Foldamer structuring by covalently bound macromolecules

Article abstract of DOI:10.1021/ja2087163

We used fluorescence and electronic absorption spectroscopy to study the molecular weight dependence of macromolecule-induced folding in a chain-centered meta-phenylene ethynylene (mPE) oligomer. Analogous to the ability of intrinsically unstructured prot

Full text of DOI:10.1021/ja2087163

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Foldamer Structuring by Covalently Bound Macromolecules
Koushik Ghosh and Jeffrey S. Moore*
Department of Chemistry and the Beckman Institute for Advanced Science and Technology, University of Illinois at
UrbanaÀChampaign, Urbana, Illinois 61801, United States
S Supporting Information
b
Scheme 1. A Foldamer, with Polymer Chains Attached at
Each End, Equilibrating between Unstructured and Helical
Conformations
ABSTRACT: We used fluorescence and electronic absorp-
tion spectroscopy to study the molecular weight depen-
dence of macromolecule-induced folding in a chain-centered
meta-phenylene ethynylene (mPE) oligomer. Analogous
to the ability of intrinsically unstructured proteins (IUPs)
to induce folding of globular proteins in cellular environ-
ments, we show that macromolecules attached to both ends
of an mPE dodecamer induce the foldamer to collapse into
a presumed helical conformation. The collapse is especially
prominent once the macromolecule segments become larger
than ca. 50 kDa. For sufficiently large macromolecules,
the conformational structuring occurs even in solvents
that normally denature the foldamer. Based on these
findings, chain-centered foldamers might find use as mod-
els to investigate the fundamental macromolecular physics
of IUPs.
weight entropic chains significantly alter a foldamer’s local
environment, facilitating the acquisition of solvophobically
derived conformational order.
To begin, we systematically monitored the behavior of
foldamer-linked polymers of varying molecular weight in differ-
ent solvents. The particular foldamer used was an mPE dodeca-
mer, selected because it contains the minimal oligomer length to
form a stable helix in solution.7 Dodecamer 3 was synthesized by
Pd-catalyzed Sonogashira coupling of two pentamer units (2)
with one dimer (1) (Scheme 2). Initiator 4 was synthesized from
3 by deprotection of the TBS groups under buffered conditions
followed by esterification with α-bromo isobutyryl bromide.
Single-Electron-Transfer-Living-Radical-Polymerization8 (SET-
LRP) of methylacrylate (MA) produces foldamer-linked poly-
mers with varying molecular weights and low polydispersities
(PDIs). Polymerizations were performed at room temperature
in DMSO with a Cu(0) catalyst, hexamethylated tris(2-amino-
ethyl) (Me6TREN) ligand, and excess MA over various reaction
times. A series of foldamer-linked poly(methylacrylate) (PMA)
polymers (PMA-mPE12-PMA) were synthesized with Gel Per-
meation Chromatography (GPC) determined molecular weights
ranging from 40 to 600 kDa. Table 1 summarizes the different
reaction times and corresponding GPC data. Photodiode array
detection monitoring the wavelength range 250À350 nm of the
GPC eluent confirmed that a foldamer was covalently attached to
the PMA polymer [see Supporting Information (SI)].
ntrinsically disordered or unstructured proteins (IDPs or
IUPs) lack well-defined secondary and/or tertiary structures
I
under physiological conditions.1 Despite their disorder, IUPs
possess diverse functional capabilities.2 While the coupling of
folding and association is a common mechanism used by IUPs for
recognition, regulation, signaling and assembly events,2 IUPs can
also serve as entropic chains carrying out functions that depend
directly on the disordered state.3 Assisted folding of globular pro-
teins is one function common to IUPs whose action depends on
entropic chains (e.g., some examples of intramolecular chaper-
one-mediated protein folding). The chaperoning mechanism is
suggested to either involve (i) local loosening of kinetically trapped
folded intermediates or (ii) solubilization and steric protection of
the partially folded protein.4 It remains unclear if other aspects of
polymer physics, such as modulation of the local solvent envi-
ronment, contribute to the mechanism of IUP chaperones.5
Foldamers are synthetic oligomers able to adopt ordered
conformations in solution.6 They are simple systems that can
serve as models to tease out principles and thus better under-
stand behavior found in their inherently more complex biomac-
romolecule counterparts. We wondered if the simple covalent
attachment of entropic chains to the ends of a meta phenylene
ethynylene (mPE) oligomer would enhance or inhibit structur-
ing of the foldamer (Scheme 1). Here we show the molecular
weight dependence of the entropic chain on its ability to impart
structure to an mPE foldamer. Surprisingly, when the entropic
chain is larger than ca. 50 kDa, structuring of the foldamer is
enhanced, even in a solvent for which the foldamer is otherwise
denatured. This observation suggests that high molecular
The conformational transition of mPE foldamers has been pre-
viously characterized by electronic absorption,9 fluorescence,10
and circular dichroism11 spectroscopy. In a ‘good’ solvent12 such
as chloroform, both the phenylacetylene backbone and triglyme
ester side chains are well-solvated so that the mPE foldamer
forms an unstructured, random conformation. In a more polar,
Received: September 15, 2011
Published: November 15, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ja2087163 J. Am. Chem. Soc. 2011, 133, 19650–19652
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Scheme 2. Synthesis of mPE Foldamer Functionalized with an SET-LRP Initiator at Each Enda
a Reagents and conditions: (a) Pd(PPh3)4, CuI, Et3N, THF, 36%; (b) (i) TBAF, THF, AcOH; (ii) α-bromo isobutyryl bromide, Et3N, THF (15% over
two steps).
Table 1. Molecular Weight Data for Polymers Obtained at
Different SET-LRP Reaction Times
Reaction
time (min)a
Mn
(kDa)b
PDI
20
30
39
69
1.3
1.2
1.2
1.1
1.1
1.2
1.3
1.3
1.4
35
86
45
104
134
178
233
365
591
55
70
90
120
160
Figure 1. Fluorescence spectra of foldamer-linked polymers in chloroform.
[Solution concentration is ca. 1.0 μM (concentration defined per mole of
foldamer). The spectra were normalized to highest emission intensity.]
a Reaction conditions: 4 equiv of Cu(0), 4 equiv of Me6TREN, 0.2À
0.5 mL of DMSO, excess methyl acrylate. b Number average molecular
weight of the PMA-mPE-PMA polymer in THF.
monitoring emission signals at 350 and 400 nm, fluorescence
spectroscopy is used to probe the conformational state of the
foldamer covalently bound to polymer chains (Figure 1). A plot of
fraction folded vs molecular weight shows asymptotic behavior that
begins to level above ca. 130 kDa (Figure 2). This behavior remains
independent of concentration in the dilute solution (i.e., μM)
regime. Furthermore, the electronic absorption spectra show the
typical signature of folded mPE oligomers. Specifically, we used the
ratio of UV absorbance at 306 nm to that at 289 nm to monitor
conformational ordering as a function of molecular weight of the
polymers. These data are consistent with the fluorescence observa-
tions (Figures S20 and S22).
The need for covalent attachment was demonstrated by
physically mixing a 104 kDa PMA homopolymer with foldamer
4 in chloroform. If noncovalent association between PMA and
the foldamer is the cause of folding, we might expect to see a rise
in the long wavelength fluorescence at sufficiently high PMA
concentration. However, no such effect was observed (i.e., no
emission was observed at 400 nm even when the concentration of
PMA was as high as 100 mg/mL). We surmise that a chain-
centered foldamer unit becomes structured in chloroform be-
cause the good solvent (chloroform) is displaced by the mono-
mer segments of the entropic chain.
‘poor’ (i.e., solvophobic) solvent such as acetonitrile, the foldamer
collapses. By adopting a helical conformation, the backbone
reduces its solvent-accessible surface area and maximizes con-
tacts between foldamer segments. Based on electronic absorp-
tion and fluorescence data collected during solvent titration
experiments, the conformational behavior follows a cooperative,
sigmoidal two-state transition.
In chloroform (a good solvent), oligomer 4 adopts an un-
folded form as supported by electronic absorption and fluorescence
data. The folding behavior of oligomer 4 was investigated by using
typical solvent-dependent denaturation experiments by following the
conformational equilibrium with electronic absorption and fluores-
cence spectroscopy. The sigmoidal shape of the resultant titration
curve is indicative of the cooperative folding process (Figure S19).
Electronic absorption and fluorescence data in acetonitrile (a poor
solvent) show that the oligomer 4 and all of the polymers behave
identically whereby both oligomer 4 and the chain-centered folda-
mers adopt a structured form. Thus, attachment of macromolecules
to the ends of the foldamer causes no apparent denaturation of the
foldamers (Figure S20 and S23). However, the foldamer-function-
alized polymers show a conformational transition that is dependent
on the molecular weight of the attached macromolecule. By
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dx.doi.org/10.1021/ja2087163 |J. Am. Chem. Soc. 2011, 133, 19650–19652

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Preston May, and Windy Turchyn for helpful discussions relating
to this project.
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Figure 2. Plots of fraction folded verses polymer molecular weight
obtained from normalized fluorescence intensity data (spectra normal-
ized to a constant optical density OD = 0.15). The fraction folded was
determined using the equation: fu = If À I/If À Iu (fu is the mole fraction
of foldamer in the unfolded state, I is the fluorescent intensity at 350 nm
from a foldamer-centered polymer of intermediate molecular weight,
and Iu and If are the intensity values characteristic of the fully unfolded in
chloroform and folded states measured on oligomer 4).
At the start of this work, it was unclear if entropic chains would
enhance or disfavor folding of a chain-centered foldamer. Rea-
sons that might disfavor the folded state are (i) steric clashing of
the two entropic chains (i.e., self-avoidance) and (ii) stretching of
the entropic chains by a good solvent; both reasons would exert
an elongational force on the foldamer’s helical structure and
could drive it to uncoil. In contrast, entropic chains might
promote the folded state by altering the solvent environment
in the vicinity of the foldamer. Whether such a perturbation
could significantly shift the equilibrium position of the folding
transition was not predictable at the outset of this investigation. We
have shown that when the entropic chain segment is larger than ca.
50 kDa, structuring of the mPE oligomer is enhanced, even in a
solvent for which the foldamer is otherwise denatured. This observa-
tion supports the notion that high molecular weight entropic chains
facilitate conformational ordering by altering a foldamer’s local
environment. On the basis of these findings, we suggest that
solvophobic forces generated by the covalent attachment of an
entropic chain may play an important role in intramolecular
chaperone-mediated protein folding.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details, synthetic
b
procedures, NMR, GPC, electronic absorption, fluorescence
data. This material is available free of charge via the Internet at
http://pubs.acs.org
’ AUTHOR INFORMATION
Corresponding Author
jsmoore@illinois.edu
’ ACKNOWLEDGMENT
This work was supported by the Army Research Office MURI
(Grant W911NF-0701-0409). The authors thank Matt Kryger,
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dx.doi.org/10.1021/ja2087163 |J. Am. Chem. Soc. 2011, 133, 19650–19652