Cooperative duplex formation by synthetic H-bonding oligomers

Article abstract of DOI:10.1039/c5sc03414k

A series of flexible oligomers equipped with phenol H-bond donors and phosphine oxide H-bond acceptors have been synthesised using reductive amination chemistry. H-bonding interactions between complementary oligomers leads to the formation of double-stranded complexes which were characterised using NMR titrations and thermal denaturation experiments. The stability of the duplex increases by one order of magnitude for every H-bonding group added to the chain. Similarly, the enthalpy change for duplex assembly and the melting temperature for duplex denaturation both increase with increasing chain length. These observations indicate that H-bond formation along the oligomers is cooperative despite the flexible backbone, and the effective molarity for intramolecular H-bond formation (14 mM) is sufficient to propagate the formation of longer duplexes using this approach. The product K EM, which is used to quantify chelate cooperativity is 5, which means that each H-bond is more than 80% populated in the assembled duplex. The modular design of these oligomers represents a general strategy for the design of synthetic information molecules that could potentially encode and replicate chemical information in the same way as nucleic acids.

Full text of DOI:10.1039/c5sc03414k

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Cooperative duplex formation by synthetic H-
bonding oligomers†
Cite this: DOI: 10.1039/c5sc03414k
*
Alexander E. Stross, Giulia Iadevaia and Christopher A. Hunter
A series of flexible oligomers equipped with phenol H-bond donors and phosphine oxide H-bond acceptors
have been synthesised using reductive amination chemistry. H-bonding interactions between
complementary oligomers leads to the formation of double-stranded complexes which were
characterised using NMR titrations and thermal denaturation experiments. The stability of the duplex
increases by one order of magnitude for every H-bonding group added to the chain. Similarly, the
enthalpy change for duplex assembly and the melting temperature for duplex denaturation both increase
with increasing chain length. These observations indicate that H-bond formation along the oligomers is
cooperative despite the flexible backbone, and the effective molarity for intramolecular H-bond
formation (14 mM) is sufficient to propagate the formation of longer duplexes using this approach. The
product K EM, which is used to quantify chelate cooperativity is 5, which means that each H-bond is
more than 80% populated in the assembled duplex. The modular design of these oligomers represents
a general strategy for the design of synthetic information molecules that could potentially encode and
replicate chemical information in the same way as nucleic acids.
Received 10th September 2015
Accepted 15th October 2015
DOI: 10.1039/c5sc03414k
www.rsc.org/chemicalscience
chemical components and still retain the duplex forming
properties of the system. These results suggest that attempts to
Introduction
develop new synthetic information molecules would benet
from a modular strategy that would allow independent opti-
misation of the different components of the monomer units.9
This report describes the rst steps towards such a system with
the synthesis of complementary oligomers that form stable
duplexes in organic solvents.
Formation of intermolecular complexes between two linear
polymers equipped with complementary recognition sites is the
molecular basis for life on Earth. As pointed out by Watson and
Crick in 1953, this supramolecular architecture provides
a robust mechanism for encoding molecular information and
for replication of this information through template-directed
synthesis.1 Although synthetic polymers bearing side-chain
recognition sites have been reported, these systems are gener-
ally of ill-dened chemical and supramolecular structure and
lack the control over length and sequence found in biological
polymers.2 Stepwise synthesis of short oligomers provides
access to more well-dened systems, and a number of synthetic
supramolecular systems that form duplex structures have been
reported.3 However, the recognition sites in these compounds
are usually integrated into the backbone of the oligomer, which
limits their versatility because a rather precise matching of
molecular geometries is required. In contrast, the modular
architecture of nucleic acids appears to be remarkably robust
with respect to chemical manipulation.4 Re-engineering exper-
iments on nucleic acids show that it is possible to substitute the
sugar,5 the phosphate,6,7 or the bases,8 for very different
Approach
Fig. 1 illustrates the DNA molecule stripped down to the basic
constituents required for duplex assembly and template-
directed synthesis: a recognition-based pairing system (blue),
chemistry for the synthesis of oligomers (red), and a linker that
allows the recognition sites on the two polymeric backbones to
reach one another (black). This basic blueprint provides
guidelines for the construction of a wide range of different types
of molecule that could function in the same way as nucleic acids
under appropriate conditions. Nucleic acids operate in water,
where stacking of the hydrophobic bases is the main driving
force for duplex assembly.10 This supramolecular architecture
requires mutual complementarity of the chemistry, linker and
recognition elements, so that the duplex is tightly packed. Here,
we target synthetic duplexes held together by H-bonding inter-
actions in organic solvents, so that a much looser structure is
possible with fewer constraints on the components. The
modular design in Fig. 1 will allow for the future optimization of
the properties of these components independently.
Department of Chemistry, University of Cambridge, Lenseld Road, Cambridge CB2
1EW, UK. E-mail: herchelsmith.orgchem@ch.cam.ac.uk
† Electronic supplementary information (ESI) available: Detailed experimental
procedures with spectroscopic characterization data, 31P and 1H NMR titration
spectra, binding isotherms, limiting chemical shis for free and bound states,
and thermal denaturation spectra. See DOI: 10.1039/c5sc03414k
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between multiple partners leads to aggregation and formation
of cross-linked supramolecular networks rather than a well-
dened duplex. Thus the criteria for high delity duplex
formation are K EM [ 1 and EM [ c.
Qualitatively, EM increases with geometric complementarity
and decreases with conformational exibility, but it is still
difficult to make quantitative predictions of the value of EM as
a function of chemical and supramolecular structure. We have
shown that for supramolecular architectures of varying
complementarity and exibility, the values of EM for intra-
molecular interactions fall in a surprisingly narrow window (10–
1000 mM)12 compared with covalent EMs, where changes in
chemical structure can lead to variations of several orders
magnitude.13 Although there is a trade-off between conforma-
tional exibility and geometric complementarity, the margin for
error in supramolecular design is much higher if more exible
molecules are used, and the associated decrease in EM is not
dramatic.14 The consequence is that if K [ 100 Mꢀ1 for the
recognition modules in Fig. 1, it should be possible to assemble
duplex structures without the need for careful design of the
linker module, provided the backbones are sufficiently exible
to ensure geometric complementarity.
This K criterion dictates the choice of recognition modules.
The simplest possible recognition system is a single H-bond
between a H-bond donor (D) and a H-bond acceptor (A), i.e.
a two-letter alphabet rather than the four-letter alphabet used in
nucleic acids. Provided the backbone does not have any polar
functional groups that could compete for the H-bonding inter-
actions, use of simple H-bonding modules will ensure selec-
tivity of the recognition-based pairing system: D will only pair
with A; D will not pair with D, and A will not pair with A.
Phosphine oxides and phenols form exceptionally stable H-
bonds in toluene (102 to 103 Mꢀ1),14g and so these functional
Fig. 1 Blueprint for a synthetic information molecule. The key design
components are the covalent chemistry used for synthesis (red), the
non-covalent chemistry used for recognition (blue), and the backbone
linker that determines the geometric complementarity of the two
chains (black).
The two key parameters that determine the efficiency of
duplex formation between two oligomers are the association
constant for the intermolecular interaction between two
recognition sites (K) and the effective molarity for formation of
the corresponding intramolecular interactions as the duplex
zips up (EM).11 Fig. 2 shows the steps involved in the assembly
of a duplex from two complementary oligomers. The rst step is
an intermolecular interaction with an association constant K.
Formation of subsequent interactions on the intramolecular
pathway leading to duplex formation requires that the product
K EM is greater than unity. There is a competing intermolecular
assembly channel, and the ratio of EM to the operating
concentration (c) determines whether uncontrolled interactions
Fig. 2 Recognition-directed assembly of a duplex between two complementary oligomers (intramolecular channel) competes with uncon-
trolled assembly of supramolecular networks (intermolecular channel). K is the association constant for formation of an intermolecular inter-
action between two recognition elements (blue bars), EM is the effective molarity for formation of an intramolecular interaction in the duplex
structure, and c is the operating concentration. For specific oligomers, the equilibrium constants have additional statistical factors reflecting the
differences in the degeneracy of the complexes.
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Fig. 3 Design of a synthetic information molecule (cf. Fig. 1), which
can be synthesised by reductive amination of aminoaldehyde mono-
mers (red) equipped with a phenol or a phosphine oxide recognition
module (blue). X is a site for attachment of solubilising groups.
Scheme 1 (i) Tri-i-propylsilyl chloride, imidazole; (ii) formaldehyde,
aq. HCl; (iii) p-toluenesulfonyl chloride, NEt3; (iv) Cs2CO3.
groups were chosen for the recognition modules in the oligomer
design shown in Fig. 3. The backbone of the oligomer in Fig. 3
contains no H-bond donors, and the backbone aniline nitrogen
and aromatic ether oxygen atoms are both weak H-bond
acceptors (the H-bond acceptor parameter b z 4 and 3
respectively) compared with phosphine oxide (b z 10).15
Competition of the backbone with the recognition sites will
therefore be negligible. The ether oxygen provides a convenient
point for addition of side-chains to control the solubility of the
oligomers (X in Fig. 3). Here we report the synthesis of phenol
and phosphine oxide oligomers from the corresponding ami-
noaldehyde monomers and NMR experiments to characterise
assembly of the corresponding duplexes.
Results and discussion
Synthesis
Scheme 2 (i) 2-Ethylhexyl bromide, K2CO3; (ii) p-toluenesulfonic acid,
ethylene glycol; (iii) H2, Pd/C; (iv) heat; (v) NaBH4.
The recognition modules were derived from p-hydrox-
ybenzaldehyde (Scheme 1). The phenol hydroxyl group was
protected as the silyl ether 1 for the synthesis of oligomers.16
Oxidation of di-t-butylchlorophosphine in the presence of
formaldehyde gave 2, which was treated with p-toluenesulfonyl
chloride to give 3.14g Alkylation of p-hydroxybenzaldehyde with 3
gave the phosphine oxide H-bond acceptor unit, 4. Phosphine
oxide 5 was prepared by alkylation of p-cresol with 3 (Scheme
1b). 5 is a single H-bond acceptor compound (A), which was
used with p-cresol, the corresponding single H-bond donor
compound (D), for determining the strength of an intermolec-
ular phenol–phosphine oxide H-bond.
The monomer units for oligomer synthesis were prepared
according to Scheme 2. 2-Hydroxy-4-nitrobenzaldehyde was
alkylated with 2-ethylhexyl bromide to give 6, which was pro-
tected as the corresponding acetal, 7, by condensation with
ethylene glycol. Reduction of 7 gave the primary aniline 8.
Oligomers were prepared by building a chain starting from 6
(Scheme 3). Reductive amination of 6 with the monomer units
(9 or 10) using NaBH(OAc)3 gave the 1-mer chains, 11 and 12.17
The acetal groups were then deprotected using aqueous HCl,
and the resulting aldehydes were coupled with a further
monomer unit to give the 2-mers, 13 and 14. 14 is the double H-
bond acceptor AA, and deprotection of 13 using tetra-n-buty-
lammonium uoride (TBAF) gave 15, the corresponding double
H-bond donor DD. The same deprotection-coupling sequence
shown in Scheme 3 was used to prepare the 3-mers, 17 (AAA)
and 18 (DDD), and the 4-mers, 20 (AAAA) and 21 (DDDD).
Binding studies
Formation of the corresponding imines using 1 or 4 and Complexation of length-complementary oligomers was studied
reduction with sodium borohydride gave the required mono- using 1H and 31P NMR titration experiments in toluene. The
mers 9 and 10.
chemical shis of the 31P and 1H NMR signals observed for the
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Scheme 3 (i) 9 or 10, NaBH(OAc)3; (ii) aq. HCl; (iii) TBAF.
oligomers are similar to the values observed for the monomer
units, which indicates that there is no signicant folding of the
free oligomers and that the H-bonding sites are displayed in an
accessible environment on the exible backbone. The 1H NMR
spectra of the oligomers are complicated with many overlapping
signals, due to the large number of chemically similar protons.
However, the 31P NMR spectra are much simpler, and large
changes in chemical shi were observed on titration of the
donor oligomers (DN, N ¼ 1–4) into the acceptor oligomers (AN).
All titration data t well to 1 : 1 binding isotherms, and the
association constants and limiting changes in 31P NMR chem-
ical shi are reported in Table 1 (see ESI† for details). The
complexation-induced changes in 31P NMR chemical shi are
similar for all of the signals in all of the complexes (+4–5 ppm).
A large positive change in 31P NMR chemical shi is indicative
of H-bond formation18 and the results in Table 1 imply that all
of the phosphine oxide groups are fully bound in all of the
complexes. In other words, fully H-bonded duplex structures are
formed (Fig. 4). Although the 1H NMR data are more difficult to
interpret, the patterns of complexation-induced changes in
chemical shi are also similar for all of the oligomers, which
suggests that the duplexes have similar structures (see ESI†).
The oligomer chains have directionality, so there are two
possible structures for the duplexes: parallel and antiparallel
Fig. 4 The AAAA$DDDD duplex structure: parallel or antiparallel
structures are possible.
(Fig. 4). The NMR data do not allow us to determine whether
one of these is preferred over the other.
Fig. 5 shows the association constants for duplex formation
(KN) plotted as a function of the number of H-bonding sites N:
the data t well to a straight line, and there is a uniform
increase in association constant of an order of magnitude for
each additional H-bond formed (eqn (1)).
log KN ¼ 1.0N + 1.5
(1)
This relationship implies that the exible backbone is able to
adopt an ideal geometry for duplex assembly, so that the
effective molarity (EM) for sequential H-bond formation in
zipping up the duplex is constant. The association constant for
duplex formation between two oligomers with N interaction
sites (KN) can therefore be expressed as the product of stepwise
equilibrium constants (cf. Fig. 2) for assembly of the duplex
using a single value of EM (eqn (2)).
Table 1 Association constants (KN), effective molarities (EM) and
limiting complexation-induced changes in 31P NMR chemical shift for
formation of duplexes in toluene at 298 Ka
Complex
log KN/Mꢀ1
EM/mM
K EM
Dd/ppm
A$D
AA$DD
AAA$DDD
AAAA$DDDD
2.5 ꢁ 0.1
3.3 ꢁ 0.1
4.2 ꢁ 0.1
5.4 ꢁ 0.5
—
—
4.9
5.3, 5.3
4.8, 4.9, 5.1
3.8, 5.1, 5.1, 5.1
8 ꢁ 3
14 ꢁ 1
21 ꢁ 8
3 ꢁ 1
5 ꢁ 1
7 ꢁ 1
KN ¼ 2KNEMNꢀ1
(2)
a
Each titration was repeated at least twice, and the average values are
reported with errors (in brackets) at the 95% condence limit.
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Fig. 6 Experimental 31P NMR chemical shift plotted as a function of
temperature for 1 : 1 mixtures (1 mM) of A$D (black), AA$DD (blue), and
AAA$DDD (red) in toluene. The lines are the best fit to eqn (9) (total
rmsd < 0.2 ppm), and the horizontal bars show the transition melting
Fig. 5 Relationship between the association constants for duplex
formation in toluene at 298 K (KN) and the number of recognition
modules in the oligomer (N). The best fit straight line is shown (r2
¼
0.99).
temperatures, Tm,N
.
where the statistical factor of 2 assumes that the parallel and
antiparallel structures in Fig. 4 are equally populated.
This relationship was used to calculate the values of EM in
Table 1. Using the A$D association constant as K in eqn (2),
gives an average value of 14 mM for the EM for duplex assembly.
The chelate cooperativity associated with duplex assembly is
quantied by the product K EM, and the data in Table 1 give an
average value of 5. This value implies that duplex assembly is
cooperative and that it should be possible to propagate the
assembly of longer structures using this architecture. However,
each H-bond is about 80% bound on average, so the duplex
structures are dynamic with some fraying of the interaction
sites.
data to a two-state model,19 which assumes only duplex and
single strands are present. At a given temperature T, the equi-
librium constant for formation of a duplex between two oligo-
mers with N binding sites, KN(T), is given by eqn (3).
½DN $AN ꢂ
KN ðTÞ ¼
(3)
½DN ꢂ½AN ꢂ
where [DN] and [AN] are the concentrations of single strand
oligomers bearing donor groups and acceptor H-bonding
groups respectively, and [DN$AN] is concentration of the
duplex.
If the two oligomers are present in equal concentrations, the
total concentration of all free strands can be written as c and the
total fraction of all bound species as a (eqn (4)).
Thermal denaturation experiments
2a
KN ðTÞ ¼
(4)
2
Thermal denaturation data were measured by making 1 : 1
solutions of length-complementary oligomers at
cð1 ꢀ aÞ
1 mM
concentrations in toluene and measuring 31P NMR spectra at
temperatures from 228 to 373 K. At low temperatures, the 31P
NMR signals moved to higher chemical shi, indicative of an
increase in the extent of H-bond formation (Fig. 6).18 At higher
temperatures, a decrease in 31P NMR chemical shi was
observed, indicative of disruption of the H-bonding interac-
tions. When a sample of phosphine oxide 5 was monitored over
the same temperature range in the absence of a H-bond donor,
the variation in chemical shi (z1 ppm) was small relative to
the changes observed for the H-bonded complexes (>4 ppm),
conrming that the temperature dependence of the 31P spectra
in Fig. 6 is related to duplex assembly and denaturation. The
signals in the 31P NMR spectra of the AAAA$DDDD duplex
become very broad and overlapped with changes in tempera-
ture, so it was not possible to extract an accurate melting prole
for this system. However, the data for AAAA$DDDD are quali-
tatively consistent with a more stable duplex that melts at
higher temperatures than the other three complexes (see ESI†).
The thermal denaturation experiment can be used to extract
thermodynamic parameters for duplex formation by tting the
Rearranging eqn (4) gives an expression for a in terms of
KN(T) and c (eqn (5)).
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
KN ðTÞc þ 1 ꢀ 2KN ðTÞc þ 1
a ¼
(5)
KN ðTÞc
The observed chemical shi for a two state system in fast
exchange, d, can be expressed in terms of a by eqn (6).
d ¼ df + (db ꢀ df)a
(6)
where db and df are the chemical shis of the duplex and single
strand states.
Assuming that the enthalpy and entropy of duplex formation
are temperature independent, and the change in heat capacity
between free and bound states is zero, the temperature depen-
dence of KN(T) can be analysed using the integrated form of the
van't Hoff equation (eqn (7)).19
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ꢀ
ꢁ
ꢂ
ꢃ
denaturation data are reasonable. The experimental data in
Fig. 6 clearly follow the theoretical sigmoidal curve corre-
sponding to the melting transition. The free-bound transitions
occur over narrower temperature ranges with increasing N, and
the point of inection occurs at higher temperatures. These
visual observations are conrmed by the calculated parameters
shown in Table 2, which show that the increased binding
constants observed for higher values of N are associated with an
increase in the enthalpy change on duplex formation and with
an increase in the transition melting temperature. These
features are characteristic of cooperative interactions between
the H-bonding sites along the duplex.10
KN ðTÞ
DHNꢃ
1
1
ln
¼ ꢀ
ꢀ
(7)
Km;N ðTÞ
R
T
Tm;N
where KN(Tm,N) is the association constant for formation of the
DN$AN duplex at the transition melting temperature, Tm,N
,
DHꢃN is the enthalpy change for formation of the DN$AN duplex,
and R is the gas constant.
At the transition melting temperature a ¼ 0.5, and using this
value in eqn (4) gives KN(Tm,N) ¼ 4/c. Substituting for KN(Tm,N) in
eqn (7) and rearranging gives eqn (8), the temperature depen-
dence of the association constant KN(T).
ꢀ
ꢁ
ꢄ
ꢅ
DHNꢃ
1
1
Tm;N
ꢀ
ꢀ
R
T
4
KN ðTÞ ¼
e
(8)
c
Conclusions
We present a general strategy for the design of synthetic infor-
mation molecules that could potentially encode and replicate
chemical information in the same way as nucleic acids. A series
of H-bond donor (phenol) and H-bond acceptor (phosphine
oxide) oligomers (DN and AN, N ¼ 1–4) have been synthesised
using reductive amination chemistry. The assembly of
duplexes, AN$DN, was characterised using NMR titrations and
thermal denaturation experiments in toluene. The stability of
the duplex increases by one order of magnitude for every H-
bonding group added to the chain. Similarly, the enthalpy
change for duplex assembly and the melting temperature for
duplex denaturation both increase with increasing chain
length. Although the oligomers have a relatively exible back-
bone, this lack of preorganisation does not signicantly impede
assembly of the duplex, and H-bond formation along the olig-
omers is cooperative. The effective molarity for intramolecular
H-bond formation (EM ¼ 14 mM) appears to be sufficient to
propagate the formation of long duplexes using this approach.
The product K EM, which is used to quantify chelate coopera-
tivity is 5, which means that each H-bond is more than 80%
populated in the assembled duplex. The modular design of
these compounds should allow us to explore variations in the
recognition and backbone linker modules to optimise the
properties of the system for selective recognition of mixed
sequence oligomers and template-directed synthesis.
Combining eqn (5), (6) and (8), allows us to express the
observed chemical shi in terms of T.
À
Á
d ¼ df þ db ꢀ df
0
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1
ꢀ
ꢁ
ꢀ
ꢁ
ꢄ
ꢅ
ꢄ
ꢅ
DHNꢃ
DHNꢃ
1
T
1
Tm;N
1
T
1
Tm;N
ꢀ
ꢀ
ꢀ
ꢀ
B
B
B
B
B
@
C
C
C
C
C
A
R
R
1 þ 4e
ꢀ
1 þ 8e
ꢄ
ꢀ
ꢁ
ꢄ
ꢅ
DHNꢃ
1
T
1
Tm;N
ꢀ
4e
ꢀ
R
(9)
The thermal denaturation data for duplexes A$D, AA$DD
and AAA$DDD were analysed by optimising the values of db,
df, DHꢃN and Tm,N to minimise the difference between the
experimentally measured values of d and the values calcu-
lated using eqn (9). The values of db and df could depend on N,
but the limiting chemical shis determined by tting the
titration data recorded at 298 K are similar for all four
duplexes (df ¼ 53.6–53.9 ppm, and db ¼ 57.9–59.0 ppm, see
ESI†). We therefore t the thermal denaturation data using
the same value of db and df for all of the duplexes, and the
results (df ¼ 52.8 ppm, and db ¼ 58.9 ppm) are consistent
with the titration data. Fig. 6 shows the lines of best t
calculated using eqn (9) (solid lines) for each thermal dena-
turation data set, and Table 2 shows the values of the tted
parameters along with the values of DSꢃN and DGNꢃ calculated
from log KN at 298 K.
Acknowledgements
The values of log KN at 298 K in Table 2 are similar to the
corresponding values from the titration data (Table 1), which
suggests that the assumptions used in tting the thermal
We thank the EPSRC and ERC for funding.
Notes and references
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Table 2 Thermodynamic parameters for formation of H-bonded
duplexes in toluene determined using 31P NMR thermal denaturation
data
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