H-Bond Self-Assembly: Folding versus Duplex Formation

Article abstract of DOI:10.1021/jacs.7b01357

Linear oligomers equipped with complementary H-bond donor (D) and acceptor (A) sites can interact via intermolecular H-bonds to form duplexes or fold via intramolecular H-bonds. These competing equilibria have been quantified using NMR titration and dilution experiments for seven systems featuring different recognition sites and backbones. For all seven architectures, duplex formation is observed for homo-sequence 2-mers (AA·DD) where there are no competing folding equilibria. The corresponding hetero-sequence AD 2-mers also form duplexes, but the observed self-association constants are strongly affected by folding equilibria in the monomeric states. When the backbone is flexible (five or more rotatable bonds separating the recognition sites), intramolecular H-bonding is favored, and the folded state is highly populated. For these systems, the stability of the AD·AD duplex is 1-2 orders of magnitude lower than that of the corresponding AA·DD duplex. However, for three architectures which have more rigid backbones (fewer than five rotatable bonds), intramolecular interactions are not observed, and folding does not compete with duplex formation. These systems are promising candidates for the development of longer, mixed-sequence synthetic information molecules that show sequence-selective duplex formation.

Full text of DOI:10.1021/jacs.7b01357

Article
pubs.acs.org/JACS
H‑Bond Self-Assembly: Folding versus Duplex Formation
Diego Nunez-Villanueva, Giulia Iadevaia, Alexander E. Stross, Michael A. Jinks, Jonathan A. Swain,
́
̃
and Christopher A. Hunter*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
S
* Supporting Information
ABSTRACT: Linear oligomers equipped with complementary
H-bond donor (D) and acceptor (A) sites can interact via
intermolecular H-bonds to form duplexes or fold via intra-
molecular H-bonds. These competing equilibria have been
quantified using NMR titration and dilution experiments for
seven systems featuring different recognition sites and back-
bones. For all seven architectures, duplex formation is observed
for homo-sequence 2-mers (AA·DD) where there are no
competing folding equilibria. The corresponding hetero-
sequence AD 2-mers also form duplexes, but the observed self-association constants are strongly affected by folding equilibria
in the monomeric states. When the backbone is flexible (five or more rotatable bonds separating the recognition sites),
intramolecular H-bonding is favored, and the folded state is highly populated. For these systems, the stability of the AD·AD
duplex is 1−2 orders of magnitude lower than that of the corresponding AA·DD duplex. However, for three architectures which
have more rigid backbones (fewer than five rotatable bonds), intramolecular interactions are not observed, and folding does not
compete with duplex formation. These systems are promising candidates for the development of longer, mixed-sequence
synthetic information molecules that show sequence-selective duplex formation.
material. In duplex-forming synthetic systems, the recognition
sites are usually integrated into the backbone, which means that
sequence variation is difficult to introduce without disrupting
the complex. Synthetic oligomers that fold into well-defined
secondary structures, i.e., foldamers, have also been widely
studied, leading to synthetic receptors, nanomachines, and
modulators of protein−protein interactions.9 Huc has shown
that it is possible to program the recognition properties of
foldamers by changing the sequence of building blocks.10
We recently reported a new class of synthetic molecules that
form stable duplexes via multiple cooperative H-bonding
interactions between recognition units appended to oligomeric
backbones.11 Figure 1 shows the systems reported to date. Four
different backbones have been prepared using either reductive
amination or photochemical thiol−ene coupling. We found that
variations in the structure of the backbone were well-tolerated,
and different backbones and recognition modules could be used
interchangeably to form stable duplexes. However, all of these
systems are homo-sequence oligomers, i.e., all H-bond donor
(D) oligomers that form duplexes with length complementary
all H-bond acceptor (A) oligomers. As a first step toward
mixed-sequence oligomers, in this paper we describe the
properties of several AD 2-mers based on the motifs shown in
Figure 1.
INTRODUCTION
■
Macromolecules of defined sequence form the molecular basis
for living systems. Selective base pairing allows the sequence
information stored in nucleic acid polymers to be read and
translated through template synthesis.1 The sequences of
proteins and nucleic acids determine how they fold into
three-dimensional structures, which in turn dictate the
structural, recognition, and catalytic properties that govern
the biochemistry of the cell.2 Synthetic supramolecular systems
that recapitulate the sequence-selective recognition or folding
properties of biomacromolecules are beginning to appear, and
in the long term, synthetic information molecules may be
expected to offer alternatives to proteins and nucleic acids,
where sequence can be used to program function. Nucleic acids
are unique in that they combine both duplex-forming and
folding properties. The double-stranded structures are the basis
for replication, and folded single strands act as receptors and
catalysts, so it is possible to use nucleic acids in directed
evolution experiments to obtain functional polymers.3 The
balance between folding and duplex formation in nucleic acids
is therefore thought to be critical in the evolution of living
systems.4 In this Article, we examine the interplay of folding
and duplex formation in prototype synthetic molecules.
Inspired by DNA, researchers have put considerable effort
into synthetic supramolecular systems that form duplex
structures held together by non-covalent interactions.5−7
Although programmable sequence-specific recognition of
DNA has been achieved with synthetic molecules,8 the
molecular features that allow nucleic acids to encode and
replicate genetic information are still not available in any other
Mixed-sequence oligomers introduce the possibility of
intramolecular H-bonds, which can lead to folding equilibria
that compete with duplex formation (Figure 2). If two
Received: February 10, 2017
© XXXX American Chemical Society
A
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Figure 2 are the effective molarities for folding (EMf) and
duplex formation (EMd), the operating concentration (c), and
the association constant for the intermolecular interaction
between two complementary H-bonding sites (K). Figure 2
shows pathways where the first H-bond formed can be
intramolecular or intermolecular: if EMf > c and KEMf > 1,
then the folding channel will be preferred over intermolecular
interactions. If the first H-bond formed is intramolecular, then
further intramolecular interactions leading to folded hairpin
loop structures will take place if the sequence is suitable and if
KEMd > 1. If the first H-bond formed is intermolecular, there
are two pathways for subsequent interactions: intramolecular
interactions leading to duplex formation, or intermolecular
interactions leading to aggregates. The duplex channel will be
preferred over aggregation if EMd > c and KEMd > 1. Therefore,
duplex formation will be the preferred self-assembly process if
EMd is greater than all of c, 1/K, and EMf.
Before we embark on the synthesis of mixed-sequence
oligomers, it is therefore important to investigate how K, EMd,
and EMf vary with the chemical structures of the monomer
units. The association constant for intermolecular H-bond
formation (K) is straightforward to predict for any choice of
recognition unit,12 but we do not yet have reliable methods for
predicting effective molarities for intramolecular interac-
tions.13−17 Here, we analyze the competing equilibria between
folding and duplex formation for the seven different AD 2-mers
shown in Figure 3. These compounds represent the six systems
Figure 1. H-bonded duplexes formed by oligomers with different
recognition units and backbones (R is a solubilizing group).11 The
oligomers are labeled according the type of recognition unit
(phosphine oxide = PO, pyridine = N, pyridine N-oxide = NO), the
site of attachment of the recognition units (C or N), and the number
of atoms separating neighboring recognition units (7, 8, or 9).
Figure 3. Chemical structures of the AD 2-mers (R1 = (2-
ethylhexyl)oxy; R2 = (2-ethylhexyl)oxy or methoxy; R3 = H or S-β-
citronelloxy).
Figure 2. Competing equilibria in the formation of a duplex between
two complementary oligomers. Intramolecular interactions between
recognition sites lead to folding, and intermolecular interactions
between multiple oligomers lead to networks of supramolecular
aggregates. The effective molarities for folding (EMf) and duplex
formation (EMd), the operating concentration (c), and the association
constant for formation of a H-bond between two recognition sites (K)
determine the distribution of oligomers between the three different
assembly channels. The equilibrium constants have additional
statistical factors that are not shown.
shown in Figure 1 for which the corresponding A·D and AA·
DD complexes have been characterized previously and one
additional system with a more rigid backbone (C7-PO), which
provides a reference compound that cannot fold. The aim is to
obtain experimental data for understanding the complex
equilibria shown in Figure 2 in order to select suitable
architectures for the development of mixed-sequence duplex-
forming oligomers.
RESULTS AND DISCUSSION
neighboring recognition units on an oligomer interact strongly,
the folding channel will be favored over the duplex assembly
channel. Thus, the selectivity of mixed-sequence duplex
formation is limited by the extent to which intramolecular
folding occurs in AD 2-mers. The key parameters that
determine the distribution of the different species shown in
■
Synthesis. Two versions of the C8-NO AD 2-mer were
prepared: one with (2-ethylhexyl)oxy solubilizing groups for
binding studies, and one with methoxy groups for X-ray
crystallography. Building blocks 4 and 7 required for synthesis
of the methoxy analogue were prepared from 5-hydroxy-2-
B
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nitrobenzaldehyde 1 as shown in Scheme 1. After methylation
of 1, the acetal-protected aldehyde 2 was obtained using
Scheme 4. Synthesis of C8-N AD 2-mer (R = (2-
Ethylhexyl)oxy)
Scheme 1. Synthesis of Building Blocks 4 and 7
with 18, afforded 19 in good yield. After the removal of the
acetal protecting group, reductive amination with 20 gave 21.
Scheme 5 shows the synthesis of the N8-PO 2-mer 26 from
p-aminophenol and compounds 23 and 24, which have been
Scheme 5. Synthesis of N8-PO AD 2-mer (R = (2-
Ethylhexyl)oxy)
ethylene glycol and catalytic tosic acid. Hydrogenation of the
nitro group yielded aniline 3. The imines of 3 formed with 4-
pyridinecarboxaldehyde N-oxide or 2-methoxy-5-nitrobenz-
aldehyde were reduced to give 4 and 5, respectively. Reduction
of the imine derived from 5 and 6,11d followed by acid removal
of the acetal protecting group, gave 7. The syntheses of the
analogues of 4 and 7 bearing (2-ethylhexyl)oxy solubilizing
groups instead of methoxy groups (8 and 9) were reported
previously.11a,c Reductive amination was used to couple the
phenol and N-oxide building blocks in good yield to give 10
and 11 (Scheme 2). TBAF-mediated deprotection of the
phenol groups afforded the C8-NO AD 2-mers 12 and 13.
reported previously.11b The statistical reaction between these
three components gave 25 in modest yield after column
chromatography. The NH H-bond donor sites of 25 could
potentially compete with the phenol recognition units, so they
were removed by capping with 2-methoxybenzaldehyde to give
26.
The N7-PO AD 2-mer 29 differs from the N8-PO AD 2-mer
26 in the position of the substituents on the backbone aromatic
ring. 29 was obtained using a synthetic strategy similar to that
used for 26 but with the isophthalaldehyde derivative 27 in
place of 23 (Scheme 6).11b The statistical reaction of 27 with
22 and 24 yielded 28 after column chromatography, and
subsequent reductive amination with 2-methoxybenzaldehyde
gave 29.
Scheme 2. Synthesis of C8-NO AD 2-mers
Scheme 6. Synthesis of N7-PO AD 2-mer (R = (2-
Ethylhexyl)oxy)
The synthesis of the C8-PO 2-mer 16 is shown in Scheme 3.
Reductive amination of aldehyde 8 with aniline 1411a yielded
15, and deprotection using TBAF gave the AD 2-mer 16. The
route to the C8-N 2-mer 21 involves previously reported
compounds 17, 18, and 20 (Scheme 4).11a,c TBAF-mediated
phenol deprotection of 17, followed by reductive amination
Photochemically initiated thiol−ene coupling was used to
synthesize the C9-PO 2-mer 33 from 30 and 32 (Scheme 7).11d
Thiol−ene-mediated capping of 30 with tert-butyl mercaptan
gave 31. Removal of the S-acetyl protecting group of 31 under
Scheme 3. Synthesis of C8-PO AD 2-mer (R = (2-
Ethylhexyl)oxy)
Scheme 7. Synthesis of C9-PO AD 2-mer
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basic conditions, and subsequent thiol−ene coupling with 32,
afforded 33 in good yield.
Lithium−halogen exchange using n-BuLi followed by reaction
with iodine gave the bromoiodobenzene derivative 42 in good
yield. A selective Sonogashira coupling between the iodide
substituent and alkyne 36 gave 43 in excellent yield, and
subsequent Sonogashira coupling between the bromine
substituent and 3-hydroxyphenylacetylene gave 44. The C7-
PO AD 2-mer 47, which does not have the solubilizing group,
was prepared from a statistical Sonogashira coupling of an
equimolar mixture of 36, 45, and 46 followed by column
chromatography (Scheme 11).
The C7-PO AD 2-mer is a new backbone architecture, so the
corresponding AA and DD 2-mers were also synthesized. The
AA·DD complex provides an important reference point for
characterizing duplex formation (see Supporting Information
for the synthesis of the corresponding A and D 1-mers). The
C7-PO compounds were all prepared using Sonogashira
coupling. The required phosphine oxide building blocks 35
and 36 were prepared as shown in Scheme 8. Palladium-
Scheme 8. Synthesis of Building Blocks 35 and 36
Scheme 11. Synthesis of C7-PO AD 2-mer for X-ray
Crystallography
mediated P-arylation of 3-bromoiodobenzene with di-n-butyl-
phosphine 3411d gave 35 in excellent yield. Sonogashira
coupling with trimethylsilylacetylene (TMSA) followed by
TBAF-mediated deprotection gave 36. Scheme 9 shows the
NMR Binding Studies. NMR titration and dilution
experiments were performed to measure the association
constants for the A·D, AA·DD, and AD·AD complexes
(Table 1). For the A·D and AA·DD complexes, association
constants were determined by fitting 1H and 31P NMR titration
data to a 1:1 binding isotherm.11 Self-association constants for
Scheme 9. Synthesis of C7-PO AA and DD 2-mers (R = S-β-
Citronelloxy)
1
the AD·AD complexes were determined by fitting H and 31P
NMR dilution data to a dimerization isotherm.
Figure 4 shows the equilibria that are possible in these
systems. Under dilute conditions, the higher order complexes
are not expected to be significantly populated. The equilibrium
constants for the A·D complexes measure the intrinsic H-bond
strength of the recognition units (K), which is similar for the
phenol·phosphine oxide and phenol·N-oxide systems and an
order of magnitude lower for the phenol·pyridine system. The
phenol·phosphine oxide recognition system was also studied in
chloroform, where the association constant is an order of
magnitude lower than in toluene (see C7-PO entry in Table 1).
For the AA·DD complexes, the product KEMd is greater than 1
in all cases, which means that the duplex shown in Figure 4a is
the most populated state for concentrations lower than EMd.
The values of Kduplex in Figure 4 are expected to be similar for
the AA·DD and corresponding AD·AD duplexes. If there were
no competing equilibria, then the observed self-association
constants for the AD systems would be similar to the
association constants measured for formation of the corre-
sponding AA·DD duplexes. For most of the systems in Table 1,
this is not the case, which means that the competing equilibria
shown in Figure 4b play an important role in the self-
association of the AD 2-mers.
Figure 4 shows that the equilibria involving the partially
bound 1:1 complex and the closed duplex are the same for the
AA·DD and AD·AD systems, so we assume that the relative
populations of these states are similar in both cases; i.e., the
closed duplex dominates. The major competing equilibrium
that leads to the lower self-association constants observed for
the AD systems must therefore be due to folding of the
monomer to form an intramolecular A·D H-bond. This
conclusion is supported by examination of the 31P NMR
chemical shifts of the free (δfree) and bound (δbound) states from
the dilution experiments (Table 2). With the exception of the
C7-PO system, the free chemical shifts for the AD 2-mers are
much higher than the values observed for the corresponding A
1-mers and AA 2-mers. The free chemical shift measured in the
synthesis of the C7-PO AA 2-mer (40) and the C7-PO DD 2-
mer (41). A Mitsunobu reaction between 3,5-dibromophenol
and S-(−)-β-citronellol yielded 38 in excellent yield. A double
Sonogashira coupling with TMSA followed by alkyne
deprotection gave 39. Double Sonogashira coupling of 39
with 3-iodophenol or 35 gave 40 and 41.
Two versions of the C7-PO AD 2-mer were prepared: one
with a (S)-β-citronelloxy solubilizing group for binding studies
(44), and one with no solubilizing groups for X-ray
crystallography (47). The C7-PO AD 2-mer (44) was
synthesized from compound 38 in three steps (Scheme 10).
Scheme 10. Synthesis of C7-PO AD 2-mer for Binding
Studies (R = S-β-Citronelloxy)
D
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Table 1. Association Constants (Kobs) and Effective Molarities (EM) Measured by NMR Titrations and Dilutions in Toluene-d8
a
at 298 K
b
complex
Kobs (M−1
)
Kfold
EMf (mM)
EMd (mM)
8 (3)
χfold
C8-PO
A·D
350 (3)
1900 (470)
45 (15)
AA·DD
AD·AD
c
2.2 (0.5)
n.d.
7 (1)
n.d.
0.69 (0.06)
C8-N
A·D
34 (1)
130 (16)
60 (20)
AA·DD
AD·AD
C8-NO
A·D
AA·DD
AD·AD
N8-PO
A·D
AA·DD
AD·AD
N7-PO
A·D
AA·DD
AD·AD
C9-PO
A·D
57 (8)
30 (10)
20 (2)
0
330 (80)
5400 (1600)
2400 (500)
n.d.
n.d.
0
250 (10)
2500 (200)
225 (13)
0.7 (0.1)
3.6 (0.9)
4.1 (1.1)
n.d.
3 (1)
15 (4)
7 (3)
n.d.
0.40 (0.03)
0.78 (0.05)
0.81 (0.05)
0
250 (10)
1200 (400)
14 (5)
10 (3)
560 (210)
9000 (4000)
85 (22)
AA·DD
AD·AD
14 (12)
130 (40)
d
C7-PO
A·D
30 (1)
240 (80)
130 (30)
AA·DD
AD·AD
e
a
Average values with errors (reported in parentheses) at the 95% confidence limit based on the variation between different signals and experiments.
Calculated from Kfold using eq 8. AD 2-mer 16. Experiments carried out in CDCl3 due to the high stability of the complexes in toluene-d8. AD 2-
b
c
d
e
mer 44. n.d. = not detected.
for the AD 2-mers indicate that the folded state with an
intramolecular H-bond is significantly populated.
The data in Tables 1 and 2 can used to determine the
equilibrium constant for folding, Kfold, in these systems.
Equations 1−3 show the relationship between the measured
self-association constant for dimerization of AD·AD, Kobs, and
the equilibrium constants, Kduplex and Kfold, illustrated in Figure
4b.
[AD·AD]
Kobs
=
[AD]2
(1)
where [AD] is the total concentration of monomeric AD 2-mer
as defined in eq 2, i.e., the sum of the open, [AD]open, and
folded, [AD]fold, states.
[AD] = [AD]open + [AD]fold = (1 + Kfold)[AD]open
(2)
Combining eqs 1 and 2 allows Kobs to be expressed as a
function of Kfold and Kduplex, the equilibrium constant for the
formation of the AD·AD duplex from the open monomeric
state (eq 3).
Figure 4. Competing equilibria in the assembly of (a) AA·DD and (b)
AD·AD duplexes. The equilibrium constants have additional statistical
factors that are not shown.
Kduplex
[AD·AD]
1
Kobs
=
=
2
2
[AD]2
(1 + Kfold
)
(1 + Kfold)
open
(3)
AD dilution experiments is the population-weighted average
over all monomeric states, so the higher values of δfree observed
Rearranging eq 3 allows Kfold to be expressed in terms of Kobs
and Kduplex (eq 4).
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3−20 mM. For the systems that do not fold, the values of EMd
are somewhat higher (30−130 mM).
Kduplex
Kfold
=
− 1
Kobs
The 31P NMR chemical shift data in Table 2 can also be used
to estimate values of χfold. The values of δbound for the A·D, AA·
DD, and AD·AD complexes are similar in all cases, indicating
that the phosphine oxides are fully H-bonded. However, there
are significant differences between the values of δfree for the AD·
AD complexes compared with the corresponding A·D and AA·
DD complexes, because the values represent the population-
weighted average of the chemical shifts of the open and folded
states of the AD monomer. If we assume that the open AD
monomer has the same chemical shift as the free state of A (δA)
and that the folded AD monomer has the same chemical shift
as the bound state A·D (δA·D), the free chemical shift observed
for the AD 2-mer (δfree) can be used to quantify the population
of the folded state via eq 9.
(4)
If we assume that the AD·AD and AA·DD duplexes make H-
bonds of similar stability, then it is possible to use the
equilibrium constant for formation of the AA·DD duplex to
estimate Kduplex. In systems where multiple H-bonding sites are
constrained in very close proximity, local secondary electro-
static interactions can alter the relative stabilities of different H-
bonding patterns, but the recognition sites in the compounds
described here are too far apart for such effects to be
significant.18 However, there is a difference in symmetry
between the AA·DD and AD·AD duplexes, which leads to a
statistical contribution to the value of Kduplex. For AA·DD, there
is one unbound state, but there are two degenerate states for
the duplex, because two different H-bonding arrangements are
possible.19 The AD·AD duplex is made from two identical
molecules, so there are two degenerate unbound states, but
there is one bound state, because only one H-bonding
arrangement is possible. Thus, the observed association
constant for formation of the AA·DD duplex, Kobs(AA·DD),
can be used to estimate Kduplex for formation of the AD·AD
duplex from the open monomeric state (eq 5), and the
δfree = χfoldδA·D + χopenδA
(9)
The limiting complexation-induced change in chemical shift
observed in dilution experiments for the AD 2-mers (ΔδAD·AD
)
can be expressed as eq 10.
δAD·AD = δfree − δbound = (1 − χfold)(δA − δA·D
)
(10)
resulting values were used in eq 4 to calculate values of Kfold
.
Rearranging eq 10 allows χfold to be obtained from the values of
ΔδAD·AD and ΔδA·D (or ΔδAA·DD) listed in Table 2 (eq 11).
Kduplex ≈ 0.25Kobs(AA·DD)
(5)
ΔδAD·AD
ΔδA·D
ΔδAD·AD
ΔδAA·DD
χfold = 1 −
= 1 −
The effective molarity for intramolecular H-bonding in the
monomeric state, EMf, is given by the ratio of Kfold to the
association constant for the corresponding intermolecular
interaction, KA·D (eq 6).
(11)
Figure 5 shows that there is a good correlation between the
values of χfold calculated from the association constants (eq 8)
and those obtained from the complexation-induced changes in
31P NMR chemical shift (eq 11). For systems with phosphine
oxide H-bond acceptors, the 31P NMR chemical shift therefore
Kfold
KA·D
EMf =
(6)
Table 2. 31P NMR Chemical Shifts (ppm) Measured in
Toluene-d8 at 298 K
The values of Kfold can also be used to estimate the relative
populations of the open (χopen) and folded states (χfold) for the
monomeric AD 2-mers (eq 7).
a
complex
δfree
δbound
Δδ
χfold
χfold + χopen = (1 + Kfold)(1 + χfold) = 1
C8-PO
A·D
AA·DD
(7)
53.7
53.6
57.0
58.7
58.8
58.1
5.0
5.2
1.1
Rearranging eq 7 allows χfold to be calculated from the value of
Kfold (eq 8).
b
AD·AD
0.78 (0.01)
0.46 (0.08)
0.76 (0.05)
0.92 (0.01)
0.18 (0.16)
N8-PO
A·D
AA·DD
AD·AD
N7-PO
A·D
AA·DD
AD·AD
C9-PO
A·D
1
χfold = 1 −
53.5
53.5
55.1
58.5
57.5
57.5
5.0
4.0
2.4
(1 + Kfold
)
(8)
The values of Kfold, EMf, and χfold are tabulated in Table 1 for
each of the seven AD 2-mers. Four systems fold to a significant
extent, and the other three systems show no evidence of
folding. No folding was observed for C7-PO, which has a rigid
backbone that holds the recognition sites apart. The other two
systems that do not fold are C8-N and C8-NO, which have a
relatively flexible backbone. A common feature of the three AD
2-mers that do not fold is that the recognition units are
connected by fewer than five rotatable bonds. Although C8-PO
shares the same backbone as C8-N and C8-NO, this system
does fold due to the additional flexibility associated with the
two atom linker that connects the phosphine oxide to the
aromatic ring. For C8-PO, C9-PO, and N7-PO, the folded state
is highly populated (χfold = 70−80%). N8-PO also folds, but the
effective molarity for folding (EMf) is lower, so the open and
folded states are equally populated (χfold ≈ 40%). The values of
EMf and EMd are similar in all four cases and fall in the range
53.5
53.4
56.5
58.5
60.3
57.9
5.0
6.9
1.4
35.4
35.6
40.6
41.8
42.0
41.1
6.4
6.4
0.5
AA·DD
AD·AD
c
C7-PO
A·D
AA·DD
AD·AD
40.3
40.2
40.2
45.8
44.4
44.1
5.5
4.2
3.9
d
a
Average value and standard deviation (reported in parentheses)
calculated from ΔδA·D and ΔδAA·DD using eq 11. AD 2-mer 16.
Experiments carried out in CDCl3 due to the high stability of the
b
c
d
complexes in toluene-d8. AD 2-mer 44.
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suggests that the C8-NO AD 2-mer folds with an intra-
molecular pyridine N-oxide·phenol H-bond, which contradicts
the results of the NMR experiments in Table 1. Analysis of the
partial atomic charges assigned by the force-field reveals that
the charge on the oxygen of the pyridine N-oxide acceptor is
significantly higher than that on the oxygen of the phosphine
oxide in the C8-PO AD 2-mer. The association constant for
formation of a phosphine oxide·phenol H-bond in the A·D
complex in Table 1 is slightly higher than the value for a
pyridine N-oxide·phenol H-bond,12 so the folded conformation
calculated for the C8-NO system may be an artifact of
unrealistic partial atomic charges that overestimate the H-bond
strength.
Figure 5. Correlation between χfold estimated using changes in
chemical shift (Δδ, eq 11) and association constants (K, eq 8). The
line corresponds to y = x.
The structures of the complexes formed by the C8-NO and
C7-PO AD 2-mers were also investigated using X-ray
crystallography. The X-ray crystal structure of the methoxy
analogue of the C8-NO AD 2-mer (12) shows formation of the
doubly H-bonded antiparallel duplex (Figure 7). Despite the
provides a convenient and straightforward method for assessing
folding. For systems where this chemical shift information is
not available, the correlation in Figure 5 provides confidence
that the method for assessing folding on the basis of
measurement of association constants is robust.
Three-Dimensional Structures. Molecular mechanics
calculations provide insight into the folding propensity of the
AD 2-mers. A conformational search using the MMFFs force-
field was performed for the monomer of each of the seven AD
2-mers (see Supporting Information for details). A folded
conformation featuring an intramolecular H-bond is the lowest
energy structure for all four systems that were found to fold in
the NMR experiments (Figure 6).
The global minimum for the C8-N system and for the C7-
PO system corresponds to an open conformation, in agreement
with the experimental results. However, molecular modeling
Figure 7. Single-crystal X-ray structure of 12, which forms a doubly H-
bonded duplex in the solid state. Three adjacent unit cells with a total
of six molecules of 12 are shown. Hydrogen atoms have been omitted
for clarity. The backbones are shown in gray, the H-bond donor
recognition units in blue, and the H-bond acceptor units in red.
molecular modeling prediction of folding for this system, X-ray
crystallography is consistent with the NMR result that the
intermolecular channel leading to bimolecular duplexes is
favored over the intramolecular channel leading to folded
structures.
The X-ray crystal structure of 47, the analogue of the C7-PO
AD 2-mer with no solubilizing group, was also determined
(Figure 8). There is no intramolecular folding in the solid state,
in agreement with the solution-phase NMR results, but a
duplex is not observed. Instead, 47 crystallizes as a linear H-
bonded polymer, the state that the AD 2-mers are likely to
populate at high concentrations (c > EMd).
Figure 8. Single-crystal X-ray structure of 47, which forms a linear H-
bonded polymer in the solid state. Three adjacent unit cells with a
total of three molecules of 47 are shown. Hydrogen atoms have been
omitted for clarity. The backbones are shown in gray, the H-bond
donor recognition units in blue, and the H-bond acceptor units in red.
Figure 6. Lowest energy conformations of AD 2-mers calculated using
molecular mechanics conformational searches (MMFFs force-field and
CHCl3 solvation implemented in Macromodel).20
G
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
probability of folding between recognition units that are remote
in sequence will increase, even for relatively rigid backbones.
These more complex folding patterns are something that we
hope to exploit in the development of synthetic information
molecules that exhibit both sequence-selective duplex for-
mation and sequence-directed folding properties. Work on the
synthesis of longer mixed-sequence oligomers using the systems
identified here that do not fold is in progress (Figure 9).
CONCLUSIONS
■
Self-complementary molecules bearing H-bond donors (D) and
acceptors (A) can make either intermolecular interactions to
form a duplex or intramolecular interactions to fold. A
quantitative analysis of these competing equilibria is described
for a family of seven different AD 2-mers, which represent the
shortest mixed-sequence systems from a family of longer
oligomers. The compounds were prepared using a variety of
coupling chemistries (reductive amination, thiol−ene, and
Sonogashira coupling) and differ in the flexibility and geometric
complementarity of the backbone and in the nature of the H-
bond recognition units.
The equilibrium constants for intramolecular folding, Kfold
,
cannot be measured directly because the folded and open
conformers are in fast exchange on the NMR time scale.
However, it is possible to infer the value of Kfold by comparing
the equilibrium constant for self-association of an AD 2-mer
with the equilibrium constant for formation of the isomeric AA·
DD duplex, where the two components cannot fold. For
systems that have phosphine oxide H-bond acceptors, the
accuracy of this method was confirmed by comparison of the
31P NMR chemical shifts of the free and bound states. For AD
2-mers that fold to a significant extent, the phosphine oxide is
involved in an intramolecular H-bond, so the free chemical shift
for the monomeric state is very similar to the bound chemical
shift for the AD·AD duplex. The free and bound chemical shifts
for the AD·AD complex can be related to the corresponding
values for the A·D and AA·DD complexes to quantify the
populations of folded and open states in the monomeric form
of AD, and hence Kfold. The results of the chemical shift and
association constant methods show good quantitative agree-
ment.
Figure 9. Structures of duplexes formed by AD 2-mers that do not fold
(R1 = (2-ethylhexyl)oxy; R2 = S-β-citronelloxy).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b01357.
■
S
1
Detailed experimental procedures and H and 13C NMR
The three key parameters that determine the behavior of
these systems are the association constant for H-bond
formation between the recognition units and the effective
molarities for intramolecular folding, EMf, and duplex
formation, EMd. In all seven systems, EMd is larger than 1/K,
which means that efficient duplex formation is observed for 1:1
complexes of two homo-oligomers, AA·DD. In four systems,
EMf is comparable to EMd, which means that the AD 2-mers
prefer folding over duplex formation at low concentrations. It is
possible to force these systems to form AD·AD duplexes at high
concentrations, but the association constants for duplex
formation are 1−2 orders of magnitude lower than those
observed for the isomeric AA·DD complexes. For the other
three systems, EMf ≪ EMd, and no evidence for intramolecular
folding was observed. This conclusion is supported by X-ray
crystal structures of two of the systems that do not fold in
solution: one forms the doubly H-bonded AD·AD duplex in the
solid state, and the other forms a supramolecular polymer
where the molecules are connected by single A·D H-bonding
interactions.
We previously found that the value of EMd for duplex
formation does not depend greatly on the conformational
flexibility of the backbone. This work shows that folding
equilibria are more sensitive to the nature of the backbone. The
three AD 2-mers that do not fold have recognition units
connected by fewer than five rotatable bonds, but when the
backbone is more flexible, folding is observed. This observation
provides a convenient rule of thumb for the design of systems
where duplex formation is favored over intramolecular folding
of neighboring recognition units. In longer oligomers, the
spectra of all compounds, NMR titration and dilution
spectra, and molecular modeling methods (PDF)
X-ray crystallographic data for compound 12 (CIF)
X-ray crystallographic data for compound 47 (CIF)
AUTHOR INFORMATION
Corresponding Author
*herchelsmith.orgchem@ch.cam.ac.uk
ORCID
■
Diego Nunez-Villanueva: 0000-0002-1005-1464
́
̃
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. We thank Mr.
Harry Adams (University of Sheffield) and Dr. Andrew Bond
(University of Cambridge) for the help with X-ray crystallo-
graphic data collection and analysis.
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