Triazolyl-donor-acceptor chromophore-decorated unnatural amino acids and peptides: FRET events in a β-turn conformation

Article abstract of DOI:10.1039/c3cc47488g

The β-turn conformation and FRET process were established in the designed tripeptide containing fluorescent triazolyl donor and acceptor-decorated unnatural amino acids separated by a natural alanine.

Full text of DOI:10.1039/c3cc47488g

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Triazolyl-donor–acceptor chromophore-decorated
unnatural amino acids and peptides: FRET events
in a b-turn conformation†
Cite this: Chem. Commun., 2014,
50, 433
Received 30th September 2013,
Accepted 13th October 2013
Subhendu Sekhar Bag,*a Subhashis Jana,a Afsana Yashmeen,a K. Senthilkumarb and
Raghunath Baga
DOI: 10.1039/c3cc47488g
www.rsc.org/chemcomm
The b-turn conformation and FRET process were established in the (FRET) process.6 FRET, being a distance-dependent phenomenon,
designed tripeptide containing fluorescent triazolyl donor and acceptor- is widely utilized in the elucidation of structures, investigating
decorated unnatural amino acids separated by a natural alanine.
protein dynamics, studying biomolecular interactions and the
conformational distribution of unstructured peptides, along with
many other sensory applications.6 Thus, we were interested in a
b-turn mimetic peptide capable of showing a FRET interaction
between two terminally placed fluorescent unnatural amino acids.
The logic behind our choice of triazole unit was its metabolic
inertness, its tendency to associate with biological targets and its
ability to modulate photophysical properties.7 Thus, the tripeptide
containing fluorescent solvatochromic Do–Ac amino acids adopted
a b-turn conformation wherein we observed the FRET process. To
the best of our knowledge, the concept of a fluorescent unnatural
peptide showing the FRET process in a b-turn conformation is new.
The synthesis of the novel triazolyl amino acids 1–4 was
carried out via a standard click reaction of the N-,C-diprotected
azido serine 5, synthesized via Weinreb’s8 amide strategy from
serine, with the various donor and/or acceptor alkynes A–D
(Fig. 1 and ESI,† Section 2 and 3). The amino acids were char-
acterized by NMR, and mass spectrometry.
In the fast-evolving research area concerned with expanding the
genetic code, several unnatural amino acids (UNAAs) have been
synthesized and incorporated within the framework of specific
proteins, thereby affording novel functionality.1 Among the various
examples of UNAAs, one that has been recently encoded with a
‘chemical warhead’ is of considerable interest.1d However, sensing of
protein’s microenvironment requires a highly sensitive fluorescence
probe, and although reports of such probes exist, these are not
sufficient to comprehensively fulfill research needs.2 Many of the
problems associated with extrinsic fluorophore-labeled proteins could
be solved by the synthesis of an intrinsically fluorescent amino acid
that could be site-specifically incorporated into a protein.3 Only a very
few of such amino acids have been synthesized or encoded geneti-
cally.3 Therefore, the synthesis of unnatural amino acids with novel
photophysical properties is currently an emerging area of research.
As part of our ongoing research efforts regarding the installation
and modulation of the emission response4a and the design of
biomolecular building blocks4b via click chemistry, we disclose
herein the conceptual design and synthesis of triazolyl donor–
acceptor (Do–Ac) unnatural amino acids with new photophysical
properties. We envisioned that incorporation of two such fluores-
cent UNAAs into the two termini of a tripeptide separated by a
natural alanine could result in the formation of a b-turn5 confor-
mation via backbone H-bonding interactions. We also expected
that the hydrophobic, p–p stacking and van der Waals interactions
in the side chain would stabilize the conformation and allow the
two terminal triazolyl aromatics to engage in dipolar photophysical
UV-visible spectra of all the amino acids presented here were
solvatochromic and significantly red-shifted from those of the parent
aromatics (ESI,† Fig. S3–S6). It was also evident from the study of
UV-visible spectra of a 1 : 1 mixture and combined spectra of indi-
vidual amino acids in acetonitrile (ESI,† Fig. S7 and S8) that the two
UNAAs, TPhenAlaDo and TCNBAlaAc (1 and 3), could take part in
stacking interactions when in close proximity. While amino acid 3
(
TCNBAlaAc) exhibited a Stokes shift of 30 nm in its emission
behaviour (lem = 310–360 nm) showing its polarity sensitivity, an
intense emission at around 400 nm was observed for amino acid 1
(
TPhenAlaDo). Increasing the solvent polarity also increased the quan-
¨
interactions, most likely the Forster resonance energy transfer
tum yields of amino acids 1 and 3. However, a decrease in quantum
yield was observed for amino acids 2 (TNDMABAlaDo) and 4 (TNBAlaAc)
which could be due to the polar solvent–solute interaction leading to
quenching of fluorescence via dipolar or H-bonding interactions.6c
We next incorporated two amino acids (TPhenAlaDo and TCNBAlaAc)
into tripeptide sequences (a) TPhenAlaDo–Ala–TCNBAlaAc (6),
(b) TPhenAlaDo–Ala–TPhenAlaDo (7), and (c) TCNBAlaAc–Ala–TCNBAlaAc
a Bio-organic Chemistry Laboratory, Department of Chemistry,
Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India.
E-mail: ssbag75@iitg.ernet.in; Fax: +91-361-258-2349
b Department of Physics, Bharathiar University, Coimbatore – 641 046, India
† Electronic supplementary information (ESI) available: Synthesis, spectroscopic
data, NMR spectra, MacroModel study. See DOI: 10.1039/c3cc47488g
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Fig. 2 (a) CD spectra of peptide 6. Inset: variable temperature CD. (b) Pre-
sentation of various interactions revealed from spectroscopic studies.
The presence of strong intramolecular H-bonding involving the
triazole sp2 C–H (of TCNBAlaAc) and the amide carbonyl (of TPhenAlaDo
)
in the side chain of the peptide was also evident from both the
IR (u = 2973 cmÀ1) and the variable temperature NMR (Dd/DT =
ꢀ
À1.5 ppb KÀ1) spectra, and was further supported by a MacroModel
optimized geometry study (ESI,† Sections 7 and 11).10 Two other amide
N–Hs were also involved in H-bonding interactions, as seen from the
VT-NMR study. The NOESY spectrum of peptide 6 revealed interactions
among the aromatic hydrogens of the TPhen and TCNB units of the
Fig. 1 (I) Schematic presentation of triazolyl UNAAs and peptides. (II) The
alkynes used in this study, along with the structures of the amino acids and
(III) peptides.
(8) following a peptide coupling protocol (Fig. 1, ESI,† Section 2 and two terminal amino acids. It was also evident that the triazolyl C–H of
3). All peptides were characterized by NMR and mass spectrometry. the N-terminal amino acid (TPhenAlaDo) interacted with the methylene
While tripeptide 6 was designed to examine the possibility of H- (–CH2–) proton of the C-terminal amino acid (TCNBAlaAc). Moreover, the
bonding, hydrophobic and/or p-stacking interactions guided for- triazolyl C–H of the C-terminal amino acid (TCNBAlaAc) interacted with
mation and stabilization of b-turns, as well as to investigate the the aromatic hydrogens (of the TPhen unit) of the N-terminal amino
photophysical dipolar interaction between the Do–Ac pair, peptides acid (TPhenAlaDo). Furthermore, tBoc–H and NMe–H of the two terminal
7 (Do–Do) and 8 (Ac–Ac) were designed to compare the results.
amino acids showed NOESY interactions (ESI,† Section 7). All these
The secondary structure of peptide 6 was estimated by recording interactions in the side chain of peptide 6 possibly played a role in
its CD spectrum in acetonitrile and methanol, which showed a stabilizing the b-turn structure and in bringing the two terminal
maximum at B205–212 nm and a sudden switch from a positive to aromatics closer for a dipole–dipole interaction. However, these side
a negative band at 240–266 nm indicating a b-turn conformation chain interactions were weak in case of other two peptides, probably
(Fig. 2a).5c,d Aromatic absorption or a p–p stacking interaction because of the steric repulsion between two bulky phenanthrenes in
between two aromatic moieties of terminal amino acids (TPhenAlaDo peptide 7 and the dipolar repulsion between two TCNB units in
and TCNBAlaAc) was also evident from the appearance of a signature peptide 8. Therefore, they also adopted similar but deformed struc-
negative band at 297–301 nm in all solvents studied. The hypso- tures, which could be seen in the corresponding CD spectra.
chromic shift in wavelength and hypochromism of the bands at 266
Geometry minimization and molecular dynamics calculations
and 301 nm in the variable temperature CD spectrum suggest a using the Merck Molecular Force Field support the presence of a
weakening of the p–p stacking interaction between the triazolyl- b-turn conformation with H-bonding between CO(i)–NH(i + 3)
phenanthrene (TPhen) unit of TPhenAlaDo and the cyanobenzene (ESI,† Section 11).5h,i,10a Therefore, the spectroscopic evidence and
(TCNB) moiety of TCNBAlaAc (Fig. 2a, inset).5e The other two peptides, the modelling study support the b-turn conformation of peptide 6,
7 and 8, also showed similar characteristics (ESI,† Section 7.1).
which primarily derives from backbone H-bonding interactions,
The IR spectrum of peptide 6 showed absorption due to intra- and which is stabilized by other side chain interactions such as
molecular H-bonding and free amide –NH stretching at 3320 and hydrophobic and p–p stacking interactions between the terminal
3450 cmÀ1, respectively.5f,g The ratio of absorption was significantly TPhen and TCNB moieties.
larger in peptide 6 than in peptides 7 and 8, indicating that the
After establishing the b-turn conformation, we next studied the
hydrogen-bonded form of amide –NH was more favourable in possibility of photophysical interactions between the terminal Do–Ac
the case of peptide 6. Thus, the IR spectrum also supported the amino acid pair in peptide 6. The UV-visible and fluorescence spectra
formation of a b-turn type structure.5f,g The presence of strong of the individual donor and acceptor amino acids revealed that the
intramolecular H-bonding involving the carbamate carbonyl at i fluorescence spectrum of TCNBAlaAc overlapped significantly with the
and the amide –NH (of TCNBAlaAc) at i + 3 was observed from the absorption spectrum of TPhenAlaDo (Fig. 3a). Moreover, the peptide (6)
variable temperature NMR experiment (VT-NMR) where the tem- containing these two amino acids can be selectively excited at 272 nm
perature coefficients of the chemical shifts were found to be closer (the maximum absorbance of TCNBAlaAc) where there is very low
to the Kessler limit9, supporting the presence of a b-turn structure absorbance of TPhenAlaDo. Therefore, these two amino acids should
in peptide 6 (Fig. 2b, ESI,† Section 7.4).
form a FRET pair in our designed tripeptide where the conceptual
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by a fluorescence lifetime measurement study (ESI,† Section 6
and 10).6f A very weak FRET emission was observed in phosphate
buffer which could be due to non-radiative deactivation.6c However,
the FRET signal could be optimized upon addition of DMSO to give a
296% enhancement (Fig. 4b). This addition of DMSO possibly fine-
tuned the donor–acceptor interaction to minimize the non-radiative
deactivation process, thereby leading to an increase in intensity.6f
¨
In summary, Forster resonance energy transfer (FRET) was
Fig. 3 (a) Overlap of the emission spectrum of TCNBAlaAc (blue) and the
absorbance spectrum of TPhenAlaDo (red). (b) Fluorescence spectra of
individual amino acids (black for 1 and blue for 3) and the tripeptide 6
(red) containing these two amino acids. The mole fractions of triazolyl
chromophoric units in the monomers and in the tripeptide were the same.
Spectra were recorded in CH3CN with a 10 mM concentration.
established in the conceptually designed novel unnatural b-turn
peptide containing a new class of fluorescent unnatural donor–
acceptor amino acids. The FRET process occurred from the TCNB
moiety of TCNBAlaAc (the FRET donor) to the TPhen unit of TPhenAlaDo
(the FRET acceptor). Our developed FRET pair, TCNBAlaAc–TPhenAlaDo
,
wherein both the partners are unnatural amino acids, is new to the
best of our knowledge. As FRET can provide information about
peptide and protein conformation, our unnatural amino acid
pair (FRET pair) and the FRET peptide could find application in
studying the conformational distribution of unstructured peptides
in solution and in FRET-based bioassays.
This work is dedicated to Professor Amit Basak on the occasion of
his 62nd birthday. Financial support from the Council of Scientific
and Industrial Research [CSIR: 01(2330)/09/EMR-II], New Delhi,
Govt. of India, is gratefully acknowledged.
donor amino acid 1 (TPhenAlaDo) and the acceptor amino acid 3
(
TCNBAlaAc) acted as a FRET acceptor and donor, respectively. With
this observation we turned our attention to study the FRET process.
It was observed that the fluorescence intensity of TPhenAlaDo
increased almost four-fold from that of the monomer emission in
the presence of TCNBAlaAc in peptide 6 when it was excited at the
maximum absorbance of TCNBAlaAc (lmax = 272 nm). On the other
hand, the fluorescence intensity of TCNBAlaAc in the peptide decreased
almost seven to eight times that of the monomer fluorescence. This
ratiometric change in fluorescence intensity provided visual evidence
of the FRET process from TCNBAlaAc to TPhenAlaDo (Fig. 3b).6e The
Notes and references
¨
Forster radius and the efficiency of energy transfer were calculated
and found to be 20 Å and 85%, respectively. The donor–acceptor
distance was found to be r = 15 Å. The time-resolved fluorescence
study revealed a decrease in donor lifetime (TCNBAlaAc; lex = 308 nm,
lem = 340 nm) from 1.9 ns to 1.2 ns in peptide 6, indicating the
occurrence of the FRET process. More interestingly, the acceptor
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TPhenAlaDo) exhibited a biexponential profile (lex = 308 nm, lem
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increases from 13.2 ns (in the absence of the donor) to 14.1 ns (in the
presence of the donor) which is also clearly an indication of FRET.11
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emission from TPhenAlaDo (at 390 nm) decreased slightly (Fig. 4a). This
could be due to the influence of the solvent polarity on the FRET
parameters and association within the TPhen–TCNB pair that led to a
slight decrease in FRET emission intensity, which was also supported
¨
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Fig. 4 (a) Solvent-assisted modulation of FRET-induced fluorescence of pep-
tide 6 (10 mM). (b) Amplification of the FRET signal in phosphate buffer (pH 7.0)
upon addition of increasing % concentration of DMSO (lex = 272 nm).
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Chem. Commun., 2014, 50, 433--435 | 435