An Environmentally Responsive Reciprocal Replicating Network

Article abstract of DOI:10.1021/jacs.7b13576

A reciprocal replication system is constructed from four building blocks, A, B, C, and D, which react in a pairwise manner through either a 1,3-dipolar cycloaddition or the condensation reaction between an amine and an aldehyde to create two templates, tr

Full text of DOI:10.1021/jacs.7b13576

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Article
An environmentally-responsive reciprocal replicating network
Craig C. Robertson, Harold W Mackenzie, Tamara Kosikova, and Douglas Philp
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13576 • Publication Date (Web): 09 May 2018
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An environmentally-responsive
reciprocal replicating network
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‡
†
Craig C. Robertson, Harold W. Mackenzie,
Tamara Kosikova and Douglas Philp*
School of Chemistry and EaStCHEM, University of St Andrews,
North Haugh St Andrews, Fife KY16 9ST, United Kingdom
*Corresponding author e-mail: d.philp@st-andrews.ac.uk
Fax: +44 (0)1334 463808; Tel: +44 (0)1334 467264.
ORCID IDs:
CCR:
HWM:
TK:
orcid.org/0000-0002-1419-9618
orcid.org/0000-0001-7575-2790
orcid.org/0000-0001-7886-9660
orcid.org/0000-0002-9198-4302
DP:
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Abstract
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A reciprocal replication system is constructed from four building blocks, A, B, C, and D,
which react in a pairwise manner through either a 1,3-dipolar cycloaddition or the
condensation reaction between an amine and an aldehyde to create two templates, trans-
TAB and TCD. These templates are equipped with complementary recognition sites—two
carboxylic acids (trans-TAB) or two 4,6-dimethylamidopyridines (TCD)—that enable each
template to direct the formation of its complementary partner through two mutually-
reinforcing crosscatalytic pathways, in which the templates trans-TAB or TCD preorganize
the appropriate building blocks within two catalytically-active ternary complexes:
[C•D•trans-TAB] and [A•B•TCD]. The template-directed processes within these complexes
generate a heteroduplex [trans-TAB•TCD], which is shown to possess significant stability
through kinetic simulation and fitting. As a consequence, the individual crosscatalytic
pathways perform more efficiently in template-directed experiments when the
concentration of the template being formed is below that of the template added as
instruction. Comprehensive analysis of the system when A, B, C, and D are mixed
together directly, using a series of 1H NMR spectroscopic kinetic experiments,
demonstrates that the behavior of the reciprocal system is more than the simple sum of its
parts—as part of the interconnected network, the product of each reaction clearly directs
the fabrication of its reciprocal partner, facilitating both higher rates of formation for both
templates and improved diastereoselectivity for trans-TAB. A simple change in
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experimental conditions (from dry to ‘wet’ CDCl ) demonstrates the sensitivity of the
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replication pathways within the network to the reaction environment, which leads to a
>10-fold increase in the contribution of a new minimal self-replicator, trans-TAB*, to the
replication network.
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Introduction
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Networks constructed from individual components that interact and react with
each other in pre-defined ways are a common feature at almost every length scale in the
world around us—from the human immune system to patterns of weather in the
atmosphere. The functions and behaviors expressed by these networks at a whole system
level are derived from the interconnections1 between their individual components. In the
last 20 years, there has been significant growth in many disciplines1,2 in the study of
complexity and complex systems,3 and the properties4 that can emerge through the
interactions of their constituent components. In chemistry, the nascent field5 of systems
chemistry has started to create synthetic chemical entities that can be assembled into
complex networks that exhibit system-level behaviors4a,b,g,5b,6 and functions by exploiting a
bottom-up approach. Central to these strategies are the processes7 that surround molecular
replication. The auto- and crosscatalytic properties of synthetic replicators, coupled with
their highly specific self- and mutual recognition properties, make them ideal building
blocks from which to construct complex networks. Proponents of the systems chemistry
approach have to date produced an array of replicating systems based on
oligonucleotides,8 peptides,9 and small synthetic molecules.10 These studies demonstrate
clearly that replication is not the sole preserve of nucleic acids augmented by extensive
enzymatic machinery. These replicators have been embedded within systems that can
exhibit error-correction,11 stereospecific replication,12 Boolean logic,13 as well as the
capacity to respond to the reaction environment.9g,h,j,10c,13d,14 The vast majority of synthetic
replicators described to date rely on self-complementary templates, especially those based
on simple organic compounds. In these so-called minimal replicating systems, a template
that is complementary to itself directs its own formation through an autocatalytic cycle. It
is, however, possible to construct a replicating system from two mutually complementary
templates in which the catalytic effects are reciprocal. In such systems, each of the two
non-identical, but mutually-complementary, templates directs the formation of its partner
through two interlinked crosscatalytic cycles. The processes involved in reciprocal
replication are encapsulated schematically in Figure 1.
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The prototypical reciprocal replicating system (Figure 1) can be constructed from
two sets of reagents, each bearing complementary recognition and reactive sites. In the
system shown in Figure 1, component A can react only with B and component C can react
only with D. The bimolecular reactions of these two pairs of reagents create two templates,
TAB and TCD (Figure 1). These templates do not have the necessary self-complementary
recognition sites required for self-replication. Instead, the reciprocal nature of the non-
covalent interactions between the two templates allows the TCD template to crosscatalyze
the formation of TAB through the assembly of the catalytically-active ternary complex
[A•B•TCD]. In the same manner, template TAB is able to crosscatalyze the formation of TCD
through the assembly of the catalytically-active ternary complex [C•D•TAB].
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Figure 1 Two templates, TAB and TCD, containing complementary recognition sites (light and dark gray) are
formed initially through the bimolecular reactions between A and B (blue) and between C and D (red). Once
formed, these templates can participate in two template-directed crosscatalytic cycles, where TAB catalyzes
the formation of TCD through the assembly of C and D in a catalytically-active ternary complex [C•D•TAB],
and vice versa. However, neither template TAB nor TCD possess the recognition sites required for self-
replication. Taken together, these two crosscatalytic cycles (TAB → TCD and TCD → TAB) represent a formal
reciprocal replication cycle.
Ultimately, these processes afford the template heteroduplex [TAB•TCD] that can
dissociate to return one molecule each of TAB and TCD back to the start of the respective
catalytic cycles. These two interlinked cross-catalytic cycles represent a formal reciprocal
replication cycle. It is important to note that the complexity of this system can increase
dramatically depending on the nature of the chemical reactions employed to create the
two templates from their constituents. In the case where the reaction between A and B and
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the reaction between C and D are chemically orthogonal, only the reciprocal replication
cycle discussed above is present. However, if the system loses this chemical orthogonality
and reaction between, for example, A and C becomes possible, then additional minimal
replication cycles can emerge through the formation of self-complementary templates.
Although several replicating networks involving oligonucleotides and peptides that
participate in crosscatalytic interactions have been described previously,8b,c,f,9a,d,h,11,14a
examples that are based on small, synthetic organic molecules are somewhat rare. Both the
Rebek15 laboratory and our own16 have reported examples of systems that exhibit
reciprocal template effects based on small organic molecules. However, in both these
cases, a lack of orthogonality between the chemical ligation reactions used to construct the
templates allowed the systems15a,16a to create selfish, autocatalytic templates and their
presence diminished the overall importance of reciprocal replication in these systems. In
order to create a purely reciprocal replicating system, unwanted reactivity between
replicator components must be eliminated. In order to achieve this goal, we developed an
orthogonal reactivity strategy for the creation of the two complementary templates shown
in Figure 2.
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Critical elements in replicator design are the recognition elements that drive the
catalytic processes within the replication cycle. We have demonstrated
previously10a-c,e,f,13c,16,17 that hydrogen bonding between an amidopyridine and a carboxylic
acid can be applied to direct autocatalytic processes. Accordingly, maleimide A and
nitrone B are equipped with carboxylic acid recognition sites. Amine C and aldehyde D
are equipped with complementary amidopyridine recognition sites, ensuring that
templates TAB and TCD are mutually complementary. The association constant (K
these recognition partners, determined18 by 1H NMR spectroscopic titration, is
3470 ± 300 M−1 in CDCl
at 283 K.
a
) for
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A key design element in our strategy is the use of the 1,3-dipolar cycloaddition
between maleimide and nitrone (Figure 2). This reaction has been applied successfully in
our laboratory10a–c,e,13c,16,17a,b to create several minimal replicating systems. Previously, we
have also shown19 that imine formation between an aniline and an aromatic aldehyde can
proceed through a recognition-mediated pathway under conditions similar to those
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required for the 1,3-dipolar cycloaddition reaction. We therefore designed the system
shown in Figure 2, in which we expected the reaction between maleimide A and nitrone B,
which forms TAB, to be chemically orthogonal to the reaction between amine C and
aldehyde D, which forms imine TCD.
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O
O
O
O
O
N
OH
N
OH
N
N
N
N
O
O
H
H
O
NH2
H
15 C7
AB CD
C8H17
[trans-T •T
]
B
A
C
D
HO
O
O
O
OH
H
H
H
H
15 C7
N
N
O
O
AB
CD
trans-T
T
C8H17
O
O
HO
O
O
N
HN
O
O
NH
N
OH
H
H
H
N
AB
cis-T
H
15 C7
N
N
C8H17
O
(a)
(b)
‡
Figure 2 Four building blocks, A, B, C, and D, react in a pairwise manner to give two templates, trans-TAB
and TCD, which are equipped with mutually complementary recognition sites (carboxylic acid and 4,6-
dimethylamidopyridien) that enable these templates to participate in two crosscatalytic pathways mediated
by the formation of two catalytically active complexes: [C•D•trans-TAB] and [A•B•TCD]. Calculated
structures of (a) the transition state (‡) accessed by [A•B•TCD] and (b) the hemiaminal intermediate on the
pathway to the corresponding template heteroduplex [trans-TAB•TCD]. Calculations performed at the
ωB97X/def2-SVP level of theory using the polarized continuum solvation model for chloroform. Hydrogen
bonds are represented using dashed blacked lines. Carbon atoms are colored gray, nitrogen atoms blue,
oxygen atoms red, and hydrogen atoms white. Most hydrogen atoms are omitted for clarity.
The 1,3-dipolar cycloaddition reaction between A and B creates two
diastereoisomeric products, trans-TAB and cis-TAB. In order to understand the influence that
template TCD might have on this process, we conducted a series of DFT calculations at the
ωB97X/def2-SVP level of theory using a continuum solvation model for CHCl . These
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calculations reveal (Figure 2) that TCD is highly complementary to trans-TAB. Further, the
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transition state leading to trans-TAB (Figure 2a) is accommodated on template TCD without
significant distortion, suggesting that imine TCD should be capable of catalyzing the
formation of trans-TAB. By contrast, cis-TAB possesses a folded structure that is not at all
complementary with TCD. This observation suggests that TCD would be incapable of
catalyzing the formation of cis-TAB and that the overall formation of TAB should proceed
diastereoselectively in the presence of TCD. Calculations also demonstrate that trans-TAB is
capable of accommodating the hemiaminal intermediate (Figure 2b), associated with the
formation of imine TCD without significant distortion, suggesting that trans-TAB may also
be capable of catalyzing the formation of imine TCD. Taken together, these results suggest
that a reciprocal replication cycle can be established using trans-TAB and TCD.
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Results and discussion
In order to identify the presence of the crosscatalytic pathways and to assess their
efficiencies, the pairwise reactions of maleimide A with nitrone B and aniline C with
aldehyde D were examined first in isolation (e.g., A and B were reacted in solution in the
absence of either C and D or preformed template). Subsequently, the reactions of the two
pairs of reagents were analyzed in the presence of the relevant reciprocal template (e.g.,
reaction of A with B was examined in the presence of TCD and vice versa). In each kinetic
experiment, an equimolar solution of the required reagents (10 mM) was prepared in
CDCl
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and the appearance of products was monitored at regular intervals at 10 °C over
1
15 h by 500 MHz H NMR spectroscopy. The concentrations of the products were
determined by deconvolution of the resonances arising either from protons in the bicyclic
ring system present in TAB in the range δ 4.5 to 6.0 or the resonance arising from the imine
proton in TCD around δ 8.2 in comparison to appropriate resonances arising from the
starting materials.
In isolation, the reaction between maleimide A and nitrone B (Figure 3a, filled
circles) proceeds slowly and with limited diastereoselectivity, reaching an overall
conversion of 23% after 15 h, with the concentrations of trans-TAB and cis-TAB reaching
1.74 mM and 0.58 mM, respectively ([trans-TAB]:[cis-TAB] = 3:1). Likewise, the reaction
between aniline C and aldehyde D (Figure 3b, filled circles) afforded imine TCD, which
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reached a concentration of 0.68 mM after 15 h at 10 °C (7% conversion). Imine formation is
a reversible process, and imine condensation is therefore expected to reach equilibrium
after a certain time. After 14 days, the condensation between aniline C with aldehyde D
had reached equilibrium at ca. 35% conversion to imine TCD. These results confirm that, in
the absence of an appropriate reciprocal template, both trans-TAB and TCD are formed
slowly, and, in the case of trans-TAB, with low diastereoselectivity.
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Figure 3 (a) Maleimide A reacts with nitrone B to give two diastereoisomeric products, trans-TAB (blue) and
cis-TAB (black) in the absence of template (filled circles) and in the presence of 20 mol% (open circles) and 46
mol% (open squares) of preformed TCD. (b) Aniline C reacts with aldehyde D to give TCD (red) in the absence
of template (filled circles) and in the presence of 20 mol% of preformed trans-TAB (open circles). The full lines
represent the best fits of the appropriate kinetic models to the experimental data (see Supporting
Information). Reaction conditions: [A] = [B] or [C] = [D] = 10 mM, 10 °C, CDCl . The reaction profiles were
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determined by deconvolution of 500 MHz H NMR spectroscopic data; all concentrations have been
corrected for the amount of template added.
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In order to examine the capacity of imine TCD to function as a template for the
formation of trans-TAB, the reaction between maleimide A and nitrone B was repeated in
the presence of 20 mol% of preformed TCD added at t = 0 h. The concentration vs. time
profile for this reaction (Figure 3a, open circles) is strikingly different to that for the
uninstructed process. The production of trans-TAB (open blue circles) is enhanced
significantly. At t = 2 h, trans-TAB reaches a concentration of 2.52 mM, i.e., a value that
exceeds the amount of trans-TAB produced after 15 h in the absence of instruction by TCD.
After 15 h, the concentrations of trans-TAB and cis-TAB had reached 4.72 mM and 0.75 mM,
respectively, an overall conversion to both diastereoisomers of 55% ([trans-TAB]:[cis-TAB] =
6.3:1).
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The reaction between maleimide A and nitrone B was also examined in the
presence of 46 mol% of preformed TCD added at t = 0 h. Again, the concentration vs. time
profile for this reaction (Figure 3a, open squares) displays a significant enhancement in the
formation of trans-TAB when compared to the uninstructed process. The production of
trans-TAB (open blue squares) is enhanced significantly. At t = 2 h, trans-TAB reaches a
concentration of 5.19 mM, a value that exceeds the amount of trans-TAB produced after 15
h in the presence of 20 mol% of TCD. After 15 h, the concentrations of trans-TAB and cis-TAB
had reached 7.08 mM and 0.35 mM, respectively, an overall conversion to both
diastereoisomers of 74% ([trans-TAB]:[cis-TAB] = 20.3:1). These results mirror the prediction
from our DFT calculations, which suggested that TCD should be an effective template for
the formation of trans-TAB, resulting in an increase in the rate and diastereoselectivity for
this reaction. By contrast, the conversion to cis-TAB exhibited no significant change in the
presence of TCD. Taken together, these observations verify our hypothesis that the imine
TCD can direct the formation of trans-TAB, but not cis-TAB.
In order to examine the efficiency of the second crosscatalytic pathway, the
condensation reaction between aniline C and aldehyde D was performed in the presence
of 20 mol% of preformed trans-TAB. The concentration–time profile for this reaction
(Figure 3b, open circles) displayed an enhancement in the rate of formation of TCD. After
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2 h, the concentration of imine TCD reached 1.36 mM. Even at this early reaction time, the
concentration of TCD formed in this reaction exceeded that created in the absence of
reciprocal template. After the initial period of ca. 3 h, the formation of TCD proceeded at a
slower rate, reaching a concentration of 3.47 mM after 15 h, which represents an overall
conversion of 35%. Clearly, the marked changes observed in the rate and extent of
formation of imine TCD in the presence of trans-TAB confirm the efficiency of the second
crosscatalytic pathway required for the establishment of the fully operational reciprocal
system. Whilst it would be desirable to explore the effect of different concentrations of TAB
on the formation of TCD more fully, unfortunately, the limited solubility of trans-TAB in
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CDCl , arising from the presence of two carboxylic acids, precluded the examination of
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this crosscatalytic pathway at higher loadings of trans-TAB template.
In order to quantify the changes observed in the two crosscatalytic pathways
following the introduction of the reciprocal templates, we fitted the experimental data
shown in Figure 3 to the appropriate kinetic models (for details, see the Supporting
Information, Section S5). The data gave excellent fits (Figure 3, solid lines) to these models
and allowed us to extract the key kinetic and thermodynamic parameters for the two
control (uninstructed) and two template-directed pathways. Specifically, employing
models that describe the key interactions and reactions leading to the formation of trans-
TAB and TCD, we were able to determine the bimolecular rate constants (kbi) for the
formation of trans-TAB and cis-TAB, the unimolecular rate constant (kcat) for the formation of
trans-TAB on TCD template (via the ternary complex [A•B•TCD]) and the heteroduplex
association constant (K Heteroduplex
) (Table 1). In addition, were able to determine the rates of
a
the forward20 reaction to give TCD, both in the absence and presence of trans-TAB as
instruction (Table 1). These parameters were used subsequently to calculate (Table 1) the
kinetic21,22 and thermodynamic23 effective molarities (EMkinetic and EMthermo, respectively) for
the two template-directed crosscatalytic pathways.
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Table 1 Overview of rate constants determined for the bimolecular and template-directed cycloaddition
reaction and imine formation to give trans-TAB and TCD, respectively. The heteroduplex association constant
(K Heteroduplex
a
) was estimated to be 3.35 × 107 M–1. The kbi for the formation of cis-TAB was determined to be
1.40 × 10–4 M–1 s–1.
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Cycloaddition Reaction
k
EMkinetic / M
Bimolecular kbi / 10–4 M–1 s–1
Templateꢀdirected kcat / 10–4 s–1
A + B → transꢀTAB
4.20 ± 0.01
21.1 ± 0.02
—
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[A•B•TCD] → [transꢀTAB•TCD
]
5.02
Imine Formation
C + D → TCD
k
EMkinetic / M
Bimolecular kbi / 10–4 M–1 s–1
Templateꢀdirected kcat / 10–4 s–1
1.61 ± 0.00
—
[C•D•transꢀTAB] → [transꢀ
5.55 ± 0.002
3.44
TAB•TCD
]
Examination of the parameters summarized in Table 1 reveals that, in the presence
TCD, the formation of trans-TAB is associated with an EMkinetic of 5.02 M, whereas, the
formation of TCD (via the key catalytically-active complex [C•D•trans-TAB]) is associated
with an EMkinetic of 3.44 M. These results demonstrate that the mutually complementary
templates trans-TAB and TCD are capable of accelerating the formation of each other
efficiently via the two crosscatalytic template-directed pathways.
A key element in this system is the heteroduplex [trans-TAB•TCD] (Figure 2)—the
stability of this duplex controls the availability of the catalytically-active templates in
solution. This duplex is expected to be very stable and an assessment of its stability was
not possible directly24 using H NMR spectroscopic methods. From our kinetic simulation
1
and fitting, we were able to confirm that the [trans-TAB•TCD] complex is, indeed, very
stable, having an association constant (K
Heteroduplex) that is in excess 107 M−1. This association
a
constant corresponds to a connection EM25 (EMthermo) of 2.8 M indicating that there is a high
level of complementarity between these two templates. The direct consequence of the
stable [trans-TAB•TCD] complex is the apparent biphasic character of the kinetic profiles for
the template-directed experiments shown in Figure 3 (blue and red open symbols). For
example, when the formation of trans-TAB is being instructed by TCD, as long as the
concentration of trans-TAB is below that of TCD added initially, there must always be
unbound TCD available to accelerate the formation of trans-TAB. This situation arises as a
result of the maximum concentration of the tightly bound complex [trans-TAB•TCD] being
determined by the concentration of trans-TAB formed. However, once the concentration of
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trans-TAB exceeds that of TCD, the efficiency of the system is reduced dramatically as the
stability of the [trans-TAB•TCD] heteroduplex now controls the availability of free TCD in
solution. This effect can be seen clearly in the data shown in Figure 3a—the change in
reaction rate occurs at [trans-TAB] = 2 mM when 20 mol% of TCD is added and at about 4.5
mM when 46 mol% of TCD is added. An identical argument can be constructed for the
reverse catalytic situation where TCD is constructed on trans-TAB.
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Having characterized the individual pathways present in the reciprocal system, we
next set out to combine the four starting materials within a single experiment. In this
situation, we envisaged that the in situ formation of trans-TAB would catalyze the
formation of imine TCD and vice versa, allowing the formation of a reciprocal, mutually
reinforcing replicating system. To this end, an equimolar mixture (10 mM) of A, B, C, and
D was prepared in CDCl
and the formation of TAB and TCD (Figure 4a) was monitored at
3
10 °C over 40 h using 500 MHz 1H NMR spectroscopy.
Examination of the concentration-time profiles of the products formed from the
four-component system (Figure 4a) reveals that the major products are the two reciprocal
templates trans-TAB and TCD. The data reveals the presence of a small initial lag period for
the formation of trans-TAB and a more pronounced initial lag period in the formation of
TCD. The presence of the sigmoidal concentration-time profiles for both templates is
directly related to the processes and reactions inherent (Figure 1) in mutually
crosscatalytic reciprocal replication. Specifically, at the start of the reaction, the reciprocal
templates can only be formed through the uncatalyzed bimolecular pathways. The rates at
which trans-TAB and TCD are formed start to increase once the template-mediated processes
begin to operate efficiently, i.e., when the concentration of each template exceeds the value
of K for the binding of the building blocks, A, B, C, and D, to the appropriate template,
d
forming the catalytically-active ternary complexes. Finally, the depletion of the starting
materials eventually leads to a decrease in the rates at which the two reciprocal templates
are produced.
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Figure 4 A reciprocal replication network is constructed by the reaction of maleimide A, nitrone B, aniline C,
and aldehyde D to give two diastereoisomeric products, trans-TAB (blue) and cis-TAB (black), and imine TCD
(red) (a) in the absence of any added template as instruction, and in the presence of (b) 30 mol% of
preformed trans-TAB, or (c) 30 mol% of preformed TCD. The impact of the added template on the formation of
trans-TAB and TCD is demonstrated by comparison of the concentration ratios calculated at t = 5 h and 40 h,
and the ratios of initial rates for the formation of the templates. Reaction conditions: [A] = [B] = [C] = [D] = 10
1
mM, 10 °C, CDCl
3
. The reaction profiles were determined by deconvolution of 500 MHz H NMR
spectroscopic data; all concentrations have been corrected for the amount of template added. A description
of the error analysis is provided in the Supporting Information.
Comparison of this experiment with the uninstructed formation of trans-TAB or TCD
in isolation (data in Figure 3, filled circles) reveals striking differences in behavior. In the
case of trans-TAB, the overall conversion to this product in the four-component system after
5 h (Figure 4a) was 20%, with the diastereoselectivity being 6.3:1 in favor of trans-TAB.
These values can be compared with those from the data in Figure 3a at t = 5 h—
conversion = 7% and a diastereoselectivity of only 3:1 in favor of trans-TAB. Similarly, the
condensation of C and D to form imine TCD was also enhanced within the four-component
system—the conversion of C and D to TCD reached 11% after 5 h (Figure 4a) in comparison
to <3% in the data shown in Figure 3b after the same time. These results demonstrate the
mutually-reinforcing effect of the two crosscatalytic cycles when they operate together
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within a shared environment and these template-directed replication processes enhance
both the rates of formation of trans-TAB and TCD and the diastereoselectivity for the
formation of trans-TAB. This situation is in direct contrast to the conditions used to acquire
the data presented in Figure 3. In these experiments, the concentration of the instructing
template remains constant throughout the entire duration of the experiment, since only
two out of the four possible building blocks are present at a time. By contrast, the data in
Figure 4 relates to experiments where all four building blocks are present and,
consequently, the concentrations of the instructing templates are variable across the time
course of the experiments. Thus, the comparison between the data shown in Figure 3 and
Figure 4 must be limited to a qualitative assessment evidencing the nature of the mutually
reinforcing nature of the reciprocal relationship between replicators TAB and TCD.
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In order to demonstrate the importance of template effects in this reciprocal
replication system, two template-instructed experiments were performed. Firstly, the four
reagents A to D were reacted at starting concentrations of 10 mM in CDCl at 10 °C in the
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presence of 30 mol% of preformed trans-TAB added at t = 0 h. These data (Figure 4b) show
a rapid increase in the concentration of imine TCD, the initial rate of formation of TCD is
1.01 mM h–1 (compared to 0.20 mM h–1 in the absence of template), with the concentration
of TCD reaching 4.48 mM after 5 h. After this point, the rate of formation of TCD slows
significantly and the concentration of TCD only increases a further 1.76 mM to 6.24 mM at
the end of the experiment after 40 h. In order to make comparisons with the uninstructed
data, we calculated the ratios of template concentrations (at 5 h and 40 h) and initial rates,
as shown in Figure 4. These ratios will take values that are >1 when enhancement occurs in
the template-instructed experiment and values <1 when reduction in a given parameter is
observed. Comparing the data in Figure 4b with that obtained under the same conditions
in the absence of added trans-TAB (Figure 4a) reveals some significant differences. The
initial rate ratio for the formation of TCD in the presence of trans-TAB is 5.18, and the
concentration ratio for TCD after 5 h is 4.14. By contrast, at 40 h, the concentration ratio for
TCD is 0.92. These observations indicate that the most significant effects arising from the
addition of template are present at early reaction times. This result is entirely consistent
with the behavior observed in the experiments where TCD is formed in isolation (data in
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Figure 3b). Once the concentration of TCD becomes comparable to that of the instructing
template, product inhibition resulting from the extremely stable [trans-TAB•TCD] duplex
becomes progressively dominant in the system. As trans-TAB does not catalyze directly its
own formation, it may seem counterintuitive to expect that the addition of trans-TAB to the
system will affect the rate and extent of formation of itself. Nevertheless, this experiment
reveals that the addition of trans-TAB does indeed have an effect on the rate of its own
formation as a result of the reciprocal nature of the entire system. The initial rate of
formation of trans-TAB = 0.71 mM h–1 (compared to 0.40 mM h–1 in the absence of template),
with the concentration of trans-TAB reaching 3.25 mM after 5 h. Once again after this point,
the rate of formation of trans-TAB slows and the concentration of trans-TAB increases a
further 4.20 mM to 7.45 mM at the end of the experiment after 40 h. Despite the fact that
trans-TAB does not template its own formation, the increase in the rate of production of
TCD, engendered by the addition of trans-TAB as an instruction, results in an initial rate
ratio for the formation of trans-TAB of 1.76 by virtue of its crosscatalytic connection to TCD.
This enhancement in the rate of formation of trans-TAB also results in a dramatic increase
in the diastereoselectivity for the formation of trans-TAB after 5 h to 19.3:1. The
enhancement in the formation of TAB in the presence of TCD was also evidenced by the
significant changes in the concentration vs. time profile of TAB observed in the presence of
preformed TAB (Figure 4b) when compared to the uninstructed scenario (Figure 4a). These
changes arise as a result of the efficient crosscatalytic formation of TAB on TCD—our kinetic
analyses demonstrate that TCD is a much better catalyst for the formation of TAB (kcat = 21.1
× 10–4 s–1) than TAB is for the formation of TCD (kcat = 5.55 × 10–4 s–1).
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Secondly, the four reagents A to D were reacted at starting concentrations of 10 mM
in CDCl
at 10 °C in the presence of 30 mol% of preformed TCD added at t = 0 h. These data
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(Figure 4c) show a rapid increase in the concentration of trans-TAB, and the initial rate of
formation of trans-TAB is 1.02 mM h–1, with the concentration of trans-TAB reaching 4.91
mM after 5 h. After this point, the rate of formation of trans-TAB slows and the
concentration of this template increases a further 4.50 mM to 9.41 mM at the end of the
experiment after 40 h. Comparing the data in Figure 4c with that obtained under the same
conditions in the absence of any preformed template (Figure 4a) once again reveals some
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significant differences. The ratio of initial rates for the formation of trans-TAB is 2.56 and
the concentration ratio calculated for trans-TAB is 2.45 after 5 h. Once again, this
enhancement in the rate of formation of trans-TAB also manifests in a dramatic increase in
the diastereoselectivity for the formation of trans-TAB after 5 h to 29:1. By contrast, at 40 h,
the concentration ratio for trans-TAB has a value of 1.10, although the diastereoselectivity
for the formation of trans-TAB remains high, at 32:1. These observations once again indicate
that the most significant effects arising from the addition of the preformed template are
most significant at early reaction times. This result is again consistent with the behavior
observed in the experiments where trans-TAB is formed in isolation (data in Figure 3a)—
once the concentration of trans-TAB becomes comparable to that of the instructing template
added initially, product inhibition arising from the extremely stable [trans-TAB•TCD]
duplex becomes progressively dominant in the system. In contrast to the previous
experiment, TCD does not benefit from either an enhancement in initial rate or, indeed,
extent of its formation in the same manner as trans-TAB when it was used as the instructing
template. The concentration ratios calculated for TCD at both 5 h and 40 h are close to one
(1.02 and 0.86, respectively). The same trend is observed for the initial rate ratio for TCD,
which is found to be 0.96. This observation can be rationalized by the fact that formation of
TCD is a dynamic process and the total concentration of TCD that can be achieved is subject
to a global equilibrium condition for the system. As we have added TCD to this reaction at
an initial concentration of 3 mM, the reaction starts at a point that is inherently closer to
this global equilibrium position for the formation of TCD. As a consequence, we would
expect the formation of TCD to behave differently in this situation compared to the other
two experiments in which the initial concentration of TCD is zero.
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It is clear from these results that the behavior of the reciprocal system is more than
the simple sum of its parts. In the absence of a reciprocal template, each of the reactions
proceeds slowly, and in the case of the cycloaddition, also with poor diastereoselectivity.
As part of an interconnected network, however, the product of each reaction is clearly
capable of facilitating the fabrication of its reciprocal partner, facilitating both higher
reaction rates and diastereoselectivity.
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The effect of reaction environment on the reciprocal replicators
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The kinetic experiments described thus far confirm conclusively that the two
reciprocal templates, trans-TAB and TCD, are the dominant products that emerge from a
reagent pool that contains A, B, C and D. However, under appropriate conditions, nitrone
B, which is used in the creation of trans-TAB, is susceptible to hydrolysis. Under hydrolytic
conditions, this nitrone affords aldehyde 1 and hydroxylamine 2 (Figure 5a) and, in this
situation, hydroxylamine 2 can react with aldehyde D to afford a new nitrone B*, which is
equipped with an amidopyridine recognition site. Nitrone B* can then react in a 1,3-
dipolar cycloaddition reaction with maleimide A to form cycloadduct trans-TAB*. However,
in contrast to trans-TAB, which can, by virtue of its two identical recognition sites, act only
as a catalyst for the formation of TCD, trans-TAB* possesses recognition sites that render this
template self-complementary. As a consequence, trans-TAB* is capable26 of directing its own
formation through an autocatalytic template-directed pathway (Figure 5b). This possibility
gives rise to an interesting scenario in which the environment in which the replicators
derived from A, B, C, and D operate can promote either the reciprocal replication cycle
mediated by trans-TAB and TCD, or, alternatively, a minimal replication pathway mediated
by trans-TAB*, or, indeed, a mixture of both of these pathways. Thus far, the environmental
conditions were selected specifically to avoid hydrolysis and thereby ensure that the
reciprocal replication cycle is dominant.
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Under these conditions, the ratio of the reciprocal template trans-TAB to the minimal
replicator template trans-TAB* was 49:1. If the rate of hydrolysis of nitrone B is enhanced in
the four-component reaction, we would expect to observe an increase in the concentration
of the amidopyridine-bearing nitrone B*. In turn, the contribution of minimal replication,
mediated by the template trans-TAB*, should also increase relative to the reciprocal
replication cycle involving trans-TAB and TCD.
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(a)
(b)
O
O
O
O
N
N
HO
HO
N
N
O
N
O
H
O
B
B*
A
AB*
AB*
trans-T
trans-T
C8H17
C7H15
C7H15
H O
2
8
9
HOHN
O
1
2
D
O
HO
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N
H
N
Minimal
Autocatalytic
Cycle
O
O
C7H15
(c)
10
8
AB
trans-T
trans-T
AB*
AB*
AB*
AB*
[A•B*•trans-T
]
[trans-T •trans-T
]
N
6
O
O
HN
4
O
AB*
trans-T
2
O
OH
H
0
H
15 C7
N
N
Dry
Wet
Wet
AB*
+ trans-T
Conditions
H
C8H17
O
Figure 5 (a) In wet CDCl , nitrone B can hydrolyze to afford aldehyde 1 and hydroxylamine 2. When these
3
conditions are employed for the reaction of A, B, C, and D, hydroxylamine 2 can react with aldehyde D to
afford nitrone B*, bearing the 4,6-dimethylamidopyridine recognition site. (b) Reaction of nitrone B* with
maleimide A produces trans-TAB* (magenta), which is capable of directing its own formation by the assembly
of A and B* into a catalytically-active ternary complex [A•B*•trans-TAB*]. (c) Under dry conditions, reagents
A to D react to produce the reciprocal replicator trans-TAB and minimal self-replicator trans-TAB* in a ratio of
49:1. Under wet conditions, the ratio of [trans-TAB]:[ trans-TAB*] decreases markedly to 5:1, and the addition of
preformed trans-TAB* decreases this ratio even further to 3:1. All concentrations have been corrected for the
amount of template added. A description of the error analysis is provided in the Supporting Information.
In order to create the environmental conditions necessary to promote nitrone
hydrolysis, we saturated CDCl
Specifically, CDCl was stirred for 20 min with D
layers were allowed to separate. Recovery of the organic layer afforded a sample of CDCl
in which the concentration of D O was >1500 ppm, as determined by Karl–Fisher titration.
This sample of CDCl was used to prepare a four-component experiment in which A, B, C,
3
with water27 to create conditions that were suitably ‘wet’.
3
2
O before the aqueous and the organic
3
2
3
and D (each at a starting concentration of 10 mM) were allowed to react with each other
1
for 40 h at 10 °C. Analysis of this experiment after this time by 500 MHz H NMR
spectroscopy revealed that the concentration of trans-TAB had decreased from 8.6 mM to
6.2 mM, while the concentration of trans-TAB* had increased from <200 µM to 1.3 mM
(Figure 5c). Under these ‘wet’ conditions, therefore, the reciprocal and minimal templates
trans-TAB and trans-TAB* were present in a ratio of 5:1—representing a 10-fold increase in
the contribution of the minimal replicator to replication in this system, a dramatic change
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induced by a simple change in the environmental conditions. It is well established that
minimal replication pathways can be promoted by the addition of an autocatalytic
template at the start of the reaction. Accordingly, we performed an experiment in which
20 mol% of preformed trans-TAB* was added to a mixture of the four building blocks, A to
D, at 10 mM in ‘wet’ CDCl
. The presence of trans-TAB* in this experiment engendered a
3
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further enhancement in the quantity of the minimal self-replicator formed and trans-TAB*
reached a concentration of 2.1 mM after 40 h—representing a 3:1 ratio of the reciprocal
template trans-TAB to the minimal template trans-TAB*.
Although the reciprocal template trans-TAB remained as the dominant species in the
system, even in the presence of added preformed trans-TAB*, the marked increase in the
contribution of the minimal self-replication pathway demonstrates the considerable power
of both template instruction and environmental conditions in determining the outcome of
replication processes. It can be envisaged that a similar reciprocal system constructed from
building blocks that are more susceptible to hydrolysis would permit the construction of a
replication system in which a change in the reaction environment could be used to steer
the system towards either reciprocal or minimal replication pathways with increased
selectivity. Such changes can be further complemented by changes in other environmental
parameters such light, temperature, or pH, which can alter9g,h,j,13d,14b the efficiencies of the
replication pathways and even turn them on28 and off.
Conclusions
In conclusion, we have designed and implemented a reciprocal replication network that is
constructed from a pair of mutually-complementary templates that are based on simple,
synthetic organic molecules. The formation of this pair of complementary templates is
driven by orthogonal 1,3-dipolar cycloaddition and imine condensation reactions. The
formation of each template is accelerated markedly in the presence of its reciprocal
partner. The fully functional replicating network can be established and maintained
simply by mixing the four reagents required to synthesize the two templates in a single
flask. In this situation, the mutually reinforcing nature of the exclusively reciprocal
replicating system enhances the formation of both templates well above the levels
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observed when these templates are examined in isolation. The reciprocal replication
network also responds to the introduction of instructional templates. When either of the
two templates are added to the reaction mixture containing all four reagents, the network
responds to this instruction by enhancing the formation of both its reciprocal partner and
the instructional template itself by virtue of the symbiotic nature of the reciprocal catalytic
relationships within the network. The creation of this reciprocal replicating system opens
the way for the implementation of the complex functions29 provided by networks
constructed from oligonucleotides and peptides using simple, synthetic organic molecules.
Critical to the success of the reciprocal replicating system reported here is the
orthogonality of the reactions that form the two templates. We envisage applying this
technology to the creation of responsive networks in which the replication pathways can
be controlled by template inputs as well as environmental factors, such as pH or water
content.
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Acknowledgements
The financial support for this work was provided by EaStCHEM and the Engineering and
Physical Sciences Research Council (Grant EP/K503162/1). The authors acknowledge the
contributions of Eleftherios Kassianidis, Evan Wood and Thomas Smith to early,
exploratory work in this area.
Competing financial interests
The authors declare no competing financial interests.
Supporting Information
The Supporting information is available free of charge on the ACS Publications website:
General experimental procedures; synthetic procedures and compound characterization;
details of kinetic analyses, fitting (including scripts), error determination, and details of
DFT calculations.
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Author Information
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Corresponding author
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* d.philp@st-andrews.ac.uk; Fax: +44 (0)1334 463808; Tel: +44 (0)1334 467264.
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Present Addresses
‡ Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, United
Kingdom
† Institute of Structural and Molecular Biology, Division of Biosciences, University College
London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom
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Kassianidis, E.; Slawin, A. M. Z.; Philp, D. Chem. — Eur. J. 2006, 12, 6829. (g)
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Soc. 2015, 137, 16074. (b) Sadownik, J. W.; Philp, D. Angew. Chem., Int. Ed. 2008, 47,
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(18) Huck, J. PhD Thesis, University of St Andrews, St Andrews, 2011.
(19) del Amo, V.; Slawin, A. M. Z.; Philp, D. Org. Lett. 2008, 10, 4589.
(20) Although imine formation is in principle reversible, the formation of imine TCD from
its constituent components (C and D) was fitted in the present work as an
irreversible reaction because any attempts to fit the imine formation as a reversible
process have afforded values of kreverse that were ca. 108 smaller than the
corresponding value of kforward under the employed conditions (dry CDCl ).
3
(21) Kinetic effective molarity provides a measure of the enhancement in the template-
directed reaction relative to the corresponding bimolecular reaction. The parameter
EMkinetic also provides information about the concentration at which the reaction
would have to be carried out in order for the bimolecular pathway to perform with
the same efficiency as the recognition-mediated pathway. As a consequence, rate
acceleration is observed even if EMkinetic is < 1 M (as long as the concentration at
which the reaction is performed is lower than EMkinetic).
(22) In 1993, von Kiedrowski introduced7e the concept of catalytic efficiency (ε) and
reaction order (p) as parameters for describing and quantifying the behavior of
replicating systems. The values of kinetic effective molarity (EMkinetic) reported in this
work are not directly comparable to the values of ε reported in the literature for
replicating systems. Values of EMkinetic are derived from the ratio of a first order rate
constant and a second order rate constant and this ratio has defined units of M, hence
its description as ‘‘effective molarity’’. By contrast, the parameter ε is derived from
the ratio of two rate constants, one of which has units that are dependent on the
value of p. Strictly, this parameter cannot be described as ‘‘effective molarity’’ since it
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does not have units of M (unless p = 1) and values of ε are not strictly comparable as
they most likely have different units.
(23) Thermodynamic effective molarity provides information about the stability of the
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template duplex relative to the strength of the ternary complex.
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(24) The estimated stability of the [trans-TAB•TCD] duplex, assuming a connection EM of
1 M, is 1.2 × 107 M–1. In order to determine the stability constant of this duplex
experimentally using NMR, we would have to operate at concentrations centered the
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putative K , which in this case is around 100 nM. Working accurately and
d
reproducibly at concentrations is very challenging and is complicated by the fact that
dynamic processes on the NMR chemical shift timescale may start to become
important at K values of this magnitude. Specifically, the off rate for the complex
a
may be coincidentally similar to the frequency separation of the bound and unbound
resonances. In these circumstances, the spectrum will become exchange-broadened,
making analysis impossible. The compounds have no chromophores that exhibit
meaningful changes on binding, thereby precluding the use of UV-vis spectroscopy.
(25) (a) Motloch, P.; Hunter, C. A. Adv. Phys. Org. Chem. 2016, 50, 77. (b) Misuraca, M. C.;
Grecu, T.; Freixa, Z.; Garavini, V.; Hunter, C. A.; van Leeuwen, P. W. N. M.; Segarra-
Maset, M. D.; Turega, S. M. J. Org. Chem. 2011, 76, 2723. (c) Hunter, C. A.; Anderson,
H. L. Angew. Chem., Int. Ed. 2009, 48, 7488. (d) Badjić, J. D.; Nelson, A.; Cantrill, S. J.;
Turnbull, W. B.; Stoddart, J. F. Acc. Chem. Res. 2005, 38, 723. (e) Hunter, C. A.; Tomas,
S. Cell Chem. Biol. 2003, 10, 1023.
(26) The molecular framework that is at the core of replicator trans-TAB* has been
investigated extensively by the Philp Laboratory. This autocatalytic framework
shows an attenuated response to added template at levels above 20 mol% as a result
of product inhibition and saturation of the catalytically-active ternary complex. See
Ref. 10b for an extensive discussion.
(27) There are siginificant experimental difficulties associated with controlling the
amount of water present in chlroform in a reproducible manner. Whilst it would be
desirable to study the effect of the concentration of water on the rates of exchange
between nitrones B and B*, in practice, it was only possible to obtain reproducible
results when saturating the chloroform with D O as described in the main text. In
2
addition, the relative stabilities of nitrones B and B* are such that exchange occurs
more readily in the direction exploited here—nitrone B* is considerably more stable
in a hydrolytic sense than B, making experiments that drive the network in the
opposite direction challenging.
(28) Turega, S. M.; Philp, D. Chem. Commun. 2006, 3684.
(29) Padirac, A.; Fujii, T.; Estévez-Torres, A.; Rondelez, Y. J. Am. Chem. Soc. 2013, 135,
14586. (b) Fujii, T.; Rondelez, Y. ACS Nano 2013, 7, 27.
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Reciprocal Replicating System
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TAB
TAB
A
B
TCD
Reciprocal
+
TCD
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TAB
Minimal
C
D
Replicator
TAB*
TCD
Dry CDCl
Wet CDCl
3
3
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AB
[C•D•T
]
D PagJeo2u9rnoafl3o3f the American Chemical Society
CD
T
C
Crosscatalytic Cycle
AB CD
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T
→ T
AB
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CD
AB CD
[T •T
T
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Crosscatalytic Cycle
CD AB
T
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AB
T
B
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CD
[A•B•T
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