Mixing End Groups in Thiol-Ene/Yne Reactions as a Simple Approach toward Multienzyme-Responsive Polymeric Amphiphiles

Article abstract of DOI:10.1055/s-0037-1611340

Taking advantage of the high fidelity of thiol-ene and thiol-yne chemistries, we used mixtures of thiols to prepare degradable PEG-dendron amphiphiles functionalized with two different types of enzymatically cleavable end groups. By tuning the feed ratios of the two thiols, we achieved mixtures of hybrids with statistically different ratios of end groups. Studies of the disassembly of statistically mixed hybrids showed that these amphiphiles have higher degrees of response when incubated with each of the activating enzymes, whereas a greater degree of selectivity was observed for a control mixture of two distinct amphiphiles, which required the presence of both types of enzyme to undergo complete disassembly. The potential to introduce different end groups by using a mixture of thiols in an efficient single thiol-ene or thiol-yne step opens the way for simple modification of various ene- or yne-containing polymers and tailoring of their structural and functional properties.

Full text of DOI:10.1055/s-0037-1611340

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Mixing End Groups in Thiol-Ene/Yne Reactions as a Simple Ap-
proach toward Multienzyme-Responsive Polymeric Amphiphiles
Assaf J. Harnoya,b
Nitsan Papoa,b
Gadi Slora,b
Roey J. Amir*a,b,c
a Department of Organic Chemistry, School of Chemistry, Fac-
ulty of Exact Sciences, Tel-Aviv University, Tel-Aviv 6997801,
Israel
amirroey@tauex.tau.ac.il
b Tel Aviv University Center for Nanoscience and Nanotechnolo-
gy, Tel-Aviv University, Tel-Aviv 6997801, Israel
c BLAVATNIK CENTER for Drug Discovery, Tel-Aviv University,
Tel-Aviv 6997801, Israel
Published as part of the Cluster Synthesis of Materials
Received: 03.10.2018
Accepted after revision: 09.11.2018
Published online: 16.11.2018
pression of specific enzymes in certain diseases.13 Design-
ing enzyme-responsive polymeric amphiphiles to respond
to these enzymes can potentially allow the selective release
of drug molecules only at the site of disease.14 However, the
heterogenic expression of such enzymes within the site of
disease have been reported to limit the potential applicability
of enzyme-responsive polymeric micellar drug carriers.15,16
Here we report the utilization of a mixture of two dif-
ferent esterase- and amidase-cleavable end groups in thiol-
ene17 or thiol-yne18 click reactions at the last step of an ac-
celerated synthesis19 of PEG-dendron amphiphiles that can
respond to two different enzymes. Taking advantage of the
high molecular precision that emerges from the use of den-
drons as the hydrophobic blocks, we were able to analyze
the molecular composition of the formed amphiphilic hy-
brids with high resolution by both HPLC and NMR. The abil-
ities of these statistically mixed amphiphiles to self-assem-
ble into micelles and disassemble upon incubation with ei-
ther one or both of the activating enzymes were studied
and compared with micelles prepared from a mixture of
two distinct esterase- and amidase-responsive amphi-
philes.
Building upon our previous experience in synthesizing
PEG-dendron amphiphiles and the applicability of thiol-ene
and thiol-yne reactions in synthesizing dendrons and den-
drimers as demonstrated exactly a decade ago by Hawker,
Campos, and Killops,20 and later by the groups of Hawker,21–23
Malkoch,24,25 Roy,26 and others, we decided to explore the
use of mixtures of two different thiol-containing mono-
mers in order to evaluate simultaneous introduction of two
different enzymatically degradable hydrophobic end
groups. We were curious to see if the resulting polymeric
amphiphiles would contain a statistical mixture of PEG-
dendron hybrids with esterase- and amidase-cleavable end
groups that corresponded directly to the monomer feed ra-
tio or if there would be bias toward the incorporation of one
DOI: 10.1055/s-0037-1611340; Art ID: st-2018-v0634-c
Abstract Taking advantage of the high fidelity of thiol-ene and thiol-
yne chemistries, we used mixtures of thiols to prepare degradable PEG-
dendron amphiphiles functionalized with two different types of enzy-
matically cleavable end groups. By tuning the feed ratios of the two thi-
ols, we achieved mixtures of hybrids with statistically different ratios of
end groups. Studies of the disassembly of statistically mixed hybrids
showed that these amphiphiles have higher degrees of response when
incubated with each of the activating enzymes, whereas a greater de-
gree of selectivity was observed for a control mixture of two distinct
amphiphiles, which required the presence of both types of enzyme to
undergo complete disassembly. The potential to introduce different
end groups by using a mixture of thiols in an efficient single thiol-ene or
thiol-yne step opens the way for simple modification of various ene- or
yne-containing polymers and tailoring of their structural and functional
properties.
Key words polymeric amphiphiles, enzyme-responsive polymers,
micelles, thiol-ene, thiol-yne
Polymeric micelles have been explored as nanocarriers
due to their ability to encapsulate small hydrophobic drug
molecules in their lipophilic cores.1,2 The search for ideal
amphiphilic polymers for the fabrication of micellar deliv-
ery systems has led to the development and study of vari-
ous PEG-based amphiphiles,3–5 such as pluronic,6 PEG-poly-
lactide7,8, and PEG-polycaprolactone,8 in addition to polyox-
azolines9 and polyglycerols10 as potential drug carriers. It is
clear that polymeric delivery systems must have high load-
ing capacity and micellar stability in addition to tunable re-
lease rates.11 Different release mechanisms have been intro-
duced in order to gain control over drug release rates, rang-
ing from simple diffusion of the hydrophobic guests to
active shrinkage or disassembly of the polymeric carriers in
response to specific stimuli.12 Among the various types of
stimuli, enzymes are highly attractive due to the overex-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, 2582–2587

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A. J. Harnoy et al.
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of the monomers. Toward this goal, we designed and syn-
thesized two types of PEG-dendron precursors with either
four allyl (1) or two propargyl end groups (2), which can be
functionalized at the last step of the synthesis by either thi-
ol-ene or thiol-yne click reactions, respectively. Both types
of precursors were prepared through a simple high-yielding
three-step synthesis starting from commercially available
5-KDa poly(ethylene glycol) methyl ether (Scheme 1). The
tetraallyl precursor 1 was synthesized by propargylation of
the hydroxyl chain end of the PEG to give PEG-propargyl 3,
followed by thiol-yne reaction with cysteamine hydrochlo-
ride to yield a PEG-diamine 4, which was then conjugated
with two diallyl branching units (5) to give the tetraallyl
precursor 1 in high overall yield.
The diyne precursor 2 was prepared through a similar
methodology (Scheme 1), but in this case the PEG was first
converted into PEG-allyl 6 by reaction with allyl bromide,
followed by thiol-ene reaction with cysteamine hydrochlo-
ride to yield a monoamine PEG 7, which was then conjugat-
ed to our previously reported diyne containing branching
unit 8 to yield the diyne precursor 2. All polymeric precur-
sors were fully characterized by 1H NMR and 13C NMR spec-
R
molar ratio
thiol 10/thiol 11
12a (100:0)
12b (75:25)
12c (50:50)
12d (25:75)
12e (0:100)
S
O
O
H
N
H
MeO
NH2
MeO
R
MeO
N
O
S
O
S
O
S
S
O
S
O
NO2
O
DIPEA
DCM:DMF
1:1 v/v
n
n
n
S
S
O
O
S
O
O
4
overnight
RT
NH2
NH
NH
mPEG5kDa-(NH2)2
95%
O
O
R
O
NH3+Cl-
O
1.
O
HS
12a (98%)
12b (92%)
12c (95%)
12d (93%)
12e (93%)
O
O
1
DMPA, UV, 2 h
DMPA
DMF
UV, 2 h
(5)
mPEG5kDa-2+2ene
91%
2. NaOH
S
MeO
R
O
n
3
mPEG5kDa-propargyl
quantitative
toluene
60 °C
Br
KOH
24 h
HS
HS
O
O
O
O
thiol 10
O
R =
or
MeO
H
and/or
O
n
O
mPEG5kDa-OH
N
H
N
H
thiol 11
toluene
Br
60 °C
KOH
24 h
MeO
O
n
6
DMPA
DMF
UV, 2 h
NO2
mPEG5kDa-Allyl
92%
R
R
NH3+Cl-
1.
O
O
HS
S
DMPA, UV, 2 h
S
2. NaOH
O
O
O
O
(8)
H
H
MeO
N
R
MeO
N
MeO
NH2
O
S
O
S
R
O
S
O
O
n
S
DIPEA
DCM:DMF
1:1 v/v
overnight
RT
molar ratio
n
O
S
n
O
thiol 10/thiol 11
13a (100:0)
13b (75:25)
13c (50:50)
13d (25:75)
13e (0:100)
2
7
13a (95%)
mPEG5kDa-2yne
91%
13b (quant.)
13c (quant.)
13d (94%)
13e (93%)
mPEG5kDa-NH2
93%
Scheme 1 Synthesis of PEG-dendron amphiphiles 12a–e and 13a–e
© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, 2582–2587

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troscopy, GPC, and MALDI-TOF MS, and the obtained results
were in excellent correlation with the expected values (see
Supporting Information). Once the precursors were pre-
pared, we reacted them with mixtures of two thiol deriva-
tives containing benzyl ester (thiol 10) or phenyl acetamide
(thiol 11) functionalities, which can serve as esterase or
amidase cleavable end groups, respectively. As both the
tetraallyl and dipropargyl precursors can each react with
four thiols through thiol-ene or thiol-yne reactions, respec-
tively, we expected to get statistically mixed amphiphilic
hybrids with four hydrophobic end groups (hybrids 12a–e
and 13a–e, Scheme 1). The ratios of the ester-to-amide-
containing monomers were altered from 100% esters to
75%:25%, 50%:50%, 25%:75%, and, eventually, 100% amides,
as feed ratios were altered. The resulting distinct amphi-
philes 12a, 12e, 13a, and 13e and the statistically mixed hy-
brids 12b–d and 13c–d were first analyzed by 1H NMR
spectroscopy (Figures S18, S19, S22, S23, S25–S27, and
S29–S31); the spectra clearly indicated that the ratios of
end groups corresponded directly to the feed ratio of the
two thiol-containing monomers that were used for the thi-
ol-ene and thiol-yne reactions. Although the 1H NMR spec-
tra indicated that both types of monomers reacted to simi-
lar extents depending only on their feed ratio, we were ex-
tremely encouraged to see the results of an HPLC analysis of
the formed amphiphilic hybrids (Figure 1). The obtained
HPLC chromatograms for the 1:1 ratio (hybrid 12c and 13c)
revealed the formation of five peaks for both thiol-ene- and
thiol-yne-based polymers that corresponded to hybrids
with perfectly symmetrical statistical distributions of es-
ter- and amide-containing end groups. When looking at
amphiphiles that were prepared with 3:1 or 1:3 ratios (hy-
brids 12b and 12d, respectively, for the ene-based hybrids,
and 13b and 13d, respectively, for the yne-based hybrids),
the HPLC results showed a biased distribution as expected
from the feed ratios of the two monomers (Table 1).
Figure 1 HPLC chromatograms for micelles composed of amphiphilic
hybrids (a) 12a–e and (b) 13a–e
Table 1 Feed Ratios and Distributions of End Groups Determined by HPLC and NMR Spectroscopy for Hybrids 12a–e and 13a–e
Hybrid
Feed ratio
thiol 10/thiol 11
4 esters (%)
1 amide
3 esters (%)
2 amides
2 esters (%)
3 amides
1 ester (%)
4 amides (%)
Molar ratio
esters/amidesa
12a
12b
12c
12d
12e
13a
13b
100:0
75:25
50:50
25:75
0:100
100:0
75:25
100
30
6
0
0
0
0
100:0
75:25
50:50
25:75
0:100
100:0
80:20
42
22
3
22
35
20
0
6
0
28
42
0
9
0
35
100
0
0
0
100
28
0
0
0
42
24
6
0
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Hybrid
Feed ratio
thiol 10/thiol 11
4 esters (%)
1 amide
3 esters (%)
2 amides
2 esters (%)
3 amides
1 ester (%)
4 amides (%)
Molar ratio
esters/amidesa
13c
13d
13e
50:50
25:75
0:100
5
0
0
20
3
37
18
0
28
44
0
10
54:46
26:74
0:100
35
0
100
a Determined by 1H NMR spectroscopy.
After we completed the synthesis and characterization
the kinetic studies. As a control, we prepared a 1:1 mixture
of amphiphiles 12a and 12e bearing four ester groups and
four amide groups, respectively.
1
of the different hybrids by H NMR and 13C NMR spectros-
copy, GPC, HPLC, and MALDI-TOF MS, we studied their self-
assembly in aqueous buffer solutions. First, we measured
their critical micellar concentrations (CMCs) by using Nile
red as a solvatochromic dye that can report the formation of
micelles based on noticeable change in its fluorescence
emission.27 The results showed CMC values of around 5–20
M for both thiol-ene and thiol-yne based amphiphiles,
12a–e and 13a–e (Figures S36–S45). In general, amphi-
philes with four amide-containing end groups showed
slightly higher CMC values, most likely due to the relative
higher hydrophilicity of amide bonds in comparison with
their ester equivalents. Next we measured the diameter of
the self-assembled structures by dynamic light scattering
(DLS). The DLS measurements showed the formation of
nanostructures with average diameters of around 20 nm for
all hybrids (Figure 2). This range of diameter is typical of
polymeric micelles and is in good correlation with previous
reports by our group on similar PEG-dendron amphi-
philes.28
Once the self-assembly of the statistically mixed amphi-
philes into polymeric micelles was confirmed, we studied
their abilities to disassemble in the presence of esterase
and/or amidase. In previous studies on yne-based amidase-
responsive hybrids, we noticed that although the micelles
disassembled in the presence of an amidase, not all end
groups were cleaved and the formation of partially cleaved
intermediates was observed.27,28 This phenomena was not
observed for dendrons bearing four ester-containing end
groups.29 Aiming to gain a high resolution understanding of
the enzymatic degradation and consequent disassembly
processes, we decided to focus on the enzymatic degrada-
tion of the ene-based amphiphiles. We selected the statisti-
cally mixed hybrids that were prepared by thiol-ene reac-
tion of the 1:1 mixture of the ester- and amide-containing
end groups (hybrid 12c). The degradation and disassembly
were studied using a combination of three different analyt-
ical tools: HPLC, florescence spectroscopy, and DLS. HPLC
was used to directly monitor the enzymatic degradation of
the different hybrids that form the statistical mixture,
whereas fluorescence spectroscopy allowed us to indirectly
follow the disassembly of the micelles by monitoring the
release of encapsulated Nile red. DLS was utilized to directly
confirm the presence or absence of micelles at the end of
Figure 2 DLS data for micelles composed of amphiphilic hybrids (a)
12a–e and (b) 13a–e
HPLC analysis of the degradation process of the statisti-
cally mixed hybrids in the presence of porcine liver esterase
(PLE) or penicillin G amidase (PGA) revealed the cleavage of
the esters or amide end groups, respectively, and the forma-
tion of mixtures of cleaved hybrids. In chromatograms of
the hybrids in the presence of the esterase, we observed full
cleavage of all the ester end groups. We also observed the
formation of a new set of peaks, which we attributed to hy-
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brids bearing mixtures of cleaved carboxylic acids and non-
cleaved amide end groups (4 acids; 3 acids and 1 amide; 2
acids and 2 amides; 1 acid and 3 amides). In addition, we
observed a peak corresponding to the hybrid functionalized
with four amide end groups, which remained intact under
these conditions (Figure S52). The analysis of the degrada-
tion in the presence of the amidase showed a more complex
mixture of peaks (Figure S51), and it was clear that hybrids
with three esters and one amide end groups were not
cleaved to a significant extent. This may be attributed to the
higher hydrophobicity of the ester end groups in compari-
son with the equivalent amides. Consequently, these more
hydrophobic hybrids showed greater micellar stability and
a lower degree of micelle–monomer exchange, which limit-
ed degradation by the enzyme outside of the micelles.28,30
When the hybrids were incubated with both enzymes,
we were encouraged to see the formation of a new set of
five peaks (Figure S50) with shorter retention times in com-
parison with the set of peaks formed in the presence of the
esterase alone (Figure S52). The peaks are assumed to cor-
respond to the more polar, fully cleaved hybrids with mix-
tures of carboxylic acid and amine end groups (4 amines; 3
amines and 1 acid; 2 amines and 2 acids; 1 amine and 3 ac-
ids; and 4 acids).
the presence of the esterase, which, as indicated by the
HPLC analysis, led to a higher degree of cleavage of end
groups in comparison with the cleavage by the amidase.
As the fluorescence results were not conclusive in
demonstrating the degree of disassembly, we used DLS to
directly monitor the presence or absence of micellar struc-
tures in the solutions of hybrid 12c after incubation with
either one of the two types of enzymes or with both of
them. The DLS results clearly showed that incubation with
either one of the two enzymes or with both led to disas-
sembly of the larger micellar assemblies, and smaller sizes
that may correlate with the degraded hydrophilic polymers
were observed (Figure 3, a). Interestingly, DLS analysis of
the 1:1 mixture of hybrids 12a and 12e (Figure 3, b) re-
vealed the presence of micellar assemblies after incubation
with just the esterase or just the amidase. For this mixture
of hybrids, only in the presence of both enzymes did we ob-
serve a complete disappearance of the peak corresponding
to the micelles and formation of peaks corresponding to
In the control experiment, in which we used a 1:1 mix-
ture of hybrids 12a and 12e with four esters and four am-
ides, respectively, we observed the expected results of full
cleavage of the ester-based hybrid 12a in the presence of
the esterase while hybrid 12e with the four amide end
groups stayed intact (Figure S56). The reverse result was
obtained in the presence of the amidase (Figure S55), which
led to cleavage only of the amide groups from 12e. The in-
cubation of the mixture with the two enzymes resulted in
the formation of 1:1 mixture of hybrids with four carboxylic
acids and hybrids with four amine end groups (Figure S54).
Next, we used fluorescence spectroscopy to follow the
release of Nile red from the micelles in the presence of ei-
ther one of the activating enzymes or with both of them
(Figures S60 and S61). In the presence of both enzymes,
both the statistically mixed 12c and the mixture of the two
distinct amphiphiles 12a and 12e showed clear decreases in
fluorescence intensity, indicative of the release of the en-
capsulated dye. Smaller changes in intensity were obtained
in the presence of only one of the activating enzymes for
hybrid 12c, and almost no change was observed for the 1:1
mixture of the four-esters- and four-amides-containing
amphiphiles 12a and 12e, respectively. Based on the HPLC
analysis, we explain these results by considering that in the
latter case, as one type of hybrids remained intact in the
presence of either one of the two enzymes, there were al-
ways micelles left in the solution, and hence there was a
nearly negligible decrease in fluorescence emission of the
dye molecules, which remained encapsulated in the mi-
celles. In the case of the statistically mixed amphiphile 12c,
a more significant change in fluorescence was observed in
Figure 3 DLS data for micelles composed of amphiphilic hybrids (a)
12a–e and (b) 13a–e after incubation with PLE, PGA, or both types of
enzymes
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A. J. Harnoy et al.
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cleaved hybrids. Once again, this result can be rationalized
by considering that the HPLC data that showed that both
enzymes are required to cleave the two types of end groups
from the two distinct esterase- and amidase-responsive hy-
brids. The difference between the statistically mixed hybrid
12c, which is actually a mixture of five distinct amphiphiles
with different numbers of ester- and amide-containing end
groups, and the 1:1 mixture of the two distinct amphiphiles
12a and 12e demonstrates the different properties of the
two mixtures. The statistically mixed hybrid 12c can clearly
undergo full disassembly by either one of the two types of
activating enzymes due to the presence of at least one
cleavable end group on nearly 95% of the hybrids in the
mixture; this leads to greater degree of response than in the
1:1 mixture of the two distinct amphiphiles. In the 1:1 mix-
ture there is an “AND” like response to enzymes. The pres-
ence of both the esterase and the amidase were required to
achieve full disassembly. Taking these different properties
and degree of response into account, we propose that sta-
tistical mixtures could be used in order to get higher de-
grees of enzymatic degradation and disassembly, while the
mixing of distinct amphiphiles can be utilized to achieve
higher selectivity as both types of amphiphiles need to be
degraded to achieve full response and disassembly of the
micellar assemblies.
To summarize, taking advantage of the high fidelity of
thiol-ene and thiol-yne chemistries, we synthesized de-
gradable PEG-dendron amphiphiles bearing two different
types of enzymatically cleavable end groups through a
highly step-efficient, accelerated synthetic methodology.
Utilizing a mixture of thiols, we achieved statistical mix-
tures of hybrids with different ratios of end groups, which
could be tuned by simply tuning the feed ratio of the two
types of thiols. Studies of the disassembly of statistically
mixed hybrids showed that such amphiphiles allow a high-
er degree of response to either one of the two activating en-
zymes than does a mixture of two distinct amphiphiles. The
mixture of two amphiphiles, which were studied as a con-
trol, showed greater degree of selectivity. The ability to effi-
ciently introduce different end groups by using a mixture of
thiols in a single synthetic step opens the way for simple
modification of various ene- or yne-containing polymers
and will allow tailoring of structural and functional proper-
ties of micelles.
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Funding Information
(28) Harnoy, A. J.; Rosenbaum, I.; Tirosh, E.; Ebenstein, Y.;
Shaharabani, R.; Beck, R.; Amir, R. J. J. Am. Chem. Soc. 2014, 136,
7531.
This research was funded by the ISRAEL SCIENCE FOUNDATION (grant
No. 1553/18).
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(29) Segal, M.; Avinery, R.; Buzhor, M.; Shaharabani, R.; Harnoy, A. J.;
Tirosh, E.; Beck, R.; Amir, R. J. J. Am. Chem. Soc. 2017, 139, 803.
(30) Azagarsamy, M. A.; Sokkalingam, P.; Thayumanavan, S. J. Am.
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Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0037-1611340.
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2018, 29, 2582–2587