H-Bonded Duplexes based on a Phenylacetylene Backbone

Article abstract of DOI:10.1021/jacs.8b08087

Complementary phenylacetylene oligomers equipped with phenol and phosphine oxide recognition sites form stable multiply H-bonded duplexes in toluene solution. Oligomers were prepared by Sonogashira coupling of diiodobenzene and bis-acetylene building blocks in the presence of monoacetylene chain terminators. The product mixtures were separated by reverse phase preparative high-pressure liquid chromatography to give a series of pure oligomers up to seven recognition units in length. Duplex formation between length complementary homo-oligomers was demonstrated by ³¹P NMR denaturation experiments using dimethyl sulfoxide as a competing H-bond acceptor. The denaturation experiments were used to determine the association constants for duplex formation, which increase by nearly 2 orders of magnitude for every phenol-phosphine oxide base-pair added. These experiments show that the phenylacetylene backbone supports formation of extended duplexes with multiple cooperative intermolecular H-bonding interactions, and together with previous studies on the mixed sequence phenylacetylene 2-mer, suggest that this supramolecular architecture is a promising candidate for the development of synthetic information molecules that parallel the properties of nucleic acids.

Full text of DOI:10.1021/jacs.8b08087

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H‑Bonded Duplexes based on a Phenylacetylene Backbone
Jonathan A. Swain, Giulia Iadevaia, and Christopher A. Hunter*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
S
* Supporting Information
ABSTRACT: Complementary phenylacetylene oligomers equipped
with phenol and phosphine oxide recognition sites form stable multiply
H-bonded duplexes in toluene solution. Oligomers were prepared by
Sonogashira coupling of diiodobenzene and bis-acetylene building
blocks in the presence of monoacetylene chain terminators. The
product mixtures were separated by reverse phase preparative high-
pressure liquid chromatography to give a series of pure oligomers up to
seven recognition units in length. Duplex formation between length
complementary homo-oligomers was demonstrated by 31P NMR
denaturation experiments using dimethyl sulfoxide as a competing H-
bond acceptor. The denaturation experiments were used to determine
the association constants for duplex formation, which increase by nearly
2 orders of magnitude for every phenol-phosphine oxide base-pair added. These experiments show that the phenylacetylene
backbone supports formation of extended duplexes with multiple cooperative intermolecular H-bonding interactions, and
together with previous studies on the mixed sequence phenylacetylene 2-mer, suggest that this supramolecular architecture is a
promising candidate for the development of synthetic information molecules that parallel the properties of nucleic acids.
a backbone with sufficient flexibility to adopt to a compatible
conformation.12b,g
INTRODUCTION
■
Nucleic acids encode information in the sequence of monomer
units, and this structure provided the molecular basis for the
evolution of biological life. The information is read through
sequence-selective duplex formation and copied through
template synthesis.1 The sequence of monomer units also
defines the three-dimensional structures and functional
properties of single-stranded nucleic acids.2 Modified ana-
logues of nucleic acids have been reported,3 where the sugar,4
the phosphate linker,5 or the base pairing system6 have been
replaced and the ability of forming duplexes was not affected,
suggesting that fully synthetic information molecules may be
able to have some or all the functions of nucleic acids. There
are some examples of synthetic oligomers that form duplexes
using metal coordination, salt bridges, or H-bonding as the
base-pairing interactions.7−11 Figure 1a shows a blueprint for
duplex forming oligomers that is currently being investigated in
our laboratory. The modular nature of this design allows the
three different components to be explored independently,
providing insights into the key requirements for architectures
that will lead to sequence-selective duplex formation.
We have shown that oligomers equipped with single H-bond
recognition modules form stable duplexes in nonpolar
solvents.12a−g It is possible to interchange different H-bond
donor−acceptor recognition motifs (D·A) on the same
backbone and maintain the duplex forming properties of the
oligomers.12c Oligomers have been prepared using reductive
amination chemistry and using thiol−ene coupling, and both
types of backbone lead to the formation of stable duplexes.
Experiments on three isomeric reductive amination backbones
indicate that an important requirement for duplex formation is
However, when mixed donor−acceptor sequences were
prepared, it became clear that the conformational properties of
the backbone also play a key role in discriminating between
intramolecular H-bonding interactions that lead to folding and
intermolecular interactions that lead to duplex formation.
Highly flexible backbones lead to folding that precludes
effective sequence-selective duplex formation in mixed
sequence oligomers.12e These studies have identified two
backbones that have promising conformational properties, and
we recently showed that one of these systems supports
sequence-selective duplex formation for mixed sequence 3-
mers.12f Figure 1b shows the other backbone that is sufficiently
rigid to preclude intramolecular folding in the mixed sequence
AD 2-mer.12e This phenylacetylene backbone is particularly
attractive for a number of reasons. Phenylacetylene oligomers
have been extensively studied by Moore,13 who developed
solid phase synthesis methods that could be used to make
oligomers with controlled length and sequence.14 Oligomers
longer than about eight repeat units fold into helical structures
in acetonitrile solution, due to stacking interactions,15 so the
single-stranded forms may have interesting properties.
Formation of phenylacetylene duplexes based on dynamic
covalent base-pairs was investigated previously, but slow
kinetics limited the fidelity.16 Here we describe the synthesis
of families of recognition-encoded oligomers based on the
phenylacetylene backbone and show that this molecular
Received: July 30, 2018
© XXXX American Chemical Society
A
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reaction mixture, and the resulting product mixture can be
separated by preparative HPLC. Sonogashira coupling of bis-
acetylene recognition modules with diiodo-linkers in the
presence of the corresponding monoacetylene recognition
modules was therefore investigated as a simple route to homo-
oligomers.
Synthesis of Building Blocks. The monoacetylene donor
recognition module (1) was commercially available, and the
monoacetylene acceptor recognition module (2) was synthe-
sized as previously described (Scheme 1).12e Recognition
Scheme 1. Mono-Acetylene Recognition Modules, and
Synthesis of Bis-Acetylene Recognition Modules
Figure 1. (a) A blueprint for duplex forming molecules. There are
three key design elements: the coupling chemistry used for the
synthesis of oligomers (red), the recognition module, which controls
intermolecular binding (blue), and the backbone module, which links
these components together (black). (b) Proposed duplex formed by
phenylacetylene oligomers equipped with phenol and phosphine
oxide recognition modules (R is a solubilizing group).
architecture leads to the formation of very stable H-bonded
duplexes in toluene.
RESULTS AND DISCUSSION
■
Stepwise synthesis of oligomeric molecules requires multiple
deprotection-coupling cycles and is labor intensive. Here we
investigate a one-step process, statistically controlled oligome-
rization, to rapidly access families of oligomers of varying
length (Figure 2). The average oligomer length can be
controlled by adding monofunctional chain stoppers to the
modules bearing two acetylene moieties were synthesized
using the route shown in Scheme 1. Treatment of
n
tribromobenzene (3) with BuLi followed by reaction with
iodine gave dibromoiodobenzene (4). The difference in
reactivity of halides allowed for selective P-arylation with di-
n-butylphosphine oxide to give 5. Sonogashira coupling with
TMSA yielded 6, and TBAF mediated deprotection yielded the
bis-acetylene acceptor recognition module (7). Acetylation of
3,5-dibromophenol (8) with acetic anhydride yielded the
acetate derivative (9). Acetylation was required to increase the
reactivity in the subsequent Sonogashira coupling with TMSA
to obtain 10. Simultaneous base-mediated deprotection of the
phenol and acetylenes yielded the bis-acetylene donor
recognition module (11). The diiodo-linker (13) was
synthesized by an EDC-mediated esterification of 3,5-
Figure 2. Oligomer synthesis using statistical coupling of mono- and
bifunctional monomers to produce mixtures that can be separated by
chromatography. H-bond donor (D) or acceptor (A) recognition
groups can be used, and R is a solubilizing group.
i
diiodobenzoic acid (12) with BuOH (Scheme 2).
B
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from the 3-mer to 6-mer were isolated by this method (Table
Scheme 2. Synthesis of Diiodo-Linker
1).18 The overall yield with respect to the diiodo-linker 13 was
Table 1. Donor homo-oligomers isolated from the
oligomerization reaction in Scheme 3
product
sequence
% mol fraction
% yield
Oligomer Synthesis. Donor homo-oligomers were
synthesized by oligomerization of the phenol recognition
modules (1 and 11) with linker 13 under Sonogashira coupling
conditions in toluene (Scheme 3).12e,17 The ratio of starting
14 (3-mer)
15 (4-mer)
16 (5-mer)
17 (6-mer)
DDD
DDDD
DDDDD
DDDDDD
40
27
21
12
6
4
3
2
Scheme 3. Oligomerization of Phenol Recognition Modules
to Yield Donor Homo-Oligomers (n = 1−4 Corresponding
to the 3-mer to 6-mer Donor Homo-Oligomers)
15%, due to the low solubility of the oligomeric products. The
most abundant oligomer isolated was the DDD 3-mer, as
expected from the stoichiometry of the two recognition
modules used in the reaction.
Acceptor homo-oligomers were synthesized by oligomeriza-
tion of the phosphine oxide recognition modules (2 and 7)
with linker 13 under Sonogashira conditions in toluene
(Scheme 4). The ratio of starting materials was chosen to
Scheme 4. Oligomerization of Phosphine Oxide
Recognition Modules to Yield Acceptor Homo-Oligomers
(n = 1−5 Corresponding to the 3-mer to 7-mer Acceptor
Homo-Oligomers)
materials was chosen to target the DDD 3-mer as the major
product (1:11:13 = 2:1:2). Although the crude reaction
mixture contained a large amount of insoluble material, it
could be dissolved in DMSO. LCMS analysis of the DMSO
solution showed the presence of oligomers up to the 6-mer
(Figure 3a). Preparative HPLC was used to separate the
products (Figure 2b), and samples of donor homo-oligomers
target the AAA 3-mer as the major product (2:7:13 = 2:1:2).
The reaction mixture was dissolved in ethanol and analyzed by
LCMS (Figure 3a). Oligomers up to the 7-mer were observed.
Preparative HPLC was used to separate the products (Figure
4b), and samples of the acceptor homo-oligomers from the 3-
mer to 7-mer were isolated (Table 2). The overall yield with
respect to the diiodo-linker 13 was 33%. The most abundant
oligomer in the mixture was the AAA 3-mer, as expected from
the stoichiometry of the two recognition modules used in the
reaction.
1
Oligomer Characterization. Mass spectrometry and H
Figure 3. Product distribution from the donor oligomerization
reaction shown in Scheme 3. (a) LCMS analysis of the crude reaction
mixture using a Hichrom C8C18 column with a water/THF (0.1%
formic acid) solvent system. (b) Preparative HPLC separation of
donor homo-oligomers using a HIRBP-6988 prep column with a
water/THF solvent system. Samples were prepared in DMSO, UV/vis
absorption was measured at 290 nm, and the peaks identified by MS
are labeled with retention time in minutes. dba = dibenzylideneace-
tone.
NMR spectroscopy were used to confirm the structures of the
oligomers. The terminal recognition modules, the internal
recognition modules, and the linkers all give rise to distinct
1
signals in the H NMR spectra, and the ratios of the integrals
of these signals were used to confirm oligomer length. For the
1
donor oligomers, the ratio of the integrals of the H NMR
signals due to the terminal linkers (blue and orange) compared
with the signals due to the internal linkers (red and green)
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due to the internal linkers (red) allowed quantification of the
number of linkers and therefore oligomer length. These results
were confirmed using the ratio of integrals of the 31P NMR
signals due to the terminal recognition modules (blue)
compared with the signals due to the internal recognition
modules (red) (Figure 6).
For both the donor homo-oligomers in THF and the
acceptor homo-oligomers in chloroform, the chemical shifts of
the signals due to equivalent protons from equivalent building
blocks do not change as the length of the oligomers increases.
Moore and co-workers have studied the properties of a related
family of phenylacetylene oligomers in acetonitrile, and they
observed substantial upfield changes in chemical shift (0.5
ppm) for the signals due to the aromatic protons as the length
of the oligomers increased.14 These chemical shift changes are
indicative of folding into a helical conformation with stacking
Figure 4. Product distribution from the acceptor oligomerization
reaction shown in Scheme 4. (a) LCMS analysis of the crude reaction
mixture using a Hichrom C8C18 column with a water/THF (0.1%
formic acid) solvent system. (b) Preparative HPLC separation of
acceptor homo-oligomers using a HIRBP-6988 prep column with a
water/THF solvent system. Samples were prepared in EtOH, UV/vis
absorption was measured at 290 nm, and the peaks identified by MS
are labeled with retention time in minutes. dba = dibenzylideneace-
tone.
1
of the aromatic rings. The H NMR data in Figures 5 and 6
show that the oligomers described in this work do not fold in
chloroform or THF, but the solubility is not sufficient to
record NMR spectra in acetonitrile.
Binding Studies. Previous studies of duplex assembly used
toluene as the solvent, because toluene is a very weak H-bond
donor and acceptor, which maximizes the strength of the
phenol-phosphine oxide interactions. The intrinsic strength of
this base-pairing interaction is important for maximizing K EM,
the parameter that governs chelate cooperativity in zipping up
the duplex.12,19 The poor solubility of the donor homo-
oligomers in toluene described here made it impossible to
measure association constants for duplex formation by titration
experiments. However, in the presence of the complementary
acceptor homo-oligomer, both DDD and DDDD are soluble at
millimolar concentrations in toluene. The longer donor homo-
oligomers do not dissolve in toluene even in the presence of
the complementary acceptor homo-oligomer.
Table 2. Acceptor Homo-Oligomers Isolated from the
Oligomerization Reaction in Scheme 4
product
sequence
% mol fraction
% yield
18 (3-mer)
19 (4-mer)
20 (5-mer)
21 (6-mer)
22 (7-mer)
AAA
AAAA
AAAAA
AAAAAA
AAAAAAA
58
27
10
4
19
9
3
1
1
<1
allowed quantification of number of linkers and therefore
oligomer length (Figure 5). Similarly, for the acceptor
The 31P NMR spectrum of a 1:1 mixture of AAA and DDD
in toluene-d8 shows one broad signal at 40−41 ppm. In
1
oligomers, the ratio of the integrals of the H NMR signals
due to the terminal linkers (blue) compared with the signals
1
1
Figure 5. (a) Chemical structures of the donor homo-oligomers. (b) Partial 500 MHz H NMR spectra in THF-d6. The signals in the H NMR
spectra are assigned to the chemical structures using color coding. The 4-mer contains traces of an impurity that could not be removed by HPLC.
D
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1
Figure 6. (a) Chemical structures of the acceptor homo-oligomers. (b) Partial 500 MHz H NMR spectra in CDCl3. (c) 162 MHz 31P NMR
1
spectra in CDCl3. The signals in the H and 31P NMR spectra are assigned to the chemical structures using color coding.
contrast in the 31P NMR spectrum of AAA in toluene-d8, there
are two signals at 35.9 ppm. These differences in 31P chemical
shift are characteristic of phosphine oxide groups that are fully
H-bonded to phenol in the mixture of AAA and DDD. In the
1H NMR spectrum of this mixture, the two signals due to the
phenol OH groups appear at 11 ppm, which indicates that the
three phenol groups of DDD are fully H-bonded to phosphine
oxide groups on AAA. These data provide good evidence that
at a concentration of 1 mM in toluene-d8 the AAA·DDD
duplex is fully assembled with three intermolecular phenol-
phosphine oxide H-bonds. Similarly for a 1:1 mixture of AAAA
and DDDD in toluene-d8, the 31P NMR spectrum has two
1
signals at 40 ppm, and the H NMR spectrum has two signals
due to the phenol OH groups at 11 ppm. Thus, the AAAA·
DDDD duplex is also fully assembled with four intermolecular
phenol-phosphine oxide H-bonds at a concentration of 1 mM
in toluene-d8. In all cases, equilibration was rapid, and no slow
exchange processes were observed.
Figure 7. 1-mers and 2-mers used to validate the DMSO denaturation
experiment.
The stabilities of the AAA·DDD and AAAA·DDDD
duplexes were characterized by denaturation experiments
with DMSO. DMSO can H-bond to the phenol groups on
the donor homo-oligomers and thus dissociate the duplexes. If
the association constant for the phenol-DMSO interaction is
known (Kd), it is possible to use the denaturation isotherm to
deduce the association constant for duplex formation between
two complementary N-mers (KN). To test the validity of this
approach, the 1-mers and 2-mers, which were reported
previously,12e were used to directly compare the results from
a titration and a denaturation experiment (Figure 7).
titration experiments. The data fit well to 1:1 binding
isotherms (see the Supporting Information, SI), and the
results are summarized in Table 3. The large downfield
changes in 31P NMR chemical shift (+6−7 ppm) are indicative
of H-bond formation, and the bound chemical shifts are very
similar to those observed for the AAA·DDD and AAAA·
DDDD duplexes described above. The association constant for
the AA·DD complex (K2 = 44 000 M−1) is 2 orders of
magnitude higher than the association constant for the A·D
complex (K1 = 760 M−1) indicating that the duplex is fully
assembled with two cooperative intermolecular phenol-
phosphine oxide H-bonds.
Association constants for formation of the A·D and AA·DD
complexes in toluene-d8 were determined using 31P NMR
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Table 3. Association Constants for Duplex Formation (KN), Effective Molarities (EM), and 31P NMR Chemical Shifts (δ)
a
Measured by NMR Titration and DMSO Denaturation Experiments in Toluene-d8 at 298 K
method
titration
titration
denaturation
denaturation
denaturation
complex
A·D
AA·DD
AA·DD
AAA·DDD
AAAA·DDDD
KN (M−1
)
EM (mM)
δf (ppm)
δb (ppm)
760
44 000
83 000
35.3
35.3
34.8
34.8
35.3
42.1
41.3
42.1
40.5
40.1
38
72
52
59
2 300 000
130 000 000
a
Errors are estimated at 30% based on two repeats of the experiment.
1
The value of Kd was measured by H NMR titration of
DMSO into the D 1-mer in toluene (Kd = 150 M−1, see SI).
For denaturation of the AA·DD duplex, an equimolar 1 mM
solution of AA and DD was prepared in toluene-d8. At this
concentration, AA is 90% bound to DD, and the 31P signal was
accordingly observed close to the bound chemical shift at 41
ppm. Addition of DMSO-d6 lead to a decrease in the chemical
shift of the 31P NMR signal to 36 ppm, due to disruption of the
H-bonding interactions (Figure 8b). However, the final
chemical shift is 1 ppm higher than the free chemical shift
from the titration experiment (35 ppm), and Figure 8b shows
that the 31P chemical shift starts to increase at very high
concentrations of DMSO at the end of the denaturation
experiment. This observation is presumably due to a change in
the nature of the solvent for DMSO concentrations of the
order 1 M. DMSO-d6 was therefore titrated into a 1 mM
sample of the A 1-mer in toluene-d8 where no H-bonding
interactions are present (Figure 8a). An increase of about 1
ppm was observed in the 31P chemical shift at very high
DMSO concentrations, confirming that the unusual shape of
the AA·DD denaturation data is due to nonspecific effects of
the change in solvent. The 31P chemical shift data for the A 1-
mer were therefore used to correct the 31P chemical shifts from
the AA·DD denaturation experiment for the effects of DMSO
on the chemical shift of the unbound phosphine oxide signal.
The proportion of duplex present at any point in the
denaturation experiment is therefore given by eq 1.
%duplex = (δobs − δA − δf)/(δb − δf)
(1)
where δobs is the observed chemical shift for the 1:1 mixture at
a specific concentration of denaturant, δA is the observed
chemical shift of the A 1-mer at the same concentration of
denaturant, δf is the free chemical shift of the acceptor homo-
oligomer in pure toluene-d8, and δb is the bound chemical shift
of the duplex.
The data from the AA·DD denaturation experiment did not
fit to a simple two-state, all-or-nothing denaturation isotherm
(see SI), which suggests that partially denatured species must
also be considered. Figure 9 shows all possible complexes
present in the AA·DD denaturation experiment. Both the fully
bound duplex and unbound species are present at the start of
the experiment. Binding of one molecule of DMSO to the
duplex leads to the partially denatured AA·DD·DMSO
complex. It is possible to estimate the equilibrium constant
for the formation of this termolecular complex as the product
of Kd (the D·DMSO association constant) and K1 (the A·D
association constant), multiplied by a statistical factor of 4. The
unbound DD 2-mer can bind one or two molecules of DMSO,
and the equilibrium constants for formation of the DD·DMSO
and DD·(DMSO)2 complexes can be estimated using the D·
Figure 8. 202 MHz 31P NMR spectra for titration of DMSO-d6 into
a) A (1 mM), and DMSO-d6 denaturation of equimolar 1 mM
solutions of b) DD and AA, c) DDD and AAA, and d) DDDD and
AAAA in toluene-d8 at 298 K.
The data for denaturation of the AA·DD duplex fit well to a
denaturation isotherm that allows for all of these species, and
this allowed determination of K2 as the only unknown
equilibrium constant. The results are shown in Table 3. The
2
DMSO association constant as 2Kd and Kd respectively.
F
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Figure 9. Equilibria involved in denaturation of AA·DD. Kd is the D·
DMSO association constant, and KN is the association constant for
duplex formation between homo-oligomers of length N in toluene-d8.
value of K2 determined in the denaturation experiment is
comparable to the value determined in the corresponding
titration experiment in toluene, which indicates that the
denaturation methodology provides a robust method for
determining duplex stability.
The 31P NMR DMSO denaturation experiments for the
AAA·DDD and AAAA·DDDD duplexes are shown in Figure 8c
and d, respectively, and the results are similar to those obtained
for AA·DD. Fitting the denaturation data for the longer
oligomers requires consideration of a larger number of partially
denatured species (Figures 10 and 11). The equilibrium
Figure 11. Equilibria involved in denaturation of AAAA·DDDD. Kd is
the D·DMSO association constant, and KN is the association constant
for duplex formation between homo-oligomers of length N in toluene-
d8. For some of the complexes, isomeric arrangements are possible
(not shown).
similar stability to the complex with DMSO bound to the
central recognition unit. By analogy with the AA·DD·DMSO
complex, one might assume that the association constant for
AAA·DDD·DMSO complex, KP, will be the product of Kd and
K2, multiplied by a statistical factor, but to avoid any bias in the
fitting, KP was optimized as a variable along with K3 in analysis
of the AAA·DDD denaturation data. The results of fitting the
proportion of duplex present determined using eq 1 to the
denaturation isotherm are shown in Figure 12, and the
resulting association constant for duplex formation is reported
in Table 3. The optimized value of KP from the fitting is equal
to 3.5 Kd K2. If the duplex preferentially denatures from the
ends, so that the central H-bond is intact in the partially
denatured AAA·DDD·DMSO complex, the statistical factor
would be 4, which is close to the optimized value of 3.5. The
AAA·DDD duplex is more than an order of magnitude more
stable than the AA·DD duplex, which confirms that the fully
assembled triply H-bonded duplex is formed in toluene.
For denaturation of the AAAA·DDDD duplex, there are two
partially denatured complexes with unknown stability, AAAA·
DDDD·DMSO and AAAA·DDDD·(DMSO)2 (Figure 11).
Therefore, fitting of the denaturation data for this system
involved optimization of three equilibrium constants, K4, KP1,
Figure 10. Equilibria involved in denaturation of AAA·DDD. Kd is the
D·DMSO association constant, and KN is the association constant for
duplex formation between homo-oligomers of length N in toluene-d8.
For some of the complexes, isomeric arrangements are possible (not
shown).
constants for formation of most of these species can also be
estimated from the values of Kd and K1, but some partially
denatured duplexes have no direct analogues. For example, a
number of different structures are possible for the AAA·DDD·
DMSO complex, and we do not know whether complexes with
DMSO bound to one of the terminal recognition units have
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Figure 12. Duplex denaturation data plotted as a function of DMSO-
d6 concentration in toluene-d8 at 298 K for AA·DD (black), AAA·
DDD (blue), and AAAA·DDDD (red). The dots represent the
experimental values obtained using eq 1, and the lines are the
calculated denaturation isotherms.
and KP2. The results are shown in Figure 12 and Table 3. The
AAAA·DDDD is more than an order of magnitude more stable
than the AAA·DDD duplex, which confirms that the fully
assembled duplex with all four H-bonds is formed in toluene.
The value of KP1 is equal to 7.5 Kd K3. If the duplex
preferentially denatures from the ends, so that the central H-
bonds are both intact in the partially denatured AAAA·DDDD·
DMSO complex, then the statistical factor would be 4, which is
smaller than the optimized value of 7.5. This result suggests
that there are some additional partially denatured states where
the central H-bonds are broken. The value of KP2 is equal to
8.0 Kd2 K2. If the duplex preferentially denatures from the ends,
then the statistical factor would be 9, which is close to the
optimized value.
Figure 13 shows the speciation of different complexes
calculated from the denaturation isotherms. In the denatura-
tion of all three duplexes, the first step is breaking of one
duplex H-bonds to form a phenol-DMSO H-bond, and this
complex is the major partially denatured state in all cases (blue
lines in Figure 13a−c). For the AA·DD duplex, this partially
denatured complex is not very stable, so the DD·DMSO
complex is also significantly populated (pink line in Figure
13a). At high concentrations of DMSO, the fully denatured
state dominates in all cases (red lines in Figure 13a−c).
1
Denaturation of the duplexes can also be monitored by H
NMR spectroscopy (Figure 14). The signals due to the
aromatic protons of the oligomers move by 0.1−0.6 ppm on
denaturation. For the AA·DD duplex, the signal due to the
phenol OH proton starts at 6.2 ppm, suggesting that the
duplex is not fully bound, and moves to 10 ppm when bound
to DMSO (Figure 14a). For both the AAA·DDD and AAAA·
DDDD duplexes, the signals due to the phenol OH protons
start at about 11 ppm, suggesting that the duplexes are fully
bound, and move to about 10 ppm when bound to DMSO
(Figure 14b and c). As observed for the 31P NMR spectra,
there are differences at very high concentrations of DMSO-d6
due to the change in the nature of the solvent.
The association constants for duplex formation, the average
effective molarities (EM) for intramolecular H-bonding
interactions in the duplexes, and the free and bound chemical
shifts are shown in Table 3. The consistency of the values
suggests that the results obtained from the denaturation
experiments are accurate. The association constants increase
Figure 13. Speciation from DMSO denaturation experiments on
length complementary homo-oligomer duplexes. a) The 2-mer
duplex: AA·DD (black), DD·DMSO (pink), AA·DD·DMSO (blue),
and DD·(DMSO)2 (red). b) The 3-mer duplex: AAA·DDD (black),
AAA·DDD·DMSO (blue), DDD·(DMSO)2 (pink), AAA·DDD·
(DMSO)2 (green), and DDD·(DMSO)3 (red). c) The 4-mer duplex:
AAAA·DDDD (black), AAAA·DDDD·DMSO (blue), AAAA·DDDD·
(DMSO)2 (cyan), DDDD·(DMSO)3 (pink), and DDDD·(DMSO)4
(red). Free acceptor homo-oligomer populations are not shown, and
all other possible complexes are not populated to a significant extent.
with each additional H-bond, and the value of EM does not
vary significantly between duplexes.
Figure 15 shows the value of log KN plotted as a function of
the number of recognition modules in the oligomers (N).
There is a uniform increase of 1.7 log units for every additional
H-bond in the duplex. The results presented here can be
compared with previous experiments on different backbone
H
DOI: 10.1021/jacs.8b08087
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Figure 14. 500 MHz 1H NMR spectra for DMSO-d6 denaturation of
equimolar 1 mM solutions of a) A, b) DD·AA, c) DDD·AAA, and d)
DDDD·AAAA in toluene-d8 at 298 K.
stuctures equipped with the same phenol-phosphine oxide
recognition modules. The data in Figure 15 show that the
phenylacetylene backbone (red) gives the most stable
duplexes, presumably because these oligomers are more rigid
than the aniline (green) and thioether (blue) backbones.
Although the increased preorganization and geometric
complementarity of the phenylacetylene backbone leads to
higher values of EM for formation of the intramolecular H-
bonds that zip up the duplex, the increase is actually quite
small compared with the more flexible backbones (50 mM
compared with 20 mM and 30 mM for the green and blue
backbones, respectively, in Figure 15). However, small
differences in H-bond strength and EM are cumulative along
an oligomer, so that the phenylacetylene 4-mer duplex is 2
orders of magnitude more stable than the corresponding
duplex with the aniline backbone.
Figure 15. Relationship between the association constant for duplex
formation between length-complementary oligomers in toluene-d8 at
298 K (KN) and the number of intermolecular H-bonds formed (N).
Data are shown for three different backbones (color coded), each
equipped with phenol-phosphine oxide recognition modules. The
lines of best fit are shown: red log KN = 1.7N + 1.3; blue log KN
=
1.2N + 1.6; green log KN = 1.0N + 1.5. R represents a solubilizing
group.
phenol-phosphine oxide H-bonding interactions in toluene
solution. NMR spectroscopy indicates that all possible base-
pairing interactions between recognition sites are fully bound
in the duplexes.
Addition of a competing H-bond acceptor, DMSO,
dissociated the duplexes, and DMSO denaturation experiments
were used to measure the association constants for duplex
formation. The association constants increase by almost 2
orders of magnitude for every additional H-bonded base-pair
added to the duplex, reaching 108 M−1 for AAAA·DDDD. The
average effective molarity for the intramolecular H-bonding
interactions responsible for zipping up the duplex is EM = 50
mM, and the corresponding value of the intramolecular
equilibrium constant that quantifies chelate cooperativity in
these systems is K EM = 40 (the product of the association
constant for formation of one intermolecular phenol-
CONCLUSIONS
■
Oligomerization of bifunctional building blocks in the presence
of monofunctional chain terminators provides a straightfor-
ward approach to access families of oligomeric molecules with
minimal synthetic effort. Here we describe the use of this
strategy to prepare phenylacetylene oligomers equipped with
phenol and phosphine oxide recognition sites. Pure samples of
homo-oligomers up to seven recognition units in length were
obtained by preparative hplc of the crude reaction mixtures.
The phenol oligomers form stable duplexes with length
complementary phosphine oxide oligomers via intermolecular
I
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Article
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40 for K EM means that in the duplex the recognition sites are
98% bound, which is consistent with the NMR data. The
uniform increase in duplex stability with oligomer length
suggests that this supramolecular architecture is likely to be
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b08087.
■
S
1
Detailed experimental procedures and H and 13C of all
compounds, NMR titration and dilution spectra, details
of fitting the binding isotherm, and detailed HPLC
methods used for separation of oligomers (PDF)
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AUTHOR INFORMATION
Corresponding Author
*herchelsmith.orgchem@ch.cam.ac.uk
ORCID
■
Christopher A. Hunter: 0000-0002-5182-1859
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Engineering and Physical Sciences Research
Council (EP/J008044/2) and European Research Council
(ERC-2012-AdG 320539-duplex) for funding.
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