Design of Turn-On Near-Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Article abstract of DOI:10.1002/anie.202000108

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn-on near-infrared (NIR) fluorescent probes with intrinsically high signal-to-background ratio

Full text of DOI:10.1002/anie.202000108

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Accepted Article
Title: Design of Turn-On Near-Infrared Fluorescent Probes for Highly
Sensitive and Selective Monitoring of Biopolymers
Authors: Gerard T. Ducharme, Zane LaCasse, Tanya Sheth, Irina V.
Nesterova, and Evgueni E. Nesterov
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202000108
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Design of Turn-On Near-Infrared Fluorescent Probes for Highly
Sensitive and Selective Monitoring of Biopolymers
Gerard T. Ducharme,[a] Zane LaCasse,[b] Tanya Sheth,[b] Irina V. Nesterova,*[b] and Evgueni E.
Nesterov*[a], [b]
[a]
Dr. G. T. Ducharme and Prof. E. E. Nesterov*
Department of Chemistry
Louisiana State University
Baton Rouge, LA 70803, USA
E-mail: een@niu.edu
[b]
Z. LaCasse, T. Sheth, Prof. I. V. Nesterova* and Prof. E. E. Nesterov*
Department of Chemistry and Biochemistry
Northern Illinois University
DeKalb, IL 60115, USA
E-mail: inesterova@niu.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Simple, sensitive and selective detection of specific
biopolymers is critical in broad range of biomedical and
probe. Thus, increasing S/B ratio commonly requires additional
a
step(s) such as washing to remove unbound probes or reliance
[3a, 3b, 5]
technological areas. We present a design of turn-on near-infrared
(NIR) fluorescent probes with intrinsically high signal-to-background
ratio. The fluorescent signal generation mechanism is based on the
aggregation/de-aggregation of phthalocyanine chromophores
controlled by selective binding of small-molecule “anchor” groups to a
specific binding site of a target biopolymer. As a proof-of-concept, we
demonstrate a design of a sensor for EGFR tyrosine kinase – an
on multistep bioconjugation protocols
With an intent to
make possible a simple single-step detection of biopolymers, we
report near-infrared (NIR) turn-on fluorescent sensing systems for
selective and highly sensitive detection of protein targets.
important target in cancer research.
The universality of the
fluorescent signal generation mechanism, as well as the response
selectivity independently coming from a proper choice of a small-
molecule “anchor” group, make possible to use this approach in
designing reliable turn-on NIR fluorescent sensors for detecting
specific protein targets present in low-nanomolar concentration range.
Reliable tools for efficient detection and visualization of
biomolecules are essential for a wide range of areas spanning
from life sciences research to routine applications in point-of-care
settings. Among various methodologies, fluorescence-based
techniques enable simple, robust, and selective target monitoring
over complex biological backgrounds.[1] Therefore, the field of
fluorescent biosensor development has been continuously
expanding striving to improve sensor design.[2] As for any
detection platform, the ultimate goal is to have a simple, possibly
one-component, biocompatible system that is sensitive and
selective toward a specific target, i.e. it generates a high signal-
to-background (S/B) ratio upon target binding over complex
biochemical environments.[3] A traditional fluorescent sensor
design scheme relies on linking together a fluorescent reporter
with a recognition domain.[4] This approach works well for small-
molecule reactive analytical targets that can sufficiently alter the
electronic structure or properties of the recognition domain (e.g.
as in case of fluorescence-quenching analytes); it is, however,
less applicable for biopolymer detection since many large
biomolecules do not produce significant electronic perturbations
upon interaction with recognition domain. In addition, the
traditional approach typically yields low S/B ratios for biopolymer
detection due to high fluorescent background of an unbound
Figure 1. General schematics of a selective turn-on fluorescent probe. The
probe consists of a Pc reporter and a target recognition “anchor” domain. When
unbound, the probe is non-fluorescent due to the extensive aggregation of Pc
chromophores. Binding the “anchor” domain to a specific protein target
decreases the propensity towards aggregation, and Pc’s fluorescence turns on.
We based our probe design on deliberate control over
aggregation/de-aggregation of phthalocyanines (Pc’s).[6] Various
strategies utilizing disaggregation of small molecular
chromophores enable a simple and selective analytical detection
format. This general approach relies on difference in emission
pattern of the chromophores that are assembled in solution and
get disassembled upon the probe binding to a target.[7] The
required ultimate selectivity of the probe is achieved via
engineering
a
target-specific fragment into the probe’s
structure.[5d] Several promising single-step disassembly-driven
approaches have been previously reported.[7] While in some
cases they demonstrate sufficient detection selectivity, they are
typically capable of detecting biopolymer targets present in
1
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10.1002/anie.202000108
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COMMUNICATION
relatively high (in the micromolar range) concentrations.[7d, 8] The
currently missing ability to assess biomolecules at much lower
concentration levels may be required for some practical
applications; furthermore, intrinsic availability of turn-on sensors
capable of operating within low-nanomolar concentration range
would enhance fundamental appeal of fluorescent biosensing.
Water-soluble Pc’s are known as biocompatible, bright, and
characteristic NIR fluorescence would lead to further
improvement of the S/B ratio[12] due to a lower photon absorption
and scattering and reduced auto-fluorescence from both artificial
[13] and biological environments.[12d, 14]
To make use of the Pc aggregation/de-aggregation for the
design of selective turn-on fluorescent sensors for proteins, we
functionalize a Pc fluorescent reporter with a small-molecule
(“anchor”) group that can selectively bind to a target biopolymer.
We assume that in the absence of the specific target, the small
“anchor” group would be insufficient to preclude Pc aggregation
in aqueous conditions (and indeed, due to its hydrophobicity,
would facilitate Pc aggregation), and thus the probe will be non-
fluorescent. In the presence of a target protein, however,
interaction of the “anchor” with the target’s binding site would “pull”
a sensor molecule out of an aggregate, and thus turn the
fluorescence on (Figure 1). This conceptually simple design relies
on a general, protein-independent signal generation mechanism
(Pc aggregation/de-aggregation), whereas the detection
selectivity comes from a proper choice of a small-molecule
binding group.
stable NIR fluorescent imaging agents.[9]
Phthalocyanines
intrinsically aggregate in aqueous conditions (with dimerization
constant as high as 109) because of the attractive π-π interactions
between Pc planar hydrophobic aromatic cores,[10] and form
essentially non-fluorescent H-aggregates.
Importantly, the
propensity towards aggregation decreases when Pc’s are
associated with large biopolymers.[11] As a consequence of the
de-aggregation, a Pc chromophore bound to a large biomolecule
becomes fluorescent. Therefore, a turn-on fluorescent response
upon Pc de-aggregation will happen over an absolutely dark
background of aggregated, unbound Pc molecules and lead to an
intrinsically high S/B ratio without the need to remove unbound
species.
We anticipated that the absence of fluorescent
background should enable achieving a much lower detection limit
than what has been obtained so far. In addition, the Pc’s
I
HN
N
I
O
HN
N
O
OH
H
1) d
2) e
N
H2N
O2N
O2N
a
N
b, c
N
HO
OH
NH2
N
O
O
N
OH
O
3
2
O
3) f
NHBoc
NHBoc
O
Fmoc-Lys(Boc)-OH
1) g
CN
4
H
H
N
NH2
N
CN
O
NHFmoc
N
e
2) e
HOOC
H
O
O
O
CN
5
6
CN
CN
CN
OTEG
OTEG
TEGO
TEGO
NHBoc
O
NH2
O
N
Zn
N
N
Zn
N
OTEG
i
N
N
N
N
N
N
7
N
N
N
N
H
h
N
N
N
H2N
N
N
H
H
O
O
O
O
OTEG
OTEG
8
9
TEGO
TEGO
O
OTEG
O
N
N
H
N
N
NH2
N
N
N
e
N
N
N
N
h
O
N
3
+
9
N
Zn
N
N
7
Zn
N
N
HN
O
N
HN
I
HN
N
OTEG
TEGO
O
OTEG
TEGO
O
1
10
Pc fluorescent reporter
EGFR binding "anchor"
O
OTEG =
O
O
Scheme 1. Synthesis and Structure of the EGFR Kinase Sensor 1 and Control 10. Reagents and conditions: (a) formamide, 160 °C, 6 h; (b) SOCl2, DMF (cat.),
reflux, 3 h; (c) 3-iodoaniline, DIPEA, i-PrOH, reflux, 3 h; (d) SnCl2 × 2H2O, EtOH, reflux, 2 h; (e) HOBt, HBTU, DIPEA, CH2Cl2 or DMF, 12-36 h; (f) LiOH, MeOH-
H2O, 12 h, followed by HCl; (g) 4-methylpiperidine, DMF; (h) Zn(OAc)2, DBU, n-BuOH, DMF, 106 °C, 24 h; (i) TFA, TIPS-H, CH2Cl2, 3-5 h.
2
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As a proof-of-concept, we designed a turn-on fluorescent
detector for EGFR kinase. Abnormal expression of epidermal
growth factor receptor (EGFR) tyrosine kinase leads to an array
of mitogenic cellular changes making this enzyme one of the most
attractive targets for anticancer therapeutics. This kinase
exemplifies a need for developing simple sensory platforms for
selective detection of nanomolar concentrations of endogenous
EGFR in biological systems over the exceeding backgrounds of
other biopolymers. There are a number of EGFR-selective
fluorescent sensors and imaging agents, including ones with
genetically encoded functionalities[15] as well as based on linked
anti-EGFR antibody as a receptor and various fluorescent
reporters (small-molecule dyes, nanoparticles/quantum dots, and
conjugated polymers).[4b, 12f, 16] However, none of the previous
systems offer a selective, simple, single-step platform with high
S/B ratio of a turn-on fluorescent detection.
crystallographic data[21] indicated that the 6-NH2 group of the
quinazoline core 2 would be the most appropriate place for the
attachment of a Pc fluorescent reporter because it extends toward
the solvent when the molecule is bound to the EGFR tyrosine
kinase domain. An additional acrylamido group attached at the 6
position of quinazoline was demonstrated to enhance binding of
this compound to the same ATP-binding site of the activated
EGFR kinase.[20e, 22] As a Pc fluorescent reporter, we chose TEG-
functionalized Zn-phthalocyanine (Zn-Pc) as this dye is water-
soluble and has been previously shown to aggregate in aqueous
media.
The quinazoline “anchor” fragment 3 was prepared in six steps
starting from 5-nitroantranilic acid (Scheme 1).
Although
preparation of symmetric phthalocyanines is readily
accomplished by condensation of four phthalonitrile molecules,
the non-symmetric Zn-Pc 9 with a single lysine amino group for
linking with the quinazoline “anchor” 3 required a more elaborate
approach. We utilized a previously developed solid-phase
synthesis to accomplish this preparation.[23] First, we prepared
amine-functionalized polymer resin by deprotecting Fmoc-
protected Rink amide resin. It was then coupled with protected
lysine 4, followed by removal of the Fmoc protecting group and
subsequent coupling with benzoic acid functionalized
phthalonitrile 5 to form a resin-bound phthalonitrile precursor 6.
Condensation of 6 with excess phthalonitrile 7 gave resin-bound
Zn-Pc 8. Trifluoroacetic acid promoted cleavage from the resin
occurred simultaneously with the deprotection of the lysine NH2
group, and furnished Zn-Pc building block 9. Finally, the two
building blocks 3 and 9 were coupled to form the EGFR sensor 1.
Spectroscopic studies of the sensor 1 indicated that it was
suitable for NIR turn-on sensing. Thus, it exhibited a high
extinction coefficient (78,400 cm–1 M–1) and
a strong NIR
emission from the non-aggregated state (quantum yield 12.5% in
DMSO). Importantly, although it was soluble in aqueous media,
it showed an extensive aggregation as could be seen from the
hypsochromic shift of the Q band in absorption spectrum.
Dynamic light scattering (DLS) experiments also indicated
formation of nanoscale aggregates in aqueous media with an
average size 8.0 ± 1.8 nm (Figure S2). This size approximately
corresponds to 6-7 molecules of Pc 1 per aggregate. As a
consequence of aggregation, 1 was non-fluorescent in aqueous
solutions (Figure 2).
To determine the sensor 1 response towards EGFR, we
exposed it to low-nanomolar concentrations of the kinase in
aqueous solutions. Addition of EGFR resulted in an immediate
appearance of the fluorescent emission with a maximum at 692
nm (Figure 3A); the equilibrium was reached within several
minutes (Figure S3). As expected, the emission intensity
increased linearly upon increasing EGFR concentration, with the
limit of detection (LOD) estimated at 3 nM. The observed
behavior was consistent with our hypothesis that Pc’s emission
was restored in the presence of EGFR. We attribute the turn-on
fluorescent response to de-aggregation of the phthalocyanine
reporter upon quinazoline “anchor” binding to the EGFR kinase
site. We confirmed this assumption through comparison of the
absorption spectra of 1 in the presence of EGFR and without the
enzyme. In aqueous solution, the Q band (black trace in Figure
3B) was hypsochromically shifted indicating an extensive H-
aggregation. Addition of EGFR resulted in appearance of the
bathochromically shifted “non-aggregated” Q band at 680 nm
(blue trace in Figure 3B) indicating that at least a fraction of the
Figure 2. (A) When non-aggregated, Pc sensor
1 exhibits strong NIR
fluorescence (black trace, DMSO solution). When aggregated, 1 is completely
non-fluorescent (red trace, solution in 10% DMSO in aqueous buffer).
Concentration of Pc 1 400 nM, excitation at 665 nm. (B) Absorption spectra
indicate no aggregation in DMSO solution (black trace) and extensive
aggregation (low extinction coefficient, hypsochromic shift of the Q band) in
aqueous solution (10% DMSO in aqueous buffer). Concentration of 1 400 nM.
Inserts show expanded spectra for the respective aggregated traces.
The EGFR sensor 1 (Scheme 1) incorporates an “anchor”
which is structurally related to Gefitinib that is a known small-
molecule inhibitor of the active EGFR kinase domain.[17] IC50 of
this compound is 250-fold higher for EGFR than for any other
related tyrosine kinase.[18]
The variations of this 4-(3-
iodoanilino)quinazoline structural motif have been used in a
number of previously reported PET contrast agents.[19] Numerous
computational docking studies[20] as well as experimental X-ray
3
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Pc chromophore population was not aggregated in the presence
of EGFR. We also found that absorbance of the “non-aggregated”
band at 680 nm was proportionally increasing with an increase in
EGFR concentration (Figure S4).
S5). This indicated the key role of “anchor” specific binding in
causing de-aggregation of Pc 1.
In conclusion, we developed
a selective and sensitive
fluorescent probe that turns on upon binding to a specific protein
target and enables reliable detection at low-nanomolar
concentrations – the range that is significantly lower than can be
achieved with other reported long-wavelength fluorescent probes
for biopolymers.[5a] The turn-on response is based on the Pc
aggregation/de-aggregation mechanism, and thus independent
on the specific protein target. Binding selectivity originates form
including a well characterized small-molecule inhibitor fragment
into the sensor’s structure. While other sensors with specificity
driven by inhibitor-based domains are known,[3a, 5b, 5c, 5e, 16a, 24] our
approach has an intrinsic turn-on response (with high S/B ratio
over completely dark background), does not require any washing
steps, and does not suffer from non-target reactivity in biological
environments ultimately leading to simplified analytical
protocols.[5d] As a proof-of-concept demonstration, we prepared
a selective turn-on fluorescent sensor for EGFR kinase. It
illustrated that Pc’s aggregation/de-aggregation offers a perfect
balance between the possibility to affect this process via binding
to a specific biopolymer target while remaining unaffected by non-
specific binding.
Therefore, this approach offers broad
capabilities in designing turn-on sensors for protein targets.
Acknowledgements
We acknowledge partial support of this research by the National
Science Foundation (grant number CHE-1362686). We thank Mr.
Eric Sarnello and Prof. Tao Li for assistance with DLS
experiments, and Dr. Serhii Pakhomov for helping with solid-
phase synthesis.
Figure 3. (A) Addition of increasing amounts of EGFR results in a turn-on
fluorescent response from Pc 1 in aqueous conditions. No emission from Pc 1
is observed in the presence of BSA protein or AKT kinase. (B) Absorption profile
of Pc 1 in the presence of EGFR (blue trace) indicates at least partial de-
aggregation of the compound (appearance of the Q band at approx. 685 nm
corresponding to a non-aggregated state of the Pc chromophore); while the
aggregation remains extensive (Q band is shifted to a shorter wavelength) in
aqueous conditions (black trace). Addition of BSA protein (red trace) or AKT
kinase (green trace) does not cause de-aggregation. Experimental conditions
for (A) and (B): conc. of Pc 1 400 nM in 10% DMSO in aqueous buffer, excitation
at 665 nm.
Conflict of interest
The authors declare no conflict of interest.
Keywords: fluorescent probes • biosensors • EGFR tyrosine
kinase • phthalocyanines • aggregation
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10.1002/anie.202000108
Angewandte Chemie International Edition
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Highly sensitive and selective turn-on near-infrared fluorescent probes for detection and monitoring of specific biopolymers can be
designed based on aggregation/de-aggregation of phthalocyanine chromophores controlled by specific binding. As a proof-of-
concept example, an EGFR tyrosine kinase sensor with 3 nM limit of detection of the protein concentration is reported.
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