Folded short azapeptide for conformation switching-based fluorescence sensing

Article abstract of DOI:10.1039/c7cc06915d

Herein, we report a conformation switching-based fluorescence sensing scheme using dipeptide-based amidothioureas (azapeptides) that contain a folded β-turn structure. Amidothiourea is equipped at its two termini with an electron acceptor and an electron donor or two fluorophores, such that it exhibits enhanced exciplex or excimer emission because of the turn structure in which the two termini are brought into close proximity; on the other hand, it exhibits a dramatic ratiometric fluorescence response upon anion binding to the thiourea moiety because of the resultant extended conformation of the anion binding complex.

Full text of DOI:10.1039/c7cc06915d

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Folded short azapeptide for conformation switching based
fluorescent sensing
Received 00th January 20xx,
Accepted 00th January 20xx
Yuan Yuan, Xiao-Sheng Yan, Xiao-Rui Li, Jin-Lian Cao, Zhao Li* and Yun-Bao Jiang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a conformation switching based fluorescent sensing structure is that within the turn structure the two thioureido -
scheme by using dipeptided based amidothioureas (azapeptides) NHs are in the trans-conformation.3 It was therefore expected
that contain a folded β-turn. The amidothiourea was equipped at that a conformation change in the thiourea moiety will take
its two termini with an electron acceptor and an electron donor or place to bind to anion that leads to an extended structure
two fluorophores, respectively, that it exhibits enhanced exciplex (Scheme 1a), a conformation change similar to that operates in
or excimer emission because of the turn structure in which the the nucleic acid beacons.1 Those folded azapeptides were thus
two termini are brought into close proximity, while it undergoes a assumed to be potential candidates for short peptide based
dramatic ratiometric fluorescent response upon anion binding to beacons. Here we report our proof-of-concept explorations by
the thiourea moiety because of the resultant extended equipping the two termini of the azapeptide backbone with
conformation of the anion binding complex.
one pyrene fluorophore and one dimethylaniline (DMA)
electron donor, or two pyrene fluorophores, respectively
(Scheme 1b). The distance dependent exciplex or excimer
formation5 and their respective dual emission were expected
to undergo changes upon binding to the model anions to the
thiourea moiety designed to locate within the turn, that leads
to a conformation change.
Hairpin structured nucleic acid beacon1 has been well applied
in the detection of nucleic acids, and recently it has been
applied to other species such as metal ions as well. The basic
mechanism for the fluorescent sensing is a conformation
change upon its interacting in the loop part with the nucleic
acid analyte. In principle this could apply to build a peptide
based beacon as well, yet the fact is that not much progress
has been made in this regard,2 probably because that the
peptide-peptide interactions are not of that high affinity and
selectivity as for the nucleic acid counterparts that are govern
by the complimentary nucleic base pairing. We thus attempted
to an alternative strategy by using short peptides designed to
(a)
H
H
N
CH3
O
N
F
R1
R2
∗
O
CH3COO-
∗
O
O
O
O
-turn
β
N
H
N
N
H
N
H
N
O
H
H
N
H
N
N
H
N
H
Q
H
Q
∗
S
N
S
S
H
-turn
γ
O
contain
a structural unit that promotes the folded
F
conformation of the peptides while it is able to bind to the
analyte to lead to an extended structure. As the first attempt,
we came to employ our recently discovered azapeptides, the
dipeptide based amidothioureas, that contain a folded β-turn
structure (Scheme 1a).3 It is noted that in these
amidothioureas the two terminal groups R1 and R2 are indeed
made in close proximity because of the folded structure.
Within the turn structure of our developed azapeptides,
thiourea is a known anion-receptor.4 Noted from the crystal
(b)
Ha
H
N
N
N
∗
∗
∗
O
O
O
O
O
O
(H3C)2
N
(H3C)2
N
N
N
N
Hd
N
Hd
N
H
N
H
Hb
H
N
N
N
H
Hc
H
Hf
He
S
S
S
L
-Phe1/
D
-Phe1
L
-Phe3
L
-Pro1/
D
-Pro1
H
N
R4
N
R3
O
∗
CH3
H
N
O
(H3C)2
N
N
Hd
N
∗
∗
H
O
O
N
(H3C)2
N
H
N
N
H
N
H
N
H
H
S
N
N
Department of Chemistry, College of Chemistry and Chemical Engineering, the MOE
Key Laboratory of Spectrochemical Analysis and Instrumentation, and iChEM,
Xiamen University, Xiamen 361005, China. E-mail: lizhao@xmu.edu.cn,
ybjiang@xmu.edu.cn; Tel: +86 592 218 8372; Fax: +86 592 218 6401
H
H
S
S
L
L
L
-Ala1 (R3 = H, R4 = CH3
)
Leu1
-
(R3 = H, R4 = CH2CH(CH3) )
2
-Gly1 (R3 = H, R4 = H)
L
-Phe2
L
-Phe4
Electronic Supplementary Information (ESI) available: Synthesis, characterization,
X-ray structures and spectra. CCDC 1583614. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/x0xx00000x
Scheme 1 (a) β-Turn structure in dipeptide based N-amidothioureas (azapeptides) and
sensing mechanism of the folded azapeptide. “F” represents the fluorophore and “Q”
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the quencher, while the thiourea moiety highlighted in red is the binding receptor for
analyte, anion here. (b) Molecular structures of -/ -Phe1, -Phe2, -Pro1, -Ala1, -Leu1,
-Gly1, -Phe3 and -Phe4. -Phe2 and -Phe4 are control compounds of -Phe1 and
of
L
-
Phe1 at 10 µM in CH3CN in the presence of acetate anion
L
D
L
L
L
L
of increasing concentration. It was observed that, with
increasing anion concentration, the exciplex emission is
quenched while the monomer emission is enhanced, with an
isoemissive point, suggesting that the formation of exciplex is
L
L
L
L
L
L
L-
Phe3, respectively, without turn structure. “*” in the structures indicate chiral carbon
in the amino acid residue. Dashed green lines highlight intramolecular hydrogen bonds.
blocked. This was attributed to an extended conformation of L-
L
-/D-Phe1 (Scheme 1b) were designed by attaching one
Phe1 in its anion binding complex in which the pyrene
fluorophore locates far away from the DMA donor (Scheme
1a). This led to a decrease in the exciplex to monomer
pyrene fluorophore and an electron donor group DMA into the
two termini of the assumed β-turn structure of phenylalanine
based azapeptide, following the fact that DMA is known to
form exciplex with pyrene.5 Syntheses of them from
phenylalanine were carried out following the procedures
described in the ESI†. Calculated structure (Fig. S1, ESI†) shows
intensity ratio and established
a
ratiometric fluorescent
sensing of anion. Profile for
D
-Phe1 is the same as that of
L-
Phe1 (Fig. S4, ESI†).
β-turn in
close proximity, enabling pyrene-DMA exciplex formation. This
was confirmed by the observed dual emission of Phe1 in
L-Phe1 that brings pyrene fluorophore and DMA into
L
-
CH3CN (Fig. 1). The short-wavelength structured emission
between 370-400 nm is assigned to pyrene monomer, while
the long-wavelength structureless emission around 550 nm is
assigned to the exciplex.5 We found that, with increasing
concentration of L-Phe1, both the intensities of the monomer
and exciplex increase, while the intensity ratio of the exciplex
to monomer remains unchanged over 1 to 20 µM (Fig. 1 inset),
demonstrating that the exciplex is of the intramolecular nature.
This means that within Phe1 molecule the pyrene and DMA
moieties are indeed brought into close proximity that the
formation of exciplex is facilitated, since otherwise the
exciplex formation would be much less favourable because the
pyrene fluorophore is away from DMA by 10 atoms. This
observation thus strongly supports the folded structure of the
Fig. 2 Fluorescence spectra of
acetate anion of 0 to 3 equivalents. Inset in (b) shows the ratio of the exciplex (550 nm)
to monomer (376 nm) intensity of Phe1 and Phe2 versus concentration of acetate
as n-Bu4N+ salt. [
-Phe1] = [ Phe2] = 10 µM, λex = 340 nm.
L-Phe1 (a) and L-Phe2 (b) in CH3CN in the presence of
L-
L-
L
L-
azapeptide L-Phe1, which was further confirmed by the
variable-temperature 1H NMR spectra that show a smaller
temperature coefficient6 of the thioureido -NHd (-4.4 ppb/K)
compared to those of the other three -NH protons (from -7.3
to -10.9 ppb/K, Fig. S2, ESI†), and later by the crystal structure
To confirm if the turn structure does promote the formation
of exciplex and the ratiometric fluorescent response toward
anion, we designed a control compound of L-Phe1, L-Phe2
(Scheme 1b), in which no β-turn structure exists due to the
absence of the hydrogen bonding acceptor. We found that the
of L-Pro1 (see Fig. 4). With D-Phe1, similar observation of the
concentration dependent fluorescence spectra was made (Fig.
S3, ESI†).
exciplex emission of
from the long distance between the electron donor and
acceptor, and the ratio of exciplex to monomer intensity of
Phe2 is much lower than that of Phe1 (Fig. 2). This
observation supports the promotion of the β-turn structure in
Phe1 for the formation of pyrene-DMA exciplex. As a
consequence, the response of the dual emission of Phe2
toward acetate anion (Fig. 2) is much weaker than that of
Phe1, indicating a higher sensitivity of Phe1 in its fluorescent
ratiometric sensing of anion. It was therefore demonstrated
L-Phe2 is extremely weak, likely resulting
L
-
L
-
L
-
L
-
L
-
L
-
that the folded β-turn structure in
sensing performance.
L-Phe1 enhances the
The anion quenching the exciplex emission of
enhancing the monomer emission suggests a conformation
change in Phe1 upon its binding to anion, for example the
L-Phe1 while
L
-
breaking of the β-turn structure (Scheme 1b).3a We therefore
monitored the absorption and CD spectra of Phe1 upon
addition of acetate anion (Fig. 3). It was found that the
absorbances around 270 nm and 290 nm increase, while that
of the pyrene chromophore (320-360 nm) only drops slightly.
The absorptions around 270 and 290 nm were previously
assigned to the chromophores of thiourea and thiourea-anion
Fig. 1 Fluorescence spectra of
L-Phe1 of increasing concentration in CH3CN. Inset shows
the concentration dependent ratio of the exciplex to monomer intensity. λex = 340 nm.
We next examined the fluorescent response of L- and D-Phe1
toward model anion acetate that is known to bind to thiourea
via double hydrogen bonds.4 Fig. 2 shows fluorescence spectra
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binding complex, respectively.3a,4d The CD spectra of
and Phe1 undergo mirror-imaged variations, confirming the
L
-
Phe1 the ten-membered ring intramolecular hydrogen bond in, for
D
-
example, the crystal structure of L-Pro1 (Fig. 4a, NHd···O1, 2.12
Å in distance and 157.48º in angle). The distance between
pyrene and DMA centre is 4.82 Å while the angle of the two π-
planes is 76.78º, both favourable for the formation of exciplex
in the β-turn afforded conformation that the electron donor
stands above pyrene fluorophore (Fig 4a). We also measured
the temperature coefficient of the chemical shift of -NHd as an
indication of the stability of the β-turn. We observed a positive
dependence of the ratio of exciplex to monomer intensity and
the temperature coefficient6 of the -NHd chemical shift (Fig.
4b). This means that a more stable β-turn structure promotes
more the formation of exciplex. It was interesting to note all
the folded azapeptides showed ratiometric fluorescent
response to acetate (Figs. S10-S14, ESI†), exhibiting increasing
sensitivity with the stability of its β-turn. Data in Fig. S14 show
that rigid proline based turn structure affords the best sensing
performance.
Fig. 3 Absorption (a,
L-Phe1) and CD (b, D-Phe1; c, L-Phe1) spectra in CH3CN in the
presence of acetate anion. [Phe1] = 10 µM.
origin of the chirality from the phenylalanine residue. It was
found that the CD signals around 280 nm of Phe1, from the
thiourea and thiourea-anion complex, were enhanced while a
new Cotton effect developed at ca.310 nm, indicating a
structural change that results in an altered chirality
communication in the AcO--Phe1 complex than in Phe1 itself.
Note CD signals were also observed at the absorption window
of the pyrene chromophore beyond 330 nm, which, upon
addition of acetate anion, undergo mirror-imaged variations as
Fig.
4 (a) X-ray crystal structure of L-Pro1. Dashed green line highlights the
intramolecular hydrogen bonding indicative of the β-turn. (b) The temperature
well, suggesting that the pyrene chromophore in Phe1 coefficients (red columns) of the chemical shift of -NHd and the Ie/Im ratio value (blue
lines and dots) of the five folded azapeptides.
experiences a changed chiral environment in the anion binding
complex while it remains within the network containing the
The same protocol was next extended to the case of pyrene
excimer formation, that the two termini of the azapeptide
were both equipped with pyrene fluorophores that would be
brought into close proximity by the turn that the pyrene
chiral phenylalanine residue (Scheme 1b). Furthermore,
switching of the hydrogen bonding in the anion binding
complex was indicated by the changes of the -NH protons
Phe1 by AcO- (Fig. S5, ESI†).
1
signals in H NMR titrations of
L
-
excimer emission would be observed.7 We thus obtained
Phe3 and its control compound Phe4 (Scheme 1b). We
observed pyrene excimer emission from both Phe3 and
Phe4 but is much stronger in Phe3 than in Phe4 (Fig. 5),
confirming that the folded β-turn structure in Phe3 promote
the formation of pyrene excimer too. Moreover, the response
of the dual emission of Phe3 toward acetate anion is much
stronger than that of Phe4 (Fig. 5 inset), similar to the case of
Phe1 versus Phe2 (Fig. 2 inset). Ratiometric fluorescent
response of Phe3 was also observed for fluoride anion,
L-
Broadening of the thioureido -NHc and -NHd protons’ signals
suggested their involving in the intermolecular hydrogen
bonding with AcO-. The splitting of the signals of the geminal -
CH2 protons He and Hf became smaller (Fig. S6, ESI†),
suggesting a weakened chiral communication between the
chiral phenylalanine residue and the methylene -CHeHf. In view
of the observed dramatically quenched exciplex emission,
Phe1 was concluded to experience a conformation switching
from the folded to the extended, upon the addition of AcO-
(Scheme 1b), resulting in therefore a reduced magnetic
nonequivalence of the geminal protons in -CHeHf.
L
-
L
-
L-
L
-
L-
L
-
L
-
L
-
L
-
L-
L-
whereas it is much weaker for other anions such as H2PO4- and
Br-, in agreement with that expected from the thiourea-anion
To explore if and how the amino acid residue would
influence the β-turn structure and the exciplex emission, we
binding mode (Figs. S15 and S16, ESI†).4 Importantly,
L-Phe3
was found to exhibit ratiometric fluorescent response toward
acetate anion in aqueous CTAB solutions, showing again a
developed four derivatives of L-Phe1, containing proline,
alanine, leucine and glycine residues, respectively (Scheme 1b).
All these four azapeptides showed dual emission featuring the
pyrene-DMA exciplex, despite with different ratios of Ie to Im
(Fig. S7, ESI†). This means that β-turn may exist in these four
derivatives as well. We obtained crystals from the racemic
higher sensitivity than
L-Phe4 (Fig. S17, ESI†), and therefore
approving its applicability in aqueous solutions as well.
mixture of L-Pro1 and D-Pro1 in CH3CN through solvent
evaporation at room temperature (Tables S1 and S2, Figs. S8
and S9, ESI†). Both feature folded β-turn structures, shown by
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Chem. Soc. Rev., 2010, 39, 3936; (c) A.-F. Li, J.-H. Wang, F.
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Wang, W. B. He, J. H. Wang, X. S. Yan, Y. Zhan, Y. Y. Ma, L. C.
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Fig. 5 Fluorescence spectra of L-Phe3 (a) and L-Phe4 (b) in CH3CN in the presence of
acetate of 0 to 2.5 eq. Inset in (b) shows the ratio of excimer (480 nm) to monomer
(395 nm) intensity of -Phe3 and -Phe4 versus concentration of acetate. [ -Phe3] = [
Phe4] = 10 µM, λex = 340 nm.
L
L
L
L-
In conclusion, we showed that the folded short azapeptides
exhibited fluorescent response towards species able to bind to
the structural moiety placed within the turn structure. The
response was shown to result from a conformation change
similar to that occurs in the nucleic acid beacons, which
affords a much higher response sensitivity than that of the
8
(a) O. Koch, Mol. Inf., 2012, 31, 624; (b) B. Song, M. G. Bomar,
,
P. Kibler, K. Kodukula and A. K. Galande, Org. Lett., 2012, 14
732.
control compound (L-Phe2 for L-Phe1 and L-Phe4 for L-Phe3)
Graphic Abstract:
that, despite differing in molecular structure by only one less O
atom, has no such folded structure. As folded turn structures,
e.g. α-, β-, or γ-turn, can be made available in a variety of short
peptides,8 the protocol reported here is expected to be well
applied for creating short peptide based beacon-like sensors,
by incorporating target binding site and/or signalling unit into
or around the folded structure, for a broader range of targets.
Dipeptide based amidothiourea (azapeptides) bearing folded β-
turn structure were employed to build beacon-like fluorescent
sensors, signalling by pyrene exciplex or excimer dual emission
to allow highly sensitive ratiometric fluorescent sensing of anion
able to bind to the structural moiety placed within the turn.
We greatly appreciate the support of this work by the NSF of
China (Grants 21435003, 21521004, and J1310024), and the
Program for Changjiang Scholars and Innovative Research
Team in University, administrated by the MOE of China (Grant
IRT13036). We thank Professor Xiang-Jian Kong for his
suggestions on the analysis of crystal structure data.
Notes and references
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4 | J. Name., 2012, 00, 1-3
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