Uracil-amino acid as a scaffold for β-sheet peptidomimetics: Study of photophysics and interaction with BSA protein

Article abstract of DOI:10.1016/j.bmcl.2017.11.017

We report herein the uracil-di-aza-amino acid (U^rAA) as a new family of molecular scaffold to induce β-hairpin structure with H-bonded β-sheet conformation in a short peptide. This has been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine and/or triazolylmethoxynapthyl alanine (^TPyAla_Do and/or ^TMNapAla_Do) are embedded into two arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR, variable temperature and 2D NMR spectroscopy reveals the β-hairpin structures for both the peptides. Study of photophysical property reveals that the pentapeptide containing fluorescent triazolyl unnatural amino acids ^TMNapAla_Do and ^TPyAla_Do at the two termini exhibits dual path entry to exciplex emission-either via FRET from ^TMNapAla_Do to ^TPyAla_Do or via direct excitation of a FRET acceptor, ^TPyAla_Do. The other pentapeptide with ^TPyAla_Do/^TPyAla_Do pair shows excimer emission. Furthermore, both the peptides maintaining their fundamental photophysics are found to interact with BSA as only a test biomolecule.

Full text of DOI:10.1016/j.bmcl.2017.11.017

Accepted Manuscript
Uracil-Amino Acid as a Scaffold for β-Sheet Peptidomimetics: Study of Pho-
tophysics and interaction with BSA Protein
Subhendu Sekhar Bag, Afsana Yashmeen
PII:
S0960-894X(17)31104-6
https://doi.org/10.1016/j.bmcl.2017.11.017
BMCL 25423
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
30 May 2017
5 November 2017
8 November 2017
Please cite this article as: Bag, S.S., Yashmeen, A., Uracil-Amino Acid as a Scaffold for β-Sheet Peptidomimetics:
Study of Photophysics and interaction with BSA Protein, Bioorganic & Medicinal Chemistry Letters (2017), doi:
https://doi.org/10.1016/j.bmcl.2017.11.017
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Graphical Abstract
Uracil-Amino Acid as a Scaffold for β-Sheet
Peptidomimetics: Study of Photophysics and
interaction with BSA Protein
Leave this area blank for abstract info.
SubhenduSekhar Bag* and Afsana Yashmeen
1

Bioorganic and Medicinal Chemistry Letter
journal h omepage: www.els evier. com
Uracil-Amino Acid as a Scaffold for β-Sheet Peptidomimetics: Study of
Photophysics and interaction with BSA Protein
Subhendu Sekhar Bag* and Afsana Yashmeen
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati, North Guwhati-781039, Assam, India. Fax: +91-361-
258-2349; Tel: +91-361-258-2324.E-mail: ssbag75@iitg.ernet.in
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report herein the uracil-di-aza-amino acid (UrAA) as a new family of molecular scaffold to
induce β-hairpin structure with H-bonded β-sheet conformation in a short peptide. This has
been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine
and/or triazolylmethoxynapthyl alanine (TPyAlaDo and/or TMNapAlaDo) are embedded into two
arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR,
variable temperature and 2D NMR spectroscopy reveals the β-hairpin structures for both the
peptides. Study of photophysical property reveals that the pentapeptide containing fluorescent
triazolyl unnatural amino acids TMNapAlaDo and TPyAlaDo at the two termini exhibits dual path
entry to exciplex emission-either via FRET from TMNapAlaDo to TPyAlaDo or via direct excitation
Available online
Keywords:
Uracil-amino acid scaffold,
β-sheet peptidomimetic,
dual path to exciplex,
excimer peptide,
of a FRET acceptor, TPyAlaDo. The other pentapeptide with TPyAlaDo TPyAlaDo pair shows
/
excimer emission. Furthermore, both the peptides maintaining their fundamental photophysics
are found to interact with BSA as only a test biomolecule.
2017 Elsevier Ltd. All rights reserved.
BSA-peptide interaction
A great deal of research efforts have been put forth to synthesise
constrained small molecule scaffold that mimic a protein’s particular
secondary structure, thereby, restricting peptides into a specific
conformation such as β-sheet.1-2 Among the various bioactive
conformations, the β-sheet is the second most common structural
motif of protein that not only plays a scaffolding role, but also serves
as a key recognition motif in many important interbiomolecular
interactions processes.1 Therefore, the field of β-sheet
peptidomimetic encompases from several successful design of
constrained molecular scaffolds that nucleate β-sheet conformation1
to the mimicry of β-sheet quaternary interactions2 with simple model
systems. However, β-strand mimicking and β -sheet like structures in
a designed fluorescent peptide aiming at the fundamental aspects that
a particular conformation can have an impact on the photophysics
has received little attention.3 Investigating such an impact might find
widespread applications in studying interbiomolecular interactions in
cell and the design of new peptide based drug candicates.3
Considerable efforts, though separately have been invested in the
design of scaffolded β-sheet peptidomimetics in one hand1 and
fluorescent peptide probes for studying peptide-protein interaction on
the other hand,3 less efforts have been undertaken simultaneously to
consider the both. Therefore, the generation of scaffolded
peptidomimetics with interesting fluorescence property of
fundamental importance is highly demanding. Such probes could be
of immense importance which can be applied dually-for mimicking a
protein’s particular secondary structure, such as β-sheet,1 of
pharmacological importance and studying protein-peptide
interaction3 at least at the fundamental level in a model system.
of β-hairpins in a
Orn-containing analogue peptide.4c-d In
continuation of our current research efforts on the design of
DNA/protein’s building blocks5a-c and constrained molecular scaffold
for peptidomimetics,5d we wish to report on the design of uracil-di-
aza-amino acid (1, UrAA) as a new family of novel molecular
scaffold for β-hairpin mimic (Fig. 1). In this report, efforts have been
taken to design fluorescent peptides whose imposed conformation
could have an impact on the fluorescence photophysics and thereby
protein-peptide interaction. The logics behind our choice of uracil
nucleobase to be the scaffold, for the first time, are (a) its easy
associability with biological targets, (b) expectedly high propensity
to nucleate β-sheet conformation owing to its conformational
restriction at i angle, (c) better understandable pharmacological
implications of uracil than those of other heterocycles.6 Moreover,
the nucleobase-amino acid hybrid peptide would be valuable for
studying peptide-protein/peptide-DNA interaction.6d Therefore, the
simple uracil scaffold would be advantageous over the reported other
scaffolds for the nucleation of β-sheet structure in a short peptide
sequence because it is very simple, easy to synthesise, low cost, less
time consuming which is an issue for the success of the design of a
β-sheet nucleator. There are also no precedented examples of
utilizing nucleobase-peptide hybrid construct with the nucleobase
acting as a β-sheet inducer. This type of hybrid construct might
interact with the biomolecules efficiently through the uracil base via
H-bonding interaction.
The concept of our design of fluorescent pentapeptides is based
on the fact that upon embedding two fluorescent triazolo amino acids
(
TPyAlaDo and/or TMNapAlaDo) into two arms of the uracil-amino acid
Smith, Gellman and others have shown that a good turn mimetic
moiety is critical to induce a β-hairpin structure in a peptide.4a-b
Nowick have shown that ornithine turns are very efficient stabilizer
via an intervening leucine might adopt β-sheet conformation. We
also expected that the particular conformation induced by
constrained uracil scaffold would allow the two terminal fluorescent
2

triazolyl amino acids to come closer and to involve in photophysical
interaction, most likely via Förster resonance energy transfer (FRET)
process as well as exciplex emission in hetero-fluorophoric pair
(ExcipFRET-peptide 2, TMNapAlaDo/TPyAlaDo) and/or excimer
formation in homo-fluorophoric pair (Excim-peptide 3,
These observations indicated that the uracil amino acid scaffold
(UrAA) induced folding between two arms bringing two terminal
fluorescent amino acids closer.
TPyAla
TPyAla
/
Do
Do). Furthermore, we envisaged that the exciplex
emission could be achieved via dual mechanism-either via FRET or
direct excitation of a FRET acceptor in ExcipFRET-peptide 2.
Though, all the photophysical phenomena, FRET7a-b/Eciplex7c-d
/Excimer7e-f, are well known and exploited in biology, design of a
fluorescent peptide with a predefined secondary structure together
with predicted photophysical properties is fully a new and novel
concept.
FIGURE 2. (a) CD and UV spectra of pentapeptide 2-3 [10 µM,
ethanol]. (b-c) MD simulated cluster of conformers within 21
kJ/mole from the global minima of pentapeptide 2-3.
The presence of intramolecular H-bonding between two peptide
chains in two arms through amide NHs was evident from both IR
and variable temperature NMR (VT NMR) study (SI, Section 5).9
Moreover, the pentapeptides 2-3 showed amide I band absorptions at
1653-1655 cm-1 supported the predominant β-sheet conformation.9c-d
While strong H-bonds involving the amide NHs of C-terminal-
TMNapAlaDo, Leu and UrAA-NH were observed in ExcipFRET-
peptide 2, all the amide NHs of Excim-peptide 3 showed strong to
moderate intramolecular H-bonding as was evident from Kessler’s
FIGURE 1. Synthesis of uracil-di-aza-amino acid, UrAA (1) as
constrained molecular scaffold and structures of designed
pentapeptides 2-3 with predefined conformation and predicted
photophysics.
limit of δ/T (SI, section 5.3) supporting the predominant β-sheet
9a-b
With this above idea, herein we demonstrate the synthesis, β-
sheet nucleating property of uracil-di-aza-amino acid scaffold, UrAA
(1) and impact of the adopted conformation on the possibility of dual
mechanism of exciplex emission in a design UrAA-scaffolded short
peptide 2, (Boc-TPyAlaDo-Leu-UrAA -Leu-TMnapAlaDo-CO2Me), and
excimer emission in peptide 3 (Boc-TPyAlaDo-Leu-UrAA -Leu-
TPyAlaDo-CO2Me). The peptides 2 and 3 with UrAA in the backbone
are found to adopt predominantly β-sheet structures. Based on our
conceptual understanding, we were able to establish a dual
mechanism of exciplex emission-either via FRET (from TMNapAlaDo
to TPyAlaDo) or upon direct excitation of the FRET acceptor,
triazolylpyrene (TPy) of TPyAlaDo, in peptide 2 and excimer emission
in peptide 3 in aqueous buffer. To the best of our knowledge this is a
start of a new generation peptide probe of fundamental spectroscopic
importance and might find widespread application in chemical
biology for monitoring cellular events and other biotechnological
applications.
like conformation.
The analysis of solution conformations from
NOESY and ROESY spectra in d6-DMSO of both the pentapeptides
2-3 revealed that the scaffold adopted a rigid hairpin shape. The
backbone H-bonding and hydrophobic/stacking interactions between
terminal chromophores helped the scaffold nucleating the peptide
strands to adopt overall β-sheet conformation (SI, Section 5). The 2D
NMR supported the close proximity between the two terminal
fluorescent unnatural amino acids (TMNapAlaDo/TPyAlaDo for 2 and
TPyAla
TPyAla
/
Do
for 3). Molecular dynamics (MD) simulations
Do
carried out using Schrodinger Macromodel (Maestro vs. 9.0)
software package with OPLS 2005 force field also supported the β-
sheet conformation and the close proximity of the terminal
chromophores in both the pentapeptides (Fig. 2b-c).
We, next, examined the photophysical interaction between the
terminal triazolyl unnatural amino acids in both the fluorescent
pentapeptides in aqueous buffer. The UV-visible and fluorescence
photophysical properties of the fluorescent triazolyl amino acid
monomers indicated a possibility of FRET process from donor,
TMNapAlaDo to acceptor, TPyAlaDo in ExcipFRET-peptide 2 (SI, Fig.
The synthesis of the BocNH-uracil-diaza-amino acid, UrAA (1,
Fig. 1) proceeded through a sequential coupling of 3-bromo-methyl
propionate and 2-iodo-N-Boc-ethylamine with uracil (4) (Fig. 1).
The amino acid scaffold was characterised by NMR and mass
spectrometry. To explore the constrained property and to determine
the effect exerts by the scaffold 1 on the conformation of a peptide,
we, next, incorporated it into two pseudo-symmetric fluorescent
pentapeptides 2-3 differing only in the C-terminal fluorescent amino
acid (Fig. 1). All the peptides were synthesized via a solution phase
peptide coupling protocol5c-d and characterised by 1D, 2D NMR and
mass spectrometry (SI, Section 2-4).
S18). Moreover, at the absorption wavelength (
= 300 nm) of
TMNapAlaDo, the amino acid TPyAlaDo absorb minimally indicating
that the ExcipFRET-peptide 2 could be excited at 300 nm to test the
possibility of FRET. Thus, based on our designing concept and the
above observations we turned our attention to study the FRET
process/exciplex emission in detail in ExcipFRET-peptide 2.7a-b
Upon excitation at the absorption maximum of the donor,
TMNapAlaDo (ex = 300 nm), the ExcipFRET-peptide 2 showed an
weak and overlapped emission at 365 nm corresponding to the
emission from TMNapAlaDo, along with another broad emission which
was resolved into two bands, 405 and 465 nm. The band at 405 nm
was the emission from TPy via FRET and the broad long wavelength
emission at 465 nm was most likely from a complex/exciplex of
TMNap-TPy. Further analysis revealed that the emission intensity
of the donor, TMNapAlaDo, decreased seven times of that of the free
donor fluorescence in buffer. While in methanol, a two times
increase in emission intensity of the acceptor, TPyAlaDo was observed
The secondary structure determination in methanol using CD
spectropolarimeter revealed a negative band at around 208-215 nm
and a positive band at 195 nm indicating a predominantly β-sheet
conformation in both the pentapeptides 2-3 (Fig. 2a).8 The signature
of aromatic - stacking interaction between two terminal
chromophores in both the pentapeptides were also evident from the
appearance of the induced CD signal in the range of 325-425 nm.8b-c
3

with no band at 465 nm, in buffer the TPyAlaDo monomer emission
from ExcipFRET-peptide 2 decreased compared to the free
acceptor emission along with a new emission (exciplex) band at 465
nm. Therefore, the change in donor/acceptor emissions supported a
FRET process from TMNapAlaDo to TPyAlaDo in ExcipFRET-peptide
2 in both solvents (Fig. 3a).7b The occurrence of a FRET process was
also evident from a time resolved fluorescence study wherein we
stacked TPy-TMNap complex, respectively. Thus, the life time
component of pyrene came out to 14.3 ns while that of exciplex at
2.5 ns (SI, Table 9, λex = 375 nm) which was almost same as that was
observed while exciting the ExcipFRET-peptide 2 at donor
absorbance at 290 nm. The second path of exciplex emission was
further supported both from a variable temperature steady state as
well as time resolve fluorescence experiment (Fig. 3d and SI, Fig. S
19).5a With increasing the temperature the exciplex life time
component was found to decrease slowly but gradually. The
nucleation by the UrAA scaffold into a β-sheet conformation played
an important role in bringing the two terminal fluorescent amino
acids closer and to interact photophysically leading to an exciplex
emission in ExcipFRET-peptide 2 (ExciFRET-peptide) via two
path-either via FRET or via direct excitation of the FRET acceptor.
To the best of our knowledge this fundamental phenomenon is new.
observed a decrease in donor life time component (TMNapAlaDo; λex
=
290 nm, λem = 365 nm) from 8.8 to 6.4 ns (SI, Table 7.3). However,
we observed an almost similar intense emission from the acceptor
(TPy) as well as life time component (TPyAlaDo; λex = 290 nm, λem
=
405 nm) from 7.9 ns to 7.0 ns with an appearance of exciplex band at
465 nm of life time component of 1.7 ns (SI, Table 8, 10). The slight
decrease in acceptor’s life time could be explained considering n-*
quenching of TPy monomer (aaceptor) emission by closely spaced
donor TMNap through –OMe group leading to decrease in monomer
emission and formation of TPy-TMNap exciplex complex.7c-d
A
After establishing the fundamental concept of dual door entry to
exciplex emission in ExcipFRET-peptide 2, we next studied the
photophysical interaction between the two terminal TPy moieties in
Excim-peptide 3. The UV-visible absorption spectra of Excim-
peptide 3 showed bands at 268, 277 and 346 nm corresponding to
the uracil and TPy absorptions, respectively, in methanol. Upon
excitation at long wavelength absorption band (ex = 350 nm), the
pentapeptide showed dual emission centred at 405 nm corresponds to
the structured triazolyl pyrene monomer emission and unstructured
broad emission at 475 nm which was unambiguously assigned as an
excimer emission. Support for an excimer emission also came from a
fluorescence life time experiment (SI, Fig. S14, Table S5). The H-
bonding and side chain hydrophobic interaction between the two
dipeptide arms of the scaffold in the β-sheet conformation allowed
the terminal TPyAlaDo to come closer to involve in --stacking
interaction leading to TPy-TPy excimer emission. This was also
supported by CD (Figure 2a), 2D NMR spectroscopy (SI, section
5.4) as well as a molecular dynamics (MD, Figure 2b-c) simulation
study.
gradual decrease in the emission intensity as well as life time of
emission at 465 nm with increasing temperature indicated a
breakdown of -stacked excited state complex (exciplex) between
TMNap and TPy confirming the exciplex emission (Fig. 3b-c and
SI, Table 10-11).5a, 7c-d The close proximity guided high possibility of
formation of - stacked complex between excited state
triazolylpyrene (TPy) at the C-terminus and ground state triazolyl
methoxynaphthalene (TMNap) at the N-terminus was supported
from a 2D NMR as well as by a molecular dynamics (MD)
simulation (Fig 2b).10 Moreover, the excitonic interactions among the
terminal fluorescent amino acids as was seen in the
absorption/emission spectra further supporting - stacked
complexation between TPy and TMNap (SI, Section 6). All the
observations evident our fundamental concept of FRET mediated
exiplex emission in a β-sheet fluorescent ExcipFRET-peptide 2.
FIGURE 3. Steady state (a) variable temperature emission (ex
=
300 nm) and (b) time resolved (c-d) fluorescence spectra of
ExciFRET-peptide 2 in buffer showing FRET-mediated exciplex
emission.
FIGURE 4. (c) Fluorescence titration of ExcipFRET-peptide probe
2 (a-b) and Exim-peptide 3 (c-d) in presence and absence of
increasing concentration of BSA showing dual path entry to exciplex
in 2 and excimer in 3 [λex = 280 nm; for a,c and λex = 350 nm for b,d;
concentration of pentapeptide = 10uM]. All the experiments were
carried out in 5 mM phosphate buffer, pH 7.0, r.t.
After investigating the photophycal property of the ExcipFRET-
peptide 2, we turned our attention to prove the second path of
exciplex formation via direct excitation at the FRET acceptor, TPy.
Thus, upon excitation at the absorption wavelength of FRET
acceptor, TPy, of N-terminal TPyAlaDo (ex = 350 nm), the
ExcipFRET-peptide 2 showed two emission bands at 405 and 465
nm which were superimposable to the spectrum obtained when
excited at the donor absorbance maximum (ex = 300 nm of TMNap,
of C-terminal TMNapAlaDo) and were unambiguously assigned as
monomer emission from TPy and the exciplex emission from -
Therefore, we established the dual door entry to exciplex
emission in ExcipFRET-peptide 2 and an excimer emission in
Excim-peptide 3 containing uracil amino acid scaffold in the
backbone as β-sheet nucleator. The exciplex and excimer emissive
pentapeptides are both fundamentally and conceptually new and
novel which could serve as sensitive probes for studying protein-
4

peptide/peptide-DNA interaction and in other wider applications.3
We, therefore, thought that the monomer/exciplex and
monomer/excimer emission from pentapeptide 2 and 3, respectively,
might offer some interesting insight into the interaction with a
biomolecule such as a model protein BSA. Thoughtful knowledge on
the mode of interactions between a drug and serum albumins is
needed to assess the therapeutic effectiveness of a drug.11 This would
correlate with its bioavailability, metabolism, excretion and
interaction with the target tissues and thereby its biological activity.12
Therefore, immense biological and clinical importance of peptide
based drug analogues enforce to accumulate knowledge how they are
being recognized and transported into the cell by transporter
membrane protein. In this respect, we thought that it would be
worthwhile to study the interaction behaviour of our designed
peptides with bovine serum albumin (BSA) which has 76% sequence
identity to human serum albumin (HSA) and is widely used as a
model protein in investigating protein-drug interaction.13 Thus, we,
finally, studied the interaction with model protein BSA using UV-
visible and fluorescence spectroscopy. Our main aim was to test
whether the fluorescent peptides could interact with any
biomolecule, such as a protein biomolecule. Moreover, we wanted to
know that in protein’s microenvironment these peptide probes
whether could retain their photophysical outcome-such as dual door
entry to exciplex emission in case of ExcipFRET-peptide 2 and an
excimer emission in case of Excim-peptide 3. Therefore, at this
stage, we did not consider the probes for studying interaction with
other proteins to test their protein binding specificity. This extended
study along with the study with DNA biomolecule is under
consideration of our laboratory.
formation of -- stacked TPy-TMNap complex leading to an
ultimate increase in TPy monomer emission. This explanation was
also supported from an anisotropy experiment,14
a
small
excimer/monomer intensity ratio, life time experiment. That the
probe resided in the hydrophobic site of BSA was also supported
from the increase in steady state anisotropy from 0.01 to 0.17 (λex
=
300 nm, λem = 365); 0.03 to 0.13 (λex = 350 nm, λem = 365) and from
0.015 to 0.06 (λex = 350 nm, λem = 465 nm) (SI, Table 16). Therefore,
the Excip-peptide
2 maintained its photophysics in BSA
microenvironment and behaved as a system of dual path to exciplex
emission-either via FRET (from BSA or TMNap to TPy) or direct
excitation of a FRET acceptor (TPy). The backbone H-bond
mediated intrastrand β-sheet conformation in both the pentapeptides
nucleated by the scaffold UrAA played an important role in bringing
two terminal chromophoric amino acids closer leading to predefined
photophysical interactions and exciplex/excimer formation.
On the other hand, addition of an increasing concentration of
BSA, both the monomer at 405 nm and the excimer emission at 485
nm of the probe 3 increased gradually (ex = 280 nm). Closer look at
emission of only BSA, only probe 3 and a 1:1 mixture indicated a
decrease in emission of BSA and an increase in both monomer
emission from TPy ( 405 nm) as well as TPy-TPy excimer emission
at 485 nm when excited at BSA absorption (280 nm) (Fig. 4c).
Moreover, excitation at TPy (ex = 350 nm), both the monomer at
405 nm and the excimer emission at 485 nm of the probe 3 increased
gradually as BSA concentration increases (Fig. 4d). The time
resolved fluorescence study also supported our observation. Thus,
life time of the minor component in Excim-peptide 3 increased from
3.3 to 5.5 ns upon addition of 1.0 equivalent of BSA (λex = 290, λem
= 405 nm) while the major component was also increased from 27.5
to 31.8 ns (SI, Table 19). While monitoring the excimer emission at
470 nm, both the component lifetimes increased upto addition of 1.0
equivalent of BSA. However, further increase in BSA concentration
had resulted in slight decrease in life time supporting the steady state
fluorescence study (SI, Table 15). Similar results were observed
when excited at 350 nm. Thus, life time of the minor component
increased from 4.8 to 5.8 to 6.0 to 6.4 ns upon gradual addition of
The UV-visible spectra in phosphate buffer, thus, revealed that
the probe pentapeptides 2-3 exhibited weak and structureless
absorptions at around 300 and 345 nm, corresponding to TMNap
and TPy, respectively. Upon gradual addition of BSA to a solution
of the ExcipFRET-peptide 2 and/or Excim-peptide 3, the absorption
bands experienced strong hyperchromicity along with
a
hypsochromic shift of 18 and 10 nm, respectively, indicating a strong
binding interaction between both the probes and BSA in the
hydrophobic region (SI, Section 8). The Job’s plots suggested a 1:1
probe : BSA binding events in all the cases with association
constants ranging from 0.5-1.3 x 105 M-1 and binding free energy -6
to -7 kcal/mole-1.
0.5, 1.0, and 1.5 equivalent of BSA, respectively, (λex = 375, λem
=
405 nm) while the major component was also increased from 28.2 to
39.7 to 41.6 to 44.7 ns (SI, Table 20). While monitoring the excimer
emission at 470 nm, both the component lifetimes increased upto
addition of 1.0 equivalent of BSA but slowly decreased upon
addition of much amount of BSA (SI, Table 20). All spectroscopic
outcomes unambiguously indicated the excimer emission via FRET
from BSA to TPy in Excim-peptide 3 when excited at BSA (280
nm) (SI, Section 8).
Next, a fluorescence titration experiment was carried out to
investigate the protein sensing ability and to gain insight into the
interaction of the probes with BSA. Thus, the ExcipFRET-peptide 2
showed a regularly increased TPy monomer emission (405 nm) and
exciplex emission (470 nm) when excited either at BSA (280 nm) or
TMNap (300 nm) upon gradual addition of an increasing amount of
BSA (Fig 4a). Analysis of emission of BSA, probe 2 and a 1:1
mixture indicated a decrease in emission of BSA or TMNap along
with an enhancement of TPy monomer emission and of TPy-
TMNap exciplex emission clearly indicating a FRET mediated
exciplex emission. Similar increase in both the emission at 405 nm
(TPy emission) and at 470 nm (exciplex) was observed when excited
at TPy (350 nm) upon gradual increase in the concentration of BSA
(Fig. 4b). All the observations were further supported from a time
resolve fluorescence study (SI, Section 8). Thus, life time of the
minor component in ExcipFRET-peptide 2 increased from 5.6 to 6.7
to 6.8 to 7.1 ns upon gradual addition of 0.5, 1.0, and 1.5 equivalent
of BSA, respectively, (λex = 375, λem = 405 nm) while the major
component was also increased from 26.9 to 38.2 to 41.3 to 43.7 ns
(SI, Table 15). While monitoring the exciplex emission at 465 nm,
both the component lifetimes increased upto addition of 0.5
equivalent of BSA. However, further increase in BSA concentration
had resulted in slight decrease in life time supporting the steady state
fluorescence study (SI, Table 15). Similar results were observed
when excited at 290 nm (SI, Table 14). The exciplex emission
intensity decreased slightly at high BSA concentration possibly due
to encumbering TPy moiety in the hydrophobic pocket restricting
FIGURE 5. (a) Integrated heat profile of the ITC with non-linear-
squares fit to a one-site binding model and (b) docking pose of
ExcipFRET-peptide 2 in presence of BSA.
Therefore, in BSA microenvironment, the probe 3 behaved as a
system of dual path to excimer emission-eitheir via FRET (from
BSA to TPy) or direct excitation of a FRET acceptor (TPy). The
slight decrease in excimer emission at high BSA concentration, a
small change in anisotropy and excimer/monomer intensity ratio
indicated that the TPy-TPy excimer formation was restricted as the
concentration of BSA increased which was also supported from a life
time experiment.14 The steady state anisotropy increased from 0.005
5

Burgess K. Universal Peptidomimetics. J. Am. Chem. Soc. 2011;133:462. (d)
Meunier B. Chem. Rev. 2008; 104: 69.
to 0.103 (λex = 350 nm, λem = 405); and from 0.011 to 0.051 (λex
=
350 nm, λem = 475 nm) indicated that the chromophore resided in the
hydrophobic site of BSA. All the observations could be explained if
we consider single site binding of the probe 3 leading to
encumbering TPy moiety in the hydrophobic pocket of BSA which
might be the possible cause of low FRET efficiency as the
concentration of BSA increased ultimately leading to a restricted
TPy-TPy excimer formation. The binding event was also supported
from an ITC experiment15 as well as from a molecular docking16
calculation. The binding events from an ITC experiment indicated a
single event binding with affinity of 0.5 ×105 M−1 and a free energy
change of -6 to -6.9 kcal/mol (Fig. 5a and SI section 8.2.3). The
docking study showed the binding of the probes in hydrophobic
pocket of site I in the vicinity of Trp 134 supporting the close
proximity and FRET incidences in both the cases (Fig 5b and SI
section 8.2.9). The CD spectral titration revealed that both the probes
did not perturb the α-helicity of the BSA upon binding. The intensity
slightly increases as well as the slight increase in the % α-helicity of
BSA was observed which is due to possible conformational
adjustment of BSA upon association (SI, Figure S32, 42).17
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In summary, the newly designed uracil-amino acid (UrAA) in the
peptide backbone marked a novel class of unnatural peptide building
block with ability to induce β-sheet conformation. We established
the dual mechanism of exciplex emission in a designed ExcipFRET-
peptide 2 and excimer emission in an Excim-peptide 3. Both the
peptides maintainning their predefined photophysics were found to
interact with a model biomolecule with fluorescence swith-on
response. This is the first report of design of a fluorescent peptide
with a predefined secondary structure together with predicted
photophysical properties. Under consideration is the study of
exploration of sequence specific DNA binding event of uracil-amino
acid (UrAA) scaffold and study of interaction with other protein
biomolecules.
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Acknowledgments
SSB is thankful the DBT [BT/PR5169/BRB/10/1065/2012], Govt. of
India, for a research grant. Author AY is thankful to CSIR, New
Delhi, for a fellowship. Authors are also thankful to CIF, IIT Guwahati
for NMR facility.
Supplementary data
Supplementary data (Experimental details, spectroscopic data, macromodel
study and copy of NMR spectra) associated with this article can be found in
the online version, at http://dx.doi.org/10.xx/j.bmcl.2017.xx.
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6

Uracil-Amino Acid as a Scaffold for β-
Sheet Peptidomimetics: Study of
Photophysics and interaction with BSA
Protein
SubhenduSekhar Bag* and Afsana Yashmeen
Bioorganic Chemistry Laboratory, Department of
Chemistry, Indian Institute of Technology
Guwahati-781039, India. Fax: +91-258-2349; Te.:
+91-258-2324; Email: ssbag75@iitg.ernet.in.
HIGHLIGHTS
Novel Uracil amino acid (UrAA) is
uncovered for the first time as a potential -sheet
nucleator
UrAA constitutes a new family of
conformationally constrained molecular scaffold
Established dual path entry to exciplex
emission in a UrAA scaffolded dual labeled β-sheet
pentapeptide
Excimer emission in another UrAA
scaffolded pentapeptide with TPyAlaDo/ TPyAlaDo at the
two termini
Swith-on fluorescence sensing of BSA by
these pentapeptides maintaining their fundamental
photophysics
7