Synthesis, Absorption, and Fluorescence Studies of Coumaryl-Labelled Amino Acids and Dipeptides Linked Via Triazole Ring

Article abstract of DOI:10.1071/CH14708

Fluorophores based on 4-triazolyl, 7-hydroxy-4-triazolylmethyl, 4-O-triazolylmethyl, and 7-O-triazolylmethyl coumaryl-tagged amino acids and dipeptides were synthesized by copper-catalyzed [3+2] cycloaddition reaction between azido- or alkynyl-functionalized coumarins with alkynyl- or azido-functionalized amino acid and dipeptides in good-to-excellent yields. Steady-state absorption and the fluorescence properties of the synthesized conjugates were studied. The chemical applicability of these amino acid and peptide-based fluorophores was successfully demonstrated by their linear elongation by further tagging them with appropriate C- or N-terminus amino acid.

Full text of DOI:10.1071/CH14708

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH14708
Synthesis, Absorption, and Fluorescence Studies
of Coumaryl-Labelled Amino Acids and Dipeptides
Linked Via Triazole Ring
A
B
A
Santosh Kumari, Sunita Joshi, S. M. Abdul Shakoor,
A
C
B
Devesh S. Agarwal, Siva S. Panda, Debi D. Pant,
and Rajeev Sakhuja
A D
,
A
Department of Chemistry, Birla Institute of Technology and Science, Pilani 333 031,
Rajasthan, India.
B
Department of Physics, Birla Institute of Technology and Science, Pilani 333 031,
Rajasthan, India.
C
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida,
Gainesville, FL 32611-7200, USA.
D
Corresponding author. Email: sakhuja.rajeev@gmail.com
Fluorophores based on 4-triazolyl, 7-hydroxy-4-triazolylmethyl, 4-O-triazolylmethyl, and 7-O-triazolylmethyl coumaryl-
tagged amino acids and dipeptides were synthesized by copper-catalyzed [3 þ 2] cycloaddition reaction between azido- or
alkynyl-functionalized coumarins with alkynyl- or azido-functionalized amino acid and dipeptides in good-to-excellent
yields. Steady-state absorption and the fluorescence properties of the synthesized conjugates were studied. The chemical
applicability of these amino acid and peptide-based fluorophores was successfully demonstrated by their linear elongation
by further tagging them with appropriate C- or N-terminus amino acid.
Manuscript received: 11 December 2014.
Manuscript accepted: 9 February 2015.
Published online: 7 April 2015.
Introduction
and signalling unit in sensors and in sophisticated photo-
physical systems.[8] Excellent fluorescence quantum yields have
been observed in coumarins bearing electron-donating substi-
tuents at the 7-position, such as 7-aminocoumarin, 7-(dialkyl-
amino)coumarins, 7-hydoxycoumarin that makes them suitable
candidates in the application of fluorescence labelling of living
cells.[9] For example, a-L-(2-(7-hydroxycoumarin-4-yl)ethyl)
glycine (Fig. 1, I) was genetically incorporated at a defined site
in proteins in living organisms for the first time by Schultz and
coworkers.[10] Coumarin-labelled lysines (Fig. 1, II) have also
been designed for the synthesis of n˜uorogenic triple-helical
substrates for the analysis of matrix metalloproteinase family
members.[11] Unnatural amino acid linked to 7-aminocoumarin
substrate (Fig. 1, III) has been recently developed as a fluores-
cent ligase substrate for specific protein labelling in living
cells.[12] Katritzky et al. have explored the application of
benzotriazole methodology for the synthesis of fluorescent
coumarin-labelled amino acids and dipeptides as building
blocks for solid phase peptide synthesis (SPPS).[13]
Amino acids, peptides, and proteins are the most abundant
molecules in living cells that exhibit various physiological
functions in all living organisms. Impairing of amino acid
metabolism in the body may lead to various disease conditions
(e.g. phenylketonuria). Biologically active peptides serve as an
interesting starting point in drug discovery processes. Several
peptide-based pharmaceuticals have been developed as useful
therapeutic agents, which offer advantages such as high potency
and selectivity as well as low accumulation in tissues. Thus,
investigation and understanding biologically relevant inter-
actions and processes, such as receptor–ligand binding, protein
structures, protein folding, protein–protein interactions, and
enzyme activity using labelling studies, have become an
established exercise in biochemical research.[1] For studying
these interactions, tagging C- and N-terminal domains of a
peptide with a fluorescent label and probe or incorporating a
natural fluorescent amino acid, such as tyrosine (Tyr) or tryp-
tophan (Trp) in peptide synthesis, are two interactive modes of
detecting a peptide without allowing any changes in its con-
formational and biological characteristics.[2]
Copper-catalyzed [3 þ 2] cycloaddition reaction between an
azide and an alkyne is an established and attractive labelling
methodology for linking two chemical moieties via a triazole
ring.[14] Triazoles themselves possess interesting chemical
properties that include tolerance to acidic, basic, oxidative,
and reductive conditions. A triazole ring has been considered
as a non-classical bioisostere of the peptide bond.[15] Its function
Coumarin is an excellent example of a fluorescent label with
advantages of extended spectral ranges, high emission quantum
yields, photo-stability, and good solubility in many solvents.[3]
Coumarin derivatives are widely used as fluorescent probes,[4]
labels[5] pigments,[6] and laser dyes[7] for the blue–green region
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

B
S. Kumari et al.
O
O
HO
O
O
O
O
O
H2N
H
N
H
COOH
COOH
N
4
O
COOH
H2N
NH
Fmoc
I
II
III
Fig. 1. Coumarin-tagged amino acids used in labelling studies.
Table 1. Synthesis of protected aminoacyl and dipeptidoyl O-propargyl esters 5 and 7
O
H
N
Pg
OH
R
O
4a–g
H
N
Pg
O
K2CO3/DMF
R
20Њ
C, 8–12 h
5a–g
Br
O
H
N
Cbz
3
N
H
OH
R1
O
O
6a, 6b
H
N
Cbz
K2CO3/DMF
20 C, 10–12 h
N
O
Њ
H
R1
O
7a, 7b
Serial no.
Protected amino acid and dipeptide
Product
Yield [%]
mp [8C]
1
2
3
4
5
6
7
8
9
Boc-L-Phe-OH (4a)
Cbz-L-Ala-OH (4b)
Cbz-L-Phe-OH (4c)
Fmoc-L-Phe-OH (4d)
Fmoc-L-Leu-OH (4e)
Fmoc-L-Ile-OH (4f)
Boc-L-Ala-OH (4g)
Cbz-L-Phe-L-Trp-OH (6a)
Cbz-L-Phe-L-Ala-OH (6b)
5a
5b
5c
5d
5e
5f
90
86
88
96
98
97
87
98
96
Oil[24]
58–60[25]
64–66[24]
90–92[25]
96–97[25]
102–103
Oil[26]
5g
7a
7b
120–122
126–128
to act as a helical component,[16] a b-turn unit,[17] and a cis or
trans-prolyl ratio modifier[18] in linear peptidomimetics has
been studied by few researchers. Construction of a triazole ring
has facilitated an increase in the emission intensity and variation
in emission wavelength properties, desirable for molecules to
act as fluorogenic probes.[19] Moreover, the beneficial effects on
fluorescence properties of molecules acting as selective chemo-
sensors have been produced by attaching triazole moiety as a
linker[20] and as a coordinating ligand.[21] Thus, it was envisaged
that augmenting triazole moiety to coumaryl-amino acid labels
will yield candidates with fluorescence-emission properties that
may be used for studying various biological interactions in
labelling studies.
strategy involves clicking alkynyl-functionalized amino acids
with coumarin-based azides. The second strategy involves
clicking azide-functionalized amino acids with coumarin-based
alkyne derivatives. Thus, azido-functionalized coumarins viz.
7-hydroxy-4-azidomethyl-2H-chromen-2-one (1a) and 4-azido-
2H-chromen-2-one (1b) were synthesized by the reaction of
NaN3/DMF with 7-hydroxy-4-chloromethyl-2H-chromen-2-
one and 4-chloro-2H-chromen-2-one, respectively, according to
literature procedures.[22,23]
In order to employ the first strategy, alkynyl-functionalized
amino acids-based clickable agents were prepared by esterifica-
tion of different protected amino acids 4a–g with propargyl
bromide (3) using K2CO3 in DMF at 208C to yield protected
amino acyl O-propargyl esters 5a–g (Table 1). Two novel
dipeptidoyl O-propargyl esters, 7a and 7b, were envisaged
and synthesized using the same method starting from their
corresponding Z-protected dipeptides 6a and 6b, respectively
(Table 1).
Results and Discussion
We plan to explore the application of click chemistry for syn-
thesizing targeted molecules via two strategies. The first

Fluorescent-Labelled Amino Acids and Peptides
C
Table 2. Synthesis of 7-hydroxy-4-triazolylmethyl coumaryl-labelled amino acids and dipeptides 8 and 9
O
H
N
O
Pg
R
O
HO
O
5a–f
Pg
NH
R
CuSO4·5H2O, sodium ascorbate
EtOH/water (1:1), 20ЊC, 8–14 h
O
O
HO
N
O
O
N
N
8a–f
Ph
O
H
Cbz
N
N3
1a
N
H
O
Cbz
HN
Ph
R1
HO
O
O
O
O
7a, 7b
R1
CuSO4·5H2O, sodium ascorbate
EtOH/water (1:1), 20ЊC, 12–14 h
NH
O
N
O
N
N
9a, 9b
Serial no.
Protected amino acyl-O-propargyl esters
Product
Yield [%]A
mp [8C]
1
2
3
4
5
6
7
8
Boc-L-Phe-OCH2CꢁCH (5a)
Cbz-L-Ala-OCH2CꢁCH (5b)
Cbz-L-Phe-OCH2CꢁCH (5c)
Fmoc-L-Phe-OCH2CꢁCH (5d)
Fmoc-L-Leu-OCH2CꢁCH (5e)
Fmoc-L-Ile-OCH2CꢁCH (5f)
Cbz-L-Phe-L-Trp-OCH2CꢁCH (7a)
Cbz-L-Phe-L-Ala-OCH2CꢁCH (7b)
8a
8b
8c
8d
8e
8f
92
87
86
85
72
74
85
85
143–145
147–149
150–152
148–150
146–148
143–146
155–158
153–155
9a
9b
AIsolated yield (before recrystallization).
We attempted the copper-catalyzed azide–alkyne cycloaddi-
tion (CuAAC) with clickable alkynyl-functionalized amino acid
and dipeptide and azido-functionalized coumarin reagents. Due
to the high fluorescence quantum yield, relatively large Stokes
shift, small size, and pH sensitivity, the methodology was
attempted on 7-hydroxycoumarin moiety. Thus, 7-hydroxy-4-
azidomethyl-2H-chromen-2-one (1a) on cycloaddition with
protected a-amino acyl O-propargyl esters 5a–f, using
CuSO4ꢀ5H2O and sodium ascorbate, yielded a series of
7-hydroxy-4-triazolylmethyl coumaryl-labelled amino acids
8a–f in 72–92 % yield (Table 2). Furthermore, 7-hydroxy-4-
azidomethyl-2H-chromen-2-one (1a) on cycloaddition with
dipeptidoyl O-propargyl ester 7a or 7b using CuSO4ꢀ5H2O
and sodium ascorbate yielded 7-hydroxy-4-triazolylmethyl
coumaryl-labelled dipeptide 9a or b, respectively (Table 2).
In order to make a comparison on the fluorescent behaviour
of 7-substituted coumaryl derivatives with unsubstituted deri-
vatives, we extended the methodology for synthesizing few
derivatives of 1b. Thus, 4-azido-2H-chromen-2-one (1b) on
cycloaddition with few protected amino acyl O-propargyl esters,
5a–d and 5g, using CuSO4ꢀ5H2O and sodium ascorbate yielded
a series of 4-triazolyl coumaryl-labelled amino acids, 10a–d and
10g, respectively, in 81–93 % yields (Table 3). 4-Azido-2H-
chromen-2-one (1b) on cycloaddition with dipeptidoyl O-pro-
pargyl ester 7a under similar conditions yielded 4-triazolyl
coumaryl-labelled dipeptide 11a in 78 % yield (Table 3).
In the second strategy, 4-methyl-7-(prop-2-ynyloxy)-2H-
chromen-2-one (2a) and 4-(prop-2-ynyloxy)-2H-chromen-2-one
(2b) were prepared by O-alkylation of 4-methyl-7-hydroxy-2H-
chromen-2-one and 4-hydroxy-2H-chromene-2-one, respec-
tively, with propargyl bromide using K2CO3/CH3CN following
literature procedures.[27] a-Azido esters 12a–c were also synthe-
sized by the reaction of triflic azide (prepared in situ) with
a-amino esters following literature procedures.[28–30]
Copper-catalyzed cycloaddition was then attempted with
clickable alkynyl-functionalized coumarin and azido-
functionalized amino acids. The cycloaddition of 4-methyl-7-
(prop-2-ynyloxy)-2H-chromen-2-one (2a) and 4-(prop-2-
ynyloxy)-2H-chromen-2-one (2b) and with clickable a-azido
esters 12a–c using CuSO4ꢀ5H2O and sodium ascorbate yielded
two series of 7-O-triazolylmethyl and 4-O-triazolylmethyl
coumaryl-labelled amino acids, 13a–f, in 82–90 % yields
(Table 4).
All the products were characterized by their detailed spectral
studies (1H NMR, 13C NMR, and high-resolution mass spec-
trometry (HRMS)). The absence of any minor or duplicate
1
signals in the H NMR spectra of triazolyl-linked coumaryl-
labelled dipeptides, 9a, 9b, 11a, and their univocal correspon-
dence to the expected structure showed them not to be a mixture
of any possible diastereoisomers (that might have resulted due to
two chiral centres of dipeptide).
The steady-state absorption and emission parameters of
several newly synthesized derivatives of coumarins have been
studied thoroughly in polar aprotic tetrahydrofuran (THF)
solvent. As coumarin molecules are fluorescent probes, they
are quite sensitive to their environment and display solvato-
chromism. Therefore, some sort of comparison with a more
polar solvent than THF would be important. However, in the
current investigation, these derivatives are insoluble in water.
Thus, we have recorded UV-visible absorption and fluorescence
spectra of some representative conjugates in polar protic meth-
anol (MeOH) solvent. The absorption maxima (lAbs), emission

D
S. Kumari et al.
Table 3. Synthesis of 4-triazolyl-linked coumaryl-labelled amino acids and dipeptide 10 and 11
O
H
N
O
O
O
Pg
R
5a–d, 5g
Pg
N
HN
O
CuSO4·5H2O, sodium ascorbate
N
EtOH/water (1:1), 20ЊC, 6–10 h
N
O
O
R
O
10a–d, 10g
Ph
O
N3
H
Cbz
N
O
O
N
Ph
Cbz
NH
N
H
O
1b
R1
O
7a
CuSO4·5H2O, sodium ascorbate
EtOH/water (1:1), 20ЊC, 10 h
HN
R1
O
O
N
N
O
11a
Serial no.
Protected amino acyl-O-propargyl esters
Product
Yield [%]A
mp [8C]
1
2
3
4
5
6
Boc-L-Phe-OCH2CꢁCH (5a)
Cbz- L-Ala-OCH2CꢁCH (5b)
Cbz-L-Phe-OCH2CꢁCH (5c)
Fmoc-L-Phe-OCH2CꢁCH (5d)
Boc-L-Ala-OCH2CꢁCH (5g)
Cbz-L-Phe-L-Trp-OCH2CꢁCH (7a)
10a
10b
10c
10d
10g
11a
85
91
93
89
81
78
138–140
139–142
143–145
134–136
133–135
150–152
AIsolated yield (before recrystallization).
Table 4. Synthesis of 7-O-triazolylmethyl and 4-O-triazolylmethyl coumaryl-labelled amino acids 13
O
O
O
O
2a
R1O
N
N
N
O
O
O
CuSO4·5H2O, sodium ascorbate
EtOH/water (1:1), 20ЊC, 8–10 h
R2
O
N3
13a–c
OR1
O
O
O
R2
12a–c
OR1
O
R2
O
O
O
2b
CuSO4·5H2O, sodium ascorbate
EtOH/water (1:1), 20ЊC, 6–8 h
N
N
N
13d–f
Serial no.
a-Azido ester (Reactant 1)
Reactant 2
Product
Yield [%]A
mp [8C]
1
2
3
4
5
6
N3-L-Gly-OEt (12a)
N3-L-Phe-OMe (12b)
N3-L-Trp-OMe (12c)
N3-L-Gly-OEt (12a)
N3-L-Phe-OMe (12b)
N3-L-Trp-OMe (12c)
2a
2a
2a
2b
2b
2b
13a
13b
13c
13d
13e
13f
90
87
85
88
85
82
128–130
130–132
134–136
129–132
133–135
138–140
AIsolated yield (before recrystallization).

Fluorescent-Labelled Amino Acids and Peptides
E
Table 5. Spectroscopic steady-state data of 7-hydroxy-4-triazolylmethyl and 4-triazolyl coumaryl-labelled amino acids and dipeptide
Compound
Solvent
lAbs (nm)
lEm (nm)
ꢀva ꢂ ꢀvf [cmꢂ1
]
e [Mꢂ1 cmꢂ1
]
Quantum yield, F
8a
8b
8c
8d
THF
THF
THF
THF
MeOH
THF
THF
THF
THF
THF
THF
THF
THF
MeOH
THF
THF
323
323
323
266
265
266
266
324
275
280
280
280
267
265
280
281
388
388
387
389
411
388
387
388
388
383
383
383
381
411
413
383
5186.6
5199.9
5120.0
5252.8
6250.1
5119.9
5119.9
5120.0
5186.6
5003.3
5071.9
5101.2
5042.9
6343.1
7036.9
5476.8
7860
18500
9120
0.136
0.140
0.162
0.184
0.220
0.150
0.143
0.190
0.161
0.018
0.025
0.048
0.091
0.140
0.009
0.096
16460
13840
20860
19700
4960
8e
8f
9a
9b
13780
27580
9620
10a
10b
10c
10d
26900
12860
5820
10g
11a
17640
9400
1.2
1.0
0.8
0.6
0.4
0.2
0
(a)
(b)
160
8a
8b
8c
8d
8e
8f
8a
8b
8c
8d
8e
8f
140
120
100
80
9a
9b
9a
9b
60
40
20
0
250
300
350
400
400
450
500
550
600
350
Wavelength [nm]
Wavelength [nm]
Fig. 2. (a) Absorption spectra and (b) emission spectra of compounds 8a–f, 9a, 9b (5 ꢄ 10ꢂ5 M) in THF; lex ¼ 325 nm.
(a)
(b)
30
20
10
0
1.6
10a
10b
10c
10d
10g
11a
10a
10b
10c
10d
10g
11a
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
400
450
500
550
600
350
250
300
350
400
Wavelength [nm]
Wavelength [nm]
Fig. 3. (a) Absorption spectra and (b) emission spectra of compound 10a–d, 10g, 11a (5 ꢄ 10ꢂ5 M) in THF; lex ¼ 325 nm.
maxima (lEm), Stokes shift (ꢀva ꢂ ꢀvf ), extinction coefficient (e),
and quantum yield (F) of compounds 8a–f, 9a, 9b, 10a–d, 10g,
11a in THF are listed in Table 5. The corresponding absorption
and emission spectra are shown in Figs 2 and 3.
In general, 7-hydroxytriazolylmethyl- and 4-triazolyl-linked
coumaryl amino acids and dipeptide conjugates (8, 9, 10, 11)
showed absorption maxima in the wavelength region of
265–324 nm and are shown in Table 5. The well-resolved
absorption band around 325 nm is due to a p ! pꢃ transition
to the S1 state. The absorbance of 4-triazolyl-linked coumaryl-
labelled amino acids and dipeptides was less than that for
7-hydroxytriazolylmethyl-linked coumaryl amino acids and

F
S. Kumari et al.
(a)
(b)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
80
60
40
20
0
13a
13b
13c
13d
13e
13f
13a
13b
13c
13d
13e
13f
250
300
350
400
350
400
450
500
550
600
Wavelength [nm]
Wavelength [nm]
Fig. 4. (a) Absorption spectra and (b) emission spectra of compounds 13a–f (5 ꢄ 10ꢂ5 M) in THF; lex ¼ 325 nm.
Table 6. Spectroscopic steady-state data of 7-O- triazolylmethyl and 4-O-triazolylmethyl coumaryl-labelled amino acids
Compound
Solvent
lAbs [nm]
lEm [nm]
ꢀva ꢂ ꢀvf [cmꢂ1
]
e [Mꢂ1 cmꢂ1
]
Quantum yield, F
13a
13b
THF
318
318
316
280
275
275
265
275
380
380
392
381
382
382
401
382
4992.7
5061.3
6134.8
5336.8
5090.7
5535.9
6004.5
5535.9
6360
17500
23600
32000
31200
11600
5640
0.201
0.240
0.302
0.280
0.038
0.082
0.120
0.096
THF
MeOH
THF
13c
13d
13e
THF
THF
MeOH
THF
13f
32400
dipeptide conjugates. The absorbance maxima of 7-hydroxy-
triazolylmethyl-linked coumaryl amino acids are clearly red
shifted (50 nm) than that for 4-triazolyl-linked coumaryl-
labelled amino acids and dipeptides. The (ꢀva ꢂ ꢀvf ) is expressed
in wave number and are calculated by taking the difference
between last absorption maximum and first emission maximum
of each derivative studied. Given the different nature of the
various absorption bands due to difference in structural aspects
of the conjugates (amino acids: Gly (Glycine), Ala (Alanine),
Phe (Phenyl alanine), Trp (Tryptophan), Ile (Isoleucine), Leu
(Leucine), and protecting groups: Fmoc (Fluorenylmethyloxy-
carbonyl), Cbz (carboxybenzyl), Boc (t-butoxycarbonyl)), the
excitation wavelength in fluorescence measurements is crucial
and it will make sense to compare the various fluorescence
quantum yields of the conjugates only if excitation is performed
in the charge-transfer band region. So, the fluorescence spec-
trum measurements in the present study have been performed by
exciting at 325 nm.
Both the locally excited (LE) band around 380 nm and
intermolecular charge transfer (ICT) shoulder around 415 nm
have been observed in the fluorescence spectra of all the
coumarin derivatives 10a–d and 10g studied upon excitation
at 325 nm. The quantum yields of 7-hydroxy-4-triazolylmethyl
coumaryl-labelled amino acids and dipeptide, 8a–d and 9a,
were found be greater than the corresponding 4-triazolyl
coumaryl derivatives 10a–d and 11a.
amino acids 13a–c are clearly red shifted (50 nm) than that for
4-O-triazolyl-linked coumaryl-labelled amino acids 13d–f. The
conjugate 13c possessing the natural fluorescent amino acid
tryptophan showed the maximum value of quantum yield
(0.280) as compared with all others in THF.
The electron-donating functionalities cause an increase in
the quantum yield of molecules. The observed bathochromic
shift of fluorescence maxima and increase in quantum yield
could be possibly due to the electron-donating hydroxyl group at
position 7 of coumaryl derivatives. These effects are more
prominent for coumarin derivatives with O-substituted triazo-
lylmethyls than that for unsubstituted derivatives and possibly
due to the intermolecular hydrogen bonding in the former.
The natural tryptophan amino acid emits in the wavelength
range of 330–350 nm, whereas the different amino acid
derivatives studied in the present manuscript emit in the range
of 340–560 nm.
To further demonstrate the chemical utility of the synthe-
sized triazolyl-linked coumaryl-labelled amino acid conjugates
as fluorescent labels, their linear elongation by coupling them
with appropriate C- or N-terminus amino acid in solution phase
was attempted. Thus, 10g on reaction with dioxane/HCl yielded
unprotected 4-triazolyl-linked coumaryl-labelled amino acid
hydrochloride salt 14 in 68 %, which on further reaction with
Boc-L-phenylalanine using EDC (N-(3-Dimethylaminopropyl)-
N0-ethylcarbodiimide)ꢀHCl/HOBt (Hydroxybenzotriazole) in
DMF yielded 15 in 86 % yield (Scheme 1).
The absence of any minor or duplicate signals in the 1H NMR
spectrum of 15 and their univocal correspondence to the
expected structure revealed the absence of any possible diaster-
eoisomers or epimers mixtures.
4-O-Triazolyl- and 7-O-triazolylmethyl-linked coumaryl
amino acids 13a–f showed absorption maxima in the wave-
length region of 264–326 nm and are shown in Fig. 4. The
corresponding spectroscopic data are given in Table 6. The
absorbance maxima of 7-O-triazolylmethyl-linked coumaryl

Fluorescent-Labelled Amino Acids and Peptides
G
O
N
O
O
O
HCl/Dioxane
room temperature, 2 h
68 %
Boc
HN
N
O
N
N
O
NH2
·HCl
N
N
O
O
10g
14
O
HN
Boc
OH
EDC·HCl/HOBt/DMF
room temperature, 8 h
Ph
O
O
Boc
O
HN
Ph
N
N
HN
O
N
O
15
86 %
Scheme 1. Linear elongation of 4-triazolyl-linked coumaryl amino acid 10g with C-terminus amino acid.
EtO
HO
O
O
N
N
N
O
O
O
N
20 % NaOH/EtOH
N
O
O
O
N
room temperature, 3 h
85 %
13a
16
O
H2N
O
EDC·HCl/HOBt/DMF
room temperature, 6 h
Ph
90 %
O
O
O
HN
N
N
O
O
O
Ph
N
17
Scheme 2. Linear elongation of 7-O-triazolylmethyl-linked coumaryl amino acid 13a with N-terminus amino acid.
Similar chemical applicability of 7-O-triazolylmethyl
coumaryl-labelled amino acid conjugates as fluorescent labels
was demonstrated on one of its derivatives viz. 13a. Hydrolysis
of 13a with 20 % NaOH solution in ethanol followed by acidic
workup gave 16 in 85 % yield, which on reaction with
L-phenylalanine methyl ester using EDCꢀHCl/HOBt in DMF
gave 17 in 90 % yield (Scheme 2).
The methodology used for the linear elongation of glycine-
based 7-O-triazolylmethyl coumaryl-labelled amino acid con-
jugate 13a (Scheme 2) was very similar to that followed in
Scheme 1 and thus it was assumed that epimerization will not be
a problem if the linear elongation were to be carried on chiral
substrates.
The absorption and emission spectra of 15 and 17 were also
recorded in THF in order to compare and contrast different
spectroscopic data with 10g and 13a. The absorption and
emission data 15 and 17 are summarized in Table 7. Clearly,
there is a significant increase in quantum yields of 15 and 17
when compared with that of 10g and 13a. This observation
suggests that incorporation of another amino acid into triazolyl-
based fluorescent conjugates makes these significantly more
fluorescent.
However, biological interactions in peptides are usually
studied in aqueous solutions. Because the conjugates are water
insoluble, a comparative study using a polar protic solvent
(MeOH) of some representative conjugates (8d, 10d, 13b, and

H
S. Kumari et al.
Table 7. Spectroscopic steady-state data of 4- triazolyl and 7-O-triazolylmethyl coumaryl-linked amino acids with C-terminus amino acid and
N-terminus amino acid
Compound
Solvent
lAbs [nm]
lEm [nm]
ꢀva ꢂ ꢀvf [cmꢂ1
]
e [Mꢂ1 cmꢂ1
]
Quantum yield, F
10g
15
THF
THF
THF
THF
280
281
318
265
384
381
380
382
7036.9
5071.9
4962.7
5306.3
17640
10800
6360
0.009
0.110
0.201
0.340
13a
17
19500
13e) indicated a red shift in the absorption and emission maxima
in the UV-visible absorption and fluorescence spectra. The shift
of fluorescence maxima is more pronounced than the absorption
maxima. This behaviour is indicative of an emitting state that is
more polar than the ground state. Interestingly, the quantum
yield values in MeOH are significantly higher than that in THF
and exhibited larger Stokes shifts. The Stokes shift is consider-
ably larger than that expected on the basis of solvent polarity
alone. We believe that the hydrogen bonding reaction of the
fluorophore with the hydroxyl MeOH is responsible for the large
Stokes-shifted fluorescence. The observation of polarity-
dependent enhancement of quantum yield and bathochromic
shift of absorption spectra is consistent with our assignment of
absorption band to transition to the first excited state in these
derivatives.
time-of-flight mass spectrometry (TOF-MS) with electrospray
ionization (ESI). Electronic absorption spectra were taken using
dual beam Thermo Evolution 201 UV/Vis/NIR spectropho-
tometer and fluorescence spectra were recorded using Shimadzu
RF-5301PC spectrofluorometer. The data were analyzed using
the related software. The concentration of the coumarin deri-
vatives in all the solutions prepared in solvents THF and MeOH
was 5 ꢄ 10ꢂ5 M. The fluorescence quantum yields of all the
coumarin derivatives in THF and MeOH were estimated by
using quinine sulfate in 0.1N sulfuric acid (F ¼ 0.55) as the
standard reference.[31–32]
Synthesis
Typical Procedure for the Synthesis of Protected Amino
Acyl or Dipeptidoyl O-Propargyl Esters 5, 7
In addition, the water soluble version 14 of one of the
conjugates 10g (lEm (THF) ¼ 413 nm, F ¼ 0.009) was charac-
terized by a significant red shift of its fluorescence emission in
water (lEm ¼ 430 nm, F ¼ 0.046) when compared with THF
(lEm ¼ 383 nm, F ¼ 0.016).
N-Protected amino acid or dipeptide 4a–g or 6a, 6b (1 equiv.)
was dissolved in anhydrous DMF (10 mL) and the solution was
cooled to ꢂ108C. Anhydrous K2CO3 (1.5 equiv.) was added to
the solution and the stirring continued until a syrup-like solution
formed. Propargyl bromide (2 equiv., 80 % solution in toluene)
was added dropwise to the reaction mixture, and the stirring
continued at ꢂ108C for 1 h. The reaction mixture was then
stirred at room temperature for 8–12 h and monitored via TLC
until the disappearance of the starting material. The reaction
mixture was then added dropwise in an ice bath, and the
precipitated white solid was collected by filtration and washed
with cold water (3 ꢄ 20 mL) to yield pure 5 or 7.
Conclusion
In summary, we have reported an efficient synthesis of triazolyl-
conjugated coumaryl–amino acid conjugates by introducing
fluorophores on the C- and N-termini of natural amino acids and
few dipeptides. Spectroscopic studies showed that 7-triazolyl-
linked conjugates showed better fluorescence behaviour than
4-triazolyl-linked conjugates. On removing the protecting
group, hydrosoluble coumaryl-tagged amino acids could be
obtained, to which amino acids and peptides could be coupled.
Thus, our methodology presents a useful tool for the synthesis of
environment-sensitive fluorescent amino acid conjugates, with
potential applications in labelling studies.
Prop-2-yn-1-yl((benzyloxy)carbonyl)-L-phenylalanyl-L-tryp-
tophanate (7a) (0.110 g, 98 %), white solid, mp 120–1228C. dH
([D6]DMSO þ CDCl3, 400 MHz) 10.84 (1H, s), 8.44 (1H, br s),
7.52 (1H, s), 7.41–7.12 (13H, m), 7.11–6.95 (2H, m), 5.02–4.85
(2H, m), 4.75–4.58 (3H, m), 4.34 (1H, br s), 3.38 (1H, s),
3.18 (2H, d, J 20.1), 3.01 (1H, d, J 11.9), 2.74 (1H, s). dC
([D6]DMSO þ CDCl3, 101 MHz) 171.7, 170.8, 155.7, 137.9,
136.8, 136.0, 129.1, 128.1, 127.8, 127.5, 127.4, 127.0, 126.1,
123.7, 120.9, 118.4, 117.9, 111.3, 108.9, 77.7, 77.5, 65.3, 55.8,
52.9, 52.2, 37.5, 26.8.
Experimental
General
All chemicals were obtained from commercial suppliers and
used without further purification. Melting points were deter-
mined in open capillary tubes on a MPA120-automated melting
point apparatus and are uncorrected. Reactions were monitored
using thin layer chromatography (TLC) on 0.2 mm silica gel
F254 plates (Merck). The chemical structures of final products
and intermediates were characterized by nuclear magnetic res-
onance spectra (1H NMR, 13C NMR) determined on a Bruker
NMR spectrometer (300 (or 400) MHz, 75 (or 101 MHz). 13C
NMR spectra are fully decoupled. Chemical shifts were reported
in parts per million (ppm) using deuterated solvent peak or
tetramethylsilane as internal standard. The chemical structures
of final products were confirmed on a high-resolution Biosys-
tems QStar Elite time-of-flight electrospray mass spectrometer.
HRMS was performed on an Agilent 6210 instrument using
Prop-2-yn-1-yl((benzyloxy)carbonyl)-L-phenylalanyl-L-
alaninate (7b) (0.105 g, 96 %), white solid, mp 126–1288C. dH
([D6]DMSO, 300 MHz) 8.54 (1H, d, J 6.0), 7.48 (1H, d, J 8.1),
7.37–7.09 (10H, m), 4.90 (2H, s), 4.70 (2H, s), 4.30 (2, dd, J
13.1, 6.8), 3.55 (1H, s), 3.00 (1H, d, J 13.2), 2.71 (1H, t, J 12.1),
1.31 (3H, d, J 6.5). dC ([D6]DMSO, 75 MHz) 172.3, 156.4,
138.7, 137.5, 129.7, 128.8, 128.6, 128.2, 128.0, 126.8, 78.7,
78.5, 65.7, 56.4, 52.9, 48.1, 37.9, 17.2.
Typical Procedure for the Synthesis of Triazolyl-Linked
Coumaryl-Labelled Amino Acids or Peptides 8a–f, 9a, 9b,
10a–d, 10g, 11a
To a solution of 4-azidomethyl-7-hydroxycoumarin (1a) or
4-azido coumarin (1b) (0.100 g, 1 equiv.) in ethanol/H2O (1 : 1)

Fluorescent-Labelled Amino Acids and Peptides
I
was added protected amino acyl O-propargyl ester 5 or 7
(1 equiv.), followed by addition of CuSO4 (0.01 equiv.) and
sodium ascorbate (0.1 equiv.). The reaction mixture was stirred
at room temperature for 6–14 h and monitored via TLC. After
completion of the reaction, the reaction mixture was evaporated
and the residue was diluted with water and ammonium hydrox-
ide (2–3 mL) and extracted into dichloromethane. Organic layer
was separated, dried over anhydrous sodium sulfate to evaporate
the volatiles. The crude compound was recrystallized from
ethanol to yield pure 8a–f, 9a, 9b, 10a–d, 10g, 11a.
s), 5.92 (2H, d, J 10.5), 5.53–5.51 (1H, m), 5.28–5.18 (2H,
m), 4.36–4.19 (2H, m), 4.12–4.01 (1H, m), 3.39 (1H, q, J 6.8),
1.66–1.38 (2H, m), 1.10 (1H, t, J 6.9), 0.85 (6H, 2d, J 5.5
each). dC ([D6]DMSO, 101 MHz) 172.6, 162.1, 159.9, 156.1,
155.1, 150.4, 143.8, 142.5, 142.2, 139.6, 126.0, 125.2, 121.3,
113.3, 109.7, 109.1, 108.9, 102.5, 65.6, 64.9, 57.5, 52.2, 49.1,
46.6, 24.1, 22.7, 21.1. m/z (HRMS ESI) 631.2189; [M þ Na]þ
requires 631.2163.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-
isoleucinate (8f) (0.235 g, 85 %), white solid, mp 143–1468C.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl(tert-butoxycarbonyl)-L-phenylalaninate (8a)
(0.235 g, 85 %), white solid, mp 143–1458C. dH ([D6]DMSO,
400 MHz) 10.65 (1H, s), 8.21 (1H, s), 7.71 (1H, d, J 8.7), 7.27–
7.16 (6H, m), 6.83 (1H, dd, J 8.7, 7.1), 6.76 (1H, s), 5.90 (2H, s),
5.62 (1H, s), 5.25–5.12 (2H, m), 4.24–4.14 (1H, m), 2.98 (1H,
dd, J 13.8, 5.0), 2.86 (1H, dd, J 13.5, 10.1), 1.29 (9H, s). dC ([D6]
DMSO þ CDCl3, 101 MHz) 171.8, 161.6, 159.8, 155.3, 155.1,
150.2, 142.1, 137.3, 129.0, 128.0, 126.3, 125.9, 125.7, 113.1,
109.4, 109.3, 102.5, 78.2, 57.6, 55.2, 49.1, 36.3, 28.0. m/z
(HRMS ESI) 543.1837; [M þ Na]þ requires 543.1850.
d
H ([D6]DMSO þ CDCl3, 400 MHz) 10.58 (1H, s), 8.26 (1H,
s), 7.84 (2H, d, J 7.5), 7.76–7.69 (3H, m), 7.66 (1H, d, J 8.8),
7.40 (2H, t, J 7.4), 7.31 (2H, t, J 6.6), 6.81 (1H, dd, J 8.7, 2.3),
6.74 (1H, d, J 2.3), 5.87 (2H, s), 5.55 (1H, s), 5.24 (2H, dd, J
27.9, 12.7), 4.28 (2H, d, J 7.2), 4.24–4.18 (1H, m), 4.04–3.99
(1H, m), 1.84–1.75 (1H, m), 1.42–1.33 (1H, m), 1.24 (1H, s),
0.83–0.73 (6H, m). dC ([D6]DMSO þ CDCl3, 101 MHz) 171.5,
161.6, 159.8, 156.2, 155.1, 150.1, 143.7, 143.6, 142.2, 140.7,
127.5, 126.9, 125.8, 125.7, 125.2, 119.9, 113.1, 109.2, 109.2,
102.5, 65.7, 58.5, 57.2, 49.1, 46.6, 36.1, 24.8, 15.3, 10.9. m/z
(HRMS ESI) 631.2164; [M þ Na]þ requires 631.2164.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl((benzyloxy)carbonyl)-L-phenylalanyl-L-
tryptophanate (9a) (0.240 g, 72 %), yellow solid, mp
155–1588C. dH ([D6]DMSO, 300 MHz) 10.88 (1H, s), 8.51
(1H, d, J 7.0), 8.14 (1H, s), 7.69 (1H, d, J 8.6), 7.46 (2H, dd, J
18.6, 8.3), 7.34–7.17 (14H, m), 7.10–6.95 (2H, m), 6.84 (1H, d,
J 8.6), 6.77 (1H, s), 5.88 (2H, s), 5.54 (1H, s), 5.21–5.11 (2H,
m), 4.98–4.86 (2H, m), 4.57 (1H, dd, J 12.4, 6.0), 4.32 (1H, t, J
7.4), 3.22–3.09 (2H, m), 2.94 (1H, d, J 14.1), 2.67 (1H, t, J
11.7). dC ([D6]DMSO, 75 MHz) 172.4, 171.9, 162.3, 160.5,
156.3, 155.9, 155.6, 155.3, 151.3, 150.9, 142.7, 138.6, 137.5,
136.6, 129.7, 128.8, 128.5, 128.2, 128.0, 127.6, 126.7, 126.6,
126.3, 124.3, 121.5, 119.0, 118.5, 113.8, 112.0, 109.7, 103.10,
65.8, 58.3, 56.4, 53.7, 49.7, 38.0, 27.4. m/z (HRMS ESI)
763.2480; [M þ Na]þ requires 763.2487.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl((benzyloxy)carbonyl)-L-alaninate
(8b)
(0.200 g, 92 %), white solid, mp 147–1498C. dH (CDCl3,
300 MHz) 7.77 (1H, s), 7.30 (1H, d, J 7.5), 7.22–7.14 (6H, m),
6.70 (1H, d, J 7.6), 6.61 (1H, s), 5.62–5.55 (2H, m), 5.51 (2H, s),
5.20–5.13 (1H, m), 5.01–4.90 (2H, m), 4.27–4.17 (1H, m), 1.26
(3H, d, J 6.5). dC (CDCl3, 75 MHz) 173.0, 161.5, 161.18, 156.1,
155.2, 148.9, 143.1, 136.0, 128.6, 128.3, 128.0, 125.5, 125.0,
114.0, 110.5, 109.7, 103.5, 67.1, 58.1, 50.3, 49.8, 18.0. m/z
(HRMS ESI) 501.1390; [M þ Na]þ requires 501.1381.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl((benzyloxy)carbonyl)-L-phenylalaninate (8c)
(0.220 g, 87 %), white solid, mp 150–1528C. dH ([D6]DMSO,
400 MHz) 8.27 (1H, s), 7.87 (1H, d, J 8.1), 7.73 (1H, d, J 8.8),
7.36–7.29 (3H, m), 7.28–7.21 (8H, m), 6.86 (1H, dd, J 8.7, 2.3),
6.78 (1H, d, J 2.3), 5.94 (2H, s), 5.61 (1H, s), 5.23 (2H, s), 5.02–
4.95 (2H, m), 4.30 (1H, ddd, J 10.0, 8.1, 5.2), 3.03 (1H, dd, J
13.8, 5.1), 2.88 (1H, dd, J 13.7, 10.1). dC ([D6]DMSO,
101 MHz) 171.6, 161.8, 159.9, 155.9, 155.1, 150.4, 142.1,
142.0, 137.2, 137.1, 136.8, 129.0, 128.3, 128.2, 127.7, 127.5,
127.0, 126.5, 126.1, 125.9, 113.2, 109.3, 102.5, 65.4, 57.7, 55.5,
49.2, 36.3. m/z (HRMS ESI) 577.1693; [M þ Na]þ requires
577.1694.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl((benzyloxy)carbonyl)-L-phenylalanyl-L-ala-
ninate (9b) (0.220 g, 74 %), yellow solid, mp 153–1558C. dH
([D6]DMSO, 400 MHz) 10.79 (1H, br s), 8.57 (1H, s), 8.32 (1H,
s), 7.70 (1H, d, J 8.3), 7.48 (1H, d, J 7.9), 7.38–7.17 (10H, m),
6.85 (1H, d, J 8.2), 6.78 (1H, s), 5.91 (2H, s), 5.55 (1H, s), 5.28–
5.17 (2H, m), 4.94 (2H, s), 4.31 (2H, d, J 7.5), 2.99 (1H, d, J
13.6), 2.69 (1H, t, J 11.8), 1.31 (3H, br s). dC ([D6]DMSO,
101 MHz) 172.2, 171.7, 161.8, 159.9, 155.8, 155.1, 150.4,
142.3, 138.1, 137.0, 129.2, 128.2, 128.0, 127.6, 127.4, 127.0,
127.0, 126.2, 126.0, 125.9, 113.2, 109.3, 109.1, 102.5, 65.1,
57.6, 55.7, 49.1, 47.6, 37.3, 16.7. m/z (HRMS ESI) 648.2085;
[M þ Na]þ requires 648.2065.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,
3-triazol-4-yl)methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-
L-phenylalaninate (8d) (0.250 g, 86 %), white solid, mp 148–
1508C. dH (CDCl3, 300 MHz) 7.72 (2H, d, J 7.5), 7.61 (1H, s),
7.53–7.47 (3H, m), 7.41–7.33 (4H, m), 7.31–7.27 (2H, m), 7.22–
7.16 (3H, m), 7.04 (1H, s), 6.83 (1H, d, J 8.5), 6.76 (1H, s), 5.77
(1H, s), 5.60–5.38 (2H, m), 5.32–5.17 (2H, m), 4.61 (1H, dd,
J 13.7, 7.6), 4.34 (2H, dd, J 21.2, 7.9), 4.13 (1H, t, J 6.7),
3.15–3.00 (2H, m). dC (CDCl3, 75 MHz) 171.4, 162.0, 161.5,
160.8, 156.1, 155.8, 155.6, 148.3, 148.0, 143.6, 143.1, 141.3,
135.5, 129.3, 128.7, 127.8, 127.2, 120.0, 114.0, 111.5, 109.8,
103.9, 67.2, 58.2, 55.0, 47.1, 38.0, 29.8. m/z (HRMS ESI)
665.2021; [M þ Na]þ requires 665.2007.
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
(tert-butoxycarbonyl)-L-phenylalaninate (10a) (0.220 g, 85 %),
white solid, mp 138–1408C. dH (CDCl3, 300 MHz) 8.05 (1H, s),
7.93 (1H, d, J 7.6), 7.77 (1H, t, J 7.7), 7.56 (1H, d, J 8.3), 7.45
(1H, t, J 7.5), 7.36–7.27 (3H, m), 7.19 (2H, d, J 6.8), 6.62 (1H,
s), 5.47 (2H, s), 5.08 (1H, d, J 6.5), 4.68 (1H, d, J 6.0), 3.18 (2H,
d, J 6.8), 1.47 (9H, s). dC (CDCl3, 75 MHz) 172.0, 159.6, 155.3,
154.4, 146.7, 135.8, 133.8, 129.4, 128.7, 127.3, 125.6, 125.2,
117.7, 114.3, 110.2, 80.4, 58.1, 54.7, 38.1, 28.4. m/z (HRMS
ESI) 513.1751; [M þ Na]þ requires 513.1745.
(1-((7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,
3-triazol-4-yl)methyl(((9H-fluoren-9-yl)methoxy)carbonyl)-
L-leucinate (8e) (0.235 g, 84 %), white solid, mp 146–1488C.
d
H ([D6]DMSO, 400 MHz) 8.30 (1H, s), 7.90 (2H, d, J 7.4),
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
((benzyloxy)carbonyl)-L-alaninate (10b) (0.215 g, 91 %),
white solid, mp 139–1428C. dH (CDCl3, 300 MHz) 7.97
7.85 (1H, t, J 8.7), 7.74–7.67 (2H, m), 7.43 (2H, t, J 7.2),
7.38–7.30 (2H, m), 6.84 (1H, d, J 8.5), 6.77 (1H, s), 6.29 (1H,

J
S. Kumari et al.
(1H, s), 7.78–7.63 (1H, m), 7.59–7.46 (1H, m), 7.38–7.27 (1H,
m), 7.23–7.03 (6H, m), 6.43 (1H, s), 5.39–5.02 (3H, m), 4.94
(2H, s), 4.24 (1H, d, J 3.5), 1.29 (3H, br s). dC (CDCl3, 75 MHz)
173.1, 159.6, 156.4, 155.8, 154.4, 146.6, 136.5, 136.2, 133.7,
128.6, 128.2, 128.0, 125.6, 125.1, 117.7, 114.3, 110.2, 67.1,
58.2, 49.9, 18.2. m/z (HRMS ESI) 471.1282; [M þ Na]þ
requires 471.1275.
temperature for 6–10 h and monitored via TLC. After comple-
tion of the reaction, the reaction mixture was evaporated and the
residue was diluted with water and ammonium hydroxide (2–
3 mL) and extracted into dichloromethane. Organic layer was
separated dried over anhydrous sodium sulfate to evaporate the
volatiles. The crude compound was recrystallized from ethanol
to yield pure 13a–f.
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
((benzyloxy)carbonyl)-L-phenylalaninate (10c) (0.260 g,
93 %), white solid, mp 143–1458C. dH (CDCl3, 300 MHz)
7.84 (1H, s), 7.76 (1H, d, J 7.9), 7.61 (1H, t, J 7.7), 7.40 (1H,
d, J 8.4), 7.30 (4H, d, J 7.7), 7.19–7.16 (2H, m), 7.17–7.10 (3H,
m), 7.01 (2H, d, J 6.7), 6.46 (1H, s), 5.34 (2H, t, J 8.2), 5.20
(1H, d, J 7.9), 5.01 (2H, s), 4.61 (1H, dd, J 12.8, 6.2), 3.06 (2H,
d, J 5.4). dC (CDCl3, 75 MHz) 171.6, 159.7, 159.4, 155.8,
154.4, 146.6, 143.4, 136.1, 135.4, 133.8, 129.4, 128.7, 128.3,
128.1, 127.4, 125.6, 125.2, 121.5, 117.7, 114.2, 110.2, 67.3,
58.2, 55.0, 38.1. m/z (HRMS ESI) 547.1599; [M þ Na]þ
requires 547.1588.
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
(((9H-fluoren-9-yl)methoxy)carbonyl)-L-phenylalaninate (10d)
(0.290 g, 89 %), white solid, mp 134–1368C. dH (CDCl3,
300 MHz) 7.90 (1H, s), 7.76 (4H, m), 7.63 (1H, t, J 7.5), 7.53
(2H, d, J 7.0), 7.41 (4H, dd, J 18.5, 7.8), 7.33–7.27 (3H, m),
7.24–7.17 (2H, m), 7.14–7.04 (1H, m), 6.50 (1H, s), 5.50–5.35
(2H, m), 5.34–5.25 (1H, m), 4.71 (1H, t, J 6.1), 4.39 (2H, dd,
J 17.8, 10.5), 4.18 (1H, d, J 6.1), 3.15 (2H, d, J 6.2). dC (CDCl3,
75 MHz) 171.5, 159.5, 155.6, 154.2, 146.4, 143.6, 141.3,
135.4, 134.7, 133.8, 133.6, 129.3, 128.8, 128.6, 127.7, 127.3,
127.1, 125.4, 124.9, 120.0, 117.6, 114.1, 110.1, 67.0, 58.2, 54.9,
47.1, 37.9. m/z (HRMS ESI) 635.1919; [M þ Na]þ requires
635.1901.
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
(tert-butoxycarbonyl)-L-alaninate (10g) (0.180 g, 81 %), white
solid, mp 133–1358C. dH (CDCl3, 300 MHz) 8.16 (1H, s), 7.87
(1H, d, J 7.8), 7.67 (1H, t, J 7.8), 7.46 (1H, d, J 8.1), 7.36 (1H, t,
J 7.8), 6.58 (1H, s), 5.43 (2H, q, J 13.0), 5.01 (1H, d, J 4.8), 4.30
(1H, t, J 6.6), 1.42 (3H, br s), 1.39 (9H, s). dC (CDCl3, 75 MHz)
173.5, 159.6, 155.3, 154.4, 146.7, 143.9, 133.7, 125.6, 125.2,
125.0, 117.7, 114.3, 110.2, 80.3, 58.1, 49.5, 28.4, 18.2. m/z
(HRMS ESI) 437.1441; [M þ Na]þ requires 437.1432.
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)methyl
((benzyloxy)carbonyl)-L-phenylalanyl-L-tryptophanate (11a)
(0.295 g, 78 %), yellow solid, mp 150–1528C. dH ([D6]DMSO,
300 MHz) 11.1 (1H, s), 8.79 (2H, br s), 7.94 (2H, br s), 7.80–7.62
(4H, m), 7.49–7.33 (12H, m), 7.25–7.12 (2H, m), 7.04 (1H, s),
5.46 (2H, s), 5.09 (2H, s), 4.87–4.76 (1H, m), 4.53–4.44 (1H, m),
3.42–3.27 (2H, m), 3.13–3.04 (1H, m), 2.86–2.75 (1H, m). dC
([D6]DMSO, 101 MHz) 171.9, 171.3, 159.4, 155.8, 153.6,
145.7, 142.6, 138.0, 136.9, 136.0, 133.5, 129.1, 128.2, 127.9,
127.6, 127.4, 127.0, 126.3, 125.4, 125.0, 123.8, 123.7, 121.0,
118.4, 118.0, 117.1, 114.20, 111.4, 110.6, 109.1, 65.2, 57.3,
55.8, 53.1, 37.3, 26.8. m/z (HRMS ESI) 733.2369; [M þ Na]þ
requires 733.2381.
Ethyl 2-(4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-
1H-1,2,3-triazol-1-yl)acetate (13a) (0.140 g, 88 %), white solid,
mp 129–1328C. dH (CDCl3, 300 MHz) 7.82 (1H, s), 7.50 (1H, d, J
8.6), 6.98–6.89 (2H, m), 6.14 (1H, s), 5.28 (2H, s), 5.18 (2H, s),
4.27 (2H. q, J 7.1), 2.39 (3H, s), 1.30 (3H, t, J 7.1). dC (CDCl3,
75MHz) 161.2, 155.2, 152.6, 143.6, 126.0, 125.8, 124.5, 114.2,
112.5, 112.4, 102.2, 62.7, 62.3, 51.0, 18.8, 14.2. m/z (HRMS ESI)
366.1056; [M þ Na]þ requires 366.1060.
Methyl-(S)-2-(4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)-3-phenylpropanoate
(13b)
(0.165 g, 85 %), white solid, mp 133–1358C. dH (CDCl3,
300 MHz) 7.69 (1H, s), 7.48 (1H, d, J 6.6), 7.21–7.09 (3H, m),
7.02–6.81 (4H, m), 6.12 (1H, s), 5.62–5.52 (1H, m), 5.20 (2H, s),
3.74 (3H, s), 3.52–3.38 (2H, m), 2.37 (3H, s). dC (CDCl3,
75 MHz) 168.6, 161.3, 161.2, 155.2, 152.5, 143.4, 143.1,
134.5, 128.9, 127.7, 125.8, 123.3, 114.1, 112.6, 112.4, 102.2,
64.2, 62.4, 53.3, 39.1, 18.9. m/z (HRMS ESI) 442.1385;
[M þ Na]þ requires 442.1373.
Methyl-(S)-3-(1H-indol-3-yl)-2-(4-(((4-methyl-2-oxo-2H-
chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)propanoate (13c)
(0.175 g, 82 %), white solid, mp 138–1408C. dH ([D6]DMSO,
300 MHz) 10.85 (1H, s), 8.44 (1H, s), 7.70 (1H, d, J 8.8), 7.51
(1H, d, J 7.8), 7.32 (1H, d, J 8.0), 7.14 (1H, s), 7.10–6.94 (4H,
m), 6.24 (1H, s), 5.87 (1H, t, J 7.6), 5.24 (2H, s), 3.73 (3H, s),
3.71–3.67 (2H, m), 2.42 (3H, s). dC ([D6]DMSO, 75 MHz)
169.6, 161.6, 160.7, 155.2, 154.0, 142.3, 136.5, 127.2, 127.0,
125.7, 124.3, 121.7, 119.1, 118.5, 114.0, 113.1, 112.0, 108.6,
102.1, 63.3, 62.2, 53.5, 27.8, 18.7. m/z (HRMS ESI) 481.1496;
[M þ Na]þ requires 481.1482.
Ethyl-2-(4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)acetate (13d) (0.145 g, 90%), white solid, mp 128–
1308C. dH (CDCl3, 300MHz) 7.87 (1H, s), 7.72 (1H, d, J 7.8),
7.48 (1H, t, J 7.7), 7.24–7.14 (2H, m), 5.81 (1H, s), 5.32 (2H, s),
5.18 (2H, s), 4.24 (2H, q, J 7.0), 1.27 (3H, t, J 7.1). dC (CDCl3,
75 MHz) 166.1, 165.0, 162.7, 153.4, 142.0, 132.6, 125.0, 124.0,
123.2, 116.8, 115.5, 91.3, 62.7, 51.1, 14.2. m/z (HRMS ESI)
352.0911; [M þ Na]þ requires 352.0904.
Methyl-(S)-2-(4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-1H-
1,2,3-triazol-1-yl)-3-phenylpropanoate (13e) (0.175 g, 87 %),
white solid, mp 130–1328C. dH (CDCl3, 300 MHz) 7.80
(1H, s), 7.69 (1H, d, J 7.3), 7.48 (1H, t, J 7.7), 7.27–7.09
(5H, m), 6.93 (2H, d, J 3.2), 5.75 (1H, s), 5.65–5.56 (1H, m),
5.23 (2H, s), 3.71 (3H, d, J 6.9), 3.51–3.38 (2H, m). dC (CDCl3,
75 MHz) 168.5, 164.9, 162.6, 153.4, 134.4, 132.6, 128.9, 127.8,
124.0, 123.2, 116.8, 115.5, 91.2, 64.4, 62.7, 53.3, 39.1. m/z
(HRMS ESI) 428.1226; [M þ Na]þ requires 428.1217.
Methyl-(S)-3-(1H-indol-3-yl)-2-(4-(((2-oxo-2H-chromen-4-yl)
oxy)methyl)-1H-1,2,3-triazol-1-yl)propanoate (13f) (0.185 g,
85 %), white solid, mp 134–1368C. dH ([D6]DMSO, 300 MHz)
10.88 (1H, s), 8.52 (1H, s), 7.71–7.65 (2H, m), 7.50–7.29 (5H, m),
7.06–7.00 (1H, m), 6.95 (1H, t, J 7.4), 6.14 (1H, s), 5.90 (1H, t, J
7.6), 5.39 (2H, s), 3.75 (3H, s), 3.72–3.68 (2H, m). dC ([D6]
DMSO, 75 MHz) 169.5, 164.8, 162.1, 153.27, 141.3, 136.4, 133.4,
127.3, 126.0, 124.8, 124.4, 123.4, 121.6, 119.1, 118.4, 117.0,
115.6, 112.0, 108.6, 91.9, 63.5, 63.3, 53.7, 27.8. m/z (HRMS ESI)
462.1776; [M þ NH4]þ requires 462.1772.
Typical Procedure for the Synthesis of Triazolyl-Linked
Amino Acids 13a–f
To a solution of 4-methyl-7-(prop-2-ynyloxy)-coumarin (2a)
or 4-(prop-2-ynyloxy)-coumarin (2b) (0.100 g, 1equiv.) was
added a-azido esters 12a–c (1 equiv.) in ethanol/H2O (1 : 1),
followed by addition of CuSO4 (0.01 equiv.) and sodium ascor-
bate (0.1 equiv.). The reaction mixture was stirred at room

Fluorescent-Labelled Amino Acids and Peptides
K
Procedure for the Synthesis of (1-(2-oxo-2H-chromen-
4-yl)-1H-1,2,3-triazol-4-yl)methyl-L-alaninate
Hydrochloride (14)
the reaction mixture was stirred for 15 min at 08C. Later L-
phenylalanine methyl ester (0.029 g, 1 equiv.) was added and the
reaction was stirred at room temperature for 6 h. The completion
of the reaction was monitored by TLC. After the completion of
the reaction, crushed ice was added that resulted in the precipi-
tation of the product, which was filtrated, washed with cold
water, and recrystallized with ethanol to afford 17 (0.065 g,
90 %), white solid, mp 135–1378C. dH ([D6]DMSO 1 CDCl3,
400 MHz) 8.89 (1H, d, J 7.7), 8.09 (1H, s), 7.64 (1H, d, J 8.8),
7.31–7.25 (2H, m), 7.24–7.18 (3H, m), 7.10 (1H, d, J 2.2), 7.01
(1H, dd, J 8.8, 2.2), 6.16 (1H, s), 5.26 (2H, s), 5.14 (2H, q, J
16.2), 4.57 (1H, dd, J 13.7, 8.1), 3.64 (3H, s), 3.08 (1H, dd, J
13.8, 5.5), 2.95 (1H, dd, J 13.8, 8.7), 2.40 (3H, s). dC ([D6]
DMSO 1 CDCl3, 101 MHz) 171.3, 165.3, 161.0, 160.0, 154.7,
152.9, 141.6, 136.5, 129.0, 128.2, 126.5, 126.1, 126.0, 113.3,
112.4, 111.3, 101.4, 61.5, 53.6, 51.8, 51.3, 36.8, 18.1.
A solution of dioxane/HCl was added dropwise to 10g
(0.150 g, 1 equiv.) that was initially dissolved in dioxane at
08C. The reaction was stirred at room temperature for 2 h and
monitored via TLC. After reaction completion, the reaction
mixture was completely concentrated, washed with diethyl
ether, and filtered off to give 14 (0.085 g, 68 %), white solid,
mp 160–1628C. dH ([D6]DMSO 1 CDCl3, 300 MHz) 8.99 (1H,
s), 7.87 (1H, d, J 8.1), 7.78 (1H, t, J 7.6), 7.57 (1H, s), 7.45 (1H, t,
J 7.4), 6.95 (1H, s), 5.47 (2H, s), 4.15 (1H, dd, J 13.4, 6.5), 3.39
(2H, br s (merged with moisture of DMSO)), 1.50 (3H, d, J 6.8).
dC ([D6]DMSO 1 CDCl3, 101 MHz) 169.6, 159.3, 153.6, 145.7,
142.2, 133.4, 126.6, 125.5, 124.9, 117.1, 114.1, 110.4, 58.2,
47.9, 15.6.
Supplementary Material
(1-(2-Oxo-2H-chromen-4-yl)-1H-1,2,3-triazol-4-yl)
methyl(tert-butoxycarbonyl)-L-phenylalanyl-L-
alaninate (15)
Copies of 1H NMR, 13C NMR, and HRMS spectra of the syn-
thesized compounds are available on Journal’s website.
To the stirred solution of 14 (0.050, 1 equiv.) in DMF, triethyl
amine (2.5 equiv.) was added at 08C and subsequently EDCꢀHCl
(1.5 equiv.) and HOBt (1.2 equiv.) were added, and the reaction
mixture was stirred for 15 min at 08C. Then, Boc-L-phenylala-
nine (0.038 g, 1 equiv.) was added and the reaction was stirred at
room temperature for 8 h. The completion of the reaction was
monitored by TLC. After the completion of the reaction, crushed
ice was added that resulted in the precipitation of the product
which was filtrated, washed with cold water, and recrystallized
with ethanol to afford 15 (0.075 g, 86 %), white solid, mp 145–
1478C. dH ([D6]DMSO 1 CDCl3, 400 MHz) 8.81 (1H, s), 8.42
(1H, s), 7.83 (1H, d, J 8.4), 7.75 (1H, s), 7.54 (1H, d, J 8.1), 7.40
(1H, d, J 8.0), 7.27–7.10 (5H, m), 6.90–6.83 (1H, m), 6.71 (1H,
d, J 7.7), 5.34 (2H, s), 4.45–4.34 (1H, m), 4.25–4.18 (1H, m),
2.95 (1H, d, J 13.8), 2.68 (1H, d, J 12.0), 1.37 (3H, br s), 1.29
(9H, s). dC ([D6]DMSO 1 CDCl3, 101 MHz) 172.1, 171.8,
159.2, 155.1, 153.6, 145.7, 142.7, 137.9, 133.3, 129.1, 127.8,
126.2, 126.0, 125.5, 124.8, 117.0, 114.1, 110.3, 77.9, 57.3, 55.2,
47.6, 37.4, 28.1, 16.8.
Acknowledgements
The authors acknowledge the Department of Science and Technology
(DST), New Delhi, for providing research grant (SB/FT/CS-033/2012) for
the research work. DSA also thanks DST, New Delhi for the research fel-
lowship. SK and SMAS thank UGC, New Delhi, India for their Junior
Research Fellowship and BSR Fellowship, respectively.
References
[1] M. S. Dillingham, M. I. Wallace, Org. Biomol. Chem. 2008, 6, 3031.
doi:10.1039/B808552H
[2] H. Mihara, S. Lee, Y. Shimohigashi, H. Aoyagi, T. Kato, N. Izumiya,
T. Costa, FEBS Lett. 1985, 193, 35. doi:10.1016/0014-5793(85)
80074-0
[3] (a) S. Heiner, H. Detert, A. Kuhn, H. Kunz, Bioorg. Med. Chem. 2006,
14, 6149. doi:10.1016/J.BMC.2006.06.014
(b) Z. Zhou, C. J. Fahrni, J. Am. Chem. Soc. 2004, 126, 8862.
doi:10.1021/JA049684R
[4] X.-G. Zhou, M.-S. Peng, T.-Z. Feng, S. Afr. J. Chem. 2013, 66, 69.
[5] I. Kosiova, P. Kois, Collect. Czech. Chem. Commun. 2007, 72, 996.
doi:10.1135/CCCC20070996
2-(4-(((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-
1H-1,2,3-triazol-1-yl)acetic Acid (16)
[6] S. Zhou, J. Jia, J. Gao, L. Han, Y. Li, W. Sheng, Dyes Pigm. 2010, 86,
123. doi:10.1016/J.DYEPIG.2009.12.005
To a solution of 13a (0.100 g, 1 equiv.) in ethanol cooled to
08C was added 30 % NaOH solution in water (5 mL) dropwise.
The reaction was stirred at room temperature for 3 h and
monitored via TLC. After the completion of the reaction,
ethanol was completely concentrated under reduced pressure
to give the corresponding salt of the acid, which was acidified
using 4N HCl at 08C and the aqueous layer was extracted with
dichloromethane to give 16 (0.075 g, 85 %), white solid, mp
110–1128C. dH ([D6]DMSO 1 CDCl3, 400 MHz) 8.23 (1H, s),
7.66 (1H, d, J 8.8), 7.12 (1H, s), 7.03 (1H, d, J 8.8), 6.17 (1H, s),
5.29 (2H, s), 5.26 (2H, s), 2.41 (3H, s). dC ([D6]DMSO 1
CDCl3, 101 MHz) 168.3, 161.0, 160.0, 154.7, 152.9, 141.8,
126.2, 126.0, 113.3, 112.5, 111.3, 101.5, 61.6, 50.6, 18.1.
[7] S. Azim, S. Al-Hazmy, E. Ebeid, S. El-Daly, Opt. Laser Technol. 2005,
37, 245. doi:10.1016/J.OPTLASTEC.2004.04.003
[8] B. D. Wagner, Molecules 2009, 14, 210. doi:10.3390/
MOLECULES14010210
[9] D. Y. Yee, V. Balsanek, D. Sames, J. Am. Chem. Soc. 2004, 126, 2282.
doi:10.1021/JA039799F
[10] J. Wang, J. Xie, P. G. Schultz, J. Am. Chem. Soc. 2006, 128, 8738.
doi:10.1021/JA062666K
[11] J. L. Lauer-Fields, T. Broder, T. Sritharan, L. Chung, H. Nagase, G. B.
Fields, Biochemistry 2001, 40, 5795. doi:10.1021/BI0101190
[12] X. Jin, C. Uttamapinant, A. Y. Ting, ChemBioChem 2011, 12, 65.
doi:10.1002/CBIC.201000414
[13] A. R. Katritzky, T. Narindoshvili, P. Angrish, Synthesis 2008, 2013.
[14] M. Meldal, C. W. Tornøe, Chem. Review 2008, 108, 2952.
doi:10.1021/CR0783479
[15] G. C. Tron, T. Pirali, R. A. Billington, P. L. Canonico, G. Sorba, A. A.
Genazzani, Med. Res. Rev. 2008, 28, 278. doi:10.1002/MED.20107
[16] W. S. Horne, M. K. Yadav, C. D. Stout, M. R. Ghadiri, J. Am. Chem.
Soc. 2004, 126, 15366. doi:10.1021/JA0450408
[17] K. Oh, Z. Guan, Chem. Commun. 2006, 3069. doi:10.1039/B606185K
[18] A. Tam, U. Arnold, M. B. Soellner, R. T. Raines, J. Am. Chem. Soc.
2007, 129, 12670. doi:10.1021/JA075865S
Methyl-(2-(4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)acetyl)-L-
phenylalaninate (17)
To the stirred solution of 16 (0.050 g, 1 equiv.) in DMF,
triethyl amine (2.5 equiv.) was added at 08C and subsequently
EDCꢀHCl (1.5 equiv.) and HOBt (1.2 equiv.) were added, and

L
S. Kumari et al.
[19] K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill, Q. Wang,
Org. Lett. 2004, 6, 4603. doi:10.1021/OL047955X
[20] A. V. Moro, P. C. Ferreira, P. Migowski, F. S. Rodembusch, J. Dupont,
D. S. Ludtke, Tetrahedron 2013, 69, 201. doi:10.1016/J.TET.2009.07.
054
[21] N. Li, P. Zhao, M. E. Igartua, A. Rapakousiou, L. Salmon, S. Moya,
J. Ruiz, D. Astruc, Inorg. Chem. 2014, 53, 11802. doi:10.1021/
IC5021498
[26] G. Gao, F. Sanda, T. Masuda, Macromolecules 2003, 36, 3932.
doi:10.1021/MA021738Z
[27] N. Anand, N. Jaiswal, S. K. Pandey, A. K. Srivastava, R. P. Tripathi,
Carbohydr. Res. 2011, 346, 16. doi:10.1016/J.CARRES.2010.10.017
[28] N. J. Stanley, D. S. Pedersen, B. Nielsen, T. Kyist, J. M. Mathiesen,
H. Bra¨uner-Osborne, D. K. Taylor, A. D. Abell, Bioorg. Med. Chem.
Letters 2010, 20, 7512. doi:10.1016/J.BMCL.2010.09.139
[29] H. Li, Y. Yao, C. Han, J. Zhan, Chem. Commun. 2009, 4812.
doi:10.1039/B908761C
[22] M. Kova´cˇ, A. Sabatie´, L. Floch, ARKIVOC 2001, 2001, 100.
doi:10.3998/ARK.5550190.0002.610
[30] E. Ko, J. Liu, L. M. Perez, G. Lu, A. Schaefer, K. Burgess, J. Am.
[23] W. Lin, L. Long, J. Feng, B. Wang, C. Guo, Eur. J. Org. Chem. 2007,
2007, 4301. doi:10.1002/EJOC.200700475
[24] S. P. Bew, G. D. Hiatt-Gipson, J. Org. Chem. 2010, 75, 3897.
doi:10.1021/JO100537Q
Chem. Soc. 2011, 133, 462. doi:10.1021/JA1071916
[31] J. H. Mishra, D. Pant, T. C. Pant, H. B. Tripathi, J. Photochem.
Photobiol. Chem. 2006, 177, 197. doi:10.1016/J.JPHOTOCHEM.
2005.05.026
[25] T. Aravinda, H. S. B. Naik, H. R. P. Naik, Int. J. Pept. Res. Ther. 2009,
15, 273. doi:10.1007/S10989-009-9188-X
[32] C. Ranjith, K. K. Vijayan, V. K. Praveen, N. S. S. Kumar, Spectrochim.
Acta, Part A 2010, 75, 1610. doi:10.1016/J.SAA.2010.02.027