Orthogonal self-assembly in folding block copolymers

Article abstract of DOI:10.1021/ja310422w

We herein report the synthesis and characterization of ABA triblock copolymers that contain two complementary association motifs and fold into single-chain polymeric nanoparticles (SCPNs) via orthogonal self-assembly. The copolymers were prepared using atom-transfer radical polymerization (ATRP) and possess different pendant functional groups in the A and B blocks (alcohols in the A block and acetylenes in the B block). After postfunctionalization, the A block contains o-nitrobenzyl-protected 2-ureidopyrimidinone (UPy) moieties and the B block benzene-1,3,5-tricarboxamide (BTA) moieties. While the protected UPy groups dimerize after photoinduced deprotection of the o-nitrobenzyl group, the BTA moieties self-assemble into helical aggregates when temperature is reduced. In a two-step thermal/photoirradiation treatment under dilute conditions, the ABA block copolymer forms both BTA-based helical aggregates and UPy dimers intramolecularly. The sequential association of the two self-assembling motifs results in single-chain folding of the polymer, affording nanometer-sized particles with a compartmentalized interior. Variable-temperature NMR studies showed that the BTA and UPy self-assembly steps take place orthogonally (i.e., without mutual interference) in dilute solution. In addition, monitoring of the intramolecular self-assembly of BTA moieties into helical aggregates by circular dichroism spectroscopy showed that the stability of the aggregates is almost independent of UPy dimerization. Size-exclusion chromatography (SEC) and small-angle X-ray scattering analysis provided evidence of significant reductions in the hydrodynamic volume and radius of gyration, respectively, after photoinduced deprotection of the UPy groups; a 30-60% reduction in the size of the polymer chains was observed using SEC in CHCl₃. Molecular imaging by atomic force microscopy (AFM) corroborated significant contraction of individual polymer chains due to intramolecular association of the BTA and UPy groups. The stepwise folding process resulting from orthogonal self-assembly-induced supramolecular interactions yields compartmentalized SCPNs comprised of distinct microdomains that mimick two secondary-structuring elements in proteins.

Full text of DOI:10.1021/ja310422w

Article
pubs.acs.org/JACS
Orthogonal Self-Assembly in Folding Block Copolymers
Nobuhiko Hosono,† Martijn A. J. Gillissen,† Yuanchao Li,‡ Sergei S. Sheiko,‡ Anja R. A. Palmans,*,†
and E. W. Meijer*,†
†Institute for Complex Molecular Systems, Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of
Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands
‡Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States
S
* Supporting Information
ABSTRACT: We herein report the synthesis and characterization
of ABA triblock copolymers that contain two complementary
association motifs and fold into single-chain polymeric nano-
particles (SCPNs) via orthogonal self-assembly. The copolymers
were prepared using atom-transfer radical polymerization (ATRP)
and possess different pendant functional groups in the A and B
blocks (alcohols in the A block and acetylenes in the B block).
After postfunctionalization, the A block contains o-nitrobenzyl-
protected 2-ureidopyrimidinone (UPy) moieties and the B block benzene-1,3,5-tricarboxamide (BTA) moieties. While the
protected UPy groups dimerize after photoinduced deprotection of the o-nitrobenzyl group, the BTA moieties self-assemble into
helical aggregates when temperature is reduced. In a two-step thermal/photoirradiation treatment under dilute conditions, the
ABA block copolymer forms both BTA-based helical aggregates and UPy dimers intramolecularly. The sequential association of
the two self-assembling motifs results in single-chain folding of the polymer, affording nanometer-sized particles with a
compartmentalized interior. Variable-temperature NMR studies showed that the BTA and UPy self-assembly steps take place
orthogonally (i.e., without mutual interference) in dilute solution. In addition, monitoring of the intramolecular self-assembly of
BTA moieties into helical aggregates by circular dichroism spectroscopy showed that the stability of the aggregates is almost
independent of UPy dimerization. Size-exclusion chromatography (SEC) and small-angle X-ray scattering analysis provided
evidence of significant reductions in the hydrodynamic volume and radius of gyration, respectively, after photoinduced
deprotection of the UPy groups; a 30−60% reduction in the size of the polymer chains was observed using SEC in CHCl3.
Molecular imaging by atomic force microscopy (AFM) corroborated significant contraction of individual polymer chains due to
intramolecular association of the BTA and UPy groups. The stepwise folding process resulting from orthogonal self-assembly-
induced supramolecular interactions yields compartmentalized SCPNs comprised of distinct microdomains that mimick two
secondary-structuring elements in proteins.
formation7 or supramolecular associations8 to achieve a
INTRODUCTION
■
compartmentalized structure.9 We recently developed single-
Protein folding is a dynamic process of molecular self-assembly
chain folding of synthetic polymers bearing a single self-
during which a single-stranded polypeptide chain folds to form
assembling motif. Either benzene-1,3,5-tricarboxamide (BTA)10
a well-defined three-dimensional (3D) tertiary structure.1 One
of the defining features of this tertiary structure is the presence
of individually folded domains (α-helixes, β-sheets), which
or 2-ureido-4[1H]-pyrimidinone (UPy)11 moieties were
applied as the recognition motif to induce single-chain folding
under selected conditions. BTA is a well-explored helically self-
enable these biomacromolecules to fulfill dedicated functions in
catalysis, recognition, and signaling.2 Enzymes, for example,
assembling motif that forms aggregates through threefold-
symmetric hydrogen bonding.12 The UPy motif forms a dimer
have evolved to take full advantage of their 3D structures in
complex with a large equilibrium constant through comple-
order to realize remarkable catalytic activities and selectivities
mentary quadruple hydrogen bonding.13 When incorporated
by encapsulating multiple recognition sites close to the
catalytically active center within compartmentalized domains.3
into synthetic macromolecules, each of these self-assembling
motifs leads to controlled single-chain folding.
To fix this unique, active conformation with high fidelity, nature
In contrast to biological systems, where orthogonal self-
applies a dynamic and reversible folding mechanism that relies
to a large extent on non-covalent interactions.4
assembly is one of the essential features for the formation of
tertiary structures,1,4 examples of multiple orthogonal self-
Inspired by nature’s way, several approaches for preparing
assembling motifs in folding of synthetic macromolecules are
synthetic analogues of folded proteins using, for example,
helical polymers5 and dendrimers6 have been explored. In
addition, a number of systems have been reported in which
synthetic polymers were collapsed through covalent bond
Received: October 22, 2012
© XXXX American Chemical Society
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Figure 1. (a) Design of a triblock copolymer with BTA and UPy moieties that folds into a single-chain polymeric nanoparticle cross-linked via
orthogonal self-assembly. (b) Chemical structure of the triblock copolymers P1[UBU], P2[UBU], and P3[UBU]. (c) Helical self-assembly of chiral
BTAs via threefold-symmetric hydrogen bonding. (d) Photoinduced dimerization of o-nitrobenzyl-protected UPys via quadruple hydrogen bonding.
very rare. The only example reported to date describes the self-
folding of a polymer chain initiated by two pairwise hydrogen-
bonding interactions that self-assemble orthogonally.8e Quite
recently, we and others have applied the concept of orthogonal
self-assembly to design novel materials.14,15 For example,
materials using self-assembly of BTA and UPy motifs were
prepared in which the two motifs individually form phase-
segregated nanorods in an orthogonal fashion, affording cross-
linked polymeric materials.15 A molecular design approach
combining both of these self-assembling motifs in a folding
block copolymer would represent a significant milestone
leading to synthetic protein analogues in which orthogonal
self-assembly would induce folding of each block into internal
segregated domains. In fact, such a system would exhibit the
conformational adaptability and perfectly organized inner
structure of proteins, both of which are essential for their
effective functioning.
We herein report the synthesis and characterization of a
series of ABA triblock copolymers that fold into single-chain
polymeric nanoparticles (SCPNs) by intramolecular orthogonal
self-assembly (Figure 1). The compartmentalized architectures
were successfully realized by incorporating BTA and UPy
motifs into the B and A blocks, respectively. The use of a
photocleavable protecting group on the UPy moiety enabled a
two-step folding process triggered by a temperature decrease
for BTA self-assembly and light irradiation for UPy
dimerization. During the folding process, the selected motifs
organized into individual helical and dimeric aggregates through
mutual recognition properties, resulting in a compartmentalized
structure. The folding process was fully characterized using a
combination of 1H NMR and circular dichroism (CD)
spectroscopy, size-exclusion chromatography (SEC), and
small-angle X-ray scattering (SAXS). In addition, atomic force
microscopy (AFM) was used to monitor the folding process on
the molecular scale and provided a clear view of the single-
molecule nanostructures.
RESULTS AND DISCUSSION
■
Polymer Design. We designed the ABA triblock
copolymers P1[UBU], P2[UBU], and P3[UBU] having chiral
BTA moieties in the middle (B) block and UPy moieties in
both end (A) blocks (Figure 1). A postpolymerization
functionalization approach was employed to introduce the
BTA and UPy motifs, which reduced the synthetic effort.16
Because of its tolerance toward a variety of functional groups,
we used Cu(I)-catalyzed atom-transfer radical polymerization
(ATRP)17 to copolymerize the postfunctionalizable monomers
propargyl methacrylate (PMA) and hydroxyethyl methacrylate
(HEMA) with isobornyl methacrylate (IBMA). We first
prepared the polymeric scaffolds bearing postfunctionalizable
alkyne and hydroxyl groups (the so-called “bare” block
copolymers), poly(IBMA-co-HEMA)-block-poly(IBMA-co-
PMA)-block-poly(IBMA-co-HEMA), denoted as Pn[−−−] (n
= 1−3) (see Scheme 1). IBMA was chosen as the comonomer
to enhance the solubility of the resulting polymers. Reaction of
Pn[−−−] with azide-substituted BTA 210a through Cu(I)-
catalyzed azide−alkyne cycloaddition (CuAAC) afforded the
BTA-functionalized polymers, denoted as Pn[−B−]. Subse-
quent reaction of Pn[−B−] with isocyanate-functionalized, o-
nitrobenzyl-protected UPy 311d resulted in the desired triblock
copolymers, denoted as Pn[UBU]. This approach allowed for
convenient double functionalization of the copolymers with
both BTA and UPy moieties.
The use of a photocleavable protecting o-nitrobenzyl group
allowed us to trigger the folding processes in a stepwise fashion.
While the BTA moiety undergoes internal helical stack
formation in a cooperative manner, a process that is sensitive
to temperature,10 irradiation with UV light under diluted
conditions results in UPy dimer formation. We selected this
stepwise approach because it is reminiscent of the folding of
proteins, in which different secondary structures are first
formed and then the 3D structure is attained.1 In our case, we
B
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Scheme 1. Synthesis of Folding Triblock Copolymers Using a Postfunctionalization Approach
Figure 2. (a, d) Kinetic plots for the copolymerizations of (a) IBMA and TMS-PMA for M1 and (d) IBMA and TMS-HEMA for P1 by ATRP in the
presence of a CuBr/BPMI catalyst. (b, e) Dependence of Mn and PDI on the total monomer conversion for the copolymerizations of (b) IBMA and
TMS-PMA for M1 and (e) IBMA and TMS-HEMA for P1. (c, f) Instantaneous compositions of (c) TMS-PMA for M1 and (f) TMS-HEMA for P1
as functions of the degree of polymerization.
anticipated that sequential cooling and UV irradiation would
afford triblock copolymers folded into compartmentalized
SCPNs in a controlled manner through orthogonal self-
assembly. Both BTA and UPy self-assembly were probed by
NMR spectroscopy, and BTA self-assembly could also be
monitored by CD spectroscopy. In addition, the chain collapse
was quantified by SEC and synchrotron-radiation SAXS (SR-
SAXS).
Synthesis of Triblock Copolymers Bearing Both BTA
and UPy Motifs. The postfunctionalizable “bare” polymers
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with acetylene groups in the B block and hydroxyl groups in
both A blocks were readily prepared by two-step ATRP17
(Scheme 1). Starting from bifunctional initiator 1, the B block
consisting of poly(IBMA-co-TMS-PMA) was polymerized first,
and then the two A blocks consisting of poly(IBMA-co-TMS-
HEMA) were grown from the resulting macroinitiator. Initiator
1 is commercially available and has no absorption at the
irradiation wavelength required for the o-nitrobenzyl depro-
tection. The trimethylsilyl (TMS)-protected monomers TMS-
PMA18 and TMS-HEMA19 were used to avoid undesirable
interactions with and coordination to the catalyst, since it has
been reported that the unprotected monomers may give rather
broad polydispersity indices (PDI = Mw/Mn, where Mw and Mn
are the weight-average and number-average molecular weights,
respectively), especially in the case of PMA.18a In the present
study, the monomer feed ratio of each functionalized monomer
with respect to IBMA was fixed at 10% in both blocks. Toluene
was used as the reaction solvent, and the monomer conversion
was followed by gas chromatography (GC) using p-xylene as an
internal standard. To investigate the influence of the molecular
weight of the triblock copolymer on the folding properties, we
aimed for three different polymers, P1−P3, with degrees of
polymerization (DPs) ranging from 90 to 420.
molecular weights, P2[−−−] and P3[−−−] (Mn = 38 200 and
90700 g/mol, respectively; Scheme 1), were prepared. All of
the data for the numbers of incorporated functional groups of
both types are summarized in Table 1.
Table 1. Conditions and Results for the Triblock
Copolymerizations by ATRP
Mn (g/mol)
c
d
e
polymer initiator
monomers
calcd
exptl
PDI
1.21
nCC/nOH
3.8/−
M1
M2
M3
P1
1
IBMA /TMS-
7800
12900
33100
20500
37400
89200
8100
12500
29600
20500
39400
93000
a
PMA
1
IBMA/TMS-
1.21
1.20
1.24
1.33
6.3/−
16.6/−
3.8/6.4
a
PMA
1
IBMA/TMS-
a
PMA
M1
M2
M3
IBMA/TMS-
a
HEMA
P2
IBMA/TMS-
6.3/11.3
b
HEMA
f
P3
IBMA/TMS-
1.54
16.6/27.9
b
HEMA
a
ATRP was carried out with [CuBr]0:[I]0:[BPMI]0 = 2:1:4, where I is
the initiator. The ratio of IBMA to functionalized monomer (TMS-
b
PMA or TMS-HEMA) was 9:1. Same as a, except that [CuBr]0:[I]0:
For the synthesis of the P1 series, we first prepared the
macroinitiator M1. Copolymerization of TMS-PMA and IBMA
in the presence of a CuBr/N-butyl-2-pyridylmethanimine
(BPMI) catalyst20 showed good control, as evidenced by the
kinetic plots (Figure 2a,b). The polymerization proceeded with
a small termination reaction and a slight difference in the
reaction rates for the two monomers, with TMS-PMA being
slightly more reactive than IBMA (Figure 2a). From the
monomer conversion data, we calculated the instantaneous
composition of TMS-PMA along with the DP (Figure 2c),
since the incorporation sequence of the acetylene-function-
alized monomers ultimately determines the secondary structure
of the polymer. The sequence was expected to be crucial for the
self-assembling event, which strongly depends on the local
concentration of self-assembling motifs. The calculation was
carried out following the work of Matyjaszewski and co-workers
on spontaneous gradient copolymerization for brush poly-
mers.21 From the monomer feed ratio, the instantaneous
composition of acetylene-functionalized monomers in the
polymer backbone was calculated to be 10% in any part of
polymer. Gratifyingly, the calculated instantaneous composition
of TMS-PMA showed a constant value of ∼10% at all DPs. At
90% conversion, the reaction mixture was exposed to air and
diluted in tetrahydrofuran (THF). The resultant polymer was
precipitated from methanol, affording macroinitiator M1,
poly(IBMA-co-TMS-PMA) (Mn = 8100 g/mol), as a white
powder (Scheme 1).
In the next step, the copolymerization of TMS-HEMA and
IBMA started with the macroinitiator M1. Analysis of Figure
2d,e indicates that also in this case good control was obtained.
The calculated instantaneous composition of TMS-HEMA
maintained a constant value of ∼10% at all DPs (Figure 2f),
showing that obtaining P1 with good control over the
copolymer composition (Mn = 20 500 g/mol, PDI ≈ 1.2) is
feasible with different functional groups in the A and B blocks.
Subsequent removal of the TMS groups using tetrabutylammo-
nium fluoride (TBAF) afforded the bare triblock copolymer
P1[−−−] (Mn = 20 100 g/mol) bearing acetylene and
hydroxyl groups in the B and A blocks, respectively. By a
similar approach, the other two bare polymers with different
[BPMI]0 = 4:1:8. For details, see the Supporting Information.
Calculated as [MWI + ([M1]0/[I]0) × MW1 × conv1 + ([M2]0/
c
[I]0) × MW2 × conv2], where MWI is the molecular weight of the
initiator, MWi and convi are the molecular weight and conversion,
respectively, of monomer Mi (i = 1: IBMA; i = 2: TMS-PMA or TMS-
d
HEMA). Determined via SEC using THF as the eluent, calibrated
e
with polystyrene standards. nCC and nOH are the numbers of
acetylene and hydroxyl groups per chain, respectively, as calculated
f
from the conversions of the functionalized monomers. The molecular
weight distribution was bimodal.
Subsequently, the pendant acetylene and hydroxyl groups
were functionalized with azide-functionalized BTA 2 and
protected UPy 3, respectively, affording the desired triblock
copolymers bearing both self-assembling motifs, P1[UBU],
P2[UBU], and P3[UBU] (28 100, 52 600, and 124 000 g/mol,
respectively; Scheme 1). The numbers of incorporated BTA
and protected UPy moieties in the polymers were determined
by a combination of 1H NMR and ATRP conversion data. The
results for polymers P1−P3 are summarized in Table 2. First,
the CuAAC reactions with 2 were performed in deoxygenated
THF in the presence of the stoichiometric Cu(I) catalyst
generated in situ from CuI and N,N-diisopropylethylamine
(DIPEA), which afforded the Pn[−B−] series of polymers
bearing only BTA moieties on the B block. The Pn[−B−]
polymers were then reacted with 3 using dibutyltin dilaurate
(DBTDL) as a catalyst. The reaction mixtures were precipitated
from hexane and then methanol twice, affording the double-
functionalized Pn[UBU] series of polymers. As an example,
Figure 3 shows the representative SEC traces for the P1 series.
Side reactions and undesired associations were not observed
during the postfunctionalization reactions.
Orthogonal Self-Assembly Followed by NMR Spec-
troscopy. In the past, variable-temperature NMR (VT-NMR)
spectroscopy has been shown to be a powerful technique to
assess the orthogonality of different self-assembling system-
s.9f,14e Thus, we performed VT-NMR analysis of P2[UBU]
before and after irradiation (Figure 4). Figure 4a depicts the
temperature-dependent NMR spectra of P2[UBU] in pure d4-
dichloroethane (d4-DCE) at 1.0 mg/mL concentration before
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Table 2. Data for the Triblock Copolymer Synthesis
a
b
Mn (g/mol) (PDI) [nF]
[−−−]
[−B−]
[UBU]
P1
P2
P3
20100 (1.25) [3.8CC] [6.4OH
38200 (1.34) [6.3CC] [11.3OH
90700 (1.53) [16.6CC] [27.9OH
]
24000 (1.17) [3.8BTA] [6.4OH
46000 (1.30) [6.3BTA] [11.3OH
102000 (1.48) [16.6BTA] [27.9OH
]
28100 (1.16) [3.8BTA] [3.8UPy/2.6OH
52600 (1.26) [6.3BTA] [5.5UPy/5.8OH
]
]
]
]
]
]
124000 (1.46) [16.6BTA] [17.3UPy/10.6OH]
a
b
Determined via SEC using THF as the eluent, calibrated with polystyrene standards. Numbers of incorporated functional groups (indicated by the
subscripts), determined via H NMR with reference to the corresponding numbers determined previously.
1
proton signals sharpened, and the UPy dimer protons
broadened and disappeared above 50 °C, indicating that all
of the hydrogen bonds were broken. Under these conditions,
the polymer unfolded and adopted a random coil conformation.
Upon cooling, the BTA signals showed the expected broad-
ening and downfield shifts, and the UPy dimer signals
reappeared. Importantly, the behavior of the BTAs was
identical in the presence and absence of UPy dimers. This
indicates that the hydrogen bonds between the different
supramolecular motifs were restored and that the self-assembly
was orthogonal (i.e. the two self-assembly events occurred
independently of each other). As a result, a folded structure
comprising compartments stabilized by UPy dimers and
compartments stabilized by BTA helical aggregates was formed.
Intramolecular BTA Self-Assembly Followed by CD
Spectroscopy. The self-assembly of chiral BTA moieties on
the B block of the polymer was investigated with temperature-
dependent CD spectroscopy (Figure 5). We selected the mixed
solvent system of methylcyclohexane (MCH) and 1,2-DCE
(25/75 v/v). For hydrogen-bonding-induced polymer folding,
one has to consider the critical balance between backbone
solubility and self-assembly capability of the hydrogen-bonding
motif, which prefers less polar solvents. Previously, we
discussed the importance of solvent polarity on BTA-driven
SCPN formation.10 “Free” BTAs self-assemble in apolar
solvents such as dodecane, heptane, and MCH.12 When
attached to a polymer, however, two conflicting propensities,
aggregation of BTAs and solvation of the polymer backbone,
have to be balanced by the solvent polarity and temperature in
order to obtain SCPNs. Increasing the solvent polarity or
solution temperature brings about unfolding (denaturation) of
the SCPNs. The 25/75 (v/v) MCH/DCE mixture provides a
compromise between BTA aggregation and polymer solubility,
allowing us to follow the whole folding process in a convenient
temperature window between 80 and 20 °C.
Figure 3. SEC traces of M1, P1[−−−], P1[−B−], and P1[UBU]
recorded with a refractive index detector using THF as the eluent.
UV irradiation. A pronounced peak shift and broadening of the
protons attributed to the BTA moieties were clearly observed
upon cooling from 80 to 20 °C. The broadening of the signals
is indicative of BTA self-assembly. The peak at 6.6 ppm,
corresponding to the amide protons on the BTA moieties,
shifted downfield upon cooling, indicative of threefold
hydrogen-bond formation by BTAs.22 After 350 nm UV light
irradiation of the sample at 20 °C, the spectra showed that a
peak originating from benzyl protons on the protected UPy
moieties disappeared and other peaks corresponding to UPy
dimers simultaneously appeared at 10.1, 11.8, and 13.1 ppm
(Figure S1 in the Supporting Information).13d At the same
time, the BTA peaks remained broad, indicating that their self-
assembly was not affected. The deprotection was completed
after 20 min irradiation. These results suggest that both motifs
were self-assembled under the conditions applied.
After completion of the deprotection, we heated the sample
to 80 °C again and recorded NMR spectra as the sample was
cooled to 20 °C (Figure 4b). At higher temperature, the BTA
Figure 4. Arrays of VT-NMR spectra recorded for 1 mg/mL solutions in d4-DCE upon cooling from 80 to 20 °C for P2[UBU] (a) before and (b)
after UV irradiation (P2[UBU]UV). The solvent peak was used as an internal standard. The insets depict the assignments of protons on BTA
(blue), protected UPy (yellow), and the UPy dimer complex (red).
E
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Figure 5. (a) CD cooling curves of P1[UBU], P2[UBU], and P3[UBU] before (red) and after (blue) UV irradiation (cBTA = 50 μM) monitored at λ
= 225 nm in 25/75 (v/v) MCH/DCE at temperatures from 80 to 20 °C (cooling rate 1 °C/min). (b) CD spectra of P1[UBU], P2[UBU], and
P3[UBU] in 25/75 (v/v) MCH/DCE at 20 °C during 120 min of 350 nm UV irradiation.
Figure 6. SEC traces for (a) P1[UBU], (b) P2[UBU], and (c) P3[UBU] before and after 2 h of 350 nm UV irradiation, recorded with a UV
detector using CHCl3 as the eluent.
The CD spectra of the three triblock copolymers carrying
both BTA and protected UPy were measured on cooling from
80 to 20 °C (Figure 5a). The BTA concentration (cBTA) was
fixed at 50 μM in all polymer solutions. To ensure that self-
assembly would be under thermodynamic control, slow cooling
(1 °C/min) was applied. Above 65 °C, all of the polymers were
CD-silent, indicating that the polymers were molecularly
dissolved and adopted fully unfolded conformations. Upon
cooling, a negative Cotton effect was observed for all of the
polymers with a maximum at λ = 225 nm, in agreement with
our previous reports and indicative of the formation of left-
handed helical columnar aggregates inside the polymer.10,12
The CD signal intensity of these three polymer solutions was
proportional to the number of BTAs per chain when the molar
concentrations of BTA units were identical. This shows that the
BTA self-assembly event occurs within a single chain (Figure
5). These observations are all consistent with our previous work
on BTA-based SCPNs, in which we found that the local BTA
concentration is responsible for the magnitude of the Cotton
effect.10 Taking these results into account, we propose that
before UV irradiation, the block copolymers are partially folded
as a result of intramolecular BTA self-assembly in the middle
block.
Subsequent UV irradiation provided the polymers P1-
[UBU]UV, P2[UBU]UV, and P3[UBU]UV, in which the
UPy groups were deprotected and formed dimers (see above).
The CD spectra at 20 °C before and after irradiation (Figure
5b) were identical, confirming the NMR results that UPy dimer
formation did not interfere with the BTA self-assembly. The
stability of the BTA aggregates was investigated by heating and
cooling the samples and probing the CD effect as a function of
temperature at 225 nm. The cooling curves before and after UV
irradiation were superimposable, indicating that UPy dimeriza-
tion did not affect the stability of the BTA helical aggregates.
Only the largest polymer, P3[UBU]UV, showed a small
deviation in the CD cooling curve from that obtained before
irradiation. The decrease in CD signal could have been caused
by the fact that the BTA columnar aggregates were disrupted as
a result of large conformational changes of the polymer arising
from intramolecular UPy association. Alternatively, there may
have been some loss of the orthogonality between the BTA and
UPy self-assembly processes due to high incorporation of both
groups in the single polymer. Importantly also, in pure DCE as
the solvent, in which “free” BTAs are molecularly dissolved
under these conditions, all of the polymers showed CD signals
similar in shape albeit with reduced intensity compared with
those observed in the 25/75 (v/v) MCH/DCE mixed solvent.
In pure DCE, P2[UBU]UV also exhibited a small deviation in
the intensity of the CD signal after UV irradiation, while it did
not in the 25/75 (v/v) MCH/DCE mixed solvent (Figure S2
in the Supporting Information). These observations suggest
that the degree of orthogonality also depends on the balance
between the backbone solubility and the self-assembly
capability of the hydrogen-bonding motif.
Chain Collapse Induced by Intramolecular UPy
Association. While NMR and CD spectroscopy are useful
for gathering information on the orthogonality of the self-
assembling motifs, they do not provide information on the
effect of self-assembly on the polymer conformation. For this
purpose, we used SEC and SR-SAXS analyses. Both techniques
provide information on the volume of the polymer coil, which
can be related to the degree to which the polymer adopts a
random coil conformation or is present in a collapsed state.
After UV light irradiation, the triblock copolymers were
analyzed by SEC measurements in chloroform and THF
(Figure 6 and Table 3). The SEC samples were prepared by
F
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a
Table 3. SEC Data for the Triblock Copolymers and Photoinduced Intramolecular Collapse
b
c
CHCl3 SEC
THF SEC
before irradiation
Mn (g/mol) PDI
after irradiation for 2 h
before irradiation
Mn (g/mol) PDI
after irradiation for 2 h
polymer
Mn (g/mol)
PDI
% decrease
Mn (g/mol)
PDI
% decrease
P1[UBU]
P2[UBU]
P3[UBU]
27000
50600
1.35
1.65
2.60
17800
32500
60700
1.29
1.91
2.60
34
36
62
28100
52600
1.16
1.26
1.46
26000
43900
1.18
1.33
1.57
7.4
17
158000
124000
100200
19
a
Reaction conditions: polymer (0.5 mg/mL) dissolved in 25/75 (v/v) MCH/DCE mixed solvent was exposed to 350 nm UV irradiation for 2 h and
b
then subjected to SEC analysis without further treatment. Determined via SEC using CHCl3 as the eluent, calibrated with polystyrene standards.
c
Determined via SEC using THF as the eluent, calibrated with polystyrene standards.
Figure 7. (a) SAXS data and fitting curves (black solid lines) of P2[−−−], P2[−B−], P2[UBU], and P2[UBU] after UV irradiation. The SAXS
data were recorded in 1 mg/mL solutions in 75/25 (v/v) MCH/1,4-dioxane at 20 °C and are drawn here on the same scale but offset vertically for
clarity. (b) Rg values for P2[−−−], P2[−B−], P2[UBU], and P2[UBU]UV obtained from fitting analysis of the SAXS data. The insets
schematically illustrate the triblock copolymer in a partly folded state and the fully folded state after UV irradiation.
dissolving the protected polymers in the 25/75 (v/v) MCH/
DCE mixed solvent at 0.5 mg/mL concentration and then
irradiating them with 350 nm UV light for 2 h. The irradiated
solutions were subjected to SEC directly without any further
treatment. The volume decrease of the polymer coil was a
result of intramolecular UPy dimerization after the cleavage of
protecting group, leading to a collapse of the polymer chain.11
The chain collapse was large (34−62%) when chloroform was
used as the eluent (Figure 6). On the other hand, SEC using
THF as the eluent showed a rather small volume change for all
of the polymers, although the extent of the volume decrease
was similar to that for UPy-pendant SCPNs (18%−22% for a
PMMA backbone with 10% UPy incorporation) reported in
our previous work using THF as a SEC eluent.11c Because
P2[UBU] showed a CD signal in pure DCE but not in pure
THF, the eluent dependence of the volume decrease might
have been related to BTA self-assembly on the polymer.
Intriguingly, the largest polymer, P3[UBU], showed an
extraordinarily large volume decrease of 62% in CHCl3 SEC
measurements. This result is quite consistent with the CD
signal decrease of P3[UBU] observed upon UPy deprotection
and gives reasonable explanation for the hypothesis that such a
large collapse may disrupt internal BTA helical stacks. With the
large shift of the main SEC peak toward low-molecular-weight
region, a small shoulder at higher molecular weight could be
observed (Figure 6). This tendency became more pronounced
in higher-DP polymers, indicating the presence of some
intermolecular UPy association. In fact, we found that the
distinct shoulder became much larger when the samples were
prepared at higher concentrations of 1.0 mg/mL (Figure S3 in
the Supporting Information).23 Thus, these observations are
certainly indicative of the intermolecular polymer interactions,
which increase with the volume concentrations of polymers.
SR-SAXS measurements were done with the P2 polymer
series of [−−−], [−B−], [UBU], and [UBU]UV in 75/25 (v/
v) MCH/1,4-dioxane (Figure 7). Because chlorinated solvents
absorb a large part of the X-ray radiation, preventing useful
measurements, we changed from DCE to 1,4-dioxane. Even in
75/25 (v/v) MCH/1,4-dioxane, the BTA moieties on the
polymer self-assembled into helical columnar aggregates and
gave a typical CD signal (Figure S4 in the Supporting
Information). Fitting of the SAXS curves provided the radius
of gyration (Rg) and the excluded volume parameter (ν) of the
polymers before and after irradiation.23 The bare polymer
P2[−−−] showed Rg = 8.1 0.1 nm (ν = 0.547 0.004), and
the corresponding functionalized polymers P2[−B−] and
P2[UBU] showed Rg = 11.8 0.3 nm (ν = 0.561 0.004)
and 14.4 0.3 nm (ν = 0.567 0.003), respectively (Figure
7b). The increase in Rg is consistent with an increase in the
mass of the polymers upon functionalization.
UV irradiation of P2[UBU] to produce P2[UBU]UV
resulted in a significant decrease in Rg to 11.1 0.2 nm (ν =
0.513
0.003), corresponding to a size reduction of 22.9%.
The decrease in Rg is in good agreement with the calculated
value of 19.8% obtained from the Mn decrease in SEC analysis
24
0.5
ν
with an assumption of Rg ∼ Mn ∼ Mn
.
The ν value for
P2[UBU] also showed a decrease after UV irradiation,
indicating that the polymer adopts more compact conformation
as a result of the strong dimerization between UPys
incorporated at the two ends of the polymer. These
observations provide a clear image of the fully folded state
whereby the triblock copolymer adopts a globular shape with
both internal helical and dimeric structures (Figure 7).
Microstructure of Individual SCPNs Imaged by AFM.
AFM is a powerful tool for imaging the nanostructures of
individual macromolecules25 and polymeric nanoparticles11 on
surfaces. Since the second folding process induced by
G
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Figure 8. AFM height images of P3[UBU] capture two-steps of the molecular folding process (a) before and (b) after UV irradiation. The film
samples were prepared by spin-casting from diluted DCE solution on a mica substrate. The top-right insets show magnifications of the framed areas.
photoinduced UPy association in P3[UBU] is related to a
significant volume reduction, we anticipated that changes in the
polymer conformation before and after deprotection could be
readily followed by AFM. Thus, we prepared two thin-film
samples of P3[UBU] before and after UV irradiation by spin-
casting a highly diluted DCE solution (0.05 μg/mL) on mica
substrates.23 In both cases, AFM measurements showed the
presence of individual block copolymers (Figure 8a,b and
Figures S5 and S6 in the Supporting Information). Before UV
irradiation, some individual chains adopted a “bead−tail”
conformation. Their visual appearance is highly reminiscent
of the “partly folded” state (Figures 8a and 7b), in which only
BTA moieties in the middle block self-assemble into internal
helical stacks and partly collapse the polymer chain.
Subsequently, UV irradiation triggered intramolecular UPy
association resulting in complete collapse of individual polymer
chains into nanoparticles. The AFM images, clearly depict this
drastic chain collapse and image the “fully folded” SCPNs
(Figure 8b). Apart from visualizing the individual block
copolymers on the mica surface, the AFM measurements
provide real snapshots of the compartmentalized SCPNs before
and after UV irradiation. The results further support our
proposal that the triblock copolymers presented here
successfully demonstrate the controlled folding of synthetic
polymers into compartmentalized SCPNs via orthogonal self-
assembly.
domains mimicking an α-helix and a β-sheet were successfully
provided.
Our present system provides a rational design approach for
self-folding protein analogues and shows the broad applicability
of orthogonal self-assembly within one polymer chain as a
design principle for developing synthetic enzyme mimics with
well-defined tertiary folded structure. Evidently, our ability to
fold synthetic macromolecules into defined, compartmentalized
structures is still far from the perfection achieved in the folding
of polypeptides into conformations that display selected
functions. To mimic these natural systems, forming compart-
mentalized structures with an ordered interior is not enough.
Precise location of self-assembling motifs within the polymer
chain and enhanced control over polydispersities are of crucial
importance for improving the scope of synthetic analogues.
Recent advances in sequence-controlled polymerizations and
improvements in controlled radical polymerizations will
undoubtedly provide new opportunities to achieve this.26
Currently, we are focusing on transferring the structuring
concept presented in this paper to aqueous systems with the
aim of making available novel enzyme mimics capable of
multiple cascade reactions that show exceptionally high
reactivity and selectivity for non-natural substrates. Moreover,
on the basis of the present compartmentalizing technique,
Janus-type SCPNs are being designed to study the self-
assembly of these spherical objects.
CONCLUSION
■
ASSOCIATED CONTENT
In summary, a series of ABA-type triblock copolymers with
orthogonally self-assembling motifs, BTA and UPy, were
readily synthesized using a postfunctionalization approach.
Poly(isobornyl methacrylate) backbones having pendant alkyne
and hydroxyl groups in the B and A blocks, respectively, were
successfully prepared by ATRP. The resulting “bare” triblock
copolymers were then functionalized using the CuAAC and
isocyanate conjugation reactions, affording the triblock
copolymers with distinct self-assembling motifs in a single
chain. The postfunctionalization approach is broadly applicable
and allows easy access to other combinations of orthogonally
self-assembling moieties and polymer topologies. The triblock
copolymers can thermally fold into SCPNs through intra-
molecular self-assembly of the BTAs to form internal columnar,
helical stacks. UPy deprotection using UV light results in a
further collapse of the polymers into more compact
conformation via intramolecular UPy association. Well-defined
single-chain nanoparticles with orthogonally self-assembling
■
S
* Supporting Information
General information on materials and instrumentation,
synthetic procedures for all block copolymers, additional CD
cooling curves measured in pure DCE and 75/25 (v/v) MCH/
1,4-dioxane, NMR spectra recorded during UV irradiation, SEC
traces of the samples prepared at higher concentrations of 1.0
mg/mL, experimental setup and fitting analysis for SAXS data,
and AFM setup and height profiles. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
a.palmans@tue.nl; e.w.meijer@tue.nl
Notes
The authors declare no competing financial interest.
H
dx.doi.org/10.1021/ja310422w | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
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Mes, T.; de Greef, T. F. A.; Gillissen, M. A. J.; Besenius, P.; Palmans,
A. R. A.; Meijer, E. W. J. Am. Chem. Soc. 2011, 133, 4742−4745.
(11) (a) Foster, E. J.; Berda, E. B.; Meijer, E. W. J. Am. Chem. Soc.
2009, 131, 6964−6966. (b) Foster, E. J.; Berda, E. B.; Meijer, E. W. J.
Polym. Sci., Part A: Polym. Chem. 2011, 49, 118−126. (c) Berda, E. B.;
Foster, E. J.; Meijer, E. W. Macromolecules 2010, 43, 1430−1437.
ACKNOWLEDGMENTS
■
N.H. is thankful to the Japan Society for the Promotion of
Science (JSPS) for a Young Scientist Fellowship. Dr. Ilja K.
Voets is acknowledged for help with interpreting SAXS data
and Patrick J. M. Stals for synthesis of some compounds and
help with AFM measurements. The authors express their
gratitude to ESMI and the SLS for providing beam time and
gratefully acknowledge the technical assistance of Dr. Andreas
Menzel. The authors also thank the ICMS animation studio for
the artwork. This work was supported by the European
Research Council (ERC), the Dutch Science Foundation
(NWO), and the National Science Foundation (DMR-
1122483).
(d) Stals, P. J. M.; Gillissen, M. A. J.; Nicolay, R.; Palmans, A. R. A.;
̈
Meijer, E. W. In preparation.
(12) (a) Ogata, D.; Shikata, T.; Hanabusa, K. J. Phys. Chem. B 2004,
108, 15503−15510. (b) Smulders, M. M. J.; Schenning, A. P. H. J.;
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Everts, J. C.; de Bruijn, R.; Filot, I. A. W.; Smulders, M. M. J.; Martín-
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