Enhancing the kinetics of hydrazone exchange processes: An experimental and computational study

Article abstract of DOI:10.1039/c9ob00058e

The capacity of hydrazone bonds to readily undergo component exchange processes sees their extensive utilization in dynamic combinatorial chemistry. The kinetics of hydrazone exchange are optimal at pH ～4.5, which limits the use of hydrazone-based dynamic combinatorial libraries, particularly for biological targets which are only stable at near-neutral pH values. It would thus be advantageous if hydrazone exchange proceeded with faster rates at pH values closer to neutral. We experimentally and computationally evaluated the hypothesis that hydrazones possessing neighbouring acidic or basic functional groups within the carbonyl-derived moitety of the hydrazone would enhance exchange rates. Our work suggests that judiciously placed N- or O-hydrogen bond acceptors within the carbonyl-derived moiety of the hydrazone stabilize transition states via hydrogen bonding interactions, providing a valuable boost to exchange kinetics at near-neutral pH values. We anticipate these findings will be of interest in dynamic combinatorial chemistry, dynamic covalent polymers/materials, functionalized nanoparticles and interlocked molecules, all of which may benefit from hydrazone exchange processes able to operate at near-neutral pH values.

Full text of DOI:10.1039/c9ob00058e

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Enhancing the kinetics of hydrazone exchange
processes: an experimental and computational
study†
Cite this: DOI: 10.1039/c9ob00058e
a
Patrick L. Higgs, a Antonio J. Ruiz-Sanchez, a Milene Dalmina,
Benjamin R. Horrocks, a Andrew G. Leach *b and David A. Fulton
*
a
The capacity of hydrazone bonds to readily undergo component exchange processes sees their extensive
utilization in dynamic combinatorial chemistry. The kinetics of hydrazone exchange are optimal at
pH ∼4.5, which limits the use of hydrazone-based dynamic combinatorial libraries, particularly for biologi-
cal targets which are only stable at near-neutral pH values. It would thus be advantageous if hydrazone
exchange proceeded with faster rates at pH values closer to neutral. We experimentally and computationally
evaluated the hypothesis that hydrazones possessing neighbouring acidic or basic functional groups within
the carbonyl-derived moitety of the hydrazone would enhance exchange rates. Our work suggests that
judiciously placed N- or O-hydrogen bond acceptors within the carbonyl-derived moiety of the hydrazone
stabilize transition states via hydrogen bonding interactions, providing a valuable boost to exchange kinetics
at near-neutral pH values. We anticipate these findings will be of interest in dynamic combinatorial chem-
istry, dynamic covalent polymers/materials, functionalized nanoparticles and interlocked molecules, all of
which may benefit from hydrazone exchange processes able to operate at near-neutral pH values.
Received 9th January 2019,
Accepted 25th February 2019
DOI: 10.1039/c9ob00058e
rsc.li/obc
S-transferase. The relatively high concentrations of aniline
required (100 mM) to enhance the rate of component exchange
Introduction
The field of dynamic combinatorial chemistry1 requires chemi- can limit significantly the wider biocompatibility of the
cal bonds that readily undergo component exchange pro- organocatalyst approach, and to this end Kool et al. have devel-
cesses. One of the most utilized is the hydrazone bond, (Fig. 1) oped6 improved catalysts which can provide rate enhance-
which has optimal exchange kinetics at pH 4.5, being con- ments of up to eight times that of aniline catalysis at lower
siderably slower at neutral pH.1b,2 The requirement to operate concentrations of catalyst.
at lower pH limits significantly the scope and application of
While investigating hydrazone and oxime formation at
hydrazone-based dynamic combinatorial libraries as many neutral pH, Kool and co-workers also studied6a an alternative
interesting biological templates are only stable at near neutral approach to organocatalysis in which structural modifications
pH values, and thus it would be advantageous if hydrazone of aldehyde components can increase the rate of hydrazone or
exchange were able to operate on an experimentally useful oxime formation at neutral pH. These structural modifications
timescale at pH values closer to neutral.
involve the inclusion of neighbouring acidic or basic func-
Inspired by the work3 of Jencks in the 1960s, Dawson and tional groups or atoms within the carbonyl-derived moiety of
co-workers demonstrated4 that aniline can successfully cata- the hydrazone that assist proton transfer within the rate limit-
lyse exchange processes at neutral pH, and aniline catalysis ing step, thus lowering transition state energies and enhancing
was applied5 successfully in a hydrazone-based dynamic com- the rate of hydrazone formation. We reasoned that these struc-
binatorial library for the discovery of inhibitors of glutathione tural modifications may also help increase the rate of hydra-
zone exchange processes at neutral pH. Thus, in this work, we
aChemical Nanoscience Laboratory, Chemistry-School of Natural and Environmental
Sciences, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU,
UK. E-mail: david.fulton@ncl.ac.uk; Tel: +44(0)191 208 7065
bSchool of Pharmacy and Biomolecular Sciences, James Parsons Building, Liverpool
John Moores University, Byrom Street, Liverpool, L3 3AF, UK.
E-mail: A.G.leach@ljmu.ac.uk; Tel: +44(0)151 231 2404
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob00058e
Fig. 1 Hydrazones undergo reversible component exchange through
transimination processes where a hydrazone reacts with a hydrazide to
afford a new hydrazone and hydrazide.
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investigated exchange kinetics for a small pool of hydrazones
containing acidic or basic functional groups/atoms and ration-
alized their observed order of reactivity by computational
studies. Our computational model indicates that the rate
enhancements likely arise on account of the abilities of neigh-
bouring functional groups/atoms to form stabilizing hydrogen
bonds within the transition state. Furthermore, this model cor-
rectly identified benzodihydropyran (benzoDHP) as a candi-
date rate-enhancing group – a prediction that was initially sur-
prising given the absence of any acidic or basic moieties
within benzoDHP – which was verified by experiment to be the
fastest performing group, demonstrating that useful enhance-
ments in rate can be obtained.
Fig. 3 Relative rates of component exchange for hydrazones 1a–f,
which were obtained from the forward rate constant values kf (see ESI†
for details). Error bars show 1σ confidence intervals.
Results and discussion
Experimental hydrazone exchange studies
Hydrazones 1a–c and 1e–f contain a basic nitrogen or acidic
group either upon or within the aromatic moiety of the carbonyl
components which we postulated would likely influence
the kinetics of exchange. These specific substrates were
chosen based upon the work6a of Kool et al., where they dis-
played relatively high rate enhancements for hydrazone
formation and thus are sensible starting points to investigate
their influence on hydrazone exchange. Hydrazone 1d contains
no potential rate-enhancing structural features, and thus serves
as a control.
When considering the application of hydrazone bonds in
dynamic combinatorial chemistry, one must take account of
several important requirements. It is crucial that equilibria lie
very much on the side of product hydrazone, and thus aro-
matic aldehyde partners are often used as the extended conju-
gation of the resultant hydrazone ensures product stability,
especially important when operating in aqueous solutions.
Aliphatic aldehydes, on the other hand, tend to form hydra-
zones where the equilibrium is less towards the desired hydra-
zone. Furthermore, acyl hydrazide reaction partners are used to
ensure reasonable rates of component exchange as other classes
of hydrazides/hydrazines often form hydrazones which undergo
component exchange on too slow a timescale to be useful. With
these considerations in mind, we focused upon a small pool of
hydrazones 1a–f (Fig. 2) (for synthetic procedures see ESI†).
Component exchange to form hydrazones 2a–f was accom-
plished by reaction of hydrazones 1a–f with an excess of acyl
hydrazide 3 (see ESI† for experimental details). Acyl hydrazides
3 and 4 possess hydroxyl and quaternary ammonium groups,
respectively, which ensure water solubility of their associated
hydrazones. Exchange reactions (see ESI† for details) were
1
monitored by H NMR spectroscopy at room temperature over
a range of pD values (5.4–7.4).‡ The mole fraction of each
species in solution was determined at each time point from
the normalized integrals of diagnostic protons. We found any
diagnostic signal could be used as a spectral handle to quan-
tify the rate of component exchange, and for experimental sim-
plicity we chose to utilize signals associated with hydrazones
1a–f and exchange product 4 (Fig. 2). 1H NMR spectroscopic
integral analysis afforded kinetics traces (see ESI, Fig S10†)
from which the second order rate constants (kf and kr) were
determined (see ESI† for details), allowing for the relative rates
of hydrazone exchange (Fig. 3) to be deduced.
As anticipated, the kinetics of exchange of all examples
were faster as the pD decreased. The rates of exchange are 2–8
times faster at the lowest pD investigated (5.4) compared to the
highest pD (7.8), observations consistent with component
exchange being accelerated by protonation. Component
Fig. 2 Component exchange of hydrazones 1a–f with acyl hydrazide 3
to form hydrazones 2a–f and acyl hydrazide 4, a process which was
studied by 1H NMR spectroscopy. (A) Exchange of 1a–f was studied both
experimentally and modelled computationally as
a symmetrical
exchange process, where AcNHNH2 is both the attacking nucleophile,
and hydrazide component of the hydrazone. (B) Substrates h–j were
only studied computationally, and based upon the outcomes of this
work, g was progressed to experimental study.
‡All experimental studies are performed in D2O and thus acidities were
measured in pD. Computational studies which were modelled in H2O with
acidity measured in pH. pD can be related to pH by the simple calculation pD =
pH + 0.42.21
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Fig. 4 Three mechanisms of hydrazone exchange which were explored computationally. (A) Process (1): No protonation of hydrazone prior to
hydrazide attack (uncatalysed reaction). (B) Process (2): Protonation of proximal acid/base group within aldehyde component of hydrazone prior to
attack. (C) Process (3): Protonation of hydrazone nitrogen (NI) prior to attack. Energetics (kcal mol−1) were calculated at pH 7, whilst values for pH 5
are in brackets. Process (3) represents the most likely mechanism for hydrazone exchange, on account of having low energy barriers, relative to
Processes (1) and (2). (D–F) Transition state structures P1TS, P2TS and P3TS, respectively.
exchange was fastest with hydrazone 1a, being approximately 5 knowledge, this is the first computational study to explicitly
times faster than control hydrazone 1d at all pD values investi- examine the mechanism of hydrazone exchange. Three poss-
gated, suggesting that the inclusion of a proximal basic nitro- ible hydrazone exchange mechanisms were considered and
gen may catalyse hydrazone exchange. Surprisingly, hydra- studied at the M06-2X/6-31G* level8 of theory (a level that is
zones 1e and 1f – both of which possess proximal acid/basic expected to produce reasonable agreement with barrier
groups
–
were observed to undergo slower component heights). All calculations were performed in Gaussian099 and
exchange processes (at all pD values) than the control hydra- included implicit solvation using the PCM protocol.§ To sim-
zone 1d. This result was initially surprising as we had antici- plify calculations, the hydrazide employed in modelling the
pated that the hydrazone containing the most basic group exchange processes was AcNHNH2, which was also used as the
would best catalyse the hydrazone exchange process as it hydrazide component within the hydrazone. The overall
would exhibit the greatest likelihood of being protonated and process modelled was therefore a symmetrical exchange. We
so be able to transfer a proton in the rate limiting step; the pKa considered firstly process (1) (Fig. 4A) in which no protons
of the pyridyl nitrogen is estimated to be 5.14,7 which is were added into the system. In the calculated transition state
higher than that of the quinoline (pKa = 4.85)7 and the (P1TS), proton shuttling between the incoming nucleophile
benzoic acid (pKa = 4.20)7 suggesting that pyridine 1e should and the hydrazone was required, and a single water molecule
undergo the fastest hydrazone exchange. Our observed can fulfill this role by simultaneously removing a proton from
order of reactivity (quinoline > phenol > phenyl > pyridine ≈ the incoming hydrazine and protonating the hydrazone. This
carboxylate) does not correlate with the pKa value of the proton shuttling leads to a neutral tetrahedral intermediate
proximal acid/basic groups, an observation which suggests (P1Int) that would be expected to collapse either to reactants
that the rate enhancement is not caused by protonation of this or products through similar barriers. In process (2) (Fig. 4B),
group.
groups located within the aldehyde-derived moiety of the
hydrazone were protonated to give reaction precursors (P2Prot)
before nucleophilic attack by the hydrazide, a process that pro-
Computational hydrazone exchange studies
In order to better understand our experimental observations,
computational studies were undertaken. To the best of our §All species were characterized by frequency calculations.
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Fig. 5 Hydrogen bonding interactions stabilise the transition states for hydrazone exchange. The energy barriers (ΔG†) corresponding to each TS
structure were calculated at pH 7.4 and pH 5.4 (brackets). (A) 6-membered cyclic TS (benzoDHP 1g, quinoline 1a, phenol 1b) have the lowest ener-
gies and exhibit the fastest hydrazone exchange. (B) 5-membered cyclic TS (pyridine 1c, thiophenol 1j), 7-membered TS (carboxylate 1f). (D)
Substrates 1h–j were only studied computationally.
ceeds through a similar transition state (P2TS) to process (1).
For process (1), the lowest computed free energy barrier
This mechanism leads to a protonated intermediate (P2PI) (and therefore fastest reaction) is for carboxylate 1f ¶ (see ESI,
that can either rearrange (intramolecular proton transfer) or Table S19†) whilst the highest energy process (and therefore
return a proton to the surrounding environment. The com- slowest) involves quinoline 1a, observations that are not con-
puted energetics (see ΔG† values in Fig. 4) are those calculated sistent with experiment and therefore process (1) was dis-
for pH 7, while those for pH 5 (where different) are in brackets. counted. The energetics calculated for process (2) (ESI,
The calculated values reveal that process (2) features a high Table S20†) predict that: (i) the most reactive substrate is
energy barrier, with ΔG† values exceeding 30 kcal mol−1, indi- control compound 1d – which is absent of any acid/basic
cating that protonation of the functional group/atom within groups to catalyse the reaction; and (ii) the least reactive sub-
the aldehyde-derived moiety likely impedes the exchange strate is 1a – which was experimentally observed to have the
process. We then considered process (3) (Fig. 4C), which rep- fastest hydrazone exchange kinetics. Process (2) was not con-
resents a specific acid-catalysed reaction in which hydrazone sistent with the observed order of reactivity and was dis-
nitrogen (N1) is protonated prior to nucleophilic attack by the counted. The computed barrier heights for process (3) (ESI,
hydrazide. This protonation gives protonated hydrazone Table S21†) however, predict an order of reactivity (pH 7.4: 1a
(P3PH) that is attacked by hydrazide through transition state > 1b > 1d ≈ 1e > 1f; pH 5.4: 1a ≈ 1b > 1d ≈ 1e > 1f) that was
P3TS to give a protonated tetrahedral intermediate P3PTI. For consistent with the observed relative rates (Fig. 3), further sup-
process (3) the calculated free energy barrier (see ΔG† values)
is significantly lower than those obtained for processes (1) and
(2), indicating that process (3) constitutes the most likely
mechanism for hydrazone exchange. The pathway with the ¶Further modelling of 1f revealed that the carboxylate moiety may ring-close
upon the aminal-like intermediate to form a meta-stable 5-membered cyclic
lowest free energy barrier is likely to be the one that is oper-
ational but there are significant uncertainties in these com-
parisons and therefore the ability of each process to explain
structure (Fig. S24†), although no evidence of this species was observed during
1H NMR experiments. This meta-stable presented a local minimum on the
potential energy surface, making it challenging to reliably compute energetics
the relative reactivity of the different hydrazones was also
considered.
that were consistent with a single exchange mechanism. Energetics of all other
substrates however, were in close agreement with process (3).
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porting the idea that process (3) constitutes the most likely
mechanism of hydrazone exchange.
We then further scrutinized the key species that governs
reactivity via process (3), the transition state for hydrazide
attack (P3TS). The origin of the high reactivity of 1a was
revealed in the corresponding transition state structure
(Fig. 5A), which features two hydrogen bonds from the quino-
line nitrogen to both the incoming hydrazide (N–H distance:
3.18 Å) and the protonated hydrazone (N–H: 1.96 Å), that stabil-
ize the transition state.10 Crucially, these stabilizing interactions
help to lower the energy barrier for hydrazone exchange, thus
providing a boost in the exchange kinetics. In the analogous
transition states for pyridine 1e (N–H: 3.11 Å, 2.25 Å) and car-
boxylate 1f (O–H: 2.23 Å, 2.21 Å) (Fig. 5B and C) these distances
are longer, suggesting that of the two interactions it is the
hydrogen bond to the protonated hydrazone that governs reac-
tivity. It has been noted previously11 that 6-membered ring
intramolecular hydrogen bonding interactions (such as those
that operate for quinoline 1a) are slightly favoured over their
5-membered equivalent (as for pyridine 1e) and much favoured
compared to their 7-membered equivalent (carboxylate 1f).
Thus, it is the ideal spatial positioning and orientation of hydro-
gen bond acceptor atoms/groups within the transition state,
rather than simply the presence of acidic or basic moieties, that
leads to increased reactivity in hydrazone exchange.
Fig. 6 Hydrazone exchange kinetics of 1a, 1d and 1g were studied at
pD 7.8 by 1H NMR spectroscopy. (A) Exchange of hydrazones 1a,d,g with
hydrazide 5. (B) Kinetic traces of 1a, 1d, 1g. Experimental data and theore-
tical fit are shown as circles and solid lines, respectively. Inset: Derived rela-
tive rates for hydrazone exchange. See ESI† for absolute rate constants.
With this thought in mind, alternative oxygen-containing
substrates 1g–i, and thiophenol 1j (Fig. 5) were considered
computationally as a test of this model, as such substrates
contain hydrogen bond acceptors, but lack the suitably acidic/
basic groups required to catalyse the reaction via intra- troscopy and the relative rates of exchange were deduced (see
molecular proton transfer. Amongst these examples, ESI Table S15†). It was necessary to study this exchange
benzoDHP 1g was predicted to be faster than all the other process with morpholine hydrazide (5) instead of glycol hydra-
compounds studied experimentally and was therefore selected zide 3, as exchange of 1g with 3 resulted in product precipi-
for synthesis. The origin of this predicted rate enhancement tation that convoluted the exchange kinetics (see ESI,
was clear in the structure of the relevant transition state for 1g Fig. S12†). Gratifyingly, 1g displayed a 2-fold rate enhancement
(Fig. 5A). The heterocyclic oxygen atom in this species is posi- with respect to quinoline 1a (Fig. 6B), highlighting the predic-
tioned in such a way that it can form two stabilizing inter- tive power of our computational model of hydrazone exchange.
actions through a favoured 6-membered ring: one with the pro-
Despite mechanistic differences between hydrazone
tonated hydrazone (2.04 Å, O–H) and a second with the incom- exchange and formation processes, we speculate that the rate-
ing nucleophile (2.73 Å, O–H). Short hydrogen bonding inter- enhancing effects observed for proximal acid/base groups
actions within the transition state indicate stronger stabilizing upon hydrazone formation may also arise on account of hydro-
interactions, the likes of which lower the transition state gen bonding interactions which lower the activation energies
energy to a greater extent, thus resulting in faster hydrazone (by stabilising the transition states). The ability of those
exchange. The transition state of benzoDHP 1g features two groups to facilitate intramolecular proton transfer, as postu-
such short hydrogen bonds, which are considerably lated by Kool et al.,6a–d will also be a contributing factor.
shorter (and therefore presumably stronger) than analogous Preliminary experiments (ESI, Fig. S16 and S17†) revealed that
bond lengths calculated for quinoline 1a (N–H: 1.96 Å, 3.18 Å) chroman-8-carbaldehyde (the aldehyde from which benzoDHP
and phenol 1b (O–H: 2.03 Å, 2.93 Å). This observation suggests 1g was derived) exhibited rapid hydrazone formation, reacting
that 1g would offer significantly improved exchange kinetics 15-fold faster than quinoline-8-carbaldehyde, and 26-fold
over quinoline 1a, an already fast exchanging hydrazone. We faster than benzaldehyde, which lacks any rate-enhancing fea-
then experimentally validated this hypothesis by determining tures. The intriguing observation that the benzoDHP moiety
the hydrazone exchange kinetics of benzoDHP 1g (Fig. 6).
catalyses rapid hydrazone formation, despite its lack of a sig-
nificantly acidic or basic group to facilitate intramolecular
proton transfer processes, supports our hypothesis that hydro-
Exchange kinetics of benzoDHP (1g)
Hydrazones 1a, 1d and 1g were exchanged with hydrazide 5 gen-bonding interactions play an important role within the
(Fig. 6A) and the kinetics were monitored by 1H NMR spec- context of organocatalysed hydrazone formation, and probably
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also the mechanistically similar processes of imine and oxime
formation.
Conflicts of interest
There are no conflicts of interest.
Conclusion
Acknowledgements
We have demonstrated that the judicial placement of neigh-
bouring hydrogen-bond acceptors within the carbonyl-derived
moiety of a hydrazone does lead to enhancements in rates of
hydrazone exchange. Computational modelling identified a
likely reaction pathway for this process whose energetics were
consistent with experimentally determined exchange rates.
Modelling supported the hypothesis that the rate-determining
step in hydrazone exchange was nucleophilic attack on the pro-
tonated hydrazone, which is an important distinction between
hydrazone exchange and hydrazone formation, where the rate-
limiting step is collapse of the carbinolamine tetrahedral inter-
mediate. Crucially, modelling indicated that the origin of the
observed rate enhancements lies in the ability of neighbouring
functional groups to form a stabilizing hydrogen bonds within
the transition state, and that geometries where 6-membered
ring intramolecular hydrogen bonding interactions can be
adopted are particularly important. Our confidence in this
model was demonstrated by its prediction that a benzoDHP
group – containing a very weakly basic but optimally placed
oxygen atom that acts as a hydrogen-bond acceptor – displayed
fast exchange kinetics, which was gratifyingly supported by
experimental observation. Preliminary experiments revealed
that chroman-8-carbaldehyde (from which BenzoDHP 1g was
derived) also catalyses rapid hydrazone formation. Surprisingly,
chroman-8-carbaldehyde was found to react 15-fold faster than
previously reported quinoline-8-carbaldehyde,6a despite its lack
of an acidic/basic group. These observations suggest that the
inclusion of hydrogen-bond acceptor moieties within the alde-
hyde component may also play an important role in catalysing
hydrazone formation, alongside the previously reported6a–d cata-
lytic effect of proximal acid/base groups. At neutral pD,
benzoDHP 1g was observed to afford an 2-fold enhancement in
the rate of hydrazone exchange, compared to that of quinoline
1a, and was 10-fold faster than control hydrazone 1d. With
regards to our own interest in dynamic combinatorial chemistry,
our work suggests that valuable gains in rate of exchange can be
made that would allow the design of a polymer-scaffolded
DCLs12 operating with reasonable kinetics at near-neutral pH –
a crucial requirement for interfacing DCLs with biomacro-
molecules.13 Furthermore, given the importance of hydrazone
exchange within dynamic covalent polymers,14 materials,15 sur-
faces,16 molecular machines,17 interlocked molecules,18 cages19
and functionalized nanoparticles,20 where component exchange
processes endow structural adaptivity, we speculate this work
will offer insight to the design and optimization of new
systems. We also anticipate our work will benefit the develop-
ment of new organocatalysts for hydrazone/oxime formation
and exchange processes, indicating that computational studies,
on account of their ability to ‘pick winners’, might minimise
tedious preliminary screenings for catalytic activity.
The authors wish to thank Dr Rachael Dack (Newcastle
University) and Dr Jackie Mosely (Durham University) for
assistance with HRMS.
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