FRET events in fluorescent pentapeptides containing aliphatic triazolo amino acid scaffolds: Role of spacer lengths

Article abstract of DOI:10.1016/j.jphotochem.2019.04.024

The FRET efficiencies in donor/acceptor pairs in the two termini of designed fluorescent pentapeptides depend on the flexibility of two arms of triazolyl amino acid scaffolds positioned in the center of the backbones inducing predominant β-sheet conformations. Flexible N-Terminus of the scaffold in a pentapeptide has led to higher FRET efficiency and makes it different from other peptide with flexible C-terminus.

Full text of DOI:10.1016/j.jphotochem.2019.04.024

JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
FRET events in fluorescent pentapeptides containing aliphatic triazolo
amino acid scaffolds: Role of spacer lengths
Subhendu Sekhar Bag⁎, Afsana Yashmeen
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology, Guwahati 781039, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
The FRET efficiencies in donor/acceptor pairs in the two termini of designed fluorescent pentapeptides depend
on the flexibility of two arms of triazolyl amino acid scaffolds positioned in the center of the backbones inducing
predominant β-sheet conformations. Flexible N-Terminus of the scaffold in a pentapeptide has led to higher
FRET efficiency and makes it different from other peptide with flexible C-terminus.
FRET efficiency
Spacer length
N-terminus flexibility
Aliphatic amino acid scaffolds
β-Sheet pentapeptides
1. Introduction
orientation lead to low probability for FRET. However, under rapid
2
isotropic sampling a dynamic random average value of 2/3 for κ
Förster resonance energy transfer (FRET) is an energy transfer
phenomenon between a donor and an acceptor chromophore in a suf-
ficient proximity (10–100 Å). In general, the donor and the acceptor
with an overlapped emission and excitation spectra participate in FRET
process [1]. The efficiency of this energy transfer is inversely propor-
tional to the sixth power of the distance between donor and acceptor. A
small change in this distance is reflected in FRET efficiency. Thus, FRET
efficiency is widely used as a research tool in the field of chemistry,
biology, biomedical research and in today’s drug discovery [2]. FRET is
one of few tools available for measuring the fluctuations in nanometer
scale distances both in vitro and in vivo [3]. Due to its sensitivity, FRET
has been used to investigate molecular level interactions [2] and to
measure the end-to-end distances [4].
would led to larger probability of FRET. On the same line, very recently,
the importance of the differences in transition dipole orientation angle
in donor-acceptor pair which is again dependent on the local micro-
environment on the quantification of FRET process has been demon-
strated by Bain and coworkers [7c].
Many more results and designed chromophoric pairs have been
reported aiming at uncovering the structure, function, dynamics and
interactions involving biomolecules inside a cell [8]. However, a
number of issues need to be considered while designing an efficient
FRET system, such as-(a) close proximity and (b) enough spectral
overlap among donor/acceptor pairs. Moreover, the factors such as
concentration, cellular localization etc. also played an important role in
controlling the FRET process in biological systems. Therefore, design of
an efficient FRET pair as well as their proper placement within a mo-
lecular entity is crucial in deciphering the bimolecular conformations.
One common donor-acceptor FRET pair in biology used is a cyan
fluorescent protein (CFP) – yellow fluorescent protein (YFP) pair [5]. In
biology, FRET has been utilized to study the conformational changes of
short polypeptides upon phosphorylation [6]. Recently, Iqbal et al.
described the importance of fluorophore orientation for understanding
FRET event in terms of distances in some circumstances in nucleic acid
based scaffold [7a]. They showed Förster distance calculation resulted
in an error upto 12 Å while considering the κ2 value of 2/3. However,
increasing the flexibility of the chain containing the fluorophores can
diminish this discrepancy. The impact of heterogeneity on the FRET
process was demonstrated by Vogel et al. using Monte Carlo simulations
[7b]. The FRET is dependent on the relative orientation of transition
dipoles of the donor and acceptor and their separation vector (κ2). It
was suggested that the isotropically or pseudo-isotropically random
2. The concept
Based on our earlier concept of design of peptidomimetic scaffolds,
we thought that altering the length of amine (m) or acid terminus (n) or
both, the triazolyl aliphatic amino acid scaffold (A) in a peptide back-
bone could induce β-sheet conformations (B) with varying propensities
(Fig. 1) [9]. Furthermore, we envisioned that the extents of photo-
physical dipolar interaction, such as, FRET, between two fluorescent
amino acids (TMnapAlaDo and TPyAlaDo) at the two termini of penta-
peptides would be different due to different spacer lengths (Fig. 1).
Therefore, we would be able to establish a novel concept of distance
⁎ Corresponding author.
E-mail address: ssbag75@iitg.ac.in (S.S. Bag).
https://doi.org/10.1016/j.jphotochem.2019.04.024
Received 23 January 2019; Received in revised form 14 April 2019; Accepted 16 April 2019
Availableonline27April2019
1010-6030/©2019ElsevierB.V.Allrightsreserved.

S.S. Bag and A. Yashmeen
JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Fig. 1. Graphics of aliphatic triazolo amino acid scaffolds with different spacer lengths (A), the designed fluorescent pentapeptides (B) and the possible FRET event.
dependent FRET process in these designed peptides. Thus, with this
concept, we planned to synthesise triazolyl amino acid scaffolds A of
arm length differed by 1–2 carbon atoms on both N- and C- termini as β-
sheet inducers and to place in the middle of the backbones of designed
four fluorescent pentapeptides (Fig. 1).
1–4 in the backbones were synthesized via the standard peptide cou-
pling reaction following our earlier published protocol [9]. Thus,
the synthesis of pentapeptide 5 [BocNH-TMnapAlaDo-Leu-1/1AlTAA-
Leu-TPyAlaDo-CONMe(OMe)] containing 1/1−AlTAA scaffold (1), was
performed following Scheme 3A with 34% overall yield. The syntheses
of other three unnatural fluorescent pentapeptides 6–8 containing ali-
phatic triazolyl amino acid scaffolds 2/1-AlTAA (2), 1/2-AlTAA (3) and 2/
2-AlTAA (4), respectively, of different spacer lengths were carried out
using similar peptide coupling protocols (Schemes 3B for 6 and Scheme
4A-B for 7–8). All the intermediate and final peptides were purified by
silica-gel (60–120 mesh) column chromatography and characterized
with the help of analytical techniques such as, 1H, 13C NMR and mass
spectrometry.
Thus, we targeted to synthesise 1/1-, 2/1, 1/2, and 2/2-aliphatic
triazolyl amino acid scaffold (1–4) having 1-carbon spacer in each
terminus (1), 2-carbon spacer in N-and 1-carbon in C-terminus (2), 1-
carbon spacer in N-and 2-carbon in C-terminus (2) and 2-carbon spacer
in each terminus (4), respectively (Fig. 1). The scaffold 1 being the
highest in rigidity and the scaffold 4 being the highest in flexibility
constitute two extreme ends of our design. On the other hand, 2/1-
scaffold 2 and 1/2-scaffold 3 are the two alternates in term of flexibility
of N-and C-terminal arms, respectively. We expected that FRET effi-
ciency would vary depending on the spacer length. Thus, the FRET
efficiency would be higher and lower, respectively, at a largest and
smallest spacer length maintaining the secondary structure. We, thus,
established that increase in chain length of the scaffold enhances the
extent of FRET efficiency which is due to increase in extent of flexibility
that led to closeness of the donor/acceptor pair.
3.2. Conformational analysis
Having our target peptides in hand, we, next, studied their con-
formations using various spectroscopic techniques. Thus, the secondary
structure determination via circular dichroism (CD) spectropolarimeter
of three fluorescent pentapeptides 5 and 7–8, containing aliphatic
triazolyl amino acid scaffolds with different spacer lengths (1, and 3-4,
respectively) in the backbone, showed predominantly β-sheet like
structures (Fig. 2 and SI, Section 1) [10a–c]. The appearance of nega-
tive induced circular dichroism (ICD) bands in the chromophoric ab-
sorption region (300–350 nm) in all these peptides indicated the π-π
stacking interactions between the two terminal chromophores (Fig. 2b,
3. Results and discussion
3.1. The synthesis
The syntheses of aliphatic triazolyl amino acid scaffolds were
started via Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reac-
tion following our earlier protocol [9]. Thus, the synthesis of 1/1-
scaffold, 1, was reported earlier and we used the earlier synthesized
material [9]. In short the synthesis was started from a conversion of
ethyl bromoacetate (9) to ethyl azidoacetate (10) followed by click
reaction with propargyl alcohol (11) following Scheme 1A. Subse-
quently the triazolyl alcohol 12 was converted to BocNH-triazolyl
amino acid scaffold 1, (BocNH-1/1AlTAA) in excellent yield (Scheme
1A). Next, the synthesis of scaffolds 2 was carried out starting with a
click reaction between methyl 2-azidoacetate (17) and homopropargyl
alcohol (18) following similar strategy. The click triazolyl product 19
was then subsequently transformed into triazolyl amino acid 23, (2/
1−AlTAA) and then to scaffold 2 (BocNH-1/1AlTAA) in excellent yield
(Scheme 1B).
d) [10a–c]. The ICD correspond to triazolyl pyrene (TPy of TPyAlaDo
)
and triazolylmethoxynapthalene (TMNap of TMNapAlaDo) absorptions
appeared in the region of 345–378 nm (faint intensity) and at 287 nm
(prominent intensity), respectively. The peptide 8 exhibited more in-
tense ICD at 287 nm compared to 7 reflecting the role of more flexible
spacer in peptide 8. On the other hand, the peptide 5 with rigid scaffold
(1/1-spacer) possessed ICD bands of similar intensities.
The interesting CD spectrum was observed for peptide 6 which is
different from other three peptides. Thus, in acetonitrile, it showed
positive bands at 192 (weak), 200 and 218 nm indicating its extended
helical conformation similar to poly-L-glutamic acid [10]. Moreover,
the appearance of positive ICD bands at 290 and 305–365 nm are also
indicative of a clear opposite characteristic feature of the peptide 6
compared to other three peptides (Fig. 2b, d). Interestingly, changing
the solvent polarity from acetonitrile to MeOH, the peptide 6 showed a
negative band at 192 nm and positive bands at 200 and 218 nm in-
dicating its’ structural similarity to rat-tail collagen in the native state
(SI, Fig. S1) [10]. On the contrary, in methanol all other three peptides,
5, and 7–8 behave in a similar way both in respect of CD as well as the
Following a similar strategy, we synthesized other two triazolyl
amino acids 30 and 35 from where the BocNH-protected scaffolds 3
(BocNH-1/2−AlTAA) and 4 (BocNH-2/2-AlTAA), respectively, in very
good yields (Scheme 2A-B).
Next, the fluorescent pentapeptides with the scaffold amino acids
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S.S. Bag and A. Yashmeen
JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Scheme 1. Synthesis of aliphatic triazolyl amino acid scaffolds-(A) 1/16, 1/1−AlTAA and (B) 2/23, 2/1AlTAA.
appearance of all positive ICD bands indicating more disorder structure
in H-bonding solvent, methanol (SI, Fig. S1). These differences are re-
flecting the role of spacer length of the various scaffolds in these pep-
tides (Fig. 1). More study is necessary to ascertain the effect of spacer
length on the conformational rigidity which is our future target.
The H-bonded conformations were supported from IR and variable
temperature NMR. Thus, the IR spectra showed the presence of in-
tramolecular H-bonded and free amide ‒NH stretching absorptions at
around ῡ = 3310−3296 cm−1, C]O stretching at ῡ = 1885−1660
cm-1 for all fluorescent pentapeptides, in particular, for peptides 5, 7
and 8 which supported β-sheet like structure [11]. VT-NMR analysis
indicated the presence of weak intramolecular H-bonding between the
two peptide strands (SI, Section 1.3) [11b]. In this experiment, a linear
dependence of the change in chemical shift (Δδ) of the protons involved
in intramolecular H-bonding was observed with increase in tempera-
ture (T). Thus, the temperature coefficient (Δδ/ΔT) of the -NH proton is
generally used to study the presence of intramolecular H-bonds
[11b–d]. With increase in temperature, the –NH protons that are H-
bonded intramolecularly exhibit upfield shifting of their resonance
signals indicating the breaking of such intramolecular H-bonds. The
temperature coefficients ranging from 0- (−)4.5 ppb K−1 are con-
sidered to be the indicator of strong intramolecular H-bonds [11b,c].
The values ranging upto (-) 8 ppb/k indicated the moderate to weak
intramolecular H-bonds. A noticeable change in chemical shift was
observed for the 2/1-scaffold-NH in peptide
6 wherein Δδ/ΔT
(-5.9 ppb/k) value was lowest among all amide-NHs of all peptides
studied. This might be because of unique flexibility of the amino arm of
the 2/1-scaffold containing two carbon spacer length at the N-terminus.
This uniqueness was also reflected in the conformation adopted in CD
by the peptide 6 having 2/1-scaffold in the backbone. Thus, both the CD
and VT-NMR supported our design concept.
Next, the 2D NMR analysis in DMSO-d6 was carried out to support
the β-sheet conformation. The NOESY and ROESY spectra revealed the
backbone H-bonding interactions indicating possible β-sheet con-
formations. The 2D NMR supported the close proximity of the two
terminal fluorescent unnatural amino acids and possibility of
a
Scheme 2. Synthetic scheme for the synthesis of two triazolyl aliphatic amino acid scaffolds-(A) 1/2−AlTAA (30/3), and (B) 2/2-AlTAA (35/4).
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JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Scheme 3. Synthetic scheme for the fluorescent pentapeptides-(A) 5 and (B) 6 - containing scaffold 1/1−AlTAA (1) and 2/1-AlTAA (2), respectively, and fluorescent
unnatural amino acids, TMnapAlaDo and TPyAlaDo
.
Scheme 4. Synthetic scheme for the fluorescent pentapeptides-(A) 7 and (B) 8 - containing scaffold 1/2−AlTAA (3) and 2/2-AlTAA (4), respectively, and fluorescent
unnatural amino acids, TMnapAlaDo and TPyAlaDo
.
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JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Fig. 2. (a) CD spectra of pentapeptides 5-8 in acetonitrile and (b) the ICD bands.
Fig. 3. (a) Schematics of long range 1H-1H interactions in pentapeptide 5 as evident from 2D NMR and the (b) clustering of structures obtained from molecular
dynamics simulation.
photophysical interaction between (TMnapAlaDo
/
TPyAlaDo) for all pen-
depends on local microenvironmental heterogenity.7 The similar si-
tuations are quite frequent in most fluorescent proteins [13] In sum-
mary, we observed a decrease in donor life time (TMNapAlaDo, λex
= 290 nm, λem = 365 nm) from 6.8 to 3.38 ns in pentapeptide 5 in
acetonitrile (SI, Fig. S12a, Table S9) indicating a FRET process [9]. At
this stage, the detailed investigation of the potential of the rise time of
acceptor fluorescence to probe FRET was not done. In future, we would
like to quantify the FRET from the rise time of acceptor fluorescence
which should be equivalent to donor fluorescence lifetime [14].
Under the same experimental condition, other pentapeptide 6 also
showed a weak emission at 365 nm corresponding to the emission from
tapeptides [9b,c]. The possible observed interactions for one re-
presentative peptide 5 was shown in Fig. 3a. The MD simulations for
the pentapeptides were carried out with Schrodinger Macromodel
(Maestro vs. 9.1) software package using an OPLS 2005 force field
which supported the β-sheet conformation (Fig. 3b) [12].
3.3. Photophysical property and FRET efficiency
The UV–vis and fluorescence photophysical properties of the
fluorescent triazolyl amino acid monomers, TMnapAlaDo and TPyAlaDo
,
TMNapAlaDo along with a broad emission at 405 nm (SI, Fig. S8, Table
(SI, Fig. S5–6; Table S3/4) indicated that there is a possibility of FRET
process from donor TMNapAlaDo to acceptor TPyAlaDo in all peptides (SI,
Fig. S11a). Furthermore, from the UV visible spectra of TMNapAlaDo and
S6) when excited at the absorption wavelength of TMNapAlaDo
(
≈
max
300 nm). Similar to the case in peptide 5, when the pentapeptidea6bs was
excited at 300 nm, the donor (TMNapAlaDo) fluorescence intensity de-
creased drastically while the acceptor (TPyAlaDo) fluorescence intensity
increased from that of the bare acceptor supporting a FRET process in
pentapeptide 6 (Fig. 4b). The occurrence of FRET process was evident
from a time resolved fluorescence study wherein we observed a de-
crease in donor life time (TMNapAlaDo, λex = 290 nm, λem = 365 nm)
from 6.8 to 3.15 ns in pentapeptide 6 (SI, Fig. S12b, Table S9). The
FRET events in pentapeptide 7–8 were also evident both from steady
state as well as from time resolved fluorescence study (SI, Fig. S9-10,
Table S7–8 and Table S9-S10). However, the extents of intensities/ef-
ficiencies were different.
TPyAlaDo it was evident that at the absorption wavelength of
TMNapAlaDo
(
= 300 nm), the amino acid TPyAlaDo absorb negli-
max
gibly. Thus, it awbsas clear that all the peptides 5-8 could selectively be
9a, d
excited at 300 nm.
Upon excitation at the absorption maximum of the donor,
TMNapAlaDo (λex = 300 nm), the pentapeptide 5 showed a weak and
overlapped emission at 365 nm corresponding to the emission from
TMNapAlaDo accompanied by another broad emission at 405 nm in all
solvents (SI, Fig. S7, Table S5). The band at 405 nm is most expectedly
the emission from TPy of TPyAlaDo via FRET (SI, Fig. S11a-b). Fig. 4a
clearly indicated that when the pentapeptide 5 was excited at 300 nm,
the donor (TMNapAlaDo) fluorescence intensity decreased drastically
while the acceptor (TPyAlaDo) fluorescence intensity increased from
that of the bare acceptor. Thus, the steady state fluorescence clearly
evidenced the FRET process in pentapeptide 5 (Fig. 4a). The occurrence
of a FRET process was also evident from a time resolved fluorescence
study. The lifetime measurements reveal complex excited state popu-
lations. Thus, the observed multiexponential fluorescence decays might
be because of heterogeneous excited state population possibly arising
out of either multiple emitting states or various molecular conforma-
tions, or noninteracting subpopulations of chromophores which is again
3.4. Flexibility and the FRET efficiency
We, next, turn our attention to investigate the cause of above dif-
ferences. Is there any role of flexibility i.e. the length of the spacers in
the scaffolds? Is there any role of flexibility to bring the FRET pair
closer to each other? Spacer length of which terminus of the peptides 5-
8 is more effective in bringing more flexibility and hence FRET effi-
ciency? The analysis of relative steady state emission intensities of
donor (TMnapAlaDo)/acceptor (TPyAlaDo) in a peptide and in their bare
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JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
Fig. 4. Fluorescence spectra of individual donor amino acid, TMnapAlaDo, acceptor amino acid, TPyAlaDo and (a) the pentapeptide 5 and (b) pentapeptide 6 (10 μM
each, r.t., λex = 300 nm) in MeOH showing FRET process.
states along with the time resolved fluorescence study suggested that
the extent of FRET process from TMnapAlaDo to TPyAlaDo in all the
pentapeptides differed from each other. This might be because of the
differential proximity of the two terminal chromophores which is again
dependent on the length of the carbon spacer (no. of carbons) of C-/N-
terminus of triazolyl amino acid scaffolds in the backbone of the pep-
tides. As per our limited design, the spacer length is highest (two carbon
distance) in case of 2/2-scaffold 4 (Fig. 1). Therefore, the two peptide
arms are highest most flexible in case of pentapeptide 8 containing 2/2-
scaffold 4 allowing the two chromophores in two termini to come to a
closest distance compared to other peptides. This highest flexibility and
closest positioning of the terminal donor/acceptor chromophores ex-
pectedly would result in highest extent of FRET in peptide 8 (SI, Fig.
S12b) [7].
that two termini come close together and the extent of FRET increases.
All these results supported our expectation and explanation on the
flexibility in peptide and the extent of FRET. Thus, the hypothesis of
increase in extent of FRET via increase in two terminal flexibilities
along with our observations in the limited designs would be worthy of
further investigation [7].
4. Conclusion
In conclusion, we have successfully synthesized aliphatic triazolo
amino acid molecular scaffolds of various spacer length and in-
corporated them into the backbones of designed fluorescent penta-
peptides in order to achieve turn induced β-sheet fluorescent penta-
peptides. We established a correlation among the spacer length of the
two arms of the amino acid scaffolds, the extent of flexibility and the
extent of FRET process among the two terminally situated FRET pairs.
Our result suggested that the increase of flexibility of the molecular
scaffold helps getting turn in such a way that two termini come close
together and the extent of FRET increases. Our study might shed light to
design of peptide-based molecular rulers to probe distance-dependent
FRET processes.
On the other hand, the 1/1-scaffold amino acid 1 is most rigid
among all other scaffolds which expectedly would bring more rigidity
on to the peptide arms. That means the two terminal chromophores
would remain in a fixed and rigid distant away compared to other cases.
This rigidity brought two donor/acceptor chromophores farthest away
and expectedly would result in lowest extent of FRET in peptide 5 (SI,
Fig. S12b). Conversely, 2/1-scaffold 2 containing peptide 6 and 1/2-
scaffold 3 containing peptide 7 are the two alternates in term of flex-
ibility of N-terminus and C-terminus arms, respectively, as well as in
terms of closeness of two terminal chromophores. From the CD spec-
trum and VT-NMR study it was clear that the peptide 6 is unique in
respect of conformational adaptation and intramolecular H-bonding
interaction through scaffold-NH units (SI, Section 5). The more flexible
nature of peptide 8 compared to peptide 7 and 5 was also reflected from
an intense ICD band. Moreover, the opposite nature of the peptide 6
compared to other three peptides was also reflected in the ICD bands
(Fig. 2b). Therefore, we expected that the CD spectral behaviour and
the flexibility would be reflected in their corresponding FRET effi-
ciency. The 2-C spacer length in the N-terminus led peptide 6 com-
paratively more flexible than peptide 7 with 1-C spacer length as was
evident from more intramolecular H-bonding ability of scaffold-NH in
peptide 6. Therefore, we would expect that the terminal donor-acceptor
chromophores would be more close in peptide 6 than in peptide 7
which ultimately would lead to more FRET efficiency in peptide 6
[4–6].
5. Experimental section
5.1. General experimental
All reactions were carried out under inert atmosphere using flame-
dried glassware. Combined organic layers were dried over anhydrous
sodium sulfate. After work up solvents were removed in a rotary eva-
porator under reduced pressure. For column chromatography Silica gel
(60-120 mesh) was used. Reactions were monitored by TLC on silica gel
60 F254 (0.25).
1H NMR spectra were recorded either at 400 MHz or at 600 MHz
and 13C NMR spectra were recorded either at 100 MHz or at 150 MHz
(mentioned accordingly). Coupling constants (J value) were reported in
hertz (Hz). The chemical shifts were shown in ppm downfield form
tetramethylsilane, using residual chloroform (δ = 7.26 in 1H NMR,
δ = 77.23 in 13C NMR), DMSO (δ = 2.5 in 1H NMR, δ = 39.5 in 13C
NMR), as an internal standard. Mass spectra were recorded with a HR
mass spectrometer and data analyzed by using built-in software. IR
spectra were recorded in KBr on a FT-IR spectrometer. All 2D NMR
Experiments were carried out on 600 MHz spectrometer at room tem-
perature using 7–10 mM concentration in d6-DMSO solvent. Spectra
were acquired with 2048 × 256 in both dimension (F2 and F1) and
other parameter are given below. TOCSY : Free induction decay (FID)
with NS = 16 and DS = 32, relaxation delay (D1) 2 s, mixing time (D9)
0.08 s, acquisition time (AQ) 0.085 s, spectral width 12,019 Hz. ROESY
: Free induction decay (FID) with NS = 16 and DS = 16, relaxation
delay (D1) 2 s, mixing time (P15) 0.02 s, acquisition time (AQ) 0.085 s,
The calculated FRET efficiencies in all the peptides exactly followed
the above explanation establishing our concept of distance dependent
FRET and the logic of design. The FRET efficiency was found to be
highest for highest flexible pentapeptide 8 (96%), lowest for lowest
flexible pentapeptide 5 (85%) and moderate for moderately flexible
pentapeptide 6 (95%) and pentapeptide 7 (94%) (SI, Fig. S12). The
results clearly suggested that the increased flexibility in terms of spacer
length in the N-terminus has a more gross effect in enhancing the FRET
efficiency compared to the C-terminus. This may be due to the increase
of flexibility of the molecular scaffold to help getting turn in such a way
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spectral width (SWH) 12,019 Hz. NOESY : Free induction decay (FID)
with NS = 8 and DS = 16, relaxation delay (D1) 2 s, mixing time (D8)
0.6 s, acquisition time (AQ) 0.085 s, spectral width (SWH) 12,019 Hz.
VT-NMR: The presence of intramolecular H-bonds in the peptides was
assessed through the variable temperature (VT) NMR by determining
the variation of chemical shifts of the various -NHs with temperature
(20 °C–75 °C) in d6-DMSO solvent.
3–4 h. The solvent methanol was then evaporated and the crude mass
was dissolved in water and then filtered, filtrate is evaporated to afford
the compound with quantitative yield.
5.2.7. General procedure for the Boc protection of amine group
In a solution of amine compound (1 equivalent) in 1:1 mixture of
1,4 dioxane and water (3 ml each) was added NaOH (2 equivalent)
followed by di-tert-butyl dicarbonate (1.5 equiv.) maintaining the pH
between 7.5–8.5. The reaction mixture was stirred at room temperature
for 20 h. The reaction mixture was washed with ethyl acetate
(2 x 10 ml) and the aqueous phase was treated with dilute HCl to bring
pH 4 in cold condition. Immediately the solution was extracted with
ethyl acetate and the organic layer was washed with water, brine, dried
and evaporated under vacuum to furnish the Boc-protected amine de-
rivative as white solid in pure form.
5.2. Some general procedure for our synthetic scheme of pentapeptides
5.2.1. General procedure for the preparation of azide from bromo
To a solution of bromo compound (1 equiv.) in water/acetone (1:3)
was added NaN3 (1.5 equiv.) and the mixture was heated at 60 °C for
4 h. Then the reaction mixture was diluted with DCM and washed with
water. The organic layer was dried over anhydrous Na2SO4 and con-
centrated in vacuum. The crude material was obtained in quantitative
yield. This can be used for next step without further purification.
5.2.8. General procedure for the Peptide coupling
To a solution N-protected amino acids in 3:1 mixture of dry DCM
and DMF, 1-[3-dimethyl amino propyl]-3-ehtylcarbo-diimide hydro-
chloride (EDC.HCl) (1.2 equivalent) and HOBT (1.2 equivalent) were
added and the reaction mixture was stirred for 1 h at 0 °C. Then the
amine salt of wienreb amide or methyl ester protected corresponding
amino acids or dipeptide (1.1 equivalent) were added followed by
diisopropylethylamine (DIPEA) (2.4 equivalent). The reaction mixture
was stirred for another 18–20 h at 0 °C to room temperature. Then
solvent was dried by rotary evaporator, after which it was partitioned
between EtOAc and aqueous NaHCO3 solution (50 ml each). The or-
ganic layer was washed with brine solution. Pure product was isolated
by column chromatography.
5.2.2. General procedure for [3 + 2] cyclo-addition (Click reaction)
reaction
The prepared azido compound taken in dry THF and degassed for
5 min with nitrogen gas. After adding alkyne (1.1 equivalent) degassing
was continued for the next 5 min. Then 1 mol % powdered CuI was
added. Then 1.2 equivalent DIPEA was added and reaction mixture was
degassed and allowed to proceed for 18–20 h at room temperature.
After total consumption of the starting azide, the reaction mixture was
evaporated completely and work up was done by EtOAc and NH4Cl
solution. The organic layer was washed with brine, dried over
Na2SO4.The title trizolyl compound was separated by column chro-
matography and characterized.
5.2.9. General procedure for the deprotection of the Boc-group
5.2.3. General procedure for the preparation of mesyl derivative from
hydroxyl group
The respective both side protected amino acids and peptides was
dissolved in CH2Cl2 and cooled to 0 °C. TFA (equal amount as the sol-
vent) was added and the solution was allowed to warm to room tem-
perature. The stirring was continuing at room temperature until the
starting material was consumed (monitored by TLC). The reaction
mixture was evaporated in vacuum. The residual TFA was evaporated
by triturating the mixture with dry toluene thrice, evaporated thrice
and dried to afford the product in quantitative yield.
In a dry R.B −OH compound (1 equivalent) was loaded in a dry
CH2Cl2. Mesyl chloride (1.4 equivalent) and triethylamine (1.3
equivalent) were added to the reaction mixture at 0 °C. The reaction
mixture was stirred at 0 °C till the starting material was consumed. After
that the reaction mixture was diluted with DCM and washed with water
and dried over Na2SO4 and then evaporated in vacuum. The pure
compound was then isolated by column chromatography and utilized
immediately for the next step without further characterization.
5.3. Synthesis of aliphatic triazolo amino acid scaffolds of various spacer
lengths (1–4)
5.2.4. General procedure for the deprotection of the methyl ester
To a solution of the respective methyl ester protected peptide in
THF: H2O = 5 : 1, lithium hydroxide (1.5 equivalent) was added at 0 °C.
The reaction mixture was stirred for about 3–4 hour until starting ma-
terial was fully consumed. Reaction was monitored by TLC. After
completion of the reaction, solvent was dried by rotary evaporator.
Then water (4–5 ml) was added to the reaction mixture and cooled to
0 °C. The dilute acetic acid was added to the reaction mixture to adjust
pH- 3–4. The reaction mixture was extracted with EtOAc. The combined
organic layers were dried over Na2SO4. The hydrolyzed compound was
isolated by column chromatography (Si-gel, CHCl3:MeOH = 10:1).
Yield was 90–96%.
5.3.1. Synthesis of 1/1-Scaffold (1)
The synthesis of 1/1-scaffold (1, BocNH-1/1−AlTAA) was carried
out following the Scheme S1 A and our earlier literature protocol and
followed the following steps.
5.3.1.1. Synthesis of ethyl azido acetate (10): [8a,b]. To a solution of
ethyl bromoacetate (9, 1510 mg, 9.04 mmol) in water/acetone (1:3)
was added NaN3 (881.6 mg, 13.56 mmol) and the mixture was heated at
60 °C for 4 h. Then the reaction mixture was diluted with DCM and
washed with water. The organic layer was dried over anhydrous
Na2SO4 and evaporated in vacuum. The crude material was obtained
in quantitative yield. The product 10 is oily liquid. Yield 97%. IR (KBr)
2955, 2103, 1741, 1441, 1372, 1175, 1064 cm−1. The azide was used
for the next step without further purification and characterization.
5.2.5. General procedure for the preparation of azide from mesyl derivative
To a solution of mesyl compound (1 equivalent) in dry DMF was
added NaN3 (1.5 equiv.) and the mixture was stirred at room tem-
perature. Then the reaction mixture was diluted with DCM and washed
with water. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuum. The product was isolated by column chro-
matography and characterized. Yield was 70–75%.
5.3.1.2. Synthesis of ethyl 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)
acetate (12): [8a,b]. This compound is known compound [8a,b]. The
prepared ethyl azido acetate 10 (500 mg, 3.876 mmol) and propargyl
alcohol 11 (0.246 ml, 5.8 mmol) was undergone click reaction to get
the corresponding triazolyl alcohol compound. The title alcohol
compound 12 was isolated by column chromatography (Si-gel,
PE:EA = 1:1) in pure form as amorphous white solid (560 mg, Yield
79%). 1H NMR (CDCl3, 600 MHz) δ 1.26 (3H, t, J = 3.0 Hz), 4.21 (2H,
5.2.6. General procedure for the preparation of amine from azide derivative
To a solution of the azide compound in methanol Pd/C is added and
to it hydrogen gas balloon was set and stirred the reaction mixture for
177

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JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
q, J = 5.4 Hz), 4.74 (2H, d, J = 7.8 Hz), 5.10 (2H, s), 7.63 (1H, s).
13C{1 H} NMR (CDCl3, 150 MHz) δ 13.9, 50.7, 55.7, 62.3, 123.8, 148.2,
166.6. +APCI-MS calcd. for C7H12N3O3 [M+H]+ 186.1, found 186.1.
bring pH 4 in cold condition. Immediately the solution was extracted
with ethyl acetate and the organic layer was washed with water, brine,
dried and evaporated under vacuo to furnish the Boc-protected 2-(4-
(azidomethyl)-1H-1,2,3-triazol-1-yl) acetic acid 1 as white solid in pure
form (265.4 mg, Yield 81%). mp 105–106 °C, 1H NMR (CD3OD,
400 MHz) δ 1.43 (9H, s), 4.31 (2H, s), 5.25 (2H, s), 7.87 (1H, s).
13C{1 H} NMR NMR (CD3OD, 100 MHz) δ 28.9, 36.8, 51.8, 80.6, 125.6,
5.3.1.3. Synthesis of ethyl 2-(4-(((methylsulfonyl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)acetate (13): [8a,b]. This compound is known compound
[8a,b]. Compound 12 (100 mg, 0.54 mmol) was taken in a dry CH2Cl2.
Mesyl chloride (0.06 ml, 0.81 mmol) and triethyl amine (0.113 ml,
0.81 mmol) were added to the reaction mixture at 0 °C. The reaction
mixture was stirred at 0 °C till the starting material was consumed. After
that the reaction mixture was diluted with DCM and washed with water
and dried over Na2SO4 and then evaporated in vacuum. The pure
compound 13 was then isolated by column chromatography (si-gel,
PE:EA = 2:1) as a colourless oil (129 mg, Yield 83%) and utilized
immediately for the next step without further characterization.
147.4, 158.4, 170.0. ‒APCI-MS calcd. for
255.2540, found 255.2255.
C10H15N4O4 [M−H]-
5.3.2. Synthesis of scaffold 2/1-scaffold (2)
The synthesis of 2/1-scaffold (2, BocNH-2/1−AlTAA) was carried
out following the Scheme S1B and followed the following steps.
5.3.2.1. Synthesis of methyl 2-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)
acetate (19). Using general procedure of click reaction, we have
obtained title compound 19 (1.577 g) from methyl azido acetate 17
as colourless oil (1.90 g) [prepared from methyl bromo acetate] and
homopropergyl alcohol 18. product yield = 83%. 1H NMR (CDCl3,
400 MHz) δ 2.95 (2H, t, J = 6.0 Hz), 3.79 (3H, s), 3.92 (2H, bs), 5.14
(2H, s), 7.53 (1H, s). 13C{1 H} NMR (CDCl3, 100 MHz) δ 28.9, 50.8,
53.2, 61.6, 123.3, 146.1, 167.2. +APCI-MS calcd. for C7H12N3O3 [M
+H]+ 186.0873, found 186.0877.
5.3.1.4. Synthesis of ethyl 2-(4-(azidomethyl)-1H-1,2,3-triazol-1-yl)
8a−b
acetate (14): [8a,b]. This compound is known compound.
To a
solution of the mesyl derivative 13 (129 mg, 0.523 mmol) in dry DMF
(5 ml), NaN3 (40.7 mg, 0.627 mmol) was added and stirrer for 18 h at
50 °C. The reaction mixture was partitioned between EtOAc and water
(20 ml each). The organic layer was washed with brine solution, dried
with Na2SO4, filtered and then evaporated. The title compound 14 was
isolated by column chromatography (si-gel, PE:EA = 5:1) in pure form
as white solid (98 mg, Yield 89%). mp 43–44 °C, IR (KBr) 3447, 3138,
3006, 2108, 1739, 1634, 1464, 1407, 1259, 1021, 771 cm-1.1H NMR
(CDCl3, 600 MHz) δ 1.29 (3H, t, J = 6.6 Hz), 4.27 (2H, q, J = 7.2 Hz),
4.51 (2H, s), 5.17 (2H, s), 7.17 (1H, s). 13C{1 H} NMR (CDCl3,
150 MHz) δ 14.0, 45.5, 50.9, 62.5, 124.0, 142.9, 166.2. +APCI-MS
calcd. for C7H10N6O2 [M+H]+ 211.1, found 211.1.
Following the general procedure for the synthesis of mesylate from
alcohol, we have obtained the title compound 20 (1.215 g) in 81% yield
from the compound 19 (1.5 g). The mesylate was utilized for the next
step without further purification or characterization.
Using general procedure for the synthesis of azide from mesylate,
we have obtained the title azide 21 (1.032 g) from the mesylate 20
(1.2 g) as pale yellow gummy material in 86% yield. 1H NMR (CDCl3,
400 MHz) δ 3.03 (2H, t, J = 6.8 Hz), 3.64 (2H, t, J = 6.4 Hz), 3.81 (3H,
s), 5.17 (2H, s), 7.56 (1H, s). 13C{1 H} NMR (CDCl3, 100 MHz) δ 25.7,
50.6, 50.8, 53.1, 123.4, 144.8, 166.9. +APCI-MS calcd. for
C7H11N6O2[M+H]+ 211.0938, found 211.0943
5.3.1.5. Synthesis of 2-(4-(azidomethyl)-1H-1,2,3-triazol-1-yl)acetic acid
(15): [8a,b]. This compound is known compound. 8a−b To a solution of
compound 14 in methanol, 2.5 (M) NaOH solution was added and
stirred for about 2–3 h at room temperature until starting material was
vanished. After that in the reaction mixture, dilute HCl was added until
the pH became 4. Then it was partitioned between EtOAc and water
(20 ml each). The organic layer was washed with brine solution, dried
with Na2SO4, filtered and then evaporated. The prepared title
compound 15 was isolated as an amorphous white solid and used for
next step without further purification (Yield 95%). IR (KBr) 3153, 2101,
1725, 1557, 1447, 1414, 1362, 1333, 1231, 1068, 822 cm-1. 1H NMR
(CD3OD, 400 MHz) δ 4.49 (2H, s), 5.30 (2H, s), 8.06 (1H, s). 13C{1 H}
NMR (CD3OD, 100 MHz) δ 46.1, 51.8, 126.6, 144.0, 169.9. ‒APCI-MS
calcd. for C5H5N6O2 [M−H]- 181.1, found 181.2.
Using general procedure of methyl ester hydrolysis reaction, we
have obtained the title compound 22 (0.94 g) from methyl 2-(4-(2-
azidoethyl)-1H-1,2,3-triazol-1-yl)acetate (21, 1.0 g) as an amorphous
white solid with yield of 94%. 1H NMR (CD3OD, 600 MHz) δ 3.02 (2H,
t, J = 7.2 Hz), 3.63 (2H, t, J = 6.4 Hz), 5.16 (2H, s), 7.68 (1H, s).
13C{1 H} NMR (CDCl3, 150 MHz) δ 25.3, 50.3, 50.8, 123.7, 144.4,
168.2. +APCI-MS calcd. for C6H7N6O2 [M+H]+ 195.0636, found
195.0633.
Using general procedure for the conversion of azide to amine, we
have obtained free triazolyl amino acid scaffold 23 (0.855 g) from the
azide 22 (0.9 g) as white solid with a yield of 95%. 1H NMR (D2O,
600 MHz) δ 3.13 (2H, t, J = 6.6 Hz), 3.35 (2H, t, J = 6.6 Hz), 5.04 (2H,
s), 7.88 (1H, s). 13C{1 H} NMR (CDCl3, 100 MHz) δ 25.5, 41.7, 55.8,
127.9, 145.6, 175.9. Without further characterization the free amino
acid was utilized for the next step.
5.3.1.6. Synthesis of 2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)acetic acid
(16): [8a,b]. This compound is known compound. 8a−b To a solution of
the compound 15 in methanol (200 mg, 1.1 mmol) Pd/C is added and to
it hydrogen gas balloon was set and stir the reaction mixture for 3–4 h.
The solvent methanol was then evaporated and the crude mass was
dissolved in water and then filtered, filtrate is evaporated to afford the
title compound 16 as a white solid with quantitative yield. mp
285–286 °C, IR (KBr) 3418, 3153, 1614, 1393, 1308, 1233 cm-1. 1H
NMR (D2O, 600 MHz) δ 4.34 (2H, s), 5.06 (2H, s), 8.07 (1H, s).
13C{1 H} NMR (D2O, 150 MHz) δ 34.0, 53.2, 126.3, 139.6, 173.1.
+APCI-MS calcd. for C5H9N4O2 [M+H]+ 157.0727, found 157.0713.
5.3.2.2. Synthesis of scaffold 2/1-scaffold (2). Using general procedure
for Boc protection, we have obtained title compound 2 (0.696 g) from
amino acid 23 (0.8 g) as white solid with a yield of 87%. 1H NMR
(CDCl3, 400 MHz) δ 1.43 (9H, s), 2.91 (2H, t, J = 6.4 Hz), 3.40 (2H, q,
J = 8 Hz, J = 14 Hz), 5.14 (2H, s), 7.60 (1H, s). 13C{1 H} NMR (CDCl3,
100 MHz) δ 29.8, 32.1, 43.6, 54.6, 83.4, 127.4, 149.4, 160.5, 172.2.
C6H7N6O2 calcd. for C11H17N4O4 [M+H]+ 269.1255, found 269.1251.
5.3.1.7. Synthesis of 1/1AlTAA (1): [8a,b]. This compound is known
compound [8a,b]. In a solution of 2-(4-(azidomethyl)-1H-1,2,3-triazol-
1-yl)acetic acid 16 (200 mg, 1.28 mmol) in 1:1 mixture of 1,4 dioxane
and water (3 ml each) was added NaOH (102.6 mg, 2.56 mmol)
followed by di-t-butyl dicarbonate (0.44 ml, 1.92 mmol) maintaining
the pH between 7.5–8.5. The reaction mixture was stirred at room
temperature for 20 h. The reaction mixture was washed with ethyl
acetate (2 x 10 ml) and the aqueous phase was treated with dil. HCl to
5.3.3. Synthesis of scaffold 1/2-scaffold (3)
The synthesis of 1/2-scaffold (3, BocNH-2/1−AlTAA) was carried
out following the Scheme S2A and followed the following steps.
5.3.3.1. Synthesis of methyl 3-azidopropanoate (25). Using general
procedure for the conversion of bromo to azide, we have obtained the
title compound 25 (1.96 g) from methyl 3-bromopropanoate 24 (2.0 g)
as colourless oil. Product yield = 98%. IR (KBr) 2956, 2104, 1743
178

S.S. Bag and A. Yashmeen
JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
cm−1.The azide was used for the next step without further purification
and characterization.
Product yield = 79%. 1H NMR (CDCl3, 400 MHz) δ 2.86–2.93 (4H,
m), 3.19–3.28 (1H, bs), 3.66 (3H, s), 3.86–3.87 (2H, bd, J = 4 Hz),
4.55–4.60 (2H, q, J = 6.0 Hz, 11.2 Hz), 7.47 (1H, s). 13C{1 H} NMR
5.3.3.2. Synthesis of methyl 3-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)
propanoate (26). Using general procedure for click reaction, we have
obtained the title compound 26 (1.539 g) from methyl 3-
azidopropanoate (25, 1.90 g) as white solid. Yield = 81%. 1H NMR
(CDCl3, 400 MHz) δ 2.91 (2H, t, J = 6.4 Hz), 3.63 (3H, s), 4.36–4.37
(1H, d), 4.58 (2H, t, J = 6.4 Hz), 4.67–4.68 (2H, d, J = 5.6 Hz), 7.63
(1H, s). 13C{1 H} NMR (CDCl3, 100 MHz) δ 34.1, 45.4, 51.9, 55.5,
122.8, 147.7, 170.9. + APCI MS calcd. for C7H12N3O3 [M+H]+
186.0873, found 186.0872.
(CDCl3, 100 MHz) δ 28.8, 34.5, 45.5, 52.3, 61.5, 122.6, 145.5,
171.2. + APCI MS calcd. for C8H14N3O3 [M+H]+ 200.1029, found
200.1031.
5.3.4.2. Synthesis of methyl 3-(4-(2-((methylsulfonyl)oxy)ethyl)-1H-
1,2,3-triazol-1-yl)propanoate (32). Using general procedure for the
conversion of alcohol to mesylate, we have obtained the title
compound 32 (1.19 g) as colourless gummy material from methyl 3-
(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl) propanoate (31, 1.4 g).
Product yield = 85%. The mesylate was used for the next step
without further purification and characterization.
5.3.3.3. Synthesis of methyl 3-(4-(((methylsulfonyl)oxy)methyl)-1H-
1,2,3-triazol-1-yl)propanoate (27). Using general procedure for the
conversion of alcohol to mesylate derivative, we have obtained the
title compound 27 (1.215 g) from compound 26 (1.5 g) as yellow
gummy material. Yield = 81%. The mesylate was used for the next step
without further purification and characterization.
5.3.4.3. Synthesis of methyl 3-(4-(2-azidoethyl)-1H-1,2,3-triazol-1-yl)
propanoate (33). Using general procedure for the conversion of
mesylate to azide derivative, we have obtained the title compound 33
(0.902 g) from methyl 3-(4-(2-((methylsulfonyl) oxy)ethyl)-1H-1,2,3-
triazol-1-yl)propanoate (32, 1.1 g) as colourless gummy material.
Product yield = 82%. 1H NMR (CDCl3, 400 MHz) δ 2.87–2.91 (4H,
m), 3.52 (2H, t, J = 7.2 Hz), 3.66 (3H, s), 4.55 (2H, t, J = 6.8 Hz), 7.47
(1H, s). 13C{1 H} NMR (CDCl3, 100 MHz) δ 25.6, 34.4, 45.4, 50.5, 52.1,
122.6, 144.0, 170.9. + APCI MS calcd. for C8H13N6O2[M+H]+
225.1094, found 225.1096.
5.3.3.4. Synthesis of methyl 3-(4-(azidomethyl)-1H-1,2,3-triazol-1-yl)
propanoate (28). Using general procedure for the conversion of
mesylate to azide derivative, we have obtained the title compound 28
(1.02 g) from compound mesylate 27 (1.2 g) as white solid.
Yield = 85%. 1H NMR (CDCl3, 400 MHz) δ 2.899 (2H, t, J = 6.0 Hz),
3.59 (3H, s), 4.37 (2H, s), 4.58 (2H, t, J = 6.4 Hz), 7.66 (1H, s).
13C{1 H} NMR (CDCl3, 100 MHz) δ 34.2, 45.4, 45.6, 52.1, 123.4, 142.2,
170.9. + APCI MS calcd. for C7H11N6O2[M+H]+ 211.0938, found
211.0938.
5.3.4.4. Synthesis of 3-(4-(2-azidoethyl)-1H-1,2,3-triazol-1-yl)propanoic
acid (34). Using general procedure for the methyl ester hydrolysis,
we have obtained the title compound 34 (0.752 g) from methyl 3-(4-(2-
azidoethyl)-1H-1,2,3-triazol-1-yl) propanoate (33, 0.8 g) as white solid.
Product yield = 94%. 1H NMR (CDCl3, 400 MHz) δ 2.95–3.02 (4H, m),
3.57 (2H, t, J = 6.4 Hz), 4.62 (2H, t, J = 6.8 Hz), 7.55 (1H, s). 13C{1 H}
5.3.3.5. Synthesis of 3-(4-(azidomethyl)-1H-1,2,3-triazol-1-yl)propanoic
acid (29). Using general procedure for methyl ester hydrolysis, we
have obtained the title compound 29 (0.93 g) from methyl 3-(4-
(azidomethyl)-1H-1,2,3-triazol-1-yl)propanoate, 28 (1.0 g) as white
NMR (CDCl3, 100 MHz)
δ 25.6, 34.6, 45.8, 52.1, 123.3, 144.2,
174.2. + APCI MS calcd. for C7H9N6O2[M−H]- 209.0792, found
209.0801.
solid. Yield = 93%. 1H NMR (CDCl3, 400 MHz)
δ 2.99 (2H, t,
J = 6.4 Hz), 4.47 (2H, s), 4.69 (2H, t, J = 6.4 Hz), 7.95 (1H, s).
13C{1 H} NMR (CDCl3, 100 MHz) δ 35.2, 47.3, 48.6, 125.6, 143.9,
173.9. + APCI MS calcd. for C6H7N6O2[M+H]− 195.0636, found
195.0618.
5.3.4.5. Synthesis of 3-(4-(2-aminoethyl)-1H-1,2,3-triazol-1-yl)propanoic
acid (35). Using general procedure for the conversion of azide to
amine, we have obtained the title compound 35 (0.644 g) from3-(4-(2-
azidoethyl)-1H-1,2,3-triazol-1-yl)propanoic acid (34, 0.7 g) as white
solid. Product yield = 92%. 1H NMR (D2O, 400 MHz) δ 2.79 (4H, m),
3.11 (2H, t, J = 6.8 Hz), 3.34 (2H, t, J = 7.2 Hz), 7.88 (1H, s). 13C{1 H}
NMR (CDCl3, 100 MHz) δ 22.9, 37.6, 47.7, 57.9, 124.1, 142.9, 178.6.
The acid was used for the next step without further purification and
characterization.
5.3.3.6. Synthesis of 3-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)propanoic
acid (30). Using general procedure for the conversion of azide to
amine, we have obtained the title compound 30 (0.855 g) from azido
acid 29 (0.9 g) as white solid. Yield = 95%. 1H NMR (CDCl3, 600 MHz)
δ 2.81 (2H, t, J = 6.6 Hz), 4.33 (2H, s), 4.67 (2H, t, J = 6.6 Hz), 8.08
(1H, s). 13C{1 H} NMR (CDCl3, 150 MHz) δ 33.9, 37.4, 47.7, 125.1,
139.4, 178.4. The acid was used for the next step without further
purification and characterization.
5.3.4.6. Synthesis of 2/2AlTAA (4). Using general procedure for Boc
protection, we have obtained the title compound 4 (0.474 g) from 3-(4-
(2-aminoethyl)-1H-1,2,3-triazol-1-yl) propanoic acid (35, 0.6 g) as
white solid. Product yield = 79%. 1H NMR (CDCl3, 600 MHz) δ 1.41
(9H, s), 2.88 (2H, t, J = 6.5 Hz), 2.98 (2H, t, J = 5.8 Hz), 3.42 (2H, s),
4.63 (2H, t, J = 6.3 Hz), 5.14 (1H, s), 7.53 (1H, s). 13C{1 H} NMR
(CDCl3, 150 MHz) δ 26.2. 28.6, 34.7, 40.1, 45.9, 79.8, 122.9, 145.4,
156.5, 173.4. -APCI MS calcd. for C11H17N4O4 [M−H]− 269.1255,
found 269.1261.
5.3.3.7. Synthesis of 2/1AlTAA (3). Using general procedure for Boc
protection, we have obtained the title compound 3 (0.608 g) from 3-(4-
(aminomethyl)-1H-1,2,3-triazol-1-yl)propanoic acid (30, 0.80 g) as
white solid. Yield = 76%. 1H NMR (CDCl3, 600 MHz) δ 1.40 (9H, s),
2.96 (2H, t, J = 6.6 Hz), 4.34 (2H, d, J = 5.4 Hz), 4.67 (2H, t,
J = 5.4 Hz), 7.69 (1H, s). 13C{1 H} NMR (CDCl3, 150 MHz) δ 28.4,
34.6, 35.8, 46.0, 80.2, 123.4, 145.3, 156.4, 173.4. + APCI MS calcd. for
C
11H17N4O4 [M+H]+ 269.1255, found 269.1247.
5.3.5. Synthesis of the peptides (5–8)
Peptides 5–6 were synthesized following Scheme 3A-B and peptides
7–8 were synthesized following Scheme 4A-B.
5.3.4. Synthesis of scaffold 2/2-scaffold (4)
The synthesis of 2/2-scaffold (4, BocNH-2/2−AlTAA) was carried
out following the Scheme S2B and followed the following steps.
5.3.5.1. Synthesis of BocNH‒Leu-TPyAlaDo−CONMe(OMe) (38). This
8a−b
compound is known compound.
Using the general procedure of
5.3.4.1. Synthesis of methyl 3-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)
propanoate (31). Using general procedure for click reaction, we have
obtained the title compound 31 (1.501 g) as white solid from methyl 3-
azidopropanoate 25 (1.90 g) and the homopropargyl alcohol 18.
[3 + 2]cyclo-addition reaction, starting from 200 mg (0.52 mmol) of
8a-b
azide derivative of dipeptide 36
and 140 mg (0.62 mmol) of 1-
ethynyl pyrene 37, 247 mg (0.40 mmol) of the title compound 38 was
isolated as a light brown solid material (Si-gel, PE : EtOAc = 1:1). Yield
179

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JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
78%, IR (KBr) 3450, 2958, 2928, 2102, 1653, 1509, 1390, 1167, 1049,
848 cm-1. 1H NMR (CDCl3, 600 MHz), δ 0.89 (6H, d, J = 5.4 Hz), 1.38
(9H, s), 1.52−1.48 (1H, m), 1.67−1.59 (2H, m), 3.29 (3H, s), 3.7 (3H,
s), 4.11 (1H, bs), 4.89 (1H, dd, J = 4.2 Hz, 9.6 Hz), 4.95 (1H, d,
J = 6.6 Hz), 5.05 (1H, bs), 5.36 (1H, bs), 7.22 (1H, d, J = 5.4 Hz), 7.99
(1H, t, J = 7.2 Hz), 8.06 (2H, d, J = 4.8 Hz), 8.09 (1H, t, J = 9.0 Hz),
8.18−8.15 (3H, m), 8.30 (1H, s), 8.31 (1H, s), 8.79 (1H, d, J = 9.0 Hz).
13C{1 H} NMR (CDCl3, 150 MHz), δ 21.9, 23.1, 24.9, 28.4, 32.9, 41.1,
50.4, 50.7, 53.9, 62.1, 80.4, 124.8, 124.9, 125.0, 150.2, 125.3, 125.4,
126.1, 127.4, 127.5,127.8, 128.2, 128.7, 131.1, 131.4, 131.5, 147.8,
δ 21.4, 21.7, 22.1, 22.7, 22.9, 23.1, 23.2, 24.1, 24.2, 24.3, 28.0, 29.0,
43.4, 45.9, 49.5, 49.7, 51.4, 51.5, 51.7, 55.0, 55.3, 79.2, 123.3, 123.3,
123.9, 124.3, 125.0, 125.1, 125.4, 125.9, 126.3, 126.5, 126.8, 127.4,
127.5, 127.5, 127.6, 127.8, 127.9, 128.0, 128.7, 129.6, 130.4, 130.6,
130.6, 130.9, 132.1, 133.2, 133.9, 141.3, 141.4, 145.8, 146.0, 155.1,
165.4, 167.7, 168.8, 175.3, 177.2. + APCI MS calcd. for C61H72N15O9
[M+H]+ 1158.5632, found 1158.5646.
155.9, 168.2, 173.2. + APCI MS calcd for
613.3133, found 613.3132.
C
34H41N6O5 [M+H]+
5.3.5.2. Synthesis of TFA salt of BocNH‒Leu-TPyAlaDo−CONMe(OMe)
(39). Using the general procedure of Boc-deprotection, the compound
38 (240 mg, 0.40 mmol) was deprotected and the product 39 was
obtained in quantitative yield as light brown solid and were used
without further purification and characterization.
5.3.5.6. Synthesis of BocNH‒1/2AlTAA‒Leu‒TPyAlaDo−CONMe(OMe)
(43). Following the similar procedure of DMAP mediated peptide
coupling reaction, we have obtained tripeptide 43 from N-protected
aliphatic amino acid scaffold 2 (160 mg, 0.625 mmol) as light brown
solid compound. Yield 71%. 1H NMR (CDCl3, 600 MHz) δ 0.82 – 0.91
(6H, m), 1.37 (9H, s), 1.49–1.63 (3H, m), 2.59 (1H, s), 2.87 (1H, s),
3.28 (3H, s), 3.33–3.43 (3H, m), 3.50–3.61 (1H, m), 3.81 (3H, s),
4.42–4.50 (1H, m), 4.52–4.62 (1H, m), 4.88 (1H, s), 4.98 (2H, s), 5.04
(1H, m), 5.09–5.18 (1H, m), 5.42 (1H, s), 7.04 (3H, bs), 7.55 (2H, s),
7.97–8.12 (5H, m), 8.15–8.24 (4H, m), 8.64 (1H, d, J = 9.2 Hz).
13C{1 H} NMR (CDCl3, 150 MHz) δ 21.9, 22.9, 23.0, 24.9, 24.9, 26.2,
26.4, 28.6, 32.5, 32.8, 39.7, 40.1, 41.0, 49.8, 50.5, 51.9, 52.5, 52.8,
61.9, 62.2, 66.0, 79.3, 123.5, 123.6, 124.7, 124.7, 124.8, 124.9, 125.0,
125.1, 125.3, 125.4, 125.7, 125.8, 126.4, 126.5, 127.4, 127.5, 128.1,
128.5, 128.7, 129.9, 131.0, 131.5, 147.5, 151.6, 155.9, 165.8, 166.2,
171.8. + APCI MS calcd. for C40H49N10O6 [M+H]+ 765.3831, found
765.3839.
5.3.5.3. Synthesis of BocNH‒1/1AlTAA‒Leu-TPyAlaDo−CONMe(OMe)
(40). To a solution of N-protected aliphatic amino acid scaffold (1,
160 mg, 0.625 mmol) in dry DMF, (EDC.HCl) (178 mg, 0.937 mmol),
followed by DMAP (288 mg, 1.87 mmol) were added at 0 °C. Next, the
amine salt of Weinreb amide, the dipeptide 39 (390 mg, 0.625 mmol)
was added and the reaction mixture was stirred for 30 min at 0 °C and
then 18 h at room temperature. After completion of the reaction
(monitored by TLC) the work up was done with EtOAc and water.
The organic layer was washed with brine solution. The product
tripeptide 40 (342 mg, 0.456 mmol) was isolated in pure form by
column chromatography (Si-gel, EtOAc) as light brown solid
compound. Yield 73%. 1HNMR (CDCl3, 600 MHz) δ 0.81 – 0.91 (6H,
m), 1.37 (9H, s), 1.49–1.63 (3H, m), 3.27 (3H, s), 3.80 (3H, s), 4.22
(2H, s), 4.44–4.53 (1H, m), 4.87 (2H, s), 4.97 (1H, d, J = 8.1 Hz),
5.01–5.07 (1H, m), 5.08–5.14 (1H, m), 5.20 (1H, s), 5.41 (1H, d,
J = 6.3 Hz), 7.51 (1H, bs), 7.56 (1H, s), 8.00–8.10 (m, 5 H), 8.14–8.23
(4H, m), 8.64 (1H, d, J = 8.9 Hz). 13C{1 H} NMR (CDCl3, 100 MHz) δ
21.9, 22.9, 24.9, 29.9, 32.8, 36.3, 40.2, 40.3, 43.4, 50.5, 51.9, 52.5,
52.6, 52.7, 54.8, 61.9, 62.1, 62.3, 79.7, 113.9, 113.9, 116.3, 116.8,
123.1, 123.9, 123.9, 124.0, 124.2, 124.6, 124.7, 124.8, 124.9, 125.0,
125.1, 125.3, 125.4, 125.6, 125.7, 125.8, 125.8, 126.3, 126.4, 127.3,
127.5, 128.1, 128.4, 128.5, 128.5, 128.6, 128.7, 128.9, 131.0, 131.4,
131.5, 131.9, 135.0, 135.1, 142.0, 142.3, 147.6, 151.9, 155.9, 161.4,
166.1, 171.9. + APCI MS calcd. for C39H47N10O6 [M+H]+ 751.3675,
found 751.3675.
5.3.5.7. Synthesis of TFA salt of BocNH‒1/2AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (44). Using the general procedure of Boc-
deprotection, compound 43 (300 mg, 0.39 mmol) was reacted with
TFA to get the product 44 as brown solid material in quantitative (96%)
yield and was used for the next step without further purification and
characterization.
5.3.5.8. Synthesis of BocNH‒TMnapAlaDo‒Leu-1/2AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (6). Using general procedure of peptide coupling
reaction, the acid compound 42 (200 mg, 0.38 mmol) was taken as a
starting material then (296 mg, 0.38 mmol) of amine compound 44 was
reacted with it. After completion of reaction 151 mg (0.129 mmol)
5.3.5.4. Synthesis of TFA salt of BocNH‒1/1AlTAA‒Leu-TPyAlaDo−CONMe
(OMe) (41). Using the general procedure of Boc-deprotection,
compound 40 (300 mg, 0.4 mmol) was reacted with TFA to get the
product 41 in quantitative (95%) yield as brown solid material and was
used for the next step without further purification and characterization.
of the title compound
6
was isolated in pure form by column
EtOAc = 1:4) as light brown solid
chromatography (Si-gel, PE
:
material. Yield 34%. IR (KBr) 3308, 2924, 2105, 1680, 1657, 1549,
1465, 1246, 1163, 1024, 849 cm−1. 1H NMR (CDCl3, 600 MHz) δ 0.82 –
0.91 (12H, m), 1.29 (9H, s), 1.39–1.51 (6H, m), 3.16 (3H, s), 3.41 (3H,
s), 3.74 (2H, d, J = 22.0 Hz), 3.87 (3H, s), 4.37 (2H, s), 4.53 (1H, s),
4.55–4.63 (1H, m), 4.72–4.79 (2H, m), 4.85 (2H, d, J = 13.3 Hz), 4.98
(1H, s), 5.03 (1H, d, J = 3.4 Hz), 5.09 (2H, d, J = 8.8 Hz), 5.37 (1H, s),
7.18 (1H, d, J = 7.9 Hz), 7.83–7.92 (2H, m), 8.07–8.14 (2H, m), 8.24
(5H, dd, J = 7.9, 5.4 Hz), 8.30–8.36 (6H, m), 8.39 (1H, d, J = 7.9 Hz),
8.44 (1H, s), 8.48 (1H, s), 8.53 (1H, s), 8.68 (1H, s), 8.82 (1H, d,
J = 9.3 Hz). 13C{1 H} NMR (CDCl3, 150 MHz) δ 21.5, 21.6, 21.7, 22.7,
22.9, 23.1, 23.1, 23.2, 24.1, 28.0, 28.1, 49.4, 49.8, 51.4, 51.7, 52.4,
55.1, 55.5, 78.4, 123.9, 124.3, 124.4, 124.9, 125.2, 126.3, 126.5,
126.6, 126.7, 127.0, 127.1, 127.4, 127.6, 127.7, 127.8, 127.9, 128.0,
128.1, 128.3, 128.5, 130.4, 130.6, 130.7, 130.7, 130.9, 131.1, 133.3,
145.8, 146.0, 148.8, 149.4, 154.8, 165.2, 167.8, 170.9, 172.0. + APCI
MS calcd.for C62H74N15O9 [M+H]+ 1172.5788, found 1172.5771.
5.3.5.5. Synthesis of BocNH‒TMnapAlaDo‒Leu‒1/1AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (5). Using general procedure of peptide coupling,
the acid compound 42 (200 mg, 0.38 mmol)8a,b was taken as starting
material and amine compound 41 (290 mg, 0.38 mmol) was reacted
with it. After completion of reaction, 162 mg (0.141 mmol) of the title
compound 5 was isolated in pure form by column chromatography (Si-
gel, PE : EtOAc = 1:4) as light brown solid material. Yield 37%. IR
(KBr) 3423, 3310, 2925, 2106, 1673, 1557, 1456, 1432, 1163, 1027,
849 cm−1. 1H NMR (CDCl3, 600 MHz) δ 0.48 – 0.72 (12H, m), 1.20 (9H,
s), 1.53–1.61 (6H, m), 3.14 (3H, s), 3.31 (3H, s), 3.86 (3H, s), 4.30 (4H,
dd, J = 26.2, 11.2 Hz), 4.48–4.58 (2H, m), 4.75 (2H, dd, J = 13.9,
3.8 Hz), 4.83 (2H, d, J = 9.2 Hz), 4.97–5.04 (1H, m), 5.07–5.16 (2H,
m), 7.85 (2H, d, J = 8.6 Hz), 8.05–8.13 (2H, m), 8.23 (5H, ddd,
J = 15.8, 8.8, 4.8 Hz), 8.27–8.38 (7H, m), 8.47 (2H, s), 8.52 (1H, s),
8.70 (1H, s), 8.79 (2H, t, J = 9.2 Hz). 13C{1 H} NMR (CDCl3, 150 MHz)
180

S.S. Bag and A. Yashmeen
JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
compound. Yield 65%. 1H NMR (CDCl3, 600 MHz) δ 0.79 - 0.92 (6H, m),
1.38 (9H, s), 1.48–1.66 (3H, m), 2.31 (2H, d, J = 15.7 Hz), 2.68 (1H, dt,
J = 31.1, 12.2 Hz), 2.92 - 2.74 (1H, m), 3.20 (1H, s), 3.26 (3H, s),
3.50–3.64 (1H, m), 3.72 (1H, d, J = 1.5 Hz), 3.80 (3H, s), 4.20 (2H, d,
J = 5.5 Hz), 4.33 (1H, dd, J = 15.2, 7.6 Hz), 4.42 (1H, s), 4.57 (1H, dd,
J = 8.3, 4.2 Hz), 4.88 (2H, s), 5.42 (1H, d, J = 5.2 Hz), 7.04 (1H, d,
J = 20.9 Hz), 7.22 (1H, s), 7.49 (1H, s), 7.93–8.09 (5H, m), 8.11–8.20
(4H, m), 8.67 (1H, dd, J = 24.4, 9.0 Hz). 13C{1 H} NMR (CDCl3,
150 MHz) δ 21.8, 22.0, 23.0, 23.1, 24.8, 24.9, 28.6, 29.9, 32.8, 40.4,
41.5, 46.0, 50.6, 51.9, 52.3, 62.2, 66.0, 79.7, 124.7, 124.8, 124.9, 125.0,
125.1, 125.3, 125.5, 125.6, 126.2, 126.3, 127.4, 127.5, 127.9, 128.0,
128.3, 128.4, 128.7, 130.9, 131.0, 131.4, 131.4, 131.5, 131.5, 131.5,
132.1, 145.2, 147.5, 155.9, 168.4, 169.9, 172.6. + APCI MS calcd. for
5.3.5.9. Synthesis of BocNH‒2/1AlTAA‒Leu‒TPyAlaDo−CONMe(OMe)
(45). Following the similar procedure of DMAP mediated peptide
coupling we have obtained tripeptide 45 from N-protected aliphatic
amino acid scaffold 3 (160 mg, 0.625 mmol) as yellowish brown
gummy compound. Yield 67%. 1H NMR (CDCl3, 600 MHz) δ 0.84 –
0.94 (6H, m), 1.25 (9H, s), 1.50 (2H, m), 1.59–1.65 (1H, m), 2.69 (2H,
s), 3.31 (3H, s), 3.86 (3H, s), 4.31–4.44 (1H, m), 4.48–4.66 (2H, m),
4.74–4.85 (5H, m), 4.94 (2H, dd, J = 19.7, 9.6 Hz), 5.07 (1H, s), 5.40
(1H, d, J = 2.0 Hz), 5.56 (6H, s), 5.99 (1H, dd, J = 26.0, 7.3 Hz), 6.35
(6H, d, J = 23.5 Hz), 7.32 (1H, d, J = 8.8 Hz), 7.98–8.13 (5H, m),
8.17–8.28 (4H, m), 8.72 (3H, d, J = 9.3 Hz). +APCI MS calcd.for
C
41H51N10O6 [M+H]+ 779.3988, found 779.3991.
5.3.5.13. Synthesis of TFA salt of BocNH‒2/2AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (48). Using the general procedure of Boc-
deprotection, compound 47 (300 mg, 0.39 mmol) was reacted with
TFA to get the product 48 in quantitative (96%) yield and was used for
the next step without further purification and characterization.
C
40H49N10O6 [M+H]+ 765.3831, found 765.3826.
5.3.5.14. Synthesis of BocNH‒TMnapAlaDo‒Leu-2/2AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (8). Using general procedure of peptide coupling,
the acid compound 42 (200 mg, 0.38 mmol) was taken as starting
material then (301 mg, 0.37 mmol) of amine compound 48 was reacted
with it. After completion of reaction 166 mg (0.14 mmol) of the title
compound 8 was isolated in pure form by column chromatography (Si-
gel, PE : EtOAc = 1:4) as light brown amorphous solid material. Yield
37%. IR (KBr) 3296, 2924, 2105, 1648, 1546, 1435, 1171, 1053, 849
5.3.5.10. Synthesis of TFA salt of BocNH‒2/1AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (46). Using the general procedure of Boc-
deprotection, compound 45 (300 mg, 0.39 mmol) was reacted with
TFA to get the product 46 in quantitative (95%) yield and was used for
the next step without further purification and characterization.
cm−1
.
1H NMR (CDCl3, 600 MHz) δ 0.67 – 0.71 (6H, m), 0.83 (6H, m),
5.3.5.11. Synthesis of BocNH‒TMnapAlaDo‒Leu-2/1AlTAA‒Leu‒TPyAlaDo
−CONMe(OMe) (7). Using general procedure of peptide coupling,
the acid compound 42 (200 mg, 0.38 mmol) was taken as a starting
material then (296 mg, 0.38 mmol) of amine compound 45 was reacted
with it. After completion of reaction 138 mg (0.118 mmol) of the title
compound 7 was isolated in pure form by column chromatography (Si-
gel, PE : EtOAc = 1:4) as light brown solid material. Yield 31%. IR
(KBr) 3299, 2957, 2106, 1683, 1660, 1541, 1389, 1163, 1056, 848
1.25–1.36 (3H, m), 1.39–1.51 (3H, m), 1.73 (9H, s), 3.12 (3H, d,
J = 18 Hz), 3.15 (3H, s), 3.47–3.53 (1H, m), 3.70 (2H, s), 3.75 (5H, s),
4.19–4.35 (4H, m), 4.66 (1H, dd, J = 13.8, 7.4 Hz), 4.74 (2H, dd,
J = 13.9, 8.3 Hz,), 4.83 (3H, dt, J = 21.1, 7.7 Hz,), 5.32 (2H, s), 5.49
(1H, s), 7.99–8.04 (1H, m), 8.10 (3H, dt, J = 11.4, 7.6 Hz), 8.16–8.27
(6H, m), 8.27–8.36 (8H, m), 8.39 (1H, d, J = 7.9 Hz), 8.48 (1H, bs),
8.71 (2H, s), 8.82 (1H, d, J = 9.3 Hz). 13C{1 H} NMR (CDCl3, 150 MHz)
δ 20.3, 22.0, 22.1, 22.2, 23.0, 23.1, 23.5, 23.7, 24.7, 24.8, 25.5, 32.7,
39.3, 39.7, 39.9, 40.0, 40.1, 40.3, 40.4, 40.6, 40.7, 41.2, 41.5, 49.3,
50.1, 50.4, 51.3, 51.5, 51.9, 58.7, 62.1, 63.7, 65.6, 87.5, 124.4, 124.4,
124.6, 124.7, 124.9, 125.0, 125.3, 125.5, 125.7, 125.8, 125.9, 126.1,
126.1, 126.2, 126.5, 127.1, 127.4, 127.7, 127.8, 128.0, 128.1, 128.2,
128.3, 128.5, 128.6, 129.2, 131.0, 131.1, 131.2, 131.3, 131.6, 146.6,
154.5, 169.9, 171.3, 173.1, 173.2, 173.3. + APCI MS calcd. for
cm−1
.
1H NMR (CDCl3, 600 MHz) δ 0.82−0.87 (12H, m), 1.68 (9H, s),
1.80–1.87 (6H, m), 3.17 (3H, s), 3.41 (3H, s), 3.76 (4H, s), 3.90 (2H, d,
J = 9.3 Hz), 4.32 (3H, m), 4.68 (1H, dd, J = 13.7, 7.5 Hz), 4.76 (2H,
m), 4.78–4.93 (3H, m), 5.34 (2H, bs), 5.51 (1H, bs), 7.50 (1H, d,
J = 7.9 Hz), 8.02–8.06 (1H, m), 8.13 (3H, dt, J = 14.9, 7.8 Hz),
8.24–8.29 (6H, m), 8.33–8.39 (7H, m), 8.41 (1H, d, J = 7.9 Hz), 8.52
(1H, bs), 8.74 (1H, s), 8.85 (1H, d, J = 9.3 Hz). 13C{1 H} NMR (CDCl3,
150 MHz) δ 22.0, 22.1, 23.0, 23.5, 23.7, 24.7, 24.8, 25.2, 25.3, 28.0,
29.6, 29.7, 32.8, 50.1, 50.4, 51.5, 51.7, 51.9, 53.5, 53.7, 59.5, 62.2,
79.8, 124.4, 124.6, 124.9, 125.3, 125.7, 125.8, 125.9, 126.2, 126.3,
126.6, 127.1, 127.3, 127.7, 127.9, 128.0, 128.2, 128.3, 128.4, 128.6,
128.9, 128.9, 130.3, 131.0, 131.2, 131.4, 131.6, 141.5, 146.5, 150.7,
157.5, 169.5, 169.9, 172.3, 173.1, 173.3. + APCI MS calcd. for
C
63H76N15O9 [M+H]+ 1186.5945, found 1186.5976.
C
62H74N15O9 [M+H]+1172.5788, found 1172.5719.
5.4. Photophysical studies of the synthesized peptides: general procedures
5.4.1. UV–vis measurements
The UV –visible spectra of all final peptides and UNNAs (10 μM)
were measured in different solvents using a UV–vis spectrophotometer
(SHIMADZU, UV-2550) with a cell of 1 cm path length. The mea-
surements were done in absorbance mode. The sample solutions ab-
sorbance values were measured in the wavelength range of
200–700 nm. All the sample solutions were prepared freshly just before
doing the experiment.
5.3.5.12. Synthesis of BocNH‒2/2AlTAA‒Leu-TPyAlaDo−CONMe(OMe)
(47). Following the similar procedure of DMAP mediated peptide
coupling we have obtained tripeptide 47 from N-protected aliphatic
amino acid scaffold 4 (160 mg, 0.625 mmol) as yellowish brown gummy
181

S.S. Bag and A. Yashmeen
JournalofPhotochemistry&PhotobiologyA:Chemistry378(2019)171–183
5.4.2. Fluorescence experiments
5.4.4. Macromodel study
All the sample solutions for fluorescence measurements (HORIBA
Scientific, Fluoromax-4) were also prepared freshly just before doing
the experiment. Fluorescence spectra were obtained using a fluores-
cence spectrophotometer at 25 °C using 1 cm path length cell. The
wavelengths for excitation in all the cases were set at the absorption
maxima of each sample in each solvent. The emission spectra were
measured in the wavelength regime of 300–700 nm with an integration
time of 0.2 s. From 2.0 ml 500 μM stock solution, 2 ml of 10 μM con-
centration of solution was used for fluorescence experiment in 1 ml cell.
Fluorescence emissions were recorded by exciting the sample solutions
at their absorption maxima. Steady-state fluorescence emission spectra
were recorded at room temperature as an average of five scans using an
excitation slit of 3.0 nm, emission slit 3.0 nm, and scan speed of
120 nm/min. Using quinine sulphate as a reference, the fluorescence
quantum yields (Φf) were determined with the known Φf (0.55) in 0.1 M
solution in sulphuric acid. Following equation was used to calculate the
quantum yield,
Next, MD simulations for the pentapeptides were carried out with
Schrodinger Macromodel (Maestro vs. 9.1) software package using an
OPLS 2005 force field. The starting structures for the were the global
minimum conformers.
Acknowledgements
The authors thank DBT, [BT/PR5169/BRB/10/1065/2012 and BT/
PR5169/ BMB/2015/39] Govt. of India., for financial support.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.04.
024.
References
FlSArea
nS2
AbsR
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