Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA

Article abstract of DOI:10.1016/j.bmc.2016.05.069

The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV–visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO–LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor–acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II β-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor–acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide–protein interaction.

Full text of DOI:10.1016/j.bmc.2016.05.069

Accepted Manuscript
Synthesis, Photophysical Properties of Triazolyl-Donor/Acceptor Chomo-
phores Decorated Unnatural Amino Acids: Incorporation of a Pair into Leu-
Enkephalin Peptide and Application of Triazolylperylene Amino Acid in
Sensing BSA
Subhendu Sekhar Bag, Subhashis Jana, Manoj Kumar Pradhan
PII:
S0968-0896(16)30413-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.069
BMC 13053
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 April 2016
29 May 2016
30 May 2016
Please cite this article as: Bag, S.S., Jana, S., Pradhan, M.K., Synthesis, Photophysical Properties of Triazolyl-Donor/
Acceptor Chomophores Decorated Unnatural Amino Acids: Incorporation of a Pair into Leu-Enkephalin Peptide
and Application of Triazolylperylene Amino Acid in Sensing BSA, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.05.069
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Synthesis, Photophysical Properties of Triazolyl-Donor/Acceptor Chomophores Decorated
Unnatural Amino Acids: Incorporation of a Pair into Leu-Enkephalin Peptide and
Application of Triazolylperylene Amino Acid in Sensing BSA
Subhendu Sekhar Bag,* Subhashis Jana, and Manoj Kumar Pradhan
Bioorganic Chemistry Laboratory, Department of Chemistry
Indian Institute of Technology, Guwahati-781039, India.
Fax: (+)91-361-258-2349; Tel.: (+)91-361-258-2324.
E-mail: ssbag75@iitg.ernet.in
ABSTRACT: The research in the field of design and synthesis of unnatural amino acids is
growing at a fast space for the increasing demand of proteins of potential therapeutics and many
other diversified novel functional applications. Thus, we report herein the design and synthesis
of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated
with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized
fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular
charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical
properties and DFT/TDDFT calculation. HOMO-LUMO distribution shows that the emissive
states of some of the amino acids are characterized with more significant electron redistribution
between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated
emission property. A pair of donor-acceptor amino acid shows interesting photophysical
interaction property indicating a FRET quenching event. Furthermore, one of the amino acid,
1

triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found
that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we
incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide
which was found to adopt predominantly type II β-turn conformation. We envisage that our
investigation is of importance for the development of new fluorescent donor-acceptor unnatural
amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of
interesting photophysical property for applications in studying peptide-protein interaction.
Key Words: Triazolyl donor-acceptor unnatural amino acids, click reaction, photophysical,
BSA sensing, Leu-enkephalin analogue, peptidomimetic, b-turn
Introduction
Amino acids are central ingredients of life system and protein function. However, there exist
only 20 naturally occurring amino acids as building blocks of all translated proteins in all
organisms having more or less common structures and functions in a population.1 Therefore,
proteins generated from natural selection/evolution are highly specialized for specific functions
and thus, are not suitable for a different function other than they use to do the specific job. To
diversify protein’s function, the building blocks need additional functionalities.1b Moreover,
posttranslational modifications, cofactor-dependent catalysis, and pyrrolysine/selenocystein
incorporation in bacteria proves that the natural evolutionary movement needs extra chemical
functionalities other than those present within the 20 natural amino acids.2 Thus, the method of
“directed evolution” has been introduced to mimic the natural evolution cycle in a laboratory to
2

generate proteins of altered structural and functional properties that are different from natural
one.3
Inspired by the mother Natures’ post-translational modification to include different
functional units or molecules into the proteins, several chemical/biochemical strategies have
been developed to attach biophysical probe to proteins to facilitate the study of structure and
functions at molecular level.4 However, there are only nine canonical amino acids with a limited
number of functional groups available for protein modification. Therefore, site-specific
conjugation to a desired canonical amino acid within a protein is a difficult task.5 To circumvent
this problem, several chemical and biochemical methodologies have been developed to site-
specifically incorporate designer amino acids (the unnatural amino acids) with desired
functionalities to probe protein structure and function and to generate proteins/enzyme for
several novel biochemical applications, such as for chemical synthesis, biomedical research or
even as therapeutics.6
Furthermore, natural peptides/proteins are generally poor drug candidates due to their low
oral bioavailability and unfavorable pharmacokinetics.7 Only a limited number of successes have
been achieved to circumvent the problems and improve the bioavailability of natural peptide
based drug candidates.8 Therefore, field of peptidomimetics is evolved to replace biologically
active parts of peptides with non-peptide structures to increase bioavailability of peptide-based
drugs.9 Out of two main strategies in the development of peptidomimetic substrates,
incorporation of unnatural amino acid into peptide backbone have a great potential in mimicking
specific secondary structure with improved bioavailability.10 While conformationally constrained
amino acids/ scaffolds restrict the rotation of amide bond, side chain modified amino acids could
3

restrict conformational flexibility via steric interactions and helps adopting a folded
conformation without drastically changing the stereo-electronic properties.10 Towards this end
several unnatural amino acids (UNAA)/amino acids scaffolds with novel functionalities have
been designed and incorporated within a protein’s framework aiming to expand the genetic code
or to have enzymatically stable peptide-based drugs candidates. 6,8—10,11
Thus, interest in the design and application of novel unnatural amino acids is increasing
universally. Moreover, the synthesis of unnatural amino acids with novel functionality is highly
demanding and is currently an emerging area of research because of the currently developed
technology, “recombinant introduction” of amino acid into proteins.12 Furthermore, for
visualizing intracellular events and understanding molecular interactions inside a cell such as
elucidating protein’s structures, functions and dynamics,13j-l receptor-ligand binding,13c-e
monitoring enzyme activity,13b,f-i and proteomics,13r needs sensing of proteins’ microenvironment
with the help of highly sensitive fluorescence probe.13 Though, many of the aspects have been
studied either by exploiting intrinsic fluorescence of tryptophan14 or extrinsic fluorescent probe15
but these are not free from limitations and thereby not sufficient to fulfill the all research need.
Overcoming the shortcomings has led a strong demand for site-specific incorporation of
microenvironment sensitive fluorescent unnatural amino acids16 into a protein.15-16 Towards this
journey, a very few fluorescent amino acids have been synthesized or encoded genetically and
site-specifically incorporated into proteins.17 Therefore, there is a great demand to develop
fluorescent unnatural amino acids (FUAA) or fluorescently labelled UAA for genetic encoding
or to generate labelled proteins/peptide for studying conformational or diverse functional
realm.18 Furthermore, utilisation of these amino acids to replace aromatic amino acids of a native
4

peptide sequence and study of their effect on the conformation is also an attractive research
target.7-11
FIGURE 1. (a) Schematic presentation of the synthesis of triazolyl UNAAs. (b) The structures
of the donor/acceptor aromatic alkynes used in this study.
5

Results and Discussion
Concept and Objective
As a part of our current research efforts on synthesis of fluorescent small molecules and
biomolecular buildings blocks via azide-alkyne cycloaddition reaction we previously showed
that the triazole rings introduces interesting photophysical properties to the generated
fluorophores or the building blocks. 19 We took the same strategy to increase the list of triazolyl
amino acids with good fluorescence property which we want to report in this article.19
Previously, we have shown that the triazolyl units are capable of installation of fluorescence
property into a non-fluorescent precursor as well as electronically coupling between a triazole
19
and a fluorophore give rise to a modulated emission response to the fluorophore. Therefore,
owing to our previous result, we thought that it would be worthwhile to synthesize triazolyl
amino acids containing donor and/or acceptor aromatics so as to produce solvatochromic
photophysical response into the UNAAs. Thus, we previously reported the synthesis of four
amino acids, namely, TPhenAlaDo (5), TNDMABAlaDo (1), TCNBAlaAc (10), TNBAlaAc (11), and
exploited two of them (5 and 10) for studying β-turn peptidomimetics.20a Triazolyl pyrene amino
acid (7) in a dipeptide sequence was also reported by us to study the photophysics in a peptidic
turn conformation. However, the detail solvatochromic photophysical property of the single
monomeric amino acid was not reported. 20a
Our main aim of the synthesis of more triazolyl amino acids was here two folds i.e. to study
(a) the microenvironment sensitive photophysical properties, (b) application for studying
interaction with BSA protein, and (c) effect on conformation upon incorporation of a pair of
donor/acceptor amino acid into a short peptidomimetic. The advantages of triazole moieties were
6

mainly exploited in this class of amino acids which are: triazoles (a) are metabolically inert, (b)
act as trans-amide mimetics, (c) readily associate with biological targets, through hydrogen
bonding and dipole interactions, (d) can improve/modulate photophysical properties.20 We
envisaged that the introduction of 1,2,3-triazole residues to an amino acid might significantly
improve the interaction property via stacking/H-bonding interaction with the other amino acid
residues in a protein and thereby might improve the activity towards a broad
chemical/biochemical range.5 Thus, we wanted to explore the pseudoaromatic nature of 1,2,3-
triazole to modulate the distribution of electrons and to endow new photophysical properties to
the whole unnatural amino acids. With this background and aim we synthesized few more
unnatural triazolyl amino acids with novel photophysical properties via our previously reported
click chemistry protocol. The synthesized triazolyl amino acids containing donor/acceptor
aromatics showed interesting solvatochromic fluorescence photophysical properties. One of the
synthesised triazolyl amino acid showed good interaction with BSA which was reflected from a
fluorescence switch-on response. Finally, we incorporated a pair of donor/acceptor amino acids
into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II β-
turn conformation.
Synthesis of Triazolyl Unnatural Amino Acids
The synthesis is straightforward and follows a 1, 3-dipolar azide-alkyne cycloaddition route
similar to our earlier report.20 Thus, L-serine was treated with di-t-butyl dicarbonate in 1:1
dioxane: water under basic condition (20 mmol NaOH) to affoard Boc-protected L-serine which
upon treatment with N,O-dimethyl hydroxylamine hydrochloride under peptide coupling
7

condition yielded Weinreb amide ester of N-protected L-serine (16). The N-,C-diprotected L-
serine was then converted to the corresponding mesylate derivative upon treatment with mesyl
chloride which upon reaction with NaN3 afforded azido serine (17) in very good yield (Fig.
1a).20, 21 Finally, a click reaction of the N-,C-diprotected azido serine 17, with the various donor
and/or acceptor alkynes, A-O (Fig. 1b) in 5:1 THF:H2O afforded the novel triazolyl amino acids
1-15 in good to excellent yield (Fig.1a and Fig. 2). All the synthesized amino acids (TAlaDo/Ac
)
were purified by column chromatography and characterised by NMR and HRMS analysis.
OMe
NMe2
OMe
N
N
N
N
N
N
N
N
N
N
N
N
N
OMe
O
O
O
O
N
N
N
N
O
O
O
O
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
O
TNDMBAlaDo
TPyrAlaDo
TDMBAlaDo
TMNapAlaDo
1 (82%)
2 (75%)
3 (80%)
4 (75%)
NMe2
3
O
N
HN
N
N
N
N
N
O
O
N
N
N
N
N
N
N
O
O
O
O
N
N
O
N
O
N
H
O
O
O
N
H
N
H
N
H
O
O
O
O
O
O
O
TPhenAlaDo
T4-DMANIAlaDo
TPyAlaDo
TBAPyAlaDo
5 (84%)
6 (74%)
7 (72%)
8 (82%)
NO2
CN
N
O
CO2Me
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
N
N
N
N
O
O
O
O
N
H
N
H
N
H
N
H
O
O
O
O
O
O
O
TPerAlaDo
TCNBAlaAc
TNBAlaAc
TMBAAlaAc
9 (52%)
10 (81%)
11 (81%)
12 (78%)
CF3
NO2
O
O
H
N
N
N
N
N
N
N
C
N
O
N
O
N
O
N
N
CF3
O
O
O2N
N
N
N
O
O
O
N
H
O
N
H
O
O
O
TDNBAlaAc
TDTFMBAlaAc
T4-CPNIAlaAc
13 (71%)
14 (73%)
15 (58%)
FIGURE 2. The structures of the synthesized triazolyl UNAAs.
8

Study of Photophysical Properties of Triazolyl Donor/Acceptor Unnatural Amino Acids
After getting all the N-,C-diprotected amino acids in hand, we, next, studied the UV-visible
and fluorescence photophysical properties of each fluorescent amino acids. All the UV –visible
spectra of the amino acids (10 µm) were measured in different solvents using a UV-visible
o
spectrophotometer with a cell of 1 cm path length at 25 C. All the sample solutions were
prepared before an hour for the experiment. Emission spectra were obtained using a fluorescence
o
spectrophotometer at 25 C using 1 cm path length cell. The excitation wavelengths for the
monomers were set at ꢀꢂꢁꢄꢂꢅꢃ. The fluorescence quantum yields (Φf) were determined using
quinine sulphate as a reference with the known Φf = 0.54 in 0.1 molar solution in sulphuric
acid.22
From the UV-visible spectra it was clear that the amino acid 1 (TNDMBAlaDo) containing a
N,N-dimethylaminobenzene donor exhibited structureless broad absorption maxima at around
280-290 nm in various organic solvents. The absorption spectra experienced about ~7 nm red
shift upon increasing the polarity of the solvents. Next, excitation at absorption maxima of each
solvent showed fluorescence emission at around 360 nm with a red shift of 26 nm as the solvent
polarity was increased. More strikingly, the fluorescence intensity became negligible in polar
protic solvents like DMF and MeOH. The quantum yield in various solvents followed the same
trend as the steady state fluorescence (Fig. 3a-b, Table 1). The fluorescence quenching in
polar/protic solvents could be attributed to the radiationless decay assisted by polar solvent–
solute interaction or charge transfer or H-bonding interactions.23
Pyridyl triazolyl alanine amino acid 2 (TPyrAlaDo) showed two absorption bands at around
240 and a long wavelength of absorbance at 285 nm in various organic solvents. Interestingly,
9

the absorbance at 285 nm increases with a slight hypsocromic shift as the solvent poratity
increased indicating a solvatochromic nature of the amino acid and the band was characterised as
charge transfer band. Upon excitation at absorption maxima of each solvent the amino acid
showed negligible emission in nonpolar solvents but a strong emission at around 310-330 nm in
polar and protic solvents. The intensity of emission gradually increased with slight
hypsochromicity as the solvent polarity increases (See SI, Fig. S3, Table S1). The emission was
thus unambigueously characterised as ICT emission. It was expected that in protic solvent like
MeOH the pyridyl-N could involve in H-bonding interaction which ultimately would open the
channel of charge transfer from donor triazole moiety to the protonated pyridine unit leading to
an enhanced fluorescence intensity. 23h-i
FIGURE 3. UV-visible absorption and fluorescence emission spectra of amino acids in various
organic solvents; (a-b) for 1 (TNDMBAlaDo) and (c-d) for 3 (TDMBAlaDo). Excitation wavelength
ꢂꢄꢅ
ꢁꢂꢃ
was the l
in each solvent and the concentration of amino acids was 10 µM.
10

The UV-visible spectra of amino acid
3
(
TDMBAlaDo) containing a triazolyl
dimethoxybenzene aromatic unit displayed three absorption bands at around 260, 290 and 300
nm. The solvent polarity did not affect much on absorption property. Upon excitation at long
wavelength absorption maxima (290-300 nm) showed strong emission at around 325 nm in low
polar solvent and a red shifted (5-7 nm) emission with enhanced intensity in polar solvents like
acetonitrile or methanol (Fig. 3c-d, Table 1). On the other hand, the triazolyl 6-methoxynaphthyl
alanine amino acid 4 (TMNapAlaDo) exhibited solvatochromic and structured absorption bands at
290 and 305 nm. In low polar solvent like cyclohexane, dioxane, it exhibited broad structured
emission band at around 350-380 nm; however, incresing solvent polarity the intensity decreases
with a red shift of 15 nm indicating its ICT character when excited at 305 nm. The decrease in
fluorescence intensity might be attributed to the radiadtionless decay caused due to solvent solute
interaction, and H-bonding in protic polar solvents (Fig. 4a-b, Table 1).23 The quantum yield and
time resolved fluorescence decay in various solvents followed the same trend as the steady state
fluorescence for all the amino acids.
11

FIGURE 4. UV-visible absorption and fluorescence emission spectra of amino acids in various
organic solvents; (a-b) for 4 (TMNapAlaDo) and (c-d) for 7 (TPyAlaDo). Excitation wavelength was
ꢂꢄꢅ
ꢁꢂꢃ
the l
in each solvent and the concentration of amino acids was 10 µM.
The amino acid TPhenAlaDo (5) exhibited broad and solvatochromic absorption band at around
299-305. The charge transfer absorption band exhibited 10 nm blue shift as the solvent polarity
increases. Upon excitation at 300 nm, TPhenAlaDo showed structured emission at around 400 nm.
However, increasing solvent polarity the quantum yields increases (See SI, Fig. S4, Table S2).
On the other hand, donor-acceptor triazolyl 4-(4-N,N-dimethylaminophenylethynyl)-N-(2-
propynyl)-1,8 naphthalimide amino acid T4-DMAPNIAlaDo (6) revealed structureless absorption
bands at 300 nm and 443 nm. Changing the solvent polarity from dioxane to ethanol the
absorption band at 443 nm experienced a red shift of 9 nm with an increase in intensity (See SI,
Fig. S5, Table S2). The long wavelength and fairly broad solvatochromic band suggested that the
ground state of the molecule was significantly polar and thus was unambiguously assigned as
12

intramolecular charge transfer (ICT) band. When excited at absorption maxima, it showed a
structureless broad emission band. As the solvent polarity increases from dioxane to chloroform,
a strong red shift of about 31 nm (lmax, Dioxane 580 nm, and lmax, chloroform 611 nm) was observed
which showed a decrease in both the fluorescence intensity and the quantam yield (See SI, Fig.
S5, Table S2). Therefore, the amino acid 6 is highly solvatochromic. The conjugation between
the N,N-dimethyl aminophenyl acetylenic moiety as an electron donor and the naphthalimide
core as an electron acceptor played an important role in the large dipole change during excitation
and made it highly solvatochromic. However, in aprotic and protic polar solvents negligible
emission was observed which might be again due to solvent-solute dipolar interaction and H-
bonding mediated radiationless decay.23
TABLE 1. Summary of photophysical properties of amino acids 1 (TNDMBAlaDo), 3 (TDMBAlaDo)
and 4 (TMNapAlaDo).
Entry à
Solvents
à
1 (TNDMBAlaDo)
Toluene Diox EtOAc THF
Cyhex Hex
DMF EtOH ACN
MeOH
0.000
285
0.001
284
0.013
288
0.021 0.201
0.210 0.275 0.290 0.307
0.309
288
Df
ꢀꢂꢁꢄꢂꢅꢃ(nm)
286
22.8
358
283
20.1
361
285
28.3
357
292
23.4
369
-----
-----
-----
286
e
max X 103
22.7
345
17.5
346
22.8
359
25.82
368
22.8
-----
ꢆꢊꢇꢋꢈꢉ(nm)
0.24
0.26
0.15
0.15
0.08
0.13
0.05
-----
0.07
-----
Ff
3 (TDMBAlaDo)
ꢀꢂꢁꢄꢂꢅꢃ(nm)
258,
301
21.9
318
257,
300
18.1
318
CHCl3
257, 299
27.9
257,
256,
301
22.6
319
257,
300
29.6
317
-----
255,
300
26.2
323
257,
299
37.5
320
254,
300
25.3
323
301
24.7
318
e
max X103
-----
-----
ꢀꢌꢁꢍꢂꢃ(nm)
323
0.034
0.035
0.005
0.037 0.032
0.42
-----
0.055 0.042
0.047
Ff
4 (TMNapAlaDo)
ꢀꢂꢁꢄꢂꢅꢃ(nm)
288,
300
287,
300
291, 301 289,
302
288,
300
289,
300
290,
301
290,
300
288,
300
289,
300
13

max X 103
11.9
356,
365
9.0
355,
363
16.1
351, 389 358,
368
17.4
16.2
358,
367
16.8
359,
368
17.2
351,
393
15.8
350,
388
16.6
351,
391
14.7
350,
388
e
ꢀꢌꢁꢍꢂꢃ(nm)
0.287
0.232
0.122
0.269 0.156
0.246 0.117 0.121 0.119
0.129
Ff
---- -----
-----
8.81 -----
----- ----- ----- 7.11
7.23
<t>[ns]
-
While triazolyl-pyrenyl-alanine 7 (TPyAlaDo) showed unstructured broad absorption band at
around 348 nm (Fig. 4c-d, Table 2), amino acid 8 (TBAPyAlaDo), pyrenebutaramido triazolyl
alanine, displayed characteristic structured absorption bands of pyrene at 313, 327 and 343 nm,
in various organic solvents (See SI, Fig. S6, Table S2). As the solvent polarity increases the
absorption experienced a slight blue shift (3 nm) in case of amino acid 8. The disappearance of
pyrene structural band in 7 could easily be explained if we consider a strong electronic coupling
between the p-cloud of pseudoaromatic triazole and directly linked pyrene (Fig. 4c-d). Due to
this reason amino acid 7 showed a hypsochromic shift of 6-8 nm when solvent polarity
increased. However, when excited on their absorption maxima while amino acids 8 showed
structured emissions at 379, 399 and 418 nm, amino acid 7 showed emissions at 389, 407 and
425 nm with a red shift (8-10 nm) of all vibronic bands. The intensity of amino acid 8 did not
alter much with increasing solvent polarity. The quantum yield and time resolved fluorescence
decay in various solvents followed the same trend as the steady state fluorescence for both the
cases. On the other hand, amino acid 7 experienced a gradual decrease in emission intensity as
the solvent polarity increases indicating a solvent-solute or hydrogen bonding interaction
mediated fluorescence quenching.23
14

FIGURE 5. UV-visible absorption and fluorescence emission spectra of amino acids in various
organic solvents; (a-b) for 9 (TPerAlaDo) and (c-d) for 10 (TCNBAlaAc). Excitation wavelength was
the lꢂꢁꢄꢂꢅꢃ in each solvent and the concentration of amino acids was 10 µM.
The triazolylperylene amino acid 9 (TPerAlaDo) exhibited tuned absorption and fluorescence
property compared to perylene indicating a donor-acceptor character. The electron charge
density of triazole moiety probably redistributed with the electron deficient aromatic ring of
perylene. Because of this charge transfer it showed structured absorption at longer wavelength
(399, 422 and 449 nm) than the characteristic absorption of perylene (387, 409, 435 nm in
cyclohexane). As solvent polarity increases from chloroform to methanol, amino acid 9 showed
hypsochromic shift of 9 nm. Upon excitation at 425 nm, it exhibited less-structured emission at
longer wavelength (470 and 502 nm) than perylene (437, 462 and 497 nm). Moreover, the
emission of 9 experienced a hypsochromic shift of 9-10 nm as the polarity of the solvent
15

increases (Fig. 5a-b, Table 2). The quantum yield and time resolved fluorescence decay in
various solvents followed the same trend as the steady state fluorescence.
The amino acid 10 (TCNBAlaAc) containing a triazolyl cyanobenzene aromatic unit as an
acceptor possessed strong structure-less absorption at 287 nm in low polar solvent toluene. The
absorbance was characterized by a hypsochromic shift of about 10-15 nm as the polarity of the
solvent increases. Excitation at 280 nm it showed emission at around 315 nm in toluene which
became broad as the solvent polarity increases. An appearance of an extra band at 335 nm was
observed in dioxane which shifted to 358 nm in MeOH. The high intense solvatochromic band
was most probably the ICT emission indicating acharge transfer from donor triazolyl moiety to
acceptor cyanobenzene moiety (Fig. 5c-d, Table 2). On the other hand triazolyl nitrobenzene
alanine amino acid TNBAlaAc (11) exhibited a strong solvatochromic (hypsochromic) absorbance
at 315 nm. However, excitation upon absorbance maxima, it emited very weak emission band at
355 nm which showed a red shift (of 15 nm) and decreasing intensity of emission as the solvent
polarity increases which might be because of polar solvent-solute interaction leading to
quenching of fluorescence via dipolar/ H-bonding interaction (see SI, Fig. S7, Table S3).23
The
quantum yield and time resolved fluorescence decay in various solvents followed the same trend
as the steady state fluorescence for both the amino acids.
TABLE 2. Summary of photophysical properties of amino acids 7 (TPyAlaDo), 9 (TPerAlaDo) and
10 (TCNBAlaAc).
THF
DMF
EtOH ACN
MeOH
Entry à
7 (TPyAlaDo)
0.000
Cyhex
279, 347 280, 349
0.021
Diox
0.148
280,
349
0.201
0.210
281,
349
0.265
282,
351
0.290
279,
347
0.307
278,
346
0.309
277,
344
Solvents à
Df
CHCl3 EtOA
ꢂꢄꢅ
c
279,
ꢀ
ꢁꢂꢃ
(nm)
348
16

22.1
25.0
25.0
386,
405
24.3
385,
406
24.1
386,
406
23.7
387,
406
23.5
385,
405
23.9
385,
405
24.8
385,
404
e
max X 103
ꢌꢍ
385, 405 387, 406
ꢀ
(nm)
ꢁꢂꢃ
0.126
----
0.146
34.1
0.292
----
0.123
----
0.183
0.457
----
0.147
----
0.130
15.16
0.120
17.42
Ff
<t> [ns]
----
9 (TPerAlaDo)
ꢂꢄꢅ
----
425, 451
427,
452
22.9
471,
500
0.81
----
424,
450
24.4
468,
496
0.78
----
427,
429,
455
24.5
474,
502
0.82
----
422,
448
26.0
466,
493
0.77
----
423,
448
26.0
467,
494
0.78
4.1
420,
446
25.5
465,
492
0.81
4.2
ꢀ
(nm)
ꢁꢂꢃ
452
24.1
470,
499
0.79
----
----
----
24.4
470, 499
e
max X 103
ꢌꢍ
ꢀ
(nm)
ꢁꢂꢃ
----
----
0.82
----
Ff
<t> [ns]
10 (TCNBAlaAc)
272
29.7
311,
336
0.28
----
ꢂꢄꢅ
272
26.3
310
272
35.1
310, 336
273
33.6
314
273
276
32.5
320
271
271
270
ꢀ
e
(nm)
ꢁꢂꢃ
max X 103
29.8
306,
338
0.34
----
31.5
308,
360
0.33
----
30.4
307,
352
0.36
1.9
30.5
310,
358
0.37
3.2
ꢌꢍ
ꢀ
(nm)
ꢁꢂꢃ
0.09
----
0.30
1.4
0.32
----
0.04
----
Ff
<t> [ns]
The 3,5-trifluoromethylphenyl triazolyl alanine amino acid TDTFMBAlaAc (14) showed strong
broad absorbance at 260 nm which experienced a negligible solvatocromicity. Upon excitation at
its absorbance maxima it emitted at 315 nm in cyclohexane which shifted to 329 nm in
acetonitrile/methanol. The emission was characterised by a bathochromic shift of 14 nm and a
hyperchromic effect as the solvent polarity increases (See SI, Fig. S8, Table S3). This might be
because of internal charge transfer process from donor triazolyl unit to acceptor 3,5-
trifluoromethyl benzene in polar solvent. On the other hand, amino acid 15, (T4-CPNIAlaAc) which
is an analogue of amino acid 6 but with an acceptor cyanophenyl acetyleneic moiety exhibited
strong absorption at 351, 369 and 389 nm. The absorption spectra of 15 sowed a little
hypsochromic shift as the polarity of solvent increases. It exhibited structured emission at 404
and 424 nm in low polar solvent like dioxane, ethyl acetate, THF and structureless emission at
435 nm in polar solvents such as acetonitrile, methanol, and ethanol (lex = 370 nm). The
17

emission experienced bathochromic shift of about 10 nm with increased intensity and quantum
yield (See SI, Fig. S9, Table S3). While amino acid 6 experienced a large dipole change due to
its donor-acceptor character, the amino 15 having acceptor-acceptor character did not show such
dipole change in increasing the solvent dielectric constant. Therefore, these two amino acids
behave oppositely-which was also reflected in the emission intensity as well as in quantum yield.
Therefore, all the triazolyl aromatic amino acids showed a significantly more solvatochromic
absorption and emission properties compared to their parent donor/acceptor aromatics. This is
attributed to the electronic coupling of triazolyl moiety with p-cloud of aromatic chromophoric
unit. The quantum yield and time resolved fluorescence decay in various solvents followed the
same trend as the steady state fluorescence for all the amino acids. Next, we were interested to
know whether a mixture of donor-acceptor triazolyl amino acids involved in photophysical
interaction. Thus, we titrated a solution of a representative acceptor triazolyl amino acid
TCNBAlaAc (10) with increasing concentration of a donor triazolyl amino acid, TPhenAlaDo (5) and
measured absorption and emission properties in acetonitrile solvent. A solution of 1: 1 mixture of
donor-acceptor amino acids and various other concentration ratios showed characteristic
absorptions of both the donor and acceptor amino acids indicating that there was no possibility of
ground state charge transfer complexation (Fig. 6a). Next a fluorescence titration experiment was
carried out. Thus, upon excitation at absorption maxima of TCNBAlaAc (lex = 270 nm) the
emission at 330 nm gradually decreased as the concentration of donor amino acid TPhenAlaDo
increases (Fig. 6b). This observation clearly indicated a fluorescence quenching of TCNBAlaAc in
presence TPhenAlaDo via a dynamic quenching process as the static mode of quenching was ruled
out from the UV-visible spectroscopic study. The above result of fluorescence knocked us to
relook and we observed that emission spectra of TCNBAlaAc overlapped significantly with the
18

absorption spectra of TPhenAlaDo indicating a possibility of quenching of emission of TCNBAlaAc
via a dynamic quenching mode, the FRET.24 Therefore, in the mixture, the acceptor triazolyl
amino acid TCNBAlaAc acts as FRET donor and the donor amino acid TPhenAlaDo acts as a FRET
acceptor. Therefore as the concentration of FRET acceptor (TPhenAlaDo) increases the
fluorescence of the FRET donor (TCNBAlaAc) decreases (Fig. 6c). Stern-Volmer quenching
constant was found to be 6 x 10-2 (Fig. 6d). Therefore, a fovourable photophysical interaction via
FRET was observed between these amino acids pair which might be of great importance in
designing FRET-peptide probe usable for studying peptide-protein/protein-protein interaction.25
FIGURE 6. UV-visible (a), fluorescence (b) titration spectra of acceptor amino acid 10
(
TCNBAlaAc) with donor amino acid 5 (TPhenAlaDo). (c) Overlapped of absorption and
fluorescence spectra of 5 and 10, respectively. (d) Stern-Volmer plot.
19

Next, the solvatochromic behaviors, particularly ICT originated solvofluorochromicity, of
amino acids 1, 4, 9 and 10 was studied via the correlations of their absorption (ꢎꢏꢁꢂꢄꢂꢅꢃ) and
fluorescence maxima (ꢎꢏꢁꢌꢍꢂꢃ) with the solvent polarity function Δf. A linear correlation between
the νabs
these fluorescent amino acids are moderately polar in nature. However, much more linear
correlation was observed between νfl and Δf for all these aforementioned amino acids suggesting
̃
and Δf was reflected from the plots in figure 7a-d that indicated that the ground states of
̃
that the nature of the fluorescent states remained essentially unchanged in all the solvents though
solvent induced modulation was reflected in the spectral features.28a That the fluorescent states of
these amino acids are highly polar in nature and of ICT character was also reflected in the
reasonably high slopes of the νfl
(Δν ) in different solvents against solvent polarity parameter Δf also showed a good linear
̃
vs Δf plots (Fig. 7a-b, d-e). 26 Moreover, a plot of Stokes’ shift
̃
correlation with large slopes indicating the highly polar nature of fluorescence states of all the
amino acids said above (Fig.7c, f).
20

FIGURE 7. Plots of νf̃ l/νã bs and ∆ν̃ (Stock shift) against ∆f for amino acids 1 (a), 4 (b), 1-4 (c), 9
(c), 10 (d) and 9-10 (f) in different solvents.
Theoretical Calculation for Triazolyl Donor/Acceptor Unnatural Amino Acids
Next, to examine the effect of solvent polarity on the absorption and emission property of
donor/acceptor aromatic triazolyl amino acids, we studied the TDDFT calculation using
Gaussian 09 program package.27 From the HOMO-LUMO overlap and transition oscillator
strength (f) of the donor/acceptor aromatic triazolyl amino acids it was clear that the electronic
transition from So to S1 or other possible electronic transitions are feasible. So the reverse
transition i.e., S0←S1, was also fully allowed that revealed the fluorophoric nature of triazolyl
unnatural amino acids. Solvatochromicity and intramolecular charge transfer emissions were also
explained by redistribution of electronic charge density between HOMO-LUMO (Fig 8-9).26a, 28
21

As for example, from the TDDFT calculations the dominant orbital transitions in the low-
lying singlet excited states of the studied amino acids is the S0→S1 transition having highest
configuration interaction (CI) values. Thus, the calculated excitation energy for the transition
from S0→S1 of donor triazolyl amino acids TNDMBAlaDo (1), TMNapAlaDo (4), TPhenAlaDo (5),
TPyAlaDo (7) and TPerAlaDo (9) were found to be 328 (3.8 ev, f = 0.05, CI = 0.7); 324 nm (3.8 ev,
f = 0.15, CI = 0.67); 308 nm (4.03 ev, f = 0.19; CI = 0.63); 356 nm (3.48 ev, f = 0.55; CI = 0.69)
and 449 nm (2.76 ev, f = 0.56; CI = 0.71) (in vacuum), respectively. These values were in close
agreement with the experimental result of 285 nm (cyclohexane), 300 nm (in hexane), 310 nm
(in hexane), 348 nm (in hexane) and 449 nm (in chloroform) respectively. For amino acid 1, the
S0→S1 transition in lowest excited state appeared at much longer wavelength of 328 nm (f =
0.05) which was far from the experimental value. However, both the transitions at 273 nm (4.54
→LUMO+2); CI = 0.68];
ev; f = 0.76) in vacuum in the second excited state [S0→S3 (HOMO
→LUMO+1; CI = 0.16] correlated well with the observed abs
and [S0→S2 (HOMO
orbance at
285 nm (in cyclohexane). 26a, 28
FIGURE 8. Diagram of HOMO-LUMO of amino acids 1, 4 and 5 calculated at B3LYP//6-31G*
level of theory using Gaussian 09 program package.
22

The emission spectra for all the donor triazolyl amino acids also showed solvatochromic
effect and bathochromic shift due to electronic charge redistribution between triazole moiety and
fluorophoric unit which was also reflected from their individual HOMO-LUMO diagrams (see
SI, Section 7). The emissive states of all the donor triazolyl amino acids were found to be locally
excited (LE) states except for the case of amino acid 1 (TNDMBAlaDo) as was suggested by the
HOMO−LUMO distributions. Thus, in case of amino acid 1 the emissive state was characterized
with more significant electron redistribution, i.e., ICT feature. Calculated results of
HOMO−LUMO distribution supported our conclusion. 26a,28
FIGURE 9. Diagram of HOMO-LUMO of amino acids 7, 9 and 10 calculated at B3LYP//6-
31G* level of theory using Gaussian 09 program package.
On the other hand, the dominant orbital transitions in the low-lying singlet excited states of
TCNBAlaAc
→LUMO) for both
acceptor triazolyl amino acids were found to be S0→S1 (HOMO
(10) and TNBAlaAc (11) with transition wavelength of 268 nm (4.62 ev, f = 0.77; CI = 0.69) and
334 nm (3.71 ev, f = 0.47; CI = 0.70), respectively, in vacuum (Fig. 9 and see SI, Fig. S18, Table
23

S6). These values were in close agreement with the experimental results of 271 and 300 nm in
hexane for acceptor triazolyl amino acids 10 and 11, respectively. Thus, as revealed from the
overlapping of HOMO-LUMO as well as the transition oscillator strength (f), the electronic
→S1 are fully allowed for all the acceptor amino acids (see SI, Table S6). This
transitions of S0
means that reverse transition, i.e., S0←S1, was also fully allowed indicating their potential
fluorophoric nature. The emissive state of TCNBAlaAc (10) showed dual emission at 320 and 360
nm which were well characterized with more significant electron redistribution, i.e., ICT feature
which were again supported by the HOMO−LUMO distributions. However, the emissive state of
acceptor amino acid TNBAlaAc (11) was basically a locally excited (LE) state as was suggested by
the HOMO−LUMO distributions. 26a,28
Study of Interaction of Triazolyl Donor Unnatural Amino Acids 9 (TPerAlaDo) with BSA
As an application, we, next explored the novel triazolyl perylene amino acid 9 (TPerAlaDo) as
a possible probe for sensing BSA protein.29 We envisaged that the triazolylperylene would sense
the interaction with BSA via the generation of an enhanced fluorescence signal.30 The UV-
visible absorption of amino acid 9 in phosphate buffer showed a structureless absorption at
around 375 nm and weak bands at 440 nm. Addition of an increasing concentration of BSA to
the probe solution resulted in a structured absorption at 425 and 450 nm characteristic of
perylene which experienced strong hyperchromicity in absorbance. The BSA absorption at 280
nm also shifted to 295 nm. These observations indicated a strong binding interaction between
BSA and amino acid 9 in the hydrophobic region (Fig. 10a).
To get more insight into the binding event we next carried out fluorescence titration
experiment. In phosphate buffer the amino acid 9 showed extremely weak emission at 470 and
24

495 nm. Upon gradual addition of an increasing amount of BSA, the emission intensity of the
probe 9 gradually increased when excited at the absorption maximum of TPer (420 nm)
indicating a strong interaction between the probe and hydrophobic pocket of BSA (Fig. 10b).
The drastic enhancement of fluorescence of TPer could be explained if we consider that the
TPer moiety got accommodated in the hydrophobic pocket of BSA. This was also supported
from a solvatochromic emission of TPer in various organic solvents. That the TPer moiety of
probe 9 bound strongly inside the hydrophobic pocket of BSA and experienced a highly
restricted rotational motion was supported by a large enhancement of fluorescence anisotropy
from 0.02 to 0.3 (See SI, Section 8.4).31 The enhancement of fluorescence intensity and the
quantum yield of probe 9 upon binding with BSA were also evident from a time resolve
fluorescence study. Thus, we observed a gradual increase in TPer life time (lex = 405 nm) from
5.3 to 5.5 ns while monitoring the decay at TPer-emission (lem = 495 nm) (See SI, Section 8.3).
25

FIGURE 10. (a) UV-visible and (b) fluorescence titration of of amino acid probe 9 (TPerAlaDo)in
absence or in presence of BSA (concentration of 9 was 10 µM; lex = 420 nm). (c) The Benesi-
Hildebrand plot. (d) Docking pose of 9 in hydrophobic pocket of BSA.
The association constant of probe with BSA determined by Benesi-Hildebrand plot (Fig. 10c)
was found to be 1.6 x 104 M-1 with an experimental free energy of binding, DG = -5.8 kcal/mol.
All the results suggested that both the hydrophobic as well as electrostatic interaction played an
important role in the present BSA-peptide interaction process. The binding of the TPer-moety in
the hydrophobic pocket of BSA was also supported by a molecular docking calculation with
32
Autodoc programme which clearly showed that TPer moiety of the probe 9 was remained
26

surrounded by hydrophobic amino acids of the hydrophobic pocket of subdomain IB of site I of
BSA (Fig. 10d and see SI, Section 8.6).
Incorporation of Triazolyl Donor/Acceptor Unnatural Amino Acids into a Leu-Enkephalin
Analogue Peptide: Synthesis, Conformational Analysis, and Photophysical Study
As an extension of the work presented herein and for showcasing the synthesised amino acids
as peptide building block, we, next incorporated a pair of triazolyl donor-acceptor unnatural
33a
amino acids into a short pentapeptide sequence analogus to leu-enkephalin
peptide. Opioid
peptide, such as Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) is responsible for a variety of processes
in organisms and they showed a very broad spectrum of biological activities. Therefore such
endogenous opioid peptides are often important targets with a good potential for further
development as therapeutic agents. However, poor metabolic stability in native form forces to
design a numbers of analogs to overcome pharmaceutical and pharmacokinetic barriers in
clinical application.33b As a part of our continuous research effort in β-turn peptidomimetics we
thought that if we could replace the aromatic amino acids by our designed triazolyl dono-
acceptor aromatic amino acids (TAlaDo/Ac) it would be beneficial.16a-b Therefore we took it as a
target and herein we want to report the synthesis and conformational study.
Thus, in the pentapeptide sequence of Leu-enkephalin native peptide, the two aromatic amino
acids Tyr and Phe were replaced by a donor amino acid, TDMBAlaDo and an acceptor amino acid,
TDNBAlaAc respectively. These two amino acids were chosen considering their maximum
,
donor/acceptor character in the series reported herein. We also wanted to know whether such
replacement could lead to retaining of β-turn conformation of the native leu-enkephalin
pentapeptide. The synthesis was accomplished via a solution phase peptide coupling protocol
27

following our earlier published literature as is depicted in the below scheme 1.16a-b All the
intermediates and final target pentapeptide 18 [BocN-TDMBAlaDo-Gly-Gly-TDNBAlaAc-Leu-
CO2Me] were purified by silica gel column chromatography and characterized by 1D and 2D
NMR and HR-mass spectrometry.
Scheme 1. Synthesis of triazolyl donor/acceptor unnatural amino acids decorated Leu-
Enkephalin analogue pentapeptide.
Next, the secondary structure of the Leu-enkephalin analogue pentapeptide 18 was estimated
by recording its CD spectrum in methanol. Thus, in methanol, the peptide possessed strong
structured positive bands at 195, 200 nm and a very weak negative band at 215 nm. In
acetonitrile it showed closely similar behaviour with a slight shift in band position and decrease
in intensity with appearance of positive bands at 198, 203 nm and a weak negative band at 220
nm (Fig. 11a). Therefore, in both the solvents the Leu-enkephalin analogue peptide 18 showed
predominant characteristics of a type II β-turn conformation indicating more rigid than native
Leu-enkephalin pentapeptide. The weak positive induced CD signal was also appeared which
corresponds to the absorption of triazolyl aromatic chromophores. 16a-b, 34
The β-turn structure in the peptide 18 indicated the presence of intramolecular back bone H-
28

bonds between COi and NHi+3 residues. To probe the intramolecular H-bonding, variable
temperature NMR (VT NMR) spectroscopy was used. Thus, from the VT-NMR experiment,
strong intramolecular H-bonding involving the amide NH (of TDMBAlaDo) at i+3 (Dδ/DT = -3.8
ppb/k) was observed in peptide 18 supporting the type II β-turn structure.35 The amide NHs of
both i+1 and i+2 Gly were found to be moderate while the same of TDNBAlaAc at ith position was
weak (Fig. 11b and see SI, Section 9.1).
Figure 11. (a) CD spectra of peptides 18 in methanol/acetonile. (b) Some important interactions
observed in pentapeptide 18. (c) Clustering of conformers within 21 kJ/mole from the global
minima of pentapeptide 18 obtained from molecular dynamics simulation.
The presence of ROESY cross peak between Ha(ith, of TDMBAlaDo)/NH (i+1, of Gly1); Ha(ith,
of of Gly2)/NH (i+1, of TDNBAlaAc); Ha(ith, of TDNBAlaAc)/NH (i+1, of Leu); NH(ith, of
TDMBAlaDo)/NH (i+2, of Gly2) also revealed a type-II β-turn conformation (Fig. 11b and see SI,
1
Section 9.2).36 The solution conformation of β-turn peptide 18 was determined from H NMR
spectra in DMSO-d6. From the coupling constants in the 1H NMR it was revealed that the Leu-
side chain was very rigid and adopted predominantly one single conformation about χ1 and χ2
with an anti-relationship between βH(pro-S) and γH. Strong ROESY interaction between
29