Synthesis and characterization of amphiphilic o-phenylene ethynylene oligomers

Article abstract of DOI:10.1039/b707618e

We have previously reported the synthesis of short o-phenylene ethynylene oligomers with polar triethylene glycol side chains which adopt a helical conformation in solution with three residues per turn. Two new oligomers have been synthesized, a hexamer a

Full text of DOI:10.1039/b707618e

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PAPER
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Synthesis and characterization of amphiphilic o-phenylene ethynylene
oligomersw
Morris M. Slutsky, Jason S. Phillip and Gregory N. Tew*
Received (in Gainesville, FL, USA) 21st May 2007, Accepted 19th December 2007
First published as an Advance Article on the web 25th January 2008
DOI: 10.1039/b707618e
We have previously reported the synthesis of short o-phenylene ethynylene oligomers with
polar triethylene glycol side chains which adopt a helical conformation in solution with three
residues per turn. Two new oligomers have been synthesized, a hexamer and a nonamer,
incorporating a repeated triad motif of polar–nonpolar–polar sidechains in order to create a
hydrophobic stripe in the folded conformation which we report here for the first time. Helical
folding in solution was observed and, unlike the previously-reported oligomers, these new
oligomers are ordered solids at room temperature. Although these oligomers were designed to
assemble into helical bundle-like structures, no evidence for a quaternary-like structure was
found. The difference in polarity between alkyl and triethylene glycol side chains is likely not
strong enough to induce self-association of folded helices, especially since the molecules are not
water soluble where the driving force for association of the nonpolar stripe would be larger. We
expect that more polar side chains, granting water solubility, represent an important target for
future research.
adopt helical conformations in solution33–35 and other pheny-
Introduction
lene ethynylenes which demonstrated antimicrobial activ-
ity36–39 similar to host defense peptides like the magainins
Foldamers have been of great interest as model systems for
investigation and computational simulation of biological
macromolecules in a simple and controlled context.1–13
and defensins. Here we describe new oPE oligomers which
A
have both polar and nonpolar side chains to create an
amphiphilic structure in the helically folded conformation. It
represents our first step towards folded assemblies beyond the
secondary unit. We have successfully prepared two oligomers
containing six and nine oPE units with a repeating pattern of
polar, nonpolar, polar side chains to match the three residues
per turn of the helix. This places the polar and nonpolar
groups on distinct faces of the folded structure. As per our
earlier reports, the polar group is the ester of triethylene glycol
monomethyl ether while the nonpolar group is the ester of
(S)-2-methyl butanol.
great variety of foldamer systems that show secondary struc-
ture analogous to that of natural macromolecules have been
investigated over the past few years. Foldamers demonstrating
biomimetic tertiary and quaternary structures have been de-
signed as well,14–16 although there are significantly fewer
examples. Extensive investigations of phenylene ethynylene
foldamers have been made by various workers,17–26 due to the
simplicity and flexibility of the backbone structure.
Designed peptides have been extensively explored in order
to study determinants of secondary, tertiary, and quaternary
structure formation. Many peptides that fold into helical
bundles in solution have been described in the literature.27–32
The major driving force for this assembly is the burial of the
hydrophobic stripe created upon helix formation. Conse-
quently, natural as well as designed helical bundles are com-
posed of amphiphilic helices which display a hydrophobic face
in their folded conformation, inducing self-association in
order to bury the hydrophobic areas.
These new oligomers were shown to adopt helical confor-
mations in solution, representing an important milestone.
Since solvophobic-like driving forces were believed to promote
helix formation in our previous oligomers containing all Teg
side chains, it was unclear how the replacement of one Teg
chain per turn with a non-polar group would effect helix
formation and stability. This solvophobic effect, proposed by
Moore and Ray,25 takes advantage of solubility differences
between Teg side chains and the PE backbone. Upon changing
the solvent from CHCl3 to more polar solvents, like acetoni-
trile, the backbone folds to reduce its surface area in contact
with the polar solvent. At the same time, the polar Teg side
chains promote solubility of the oligomer. Therefore, the fact
that these oligomers still adopt a helical conformation in
acetonitrile is an important result. However, our goal remains
higher order assembly and no evidence for such assemblies was
observed for these two oligomers. This is attributed to their
lack of water solubility where the driving force for burial of the
hydrophobic stripe would be larger.
Amphiphilic foldamers are interesting both as a means of
investigating folded structure and as potential scaffolds to
modulate biological activity. We have previously developed
short ortho-phenylene ethynylene (oPE) oligomers which
Department of Polymer Science & Engineering, University of
Massachusetts, 120 Governors Drive, Amherst MA 01003, USA.
E-mail: tew@mail.pse.umass.edu
w Electronic supplementary information (ESI) available: Experimental
section; synthetic procedures for oligomers 1 and 2; COSY spectra
used for assignment of aromatic ring protons in 1 and 2. See DOI:
10.1039/b707618e
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Results and discussion
The amphiphilic oligomers 1 and 2 composed of 6 or 9 oPE
units, respectively, as shown in Fig. 1, were synthesized in order
to study their ability to fold and assemble. They are similar to
our previously reported triethylene glycol monomethyl ether
(Teg) substituted oPE oligomers, which adopt helical structures
in solution; however, a new repeating 3-residue motif of
polar–nonpolar–polar side chains creates a hydrophobic face
in the helical conformation, as shown in Fig. 2.
Fig. 2 Nonamer (2) in two potential conformations, fully extended
Oligomers 1 and 2 were synthesized using a partially con-
vergent strategy of deprotection and Sonogashira couplings.
Acetylene groups were trimethylsilyl (TMS) protected, while
triazene groups were used to provide masked aryl iodide
functionality, as in previous PE synthetic work. Protected
(S)-2-methylbutyl ester monomers 9 and 10 were prepared as
shown in Scheme 1 from previously reported intermediate 6,34
using chemistry similar to that used to prepare previously
reported Teg-ester monomers 3 and 4. Aryl iodide-terminated
oPE dimers 12 and 14 were synthesized as shown in Scheme 2,
similarly to the preparation of previously reported Teg-ester
dimer iodide 5.35 In our hands, the methyl iodide reaction to
convert the triazene to an aryl iodide provides the lowest yield.
In fact, as the oligomer length increased, this reaction became
more problematic and influenced the synthetic route. With
dimers 12 and 14 in hand, along with 5, construction of the
hexamer and nonamer was performed. Schemes 3 and 4 out-
line the synthesis of hexamer 1 and nonamer 2, respectively.
The sequential coupling steps used dimeric units instead of
trimeric units due to the very low yield obtained during the
unmasking of triazene-terminated trimers. Scheme 3 combined
(A) and fully helical (B).
4 and 12 to generate the amphiphilic trimer, 15, which was
converted to the free acetylene 16 and reacted with 14 to
generate pentamer 17. Another cycle of acetylene deprotection
to form 18 followed by Sonogashira coupling, this time with 3,
yielded the hexamer 1 in 21% overall yield for the five steps
shown in Scheme 3. Sonogashira coupling of intermediate 18
with 5 followed by another acetylene deprotection step and a
final Sonogashira coupling with 12 gave nonamer 2 in only 7%
yield over 7 steps, as shown in Scheme 4.
Folding of these two new oligomers, 1 and 2, was studied
using NMR spectroscopy. Similar to peptides, a main driving
force for folding is burial of the hydrophobic backbone. In our
case, the aromatic backbone of these foldamers is strongly
hydrophobic and only weakly solvated by acetonitrile, which
tends to promote a helically folded conformation as the oPE
attempts to minimize the surface area in contact with the
solvent. In contrast, a solvent such as chloroform, which
strongly solvates aromatic backbones, tends to promote a
more unfolded, or randomly coiled, conformation. Due to
the precise chemical synthesis, NMR spectroscopy provides an
excellent handle for studying conformational changes in these
oPE foldamers. The three protons on each ring are expected to
shift upfield in the event of ring stacking, due to ring current
effects. Our previous studies on shorter oPE foldamers con-
firmed that this upfield-shift was due to folding by using
simultaneous 2D NOE measurements.34 Therefore, since these
oligomers would represent 2 and 3 full turns upon folding into
a fully helical conformation, the entire aromatic region of each
spectra was expected to shift upfield.
Fig. 1 Triethylene glycol monomethyl ether and alkyl ester-substi-
tuted oPE oligomers, hexamer (1) and nonamer (2). Each ring has
three protons that are labeled by their NMR splitting pattern: a
(8.4 Hz, d); b (2.1 Hz, d); c (8.4 Hz and 2.1 Hz, dd).
Scheme 1 Synthesis of (S)-2-methylbutyl ester nonpolar monomers
9 and 10.
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Scheme 2 Synthesis of aryl iodide-terminated dimers 12 and 14. Previously reported dimer 5 shown as well.
One-dimensional 1H NMR spectra were collected from 1.25
mM solutions of 1 and 2 in CDCl3 and CD3CN, as shown in
Fig. 3, and a clear upfield shifting of the aromatic ring protons
is observed upon changing the solvent from CDCl3 to
CD3CN. The shift of all rings is consistent with a helical
conformation in CD3CN. Using two-dimensional correlation
Scheme 3 Synthesis of hexamer. Sequential Sonogashira coupling of 12 and 14 followed by TMS deprotection produces intermediate 18, which is
used in a final coupling reaction to yield 1. This intermediate, 18, is also used to synthesize 2.
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Scheme 4 Synthesis of nonamer 2 from 18 by sequential Sonagashira coupling of 5 and 12.
spectroscopy (COSY) methods, it is relatively straightforward
to connect each spin system, A, B and C, on the aromatic
rings. In contrast, it is more difficult to determine the exact
primary sequence by NMR; although we previously showed
that heteronuclear multiple bond correlation (HMBC) could
be used for absolute primary sequence assignment of oPE
oligomers.35 For the two oligomers reported here, we have not
performed HMBC experiments, but COSY experiments were
performed and Fig. 4 shows the average chemical shift of each
spin system for 1 and 2 in CDCl3 and CD3CN. Globally, the
upfield shift in CD3CN is clearly observed; however since the
exact primary sequence has not been determined it is difficult
to track each ring from CDCl3 to CD3CN. In other words,
COSY easily allows us to determine the a, b, and c protons of
each ring (spin system) and thus the average chemical shift of
each ring, but quantitative information on the primary se-
quence prevents definite assignment of the rings in the primary
sequence. Therefore, we cannot say that every ring proton
shifts upfield but obviously the overall trend is for substantial
upfield shifts and it therefore seems reasonable to assume all
rings are involved in the folded conformation. This is further
supported by the fact that no ring appears to shift downfield
which is the intrinsic nature of going from CDCl3 to CD3CN
for the PE unit, as shown previously with model dimer and
trimer oligomers which cannot fold.34
This helical folding of 1 and 2 enables the hydrophobic alkyl
side chains to occupy one face of the folded molecule and gives
the molecule an overall amphiphilic character which is com-
monly used in peptide design to drive self-association into a
Fig. 4 Average chemical shifts for the a, b, and c protons of each
aromatic ring in oligomers 1 and 2. The average chemical shifts of the
6 rings of 1 in CDCl3 (A) and in CD3CN (B), and the average chemical
shifts of the 9 rings of 2 in CDCl3 (C) and CD3CN (D). Due to
overlap, not all average chemical shifts are distinctly visible in all cases.
Fig. 3 1H NMR spectra showing all aromatic protons for hexamer 1
in CDCl3 (A) and CD3CN (B), and of nonamer 2 in CDCl3 (C) and
CD3CN (D).
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helical bundle or other higher order structures. The
chiral alkyl side chains were incorporated in order to
induce a chiral bias upon helical folding which might be
detectable by circular dichroism (CD) spectroscopy with the
oPE backbone acting as a chromophore.20 Unfortunately,
significant CD signals were not observed under solvent and
temperature conditions similar to those used for the NMR
experiments. Even with helical oPEs containing chiral side
chains at every position, we have never observed a significant
CD signal. Another way in which higher order association
might reasonably be detected would be by examination of the
1H NMR signals of the alkyl chains. If they were tightly
packed together, such as in the middle of a hydrophobically-
collapsed structure, their signals would be expected to be
weakened by an increase in relaxation time. However,
although shorter signal acquisition times were tried, no weak-
ening of these signals was observed. Therefore, by CD and
NMR spectroscopy, there is no evidence for assembly of these
helical foldamers into higher order structures.
Conclusions
The synthesis of two new amphiphilic oPE oligomers was
reported for the first time. Despite the amphiphilic character,
the oligomers still undergo folding into a helical conformation
when the solvent is changed from CDCl3 to CD3CN.
Although helical folding of these amphiphilic oligomers is an
important milestone, they did not show any evidence by CD,
NMR, or DLS that they further assembled into higher ordered
structures like a helical bundle. This is most likely due to the
fact that they are not water-soluble which limits the hydro-
phobic driving force. We are building amphiphilic oligomers
with more polar side chains than Teg and expect that if they
are water soluble, helical bundle-like assembly will occur. It
was observed that these oligomers formed ordered solids
unlike the homo-Teg derivatives, suggesting there is a funda-
mental difference between these new amphiphilic oligomers
and their earlier analogs. It is too early to know if the
molecules in the solid are helical in nature and if they are
ordered into bundles. X-Ray studies should help address these
questions, but they are beyond the scope of this report.
Dynamic light scattering (DLS) was used in an attempt
to detect aggregation due to amphiphilicity upon change
of solvent. Changing solvent from CHCl3 to the more
polar CH3CN would be expected to induce aggregation in
order to bury the hydrophobic side chains. As the NMR
measurements showed evidence of helical folding in CH3CN,
it would be reasonable to expect aggregation due to self-
association of the resulting amphiphilic helix, which would
appear as increased particle size. Regrettably, no evidence for
increased particle size with increasing solvent polarity was
found by DLS.
Acknowledgements
We thank the NSF for financial support (NSF CAREER
CHE-0449663). G. N. T. thanks the ARO and ONR Young
Investigator programs in addition to the PECASE program,
3M Nontenured faculty grant, and Dupont Young Faculty
Award for generous support.
Although 1 and 2 displayed similar solution behavior to our
previously reported homo-Teg oPE oligomers, their solid-
phase properties were strikingly different, possibly due to their
amphiphilic nature. The previously reported homo-Teg oligo-
mers (up to hexamer) were all viscous liquids at room tem-
perature. However, 1 and 2 are solids at room temperature
and, when solvent cast onto glass slides, showed birefringence
by polarized optical microscopy (POM) with a microcrystal-
line appearance, as shown in Fig. 5. In contrast, the homo-Teg
oligomers showed no birefringence when examined by POM.
Utilizing a heating stage, the birefringence of 1 and 2 dis-
appeared between 55–60 1C and 120–130 1C, respectively.
After cooling a melted slide of 1 under vacuum, larger
birefringence patterns with long-range order appeared,
although this was not observed with 2 which regained the
microcrystalline appearance after this treatment.
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