afforded the corresponding products 10 and 11 in
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8% and 89% yield, respectively. On the other hand, the reaction with phenyl azide was
sluggish and the desired product 8 was isolated in only 25% yield. Moreover, we have
also changed the structure of the βꢀketo ester partner and compounds 2ꢀ4 have also
been used in the reaction with 4ꢀmethoxyphenyl azide (5). The 1,2,3ꢀtriazoles 12, 13 and
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14 were obtained in 64%, 50% and 72%, respectively.
Scheme 3. Scope of the reaction
Compound 15, bearing a free NH group, was also prepared in excellent yield by
Bocꢀdeprotection using trifluoroacetic acid in CH Cl , followed by neutralization of the
2
2
2 3
resulting salt with K CO (Scheme 4).
Scheme 4. Deprotection of 11
Photophysics
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6
The absorption spectra of the amino acidꢀderived 1,2,3ꢀtriazoles (6,7 and 10ꢀ15)
ꢀ5
are shown in Figure 1 using 1,4ꢀdioxane and acetonitrile as solvents (10 M). The
relevant data from UVꢀVis absorption spectroscopy are presented in Table 2.
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Figure 1. UVꢀVis absorption spectra in solution of the amino acidꢀ
derived 1,2,3ꢀtriazoles (6, 7 and 10ꢀ15) in 1,4ꢀdioxane (left) and
acetonitrile (right)
From the analysis of the UVꢀVis absorption curves, it can be observed that in
general, the studied compounds present absorption maxima (λabs) below 250 nm. For
compounds containing chromophoric groups different from the benzene and triazole
rings, such as the nitro group (10) or the quinoline unity (11 and 15), it was observed a
shift to longer wavelengths (> 250 nm), as expected. Additionally, a very small
solvatochromic effect could be observed in the ground state for the amino acidꢀderived
1
,2,3ꢀtriazoles, indicating an almost absent intramolecular charge transfer state. It is
worth mentioning that for compound 6 the difference on the λabs location in respect to the
solvents 1,4ꢀdioxane and acetonitrile (39 nm) is believed to be related to the compound
planarity. In 1,4ꢀdioxane the quinoline and triazole moieties seems to present a higher
planarity allowing an extended conjugation and consequently an redshift absorption (260
nm). On the other hand, acetonitrile does not allow such stabilization and only the
absorption located at 229 nm was observed.
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In order to better characterize the photophysical behavior of the synthesized
compounds, the StricklerꢀBerg relation (Equation 1) was applied to relate the molar
ꢀ
1
ꢀ1
36
e
absorptivity coefficient ε (M —cm ) and the strength (f ) of a single electron oscillator:
1
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ꢀꢁ ≈ 4.3ꢂ10ꢃꢄ ꢅ ꢆꢇꢈ
̅
(1)
In this equation the integral is related to the area under the absorption curve from
ꢀ
1
ꢀ1
ꢀ1
a plot of the molar absorptivity coefficient ε (M —cm ) against wavenumber
can be defined as a quantity that measures the intensity or probability of an electronic
transition that is induced by the interaction of electrons and matter with the
ꢈ̅ (cm ) and
f
e
37
electromagnetic field of a light wave. The theoretical relationship which allow the
0
calculation of the rate constant
k
e
from the oscillator strength
f
e
is present in Equation
36
0
0 37
(
e
2). In addition, the pure radiative lifetime τ is defined as 1/k .
ꢊ
ꢃꢄ
ꢋ
ꢊ
ꢉ ≈ 2.88ꢂ10 ꢈ
̅
ꢅ ꢆꢇꢈ
̅
(2)
ꢁ
The limit values to the molar absorptivity coefficient, as well as calculated
radiative rate constant associated with an oscillator strength of unity corresponds to ε
5
ꢀ1
ꢀ1
0
9
ꢀ1
around 10 M —cm and
k
e
around 10 s . Spin and symmetry allowed electronic
0
2
ꢀ1
ꢀ1
transitions ranges over four orders of magnitude to ε and
k
e
starting at 10 M —cm and
5
ꢀ1
37
1
0 s , respectively. In this way, based on the data summarized in Table 2, all
compounds present spin and symmetry allowed electronic transitions, which could be
1
related to π−π* transitions.
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Table 2 Relevant photophysical data of the UVꢀVis spectra of the amino acidꢀderived 1,2,3ꢀ
4
ꢀ
triazoles (6, 7 and 10ꢀ15), where λabs is the absorption maxima (nm), ε is the molar absorptivity (10 M
1
ꢀ1
0
e
8
ꢀ1
0
—cm ), f
e
is the calculated oscillator strength, k
is the calculated radiative rate constant (10 s ) and τ is
the calculated pure radiative lifetime (in ns).
0
0
Compound
6
Solvent
λabs
268
229
228
223
268
267
286
285
230
230
230
229
230
229
283
281
ε
f
e
k
e
τ
1
,4ꢀdioxane
1.52
1.62
2.78
1.29
0.96
0.94
0.78
0.48
5.44
3.35
3.23
2.36
2.75
1.95
1.38
0.53
0.29
0.49
0.78
0.38
0.28
0.27
0.23
0.14
0.98
0.53
0.89
0.82
0.74
0.60
0.42
0.16
2.77
6.29
10.1
5.25
2.62
2.56
1.88
1.15
12.4
5.53
11.3
10.5
9.36
7.67
3.56
1.43
3.61
1.59
0.99
1.91
3.82
3.90
5.31
8.67
0.81
1.81
0.89
0.95
1.07
1.30
2.81
6.97
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acetonitrile
1
,4ꢀdioxane
7
acetonitrile
1
,4ꢀdioxane
10
11
12
13
14
15
acetonitrile
1
,4ꢀdioxane
acetonitrile
1
,4ꢀdioxane
acetonitrile
1
,4ꢀdioxane
acetonitrile
1
,4ꢀdioxane
acetonitrile
1
,4ꢀdioxane
acetonitrile
Due to the structural rigidity present on the chromophores in these compounds,
values close to unity could be obtained to the oscillator strength. Furthermore, an almost
0
constant radiative lifetime (τ ) can be observed for each compound changing the
0
solvent. However, different values could be calculated for τ among the studied
compounds, ranging from 0.81 to 5.31 ns or 0.95 to 8.67 ns in 1,4ꢀdioxane or
acetonitrile, respectively. Although these compounds present the similar chromophoric
and fluorophoric structures, the aminoꢀacid seems to play a significant role on the
photophysics of these dyes. In this way, despite the different calculated values, these
results are probably indicating that after the radiation absorption the amino acidꢀderived
1
,2,3ꢀtriazoles are populating the same excited state.
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After a screening based on the fluorescence emission signal from each
compound, we observed that those presenting the quinoline moiety (11 and 15) have
shown the stronger fluorescence signals. In this way, the photophysical study in the
excited state was performed for these compounds and is presented in Figure 2. The
relevant data from this study are summarized in Table 2. It is worth mentioning that the
fluorescence emission spectra were obtained using, in addition to the absorption
maxima, additional excitation wavelengths. The presence of different fluorophoric groups
allowed the obtaining of fluorescence emission spectra using different excitation
radiations. A scanning in the excitation wavelengths allowed to evaluate and to discard
the hypothesis of these compounds not to obey the Kasha’s rule.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Fluorescence emission spectra in solution of the amino
acidꢀderived 1,2,3ꢀtriazole 11 in (a) 1,4ꢀdioxane and (b) acetonitrile. In
(
c) compound 11 is in a 1,4ꢀdioxane solution, at different excitation
ꢀ
5
wavelengths (350ꢀ375 nm) ([10 M] and excitation/emission slits 5.0
nm/5.0 nm). The asterisk indicates the Raman signal.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
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4
4
4
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5
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5
5
5
5
6
The amino acidꢀderived 1,2,3ꢀtriazole 11 presents in both solvents an almost
absent fluorescence emission under excitation below 250 nm. At the absorption maxima
in 1,4ꢀdioxane (286 nm, Figure 2a) a low intense emission could be observed located in
the UV region (330 nm). Once again, in acetonitrile at 285 nm, the fluorescence
emission remains almost absent. It is worth mentioning that for both solvents, the
excitation wavelength (λexc) scanning indicated that dye 11 presents higher emission
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
intensity at λexc>300 nm. Higher intensity values were obtained under excitation at 312
nm and 368 nm in 1,4ꢀdioxane (Figure 2a) and acetonitrile (Figure 2b), ascribed to
emissions in the UV (332 nm) and cyan (441 nm) regions. The dependence on the
fluorescence maxima location with the excitation wavelength was also studied scanning
from 350ꢀ375 nm (Figure 2c). The result shows that compound 11 respect the Kasha’s
rule of excitation wavelength independence of the emission spectrum. Although less
intense, a similar behavior was observed to the compound 15 in 1,4ꢀdioxane (see
Supporting Information).
BSA interaction study
Bovine serum albumin (BSA) is widely used to study protein morphology and drug
delivery presenting high affinity to bind and transport ligands to specific sites, mainly due
38ꢀ40
to the presence of two structured and hydrophobic binding pockets in its structure.
Compounds 11 and 15 were studied as fluorescent probes for protein detection in
solution using BSA as the model system. The BSA association study was performed
with different amounts of protein (0ꢀ12 ꢁM in PBS buffer pH 7.2) added to a solution of
dyes 11 or 15 (2 ꢀM in acetonitrile). After each addition, UVꢀVis and fluorescence
emission spectra (λexc= 310 nm, Exc/Em slits of 10.0 nm/10.0 nm) were obtained. It can
be observed that the increase of BSA raised the absorption intensity at 278 nm, with
absence of redshift of maxima absorption (data not shown, see supporting information).
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The fluorescence emission spectra of both compounds in the absence and
presence of BSA are given in Figure 3. It can be observed an emission maxima
independent on the BSA amount located at 345 nm, related to the fluorophores present
in 11 and 15. Any additional fluorescence emission was observed indicating the absence
of aggregates in solution in these compounds. Both compounds already present
fluorescence emission in absence of BSA (blank). Additionally, it could be observed a
linear correlation between the fluorescence intensity and protein concentration up to 1.2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
ꢀ
M of BSA, as follows: 11 (R = 0.9987) and 15 (R = 0.9992). The first addition of BSA
(1 ꢁM) increased the fluorescence emission around 2 times. After the last BSA addition
(12 ꢀM), the fluorescence increased more than 10 times, with any shift on the maxima
location.
Figure 3. Fluorescence emission spectra of the amino acidꢀderived
1
,2,3ꢀtriazoles (a) 11 and (b) 15 at different BSA concentrations (0−12
M) in a dye concentration of 2 ꢁM in acetonitrile. In (c) the linear
ꢁ
relation of fluorescence intensity vs. BSA concentration (ꢀM) is also
depicted. (Blank: in absence of BSA).
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1
1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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4
4
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5
5
5
5
5
5
5
6
These preliminary results can probably be related to a confinement effect,
afforded by specific interaction between the studied dyes and BSA. It is reported in the
literature that aromatic and heterocyclic structures were found to bind within hydrophobic
41ꢀ44
sites of BSA in specific subdomains, soꢀcalled IIA and IIIA.
This interaction increases
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
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1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
the rigidity of the fluorophore, minimizing the nonradiative deactivation of the excited
state and consequently increasing the fluorescence emission intensity. This
photophysical behavior can be related to a significant affinity of the studied compounds
with the protein, indicating its potential application as optical probes for protein detection
in solution.
Enantiomers identification in solution
The amino acidꢀderived 1,2,3ꢀtriazoles 11 and 15 were also studied for
enantioselective sensing using
and 5). Fluorimetric titrations were performed using 11 (20 ꢀM in acetonitrile) and 15 (28
M in acetonitrile) in presence of different amounts of ꢀ(ꢀ)ꢀarabinose (18ꢀ110 ꢁM) or
Dꢀ(ꢀ)ꢀarabinose and Lꢀ(+)ꢀarabinose as models (Figure 4
ꢀ
D
Lꢀ
(+)ꢀarabinose (21ꢀ125 ꢀM) both in DMSO. After each addition, UVꢀVis and fluorescence
emission spectra (λexc= 310 nm, Exc/Em slits of 5.0 nm/10.0 nm) were obtained.
Figure 4. Fluorescence emission intensities of the 1,2,3ꢀtriazoles 11
(
20 ꢀM) (left) and 15 (28 ꢀM) (right) relative to the added volume of
the enantiomers. [Dꢀ(ꢀ)ꢀarabinose: 1.53 mM] and [
Lꢀ(+)ꢀarabinose: 1.73
o
mM]. All experiment were performed in acetonitrile at 25 C.
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It can be observed that both compounds have a different response to the
presence of the arabinose enantiomers in solution, indicating that their excited states
can be notably affected by the presence of these compounds (Figure 4). The Bocꢀ
protected 1,2,3ꢀtriazole 11 shows a more intense fluorescence signal after the addition
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
of equal amounts of
behavior can be observed for the deprotected compound 15, because a more intense
fluorescence emission was observed upon addition of the ꢀ(ꢀ)ꢀarabinose in comparison
with the opposite enantiomer. Despite the similar result, the Bocꢀprotected 1,2,3ꢀtriazole
1 presents the greatest difference between the values of the fluorescence intensities,
Dꢀ(ꢀ)ꢀarabinose, when compared to Lꢀ(+)ꢀarabinose. A similar
D
1
which might be ascribed as the result of different interactions in the excited state
between the this fluorophore and each enantiomer of the carbohydrate. It is worth
mentioning that any significant change was observed in the UVꢀVis spectra, indicating
that in the ground state, both compounds behave equally (see Supporting Information).
After observing that 1,2,3ꢀtriazoles 11 and 15 interact differently with each
enantiomer of arabinose, a plot of the relative fluorescence intensity (I
fluorophores against the concentration of the enantiomers in acetonitrile was made
Figure 5). In this plot, the fall of the ratio of the fluorescence intensity is related to the
interaction between the fluorophore and the enantiomer. For compound 11, the higher
the concentration of the carbohydrate, the lower the I /I ratio, and higher the difference
between ꢀ(ꢀ)ꢀarabinose and ꢀ(+)ꢀarabinose. On the other hand, despite the also lower
0
/I) of these
(
0
D
L
relative fluorescence intensity, compound 15 presents closer values to the I /I ratio if
0
compared to 11.
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Ratio of the fluorescence emission intensities of the 1,2,3ꢀ
triazoles 11 (20 ꢀM in acetonitrile) (top) and 15 (28 ꢀM in acetonitrile)
(bottom) at different enantiomer concentrations (18ꢀ110 ꢁM for
arabinose and 21ꢀ125 ꢀM for ꢀ(+)ꢀarabinose, respectively).
Dꢀ(ꢀ)ꢀ
L
Phenomenologically, the results depicted in Figures 4 and 5 indicate that
arabinose behaves as a fluorescence quencher, probably due to a higher interaction
with the fluorophore 11. Due to this interaction, fluorophore 11 and ꢀ(+)ꢀarabinose are in
Lꢀ(+)ꢀ
L
closer proximity, resulting in a better interaction, and thus allowing a nonꢀradiative
excited state deactivation or perhaps, due to the fluorophore emission and enantiomer
UV absorption spectra overlap, an energy transfer from the fluorophore to the Lꢀ(+)ꢀ
45
arabinose.
The fluorescent sensitivity (I
) were also obtained in the recognition of the arabinose enantiomers. The
relevant data are summarized in Table 3.
D 0 L 0 D
/I and I /I ), as well as the enantioselectivity (I ꢀ
0 L 0
I )/(I ꢀI
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Table 3 Fluorescent sensitivity (I
D
/I
0
and I
L
/I
0
) and enantioselectivity (I
D 0 L 0
ꢀI )/(I ꢀI ) of the 1,2,3ꢀ
triazoles 11 and 15 in the recognition of 60 ꢀL of
Dꢀ(ꢀ)ꢀarabinose [1.53 mM] and
L
ꢀ(+)ꢀarabinose [1.73 mM].
Fluorescent sensitivity
/I /I
Enantioselectivity
(I ꢀI )/(I ꢀI
Fluorophore
11
I
D
0
I
L
0
D
0
L 0
)
a
1.23
1.29
1.09
1.12
2.64 (2.22)
2.33 (1.25)
a
15
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Obtained using 180
ꢀ
L of the respective enantiomers.
Despite the observed variation on the fluorescent sensitivity for each enantiomer
upon the fluorophore 11 or 15, the differences between these values are lower than
33
those already published for different enantiomers. However, it is worth pointing out that
differently from other systems used in enantiomeric recognition studies, in which acidic
and basic moieties are present at the probe and analyte, neither our 1,2,3ꢀtriazoleꢀ
derived optical sensor nor arabinose bear such reactive functionalities. Therefore, the
response obtained in our enantiomeric sensing experiment are result of weaker
interactions, mainly resulting from hydrogen bonds, and the enantioselectivity values are
similar or even higher than some amino acid derivatives using different chiral optical
33
sensors.
Theoretical calculations
Density Functional Theory (DFT) calculations were performed using the Gaussian
46
1
6 package in order to obtain additional information about the interaction between the
amino acidꢀderived 1,2,3ꢀtriazoles and both enantiomers of arabinose. Initially, a
geometry optimization of 11 and 15 was performed at the PBE1PBE/6ꢀ311+G(d,p) level.
The influence of the solvent acetonitrile was considered in these calculations using
Polarizable Continuum Model (PCM). The interaction between compound 11 with
arabinose or ꢀ(+)ꢀarabinose (Figure 6) was performed at ωB97XD/ccꢀpVDZ level. The
interaction between compound 15 with ꢀ(ꢀ)ꢀarabinose or ꢀ(+)ꢀarabinose (Figure 7) was
Dꢀ(ꢀ)ꢀ
L
D
L
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
performed at PBE1PBE/6ꢀ311+G(d,p). In both cases, the thermodynamically favored βꢀ
47
arabinopyranose form of the carbohydrate was used for the calculations. It can be
observed that the main interaction of both set of compounds is due to two hydrogen
bonds occurring between the 1,2,3ꢀtriazoles 11 and the each of the enantiomers of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
arabinose. In this case, the ∆E values of 1,2,3ꢀtriazoles 11 with Dꢀ(ꢀ)ꢀarabinose is shown
ꢀ
1
to be 8.61 kcal—mol less stable than with
Lꢀ(+)ꢀarabinose.
In the interaction between 11 and
D
ꢀ(ꢀ)ꢀarabinose, the first hydrogen bond takes
place between the H19 and O47 atoms in a distance of 1.90 Å and the second one,
between the H and O56 atoms in a distance of 1.77 Å (Figure 6 top). The angles
between OꢀHꢀO in the hydrogen bonds are 164.19° for O18 47 and 164.19° for
56. The hydrogen bonds between 11 and the ꢀ(+)ꢀarabinose, which has been
shown to be more stable, are shorter than with the ꢀ(ꢀ)ꢀarabinose. The first hydrogen
8
19
H O
O
7
H
8
O
L
D
bond distance, between the H19 and O47 atoms has 1.80 Å with an angle between the
atoms O H O of 168.51°. The second hydrogen bond between the H and O atoms
18
19 47
8
56
7 8
also has a length of 1.80 Å and the angle between O H O56 is 159.64° (Figure 6,
bottom).
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Figure 6. Interaction systems of 1,2,3ꢀtriazole 11 with Dꢀ(ꢀ)ꢀarabinose
(
top) and Lꢀ(+)ꢀarabinose (bottom).
The interaction between 15 and the each enantiomer of the carbohydrate was
also studied for comparison (Figure 7). The results showed once again that the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
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5
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5
5
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
interaction between the 1,2,3ꢀtriazoles 15 and
L
ꢀ(+)ꢀarabinose is stronger than with Dꢀ(ꢀ)ꢀ
ꢀ1
arabinose, but in this case by a lower value (4.16 kcal—mol ). The first interaction,
between 15 with
Dꢀ(ꢀ)ꢀarabinose (Figure 7, top), presented only one hydrogen bond,
which causes a decrease in the interaction energy in the system. This interaction takes
place between the atoms H11 and O47 and has a distance of 1.86 Å with an angle
between O10ꢀH11ꢀO47 of 171.99°. On the other hand, the interaction of 15 with the Lꢀ(+)ꢀ
arabinose occurs through two hydrogen bonds (Figure 7 bottom). The first one between
the H20 and O48 atoms presents a distance of 1.86 Å and an angle between O19ꢀH20ꢀO48
of 178.52°. The second one is through H and N and shows a length of 1.81 Å with an
8
55
angle between O ꢀH ꢀN of 168.61°.
7
8
55
The calculations are in agreement with the experimental findings, in which the Lꢀ
(
+)ꢀarabinose enantiomer has a greater interaction with the 1,2,3ꢀtriazoles 11 and 15. It
is believed that for this reason the differences in the fluorescence intensities between
the enantiomers were observed. In both cases, it was found two hydrogen bonds
between the 1,2,3ꢀtriazoles and the arabinose enantiomers, which on average has a
smaller distance, thus increasing the interaction and decreasing the energy of the
system.
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Figure 7. Interaction systems of 15 with
and ꢀ(+)ꢀarabinose (bottom).
Dꢀ(ꢀ)ꢀarabinose (top)
L
Conclusions
In summary, we have described the synthesis of amino acidꢀderived 1,2,3ꢀ
triazoles, through a metalꢀfree, organocatalyzed enamideꢀazide [3+2]ꢀcycloaddition.
Ground state photophysical studies of these compounds by UVꢀVis absorption
spectroscopy have been performed. 1,2,3ꢀTriazoles 11 and 15 bearing a 7ꢀ
chloroquinolineꢀderived fluorophoric moiety have been selected from these studies for
further applications. Both molecules were studied to detect the presence of protein in
solution, using BSA as a model. In this study, the increase of the fluorescence emission
in presence of BSA was related to a significative affinity of the studied compounds with
the protein, indicating its potential application as optical probes. Furthermore, NꢀBoc
1,2,3ꢀtriazole 11 was successfully used for enantiomeric recognition of the carbohydrate
arabinose. We have shown that interaction of 11 with Lꢀ(+)ꢀarabinose led to a stronger
fluorescence quenching, indicating that a stronger interaction occurs with the (+)ꢀ
enantiomer of the carbohydrate, in contrast to what was observed to the (ꢀ)ꢀenantiomer.
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To better understand this behavior, computational studies have been carried out and
revealed that a stronger interaction through hydrogen bonds occurs between 11 and
+)ꢀarabinose.
Lꢀ
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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0
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7
8
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0
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0
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9
0
Experimental Section
General Information. Airꢀ and moistureꢀsensitive reactions were conducted in
flameꢀ or ovenꢀdried 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 hotplate. Column chromatography was performed using
48
silica gel (230ꢀ400 mesh) following the methods described by Still. Thin layer
chromatography (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 300 and 400 MHz or a Bruker 400 MHz instrument in CDCl
shifts are reported in ppm, referenced to the solvent peak of residual CHCl
tetramethylsilane (TMS) as reference. The data are reported as follows: chemical shift
3
solutions. Chemical
3
or
13
(
δ), multiplicity, coupling constant (J) in Hz, and integrated intensity. C NMR spectra
were recorded at 75 and 100 MHz in CDCl solutions. Chemical shifts are reported in
3
3
ppm, referenced to the solvent peak CDCl . Abbreviations to denote the multiplicity of a
particular signal are: s (singlet), d (doublet), t (triplet), dd (double doublet), m (multiplet),
q (quartet), and br (broad singlet). ESIꢀQTOFꢀMS measurements were performed in the
positive ion mode (m/z 50ꢀ2000 range). IR spectra were obtained on an FTIRꢀATR
instrument (Bruker). Melting points were obtained on a Buchi Mꢀ565. Optical rotations
were measured using a JASCO Pꢀ200 digital polarimeter using 10 cm cells and are
ꢋꢊ
reported as ꢌαꢍ , concentration (g/100 mL), and solvent. Spectroscopic grade solvents
ꢎ
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5
5
5
5
5
5
5
6
were used in the photophysical study. The UVꢀVis absorption spectra in solution were
acquired on a Shimadzu UVꢀ2450 spectrophotometer, and the steadyꢀstate fluorescence
spectra were measured on a Shimadzu spectrofluorometer model RFꢀ5301PC.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
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9
0
Procedure for the synthesis of βꢀketoesters. To the reaction flask, under inert
atmosphere, was added the appropriate amino alcohol (1 equiv, 1.70 mmol), DMAP
(
0.25 equiv., 0.42 mmol) and dry toluene (6 mL). Then, tertꢀbutyl acetoacetate (1.2
equiv., 2.04 mmol) was slowly added. The reaction system was heated under reflux for
4 h. After this period, the mixture was washed with distilled water, sat. NH Cl, and sat.
NaCl. The combined organic extracts were dried with anhydrous MgSO , filtered and
2
4
4
evaporated. The residue was purified by flash column chromatography typically eluting
with a mixture of EtOAc/hexanes, 30:70.
tertꢀbutyl
(S)ꢀ2ꢀ(((3ꢀoxobutanoyl)oxy)methyl)pyrrolidineꢀ1ꢀcarboxylate (1).
ꢋꢊ
ꢀ1
Yellow oil in 71% yield (1.460 g, 5.12 mmol). ꢌαꢍ ꢀ40 (c 0.75, CH
2
Cl
2
). IR (cm ): 2982,
ꢎ
1
1
690, 1395, 1164, 1102. Ketoꢀenol equilibrium detected. H NMR (CDCl
3
, 300 MHz):
12.02 (s, 0.1H), 4.99 (s, 0.1H), 4.33ꢀ3.90 (m, 3H), 3.48 (s, 2H), 3.42ꢀ3.29 (m, 2H), 2.28
13
(
3
s, 3H), 1.93ꢀ1.78 (m, 4H), 1.47 (s, 9H). C NMR (CDCl , 75 MHz): 199.6, 171.5, 166.1,
153.7, 153.4, 88.7, 78.7, 78.4, 64.40, 54.9, 54.6, 49.1, 45.9, 45.7, 27.6, 22.9, 22.2, 20.3.
+
HRMSꢀESI calcd for [C H NO +H] : 286.1654, found 286.1656.
14
23
5
(S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀphenylpropyl 3ꢀoxobutanoate (2). White
ꢋꢊ
ꢀ
solid in 95% yield (0.540 g, 1.61 mmol). Mp: 65ꢀ66 ºC. ꢌαꢍ ꢀ14 (c 0.6, CH Cl ). IR (cm
ꢎ
2
2
1
1
)
: 3383, 3023, 2979, 1734, 1686, 1518, 1160. Ketoꢀenol equilibrium detected. H NMR
(CDCl , 400 MHz): 11.95 (s, 0.1H), 7.32ꢀ7.15 (m, 5H), 5.05 (s, 0.1H), 4.80ꢀ4.71 (m, 1H),
4.24ꢀ4.15 (m,1H), 4.12ꢀ4.01 (m, 2H), 3.51 (s, 2H), 2.88 (dd, J = 6.0, 13.6 Hz, 1H), 2.79
3
13
(
3
dd, J = 7.6, 13.6 Hz, 1H), 2.29 (s, 3H), 1.41 (s, 9H). C NMR (CDCl , 100 MHz): 200.4,
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66.8, 155.1, 137.0, 129.1, 128.4, 126.5, 89.3, 79.4, 65.5, 50.5, 49.8, 37.7, 28.2, 21.1.
+
HRMSꢀESI calcd for [C18
H
25NO
5
+H] : 336.1811, found 336.1814.
(S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀmethylbutyl 3ꢀoxobutanoate (3). Pale
ꢋꢊ
ꢀ
yellow waxyꢀsolid in 98% yield (0.333 g, 1.16 mmol). ꢌαꢍ ꢀ29 (c 0.76, CH
2
Cl
2
). IR (cm
ꢎ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
): 3370, 2976, 1744, 1686, 1524, 1242, 1143. Ketoꢀenol equilibrium detected. H NMR
(CDCl
3
, 300 MHz): 11.97 (s, 0.1H), 5.00 (s, 0.1H), 4.68ꢀ4.57 (m, 1H), 4.23 (dd, J = 4.2,
11.1 Hz, 1H), 4.16 (dd, J = 6.3, 11.1 Hz, 1H), 3.72ꢀ3.62 (m, 1H), 3.47 (s, 2H), 2.28 (s,
13
3H), 1.79 (m, 1H), 1.44 (s, 9H), 0.96 (d, J = 5.1 Hz, 3H), 0.94 (d, J = 5.1 Hz, 3H).
C
NMR (CDCl , 75 MHz): 200.4, 166.9, 155.6, 89.4, 79.2, 65.4, 54.4, 49.8, 29.5, 28.2,
3
+
19.2, 18.2. HRMSꢀESI calcd for [C14
H
25NO
5
+H] : 288.1811, found 288.1809.
(S)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)propyl 3ꢀoxobutanoate (4). White solid in
ꢋꢊ
ꢀ1
2 2
89% yield (0.3927 g, 1.51 mmol). Mp: 45ꢀ47 ºC.ꢏꢌαꢍꢎ ꢀ19 (c 0.56, CH Cl ). IR (cm ):
1
3
4
4
1
372, 2983, 1731, 1686, 1525, 1160. Ketoꢀenol equilibrium detected. H NMR (CDCl
3
,
00 MHz): 11.97 (s, 0.1H), 5.02 (s, 0.1H), 4.62 (br, 1H), 4.16 (dd, J = 4.4, 11.2 Hz, 1H),
.08 (dd, J = 5.6 Hz, 11.2 Hz, 1H), 3.97 (br, 1H), 3.49 (s, 2H), 2.28 (s, 3H), 1.44 (s, 9H),
13
.17 (d, J = 6.8 Hz, 3H). C NMR (CDCl
3
, 75 MHz): 200.4, 166.8, 155.0, 89.3, 79.2,
+
67.8, 66.5, 49.7, 45.1, 28.2, 21.0, 17.4. HRMSꢀESI calcd for [C H NO +H] : 260.1498,
12
21
5
found 260.1469.
Procedure for the synthesis of 1,4,5ꢀtrisubstituedꢀ1,2,3ꢀtriazoles. To the
reaction flask, under inert atmosphere, was added the aryl azide (2.5 equiv, 1.25 mmol),
the appropriate βꢀketo ester (1 equiv, 0.50 mmol) in dry DMSO (1 mL). Then 10 mol% of
2
the catalyst Et NH (0.1 equiv, 0.05 mmol) was added and the reaction mixture was
stirred for 24 h at 80ºC. After reaction time, the solvent was removed in vacuum. The
crude product was purified by flash column chromatography using an appropriate
mixture of AcOEt/hexane.
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(
S
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl
1ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ
methylꢀ1Hꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (6). Pale orange solid in 80% yield (0.166 g,
ꢋꢊ
ꢀ1
0
.40 mmol). Mp: 77ꢀ79 ºC. ꢌαꢍ ꢀ36 (c 0.55, CH Cl ). IR (cm ): 3085, 2966, 1724, 1684,
ꢎ
2
2
1
1
367, 1107. H NMR (CDCl , 300 MHz): 7.36 (d, J = 9.0 Hz, 2H), 7.06 (d, J = 9.0 Hz,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2H), 4.58ꢀ4.08 (m, 3H), 3.89 (s, 3H), 3.50ꢀ3.27 (m, 2H), 2.55 (s, 3H), 2.10ꢀ1.96 (m, 3H),
13
1.94ꢀ1.79 (m, 1H), 1.53ꢀ1.42 (m, 9H). C NMR (CDCl
3
, 100 MHz): 161.5, 160.6, 154.5,
1
2
4
54.4, 139.1, 139.0, 136.3, 136.2, 128.1, 126.7, 79.7, 79.3, 64.7, 55.6, 46.7, 46.4,28.8,
+
8.6, 28.4, 27.9, 23.7, 22.8, 9.9. HRMSꢀESI calcd for [C21
17.2141.
28 4 5
H N O +H] : 417.2138, found
(S
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl
5ꢀmethylꢀ1ꢀ(pꢀtolyl)ꢀ1Hꢀ
1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (7). Beige solid in 52% yield (0.104 g, 0.26 mmol). Mp:
ꢋꢊ
ꢀ1
120ꢀ121 ºC. ꢌαꢍ ꢀ39 (c 0.50, CH
2
Cl
2
). IR (cm ): 2975, 2918, 1714, 1684, 1399, 1106.
ꢎ
1
H NMR (CDCl
3
, 400 MHz): 7.37 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 4.55ꢀ4.32
(
m, 2H), 4.28ꢀ4.13 (m, 1H), 3.46ꢀ3.30 (m, 2H), 2.57 (s, 3H), 2.46 (s, 3H), 2.07ꢀ1.84 (m,
13
4
3
H),1.51ꢀ1.43 (m, 9H). C NMR (CDCl , 100 MHz): 161.4, 154.4, 154.3, 140.3, 138.9,
1
38.8, 136.3, 136.2, 132.8, 130.1, 125.0, 79.7, 79.2, 64.7, 55.5, 46.7, 46.4,28.8, 28.4,
+
27.9,23.7, 22.8, 21.1, 9.9. HRMSꢀESI calcd for [C H N O +H] : 401.2189, found
21
28
4
4
401.2187.
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl
,2,3ꢀtriazoleꢀ4ꢀcarboxylate (8). Pale yellow waxyꢀsolid in 25% yield (0.048 g, 0.12
(S
5ꢀmethylꢀ1ꢀphenylꢀ1Hꢀ
1
ꢋꢊ
ꢀ1
mmol). ꢌαꢍ ꢀ37 (c 0.34, CH
2
Cl
2
). IR (cm ): 2975, 2952, 1718, 1677, 1399, 1166, 1102.
ꢎ
1
3
H NMR (CDCl , 400 MHz): 7.61ꢀ7.55 (m, 3H), 7.48ꢀ7.43 (m, 2H), 4.54ꢀ4.34 (m, 2H),
4.28ꢀ4.13 (m, 1H), 3.48ꢀ3.38 (m, 2H), 2.59 (s, 3H), 2.08ꢀ1.99 (m, 3H), 1.91ꢀ1.84 (m,
13
1
H),1.50ꢀ145 (m, 9H). C NMR (CDCl , 100 MHz): 161.5, 154.5, 154.4, 139.0, 138.8,
3
1
36.6, 136.4, 135.4, 130.1, 129.6, 125.3, 79.8, 79.3, 64.8, 55.5, 46.7, 46.4, 28.4, 24.0,
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+
2
3.8, 22.9, 22.6, 10.0. HRMSꢀESI calcd for [C H N O +H] : 387.2032, found
20 26 4 4
3
87.2037.
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl
methylꢀ1 ꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (9). White solid in 38% yield (0.077 g, 0.19
(S
1ꢀ(4ꢀfluorophenyl)ꢀ5ꢀ
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢋꢊ
ꢀ1
mmol). Mp: 121ꢀ122 ºC. ꢌαꢍ ꢀ37 (c 0.50, CH
2
Cl
2
). IR (cm ): 2973, 2873, 1717, 1677,
ꢎ
1
1401, 1102. H NMR (CDCl
3
, 400 MHz): 7.41ꢀ7.35 (m, 2H), 7.25ꢀ7.16 (m, 2H), 4.41 (dd,
J = 3.6, 10.8 Hz, 1H), 4.31 (dd, J = 7.2, 10.8 Hz, 1H), 4.18ꢀ4.07 (m, 1H), 3.92ꢀ3.27 (m,
13
2
7
1
3
H), 2.50 (s, 3H), 2.01ꢀ1.90 (m, 3H), 1.86ꢀ1.77 (m, 1H), 1.40 (s, 9H). C NMR (CDCl ,
5 MHz): 163.1 (d, J = 249.0 Hz), 161.3, 154.4, 139.0, 136.4, 131.4 (d, J = 2.9 Hz),
27.3 (d, J = 8.9 Hz), 116.7 (d, J = 23.1 Hz), 79.7, 79.3, 64.8, 55.5, 46.6, 46.4, 28.4,
+
23.7, 22.8, 9.9. HRMSꢀESI calcd for [C20
H
25FN
4
O
4
+H] : 405.1938, found 405.1963.
(S
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl 5ꢀmethylꢀ1ꢀ(4ꢀnitrophenyl)ꢀ
1
Hꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (10). Yellow solid in 88% yield (0.190 g, 0.44 mmol).
ꢋꢊ
ꢀ1
Mp: 148ꢀ150 ºC. ꢌαꢍ ꢀ38 (c 0.54, CH
2
Cl
2
). IR (cm ): 3088, 2978, 2880, 1728, 1691,
ꢎ
1
1389, 1344, 1109. H NMR (CDCl
3
, 400 MHz): 8.48 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.8
Hz, 2H), 4.55ꢀ4.44 (m, 1H), 4.42 (dd, J = 6.8, 10.8 Hz, 1H), 4.30ꢀ4.12 (m, 1H), 3.46ꢀ3.32
13
(m, 2H), 2.69 (s, 3H), 2.10ꢀ1.96 (m, 3H), 1.92ꢀ1.84 (m, 1H), 1.52ꢀ1.43 (m, 9H). C NMR
(CDCl
3
, 100 MHz): 160.9, 154.5, 154.2, 148.0, 140.1, 138.9, 138.8, 137.1, 136.9, 125.7,
1
25.0, 79.6, 79.2, 65.0, 55.4, 46.6, 46.4, 28.3, 23.6, 22.8, 10.1. HRMSꢀESI calcd for
+
[
C
20
H
25
N
5
O
6
+H] : 432.1883, found 432.1905.
)ꢀ(1ꢀ(tertꢀbutoxycarbonyl)pyrrolidinꢀ2ꢀyl)methyl 1ꢀ(7ꢀchloroquinolinꢀ4ꢀyl)ꢀ5ꢀ
methylꢀ1 ꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (11). Yellow solid in 89% yield (0.211 g, 0.45
(S
H
ꢋꢊ
ꢀ1
mmol). Mp: 71ꢀ73 ºC. ꢌαꢍ ꢀ26 (c 0.58, CH
2
Cl
2
). IR (cm ): 2982, 2873, 1721, 1684,
ꢎ
1
1
7
4
392, 1143, 1099. H NMR (CDCl , 400 MHz): 9.14 (d, J = 4.0 Hz, 1H), 8.40 (br, 1H),
3
.59 (dd, J = 2.0, 8.8 Hz, 1H), 7.48 (d, J = 4.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H), 4.50ꢀ
.32 (m, 2H), 4.26ꢀ4.07 (m, 1H), 3.42ꢀ3.27 (m, 2H), 2.45 (s, 3H), 2.07ꢀ1.93 (m, 3H),
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2
2
2
2
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3
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3
3
3
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3
4
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5
5
5
5
5
5
5
6
1
3
1
1
1
.88ꢀ1.81 (m, 1H),1.47ꢀ136 (m, 9H). C NMR (CDCl , 100 MHz): 160.9, 154.5,154.3,
3
51.1, 149.5, 140.5, 140.4, 139.6, 139.5, 137.3, 136.6, 136.5, 129.9, 128.6, 123.6,
22.1, 118.8, 79.7, 79.3, 65.1, 55.4, 55.3, 46.6, 46.4, 28.3, 23.6, 22.8, 9.6. HRMSꢀESI
+
calcd for [C23
H
26ClN
5
O
4
+H] : 472.1752, found 472.1739.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
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(S
)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀphenylpropyl
1ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ
methylꢀ1 ꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (12). Brown oil in 64% yield (0.148 g, 0.32
H
ꢋꢊ
ꢀ1
mmol). ꢌαꢍ ꢀ6 (c 0.98, CH
2
Cl
2
). IR (cm ): 3353, 2976, 1708, 1514, 1253, 1161, 1109.
ꢎ
1
3
H NMR (CDCl , 300 MHz): 7.36 (d, J = 9.0 Hz, 2H), 7.32ꢀ7.19 (m, 5H), 7.07 (d, J = 9.0
Hz, 2H), 5.15 (d, J = 8.4 Hz, 1H), 4.37ꢀ4.30 (m, 2H), 4.30ꢀ4.21 (m, 1H), 3.89 (s, 3H),
13
3
3
.02ꢀ2.83 (m, 2H), 2.56 (s, 3H), 1.39 (s, 9H). C NMR (CDCl , 75 MHz): 161.4, 160.6,
155.2, 139.2, 137.2, 135.9, 129.2, 129.1, 128.4, 128.2, 127.9, 126.6, 126.4, 126.1,
+
114.6, 79.2, 65.3, 55.5, 50.7, 37.8, 28.2, 9.8. HRMSꢀESI calcd for [C25
67.2294, found 467.2269.
)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ3ꢀmethylbutyl
methylꢀ1 ꢀ1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (13). Brown waxyꢀsolid in 50% yield (0.105 g,
.25 mmol). ꢌαꢍ ꢀ18 (c 0.94, CH Cl ). IR (cm ): 3370, 3080, 2965, 1693, 1513, 1243,
H
30
N
4
O
5
+H] :
4
(S
1ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ
H
ꢋꢊ
ꢀ1
0
ꢎ
2
2
1
1106. H NMR (CDCl , 400 MHz): 7.34 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 4.86
3
(d, J = 9.6 Hz, 1H), 4.48 (dd, J = 6.0, 11.2 Hz, 1H), 4.37 (dd, J = 4.0, 11.2 Hz, 1H), 3.89
(
s, 3H), 3.85ꢀ3.76 (m, 1H), 2.54 (s, 3H), 1.96ꢀ1.94 (m, 1H), 1.41 (s, 9H), 1.03ꢀ0.98 (m,
13
6
7
4
H). C NMR (CDCl
3
, 100 MHz): 161.4, 160.5, 155.6, 138.9, 136.0, 127.9, 126.5, 114.6,
+
8.9, 65.0, 55.5, 54.6, 28.2, 28.1, 19.3, 18.3, 9.8. HRMSꢀESI calcd for [C21
19.2294, found 419.2293.
H
30
N
4
O
5
+H] :
(S
)ꢀ2ꢀ((tertꢀbutoxycarbonyl)amino)propyl 1ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀmethylꢀ1
Hꢀ
1,2,3ꢀtriazoleꢀ4ꢀcarboxylate (14). Beige solid in 72% yield (0.140 g, 0.36 mmol). Mp:
ꢋꢊ
ꢀ1
91ꢀ92 ºC. ꢌαꢍ ꢀ19 (c 0.53, CH
2
Cl
2
). IR (cm ): 3386, 2978, 1701, 1501, 1209, 1126,
ꢎ
1
1062. H NMR (CDCl
3
, 400 MHz): 7.35 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H),
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.91ꢀ4.80 (m, 1H), 4.39 (dd, J = 5.2, 11.0 Hz, 1H), 4.34 (dd, J = 4.4 , 11.0 Hz, 1H), 4.19ꢀ
13
4.08 (m, 1H), 3.89 (s, 3H), 2.56 (s, 3H), 1.43 (s, 9H), 1.28 (d, J = 6.8 Hz, 3H). C NMR
(CDCl
3
, 100 MHz): 161.5, 160.6, 155.1, 139.1, 136.0, 128.1, 126.6, 114.7, 79.3,67.5,
+
5
5.6, 45.4, 28.2, 17.7, 9.9. HRMSꢀESI calcd for [C19
91.2007.
H
26
N
4
O
5
+H] : 391.1982, found
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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2
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8
9
0
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2
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4
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8
9
0
3
Procedure for the deprotection of the Bocꢀgroup. To a solution of the 1,2,3ꢀ
triazole 11 (1 equiv., 0.18 mmol, 0.085 g) in dichloromethane (0.36 mL), TFA (13 equiv.,
.34 mmol, 0.18 mL) was slowly added. The reaction mixture was stirred at room
2
temperature for 2 h. Then the volatile substances were evaporated. The residue was
dissolved in dichloromethane and the solvent was evaporated (this process was
repeated three times). The result oil was dissolved in dichloromethane (0.36 mL) and
2 3
K CO (3.6 equiv., 0.65 mmol, 0.090 g) was added. The mixture was stirred at room
temperature for 2 h. Then the solids were separated by filtration and the filtered was
evaporated. The crude product was purified by flash column chromatography using a
mixture of AcOEt/Et N (99:1).
3
(S
)ꢀpyrrolidinꢀ2ꢀylmethyl 1ꢀ(7ꢀchloroquinolinꢀ4ꢀyl)ꢀ5ꢀmethylꢀ1Hꢀ1,2,3ꢀtriazoleꢀ
4ꢀcarboxylate (15). Beige solid in 89% yield (0.060 g, 0.16 mmol). Mp: 171ꢀ173 ºC.
ꢋꢊ
ꢀ1
1
ꢌαꢍ ꢀ41 (c 0.17, CH
2
Cl
, 400 MHz): 9,15 (d, J = 4.8 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 7.58 (dd, J = 2.0,
.2 Hz, 1H), 7.43 (d, J = 4.4 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 4.91ꢀ4.84 (m, 1H), 4.57ꢀ
.49 (m, 1H), 4.48ꢀ4.39 (m, 1H), 4.17ꢀ4.08 (m, 1H), 3.86ꢀ3.76 (m, 2H), 2.46 (s, 3H),
2
). IR (cm ): 3340, 2980, 2874, 1589, 1420, 1050. H NMR
ꢎ
(
CDCl
3
9
4
2
13
3
.22ꢀ2.13 (m, 1H), 2.09ꢀ1.92 (m, 2H), 1.79ꢀ1.70 (m, 1H). C NMR (CDCl , 100 MHz):
162.8, 151.3, 150.0, 140.2, 140.0, 139.8, 137.2, 129.8, 129.1, 123.8, 122.3, 118.8, 67.2,
+
6
2.3, 50.4, 28.0, 25.0, 10.0. HRMSꢀESI calcd for [C18
72.1222.
H18ClN
5
O
2
+H] : 372.1227, found
3
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BSA association study. A dye solution in acetonitrile (11: 2 ꢀM and 15: 3 ꢀM)
was used to prepare a dye solution in PBS (12 ꢀL in 3 mL PBS). To this PBS/ dye
solution different amounts of a previously prepared PBS/BSA solution (1ꢀ12 ꢀM in PBS
pH 7.2) were added. The final solution was kept to rest for 1 h. The fluorescence spectra
0
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5
6
7
8
9
0
o
were obtained at 25 C and under excitation at 310 nm using Exc. Em. slits of 10.0
nm/10.0 nm, respectively.
Enantiomer interaction study. To a solution of 11 (20 ꢀM) or 15 (28 ꢀL) both in
acetonitrile, different amounts of DMSO solutions of
Dꢀ(ꢀ)ꢀarabinose (18ꢀ110 ꢀM) and
Lꢀ
(+)ꢀarabinose (21ꢀ125 ꢀM) were added. In this study, the enantiomer concentrations
correspond to the addition of 30 ꢀL up to 180 ꢀL (in 30 ꢀL step) of each enantiomer to
the dye solution. The working concentrations of the enantiomers were 1.53 mM and 1.73
mM for Dꢀ(ꢀ)ꢀarabinose and Lꢀ(+)ꢀarabinose, respectively. This fluorimetric titration was
performed using an excitation wavelength of 310 nm with Exc. Em. slits of 5.0 nm/10.0
o
nm, respectively. All experiments were performed at 25 C.
Acknowledgements
This work was supported by CNPq, CAPES, and INCTꢀCatálise. We would also
thank Andressa M. M. Carlos for assistance with HPLC analysis.
Supporting Information:
The Supporting Information is available free of charge on the ACS Publications
website. Copies of NMR spectra for all compounds, additional photophysical, and
computational data.
References
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Eicher, T.; Hauptmann, S. The Chemistry of Heterocycles, WileyꢀVCH, Weinheim,
2003.
Katrizky, A. R.; Pozharskii, A. F. Handbook of Heterocyclic Chemistry, 2nd ed.,
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For a recent set of reviews in this area, see the themed issue: Applications of click
chemistry. Chem. Soc. Rev. 2010, 39, 1221.
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1
1
1
1
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9
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4
5
Huisgen, R. Angew. Chem. 1963, 75, 604.
For representative examples on metalꢀcatalyzed 1,3ꢀdipolar cycloaddition see: (a)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed.
2002, 41, 2596. (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
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G.; Fokin, V. V. J. Am. Chem. Soc. 2008, 130, 8923. (d) McNulty, J.; Keskar, K.;
Vemula, R. Chem. Eur. J. 2011, 17, 14727. (e) Ding, S.; Jia, G.; Sun, J. Angew.
Chem., Int. Ed. 2014, 53, 1877.
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7
8
Baskin, J. M.; Bertozzi, C. R. QSAR Comb. Sci. 2007, 26, 1211.
Jomova, K.; Valko, M. Toxicology 2011, 283, 65.
Gierlich, J.; Burley, G. A.; Gramlich, P. M. E.; Hammond, D. M.; Carell, T. Org. Lett.
006, 8, 3639.
2
9
Lallana, E.; FernandezꢀMegia, E.; Riguera, R. J. Am. Chem. Soc. 2009, 131, 5748.
Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598.
10
11
Gaetke, L. M.; Chow, C. K. Toxicology 2003, 189, 147.
12
Kennedy, D. C.; McKay, C. S.; Legault, M. C. B.; Danielson, D. C.; Blake, J. A.;
Pegoraro, A. F.; Stolow, A.; Mester, Z.; Pezacki, J. P. J. Am. Chem. Soc. 2011, 133,
1
7993.
13
Debets, M. F.; Van Berkel, S. S.; Dommerholt, J.; Dirks, A. J.; Rutjes, F. P. J. T.; Van
Delft, F. L. Acc. Chem. Res. 2011, 44, 805.
14
Baskin, J. M.; Bertozzi, C. R. Aldrichim. Acta 2010, 43, 15.
15
Lima, C. G. S.; Ali, A.; Van Berkel, S. S.; Westermann, B.; Paixão, M. W. Chem.
Commun. 2015, 51, 10784.
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Ramasastry, S. S. V. Angew. Chem. Int. Ed. 2014, 53, 14310.
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John, J.; Thomas, J.; Dehaen, W. Chem. Commun. 2015, 51, 10797.
Danence, L. J. T.; Gao, Y.; Li, M.; Huang, Y.; Wang, J. Chem. Eur. J. 2011, 17, 3584.
Belkheira, M.; Abed, D. E.; Pons. J. ꢀM.; Bressy, C. Chem. Eur. J. 2011, 17, 12917.
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