A Bioorthogonal Small Molecule Selective Polymeric "clickase"

Article abstract of DOI:10.1021/jacs.0c06553

Synthetic polymer scaffolds may serve as gatekeepers preventing the adhesion of biomacromolecules. Herein, we use gating to develop a copper-containing single-chain nanoparticle (SCNP) catalyst as an artificial "clickase"that operates selectively on small

Full text of DOI:10.1021/jacs.0c06553

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Article
A Bioorthogonal Small Molecule Selective Polymeric “Clickase”
Junfeng Chen, Ke Li, Sarah Bonson, and Steven C. Zimmerman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c06553 • Publication Date (Web): 14 Jul 2020
Downloaded from pubs.acs.org on July 15, 2020
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A Bioorthogonal Small Molecule Selective Polymeric “Clickase”
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Junfeng Chen, Ke Li, Sarah E. Bonson, and Steven C. Zimmerman*
Supporting Information
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ABSTRACT: Synthetic polymer scaffolds may serve as gatekeepers preventing the adhesion of biomacromolecules. Herein, we use gating to
develop a copper-containing single-chain nanoparticle (SCNP) catalyst as an artificial “clickase” that operates selectively on small molecules
that are able to penetrate the polymeric shell. Whereas the analogous clickase with surface ammonium groups performs highly efficient
copper(I)-catalyzed alkyne–azide cyclo-addition (CuAAC) reactions on both alkynylated proteins and small molecule substrates, the new
SCNP clickase with polyethylene glycol (PEG) groups is only active on small molecules. Further, the new SCNP resists uptake by cells allowing
extracellular click chemistry to be performed. We describe two proof of principle applications that illustrate the utility of the bioorthogonal
activity. First, the SCNP catalyst is able to screen for ligands that bind proteins, including proteolysis targeting chimera (PROTAC)-like
molecules. Second, the non-membrane permeable SCNP can efficiently catalyze the click reaction extracellularly, thereby enabling in situ
anticancer drug synthesis and screening without the catalyst perturbing intracellular functions.
be able to perform reactions in cells.19 Previously, we reported the
◼ INTRODUCTION
development of a copper containing SCNP as a “clickase.” The
Gating has emerged as a useful strategy to control all aspects of
1
water-soluble polyacrylamide SCNP was covalently cross-linked
chemical catalysis. By caging in a shell (Scheme 1), increased catalyst
stability can be achieved and desired substrate selectivity controlled
as the surrounding scaffold determines the molecules able to diffuse
or bind to the catalytic center. The overall approach has been widely
used in multiple areas of chemistry. For example, in supramolecular
chemistry, cucurbituril has served as the gate-keeper of a nano-
reactor1 or a metal complex,2 in both cases turning on and off
catalysis by reversibly blocking access to the active site. Synthetic
organic chemists have used metal-organic cages to encapsulate
catalysts and alter their stability and accessibility.3,4 An example in
the biomaterials area involves a PEGylated polymeric micelle that
was reported to protect an enzyme from antibody binding and
protease degradation, while preserving its activity toward small
molecule substrates.5
This work is focused on a type of gating by catalytic single-chain
nanoparticles (SCNP). Catalytic SCNP have received considerable
attention recent years.6-12 The polymeric scaffold encapsulates and
solubilizes the synthetic catalyst in water,13,14 and binds substrates in
proximity to the catalytic sites in an enzyme-like manner to achieve
high efficiency.15,16 Some cationic SCNPs are taken up by cells,
retaining their activity and performing intracellular catalysis.17,18
Neutral Jeffamine functionalized SCNP catalysts have also shown to
with BTTAA-like ligands that are particularly effective at stabilizing
CuI. The artificial clickase performed CuAAC reactions at
unprecedented rates by binding small molecule substrates in interior
pockets, which was referred to as “uptake mode.”20 Surprisingly we
discovered that the same clickase performed highly efficient
reactions on protein surfaces. Mechanistic studies showed that the
macromolecule to biomacromolecule catalysis was realized through
an “attach mode,” wherein the SCNP supramolecularly attaches to
the protein surface using multivalent interactions.
Given that the CuAAC reaction is one of the most widely used
conjugation tools for organic chemistry and chemical biology,21 we
I
sought to expand the utility of Cu −SCNP as a clickase. In
1
particular, we sought an analogous SCNP that would retain the high
CuAAC activity at micromolar concentrations of small molecule
substrates in aqueous buffer,22 but be fully bioorthogonal. Beyond
I
1
the ability to bind proteins, Cu −SCNP is taken up by cells through
endocytosis, likely because the polycationic SCNP adheres to cell
I
surfaces. Herein, we report Cu −SCNP with surface PEG groups
2
for water-solubility. This new catalyst performs the bioorthogonal
CuAAC click reaction on small molecules with high efficiency, and
2
I
exhibits its own bioorthogonality. Thus, Cu -SCNP interacts with
proteins weakly and is not taken up by cells, allowing proof of
principle experiments such as in situ anticancer drug synthesis and
screening for ligand-protein binding.
◼ RESULTS AND DISCUSSION
2
Design and synthesis. SCNP was prepared following our
reported “folding and cross-linking” strategy.20 Thus,
poly(pentafluorophenyl acrylate) was post-functionalized with 6-
aminohexanoic acid, 3-azidopropylamine, and the mono-
MePEG1000 amide of 1,10-decane diamine. The resulting azido
polymer was intramolecularly cross-linked with N,N-dipropargyl-
-
(1-(tert butyl)-1H-1,2,3-triazol-4-yl)methanamine in water using
Scheme 1. Schematic illustration of catalyst (a) free in solution and
(b) gated by encapsulation.
the CuAAC reaction. The resulting covalent cross-linking groups are
N-tert-butyl-tris(triazolyl)methylamine ligands that, together with
the carboxylate groups, act analogously to BTTAA-like ligands.23
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Figure 1. Schematic illustration of the two SCNP catalysts. (a) The structure of cationic SCNP
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which binds and catalyzes reactions on protein
surfaces. (b) The structure of PEGylated SCNP which does not bind proteins and only perform reactions on small molecules.
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The decyl linker units were chosen to provide a hydrophobic
1
spectra were acquired by irradiating the mixture at 7.0 ppm, the
protein aromatic region. The STD signals corresponding to the
trimethyl ammonium on SCNP , the PEG groups on SCNP and
the hydrophobic alkyl chains for both SCNPs were measured and
the % STD calculated. As shown in Fig. 2b, the PEG and
binding capacity analogous to that found in SCNP whereas the
PEG shell conferred water-solubility and resistance to protein and
cell binding.
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The nanoparticle was purified by dialyzing against water and
characterized by dynamic light scattering (DLS, Figure 2a) and
transmission electron microscopy (TEM, Figure S1). The diameter
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hexamethylene groups on SCNP showed negligible and weaker
STD signals, respectively with BSA. The magnitude of these
differences is significant and consistent with weaker interactions.25,26
To further examine the potential interaction with proteins, a
fluorescence anisotropy experiment was performed using
2
fluorescein labeled BSA. As seen in Fig. 2c, SCNP exhibited
1
significantly lower polarization values than SCNP . To assess the
significance of this difference, the polarization of the SCNP ⋅BSA
complex was reexamined with increasing sodium chloride
concentration to lower the electrostatic binding (Figure S4). The
2
reduced polarization is similar to that see in SCNP and supports
the reduced adhesion afforded by the PEG shell.
Protein vs. small molecule CuAAC activity.
To test whether the
2
of SCNP was found to be around 11 nm by DLS, which is higher
1
than that for SCNP (6-7 nm) due to its larger molecular weight.
After the nanoparticle synthesis and purification, sufficient CuSO4
was added to give a 1:1 ratio of copper ion to the tris-triazolylmethyl-
II
amine crosslinks to give Cu −SCNP , which can be reduced by
2
sodium ascorbate (NaAsc) to generate the catalytically active
1
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Cu −SCNP in situ.
SCNP-protein binding study.
Replacing the cationic
2
trimethylammonium ion groups with PEG groups gives SCNP
with a potentially uncharged neutral surface, depending on the
location and ionization of the carboxylic acid groups. Indeed, the
2
PEG shell in SCNP with its lower protein association translates
into reduced uptake or attach mode catalysis, the rates of the
CuAAC click reaction between Al and alkynylated BSA (BSA-Al)
1
2
measured ζ-potential for SCNP and SCNP were 33.9 ± 1.7 mV
and 1.57 ± 0.64 mV, respectively (Figure 2a), indicating a nearly
neutral surface for the latter polymeric nanoparticle. Previously, we
1
were measured. BSA-Al was prepared by reacting BSA with the
NHS-ester of 4-pentynoic acid, the protein product containing on
average 13 alkyne groups as indicated by MALDI-MS.20 To monitor
the reactions at low concentration, a fluorogenic azido coumarin
1
reported that the carboxylate groups in SCNP accelerated its click
reaction analogous to that found in the small molecule ligand
BTTAA.23 The low ζ-potential for SCNP may indicate that the
carboxylic acid groups are protonated, form zwitterionic structures
with the tertiary amino groups, or, more likely that the PEG groups
provide a neutral surface layer.
2
1
1
(Az ) was used as the azide substrate. Thus, Az exhibits a large
increase in fluorescence after the click reaction.27 Fluorogenic
1
reactions were performed in PBS buffer containing Az (20 μM)
To assess its adhesive character, bovine serum albumin (BSA)
was chosen as the model protein to measure the potential for SCNP
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with Al (40 μM) or BSA-Al (2 μM) by using either Cu −SCNP (2
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binding. The interaction between BSA with Cu −SCNP or
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μM) or Cu −SCNP (2 μM). For reference, one of the fastest known
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small molecule catalysts CuI−BTTAA (20 μM) was used at the same
II
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2
Cu −SCNP was measured by using STD spectroscopy, which
uses the nuclear Overhauser effect (NOE) to assess the nature of
possible intermolecular interactions. The nanoparticles were mixed
with BSA in 1:1 molar ratio in deuterated PBS buffer, and the STD
copper concentration.23
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Cu −SCNP and Cu −SCNP both showed high efficiency in
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catalyzing the click reaction between the small molecules Az and
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Figure 2. (a) DLS and ζ-potential data of SCNP
μM CuII−SCNP or CuII−SCNP
with 100 μM BSA in deuterated PBS buffer (1x, pD = 7.4) irradiated at 7.0 ppm. Trimethyl ammonium group and
hydrophobic chains labelled with colors used in STD plot. (c) Fluorescence polarization of fluorescein labelled BSA (2 μM) with different
concentration of CuII−SCNP or CuII−SCNP
in PBS buffer (1x, pH = 7.4). (d) Initial reaction rates for small molecule and protein substrates with
CuII−SCNP (2 μM) or CuII−SCNP
(2 μM) in PBS buffer (1x, pH = 7.4) at room temperature. For small molecule substrate: Al (40 μM) and Az
(20 μM). For protein substrate: BSA-Al (2 μM) and Az (40 μM). Error bars represent the standard deviation of three independent experiments.
Data for SCNP and BTTAA from ref. 20.
and SCNP . (b) STD signal intensities of the trimethyl ammonium groups and alkyl chains on 100
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Al . Thus, both nanoparticles achieved >90% conversion within 5
min (Figure S2a), and their initial rates were around 15 times faster
than that of CuI−BTTAA (Figure 2d).23 As previously observed,
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Cu −SCNP exhibited a high rate of reaction in protein labeling,
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performing the click reaction between BSA-Al and Az 12-fold faster
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than Cu −BTTAA. However, Cu −SCNP failed to catalyze the
reactions on BSA-Al, exhibiting an 11 and 137-fold slower rate than
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that of Cu −BTTAA or Cu -SCNP , respectively. The fluorogenic
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reactions were also performed on a mixture of Al and BSA-Al, and
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Cu −SCNP showed a 60-fold preference for the small molecule
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over the protein (Figure S2b). These results suggest that the PEG
groups on the clickase block its active sites for protein substrates,
while maintaining the capability to uptake and catalyze click
reactions with small molecule substrates.
Protein-ligand binding study
.
Uptake mode catalysis by
CuI−SCNP requires the nanoparticle to bind small molecules within
their interior, the binding constants estimated to be in the micro-
molar range.16,20 If a protein binds a small molecule azide or alkyne
with a comparable or lower KD, its uptake mode click reaction would
be inhibited, but only if attach mode is not operative (Figure 3a).
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The demonstration that the PEG groups in Cu −SCNP prevent its
interaction with proteins raises the interesting possibility that the
nanoparticle might be used to screen for small molecules that are
bound to proteins of interest. To test this hypothesis, fluorogenic
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reactions were performed between Az and seven alkyne substrates
with or without adding carbonic anhydrase II.
Figure 3. Protein-ligand binding study by using CuI-SCNP
2
. (a)
Carbonic anhydrase II (CA) was chosen as an inexpensive,
readily available, and prevalent enzyme. Its inhibitors were once
commonly used therapeutic agents, but now are limited mostly to
glaucoma treatment. The proof of principle screen for CA binders
used a reactivity index to estimate the small molecule binding
capability. The reactivity index is defined as the ratio of the initial
Illustration of competitive binding with proteins making nanoparticle
only active on free substrates. (b) Chemical structure of alkyne
substrates and their reactivity index. Fluorogenic reactions in PBS
buffer: [CuI-SCNP
[Al] = 1 μM with/without [CA] = 2 μM.
] = 4 μM, [Az ] = 2 μM, [NaAsc] = 200 μM and
2 1
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prepared with either a short (Al ) or a long linker (Al ) (Figure 4a).
NAv is the deglycosylated form of avidin and exhibits a neutral
surface charge. The crystal structure of the Nav-biotin complex was
obtained from the protein data bank (PDB, ID: 2AVI) and imported
into the molecular operating environment (MOE) software. The
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biotin structure in the protein was mutated into Al or Al using the
build feature in MOE, and the structure underwent energy
minimization. Shown in Figure 4a are two identical subunits of NAv,
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one colored blue and the ligands colored red. For the NAv-Al
complex, the whole Al substrate was buried inside the protein,
whereas in NAv-Al , the linker chain reaches out to the protein
surface allowing the alkyne group to be accessed.
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The initial rates of the CuAAC click reaction between Az and
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either free ligands Al and Al or bound ligands NAv-Al or NAv-
Al catalyzed by Cu -SCNP and Cu -SCNP were measured. As
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shown in Figure 4b, both nanoparticles behaved similarly with free
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and bound Al . Thus, free ligand Al reacts in uptake mode, but the
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neutravidin-bound Al is inaccessible. Even with the longer linker
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group in NAv-Al , Cu -SCNP showed almost no activity in the
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click reaction with Az , consistent with the observation that only
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free small molecules are reactive toward the PEGylated nanoparticle.
These results indicate that protein-ligand binding can be assessed by
using the two nanoparticles as the dual logical gates. As shown in
Figure 4c, the assay would be conducted by performing fluorogenic
reactions with Cu -SCNP and Cu -SCNP separately. The protein
ligand interactions can be assessed via the fluorescence readout with
three possibilities: free (on and on), on the protein surface and
accessible (on and off) or bound to the protein interior and
inaccessible (off and off).
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Recently, proteolysis targeting chimera (PROTAC) technology
has received considerable attention because of its potential to
degrade pathogenic proteins and particularly to treat diseases
originating from “undruggable” proteins, those for which
a
traditional inhibitor strategy is not available.29 PROTAC ligands
typically contain two different protein binding groups on each end
of a linker, and thus are able to bring together the target protein and
the protein degradation machinery. However, PROTAC binding is
highly dependent on the environment and concentrations, and some
suffer from the “hook effect,” resulting in poor conjugation of the
two proteins.30 Thus, evaluating the binding status of both end-
group ligands is essential in developing PROTAC ligands.
Figure 4. Activity of CuI-SCNP
complexes. (a) The chemical structures of two alkyne functionalized
biotin compounds and the MOE simulation results of NAv-Al and
NAv-Al complexes. (b) The initial reaction rates of fluorogenic
reactions conducted in PBS buffer containing: [CuI-SCNP] = 4 μM,
[Az ] = 2 μM, [NaAsc] = 200 μM and [Al] = 1 μM with/without [CA]
= 2 μM. The error bars represent the standard deviation of three
independent experiments. (c) Schematic illustration of the SCNP
mediated fluorogenic protein-ligand binding assay.
and CuI-SCNP
1 2
on biotin-NAv
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A PROTAC-inspired model compound, Al , was synthesized
with a biotin ligand and a sulfamoyl group for targeting for NAv and
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CA, respectively as well as an alkyne group adjacent to each binding
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moiety. In the absence of proteins, both Cu -SCNP and CuI-
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SCNP catalyzed the click reaction between Al and Az as was
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observed by a fluorescence increase (on and on). Upon adding NAv,
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1
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Cu -SCNP remained active exhibiting a reactivity index of ca. 1.2,
1
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whereas Cu -SCNP showed almost no activity (Figure 5). This on
2
and off fluorescence readout suggests that Al was bound to NAv,
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most likely with the alkyne group next to the biotin group buried
inside the protein scaffold and the other alkyne group exposed on
rate of the click reaction with CA to that without CA. If an alkyne
substrate binds to CA, it would become less accessible to CuI-
2
SCNP , producing a reactivity index lower than 1. If there is no
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the surface. When both NAv and CA were added to Al , Cu -
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SCNP remained inactive, whereas the initial rate of Cu -SCNP
interaction between the substrate and protein, the index should
remain around 1, because the small molecule is free in solution to be
taken up into the nanoparticle interior for the click reaction. As
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1
decreased only ca. 20% (off and mostly on), indicating that the
alkyne group on the sulfamoyl side remained partly available to CuI-
1 6
shown in Figure 3b, Al - exhibited reactivity index closed to 1,
1
SCNP . This observation suggests that in the presence of both
proteins there is an equilibrium mixture of CA-Al -NAv and Al
7
indicating they were nonbinding or weakly bound to CA. Al was
10 10
-
used because sulfamoyl compounds are known CA inhibitors.28
Indeed, its reactivity index was significantly lower.
NAv. Overall, this model experiment demonstrates the potential use
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of Cu -SCNP and Cu -SCNP in a combination fluorogenic assay
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to test potential PROTAC ligands.
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The activity of Cu −SCNP and Cu −SCNP toward protein-
ligand complexes was studied in more detail. Thus, two biotin-
alkyne derivatives that bind to neutravidin (NAv) tightly were
CuI-SCNP2 mediated extracellular synthesis and drug screening
2
Given the weak protein absorption, we wondered whether SCNP ,
.
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groups which could subsequently react with alkynes to generate a
large library.35 The relatively low activity of traditional copper
catalysts means that the CuAAC reactions are typically performed at
millimolar concentration of substrates and catalysts, thereby
requiring an organic solvent to solubilize the reagents. Thus, the
products are purified and subsequently transferred to cells to test
their bioactivity. The bioorthogonality and high activity of the CuI-
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SCNP clickase provides an opportunity for in situ drug synthesis,
streamlining the screening. In particular, the ability to obtain near
quantitative yields of click products at micromolar concentrations20
is important because this is the effective concentration range of
many anticancer agents.
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Figure 5. The chemical structure of Al10 and the illustration of its
binding towards CA and NAv. The initial reaction rates of fluorogenic
reactions conducted in PBS buffer containing: [CuI-SCNP
2
] or
] = 4 μM, [Az ] = 2 μM, [NaAsc] = 200 μM and [Al] =
[CuI-SCNP
2 1
1 μM with/without [NAv] = 0.4 μM or [CA] = 2 μM. The error bars
represent the standard deviation of six independent experiments.
1
unlike SCNP might reside extracellularly thereby allowing drug
synthesis and screening without the nanoparticle potentially
affecting the intracellular environment. Albertazzi and Palmans
reported that catalytic Jeffamine-based SCNP could be kept largely
in the extracellular space under certain conditions.19 Therefore, the
cytotoxicity and cell permeability of the SCNP were investigated.
Figure 6. Cell uptake ability of SCNPs and extracellular synthesis. (a)
The confocal images of HeLa cells incubated with 2 µM of CuII-
2
Using HeLa cells, SCNP was found to exhibit significant
cytotoxicity at concentrations ≥ 8 μM (Fig. S3). The uptake was
SCNP -Cy5 for 24 h in DMEM media (10% FBS
-Cy5 or CuII-SCNP
1 2
1
2
assessed by labelling both SCNP and SCNP with Cy5 (see
Supporting Information for details). HeLa cells were incubated in
added). (b) Schematic illustration of extracellular CuAAC synthesis
where the nanoparticle stayed outside the cells and the reaction
products defused in. (c) The confocal images over time of fluorogenic
reaction outside HeLa cells performed in PBS buffer containing: [CuI-
the DMEM media (10% FBS added) containing 2 µM of CuII-
II
SCNP -Cy5 or Cu -SCNP -Cy5 for 24 h. As shown in Figure 6a,
the red fluorescence within the cells indicated Cu -SCNP -Cy5 was
taken up, whereas almost no fluorescence was found intracellularly
when the HeLa cells were treated with Cu -SCNP -Cy5. This
1
2
II
1
SCNP ] = 1 µM, [Az ] = 20 µM, [Al ] = 20 µM and [NaAsc] = 200
2 1 1
µM.
II
2
2
observation confirms that under these conditions SCNP is
"nonsticky" and exhibits poor cell penetration.
To test whether the extracellular catalyst is active and can
produce small molecule products that can subsequently enter the
cell through passive diffusion (Figure 6b),31 the fluorogenic click
The proof of concept, anticancer agent screening method
developed is based on the in situ, extracellular click reaction between
2 11
1 6
10 azido substrates (Az - ) and 6 alkyne substrates (Al - ).
Screening of the 60 possible triazole products was performed by
using a 96 wells plate with HeLa cells in a fast and high-throughput
manner. To each well was added 50 µL of PBS buffer containing [CuI-
1
reaction between Az and Al was performed with HeLa cells and
Cu -SCNP in PBS buffer. A concentration of 500 nM was used that
1
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2
SCNP ] = 1 µM, [Az] = 40 µM, [Al] = 40 µM and [NaAsc] = 100 µM,
2
is below the concentration where a similar Jeffamine-based SCNP
remained in the extracellular space for several hours.19 As shown in
Figure 6c, after the reaction initiation by adding NaAsc, the
fluorescence intensity of the HeLa cells gradually increased and
reached the maximum in about 10 min. This result suggests that
although the nanoparticle cannot penetrate the cell membrane, its
reaction products readily diffuse inside the cells.
Click chemistry has been successfully applied to drug screening
because it can generate a large number of compounds in high
yields.32,33 Recently, by using “SuFEx” click chemistry,34 Dong and
Sharpless reported a method to convert primary amines into azido
and the reaction was allowed to proceed outside the cell for 30 min, long
enough for full conversion (Figure S8). To each well was added 50 µL of
DMEM media (10% FBS), and the cells were incubated for 24 h. The
cell viability was measured by using the MTT assay, the results
presented in Figure 7. Different levels of cytotoxicity were observed for
the screened compounds, the most of toxic compounds were the
derivatives of triphenyl phosphonium, phenyl methoxy compound and
colchicine. We chemically synthesized and purified click product Al
Az , and its cytotoxicity towards HeLa cells was studied more carefully.
3
-
3
Thus, a dose-dependent cytotoxicity was observed with an IC50 = 24 µM,
consistent with the screening result (Figure S10). The toxicity of this
compound likely arises because the triphenyl phosphonium group (Al
3
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Figure 7. CuI-SCNP
2
mediated CuAAC synthesis and drug screening extracellularly. The chemical structures of the substrates and their
numbering are presented. Reactions were performed outside HeLa cells in a 96 wells plate, where the PBS buffer contained: [CuI-SCNP
2
] = 1
µM, [Az] = 40 µM, [Al = 40 µM and [NaAsc] = 100 µM. The data was presented as duplicated experiments.
]
brings the phenyl methoxy unit (Az
disrupts its function.36-38
3
) into cell mitochondria and
AUTHOR INFORMATION
Corresponding Author
Steven C. Zimmerman - Department of Chemistry, Center for
Biophysics and Quantitative Biology, University of Illinois, Urbana,
Illinois 61801, United States; orcid.org/0000-0002-5333-3437; E-
mail: sczimmer@illinois.edu.
◼ CONCLUSION
We previously demonstrated the high CuAAC activity of CuI-
SCNP in performing the click reaction between small molecules
1
(uptake mode). Surprisingly the utility of this catalyst extended to
protein surface reactions because of the unexpected discovery of an
"attach mode" wherein the SCNP binds protein surfaces using both
electrostatic and hydrophobic interactions. Although this added
functionality can be useful, it limits some potential applications. The
Authors
Junfeng Chen - Department of Chemistry, University of Illinois,
Urbana, Illinois 61801, United States; orcid.org/0000-0002-
7100-0839
Ke Li - Department of Chemistry, University of Illinois, Urbana,
Illinois 61801, United States; orcid.org/0000-0003-3851-7710
Sarah E. Bonson - Department of Chemistry, University of Illinois,
Urbana, Illinois 61801, United States; orcid.org/0000-0003-
4167-3730
CuI-SCNP
2
clickase developed here, retains the high reactivity and
selectivity in performing the CuAAC (click) reaction on smallmolecules,
but its PEGylated shell serves as a protein and cell membrane gate. Thus,
the bioorthogonal click reaction is performed by a polymeric catalyst
that is itself bioorthogonal. This new selectivity enables a fluorescent
assay for studying protein-ligand binding, with CuI-SCNP
SCNP acting in combination as a dual logical gate. The PEG groups
also prevent the cell uptake of the nanoparticle. As a result, CuI-SCNP
1
and CuI-
Complete contact information is available at:
https://
2
2
resides extracellularly and serves as a nanoscale factory to produce
bioactive compounds in situ at the low concentrations often used in
bioassays. The application to a potential anticancer agent screening
method was demonstrated. More broadly, this work points to the utility
of synthetic polymers as artificial enzymes with versatile, non-natural
functions for bioapplications.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation
(NSF CHE-1709718, NSF GRFP DGE-1746047) and the
National Institutes of Health (R01 AR069645).
ASSOCIATED CONTENT
REFERENCES
Supporting Information
.
The Supporting Information is available free of charge at:
(1) Tonga, G. Y.; Jeong, Y.; Duncan, B.; Mizuhara, T.; Mout, R.; Das,
R.; Kim, S. T.; Yeh, Y.-C.; Yan, B.; Hou, S.; Rotello, V. M.,
Supramolecular regulation of bioorthogonal catalysis in cells using
nanoparticle-embedded transition metal catalysts. Nat. Chem. 2015, 7,
597-603.
General experimental procedures and detailed synthetic
procedures and characterization data for small molecules and
polymers, and additional kinetic data along with details of the
computational methods (PDF)
ACS Paragon Plus Environment
6

Page 7 of 8
Journal of the American Chemical Society
(2) Brevé, T. G.; Filius, M.; Araman, C.; van der Helm, M. P.; Click Reaction of Small Molecules, Proteins, and Cells. J. Am. Chem.
1
2
3
4
5
6
7
8
Hagedoorn, P.-L.; Joo, C.; van Kasteren, S. I.; Eelkema, R., Conditional
Copper-Catalyzed Azide–Alkyne Cycloaddition by Catalyst
Encapsulation. Angew. Chem. Int. Ed. 2020, 59, 9340-9344.
(3) Kaphan, D. M.; Levin, M. D.; Bergman, R. G.; Raymond, K. N.;
Toste, F. D., A supramolecular microenvironment strategy for transition
metal catalysis. Science 2015, 350, 1235-1238.
(4) Yang, Q.; Xu, Q.; Jiang, H.-L., Metal–organic frameworks meet
metal nanoparticles: synergistic effect for enhanced catalysis. Chem. Soc.
Rev. 2017, 46, 4774-4808.
(5) Blackman, L. D.; Varlas, S.; Arno, M. C.; Houston, Z. H.; Fletcher,
N. L.; Thurecht, K. J.; Hasan, M.; Gibson, M. I.; O’Reilly, R. K.,
Confinement of Therapeutic Enzymes in Selectively Permeable
Polymer Vesicles by Polymerization-Induced Self-Assembly (PISA)
Reduces Antibody Binding and Proteolytic Susceptibility. ACS Cent.
Sci. 2018, 4, 718-723.
(6) Rubio-Cervilla, J.; González, E.; Pomposo, J., Advances in Single-
Chain Nanoparticles for Catalysis Applications. Nanomaterials 2017, 7,
341-360.
(7) Mavila, S.; Eivgi, O.; Berkovich, I.; Lemcoff, N. G., Intramolecular
Cross-Linking Methodologies for the Synthesis of Polymer
Nanoparticles. Chem. Rev. 2016, 116, 878-961.
(8) Ouchi, M.; Badi, N.; Lutz, J.-F.; Sawamoto, M., Single-chain
technology using discrete synthetic macromolecules. Nat. Chem. 2011,
3, 917-924.
(9) Huurne, G. M. t.; Palmans, A. R. A.; Meijer, E. W., Supramolecular
Single-Chain Polymeric Nanoparticles. CCS Chemistry 2019, 1, 64-82.
(10) Chen, J.; Garcia, E. S.; Zimmerman, S. C., Intramolecularly
Cross-Linked Polymers: From Structure to Function with Applications
Soc. 2019, 141, 9693-9700.
(21) Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry:
Diverse Chemical Function from a Few Good Reactions. Angew. Chem.
Int. Ed. 2001, 40, 2004-2021.
(22) Blümmel, J.; Perschmann, N.; Aydin, D.; Drinjakovic, J.; Surrey,
T.; Lopez-Garcia, M.; Kessler, H.; Spatz, J. P., Protein repellent
properties of covalently attached PEG coatings on nanostructured
SiO2-based interfaces. Biomaterials 2007, 28, 4739-4747.
(23) Besanceney-Webler, C.; Jiang, H.; Zheng, T.; Feng, L.; Soriano
del Amo, D.; Wang, W.; Klivansky, L. M.; Marlow, F. L.; Liu, Y.; Wu, P.,
Increasing the Efficacy of Bioorthogonal Click Reactions for
Bioconjugation: A Comparative Study. Angew. Chem. Int. Ed. 2011, 50,
8051-8056.
(24) Mayer, M.; Meyer, B., Characterization of Ligand Binding by
Saturation Transfer Difference NMR Spectroscopy. Angew. Chem. Int.
Ed. 1999, 38, 1784-1788.
(25) Vasile, F.; Menchi, G.; Lenci, E.; Guarna, A.; Potenza, D.;
Trabocchi, A., Insight to the binding mode of triazole RGD-
peptidomimetics to integrin-rich cancer cells by NMR and molecular
modeling. Biorg. Med. Chem. 2016, 24, 989-994.
(26) Neffe, A. T.; Bilang, M.; Meyer, B., Synthesis and optimization of
peptidomimetics as HIV entry inhibitors against the receptor protein
CD4 using STD NMR and ligand docking. Org. Biomol. Chem. 2006, 4,
3259-3267.
(27) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.;
Wang, Q., A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-
Azidocoumarins and Acetylenes. Org. Lett. 2004, 6, 4603-4606.
(28) Supuran, C. T., Carbonic anhydrases: novel therapeutic
applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008,
7, 168-181.
(29) Sakamoto, K. M.; Kim, K. B.; Kumagai, A.; Mercurio, F.; Crews,
C. M.; Deshaies, R. J., Protacs: Chimeric molecules that target proteins
to the Skp1–Cullin–F box complex for ubiquitination and degradation.
Proc. Natl. Acad. Sci. 2001, 98, 8554-8559.
(30) An, S.; Fu, L., Small-molecule PROTACs: An emerging and
promising approach for the development of targeted therapy drugs.
EBioMedicine 2018, 36, 553-562.
(31) Clavadetscher, J.; Hoffmann, S.; Lilienkampf, A.; Mackay, L.;
Yusop, R. M.; Rider, S. A.; Mullins, J. J.; Bradley, M., Copper Catalysis
in Living Systems and In Situ Drug Synthesis. Angew. Chem. Int. Ed.
2016, 55, 15662-15666.
(32) Kolb, H. C.; Sharpless, K. B., The growing impact of click
chemistry on drug discovery. Drug Discov. Today 2003, 8, 1128-1137.
(33) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K., Click Chemistry
for Drug Development and Diverse Chemical–Biology Applications.
Chem. Rev. 2013, 113, 4905-4979.
(34) Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B., Sulfur(VI)
Fluoride Exchange (SuFEx): Another Good Reaction for Click
Chemistry. Angew. Chem. Int. Ed. 2014, 53, 9430-9448.
(35) Meng, G.; Guo, T.; Ma, T.; Zhang, J.; Shen, Y.; Sharpless, K. B.;
Dong, J., Modular click chemistry libraries for functional screens using a
diazotizing reagent. Nature 2019, 574, 86-89.
(36) Sabharwal, S. S.; Schumacker, P. T., Mitochondrial ROS in
cancer: initiators, amplifiers or an Achilles' heel? Nat. Rev. Cancer 2014,
14, 709-721.
(37) Pagliai, F.; Pirali, T.; Del Grosso, E.; Di Brisco, R.; Tron, G. C.;
Sorba, G.; Genazzani, A. A., Rapid Synthesis of Triazole-Modified
Resveratrol Analogues via Click Chemistry. J. Med. Chem. 2006, 49,
467-470.
(38) Wang, F.; Zhang, Y.; Liu, Z.; Du, Z.; Zhang, L.; Ren, J.; Qu, X., A
Biocompatible Heterogeneous MOF–Cu Catalyst for In Vivo Drug
Synthesis in Targeted Subcellular Organelles. Angew. Chem. Int. Ed.
2019, 58, 6987-6992.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
as Artificial Antibodies and Artificial Enzymes. Acc. Chem. Res. 2020
.
(11) Cole, J. P.; Hanlon, A. M.; Rodriguez, K. J.; Berda, E. B., Protein-
like structure and activity in synthetic polymers. J. Polym. Sci., Part A:
Polym. Chem. 2017, 55, 191-206.
(12) Pomposo, J. A., Ed. Single-Chain Polymer Nanoparticles:
Synthesis, Characterization, Simulations, and Applications; John Wiley
& Sons, 2017
.
(13) Terashima, T.; Mes, T.; De Greef, T. F. A.; Gillissen, M. A. J.;
Besenius, P.; Palmans, A. R. A.; Meijer, E. W., Single-Chain Folding of
Polymers for Catalytic Systems in Water. J. Am. Chem. Soc. 2011, 133,
4742-4745.
(14) Liu, Y.; Pauloehrl, T.; Presolski, S. I.; Albertazzi, L.; Palmans, A.
R. A.; Meijer, E. W., Modular Synthetic Platform for the Construction of
Functional Single-Chain Polymeric Nanoparticles: From Aqueous
Catalysis to Photosensitization. J. Am. Chem. Soc. 2015, 137, 13096-
13105.
(15) Huerta, E.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A.,
Consequences of Folding
a Water-Soluble Polymer Around an
Organocatalyst. Angew. Chem. 2013, 125, 2978-2982.
(16) Chen, J.; Wang, J.; Bai, Y.; Li, K.; Garcia, E. S.; Ferguson, A. L.;
Zimmerman, S. C., Enzyme-like Click Catalysis by
a Copper-
Containing Single-Chain Nanoparticle. J. Am. Chem. Soc. 2018, 140,
13695-13702.
(17) Bai, Y.; Feng, X.; Xing, H.; Xu, Y.; Kim, B. K.; Baig, N.; Zhou, T.;
Gewirth, A. A.; Lu, Y.; Oldfield, E.; Zimmerman, S. C., A Highly
Efficient Single-Chain Metal–Organic Nanoparticle Catalyst for
Alkyne–Azide“Click” Reactions in Water and in Cells. J. Am. Chem. Soc.
2016, 138, 11077-11080.
(18) Chen, J.; Li, K.; Shon, J. S. L.; Zimmerman, S. C., Single-Chain
Nanoparticle Delivers a Partner Enzyme for Concurrent and Tandem
Catalysis in Cells. J. Am. Chem. Soc. 2020, 142, 4565-4569.
(19) Liu, Y.; Pujals, S.; Stals, P. J. M.; Paulöhrl, T.; Presolski, S. I.;
Meijer, E. W.; Albertazzi, L.; Palmans, A. R. A., Catalytically Active
Single-Chain Polymeric Nanoparticles: Exploring Their Functions in
Complex Biological Media. J. Am. Chem. Soc. 2018, 140, 3423-3433.
(20) Chen, J.; Wang, J.; Li, K.; Wang, Y.; Gruebele, M.; Ferguson, A.
L.; Zimmerman, S. C., Polymeric “Clickase” Accelerates the Copper
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