Enzyme-like Click Catalysis by a Copper-Containing Single-Chain Nanoparticle

Article abstract of DOI:10.1021/jacs.8b06875

A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the micromolar range. We report that copper cross-linked single-chain nanoparticles (SCNPs) are able to significantly increase the efficiency of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions at low substrate concentration in aqueous buffer by promoting substrate binding. Using a fluorogenic click reaction and dye uptake experiments, a structure-activity study is performed with SCNPs of different size and copper content and substrates of varying charge and hydrophobicity. The high catalytic efficiency and selectivity are attributed to a mechanism that involves an enzyme-like substrate binding process. Saturation-transfer difference (STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentrations, and computational simulations are consistent with a Michaelis-Menten, two-substrate, random-sequential enzyme-like kinetic profile. This general approach may prove useful for developing more-sustainable catalysts and agents for biomedicine and chemical biology.

Full text of DOI:10.1021/jacs.8b06875

Subscriber access provided by University of South Dakota
Article
Enzyme-Like Click Catalysis by a Copper-
Containing Single-Chain Nanoparticle
Junfeng Chen, Jiang Wang, Yugang Bai, Ke Li, Edzna
S. Garcia, Andrew L. Ferguson, and Steven Zimmerman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06875 • Publication Date (Web): 07 Sep 2018
Downloaded from http://pubs.acs.org on September 7, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Enzyme-Like Click Catalysis by a Copper-Containing Single-Chain
Nanoparticle
§
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
Junfeng Chen,† Jiang Wang,‡ Yugang Bai,† Ke Li,† Edzna S. Garcia, † Andrew L. Ferguson, and
Steven C. Zimmerman*,†
†Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
‡Department of Physics, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
§Institute for Molecular Engineering, 5640 South Ellis Avenue, Chicago, Illinois 60637, United States
Supporting Information
ABSTRACT: A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the
micromolar range. We report that copper crosslinked single-chain
polymeric nanoparticles (SCNPs) are able to significantly increase
the efficiency of copper(I)-catalyzed alkyne-azide cycloaddition
(CuAAC) reactions at low substrate concentration in aqueous
buffer by promoting substrate binding. Using a fluorogenic click
reaction and dye uptake experiments, a structure-activity study is
performed with SCNPs of different size and copper content and
substrates of varying charge and hydrophobicity. The high catalytic
efficiency and selectivity is attributed to a mechanism that involves
an enzyme-like binding process. Saturation transfer difference
(STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentration, and computational simulations are
consistent with a Michaelis-Menten, two-substrate random sequential enzyme-like kinetic profile. This general approach may prove useful for
developing more sustainable catalysts and as agents for biomedicine and chemical biology.
INTRODUCTION
Metalloenzymes often achieve their remarkable catalytic efficiency
and selectivity through an architecture that places a substrate binding
site in close proximity to a reactive metal center. The result is an
enzymatic reaction that is fast, clean, and selective despite the complex
and competitive aqueous bioenvironment. The protein scaffold plays a
key role in protecting the reactive metal center. Not surprisingly,
considerable effort has focused on developing artificial
metalloenzymes.1 In parallel, an increasing number of transition metal
catalysts have been developed that function in aqueous media, some
sufficiently biocompatible to operate inside the competitive
environment of living cells.2 These advances have exciting implications
for sustainable chemistry and as powerful new tools for chemical biology
and medicinal chemistry.2d Nonetheless, significant hurdles remain
especially in living systems where the required low substrate
concentration and physiological pH and temperature often results in
low reaction rates. The further demands for low toxicity and
compatibility with a broad range of redox-active and coordinating
functionality suggests that improvements in catalytic efficiency and bio-
compatibility may require a protein-like shell for shielding the metal
center and for substrate binding.
SCNPs have been reported for copper(I)-catalyzed alkyne-azide
cycloaddition (CuAAC),4 enantio- and diastereo-selective aldol
reaction,5 ketone reduction,6 palladium-mediated depropargylation,7
enantio-selective sulfur oxidation8 and phenol hydroxylation9 reactions
as well as for living radical polymerization processes.10 As impressive as
these examples are, there have been very limited demonstrations of
enzyme-like kinetics11 and only few explorations of the putative
hydrophobic binding sites, for example, through structure-activity
relationships.12
Our interest in cross-linked polymers as organic nanoparticles13
and their host-guest capabilities14 has led us to study their cell uptake15
and the possibility of creating selective, nanoscale intracellular catalysts.3
We recently reported a single-chain metal-organic nanoparticle, cross-
linked by copper coordination chemistry that effected the well-known
CuAAC click reaction16 at ppm levels of copper, both in water and in
mammalian and bacterial cells.4 Herein, we report the synthesis of
SCNPs with different structures that has allowed us to develop a
structure-activity relationship as well as to shed light on the overall
reaction mechanism. The copper containing SCNPs show enzyme-like
behavior, in particular substrate binding that increases the reaction rate
and selectivity. The results suggest that the high catalytic efficiency of
SCNPs may be attributed to their enzyme-like structure. As the first
demonstration of dual saturation kinetics, this approach to metallo-
enzyme-mimicry that combines metal centers and a polymeric scaffold
should provide a useful strategy for future catalyst design and
development.
Recently, there has been intense interest in metal-containing,
catalytic, single-chain nanoparticles (SCNPs) formed by intramolecular
crosslinking.3 The cross-linked polymers loosely resemble the folded
polypeptide structure of bioactive enzymes. More importantly, the wide
array of polymerization methods and cross-linking chemistries available
to produce SCNPs opens the door to a remarkably broad range of
structures and structural tunability. To date, water-soluble, catalytic
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 8
1
2
3
4
5
6
7
8
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
Figure 1. Illustration of the synthesis of the copper crosslinked single-chain organic nanoparticles (SCNPs)
EXPERIMENTAL SECTION
removed using a syringe. A UV-vis spectrophotometer was used to
measure the absorbance of pyrene in the aqueous layer, a direct measure
of the amount solubilized by the SCNP.
Materials and Instrumentation. Details regarding the chemical
reagents used, synthetic procedures for polymers and substrates used in
reactions with SCNP catalysts, instrumentation used in this work, and
additional details on the computational methods can be found in the
Supporting Information.
STD NMR Method. SCNP-2a was dissolved in D2O at a
concentration of 100 μM, and the substrate was added as a 200 mM
DMSO-d6 solution to reach a final concentration of 2 mM (20
equivalents). Spectra were collected after selectively irradiating SCNP-
2a to saturation at δ 1.2 ppm a spectral region where there were no
substrate signals. During the saturation period, the magnetization was
transferred through intra/intermolecular spin diffusion to other protons
on the SCNP as well as to the bound substrates, which further
transferred to free molecules due to the exchange of free and bound
substrates.
Molecular Dynamics Simulation of SCNP-Substrate Binding. All
the SCNP-2a simulations were conducted using the GROMACS 4.6
simulation suite.20 The binding process was studied with different
substrates. The linear polymers were first crosslinked by connecting the
copper ions and amino acid groups, and then placed in a 11 nm cubic
box of water molecules. The system was simulated at 300 K and 1 bar
for 100 ns at which time the SCNP had folded into a stable globular
structure. Subsequently 20 copies of one specific substrate molecule
were randomly placed into the box and the simulation continued for 20
ns. After discarding the first 10 ns for equilibration, substrate binding
was quantified by counting the number of substrate molecules within
the nanoparticle over the course of the terminal 10 ns.
Procedure for Fluorogenic Click Reactions and Kinetic Analysis.
The CuAAC click reaction was monitored using fluorescence-quenched
coumarin azide
compound with restored fluorescence (Scheme 1). In a 0.7 mL
and alkyne 2 17
. After the click reaction, forms
1 1
3
fluorimeter cuvette, SCNPs, sodium ascorbate and a DMSO solution of
substrate was added in 0.5 mL PBS buffer at pH = 7.4. The ascorbic acid
concentration was 2 mM and the final amount of DMSO was 2% (v/v).
The intensity was monitored by fluorimeter every 10 s at λem = 488 nm
with λex = 410 nm. The reaction conversion was calculated from the
observed fluorescence intensity using pure
3
as the standard. Relative
rates were determined as follows. Approximately 1 min after initiating
the reaction the increase in fluorescence stabilized and became linear
over time. The slope of the fluorescence vs time plot starting at ca. 2 min
and using 10-15 data points collected every 10 s was used for the
calculation of relative rates.
For kinetic studies, a 4 μM aqueous solution of SCNP-2a was used.
The concentration of
1
was varied from 10 μM to 40 μM, whereas the
concentration of was varied from 12.5 μM to 500 μM due to its higher
2
water solubility. The collected kinetic data was fit to various models but
a random sequential two-substrates enzyme kinetics equation gave the
best fit.18
RESULT AND DISCUSSION
Synthesis and Characterization of SCNPs. Using a modified
procedure based on our original report,4 P1 were prepared by ring-
opening metathesis polymerization (ROMP) of monomers M1 and M2
with pyridine-modified Grubbs third-generation catalyst (Figure 1).21
Monomer content and degrees of polymerization (DPs) were
controlled by adjusting the feed ratios of monomers and the amount of
Grubbs catalyst during the ROMP.
Scheme 1
Formation of P1 was confirmed by gel-permeation chromato-
graphy (GPC), which showed a good correlation between the measured
molecular weights and the catalyst and monomer feed ratios, as well as
low polydispersity indices (PDI) that ranged from 1.01 to 1.05. The P1
were post-functionalized by treatment with N-butyl-imidazole
providing imidazolium groups that afforded water-soluble, amphiphilic
Pyrene Uptake Experiments. The uptake of pyrene as a
hydrophobic guest by SCNPs was quantified by shaking a vial
containing 0.2 mL of a 1 M pyrene solution in chloroform with 1 mL of
a 5 μM aqueous solution of SCNP-1b (or the weight equivalent of other
SCNPs).19 The vial underwent centrifugation and the aqueous layer
ACS Paragon Plus Environment

Page 3 of 8
Journal of the American Chemical Society
polymers. Finally, the amino acid residues were deprotected with
computationally determined diameter of 5-6 nm (vide infra). Overall,
the data are consistent with a SCNP. Because of their greater stability,
SCNPs were kept in aqueous solution as Cu(II) complexes. For
performing CuAAC click reactions, sodium ascorbate was added to
produce the Cu(I) SCNPs in situ.
1
2
3
trifluoroacetic acid (TFA). The resulting P2 were purified by
precipitation in ether followed by dialysis against water. The post-
functionalized polymers P2 were characterized by NMR spectroscopy.
4
5
6
7
8
9
Rate of CuAAC Reactions and Substrate Selectivity. To test
whether substrate charge or hydrophobicity affect the SCNP-catalyzed
fluorogenic click reaction, alkyne substrates
2
- were prepared and
4 6
SCNP-2a, and ascorbic acid. For comparison, the
mixed with azide
,
reactivity of the same substrates was examined using the most highly
active tris(triazolylmethyl)amine-based ligand for Cu(I) developed by
Liu, Marlowe, Wu, and coworkers known as BTTAA23 (see Figure S11).
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
In general,
In contrast, with SCNP-2a, along each homologous series of substrates,
4a 4d 5a 5d, and 6a 6c, the rate of click reaction increased dramatically
with increasing length of the aliphatic chain indicating the importance
of substrate hydrophobicity (Figure 3). Substrates 4d
5d and 6c with
the longest aliphatic chains were on average 25 times faster than
substrates 4a
5a and 6a with the shortest chains. The importance of
hydrophobic binding by the SCNP is further illustrated by the 6-fold
rate increase for 6a seen with SCNP-2a relative to BTTAA despite the
amino acid-Cu(I) complex being a comparatively poor catalyst.
- show only small (<2-fold) rate differences with BTTAA.
4 6
-
,
-
-
,
Figure 2. TEM images of SCNP-2a
.
,
Given that several α-amino acids are reported to form stable
complexes with Cu(II) and Cu(I) with 2:1 stoichiometries,22 0.5 eq of
CuSO4 relative to the aspartate units was added to P2 to form Cu(II)-
containing SCNP. The resulting nanoparticles were characterized by
transmission electron microscopy (TEM) (Figure 2) and dynamic light
scattering (DLS, Figure S10). For the representative SCNP-2a, the
TEM-determined diameter was ca. 8-10 nm, which was larger than the
5.8 nm size determined by DLS. Despite cross-linking, the polymeric
nanoparticles can flatten when absorbed on the grid surface13c and the
ammonium molybdate negative staining further increases the observed
diameter. The DLS-measured hydrodynamic size of 5.8 nm is quite
reasonable for a 37 kDa polymeric nanoparticle and close to the
Figure 4. Structure of cationic, zwitterionic, and anionic SCNPs 2a
respectively and relative reaction rates of substrates 4d and 6c in the
fluorogenic click reaction. Conditions were SCNP (4 μM), (20 μM), 4d and
, , and ,
3 4
1
6c (40 μM) and sodium ascorbate (2 mM) in PBS buffer pH = 7.4. Rates are
relative to that of SCNP-4 with 4d. Error bars are standard deviations of three
The other trend evident in the Figure 3 data is that charge
significantly influences substrate reactivity. Thus, the click reaction of
negatively charged substrate 6c is two times faster than neutral substrate
5d, and 20 times faster than cationic substrate 4d. With BTTAA there is
a small advantage for the cationic alkynes , but the largest rate
4
difference (4a vs. 6a) is three-fold and as indicated above, most rates are
<2-fold different. The structure activity relationship that emerges from
Figure 3 is that polycationic SCNP-2a selectively takes up hydrophobic
substrates with a preference for anionic over neutral guests whereas an
electrostatic repulsion significantly disfavors cationic substrates.
To further study the effect of charge and broaden the substrate
selectivity, two additional SCNPs (SCNP-3 and SCNP-4) were
prepared (see Figure 4 and Supporting Information). Thus, zwitterionic
Figure 3. Structures of substrates with hydrophobicity and charge. Fluorogenic
1 4 6
reaction rate of different substrates with of SCNP-2a (4 μM), (20 μM), - (40
μM) and sodium ascorbate (2 mM) in PBS buffer pH = 7.4. Rates are relative to
that of 4a. Error bars are standard deviations of three independent runs.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 8
support for the substrate binding model, we examined the binding both
computationally and experimentally.
1
Molecular Dynamics Simulation of Substrate Binding Within
SCNP. The potential binding process was studied by molecular
dynamics simulation (see Experimental Section and Supporting
Information for additional details). When the SCNP-2a structure was
built and modeled computationally, it was found to adopt a globular
shape with a diameter of ca. 5-6 nm. The substrate-nanoparticle
interaction was evaluated by calculating the percentage of small
molecules that reside inside the nanoparticle. As shown in Figure 5 and
2
3
4
5
6
7
8
9
Supporting Movie 1, coumarin azide
2
is mostly bound and differential
4d 5d, and 6c that is consistent
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
substrate uptake was observed for 4a
,
,
with the dependence on the hydrophobicity and charge seen
experimentally in Figure 3.
Substrate Binding is Detected by NMR. To obtain direct
experimental evidence of substrate binding, saturation transfer
difference (STD) spectroscopy was applied to SCNP-2a and alkynes
2
and 4a to observe possible nuclear Overhauser effect (NOE) between
catalyst and substrate. STD has been commonly used to study the
interaction between proteins and guest ligands.24 STD spectra were
measured separately for 20 equivalents of
2
and 4a mixed with SCNP-
exhibited
2a. As shown in Figure 6, the hydrophobic substrate
2
relatively strong signals indicating residence within the polymeric
nanoparticle. In contrast, the positively charged and hydrophilic
substrate 4a showed negligible signal. These results support substrate
binding and are consistent with the observed reaction kinetics.
Figure 5. (a) Snapshot of SCNP-2a and
2
in an 11 Å water box (water not
The binding between substrate
elucidated by two-dimensional nuclear Overhauser effect spectroscopy
(2D-NOESY). As seen in Figure 7, substrate gives signals at δ 7.3, 6.8,
2
and SCNP-2a was further
shown) before and after equilibrium. Polymer atoms were colored as blue,
substrate molecule were colored red and solvent molecules were turned off. (b)
Percentage of substrate molecules within the nanoparticle averaged over the
terminal 10 ns of the simulation. Error bars represent standard deviations of the
percentage measured over the equilibrium portion.
2
and 3.7 ppm all three of which exhibit strong cross-peaks to SCNP peaks
appearing between δ 0.5 - 2.0 ppm. This region contains the signals of
hydrophobic aliphatic chains. In contrast, the alkene region from the
polymer backbone showed almost no NOE signal although it is also
hydrophobic. These results suggest that hydrophobic substrates may
preferentially bind within pockets formed by the aliphatic side-chains
and the imidazolium groups.
Probing the Importance of the SCNP MW, Copper Content, and
Amphiphilic Structure. If hydrophobic binding by the amphiphilic side-
chains of the SCNP is important to the catalysis then changing the ratio
of the aliphatic to imidazolium content within the side-chains would be
expected to alter the rate of the click reaction. Thus, SCNP-5 and SCNP-
SCNP-3 and anionic SCNP-4 were designed to test whether the
substrate preference in Figure 3 could be altered. Their catalytic
performance in the fluorogenic CuAAC reaction was tested with
cationic alkyne 4d and anionic alkyne 6c and compared to that with
SCNP-2a. As shown in Figure 4, SCNP-4 processed cationic alkyne 4d
significantly faster than did SCNP-2a whereas almost no reaction was
measured with 6c which reversed the selectivity profile of SCNP-2a. On
the other hand, the zwitterionic SCNP-3 exhibited similar rates towards
both cationic and anionic substrates.
The SCNP is critical for the catalysis observed. Thus, BTTAA was
used to test the role of the polymer in the control experiments (Figure
S11) because no reaction could be observed with the copper complex of
glycine. The data presented in Figures 3 and 4 suggest clearly that one
key role played by the polymeric nanoparticle is in binding the substrates
in proximity to the metal catalyst. To obtain more than inferential
Figure 6. STD spectrums for SCNP-2a (100 μM) with
(2mM) in D2O.
2
(2 mM) or 4a
Figure 7. 2D-NOESY spectrum of SCNP-2a (100 μM) with 2
(2 mM) in D2O.
ACS Paragon Plus Environment

Page 5 of 8
Journal of the American Chemical Society
ratios. An increase in the M2:
M1 ratio increases both the crosslinking
1
2
3
4
5
6
7
8
density and the number of copper complexes per SCNP. To determine
the role of these two variables, the rate of the fluorogenic CuAAC click
reaction was measured at 1 μM of each SCNP. Using a constant SCNP
concentration means that the solution of SCNP-1d contained six times
more copper than did SCNP-1a. As seen in Figure 9a, the reaction rates
of all four SCNP were very similar. Despite SCNP-1d containing the
largest amount of copper ion, it showed the slowest rate. Dye uptake
experiments were also conducted and as shown in Figure 9b. The four
SCNPs exhibited comparable results, each SCNP solubilizing the
hydrophobic pyrene structure in water between 43-57% of that
solubilized by SCNP-2a. The increased copper content is expected to be
accompanied by a more tightly cross-linked and more polar polymer
interior and this is reflected in the regular, albeit small, decrease in
pyrene uptake. Overall, the results suggest that the copper content is less
important than the SCNP capacity for hydrophobic binding.
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
The same general approach was used to determine the importance
of nanoparticle size, the size decreasing along the series SCNP-1b
<
SCNP-2a SCNP-2b SCNP-2c (Figure 9c and 9d). The dye uptake
<
<
experiments were performed at the same mass concentrations. SCNP
size appears to matter more than copper content, although the effect
both on rate and pyrene uptake is not large. The results are again
consistent with the rate correlating with SCNP pyrene uptake and
further suggesting that intermediate sized SCNP will give the fastest
catalysts. It is possible that small polymers might not have enough
flexibility to form hydrophobic pockets whereas large polymers might
pack too tightly.
Enzyme-Mimetic Behavior by SCNPs as Revealed by Kinetic
Analysis. Given that the combined results of the STD, 2D-NOESY, and
pyrene uptake experiments, suggest that the SCNPs catalyze the
CuAAC click reaction by binding the azide and alkyne in proximity to
the metal catalytic site, we sought to apply enzyme kinetics to the SCNP-
Figure 8. (a) Polymer parent structures of SCNPs with different water
solubilization structure. (b) Reaction kinetics of SCNPs with SCNPs (4 μM),
(20 μM), (40 μM) and sodium ascorbate (2 mM) in PBS buffer pH = 7.4. (c)
1
2
6
were designed with progressively shorter aliphatic chains but
otherwise a structure directly analogous to SCNP-2a (Figure 8a). A
neutral, but very hydrophilic nanoparticle (SCNP-7) was also prepared.
To have a more quantitative measure of the SCNP’s ability to bind
hydrophobic substrates, pyrene uptake experiments were performed,
where the percent of pyrene extracted from chloroform into the aqueous
layer provides a direct measure of the ability of the SCNP to bind a
hydrophobic substrate. As seen in Figure 8b and 8c, there is an excellent
correlation between catalytic activity and the ability of the SCNP to take
up pyrene. Replacing the butyl group to methyl minimally decreases the
rate of the click reaction and the pyrene uptake (compare SCNP-2a and
SCNP-5) suggesting that the peripheral butyl groups minimally
and . Because the CuAAC click
2a catalyzed CuAAC click reaction of 1 2
reaction is a two-substrate reaction that requires binding of both
participate in binding and catalysis. In contrast, SCNP-6 and SCNP-7
,
show little binding and little catalysis. It is likely that these two polymeric
nanoparticles are too hydrophilic to significantly bind substrate.
As seen in Figure 1, four nanoparticles, SCNP-1a-d, were prepared
with roughly the same degree of polymerization but different M1 to M2
Figure 9. (a) Rate of fluorogenic CuAAC click reactions performed with
μM), (40 μM), sodium ascorbate (2 mM) and 1 μM SCNP. (b) Pyrene uptake
by SCNP. (d) Fluorogenic CuAAC click reaction performed with (20 μM),
1
(20
2
1
2
(40 μM), sodium ascorbate (2 mM) and 1 μM SCNP-1b. Other SCNP run at
same mass concentration. (d) Pyrene uptake by SCNP. Pyrene uptake by
SCNP-2a was set at 100% relative uptake here and (b). The molecular weight of
Figure 10. (a) Random-sequential two substrates enzyme kinetics equation
fitting of SCNP-2a kinetics data. (b) Fitting equation and parameters.
, ,
SCNP-1b SCNP-2a SCNP-2b and SCNP-2c were 76 kDa, 38 kDa, 19 kDa and
ACS Paragon Plus Environment
8 kDa respectively.

Journal of the American Chemical Society
Page 6 of 8
1
2
3
4
5
6
7
8
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
Figure 11. (a) Illustration of selective catalysis on free substrates by SCNPs. (b) Fluorogenic CuAAC click reaction on 7
(10 μM) with SCNP-2a (4 μM) at different
7
avidin concentration in PBS buffer pH=7.4. The ratio of avidin to was varied from 0:5 to 2:5. (c) Reaction rate over the concentration of avidin.
substrates in random sequence, the kinetics data were analyzed using the
random-sequential Bi-Bi model.18
By varying both substrate concentrations and measuring the rate
avidin molecule offers four binding sites (Figure 11). Alkyne substrate
7
was prepared with a short linker to the biotin unit, which ensures the
alkyne group will be deeply buried inside the protein (Supporting
Information). During the fluorogenic click reaction, the concentrations
of the CuAAC click reaction between and , a three-dimensional rate
1 2
surface was generated as shown in Figure 10. Looking along each
concentration axis separately, i.e., holding one component constant and
increasing the other, it can be seen that the reaction rate gradually
reaches saturation, consistent with Michaelis-Menton-like kinetics. The
surface in Figure 9 could be fit to the equation describing a random-
sequential Bi-Bi model with the R-square as high as 0.99. Palmans,
Meijer, and coworkers have reported11 one of the few examples of
Michaelis-Menten kinetics, but to our knowledge, this represents the
first demonstration of two substrate enzyme kinetic behavior for an
SCNP-based catalyst.
Toward The Use of SCNP in Bioapplications. We previously
demonstrated that copper-containing SCNP could perform click
reactions both in bacterial and mammalian cells. Here the interest was
in exploring the potential use of SCNP in an additional application. As
supported by the results above, substrate binding is the key step during
the catalysis performed by the SCNP. If the substrate has a higher
binding affinity toward another macro-molecular scaffold and is deeply
buried inside a binding pocket, the reactive group on the substrate would
be unreactive toward the SCNP because of the steric effect of the
polymeric structure. Thus, free molecules would readily undergo the
click transformation whereas bound molecules would not. Such an
approach might provide a simple click-based alternative to fragment
based drug discovery.25
of SCNP-2a
measured with increasing concentration of avidin. Thus, the
concentration of free that is accessible to the nanoparticle
progressively decreased with a concomitant reduction in reaction rate
that is linearly correlated with the avidin concentration (Figure 11c)
consistent with the experimental design.
, and were kept constant, and reaction kinetics were
1 7
7
CONCLUSION
Copper crosslinked single-chain organic nanoparticles were
designed and synthesized with cationic, anionic, zwitterion, and neutral
water-solubilizing groups. By using alkyne substrates with different
charges and varying alkyl chain length, a structure-activity relationship
was developed. In addition, the size of the polymeric nanoparticle and
number of copper centers per particle were varied. The overall picture
that emerges is that the rate of the copper-containing SCNP is governed
primarily by the hydrophobic character of the substrate and polymer and
the charge complementarity. The other factors appear to be less critical
although an intermediate-sized polymer appears to have some
advantages over larger and smaller SCNPs.
The structure-activity relationship combined with the STD
spectroscopy, 2D-NOESY, and computational experiments strongly
support the binding of both the alkyne and azide as critical for providing
the enhanced rate and substrate selectivity. Indeed, the synthetic single-
chain nanoparticle catalysts exhibited two-substrate enzyme kinetics
behavior, making the analogy to a metalloenzyme apt. Model studies
with avidin and alkyne-labeled biotin show the potential use of SCNP in
To test the general principle of binding-inhibited SCNP-click,
biotin and avidin was chosen because of its strong noncovalent binding,
relatively buried binding site, and utility in a range of applications.26 Each
ACS Paragon Plus Environment

Page 7 of 8
Journal of the American Chemical Society
drug discovery. We are actively working on extending this system to
(7) Liu, Y.; Pauloehrl, T.; Presolski, S. I.; Albertazzi, L.; Palmans, A.
R. A.; Meijer, E. W. J. Am. Chem. Soc. 2015, 137, 13096–13105.
1
2
other metal centers, and modifying the macromolecular scaffolds to
afford greater rate enhancements for biolabeling both in vitro and in
cellulo and the results of these efforts will be report in due course.
(8) Zhang, Y.; Tan, R.; Gao, M.; Hao, P.; Yin, D. Green Chem. 2017
19, 1182–1193.
,
3
4
5
6
7
8
(9) Thanneeru, S.; Duay, S. S.; Jin, L.; Fu, Y.; Angeles-Boza, A. M.;
He, J. ACS Macro. Lett. 2017, 6, 652–656.
(10) (a) Sanchez-Sanchez, A.; Arbe, A.; Kohlbrecher, J.; Colmenero,
J.; Pomposo, J. A. Macromol. Rapid Commun. 2015, 36, 1592–1597. (b)
Azuma, Y.; Terashima, T.; Sawamoto, M. ACS Macro. Lett. 2017, 6,
830–835.
ASSOCIATED CONTENT
Supporting Information
.
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
9
(11) Huerta, E.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A.
Angew. Chem. Int. Ed. 2013, 52, 2906–2910.
(12) (a) Artar, M.; Souren, E. R. J.; Terashima, T.; Meijer, E. W.;
Palmans, A. R. A. ACS Macro. Lett. 2015, 4, 1099–1103. (b) Stals, P. J.
M.; Cheng, C.-Y.; van Beek, L.; Wauters, A. C.; Palmans, A. R. A.; Han,
S.; Meijer, E. W. Chem. Sci. 2016, 7, 2011–2015.
(13) (a) Wendland, M. S.; Zimmerman, S. C. J. Am. Chem. Soc.
1999, 121, 1389–1390. (b) Schultz, L. G.; Zhao, Y.; Zimmerman, S. C.
Angew. Chem. Int. Ed. 2001, 40, 1962–1966. (c) Lemcoff, N. G.;
Spurlin, T. A.; Gewirth, A. A.; Zimmerman, S. C.; Beil, J. B.; Elmer, S. L.;
Vandeveer, H. G. J. Am. Chem. Soc. 2004, 126, 11420–11421. (d) Bai,
Y.; Xing, H.; Vincil, G. A.; Lee, J.; Henderson, E. J.; Lu, Y.; Lemcoff, N.
G.; Zimmerman, S. C. Chem. Sci. 2014, 5, 2862–2868.
(14) (a) Zimmerman, S. C.; Wendland, M. S.; Rakow, N. A.; Zharov,
I.; Suslick, K. S. Nature 2002, 418, 399–403. (b) Zimmerman, S. C.;
Quinn, J. R.; Burakowska, E.; Haag, R. Angew. Chem. Int. Ed. 2007, 46,
8164–8167. (c) Burakowska, E.; Quinn, J. R.; Zimmerman, S. C.; Haag,
R. J. Am. Chem. Soc. 2009, 131, 10574–10580.
(15) (a) Bai, Y.; Xing, H.; Wu, P.; Feng, X.; Hwang, K.; Lee, J. M.;
Phang, X. Y.; Lu, Y.; Zimmerman, S. C. ACS Nano 2015, 9, 10227–
10236. (b) Li, Y.; Bai, Y.; Zheng, N.; Liu, Y.; Vincil, G. A.; Pedretti, B. J.;
Cheng, J.; Zimmerman, S. C. Chem. Commun. 2016, 52, 3781–3784.
(16) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K.
B. Angew. Chem. Int. Ed. 2002, 41, 2596–2599. (b) Tornoe, C. W.;
Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064.
(17) (a) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H.
N.; Wang, Q. Org. Lett. 2004, 6, 4603–4606. (b) Zhou, Z.; Fahrni, C. J.
J. Am. Chem. Soc. 2004, 126, 8862–8863. (c) Le Droumaguet, C.; Wang,
C.; Wang, Q. Chem. Soc. Rev. 2010, 39, 1233–1239.
(18) (a) Cleland, W. W. Biochim. Biophys. Acta 1963, 67, 104–137.
(b) Ivanetich, K. M.; Goold, R. D. Biochim. Biophys. Acta, Protein
Struct. Mol. Enzymol. 1989, 998, 7–13.
(19) (a) Diederich, F.; Dick, K. J. Am. Chem. Soc. 1984, 106, 8024–
8036. (b) Denti, T. Z. M.; vanGunsteren, W. F.; Diederich, F. J. Am.
Chem. Soc. 1996, 118, 6044–6051. (c) Basu Ray, G.; Chakraborty, I.;
Moulik, S. P. J. Coll. Interf. Sci. 2006, 294, 248–254.
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)
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
AUTHOR INFORMATION
Corresponding Author
*sczimmer@illinois.edu
ORCID
Junfeng Chen: 0000-0002-7100-0839
Yugang Bai: 0000-0002-9834-7517
Ke Li: 0000-0003-3851-7710
Edzna S. Garcia: 0000-0003-4935-4988
Steven C. Zimmerman: 0000-0002-5333-3437
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation (NSF
CHE-1709718) and the Binational Science Foundation (2014116).
REFERENCES
(1) For lead references see: (a) Ilie, A.; Reetz, M. T. Isr. J. Chem.
2015, 55, 51–60. (b) Schwizer, F.; Okamoto, Y.; Heinisch, T.; Gu, Y.;
Pellizzoni, M. M.; Lebrun, V.; Reuter, R.; Köhler, V.; Lewis, J. C.; Ward,
T. R. Chem. Rev. 2018, 118, 142–231.
(2) Selected lead reviews: (a) Sasmal, P. K.; Streu, C. N.; Meggers,
E. Chem. Commun. 2013, 49, 1581–1587. (b) Chankeshwara, S. V.;
Indrigo, E.; Bradley, M. Curr. Opin. Chem. Biol. 2014, 21, 128–135. (c)
Yang, M. Y.; Li, J.; Chen, P. R. Chem. Soc. Rev. 2014, 43, 6511–6526.
(d) Bai, Y.; Chen, J.; Zimmerman, S. C. Chem. Soc. Rev. 2018, 47,
1811–1821. (e) Martinez–Calvo, M.; Mascarenas, J. L. Coord. Chem.
Rev. 2018, 359, 57–79.
(20) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.;
Hess, B.; Lindahl, E. SoftwareX 2015, 1, 19–25.
(21) Choi, T.-L.; Grubbs, R. H. Angew. Chem. Int. Ed. 2003, 115,
1785–1788.
(3) (a) Gonzalez-Burgos, M.; Latorre-Sanchez, A.; Pomposo, J. A.
Chem. Soc. Rev. 2015, 44, 6122–6142. (b) Altintas, O.; Barner‐
Kowollik, C. Macromol. Rapid Commun. 2016, 37, 29–46. (c) Mavila,
S.; Eivgi, O.; Berkovich, I.; Lemcoff, N. G. Chem. Rev. 2016, 116, 878–
(22) (a) Van Heeswijk, W. A. R.; Eenink, M. J. D.; Feijen, J.
Synthesis 1982, 1982, 744–747. (b) Thomas, G.; Zacharias, P. S.
Polyhedron 1985, 4, 811–816. (c) Cerda, B. A.; Wesdemiotis, C. J. Am.
Chem. Soc. 1995, 117, 9734–9739.
(23) (a) Soriano del Amo, D.; Wang, W.; Jiang, H.; Besanceney, C.;
Yan, A. C.; Levy, M.; Liu, Y.; Marlow, F. L.; Wu, P. J. Am. Chem. Soc.
2010, 132, 16893–16894. (b) 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. Angew. Chem. Int. Ed. 2011, 50, 8051–8056. (c)
See also: Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem.
Int. Ed. 2009, 48, 9879–9883.
(24) (a) Mayer, M.; Meyer, B. Angew. Chem. Int. Ed. 1999, 38,
1784–1788. (b) Mayer, M.; Meyer, B. J. Am. Chem. Soc. 2001, 123,
6108–6117.
(25) Selected lead reviews: (a) Kolb, H. C.; Sharpless, K. B. Drug
Discov. Today 2003, 8, 1128–1137. (b) Lipinski, C.; Hopkins, A.
961. (d) Rubio-Cervilla, J.; González, E.; Pomposo, J. Nanomater. 2017
,
7, 341–360. (e) Pomposo J. A., ed. Single-Chain Polymer Nanoparticles:
Synthesis, Characterization, Simulations, and Applications. John Wiley
& Sons: 2017.
(4) (a) 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. J. Am. Chem.
Soc. 2016, 138, 11077–11080.
(5) Huerta, E.; Genabeek, B. v.; Stals, P. J. M.; Meijer, E. W.;
Palmans, A. R. A. Macromol. Rapid Commun. 2014, 35, 1320–1325.
(6) Terashima, T.; Mes, T.; De Greef, T. F. A.; Gillissen, M. A. J.;
Besenius, P.; Palmans, A. R. A.; Meijer, E. W. J. Am. Chem. Soc. 2011
133, 4742–4745.
,
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 8
Nature 2004, 432, 855–861. (c) Hajduk, P. J.; Greer, J. Nat. Rev. Drug
Discov. 2007, 6, 211–219.
5429. (c) Laitinen, O. H.; Nordlund, H. R.; Hytonen, V. P.; Kulomaa,
M. S. Trends Biotechnol. 2007, 25, 269–277.
1
2
3
(26) (a) Wilchek, M.; Bayer, E. A. Method Enzym. 1990, 184, 14–
45. (b) DeChancie, J.; Houk, K. N. J. Am. Chem. Soc. 2007, 129, 5419–
4
5
6
7
8
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
ACS Paragon Plus Environment