Orthogonal breaking and forming of dynamic covalent imine and disulfide bonds in aqueous solution

Article abstract of DOI:10.1039/c5cc02716k

Orthogonal bond-breaking and forming of dynamic covalent disulfide and imine bonds in aqueous solution is demonstrated. Through judicious choice of reaction partners and conditions, it is possible to cleave and reform selectively these bonds in the presence of each other in the absence of unwanted competing processes.

Full text of DOI:10.1039/c5cc02716k

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Orthogonal Breaking and Forming of Dynamic Covalent Imine and
Disulfide Bonds in Aqueous Solution
Michael E. Bracchi and David A. Fulton*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
with no undesired reactions occurring between the molecules in
Orthogonal bond-breaking and forming of dynamic covalent
disulfide and imine bonds in aqueous solution is
demonstrated. Through judicious choice of reaction partners
and conditions, it is possible to cleave and reform selectively
10 these bonds in the presence of each other in the absence of
unwanted competing processes.
50 the mixture i.e. alternative combinations of the reaction partners
1
are not detectable by H NMR spectroscopy. In particular, the
reaction between thiols and aldehydes to form hemithioacetals
(Fig. 1c) was identified as a potential competing process, and our
investigation showed that careful choice of reaction partners is
55 necessary to avoid this problem.
In this work we investigate the orthogonality of the bond
breaking and forming of imine and disulfide DCBs by
The design and study of functional systems of molecules is an
area of interest within the growing field of systems chemistry.1
An approach to increase system complexity is to exploit multiple
15 orthogonal supramolecular interactions, yet only in recent years
have chemists made significant efforts2 to emulate this feature
which is ubiquitous in natural systems. Orthogonal interactions
in functional systems3 have been exploited in increasingly
complex systems e.g. molecular machines,4 interlocked
20 molecules5 and responsive materials,6 and to continue this
development there is a clear need for wellꢀunderstood orthogonal
interactions.
Chemical bonds which can reversibly break and reform in
response to stimuli are wellꢀknown7 in chemistry, and these soꢀ
25 called “dynamic covalent bonds” (DCBs) can be utilized as
“modules”8 to introduce stimuliꢀresponsiveness into functional
systems. Of particular interest to us is reversible imine and
disulfide bonds. Imine bonds are formed from the condensation
of amines and carbonyls (Fig. 1a), and the position of the
30 equilibrium is pH dependent, with work by Lehn demonstrating9
that the position of the imine equilibrium can be shifted from
almost complete imine to starting materials over about three pH
units. Redoxꢀsensitive disulfide bonds can be reduced to their
corresponding thiols in the presence of a reducing stimulus, and
35 reꢀoxidized to form the disulfide (Fig. 1b).10 Since pH and redox
can be controlled independent of each other it should be possible
to selectively cleave and reform one of these bonds in the
presence of the other, and thus these bonds can be considered to
be orthogonal.[‡] Assuming complete orthogonality, and
40 considering only situations in which DCBs are in either “broken”
or “formed” states, there are four distinct scenarios. It is
convenient to map these states onto a fourꢀnode network (Fig. 2)
where each node represents one of four possible scenarios
regarding whether the disulfide and imine bonds are “broken” or
45 “formed” and the vertices display the orthogonal stimuli required
to drive the bond forming and breaking processes. The condition
for orthogonality is that it is possible to successfully navigate
between all nodes through the application of orthogonal stimuli
establishing
a set of conditions under which sequential
application of stimuli allows interconversion between nodes
60 within a 4ꢀnode network in the absence of unwanted, interfering
processes.11 We also highlight how careful choice of reaction
partners is required in order to avoid formation of undesired
products and ‘crossꢀtalk’ between our chosen DCB motifs.
As a redoxꢀsensitive DCB, we chose the disulfide 1 whose
65 quaternary ammonium groups impart water solubility. It is
possible to reversibly interconvert this species with thiol 4 upon
application of reducing and oxidising agents. As a pHꢀsensitive
DCB we chose imine 5, which can be reversibly interconverted
into its waterꢀsoluble reaction partners amine 2 and aldehyde 3 by
70 modulation of pH. The imine is formed almost exclusively at pH
12.0, and the reaction partners at pH 6.5.
Fig. 1. (a) pHꢀsensitive imine formation and hydroysis, (b) redoxꢀ
sensitive disulfide formation and cleavage, (c) hemithioacetal formation.
75
The fourꢀnode network can be analyzed starting at any node,
and for the sake of experimental simplicity we started at node A.
A solution of disulfide 1, amine 2 and aldehyde 3 in D2O (15mM
of each of these three species) at pH 6.5 was prepared and
1
analyzed by H NMR spectroscopy (Fig. 3a). The presence of
80 aldehyde is confirmed by a singlet at δ = 9.9 ppm and a pair of
aromatic doublets at δ = 7.7 – 8.0 ppm. Signals at δ = 7.4 ppm
indicates the presence of the aromatic disulfide 1. Importantly,
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the spectrum indicates disulfide 1 is stable at pH 6.5 and the
absence of a signal at δ = 8.4 ppm indicates that there is no
change was accompanied by the appearance of a new pair of
aromatic doublets at δ = 6.8 – 7.2 ppm corresponding to the
unwanted imine formed at this pH. Closer examination of this 50 aromatic protons of imine 5 and the disappearance of the
spectrum does reveal a second pair of doublets of extremely low
intensity between δ = 7.5 – 7.7 ppm and a singlet at ~δ = 6.0 ppm
suggesting the presence of a small amount of hydrate. The
hydrate of 3 exists merely as a “spectating” species in low
aromatic protons associated with aldehyde 3. These observations
suggest nearꢀquantitative formation of imine 5 from aldehyde 3
and amine 2 at pH 12.0 without the formation of any unwanted
sideꢀproducts. In particular, there is no evidence for the
5
concentration at pH 6.5 and does not influence the orthogonality 55 formation of unwanted hemithioacetal as observed by the absence
of the imine and disulfide bonds.
of a singlet at ~δ = 6.1 – 6.2 ppm. Because the increase in pH
causes deprotonation of the aromatic thiol 4 resulting in thiolate
4’, its aromatic signals appear as a pair of doublets at δ = 6.7 –
7.2 ppm. It is also worthwhile to note that at pH 12 aldehyde 3 is
60 not susceptible to hydrate formation, as evidenced by the lack of
a signal at ~δ = 6.0 – 6.1 ppm .
10
65
70
15 Fig. 2 Considering only situations in which imine and disulfide DCBs are
in either “broken” or “formed” states, there are four distinct scenarios
which can be mapped onto a fourꢀnode network. In node A, the disulfide
bond is “formed” and the imine “broken”. In node B both the disulfide
and imine bonds are “broken”. In node C both the disulfide and imine
75
20
bonds are “formed”, and in node D the disulfide bond is “broken” the
imine “formed”. It is possible to successfully navigate between all nodes
through the application of the appropriate orthogonal stimuli.
The transition from node D to node C was completed by slow
addition of 0.25M H2O2 to drive the oxidation of the thiolate 4’
80 whilst maintaining pH at 11.8. The resulting H NMR spectrum
To drive the transition from node A to node B, a reductive
stimulus was applied through the addition of a slight excess of the
1
(Fig. 3c) displays a pair of aromatic doublets at δ = 7.3 ppm
corresponding to aromatic protons in 1 and the disappearance of
the pair of doublets corresponding to the thiolate 4’. A singlet
corresponding to the imine proton at δ = 8.4 ppm of 5
85 accompanied by the characteristic pair of aromatic doublets at δ =
7.6 ppm suggests the imine bond has been successfully retained.
The presence of lowꢀintensity doublets at ~δ = 8.0 ppm suggest
the presence of a very small fraction of unreacted aldehyde.
These observations indicate that successful oxidation of thiol 4 to
90 form stable disulfide 1 at pH 11.8 in the presence of imine 5.
To ensure the complete reversibility of every step within the
network, an antiꢀclockwise cycle was performed. 1H NMR
spectroscopic analysis of the system at each node displayed the
expected resonances, suggesting that the system is reversible at
95 each transition (see ESI Fig. S4).
25 organic reductant dithiothreitol (DTT). Analysis by 1H NMR
spectroscopy (Fig. 3b) reveals the appearance of a broad multiplet
at δ = 7.8 ppm associated with thiol 4 and the disappearance of
the pair of doublets at δ = 7.3 – 7.5 ppm associated with disulfide
1. This observation indicates the successful and complete
30 reduction of the disulfide
1 into thiol 4. The signals
corresponding to aldehyde 3 remain unchanged suggesting no
unwanted hemithioacetal formation has occurred as through
reaction of thiol 4 with aldehyde 3. Furthermore, signals
corresponding to amine 2 remain unchanged between nodes A to
35 B (see ESI for expanded spectra), indicating no unwanted
processes occur involving 2. A degree of unexpected spectra
broadening was observed which further NMR work indicates is
attributable to dynamic processes (see ESI for full details),
however, this spectrum is still sufficiently informative to fully
40 support the conclusions drawn.
The transition from node B to node D was driven by applying
an increase in pH, serving as a stimulus to favor the condensation
of 2 with 3 to form imine 5. The pH was raised from 6.5 to 12.0
using 10 µl aliquots of 0.1 M NaOH, and subsequent analysis by
45 1H NMR spectroscopy revealed (Fig. 3d) the disappearance of the
aldehyde proton signal at δ = 9.9 ppm and emergence of a new
singlet at δ = 8.4 ppm corresponding to imine proton in 5. This
Taken together, these experiments demonstrate the high degree
of orthogonality which can be displayed in the bond breaking and
forming processes of disulfide and imine DCBs.
To further explore the limitations of orthogonality, additional
100 experiments were performed suggesting that the disulfide and
imine bonds are orthogonal only below pH 12.0. At pH values
higher than 12.0 unwanted decomposition of disulfide 1 to yield
the thiolate 4’ occurs i.e. the stimuli which modulates the
2
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formation and hydrolysis of the imine bond is actually interfering
invaluable help and guidance with NMR spectroscopy.
with the disulfide bond. Direct attack by hydroxide resulting in
cleavage of aromatic disulfide bonds has been reported by
Danehy,12 and this undesired process sets an upper operational
limit regarding pH upon the system.
5
Alternative
reaction
partners
investigated
by
us
failed to deliver orthogonality. When electronꢀrich aldehydes
such as 4ꢀhydroxybenzaldehyde were used, it was found that
there is very little reaction with amine 2 to form imine at pH 12.0.
10 The chosen electronꢀdeficient aromatic aldehyde 3 appears to be
both relatively resistant to hydrate formation and is capable of
forming imines at pH 12.0. A series of alkyl thiols were also
investigated as substitutes for thiol 4. At pH 6.5 in D2O these
thiols engaged in nucleophilic attack at aldehyde 3, with further
15 1H NMR spectroscopic studies providing evidence for the
formation of unwanted hemithioacetals (Fig. 4). The propensity
to form hemithioacetals with electronꢀdeficient aldehydes rules
alkyl thiols out as potential thiolꢀdisulfide system components.
The chosen aromatic thiol 4 does not form hemithioacetal with
20 aldehyde 3, probably on account of aromatic thiols being poorer
nucleophiles than alkyl thiols and thus less likely to form
hemithioacetals.
45
50
Fig. 4 1H NMR spectra (300 MHz, D2O) demonstrating formation of
hemithioacetal at pH 6.5. Aldehyde 3 and RSH present in 15 mM
concentration (30 mM total). RSH = (a) 2ꢀmercaptoethyl acetate, (b)
sodium 3ꢀmercaptoꢀ1ꢀpropanesulfonate, (c) 2ꢀmercaptoethanol. The low
intensity singlet observed just upfield of the diagnostic hemithioacetal
55 signal at δ = 6.2 ppm indicates the presence of expected hydrate formed
by reaction the aldehyde with water.
Notes and references
a A Chemical Nanoscience Laboratory, School of Chemistry, Bedson
60 Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
Fax: +44 (0)191 208 6929 ; Tel: +44(0)191 208 7065; E-mail:
david.fulton@ncl.ac.uk
25
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
65 DOI: 10.1039/b000000x/
‡ We follow the definition of orthogonality as proposed2 by
Schmittel et al. as “two (or more) dynamic interactions without
crosstalk”, where without crossꢀtalk denotes that “two or more
interactions are mutually compatible and that alternative
70 combinations are not detectable by means of the applied
spectroscopic techniques”.
Fig. 3 1H NMR spectra (300 MHz, D2O) collected at each ‘node’ in the 4ꢀ
node network. (a) In node A, the disulfide bond is “formed” and the imine
30 “broken”. (b) In node B both the disulfide and imine bonds are “broken”.
(c) In node C both the disulfide and imine bonds are “formed”, (d) in
node D the disulfide bond is “broken” the imine “formed”.
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