Orthogonal or simultaneous use of disulfide and hydrazone exchange in dynamic covalent chemistry in aqueous solution

Article abstract of DOI:10.1039/b808725c

Hydrazone and disulfide exchange have been combined in a single system, but can be addressed independently: by adjusting the pH of the solution from acidic to mildly basic it is possible to switch from exclusively hydrazone exchange to exclusively disulfide exchange, while at intermediate pH both reactions occur simultaneously. The Royal Society of Chemistry.

Full text of DOI:10.1039/b808725c

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Orthogonal or simultaneous use of disulfide and hydrazone exchange in
dynamic covalent chemistry in aqueous solutionw
a
ab
Zaida Rodriguez-Docampo and Sijbren Otto*
Received (in Cambridge, UK) 22nd May 2008, Accepted 16th July 2008
First published as an Advance Article on the web 11th September 2008
DOI: 10.1039/b808725c
Hydrazone and disulfide exchange have been combined in a
single system, but can be addressed independently: by adjusting
the pH of the solution from acidic to mildly basic it is possible to
switch from exclusively hydrazone exchange to exclusively
disulfide exchange, while at intermediate pH both reactions
occur simultaneously.
Dynamic covalent chemistry combines the robustness of
covalent bonds with the proof-reading ability of noncovalent
1
self-assembly. This makes it a powerful tool for the synthesis
2
of impressively complex structures. Reversible covalent reac-
tions also form the core of the topical field of dynamic
3
combinatorial chemistry and provide an exciting entry into
4
the emerging field of systems chemistry.
With the number of reversible covalent chemistries at our
disposal increasing steadily, studies are starting to appear
which combine two or more reversible chemistries in a single
Scheme 1 Reactions involved in disulfide and hydrazone chemistry.
5
system. Some of the most striking complex molecular
requirements and experimental conditions that would enable
the orthogonal use of the two chemistries.
2a
architectures such as interlocked structures or supramolecu-
2
b,c
lar assemblies
of covalent exchange reactions and more labile metal
are the result of the successful combination
Compound 1 (Fig. 1) is a suitable model system containing
two hydrazide termini that can be reacted with two equivalents
of aldehyde to give the desired hydrazone product, which can
subsequently undergo hydrazone exchange upon introducing a
second aldehyde. Reaction of the central disulfide bond with a
thiol would allow us to probe disulfide exchange. In the
presence of air irreversible thiol oxidation will also occur.
Through a combination of these four different reactions
2
a,c
2b,5c
coordination
or hydrogen bonds.
If two or more
reversible chemistries could be switched on and off indepen-
dently this would offer enhanced control and create new
opportunities for evolving the system by alternating use of
the different chemistries. We now report the first example of
two dynamic covalent chemistries in a single system that can
be either operated fully orthogonallyz or occur simultaneously
(Scheme 1), a mixture of hydrazide/disulfide 1, aldehydes 2
and 4 and thiol 3 can potentially produce products 5–13 depicted
in Fig. 2. With four simple experiments (A–D) we show how the
(
albeit slowly), depending on the pH of the solution.
We focussed our efforts on combining disulfide and hydra-
zone exchange, which are currently two of the most popular
reversible covalent exchange reactions. In general, the optimum
3
pH for disulfide exchange in water is 7–9 (Scheme 1), while in
the same solvent hydrazone exchange requires acid catalysis
3
pH 2.5 to 5; Scheme 1). Outside these ranges the efficiencies of
(
the reactions generally decrease. However, exceptions to these
rules have been reported where hydrazone exchange occurs at
6
a
6b
higher pH and disulfide exchange at lower pH. The goal
of the present investigation was to establish the structural
a
Department of Chemistry, University of Chemistry, Lensfield Road,
Cambridge, UK CB2 1EW. Fax: +44 1223 336017;
Tel: +44 1223 336509
Stratingh Institute for Chemistry, University of Groningen,
b
Nijenborgh 4, 9747 AG Groningen, The Netherlands.
E-mail: s.otto@rug.nl
w Electronic supplementary information (ESI) available: Materials
and experimental procedures; LC-MS characterisation data; data for
disulfide exchange with various thiols at pH 4.5–2.5. See DOI:
1
0.1039/b808725c
Fig. 1 Building blocks and their schematic representations.
Chem. Commun., 2008, 5301–5303 | 5301
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oxidation and disulfide exchange depend on the pH of the
solution.
In experiment A we mixed disulfide 1, aldehyde 2 and thiol 3
at pH 8.5. We analysed the mixture after 3 days by HPLC,
using two different wavelengths: l = 290 nm corresponding to
the maximum UV absorbance of the hydrazone moiety and
l = 245 nm which allows monitoring of furanmethanethiol 3.y
We were able to directly observe thiol oxidation giving rise to
disulfide 7 (Fig. 3a). Disulfide exchange could only be
confirmed indirectly, as we were unable to detect any free
hydrazides including 8 and 10 (and parent compound 1) with
our chromatographic methods. Thus, within 48 h after
acidification to pH 2.5 we observed the formation of 6 and
Fig. 2 Schematic representations of possible products.
9, which are the expected hydrazone derivatives of disulfide
exchange product 8 and thiol 10, respectively. No change in
the concentrations of 3 and 7 was observed after addition of
acid, suggesting disulfide oxidation and exchange no longer
occurred at pH 2.5.
formation of these products can be controlled by activa-
ting or deactivating the disulfide and hydrazone formation and
exchange processes. We first set out to establish how thiol
Fig. 3 HPLC analyses recorded at l = 290 nm (green traces) and l = 245 nm (black traces) for the mixtures prepared from hydrazide 1
5.0 mM); aldehyde 2 (10 mM) and thiol 3 (10 mM) in (a) 1 : 1 CH CN–ammonium acetate buffer at pH 8.5 after 3 days (top two chromatograms)
and 2 days after the addition of formic acid to reach pH 2.5 (bottom two chromatograms); (b) 1 : 1 CH CN–ammonium formate buffer at pH 2.5
after 3 days (top two chromatograms) and 2 days after the addition of Et N to reach pH 8.5 (bottom two chromatograms); (c) HPLC traces
recorded at l = 290 nm for the mixtures containing hydrazide 1 (5.0 mM) and aldehyde 2 (10 mM) (top chromatogram) and 3 days after the
addition of 1 equiv. (5.0 mM) of aldehyde 4 in 1 : 1 CH CN–ammonium formate buffer at pH 2.5 and in 1 : 1 CH CN–ammonium acetate buffer at
pH 4.5 and 8.5; (d) HPLC analyses of the mixture prepared from hydrazide 1 (5.0 mM); aldehyde 2 (10 mM) and thiol 3 (10 mM) in 1 : 1
CH CN–ammonium acetate buffer at pH 4.5 after 5 days. All samples, except those at pH 4.5, had reached stable product distributions well within
the specified reaction times.
(
3
3
3
3
3
3
5
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In experiment B the same three starting materials (1–3) were
mixed at pH 2.5 and analysed after 3 days. Only thiol 3 and
hydrazone 5 are observed (Fig. 3b). The absence of disulfide 6
and thiol 9 proves that disulfide exchange does not take place
and the absence of disulfide 7 indicates also that disulfide
oxidation does not happen at this low pH. However, upon
raising the pH to 8.5 disulfide oxidation and exchange were
activated: after 48 h thiol 3 was completely consumed and
disulfide exchange product 6 had appeared.
significant rate of exchange even at pH 2.5 (see ESIw). This
a
difference reflects the higher pK of aliphatic thiols (ca. 9) as
8
compared to that of their aromatic counterparts (ca. 6.5).
In conclusion, we have identified conditions under which
two different covalent exchange reactions can be used
orthogonally or simultaneously (albeit slowly) in the same
system. Depending on the pH, hydrazone or disulfide
exchange can be selectively activated or deactivated. Different
product distributions are obtained depending on the order in
which the two exchange processes are activated, indicating
that the equilibrium of the system can be shifted in directions
different from the global energy minimum attained when both
reactions occur simultaneously.8 These findings pave the way
to future construction of more complex molecular architec-
The same two experiments also provide information about
the pH dependence of hydrazone formation. Experiment A
demonstrates that with aldehyde 2 no significant hydrazone
formation occurs at pH 8.5, while it is rapid at pH 2.5
(
(
Fig. 3a). The results of experiment B confirm this conclusion
Fig. 3b). At pH 2.5 building blocks 1 and 2 react rapidly to
2
tures and chemical systems capable of being evolved by
give dihydrazone 5, although some free aldehyde 2 can still be
observed. After raising the pH to 8.5, the amount of 5
decreased solely as a result of disulfide exchange. The fact
that the peak for aldehyde 2 remained constant indicates that
at pH 8.5 no additional hydrazone is produced.
alternating use of the two reversible covalent chemistries.
This work was supported by the EU (Marie Curie EST 8303;
ChemBioCam); the EPSRC and the Royal Society. We thank
Dr Ana Belenguer for help with the HPLC/ESI-MS analysis.
In experiment C we probed the pH dependence of the
hydrazone exchange reaction (Fig. 3c). We mixed building
block 1 with aldehyde 2 at pH 2.5, giving the dihydrazone 5.
The pH of the mixture was then raised to 8.5, followed by
addition of 1 equiv. of 3,5-dihydroxybenzaldehyde 4. Compar-
ison of the HPLC traces before and 3 days after the addition of
Notes and references
z With orthogonal we imply the ability to selectively activate one
exchange process while the other occurs slowly enough to be negligible
on the same timescale.
y The maximum of UV absorbance for furanmethanethiol 3 (l =
2
39 nm) is close to the acetonitrile absorbance (l = 230 nm).
4
showed that the peaks corresponding to compounds 2 and 5
However, monitoring at 245 nm allowed the analysis of the mixtures,
albeit with a considerable drift in the baseline reflecting the increasing
amount of acetonitrile during gradient elution.
were not altered, indicating that no hydrazone exchange took
place at this pH. We did detect a small amount of hydrazone 11,
which therefore can only have formed through reaction of
aldehyde 4 with monohydrazide 13 (itself not detectable using
our HPLC method). We then reduced the pH back to 2.5 and
observed rapid hydrazone exchange to give the new hydrazone
z Aldehyde 2 is deactivated by the para-OH group (electron donation
through resonance; Hammett s
activated by the two meta-OH groups (electron withdrawal by induc-
tion, Hammett s = +0.13).
p
= ꢁ0.38), while aldehyde 4 is
m
8 This difference most likely results from the different environments
experienced by the different exchange processes, with respect to both pH
and the chemical nature of the non-exchanging end of the molecule.
12 together with more hydrazone 11, at the expense of 5.
While experiments A–C demonstrate that hydrazone exchange
and disulfide exchange are orthogonal when operating at either
pH 2.5 or 8.5, respectively, it is also possible to operate both
chemistries simultaneously. Mixing building blocks 1, 2 and 3 at
pH 4.5 generated all the possible products expected from the
simultaneous occurrence of thiol oxidation, disulfide exchange
and hydrazone formation: 5, 6, 7 and 9 (experiment D; Fig. 3d).
Hydrazone exchange at this pH was studied in an independent
experiment. The addition of aldehyde 4 to a pre-formed mixture
of 1 and 2 at pH 4.5 resulted in reaction with dihydrazone 5
displacing aldehyde 2 to produce 11 and 12. Hydrazone
exchange at pH 4.5 was much slower than at pH 2.5, as noticed
1
2
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3
previously, so that after 3 days the mixture had not yet reached
equilibrium (third trace in Fig. 3c). However, the rate of
7
exchange can be increased by the addition of aniline, producing
within 4 h the same product distribution as that obtained at
pH 2.5 (see fourth trace in Fig. 3c and ESIw Fig. S14).
Finally, we briefly investigated the generality of the experi-
mental protocol using different building blocks. We noticed that
aromatic aldehydes activated by electron withdrawing groups
form hydrazones more readily than those with electron
donating groups, as apparent from the different tendency for
hydrazone formation exhibited by aldehydes 2 and 4 in experi-
ments A and C.z Also the aliphatic nature of thiol 3 turned out
to be crucial for orthogonality. Aromatic thiols exhibited a
6
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8
(a) F. G. Bordwell and D. L. Hughes, J. Org. Chem., 1982, 47,
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3
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