Orthogonal Recognition Processes Drive the Assembly and Replication of a [2]Rotaxane

Article abstract of DOI:10.1021/jacs.5b09738

Within a small, interconnected reaction network, orthogonal recognition processes drive the assembly and replication of a [2]rotaxane. Rotaxane formation is governed by a central, hydrogen-bonding-mediated binding equilibrium between a macrocycle and a linear component, which associate to give a reactive pseudorotaxane. Both the pseudorotaxane and the linear component undergo irreversible, recognition-mediated 1,3-dipolar cycloaddition reactions with a stoppering maleimide group, forming rotaxane and thread, respectively. As a result of these orthogonal recognition-mediated processes, the rotaxane and thread can act as auto-catalytic templates for their own formation and also operate as cross-catalytic templates for each other. However, the interplay between the recognition and reaction processes in this reaction network results in the formation of undesirable pseudorotaxane complexes, causing thread formation to exceed rotaxane formation in the current experimental system. Nevertheless, in the absence of competitive macrocycle-binding sites, realization of a replicating network favoring formation of rotaxane is possible.

Full text of DOI:10.1021/jacs.5b09738

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Article
Orthogonal recognition processes drive the
assembly and replication of a [2]rotaxane
Tamara Kosikova, Nurul Izzaty Hassan, David B. Cordes, Alexandra M. Z. Slawin, and Douglas Philp
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b09738 • Publication Date (Web): 16 Oct 2015
Downloaded from http://pubs.acs.org on October 16, 2015
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Orthogonal recognition processes
drive the assembly and replication of
a [2]rotaxane
Tamara Kosikovaa, Nurul Izzaty Hassanab, David B. Cordesa,
Alexandra M. Z. Slawina and Douglas Philp*a
aSchool of Chemistry and EaStCHEM, University of St Andrews, North Haugh, St Andrews, KY16 9ST, United
Kingdom
bSchool of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 Bangi, Selangor, Malaysia
Corresponding author: d.philp@st-andrews.ac.uk
Abstract
Within a small interconnected reaction network, orthogonal recognition processes drive the assembly and
replication of a [2]rotaxane. Rotaxane formation is governed by a central, hydrogen bonding-mediated
binding equilibrium between a macrocycle and a linear component, which associate to give a reactive
pseudorotaxane. Both the pseudorotaxane and the linear component undergo irreversible, recognition-
mediated 1,3-dipolar cycloaddition reactions with a stoppering maleimide group, forming rotaxane and
thread, respectively. As a result of these orthogonal recognition-mediated processes, the rotaxane and
thread can act as autocatalytic templates for their own formation and also operate as crosscatalytic
templates for each other. However, the interplay between the recognition and reaction processes in this
reaction network results in the formation of undesirable pseudorotaxane complexes, causing thread
formation to exceed rotaxane formation in the current experimental system. Nevertheless, in the absence
of competitive macrocycle-binding sites, realization of a replicating network favoring formation of rotaxane
is possible.
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Introduction
Molecular recognition plays a pivotal role in the self-assembly1 and self-organization2 of both synthetic
chemical systems and those found in Nature. The constitution of individual components within
interconnected networks present in such systems governs their capacity to perform specific functions and
to interact with other species in a recognition-mediated fashion. Small changes in the identity or
orientation of recognition-enabling structural elements can have profound effects3 on the recognition
processes within a network. Therefore, careful consideration has to be given to both the desirable
function-encoding elements, as well as their relative position within a given molecular structure. Through
controlled design, we can exploit the potential of molecular recognition and self-assembly as driving
forces for the fabrication4 of complex systems in a pre-organized manner. In the past, molecular
recognition has been employed successfully in the formation of numerous molecular assemblies5,
mechanically-interlocked architectures6, nanoscale machines7 and systems capable of transferring
structural information8 through self-replication. Specifically, we are interested in exploring the underlying
requirements that give rise to chemical systems capable of exhibiting phenomena such as self-replication.
A better understanding of the principles governing assembly and replication in chemical systems will
allow us to probe the requirements for molecular evolution9 and emergence10 of complex function. The
minimal requirements for non-enzymatic, recognition-mediated replication are well understood, with a
number of small-molecule examples reported11 in the literature. However, the requirements for integrating
self-replication with other recognition-mediated processes, such as those required for assembly of
mechanically-interlocked architectures, remains a largely unexplored area. Previously, we have
described12 a kinetic framework for integrating replication with the synthesis of a mechanically-interlocked
architecture, namely a rotaxane. However, the experimental implementations of this design have not yet
afforded a functional self-replicating rotaxane. Here, we propose an alternative kinetic model for the
creation of a system where the assembly and replication of a [2]rotaxane is driven by two orthogonal
recognition processes.
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Designing a molecule capable of self-replication requires two building blocks, here termed the linear
component L and the stoppering species S, equipped with complementary recognition and reactive sites.
Reaction of these components (Figure 1, centre) produces a template T. This template is capable of
binding components L and S in a recognition-mediated, catalytically-active ternary complex [TꢀLꢀS],
thereby accelerating the reaction to form T (Figure 1, top left). Ultimately, the resulting product duplex
[TꢀT] dissociates to afford two template molecules capable of taking part in further template-mediated
replication cycles. Formation of a rotaxane with the capacity to replicate, however, requires an additional
binding site in order to enable association between macrocyclic component M and one of the building
blocks required for self-replication. In this case, the linear component L bears the additional macrocycle-
binding site, enabling formation of a reactive pseudorotaxane complex [LꢀM] (Figure 1, centre). The
replicating network radiates from this central equilibrium, established through the association of the
macrocycle M and the linear component L, into separate, yet interconnected thread and rotaxane forming
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catalytic cycles. Both linear component L and pseudorotaxane complex [LꢀM] can react with stoppering
species S to afford thread T and rotaxane R, respectively. T and R can both establish autocatalytic
cycles, proceeding via the formation of catalytically active ternary complex, [TꢀLꢀS] to make thread T
(Figure 1, top left) and quaternary complex, [RꢀLꢀMꢀS], to create rotaxane R (Figure 1, bottom left). The
rotaxane and thread templates also satisfy the structural and recognition requirements necessary for
taking part in crosscatalytic cycles, proceeding through [RꢀLꢀS] and [TꢀLꢀMꢀS] complexes (Figure 1, top
right and bottom right, respectively). Overall, this interconnected replicating network can operate through
two autocatalytic and two crosscatalytic cycles. We envisaged that implementation of orthogonal
recognition processes would permit the formation of a system where all catalytic cycles are active.
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Figure 1. A kinetic model describing the main interactions in a replicating network with orthogonal recognition processes, leading to
the formation of thread T and rotaxane R. The interconnected network is based around a central equilibrium (solid rectangle)
between linear component L and macrocycle M, to give reactive pseudorotaxane complex [LꢀM]. The network branches out into
autocatalytic thread (top left) and rotaxane (bottom left) cycles, and crosscatalytic thread (top right) and rotaxane (bottom right)
cycles. For simplicity, equilibrium arrows are not shown outside of the central equilibrium. Reactive and recognition elements are
represented in cartoon form: blue and yellow represent complementary recognition sites, orange and green denote reactive
elements, grey represent the reaction product and macrocycle-binding site is shown in dark red.
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Our design (Figure 2) of the key quaternary [RꢀLꢀMꢀS] complex exploits H-bonding mediated recognition
for both macrocycle binding and the complementary recognition units required for self-replication.
Specifically, we exploited the recognition between a phenylacetic acid, located on the stoppering
component S, and a 6-methyl-2-amidopyridine unit on linear component L (Figure 2a), which we have
successfully employed8d,13 in several self-replicating systems in the past. Similarly, we have utilized a 1,3-
dipolar cycloaddition reaction between a nitrone, here positioned on linear component L, and a maleimide
on stoppering element S, as the means of irreversibly connecting the starting building blocks into a
template Thread molecule (Figure 2b). From previous experience, we believed that a biphenyl spacer
between the nitrone reactive site and the macrocycle binding site would be sufficient for isolating the
macrocycle from the reactive site, thus preventing potential steric issues12, which would hinder the
cycloaddition. The recognition event used to associate the macrocyclic and linear component in the
pseudorotaxane complex [LꢀM] is provided by H-bonding mediated recognition between an amide group
on the linear component and complementary recognition units on the macrocycle. We utilized two
structurally similar macrocycles for the formation of our rotaxane (Figure 2): macrocycle GM employed14
previously, containing a glycol spacer, and macrocycle PM15, incorporating a pyridine linker. Once the
pseudorotaxane complex is formed, the irreversible reaction locks the macrocycle on the linear
component through the stoppering approach, resulting in GM [2]Rotaxane and PM [2]Rotaxane (Figure
2b). Once the concentration of template present in the reaction mixture is sufficient to initiate a catalytic
cycle, the reaction proceeds through the recognition-mediated replication channel (Figure 2c), with high
diastereoselectivity.
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Results and Discussion
We, and others, have established12,16 previously that various substituted diarylamides are capable of
associating with macrocycle GM, demonstrating that the amide-containing linear component L is likely to
be a suitable binding partner. Macrocycle PM contains a second pyridine ring with H-bond acceptor
abilities, which we expected to bind more strongly, affording a system more selective towards the
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formation of a rotaxane. Initially, we performed H NMR spectroscopic binding studies examining the
association between each macrocycle and a model compound N, equipped with a terminal –NO2 group in
place of the nitrone reactive site (Figure 3). The desired amide-binding site is therefore present in
isolation from any other potential recognition or reactive moieties. By reducing the temperature to 233 K,
the macrocycle-linear component equilibrium enters the slow exchange regime on the NMR chemical shift
timescale, thus enabling direct observation of resonances corresponding to unbound species and the
pseudorotaxane complex. For illustrative purposes, we will discuss only the complex formed between the
compound N and the macrocycle GM (the formation of other pseudorotaxane complexes is discussed in
the SI). Particularly diagnostic of pseudorotaxane formation is the downfield shift of the resonance arising
from the macrocycle NH protons (Figure 3b, Hg) (+1.49 ppm). The chemical shift of the macrocycle NH
resonance in the bound complex (9.42 ppm) suggests that these protons are hydrogen bonded to the
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carbonyl of the amide present in N. Macrocycle resonances arising from Hd and He are also shifted upfield
(–0.66 and –0.56 ppm respectively)
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Figure 2. (a) Cartoon representation of the chemical building blocks employed in the design of a self-replicating [2]rotaxane. The
amide macrocycle-binding site (dark red) enables formation of a pseudorotaxane [LꢀM]. Amidopyridine (blue) and a carboxylic acid
(yellow) recognition sites facilitate recognition-mediated self-replication through the formation of a quaternary complex [RꢀLꢀMꢀS],
while the nitrone reactive site (orange) on the pseudorotaxane reacts with the maleimide (green) on the stoppering element to give
the final [2]rotaxane. (b) Chemical structures of Thread, GM [2]Rotaxane and PM [2]Rotaxane. (c) Computed transition state
geometry (RM1, MOPAC 2012) for Thread being made from L and S Thread (shown as stick representation, where carbon atoms
are shown in dark grey, oxygen atoms in red, nitrogen atoms are in blue and hydrogen atoms in pale grey). Most hydrogen atoms
are omitted for clarity. Red dashed lines represent H-bonds mediating the assembly of the ternary complex. Black dashed lines
denote the C–O and C–C bonds partially formed in the transition state (For details, see ESI).
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Figure 3. Partial 1H NMR (500.1 MHz, CDCl3) of (a) compound N (RT), (b) equimolar mixture of N compound and GM (233 K), (c)
GM (RT). Dashed lines are shown to connect resonances belonging to specific protons in bound and unbound states. Unassigned
resonances arise from the unbound species.
The inequivalence of the resonances arising from the methylene protons in the CH2 group (Ha, Hb and Hc)
on the macrocycle in the pseudorotaxane is a result of the end-to-end asymmetry imposed on the
macrocycle by complexation with the model linear compound N. Low temperature 1H NMR spectroscopy
further allowed single-point determination of association constants with both macrocycles, affording Ka
values of 3750 M–1 for complex [NꢀGM] and 5100 M–1 for complex [NꢀPM] at 223 K. For pseudorotaxane
[NꢀPM], we confirmed the location of the macrocycle at the expected binding site on N by low
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temperature (223 K) H-1H ROESY experiments (for details, see SI), which showed rOe cross peaks
between the resonances associated with the NH protons (H7’) of the macrocycle and the two phenyl rings
(H3 and H5) on each side of the amide present in N, and the resonances arising from the macrocycle CH2
protons and the resonance associated with the NH of N. Next, we examined the association of each
macrocycle with linear component L, which, in addition to the macrocycle-binding amide site, also
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contains a nitrone and an amidopyridine, to which the macrocycles may potentially bind. The nitrone
moiety in particular possesses a negatively charged oxygen atom with the potential17 to be a hydrogen
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bond acceptor in the presence of complementary recognition units. The low temperature H-1H ROESY
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NMR experiments, however, did not afford sharp resonances and calculation of association constants
was therefore not possible from these data. Despite these limitations, there is clear evidence for
association between L and M, suggesting that the system may be suitable for rotaxane formation.
In order to confirm successful formation of thread T and the two rotaxanes, GM [2]rotaxane (GM-R)
and PM [2]rotaxane (PM-R), we synthesized these products on a preparative scale by low temperature
reaction of linear component L and stopper S in the presence of excess macrocycle. All three
components, T, GM-R and PM-R, were isolated successfully by reverse phase flash column
chromatography. Initially, we confirmed the identity of our products by MALDI-TOF mass spectrometry
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and H and 13C NMR spectroscopy. In order to further confirm that GM-R and PM-R contained the
macrocycles bound at the expected site, we obtained single crystals18 suitable for X-ray crystallographic
analysis (for details, see ESI). The X-ray analyses confirmed the expected molecular structures in both
GMꢀR and PMꢀR, in particular the trans stereochemistry of the isoxazolidine ring, and the location of each
macrocycle on the linear component, mediated by H-bonding interactions between the macrocycle NH
and the carbonyl of the linear component (Figure 4). For both GM-R and PM-R, The crystal lattice of both
rotaxanes contained a racemic mixture of the trans diastereoisomeric cycloadduct (Figure 4), where two
molecules typically interact through hydrogen bonding between one –COOH and one amidopyridine
recognition site, forming H-bonded chains running along the b-axis (GMꢀR) or c-axis (PMꢀR). Interestingly,
the phenylacetic acid moiety present in the rotaxanes was directed towards the pyridine ring of each
macrocycle in both GM-R and PM-R.
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(a)
(i)
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(b)
(ii)
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Figure 4. Stick (i) and space-fill (ii) representations of the X-ray crystal structure of (a) GM [2]rotaxane and (b) PM [2]rotaxane as
determined by single-crystal X-ray crystallography. In the stick representations, carbon atoms are shown in grey, oxygen atoms in
red, nitrogen atoms in blue and hydrogen atoms are shown in white. Most hydrogen atoms are omitted for clarity. Black circles in the
rotaxane stick representations highlight the relative trans stereochemistry of the isoxazolidine ring protons. In the space fill
representations, the thread component is colored grey and the macrocycle is colored either orange (GM) or blue (PM).
We conducted two-dimensional 1H-1H ROESY experiments at room temperature in order to establish the
location of the macrocycle in both GM-R and PM-R on the linear component in solution. Figure 5 shows
the rOe cross peaks observed in the 2D ROESY NMR spectrum of GM-R. Particularly diagnostic are the
highlighted cross peaks associating the resonances arising from the macrocycle Ha, Hb and Hc protons
with the resonances arising from the H1 and H3 protons on the linear component, confirming the expected
location of GM macrocycle on the thread in solution. Similar results were obtained for the PM-R (for
details, see ESI).
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H1
H1'
H3
H3'
H2
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3.8
Hb
Hb'
Hc'
Hc
δH
9.2
Ha
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ppm
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Figure 5. Partial 1H-1H ROESY correlation (500.1 MHz, CDCl3, RT, 500 ms mixing time) of GM [2]Rotaxane. Highlighted rOe cross
peaks represent the through space interactions between linear component and the macrocycle. Unassigned cross peaks represent
intra-component interactions.
Having synthesized and fully characterized thread
T
and both rotaxanes GM-R and
PM-R successfully, we were ready to undertake a comprehensive kinetic analysis in order to ascertain
which catalytic cycles (see Figure 1) are active in our rotaxane:thread system. Initially, we examined the
formation of thread T in the absence of any added template. An equimolar mixture containing the desired
components ([L] = [S] = 10 mM) was prepared in CDCl3 and the reaction progress at 5 °C was monitored
by 1H NMR (500.1 MHz) spectroscopy every 15 minutes over 8 hours. This low reaction temperature was
selected for the kinetic experiments because it maximizes the strength of recognition-mediated processes
within the system while simultaneously allowing accurate monitoring of the reaction progress on a
reasonable timescale by 1H NMR spectroscopy. The reaction time course (Figure 6a) for this reaction was
determined by examining the resonances for the three protons associated with the trans isoxazolidine
ring of the cycloadduct relative to 2,4-dinitrotoluene as an internal standard. We found that, following an
initial lag period, typical for self-replicating systems, the thread replicated very efficiently, exhibiting a clear
sigmoidal rate profile where the thread concentration reached 9.1 mM after 4 hours (91% conversion).
We determined the rate for this reaction (d[T]/dt) by computing the first derivative of a seventh-order
polynomial fitted to the concentration vs time data. The trans diastereoisomer was formed exclusively,
with the rate maximum observed after 63 minutes (4.1 mM h–1). Next, we examined the autocatalytic
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formation of thread in the presence of instructing thread template. The required starting components ([L]
= [S] = 10 mM) and pre-formed thread (10 mol%) were dissolved in CDCl3 and the reaction progress was
followed by 1H NMR spectroscopy as described previously. Addition of pre-formed thread at t = 0 resulted
in the disappearance of the lag period (Figure 6a), allowing the system to reach its maximum rate (6.0
mM h–1) from the beginning of the reaction (Figure 6b), thereby confirming the successful design of our
self-replicating system19. With this information in hand, we analyzed thread formation in the presence of
10 mol% preformed GM-R and PM-R (Figure 6a).
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Figure 6. (a) Concentration and (b) rate profile for the formation of Thread in the absence of template (black) and in the presence of
pre-formed thread (grey), 10 mol% GM-R (orange) or 10 mol% PMꢀR (blue) as determined by 1H NMR spectroscopy (500.1 MHz,
CDCl3, 5 °C, all components at 10 mM). Concentration for each product was determined relative to 2,4-dinitrotoluene as an internal
standard.
The concentration-time data for the GMꢀR and PMꢀR instructed thread kinetic experiments closely
mirrored formation of thread in the presence of pre-formed thread template, showing clear disappearance
of the lag period and shift in the time of maximum reaction rate (Figure 6b). The closely matching reaction
profiles determined for the thread and GMꢀR and PMꢀR instructed kinetic experiments (max. rate 6.8 and
6.3 mM h–1, respectively) revealed that the thread is autocatalytic and the two rotaxanes are capable of
crosscatalyzing thread formation with efficiency equal to that of thread.
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Having explored the thread kinetic pathways fully, we next examined the individual, uninstructed
kinetics of GM-R (Figure 7) and PM-R (Figure 8). The progress of the rotaxane kinetic experiments was
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again followed by H NMR spectroscopy, allowing us to deconvolute the individual proton resonances
associated with the isoxazolidine ring of each thread and rotaxane product (for details, see ESI), which
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were formed with high diastereoselectivity for the trans cycloadducts.
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Figure 7. Concentration and rate profiles for the formation of (a) Thread (circles) and (b) GMꢀR (squares) in the GM [2]rotaxane
kinetic experiments carried out in the absence of template (black) and in the presence of pre-formed 10 mol% thread (grey) or 10
mol% GM-R (orange) as determined by 1H NMR spectroscopy (500.1 MHz, CDCl3, 5 °C, all components at 10 mM). Concentration
for each product was determined relative to 2,4-dinitrotoluene as an internal standard.
After
4
hours,
T
reached
a
higher concentration than either rotaxane, resulting in
a
1:3 ratio of GM-R:T and 1:2 of PM-R:T, reflecting the higher affinity of the linear guest for the pyridine
macrocycle PM. As observed in the uninstructed thread experiment, the rotaxane reaction profiles in the
absence of added template revealed catalysis, exhibiting an initial lag period and sigmoidal reaction
profile. In the GM-R kinetic experiment, the maximum rate of product formation (Figure 7) was observed
at 90 minutes (1.78 mM h–1) for thread and 57 minutes (0.47 mM h–1) for the rotaxane. Similarly, the
maximum rate of thread formation in the PM-R experiment was determined at 87 minutes (1.57 mM h–1),
and at 102 minutes (0.60 mM h–1) for the rotaxane (Figure 8).
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Figure 8. Concentration and rate profiles for the formation of (a) Thread (circles) and (b) PMꢀR (squares) in the PM [2]rotaxane
kinetic experiments carried out in the absence of template (black) and in the presence of pre-formed 10 mol% thread (grey) or 10
mol% PM-R (blue) as determined by 1H NMR spectroscopy (500.1 MHz, CDCl3, 5 °C, all components at 10 mM). Concentration for
each product was determined relative to 2,4-dinitrotoluene as an internal standard.
The concentration-time profiles for the GMꢀR (Figure 7, top) and PMꢀR (Figure 8, top) rotaxane kinetic
experiments in the presence of 10 mol% of pre-formed instructing thread mirrored the corresponding
profiles obtained in the rotaxane kinetic experiments instructed with GM-R or PM-R template very closely,
exhibiting a nearly identical ratio of R:T and shortened lag period across each rotaxane set, as evidenced
by the shift in maximum rate for the formation of thread and rotaxanes to an earlier time point (Figure 7
and 8, bottom) in all instructed kinetic experiments. The outcome of the rotaxane kinetic experiments
confirmed that both auto- and crosscatalytic cycles are operating in each rotaxane system with equal
efficiency. Satisfyingly, results of the kinetic analyses confirmed that the orthogonal recognition processes
enable formation of a replicating network where thread and rotaxane are matched in catalytic efficiencies
as templates, in both auto- and crosscatalytic cycles. Nevertheless, the ratios of R:T determined in our
kinetic experiments showed a strong preference for the formation of thread over the rotaxane in both
replicating networks. This lower ratio indicates that while the pre-formed rotaxane templates are capable
of catalyzing formation of both thread and rotaxane products efficiently, the rotaxane formation itself
proceeds less effectively than we had hoped based on the low temperature pseudorotaxane experiments.
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One plausible explanation for the low efficiency of rotaxane formation and thus the low R:T ratio is the
insufficient proportion of each reactive pseudorotaxane complex [LꢀM]amide (Figure 9a), containing the
macrocycle at the desired amide binding site, in each reaction as a result of the nitrone reactive site and
amidopyridine recognition unit also associating with the macrocycles. The reactive pseudorotaxane
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complex [LꢀM]amide can react with stopper S through a bimolecular reaction (k1
= kbi) and, in the presence
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of template, also through a recognition-mediated unimolecular reaction (k2 kuni). By contrast, macrocycle
=
bound at the nitrone reactive site results in formation of a pseudorotaxane [LꢀM]nitrone (Figure 9b) that
sequesters the L and M components in a co-conformation that hinders formation of any product. By
contrast, association of the macrocycle with the amidopyridine recognition site, required for recognition-
mediated self-replication, affords a pseudorotaxane complex [LꢀM]amidopyridine (Figure 9c) that can only
react through the bimolecular pathway (k4
= kbi). Ultimately, the presence of these two additional sites on
the linear component, capable of associating with the macrocycle, results in a smaller proportion of the
desired pseudorotaxane complex [LꢀM]amide, and, thus, a lower apparent Ka for the pseudorotaxane
formation. The apparent Ka reflects binding of the macrocycle to three different binding sites. Similarly, the
observed unimolecular rate constant (kuni) associated with the template-mediated formation of each
rotaxane is lower than it would be if the macrocycle was bound solely at the amide binding site.
Figure 9. Model illustrating the association between macrocycle M and linear component L, affording formation of three different
pseudorotaxane complexes, only one of which results in the formation of the desired rotaxane.
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In order to gain a better understanding of the effect of additional binding sites on this highly
interconnected system, we performed fitting of all of the concentration-time data available from the related
thread and rotaxane kinetic experiments to kinetic models encompassing all interactions and reactions.
These models included the formation of the three different pseudorotaxane complexes, in each system.
Exploiting an appropriate fitting procedure (for details, see ESI), we were able to extract normally
inaccessible kinetic parameters, namely, the template-mediated unimolecular rate constants (kuni) and the
product duplex association constants (KDuplex) for the formation of Thread, GM-R and PM-R (Table 1). In
order to avoid fitting a larger number of constants than the data sets available, we implemented two
assumptions throughout the fitting procedure: (i) kbi was assumed to be identical for all three templates
and (ii) only a single kuni was fitted for each template across the auto- and crosscatalytic kinetic
experiments forming a particular template. The latter assumption was based on the experimental
observation of closely matched catalytic efficiencies determined during the kinetic experiments, and the
fact that attempts to fit different unimolecular rate constants for the individual auto- and crosscatalytic
pathways revealed little variation in the fitted values. Interestingly, the fitting revealed that kuni for the
formation of both rotaxanes are, as a result of the binding between each macrocycle and the linear
component, almost four times smaller that the kuni of thread. This difference reflects a decrease in the
reactivity of the rotaxane-forming, catalytically-active quaternary complexes relative to the thread-forming
ternary complexes. Using the fitted values of kbi and kuni, the effective molarity20 for the formation of each
template can be calculated. This parameter provides a measure of the efficiencies of the template-
mediated catalytic pathways leading to the formation of thread and both rotaxanes, relative to the
bimolecular pathways (EM = kuni/kbi). The apparent Ka values determined21 for the formation of [LꢀGM]
and [LꢀPM] pseudorotaxane complexes (Table 1) reflect the trend in association constants previously
determined through low temperature experiments for [NꢀM].
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Table 1. Kinetic parameters and association constants determined for the (i) formation of thread, (ii) formation of GM-R and
(iii) PM-R, through kinetic simulation and fitting of experimental kinetic data (at 278 K) using the SimFit software (Günter von
Kiedrowski, University of Bochum, 2008). In all cases, the kbi and Ka Duplex were determined as 1.04×10–4 M–1s–1 and 10.3×106 M–
1 for thread, GM-R and PM-R.
Analysis of the fitted kinetic parameters for thread and rotaxanes shows the detrimental effect of
competitive macrocycle-binding sites on the apparent Ka for the formation of [LꢀM] and the unimolecular
rate constant and consequently, the ratio of R:T formed in the system. In order to demonstrate how a
similar system, integrating the formation of a rotaxane with self-replication, would work in the absence of
competitive binding sites, we performed kinetic simulations. These simulations employed the parameters
determined for PMꢀR system, which exhibits more efficient rotaxane formation than GMꢀR as a result of a
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higher Ka for the [LꢀM]. We specifically explored the effect of varying the strength of [LꢀM] associations
constants from 102 to 105 M–1 on the ratio of rotaxane:thread under two different conditions: condition A
employed kinetic parameters determined for our experimental system (kuni rotaxane < kuni thread);
condition B examined an ideal system with no competitive macrocycle-binding sites on the linear
component (kuni rotaxane = kuni thread). Simulating condition B (Figure 10), where the effective molarity for
thread and rotaxane are identical, allowed us to elucidate how the experimental system would behave in
the absence of competitive binding sites – a situation where the ratio of products formed depends solely
on the Ka governing the formation of [LꢀM].
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Figure 10.
Outcome of kinetic simulations examining the effect of increasing Ka for the formation of pseudorotaxane
complex [LꢀM] on the ratio of rotaxane:thread formed in: condition A (ꢁ) employing kinetic parameters determined for our
experimental system (kuni rotaxane < kuni thread) and condition B (ꢀ) examining an ideal system with no competitive macrocycle-
binding sites on the linear component (kuni rotaxane = kuni thread). The blue rectangle represents the outcome of the simulation
employing kinetic parameters determined for T and PM-R. Simulations were performed using SimFit software package (Günter von
Kiedrowski, University of Bochum, 2008).
The results of the simulation (Figure 10, condition A) revealed that an increase in the Ka for the
association between the linear component and macrocycle does not afford an R:T ratio greater than 8:1
in the condition employing experimental parameters, unless Ka > 105 M–1. While a considerable
improvement over the current product ratios, it is possible that a larger Ka value for the formation of
pseudorotaxane [LꢀM] might also result in a concurrent increase in the strength of association for the
macrocycle binding sites (see Figure 9), therefore, resulting in a lower R:T ratio than predicted from this
simulation. Whilst the current experimental design, incorporating two additional competing macrocycle-
binding sites, does not favor rotaxane formation, the same kinetic model can afford significantly higher
R:T ratios (>15:1) if only a single, desired macrocycle binding site is present (Condition B), demonstrating
that rotaxane can be formed selectively in a replicating network operating simultaneously through both
auto- and crosscatalytic pathways.
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In conclusion, we have described a successful experimental implementation and full characterization of a
reaction network integrating replication processes with the assembly of a [2]rotaxane. Through the
application of orthogonal recognition processes, we were able to effectively direct formation of a
replicating network where thread and rotaxane products are matched in catalytic efficiencies as
templates, in both auto- and crosscatalytic cycles. However, interplay between the different recognition
and reactive processes in the complex network, specifically the nitrone and amidopyridine moieties acting
as competitive binding sites for the macrocyclic component, resulted in thread formation surpassing
rotaxane formation in the current experimental system. Exploiting kinetic simulations, we demonstrated
that orthogonal recognition processes are capable of driving autocatalytic rotaxane formation in the
absence of competitive macrocycle binding sites. The realization of this goal is currently under
investigation in our laboratory.
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Acknowledgements
The financial support for this work was provided by EPSRC (Grant EP/K503162/1 and EP/E017851/1)
and the Ministry for Higher Education Malaysia. The research data supporting this publication can be
accessed at http://dx.doi.org/10.17630/d115d058-349e-4300-94e3-76a3181a9fc7.
Electronic Supplementary Information (ESI) available: experimental procedures, details of kinetic
measurements, binding studies, computational modeling of the thread:rotaxane network, example fitting
and simulation scripts.
References and Footnotes
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(18) Suitable single crystals were grown as colorless needles by slow evaporation of acetonitrile/CDCl3
(GM [2]rotaxane) or as plates by slow evaporation from acetonitrile (PM [2]rotaxane). For more details,
see ESI.
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(19) The recognition-disabled, bimolecular reactions to form thread and both rotaxane from the linear
component L and a p-methyl substituted maleimide (Sdis) showed low conversion and low
diastereoselectivity (for details, see ESI).
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(21) Throughout the fitting process, only a single Ka value was fitted for all three processes leading to the
formation of the different pseudorotaxane complexes: [LꢀM]amide, [LꢀM]nitrone, [LꢀM]amidopyridine. While the Ka
governing the formation of each pseudorotaxane complex might be different, we were unable to
deconvolute the three individual binding events. Therefore, in order to avoid using an inappropriately
large number of variable parameters during fitting, only one association constant for the three binding
processes was fitted (see fitting script in the ESI).
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Journal of the American Chemical Society
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ACS Paragon Plus Environment

Journal of the American ChemPiacgael S20ocoifet3y1
ACS Paragon Plus Environment
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Page 21 of 31
Journal of the American Chemical Society
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ACS Paragon Plus Environment

Journal of the American Chemical SoPcaiegtey 22 of 31
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ACS Paragon Plus Environment

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Page 23 of 31
Journal of the American Chemical Society
h
O
O
N
1
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5
6
7
8
9
10
11
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f
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14
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a
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3
8
5
7
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(a)
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5 + 6
h
8
7
c + f
d + e
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1 + a + b
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ACS Paragon Plus Environment
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(a)
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(i)
Journal of the American Chemical Society
Page 24 of 31
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ACS Paragon Plus Environment

Page 25 of 31
Journal of the American Chemical Society
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H1
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ACS Paragon Plus Environment
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Journal of the American Chemical Society
Page 26 of 31
(a)
10
8
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(3b2 )
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4
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
+ 10 mol% PM-R
2
0
0
1
2
3
4
Time / h
7
6
5
4
3
2
1
0
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
+ 10 mol% PM-R
0
1
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ACS Paragon Plus Environment
Time / h

Page 27 of 31
Journal of the American Chemical Society
(a)
(b)
Thread
GM-R
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2
3
10
10
4
5
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9
10
11
12
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
8
8
6
4
2
0
6
13
14
15
16
17
4
18
19
20
21
22
2
23
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27
0
28
29
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33
34
35
0
1
2
3
4
0
1
2
3
4
Time / h
Time / h
3
2
1
0
36
3
37
38
39
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43
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
No template added
+ 10 mol% Thread
+ 10 mol% GM-R
44
2
45
46
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48
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50
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1
53
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0
0
1
2
3
4
0
1
2
3
4
ACS Paragon Plus Environment
Time / h
Time / h

Journal of the American Chemical Society
Page 28 of 31
(a)
(b)
Thread
PM-R
1
2
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5
6
7
8
9
10
8
10
No template added
+ 10 mol% Thread
+ 10 mol% PM-R
No template added
+ 10 mol% Thread
+ 10 mol% PM-R
8
6
4
2
0
10
11
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6
4
2
0
0
1
2
3
4
0
1
2
3
4
Time / h
Time / h
3
2
1
0
3
2
1
0
No template added
+ 10 mol% Thread
+ 10 mol% PM-R
No template added
+ 10 mol% Thread
+ 10 mol% PM-R
0
1
2
3
4
0
1
2
3
4
ACS Paragon Plus Environment
Time / h
Time / h

Page 29 of 31
Journal of the American Chemical Society
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ACS Paragon Plus Environment