High-Fidelity End-Functionalization of Poly(ethylene glycol) Using Stable and Potent Carbamate Linkages

Article abstract of DOI:10.1002/anie.202006687

Commercial PEG-amine is of unreliable quality, and conventional PEG functionalization relies on esterification and etherification steps, suffering from incomplete conversion, harsh reaction conditions, and functional-group incompatibility. To solve these challenges, we propose an efficient strategy for PEG functionalization with carbamate linkages. By fine-tuning terminal amine basicity, stable and high-fidelity PEG-amine with carbamate linkage was obtained, as seen from the clean MALDI-TOF MS pattern. The carbamate strategy was further applied to the synthesis of high-fidelity multi-functionalized PEG with varying reactive groups. Compared to with an ester linkage, amphiphilic PEG-PS block copolymers bearing carbamate junction linkage exhibits preferential self-assembly tendency into vesicles. Moreover, nanoparticles of the latter demonstrate higher drug loading efficiency, encapsulation stability against enzymatic hydrolysis, and improved in vivo retention at the tumor region.

Full text of DOI:10.1002/anie.202006687

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: High-Fidelity End-Functionalization of Poly(ethylene glycol) Using
Stable and Potent Carbamate Linkages
Authors: Shengyu Shi, Chenzhi Yao, Jie Cen, Lei Li, Guhuan Liu,
Jinming Hu, and Shiyong Liu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006687
Link to VoR: https://doi.org/10.1002/anie.202006687

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
High-Fidelity End-Functionalization of Poly(ethylene glycol) Using
Stable and Potent Carbamate Linkages
Shengyu Shi, Chenzhi Yao,+ Jie Cen,+ Lei Li, Guhuan Liu, Jinming Hu, and Shiyong Liu*
Abstract: It is an open “secret” that commercial PEG-amine is of
unreliable quality, for which we ascribe to both flawed manufacturing
protocols and storage stability issues. In addition, conventional PEG
functionalization relies on esterification and etherification steps,
suffering from incomplete conversion, harsh reaction condition, and
functional group incompatibility. To solve these practical challenges,
we propose an efficient strategy for PEG functionalization through
carbamate linkages. By finely tuning terminal amine basicity, stable
and high-fidelity PEG-amine with carbamate linkage was successfully
obtained, as evidenced from the clean set of MALDI-TOF MS pattern.
The “carbamate” strategy was further applied to the synthesis of high-
fidelity multi-functionalized PEG with varying reactive groups.
Compared to that with ester linkage, amphiphilic PEG-PS block
copolymers bearing carbamate junction linkage exhibits preferential
self-assembly tendency into vesicles. Moreover, nanoparticles of the
latter demonstrate higher drug loading efficiency, encapsulation
stability against enzymatic hydrolysis, and improved in vivo retention
at the tumor region.
(Figure S1).[7] mPEG-NH2 is typically manufactured by converting
hydroxyl group into sulfonyl ester (mesylate or tosylate), followed
by treating with an excess of ammonium.[5a-c,8] As already
revealed in the original work published when MALDI-TOF MS
technique was not available,[5] this protocol inevitably leads to the
formation PEG dimers and branched trimers. Note that either
hydrogenation reaction of mPEG-azide or Staudinger reaction
with PPh3 is associated with similar side reactions,[9] which are
implicated by stronger basicity/nucleophilicity of newly generated
secondary amines (Figure S1b). Impurities from these side
reactions are clearly evident from GPC shoulder peaks of mPEG-
NH2 purchased from Fluka, Laysan Bio, and Nanocs (Figure S1).
Moreover, we also found that the quality of commercial PEG-NH2
o
gradually worsen up even when stored at -20 C in a glovebox,
implying functional group incompatibility between EG repeating
units and terminal amines.
In general, end-group functionalization of PEG has been
conventionally conducted through esterification or etherification of
hydroxyl moiety (Scheme 1a). Esterification reaction is a
reversible process and complete conversion could be only
obtained under extreme conditions (anhydrous, excess reactant,
elevated temperature, and potent condensation reagent).[5b,10] In
addition to its intrinsic hydrolytic tendency, ester linkage is also
incompatible with primary amine moieties due to possible
amidation reactions.[11] Etherification reaction of PEG involves the
use of strong bases (e.g., NaH) and might lead to PEG chain
scission.[12] On the other hand, previous studies revealed that
carbamate linkage possesses superior stability towards both
hydrolysis and aminolysis, as compared to ester and ether
linkages.[2c,13]
To solve these practical challenges associated with the quality
and storage stability issues of PEG-NH2, we hypothesize that
carbamate linkage might be utilized for the synthesis of PEG-NH2,
and further generalized for various PEG derivatives (Scheme 1a).
We propose that the storage stability issue is due to amine
basicity based on a series of PEG-amine derivatives with varying
spacers (Scheme 1b). The successful synthesis of stable PEG
derivatives with high-fidelity terminal functionality are evidenced
from the clean set of MALDI-TOF MS patterns. Moreover,
nanostructures self-assembled from amphiphilic BCPs with
carbamate junction linkage possess improved drug loading
efficiency, encapsulation stability, and enhanced in vivo retention
at the tumor region, as compared to that of BCPs with ester
junction linkage (Scheme 1c).[14]
Poly(ethylene glycol) (PEG) has been the most well-known
synthetic polymer across both academics and industry, with
applications ranging from pharmaceutics, biomaterials, cosmetics,
and analytical science.[1] Due to its biologically inert and
biocompatible nature, PEG has been covalently conjugated with
proteins, peptides, oligonucleotides, drugs, and lipids to extend in
vivo circulation lifetime and stability, optimize pharmacokinetics,
and reduce systemic toxicity.[2] Up to date, over 10 pegylated
proteins and peptides have received FDA approval.[3] In addition,
amphiphilic block copolymers (BCPs) synthesized using PEG-
based initiators have been increasingly utilized to self-assemble
into drug delivery nanocarriers, exhibiting extended blood
circulation lifetime and reduced uptake by the reticuloendothelial
system (RES).[4]
End-group functionalized PEG is closely relevant to its
functional applications. Due to its superior reactivity, primary
amine-functionalized PEG has been extensively utilized for
conjugation reactions and polymerization initiators.[5] For example,
amphiphilic PEG-polypeptide BCPs are synthesized using PEG-
NH2 as initiator.[6] However, when the project was started four
years ago, we soon realized that commercial PEG-NH2 materials
from main reagent suppliers and PEG-specialized manufacturers,
either domestic or abroad, are of poor quality. GPC analysis gave
multimodal elution peaks, and MALDI-TOF MS analysis revealed
the presence of impurities unassignable to the specified structure
When two commercial mPEG45-NH2 samples (Laysan Bio and
Nanocs) were stored for one week under open air at RT and 60
oC, GPC analysis revealed apparent shift to lower and higher MW
side, respectively. These amine-mediated chain scission and
coupling events are further implicated by air oxidation and amine
carboxylation with CO2 (Figure S1, c-d).[15] Accordingly, MALDI-
TOF MS analysis revealed increasing amount of deamination
impurities (II, III) upon accelerated aging (Figure S1, e-g).[12b,16] As
these side reactions also occur when stored at -20 oC inside
glovebox, we surmise that the instability mainly originates from
amine basicity.[17] Additionally, NH-O hydrogen bonding and
superimposed O/N inductive effects might also contribute to
[] S. Shi, Dr. C. Yao, J. Cen, L. Li, Dr. G. Liu, Dr. J. Hu, Prof. Dr. S. Liu
CAS Key Laboratory of Soft Matter Chemistry, Hefei National
Laboratory for Physical Sciences at the Microscale, Department of
Polymer Science and Engineering
University of Science and Technology of China
96 Jinzhai Road, Hefei, Anhui Province, 230026, China
E-mail: sliu@ustc.edu.cn
[+] Co-first authors
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.202006687
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
instability of terminal -OCH2CH2NH2 moiety.[12b,16]
Terminal Functionalities
High-Fidelity
Multifunctional PEG
Previous
Approaches
& Deprotection
a)
Harsh Condition
& Deprotection
Example: PEG-2Mal
Inadequate Efficiency
This Work
& Deprotection
 Modular Design
 High Efficiency
 Facile Purification
Example: 3-Arm PEG-3Mal
mPEG-Amine Stability
mPEG45-b-PSm with Carbamate & Ester Block Junctions
b)
c)
m = 46, 101
Commercial mPEG-NH2
Deamination in
Open Air (< 1 Week)
Vesicles
Self-
Assembly
Stable
Micelles
Micelles
m = 6, 21
mPEG45-Carbamate-PSm
mPEG-B-NH2
Stable in Glovebox
(> 1 Year)
Esterase
Self-Assembly
Micellar
into Micelles
Disassembly
mPEG-C3-G-NH2
n = 6, 25, 43, 103
mPEG45-Ester-PSn
Stable in Open Air
Scheme 1. a) Schematics of the synthesis of high-fidelity amine-functionalized PEG. The introduction of carbamate linkage via in situ generation of isocyanate from
acyl azide moiety provides advantages over conventional ether and ester linkages in terms of linker stability, conjugation efficiency, and mildness of reaction
conditions. The design was successfully applied to diverse terminal functionalities including hydrazide, thiol, aldehyde, ketone, maleimide, and bromine, and the
fabrication of multi-functionalized PEG. This strategy could be further generalized to the post-functionalization of any hydroxyl and amine-containing precursors. b)
The quality of commercial mPEG-NH2 is less reliable due to side reactions (i.e., interchain coupling and branching) associated with manufacturing process and
storage stability resulting from deamination reaction caused by amine basicity (pKa ~ 11). Using the new strategy, the efficiency of end group functionalization is
guaranteed and storage stability much improved for carbamate-incorporated mPEG-B-NH2 and mPEG-C3-G-NH2 with decreasing amine basicities (pKa ~ 10.2 and
8.4, respectively). c) For amphiphilic block copolymers with different block junction linkages, mPEG45-Carbamate-PSm and mPEG45-Ester-PSn, the former prefers
to self-assemble in aqueous media into vesicles, whereas the latter self-assembles into micellar nanoparticles at comparable PS chain lengths. Micellar nanocarriers
of mPEG45-Carbamate-PSm possess excellent stability towards hydrolysis and enzymatic degradation, as compared to that of mPEG45-Ester-PSn.
Based on chemical intuition, we reasoned that benzyl amine-
functionalized PEG with carbamate linker might possess
improved stability due to decreased amine basicity (pKa ~ 10.2 in
DMSO) and decoupled N/O inductive effects in conventional
PEG-NH2 terminal (Figure 1a). Considering the moisture-
sensitive and highly reactive nature of isocyanates, acyl azide
derivative bearing benzyloxycarbonyl (Cbz)-protected benzyl
amine moiety was synthesized at first (2, Scheme S1a and Figure
S2). Heating of 2 leads to clean and quantitative transformation
into isocyanate derivative,[18] which in situ react with high quality
mPEG45-OH (Figure S1i) under DBTL catalysis to afford mPEG45-
B-NH-Cbz (Scheme S1a). Upon hydrogenation, as-synthesized
mPEG45-B-NH2 in the native unprotonated state could serve as a
model compound to interrogate the effects of amine basicity and
spacer linker on PEG-amine storage stabilities.
Both GPC and 1H NMR confirmed quantitative transformation
of mPEG45-OH into mPEG45-B-NH-Cbz (Figures 1b and S3, Table
S1). MALDI-TOF MS revealed high-fidelity terminal functionality
by the presence of only one set of patterns corresponding to
[M+Na]+ (Figure S3b). Upon hydrogenation, GPC analysis of
freshly synthesized mPEG-B-NH2 revealed symmetric elution
trace, with peak elution time intermediate between those of
mPEG45-OH and mPEG45-B-NH-Cbz precursors (Figure S1b).
The symmetric GPC elution trace of mPEG-B-NH2 is quite
amazing as PEG-specialized manufacturers tend to interpret
multimodal GPC traces of mPEG-NH2 (Figure S1) to nonspecific
interactions between terminal amine and GPC column packing
materials. These results confirm that multimodal GPC traces are
correlated with uncontrolled quality during both manufacturing
and storage period afterwards. Two sets of MS peaks are present
on the MALDI-TOF MS of mPEG-B-NH2, which are assigned
[M+Na]+ and [M+Na+184.1]+ (Figures 1c and S4b). Note that the
latter main MS peak is due to in situ addition reaction of terminal
amine with DCTB matrix during MALDI sample preparation, as
revealed by Meijer and coworkers.[19] We further examined
storage stability of as-prepared mPEG-B-NH2. Upon storage at -
o
o
20 C inside a glovebox for 7 d and accelerated aging at 60 C
under vacuum for 7 d, respectively, we could observe the
evolution of a slight shoulder peak at the high MW side and tailing
at the low MW side; note that the total content of newly formed
species is <5% (Figure S5). On the other hand, MALDI-TOF MS
analysis revealed almost no changes, suggesting the
complementarity between GPC and MALDI-TOF MS techniques.
Upon storage in a glovebox at -20 oC for ~1 year, GPC analysis
revealed prominent broadening and emergence of at least two
new peaks at higher MW side, implying interchain coupling and
degradation reactions. Notably, MALDI-TOF MS analysis also
revealed the shift to higher MW side, although MS peak
assignments remained unchanged (Figure S5). These results
suggested that in addition to flawed manufacturing protocols,
storage stability issue of conventional mPEG-NH2 originating from
amine basicity remains a serious concern. mPEG-B-NH2 could
partially solve the stability issue due to decreased basicity of
terminal benzyl amine and N-phenylcarbamate spacer. If
applicable, storage of PEG-amine in the primary ammonium form
provides to be a better alternative (refer to Figure S6 for the
+
storage stability of mPEG45-B-NH3 Cl-).
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
mPEG45-B-NH-Cbz
a)
b)
e)
h)
c)
mPEG45-OH
[M+Na]+
2142.45
Δm = 44.026
mPEG45-B-NH-Cbz
mPEG45-B-NH2
2100 2160 2220
[M+Na+184.1]+
mPEG45-B-NH2
2236.27
2280.53
Δm = 44.026
[M+Na]+
2271.54
2227.73
2240 2280
14 15 16 17 18 19 20
1200 1800 2400 3000 3600
Elution Time / min
m/z
mPEG45-OH
d)
f) mPEG45-C3-G-NH-Cbz
Δm = 44.026
[M+Na]+
2327.43
mPEG45-C3-G-NH-Cbz
mPEG45-C3-G-NH2
2280 2340 2400
[M+Na+184.1]+
2377.49
mPEG45-C3-G-NH2
Δm = 44.026
2320 2400
14
15
16
17
18
19
20 1200
1800
2400
m/z
3000
3600
Elution Time / min
As-
As-Prepared
1 Week
4 Weeks
6 Weeks
Storage at RT; Open to Air
Storage at RT; Open to Air
g)
mPEG45-C3-G-NH2
i)
Prepared
As-Prepared
1 Week
1 Week
4 Weeks
6 Weeks
4 Weeks
6 Weeks
2200
2240
2280
14
15
16
17
18
19
20 1600
2000
2400
2800
m/z
m/z
Elution Time / min
Figure 1. Schematics of the preparation of a) mPEG45-B-NH2 and d) mPEG45-C3-NH2. b) GPC traces recorded in THF for mPEG45-B-NH-Cbz and mPEG45-B-NH2.
c) MALDI-TOF mass spectra of mPEG45-B-NH-Cbz and mPEG45-B-NH2. e) GPC traces recorded in DMF for mPEG45-C3-G-NH-Cbz and mPEG45-C3-G-NH2. f)
MALDI-TOF mass spectra of mPEG45-C3-G-NH-Cbz and mPEG45-C3-G-NH2. The storage stability of mPEG45-C3-G-NH2 when exposed to air at room temperature,
as assayed by g) GPC and h-i) MALDI-TOF MS characterization. For comparison, the GPC trace of mPEG45-OH is also shown in b) and e).
a)
b)
c)
mPEG45-Hydrazide
mPEG45-OH
mPEG45-Hydrazide
mPEG45-SH
Δm = 44.026
[M+Na+184.1]+
2397.4549
mPEG45-CHO
mPEG45-Ketone
mPEG45-Br
2350
2400
2450
17
19
1200
1800
2400
m/z
3000
3600
1E6 lution Time 1/8min
d)
e)
f)
mPEG45-Ketone
mPEG45-SH
mPEG45-CHO
Δm = 44.026
[M+Na]+
Δm = 44.026
[M+Na]+
[M39+DCTB+Na]+
2183.2556
2198.620
Δm = 44.026
2187.15
[M45+Na]
+ [M46+H]+
2223.11
2201.19
2160
2220
2150
2200
2200
2220
2000 2500 3000 3500
1200 1800 2400 3000 3600
1500
2000
3000
m/2z500
m/z
m/z
Figure 2. a) Synthetic routes and b) relevant GPC traces recorded in THF for mPEG45-Hydrazide, mPEG45-SH, mPEG45-CHO, mPEG45-Ketone, and mPEG45-Br.
MALDI-TOF mass spectra recorded for c) mPEG45-Hydrazide, d) mPEG45-SH, e) mPEG45-CHO, and f) mPEG45-Ketone.
We envisage that further decreasing terminal amine basicity
could help enhance or even solve the stability issue of PEG-amine
(Scheme 1b). N-Terminal amine of peptides typically possess
lower much lower pKa compared to ethylamine or benzyl amine
moieties. Moreover, decreased basicity could also elevate the
efficacy of amine-relevant conjugation reactions under neutral or
even mildly acidic conditions.[5b] mPEG23-G-NH-Cbz was facilely
synthesized in one-pot via reaction of mPEG23-OH with in situ
generated isocyanate derivative from acyl azide-containing
However, the postulated hydrogenation product, mPEG23-G-NH2,
is unstable and accompanied with spontaneous deamination and
rearrangement (Scheme S1b). The undesired formation of PEG
derivative with carbamate terminal was confirmed by GPC, NMR,
and MALDI-TOF MS characterization (Figures S7-S8), and
further corroborated by studies on small molecule model
compounds (Scheme S1c and Figure S9).
We further attempted to incorporate spacer linker by starting
with Cbz-GG-acyl azide (8, Scheme S2a). Unfortunately, upon
precursor 3 upon heating (Scheme S1b, Figures S7-S8, Table S1). hydrogenation, mPEG45-C1-G-NH2 exhibits spontaneous amine-
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
mediated cyclization side reactions, although GPC elution traces
remained monomodal and symmetric upon long-term storage
under open air (Figures S10-S11). Considering that further
extending the spacer length could inhibit cyclization and eliminate
deamination reactions, mPEG45-C3-G-NH2-Cbz with C3 spacer
was synthesized using 10 as the isocyanate precursor (Scheme
S2b; Figure 1d; Figures S12-S13). Upon hydrogenation, high-
fidelity fabrication of mPEG45-C3-G-NH2 was accomplished, as
revealed by the monomodal and symmetric GPC elution trace
(Figure 1e) and the clean set of MALDI-TOF MS pattern (Figures
1f and S13). Furthermore, when exposed under open air for 1-6
weeks, both GPC and MALDI-TOF MS characterization revealed
essentially no changes (Figure 1g-i), which is in stark contrast to
commercial mPEG45-NH2 (Figure S1) and mPEG-B-NH2 (Figure
S5). These results indicate that the PEG-amine stability issue
could be solved by finely tuning amine basicity/nucleophilicity
(Scheme 1). The excellent storage stability of mPEG45-C3-G-NH2
towards air oxidation and spontaneous chain degradation augurs
well for its commercial manufacturing and storage. Under certain
application circumstances, PEG-amine in the native form has to
be used (e.g., amphiphilic PEG-polypeptide synthesis via NCA
polymerization), as in situ deprotonation with organic bases will
lead to alternate polymerization mechanisms.[6c,20]
maleimide functionality. Next, mPEG45-Mal, PEG45-2Mal, and 3-
arm PEG21-3Mal were obtained by deprotection at 110 oC
(Schemes 1 and S4). Again, 1H NMR, GPC, and MALDI-TOF MS
analysis indicated high-efficiency conversion and high-fidelity
terminal functionality (Figure 3, Figures S24-S28). Notably, furan-
protected maleimide moieties are unstable under the MALDI-TOF
MS conditions and main MS peaks are assigned to [M+Na-Fu]+
(Figure S25b). The MALDI-TOF mass spectra of mPEG45-Mal,
PEG45-2Mal, and 3-arm PEG21-3Mal are extremely clean,
exhibiting no discernible impurity peaks. These results confirmed
high-fidelity functionalization and the feasibility of fabricating PEG
derivatives with nonlinear chain topologies (Figure 3d,e,f; Figures
S26-S28).
Based on the facile fabrication of PEG derivatives with
carbamate linkage and incubation stability towards enzymatic
hydrolysis,[2c,13] we are curious about the effects of carbamate
linkage on BCP self-assembly and relevant biomedical functions.
Previously, mPEG-based ATRP and RAFT macroinitiators with
ester bonds have been exclusively utilized to synthesize
amphiphilic BCPs.[4] We envisage that the use of alternate
carbamate junction linkage could endow enhanced nanostructure
stability in aqueous media and a new approach to PEGylated
nanomaterials. We then synthesized a new PEG-based ATRP
macroinitiator bearing carbamate linkage (mPEG45-Carbamate-Br)
through in situ reaction of 4-(bromomethyl)benzoyl azide (21) with
mPEG45-OH (Figure S29). For comparison, conventional ester-
based mPEG45-Ester-Br macroinitiator was also synthesized.
Subsequent ATRP of styrene (St) using these two types of
macroinitiators afforded PEG45-Carbamate-PSm and mPEG45-
Ester-PSn with varying polystyrene (PS) block lengths (Scheme
S5, Figures S30-S32; Table S2). Quite unexpectedly, both
mPEG45-Carbamate-PS101 and mPEG45-Carbamate-PS46 self-
assembled into vesicular nanostructures, whereas mPEG45-
Building on the successful synthesis of high-fidelity and air-
stable PEG-amine derivatives, we further explored the utilization
of carbamate linkage to fabricate PEG-derivatives with a diverse
range of terminal functionalities including hydrazide, thiol,
aldehyde, ketone, maleimide, and bromine (Scheme 1). Note that
these reactive groups are important for protein PEGylation and
fabrication of drug conjugates.[2] Due to functional compatibility
between aldehyde/ketone and isocyanates, high-fidelity mPEG45-
CHO and mPEG45-Ketone were facilely synthesized in one-step
by directly reacting mPEG45-OH with 4-formylbenzoyl azide (16a),
and 4-acetylbenzoyl azide (16b), respectively (Figure 2b,e,f and
Figures S20-S23). For the synthesis of PEG45-Hydrazide and
PEG45-SH, a three-step approach was employed: i) synthesis of
acyl azide derivative bearing Boc-protected benzoyl hydrazide
(13) and Trt-protected benzyl mercaptan (15); ii) in situ reaction
Ester-PS103
and
mPEG45-Ester-PS43 with
comparable
hydrophobic PS lengths only self-assembled into micellar
nanoparticles (Figure 4b-f and Figure S33). Previously, we
reported that amphiphilic BCPs with the hydrophobic block
bearing side chain or main chain carbamate moieties could
of 13 and 15 with mPEG45-OH upon heating under DBTL catalysis; promote self-assembly into higher-order nanostructures other
iii) deprotection by TFA treatment (Schemes S2-S3; Figure 2b-
2d). All intermediates and products were thoroughly characterized
by GPC, MALDI-TOF MS, and H NMR, revealing quantitative
than nanospheres.[4f,4g,11a,21] In the current study, we discovered
that the replacement of the single ester linkage into carbamate
one at the diblock junction position could render a huge difference
in self-assembling behaviors. We ascribe this phenomenon to
interchain carbamate-mediated hydrogen bonding interactions at
the core-shell interface (Scheme 1c, Figures 4b and S34), which
facilitate morphological transition from spherical micelles into
vesicles and provide additional stabilization of corresponding
nanostructures. Note that carbamate-relevant hydrogen bonding
interactions at the core-shell interface were further verified by
temperature-dependent FT-IR for the corresponding micellar
dispersion in D2O, and this feature was completely absent for the
counterpart BCP with ester linkage at the block junction (Figure
S34).
Considering the use of self-assembled nanostructures as drug
nanocarriers, we further evaluated the stability of self-assembled
nanostructures against esterase, which is abundant in biological
fluids such as blood serum.[22] For direct comparison and clarity
of GPC data interpretation, spherical micelles self-assembled
from mPEG45-Carbamate-PS6 and mPEG45-Ester-PS6 BCPs
were examined. Upon incubating the micellar dispersion with
esterase for 0-120 h, esterase-mediated degradation of mPEG45-
Ester-PS6 amphiphiles were clearly apparent, as evidenced from
time-dependent GPC elution traces (Figure 4g). In sharp contrast,
1
transformation of mPEG45-OH into corresponding end-
functionalized mPEG45 and high-fidelity terminal functionality
(Figure 2, Figures S16-S23; Table S1). It is worthy of noting that
MALDI-TOF MS patterns of PEG45-Hydrazide and PEG45-SH are
very clean, with all MS peaks assignable to specified chemical
structures (Figure 2c,d). Therefore, the “carbamate” strategy
provides a facile and universal strategy for end-functionalized
PEG with diverse reactive groups. In principle, it could be further
generalized to the functionalization of any small molecules and
polymer precursors bearing hydroxyl and amine moieties.
Maleimide functionality is also of vital importance towards the
synthesis of functional bioconjugates.[3] On the other hand, ,-
bifunctional and 3-arm PEG have been extensively utilized for
hydrogel and network fabrication.[12c] We then utilized the
“carbamate” strategy to synthesize high-fidelity multifunctional
PEG derivatives bearing one, two, and three maleimide moieties.
Considering the highly reactive nature of maleimide, acyl azide
derivative containing furan-protected maleimide moiety, 20, was
prepared at first, followed by in situ reactions with mPEG45-OH,
PEG45 diol, and 3-arm PEG21-3OH upon heating to 85 oC,
affording corresponding functionalized PEG with furan-protected
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
micellar nanoparticles assembled from mPEG45-Carbamate-PS6
intravenous injection into mice. For mPEG45-Ester-PS6 micelles,
are resistant to both esterase hydrolysis and spontaneous
ICG emission completely diminished after 24 h under identical
hydrolysis under otherwise identical conditions (inset in Figure 4g), ICG injection levels (Figure 4j,k). These results demonstrated that
verifying the microstructural stability of carbamate linkages over
ester bonds.[2c,13]
the carbamate linkage provides extra advantages compared to
conventional BCPs with ester junction linkage, synthesized via
typical controlled radical polymerizations, due to cooperative
hydrogen bonding interactions at the core-shell interface (Figures
4b and Figure S34). Overall, polymeric amphiphiles with
carbamate moieties, located either in side linkages or at block
junction point, could lead to robust self-assembled nanostructures
with high drug loading capacity, enhanced encapsulation stability,
and extended blood circulation lifetimes.[4f,4g,11a,21a-e] In addition,
the replacement of single ester junction linkage in amphiphilic
BCPs with carbamate moiety, and its effects on both self-
assembled nanoassemblies and relevant drug delivery functions
are also reminiscent of “single point mutation” in the field of
genetics and epigenetics.
Using clinically applied indocyanine green (ICG) as a modal
functional agent, it was found that mPEG45-Carbamte-PS6
micelles could efficiently load ICG with an encapsulation
efficiency (EE) up to ~90% and loading content (LC) ~8.1%, in
contrast to ~60% and ~5.7% for micelles of the ester counterpart,
mPEG45-Ester-PS6 (Figure 4h). Due to improved stability against
esterase, ICG release from mPEG45-Carbamte-PS6 micelles was
remarkably prohibited, and less than ~10% ICG was released
within 72 h incubation duration. On the other hand, >90% ICG was
released from mPEG45-Ester-PS6 micelles. Under in vivo
conditions, fluorescence emission of ICG-loaded mPEG45-
Carbamte-PS6 micelles could still be detected 48 h after
a)
b)
3-Arm PEG21-OH
3-Arm PEG21-3Mal-Fu
3-Arm PEG21-3Mal
PEG45-OH
PEG45-2Mal-Fu
PEG45-2Mal
mPEG45-OH
mPEG45-Mal-Fu
mPEG45-Mal
15
16
17
18
19
20
Elution Time / min
c)
d)
e)
3-Arm PEG21-3Mal
PEG45-2Mal
mPEG45-Mal
Δm = 44.026
[M+Na]+
Δm = 44.026
[M+Na]+
2132.1948
Δm = 44.026
[M+Na]+
2478.1883
1723.8556
2450
2500
1700
1750
2100
2150
2000 2500 3000 3500
1500 2000 2500 3000 3500
1500
2000
2500
m/z
m/z
m/z
Figure 3. a) Synthetic routes and b) GPC traces recorded in THF for mPEG45-Mal, PEG45-2Mal, and 3-arm PEG21-3Mal; GPC traces of PEG precursors and relevant
intermediates are also shown for comparison. MALDI-TOF mass spectra recorded for c) mPEG45-Mal, d) PEG45-2Mal, and e) 3-arm PEG21-3Mal.
0 h
6 h
a)
b)
g)
0 h
120 h
24 h
48 h
72 h
120 h
17
18
Elution Time (min)
15
16
18
E1lu7tion Time / min
19
20
21
c)
d)
h)
100
10
8
Encapsulation Efficiency %
Loading Content %
80
60
40
20
0
6
4
2
0.2 μm
0.2 μm
0
mPEG45
-
mPEG45
-
e)
f)
Carbamate-PS6
Ester-PS6
i)
PEG45-Carbamate-PS6 + Esterase
PEG45-Ester-PS6 + Esterase
100
80
60
40
20
0
0.2 μm
0.2 μm
j)
0 h
2 h
4 h
8 h
12 h
24 h
48 h
0
10
20
30
40
50
60
70
Incubation Time / h
k)
106 (p/sec/cm2/sr) / (μW/cm2
)
ICG@PEG45-Ester-PS6
ICG@PEG45-Carbamate-PS6
250
200
150
100
50
0
0
10
20
30
40
50
p/sec/cm2/sr
Time / h
 108
5.0
μW/cm2
1.0
2.0
4.0
3.0
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
Figure 4. a) Chemical structures of amphiphilic BCPs with different junction linkages: mPEG45-Ester-PSn versus mPEG45-Carbamate-PSm. b) Schematics of vesicle
formation from mPEG45-Carbamate-PSm and nanostructure stabilization facilitated by interchain hydrogen bonding interactions of carbamate linkages at diblock
junctions; note that mPEG45-Ester-PSn with comparable PS lengths only self-assembles into micellar nanoparticles. Typical TEM images recorded for c) mPEG45
-
Carbamate-PS101, d) mPEG45-Carbamate-PS46, e) mPEG45-Ester-PS103, and f) mPEG45-Ester-PS43 nanoassemblies. g) Evolution of GPC traces recorded for
mPEG45-Ester-PS6 and mPEG45-Carbamate-PS6 micelles upon incubation with esterase in PB buffer (pH 7.4, 10 mM). h) Comparison of loading efficiencies and
loading contents of ICG by mPEG45-Carbamate-PS6 and mPEG45-Ester-PS6 micelles. i) Cumulative release profiles of ICG-loaded mPEG45-Ester-PS6 micelles and
mPEG45-Carbamate-PS6 micelles upon incubation with esterase in PB buffer (pH 7.4, 10 mM). j) In vivo NIR fluorescence tumor imaging and k) evolution of average
emission intensities at the tumor region for 4T1 tumor-bearing Balb/c mice at 2, 4, 8, 12, 24, and 48 h after intravenous injection of ICG-loaded mPEG45-Ester-PS6
micelles and mPEG45-Carbamate-PS6 micelles at an equivalent ICG dosage of 80 μg/kg.
Hu, S. Liu, Angew. Chem. Int. Ed. 2016, 55, 1760-1764; Angew. Chem.
2016, 128, 1792-1796; g) Z. Deng, Y. Qian, Y. Yu, G. Liu, J. Hu, G. Zhang,
In summary, we developed a highly efficient strategy to
synthesize PEG derivatives with varying terminal with high-fidelity
terminal functionalities including primary amine, hydrazide, thiol,
aldehyde, ketone, maleimide, and bromide. The functionalization
process is conducted in a one-pot manner by taking advantage of
acyl azide chemistry, which quantitatively transforms in situ into
isocyanate derivatives upon heating. The high-fidelity of PEG
functionalization is verified by the clean set of MALDI-TOF MS
patterns and monomodal/symmetric GPC elution traces. Storage-
stable and air-insensitive PEG-amine was fabricated for the first
time by integrating the “carbamate” strategy with finely tuned
terminal amine basicity/nucleophilicity, together with appropriate
choice of spacer linkers. Besides linear PEG, non-linear ones
(e.g., star-shaped) could also be quantitatively functionalized
without compromising conjugation efficiency. Notably, the
incorporation of carbamate linkage instead of conventional ester
bond at the diblock junction could not only facilitate higher-order
nanostructure formation, but also remarkably improve
nanostructure (encapsulation) stability under in vitro and in vivo
conditions. In principle, the reported “carbamate” strategy could
be further generalized to the functionalization of any small
molecules and polymer precursors bearing hydroxyl and amine
moieties.
S. Liu, J. Am. Chem. Soc. 2016, 138, 10452-10466.
[5]
[6]
a) A. F. Bückmann, M. Morr, G. Johansson, Makromol. Chem. 1981, 182,
1379-1384; b) S. Zalipsky, Bioconjugate Chem. 1995, 6, 150-165; c) M.
Leonard, E. Dellacherie, Makromol. Chem. 1988, 189, 1809-1817; d) J.
M. Harris, J. Macromol. Sci.: Part C 1985, 25, 325-373.
a) T. J. Deming, Chem. Rev. 2016, 116, 786-808; b) N. Hadjichristidis, H.
Iatrou, M. Pitsikalis, G. Sakellariou, Chem. Rev. 2009, 109, 5528-5578;
c) Z. Y. Song, Z. Y. Han, S. X. Lv, C. Y. Chen, L. Chen, L. C. Yin, J. J.
Cheng, Chem. Soc. Rev. 2017, 46, 6570-6599.
[7]
[8]
P.-C. Chen, Y. Liu, J. S. Du, B. Meckes, V. P. Dravid, C. A. Mirkin, J. Am.
Chem. Soc. 2020, 142, 7350-7355.
a) J. Loccufier, J. Crommen, J. Vandorpe, E. Schacht, Makromol. Chem.
Rapid Commun. 1991, 12, 159-165; b) D. L. Elbert, J. A. Hubbell,
Biomacromolecules 2001, 2, 430-441; c) A. Karatzas, J. S. Haataja, D.
Skoulas, P. Bilalis, S. Varlas, P. Apostolidi, S. Sofianopoulou, E. Stratikos,
N. Houbenov, O. Ikkala, H. Iatrou, Biomacromolecules 2019, 20, 4546-
4562; d) N. J. Warren, O. O. Mykhaylyk, D. Mahmood, A. J. Ryan, S. P.
Armes, J. Am. Chem. Soc. 2014, 136, 1023-1033.
[9]
B. Banasik, C. Nadala, M. Samadpour, Org. Prep. Proced. Int. 2018, 50,
95-99.
[10] a) S. Zalipsky, C. Gilon, A. Zilkha, J. Macromol. Sci.: Part A 2006, 21,
839-845; b) Y. Wei, R. Zhuo, X. Jiang, J. Sep. Sci. 2016, 39, 4305-4313.
[11] a) X. Wang, G. Liu, J. Hu, G. Zhang, S. Liu, Angew. Chem. Int. Ed. 2014,
53, 3138-3142; Angew. Chem. 2014, 126, 3202-3206; b) F. Z. Lu, X. Y.
Xiong, Z. C. Li, F. S. Du, B. Y. Zhang, F. M. Li, Bioconjugate Chem. 2002,
13, 1159-1162; c) G. Liu, C. M. Dong, Biomacromolecules 2012, 13,
1573-1583.
[12] a) K. Maranski, Y. G. Andreev, P. G. Bruce, Angew. Chem. Int. Ed. 2014,
53, 6411-6413; Angew. Chem. 2014, 126, 6529-6531; b) N. Boden, R. J.
Bushby, S. Clarkson, S. D. Evans, P. F. Knowles, A. Marsh, Tetrahedron
1997, 53, 10939-10952; c) Y. Z. Wei, R. X. Zhuo, X. L. Jiang, J.
Chromatogr. 2016, 1447, 122-128.
[13] a) C. Y. Cho, E. J. Moran, S. R. Cherry, J. C. Stephans, S. P. A. Fodor,
C. L. Adams, A. Sundaram, J. W. Jacobs, P. G. Schultz, Science 1993,
261, 1303-1305; b) M. Shamis, H. N. Lode, D. Shabat, J. Am. Chem. Soc.
2004, 126, 1726-1731; c) X. Hu, S. Liu, Y. Huang, X. Chen, X. Jing,
Biomacromolecules 2010, 11, 2094-2102; d) D. Aydin, M. Arslan, A.
Sanyal, R. Sanyal, Bioconjugate Chem. 2017, 28, 1443-1451.
[14] “End-group functionalization of hydroxyl-relevant compounds and
polymers and methods thererof”: S. Y. Liu, S. Y. Shi, Patent Application
Filed, CN202010578627.0, 2020.
Acknowledgements
The financial support from National Natural Science Foundation
of China (NNSFC) Project (51690150, 51690154, 21674103, and
U19A2094) and International S&T Cooperation Program of China
(ISTCP) of MOST (2016YFE0129700) is gratefully acknowledged.
[15] a) J. Glastrup, Polym. Degrad. Stab. 1996, 52, 217-222; b) A. Sayari, A.
Heydari-Gorji, Y. Yang, J. Am. Chem. Soc. 2012, 134, 13834-13842.
[16] P. Bollini, S. Choi, J. H. Drese, C. W. Jones, Energy Fuels 2011, 25, 2416-
2425.
Conflict of interest
[17] C. Godoy-Alcántar, A. K. Yatsimirsky, J. M. Lehn, J. Phys. Org. Chem.
2005, 18, 979-985.
[18] M. V. Zabalov, R. P. Tiger, Russ. Chem. Bull. 2007, 56, 7-13.
[19] X. Lou, B. F. de Waal, J. L. van Dongen, J. A. Vekemans, E. W. Meijer, J.
Mass Spectrom. 2010, 45, 1195-1202.
S.Y.L. and S.Y.S are inventors on a patent application filed by
University of Science and Technology of China that covers end-
group functionalization of hydroxyl-relevant compounds.
[20] H. R. Kricheldorf, Angew. Chem. Int. Ed. 2006, 45, 5752-5784; Angew.
Chem. 2006, 118, 5884-5917.
Keywords: functionalized PEG • carbamate linker • amphiphilic
block copolymers • self-assembly • encapsulation stability
[21] a) G. Liu, X. Wang, J. Hu, G. Zhang, S. Liu, J. Am. Chem. Soc. 2014, 136,
7492-7497; b) X. Wang, J. Hu, G. Liu, J. Tian, H. Wang, M. Gong, S. Liu,
J. Am. Chem. Soc. 2015, 137, 15262-15275; c) X. R. Wang, C. Z. Yao,
G. Y. Zhang, S. Y. Liu, Nature Commun. 2020, 11, 13; d) C. Z. Yao, X. R.
Wang, J. M. Hu, S. Y. Liu, Acta Polym. Sin. 2019, 50, 553-566; e) J. M.
Hu, S. Y. Liu, Sci. China Chem. 2018, 61, 1110-1122; f) Z. Y. Deng, S.
Yuan, R. X. Xu, H. J. Liang, S. Y. Liu, Angew. Chem. Int. Ed. 2018, 57,
8896-8900; Angew. Chem. 2018, 130, 9034-9038.
[22] a) F. Liu, Z. Zhao, J. Yang, J. Wei, S. Li, Polym. Degrad. Stab. 2009, 94,
227-233; b) R. J. Amir, S. Zhong, D. J. Pochan, C. J. Hawker, J. Am.
Chem. Soc. 2009, 131, 13949-13951; c) X. Zhu, M. Fryd, B. D. Tran, M.
A. Ilies, B. B. Wayland, Macromolecules 2012, 45, 660-665; d) J.
Rosselgong, E. G. L. Williams, T. P. Le, F. Grusche, T. M. Hinton, M.
Tizard, P. Gunatillake, S. H. Thang, Macromolecules 2013, 46, 9181-
9188; e) N. Qiu, X. Liu, Y. Zhong, Z. Zhou, Y. Piao, L. Miao, Q. Zhang, J.
Tang, L. Huang, Y. Shen, Adv. Mater. 2016, 28, 10613-10622; f) Y. Zheng,
B. Yu, Z. Li, Z. Yuan, C. L. Organ, R. K. Trivedi, S. Wang, D. J. Lefer, B.
Wang, Angew. Chem. Int. Ed. 2017, 56, 11749-11753; Angew. Chem.
2017, 129, 11911-11915; g) A. Tatsumi, S. Inoue, T. Hamaguchi, S.
[1]
[2]
a) S. Zalipsky, Adv. Drug Del. Rev. 1995, 16, 157-182; b) J. M. Harris, S.
Zalipsky, Poly(ethylene glycol): Chemistry and Biological Applications,
ACS, Washington, DC, 1997.
a) A. Abuchowski, J. R. Mccoy, N. C. Palczuk, T. Van Es, F. F. Davis, J.
Biol. Chem. 1977, 252, 3582-3586; b) R. Haag, F. Kratz, Angew. Chem.
Int. Ed. 2006, 45, 1198-1215; Angew. Chem. 2006, 118, 1218-1237; c) R.
B. Greenwald, Y. H. Choe, J. McGuire, C. D. Conover, Adv. Drug Del.
Rev. 2003, 55, 217-250; d) G. Liu, G. Shi, H. Sheng, Y. Jiang, H. Liang,
S. Liu, Angew. Chem. Int. Ed. 2017, 56, 8686-8691; Angew. Chem. 2017,
129, 8812-8817.
[3]
[4]
S. N. S. Alconcel, A. S. Baas, H. D. Maynard, Polym. Chem. 2011, 2,
1442-1448.
a) H. Otsuka, Y. Nagasaki, K. Kataoka, Adv. Drug Del. Rev. 2003, 55,
403-419; b) J. W. Singer, J. Controlled Release 2005, 109, 120-126; c) K.
Osada, K. Kataoka, Adv. Polym. Sci. 2006, 202, 113-153; d) K. Knop, R.
Hoogenboom, D. Fischer, U. S. Schubert, Angew. Chem. Int. Ed. 2010,
49, 6288-6308; Angew. Chem. 2010, 122, 6430-6452; e) Z. S. Ge, S. Y.
Liu, Chem. Soc. Rev. 2013, 42, 7289-7325; f) Y. Li, G. Liu, X. Wang, J.
Iwakawa, Biol. Pharm. Bull. 2018, 41, 277-280; h) C. Yao, Y. Li, Z. Wang,
C. Song, X. Hu, S. Liu, ACS Nano 2020, 14, 1919-1935.
This article is protected by copyright. All rights reserved.

10.1002/anie.202006687
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Terminal Functionalities
Curtius
Reaction
& Deprotection



Modular Design
High Efficiency
Facile Purification
Self-Assembly of mPEO45-Carbamate-PSm
MALDI-TOF MS Data
Tendency into
Vesicles
Esterase
Stable
Micelles
2000
2400
m/z
Storage-stable and air-insensitive PEG-amine with high-fidelity functionality was constructed via integrating carbamate linkage with
finely tuned amine basicity. The “carbamate” strategy provides a general approach towards PEG derivatives with diverse functionalities.
Carbamate linkage at diblock junction facilitates self-assembly into higher-order nanostructures with improved stability towards both
hydrolysis and drug encapsulation.
S. Shi, C. Yao, J. Cen, L. Li, G. Liu, J. Hu, S. Liu*
Page No. – Page No.
High-Fidelity End-Functionalization of Poly(ethylene glycol) Using Stable and Potent Carbamate Linkages
This article is protected by copyright. All rights reserved.