Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence

Article abstract of DOI:10.1021/jacs.1c06435

Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine

Full text of DOI:10.1021/jacs.1c06435

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Article
Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-
Assembly and Fluorescence
Binbin Hu, Na Song, Yawei Cao, Mingming Li, Xin Liu, Zhifei Zhou, Linqi Shi, and Zhilin Yu*
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ABSTRACT: Design of endogenous stimuli-responsive amino acids
allows for precisely modulating proteins or peptides under a biological
microenvironment and thereby regulating their performance. Herein we
report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and
explore its functions in creation of the nitroreductase (NTR)-responsive
peptide-based supramolecular probes for efficient hypoxia imaging. On
the basis of the reduction potential of the nitroimidazole unit, the amino
acid was synthesized via the Mitsunobu reaction between 2-nitro-
imidazole and a serine derivate. We elucidated the relationship between
the NTR-responsiveness of the amino acid and the structural feature of
peptides involving a series of peptides. This eventually facilitates
development of aromatic peptides undergoing NTR-responsive self-
assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created
a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the
resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in
hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties
in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for
synthetic biology and development of novel biomaterials.
scarce, thus limiting the development of NTR-responsive
peptide-based biomaterials. To address the concern, we
designed and synthesized a noncanonical NTR-responsive
amino acid for regulating self-assembly of peptides into
morphology-transformable supramolecular probes for effi-
ciently imaging hypoxic tumor cells.
Inspired by the imidazole moiety within natural histidine
and the NTR-responsive property of nitroimidazole, the NTR-
responsive amino acid was designed based on rationally
integrating a nitroimidazole unit with alanine at its side chain.
The reduction propensity of the nitroimidazole is intimately
associated with the position of the nitro group, in which 2-
nitroimidazole exhibits the highest reduction potential and is
the most readily reduced counterpart compared to 4- and 5-
nitroimidazole.45 In addition, alkylation of 2-nitroimidazole at
the 1-position of the imidazole unit allows for further
improvement of the reduction tendency compared to the 4-
and 5-position of the imidazole.46 These considerations
INTRODUCTION
■
The intimate relationship between the structural features of
peptide nanostructures and amino acid sequences has been
thoroughly elucidated over the past few decades.1−6 This has
inspired establishment of various controllable self-assembling
peptide systems based on stimuli-responsive reactions of
natural or noncanonical amino acids, including redox-,7−13
pH-,14−17 photo-,18 and enzyme-responsive reactions,19−25 and
among others,26,27 for creation of functional materials with
great potential in disease diagnosis and treatment.28−30 Among
the internal or external stimuli, nitroreductase (NTR) is a
flavin-containing enzyme conventionally overexpressed in the
hypoxic region of solid tumors and enables one to reduce the
nitro groups to (hydroxy)amino groups using NAD(P)H as
the electron source.31 Thus far, a considerable number of
NTR-responsive hydrogels,32,33 imaging probes,34−37 drug
delivery vehicles,38−40 or activatable prodrugs41−43 have been
developed and exhibit broad biomedical applications. For
instance, the 1,6-elimination reaction of the 4-nitrophenyl
group induced by NTR reduction has been utilized to create
smart supramolecular hydrogels or the target release of
hydrogen sulfide as demonstrated by the Hamachi or Matson
laboratory, respectively.33,44 Despite the remarkable progress
achieved in stimulus-responsive self-assembly of peptides,
reliable strategies for manipulating the self-assembly of
peptides through NTR-reduction of amino acids remain
Received: June 21, 2021
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A

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a
Scheme 1. Schematic Illustration of the Design Principle for Amino Acid 2-Nitroimidazol-1-yl Alanine (A(2NI))
a
(Top) The reduction potential (vs standard H-electrode) of nitroimidazole units associated with the position of the nitro-group or the alkylation
(methylation) of 2-nitroimidazole. (Bottom) The route for design of A(2NI) starting from histidine via considering the positions for incorporation
of the nitro group and the alkylation of 2-nitroimidazole.
Figure 1. (A) Synthetic route for Trt-A(2NI)-OMe and Fmoc-A(2NI). (B) UPLC traces of Fmoc-A(2NI) upon addition of NTR and NADPH at
different time points and the corresponding reduction reaction induced by NTR or Na2S2O4.
instruct us to design and synthesize a 2-nitroimidazol-1-yl
alanine, abbreviated as A(2NI), as the NTR-responsive amino
acid (Scheme 1). The resulting amino acid was rationally
incorporated into peptide sequences, thereby leading to a
fluorenylmethyloxycarbonyl (Fmoc)-terminated aromatic pep-
tide undergoing the NTR-responsive self-assembly between
nanofibers and nanoparticles. In addition, the nitroimidazole
unit also serves as the fluorescence quencher for many
fluorophores.47,48 Combining this with the potential advan-
tages of morphology-transformable nanoprobes in tumor
accumulation and penetration for efficiently imaging hypoxic
tumor cells, we further designed and synthesized a dye-
containing aromatic peptide and created a NTR-responsive
supramolecular probe for hypoxia imaging via coassembling
the two aromatic peptides. Reduction of nitro-groups within
the aromatic peptides into (hydroxy)amino groups upon
exposure to NTR allows for promotion of the morphological
transition and turning on the fluorescence of the IR780 moiety
via inhibiting the quenching pathway.
diisopropyl azodicarboxylate (DIAD) and triphenylphosphine
(PPh3), leading to Trt-A(2NI)-OMe (Figure 1A).49 The
corresponding intermediate was subsequently treated with
TFA and LiOH to remove the trityl group and hydrolyze the
methyl ester group. Subsequently, Fmoc-protection of the
amine group of A(2NI) was eventually carried out to generate
Fmoc-A(2NI), which was utilized for the standard Fmoc solid-
phase peptide synthesis. The structure and purity of Trt-
A(2NI)-OMe and Fmoc-A(2NI) were characterized by
electrospray ionization-mass spectrometer (ESI-MS) and
nuclear magnetic resonance (NMR) (Figures S1−S4). Upon
addition of NTR and NADPH into amino acid Fmoc-A(2NI),
ultra-performance liquid chromatography (UPLC) trace
showed the gradual conversion of the nitroimidazole to
hydroxyamino-imidazole group and a quantitative conversion
within 1 h (Figure 1B and Figure S5). This suggests the NTR-
response of Fmoc-A(2NI) and its implication in creating NTR-
responsive peptide systems. Reduction of the nitro to amine
group in Fmoc-A(2NI) was also carried out by using an
inorganic reductase sodium dithionite (Na2S2O4) (Figure S6).
However, the NTR-reduction property was not observed for
the unprotected amino acid A(2NI) (Figure S7). This result
implies the critical role of the aromatic protection group in the
RESULTS AND DISCUSSION
■
Amino acid A(2NI) was synthesized by initially attaching 2-
nitroimidazole unit to trityl-protected serine methyl ester
(Trt-L-Ser-OMe) via Mitsunobu reaction in the presence of
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Figure 2. (A) Chemical structures of the investigated peptides containing A(2NI) residue and the corresponding conversion yield of the peptides
in the presence of NTR and NADPH. a: 20 μg/mL for NTR; b: 60 μg/mL for NTR. (B) UPLC traces of aromatic peptide Fmoc-A(2NI)IE in the
absence or presence of NTR and NADPH. (C) The graphical illustration of the association between peptide Fmoc-A(2NI)IE and enzyme NTR, in
which cofactor FMN and the peptide were labeled in dark green and red, respectively.
NTR-reduction process for Fmoc-A(2NI) and the design of
aromatic peptides for the NTR-responsive property.
aggregation concentration (CAC) of the synthesized peptides
via recording the shift of the maximal emission of Nile Red in
the presence of the peptides at various concentrations (Figure
S30), which is one of conventional approaches for determining
the CAC values.50 We found that peptide Fmoc-A(2NI)IE,
Fmoc-IA(2NI)E, or Fmoc-A(2NI)IK exhibited a CAC value of
approximately 10 μM. In contrast, peptide A(2NI)IE,
IA(2NI)IE, or Cbz-IA(2NI)E did not show the aggregating
behavior until the concentration of 1 mM. On the basis of
these results, peptides Fmoc-A(2NI)IE, Fmoc-IA(2NI)E, and
Fmoc-A(2NI)IK with a concentration of 50 μM in the NTR-
conversion studies are under the assembled state. However,
only peptides Fmoc-A(2NI)IE and Fmoc-IA(2NI)E exhibited
the NTR-responsiveness, thus implying no apparent correla-
tion between the NTR-responsiveness and the assembled state
of peptides. In particular, the replacement of the negative
glutamic acid with the positively charged lysine within the
aggregated Fmoc-A(2NI)IK led to the complete loss of the
NTR-responsiveness. In addition, we also estimated the
aggregating behavior of amino acid Fmoc-A(2NI) via the
Nile Red assay, which revealed no aggregation for Fmoc-
A(2NI) until a concentration of 1 mM. Therefore, the
quantitative conversion of Fmoc-A(2NI) by NTR-reduction
excludes the correlation between the NTR-responsiveness and
the assembled state as well. Hence, combining the CAC results
with the NTR-responsiveness of the peptides, our findings
clearly demonstrate the critical role of the structural features of
the peptides (aromatic unit and negative residue) in the NTR-
reducing process, instead of their assembling propensity. In
principle, the flavoprotein nature of NTR demands the
We incorporated amino acid A(2NI) into peptides and
elucidated the relationship between the NTR-responsiveness
of A(2NI) and the structural features of peptides via designing
and synthesizing a series of peptides (Figure 2A). Inspired by
the NTR-response of Fmoc-A(2NI), we initially designed and
synthesized an aromatic peptide Fmoc-A(2NI)IE. Addition of
NTR and NADPH into the solution of Fmoc-A(2NI)IE (50
μM) led to quantitative conversion of the nitroimidazole to the
hydroxyamino-imidazole group (Figure 2B and Figure S18).
Analogue to amino acid A(2NI), peptides A(2NI)IE and
IA(2NI)IE without the Fmoc group did not show the NTR-
induced reduction in the presence of NTR and NADPH
(Figures S19−S20). In addition, distancing A(2NI) from the
terminal aromatic group via localizing A(2NI) at the center
position of peptide Fmoc-IA(2NI)E decreased the NTR-
response of A(2NI), which was converted to A(2NH2I) with a
percentage of 55% in the presence of three folds of NTR
compared to peptide Fmoc-A(2NI)IE (Figure S21). Either
replacing the N-terminal Fmoc group with a benzyloxycar-
bonyl (Cbz) group or the C-terminal negative glutamic acid
with positive lysine residue resulted in the loss of the NTR-
response of peptides Cbz-IA(2NI)E and Fmoc-A(2NI)IK,
respectively (Figure S22). These results clearly demonstrate
the critical structural features for the NTR-responsiveness of
the peptides.
We sought to gain insight into the mechanism for the NTR-
responsiveness of the peptides by investigating the assembling
propensity of the peptides. We estimated the critical
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Figure 3. (A) Chemical structures of aromatic peptides with optimized central residues including Ile, Val, and Ala, and the graphic illustration of
the morphological transition of the assemblies upon NTR-reduction. (B) CD spectra of peptide Fmoc-A(2NI)IE before and after reduction of the
nitro to amine group. (C,D) AFM image of the nanofibrils formed by Fmoc-A(2NI)IE (C) or Fmoc-A(2NH2I)IE (D). (E) CD spectra of peptide
Fmoc-A(2NI)VE before and after reduction of the nitro to amine group. (F,G) AFM images of the nanofibrils and nanoparticles formed by Fmoc-
A(2NI)VE (F) or Fmoc-A(2NH2I)VE (G). The inset panels in AFM images showed the height of the selected nanofibrils or the distribution of the
nanoparticle diameter.
presence of the aromatic cofactor flavin mononucleotide
(FMN) at the catalytic site of NTR.51 We hypothesize that
the aromatic Fmoc group of peptides facilitates the association
between peptides and enzyme NTR via the π,π-stacking
interactions involving FMN cofactors (Figure 2C). In addition,
the negative glutamic acid residue potentially favors the
catalytic process through the electrostatic interaction between
the peptides and enzymes. Nevertheless, our results demon-
strate development of NTR-reducible amino acid for creating
enzyme-responsive peptides via rationally tailoring the
structural feature of peptides.
We further optimized the sequence of the A(2NI)-
containing aromatic peptides to establish the NTR-regulated
self-assembling peptide system via thoroughly investigating the
conformation of peptides and the morphology of their
assemblies prior to or after NTR-reduction (Figure 3).
Considering the critical roles of the N-terminal Fmoc and
the C-terminal negatively charged groups for the NTR-
response, we optimized the second residue within the aromatic
tripeptides to tune the assembling propensity of peptides,52
leading to NTR-reducible peptides Fmoc-A(2NI)IE, Fmoc-
A(2NI)VE, and Fmoc-A(2NI)AE (Figure 3A). The NTR-
induced conversion studies revealed that addition of NTR and
NADPH into the solution of peptides Fmoc-A(2NI)IE, Fmoc-
A(2NI)VE, and Fmoc-A(2NI)AE led to the quantitative
conversion of the nitro to hydroxyamino groups, indicating
the maintained NTR-responsiveness for the three peptides. It
is worth noting that peptide Fmoc-A(2NI)VE was efficiently
converted by NTR at a broad concentration range from 4 to 20
μg/mL, thus endowing its NTR-responsiveness in the tumor
hypoxic regions (Figure S28).35,53 Subsequently, we inves-
tigated the conformation of the peptides as well as the
morphology of their assemblies prior to and after quantitative
nitro-reduction via utilization of Na2S2O4. Circular dichroism
(CD) spectra of peptide Fmoc-A(2NI)IE and its reduced
counterpart Fmoc-A(2NH2I)IE displayed two positive bands
at approximately 210 and 270 nm, one negative peak at 223
nm (Figure 3B). The positive and negative bands at 210 and
223 nm indicate formation of β-sheets by peptides Fmoc-
A(2NI)IE and Fmoc-A(2NH2I)IE.54 The positive Cotton
effect at 270 nm in the CD spectra is associated with the
absorption of the Fmoc group.55 CD results suggest the
maintained ordered secondary structures of peptide Fmoc-
A(2NI)IE before and after nitro-reduction. We also carried out
the morphological studies of the assemblies formed by
peptides Fmoc-A(2NI)IE and Fmoc-A(2NH2I)IE. Both
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Figure 4. (A) The conversion percentage of peptides Fmoc-A(2NI)VE and Fmoc-A(2NI)AE at a concentration of 10 or 50 μM induced by
enzyme NTR and NADPH as a function of time. (B−F) The morphological transition of the aggregates formed by quantitatively reduced Fmoc-
A(2NI)VE, i.e., Fmoc-A(2NH2I)VE, at various time points as revealed by AFM: (B) 0 min; (C) 5 min; (D) 15 min; (E) 30 min; and (F) 60 min.
atomic force microscopy (AFM) images of peptides Fmoc-
A(2NI)IE and Fmoc-A(2NH2I)IE showed the defined micro-
scale nanofibrils with an average height of approximately 10
nm (Figure 3C,D). The morphology of the resulting
assemblies was confirmed by transmission electron microscopy
(TEM), in which nanofibrils with an average width of 10 nm
were observed for both peptides Fmoc-A(2NI)IE and Fmoc-
A(2NH2I)IE (Figure S34). These results suggest the
maintained conformation of peptide Fmoc-A(2NI)IE and the
morphology of its assemblies prior to or after the NTR-
reduction.
In the case of peptides Fmoc-A(2NI)VE and Fmoc-
A(2NI)AE that contain a hydrophobic residue with less
tendency to form β-sheets compared to Fmoc-A(2NI)IE, CD
spectra of peptide Fmoc-A(2NI)AE indicates formation of
random coil conformation prior to and after reduction of the
nitro to amine group (Figure S31). This result is consistent
with no aggregation of peptide Fmoc-A(NI)AE as revealed by
the CAC studies (Figure S30). In contrast, CD signals of
peptide Fmoc-A(2NI)VE before and after nitro-reduction were
distinct (Figure 3E), in which peptide Fmoc-A(2NI)VE
showed two positive bands at approximately 215 and 270
nm and one negative band at 195 nm, while peptide Fmoc-
A(2NH2I)VE only exhibited one negative peak at 195 nm.
Despite the absence of the standard CD signals for β-sheets,
the CD signals of peptide Fmoc-A(2NI)VE are consistent with
those of aromatic peptides forming ordered nanostructures.12
These results suggest that incorporation of valine residue into
the aromatic tripeptide leads to the NTR-responsive change of
the ordered structures formed by Fmoc-A(2NI)VE. In
addition, AFM images showed peptides Fmoc-A(2NI)VE
and Fmoc-A(2NH2I)VE assembled into nanofibers with an
average height of around 9 nm and nanoparticles with an
average diameter of ca. 90 nm, respectively (Figure 3F,G). The
different morphology of the assemblies formed by peptides
Fmoc-A(2NI)VE and Fmoc-A(2NH2I)VE was confirmed by
TEM studies, in which nanofibers with a width of 9 nm and
nanoparticles with a diameter of 40 nm were observed for
peptides Fmoc-A(2NI)VE and Fmoc-A(2NH2I)VE, respec-
tively (Figure S35). The difference between the diameter
values estimated by AFM and TEM potentially is attributed to
the broadening effect of AFM tips or the deposition and
collapse of the nanoparticles on the mica surface. TEM images
of the nanoparticles did not show the typical bilayered
structures, thus excluding the formation of the vesicles by
the reduced peptides. In addition, the maximal length of
approximately 2.2 nm for the peptides rules out the formation
of the core−shell spherical micelles. Hence, we hypothesize
that the reduced peptide Fmoc-A(2NH2I)VE potentially forms
the multicomponent nanoparticles with complicated internal
structures. The conformational and morphological studies
suggest that peptide Fmoc-A(2NI)VE undergoes the NTR-
responsive morphological transition, whereas the morphology
of the peptide Fmoc-A(NI)IE is retained prior to and after
NTR-reduction. Considering the quantitative conversion of the
nitro groups within both the peptides, their distinct
morphological transition is attributed to the different hydro-
phobicity of the second residues, in which valine exhibits a
lowered hydrophobicity compared to isoleucine, thereby
preventing formation of ordered nanofibers for Fmoc-
A(2NH2I)VE. Nevertheless, these results demonstrate that
optimizing the sequence of the aromatic peptides leads to the
development of an aromatic peptide undergoing the NTR-
responsive morphological transition.
We further investigated the kinetics of the conversion of
peptide Fmoc-A(2NI)VE to the hydroxyamino product and
the morphological transition of its assemblies to nanoparticles
promoted by NTR-reduction (Figure 4). With respect to the
conversion of Fmoc-A(2NI)VE, we sought to uncover the
preference of the monomeric or assembled peptides for the
enzyme-responsiveness. Hence, we carried out the kinetic
studies of the NTR-conversion of peptide Fmoc-A(2NI)VE at
the concentrations below (10 μM) and above (50 μM) its
CAC value (Figure 4A). Simultaneously, the kinetic studies of
the NTR-induced conversion of the nonaggregating peptide
Fmoc-A(2NI)AE at the two concentrations were also carried
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Figure 5. (A) Illustration of the fluorescence off or on state of IR780 moieties in peptide IR780-A(2NI)VE arising from the nitro-reduction and the
corresponding UPLC traces of IR780-A(2NI)VE before and after reduction by NTR and NADPH. (B) Fluorescence spectra of peptide IR780-
A(2NI)VE and the supramolecular probe FI-A(2NI)VE before and after NTR-reduction excited at 780 nm. (C,D) AFM images of FI-A(2NI)VE
(C) and FI-A(2NH2I)VE (D), in which the inset panels showed the height of the selected nanofibrils or the distribution of the nanoparticle
diameter. (E) TEM image of the nanofibrils formed by FI-A(2NI)VE. (F) Maximal fluorescence intensity of FI-A(2NI)VE in the presence of
NADPH and interference agents or NTR. (G) Schematic representation of the morphology-transformable supramolecular probes via coassembling
Fmoc-A(2NI)VE and IR780-A(2NI)VE, in which NTR-reduction of A(2NI) in Fmoc-A(2NI)VE and IR780-A(2NI)VE lead to the morphological
transition of the nanostructures and recovery of the emission of IR780 moieties, respectively. The gray dots along the nanofibers and the red circles
around the particles denote the quenched and luminescent IR780 moieties, respectively.
out to exclude the concentration effect on the kinetic rates.
Our results showed a time lag of the quantitative conversion of
the assembled Fmoc-A(2NI)VE compared to the monomeric
ones. In contrast, the conversion of the nonaggregating peptide
Fmoc-A(2NI)AE displayed an almost identical rate for the
samples at different concentrations. These results clearly
demonstrate the preference of the NTR-induced reduction of
the peptides in the monomeric state. However, it is challenging
to exclude the NTR-responsiveness of the peptide assemblies
by our results. In addition, to reveal the kinetics of the
morphological transition of the assemblies of Fmoc-A(2NI)VE
promoted by nitro-reduction, we performed AFM studies to
monitor the morphology of peptide assemblies at different
time points after quantitative conversion of Fmoc-A(2NI)VE
via utilization of inorganic reductase Na2S2O4 (Figure 4B−F).
AFM images of the reduced assemblies showed pretty short
nanofibers at 5 min after nitro-reduction, indicating the break
of the microscale nanofibers formed by Fmoc-A(2NI)VE.
While the quantity of the shortened nanofibers became less at
15 min, short nanofibers and nanoparticles were coexistent at
30 min. Eventually, only nanoparticles were observed in the
AFM image of the sample at 60 min, thus implying the
completion of the morphological transition from nanofibers to
nanoparticles. These results demonstrate the efficient morpho-
logical transition for peptide Fmoc-A(2NI)VE promoted by
nitro-reduction.
In this context, the morphology-transformable nanostruc-
tures have been demonstrated as one of ideal candidates for
targeting delivery of therapeutics or probes to pathological
lesions, due to the advantages in overcoming the delivery
barriers.56,57 To explore the implications of the NTR-
responsive peptide systems as biomaterials, we designed and
synthesized a fluorescent dye-functionalized tripeptide IR780-
A(2NI)VE and created a morphology-transformable supra-
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Figure 6. (A) CLSM images of breast tumor cell 4T1 treated with FI-A(2NI)VE under the hypoxic or normoxic condition or FI-A(2NH2I)VE
under normoxic condition. Scale bar: 20 μm. (B,C) In vivo or ex vivo fluorescence imaging of 4T1 breast tumor-bearing mice (B) or the tumor
tissues and major organs dissected from the mice at 12 h postadministration (C) with FI-A(2NI)VE (I), FI-A(2NH2I)VE (II), or FI-A(2NI)VE +
dicoumarin (III). (D,E) Quantitative mean fluorescence intensity of the in vivo signals at tumor sites in the mice (C) or ex vivo signals at tumor
sites or major organs from the mice (E) administrated with treatments I−III. H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, and T: tumor. (F)
CLSM images of the frozen sections of the tumor tissues from the mice administrated with treatment FI-A(2NI)VE (I), FI-A(2NH2I)VE (II), FI-
A(2NI)VE + dicoumarin (III), or FI-A(2NI/2NH2I)VE + dicoumarin (IV), accompanied by the graphical illustration of the imaging mechanism
for different probes for hypoxic tumor cells. Scale bar: 250 μm.
molecular probe FI-A(2NI)VE via coassembling IR780-
A(2NI)VE with Fmoc-A(2NI)VE in a molar ratio of 5:95
for imaging hypoxic tumor cells. 2-Nitroimidazole is one
conventional quencher for cyanine dye IR780, which exhibits
near-IR fluorescence and has been broadly utilized in
fluorescence bioimaging, through a photoinduced electron
transfer (PET) mechanism.58 We attached dye IR780 to a
peptide via a thiol-involving nucleophilic reaction, leading to
peptide IR780-A(2NI)VE. Addition of NTR and NADPH into
the solution of peptide IR780-A(2NI)VE resulted in
conversion of nitroimidazole to aminoimidazole in peptide
IR780-A(2NH2I)VE (Figure 5A and Figure S27), different
with production of the hydroxyamino group within peptide
Fmoc-A(2NI)VE. Fluorescence spectroscopy revealed that
addition of enzyme NTR and cofactor NADPH into IR780-
A(2NI)VE led to a 5-fold increase of the fluorescence intensity
of IR780 moieties (Figure 5B), arising from the conversion
from the nitroimidazole to the aminoimidazole group. This
result directly suggests that residue A(2NI) within the
aromatic peptide serves as the quencher for the IR780
conventionally through a PET mechanism. Hence, converting
the nitroimidazole to the aminoimidazole group prevents the
PET process, thereby recovering the fluorescence intensity of
the IR780 moiety within peptide IR780-A(2NH2I)VE.
We characterized the morphology of the resulting supra-
molecular probe before and after nitro-reduction (Figure 5C−
E). While AFM and TEM images of FI-A(2NI)VE displayed
nanofibrils with a height and width of appropriately 10 and 10
nm, respectively (Figure 5C,E), coassembly FI-A(2NH2I)VE
by Fmoc-A(2NH2I)VE and IR780-A(2NH2I)VE at a molar
ratio of 95:5 aggregated into nanoparticles with an average
diameter of 85 and 38 nm as estimated by AFM and TEM
studies (Figure 5D and Figure S36). These results directly
demonstrate development of a NTR-responsive morphology-
transformable coassembly via integrating the two aromatic
peptides. It is worth noting that the presence of a slight
amount of dimethyl sulfoxide for assisting the solubility of
IR780-A(2NI)VE prevents the CD studies of FI-A(2NI)VE as
a result of the abnormal high tension voltage values.
Fluorescence studies revealed that reduction of the nitro
groups within coassemblies FI-A(2NI)VE by NTR and
NADPH gave rise to the increase of the fluorescence intensity
of IR780 moieties with a fold comparable to that of peptide
IR780-A(2NI)VE (Figure 5B). The slight decrease of the
fluorescence intensity and the shift of the maximal emission
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wavelength of FI-A(2NH2I)VE compared to IR780-A-
(2NH2I)VE are potentially attributed to the organization of
IR780 moiety along the nanofibrils. Combining the fluo-
rescence results with morphological studies demonstrates
creation of a morphology-transformable supramolecular
probe FI-A(2NI)VE, in which the NTR-reduction of A(2NI)
in Fmoc-A(2NI)VE and IR780-A(2NI)VE is attributed to the
morphological transition of the nanostructures and recovery of
the emission of IR780 moieties, respectively (Figure 5G). In
addition, the selectivity of the probe toward NTR was
estimated by carrying out the fluorescence studies in the
presence of biological internal interference agents (Figure 5F
and Figure S39). Addition of reductants glutathione and
ascorbic acid, glucose, hydrogen peroxide, or metal ions Na+,
K+, Ca2+, and Mg2+ into the solution of FI-A(2NI)VE induced
negligible change of the fluorescence intensity of the IR780
moiety within the coassemblies. These results suggest the
selectivity of the supramolecular probe responsive to enzyme
NTR, thus demonstrating great potential in NTR detection.
The NTR-specific fluorescence of the supramolecular probe
inspired us to estimate its capability for the bioimaging of
tumor cells under hypoxic condition based on overexpression
of NTR within the hypoxia region (Figure 6). MTT assays
revealed that treating normal mouse fibroblast 3T3 and breast
tumor 4T1 cells with probe FI-A(2NI)VE and reduced
counterpart FI-A(2NH2I)VE did not lead to an apparent
decrease of cell viability, indicating the excellent biocompat-
ibility of the supramolecular probe (Figure S41). During the
bioimaging studies of tumor cells, 4T1 cells were incubated
under the hypoxic or normoxic condition in the presence of
to the inhibited activity of enzyme NTR. The remaining
fluorescence signals might be attributed to the remaining
activity of NTR in the presence of dicoumarin. The results
suggest that the fluorescence signal in the mice administrated
with FI-A(2NI)VE is attributed to the NTR-reduction of the
A(2NI) residue. At 12 h after subcutaneous injection, the
tumor tissues and major organs were dissected from the mice
and ex vivo fluorescence imaging was performed (Figure 6C).
The primary fluorescence signals were detected at the tumor
and liver tissues dissected from the mice administrated with FI-
A(2NI)VE. In contrast, the ex vivo images showed pretty weak
fluorescence signals at the tumor tissues from the mice
administrated with FI-A(2NH2I)VE or FI-A(2NI)VE +
dicoumarin (Figure 6E), arising from the poor tumor
accumulation and inhibited NTR-activity, respectively. In
particular, CLSM images of the frozen sections of the tumor
tissues from the mice administrated with FI-A(2NI)VE
showed strong fluorescence signals associated with IR-780
moieties at the deep region (Figure 6F). However, the tumor
tissues from the mice treated with FI-A(2NH2I)VE displayed
pretty weak fluorescence signals around the tumor tissues.
These results suggest the improved tumor accumulation and
penetration of the supramolecular probe. Eventually, the in vivo
and ex vivo fluorescence imaging studies demonstrate the
excellent capability of the supramolecular probes for efficient
fluorescence imaging of the solid tumors in mice.
We further unraveled the mechanism for the tumor
accumulation and penetration of the supramolecular probes
involving one counterpart probe FI-A(2NI/2NH2I)VE (Figure
6F and Figure S45). The probe FI-A(2NI/2NH2I)VE was
created via coassembling Fmoc-A(2NI)VE with IR780-A-
(2NH2I)VE with a constant ratio for the assembling and
fluorescent components in FI-A(2NI)VE. AFM and TEM
images of FI-A(2NI/2NH2I)VE revealed the nanofibrous
morphology for FI-A(2NI/2NH2I)VE (Figures S33 and
S37), which is attributed to the strong assembling propensity
of the Fmoc-A(2NI)VE component. The emission intensity of
the IR780 moieties within FI-A(2NI/2NH2I)VE is comparable
to that of FI-A(2NI)VE treated with NTR and NADPH
(Figure S40), indicating the negligible intermolecular PET
process between the IR780 and nitroimidazole moieties within
the coassemblies and thereby serving as the intrinsic
fluorescence nanofibrous probe. CLSM image of FI-A(2NI/
2NH2I)VE confirmed formation of nanofibrils with strong red
fluorescence signals (Figure S38), directly supporting the
coassembly of Fmoc-A(2NI)VE with IR780-A(2NH2I)VE into
the nanofibrils. Therefore, we hypothesize that simultaneously
administrating the tumor-bearing mice with the nanofiber
probe FI-A(2NI/NH2I)VE and NTR-inhibitor dicoumarin
potentially allows for maintenance of the in vivo property of the
nanofibrous probe and also prevention of its morphological
transformation due to the inhibited NTR activity. As a
consequence, CLSM images of the frozen sections of the
tumor tissues dissected from the mice administrated with FI-
A(2NI)VE + dicoumarin only showed poor signals around the
edge of the tumor tissues (Figure 6F). This result confirms the
critical role of the nanofibrous morphology in tumor
accumulation of the nanofiber probes, as well as the
mechanism of the morphological transition for the tumor
penetration of the nanofibrous probe promoted by the NTR-
reduction of nitro groups. Hence, these CLSM results directly
demonstrate the tumor accumulation and penetration of the
probe FI-A(2NI)VE, in which the hypoxic condition was
59,60
generated by a conventional chemical inducer CoCl2
and
thereby allowing for the expression of NTR by 4T1 cells and
rendering the enzyme responsive for the probe. Incubation of
4T1 cells with probe FI-A(2NI)VE under the hypoxic
condition resulted in strong red fluorescence signals associated
with the IR780 moiety detected by confocal laser scanning
microscopy (CLSM), whereas the cells treated the probe
under the normoxic condition did not give rise to apparent
fluorescence signals (Figure 6A and Figure S42). However,
incubation of 4T1 cells with FI-A(2NH2I)VE showed apparent
red signals associated with IR780 moieties due to the
recovered fluorescence of IR 780 (Figure S43). These results
directly suggest the internalization of the probe by tumor cells
and the fluorescence emission of the probe induced by NTR-
reduction for selective bioimaging of hypoxic tumor cells.
We subsequently carried out the in vivo bioimaging of tumor
cells in the hypoxic region via subcutaneous injection of the
probe into 4T1 breast tumor-bearing mice (Figure 6B). In vivo
fluorescence imaging of the mice administrated with FI-
A(2NI)VE detected apparent fluorescence signals associated
with IR780 moieties around tumor tissues during a long period
(Figure 6D), indicating the efficient fluorescence imaging of
the solid tumors by the supramolecular probes. However, the
tumor tissues in the mice treated with FI-A(2NH2I)VE did not
exhibit obvious fluorescence signals, suggesting its poor
accumulation at tumor sites. These results imply the improved
accumulation of the NTR-responsive supramolecular probes at
tumor sites compared to the intrinsic fluorescence probes. In
addition, in the group of the mice simultaneously adminis-
trated with FI-A(2NI)VE and dicoumarin that is a conven-
tional inhibitor for NTR,61 the tumor tissues displayed rather
poor fluorescence signals associated with IR780 moieties, due
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https://doi.org/10.1021/jacs.1c06435
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
pubs.acs.org/JACS
Article
morphology-transformable supramolecular probes for efficient
hypoxic imaging compared to other counterpart probes.
Mingming Li − Key Laboratory of Functional Polymer
Materials, Ministry of Education, State Key Laboratory of
Medicinal Chemical Biology, Institute of Polymer Chemistry,
College of Chemistry, Nankai University, Tianjin 300071,
China
Xin Liu − Key Laboratory of Functional Polymer Materials,
Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China
Zhifei Zhou − Key Laboratory of Functional Polymer
Materials, Ministry of Education, State Key Laboratory of
Medicinal Chemical Biology, Institute of Polymer Chemistry,
College of Chemistry, Nankai University, Tianjin 300071,
China
CONCLUSION
■
In summary, we have designed and synthesized a noncanonical
amino acid 2-nitroimidazol-1-yl alanine for development of the
NTR-responsive peptide nanostructures and the morphology-
transformable hypoxic fluorescence probes. The amino acid
was synthesized via the Mitsunobu reaction between 2-
nitroimidazole and a serine derivate and exhibited NTR-
reduction property when protecting the amine group with a
bulky aromatic moiety. This allows us to create aromatic
peptides undergoing NTR-responsive self-assembly between
nanofibers and nanoparticles by rationally optimizing the
sequences. We also designed and synthesized a dye-function-
alized aromatic peptide and created a morphology-trans-
formable supramolecular probe for imaging tumor cells based
on the NTR-dependent role of 2-nitroimidazole in fluores-
cence quench. We found that the resulting supramolecular
probes primarily accumulated at tumor sites and penetrated
into solid tumors arising from the morphological trans-
formation, thus allowing for efficient fluorescence imaging of
tumor cells in hypoxic regions. Our results demonstrate the
versatile property of the new amino acid in regulating
assembling propensity of peptides and the optical property of
nanostructures, which could in principle be further expanded
to the other fields including peptide ligation and radiation
sensitizers, thus serving as a powerful toolkit for synthetic
biology and disease theranostics.
Linqi Shi − Key Laboratory of Functional Polymer Materials,
Ministry of Education, State Key Laboratory of Medicinal
Chemical Biology, Institute of Polymer Chemistry, College of
Chemistry, Nankai University, Tianjin 300071, China;
orcid.org/0000-0002-9534-795X
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c06435
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (21774065, 81972903, and 51933006)
and “the Fundamental Research Funds for the Central
Universities”, Nankai University (63213070).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c06435.
■
sı
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Na Song − Key Laboratory of Functional Polymer Materials,
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