Doubly Caged Linker for AND-Type Fluorogenic Construction of Protein/Antibody Bioconjugates and In Situ Quantification

Article abstract of DOI:10.1002/anie.201702748

In situ quantification of the conjugation efficiency of azide-terminated synthetic polymers/imaging probes and thiol-functionalized antibodies/proteins/peptides was enabled by a doubly caged profluorescent and heterodifunctional core molecule C1 as a self-sorting bridging unit. Orthogonal dual “click” coupling of C1 with azide- and thiol-functionalized precursors led to highly fluorescent bioconjugates, whereas single-click products remained essentially nonfluorescent. Integration with FRET processes was also possible. For the construction of antibody–probe conjugates from an anti-carcinoembryonic antigen and a quinone-caged profluorescent naphthalimide derivative, the dual “click” coupling process with C1 was monitored on the basis of the emission turn-on of C1, whereas prominent changes in FRET ratios occurred for antibody–imaging-probe conjugates when specifically triggered by quinone oxidoreductase (NQO1), which is overexpressed in various types of cancer cells.

Full text of DOI:10.1002/anie.201702748

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Accepted Article
Title: Double-Caging Linker for AND-Type Fluorogenic Construction of
Protein/Antibody Bioconjugates and in situ Quantification
Authors: Shiyong Liu, Guhuan Liu, Guohai Shi, Haoyue Sheng,
Yanyan Jiang, and Haojun Liang
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201702748
Angew. Chem. 10.1002/ange.201702748
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Double-Caging Linker for AND-Type Fluorogenic Construction of
Protein/Antibody Bioconjugates and in situ Quantification
Guhuan Liu,[a] Guohai Shi,[b] Haoyue Sheng,[b] Yanyan Jiang,[a] Haojun Liang,[a] and Shiyong Liu*[a]
Abstract: We report on in situ fluorescent quantification of the
conjugation efficiency between azide-terminated synthetic polymers/
imaging probes and thiol-functionalized antibodies/proteins/peptides,
by utilizing a doubly caged profluorescent and heterodifunctional core
molecule (C1) as the self-sorting bridging unit. Orthogonal dual ‘click’
coupling of C1 with azide- and thiol-functionalized precursors leads to
highly fluorescent bioconjugates, whereas single click products of C1
remain essentially nonfluorescent. This ‘AND’ logic gate-type
fluorogenic feature also enables further integration with FRET
processes. For the construction of antibody-probe conjugates from an
anti-carcinoembryonic antigen and a quinone-caged profluorescent
naphthalimide derivative, the dual ‘click’ coupling process with C1 can
be conveniently monitored via emission turn-on of C1, whereas
prominent changes in FRET ratios occur for antibody-imaging probe
conjugates when specifically triggered by quinone oxidoreductase
(NQO1), which is overexpressed in various types of cancer cells.
in diagnostic/imaging functions. The introduction of fluorogenic
reactions into bioconjugates synthesis should enable the
convenint monitoring of coupling efficiency and further integration
with other photophysical processes. Previously, a variety of
fluorogenic ‘click’ reactions between small molecule caged
profluorophores and complementary reactive activators have
been developed.[4] As fluorescence emission was only switched
on when triggered by bioorthogonal reactions, background
signals from excess probes were largely eliminated.
In the context of protein-polymer conjugates fabrication with
fluorogenic coupling features, we are only aware of two relevant
reports up to date.[5] In 2009, Cornelissen et al.[5a] reported
fluorogenic CuAAC reaction between profluorescent 3-azido-
coumarin-terminated PEG and alkyne-functionalized bovine
serum albumin (BSA). O’Reilly and coworkers[5b] reported the
fluorogenic conjugation of 2,3-dibromomaleimide-terminated
PEG2k onto salmon calcitonin (sCT). In both cases, the extent of
bioconjugation could be in situ determined via emission changes
resulting from ‘single click’ fluorogenic reactions. However, as
commonly practiced during ‘grafting-to’ fabrication of protein-
polymer conjugates,[6] the chemical design is not modular as the
reactive functionality need to be preinstalled on the PEG terminus.
Covalent functionalization of proteins, peptides, and antibodies
with synthetic polymers, drugs, and imaging probes affords
clinically important protein-polymer conjugates[1] and antibody-
drug conjugates.[2] The fabrication of these therapeutic
bioconjugates relies on the appropriate choice of linker
chemistries and high-efficiency ‘click’-type conjugation reactions
such as Staudinger ligation, Michael addition, copper-catalyzed
azide-alkyne cycloaddition (CuAAC), and strain-promoted azide-
cycloalkyne cycloaddition (SPAAC).[3] Further advancement in
this field requires the introduction of new design criteria with
features such as modular design, facile synthesis, and capability
of being optically traced and multifunctional integration.
Traditionally, monitoring the formation of protein/antibody
bioconjugates mainly relies on ex situ techniques such as sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
mass spectrometry, high-performance liquid chromatography
(HPLC), and gel permeation chromatography (SEC). This
prohibits in situ real-time monitoring of bioconjugation processes.
We envisage that endowing fluorescent monitoring and optically
trackable features with protein/antibody bioconjugates would
allow for the construction of theranostic bioconjugates with built-
Scheme 1. Design strategy for in situ fluorescent quantification of the coupling
efficiency between azide-terminated functional agents (e.g., molecular probes
and PEG) and thiol-functionalized proteins/peptides/antibodies by utilizing
doubly caged heterodifunctional C1 as the self-sorting bridging unit. Orthogonal
dual ‘click’ conjugation of C1 with azide and thiol moieties leads to highly
fluorescent functional bioconjugates, whereas single click products of C1
remain essentially nonfluorescent.
[a]
Dr. G. Liu, Dr. Y. Jiang, Prof. Dr. H. Liang, Prof. Dr. S. Liu
CAS Key Laboratory of Soft Matter Chemistry, Hefei National
Laboratory for Physical Sciences at the Microscale, iChem
(Collaborative Innovation Center of Chemistry for Energy Materials),
Department of Polymer Science and Engineering
University of Science and Technology of China
96 Jinzhai Road, Hefei, Anhui Province 230026, China
E-mail: sliu@ustc.edu.cn
We speculate that if heterodifunctional profluorophore
containing two orthogonal ‘click’ motifs was utilized as the
bridging linker, modular coupling of proteins/antibodies with
polymers/imaging probes could be achieved if complementary
reactive handles were installed. To introduce the fluorogenic
feature for in situ assessment of the conjugation efficiency, the
heterodifunctional profluorophore has to be doubly caged and
only exhibits emission turn-on upon dual ‘click’-enabled formation
[b]
Prof. Dr. G. Shi, H. Sheng
Department of Urology, Fudan University Shanghai Cancer Center,
Department of Oncology, Shanghai Medical College
Fudan University
270 Dong'An Road, Shanghai 200032, China
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
http://dx.doi.org/10.1002/anie.201702748.
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10.1002/anie.201702748
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of conjugates. Herein, we report on the fluorogenic construction
of protein-polymer and antibody-probe conjugates via orthogonal
dual ‘click’ coupling with doubly caged profluorophore (Scheme
1). After preliminary structural screening and optimization, core
molecule C1 was chosen. This coumarin derivative contains
heterodifunctional reactive moieties, which undergo CuAAC and
Michael addition reactions with azide- and thiol-functionalized
precursors, respectively. Serendipitously, 7-ethynyl and 3-alkenyl
amide/ester functionalities of C1 could serve as double
fluorescence quenchers, and coumarin emission could only be
switched-on upon ‘AND’-type dual ‘click’ reactions. Since single
‘click’ reaction of C1 with either thiol or azide-anchored precursors
cannot lead to emission turn-on, C1-mediated coupling between
azide-functionalized imaging probes/polymers and thiol-
containing antibodies/proteins could be in situ optically monitored
(Scheme 1).
both CuAAC and thiol-ene Michael addition fluorogenic reactions
(Figure 1d and Figure S7f).
The screening of doubly caged profluorescent core molecule
with two distinct types of reactive moieties was a process of trial-
and-error. At first, we came across with C3 as a synthetic
intermediate (Figure S1).[7] We observed that only dual CuAAC
and Michael addition ‘click’ reactions of C3 with azide- and thiol-
containing molecules lead to a highly fluorescent product,
whereas single click products of C3 remain essentially
nonfluorescent (Figure S1). Thus, C3 obviously exhibits doubly
caged fluorogenic behavior. However, we later found that the
Michael addition reactivity of -unsaturated ketone moiety in C3
was quite low towards structurally hindered thiol moieties such as
those in BSA proteins (Figure S1c).
We then designed heterodifunctional C2 containing both
propargyl ether and highly thiol-reactive alkenyl amide/ester
moieties[8] as a possible candidate (Figure S2). Unfortunately, C2
lacks double-caging fluorogenic behavior because Michael
addition reaction of C2 with mercaptoethanol (ME) already leads
to emission turn-on (Figure S2b). In 2004, Fahrni and
coworkers[4b] reported that 7-ethynyl-coumarin derivative could
serve as a fluorogenic substrate and its click reaction with azide-
containing molecules could switch on coumarin emission. This
feature prompted us to design C1 containing both 7-ethynyl and
3-alkenyl amide/ester moieties. Note that all screened core
molecules (C1-C3) and synthetic intermediates were synthesized
from commercially available starting materials (Scheme S1) and
fully characterized (Figures S3-S6).
The orthogonal ‘click’ reactivity and fluorogenic feature of C1
were then investigated (Figure 1a). Upon treating with both AP
and ME, the emission intensity of C1 at 415 nm increased ~77.8-
fold within 2 h (Figures 1b). Meanwhile, coumarin emission
exhibited no changes when C1 was separately reacted with either
AP or ME (Figure 1b). Thus, C1 displays the desired double-
caging fluorogenic feature. To further examine whether the
emission enhancement of C1 resulting from double ‘click’
reactions could be utilized for in situ quantifying the conjugation
efficiency, single click products (C1-ME and AP-C1) were
synthesized (Figures S7a-d), which are nonfluorescent (Figure 1c
and S7f). For the CuAAC reaction between C1-Me and AP, HPLC
analysis revealed the gradual formation of double ‘click’ product
AP-C1-ME; meanwhile, C1 emission intensities also increased
and reached a maximum value (Figure 1c). On the other hand,
thiol-ene reaction of AP-C1 with ME was much faster (Figure S7f),
indicating that CuAAC reaction is the rate-determining step during
one-pot double ‘click’ reactions. Most importantly, emission
intensities are linearly correlated with the coupling efficiency for
Figure 1. (a) Schematics for ‘AND’ logic-type fluorogenic reactions of doubly
caged profluorescent C1 with thiol- and azide-containing model compounds. (b)
Emission intensities and fluorescence emission spectra (inset in b, λex = 365 nm;
with both ME and AP) recorded for C1 (200 μM; PBS buffer containing 10 v/v%
DMSO) upon reacting with or without ME (400 μM) in the presence or absence
of AP (200 μM). Emission spectra (c, left), HPLC traces (c, right, methanol/H2O
6/4 v/v; 254 nm absorbance), and correlation of emission intensities (λem = 420
nm) with conjugation efficiency (d) recorded for C1-ME upon reacting with AP
in the presence of CuSO4/Na-ascorbate. (e) DMF GPC traces (PEG20K as
external standard), (f) correlation of normalized emission intensities (~420 nm)
with conjugation efficiency, and emission spectra (inset in f, λex = 365 nm)
recorded for HS-PDMA37 (0.76 g/L) upon reacting with C1 (200 μM) and PEG45
-
N3 (200 μM) in the presence of CuSO4/Na-ascorbate.
The above results prompted us to further utilize doubly caged
fluorogenic C1 as a dually reactive core molecule for the modular
fabrication of functional protein/antibody bioconjugates and in situ
optically monitoring the conjugation process. As a model system,
we then attempted to in situ quantify the ‘click’ coupling efficiency
between azide- and thiol-functionalized polymer precursors by
utilizing doubly caged C1 as double-caging fluorogenic bridging
linker. Thiol-terminated poly(dimethyl acrylamide) (HS-PDMA37)
(Figure S8) and azide end-functionalized PEG (PEG45-N3) were
synthesized first. The coupling efficiency was quantified from
traditional ex situ GPC results. C1-mediated double ‘click’
conjugation between PEG45-N3 and HS-PDMA required ~2-3 h to
complete (Figure 1e), which was accompanied with a cumulative
~72-fold increase in emission intensity (Figure 1f inset).
Intriguingly, linear correlation between the coupling efficiency and
emission intensity was obtained (Figure 1f). Note that PEG45-N3,
HS-PDMA37, and C1 linker were at 1:1:1 molar ratio, and the
complete consumpution of PEG45-N3 and HS-PDMA indicated
that there was no need for further purification. To the best of our
knowledge, this represents the first example of modular and
fluorogenic construction of synthetic diblock copolymers,[9] and
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the coupling efficiency could be monitored in situ using
initially caged by covalently linked trimethyl-locked quinone
(Q3PA), but could be selectively switched on via quinone
oxidoreductase (NQO1)-triggered cleavage.[10] Thus, for the
antibody-imaging probe ensemble, FRET between bridging
coumarin chromophore and naphthalimide (NAM) will occur.
NQO1, overexpressed up to ~50-fold in the cytosolic milieu of
various human tumor cells could then be detected and quantified
in a ratiometric fluorescent manner. ACEA, anti-carcinoembryonic
antigen, was chosen to construct antibody-probe conjugates, as
CEA is also overexpressed in most types of carcinomas (Scheme
1 and Figure 3a).
fluorescence technique (Scheme 1).
Figure 2. (a) Schematics for the conjugation of bovine serum albumin (BSA) or
reduced BSA (BSAred) with PEG227-N3 bridged by C1. (b) SDS-PAGE results
recorded for 60 μM BSA (left) and reduced BSA (right) upon reacting with C1
(600 μM) and PEG227-N3 (600 μM) in the presence of CuSO4/Na-ascorbate. The
upper row (with Coomassie blue staining) and lower row images in (b) were
taken under white light and UV irradiation, respectively. (c) Time-dependent
emission intensities (λex = 365 nm, λem = 420 nm) recorded for original and
reduced BSA (4 g/L, 60 μM) upon reacting with C1 (600 μM) and PEG227-N3
(600 μM) in the presence of CuSO4/Na-ascorbate. (d) Correlation of emission
intensities (~420 nm) with protein conjugation efficiency determined via
densitometric quantification of the SDS-PAGE data (b).
Next, we utilized C1-mediated double ‘click’ fluorogenic
reactions to construct protein-polymer conjugates and quantify
the conjugation efficiency. Thus, BSA was selected as a model
protein, which contains a single free surface-exposed thiol moiety
at Cys-34. The conjugation of PEG227-N3 with BSA by C1 was
then investigated (Figure 2a). Upon reacting with PEG-N3 and
native BSA in the presence of C1, the emission intensity gradually
increased and tended to stabilize within ~4 h (Figure 2c). SDS-
PAGE results revealed partial formation of fluorescent BSA-PEG
conjugates and presence of considerable unreacted native BSA
(Figure 2b, Figures S9a-S10). Densitometric quantification of gel
electrophoresis bands revealed that >70% BSA remained intact
after 4 h reaction time (Figure 2b). This should be due to the fact
that the majority of Cys-34 residues in commercial BSA batches
are in the oxidized form (~50% as quantified by Ellman's reagent).
We further investigated the fluorogenic conjugation between
PEG227-N3 and reduced BSA, in which the reduction of disulfide
linkages by tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
liberates more free thiol moieties. Significantly higher emission
intensities were observed within ~4 h (Figure 2c). Complete
consumption of reduced BSA and formation of BSA-PEG
conjugates could be achieved within ~3-4 h (Figure 2b and Figure
S9c). Not that only the gel electrophoresis bands of PEG-C1-BSA
conjugates emit fluorescence (Figure 2b). Most importantly, a
linear correlation between the conjugation efficiency and emission
intensities was obtained (Figure 2d).
Figure 3. (a) Schematics for the fabrication of antibody-imaging probe
conjugates from ACEA and fluorescently caged naphthalimide (QNAM-N3) via
double click reactions with C1, and subsequent enzyme-triggered turn-on of
FRET process. Emission spectra (b, λex = 365 nm) and emission intensities
(inset in b, λem = 435 nm) recorded for QNAM-N3 (2.5 mM) upon reacting with
C1 (2.5 mM) and ACEA (1 mg/mL) in the presence of CuSO4/Na-ascorbate. (c)
Emission spectra (λex = 365 nm) and (inset in c) time-dependent emission
intensity ratios, I530nm/I435nm, recorded for QNAM-C1-ACEA (1 mg/L) upon
treating with NQO1 enzyme (20 ng/mL) and NADPH (10 M). (d)
Representative blue channel (coumarin, 430±20 nm) and green channel
(naphthalimide, 530±20 nm) CLSM images (scar bar: 10 m) recorded for
HepG2 (CEA-deficient, NQO1-deficient), LS180 (CEA-positive, NOQ1-
deficient), and HT29 (CEA-positive, NQO1-positive) cells upon incubating with
QNAM-C1-ACEA (0.05 mg/mL) for 12 h; the bottom row represents an overlay
of blue and green channel images.
During C1-mediated ‘AND’-type fluorogenic conjugation of
ACEA with QNAM-N3, we could only observe the gradual
enhancement of emission band at ~435 nm, corresponding to the
emission turn-on of C1 linker (Figure 3b). Note that NAM emission
at ~530 nm was still caged by Q3PA. A cumulative ~66-fold
increase in emission intensity was recorded during conjugation.
Thus, we could in situ quantify the conjugation efficiency based
on emission intensity changes of C1.
Upon co-incubating with NADPH/NQO1, emission intensity of
QNAM-N3 precursor at ~540 nm increased ~52 times (Figures
S12). Moreover, the emission band of AP-C1-ME overlaps with
the excitation band of NQO1-treated QNAM-N3, but not with that
of original QNAM-N3 (Figure S13). During co-incubating of
QNAM-C1-ACEA bioconjugates with NADPH/NQO1, a prominent
increase of NAM emission intensities (~530 nm) was observed,
which was accompanied with a considerable reduction in C1
emission at ~435 nm. Within ~1 h, ~20-fold change in FRET ratios
(I530nm/I435nm) was recorded (Figure 3c inset). The above results
Antibody-directed molecular imaging offers huge potentials
for medical diagnosis and evaluation of therapeutic responses,
and could be directly integrated with surgical or endoscopic
procedures.[2] As
a further demonstration, we constructed
functional antibody-imaging probe bioconjugates via C1-mediated
modular approach (Scheme 1 and Figure 3). Fluorescence
emission of the conjugated probe, QNAM (Figure S11), was
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indicated effective FRET between C1 linker and enzymatically
generated NAM residues. The abrupt changes in FRET intensity
ratios suggested that the antibody-probe conjugates could serve
as an excellent probe for the assay of NQO1 enzyme levels and
bioactivities. Compared to previous works by McCarley and
coworkers[10] concerning single emission band-based NQO1
molecular probes, fluorogenically fabricated QNAM-C1-ACEA
hybrid probes provide advantages including self-calibration,
ratiometric fluorescent detection, and enhanced selectivity and
site-specificity.
Keywords: bioconjugates • prodrugs • click chemistry • FRET •
fluorescent probes
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hybrid probes prompted us to further explore the possibility of
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level and NQO1 level/bioactivity. We exposed live HepG2 (CEA-
deficient, NQO1-deficient), LS180 (CEA-positive, NOQ1-
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(Scheme 1 and Figures 3a-3b).
In summary, we reported the screening of doubly caged
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modular construction of functional conjugates allows for facile in
situ quantification of the coupling efficiency via single band
emission intensity changes. Fluorogenically constructed
antibody-imaging probe conjugates, QNAM-C1-ACEA, could
selectively detect oxidoreductase such as NQO1 in a ratiometric
fluorescent manner. The reported double-caging strategy and
associated modular/fluorogenic fabrication of functional
protein/antibody conjugates could further be generalized to
integrate with other types of bioorthogonal chemistries.
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Acknowledgements
The financial support from National Natural Science Foundation
of China (NNSFC) Project (51690150, 51690154, 21674103, and
21504087) and International S&T Cooperation Program of China
(ISTCP) of MOST (2016YFE0129700) is gratefully acknowledged.
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10.1002/anie.201702748
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Entry for the Table of Contents
COMMUNICATION
Fluorogenic Bioconjugates: Doubly
caged profluorescent and
G. Liu, G. Shi, H. Sheng, Y. Jiang, H.
Liang, S. Liu*
heterodifunctional core molecules
were screened, and used for
orthogonal dual ‘click’ reactions to
fabricate functional protein/antibody
bioconjugates. Both conjugate
formation and subsequent imaging
probe activation could be in situ
quantified via fluorescence emission
intensity and FRET ratio changes,
respectively.
Page No. – Page No.
Double-Caging Linker for AND-Type
Fluorogenic Construction of Protein/
Antibody Bioconjugates and in situ
Quantification
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