A dual wavelength polymerization and bioconjugation strategy for high throughput synthesis of multivalent ligands

Article abstract of DOI:10.1021/jacs.9b09899

Structure–function relationships for multivalent polymer scaffolds are highly complex due to the wide diversity of architectures offered by such macromolecules. Evaluation of this landscape has traditionally been accomplished case-by-case due to the experimental difficulty associated with making these complex conjugates. Here, we introduce a simple dual-wavelength, two-step polymerize and click approach for making combinatorial conjugate libraries. It proceeds by incorporation of a polymerization friendly cyclopropenone-masked dibenzocyclooctyne into the side chain of linear polymers or the α-chain end of star polymers. Polymerizations are performed under visible light using an oxygen tolerant porphyrin-catalyzed photoinduced electron/energy transfer-reversible addition–fragmentation chain-transfer (PET-RAFT) process, after which the deprotection and click reaction is triggered by UV light. Using this approach, we are able to precisely control the valency and position of ligands on a polymer scaffold in a manner conducive to high throughput synthesis.

Full text of DOI:10.1021/jacs.9b09899

Subscriber access provided by JAMES COOK UNIVERSITY LIBRARY
Article
A Dual Wavelength Polymerization and Bioconjugation
Strategy for High Throughput Synthesis of Multivalent Ligands
Zihao Li, Shashank Kosuri, Henry Foster, Jarrod Cohen, Coline
Jumeaux, Molly M. Stevens, Robert Chapman, and Adam J. Gormley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b09899 • Publication Date (Web): 19 Nov 2019
Downloaded from pubs.acs.org on November 22, 2019
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 13
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
A Dual Wavelength Polymerization and Bioconjugation Strategy for
High Throughput Synthesis of Multivalent Ligands
Zihao Li,1‡ Shashank Kosuri,2‡ Henry Foster,1 Jarrod Cohen,3 Coline Jumeaux,4,5 Molly M. Stevens,4,5
Robert Chapman,*1 Adam J. Gormley*2
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
1Centre for Advanced Macromolecular Design (CAMD) and the Australian Centre for Nanotechnology (ACN), School of
Chemistry, UNSW, Sydney, Australia
2Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
3New Jersey Center for Biomaterials, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
4Department of Materials, Department of Bioengineering, and the Institute for Biomedical Engineering, Imperial Col-
lege London, London SW7 2AZ, United Kingdom
5Division of Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm, Sweden
ABSTRACT: Structure-function relationships for multivalent polymer scaffolds are highly complex due to the wide diversity
of architectures offered by such macromolecules. Evaluation of this landscape has traditionally been accomplished case-by-
case due to the experimental difficulty associated with making these complex conjugates. Here, we introduce a simple dual-
wavelength, two-step polymerize & click approach for making combinatorial conjugate libraries. It proceeds by incorpora-
tion of a polymerization friendly cyclopropenone-masked dibenzocyclooctyne (cp-DIBAC) into the side chain of linear pol-
ymers or the α-chain end of star polymers. Polymerizations are performed under visible light using an oxygen tolerant por-
phyrin-catalyzed photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT)
process, after which the deprotection and click reaction is triggered by UV light. Using this approach, we are able to precise-
ly control the valency and position of ligands on a polymer scaffold in a manner conducive to high throughput synthesis.
23
There are now several additive-free click reactions in-
INTRODUCTION
cluding Diels-Alder,24 thiol-ene,25,
tetrazine-based cy-
26
cloadditions,27-29 and strain promoted azide-alkyne cy-
cloadditions (SPAAC) using cyclooctynes and dibenzocy-
clooctynes,30-37 which avoid the use of potentially toxic
catalysts such as copper. Many of these catalyst-free click
reactions proceed very quickly in a wide range of solvents
and do not produce reaction by-products. The overall sta-
bility of cyclooctynes and specificity for the azide reactive
handle make SPAAC the most widely used catalyst-free
click reaction in bioconjugations.32 Cyclooctynes such as
difluorocyclooctyne (DIFO),31 as well as the more syntheti-
cally tractable dibenzylcyclooctynes (DBCO or DIBAC) and
biarylazacyclooctynones (BARAC),33-35 yield reaction rates
of ~0.1 - 0.6 M-1 thanks to the electron withdrawing groups
adjacent to the octyne. While these rates are lower than
many tetrazine and Diels-Alder reactions, they are compa-
rable with copper catalyzed cycloadditions and sufficient
to give quantitative click within hours at concentrations as
low as 0.1-1 mM.
Multivalent polymer and nanoparticle scaffolds are in-
creasingly important in drug delivery, glycoscience, immu-
nology, cancer therapy, and regenerative medicine because
of their ability to interact with and control protein func-
tion.1-14 The specificity and avidity of these large molecules
comes not only from their size, but also their polyvalent
display of ligands.6, 8, 14-16 One challenge associated with the
design of multivalent scaffolds is the very large diversity of
available physicochemical characteristics. The size, valen-
cy, position of ligands, polymer chemistry, and architecture
of the material will all dramatically affect function. Point
substitution and chemical optimization of ligands on small-
molecule scaffolds has been common practice for develop-
ing structure-activity relationships in medicinal chemistry
for many years.17-19 However, while modest efforts to ex-
plore the macromolecular landscape have been attempted,
they are severely restricted in synthetic throughput.3-5, 20
Because of the incompatibility of many biologically rele-
vant ligands, such as peptides, with controlled-radical
polymerization (CRP) processes, complex polymer scaf-
folds are often functionalized after polymerization. To do
this requires a conjugation strategy that is both efficient
and compatible with high throughput polymer synthesis.21-
Unfortunately, these ‘spring-loaded’ chemical moieties
tend not to be stable during free radical polymerization,
resulting in both loss of click functionality and control over
the polymerization. While CRPs can tolerate low concen-
tration norbornene, its reactivity towards radicals will
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 13
Linear
Linear
O
1
2
3
R
N
H
M1
4
5
6
7
2-Arm
3-Arm
3-Arm
R2
560 nm
290-350 nm
Deprotect
8
Polymerize
Click
9
O
N
R3
N
N
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
4-Arm
R =
O
O
O
O
O
O
N
O
N
N
O
R4
O
Figure 1. Reaction schematic. Cyclopropenone-masked DIBAC (cp-DIBAC) was synthesized either as a monomer (M1) or at the end of 2-
arm (R2), 3-arm (R3), or 4-arm (R4) RAFT agents. Polymerization by PET-RAFT with 560 nm light followed by cp deprotection with UV pro-
duced SPAAC-ready polymer scaffolds for ligand clicking.
always result in some norbornene consumption during
procedures creates an overall versatile and robust tool for
making biofunctional polymers for the non-expert.
polymerization,
particularly
at
high
monomer
conversion.38, For this reason most click functionalities
need to be either chemically deprotected or introduced
after polymerization, neither of which are compatible with
high throughput approaches.40, 41 Popik et al. recently de-
veloped a cyclopropenone-masked DIBAC (cp-DIBAC),
which becomes photoactivated following UV deprotection
of the cyclopropenone and generation of the strained al-
kyne.42 The ability to unmask DIBAC with UV creates a
photo-triggered click reaction that has since been used in
several applications.43-47 While originally introduced to
simplify the synthesis of the molecule and to provide a
photo-trigger, masking of DIBAC with the bulky cyclopro-
penone also renders the alkene compatible with free radi-
cal polymerizations. Zhang et al. leveraged this compatibil-
ity to incorporate a cp-DIBAC into the chain-end of linear
polymers made by RAFT and atom transfer radical
polymerization (ATRP) which they used to photo-trigger
polymer cyclization.48-51 Building on Zhang’s encouraging
findings, we sought to expand on cp-DIBAC’s potential as a
functional tool in polymer click chemistry.
39
EXPERIMENTAL
Materials and synthetic procedures: Detailed synthetic
methods and characterization of the cp-DIBAC, cp-DIBAC mono-
mer (Figures S1-S5) and the star RAFT agents are given in the
supporting information. The peptide GEVC (GEVCLTSCSRLR-
PEG2-K(N3)-CONH2),63 was purchased from China Peptides with
>95% purity as measured by LCMS. The remaining two peptides
BMP (K(N3)-PEG2-KIPKASSVPTELSAISTLYL-CONH2),64 and QK
(KLTWQELYQLKYKGI-PEG2-K(N3)-CONH2),65 were synthesized on
the solid phase (rink-amid resin) using a CEM Liberty Blue auto-
mated microwave peptide synthesizer (CEM Corporation). Fol-
lowing deprotection and cleavage from the resin, peptides were
purified using a C18 column and a gradient of acetonitrile. Purity
and mass were confirmed by HPLC and MS.
General procedure to prepare linear polymers with cp-
DIBAC monomer (M1): Stock solutions of monomer (2 M), cp-
DIBAC (45 mM), 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfa-
nyl]pentanoic acid (RAFT agent, 0.05 M) and ZnTPP (2 mM) were
prepared in DMSO and pipetted into 384-well clear flat-bottom
white plates (Greiner bio-one) while keeping the ZnTPP/CTA
ratio at 0.01, varying monomer/CTA ratio and monomer/cp-
DIBAC ratio depending on the target degree of polymerization
(DP), as well as the amount of cp-DIBAC incorporation desired in
the final polymer composition. The mixtures were then diluted
with DMSO to a final volume of 100 µL and final monomer con-
centration at 0.5 M. Mixtures were covered with well-plate sealing
tape to prevent evaporation and irradiated under 560 nm LED
light for 18 h (5 mW/cm2, TCP 12 Watt Yellow LED BR30 bulb).
Despite non-uniform lighting of each well from the bulb, we find
that full conversion is obtained for each polymer after an over-
night reaction.
Polymer purification: Linear polymers were purified using Se-
phadex G-25 spin columns to remove any unreacted cp-DIBAC
before deprotection and click addition. Briefly, Sephadex G-25
super fine powder (Sigma Aldrich) was dissolved in DMSO (37.5
mg/mL) and allowed to sit for at least 3 h at room temperature
for the resin to swell. 0.5 mL Zeba spin columns (Thermo Fisher)
were loaded manually with the resin and washed three times with
DMSO at 1000 g for 1 min. After washing, 60 µL of polymer crude
was added on top of the resin and centrifuged at 1000 g for 2 min
to obtain the purified sample. UV-Vis spectral absorbance (250-
400 nm) of polymer sample before and after purification (2 µL in
Here, we present a simple one-pot, dual-wavelength pro-
cedure for preparing SPAAC-ready linear or star-shaped
polymers for bioconjugation (Figure 1). This study builds
on our previous work in the development of oxygen-
tolerant CRP techniques for combinatorial polymer library
production.52-57 In this work we use the PET-RAFT
polymerization method developed by Boyer and cowork-
ers to form libraries in well-plates using 560 nm photoex-
citation.57-62 We incorporate cp-DIBAC either into the side
chain of linear polymers by statistical copolymerization, or
into the Z group of star RAFT agents prior to polymeriza-
tion, thus enabling the synthesis of end-functional star
polymers. Because star polymers present exactly the same
number of ligands from each scaffold the biological effect
of 2, 3 and 4 ligands can be compared, alongside the effect
of polymer size. Subsequent deprotection of the cp group
at an orthogonal wavelength (350 nm) followed by click
addition of azido ligands results in the preparation of bio-
functional polymers. The combination of these simplified
ACS Paragon Plus Environment

Page 3 of 13
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
Figure 2. UV-Vis of cp-DIBAC and polymerization kinetics. a) UV-Vis of cp-DIBAC monomer (M1) before and after deprotection showing
blue-shift of the characteristic peak, followed by a loss in absorbance after click to 2kDa PEG-N3. b) UV-Vis traces of pDMA with 5% (mol) cp-
DIBAC monomer (M1) incorporated at DP 200 and 400 before and after deprotection, and following click to 2kDa PEG-N3. c) Kinetics of PET-
RAFT polymerization with and without cp-DIBAC monomer (M1) showing complete conversion in both cases after 16h. d) GPC traces for
pDMA with 5% (mol) cp-DIBAC monomer (M1) after 8, 12 and 16h of polymerization, showing incorporation of the cp-DIBAC by UV detection
at 265 nm.
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
98 µL DMSO for measurement) were collected to calculate the
percentage of cp-DIBAC removed.
linear polymers by a statistical copolymerization. This
monomer was prepared following a procedure modified
from literature (Scheme S1),37 by addition of acryloyl
chloride to the dibenzyl precursor (2) followed by cycliza-
tion of the dibenzocyclooctyne (3) with tetrachlorocyclo-
propene. The linker between cp-DIBAC and the acrylamide
group was necessary to enhance DMSO solubility and pro-
vide space from the polymer chain. Most of the steps pro-
ceeded with good yield except the last cyclization step
which generally provided 30% yield which is consistent
with previously reported values,42 except in cases of ultra-
conservative purification of M1 (11%). In general, we
found that monomer purity was critical as the presence of
trace contaminants led to reduced polymerization kinetics.
Despite this low yield, we do not find that this limits
throughput and scalability due to the low volume of each
reaction. Detailed synthesis information can be found in
the supporting information.
Deprotection: Purified polymer was further diluted in DMSO to
a final cp-DIBAC concentration of 5 mM into a 96-well polysty-
rene flat bottom plate (Denville) with a total volume of 40 µL.
Deprotection of cp-DIBAC was completed by placing the 96-well
plate under a 365 nm Spectroline (R) E-series UV-lamp (Sigma
Aldrich) for 15 min. Deprotection was confirmed by UV-Vis spec-
troscopy.
Clicking: Conjugation of 2kDa PEG-N3 onto polymers was
achieved following a similar procedure to the deprotection, but in
the presence of one equivalence of PEG-N3 (50 mM in DMSO) with
respect to cp-DIBAC. Solutions were left overnight at room tem-
perature to allow complete conjugation. A UV-Vis spectrum was
collected at the end of reaction time to confirm PEG addition.
General procedures to prepare star polymers: Stock solutions
of star RAFT agents (0.1 M for CTA), monomer (2 M) and ZnTPP
(4 mM) were prepared in DMSO and pipetted into 384 well clear
flat-bottom black plates (Corning) to keep ZnTPP/CTA ratio at
0.01 and varying monomer/star RAFT agent ratio with desired
targeting DP. The mixtures were then diluted with DMSO to make
the total volume 40 µL and final DMA concentration 0.5 M. For 4-
armed polymers, N-methyl-2-pyrrolidone (NMP, 8 µL) was added
to the mixture before dilution with DMSO in order to suppress
star-star coupling during polymerization. Mixtures were covered
with well-plate sealing tape to prevent evaporation and irradiated
under 560 nm LED light (Thorlabs M565L3, 2 mW/cm2, Figure
S28) for 18 h. A collimated lens was used to ensure even light
irradiation across all samples. The resulting polymer solutions
were processed directly for deprotection and click reactions
without purification.
Deprotection: Aliquots of polymer solutions were transferred
into PCR tubes and diluted with DMF to get cp-DIBAC concentra-
tion at 3 mM with total volume of 20 µL. Deprotection of cp-
DIBAC was completed by exposing the PCR tubes to 290-350 nm
UV light (Cosmedico ARIMED B6, 0.372 mW/cm2, Figure S28) for
15 min.
Click: Conjugation of 2kDa PEG-N3 onto polymers was achieved
following a similar procedure to the deprotection, but in the pres-
ence of one equivalence of PEG-N3 (100 mM in DMSO) with re-
spect to cp-DIBAC. After UV deprotection, click samples were left
in ambient environment overnight to allow complete conjugation.
The spectra of cp-DIBAC, deprotected DIBAC and DIBAC-
azide allow for monitoring of these steps by UV-Vis.
cp-DIBAC has a characteristic, two-hump peak between
340-360 nm which quickly drops off above 370 nm (Fig-
ure 2a). Deprotection of the cyclopropenone group with
UV (290-350 nm) results in a 30 nm blue shift of this spec-
tra with a new peak between 310-330 nm. Complete con-
version to the deprotected DIBAC can be followed by mon-
itoring for remaining absorbance above 350 nm. Subse-
quent addition of azide results in a fast click reaction and
near complete loss of these peaks (Figure 2a). This con-
venient method therefore allows for online monitoring of
the click reaction and for calculating the final concentra-
tion of cp-DIBAC against calibrated concentration stand-
ards.
Linear Polymers
Linear polymers with cp-DIBAC in the side chains were
prepared in 384 or 96 well plates by PET-RAFT copoly-
merization using ZnTPP as the photoinitiator in DMSO.57
Monomer concentration was varied between 1.0 and
0.5 M, and ZnTPP/CTA ratio was fixed at 0.01 or 0.02 de-
pending on the desired kinetics and conversion. The stock
concentration of cp-DIBAC monomer (M1) was 45 mM as
this is the solubility limit in DMSO. PET-RAFT polymeriza-
RESULTS AND DISCUSSION
We began by exploring the synthesis of a cp-DIBAC func-
tionalized monomer (M1), which was incorporated into
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 13
tion was conducted under a yellow lamp overnight to one unprotected). The first addition of PEG-N3 by clicking
1
2
3
4
5
6
7
8
achieve full conversion. If desired, relative polymerization
conversion can be followed by the ratio of the ZnTPP fluo-
rescence emission intensities at 632 and 615 nm.66 After
polymerization, the polymers were purified to remove any
free cp-DIBAC and deprotected under UV for 15-25
minutes. During this time, deprotection was followed eve-
ry five minutes by UV-Vis to verify complete deprotection.
After deprotection, the concentration of DIBAC was calcu-
lated by UV-Vis and 1:1 equivalents of azide / DIBAC was
added. Click was verified by UV-Vis and was typically com-
plete within one hour.
to DBCO provided a predictable shift in molecular weight
(Figure 4a) as well as a drop in the UV signal for DBCO at
318 nm (Figure 4b). Deprotection of cp-DIBAC with UV
followed by the second PEG-N3 addition resulted in anoth-
er molecular weight shift and reduction in UV absorption
from DIBAC. This indicates that we were able to dual func-
tionalize polymers using SPAAC in both cases. This capabil-
ity is very interesting as it allows for polymers to be func-
tionalized with multiple ligands using by SPAAC. For ex-
ample, when designing mucin mimetics, it is often desira-
ble to label multivalent polymers with several different
glycans in order to mimic the complex chemistry of most
glycoproteins.67
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 PET-RAFT copolymerization had no effect on cp-
DIBAC and its subsequent deprotection and click as veri-
fied by UV-Vis (Figure 2b) and NMR (data not shown).
Copolymerization showed adequate evolution of molecular
weight with slightly delayed kinetics (Figure 2c). We hy-
pothesize that this slight delay may be due to residual and
difficult to remove impurities, but this has not been con-
firmed. Nonetheless, if left overnight, copolymerization
with cp-DIBAC proceeds to >90% conversion. For example,
when N,N-dimethyacrylamide (DMA) was copolymerized
(DP 200 or 400) with 5 mol% cp-DIBAC, molecular weights
were 29,091 (Đ = 1.15) and 55,032 (Đ = 1.27) Daltons, re-
spectively (Figure 3a, Table S1). After clicking with 2kDa
PEG-N3, these molecular weights shifted to 41,758 (Đ =
1.29) and 82,041 (Đ = 1.34) Daltons. This indicates that
approximately 7 and 14 PEGs were clicked to the DP 200
and DP 400 polymers, respectively. These click additions
match the theoretical number of PEGs that would be
clicked at 70% functionalization indicating remarkable
control of post-polymerization modification. We also found
that cp-DIBAC was tolerant to a number of monomers in-
cluding 2-hydroxyethyl acrylate (HEA) (Figure 3b), 4-
acryloyl morpholine (NAM) and N-isopropylacrylamide
(NIPAM) (Figure S22). Evaluation of 2.5, 5, and 7.5% cp-
DIBAC copolymerization also shows a reasonable range of
obtainable valency with increasing co-monomer input
(Figure 3c). We were not able to go higher than 7.5% due
to solubility limitations in DMSO, however, sulfonation of
the cp-DIBAC could be used to facilitate greater solubility if
desired.44 Because of the strong UV extinction coefficient of
cp-DIBAC, the UV trace from the GPC provided a reliable
indicator of incorporation into the polymer chain (Figure
3d). We assume that the earlier GPC elution time for poly-
mers with greater valency was due to collapse of the poly-
mer chain in GPC eluent (DMF) due to the increased cp-
DIBAC content. By NMR, all polymers (0, 2.5, 5, and 7.5%)
proceeded to full conversion with narrow dispersity and
should therefore have similar molecular weights.
Figure 3. GPC traces showing the copolymerization of cp-
DIBAC monomer (1) into side-chain functionalized linear pol-
ymers followed by click with 2kDa PEG-N3. a) DMA with 5% cp-
DIBAC, b) HEA with 5% cp-DIBAC, and c) DMA DP 200 with 2.5 –
7.5% cp-DIBAC. d) UV signal at 265 nm from GPC showing in-
creasing incorporation of cp-DIBAC as a function of feed ratio.
Taking advantage of the cp protected DIBAC, we decided to
see if this strategy could allow for dual ligand functionali-
zation using the same SPAAC chemistry. This follows pre-
vious work by the authors in which we first copolymerized
NHS-acrylate into a polymer by PET-RAFT, and then chem-
ically conjugated DBCO-NH2 to provide a polymer-DBCO
conjugate.57 Note that in this case, polymer-DBCO is SPAAC
ready. To extend this in the present work we copolymer-
ized 95% (mol) of a non-active monomer (DMA) with 2.5%
NHS-acrylate and 2.5% cp-DIBAC to make a random heter-
opolymer with two different DIBACs (one protected and
Figure 4. Dual functionalization by SPAAC. a) GPC traces after
copolymerization of DMA / NHS-acrylate / cp-DIBAC (95 / 2.5 / 2.5
mol%) and addition of DBCO-NH2 to create dual SPAAC-ready
polymers. Subsequent click to DBCO, deprotection and secondary
click of 2kDa PEG-N3 to cp-DIBAC are shown in the green and blue
lines respectively. b) UV-Vis traces of the protected polymer before
ACS Paragon Plus Environment

Page 5 of 13
Journal of the American Chemical Society
and after the first DBCO click, after deprotection of the cp-DIBAC
and after subsequent click to the DIBAC.
agents. 2- and 3-arm RAFT agents were polymerized in
DMSO, but the same conditions for the 4-arm RAFT agent
resulted in a large degree of star-star coupling. We believe
this is due to coordination of the ZnTPP by the polymer, as
introduction
1
2
Star Polymers
3
4
5
6
7
8
9
Having demonstrated the chemistry in linear polymers we
moved on to using cp-DIBAC to prepare end-functional
star polymers. By placing cp-DIBAC at the ends of the star
architecture, exact control over the number of ligands pre-
sented per polymer is achieved. The synthetic scheme for
accessing star RAFT agents end functionalized with cp-
DIBAC at the R group is depicted in Scheme S4. An alkyne
group was first installed on cp-DIBAC via a similar synthet-
ic route to that of the monomer M1 (Scheme S2). Succinic
anhydride was first ring-opened with propargyl alcohol
and then coupled to the dibenzyl precursor (1) by ami-
dation. Cyclization of the dibenzocyclooctyne with tetra-
chlorocyclopropene resulted in the alkyne functionalized
cp-DIBAC (7) which could be attached to the star RAFT
agents. The precursor azido-ended star RAFT agents (10,
12 and 14) were synthesized by substitution of bromo
groups with thiols in the corresponding bromomethyl ben-
zene molecules68 followed by sequential addition of car-
bon disulfide and 1-azido-4-bromobutane. Bromo-
tris(triphenylphosphine) copper (I) catalyzed click reac-
tions between the star RAFT agent’s alkyne and cp-DIBAC
(7) produced the desired star RAFT agents (R2, R3 and
R4). Detailed information about the synthesis optimization
can be found in the supporting information.
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 5. Deprotection and click of 3-arm star RAFT agent (3)
by 1H NMR (d6-DMSO). a) Before and b) after deprotection (3h
UV) and c) after addition of 1.0 eq of 400 g.mol-1 PEG-N3 with re-
spect to DIBAC.
The quantitative deprotection and click of the RAFT agents
1
was observed by H-NMR spectroscopy, as shown in Fig-
ure 5 for the 3-arm example (R3), and in the supporting
information (Figures S19 and S20) for the 2- and 4-arm
examples (R2 and R4). Upon irradiation (290-350 nm, 3 h)
at 15 mM in d6-DMSO, the phenol peaks within the cp-
DIBAC group shifted downfield by >0.5 ppm. Interestingly
the triazole protons, despite their distance from the cp-
DIBAC groups, shifted from 8.02 ppm to 8.06 ppm (Figure
5a and 5b). The results suggested a significant change in
chemical environment due to the deprotection of cp-DIBAC
from the cyclopropenone mask. After deprotection, quanti-
tative click was observed after addition of 1 equivalence of
O-(2-azidoethyl) heptaethylene glycol (400 PEG-N3) rela-
1
tive to DIBAC, as evidenced again by H NMR. The signal
from the triazole protons at 8.06 ppm split into two peaks
at 7.99 and 8.09 ppm with approximately the equal inte-
gration (Figure 5c) due to the mixture of regioisomers,
45
consistent with previous reports.30,
It is important to
note that the trithiolcarbonate groups responsible for
polymerizations were undamaged during these processes,
as evidenced by the unaffected peak of the adjacent pro-
tons at 4.59 ppm (Figure 5). The same experiments were
conducted with 2- and 4-arm star RAFT agents (R2 and
R4) and displayed consistent results (Figures S19 and
S20).
ZnTPP initiated PET-RAFT polymerizations of star RAFT
agents were prepared in a 384 well plate with total volume
of 40 µL for each reaction mixture. The ZnTPP/CTA ratio
was kept at 0.01, and 0.5 M DMA was used as the mono-
mer. By controlling the ratio of monomer to star RAFT
agents, two sets of polymerizations with targeting total
DPs of 200 and 400 were prepared for each star RAFT
Figure 6. Polymerization, deprotection and SPAAC on star
polymers. a) GPC and b) UV-Vis traces of DPtarg 200 and 400
pDMA using 2 (top), 3 (middle) and 4 (bottom) armed RAFT agents
before and after deprotection and after addition of 1.0 eq of 2kDa
PEG-N3 with respect to DIBAC.
of 20% (v/v) N-methyl-2-pyrrolidone (NMP) suppressed
this phenomenon (Figure 6a) resulting in star polymers of
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 13
nearly monomodal molecular weight distributions. In a concurrent loss of signal from the substrate (Figure 7a).
1
2
3
4
5
6
7
8
384 well plate, the reaction mixtures were photoinitiated
and allowed to polymerize for 18 hours. After polymeriza-
tion, fractions of each sample were taken and diluted into
two solutions with the final cp-DIBAC concentrations as
2.5 mM. The first solution was added with 1 equivalent
2kDa PEG-N3 relative to cp-DIBAC prior to dilution and
labeled as “click”. The second solution contains the same
mixture but without 2kDa PEG-N3 and labeled as “depro-
tection”. Deprotection of cp-DIBAC was carried out by ra-
diating all solutions under UV light (290-350 nm) for 15
min while the click solutions were kept in ambient envi-
ronment overnight to allow complete conjugation before
measurements. GPC and UV-Vis measurements for pol-
ymerized, deprotected and clicked samples were conduct-
ed and compared accordingly (Figure 6 and Table S2).
The identification of the polymer peaks was confirmed
from their mass spectra (Figure S23). In the PDA spectra,
the polymer peak can be seen to lose its characteristic ab-
sorbance at 350 nm (which comes from the protected cp-
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
PET-RAFT polymerizations with the star RAFT agents in
well plates produced well controlled star polymers with
narrow dispersities (Đ < 1.35), particularly for the 3-arm
polymers, having dispersities of 1.04 and 1.10 for target
DPs of 200 and 400 respectively (Table S2). With the dilu-
tion of cp-DIBAC concentrations to 2.5 mM, the time re-
quired for complete deprotection was significantly re-
duced to 15 minutes as shown by UV-Vis (Figure 6b),
compared to 3 hours for 15 mM cp-DIBAC from the NMR
studies. More importantly, UV deprotection of cp-DIBAC
does not re-initiate polymerization with the excess mono-
mers presented in the mixtures, since there is no observa-
ble shift of GPC traces and only minor star-star coupling as
indicated by small increases in higher molecular weight
shoulders (Figure 6a). After deprotection and overnight
incubation in ambient environment, the click samples dis-
played quantitative conjugation for polymers as loss of
DIBAC peaks on UV-Vis (Figure 6b) and complete shift of
GPC traces to higher molecular weight (Figure 6a). The
new peaks displayed in the lower molecular weight re-
gions are due to small amounts of excess unreacted PEG-N3
in the mixture. This is because it is difficult to control 1:1
stoichiometry of 2 kDa PEG-N3, which is a polymer, to
DIBAC. DMSO solutions of star polymer-ligand conjugates
can be purified easily by spin filtration with Sephadex
where necessary.
Figure 7. Scope of click and high throughput reactions a) Total
positive ion count (TIC+) LC-MS data for mixtures of a 2-arm DP20
pDMA with 400 Da PEG-N3 and three different peptides, with and
without irradiation with UV light. Peaks corresponding to the poly-
mer before (○) and after (●) click as well as the substrate peaks (*)
are highlighted. b) Summary of GPC Mn and Đ data high through-
put polymer library of pDMA, pNAM and pHEAm linear copolymers
(5% cp-DIBAC) and 3-arm star polymers before and after click with
2 kDa PEG-N3. Full LC-MS data and GPC traces for each polymer
are shown in the supporting information.
Click Versatility and High Throughput
To show the versatility of this technique for preparing
functional polymer conjugates we explored the efficiency
of the click reaction with a range of more complicated pep-
tides. We chose to test three different bioactive peptides
(12-20 amino acids) which were modified to contain an
azide at either the C or N terminus: a TRAIL mimicking
DIBAC) after deprotection and click (Figure S24). 1H-NMR
(Figure S25) confirms these results for each substrate, by
the disappearance of the cp-DIBAC peaks associated with
both the protected and deprotected polymers, and appear-
ance of peaks corresponding to the clicked products.
peptide ‘GEVC’ (GEVCLTSCSRLR-PEG2-K(N3)-CONH2),63
bone morphogenic protein mimicking peptide ‘BMP’
(K(N3)-PEG2-KIPKASSVPTELSAISTLYL-CONH2),64 and
a
a
VEGF mimicking peptide ‘QK’ (KLTWQELYQLKYKGI-PEG2-
K(N3)-CONH2).65 A 2-arm pDMA (DP20) polymer was pre-
pared following the general procedures for star polymers
from the 2-arm RAFT agent (R2), using 200 µL d6-DMSO as
the solvent and 10 h reaction time. This resulted in a con-
version of 86% and a dispersity of 1.05. After mixing with
1.0 equiv peptide / cp-DIBAC and UV deprotection, quanti-
tative conjugation of all four substrates was observed by a
complete shift of the polymer peak in the LC-MS with a
Finally, we applied this system to synthesize a library of
over 80 polymers and polymer conjugates to validate its
high throughput capability (Figure 7b). Polymerization
and click of linear and multi-arm polymers were per-
formed in single wells of 384 well plates. The resulting
polymers and their conjugates demonstrated excellent
control of molecular weight, dispersity, conversion and
click (Figures S26-S27 and Table S6). Overall, the meth-
ACS Paragon Plus Environment

Page 7 of 13
Journal of the American Chemical Society
addition-fragmentation chain-transfer; SPAAC, strain promot-
ed azide-alkyne cycloaddition.
odology to produce this large library was not manually
1
2
3
4
5
6
7
8
intensive and required small amounts of starting material
(M1, R2-4) due to the small scale of this reaction. Further
scaling of library size would therefore be straightforward
to implement if desired.
REFERENCES
1.
Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel,
P. C.; Mirkin, C. A. Polyvalent DNA Nanoparticle Conjugates
Stabilize Nucleic Acids. Nano Letters 2009, 9 (1), 308-311 DOI:
10.1021/nl802958f.
CONCLUSIONS
We have presented a simplified, dual wavelength, one pot
strategy for making multivalent polymers. It takes ad-
vantage of the tolerance of cp-DIBAC to radical polymeri-
zations as well as well plate format PET-RAFT to make a
highly versatile synthetic platform. We have shown this to
work with linear polymers whose ligands are in the side
chains as well as star polymers that are end-functionalized.
Additional use of DBCO provides a simple avenue for func-
tionalizing multiple ligands to single polymers using the
same SPAAC click chemistry for each ligand. Therefore,
using this synthetic technique, one can imagine making
very large and diverse libraries of multivalent polymers
with relative ease. Moving forward, we anticipate that this
will enable non-experts to attempt this simple chemistry
once cp-DIBAC becomes accessible to non-chemists.
2.
Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone,
J. E.; Rosi, N. L.; Mirkin, C. A. Oligonucleotide Loading
Determines Cellular Uptake of DNA-Modified Gold
Nanoparticles. Nano Letters 2007, 7 (12), 3818-3821 DOI:
10.1021/nl072471q.
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
3.
Lynn, G. M.; Laga, R.; Darrah, P. A.; Ishizuka, A. S.;
Balaci, A. J.; Dulcey, A. E.; Pechar, M.; Pola, R.; Gerner, M. Y.;
Yamamoto, A. In vivo characterization of the physicochemical
properties of polymer-linked TLR agonists that enhance vaccine
immunogenicity. Nature biotechnology 2015, 33 (11), 1201 DOI:
https://doi.org/10.1038/nbt.3371.
4.
Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L.
L. Control of multivalent interactions by binding epitope density.
Journal of the American Chemical Society 2002, 124 (8), 1615-
1619 DOI: https://doi.org/10.1021/ja016727k.
5.
Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K.
A.; Kiessling, L. L. Influencing receptor-ligand binding
mechanisms with multivalent ligand architecture. Journal of the
American Chemical Society 2002, 124 (50), 14922-14933 DOI:
https://doi.org/10.1021/ja027184x.
ASSOCIATED CONTENT
Supporting Information
Detailed methods, cp-DIBAC characterization and additional
results can be found in Supporting Information. The Support-
ing Information is available free of charge on the ACS Publica-
tions website.
6.
Mammen, M.; Choi, S.-K.; Whitesides, G. M.
Polyvalent Interactions in Biological Systems: Implications for
Design and Use of Multivalent Ligands and Inhibitors.
Angewandte Chemie International Edition 1998, 37 (20), 2754-
Supporting_Information.pdf
2794
DOI:
https://doi.org/10.1002/(SICI)1521-
3773(19981102)37:20%3C2754::AID-ANIE2754%3E3.0.CO;2-
3.
AUTHOR INFORMATION
Corresponding Author
7.
Kwon, S.-J.; Na, D. H.; Kwak, J. H.; Douaisi, M.;
Zhang, F.; Park, E. J.; Park, J.-H.; Youn, H.; Song, C.-S.; Kane,
R. S.; Dordick, J. S.; Lee, K. B.; Linhardt, R. J. Nanostructured
glycan architecture is important in the inhibition of influenza A
virus infection. Nat Nano 2017, 12 (1), 48-54 DOI:
10.1038/nnano.2016.181.
* Adam J. Gormley: Adam.Gormley@rutgers.edu
* Robert Chapman: r.chapman@unsw.edu.au
Present Addresses
Author Contributions
8.
Krishnamurthy, V. M.; Estroff, L. A.; Whitesides, G.
M., Multivalency in Ligand Design. In Fragment‐based
Approaches in Drug Discovery, Mannhold, R., Kubinyi, H.,
Folkers, G., Jahnke, W., Erlanson, D. A., Eds. 2006; pp 11-53.
‡These authors contributed equally to the work.
Funding Sources
9.
Harrison, R. H.; Steele, J. A. M.; Chapman, R.;
This work was supported by a Rutgers Global Collaborative
Research Grant and a Busch Biomedical Award to AJG. RC is
grateful to the Australian Research Council for funding
through a Discovery Early Career Researcher Award (DECRA,
DE170100315).
Gormley, A. J.; Chow, L. W.; Mahat, M. M.; Podhorska, L.;
Palgrave, R. G.; Payne, D. J.; Hettiaratchy, S. P.; Dunlop, I. E.;
Stevens, M. M. Modular and Versatile Spatial Functionalization
of Tissue Engineering Scaffolds through Fiber-Initiated
Controlled Radical Polymerization. Advanced Functional
Materials 2015, 25 (36), 5748-5757.
Notes
10.
Conway, A.; Vazin, T.; Spelke, D. P.; Rode, N. A.;
Any additional relevant notes should be placed here.
Healy, K. E.; Kane, R. S.; Schaffer, D. V. Multivalent ligands
control stem cell behaviour in vitro and in vivo. Nature
Nanotechnology 2013, 8 (11), 831-838.
ACKNOWLEDGMENT
A.J.G. would like to thank Inês Albino, Laura Tiemeijer and
Chris Spicer for their contributions to cp-DIBAC synthesis and
characterization. Jarrod Cohen is a graduate student in the
laboratory of Professor Joachim Kohn at Rutgers University
and was supported by the New Jersey Center for Biomaterials.
11.
Chu, T.-W.; Kopecek, J. Drug-free macromolecular
therapeutics - a new paradigm in polymeric nanomedicines.
Biomaterials Science 2015,
3
(7), 908-922 DOI:
10.1039/C4BM00442F.
12.
Bennett, N. R.; Zwick, D. B.; Courtney, A. H.;
Kiessling, L. L. Multivalent antigens for promoting B and T cell
activation. ACS Chemical Biology 2015, 10 (8), 1817-1824.
ABBREVIATIONS
CRP, controlled radical polymerization; cp-DIBAC, cyclopro-
penone-masked dibenzocyclooctyne; PET-RAFT, porphyrin-
catalyzed photoinduced electron/energy transfer-reversible
13.
Clayton, R.; Hardman, J.; LaBranche, C. C.;
McReynolds, K. D. Evaluation of the Synthesis of Sialic Acid-
PAMAM Glycodendrimers without the Use of Sugar Protecting
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 13
Groups, and the Anti-HIV-1 Properties of These Compounds.
29.
Devaraj, N. K.; Weissleder, R.; Hilderbrand, S. A.
1
2
3
4
5
6
7
8
Bioconjugate Chemistry 2011, 22 (10), 2186-2197 DOI:
10.1021/bc200331v.
Tetrazine-based cycloadditions: application to pretargeted live
cell imaging. Bioconjugate chemistry 2008, 19 (12), 2297-2299
DOI: https://doi.org/10.1021/bc8004446.
14.
Delivery–When Is It Too Much of a Good Thing? Bioconjugate
Chemistry 2019, 30, 503-514 DOI:
10.1021/acs.bioconjchem.8b00804.
15. Ercolani, G.; Schiaffino, L. Allosteric, Chelate, and
Tjandra, K. C.; Thordarson, P. Multivalency in Drug
30.
Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-
Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent
Modification of Biomolecules in Living Systems. Journal of the
American Chemical Society 2004, 126 (46), 15046-15047 DOI:
10.1021/ja044996f.
Interannular Cooperativity: A Mise au Point. Angewandte Chemie
International Edition 2011, 50 (8), 1762-1768 DOI:
10.1002/anie.201004201.
31.
Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C.
R. Second-generation difluorinated cyclooctynes for copper-free
click chemistry. Journal of the American Chemical Society 2008,
130 (34), 11486-11493 DOI: 10.1021/ja803086r.
9
16.
Curk, T.; Dobnikar, J.; Frenkel, D., Design principles
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
for super selectivity using multivalent interactions. In
Multivalency, Huskens, J., Prins, L. J., Haag, R., Ravoo, B. J.,
Eds. John Wiley & Sons Ltd.: 2017.
32.
Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes,
F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Aza-
dibenzocyclooctynes for fast and efficient enzyme PEGylation via
copper-free (3+2) cycloaddition. Chemical Communications
2010, 46 (1), 97-99 DOI: 10.1039/B917797C.
17.
Zhang, K. Y. J.; Milburn, M. V.; Artis, D. R., Scaffold-
Based Drug Discovery. In Structure-Based Drug Discovery,
Springer Netherlands: Dordrecht, 2007; pp 129-153.
18.
Welsch, M. E.; Snyder, S. A.; Stockwell, B. R.
33.
Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J.
Privileged scaffolds for library design and drug discovery.
Current Opinion in Chemical Biology 2010, 14 (3), 347-361 DOI:
https://doi.org/10.1016/j.cbpa.2010.02.018.
Visualizing Metabolically Labeled Glycoconjugates of Living
Cells by Copper-Free and Fast Huisgen Cycloadditions.
Angewandte Chemie International Edition 2008, 47 (12), 2253-
2255 DOI: 10.1002/anie.200705456.
19.
Lounnas, V.; Ritschel, T.; Kelder, J.; McGuire, R.;
Bywater, R. P.; Foloppe, N. Current progress in structure-based
rational drug design marks a new mindset in drug discovery.
Computational and Structural Biotechnology Journal 2013, 5 (6),
e201302011 DOI: https://doi.org/10.5936/csbj.201302011.
34.
Mbua, N. E.; Guo, J.; Wolfert, M. A.; Steet, R.; Boons,
G.-J. Strain-Promoted Alkyne–Azide Cycloadditions (SPAAC)
Reveal New Features of Glycoconjugate Biosynthesis.
ChemBioChem
2011,
12
(12),
1912-1921
DOI:
20.
Danial, M.; Root, M. J.; Klok, H.-A. Polyvalent side
10.1002/cbic.201100117.
chain peptide–synthetic polymer conjugates as HIV-1 entry
inhibitors. Biomacromolecules 2012, 13 (5), 1438-1447 DOI:
https://doi.org/10.1021/bm300150q.
35.
Free
Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. Rapid Cu-
Click Chemistry with Readily Synthesized
Biarylazacyclooctynones. Journal of the American Chemical
Society 2010, 132 (11), 3688-3690 DOI: 10.1021/ja100014q.
21.
Nwe, K.; Brechbiel, M. W. Growing Applications of
“Click Chemistry” for Bioconjugation in Contemporary
36.
Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks,
Biomedical
Research.
Cancer
Biotherapy
and
A. J.; Rutjes, F. P. J. T.; van Delft, F. L. Bioconjugation with
Strained Alkenes and Alkynes. Accounts of Chemical Research
2011, 44 (9), 805-815 DOI: 10.1021/ar200059z.
Radiopharmaceuticals
2009, 24
(3),
289-302
DOI:
10.1089/cbr.2008.0626.
22.
click chemistry on drug discovery. Drug Discovery Today 2003, 8
(24), 1128-1137 DOI: https://doi.org/10.1016/S1359-
6446(03)02933-7.
23. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click
Kolb, H. C.; Sharpless, K. B. The growing impact of
37.
Debets, M. F.; Prins, J. S.; Merkx, D.; van Berkel, S. S.;
van Delft, F. L.; van Hest, J. C. M.; Rutjes, F. P. J. T. Synthesis of
DIBAC analogues with excellent SPAAC rate constants. Organic
& Biomolecular Chemistry 2014, 12 (27), 5031-5037 DOI:
https://doi.org/10.1039/C4OB00694A.
Chemistry: Diverse Chemical Function from a Few Good
Reactions. Angewandte Chemie International Edition 2001, 40
38.
Dong, Z.-m.; Liu, X.-h.; Tang, X.-l.; Li, Y.-s. Synthesis
of hyperbranched polymers with pendent norbornene
functionalities via RAFT polymerization of a novel asymmetrical
divinyl monomer. Macromolecules 2009, 42 (13), 4596-4603
DOI: https://doi.org/10.1021/ma9005796.
(11),
2004-2021
DOI:
https://doi.org/10.1002/1521-
3773(20010601)40:11%3C2004::AID-ANIE2004%3E3.0.CO;2-
5.
24.
Tasdelen, M. A. Diels–Alder “click” reactions: recent
39.
Hansell, C. F.; O’Reilly, R. K. A “Mix-and-Click”
applications in polymer and material science. Polymer Chemistry
2011, 2 (10), 2133-2145 DOI: 10.1039/C1PY00041A.
Approach to Double Core–Shell Micelle Functionalization. ACS
Macro Letters 2012, 1 (7), 896-901 DOI: 10.1021/mz300230c.
25.
Hoyle, C. E.; Bowman, C. N. Thiol–Ene Click
40.
Goldmann, A. S.; Glassner, M.; Inglis, A. J.; Barner‐
Chemistry. Angewandte Chemie International Edition 2010, 49
(9), 1540-1573 DOI: 10.1002/anie.200903924.
Kowollik, C. Post‐Functionalization of Polymers via Orthogonal
Ligation Chemistry. Macromolecular rapid communications
26.
Uygun, M.; Tasdelen, M. A.; Yagci, Y. Influence of
2013,
https://doi.org/10.1002/marc.201300017.
41. Gauthier, M. A.; Gibson, M. I.; Klok, H. A. Synthesis of
34
(10),
810-849
DOI:
Type of Initiation on Thiol–Ene “Click” Chemistry.
Macromolecular Chemistry and Physics 2010, 211 (1), 103-110
DOI: 10.1002/macp.200900442.
Functional Polymers by Post‐Polymerization Modification.
Angewandte Chemie International Edition 2009, 48 (1), 48-58
DOI: https://doi.org/10.1002/anie.200801951.
27.
Hansell, C. F.; Espeel, P.; Stamenovic, M. M.; Barker, I.
A.; Dove, A. P.; Du Prez, F. E.; O’Reilly, R. K. Additive-free
clicking for polymer functionalization and coupling by tetrazine–
norbornene chemistry. Journal of the American Chemical Society
42.
Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.;
Boons, G.-J.; Popik, V. V. Selective labeling of living cells by a
photo-triggered click reaction. Journal of the American Chemical
2011,
https://doi.org/10.1021/ja203957h.
28. Karver, M. R.; Weissleder, R.; Hilderbrand, S. A.
133
(35),
13828-13831
DOI:
Society
https://doi.org/10.1021/ja9054096.
43. Nainar, S.; Kubota, M.; McNitt, C.; Tran, C.; Popik, V.
2009,
131
(43),
15769-15776
DOI:
Synthesis and evaluation of a series of 1, 2, 4, 5-tetrazines for
bioorthogonal conjugation. Bioconjugate chemistry 2011, 22 (11),
2263-2270 DOI: https://doi.org/10.1021/bc200295y.
V.; Spitale, R. C. Temporal Labeling of Nascent RNA Using
Photoclick Chemistry in Live Cells. Journal of the American
ACS Paragon Plus Environment

Page 9 of 13
Journal of the American Chemical Society
Chemical Society 2017, 139 (24), 8090-8093 DOI:
10.1021/jacs.7b03121.
Controlled/Living Radical Polymerization. Macromolecules 2019,
52 (1), 3-23 DOI: 10.1021/acs.macromol.8b01864.
57. Gormley, A. J.; Yeow, J.; Ng, G.; Conway, Ó.; Boyer,
C.; Chapman, R. An Oxygen‐Tolerant PET‐RAFT Polymerization
for Screening Structure–Activity Relationships. Angewandte
1
2
3
4
5
6
7
8
44.
Friscourt, F.; Ledin, P. A.; Mbua, N. E.; Flanagan-Steet,
H. R.; Wolfert, M. A.; Steet, R.; Boons, G.-J. Polar
dibenzocyclooctynes for selective labeling of extracellular
glycoconjugates of living cells. Journal of the American Chemical
Chemie
https://doi.org/10.1002/anie.201711044.
58. Ng, G.; Yeow, J.; Xu, J.; Boyer, C. Application of
2018,
130,
1573-1578
DOI:
Society
https://doi.org/10.1021/ja3002666.
45. McNitt, C. D.; Cheng, H.; Ullrich, S.; Popik, V. V.;
2012,
134
(11),
5381-5389
DOI:
oxygen tolerant PET-RAFT to polymerization-induced self-
assembly. Polymer Chemistry 2017, 8 (18), 2841-2851 DOI:
10.1039/C7PY00442G.
Bjerknes, M. Multiphoton Activation of Photo-Strain-Promoted
Azide Alkyne Cycloaddition “Click” Reagents Enables in Situ
Labeling with Submicrometer Resolution. Journal of the
American Chemical Society 2017, 139 (40), 14029-14032 DOI:
10.1021/jacs.7b08472.
9
59.
Corrigan, N.; Rosli, D.; Jones, J. W. J.; Xu, J.; Boyer, C.
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
Oxygen Tolerance in Living Radical Polymerization:
Investigation of Mechanism and Implementation in Continuous
Flow Polymerization. Macromolecules 2016, 49 (18), 6779-6789
DOI: https://doi.org/10.1021/acs.macromol.6b01306.
46.
Orski, S. V.; Poloukhtine, A. A.; Arumugam, S.; Mao,
L.; Popik, V. V.; Locklin, J. High density orthogonal surface
immobilization via photoactivated copper-free click chemistry.
Journal of the American Chemical Society 2010, 132 (32), 11024-
11026 DOI: https://doi.org/10.1021/ja105066t.
60.
Xu, J.; Jung, K.; Boyer, C. Oxygen Tolerance Study of
Electron Transfer–Reversible Addition–
Photoinduced
Fragmentation Chain Transfer (PET-RAFT) Polymerization
Mediated by Ru(bpy)3Cl2. Macromolecules 2014, 47 (13), 4217-
4229 DOI: 10.1021/ma500883y.
47.
Bjerknes, M.; Cheng, H.; McNitt, C. D.; Popik, V. V.
Facile quenching and spatial patterning of cylooctynes via strain-
promoted alkyne–azide cycloaddition of inorganic azides.
Bioconjugate Chemistry 2017, 28, 1560-1565 DOI:
https://doi.org/10.1021/acs.bioconjchem.7b00201.
61.
Yeow, J.; Chapman, R.; Xu, J.; Boyer, C. Oxygen
tolerant photopolymerization for ultralow volumes. Polymer
Chemistry 2017, 8, 5012-5022 DOI: 10.1039/C7PY00007C.
48.
defined
Sun, P.; Yan, G.; Tang, Q.; Chen, Y.; Zhang, K. Well-
cyclopropenone-masked dibenzocyclooctyne
62.
Xu, J.; Jung, K.; Atme, A.; Shanmugam, S.; Boyer, C.
A Robust and Versatile Photoinduced Living Polymerization of
Conjugated and Unconjugated Monomers and Its Oxygen
Tolerance. Journal of the American Chemical Society 2014, 136
(14), 5508-5519 DOI: 10.1021/ja501745g.
functionalized polymers from atom transfer radical
polymerization. Polymer 2015, 64, 202-209 DOI:
https://doi.org/10.1016/j.polymer.2014.10.041.
49. Tang, Q.; Chen, J.; Zhao, Y.; Zhang, K. A ring-closure
method for preparing cyclic polymers from unconjugated vinyl
monomers. Polymer Chemistry 2015, 6 (37), 6659-6663 DOI:
https://doi.org/10.1039/C5PY01049G.
63.
Li, B.; Russell, S. J.; Compaan, D. M.; Totpal, K.;
Marsters, S. A.; Ashkenazi, A.; Cochran, A. G.; Hymowitz, S. G.;
Sidhu, S. S. Activation of the proapoptotic death receptor DR5 by
oligomeric peptide and antibody agonists. Journal of Molecular
Biology 2006, 361 (3), 522-536.
50.
Qu, L.; Wu, Y.; Sun, P.; Zhang, K.; Liu, Z.
Cyclopropenone-masked dibenzocyclooctyne end-functionalized
polymers from reversible addition-fragmentation chain transfer
64.
Saito, A.; Suzuki, Y.; Ogata, S.-i.; Ohtsuki, C.;
Tanihara, M. Activation of osteo-progenitor cells by a novel
synthetic peptide derived from the bone morphogenetic protein-2
knuckle epitope. Biochimica et Biophysica Acta (BBA)-Proteins
and Proteomics 2003, 1651 (1), 60-67.
polymerization.
http://dx.doi.org/10.1016/j.polymer.2017.02.071.
51. Sun, P.; Tang, Q.; Wang, Z.; Zhao, Y.; Zhang, K.
Polymer
2017,
114,
36-43
DOI:
Cyclic polymers based on UV-induced strain promoted azide-
alkyne cycloaddition reaction. Polymer Chemistry 2015, 6 (22),
4096-4101.
65.
D'Andrea, L. D.; Iaccarino, G.; Fattorusso, R.;
Sorriento, D.; Carannante, C.; Capasso, D.; Trimarco, B.; Pedone,
C. Targeting angiogenesis: structural characterization and
biological properties of a de novo engineered VEGF mimicking
peptide. Proceedings of the National Academy of Sciences 2005,
102 (40), 14215-14220.
52.
Chapman, R.; Gormley, A. J.; Stenzel, M. H.; Stevens,
M. M. Combinatorial Low-Volume Synthesis of Well-Defined
Polymers by Enzyme Degassing. Angewandte Chemie
International Edition 2016, 55 (14), 4500-4503 DOI:
10.1002/anie.201600112.
66.
Yeow, J.; Joshi, S.; Chapman, R.; Boyer, C. A. J. M. A
Self‐Reporting Photocatalyst for Online Fluorescence Monitoring
of High Throughput RAFT Polymerization. Angewandte Chemie
53.
Gormley, A. J.; Chapman, R.; Stevens, M. M.
Polymerization Amplified Detection for Nanoparticle-Based
Biosensing. Nano Letters 2014, 14 (11), 6368-6373 DOI:
10.1021/nl502840h.
International
https://doi.org/10.1002/anie.201802992.
67. Werlang, C.; Cárcarmo-Oyarce, G.; Ribbeck, K.
Engineering mucus to study and influence the microbiome.
Edition
2018, 57, 10102-10106
DOI:
54.
Chapman, R.; Gormley, A. J.; Herpoldt, K.-L.; Stevens,
M. M. Highly Controlled Open Vessel RAFT Polymerizations by
Enzyme Degassing. Macromolecules 2014, 47 (24), 8541-8547
DOI: 10.1021/ma5021209.
Nature Reviews Materials 2019,
4
(2), 134-145 DOI:
10.1038/s41578-018-0079-7.
68.
Kramer, J. J.; Nieger, M.; Bräse, S. Synthesis of Planar
55.
Yeow, J.; Chapman, R.; Gormley, A. J.; Boyer, C. Up in
Chiral N‐Heterocyclic‐Substituted Pyridinophanes. European
Journal of Organic Chemistry 2013, 2013 (3), 541-549 DOI:
https://doi.org/10.1002/ejoc.201201286.
the air: oxygen tolerance in controlled/living radical
polymerization. Chemical Society Reviews 2018, 47, 4357-4387
DOI: https://doi.org/10.1039/C7CS00587C.
56.
Oliver, S.; Zhao, L.; Gormley, A. J.; Chapman, R.;
Boyer, C. Living in the Fast Lane—High Throughput
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 13
1
2
FIGURES
3
4
5
Linear
Linear
O
6
7
R
N
H
M1
8
2-Arm
3-Arm
9
3-Arm
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
R2
560 nm
290-350 nm
Deprotect
Polymerize
Click
O
N
R3
N
N
4-Arm
R =
O
O
O
O
O
O
N
O
N
N
O
R4
O
Figure 1. Reaction schematic. Cyclopropenone-masked DIBAC (cp-DIBAC) was synthesized either as a monomer (M1) or at the end of 2-
arm (R2), 3-arm (R3), or 4-arm (R4) RAFT agents. Polymerization by PET-RAFT with 560 nm light followed by cp deprotection with UV pro-
duced SPAAC-ready polymer scaffolds for ligand clicking.
Figure 2. UV-Vis of cp-DIBAC and polymerization kinetics. a) UV-Vis of cp-DIBAC monomer (M1) before and after deprotection showing
blue-shift of the characteristic peak, followed by a loss in absorbance after click to 2kDa PEG-N3. b) UV-Vis traces of pDMA with 5% (mol) cp-
DIBAC monomer (M1) incorporated at DP 200 and 400 before and after deprotection, and following click to 2kDa PEG-N3. c) Kinetics of PET-
RAFT polymerization with and without cp-DIBAC monomer (M1) showing complete conversion in both cases after 16h. d)∫ GPC traces for
pDMA with 5% (mol) cp-DIBAC monomer (M1) after 8, 12 and 16h of polymerization, showing incorporation of the cp-DIBAC by UV detection
at 265 nm.
10
ACS Paragon Plus Environment

Page 11 of 13
Journal of the American Chemical Society
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 3. GPC traces showing the copolymerization of cp-DIBAC monomer (1) into side-chain functionalized linear polymers fol-
lowed by click with 2kDa PEG-N3. a) DMA with 5% cp-DIBAC, b) HEA with 5% cp-DIBAC, and c) DMA DP 200 with 2.5 – 7.5% cp-DIBAC.
d) UV signal at 265 nm from GPC showing increasing incorporation of cp-DIBAC as a function of feed ratio.
Figure 4. Dual functionalization by SPAAC. a) GPC traces after copolymerization of DMA / NHS-acrylate / cp-DIBAC (95 / 2.5 / 2.5 mol%)
and addition of DBCO-NH2 to create dual SPAAC-ready polymers. Subsequent click to DBCO, deprotection and secondary click of 2kDa
PEG-N3 to cp-DIBAC are shown in the green and blue lines respectively. b) UV-Vis traces of the protected polymer before and after the first
DBCO click, after deprotection of the cp-DIBAC and after subsequent click to the DIBAC.
11
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 13
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 5. Deprotection and click of 3-arm star RAFT agent (3) by 1H NMR (d6-DMSO). a) Before and b) after deprotection (3h UV) and c)
after addition of 1.0 eq of 400 g.mol-1 PEG-N3 with respect to DIBAC.
Figure 6. Polymerization, deprotection and SPAAC on star polymers. a) GPC and b) UV-Vis traces of DPtarg 200 and 400 pDMA using 2
(top), 3 (middle) and 4 (bottom) armed RAFT agents before and after deprotection and after addition of 1.0 eq of 2kDa PEG-N3 with respect to
DIBAC.
12
ACS Paragon Plus Environment

Page 13 of 13
Journal of the American Chemical Society
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 7. Scope of click and high throughput reactions a) Total positive ion count (TIC+) LC-MS data for mixtures of a 2-arm DP20 pDMA
with 400 Da PEG-N3 and three different peptides, with and without irradiation with UV light. Peaks corresponding to the polymer before (○)
and after (●) click as well as the substrate peaks (*) are highlighted. b) Summary of GPC Mn and Đ data high throughput polymer library of
pDMA, pNAM and pHEAm linear copolymers (5% cp-DIBAC) and 3-arm star polymers before and after click with 2 kDa PEG-N3. Full LC-MS
data and GPC traces for each polymer are shown in the supporting information.
Insert Table of Contents artwork here
13
ACS Paragon Plus Environment