Optimizing the reversibility of hydrazone formation for dynamic combinatorial chemistry

Article abstract of DOI:10.1039/b211645f

Hydrazones from hydrazines bearing electron withdrawing groups, and aromatic or aliphatic aldehydes form and hydrolyse rapidly in water at neutral pH.

Full text of DOI:10.1039/b211645f

Optimizing the reversibility of hydrazone formation for dynamic
combinatorial chemistry
Régis Nguyen and Ivan Huc*
Institut Européen de Chimie et Biologie, 16 Av. Pey Berland, 33607 Pessac Cedex, France.
E-mail: i.huc@iecb-polytechnique.u-bordeaux.fr
Received (in Cambridge, UK) 22nd November 2002, Accepted 7th March 2003
First published as an Advance Article on the web 12th March 2003
Hydrazones from hydrazines bearing electron withdrawing
groups, and aromatic or aliphatic aldehydes form and
hydrolyse rapidly in water at neutral pH.
When these compounds are mixed with a model aliphatic
aldehyde (isobutyraldehyde) or a model aromatic aldehyde
(4-carboxy-benzaldehyde) in water under close to neutral
conditions (6 < pH < 8), hydrazone formation takes place
within minutes. The 1H NMR spectra show a rapid decrease of
the signals of the aldehyde and, when present, of its hydrate,‡
with the concomitant appearance of the signals of the hydra-
zones. The hemiaminals are sometimes observed but they
remain minor species. At high hydrazine concentration, an
equilibrium with the aminal is observed. Integration of these
signals at various concentrations allows the calculation of the
equilibrium constants of hydrazone formation reported in Table
1. These values range over almost three orders of magnitude and
do not correlate with the pKBH+’s of the hydrazines.† In most
cases, the constant of hydrazone formation of a given hydrazine
is several times higher with the aromatic aldehyde than with the
aliphatic aldehyde. Within each series, the most stable hydra-
zone is formed from the amine bearing a single EWD group (1),
followed by the imine formed from the amine bearing one
For the purpose of synthetic chemistry, reversible reactions
generally inspire scepticism and are often left aside when they
cannot be improved to limit product instability. Yet, interest for
reversible reactions has been rising during the past few years,
motivated by the development of dynamic combinatorial
chemistry (DCC). This technique takes advantage of the
continuous variation of the proportions of products formed
reversibly from a set of precursors.1 For example, the presence
of a target substance in a dynamic mixture may result in an
increase of the mole fraction of the products which bind to it,
thus facilitating their identification.
For pharmaceutical applications of DCC, dynamic libraries
are directly mixed with biological targets.2 Thus, useful
reactions should be reversible under physiological conditions
and should not interfere with biological functional groups.1,3
Several reactions have been proposed for this purpose, but very
few fulfil these strict requirements. Disulfide exchange3b,3d,4 for
example may lead to cross-reaction with cysteine residues of the
target. Olefin metathesis is promising but has not yet been
optimized in water in the presence of biological targets.5
Examples of boronate ester formation5,6 and coordination to
transition metals7 have also been presented. In this communica-
tion, we show that one of the most typical reversible reactions,
namely hydrazone formation, can be optimized for DCC in
neutral water when using a particular class of hydrazines.
Imines from aliphatic amines and aromatic or aliphatic
aldehydes form and hydrolyse rapidly, but are of low stability in
neutral water. At pH 8 or below, most aliphatic amines are
predominantly protonated and the equilibrium is shifted in the
direction of hydrolysis to the point that the imines are hardly
detectable. This problem has usually been circumvented by
reducing this small percentage of imine with NaBH3CN and
assuming that the proportion of amine products reflect the
proportion of imines.3a,3e,8 On the other hand, hydrazones, acyl
hydrazones, semicarbazones and oximes formed by hydrazines,
hydrazides, semicarbazides or hydroxylamines are stable even
at low pH. However, they prove to be kinetically inert under
neutral conditions. The mesomeric effects which lead to their
high stability decrease the electrophilicity of the imine, slowing
down hydrolysis and transimination.9 Only under acidic
conditions (at pH 4 or below) or at high temperatures do
hydrolysis and transimination reach significant rates.10
Fig. 1 Structures of amines 1–6.
Table 1 Values of the pKBH+’s of 1–6 and of their equilibrium constants of
hydrazone formation with an aliphatic and an aromatic aldehyde.§
K/L mol21b
Isobutyraldehyde
K/L mol21b
4-Carboxybenzaldehyde
We speculated that hydrazine derivatives bearing electron
withdrawing (EWD) groups may form hydrazones sufficiently
activated to be hydrolysed at neutral pH (Fig. 1).† Compounds
1–6, which bear EWD groups of various strengths were selected
to test this hypothesis. Several methods give access to such
compounds and in principle, a series of similar hydrazines could
easily be prepared. Some are available from commercial
sources (3, 4). Others can be synthesised by acylation of
hydrazine precursors (1, 2),11 by aromatic nucleophilic substitu-
tion (5),12 or by amination of endocyclic nitrogens (6).13 The
effect of the electron withdrawing substituents is illustrated by
the low basicity of most of these amino groups (Table 1).
a
pKBH+
1
2
3
4
5
6
5.6
3.0
6.0
4.1
3.9c
1.9
4700
1500
440
470
164
33
29000
9900
2500
870
d
—
44
a In H2O. b In 9+1 H2O+D2O at pH 7 (20 mM phosphate buffer). The effect
of 10% D2O on the pH was neglected. c This pKBH+ is probably that of the
amine in position 6, and not that of the amine in position 9. d This value
could not be measured due to the precipitation of the product.
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CHEM. COMMUN., 2003, 942–943
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carbonyl EWD group and one phenyl ring (2). Amines having
two (thio)-carbonyl EWD groups either directly connected (3,
4), or conjugated (5, 6) give rise to significantly less stable
hydrazones.
the examples presented here are aimed at widening the class of
compounds useful for DCC applications and do not represent an
ensemble of readily compatible hydrazines. Using these
findings, we are currently developing a DCC of nucleic acids
involving aminobases such as 1-aminouracil 6 and 9-aminoade-
nine 5.
This work was supported by the CNRS and by the Ecole
Polytechnique (predoctoral fellowship to RN). We thank Dr C.
Crean for proof reading the manuscript.
The reversibility of these condensations is easily demon-
strated upon diluting a solution of hydrazone and observing its
hydrolysis into the original hydrazine and aldehyde. Equilibria
are reached within minutes at pH 6 and in less than an hour at
pH 8. An illustration of this reversibility and of the potential of
these reactions for applications in DCC is given in Fig. 2. On the
400 MHz 1H NMR spectrum of a mixture of isobutyraldehyde
and of hydrazines 2–6 (Fig. 2a), the signals of most CH(CH3)2
protons do not overlap and can be assigned to the five
hydrazones, the aldehyde and its hydrate according to their
chemical shifts when only one amine is present. Minor signals
presumably arise from the 15 possible aminals and 5 hemiami-
nals. Upon addition of an excess of 1 (Fig. 2b) the equilibria are
shifted in favour of the hydrazone, the hemiaminal and the
aminal this hydrazide forms with isobutyraldehyde.
The compatibility of hydrazones formed by 1–6 with
biological functions, and in particular with N-terminal amino
groups and lysine residues of peptides and proteins was
demonstrated by showing that the equilibrium in Fig. 2a is
unaffected by the addition of 10 equivalents (with respect to
isobutyraldehyde) of lysine hydrochloride.
Notes and references
† The hydrolysis of hydrazones is rate limited by the hydration step above
pH 6 (Fig. 1). Studies on semicarbazones, acethydrazones and p-
toluenesulfonylhydrazones14 show that this rate increases with the EWD
strength of nitrogen substituents, presumably because of an enhanced
electrophilicity of the hydrazone. Conversely, hydrazone formation is rate
limited by the dehydration step above pH 6. At pH 6–7, this step is acid
catalysed and is not affected by the EWD strength of hydrazine substituents.
Above pH 8, this step is faster for strongly EWD groups which promote a
base catalysed pathway. The amination/deamination steps are also expected
to depend upon the strength of EWD groups. These steps are not rate
determining but they directly influence the overall equilibrium constant,
which thus cannot be predicted in an simple way.
§ We observed that high phosphate buffer concentration promotes aldehyde
Cannizzaro disproportionation.
In conclusion, a delicate combination of hydrazine nitrogens
and EWD substituents allows the rapid formation and hydroly-
sis of some hydrazone derivatives under neutral conditions.
That equilibrium constants vary significantly with the nature of
the substituents may strongly bias dynamic combinatorial
mixtures in favour of the most stable products. To some extent,
this may be compensated by tuning the initial proportion of
amines accordingly, as in the experiments depicted in Fig. 2.
Alternatively, one may prefer to build libraries from hydrazines
having similar reactivities, and to keep the groups by which they
differ at positions remote from the reactive site. In this respect,
‡ Isobutyraldehyde is 45% hydrated in neutral water; the hydrate of
4-carboxy-benzaldehyde was not detected.
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1
Fig. 2 Part of 400 MHz H NMR spectra of mixtures of hydrazines and
isobutyraldehyde in H2O+D2O at pH 6 (20 mM phosphate buffer§) showing
the resonances of the CH(CH3)2 protons. The upper spectrum is obtained
upon mixing hydrazines 2 (0.33 mM), 3 (1 mM), 4 (1 mM), 5 (2.5 mM), 6
(12.5 mM) and isobutyraldehyde (1 mM). The lower spectrum is obtained
upon adding 1 (20 mM) to the above mixture.
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