do not originate from the analytical technique itself, but rather
from the fact that near stoichiometric amounts of hydrazides
were required to allow the simultaneous coexistence of the
thermodynamically much less stable imines.17 The absence of a
buffering amount of free hydrazide introduces a cross-talk
between the various equilibria in the sense that the preferred
selection of H+ over H by one scaffold will also affect the
hydrazone ratio for the other scaffolds. Such a cross-talk is
detrimental for a proper quantification of the relative thermo-
dynamic stability of each species. For that reason, the analysis
was repeated on the separate hydrazone and imine libraries,
containing 18 and 8 components, respectively. In addition,
previous NMR study on a similar non-labelled system,
13C-labelling permits an increase in complexity from 8 to 26
signals without the need for bidimensional 1H–13C HSQC
spectroscopy, specialized NMR cryo-probes or complex
data-processing protocols.5 Most importantly, much lower
compound concentrations were required (0.1 mM compared
to 10 mM) and a total of 18 out of 26 signals could be
quantified in just 20 minutes, implying that the kinetics of
these systems can be studied with enhanced time resolution,
too. It is anticipated that the combination of advanced
NMR protocols with 13C-labelling will enable the analysis of
mixtures of even higher complexity than reported here. As we
have shown, the ability to quantify the concentrations of all
components of the system provides a unique possibility to
determine quantitative structure–activity relationships in a
straightforward manner.
individual competition experiments between P1–4A and P1–4
H
were used to correlate the energy scales of the imines and
hydrazones (see ESIw). From these studies it emerges in a
straightforward manner how substituents on the scaffolds
P1–P4 affect the thermodynamic stability of the corresponding
hydrazones and imines (Fig. 1). The interpretation of these
results requires some considerations. First, in contrast to our
previous studies using P1 and P2,5,11,19 the competition experi-
ments involving different scaffolds now occur simultaneously
in the same NMR tube in a parallel set of equilibria. On one
hand, this guarantees identical screening conditions but, on
the other hand, it may complicate the analysis in the case of
crosstalk between the platforms (see above). This appears not
to be the case, since the measured differences in thermodynamic
stabilities between hydrazones P1H–P1H+ (0.0 kJ molꢀ1) and
P2H–P2H+ (0.8 kJ molꢀ1) are nearly identical to those
obtained previously from separate experiments (0.2 kJ molꢀ1
and 0.7 kJ molꢀ1).5,11 Second, it should be noted that these
experiments do not reveal information on the thermodynamic
stabilities between the different scaffolds.z In fact, all energy
levels have been arbitrarily normalized on imines P1–4A, which
are taken as a reference within each scaffold series. Third, in
this preliminary analysis we have not taken hydrazone isomerism
into account and have treated each component P1–4X as a single
species.y
Financial support from the European Research Council under
the European Community’s Seventh Framework Programme
(FP7/2007–2013)/ERC Starting Grant agreement no. 239898
and COST (CM0703) is acknowledged.
Notes and references
z In the presence of an excess of amines and hydrazides there is
no competition between scaffolds for imine- or hydrazone bond
formation.
y A full account including a treatment of hydrazone isomerism and
all energetic contributions involved will be reported in due course.
Preliminary results indicate that steric hindrance in scaffold P4 induces
the formation of two non-planar isomers in P4H in addition to the
regular two isomers originating from E/Z isomerism of the amide
bond, which are common to all hydrazones.
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This analysis provides a quantitative insight into the correlation
between structure and thermodynamic stability. A first glance
immediately reveals some key features. First, the anticipated
stabilizing interactions between phosphonate and ammonium
groups in both P2A+ (2.6 kJ molꢀ1) and P2H+ (0.8 kJ molꢀ1),
which is further enhanced upon the insertion of a methyl group
in the ortho-position to the phosphonate group (scaffold P3).
Second, the anticipated much higher intrinsic stability of hydra-
zones compared to imines. Nonetheless, after a closer look it
emerges that the overall picture is actually rather complicated. For
example, substituents affect the intrinsic stability of imines and
hydrazones (DGP H–P A = 16.8 kJ molꢀ1 against 13.6 kJ molꢀ1
2
2
for DGP H–P A) and the presence of the methoxy substituent in
1
1
P4 has a destabilizing effect in particular on the hydrazones P4H
and P4H+.y It is evident that such an interplay between
stabilizing and destabilizing energetic contributions always plays
a role in determining the composition of a dynamic system.
However, such information can only emerge if the thermodynamic
stability of all system components can be determined.
In summary, these results show that 13C isotopic labelling of
a dynamic library is a very attractive tool to study complex
dynamic systems by NMR spectroscopy. Compared to a
19 G. Gasparini, F. Bettin, P. Scrimin and L. J. Prins, Angew. Chem.,
Int. Ed., 2009, 48, 4546–4550.
c
12478 Chem. Commun., 2011, 47, 12476–12478
This journal is The Royal Society of Chemistry 2011