A Ligand-Directed Nitrophenol Carbonate forTransient in situ Bioconjugation and Drug Delivery

Article abstract of DOI:10.1002/cmdc.202000655

Here we report the first use of ligand-directed proximity accelerated bioconjugation chemistry in the tandem delivery and release of a therapeutic payload. To do this, we designed a nitrophenol carbonate for ligand-directed in situ bioconjugation of a prodrug payload to a protein. The transient nature of our conjugation chemistry renders the protein a depot for time-dependent release of active drug following hydrolysis and self-immolation. In our model system, using an immunostimulant prodrug, biotin ligand, and avidin protein, we observe release of bioavailable immunostimulant both spectroscopically and with an immune cell line over 48 h. Avidin co-crystalized with the nitrophenolate directing group verified the binding pose of the ligand and offered insight into the mechanism of in situ bioconjugation. Overall, this scaffold warrants further investigation for the time-dependent delivery of therapeutics and use in protein ligand pairs beyond biotin and avidin used for this work.

Full text of DOI:10.1002/cmdc.202000655

Communications
doi.org/10.1002/cmdc.202000655
ChemMedChem
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A Ligand-Directed Nitrophenol Carbonate for Transient
in situ Bioconjugation and Drug Delivery
Anthony J. Burt,[a] Parvaneh Ahmadvand,[a] Larissa K. Opp,[a] Austin T. Ryan,[a] ChulHee Kang,[a]
and Rock J. Mancini*[a, b]
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ologies in bioconjugate drug delivery. For example, in situ
bioconjugates have been explored using ligand-directed small-
molecule probes whereby ligand-protein complexation leads to
covalent affinity-labeling resulting in a bioconjugate.[6] We
envisioned that if this concept could be expanded to develop
an affinity labeling platform that yields in situ bioconjugates
capable of time-dependent drug release, this could present a
new way to bypass the drawbacks of producing bioconjugate
drugs while still harnessing the therapeutic benefits by
producing them in situ.
Ligand-directed affinity labeling techniques have been
pioneered by Hamachi and coworkers.[7] They have developed
multiple electrophilic moieties over the past decade capable of
labeling nucleophilic amino acid side chains on proteins with
small-molecule probes peripheral to the ligand binding site.[8–16]
It is hypothesized that upon ligand binding, an electrophilic
moiety on the ligand-directed reagent is attacked by a
nucleophilic side chain, leading to covalent attachment of the
probe to the target protein. Ligand-directed affinity labeling has
demonstrated success with soluble proteins such as carbonic
anhydrase as well as membrane bound proteins such as the
folate receptor with examples of in situ, in vitro, ex vivo, and
in vivo labeling.[11,17] Building on these concepts, we designed a
scaffold (Figure 1) to allow for time-dependent drug delivery via
a transient in situ bioconjugate formed by ligand-directed
affinity labeling.
Our ligand-directed nitrophenol carbonate (LDNPC)
chemistry was built around the leaving group ability of nitro-
phenols paired with the modest stability of nitrophenol
carbonates at physiological conditions. To adapt the affinity
probe to drug delivery, we included a self-immolative spacer[18]
between the carbonate electrophile and drug payload. We
hypothesized that following affinity labeling of the payload and
spacer to the target protein, hydrolysis would lead to self-
immolation to liberate the active payload. Importantly, this
scaffold can be built from inexpensive abundant precursors: p-
nitrophenol, formaldehyde, cyanide, and ethylene glycol.
LDNPC reagents exhibit excellent bench stability as a lyophi-
lized powder and modest solution stability. Thus, we envision
LDNPC reagents could mitigate many drawbacks of conven-
tional bioconjugate drug production while retaining their
alluring benefits via in situ bioconjugate synthesis.
Here we report the first use of ligand-directed proximity
accelerated bioconjugation chemistry in the tandem delivery
and release of a therapeutic payload. To do this, we designed a
nitrophenol carbonate for ligand-directed in situ bioconjugation
of a prodrug payload to a protein. The transient nature of our
conjugation chemistry renders the protein a depot for time-
dependent release of active drug following hydrolysis and self-
immolation. In our model system, using an immunostimulant
prodrug, biotin ligand, and avidin protein, we observe release
of bioavailable immunostimulant both spectroscopically and
with an immune cell line over 48 h. Avidin co-crystalized with
the nitrophenolate directing group verified the binding pose of
the ligand and offered insight into the mechanism of in situ
bioconjugation. Overall, this scaffold warrants further investiga-
tion for the time-dependent delivery of therapeutics and use in
protein ligand pairs beyond biotin and avidin used for this
work.
Bioconjugates are an alluring therapeutic modality that, relative
to small molecules, suffer drawbacks in production including
high cost, poor batch reproducibility, and poor shelf life; all of
which hinder the development of bioconjugates as drugs.[1]
Despite these challenges, antibody drug conjugates such as
brentuximab vedotin and trastuzmab emtansine which have
found clinical success and FDA approval (2011 and 2013
respectively) exemplify the benefits of bioconjugates including
tissue specificity,[2] synergistic effects of the biomolecule and
drug,[3,4] and increased biological half-life.[5] These examples and
benefits have pushed researchers to explore further method-
[a] A. J. Burt, P. Ahmadvand, L. K. Opp, A. T. Ryan, Prof. C. Kang,
Prof. R. J. Mancini
Department of Chemistry, Washington State University
1470 NE College Ave
Pullman, WA 99164 (USA)
[b] Prof. R. J. Mancini
The Gene & Linda Voiland School of Chemical Engineering and Bioengineer-
ing
Washington State University
1470 NE College Ave
Pullman WA 99164 (USA)
E-mail: Rmancini@wsu.edu
For this proof-of-concept work, the protein ligand pair of
avidin-biotin was chosen because: 1) the avidin-biotin crystal
structure is known, and verified the presence of possible
peripheral nucleophiles (Nu) to the biotin binding site.[19] 2) The
high affinity of biotin for avidin (Kd �10À 15 M)[20] was hypothe-
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000655
© 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
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ChemMedChem
avidin complex to serve as a prodrug of IMQ. Furthermore, the
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IMQ payload is an active immunostimulant for the RAW-Blue
murine macrophage cell line (RB Cells) which links activation of
the inflammatory transcription factor NF-kB to secreted embry-
onic alkaline phosphatase which can be readily quantified by
colorimetric assay. For these reasons, the utility of LDNPC
chemistry as a drug delivery technique was demonstrated using
a biotin directing group to render avidin as a depot for time-
dependent IMQ release in situ.
The biotin-LDNPC-IMQ reagent (8) was synthesized via the
following convergent synthetic approach (Figure 2a) in which
ligand (Fragment A) can be combined with nitrophenol moiety
(Fragment B) and the resulting biotin-phenol (Fragment A+B)
is then attached to IMQ payload (Fragment C) to yield LDNPC
(8). Fragment A is prepared in one step by the known
procedure of activating the carboxylate of biotin as succinimidyl
ester (1) with EDC.[29] Fragment B is prepared over three steps
from p-nitrophenol (PNP). Briefly, chloromethylation of PNP is
accomplished under acidic conditions with methylal and HCl
gas.[30] The resultant benzyl chloride (2) is displaced by cyanide
to yield benzyl cyanide (3) which is subsequently reduced with
borane to yield primary amine (4).[31] Fragment C is synthesized
over two steps from ethylene glycol and p-nitrophenyl chlor-
oformate, yielding the ethylene glycol dicarbonate (5). Symme-
try of (5) is broken under microwave conditions with IMQ to
yield (6), the intermediate self-immolative spacer (Fragment C).
Fragments A and B are combined in DMF with tertiary amine
base to yield biotin-phenol (7), and then reacted with Fragment
C under basic conditions. After 24 h, equilibrium is reached and
LDNPC (8) is obtained in 8.7% yield over 5 synthetic steps from
PNP. Complete synthetic procedures and characterization may
be found in the Supporting Information.
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With LDNPC (8) in hand, we next characterized aqueous
stability. First, the pKa of biotin-phenol leaving group (7) was
determined to be 8.02 by titration with sodium hydroxide in
water (Figure S1) suggesting that LDNPC (8) should be more
resistant to base mediated hydrolysis at lower pH values.
Figure 1. Drug delivery via ligand-directed nitrophenol carbonate (LDNPC)
chemistry involves 3 steps to release active payload. Step 1. Addition of
LDNPC reagent to target protein results in protein-ligand complex. Step 2.
Nucleophile (Nu) peripheral to the binding site attacks the LDNPC reagent
displacing the nitrophenolate-modified ligand, resulting in a covalently
modified target protein. The nitrophenolate-modified ligand is free to
dissociate. Step 3. Hydrolysis and self-immolation result in release of
bioavailable payload at the location of the covalently modified protein. In
this work X=NH.
°
Aqueous stability of (8) was monitored over 48 h at 37 C in
citrate phosphate buffer at pH: 4, 5, 6, and 7, typical of
cellular organelles,[32] and release of IMQ from direct
hydrolysis was quantified by HPLC. Here, LDNPC (8) displayed
a trend of increasing aqueous stability with decreasing pH.
However, even at pH 7, total IMQ released from direct
hydrolysis over 4, 12, and 48 h was determined to be 0, 5,
and 77%, respectively (Figure S2). Stability at pH 7 was
adequate over 12 h considering the high, albeit attenuated,
affinity of our LDNPC reagent for avidin. This is also similar to
Hamachi et al. who observe saturated covalent labelling with
their ligand-directed acyl imidazole chemistry within 7 h for
carbonic anhydrase I.[17]
sized to lead to rapid covalent labeling. 3) We believe that
LDNPC chemistry could add a new tool to the many established
applications that utilize avidin-biotin biotechnology.
The
imidazoquinoline
immunostimulant
Imiquimod
(IMQ)[21–24] was chosen as payload for our proof-of-concept
LDNPC reagent because our previous work with enzyme-
We next examined the influence of avidin on the degrada-
tion of LDNPC (8). Due to the 4-nitrophenolate leaving group of
(7), it was observed that cleavage of the carbonate phenol
bond liberates a colorimetric indicator of degradation. We
exploited this property to follow degradation of (8) in pH 7
buffer, with avidin or bovine serum albumin (negative control).
In the presence of avidin, significant (80 μM) release of (7) was
directed IMQ allowed us to estimate its behavior as
a
prodrug.[25,26] Based on this, and the well-defined structure
activity relationship of the imidazoquinoline drug class,[27,28] we
hypothesized that linkage at the aminoquinoline nitrogen
would lead to abrogated immunostimulatory activity and there-
fore allow the LDNPC reagent, as well as the covalently labeled
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Figure 2. a) Synthetic route to ligand-directed nitrophenol carbonate (LDNPC) (8). Fragment A i: EDC-HCl, NHS, DMF, RT 24 h. Fragment B ii: Methylal, HCl (g),
°
°
°
°
°
HCl conc., H2SO4 conc., 72 C, 4 h. iii: Acetonitrile, 5 M KCN (aq), 0 C 30 min, 60 C 30 min. iv: 1) BH3-THF 1 M in THF, 100 C, 4 h. 2) HCl 0.8 M in MeOH, 100 C,
°
°
12 h. Fragment C v: p-Nitrophenyl chloroformate, pyridine, DCM, 90 C, 24 h . vi: Imiquimod, THF, MW Irradiation: 90 C, 55 min, 1 bar. (A+B) vii: DIPEA, DMF,
RT, 18 h. (A+B)+C viii: DIPEA, DMF, RT, 24 h b) Degradation kinetics of 120 μM LDNPC (8) was measured as liberated nitrophenolate (7) in the presence of
Avidin (Red, 0.42 mg/mL), Avidin+Biotin (Blue, 0.42 mg/mL, 12 μM), Bovine Serum Albumin (BSA) (Black, 0.42 mg/mL) or citrate phosphate buffer (Purple,
50 mM pH 7). The effects of avidin on LDNPC (8) are significant compared to BSA and Buffer (p <0.05 calculated at 2, 5, 10, 15, 20 min; error bars are standard
deviation of the mean for triplicate experiments). See the Supporting Information for longer timepoints.
measured compared to minimal hydrolytic degradation with
BSA or buffer alone (<20 μM). Expectedly, competition from
biotin attenuates degradation emphasising the requirement for
ligand complexation and nucleophilic attack (Figure 2b, S3).
After using colorimetry to observe the initial formation of
bioconjugate over 90 min, we next used HPLC to quantify the
release of IMQ over 48 h with and without avidin. Both solutions
showed measurable amounts of IMQ release. Interestingly, the
sample with avidin showed a significant reduction in IMQ
released (Figure S4), and these measurements combined with
the colorimetric differences lead us to conclude that avidin is
successfully labelled by LDNPC (8) and that the transient in situ
bioconjugate degrades more slowly than (8) alone. Based on
the possible nucleophilic side chain addition to (8), we expect
the resulting bioconjugate to be considerably more stable than
the carbonic acid formed from direct hydrolysis. Therefore,
although subsequent experiments were unable to determine
the exact Nu that participates in conjugation, we reasoned that
the results of this experiment are consistent with transient
covalent attachment of the self-immolative spacer and IMQ to
avidin.
biotin-phenol leaving group (7). This approach resulted in a
1.58 Å resolution crystal structure of the avidin-biotin-phenol
(7) complex (PDB: 6XND, Figure 3, Table S1).
As expected, the biotin moiety of (7) is situated in a
similar binding pose as biotin,[19] buried within the binding
pocket of the eight-stranded anti-parallel β-barrel. Emerging
from the pocket and surrounded by solvent accessible
flexible loop regions is the nitrophenol moiety (Figure 3a).
Isothermal titration calorimetry (ITC) shows the dissociation
constant of the ligand to be Kd =2.25×10À 7 M (Figure S5),
considerably weaker than biotin. We also determined con-
ditions to directly compare the binding of (7) and (8) by ITC,
and the results show binding affinities within one order of
magnitude (Figure S6) in agreement with binding observed
by HABA assay (Figure S7).
The nitrophenolate appears to be stabilized by hydrogen
bonds from S101 and R114 to the phenol, as well as a Van der
Waals interaction between A39 and the nitro group (Figure 3b).
Possible Nu in the region (within 12 Å of phenolate) include
T40, S41, S101, S102, T113 and K111. All are constituents of
dynamic loop regions and therefore all potentially capable of
interacting with LDNPC (8). Interestingly, the interface between
the monomers of avidin position two equivalents of ligand
within the same solvent accessible pocket, with the aromatic
rings separated by only 8.4 Å in the static crystal structure
(Figure 3c). This overcrowded region could also drive hydrolysis
to release the payload thereby relieving steric strain and
returning the protein to its native state. Taken together, this
data concretely proves that the modified biotin reagent remains
After confirming that our LDNPC reagent could create an
in situ bioconjugate with avidin, we turned to structural biology
to gain insight into the possible residues acting as Nu. Due to
hydrolysis of the bioconjugate over the crystallization period
we were unable to obtain co-crystals of avidin with (8). We also
attempted to crystalize avidin and soak in LDNPC (8), however,
these crystals did not provide complete electron density maps.
In lieu of this, we proceeded to co-crystalize avidin with the
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We next sought to demonstrate the prodrug nature of
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avidin labeled with LDNPC (8) and time-dependent release of
IMQ from the resultant in situ bioconjugate. First, to compare
the abrogated immunogenicity from LDNPC (8) to IMQ, we
treated RB cells with equimolar concentrations (1–100 μM) of
each respective compound (Figure S8). Because the RB assay
itself is conducted over 16 h, we observed some immunoge-
nicity resulting from expected hydrolysis of (8) in the cell media.
Regardless, the results show abrogated activity of LDNPC
reagent compared to IMQ, with significantly reduced immuno-
genicity for the LDNPC reagent at 50 and 100 μM (p<0.001).
Even at lower concentrations (1, 5, and 10 μM) the trend of
reduced immunogenicity is conserved. With proof of abrogated
activity, we next designed an experiment to capture the time-
dependent immunogenicity that results from release of IMQ
while simultaneously highlighting that in situ bioconjugation
results in ligand-directed immunogenicity. To accomplish these
aims, we utilized a 10 kDa molecular weight cut off centrifugal
filter device (CFD). The workflow of the assay (Figure 4a), begins
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°
by incubating avidin with LDNPC (8) for 60 s at 37 C to allow
complexation. Next, the solution was spun through the CFD
effectively isolating the IMQ-avidin bioconjugate. The bioconju-
gate was then washed, concentrated, reconstituted in buffer,
and incubated for the indicated time (0–72 h at 12 h incre-
ments) before being used in a RB cell assay that was developed
for 16 h (t=0 h was still incubated with RB cells for 16 h). The
results of the assay show increased immunogenicity over time
caused by transient bioconjugation of the LDNPC reagent to
avidin. Any uncomplexed LDNPC reagent, and likewise any IMQ
released by nonspecific hydrolysis (both <1 kDa) is washed
through the CFD. Therefore, the observed immunogenicity in
this assay results from in situ bioconjugation and subsequent
hydrolysis from avidin. We anticipated that longer incubation
times of the bioconjugate in buffer would result in greater IMQ
release and therefore higher immunogenicity approaching the
theoretical loading target of 50 μM. Indeed, increasing immuno-
genicity with increasing incubation time was observed, match-
ing the 50 μM IMQ standard by 24 h (Figure 4b). Additionally,
the avidin concentration required to deliver 50 μM of IMQ from
the bioconjugate showed small but non-negligible immunoge-
nicity on its own and we attribute this to the later time points
exceeding the IMQ standards. Overall, this experiment high-
lighted the ability of the bioconjugate to release a biologically
relevant substrate, in a time-dependent manner, sustained over
several days after in situ labeling with LDNPC (8).
Figure 3. X-ray crystal structure (1.58 Å resolution) of biotin-phenol leaving
group (7, grey) bound to avidin. a) Flexible loop regions around nitro-
phenolate moiety with proximal (<12 Å from phenolate) nucleophiles (Nu)
on a single monomer unit shaded in purple including residues S41,102,
T40,113, and K111. Monomer A and C are shaded lavender and light green,
respectively. b) Highlight of residues A39, S101, R114 (lime green) directly
implicated in stabilizing nitrophenolate in the avidin binding pocket. c) View
showing electrostatic surface. Distance between ligand bound to monomer
A and C is only 8.4 Å between the aryl rings. This view also shows how the
nitrophenolate moiety is positioned just outside the binding pocket in a
solvent accessible region.
In conclusion, we demonstrated ligand-directed nitro-
phenol carbonate (LDNPC) chemistry in a proof-of-concept
example that forms a transient in situ bioconjugate between
avidin and a self-immolative spacer with imidazoquinoline
payload. We have shown, using structural biology and ITC,
that the biotin modified with nitrophenolate, remains a
viable ligand (Kd =2.25×10À 7 M) for avidin. Although we have
yet to conclusively prove if IMQ is conjugated through a
carbamate or carbonate bond, the crystal structure of avidin
with the biotin-phenol leaving group (7) demonstrates the
abundance of nucleophiles proximal to the biotin binding
site. With S101 and K111 the two most spatially rational leads
a ligand for avidin and that proximal Nu to the carbonate
moiety of LDNPC (8) are abundant.
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Acknowledgements
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Research reported in this publication was supported by the
National Cancer Institute of the National Institutes of Health under
Award Number R01CA234115. The content is solely the responsi-
bility of the authors and does not necessarily represent the official
views of the National Institutes of Health. Crystallography was
supported by grants from NSF (CHE 1804699) and Murdock
Charitable Trust. X-ray data collection used resources of the
Advanced Light Source (beamline 5.0.2), which is a DOE Office of
Science User Facility under contract no. DE-AC02-05CH11231. We
also acknowledge the NMR facility at Washington State University.
The WSU NMR Center equipment is supported by NIH grants
RR0631401 and RR12948, NSF grants CHE-9115282 and DBI-
9604689, the Murdock Charitable Trust, and private donors Don
and Marianna Matteson.
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: Affinity Labelling
Immunochemistry · Prodrugs
· Avidin · Bioconjugation ·
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Figure 4. a) Workflow of time-dependent immunogenicity assay. Avidin was
°
incubated with (8) for 60 s, at 37 C, before separation with 10 kDa MWCO
°
centrifuge device (14,000 RCF, 5 min, 37 C). Avidin was washed (PBS
3×300 μL) and then incubated for indicated time prior to running a RB Cell
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bioconjugate following 12–24 h of incubation. Avidin has small, but
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bars are standard deviation of the mean for experiments performed in
triplicate.
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Manuscript received: August 26, 2020
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Revised manuscript received: September 21, 2020
Accepted manuscript online: October 8, 2020
Version of record online: October 14, 2020
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