Click chemistry to fluorescent amino esters: Synthesis and spectroscopic studies

Article abstract of DOI:10.1002/ejoc.201000042

A new series of fluorescent amino esters have successfully been prepared by click chemistry by introducing different fluorophores (fluorescein, dansyl, 4-nitro-2,1,3-benzoxadiazole, coumarin and benzothiadlazole) into the side-chain of either serine, lysine or phenylalanine. The newly synthesized fluorescent amino esters displayed spectroscopic properties similar to their native fluorophores, with high quantum yields and fluorescence in the visible region (420-520 nm). Steadystate as well as time-resolved studies indicated, that benzothiadiazole derivatives 22-25 are promising probes for studying protein dynamics or molecular interactions, especially in the cell, due to their emissions in the visible region, their high, quantum yields and their relatively long lifetimes (14.7-18.3 ns). Moreover, the fluorescence of 7-hydroxycoumarinsubstituted amino ester 27 was very sensitive to the solvent proticity and pH value. This compound appears to be a suitable pH-sensitive amino acid for probing the local pH in cell compartments.

Full text of DOI:10.1002/ejoc.201000042

FULL PAPER
DOI: 10.1002/ejoc.201000042
Click Chemistry to Fluorescent Amino Esters: Synthesis and Spectroscopic
Studies
Chun Li,[a,b] Etienne Henry,[c] Naresh Kumar Mani,[c] Jie Tang,[b] Jean-Claude Brochon,[c]
Eric Deprez,*[c] and Juan Xie*[a]
Keywords: Amino acids / Click chemistry / Fluorescent probes / Fluorescence / Solvent effects
A new series of fluorescent amino esters have successfully
been prepared by click chemistry by introducing different
fluorophores (fluorescein, dansyl, 4-nitro-2,1,3-benzoxadi-
azole, coumarin and benzothiadiazole) into the side-chain of
either serine, lysine or phenylalanine. The newly synthesized
fluorescent amino esters displayed spectroscopic properties
similar to their native fluorophores, with high quantum yields
and fluorescence in the visible region (420–520 nm). Steady-
thiadiazole derivatives 22–25 are promising probes for study-
ing protein dynamics or molecular interactions, especially in
the cell, due to their emissions in the visible region, their
high quantum yields and their relatively long lifetimes (14.7–
18.3 ns). Moreover, the fluorescence of 7-hydroxycoumarin-
substituted amino ester 27 was very sensitive to the solvent
proticity and pH value. This compound appears to be a suit-
able pH-sensitive amino acid for probing the local pH in cell
state as well as time-resolved studies indicated that benzo- compartments.
cently been proposed to circumvent the problem of non-
Introduction
specific labelling, mainly based on the use of specific and
small peptidyl tags fused to the protein of interest. The first
one allows a specific and enzyme-mediated labelling of a
target protein at a single and predefined position. This en-
zymatic labelling, by transglutaminase, occurs specifically
on a Gln residue of the PKPQQFM peptide substrate by
using various commercially available fluorophore–cadaver-
ine compounds such as fluorescein–cadaverine or
TAMRA–cadaverine.[4] Specific covalent labelling can also
occur in an enzymatic-independent process by using a spe-
cific peptide tag containing four Cys residues and a bis-
(arsenic) fluorescein derivative. The latter strategy is com-
patible with labelling in a cellular environment.[5] Alterna-
tively, synthetic fluorescent amino acids are useful tools for
protein labelling because they can be introduced into pep-
tide sequences by chemical synthesis[6] or into proteins by
translational machinery.[7] Increasing effort has been de-
voted to the chemical synthesis of fluorescent amino acids[8]
as a result of their various biological applications.[9] How-
ever, there are no general methods for introducing a fluoro-
phore into the amino acid sequence: different strategies
have to be employed to obtain each fluorescent amino
acid.[8]
The fluorescent labelling of biomolecules is a powerful
method for investigating various biological events at a mo-
lecular level.[1] With fluorescence spectroscopy, different as-
pects of fluorescence output, for example, steady-state in-
tensity, Stokes shifts, lifetimes, steady-state and time-
resolved anisotropies and resonance energy transfer, can be
used for studying biological structures, dynamics and func-
tions and for visualizing intracellular processes or molecu-
lar interactions.[2] Considerable progress has been realized
since the discovery of green fluorescent protein (GFP) and
its derivatives, which allowed the labelling of a protein of
interest by a fluorescent protein.[3] However, such fusion
proteins, obtained by molecular biology techniques, can
lead to steric hindrance problems and, consequently, to per-
turbation of the natural function of the protein of interest.
Proteins can also be chemically labelled in vitro by using
extrinsic small organic fluorophores. Depending on the
number of reactive groups available on the protein, chemi-
cal labelling may lead to heterogeneous populations of
fluorescently labelled proteins. Several strategies have re-
[a] PPSM, Institut d’Alembert, ENS de Cachan, CNRS UMR
8531,
CuI-catalysed
Huisgen
1,3-dipolar cycloaddition
61 Avenue du Pt Wilson, 94235 Cachan, France
Fax: +33-1-47402454
(CuAAC) between terminal alkynes and organic azides
(also known as “click chemistry”) has recently been exten-
sively used and has found wide applications in organic syn-
thesis, medicinal chemistry, molecular biology and polymer
and materials science because of its high efficiency, versati-
lity, regioselectivity and excellent functional-group compati-
bility.[10] Furthermore, the 1,2,3-triazole ring formed is re-
E-mail: joanne.xie@ens-cachan.fr
[b] Institute of Medicinal Chemistry, Department of Chemistry,
East China Normal University,
Shanghai 200062, China
[c] LBPA, Institut d’Alembert, ENS de Cachan, CNRS UMR
8113,
61 Avenue du Pt Wilson, 94235 Cachan,France
Fax: +33-147407671
E-mail: deprez@lbpa.ens-cachan.fr
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E. Deprez, J. Xie et al.
FULL PAPER
sistant to hydrolysis, oxidation, reduction, and other modes cent amino acids. As amino acids, we used Ser, Lys and Phe
of cleavage. Owing to their electronic properties, 1,2,3-tri- as the starting materials. To obtain dansyl amino acids, O-
azoles have been employed as rigid linking units to mimic propargyl-Boc-Ser-OBn (2)[15] was treated with the dansyl
amide and ester bonds.[10d,11] Click chemistry has been used azide 1[16] in the presence of CuSO4/Na ascorbate in a mix-
for the fluorescent labelling of alkyne- or azide-function- ture of CH2Cl2/H2O at room temperature (Scheme 1). The
alized proteins[12] or for profiling enzyme activities.[13] Tri- dansyl serine derivative 3 was obtained in 94% yield. Simi-
azole amino acids and peptides have also been reported.[14] larly, the click reaction of the dansyl alkyne 4[17] with azido
However, to the best of our knowledge, the synthesis of amino esters 5,[18] 7[15] and 9[19] afforded the corresponding
fluorescent amino acids by click chemistry has never been compounds 6 (76%), 8 (94%) and 10 (93%).
reported. We have successfully used natural amino acids as
Fluorescein-substituted amino esters 12, 13 and 14 were
the starting materials to obtain their fluorescent derivatives, prepared in excellent yields by CuAAC between the alkyne
and we report herein the preparation and basic photophysi- 11[20] and the azido amino esters 5, 7 and 9, respectively
cal characterization of these molecules in the amino ester (Scheme 2).
forms. Several of these molecules were found to be suitable
To synthesize NBD derivatives [7-(alkylamino)-substi-
for biological applications, especially compounds 22–25 and tuted 4-nitro-2,1,3-benzoxadiazole], we first prepared azi-
27. It is important to note that the amino ester derivatives do- or alkyne-substituted NBDs 15 and 17 by treating
are characterized by poor water solubility. However, the NBD-Cl with 2-azidoethylamine or propargylamine under
CO2Me group can be easily deprotected by saponification. basic conditions (Scheme 3). The click reaction of 2 with 15
The saponification procedure was tested on compound 22, was realized with CuI as catalyst at 90 °C to afford com-
and its soluble version 23 was successfully obtained and pound 16. No reaction was observed with CuSO4/Na as-
characterised.
corbate at room temperature. Propargyl-NBD 17 reacted
with azides 5 and 7 at room temperature to give the corre-
sponding alanine and lysine derivatives 18 and 19, respec-
tively. The phenylalanine derivative 20 was obtained after
reaction under reflux.
Results and Discussion
Synthesis
We have also prepared benzothiadiazole-substituted
Fluorophores like dansyl, fluorescein, 4-nitro-2,1,3- amino acid derivatives (Scheme 4). The reaction of TMS-
benzoxadiazole, benzothiadiazole and coumarin with emis- alkyne 21[21] with azide 5 in the presence of CuSO4/Na as-
sion at around 500 nm were chosen to prepare the fluores- corbate/TBAF at room temperature led to 22 in 81% yield.
Scheme 1. Synthesis of dansyl-substituted amino esters 3, 6, 8 and 10.
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Click Chemistry to Fluorescent Amino Esters
Scheme 2. Synthesis of fluorescein-substituted amino esters 12–14.
Scheme 3. Synthesis of NBD-substituted amino esters 16, 18–20.
Saponification of 22 led to the free acid 23 in 97% yield.
It was also convenient to prepare coumarin-substituted
The corresponding Lys and Phe derivatives 24 and 25 were serine derivatives 27 and 29 by CuAAC by treating 2 with
synthesized in a similar way.
the azidocoumarins 26[22] and 28[22] (Scheme 5).
Scheme 4. Synthesis of benzothiadiazole-substituted amino acid derivatives 22–25.
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Scheme 5. Synthesis of coumarin-substituted amino esters 27 and 29.
Spectroscopic Properties
beit with a dramatic decrease in the quantum yield, as
shown previously.[23] Identical results were obtained with
compounds 13 and 14. Thus, compounds 12–14 were
studied under conditions that favour the anionic forms,[23]
that is, in DMSO/buffer solutions of increasing pH (see
Table 1). In the pH range of 7–9, their quantum yields were
significantly higher (ΦF = 0.31–0.36) than those obtained
in ethanol or DMSO, which shows that the low quantum
yields in ethanol and DMSO are due to the prevalence of
the non-fluorescent lactone form.
General Spectroscopic Properties
First, the steady-state fluorescence parameters of several
fluorescent amino acids were determined in two solvents,
DMSO and ethanol, characterized by different polarities
(Table 1). The fluorescence quantum yields were deter-
mined by using fluorescein or quinine sulfate as standards
for compounds 12 and 18 and for compounds 6 and 22–29,
respectively (see the Exp. Sect.).
The fluorescence emissions of the different compounds
presented in Table 1 were affected by solvent polarity to
different extents, with the most marked redshifts in emis-
sion being observed for compounds 6 and 18. Note that
compound 23, the water-soluble version of compound 22,
With the exception of the fluorescein derivative (com-
pound 12), the spectroscopic properties of the newly syn-
thesized fluorophores in EtOH and DMSO were similar to
the native molecules: they exhibited large Stokes shifts and
high quantum yields. Compound 12 (lactone form of fluo-
rescein) displayed excitation and emission characteristics
qualitatively similar to the standard anionic fluorescein, al-
was characterized by a significant redshift of its fluores-
F
cence emission in water compared with DMSO (λmax
=
Table 1. Spectroscopic steady-state data of the fluorescent amino acid derivatives.[a,b]
∆νA–F [cm–1]
ε00 [Lmol–1 cm–1]
ΦF
A
F
Compound
Solvent
λmax [nm]
λmax [nm]
6
EtOH
DMSO
EtOH
DMSO
T7D[d]
T8D[d]
T9D[d]
EtOH
DMSO
EtOH
DMSO
EtOH
DMSO
Water
EtOH
DMSO
EtOH
DMSO
EtOH
DMSO
EtOH
DMSO
336
342
276/480
277
517
532
512
538
516
516
516
527
537
476
473
473
475
500
481
479
478
480
421
426
416
419
10420
10443
16701/1302
17514
3600
3000
6400/720
6300
0.40
0.64
0.02
0.005
0.31
0.35
0.36
0.57
0.83
0.86
0.73
0.39
0.35
0.22
0.82
0.75
0.49
0.46
0.65
0.59
0.80
0.53
12[c]
455/480
455/480
455/480
326/456
341/471
316/360
318/366
316/359
316/360
316/350
316/360
317/359
316/360
319/362
346
2598/1454
2598/1454
2598/1454
11700/2954
10704/2609
10637/6769
10305/6181
10504/6714
10593/6725
11646/8571
10856/6988
10669/6978
10725/6857
10515/6791
4875
27500/25500
30000/27000
30000/27000
20000/7600
21000/6600
14000/5200
10000/3600
14800/5400
10400/3600
10200/3200
14000/6100
11500/4100
15000/6300
14800/7800
20000
18
22
23
24
25
27
29
346
344
344
5289
5031
5203
19000
20000
18000
[a] λmaxA: absorbance wavelength, λmaxF: emission wavelength, ∆υA–F: Stokes shift, ε00: molar absorbance coefficient, ΦF: fluorescence
quantum yield. [b] When molecules are characterized by two absorption modes (12 in EtOH and 18–25 in both EtOH and DMSO), both
F
of the absorption modes are responsible for fluorescence emission and the same λmax was obtained regardless of the excitation wave-
length. For such compounds, ΦF was measured by using the highest absorption wavelength. [c] Compounds 13 and 14 have spectroscopic
and solubility properties identical to 12 (data not shown). [d] T7D, T8D and T9D correspond to DMSO/Tris buffer mixtures (2:8, v/v)
at pH = 7, 8 and 9, respectively.
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Click Chemistry to Fluorescent Amino Esters
500 and 475 nm, respectively). Importantly, all the amino
acid derivatives emitted fluorescence in the visible region of
the spectrum, from 420 nm for compounds 27 and 29 to
520 nm for compounds 6, 12–14 and 18. Thus, the different
amino acid derivatives tested appear to be suitable fluores-
cent probes for protein/peptide labelling and cellular im-
aging applications, in contrast to the natural tryptophan
amino acid which emits at 330–350 nm.
Time-Resolved Fluorescence
To compare the fluorescence lifetimes of the non-natural
amino esters and tryptophan, we next performed time-re-
solved fluorescence measurements. The influence of solvent
polarity on the lifetimes was also assessed, because fluores-
cence lifetimes are known to be highly sensitive to environ-
ment, with particularly high polar solvents responsible for
a substantial decrease in the lifetimes because of solvent
relaxation. Most of the compounds are not fully soluble in
water due to the presence of Boc and methyl groups (except
for compound 23 which is in the free acid form). The fluo-
rescence lifetimes were thus measured in pure DMSO and
in a DMSO/water mixture (2:8, v/v) (Table 2).
Figure 1. Fluorescence intensity decays of compounds 6, 18 and
22–25 in DMSO (5 µ). Acquisition and analysis of the intensity
decays were performed as described in the Exp. Sect.
cence lifetime imaging (FLIM) by enhancing image con-
trasts and accuracy in the determination of FRET effi-
ciency when measuring the decrease in the donor lifetime
upon interaction between donor- and acceptor-labelled
partners. In addition, another application of such long life-
time probes is related to the enhancement of image contrast
by time-gated fluorescence imaging, which reduces cellular
auto-fluorescence characterized by short fluorescence life-
times.
Furthermore, long lifetimes are also of particular interest
for time-resolved fluorescence anisotropy experiments. This
approach allows rotational correlation times to be deter-
mined. A long correlation time is related in a first approxi-
mation to the hydrated volume of an entire particle by the
Stokes–Einstein relationship and, for example, can be used
to discriminate between different higher-order oligomeric
states of a protein. However, by using the intrinsic trypto-
phan fluorescence, this approach suffers from the short life-
time of tryptophan, which is severely limited for estimating
precisely the long rotational correlation times characteriz-
ing large molecules (typically above 80–100 ns) due to a lim-
ited time window for measuring the fluorescence anisotropy
decay.[24] Therefore, extrinsic fluorescent probes with long
lifetimes could be suitable for improving the accurate recov-
ery of long rotational correlation times in protein studies. In
this context, the benzothiadiazole-substituted amino acid
derivatives 22–25 represent promising fluorophores for both
in vitro and in vivo biological applications based on fluores-
cence anisotropy or FLIM methodologies.
Table 2. Analysis of fluorescence decay.
Compound
τ [nm]
DMSO/H2O[a]
DMSO
6
17
7.1
3.4
2.0
18.3
15.6[b]
18.6
14.7
4.2
12
18
22
23
24
25
27
29
0.75
7.77
14.0
13.6
15.4
9.47
2.4
2.17
3.1
[a] DMSO/H2O in a ratio of 2:8 (v/v). [b] τ measured in Tris buffer
at pH = 7.
All tested compounds exhibited a monoexponential I(t)
decay (Figure 1). Moreover, compounds 6, 18 and 22–25
were characterized by long fluorescence lifetimes. However,
compounds 6 and 18 were highly sensitive to solvent po-
larity, as judged by the significant decrease in their lifetimes
upon the addition of water (from 17 to 7.1 ns and from
7.77 to 2.0 ns, respectively). In contrast, the lifetimes of the
benzothiadiazole-substituted amino acid derivatives 22–25
were long, above 14 ns in DMSO/water mixtures, much
higher than the fluorescence lifetime of tryptophan, which
is about 3 ns.
In conclusion, the steady-state and time-resolved studies
indicate that compounds 22–25 are promising probes for
studying protein dynamics or molecular interactions, espe-
cially in the cell environment due to their emissions in the
Insight into the Properties of Coumarin Derivatives 27 and
29
7-Hydroxycoumarin is a well-known pH-sensitive blue-
visible region of the spectrum and their high quantum fluorescent fluorophore with a pKa of 7.8.[25] Indeed, pro-
yields. More specifically, owing to their relative long life- tonation of the 7-hydroxy group induces significant changes
times, they could be suitable as donor molecules in time- in both the emission and absorption bands depending on
resolved fluorescence resonance energy transfer (FRET) ex- the proton-donating property of the solvent or pH.[26] Thus,
periments (i.e., when FRET efficiency is determined by me- we assessed whether compound 27 behaves as a 7-hydroxy-
asuring the donor lifetime) and, more generally, in fluores- coumarin. The spectroscopic properties of 27 were then fur-
Eur. J. Org. Chem. 2010, 2395–2405
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E. Deprez, J. Xie et al.
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Table 3. Spectral fluorescence characteristics of coumarin derivatives 27 and 29.[a]
A1
A2
F1
F2
F2
Entry
Solvent
λmax [nm]
λmax [nm]
λmax [nm]
λmax [nm]
ImaxF1/Imax
Compound 27
1
2
3
4
5
6
7
CH3CN
EtOH
DMSO
341
346
346
346
346
346
346
346
–
–
–
–
–
–
–
–
400
400
–
436
436
420
421
426
426
426
426
426
423
–
–
–
–
483
481
479
478
478
476
–
DMSO/H2O (8:2, v/v)
DMSO/H2O (6:4, v/v)
DMSO/H2O (4:6, v/v)
DMSO/H2O (2:8, v/v)
0.567
0.164
0.08
0.06
8
9
10
11
12
DMSO/Tris buffer pH = 7 (2:8, v/v)[b]
DMSO/Tris buffer pH = 9 (2:8, v/v)
DMSO + 10 µ NaOH[c]
345
345
–
424
424
–
DMSO + 50 µ NaOH[c,d]
485
485
DMSO + 100 µ NaOH[c]
Compound 29
13
14
15
16
17
DMSO
DMSO/H2O (2:8, v/v)
DMSO/Tris buffer pH = 7 (2:8, v/v)
DMSO/Tris buffer pH = 9 (2:8, v/v)
DMSO + 100 µ NaOH[c]
344
346
346
346
344
–
–
–
–
–
419
423
423
423
419
–
–
–
–
–
[a] ImaxF1/ImaxF2 corresponds to the intensity ratio of the emission maxima. [b] Excitation at 346 nm is responsible for the two fluorescence
emissions at 423 and 478 nm, whereas excitation at 400 nm is responsible for fluorescence emission at 478 nm only. [c] 10, 50 and 100 µ
NaOH corresponds to an NaOH/compound molar ratio of 2.8, 13.9 and 28, respectively. [d] Excitation at 345 nm is responsible for the
fluorescence emission at 424 nm only, and excitation at 436 nm is responsible for the fluorescence emission at 485 nm only.
ther studied by varying (1) the proticity of solvents by using 400 nm (7-O–). At pH = 9 or upon addition of 100 µ
aprotic solvents (acetonitrile, DMSO) and solvents with NaOH, only the excitation mode above 400 nm was ob-
either low (ethanol) or high (DMSO/water mixtures) protic- served (Entries 9 and 12, respectively). Under equilibrium
ity or (2) the pH by adding NaOH or by using buffered conditions and high proticity (Entry 8), excitation at
solutions (Table 3). Compound 29 was used as a control, 346 nm (selective excitation of the 7-OH form) leads to dual
because the 7-OH group in 27 is substituted by a methyl emission, that is, at 423 (7-OH*) and 478 nm (7-O–*) (as
group in 29.
found in Entries 4–7), whereas excitation at 400 nm (selec-
In aprotic solvents or solvents with low proticity, only tive excitation of the 7-O– form) only leads to emission at
slight differences appeared in the absorption and fluores- 478 nm (7-O–*). In contrast, different results were obtained
cence spectra of compound 27 (Table 3, Entries 1–3). In under aprotic conditions (Entry 11): excitation at 346 nm
sharp contrast, the emission spectra of compound 27 yields a single emission (at 424 nm, as found in Entries 1–
changed dramatically in solvents with higher proticity 3). The emission at 485 nm can be observed only with exci-
(DMSO/water mixtures): by increasing the solvent proticity tation above 400 nm. Again, the dual absorption was not
(achieved by decreasing the DMSO/water ratio), the fluo- observed with compound 29 (Entries 16 and 17). The 7-
rescence emission band intensity decreased at 426 nm, and
a second emission band appeared at 478 nm (Figure 2;
Table 3, Entries 3–7). This ratiometric behaviour is due to
the presence of the 7-OH group, because compound 29 was
unaffected by modulation of the DMSO/water ratio
(Table 3, Entries 13 and 14). According to the prototype
compound 7-hydroxycoumarin,[25] this dual emission of
compound 27 is likely due to a protonation equilibrium in
the excited state, [7-OH]* h [7-O–]*, with the 7-OH* form
responsible for the emission band at 426 nm and the 7-O–*
form responsible for the emission band at 478 nm upon ex-
citation of the 7-OH form at 346 nm (see Figure 3). More-
over, by increasing the pH, a dual absorption is observed
that originates in a protonation equilibrium in the ground
state, 7-OH h 7-O–: increasing the pH of the Tris buffer
(Table 3, Entries 8 and 9) or directly adding NaOH in
DMSO (Table 3, Entries 10–12) favours the 7-O– ground-
state form. Consequently, the absorbance at 346 nm (7-OH)
decreased, and a second absorption band appeared at above of the DMSO/water ratio. λexc = 346 nm.
Figure 2. Emission spectra of compound 27 (2.6 µ) as a function
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Click Chemistry to Fluorescent Amino Esters
spectrofluorimeter (Varian). For the emission measurements, the
absorbance at the excitation wavelengths was kept below 0.05 to
avoid inner filter effects. DMSO, ethanol, acetonitrile as well as Tris
buffer (20 m), prepared with Millipore-filtered water (resistivity
Ͼ 18 MΩcm–1 at 20 °C), were from Sigma–Aldrich (spectrometric
grade). Quantum yields were estimated by using fluorescein in
0.1  NaOH (ΦF = 0.95) and quinine sulfate in 0.1  H2SO4 (ΦF
= 0.58 for λex = 350 nm) as standards, taking into account the
refractive index of the standard and the sample solvent according
to ref.[2] Time-resolved fluorescence experiments were performed to
determine the fluorescence lifetimes, which were obtained from the
fluorescence intensity decays, I(t), by using the time-correlated sin-
gle photon counting technique. The instrumentation setup was es-
sentially the same as those previously described[27] with modifica-
tions: the excitation light pulse source was a Ti:sapphire laser (Mai-
tai femtosecond laser, Spectra Physics) associated with either a sec-
ond or a third harmonic generator tuned at 420 or 300 nm, respec-
tively. The polarizer was set at the magic angle (54.7°), and the
emission monochromator (ARC SpectraPro-150) was set at the
maximum emission pick (∆λ = 15 nm). To determine the lifetime
(τ) distribution, I(t) was analysed by the maximum entropy
method.[24,28]
hydroxycoumarin derivative thus appears to be a suitable
pH-sensitive amino acid to probe local pH in cell compart-
ments.
Figure 3. Model for the influence of proticity and pH on the ab-
sorption and emission bands of compound 27.
Conclusions
We have reported an efficient synthesis of new fluores-
cent amino ester derivatives by introducing fluorophores
into the side-chain of natural amino acids like serine, lysine
General Procedure A for the Click Reaction: CuSO4·5H2O (0.008 to
0.02 mmol) and Na ascorbate (0.0017 to 0.04 mmol) were added
and phenylalanine by click chemistry. Dansyl, NBD, fluo- successively to
a solution of alkyne (0.1 mmol) and azide
(0.1 mmol) in CH2Cl2 (2 mL) and water (2 mL). The reaction mix-
ture was vigorously stirred at room temp. for 12 h. CH2Cl2 (5 mL)
was then added. After separation of the organic layer, the aqueous
layer was extracted with CH2Cl2 (3ϫ10 mL). The combined or-
ganic layers were washed with brine (20 mL), dried with MgSO4
and the solvents evaporated.
rescein, coumarin and benzothiadiazole derivatives have
been prepared by this approach. Spectroscopic studies
showed that all the newly synthesized fluorophores exhibit
similar fluorescence properties compared with the native
molecules, with large Stokes shifts and high quantum yields.
Time-resolved fluorescence measurements demonstrated
that all compounds exhibited a monoexponential I(t) decay.
In particular, the benzothiadiazole-substituted amino ester
derivatives 22–25 showed long fluorescence lifetimes (above
14 ns in DMSO/water mixtures), much longer than that of
tryptophan (about 3 ns). Consequently, compounds 22–25
represent promising fluorophores for both in vitro and in
vivo biological applications based on fluorescence anisot-
ropy or FLIM methodologies. Moreover, the fluorescence
properties of 7-hydroxycoumarin-substituted amino ester
27 appeared to be very sensitive to the proticity of the sol-
vent and the pH, which makes this molecule a suitable pH-
sensitive amino acid for probing the local pH in cell com-
General Procedure B for the Click Reaction: CuI (0.03 mmol) was
added to a solution of alkyne (0.1 mmol) and azide (0.1 mmol) in
tBuOH (2 mL) and water (2 mL). The reaction mixture was vigor-
ously stirred under argon at 90 °C for 48 h. Then water (15 mL)
was added. After separation of the organic layer, the aqueous layer
was extracted with CH2Cl2 (3ϫ10 mL). The combined organic lay-
ers were washed with brine (20 mL), dried with MgSO4 and the
solvents evaporated.
Benzyl
(S)-2-(tert-Butoxycarbonylamino)-3-[(1-{2-[5-(dimethyl-
amino)naphthalen-1-ylsulfonamido]ethyl}-1H-1,2,3-triazol-4-yl)meth-
oxy]propanoate (3): From alkyne 2 (23 mg, 0.07 mmol) and azide 1
(21.8 mg, 0.07 mmol), the click reaction according to the General
Procedure A led to 3 as a light-green solid (42 mg, 94%). [α]D
=
1
partments. Finally, the different amino esters can be easily –1.0 (c = 0.5, CH2Cl2). H NMR (400 MHz, CDCl3): δ = 8.57 (d,
3
3JH,H = 8.7 Hz, 1 H, Ar-H), 8.23 (d, JH,H = 7.3 Hz, 1 H, Ar-H),
deprotected to yield hydrosoluble amino acids, as testified
3
8.17 (d, JH,H = 8.7 Hz, 1 H, Ar-H), 7.53–7.48 (m, 2 H, Ar-H),
by the successful conversion of ester 22 into carboxylic acid
7.30–7.27 (m, 5 H, Ph), 7.23 (s, 1 H, Ar-H), 7.17 (d, 3JH,H = 7.4 Hz,
23 by saponification.
3
3
1 H, Ar-H), 5.69 (t, JH,H = 6.2 Hz, 1 H, NH), 5.44 (d, JH,H
=
2
8.7 Hz, 1 H, NH), 5.23 (d, JH,H = 12.4 Hz, 1 H, CHPh), 5.13 (d,
2JH,H = 12.4 Hz, 1 H, CHPh), 4.55–4.44 (m, 3 H, 2-H, CH2), 4.35
(t, 3JH,H = 5.3 Hz, 2 H, CH2), 3.90 (dd, 3JH,H = 2.8, 2JH,H = 9.2 Hz,
Experimental Section
1 H, 3-H), 3.71 (dd, 3JH,H = 3.2, JH,H = 9.6 Hz, 1 H, 3Ј-H), 3.39–
2
General: 1H and 13C NMR spectra were recorded with a Jeol
ECS400 spectrometer. Column chromatography was performed
with silica gel 60 (230–400 mesh). Analytical thin-layer chromatog-
raphy was performed with aluminium precoated plates of silica gel
60F-254 with detection carried out by using UV light and ninhyd-
rin. ESI-HRMS were recorded with a Thermo LTQ-Orbitrap spec-
trometer at the Service of Mass Spectrometry of the University
Pierre and Marie Curie. UV/Vis absorption spectra were recorded
with a double-beam Uvikon 933 spectrophotometer. Corrected ex-
citation and emission spectra were obtained with a Cary Eclipse
3.35 (m, 2 H, N-CH2), 2.86 (s, 6 H, 2 N-Me), 1.41 (s, 9 H, Boc)
ppm. 13C NMR (100 MHz, CDCl3): δ = 170.7, 155.6, 152.2, 135.6,
134.4, 130.9, 130.0, 129.6, 128.8, 128.6, 128.4, 128.2, 123.2, 118.6,
115.5, 80.2, 70.1, 67.3, 64.6, 54.2, 50.4, 45.5, 42.9, 28.4 ppm.
HRMS (ESI): calcd. for C32H41N6NaO7S 675.2577; found
675.2571.
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-(4-{[5-(dimethylamino)-
naphthalen-1-ylsulfonamido]methyl}-1H-1,2,3-triazol-1-yl)propano-
ate (6): From alkyne 4 (36 mg, 0.13 mmol) and azide 5 (30 mg,
Eur. J. Org. Chem. 2010, 2395–2405
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2401

E. Deprez, J. Xie et al.
FULL PAPER
0.13 mmol), the click reaction according to the General Pro-
cedure A followed by purification by column chromatography (pe-
troleum ether/EtOAc, 1:2) afforded 6 as a colourless liquid
(134 mg, 76%). Rf = 0.1 (petroleum ether/EtOAc, 1:2). [α]D = +26.4
(c = 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3): δ = 8.57 (d, 3JH,H
= 8.7 Hz, 1 H, Ar-H), 8.27–8.22 (m, 2 H, Ar-H), 7.59–7.52 (m, 2
153.1, 152.5, 152.4, 143.5, 135.2, 129.8, 129.2, 126.7, 125.0, 124.4,
124.1, 112.7, 111.9, 111.8, 110.5, 103.1, 102.1, 77.4, 61.8, 53.7, 53.3,
51.2, 28.3 ppm. HRMS (ESI): calcd. for C32H30N4NaO9 637.1911;
found 637.1915.
Methyl (2S)-2-(Benzyloxycarbonylamino)-6-{4-[(3Ј-hydroxy-3-oxo-
3H-spiro[isobenzofuran-1,9Ј-xanthen]-6Ј-yloxy)methyl]-1H-1,2,3-tri-
azol-1-yl}hexanoate (13): From alkyne 11 (30 mg, 0.08 mmol) and
azide 7 (25 mg, 0.08 mmol), the click reaction according to the Ge-
3
H, Ar-H), 7.28 (s, 1 H, CH=C), 7.20 (d, JH,H = 6.8 Hz, 1 H, Ar-
3
3
H), 5.29 (d, JH,H = 6.4 Hz, 1 H, NH), 5.15 (t, JH,H = 6.0 Hz, 1
H, NH), 4.73–4.63 (m, 3 H, 2-H, N-CH2), 4.19 (d, 3JH,H = 6.4 Hz,
2 H, CH2), 3.76 (s, 3 H, OMe), 2.90 (s, 6 H, 2 N-CH3), 1.45 (s, 9
H, Boc) ppm. 13C NMR (100 MHz, CDCl3): δ = 169.4, 155.1,
152.1, 143.8, 134.4, 130.8, 129.8, 128.8, 123.5, 123.2, 118.6, 115.4,
80.9, 53.7, 53.3, 50.9, 45.5, 38.9, 28.3 ppm. HRMS (ESI): calcd. for
C24H32N6NaO6S 555.2002; found 555.2003.
neral Procedure A led to 13 as a brown solid (50 mg, 91%). [α]D
=
1
+3.0 (c = 0.5, CH2Cl2). H NMR (400 MHz, CDCl3): δ = 8.13 (s,
1 H, OH), 7.98 (dd, 4JH,H = 0.9, 3JH,H = 7.3 Hz, 1 H, Ar-H), 7.66–
3
3
7.56 (m, 2 H, Ar-H), 7.57 (dd, JH,H = 0.9, JH,H = 7.4 Hz, 1 H,
3
Ar-H), 7.32–7.25 (m, 5 H, Ph), 7.12 (d, JH,H = 7.3 Hz, 1 H, Ar-
H), 6.82 (d, JH,H = 2.3 Hz, 1 H, Ar-H), 6.74 (d, JH,H = 1.8 Hz,
1 H, Ar-H), 6.64–6.58 (m, 2 H, Ar-H), 6.56–6.53 (m, 2 H, Ar-H),
4
4
Methyl (S)-2-(Benzyloxycarbonylamino)-6-(4-{[5-(dimethylamino)-
naphthalen-1-ylsulfonamido]methyl}-1H-1,2,3-triazol-1-yl)hexano-
ate (8): From alkyne 4 (23 mg, 0.08 mmol) and azide 7 (25 mg,
0.08 mmol), the click reaction according to the General Pro-
cedure A led to 8 as a colourless liquid (45 mg, 94%). [α]D = +6.2
(c = 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3): δ = 8.54 (d, 3JH,H
3
5.43 (d, JH,H = 7.8 Hz, 1 H, NH), 5.16 (s, 2 H, CH2), 5.07 (s, 2
H, CH2), 4.36–4.28 (m, 3 H, 2-H, CH2), 3.69 (s, 3 H, OMe), 1.93–
1.30 (m, 6 H, 3 CH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
172.6, 169.9, 159.7, 158.7, 156.1, 153.1, 152.5, 152.4, 143.3, 136.1,
135.2, 12.7, 129.2, 128.6, 128.3, 128.1, 123.1, 112.8, 111.9, 111.8,
110.5, 103.1, 102.2, 67.2, 61.8, 53.5, 52.6, 50.2, 32.0, 29.5,
22.2 ppm. HRMS (ESI): calcd. for C38H34N4NaO9 713.2224; found
713.2222.
4
3
= 8.7 Hz, 1 H, Ar-H), 8.25 (dd, JH,H = 1.4, JH,H = 7.8 Hz, 2 H,
Ar-H), 7.53–7.47 (m, 2 H, Ar-H), 7.35–7.27 (m, 5 H, Ph), 7.21 (s,
3
3
1 H, CH=C), 7.16 (d, JH,H = 7.4 Hz, 1 H, Ar-H), 5.58 (t, JH,H
=
6.2 Hz, 1 H, NH), 5.42 (d, 3JH,H = 8.2 Hz, 1 H, NH), 5.07 (s, 2 H,
Methyl (2S)-2-(tert-Butoxycarbonylamino)-3-(4-{4-[(3Ј-hydroxy-3-
oxo-3H-spiro[isobenzofuran-1,9Ј-xanthen]-6Ј-yloxy)methyl]-1H-
1,2,3-triazol-1-yl}phenyl)propanoate (14): From alkyne 11 (25 mg,
0.07 mmol) and azide 9 (22 mg, 0.07 mmol), the click reaction ac-
cording to the General Procedure A followed by purification by
column chromatography (petroleum ether/EtOAc, 1:2) afforded 14
as a yellow solid (45 mg, 94%). [α]D = +24.8 (c = 0.3, CH2Cl2). 1H
PhCH2), 4.34–4.30 (m, 1 H, 2-H), 4.18 (d, 3JH,H = 6.4 Hz, 2 H, N-
3
CH2), 4.14 (t, JH,H = 6.8 Hz, 2 H, N-CH2), 3.71 (s, 3 H, OMe),
2.86 (s, 6 H, 2 N-CH3), 1.82–1.19 (m, 6 H, 3 CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 172.7, 155.9, 152.1, 143.9, 136.2, 134.7,
130.7, 129.9, 129.7, 128.6, 128.3, 128.2, 122.2, 118.8, 115.4, 67.2,
53.5, 52.6, 49.9, 45.5, 38.9, 31.9, 29.5, 22.1 ppm. HRMS (ESI):
calcd. for C30H36N6NaO6S 631.2315; found 631.2292.
3
NMR (400 MHz, CDCl3): δ = 8.06 (s, 1 H, Ar-H), 8.01 (d, JH,H
3
= 7.3 Hz, 1 H, Ar-H), 7.66–7.60 (m, 5 H, Ar-H), 7.29 (d, JH,H
=
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-[4-(4-{[5-(dimeth-
ylamino)naphthalen-1-ylsulfonamido]methyl}-1H-1,2,3-triazol-1-yl)-
phenyl]propanoate (10): From alkyne 4 (30 mg, 0.1 mmol) and azide
9 (33.8 mg, 0.1 mmol), the click reaction according to the General
3
8.2 Hz, 1 H, Ar-H), 7.16 (d, JH,H = 7.3 Hz, 1 H, Ar-H), 6.86 (s, 1
H, Ar-H), 6.76–6.74 (m 1 H, Ar-H), 6.67–6.66 (m, 2 H, Ar-H),
6.59 (s, 1 H, Ar-H), 6.58 (dd, 4JH,H = 2.3, 3JH,H = 8.7 Hz, 1 H, Ar-
3
H), 5.28 (s, 2 H, CH2), 5.11 (d, JH,H = 7.8 Hz, 1 H, NH), 4.64–
Procedure A led to 10 as a colourless liquid (60 mg, 93%). [α]D
=
4.60 (m, 1 H, 2-H), 3.70 (s, 3 H, OMe), 3.23 (dd, 3JH,H = 5.5, 2JH,H
+30.6 (c = 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3): δ = 8.49
3
2
= 13.8 Hz, 1 H, 3-H), 3.11 (dd, JH,H = 6.4, JH,H = 13.8 Hz, 1 H,
3Ј-H), 1.41 (s, 9 H, Boc) ppm. 13C NMR (100 MHz, CDCl3): δ =
172.1, 169.9, 159.8, 158.6, 155.2, 153.2, 152.6, 152.5, 144.2, 137.5,
135.8, 135.2, 130.8, 129.8, 129.3, 126.8, 125.1, 124.1, 121.3, 120.8,
112.7, 111.9, 110.7, 103.2, 102.1, 80.4, 61.7, 54.4, 52.5, 38.1,
28.4 ppm. HRMS (ESI): calcd. for C38H34N4NaO9 713.2224; found
713.2218.
3
(d, JH,H = 8.7 Hz, 1 H, Ar-H), 8.27–8.23 (m, 2 H, Ar-H), 7.53 (s,
3
1 H, Ar-H), 7.51–7.43 (m, 4 H, Ar-H), 7.22 (d, JH,H = 7.8 Hz, 2
4
3
H, Ar-H), 7.13 (dd, JH,H = 1.4, JH,H = 7.8 Hz, 2 H, Ar-H), 5.74
3
(s, 1 H, NH), 5.07 (d, JH,H = 7.4 Hz, 1 H, NH), 4.62–4.57 (m, 1
H, 2-H), 4.30 (d, JH,H = 6.0 Hz, 2 H, CH2), 3.71 (s, 3 H, OMe),
3
3
2
3
3.20 (dd, JH,H = 5.5, JH,H = 13.3 Hz, 1 H, 3-H), 3.09 (dd, JH,H
= 6.4, 2JH,H = 13.8 Hz, 1 H, 3Ј-H), 2.81 (s, 6 H, 2 N-CH3), 1.40 (s,
9 H, Boc) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.1, 155.1,
152.1, 144.6, 137.3, 135.7, 134.7, 130.8, 130.6, 129.9, 129.8, 129.6,
128.7, 123.2, 120.5, 120.3, 118.7, 115.4, 80.3, 54.4, 52.5, 45.4, 38.8,
38.0, 28.4 ppm. HRMS (ESI): calcd. for C30H37N6O6 609.2495;
found 609.2490.
N-(2-Azidoethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (15): A
solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole (200 mg,
1 mmol) in methanol (3.9 mL) was added to a solution of (2-azido-
ethyl)amine (90 mg, 1 mmol) in 0.3  NaHCO3 (3.9 mL), and the
reaction mixture was stirred at 50 °C for 3 h. The solvent was evap-
orated under reduced pressure. The residue was purified by column
chromatography to afford 15 as a black solid (90 mg, 36%). 1H
Methyl (2S)-2-(tert-Butoxycarbonylamino)-3-{4-[(3Ј-hydroxy-3-oxo-
3H-spiro[isobenzofuran-1,9Ј-xanthen]-6Ј-yloxy)methyl]-1H-1,2,3-tri-
azol-1-yl}propanoate (12): From alkyne 11 (37 mg, 0.1 mmol) and
azide 5 (24 mg, 0.1 mmol), the click reaction according to the Ge-
3
NMR (400 MHz, CDCl3): δ = 8.52 (d, JH,H = 8.7 Hz, 1 H, 6-H),
3
6.30 (s, 1 H, NH), 6.27 (d, JH,H = 8.7 Hz, 1 H, 5-H), 3.78–3.76
(m, 2 H, CH2), 3.73–3.69 (m, 2 H, CH2) ppm. 13C NMR
(100 MHz, [D6]DMSO): δ = 145.7, 145.0, 138.4, 121.9, 100.1, 49.4,
43.4 ppm. HRMS (ESI): calcd. for C8H7N7NaO3 272.0508; found
272.0503.
neral Procedure A led to 12 as a brown solid (55 mg, 94%). [α]D
=
1
–16.5 (c = 0.3, MeOH). H NMR (400 MHz, CDCl3): δ = 8.01 (d,
3JH,H = 7.4 Hz, 1 H, Ar-H), 7.91 (s, 1 H, OH), 7.67–7.60 (m, 3 H,
3
Ar-H), 7.15 (d, JH,H = 7.8 Hz, 1 H, Ar-H), 6.82 (m, 1 H, Ar-H),
3
6.74 (s, 1 H, Ar-H), 6.67 (d, JH,H = 8.7 Hz, 1 H, Ar-H), 6.63–6.60
Benzyl (S)-2-(tert-Butoxycarbonylamino)-3-({1-[2-(7-nitrobenzo-
[c][1,2,5]oxadiazol-4-ylamino)ethyl]-1H-1,2,3-triazol-4-yl}methoxy)-
(m, 1 H, Ar-H), 6.58–6.55 (m, 2 H, Ar-H), 5.45 (d, 3JH,H = 6.4 Hz,
1 H, NH), 5.19 (s, 2 H, O-CH2), 4.83–4.81 (m, 2 H, N-CH2), 4.74– propanoate (16): From alkyne 2 (40 mg, 0.12 mmol) and azide 15
4.70 (m, 1 H, 2-H), 3.76 (s, 3 H, OMe), 1.42 (s, 9 H, Boc) ppm. (30 mg, 0.12 mmol), the click reaction according to the General
13C NMR (100 MHz, CDCl3): δ = 169.9, 169.4, 159.7, 158.6, 155.2, Procedure B followed by purification by column chromatography
2402
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2395–2405

Click Chemistry to Fluorescent Amino Esters
(petroleum ether/EtOAc, 1:2) afforded 16 as a yellow solid (66 mg,
Procedure B followed by purification by column chromatography
93%). [α]D = –2.0 (c = 0.5, CH2Cl2). 1H NMR (400 MHz, CDCl3): (petroleum ether/EtOAc, 1:2) afforded 20 as a yellow solid (50 mg,
3
δ = 8.40 (d, JH,H = 8.3 Hz, 1 H, Ar-H), 7.41–7.29 (m, 6 H, Ar-
69%). [α]D = +1.2 (c = 0.3, MeOH). 1H NMR (400 MHz, CDCl3):
3
3
3
H), 6.17 (d, JH,H = 8.2 Hz, 1 H, Ar-H), 5.43 (d, JH,H = 8.3 Hz,
δ = 8.46 (d, JH,H = 9.9 Hz, 1 H, Ar-H), 8.04 (s, 1 H, Ar-H), 7.62
2
2
3
3
1 H, NH), 5.21 (d, JH,H = 12.4 Hz, 1 H, CHPh), 5.09 (d, JH,H
=
(d, JH,H = 8.3 Hz, 2 H, Ar-H), 7.27 (d, JH,H = 8.3 Hz, 2 H, Ar-
3
12.4 Hz, 1 H, CHPh), 4.68 (s, 2 H, CH2), 4.62–4.52 (m, 2 H, CH2), H), 7.03 (s, 1 H, NH), 6.39 (d, JH,H = 8.7 Hz, 1 H, Ar-H), 5.04
4.46 (m, 1 H, 2-H), 4.09 (s, 2 H, CH2), 3.92 (dd, 3JH,H = 2.8, 2JH,H
(d, JH,H = 7.8 Hz, 1 H, NH), 4.90 (d, JH,H = 5.5 Hz, 2 H, N-
3
3
3
2
3
= 9.2 Hz, 1 H, 3-H), 3.75 (dd, JH,H = 2.8, JH,H = 9.2 Hz, 1 H, CH2), 4.58 (m, 1 H, 2-H), 3.72 (s, 3 H, OMe), 3.18 (dd, JH,H
=
=
3Ј-H), 1.40 (s, 9 H, Boc) ppm. 13C NMR (100 MHz, CDCl3): δ = 5.5, JH,H = 13.8 Hz, 1 H, 3-H), 3.05 (dd, JH,H = 6.4, JH,H
2
3
2
170.7, 155.6, 145.1, 144.3, 143.9, 143.5, 136.2, 135.5, 128.6, 128.4,
13.7 Hz, 1 H, 3Ј-H), 1.39 (s, 9 H, Boc) ppm. 13C NMR (100 MHz,
128.0, 124.6, 123.8, 80.3, 76.8, 70.4, 64.7, 54.2, 48.6, 43.6, CDCl3): δ = 172.0, 155.2, 144.4, 143.9, 143.2, 143.1, 138.0, 136.3,
28.4 ppm. HRMS (ESI): calcd. for C26H30N8NaO8 605.2084; found 135.0, 130.9, 124.8, 120.7, 120.4, 99.8, 80.3, 54.4, 52.6, 39.5, 38.1,
605.2079.
28.4 ppm. HRMS (ESI): calcd. for C24H26N8NaO7 561.1822; found
561.1817.
7-Nitro-N-(prop-2-ynyl)benzo[c][1,2,5]oxadiazol-4-amine (17): A
solution of 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole (124.3 mg,
0.623 mmol) in methanol (10 mL) was added to a solution of pro-
pargylamine (44 µL, 0.64 mmol) in 0.3  NaHCO3 (2.5 mL), and
the reaction mixture was stirred at 50 °C for 3 h. After solvent
evaporation, the residue was purified by column chromatography
Methyl (S)-3-[4-(Benzo[c][1,2,5]thiadiazol-4-yl)-1H-1,2,3-triazol-1-
yl]-2-(tert-butoxycarbonylamino)propanoate (22): From 4-(tri-
methylsilylethynyl)benzo[c][1,2,5]thiadiazole (21; 46.8 mg,
0.2 mmol) and azide 5 (49 mg, 0.2 mmol), the click reaction accord-
ing to the General Procedure A in the presence of Bu4NF (253 mg,
0.8 mmol) followed by purification by column chromatography (pe-
troleum ether/EtOAc, 1:1) afforded 22 as a yellow solid (65 mg,
81 %). [α]D = +38.8 (c = 0.5, CH2Cl2). 1H NMR (400 MHz,
(petroleum ether/EtOAc, 7:3) to afford 35 mg (30%) of 17 as a
3
black solid. 1H NMR (400 MHz, CDCl3): δ = 8.55 (d, JH,H
=
8.7 Hz, 1 H, 6-H), 6.36 (d, 3JH,H = 8.7 Hz, 1 H, 5-H), 6.32 (s, 1 H,
NH), 4.3 (dd, 4JH,H = 2.3, 3JH,H = 5.5 Hz, 2 H, CH2), 2.44 (t, 4JH,H
= 2.5 Hz, 1 H, CHϵC) ppm. 13C NMR (100 MHz, CDCl3): δ =
144.6, 144.0, 136.6, 123.5, 99.7, 77.4, 72.8, 32.2 ppm. HRMS (ESI):
calcd. for C9H6N4NaO3 241.0338; found 241.0332.
4
3
CDCl3): δ = 8.71 (s, 1 H, Ar-H), 8.52 (dd, JH,H = 1.4, JH,H
=
7.3 Hz, 1 H, Ar-H), 7.97 (dd, 4JH,H = 1.4, 3JH,H = 9.2 Hz, 1 H, Ar-
3
H), 7.71–7.67 (m, 1 H, Ar-H), 5.48 (d, JH,H = 6.9 Hz, 1 H, NH),
3
4.97 (t, JH,H = 3.7 Hz, 2 H, CH2), 4.80–4.76 (m, 1 H, 2-H), 3.82
(s, 3 H, OMe), 1.44 (s, 9 H, Boc) ppm. 13C NMR (100 MHz,
CDCl3): δ = 169.6, 155.4, 155.2, 151.9, 143.2, 130.0, 125.5, 125.4,
123.3, 120.9, 80.8, 53.9, 53.3, 51.0, 28.3 ppm. HRMS (ESI): calcd.
for C17H20N6NaO4S 427.1164; found 427.1166.
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-{4-[(7-nitrobenzo-
[c][1,2,5]oxadiazol-4-ylamino)methyl]-1H-1,2,3-triazol-1-yl}-
propanoate (18): From alkyne 17 (35 mg, 0.16 mmol) and azide 5
(39 mg, 0.16 mmol), the click reaction according to the General
Procedure B followed by purified by column chromatography (pe-
troleum ether/EtOAc, 1:2) afforded 18 as a yellow solid (54 mg,
(S)-3-[4-(Benzo[c][1,2,5]thiadiazol-4-yl)-1H-1,2,3-triazol-1-yl]-2-
(tert-butoxycarbonylamino)propanoic Acid (23): LiOH (3.6 mg,
0.149 mmol) was added to a solution of methyl ester 22 (50 mg,
0.124 mmol) in MeOH (3 mL) and water (1 mL). The reaction mix-
ture was stirred at room temp. for 24 h, then acidified with H+
resin, filtered, and concentrated directly to give the free acid 23
(46 mg, 97%). 1H NMR (400 MHz, CD3OD): δ = 8.49 (s, 1 H, Ar-
l
73%). [α]D = –23.1 (c = 0.3, MeOH). H NMR (400 MHz, [D6]-
3
DMSO): δ = 9.86 (s, 1 H, NH), 8.49 (d, JH,H = 8.7 Hz, 1 H, Ar-
3
H), 8.03 (s, 1 H, Ar-H), 7.38 (d, JH,H = 7.8 Hz, 1 H, NH), 6.48
3
(d, JH,H = 9.2 Hz, 1 H, Ar-H), 4.71–4.65 (m, 3 H, 3-H, CH2),
4.54–4.42 (m, 2 H, 2,3-H), 3.60 (s, 3 H, OMe), 1.20 (s, 9 H, Boc)
ppm. 13C NMR (400 MHz, [D6]DMSO): δ = 170.5, 155.6, 145.0,
144.7, 143.0, 138.3, 124.7, 122.0, 100.4, 79.2, 54.1, 52.8, 49.9, 38.9,
28.4 ppm. HRMS (ESI): calcd. for C18H22N8NaO7 485.1509; found
485.1511.
3
4
H), 8.36 (d, JH,H = 7.4 Hz, 1 H, Ar-H), 7.97 (dd, JH,H = 0.9,
3JH,H = 10.1 Hz, 1 H, Ar-H), 7.72 (dd, 3JH,H = 6.9, 3JH,H = 8.7 Hz,
1 H, Ar-H), 5.02 (dd, 3JH,H = 4.1, 2JH,H = 13.8 Hz, 1 H, 3-H), 4.77
3
2
(dd, JH,H = 8.2, JH,H = 14.2 Hz, 1 H, 3Ј-H), 4.61 (m, 1 H, 2-H),
1.30 (s, 9 H, Boc) ppm. 13C NMR (100 MHz, CD3OD): δ = 171.9,
155.3, 151.8, 129.7, 125.6, 124.8, 123.4, 120.5, 79.5, 54.7, 51.0,
27.2 ppm.
Methyl (S)-2-(Benzyloxycarbonylamino)-6-{4-[(7-nitrobenzo-
[c][1,2,5]oxadiazol-4-ylamino)methyl]-1H-1,2,3-triazol-1-yl}-
hexanoate (19): From alkyne 17 (17.6 mg, 0.081 mmol) and azide 7
(25 mg, 0.081 mmol), the click reaction according to the General
Procedure B followed by purification by column chromatography
(petroleum ether/EtOAc, 1:2) afforded 19 as a yellow solid (40 mg,
Methyl (S)-6-[4-(Benzo[c][1,2,5]thiadiazol-4-yl)-1H-1,2,3-triazol-1-
yl]-2-(benzyloxycarbonylamino)hexanoate (24): From alkyne 21
(19 mg, 0.08 mmol) and azide 7 (25 mg, 0.08 mmol), the click reac-
tion was carried out according to the General Procedure A in the
presence of Bu4NF (202 mg, 0.64 mmol). After 12 h, the reaction
mixture was treated with a CH2Cl2/0.1  EDTA solution (1:1,
16 mL). The water layer was extracted with CH2Cl2 (3ϫ10 mL).
The organic layers were combined, washed with brine, dried with
MgSO4 and the solvents evaporated under vacuum. Purification on
silica gel with EtOAc/petroleum ether (1:1) gave 24 as a light-yellow
solid (40 mg, 79 %). [α]D = +6.0 (c = 0.3, CH2Cl2). 1H NMR
(400 MHz, CDCl3): δ = 8.72 (s, 1 H, Ar-H), 8.54 (d, 3JH,H = 6.9 Hz,
94%). [α]D = –2.0 (c = 0.5, CH2Cl2). Rf = 0.1 (petroleum ether/
3
EtOAc, 1:2). 1H NMR (400 MHz, CDCl3): δ = 8.42 (d, JH,H
=
8.7 Hz, 1 H, Ar-H), 7.64 (s, 1 H, Ar-H), 7.33–7.25 (m, 5 H, Ph),
3
3
6.32 (d, JH,H = 8.7 Hz, 1 H, Ar-H), 5.44 (d, JH,H = 8.3 Hz, 1 H,
3
NH), 5.05 (s, 2 H, OCH2), 4.78 (d, JH,H = 4.1 Hz, 2 H, CH2),
4.36–4.29 (m, 3 H, 2-H, N-CH2), 3.70 (s, 3 H, OMe), 1.97–1.36 (m,
6 H, 3 CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.0, 156.0,
144.3, 143.9, 143.4, 142.3, 136.4, 136.1, 128.6, 128.3, 128.0, 124.4,
122.3, 99.7, 67.1, 53.5, 52.6, 50.2, 39.4, 32.0, 29.5, 22.2 ppm.
HRMS (ESI): calcd. for C24H26N8NaO7 561.1822; found 561.1817.
3
3
1 H, Ar-H), 7.97 (d, JH,H = 8.7 Hz, 1 H, Ar-H), 7.72 (dd, JH,H
= 6.9, 3JH,H = 8.7 Hz, 1 H, Ar-H), 7.34–7.27 (m, 5 H, Ph), 5.33 (d,
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-(4-{4-[(7-nitrobenzo- 3JH,H = 8.2 Hz, 1 H, NH), 5.06 (s, 2 H, CH2), 4.48 (t, JH,H
=
3
[c][1,2,5]oxadiazol-4-ylamino)methyl]-1H-1,2,3-triazol-1-yl}phenyl)-
propanoate (20): From alkyne 17 (29 mg, 0.14 mmol) and azide 9
(43 mg, 0.14 mmol), the click reaction according to the General
7.1 Hz, 2 H, N-CH2), 4.41–4.36 (m, 1 H, 2-H), 3.71 (s, 3 H, OMe),
2.09–1.38 (m, 6 H, 3 CH2) ppm. 13C NMR (100 MHz, CDCl3): δ
= 172.7, 155.9, 155.4, 151.9, 143.3, 136.2, 130.1, 128.6, 128.3, 128.2,
Eur. J. Org. Chem. 2010, 2395–2405
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2403

E. Deprez, J. Xie et al.
FULL PAPER
125.4, 123.9, 123.6, 120.7, 67.1, 53.5, 52.6, 50.1, 32.2, 29.8,
22.3 ppm. HRMS (ESI): calcd. for C23H24N6NaO4S 503.1477;
found 503.1472.
Acknowledgments
C. L. thanks the China Scholarship Council for a doctorate schol-
arship.
Methyl (S)-3-{4-[4-(Benzo[c][1,2,5]thiadiazol-4-yl)-1H-1,2,3-triazol-
1-yl]phenyl}-2-(tert-butoxycarbonylamino)propanoate (25): From al-
kyne 21 (23 mg, 0.1 mmol) and azide 9 (34 mg, 0.1 mmol), the click
reaction was performed according to the General Procedure A in
the presence of Bu4NF (252 mg, 0.8 mmol). After 12 h, the reaction
mixture was treated with a CH2Cl2/0.1  EDTA solution (1:1,
20 mL). The aqueous layer was extracted with CH2Cl2 (3ϫ10 mL).
The organic layers were combined, washed with brine, dried with
MgSO4 and the solvents evaporated under vacuum. Purification on
silica gel with EtOAc/petroleum ether (1:4) gave 25 as a light-green
solid (45 mg, 80 %). [α]D = +42.8 (c = 0.5, CH2Cl2). 1H NMR
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4
(400 MHz, CDCl3): δ = 9.17 (s, 1 H, Ar-H), 8.60 (dd, JH,H = 0.9,
4
3
3JH,H = 7.3 Hz, 1 H, Ar-H), 7.98 (dd, JH,H = 0.9, JH,H = 8.3 Hz,
3
3
1 H, Ar-H), 7.81 (d, JH,H = 8.2 Hz, 2 H, Ar-H), 7.72 (dd, JH,H
3
3
= 6.9, JH,H = 8.7 Hz, 1 H, Ar-H), 7.32 (d, JH,H = 8.2 Hz, 2 H,
Ar-H), 5.07 (d, 3JH,H = 7.8 Hz, 1 H, NH), 4.62 (m, 1 H, 2-H), 3.74
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3
2
(s, 3 H, OMe), 3.21 (dd, JH,H = 6.0, JH,H = 13.8 Hz, 1 H, 3-H),
3
2
3.10 (dd, JH,H = 2.4, JH,H = 13.8 Hz, 1 H, 3Ј-H), 1.40 (s, 9 H,
Boc) ppm. 13C NMR (100 MHz, CDCl3): δ = 172.1, 155.4, 155.1,
151.9, 143.8, 137.2, 136.1, 130.8, 130.0, 125.8, 123.2, 121.9, 121.1,
120.7, 80.3, 54.4, 52.5, 38.1, 28.4 ppm. HRMS (ESI): calcd. for
C23H24N6NaO4S 503.1477; found 503.1472.
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Benzyl (S)-2-(tert-Butoxycarbonylamino)-3-{[1-(7-hydroxy-2-oxo-
2H-chromen-3-yl)-1H-1,2,3-triazol-4-yl]methoxy}propanoate (27):
From alkyne 2 (50 mg, 0.15 mmol) and azidocoumarin 26 (30.5 mg,
0.15 mmol), the click reaction according to the General Pro-
cedure A followed by purification by column chromatography (pe-
troleum ether/EtOAc, 1:1) afforded 27 as a colourless liquid
(70 mg, 88%). [α]D = –11.4 (c = 0.5, CH2Cl2). lH NMR (400 MHz,
CDCl3): δ = 8.46 (s, 1 H, Ar-H), 8.39 (s, 1 H, Ar-H), 7.49 (d, 3JH,H
= 8.2 Hz, 1 H, Ar-H), 7.30–7.24 (m, 5 H, Ph), 6.93–6.90 (m, 2 H,
Ar-H), 5.63 (d, 3JH,H = 8.7 Hz, 1 H, NH), 5.25 (d, 2JH,H = 12.4 Hz,
2
1 H, CHPh), 5.14 (d, JH,H = 12.4 Hz, 1 H, CHPh), 4.70–4.61 (m,
3
2
2 H, CH2), 4.52–4.50 (m, 1 H, 2-H), 4.02 (dd, JH,H = 3.7, JH,H
3
2
= 9.6 Hz, 1 H, 3-H), 3.82 (dd, JH,H = 3.2, JH,H = 9.6 Hz, 1 H,
3Ј-H), 1.43 (s, 9 H, Boc) ppm. 13C NMR (100 MHz, CDCl3): δ =
170.9, 161.6, 156.2, 155.8, 154.7, 144.2, 135.3, 134.3, 130.4, 128.6,
128.4, 128.1, 123.9, 119.5, 114.9, 103.2, 80.5, 70.5, 67.4, 64.4, 54.2,
28.4 ppm. HRMS (ESI): calcd. for C27H28N4NaO8 559.1805; found
559.1800.
Benzyl (S)-2-(tert-Butoxycarbonylamino)-3-{[1-(7-methoxy-2-oxo-
2H-chromen-3-yl)-1H-1,2,3-triazol-4-yl]methoxy}propanoate (29):
From alkyne 2 (61 mg, 0.19 mmol) and azidocoumarin 28 (40 mg,
0.19 mmol), the click reaction according to the General Pro-
cedure A followed by purification by column chromatography (pe-
troleum ether/EtOAc, 3:2) afforded 29 as a colourless liquid
1
(80 mg, 80%). [α]D = –8.0 (c = 0.5, CH2Cl2). H NMR (400 MHz,
CDCl3): δ = 8.47 (s, 1 H, Ar-H), 8.46 (s, 1 H, Ar-H), 7.54 (d, 3JH,H
4
= 8.7 Hz, 1 H, Ar-H), 7.34–7.26 (m, 5 H, Ph), 6.95 (dd, JH,H
=
3
4
2.3, JH,H = 8.2 Hz, 1 H, Ar-H), 6.90 (d, JH,H = 2.3 Hz, 1 H, Ar- [9] a) C. C. Knight, Biochem. J. 1991, 274, 45–48; b) S. J. Haw-
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3
2
thorne, P. Harriott, J. Lim, A. J. Turner, B. Walker, C. H. Wil-
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2
H, CHPh), 5.11 (d, JH,H = 12.4 Hz, 1 H, CHPh), 4.69–4.60 (m, 2
H, CH2), 4.47 (m, 1 H, 2-H), 3.97 (dd, 3JH,H = 3.2, 2JH,H = 9.6 Hz,
3
2
1 H, 3-H), 3.91 (s, 3 H, OMe), 3.76 (dd, JH,H = 3.2, JH,H
=
9.6 Hz, 1 H, 3Ј-H), 1.42 (s, 9 H, Boc) ppm. 13C NMR (100 MHz,
CDCl3): δ = 170.5, 163.9, 156.1, 155.6, 154.8, 144.5, 135.5, 133.9,
130.0, 128.6, 128.4, 128.1, 123.8, 120.2, 114.2, 111.6, 100.8, 80.1,
70.5, 67.2, 64.6, 56.1, 54.2, 28.4 ppm. HRMS (ESI): calcd. for
C28H30N4NaO8 573.1961; found 573.1956.
2404
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2395–2405

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Published Online: March 11, 2010
Eur. J. Org. Chem. 2010, 2395–2405
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2405